Modified Alphaviruses Having Heterologous Nonstructural Proteins - Patent application

Abstract

The present disclosure relates to the field of molecular virology, including nucleic acid molecules comprising modified viral genomes or replicons (e.g., self-replicating RNA), pharmaceutical compositions containing same, and the use of such nucleic acid molecules and compositions to produce desired products in cell culture or in vivo. Also provided are methods for inducing an immune response in a subject in need thereof, as well as methods for 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 Patent Application No. 63 / 240,297, filed September 2, 2021. The disclosure of the above application, including any drawings, is expressly incorporated herein by reference in its entirety.

[0002] 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 such nucleic acid molecules and compositions to produce desired products in cell culture or in vivo. Also provided are methods for inducing a pharmacodynamic effect in a subject in need thereof, e.g., eliciting an immune response in a subject in need thereof, as well as methods for preventing and / or treating various health conditions.

[0003] Incorporating sequence tables The material in the attached Sequence Listing is incorporated herein by reference. The attached Sequence Listing XML file entitled 058462_504001WO_SequenceListing.XML was created on Aug. 28, 2022 and is 95 KB. [Background technology]

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

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

[0006] Therefore, 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

[0007] 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 diseases and microbial infections. In particular, as described in more detail below, some embodiments of the present disclosure provide, inter alia, a nucleic acid construct encoding a recombinant alphavirus comprising a coding sequence for at least one heterologous nonstructural protein (nsP) or a portion thereof. Also provided are recombinant cells comprising the nucleic acid constructs disclosed herein, transgenic animals comprising the nucleic acid constructs disclosed herein, methods for generating the nucleic acid constructs disclosed herein, methods for generating a recombinant polypeptide of interest ex vivo or in vivo, and the recombinant polypeptides generated thereby, as well as pharmaceutical compositions comprising the nucleic acid constructs, the recombinant cells, and the recombinant polypeptides. Also provided herein are methods for inducing an immune response, and methods for preventing and / or treating a health condition in a subject in need thereof, comprising administering a nucleic acid construct of the present disclosure, a recombinant cell of the present disclosure, a recombinant polypeptide of the present disclosure, and / or a pharmaceutical composition of the present disclosure.

[0008] In one aspect of the disclosure, provided herein is a nucleic acid construct comprising a modified genome or RNA replicon (e.g., a self-replicating RNA) of an alphavirus species, wherein at least one nonstructural protein (nsP) or portion thereof of the modified alphavirus genome or RNA replicon is heterologous to the remainder of the modified alphavirus genome or RNA replicon.

[0009] Non-limiting embodiments of the nucleic acid constructs of the present disclosure may include one or more of the following features: In some embodiments, at least one heterologous nsP or portion thereof is nsP1, nsP2, nsP3, nsP4, or any portion thereof, or any combination of the above. In some embodiments, at least one heterologous nsP or portion thereof is from a different strain of the same alphavirus species. In some embodiments, at least one heterologous nsP or portion thereof is from a different alphavirus species.

[0010] In some embodiments, the modified alphavirus genome or RNA replicon (e.g., a self-replicating RNA) does not include at least a portion of a nucleic acid sequence encoding one or more viral structural proteins. In some embodiments, the modified viral genome or RNA replicon does not include a substantial portion of a nucleic acid sequence encoding one or more viral structural proteins. In some embodiments, the modified viral genome or RNA replicon does not include a nucleic acid sequence encoding a viral structural protein.

[0011] In some embodiments, a modified alphavirus genome or RNA replicon (e.g., a self-replicating RNA) of the disclosure further comprises one or more expression cassettes, each comprising a promoter operably linked to a heterologous nucleic acid sequence. In some embodiments, at least one of these 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, a modified alphavirus genome or RNA replicon of the disclosure further comprises one or more untranslated regions (UTRs). In some embodiments, at least one of these UTRs is a heterologous UTR.

[0012] 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 for 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 for the GOI is optimized for enhanced RNA stability.

[0013] In some embodiments, the modified alphavirus genome or RNA replicon (e.g., self-replicating RNA) of the present disclosure is selected from the group consisting of Aura virus (AURAV), Babanki virus (BABV), Barmah Forest virus (BFV), Bebaru virus (BEBV), Buggy Creek virus, Caaingua virus, Cabassou virus, Chikungunya virus (CHIKV), Eastern equine encephalitis virus (EEEV), Eilat virus, Everglades virus (EVEV), Fort Morgan virus (FMV), Getah virus (GETV), Highlands J virus, and the like. These include: Hymenovirus (HJV), Kyzylagach virus (KYZV), Madariaga virus (MADV), Mayaro virus (MAYV), Middelburg virus (MIDV), Mosso das Pedras virus, Mucambo virus (MUCV), Ndumu virus (NDUV), O'nyong'nyong virus (ONNV), Pixuna virus (PIXV), Rio Negro virus (RNV), Ross River virus (RRV), Salmon pancreas disease virus (SPDV), and Semliki Forest virus (SEV). Sindbis virus (SFV)The virus is of an alphavirus species selected from the group consisting of Sleeping disease virus (SINV), Sleeping disease virus (SDV), Southern elephant seal virus (SESV), Tai Forest virus (TFV), Tonate virus, Trocara virus, Una virus (UNAV), Venezuelan equine encephalitis virus (VEEV), Western equine encephalitis virus (WEEV), and Whataroa virus (WHAV).

[0014] In some embodiments, the modified alphavirus genome or RNA replicon (e.g., self-replicating RNA) of the disclosure is of Sindbis virus (SINV). In some embodiments, the modified alphavirus genome or RNA replicon of the disclosure is of the Girdwood strain of SINV.

[0015] In some embodiments, at least one heterologous nsP or portion thereof of the modified genome or RNA replicon (e.g., a self-replicating RNA) is derived from the AR86 strain of SINV. In some embodiments, at least one heterologous nsP or portion thereof of the modified genome or RNA replicon is derived from the Girdwood strain of SINV.

[0016] In some embodiments, at least one heterologous nsP or portion thereof is nsP1, nsP3, nsP4, or any portion thereof, or any combination of the above. In some embodiments, the modified genome or RNA replicon (e.g., self-replicating RNA) is of the AR86 strain of SINV. In some embodiments, at least one heterologous nsP or portion thereof of the modified SINV-AR86 genome or RNA replicon is from the Girdwood strain of SINV. In some embodiments, at least one heterologous nsP or portion thereof of the modified SINV-AR86 genome or RNA replicon is from nsP2 of the Girdwood strain of SINV.

[0017] In some embodiments, the nucleic acid construct of the present disclosure is incorporated into a vector, hi some embodiments, the vector is a self-replicating RNA (srRNA) vector.

[0018] In some embodiments of the present disclosure, the nucleic acid construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1-4.

[0019] In one aspect, provided herein is a recombinant cell comprising a nucleic acid construct as 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 animal cell is an insect cell. In some embodiments, the insect cell is a mosquito cell. In some embodiments, the recombinant cell is a mammalian cell. In some embodiments, the recombinant cell is a SV40 transformed monkey kidney CV1 cell (COS-7), human embryonic kidney cell (e.g., HEK293 or HEK293 cell), baby hamster kidney cell (BHK), mouse Sertoli cell (e.g., TM4 cell), monkey kidney cell (CV1), human cervical carcinoma cell (HeLa), canine kidney cell (MDCK), buffalo rat liver cell (BRL3A), human lung cell (W138), human liver cell (Hep G2), mouse mammary tumor (MMT060562), 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 laryngeal epidermoid cells, human fibroblast cells, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocytes, human macrophage cells, human RAW264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells. Also provided in a related aspect is a cell culture comprising at least one recombinant cell as disclosed herein and a medium.

[0020] In another aspect, provided herein is a transgenic animal comprising a nucleic acid construct as described herein. In some embodiments, the animal is a vertebrate or an invertebrate. In some embodiments, the animal is an insect. In some embodiments, the animal is a mammal. In some embodiments, the mammal is a non-human mammal. In another aspect, provided herein is a method for producing a polypeptide of interest, 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 transgenic animal or recombinant cell produces a polypeptide encoded by the GOI.

[0021] In another aspect, provided herein is a method for producing a polypeptide of interest in a subject, comprising administering to the subject a nucleic acid construct as disclosed herein. In some embodiments, the subject is a vertebrate or an invertebrate. In some embodiments, the subject is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the subject is a 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.

[0022] In yet another aspect, provided herein is a pharmaceutical composition comprising a pharma- ceutical 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.

[0023] Non-limiting exemplary embodiments of the pharmaceutical composition 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 liposomal, lipid nanoparticle (LNP), or polymeric nanoparticle formulated composition. 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, intranodal, transdermal, intraperitoneal, intramuscular, intratumoral, intraarticular, intravenous, subcutaneous, intravaginal, intraocular, oral, and rectal administration.

[0024] In another aspect, provided herein is a method for functionalizing / engineering an alphavirus genome or RNA replicon (e.g., a self-replicating RNA), comprising: (a) providing a non-functional alphavirus genome or RNA replicon; (b) replacing a non-structural protein (nsP) or portion thereof of said non-functional alphavirus genome or RNA replicon with a heterologous coding sequence of a corresponding nsP or portion thereof from a different alphavirus strain, thereby generating a modified alphavirus genome or RNA replicon; (c) evaluating the functionality of said modified alphavirus genome or RNA replicon; and (d) considering said modified alphavirus genome or RNA replicon functional if said modified alphavirus genome or RNA replicon is capable of RNA replication and / or expression.

[0025] Non-limiting and exemplary embodiments of the disclosed methods for functionalizing / engineering an alphavirus genome or RNA replicon (e.g., self-replicating RNA) may include one or more of the following features: In some embodiments, the heterologous nsP or portion thereof is from another strain of the same alphavirus species. In some embodiments, the heterologous nsP or portion thereof is from another alphavirus species. In some embodiments, the heterologous nsP or portion thereof is nsP1, nsP2, nsP3, nsP4, or any portion thereof. In some embodiments, the non-functionality of the alphavirus genome or RNA replicon is determined by insufficient self-replication within the host cell. In some embodiments, assessing the functionality of the modified alphavirus genome or RNA replicon comprises an assay selected from the group consisting of detection of RNA replication, detection of viral protein expression, detection of cytopathic effect (CPE), and detection of heterologous transgene expression.

[0026] In another aspect, provided herein is a method for inducing a pharmacodynamic effect in a subject, in particular a method for eliciting an immune response in a subject in need thereof, comprising administering to said 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.

[0027] In yet another aspect, provided herein is a method for preventing and / or treating a health condition in a subject in need thereof, comprising administering to said subject, for prophylactic or therapeutic purposes, 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 composition comprising any one of the pharmaceutical compositions of the present disclosure.

[0028] Non-limiting and exemplary embodiments of the disclosed methods may include one or more of the following features: In some embodiments, the condition is a proliferative disease or a microbial infection. In some embodiments, the subject has or is suspected of having a condition associated with a proliferative disease or a microbial infection. In some embodiments, the administered composition results in increased interferon production in the subject. In some embodiments, the composition is administered to the subject individually as a monotherapy or in combination with at least one additional therapy as a first treatment. 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.

[0029] In yet another aspect, provided herein is a kit for inducing a pharmacodynamic effect, eliciting an immune response, preventing a condition or a microbial infection, and / or treating a condition or a microbial infection 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.

[0030] Each of the aspects and embodiments described herein can be used together unless expressly or unambiguously excluded from the context of the embodiment or aspect.

[0031] The above summary is illustrative only and is not intended to be limiting in any way. In addition to the exemplary embodiments and features described herein, further aspects, embodiments, objects, and features of the present disclosure will be readily apparent from the drawings and detailed description and claims. [Brief description of the drawings]

[0032] [Figure 1] FIG. 1 is a schematic diagram of four non-limiting examples of alphavirus genome designs according to some embodiments of the present disclosure. Non-structural proteins nsP1, nsP2, nsP3, and nsP4 are shown. Each of these alphavirus designs contains (i) a heterologous gene of interest (GOI) under the control of a 26S subgenomic promoter; and (ii) native 5'UTR and 3'UTR sequences from the AR86 strain of SINV. In AR86-Girdwood Hybrid 1, structural proteins nsP1, nsP3, and nsP4 are from the AR86 strain of SINV, and nsP2 is from the Girdwood strain of SINV. In AR86-Girdwood Hybrid 2, nsP4 is from the AR86 strain, and nsP1, nsP2, and nsP3 are from the Girdwood strain. In AR86-Girdwood Hybrid 3, nsP3 is from the AR86 strain, and nsP1, nsP2, and nsP4 are from the Girdwood strain. In AR86-Girdwood hybrid 4, nsP1 is from strain AR86, and nsP2, nsP3, while nsP4 are from Girdwood. [Figure 2A] FIG. 2A is a schematic structure of the basic Sindbis AR86-Girdwood Hybrid 1 vector described in FIG. 1, which does not contain the coding sequence of the gene of interest (GOI). [Figure 2B]FIG. 2B is a schematic structure of the Sindbis AR86-Girdwood Hybrid 1 vector described in FIG. 1, containing the coding sequence of an exemplary GOI, e.g., the hemagglutinin precursor (HA) of influenza A virus H5N1 (H5N1 HA), placed under the control of a 26S subgenomic promoter. [Figure 3A] FIG. 3A is a schematic structure of the underlying Sindbis AR86-Girdwood Hybrid 2 vector described in FIG. 1, which does not contain the coding sequence of the GOI. [Figure 3B] FIG. 3B is a schematic structure of the Sindbis AR86-Girdwood Hybrid 2 vector described in FIG. 1, containing the coding sequence of an exemplary GOI, e.g., H5N1 HA, placed under the control of a 26S subgenomic promoter. [Figure 4A] FIG. 4A is a schematic structure of the basic Sindbis AR86-Girdwood Hybrid 3 vector described in FIG. 1, which does not contain the coding sequence of the GOI. [Figure 4B] FIG. 4B is a schematic structure of the Sindbis AR86-Girdwood Hybrid 3 vector described in FIG. 1, containing the coding sequence of an exemplary GOI, e.g., H5N1 HA, placed under the control of a 26S subgenomic promoter. [Figure 5A] FIG. 5A is a schematic structure of the underlying Sindbis AR86-Girdwood Hybrid 4 vector described in FIG. 1, which does not contain the coding sequence of the GOI. [Figure 5B] FIG. 5B is a schematic structure of the Sindbis AR86-Girdwood Hybrid 4 vector described in FIG. 1, containing the coding sequence of an exemplary GOI, e.g., H5N1 HA, placed under the control of a 26S subgenomic promoter. [Diagram 2]Figure 6 graphically summarizes the results of experiments performed to demonstrate that non-functional alphavirus genomes or RNA replicons (e.g., self-replicating RNA) can be functionalized by replacing defective nsP sequences with corresponding functional nsPs from a heterologous alphavirus genome or RNA replicon. Figure 6 shows contour plots of BHK-21 cells transformed with exemplary alphavirus genome designs according to some embodiments of the present disclosure. In these experiments, these alphavirus genome designs were each introduced into BHK-21 cells by electroporation, and 20 hours after transformation, cells were fixed, permeabilized, and stained with a PE-conjugated anti-double stranded RNA (dsRNA) mouse monoclonal antibody (J2, Scicons) to quantify the frequency of dsRNA+ cells by fluorescent flow cytometry. The ability of these alphavirus genome designs to undergo RNA replication leading to the production of dsRNA is shown. [Diagram 3] Figure 7 graphically summarizes the results of experiments performed to demonstrate the ability to detect expression of a GOI from srRNA vectors containing heterologous nonstructural protein genes. Figure 7 is a bar graph showing quantification of relative expression of avian influenza A H5N1 HA polypeptide in cells transformed with srRNA vector designs according to some embodiments of the present disclosure. In these experiments, these alphavirus srRNA designs were each electroporated into BHK-21 cells, and 20 hours after transformation, cells were fixed, permeabilized, and stained with an APC-conjugated anti-H5N1 mouse monoclonal antibody (2B7, Abcam; APC: allophycocyanin) to quantify the mean fluorescence intensity (MFI) of H5N1+ cells by fluorescent flow cytometry. [Figure 8A]8A-8B are schematic summaries of the results of experiments demonstrating that modified srRNA vectors containing heterologous nonstructural protein genes 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. 8A-8B are bar graphs showing quantification of the bioactivity of proteins secreted from BHK-21 cells transformed with these srRNAs. The srRNAs shown in FIGs. 8A-8B are SINV AR86-Girdwood Hybrid 1 srRNAs (RBI307, RBI308), which encode two proteins, IL-1RA and IL-12, respectively, in two different configurations. Four VEEV-based srRNA controls were also included in these experiments: VEEV srRNAs encoding both IL-1RA and IL-12 in two configurations (RBI299, RBI300), and VEEV srRNAs containing a control transgene (RBI296, RBI298). Two SINV Girdwood-based srRNA controls (RBI309, RBI310), encoding the two proteins IL-1RA and IL-12, respectively, in two different configurations, were also included in these experiments. Figure 8A shows the quantification of bioactive IL-1RA in cell culture media 24 and 48 hours after transfection with srRNAs. [Figure 8B]8A-8B are schematic summaries of the results of experiments demonstrating that modified srRNA vectors containing heterologous nonstructural protein genes 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. 8A-8B are bar graphs showing quantification of the bioactivity of proteins secreted from BHK-21 cells transformed with these srRNAs. The srRNAs shown in FIGs. 8A-8B are SINV AR86-Girdwood Hybrid 1 srRNAs (RBI307, RBI308), which encode two proteins, IL-1RA and IL-12, respectively, in two different configurations. Four VEEV-based srRNA controls were also included in these experiments: VEEV srRNAs encoding both IL-1RA and IL-12 in two configurations (RBI299, RBI300), and VEEV srRNAs containing a control transgene (RBI296, RBI298). Two SINV Girdwood-based srRNA controls (RBI309, RBI310), encoding the two proteins IL-1RA and IL-12, respectively, in two different configurations, were also included in these experiments. Figure 8B shows the quantification of bioactive IL-12 in cell culture media 24 and 48 hours after transfection with srRNAs. [Figure 9A]9A-9B are bar graphs showing the in vivo immunogenicity of an exemplary panel of srRNAs encoding viral antigens, the exemplary viral antigen being 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 AR86-Girdwood hybrid 1 (SIN.AR86), and Eastern equine encephalitis virus (EEE.FL93). FIG. 9A shows quantification of antigen-specific splenic T cell responses assessed by ELISpot after two immunizations. [Figure 9B] 9A-9B are bar graphs showing the in vivo immunogenicity of an exemplary panel of srRNAs encoding viral antigens, the exemplary viral antigen being 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 AR86-Girdwood hybrid 1 (SIN.AR86), and Eastern equine encephalitis virus (EEE.FL93). FIG. 9B shows the titers of anti-rabies neutralizing antibodies from sera after two immunizations. [Figure 10A]10A-10C 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 Sindbis AR86-Girdwood hybrid 1 (SIN.AR86) and five other alphaviruses, Venezuelan equine encephalitis virus (VEE.TC83), S27 strain (CHIK.S27) and DRDE-06 strain (CHIK.DRDE) of Chikungunya virus, Girdwood strain of Sindbis virus (SIN.GW), and Eastern equine encephalitis virus (EEE.FL93). Each srRNA contains sequences encoding three polypeptides, estrogen receptor 1 (ESR1), human epidermal growth factor 2 (HER2), and human epidermal growth factor 2 (HER3). Figures 10A-C show splenic T cell responses to these three antigens measured using ELISpot analysis in mice that received two immunizations, including statistical comparisons between each of the antigens tested. [Figure 10B] 10A-10C 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 Sindbis AR86-Girdwood hybrid 1 (SIN.AR86) and five other alphaviruses, Venezuelan equine encephalitis virus (VEE.TC83), S27 strain (CHIK.S27) and DRDE-06 strain (CHIK.DRDE) of Chikungunya virus, Girdwood strain of Sindbis virus (SIN.GW), and Eastern equine encephalitis virus (EEE.FL93). Each srRNA contains sequences encoding three polypeptides, estrogen receptor 1 (ESR1), human epidermal growth factor 2 (HER2), and human epidermal growth factor 2 (HER3). Figures 10A-C show splenic T cell responses to these three antigens measured using ELISpot analysis in mice that received two immunizations, including statistical comparisons between each of the antigens tested. [Figure 10C] 10A-10C 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 Sindbis AR86-Girdwood hybrid 1 (SIN.AR86) and five other alphaviruses, Venezuelan equine encephalitis virus (VEE.TC83), S27 strain (CHIK.S27) and DRDE-06 strain (CHIK.DRDE) of Chikungunya virus, Girdwood strain of Sindbis virus (SIN.GW), and Eastern equine encephalitis virus (EEE.FL93). Each srRNA contains sequences encoding three polypeptides, estrogen receptor 1 (ESR1), human epidermal growth factor 2 (HER2), and human epidermal growth factor 2 (HER3). Figures 10A-C show splenic T cell responses to these three antigens measured using ELISpot analysis in mice that received two immunizations, including statistical comparisons between each of the antigens tested. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Provided herein are viral expression systems with excellent expression potential, particularly suitable for expressing recombinant polypeptides, such as vaccine and therapeutic polypeptides, in recombinant cells. For example, some embodiments of the present disclosure relate to nucleic acid constructs, such as expression constructs and expression vectors, containing modified genome or replicon RNA (e.g., self-replicating RNA) of an alphavirus species, where at least one nonstructural protein (nsP) or portion thereof of the modified alphavirus genome or RNA replicon is heterologous to the remainder of the modified alphavirus genome or RNA replicon. Also provided in some embodiments of the present disclosure are viral-based expression vectors that include one or more expression cassettes encoding a polypeptide of interest encoded by a gene of interest (GOI). Additionally, provided are recombinant cells genetically engineered to contain one or more of the nucleic acid constructs disclosed herein. Biological materials and recombinant products resulting from such recombinant cells are also within the scope of the present application. Also provided are compositions and methods useful for inducing a pharmacodynamic effect in a subject in need thereof, e.g., for eliciting an immune response in a subject in need thereof, as well as methods for preventing and / or treating various health conditions.

