Modified modaria virus, self-replicating RNA constructs and uses thereof

Abstract

The present disclosure relates to the field of molecular virology, including nucleic acid molecules comprising a modified viral genome or self-replicating RNA, pharmaceutical compositions containing said nucleic acid molecules, and the use of such nucleic acid molecules and compositions for producing a desired product in a cell culture or in vivo. Also provided are methods for eliciting a pharmacodynamic effect in a subject in need thereof, as well as methods for preventing and / or treating a variety of health conditions.

Description

Cross - Reference to Related Applications

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application Serial No. 63 / 385,556, filed on November 30, 2022. The disclosure of the above - cited application is hereby incorporated by reference in its entirety (including any drawings). Incorporation of Sequence Listing

[0002] The material in the accompanying sequence listing is hereby incorporated by reference into this application. The accompanying sequence listing XML file, named 2023 - 11 - 27Sequence_Listing_ST26 058462 - 513001WO.xml, was created on November 27, 2023, and is 26,590 bytes in size. Technical Field

[0003] The present disclosure relates to the fields of molecular virology and immunology, and more particularly to nucleic acid molecules encoding modified viral genomes and replicons (e.g., self - replicating RNA (srRNA) molecules), pharmaceutical compositions containing such nucleic acid molecules, and the use of such nucleic acid molecules and compositions for the production of desired products in cell culture or in vivo. Also provided are methods for eliciting a pharmacodynamic effect in a subject in need thereof and methods for preventing and / or treating a variety of health conditions. Background Art

[0004] In recent years, several different groups of animal viruses have been genetically manipulated by homologous recombination or direct engineering of their genomes. The availability of reverse genetic systems for both DNA and RNA viruses has created new prospects for the use of recombinant viruses (e.g., as vaccines, expression vectors, anti - tumor agents, gene therapy vectors, and drug delivery vehicles).

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

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

[0007] Accordingly, there remains a need for more effective methods and systems for expressing a product of interest in an srRNA-based expression platform. SUMMARY OF THE INVENTION

[0008] The present disclosure generally relates to the development of immunotherapeutic agents, such as recombinant nucleic acid constructs and pharmaceutical compositions comprising the recombinant nucleic acid constructs, for use in the prevention and management of a variety of health conditions, such as proliferative disorders and microbial infections. In particular, as described in more detail below, some embodiments of the present disclosure provide nucleic acid constructs containing sequences encoding a modified genome or replicon of Madariaga virus (MADV) (e.g., a self-replicating RNA (srRNA), such as a replicon), wherein the modified genome or replicon lacks at least a portion of the viral nucleic acid sequence encoding one or more structural proteins of the virus. Also disclosed are recombinant cells and transgenic animals that have been engineered to contain one or more of the nucleic acid constructs disclosed herein; methods for producing a molecule of interest (e.g., a polypeptide of interest); pharmaceutical compositions comprising one or more of the following: (a) the nucleic acid constructs of the present disclosure, (b) the polypeptides of the present disclosure, (c) the recombinant cells of the present disclosure. In certain aspects of the present disclosure, compositions and methods are further provided for eliciting a pharmacodynamic effect in a subject in need thereof and / or for the prevention and / or treatment of a variety of health conditions, including proliferative disorders (e.g., cancer) and infections.

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

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

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

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

[0013] In some embodiments, at least one of the expression cassettes comprises a coding sequence of a gene of interest (GOI). In some embodiments, the GOI encodes a polypeptide selected from the group consisting of therapeutic polypeptides, prophylactic polypeptides, diagnostic polypeptides, nutraceutical polypeptides, industrial enzymes, and reporter polypeptides. In some embodiments, the GOI encodes a polypeptide selected from the group consisting of antibodies, antigens, immunomodulators, enzymes, signaling proteins, and cytokines. In some embodiments, the coding sequence of the GOI is optimized for expression at a level higher than the expression level of a reference coding sequence. In some embodiments, the coding sequence of the GOI is optimized for enhancing RNA stability.

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

[0015] In one aspect, the present disclosure provides recombinant cells that comprise a nucleic acid construct as disclosed herein. In some embodiments, the recombinant cells are eukaryotic cells. In some embodiments, the recombinant cells are animal cells. In some embodiments, the animal cells are vertebrate cells or invertebrate cells. In some embodiments, the recombinant cells are insect cells. In some embodiments, the recombinant cells are mosquito cells. In some embodiments, the recombinant cells are mammalian cells. In some embodiments, the recombinant cells are selected from the group consisting of or derivatives thereof: monkey kidney CV1 cells transformed by SV40, human embryonic kidney cells (HEK), baby hamster kidney cells (BHK), mouse Sertoli cells, monkey kidney cells, human cervical cancer cells, dog kidney cells, buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumors, TRI cells, FS4 cells, Chinese hamster ovary cells (CHO), African green monkey kidney cells, human A549 cells, human cervical cells, human CHME5 cells, human PER.C6 cells, NS0 mouse myeloma cells, human epidermoid laryngeal cells, human fibroblasts, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocytes, human macrophages, human RAW264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells.

[0016] In some embodiments, the recombinant cells are selected from 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 epidermoid laryngeal cells, human fibroblasts, human HEK-293 cells, human HeLa cells, human HepG2 cells, human HUH-7 cells, human MRC-5 cells, human muscle cells, mouse 3T3 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells. In some embodiments, the recombinant cells are cells derived from the cells described above (i.e., derivative cells of the original cells described herein), such as cells amplified from a clone of the original cell (an engineered form of the original cell) or cells reclassified after the original cell has undergone extensive passage or has been passaged through another host.

[0017] In another aspect, the present disclosure provides a cell culture that comprises at least one recombinant cell as disclosed herein and a culture medium.

[0018] In another aspect, the present disclosure provides transgenic animals that comprise a nucleic acid construct as described herein. In some embodiments, the transgenic animal is a vertebrate or an invertebrate. In some embodiments, the transgenic animal is a mammal. In some embodiments, the transgenic mammal is a non-human mammal. In some embodiments, the transgenic animal is an insect. In some embodiments, the transgenic insect is a transgenic mosquito. In another aspect, the present disclosure provides methods for producing a polypeptide of interest, wherein the method comprises (i) raising an animal as disclosed herein under conditions such that; or (ii) culturing a recombinant cell comprising a nucleic acid construct as disclosed herein under conditions such that the transgenic animal or recombinant cell produces a polypeptide encoded by the GOI.

[0019] In another aspect, the present disclosure provides methods for producing a polypeptide of interest in a subject, wherein the method comprises 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, the present disclosure provides recombinant polypeptides produced by the methods of the present disclosure.

[0020] In yet another aspect, the present disclosure provides pharmaceutical compositions that comprise a pharmaceutically acceptable excipient and: a) a nucleic acid construct of the present disclosure; b) a recombinant cell of the present disclosure; and / or c) a recombinant polypeptide of the present disclosure.

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

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

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

[0024] Non-limiting exemplary embodiments of the methods of the present disclosure may include one or more of the following features. In some embodiments, the disorder is a proliferative disorder or a microbial infection. In some embodiments, the subject has or is suspected of having a disorder associated with a proliferative disorder or a microbial infection. In some embodiments, the administered composition results in an increase in the production of interferon in the subject. In some embodiments, the composition is administered to the subject alone as a single therapy (monotherapy) or in combination with at least one additional therapy as a first therapy. In some embodiments, the at least one additional therapy is selected from chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery.

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

[0026] Unless explicitly or clearly excluded from the context of an embodiment or aspect, each aspect and embodiment described herein can be used together.

[0027] The foregoing summary is merely illustrative and is not intended to be limiting in any way. In addition to the illustrative embodiments and features described herein, other aspects, embodiments, objects, and features of the present disclosure will become fully apparent from the drawings and the detailed description and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0029] Figures 2A - 2B is a graphical presentation of a non-limiting exemplary MADV srRNA design according to some embodiments of the present disclosure, in which the sequence encoding the modified MADV genome from the BeAr300851 strain is incorporated into a plasmid DNA vector ( Figure 2A), the latter of which further contains the coding sequence of an exemplary gene of interest (GOI) (e.g., the hemagglutinin precursor (HA) of influenza A virus H5N1). Figure 2B )

[0030] Figures 3A - 3B is a contour plot of BHK-21 that has been transformed with MADV srRNA. In Figure 3A , MADV srRNA without GOI was transformed by electroporation, and 20 hours after transformation, the cells were fixed and permeabilized, and co-stained with two antibodies: a PE-conjugated anti-dsRNA mouse monoclonal antibody (J2, Scicons) to identify the frequency of dsRNA+ cells, and a DyLight650-conjugated anti-HA mouse monoclonal antibody (C102, Novus Bio; catalog number NB100-65047C) to identify the frequency of HA+ cells and determine the expression level of the HA transgene by fluorescence flow cytometry. In Figure 3B , MADV srRNA containing the coding sequence of HA was similarly transfected into BHK-21 cells and co-stained similarly using the same antibodies. Positive staining of individual cells with both anti-dsRNA and anti-HA antibodies demonstrated that the modified MADV srRNA design described herein is a viable synthetic srRNA and is capable of undergoing RNA replication and expressing the transgene.

[0031] Figure 4 is a graph showing the average expression of HA in cells transfected with the srRNA construct. BHK-21 cells were transfected by electroporation (Lonza 4D-Nucleofector) with 200 ng of an srRNA vector encoding influenza H1N1 HA, and 24 hours later, the cells were fixed, permeabilized and stained using a DyLight650-conjugated mouse monoclonal antibody (Novus Bio; catalog number NB100-65047C) that binds to H1N1 HA. The cells were analyzed by fluorescence flow cytometry to identify the fluorescence intensity of the cells, which corresponds to the HA expression level. The average HA expression in cells transfected with MADV-HA is similar to or higher than that of other srRNA vectors encoding the same transgene. VEEV: Venezuelan equine encephalitis virus, CHIKV-S27: Chikungunya virus strain S27, CHIKV-DRDE: Chikungunya virus strain DRDE-06, SINV-G: Sindbis virus strain Girdwood, SINV-AR86: Sindbis virus strain AR86, WEEV: Western equine encephalitis virus, MADV: Madariaga virus BeAr300851.

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

[0033] Figures 6A - 6B Summarizes the results of experiments conducted to demonstrate that the MADV srRNA-based vector can express a biotherapeutic protein in vivo. Seven days after administration of MADV-srRNA (Rep-657) co-encoding the two genes, the protein levels of murine IL-1RA ( Figure 6A ) and murine IL-18BP ( Figure 6B ) were measured using ELISA and compared to the srRNA encoding the irrelevant protein red firefly luciferase ("irrelevant control") for the basal protein expression level. Mann-Whitney statistics between groups are shown (* < 0.05). Detailed Description

[0034] The present disclosure particularly provides a viral expression system with excellent expression potential, which is suitable for expressing heterologous molecules in recombinant cells, such as vaccines and therapeutic polypeptides. For example, some embodiments of the present disclosure relate to nucleic acid constructs (such as expression constructs and vectors) that contain a modified genome or replicon of Madariaga virus (MADV) (e.g., self-replicating RNA (srRNA)), wherein at least some of the original viral sequences encoding the structural proteins of the virus have been deleted. Some embodiments of the present disclosure also provide virus-based expression vectors that contain one or more expression cassettes encoding heterologous polypeptides. Recombinant cells genetically engineered to contain one or more of the nucleic acid molecules disclosed herein are further provided. Biomaterials and recombinant products derived from such recombinant cells are also within the scope of this application. Compositions and methods useful for eliciting a pharmacodynamic effect in a subject in need thereof and methods for preventing and / or treating a variety of health conditions are also provided.

[0035] Self-replicating RNA (srRNA) based on RNA viruses (e.g., alphaviruses) can be used as a robust expression system. For example, it has been reported that the advantage of using alphaviruses (such as MADV) as viral expression vectors is that they can direct the synthesis of large amounts of heterologous proteins in recombinant host cells. Among other advantages, polypeptides (such as therapeutic single-chain antibodies) may be most effective if expressed at high levels in vivo. In addition, for the production of recombinant antibodies purified from cultured (ex vivo) cells, high protein expression from srRNA can increase the total yield of antibody products. Further, if the protein expressed is a vaccine antigen, high-level expression can induce the most robust pharmacodynamic effect in vivo.

[0036] Alphaviruses utilize motifs contained within their UTRs, structural regions, and non-structural regions to influence their replication in host cells. These regions also contain mechanisms to evade the host cell innate immunity. However, significant differences have been reported among alphavirus species. For example, New World and Old World alphaviruses have evolved different components to utilize intracellular stress granules, JAK-STAT signaling, farnesoid X receptor (FXR), and Ras-GTPase activating protein (SH3 domain)-binding protein (G3BP) proteins to assemble the viral replication complex. Which part of the genome contains these components also varies among alphaviruses. For example, bypassing the activation of PKR and subsequent phosphorylation of eIF2-alpha (eIF2α) is accomplished via a downstream loop in some Old World alphaviruses (e.g., Sindbis), but bypassing this pathway in chikungunya lacking an identifiable DLP is thought to be via nsP4. In addition, in addition to the variation among individual alphaviruses, differences often exist within alphavirus strains, which can account for changes in characteristics such as virulence. As an example, sequence variation between North American and South American strains of New World eastern equine encephalitis virus (EEEV) altered the ability to regulate the STAT1 pathway, resulting in differential induction of type I interferon and changes in virulence. As another example, mouse studies with a group of North American MADV isolates with less than 2.7% genomic sequence divergence had outcomes ranging from zero to complete lethality.

