Modified madagascar viruses, self-replicating RNA constructs, and uses thereof

Abstract

The present disclosure relates to the field of molecular virology, including nucleic acid molecules comprising modified viral genomes or self-replicating RNA, pharmaceutical compositions comprising the same, and the use of such nucleic acid molecules and compositions to produce desired products in cell culture or in an organism. Methods for inducing a pharmacodynamic effect in a subject in need thereof, as well as methods for preventing and / or treating various health conditions, are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 385,556, filed November 30, 2022. The disclosure of the above application is expressly incorporated herein by reference in its entirety, including any drawings.

[0002] Incorporating a sequence listing The contents of the accompanying Sequence Listing are incorporated herein by reference. The accompanying Sequence Listing XML file entitled 2023-11-27Sequence_Listing_ST26058462-513001WO.xml was created on November 27, 2023, and is 26,590 bytes.

[0003] The present invention relates to the fields of molecular virology and immunology, and in particular to nucleic acid molecules, e.g., self-replicating RNA (srRNA) molecules, encoding modified viral genomes and replicons, pharmaceutical compositions containing the same, and the use of such nucleic acid molecules and compositions to produce desired products in cell culture or in living organisms. Methods for inducing a pharmacodynamic effect in a subject in need thereof, as well as methods for preventing and / or treating various health conditions, are also provided. [Background technology]

[0004] In recent years, several different groups of animal viruses have been subjected to genetic manipulation either by homologous recombination or by direct engineering of their genomes. The availability of reverse genetics systems for both DNA and RNA viruses has opened up new perspectives for the use of recombinant viruses, e.g., as vaccines, expression vectors, antitumor agents, gene therapy vectors, and drug delivery vehicles.

[0005] For example, many virus-based expression vectors have been developed for the expression of heterologous proteins in cultured recombinant cells. For example, the application of modified viral vectors for gene expression in host cells continues to expand. Recent advances in this regard include further development of techniques and systems for producing multi-subunit protein complexes and co-expression of protein-modifying enzymes to improve the production of heterologous proteins. Other recent advances in viral expression vector technology include the application of many advanced genome engineering techniques for controlling gene expression, preparing viral vectors, applying in vivo gene therapy, and creating vaccine delivery vectors.

[0006] However, it has been reported that host cells can develop complex and powerful mechanisms to detect and counter pathogen invasion. Furthermore, it has been reported that viruses, especially pathogenic viruses, have co-evolved with host cells to counter these cellular defenses against infection and replication. As a result of infection, many host cells shut down the cellular protein translation machinery to control viral replication and / or the production of progeny viruses that could potentially spread to additional cells. This phenomenon is commonly referred to as the "innate immune response." Infected cells also send danger signals to other cells locally and systemically, creating an antiviral state to control the infection. While these cellular antiviral systems benefit host cells, they can also adversely affect self-replicating RNAs (srRNAs), e.g., replicons, designed to express beneficial vaccine antigens and therapeutic agents. For example, if a cell detects srRNA expressing a beneficial protein and activates its innate immune defense mechanism, the expression of the beneficial protein in such cells may be affected, compromising the efficacy of the srRNA.

[0007] Therefore, there remains a need for more efficient methods and systems for expressing products of interest in srRNA-based expression platforms. Summary of the Invention

[0008] The present disclosure generally relates to the development of immunotherapies, such as recombinant nucleic acid constructs and pharmaceutical compositions comprising the same, for use in the prevention and management of various health conditions, including proliferative diseases and microbial infections. In particular, as described in more detail below, some embodiments of the present disclosure provide nucleic acid constructs comprising a modified Madariaga virus (MADV) genome or replicon, e.g., a self-replicating RNA (srRNA), e.g., a sequence encoding a replicon, lacking at least a portion of the viral nucleic acid sequence encoding one or more viral structural proteins. Also disclosed are recombinant cells and transgenic animals engineered to contain one or more of the nucleic acid constructs disclosed herein, methods for producing molecules of interest (e.g., polypeptides of interest), and pharmaceutical compositions comprising one or more of: (a) a nucleic acid construct of the present disclosure; (b) a polypeptide of the present disclosure; or (c) a recombinant cell of the present disclosure. Additionally, certain aspects of the present disclosure provide compositions and methods for inducing a pharmacodynamic effect in a subject in need thereof, and / or for the prevention and / or treatment of various health conditions, including proliferative diseases (e.g., cancer) and infectious diseases.

[0009] In one aspect of the present disclosure, provided herein is a nucleic acid construct comprising a nucleic acid sequence encoding a modified Madagascar virus (MADV) genome or replicon, e.g., a self-replicating RNA (srRNA), wherein the modified MADV genome or srRNA lacks at least a portion of a nucleic acid sequence encoding one or more viral structural proteins.

[0010] Non-limiting exemplary embodiments of nucleic acid constructs of the present disclosure can include one or more of the following features: In some embodiments, the modified viral genome or srRNA lacks a substantial portion of a nucleic acid sequence encoding one or more viral structural proteins. In some embodiments, the modified viral genome or srRNA does not include a nucleic acid sequence encoding a viral structural protein. In some embodiments, the nucleic acid molecule of the present disclosure further comprises one or more expression cassettes, each of the expression cassettes comprising a promoter operably linked to a heterologous nucleic acid sequence. In some embodiments, at least one of the expression cassettes comprises a subgenomic (sg) promoter operably linked to a heterologous nucleic acid sequence. In some embodiments, the sg promoter is a 26S subgenomic promoter.

[0011] In some embodiments, at least one nonstructural protein (nsP) or portion thereof of the modified MADV genome or srRNA is heterologous to the remainder of the modified MADV genome or srRNA, hi some embodiments, the modified MADV genome or srRNA further comprises a nucleic acid sequence encoding the heterologous nsP or portion thereof.

[0012] In some embodiments, the nucleic acid construct of the present disclosure further comprises one or more untranslated regions (UTRs). In some embodiments, at least one of the UTRs is a heterologous UTR.

[0013] In some embodiments, at least one of the expression cassettes comprises a coding sequence for a gene of interest (GOI). In some embodiments, the GOI encodes a polypeptide selected from the group consisting of a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide, an industrial enzyme, and a reporter polypeptide. In some embodiments, the GOI encodes a polypeptide selected from the group consisting of an antibody, an antigen, an immunomodulator, an enzyme, a signaling protein, and a cytokine. In some embodiments, the coding sequence of the GOI is optimized for a higher level of expression than the expression level of a reference coding sequence. In some embodiments, the coding sequence of the GOI is optimized for increased RNA stability.

[0014] In some embodiments, a nucleic acid construct of the present disclosure comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:1.

[0015] In one aspect, provided herein is a recombinant cell comprising a nucleic acid construct disclosed herein. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a vertebrate cell or an invertebrate cell. In some embodiments, the recombinant cell is an insect cell. In some embodiments, the recombinant cell is a mosquito cell. In some embodiments, the recombinant cell is a mammalian cell. In some embodiments, the recombinant cell is selected from the group consisting of SV40-transformed monkey kidney CV1 cells, human embryonic kidney cells (HEK), baby hamster kidney cells (BHK), mouse Sertoli cells, monkey kidney cells, human cervical carcinoma cells, canine kidney cells, buffalo rat hepatocytes, human lung cells, human hepatocytes, mouse mammary tumor, TRI cells, FS4 cells, Chinese hamster ovary cells (CHO), African green monkey kidney cells, human A549 cells, human cervical cells, human CHME5 cells, human PER.C6 cells, NS0 mouse myeloma cells, human epidermoid laryngeal cells, human fibroblasts, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocyte cells, human macrophage cells, human RAW264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells.

[0016] In some embodiments, the recombinant cells are selected from the group consisting of African green monkey kidney cells (Vero cells), baby hamster kidney cells (BHK cells), Chinese hamster ovary cells (CHO cells), human A549 cells, human cervical cells, human CHME5 cells, human epidermoid laryngeal cells, human fibroblast cells, 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. In some embodiments, the recombinant cells are cells derived from the above-mentioned cells (i.e., derivatives of the original cells described herein), such as cells propagated from clones of the original cells, engineered forms of the original cells, or reassortments of the original cells after extensive passaging or passaging through another host.

[0017] In another aspect, a cell culture is provided comprising at least one recombinant cell disclosed herein and a culture medium.

[0018] In another aspect, provided herein is a transgenic animal comprising a nucleic acid construct described herein. 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. In another aspect, provided herein is a method for producing a polypeptide of interest, comprising (i) raising a transgenic animal disclosed herein, or (ii) culturing a recombinant cell comprising a nucleic acid construct disclosed herein, under conditions wherein the transgenic animal or recombinant cell produces the polypeptide encoded by the GOI.

[0019] In another aspect, provided herein is a method for producing a polypeptide of interest in a subject, the method comprising administering to 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 mammal. In some embodiments, the mammalian subject is a human subject. In yet another aspect, provided herein is a recombinant polypeptide produced by the methods of the present disclosure.

[0020] In yet another aspect, provided herein is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a) a nucleic acid construct of the present disclosure, b) a recombinant cell of the present disclosure, and / or c) a recombinant polypeptide of the present disclosure.

[0021] Non-limiting exemplary embodiments of pharmaceutical compositions of the present disclosure can include one or more of the following features. In some embodiments, provided herein are compositions comprising a nucleic acid construct disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, provided herein are compositions comprising a recombinant cell disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, a composition comprises a recombinant polypeptide disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, provided herein are compositions formulated in liposomes, lipid-based nanoparticles (LNPs), polymeric nanoparticles, polyplexes, viral replicon particles (VRPs), microspheres, immune stimulating complexes (ISCOMs), conjugates of bioactive ligands, or any combination thereof. In some embodiments, the composition is an immunogenic composition. In some embodiments, the immunogenic composition is formulated as a vaccine. In some embodiments, the immunogenic composition is substantially non-immunogenic to a subject. In some embodiments, the pharmaceutical composition is formulated as an adjuvant. In some embodiments, the pharmaceutical composition is formulated for one or more of intranasal, intrathecal, transdermal, intraperitoneal, intramuscular, intratracheal, intralymphatic, intratumoral, intraarticular, intravenous, subcutaneous, intravaginal, intraocular, rectal, and oral administration.

[0022] In another aspect, provided herein are methods for inducing a pharmacodynamic effect in a subject in need thereof, comprising administering to the subject a composition comprising: a) a nucleic acid construct of the present disclosure, b) a recombinant cell of the present disclosure, c) a recombinant polypeptide of the present disclosure, and / or d) a pharmaceutical composition of the present disclosure. In some embodiments, the pharmacodynamic effect includes one or more of an immunogenic effect, a biomarker response, a therapeutic effect, a prophylactic effect, a desired effect, an undesired effect, an adverse effect, and an effect in a disease model. In some embodiments, the pharmacodynamic effect includes eliciting an immune response in the subject.

[0023] In yet another aspect, provided herein are methods for preventing and / or treating a condition in a subject in need thereof, comprising prophylactically or therapeutically administering to the subject a composition comprising: a) a nucleic acid construct of the present disclosure; b) a recombinant cell of the present disclosure; c) a recombinant polypeptide of the present disclosure; and / or d) any one of the pharmaceutical compositions of the present disclosure. In some embodiments, the administered composition elicits a pharmacodynamic effect. In some embodiments, the pharmacodynamic effect includes eliciting an immune response in the subject.

[0024] Non-limiting exemplary embodiments of the disclosed methods can include one or more of the following features: In some embodiments, the condition is a proliferative disease or a microbial infection. In some embodiments, the subject has or is suspected of having a condition associated with a proliferative disease or a microbial infection. In some embodiments, the administered composition increases interferon production in the subject. In some embodiments, the composition is administered to the subject individually as a single therapy (monotherapy) or as a first therapy in combination with at least one additional therapy. In some embodiments, the at least one additional therapy is selected from the group consisting of chemotherapy, radiation therapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery.

[0025] In yet another aspect, provided herein is a kit for eliciting a pharmacodynamic response, eliciting an immune response, and / or preventing and / or treating a condition or a microbial infection, the kit comprising: a) a nucleic acid construct of the present disclosure; b) a recombinant cell of the present disclosure; c) a recombinant polypeptide of the present disclosure; and / or d) a pharmaceutical composition of the present disclosure.

[0026] Each aspect and embodiment described herein can be used together unless expressly or specifically excluded from the context of the embodiment or aspect.

[0027] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the exemplary embodiments and features described herein, further aspects, embodiments, objects, and features of the present disclosure will become more fully apparent from the drawings, detailed description, and claims. [Brief explanation of the drawings]

[0028] [Figure 1] Figure 1 is a diagram of a non-limiting example of a modified MADV genome design according to some embodiments of the present disclosure, in which the nucleic acid sequences encoding the viral structural proteins of the original virus have been completely deleted. The modified MADV design depicted in this figure contains the native 5' and 3' UTRs from MADV strain BeAr300851 and further contains a heterologous gene of interest (GOI) placed under the control of a 26S subgenomic promoter. The coding sequences for the nonstructural proteins nsP1, nsP2, nsP3, and nsP4 are shown.

[0029] [Figure 2A] 2A-2B are illustrations of non-limiting exemplary MADV srRNA designs according to some embodiments of the present disclosure, in which sequences encoding a modified MADV genome from the BeAr300851 strain are incorporated into a plasmid DNA vector (FIG. 2A) and include the coding sequence for an exemplary gene of interest (GOI), e.g., the hemagglutinin precursor (HA) of influenza A virus H5N1 (FIG. 2B). [Figure 2B] 2A-2B are illustrations of non-limiting exemplary MADV srRNA designs according to some embodiments of the present disclosure, in which sequences encoding a modified MADV genome from the BeAr300851 strain are incorporated into a plasmid DNA vector (FIG. 2A) and include the coding sequence for an exemplary gene of interest (GOI), e.g., the hemagglutinin precursor (HA) of influenza A virus H5N1 (FIG. 2B).

[0030] [Figure 3]Figures 3A-3B show contour maps of BHK-21 cells transformed with MADV srRNA. In Figure 3A, MADV srRNA containing no GOI was transfected by electroporation. 20 hours after transfection, the cells were fixed and permeabilized and co-stained with two antibodies: a PE-conjugated anti-dsRNA mouse monoclonal antibody (J2, Scicons) to quantify the abundance of dsRNA+ cells, and a DyLight650-conjugated anti-HA mouse monoclonal antibody (C102, NovusBio, catalog no. NB100-65047C) to quantify the abundance of HA+ cells and the expression level of the HA transgene by fluorescence flow cytometry. In Figure 3B, BHK-21 cells were similarly transfected with MADV srRNA containing the HA coding sequence and co-stained with the same antibodies. Positive staining of individual cells with both anti-dsRNA and anti-HA antibodies indicates that the modified MADV srRNA designs described herein are viable synthetic srRNAs capable of RNA replication and transgene expression.

[0031] [Figure 4]Figure 4 shows the average HA expression in cells transfected with srRNA constructs. BHK-21 cells were transfected with 200 ng of srRNA vector encoding influenza H1N1 HA by electroporation (Lonza 4D-Nucleofector). 24 hours later, the cells were fixed, permeabilized, and stained with a DyLight650-conjugated mouse monoclonal antibody (NovusBio, catalog no. NB100-65047C) that binds to H1N1 HA. Cells were analyzed by fluorescent flow cytometry to quantify cellular fluorescence intensity, which corresponds to HA expression levels. The average HA expression in cells transfected with MADV-HA was similar or higher than that of other srRNA vectors encoding the same transgene. VEEV: Venezuelan equine encephalitis virus, CHIKV-S27: Chikungunya virus strain S27, CHIKV-DRDE: Chikungunya virus strain DRDE-06, SINV-G: Sindbis virus Girdwood strain, SINV-AR86: Sindbis virus strain AR86, WEEV: Western equine encephalitis virus, MADV: Madaraga virus strain BeAr300851.

[0032] [Figure 5A-B]Figures 5A-5D summarize the results of experiments conducted to demonstrate that a MADV srRNA-based influenza vaccine against a viral antigen (influenza H1 hemagglutinin) can generate neutralizing antibody responses as measured by a hemagglutinin inhibition (HAI) assay (Figure 5A) and in vivo T cell responses as measured by ELISpot (Figures 5B-5D). Antibody and T cell responses measured by an HAI assay and IFNγ ELISpot, respectively, 14 days after a prime-boost of 0.01 μg of MADV-srRNA (Rep-631) LNP are shown. Figure 5A shows serum HAI titers against H1N1. Figure 5B shows the results of splenocytes stimulated with an HA peptide library. Figure 5C shows the results of CD4 T cell epitope-specific peptide stimulation. Figure 5D shows the results of CD8 T cell epitope-specific peptide stimulation. Mann-Whitney statistics between groups are shown (**<0.01; *<0.05). [Figure 5C-D] Figures 5A-5D summarize the results of experiments conducted to demonstrate that a MADV srRNA-based influenza vaccine against a viral antigen (influenza H1 hemagglutinin) can generate neutralizing antibody responses as measured by a hemagglutinin inhibition (HAI) assay (Figure 5A) and in vivo T cell responses as measured by ELISpot (Figures 5B-5D). Antibody and T cell responses measured by an HAI assay and IFNγ ELISpot, respectively, 14 days after a prime-boost of 0.01 μg of MADV-srRNA (Rep-631) LNP are shown. Figure 5A shows serum HAI titers against H1N1. Figure 5B shows the results of splenocytes stimulated with an HA peptide library. Figure 5C shows the results of CD4 T cell epitope-specific peptide stimulation. Figure 5D shows the results of CD8 T cell epitope-specific peptide stimulation. Mann-Whitney statistics between groups are shown (**<0.01; *<0.05).