[0034] Self-amplifying RNA (e.g., self-replicating RNA or replicons) based on RNA viruses (e.g., alphaviruses) can be used as robust expression systems. For example, it has been reported that an advantage of using alphaviruses, such as SINV, as viral expression vectors is that they can direct the synthesis of large amounts of recombinant protein in recombinant host cells. Among other advantages, polypeptides such as therapeutic single-chain antibodies can be most effective if expressed at high levels in vivo. Additionally, in generating recombinant antibodies purified from cells in culture (ex vivo), high protein expression from replicon RNA can increase the overall yield of antibody product. Furthermore, if the protein being expressed is a vaccine antigen, high level expression can induce the most robust immune response in vivo.

[0035] Alphaviruses use motifs contained in their UTRs, structural regions, and nonstructural regions to influence their replication in host cells. These regions also contain mechanisms to evade the innate immunity of host cells. However, significant differences have been reported among alphavirus species, for example, in mechanisms of immune evasion, tissue tropism, xenotropic hosts, and disease symptoms and severity.

[0036] Considering the differences in the presence or absence of host cell attenuation factors in the nonstructural and structural regions of alphaviruses, deletion of structural genes to allow expression of a gene of interest in synthetic vectors will have varying effects on individual vectors. Synthetic replicons (e.g., self-replicating RNAs) with different host attenuation factors in the nonstructural regions are better or worse at inducing an immune response to the expressed gene of interest. The inability of the nonpathogenic Sindbis Girdwood strain to inhibit STAT1 makes this strain an advantageous vector for expressing recombinant proteins without generating a robust immune response to the encoded protein. The advantages of these individual vectors have been entirely unknown and unpredicted until now.

[0037] As described in more detail below, early findings indicate that published alphavirus genomic data do not necessarily provide nucleotide sequences that allow for direct replacement of nucleic acid sequences encoding structural proteins with a gene of interest (GOI) to obtain self-replicating RNAs and transgene-expressing replicons. In particular, many of the published alphavirus genomes have been found to be non-functional, e.g., unable to replicate and / or express a transgene. As described in more detail below in the Examples, novel procedures are provided herein that are useful for functionalizing defective (e.g., non-functional) alphavirus genomes or RNA replicons (e.g., self-replicating RNAs), in particular by replacing the non-structural protein (nsP) sequences of the defective alphavirus genome or RNA replicon with corresponding nsP sequences from a different alphavirus strain to generate modified alphavirus genomes or RNA replicons that are functional.

[0038] Although it is not fully understood, full-length viruses and synthetic replicons (e.g., self-replicating RNA) do not have the same replication capabilities. In particular, many of the full-length viruses and replicons available from public sources are defective in function. Currently, the engineering approach to functionalize (e.g., make functional) defective alphavirus genomes or RNA replicons is to revert one or more critical point mutations associated with virulence that have diverged between strains, e.g., mutations that represent diversity between functional strains (e.g., Girdwood) and non-functional strains (e.g., AR86). However, this strategy often fails, or a solution is arrived at arbitrarily, indicating that there is a great deal of uncharacterized and therefore unpredictable sequence divergence between strains. Thus, there is a need for more rapid and efficient methods to identify functional alphavirus strains (rather than simply reverting point mutations or selecting arbitrary regions to create chimeras).

[0039] As noted above, during viral replication, each of the nsP subunits (e.g., nsP1, nsP2, nsP3, nsP4) of the nsP polyprotein complex is processed separately into individual proteins. These proteins then assemble to form the nsP polyprotein complex, which performs the transcriptional functions of the genome and subgenome. Without wishing to be bound by theory, it is hypothesized that each nsP is biologically independent in its own right, in that it contributes to the overall function of the replicon (e.g., a self-replicating RNA), and should be treated as a separate modular unit. As described in more detail below, some embodiments of the present disclosure relate to methods of functionalizing a non-functional alphavirus genome or RNA replicon, where each of the nsP subunits can be treated as a separate modular unit and replaced (swapped) by a corresponding modular unit from another virus (e.g., another species or another strain of the same species), resulting in a chimeric virus with novel characteristics. The disclosed method allows a novel combinatorial view of the effect of swapping nsPs in a minimal set of: (1) replicons that are non-functional in vitro; and (2) replicons that are non-functional in vitro. This approach rapidly provides information about which nsPs are problematic in any given strain, without the need to generate a large number of new constructs. Thus, the novel procedure described herein is a rapid, technically feasible method that represents a significant improvement over any currently known method.

[0040] As described in more detail below, some embodiments of the present disclosure relate to self-replicating RNA (srRNA) vectors comprising heterologous nonstructural protein genes engineered to express one or more heterologous genes of interest (GOIs). For example, it has been found possible to replace the structural polyprotein genes in a srRNA vector comprising one or more heterologous nonstructural protein genes with one or more GOIs. In one example, a Sindbis srRNA vector comprising heterologous nonstructural protein genes (SINV AR86-Girdwood Hybrid 1) was engineered to replace the structural polyprotein genes with synthetic human IL-1RA gene or IL-12 gene cassettes to generate a self-replicating vector capable of RNA replication and transgene expression in transfected BHK-21 cells (see, e.g., FIG. 8). In addition, as described in more detail below, some SINV AR86-Girdwood Hybrid 1-based srRNA constructs as described herein could be employed to express antigenic molecules of interest and formulated as vaccines with measurable pharmacodynamic effects in vivo (see, e.g., FIG. 9 and FIG. 10). Furthermore, the experimental data presented in Figures 7A-7B indicate that SINV AR86-Girdwood Hybrid 1-based srRNA vectors may be useful in expressing multiple proteins whose coding sequences are operably linked to each other in one open reading frame (e.g., in a polycistronic ORF) and have biological activity as measured by pharmacodynamic effects in vivo (see, e.g., Figure 10). Collectively, these studies provide further validation of the use of srRNA vectors carrying heterologous nonstructural protein genes and SINV AR86-Girdwood Hybrid 1-based srRNA vectors in therapeutic and vaccine applications.

[0041] definition Unless otherwise defined, all terms, symbols, 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 commonly understood meanings are defined herein for clarity and / or ease of reference, but the inclusion of such definitions herein should not necessarily be interpreted as representing a significant departure from what is commonly understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those of ordinary skill in the art using conventional methodology.

[0042] The singular forms "a," "an," and "the" include the plural forms unless the context clearly indicates otherwise. For example, the term "a cell" includes a plurality of cells, including a cell or a mixture thereof. "A and / or B" is used herein to include all of the following options: "A," "B," "A or B," and "A and B."

[0043] Aspects and embodiments of the disclosure described herein are understood to encompass aspects and embodiments "comprising," "consisting of," 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 in a description of a step of a method, is understood to encompass compositions and methods that consist essentially of or consist of the recited components or steps.

[0044] The term "administration," and any grammatical variations thereof, as used herein, refers to the delivery of a biologically active 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 term includes, but is not limited to, administration by a healthcare professional and self-administration.

[0045] The terms "cell," "cell culture," and "cell line" refer not only to a particular subject cell, cell culture, or cell line, but also to the progeny or potential progeny of such a cell, cell culture, or cell line, regardless of the number of transplants or passages in culture. It should be understood that not all progeny are exactly identical to the parent cell. This is because certain changes may occur in the progeny due to mutations (e.g., deliberate or inadvertent mutations) or environmental influences (e.g., methylation or other epigenetic modifications), and thus the progeny may not in fact be identical to the parent cell, but are included within the scope of the term as used herein so long as the progeny retains the same functionality as that of the original cell, cell culture, or cell line.

[0046] The terms "effective amount", "therapeutically effective amount", or "pharmaceutical effective amount" of a composition of the present disclosure, e.g., a nucleic acid construct, a recombinant cell, a recombinant polypeptide, and / or a pharmaceutical composition, generally refer to an amount sufficient for the composition to achieve a stated purpose (e.g., achieve the effect for which it is administered, stimulate an immune response, prevent or treat a disease, or reduce one or more symptoms of a disease, disorder, infection, or condition) compared to the absence of the composition. An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or reduction of one or more symptoms of a disease, which may also be referred to as a "therapeutically effective amount". A "reduction" of a symptom refers to a decrease in the severity or frequency of the symptom, or the elimination of the symptom. The precise amount of the composition, including a "therapeutically effective amount," will depend on the purpose of the treatment, and can be ascertained by one of 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).

[0047] The term "nucleic acid construct" refers to a recombinant nucleic acid molecule that includes one or more isolated nucleic acid sequences from a heterologous source. For example, a nucleic acid construct of the present disclosure can be a chimeric nucleic acid molecule, in which two or more nucleic acid sequences from different sources are assembled into one nucleic acid molecule. That is, representative nucleic acid constructs include any construct that contains (1) a nucleic acid sequence that includes a regulatory sequence and a coding sequence that are not naturally contiguous with 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 portion of a promoter 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 from any source, such as a plasmid, cosmid, virus, self-replicating polynucleotide molecule, phage, etc., that includes a nucleic acid molecule to which one or more nucleic acid sequences are operably linked, capable of genome integration or autonomous replication. A construct of the present disclosure can include the elements necessary to direct the expression of a nucleic acid sequence of interest contained in the construct. Such elements can include regulatory elements, such as a promoter operably linked to the nucleic acid sequence of interest (to direct transcription of the nucleic acid sequence of interest), and optionally, a polyadenylation sequence.

[0048] 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 introducing or transporting another nucleic acid molecule. That is, the term "vector" encompasses both DNA-based vectors 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 and is thereby capable of expressing DNA sequences and fragments in vitro, ex vivo, and / or in vivo. In some embodiments, the vector may include a sequence that directs autonomous replication in a cell, such as a plasmid (a DNA-based vector) or a self-replicating RNA vector. In some embodiments, the vector may include a sequence sufficient to allow integration into a host cell DNA. In some embodiments, the 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 or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. In some embodiments, the vectors of the present disclosure may be single-stranded vectors (e.g., ssDNA or ssRNA). In some embodiments, the vectors of the present disclosure may be double-stranded vectors (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 introduce genes into cells.

[0049] 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 a prokaryotic origin and a eukaryotic origin, at least one multiple cloning site, and / or elements that facilitate stable integration of the construct into the genome of a cell. Two or more constructs may be incorporated into one nucleic acid molecule, such as one vector, or may be contained in two or more separate nucleic acid molecules, such as two or more separate vectors. An "expression construct" typically includes at least a control sequence operably linked to a nucleotide sequence of interest. Thus, for example, a promoter operably linked to the nucleotide sequence to be expressed is provided in an expression construct for intracellular expression. Compositions and methods for making and using constructs and cells in the practice of the present disclosure are known to those of skill in the art.

[0050] The terms "effective amount", "therapeutically effective amount", or "pharmaceutical effective amount" of a composition of the present disclosure, e.g., a nucleic acid construct, a recombinant cell, a recombinant polypeptide, and / or a pharmaceutical composition, generally refer to an amount sufficient for the composition to achieve a stated purpose (e.g., achieve the effect for which it is administered, stimulate an immune response, prevent or treat a disease, or reduce one or more symptoms of a disease, disorder, infection, or condition) compared to the absence of the composition. An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or reduction of one or more symptoms of a disease, which may also be referred to as a "therapeutically effective amount". A "reduction" of a symptom refers to a decrease in the severity or frequency of the symptom, or the elimination of the symptom. The precise amount of the composition, including a "therapeutically effective amount," will depend on the purpose of the treatment, and can be ascertained by one of 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).

[0051] The term "operably linked" as used herein refers to a physical or functional linkage between two or more elements, e.g., between polypeptide sequences or polynucleotide sequences, that allows them to operate in their intended manner. For example, the term "operably linked" when used in the context of a nucleic acid molecule or a coding sequence and a promoter sequence within a nucleic acid molecule described herein means that the coding sequence and the promoter sequence are in frame and appropriately separated in space and distance to allow binding by a transcription factor or RNA polymerase, respectively, to affect transcription. It will 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 linkage (e.g., direct or indirect linkage) between amino acid sequences (e.g., between different segments, portions, regions, or domains) that results in the described activity of the construct. Functionally 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).

[0052] The term "percent identity," as used herein in connection with two or more nucleic acids or proteins, refers to two or more sequences or subsequences being identical or having a specified percentage of identical nucleotides or amino acids (e.g., about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity over a specified region when compared and aligned for maximum correspondence over a comparison window or specified region) when measured using the BLAST or BLAST 2.0 sequence comparison algorithm using the default parameters set forth 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 be applied to the complementary sequence of a sequence. This definition also includes sequences with deletions and / or additions, as well as sequences with 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, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705, using its default parameters.

[0053] The term "portion" as used herein may refer to a sub-portion. With respect to a particular structure, such as an amino acid sequence or a protein, the term "portion" may refer to a continuous or non-contiguous sub-portion of the structure. For example, a portion of an amino acid sequence may include at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% of the amino acids of the amino acid sequence. Additionally, or alternatively, when the portion is a non-contiguous sub-portion, the non-contiguous sub-portion may be composed of 2, 3, 4, 5, 6, 7, 8, or more portions of the structure, each of which is a continuous element of the structure. For example, a non-contiguous portion of an amino acid sequence may consist of 2, 3, 4, 5, 6, 7, 8 or more, e.g., 4 or less, portions of the amino acid sequence, each of which may contain at least 2, 3, 4, 5 consecutive amino acids, at least 10 consecutive amino acids, at least 20 consecutive amino acids, at least 30 consecutive amino acids of the amino acid sequence.

