Modified western equine encephalitis virus and uses thereof

Abstract

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

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 332,606, filed April 19, 2022. The disclosures of the above-referenced applications are expressly incorporated herein by reference in their entirety, including any drawings. Importing sequence listings

[0002] The attached sequence listing material is incorporated by reference into this application. The attached sequence listing XML file (2023-04-13 Sequence_Listing_ST26 058462-511001WO.xml) was created on April 13, 2023 and is 26,362 bytes in size. Field

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

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

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

[0006] However, it has been reported that host cells can develop complex and powerful mechanisms to detect and combat pathogen invasion. In addition, it has been reported that viruses, especially pathogenic viruses, have evolved along with host cells to counter the host cells' 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 viral production of progeny that can spread to other 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 provide benefits to the host cells, they can also adversely affect self-replicating RNAs (srRNAs) designed to express beneficial vaccine antigens or therapeutic substances. For example, when a cell detects srRNA expressing a beneficial protein and activates its innate immune defense mechanism, the expression of the beneficial protein in such cells may be affected, compromising the effectiveness of the srRNA. Summary of the Invention [Problem to be solved by the invention]

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

[0008] overview The present disclosure relates to the development of immunotherapeutic agents, such as recombinant nucleic acid constructs and pharmaceutical compositions comprising the same, for use in the prevention and management of various health conditions, including proliferative diseases and microbial infections. In particular, as described in more detail below, some embodiments of the present disclosure provide modified genomes or replicons, such as nucleic acid constructs comprising sequences encoding the self-replicating RNA (srRNA) of the Alphavirus Western Equine Encephalitis Virus (WEEV), lacking at least a portion of the viral nucleic acid sequences encoding one or more structural proteins of the virus. Also disclosed are recombinant cells and transgenic animals engineered to include one or more of the nucleic acid constructs disclosed herein, methods of producing a molecule of interest (e.g., a polypeptide of interest), and pharmaceutical compositions comprising one or more of the following: (a) a nucleic acid construct of the present disclosure, (b) a polypeptide of the present disclosure, or (c) a recombinant cell of the present disclosure. Additionally, in certain embodiments of the present disclosure, compositions and methods are provided for eliciting a pharmacodynamic effect in a subject in need thereof and / or for the prevention and / or treatment of various health conditions, including proliferative diseases (e.g., cancer) and infectious diseases.

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

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

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

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

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

[0014] In some embodiments, a nucleic acid construct of the present disclosure comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0015] In one embodiment, provided herein is a recombinant cell comprising a nucleic acid construct disclosed herein. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a vertebrate cell or an invertebrate cell. In some embodiments, the recombinant cell is an insect cell. In some embodiments, the recombinant cell is a mosquito cell. In some embodiments, the recombinant cell is a mammalian cell. In some embodiments, the recombinant cell is a monkey kidney CV1 cell transformed by SV40 (COS-7), human embryonic kidney cell (e.g., HEK293 or HEK293 cell), baby hamster kidney cell (BHK), mouse Sertoli cell (e.g., TM4 cell), monkey kidney cell (CV1), human cervical carcinoma cell (HeLa), canine kidney cell (MDCK), buffalo rat hepatocyte (BRL3A), human lung cell (W138), human hepatocyte (HepG2), mouse mammary tumor (MMT060562), TRI cell, FS4 cell, The recombinant cell is selected from the group consisting of Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (Vero cells), human A549 cells, human cervical cells, human CHME5 cells, human PER.C6 cells, NS0 mouse myeloma cells, human epithelial laryngeal cells, human fibroblast cells, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocytes, human macrophage cells, human RAW264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells. Also provided in related aspects is a cell culture comprising at least one recombinant cell as disclosed herein and a culture medium.

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

[0017] In another aspect, provided herein is a method of producing a polypeptide of interest in a subject, the method comprising administering to the subject a nucleic acid construct disclosed herein. In some embodiments, the subject is a vertebrate or an invertebrate. In some embodiments, the subject is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the subject is a mammalian subject. In some embodiments, the mammalian subject is a human subject. In yet another aspect, provided herein is a recombinant polypeptide produced by the method of the present disclosure.

[0018] In yet another aspect, provided herein is a pharmaceutical composition comprising a pharma- ceutically acceptable excipient and a) a nucleic acid construct of the present disclosure, b) a recombinant cell of the present disclosure, and / or c) a recombinant polypeptide of the present disclosure.

[0019] Non-limiting exemplary embodiments of the pharmaceutical composition of the present disclosure may include one or more of the following features. In some embodiments, provided herein is a composition comprising a nucleic acid construct disclosed herein and a pharma- ceutically acceptable excipient. In some embodiments, provided herein is a composition comprising a recombinant cell disclosed herein and a pharma- ceutically acceptable excipient. In some embodiments, a composition comprises a recombinant polypeptide disclosed herein and a pharma- ceutically acceptable excipient. In some embodiments, provided herein is a composition formulated in a liposome, a lipid-based nanoparticle (LNP), a polymeric nanoparticle, a polyplex, a viral replicon particle (VRP), a microsphere, an immune stimulating complex (ISCOM), a conjugate of a bioactive ligand, or any combination thereof. In some embodiments, the composition is an immunogenic composition. In some embodiments, the immunogenic composition is formulated as a vaccine. In some embodiments, the immunogenic composition is substantially non-immunogenic to a subject. In some embodiments, the pharmaceutical composition is formulated as an adjuvant. In some embodiments, the pharmaceutical composition is formulated for one or more of intranasal, intrathecal, transdermal, intraperitoneal, intramuscular, intratracheal, intranodal, intratumoral, intraarticular, intravenous, subcutaneous, intravaginal, intraocular, rectal, and oral administration.

[0020] In another aspect, provided herein is a method of inducing a pharmacodynamic effect in a subject in need thereof, comprising administering to the subject a composition comprising: a) a nucleic acid construct of the present disclosure; b) a recombinant cell of the present disclosure; c) a recombinant polypeptide of the present disclosure; and / or d) a pharmaceutical composition of the present disclosure. In some embodiments, the pharmacodynamic effect comprises one or more of the following: immunogenic effect, biomarker response, therapeutic effect, prophylactic effect, desired effect, undesirable effect, adverse effect, and effect in a disease model. In some embodiments, the pharmacodynamic effect comprises eliciting an immune response in the subject.

[0021] In yet another aspect, provided herein is a method for preventing and / or treating a condition in a subject in need thereof, comprising prophylactically or therapeutically administering to the subject a) a nucleic acid construct of the present disclosure, b) a recombinant cell of the present disclosure, c) a recombinant polypeptide of the present disclosure, and / or d) a composition comprising any one of the pharmaceutical compositions of the present disclosure. In some embodiments, the administered composition induces a pharmacodynamic effect. In some embodiments, the pharmacodynamic effect comprises eliciting an immune response in the subject.

[0022] Non-limiting exemplary embodiments of the disclosed methods may include one or more of the following features. In some embodiments, the condition is a proliferative disorder or a microbial infection. In some embodiments, the subject has or is suspected of having a condition associated with a proliferative disorder or a microbial infection. In some embodiments, the administered composition results in increased interferon production in the subject. In some embodiments, the composition is administered to the subject individually as a single therapy (monotherapy) or as a first therapy in combination with at least one additional therapy. In some embodiments, the at least one additional therapy is selected from the group consisting of chemotherapy, radiation therapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery.

[0023] In yet another aspect, provided herein is a kit for inducing a pharmacodynamic response, for inducing an immune response, and / or for the prevention and / or treatment of a condition or a microbial infection, the kit comprising a) a nucleic acid construct of the present disclosure, b) a recombinant cell of the present disclosure, c) a recombinant polypeptide of the present disclosure, and / or d) a pharmaceutical composition of the present disclosure.

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

[0025] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the exemplary embodiments and features described herein, further aspects, embodiments, objects, and features of the present disclosure will become more fully apparent from the drawings, detailed description, and claims. [Brief description of the drawings]

[0026] [Figure 1] Figure 1 is a graphical representation of a non-limiting example of a modified WEEV 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 WEEV design depicted in this figure contains the native 5'UTR and 3'UTR from WEEV strain Imperial, and further contains a heterologous gene of interest (GOI) placed under the control of the 26S subgenomic promoter. The coding sequences for the nonstructural proteins nsP1, nsP2, nsP3, and nsP4 are shown.

[0027] [Figure 2A-2B] 2A-2B are graphical representations of non-limiting exemplary WEEV srRNA designs according to some embodiments of the present disclosure, in which sequences encoding a modified WEEV genome from the Imperial strain have been incorporated into a plasmid DNA vector (FIG. 2A), which also includes the coding sequence for an exemplary gene of interest (GOI), such as the hemagglutinin precursor (HA) of influenza A virus H5N1 (FIG. 2B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Detailed Description of the Disclosure Provided herein are, inter alia, viral expression systems with superior expression capabilities suitable for expressing heterologous molecules, such as vaccines and therapeutic polypeptides, in recombinant cells. For example, some embodiments of the present disclosure relate to nucleic acid constructs, such as expression constructs and vectors, including modified genomes or replicons, such as srRNA of Western Equine Encephalitis Virus (WEEV), from which at least a portion of the original viral sequence encoding structural proteins has been deleted. Also provided in some embodiments of the present disclosure are viral-based expression vectors including one or more expression cassettes encoding heterologous polypeptides. Additionally, provided are recombinant cells genetically engineered to include one or more nucleic acid molecules disclosed herein. Biomaterials and recombinant products obtained from such recombinant cells are also within the scope of the present application. Also provided are compositions and methods useful for inducing a pharmacodynamic effect in a subject in need thereof, as well as methods for preventing and / or treating various health conditions.

[0029] Self-replicating RNA (srRNA) based on RNA viruses (e.g., alphaviruses) can be used as robust expression systems. For example, it has been reported that an advantage of using alphaviruses such as WEEV as viral expression vectors is that they can induce the synthesis of large amounts of heterologous proteins in recombinant host cells. Among other advantages, polypeptides such as therapeutic single-chain antibodies may be most effective when expressed at high levels in vivo. Furthermore, when producing purified recombinant antibodies from cultured cells (ex vivo), high protein expression from srRNA can increase the overall yield of antibody product. Furthermore, when the expressed protein is a vaccine antigen, high levels of expression can induce the most robust pharmacodynamic effects in vivo.

[0030] Alphaviruses utilize motifs contained in UTRs, structural regions, and nonstructural regions to affect their replication within host cells. These regions also contain mechanisms to evade the innate immunity of the host cell. However, significant differences have been reported between alphavirus species. For example, New World and Old World alphaviruses have evolved different components to assemble viral replication complexes utilizing intracellular stress granules, JAK-STAT signaling, FXR, and G3BP proteins. Which parts of the genome contain these components differs between alphaviruses. For example, avoiding PKR activation and subsequent phosphorylation of EIF2alpha is achieved through a downstream loop in some Old World alphaviruses, such as Sindbis, whereas avoiding this pathway is thought to be achieved through nsP4 in Chikungunya, which lacks a recognizable DLP. Furthermore, beyond the variation between individual alphaviruses, differences are also common within alphavirus strains that may account for changes in properties such as virulence. As one example, sequence variation between North American and South American strains of the New World Eastern Equine Encephalitis virus (EEEV) alters their ability to regulate the STAT1 pathway, leading to differential induction of type I interferon and consequently altered virulence. As a further example, in a mouse study with a set of North American WEEV isolates that shared less than 2.7% genomic sequence divergence, outcomes ranged from null to complete lethality.

[0031] Considering the different presence of host cell attenuation factors in the nonstructural and structural regions of alphaviruses, deleting structural genes to enable heterologous gene expression in synthetic vectors will have different effects on individual vectors. Synthetic replicons with different host attenuation factors in the nonstructural regions will have different advantages in inducing immune responses against the expressed heterologous genes. For example, in Old World viruses, shutting down of host cell functions is associated with nsP2, whereas in New World viruses (e.g., EEEV, VEEV, and WEEV), this activity is mainly associated with the capsid protein (C), which is partially or completely deleted in synthetic vectors. The hypervariable domain (HVD) of the nsP3 protein has host interactions specific for each alphavirus. In particular, EEEV nsP3 has been shown to interact with the cellular FXR and G3BP protein families, DDX3, S100A4, IKKβ, PGAM5, and cytoskeleton remodeling proteins, and vesicle transport proteins. Few studies have specifically detailed the nonstructural proteins of WEEV, but WEEV genome sequencing has revealed that it is the result of recombination between EEEV and SINV ancestors. Although the amino acid identity of WEEV nsPs has been reported to be over 80% identical to EEEV nsPs, replacement of EEEV nsPs with WEEV nsPs has been demonstrated to produce attenuated chimeras with significant differences in biological activity, although the nucleotide and coding sequence changes retain some activities essential for the viral life cycle. The known and undescribed mechanisms by which EEEV and WEEV nsPs contribute to the broad spectrum of pathogenicity suggest that WEEV-based srRNA vectors may be unique and advantageous vectors for the expression of heterologous proteins for vaccine or biotherapeutic applications. The advantages that these previously undescribed srRNA vectors may bring are entirely unexplored and unpredicted.

[0032] definition Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used herein are intended to have the meaning commonly understood by those of ordinary skill in the art to which this application pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art. Many of the techniques and operations described or referenced herein are well understood and commonly used using conventional methodology by those of ordinary skill in the art.

