Compositions and methods for the expression of IL-1RA and IL-18BP

Abstract

This disclosure relates to modified viral genomes or self-replicating RNA (srRNA) and pharmaceutical compositions containing the same, as well as the use of such nucleic acid molecules and compositions for producing desired products in cell culture or in vivo. It also provides methods for modulating pharmacodynamic effects in subjects requiring such modification, as well as methods for preventing and / or treating various health conditions and diseases.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 504,564, filed on 26 May 2023. The disclosure of the above-referenced application, including all drawings, is expressly incorporated herein by reference.

[0002] Sequence listing inclusion The materials listed in the attached sequence listing are incorporated into this application by reference. The attached sequence listing XML file (named 058462_515001WO_Sequence_Listing.XML) was created on May 21, 2024, and is approximately 40,960 bytes in size.

[0003] This disclosure relates to the field of immunology, and more particularly to modified viral genomes or self-replicating RNA (srRNA) and pharmaceutical compositions containing them, as well as the use of such nucleic acid molecules and compositions for cell culture or the production of desired products in vivo. Also provided are methods for modulating pharmacodynamic effects in subjects requiring such modification, and methods for preventing and / or treating various health conditions and diseases. [Background technology]

[0004] Autoimmune and inflammatory diseases are mediated by several pro-inflammatory cytokines. Blocking one or more of these cytokines can have a significant effect on the course and symptoms of these diseases. Many therapeutic compositions and methods have been developed for the treatment of these diseases. However, many of them are unsatisfactory due to insufficient efficacy, side effects, and / or instability.

[0005] For example, interleukin-1 receptor antagonist protein (IL-1RA) 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 crucial role, reducing disease-related inflammation and cartilage degradation. 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 thought to function as a natural anti-inflammatory and immunosuppressive molecule that neutralizes the effects of high IL-18 levels during inflammation. Recombinant human IL-18BP has been used in a Phase II clinical trial in Europe to treat adult-onset Still's disease patients, and in a Phase III clinical trial using the experimental drug IL-18BP in patients with NOD-like receptor C4 (NLRC4) gene mutations characterized by severe, life-threatening systemic inflammation associated with extremely high levels of IL-18.

[0006] However, many existing methods and compositions are associated with problems related to the stability and half-life of IL-1RA and IL-18BP, as well as the quantity and rate of IL-1RA and / or IL-18BP delivered. Consequently, improved compositions and methods for delivering IL-1RA and / or IL-18BP are desirable and useful for treating conditions and pathologies mediated by interleukin-1 and interleukin-18 signaling, or abnormal inflammasome activation.

[0007] The disclosures provided herein, among other things, provide solutions to various problems that exist in previous attempts to deliver IL-1RA and / or IL-18BP, and provide improved methods for treating health conditions, including autoimmune diseases and inflammatory diseases. [Overview of the project]

[0008] This disclosure relates, in general, to the development of immunotherapeutic agents, such as recombinant nucleic acid constructs and pharmaceutical compositions containing them, for use in the prevention and management of various health conditions, including autoimmune diseases, inflammatory diseases, and cardiovascular diseases. In particular, as will be described in more detail below, some embodiments of this disclosure provide nucleic acid constructs comprising a modified genome or replicon, e.g., a sequence encoding a self-replicating RNA (srRNA), e.g., a replicon, wherein at least a portion of the nucleic acid sequence encoding the viral structural protein of the modified alphaviral genome or replicon (e.g., srRNA) is replaced with a coding sequence of a polypeptide construct comprising: (a) the coding sequence of an interleukin-1 receptor antagonist (IL-1RA) protein or a functional variant thereof, and (b) the coding sequence of an interleukin-18 binding protein (IL-18BP), wherein the coding sequences of IL-1RA and IL-18BP are functionally linked to each other, and the polypeptide construct does not contain a dimerization domain. Also disclosed are recombinant cells manipulated to contain one or more nucleic acid constructs disclosed herein, as well as pharmaceutical compositions comprising one or more of the following: (a) nucleic acid constructs of the Disclosure and / or (b) recombinant cells of the Disclosure. In certain aspects of the Disclosure, compositions and methods for modulating at least one pharmacodynamic effect in a subject, as well as methods for preventing and / or treating a variety of health conditions, including autoimmune diseases, inflammatory diseases, and cardiovascular diseases, are further provided.

[0009] In one aspect of the present disclosure, the Spectrum provides a nucleic acid construct comprising a nucleic acid sequence encoding a modified alphaviral genome or replicon (e.g., self-replicating RNA (srRNA)), wherein at least a portion of the nucleic acid sequence encoding one or more viral structural proteins of the modified alphaviral genome or replicon (e.g., srRNA) is replaced with a coding sequence of a polypeptide construct comprising (a) a coding sequence of an interleukin-1 receptor antagonist (IL-1RA) protein or a functional variant thereof, and (b) a coding sequence of an interleukin-18 binding protein (IL-18BP) or a functional variant thereof, wherein the coding sequences of IL-1RA and IL-18BP are functionally linked to each other, and the polypeptide construct does not contain a dimerization domain.

[0010] Non-limiting exemplary embodiments of the nucleic acid constructs of this disclosure (e.g., replicon constructs, e.g., srRNA constructs) may include one or more of the following features: In some embodiments, the polypeptide construct does not contain a fragment crystallization region (Fc region) of immunoglobulin. In some embodiments, the coding sequence of IL-1RA is N-terminally ligated to the coding sequence of IL-18BP. In some embodiments, the coding sequence of IL-1RA is C-terminally ligated to a second coding sequence of IL-18BP. In some embodiments, the coding sequences of IL-1RA and IL-18BP express a protein that is functional in a bioactivity assay. In some embodiments, the IL-18BP protein is IL-18BP isoform a (IL-18BPa). In some embodiments, the IL-1RA protein and / or IL-18BP protein are derived from a mammalian subject. In some embodiments, the mammalian subject is a human subject.

[0011] In some embodiments of this disclosure, the coding sequences of IL-1RA and / or IL-18BP are as follows: It is independently optimized for one or more of the following: (a) enhancing RNA stability and / or expression levels; (b) minimizing the use of rare codons and / or secondary structures; and (c) facilitating better srRNA replication and RNA production processes.

[0012] In some embodiments, the coding sequences of IL-1RA and IL-18BP in the nucleic acid constructs disclosed herein are functionally linked to each other via connector sequences encoding autoproteolytic peptides and / or internal ribosome entry sites (IRESs). In some embodiments, the autoproteolytic peptides include one or more autoproteolytic cleavage sequences from calcium-dependent serine endoprotease (Fulin), porcine rhinitis virus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), Thosea asigna virus 2A (T2A), cytoplasmic polyhedron virus 2A (BmCPV2A), Flacherie virus 2A (BmIFV2A), or combinations thereof. In some embodiments, the internal ribosome entry sites (IRESs) are derived from Kaposi's sarcoma-associated herpesvirus (KSHV) IRES, hepatitis virus IRES, pestivirus IRES, Kripavirus IRES, roparosifanpadivirus IRES, fibroblast growth factor IRES, platelet-derived growth factor IRES, vascular endothelial growth factor IRES, insulin-like growth factor IRES, picornavirus IRES, encephalomyocarditis virus (EMCV) IRES, Pim-1 IRES, p53 IRES, Apaf-1 IRES, TDP2 IRES, L-myc IRES, and c-myc IRES.

[0013] In some embodiments of the nucleic acid constructs disclosed herein, the modified viral genome or srRNA lacks a substantial portion of the nucleic acid sequence encoding one or more viral structural proteins. In some embodiments, the modified alphaviral genome or srRNA does not contain any nucleic acid sequence encoding a viral structural protein. In some embodiments, the coding sequences of IL-1RA and IL-18BP are functionally linked to each other 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 functionally ligated to a promoter sequence. In some embodiments, the promoter sequence is a subgenome (sg) promoter. In some embodiments, the sg promoter sequence is a 26S subgenome promoter. In some embodiments, the subgenome promoter is heterologous to the modified viral genome or the rest of the srRNA. In some embodiments, the subgenome promoter is an alphavirus subgenome promoter.

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

[0016] In some embodiments of this disclosure, the modified alphavirus genome or srRNA is an alphavirus belonging to the Venezuelan Encephalitis Virus / Eastern Equine Encephalitis Virus (VEEV / EEEV) group, the Semlik Forest Virus (SFV) group, or the Sindbis virus (SINV) group. In some embodiments, the modified alphavirus genome or srRNA is an alphavirus belonging to the BFV complex, EEEV complex, MIDV complex, NDUV complex, SFV complex, VEEV complex, or WEEV complex. In some embodiments, the alphavirus is Eastern Equine Encephalitis Virus (EEEV), Venezuelan Encephalitis Virus (VEEV), Everglades Virus (EVEV), Mukambo Virus (MUCV), Pixna Virus (PIXV), Middleberg Virus (MIDV), Chikungunya Virus (CHIKV), Onyonnyon Virus (ONNV), Ross River Virus (RRV), Burma Forest Virus (BF), Geta Virus (GET), Sagiyama Virus (SAGV), These include Beval virus (BEBV), Mayarovirus (MAYV), Unavirus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Wataroa virus (WHAV), Bavanchi virus (BABV), Kyzilagati virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Nudum virus (NDUV), Madariaga virus (MADV), or Buggy Creek virus. In some embodiments, the alphavirus is VEEV, EEEV, CHIKV, or SINV.

[0017] In some embodiments of the present disclosure, the coding sequence of the polypeptide construct includes, in the 5' to 3' direction, i.e., from the N-terminus to the C-terminus of the polypeptide sequence, (a) (i) the coding sequence of IL-1RA, (ii) a connector sequence encoding IRES, and (iii) the coding sequence of IL-18BP, (b) (i) the coding sequence of IL-1RA, (ii) a connector sequence encoding P2A autoproteolytic peptide, and (iii) the coding sequence of IL-18BP, (c) (i) the coding sequence of IL-18BP, (ii) a connector sequence encoding IRES, and (iii) the coding sequence of IL-1RA, or (d) (i) the coding sequence of IL-18BP, (ii) a connector sequence encoding P2A autoproteolytic peptide, and (iii) the coding sequence of IL-1RA.

[0018] In some embodiments of the present disclosure, 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 with an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 10.

[0019] In some embodiments, the coding sequence of 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 with an amino acid sequence selected from the group consisting of SEQ ID NOs.11 to 14. In some embodiments, the nucleic acid construct of the present disclosure 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 with an amino acid sequence selected from the group consisting of SEQ ID NOs.15.

[0020] In one embodiment, this specification provides recombinant cells comprising nucleic acid constructs disclosed herein. In some embodiments, the recombinant cells are eukaryotic cells. In some embodiments, the eukaryotic cells are animal cells. In some embodiments, the animal cells are vertebrate cells or invertebrate cells. In some embodiments, the animal cells are mammalian cells. In some embodiments, the animal cells are insect cells. In some embodiments, the insect cells are mosquito cells. In some embodiments, the recombinant cells are immune cells. In some embodiments, the immune cells include B cells, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, basophils, eosinophils, neutrophils, dendritic cells (DCs), macrophages, regulatory T cells, and helper T cells (T). H ), cytotoxic T cells (T CTL These include memory T cells, gamma delta (γδ) T cells, hematopoietic stem cells, or hematopoietic stem cell progenitor cells. In some embodiments, the immune cells are B cells, T cells, macrophages, or dendritic cells (DCs). In related embodiments, a cell culture is also provided, comprising at least one recombinant cell as disclosed herein and a culture medium.

[0021] In another embodiment, a transgenic animal comprising a nucleic acid construct disclosed herein is provided. In some embodiments, the transgenic animal is a vertebrate or an invertebrate. 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 other embodiments, compositions comprising a) a nucleic acid construct of the Disclosure and / or b) recombinant cells of the Disclosure are provided. Non-limiting exemplary embodiments of the pharmaceutical compositions of the Disclosure may include one or more of the following features: In some embodiments, the Spec. provides compositions comprising a nucleic acid construct of the Disclosure and a pharmaceutically acceptable excipient. In some embodiments, the Spec. provides compositions comprising recombinant cells of the Disclosure and a pharmaceutically acceptable excipient. In some embodiments, the composition is formulated into a delivery system together with a delivery vehicle, the delivery system comprising polymer nanoparticles, lipid-based nanoparticles (LNPs), liposomes, viral replicon particles (VRPs), physiological buffers, microspheres, immunostimulatory complexes (ISCOMs), conjugates of bioactive ligands, or any combination thereof.

[0023] In some embodiments, the polymer nanoparticles include cationic polymers, non-cationic polymers, or combinations thereof. In some embodiments, the cationic polymer includes naturally derived cationic polymers. In some embodiments, the naturally derived cationic polymer includes chitosan, gelatin, dextran, cellulose, cyclodextrin, or combinations thereof. In some embodiments, the cationic polymer includes synthetic cationic polymers. In some embodiments, the synthetic cationic polymers include poly(ethyleneimine) (PEI), poly-L-lysine (PLL), poly(amino) acid (PAA), poly(amidoamine) (PAMAM), poly(cystamine bisacrylamide-co-4-amino-1-butanol) (pABOL), poly(amino-co-ester) (PAE), poly(2-N,N-dimethylaminoethyl methacrylate), poly(beta-aminoester) (PBAE), imidazole-containing polymers, tertiary amine-containing polymers, poly(2-(dimethylamino)ethyl methacrylate), poly-N-(2-hydroxypropyl)methacrylamide, polyamidoamine dendrimers, cationic glycopolymers, 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 polyethylene glycol (PEG), polyester (e.g., polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyglycolic 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 present disclosure, the polymer nanoparticles include one or more of the following: poly-(γ-L-glutamylglutamine) (PGGA), poly-(γ-L-aspartylglutamine) (PGAA), poly-L-lactic acid (PLLA), poly-(lactic-co-glycolic acid) (PLGA), polyalkylcyanoacrylate (PACA), polyanhydride, polyhydroxy acid, polypropyl fumerate, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, [N-(2-hydroxypropyl)methacrylamide] (HPMA) copolymer, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polyurea, polyamine poly-epsilon-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 at a lipid-to-RNA mass ratio of 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 compositions disclosed herein are formulated as biological agents.

[0027] In another embodiment, the Specified Publicly Provided is a method for modulating pharmacodynamic effects in a subject requiring such modification, comprising administering to the subject a composition comprising: (a) a composition comprising a nucleic acid construct of the Disclosure, (b) recombinant cells 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, biomarker response, therapeutic effect, prophylactic effect, desired effect, undesirable effect, adverse effect, and effect in a disease model. In some embodiments, the pharmacodynamic effect includes inducing an immune response in the subject.

[0028] In yet another aspect, the Specified provides a method for improving / extending the expression dynamics of IL-1RA and / or IL-18BP, or for enhancing the endogenous expression of IL-1RA and / or IL-18BP in a subject, comprising administering to the subject a composition comprising: (a) a nucleic acid construct of the Disclosure, (b) recombinant cells of the Disclosure, and / or (c) any one of the pharmaceutical compositions of the Disclosure.

[0029] In yet another embodiment, the Specified Public Service provides a method for preventing and / or treating a health condition in a subject that requires such treatment, comprising administering to the subject, prophylactically or therapeutically, a composition comprising: (a) nucleic acid constructs of the Disclosure, (b) recombinant cells of the Disclosure, and / or (c) any one of the pharmaceutical compositions of the Disclosure. In some embodiments, the administered composition induces a pharmacodynamic effect. In some embodiments, the pharmacodynamic effect includes inducing an immune response in the subject.

[0030] Non-limiting exemplary embodiments of the methods of the present disclosure may include one or more of the following features: In some embodiments, the health condition is an autoimmune 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 induces an immune response in the subject. In some embodiments, the administered composition modulates the production of one or more pro-inflammatory molecules in the subject. In some embodiments, one or more pro-inflammatory molecules include interleukin-1α (IFNα), interferon-1 beta (IFNβ), interleukin-18 (IL-18), interleukin-6 (IL-6), interferon-gamma (IFNγ), cytokines, TNF-α, GM-CSF, and MIP1α, granzyme B, granzyme A, perforin, or any combination thereof. In some embodiments, the compositions are administered to the subject individually as a single treatment (monotherapy) or as a first-line treatment in combination with at least one additional treatment. In some embodiments, at least one additional treatment is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery.

[0031] In yet another aspect, the Specified provides a method for improving / extending the expression dynamics of IL-1RA and / or IL-18BP, or for enhancing the endogenous expression of IL-1RA and / or IL-18BP in a subject, comprising administering to the subject a composition comprising: (a) a nucleic acid construct of the Disclosure, (b) recombinant cells of the Disclosure, and / or (c) any one of the pharmaceutical compositions of the Disclosure.

[0032] In yet another embodiment, the Specified provides a kit for inducing a pharmacodynamic response, for inducing an immune response, and / or for the prevention and / or treatment of a health condition, comprising: (a) a nucleic acid construct of the Disclosure, (b) recombinant cells of the Disclosure, and / or (c) a pharmaceutical composition of the Disclosure.

[0033] Each of the embodiments and models described herein can be used together unless explicitly or expressly excluded from the context of the embodiment or model.

[0034] The above summary is illustrative and not intended to limit the scope of this invention. In addition to the exemplary embodiments and features described herein, further aspects, embodiments, purposes and features of this disclosure will be fully apparent from the drawings, detailed description and claims. [Brief explanation of the drawing]

[0035] [Figure 1] This is a schematic diagram of a non-limiting example of an expression vector containing the modified alphavirus genome described herein. In this example, the EEEV-based vector includes a bigenic expression cassette for the expression of IL-1RA and IL-18BP. The coding sequences for IL-1RA and IL-18BP are linked to each other via connector sequences encoding internal ribosome entry sites (IRESs).

[0036] [Figure 2] This is a schematic diagram of exemplary EEEV srRNA designs according to some non-limiting embodiments of the present disclosure, in which the sequence encoding the modified EEEV genome was incorporated into a plasmid DNA vector, which also included the coding sequence for the IL-1RA protein, the coding sequence for the IL-18BP protein, and the coding sequence for an exemplary polypeptide construct including an internal ribosome entry site (IRES).

[0037] [Figure 3] This figure summarizes the results of ELISA experiments conducted to measure in vitro protein expression of IL-18BP in monogenic and digenic expression cassettes. In these experiments, the protein concentration of secreted IL-18BP was measured using culture supernatant derived from BHK-21 cells transfected with each srRNA construct.

[0038] [Figure 4] This figure summarizes the results of experiments conducted to measure the in vitro protein levels of bioactive IL-18BP in monogenic and digenic expression cassettes. In these experiments, the bioactivity of IL-18BP was measured by inhibiting IL-18 signaling on reporter cells expressing the congeneral IL-18 receptor, using culture supernatants derived from BHK-21 cells transfected with each srRNA construct.

[0039] [Figure 5] This is a schematic diagram of the results from ELISA experiments conducted to measure in vitro protein expression of IL-1RA in a bigenic expression cassette. In these experiments, the concentration of secreted IL-1RA was measured using the culture supernatant derived from BHK-21 cells transfected with each srRNA construct.

[0040] [Figure 6] This is a schematic diagram of the results from ELISA experiments conducted to measure the in vitro protein levels of bioactive IL-1RA in a bigenic expression cassette. In these experiments, the bioactivity of IL-1RA was measured using culture supernatants derived from BHK-21 cells transfected with each srRNA construct, via inhibition of recombinant IL-1β signaling on reporter cells expressing the congener receptor.