[0037] Given the differential presence of host cell attenuating factors in the non-structural and structural regions of alphaviruses, deletion of the structural genes to permit heterologous gene expression in a synthetic vector will have different effects on individual vectors. Synthetic replicons with different host attenuating factors in the non-structural region will have different advantages in inducing an immune response to the heterologous gene being expressed. For example, while in Old World viruses, the shutdown of host cell functions has been associated with nsP2, in New World viruses (e.g., EEEV, VEEV, and MADV), this activity is primarily associated with the capsid protein (C), which is partially or fully deleted in the synthetic vector. The hypervariable domain (HVD) of the nsP3 protein has host interactions unique to each alphavirus. Among them, it has been shown that EEEV nsP3 can interact with cellular FXR and G3BP protein families, DDX3, S100A4, IKKβ, PGAM5, and cytoskeletal reorganization and vesicle trafficking proteins. Few studies have explicitly detailed the non-structural proteins of MADV, yet genome sequencing of MADV revealed that it is the result of recombination between the progenitor genotypes of EEEV and SINV. It has been reported that the amino acid identity of MADV nsP is >80% identical to EEEV nsP, yet substitution of EEEV nsP with MADV nsP produces an attenuated chimera, demonstrating that changes in the nucleotide and coding sequences retain some activities crucial for the viral life cycle but confer significant differences in biological activity. The known and undescribed mechanisms by which EEEV and MADV nsP contribute to their broad range of pathogenicity suggest that MADV-based srRNA vectors can be unique and advantageous vectors for expressing heterologous proteins for vaccine and biotherapeutic applications. The advantages conferred by these previously undescribed srRNA vectors have remained completely unexplored and unforeseen. Definitions

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

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

[0040] As used herein, the terms "administration" and "administering" refer to the delivery of a bioactive composition or formulation by an administration route, including but not limited to intranasal, transdermal, intravenous, intraarterial, intramuscular, intranodal, intraperitoneal, subcutaneous, intramuscular, oral, intravaginal, and topical administration or combinations thereof. The terms include, but are not limited to, administration by a medical professional and self-administration.

[0041] The terms "cell", "cell culture", and "cell line" refer not only to a particular subject cell, cell culture, or cell line, but also to progeny or potential progeny of such a cell, cell culture, or cell line, regardless of the number of transfers or passages in culture. It should be understood that not all progeny are identical to the parental cell. This is because certain modifications may occur in the progeny due to mutations (e.g., intentional or unintentional mutations) or environmental influences (e.g., methylation or other epigenetic modifications), such that the progeny may actually be different from the parental cell, but are still included within the scope of the terms as used herein, provided that the progeny retain the same function as the original cell, cell culture, or cell line.

[0042] "Effective amount", "therapeutically effective amount", or "pharmaceutically effective amount" of a composition of the present disclosure (e.g., a nucleic acid construct (e.g., a replicon construct, e.g., an srRNA construct), a recombinant cell, a recombinant polypeptide, and / or a pharmaceutical composition) generally refers to an amount sufficient, relative to the absence of the composition, to enable the composition to achieve the stated purpose (e.g., to achieve the effect for which it is administered, to stimulate an immune response, to prevent or treat a disease or to alleviate one or more symptoms of a disease, disorder, infection, or health condition). An example of an "effective amount" is an amount sufficient to effect treatment, prevention, or alleviation of one or more symptoms of a disease, which may also be referred to as a "therapeutically effective amount". "Alleviation" of a symptom means a reduction in the severity or frequency of one or more symptoms or the elimination of one or more symptoms. The exact amount of the composition (including a "therapeutically effective amount") will depend on the purpose of the treatment and will be determinable by those skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (volumes 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th edition, 2003, Gennaro ed., Lippincott, Williams & Wilkins).

[0043] The term "construct" refers to a recombinant molecule that contains one or more isolated nucleic acid sequences or amino acid sequences from heterologous sources, such as a recombinant nucleic acid or polypeptide. For example, a polypeptide construct can be a chimeric polypeptide molecule in which two or more amino acid sequences of different origin are operably linked to each other in a single polypeptide construct. Similarly, a nucleic acid construct can be a chimeric nucleic acid molecule in which two or more nucleic acid sequences of different origin are assembled into a single nucleic acid molecule. Thus, representative nucleic acid constructs include any construct that contains: (1) nucleic acid sequences that include regulatory and coding sequences not found adjacent to each other in nature (e.g., at least one of the nucleotide sequences is heterologous relative to at least one of its other nucleotide sequences); or (2) sequences encoding portions of functional RNA molecules or proteins that are not naturally adjacent; or (3) portions of promoters that are not naturally adjacent. Representative nucleic acid constructs can contain any recombinant nucleic acid molecule (linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule) derived from any source capable of genomic integration or autonomous replication (such as plasmids, cosmids, viruses, autonomously replicating polynucleotide molecules, bacteriophages), including nucleic acid molecules in which one or more nucleic acid sequences have been operably linked. The nucleic acid constructs of the present disclosure can contain elements necessary to direct the expression of a nucleic acid sequence of interest also contained in the construct. Such elements can include control elements (such as a promoter operably linked to the nucleic acid sequence of interest (to direct its transcription)), and optionally include a polyadenylation sequence.

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

[0045] In addition to the components of the construct, the vector can also contain, for example, one or more selectable markers, one or more origins of replication (such as prokaryotic and eukaryotic origins), at least one multiple cloning site, and / or elements that facilitate stable integration of the construct into the genome of the cell. Two or more constructs can be incorporated within a single nucleic acid molecule (such as a single vector), or can be incorporated within two or more separate nucleic acid molecules (such as two or more separate vectors). An "expression construct" generally contains at least a control sequence operably linked to a nucleotide sequence of interest. In this way, for example, a promoter operably linked to the nucleotide sequence to be expressed is provided in the expression construct for expression in a cell. For the practice of the present disclosure, the compositions and methods for preparing and using the constructs and cells are known to those of skill in the art.

[0046] As used herein, the term "operably linked" refers to a physical or functional connection between two or more elements (e.g., polypeptide sequences or polynucleotide sequences) that permits them to operate in their intended manner. For example, when used in the context of a nucleic acid molecule or the coding sequence and promoter sequence in a nucleic acid molecule described herein, the term "operably linked" means that the coding sequence and the promoter sequence are in-frame and in appropriate spatial and distance relationships to permit the corresponding binding of transcription factors or RNA polymerase to exert an effect on transcription. It should be understood that operably linked elements can be continuous or non-continuous (e.g., linked to each other by a linker). In the context of a polypeptide construct, "operably linked" refers to a physical connection (e.g., directly or indirectly linked) between amino acid sequences (e.g., different segments, moieties, regions, or domains) to provide the described activity of the construct. The operably linked segments, moieties, regions, and domains of a polypeptide or nucleic acid molecule disclosed herein can be continuous or non-continuous (e.g., linked to each other by a linker).

[0047] The term "portion" as used herein refers to a fraction. With respect to a particular structure (such as a polynucleotide sequence, an amino acid sequence, or a protein), a "portion" thereof can represent a continuous or discontinuous fraction of the structure. For example, a portion of an amino acid sequence comprises at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% of the amino acids of the amino acid sequence. Additionally or alternatively, if the portion is a discontinuous fraction, the discontinuous fraction is composed of 2, 3, 4, 5, 6, 7, 8, or more sub-portions of the structure (e.g., a domain of a protein), each sub-portion being a continuous element of the structure. For example, a discontinuous fraction of an amino acid sequence can be composed of 2, 3, 4, 5, 6, 7, 8, or more (e.g., no more than 4) sub-portions of the amino acid sequence, wherein each sub-portion comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, or at least 30 consecutive amino acids of the amino acid sequence.

[0048] When used in reference to a cell, nucleic acid, protein, or vector, the term "recombinant" indicates that the cell, nucleic acid, protein, or vector has been altered or produced by human intervention, such as being modified by laboratory methods or being the result of laboratory methods. Thus, for example, recombinant proteins and nucleic acids include those produced by laboratory methods. A recombinant protein may contain amino acid residues not found 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 the following: 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 in a peptide or protein; production of a fusion protein (e.g., a fusion protein containing an antibody fragment); and addition, deletion, and / or substitution of one or more nucleic acids in a nucleic acid sequence. When used in reference to a cell, the term "recombinant" is not intended to include naturally occurring cells, but encompasses cells that have been engineered / modified to contain or express a polypeptide or nucleic acid that is not present in the cell when the cell is not engineered / modified.

[0049] In the context of two or more nucleic acids or proteins, the term "percent identity" as used herein refers to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotides or amino acids (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned to maximize correspondence over a comparison window or specified region), as measured using the BLAST or BLAST 2.0 sequence comparison algorithms with the default parameters described below or by manual alignment and visual inspection. See, e.g., the NCBI website ncbi.nlm.nih.gov / BLAST. Such sequences are then said to be "substantially identical". This definition also pertains to or can be applied to the complement of a query sequence. This definition includes sequence comparisons performed by the BLAST algorithm, where the parameters of the algorithm are selected to give the maximum match between the respective sequences over the full length of the respective reference sequences. This definition also includes sequences with deletions and / or additions and those with substitutions. Sequence identity can be calculated over a region of at least about 20 amino acids or nucleotides, or over a region of 10 - 100 amino acids or nucleotides, or over the entire length of a given sequence. Sequence identity can be calculated using published techniques and widely available computer programs such as the GCS program package (Devereux et al., Nucleic Acids Res (1984) 12:387), BLASTP, BLASTN, FASTA (Atschul et al., J Mol Biol (1990) 215:403). Sequence identity can be measured using sequence analysis software with its default parameters, such as the sequence analysis software package of the Genetics Computer Group of the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wisconsin 53705). Additional methods that can be suitably used to determine the similarity or identity of amino acid sequences include those that rely on position-specific structure scoring matrices (P3SM) incorporating structure prediction scores from Rosetta, and those based on length-normalized edit distances, as previously described, e.g., in Setcliff et al., Cell Host & Microbe 23(6), May 2018.

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

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

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

[0053] It should be understood that certain features of the present disclosure that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, the various features of the present disclosure that are described in the context of a single embodiment for brevity may also be provided separately or in any suitable sub-combination. All combinations of embodiments belonging to the present disclosure are exactly covered by the present disclosure and are disclosed herein as if each combination were separately and explicitly disclosed. In addition, all sub-combinations of the various embodiments and their elements are exactly covered by the present disclosure and are disclosed herein as if each such sub-combination were separately and explicitly disclosed herein.

[0054] Where a range of values is provided, it should be understood that, unless the context clearly dictates otherwise, each intermediate value between the upper and lower limits of that range (to one-tenth of the unit of the lower limit) and any other stated or intermediate value within the stated range is covered by the present disclosure. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also covered by the present disclosure, subject to any exact excluded limits within the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present disclosure.

[0055] Certain ranges are presented herein with numerical values preceded by the term "about", and as used herein, the term "about" has its ordinary meaning of approximately. The term "about" is used herein to provide literal support for the exact number that follows and for numbers that are close to or approximate the number that follows. In determining whether a number is close to or approximates a specifically recited number, an unrecited number that is close to or approximates the specifically recited number may be a number that is a substantially equivalent form of the specifically recited number in the context in which it is presented. If the degree of approximation is not clear from the context, "about" means within plus or minus 10% of the value provided, or rounded to the nearest significant digit, including in all cases the value provided. In some embodiments, the term "about" indicates plus or minus at most 10%, at most plus or minus 5%, or at most plus or minus 1% of a specified value.

[0056] Where a range of values is provided, those skilled in the art will appreciate that all ranges disclosed herein cover any and all possible sub-ranges and combinations of those sub-ranges. Any listed range can be readily identified as fully describing the same range and enabling the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. By way of non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, and upper third, etc. As will also be understood by those skilled in the art, all language such as "at most", "at least", "greater than", "less than", etc. includes the recited numbers and refers to ranges that can then be broken down into sub-ranges as discussed above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 - 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 - 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

[0057] The presentation of headings (e.g., (a), (b), (i), etc.) is for ease of reading the specification and claims only. The use of headings in the specification or claims does not require steps or elements to be in alphabetical or numerical order or the order in which they are presented. Madariaga virus (MADV)

[0058] Mayaro virus (MADV), previously known as South American eastern equine encephalitis virus (SAEEEV), is an arbovirus belonging to the genus Alphavirus, which includes a group of genetically, structurally, and serologically related viruses of the family Togaviridae and is classified as a Group IV positive-sense single-stranded RNA virus. Currently, the genus Alphavirus particularly includes 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 capable of infecting various vertebrates (such as mammals, rodents, fish, bird species, and large mammals (such as humans and horses)) as well as invertebrates (such as insects).

[0059] The EEEV complex consists of four different genetic lineages: one that circulates in North America (NA EEEV) and the Caribbean, and three that circulate in Central and South America (i.e., MADV). In different studies, the evolutionary and ecological diversity between NA and SA EEEV has suggested that the NA and SA lineages can be considered separate species within the EEEV complex. The single monophyletic NA EEEV lineage mainly exhibits temporally associated relationships and is highly conserved within its geographical range. In contrast, MADV (i.e., SA EEEV) consists of three different lineages, two of which are composed of highly conserved geographical groupings that lack any temporal association completely. The phylogenetic comparison of MADV (i.e., SA EEEV) and VEEV shows similar genetic and evolutionary patterns, which is consistent with the known use of mammalian reservoirs by VEEV. Although they are associated with equine diseases, the MADV species are not clearly associated with human diseases. This lack of human pathogenicity limits our ability to expand our knowledge of the epidemiology and ecology of MADV strains. MADV isolated from species of the Culex (Melanoconion) genus, Spissipes section (Culex pedroi in South America and Culex taeniopus in Central America) indicates that they are the main enzootic vectors and may become epizootic vectors. The movement of these vectors outside their tropical forest habitats is usually restricted, which may affect the limitations of transmission. However, the foraging behavior of these species is relatively extensive, which broadens the transmission cycles that MADV may exploit. However, the higher diversity of vectors in the tropics may also contribute to the genetic diversity within the MADV lineages, although data on vector transmission capacity are limited. More information in this regard can be found in Arrigo NC et al., Journal of Virology, January 2010, pp. 1014 - 1025, which is incorporated herein by reference.

[0060] The genus Alphavirus (including MADV and EEEV) has been extensively studied, and the life cycle, replication patterns, etc. of these viruses are well characterized. More information in this regard can be found, for example, in Arrigo NC et al., ibid 2010 and Corrin T. et al., Vector - Borne and Zoonotic Diseases, Vol. 21, No. 5, 2021. Additionally, it has been shown that alphaviruses can replicate very efficiently in animal cells, which makes them valuable as vectors for producing proteins and nucleic acids in such cells. Transmission between species and individuals occurs mainly via mosquitoes, making alphaviruses contributors to the collection of arboviruses or arthropod - borne viruses.