[0054] The term "pharmaceutical acceptable excipient" as used herein refers to any suitable substance that serves as a pharmaceutical acceptable carrier, additive, or diluent for administration of the desired compound to a subject. Thus, "pharmaceutical acceptable excipient" can include substances referred to as pharmaceutical acceptable diluents, substances referred to as pharmaceutical acceptable additives, and substances referred to as pharmaceutical acceptable carriers. As used herein, the term "pharmaceutical acceptable carrier" includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, and absorption delaying agents, etc., that are compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics and additional therapeutic agents) may also be incorporated into the composition.

[0055] The term "recombinant" when used in reference to a cell, nucleic acid, protein, or vector, refers to the cell, nucleic acid, protein, or vector having been altered or produced through human intervention, e.g., modified by or the result of a laboratory method. Thus, for example, recombinant proteins and recombinant nucleic acids encompass proteins and nucleic acids produced by laboratory methods. Recombinant proteins may contain amino acid residues that are not present in the native (non-recombinant or wild-type) form of the protein, or may contain amino acid residues that have been modified, e.g., labeled. The term may include any modification to a peptide, protein, or nucleic acid sequence. Such modifications may include: any chemical modification of a peptide, protein, or nucleic acid sequence, including any chemical modification of one or more amino acids, deoxyribonucleotides, or ribonucleotides; addition, deletion, and / or substitution of one or more amino acids of a peptide or protein; creation of fusion proteins, e.g., fusion proteins including antibody fragments; and addition, deletion, and / or substitution of one or more nucleic acids within a nucleic acid sequence. The term "recombinant," when used in reference to a cell, is not intended to include naturally occurring cells, but does include cells that have been engineered / modified to contain or express a polypeptide or nucleic acid that would not be present in the cell if the cell had not been engineered / modified.

[0056] As used herein, the term "replicon RNA" or "RNA replicon" refers to an RNA that contains all of the genetic information required to direct its own amplification, i.e., self-replication, in a permissive cell. Thus, replicon RNA may also be referred to as "self-amplifying RNA" (saRNA) or "self-replicating RNA" (srRNA). To direct its own replication, an RNA molecule 1) encodes a polymerase, replicase, or other protein that can catalyze the RNA amplification process by interacting with viral or host cell-derived proteins, nucleic acids, or ribonucleoproteins; and 2) contains cis-acting RNA sequences required for the replication and transcription of the subgenomic replicon-encoded RNA. These sequences may be bound to its self-encoded proteins, or non-self-encoded cell-derived proteins, nucleic acids, or ribonucleoproteins, or complexes of any of these components during the replication process. For purposes of this disclosure, an alphavirus replicon RNA molecule (e.g., an srRNA or saRNA molecule) typically contains the following ordered elements: 5' viral RNA sequences 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 polyadenylate tract (poly(A)). Furthermore, the term replicon RNA (e.g., an srRNA or saRNA molecule) typically refers to a molecule of positive polarity, or "message" sense, and the replicon RNA may have a length that differs from that of any known naturally occurring alphavirus. In some embodiments of the present disclosure, the replicon RNA does not contain at least one sequence of a structural viral protein; and / or the sequence encoding a structural gene may be replaced with a heterologous sequence. If the replicon RNA is to be packaged into recombinant alphavirus particles, it may contain one or more sequences, so-called packaging signals, that serve to initiate interactions with alphavirus structural proteins resulting in particle formation.

[0057] 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 receiving medical treatment from a physician. That is, a subject may 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 said health condition. A subject may also be an individual who has been diagnosed at or after the time of diagnosis as being at risk for a health condition and / or disease of interest. The term "non-human animal" includes all vertebrates, such as mammals, e.g., rodents, e.g., mice, non-human primates, and other mammals, such as sheep, dogs, cows, chickens, etc., as well as non-mammals, such as amphibians, reptiles, etc.

[0058] When a range of values ​​is provided, it will be understood by one of ordinary skill in the art that all ranges disclosed herein encompass any and all possible subranges and combinations thereof. Any range recited can be readily recognized as fully descriptive and allowing the range to be broken down into at least one-half, at least one-third, at least one-quarter, at least one-fifth, at least one-tenth, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, a middle third, and an upper third, etc. Also, as will be understood by one of ordinary skill in the art, terms such as "up to," "at least," "greater than," "less than," etc. all refer to ranges that are inclusive of the recited numbers and can then be broken down into subranges as previously described. Moreover, as will be understood by one of ordinary skill in the art, a range includes each individual component. That is, for example, a group having 1-3 items refers to a group having 1 item, 2 items, or 3 items. Similarly, a group having 1-5 items refers to a group having 1 item, 2 items, 3 items, 4 items, or 5 items, etc.

[0059] In this specification, the term "about" is used in front of a numerical value to indicate a certain range, and this term has its usual meaning of approximately when used herein. The term "about" is used to support not only the exact number that it precedes, but also a number that is close to or approximately the number that it precedes. When determining whether a number is close to or approximately a specifically stated number, the unstated number that is close or approximately can be a number that is substantially equivalent to the specifically stated number in the context in which the number is presented. If the degree of approximation is not clear from the context, "about" means within ±10% of the provided value, or rounded to the nearest significant figure in all cases including the provided value. In some embodiments, the term "about" refers to up to ±10%, up to ±5%, or up to ±1% of the specified value.

[0060] 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 presented.

[0061] It should be understood that certain features of the present disclosure, which are described in connection with separate embodiments for clarity, may also be provided in combination in one embodiment. Conversely, various features of the present disclosure, which are described in connection with one embodiment for brevity, may also be provided separately or in any suitable subcombination. All combinations of the embodiments according to the present disclosure are specifically included in the present disclosure and are disclosed herein as if each and every combination were individually and explicitly disclosed herein. In addition, all subcombinations of the various embodiments and elements thereof are also specifically included in the present disclosure and are disclosed herein as if each and every such subcombination were individually and explicitly disclosed herein.

[0062] Alphaviruses Alphaviruses are small enveloped RNA viruses with a single-stranded positive-sense RNA genome. The alphavirus genus includes, among others, Sindbis virus (SINV), Semliki Forest virus (SFV), Ross River virus (RRV), Venezuelan equine encephalitis virus (VEEV), and Eastern equine encephalitis virus (EEEV), all of which are closely related and can infect a variety of vertebrates, such as mammals, rodents, fish, birds, and larger mammals, such as humans and horses, as well as invertebrates, such as insects. In particular, Sindbis virus and Semliki Forest virus have been extensively studied, and the life cycle, replication mode, etc. of these viruses have been well characterized. Non-limiting exemplary alphavirus species include Aura virus (AURAV), Babanki virus (BABV), Barmah Forest virus (BFV), Bebaru virus (BEBV), Buggy Creek virus, Caaingua virus, Cabassou virus, Chikungunya virus (CHIKV), Eastern equine encephalitis virus (EEEV), Eilat virus, Everglades virus (EVEV), Fort Morgan virus (FMV), Getah virus (GETV), Highlands J virus (HJV), Kyzylagach virus (KYV), and the like. KYZV, Madariaga virus (MADV), Mayaro virus (MayaroMAYV, Middelburg virus (MIDV), Mosso das Pedras virus, Mucambo virus (MUCV), Ndumu virus (NDUV), O'nyong'nyong virus (ONNV), Pixuna virus (PIXV), Rio Negro virus (RNV), Ross River virus (RRV), Salmon pancreas disease virus (SPDV), Semliki Forest virus (SFV), Sindbis virus (SINV), Sleeping disease virus (SDV), Southern elephant seal virus (SESV), Tai Forest virus (Tai Examples of viruses that cause encephalitis include: Trocara virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, and Whataroa virus.

[0063] The alphavirus genome is approximately 12 kb long and consists of two open reading frames (ORFs): a 7 kb frame encoding the nonstructural proteins (nsPs) and a 4 kb frame encoding the structural polyprotein, which is cleaved into four distinct proteins (nsP1, nsP2, nsP3, and nsP4) required for the transcription and translation of viral mRNA in the cytoplasm of the host cell.

[0064] The nsP1 protein is an mRNA capping enzyme with both guanine-7-methyltransferase (MTase) and guanylyltransferase (GTase) activities that direct the methylation and capping of newly synthesized viral genomic and subgenomic RNAs. An MTase motif in the N-terminal domain of nsP1 catalyzes the transfer of a methyl group from S-adenosylmethionine (AdoMet) to the N7 position of a GTP molecule (m7Gppp). The GTase then binds m7Gppp, forming a covalent bond with the catalytic histidine (m7Gp-GTase) and releasing PPi. The GTase then transfers the m7Gp molecule to the 5'-diphosphate RNA, generating m7GpppNp-RNA. The resulting cap structure is essential for the translation of viral mRNA and protects this mRNA from degradation by cellular 5' exonucleases. Following this N-terminal domain are features that allow the nsP1 protein to bind to cell membranes. The presence of an α-helical amphipathic loop and a palmitoylation site allows the nsP1 protein and nsP1-containing replication complexes to be anchored to the plasma membrane, presumably through interactions of nsP1 with anionic membrane phospholipids.

[0065] The nsP2 protein has multiple enzymatic activities and functional roles. The N-terminal region contains a helicase domain with seven signature motifs of superfamily 1 (SF1) helicases. This helicase domain functions as an RNA triphosphatase, which is responsible for the initiation of the viral RNA capping reaction. This helicase domain also functions as a nucleotide triphosphatase (NTPase) to fuel the RNA helicase activity. The C-terminal region of nsP2 contains a papain-like cysteine ​​protease, which is involved in the processing of the viral nonstructural polyprotein. The protease recognizes conserved motifs within the polyprotein. This proteolytic function is highly regulated and is modulated by other domains of nsP2. The alphavirus nsP2 protein has also been described as a virulence factor involved in the shutoff of transcription and translation in infected host cells, as well as the inhibition of interferon (IFN)-mediated antiviral responses, which contribute to the control of the translation machinery by viral factors.

[0066] The precise role of the alphavirus nsP3 protein in the replication complex is less clear. Three domains have been recognized in the nsP3 protein: a large N-terminal domain with phosphatase activity and nucleic acid binding activity, an alphavirus unique domain (AUD), and a C-terminal hypervariable domain. Deletion of this domain in SFV nsP3 has been shown to result in reduced viral pathogenicity, suggesting its importance in regulating viral RNA transcription.

[0067] The nsP4 polymerase is the most highly conserved protein in alphaviruses, with the most divergent nsP4 sharing >50% amino acid sequence identity with other alphavirus nsP4s. nsP4 contains a core RNA-dependent RNA polymerase (RdRp) domain at its C-terminus that is solely responsible for the RNA synthesis properties of the viral replication complex. This RdRp is involved in negative-strand RNA-mediated replication of genomic RNA and transcription of 26S subgenomic RNA. The N-terminal domain is unique to alphaviruses and may be partially disordered.

[0068] The 5' two-thirds of the alphavirus genome encodes a number of nonstructural proteins (nSPs) necessary for transcription and replication of viral RNA. When these proteins are directly translated from RNA, they combine with cellular proteins to form the RNA-dependent RNA polymerase essential for viral genome replication and transcription of subgenomic RNA. Four nonstructural proteins (nsP1, nsP2, nsP3, nsP4) are generated as a polyprotein and constitute the viral replication machinery. Processing of this polyprotein occurs in a highly regulated manner, and cleavage of the P2 / 3 junction site affects the use of the RNA template during genome replication. This site is located at the bottom of a narrow cleft and is not easily accessible. Once cleaved, nsP3 generates a ring structure that surrounds nsP2. These two proteins have an extensive interface. Mutations in nsP2 that produce noncytopathic viruses or temperature-sensitive phenotypes are clustered at the P2 / P3 interface region. P3 mutations opposite the location of the nsP2 noncytopathic mutations prevent efficient cleavage of P2 / 3. This in turn may affect RNA infectivity and alter the levels of viral RNA production.

[0069] The 3' third of the genome contains a subgenomic RNA that serves as a template for the translation of all structural proteins required to form viral particles (the core nucleocapsid protein C and the envelope proteins P62 and E1, which assemble as a heterodimer). These viral membrane-anchored surface glycoproteins are involved in entry into target cells via receptor recognition and membrane fusion. This subgenomic RNA is transcribed from the p26S subgenomic promoter located at the 3' end of the RNA sequence encoding the nsP4 protein. The proteolytic maturation of P62 into E2 and E3 results in changes to the viral surface. Together, E1, E2, and sometimes E3 form glycoprotein "spikes" that form E1 / E2 dimers or E1 / E2 / E3 trimers, with E2 extending from the center to each vertex, E1 filling the space between the vertices, and E3, when present, located at the distal end of the spike. When the virus is exposed to the acidity of the endosome, E1 dissociates from E2 to form the E1 homotrimer, which is required for the fusion step that brings the cellular and viral membranes together. The alphavirus glycoprotein E1 is a class II viral fusion protein, which differs in structure from the class I fusion proteins present in influenza viruses and HIV. The E2 glycoprotein functions to interact with the nucleocapsid through its cytoplasmic domain, while its ectodomain is involved in binding to cellular receptors. Most alphaviruses have lost the peripheral protein E3, but in Semlikivirus, E3 remains associated with the viral surface.

[0070] Alphavirus replication has been reported to occur on the membrane surface within the host cell. In the first step of the infection cycle, the 5' end of the genomic RNA is translated into polyproteins (nsP1-4) with RNA polymerase activity, which generate a minus strand complementary to the genomic RNA. In the second step, this minus strand is used as a template for the production of two RNAs: (1) a positive strand genomic RNA, which corresponds to the genome of a secondary virus, which translates to produce other nsPs and serves as the genome of this virus; and (2) a subgenomic RNA, which codes for the structural proteins of the virus that form the infectious particle. The ratio of positive strand genomic RNA / subgenomic RNA is controlled by the proteolytic autocleavage of the polyprotein into nsP1, nsP2, nsP3, and nsP4. In reality, viral gene expression occurs in two stages. In the first stage, there is the primary synthesis of the positive strand genome and the minus strand. In the second stage, the synthesis of the subgenomic RNA is virtually exclusive, which results in the production of a large amount of structural proteins.

[0071] Compositions of the Disclosure As described in more detail below, one aspect of the disclosure relates to nucleic acid constructs encoding modified viral genomes or replicon RNAs (e.g., self-replicating RNAs) of alphavirus species, nucleic acid sequences, recombinant cells comprising the nucleic acid constructs, transgenic animals comprising the nucleic acid constructs, and recombinant polypeptides produced by the methods of the disclosure.

[0072] Some embodiments of the present disclosure provide a modified alphavirus genome or replicon RNA (e.g., a self-replicating RNA) wherein at least one structural protein (nsP) or portion thereof of said modified alphavirus genome or RNA replicon is heterologous to the remainder of said modified alphavirus genome or RNA replicon, and wherein said at least one heterologous nsP or portion thereof is nsP1, nsP2, nsP3, nsP4, or any portion thereof, or any combination of the above. In some embodiments, nsP1 or a portion thereof is heterologous to the remainder of said modified alphavirus genome or RNA replicon. In some embodiments, nsP2 or a portion thereof is heterologous to the remainder of said modified alphavirus genome or RNA replicon. In some embodiments, nsP3 or a portion thereof is heterologous to the remainder of said modified alphavirus genome or RNA replicon. In some embodiments, nsP4 or a portion thereof is heterologous to the remainder of the modified alphavirus genome or RNA replicon. In some embodiments, two nsP proteins are heterologous to the remainder of the modified alphavirus genome or RNA replicon. In some embodiments, three nsP proteins are heterologous to the remainder of the modified alphavirus genome or RNA replicon. In some embodiments, nsP1, nsP2, and nsP3, or portions thereof, are heterologous to the remainder of the modified alphavirus genome or RNA replicon. In some embodiments, nsP1, nsP2, and nsP4, or portions thereof, are heterologous to the remainder of the modified alphavirus genome or RNA replicon. In some embodiments, nsP2, nsP3, and nsP4, or portions thereof, are heterologous to the remainder of the modified alphavirus genome or RNA replicon.

[0073] A. Nucleic acid constructs 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 Sindbis virus (SINV), wherein at least one nonstructural protein (nsP) or portion thereof of the modified alphavirus genome or RNA replicon is heterologous to the remainder of the modified alphavirus genome or RNA replicon. For example, at least one heterologous nsP or portion thereof is nsP1, nsP2, nsP3, nsP4, or any portion thereof, or any combination of the above. As noted above, it will be understood by those skilled in the art that a portion of a nucleic acid sequence encoding a nonstructural polypeptide may contain sufficient nucleic acid sequence encoding a nonstructural polypeptide to obtain putative identification of the polypeptide by manual evaluation of the sequence by one of skill in the art 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 portion of a nucleotide sequence contains sufficient sequence to obtain specific identification and / or isolation of a nucleic acid fragment comprising the sequence. For example, a portion of a nucleic acid sequence may 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.

[0074] Non-limiting exemplary alphavirus species suitable for the compositions and methods of the present disclosure include Aura virus (AURAV), Babanki virus (BABV), Barmah Forest virus (BFV), Bebaru virus (BEBV), Buggy Creek virus, Caaingua virus, Cabassou virus, Chikungunya virus (CHIKV), Eastern equine encephalitis virus (EEEV), Eilat virus, Everglades virus (EVEV), Fort Morgan virus (FMV), Getah virus (GETV), Highlands J virus (HJV), Kyzylagach virus (KYV), and others. These include: Kyzvirus (KYZV), Madariaga virus (MADV), Mayaro virus (MAYV), Middelburg virus (MIDV), Mosso das Pedras virus, Mucambo virus (MUCV), Ndumu virus (NDUV), O'nyong'nyong virus (ONNV), Pixuna virus (PIXV), Rio Negro virus (RNV), Ross River virus (RRV), Salmon pancreas disease virus (SPDV), Semliki Forest virus (SFV), Sindbis virus (SINV), and Sleeping disease virus (SSDV).Examples of suitable alphaviruses include: Southern elephant seal virus (SDV), Southern elephant seal virus (SESV), Tai Forest virus (TFV), Tonate virus, Trocara virus, Una virus (UNAV), Venezuelan equine encephalitis virus (VEEV), Western equine encephalitis virus (WEEV), and Whataroa virus (WHAV). Both pathogenic and non-pathogenic alphavirus strains are suitable. In some embodiments, the alphavirus is Venezuelan equine encephalitis virus (VEEV). In some embodiments, the alphavirus is Eastern equine encephalitis virus (EEEV). In some embodiments, the alphavirus is Western equine encephalitis virus (WEEV).