[0041] [Figure 7] This is a schematic diagram of the ratio of in vitro protein levels to IL-1RA expressed from a bigenic expression cassette using ELISA. In these experiments, the concentration of secreted IL-1RA was measured using the culture supernatant derived from BHK-21 cells transfected with each srRNA construct.

[0042] [Figure 8]This is a schematic diagram of the results from ELISA experiments conducted to evaluate the order of constituents by measuring in vitro protein expression of IL-1RA in 17 multigenetic expression cassettes. Here, the expression cassettes contain IL-1RA and one or more multigenetic addition polypeptides. See also Table 2 for a more detailed description of the expression cassettes and alphaviral vectors used in these experiments. The concentration of secreted IL-1RA was measured using the culture supernatant from BHK-21 cells transfected with each srRNA construct.

[0043] [Figure 9] Figure 8 is a schematic diagram of the results from ELISA experiments performed to measure the in vitro protein levels of bioactive IL-1RA in the multigeneic expression cassette. In these experiments, the bioactivity of IL-1RA was measured by inhibiting recombinant IL-1β signaling on reporter cells expressing the congenital receptor, using culture supernatants derived from BHK-21 cells transfected with each srRNA construct.

[0044] [Figure 10] This is a schematic diagram of in vitro protein expression of IL-1RA in different alphaviral vectors, as determined by ELISA. See Table 1 for a more detailed description of the alphaviral vectors used in these experiments. In these experiments, the concentration of secreted IL-1RA was measured using the culture supernatant derived from BHK-21 cells transfected with each srRNA construct.

[0045] [Figure 11] Figure 10 shows a schematic diagram of the in vitro protein levels of bioactive IL-1RA in the modified alphavirus vector described. In these experiments, the bioactivity of IL-1RA was measured using culture supernatants derived from BHK-21 cells transfected with each srRNA construct, mediated by the inhibition of recombinant IL-1β signaling on reporter cells expressing the congener receptor.

[0046] [Figure 12] This figure summarizes the results of ELISA experiments conducted to measure the in vitro protein expression of IL-18BP in different alphavirus vectors. The concentration of secreted IL-18BP was measured using the culture supernatant derived from BHK-21 cells transfected with each srRNA construct.

[0047] [Figure 13] Figure 12 summarizes the in vitro protein levels of bioactive IL-18BP in the modified alphavirus vectors described. The bioactivity of IL-18BP was measured by inhibiting IL-18 signaling on reporter cells expressing the congeneral IL-18 receptor using the culture supernatant from BHK-21 cells transfected with each srRNA construct.

[0048] [Figure 14] This figure summarizes the results from in vivo experiments conducted to evaluate a panel of modified alphaviral vectors for in vivo expression of IL-1RA. Serum from mice administered a single dose of srRNA encapsulated in LNPs encoding either both IL-1RA and IL-18BP (digenic) or IL-1RA only (monogenic) as a control was used to measure the concentration of secreted IL-1RA and compared to vehicle-treated control animals.

[0049] [Figure 15] This figure summarizes the results from in vivo experiments conducted to evaluate the in vivo expression of IL-18BP from the modified alphavirus vector shown in Figure 14, with the exception of one gene, which is an srRNA encoding only IL-18BP and was formulated with LNP and used as a control.

[0050] [Figure 16]Schematic diagrams of in vivo expression of IL-1RA in animal serum from two different delivery vehicles, namely (i) a bigenic EEEV vector formulated with LNPs and (ii) polymer nanoparticles, according to some non-limiting embodiments of the present disclosure. In these experiments, only unformulated alphavirus vectors and delivery vehicles were used as controls.

[0051] [Figure 17] This figure shows the in vivo expression of IL-18BP in the formulations described in Figure 16. In these experiments, only unformulated alphavirus vectors and delivery vehicles were used as controls.

[0052] [Figure 18] Figure 18A summarizes the results of in vivo pharmacokinetic studies conducted to demonstrate that srRNA constructs expressing IL-1RA and IL-18BP as described herein (e.g., in LNP formulation vectors) can extend the pharmacokinetics of target proteins with short half-lives (e.g., recombinant IL-1RA). Figure 18A is a schematic diagram of an in vivo study design (top) measuring serum levels of human IL-1RA after administration of the recombinant protein version at t=0, or a schematic diagram (bottom) measuring serum levels of human IL-1RA after administration of LNP formulation srRNA (the human IL-1RA gene) encoding IL1RN together with IL-18BPa, 96 hours before achieving Tmax. Figure 18B shows serum levels of human IL-1RA measured 2 and 8 hours after administration of the recombinant protein version of IL-1RA or IL-1RA encoded in srRNA using standard ELISA. [Modes for carrying out the invention]

[0053] This specification provides viral expression systems comprising self-replicating RNA (srRNA) based on RNA viruses (e.g., alphaviruses) having excellent expression capabilities, particularly suitable for expressing heterologous molecules, such as therapeutic polypeptides, in recombinant cells. For example, some embodiments of this disclosure relate to nucleic acid constructs that express (i) interleukin-1 receptor antagonist (IL-1RA) protein and / or functional variants thereof, and (ii) interleukin-18 binding protein (IL-18BP) or functional variants thereof, for the purpose of therapeutic treatment of human health conditions or diseases, such as autoimmune diseases, inflammatory diseases, and cardiovascular diseases. These constructs address problems with therapeutic modes, such as recombinant cytokine administration, due to previously demonstrated dose-limiting toxicity issues. In some embodiments, this specification provides gene expression systems having excellent expression capabilities, particularly suitable for expressing the coding sequences of IL-1RA or its functional variants and IL-18BP or its functional variants in recombinant cells. For example, some embodiments of the present disclosure relate to nucleic acid constructs, such as expression constructs and vectors, which contain a modified alphavirus genome or srRNA, wherein at least a portion of the nucleic acid sequence encoding the viral structural protein of the modified alphavirus genome or srRNA is replaced by a polypeptide construct coding sequence comprising (a) the coding sequence of an interleukin-1 receptor antagonist (IL-1RA) protein or a functional variant thereof, and (b) the coding sequence of an interleukin-18 binding protein (IL-18BP) or a functional variant thereof, and the coding sequences of IL-1RA and IL-18BP are functionally linked to each other. In some embodiments, the polypeptide construct does not contain a dimerization domain. This is because the presence of a dimerization domain in the polypeptide constructs disclosed herein is likely to result in undesirable properties, such as changes in protein structure, decreased stability, unstable protein folding, loss of function, or acquisition of undesirable signaling function.Furthermore, recombinant cells and transgenic animals genetically engineered to contain one or more nucleic acid constructs disclosed herein are provided. Biomaterials and recombinant products derived from such recombinant cells are also within the scope of this application. Also provided are compositions and methods useful for (i) modulating pharmacodynamic effects, (ii) improving / extending the dynamics of IL-1RA and / or IL-18BP expression in a subject, (iii) or enhancing the endogenous expression of IL-1RA and / or IL-18BP in a subject, and / or (iv) doing so in a subject where prevention and / or treatment of a health condition is required.

[0054] As will be described in more detail below, IL-1B and IL-18 are inflammatory cytokines that are cleaved into their bioactive forms upon inflammasome activation. Both cytokines are downstream mediators of inflammasome activation and can act synergistically to promote inflammation. Therefore, the antagonistic effects of both IL-1 and IL-18 signaling can act as downstream inhibitors of the inflammasome, which is a central mediator in several autoimmune and inflammatory diseases (both immune system and other conditions such as cardiovascular diseases). Each cytokine has an endogenous antagonist, IL-1RA and IL-18BP, respectively. Since both IL-1A and IL-1B share the same receptor IL-1R1, which is the target of IL-1RA, IL-1RA can also block IL-1A function. Inflammasome activation by caspase-1 results in the cleavage of pro-IL-1B and pro-IL-18 into their active forms. Several existing assets targeting specific aspects of this pathway (e.g., glibenclamide, curcumin, MCC950, BHB, resveratrol, pralunasane, vernasane, lilonacept, P2D7KK, LY2189102, gevokizumab, canakinumab, and GSK-1070806) have been used to treat a limited number of autoimmune and autoinflammatory diseases, such as cryopyrin-associated periodic syndromes (CAPS), interleukin-1 receptor antagonist deficiency (DIRA), septic arthritis, pyoderma gangrenosum, and acne (PAPA syndrome), familial Mediterranean fever (FMF), and mevalonate kinase deficiency (MKD). Co-expression of these two endogenous antagonists, IL-1RA and IL-18BP, may help mitigate inflammation associated with inflammasome activation in several autoimmune and inflammatory indications where single agents may be limited. As discussed above, the short in vivo half-life of IL-1RA (commercially available as a recombinant protein under the Anakinra / Kineret® trademark) is less than 4 hours in humans and even shorter in rodents, so strategies to lengthen its half-life have been explored.While some of these strategies have shown success, administration of recombinant IL-1RA still requires frequent dosing to achieve therapeutic levels of the encoded protein. As will be described in more detail below, the use of srRNA platforms is expected to surpass what has been observed with these existing methodologies, as it can continue to express the protein for several weeks. Furthermore, various modifications of recombinant proteins, such as the addition of the Fc region of immunoglobulins (even when humanized), are immunogenic and can generate anti-drug antibodies (ADAs) with repeated dosing in the context of chronic disease. The compositions and methods disclosed herein provide approaches that enable endogenous expression of IL-1RA and IL-18BP without the involvement of additional motifs that can result in anti-drug antibodies.

[0055] While combinations of multiple cytokines into a single expression vector have been proposed, the ability to express multiple functional cytokines in a single srRNA vector requires something far beyond simply assembling individual cytokine coding sequences in a random order. Furthermore, since protein expression does not always correlate with the product of a functional protein, the experimental results presented herein demonstrate that the combination of IL1-RA and IL-18BP in srRNA vectors is not an easily solved problem in the field of srRNA vector design.

[0056] The disclosures provided herein, in particular, offer solutions to the various problems present in conventional attempts to deliver IL-1RA and / or IL-18BP, thereby providing improved therapeutic methods for health conditions, including, but not limited to, autoimmune and inflammatory diseases in immunological and cardiovascular diseases. As illustrated in Examples 2-4 below, srRNA vectors capable of expressing both functional IL-1RA and functional IL-18BP can be produced by using the compositions and methods disclosed herein.

[0057] definition Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used herein are intended to have meanings that are generally understood by those skilled in the art to which this application pertains. In some cases, terms that have generally understood meanings are defined herein for clarity and / or for easy reference, and the inclusion of such definitions herein should not necessarily be construed as meaning that they differ substantially from the generally understood meanings in the art. Many of the techniques and procedures described or referenced herein are well understood by those skilled in the art and are commonly employed using conventional methodologies.

[0058] The singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. For example, the term “a cell” includes one or more cells, including a mixture thereof. “A and / or B” is used herein to include all options of “A,” “B,” “A or B,” and “A and B.”

[0059] As used herein, the terms “administer” and “to administer” refer to the delivery of a bioactive composition or formulation by a route of administration including, but not limited to, intranasal, transdermal, intravenous, intra-arterial, intramuscular, intranodal, intraperitoneal, subcutaneous, intramuscular, oral, vaginal, and topical administration, or a combination thereof. This term includes, but is not limited to, administration by a healthcare professional and self-administration.

[0060] The terms “cell,” “cell culture,” and “cell line” refer not only to specific target cells, cell cultures, or cell lines, but also to the offspring or potential offspring of such cells, cell cultures, or cell lines, regardless of the number of transfers or passages during culture. It should be understood that not all offspring are strictly identical to the parent cells. This is because certain modifications may occur in subsequent generations due to either mutations (e.g., intentional or unintentional mutations) or environmental influences (e.g., methylation or other epigenetic modifications), so offspring may not actually be identical to the parent cells, but as long as the offspring retain the same function as the original cells, cell culture, or cell line, they are still included in the scope of the terms used herein.

[0061] The term "construct" refers to a recombinant molecule, such as a recombinant nucleic acid or polypeptide, comprising one or more isolated nucleic acid sequences or amino acid sequences from heterogeneous sources. For example, a polypeptide construct may be a chimeric polypeptide molecule in which two or more amino acid sequences of different origins are functionally linked to one another in a single polypeptide construct. Similarly, a nucleic acid construct may be a chimeric nucleic acid molecule in which two or more nucleic acid sequences of different origins are assembled into a single nucleic acid molecule. Typical nucleic acid constructs include any recombinant nucleic acid molecule, linear or cyclic, single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source such as plasmids, cosmids, viruses, autonomously replicating polynucleotide molecules, or phages, comprising a nucleic acid molecule in which one or more nucleic acid sequences are functionally linked, and capable of genome integration or autonomous replication. Two or more nucleic acid constructs may be contained in a single nucleic acid molecule, such as a single vector, or in two or more separate nucleic acid molecules, such as two or more separate vectors.

[0062] In some embodiments of this disclosure, one or more nucleic acid constructs may be incorporated into a single nucleic acid molecule, for example, a single vector (e.g., insertion), or into two or more separate nucleic acid molecules, for example, two or more separate vectors (e.g., insertion). The term “vector,” as used herein, refers to a nucleic acid molecule or sequence capable of transferring or transporting another nucleic acid molecule. Therefore, 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 integration vectors. An “expression vector” is a vector comprising a regulatory region, thereby capable of expressing DNA sequences and fragments in vitro, ex vivo, and / or in vivo. In some embodiments, the vector may include sequences that induce autonomous replication within a cell, such as plasmids (DNA-based vectors) or self-replicating RNA vectors. In some embodiments, the vector may include sequences sufficient to enable 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 may be a single-stranded vector (e.g., ssDNA or ssRNA). In some embodiments, the vector of the Disclosure may be a double-stranded vector (e.g., dsDNA or dsRNA). In some embodiments, the expression vector is a gene delivery vector. In some embodiments, the vector is used as a gene delivery vehicle for transferring 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 replication origins, e.g., prokaryotic and eukaryotic origins, at least one multicloning site, and / or elements that facilitate the stable integration of the construct into the cell's genome. As described above, two or more constructs may be incorporated into a single nucleic acid molecule, e.g., a single vector, or into two or more separate nucleic acid molecules, e.g., two or more separate vectors. The “expression construct” generally includes at least one regulatory sequence functionally linked to the nucleotide sequence of interest. Thus, for example, a promoter functionally linked to the nucleotide sequence to be expressed is provided in the expression construct for expression in a cell. Compositions and methods for preparing and using constructs and cells for the implementation of this disclosure are known to those skilled in the art.

[0064] The terms “effective amount,” “therapeutic effective amount,” or “pharmaceutical effective amount” of compositions, for example, nucleic acid constructs (e.g., srRNA constructs), recombinant cells, and / or pharmaceutical compositions in this disclosure generally refer to an amount sufficient to achieve a given purpose (e.g., to achieve an effect to be administered, to stimulate an immune response, to prevent or treat a disease, or to alleviate one or more symptoms of a disease, disorder, infection, or health condition) compared to the absence of the composition. An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or alleviation of one or more symptoms of a disease, which may also be called a “therapeutic effective amount.” “Alleviation” of symptoms means a reduction in the severity or frequency of symptoms, or the disappearance of symptoms. The exact amount of a composition containing the "therapeutic dose" depends on the therapeutic purpose and can be determined by those skilled in the art using known techniques (see, for example, 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, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0065] As used herein, the term “naked” refers to nucleic acids that are substantially free of other macromolecules such as lipids, polymers, and proteins. “Naked” nucleic acids, such as self-replicating RNA, are not formulated with other macromolecules to improve cellular uptake. Therefore, naked nucleic acids are not encapsulated, absorbed, or bound to liposomes, microparticles, nanoparticles, cationic emulsions, etc.

[0066] The term "functionally linked," as used herein, means a physical or functional linkage between two or more elements, such as polypeptide sequences or polynucleotide sequences, that enables them to function in the manner intended. For example, when the term "functionally linked" is used in the context of a nucleic acid molecule or coding and promoter sequences within a nucleic acid molecule described herein, it means that the coding and promoter sequences are in a frame and separated by appropriate space and distance so as to enable the effect of their respective binding on transcription by a transcription factor or RNA polymerase. It should be understood that functionally linked elements may or may not be adjacent (e.g., they may be linked to each other via linkers). In the context of polypeptide constructs, "functionally linked" refers to a physical linkage (e.g., direct or indirect linkage) between amino acid sequences (e.g., different segments, parts, regions, or domains) to provide the described activity of the construct. Functionally linked segments, parts, regions, and domains of polypeptides or nucleic acid molecules disclosed herein may or may not be adjacent (e.g., they may be linked to each other via linkers).

[0067] As used herein, the term “part” refers to a small fragment. With respect to a particular structure such as a polynucleotide sequence, an amino acid sequence, or a protein, the term “part” may refer to a continuous or discontinuous fragment of the structure. For example, a part of an amino acid sequence contains 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 the amino acid sequence. In addition, or instead, if a part is a discontinuous fragment, the discontinuous fragment consists of two, three, four, five, six, seven, eight, or more parts of the structure (e.g., a protein domain), each part being a continuous element of the structure. For example, a discontinuous small fragment of an amino acid sequence may consist of 2, 3, 4, 5, 6, 7, 8 or more parts of the amino acid sequence, for example, 4 or fewer parts, each part containing at least 1, at least 2, at least 3, at least 4, at least 5 consecutive amino acids, at least 10 consecutive amino acids, at least 20 consecutive amino acids, or at least 30 consecutive amino acids of the amino acid sequence.

[0068] As used herein in the context of two or more nucleic acids or proteins, the term “percent identity” means two or more sequences or subsequences that are the same or have a specific percentage of identical nucleotides or amino acids, as measured using the BLAST or BLAST2.0 sequence comparison algorithm with the default parameters described below, or by manual alignment and visual inspection (for example, approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity across a specific region when compared and aligned for maximum match across a comparison window or designated region). See, for example, the NCBI website at ncbi.nlm.nih.gov / BLAST. Such sequences are said to be “substantially identical.” This definition also refers to, or may apply to, a complement of the sequence of interest. This definition includes sequence comparison performed by the BLAST algorithm, the algorithm's parameters being selected to provide the greatest possible match between each sequence over the entire length of each reference sequence. This definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. Sequence identity can be calculated over regions of at least approximately 20 amino acids or nucleotides in length, over regions of 10 to 100 amino acids or nucleotides in length, or over the entire length of a given sequence. Sequence identity can be calculated using publicly available techniques and widely available computer programs such as the GCS program package (Devereux et al., Nucleic Acids Res (1984) 12:387), BLASTP, BLASTN, and FASTA (Atschul et al., J Mol Biol (1990) 215:403).Sequence identity can be measured using the default parameters of 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). Further methodologies that may be appropriately used to determine amino acid sequence similarity or identity include those that rely on a position-specific structure scoring matrix (P3SM) incorporating structural prediction scores from Rosetta, and those based on length-normalized edit distance, as previously described, for example, by Setcliff et al., Cell Host & Microbe 23(6), May 2018.

[0069] As used herein, the term “pharmaceutically acceptable excipient” refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for administering the compound of interest to a subject. Thus, “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, antimicrobial and antifungal agents, isotonic agents, and absorption retarders that are suitable for pharmaceutically effective administration. Auxiliary active compounds (e.g., antibiotics and additional therapeutic agents) can also be incorporated into the composition.

