Modified eastern equine encephalitis viruses, self-replicating rna constructs, and uses thereof

Abstract

The present disclosure relates to the field of molecular virology, including nucleic acid molecules encoding modified viral genomes or replicons (e.g., self-replicating RNA), pharmaceutical compositions containing them, and the use of the nucleic acid molecules and compositions to produce desired products in cell culture or in an organism. Also provided are methods of eliciting an immune response in a subject in need thereof, and methods of preventing and / or treating various health conditions.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 220,139, filed July 9, 2021. The disclosures of the above-referenced applications are hereby incorporated by reference in their entirety, including any drawings.

[0002] Field The present disclosure relates to the fields of molecular virology and immunology, in particular to nucleic acid molecules encoding modified viral genomes and replicons (e.g., self-replicating RNAs), pharmaceutical compositions containing the same, and the use of the nucleic acid molecules and compositions to produce desired products in cell culture or in an organism. Also provided are methods for inducing a pharmacodynamic effect, e.g., eliciting an immune response, as well as methods for preventing and / or treating various health conditions in a subject in need thereof.

[0003] Incorporating sequence tables The contents of the accompanying sequence listing are hereby incorporated by reference into this application. The attached sequence listing text file, named _Sequence_Listing_058462-502001WO ST26.xml, was created on July 8, 2022 and is 93KB. [Background technology]

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

[0005] For example, many virus-based expression vectors have been deployed for the expression of heterologous proteins in cultured recombinant cells. For example, the use 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 the production of multi-subunit protein complexes, and co-expression of protein-modifying enzymes to improve heterologous protein production. Other recent advances in viral expression vector technology include many advanced genome engineering applications for controlling gene expression, preparation of viral vectors, in vivo gene therapy applications, and creation of vaccine delivery vectors.

[0006] However, it has been reported that host cells can develop complex and powerful mechanisms to detect and combat pathogen invasion. Furthermore, it has been reported that viruses, especially pathogenic viruses, have evolved along with host cells to combat 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 progeny viral production that can 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 to establish an antiviral state and control the infection. Although these cellular antiviral systems benefit host cells, they can also have a detrimental effect on self-amplifying RNAs (called replicons or self-replicating RNAs) designed to express beneficial vaccine antigens or therapeutics. For example, when a cell detects a replicon RNA (e.g., a self-replicating RNA) 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 effectiveness of the replicon.

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

[0008] overview The present disclosure generally relates to the development of immunotherapeutic agents, such as recombinant nucleic acid constructs and pharmaceutical compositions comprising the same, for use in the prevention and management of various health conditions, such as proliferative disorders and microbial infections. In particular, as described in more detail below, some embodiments of the present disclosure provide nucleic acid constructs comprising sequences encoding a modified genome or replicon (e.g., self-replicating RNA) of the alphavirus Eastern Equine Encephalitis Virus (EEEV) lacking at least a portion of the viral nucleic acid sequence encoding one or more structural proteins of the virus. Also disclosed are recombinant cells and transgenic animals engineered to include one or more nucleic acid constructs (e.g., vectors or srRNA molecules) disclosed herein, methods of producing molecules of interest, and pharmaceutical compositions comprising one or more of (a) the nucleic acid constructs of the present disclosure, (b) the polypeptides of the present disclosure, and (c) the recombinant cells of the present disclosure. In certain embodiments of the present disclosure, compositions and methods are further provided for inducing a pharmacodynamic effect, e.g., eliciting an immune response in a subject in need thereof, and / or for preventing and / or treating various health conditions, including proliferative disorders (e.g., cancer) and chronic infectious diseases, in a subject in need thereof.

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

[0010] Non-limiting exemplary embodiments of the nucleic acid constructs of the present disclosure can include one or more of the following features: In some embodiments, the modified viral genome or replicon RNA (e.g., a self-replicating RNA) lacks a substantial portion of a nucleic acid sequence encoding one or more viral structural proteins. In some embodiments, the modified viral genome or replicon RNA 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. In some embodiments, the 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.

[0011] 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 expression at a level higher than the expression level of a reference coding sequence. In some embodiments, the coding sequence of the GOI is optimized for enhanced RNA stability.

[0012] In some embodiments, the nucleic acid construct of the present disclosure is incorporated into a vector. In some embodiments, the vector is a self-replicating RNA (srRNA) vector.

[0013] 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, SEQ ID NO:2, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.

[0014] 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 a mammalian cell. In some embodiments, the recombinant cell is a monkey kidney CV1 cell transformed with SV40 (COS-7), a human embryonic kidney cell (e.g., HEK 293 or HEK 293 cells), a baby hamster kidney cell (BHK), a mouse Sertoli cell (e.g., TM4 cells), a monkey kidney cell (CV1), a human cervical carcinoma cell (HeLa), a canine kidney cell (MDCK), a buffalo rat hepatocyte (BRL3A), a human lung cell (W138), a human hepatocyte (Hep G2), a mouse mammary tumor (MMT 060562), a TRI cell, a FS4 cell, a Chinese hamster ovary cell (CHO cell), an African green monkey kidney cell (Vero cell), a human A549 cell, a human cervical cell, Human CHME5 cells, human PER.C6 cells, NS0 mouse myeloma cells, selected from the group consisting of human epidermal laryngeal cells, human fibroblast cells, human HUH-7 cells, human MRC-5 cells, human muscle cells, human lymphatic 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, rabbit kidney cells. Also provided in related embodiments is a cell culture comprising at least one recombinant cell disclosed herein and a culture medium.

[0015] In some embodiments, the recombinant cell is an insect cell, hi some embodiments, the recombinant cell is a mosquito cell.

[0016] In another aspect, provided herein is a transgenic animal comprising a nucleic acid construct as described herein. In some embodiments, the transgenic animal is a vertebrate or invertebrate animal. In some embodiments, the transgenic animal is a mammal. In some embodiments, the transgenic mammal is a non-human mammal. In some embodiments, the transgenic animal is an insect. In some embodiments, the transgenic insect is a transgenic mosquito. In another aspect, provided herein is a method for producing a polypeptide of interest, the method comprising: (i) rearing a transgenic animal as disclosed herein; or (ii) culturing a recombinant cell comprising a nucleic acid construct as disclosed herein, under conditions in which the recombinant cell or recombinant cell produces a polypeptide encoded by the GOI.

[0017] In another aspect, provided herein is a method of 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 mammalian subject. 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.

[0018] In yet another aspect, provided herein is a pharmaceutical composition comprising a pharma- ceutically 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.

[0019] Non-limiting exemplary embodiments of the pharmaceutical compositions of the present disclosure may include one or more of the following features. In some embodiments, provided herein is a composition comprising a nucleic acid construct as disclosed herein and a pharma- ceutically acceptable excipient. In some embodiments, provided herein is a composition comprising a recombinant cell as disclosed herein and a pharma- ceutically acceptable excipient. In some embodiments, the composition comprises a recombinant polypeptide as disclosed herein and a pharma- ceutically acceptable excipient. In some embodiments, provided herein is a composition formulated in a liposome, lipid-based nanoparticle (LNP), or polymeric nanoparticle. 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, transdermal, intraperitoneal, intramuscular, intranodal, intratumoral, intraarticular, intravenous, subcutaneous, intravaginal, ocular, oral, and rectal administration.

[0020] In another aspect, provided herein is a method of inducing a pharmacodynamic effect in a subject, in particular a method of eliciting an immune response in a subject in need thereof, the method 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.

[0021] In yet another aspect, provided herein is a method for preventing and / or treating a condition in a subject in need thereof, the method 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) a pharmaceutical composition described in any one of the present disclosure.

[0022] 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 disorder or a microbial infection. In some embodiments, the subject has or is suspected of having a condition associated with a proliferative disorder or a microbial infection. In some embodiments, the administered composition results in increased production of interferon 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.

[0023] In yet another aspect, provided herein is a kit for inducing a pharmacodynamic effect, eliciting an immune response, for prophylaxis, and / or treatment of 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.

[0024] Each of the aspects and embodiments described herein can be used together unless expressly or specifically excluded from the context of the embodiment or aspect.

[0025] 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 and detailed description, and from the claims. [Brief description of the drawings]

[0026] [Figure 1]Figure 1 illustrates a non-limiting example of a modified EEEV 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 are completely deleted. The modified EEEV design depicted in this figure contains the native 5'UTR and 3'UTR from EEEV strain FL93-939, and may further contain a heterologous gene of interest (GOI) placed under the control of the 26S subgenomic promoter. The coding sequences for the nonstructural proteins nsP1, nsP2, nsP3, and nsP4 are shown. [Figure 2-1] 2A-2B are schematic illustrations of non-limiting exemplary EEEV RNA replicon-based designs according to some embodiments of the present disclosure, in which sequences encoding a modified EEEV genome from the FL93-939 strain are incorporated into a plasmid DNA vector (FIG. 2A), which also contains the coding sequence of an exemplary gene of interest (GOI), such as the hemagglutinin precursor (HA) of influenza A virus H5N1 (FIG. 2B). [Figure 2-2] 2A-2B are schematic illustrations of non-limiting exemplary EEEV RNA replicon-based designs according to some embodiments of the present disclosure, in which sequences encoding a modified EEEV genome from the FL93-939 strain are incorporated into a plasmid DNA vector (FIG. 2A), which also contains the coding sequence of an exemplary gene of interest (GOI), such as the hemagglutinin precursor (HA) of influenza A virus H5N1 (FIG. 2B). [Diagram 3]3A-3B are contour maps of BHK-21 cells transformed with an EEEV RNA replicon (e.g., a self-replicating RNA). In FIG. 3A, EEEV replicon RNA without a GOI was transformed by electroporation, and 20 hours after transformation, the cells were fixed and permeabilized and stained using a PE-conjugated anti-dsRNA mouse monoclonal antibody (J2, Scicons) to quantitate the frequency of dsRNA+ cells by fluorescent flow cytometry. In FIG. 3B, EEEV replicon RNA encompassing the coding sequence for HA was similarly transformed into BHK21 cells, and in addition to dsRNA detection, an APC-conjugated anti-HA mouse monoclonal antibody (2B7, Abcam) was used to detect transgene expression. Positive staining of individual cells with both anti-dsRNA and anti-HA antibodies demonstrates that the modified EEEV design described herein is a viable synthetic replicon and can undergo RNA replication and express a transgene. [Figure 4-1]4A-4B graphically summarize the results of experiments demonstrating that modified EEEV vectors designed according to some embodiments of the present disclosure can be used to express two exemplary bioactive proteins: (i) interleukin 1 receptor antagonist protein (IL-1RA) and (ii) interleukin-12 (IL-12). FIGs. 4A-4B are bar graphs illustrating the quantification of secreted protein bioactivity from BHK-21 transformed with EEEV self-replicating RNA (srRNA). The srRNAs shown in FIGs. 4A-4B are EEEV srRNAs (RBI305, RBI306) that encode two distinct forms of the two proteins IL-1RA and IL-12, respectively. Four control VEEV-based srRNAs were also included in these experiments: VEEV srRNA encoding both IL-1RA and IL-12 in two forms (RBI299, RBI300), and VEEV srRNA with a control transgene (RBI296, RBI298). Figure 4A shows quantification of bioactive IL-1RA in cell culture medium 24 and 48 hours after srRNA transformation. Figure 4B shows quantification of bioactive IL-12 in cell culture medium 24 and 48 hours after srRNA transformation. [Figure 4-2]4A-4B graphically summarize the results of experiments demonstrating that modified EEEV vectors designed according to some embodiments of the present disclosure can be used to express two exemplary bioactive proteins: (i) interleukin 1 receptor antagonist protein (IL-1RA) and (ii) interleukin-12 (IL-12). FIGs. 4A-4B are bar graphs illustrating the quantification of secreted protein bioactivity from BHK-21 transformed with EEEV self-replicating RNA (srRNA). The srRNAs shown in FIGs. 4A-4B are EEEV srRNAs (RBI305, RBI306) that encode two distinct forms of the two proteins IL-1RA and IL-12, respectively. Four control VEEV-based srRNAs were also included in these experiments: VEEV srRNA encoding both IL-1RA and IL-12 in two forms (RBI299, RBI300), and VEEV srRNA with a control transgene (RBI296, RBI298). Figure 4A shows quantification of bioactive IL-1RA in cell culture medium 24 and 48 hours after srRNA transformation. Figure 4B shows quantification of bioactive IL-12 in cell culture medium 24 and 48 hours after srRNA transformation. [Figure 5-1] 5A-5B are bar graphs illustrating the in vivo immunogenicity of a panel of srRNAs encoding an exemplary viral antigen, the envelope glycoprotein G of rabies virus (RABV-G). The panel included srRNAs from Venezuelan equine encephalitis virus (VEE.TC83), chikungunya virus strains S27 (CHIK.S27) and DRDE-06 (CHIK.DRDE), Sindbis virus strains Girdwood (SIN.GW) and AR87 (SIN.AR86), and Eastern encephalitis virus (EEE.FL93). FIG. 5A shows quantification of antigen-specific splenic T cell responses as assessed by ELISpot after two immunizations. FIG. 5B shows anti-rabies neutralizing antibody titers from serum after two immunizations. [Figure 5-2]5A-5B are bar graphs illustrating the in vivo immunogenicity of a panel of srRNAs encoding an exemplary viral antigen, the envelope glycoprotein G of rabies virus (RABV-G). The panel included srRNAs from Venezuelan equine encephalitis virus (VEE.TC83), chikungunya virus strains S27 (CHIK.S27) and DRDE-06 (CHIK.DRDE), Sindbis virus strains Girdwood (SIN.GW) and AR87 (SIN.AR86), and Eastern encephalitis virus (EEE.FL93). FIG. 5A shows quantification of antigen-specific splenic T cell responses as assessed by ELISpot after two immunizations. FIG. 5B shows anti-rabies neutralizing antibody titers from serum after two immunizations. [Figure 6-1] 6A-6C are bar graphs showing the in vivo immunogenicity of a panel of srRNAs encoding exemplary tumor-associated antigens for use as a vaccine, e.g., to elicit an immune response in a subject. The panel included srRNAs from Eastern Encephalitis Virus (EEE.FL93) and five other alphaviruses: Venezuelan Equine Encephalitis Virus (VEE.TC83), Chikungunya Virus strains S27 (CHIK.S27) and DRDE-06 (CHIK.DRDE), Sindbis Virus strains Girdwood (SIN.GW), and AR87 (SIN.AR86). Each srRNA includes sequences encoding three polypeptides: estrogen receptor 1 (ESR1), human epidermal growth factor 2 (HER2), and human epidermal growth factor 2 (HER3). 6A-6C show splenic T cell responses to these three antigens measured using ELISpot analysis in mice immunized twice, with statistical comparisons between each antigen tested. [Figure 6-2]6A-6C are bar graphs showing the in vivo immunogenicity of a panel of srRNAs encoding exemplary tumor-associated antigens for use as a vaccine, e.g., to elicit an immune response in a subject. The panel included srRNAs from Eastern Encephalitis Virus (EEE.FL93) and five other alphaviruses: Venezuelan Equine Encephalitis Virus (VEE.TC83), Chikungunya Virus strains S27 (CHIK.S27) and DRDE-06 (CHIK.DRDE), Sindbis Virus strains Girdwood (SIN.GW), and AR87 (SIN.AR86). Each srRNA includes sequences encoding three polypeptides: estrogen receptor 1 (ESR1), human epidermal growth factor 2 (HER2), and human epidermal growth factor 2 (HER3). 6A-6C show splenic T cell responses to these three antigens measured using ELISpot analysis in mice immunized twice, with statistical comparisons between each antigen tested. [Figure 6-3] 6A-6C are bar graphs showing the in vivo immunogenicity of a panel of srRNAs encoding exemplary tumor-associated antigens for use as a vaccine, e.g., to elicit an immune response in a subject. The panel included srRNAs from Eastern Encephalitis Virus (EEE.FL93) and five other alphaviruses: Venezuelan Equine Encephalitis Virus (VEE.TC83), Chikungunya Virus strains S27 (CHIK.S27) and DRDE-06 (CHIK.DRDE), Sindbis Virus strains Girdwood (SIN.GW), and AR87 (SIN.AR86). Each srRNA includes sequences encoding three polypeptides: estrogen receptor 1 (ESR1), human epidermal growth factor 2 (HER2), and human epidermal growth factor 2 (HER3). 6A-6C show splenic T cell responses to these three antigens measured using ELISpot analysis in mice immunized twice, with statistical comparisons between each antigen tested. [Figure 7]7 is a bar graph illustrating the in vivo expression levels of interleukin-12 (IL-12) from a panel of srRNAs encoding an exemplary biotherapeutic protein (human IL-12). The panel included srRNAs from Venezuelan Equine Encephalitis Virus (VEE.TC83), Chikungunya Virus strains S27 (CHIK.S27) and DRDE-06 (CHIK.DRDE), Sindbis Virus strains Girdwood (SIN.GW) and AR87 (SIN.AR86), and Eastern Encephalitis Virus (EEE.FL93). srRNAs were administered intramuscularly in mice and serum was collected on day 7 to detect systemic levels of protein. [Figure 8] 8 is a bar graph illustrating the in vivo activity of IL-12 expressed from an EEEV srRNA vector encoding another exemplary biotherapeutic protein (murine IL-12). The functionality of IL-12 was measured by assessing the induction of IFNγ as a downstream pharmacodynamic marker. Serum from mice 3 days after srRNA administration showed detectable levels of IFNγ. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description of the Disclosure Provided herein are, inter alia, viral expression systems with excellent expression capabilities suitable for expressing heterologous molecules, such as vaccines 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, containing a modified genome or replicon RNA (e.g., self-replicating RNA) of Eastern Equine Encephalitis virus, in which at least some of its original viral sequences encoding structural proteins have been deleted. Also provided in some embodiments of the present disclosure are viral-based expression vectors comprising one or more expression cassettes encoding heterologous polypeptides. Further provided are recombinant cells genetically engineered to contain one or more of the nucleic acid molecules disclosed herein. Biomaterials and recombinant products derived from the recombinant cells are also within the scope of the present application. Also provided are compositions and methods useful for inducing a pharmacodynamic effect, such as eliciting an immune response in a subject in need thereof, as well as methods for preventing and / or treating various health conditions in a subject in need thereof.