[0075] In some embodiments, the alphavirus is chikungunya virus (CHIKV). Non-limiting examples of CHIKV strains suitable for the compositions and methods of the present disclosure include CHIKV S27, CHIKV LR2006-OPY-1, CHIKV YO123223, CHIKV DRDE, CHIKV 37997, CHIKV 99653, CHIKV Ag41855, and Nagpur (India) 653496 strains. Both pathogenic and non-pathogenic CHIKV strains are suitable. Further examples of CHIKV strains suitable for the compositions and methods of the present disclosure include, but are not limited to, those described in Afreen et al. Microbiol. Immunol. 2014, 58:688-696, Lanciotti and Lambert ASTMH 2016, 94(4):800-803 and Langsjoen et al. mBio. 2018, 9(2):e02449-17. In some embodiments, the modified CHIKV genome or replicon RNA (e.g., self-replicating RNA) is from a CHIKV S27 strain. In some embodiments, the modified CHIKV genome or replicon RNA is from a CHIKV DRDE strain. In some embodiments, the modified CHIKV genome or replicon RNA is from a CHIKV DRDE-06 strain. In some embodiments, the modified CHIKV genome or replicon RNA is from a CHIKV DRDE-07 strain.

[0076] In some embodiments, the alphavirus is Eastern Equine Encephalitis Virus (EEEV). Non-limiting examples of EEEV strains suitable for the compositions and methods of the present disclosure include EEEV 792138, 783372, BeAn5122, BeAr300851, BeAr436087, C-49, FL91-4679, FL93-939, GML903836, MP-9, PE6, and V105-00210. Both pathogenic and non-pathogenic EEEV strains are suitable. Additional suitable EEEV strains include, but are not limited to, those listed on the Virus Pathogen Resource website (ViPR; publicly available at www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=868&decorator=toga). In some embodiments, the modified EEEV genome or replicon RNA (e.g., a self-replicating RNA) is derived from EEEV strain FL93-939.

[0077] In some embodiments, the alphavirus is Sindbis virus (SINV). In some embodiments, the modified genome or RNA replicon (e.g., self-replicating RNA) is of a SINV strain. Non-limiting examples of SINV strains suitable for the compositions and methods of the present disclosure include the AR339, AR86, and Girdwood strains of SINV. Examples of SINV strains suitable for the compositions and methods of the present disclosure include, but are not limited to, those described in Sammels et al. J. Gen. Virol. 1999, 80(3):739-748, Lundstrom and Pfeffer Vector Borne Zoonotic Dis. 2010, 10(9):889-907, Sigei et al. Arch. of Virol. 2018, 163:2465-2469 and Ling et al. J. Virol. 2019, 93:e00620-19. Further suitable SINV strains include, but are not limited to, those listed on the Virus Pathogen Resource website (ViPR; publicly available at www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=868&decorator=toga). Both pathogenic and non-pathogenic SINV strains are suitable. In some embodiments, the modified genome or RNA replicon is of the Girdwood strain of SINV. In some embodiments, the modified genome or RNA replicon is of the AR86 strain of SINV. In some embodiments, the modified SINV genome or replicon RNA is from the Girdwood strain of SINV. In some embodiments, the modified SINV genome or replicon RNA is from the AR86 strain of SINV. In some embodiments, at least one heterologous nsP or portion thereof of the modified genome or RNA replicon is from the AR86 strain of SINV. In some embodiments, at least one heterologous nsP or portion thereof is nsP1, nsP3, nsP4, or any portion thereof, or any combination of the above.In some embodiments, the modified genome or RNA replicon is of the AR86 strain of SINV.

[0078] In some embodiments, the alphavirus is Western Equine Encephalitis Virus (WEEV). Non-limiting examples of WEEV strains suitable for the compositions and methods of the present disclosure include WEEV California, MacMillan, IMP181, Imperial, Imperial 181, IMPR441, 71V-1658, AG80-646, BFS932, COA592, EP-6, E1416, BFS1703, BFS2005, BSF3060, BSF09997, CHLV53, KERN5547, 85452NM, Montana-64, S8-122, and TBT-235. Further examples of WEEV strains suitable for the compositions and methods of the present disclosure include 5614, 93A27, 93A30, 93A38, 93A79, B628(Cl 15), CBA87, CNTR34, CO921356, Fleming, Lake 43, PV012357A, PV02808A, PV72102, R02PV001807A, R02PV002957B, R02PV003422B, R05PV003422B, R0PV003814A, and R0PV00384A. Both pathogenic and non-pathogenic WEEV strains are suitable. Additional suitable WEEV strains include, but are not limited to, those described in Berggren NA et al., J. Virol. 88(16): 9260-9267, Aug 2014, and on the Virus Pathogen Resource website (ViPR; publicly available at https: / / www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=57240&decorator=toga). In some embodiments, the modified WEEV genome or srRNA is from the Imperial strain of WEEV. In some embodiments, the modified WEEV genome or srRNA is from the MacMillan strain of WEEV.

[0079] In some embodiments, at least one heterologous nsP or portion thereof is from another strain of the same alphavirus species. In some embodiments, at least one heterologous nsP or portion thereof is from another alphavirus species. For example, in some embodiments, at least one heterologous nsP or portion thereof of the modified SINV-AR86 genome or RNA replicon (e.g., self-replicating RNA) is from the Girdwood strain of SINV. In some embodiments, at least one heterologous nsP or portion thereof of the modified SINV-AR86 genome or RNA replicon is from nsP2 of the Girdwood strain of SINV. In some embodiments, the structural proteins nsP1, nsP3, and nsP4 are from the AR86 strain of SINV, and nsP2 is from the Girdwood strain of SINV. In some embodiments, nsP4 is from the AR86 strain, and nsP1, nsP2, and nsP3 are from the Girdwood strain. In some embodiments, nsP3 is from the AR86 strain, and nsP1, nsP2, and nsP4 are from the Girdwood strain. In some embodiments, nsP1 is from the AR86 strain, and nsP2, nsP3, while nsP4 are from the Girdwood strain (see, eg, Figures 1 and 6).

[0080] In some embodiments, a substantial portion of the nucleic acid sequence encoding one or more viral structural proteins has been removed. In some embodiments, the modified viral genome or replicon RNA (e.g., a self-replicating RNA) does not include 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 unmodified viral genome or replicon RNA. In some embodiments, the modified alphavirus genome or RNA replicon does not include at least a portion of the nucleic acid sequence encoding one or more viral structural proteins. In some embodiments, the modified viral genome or RNA replicon does not include a substantial portion of the nucleic acid sequence encoding one or more viral structural proteins. In some embodiments, the modified viral genome or RNA replicon does not include the nucleic acid sequence encoding the viral structural protein. It will be understood by those skilled in the art that a substantial portion of a nucleic acid sequence encoding a viral structural polypeptide may contain sufficient nucleic acid sequence encoding a viral structural polypeptide to obtain putative identification of the polypeptide, either by manual evaluation of the sequence by those skilled in the art 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 contains sufficient sequence to obtain specific identification and / or isolation of a nucleic acid fragment comprising the sequence. For example, a substantial portion of a nucleic acid sequence may comprise at least about 20%, e.g., about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% of the full-length nucleic acid sequence. As noted above, the present disclosure provides nucleic acid molecules and nucleic acid constructs that are partially or completely free of nucleic acid sequence encoding one or more viral structural proteins.

[0081] In some embodiments, the nucleic acid constructs of the present disclosure comprise a nucleic acid sequence encoding a modified alphavirus genome or replicon RNA (e.g., a self-replicating RNA) from which a significant portion of the nucleic acid sequence encoding one or more structural proteins of the modified alphavirus genome or replicon RNA has been removed, e.g., the modified alphavirus genome or replicon RNA does not include at least a portion of the coding sequence for one or more of the alphavirus structural proteins CP, E1, E2, E3, and 6K.

[0082] Non-limiting and exemplary embodiments of the nucleic acid constructs of the present disclosure may include one or more of the following features: In some embodiments, at least a portion of the nucleic acid sequence encoding one or more of the viral structural proteins CP, E1, E2, E3, and 6K of the unmodified viral genome or replicon RNA (e.g., a self-replicating RNA) has been deleted. In some embodiments, a portion or all of the sequence encoding CP has been deleted. In some embodiments, a portion or all of the sequence encoding E1 has been deleted. In some embodiments, a portion or all of the sequence encoding E2 has been deleted. In some embodiments, a portion or all of the sequence encoding E3 has been deleted. In some embodiments, a portion or all of the sequence encoding 6K has been deleted. In some embodiments, a portion or all of the sequence encoding a combination of CP, E1, E2, E3, and 6K has been deleted. In some embodiments, the entire sequence encoding the viral structural proteins has been deleted, e.g., the modified viral genome or replicon RNA does not include the nucleic acid sequence encoding the structural proteins of the unmodified viral genome or replicon RNA.

[0083] Non-limiting and exemplary, the nucleic acid construct of the present disclosure may comprise 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 any one of the nucleic acid sequences selected from the group consisting of SEQ ID NO: 1 to 4. In some embodiments, the nucleic acid construct of the present disclosure may comprise 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 any one of the nucleic acid sequences selected from the group consisting of SEQ ID NO: 1. In some embodiments, the nucleic acid construct of the present disclosure may comprise 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 any one of the nucleic acid sequences selected from the group consisting of SEQ ID NO: 2. In some embodiments, a nucleic acid construct of the present disclosure may comprise 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 any one of the nucleic acid sequences selected from the group consisting of SEQ ID NO: 3. In some embodiments, a nucleic acid construct of the present disclosure may comprise 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 any one of the nucleic acid sequences selected from the group consisting of SEQ ID NO: 4.

[0084] A nucleic acid construct (e.g., a vector construct or srRNA construct) of the present disclosure typically has a length of at least about 2 kb. For example, a nucleic acid construct (e.g., a vector or srRNA) can have a length of at least about 2 kb, at least about 3 kb, at least about 4 kb, at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 11 kb, at least about 12 kb, or greater than 12 kb. In some embodiments, the 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 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) may have a length of about 6 kb to about 14 kb. In some embodiments, the nucleic acid construct (e.g., vector or srRNA) may have a length of about 6 kb to about 16 kb.

[0085] 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 may comprise at least two, at least three, at least four, at least five, or at least six expression cassettes. As will be understood by those skilled in the art, the term "expression cassette" refers to a construct of genetic material that contains a coding sequence and sufficient control information to direct the proper transcription and / or translation of the coding sequence in a cell in vivo and / or ex vivo. The expression cassette may be inserted into a vector and / or into a subject to target a desired host cell. Thus, in some embodiments, the term expression cassette may be used synonymously 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 any or a combination of other nucleic acid sequences that affect the transcription or translation of the gene.

[0086] In some embodiments, at least one of the expression cassettes may include a promoter operably linked to a heterologous nucleic acid sequence. Thus, a nucleic acid construct as provided herein may be used, for example, as an expression vector that may affect expression of a heterologous nucleic acid sequence when it includes a regulatory element (e.g., a promoter) operably linked to the heterologous nucleic acid sequence. In some embodiments, at least one of these expression cassettes includes 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 includes one or more untranslated regions (UTRs). In some embodiments, at least one of these UTRs is a heterologous UTR. In some embodiments, the 5'UTR sequence of the modified alphavirus genome or RNA replicon (e.g., self-replicating RNA) is a heterologous 5'UTR sequence, for example, from a heterologous source, for example, from a different strain of the same alphavirus species or from a different alphavirus species, for example, a UTR sequence from a chikungunya virus. In some embodiments, the 3'UTR sequence of the modified alphavirus genome or RNA replicon is a heterologous 3'UTR sequence. In some embodiments, both the 5'UTR sequence and the 3'UTR sequence of the modified alphavirus genome or RNA replicon are heterologous UTR sequences. In some embodiments, these heterologous 5'UTR sequence and / or heterologous 3'UTR sequence may be from a Chikungunya virus. In some embodiments, these heterologous 5'UTR sequence and / or heterologous 3'UTR sequence may be from a Chikungunya S27 strain. In some embodiments, these heterologous 5'UTR sequence and / or heterologous 3'UTR sequence may be from a Chikungunya DRDE strain.

[0087] 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 to obtain desired properties, such as increased stability, potency, and expression (e.g., translation efficiency), which may in turn maximize the impact of the production, delivery, and administration of the biotherapeutic. For example, in some embodiments, the coding sequence of the GOI is optimized to obtain expression at a level higher than the expression level of a reference coding sequence, e.g., 20% higher, 30% higher, 40% higher, 50% higher, 60% higher, 70% higher, 80% higher, 90% higher, or 95% higher than the reference coding sequence. In some embodiments, the reference coding sequence is a wild-type, unoptimized sequence. With regard to sequence optimization of a nucleotide sequence, the degeneracy of the genetic code creates the possibility of replacing at least one base of a protein coding sequence of a gene with a different base without changing the amino acid sequence of a polypeptide produced from said gene. Thus, the nucleic acid constructs of the present disclosure may have any base sequence that varies from any polynucleotide sequence disclosed herein by substitutions according to the degeneracy of the genetic code. References describing codon usage are readily available in the public domain. In some embodiments, variants of polynucleotide sequences may be generated for various reasons, such as to optimize expression for a particular host (e.g., changing the codon usage in alphavirus mRNA to that preferred by other organisms, such as humans, non-human primates, hamsters, mice, or monkeys). Thus, in some embodiments, the coding sequence of the GOI is optimized to obtain a higher level of expression in the target host cell through the use of expression-optimized codons than that of a reference coding sequence, such as a coding sequence that is not codon-optimized. In some embodiments, the codon-optimized GOI sequence results in an increase in expression level of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% compared to a reference coding sequence that is not codon-optimized.In some embodiments, the codon-optimized GOI sequence provides at least a 2-fold, at least a 3-fold, at least a 4-fold, or at least a 5-fold increase in expression level compared to a non-codon-optimized reference coding sequence.

[0088] In some embodiments, the coding sequence of the GOI is optimized to enhance RNA stability and / or expression. RNA stability is usually 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 a molecule. In the context of the present disclosure, the half-life of an RNA refers to the stability of that RNA. The half-life of an RNA may affect the "duration of expression" of that RNA. Several methodologies and techniques are known that are useful for assessing RNA stability, including the storage of replicons (e.g., self-replicating RNAs) with different GOI codon usage, and various in silico methodologies and / or empirical stress testing of the potency of the replicon (e.g., testing dsRNA in cells after transfection) and its effect on gene expression. Further information on this can be found, for example, in Wayment-Steele, H. et al. (2021). Cold Spring Harbor Laboratory (doi.org / 10.1101 / 2020.08.22.262931). Further 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.

[0089] 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 encodes a polypeptide that can be an antibody, an antigen, an immunomodulator, an enzyme, a signaling protein, or 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. Non-limiting examples of GOIs include interleukins and interacting proteins such as 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.Further suitable GOIs include interferons (e.g., IFN-α, IFN-β, IFN-γ), TNF (e.g., CD154, LT-β, ​​TNF-α, TNF-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, and TRANCE), TGF-β (e.g., TGF-β1, TGF-β2, and TGF-β3), hematopoietins (e.g., Epo, Tpo, Flt-3L, SCF, M-CSF, MSP), chemokines and their receptors (e.g., XCL1, XCL2, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL9, CCL10, CCL112, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL210, CCL220, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL29, CCL30, CCL310, CCL320, CCL333, CCL34, CCL35, CCL36, CCL37, CCL38, CCL39 ... L11, 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 associated transcription factors (e.g., PECAM1, FCGR3A, FOS, NFKB1, JUN, HIF1A, PD-L1, mTOR, STAT5B, and STAT4).

[0090] 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.

[0091] In some embodiments, the GOI may encode an antibody or antibody variant (e.g., single chain Fv antibodies, bispecific antibodies, camelid antibodies, Fab antibodies, and heavy chain antibodies). In some embodiments, the antibody targets a surface molecule associated with or upregulated by cancer, or associated with infectious disease. In some embodiments, the antibody targets a surface molecule with immunostimulatory or immunosuppressive function.

[0092] In some embodiments, the GOI may encode an enzyme whose deficiency or mutation is associated with a disease or condition, such as, for example, agalsidase beta, agalsidase alpha, imiglucerase, taliglucerase alpha, velaglucerase alpha, alglucerase, sebelipase alpha, laronidase, idursulfase, elosulfase alpha, galsulfase, alglucosidase alfa, and CTFR.

[0093] 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 neuromodulator, 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 coat a biotherapeutic polypeptide selected from IL-12A, IL-12B, IL-1RA, and any combination thereof.

[0094] 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 generated 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.

[0095] In some embodiments, the polypeptide encoded by the GOI is a recombinant polypeptide. In some embodiments, the GOI encodes an antigenic HA polypeptide of avian influenza A H5N1. In some embodiments, the GOI encodes an oncology-related protein, or a portion thereof, such as ESR1, HER2, and HER3. In some embodiments, the GOI encodes a cytokine, such as IL-1RA or IL-12.