[0070] The term “recombinant,” when used in relation to cells, nucleic acids, proteins, or vectors, indicates that the cells, nucleic acids, proteins, or vectors have been modified or produced by human intervention, such as by laboratory methods, or are a result of such modifications. Therefore, recombinant proteins and nucleic acids, for example, include proteins and nucleic acids produced by laboratory methods. Recombinant proteins may contain amino acid residues not found in the natural (non-recombinant or wild-type) form of the protein, or may contain modified, for example, labeled amino acid residues. This term can include any modification of a peptide, protein, or nucleic acid sequence. Such modifications include any chemical modification of a peptide, protein, or nucleic acid sequence containing one or more amino acids, deoxyribonucleotides, or ribonucleotides; the addition, deletion, and / or substitution of one or more amino acids in a peptide or protein; the creation of fusion proteins, such as fusion proteins containing antibody fragments; and the addition, deletion, and / or substitution of one or more nucleic acids in a nucleic acid sequence. When used in relation to cells, the term "recombinant" is not intended to include naturally occurring cells, but rather to include cells that have been manipulated / modified to contain or express polypeptides or nucleic acids that would not be present in the cell if it were not manipulated / modified.

[0071] As used herein, “subject” or “individual” includes humans (e.g., human individuals) and animals such as non-human animals. In some embodiments, “subject” or “individual” is a patient under the care of a physician. Thus, a subject may be a human patient or individual who has, is at risk of having, or is suspected of having, one or more signs of the health condition in question (e.g., autoimmune disease, inflammatory disease, or cardiovascular disease) and / or the health condition in question. A subject may also be an individual diagnosed at the time of diagnosis or thereafter as being at risk of the condition in question. The term “non-human animal” 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 sheep, dogs, cats, cattle, chickens, etc., as well as non-mammals such as amphibians and reptiles.

[0072] The aspects and embodiments of the disclosure described herein are understood to include aspects and embodiments of “comprising,” “consisting,” and “consisting essentially of.” As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is comprehensive or open-ended and does not exclude additional unlisted elements or steps of a method. As used herein, “consisting of” excludes any elements, steps, or components not specified in the claimed composition or method. As used herein, “consisting essentially of” does not exclude materials or steps that do not substantially affect the basic and novel features of the claimed composition or method. Any enumeration of the term “comprising” herein, particularly in descriptions of composition components or steps of a method, is understood to include compositions and methods that are essentially composed of, and comprise, the enumerated components or steps.

[0073] Where a range of values ​​is provided, unless otherwise explicitly indicated in the context, each value between the upper and lower limits of that range, up to one-tenth of the lower limit unit, and any other stated or existing values ​​within that stated range are understood to be included in this disclosure. The upper and lower limits of these smaller ranges may independently be included in smaller ranges and are also included in this disclosure, subject to any restrictions specifically excluded in the stated range. Where a stated range includes one or both of the limit values, ranges excluding one or both of those limit values ​​are also included in this disclosure.

[0074] In this specification, a particular range is indicated by a number preceded by the term “about,” which, when used herein, has its usual meaning of “approximately.” In this specification, the term “about” is used to provide literal support for the exact number preceded by it, as well as for any number that is close to or approximates the number preceded by the term. When determining whether a number is close to or approximates a specifically enumerated number, an unenumerated number that is close to or approximates it may be a number that, in the context in which it is presented, provides a substantially equivalent to the specifically enumerated number. Where the degree of approximation is not evident from the context, “about” means either within plus or minus 10% of the given value, or rounded to the nearest significant figure, including in all cases the given value. In some embodiments, the term “about” indicates up to ±10%, ±5%, or ±1% of a given value.

[0075] Where a range of values ​​is provided, it will be understood by those skilled in the art that all ranges disclosed herein encompass all possible subranges and combinations thereof. Any enumerated range can be readily recognized as sufficient to explain and enable that the same range can be decomposed into at least equal 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, etc. As a non-limiting example, each range discussed herein can be readily decomposed into the lower third, middle third, upper third, etc. Similarly, as will be understood by those skilled in the art, all terms such as “up to / at most,” “at least,” “more,” and “less than” include the enumerated numbers and refer to ranges that can be later decomposed into subranges as described above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

[0076] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species to which the compositions and methods disclosed herein are applicable. Accordingly, this term includes, but is not limited to, genes and gene products from humans and mice. Where a gene or gene product from a particular species is disclosed, this disclosure is intended to be illustrative only and should not be construed as limiting unless the context in which it appears is explicitly indicated. Therefore, for example, with respect to genes or gene products disclosed herein, in some embodiments they relate to mammalian nucleic acid sequences and amino acid sequences, and are intended to include 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 its variants.

[0077] Headings, such as (a), (b), (i), etc., are provided solely for the purpose of making the specification and claims easier to read. Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein. The use of headings in this specification or claims does not require that the processes or elements be performed in alphabetical or numerical order, or in the order in which they are presented.

[0078] For clarity, it is understood that certain features of the Disclosure described in the context of separate embodiments may be provided in combination in a single embodiment. Conversely, for brevity, various features of the Disclosure described in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of embodiments relating to the Disclosure are specifically encompassed by the Disclosure and are disclosed herein as if any combination were explicitly disclosed individually. In addition, all subcombinations of various embodiments and their elements are also specifically encompassed by the Disclosure and are disclosed herein as if any such subcombination were explicitly disclosed individually.

[0079] IL-1RA Interleukin-1 receptor antagonist protein (IL-1RA) is a member of the interleukin-1 cytokine family. IL-1RA protein is secreted by various cell types, including immune cells, epithelial cells, and adipocytes, and is a natural inhibitor of the pro-inflammatory effects of IL-1 beta (IL-1B) and IL-1 alpha (IL-1A).

[0080] In humans, IL-1RA acts as a natural inhibitor of the IL-1 receptor. The IL-1RA protein is used to suppress the biological activity caused by interleukin-1 alpha and beta (IL-1A, IL-1B). It binds to the cell membrane-bound IL-1 receptor (IL-1R), preventing IL-1 from binding to the same IL-1R, thereby preventing IL-1 from signaling to that cell. IL-1RA inhibits the activity of IL-1 alpha (IL-1A) and IL-1 beta (IL-1B), modulating various IL-1-related immune and inflammatory responses.

[0081] In humans, IL-1RA is encoded by the IL1RN gene. KINERET®-anakinra, a recombinant nonglycosylated version of human IL-1RA, 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 the site of inflammation by immune cells and local tissue cells. KINERET® is approved in Europe for the treatment of rheumatoid arthritis (RA) and cryopyrin-associated periodic syndromes (CAPS), and in the United States for the most severe form of CAPS, namely chronic infantile neurocutaneous and arthral syndrome (CINCA). Anakinra has a short half-life of 4-6 hours, and therefore, the general pharmacologic requirement is 100 mg / day for RA and 1-2 mg / kg / day daily subcutaneous injection for CINCA.

[0082] IL-18BP Interleukin-18 binding protein (IL-18BP) is a natural antagonist of IL-18 associated with arthritis and many other inflammatory and / or autoimmune diseases. IL-18BP has been reported to bind to IL-18 ligands with high affinity, and this high-affinity binding effectively blocks the interaction between IL-18 and the IL-18Rα ligand-binding chain on the cell surface, ultimately inhibiting the IL-18 signaling pathway. Therefore, IL-18BP is considered a potent IL-18 inhibitor. In particular, IL-18BP reduced the severity of autoimmune diseases in several experiments. Thus, IL-18BP is thought to function as a natural anti-inflammatory and immunosuppressive molecule that neutralizes the effects of high IL-18 levels during inflammation. IL-18BP is specifically induced by IFNγ as part of a negative feedback loop that controls IL-18-mediated induction of IFNγ.

[0083] IL-18BP has four isoforms (a, b, c, and d) in humans and two isoforms (c and d) in mice. Human isoforms a and c, and mouse isoforms c and d, can bind to the receptor binding site of IL-18 and inhibit IL-18 in equimolar proportions. In humans, IL-18BP isoform a (IL-18BPa) has the highest affinity for IL-18 (e.g., higher than isoform c). Isoforms b and d lack IL-18 neutralizing activity. IL-18BP binds to IL-18 with high affinity, blocking the formation of a complex between IL-18 and IL-18R, and subsequently inhibiting IL-18 activation. Under normal conditions, there is enough naturally occurring IL-18BP to maintain low levels of free IL-18. However, in patients with certain inflammatory diseases, high levels of IL-18 can disrupt the IL-18 / IL-18BP balance, leading to increased free and active IL-18, which can result in pathological inflammation. This IL-18 / IL-18BP imbalance may be associated with increased disease severity. The absence of IL-18BP has been reported to cause an IL-18 / IL-18BP imbalance, and excessive NK cell activation by high levels of IL-18 has been reported to lead to uncontrolled death of human hepatocytes. IL-18 / IL-18BP imbalance is highly associated with immunologically mediated diseases, particularly those involving a pathological role of IFNγ, such as macrophage activation syndrome (MAS).

[0084] IL-18 is involved in numerous diseases in clinical and animal studies, including autoimmune diseases, cardiovascular diseases, neurovascular EC injury, occlusive renal injury mediated by lower aortic occlusion, hemophagocytic lymphohistiocytosis, and inflammatory bowel disease. IL-18BP and anti-IL-18 antibodies are used to neutralize IL-18 and treat these diseases. At least 33 disease models have been reported in which inhibition of IL-18 activity by administration of either a neutralizing anti-IL-18 antibody or IL-18BP has resulted in a reduction in disease severity. Recently, recombinant human IL-18BP (Tadekinig Alfa) has been used in a Phase II clinical trial in Europe to treat adult-onset Still's disease patients, and in a Phase III clinical trial using the experimental drug IL-18BP in patients with NOD-like receptor C4 (NLRC4) gene mutations characterized by severe, life-threatening systemic inflammation associated with extremely high levels of IL-18.

[0085] self-replicating RNA As will be understood by those skilled in the art, the term “self-replicating RNA” refers to an RNA molecule containing all the genetic information necessary to induce its own amplification or self-replication within a permissible cell. Therefore, srRNA is sometimes called “self-amplifying RNA” (saRNA). In some embodiments, srRNA is a “replicon,” which can be a linear or circular section of DNA or RNA that replicates sequentially as a unit. Non-limiting examples of replicons include “replicon RNA” or “RNA replicon.” To induce its own replication, srRNA generally (1) encodes a polymerase, replicase, or other protein that can interact with a viral or host cell-derived protein, nucleic acid, or ribonucleoprotein to catalyze the RNA amplification process, and (2) contains a cis-acting RNA sequence necessary for the replication and transcription of subgenomic RNA. These sequences can, during the replication process, bind to its self-coding protein, or to a non-self-coding cell-derived protein, nucleic acid, or ribonucleoprotein, or to a complex between any of these components. In some embodiments of this disclosure, the replicon, e.g., srRNA, is derived from Madariaga virus (MADV). In some embodiments of this disclosure, the MADV, srRNA construct (e.g., srRNA, saRNA, or replicon molecule) generally comprises the following elements: a 5' viral or defective interfering RNA sequence required in cis replication; a sequence encoding a biologically active alphaviral nonstructural protein (e.g., nsP1, nsP2, nsP3, and nsP4); a subgenome promoter (sg) for the subgenomic RNA (sgRNA); a 3' viral sequence required in cis replication; and optionally a polyadenylate tract (Poly(A)). In some examples, a subgenome promoter (sg) that induces the expression of heterologous sequences may be included in the srRNA construct of this disclosure.

[0086] Furthermore, the term srRNA molecule (e.g., srRNA, saRNA, or replicon molecule) generally refers to a positively polarized molecule, or "message" sense, and the srRNA may be of a length different from that of all known naturally occurring alphaviruses. In some embodiments of this disclosure, the srRNA may not contain at least a portion of the coding sequence for one or more alphavirus structural proteins, and / or the sequence encoding the structural gene may be replaced with a heterologous sequence. In these examples, if the srRNA is packaged into a recombinant alphavirus particle, it may contain one or more sequences, so-called packaging signals, that work to initiate interaction with the alphavirus structural proteins that cause the particle to form.

[0087] The nucleic acid molecules of this disclosure may be nucleic acid molecules of any length, generally including nucleic acid molecules of about 2kb to about 50kb, for example, about 5kb to about 40kb, about 5kb to about 30kb, about 5kb to about 20kb, or about 10kb to about 50kb, for example, about 15kb to about 30kb, about 20kb to about 50kb, about 20kb to about 40kb, about 5kb to about 25kb, or about 30kb to about 50kb in length. In some embodiments, the nucleic acid molecules are at least 6kb in length. In some embodiments, the nucleic acid molecules are about 6kb to about 20kb in some embodiments of this disclosure. The srRNA constructs of this disclosure generally have a length of at least about 2kb. For example, srRNA may have a length of at least about 2kb, at least about 3kb, at least about 4kb, at least about 5kb, at least about 6kb, at least about 7kb, at least about 8kb, at least about 9kb, at least about 10kb, at least about 11kb, at least 12kb, or greater than 12kb. In some embodiments, srRNA is approximately 4kb to 20kb, 4kb to 18kb, 5kb to 16kb, 6kb to 14kb, 7kb to 12kb, 8kb to 16kb, 9kb to 14kb, 10kb to 18kb, 11kb to 16kb, 5kb to 18kb, 6kb to 20kb, 5kb to 10kb, 5kb to 8kb, 5kb to 7kb, and 5kb to 6kb. , srRNA may have lengths of approximately 6kb to 12kb, 6kb to 11kb, 6kb to 10kb, 6kb to 9kb, 6kb to 8kb, 6kb to 7kb, 7kb to 11kb, 7kb to 10kb, 7kb to 9kb, 7kb to 8kb, 8kb to 11kb, 8kb to 10kb, 8kb to 9kb, 9kb to 11kb, 9kb to 10kb, or 10kb to 11kb. In some embodiments, srRNA may have lengths of approximately 6kb to 14kb. In some embodiments, srRNA may have lengths of approximately 6kb to 16kb.

[0088] Composition of the present disclosure As described in more detail below, one aspect of the present disclosure relates to a nucleic acid construct sequence encoding a modified alphaviral genome or srRNA, wherein at least a portion of the nucleic acid sequence encoding the viral structural protein of the modified alphaviral genome or srRNA is replaced by a polypeptide construct coding sequence comprising (a) the coding sequence of the IL-1RA protein or a functional variant thereof, and (b) the coding sequence of IL-18BP or a functional variant thereof, wherein the coding sequences of IL-1RA and IL-18BP are functionally linked to each other, and the polypeptide construct does not contain a dimerization domain. Recombinant cells and cell cultures manipulated to include the nucleic acid constructs disclosed herein are also provided.

[0089] A. Nucleic acid constructs As described in more detail below, one aspect of the present disclosure relates to a nucleic acid construct comprising a nucleic acid sequence encoding a modified alphaviral genome or replicon (e.g., srRNA), wherein at least a portion of the nucleic acid sequence encoding the viral structural protein of the modified alphaviral genome or replicon (e.g., srRNA) is replaced by a polypeptide construct encoding sequence comprising (a) the encoding sequence of the IL-1RA protein or a functional variant thereof, and (b) the encoding sequence of IL-18BP or a functional variant thereof, wherein the encoding sequences of IL-1RA and IL-18BP are functionally linked to each other, and the polypeptide construct does not contain a dimerization domain. In some embodiments, the sequence encoding the srRNA construct may be functionally linked and, for example, positioned under the control of expression-necessary elements (e.g., promoter sequences) that enable the expression of the srRNA construct in a host cell, a subject, or 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 containing cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. Nucleic acid molecules may be double-stranded or single-stranded (e.g., sense strand or antisense strand). Nucleic acid molecules may contain non-natural or modified nucleotides. The terms “polynucleotide sequence” and “nucleic acid sequence” are used interchangeably herein and refer to sequences of polynucleotide molecules. The nomenclature for nucleotide bases described in 37 CFR §1.822 is used herein.

[0091] The nucleic acid molecules of this disclosure may be of any length, including, for example, lengths of about 1.5Kb to about 50Kb, for example, about 5Kb to about 40Kb, about 5Kb to about 30Kb, about 5Kb to about 20Kb, or about 10Kb to about 50Kb, for example, about 15Kb to about 30Kb, about 20Kb to about 50Kb, about 20Kb to about 40Kb, about 5Kb to about 25Kb, or about 30Kb to about 50Kb.

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

[0093] In some embodiments of this disclosure, the coding sequences of IL-1RA and / or IL-18BP are optimized for one or more desired features. In some embodiments, the coding sequences of IL-1RA and / or IL-18BP are independently optimized for one or more of the following: (a) enhancing RNA stability, (b) enhancing expression levels, (c) minimizing the use of rare codons, (d) minimizing secondary structures, (d) promoting better srRNA replication, and (e) promoting a better RNA manufacturing process.

[0094] In some embodiments, the modified alphavirus genome or replicon (e.g., srRNA vector) lacks at least a portion of the nucleic acid sequence encoding one or more of the viral structural proteins CP, E1, E2, E3, and 6K of the alphavirus genome or srRNA vector. In some embodiments, the modified alphavirus genome or srRNA vector lacks a portion or all of the sequence encoding CP. In some embodiments, the modified alphavirus genome or srRNA vector lacks a portion or all of the sequence encoding E1. In some embodiments, the modified alphavirus genome or srRNA vector lacks a portion or all of the sequence encoding E2. In some embodiments, the modified alphavirus genome or srRNA vector lacks a portion or all of the sequence encoding E3. In some embodiments, the modified alphavirus genome or srRNA vector lacks a portion or all of the sequence encoding 6K. In some embodiments, the modified alphavirus genome or srRNA vector lacks a portion or all of the sequence encoding a combination of CP, E1, E2, E3, and 6K. In some embodiments of this disclosure, coding sequences for the non-structural proteins nsP1, nsP2, nsP3, and nsP4 of the modified alphavirus genome or srRNA vector are present, but at least a portion or all of the coding sequences for one or more structural proteins (e.g., CP, E1, E2, E3, and 6K) of the modified alphavirus genome or srRNA vector are absent.

[0095] In some embodiments, the modified alphaviral genome or srRNA vector lacks a substantial portion of the nucleic acid sequence encoding one or more viral structural proteins. Those skilled in the art will understand that a substantial portion of the nucleic acid sequence encoding a viral structural polypeptide may contain enough of the sequence to provide an estimated identification of that polypeptide, either by manual sequence evaluation by those skilled in the art or by computer-automated sequence comparison and identification using an algorithm such as BLAST (e.g., "Basic Local Alignment Search Tool"; see Altschul SF, et al., J.Mol.Biol.215:403-410, 1993). Thus, a substantial portion of the nucleotide sequence contains enough of the sequence to provide specific identification and / or isolation of the nucleic acid fragment containing that sequence. For example, a substantial portion of the nucleic acid sequence may contain at least about 20% of the full-length nucleic acid sequence, e.g., about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%.

[0096] In some embodiments, the modified alphaviral genome or srRNA vector of this disclosure lacks the entire sequence encoding the viral structural protein; for example, the modified alphaviral genome or srRNA vector does not contain the nucleic acid sequence encoding the viral structural protein.