[0028] Self-amplifying RNA (e.g., replicon or self-replicating RNA) based on RNA viruses (e.g., alphaviruses) can be used as powerful expression systems. For example, an advantage of using alphaviruses, such as EEEV, as viral expression vectors has been reported 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 replicon RNA may increase the overall yield of antibody product. Furthermore, if the protein being expressed is a vaccine antigen, high levels of expression may induce the strongest immune response in vivo.

[0029] Alphaviruses utilize motifs contained in their UTRs, structural regions, and nonstructural regions to affect their replication in host cells. These regions also contain mechanisms to evade the innate immunity of the host cell. However, significant differences between alphavirus species have been reported. For example, New World and Old World alphaviruses have evolved different components to utilize intracellular stress granules, JAK-STAT signaling, FXR, and G3BP proteins for the assembly of viral replication complexes. What parts of the genome contain these components also differ among alphaviruses. For example, bypass of PKR activation and subsequent phosphorylation of EIF2α is achieved through a downstream loop in some Old World alphaviruses, such as Sindbis, whereas bypassing this pathway is thought to be achieved through nsP4 in chikungunya, which lacks a recognizable DLP. Furthermore, beyond the variation between individual alphaviruses, there are often differences within alphavirus strains that can explain changes in properties such as pathogenicity. 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, leading to differential induction of type I interferons and resulting changes in virulence.

[0030] Given the specific presence of host cell attenuation factors in the nonstructural and structural regions of alphaviruses, deleting structural genes to allow heterologous gene expression in synthetic vectors has different effects on individual vectors. Synthetic replicons (e.g., self-replicating RNAs) with different host attenuation factors in the nonstructural regions are differentially better at inducing immune responses against the expressed heterologous genes. For example, blocking of host cell functions was related to nsP2 in Old World viruses, whereas in New World viruses such as VEEV and EEEV, this is mainly related to the capsid protein (C), which is partially or completely deleted in synthetic vectors. The hypervariable region (HVD) of the nsP3 protein has host interactions that are specific for each alphavirus. In particular, EEEV nsP3 has been shown to interact with the cellular FXR and G3BP protein families, DDX3, S100A4, IKKβ, PGAM5, as well as cytoskeletal remodeling and vesicle transport proteins. In particular, EEEV is considered to be the most highly pathogenic of the alphaviruses, and the characterized EEEV strain, that of FL93-939, induces the highest IFN levels in mice. Although FL939-939 and other North American strains induce high levels of IFN, there are only minor differences in viremia or mortality in infected wild-type IFN-deficient mice, suggesting that IFN plays little role in protection against these strains, in contrast to the South American EEEV strains. Chimeras of the Northern (FL93-939) and South American (BeAr436087) strains show intermediate sensitivity to IFN and differences in neurovirulence and tissue tropism in mice, demonstrating that both structural and nonstructural proteins contribute to phenotypic variation. Overall, the ability of EEEV to interfere with stress granules, its strain-dependent agnosticism to IFN, and other yet unexplained mechanisms that contribute to its robust pathogenicity suggest that EEEV may make a useful vector for the expression of heterologous proteins for vaccine biotherapeutic applications. The advantages that this vector offers have never before been explored or predicted.

[0031] As described in more detail below, an initial observation was made that publicly available alphavirus genome data does not always provide nucleotide sequences that allow direct replacement of nucleic acid sequences encoding structural proteins with a gene of interest (GOI) resulting in a self-replicating RNA and transgene expression replicon. For example, simple replacement of EEEV structural proteins with heterologous genes using available published sequences is not always sufficient to generate a functional replicon. In other words, further manipulations such as using heterologous 5' and / or 3' UTR sequences are required to create a replicon system suitable for use in vaccines and therapeutics.

[0032] As described in more detail below, some embodiments of the present disclosure relate to modified EEEV genomes or replicon RNAs (e.g., self-replicating RNAs) engineered to express one or more heterologous genes of interest (GOIs). For example, it has been found that the structural polyprotein gene of EEV strain FL93-939 can be replaced with a synthetic hemagglutinin precursor (HA) from the influenza A virus H5N1 gene (see, e.g., FIG. 2B) to generate a self-replicating vector capable of RNA replication and transgene expression in transfected BHK21 cells (see, e.g., FIG. 3B). In addition, as described in more detail below, some EEEV-based srRNA constructs described herein can be utilized for the expression of antigens and formulated as vaccines with pharmacodynamic effects measured in vivo (see, e.g., FIG. 5 and FIG. 6). Furthermore, the experimental data depicted in Figures 4A-4B demonstrate that EEEV-based srRNA vectors can be effective for the expression of multiple proteins whose coding sequences are operably linked to each other in a single open reading frame (e.g., a polycistronic ORF) and have bioactivity as measured by pharmacodynamic effects in vivo (see, e.g., Figure 6). Furthermore, the EEEV-based srRNA vectors of the present disclosure are also useful for the expression of biological therapeutic proteins (see, e.g., Figure 7) with confirmed bioactivity in vivo (see, e.g., Figure 8). Taken together, these studies further demonstrate the use of EEEV srRNA in therapeutic and vaccine applications. definition

[0033] Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used herein are intended to have the meaning commonly understood by those 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 ready reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference to 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.

[0034] 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. "A and / or B" is used herein to include all alternatives of "A," "B," "A or B," and "A and B."

[0035] 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, intranodal, intraperitoneal, subcutaneous, intramuscular, oral, intravaginal, and topical administration, or a combination thereof. The terms include, but are not limited to, administration by a medical professional and self-administration.

[0036] 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 that cell, cell culture, or cell line, regardless of the number of transplants or passages in culture. It is understood that not all progeny are exactly identical to the parent cell. This is because certain modifications may occur in later generations, either due to mutations (e.g., deliberate or inadvertent mutations) or environmental influences (e.g., methylation or other epigenetic modifications), so that the progeny may not, in fact, be identical to the parent cell, but still be within the scope of the term as used herein, so long as the progeny retains the same function as the original cell, cell culture, or cell line.

[0037] The term "effective amount", "therapeutically effective amount", or "pharmaceutical effective amount" of a composition of the present disclosure, e.g., a nucleic acid construct (e.g., a vector or srRNA molecule), a recombinant cell, a recombinant polypeptide, and / or a pharmaceutical composition generally refers to an amount sufficient for the composition to achieve a given purpose compared to the absence of the composition (e.g., achieve the effect for which it is administered, stimulate an immune response, prevent or treat a disease, or alleviate one or more symptoms of a disease, disorder, infection, or condition). An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or alleviation of a symptom(s) of a disease, which may also be referred to as a "therapeutically effective amount". "Alleviation" of a symptom refers to a decrease in the severity or frequency of the symptom, or 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 of ordinary skill 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).

[0038] When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limit of that range (to one-tenth of the unit of the lower limit unless the context clearly dictates otherwise) is encompassed within the disclosure, as well as any other stated or intervening value in that stated range. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0039] In this specification, a particular range, when used herein, is presented with the term "about", which usually has the meaning of approximately, preceding the numerical value. The term "about" is used to literally support the exact number preceded by the term, as well as a number that is close to or approximately the number preceded by the term. In determining whether a number is close to or approximately a specifically recited number, a number that is close to or approximately a non-recited number may be a number that provides a substantial equivalent to the specifically recited number in the context in which it is presented. If the degree of approximation is not clear from the context, "about" is within plus or minus 10% of the provided value in all cases, including the provided value, or rounded to the nearest significant figure. In some embodiments, the term "about" refers to the specified value ± up to 10%, up to ± 5%, or up to ± 1%.

[0040] The term "construct" refers to a recombinant molecule that includes one or more isolated nucleic acid sequences from a heterologous source. For example, a nucleic acid construct can be a chimeric nucleic acid molecule in which two or more nucleic acid sequences from different sources are assembled into a single nucleic acid molecule. Thus, representative nucleic acid constructs include any construct that contains (1) a nucleic acid sequence that includes regulatory and coding sequences that are not naturally contiguous to each other (e.g., at least one of the nucleotide sequences is heterologous to at least one of the other nucleotide sequences), or (2) a sequence that encodes a portion of a functional RNA molecule or protein that is not naturally contiguous, or (3) a promoter portion that is not naturally contiguous. Representative nucleic acid constructs can include any recombinant nucleic acid molecule, linear or circular, single-stranded or double-stranded DNA or RNA nucleic acid molecule, including nucleic acid molecules derived from any source capable of genomic integration or autonomous replication, such as plasmids, cosmids, viruses, autonomously replicating polynucleotide molecules, phages, to which one or more nucleic acid sequences are operably linked. Constructs of the present disclosure can include elements necessary to direct expression of a nucleic acid sequence of interest that is also included in the construct. Such elements can include regulatory elements, such as a promoter, operably linked to (to direct transcription of) the nucleic acid sequence of interest, and optionally includes a polyadenylation sequence.

[0041] In some embodiments of the present disclosure, the nucleic acid construct may be incorporated into a vector. The term "vector" is used herein to refer to a nucleic acid molecule or sequence capable of transferring or transporting another nucleic acid molecule. Thus, the term "vector" encompasses both DNA-based and RNA-based vectors. The term "vector" encompasses cloning and expression vectors, as well as viral and integrating vectors. An "expression vector" is a vector that includes a regulatory region such that it can express DNA sequences and fragments in vitro, ex vivo, and / or in vivo. In some embodiments, a vector may include a sequence that directs autonomous replication in a cell, such as, for example, a plasmid (a DNA-based vector) or a self-replicating RNA vector. In some embodiments, a vector may include a sequence sufficient to allow integration into a host cell DNA. In some embodiments, a vector may include a DNA sequence that can be transcribed into RNA in vitro and / or in vivo. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. In some embodiments, a vector of the present disclosure may be a single-stranded vector (e.g., ssDNA or ssRNA). In some embodiments, the 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 genes into cells.