[0033] The singular forms "a," "an," and "the" include the plural forms unless the context clearly indicates otherwise. For example, the term "a cell" includes one or more cells, including mixtures thereof. "A and / or B" is used herein to include all of the following options: "A," "B," "A or B," and "A and B."

[0034] The terms "administration" and "administering" as used herein refer to the delivery of a bioactive composition or formulation by a route of administration including, but not limited to, intranasal, transdermal, intravenous, intraarterial, intramuscular, intranodal, intraperitoneal, subcutaneous, intramuscular, oral, intravaginal, and topical administration, or a combination thereof. The terms include, but are not limited to, administration by a medical professional and self-administration.

[0035] The terms "cell," "cell culture," and "cell line" refer not only to a particular subject cell, cell culture, or cell line, but also to the progeny or potential progeny of such a cell, cell culture, or cell line, regardless of the number of transplants or passages in culture. It should be understood that not all progeny are completely identical to the parent cell. This is because certain modifications may occur in subsequent generations, either due to mutations (e.g., intentional or unintentional mutations) or environmental influences (e.g., methylation or other epigenetic modifications), and thus the progeny may not actually be identical to the parent cell, but are within the scope of the terms used herein so long as the progeny retain the same function as the original cell, cell culture, or cell line.

[0036] The terms "effective amount", "therapeutically effective amount", or "pharmaceutical effective amount" of a composition of the present disclosure, such as a nucleic acid construct (e.g., a replicon construct, e.g., an srRNA construct), recombinant cell, recombinant polypeptide, and / or pharmaceutical composition, generally refer to an amount of the composition sufficient to achieve a stated purpose (e.g., achieve the intended effect of administration, stimulate an immune response, prevent or treat a disease, or alleviate one or more symptoms of a disease, disorder, infection, or condition) compared to the absence of the composition. An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or alleviation of a symptom of a disease, also referred to as a "therapeutically effective amount". "Alleviation" of a symptom refers to a decrease in the severity or frequency of the symptom, or elimination of the symptom. The precise amount of a composition that comprises a "therapeutically effective amount" will depend on the purpose of the treatment, and can be ascertained by one of ordinary skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0037] The term "construct" refers to a recombinant molecule, e.g., a recombinant nucleic acid or polypeptide, that includes one or more isolated nucleic acid or amino acid sequences of heterologous origin. For example, a polypeptide construct can be a chimeric polypeptide molecule in which two or more amino acid sequences of different origins are operably linked to each other within a single polypeptide construct. Similarly, a nucleic acid construct can be a chimeric nucleic acid molecule in which two or more nucleic acid sequences of different origins are assembled into a single nucleic acid molecule. Thus, representative nucleic acid constructs include any construct that includes (1) a nucleic acid sequence that includes regulatory and coding sequences that are not found contiguous to each other in nature (e.g., at least one nucleotide sequence is heterologous with respect to at least one other nucleotide sequence), or (2) a sequence that encodes portions of a functional RNA molecule or protein that are not contiguous in nature, or (3) portions of a promoter that are not contiguous in nature. Exemplary nucleic acid constructs include any recombinant nucleic acid molecule, linear or circular, single-stranded or double-stranded DNA or RNA nucleic acid molecule, from any source, such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, capable of genomic integration or autonomous replication, including a nucleic acid molecule operably linked to one or more nucleic acid sequences. The nucleic acid constructs of the present disclosure can include elements necessary to induce expression of a nucleic acid sequence of interest that is also included in the construct. Such elements can include regulatory elements, such as a promoter operably linked (to induce transcription) to the nucleic acid sequence of interest, and can optionally include a polyadenylation sequence.

[0038] In some embodiments of the present disclosure, one or more nucleic acid constructs can be incorporated (e.g., inserted) into a single nucleic acid molecule, such as a single vector, or into two or more separate nucleic acid molecules, such as two or more separate vectors. As used herein, the term "vector" refers to a nucleic acid molecule or sequence capable of transferring or transporting another nucleic acid molecule. Thus, the term "vector" encompasses both DNA-based and RNA-based vectors. The term "vector" includes cloning and expression vectors, as well as viral and integrating vectors. An "expression vector" is a vector that includes a regulatory region, which allows for the expression of DNA sequences and fragments in vitro, ex vivo, and / or in vivo. In some embodiments, the vector includes a sequence that directs autonomous replication in a cell, such as, for example, a plasmid (a DNA-based vector) or a self-replicating RNA vector. In some embodiments, the vector includes a sequence sufficient to allow integration into the host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. In some embodiments, the vector of the present disclosure can be a single-stranded vector (e.g., ssDNA or ssRNA). In some embodiments, the vector of the present disclosure can be a double-stranded vector (e.g., dsDNA or dsRNA). In some embodiments, the vector is a gene delivery vector. In some embodiments, the vector is used as a gene delivery vehicle to transfer genes to cells. In some embodiments, the vector of the present disclosure is a self-replicating RNA (srRNA) vector.

[0039] In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication of prokaryotic and eukaryotic origin, at least one multiple cloning site, and / or elements that facilitate stable integration of the construct into the genome of the cell. Two or more constructs can be incorporated into a single nucleic acid molecule, such as a single vector, or into two or more separate nucleic acid molecules, such as two or more separate vectors. An "expression construct" generally includes at least one control sequence operably linked to a nucleotide sequence of interest. 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 the cell. Compositions and methods for preparing and using the constructs and cells for the practice of the present disclosure are known to those of skill in the art.

[0040] The term "operably linked" as used herein refers to a physical or functional connection between two or more elements, e.g., polypeptide or polynucleotide sequences, that allows these elements to operate in an intended manner. For example, the term "operably linked" as used in the context of a nucleic acid molecule or a coding sequence and a promoter sequence within a nucleic acid molecule described herein means that the coding sequence and the promoter sequence are in frame and separated by an appropriate spatial and distance to allow binding of each by a transcription factor or RNA polymerase to affect transcription. It should be understood that operably linked elements may be contiguous or non-contiguous (e.g., linked to each other via a linker). In the context of a polypeptide construct, "operably linked" refers to a physical connection (e.g., directly or indirectly linked) between amino acid sequences (e.g., different segments, portions, regions, or domains) that provides the described activity of the construct. The operably linked segments, portions, regions, and domains of the polypeptides or nucleic acid molecules disclosed herein may be contiguous or non-contiguous (e.g., linked to each other via a linker).

[0041] The term "portion" as used herein refers to a proportion. With respect to a particular structure, such as a polynucleotide sequence, an amino acid sequence, or a protein, the term "portion" may refer to a contiguous portion or a discontinuous portion of said structure. For example, a portion of an amino acid sequence includes at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% of the amino acids of the amino acid sequence. Additionally or alternatively, when a portion is a discontinuous portion, the discontinuous portion may be composed of 2, 3, 4, 5, 6, 7, 8, or more portions of the structure (e.g., a domain of a protein), each portion being a contiguous element of the structure. For example, the non-contiguous portion of an amino acid sequence may consist of 2, 3, 4, 5, 6, 7, 8 or more, e.g., up to 4, portions of said amino acid sequence, each portion comprising at least 1, at least 2, at least 3, at least 4, at least 5 consecutive amino acids, at least 10 consecutive amino acids, at least 20 consecutive amino acids, or at least 30 consecutive amino acids of the amino acid sequence.

[0042] The term "recombinant" as used with respect to a cell, nucleic acid, protein, or vector indicates that the cell, nucleic acid, protein, or vector has been modified or produced by human intervention, e.g., modified by or is the result of a laboratory method. Thus, for example, recombinant proteins and nucleic acids include proteins and nucleic acids produced by laboratory methods. Recombinant proteins may include amino acid residues that are not present in the native (non-recombinant or wild-type) form of the protein, or may include amino acid residues that have been modified (e.g., labeled). The term includes any modification to a peptide, protein, or nucleic acid sequence. Such modifications include: any chemical modification of a peptide, protein, or nucleic acid sequence that includes one or more amino acids, deoxyribonucleotides, or ribonucleotides; the addition, deletion, and / or substitution of one or more amino acids within a peptide or protein; the creation of fusion proteins (e.g., fusion proteins with antibody fragments); and the addition, deletion, and / or substitution of one or more nucleic acids within a nucleic acid sequence. The term "recombinant" as used with respect to cells is not intended to include naturally occurring cells, but rather to encompass cells that have been engineered / modified to contain or express a polypeptide or nucleic acid that is not present in the cell unless engineered / modified.

[0043] The term "percent identity" as used herein in the context of two or more nucleic acids or proteins refers to two or more sequences or subsequences that are the same or have a certain percentage of the same nucleotides or amino acids (e.g., about 60% sequence identity over a particular region, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a comparison window or designated region when compared and aligned for maximum correspondence) as determined using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters as described below, or by manual alignment and visual inspection. See, e.g., the NCBI website ncbi.nlm.nih.gov / BLAST. Such sequences are then said to be "substantially identical". This definition also refers to or applies to the complement of a query sequence. This definition includes sequence comparisons performed by the BLAST algorithm, where the parameters of this algorithm are selected to obtain the maximum match between the respective sequences over the entire length of each reference sequence. This definition also includes sequences containing deletions and / or additions, as well as sequences containing substitutions. Sequence identity can be calculated over a region of at least about 20 amino acids or nucleotides in length, or over a region of 10-100 amino acids or nucleotides in length, or over the entire length of a particular 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, such as the sequence analysis software package of the Genetics Computer Group at the Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, using its default parameters. Additional methodologies that can be suitably used to determine amino acid sequence similarity or identity include methodologies that rely on position-specific structural scoring matrices (P3SMs) incorporating structural prediction scores from Rosetta, as well as methodologies based on length-normalized edit distances, as previously described, for example, in Setcliff et al., Cell Host & Microbe 23(6), May 2018.

[0044] The term "pharmaceutical acceptable excipient" as used herein refers to any suitable substance that provides a pharmaceutical acceptable carrier, additive, or diluent for administering a compound of interest to a subject. Thus, "pharmaceutical acceptable excipient" can include substances that are called pharmaceutical acceptable diluents, pharmaceutical acceptable additives, and pharmaceutical acceptable carriers. The term "pharmaceutical acceptable carrier" as used herein includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics and additional therapeutic agents) can also be incorporated into the composition.

[0045] As used herein, a "subject" or "individual" includes animals, such as humans (e.g., human individuals) and non-human animals. In some embodiments, a "subject" or "individual" is a patient under the care of a physician. Thus, a subject can be a human patient or individual who is suffering from, suspected of suffering from, or at risk of suffering from the relevant health condition (e.g., cancer or infectious disease) and / or one or more symptoms of the health condition. A subject can also be an individual who has been diagnosed as being at risk for the relevant health condition at the time of diagnosis or thereafter. The term "non-human animal" includes all vertebrates, such as mammals, such as rodents, e.g., mice, livestock, domesticated animals and pets, non-human primates, and other mammals, such as sheep, cats, dogs, cows, chickens, and non-mammals, such as amphibians, reptiles, and the like.

[0046] Aspects and embodiments of the disclosure described herein are understood to include "comprising," "consisting," and "consisting essentially of" aspects and embodiments. As used herein, "comprising" is synonymous with "including," "containing," or "characterized by" and is not inclusive or limiting and does not exclude additional, unrecited elements or method steps. As used herein, "consisting" excludes elements, steps, or ingredients not specified in the claimed composition or method. As used herein, "consisting essentially" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. When the term "comprising" is referred to herein specifically in the description of a component of a composition or in the description of a step of a method, it is understood to include compositions and methods that consist essentially of and consist of the recited components or steps.

[0047] It is understood that certain features of the present disclosure that are described for clarity in the context of separate embodiments can be provided in combination in a single embodiment. Conversely, various features of the present disclosure that are described for brevity in the context of a single embodiment may be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present disclosure are specifically embraced by the present disclosure and are disclosed herein as if all combinations were individually and expressly disclosed. Moreover, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein as if all such subcombinations were individually and expressly disclosed herein.

[0048] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value between the upper and lower limit of that range, and any other specified or intervening value in that stated range, to the tenth of the unit of the lower limit, is included within the disclosure. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also included within the disclosure, subject to any expressly excluded limit in the stated range. Where one or both of the limits are included in the stated range, ranges excluding one or both of those included limits are also included in the disclosure.

[0049] In this specification, a particular range is provided with the term "about" preceding the numerical values, which, as used herein, has its ordinary meaning of approximation. The term "about" is used to literally support the exact numerical value followed by it, and a numerical value that is close to or approximately the numerical value followed by the term. In determining whether a numerical value is close to or approximately a specifically stated numerical value, a near or approximately unstated numerical value may be a numerical value that is substantially equivalent to the specifically stated numerical value in the context in which it is presented. If the degree of approximation is not clear from the context, "about" means a numerical value that is within plus or minus 10% of the provided value, or in all cases rounded to the nearest significant figure including the provided value. In some embodiments, the term "about" refers to the specified value ±10%, up to ±5%, or up to ±1%.

[0050] When a range of values ​​is provided, one of ordinary skill in the art will understand that all ranges disclosed herein encompass any and all subranges and combinations thereof. Any range described can be readily recognized as fully descriptive and allowing for at least one-half, one-third, one-quarter, one-fifth, one-tenth, etc., of the same range. As a non-limiting example, each range described herein can be readily divided into a lower third, a middle third, an upper third, etc. One of ordinary skill in the art will also understand that all terms such as "up to," "at least," "greater than," "less than," etc., are inclusive of the recited numbers and refer to ranges that can be subsequently divided into subranges as described above. Finally, one of ordinary skill in the art will understand that ranges include individual members. Thus, for example, a group containing 1-3 items refers to a group containing 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.