[0097] The nucleic acid constructs of this disclosure further include coding sequences of polypeptide constructs that replace at least a portion of the nucleic acid sequence encoding a viral structural protein of a modified alphaviral genome or srRNA. In principle, the nucleic acid constructs disclosed herein may generally include any number of coding sequences of polypeptide constructs. In some embodiments, the nucleic acid constructs disclosed herein may include at least one, at least two, at least three, at least four, at least five, or at least six coding sequences of polypeptide constructs. The coding sequence of a polypeptide construct may be a construct of genetic material comprising the coding sequence and sufficient regulatory information to induce proper transcription and / or translation of the coding sequence in vivo and / or ex vivo within the cell. The coding sequence of a polypeptide construct can be inserted into a vector to target a desired host cell and / or subject or individual. Accordingly, in some embodiments, the term “coding sequence of polypeptide construct” may be used interchangeably with the term “expression construct.” In some embodiments, the coding sequence of the polypeptide construct may be a nucleic acid construct comprising, for example, a gene encoding a protein or functional RNA functionally linked to regulatory elements such as promoters and / or termination signals, 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 the coding sequence of an interleukin-1 receptor antagonist (IL-1RA) protein or a functional variant thereof, and the coding sequence of an interleukin-18 binding protein (IL-18BP) or a functional variant thereof, wherein the coding sequences of IL-1RA and IL-18BP are functionally linked to each other, and the polypeptide construct does not contain a dimerization domain. In some embodiments, the nucleic acid construct encodes IL-1RA and IL-18BP polypeptides that can induce pharmacodynamic effects in a subject. Functional variants of IL-1RA and IL-18BP may include the coding sequence of a polypeptide having an amino acid sequence that is the same as or essentially the same as the amino acid sequence of a reference protein (e.g., IL-1RA and IL-18BP), except that at least one amino acid is modified, e.g., deleted, inserted, or substituted. The amino acid substitution may be a conserved amino acid substitution, preferably at a non-essential amino acid residue in the protein. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by 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), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Modified proteins may have an amino acid sequence that is 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, the variant is a functional variant of the protein that retains the same or essentially the same function as the original protein.When used in relation to nucleic acid sequences, the term “modified” refers to a nucleic acid sequence in which one or more nucleotides differ from another, usually associated, nucleotide acid sequence. Therefore, the term “modified” can refer to a change in one or more nucleotides of a reference nucleic acid, including the insertion of one or more new nucleotides, the deletion of one or more nucleotides, and the substitution of one or more existing nucleotides. Modifieds may also include point mutations, multiple mutations, single nucleotide polymorphisms (SNPs), deletions, insertions, and translocations. Thus, modified coding sequences described herein include nucleic acids encoding polypeptides that may be, for example, the full-length, variant, shortened, inactivated, peptide / epitope, or combinations thereof of IL-1RA and IL-18BP.

[0099] The full-length amino acid sequence of human IL-1RA is shown in SEQ ID NO: 1(177aa) below. MEICRGLRSHLITLLLFLFHSETICRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE

[0100] In some embodiments, the coding sequence for the IL-1RA protein in the nucleic acid construct described herein codes for the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid construct of the present disclosure includes a nucleic acid sequence that codes for the IL-1RA 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 with respect to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the coding sequence for the IL-1RA protein codes for a smaller portion of the amino acid sequence of SEQ ID NO: 1. These smaller portions may 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 of the polypeptide construct in nucleic acid described herein encodes a variant of the IL-1RA protein.

[0102] As described above, the nucleic acid constructs described herein also include coding sequences for IL-18BP or its functional variants. In some embodiments, the nucleic acid constructs described herein also include coding sequences for IL-18BP isoform A (IL-18BPa) or its functional variants.

[0103] The full amino acid sequence of human IL-18BPa, along with its signal sequence, is shown in Sequence ID No. 2 (194aa) as follows. MTMRHNWTPDLSPLWVLLLCAHVVTLLVRATPVSQTTTAATASVRSTKDPCPSQPPVFPAAKQCPALEVTWPEVEVPLNGTLSLSCVACSRFPNFSILYWLGNGSFIEHLPGRLWEGSTSRERGSTGTQLCKALVLEQLTPALHSTNFSCVLVDPEQVVQRHVVLAQLWAGLRATLPPTQEALPSSHSSPQQQG

[0104] In some embodiments, the coding sequence for IL-18BP in the nucleic acid construct described herein codes for 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 with respect to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the coding sequence for IL-18BP codes for a smaller portion of the amino acid sequence of SEQ ID NO: 2. These smaller portions may 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 sequences of the polypeptide constructs in nucleic acids described herein encode variants of IL-18BP.

[0106] In some embodiments, the coding sequences of the IL-1RA protein or its functional variant, and IL-18BP or its functional variant, comprise a single polypeptide coding sequence (e.g., monogenic). In some embodiments, the coding sequences of the IL-1RA protein or its functional variant, and IL-18BP or its functional variant, comprise multiple polypeptide coding sequences (e.g., polygenic (e.g., digenic or trigenic)). In some embodiments, each of the coding sequences of the IL-1RA protein or its functional variant, and IL-18BP or its functional variant, is functionally linked to a separate promoter sequence. In some embodiments, the coding sequences of the IL-1RA protein or its functional variant, and IL-18BP or its functional variant, are functionally linked to each other within a single open reading frame (e.g., in a polycistronic ORF). In some embodiments, the nucleic acid sequence encoding the polypeptide construct is functionally linked to a promoter sequence. In some embodiments, the coding sequence of a polycistronic ORF is functionally linked to a promoter sequence. In some embodiments, the promoter sequence is a subgenome sg promoter. In some embodiments, the sg promoter sequence is a 26S subgenome promoter. In some embodiments, the subgenome promoter is heterologous to the rest of the modified viral genome or srRNA. In some embodiments, the subgenome promoter is an alphavirus subgenome promoter.

[0107] In some embodiments of this disclosure, at least one nonstructural protein (nsP) or portion thereof of the modified viral genome or srRNA is heterologous to the rest of the modified viral genome or srRNA. In some embodiments, the nucleic acid construct disclosed herein further comprises a nucleic acid sequence encoding the heterologous nsP or portion thereof. In some embodiments, the nucleic acid construct disclosed herein further comprises 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 from a virus belonging to the genus Alphavirus of the family Togaviridae. In some embodiments of the present disclosure, the modified alphavirus genome or srRNA is from an alphavirus belonging to the Venezuelan horse encephalitis virus / eastern equine encephalitis virus (VEEV / EEEV) group, the Semryqui Forest virus (SFV) group, or the Sindbis virus (SINV) group. In some embodiments, the modified alphavirus genome or srRNA is from an alphavirus belonging to the BFV complex, EEEV complex, MIDV complex, NDUV complex, SFV complex, VEEV complex, or WEEV complex. In some embodiments, alphaviruses include Eastern Equine Encephalitis Virus (EEEV), Venezuelan Equine Encephalitis Virus (VEEV), Everglades Virus (EVEV), Mukambo Virus (MUCV), Pixna Virus (PIXV), Middleberg Virus (MIDV), Chikungunya Virus (CHIKV), Onyonnyon Virus (ONNV), Ross River Virus (RRV), Burma Forest Virus (BF), Geta Virus (GET), Sagiyama Virus (SAGV), The alphaviruses are Beval virus (BEBV), Mayarovirus (MAYV), Unavirus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Wataroa virus (WHAV), Babanki virus (BABV), Kyzilagati virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Nudum 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 this disclosure include CHIKV S27, CHIKV LR2006-OPY-1, CHIKV YO123223, CHIKV DRDE, CHIKV 37997, CHIKV 99653, CHIKV Ag41855, and Nagpur(India)653496 strains. Both toxic and non-toxic CHIKV strains are suitable. Additional examples of CHIKV strains suitable for the compositions and methods of the present invention include, but are not limited to, those described in AfreenBarton et al. Microbiol.Immunol.2014,58:688-696, Lanciotti and Lambert ASTMH 2016,94(4):800-803, and Langsjoen et al. 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 this disclosure include EEEV 792138, 783372, BeAn5122, BeAr300851, BeAr436087, C-49, FL91-4679, FL93-939, GML903836, MP-9, PE6, and V105-00210. Both toxic and non-toxic EEEV strains are suitable. Additional suitable EEEV strains include, but are not limited to, those listed on the Virus Pathogen Resource website (ViPR; 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.

[0111] In some embodiments, the alphavirus is a 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 this disclosure include SINV strains AR339, AR86, and Girdwood. Examples of SINV strains suitable for the compositions and methods of this disclosure, but not limited to those described in Sammelset al. J. Gen. Virol. 1999, 80(3):739-748, Lundstrom and Pfeffer Vector Borne Zoonotic Dis. 2010, 10(9):889-907, Sigei et al. 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 to, those listed on the Virus Pathogen Resource website (ViPR; publicly available at www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=868&decorator=toga). Both toxic and non-toxic SINV strains are suitable. In some embodiments, the modified genome or RNA replicon is from the SINV strain Girdwood. In some embodiments, the modified genome or RNA replicon is from the SINV strain AR86. In some embodiments, the modified SINV genome or replicon RNA is derived from the SINV strain Girdwood. In some embodiments, the modified SINV genome or replicon RNA is derived from the SINV strain AR86. In some embodiments, at least one heterologous nsP or a portion thereof of the modified genome or RNA replicon is derived from the SINV strain AR86. In some embodiments, at least one heterologous nsP or a portion thereof is nsP1, nsP3, nsP4, or a portion of any of them, or any combination thereof. In some embodiments, the modified genome or RNA replicon is from the 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 this disclosure include WEEV California, McMillan, IMP181, Imperial, Imperial181, 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 this disclosure include 5614, 93A27, 93A30, 93A38, 93A79, B628(C 115), CBA87, CNTR34, CO921356, Fleming, Lake 43, PV012357A, PV02808A, PV72102, R02PV001807A, R02PV002957B, R02PV003422B, R05PV003422B, R0PV003814A, and R0PV00384A. Both toxic and non-toxic WEEV strains are 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 the Virus Pathogen Resource website (ViPR; publicly available at https: / / www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=57240&decorator=toga). In some embodiments, the modified WEEV genome or srRNA is derived from the WEEV strain Imperial. In some embodiments, the modified WEEV genome or srRNA is derived from the WEEV strain McMillan.

[0113] In some embodiments, the alphavirus is Madariaga virus (MADV), formerly known as East South American equine encephalitis virus (SA EEEV). Non-limiting examples of MADV strains suitable for the compositions and methods of the present invention 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 E 1 Delirio (Arrigo NC et al., 2010). Additional examples of MADV strains suitable for the compositions and methods of the present invention include 76V25343, 77U1 (BR77), BeAr 348998, IVICPan57151, BeaN 416361, 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., 2010). Additional suitable MADV strains include those described in Arrigo NC et al., 2010, and those publicly available on the Virus Pathogen Resource website (ViPR; 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 the MADV strain BeAr300851.

[0114] In some embodiments of the present disclosure, the coding sequence of the polypeptide construct includes, in the 5' to 3' direction, i.e., from the N-terminus to the C-terminus of the polypeptide sequence, (a) (i) the coding sequence of IL-1RA, (ii) a connector sequence encoding IRES, and (iii) the coding sequence of IL-18BP, (b) (i) the coding sequence of IL-1RA, (ii) a connector sequence encoding P2A autoproteolytic peptide, and (iii) the coding sequence of IL-18BP, (c) (i) the coding sequence of IL-18BP, (ii) a connector sequence encoding IRES, and (iii) the coding sequence of IL-1RA, or (d) (i) the coding sequence of IL-18BP, (ii) a connector sequence encoding P2A autoproteolytic peptide, and (iii) the coding sequence of IL-1RA.

[0115] In some embodiments of this 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 with an amino acid sequence selected from the group consisting of SEQ ID NOs. 7 to 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 with 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 with the amino acid sequence 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 with respect 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 with respect 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 with the amino acid sequence of SEQ ID NO: 7, and furthermore, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in the amino acid sequence are substituted with different amino acids. In some embodiments, the polypeptide construct includes an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO: 8, and furthermore, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in the amino acid sequence are substituted with different amino acids. In some embodiments, the polypeptide construct includes an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO: 9, and furthermore, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in the amino acid sequence are substituted with different amino acids. In some embodiments, the polypeptide construct comprises an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO: 10, and furthermore, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in the amino acid sequence are substituted with different amino acids.

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

[0118] In some embodiments of this disclosure, the coding sequence of 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 with a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 11 to 14. In some embodiments, the coding sequence of 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 with the nucleic acid sequence of SEQ ID NO. 11. In some embodiments, the coding sequence of 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 with the nucleic acid sequence of SEQ ID NO. 12. In some embodiments, the coding sequence of 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 with respect to the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the coding sequence of 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 with respect to the nucleic acid sequence of SEQ ID NO: 14.

[0119] In some embodiments, the coding sequence of the polypeptide construct includes a nucleic acid sequence having 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 11, and furthermore, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in the nucleic acid sequence are substituted with different nucleotides. In some embodiments, the coding sequence of 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 furthermore, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in the nucleic acid sequence are substituted with different nucleotides. In some embodiments, the coding sequence of the polypeptide construct includes a nucleic acid sequence having 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 13, and furthermore, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in the nucleic acid sequence are substituted with different nucleotides. In some embodiments, the coding sequence of the polypeptide construct includes a nucleic acid sequence having 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 14, and furthermore, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in the nucleic acid sequence are substituted with different nucleotides.

[0120] In some embodiments, the coding sequence of 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 with respect to the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the coding sequence of the polypeptide construct includes a nucleic acid sequence having 100% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 15, and furthermore, nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 in the nucleic acid sequence are substituted with different nucleotides. In some embodiments, the coding sequence of the polypeptide construct includes a nucleic acid sequence having 100% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 15.

[0121] In some embodiments, the coding sequences of IL-1RA and IL-18BP in the nucleic acid constructs disclosed herein are functionally linked to each other via one or more connector sequences. In some embodiments, the length and amino acid composition of the connector sequences can be optimized to alter the orientation, flexibility, and / or proximity of the polypeptides to each other, thereby achieving the desired activity or properties of the polypeptide construct. In some embodiments, the connector sequences include coding sequences for autoproteolytic peptides and / or internal ribosome entry sites (IRESs). Several autoproteolytic peptides, first identified in foot-and-mouth disease virus (FMDV), a member of the picornavirus group, have since been identified, such as “2A-like” peptides derived from equine rhinitis A virus (E2A), porcine rhinitis virus-1 (P2A), and Tosea signalavirus (T2A), and their activity in proteolytic cleavage has been demonstrated in various ex-vitro, in-vitro, ex-vivo, and in-vivo eukaryotic systems. Thus, the concept of autoproteolytic peptides is available to those skilled in the art, and many naturally occurring autoprotease systems have been identified. Well-tested autoprotease systems include, for example, viral proteases, developmental proteins (e.g., HetR, Hedgehog protein), RumA autoprotease domain, UmuD, etc. Non-limiting examples of autoproteolytic peptides suitable for the compositions and methods of this disclosure include one or more autoproteolytic cleavage sequences from calcium-dependent serine endoprotease (Fulin), porcine rhinitis virus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), Tosea signalavirus 2A (T2A), cytoplasmic polyhedron virus 2A (BmCPV2A), flachius virus 2A (BmIFV2A), or combinations thereof. In some embodiments, the autoproteolytic peptide comprises a P2A sequence.

[0122] In some embodiments, the coding sequences of the IL-1RA protein or its functional variant, and IL-1BP or its functional variant, are functionally linked to one or more internal ribosome entry sites (IRESs) coding sequences. An IRES, or "internal ribosome entry site," is a sequence located between polycistronic genes that enables the production of an expression product derived from a second gene by internal initiation of translation of a dicistronic mRNA. It facilitates direct internal ribosome entry into the start codon of the cistron (protein coding region), e.g., ATG, thereby resulting in cap-independent translation of the gene. See, for example, Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. In some embodiments, the IRES may be a viral IRES, a cellular IRES, or an artificial IRES. Examples of IRESs commonly used by those skilled in the art include those described in U.S. Patent No. 6,692,736. In some embodiments, the IRES is selected from Kaposi's sarcoma-associated herpesvirus (KSHV) IRES, hepatitis virus IRES, pestivirus IRES, Kripa virus IRES, roparosifanpadi virus IRES, fibroblast growth factor IRES, platelet-derived growth factor IRES, vascular endothelial growth factor IRES, insulin-like growth factor IRES, picornavirus IRES, encephalomyocarditis virus (EMCV) IRES, Pim-1 IRES, p53 IRES, Apaf-1 IRES, TDP2 IRES, L-myc IRES, and c-myc IRES. In some embodiments, the IRES is derived from EMCV.

[0123] Those skilled in the art will recognize that different configurations of the coding sequences of the IL-1RA protein or its functional variant, IL-1BP or its functional variant, and IRES can be used, provided that the expression and / or biological activity of the IL-1RA protein or its functional variant and IL-1BP or its functional variant are adequately maintained. These sequences are typically functionally linked and configured so that the polypeptide encoded by the gene of interest can be released from the ribosome and other sequences after self-cleavage or ribosome skipping.

[0124] Methodologies and techniques for functionally ligating two or more DNA sequences are well known to those skilled in the art, and such methods are described in many books on standard molecular biological operations (see, for example, Maniatis et al., "Molecular Cloning: A Laboratory Manual," 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Gibson et al., Nature Methods 6:343-45, 2009).

[0125] As described in further detail in Examples 1-3 below, the expression and biological activity of IL-1RA and IL-18BP were measured from different monogenic and digenic constructs in different modified alphaviral vectors. Exemplary compositions of nucleic acid constructs described herein are shown in Table 1 below.

[0126] In some embodiments, the coding sequences for the IL-1RA protein and / or IL-1BP are redesigned, refactored, and / or optimized for desired properties, such as increased stability, potency, and expression (e.g., translation efficiency), thereby maximizing the impact of the production, delivery, and administration of the biological agent IL-1RA protein and / or IL-1BP. For example, in some embodiments, the coding sequences for the IL-1RA protein and / or IL-1BP are optimized for expression at levels higher than the expression level of the reference coding sequence, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% higher than the reference coding sequence. In some embodiments, the reference coding sequence is a wild-type, unoptimized sequence. In some embodiments, the coding sequences of the IL-1RA protein and / or IL-1BP are optimized for one or more of the following: (a) enhancing RNA stability, (b) enhancing expression levels, (c) minimizing the use of rare codons, (d) minimizing secondary structures, (d) promoting better srRNA replication, and (e) promoting a better RNA manufacturing process.

[0127] With regard to the sequence optimization of nucleotide sequences, genetic coding degeneracy offers the possibility of substituting at least one base of a gene's protein-coding sequence with a different base without altering the amino acid sequence of the polypeptide produced from the gene. Thus, nucleic acid constructs of this disclosure may also have any base sequence modified from any polynucleotide sequence disclosed herein by substitution according to genetic coding degeneracy. References describing codon usage frequencies are readily available in the public. In some embodiments, polynucleotide sequence modifiers may be produced for various reasons, for example, to optimize expression for a specific host (e.g., changing the codon usage frequency in alphavirus mRNA to one preferred by humans, non-human primates, hamsters, mice, or other organisms such as monkeys). Thus, in some embodiments, coding sequences are optimized for expression in target host cells through the use of codons optimized for expression. Techniques for constructing synthetic nucleic acid sequences encoding genes with preferred codons optimized for host cell expression can be determined by computer methods that analyze the commonality and relative abundance of codon usage frequencies encoding native proteins in the host cell genome using techniques known in the art. Various codon frequency databases (e.g., www.kazusa.or.jp / codon) can be used to generate codon-optimized sequences in the mammalian cell environment. Furthermore, various software tools are available to convert sequences from one organism to codon frequencies optimized for different host organisms; for example, the JCat Codon Optimization Tool (www.jcat.de), the Integrated DNA Technologies (IDT) Codon Optimization Tool (https: / / www.idtdna.com / CodonOpt), or the Optimizer online codon optimization tool (http: / / genomes.urv.es / OPTIMIZER) are available. Such synthetic sequences can be constructed using techniques known in the art for the construction of synthetic nucleic acid molecules, or they can be obtained from various commercial suppliers.