[0042] In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication, such as prokaryotic and eukaryotic origins, at least one multiple cloning site, and / or elements that facilitate stable integration of the construct into the genome of the cell. Two or more constructs can be incorporated into a single nucleic acid molecule, such as a single vector, or can be contained in two or more separate nucleic acid molecules, such as two or more separate vectors. An "expression construct" generally includes at least one 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 the cell. Compositions and methods for preparing and using the constructs and cells for the practice of the present disclosure are known to those of skill in the art.

[0043] The term "operably linked" as used herein means a physical or functional connection between two or more elements, e.g., polypeptide or polynucleotide sequences, so that they can operate in their intended manner. For example, when used in the context of a nucleic acid molecule described herein, or a coding sequence and a promoter sequence in a nucleic acid molecule, the term "operably linked" means that the coding sequence and the promoter sequence are in frame and at an appropriate space and distance to allow the effect of each binding by a transcription factor or RNA polymerase on transcription. It should be 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) that provides 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).

[0044] The term "portion" as used herein refers to a fraction. With respect to a particular structure, such as a polynucleotide sequence or an amino acid sequence or a protein, the term "portion" may refer to a continuous or discontinuous fraction of said structure. For example, a portion of an amino acid sequence comprises at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% of the amino acids of said amino acid sequence. Additionally or alternatively, when the portion is a discontinuous fraction, the discontinuous fraction is composed of 2, 3, 4, 5, 6, 7, 8, or more portions of the structure (e.g., domains of a protein), each portion being a continuous element of the structure. For example, the non-contiguous fraction of an amino acid sequence may consist of 2, 3, 4, 5, 6, 7, 8 or more, e.g. up to 4 portions of said amino acid sequence, 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.

[0045] When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limit of that range (to one-tenth of the unit of the lower limit unless the context clearly dictates otherwise) is encompassed within the disclosure, as well as any other stated or intervening value in that stated range. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0046] In this specification, certain ranges are presented with the term "about" preceding the numerical values. The term "about" is used to literally support the exact number preceded by the term, as well as a number that is close to or approximately the number preceded by the term. In determining whether a number is close to or approximately a specifically recited number, the number that is close to or approximately the unrecited number may be a number that, in the context in which it is presented, provides a substantial equivalent to the specifically recited number.

[0047] 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 having a specified percentage of nucleotides or amino acids that are identical or identical (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a particular 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 at ncbi.nlm.nih.gov / BLAST. Such sequences are said to be "substantially identical." This definition also refers to or may apply to the complementation of a sequence. This definition also includes sequences that have deletions and / or additions, as well as sequences that have substitutions. 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. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J Mol Biol 215:403, 1990). Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705) using its specified parameters.

[0048] The term "pharmaceutically acceptable excipient" as used herein refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for administration of the compound(s) of interest to a subject. Thus, "pharmaceutically acceptable excipient" can include substances that are called 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, dispersions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics and additional therapeutic agents) can also be incorporated into the composition.

[0049] 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. A recombinant protein is one that includes amino acid residues not found in the native (non-recombinant or wild-type) protein, or that includes modified, e.g., labeled, amino acid residues. The term can include any modification to a peptide, protein, or nucleic acid sequence. Such modifications can include: any chemical modification of a peptide, protein, or nucleic acid sequence, including 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 including antibody fragments; and in addition, the addition, deletion, and / or substitution of one or more nucleic acids in a nucleic acid sequence. The term "genetically recombinant," when used in reference to a cell, is not intended to include naturally occurring cells, but includes cells that have been genetically engineered or modified to contain or express a polypeptide or nucleic acid that would not be present in the cell if it had not been genetically engineered or modified.

[0050] As used herein, the term "replicon RNA" refers to an RNA that contains all the genetic information required to direct its own amplification or self-replication in a permissive cell. Therefore, sometimes replicon RNA is also called "self-amplifying RNA" (saRNA) or "self-replicating RNA" (srRNA). To direct its own replication, the RNA molecule 1) encodes a polymerase, replicase, or other protein that can interact with proteins, nucleic acids, or ribonucleoproteins from the virus or host cell and catalyze the RNA amplification process; 2) contains cis-acting RNA sequences required for the replication and transcription of the RNA encoded in the subgenomic replicon. These sequences can be bound to its self-encoded proteins, or to proteins, nucleic acids, or ribonucleoproteins from non-self-encoded cells, or to complexes between any of these components during the process of replication. In some embodiments of the present disclosure, an alphavirus replicon RNA molecule (e.g., srRNA or saRNA molecule) generally contains the following ordered elements: 5' viral RNA sequence(s) required in cis for replication, sequences encoding biologically active alphavirus nonstructural proteins (e.g., nsP1, nsP2, nsP3, and nsP4), a promoter for the subgenomic RNA (sgRNA), 3' viral sequences required in cis for replication, and a continuous polyadenylate tract (poly(A)). In some instances, a subgenomic promoter (sg) directing expression of a heterologous sequence may be included within the srRNA construct of the present disclosure. Additionally, the term RNA replicon (e.g., srRNA or saRNA) generally refers to a molecule of positive polarity or "message" sense, and the replicon RNA may be of a length different from that of any known naturally occurring alphavirus. In some embodiments of the present disclosure, the replicon RNA does not include sequences for at least one structural viral protein; sequences encoding structural genes may be replaced with heterologous sequences.In those cases where the replicon RNA is packaged into recombinant alphavirus particles, it may contain one or more sequences, so-called packaging signals, that function to initiate interactions with alphavirus structural proteins that lead to particle formation.

[0051] As used herein, a "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 under the care of a physician. 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 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, such as mammals, e.g., rodents, e.g., mice, non-human primates, and other mammals, e.g., sheep, dogs, cows, chickens, and non-mammals, e.g., amphibians, reptiles, and the like.

[0052] Aspects and embodiments of the disclosure described herein are understood to include aspects and embodiments that "comprising," "consisting," and "consisting essentially of." 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, is understood to encompass compositions and methods that consist essentially of, and consist of, the recited components or steps.

[0053] Headings, e.g., (a), (b), (i), etc., are provided solely to facilitate reading of the specification and claims. The use of headings in the specification or claims does not require that the steps or elements be performed in alphabetical or numerical order or in the order in which they are presented.

[0054] It should be understood that certain features of the present disclosure are described for clarity in the context of separate embodiments, and can also be provided in combination in a single embodiment. Conversely, various features of the present disclosure are described for brevity in the context of a single embodiment, and can also be provided separately or in any suitable subcombination. All combinations of the embodiments relating to the present disclosure are specifically embraced by the present disclosure and are disclosed herein as if all combinations were individually and expressly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein as if each such subcombination was individually and expressly disclosed herein. Eastern Equine Encephalitis Virus (EEEV)

[0055] Eastern equine encephalitis virus (EEEV) belongs to the mosquito-borne genus Alphavirus, which includes a group of genetically, structurally, and serologically related viruses of the Togaviridae family. Currently, the Alphavirus genus includes Sindbis virus (SINV), Semliki Forest virus (SFV), Ross River virus (RRV), Venezuelan equine encephalitis virus (VEEV), and Eastern equine encephalitis virus (EEEV), among others, all of which are closely related and can infect a variety of vertebrates, such as mammals, rodents, fish, and birds, as well as large mammals, such as humans and horses, and invertebrates, such as insects. In particular, EEEV has been widely studied, and the life cycle, mode of replication, etc. of these viruses are well characterized. Further information in this regard can be found, for example, in Corrin T. et al., Vector-Borne and Zoonotic Diseases, Vol. 21, No. 5, 2021. In addition, alphaviruses have been shown to replicate very efficiently in animal cells, making them useful as 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.

[0056] Each of these alphaviruses has a single-stranded RNA genome of positive polarity surrounded by an enveloped nucleocapsid containing the viral spike protein. Alphavirus particles are enveloped and tend to be spherical (although slightly polymorphic) with an isometric nucleocapsid. The alphavirus genome is a single-stranded RNA of positive polarity approximately 11-12 kb in length with a 5' cap, a 3' polyA tail, and two open reading frames with a first frame encoding nonstructural proteins with enzymatic function, and a second frame encoding viral structural proteins (e.g., capsid protein CP, E1 glycoprotein, E2 glycoprotein, E3 protein, and 6K protein). For example, EEEV has a single-stranded, positive-sense RNA genome of approximately 11.7 kb that is capped at the 5' end and polyadenylated at the 3' end. EEEV is transmitted by the bite of infected mosquitoes, and most spillover transmission occurs in low-lying areas with hardwood trees and wetlands that support mosquito larval development. As its name suggests, EEEV infects horses and causes fever, behavioral changes, and other symptoms of encephalitis. Wild birds are the primary reservoir for EEEV. However, infection is often fatal to horses.

[0057] The 5' two-thirds of the alphavirus genome encodes a number of nonstructural proteins necessary for viral RNA transcription and replication. These proteins are directly translated from RNA and, together with cellular proteins, form the RNA-dependent RNA polymerase that is essential for viral genome replication and transcription of subgenomic RNA. Four nonstructural proteins (nsP1-4) are produced as a single polyprotein that constitutes the viral replication machinery. Polyprotein processing is highly regulated and cleavage at the P2 / 3 junction affects the use of the RNA template during genome replication. This site is at the bottom of a narrow depression and is not easily accessible. Upon cleavage, nsP3 creates a ring structure that surrounds nsP2. These two proteins have an extensive interface. Mutations in nsP2 that produce noncytopathic viruses or temperature-sensitive phenotypes cluster at the P2 / P3 interface region. P3 mutations opposite the location of nsP2 noncytopathic mutations prevent efficient cleavage of P2 / 3. This can affect RNA infectivity and alter viral RNA production levels.

[0058] The 3' third of the genome contains the subgenomic RNA that serves as a template for the translation of all structural proteins required for the formation of viral particles: the core nucleocapsid protein C, and the envelope proteins P62 and E1, which assemble as a heterodimer. The viral membrane-anchored surface glycoproteins are involved in receptor recognition and entry into target cells by membrane fusion. The subgenomic RNA is transcribed from the p26S subgenomic promoter, which is present at the 3' end of the RNA sequence encoding the nsP4 protein. Proteolytic maturation of P62 into E2 and E3 causes changes in the viral surface. Together, E1, E2, and sometimes E3 glycoprotein "spikes" form E1 / E2 dimers or E1 / E2 / E3 trimers, with E2 extending from the center to the apex, E1 filling the space at the apex, and E3, if present, at the head of the spike. When the virus is exposed to the acidity of the endosome, E1 dissociates from E2 to form the E1 homotrimer. This 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, structurally distinct from the class I fusion proteins found in influenza viruses and HIV. The E2 glycoprotein functions to interact with the nucleocapsid through its cytoplasmic domain, while its ectodomain is responsible for binding to cellular receptors. Most alphaviruses lose the peripheral protein E3, but in Semlikivirus it remains associated with the viral surface.

[0059] Alphavirus replication has been reported to occur on membranous surfaces within the host cell. In the first step of the infection cycle, the 5' end of the genomic RNA is translated into a polyprotein (nsP1-4) with RNA polymerase activity that generates a negative strand complementary to the genomic RNA. In the second step, the negative strand is used as a template to produce two RNAs: (1) a positive genomic RNA corresponding to the genome of a secondary virus that produces other nsP proteins by translation and acts as the genome of the virus; (2) a subgenomic RNA that codes for the structural proteins of the virus that form the infectious particle. The positive genomic RNA / subgenomic RNA ratio is regulated by proteolytic autocleavage of the polyprotein into nsP1, nsP2, nsP3, and nsP4. In reality, viral gene expression occurs in two stages. The first stage is mainly the synthesis of the positive and negative genomic strands. In the second stage, the synthesis of the subgenomic RNA is virtually exclusive, so that a large amount of structural proteins are produced. Compositions of the Disclosure

[0060] As described in more detail below, one aspect of the disclosure relates to nucleic acid constructs comprising a nucleic acid sequence encoding a modified viral genome or replicon RNA (e.g., a self-replicating RNA), where the modified genome or replicon RNA lacks (e.g., does not include) at least a portion of the nucleic acid sequence encoding one or more structural proteins of the corresponding unmodified viral genome or replicon RNA. Some embodiments of the disclosure provide modified alphavirus genomes or replicon RNAs in which the coding sequences for nonstructural proteins nsP1, nsP2, nsP3, and nsP4 are present, but at least a portion or the entirety of the sequence encoding one or more structural proteins is absent. Also provided are recombinant cells and cell cultures engineered to contain the nucleic acid constructs disclosed herein. A. Nucleic acid constructs

[0061] As described in more detail below, one aspect of the disclosure relates to novel nucleic acid constructs that include a nucleic acid sequence encoding a modified genome or replicon RNA (e.g., a self-replicating RNA) of an alphavirus, such as Equine Encephalitis Virus (EEEV). For example, in some embodiments, the modified alphavirus genome can include deletions, substitutions, and / or insertions in one or more genomic regions of the parent alphavirus genome.