[0033] [Figure 6]Figures 6A-6B summarize the results of experiments conducted to demonstrate the ability of MADV srRNA-based vectors to express biotherapeutic proteins in vivo. Protein levels of murine IL-1RA (Figure 6A) and murine IL-18BP (Figure 6B) were measured using ELISA 7 days after administration of MADV-srRNA (Rep-657) co-encoding these two genes. Basal protein expression levels were compared with those of an unrelated protein, red firefly luciferase ("unrelated"). Mann-Whitney statistics between groups are shown (*<0.05). DETAILED DESCRIPTION OF THE INVENTION

[0034] Provided herein are viral expression systems with excellent expression capabilities that are particularly suitable for expressing heterologous molecules, such as vaccine and therapeutic polypeptides, in recombinant cells. For example, some embodiments of the present disclosure relate to nucleic acid constructs, such as expression constructs and vectors, that contain a modified genome or replicon, e.g., a self-replicating RNA (srRNA), of Madaraga virus (MADV) in which at least a portion of the original viral sequence encoding the structural proteins has been deleted. Some embodiments of the present disclosure also provide viral-based expression vectors that contain one or more expression cassettes encoding heterologous polypeptides. Additionally, provided are recombinant cells genetically engineered to contain one or more of the nucleic acid molecules disclosed herein. Biomaterials and recombinant products obtained from such recombinant cells are also within the scope of the present application. Compositions and methods useful for inducing a pharmacodynamic effect in a subject in need thereof, as well as methods for preventing and / or treating various health conditions, are also provided.

[0035] Self-replicating RNA (srRNA) derived from RNA viruses (e.g., alphaviruses) can be used as robust expression systems. For example, a reported advantage of using alphaviruses, such as MADV, as viral expression vectors is their ability to direct the synthesis of large amounts of heterologous proteins in recombinant host cells. Among other advantages, polypeptides such as therapeutic single-chain antibodies may be most effective when expressed at high levels in vivo. Furthermore, for producing recombinant antibodies purified from cells in culture (ex vivo), high protein expression from srRNA can increase the total yield of antibody product. Furthermore, if the expressed protein is a vaccine antigen, high levels of expression may induce the most robust pharmacodynamic effects in vivo.

[0036] Alphaviruses utilize motifs contained in UTRs, structural regions, and nonstructural regions to influence their replication in host cells. These regions also contain mechanisms for evading the host cell's innate immune system. However, significant differences have been reported between alphavirus species. For example, New World and Old World alphaviruses have evolved different components to utilize stress granules, JAK-STAT signaling, farnesoid X receptor (FXR), and Ras-GTPase-activating protein (SH3 domain)-binding protein (G3BP) proteins within the cell for assembly of viral replication complexes. The portions of the genome that contain these components also differ among alphaviruses. For example, in some Old World alphaviruses, such as Sindbis, bypass of PKR activation and subsequent eIF2-alpha (eIF2α) phosphorylation occurs via a downstream loop, whereas in chikungunya, which lacks a recognizable DLP, bypass of this pathway is thought to occur via nsP4. Furthermore, beyond variation between individual alphaviruses, differences often exist within alphavirus strains that contribute to variations in properties such as virulence. As an example, sequence variation between North American and South American strains of New World Eastern Equine Encephalitis Virus (EEEV) alters their ability to regulate the STAT1 pathway, which leads to differential induction of type I interferon and altered virulence. As a further example, mouse studies using a set of North American MADV isolates with less than 2.7% genomic sequence divergence resulted in outcomes ranging from zero to complete lethality.

[0037] Given the differential presence of host cell attenuating factors in the nonstructural and structural regions of alphaviruses, deleting structural genes to enable heterologous gene expression in synthetic vectors has different effects on individual vectors. Synthetic replicons with different host attenuating factors in the nonstructural regions are differentially superior in eliciting immune responses against the expressed heterologous gene. For example, in Old World viruses, shutting down host cell functions has been linked to nsP2, whereas in New World viruses (e.g., EEEV, VEEV, and MADV), this activity is primarily associated with the capsid protein (C), which is partially or completely deleted in synthetic vectors. The hypervariable domain (HVD) of the nsP3 protein possesses host interactions specific to each alphavirus. In particular, EEEV nsP3 has been shown to interact with cellular FXR and G3BP protein families, DDX3, S100A4, IKKp, PGAM5, and cytoskeletal reorganization and vesicle transport proteins. Although few studies have specifically detailed the nonstructural proteins of MADV, genome sequencing of MADV revealed that it is the result of recombination between EEEV and SINV ancestors. The amino acid identity of MADV nsPs was reported to be over 80% identical to EEEV nsPs, but replacement of EEEV nsPs with MADV nsPs resulted in attenuated chimeras, indicating that changes in nucleotide and coding sequences maintain some essential activities for the viral life cycle but result in significant differences in biological activity. The known yet unexplained mechanisms by which EEEV and MADV nsPs contribute to broad pathogenicity suggest that MADV-based srRNA vectors may be distinct and advantageous vectors for expressing heterologous proteins for vaccine or biotherapeutic applications. The previously undescribed advantages of these srRNA vectors have never been explored or predicted.

[0038] definition Unless otherwise defined, all industry terms, notation, and other scientific or technical terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this application pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial departure from what is commonly understood in the art. Many of the techniques and procedures described or referenced herein are well understood by those of ordinary skill in the art and are commonly employed using conventional methodology.

[0039] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes one or more cells, including mixtures thereof. As used herein, "A and / or B" is used to include all of the alternatives "A," "B," "A or B," and "A and B."

[0040] As used herein, the terms "administration" and "administering" refer to the delivery of a bioactive composition or formulation by a route of administration including, but not limited to, intranasal, transdermal, intravenous, intraarterial, intramuscular, intralymphatic, intraperitoneal, subcutaneous, intramuscular, oral, intravaginal, and topical administration, or a combination thereof. This term includes, but is not limited to, administration by a medical professional and self-administration.

[0041] The terms "cell," "cell culture," and "cell line" refer not only to a particular subject cell, cell culture, or cell line, but also to the progeny or potential progeny of such a cell, cell culture, or cell line, regardless of the number of transplants or passages in culture. It is understood that all progeny may not be completely identical to the parent cell. This is because certain modifications may occur in later generations due to mutations (e.g., intentional or inadvertent mutations) or environmental influences (e.g., methylation or other epigenetic modifications), such that the progeny may not actually be identical to the parent cell; however, so long as the progeny retain the same functionality as the original cell, cell culture, or cell line, they are still within the scope of the terms as used herein.

[0042] The terms "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" of a composition of the present disclosure, e.g., a nucleic acid construct (e.g., a replicon construct, e.g., an srRNA construct), a recombinant cell, a recombinant polypeptide, and / or a pharmaceutical composition, generally refer to an amount sufficient for the composition to achieve a stated purpose (e.g., achieve the effect for which the composition is administered, stimulate an immune response, prevent or treat a disease, or alleviate one or more symptoms of a disease, disorder, infection, or 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 symptoms of a disease, which is also referred to as a "therapeutically effective amount." "Alleviation" of a symptom refers to a decrease in the severity or frequency of the symptom, or the elimination of the symptom. The precise amount of a composition that comprises a "therapeutically effective amount" will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0043] The term "construct" refers to a recombinant molecule, e.g., a recombinant nucleic acid or polypeptide, that contains one or more isolated nucleic acid or amino acid sequences from heterologous sources. For example, a polypeptide construct can be a chimeric polypeptide molecule in which two or more amino acid sequences of different origins are operably linked in a single polypeptide construct. Similarly, a nucleic acid construct can 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. Thus, exemplary nucleic acid constructs include (1) a nucleic acid sequence containing regulatory and coding sequences that are not found contiguous to each other in nature (e.g., at least one of the nucleotide sequences is heterologous to at least one of the other nucleotide sequences), or (2) a sequence encoding portions of a functional RNA molecule or protein that are not contiguous in nature, or (3) any construct that contains portions of a promoter that are not contiguous in nature. Exemplary nucleic acid constructs include any recombinant nucleic acid molecule, linear or circular, single- or double-stranded DNA or RNA nucleic acid molecule, derived from any source, such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, etc., capable of genomic integration or autonomous replication, and including nucleic acid molecules to which one or more nucleic acid sequences are operably linked. The nucleic acid constructs of the present disclosure can include the necessary elements for directing expression of a nucleic acid sequence of interest also included in the construct. Such elements may include regulatory elements, such as a promoter, operably linked to (to direct transcription of) the nucleic acid sequence of interest, and optionally including a polyadenylation sequence.

[0044] In some embodiments of the present disclosure, one or more nucleic acid constructs may be incorporated (e.g., inserted) into a single nucleic acid molecule, such as a single vector, or may be incorporated (e.g., inserted) into two or more separate nucleic acid molecules, such as two or more separate vectors. The term "vector" is used herein to refer to a nucleic acid molecule or sequence capable of transferring or transporting another nucleic acid molecule. Thus, the term "vector" includes both DNA-based vectors and RNA-based vectors. The term "vector" includes cloning vectors and expression vectors, as well as viral vectors and integrating vectors. An "expression vector" is a vector that contains regulatory regions and is thereby capable of expressing DNA sequences and fragments in vitro, ex vivo, and / or in vivo. In some embodiments, a vector may contain sequences that direct autonomous replication in a cell, such as a plasmid (a DNA-based vector) or a self-replicating RNA vector. In some embodiments, a vector may contain sufficient sequences to allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. In some embodiments, a vector of the present disclosure can be a single-stranded vector (e.g., ssDNA or ssRNA). In some embodiments, a vector of the present disclosure can be a double-stranded vector (e.g., dsDNA or dsRNA). In some embodiments, the vector is a gene delivery vector. In some embodiments, the vector is used as a gene delivery vehicle to transfer a gene into a cell. In some embodiments, the vector of the present disclosure is a self-replicating RNA (srRNA) vector.

[0045] In addition to the components of the construct, a vector may contain, for example, one or more selectable markers, one or more origins of replication, such as prokaryotic and eukaryotic origins of replication, at least one multiple cloning site, and / or elements that facilitate stable integration of the construct into a cellular genome. Two or more constructs can be incorporated into a single nucleic acid molecule, such as a single vector, or into two or more separate nucleic acid molecules, such as two or more separate vectors. An "expression construct" generally includes at least a control sequence operably linked to a nucleotide sequence of interest. Thus, for example, a promoter operably 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 the constructs and cells for practicing the present disclosure are known to those of skill in the art.

[0046] As used herein, the term "operably linked" refers to a physical or functional connection between two or more elements, e.g., polypeptide sequences or polynucleotide sequences, that allows those elements to operate in their intended manner. For example, when used in the context of a nucleic acid molecule described herein or a coding sequence and promoter sequence therein, the term "operably linked" means that the coding sequence and promoter sequence are in frame and at a suitable space and distance to allow the binding of a transcription factor or RNA polymerase, respectively, to effect transcription. It is understood that operably linked elements can be contiguous or non-contiguous (e.g., linked to each other via a linker). In the context of a polypeptide construct, "operably linked" refers to a physical connection (e.g., directly or indirectly linked) between amino acid sequences (e.g., different segments, portions, regions, or domains) to result in the described activity of the construct. The operably linked segments, portions, regions, and domains of the polypeptides or nucleic acid molecules disclosed herein can be contiguous or non-contiguous (e.g., linked to each other via a linker).

[0047] As used herein, the term "portion" refers to a fraction. With respect to a particular structure, such as a polynucleotide sequence, an amino acid sequence, or a protein, the term "portion" may refer to a contiguous or discontinuous fraction of the structure. For example, a portion of an amino acid sequence includes 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. Additionally or alternatively, when a portion is a discontinuous fraction, the discontinuous fraction may be comprised of 2, 3, 4, 5, 6, 7, 8, or more portions of the structure (e.g., a domain of a protein), where each portion is a contiguous element of the structure. For example, a non-contiguous segment of an amino acid sequence may consist of 2, 3, 4, 5, 6, 7, 8 or more portions of the amino acid sequence, e.g., 4 or fewer portions, each portion comprising 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.

[0048] The term "recombinant," when used with respect to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been altered or produced by human intervention, e.g., modified by or is the result of a laboratory method. Thus, for example, recombinant proteins and nucleic acids include proteins and nucleic acids produced by laboratory methods. Recombinant proteins can contain amino acid residues not found in the native (non-recombinant or wild-type) form of the protein or can contain modified, e.g., labeled, amino acid residues. The term includes any modification to a peptide, protein, or nucleic acid sequence. Such modifications may include any chemical modification of a peptide, protein, or nucleic acid sequence (including any chemical modification of 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, e.g., fusion proteins with antibody fragments; and the addition, deletion, and / or substitution of one or more nucleic acids in a nucleic acid sequence. The term "recombinant," when used in reference to a cell, is not intended to include naturally occurring cells, but is intended to encompass cells that have been modified / altered to contain or express a polypeptide or nucleic acid that is not present in the cell if not modified / altered.

[0049] The term "percent identity," as used herein in the context of two or more nucleic acids or proteins, refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides or amino acids (about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters, as described below, or by manual alignment and visual inspection. See, e.g., the NCBI website (ncbi.nlm.nih.gov / BLAST). Such sequences are said to be "substantially identical." This definition may also refer to or apply to the complement of a query sequence. This definition includes sequence comparisons performed by the BLAST algorithm, where the algorithm parameters are selected to maximize the 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 a region of at least about 20 amino acids or nucleotides in length, or over a region of 10-100 amino acids or nucleotides in length, or over the entire length of a given sequence. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux 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 sequence analysis software, for example, the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), using its default parameters.Additional methodologies that may be suitably employed to determine amino acid sequence similarity or identity include those that rely on position-specific structural scoring matrices (P3SMs) incorporating Rosetta structural prediction scores, and those based on length-normalized edit distances as previously described, e.g., in Setcliff et al., Cell Host & Microbe 23(6), May 2018.

[0050] As used herein, the term "pharmaceutically acceptable excipient" refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for administration of a compound of interest to a subject. Thus, "pharmaceutically acceptable excipient" can include substances that are referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. Supplementary active compounds (such as antibiotics and additional therapeutic agents) can also be incorporated into the composition.

[0051] As used herein, "subject" or "individual" includes animals, such as humans (e.g., human individuals) and non-human animals. In some embodiments, a "subject" or "individual" is a patient receiving medical care. Thus, a subject can be a human patient or individual who has, is at risk of, or is suspected of having a health condition of interest (e.g., cancer or an infectious disease) and / or one or more symptoms of a health condition. A subject can also be an individual who has been diagnosed as being at risk for a health condition of interest at the time of diagnosis or thereafter. The term "non-human animal" includes all vertebrates, e.g., mammals (e.g., rodents, e.g., mice, livestock, poultry, and pets, non-human primates, and other mammals, e.g., sheep, dogs, cats, cows, chickens) and non-mammals (e.g., amphibians, reptiles, etc.).

[0052] It is understood that aspects and embodiments of the disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments. As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claimed composition or method. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation of the term "comprising" herein, particularly in a description of a component of a composition or a description of a step of a method, should be understood to encompass compositions and methods that consist essentially of and consist of the recited components or steps.

[0053] It should be understood that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination. All combinations of the embodiments of the present disclosure are specifically embraced by the present disclosure and are disclosed herein as if each and every combination were individually and expressly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein as if each and every such subcombination were individually and expressly disclosed herein.

[0054] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limit of that range, and each intervening value (to the tenth of the unit of the lower limit, unless the context clearly indicates otherwise) between any other stated or intervening value in that stated range, is included within the scope of the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, which are also included within the scope of the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding one or both of those included limits are also included in the disclosure.

[0055] In this specification, certain ranges are presented with the term "about" preceding the numerical values, which, as used herein, have the ordinary meaning of approximating. The term "about" is also used herein to provide literal support for the numerical value it precedes and for numerical values ​​that are close to or approximately the numerical value it precedes. When determining whether a number is close to or approximately a specifically stated numerical value, the close or approximately unstated numerical value may be a numerical value that, in the context in which it is presented, provides a substantial equivalent to the specifically stated numerical value. Unless the degree of approximation is otherwise clear from the context, "about" means within plus or minus 10% of the provided value or rounded to the nearest significant figure, and in all cases includes the provided value. In some embodiments, the term "about" refers to ± up to 10%, ± 5%, or ± 1% of the specified value.