[0128] Therefore, in some embodiments, the coding sequence of the IL-1RA protein and / or IL-1BP is optimized for expression at a higher level than the expression level of a reference coding sequence, such as a non-codon optimized coding sequence. In some embodiments, the codon-optimized sequence of the IL-1RA protein and / or IL-1BP results in an increase in expression level of 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 non-codon-optimized reference coding sequence. In some embodiments, the codon-optimized sequence of the IL-1RA protein and / or IL-1BP results in an increase in expression level of at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold compared to a non-codon-optimized reference coding sequence.

[0129] In some embodiments, the coding sequences of the IL-1RA protein and / or IL-1BP are optimized for enhanced RNA stability and / or expression. RNA stability is generally related to the RNA's "half-life," which refers to the time required to eliminate half of the activity, quantity, or number of a molecule. In the context of this disclosure, the RNA's half-life indicates its stability. The RNA's half-life can affect the RNA's "duration of expression." Further information on principles, strategies, and methods for use in enhancing RNA stability can be found, for example, in Leppek K. et al., Nature Communications, 22 Mar 2022, 13(1):1536.

[0130] In some embodiments, the nucleic acid construct of the Disclosure comprises a nucleic acid sequence encoding a polypeptide construct of the Disclosure, the 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 with the 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 present disclosure include a 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 with different nucleotides.

[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%) with respect to the target modified alphavirus genome or srRNA sequence can be identified and / or isolated by genome sequencing analysis, hybridization, and / or PCR using degenerate primers or gene-specific primers from the sequence identified in each alphavirus genome or srRNA, using the sequence identified herein (e.g., SEQ ID NO: 1) or any other sequence known in the art.

[0133] Molecular techniques and methods for assembling and characterizing these new nucleic acid constructs are described in more detail in the embodiments of this 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, polypeptides) that results from, but indirectly from, human manipulation. As a non-limiting example, cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that is produced by an in vitro polymerase reaction, or to which a linker is attached, or incorporated into a vector such as a cloning vector or an expression vector. As a non-limiting example, recombinant nucleic acid molecules are 1) synthesized or modified in vitro using, for example, chemical or enzymatic methods (e.g., by the use of chemical nucleic acid synthesis, or by the use of enzymes for replication, polymerization, exonuclease digestion, endonuclease digestion, ligation, reverse transcription, transcription, base modification (e.g., including methylation), or recombination (including homologous and site-directed recombination) of nucleic acid molecules), 2) contain conjugation nucleotide sequences that are not conjugated in nature, 3) manipulated using molecular cloning techniques to lack one or more nucleotides from a naturally occurring nucleotide sequence, and / or 4) manipulated using molecular cloning techniques to have one or more sequence changes or rearrangements from a naturally occurring nucleotide sequence.

[0134] In some embodiments, the nucleic acid molecules disclosed herein are produced using recombinant DNA techniques (e.g., polymerase chain reaction (PCR) amplification, cloning, etc.) or chemical synthesis. Nucleic acid molecules disclosed herein include, but are not limited to, natural nucleic acid molecules and their homologs, including, but not limited to, natural allele variants, and modified nucleic acid molecules in which one or more nucleotide residues are inserted, deleted, and / or substituted in a manner that provides desired properties when such modification affects the biological activity described herein.

[0135] Nucleic acid molecules, including variants of naturally occurring nucleic acid sequences, can be produced using many 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, NY (1989)). The sequences of nucleic acid molecules can be modified from their naturally occurring sequences using a variety of techniques, including, but not limited to, classical mutagenesis and recombinant DNA techniques, such as, but not limited to, site-directed mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, restriction enzyme cleavage of nucleic acid fragments, ligation of nucleic acid fragments, PCR amplification and / or mutagenesis of selected regions of nucleic acid sequences, recombinant cloning, and chemical synthesis including the chemical synthesis of oligonucleotide mixtures and the "construction" of nucleic acid molecule mixtures by ligating groups of mixtures, as well as 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 srRNA encoded by the nucleic acid molecule, and / or by hybridization with the wild-type gene or its fragment, or by PCR using primers homologous to the target or wild-type nucleic acid molecule or sequence.

[0136] B. Recombinant cells The nucleic acid constructs of this disclosure can be introduced into host cells to generate recombinant cells containing nucleic acid molecules. Therefore, prokaryotic or eukaryotic cells containing nucleic acid constructs encoding modified alphaviral genomes or srRNAs as described herein are also features of this disclosure. In relevant embodiments, some embodiments disclosed herein relate to a method for transforming cells, comprising the steps of introducing the nucleic acid constructs provided herein into host cells such as animal cells, and then selecting or screening transformed cells. The introduction of the nucleic acid constructs of this disclosure into cells can be carried out by methods known to those skilled in the art, such as viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI) transfection, DEAE-dextran transfection, liposome transfection, particle gun technology, direct microinjection, and nucleic acid delivery by nanoparticles.

[0137] In one embodiment, several embodiments of the present disclosure relate to recombinant cells, for example, recombinant animal cells containing nucleic acid constructs described herein. The nucleic acid constructs can be stably incorporated into the host genome, or replicated episomatically, or present in recombinant host cells as minicircle expression vectors for stable or transient expression. Thus, in several embodiments of the present disclosure, the nucleic acid constructs are maintained and replicated in recombinant host cells as episomatic units. In several embodiments, the nucleic acid constructs are stably incorporated into the genome of recombinant cells. Stable integration can be achieved using classical random genome recombination techniques or using more precise genome editing techniques, such as guide RNA-guided CRISPR / Cas9 or TALEN genome editing. In several embodiments, the nucleic acid constructs are present in recombinant host cells as minicircle expression vectors for stable or transient expression.

[0138] The host cell may be either an untransformed cell or a cell already transfected with at least one nucleic acid molecule. Therefore, in some embodiments, the host cell may be genetically engineered with at least one nucleic acid molecule (e.g., transfected, transformed, or transfected).

[0139] Suitable host cells for cloning or expressing the target polypeptides described herein include prokaryotic or eukaryotic cells as described herein. In some embodiments, the recombinant cells are prokaryotic cells such as Escherichia coli, or eukaryotic cells such as insect cells (e.g., mosquito cells or Sf21 cells) or mammalian cells (e.g., COS cells, NIH 3T3 cells or HeLa cells). In some embodiments, the cells are in vivo, for example, recombinant cells in a living organism, e.g., cells to be transfected. In some embodiments, the subject is a vertebrate or invertebrate. In some embodiments, the subject is an insect. In some embodiments, the subject is a mammalian subject. In some embodiments, the cells are ex vivo, for example, extracted from a living organism or organism as individual cells or as part of an organ or tissue for therapeutic or procedural purposes and then returned to the living organism or organism. In some embodiments, the cells are in vitro, for example, obtained from a repository. In some embodiments, the recombinant cells are eukaryotic cells. In some embodiments, the recombinant cells are animal cells. In some embodiments, the animal cells are vertebrate cells or invertebrate cells. In some embodiments, the recombinant cells are mammalian cells.Non-limiting examples of recombinant cells suitable for the methods and compositions of this disclosure include SV40-transformed monkey kidney CV1 cells (e.g., COS-7 cells), human fetal kidney cells (e.g., HEK 293 or HEK 293 cells) or their derivatives (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 cancer cells (e.g., HeLa cells), canine kidney cells (MDCK cells), buffalo rat hepatocytes (e.g., BRL 3A cells), human lung cells (e.g., W138 cells), human hepatocytes (e.g., Hep G2 cells), and mouse mammary tumor cells (e.g., MMT cells). Examples include 060562 cells, TRI cells, FS4 cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (e.g., Vero cells), human A549 cells, human cervical cells, human CHME5 cells, human PER.C6 cells, NS0 mouse myeloma cells, human laryngeal squamous epithelial cells, human fibroblasts, 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, recombinant cells are selected from the group consisting of African green monkey kidney cells (Vero cells), baby hamster kidney (BHK) cells, Chinese hamster ovary cells (CHO cells), human A549 cells, human cervical cells, human CHME5 cells, human laryngeal squamous epithelial cells, human fibroblasts, human HEK-293 cells, human HeLa cells, human HepG2 cells, human HUH-7 cells, human MRC-5 cells, human muscle cells, mouse 3T3 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells.

[0141] In some embodiments, recombinant cells are immune cells. In some embodiments, immune cells include B cells, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, basophils, eosinophils, neutrophils, dendritic cells (DCs), macrophages, regulatory T cells, and helper T cells (T). H ), cytotoxic T cells (T CTL ), memory T cells, gamma delta (γδ) T cells, hematopoietic stem cells, or hematopoietic stem cell progenitor cells. In some embodiments, the immune cells are B cells, T cells, macrophages, or dendritic cells (DCs). In some embodiments, the immune cells are B cells. In some embodiments, the immune cells are T cells.

[0142] In some embodiments, recombinant cells are the cells described above (i.e., cells derived from the original cells described herein), such as clones of the original cells, manipulated versions of the original cells, or cells grown from a reclassification of the original cells after extensive passage, or passaged through another host.

[0143] In some embodiments, the recombinant cells are insect cells, for example, cells from an insect cell line. In some embodiments, the recombinant cells are Sf21 cells. Additional suitable insect cell lines, but not limited to these, include lines established from insects of the orders Diptera, Lepidoptera, and Hemiptera, and may originate from a variety of tissue sources. In some embodiments, the recombinant cells are cells from a Lepidoptera insect cell line. Over the past few decades, the number of available Lepidoptera insect cell lines has increased by approximately 50 per decade. Further information on available Lepidoptera insect cell lines can be found, for example, in Lynn DE, Available lepidoptera insect cell lines. Methods Mol Biol. 2007;388:117-38, which is incorporated herein by reference. In some embodiments, the recombinant cells are mosquito cells, for example, cells of mosquito species of the genera Anopheles (An.), Culex (Cexus pipiens) (Cx.), and Aedes (Aedes mosquitoes, Zebra mosquitoes) (Ae.). Examples of mosquito cell lines suitable for the compositions and methods described herein include the following mosquito species, namely, Aedes aegyptii (Aedes aegypti), Aedes arbopictus (Aedes albopictus), Aedes pseudoscutellaris, Aedes triseriatus, Aedes bexans, Anopheles gambier (Anopheles gambier), Anopheles stepens (Anopheles stepens), Anopheles albimanus, Crex quinquephassiatus, Crex tyrellii, Crex tritaeniolynchus (Culex tritaeniolynchus), Crex bitaeniolinchus (Culex cuvieri), and cell lines derived from Toxolincites 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, AtGRIP-1, AtGRIP-2, UM-AVE1, Mos.55, Sua1B, 4a-3B, Mos.43, MSQ43, and LSB-AA695BB. In some embodiments, the mosquito cells are from the C6 / 26 cell line.

[0144] In another aspect, this specification provides a cell culture comprising at least one recombinant cell and a culture medium disclosed herein. Generally, the culture medium may be any culture medium suitable 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 are described in the technical and scientific literature. Therefore, a cell culture comprising at least one recombinant cell disclosed herein is also within the scope of this application. Suitable methods and systems for generating and maintaining cell cultures are known in the art.

[0145] C. Transgenic Animals In other embodiments, transgenic animals comprising nucleic acid constructs (e.g., vectors, replicons, or srRNA molecules) described herein are also provided. In some embodiments, the transgenic animal is a vertebrate or an invertebrate. 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 mammal is a non-human mammal. Generally, the transgenic animals of this disclosure may be any non-human animal known in the art. In some embodiments, the non-human animals of this disclosure are non-human primates. Other animal species suitable for the compositions and methods of this 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, but not limited to, include mice, rats, hamsters, rabbits, chickens, goats, pigs, sheep, and cattle. Additional examples of non-human animals suitable for the compositions and methods of this disclosure include, but are not limited to, 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, minks, beavers, ermine, otters, sables, 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 animal of the Disclosure is a chimeric transgenic animal. In some embodiments, the transgenic animal of the Disclosure is a transgenic animal having 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 Disclosure. In some embodiments, one or more nucleic acid constructs are stably incorporated into the genome of the transgenic animal. In some embodiments, the genome of the transgenic animal of the Disclosure may contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, four, or more copies of one or more nucleic acid constructs of the Disclosure.

[0147] Approaches and methods for producing transgenic non-human animals are known in the art. Exemplary methods include pronuclear microinjection, DNA microinjection, lentiviral vector-based DNA transfer into early embryos and sperm-based transgenesis, adenovirus-based DNA transfer into animal sperm (e.g., in pigs), retroviral vectors (e.g., in birds), and somatic cell nuclear transfer (e.g., in goats). The latest techniques in the production of transgenic livestock are reviewed in Niemann, H. et al. (2005), Rev. Sci. Tech. 24:285-298. In some embodiments, the transgenic non-human host animals of this disclosure are produced using standard methods known in the art for introducing exogenous nucleic acids into the genomes of non-human animals. In some embodiments, the transgenic animals of this disclosure can be prepared using classical random genome recombination techniques, or using more precise techniques such as guide RNA-induced CRISPR / Cas genome editing, DNA-induced endonuclease genome editing with NgAgo (Natronobacterium gregoryi Argonaute), or TALEN genome editing (transcription activator-like effector nucleases). In some embodiments, the transgenic animals of this disclosure can be prepared using transgenic microinjection techniques, which do not require the use of homologous recombination techniques and are therefore considered easier to prepare and select than approaches using homologous recombination. In some embodiments, the transgenic animals produce polypeptide constructs described herein.

[0148] The transgenic non-human host animals of this disclosure are prepared using standard methods known in the art for introducing exogenous nucleic acids into the genome of non-human animals. In some embodiments, the non-human animals of this disclosure are non-human primates. Other animal species suitable for the compositions and methods of this 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, but not limited to, include mice, rats, hamsters, rabbits, chickens, goats, pigs, sheep, and cattle. Approaches and methods for preparing transgenic non-human animals are known in the art. Exemplary methods include pronuclear microinjection, DNA microinjection, DNA transfer into early embryos and transgenesis by sperm using lentiviral vectors, DNA transfer into animal sperm using adenoviruses (e.g., in pigs), retroviral vectors (e.g., in bird species), and somatic cell nuclear transfer (e.g., in goats). The latest technologies for creating transgenic livestock are reviewed in Niemann, H. et al. (2005), Rev. Sci. Tech. 24:285-298.

[0149] In some embodiments, the animal is a vertebrate or an invertebrate. In some embodiments, the animal is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the animal is a mammal. In some embodiments, the mammal is a non-human animal. In some embodiments, the mammal is a non-human primate. In some embodiments, the transgenic animals of this disclosure can be prepared using classical random genome recombination techniques, or using more precise techniques such as guide RNA-induced CRISPR / Cas genome editing, DNA-induced endonuclease genome editing with NgAgo (Natronobacterium gregoryi Argonaute), or TALEN genome editing (transcription activator-like effector nuclease). In some embodiments, the transgenic animals of this disclosure can be prepared using transgenic microinjection techniques, which do not require the use of homologous recombination techniques and are therefore considered easier to prepare and select than approaches using homologous recombination. In another embodiment, a method for producing a target polypeptide is provided herein, comprising (i) raising a transgenic animal disclosed herein, or (ii) culturing recombinant cells containing a nucleic acid construct disclosed herein under conditions that cause the transgenic animal or recombinant cells to produce a polypeptide construct encoded by the nucleic acid construct disclosed herein. In another embodiment, a method for producing a target polypeptide in a subject is provided herein, comprising administering the subject a nucleic acid construct disclosed herein. In some embodiments, the subject is a vertebrate or an invertebrate. In some embodiments, the subject is an insect. In some embodiments, the insect is a mosquito. In some embodiments, 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.

[0150] D. Pharmaceutical Compositions The nucleic acid constructs (e.g., replicon constructs, e.g., srRNA constructs) and recombinant cells of this disclosure can be incorporated into compositions comprising pharmaceutical compositions. Such compositions generally comprise one or more nucleic acid constructs (e.g., replicon constructs, e.g., srRNA constructs) and recombinant cells described and provided herein, and pharmaceutically acceptable excipients, e.g., carriers. In some embodiments, the compositions of this disclosure are formulated for the prevention, treatment, or management of health conditions such as autoimmune diseases, inflammatory diseases, or cardiovascular diseases. For example, the compositions of this disclosure can be formulated as prophylactic compositions, therapeutic compositions, or pharmaceutical compositions, or mixtures thereof, comprising pharmaceutically acceptable excipients. In some embodiments, the compositions of this disclosure are formulated for use as adjuvants.

[0151] Accordingly, in one embodiment, a pharmaceutical composition is provided herein comprising a pharmaceutically acceptable excipient and: a) a nucleic acid construct of the Disclosure, and / or b) recombinant cells of the Disclosure.

[0152] Non-limiting exemplary embodiments of the pharmaceutical compositions of this disclosure may include one or more of the following features. In some embodiments, this specification provides a composition comprising a nucleic acid construct disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, this specification provides a composition comprising recombinant cells disclosed herein and a pharmaceutically acceptable excipient.

[0153] In some embodiments, the nucleic acid constructs of this disclosure (e.g., vectors or srRNA molecules) can be used in their bare form or formulated with a delivery vehicle. Examples of suitable delivery vehicles for the compositions and methods of this disclosure include, but are not limited to, liposomes (e.g., neutral or anionic liposomes), microspheres, immunostimulatory complexes (ISCOMs), lipid-based nanoparticles (LNPs), solid lipid nanoparticles (SLNs), polyplexes, polymer nanoparticles, viral replicon particles (VRPs), or those conjugated with bioactive ligands that can facilitate delivery and / or enhance the immune response. These compounds are readily available to those skilled in the art. See, for example, Liposomes: A Practical Approach, RCP New Ed, IRL press (1990). Adjuvants other than liposomes are also used and are known in the art. Adjuvants can protect antigens (e.g., nucleic acid constructs, vectors, srRNA molecules) from rapid dispersion by sequestering them within local deposits, or they may include substances that stimulate the host to secrete factors that are chemotactic to macrophages and other components of the immune system. Those skilled in the art can make appropriate selections from, for example, those described below.

[0154] The compositions of this disclosure can be formulated in a form compatible with the intended route of administration, such as liposomes, lipid-based nanoparticles (LNPs), polymer nanoparticles, polyplexes, viral replicon particles (VRPs), microspheres, immunostimulatory complexes (ISCOMs), bioactive ligand conjugates, or any combination thereof. Accordingly, in some embodiments, the compositions of this disclosure can be formulated in liposomes.

[0155] In some embodiments, the compositions of this disclosure are formulated with lipid-based nanoparticles (LNPs). 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 LNPs of this disclosure may comprise one or more ionizable lipids. Examples of ionizable lipids suitable for the compositions and methods of this disclosure are described in International Publication Nos. 2020252589(A1) and 2021000041(A1), and in Love KT et al., Proc Natl Acad Sci USA, Feb. 2, 2010 107(5)1864-1869, the entire contents of which are incorporated herein by reference.

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

[0158] In some embodiments, the LNPs of this disclosure comprise 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.

[0159] In some embodiments, the LNPs of this disclosure comprise one or more neutral lipids. As described above, neurolipids, also known as “structural lipids” or “helper lipids,” may also be incorporated into lipid formulations and lipid particles in some embodiments. Lipid formulations and lipid particles may contain one or more structural lipids in about 10 to 40 mol% of the composition. Appropriate structural lipids support the formation of particles during manufacturing. Structural lipids refer to one of a number of lipid species that exist in either anionic, uncharged, or neutral zwitterionic forms at physiological pH. Representative structural lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, diacylphosphatidylglycerol, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebroside.