[0096] In some embodiments, a nucleic acid construct of the disclosure comprises a nucleic acid sequence encoding a modified genome or RNA replicon (e.g., a self-replicating RNA) of an alphavirus species that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-4. In some embodiments, a nucleic acid construct of the disclosure comprises a nucleic acid sequence encoding a modified genome or RNA replicon of an alphavirus species that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, a nucleic acid construct of the disclosure comprises a nucleic acid sequence encoding a modified genome or RNA replicon of an alphavirus species 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 disclosure comprises a nucleic acid sequence encoding a modified genome or RNA replicon of an alphavirus species 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: 3. In some embodiments, a nucleic acid construct of the present disclosure comprises a nucleic acid sequence encoding a modified genome or RNA replicon of an alphavirus species 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:4. Table 1: Brief description of the sequences in the sequence listing [Table 1]

[0097] 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 genome or RNA replicon (e.g., self-replicating RNA) of the alphavirus species of interest can be identified and / or isolated from the sequences identified within the alphavirus species genome by genomic sequence analysis, hybridization, and / or PCR using degenerate or gene-specific primers, using the sequences identified herein (e.g., SEQ ID NOs: 1-4) or any others as known in the art.

[0098] The molecular techniques and methods used to construct and characterize these novel nucleic acid constructs are described in more detail in the Examples herein of this application.

[0099] In some embodiments, the nucleic acid molecule is a recombinant nucleic acid molecule. As stated above, the term recombinant nucleic acid molecule refers to any nucleic acid molecule (e.g., DNA, RNA) that is obtained from or that is the result, even indirectly, of human manipulation. As a non-limiting example, cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that is produced by an in vitro polymerase reaction, or that is attached with a linker, or that is incorporated into a vector, such as a cloning vector or an expression vector. As non-limiting examples, a recombinant nucleic acid molecule is one that: 1) has been synthesized or modified in vitro, for example, using chemical or enzymatic techniques (e.g., by the use of chemical nucleic acid synthesis or by the use of enzymes for replication, polymerization, exonuclease digestion, endonucleolytic digestion, ligation, reverse transcription, transcription, base modification (including, for example, methylation), or recombination (including homologous recombination and site-specific recombination) of nucleic acid molecules; 2) contains linked nucleotide sequences that are not linked in nature; 3) has been engineered using molecular cloning techniques to lack one or more nucleotides compared to the native nucleotide sequence; and / or 4) has been engineered using molecular cloning techniques to have one or more sequence changes or sequence rearrangements compared to the native nucleotide sequence.

[0100] In some embodiments, the nucleic acid molecules disclosed herein are produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning, etc.) or chemical synthesis. Nucleic acid molecules as disclosed herein include naturally occurring nucleic acid molecules and their homologues, including, but not limited to, naturally occurring allelic variants, and modified nucleic acid molecules in which one or more nucleotide residues have been inserted, deleted, and / or substituted such that the modification provides desired properties in exerting a biological activity as described herein.

[0101] Nucleic acid molecules, including variants of naturally occurring nucleic acid sequences, can be generated using many methods known to those of skill in the art (see, e.g., Sambrook et al., In: Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989)). The sequence of a nucleic acid molecule can be altered from the naturally occurring sequence from which it is derived using a variety of techniques, including, but not limited to, classical mutagenesis and recombinant DNA techniques, such as, but not limited to, site-directed mutagenesis, mutagenesis by chemical treatment of a nucleic acid molecule, restriction enzyme cleavage of nucleic acid fragments, ligation of nucleic acid fragments, PCR amplification and / or mutagenesis of selected regions of a nucleic acid sequence, recombinational cloning, and chemical synthesis, including "assembly" of mixtures of nucleic acid molecules by chemical synthesis of mixtures of oligonucleotides and ligation of mixtures, and combinations thereof. Nucleic acid molecule homologs can be selected from a mixture of modified nucleic acid molecules by screening for the function of the protein or 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 a target or wild-type nucleic acid molecule or sequence.

[0102] B. Recombinant Cells As described in more detail below, one aspect of the disclosure relates to recombinant cells engineered to contain (e.g., express) a nucleic acid construct as described herein. In some embodiments, a nucleic acid construct (e.g., a vector or srRNA) of the disclosure may be introduced into a host cell to generate a recombinant cell containing the nucleic acid construct and / or srRNA construct. For example, a nucleic acid construct of the disclosure may be introduced into a host cell to generate a recombinant cell containing the nucleic acid construct. Thus, prokaryotic or eukaryotic cells containing a nucleic acid construct encoding a modified genome or RNA replicon (e.g., a self-replicating RNA) of an alphavirus species as 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 as provided herein into a host cell, such as an animal cell, and then selecting or screening for transformed cells. Introduction of the nucleic acid constructs of the present disclosure into cells can be achieved by methods known to those of skill in the art, such as, for example, viral infection, transfection, conjugative transfer, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethylenimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, direct microinjection, and nanoparticle-mediated nucleic acid delivery.

[0103] In one aspect, some embodiments of the present disclosure relate to a recombinant cell, e.g., a recombinant eukaryotic cell, e.g., an insect cell or an animal cell, comprising a nucleic acid construct as described herein. The nucleic acid construct may be stably integrated into the host genome or may be episomally replicated, i.e., present in the recombinant host cell as a minicircle expression vector for stable or transient expression. Thus, in some embodiments of the present disclosure, the nucleic acid construct is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid construct is stably integrated into the genome of the recombinant cell. Stable integration can be achieved using classical random genome recombination methods or by more precise genome editing methods, such as using guide RNA-guided CRISPR / Cas9 or TALEN genome editing. In some embodiments, the nucleic acid construct is present in the recombinant host cell as a minicircle expression vector for stable or transient expression.

[0104] A host cell may be an untransformed cell or a cell that has been transfected with at least one nucleic acid molecule. Thus, in some embodiments, a host cell may be genetically modified (e.g., transduced or transformed or transfected) with at least one nucleic acid molecule.

[0105] Suitable host cells for cloning or expressing a protein of interest as described herein include prokaryotic or eukaryotic cells as described herein. Thus, in some embodiments, the recombinant cell is a prokaryotic cell, such as E. coli, or a eukaryotic cell, such as an insect cell (e.g., a mosquito cell or an Sf21 cell), or a mammalian cell (e.g., a COS cell, an NIH 3T3 cell, or a HeLa cell). In some embodiments, the cell is an in vivo cell, e.g., a recombinant cell in a living organism, e.g., a cell of a transgenic subject. In some embodiments, the subject is a vertebrate or an invertebrate. In some embodiments, the subject is an insect. In some embodiments, the subject is a mammalian subject. In some embodiments, the mammalian subject is a human subject. In some embodiments, the cell is an ex vivo cell, e.g., extracted as an individual cell or as part of an organ or tissue from a living organism or organism to be subjected to a treatment or procedure, and then returned to the living organism or organism. In some embodiments, the cell is an in vitro cell, 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.

[0106] For expression of glycosylated proteins, suitable host cells may be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include insect cells. Vertebrate cells may also be used as hosts. In this regard, mammalian cell lines adapted to grow in suspension may be useful. 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., HEK293 or HEK293 cells), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells), human cervical carcinoma cells (HeLa), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT060562), 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 laryngeal epidermoid cells, human fibroblast cells, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocytes, human macrophage cells, human RAW264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells.

[0107] In some embodiments, the recombinant cell is selected from the group consisting of African green monkey kidney cells (Vero cells), baby hamster kidney (BHK) cells, Chinese hamster ovary cells (CHO cells), human A549 cells, human cervical cells, human CHME5 cells, human laryngeal epidermoid cells, human fibroblast cells, human HEK-293 cells, human HeLa cells, human HepG2 cells, human HUH-7 cells, human MRC-5 cells, human muscle cells, mouse 3T3 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells. In some embodiments, the recombinant cell is a BHK cell. In some embodiments, the BHK cell is a BHK-21 cell. In some embodiments, the recombinant cell is a Vero cell.

[0108] In some embodiments, the recombinant cell is an insect cell, for example, a cell of an insect cell line. In some embodiments, the recombinant cell is an Sf21 cell. Additional suitable insect cell lines include, but are not limited to, cell lines established from insect Diptera, Lepidoptera, and Hemiptera, and can be obtained from a variety of tissue sources. In some embodiments, the recombinant cell is a cell of a lepidopteran insect cell line. Over the past few decades, the availability of lepidopteran insect cell lines has increased at a rate of about 50 lines / 10 years. Further 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 included in the genera Anopheles (An.), Culex (Cx.), and Aedes (Ae.). Exemplary mosquito cell lines suitable for the compositions and methods described herein include cell lines obtained 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 quinquefasciat ... bitaeniorhynchus, and Toxorhynchites amboinensis.Suitable mosquito cell lines include, but are not limited to, CCL-125, Aag-2, RML-12, C6 / 26, C6 / 36, C7-10, AP-61, AtGRIP-1, AtGRIP-2, UM-AVE1, Mos.55, Sua1B, 4a-3B, Mos.43, MSQ43, and LSB-AA695BB. In some embodiments, the mosquito cell is a cell of the C6 / 26 cell line.

[0109] C.Cell culture In another aspect, provided herein is a cell culture comprising at least one recombinant cell as disclosed herein and a medium. Typically, the medium may be any medium suitable for culturing the cells described herein. Techniques for transforming the various host cells and species mentioned above are known in the art and described in the technical and scientific literature. Thus, also within the scope of the present application is a cell culture comprising at least one recombinant cell as disclosed herein. Suitable methods and systems for producing and maintaining cell cultures are known in the art.

[0110] D. Transgenic Animals Also provided in another aspect is a transgenic animal comprising a nucleic acid construct (e.g., a vector or srRNA molecule) 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 general, the transgenic animal of the present disclosure can be any non-human animal known in the art. In some embodiments, the non-human animal of the present disclosure is a non-human primate. Other animal species suitable for the compositions and methods of the present disclosure include those that are (i) suitable for genetic recombination and (ii) capable of rearranging immunoglobulin gene segments to generate an antibody response. Examples of such species include, but are not limited to, mice, rats, hamsters, rabbits, chickens, goats, pigs, sheep, and cows. Further examples of non-human animals suitable for the compositions and methods of the present disclosure may include, but are not limited to, laboratory animals (e.g., mice, rats, hamsters, gerbils, guinea pigs, etc.), farm animals (e.g., horses, cows, pigs, sheep, goats, ducks, geese, chickens, etc.), non-human primates (e.g., apes, chimpanzees, orangutans, monkeys, etc.), fish, amphibians (e.g., frogs, salamanders, etc.), reptiles (e.g., snakes, lizards, etc.), and other animals (e.g., foxes, weasels, rabbits, minks, beavers, ermines, otters, sables, seals, coyotes, chinchillas, deer, musk rats, possums, etc.).

[0111] In some embodiments, the transgenic animal is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the transgenic animal of the present disclosure is a chimeric transgenic animal. In some embodiments, the transgenic animal of the present disclosure is a transgenic animal that comprises germ cells and somatic cells that contain one or more (e.g., one or more, two or more, three or more, four or more, etc.) nucleic acid constructs of the present disclosure. In some embodiments, the one or more nucleic acid constructs are stably integrated into the genome of the transgenic animal. In some embodiments, the genome of the transgenic animal of the present disclosure may comprise any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more copies of one or more nucleic acid constructs of the present disclosure.

[0112] Approaches and methods for producing transgenic non-human animals are known in the art. Exemplary methods include pronuclear microinjection, DNA microinjection, DNA transfer into early embryos via lentivirus vectors, sperm-mediated gene recombination, adenovirus-mediated DNA transfer into animal sperm (e.g., pigs), retrovirus vectors (e.g., birds), and somatic cell nuclear transfer (e.g., goats). The current state of the art in producing transgenic livestock animals is reviewed in Niemann, H. et al. (2005) Rev. Sci. Tech. 24:285-298. In some embodiments, the transgenic non-human host animals of the present disclosure are produced using standard methods known in the art for introducing foreign nucleic acid into the genome of a non-human animal. In some embodiments, the transgenic animals of the present disclosure may be generated using classical random genome recombination methods, or using more precise techniques such as guide RNA-guided CRISPR / Cas genome editing, or DNA-guided endonuclease genome editing using NgAgo (Natronobacterium gregoryi argonaute), or TALEN genome editing (transcription activator-like effector nuclease). In some embodiments, the transgenic animals of the present disclosure may be generated using transgenic microinjection techniques, which do not require the use of homologous recombination techniques and may be easier to generate and screen than approaches using homologous recombination. In some embodiments, the transgenic animals produce a protein of interest as described herein.

[0113] E. Pharmaceutical Compositions The nucleic acid constructs, recombinant cells, recombinant polypeptides of the present disclosure may be incorporated into compositions, including pharmaceutical compositions. Such compositions typically include one or more of the nucleic acid constructs, recombinant cells, recombinant polypeptides described and provided herein, and a pharma- ceutically acceptable excipient, such as a carrier. In some embodiments, the compositions of the present disclosure are formulated for the prevention, treatment, or management of a health condition, such as an immune disorder or a microbial infection. For example, the compositions of the present disclosure may be formulated as a prophylactic composition, a therapeutic composition, or a pharmaceutical composition, including a pharma- ceutically acceptable excipient or mixtures thereof. In some embodiments, the compositions of the present disclosure are formulated for use as a vaccine. In some embodiments, the compositions of the present application are formulated for use as an adjuvant.

[0114] Thus, in one aspect, provided herein is a pharmaceutical composition comprising a pharma- ceutically acceptable excipient and a) a nucleic acid construct (e.g., a vector or srRNA molecule) of the present disclosure; b) a recombinant cell of the present disclosure; and / or c) a recombinant polypeptide of the present disclosure. Non-limiting and exemplary embodiments of the pharmaceutical composition 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 (e.g., a vector or srRNA molecule) 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.

[0115] In some embodiments, the nucleic acid constructs (e.g., vectors or srRNA molecules) of the present disclosure 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 nanoparticles (LNPs), solid lipid nanoparticles (SLNs), polyplexes, polymeric nanoparticles, viral replicon particles (VRPs), or conjugated with bioactive ligands that can facilitate delivery and / or enhance immune responses. These compounds are readily available to those skilled in the art; see, for example, Liposomes: A Practical Approach, RCP New Ed, IRL press (1990). Adjuvants other than liposomes and the like are also used and are known in the art. Adjuvants may protect antigens (e.g., nucleic acid constructs, vectors, srRNA molecules) from rapid dispersion by sequestering them in a localized precipitate, or may contain substances that stimulate the host to secrete immune system components, such as macrophage chemotactic factors. An appropriate selection from, for example, the following, can be made by one skilled in the art.

[0116] In some embodiments, the compositions of the present disclosure may include one or more of the following: physiological buffer, liposomes, lipid nanoparticles (LNPs), solid lipid nanoparticles (SLNs), polyplexes, polymeric nanoparticles, viral replicon particles (VRPs), microspheres, immune stimulating complexes (ISCOMs), conjugates of bioactive ligands, or any combination thereof. In some embodiments, the compositions of the present disclosure are formulated as liposomes. In some embodiments, the compositions of the present disclosure are formulated as lipid nanoparticles (LNPs). LNPs are generally less immunogenic than viral particles. Many humans have pre-existing immunity to viral particles, but not to LNPs. In addition, adaptive immune responses to LNPs are unlikely to occur, which allows for repeated administration of LNPs.

[0117] Lipids suitable for the compositions and methods described herein can be cationic lipids, ionizable cationic lipids, anionic lipids, or neutral lipids.

[0118] 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 becomes cationic or ionic (protonated) when the pH is lower than the pKa of the ionizable group of the lipid, but becomes more neutral at higher pH values. At pH values ​​below the pKa, the lipid is capable of associating with negatively charged nucleic acids (e.g., oligonucleotides). As used herein, the term "ionizable lipid" encompasses lipids that assume a positive charge when the pH is lowered from physiological pH, and any of a 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 been found to be too toxic for clinical use. Ionizable lipids may be present in the lipid formulation in other embodiments, preferably in the range of about 30 to about 70 mol% in some embodiments, about 30 mol% in other embodiments, about 40 mol% in other embodiments, about 45 mol% in other embodiments, about 47.5 mol% in other embodiments, about 50 mol% in still other embodiments, and about 60 mol% in still other embodiments ("mol%" refers to the percentage of the total moles of a particular component). The term "about" in this paragraph refers to a range of ±5 mol%. DODMA, i.e. 1,2-dioleyloxy-3-dimethylaminopropane, is an ionizable lipid, as is DLin-MC3-DMA or 0-(Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N-dimethylamino) ("MC3").

[0119] Exemplary ionizable lipids suitable for the compositions and methods of the present disclosure include those described in WO 2020252589 and WO 2021000041, U.S. Patent Nos. 8,450,298 and 10,844,028, and Love KT et al., Proc Natl Acad Sci USA, Feb. 2, 2010 107 (5) 1864-1869, all of which are incorporated herein by reference in their entirety. Thus, in some embodiments, the LNPs of the present disclosure comprise one or more lipid compounds described in Love KT et al. (2010, supra), such as C16-96, C14-110, and C12-200. In some embodiments, the LNPs comprise an ionizable cationic lipid selected from the group consisting of ALC-0315, C12-200, LN16, MC3, MD1, SM-102, and any combination thereof. In some embodiments, the LNPs of the present disclosure comprise a C12-200 lipid. The structure of the C12-200 lipid is known in the art and described, for example, in U.S. Patent 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.

[0120] 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 outside of the LNP to act as a ligand for uptake into the liver via the asialoglycoprotein receptor. Any of these cationic lipids can be used in the LNP formulation to deliver the srRNA constructs and nucleic acid constructs of the present disclosure.

[0121] In some embodiments, the LNPs of the present disclosure comprise 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 comprise one or more ionizable lipid compounds described in WO2020252589 and WO2021000041.