[0061] Each of these alphaviruses has a positive-sense single-stranded RNA genome, which is encapsulated in a nucleocapsid surrounded by an envelope containing viral spike proteins. Alphavirus particles are enveloped, tend to be spherical (although slightly pleomorphic), and have an icosahedral nucleocapsid. The alphavirus genome is a positive-sense single-stranded RNA approximately 11-12 kb in length, containing a 5’ cap, a 3’ polyA tail, and two open reading frames, where the first open reading frame encodes non-structural proteins with enzymatic functions and the second open reading frame encodes viral structural proteins (e.g., capsid protein CP, E1 glycoprotein, E2 glycoprotein, E3 protein, and 6K protein). For example, MADV has a single-stranded positive-sense RNA genome, which contains two open reading frames (ORFs) flanked by 5'- and 3'-untranslated regions (UTRs), and is capped at the 5' end and polyadenylated at the 3' end.

[0062] The 5′-terminal two-thirds of the alphavirus genome encodes many non-structural proteins required for viral RNA transcription and replication. These proteins are translated directly from the RNA and, together with cellular proteins, form an RNA-dependent RNA polymerase that is essential for viral genome replication and subgenomic RNA transcription. Four non-structural proteins (nsP1-4) are produced as a single polyprotein, which constitutes the viral replication machinery. Processing of the polyprotein occurs in a highly regulated manner, where cleavage at the P2 / 3 junction affects the use of the RNA template during genome replication. This site is at the bottom of a narrow cleft and is not easily accessible. Once cleaved, nsP3 forms a circular structure that surrounds nsP2. These two proteins have an extensive interface. Mutations in nsP2 that produce non-cytopathic viruses or temperature-sensitive phenotypes cluster in the P2 / P3 interface region. P3 mutations opposite the positions of nsP2 non-cytopathic mutations prevent efficient cleavage of P2 / 3. This, in turn, may affect RNA infectivity and alter the level of viral RNA production.

[0063] The 3′-terminal third of the genome contains subgenomic RNA, which serves as the translation template for all the structural proteins required for virus particle formation (the core nucleocapsid protein C and the envelope proteins P62 and E1 that associate as a heterodimer). The virus membrane-anchored surface glycoproteins are responsible for receptor recognition and entry into target cells by membrane fusion. The subgenomic RNA is transcribed from the p26S subgenomic promoter present at the 3′ end of the RNA sequence encoding the nsP4 protein. Proteolytic maturation of the P62 protein into E2 and E3 causes changes on the virus surface. The E1, E2, and sometimes E3 glycoprotein “spikes” together form an E1 / E2 dimer or an E1 / E2 / E3 trimer, where E2 extends from the center to the apex, E1 fills the space between the apices, and E3 (if present) is located distally on the spike. When the virus is exposed to the acidity of the endosome, E1 dissociates from E2 to form an E1 homotrimer, which is required for the fusion step that drives the apposition of the cell membrane and the virus membrane. The alphavirus glycoprotein E1 is a class II viral fusion protein, which is structurally distinct from the class I fusion proteins found in influenza virus and HIV. The E2 glycoprotein functions through its cytoplasmic domain to interact with the nucleocapsid, while its extracellular domain is responsible for binding to cell receptors. Most alphaviruses, including MADV, lose the peripheral protein E3, while in Semliki Forest virus, it remains associated with the virus surface.

[0064] Similar to most alphaviruses, replication of MADV occurs on the surface of membranes within the host cell. In the first step of the infection cycle, the 5′ end of the genomic RNA is translated into a polyprotein (nsP1-4) with RNA polymerase activity, which generates a negative strand complementary to the genomic RNA. In the second step, the negative strand is used as a template to produce two types of RNA: (1) the positive genomic RNA corresponding to the genome of the secondary virus that produces the other nsP proteins by translation and serves as the positive genome of the virus; and (2) the subgenomic RNA encoding the structural proteins of the virus that form the infectious particles. The ratio of positive genomic RNA / subgenomic RNA is regulated by proteolytic self-cleavage of the polyprotein into nsP1, nsP2, nsP3, and nsP4. In fact, viral gene expression occurs in two phases. In the first phase, mainly the positive genomic strand and the negative strand are synthesized. During the second phase, the synthesis of subgenomic RNA is virtually exclusive, and thus leads to the production of large amounts of structural proteins. Self - replicating RNA

[0065] As will be appreciated by those skilled in the art, the term "self-replicating RNA" (srRNA) refers to an RNA molecule that contains all of the genetic information required to direct its own amplification or self-replication within a permissive cell. Thus, srRNA is sometimes also referred to as "self-amplifying RNA" (saRNA). In some embodiments, the srRNA is a "replicon", which can be a linear or circular portion of DNA or RNA that is replicated sequentially as a unit. Non-limiting examples of replicons include "replicon RNA" or "RNA replicon". To direct its own replication, the srRNA typically (1) encodes a polymerase, replicase, or other protein that can interact with proteins, nucleic acids, or ribonucleoproteins of viral or host cell origin to catalyze the RNA amplification process; and (2) contains cis-acting RNA sequences required for the replication and transcription of subgenomic RNA. These sequences can bind to proteins encoded by themselves or proteins, nucleic acids, or ribonucleoproteins of cellular origin not encoded by themselves or complexes between any of these components during the replication process. In some embodiments of the present disclosure, the replicon (e.g., srRNA) is derived from Madariaga virus (MADV). In some embodiments of the present disclosure, the MADV srRNA construct (e.g., srRNA, saRNA, or RNA replicon molecule) typically contains the following elements: one or more cis 5' viral or defective interfering RNA sequences required for replication, sequences encoding biologically active alphavirus nonstructural proteins (e.g., nsP1, nsP2, nsP3, and nsP4), a subgenomic promoter (sg) for subgenomic RNA (sgRNA), cis 3' viral sequences required for replication, and optionally a polyadenylate tract (poly(A)). In some cases, a subgenomic promoter (sg) that directs the expression of a heterologous sequence can be included in the srRNA construct of the present disclosure.

[0066] In addition, the term srRNA molecule (e.g., srRNA, saRNA, or RNA replicon molecule) generally refers to a molecule of positive polarity or sense ("message" sense), and the length of the srRNA can be different from the length of any known naturally occurring alphavirus. In some embodiments of the present disclosure, the srRNA does not contain at least a portion of the coding sequence of one or more alphavirus structural proteins; and / or the sequence encoding the structural gene can be replaced by a heterologous sequence. In those cases, where the srRNA is to be packaged into recombinant alphavirus particles, it can contain one or more sequences (so-called packaging signals) for initiating interaction with alphavirus structural proteins, leading to particle formation.

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

[0068] As described in more detail below, one aspect of the present disclosure relates to nucleic acid constructs comprising nucleic acid sequences encoding a modified viral genome or srRNA, wherein the modified genome or srRNA lacks (e.g., does not contain) at least a portion of the nucleic acid sequence encoding one or more structural proteins of the corresponding unmodified viral genome or srRNA. Some embodiments of the present disclosure provide a modified alphavirus genome or srRNA, wherein the coding sequences for the non-structural proteins nsP1, nsP2, nsP3, and nsP4 are present, yet at least a portion or the entire sequence encoding one or more structural proteins is absent. Recombinant cells and cell cultures are also provided that have been engineered to contain the nucleic acid constructs as disclosed herein. A. Nucleic acid constructs

[0069] As described in more detail below, one aspect of the present disclosure relates to novel nucleic acid constructs that comprise nucleic acid sequences encoding a modified genome or replicon (e.g., srRNA) of an alphavirus (such as Madariaga virus (MADV)). For example, in some embodiments, the modified alphavirus genome can comprise one or more deletions, one or more substitutions, and / or one or more insertions in one or more genomic regions of the parental alphavirus genome.

[0070] Non-limiting exemplary embodiments of the nucleic acid constructs (e.g., replicon constructs, such as srRNA constructs) of the present disclosure may include one or more of the following features. In some embodiments, the nucleic acid construct comprises a nucleic acid sequence encoding a modified MADV genome or replicon (e.g., srRNA), wherein the modified MADV genome or replicon (e.g., srRNA) lacks at least a portion of the nucleic acid sequence encoding one or more structural proteins of the unmodified MADV genome or srRNA, e.g., the modified MADV genome or srRNA does not comprise at least a portion of the coding sequence of one or more of the MADV structural proteins CP, E1, E2, E3, and 6K. Both virulent and avirulent MADV strains are suitable. Non-limiting examples of MADV strains suitable for the compositions and methods of the present disclosure include ArgLL, ArgB, BeAn-5122, ArgM, 24443 (TR59), 25714 (BG60), BeAr 18205, 900188 (PA62), BeAr 81828, BeAr 126650, 68U231, 77U1104 (PE70), 75V1496, BeAr 300851, 75U40, and El Delirio (Arrigo NC et al., supra 2010). Additional examples of MADV strains suitable for the compositions and methods of the present disclosure include 76V25343, 77U1 (BR77), BeAr 348998, IVICPan57151, BeAn416361, 903836 (PA84), BeAr436087, 435731 (PA86), C49 (CO92), PE-0.0155-96 (0.0155), PE-3.0815-96 (3.0815), PE-16.0050-98 (16.0050), PE-18.0140-99 (18.0140), and PE-18.0172-99 (18.0172) (Arrigo NC et al., supra 2010). Additional suitable MADV strains include, but are not limited to, those described in Arrigo NC et al., supra 2010, and the Virus Pathogen Resource website (ViPR, which is publicly available at www.viprbrc.org / brc / vipr_genome_search.spg?method = SubmitForm&blockId = 868& decorator = to ga public access). In some embodiments, the modified MADV genome or srRNA is derived from the MADV strain BeAr300851.

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

[0072] In some embodiments, the modified viral genome or srRNA lacks a substantial portion of the nucleic acid sequence encoding one or more viral structural proteins. One of ordinary skill in the art will understand that a substantial portion of the nucleic acid sequence encoding a viral structural polypeptide can include sufficient nucleic acid sequence encoding the viral structural polypeptide to provide a putative identification of the polypeptide by manual evaluation of the sequence by one of ordinary skill in the art or by computer automated sequence comparison and identification using an algorithm 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 the nucleotide sequence contains sufficient sequence to provide for the specific identification and / or isolation of the nucleic acid fragment containing the sequence. For example, a substantial portion of the nucleic acid sequence can include at least about 20% of the full-length nucleic acid sequence, such as about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%. As described above, the present disclosure provides nucleic acid molecules and constructs that lack a partial or complete nucleic acid sequence encoding one or more viral structural proteins. One of ordinary skill in the art can readily use all or a substantial portion of the disclosed sequences for the compositions and methods of the present disclosure. Accordingly, this application includes the complete sequences disclosed herein, such as those shown in the accompanying sequence listing, and substantial portions of those sequences as defined above.

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

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

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

[0076] 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 comprises a coding sequence of a polypeptide construct of interest, the polypeptide construct of interest comprising a single polypeptide (e.g., a single-gene PCI). In some embodiments, the coding sequence of the PCI comprises coding sequences of multiple polypeptides (e.g., a multi-gene (e.g., two-gene, three-gene, or four-gene, etc.) PCI). In some embodiments, each of the coding sequences of the multiple polypeptides is operably linked to a separate promoter sequence. In some embodiments, the coding sequences of the multiple polypeptides are operably linked to each other within a single open reading frame (e.g., in a polycistronic ORF). In some embodiments, the coding sequence of the polycistronic ORF is operably linked to a promoter sequence. In some embodiments, at least one of the promoter sequences is a subgenomic (sg) promoter. In some embodiments, the sg promoter is the 26S genomic promoter.

[0077] In some embodiments, the multiple polypeptides can be linked to each other directly or indirectly (e.g., via one or more linker sequences). For example, in some embodiments, the multiple polypeptides can be directly linked to each other, such as being linked adjacent to each other. In some embodiments, at least two (e.g., 2, 3, 4, or 5) of the multiple polypeptides are operably linked to each other via one or more linker sequences. In some embodiments, the length and amino acid composition of the linker sequences can be optimized to alter the orientation, flexibility, and / or proximity of the polypeptides relative to each other to achieve the desired activity or properties of the PCI. In some embodiments, the linker sequences among the multiple linker sequences comprise one or more self-cleaving peptide sequences. Non-limiting examples of self-cleaving peptide sequences applicable to the methods and compositions of the present disclosure include self-cleaving cleavage sequences derived from calcium-dependent serine endoprotease (furin), porcine teschovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), Plutella xylostella betatetravirus 2A (T2A), Bombyx mori cytoplasmic polyhedrosis virus 2A (BmCPV2A), and Bombyx mori infectious flacherie virus 2A (BmIFV2A). In some embodiments, at least two of the multiple polypeptides are operably linked to each other via a P2A self-cleaving cleavage sequence.

[0078] In some embodiments, the coding sequences of multiple polypeptides are operably linked to each other via one or more internal ribosome entry sites (IRESs). Non-limiting examples of IRESs suitable for the methods and compositions of the present disclosure include viral IRES sequences, cellular IRES sequences, and artificial IRES sequences. Examples of suitable IRES sequences include, but are not limited to, Kaposi's sarcoma-associated herpesvirus (KSHV) IRES, hepatitis virus IRES, Pestivirus IRES, Cripavirus IRES, Rhopalosiphum padi virus IRES, fibroblast growth factor IRES, platelet-derived growth factor IRES, vascular endothelial growth factor IRES, insulin-like growth factor IRES, picornavirus IRES, encephalomyocarditis virus (EMCV) IRES, Pim-1 IRES, p53 IRES, Apaf-1 IRES, TDP2 IRES, L-myc IRES, and c-myc IRES.