[0062] Non-limiting exemplary embodiments of the nucleic acid constructs of the present disclosure may include one or more of the following features: In some embodiments, the nucleic acid construct comprises a nucleic acid sequence encoding a modified EEEV genome or replicon RNA, where the modified EEEV genome or replicon RNA lacks at least a portion of the nucleic acid sequence encoding one or more structural proteins of the unmodified EEEV genome or replicon RNA, e.g., the modified EEEV genome or replicon RNA does not include at least a portion of the coding sequence for one or more EEEV structural proteins, CP, E1, E2, E3, and 6K. Both pathogenic and non-pathogenic EEEV strains are suitable. Non-limiting examples of EEEV strains suitable for the compositions and methods of the present disclosure include EEEV792138, 783372, BeAn5122, BeAr300851, BeAr436087, C-49, FL91-4679, FL93-939, GML903836, MP-9, PE6, and V105-00210. Additional suitable EEEV strains include, but are not limited to, those listed on the Virus Pathogen Resource website (ViPR; publicly available at www.viprbrc.org / brc / vipr_genome_search.spg? method= SubmitForm& blockId= 868&decorator=toga). In some embodiments, the modified EEEV genome or replicon RNA is from EEEV strain FL93-939.

[0063] Non-limiting exemplary embodiments of the nucleic acid constructs of the present disclosure can include one or more of the following features: In some embodiments, the modified viral genome or replicon RNA (e.g., a self-replicating RNA) lacks at least a portion of the nucleic acid sequences encoding one or more viral structural proteins CP, E1, E2, E3, and 6K of the unmodified viral genome or replicon RNA. In some embodiments, the modified viral genome or replicon RNA lacks a portion or all of the sequence encoding CP. In some embodiments, the modified viral genome or replicon RNA lacks a portion or all of the sequence encoding E1. In some embodiments, the modified viral genome or replicon RNA lacks a portion or all of the sequence encoding E2. In some embodiments, the modified viral genome or replicon RNA lacks a portion or all of the sequence encoding E3. In some embodiments, the modified viral genome or replicon RNA lacks a portion or all of the sequence encoding 6K. In some embodiments, the modified viral genome or replicon RNA lacks a portion or all of the sequence encoding a combination of CP, E1, E2, E3, and 6K. Some embodiments of the present disclosure provide modified EEEV genome or replicon RNA in which the coding sequences for the nonstructural proteins nsP1, nsP2, nsP3, and nsP4 of the unmodified EEEV genome or replicon RNA are present, but at least a portion or all of the sequences encoding one or more structural proteins (e.g., CP, E1, E2, E3, and 6K) of the EEEV genome or replicon RNA are absent. Some embodiments of the present disclosure provide modified EEEV genome or replicon RNA in which the coding sequences for the nonstructural proteins nsP1, nsP2, nsP3, and nsP4 of the unmodified EEEV genome or replicon RNA are present, but at least a portion or all of the sequences encoding one or more structural proteins (e.g., CP, E1, E2, E3, and 6K) of the EEEV genome or replicon RNA are absent.

[0064] In some embodiments, the modified viral genome or replicon RNA (e.g., self-replicating RNA) lacks a substantial portion of the nucleic acid sequence encoding one or more viral structural proteins. Those skilled in the art will understand that a substantial portion of a nucleic acid sequence encoding a viral structural polypeptide can include sufficient viral structural polypeptide-encoding nucleic acid sequence to allow for putative identification of the polypeptide, either by manual evaluation of the sequence by a skilled artisan or by computer-automated sequence comparison and identification using algorithms such as BLAST. (See, e.g., "Basic Local Alignment Search Tool"; Altschul SF et al., J. Mol. Biol. 215:403-410, 1993). Thus, a substantial portion of a nucleotide sequence includes sufficient sequence to allow for specific identification and / or isolation of a nucleic acid fragment comprising the sequence. For example, a substantial portion of a nucleic acid sequence can include at least about 20%, e.g., about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% of the full-length nucleic acid sequence. As mentioned above, the present disclosure provides nucleic acid molecules and constructs that lack 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 in the compositions and methods of the present disclosure. Thus, the present application includes the complete sequences disclosed herein, e.g., those set forth in the attached sequence listing, as well as substantial portions of those sequences as defined above.

[0065] In some embodiments, the modified viral genome or replicon RNA (e.g., a self-replicating RNA) lacks the entire sequence encoding a viral structural protein, e.g., the modified viral genome or replicon RNA does not include the nucleic acid sequence encoding the structural protein of the viral unmodified genome or replicon RNA.

[0066] The nucleic acid constructs (e.g., vectors or srRNA constructs) of the present disclosure generally have a length of at least about 2 kb. For example, the nucleic acid constructs (e.g., vectors or srRNA) have a length of at least about 2 kb, at least about 3 kb, at least about 4 kb, at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 11 kb, at least about 12 kb, or more than 12 kb. In some embodiments, the nucleic acid construct (e.g., a vector or srRNA) may be about 4 kb to about 20 kb, about 4 kb to about 18 kb, about 5 kb to about 16 kb, about 6 kb to about 14 kb, about 7 kb to about 12 kb, about 8 kb to about 16 kb, about 9 kb to about 14 kb, about 10 kb to about 18 kb, about 11 kb to about 16 kb, about 5 kb to about 18 kb, about 6 kb to about 20 kb, about 5 kb to about 10 kb, about 5 kb to about 8 kb, about 5 kb to about 7 kb, , about 5 kb to about 6 kb, about 6 kb to about 12 kb, about 6 kb to about 11 kb, about 6 kb to about 10 kb, about 6 kb to about 9 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, about 7 kb to about 11 kb, about 7 kb to about 10 kb, about 7 kb to about 9 kb, about 7 kb to about 8 kb, about 8 kb to about 11 kb, about 8 kb to about 10 kb, about 8 kb to about 9 kb, about 9 kb to about 11 kb, about 9 kb to about 10 kb, or about 10 kb to about 11 kb in length. In some embodiments, the nucleic acid construct (e.g., vector or srRNA) has a length of about 6 kb to about 14 kb. In some embodiments, the nucleic acid construct (eg, a vector or srRNA) has a length of about 6 kb to about 16 kb.

[0067] In some embodiments, the nucleic acid construct of the present disclosure further comprises one or more expression cassettes. In principle, the nucleic acid construct disclosed herein can generally comprise any number of expression cassettes. In some embodiments, the nucleic acid construct 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 appreciate that the term "expression cassette" refers to a construct of genetic material that includes a coding sequence and sufficient control information to direct proper transcription and / or translation of the coding sequence in a cell, in vivo and / or ex vivo. The expression cassette can be inserted into a vector for targeting to a desired host cell and / or subject. Thus, in some embodiments, the term expression cassette can be used interchangeably with the term "expression construct." In some embodiments, the term "expression cassette" refers to a nucleic acid construct that includes a gene encoding a protein or functional RNA, operably linked to regulatory elements, such as a promoter and / or a termination signal, and optionally other nucleic acid sequences or combinations thereof that affect the transcription or translation of the gene.

[0068] In some embodiments, at least one of the expression cassettes comprises a promoter operably linked to a heterologous nucleic acid sequence. 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 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. In some embodiments, the nucleic acid molecules of the present disclosure further comprise one or more untranslated regions (UTRs). In some embodiments, at least one of the UTRs is a heterologous UTR.

[0069] In some embodiments, at least one of the expression cassettes comprises a coding sequence of a gene of interest (GOI). In some embodiments, the coding sequence of the GOI is redesigned and / or optimized for desired properties, such as enhanced stability, potency, and expression (e.g., translation efficiency), which may progressively maximize the impact of the manufacture, delivery, and administration of the biological therapeutic. For example, in some embodiments, the coding sequence of the GOI is optimized for expression at a level higher than the expression level of a reference coding sequence. With regard to sequence optimization of a nucleotide sequence, the degeneracy of the genetic code provides the possibility to replace at least one base of the protein encoding sequence of the gene with a different base without changing the amino acid sequence of the polypeptide produced from the gene. Thus, the nucleic acid construct of the present disclosure may also have any base sequence that is altered from any polynucleotide sequence disclosed herein by substitutions related to the degeneracy of the genetic code. References describing codon usage are readily available. In some embodiments, polynucleotide sequence variants may be made for a variety of reasons, such as to optimize expression for a particular host (e.g., to change the codon usage in an alphavirus mRNA to one that is preferred for humans, non-human primates, hamsters, mice, or other organisms, such as monkeys). Thus, in some embodiments, the coding sequence of the GOI is optimized for expression at a level higher than the expression level of a reference coding sequence, such as a coding sequence that is not codon-optimized, in a target host cell, for example, through the use of codons optimized for expression. In some embodiments, the codon-optimized sequence of the GOI results in an enhanced 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 the reference coding sequence that is not codon-optimized. In some embodiments, the codon-optimized sequence of the GOI results in an enhanced expression level of at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold compared to the reference coding sequence that is not codon-optimized.

[0070] Techniques for constructing synthetic nucleic acid sequences encoding GOIs with optimal preferred codons for host cell expression can be determined by techniques well known in the art, computational methods that analyze the commonality of codon usage and their relative abundance for encoding natural proteins in the host cell genome. The codon usage database (http: / / www.kazusa.or.jp / codon) can be used for the generation of codon-optimized sequences in a mammalian cell environment. In addition, various software tools are available for converting sequences of one organism to the optimal codon usage of another host organism, such as the JCat codon optimization tool (www.jcat.de), the Integrated DNA Technology (IDT) codon optimization tool (https: / / www.idtdna.com / CodonOpt), and the Optimizer online codon optimization tool (http: / / genomes.urv.es / OPTIMIZER). Such synthetic sequences can be constructed by techniques well known in the art for the construction of synthetic nucleic acid molecules and may be obtained from various commercial vendors.

[0071] In some embodiments, the coding sequence of the GOI is optimized for enhanced RNA stability and / or expression. RNA stability is generally related to the "half-life" of the RNA. "Half-life" relates to the period required to remove half of the activity, amount, or number of molecules. In the context of the present disclosure, the half-life of an RNA is an indication of the stability of said RNA. The half-life of an RNA may affect the "expression period" of the RNA. Additional information on principles, strategies, and methods for use in enhancing RNA stability can be found, for example, in Leppek K. et al., Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics. bioRxiv. (Preprint). Mar 30, 2021. doi: 10.1101 / 2021.03.29.437587.

[0072] The polypeptide encoded by the GOI can generally be any polypeptide, for example, 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 the influenza A virus H5N1. Non-limiting examples of GOIs include the following: interleukins and interacting proteins, including 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.Additional suitable GOIs include, but are not limited to, 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), hematopoietins (e.g., Epo, Tpo, Flt-3L, SCF, M-CSF, MSP), chemokines and their receptors (e.g., XCL1, XCL2, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL210, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL29, CCL30, CCL310, CCL32, CCL33, CCL34, CCL35, CCL36, CCL37, CCL38, CCL39 ... CL5, CCL7, CCL8, CCL11, 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, such as 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.

[0073] In some embodiments, the GOI may 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 in cancer, or associated with infectious disease. In some embodiments, the antibody targets a surface molecule with immunostimulatory or immunosuppressive function.

[0074] In some embodiments, the GOI may encode an enzyme whose deficiency or mutation is associated with a disease or condition, such as agalsidase beta, agalsidase alpha, imiglucerase, taliglucerase alpha, velaglucerase alpha, alglucerase, sebelipase alpha, laronidase, idursulfase, elosulfase alpha, galsulfase, alglucosidase alpha, and CTFR.

[0075] In some embodiments, the GOI may encode a polypeptide selected from an antigenic molecule, a biotherapeutic molecule, or any combination thereof. In some embodiments, the GOI may encode a polypeptide selected from a tumor-associated antigen, a tumor-specific antigen, a neoantigen, and any combination thereof. In some embodiments, the GOI may encode a polypeptide selected from an estrogen receptor, an intracellular signal transduction enzyme, and a human epidermal growth receptor. In some embodiments, the GOI may encode a biotherapeutic polypeptide selected from an immunomodulator, an angiogenesis modulator, an extracellular matrix modulator, a metabolic modulator, a neurological modulator, and any combination thereof. In some embodiments, the GOI may encode a cytokine selected from a chemokine, an interferon, an interleukin, a lymphokine, and a tumor necrosis factor. In some embodiments, the GOI may 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-15, IL-17, IL-23, IL-27, IL-35, IFNγ, and any subunits thereof. In some embodiments, the GOI may encode a biotherapeutic polypeptide selected from IL-12A, IL-12B, IL-1RA, and any combination thereof.

[0076] In some embodiments, the nucleic acid construct of the present disclosure may be incorporated into a vector. In some embodiments, the vector of the present disclosure may be a single-stranded vector, such as a ssDNA vector or a ssRNA vector. In some embodiments, the vector of the present disclosure may be a double-stranded vector, such as a dsDNA vector or a dsRNA vector. In some embodiments, the vector of the present disclosure may be a plasmid. As described in more detail below, the vector of the present disclosure may be produced using recombinant DNA technology, such as polymerase chain reaction (PCR) amplification, rolling circle amplification (RCA), molecular cloning, etc., or chemical synthesis. Thus, in some embodiments, the vector of the present disclosure may be a fully synthetic vector, such as a fully synthetic ssDNA vector. In some embodiments, the vector of the present disclosure may be a fully synthetic dsDNA vector. In some embodiments, the vector of the present disclosure may be the product of a PCR reaction. In some embodiments, the vector of the present disclosure may be the product of an RCA reaction. In some embodiments, the vector may be a gene delivery vector. In some embodiments, the vector may be used as a gene delivery vehicle to transfer genes into cells.

[0077] In some embodiments, a nucleic acid construct of the present disclosure comprises a nucleic acid sequence encoding a modified EEEV 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. In some embodiments, a nucleic acid construct of the present disclosure comprises a nucleic acid sequence encoding a modified EEEV 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:2. In some embodiments, a nucleic acid construct of the present disclosure comprises a nucleic acid sequence encoding a modified EEEV having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:15. In some embodiments, a nucleic acid construct of the present disclosure comprises a nucleic acid sequence encoding a modified EEEV 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:16. In some embodiments, a nucleic acid construct of the present disclosure comprises a nucleic acid sequence encoding a modified EEEV 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:17. In some embodiments, a nucleic acid construct of the present disclosure comprises a nucleic acid sequence encoding a modified EEEV 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:18. Table 1: Brief description of the sequences according to the sequence listing [Table 1]

[0078] 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 EEEV of interest can be identified and / or isolated by using the sequences identified herein (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18) or others known in the art by genomic sequence analysis, hybridization, and / or PCR using degenerate or gene-specific primers from sequences identified in the respective EEEV genome.