[0160] Examples of structural lipids include zwitterionic lipids, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane. Examples include -1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-dierydoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE).

[0161] In another embodiment, the structural lipid may be any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerols, such as dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleylphosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, and other anionic modifying groups attached 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 this disclosure include DPSC, DPPC, POPC, DOPE, and SM. In some embodiments, the LNPs of this disclosure comprise one or more ionizable lipid compounds described in International Publication Nos. 2020252589(A1) and 2021000041(A1), which are incorporated herein by reference in whole.

[0163] In some embodiments, the LNPs of the present disclosure include 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-DMOLIE-DPyPE, and GL67A-DOPE-DMP-polyethylene glycol (PEG).

[0164] In some embodiments of the delivery system described herein, which includes LNPs, the mass ratio of lipids to nucleic acids 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 lipids to nucleic acids in the LNP delivery system is about 16:1 to 4:1. In some embodiments, the mass ratio of lipids to nucleic acids in the LNP delivery system is about 20:1. In some embodiments, the mass ratio of lipids to nucleic acids in the LNP delivery system is about 8:1. In some embodiments, the lipid-based nanoparticles (LNPs) have an average diameter of 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 less than about 25 nm. In some embodiments, the LNPs have an average diameter in the range of about 70 nm to 100 nm. In some embodiments, the LNP has an average diameter in the range of approximately 88 nm to approximately 92 nm, 82 nm to approximately 86 nm, or approximately 80 nm to approximately 95 nm.

[0165] Stabilizers may be included in embodiments of lipid formulations to ensure the integrity of the mixture. Stabilizers are a class of molecules that disrupt or help form intermolecular hydrophobic-hydrophilic interactions. Suitable stabilizers include, but are not limited to, polysorbate 80 (also known as Tween 80, IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyloctadeca-9-enoate), Myrj52 (polyoxyethylene(40) stearate), and Brij® S10 (polyoxyethylene(10) stearyl ether). Polyethylene glycol-bound lipids may also be used. Stabilizers may be used alone or in combination with each other.

[0166] In some embodiments, the stabilizer constitutes about 0.1 to 3 mol% of the total lipid mixture. In some embodiments, the stabilizer constitutes about 0.5 to 2.5 mol% of the total lipid mixture. In some embodiments, the stabilizer is present at more than 2.5 mol%. In some embodiments, the stabilizer is present at 5 mol%. In some embodiments, the stabilizer is present at 10 mol%. In some embodiments, the stabilizer is present at 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, etc. In other embodiments, the stabilizer is 2.6 to 10 mol% of the lipid mixture. In other embodiments, the stabilizer is present at more than 10 mol% of the lipid mixture.

[0167] Steroids may also be included in lipid compositions for specific applications, and the lipid particles produced therefrom may contain sterols (e.g., cholesterol and phytosterols).

[0168] Polymer nanoparticles In some embodiments, the compositions of the present disclosure can be formulated with polymer nanoparticles. In some embodiments, the polymer nanoparticles include cationic polymers, non-cationic polymers, or combinations thereof. In some embodiments, the cationic polymer includes naturally derived cationic polymers. In some embodiments, the naturally derived cationic polymer includes chitosan, gelatin, dextran, cellulose, cyclodextrin, or combinations thereof. In some embodiments, the cationic polymer includes synthetic cationic polymers. In some embodiments, examples of synthetic cationic polymers include poly(ethyleneimine) (PEI), poly-L-lysine (PLL), poly(amino) acid (PAA), poly(amidoamine) (PAMAM), poly(cystamine bisacrylamide-co-4-amino-1-butanol) (pABOL), poly(amino-co-ester) (PAE), poly(2-N,N-dimethylaminoethyl methacrylate), poly(β-aminoester) (PBAE), imidazole-containing polymers, tertiary amine-containing polymers, poly(2-(dimethylamino)ethyl methacrylate), poly-N-(2-hydroxypropyl)methacrylamide, polyamidoamine dendrimers, cationic glycopolymers, or derivatives thereof.

[0169] In some embodiments, the noncationic polymer is negatively charged (i.e., anionic) or electronically neutral. In some embodiments, the noncationic polymer includes polyethylene glycol (PEG), polyesters (e.g., polylactic acid (PLA), poly(lactic acid-coglycolic acid) (PLGA), polyglycolic 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 this disclosure, the polymer nanoparticles include one or more of the following: poly-(γ-L-glutamylglutamine) (PGGA), poly-(γ-L-aspartylglutamine) (PGAA), poly-L-lactic acid (PLLA), poly-(lactic acid-coglycolic acid) (PLGA), polyalkylcyanoacrylate (PACA), polyanhydride, polyhydroxy acid, polypropyl fumerate, 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.

[0171] Lipid-based nanoparticles (LNPs) In some embodiments, the compositions of the Disclosure can be formulated with lipid-based nanoparticles (LNPs). For example, the nucleic acid constructs of the Disclosure can be delivered to cells or targets by lipid-based nanoparticles (LNPs). LNPs are generally less immunogenic than viral particles. Many humans have pre-existing immunity to viral particles, but not to LNPs. Furthermore, the likelihood of an adaptive immune response to LNPs is low, which allows for repeated administration of LNPs.

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

[0173] In some embodiments, the LNPs of this disclosure may comprise one or more ionizable lipids. As used herein, the term “ionizable lipid” refers to a lipid that is cationic or becomes ionizable (protonated) when the pH drops below the pKa of the lipid’s ionic group, but becomes more neutral at higher pH values. At pH values ​​below pKa, lipids can associate with negatively charged nucleic acids (such as oligonucleotides). As used herein, the term “ionizable lipid” includes lipids that are presumed to become positively charged when the pH drops from physiological pH, and any of several lipid species that have a net positive charge at a selective pH such as physiological pH. Persistently cationic lipids such as DOTMA have been proven to be too toxic for clinical use. Ionizable lipids may be present in lipid formulations according to other embodiments, preferably in a ratio of about 30 to about 70 mol%, about 30 mol% in some embodiments, about 40 mol% in other embodiments, about 45 mol% in other embodiments, about 47.5 mol% in other embodiments, about 50 mol% in yet another embodiment, and about 60 mol% in yet another embodiment ("mol%" means the percentage of total moles that belong to a particular component). The LNPs of this disclosure may include DODMA, which is an ionizable lipid, as well as DLin-MC3-DMA or O-(Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N-dimethylamino) ("MC3"), or 1,2-dioleyloxy-3-dimethylaminopropane.

[0174] Examples of ionizable lipids suitable for the compositions and methods of this disclosure include those described in International Publication Nos. 2020252589(A1) and 2021000041(A1), U.S. Patent Nos. 8,450,298 and 10,844,028, and Love KT et al., Proc Natl Acad Sci USA, Feb. 2, 2010 107(5)1864-1869, all of which are incorporated herein by reference in their entirety. Accordingly, in some embodiments, the LNPs of this disclosure include one or more lipid compounds described above in Love KT et al., 2010, such as C16-96, C14-110, and C12-200. In some embodiments, the LNP comprises an ionizable cationic lipid selected from the group consisting of ALC-0315, C12-200, LN16, MC3, MD1, SM-102, and any combination thereof. In some embodiments, the LNP of this disclosure comprises C12-200. The structure of the C12-200 lipid is known in the art and is described, for example, in U.S. Patent Nos. 8,450,298 and 10,844,028, which are incorporated herein by reference in whole. In some embodiments, C12-200 is combined with cholesterol, C14-PEG2000, and DOPE. In some embodiments, C12-200 is combined with DSPC and DMG-PEG2000.

[0175] In some embodiments, the LNPs of this disclosure comprise 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 LNPs of this disclosure comprise one or more neutral lipids. Non-limiting neutral lipids suitable for the compositions and methods of this disclosure include DPSC, DPPC, POPC, DOPE, and SM. In some embodiments, the LNPs of this disclosure comprise one or more ionizable lipid compounds described in International Publication Nos. 2020252589(A1) and 2021000041(A1), which are incorporated herein by reference in whole.

[0176] LNPs can be prepared using many other lipids or combinations of lipids known in the art. Non-limiting examples of lipids suitable for use in LNP preparation include DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DPPE-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 any combination 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 present disclosure comprises 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-DMOLIE-DPyPE, and GL67A-DOPE-DMP-polyethylene glycol (PEG). In some embodiments, C12-200 is combined with cholesterol, C14-PEG2000, and DOPE. In some embodiments, C12-200 is combined with DSPC and DMG-PEG2000.

[0178] In some embodiments, the mass ratio of lipids to nucleic acids 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 lipids to nucleic acids in the LNP delivery system is about 16:1 to 4:1. In some embodiments, the mass ratio of lipids to nucleic acids in the LNP delivery system is about 20:1. In some embodiments, the mass ratio of lipids to nucleic acids in the LNP delivery system is about 8:1. In some embodiments, the lipid-based nanoparticles have an average diameter of 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 less than about 25 nm. In some embodiments, the LNPs have an average diameter in the range of about 70 nm to 100 nm. In some embodiments, the LNP has an average diameter in the range of approximately 88 nm to approximately 92 nm, 82 nm to approximately 86 nm, or approximately 80 nm to approximately 95 nm.

[0179] As described above, neurolipids, also known as "structural lipids" or "helper lipids," can also be incorporated into lipid formulations and lipid particles in some embodiments. Lipid formulations and lipid particles may contain one or more structural lipids in about 10-40 mol% of the composition. Appropriate structural lipids support particle formation during manufacturing. Structural lipids refer to one of a number of lipid species that exist in either anionic, uncharged, or neutral zwitterionic forms at physiological pH. Representative structural lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, diacylphosphatidylglycerol, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebroside.

[0180] Examples of structural lipids include zwitterionic lipids, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane. Examples include -1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-dierydoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE).

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

[0182] Stabilizers may be included in embodiments of lipid formulations to ensure the integrity of the mixture. Stabilizers are a class of molecules that disrupt or help form intermolecular hydrophobic-hydrophilic interactions. Suitable stabilizers include, but are not limited to, polysorbate 80 (also known as Tween 80, IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyloctadeca-9-enoate), Myrj52 (polyoxyethylene(40) stearate), and Brij® S10 (polyoxyethylene(10) stearyl ether). Polyethylene glycol-bound lipids may also be used. Stabilizers may be used alone or in combination with each other.

[0183] In some embodiments, the stabilizer constitutes about 0.1 to 3 mol% of the total lipid mixture. In some embodiments, the stabilizer constitutes about 0.5 to 2.5 mol% of the total lipid mixture. In some embodiments, the stabilizer is present at more than 2.5 mol%. In some embodiments, the stabilizer is present at 5 mol%. In some embodiments, the stabilizer is present at 10 mol%. In some embodiments, the stabilizer is present at 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, etc. In other embodiments, the stabilizer is 2.6 to 10 mol% of the lipid mixture. In other embodiments, the stabilizer is present at more than 10 mol% of the lipid mixture.

[0184] Steroids may also be included in lipid compositions for specific applications, and the lipid particles produced therefrom may contain sterols (e.g., cholesterol and phytosterols).

[0185] In some embodiments, the immunogenic composition is substantially non-immunogenic or minimally immunogenic (e.g., a composition that minimally stimulates an immune response in a subject). In some embodiments, the non-immunogenic or minimally immunogenic composition is formulated as a biological agent. In some embodiments, the pharmaceutical composition is formulated for one or more of the following methods of administration: intranasal, transdermal, intraperitoneal, intramuscular, intranodal, intratumoral, intraarticular, intravenous, subcutaneous, vaginal, cardiac, and oral.

[0186] Suitable pharmaceutical compositions for injection include sterile aqueous solutions or dispersions, and sterile powders for the preparation of sterile injection solutions or dispersions on demand. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS), Tris (tromethamine), and HEPES. In these cases, the composition should be sterile and fluid enough to be easily injected. It should be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of a surfactant, such as sodium dodecyl sulfate. The action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, sucrose, and trehalose, and / or sodium chloride are generally contained in the composition. In some embodiments, the composition contains tris and sucrose. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate or gelatin, in the composition.

[0187] Sterile injection solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent, along with one or a combination of the components listed above as needed, followed by sterile filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a base dispersion medium and other necessary components from those listed above.

[0188] In some embodiments, the pharmaceutical composition is formulated for one or more of the following administration methods: intranasal administration, transdermal administration, intrameningeal administration, intraperitoneal administration, intramuscular administration, intratracheal administration, intranodal administration, intratumoral administration, intraarticular administration, intravenous administration, subcutaneous administration, vaginal administration, intraocular administration, rectal administration, and oral administration.

[0189] In some embodiments, the pharmaceutical compositions of the present disclosure are formulated for inhalation as an aerosol, spray, mist, liquid, or powder. Inhalation administration may be in the form of a dry powder or aerosol formulation, which is inhaled by the subject (e.g., a patient) via the use of an inhalation device, such as a microspray, pressurized metered-dose inhaler, or nebulizer.

[0190] Method of Disclosure Any administration of any of the therapeutic compositions described herein, such as nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions, can be used to modulate at least one pharmacodynamic effect in a subject, or can be used in the treatment of related health conditions such as autoimmune diseases and / or inflammatory diseases.

[0191] In some embodiments, the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions described herein may be useful in subjects requiring the modulation, for example, induction or suppression of pharmacodynamic effects. In some embodiments, the pharmacodynamic effect includes inducing an immune response in a subject. Non-limiting examples of pharmacodynamic effects include immunogenicity, biomarker responses, therapeutic effects, prophylactic effects, desired effects, undesirable effects, adverse effects, and effects in disease models.

[0192] Accordingly, one aspect of the present disclosure is a method for modulating pharmacodynamic effects in a subject requiring such modification, comprising administering to the subject a composition comprising one or more of the following: (a) nucleic acid constructs as described herein, (c) recombinant cells as described herein, and (d) pharmaceutical compositions as described herein. In some embodiments, the pharmacodynamic effect includes one or more of the following: immunogenicity, biomarker response, therapeutic effect, prophylactic effect, desired effect, undesirable effect, adverse effect, and effect in a disease model. In some embodiments, the pharmacodynamic effect includes inducing an immune response in the subject.

[0193] In some embodiments, the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions described herein can be used in methods to improve and / or extend the dynamics of IL-1RA and / or IL-18BP expression in a subject. In this case, the use of an srRNA-based expression system enables the production of IL-1RA and / or IL-18BP over a longer period compared to expression systems that depend on the administration of mRNA or recombinant protein. Thus, the use of an srRNA-based expression system results in a longer effective "half-life" of IL-1RA and IL-18BP in a subject compared to the direct administration of mRNA or recombinant protein. In relevant embodiments, the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions described herein can be used in methods to enhance the endogenous expression of IL-1RA and / or IL-18BP in a subject. In some embodiments, the method involves administering a composition to a subject comprising one or more of the following: (a) nucleic acid constructs described herein, (c) recombinant cells described herein, and (d) pharmaceutical compositions described herein.

[0194] In some embodiments, the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions described herein can be incorporated into therapeutic agents for use in methods of treating subjects who have, are suspected of having, or are at high risk of developing one or more relevant health conditions or diseases (e.g., autoimmune diseases). Exemplary health conditions or diseases include, but are not limited to, immune disorders, autoimmune diseases, and inflammatory diseases. In some embodiments, the subjects are patients under the care of a physician.

[0195] Accordingly, in another embodiment, a method for preventing or treating a health condition in a subject is provided herein, comprising administering to the subject prophylactically or therapeutically a composition comprising one or more of the following: (a) a replicon (e.g., a self-replicating RNA construct (srRNA) as described herein), (b) a nucleic acid as described herein, (c) at least one engineered T cell as described herein, and (d) a pharmaceutical composition as described herein. In some embodiments, the administered composition induces an immune response in the subject. In some embodiments, the administered composition prolongs the pharmacokinetics of a protein having a short in vivo half-life. In some embodiments, the protein having a short in vivo half-life is an endogenous protein. In some embodiments, the protein having a short in vivo half-life is an exogenous protein. In some embodiments, the administered composition induces the production of one or more pro-inflammatory molecules in the subject. In some embodiments, one or more pro-inflammatory molecules include interleukin-1α (IFNα), interferon-1 beta (IFNβ), interleukin-18 (IL-18), interleukin-6 (IL-6), interferon-gamma (IFNγ), cytokines, TNF-α, GM-CSF, and MIP1α, granzyme B, granzyme A, perforin, or any combination thereof. In some embodiments, the subject has been previously treated with one or more therapies and has developed at least partial resistance to those therapies.

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

[0197] Non-limiting examples of inflammation suitable for the methods disclosed herein include inflammatory diseases such as asthma, inflammatory bowel disease (IBD), chronic colitis, splenomegaly, and rheumatoid arthritis. In one embodiment, a method for inducing a pharmacodynamic effect in a subject is provided herein, comprising administering to the subject a composition comprising one or more of the following: (a) nucleic acid constructs as described herein, (b) recombinant cells as described herein, and (b) pharmaceutical compositions as described herein. In some embodiments, the pharmacodynamic effect includes inducing an immune response in the subject.

[0198] Examples of autoimmune diseases suitable for the methods of this disclosure include, but are not limited to, rheumatoid arthritis, osteoarthritis, Still's disease, familial Mediterranean fever, systemic scleroderma, multiple sclerosis, ankylosing spondylitis, systemic lupus erythematosus, Sjögren's syndrome, diabetic retinopathy, diabetic vascular disease, diabetic neuropathy, insulitis, psoriasis, alopecia areata, warm and cold autoimmune hemolytic anemia (AIHA), pernicious anemia, acute inflammatory diseases, autoimmune adrenal inflammatory disease, chronic inflammatory demyelinating polyneuropathy (CIDP), Lambert-Eaton syndrome, lichen sclerosing, Lyme disease, Graves' disease, Behçet's disease, Meniere's disease, reactive arthritis (Reiter's syndrome), Churg-Strauss syndrome, Cogan syndrome, Crest syndrome, pemphigus vulgaris and pemphigus foliaceus, bullous pemphigoid, and polymyalgia rheumatica. Pain, polymyositis, primary biliary cirrhosis, pancreatitis, peritonitis, psoriatic arthritis, rheumatic fever, sarcoidosis, Sjögrensen syndrome, scleroderma, celiac disease, Stiffman syndrome, Takayasu arteritis, transient gluten intolerance, autoimmune uveitis, vitiligo, polychondritis, dermatitis herpetiformis (DH) or Duhring's disease, fibromyalgia, Goodpasture syndrome, Guillain-Barré syndrome These include Hashimoto's thyroiditis, autoimmune hepatitis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, myasthenia gravis, immune complex disease, glomerulonephritis, polyarteritis nodosa, antiphospholipid 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 this 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, an administration of any one of the therapeutic compositions described herein, e.g., nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions, can be used to induce 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 ability to confer at least one pharmacodynamic effect, either in vivo or ex vivo. Examples of pharmacodynamic effects that can be analyzed include immunogenic effects (e.g., inducing an immune response in vivo), biomarker responses, therapeutic effects, prophylactic effects, desired effects, undesirable effects, adverse effects, and effects in disease models. In some embodiments, the evaluation of pharmacodynamic effects includes evaluating the induction of an in vivo immune response. In some embodiments, the evaluation of pharmacodynamic effects includes evaluating the induction of cytokine pathways that can enhance immune responses and prevent angiogenesis and metastasis.