[0122] Any number of other lipids or lipid combinations known in the art can be used to generate LNPs. Non-limiting examples of lipids suitable for use in generating 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.

[0123] In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 100:1 to about 3:1, about 70:1 to 10:1, or 16:1 to 4:1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 16:1 to 4:1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 20:1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 8:1. In some embodiments, the lipid nanoparticles have an average diameter of less than about 1000 nm, less than about 500 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm, less than about 100 nm, less than about 75 nm, less than about 50 nm, or less than about 25 nm. In some embodiments, the LNPs have an average diameter in the range of about 70 nm to 100 nm. In some embodiments, the LNPs have an average diameter ranging from about 88 nm to about 92 nm, 82 nm to about 86 nm, or about 80 nm to about 95 nm.

[0124] In some embodiments, the compositions of the present disclosure are formulated as 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.

[0125] In some embodiments, the immunogenic composition is substantially non-immunogenic to a subject, 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, intralymphatic, intratumoral, intraarticular, intravenous, subcutaneous, intravaginal, intraocular, rectal, and oral administration.

[0126] Pharmaceutical compositions suitable for injectable use 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 it exhibits easy needle passability. The composition can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing 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 dispersions, 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, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is common to include isotonic agents in the composition, such as sugars, polyalcohols such as mannitol, sorbitol, and / or sodium chloride. Prolonged absorption of an injectable composition can be achieved by including in the composition an agent that delays absorption, such as aluminum stearate and gelatin.

[0127] Sterile injectable solutions can be prepared by mixing the required amount of the active compound in an appropriate solvent with one or a combination of ingredients as required above, followed by filtered sterilization. Typically, dispersions are prepared by mixing the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0128] In some embodiments, the pharmaceutical compositions of the present disclosure are formulated for inhalation, such as an aerosol, spray, mist, liquid, or powder. Administration by inhalation may be in the form of a dry powder or aerosol formulation that is inhaled by a subject (e.g., patient) through the use of an inhalation device, such as a microspray, pressurized metered dose inhaler, or nebulizer.

[0129] In some embodiments, the composition is formulated for one or more of intranasal, transdermal, intramuscular, intranodal, intratumoral, intraarticular, intravenous, intraperitoneal, oral, intravaginal, intraocular, intrarectal, or intracranial administration. In some embodiments, the administered composition results in increased interferon production in the subject.

[0130] Methods of the Disclosure As described in more detail below, one embodiment of the disclosure relates to, inter alia, methods of functionalizing an alphavirus by replacing at least a portion of the nsP coding sequence, methods of producing a polypeptide of interest encoded by a gene of interest (GOI), methods of eliciting an immune response in a subject in need thereof, and methods of preventing and / or treating a health condition in a subject in need thereof.

[0131] Methods for functionalizing alphaviruses As outlined above, certain aspects of the present disclosure relate to methods for functionalizing / engineering an alphavirus genome or RNA replicon (e.g., a self-replicating RNA), comprising: (a) providing a non-functional alphavirus genome or RNA replicon; (b) replacing non-structural proteins (nsPs) or portions thereof of said non-functional alphavirus genome or RNA replicon with heterologous coding sequences of corresponding nsPs or portions thereof from a different alphavirus strain, thereby generating a modified alphavirus genome or RNA replicon; (c) assessing the functionality of said modified alphavirus genome or RNA replicon; and (d) considering said modified alphavirus genome or RNA replicon functional if said modified alphavirus genome or RNA replicon is capable of RNA replication and / or expression.

[0132] In some embodiments, the heterologous nsP or portion thereof is from another strain of the same alphavirus species. In some embodiments, the heterologous nsP or portion thereof is from another alphavirus species. In some embodiments, the heterologous nsP or portion thereof is nsP1, nsP2, nsP3, nsP4, or any portion thereof. In some embodiments, the non-functionality of an alphavirus genome or RNA replicon (e.g., a self-replicating RNA) is determined by insufficient self-replication in a host cell. Generally, the functionality of a modified alphavirus genome or RNA replicon of the present disclosure can be assessed by using one or more assays and methodologies known in the art, such as detection of RNA replication, detection of viral protein expression, detection of cytopathic effect (CPE), and detection of heterologous transgene expression. In particular, a non-functional alphavirus can be identified as being incapable of self-replication in cell culture or primary cell lines, such as, for example, but not limited to, BHK, VERO, or HEK293. As described above, non-functionality of an alphavirus can be determined if a deposited alphavirus sequence (e.g., a sequence retrieved from a public database), when synthetically reproduced, is found to be insufficient for autonomous replication.

[0133] Methods for Producing a Polypeptide of Interest In one aspect, provided herein is a method for producing a polypeptide of interest, comprising culturing a recombinant cell comprising a nucleic acid construct as disclosed herein under conditions in which the recombinant cell produces a polypeptide encoded by a GOI. In another aspect, provided herein is a method for producing a polypeptide of interest in a subject, comprising administering to the subject a nucleic acid construct as disclosed herein. In some embodiments, the subject is a vertebrate or invertebrate. 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 the present disclosure.

[0134] Non-limiting and exemplary embodiments of the disclosed method for producing a recombinant polypeptide may include one or more of the following features. In some embodiments, the disclosed method for producing a recombinant polypeptide further comprises isolating and / or purifying the produced polypeptide. In some embodiments, the disclosed method for producing a polypeptide further comprises structurally modifying the produced polypeptide to extend its half-life. In some embodiments, the N-terminus of the produced polypeptide may be further modified chemically or enzymatically to extend its half-life. In some embodiments, the C-terminus of the produced polypeptide is chemically and / or enzymatically modified to extend its half-life. Non-limiting examples of chemical and enzymatic modifications suitable for the methods described herein include PEGylation, XTENylation, PASylation, ELPylation, and HAPylation. Suitable techniques, systems, and reagents for these modifications are known in the art. Thus, in some embodiments, the polypeptide produced by the methods described herein may be PEGylated, XTENylation, PASylation, ELPylation, and / or HAPylation to extend its half-life. In some embodiments, the generated polypeptide is conjugated to another protein or peptide (e.g., serum albumin, the Fc domain of an antibody, transferrin, GLK, or CTP peptide) to extend half-life.

[0135] In one embodiment, a method for producing a polypeptide of interest comprises (i) raising a transgenic animal of the present disclosure, or (ii) culturing a recombinant cell comprising a nucleic acid construct of the present disclosure under conditions in which the recombinant cell produces the polypeptide encoded by the GOI.

[0136] In some embodiments, a method for producing a polypeptide of interest in a subject comprises administering to the subject a nucleic acid construct of the present disclosure. In some embodiments, the subject is a vertebrate or invertebrate animal. In some embodiments, the animal is an insect. In some embodiments, the subject is a mammalian subject. In some embodiments, the mammalian subject is a human subject.

[0137] Methods for inducing a pharmacodynamic effect, for inducing an immune response, for preventing a condition, or for treating a condition Administration of any one of the therapeutic compositions described herein, e.g., nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions, may be used in the treatment of relevant health conditions, such as proliferative diseases (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 as described herein may be incorporated into a therapeutic agent for use in a method of treating an individual who has, is suspected of having, or may be at high risk of developing one or more relevant health conditions or diseases. Exemplary health conditions or diseases may include, but are not limited to, cancer, immune diseases, gene therapy, gene replacement, cardiovascular disease, age-related conditions, acute infections, and chronic infections. In some embodiments, the individual is a patient under the care of a physician.

[0138] In some embodiments, the nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions as 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 as described herein may be useful for modulating, e.g., inducing or suppressing, an immune response in a subject in need thereof. Thus, in one aspect, provided herein is a method for inducing an immune response in a subject in need thereof, comprising administering to said subject a composition comprising: a) a nucleic acid construct (e.g., vector or srRNA molecule) 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.

[0139] Analysis of the ability of the compositions described herein 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., eliciting an immune response in vivo), biomarker responses, therapeutic effects, prophylactic effects, desired effects, undesirable effects, adverse effects, and effects in disease models. In some embodiments, evaluation of the pharmacodynamic effect includes evaluating the induction of an immune response in vivo. In some embodiments, evaluation of the pharmacodynamic effect includes evaluating the induction of cytokine pathways that can enhance immune responses and inhibit angiogenesis and metastasis.

[0140] In another aspect, provided herein is a method for preventing and / or treating a health condition in a subject in need thereof, comprising administering to said subject, for prophylactic or therapeutic purposes, a composition comprising: a) a nucleic acid construct (e.g., a vector or an srRNA molecule) of the present disclosure; b) a recombinant cell of the present disclosure; c) a recombinant polypeptide of the present disclosure; and / or d) any one of the pharmaceutical compositions of the present disclosure.

[0141] In some embodiments, the condition is a proliferative disease or a microbial infection. In some embodiments, the subject has or is suspected of having a condition associated with a proliferative disease or a microbial infection.

[0142] In some embodiments, the compositions of the present disclosure are formulated to be compatible with their intended route of administration. For example, the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure may be given orally or by inhalation, but are more often administered via parenteral routes. Examples of parenteral routes of administration include, for example, intravenous, intranodal, intradermal, subcutaneous, transdermal (topical), transmucosal, intravaginal, and intrarectal 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 glucose. The pH may be adjusted (e.g., to about pH 7.2-7.8, e.g., pH 7.5) with acids or bases such as mono- and / or di-sodium phosphate, hydrochloric acid, or sodium hydroxide. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0143] Dosage, toxicity, and therapeutic efficacy of such subject nucleic acid constructs, 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 LD (the dose that is therapeutically effective in 50% of a population) can be determined by standard pharmaceutical procedures in cell culture or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50The therapeutic index can be expressed as a ratio of . Compounds that exhibit high therapeutic indices are usually preferred. Compounds that exhibit toxic side effects can be used, but care should be taken to design a delivery system that targets such compounds to the site of the affected tissue in order to minimize the possibility of damaging uninfected cells, thereby reducing side effects.

[0144] For example, 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 such compounds is generally administered at or above the ED 50 The IC50 concentration range includes 0.1 to 0.5% by weight of the compound, and is associated with little toxicity. Dosages may vary within this range depending on the dosage form employed and the route of administration used. For any compound used in the methods of the present disclosure, the therapeutically effective dose may be estimated initially from cell culture assays. The IC50 concentration range includes 0.1 to 0.5% by weight of the compound, and is associated with little toxicity. 50 A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes (e.g., the concentration of the test compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0145] The therapeutic compositions, e.g., nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions described herein, may be administered one or more times per day to one or more times per week; including once every other day. It will be appreciated by those of skill in the art that certain factors may affect the dosage and timing required to effectively treat a subject, including, but not limited to, severity of the disease, previous treatments, the overall health and / or age of the subject, and other diseases present. Furthermore, treatment of a subject with a therapeutically effective amount of the multivalent polypeptides and multivalent antibodies of the presently disclosed subject matter may include one treatment or may include a series of treatments. In some embodiments, the composition is administered every 8 hours for 5 days, followed by a rest period of 2 to 14 days (e.g., 9 days), followed by administration every 8 hours for an additional 5 days. With respect to nucleic acid constructs and recombinant polypeptides, the therapeutically effective amount (e.g., effective dose) 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 within the range of about 0.001-0.1 mg / kg of patient body weight may be administered. In some embodiments, about 0.005 mg / kg, about 0.01 mg / kg, about 0.05 mg / kg may be administered. In some embodiments, a single dose within the range of about 0.03 μg-300 μg / kg of patient body weight may be administered. In some embodiments, a single dose within the range of about 0.3 mg-3 mg / kg of patient body weight may be administered.

[0146] As discussed above, a therapeutically effective amount includes an amount of a therapeutic composition sufficient to promote a particular effect when administered to a subject, such as a subject having, suspected of having, or at risk for a health condition (e.g., a disease or infection). In some embodiments, an effective amount includes an amount sufficient to prevent or delay the onset of a symptom of a disease or infection, alter the course of a symptom of a disease or infection (e.g., 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 should be understood that the appropriate "effective amount" in any given case can be determined by one of ordinary skill in the art using routine experimentation.

[0147] The efficacy of a treatment, including the therapeutic composition of the present disclosure 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 the individual not getting worse (e.g., the progression of the disease or infection is stopped or at least slowed) as assessed by the need for hospitalization or medical intervention. Methods for measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease or infection in a subject or animal (some non-limiting examples include humans or mammals), including (1) inhibiting the disease or infection, e.g., stopping or slowing the progression of the symptoms; or (2) relieving the disease or infection, e.g., causing regression of the symptoms; and (3) inhibiting the onset of the symptoms or reducing the likelihood of the onset of the symptoms.

[0148] 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 comprising a pharma- ceutically acceptable carrier and in an amount effective to stimulate an immune response. Typically, a subject is immunized through an initial series of injections (or administration via one of the other routes described below), after which boosters may be given to augment the protection provided by the initial series. The initial series of injections and subsequent boosters are administered at doses and for periods of time necessary to stimulate an immune response in the subject, for example, 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 interferon production in a subject not administered the composition. In some embodiments, the administered composition results in increased interferon production in the subject. In some embodiments of the methods of the present disclosure, the subject is a mammal. In some embodiments, the mammal is a human.

[0149] As mentioned above, pharma- ceutically acceptable carriers suitable for injectable use 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 fluid to the extent that they exhibit easy syringability. They must also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carriers can be, for example, solvents or dispersion media containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.

[0150] Sterile injectable solutions can be prepared by mixing the required amount of nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, and / or recombinant polypeptides in an appropriate solvent, with one or a combination of above ingredients, as required, followed by filtered sterilization.

[0151] The nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions, when properly protected, can be administered orally, for example, with an inert diluent or an assimilable edible carrier, as described above. The nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions, as well as other ingredients, can also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or mixed directly into the diet of an individual. For oral therapeutic administration, the active compounds can be mixed with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0152] 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 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, which allows for repeated administration of LNPs.

[0153] As mentioned above, several different ionizable cationic lipids have been developed for use in LNPs. These include, among others, C12-200, MC3, LN16, and MD1. For example, in one type of LNP, a GalNAc moiety is attached to the exterior of the LNP and acts as a ligand for uptake into the liver via the asialoglycoprotein receptor. Any of these cationic lipids can be used in LNP formulations to deliver the nucleic acid constructs and recombinant polypeptides of the present disclosure to the liver.

[0154] In some embodiments, LNP refers to any particle having a diameter of less than 1000 nm, less than 500 nm, less than 250 nm, less than 200 nm, less than 150 nm, less than 100 nm, less than 75 nm, less than 50 nm, or less than 25 nm. Alternatively, nanoparticles can range in size from 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

[0155] LNPs can be prepared from cationic, anionic, or neutral lipids. Neutral lipids such as DOPE, a fusogenic phospholipid, and cholesterol, a membrane component, can be included in LNPs as "helper lipids" to enhance transfection activity and nanoparticle stability. The limitations of cationic lipids include poor efficacy due to poor stability and rapid clearance, as well as the generation of inflammatory or anti-inflammatory responses. LNPs can also have hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.

[0156] As described above, any number of lipids or combinations of lipids developed for use in LNPs can be used to generate the LNPs of the present disclosure. Non-limiting examples of lipids suitable for use in generating 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 suitable for use in generating LNPs include 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Non-limiting examples of neutral lipids suitable for use in generating LNPs include DPSC, DPPC, POPC, DOPE, and SM. Non-limiting examples of PEG-modified lipids suitable for use in generating LNPs include PEG-DMG, PEG-CerC14, and PEG-CeraC20.

[0157] In some embodiments, these lipids may be combined in any molar ratio to produce LNPs. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 100:1 to about 3:1, about 70:1 to 10:1, or about 16:1 to 4:1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 16:1 to 4:1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 20:1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 8:1. Additionally, polynucleotides may be combined with lipids in a wide range of molar ratios to produce LNPs.

[0158] 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 a subject having, suspected of having, or who may be at high risk for developing one or more relevant health conditions or diseases. 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.

[0159] 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 an immune disease, an autoimmune disease, or an inflammatory disease, such as glomerulonephritis, inflammatory bowel disease, nephritis, peritonitis, psoriatic arthritis, osteoarthritis, Still's disease, familial mediterranean fever, or inflammatory bowel disease. fever), systemic sclerosis and sclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, acute lung injury, meningitis, encephalitis, uveitis, multiple myeloma, glomerulonephritis, nephritis, asthma, atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud's disease, Sjogren's syndrome, early-onset diabetes mellitus, Reiter's syndrome, Behçet's disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, immune-mediated thrombocytopenia, hemolytic anemia, myasthenia gravis, lupus nephritis, lupus erythematosus, rheumatoid arthritis (RA), ankylosing spondylitis, pemphigus, Graves' disease, Hashimoto's thyroiditis, small vessel vasculitis, Omen's syndrome syndrome), chronic renal failure, autoimmune thyroid disease, acute infectious mononucleosis, HIV, herpes virus-related diseases, human viral infections, coronaviruses, other enteric viruses, herpes viruses, influenza viruses, parainfluenza viruses, respiratory syncytial viruses, or adenovirus infections, bacterial pneumonia, wounds, sepsis, stroke / cerebral edema, ischemia-reperfusion injury, and hepatitis C.

[0160] Non-limiting examples of inflammatory conditions suitable for the methods of the present disclosure include inflammatory diseases such as asthma, inflammatory bowel disease (IBD), chronic colitis, splenomegaly, and rheumatoid arthritis.