[0051] Headings such as (a), (b), (i), etc. are provided solely to facilitate the reading of the specification and claims. The use of headings in the specification or claims does not require that the steps or elements be performed in alphabetical or numerical order or in the order in which they are presented.

[0052] Western Equine Encephalitis Virus (WEEV) Western equine encephalitis virus (WEEV) is a mosquito-borne virus belonging to the Alphavirus genus, which includes a group of genetically, structurally, and serologically related viruses in the family Togaviridae, and is classified as a group IV positive-sense single-stranded RNA virus. Currently, the Alphavirus genus includes Sindbis virus (SINV), Semliki Forest virus (SFV), Ross River virus (RRV), Venezuelan equine encephalitis virus (VEEV), and Eastern equine encephalitis virus (EEEV), which are all closely related and can infect a variety of vertebrates, including mammals, rodents, fish, birds, large mammals such as humans and horses, and invertebrates such as insects. One unusual feature of WEEV is that it is the descendant of an ancient recombination event between a Sindbis virus (SINV)-like ancestor and an Eastern equine encephalitis virus (EEEV)-like ancestor.

[0053] The alphavirus genus, which includes WEEV and EEEV, has been widely studied and the life cycle, mode of replication, etc. of these viruses have been well characterized. More information on this point can be found, for example, in Corrin T. et al., Vector-Borne and Zoonotic Diseases, Vol. 21, No. 5, 2021. Moreover, it has been shown that alphaviruses replicate very efficiently in animal cells, making them useful as vectors for the production of proteins and nucleic acids in cells. Since transmission between species and individuals occurs mainly via mosquitoes, alphaviruses contribute to the collection of arboviruses (arthropod-borne viruses).

[0054] Each of these alphaviruses has a single-stranded RNA genome of positive polarity that is enclosed in a nucleocapsid surrounded by an envelope that contains the viral spike protein. Alphavirus particles are enveloped, tend to be spherical (although slightly polymorphic), and have an isometric nucleocapsid. The alphavirus genome is a single-stranded RNA of positive polarity approximately 11-12 kb in length, containing a 5' cap, a 3' polyA tail, and two open reading frames, the first of which encodes nonstructural proteins with enzymatic functions, and the second of which encodes viral structural proteins (e.g., capsid protein CP, E1 glycoprotein, E2 glycoprotein, E3 protein, and 6K protein). For example, WEEV has a single-stranded, positive-sense RNA genome of approximately 11.5 kb that contains two open reading frames (ORFs) flanked by 5' and 3' untranslated regions (UTRs), which are capped at the 5' end and polyadenylated at the 3' end. WEEV is transmitted primarily in agricultural habitats by its main mosquito vector, the Culex tarsalis. Mammals can participate in the secondary cycle. Both humans and horses are considered definitive hosts, but some equids, such as donkeys and ponies, can develop low-to-moderate viremia (slightly below 104 PFU / ml), and these hosts may contribute to amplifying the epizootic. Human infections with WEEV range from asymptomatic (in the majority of cases), to influenza-like syndromes, to life-threatening encephalitis and meningitis. Symptomatic infections typically present with a sudden onset of fever, headache, nausea, vomiting, anorexia, and malaise, followed by cognitive symptoms, weakness, and meningeal involvement.

[0055] The 5'-two-thirds of the alphavirus genome encodes a number of nonstructural proteins required for viral RNA transcription and replication. These proteins are directly translated from RNA and, together with cellular proteins, form the RNA-dependent RNA polymerase essential for viral genome replication and subgenomic RNA transcription. Four nonstructural proteins (nsP1-4) are produced as a single polyprotein and constitute the viral replication machinery. Polyprotein processing is highly regulated, and cleavage at the P2 / 3 junction affects the use of the RNA template during genome replication. This site is located at the bottom of a narrow groove and is not easily accessible. Upon cleavage, nsP3 forms a ring structure surrounding nsP2. These two proteins have an extensive interface. Mutations in nsP2 that produce noncytopathic viruses or temperature-sensitive phenotypes are concentrated in the P2 / P3 interface region. P3 mutations opposite the location of nsP2 noncytopathic mutations prevent efficient cleavage of P2 / 3. This in turn can affect RNA infectivity and alter the levels of viral RNA production.

[0056] The 3' third of the genome contains the subgenomic RNA that serves as a template for the translation of all structural proteins required for the formation of viral particles (the core nucleocapsid protein C, and the envelope proteins P62 and E1, which assemble as a heterodimer). Surface glycoproteins, anchored in the viral membrane, are responsible for receptor recognition and entry into target cells by membrane fusion. The subgenomic RNA is transcribed from the p26S subgenomic promoter at the 3' end of the RNA sequence encoding the nsP4 protein. Proteolytic maturation of P62 to E2 and E3 results in a change in the viral surface. E1, E2, and sometimes E3 glycoprotein "spikes" join together to form E1 / E2 dimers or E1 / E2 / E3 trimers, where E2 extends from the center to the vertices, E1 fills the space between the vertices, and E3, if present, is at the distal end of the spike. When the virus is exposed to the acidic environment of the endosome, E1 dissociates from E2 to form the E1 homotrimer, which is required for the fusion step that moves the cellular and viral membranes together. The alphavirus glycoprotein E1 is a class II viral fusion protein, which is structurally distinct from the class I fusion proteins found in influenza viruses and HIV. The E2 glycoprotein functions to interact with the nucleocapsid through its cytoplasmic domain, while its extracellular domain is responsible for binding to cellular receptors. Most alphaviruses, including WEEV, lose the peripheral protein E3, but in Semlikivirus, E3 remains associated with the viral surface.

[0057] Like most alphaviruses, WEEV replication occurs on membranous surfaces within the host cell. In the first phase of the infection cycle, the 5' end of the genomic RNA is translated into polyproteins (nsP1-4) with RNA polymerase activity, generating a minus strand complementary to the genomic RNA. In the second phase, the minus strand is used as a template to generate two RNAs, respectively: (1) a positive genomic RNA, which corresponds to the genome of a secondary virus that produces other nsP proteins by translation and acts as the genome of the virus; and (2) a subgenomic RNA, which codes for the structural proteins of the virus that form the infectious particle. The positive genomic RNA / subgenomic RNA ratio is regulated by the proteolytic autocleavage of the polyprotein into nsP1, nsP2, nsP3, and nsP4. In fact, the expression of viral genes occurs in two phases. In the first phase, the main synthesis of the positive and negative genomic strands takes place. In the second phase, the synthesis of the subgenomic RNA is virtually exclusive, resulting in the production of large amounts of structural proteins.

[0058] self-replicating RNA As will be appreciated by those skilled in the art, the term "self-replicating RNA" refers to an RNA molecule that contains all the genetic information necessary to induce self-amplification or self-replication in a permissive cell. Thus, srRNA may also be referred to as "self-amplifying RNA" (saRNA). In some embodiments, srRNA is a "replicon," which is a linear or circular section of DNA or RNA that is replicated continuously as a unit. Non-limiting examples of replicons include "replicon RNA" or "RNA replicons." To induce its own replication, srRNA generally (1) encodes a polymerase, replicase, or other protein that may interact with a protein, nucleic acid, or ribonucleoprotein from a virus or host cell to catalyze the RNA amplification process, and (2) contains cis-acting RNA sequences necessary for the replication and transcription of subgenomic RNA. These sequences may bind to self-encoded proteins, or proteins, nucleic acids, or ribonucleoproteins from a non-self-encoded cell, or a complex between any of these components during the replication process. In some embodiments of the present disclosure, the replicon (e.g., srRNA) is derived from an alphavirus (e.g., WEEV). In some embodiments of the present disclosure, an alphavirus srRNA construct (e.g., a srRNA, saRNA, or replicon molecule) generally contains the following elements: 5' viral or defective interfering RNA sequences required in cis for replication, sequences encoding biologically active alphavirus nonstructural proteins (e.g., nsP1, nsP2, nsP3, and nsP4), a subgenomic promoter (sg) for the subgenomic RNA (sgRNA), 3' viral sequences required in cis for replication, and optionally a polyadenylation region (poly(A)). Optionally, a subgenomic promoter (sg) directing expression of a heterologous sequence can be included in the srRNA construct of the present disclosure.

[0059] Additionally, the term srRNA molecule (e.g., srRNA, saRNA, or replicon molecule) generally refers to a molecule of positive polarity or "message" sense, and the srRNA may be of a length different from that of known naturally occurring alphaviruses. In some embodiments of the present disclosure, the srRNA does not include at least a portion of the coding sequence for one or more alphavirus structural proteins, and / or the sequence encoding the structural genes can be replaced with a heterologous sequence. In these cases, when the srRNA is packaged into recombinant alphavirus particles, it can include one or more sequences, so-called packaging signals, useful for initiating interactions with alphavirus structural proteins leading to particle formation.

[0060] The nucleic acid molecule of the present disclosure may be a nucleic acid molecule of any length, and generally includes a nucleic acid molecule of about 2 kb to about 50 kb in length, such as about 5 kb to about 40 kb, about 5 kb to about 30 kb, about 5 kb to about 20 kb, or about 10 kb to about 50 kb, such as about 15 kb to about 30 kb, about 20 kb to about 50 kb, about 20 kb to about 40 kb, about 5 kb to about 25 kb, or about 30 kb to about 50 kb. In some embodiments, the nucleic acid molecule is at least 6 kb in length. In some embodiments, the nucleic acid molecule is about 6 kb to about 20 kb. The replicon construct (e.g., srRNA construct) of the present disclosure generally has a length of at least about 2 kb. For example, the srRNA can have a length of at least about 2 kb, at least about 3 kb, at least about 4 kb, at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 11 kb, at least about 12 kb, or greater than 12 kb. In some embodiments, the srRNA is about 4 kb to about 20 kb, about 4 kb to about 18 kb, about 5 kb to about 16 kb, about 6 kb to about 14 kb, about 7 kb to about 12 kb, about 8 kb to about 16 kb, about 9 kb to about 14 kb, about 10 kb to about 18 kb, about 11 kb to about 16 kb, about 5 kb to about 18 kb, about 6 kb to about 20 kb, about 5 kb to about 10 kb, about 5 kb to about 8 kb, about 5 kb to about 7 kb, about 5 kb to about 6 kb, b, about 6 kb to about 12 kb, about 6 kb to about 11 kb, about 6 kb to about 10 kb, about 6 kb to about 9 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, about 7 kb to about 11 kb, about 7 kb to about 10 kb, about 7 kb to about 9 kb, about 7 kb to about 8 kb, about 8 kb to about 11 kb, about 8 kb to about 10 kb, about 8 kb to about 9 kb, about 9 kb to about 11 kb, about 9 kb to about 10 kb, or about 10 kb to about 11 kb in length. In some embodiments, the srRNA may have a length of about 6 kb to about 14 kb. In some embodiments, the srRNA may have a length of about 6 kb to about 16 kb.

[0061] Compositions of the Disclosure As described in more detail below, one aspect of the disclosure relates to nucleic acid constructs having a nucleic acid sequence encoding a modified viral genome or srRNA, where the modified genome or srRNA lacks (e.g., does not include) at least a portion of the nucleic acid sequence encoding one or more structural proteins of the corresponding unmodified viral genome or srRNA. Some embodiments of the disclosure provide modified alphavirus genomes or srRNAs in which the coding sequences for the nonstructural proteins nsP1, nsP2, nsP3, and nsP4 are present, but at least a portion or the entire sequence encoding one or more structural proteins is absent. Also provided are recombinant cells and cell cultures engineered to contain the nucleic acid constructs disclosed herein.

[0062] A. Nucleic acid constructs As described in more detail below, one aspect of the disclosure relates to novel nucleic acid constructs that include nucleic acid sequences encoding a modified genome or srRNA of an alphavirus, such as Western Equine Encephalitis Virus (WEEV). For example, in some embodiments, the modified alphavirus genome can include deletions, substitutions, and / or insertions in one or more of the genomic regions of the parent alphavirus genome.

[0063] Non-limiting exemplary embodiments of the nucleic acid constructs (e.g., replicon constructs, e.g., srRNA constructs) of the present disclosure can include one or more of the following features. In some embodiments, the nucleic acid construct can include a nucleic acid sequence encoding a modified WEEV genome or srRNA, where the modified WEEV genome or srRNA lacks at least a portion of the nucleic acid sequence encoding one or more structural proteins of the unmodified WEEV genome or srRNA, e.g., the modified WEEV genome or srRNA does not include at least a portion of the coding sequence for one or more of the WEEV structural proteins CP, E1, E2, E3, and 6K. Both virulent and non-virulent WEEV strains are suitable. Non-limiting examples of WEEV strains suitable for the compositions and methods of the present disclosure include WEEV California, McMillan, IMP181, Imperial, Imperial181, IMPR441, 71V-1658, AG80-646, BFS932, COA592, EP-6, E1416, BFS1703, BFS2005, BSF3060, BSF09997, CHLV53, KERN5547, 85452NM, Montana-64, S8-122, and TBT-235. Further examples of WEEV strains suitable for the compositions and methods of the present disclosure include 5614, 93A27, 93A30, 93A38, 93A79, B628(Cl15), CBA87, CNTR34, CO921356, Fleming, Lake43, PV012357A, PV02808A, PV72102, R02PV001807A, R02PV002957B, R02PV003422B, R05PV003422B, R0PV003814A, and R0PV00384A. Further suitable WEEV strains include, but are not limited to, strains described in Berggren NA et al., J. Virol. 88(16): 9260-9267, Aug 2014, and strains available from the Virus Pathogen Resource website (ViPR; www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=868&decorator=toga In some embodiments, the modified WEEV genome or srRNA is derived from the WEEV Imperial strain.