[0201] In some embodiments, the disclosed pharmaceutical compositions are formulated to suit their intended route of administration. For example, the nucleic acid constructs (e.g., srRNA constructs), recombinant cells, and / or pharmaceutical compositions of this disclosure may be administered orally or by inhalation, but they are more likely to be administered via parenteral routes. Examples of parenteral routes include, for example, intramuscular, intratumoral, intraocular, intravenous, intranodal, intradermal, subcutaneous, transdermal (topical), transmucosal, vaginal, and rectal administration. In some embodiments, the compositions are administered intramuscularly. In some embodiments, the compositions are administered via intratumoral or cardiac administration. Solutions or suspensions used for parenteral administration may contain the following components: sterile diluents such as water for injection, physiological saline, non-volatile oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffering agents such as acetates, citrates, or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with an acid or base such as monobasic and / or dibasic sodium phosphate, hydrochloric acid, or sodium hydroxide (for example, to about 7.2 to 7.8, e.g., pH 7.5). Parenteral preparations may be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0202] The dosage, toxicity, and therapeutic efficacy of nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions that are the subject matter of this disclosure are, for example, LD50 (lethal dose for 50% of the population) and ED 50 The therapeutic dose (the dose that is therapeutically effective in 50% of the population) can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxicity and therapeutic effect is the therapeutic index, or LD50. 50 / ED 50It can be expressed as a ratio. Compounds showing a high therapeutic index are generally appropriate. Compounds showing toxic side effects can be used, but care should be taken to design a delivery system that targets such compounds to the site of the diseased tissue in order to minimize potential damage to uninfected cells and thereby reduce side effects.

[0203] For example, data obtained from cell culture assays and animal tests can be used in formulating a dosage range for use in humans. The dosage of such a compound is generally within the range of circulating concentrations that have little or no toxicity, including the ED 50 The dosage can be varied within this range depending on the dosage form used and the route of administration utilized. For all compounds used in the methods of the present disclosure, a therapeutically effective dose can first be estimated from cell culture assays. The dosage can be formulated in an animal model such that a range of circulating plasma concentrations is obtained, including the IC 50 (e.g., the concentration of the test compound that achieves half-maximal inhibition of symptoms). Such information can be used to more accurately determine a useful dosage in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0204] The therapeutic compositions described herein, such as nucleic acid constructs (e.g., srRNA constructs), recombinant cells, and / or pharmaceutical compositions, may be administered at doses ranging from once daily to once every other day, or at least once weekly. Those skilled in the art will understand that the dosage and timing required to effectively treat a subject may be influenced by certain factors, including but not limited to the severity of the disease, previous treatments, the subject's overall health and / or age, and other pre-existing diseases. Furthermore, the treatment of a subject with the polyvalent polypeptides and polyvalent antibodies of this disclosure in a therapeutically effective dose may consist of a single treatment or a series of treatments. In some embodiments, the composition is administered every 8 hours for 5 days, followed by a rest period of 2 to 14 days, for example, 9 days, followed by another 5 days of administration every 8 hours. With respect to nucleic acid constructs (e.g., replicon constructs, e.g., srRNA constructs), the therapeutically effective dose (e.g., effective dosage) of the nucleic acid constructs of this disclosure depends on the selected nucleic acid construct.

[0205] As described above, a therapeutically effective dose includes an amount of the therapeutic composition sufficient to promote a specific effect when administered to a subject who has, is suspected of having, or is at risk of having, a health condition such as an autoimmune disease, an inflammatory disease, or a cardiovascular disease. In some embodiments, the effective dose includes an amount sufficient to prevent or delay the onset of disease symptoms, alter the course of disease symptoms (for example, slow the progression of disease symptoms, but not limited to), or reverse disease symptoms.

[0206] The effectiveness of a treatment comprising a disclosed therapeutic composition for the treatment of a health condition or disease can be determined by a skilled clinician. However, a treatment is considered effective if at least one or all of the signs or symptoms of the disease are improved or remission. Effectiveness can also be determined by whether the individual does not worsen (e.g., the progression of the health condition or disease is stopped or at least slowed), if assessed by the need for hospitalization or medical intervention. Methods for measuring these indicators are known to those skilled in the art and / or are described herein. Treatment includes any treatment of a health condition or disease in a subject or animal (some non-limiting examples include humans or mammals), and includes (1) inhibiting the health condition or disease, e.g., stopping or slowing the progression of symptoms; or (2) alleviating the health condition or disease (e.g., reversing symptoms); and (3) preventing or reducing the onset of symptoms.

[0207] In some embodiments, nucleic acid constructs (e.g., 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 in an amount effective to stimulate an immune response. Generally, a subject can be immunized by an initial series of injections (or administration via one of the other routes described below), and then a booster can be administered to enhance the protection obtained by the original series of administrations. The initial series of injections and the subsequent booster are administered in doses and for a duration necessary to stimulate an immune response in the subject. In some embodiments, the administered composition increases interferon production in the subject. In some embodiments of the methods disclosed, the subject is a mammal. In some embodiments, the mammal is a human subject.

[0208] When nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions are adequately protected as described above, they may be administered orally, for example, with an inert diluent or an absorbable food carrier. Nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions, as well as other components, may also be encapsulated in hard-shell or soft-shell gelatin capsules, compressed into tablets, or directly incorporated into the diet of an individual. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, and wafers.

[0209] In some embodiments, the nucleic acid constructs of this disclosure can be delivered to cells or targets by lipid-based nanoparticles (LNPs). LNPs are generally less immunogenic than viral particles. Many humans have pre-existing immunity to viral particles, but not to LNPs. Furthermore, adaptive immune responses to LNPs are unlikely to occur, which allows for repeated administration of LNPs.

[0210] Additional therapy In some embodiments, the compositions of the Disclosure are administered to a subject individually as a single therapy (monotherapy) or as a primary therapy in combination with at least one additional therapy (e.g., a second-line therapy). In some embodiments, the second-line therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery. In some embodiments, the second-line therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, or surgery. In some embodiments, the primary and second-line therapies are administered synchronously. In some embodiments, the primary therapy is administered simultaneously with the second-line therapy. In some embodiments, the primary and second-line therapies are administered sequentially. In some embodiments, the primary therapy is administered before the second-line therapy. In some embodiments, the primary therapy is administered after the second-line therapy. In some embodiments, the primary therapy is administered before and / or after the second-line therapy. In some embodiments, the primary and second-line therapies are administered alternately. In some embodiments, the primary and second-line therapies are administered together as a single formulation.

[0211] kit Furthermore, various kits for carrying out the methods described herein, as well as written instructions for preparing and using them, are also provided herein. In particular, some embodiments of this disclosure provide kits for modulating (e.g., inducing, triggering, or suppressing) pharmacodynamic effects. Some other embodiments of this disclosure provide kits for inducing an immune response in a subject. Some other embodiments relate to kits for doing so in a subject that requires prevention of a health condition (e.g., an autoimmune disease). Some other embodiments relate to kits for methods for doing so in a subject that requires treatment of a health condition (e.g., an autoimmune disease). For example, some embodiments provide kits comprising one or more nucleic acid constructs, vectors, recombinant cells, and / or pharmaceutical compositions described and provided herein, and instructions for preparing and using them.

[0212] In some embodiments, the kits of the present disclosure further include one or more means useful for administering any one of the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions provided to a subject. For example, in some embodiments, the kits of the present disclosure further include one or more syringes (including pre-filled syringes) and / or catheters (including pre-filled syringes) used for administering any one of the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions provided to a subject. In some embodiments, the kits may have one or more additional therapeutic agents that can be administered simultaneously with or in conjunction with components of other kits for a desired purpose, for example, in a subject requiring the diagnosis, prevention, or treatment of a condition.

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

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

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

[0216] If the kit contains a pharmaceutical composition for parenteral administration to a subject, the kit may include a device for making such administration (e.g., an injection device or catheter). For example, the kit may include one or more subcutaneous needles or other injection devices considered above, containing one or more nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions of the present disclosure.

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

[0218] In some embodiments, the kit may further include instructions for use of the components of the kit to carry out the methods disclosed herein. For example, the kit may include a package insert containing information about the pharmaceutical composition and dosage form. Generally, such information helps patients and physicians to use the enclosed pharmaceutical composition and dosage form effectively and safely. For example, the package insert may provide information relating to the combination of the disclosures, including: pharmacokinetics, pharmacodynamics, clinical trials, efficacy parameters, indications and frequency of use, contraindications, warnings, precautions, adverse reactions, overdose, appropriate dosage and administration, supply methods, appropriate storage conditions, reference materials, manufacturer / distributor information, and intellectual property information.

[0219] Instructions for carrying out this method are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. The instructions can be included as an accompanying document within the kit, on the label of the kit or its component containers (e.g., attached to the package or subpackage). The instructions can also exist as an electronic storage data file on a suitable computer-readable storage medium, such as a CD-ROM, diskette, or flash drive. In some examples, the actual instructions are not present within the kit, but a 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 from which the instructions can be viewed and / or downloaded. Similar to the instructions, this means for obtaining the instructions may be recorded on a suitable substrate.

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

[0221] None of the references cited herein constitute prior art. The discussion of the references represents the claims of their authors, and the applicant reserves the right to challenge the accuracy and validity of the cited documents. While several sources of information are mentioned herein, including scientific journal articles, patent documents, and textbooks, it is clearly understood that none of these documents constitute part of the common general knowledge in the art.

[0222] The general methods described herein are for illustrative purposes only. Other alternative methods and substitutes will be apparent to those skilled in the art upon consideration of this disclosure and are included in the spirit and scope of this application.

[0223] Further embodiments are disclosed in more detail in the following embodiments, which are provided for illustrative purposes only and are not intended in any way to limit the scope of this disclosure or the claims. [Examples]

[0224] In carrying out the present invention, unless otherwise indicated, the prior art of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology known to those skilled in the art will be used. Such techniques are described in Sambrook, J., & Russell, DW (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russell, DW (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (collectively referred to as "Sambrook" in this specification); Ausubel, FM (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements up to 2014); Bollag, D. 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. Get 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, KB, Ferre, F. & Gibbs, R. (1994).This topic is fully explained in literature such as PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, EA (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. Let al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley (including supplements up to 2014); and Makrides, SC (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences BV, and their disclosures are incorporated herein by reference.

[0225] Further embodiments are disclosed in more detail in the following embodiments, which are provided for illustrative purposes only and are not intended in any way to limit the scope of this disclosure or the claims.

[0226] Example 1 Construction of a modified alpha virus vector This embodiment describes experiments performed to construct a base alphaviral vector (e.g., one that does not contain heterologous genes) which was subsequently used to construct a vector expressing the target gene or multiple genes (e.g., (i) IL-1RA protein or a functional variant thereof and / or (ii) IL-18BP or a functional variant thereof).

[0227] A base EEEV vector (i.e., without the target heterologous gene) was constructed as follows: The base EEEV vector was de novo synthesized from a reference sequence with several modifications (Genbank EF151502) into four approximately 4kb segments (Twist Bioscience). Silent mutations G301A, A3550C, G4516A, G5725A, and G7399A were incorporated to remove restriction enzyme cleavage sites. A unique restriction enzyme cleavage site (SpeI, 5'-A'CTAG,T-3') was incorporated in place of the coding sequence of the native EEEV structural gene (5'A corresponds to the position of the structural polyprotein ATG start codon, and 3'T corresponds to the position of the structural polyprotein stop codon TAA). A 5' adapter sequence (5'-CTGGAGACGTGGAGGAGAACCCTGGACCT-3', SEQ ID NO: 3) was inserted upstream of the SpeI site, and a 3' adapter sequence (5'-GACCGCTACGCCCCAATGACCCGACCAGC-3', SEQ ID NO: 4) was inserted downstream of the SpeI site for the subsequent Gibson Assembly® procedure (Gibson et al. Nat. Methods 6, 343-345, 2009). The bacteriophage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3', SEQ ID NO: 5) was included upstream of the EEEV genome sequence, and downstream of that, a poly(A) sequence followed by a Sap I site that cleaves upstream of the recognition site. Immediately downstream of the SapI site is the T7 terminator sequence 5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3' (SEQ ID NO: 6), followed by the unique restriction enzyme cleavage site NotI (5'-GC'GGCC, GC-3'). These segments were combined in a five-piece Gibson Assembly® reaction (linearized pYL skeleton and four synthetic fragments) to obtain the base vector for EEEV.

[0228] Other base srRNA vectors (VEEV, CHIKV, and SINV) were assembled in the same manner as the EEEV vectors based on the above-mentioned bases.

[0229] The construction of vectors containing heterologous genes was carried out as follows: an empty base vector was linearized by SpeI digestion. The IL-1RN and IL-18BP genes were codon-optimized / refactored in silico for human expression and de novo synthesized (IDT) together with encephalomyocarditis virus (EMCV) IRES. The synthesized product was amplified using primers that added either a 5' or 3' adapter sequence to the gene's end, or primers that added a P2A sequence and / or homologous sequence to adjacent gene insertions. The digested product and PCR product were combined using the Gibson Assembly® procedure to obtain the final vector. A summary of the modified alphavirus vectors prepared in these experiments is shown in Table 1 below. The amino acid sequences of the gene cassettes are also shown in the sequence listing.

[0230] [Table 1]

[0231] Example 2 In vitro evaluation of modified alphavirus vectors This example describes the results of in vitro experiments conducted to evaluate the expression level of the synthetic srRNA construct described in Example 1 and to investigate its arbitrary differential behavior (e.g., replication and protein expression).

[0232] In vitro transcription: RNA was prepared by in vitro transcription from a SapI linearized plasmid template using bacteriophage T7 RNA polymerase with a 5' ARCA cap (HiSscribe® T7 ARCA mRNA Kit, NEB), or by uncapped transcription (HiSscribe® T7 High Yield RNA Synthesis Kit, NEB), followed by addition of 5' cap 1 (Vaccinia Capping System, mRNA Cap 2'-O-methyltransferase, 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).

[0233] Replication: RNA was transformed into BHK-21 or Vero cells (e.g., 4D-Nucleofector®, Lonza) by electroporation. 15–22 hours after transformation, the cells were fixed and permeabilized (eBioscience® Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen), stained with PE conjugate anti-dsRNA mouse monoclonal antibody (J2, Scicons), and the frequency of dsRNA+ cells was quantified by fluorescence flow cytometry.

[0234] Protein expression by ELISA: Human IL-1RA and IL-18BP were detected from electroporated BHK-21 cells using monogenic or multigenic srRNA constructs. Supernatants were collected approximately 24 and 48 hours after transfection and assayed using human IL-1ra / IL-1F3 DuoSet ELISA (R&D Systems, catalog no. DY280) or human IL-18 Bpa DuoSet ELISA (R&D Systems, catalog no. DY119).

[0235] Figures 3 and 5 summarize the results of ELISA experiments performed to measure the in vitro expression of IL-18BP and IL-1RA in monogenic and / or digenic expression cassettes. IL-18BP expression was observed in monogenic and all digenic expression cassettes at 24 and 48 hours post-transformation (see, for example, Figure 3), with the highest expression level observed in the IL1RA-IRES-IL18BP cassette. IL-1RA expression was observed in all digenic cassettes at 24 and 48 hours post-transformation (see, for example, Figure 5), with the highest expression level observed in the IL18BP-IRES-IL1RA cassette.

[0236] Bioactivity assays: Functional human IL-1RA and IL-18BP were detected from electroporated BHK-21 cells using monogenic or multigenic srRNA constructs. Supernatants were collected approximately 24 and 48 hours after transfection. IL-1RA was assayed using IL-1β reporter HEK 293 cells (Invivogen, catalog number hkb-il1bv2) by pre-incubating the cells with a dilution of BHK-21 supernatant, and then incubating the cells with 1 ng / mL IL-1β according to the manufacturer's protocol to generate a signal. IL-18BP was assayed using IL-18 reporter HEK 293 cells (Invivogen, catalog number hkb-hmil18) by preparing a dilution of BHK-21 supernatant to 200 pg / mL of final IL-18, and then incubating according to the manufacturer's protocol to generate a signal.

[0237] Figures 4 and 6 summarize the results of experiments conducted to measure the in vitro expression of bioactive IL-18BP and bioactive IL-1RA in monogenetic and / or digenetic expression cassettes. Expression of bioactive IL-18BP was observed in monogenetic and all digenetic expression cassettes at 24 and 48 hours post-transformation (see, for example, Figure 4), with the highest expression level observed in the IL1RA-IRES-IL18BP cassette. Expression of bioactive IL-1RA was observed in all digenetic cassettes at 24 and 48 hours post-transformation (see, for example, Figure 6), with the highest expression level observed in the IL18BP-IRES-IL1RA cassette.

[0238] Figure 7 shows the ratio of in vitro bioactive IL-1RA to ELISA IL-1RA bigeneic expression cassette expressed from a bigeneic expression cassette. In particular, when the amino acid sequence of IL1-RA was C-terminally linked to the P2A sequence (in the case of the IL1RA-P2A-IL18BP cassette), the bioactivity of IL-1RA was disproportionately lower than the measured total IL-1RA protein. Since protein expression does not always correlate with functional protein products, the experimental results presented herein indicate that the combination of IL1-RA and IL-18BP in srRNA vectors is not an easily resolved issue in the field of srRNA vector design.

[0239] Example 3 In vitro evaluation of modified alphavirus vectors This example describes the results of in vitro experiments conducted to evaluate the IL-1RA expression and bioactivity levels of synthetic srRNA multigeneic constructs encoding IL-1RA and one or more additional polypeptides, and to investigate their arbitrary differential behavior (e.g., replication and protein expression). In these experiments, IL-1RA protein expression and bioactivity were measured from different multigeneic constructs with different gene orders. A summary of the nucleic acid constructs used in these experiments is shown in Table 2 below.

[0240] [Table 2]

[0241] In vitro transcription: RNA was prepared by in vitro transcription from a SapI linearized plasmid template using bacteriophage T7 polymerase with a 5' ARCA cap (HiSscribe® T7 ARCA mRNA Kit, NEB). The RNA was then purified by 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 into BHK-21 or Vero cells (e.g., 4D-Nucleofector®, Lonza) by electroporation. 15–22 hours after transformation, the cells were fixed and permeabilized (eBioscience® Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen), and stained with PE conjugate anti-dsRNA mouse monoclonal antibody (J2, Scicons). The frequency of dsRNA+ cells and the mean fluorescence intensity (MFI) of dsRNA in individual cells were quantified by fluorescence flow cytometry.

[0243] Protein expression by ELISA: Human IL-1RA was detected from electroporated BHK-21 cells using a 500 ng srRNA multigene construct. Supernatants were collected approximately 24 and 48 hours after transfection and assayed using human IL-1ra / IL-1F3 DuoSet ELISA from RnD Systems (catalog no. DY280).

[0244] Bioactivity assay: Functional human IL-1RA was detected from electroporated BHK-21 cells using a 500 ng srRNA multigene construct. Supernatants were collected approximately 24 and 48 hours after transfection and assayed according to the manufacturer's protocol using IL-1β reporter HEK 293 cells (Invivogen, catalog no. hkb-il1bv2) pre-incubated with 4 ng / mL IL1β (1 ng / mL final) and the supernatant from transfected BHK cells.

[0245] Evaluation of gene order: Figure 8 shows the results of IL-1RA detection ELISA experiments measured from transfected BHK-21 cells. Seventeen multigeneic constructs with different orders of IL-12 subunit p35, IL-12 subunit p40, IL-1RA, P2A, and IRES were tested to determine which gene configuration in the construct produced the most robust in vitro expression of IL-1RA. The Y axis shows the IL-1RA concentration in ng / mL. In these experiments, IL-1RA expression was quantified from all multigeneic cassettes at 24 and 48 hours post-transformation.