[0056] When a range of values ​​is provided, it will be understood by one of ordinary skill in the art that all ranges disclosed herein include any and all possible subranges and combinations of subranges. Any recited range can be readily recognized as fully descriptive and allows for division of that same range into at least one half, third, quarter, fifth, tenth, etc. As a non-limiting example, each range discussed herein can be easily divided into a lower third, middle third, upper third, etc. As will be understood by one of ordinary skill in the art, all terms such as "up to," "at least," "greater than," "less than," etc., refer to ranges that are inclusive of the recited number and can subsequently be divided into the subranges discussed above. Finally, as will be understood by one of ordinary skill in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 events refers to a group having 1, 2, or 3 events. Similarly, a group having 1 to 5 events refers to a group having 1, 2, 3, 4, or 5 events.

[0057] Headings, e.g., (a), (b), (i), etc., are provided solely to facilitate the reading of the specification and claims. The use of headings in the specification or claims does not require the steps or elements to be performed in alphabetical or numerical order or in the order in which they are presented.

[0058] Madaraga virus (MADV) Madaraga virus (MADV), formerly known as South American Eastern Equine Encephalitis virus (SA EEEV), is a mosquito-borne virus belonging to the Alphavirus genus, which includes a group of genetically, structurally, and serologically related viruses in the Togaviridae family. It is classified as a group IV positive-sense single-stranded RNA virus. The Alphavirus genus currently includes, among others, Sindbis virus (SINV), Semliki Forest virus (SFV), Ross River virus (RRV), Venezuelan equine encephalitis virus (VEEV), and Eastern equine encephalitis virus (EEEV), all of which are closely related and can infect a variety of vertebrates (e.g., mammals, rodents, fish, avian species, and large mammals such as humans and horses) and invertebrates (e.g., insects).

[0059] The EEEV complex consists of four distinct genetic lineages, one widespread in North America (NA EEEV) and the Caribbean, and three widespread in Central and South America (e.g., MADV). Various studies have demonstrated evolutionary and ecological diversity between NA EEEV and SA EEEV, suggesting that the NA and SA lineages may be considered separate species within the EEEV complex. The single, monophyletic NA EEEV lineage exhibited primarily temporal relationships and was highly conserved throughout its geographic range. In contrast, MADV (i.e., SA EEEV) contained three distinct lineages, two of which were composed of highly conserved geographic groupings with no temporal relationship. Phylogenetic comparison of MADV (SA EEEV) and VEEV showed similar genetic and evolutionary patterns, consistent with the well-documented use of mammalian reservoirs by VEEV. Although they are associated with equine disease, MADV species have no clear association with human disease. This lack of human pathogenicity has limited research to better understand the epidemiology and ecology of MADV strains. MADV isolates from Culex (Melanoconiou) mosquitoes in the Spissipes section (Culex pedroi in South America and Culex taeniopus in Central America) suggest that they are the primary endemic and potentially zoonotic vectors. These vectors typically have limited movement outside tropical forest habitats, which may affect the focality of infection. However, these species are relatively catholic in their feeding habits, broadening the potential transmission cycle used by MAD. The greater diversity of vectors in tropical regions may also contribute to genetic diversity among MADV lineages, although data on vector competence are limited.More information on this point can be found in Arrigo NC et al., Journal of Virology, Jan 2010, pp. 1014-1025, which is incorporated herein by reference.

[0060] The alphavirus genus, including MADV and EEEV, has been extensively studied, and the life cycles, replication modes, etc. of these viruses have been well characterized. More information on these can be found, for example, in Arrigo NC et al., 2010 (supra) and Corrin T. et al., Vector-Borne and Zoonotic Diseases, Vol. 21, No. 5, 2021. Furthermore, alphaviruses have been shown to replicate very efficiently in animal cells, making them valuable vectors for the production of proteins and nucleic acids in such cells. Transmission between species and individuals occurs primarily via mosquitoes, contributing alphaviruses to the collection of arboviruses or arthropod-borne viruses.

[0061] Each of these alphaviruses has a single-stranded RNA genome of positive polarity enclosed in a nucleocapsid surrounded by an envelope containing viral spike proteins. Alphavirus particles tend to be enveloped and spherical (although somewhat polymorphic) with an isometric nucleocapsid. The genome of an alphavirus is a single-stranded RNA of positive polarity approximately 11-12 kb in length, containing a 5' cap, a 3' poly(A) tail, and two open reading frames: the first encoding nonstructural proteins with enzymatic functions, and the second encoding viral structural proteins (e.g., capsid protein CP, E1 glycoprotein, E2 glycoprotein, E3 protein, and 6K protein). For example, MADV has a single-stranded, positive-sense RNA genome that is capped at the 5' end and polyadenylated at the 3' end, containing two open reading frames (ORFs) flanked by 5' and 3' untranslated regions (UTRs).

[0062] The 5' two-thirds of the alphavirus genome encodes numerous nonstructural proteins required for viral RNA transcription and replication. These proteins are translated directly from RNA and, together with cellular proteins, form the RNA-dependent RNA polymerase, essential for viral genome replication and subgenomic RNA transcription. Four nonstructural proteins (nsP1-4) are generated as a single polyprotein that constitutes the viral replication machinery. Polyprotein processing occurs in a highly regulated manner, and cleavage at the P2 / 3 junction affects the use of the RNA template during genome replication. This site is located at the bottom of a narrow cavity and is not easily accessible. Upon cleavage, nsP3 forms a ring structure surrounding nsP2. These two proteins have an extensive interface. Mutations in nsP2 that produce noncytopathic or temperature-sensitive phenotypes are clustered at the P2 / P3 interface region. P3 mutations opposite the location of nsP2 noncytopathic mutations prevent efficient P2 / 3 cleavage. This can then affect RNA infectivity and alter the level of viral RNA production.

[0063] The 3' third of the genome contains a subgenomic RNA that serves as a template for translation of all structural proteins required for viral particle formation (core nucleocapsid protein C and envelope proteins P62 and E1, which assemble as a heterodimer). Viral membrane-anchored surface glycoproteins are involved in receptor recognition and membrane fusion to enter target cells. The subgenomic RNA is transcribed from the p26S subgenomic promoter at the 3' end of the RNA sequence encoding the nsP4 protein. The proteolytic maturation of P62 into E2 and E3 results in changes to the viral surface. E1, E2, and occasionally E3 glycoprotein "spikes" combine to form E1 / E2 dimers or E1 / E2 / E3 trimers, with E2 extending from the center to the vertices, E1 filling the space between the vertices, and E3 (if present) at the distal end of the spike. When the virus is exposed to the acidic environment of the endosome, E1 dissociates from E2 to form the E1 homotrimer, which is necessary for the fusion step to drive the cellular and viral membranes together. The alphavirus glycoprotein E1 is a class II viral fusion protein, which is structurally distinct from the class I fusion proteins found in influenza virus and HIV. The E2 glycoprotein functions to interact with the nucleocapsid via its cytoplasmic domain, while the extracellular domain is involved in binding to cellular receptors. Most alphaviruses, including MADV, lose the peripheral protein E3, but in Semliki virus, it remains associated with the viral surface.

[0064] Like most alphaviruses, MADV replication occurs on the membranous surface of the host cell. In the first step of the infection cycle, the 5' end of the genomic RNA is translated into polyproteins (nsP1-4) with RNA polymerase activity, which generate a negative strand complementary to the genomic RNA. In the second step, the negative strand is used as a template to generate two RNAs: (1) a positive genomic RNA corresponding to the secondary viral genome, which translates to generate other nsP proteins and functions as the viral genome, and (2) a subgenomic RNA encoding the viral structural proteins that form the infectious particle. The positive genomic RNA / subgenomic RNA ratio is regulated by proteolytic self-cleavage of the polyprotein into nsP1, nsP2, nsP3, and nsP4. In fact, viral gene expression occurs in two phases. In the first phase, the primary synthesis of the positive and negative genomic strands occurs. In the second phase, synthesis of the subgenomic RNA is virtually exclusive, and therefore, large amounts of structural proteins are produced.

[0065] self-replicating RNA As understood by those skilled in the art, the term "self-replicating RNA" (srRNA) refers to an RNA molecule that contains all the genetic information necessary to direct its own amplification or self-replication within a permissive cell. Therefore, srRNA is sometimes referred to as "self-amplifying RNA" (saRNA). In some embodiments, srRNA is a "replicon," which can be a linear or circular section of DNA or RNA that is continuously replicated as a unit. Non-limiting examples of replicons include "replicon RNA" or "RNA replicon." To direct its own replication, srRNA generally (1) encodes a polymerase, replicase, or other protein that can interact with viral or host cell-derived proteins, nucleic acids, or ribonucleoproteins to catalyze the RNA amplification process, and (2) contains cis-acting RNA sequences necessary for the replication and transcription of subgenomic RNA. These sequences may bind to self-encoded proteins, or non-self-encoded cell-derived proteins, nucleic acids, or ribonucleoproteins, or complexes between any of these components, during the replication process. In some embodiments of the present disclosure, the replicon, e.g., srRNA, is derived from Madaraga virus (MADV). In some embodiments of the present disclosure, MADV srRNA constructs (e.g., srRNA, saRNA, or RNA replicon molecules) generally contain the following elements: a 5' viral or defective-interfering RNA sequence required in cis for replication, sequences encoding biologically active alphavirus nonstructural proteins (e.g., nsP1, nsP2, nsP3, and nsP4), a subgenomic promoter (sg) for the subgenomic RNA (sgRNA), 3' viral sequences required in cis for replication, and, optionally, a polyadenylate tract (poly(A)). In some cases, a subgenomic promoter (sg) directing expression of a heterologous sequence can be included in the srRNA constructs of the present disclosure.

[0066] Furthermore, the term srRNA molecule (e.g., srRNA, saRNA, or RNA replicon molecule) generally refers to a molecule of positive polarity or "message" sense, and the srRNA may be of a length different from that of any known naturally occurring alphavirus. In some embodiments of the present disclosure, the srRNA does not include 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 those cases, when the srRNA is packaged into recombinant alphavirus particles, the srRNA may include one or more sequences, so-called packaging signals, that serve to initiate the interaction with the alphavirus structural proteins that results in particle formation.

[0067] Nucleic acid molecules of the present disclosure can be nucleic acid molecules of any length, generally between about 2 kb and 50 kb, e.g., between about 5 kb and about 40 kb, between about 5 kb and about 30 kb, between about 5 kb and about 20 kb, or between about 10 kb and about 50 kb, e.g., between about 15 kb and 30 kb, between about 20 kb and about 50 kb, between about 20 kb and about 40 kb, between about 5 kb and about 25 kb, or between about 30 kb and about 50 kb. In some embodiments, the nucleic acid molecule is at least 6 kb in length. In some embodiments, the nucleic acid molecule is between about 6 kb and about 20 kb. In some embodiments of the present disclosure, the replicon constructs (e.g., srRNA constructs) of the present disclosure generally have a length of at least about 2 kb. For example, the srRNA can have a length of at least about 2 kb, at least about 3 kb, at least about 4 kb, at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 11 kb, at least about 12 kb, or greater than 12 kb. In some embodiments, the srRNA is about 4 kb to about 20 kb, about 4 kb to about 18 kb, about 5 kb to about 16 kb, about 6 kb to about 14 kb, about 7 kb to about 12 kb, about 8 kb to about 16 kb, about 9 kb to about 14 kb, about 10 kb to about 18 kb, about 11 kb to about 16 kb, about 5 kb to about 18 kb, about 6 kb to about 20 kb, about 5 kb to about 10 kb, about 5 kb to about 8 kb, about 5 kb to about 7 kb, about 5 kb to about 6 kb, or about 6 kb. In some embodiments, the srRNA can have a length of about 6 kb to about 12 kb, about 6 kb to about 11 kb, about 6 kb to about 10 kb, about 6 kb to about 9 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, about 7 kb to about 11 kb, about 7 kb to about 10 kb, about 7 kb to about 9 kb, about 7 kb to about 8 kb, about 8 kb to about 11 kb, about 8 kb to about 10 kb, about 8 kb to about 9 kb, about 9 kb to about 11 kb, about 9 kb to about 10 kb, or about 10 kb to about 11 kb. In some embodiments, the srRNA can have a length of about 6 kb to about 14 kb. In some embodiments, the srRNA can have a length of about 6 kb to about 16 kb.

[0068] Compositions of the present disclosure As described in more detail below, one aspect of the present disclosure relates to nucleic acid constructs comprising nucleic acid sequences encoding modified viral genomes or srRNAs, where the modified genomes or srRNAs lack (e.g., do not contain) at least a portion of the nucleic acid sequences encoding one or more structural proteins of the corresponding unmodified viral genomes or srRNAs. Some embodiments of the present disclosure provide modified alphavirus genomes or srRNAs in which the coding sequences for nonstructural proteins nsP1, nsP2, nsP3, and nsP4 are present, but at least a portion or all of the sequences encoding one or more structural proteins are absent. Recombinant cells and cell cultures engineered to contain the nucleic acid constructs disclosed herein are also provided.

[0069] A. Nucleic acid constructs As described in more detail below, one aspect of the present disclosure relates to novel nucleic acid constructs comprising nucleic acid sequences encoding modified genomes or replicons (e.g., srRNAs) of alphaviruses, such as Madaraga virus (MADV). For example, in some embodiments, the modified alphavirus genomes can comprise deletions, substitutions, and / or insertions in one or more genomic regions of the parent alphavirus genome.

[0070] Non-limiting exemplary embodiments of nucleic acid constructs (e.g., replicon constructs, e.g., srRNA constructs) of the present disclosure can include one or more of the following features: In some embodiments, the nucleic acid construct comprises a nucleic acid sequence encoding a modified MADV genome or replicon, e.g., srRNA, wherein the modified MADV genome or replicon, e.g., srRNA, lacks at least a portion of the nucleic acid sequence encoding one or more structural proteins of the unmodified MADV genome or srRNA, e.g., the modified MADV genome or srRNA does not include at least a portion of the coding sequence for one or more of the MADV structural proteins CP, E1, E2, E3, and 6K. Both virulent and non-virulent MADV strains are suitable. Non-limiting examples of MADV strains suitable for the compositions and methods of the present disclosure include ArgLL, ArgB, BeAn-5122, ArgM, 24443 (TR59), 25714 (BG60), BeAr 18205, 900188 (PA62), BeAr 81828, BeAr 126650, 68U231, 77U1104 (PE70), 75V1496, BeAr 300851, 75U40, and El Delirio (Arrigo NC et al., 2010, supra). Further examples of MADV strains suitable for the compositions and methods of the present disclosure include 76V25343, 77U1 (BR77), BeAr 348998, IVICPan57151, BeAn416361, 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 (supra)).Additional suitable MADV strains include, but are not limited to, Arrigo NC et al., 2010 (supra) and the Viral Pathogens 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 MADV genome or srRNA is derived from MADV strain BeAr300851.

[0071] Non-limiting exemplary embodiments of nucleic acid constructs (e.g., replicon constructs, e.g., srRNA constructs) of the present disclosure can include one or more of the following features: In some embodiments, the modified viral genome or srRNA 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 unmodified viral genome or srRNA. In some embodiments, the modified viral genome or srRNA lacks some or all of the sequence encoding CP. In some embodiments, the modified viral genome or srRNA lacks some or all of the sequence encoding E1. In some embodiments, the modified viral genome or srRNA lacks some or all of the sequence encoding E2. In some embodiments, the modified viral genome or srRNA lacks some or all of the sequence encoding E3. In some embodiments, the modified viral genome or srRNA lacks some or all of the sequence encoding 6K. In some embodiments, the modified viral genome or srRNA lacks some or all of the sequences encoding a combination of CP, E1, E2, E3, and 6K. Some embodiments of the present disclosure provide modified MADV genomes or srRNAs in which the coding sequences for nonstructural proteins nsP1, nsP2, nsP3, and nsP4 of the unmodified MADV genome or srRNA are present, but at least some or all of the sequences encoding one or more structural proteins (e.g., CP, E1, E2, E3, and 6K) of the MADV genome or srRNA are absent. Some embodiments of the present disclosure provide modified MADV genomes or srRNAs in which the coding sequences for nonstructural proteins nsP1, nsP2, nsP3, and nsP4 of the unmodified MADV genome or srRNA are present, but at least some or all of the sequences encoding one or more structural proteins (e.g., CP, E1, E2, E3, and 6K) of the MADV genome or srRNA are absent.