[0079] The molecular techniques and methods by which these new nucleic acid constructs are constructed and characterized are described more fully in the Examples herein.

[0080] In some embodiments, the nucleic acid molecule disclosed herein is a recombinant nucleic acid molecule. As described above, the term recombinant nucleic acid molecule refers to any nucleic acid molecule (e.g., DNA, RNA, etc.) that originates or is indirectly generated by human manipulation of polynucleotides. As a non-limiting example, cDNA is a recombinant DNA molecule, like any nucleic acid molecule made by in vitro polymerase reaction, or connected to it by a linker, or incorporated into a vector (e.g., a cloning vector or an expression vector). As non-limiting examples, a recombinant nucleic acid molecule is one that is (1) synthesized or modified in vitro, e.g., by the use of chemical or enzymatic techniques (e.g., chemical nucleic acid synthesis or using replication, polymerization, exonuclease digestion, endonuclease digestion, ligation, reverse transcription, transcription, base modification (including, e.g., methylation), or recombination of nucleic acid molecules (including homologous recombination and site-specific recombination); (2) contains the linkage of nucleotide sequences that are not linked in nature; (3) has been engineered using molecular cloning techniques to lack one or more nucleotides with respect to a naturally occurring nucleotide sequence; and / or (4) has been engineered using molecular cloning techniques to have one or more sequence changes or rearrangements with respect to a naturally occurring nucleotide sequence.

[0081] Preferably, the nucleic acid molecules disclosed herein are made using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning, etc.) or chemical synthesis. The nucleic acid molecules disclosed herein include naturally occurring nucleic acid molecules and their homologues, including, but not limited to, naturally occurring allelic variants, modified nucleic acid molecules in which one or more nucleotide residues have been inserted, deleted, and / or substituted in such a manner that such modification confers desirable properties in achieving a biological activity as described herein.

[0082] Nucleic acid molecules, including variants of naturally occurring nucleic acid sequences, can be produced 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 a naturally occurring sequence, from which it is derived using a variety of techniques, including, but not limited to, traditional mutagenesis techniques and recombinant DNA techniques (e.g., but not limited to, site-directed mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, cleavage of nucleic acid fragments with restriction enzymes, ligation of nucleic acid fragments, PCR amplification and / or mutagenesis of selected regions of a nucleic acid sequence, recombinational cloning, 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 homologues can be selected from a mixture of modified nucleic acid molecules by screening for the function of the protein or replicon (e.g., 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. B. Recombinant Cells

[0083] As described in more detail below, one aspect of the disclosure relates to recombinant cells that contain a nucleic acid construct described herein and / or that have been engineered to contain (e.g., express) a nucleic acid construct described herein. In some embodiments, a nucleic acid construct of the disclosure (e.g., a vector or srRNA) can be introduced into a host cell to produce a recombinant cell that contains the nucleic acid construct and / or srRNA construct. For example, a nucleic acid construct of the disclosure can be introduced into a host cell to produce a recombinant cell that contains the nucleic acid molecule. Thus, prokaryotic or eukaryotic cells that contain a nucleic acid construct encoding a modified EEEV genome described herein are also a feature of the disclosure. In a related aspect, some embodiments disclosed herein relate to methods of transforming a cell, comprising introducing a nucleic acid construct provided herein into a host cell, such as an animal cell, followed by a method of selecting or screening for the transformed cell. Introduction of the nucleic acid constructs of the present disclosure into cells can be accomplished by methods known to those of skill in the art, such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0084] In one aspect, some embodiments of the present disclosure relate to recombinant cells, such as genetically modified eukaryotic cells, such as animal cells, that comprise the nucleic acid constructs disclosed herein. The nucleic acid constructs can be stably integrated into the host genome, can be episomally replicated, or 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 constructs are maintained and replicated in the recombinant host cell as episomal units. In some embodiments, the nucleic acid constructs are stably integrated into the genome of the recombinant cell. Stable integration can be accomplished by using classical 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 constructs exist in the recombinant host cell as a minicircle expression vector for stable or transient expression.

[0085] A host cell can be either an untransformed cell or a cell that has already been transfected with at least one nucleic acid molecule. Thus, in some embodiments, a host cell can be genetically engineered (e.g., transduced, transformed, or transfected) with at least one nucleic acid molecule.

[0086] Suitable host cells for cloning or expression of the proteins of interest described herein include prokaryotic or eukaryotic cells as described herein. Thus, in some embodiments, the recombinant cell is a prokaryotic cell, such as the bacterium E. coli, or a eukaryotic cell, such as an insect cell (e.g., a mosquito cell or an Sf21 cell), or a mammalian cell (e.g., a COS cell, an NIH 3T3 cell, or a HeLa cell). In some embodiments, the cell is in vivo. 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., obtained from a repository. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a vertebrate cell or an invertebrate cell. In some embodiments, the recombinant cell is a mammalian cell. Suitable host cells for expression of glycosylated proteins can be obtained from multicellular organisms (invertebrates and vertebrates). Examples of vertebrate cells include insect cells.

[0087] Vertebrate cells can also be used as hosts. Mammalian cell lines adapted to growth in suspension can be useful in this regard. 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. In some embodiments, the animal cell is a human cell. In some embodiments, the animal cell is a non-human animal cell. In some embodiments, the cell is a non-human primate cell. Further examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 cells (COS-7), human embryonic kidney cells (e.g., HEK 293 or HEK 293 cells), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells), monkey kidney cells (CV1), human cervical carcinoma cells (HeLa), canine kidney cells (MDCK), buffalo rat hepatocytes (BRL3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor (MMT 060562), TRI cells, FS4 cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (Vero cells), human A549 cells, human cervical cells, human CHME5 cells, human PER.C6 cells, NS0 mouse myeloma cells, selected from the group consisting of human epidermal laryngeal cells, human fibroblast cells, human HUH-7 cells, human MRC-5 cells, human muscle cells, human lymphatic 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, rabbit kidney cells.

[0088] In some embodiments, the recombinant cell is selected from the group consisting of African green monkey kidney cells (Vero cells), baby hamster kidney cells (BHK), Chinese hamster ovary cells (CHO cells), human A549 cells, human cervical cells, human CHME5 cells, human epidermal laryngeal cells, human fibroblast cells, human HEK 293 cells, human HeLa cells, human Hep G2 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.

[0089] 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. Further suitable insect cell lines include, but are not limited to, established cell lines from the insect orders Diptera, Lepidoptera, and Hemiptera, and can be derived from different tissue sources. In some embodiments, the recombinant cell is a cell of a lepidopteran insect cell line. In the past decades, the availability of lepidopteran insect cell lines has increased by about 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 cell is a mosquito cell, e.g., a cell of a mosquito species within the genera Anopheles (An), Culex (Cx.), and Stegomyia aegypti. 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 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, At GRIP-1, At GRIP-2, UM-AVE1, Mos.55, Sua1B, 4A-3B, Mos.43, MSQ43, and LSB-AA695BB. In some embodiments, the mosquito cell is a cell of the C6 / 26 cell line.

[0090] In another aspect, provided herein is a cell culture comprising at least one recombinant cell disclosed herein, and a culture medium. In general, the culture medium can be any suitable culture medium for culturing the cells described herein. Techniques for transforming a wide variety of the above host cells and species are known in the art and described in the technical and scientific literature. Thus, a cell culture comprising at least one recombinant cell disclosed herein is also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art. C. Transgenic Animals

[0091] Also provided in another aspect is a transgenic animal comprising a nucleic acid construct as described herein. 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. In some embodiments, the transgenic animal produces a protein of interest as described herein.

[0092] 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, the non-human animals of the present disclosure are non-human primates. Other animal species suitable for the compositions and methods of the present disclosure include animals that are (i) suitable for genetic recombination and (ii) capable of rearranging immunoglobulin gene segments to generate antibody responses. Examples of such species include, but are not limited to, mice, rats, hamsters, rabbits, chickens, goats, pigs, sheep, and cows. Approaches and methods for preparing transgenic non-human animals are known in the art. Exemplary methods include pronuclear microinjection, DNA microinjection, lentiviral vector-mediated DNA transfer and sperm-mediated transfer into early embryos, adenovirus-mediated DNA transfer into animal sperm (e.g., pigs), retroviral vectors (e.g., avian species), somatic cell nuclear transfer (e.g., in goats). The state of the art in the preparation of transgenic livestock is reviewed in Niemann, H. et al. (2005) Rev. Sci. Tech. 24:285-298.

[0093] In some embodiments, the animal is a vertebrate or invertebrate. In some embodiments, the animal is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the animal is a mammalian subject. In some embodiments, the 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 produced 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 gregorii Argonaute), TALENs genome editing (Transcription Activator-Like Effector Nucleases), etc. In some embodiments, the transgenic animals of the present disclosure can be produced using transgenic microinjection techniques, which do not require the use of homologous recombination techniques and are therefore considered easier to prepare and select than approaches using homologous recombination. In another aspect, provided herein is a method for producing a polypeptide of interest, comprising: (i) raising a transgenic animal as disclosed herein; (ii) culturing a recombinant cell comprising a nucleic acid construct as disclosed herein under conditions whereby the transgenic animal or recombinant cell produces the polypeptide encoded by the GOI.

[0094] In another aspect, provided herein is a method of 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 invertebrate. In some embodiments, the subject is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the subject is a mammalian subject. In some embodiments, the mammalian subject is a human subject. Thus, recombinant polypeptides produced by the methods disclosed herein are also within the scope of this disclosure.

[0095] Non-limiting exemplary embodiments of the disclosed methods for producing recombinant polypeptides may 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 its half-life. In some embodiments, the N-terminus of the produced polypeptide may be further chemically or enzymatically modified to increase its half-life. In some embodiments, the C-terminus of the produced polypeptide is chemically or enzymatically modified to increase its half-life. Non-limiting examples of chemical and enzymatic modifications suitable for the methods described herein include PEGylation, XTENylation, PASylation, ELPylation, and HAPylation. Techniques, systems, and reagents suitable for these modifications are known in the art. Thus, in some embodiments, the polypeptides produced by the methods described herein may be PEGylated, XTENylation, PASylation, ELPylation, and / or HAPylation to increase their half-life. In some embodiments, the produced polypeptide is conjugated to another protein or peptide (eg, serum albumin, an antibody Fc domain, transferrin, GLK, or CTP peptide) to increase half-life. D. Pharmaceutical Compositions

[0096] The nucleic acid constructs, recombinant cells, recombinant polypeptides of the present disclosure can be incorporated into compositions, including pharmaceutical compositions. Such compositions generally include one or more of the nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides described and provided herein, and pharma- ceutically acceptable excipients, such as carriers. In some embodiments, the compositions of the present disclosure are formulated for the prevention, treatment, or management of health conditions, such as immune disorders or microbial infections (e.g., viral, microfungal, or bacterial infections). For example, the compositions of the present disclosure can be formulated as prophylactic compositions, therapeutic compositions, or pharmaceutical compositions that include pharma-ceutically acceptable excipients or mixtures thereof. In some embodiments, the compositions of the present disclosure are formulated for use as vaccines. In some embodiments, the compositions of the present application are formulated for use as adjuvants.

[0097] Thus, in one aspect, provided herein is a pharmaceutical composition comprising a pharma- ceutically acceptable excipient and: a) a nucleic acid construct of the present disclosure (e.g., a vector or an srRNA molecule); b) a recombinant cell of the present disclosure; and / or c) a recombinant polypeptide of the present disclosure.

[0098] Non-limiting exemplary embodiments of pharmaceutical compositions of the present disclosure may include one or more of the following features: In some embodiments, provided herein are compositions comprising a nucleic acid construct disclosed herein (e.g., a vector or srRNA molecule) and a pharma- ceutically acceptable excipient. In some embodiments, provided herein are compositions comprising a recombinant cell disclosed herein and a pharma- ceutically acceptable excipient. In some embodiments, a composition comprises a recombinant polypeptide disclosed herein and a pharma- ceutically acceptable excipient. In some embodiments, a nucleic acid construct disclosed herein (e.g., a vector or srRNA molecule) may be used in naked form or may 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, immune stimulating complexes (ISCOMS), lipid-based nanoparticles (LNPs), solid lipid nanoparticles (SLNs), polyplexes, polymeric nanoparticles, viral replicon particles (VRPs), or conjugated with bioactive ligands (to 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 known in the art. Adjuvants may protect the antigen (e.g., nucleic acid construct, vector, srRNA molecule) from rapid dispersion by its sequestration by localized deposition, or they may include substances that stimulate the host to secrete factors that are chemotactic for macrophages and other immune system components. An appropriate selection can be made by the skilled artisan, for example, from those described below.

[0099] In some embodiments, a composition of the present disclosure includes one or more of the following: a physiological buffer, a liposome, a lipid-based nanoparticle (LNP), a solid lipid nanoparticle (SLN), a polyplex, a polymeric nanoparticle, a viral replicon particle (VRP), a microsphere, an immune stimulating complex (ISCOM), a conjugate of a bioactive ligand, or any combination thereof.

[0100] The composition of the present disclosure may be formulated in a form that is compatible with its intended route of administration, such as liposomes, lipid-based nanoparticles (LNPs), or polymeric nanoparticles. Thus, in some embodiments, the composition of the present disclosure is formulated in liposomes. In some embodiments, the composition of the present disclosure is formulated in 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. In addition, adaptive immune responses to LNPs are unlikely to occur, allowing for repeated dosing of LNPs.

[0101] Lipids suitable for the compositions and methods described herein can be cationic lipids, ionized cationic lipids, anionic lipids, or neutral lipids.