[0246] Figure 9 summarizes the results of experiments conducted to measure the in vitro expression of bioactive IL-1RA in the multigeneic expression cassettes described in Table 2 and Figure 8 above. The bioactivity of expressed IL-1RA was quantified from all multigeneic cassettes at 24 and 48 hours post-transformation.

[0247] Similar to the differences observed in relative IL-1RA expression and bioactivity from bigenic vectors containing IL-1RA and IL-18BP (Figures 5-7), the bioactivity of IL-1RA does not always correspond to the level of IL-1RA expression. This further indicates that the combination of IL-1RA with one or more additional polypeptides is not an easily solved problem in the field of srRNA vector design.

[0248] Example 4 In vivo evaluation of modified alpha virus vectors This example describes the results of in vivo experiments conducted to evaluate the srRNA constructs described herein (e.g., both unformulated vectors 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 the day of administration, 0.01–40 μg of the material was administered intramuscularly either in a single dose or in divided doses into both quadriceps femoris muscles. The vector was administered either without formulation in saline or formulated with LNP or polymer. Animals were monitored for body weight and other general observations throughout the study. In the pharmacokinetic study, animals were administered only on day 0. Serum was collected on day 3 for expression analysis.

[0252] LNP formulations: srRNAs were formulated into lipid nanoparticles using a microfluidic mixer, and particle size, polydispersity, and encapsulation efficiency were analyzed using dynamic light scattering and a dye exclusion assay (Ribogreen). Lipids were suspended in ethanol. Each srRNA was suspended at a concentration of 82 μg / ml in 100 mM NaOAc pH 4.0 and mixed at a flow rate of 3:1 (aqueous:organic).

[0253] Polymer formulation:

[0254] srRNA is formulated into polymer complexes (polyplexes) by mixing equal amounts of anionic components (srRNA and shielding components [e.g., PEG]) with a cationic component (nitrogen-containing ionized polymer). Mixing on a small scale is achieved by pipetting, followed by optional short-duration vortexing. On a larger scale, microfluidic mixing can be utilized. The complexes are characterized for size and polydispersity using dynamic light scattering, and srRNA encapsulation efficiency is measured using a dye exclusion assay (Ribogreen). Particle properties can be optimized by varying the polymer-to-RNA weight ratio, as well as through the selection of buffers and mixing conditions. Buffers are typically in the pH range of 6.5–7.5 and include low-salt buffers for unshielded polyplexes (e.g., 10–20 mM Tris / His) or standard buffers such as PBS for PEGylated / shielded polyplexes.

[0255] Protein expression by ELISA: To measure serum concentrations of IL-1RA and IL-18BP, ELISA analysis was performed using the human IL-1RA ELISA kit (Abcam, catalog number ab211650) and the human IL-18BP ELISA kit (Invitrogen, catalog number EHIL18BP), following the manufacturer's protocol. Due to the small volume, serum samples were pooled before measurement from animals injected with unformulated srRNA.

[0256] Figures 14 and 15 summarize the results of ELISA experiments conducted to measure the in vivo expression of IL-1RA and IL-18BP in different modified alphavirus vectors formulated within LNP delivery systems. IL-1RA was detected in animal serum 3 days after administration of monogenic vectors and most bigenic vectors (Figure 14). Expression levels were below the detection limit of the assay for SINV vectors, with the highest expression observed from LNP-formulated bigenic EEEV vectors. IL-18BP expression was observed 3 days after administration of LNP-formulated bigenic EEEV vectors, while expression levels for other bigenic vectors were below the detection limit of the assay (Figure 15).

[0257] Figures 16 and 17 summarize the results of ELISA experiments conducted to measure the in vivo expression of IL-1RA and IL-18BP from bigenic EEEV vectors formulated with two different delivery vehicles: (i) LNP and (ii) polymer nanoparticles. IL-1RA expression was observed 3 and 7 days after administration of the LNP-formulated vector, and expression was detected 7 days after administration of the polymer-formulated vector (Figure 16). IL-18BP expression was observed 3 and 7 days after administration of the LNP-formulated vector, and expression was detected 3 and 7 days after administration of the polymer-formulated vector (Figure 17). These data demonstrate that bigenic srRNA vectors encoding IL-1RA and IL-18BP can indeed be formulated in multiple ways to express these proteins in vivo.

[0258] Example 5 srRNA constructs expressing IL-1RA and IL-18BP extend the pharmacokinetics of target proteins with short half-lives. This example describes the results of in vivo experiments conducted to demonstrate that srRNA constructs expressing IL-1RA and IL-18BP as described herein (e.g., LNP formulation vectors) can extend the pharmacokinetics of a target protein (IL-1RA) with a short half-life.

[0259] In these experiments, as shown in Figures 18A-18B, mice were either administered human IL-1RA recombinant protein at t=0 or LNP-formulated srRNA encoding IL-1RA and IL-18BP 96 hours prior to achieving Tmax for srRNA-based protein expression. Human IL-1RA levels were measured at t=2 and 8 hours by standard serum ELISA. As shown in Figure 18, srRNA-based expression of IL-1RA and IL-18BP shows less decay in systemic protein levels compared to recombinant protein.

[0260] Previous studies have reported that systemic levels of human IL-1RA rapidly decline from 2 to 8 hours in mice administered recombinant protein due to its short half-life. However, in this study, mice administered LNP-formulated sRNA co-encoding IL-1RA and IL-18BP four days prior to achieving maximum protein expression from this platform exhibited stable levels of encoded human IL-1RA at 2 and 8 hours. Therefore, systemic expression of proteins with short half-lives, such as human IL-1RA, can be extended using an srRNA-based approach, resulting in longer-term protein expression dynamics from this technology.

[0261] While certain alternatives to those described herein are disclosed, various modifications and combinations are possible and intended to be construed within the precise intent and scope of the attached claims. Therefore, there is no intention to limit ourselves to the exact abstract and disclosures presented herein.

Claims

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 is as follows: a) The coding sequence of the interleukin-1 receptor antagonist (IL-1RA) protein or a functional variant thereof, and b) Replaced with the coding sequence of a polypeptide construct containing the coding sequence of interleukin-18 binding protein (IL-18BP) or a functional variant thereof, A nucleic acid construct in which the coding sequences of IL-1RA and IL-18BP are functionally linked to each other, and the polypeptide construct does not contain a dimerization domain.

2. The nucleic acid construct according to claim 1, wherein the polypeptide construct does not contain a fragment crystallization region (Fc region) of immunoglobulin.

3. The nucleic acid construct according to claim 1 or 2, wherein the coding sequence of IL-1RA is N-terminally linked to the coding sequence of IL-18BP.

4. The nucleic acid construct according to claim 1 or 2, wherein the coding sequence of IL-1RA is C-terminally linked to the second coding sequence of IL-18BP.

5. A nucleic acid construct according to any one of claims 1 to 4, wherein the coding sequences of IL-1RA and IL-18BP express proteins that are functional in a biological activity assay.

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

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

8. The nucleic acid according to claim 7, wherein the mammalian target is a human target.

9. The code sequences for IL-1RA and / or IL-18BP are as follows: a) To enhance the stability and / or expression level of RNA, b) Minimizing the use of rare codons and / or minimizing secondary structures, c) To promote better srRNA replication and RNA production processes, A nucleic acid construct according to any one of claims 1 to 8, which is optimized for one or more of the following.

10. A nucleic acid construct according to any one of claims 1 to 9, wherein the coding sequences of IL-1RA and IL-18BP are functionally linked to each other via connector sequences encoding autoproteolytic peptides and / or internal ribosome entry sites (IRES).

11. The nucleic acid construct according to claim 10, wherein the autoproteolytic peptide comprises one or more autoproteolytic cleavage sequences from calcium-dependent serine endoprotease (Fulin), porcine rhinitis virus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), Tosea signalavirus 2A (T2A), cytoplasmic polyhedra virus 2A (BmCPV2A), flakily virus 2A (BmIFV2A), or a combination thereof.

12. The nucleic acid construct according to claim 11, wherein the internal ribosome entry sites (IRES) are derived from Kaposi's sarcoma-associated herpesvirus (KSHV) IRES, hepatitis virus IRES, pestivirus IRES, Kripa virus IRES, roparocyphanpadi virus IRES, fibroblast growth factor IRES, platelet-derived growth factor IRES, vascular endothelial growth factor IRES, insulin-like growth factor IRES, picornavirus IRES, encephalomyocarditis virus (EMCV) IRES, Pim-1 IRES, p53 IRES, Apaf-1 IRES, TDP2 IRES, L-myc IRES, and c-myc IRES.

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

14. The nucleic acid construct according to any one of claims 1 to 12, wherein the modified alphavirus genome or srRNA does not contain a nucleic acid sequence encoding a viral structural protein.

15. A nucleic acid construct according to any one of claims 1 to 14, wherein the coding sequences of IL-1RA and IL-18BP are functionally linked to each other within a single open reading frame (i.e., in a polycistronic ORF).

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

17. The nucleic acid construct according to claim 16, wherein the promoter sequence is a subgenome (sg) promoter.

18. The nucleic acid construct according to claim 17, wherein the sg promoter sequence is a 26S subgenome promoter.

19. The nucleic acid construct according to claim 17 or 18, wherein the subgenome promoter is heterogeneous with respect to the rest of the modified viral genome or srRNA.

20. The nucleic acid construct according to any one of claims 17 to 19, wherein the subgenome promoter is an alphavirus subgenome promoter.

21. The nucleic acid construct according to any one of claims 1 to 20, wherein at least one non-structural protein (nsP) or a portion thereof of the modified viral genome or srRNA is heterogeneous with respect to the rest of the modified viral genome or srRNA.

22. A nucleic acid construct according to any one of claims 1 to 21, further comprising a nucleic acid sequence encoding a heterogeneous nsP or a portion thereof.

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

24. The nucleic acid construct according to claim 23, wherein at least one of the UTRs is a heterogeneous UTR.

25. The nucleic acid construct according to any one of claims 1 to 24, wherein the modified alphavirus genome or srRNA is of an alphavirus belonging to the Venezuelan horse encephalitis virus / eastern equine encephalitis virus (VEEV / EEEV) group, the Semliki forest virus (SFV) group, or the Sindbis virus (SINV) group.

26. The nucleic acid construct according to 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, or WEEV complex.

27. The aforementioned alphaviruses include Eastern Equine Encephalitis Virus (EEEV), Venezuelan Equine Encephalitis Virus (VEEV), Everglades Virus (EVEV), Mukambo Virus (MUCV), Pixna Virus (PIXV), Middleberg Virus (MIDV), Chikungunya Virus (CHIKV), Onyonnyon Virus (ONNV), Ross River Virus (RRV), Burma Forest Virus (BF), Geta Virus (GET), Sagiyama Virus (SAGV), and Beval Virus (BEBV). The nucleic acid construct according to claim 26, which is Mayarovirus (MAYV), Unavirus (UNAV), Sindbisvirus (SINV), Auravirus (AURAV), Wataroavirus (WHAV), Bavanchivirus (BABV), Kyzilagativirus (KYZV), Western Equine Encephalitis Virus (WEEV), Highland J Virus (HJV), Fort Morgan Virus (FMV), Nudum Virus (NDUV), Madariagavirus (MADV), or Buggy Creek Virus.

28. The nucleic acid construct according to claim 27, wherein the alphavirus is VEEV, EEEV, CHIKV, or SINV.

29. The coding sequence of the polypeptide construct is as follows, from the N-terminus to the C-terminus: (a) (i) the code sequence for IL-1RA, (ii) the connector sequence for encoding IRES, and (iii) the code sequence for IL-18BP; (b) (i) coding sequence for IL-1RA, (ii) connector sequence encoding P2A autoprotein degradation peptide, and (iii) coding sequence for IL-18BP, (c) (i) code sequence for IL-18BP, (ii) connector sequence for encoding IRES, and (iii) code sequence for IL-1RA, or (d) (i) coding sequence for IL-18BP, (ii) connector sequence encoding P2A autoprotein degradation peptide, and (iii) coding sequence for IL-1RA, A nucleic acid construct according to any one of claims 1 to 28, comprising:

30. The nucleic acid construct according to 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 with an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 10.

31. The nucleic acid construct according to any one of claims 1 to 30, wherein the coding sequence of 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 with an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 14.

32. The nucleic acid construct according to 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 with respect to an amino acid sequence selected from the group consisting of SEQ ID NO:

15.

33. Recombinant cells comprising a nucleic acid construct according to any one of claims 1 to 32.

34. The recombinant cell according to claim 33, wherein the recombinant cell is a eukaryotic cell.

35. The recombinant cell according to claim 34, wherein the eukaryotic cell is an animal cell.

36. The recombinant cell according to claim 35, wherein the animal cell is a vertebrate cell or an invertebrate cell.

37. The recombinant cell according to claim 35, wherein the animal cell is a mammalian cell.

38. The recombinant cell according to claim 35, wherein the animal cell is an insect cell.

39. The recombinant cell according to claim 38, wherein the insect cell is a mosquito cell.

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

41. The aforementioned immune cells include B cells, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, basophils, eosinophils, neutrophils, dendritic cells (DCs), macrophages, regulatory T cells, and helper T cells (T). Ｈ ), cytotoxic T cells (T ＣＴＬ Recombinant cells according to claim 40, which are memory T cells, gamma delta (γδ) T cells, hematopoietic stem cells, or hematopoietic stem cell progenitor cells.

42. The recombinant cell according to 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 recombinant cells according to at least 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 according to claim 44, wherein the animal is a vertebrate or an invertebrate.

46. The transgenic animal according to claim 44, wherein the animal is a mammal.

47. The transgenic animal according to claim 46, wherein the mammal is a non-human mammal.

48. The transgenic animal according to claim 44, wherein the animal is an insect.

49. The transgenic animal according to claim 48, wherein the transgenic insect is a transgenic mosquito.

50. Pharmacologically acceptable excipients, a) A nucleic acid construct according to any one of claims 1 to 32, and / or b) A pharmaceutical composition comprising recombinant cells according to any one of claims 33 to 42.

51. The pharmaceutical composition according to claim 50, wherein the composition is formulated into a delivery system together with a delivery vehicle, the delivery system comprising polymer nanoparticles, lipid-based nanoparticles (LNPs), liposomes, viral replicon particles (VRPs), physiological buffers, microspheres, immunostimulatory complexes (ISCOMs), conjugates of bioactive ligands, or any combination thereof.

52. The pharmaceutical composition according to claim 51, wherein the polymer nanoparticles include a cationic polymer, a non-cationic polymer, or a combination thereof.

53. The pharmaceutical composition according to claim 52, wherein the cationic polymer includes a naturally derived cationic polymer.

54. The pharmaceutical composition according to claim 53, wherein the naturally derived cationic polymer comprises chitosan, gelatin, dextran, cellulose, cyclodextrin, or a combination thereof.

55. The pharmaceutical composition according to claim 52, wherein the cationic polymer includes a synthetic cationic polymer.

56. The pharmaceutical composition according to claim 55, wherein the synthetic cationic polymer comprises poly(ethyleneimine) (PEI), poly-L-lysine (PLL), poly(amino) acid (PAA), poly(amidoamine) (PAMAM), poly(cystaminebisacrylamide-co-4-amino-1-butanol) (pABOL), poly(amino-co-ester) (PAE), poly(2-N,N-dimethylaminoethyl methacrylate), poly(beta-aminoester) (PBAE), imidazole-containing polymer, tertiary amine-containing polymer, poly(2-(dimethylamino)ethyl methacrylate), poly-N-(2-hydroxypropyl)methacrylamide, polyamidoamine dendrimer, cationic glycopolymer, or derivatives thereof.

57. The pharmaceutical composition according to claim 52, wherein the noncationic polymer is negatively charged (i.e., anionic) or electronically neutral.

58. The pharmaceutical composition according to claim 57, wherein the noncationic polymer comprises polyethylene glycol (PEG), polyester (e.g., polylactic acid PLA, poly(lactic acid-coglycolic acid) (PLGA), polyglycolic acid (PGA), polycaprolactone (PCL)), and polysarcosine (pSar), or derivatives thereof.

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

60. The pharmaceutical composition according to any one of claims 52 to 58, wherein the polymer nanoparticles comprise one or more of the following: poly-(γ-L-glutamylglutamine) (PGGA), poly-(γ-L-aspartylglutamine) (PGAA), poly-L-lactic acid (PLLA), poly-(lactic acid-coglycolic acid) (PLGA), polyalkylcyanoacrylate (PACA), polyanhydride, polyhydroxy acid, polypropyl fumerate, 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 according to claim 51, wherein the delivery system for the LNP includes a cationic lipid, an ionizable cationic lipid, an anionic lipid, or a neutral lipid.

62. The pharmaceutical composition according to claim 51, wherein the lipid is present in a lipid-to-RNA mass ratio of about 100:1 to about 4:

1.

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

64. The pharmaceutical composition according to any one of claims 50 to 63, wherein the composition is formulated as a biological agent.

65. A method for adjusting the pharmacodynamic effect in a subject, wherein the subject a) A nucleic acid construct according to any one of claims 1 to 32, b) Recombinant cells according to any one of claims 33 to 42, and / or c) A method comprising administering a composition, which includes the pharmaceutical composition according to any one of claims 50 to 64.

66. The method according to claim 65, wherein the pharmacodynamic effect includes inducing an immune response in the subject.

67. The method according to claim 65, wherein the pharmacodynamic effect includes one or more of the following: immunogenicity, biomarker response, therapeutic effect, preventive effect, desired effect, undesirable effect, adverse effect, and effect in a disease model.

68. A method for improving / extending the dynamics of IL-1RA and / or IL-18BP expression in a subject, or for enhancing the endogenous expression of IL-1RA and / or IL-18BP, wherein the subject a) A nucleic acid construct according to any one of claims 1 to 32, b) Recombinant cells according to any one of claims 33 to 42, and / or c) A method comprising administering a composition, which includes the pharmaceutical composition according to any one of claims 50 to 64.

69. A method for preventing and / or treating a health condition in a person who needs such treatment, a) A nucleic acid construct according to any one of claims 1 to 32, b) Recombinant cells according to any one of claims 33 to 42, and / or c) A method comprising administering a composition, which includes the pharmaceutical composition according to any one of claims 50 to 64.

70. The method according to claim 69, wherein the health condition is an autoimmune disease, an inflammatory disease, or a cardiovascular disease.

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

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

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

74. The method according to claim 73, wherein the one or more pro-inflammatory molecules include interleukin-1 alpha (IFNα), interferon-1 beta (IFNβ), interleukin-18 (IL-18), interleukin-6 (IL-6), interferon gamma (IFNγ), cytokines, TNF-α, GM-CSF, and MIP1α, granzyme B, granzyme A, perforin, or any combination thereof.

75. The method according to any one of claims 68 to 74, wherein the composition is administered to the subject individually as a monotherapy (alone therapy) or as a first-line treatment in combination with at least one additional therapy.

76. The method according to claim 75, wherein the at least one additional therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery.

77. A kit for modulating pharmacodynamic effects, for inducing immune responses, and / or for preventing and / or treating health conditions, a) A nucleic acid construct according to any one of claims 1 to 32, b) Recombinant cells according to any one of claims 33 to 42, and / or c) A kit comprising one or more pharmaceutical compositions according to any one of claims 50 to 64.

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