[0161] Examples of autoimmune diseases suitable for the methods of the present disclosure include rheumatoid arthritis, osteoarthritis, Still's disease, familiar mediterranean fever, systemic sclerosis, multiple sclerosis, ankylosing spondylitis, Hashimoto's thyroiditis, systemic lupus erythematosus, Sjogren's syndrome, diabetic retinopathy, diabetic vasculopathy, diabetic neuralgia, insulitis, psoriasis, alopecia areata, warm and cold autoimmune hemolytic anemia (AIHA), pernicious anemia, acute inflammatory diseases, autoimmune adrenalitis, chronic inflammatory demyelinating polyneuropathy (CIDP), Lambert-Eaton syndrome, Lichen sclerosus, Lyme disease, Graves' disease, Behcet's disease, Meniere's disease, Reactive arthritis (Reiter's syndrome), Churg-Strauss syndrome, Cogan's syndrome, Crest syndrome, Pemphigus vulgaris and pemphigus foliaceus, Bullous pemphigoid, Polymyalgia rheumatica, Polymyositis, Primary biliary cirrhosis, Pancreatitis, Peritonitis, Psoriatic arthritis, Rheumatic fever, Sarcoidosis, Sjorgensen's syndrome These include, but are not limited to, scleroderma, celiac 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-Barre syndrome, Hashimoto's thyroiditis, autoimmune hepatitis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, myasthenia gravis, immune complex disease, glomerulonephritis, polyarteritis nodosa, antiphospholipid syndrome, autoimmune polyglandular syndrome, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), urticaria, autoimmune infertility, juvenile rheumatoid arthritis, sarcoidosis, and autoimmune cardiomyopathy.

[0162] 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 a subject having, suspected of having, or potentially at risk for developing a microbial infection (e.g., a bacterial, microbacterial, or viral infection). Non-limiting examples of infectious diseases suitable for the methods of the present disclosure include infections caused by viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis B virus (HCV), cytomegalovirus (CMV), respiratory syncytial virus (RSV), human papillomavirus (HPV), Epstein-Barr virus (EBV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome (MERS), influenza virus, and Ebola virus. Additional infections suitable for the methods of the present disclosure include infections with intracellular parasites such as Leishmania, Rickettsia, Chlamydia, Coxiella, Plasmodium, Brucella, Mycobacteria, Listeria, Toxoplasma, and Trypanosoma. 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.

[0163] In some embodiments, the condition is a rare disease, e.g., a disease or condition that affects fewer than 200,000 people in the United States, and / or an inflammatory and / or autoimmune disorder, as defined by the Rare Diseases Act (www.fda.gov / patients / rare-diseases-fda). In some embodiments, the subject has or is suspected of having a condition associated with an inflammatory and / or autoimmune disorder, and / or a rare disease (e.g., but not limited to, familial Mediterranean fever or adult-onset Still's disease).

[0164] Further treatments In some embodiments, the compositions of the present disclosure are administered to a subject individually as a monotherapy (monotherapy) or in combination with at least one additional therapy (e.g., a second therapy) as a first 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 simultaneously. In some embodiments, the first therapy and the second therapy are administered simultaneously. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy is administered before the second therapy. In some embodiments, the first therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and / or after the second therapy. In some embodiments, the first and second therapies are administered sequentially, hi some embodiments, the first and second therapies are administered together in a single formulation.

[0165] kit Also provided herein are various kits for carrying out the methods described herein. In particular, some embodiments of the present disclosure provide kits for inducing an immune response in a subject. Some other embodiments relate to kits for the prevention of a health condition in a subject in need. Some other embodiments relate to kits for methods of treating a health condition in a subject in need. 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 as provided and described herein, and written instructions for making and using the same.

[0166] 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 may 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 condition in a subject in need.

[0167] Any of the foregoing kits may further comprise one or more additional reagents, which may be selected from the following: dilution buffers, reconstitution solutions, wash buffers, control reagents, control expression vectors, negative controls, positive controls, reagents suitable for the in vitro production of the provided nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure.

[0168] In some embodiments, the components of the kit may be in separate containers. In some other embodiments, the components of the kit may be combined in a single container. Thus, in some embodiments of the present disclosure, the kit includes one or more of the nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions as provided and described herein in one container (e.g., in a sterile glass or plastic vial) and an additional therapeutic agent in another container (e.g., in a sterile glass or plastic vial).

[0169] In another embodiment, the kit comprises 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, combined with one or more additional therapeutic agents, optionally formulated together in a pharmaceutical composition, in a single common container.

[0170] Where the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit may include a device for performing such administration (e.g., an injection device or catheter). For example, the kit may include one or more hypodermic needles or other injection devices as described above that contain one or more of the nucleic acid constructs (e.g., vectors or srRNA molecules), recombinant cells, and / or recombinant polypeptides of the present disclosure.

[0171] In some embodiments, the kit may further include instructions for carrying out the methods disclosed herein using the components of the kit. For example, the kit may include a package insert containing information about 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 about the combinations disclosed herein may be supplemented in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, appropriate dosage and administration, dosage forms, appropriate storage conditions, literature references, manufacturer / distributor information, and intellectual property information.

[0172] The instructions for carrying out the above-described methods are typically recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. The instructions may be present in the kit as a package insert, in a label on the container of the kit or a component thereof (e.g., associated with packaging or subpackaging), etc. The instructions may be present as an electronic storage data file present on a suitable computer-readable storage medium, such as a CD-ROM, diskette, flash drive, etc. In some cases, the actual instructions are not present in the kit, but a means for obtaining the instructions from a remote source (e.g., via the Internet) may be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions may be written on a suitable substrate.

[0173] 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.

[0174] No admission is made that any reference cited herein constitutes prior art. The discussion of a reference states what its author asserts, and applicants reserve the right to challenge the accuracy and pertinence of the cited documents. Although a number of sources of information, including scientific journal articles, patent documents, and textbooks, have been referenced herein, it will be clearly understood that this reference is not an admission that any of these documents form part of the common general knowledge in the art.

[0175] The general method discussion provided herein is intended for illustrative purposes only: other alternative methods and substitutes will be apparent to those of skill in the art upon review of this disclosure, and are intended to be within the spirit and scope of this application.

[0176] Further embodiments are disclosed in more 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 in any way. EXAMPLES

[0177] 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 fully described in such references as: Sambrook, J., & Russell, DW (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russell, DW (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (collectively referred to herein as "Sambrook"); Ausubel, FM (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (with 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. 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.

[0178] Further embodiments are disclosed in more 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 in any way.

[0179] Example 1 Design and construction of the base Vector SINV AR86-Girdwood This example describes the results of experiments performed to construct several basic alphavirus vectors (e.g., containing no heterologous genes) that were subsequently used to express genes of interest (e.g., the hemagglutinin (HA) gene from influenza).

[0180] The Sindbis AR86-Girdwood Hybrid 1 vector, described in Figure 2A, was constructed as follows: The basic SINV AR86-Girdwood Hybrid 1 vector was synthesized de novo as four approximately 4 kb fragments (Twist Biosciences) from the AR-86 reference sequence (Genbank U38305) with unique restriction enzyme cleavage sites (SpeI, 5'-A'CTAG,T-3') in place of the coding sequence of the SINV structural genes (where 5'A is the next nucleotide in the P2A sequence following nucleotide 93 of the structural polyprotein gene and 3'T coincides with the location of the structural polyprotein stop codon TGA). A bacteriophage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO: 12) was included upstream of the SINV genomic sequence, and downstream, a polyA sequence, followed by a unique restriction enzyme site (SapI, 5'-GCTCTTC(N)1'(N)3,-3'), followed by a T7 transcription termination sequence (5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO: 13), followed by a unique restriction enzyme cleavage site (NotI, 5'-GC'GGCC,GC-3'). These parts were joined in a five-piece Gibson Assembly® reaction (e.g., linearized pYL backbone and four synthetic fragments). In the resulting vector, the AR86 nsP2 gene was replaced with the Girdwood nsP2 gene (Genbank MF459683), resulting in the final SINV AR86-Girdwood Hybrid 1 base vector (SEQ ID NO: 1).

[0181] The Sindbis AR86-Girdwood hybrid 2 vector, depicted in Figure 3A, was constructed as follows: The basic SINV Girdwood vector was synthesized de novo as four approximately 4 kb segments (Twist Biosciences, Thermo Fisher GeneArt) from the Girdwood strain reference sequence (Genbank MF459683) with unique restriction enzyme cleavage sites (SpeI, 5'-A'CTAG, T-3') in place of the coding sequence of the SINV structural genes (where 5'A is the next nucleotide in the P2A sequence following nucleotide 93 of the structural polyprotein gene and 3'T coincides with the location of the structural polyprotein stop codon TGA). A bacteriophage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO: 12) was included upstream of the SINV genomic sequence, followed downstream by a polyA sequence, followed by a unique restriction enzyme site (SapI, 5'-GCTCTTC(N)1'(N)3,-3'), followed by a T7 transcription termination sequence (5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO: 13), followed by a unique restriction enzyme cleavage site (NotI, 5'-GC'GGCC,GC-3'). These pieces were joined in a five-piece Gibson Assembly® reaction (e.g., linearized pYL backbone and four synthetic fragments). In the resulting vector, the Girdwood nsP4 genes were replaced with the AR86 nsP4 genes, resulting in the final SINV AR86-Girdwood Hybrid 2 base vector (SEQ ID NO: 2).

[0182] The Sindbis AR86-Girdwood Hybrid 3 vector depicted in Figure 4A was constructed as follows: The Sindbis AR86-Girdwood Hybrid 3 base vector was constructed similar to the Sindbis AR86-Girdwood Hybrid 2 base vector, but instead replacing the Girdwood nsP3 genes with the AR86 nsP3 genes (SEQ ID NO:3).

[0183] The Sindbis AR86-Girdwood Hybrid 4 vector depicted in Figure 5A was constructed as follows: The Sindbis AR86-Girdwood Hybrid 4 base vector was constructed similar to the Sindbis AR86-Girdwood Hybrid 2 base vector, but instead replacing the Girdwood nsP1 gene with the AR86 nsP1 gene (SEQ ID NO: 4).

[0184] Example 2 Construction of modified alphavirus vectors for expression of genes of interest The alphavirus vectors of Figures 2B, 3B, 4B, and 5B were constructed by linearization of the empty base vectors of Figures 2A, 3A, 4A, and 5A, respectively, by SpeI endonuclease digestion. The hemagglutinin (HA) gene from influenza (Genbank#AY651334) was codon refactored in silico for human expression and synthesized (IDT). This synthetic product was amplified using the following primers, which add 30 bp flanking homology ends to the base vector on the PCR product:

[0185] Forward primer:

[0186] (5'-GCTGGAGACGTGGAGGAGAACCCTGGACCTATGGAGAAAATAGTGCTTCTTTTTG-3'; sequence number 10).

[0187] Reverse primer:

[0188] (5'-GCTGGTCGGTCATTGGGGCGTAGCGGTCAAATGCAAATTCTGCATTGTAACG-3'; sequence number 11).

[0189] The digestion products (i.e., linearized vectors) and PCR products were ligated in a two-fragment Gibson Assembly® reaction to yield the final vectors, each containing the H5N1 HA coding sequence under the control of the 26S subgenomic promoter (SEQ ID NOs: 5-8 for AR86-Girdwood Hybrids 1-4, respectively).

[0190] Example 3 Improving the functionality of defective alphavirus genomes and RNA replicons This example describes the results of experiments performed to demonstrate that it is possible to functionalize (e.g., make functional) a defective (non-functional) RNA replicon (e.g., a self-replicating RNA) by replacing the defective nsP sequences with corresponding nsP sequences from a functional alphavirus.

[0191] In vitro transcription: Self-replicating RNA (srRNA) was generated by in vitro transcription using plasmid template DNA linearized by enzymatic digestion. In these examples, DNA was linearized with NotI, which cuts downstream of the T7 terminator, or cut with SapI, which cuts at the end of poly(A). In vitro transcription was performed using bacteriophage T7 polymerase and either a 5'ARCA cap (HiScribe™ T7 ARCA mRNA Kit, NEB) or uncapping transcription (HiScribe™ T7 High Yield RNA Synthesis Kit, NEB) was followed by the addition of a 5' Cap 1 (Vaccinia Capping System, mRNA Cap 2'-O-Methyltransferase, NEB). srRNA was purified using phenol / chloroform extraction, LiCl precipitation, or column purification (Monarch® RNA Cleanup Kit, NEB). srRNA concentrations were measured by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific).

[0192] BHK-21 or Vero cells were transformed with the replicated srRNA by electroporation (e.g., 4D-Nucleofector™, Lonza). 17-20 hours after transformation, cells were fixed and permeabilized (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen) and stained with a PE-conjugated anti-double-stranded RNA (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.

[0193] Non-functional RNA replicons (e.g., self-replicating RNAs) show no signal after staining of srRNA-transfected cells (see RNA replicons (e.g., self-replicating RNAs), marked with an X), whereas transfected functional RNA replicons (e.g., self-replicating RNAs), indicative of RNA replication, produce detectable dsRNA, necessary for 26S RNA transcription and subsequent transgene expression (see symbol "

number

[0194] Example 4 Evaluation of modified alphavirus vectors in vitro These describe the results of in vitro experiments conducted to evaluate the expression levels of the modified alphavirus vector constructs described in Examples 1 and 2 above and to examine differences in their behavior (e.g., replication and protein expression).

[0195] In these experiments, the functionality of the modified alphavirus designs depicted in Figures 2B, 3B, 4B, and 5B was evaluated using the following assays: In these experiments, modified alphavirus srRNA vectors encoding the hemagglutinin precursor (HA) of influenza A virus H5N1 (H5N1 HA) (SINV Girdwood with heterologous AR86 nsP1, or nsP2, or nsP3, or nsP4) were also evaluated utilizing the following assays.

[0196] In vitro transcription: Self-replicating RNA (srRNA) was generated by in vitro transcription using plasmid template DNA linearized by enzymatic digestion. In these examples, DNA was linearized with NotI, which cuts downstream of the T7 terminator, or cleaved with SapI, which cuts at the end of poly(A). In vitro transcription was performed using bacteriophage T7 polymerase and either a 5' ARCA cap (HiScribe™ T7 ARCA mRNA Kit, NEB) or uncapping transcription (HiScribe™ T7 High Yield RNA Synthesis Kit, NEB) was followed by the addition of a 5' Cap 1 (Vaccinia Capping System, mRNA Cap 2'-O-Methyltransferase, NEB). Eppendorf / chloroform extraction, LiCl precipitation, or column purification (Monarch® RNA Cleanup Kit, NEB) were used. srRNA was purified using a PCR-Based Reagent Kit (NEB). RNA concentration was measured by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific).

[0197] BHK-21 or Vero cells were transformed with the replicated srRNA by electroporation (e.g., 4D-Nucleofector™, Lonza). 17-20 hours after transformation, cells were fixed and permeabilized (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen) and stained with a PE-conjugated 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.

[0198] Protein expression RNA was transfected into BHK-21 or Vero cells by electroporation (e.g., 4D-Nucleofector™, Lonza). 17-20 hours after transfection, cells were fixed and permeabilized (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen) and stained with an APC-conjugated anti-HA mouse monoclonal antibody (2B7, Abcam; APC: allophycocyanin) to quantify the frequency of HA protein + cells and the mean fluorescence intensity (MFI) of HA protein in individual cells by fluorescent flow cytometry (Figure 7).

[0199] Further Experiments BHK-21 or Vero cells are pretreated with a titrated curve of recombinant IFN prior to RNA electroporation, and the effect of each vector on replication and protein expression is measured using the assays described above.

[0200] Non-functional srRNA vectors show no signal after srRNA-transfected cell staining to detect GOI expression, while functional srRNA vectors result in detectable GOI expression. In this experiment, GOI expression is quantified by the mean fluorescence intensity (MFI) of cells that stained positively using an APC-conjugated anti-HA mouse monoclonal antibody. Figure 7 shows that basic srRNA vectors that showed RNA replication (Figure 6) also showed expression after GOI insertion. These functional, GOI-expressing srRNAs can be used as practical vectors to induce pharmacokinetic effects (e.g., elicit an immune response in the host).

[0201] Example 5 Evaluation of modified alphavirus vectors in vivo This example describes the results of in vivo experiments conducted to evaluate differences in immune responses following vaccination with the modified alphavirus vector constructs in Examples 1 and 2 above (e.g., both unformulated and LNP-formulated vectors).

[0202] In these experiments, the functionality of the modified alphavirus designs depicted in Figures 2B, 3B, 4B, and 5B is assessed using the following assays.

[0203] Mice and injections Female C57BL / 6 or BALB / c mice are purchased from Charles River Laboratories or Jackson Laboratories. On the day of dosing, 0.1-10 μg of material is injected intramuscularly in split doses into both quadriceps. Vectors are administered unformulated in saline or in LNP formulations. Animals are monitored for weight and other general observations throughout the study. For immunogenicity studies, animals are dosed on days 0 and 21. Spleens were removed on day 35 and serum was isolated on days 0, 14, and 35. For protein expression studies, animals are dosed on day 0 and bioluminescence is assessed on days 1, 3, and 7. In vivo imaging of luciferase activity is performed using the IVIS system at the indicated time points.

[0204] LNP formulation The replicon RNA (e.g., self-replicating RNA) is formulated into lipid nanoparticles using a microfluidic mixer for analysis of particle size, polydispersity using dynamic light scattering, and encapsulation efficiency. The lipid molar ratios used in formulating the LNP particles are 30% C12-200, 46.5% cholesterol, 2.5% PEG-2K, and 16% DOPE.

[0205] ELISpot To measure the magnitude of influenza-specific T cell responses, IFNγ ELISpot assays are performed using the Mouse IFNγ ELISpot PLUS Kit (HRP) (MabTech) according to the manufacturer's instructions. Briefly, splenocytes are isolated and cultured at 5×10 in medium containing peptides presenting either CD4+ or CD8+ T cell epitopes against HA, PMA / ionomycin as a positive control, or DMSO as a mock stimulation. 6 Resuspend to a concentration of 100 cells / mL.