[0102] In some embodiments, the LNPs of the present disclosure may include one or more ionizable lipids. As used herein, the term "ionizable lipid" refers to a lipid that is cationic or that ionizes (protonates) when the pH falls below the pKa of the lipid's ionizable group, but is more neutral at higher pH values. At pH values ​​below the pKa, the lipid can then bind to negatively charged nucleic acids (e.g., oligonucleotides). As used herein, the term "ionizable lipid" includes lipids that assume a positive charge upon pH reduction from physiological pH, as well as any number of lipid species that carry a net positive charge at a selected pH, such as physiological pH. Permanently cationic lipids, such as DOTMA, have proven too toxic for clinical use. The ionizable lipid is preferably present in the lipid formulation in accordance with other embodiments in a ratio of about 30 to about 70 mol%, in other embodiments about 30 mol%, in other embodiments about 40 mol%, in other embodiments about 45 mol%, in other embodiments about 47.5 mol%, in still other embodiments about 50 mol%, and in still others about 60 mol% ("mol%" refers to the percentage of the total moles that are of the particular component). The term "about" in this paragraph refers to a range of plus or minus 5 mol%. DODMA, or 1,2-dioleyloxy-3-dimethylaminopropane, is an ionizable lipid such as DLin-MC3-DMA or 0-(Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N-dimethylamino) ("MC3").

[0103] Exemplary ionizable lipids suitable for the compositions and methods of the present disclosure include those described in PCT Publications WO2020252589A1 and WO2021000041A1, U.S. Patent Nos. 8,450,298 and 10,844,028, and Love KT et al., Proc Natl Acad Sci USA, Feb. 2, 2010 107 (5) 1864-1869, which are incorporated herein by reference in their entirety. Thus, in some embodiments, the LNPs of the present disclosure include 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, the LNPs include an ionized cationic lipid selected from the group consisting of ALC-0315, C12-200, LN16, MC3, MD1, SM-102, and any combination thereof. In some embodiments, the LNPs of the present disclosure include a C12-200 lipid. The structure of the C12-200 lipid is known in the art and described, for example, in U.S. Pat. Nos. 8,450,298 and 10,844,028, which are incorporated herein by reference in their entirety. In some embodiments, the C12-200 is combined with cholesterol, C14-PEG2000, and DOPE. In some embodiments, the C12-200 is combined with DSPC and DMG-PEG2000.

[0104] In some embodiments, the LNPs of the present disclosure include one or more cationic lipids. Several different ionizable cationic lipids have been developed for use in LNPs. Suitable cationic lipids include, but are not limited to, 98N12-5, C12-200, C14-PEG2000, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. In one type of LNP, a GalNAc moiety is attached to the exterior of the LNP and 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 srRNA constructs and nucleic acid constructs of the present disclosure.

[0105] In some embodiments, the LNPs of the present disclosure include one or more neutral lipids. 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 include one or more ionized lipid compounds described in PCT Publications WO2020252589A1 and WO2021000041A1.

[0106] Many other lipids or lipid combinations known in the art can be used to prepare LNPs. Non-limiting examples of lipids suitable for use in preparing LNPs include DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Further non-limiting examples of cationic lipids include 98N12-5, C12-200, C14-PEG2000, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, 7C1, and any combination thereof. Further 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.

[0107] In some embodiments, the lipid to nucleic acid mass ratio in the LNP delivery system is about 100:1 to about 3:1, about 70:1 to about 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 have an average diameter of about 1000 nm, about 500 nm, about 250 nm, about 200 nm, about 150 nm, about 100 nm, about 75 nm, about 50 nm, or less than about 25 nm. In some embodiments, the LNPs have an average diameter ranging from about 70 nm to 100 nm. In some embodiments, the LNPs have an average diameter ranging from about 88 nm to about 92 nm, 82 nm to about 86 nm, or about 80 nm to about 95 nm.

[0108] In some embodiments, the compositions of the present disclosure are formulated in polymeric nanoparticles. In some embodiments, the compositions are immunogenic compositions, e.g., compositions that can stimulate an immune response in a subject. In some embodiments, the immunogenic compositions are formulated as vaccines. In some embodiments, the pharmaceutical compositions are formulated as adjuvants. In some embodiments, the immunogenic compositions are formulated as biotherapeutics, e.g., vehicles for gene delivery of different biologically active molecules. Non-limiting examples of biotherapeutics 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 compositions are formulated as adjuvants.

[0109] 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, intraperitoneal, intramuscular, intranodal, intratumoral, intraarticular, intravenous, subcutaneous, intravaginal, and oral administration.

[0110] 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 should be sterile and fluid to the extent that easy syringability exists. The composition is stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants, for example, 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 the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0111] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or a combination of the ingredients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those listed above.

[0112] In some embodiments, the pharmaceutical compositions of the present disclosure are formulated for inhalation, such as, for example, an aerosol, spray, mist, liquid, or powder. Administration by inhalation can be in the form of either a dry powder or an aerosol formulation, which is inhaled by any subject (e.g., patient) through the use of an inhalation device, such as a microspray, pressurized metered dose inhaler, or nebulizer.

[0113] In some embodiments, the composition is formulated for one or more of intranasal, transdermal, intramuscular, intranodal, intravenous, intraperitoneal, oral, intravaginal, or intracranial administration. In some embodiments, the administered composition results in increased production of interferon in the subject. Methods of the Disclosure

[0114] Administration of any one of the therapeutic compositions, e.g., nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions described herein, may 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 (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions described herein may be useful for inducing a pharmacodynamic effect in a subject. In some embodiments, the nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions described herein may be effective to modulate, e.g., elicit or suppress, an immune response in a subject in need thereof.

[0115] Analysis of the compositions described herein for their ability to provide a pharmacodynamic effect can be performed in vivo and / or ex vivo. Examples of pharmacodynamic effects that can be analyzed include: immunogenic effects (e.g., elicitation of an immune response in vivo), biomarker responses, therapeutic effects, preventive effects, desired effects, undesirable effects, adverse effects, and effects in disease models. In some embodiments, evaluation of the pharmacodynamic effect includes evaluating induction of an immune response in vivo (see, e.g., Examples 1-4). In some embodiments, evaluation of the pharmacodynamic effect includes evaluating induction of cytokine pathways that can enhance immune responses and prevent angiogenesis and metastasis (see, e.g., Examples 1-4).

[0116] In some embodiments, the nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions described herein may be incorporated into therapeutic agents for use in methods of treating subjects who have, are suspected of having, or may be at high risk of developing one or more relevant health conditions. Exemplary health conditions or diseases may 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.

[0117] Examples of autoimmune diseases suitable for the methods of the present disclosure include, but are not limited to, rheumatoid arthritis, osteoarthritis, Still's disease, familial Mediterranean fever, systemic sclerosis, multiple sclerosis, ankylosing spondylitis, Hashimoto's thyroiditis, systemic lupus erythematosus, Sjogren's syndrome, diabetic retinopathy, diabetic vascular disease, diabetic neuralgia, insulitis, psoriasis, alopecia areata, warm and cold autoimmune hemolytic anemia (AIHA), pernicious anemia, acute inflammatory disease, autoimmune adrenalitis, chronic inflammatory demyelinating polyneuropathy (CIDP), Lambert-Eaton syndrome, lichen sclerosis, Lyme disease, Grave's disease, Behcet's disease, Meniere's disease, reactive arthritis (Reiter's syndrome), Churg-Strauss syndrome, Cogan's syndrome, Crest's syndrome, pemphigus vulgaris and pemphigus foliaceus, pemphigoid, rheumatoid arthritis ... Polymyalgia ulcerata, polymyositis, primary biliary cirrhosis, pancreatitis, peritonitis, psoriatic arthritis, rheumatic fever, sarcoidosis, Sjorgensen's syndrome, scleroderma, coeliac disease, stiff man syndrome, Takayasu's arteritis, transient gluten intolerance, autoimmune uveitis, vitiligo polychondritis, dermatitis herpetiformis (DH) or Duhring'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, polyglandular autoimmune syndrome, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), urticaria, autoimmune infertility, juvenile rheumatoid arthritis, sarcoidosis, and autoimmune cardiomyopathy.

[0118] Non-limiting examples of infectious diseases suitable for the methods of the present disclosure include viral infections 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 infectious diseases suitable for the methods of the present disclosure include infections with intracellular parasites such as Leishmania, Rickettsia, Chlamydia, Coxella, Plasmodium, Brucella, Mycobacteria, Listeria, Toxoplasma, and Trypanosoma.

[0119] In some embodiments, the nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions are used to treat, for example, glomerulonephritis, inflammatory bowel disease, nephritis, peritonitis, psoriatic arthritis, osteoarthritis, Still's disease, familial Mediterranean fever, generalized scleroderma 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 disease, Behcet's disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, immune cytoplasmic hematopoietic syndrome, pulmonary fibrosis ... In some embodiments, the therapeutic agent may be effective in the treatment and / or prevention of immune, autoimmune, or inflammatory diseases such as thrombocytopenia, hemolytic anemia, myasthenia gravis, lupus nephritis, 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 glandular fever, HIV, herpes virus related diseases, human viral infections, coronaviruses, other enteroviruses, herpes viruses, influenza viruses, parainfluenza viruses, respiratory syncytial virus or adenovirus infections, bacterial pneumonia, trauma, sepsis, stroke / cerebral edema, ischemia-reperfusion injury, and hepatitis C.

[0120] Non-limiting examples of inflammatory diseases suitable for the methods of the present disclosure include inflammatory diseases such as, for example, asthma, inflammatory bowel disease (IBD), chronic colitis, splenomegaly, and rheumatoid arthritis.

[0121] Thus, in one aspect, provided herein is a method of inducing an immune response in a subject in need of modulating (e.g., inducing) an immune response, the method 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.

[0122] In another aspect, provided herein is a method of preventing and / or treating a condition in a subject in need thereof, the method 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) a pharmaceutical composition described in any one of the present disclosure.

[0123] 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).

[0124] 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 and / or autoimmune disease. In some embodiments, the subject has or is suspected of having a condition associated with an inflammatory and / or autoimmune disease, and / or a rare disease (e.g., including but not limited to, Familial Mediterranean Fever or Adult-Onset Still's Disease).

[0125] In some embodiments, the disclosed compositions are formulated to be compatible with its intended route of administration. For example, the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure can be given orally or by inhalation, but they are more likely to be administered via parenteral routes. Examples of parenteral routes of administration include, for example, intravenous, intranodal, intradermal, subcutaneous, transdermal (topical), transmucosal, vaginal, and rectal administration. Solutions or suspensions used for parenteral application may contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; and tonicity adjusters such as sodium chloride or dextrose. The pH may be adjusted with acids or bases such as monobasic and / or dibasic sodium phosphate, hydrochloric acid or sodium hydroxide (e.g., to a pH of about 7.2 to 7.8, e.g., 7.5). Parenteral preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0126] Dosage, toxicity and therapeutic efficacy of such subject nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure can be determined, for example, by measuring the LD50 (the dose lethal to 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50 The therapeutic index can be expressed as a ratio of 0 to 1. Compounds that exhibit a high therapeutic index are generally suitable. While compounds that exhibit toxic side effects may be used, care must be taken to design a delivery system that targets the compound to the site of the affected tissue in order to minimize potential damage to uninfected cells and reduce side effects.

[0127] For example, the data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of the compound is generally administered at an ED 50 The therapeutically effective dose for any compound used in the methods of the present disclosure can be estimated initially from cell culture assays. The dose is determined based on the IC 50 The test compound may 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 can be measured, for example, by high performance liquid chromatography.

[0128] Therapeutic compositions, e.g., nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions described herein, can be administered one or more times per day to one or more times per week (including once every other day). One of skill in the art will appreciate that certain factors, including but not limited to the severity of the disease, previous treatments, the general health and / or age of the subject, and other diseases present, may affect the dosage and timing required to effectively treat the subject. Furthermore, treatment of a subject with a therapeutically effective amount of the 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 2-14 days, e.g., 9 days, followed by administration every 8 hours for an additional 5 days. With respect to nucleic acid constructs (e.g., vectors or srRNA molecules) and recombinant polypeptides, the therapeutically effective amount (e.g., effective dosage) of the nucleic acid construct or recombinant polypeptide of the present disclosure will depend on the nucleic acid construct or recombinant polypeptide selected. For example, a single dose in the range of about 0.001-0.1 mg / kg of patient weight may be administered. In some embodiments, about 0.005, 0.01, 0.05 mg / kg may be administered. In some embodiments, a single dose in the range of about 0.03 μg-300 μg / kg of patient weight may be administered. In some embodiments, a single dose in the range of about 0.3 mg-3 mg / kg of patient weight may be administered.

[0129] As discussed above, a therapeutically effective amount includes an amount of a therapeutic composition that is sufficient to promote a particular effect when administered to a subject, such as a person having, suspected of having, or at risk for a health condition, such as a disease or infection. In some embodiments, an effective amount includes an amount sufficient to prevent or delay the onset of a symptom of a disease or infection, alter the course of a symptom of a disease or infection (such as, but not limited to, slow the progression of a symptom of a disease or infection), or reverse a symptom of a disease or infection. It will be understood that for any given case, the appropriate effective amount can be determined by one of skill in the art using routine experimentation.

[0130] The efficacy of a treatment, including the disclosed therapeutic compositions, for the treatment of a disease or infection can be determined by a skilled clinician. However, a treatment is considered an effective treatment if at least any one or all of the signs or symptoms of the disease or infection are improved or ameliorated. Efficacy can also be measured by a reduction in the deterioration of an individual, as assessed by the need for hospitalization or medical intervention (e.g., the progression of the disease or infection is stopped or at least slowed). 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., stopping or slowing the progression of the symptoms; or (2) relieving the disease or infection, e.g., causing regression of the symptoms; and (3) preventing or reducing the likelihood of the symptoms developing.