[0072] In some embodiments, the modified viral genome or srRNA lacks a substantial portion of the nucleic acid sequence encoding one or more viral structural proteins. One skilled in the art will understand that a substantial portion of the nucleic acid sequence encoding a viral structural protein comprises sufficient nucleic acid sequence to encode a viral structural polypeptide, allowing putative identification of that polypeptide by manual evaluation of the sequence by one skilled in the art or by computer-automated sequence comparison and identification using algorithms such as BLAST (e.g., "Basic Local Alignment Search Tool"; see Altschul SF et al., J. Mol. Biol. 215:403-410, 1993). Thus, a substantial portion of a nucleotide sequence comprises sufficient sequence to permit specific identification and / or isolation of a nucleic acid fragment comprising the sequence. For example, a substantial portion of a nucleic acid sequence may comprise at least about 20%, e.g., about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of the full-length nucleic acid sequence. As noted above, the present disclosure provides nucleic acid molecules and constructs lacking partial or complete nucleic acid sequences encoding one or more viral structural proteins. Those skilled in the art, having the benefit of the sequences disclosed herein, can readily use all or a substantial portion of the disclosed sequences for the compositions and methods of the present disclosure. Accordingly, the present application includes the complete sequences disclosed herein, e.g., the sequences set forth in the attached sequence listing, and substantial portions of those sequences as defined above.

[0073] In some embodiments, the modified viral genome or srRNA does not include the entire sequence encoding the viral structural proteins, e.g., the modified viral genome or srRNA does not include the nucleic acid sequences encoding the structural proteins of the unmodified viral genome or srRNA.

[0074] In some embodiments, the nucleic acid constructs of the present disclosure further comprise one or more expression cassettes. In principle, the nucleic acid constructs disclosed herein can generally comprise any number of expression cassettes. In some embodiments, the nucleic acid constructs disclosed herein can comprise at least two, at least three, at least four, at least five, or at least six expression cassettes. Those skilled in the art will understand that the term "expression cassette" refers to a construct of genetic material containing a coding sequence and sufficient regulatory information to direct the precise transcription and / or translation of the coding sequence in a cell, in vivo, and / or in vitro. The expression cassette may be inserted into a vector for targeting to a desired host cell and / or subject or individual. Thus, in some embodiments, the term expression cassette may be used interchangeably with the term "expression construct." In some embodiments, the term "expression cassette" refers to a nucleic acid construct comprising either or a combination of a gene encoding a protein or functional RNA operably linked to regulatory elements, such as a promoter and / or termination signal, and optionally other nucleic acid sequences that affect the transcription or translation of the gene.

[0075] In some embodiments, at least one of the expression cassettes comprises a promoter operably linked to a heterologous nucleic acid sequence. In some embodiments, the heterologous nucleic acid sequence may encode a polypeptide. In some embodiments, the heterologous nucleic acid sequence may be or comprise a non-coding RNA or functional RNA that is not translated into a protein. Non-limiting examples of non-coding RNA genes include transfer RNA (tRNA), ribosomal RNA (rRNA), and small RNAs such as snoRNA, microRNA, siRNA, tmRNA, and piRNA. Non-limiting examples of functional RNA include guide RNA (gRNA) that functions in RNA editing. Thus, the nucleic acid constructs provided herein can find use as expression vectors that can affect expression of a heterologous nucleic acid sequence, for example, when they comprise a regulatory element (e.g., a promoter) operably linked to the heterologous nucleic acid sequence. In some embodiments, the promoter is or comprises a naturally occurring sequence isolated from or derived from the genomic sequence of the gene. In some embodiments, the promoter can be synthetically generated or engineered by modifying known DNA elements. In some embodiments, at least one of the expression cassettes comprises a subgenomic (sg) promoter operably linked to a heterologous nucleic acid sequence. In some embodiments, the sg promoter is a 26S subgenomic promoter. In some embodiments, at least one nonstructural protein (nsP) or portion thereof of the modified MADV genome or srRNA is heterologous to the remainder of the modified MADV genome or srRNA. In some embodiments, the modified MADV genome or srRNA further comprises a nucleic acid sequence encoding a heterologous nsP or portion thereof. In some embodiments, a nucleic acid molecule of the present disclosure further comprises one or more untranslated regions (UTRs). In some embodiments, at least one of the UTRs is a heterologous UTR.

[0076] In some embodiments, at least one of the expression cassettes comprises a coding sequence for a gene of interest (GOI). In some embodiments, the coding sequence for the GOI comprises a coding sequence for a polypeptide construct of interest (PCI), which comprises a single polypeptide (e.g., a monogenic PCI). In some embodiments, the coding sequence for the PCI includes coding sequences for multiple polypeptides, e.g., a multigenic PCI (e.g., a bigenic, trigenic, tetragenic, etc.). In some embodiments, each of the coding sequences for the multiple polypeptides is operably linked to a separate promoter sequence. In some embodiments, the coding sequences for the multiple polypeptides are operably linked to each other in a single open reading frame (e.g., in a polycistronic ORF). In some embodiments, the coding sequence of the polycistronic ORF is operably linked to a promoter sequence. In some embodiments, at least one of the promoter sequences is a subgenomic (sg) promoter. In some embodiments, the sg promoter is a 26S genomic promoter.

[0077] In some embodiments, multiple polypeptides can be linked to each other directly or indirectly (e.g., via one or more connector sequences). For example, in some embodiments, multiple polypeptides can be linked directly to each other, e.g., adjacent to each other. In some embodiments, at least two (e.g., 2, 3, 4, or 5) of the multiple polypeptides are operably linked to each other by one or more connector sequences. In some embodiments, the length and amino acid composition of the connector sequences can be optimized to vary the orientation, flexibility, and / or proximity between the polypeptides relative to each other to achieve a desired activity or property of the PCI. In some embodiments, one connector sequence of the multiple connector sequences comprises one or more autoproteolytic peptide sequences. Non-limiting examples of autoproteolytic peptide sequences suitable for the methods and compositions of the present disclosure include autoproteolytic sequences derived from calcium-dependent serine endoprotease (furin), porcine teschovirus-12A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), Thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), and flacherie virus 2A (BmIFV2A). In some embodiments, at least two of the plurality of polypeptides are operably linked to each other via a P2A autoproteolytic sequence.

[0078] In some embodiments, the coding sequences of the multiple polypeptides are operably linked to each other by one or more internal ribosome entry sites (IRES). Non-limiting examples of IRES suitable for the methods and compositions of the present disclosure include viral IRES sequences, cellular IRES sequences, and artificial IRES sequences. Examples of suitable IRES sequences include, but are not limited to, Kaposi's sarcoma-associated herpesvirus (KSHV) IRES, hepatitis virus IRES, pestivirus IRES, Cripavirus IRES, Rhopalosiphum padi 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.

[0079] In principle, there are no particular limitations regarding suitable polypeptides and PCIs that can be expressed by the replicon constructs (e.g., srRNA constructs) of the present disclosure. Exemplary types of polypeptides suitable for the compositions and methods of the present disclosure include microbial proteins, viral proteins, bacterial proteins, fungal proteins, mammalian proteins, and any combination thereof. For example, the PCI can include one or more antigenic molecules and / or biological therapeutic molecules, such as cytokines, cytotoxins, chemokines, immunomodulators, pro-apoptotic factors, anti-apoptotic factors, hormones, differentiation factors, de-differentiation factors, immune cell receptors, or reporters, or combinations thereof.

[0080] In some embodiments, the coding sequence of a GOI can be redesigned, refactored, and / or optimized for desired properties, such as increased stability, potency, and expression (e.g., translation efficiency), thereby maximizing the impact of biotherapeutic manufacturing, delivery, and administration. For example, in some embodiments, the coding sequence of a GOI is optimized for expression at a level higher than the expression level of a 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, non-optimized sequence. With regard to sequence optimization of nucleotide sequences, the degeneracy of the genetic code provides the possibility to replace at least one base in the protein-coding sequence of a gene with a different base without changing the amino acid sequence of the polypeptide produced from the gene. Thus, the nucleic acid constructs of the present disclosure can also have any base sequence that has been altered from any polynucleotide sequence disclosed herein by substitution in accordance with the degeneracy of the genetic code. References describing codon usage are readily available to the public. In some embodiments, polynucleotide sequence variants can be generated for a variety of reasons, such as to optimize expression for a particular host (e.g., changing the codon usage in an alphavirus mRNA to one preferred for humans, non-human primates, hamsters, mice, or other organisms, such as monkeys). Thus, in some embodiments, the coding sequence of a GOI is optimized for expression in a target host cell by using codons optimized for expression. Techniques for constructing synthetic nucleic acid sequences encoding a GOI using optimally preferred codons for host cell expression can be determined by techniques known in the art through computational methods that analyze the commonality of codon usage and their relative abundance in coding for native proteins in a host cell genome. Various codon usage databases (e.g., http: / / www.kazusa.or.jp / codon) can be used for generating codon-optimized sequences in a mammalian cellular environment.Additionally, various software tools are available for converting sequences from one organism to optimal codon usage for a different host organism, such as 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). Such synthetic sequences may be constructed by techniques known in the art for constructing synthetic nucleic acid molecules and are available from various commercial vendors. Thus, in some embodiments, the coding sequence of a GOI is optimized for expression at a level greater than that of a reference coding sequence, e.g., a coding sequence that is not codon-optimized. In some embodiments, the codon-optimized sequence of a GOI 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 reference coding sequence that is not codon-optimized. In some embodiments, the codon-optimized sequence of a GOI results in at least a 2-fold, at least a 3-fold, at least a 4-fold, or at least a 5-fold increase in expression level compared to a reference coding sequence that is not codon-optimized.

[0081] In some embodiments, the coding sequence of a GOI is optimized for improved RNA stability and / or expression. RNA stability is generally associated with the "half-life" of the RNA. "Half-life" refers to the time required for a molecule to lose half of its activity, amount, or number. In the context of the present disclosure, the half-life of an RNA is an indicator of the stability of the RNA. The half-life of an RNA can affect the "expression" period of the RNA. For additional information regarding principles, strategies, and methods used to enhance RNA stability, see, for example, Leppek K. et al., Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics (Nature Communications. Vol. 13, Article No. 1536, March 2022).

[0082] The polypeptide encoded by the GOI can generally be any polypeptide, such as a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide, an industrial enzyme, and a reporter polypeptide. In some embodiments, the GOI encodes a polypeptide selected from the group consisting of an antibody, an antigen, an immunomodulator, an enzyme, a signaling protein, and a cytokine. In some embodiments, the GOI can encode a microbial protein, a viral protein, a bacterial protein, a fungal protein, a mammalian protein, and any combination thereof. In some embodiments, the GOI encodes the hemagglutinin precursor (HA) of influenza A virus H5N1. Non-limiting examples of GOIs include interleukins and interacting proteins (G-CSF, GM-CSF, IL-1, IL-10, IL-10-like, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-18BP, IL -1-like, IL-1RA, IL-1α, IL-1β, IL-2, IL-20, IL-3, IL-4, IL-5, IL-6, IL-6-like, IL-7, IL-9, IL-21, IL-22, IL-33, IL-37, IL-38, LIF, and OSM).Other suitable GOIs include interferons (e.g., IFN-α, IFN-β, IFN-γ), TNF (e.g., CD154, LT-β, ​​TNF-α, TNF-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, and TRANCE), TGF-β (e.g., TGF-β1, TGF-β2, and TGF-β3), hematopoietic factors (e.g., Epo, Tpo, Flt-3L, SCF, M-CSF, MSP), chemokines and their receptors (e.g., XCL1, XCL2, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL1), and the like. 1, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, and CX3CL1), immunosuppressive gene products and related transcription factors (e.g., PECAM1, FCGR3A, FOS, NFKB1, JUN, HIF1A, PD-L1, mTOR, STAT5B, and STAT4). Additional GOIs suitable for the compositions and methods of the present disclosure include, but are not limited to, immunostimulatory gene products (e.g., CD27 / CD70, CD40, CD40L, B7.1, BTLA, MAVS, OX40, OX40L, RIG-I, and STING), drug-resistant mutants / variants of genes (e.g., ABCB1, ABCC1, ABCG2, AKT1, ALK, BAFF, BCR-ABL, BRAF, CCND1, cMET, EGFR, ERBB2, ERBB3, ERK2, ESR1, GRB2, KRAS, MDR1, MRP1, NTRK1, PDC4, P-gp, PI3K, PTEN, RET, ROS1, RSK1, RSK2, SHIP, and STK11). Also suitable for the compositions and methods of the present disclosure are sequences encoding viral proteins, particularly spike proteins, fiber proteins, structural proteins, and attachment proteins.

[0083] In some embodiments, the GOI can encode an antibody or antibody variant (e.g., single chain Fv, bispecific, camelid, Fab, and HCAb). In some embodiments, the antibody targets a surface molecule associated with or upregulated by cancer, or a surface molecule associated with infectious disease. In some embodiments, the antibody targets a surface molecule with immunostimulatory or immunosuppressive function.

[0084] In some embodiments, the GOI can encode an enzyme whose deficiency or mutation is associated with a disease or condition, such as agalsidase beta, agalsidase alfa, imiglucerase, taliglucerase alfa, velaglucerase alfa, alglucerase, sebelipase alfa, laronidase, idursulfase, elosulfase alfa, galsulfase, alglucosidase alfa, and CTFR.

[0085] In some embodiments, the GOI can encode a polypeptide selected from an antigen molecule, a biological therapeutic molecule, or any combination thereof. In some embodiments, the GOI can encode a polypeptide selected from a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a neoantigen, and any combination thereof. As will be understood by those skilled in the art, a TAA can generally comprise a molecule, e.g., a protein, that is present in tumor cells and normal cells or in many normal cells but at a much lower concentration than on tumor cells. In contrast, a TSA can generally comprise a molecule, e.g., a protein, that is present in tumor cells but not in normal cells. The tumor-associated antigen may be an antigen associated with cancer cells, such as breast cancer cells, B-cell lymphoma, pancreatic cancer, Hodgkin's lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma, lung cancer cells, non-Hodgkin's B-cell lymphoma (B-NHL) cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, glioma, glioblastoma, and colorectal cancer cells. It will also be understood that in some cases, tumor-associated antigens may also be expressed by non-cancer cells. In some embodiments, the GOI may encode a polypeptide selected from an estrogen receptor, an intracellular signaling enzyme, and a human epidermal growth receptor. In some embodiments, the GOI may encode a biotherapeutic polypeptide selected from an immunomodulator, an angiogenesis regulator, an extracellular matrix regulator, a metabolic regulator, a neuromodulator, and a combination thereof. In some embodiments, the GOI can encode a cytokine selected from a chemokine, an interferon, an interleukin, a lymphokine, and a tumor necrosis factor, hi some embodiments, the GOI can encode an interleukin selected from IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-17, IL-23, IL-27, IL-35, IFNγ, and any subunits thereof.In some embodiments, the GOI can encode a biotherapeutic polypeptide selected from IL-12A, IL-12B, IL-1RA, and any combination thereof.

[0086] In some embodiments, the nucleic acid construct of the present disclosure comprises a nucleic acid sequence encoding a modified MADV that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:1.

[0087] In some embodiments, the nucleic acid construct of the present disclosure comprises a nucleic acid sequence encoding a modified MADV that has 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 1, wherein 1, 2, 3, 4, 5, or more nucleotides of the nucleic acid sequence may be replaced by different nucleotides.

[0088] Nucleic acid sequences having a high degree of sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the sequence of the modified MADV of interest can be identified and / or isolated by genome sequence analysis, hybridization, and / or PCR using degenerate or gene-specific primers derived from the sequences identified in each MADV genome, using the sequences identified herein (e.g., SEQ ID NO: 1) or any other sequences known in the art.

[0089] In some embodiments, a nucleic acid construct of the present disclosure can include one or more adapter sequences, e.g., cloning adapter sequences. In some embodiments, the one or more adapter sequences include the following sequence: 5'-CTGGAGACGTGGAGGAGAACCCTGGACCT-3' (SEQ ID NO: 3). In some embodiments, the one or more adapter sequences include the following sequence: 5'-GACCGCTACGCCCCAATGACCCGACCAGC-3' (SEQ ID NO: 4).

[0090] In some embodiments, mutations (e.g., silent mutations) may be incorporated into nucleic acid constructs of the present disclosure to remove restriction sites, such as SapI, SpeI, and BspQI restriction sites (see, e.g., Examples 1-3). For example, a unique restriction site (SpeI, 5'-A'CTAG, T-3') (5'A corresponds to the position of the ATG start codon of the structural polyprotein, and 3'T corresponds to the position of the TAA stop codon of the structural polyprotein) may be incorporated in place of the coding sequence of the native MADV structural gene. In some embodiments, a 5' adapter sequence (e.g., SEQ ID NO: 3) may be inserted upstream of the SpeI site, and a 3' adapter sequence (SEQ ID NO: 4) may be inserted downstream of the SpeI site for subsequent Gibson Assembly® procedures (Gibson et al., Nat. Methods 6, 343-345, 2009). In some embodiments, a promoter sequence (e.g., a bacteriophage T7 RNA polymerase promoter) may be included upstream of the MADV genomic sequence, and downstream may be a poly(A) sequence followed by a restriction enzyme cleavage site (e.g., SapI) that cuts upstream of the recognition site. Immediately downstream of this restriction enzyme cleavage site (SapI), a terminator sequence (e.g., a T7 terminator sequence) may be included, followed by a unique restriction enzyme cleavage site (e.g., NotI').