[0064] 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 some or all of the sequence encoding CP. In some embodiments, the modified viral genome or srRNA lacks some or all of the sequence encoding E1. In some embodiments, the modified viral genome or srRNA lacks some or all of the sequence encoding E2. In some embodiments, the modified viral genome or srRNA lacks some or all of the sequence encoding E3. In some embodiments, the modified viral genome or srRNA lacks some or all of the sequence encoding 6K. In some embodiments, the modified viral genome or srRNA lacks some or all of the sequence encoding a combination of CP, E1, E2, E3, and 6K. Some embodiments of the disclosure provide modified WEEV genomes or srRNAs in which the coding sequences for the nonstructural proteins nsP1, nsP2, nsP3, and nsP4 of the unmodified WEEV genome or srRNA are present, but in which at least some or all of the sequences encoding one or more structural proteins (e.g., CP, E1, E2, E3, and 6K) of the WEEV genome or srRNA are absent. Some embodiments of the disclosure provide modified WEEV genomes or srRNAs in which the coding sequences for the nonstructural proteins nsP1, nsP2, nsP3, and nsP4 of the unmodified WEEV genome or srRNA are present, but in which at least some or all of the sequences encoding one or more structural proteins (e.g., CP, E1, E2, E3, and 6K) of the WEEV genome or srRNA are absent.

[0065] In some embodiments, the modified viral genome or srRNA lacks a majority of the nucleic acid sequence encoding one or more viral structural proteins. Those skilled in the art will understand that a majority of the nucleic acid sequence encoding a viral structural polypeptide may include sufficient nucleic acid sequence encoding a viral structural polypeptide to allow for putative identification of the polypeptide by manual evaluation of the sequence by a skilled artisan or by computer-automated sequence comparison and identification using algorithms such as BLAST (e.g., "Basic Local Alignment Search Tool"; see Altschul SF et al., J. Mol. Biol. 215:403-410, 1993). Thus, a majority of the nucleotide sequence includes sufficient sequence to allow specific identification and / or isolation of a nucleic acid fragment comprising the sequence. For example, a majority of the nucleic acid sequence may include at least about 20%, such as about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% of the full-length nucleic acid sequence. As mentioned above, the present disclosure provides nucleic acid molecules and constructs that lack some or all of the nucleic acid sequences encoding one or more viral structural proteins. A skilled artisan having the benefit of the sequences disclosed herein can readily use all or most of the disclosed sequences for the compositions and methods of the present disclosure. Thus, the present application includes the complete sequences disclosed herein, such as those set forth in the attached sequence listing, as well as most of the sequences defined above.

[0066] In some embodiments, the modified viral genome or srRNA lacks the entire sequence encoding a viral structural protein, e.g., the modified viral genome or srRNA does not include the nucleic acid sequences encoding the structural proteins of the unmodified genome or srRNA of the virus.

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

[0068] In some embodiments, at least one expression cassette comprises a promoter operably linked to the heterologous nucleic acid sequence. Thus, the nucleic acid constructs provided herein can be used as expression vectors that can affect expression of a heterologous nucleic acid sequence, for example, when they comprise a regulatory element (e.g., a promoter) operably linked to the heterologous nucleic acid sequence. In some embodiments, at least one expression cassette comprises a subgenomic (sg) promoter operably linked to the heterologous nucleic acid sequence. In some embodiments, the sg promoter is a 26S subgenomic promoter. In some embodiments, at least one nonstructural protein (nsP) or portion thereof of the modified WEEV genome or srRNA is heterologous to the remainder of the modified WEEV genome or srRNA. In some embodiments, the modified WEEV genome or srRNA further comprises a nucleic acid sequence encoding a heterologous nsP or 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 UTR is a heterologous UTR.

[0069] In some embodiments, at least one expression cassette 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 (PCI) comprising a single polypeptide (e.g., a monogenic PCI). In some embodiments, the coding sequence of the PCI comprises a coding sequence of a plurality of polypeptides, e.g., a multigenic PCI (e.g., a bigenic, trigenic, or tetragenic, etc.). In some embodiments, each of the coding sequences of the plurality of polypeptides is operably linked to a separate promoter sequence. In some embodiments, the coding sequences of the plurality of polypeptides are operably linked to each other within a single open reading frame (e.g., within a polycistronic ORF). In some embodiments, the coding sequence of the polycistronic ORF is operably linked to a promoter sequence. In some embodiments, at least one of the promoter sequences is a subgenomic (sg) promoter. In some embodiments, the sg promoter is a 26S genomic promoter.

[0070] In some embodiments, the multiple polypeptides can be linked to each other directly or indirectly (e.g., via one or more connector sequences). For example, in some embodiments, the multiple polypeptides can be linked to each other directly, e.g., adjacent to each other. In some embodiments, at least two (e.g., 2, 3, 4, or 5) of the multiple polypeptides are operably linked to each other by one or more connector sequences. In some embodiments, the length and amino acid composition of the connector sequence can be optimized to change the orientation, flexibility, and / or proximity of the polypeptides relative to each other to achieve the desired activity or properties of PCI. In some embodiments, one connector sequence of the multiple connector sequences comprises one or more autoproteolytic peptide sequences. Non-limiting examples of autoproteolytic peptide sequences suitable for the methods and compositions of the present disclosure include autoproteolytic sequences derived from calcium-dependent serine endoprotease (Furin), porcine Teschovirus-1 2A (P2A), foot and mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), Thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), and Flacherie Virus 2A (BmIFV2A). In some embodiments, at least two of the multiple polypeptides are operably linked to each other via a P2A autoproteolytic sequence.

[0071] In some embodiments, the coding sequences of the multiple polypeptides are operably linked to each other by one or more internal ribosome entry sites (IRES).Non-limiting examples of IRES suitable for the methods and compositions of the present disclosure include viral IRES sequences, cellular IRES sequences, and artificial IRES sequences.Examples of suitable IRES sequences include, but are not limited to, Kaposi's sarcoma-associated herpesvirus (KSHV) IRES, Hepatitis virus IRES, Pestivirus IRES, Cripavirus IRES, Rhopalosiphum padi virus IRES, fibroblast growth factor IRES, platelet-derived growth factor IRES, vascular endothelial growth factor IRES, insulin-like growth factor IRES, picornavirus IRES, encephalomyocarditis virus (EMCV) IRES, Pim-1 IRES, p53 IRES, Apaf-1 IRES, TDP2 IRES, L-myc IRES, and c-myc IRES.

[0072] In principle, there are no particular limitations regarding suitable polypeptides and PCIs that can be expressed by the replicon constructs (e.g., srRNA constructs) of the present disclosure. Exemplary types of polypeptides suitable for the compositions and methods of the present disclosure include microbial proteins, viral proteins, bacterial proteins, fungal proteins, mammalian proteins, and any combination thereof. For example, the PCI may 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, de-differentiation factors, immune cell receptors or reporters, or any combination thereof.

[0073] In some embodiments, the coding sequence of the GOI is redesigned, refactored, and / or optimized for desired properties, such as improved stability, potency, and expression (e.g., translation efficiency), so as to maximize the impact of biotherapeutic production, delivery, and administration. For example, in some embodiments, the coding sequence of the GOI is optimized for expression at a level higher than the expression level of the reference coding sequence, e.g., 20% higher, 30% higher, 40% higher, 50% higher, 60% higher, 70% higher, 80% higher, 90% higher, or 95% higher than the reference coding sequence. In some embodiments, the reference coding sequence is a wild-type, unoptimized sequence. With respect to sequence optimization of nucleotide sequences, the degeneracy of the genetic code allows for the substitution of at least one base of the protein coding sequence of a gene with a different base without changing the amino acid sequence of the polypeptide produced from the gene. Thus, the nucleic acid construct of the present disclosure may have any base sequence that is altered from any polynucleotide sequence disclosed herein by substitution according to the degeneracy of the genetic code. References describing codon usage are readily available to the public. In some embodiments, polynucleotide sequence variants may be generated for a variety of reasons, such as optimizing expression for a particular host (e.g., changing the codon usage of alphavirus mRNA to that preferred by other organisms, such as humans, non-human primates, hamsters, mice, or monkeys). Thus, in some embodiments, the coding sequence of the GOI is optimized for expression in the target host cell by using codons optimized for expression. Techniques for constructing synthetic nucleic acid sequences encoding the GOI using optimal preferred codons for host cell expression can be determined by techniques known in the art, computational methods that analyze the commonality of codon usage and their relative abundance for encoding natural proteins in the host cell genome. Codon usage databases (e.g., http: / / www.kazusa.or.jp / codon) can be used to generate codon-optimized sequences in a mammalian cell environment.Furthermore, various software tools are available for converting sequences of one organism to the optimal codon usage of another host organism, such as the JCat Codon Optimization Tool (www.jcat.de), the Integrated DNA 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 are available from various commercial vendors. Thus, in some embodiments, the coding sequence of the GOI is optimized to be expressed at a higher level than the expression level of a reference coding sequence, such as a coding sequence that is not codon optimized. 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 a reference coding sequence that is not codon optimized. In some embodiments, the codon-optimized sequence of the GOI results in at least a 2-fold, at least a 3-fold, at least a 4-fold, or at least a 5-fold increase in expression level compared to a non-codon-optimized reference coding sequence.

[0074] In some embodiments, the coding sequence of the GOI is optimized for enhancing RNA stability and / or expression. RNA stability is generally related to the "half-life" of the RNA. "Half-life" refers to the period required to remove half of the activity, amount, or number of a molecule. In the context of the present disclosure, the half-life of an RNA refers to the stability of said RNA. The half-life of an RNA may affect the "expression period" of the RNA. Additional information on the principles, strategies, and methods used to enhance RNA stability is described, 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).

[0075] The polypeptide encoded by the GOI can generally be any polypeptide, for example, a therapeutic polypeptide, a prophylactic polypeptide, a diagnostic polypeptide, a nutraceutical polypeptide, an industrial enzyme, and a reporter polypeptide. In some embodiments, the GOI encodes a polypeptide selected from the group consisting of an antibody, an antigen, an immunomodulator, an enzyme, a signaling protein, and a cytokine. In some embodiments, the GOI can encode a microbial protein, a viral protein, a bacterial protein, a fungal protein, a mammalian protein, and any combination thereof. In some embodiments, the GOI encodes the hemagglutinin precursor (HA) of the influenza A virus H5N1. Non-limiting examples of GOIs include interleukins and interacting proteins (G-CSF, GM-CSF, IL-1, IL-10, IL-10-like, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-18BP, I L-1-like, IL-1RA, IL-1α, IL-1β, IL-2, IL-20, IL-3, IL-4, IL-5, IL-6, IL-6-like, IL-7, IL-9, IL-21, IL-22, IL-33, IL-37, IL-38, LIF, and OSM).Other suitable GOIs include, but are not limited to, interferons (e.g., IFN-α, IFN-β, IFN-γ), TNF (e.g., CD154, LT-β, ​​TNF-α, TNF-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, and TRANCE), TGF-β (e.g., TGF-β1, TGF-β2, and TGF-β3), hematopoietin (e.g., Epo, Tpo, Flt-3L, SCF, M-CSF, MSP), chemokines and their receptors (XCL1, XCL2, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL210, CCL220, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL29, CCL30, CCL310, CCL320, CCL33, CCL34, CCL35, CCL36, CCL37, CCL38, CCL39 ... CL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, and CX3CL1), immunosuppressive gene products and related transcription factors (e.g., PECAM1, FCGR3A, FOS, NFKB1, JUN, HIF1A, PD-L1, mTOR, STAT5B, and STAT4). Additional GOIs suitable for the compositions and methods of the present disclosure include, but are not limited to, immune stimulatory gene products (e.g., CD27 / CD70, CD40, CD40L, B7.1, BTLA, MAVS, OX40, OX40L, RIG-I, and STING), drug resistance mutants / variants of genes, such as ABCB1, ABCC1, ABCG2, AKT1, ALK, BAFF, BCR-ABL, BRAF, CCND1, cMET, EGFR, ERBB2, ERBB3, ERK2, ESR1, GRB2, KRAS, MDR1, MRP1, NTRK1, PDC4, P-gp, PI3K, PTEN, RET, ROS1, RSK1, RSK2, SHIP, and STK11. Also suitable for the compositions and methods of the present disclosure include sequences encoding viral proteins, particularly spike proteins, fiber proteins, structural proteins, and attachment proteins.

[0076] In some embodiments, the GOI can encode an antibody or antibody variant (e.g., single chain Fv, bispecific, camelid antibody, Fab, and HCAb). In some embodiments, the antibody targets a surface molecule associated with cancer or upregulated, or associated with infectious disease. In some embodiments, the antibody targets a surface molecule with immunostimulatory or immunosuppressive function.

[0077] In some embodiments, the GOI can encode an enzyme whose deficiency or mutation is associated with a disease or condition, such as, for example, agalsidase beta, agalsidase alpha, imiglucerase, taliglucerase alpha, velaglucerase alpha, alglucerase, sebelipase alpha, laronidase, idursulfase, elosulfase alpha, galsulfase, alglucosidase alpha, and CTFR.