[0206] Intracellular cytokine staining Spleens were harvested as outlined in ELISpot and cultured at 1 × 10 6Cells are added to cell-containing media in a total volume of 200 μL / well. Each well contains a peptide presenting either a CD4+ or CD8+ T cell epitope for HA, PMA / ionomycin as a positive control, or DMSO as a mock stimulation. After 1 hour, GolgiPlug™ protein transport inhibitor (BD Biosciences) is added to each well. Cells are incubated for an additional 5 hours. After incubation, cells are stained for CD8+ (53-6.7), CD4+ (GK1.5), B220 (B238128), Gr-1 (RB6-8C5), and CD16 / 32 (M93) on the cell surface using standard methods. After surface staining, cells are fixed and intracellular protein staining for IFNγ (RPA-T8), IL-2 (JES6-5H4), and TNF (MP6-XT22) is performed according to standard methods. Cells were then subjected to flow cytometer analysis and the resulting FCS files were analyzed using FlowJo software version 10.4.1.

[0207] antibody Antibody responses to measure total HA-specific IgG are measured using an ELISA kit from Alpha Diagnostic International according to the manufacturer's instructions.

[0208] Example 6 In vitro evaluation of modified srRNA vectors containing heterologous nonstructural protein genes This example describes the results of in vitro experiments performed to assess the expression levels of synthetic self-replicating RNAs (srRNAs) containing heterologous nonstructural proteins and to examine differences in their behavior (e.g., replication and protein expression).

[0209] In these experiments, synthetic srRNAs derived from the Girdwood and AR86 strains of SINV were designed and subsequently evaluated, including a control VEEV srRNA containing an unrelated transgene (RBI296, RBI298), a VEEV srRNA encoding both IL-1RA and IL-12 in two configurations (RBI299, RBI300), a SINV AR86-Girdwood Hybrid 1 srRNA encoding both IL-1RA and IL-12 in two configurations (RBI307, RBI308), and a SINV Girdwood srRNA encoding both IL-1RA and IL-12 in two configurations (RBI309, RBI310).

[0210] In vitro transcription: srRNA was prepared by in vitro transcription with bacteriophage T7 polymerase (HiScribe™ T7 High Yield RNA Synthesis Kit, NEB) from a plasmid template linearized with SapI, followed by the addition of 5' Cap 1 (Vaccinia Capping System, mRNA Cap 2´-O-Methyltransferase, NEB). srRNA was then purified by LiCl precipitation. srRNA concentration was measured by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific).

[0211] Protein expression: srRNA was transfected into BHK-21 cells by electroporation (4D-Nucleofector™, Lonza). Culture supernatants were harvested from cells 24 and 48 hours after transfection. IL-1RA secretion was assessed in a bioactivity assay by pre-incubating HEK-Blue™ IL-1R cells (InvivoGen) with a range of concentrations of recombinant IL-1RA (Peprotech) or culture supernatant. Recombinant IL-1B (Invivogen) was added to the cells and after overnight incubation, the SEAP reporter was quantified using QUANTI-Blue™ (Invivogen) (Figure 8A).

[0212] A range of concentrations of recombinant IL-12 (Peprotech) or culture supernatants were incubated overnight on IL-12 Bioassay cells (Promega) in DMEM, after which IL-12 secretion was assessed in a bioactivity assay by quantifying the luciferase reporter using Bio-Glo™ Luciferase (Promega) (Figure 8B).

[0213] Example 8 In vivo evaluation of modified srRNA vectors containing heterologous nonstructural protein genes This example describes the results of in vivo experiments conducted to evaluate differences in immune responses following vaccination with self-replicating RNA (srRNA) containing heterologous nonstructural proteins, both as unformulated and LNP-formulated vectors.

[0214] In these experiments, synthetic srRNA constructs derived from SINV Girdwood-AR86 hybrid 1 were designed and subsequently evaluated.

[0215] Mice and injections Female C57BL / 6 or BALB / c mice were purchased from Charles River Laboratories or Jackson Laboratories. On the day of dosing, 0.1-10 μg of material was injected intramuscularly in split doses into both quadriceps. Vectors were administered unformulated in saline or in the LNP formulation. Animals were monitored throughout the study for weight and other general observations. For immunogenicity studies, animals were dosed on days 0 and 21. Spleens were harvested on days 14 and / or 35, and serum was isolated on days 14 and / or 35.

[0216] LNP formulation In some studies, srRNA was formulated into lipid nanoparticles (LNPs) using a microfluidic mixer for particle size analysis, polydispersity analysis using dynamic light scattering, and encapsulation efficiency analysis. The LNPs are composed of ionizable lipids, cholesterol, PEG-2K, and DOPE.

[0217] ELISpot To measure the magnitude of antigen-specific T cell responses, IFNγ ELISpot assays were performed using the Mouse IFNγ ELISpot PLUS Kit (HRP) (MabTech) according to the manufacturer's instructions. Briefly, splenocytes were isolated and cultured at 2–5 × 10 cells in medium containing peptides presenting either peptide pools corresponding to rabies virus glycoprotein G, ESR1, HER2, or HER3, PMA / ionomycin as a positive control, or DMSO as a mock stimulation. 6 The cells were resuspended to a concentration of 1000 cells / mL.

[0218] antibody The rapid fluorescent focus inhibition test is used to measure neutralizing antibody responses to rabies virus. Briefly, serum dilutions are mixed with a standard amount of live rabies virus and incubated. If neutralizing anti-rabies antibodies are present, they will neutralize the virus. Cultured cells are then added and the serum / virus / cells are incubated together. Uncoated (i.e., not neutralized by antibodies) rabies virus will infect the cells, which can be visualized by microscopy. The percentage of virus-infected cells identified on the slide is used to calculate the endpoint titer.

[0219] The in vivo immunogenicity of the viral antigen, srRNA encoding rabies virus glycoprotein G, was evaluated by assessing antigen-specific splenic T cell responses by ELISpot (Figure 9A) and titers of anti-rabies neutralizing antibodies from serum after two immunizations (Figure 9B). Compared to saline controls, all srRNA-immunized groups demonstrated robust T cell responses (Figure 9A), but differences in responses were observed between srRNA vaccines. Similarly, all srRNA-immunized groups demonstrated protective neutralizing antibody titers, with some variability between srRNA vaccines (Figure 9B). In addition to infectious disease antigens, the immunogenicity of srRNA-based vaccines against cancer antigens was evaluated (Figure 10). 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, although the patterns of responses differed between the srRNA vectors (Figure 10).

[0220] While certain alternatives of the present disclosure have been disclosed, it is to be understood that various modifications and combinations are possible and contemplated within the true spirit and scope of the appended claims, and therefore, it is not intended to be limited to the precise scope and disclosure presented herein.

Claims

1. A nucleic acid construct encoding a modified genome or RNA replicon of an alphavirus species, wherein at least one nonstructural protein (nsP) or portion thereof of the modified alphavirus genome or RNA replicon is heterologous to the remainder of the modified alphavirus genome or RNA replicon.

2. 2. The nucleic acid construct of claim 1, wherein the at least one heterologous nsP or portion thereof is nsP1, nsP2, nsP3, nsP4, or a portion of any of them, or any combination thereof.

3. 2. The nucleic acid construct of claim 1, wherein the at least one heterologous nsP or portion thereof is derived from a different strain of the same alphavirus species.

4. 2. The nucleic acid construct of claim 1, wherein the at least one heterologous nsP or portion thereof is derived from another alphavirus species.

5. 2. The nucleic acid construct of claim 1, wherein the modified alphavirus genome or RNA replicon lacks at least a portion of a nucleic acid sequence encoding one or more viral structural proteins.

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

7. 2. The nucleic acid construct of claim 1, wherein the modified viral genome or RNA replicon does not contain nucleic acid sequences encoding viral structural proteins.

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

9. 9. The nucleic acid construct of claim 8, wherein at least one of the expression cassettes comprises a subgenomic (sg) promoter operably linked to a heterologous nucleic acid sequence.

10. 10. The nucleic acid construct of claim 9, wherein the sg promoter is a 26S subgenomic promoter.

11. The nucleic acid construct of claim 1 , further comprising one or more untranslated regions (UTRs).

12. 12. The nucleic acid construct of claim 11, wherein at least one of the UTRs is a heterologous UTR.

13. 9. The nucleic acid construct of claim 8, wherein at least one of the expression cassettes comprises a coding sequence for a gene of interest (GOI).

14. 14. The nucleic acid construct of claim 13, 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.

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

16. 14. The nucleic acid construct of claim 13, wherein the coding sequence of the GOI is optimized to be expressed at a level higher than the expression level of a reference coding sequence.

17. 14. The nucleic acid construct of claim 13, wherein the coding sequence of the GOI is optimized to enhance RNA stability.

18. The alphavirus species include Aura virus (AURAV), Babanki virus (BABV), Barmah Forest virus (BFV), Bebaru virus (BEBV), Buggy Creek virus, Caaingua virus, Cabassou virus, Chikungunya virus (CHIKV), Eastern equine encephalitis virus (EEEV), Eilat virus (Eilat encephalitis virus), and the like. virus), Everglades virus (EVEV), Fort Morgan virus (FMV), Getah virus (GETV), Highlands J virus (HJV), Kyzylagach virus (KYZV), Madariaga virus (MADV), Mayaro virus (MAYV), Middleburg virus (MIDV), Mosso das Pedras virus virus), Mucambo virus (MUCV), Ndumu virus (NDUV), O'nyong'nyong virus (ONNV), Pixuna virus (PIXV), Rio Negro virus (RNV), Ross River virus (RRV), Salmon pancreas disease virus (SPDV), Semliki Forest virus (SFV), Sindbis virus virus) (SINV), sleeping disease virusvirus (SDV), Southern elephant seal virus (SESV), Tai Forest virus (TFV), Tonate virus, Trocara virus, Una virus (UNAV), Venezuelan equine encephalitis virus (VEEV), Western equine encephalitis virus (WEEV), and Whataroa virus 2. The nucleic acid construct of claim 1, wherein the nucleic acid construct is selected from the group consisting of:

19. 2. The nucleic acid construct of claim 1, wherein the modified genome or RNA replicon is of Sindbis virus (SINV).

20. 20. The nucleic acid construct of claim 19, wherein the modified genome or RNA replicon is of the Girdwood strain of SINV.

21. 21. The nucleic acid construct of claim 20, wherein at least one heterologous nsP or part thereof of said modified genome or RNA replicon is derived from the AR86 strain of SINV.

22. 21. The nucleic acid construct of claim 20, wherein the at least one heterologous nsP or portion thereof is nsP1, nsP3, nsP4, or a portion of any of them, or any combination thereof.

23. 20. The nucleic acid construct of claim 19, wherein the modified genome or RNA replicon is of the AR86 strain of SINV.

24. 24. The nucleic acid construct of claim 23, wherein at least one heterologous nsP or portion thereof of said modified SINV-AR86 genome or RNA replicon is derived from the Girdwood strain of SINV.

25. 24. The nucleic acid construct of claim 23, wherein at least one heterologous nsP or part thereof of said modified SINV-AR86 genome or RNA replicon is derived from nsP2 of the Girdwood strain of SINV.

26. The nucleic acid construct of claim 1 , which is incorporated into a vector.

27. 27. The nucleic acid construct of claim 26, wherein the vector is a self-replicating RNA (srRNA) vector.

28. 2. The nucleic acid construct of claim 1, comprising 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 a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-4.

29. A recombinant cell comprising the nucleic acid construct of claim 1.

30. 30. The recombinant cell of claim 29, which is a eukaryotic cell.

31. The recombinant cell of claim 30, which is an animal cell.

32. 32. The recombinant cell of claim 31, wherein the animal cell is a vertebrate or invertebrate cell.

33. 32. The recombinant cell of claim 31 , wherein the animal cell is an insect cell.

34. 34. The recombinant cell of claim 33, wherein the insect cell is a mosquito cell.

35. 33. The recombinant cell of claim 32, which is a mammalian cell.

36. SV40-transformed monkey kidney CV1 cells (COS-7), human embryonic kidney cells (e.g., HEK293 or HEK293 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 liver cells (BRL3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT060562), 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.

33. The recombinant cell of claim 32, wherein the recombinant cell is selected from the group consisting of C6 cells, NS0 mouse myeloma cells, human laryngeal epidermoid cells, human fibroblasts, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocytes, human macrophage cells, human RAW264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells.

37. 30. A cell culture comprising at least one recombinant cell of claim 29 and a culture medium.

38. 1. A method for functionalizing / engineering an alphavirus genome or RNA replicon, comprising: (a) providing a non-functional alphavirus genome or RNA replicon; (b) replacing a nonstructural protein (nsP) or portion thereof of said non-functional alphavirus genome or RNA replicon with a heterologous coding sequence of a corresponding nsP or portion thereof from a different alphavirus strain, thereby generating a modified alphavirus genome or RNA replicon; (c) assessing the functionality of the modified alphavirus genome or RNA replicon; (d) considering the modified alphavirus genome or RNA replicon functional if the modified alphavirus genome or RNA replicon is capable of RNA replication and / or expression; A method comprising:

39. 39. The method of claim 38, wherein the heterologous nsP or portion thereof is from a different strain of the same alphavirus species.

40. 39. The method of claim 38, wherein the heterologous nsP or portion thereof is from another alphavirus species.

41. 39. The method of claim 38, wherein the heterologous nsP or portion thereof is nsP1, nsP2, nsP3, nsP4, or any portion thereof.

42. 39. The method of claim 38, wherein the alphavirus genome or RNA replicon is non-functional as determined by insufficient autonomous replication within a host cell.

43. The method of claim 38, wherein the evaluation of the functionality of the modified alphavirus genome or RNA replicon comprises an assay selected from the group consisting of detection of RNA replication, detection of viral protein expression, detection of cytopathic effect (CPE), and detection of heterologous introduced gene expression.

44. A transgenic non-human animal comprising the nucleic acid construct of claim 1.

45. 45. The transgenic non-human animal of claim 44, which is a vertebrate or an invertebrate.

46. 46. ​​The transgenic non-human animal of claim 45, which is an insect.

47. 46. ​​The transgenic non-human animal of claim 45, which is a mammal.

48. 47. The transgenic non-human animal of claim 46, wherein the mammal is a mammal other than a human.

49. A non-therapeutic method for producing a polypeptide of interest, comprising: (i) culturing a recombinant cell comprising the nucleic acid construct of claim 1; and raising a transgenic non-human animal comprising the recombinant cell of (i) under conditions in which the recombinant cell or the transgenic non-human animal produces the polypeptide encoded by the GOI.

50. A non-therapeutic method for producing a polypeptide of interest in a subject, comprising administering to the subject the nucleic acid construct of claim 1.

51. 50. The method of claim 49, wherein the subject is a vertebrate or an invertebrate.

52. 50. The method of claim 49, wherein the animal is an insect.

53. 50. The method of claim 49, wherein the subject is a mammalian subject.

54. 54. The method of claim 53, wherein the mammalian subject is a human subject.

55. 50. A recombinant polypeptide produced by the method of claim 49.

56. a pharmaceutically acceptable excipient, and: (a) the nucleic acid construct of 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); A pharmaceutical composition comprising:

57. 57. The pharmaceutical composition of claim 56, comprising the nucleic acid construct of claim 1 and a pharmaceutically acceptable excipient.

58. 57. The pharmaceutical composition of claim 56, comprising the recombinant cell of claim 29 and a pharmaceutically acceptable excipient.

59. 57. The pharmaceutical composition of claim 56, comprising the recombinant polypeptide of claim 55 and a pharmaceutically acceptable excipient.

60. 57. The pharmaceutical composition of claim 56, formulated as a liposome, lipid nanoparticle (LNP), or polymer nanoparticle.

61. 57. The pharmaceutical composition of claim 56, which is an immunogenic composition.

62. 62. The pharmaceutical composition of claim 61, wherein the immunogenic composition is formulated as a vaccine.

63. 57. The pharmaceutical composition of claim 56, which is substantially non-immunogenic to a subject.

64. 57. The pharmaceutical composition of claim 56 formulated as an adjuvant.

65. 57. The pharmaceutical composition of claim 56, formulated for one or more of intranasal, transdermal, intraperitoneal, intramuscular, intralymphatic, intratumoral, intraarticular, intravenous, subcutaneous, intravaginal, and oral administration.

66. 1. A method for inducing a pharmacodynamic effect in a subject in need thereof, comprising: (a) the nucleic acid construct of 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 nucleic acid construct of (a), the recombinant cell of (b), and the recombinant polypeptide of (c); administering to said subject a composition comprising:

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

68. 1. Use of a composition in the manufacture of a medicament for preventing and / or treating a condition in a subject in need thereof, said composition comprising: (a) the nucleic acid construct of 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 nucleic acid construct of (a), the recombinant cell of (b), and the recombinant polypeptide of (c); The use.

69. 67. The use of claim 66, wherein the condition is a proliferative disease or a microbial infection.

70. 67. The use of claim 66, wherein the subject has or is suspected of having a condition associated with a proliferative disease or microbial infection.

71. 67. The use of claim 66, wherein the administered composition results in increased interferon production in the subject.

72. 67. The use of claim 66, wherein the composition is administered to the subject individually as a monotherapy (monotherapy) or in combination with at least one additional therapy as a first line of therapy.

73. 73. The use of claim 72, 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.

74. 1. A composition for use in the prevention and / or treatment of a condition in a subject in need thereof, the composition comprising: (a) the nucleic acid construct of 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 nucleic acid construct of (a), the recombinant cell of (b), and the recombinant polypeptide of (c); The composition comprising:

75. 1. A kit for inducing a pharmacodynamic effect, eliciting an immune response, preventing a condition or a microbial infection, and / or treating a condition or a microbial infection, comprising: (a) the nucleic acid construct of 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 nucleic acid construct of (a), the recombinant cell of (b), and the recombinant polypeptide of (c); Includes a kit.

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