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

[0080] In some embodiments, the coding sequence of the GOI is redesigned, reconstructed, and / or optimized to obtain desired properties such as increased stability, potency, and expression (e.g., translational efficiency), which in turn can maximize the impact of generating, delivering, and administering a biotherapeutic agent. For example, in some embodiments, the coding sequence of the GOI is optimized for expression at a level higher than the expression level of a reference coding sequence (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% higher than the reference coding sequence). In some embodiments, the reference coding sequence is a wild-type non-optimized sequence. With respect to sequence optimization of nucleotide sequences, the degeneracy of the genetic code provides the possibility of substituting at least one base of the gene sequence encoding a protein with a different base without resulting in a change in the amino acid sequence of the polypeptide produced by the gene. Thus, the nucleic acid constructs of the present disclosure can also have any base sequence altered from any of the polynucleotide sequences disclosed herein substituted according to the degeneracy of the genetic code. References describing codon usage are readily publicly available. In some embodiments, polynucleotide sequence variants can be generated for a variety of reasons, such as to optimize expression in a particular host (e.g., changing the codon usage in alphavirus mRNA to those codon usages 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 for expression in a target host cell by using codons optimized for expression. Techniques for constructing a synthetic nucleic acid sequence encoding the GOI using the preferred codons that are optimal for host cell expression can be determined by computational methods that analyze the prevalence and relative abundance of codon usage in the native proteins encoding the host cell genome via techniques known in the art. A variety of codon usage databases (e.g., http: / / www.kazusa.or.jp / codon) can be used to generate codon-optimized sequences in a mammalian cell environment. In addition, a variety of software tools are available for converting the sequence of one organism to the optimal codon usage for a different host organism, such as the JCat codon optimization tool (www.jcat.de), the Integrated DNA Technologies (IDT) codon optimization tool (https: / / www.idtdna.com / CodonOpt), or the Optimizer online codon optimization tool (http: / / genomes.urv.es / OPTIMIZER). Such synthetic sequences can be constructed by techniques known in the art for constructing synthetic nucleic acid molecules and can be obtained from a number of commercial suppliers. Thus, in some embodiments, the coding sequence of the GOI is optimized for expression at a level higher than the expression level of a reference coding sequence (e.g., an un-codon-optimized coding sequence).In some embodiments, the codon-optimized sequence of the GOI results in an increase in expression level of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% compared to the non-codon-optimized reference coding sequence. In some embodiments, the codon-optimized sequence of the GOI results in an increase in expression level of at least 2-fold, at least 3-fold, at least 4-fold or at least 5-fold compared to the non-codon-optimized reference coding sequence.

[0081] In some embodiments, the coding sequence of the GOI is optimized for enhanced RNA stability and / or expression. The stability of RNA is generally related to the "half-life" of the RNA. The "half-life" is the period of time required to eliminate half of the activity, amount or quantity of the molecule. In the context of the present disclosure, the half-life of the RNA indicates the stability of the RNA. The half-life of the RNA can affect the "duration of expression" of the RNA. Additional information regarding the 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 (Nature Communications. Vol. 13, Article No. 1536, March 2022).

[0082] A polypeptide encoded by a GOI can generally be any polypeptide and can be, 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 following: an antibody, an antigen, an immunomodulator, an enzyme, a signaling protein, and a cytokine. In some embodiments, the GOI can encode a microbial protein, a viral protein, a bacterial protein, a fungal protein, a mammalian protein, and any combination thereof. In some embodiments, the GOI encodes the hemagglutinin precursor (HA) of influenza A virus H5N1. Non-limiting examples of GOIs include interleukins and interacting proteins, including: G-CSF, GM-CSF, IL-1, IL-10, IL-10-like protein, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-18BP, IL-1-like protein, IL-1RA, IL-1α, IL-1β, IL-2, IL-20, IL-3, IL-4, IL-5, IL-6, IL-6-like protein, IL-7, IL-9, IL-21, IL-22, IL-33, IL-37, IL-38, LIF, and OSM. Additional suitable GOIs include, but are not limited to, interferons (e.g., IFN-α, IFN-β, IFN-γ), TNFs (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-βs (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, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, and CX3CL1), immunosuppressive gene products, and related transcription factors (e.g., PECAM1, FCGR3A, FOS, NFKB1, JUN, HIF1A, PD-L1, mTOR, STAT5B, and STAT4).Additional GOIs applicable to 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 resistance gene mutants / variants, 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 applicable to the compositions and methods of the present disclosure are sequences encoding viral proteins, particularly spike proteins, fibronectin, structural proteins, and attachment proteins.

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

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

[0085] In some embodiments, the GOI can encode a polypeptide selected from: an antigen molecule, a biotherapeutic molecule, or any combination thereof. In some embodiments, the GOI can encode a polypeptide selected from: a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), a neoantigen, and any combination thereof. As will be understood by those skilled in the art, TAAs can generally include molecules that are present on tumor cells and normal cells or many normal cells but at concentrations far lower than those on tumor cells, such as proteins. In contrast, TSAs can generally include molecules that are present on tumor cells but not on normal cells, such as proteins. Tumor-associated antigens can be antigens associated with, for example, breast cancer cells, B-cell lymphoma, pancreatic cancer, Hodgkin lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma, lung cancer cells, non-Hodgkin B-cell lymphoma (B-NHL) cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, glioma, glioblastoma, colorectal cancer cells, and the like. It will also be understood that in some cases, tumor-associated antigens can also be expressed by non-cancer cells. In some embodiments, the GOI can encode a polypeptide selected from: estrogen receptor, intracellular signal transduction enzymes, and human epidermal growth receptor. In some embodiments, the GOI can encode a biotherapeutic polypeptide selected from: immunomodulators, angiogenesis regulators, extracellular matrix regulators, metabolic regulators, neuromodulators, and any combination thereof. In some embodiments, the GOI can encode a cytokine selected from: chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. In some embodiments, the GOI can encode an interleukin selected from IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-15, IL-17, IL-23, IL-27, IL-35, IFNγ, and any subunit thereof. In some embodiments, the GOI can encode a biotherapeutic polypeptide selected from: IL-12A, IL-12B, IL-1RA, and any combination thereof.

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

[0087] In some embodiments, the nucleic acid construct of the present disclosure comprises a nucleic acid sequence encoding a modified MADV that has 100% sequence identity with the nucleic acid sequence of SEQ ID NO:1, wherein one, two, three, four, five, or more nucleotides of the nucleic acid sequence may be replaced with different nucleotides.

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

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

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

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

[0092] The molecular techniques and methods for assembling and characterizing these new nucleic acid constructs are described more fully in the examples of the present application. In some embodiments, the nucleic acid molecule is a recombinant nucleic acid molecule. As used herein, the term recombinant means any molecule (e.g., DNA, RNA, polypeptide) derived from human manipulation or produced from (albeit indirectly) human manipulation. As a non - limiting example, cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been produced by one or more in vitro polymerase reactions or has been attached to a linker or has been integrated into a vector (such as a cloning vector or an expression vector). As non - limiting examples, a recombinant nucleic acid molecule: 1) has been synthesized or modified in vitro, for example, using chemical or enzymatic techniques, such as by using chemical nucleic acid synthesis, or by using enzymes for nucleic acid molecule replication, polymerization, exonuclease digestion, endonuclease digestion, ligation, reverse transcription, transcription, base modification (including, for example, methylation) or recombination (including homologous recombination and site - specific recombination); 2) contains linked nucleotide sequences that are not linked in nature; 3) has been engineered using molecular cloning techniques such that it lacks one or more nucleotides relative to the naturally occurring nucleotide sequence; and / or 4) has been manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements relative to the naturally occurring nucleotide sequence.

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

[0094] Nucleic acid molecules (including variants of naturally occurring nucleic acid sequences) can be generated using a variety of methods known to those skilled in the art (see, e.g., Sambrook et al., in: Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)). The sequence of a nucleic acid molecule can be modified relative to the naturally occurring sequence from which it is derived using a variety of techniques, including but not limited to classical mutagenesis techniques and recombinant DNA techniques such as, but not limited to, site-directed mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, restriction enzyme cleavage of nucleic acid fragments, ligation of nucleic acid fragments, PCR amplification and / or mutagenesis of selected regions of nucleic acid sequences, recombinant cloning; and chemical synthesis, including chemical synthesis of mixtures of oligonucleotides and ligation of mixed groups to "construct" mixtures of nucleic acid molecules; and combinations thereof. Nucleic acid molecule homologs can be selected from mixtures of modified nucleic acid molecules by screening for the function of the protein or srRNA encoded by the nucleic acid molecule, and / or by hybridization to the wild-type gene or a fragment thereof, or by PCR using primers that are homologous to the target or wild-type nucleic acid molecule or sequence. B. Recombinant cells

[0095] The nucleic acid constructs of the present disclosure can be introduced into host cells to generate recombinant cells containing the nucleic acid molecules. Accordingly, prokaryotic or eukaryotic cells containing a nucleic acid construct encoding a modified MADV genome as described herein are also a feature of the present disclosure. In related aspects, some embodiments disclosed herein relate to methods of transforming cells, the methods comprising introducing a nucleic acid construct as provided herein into a host cell (such as an animal cell), and then selecting or screening the transformed cells. Introduction of the nucleic acid constructs of the present disclosure into cells can be achieved by methods known to those skilled in the art, such as viral infection, transfection, conjugation, protoplast fusion, liposome transfection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0096] In one aspect, some embodiments of the present disclosure relate to recombinant cells comprising the nucleic acid constructs described herein, such as recombinant animal cells. The nucleic acid constructs can be stably integrated into the host genome, or can replicate episomally, or exist as minicircle expression vectors in the recombinant host cell for stable or transient expression. Thus, in some embodiments of the present disclosure, the nucleic acid constructs are maintained and replicated as episomal units in the recombinant host cell. In some embodiments, the nucleic acid constructs are stably integrated into the genome of the recombinant cell. Stable integration can be accomplished using classical random genomic recombination techniques or with more precise genome editing techniques (such as CRISPR / Cas9 or TALEN genome editing guided by guide RNAs). In some embodiments, the nucleic acid constructs exist as minicircle expression vectors in the recombinant host cell for stable or transient expression.

[0097] The host cell can be an untransformed cell or a cell that has been transfected with at least one nucleic acid molecule. Thus, in some embodiments, the host cell can be genetically engineered (e.g., transduced or transformed or transfected) with at least one nucleic acid molecule.

[0098] Suitable host cells for cloning or expressing the polypeptides of interest as described herein include the prokaryotic or eukaryotic cells described herein. Thus, in some embodiments, the recombinant cell is a prokaryotic cell, such as the bacterium Escherichia coli (E. coli); or a eukaryotic cell, such as an insect cell (e.g., mosquito cell or Sf21 cell) or a mammalian cell (e.g., COS cell, NIH 3T3 cell or HeLa cell). In some embodiments, the cell is in vivo, e.g., a recombinant cell in a living body, such as 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 cell is ex vivo, e.g., having been removed from a living body or organism as a single cell or as part of an organ or tissue for treatment or procedure and then returned to the living body or organism. In some embodiments, the cell is in vitro, e.g., obtained from a repository. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a vertebrate cell or an invertebrate cell. In some embodiments, the recombinant cell is a mammalian cell. Non-limiting examples of recombinant cells suitable for the methods and compositions of the present disclosure include monkey kidney CV1 cells transformed by SV40 (e.g., COS-7 cells), human embryonic kidney cells (e.g., HEK293 or HEK 293 cells) or their derivative cells (e.g., BHK-21 or BHK-570 cells), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 cells), human cervical cancer cells (e.g., HeLa cells), dog kidney cells (e.g., MDCK cells), buffalo rat hepatocytes (e.g., BRL 3A cells), human lung cells (e.g., W138 cells), human hepatocytes (e.g., Hep G2 cells), mouse mammary tumor (e.g., MMT 060562 cells), TRI cells, FS4 cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (e.g., Vero cells), human A549 cells, human cervical cells, human CHME5 cells, human PER.C6 cells, NS0 mouse myeloma cells, human epidermoid laryngeal cells, human fibroblasts, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocytes, human macrophages, human RAW264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells and rabbit kidney cells.In some embodiments, the recombinant cell is a cell derived from the cell described above (i.e., a derivative cell of the original cell described herein), such as a cell amplified from a clone of the original cell (an engineered form of the original cell) or a cell reclassified after the original cell has undergone extensive passaging or has been passaged through another host.

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

[0100] In another aspect, the present disclosure provides cell cultures comprising at least one recombinant cell as disclosed herein and a culture medium. Generally, the culture medium can be any suitable medium 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. Accordingly, cell cultures comprising at least one recombinant cell as disclosed herein are also within the scope of the present application. Methods and systems suitable for generating and maintaining cell cultures are known in the art. C. Transgenic animals

[0001] In another aspect, transgenic animals are also provided, which comprise a nucleic acid construct (e.g., a vector, replicon, or srRNA molecule) as described herein. In some embodiments, the transgenic animal is a vertebrate or invertebrate. In some embodiments, the transgenic animal is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the transgenic animal is a mammal. In some embodiments, the transgenic mammal is a non-human mammal. Generally, the transgenic animals of the present disclosure can be any non-human animals known in the art. In some embodiments, the non-human animals of the present disclosure are non-human primates. Other animal species suitable for the compositions and methods of the present disclosure include those animals that (i) are suitable for transgenesis and (ii) are 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 cattle. Additional 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.), livestock (e.g., horses, cattle, pigs, sheep, goats, ducks, geese, chickens, etc.), domestic animals and pets (e.g., cats, dogs, etc.), non-human primates (e.g., apes, chimpanzees, orangutans, monkeys, etc.), fish, amphibians (e.g., frogs, salamanders, etc.), reptiles (e.g., snakes, lizards, etc.), and other animals (e.g., foxes, weasels, rabbits, minks, beavers, ferrets, otters, sables, seals, coyotes, chinchillas, deer, muskrats, opossums, etc.).

[0002] In some embodiments, the transgenic animal is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the transgenic animal of the present disclosure is a chimeric transgenic animal. In some embodiments, the transgenic animal of the present disclosure is a transgenic animal having germ cells and somatic cells that 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, one or more nucleic acid constructs are stably integrated into the genome of the transgenic animal. In some embodiments, the genome of the transgenic animal of the present disclosure can contain any one 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.