[0078] 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, a tumor-specific antigen, a neoantigen, and any combination thereof. As will be understood by those skilled in the art, TAAs include molecules, e.g., proteins, that are present on tumor cells and on normal cells, or on many normal cells, but at a much lower concentration than on tumor cells. In contrast, TSAs generally include molecules, e.g., proteins, that are present on tumor cells but not on normal cells. Tumor-associated antigens can be antigens associated with cancer cells, e.g., breast cancer cells, B-cell lymphoma, pancreatic cancer, Hodgkin's lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma, lung cancer cells, non-Hodgkin's B-cell lymphoma (B-NHL) cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, gliomas, glioblastomas, colorectal cancer cells, and the like. It will also be understood that in some cases, tumor-associated antigens may be expressed by non-cancer cells. In some embodiments, the GOI may encode a polypeptide selected from an estrogen receptor, an intracellular signaling enzyme, and a human epidermal growth receptor. In some embodiments, the GOI may encode a biotherapeutic polypeptide selected from an immunomodulator, an angiogenesis regulator, an extracellular matrix regulator, a metabolic regulator, a neuromodulator, and any combination thereof. In some embodiments, the GOI may encode a cytokine selected from a chemokine, an interferon, an interleukin, a lymphokine, and a tumor necrosis factor. In some embodiments, the GOI may encode an interleukin selected from IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-15, IL-17, IL-23, IL-27, IL-35, IFNγ, and any subunit thereof.In some embodiments, the GOI can encode a biotherapeutic polypeptide selected from IL-12A, IL-12B, IL-1RA, and any combination thereof.

[0079] In some embodiments, a nucleic acid construct of the disclosure comprises a nucleic acid sequence encoding a modified WEEV having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, a nucleic acid construct of the disclosure comprises a nucleic acid sequence encoding a modified WEEV having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:2.

[0080] In some embodiments, a nucleic acid construct of the disclosure comprises a nucleic acid sequence encoding a modified WEEV having 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 1, wherein 1, 2, 3, 4, 5, or more nucleotides of the nucleic acid sequence may be replaced with a different nucleotide. In some embodiments, a nucleic acid construct of the disclosure comprises a nucleic acid sequence encoding a modified WEEV having 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 2, wherein 1, 2, 3, 4, 5, or more nucleotides of the nucleic acid sequence may be replaced with a different nucleotide.

[0081] Nucleic acid sequences having high 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 WEEV of interest can be identified and / or isolated by genomic sequence analysis, hybridization, and / or PCR using degenerate or gene-specific primers from the sequences identified in the respective WEEV genome using the sequences identified herein (e.g., SEQ ID NO:1 or SEQ ID NO:2) or any other sequences known in the art.

[0082] In some embodiments, the nucleic acid construct of the present disclosure comprises one or more adaptor sequences, such as cloning adaptor sequences. In some embodiments, the one or more adaptor sequences comprise the following sequence: 5'-CTGGAGACGTGGAGGAGAACCCTGGACCT-3' (SEQ ID NO: 3). In some embodiments, the one or more adaptor sequences comprise the following sequence: 5'-GACCGCTACGCCCCAATGACCCGACCAGC-3' (SEQ ID NO: 4).

[0083] In some embodiments, mutations (e.g., silent mutations) can be incorporated into the nucleic acid constructs of the present disclosure to eliminate restriction sites, such as SapI and SpeI restriction sites (see, e.g., Examples 1-3). For example, a unique restriction site (SpeI, 5'-A'CTAG,T-3') can be incorporated in place of the coding sequence of the native WEEV structural gene (where 5'A corresponds to the position of the structural polyprotein ATG start codon and 3'T corresponds to the position of the structural polyprotein stop codon TAA). In some embodiments, a 5' adapter sequence (e.g., SEQ ID NO:3) can be inserted upstream of the SpeI site and a 3' adapter sequence (SEQ ID NO:4) can be inserted downstream of the SpeI site for use in a subsequent Gibson Assembly® operation (Gibson et al., Nat. Methods 6, 343-345, 2009). In some embodiments, a promoter sequence (e.g., a bacteriophage T7 RNA polymerase promoter) is included upstream of the WEEV genomic sequence, and downstream is a poly(A) sequence followed by a restriction site (e.g., SapI), which cuts upstream of the recognition site. Immediately downstream of this restriction site (SapI) can be a terminator sequence (e.g., a T7 terminator sequence) followed by a unique restriction site (e.g., NotI').

[0084] In some experiments, the sequence encoding one or more nsPs is replaced with a heterologous nsP, while in other experiments the sequence encoding one or more UTRs is replaced with a heterologous UTR.

[0085] Molecular techniques and methods for assembling and characterizing these new nucleic acid constructs are described in more detail in the Examples of this application. In some embodiments, the nucleic acid molecule is a recombinant nucleic acid molecule. As used herein, the term "recombinant" refers to a molecule (e.g., DNA, RNA, polypeptide) that is the result, even if indirectly, of human manipulation. As a non-limiting example, a cDNA is a recombinant DNA molecule, as is a nucleic acid molecule that has been produced by an in vitro polymerase reaction, or that has been linked or incorporated into a vector, such as a cloning vector or an expression vector. As non-limiting examples, a recombinant nucleic acid molecule is one that has been 1) synthesized or modified in vitro, for example, using chemical or enzymatic techniques of nucleic acid molecules (e.g., using chemical nucleic acid synthesis and the use of enzymes for replication, polymerization, exonuclease digestion, endonuclease digestion, ligation, reverse transcription, transcription, base modification (including, for example, methylation), or recombination (including homologous recombination and site-specific recombination); 2) contains linked nucleotide sequences that are not linked in nature; 3) has been engineered using molecular cloning techniques such that a naturally occurring nucleotide sequence is missing one or more nucleotides; and / or 4) has been engineered using molecular cloning techniques such that a naturally occurring nucleotide sequence has been modified by one or more sequence changes or rearrangements.

[0086] In some embodiments, the nucleic acid molecules disclosed herein are produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning, etc.) or chemical synthesis. The nucleic acid molecules disclosed herein include naturally occurring nucleic acid molecules and their homologues, including but not limited to naturally occurring allelic variants and modified nucleic acid molecules in which one or more nucleotide residues have been inserted, deleted, and / or substituted, such modifications providing desired properties in conferring biological activity as described herein.

[0087] Nucleic acid molecules containing variants of naturally occurring nucleic acid sequences can be prepared using a number of methods known to those of skill in the art (see, e.g., Sambrook et al., In: Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989)). The sequence of a nucleic acid molecule can be altered 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, including, 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 a nucleic acid sequence, recombinant cloning, and chemical synthesis (chemical synthesis of mixtures of oligonucleotides, ligation of mixtures to "construct" a mixture of nucleic acid molecules), and combinations thereof. Nucleic acid molecule homologues can be selected from a mixture of modified nucleic acid molecules by screening for the function of the protein or srRNA encoded by the nucleic acid molecule and / or by hybridization with the wild-type gene or a fragment thereof, or by PCR using primers having homology to the target or wild-type nucleic acid molecule or sequence.

[0088] B. Recombinant Cells The nucleic acid constructs of the present disclosure can be introduced into a host cell to generate a recombinant cell comprising the nucleic acid molecule. Thus, prokaryotic or eukaryotic cells comprising a nucleic acid construct encoding the modified WEEV genome described herein are also a feature of the present disclosure. In a related aspect, some embodiments disclosed herein relate to a method of transforming a cell, comprising introducing a nucleic acid construct provided herein into a host cell, such as an animal cell, and then selecting or screening for transformed cells. Introduction of the nucleic acid constructs of the present invention into a cell can be accomplished by methods known to those of skill in the art, such as viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0089] In one aspect, some embodiments of the present disclosure relate to recombinant cells, such as recombinant animal cells, comprising the nucleic acid constructs described herein. The nucleic acid constructs may be stably integrated into the host genome, may be episomally replicated, or may exist in the recombinant host cell as a minicircle expression vector for stable or transient expression. Thus, in some embodiments of the present disclosure, the nucleic acid constructs are maintained and replicated in the recombinant host cell as episomal units. In some embodiments, the nucleic acid constructs are stably integrated into the genome of the recombinant cell. Stable integration can be accomplished using classical random genome recombination techniques, or using more precise genome editing techniques such as guide RNA-guided CRISPR / Cas9 or TALEN genome editing. In some embodiments, the nucleic acid constructs exist in the recombinant host cell as a minicircle expression vector for stable or transient expression.

[0090] A host cell can be either an untransformed cell or a cell that has already been transfected with at least one nucleic acid molecule. Thus, in some embodiments, a host cell can be genetically engineered (e.g., transduced or transformed or transfected) with at least one nucleic acid molecule.

[0091] Suitable host cells for cloning or expressing the polypeptide of interest described herein include prokaryotic or eukaryotic cells described herein. Thus, in some embodiments, the recombinant cell is a prokaryotic cell, such as bacteria, E. coli, or a eukaryotic cell, such as an insect cell (e.g., a mosquito cell or an Sf21 cell), or a mammalian cell (e.g., a COS cell, an NIH3T3 cell, or a HeLa cell). In some embodiments, the cell is an in vivo, e.g., recombinant cell in a living body, e.g., a cell of a transgenic subject. In some embodiments, the subject is a vertebrate or an invertebrate. In some embodiments, the subject is an insect. In some embodiments, the subject is a mammal. In some embodiments, the cell is ex vivo, e.g., extracted as an individual cell or as part of an organ or tissue from a living body or organism for therapy or treatment, and then returned to the living body or organism. In some embodiments, the cell is in vitro, e.g., obtained from a repository.

[0092] In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a vertebrate cell or an invertebrate cell. In some embodiments, the recombinant cell is a mammalian cell. In some embodiments, the recombinant cell is a SV40 transformed monkey kidney CV1 cell (e.g., COS-7), human embryonic kidney cell (e.g., HEK293 or HEK293 cell), baby hamster kidney cell (BHK), mouse Sertoli cell (e.g., TM4 cell), monkey kidney cell (e.g., CV1), human cervical carcinoma cell (e.g., HeLa), canine kidney cell (e.g., MDCK), buffalo rat hepatocyte cell (e.g., BRL3A), human lung cell (e.g., W138), human liver cell (e.g., HepG2), mouse mammary tumor (e.g., MMT060562), human ovarian tumor (HTV) cell (HTV ... ), 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 fibroblast cells, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocytes, human macrophage cells, human RAW264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells.

[0093] In some embodiments, the recombinant cell is selected from the group consisting of African green monkey kidney cells (Vero cells), baby hamster kidney (BHK) cells, Chinese hamster ovary cells (CHO cells), human A549 cells, human cervical cells, human CHME5 cells, human epithelial laryngeal cells, human fibroblast cells, human HEK-293 cells, human HeLa cells, human HepG2 cells, human HUH-7 cells, human MRC-5 cells, human muscle cells, mouse 3T3 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells. In some embodiments, the recombinant cell is a cell derived from the above cells (i.e., a derivative of the original cell described herein), such as a cell propagated from a clone of the original cell, an engineered version of the original cell, or a cell that has been reassorted after the original cell has been passaged extensively or through another host.

[0094] In some embodiments, the recombinant cell is an insect cell, for example, a cell of an insect cell line. In some embodiments, the recombinant cell is an Sf21 cell. Additional suitable insect cell lines include, but are not limited to, cell lines established from 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 lepidopteran insect cell line. Over the past few decades, the availability of lepidopteran insect cell lines has increased by about 50 lines per decade. Further information on available lepidopteran insect cell lines can be found, for example, in Lynn DE, Available lepidopteran insect cell lines. Methods Mol Biol. 2007;388:117-38, which is incorporated herein by reference. In some embodiments, the recombinant cell is a mosquito cell, e.g., a cell of a mosquito species of 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 of the following mosquito species: Aedes aegypti, Aedes albopictus, Aedes pseudoscutellaris, Aedes triseriatus, Aedes vexans, Anopheles gambiae, Anopheles stephensi, Anopheles albimanus, Culex quinquefasciatus, Culex theileri, Culex tritaeniorhynchus, Culex bitaeniorhynchus, and Toxorhynchites amboinensis. Suitable mosquito cell lines include, but are not limited to, CCL-125, Aag-2, RML-12, C6 / 26, C6 / 36, C7-10, AP-61, At GRIP-1, At GRIP-2, UM-AVE1, Mos.55, Sua1B, 4a-3B, Mos.43, MSQ43, and LSB-AA695BB. In some embodiments, the mosquito cell is a cell of the C6 / 26 cell line.

[0095] In another aspect, a cell culture is provided that comprises at least one recombinant cell disclosed herein and a culture medium. In general, the culture medium can be any suitable culture medium for culturing the cells described herein. Techniques for transforming a wide variety of the above host cells and species are known in the art and described in the technical and scientific literature. Thus, a cell culture that comprises at least one recombinant cell disclosed herein is also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.