[0131] In some embodiments, the nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure may be administered to a subject in a composition with a pharma- ceutically acceptable carrier and in an amount effective to stimulate an immune response. Generally, a subject is immunized with an initial series of injections (or administered via one of the other routes described below) and then given boosters to enhance the protection provided by the original series of injections. The initial series of injections and subsequent boosters are administered at such doses and for such periods as are necessary to stimulate an immune response in the subject. In some embodiments, the administered composition results in an increase in interferon production in the subject, e.g., by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100%, as compared to interferon production in a subject to which the composition is not administered. In some embodiments of the disclosed methods, the subject is a mammal. In some embodiments, the mammal is a human subject.

[0132] As mentioned above, pharma- ceutically 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 further 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, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. 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.

[0133] Sterile injectable solutions can be prepared by incorporating the nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, and / or recombinant polypeptides, as required with one or a combination of ingredients enumerated above, in the required mount in an appropriate solvent, followed by filtered sterilization.

[0134] When the nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions are properly protected, as described above, they may be administered orally, for example, with an inert diluent or an assimilable edible carrier. The nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions and other ingredients may 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 compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0135] In some embodiments, the nucleic acid constructs (e.g., vectors or srRNA molecules) and recombinant polypeptides of the present disclosure can be delivered to cells or subjects by lipid-based nanoparticles (LNPs). LNPs are generally less immunogenic than viral particles. While many humans have pre-existing immunity to viral particles, they do not have pre-existing immunity to LNPs. Furthermore, adaptive immune responses to LNPs are less likely to occur, allowing for repeated administration of LNPs.

[0136] Several different ionizable cationic lipids have been developed for use in LNPs. These include C12-200, MC3, LN16, and MD1, among others. For example, in one type of LNP, a GalNAc moiety is attached to the outside of the LNP and serves as a ligand for uptake into the liver via the Asia riloglycoprotein receptor. Any of these cationic lipids can be used to formulate LNPs for delivery of the nucleic acid constructs (e.g., vectors or srRNA molecules) and recombinant polypeptides of the present disclosure to the liver.

[0137] 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, the size of the nanoparticles can range from 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

[0138] LNPs can be made from cationic, anionic, or neutral lipids. Neutral lipids such as the fusogenic phospholipid DOPE and the membrane component cholesterol can be included in LNPs as "helper lipids" to enhance transfection activity and nanoparticle stability. Limitations of cationic lipids include poor stability, low efficacy due to rapid clearance, and the generation of inflammatory or anti-inflammatory responses. LNPs can have hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.

[0139] Many 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.

[0140] In some embodiments, lipids can be combined in any number of molar ratios to produce LNPs. Additionally, polynucleotides can be combined with lipids in a wide range of molar ratios to produce LNPs.

[0141] In some embodiments, the therapeutic compositions described herein, e.g., nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions, are incorporated into therapeutic compositions for use in methods of preventing or treating subjects suffering from, suspected of suffering from, or at high risk of developing, cancer, an autoimmune disease, and / or an infectious disease.

[0142] In some embodiments, the therapeutic compositions described herein, e.g., nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions, are incorporated into a therapeutic composition for use in a method of preventing or treating a subject having, suspected of having, or potentially at high risk of 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. Additional Treatments

[0143] In some embodiments, the compositions described herein are 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 simultaneously with the second therapy. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first treatment is administered before the second therapy. In some embodiments, the first therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and / or after the second therapy. In some embodiments, the first therapy and the second therapy are administered in alternation. In some embodiments, the first and second therapies are administered together in a single formulation. kit

[0144] Also provided herein are various kits for carrying out the methods described herein, as well as instructions for producing and using them. In particular, some embodiments of the present disclosure provide kits for modulating immune responses in a subject. Some other embodiments relate to kits for preventing a condition in a subject in need of prevention. Some other embodiments relate to kits for methods of treating a condition in a subject in need of treatment. For example, in some embodiments, kits are provided herein that include one or more of the nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions provided and described herein, as well as written instructions for producing and using them.

[0145] In some embodiments, the kit of the present disclosure further comprises one or more means useful for administering any one of the provided nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions to a subject. For example, in some embodiments, the kit of the present disclosure further comprises 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 (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions to a subject. In some embodiments, the kit can have one or more additional therapeutic agents that can be administered simultaneously or sequentially with other kit components for a desired purpose, for example, to diagnose, prevent, or treat a health condition of a subject in need of diagnosis, prevention, or treatment.

[0146] Any of the above-mentioned kits can further comprise one or more additional reagents, where the additional reagents can be selected from a dilution buffer; a reconstitution solution, a wash buffer, a control reagent, a control expression vector, a negative control, a positive control, a reagent suitable for in vitro production of a provided nucleic acid construct (e.g., a vector or an srRNA molecule), a recombinant cell, a recombinant polypeptide, and / or a pharmaceutical composition of the present disclosure.

[0147] In some embodiments, the kit components can be in separate containers. In some other embodiments, the kit components can be combined in a single container. Thus, in some embodiments of the present disclosure, the kit includes one or more nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions provided and described herein in one container (e.g., a sterile glass or plastic vial), and an additional therapeutic agent in another container (e.g., a sterile glass or plastic vial).

[0148] In another embodiment, the kit includes a combination of compositions described herein, including one or more nucleic acid constructs (e.g., vectors or srRNA molecules), 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, in a single, common container.

[0149] Where the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit includes a device for performing such administration (e.g., an infusion device or catheter). For example, the kit includes one or more hypodermic needles or other infusion devices discussed above that contain one or more nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, and / or recombinant polypeptides of the present disclosure.

[0150] In some embodiments, the kit can further include instructions for using the components of the kit to practice the methods disclosed herein. For example, the kit can include a package insert containing information regarding the pharmaceutical compositions and dosage forms in the kit. Typically, such information will assist patients and physicians in effectively and safely using the enclosed pharmaceutical compositions and dosage forms. For example, the following information regarding the combinations of the present disclosure may be provided in the package insert: pharmacokinetics, pharmacodynamics, clinical trials, efficacy parameters, indications and usage, contraindications, warnings, cautions, adverse reactions, overdosage, appropriate usage and dosage, how it is supplied, suitable storage conditions, references, manufacturer / supplier information, and intellectual property information.

[0151] The instruction manual for carrying out the method is generally recorded on a suitable recording medium. For example, the instruction manual can be printed on a substrate such as paper or plastic. The instruction manual can be present in the kit as a package insert, in the label of the container of the kit or its components (e.g., associated with the packaging or subpackaging), etc. The instruction manual can be present as an electronic storage data file present on a suitable computer-readable storage medium, such as a CD-ROM, a floppy disk, a flash drive, etc. In some instances, the actual instruction manual is not present in the kit, but a means can be provided for obtaining the instruction manual from a remote information source (e.g., via the Internet). An example of this embodiment is a kit that includes a web address where the instruction manual can be viewed and / or where the instruction manual can be downloaded. As with the instruction manual, this means for obtaining the instruction manual can be recorded on a suitable substrate.

[0152] 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.

[0153] Any reference cited herein is not admitted to constitute prior art. The discussion of the references states what the authors assert, and applicants reserve the right to challenge the accuracy and relevance of the cited documents. Although many sources of information, including scientific journal articles, patent documents, and textbooks, are referenced herein, it is expressly understood that this reference is not an admission that any of these documents form part of the common general knowledge in the art.

[0154] The general method discussion provided herein is for illustrative purposes only: other alternative methods and substitutions will be apparent to those of skill in the art upon review of this disclosure, and are to be within the spirit and scope of this application.

[0155] Further embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not intended to limit the scope of the disclosure or the claims. EXAMPLES

[0156] 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 of skill in the art. Such techniques are described, for example, 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). “Sambrook”); Ausubel, FM (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, DM et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, MG et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998).Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, KB, Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, EA (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, SL et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, SC (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences BV, the disclosures of which are incorporated herein by reference.

[0157] Further embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not intended to limit the scope of the disclosure or claims. Example 1 Construction of EEEV vector

[0158] This example describes the results of experiments performed to construct a base EEEV vector (e.g., without a heterologous gene) that is subsequently used for the construction of an EEEV vector with expression of a gene of interest (e.g., the hemagglutinin precursor (HA) of influenza A virus H5N1).

[0159] An initial observation was made that publicly available alphavirus genome data does not always provide nucleotide sequences in which the nucleic acid sequences encoding structural proteins can be directly replaced with a gene of interest (GOI) resulting in a self-replicating RNA and transgene expression replicon. As described in more detail below, it is possible to replace the structural polyprotein genes of EEEV strain FL93-939 with the hemagglutinin (HA) gene from influenza A virus H5N1 (see, e.g., FIG. 2B) to generate a replicon (e.g., self-replicating RNA) capable of RNA replication and transgene expression in transfected BHK-21 cells (see, e.g., FIG. 3B). A base EEEV vector (i.e., without a heterologous gene of interest) was constructed as follows: the base EEEV vector (see, e.g., FIG. 2A) was de novo synthesized with four ~4 kb portions (Twist Bioscience) from a reference sequence (Genbank EF151502) with some modifications. Silent G301A, G4516A, and G7399A mutations were incorporated to eliminate a SapI restriction site. A silent A3550C mutation was incorporated to eliminate a SpeI restriction site. A silent G5725A mutation was incorporated to eliminate an Esp3I restriction site. A unique restriction site (SpeI, 5'-A'CTAG,T-3') was incorporated in place of the coding sequence of the native EEEV structural gene (where 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). 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) was inserted upstream of the SpeI site and a 3' adapter sequence (5'-GACCGCTACGCCCCAATGACCCGACCAGC -3'; SEQ ID NO:4) was inserted downstream of the SpeI site.A bacteriophage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO:5) was included upstream of the EEEV genomic sequence and downstream containing a poly(A) sequence followed by a SapI site (which cuts upstream of the recognition site). Immediately downstream of the SapI site is a T7 termination sequence (5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO:6) followed by a unique restriction enzyme cleavage site (NotI, 5'-GC'GGCC,GC-3'). The parts were combined in a five-piece Gibson Assembly® reaction: the linearized pYL backbone and the four synthetic fragments, resulting in the EEEV-based vector.

[0160] Construction of EEEV vectors containing heterologous genes was performed as follows: The EEEV vector depicted in Figure 2B was constructed by linearization of the empty EEEV vector of Figure 2A by SpeI digestion. The hemagglutinin (HA) gene from influenza (Genbank AY651334) was codon-optimized / refactored in silico for human expression and synthesized de novo (IDT). The synthesis product was amplified using primers that added 5' and 3' adapter sequences to the ends of the HA gene. The digestion and PCR products were combined by the Gibson Assembly® procedure to obtain the final vector. Example 2 In vitro evaluation of modified EEEV vectors

[0161] This example describes the results of in vitro experiments performed to evaluate the expression levels of the synthetic EEEV replicon constructs (e.g., self-replicating RNAs) described in Example 1 above, and to investigate their various differential behaviors (e.g., replication and protein expression).

[0162] In these experiments, synthetic replicon constructs (eg, self-replicating RNA) derived from EEEV strain FL93-939 were designed and subsequently evaluated.

[0163] In vitro transcription: srRNA was prepared by in vitro transcription from SapI-linearized plasmid templates using bacteriophage T7 polymerase with either a 5'ARCA cap (HiScribe™ T7 ARCA mRNA kit, NEB) or uncapped transcription (HiScribe™ T7 High Yield RNA Synthesis Kit, NEB) followed by addition of a 5' Cap 1 (Vaccinia Capping System, mRNA Cap 2'-O-Methyltransferase, NEB). srRNA was then purified using phenol / chloroform extraction, LiCl precipitation, or column purification (Monarch® RNA Cleanup Kit, NEB). srRNA concentrations were determined by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific).

[0164] Replication: srRNA was transformed into BHK-21 or Vero cells (e.g., 4D-Nucleofector™, Lonza) by electroporation. 18–20 h after transformation, cells were fixed and permeabilized (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen) and subsequently stained with a PE-labeled anti-dsRNA mouse monoclonal antibody (J2, Scicons) to quantify the frequency of dsRNA+ cells and the mean fluorescence intensity (MFI) of dsRNA in individual cells by fluorescent flow cytometry.

[0165] Protein expression: RNA was transfected into BHK-21 or Vero cells (e.g., 4D-Nucleofector™, Lonza) by electroporation. 18-20 hours after transfection, cells were fixed and permeabilized (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen) and stained using an APC-conjugated anti-HA mouse monoclonal antibody (2B7, Abcam) to quantify the frequency of cells expressing HA protein in individual cells and the mean fluorescence intensity (MFI) of HA protein by fluorescence flow cytometry.

[0166] Additional experiments: BHK-21 or Vero cells are pretreated with a titration curve of recombinant interferon (IFN) prior to electroporation of RNA and the effect on vector replication and protein expression is measured using the assays described above.

[0167] The experimental results described above illustrate that self-replicating RNA (srRNA) constructs exhibiting RNA replication, e.g., as detected by flow cytometry as described above, can be considered as promising and practical vectors for inducing pharmacokinetic effects. In addition, srRNA constructs exhibiting protein expression of transgene(s) can be considered as promising and practical vectors for inducing additional pharmacokinetic effects (e.g., eliciting an immune response in the host). Example 3 In vitro evaluation of modified EEEV vectors

[0168] This example describes the results of in vitro experiments carried out to assess the expression levels of synthetic EEEV self-replicating RNAs (srRNAs) and to investigate their various differential behaviors (e.g., replication and protein expression).

[0169] In these experiments, synthetic srRNAs derived from EEEV strain FL93-939 were designed and subsequently evaluated, including a control VEEV srRNA expressing two reference transgenes (RBI296, RBI298), a VEEV srRNA encoding both IL-1RA and IL-12 in two forms (RBI299, RBI300), and an EEEV srRNA encoding both IL-1RA and IL-12 in two forms (RBI305, RBI306).