[0003] Methods and techniques for preparing transgenic non-human animals are known in the art. Exemplary methods include pronuclear microinjection, DNA microinjection, lentiviral vector-mediated transfer of DNA into early embryos, and sperm-mediated transgenesis, adenovirus-mediated introduction of DNA into animal sperm (e.g., in pigs), retroviral vectors (e.g., avian species), somatic cell nuclear transfer (e.g., in goats). A review of the state of the art in the preparation of transgenic farm animals is provided in Niemann, H. et al. (2005) Rev. Sci. Tech. 24:285-298. In some embodiments, standard methods known in the art for introducing exogenous nucleic acid into the genome of a non-human animal are used to prepare the transgenic non-human host animal of the present disclosure. In some embodiments, the transgenic animals of the present disclosure can be generated using classical random genomic recombination techniques or with 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 nucleases). In some embodiments, the transgenic animals of the present disclosure can be prepared using transgenic microinjection techniques and do not require the use of homologous recombination techniques and are thus considered easier to prepare and select than methods using homologous recombination. In some embodiments, the transgenic animals produce the desired protein as described herein.

[0004] The transgenic non-human host animals of the present disclosure are prepared using standard methods known in the art for introducing exogenous nucleic acids into the genome of non-human animals. In some embodiments, the non-human animals of the present disclosure are non-human primates. Other animal species suitable for the compositions and methods of the present disclosure include animals that (i) are suitable for transgenesis and (ii) are capable of rearranging immunoglobulin gene segments to produce an antibody response. Examples of such species include, but are not limited to, mice, rats, hamsters, rabbits, chickens, goats, pigs, sheep, and cattle. The means and methods for preparing transgenic non-human animals are known in the art. Exemplary methods include pronuclear microinjection, DNA microinjection, lentiviral vector-mediated transfer of DNA into early embryos, and sperm-mediated transgenesis, adenovirus-mediated introduction of DNA into animal sperm (e.g., in pigs), retroviral vectors (e.g., avian species), somatic cell nuclear transfer (e.g., in goats). A review of the state of the art in the preparation of transgenic domestic farm animals is provided in Niemann, H. et al. (2005) Rev. Sci. Tech. 24:285-298.

[0005] In some embodiments, the animal is a vertebrate or an invertebrate. In some embodiments, the animal is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the animal is a mammalian subject. In some embodiments, the mammal is a non-human animal. In some embodiments, the mammal is a non-human primate. In some embodiments, the transgenic animals of the present disclosure can be prepared using classical random genomic recombination techniques or with 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 nucleases). In some embodiments, the transgenic animals of the present disclosure can be prepared using transgenic microinjection techniques and do not require the use of homologous recombination techniques and are thus considered easier to prepare and select than methods using homologous recombination. In another aspect, the present disclosure provides methods for producing a polypeptide of interest, wherein the methods comprise (i) raising an animal as disclosed herein under certain conditions; or (ii) culturing recombinant cells comprising a nucleic acid construct as disclosed herein under conditions under which the transgenic animal or recombinant cells produce the polypeptide encoded by the GOI. In another aspect, the present disclosure provides methods for producing a polypeptide of interest in a subject, wherein the methods comprise 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. Accordingly, recombinant polypeptides produced by the methods disclosed herein are also within the scope of the present disclosure.

[0006] Non-limiting exemplary embodiments of the disclosed methods for producing recombinant polypeptides can include one or more of the following features. In some embodiments, the methods for producing the recombinant polypeptides of the present disclosure further comprise isolating and / or purifying the produced polypeptide. In some embodiments, the methods for producing the polypeptides of the present disclosure further comprise structurally modifying the produced polypeptide to increase its half-life. D. Pharmaceutical compositions

[0007] The nucleic acid constructs, recombinant cells, and recombinant polypeptides of the present disclosure can be incorporated into compositions, including pharmaceutical compositions. Such compositions typically comprise one or more of the nucleic acid constructs, recombinant cells, and recombinant polypeptides described and provided herein and a pharmaceutically acceptable excipient (e.g., a carrier). In some embodiments, the compositions of the present disclosure are formulated for preventing, treating, or managing a health condition, such as an immune disorder or a microbial infection (e.g., a viral infection, a fungal infection, or a bacterial infection). For example, the compositions of the present disclosure can be formulated as a prophylactic composition, a therapeutic composition, or a pharmaceutical composition or a mixture thereof comprising a pharmaceutically acceptable excipient. In some embodiments, the compositions of the present disclosure are formulated to be used as a vaccine. In some embodiments, the compositions of the present application are formulated to be used as an adjuvant.

[0008] Accordingly, in one aspect, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and: a) a nucleic acid construct of the present disclosure; b) a recombinant cell of the present disclosure; and / or c) a recombinant polypeptide of the present disclosure.

[0009] Non-limiting exemplary embodiments of the pharmaceutical compositions of the present disclosure can include one or more of the following features. In some embodiments, the present disclosure provides a composition comprising a nucleic acid construct as disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, the present disclosure provides a composition comprising a recombinant cell as disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, the composition comprises a recombinant polypeptide as disclosed herein and a pharmaceutically acceptable excipient.

[0010] In some embodiments, the nucleic acid constructs (e.g., vectors or srRNA molecules) of the present disclosure can be used in naked form or formulated with a delivery vehicle. Exemplary delivery vehicles suitable for the compositions and methods of the present disclosure include, but are not limited to, liposomes (e.g., neutral or anionic liposomes), microspheres, immunostimulating complexes (ISCOMs), lipid-based nanoparticles (LNPs), solid lipid nanoparticles (SLNs), polyplexes, polymeric nanoparticles, virus replicon particles (VRPs), or conjugates with bioactive ligands, which can facilitate delivery and / or enhance the immune response. These compounds are readily available to those skilled in the art; see, for example, Liposomes: A Practical Approach, RCP New Ed, IRL press (1990). Adjuvants other than liposomes, etc. are also used, and such adjuvants are known in the art. Adjuvants can protect an antigen (e.g., nucleic acid construct, vector, srRNA molecule) from rapid diffusion by sequestering it in a local deposit, or they can contain substances that stimulate the host to secrete factors chemotactic for macrophages and other components of the immune system.

[0011] The compositions of the present disclosure can be formulated in a form compatible with their intended route of administration, such as liposomes, lipid-based nanoparticles (LNPs), polymeric nanoparticles, polyplexes, virus replicon particles (VRPs), microspheres, immunostimulating complexes (ISCOMs), conjugates of bioactive ligands, or any combination thereof. Thus, in some embodiments, the compositions of the present disclosure are formulated in liposomes.

[0012] In some embodiments, the compositions of the present disclosure are formulated in lipid-based nanoparticles (LNPs). Exemplary types of lipids suitable for the delivery systems described herein include cationic lipids, ionizable cationic lipids, anionic lipids, neutral lipids, and combinations thereof.

[0013] In some embodiments, the LNPs of the present disclosure can comprise one or more ionizable lipids. Exemplary ionizable lipids suitable for the compositions and methods of the present disclosure include those described in PCT Publication Nos. WO 2020252589A1 and WO 2021000041A1, and Love K.T. et al., Proc Natl Acad Sci USA, February 2, 2010, 107(5) 1864 - 1869, which are incorporated herein by reference in their entirety.

[0014] Thus, in some embodiments, the LNPs of the present disclosure include one or more lipid compounds described in Love K.T. et al. (2010, supra), such as C16-96, C14-110, and C12-200. In some embodiments, the LNP comprises an ionizable cationic lipid selected from: ALC-0315, C12-200, LN16, MC3, MD1, SM-102, and any combination thereof. In some embodiments, the LNP of the present disclosure comprises C12-200.

[0015] In some embodiments, the LNP of the present disclosure comprises one or more cationic lipids. Suitable cationic lipids include, but are not limited to, 98N12-5, C12-200, C14-PEG2000, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1.

[0016] In some embodiments, the LNP of the present disclosure comprises one or more neutral lipids. As described above, in some embodiments, neutral lipids (also referred to as "structural lipids" or "helper lipids") can also be incorporated into lipid formulations and lipid particles. Lipid formulations and lipid particles can comprise one or more structural lipids in an amount of about 10 Mol% to 40 Mol% of the composition. Suitable structural lipids support the formation of particles during manufacture. Structural lipids refer to any of a number of lipid species that exist in anionic, uncharged, or neutral zwitterionic form at physiological pH. Representative structural lipids include diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, diacyl phosphatidylglycerol, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0017] Exemplary structural lipids include zwitterionic lipids such as distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl phosphatidylethanolamine (POPE), and dioleoyl phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE).

[0018] In another embodiment, the structural lipid can be any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol such as dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacyl phosphatidylserine, diacyl phosphatidic acid, and other anionic modifying groups attached to neutral lipids. Other suitable structural lipids include glycolipids (e.g., monosialotetrahexosylganglioside GM1).

[0019] 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 LNP of the present disclosure comprises one or more ionizable lipid compounds described in PCT Publication Nos. WO2020252589A1 and WO 2021000041 A1, which are incorporated herein by reference in their entirety.

[0020] In some embodiments, the LNP of the present disclosure comprises at least one lipid selected from the group consisting of C12-200, C14-PEG2000, DOPE, DMG-PEG2000, DSPC, DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG).

[0021] In some embodiments of the delivery systems described herein that comprise LNP, the mass ratio of lipid to nucleic acid in the LNP delivery system is from 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 from 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 average diameter of the lipid-based nanoparticle (LNP) is less than about 1000 nm, about 500 nm, about 250 nm, about 200 nm, about 150 nm, about 100 nm, about 75 nm, about 50 nm, or about 25 nm. In some embodiments, the average diameter of the LNP ranges from about 70 nm to 100 nm. In some embodiments, the average diameter of the LNP ranges from about 88 nm to about 92 nm, 82 nm to about 86 nm, or about 80 nm to about 95 nm.

[0022] Stabilizers can be included in lipid formulation embodiments to ensure the integrity of the mixture. Stabilizers are a class of molecules that disrupt or assist in forming hydrophobic-hydrophilic interactions between molecules. Suitable stabilizers include, but are not limited to, polysorbate 80 (also known as Tween 80, IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl octadec-9-enoate), Myrj 52 (polyoxyethylene(40) stearate), and Brij TM S10 (polyoxyethylene(10) stearyl ether). PEG-conjugated lipids can also be used. Stabilizers can be used alone or in combination with each other.

[0023] In some embodiments, the stabilizer comprises from about 0.1 Mol% to 3 Mol% of the total lipid mixture. In some embodiments, the stabilizer comprises from about 0.5 Mol% to 2.5 Mol% of the total lipid mixture. In some embodiments, the stabilizer is present at greater than 2.5 Mol%. In some embodiments, the stabilizer is present at 5 Mol%. In some embodiments, the stabilizer is present at 10 Mol%. In some embodiments, the stabilizer is about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, etc. In other embodiments, the stabilizer is 2.6 Mol% - 10 Mol% of the lipid mixture. In other embodiments, the stabilizer is present at greater than 10 Mol% of the lipid mixture.

[0024] Steroids can also be included in the lipid composition for certain applications, and the lipid particles prepared therefrom contain sterols (such as cholesterol and phytosterols).

[0025] In some embodiments, the compositions of the present disclosure are formulated in polymeric nanoparticles. Examples of polymers suitable for the compositions and methods of the present disclosure can be found in Jiang X et al. (Polymeric nanoparticles for RNA delivery. Encyclopedia of Nanomaterials, 2021), which is incorporated herein by reference. Exemplary polymers suitable for the compositions and methods of the present disclosure include cationic polymers, non-cationic polymers, and combinations thereof. In some embodiments, the polymeric nanoparticles of the present disclosure may comprise cationic polymers of natural origin. In some embodiments, cationic polymers of natural origin may include chitosan, gelatin, dextran, cellulose, cyclodextrin, or combinations thereof. In some embodiments, the cationic polymer may be a synthetic cationic polymer. In some embodiments, synthetic cationic polymers may include polyethyleneimine (PEI), poly-L-lysine (PLL), poly(amino acid) (PAA), poly(amidoamine) (PAMAM), poly(amino-co-ester) (PAE), poly(2-N,N-dimethylaminoethyl methacrylate), poly(β-amino ester) (PBAE), imidazole-containing polymers, tertiary amine-containing polymers, poly(2-(dimethylamino)ethyl methacrylate), poly-N-(2-hydroxypropyl)methacrylamide, polyamidoamine dendrimers, cationic sugar-containing polymers, or derivatives thereof.

[0026] In some embodiments, the non-cationic polymer is negatively charged (i.e., anionic) or electrically neutral. In some embodiments, non-cationic polymers include polyethylene glycol (PEG), polyesters (e.g., polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyglycolic acid (PGA), polycaprolactone (PCL)), and poly(sarcosine) (pSar) or derivatives thereof.

[0027] In some embodiments, the polymer may be water-soluble and / or biodegradable. In some embodiments, the polymeric nanoparticles comprise one or more of the following: poly-(γ-L-glutamylglutamine) (PGGA), poly-(γ-L-aspartylglutamine) (PGAA), poly-L-lactic acid (PLLA), poly-(lactic-co-glycolic acid) (PLGA), polyalkylcyanoacrylate (PACA), polyanhydrides, polyhydroxy acids, polypropyl fumarate, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, [N-(2-hydroxypropyl)methacrylamide] (HPMA) copolymers, polyvinyl alcohol, polyurethanes, polyphosphazenes, polyacrylates, polyureas, polyamines, poly-ε-caprolactone (PCL), and copolymers thereof.

[0028] In some embodiments, the composition is an immunogenic composition, e.g., a composition that can stimulate an immune response in a subject. In some embodiments, the immunogenic composition is formulated as a vaccine. In some embodiments, the pharmaceutical composition is formulated as an adjuvant.

[0029] In some embodiments, the immunogenic composition is formulated as a biotherapeutic agent, e.g., a vehicle for gene delivery of different bioactive molecules. Non-limiting examples of biotherapeutic agents include cytokines, chemokines, and other soluble immunomodulators, enzymes, peptide and protein agonists, peptide and protein antagonists, hormones, receptors, antibodies and antibody derivatives, growth factors, transcription factors, and gene silencing / editing molecules. In some embodiments, the pharmaceutical composition is formulated as an adjuvant.