[0096] C. Transgenic Animals Also provided in another aspect is a transgenic animal comprising a nucleic acid construct (e.g., a vector, a replicon, or a srRNA molecule) described herein. In some embodiments, the transgenic animal is a vertebrate or an invertebrate. In some embodiments, the transgenic animal is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the transgenic animal is a mammal. In some embodiments, the transgenic mammal is a non-human mammal. In general, the transgenic animal of the present disclosure can be any non-human animal known in the art. In some embodiments, the non-human animal of the present disclosure is a non-human primate. Other animal species suitable for the compositions and methods of the present disclosure include animals that are (i) suitable for transgenesis and (ii) capable of rearranging immunoglobulin gene segments to generate an antibody response. Examples of such species include, but are not limited to, mice, rats, hamsters, rabbits, chickens, goats, pigs, sheep, and cows. Further examples of non-human animals suitable for the compositions and methods of the present disclosure include, but are not limited to, laboratory animals (e.g., mice, rats, hamsters, gerbils, guinea pigs, etc.), farm animals (e.g., horses, cows, pigs, sheep, goats, ducks, geese, chickens, etc.), domesticated animals and pets (e.g., cats, dogs, etc.), non-human primates (e.g., apes, chimpanzees, orangutans, monkeys, etc.), fish, amphibians (e.g., frogs, salamanders, etc.), reptiles (e.g., snakes, lizards, etc.), and other animals (e.g., foxes, weasels, rabbits, minks, beavers, ermines, otters, sables, seals, coyotes, chinchillas, deer, muskrats, opossums, etc.).

[0097] 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 of the present disclosure 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. In some embodiments, the one or more nucleic acid constructs are stably integrated into the genome of the transgenic animal. In some embodiments, the genome of the transgenic animal of the present disclosure can contain any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more copies of one or more nucleic acid constructs of the present disclosure.

[0098] Approaches and methods for preparing transgenic non-human animals are known in the art. Exemplary methods include pronuclear microinjection, DNA microinjection, lentiviral vector-mediated DNA transfer into early embryos, and sperm-mediated genetic recombination, adenovirus-mediated DNA transfer into animal sperm (e.g., pigs), retroviral vectors (e.g., avian species), somatic cell nuclear transfer (e.g., goats). The state of the art in preparing transgenic livestock is reviewed in Niemann, H. et al. (2005) Rev. Sci. Tech. 24:285-298. In some embodiments, the transgenic non-human host animals of the present disclosure are prepared using standard methods known in the art for introducing exogenous nucleic acid into the genome of non-human animals. In some embodiments, the transgenic animals of the present disclosure can be produced using classical random genome recombination techniques, or more precise techniques such as guide RNA-guided CRISPR / Cas genome editing, or DNA-guided endonuclease genome editing using NgAgo (Natronobacterium gregoryi Argonaute), or TALEN genome editing (transcription activator-like effector nuclease). In some embodiments, the transgenic animals of the present disclosure can be produced using transgenic microinjection techniques, and do not require the use of homologous recombination techniques, and therefore are considered easier to prepare and select than approaches using homologous recombination. In some embodiments, the transgenic animals produce proteins of interest as described herein.

[0099] The transgenic non-human host animals of the present disclosure are prepared using standard methods known in the art for introducing exogenous nucleic acids into the genome of non-human animals. In some embodiments, the non-human animals of the present disclosure are non-human primates. Other animal species suitable for the compositions and methods of the present disclosure include animals that are (i) suitable for transgenesis and (ii) capable of rearranging immunoglobulin gene segments to generate antibody responses. Examples of such species include, but are not limited to, mice, rats, hamsters, rabbits, chickens, goats, pigs, sheep, and cows. Approaches and methods for preparing transgenic non-human animals are known in the art. Exemplary methods include pronuclear microinjection, DNA microinjection, lentiviral vector-mediated DNA transfer into early embryos, and sperm-mediated genetic recombination, adenoviral DNA transfer into animal sperm (e.g., pigs), retroviral vectors (e.g., avian species), somatic cell nuclear transfer (e.g., goats). The state of the art in producing transgenic livestock is reviewed in Niemann, H. et al. (2005) Rev. Sci. Tech. 24:285-298.

[0100] In some embodiments, the animal is a vertebrate or invertebrate. In some embodiments, the animal is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the animal is a mammalian subject. In some embodiments, the mammal is a non-human animal. In some embodiments, the mammal is a non-human primate. In some embodiments, the transgenic animal of the present disclosure can be produced using classical random genome recombination techniques, or using more precise techniques such as guide RNA-guided CRISPR / Cas genome editing, or DNA-guided endonuclease genome editing by NgAgo (Natronobacterium gregoryi Argonaute), or TALEN genome editing (transcription activator-like effector nuclease). In some embodiments, the transgenic animal of the present disclosure can be produced using transgenic microinjection techniques, which do not require the use of homologous recombination techniques, and therefore are believed to be easier to prepare and select than approaches that use homologous recombination. In another aspect, provided herein is a method for producing a polypeptide of interest, comprising (i) raising a transgenic animal as disclosed herein, or (ii) culturing a recombinant cell comprising a nucleic acid construct as disclosed herein under conditions in which the transgenic animal or recombinant cell produces a polypeptide encoded by the GOI. In another aspect, provided herein is a method for producing a polypeptide of interest in a subject, comprising administering to the subject a nucleic acid construct as disclosed herein. In some embodiments, the subject is a vertebrate or invertebrate. In some embodiments, the subject is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the subject is a mammalian subject. In some embodiments, the mammalian subject is a human subject. Thus, recombinant polypeptides produced by the methods disclosed herein are also within the scope of the present disclosure.

[0101] Non-limiting exemplary embodiments of the disclosed methods for producing a recombinant polypeptide may include one or more of the following features: In some embodiments, the methods for producing a recombinant polypeptide of the present disclosure further include isolating and / or purifying the produced polypeptide. In some embodiments, the methods for producing a polypeptide of the present disclosure further include modifying the structure of the produced polypeptide to extend its half-life.

[0102] D. Pharmaceutical Compositions The nucleic acid constructs, recombinant cells, recombinant polypeptides of the present disclosure can be incorporated into compositions, including pharmaceutical compositions. Such compositions generally include one or more of the nucleic acid constructs, recombinant cells, recombinant polypeptides described and provided herein, and a pharma- ceutically acceptable excipient, such as a carrier. In some embodiments, the compositions of the present disclosure are formulated for the prevention, treatment, or management of a health condition, such as an immune disorder or a microbial infection (e.g., a viral infection, a microfungal infection, or a bacterial infection). For example, the compositions of the present disclosure can be formulated as a prophylactic composition, a therapeutic composition, or a pharmaceutical composition with a pharma-ceutically acceptable excipient, or a mixture thereof. In some embodiments, the compositions of the present disclosure are formulated for use as a vaccine. In some embodiments, the compositions of the present application are formulated for use as an adjuvant.

[0103] Accordingly, in one aspect, provided herein is a pharmaceutical composition comprising a pharma- ceutical acceptable excipient and a) a nucleic acid construct of the present disclosure, b) a recombinant cell of the present disclosure, and / or c) a recombinant polypeptide of the present disclosure.

[0104] Non-limiting exemplary embodiments of the pharmaceutical compositions of the present disclosure may include one or more of the following features: In some embodiments, provided herein is a composition comprising a nucleic acid construct disclosed herein and a pharma- ceutically acceptable excipient. In some embodiments, provided herein is a composition comprising a recombinant cell disclosed herein and a pharma-ceutically acceptable excipient. In some embodiments, a composition comprises a recombinant polypeptide disclosed herein and a pharma-ceutically acceptable excipient.

[0105] In some embodiments, the nucleic acid constructs (e.g., vectors or srRNA molecules) of the present disclosure can be used in naked form or can be formulated with a delivery vehicle. Exemplary delivery vehicles suitable for the compositions and methods of the present disclosure include, but are not limited to, liposomes (e.g., neutral or anionic liposomes), microspheres, immune stimulating complexes (ISCOMS), lipid-based nanoparticles (LNPs), solid lipid nanoparticles (SLNs), polyplexes, polymeric nanoparticles, viral replicon particles (VRPs), or conjugates with bioactive ligands that can facilitate delivery and / or enhance 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). Other adjuvants other than liposomes and the like are also used and are known in the art. Adjuvants can prevent antigens (e.g., nucleic acid constructs, vectors, srRNA molecules) from dispersing rapidly by sequestering them in localized deposits, or adjuvants can include substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system.

[0106] The compositions of the present disclosure can be formulated in a format compatible with the intended route of administration, such as liposomes, lipid-based nanoparticles (LNPs), polymeric nanoparticles, polyplexes, viral replicon particles (VRPs), microspheres, immune stimulating complexes (ISCOMs), conjugates of bioactive ligands, or any combination thereof. Thus, in some embodiments, the compositions of the present disclosure are formulated in liposomes.

[0107] In some embodiments, the compositions of the present disclosure are formulated into 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.

[0108] In some embodiments, the LNPs of the present disclosure can include one or more ionizable lipids. Exemplary ionizable lipids suitable for the compositions and methods of the present disclosure include those described in PCT Publications WO2020252589A1 and WO2021000041A1, and Love KT et al., Proc Natl Acad Sci USA, Feb. 2, 2010 107 (5) 1864-1869, which are incorporated herein by reference in their entireties.

[0109] Thus, in some embodiments, the LNPs of the present disclosure comprise one or more lipid compounds described in Love KT et al., 2010, supra, such as C16-96, C14-110, and C12-200. In some embodiments, the LNPs comprise an ionizable cationic lipid selected from the group consisting of ALC-0315, C12-200, LN16, MC3, MD1, SM-102, and any combination thereof. In some embodiments, the LNPs of the present disclosure comprise C12-200.

[0110] In some embodiments, the LNPs of the present disclosure comprise one or more cationic lipids. Suitable cationic lipids include, but are not limited to, 98N12-5, C12-200, C14-PEG2000, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1.

[0111] In some embodiments, the LNPs of the present disclosure include one or more neutral lipids. As mentioned above, in some embodiments, neurolipids, also known as "structured lipids" or "helper lipids", can also be incorporated into the lipid formulations and lipid particles. The lipid formulations and lipid particles can include one or more structured lipids at about 10-40 mole % of the composition. Suitable structured lipids support the formation of particles during manufacture. Structured lipids refer to any of a number of lipid species that exist in either anionic, uncharged, or neutral zwitterionic form at physiological pH. Exemplary structured lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, diacylphosphatidylglycerols, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, and cerebrosides.

[0112] Exemplary structured lipids are zwitterionic lipids, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 -carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (trans DOPE).

[0113] In another embodiment, the structured lipid can be any lipid that is negatively charged at physiological pH.These lipids include phosphatidylglycerols, such as dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleylphosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, and other anionic modification groups attached to neutral lipids.Other suitable structured lipids include glycolipids (e.g., monosialoganglioside GM1).

[0114] Non-limiting neutral lipids suitable for the compositions and methods of the present disclosure include DPSC, DPPC, POPC, DOPE, and SM. In some embodiments, the LNPs of the present disclosure include one or more ionizable lipid compounds described in PCT Publications WO2020252589A1 and WO2021000041A1, which are incorporated herein by reference in their entireties.

[0115] In some embodiments, the LNPs of the disclosure include at least one lipid selected from the group consisting of C12-200, C14-PEG2000, DOPE, DMG-PEG2000, DSPC, DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG).

[0116] In some embodiments, where the delivery system described herein comprises an LNP, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 100:1 to about 3:1, about 70:1 to 10:1, or 16:1 to 4:1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 16:1 to 4:1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 20:1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 8:1. In some embodiments, the mean diameter of the lipid-based nanoparticles (LNPs) is about 1000 nm, about 500 nm, about 250 nm, about 200 nm, about 150 nm, about 100 nm, about 75 nm, about 50 nm, or less than about 25 nm. In some embodiments, the mean diameter of the LNPs is in the range of about 70 nm to 100 nm. In some embodiments, the average diameter of the LNPs ranges from about 88 nm to about 92 nm, 82 nm to about 86 nm, or about 80 nm to about 95 nm.

[0117] An embodiment of the lipid formulation may include a stabilizer to ensure the integrity of the mixture. Stabilizers are a class of molecules that disrupt or aid in the formation of 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), Myrj52 (polyoxyethylene(40) stearate), and Brij™ S10 (polyoxyethylene(10) stearyl ether). Polyethylene glycol-linked lipids may also be used. Stabilizers may be used alone or in combination with each other.

[0118] In some embodiments, the stabilizer comprises about 0.1-3 mol% of the total lipid mixture. In some embodiments, the stabilizer comprises about 0.5-2.5 mol% of the total lipid mixture. In some embodiments, the stabilizer is present at greater than 2.5 mol%. In some embodiments, the stabilizer is present at 5 mol%. In some embodiments, the stabilizer is present at 10 mol%. In some embodiments, the stabilizer is present at about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, etc. In other embodiments, the stabilizer is present at 2.6-10 mol% of the lipid mixture. In other embodiments, the stabilizer is present at greater than 10 mol% of the lipid mixture.

[0119] In certain applications, steroids may also be included in the lipid composition and lipid particles made therefrom may contain sterols, such as cholesterol or plant sterols.

[0120] In some embodiments, the compositions of the present disclosure are formulated into polymeric nanoparticles. Examples of polymers suitable for the compositions and methods of the present disclosure are described 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 can include naturally occurring cationic polymers. In some embodiments, the naturally occurring cationic polymers can include chitosan, gelatin, dextran, cellulose, cyclodextrin, or combinations thereof. In some embodiments, the cationic polymer can be a synthetic cationic polymer. In some embodiments, the synthetic cationic polymer may include polyethyleneimine (PEI), poly-L-lysine (PLL), poly(amino acids) (PAA), poly(amidoamines) (PAMAM), poly(amino-co-esters) (PAE), poly(2-N,N-dimethylaminoethyl methacrylate), poly(beta amino esters) (PBAE), imidazole-containing polymers, tertiary amine-containing polymers, poly(2-(dimethylamino)ethyl methacrylate), poly-N-(2-hydroxypropyl) methacrylamide, polyamidoamine dendrimers, cationic glycopolymers, or derivatives thereof.