[0091] In some embodiments, the sequence encoding one or more of the nsPs is replaced with a heterologous nsP. In some other experiments, the sequence encoding one or more of the UTRs is replaced with a heterologous UTR.

[0092] Molecular techniques and methods for assembling and characterizing these new nucleic acid constructs are more fully described in the Examples of this application. In some embodiments, the nucleic acid molecule is a recombinant nucleic acid molecule. As used herein, the term "recombinant" means any molecule (e.g., DNA, RNA, polypeptide, etc.) that results from, or indirectly from, human manipulation. As a non-limiting example, cDNA is a recombinant DNA molecule, as is any nucleic acid molecule produced by an in vitro polymerase reaction, or that has been linked, or that has been incorporated into a vector, such as a cloning vector or an expression vector. By way of non-limiting example, a recombinant nucleic acid molecule is 1) synthesized or modified in vitro, for example, using chemical or enzymatic techniques (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 (including, for example, methylation), or recombination (including homologous recombination and site-specific recombination) of nucleic acid molecules); 2) contains linked nucleotide sequences that are not linked in nature; 3) has been engineered using molecular cloning techniques to delete one or more nucleotides relative to a naturally occurring nucleotide sequence; and / or 4) has been engineered using molecular cloning techniques to have one or more sequence alterations or rearrangements relative to a naturally occurring nucleotide sequence.

[0093] In some embodiments, the nucleic acid molecules disclosed herein are produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning, etc.) or chemical synthesis. The nucleic acid molecules disclosed herein include, but are not limited to, naturally occurring nucleic acid molecules and their homologs, including naturally occurring allelic variants and modified nucleic acid molecules in which one or more nucleotide residues have been inserted, deleted, and / or substituted in a manner that confers desired properties in the performance of the biological activities described herein.

[0094] Nucleic acid molecules containing variants of naturally occurring nucleic acid sequences can be generated using many methods known to those of skill in the art (see, e.g., Sambrook et al., In: Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989)). The sequence of a nucleic acid molecule can be modified relative to the naturally occurring sequence from which it is derived using a variety of techniques, including, but not limited to, classical mutagenesis techniques and recombinant DNA techniques, such as 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 a nucleic acid sequence, recombinational cloning, and chemical synthesis (including chemical synthesis of mixtures of oligonucleotides and ligation of mixtures to "assemble" a mixture of nucleic acid molecules), and combinations thereof. Nucleic acid molecule homologs can be selected from a mixture of modified nucleic acid molecules by screening for the function of the protein or srRNA encoded by the nucleic acid molecule and / or by hybridization with the wild-type gene or a fragment thereof, or by PCR using primers having homology to the target or wild-type nucleic acid molecule or sequence.

[0095] B. Recombinant Cells The nucleic acid constructs of the present disclosure can be introduced into a host cell to generate a recombinant cell containing the nucleic acid molecule. Accordingly, prokaryotic or eukaryotic cells containing a nucleic acid construct encoding a modified MADV genome described herein are also a feature of the present disclosure. In a related aspect, some embodiments disclosed herein relate to methods of transforming cells, including introducing a nucleic acid construct provided herein into a host cell, such as an animal cell, and then selecting or screening for transformed cells. Introduction of the nucleic acid constructs of the present invention into cells can be achieved 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)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0096] In one aspect, some embodiments of the present disclosure relate to recombinant cells, e.g., recombinant animal cells, comprising a nucleic acid construct described herein. The nucleic acid construct can be stably integrated into the host genome, or can be episomally replicated, or can exist in the recombinant host cell as a minicircle expression vector for stable or transient expression. Thus, in some embodiments of the present disclosure, the nucleic acid construct is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid construct is stably integrated into the genome of the recombinant cell. Stable integration can be accomplished using traditional random genome recombination techniques or more precise genome editing techniques, such as guide RNA-directed CRISPR / Cas9 or TALEN genome editing. In some embodiments, the nucleic acid construct exists in the recombinant host cell as a minicircle expression vector for stable or transient expression.

[0097] Host cells can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule. Thus, in some embodiments, host cells can be genetically engineered (e.g., transduced, transformed, or transfected) with at least one nucleic acid molecule.

[0098] Suitable host cells for cloning or expressing a polypeptide of interest described herein include prokaryotic or eukaryotic cells described herein. Thus, in some embodiments, the recombinant cell is a prokaryotic cell, such as E. coli, or a eukaryotic cell, such as an insect cell (e.g., a mosquito cell or an Sf21 cell), or a mammalian cell (e.g., a COS cell, an NIH3T3 cell, or a HeLa cell). In some embodiments, the cell is in vivo, e.g., a recombinant cell in a living organism, e.g., a cell of a transgenic subject. In some embodiments, the subject is a vertebrate or an invertebrate. In some embodiments, the subject is an insect. In some embodiments, the subject is a mammal. In some embodiments, the cell is ex vivo, e.g., extracted as an individual cell or as part of an organ or tissue from a living organism or organism for a treatment or procedure and then returned to the living organism or organism. In some embodiments, the cell is in vitro, e.g., taken from a repository. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a vertebrate cell or an invertebrate cell. In some embodiments, the recombinant cell is a mammalian cell.Non-limiting examples of recombinant cells suitable for the methods and compositions of the present disclosure include SV40-transformed monkey kidney CV1 cells (e.g., COS-7 cells), human embryonic kidney cells (e.g., HEK293 or HEK293 cells) or derivatives thereof (e.g., BHK-21 or BHK-570 cells), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 cells), human cervical carcinoma cells (e.g., HeLa cells), canine kidney cells (MDCK cells), buffalo rat hepatocytes (e.g., BRL3A cells), human lung cells (e.g., W138 cells), human liver cells (e.g., Hep G2 cells), mouse mammary tumor (e.g., MMT060562 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 epidermoid laryngeal cells, human fibroblasts, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocyte cells, human macrophage cells, human RAW264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells. In some embodiments, the recombinant cells are derived from the above-mentioned cells (i.e., derivatives of the original cells described herein), such as cells propagated from clones of the original cells, engineered forms of the original cells, or reassortments of the original cells after extensive passaging or passaging through another host.

[0099] In some embodiments, the recombinant cell is an insect cell, e.g., a cell of an insect cell line. In some embodiments, the recombinant cell is an Sf21 cell. Additional suitable insect cell lines include, but are not limited to, cell lines established from insects of the orders Diptera, Lepidoptera, and Hemiptera, and can be obtained from a variety of tissue sources. In some embodiments, the recombinant cell is a cell of a lepidopteran insect cell line. Over the past few decades, the availability of lepidopteran insect cell lines has increased by approximately 50 lines per decade. More information on available lepidopteran insect cell lines can be found, for example, in Lynn DE, Available lepidopteran 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, i.e., cells of mosquito species within the genera Anopheles (An.), Culex (Cx.), and Aedes (Slegomyia) (Ae.). Exemplary mosquito cell lines suitable for the compositions and methods described herein include cell lines from the following mosquito species: Aedes aegypti, Aedes albopictus, Aedes pseudoscutellaris, Aedes triseriatus, Aedes vexans, Anopheles gambiae, Anopheles stephensi, Anopheles albimanus, Culex quinquefasciatus, Culex theileri, Culex tritaeniorhynchus, Culex bitaeniorhynchus, and Culex quinquefasciatus. bitaeniorhynchus, and Toxorhynchites amboinensis.Suitable mosquito cell lines include, but are not limited to, CCL-125, Aag-2, RML-12, C6 / 26, C6 / 36, C7-10, AP-61, AtGRIP-1, AtGRIP-2, UM-AVE1, Mos.55, Sua1B, 4a-3B, Mos.43, MSQ43, and LSB-AA695BB. In some embodiments, the mosquito cells are cells of the C6 / 26 cell line.

[0100] In another aspect, a cell culture is provided comprising at least one recombinant cell disclosed herein and a culture medium. Generally, the culture medium can be any culture medium suitable for culturing the cells described herein. Techniques for transforming the wide variety of host cells and species described above are known in the art and described in the technical and scientific literature. Accordingly, 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.

[0101] C. Transgenic Animals In another aspect, transgenic animals comprising a nucleic acid construct (e.g., a vector, replicon, or srRNA molecule) 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 animal of the present disclosure is any non-human animal known in the art. In some embodiments, the non-human animal of the present disclosure is a non-human primate. Other animal species suitable for the compositions and methods of the present disclosure include animals that are (i) suitable for gene transfer and (ii) capable of rearranging immunoglobulin gene segments to generate an antibody response. Examples of such species include, but are not limited to, mice, rats, hamsters, rabbits, chickens, goats, pigs, sheep, and cows. Additional examples of non-human animals suitable for the compositions and methods of the present disclosure include, but are not limited to, laboratory animals (e.g., mice, rats, hamsters, gerbils, guinea pigs, etc.), farm animals (e.g., cows, horses, sheep, goats, ducks, geese, chickens, etc.), poultry and pets (e.g., cats, dogs, etc.), non-human primates (e.g., apes, chimpanzees, orangutans, monkeys, etc.), fish, amphibians (e.g., frogs, salamanders, etc.), reptiles (e.g., snakes, lizards, etc.), and other animals (e.g., foxes, weasels, rabbits, mink, beavers, ermine, otters, sables, seals, coyotes, chinchillas, deer, muskrats, possums, etc.).

[0102] 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 present disclosure is a chimeric transgenic animal. In some embodiments, the transgenic animal of the present disclosure is a transgenic animal having germ cells and somatic cells that comprise one or more (e.g., one or more, two or more, three or more, four or more) nucleic acid constructs of the present disclosure. In some embodiments, the one or more nucleic acid constructs are stably integrated into the genome of the transgenic animal. In some embodiments, the genome of the transgenic animal of the present disclosure can comprise any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more copies of one or more nucleic acid constructs of the present disclosure.

[0103] Techniques and methods for producing transgenic non-human animals are known in the art. Exemplary methods include pronuclear microinjection, DNA microinjection, lentiviral vector-mediated DNA transfer and sperm-mediated gene transfer into early embryos, adenovirus-mediated DNA transfer into animal sperm (e.g., pigs), retroviral vectors (e.g., avian species), and somatic cell nuclear transfer (e.g., goats). The state-of-the-art in producing transgenic domestic animals is reviewed in Niemann, H. et al. (2005) Rev. Sci. Tech. 24:285-298. In some embodiments, the transgenic non-human host animals of the present 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, transgenic animals of the present disclosure can be generated using classical random genome recombination techniques, or more precise techniques such as guide RNA-directed CRISPR / Cas genome editing, DNA-guided endonuclease genome editing with NgAgo (Natronobacterium gregoryi Argonaute), or TALEN genome editing (transcription activator-like effector nuclease). In some embodiments, transgenic animals of the present disclosure can be generated using transgenic microinjection techniques, which do not require the use of homologous recombination techniques and are therefore believed to be easier to prepare and select than approaches using homologous recombination. In some embodiments, the transgenic animals produce a protein of interest as described herein.

[0104] The transgenic non-human host animals of the present disclosure are produced using standard methods known in the art for introducing exogenous nucleic acids into the genome of a non-human animal. In some embodiments, the non-human animal of the present disclosure is a non-human primate. Other animal species suitable for the compositions and methods of the present disclosure include animals that are (i) suitable for gene transfer and (ii) capable of rearranging immunoglobulin gene segments to generate an antibody response. Examples of such species include, but are not limited to, mice, rats, hamsters, rabbits, chickens, goats, pigs, sheep, and cows. Techniques and methods for producing transgenic non-human animals are known in the art. Exemplary methods include pronuclear microinjection, DNA microinjection, lentiviral vector-mediated DNA transfer and sperm-mediated gene transfer into early embryos, adenovirus-mediated DNA transfer into animal sperm (e.g., pigs), retroviral vectors (e.g., avian species), and somatic cell nuclear transfer (e.g., goats). The state of the art in the production of transgenic domestic animals is reviewed in Niemann, H. et al. (2005) Rev. Sci. Tech. 24:285-298.

[0105] 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 the present disclosure can be generated using classical random genome recombination techniques, or more precise techniques such as guide RNA-directed CRISPR / Cas genome editing, or DNA-guided endonuclease genome editing with NgAgo (Natronobacterium gregorii argonaute), or TALENs genome editing (Transcription Activator-Like Effector Nucleases). In some embodiments, the transgenic animals of the present disclosure can be generated using transgenic microinjection techniques, which do not require the use of homologous recombination techniques and are therefore believed to be easier to generate and select for than approaches using homologous recombination. In another aspect, the present invention provides a method of producing a polypeptide of interest, comprising (i) raising a transgenic animal as disclosed herein, or (ii) culturing a recombinant cell comprising a nucleic acid construct as disclosed herein, under conditions such that the transgenic animal or recombinant cell produces the polypeptide encoded by the GOI. In another aspect, the present invention provides a method of producing a polypeptide of interest in a subject, comprising administering to the subject a nucleic acid construct as 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 mammal. In some embodiments, the mammalian subject is a human subject. Thus, recombinant polypeptides produced by the methods disclosed herein are also within the scope of the present invention.

[0106] Non-limiting exemplary embodiments of the disclosed methods for producing a recombinant polypeptide can include one or more of the following features: In some embodiments, the methods for producing a recombinant polypeptide of the present disclosure further include isolating and / or purifying the produced polypeptide. In some embodiments, the methods for producing a polypeptide of the present disclosure further include structurally modifying the produced polypeptide to increase half-life.

[0107] D. Pharmaceutical Compositions The nucleic acid constructs, recombinant cells, and recombinant polypeptides of the present invention can be incorporated into compositions (including pharmaceutical compositions). Such compositions generally comprise one or more of the nucleic acid constructs, recombinant cells, and recombinant polypeptides described and provided herein and a pharmaceutically acceptable excipient, e.g., a carrier. In some embodiments, the compositions of the present disclosure are formulated for the prevention, treatment, or management of a health condition, such as an immune disorder or a microbial infection (e.g., a viral infection, a microfungal infection, or a bacterial infection). For example, the compositions of the present disclosure can be formulated as a prophylactic composition, a therapeutic composition, or a pharmaceutical composition comprising a pharmaceutically acceptable excipient, or a mixture thereof. In some embodiments, the compositions of the present disclosure are formulated for use as a vaccine. In some embodiments, the compositions of the present application are formulated for use as an adjuvant.

[0108] Thus, in one aspect, provided herein is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a) a nucleic acid construct of the present disclosure, b) a recombinant cell of the present disclosure, and / or c) a recombinant polypeptide of the present disclosure.

[0109] Non-limiting exemplary embodiments of pharmaceutical compositions of the present disclosure can include one or more of the following features: In some embodiments, provided herein are compositions comprising a nucleic acid construct disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, provided herein are compositions comprising a recombinant cell disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, a composition comprises a recombinant polypeptide disclosed herein and a pharmaceutically acceptable excipient.

[0110] In some embodiments, the nucleic acid constructs (e.g., vectors or srRNA molecules) of the present disclosure can be used in naked form or can be formulated with a delivery vehicle. Exemplary delivery vehicles suitable for the compositions and methods of the present disclosure include, but are not limited to, liposomes (e.g., neutral or anionic liposomes), microspheres, immunostimulating complexes (ISCOMS), lipid-based nanoparticles (LNPs), solid lipid nanoparticles (SLNs), polyplexes, polymeric nanoparticles, viral replicon particles (VRPs), or conjugates 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 and the like are also used and are known in the art. Adjuvants may include substances that protect antigens (e.g., nucleic acid constructs, vectors, srRNA molecules) from rapid diffusion by sequestering the antigen in local deposits or that stimulate the host to secrete chemotactic factors for macrophages or other immune system components.

[0111] The compositions of the present disclosure can be formulated in a format compatible with the intended route of administration, such as liposomes, lipid-based nanoparticles (LNPs), polymeric nanoparticles, polyplexes, viral replicon particles (VRPs), microspheres, immune stimulating complexes (ISCOMs), conjugates of bioactive ligands, or any combination thereof. Thus, in some embodiments, the compositions of the present disclosure are formulated in liposomes.

[0112] In some embodiments, the compositions of the present disclosure are formulated in lipid-based nanoparticles (LNPs). Exemplary lipid types suitable for the delivery systems described herein include cationic lipids, ionizable cationic lipids, anionic lipids, neutral lipids, and combinations thereof.

[0113] In some embodiments, the LNPs of the present disclosure can comprise one or more ionizable lipids. Exemplary ionizable lipids suitable for the compositions and methods of the present disclosure include those described in PCT Publications WO2020252589A1 and WO2021000041A1 and Love KT et al., Proc Natl Acad Sci USA, February 2, 2010 107(5)1864-1869, which are incorporated by reference in their entireties.

[0114] Thus, in some embodiments, LNPs of the present disclosure comprise one or more lipid compounds described in Love KT et al., 2010, supra, such as C16-96, C14-110, and C12-200. In some embodiments, LNPs comprise 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, LNPs of the present disclosure comprise C12-200.

[0115] In some embodiments, LNPs of the present 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.