[0030] In some embodiments, the immunogenic composition is substantially non-immunogenic or minimally immunogenic (e.g., a composition that minimally stimulates an immune response in a subject). In some embodiments, the non-immunogenic or minimally immunogenic composition is formulated as a biotherapeutic agent. In some embodiments, the pharmaceutical composition is formulated for one or more of intranasal administration, transdermal administration, intrathecal administration, intraperitoneal administration, intramuscular administration, intratracheal administration, intranodal administration, intratumoral administration, intra-articular administration, intravenous administration, subcutaneous administration, intravaginal administration, intraocular administration, rectal administration, and oral administration.

[0031] Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In these cases, the composition should be sterile and should be a fluid to the extent that it is readily injectable. It can be stable under the conditions of manufacture and storage, and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or a dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, the appropriate fluidity can be maintained by using coatings such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants (e.g., sodium dodecyl sulfate). The action of preventing microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, isotonic agents such as sugars, polyols (e.g., mannitol, sorbitol) and / or sodium chloride will generally be included in the composition. The extended absorption of injectable compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0032] Sterile injectable solutions can be prepared by incorporating the active compound in an appropriate solvent, which may have one or a combination of the ingredients enumerated above as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0033] In some embodiments, the pharmaceutical compositions of the present disclosure are formulated for inhalation, such as aerosols, sprays, mists, liquids or powders. Administration by inhalation can be in the form of a dry powder or aerosol formulation which is inhaled by a subject (e.g., a patient) using an inhalation device (e.g., a nebulizer, a pressurized metered dose inhaler or an atomizer).

[0034] In some embodiments, the composition is formulated for one or more of intranasal administration, intrathecal administration, transdermal administration, intramuscular administration, intranodal administration, intravenous administration, intraperitoneal administration, oral administration, vaginal administration or intracranial administration. In some embodiments, the administered composition results in an increase in the production of interferon in the subject. In some embodiments, the administered composition induces the production of one or more pro-inflammatory molecules in the subject. In some embodiments, one or more pro-inflammatory molecules include interferon gamma (IFNγ), cytokines, TNF-α, GM-CSF, and MIP1α, granzyme B, granzyme A, perforin or any combination thereof. Methods of the present disclosure

[0035] Administration of any of the therapeutic compositions described herein (e.g., nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions) can be used to treat related health conditions such as proliferative disorders (e.g., cancer), infectious diseases (e.g., acute infections, chronic infections, or viral infections), rare diseases, and / or autoimmune diseases, and / or inflammatory diseases. In some embodiments, the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions as described herein can be used to modulate (e.g., elicit or inhibit) a pharmacodynamic effect in a subject in need thereof. In some embodiments, the pharmacodynamic effect includes eliciting an immune response in the subject. Non-limiting examples of pharmacodynamic effects include immunogenic effects, biomarker responses, therapeutic effects, prophylactic effects, desired effects, undesired effects, side effects, and effects in disease models. Accordingly, one aspect of the present disclosure relates to a method for modulating a pharmacodynamic effect in a subject in need thereof, the method comprising administering to the subject a composition comprising one or more of the following: (a) a nucleic acid construct as described herein; (b) a recombinant cell as described herein; (c) a recombinant polypeptide as described herein; and (d) a pharmaceutical composition as described herein. In some embodiments, the pharmacodynamic effect includes one or more of the following: immunogenic effects, biomarker responses, therapeutic effects, prophylactic effects, desired effects, undesired effects, side effects, and effects in disease models.

[0036] In some embodiments, the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions as described herein can be incorporated into a therapeutic agent for use in a method of treating a subject having, suspected of having, or at risk of having one or more related health conditions or diseases. Accordingly, in another aspect, the present disclosure provides a method for preventing or treating a health condition of a subject, the method comprising prophylactically or therapeutically administering to the subject a composition comprising one or more of the following: (a) a replicon as described herein, such as a self-replicating RNA (srRNA) construct; (b) a nucleic acid as described herein; (c) a recombinant cell as described herein; and (d) a pharmaceutical composition as described herein. In some embodiments, the administered composition elicits an immune response in the subject. In some embodiments, the administered composition induces the production of one or more pro-inflammatory molecules in the subject. In some embodiments, the one or more pro-inflammatory molecules include interferon gamma (IFNγ), cytokines, TNF-α, GM-CSF, and MIP1α, granzyme B, granzyme A, perforin, or any combination thereof. In some embodiments, the subject has been previously treated with one or more therapies and has developed at least partial resistance to the one or more therapies.

[0037] Exemplary health conditions or diseases can include, but are not limited to, cancer, immune diseases, autoimmune diseases, inflammatory diseases, gene therapy, gene replacement, cardiovascular diseases, age-related conditions, rare diseases, acute infections, and chronic infections. In some embodiments, the subject is a patient under the care of a physician.

[0038] Examples of autoimmune diseases suitable for the methods of the present disclosure include, but are not limited to, rheumatoid arthritis, osteoarthritis, Still's disease, familial Mediterranean fever, systemic sclerosis, multiple sclerosis, ankylosing spondylitis, systemic lupus erythematosus, Sjögren's syndrome, diabetic retinopathy, diabetic angiopathy, diabetic neuropathy, insulitis, psoriasis, alopecia areata, warm and cold autoimmune hemolytic anemia (AIHA), pernicious anemia, acute inflammatory diseases, autoimmune adrenitis, chronic inflammatory demyelinating polyneuropathy (CIDP), Lambert-Eaton syndrome, lichen sclerosus, Lyme disease, Graves' disease, Behçet's disease, Ménière's disease, reactive arthritis (Reiter's syndrome), Churg-Strauss syndrome, Cogan syndrome, CREST syndrome, pemphigus vulgaris and pemphigus foliaceus, bullous pemphigoid, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, pancreatitis, peritonitis, psoriatic arthritis, rheumatic fever, sarcoidosis, Sjogren syndrome( syndrome), scleroderma, celiac disease, stiff-person syndrome, Takayasu arteritis, transient gluten intolerance, autoimmune uveitis, vitiligo, polychondritis, dermatitis herpetiformis (DH) or Duhring's disease, fibromyalgia, Goodpasture syndrome, Guillain-Barré syndrome, Hashimoto's thyroiditis, autoimmune hepatitis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, myasthenia gravis, immune complex disorders, glomerulonephritis, polyarteritis nodosa, antiphospholipid syndrome, polyglandular autoimmune syndrome, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), urticaria, autoimmune infertility, juvenile rheumatoid arthritis, sarcoidosis, and autoimmune cardiomyopathy.

[0039] Non-limiting examples of infections for the methods applicable to the present disclosure include infections by viruses such as: human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus (CMV), respiratory syncytial virus (RSV), human papillomavirus (HPV), Epstein-Barr virus (EBV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome (MERS), influenza virus, and Ebola virus. Additional infections for the methods applicable to the present disclosure include infections by intracellular parasites such as: Leishmania, Rickettsia, Chlamydia, Coxiella, Plasmodium, Brucella, Mycobacterium, Listeria, Toxoplasma, and Trypanosoma. In some embodiments, replicon constructs, srRNA constructs, nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions can be used to treat and / or prevent immune diseases, autoimmune diseases, or inflammatory diseases such as glomerulonephritis, inflammatory bowel disease, nephritis, peritonitis, psoriatic arthritis, osteoarthritis, Still's disease, familial Mediterranean fever, systemic scleroderma and sclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, acute lung injury, meningitis, encephalitis, uveitis, multiple myeloma, glomerulonephritis, nephritis, asthma, atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud's syndrome, Sjögren's syndrome, juvenile diabetes, Reiter's disease, 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, Omenn's syndrome, chronic renal failure, autoimmune thyroid disease, acute infectious mononucleosis, HIV, herpes virus-related diseases, human viral infections, coronaviruses, other enteroviruses, herpes viruses, influenza virus, parainfluenza virus, respiratory syncytial virus, or adenovirus infections, bacterial pneumonia, trauma, sepsis, stroke / brain edema, ischemia-reperfusion injury, and hepatitis C.

[0040] Non-limiting examples of inflammatory diseases for the methods applicable to the present disclosure include inflammatory diseases such as asthma, inflammatory bowel disease (IBD), chronic colitis, splenomegaly, and rheumatoid arthritis.

[0041] In some embodiments, the health condition is a proliferative disorder or a microbial infection (e.g., a bacterial infection, a microfungal infection, or a viral infection). In some embodiments, the subject has or is suspected of having a condition associated with a proliferative disorder or a microbial infection (e.g., a bacterial infection, a microfungal infection, or a viral infection).

[0042] In some embodiments, the health condition is a rare disease (e.g., a disease or disorder affecting fewer than 200,000 people in the United States) and / or an inflammatory disorder and / or an autoimmune disorder as defined under the Orphan Drug 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 disorder and / or an autoimmune disorder and / or a rare disease (e.g., including but not limited to familial Mediterranean fever or adult-onset Still's disease).

[0043] In some embodiments, the disclosed compositions are formulated to be compatible with their intended route of administration. For example, the nucleic acid constructs (e.g., replicon constructs, e.g., srRNA constructs), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure can be administered orally or by inhalation, but more likely they will be administered by a parenteral route. Examples of parenteral routes of administration include, for example, intramuscular, intratumoral, intraocular, intravenous, intranodal, intradermal, subcutaneous, transdermal (topical), transmucosal, intravaginal, and rectal administration. In some embodiments, the composition is administered intramuscularly. In some embodiments, the composition is administered intratumorally. Solutions or suspensions for parenteral application can contain the following components: sterile diluents, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetate, citrate, phosphate, tris, sucrose; and agents for adjusting tonicity, such as sodium chloride or dextrose. The pH can be adjusted (e.g., adjusted to a pH of about 7.2 - 7.8, e.g., 7.5) with an acid or a base (such as sodium dihydrogen phosphate and / or disodium hydrogen phosphate, hydrochloric acid, or sodium hydroxide). Parenteral preparations can be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0044] It can be determined in cell cultures or experimental animals, for example, for determining LD 50 (the dose lethal to 50% of the population) and ED 50(The dose effective for treatment in 50% of the population) of standard pharmaceutical procedures to determine the dose, toxicity, and therapeutic efficacy of such subject nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure. The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD 50 / ED 50 . Compounds that exhibit high therapeutic indices are generally suitable. Although compounds that exhibit toxic side effects can be used, delivery systems that target such compounds to the site of the affected tissue should be carefully designed to minimize potential damage to uninfected cells and thereby reduce side effects.

[0045] For example, data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of such compounds is generally within a circulating concentration range that includes ED 50 with little or no toxicity. The dosage can vary within this range depending on the dosage form employed and the route of administration utilized. For any compound used in the methods of the present disclosure, the therapeutically effective dose can initially be estimated from cell culture assays. Dosages can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (e.g., the concentration of the test compound that achieves half-maximal inhibition of the symptoms) as determined in cell culture. Such information can be used to more accurately determine the useful dose in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0046] The therapeutic compositions described herein, such as nucleic acid constructs (e.g., srRNA constructs), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions, can be administered once or multiple times per day to once or multiple times per week (including once every other day). Those of ordinary skill in the art will understand that certain factors may affect the dosage and schedule required to effectively treat a subject, including but not limited to the severity of the disease, previous treatments, the general health and / or age of the subject, and other diseases present. In addition, treating a subject with a therapeutically effective amount of the multivalent polypeptides and multivalent antibodies of the present disclosure can include a single treatment or can include a series of treatments. In some embodiments, the composition is administered every 8 hours for five days, followed by a rest period of 2 to 14 days (e.g., 9 days), followed by another five days of administration every 8 hours. With respect to nucleic acid constructs (e.g., replicon constructs, such as srRNA constructs) and recombinant polypeptides, the therapeutically effective amount (e.g., effective dose) of the nucleic acid construct or recombinant polypeptide of the present disclosure depends on the nucleic acid construct or recombinant polypeptide selected. For example, a single dose amount in the range of about 0.001 to 0.1 mg / kg of patient body weight can be administered. In some embodiments, about 0.005, 0.01, 0.05 mg / kg can be administered. In some embodiments, a single dose amount in the range of about 0.03 μg to 300 μg / kg of patient body weight can be administered. In some embodiments, a single dose amount in the range of about 0.3 mg to 3 mg / kg of patient body weight can be administered.

[0047] As discussed above, a therapeutically effective amount includes an amount of a therapeutic composition that is sufficient to effect a particular result when administered to a subject, such as a subject having, suspected of having, or at risk of developing a health condition (e.g., a disease or an infection). In some embodiments, the effective amount includes an amount sufficient to prevent or delay the development of symptoms of a disease or infection, alter the course of symptoms of a disease or infection (e.g., but not limited to slowing the progression of symptoms of a disease or infection), or reverse the symptoms of a disease or infection. It should be understood that for any given case, a person of ordinary skill in the art can determine the appropriate effective amount using routine experimentation.

[0048] The efficacy of a treatment comprising the disclosed therapeutic composition for treating 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 failure of the individual to deteriorate as evaluated by, for example, hospitalization or need for medical intervention (e.g., halt or at least slowdown of disease or infection progression). Methods of measuring these metrics are known to those of skill in the art and / or are described herein. Treatment includes any treatment of a disease or infection in a subject or animal (some non-limiting examples include humans or mammals) and includes: (1) inhibiting the disease or infection, e.g., halting or slowing the progression of symptoms; or (2) alleviating the disease or infection, e.g., causing symptoms to resolve; and (3) preventing the development of symptoms or reducing their likelihood.

[0049] In some embodiments, the nucleic acid constructs (e.g., replicon constructs, e.g., srRNA constructs), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure can be administered to a subject in a composition having a pharmaceutically acceptable carrier and in an amount effective to stimulate an immune response. Generally, a subject can be immunized by an initial series of injections (or administered by one of the other routes described below), followed by a booster to increase the protection provided by the initial series of administrations. The initial series of injections and subsequent boosters are administered in a dose necessary to stimulate the subject's immune response and for a period of time necessary to stimulate the subject's immune response. In some embodiments, the administered composition results in an increase in the production of interferon in the subject. In some embodiments of the disclosed methods, the subject is a mammal. In some embodiments, the mammal is a human subject.