[0170] In vitro transcription: srRNA was prepared by in vitro transcription from a SapI-linearized plasmid template with bacteriophage T7 polymerase by uncapped transcription (HiScribe™ T7 High Yield RNA Synthesis Kit, NEB) followed by addition of a 5' Cap 1 (Vaccinia Capping System, mRNA Cap 2´-O-Methyltransferase, NEB). srRNA was then purified by LiCl precipitation. srRNA concentration was determined by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific).

[0171] Protein expression: RNA was transfected into BHK-21 cells (4D-Nucleofector™, Lonza) by electroporation. Conditioned medium was harvested from the cells at 24 and 48 hours post-transfection. Secreted IL-1RA was assessed in a bioactivity assay by pre-incubating HEK-Blue™ IL-1R cells (InvivoGen) with various concentrations of recombinant IL-1RA (Peprotech) or conditioned medium. Recombinant IL-1B (Invivogen) was added to the cells and incubated overnight, after which the SEAP reporter was quantified using QUANTI-blue™ (Invivogen) (see, e.g., FIG. 4A).

[0172] Secreted IL-12 was assessed by a bioactivity test by incubating various concentrations of recombinant IL-12 (Peprotech) or conditioned medium overnight on IL-12 bioassay cells (Promega) in DMEM, and then the luciferase reporter was quantified using Bio-Glo™ Luciferase (Promega) (see, e.g., FIG. 4B).

[0173] The experimental results described above illustrate that srRNA constructs that exhibit protein expression of transgene(s) can be considered as promising practical vectors to induce additional pharmacokinetic effects (e.g., elicit an immune response in the host). Evaluation of the activity of proteins expressed from srRNA vectors is important to ensure that the expressed proteins possess the intended function to elicit pharmacokinetic effects. Differences in relative protein expression between vectors can provide advantages (e.g., lower doses to achieve comparable levels of protein expression). Example 4 In vivo evaluation of modified EEEV vectors

[0174] This example describes the results of in vivo experiments performed to evaluate any differential immune responses following vaccination with the synthetic EEEV replicon constructs (e.g., self-replicating RNA) described in Examples 1 and 2 above (e.g., both unformulated and LNP-formulated vectors).

[0175] In these experiments, a synthetic replicon construct derived from EEEV strain FL93-939 was designed and subsequently evaluated.

[0176] Mice and injections: Female C57BL / 6 or BALB / c mice were purchased from Charles River Labs or Jackson Laboratories. On the day of dosing, 10 μg of material is injected intramuscularly into both quadriceps. Vectors are administered unformulated in saline or LNP formulated. 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 days 14 and / or 35 and serum is isolated on days 14 and / or 35. The in vivo immunogenicity of srRNA encoding the viral antigen, rabies glycoprotein G, was assessed by determining antigen-specific splenic T cell responses by ELISpot (FIG. 5A) and anti-rabies neutralizing antibody titers from serum (FIG. 5B) after two immunizations. All srRNA immunized groups showed robust T cell responses compared to saline controls (see, e.g., FIG. 5A), but differential responses were observed between the srRNA vaccines. Similarly, all srRNA immunized groups showed protective neutralizing antibody titers, with some variation between the srRNA vaccines (see, e.g., FIG. 5B). In addition to infectious disease antigens, the immunogenicity of srRNA-based vaccines against cancer antigens was evaluated (FIGS. 6A-6C). Each srRNA vaccine co-encoded sequences from ESR1, HER2, and HER3. Splenic T cell responses against these three antigens were measured using ELISpot analysis in mice that received two immunizations. Robust T cell responses were observed against all three targets, but response patterns differed between the srRNA vectors (see, e.g., FIGS. 6A-6C).

[0177] For protein expression studies, animals are dosed on day 0 and protein expression is assessed by serum ELISA on days 3 and / or 7. srRNA encoding human IL12A and IL12B to form IL-12p70 was administered intramuscularly in mice. Serum was collected on day 7 to detect systemic levels of protein (see, e.g., FIG. 7). To confirm the functionality of srRNA encoding biotherapeutic proteins, animals were administered srRNA from EEEV FL93-939 carrying the coding sequences of species-matched mouse Il12a and Il12b genes. IL-12 functionality is measured by assessing induction of IFNγ as a downstream pharmacodynamic marker. Serum from mice 3 days after srRNA administration showed detectable levels of IFNγ (see, e.g., FIG. 8).

[0178] LNP formulation: For some studies, replicon RNA (e.g., self-replicating RNA) was formulated into lipid nanoparticles using a microfluidic mixer and analyzed for particle size, polydispersity using dynamic light scattering and encapsulation efficiency. LNPs are constructed from ionizable lipids, cholesterol, PEG-2K, and DOPE.

[0179] ELISpot. To measure the magnitude of antigen-specific T cell responses, IFNγ ELISpot analysis is performed using the Mouse IFNγ ELISpot PLUS Kit (HRP) (MabTech) according to the manufacturer's instructions. Briefly, splenocytes are isolated and plated at 2–5 × 10 in medium containing either peptide pools representing rabies glycoprotein G, ESR1, HER2, or HER3, PMA / ionomycin as a positive control, or DMSO as a mock stimulation. 6 The cells were resuspended at a concentration of 1000 cells / mL.

[0180] Antibodies. Neutralizing antibody responses to rabies virus are measured using a rapid fluorescent focus inhibition test. Briefly, serum dilutions are mixed with a standard amount of live rabies virus and incubated. If neutralizing anti-rabies antibodies are present, they neutralize the virus. Cultured cells are then added and the serum / virus / cells are incubated together. Uncoated rabies virus (i.e., not neutralized by the antibodies) will infect the cells and this can be visualized by microscopy. Endpoint titer calculations were performed from the percentage of virus-infected cells observed on the slide.

[0181] ELISA. Detection of human IL-12p70 was performed using a commercial kit from R&D Systems (DY1270), Human IL-12p70 DuoSet ELISA. Detection of mouse serum IFN-gamma was performed using a commercial kit from R&D Systems (Mouse IFN-gamma DuoSet).

[0182] Taken together, these data demonstrate that EEEV-based srRNA vectors can be used to encode antigen(s) and biotherapeutic proteins and can be used as vectors for both vaccines and therapeutics, respectively, to produce desired pharmacodynamic effects in vivo.

[0183] While certain alternatives of the present disclosure have been disclosed, it is to be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. Accordingly, no limitations are intended to the precise summary and disclosure presented herein.

Claims

**Claim 1** A nucleic acid construct comprising a nucleic acid sequence encoding a modified eastern equine encephalitis virus (EEEV) genome or replicon RNA, wherein the modified EEEV genome or replicon RNA lacks at least a part of the nucleic acid sequence encoding one or more viral structural proteins. **Claim 2** The nucleic acid construct according to claim 1, wherein the modified viral genome or replicon RNA lacks a substantial part of the nucleic acid sequence encoding one or more viral structural proteins. **Claim 3** The nucleic acid construct according to any one of claims 1 to 2, wherein the modified viral genome or replicon RNA does not contain a nucleic acid sequence encoding a viral structural protein. **Claim 4** The nucleic acid construct according to claim 1, further comprising one or more expression cassettes, each of the expression cassettes comprising a promoter operably linked to a heterologous nucleic acid sequence. **Claim 5** The nucleic acid construct according to claim 4, wherein at least one of the expression cassettes comprises a subgenomic (sg) promoter operably linked to a heterologous nucleic acid sequence. **Claim 6** The nucleic acid construct according to claim 5, wherein the sg promoter is a 26S subgenomic promoter. **Claim 7** The nucleic acid construct according to claim 1, further comprising one or more untranslated regions (UTRs). **Claim 8** The nucleic acid construct according to claim 7, wherein at least one of the UTRs is a heterologous UTR. **Claim 9** The nucleic acid construct according to claim 4, wherein at least one of the expression cassettes comprises a coding sequence of a gene of interest (GOI). **Claim 10** The nucleic acid construct according to claim 9, 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. **Claim 11** The nucleic acid construct according to claim 9, wherein the GOI encodes a polypeptide selected from the group consisting of an antibody, an antigen, an immunomodulatory substance, an enzyme, a signal transduction protein, and a cytokine. **Claim 12** The nucleic acid construct according to claim 9, wherein the coding sequence of the GOI is optimized for expression at a level higher than the expression level of a reference coding sequence. **Claim 13** The nucleic acid construct according to claim 9, wherein the coding sequence of the GOI is optimized for enhanced RNA stability. **Claim 14** The nucleic acid construct according to any one of claims 1, wherein the nucleic acid construct of the present disclosure is incorporated into a vector.

15. The nucleic acid construct according to claim 14, wherein the vector is a self-replicating RNA (srRNA) vector.

16. The nucleic acid construct according to claim 1, 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, SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO:

18.

17. A recombinant cell comprising the nucleic acid construct according to claim 1.

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

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

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

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

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

23. The recombinant cell according to claim 20, wherein the recombinant cell is a mammalian cell.

24. The recombinant cell is selected from the group consisting of monkey kidney CV1 cells (COS-7) transformed by SV40, human embryonic kidney cells (e.g., HEK 293 or HEK 293 cells), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells), monkey kidney cells (CV1), human cervical cancer cells (HeLa), dog kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562), TRI cells, FS4 cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (Vero cells), human A549 cells, human cervical cells, human CHME5 cells, human PER.C6 cells, NS0 mouse myeloma cells, human epidermal laryngeal cells, human fibroblasts, human HUH-7 cells, human MRC-5 cells, human muscle cells, human lymphatic 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, rabbit kidney cells, the recombinant cell according to claim 20.

25. A cell culture comprising at least one recombinant cell according to claim 17 and a culture medium.

26. A transgenic non-human animal comprising the nucleic acid construct according to claim 1.

27. The transgenic non-human animal according to claim 26, wherein the animal is a vertebrate or an invertebrate.

28. The transgenic non-human animal according to claim 26, wherein the animal is an insect.

29. The transgenic non-human animal according to claim 27, wherein the animal is a mammal.

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

31. Under the condition that the transgenic animal or the recombinant cell produces a polypeptide encoded by the GOI, (i) culturing a recombinant cell comprising the nucleic acid construct according to claim 10, (ii) breeding a transgenic non-human animal comprising the recombinant cell of (i), A method for producing a polypeptide of interest.

32. A method for producing a polypeptide of interest in a subject, the method comprising introducing the nucleic acid construct according to any one of claims 10 to 16 into the subject.

33. The method according to claim 29, wherein the subject is a vertebrate or an invertebrate.

34. The method according to claim 29, wherein the subject is an insect.

35. The method according to claim 29, wherein the subject is a mammalian subject.

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

37. A recombinant polypeptide produced by the method according to claim 29.

38. A pharmaceutically acceptable excipient, and the following: a) The nucleic acid construct according to claim 1; b) A recombinant cell comprising the nucleic acid construct of (a); and / or c) A recombinant polypeptide encoded by the nucleic acid construct of (a), comprising a pharmaceutical composition.

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

40. The pharmaceutical composition according to claim 38, comprising the recombinant cell according to claim 17 and a pharmaceutically acceptable excipient.

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

42. The pharmaceutical composition according to claim 38, wherein the composition is formulated in liposomes, lipid-based nanoparticles (LNP), or polymeric nanoparticles.

43. The pharmaceutical composition according to claim 38, wherein the composition is an immunogenic composition.

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

45. The pharmaceutical composition according to claim 38, wherein the composition is substantially non-immunogenic to the subject.

46. The pharmaceutical composition according to claim 38, wherein the pharmaceutical composition is formulated as an adjuvant.

47. The pharmaceutical composition according to claim 38, wherein the pharmaceutical composition is formulated for one or more of intranasal administration, transdermal administration, intraperitoneal administration, intramuscular administration, intra-articular administration, intratumoral administration, intra-articular administration, intravenous administration, subcutaneous administration, intravaginal administration, and rectal administration.

48. A method of inducing a pharmacodynamic effect in a subject in need thereof, comprising the following: a) The nucleic acid construct according to claim 1; b) A recombinant cell comprising the nucleic acid construct of (a); c) A recombinant polypeptide encoded by the nucleic acid construct of (a); and / or d) The following: a pharmaceutical composition comprising one or more of the nucleic acid construct of (a), the recombinant cell of (b), and the recombinant polypeptide of (c) The method comprising administering to the subject a composition comprising **Claim 49** The method according to claim 48, wherein the pharmacodynamic effect comprises inducing an immune response in the subject. **Claim 50** A method of preventing and / or treating the health condition of a subject in need thereof, comprising: a) the nucleic acid construct according to claim 1; b) a recombinant cell comprising the nucleic acid construct of (a); c) a recombinant polypeptide encoded by the nucleic acid construct of (a); and / or d) a pharmaceutical composition comprising one or more of the following: the nucleic acid construct of (a), the recombinant cell of (b), and the recombinant polypeptide of (c), The method comprising prophylactically or therapeutically administering to the subject a composition comprising **Claim 51** The method according to claim 48, wherein the condition is a proliferative disorder or a microbial infection. **Claim 52** The method according to claim 48, wherein the subject has or is suspected of having a condition associated with a proliferative disorder or a microbial infection. **Claim 53** The method according to claim 48, wherein the administered composition results in an increased production of interferon in the subject. **Claim 54** The method according to claim 48, 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. **Claim 55** The method according to claim 54, 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. **Claim 56** A kit for inducing a pharmacodynamic effect, inducing an immune response, preventing, and / or treating a health condition or a microbial infection, comprising: a) the nucleic acid construct according to claim 1; b) a recombinant cell comprising the nucleic acid construct of (a); c) a recombinant polypeptide encoded by the nucleic acid construct of (a); and / or d) a pharmaceutical composition comprising one or more of the following: the nucleic acid construct of (a), the recombinant cell of (b), and the recombinant polypeptide of (c), The kit comprising

Images