[0121] In some embodiments, the non-cationic polymer is negatively charged (i.e., anionic) or electronically neutral. In some embodiments, the non-cationic polymer comprises polyethylene glycol (PEG), polyester (e.g., polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyglycolic acid (PGA), polycaprolactone (PCL)), and polysarcosine (pSar), or derivatives thereof.

[0122] In some embodiments, the polymer may be water soluble and / or biodegradable. In some embodiments, the polymeric nanoparticles include one or more of the following: poly-(?-L-glutamylglutamine) (PGGA), poly-(?-L-aspartylglutamine) (PGAA), poly-L-lactic acid (PLLA), poly-(lactic acid-co-glycolic acid) (PLGA), polyalkylcyanoacrylates (PACA), polyanhydrides, polyhydroxy acids, polypropylfumarates, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, [N-(2-hydroxypropyl)methacrylamide] (HPMA) copolymers, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polyureas, polyamines, polyepsilon-caprolactone (PCL), and copolymers thereof.

[0123] 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.

[0124] In some embodiments, the immunogenic composition is formulated as a vehicle for gene delivery of biotherapeutic agents, such as different molecules with biological activity.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.

[0125] In some embodiments, the immunogenic composition is substantially non-immunogenic or minimally immunogenic (e.g., a composition that minimally stimulates an immune response in a subject). In some embodiments, the non-immunogenic or minimally immunogenic composition is formulated as a biotherapeutic. In some embodiments, the pharmaceutical composition is formulated for one or more of intranasal, transdermal, intrathecal, intraperitoneal, intramuscular, intratracheal, intranodal, intratumoral, intraarticular, intravenous, subcutaneous, intravaginal, intraocular, rectal, and oral administration.

[0126] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, sterile water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In these cases, the composition must be sterile and fluid to the extent that easy syringability exists. It is stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants such as sodium dodecyl sulfate. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, the composition will typically contain an isotonic agent, for example, sugar, polyalcohol such as mannitol, sorbitol, and / or sodium chloride. Prolonged absorption of the injectable composition can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

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

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

[0129] In some embodiments, the composition is formulated for one or more of intranasal, intrathecal, transdermal, intramuscular, intranodal, intravenous, intraperitoneal, oral, intravaginal, or intracranial administration. In some embodiments, the administered composition causes an increase in the production of interferon in the subject. In some embodiments, the administered composition induces the production of one or more proinflammatory molecules in the subject. In some embodiments, the one or more proinflammatory molecules include interferon gamma (IFNγ), cytokines, TNF-α, GM-CSF, and MIP1α, granzyme B, granzyme A, perforin, or any combination thereof.

[0130] Method of Disclosure Administration of any one of the therapeutic compositions, e.g., nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions described herein, can be used to treat relevant health conditions, e.g., proliferative diseases (e.g., cancer), infectious diseases (e.g., acute, chronic, 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 described herein can be useful for modulating, e.g., inducing or suppressing, a pharmacodynamic effect in a subject in need thereof. In some embodiments, the pharmacodynamic effect includes eliciting an immune response in a subject. Non-limiting examples of pharmacodynamic effects include immunogenic effects, biomarker responses, therapeutic effects, preventive effects, desired effects, undesirable effects, adverse effects, and effects in disease models. Accordingly, one aspect of the disclosure relates to a method for modulating a pharmacodynamic effect in a subject in need thereof, comprising administering to the subject a composition comprising one or more of the following: (a) a nucleic acid construct described herein; (c) a recombinant cell described herein; (c) a recombinant polypeptide described herein; and (d) a pharmaceutical composition described herein. 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 undesirable effect, an adverse effect, and an effect in a disease model.

[0131] In some embodiments, the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions described herein can be incorporated into therapeutic agents for use in methods of treating a subject suffering from, suspected of suffering from, or at risk of suffering from one or more relevant health conditions or diseases. Thus, in another aspect, a method of preventing or treating a health condition in a subject is provided herein, comprising administering to the subject, prophylactically or therapeutically, a composition comprising one or more of the following: (a) a self-replicating RNA construct described herein; (b) a nucleic acid described herein; (c) a recombinant cell described herein; and (d) a pharmaceutical composition described herein. In some embodiments, the administered composition induces an immune response in the subject. In some embodiments, the administered composition induces the production of one or more proinflammatory molecules in the subject. In some embodiments, the one or more proinflammatory molecules comprise 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 previously been treated with one or more therapies and has developed at least partial resistance to said one or more therapies.

[0132] Exemplary conditions or diseases include, but are not limited to, cancer, immune diseases, autoimmune diseases, inflammatory diseases, gene therapy, gene replacement, cardiovascular diseases, age-related conditions, rare diseases, acute infections, and chronic infections. In some embodiments, the subject is a patient under the care of a physician.

[0133] Examples of autoimmune diseases suitable for the methods of the present disclosure include, but are not limited to, rheumatoid arthritis, osteoarthritis, Still's disease, familial Mediterranean fever, systemic sclerosis, multiple sclerosis, ankylosing spondylitis, Hashimoto's thyroiditis, systemic lupus erythematosus, Sjogren's syndrome, diabetic retinopathy, diabetic vasculopathy, diabetic neuropathy, insulitis, psoriasis, alopecia major, warm-cold autoimmune hemolytic anemia (AIHA), pernicious anemia, acute inflammatory disease, autoimmune adrenalitis, chronic inflammatory demyelinating polyneuropathy (CIDP), Lambert-Eaton syndrome, lichen sclerosis, Lyme disease, Graves' disease, Behcet's disease, Meniere's disease, reactive arthritis (Reiter's syndrome), Churg-Strauss syndrome, Cogan's syndrome, CREST syndrome, pemphigus vulgaris and pemphigus foliaceus, bullous pemphigoid, rheumatic polyneuropathy, and psoriasis. Myalgia, Polymyositis, Primary Biliary Cirrhosis, Pancreatitis, Peritonitis, Psoriatic Arthritis, Rheumatic Fever, Sarcoidosis, Sjögrensen's Syndrome, Scleroderma, Celiac Disease, Stiff Man Syndrome, Takayasu's Arteritis, Transient Gluten Intolerance, Autoimmune Uveitis, Vitiligo, Polychondritis, Dermatitis Herpetiformis (DH) or Duhring's Disease, Fibromyalgia, Goodpasture's Syndrome, Guillain-Barré Syndrome These include: autoimmune syndrome, Hashimoto's thyroiditis, autoimmune hepatitis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, myasthenia gravis, immune complex disease, glomerulonephritis, polyarteritis nodosa, antiphospholipid syndrome, autoimmune polyglandular syndrome, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), urticaria, autoimmune infertility, juvenile rheumatoid arthritis, sarcoidosis, and autoimmune cardiomyopathy.

[0134] Non-limiting examples of infectious diseases suitable for the methods of the present invention include infections caused by viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis B virus (HCV), cytomegalovirus (CMV), respiratory syncytial virus (RSV), human papillomavirus (HPV), Epstein-Barr virus (EBV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome (MERS), influenza virus, Ebola virus, etc. Additional infectious diseases suitable for the methods of the present disclosure include infections caused by intracellular parasites such as Leishmania, Rickettsia, Chlamydia, Coxiella, Plasmodium, Brucella, Mycobacteria, Listeria, Toxoplasma, Trypanosoma, etc. In some embodiments, the replicon construct, srRNA construct, nucleic acid construct, recombinant cell, and / or pharmaceutical composition is an antibody for treating an autoimmune or inflammatory disease, such as glomerulonephritis, inflammatory bowel disease, nephritis, peritonitis, psoriatic arthritis, osteoarthritis, Still's disease, familial Mediterranean fever, systemic sclerosis and sclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, acute lung injury, meningitis, encephalitis, uveitis, multiple myeloma, glomerulonephritis, nephritis, asthma, atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud's syndrome, Sjogren's syndrome, juvenile onset diabetes, Reiter's disease, Behcet's disease, immune complex nephritis, IgA nephropathy, IgM polyposis nephropathy ... In some embodiments, the compositions may be useful for the treatment and / or prevention of: acute neuropathy, 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, Omens' syndrome, chronic renal failure, autoimmune thyroid disease, acute infectious mononucleosis, HIV, herpes virus related diseases, human viral infections, coronaviruses, other enteroviruses, herpes viruses, influenza viruses, parainfluenza viruses, respiratory syncytial virus or adenovirus infections, bacterial pneumonia, wounds, sepsis, stroke / cerebral edema, ischemia-reperfusion injury, and hepatitis C.

[0135] Non-limiting examples of inflammatory diseases suitable for the methods of the present disclosure include inflammatory diseases such as asthma, inflammatory bowel disease (IBD), chronic colitis, splenomegaly, rheumatoid arthritis, and the like.

[0136] In some embodiments, the condition is a proliferative disorder or a microbial infection (e.g., a bacterial infection, a microfungal infection, or a viral infection). In some embodiments, the subject has or is suspected of having a condition associated with a proliferative disorder or a microbial infection (e.g., a bacterial infection, a microfungal infection, or a viral infection).

[0137] In some embodiments, the health condition is a rare disease, e.g., a disease or condition affecting fewer than 200,000 people in the United States as defined by the Orphan Drug Act (www.fda.gov / patients / rare-diseases-fda), and / or an inflammatory disease, and / or an autoimmune disease. In some embodiments, the subject has or is suspected of having a condition associated with an inflammatory disease and / or an autoimmune disease and / or a rare disease (e.g., including but not limited to, familial Mediterranean fever or adult-onset Still's disease).

[0138] In some embodiments, the disclosed compositions are formulated to be compatible with the intended route of administration. For example, the disclosed nucleic acid constructs (e.g., replicon constructs, e.g., srRNA constructs), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions may be administered orally or by inhalation, but are more likely to be administered by parenteral routes. Examples of parenteral administration routes include, for example, intramuscular, intratumoral, intraocular, intravenous, intranodal, intradermal, subcutaneous, transdermal (topical), transmucosal, intravaginal, and rectal administration. In some embodiments, the compositions are administered intramuscularly. In some embodiments, the compositions are administered intratumorally. Solutions or suspensions used for parenteral administration may contain the following components: a sterile diluent, such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, phosphates, Tris, sucrose; and tonicity adjusters, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as monobasic and / or dibasic sodium phosphate, hydrochloric acid, or sodium hydroxide (e.g., pH adjusted to about 7.2 to 7.8, e.g., 7.5). Parenteral formulations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple dose vials.

[0139] Dosage, toxicity, and therapeutic efficacy of such subject nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50It can be expressed as a ratio. Compounds that exhibit a high therapeutic index are generally suitable. Compounds that exhibit toxic side effects may also be used, but care must be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells, thereby reducing side effects.

[0140] For example, the data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds is generally determined to be sufficient to induce ED symptoms with little or no toxicity. 50 The dosage may vary within this range depending on the dosage form used and the route of administration used. For any compound used in the disclosed methods, the therapeutically effective dose can be estimated initially from cell culture assays. Dosages are determined based on the IC 50 The test compound can be formulated in animal models to achieve a circulating plasma concentration range that includes (e.g., the concentration of the test compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0141] The therapeutic compositions, e.g., nucleic acid constructs, e.g., srRNA constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions described herein, can be administered from once or more per day to once or more per week (including once every other day). One of skill in the art will appreciate that certain factors may affect the dosage and timing required to effectively treat a subject. Such factors include, but are not limited to, severity of the disease, previous treatments, the general health and / or age of the subject, and other diseases present. Furthermore, treatment of a subject with a therapeutically effective amount of the subject multivalent polypeptides and multivalent antibodies of the present disclosure can include a single treatment or can include a series of treatments. In some embodiments, the composition is administered every 8 hours for 5 days, followed by a rest period of 2-14 days, e.g., 9 days, and then every 8 hours for another 5 days. With respect to nucleic acid constructs (e.g., replicon constructs, e.g., srRNA constructs) and recombinant polypeptides, a therapeutically effective amount (e.g., an effective dosage) of a nucleic acid construct or recombinant polypeptide of the present disclosure will depend on the nucleic acid construct or recombinant polypeptide selected. For example, a single dosage in the range of about 0.001-0.1 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 dosage in the range of about 0.03 μg-300 μg / kg of patient body weight can be administered. In some embodiments, a single dosage in the range of about 0.3 mg-3 mg / kg of patient body weight can be administered.

[0142] As stated above, a therapeutically effective amount includes an amount of a therapeutic composition sufficient to promote a particular effect when administered to a subject, for example, a subject suffering from, suspected of suffering from, or at risk of suffering from a health condition, such as, for example, a disease or infection. In some embodiments, an effective amount includes an amount sufficient to prevent or delay the onset of a disease or infection symptom, alter the course of a disease or infection symptom (for example, but not limited to, slow the progression of a disease or infection symptom), or reverse a disease or infection symptom. It is understood that for any particular case, the appropriate effective amount can be determined by one skilled in the art using routine experimentation.