[0116] In some embodiments, the LNPs of the present disclosure comprise one or more neutral lipids. As noted above, in some embodiments, neutral lipids, also known as "structural lipids" or "helper lipids," can also be incorporated into lipid formulations and lipid particles. Lipid formulations and lipid particles can comprise one or more structured lipids at about 10-40 mol % of the composition. Suitable structured lipids support particle formation during manufacturing. A structured lipid refers to any one of a number of lipid species that exist in either anionic, uncharged, or neutral zwitterionic form at physiological pH. Exemplary structured lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, diacylphosphatidylglycerol, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0117] Exemplary structured lipids include zwitterionic lipids, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-ol. These include carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (trans DOPE).

[0118] In another embodiment, the structured lipid can be any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerols such as dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoylphosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, and other anionic modifying groups attached to neutral lipids. Other suitable structured lipids include glycolipids (e.g., monosialoganglioside GM1).

[0119] Non-limiting neutral lipids suitable for the compositions and methods of the present disclosure include DPSC, DPPC, POPC, DOPE, and SM. In some embodiments, the LNPs of the present disclosure comprise one or more ionizable lipid compounds described in PCT Publications WO2020252589A1 and WO2021000041A1, which are incorporated by reference in their entireties.

[0120] In some embodiments, the LNPs of the present disclosure comprise 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-DMRIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG).

[0121] In some embodiments where the delivery systems described herein comprise LNPs, the lipid to nucleic acid mass ratio 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 lipid to nucleic acid mass ratio in the LNP delivery system is about 16:1 to 4:1. In some embodiments, the lipid to nucleic acid mass ratio in the LNP delivery system is about 20:1. In some embodiments, the lipid to nucleic acid mass ratio in the LNP delivery system is about 8:1. In some embodiments, the lipid-based nanoparticles (LNPs) have an average diameter of less than about 1000 nm, less than about 500 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm, less than about 100 nm, less than about 75 nm, less than 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 LNPs have an average diameter ranging from about 88 nm to about 92 nm, 82 nm to about 86 nm, or about 80 nm to about 95 nm.

[0122] To ensure the integrity of the mixture, stabilizers can be included in the lipid formulation. Stabilizers are a type of molecule that disrupts or promotes hydrophobic-hydrophilic interactions between molecules. 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]ethyl octadec-9-enoate), Myrj 52 (polyoxyethylene (40) stearate), and Brij™ S10 (polyoxyethylene (10) stearyl ether). Polyethylene glycol-conjugated lipids can also be used. Stabilizers can be used alone or in combination with each other.

[0123] In some embodiments, the stabilizer comprises about 0.1-3 mol% of the total lipid mixture. In some embodiments, the stabilizer comprises about 0.5-2.5 mol% of the total lipid mixture. In some embodiments, the stabilizer is present at greater 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 present at 2.6-10 mol% of the lipid mixture. In other embodiments, the stabilizer is present at greater than 10 mol% of the lipid mixture.

[0124] Steroids can also be included in the lipid composition for certain applications, and lipid particles produced therefrom contain sterols, such as cholesterol and plant sterols.

[0125] In some embodiments, the compositions of the present disclosure are formulated in polymeric nanoparticles. Examples of polymers suitable for the compositions and methods of the present disclosure can be found in Jiang X et al. (Polymeric nanoparticles for RNA delivery. Encyclopedia of Nanomaterials, 2021), which is incorporated herein by reference. Exemplary polymers suitable for the compositions and methods of the present disclosure include cationic polymers, non-cationic polymers, and combinations thereof. In some embodiments, the polymeric nanoparticles of the present disclosure may comprise naturally occurring cationic polymers. In some embodiments, naturally occurring cationic polymers may include chitosan, gelatin, dextran, cellulose, cyclodextrin, or combinations thereof. In some embodiments, the cationic polymer may be a synthetic cationic polymer. In some embodiments, synthetic cationic polymers can include polyethyleneimine (PEI), poly-L-lysine (PLL), poly(amino acids) (PAA), poly(amidoamine) (PAMAM), poly(amino-co-ester) (PAE), poly(2-N,N-dimethylaminoethyl methacrylate), poly(beta amino ester) (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.

[0126] In some embodiments, the non-cationic polymer is negatively charged (i.e., anionic) or electronically neutral. In some embodiments, the non-cationic polymer comprises 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.

[0127] In some embodiments, the polymer may be water soluble and / or biodegradable. In some embodiments, the polymeric nanoparticles comprise one or more of 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, polypropylfumerate, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, [N-(2-hydroxypropyl)methacrylamide] (HPMA) copolymer, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polyurea, polyamine polyepsilon-caprolactone (PCL), and copolymers thereof.

[0128] In some embodiments, the composition is an immunogenic composition, e.g., a composition that can stimulate an immune response in a subject. In some embodiments, the immunogenic composition is formulated as a vaccine. In some embodiments, the pharmaceutical composition is formulated as an adjuvant.

[0129] In some embodiments, the immunogenic composition is formulated as a vehicle for the gene delivery of biological therapeutic agents, such as different biologically active molecules. Non-limiting examples of biological therapeutic agents include cytokines, chemokines, and other soluble immunomodulators, enzymes, peptide and protein agonists, peptide and protein antagonists, hormones, receptors, antibodies and antibody derivatives, growth factors, transcription factors, and gene silencing / editing molecules. In some embodiments, the pharmaceutical composition is formulated as an adjuvant.

[0130] 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 biotherapeutic. In some embodiments, the pharmaceutical composition is formulated for one or more of intranasal, transdermal, intrathecal, intraperitoneal, intramuscular, intratracheal, intranodal, intratumoral, intraarticular, intravenous, subcutaneous, intravaginal, intraocular, rectal, and oral administration.

[0131] Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In these cases, the composition must be sterile and fluid to the extent that easy syringability exists. The composition can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants such as sodium dodecyl sulfate. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is common to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and / or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0132] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent containing one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0133] In some embodiments, the pharmaceutical compositions of the present disclosure are formulated for inhalation, such as an aerosol, spray, mist, liquid, or powder. Administration by inhalation may be in the form of either a dry powder or an aerosol formulation, which is inhaled by a subject (e.g., a patient) using an inhalation device, such as a microspray, pressurized metered dose inhaler, or nebulizer.

[0134] In some embodiments, the composition is formulated for one or more of intranasal, intrathecal, transdermal, intramuscular, intranodal, intravenous, intraperitoneal, oral, intravaginal, or intracranial administration. In some embodiments, the administered composition increases interferon production in the subject. In some embodiments, the administered composition induces the production of one or more pro-inflammatory molecules in the subject. In some embodiments, the one or more pro-inflammatory molecules include interferon gamma (IFNγ), cytokines, TNF-α, GM-CSF, and MIP1α, granzyme B, granzyme A, perforin, or any combination thereof.

[0135] Methods of the present disclosure Administration of any one of the therapeutic compositions described herein, e.g., nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions, can be used to treat related health conditions, such as proliferative disorders (e.g., cancer), infectious diseases (e.g., acute, chronic, or viral infections), rare diseases, and / or autoimmune and / or inflammatory diseases. In some embodiments, the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions described herein can be useful for modulating, e.g., inducing or suppressing, a pharmacodynamic effect in a subject in need thereof. In some embodiments, the pharmacodynamic effect includes eliciting an immune response in the subject. Non-limiting examples of pharmacodynamic effects include immunogenic effects, biomarker responses, therapeutic effects, prophylactic effects, desired effects, undesired effects, adverse effects, and effects in disease models. Accordingly, one aspect of the present disclosure relates to a method for modulating a pharmacodynamic effect in a subject in need thereof, the method comprising administering to the subject a composition comprising one or more of: (a) a nucleic acid construct described herein, (b) a recombinant cell described herein, (c) a recombinant polypeptide described herein, and (d) a pharmaceutical composition described herein, in some embodiments, the pharmacodynamic effect includes one or more of an immunogenic effect, a biomarker response, a therapeutic effect, a prophylactic effect, a desired effect, an undesired effect, an adverse effect, and an effect in a disease model.

[0136] In some embodiments, the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions described herein can be incorporated into therapeutic agents for use in methods of treating a subject who may have, be suspected of having, or be at high risk for developing one or more relevant health conditions or diseases. Accordingly, in another aspect, provided herein are methods of preventing or treating a health condition in a subject, comprising prophylactically or therapeutically administering to the subject a composition comprising one or more of: (a) a replicon, e.g., a self-replicating RNA construct (srRNA), described herein; (b) a nucleic acid described herein; (c) a recombinant cell described herein; and (d) a pharmaceutical composition described herein. In some embodiments, the administered composition induces an immune response in the subject. In some embodiments, the administered composition induces the production of one or more pro-inflammatory molecules in the subject. In some embodiments, the one or more pro-inflammatory molecules include 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 said one or more therapies.

[0137] Examples of conditions or diseases include, but are not limited to, cancer, immune diseases, autoimmune diseases, inflammatory diseases, gene therapy, gene replacement, cardiovascular diseases, age-related conditions, rare diseases, acute infections, and chronic infections. In some embodiments, the subject is a patient under the care of a physician.

[0138] Examples of autoimmune diseases suitable for the methods of the present disclosure include rheumatoid arthritis, osteoarthritis, Still's disease, familial Mediterranean fever, systemic sclerosis, multiple sclerosis, ankylosing spondylitis, systemic lupus erythematosus, Sjogren's syndrome, diabetic retinopathy, diabetic vasculopathy, diabetic neuralgia, insulitis, psoriasis, alopecia areata, warm-cold autoimmune hemolytic anemia (AIHA), pernicious anemia, acute inflammatory disease, autoimmune adrenalitis, chronic inflammatory demyelinating polyneuropathy (CIDP), and Lambert-Eaton syndrome. syndrome, lichenoid sclerosis, Lyme disease, Graves' disease, Behcet's disease, Meniere's disease, reactive arthritis (Reiter's syndrome), Churg-Strauss syndrome, Cogan's syndrome, CREST syndrome, pemphigus vulgaris and pemphigus foliaceus, bullous pemphigoid, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, pancreatitis, peritonitis, psoriatic arthritis, rheumatic fever, sarcoidosis, Sjögren's syndrome, scleroderma, celiac disease, stiff-man syndrome syndrome), Takayasu's arteritis, transient gluten intolerance, autoimmune uveitis, vitiligo, polychondritis, dermatitis herpetiformis (DH) or Juhring's disease, fibromyalgia, Goodpasture's syndrome, Guillain-Barré syndrome, Hashimoto's thyroiditis, autoimmune hepatitis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, myasthenia gravis, immune complex disorders, glomerulonephritis, polyarteritis nodosa, antiphospholipid syndrome, autoimmune polyglandular syndrome, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), urticaria, autoimmune infertility, juvenile rheumatoid arthritis, sarcoidosis, and autoimmune cardiomyopathy.

[0139] Non-limiting examples of infections suitable for the methods of the present disclosure include infections caused by viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus (CMV), respiratory syncytial virus (RSV), human papillomavirus (HPV), Epstein-Barr virus (EBV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome (MERS), influenza virus, and Ebola virus. Additional infections suitable for the methods of the present disclosure include infections caused by intracellular parasites such as Leishmania, Rickettsia, Chlamydia, Coxiella, Plasmodium, Brucella, Mycobacteria, Listeria, Toxoplasma, and Trypanosoma. In some embodiments, the replicon constructs, srRNA constructs, nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions are used to treat an immune disease, autoimmune disease, or inflammatory disease, such as glomerulonephritis, inflammatory bowel disease, nephritis, peritonitis, psoriatic arthritis, osteoarthritis, Still's disease, familial Mediterranean fever, systemic sclerosis and sclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, acute lung injury, meningitis, encephalitis, uveitis, multiple myeloma, glomerulonephritis, nephritis, asthma, atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud's syndrome, Sjogren's syndrome, juvenile onset diabetes, Reiter's syndrome, Behcet's disease, immune complex nephritis, IgA nephropathy ... It may be useful for the treatment and / or prevention of gM polyneuropathy, immune-mediated thrombocytopenia, hemolytic anemia, myasthenia gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis (RA), ankylosing spondylitis, pemphigus, Graves' disease, Hashimoto's thyroiditis, small-vessel vasculitis, Omenn's syndrome, chronic renal failure, autoimmune thyroid disease, acute infectious mononucleosis, HIV, herpesvirus-related diseases, human viral infections, coronavirus, other enterovirus, herpesvirus, influenza virus, parainfluenza virus, respiratory syncytial virus, or adenovirus infection, bacterial pneumonia, wounds, sepsis, stroke / cerebral edema, ischemia-reperfusion injury, and hepatitis C.

[0140] Non-limiting examples of inflammatory diseases suitable for the methods of the present disclosure include inflammatory diseases such as asthma, inflammatory bowel disease (IBD), chronic colitis, splenomegaly, and rheumatoid arthritis.

[0141] In some embodiments, the condition is a proliferative disorder or a microbial infection (e.g., a bacterial infection, a microfungal infection, or a viral infection). In some embodiments, the subject has or is suspected of having a condition associated with a proliferative disorder or a microbial infection (e.g., a bacterial infection, a microfungal infection, or a viral infection).

[0142] In some embodiments, the health condition is a rare disease, e.g., a disease or condition affecting fewer than 200,000 people in the United States as defined by the Orphan Drug Act (www.fda.gov / patients / rare-diseases-fda), and / or an inflammatory disease and / or an autoimmune disease. In some embodiments, the subject has or is suspected of having symptoms associated with an inflammatory disease and / or an autoimmune disease and / or a rare disease (e.g., including, but not limited to, familial Mediterranean fever or adult-onset Still's disease).

[0143] In some embodiments, compositions of the present disclosure are formulated to be compatible with the intended route of administration. For example, nucleic acid constructs (e.g., replicon constructs, e.g., srRNA constructs), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure may be given orally or by inhalation, but are more likely to be administered by parenteral routes. Examples of parenteral administration routes include, for example, intramuscular, intratumoral, intraocular, intravenous, intranodal, intradermal, subcutaneous, transdermal (topical), transmucosal, intravaginal, and rectal administration. In some embodiments, the composition is administered intramuscularly. In some embodiments, the composition is administered intratumorally. Solutions or suspensions used for parenteral administration contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citrate, phosphate, Tris, or sucrose; and a tonicity adjuster such as sodium chloride or dextrose. The pH can be adjusted (e.g., to about pH 7.2 to 7.8, e.g., 7.5) using an acid or base such as monobasic and / or dibasic sodium phosphate, hydrochloric acid, or sodium hydroxide. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials.

[0144] Dosage, toxicity, and therapeutic efficacy of such subject nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure may be determined, for example, by the LD 50 (a lethal dose for 50% of the population) and ED 50 The LD (the dose therapeutically effective in 50% of the population) can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50It can be expressed as a ratio. Compounds that exhibit a high therapeutic index are generally preferred. Compounds that exhibit toxic side effects can be used, but care must be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells, thereby reducing side effects.

[0145] For example, the data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds is generally determined to be within the range of ED, with little or no toxicity. 50 The circulating concentration of the compound is within a range including the IC of the compound. The dosage may vary within this range depending on the dosage form and route of administration used. For any compound used in the methods of the present disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. The IC of the compound measured in cell culture 50 A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes (e.g., the concentration of the test compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0146] Therapeutic compositions, e.g., nucleic acid constructs, e.g., srRNA constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions described herein, can be administered from one or more times daily to one or more times weekly (including once every other day). One of skill in the art will understand that certain factors, including, but not limited to, the severity of the disease, previous treatments, the subject's general health and / or age, and other diseases present, can affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of the subject multivalent polypeptides and multivalent antibodies of the present disclosure can include a single treatment or can include 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, e.g., 9 days, followed by administration every 8 hours for an additional 5 days. With respect to nucleic acid constructs (e.g., replicon constructs, e.g., srRNA constructs) and recombinant polypeptides, the therapeutically effective amount (e.g., effective dosage) of the nucleic acid construct or recombinant polypeptide of the present disclosure will vary depending on the nucleic acid construct or recombinant polypeptide selected. For example, a single dose may be administered in the range of about 0.001 to 0.1 mg / kg of patient body weight. In some embodiments, about 0.005, 0.01, or 0.05 mg / kg may be administered. In some embodiments, a single dose may be administered in the range of about 0.03 μg to 300 μg / kg of patient body weight. In some embodiments, a single dose may be administered in the range of about 0.3 mg to 3 mg / kg of patient body weight.

[0147] As discussed above, a therapeutically effective amount includes an amount of a therapeutic composition sufficient to promote a particular effect when administered to a subject having, suspected of having, or at risk for a health condition, e.g., a disease or infection. In some embodiments, an effective amount includes an amount sufficient to prevent or delay the onset of symptoms of a disease or infection, alter the course of symptoms of a disease or infection (e.g., without limitation, delay the progression of symptoms of a disease or infection), or reverse symptoms of a disease or infection. It will be understood that the appropriate effective amount in any given case can be determined by one of ordinary skill in the art using routine experimentation.