[0050] As described above, pharmaceutically acceptable carriers suitable for injection include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In these cases, the composition must be sterile and must be fluid to the extent that easy injection is possible. The composition must also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. For example, the proper fluidity can be maintained by using a coating, such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.).

[0051] A sterile injectable solution can be prepared by incorporating the nucleic acid construct, recombinant cell, and / or recombinant polypeptide in a suitable solvent, which may or may not have one or a combination of the ingredients enumerated above, in the required amounts and then filter sterilizing.

[0052] When the nucleic acid construct, recombinant cell, recombinant polypeptide, and / or pharmaceutical composition are appropriately protected, as described above, they can be administered orally, for example, with an inert diluent or an assimilable edible carrier. The nucleic acid construct, recombinant cell, recombinant polypeptide, and / or pharmaceutical composition and other ingredients can also be encapsulated in hard or soft shell gelatin capsules, compressed into tablets, or directly incorporated into the diet of an individual. For oral therapeutic administration, the active compound can be combined with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc.

[0053] In some embodiments, the nucleic acid constructs and recombinant polypeptides of the present disclosure can be delivered to cells or a subject via lipid-based nanoparticles (LNPs). The immunogenicity of LNPs is generally lower than that of viral particles. While many people have pre-existing immunity to viral particles, they do not have pre-existing immunity to LNPs. In addition, an adaptive immune response against LNPs is unlikely to occur, which enables repeated dosing of LNPs.

[0054] Several different ionizable cationic lipids have been developed for use in LNPs. These include, in particular, 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 serves as a ligand for uptake into the liver via the asialoglycoprotein receptor. Any of these cationic lipids can be used to formulate LNPs to deliver the nucleic acid constructs and recombinant polypeptides of the present disclosure to the liver.

[0055] In some embodiments, an LNP refers to any particle having a diameter less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. Alternatively, the size of the nanoparticles can range from 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

[0056] LNPs can be made from cationic, anionic, or neutral lipids. Neutral lipids such as fusogenic phospholipids DOPE or membrane component cholesterol can be included in LNPs as "helper lipids" to enhance transfection activity and nanoparticle stability. Limitations of cationic lipids include low efficacy due to 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 lipids that are both hydrophobic and hydrophilic.

[0057] Many lipids or combinations of lipids known in the art can be used to produce LNPs. Non-limiting examples of lipids suitable for producing LNPs include DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Non-limiting examples of cationic lipids include 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Non-limiting examples of neutral lipids include DPSC, DPPC, POPC, DOPE, and SM. Non-limiting examples of PEG-modified lipids include PEG-DMG, PEG-CerC14, and PEG-CerC20.

[0058] In some embodiments, the lipids can be combined in any number of molar ratios to produce LNPs. Additionally, one or more polynucleotides can be combined with one or more lipids in a wide range of molar ratios to produce LNPs.

[0059] In some embodiments, the therapeutic compositions described herein (e.g., nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions) are incorporated into a therapeutic composition for use in methods of preventing or treating a subject having, suspected of having, or at risk of having cancer, an autoimmune disease, and / or an infection.

[0060] In some embodiments, the therapeutic compositions described herein (e.g., nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions) are incorporated into a therapeutic composition for use in methods of preventing or treating a subject having, suspected of having, or at risk of having a microbial infection. In some embodiments, the microbial infection is a bacterial infection. In some embodiments, the microbial infection is a fungal infection. In some embodiments, the microbial infection is a viral infection. Additional therapies

[0061] In some embodiments, the compositions according to the present disclosure are administered to a subject either alone as a single therapy (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 chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery. In some embodiments, the second therapy is selected from chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, or surgery. In some embodiments, the first therapy and the second therapy are administered concomitantly. In some embodiments, the first therapy 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 therapy and the second therapy are administered in alternation. In some embodiments, the first therapy and the second therapy are administered together in a single formulation. Kits

[0062] The present disclosure also provides various kits for practicing the methods described herein, as well as written instructions for the preparation and use of the kits. Specifically, some embodiments of the present disclosure provide kits for modulating a pharmacodynamic effect. Some embodiments of the present disclosure provide kits for eliciting an immune response in a subject. Some other embodiments relate to kits for preventing a health condition in a subject in need thereof. Some other embodiments relate to kits for methods of treating a health condition in a subject in need thereof. For example, in some embodiments, the present disclosure provides kits that comprise one or more of the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions provided and described herein, as well as written instructions for the preparation and use thereof.

[0063] In some embodiments, the kits of the present disclosure further comprise one or more means for administering any of the provided nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions to a subject. For example, in some embodiments, the kits of the present disclosure further comprise one or more syringes (including drug-loaded syringes) and / or catheters (including drug-loaded syringes) for administering any of the provided nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions to a subject. In some embodiments, the kit can have one or more additional therapeutic agents that can be administered concomitantly or sequentially with the other kit components for a desired purpose, such as for diagnosing, preventing, or treating a disorder in a subject in need thereof.

[0064] Any of the kits described above can further comprise one or more additional reagents, where such additional reagents can be selected from: dilution buffers, reconstitution solutions, wash buffers, control reagents, control expression vectors, negative controls, positive controls, reagents suitable for in vitro generation of the provided nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure.

[0065] In some embodiments, the components of the kit can be in separate containers. In some other embodiments, the components of the kit can be combined in a single container. Thus, in some embodiments of the present disclosure, the kit comprises one or more of the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions provided and described herein in one container (e.g., in a sterile glass or plastic vial) and another therapeutic agent in another container (e.g., in a sterile glass or plastic vial).

[0066] In another embodiment, the kit comprises a combination of the compositions described herein (comprising one or more nucleic acid constructs, recombinant cells, and / or recombinant polypeptides of the present disclosure), optionally formulated together in the form of a pharmaceutical composition, in combination with one or more other therapeutic agents, in a single common container.

[0067] If the kit comprises a pharmaceutical composition for parenteral administration to a subject, the kit can comprise a device for performing such administration (e.g., an injection device or a catheter). For example, the kit can comprise one or more subcutaneous injection needles or other injection devices as discussed above containing one or more of the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure.

[0068] In some embodiments, the components of the kit can be in separate containers. In some other embodiments, the components of the kit can be combined in a single container.

[0069] In some embodiments, the kit can further comprise instructions for practicing the methods disclosed herein using the components of the kit. For example, the kit can comprise a package insert containing information about the pharmaceutical composition and dosage form in the kit. Generally, such information helps patients and physicians use the encapsulated pharmaceutical composition and dosage form effectively and safely. For example, the following information about the combinations of the present disclosure can be provided in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdose, correct dosage and administration, supply specifications, correct storage conditions, references, manufacturer / dealer information, and intellectual property information.

[0070] Instructions for practicing the described method are typically recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. The instructions can be present as a package insert in a kit, can be present in the label of the container of the kit or its components (e.g., associated with the packaging or sub-packaging), etc. The instructions can be present as an electronic storage data file on a suitable computer-readable storage medium (e.g., CD-ROM, floppy disk, flash drive, etc.). In some cases, the actual instructions are not present in the kit, but means can be provided for obtaining the instructions from a remote source (e.g., via the Internet). An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or downloaded. Like the instructions, such means for obtaining the instructions can be recorded on a suitable substrate.

[0071] All publications and patent applications mentioned in this disclosure are hereby incorporated by reference herein to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0072] No admission is made that any reference cited herein constitutes prior art. The discussion of references states the claims of their authors, and the applicant reserves the right to challenge the accuracy and relevance of the cited documents. It will be clearly understood that although many sources of information are mentioned herein, including scientific journal articles, patent documents, and textbooks; such mention does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0073] The discussion of the general methods given herein is for illustrative purposes only. After reviewing this disclosure, other alternative methods and alternatives will be apparent to those skilled in the art and will be included within the spirit and scope of this application.

[0074] Additional embodiments are further disclosed in the following examples, which are provided by way of illustration only and are not intended to limit the scope of this disclosure or the claims in any way. Examples

[0075] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology that are well known to those of skill in the art. Such techniques are fully explained in the following references, such as Sambrook, J. and Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J. and Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (collectively referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferré, F. and Gibbs, R. (1994). PCR: The Polymerase Chain Reaction.The disclosures of Boston: Birkhauser Publisher; Greenfield, E.A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S.L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S.C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V. are hereby incorporated by reference in their entireties.

[0076] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration only and are not intended to limit the scope of the present disclosure or claims in any way. Example 1 Construction of the basic MADV vector

[0077] This example describes experiments conducted to construct a basic MADV vector (e.g., without a heterologous gene), which was subsequently used to construct a MADV vector expressing a gene of interest (e.g., the hemagglutinin precursor HA of influenza A virus H5N1).

[0078] The basic MADV vector (i.e., without a heterologous gene of interest) was constructed as follows: The basic MADV vector was de novo synthesized in four approximately 4 kb segments from the reference sequence of the MADV strain BeAr300851 having several modifications (Genbank accession number KJ469641) (see, for example, Figure 2A)。Ambiguous nucleotide assignments in the reference sequences were assigned to nucleotides that matched the synonymous codons of the encoded residues. Silent mutations were incorporated to eliminate SapI and SpeI restriction endonuclease cleavage sites. A unique restriction endonuclease cleavage site (SpeI, 5’-A’CTAG,T-3’) could be incorporated in place of the coding sequence of the native MADV structural gene (where 5’A matched the position of the ATG start codon of the structural polyprotein and 3’T matched the position of the TAA stop codon of the structural polyprotein). A 5’ adapter sequence (5’-CTGGAGACGTGGAGGAGAACCCTGGACCT-3’; SEQ ID NO:3) was inserted upstream of the SpeI site and a 3’ adapter sequence (5’-GACCGCTACGCCCCAATGACCCGACCAGC-3’; SEQ ID NO:4) was inserted downstream of the SpeI site for subsequent Gibson assembly (Gibson et al., Nat. Methods 6, 343 - 345, 2009). The bacteriophage T7 RNA polymerase promoter (5’-TAATACGACTCACTATAG-3’; SEQ ID NO:5) was included upstream of the MADV genomic sequence and could contain a poly(A) sequence downstream, followed by a SapI site that cuts upstream of the recognition site. Immediately downstream of the SapI site was the T7 terminator sequence (5’-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3’; SEQ ID NO:6), followed by a unique restriction endonuclease cleavage site (NotI, 5’-GC’GGCC,GC-3’). These parts were combined in a five-piece Gibson reaction (linearized pYL backbone and four synthetic fragments) to generate the MADV backbone vector.

[0079] In some experiments, the sequences encoding one or more nsPs were replaced with heterologous nsPs. In some other experiments, the sequences encoding one or more UTRs were replaced with heterologous UTRs.

[0080] Construction of the MADV vector containing a heterologous gene was performed as follows: The MADV vector described in Figure 2A was constructed by linearizing the empty MADV vector in Figure 2B via SpeI digestion. The hemagglutinin (HA) gene from influenza virus (Genbank AY651334) was codon-optimized / refactored for in silico human expression and de novo synthesized (IDT). The synthetic product was PCR amplified using primer pairs that added 5' and 3' adapter sequences to the ends of the HA gene. The digested product and the PCR product were combined by Gibson A program combination to generate the final vector. Example 2 Construction of the MADV vector containing a heterologous gene

[0081] The construction of the MADV vector containing one or more heterologous genes was carried out as follows: The empty basic vector as shown was linearized by SpeI digestion. Two transgene cassettes were codon-optimized / refactored by computer simulation for human expression and de novo synthesized with the above 5' and 3' linker sequences. The digested product and the synthesized product were combined by the Gibson Figure 2A program combination to generate the final vector. A first synthetic transgene cassette encoding the hemagglutinin (HA) gene from influenza (based on Genbank ATI21640) was synthesized and used to generate the template plasmid for the Rep-631 MADV srRNA described in

[0082] Figures 5A - 5D A second synthetic transgene cassette encoding the following was synthesized: murine interleukin 1 receptor antagonist (IL-1RA) (Genbank AAH18332), followed by an IRES, followed by murine interleukin 18 binding protein (IL-18BP) (GenPept NP_001034790) to generate the template plasmid for the Rep-657 MADV srRNA described in Figures 5A - 5D

[0083] Figures 6A - 6B Figures 6A - 6B Example 3 In vitro evaluation of the modified MADV vector

[0084]

[0084] This example describes in vitro experiments carried out to evaluate the expression levels of the synthetic MADV srRNA constructs described in Example 1 above and to study any differential behavior thereof (e.g., replication and protein expression).

[0085] In these experiments, synthetic srRNA constructs derived from the MADV strain BeAr300851 were designed and subsequently evaluated.

[0086] In vitro transcription : Using the 5' ARCA cap (HiScribe TM T7 ARCA mRNA Kit, NEB) or by capless transcription (HiScribe TMT7 High Yield RNA Synthesis Kit, NEB), followed by addition of 5’ Cap 1 (Vaccinia Virus Capping System, mRNA Cap 2′-O-Methyltransferase, NEB), and preparation of RNA by in vitro transcription using phage T7 polymerase from a SapI-linearized plasmid template. Then RNA was purified using phenol / chloroform extraction or column purification ( RNA Cleanup Kit, NEB). RNA concentration was determined by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific).

[0087] Replication : RNA was transfected into BHK-21 or Vero cells by electroporation (e.g., 4D-Nucleofector TM , Lonza). At 15 - 20 hours post-transfection, cells were fixed and permeabilized (eBioscience TM Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen), followed by staining with a PE-conjugated mouse monoclonal antibody against dsRNA (J2, Scicons) to quantify the frequency of dsRNA+ cells and the mean fluorescence intensity (MFI) of dsRNA in individual cells by fluorescence flow cytometry.

[0088] Protein expression : RNA was transfected into BHK-21 or Vero cells by electroporation (e.g., 4D-Nucleofector TM , Lonza). At 15 - 20 hours post-transfection, cells were fixed and permeabilized (eBioscience TM Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen), and stained with a DyLight650-conjugated mouse monoclonal antibody against HA (C102, Novus Bio; catalog number NB100-65047C) to quantify the frequency of cells expressing HA protein and the mean fluorescence intensity (MFI) of HA protein in individual cells by fluorescence flow cytometry.

[0089] In some experiments, BHK-21 cells were transfected with the MADV srRNA construct. MADV srRNA without the target GOI was transfected by electroporation, and 20 hours post-transfection, the transfected cells were fixed, permeabilized, and co-stained with two antibodies: a PE-conjugated mouse monoclonal anti-dsRNA antibody (J2, Scicons) to identify the frequency of dsRNA+ cells, and a DyLight650-conjugated mouse monoclonal anti-HA antibody (C102, Novus Bio; catalog number NB100-65047C) to identify the frequency of HA+ cells and determine the expression level of the HA transgene by fluorescence flow cytometry. Positive staining of individual cells with both the anti-dsRNA and anti-GOI antibodies demonstrated that the modified MADV design described herein is a viable synthetic srRNA and is capable of undergoing RNA replication and expressing the transgene.

[0090] Additional experiments: Prior to RNA electroporation, BHK-21 or Vero cells were pretreated with a titration curve of recombinant interferon (IFN), and the assays described above were used to measure the effects on vector replication and protein expression.

[0091] In some experiments, BHK-21 cells were transfected with the MADV srRNA construct. MADV srRNA without the target GOI was transfected by electroporation, and 20 hours post-transfection, the transfected cells were fixed, permeabilized, and co-stained with two antibodies: a PE-conjugated mouse monoclonal anti-dsRNA antibody (J2) to identify the frequency of dsRNA+ cells, and a DyLight650-conjugated mouse monoclonal anti-HA antibody (C102, Novus Bio; catalog number NB100-65047C) to identify the frequency of HA+ cells and determine the expression level of the HA transgene by fluorescence flow cytometry. As Figure 4 shown, positive staining of individual cells with both the anti-dsRNA and anti-GOI antibodies demonstrated that the modified MADV design described herein is a viable synthetic srRNA and is capable of undergoing RNA replication and expressing the transgene. Example 4 In vitro evaluation of the modified MADV vector

[0092] This example describes in vitro experiments conducted to evaluate the relative GOI expression of the synthetic MADV srRNA constructs described in Examples 1 and 2 above and other srRNA constructs expressing the same GOI.

[0093] Figure 4This is a graph showing the average expression of HA in cells transfected with the srRNA construct for display. BHK-21 cells were transfected by electroporation (Lonza 4D-Nucleofector) with 200 ng of an srRNA vector encoding influenza H1N1 HA, and the cells were fixed, permeabilized, and stained 24 hours later with a mouse monoclonal antibody conjugated to DyLight650 that binds to H1N1 HA (Novus Bio; catalog number NB100-65047C). Cells were analyzed by fluorescence flow cytometry to identify the fluorescence intensity of the cells, which corresponds to the HA expression level. The average HA expression in cells transfected with MADV-HA was similar to or higher than that of other srRNA vectors encoding the same transgene. VEEV: Venezuelan equine encephalitis virus, CHIKV-S27: Chikungunya virus strain S27, CHIKV-DRDE: Chikungunya virus strain DRDE-06, SINV-G: Sindbis virus strain Girdwood, SINV-AR86: Sindbis virus strain AR86, WEEV: Western equine encephalitis virus, MADV: Madariaga virus BeAr300851. Example 5 In vivo evaluation of the modified MADV vector

[0094] This example describes in vivo experiments conducted to evaluate the immune response after vaccination with the synthetic MADV srRNA constructs described in Examples 1 and 2 above (e.g., both unformulated vectors and LNP-formulated vectors).

[0095] In these experiments, synthetic srRNA constructs derived from the MADV strain BeAr300851 were designed and subsequently evaluated.

[0096] Mice and injectionFemale C57BL / 6 or BALB / c mice were purchased from Envigo, Charles River Labs, or Jackson Laboratories. On the day of dosing, between 0.1 - 10 μg of the material was injected intramuscularly into two quadriceps muscles. The vehicle was administered in a form not formulated in saline or in an LNP-formulated or polymer-formulated form. Throughout the study, the animals were monitored for body weight and other general observations. For immunogenicity studies, the animals were dosed on Day 0 and Day 21. Spleens were collected on Day 35, and sera were isolated on Day 14 and Day 35. For protein expression studies, the animals were dosed on Day 0, and protein expression or bioluminescence was evaluated on Day 1, Day 3, and / or Day 7. In some experiments where the MADVsrRNA construct encoded a reporter protein (such as luciferase), in vivo imaging of luciferase activity was performed using the IVIS system at the designated time points. In some experiments where the MADV srRNA construct encoded a secreted protein, the systemic levels were determined by serum ELISA.

[0097] LNP formulation In some experiments, the srRNA was formulated in lipid nanoparticles using a microfluidic mixer, and the particle size, polydispersity (using dynamic light scattering), and encapsulation efficiency were analyzed. In these experiments, a wide range of lipid molar ratios were used in formulating the LNP particles. Exemplary molar ratios of the lipids used in these experiments could be 35% C12 - 200, 46.5% cholesterol, 2.5% PEG-2K, and 16% DOPE.

[0098] ELISpot. To measure the strength of the HA-specific T cell response, IFNγ ELISpot assays were performed using the Mouse IFNγ ELISpot PLUS Kit (HRP) (MabTech) according to the manufacturer's instructions. In these experiments, splenocytes were isolated and resuspended in medium to an appropriate concentration (such as 5×10 6 cells / mL), and the medium contained peptides representing the T cell epitopes of the target protein encoded by the MADV srRNA construct. One or more positive controls (such as PMA / ionomycin) and DMSO used as mock stimulation were also included in these experiments.

[0099] Antibodies The antibody response for measuring total HA-specific IgG was measured using an ELISA kit from Alpha Diagnostic International according to the manufacturer's instructions. Example 6 In vivo evaluation of the modified MADV vaccine vector

[0100] This example describes in vivo experiments conducted to evaluate the immune response after vaccination with the synthetic MADV srRNA influenza constructs described in Examples 1 and 2 above.

[0101] In these experiments, synthetic srRNA constructs derived from the MADV strain BeAr300851 were designed and subsequently evaluated. As Figures 5A - 5D shown, the results of these experiments demonstrated that the MADV srRNA-based influenza vaccine against the viral antigen (influenza H1 hemagglutinin) could generate a neutralizing antibody response ( Figure 5A ) measured by hemagglutination inhibition (HAI) assay and an in vivo T cell response ( Figures 5B - 5D ) measured by ELISpot.

[0102] Mice and injection Female C57BL / 6 or BALB / c mice were purchased from Envigo, Charles River Labs, or Jackson Laboratories. On the dosing day, 0.01 μg of the LNP formulation was intramuscularly injected into the quadriceps muscle. Throughout the study, the animals were monitored for body weight and other comprehensive observations. The animals were dosed on Day 0 and Day 56. Spleens were collected on Day 70.

[0103] LNP formulation The srRNA was formulated in lipid nanoparticles using a microfluidic mixer, and the particle size, polydispersity (using dynamic light scattering), and encapsulation efficiency were analyzed. The lipid was suspended in ethanol. The RNA was suspended at a concentration of 82 μg / ml in 250 mM NaOAc (pH 4.0) and mixed at a flow rate of 3:1 (aqueous:organic).

[0104] Antibodies The standard hemagglutinin inhibition assay (HAI) was used to measure virus neutralization using the sera from immunized mice for the neutralizing antibody response.

[0105] The results of these experiments (shown in Figures 5A - 5D ) demonstrated that the modified MADV vaccine vector could be used to elicit an in vivo antigen-specific immune response. In these experiments, antibody and T cell responses were measured by (HAI) assay and IFNγ ELISpot 14 days after prime-boost with 0.01 μg of MADV-srRNA (Rep-631) LNP. Specifically, Figure 5A shows the serum HAI titers against H1N1. Figure 5B shows the results of splenocytes stimulated with the HA peptide library. Figure 5C shows the results from stimulation with CD4 T cell epitope-specific peptides. Figure 5DShows the results of CD8 T cell epitope-specific peptide stimulation. Mann-Whitney statistics between groups are shown (**<0.01; *<0.05). Example 7 In vivo evaluation of the modified MADV biotherapeutic vector

[0106] This example describes in vivo experiments conducted to evaluate biotherapeutic protein expression after administration of the synthetic MADV srRNA biotherapeutic constructs described in Examples 1 and 2 above.

[0107] In these experiments, synthetic srRNA constructs derived from the MADV strain BeAr300851 were designed and subsequently evaluated. An srRNA encoding an irrelevant transgene (luciferase) was also included in the experiments as a control.

[0108] Mice and injection Female C57BL / 6 or BALB / c mice were purchased from Envigo, Charles River Labs, or Jackson Laboratories. On the dosing day, 10 μg of the LNP formulation was intramuscularly injected into the quadriceps muscle. Throughout the study, the animals were monitored for body weight and other comprehensive observations. The animals were dosed on day 0, and sera were collected on day 7.

[0109] LNP formulation The srRNA was formulated in lipid nanoparticles using a microfluidic mixer, and the particle size, polydispersity (using dynamic light scattering), and encapsulation efficiency were analyzed. The lipids were suspended in ethanol. The RNA was suspended at a concentration of 82 μg / ml in 250 mM NaOAc (pH 4.0) and mixed at a flow rate of 3:1 (aqueous:organic).

[0110] Protein expression determined by ELISA : To measure the serum concentrations of IL-1RA and IL-18BP, ELISA assays were performed using a mouse IL-1RA ELISA kit (R&D Systems, catalog number MRA00) and a mouse IL-18BP ELISA kit (R&D Systems, catalog number DY122-05) according to the manufacturer's protocol.

[0111] The results of these experiments ( Figures 6A - 6B ) demonstrate that the modified MADV biotherapeutic vector can be used to express biotherapeutic proteins in vivo for at least 7 days. In these experiments, 7 days after administration of MADV-srRNA (Rep-657) co-encoding both genes, mouse IL-1RA ( Figure 6A ) and mouse IL-18BP (Figure 6B ) protein levels and compared them to srRNAs encoding an irrelevant protein red firefly luciferase for basal protein expression levels (“irrelevant control”). Mann-Whitney statistics between groups are shown (* < 0.05).

[0112] Although specific alternatives of the present disclosure have been disclosed, it should be understood that various modifications and combinations are possible and contemplated within the true spirit and scope of the appended claims. Accordingly, there is no intention to be limited to the exact abstract and disclosure presented herein.

Claims

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

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

3. The nucleic acid construct according to any one of claims 1-2, wherein the modified viral genome or srRNA does not contain a nucleic acid sequence encoding a viral structural protein.

4. The nucleic acid construct according to any one of claims 1-3, the nucleic acid construct further comprising one or more expression cassettes, wherein each of the expression cassettes comprises a promoter operably linked to a heterologous nucleic acid sequence.

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

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

7. The nucleic acid construct according to any one of claims 1-6, wherein at least one non-structural protein (nsP) or a portion thereof of the modified MADV genome or srRNA is heterologous relative to the remainder of the modified MADV genome or srRNA.

8. The nucleic acid construct according to any one of claims 1-7, the nucleic acid construct further comprising a nucleic acid sequence encoding a heterologous nsP or a portion thereof.

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

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

11. The nucleic acid construct according to any one of claims 4-10, wherein at least one of the expression cassettes comprises a coding sequence of a gene of interest (GOI).

12. The nucleic acid construct according to claim 11, wherein the GOI encodes a polypeptide selected from the group consisting of: therapeutic polypeptides, prophylactic polypeptides, diagnostic polypeptides, nutraceutical polypeptides, industrial enzymes, and reporter polypeptides.

13. The nucleic acid construct according to any one of claims 11-12, wherein the GOI encodes a polypeptide selected from the group consisting of: antibodies, antigens, immunomodulators, enzymes, signaling proteins, and cytokines.

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

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

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

1.

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

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

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

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

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

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

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

24. The recombinant cell according to claim 20, wherein the recombinant cell is selected from: monkey kidney CV1 cells transformed by SV40, human embryonic kidney cells (HEK), baby hamster kidney cells (BHK) or their derivative cells, mouse Sertoli cells, monkey kidney cells, human cervical cancer cells, dog kidney cells, buffalo rat hepatocytes, human lung cells, human hepatocytes, mouse mammary tumors, TRI cells, FS4 cells, Chinese hamster ovary cells (CHO), African green monkey kidney cells, human A549 cells, human cervical cells, human CHME5 cells, human PER.C6 cells, NS0 mouse myeloma cells, human epidermoid laryngeal cells, human fibroblasts, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocytes, human macrophages, human RAW 264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells and rabbit kidney cells.

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

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

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

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

29. The transgenic animal according to claim 27, wherein the animal is a mammalian.

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

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

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

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

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

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

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

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

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

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

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

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

42. The pharmaceutical composition according to any one of claims 38 - 41, wherein the composition is formulated in liposomes, lipid - based nanoparticles (LNP), polymer nanoparticles, polyplexes, virus replicon particles (VRP), microspheres, immunostimulating complexes (ISCOM), conjugates of bioactive ligands, or any combination thereof.

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

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

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

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

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

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

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

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

51. A method for preventing and / or treating a health condition in a subject in need thereof, the method comprising prophylactically or therapeutically administering to the subject a composition comprising: a) a nucleic acid construct according to any one of claims 1-16; b) a recombinant cell according to any one of claims 17-24; c) a recombinant polypeptide according to claim 37; and / or d) a pharmaceutical composition according to any one of claims 38-47, 52. The method according to claim 51, wherein the administered composition elicits an immune response in the subject.

53. The method according to any one of claims 51-52, wherein the condition is a proliferative disorder or a microbial infection.

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

55. The method according to any one of claims 51-54, wherein the administered composition results in an increase in the production of interferon in the subject.

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

57. The method according to claim 56, wherein the at least one additional therapy is selected from chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery.

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

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