[0148] The efficacy of a treatment involving the disclosed therapeutic compositions for the treatment of a disease or infection can be determined by a skilled physician. However, a treatment is considered effective if at least one or all of the signs and symptoms of the disease or infection are improved or alleviated. Efficacy can also be measured by the individual not getting worse (e.g., the progression of the disease or infection is stopped or at least slowed) as assessed by the need for hospitalization or medical intervention. Methods for measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease or infection in a subject or animal (some non-limiting examples include humans or mammals), including (1) inhibiting the disease or infection, e.g., halting or slowing the progression of symptoms, or (2) relieving the disease or infection, e.g., causing regression of symptoms, and (3) preventing or reducing the likelihood of onset of symptoms.

[0149] In some embodiments, the nucleic acid constructs (e.g., replicon constructs, e.g., srRNA constructs), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure can be administered to a subject in a composition with a pharmaceutically acceptable carrier and in an amount effective to stimulate an immune response. Generally, a subject is immunized with an initial series of injections (or administration by any of the other routes described below), after which boosters can be provided to enhance the protection provided by the initial series. The initial series of injections and subsequent boosters are administered in amounts and for periods of time necessary to stimulate an immune response in the subject. In some embodiments, the administered composition increases the production of interferon in the subject. In some embodiments of the disclosed methods, the subject is a mammal. In some embodiments, the mammal is a human subject.

[0150] As mentioned above, pharmaceutically acceptable carriers suitable for injection include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In these cases, the compositions must be sterile and must be fluid to the extent that easy syringability exists. The compositions must also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.

[0151] Sterile injectable solutions can be prepared by incorporating the nucleic acid constructs, recombinant cells, and / or recombinant polypeptides in the required amount in an appropriate solvent containing one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

[0152] When the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions are suitably protected, as described above, they may be orally administered, for example, with an inert diluent or a digestible edible carrier. The nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the individual's diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0153] In some embodiments, the nucleic acid constructs and recombinant polypeptides of the present disclosure can be delivered to cells or subjects via lipid-based nanoparticles (LNPs). LNPs are generally less immunogenic than viral particles. Many people have pre-existing immunity to viral particles, but not to LNPs. Furthermore, adaptive immune responses to LNPs are unlikely to occur, allowing for repeated administration of LNPs.

[0154] Several different ionizable cationic lipids have been developed for use in LNPs. These include, among others, C12-200, MC3, LN16, and MD1. For example, in one type of LNP, a GalNAc moiety is attached to the exterior of the LNP, which serves as a ligand for uptake into the liver via the asialoglycoprotein receptor. Any of these cationic lipids can be used to formulate LNPs for delivery of the nucleic acid constructs and recombinant polypeptides of the present invention to the liver.

[0155] In some embodiments, LNP refers to any particle having a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. Alternatively, nanoparticles can be in the size range of 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

[0156] LNPs can be made from cationic, anionic, or neutral lipids. Neutral lipids, such as the membrane-fusogenic phospholipid DOPE or the membrane component cholesterol, can be included in LNPs as "helper lipids" to enhance transfection activity and nanoparticle stability. Limitations of cationic lipids include low efficacy due to low stability and rapid clearance, and the generation of inflammatory or anti-inflammatory responses. LNPs can also contain hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.

[0157] Numerous lipids or lipid combinations known in the art can be used to produce LNPs. Non-limiting examples of lipids suitable for use in producing LNPs include DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Non-limiting examples of cationic lipids include 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. 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.

[0158] In some embodiments, lipids can be combined in any number of molar ratios to form LNPs. Additionally, polynucleotides can be combined with lipids in a wide range of molar ratios to form LNPs.

[0159] In some embodiments, the therapeutic compositions described herein, e.g., nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions, are incorporated into therapeutic compositions for use in methods of preventing or treating subjects who may have, be suspected of having, or be at high risk for developing cancer, an autoimmune disease, and / or an infectious disease.

[0160] In some embodiments, the therapeutic compositions described herein, e.g., nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions, are incorporated into therapeutic compositions for use in methods of preventing or treating a subject who may have, be suspected of having, or be at high risk for developing a microbial infection. In some embodiments, the microbial infection is a bacterial infection. In some embodiments, the microbial infection is a fungal infection. In some embodiments, the microbial infection is a viral infection.

[0161] Additional therapy In some embodiments, a composition according to the present disclosure is administered to a subject individually as a single therapy (monotherapy) or as a first therapy in combination with at least one additional therapy (e.g., a second therapy). In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiation therapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery. In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiation therapy, immunotherapy, hormone therapy, toxin therapy, or surgery. In some embodiments, the first therapy and the second therapy are administered concomitantly. In some embodiments, the first therapy is administered at the same time as the second therapy. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy is administered before the second therapy. In some embodiments, the first therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and / or after the second therapy. In some embodiments, the first therapy and the second therapy are administered alternatingly. In some embodiments, the first and second therapies are administered together in a single formulation.

[0162] kit Also provided herein are various kits for carrying out the methods described herein, as well as instructions for making and using the same. In particular, some embodiments of the present disclosure provide kits for modulating a pharmacodynamic effect. Some embodiments of the present disclosure provide kits for inducing an immune response in a subject. Other embodiments relate to kits for preventing a condition in a subject in need thereof. Other embodiments relate to kits for methods of treating a condition in a subject in need thereof. For example, in some embodiments, provided herein are kits that include one or more of the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions provided and described herein, and instructions for making and using them.

[0163] In some embodiments, the kits of the present disclosure further comprise one or more means useful for administering any one of the provided nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions to a subject. For example, in some embodiments, the kits of the present disclosure further comprise one or more syringes (including pre-filled syringes) and / or catheters (including pre-filled syringes) used to administer any one of the provided nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions to a subject. In some embodiments, the kits can have one or more additional therapeutic agents that can be administered simultaneously or sequentially with other kit components for a desired purpose, e.g., to diagnose, prevent, or treat a condition in a subject in need thereof.

[0164] Any of the above kits can further comprise one or more additional reagents, which can be selected from dilution buffers, reconstitution solutions, wash buffers, control reagents, control expression vectors, negative controls, positive controls, reagents suitable for producing in vitro the provided nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure.

[0165] In some embodiments, the components of the kit may be in separate containers. In other embodiments, the components of the kit may be combined in a single container. Thus, in some embodiments of the present disclosure, the kit includes one or more of the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions provided and described herein in one container (e.g., in a sterile glass or plastic vial) and an additional therapeutic agent in another container (e.g., in a sterile glass or plastic vial).

[0166] In another embodiment, the kit comprises, in a single common container, a combination of compositions described herein, including one or more nucleic acid constructs, recombinant cells, and / or recombinant polypeptides of the present disclosure in combination with one or more additional therapeutic agents, optionally formulated together in a pharmaceutical composition.

[0167] Where the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can include a device for performing such administration (e.g., an injection device or catheter). For example, the kit can include one or more hypodermic needles or other injection devices discussed above that contain one or more nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure.

[0168] In some embodiments, the kit components may be in separate containers. In other embodiments, the kit components may be combined in a single container.

[0169] In some embodiments, the kit can further include instructions for using the kit components to perform the methods disclosed herein. For example, the kit can include a package insert containing information about the pharmaceutical compositions and dosage forms in the kit. Generally, such information will assist patients and physicians in the effective and safe use of the enclosed pharmaceutical compositions and dosage forms. For example, the following information about the combinations of the present disclosure may be provided in the package insert: pharmacokinetics, pharmacodynamics, clinical trials, efficacy evaluation criteria, indications and usage, contraindications, warnings, precautions, side effects, overdose, appropriate dosage and method of administration, method of supply, appropriate storage conditions, references, manufacturer / distributor information, and intellectual property information.

[0170] The instructions for carrying out the 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 provided in the kit as a package insert, on a label on the container of the kit or its components (e.g., associated with the packaging or subpackaging). The instructions can be present as an electronic storage data file on a suitable computer-readable storage medium, such as a CD-ROM, diskette, flash drive, or the like. In some cases, the actual instructions are not provided in 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 where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.

[0171] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0172] No admission is made that any reference cited herein constitutes prior art. The discussion of references states the assertions of their authors, and applicants reserve the right to challenge the accuracy and pertinence of the cited documents. Although numerous sources of information have been referenced herein, including scientific journal articles, patent documents, and textbooks, it will be expressly understood that this reference is not an admission that any of these documents form part of the general knowledge in the art.

[0173] The general method discussion provided herein is intended for illustrative purposes only. Other alternative methods and means will be apparent to those of skill in the art upon review of this disclosure and are intended to be within the spirit and scope of this application.

[0174] Additional embodiments are disclosed in more detail in the following examples, which are provided for illustrative purposes and are not intended to limit the scope of the disclosure or claims in any way. [Example]

[0175] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are 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.). “Sambrook”);Ausubel,FM(1987).Current Protocols in Molecular Biology.New York,NY:Wiley(including supplements through 2014);Bollag,DMet al.(1996).Protein Methods.New York,NY:Wiley-Liss;Huang,L.et al.(2005).Nonviral Vectors for Gene Therapy.San Diego:Academic Press;Kaplitt,MGet 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). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publishers; Greenfield, E. A. (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 through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences BV, the disclosures of which are incorporated herein by reference.

[0176] Additional embodiments are disclosed in more detail in the following examples, which are provided for illustrative purposes and are not intended to limit the scope of the disclosure or claims in any way.

[0177] Example 1 Construction of base MADV vector This example describes experiments performed to construct a base MADV vector (e.g., containing no heterologous genes) that is subsequently used to construct MADV vectors expressing a gene of interest (e.g., the hemagglutinin precursor HA of influenza A virus H5N1).

[0178] A base MADV vector (i.e., without a heterologous gene of interest) is constructed as follows. The base MADV vector (see, e.g., Figure 2A) is synthesized de novo in four approximately 4 kb segments derived from the reference sequence of MADV strain BeAr300851 (Genbank accession number KJ469641), with some modifications. Ambiguous nucleotide assignments in the reference sequence are assigned to nucleotides corresponding to the synonymous codons for the encoded residues. Silent mutations are incorporated to remove SapI and SpeI restriction sites. A unique restriction site (SpeI, 5'-A'CTAG, T-3') (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) can be incorporated in place of the coding sequence of the native MADV structural gene. For the subsequent Gibson Assembly® procedure (Gibson et al., Nat. Methods 6, 343-345, 2009), a 5' adapter sequence (5'-CTGGAGACGTGGAGGAGAACCCTGGACCT-3'; SEQ ID NO: 3) is inserted upstream of the SpeI site, and a 3' adapter sequence (5'-GACCGCTACGCCCCAATGACCCGACCAGC-3'; SEQ ID NO: 4) is inserted downstream of the SpeI site. A bacteriophage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO: 5) is included upstream of the MADV genomic sequence, with a poly(A) sequence downstream followed by an SpeI site that cuts upstream of the recognition site. Immediately downstream of the SapI site is a T7 terminator sequence (5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO: 6), followed by a unique restriction enzyme cleavage site (NotI, 5'-GC'GGCC,GC-3'). These parts are combined in a five-piece Gibson Assembly® reaction (linearized pYL backbone and four synthetic fragments) to generate the base MADV vector.

[0179] In some experiments, one or more of the sequences encoding the nsPs are replaced with heterologous nsPs. In some other experiments, the sequences encoding one or more of the UTRs are replaced with heterologous UTRs.

[0180] Construction of MADV vectors containing heterologous genes is performed as follows: The empty MADV vector in Figure 2A is linearized by SpeI digestion to construct the MADV vector depicted in Figure 2B. The hemagglutinin (HA) gene from influenza (Genbank AY651334) was codon-optimized / refactored for human expression in silico and synthesized de novo (IDT). The synthesis product is PCR-amplified using primers that add 5' and 3' adapter sequences to the ends of the HA gene. The digestion and PCR products are joined using the Gibson Assembly® procedure to generate the final vector.

[0181] Example 2 Construction of MADV vectors containing heterologous genes Construction of MADV vectors containing one or more heterologous genes was performed as follows: The empty base MADV vector shown in Figure 2A was linearized by SpeI digestion. Two transgene cassettes were codon-optimized / refactored for human expression in silico and synthesized de novo using the 5' and 3' adapter sequences described above. The digestion and synthesis products were joined by the Gibson Assembly® procedure to generate the final vector.

[0182] The first synthetic transgene cassette was synthesized encoding the hemagglutinin (HA) gene from influenza (based on Genbank ATI21640) and used to generate the template plasmid for Rep-631 MADV srRNA, as described in Figures 5A-5D.

[0183] A second synthetic transgene cassette was synthesized encoding mouse interleukin-1 receptor antagonist (IL-1RA) (Genbank AAH18332), followed by an IRES, followed by mouse interleukin-18 binding protein (IL-18BP) (GenPept NP_001034790), to generate the template plasmid for Rep-657 MADV srRNA described in Figures 6A-6B.

[0184] Example 3 In vitro evaluation of modified MADV vectors This example describes in vitro experiments performed to assess the expression levels of the synthetic MADV srRNA constructs described in Example 1 above and to examine their differential behavior (e.g., replication and protein expression).

[0185] In these experiments, synthetic srRNA constructs derived from MADV strain BeAr300851 were designed and subsequently evaluated.

[0186] In vitro transcription. RNA was prepared by in vitro transcription with a 5' ARCA cap (HiScribe™ T7 ARCA mRNA Kit, NEB) or by uncapped transcription (HiScribe™ T7 High Yield RNA Synthesis Kit, NEB) from a SapI-linearized plasmid template using bacteriophage T7 polymerase, followed by addition of a 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, ThermoFisher Scientific).

[0187] Replication: RNA was transfected into BHK-21 or Vero cells by electroporation (e.g., 4D-Nucleofector™, Lonza). 15–20 hours after transfection, cells were fixed and permeabilized (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen) and then stained with a PE-conjugated anti-dsRNA mouse monoclonal antibody (J2, Scicons). The abundance of dsRNA+ cells and the mean fluorescence intensity (MFI) of dsRNA in individual cells were quantified by fluorescent flow cytometry.

[0188] Protein expression. RNA was transfected into BHK-21 or Vero cells by electroporation (e.g., 4D-Nucleofector™, Lonza). 15–20 hours after transfection, cells were fixed and permeabilized (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen) and stained with DyLight650-conjugated anti-HA mouse monoclonal antibody (C102, NovusBio, catalog no. NB100-65047C). The abundance of cells expressing HA protein and the mean fluorescence intensity (MFI) of HA protein in individual cells were quantified by fluorescence flow cytometry.

[0189] In some experiments, BHK-21 cells were transformed with MADV srRNA constructs. MADV srRNA without a target GOI was transfected by electroporation. 20 hours posttransfection, transformed cells were fixed, permeabilized, and co-stained with two antibodies: a PE-conjugated anti-dsRNA mouse monoclonal antibody (J2, Scicons) to quantify the abundance of dsRNA+ cells, and a DyLight650-conjugated anti-HA mouse monoclonal antibody (Cl02, NovusBio, catalog no. NB100-65047C) to quantify the abundance of HA+ cells and the expression level of the HA transgene by fluorescent flow cytometry. Positive staining of individual cells with both the anti-dsRNA and anti-GOI antibodies indicates that the modified MADV design described herein is a viable synthetic srRNA capable of RNA replication and transgene expression.

[0190] Additional experiments: BHK-21 or Vero cells are pretreated with a titration curve of recombinant interferon (IFN) prior to RNA electroporation, and the effect on vector replication and protein expression is measured using the assays described above.

[0191] In some experiments, BHK-21 cells were transformed with MADV srRNA constructs. MADV srRNA without a target GOI was transfected by electroporation. 20 hours posttransfection, transformed cells were fixed, permeabilized, and co-stained with two antibodies: a PE-conjugated anti-dsRNA mouse monoclonal antibody (J2) to quantify the abundance of dsRNA+ cells, and a DyLight650-conjugated anti-HA mouse monoclonal antibody (Cl02, NovusBio, catalog no. NB100-65047C) to quantify the abundance of HA+ cells and the expression level of the HA transgene by fluorescent flow cytometry. As shown in Figure 4, positive staining of individual cells with both the anti-dsRNA and anti-GOI antibodies indicates that the modified MADV design described herein is a viable synthetic srRNA capable of RNA replication and transgene expression.

[0192] Example 4 In vitro evaluation of modified MADV vectors This example describes in vitro experiments performed to assess relative GOI expression from the synthetic MADV srRNA constructs described above in Examples 1 and 2, as well as other srRNA constructs expressing the same GOI.

[0193] Figure 4 shows the average HA expression in cells transfected with srRNA constructs. BHK-21 cells were transfected with 200 ng of srRNA vector encoding influenza H1N1 HA by electroporation (Lonza 4D-Nucleofector). 24 hours later, the cells were fixed, permeabilized, and stained with a DyLight650-conjugated mouse monoclonal antibody (NovusBio, catalog no. NB100-65047C) that binds to H1N1 HA. Cells were analyzed by fluorescent flow cytometry, and the cellular fluorescence intensity, which corresponds to the HA expression level, was quantified. The average HA expression in cells transfected with MADV-HA was similar or higher than that of other srRNA vectors encoding the same transgene. VEEV: Venezuelan equine encephalitis virus, CHIKV-S27: Chikungunya virus strain S27, CHIKV-DRDE: Chikungunya virus strain DRDE-06, SINV-G: Sindbis virus Girdwood strain, SINV-AR86: Sindbis virus strain AR86, WEEV: Western equine encephalitis virus, MADV: Madaraga virus strain BeAr300851.

[0194] Example 5 In vivo evaluation of modified MADV vectors This example describes in vivo experiments performed to evaluate immune responses following vaccination with the synthetic MADV srRNA constructs described in Examples 1 and 2 above (e.g., both unformulated and LNP-formulated vectors).

[0195] In these experiments, synthetic srRNA constructs derived from MADV strain BeAr300851 will be designed and subsequently evaluated.

[0196] Mice and Injections. Female C57BL / 6 or BALB / c mice are purchased from Envigo, Charles River Labs, or Jackson Laboratories. On the day of dosing, 0.1–10 μg of material is injected intramuscularly in split doses into both quadriceps muscles. Vectors are administered unformulated in saline, or formulated as LNPs or polymers. Animals are monitored for weight and other general observations throughout the course of the study. For immunogenicity studies, animals are dosed on days 0 and 21. Spleens are harvested on day 35, and serum is isolated on days 14 and 35. For protein expression studies, animals are dosed on day 0, and protein expression or bioluminescence is assessed on days 1, 3, and / or 7. In some experiments, where the MADV srRNA construct encodes a reporter protein such as luciferase, in vivo imaging of luciferase activity is performed at designated time points using an IVIS system. In some experiments where the MADV srRNA construct encodes a secreted protein, systemic levels are measured by serum ELISA.

[0197] LNP Formulation. In some experiments, srRNA is formulated into lipid nanoparticles using a microfluidic mixer and analyzed for particle size, polydispersity using dynamic light scattering, and encapsulation efficiency. In these experiments, a wide range of lipid molar ratios are used to formulate the LNP particles. An exemplary lipid molar ratio used in these experiments can be 35% C12-200, 46.5% cholesterol, 2.5% PEG-2K, and 16% DOPE.

[0198] ELISpot. To measure the magnitude of HA-specific T cell responses, IFNγ ELISpot analysis is performed using the Mouse IFNγ ELISpot PLUS Kit (HRP) (MabTech) according to the manufacturer's instructions. For these experiments, splenocytes are isolated and resuspended in medium containing a peptide corresponding to the T cell epitope of the protein of interest encoded by the MADV srRNA construct, e.g., 5 × 10 6 The experiments should also include one or more positive controls, such as PMA / ionomycin, and DMSO, which is used as a mock stimulus.

[0199] Antibodies. Measure antibody responses to measure total HA-specific IgG using an ELISA kit from Alpha Diagnostic International, according to the manufacturer's instructions.

[0200] Example 6 In vivo evaluation of modified MADV vaccine vectors This example describes in vivo experiments performed to evaluate immune responses following vaccination with the synthetic MADV srRNA influenza constructs described in Examples 1 and 2 above.

[0201] In these experiments, synthetic srRNA constructs derived from MADV strain BeAr300851 were designed and subsequently evaluated. As shown in Figures 5A-5D, the results of these experiments demonstrate that a MADV srRNA-based influenza vaccine against a viral antigen (influenza H1 hemagglutinin) can generate neutralizing antibody responses as measured by hemagglutinin inhibition (HAI) assays (Figure 5A) and in vivo T cell responses as measured by ELISpot (Figures 5B-5D).

[0202] Mice and Injections. Female C57BL / 6 or BALB / c mice were purchased from Envigo, Charles River Labs, or Jackson Laboratories. On the day of dosing, 0.01 μg of LNP-formulated material was injected intramuscularly into the quadriceps. Animals were monitored for weight and other general observations throughout the course of the study. Animals were dosed on days 0 and 56. Spleens were harvested on day 70.

[0203] LNP Formulation. srRNA was formulated into lipid nanoparticles using a microfluidic mixer and analyzed for particle size, polydispersity using dynamic light scattering, and encapsulation efficiency. Lipids were suspended in ethanol. RNA was suspended in 250 mM NaOAc (pH 4.0) at a concentration of 82 μg / ml and mixed at a flow rate of 3:1 (aqueous:organic).

[0204] Antibodies. A standard hemagglutinin inhibition assay (HAI) using sera from immunized mice was used to perform neutralizing antibody responses to measure virus neutralization.

[0205] The results of these experiments (shown in Figures 5A-5D) demonstrate that the modified MADV vaccine vector can be used to elicit antigen-specific immune responses in vivo. In these experiments, antibody and T cell responses were measured by HAI (high-intensity antibody) assay and IFNγ ELISpot 14 days after a prime-boost of 0.01 μg of MADV-srRNA (Rep-631) LNP. Specifically, Figure 5A shows serum HAI titers against H1N1. Figure 5B shows the results of splenocytes stimulated with an HA peptide library. Figure 5C shows the results of CD4 T cell epitope-specific peptide stimulation. Figure 5D shows the results of CD8 T cell epitope-specific peptide stimulation. Mann-Whitney statistics between groups are shown ( ** <0.01;*<0.05).

[0206] Example 7 In vivo evaluation of modified MADV biological therapeutic vectors This example describes in vivo experiments performed to assess biotherapeutic protein expression following administration of the synthetic MADV srRNA biotherapeutic constructs described in Examples 1 and 2 above.

[0207] In these experiments, a synthetic srRNA construct derived from MADV strain BeAr300851 was designed and subsequently evaluated. An srRNA encoding an unrelated transgene (luciferase) was also included in the experiment as a control.

[0208] Mice and Injections. Female C57BL / 6 or BALB / c mice were purchased from Envigo, Charles River Labs, or Jackson Laboratories. On the day of dosing, 10 μg of LNP-formulated material was injected intramuscularly into the quadriceps. Animals were monitored for weight and other general observations throughout the course of the study. Animals were dosed on day 0 and serum was collected on day 7.

[0209] LNP formulation. srRNA was formulated into lipid nanoparticles using a microfluidic mixer and analyzed for particle size, polydispersity using dynamic light scattering, and encapsulation efficiency. Lipids were suspended in ethanol. RNA was suspended in 250 mM NaOAc (pH 4.0) at a concentration of 82 μg / ml and mixed at a flow rate of 3:1 (aqueous:organic).

[0210] Protein Expression by ELISA: To measure serum concentrations of IL-1RA and IL-18BP, ELISA analysis was performed using a mouse IL-1RA ELISA kit (R&D Systems, catalog number MRA00) and a mouse IL-18BP ELISA kit (R&D Systems, catalog number DY122-05) according to the manufacturer's protocol.

[0211] The results of these experiments (shown in Figures 6A-6B) demonstrate that modified MADV biotherapeutic vectors can be used to express biotherapeutic proteins in vivo for at least 7 days. In these experiments, protein levels of murine IL-1RA (Figure 6A) and murine IL-18BP (Figure 6B) were measured using ELISA 7 days after administration of MADV-srRNA (Rep-657) co-encoding these two genes, and compared with srRNA encoding an unrelated protein, red firefly luciferase ("unrelated"), for basal protein expression levels. Mann-Whitney statistics between groups are shown ( * <0.05).

[0212] While certain alternative embodiments of the present disclosure have been disclosed, it should be understood that various modifications and combinations are possible and contemplated within the true spirit and scope of the appended claims, and therefore, no limitation to the precise summary and disclosure provided herein is intended.

Claims

1. A nucleic acid construct comprising a nucleic acid sequence encoding a modified Madagascar virus (MADV) genome or self-replicating RNA (srRNA), wherein the modified MADV genome or srRNA lacks at least a portion of a nucleic acid sequence encoding one or more viral structural proteins.

2. 2. The nucleic acid construct of claim 1, wherein the modified viral genome or srRNA lacks a substantial portion of a nucleic acid sequence encoding one or more viral structural proteins.

3. 3. The nucleic acid construct of claim 1, wherein the modified viral genome or srRNA does not contain a nucleic acid sequence encoding a viral structural protein.

4. 4. The nucleic acid construct of claim 1, further comprising one or more expression cassettes, each expression cassette comprising a promoter operably linked to a heterologous nucleic acid sequence.

5. 5. The nucleic acid construct of claim 4, wherein at least one of the expression cassettes comprises a subgenomic (sg) promoter operably linked to a heterologous nucleic acid sequence.

6. The nucleic acid construct of claim 5 , wherein the sg promoter is a 26S subgenomic promoter.

7. A nucleic acid construct according to any one of claims 1 to 6, wherein at least one nonstructural protein (nsP) or part thereof of the modified MADV genome or srRNA is heterologous to the remainder of the modified MADV genome or srRNA.

8. A nucleic acid construct according to any one of claims 1 to 7, further comprising a nucleic acid sequence encoding a heterologous nsP or a part thereof.

9. The nucleic acid construct of any one of claims 1 to 8, further comprising one or more untranslated regions (UTRs).

10. 10. The nucleic acid construct of claim 9, wherein at least one of the UTRs is a heterologous UTR.

11. 11. The nucleic acid construct of claim 4, wherein at least one of the expression cassettes comprises a coding sequence for a gene of interest (GOI).

12. 12. The nucleic acid construct of claim 11, wherein the GOI encodes a polypeptide selected from the group consisting of a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide, an industrial enzyme, and a reporter polypeptide.

13. 13. The nucleic acid construct of claim 11 or 12, wherein the GOI encodes a polypeptide selected from the group consisting of an antibody, an antigen, an immunomodulator, an enzyme, a signaling protein, and a cytokine.

14. 14. The nucleic acid construct of any one of claims 11 to 13, wherein the coding sequence of the GOI is optimized for expression at a level higher than the expression level of a reference coding sequence.

15. 15. The nucleic acid construct of any one of claims 11 to 14, wherein the coding sequence of the GOI is optimized to increase RNA stability.

16. 16. The nucleic acid construct of any one of claims 1 to 15, wherein the nucleic acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:

1.

17. A recombinant cell comprising the nucleic acid construct of any one of claims 1 to 16.

18. 18. The recombinant cell of claim 17, wherein the recombinant cell is a eukaryotic cell.

19. The recombinant cell of claim 18, wherein the recombinant cell is an animal cell.

20. 20. The recombinant cell of claim 19, wherein the animal cell is a vertebrate or invertebrate cell.

21. 21. The recombinant cell of claim 20, wherein the recombinant cell is an insect cell.

22. 22. The recombinant cell of claim 21, wherein the recombinant cell is a mosquito cell.

23. 21. The recombinant cell of claim 20, wherein the recombinant cell is a mammalian cell.

24. The recombinant cells include SV40 transformed monkey kidney CV1 cells, human embryonic kidney cells (HEK), baby hamster kidney cells (BHK) or their derivatives, mouse Sertoli cells, monkey kidney cells, human cervical carcinoma cells, canine kidney cells, buffalo rat hepatocytes, human lung cells, human hepatocytes, mouse mammary tumor cells, TRI cells, FS4 cells, Chinese hamster ovary cells (CHO), African green monkey kidney cells, human A549 cells, human cervical cells, human CHME5 cells, human PER.

21. The recombinant cell of claim 20, wherein the recombinant cell is selected from the group consisting of C6 cells, NS0 mouse myeloma cells, human epidermoid larynx cells, human fibroblast cells, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocyte cells, human macrophage cells, human RAW264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells.

25. A cell culture comprising at least one recombinant cell according to any one of claims 17 to 24 and a culture medium.

26. A transgenic animal comprising the nucleic acid construct of any one of claims 1 to 16.

27. 27. The transgenic animal of claim 26, wherein the animal is a vertebrate or an invertebrate.

28. 27. The transgenic animal of claim 26, wherein the animal is an insect.

29. 28. The transgenic animal of claim 27, wherein the animal is a mammal.

30. 30. The transgenic animal of claim 29, wherein the mammal is a non-human mammal.

31. 31. A method for producing a polypeptide of interest, comprising (i) rearing a transgenic animal according to any one of claims 26 to 30, or (ii) culturing a recombinant cell comprising the nucleic acid construct according to any one of claims 12 to 16, under conditions in which the transgenic animal or recombinant cell produces the polypeptide encoded by the GOI.

32. A method for producing a polypeptide of interest in a subject, the method comprising administering to the subject a nucleic acid construct according to any one of claims 12 to 16.

33. 33. The method of any one of claims 29 to 32, wherein the subject is a vertebrate or an invertebrate.

34. The method of any one of claims 29 to 31, wherein the subject is an insect.

35. The method of any one of claims 29 to 33, wherein the subject is a mammal.

36. 36. The method of claim 35, wherein the mammalian subject is a human subject.

37. A recombinant polypeptide produced by the method of any one of claims 29 to 36.

38. a pharmaceutically acceptable excipient, and a) a nucleic acid construct according to any one of claims 1 to 16, b) a recombinant cell according to any one of claims 17 to 24, and / or c) a recombinant polypeptide according to claim 37 A pharmaceutical composition comprising:

39. 39. The pharmaceutical composition according to claim 38, comprising the nucleic acid construct according to any one of claims 1 to 16 and a pharmaceutically acceptable excipient.

40. 39. The pharmaceutical composition of claim 38, comprising the recombinant cell of any one of claims 17 to 24 and a pharmaceutically acceptable excipient.

41. 39. The pharmaceutical composition of claim 38, comprising the recombinant polypeptide of claim 37 and a pharmaceutically acceptable excipient.

42. 42. The pharmaceutical composition of any one of claims 38 to 41, wherein the composition is formulated in a liposome, a lipid-based nanoparticle (LNP), a polymeric nanoparticle, a polyplex, a viral replicon particle (VRP), a microsphere, an immunostimulating complex (ISCOM), a conjugate of a bioactive ligand, or any combination thereof.

43. The pharmaceutical composition of any one of claims 38 to 42, wherein the composition is an immunogenic composition.

44. 44. The pharmaceutical composition of claim 43, wherein the immunogenic composition is formulated as a vaccine.

45. 43. The pharmaceutical composition of any one of claims 38 to 42, wherein the composition is substantially non-immunogenic to a subject.

46. The pharmaceutical composition of any one of claims 38 to 45, wherein the pharmaceutical composition is formulated as an adjuvant.

47. 47. The pharmaceutical composition of any one of claims 38 to 46, wherein the pharmaceutical composition is formulated for one or more of intranasal, intrathecal, transdermal, intraperitoneal, intramuscular, intratracheal, intralymphatic, intratumoral, intraarticular, intravenous, subcutaneous, intravaginal, intraocular, rectal, and oral administration.

48. 1. A method for inducing a pharmacodynamic effect in a subject in need thereof, comprising: a) a nucleic acid construct according to any one of claims 1 to 16, b) a recombinant cell according to any one of claims 17 to 24, c) a recombinant polypeptide according to claim 37, and / or d) A pharmaceutical composition according to any one of claims 38 to 47. administering to said subject a composition comprising:

49. 49. The method of claim 48, wherein the pharmacodynamic effect comprises one or more of an immunogenic effect, a biomarker response, a therapeutic effect, a prophylactic effect, a desired effect, an undesired effect, an adverse effect, and an effect in a disease model.

50. 50. The method of claim 49, wherein the pharmacodynamic effect comprises eliciting an immune response in the subject.

51. 1. A method for preventing and / or treating a health condition in a subject in need thereof, comprising: a) a nucleic acid construct according to any one of claims 1 to 16, b) a recombinant cell according to any one of claims 17 to 24, c) a recombinant polypeptide according to claim 37, and / or d) A pharmaceutical composition according to any one of claims 38 to 47. a composition comprising:

52. 52. The method of claim 51, wherein the administered composition elicits an immune response in the subject.

53. 53. The method of claim 51 or 52, wherein the condition is a proliferative disease or a microbial infection.

54. 54. The method of any one of claims 51 to 53, wherein the subject has or is suspected of having a condition associated with a proliferative disease or microbial infection.

55. 55. The method of any one of claims 51-54, wherein the administered composition increases interferon production in the subject.

56. 56. The method of any one of claims 51 to 55, wherein the composition is administered to the subject individually as a single therapy (monotherapy) or as a first therapy in combination with at least one additional therapy.

57. 57. The method of claim 56, wherein the at least one additional therapy is selected from the group consisting of chemotherapy, radiation therapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery.

58. 1. A kit for inducing a pharmacodynamic effect, inducing an immune response, and / or preventing and / or treating a condition or a microbial infection, comprising: a) a nucleic acid construct according to any one of claims 1 to 16, b) a recombinant cell according to any one of claims 17 to 24, c) a recombinant polypeptide according to claim 37, and / or d) a pharmaceutical composition according to any one of claims 38 to 47, and A kit comprising instructions for carrying out the method of any one of claims 48 to 57.

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