[0143] The efficacy of a treatment, including the disclosed therapeutic compositions for the treatment of a disease or infection, can be determined by a skilled clinician. However, a treatment is considered an effective treatment if at least any one or all of the signs or symptoms of the disease or infection are improved or alleviated. Efficacy can also be measured by the absence of worsening of the individual's condition (e.g., the progression of the disease or infection is stopped or at least slowed) as assessed by the need for hospitalization or medical intervention. Methods for measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease or infection in a subject or animal (some non-limiting examples include humans or mammals) and includes (1) inhibiting the disease or infection, e.g., halting or slowing the progression of the symptoms, or (2) alleviating the disease or infection, e.g., causing regression of the symptoms, and (3) preventing or reducing the likelihood of the onset of the symptoms.

[0144] In some embodiments, the nucleic acid constructs (e.g., replicon constructs, e.g., srRNA constructs), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure can be administered to a subject in a composition with a pharma- ceutically acceptable carrier in an amount effective to stimulate an immune response. In general, a subject is immunized with an initial series of injections (or administered by one of the other routes described below), and then a booster can be administered to enhance the protection provided by the initial series. The initial series of injections and subsequent boosters are administered at doses and for periods of time necessary to stimulate the subject's immune response. In some embodiments, the administered composition increases the production of interferon in the subject. In some embodiments of the disclosed methods, the subject is a mammal. In some embodiments, the mammal is a human subject.

[0145] As mentioned above, pharma- ceutically acceptable carriers suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In these cases, the compositions must be sterile and fluid to the extent that easy syringability exists. The compositions must further be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.

[0146] Sterile injectable solutions can be prepared by incorporating the required amount of the nucleic acid constructs, recombinant cells, and / or recombinant polypeptides in an appropriate solvent containing one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

[0147] When the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions are appropriately protected as described above, they can be orally administered, for example, with an inert diluent or an assimilable edible carrier. The nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions and other ingredients can also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the individual's diet. For oral therapeutic administration, the active compounds can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0148] In some embodiments, the nucleic acid construct and recombinant polypeptide of the present disclosure can be delivered to cells or subjects by lipid-based nanoparticles (LNPs).LNPs are generally less immunogenic than viral particles.Many people have pre-existing immunity to viral particles, but not to LNPs.Furthermore, adaptive immune responses to LNPs are unlikely to occur, allowing repeated administration of LNPs.

[0149] Several different ionizable cationic lipids have been developed for use in LNPs. These include C12-200, MC3, LN16, and MD1, among others. For example, in one type of LNP, a GalNAc moiety is attached to the outside of the LNP, which acts as a ligand for uptake into the liver via the asialoglycoprotein receptor. Any of these cationic lipids can be used to formulate LNPs for delivery of the nucleic acid constructs and recombinant polypeptides of the present disclosure to the liver.

[0150] In some embodiments, LNP refers to any particle with a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. Alternatively, the size of the nanoparticles can range from 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

[0151] LNPs can be made from cationic, anionic, or neutral lipids. Neutral lipids, such as the fusogenic phospholipid DOPE and the membrane component cholesterol, can be included in LNPs as "helper lipids" to enhance transfection activity and nanoparticle stability. Limitations of cationic lipids include poor efficacy due to poor stability and rapid clearance, as well as the generation of inflammatory or anti-inflammatory responses. LNPs can also have hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.

[0152] Numerous lipids or lipid combinations known in the art can be used to generate LNPs. Non-limiting examples of lipids suitable for use in generating LNPs include DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). 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.

[0153] In some embodiments, lipids can be combined in any number of molar ratios to produce LNPs. Additionally, polynucleotides can be combined with lipids in a wide range of molar ratios to produce LNPs.

[0154] In some embodiments, the therapeutic compositions described herein, e.g., nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions, are incorporated into therapeutic compositions for use in methods of preventing or treating a subject suffering from, suspected of suffering from, or who may be at high risk for suffering from, cancer, an autoimmune disease, and / or an infectious disease.

[0155] In some embodiments, the therapeutic compositions described herein, such as nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions, are incorporated into therapeutic compositions for use in methods for preventing or treating subjects suffering from, suspected of suffering from, or at risk of suffering from, 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.

[0156] Additional Treatments In some embodiments, the compositions of the present disclosure are administered to a subject individually as a single therapy (monotherapy) or as a first therapy in combination with at least one additional therapy (e.g., a second therapy). In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiation therapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery. In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiation therapy, immunotherapy, hormone therapy, toxin therapy, or surgery. In some embodiments, the first therapy and the second therapy are administered simultaneously. In some embodiments, the first therapy and the second therapy are administered simultaneously. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy is administered before the second therapy. In some embodiments, the first therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and / or after the second therapy. In some embodiments, the first therapy and the second therapy are administered alternately. In some embodiments, the first and second therapies are administered together in a single formulation.

[0157] kit Also provided herein are various kits for carrying out the methods described herein, as well as instructions for their manufacture and use. In particular, some embodiments of the present disclosure provide kits for modulating a pharmacodynamic effect. Some embodiments of the present disclosure provide kits for inducing an immune response in a subject. Some other embodiments relate to kits for the prevention of a condition in a subject in need thereof. Some other embodiments relate to kits for methods of treating a condition in a subject in need thereof. For example, in some embodiments, provided herein are kits that include one or more of the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions provided and described herein, as well as instructions for their manufacture and use.

[0158] In some embodiments, the kit of the present disclosure further comprises one or more means useful for administering any one of the provided nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions to a subject. For example, in some embodiments, the kit of the present disclosure further comprises one or more syringes (including pre-filled syringes) and / or catheters (including pre-filled syringes) used to administer any one of the provided nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions to a subject. In some embodiments, the kit may have one or more additional therapeutic agents that can be administered simultaneously or sequentially with other kit components for a desired purpose, such as diagnosing, preventing, or treating a condition in a subject in need thereof.

[0159] Any of the above kits may further comprise one or more additional reagents, where such additional reagents may be selected from dilution buffers, reconstitution solutions, wash buffers, control reagents, control expression vectors, negative controls, positive controls, reagents suitable for in vitro production of the provided nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure.

[0160] In some embodiments, the components of the kit may be in separate containers. In some other embodiments, the components of the kit may be combined in a single container. Thus, in some embodiments of the present disclosure, the kit includes one or more of the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions provided and described herein in one container (e.g., a sterile glass or plastic vial), and another therapeutic agent in another container (e.g., a sterile glass or plastic vial).

[0161] In another embodiment, the kit includes a combination of compositions as described herein, which comprises one or more nucleic acid constructs, recombinant cells, and / or recombinant polypeptides of the present disclosure in combination with one or more additional therapeutic agents, optionally formulated together in a pharmaceutical composition, contained within a single common container.

[0162] Where the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit may include a device for performing such administration (e.g., an injection device or catheter). For example, the kit may include one or more hypodermic needles or other injection devices described above that include one or more nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure.

[0163] In some embodiments, the kit components may be in separate containers, hi other embodiments, the kit components may be combined in a single container.

[0164] In some embodiments, the kit may further include instructions for using the components of the kit to carry out the methods disclosed herein. For example, the kit may include a package insert containing information about the pharmaceutical compositions and dosage forms in the kit. In general, such information will help patients and physicians to effectively and safely use the enclosed pharmaceutical compositions and dosage forms. For example, the following information about the disclosed combination may be provided in the package insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, side effects, overdosage, appropriate dosage and administration, method of supply, appropriate storage conditions, references, manufacturer / distributor information, and intellectual property information.

[0165] The instructions for carrying out the method are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. The instructions can be present as a kit insert, such as on a label on the container of the kit or a component thereof (e.g., associated with the package or subpackage). The instructions can be present as an electronic storage data file present on a suitable computer-readable storage medium, such as a CD-ROM, diskette, flash drive, etc. In some cases, the actual instructions are not included in the kit, and a means for obtaining the instructions from a remote source (e.g., via the Internet) is provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.

[0166] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0167] No admission is made that the references cited herein constitute prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinence of the cited documents. Although a number of sources of information have been referenced herein, such as scientific journal articles, patent documents, textbooks, and the like, it will be clearly understood that this reference does not constitute an admission that any of these documents constitutes part of the general knowledge in the art.

[0168] The discussion of the general methods presented herein is for illustrative purposes only: other alternative methods and substitutes will be apparent to those of skill in the art upon review of this disclosure, and are intended to be included within the spirit and scope of this application.

[0169] Further embodiments are disclosed in more detail in the following examples, which are provided by way of illustration and are not intended to limit the scope of the disclosure or the claims. EXAMPLES

[0170] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are described in the literature, e.g., Sambrook, J., & Russell, DW (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, DW (2001). “Sambrook”); Ausubel, FM (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, DM et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, MG et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998).Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, KB, Ferree, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, EA (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, SL et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley (including supplements up to 2014); and Makrides, SC (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences BV, the disclosures of which are incorporated herein by reference.

[0171] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not intended to limit the scope of the disclosure or claims in any way.

[0172] Example 1 Construction of WEEV vector This example describes experiments performed to construct a basic WEEV vector (e.g., containing no heterologous genes), which is then used to construct a WEEV vector for expression of a gene of interest (e.g., the hemagglutinin precursor HA of influenza A virus H5N1).

[0173] A basic WEEV vector (i.e., without the heterologous gene of interest) is constructed as follows: The basic WEEV vector (see, for example, Figure 2A) is synthesized de novo in four approximately 4 kb pieces with some modifications to the reference sequence (Genbank GQ287641). Ambiguous nucleotide assignments in the reference sequence are assigned to nucleotides that match the synonymous codons for the encoded residues. Silent mutations are incorporated to remove SapI and SpeI restriction sites. A unique restriction site (SpeI, 5'-A'CTAG,T-3') can be incorporated in place of the coding sequence of the native WEEV structural gene (where 5'A corresponds to the position of the structural polyprotein ATG start codon and 3'T corresponds to the position of the structural polyprotein stop codon TAA). A 5' adaptor sequence (5'-CTGGAGACGTGGAGGAGAACCCTGGACCT-3'; SEQ ID NO:3) is inserted upstream of the SpeI site and a 3' adaptor sequence (5'-GACCGCTACGCCCCAATGACCCGACCAGC-3'; SEQ ID NO:4) is inserted downstream of the SpeI site for subsequent Gibson Assembly® manipulation (Gibson et al., Nat. Methods 6, 343-345, 2009). A bacteriophage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO:5) may be included upstream of the WEEV genomic sequence and downstream a poly(A) sequence followed by a SapI site, which cuts upstream of the recognition site. Immediately downstream of the SapI site is a T7 terminator sequence (5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO:6), followed by a unique restriction site (NotI, 5'-GC'GGCC,GC-3'). These parts are joined in a five-piece Gibson Assembly® reaction: the linearized pYL backbone and the four synthetic fragments to give the WEEV base vector.

[0174] In some experiments, the sequence encoding one or more nsPs is replaced with a heterologous nsP, while in other experiments the sequence encoding one or more UTRs is replaced with a heterologous UTR.

[0175] The construction of WEEV vectors containing heterologous genes is carried out as follows: The WEEV vector depicted in FIG. 2B is constructed by linearizing the empty WEEV vector of FIG. 2A by SpeI digestion. The hemagglutinin (HA) gene from influenza (Genbank AY651334) is computationally codon-optimized / refactored for human expression and synthesized de novo (IDT). The synthesis product is amplified using primers that add 5' and 3' adapter sequences to the ends of the HA gene. The digestion and PCR products are joined by the Gibson Assembly® procedure to generate the final vector.

[0176] Example 2 In vitro evaluation of modified WEEV vectors This example describes in vitro experiments performed to assess the expression levels of the synthetic WEEV srRNA constructs described in Example 1 above and to examine their differential behavior (e.g., replication and protein expression).

[0177] In these experiments, synthetic srRNA constructs derived from WEEV strain Imperial are designed and then evaluated.

[0178] In vitro transcription: RNA is prepared by in vitro transcription from SapI-linearized plasmid templates using bacteriophage T7 polymerase with a 5'ARCA cap (HiScribe™ T7 ARCA mRNA Kit, NEB) or by uncapped transcription (HiScribe™ T7 High Yield RNA Synthesis Kit, NEB) followed by addition of a 5' Cap 1 (Vaccinia Capping System, mRNA Cap 2'-O-Methyltransferase, NEB). RNA is then purified using phenol / chloroform extraction or column purification (Monarch® RNA Cleanup Kit, NEB). RNA concentration is measured by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific).

[0179] Reproduction : RNA is transfected into BHK-21 cells or Vero cells (e.g., 4D-Nucleofector™, Lonza) by electroporation. 15-20 hours after transfection, cells are fixed and permeabilized (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen) and then stained using a PE-conjugated anti-dsRNA mouse monoclonal antibody (J2, Scicons) and the frequency of dsRNA+ cells and the mean fluorescence intensity (MFI) of dsRNA in individual cells are quantified by fluorescent flow cytometry.

[0180] Protein expression : RNA is transfected into BHK-21 cells or Vero cells (e.g., 4D-Nucleofector™, Lonza) by electroporation. 15-20 hours after transfection, cells are fixed, permeabilized (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen), and stained using APC-conjugated anti-HA mouse monoclonal antibody (2B7, Abcam) to quantify the frequency of cells expressing HA protein in individual cells and the mean fluorescence intensity (MFI) of HA protein by fluorescent flow cytometry.

[0181] Additional Experiments BHK-21 or Vero cells are pretreated with a titration curve of recombinant interferon (IFN) followed by RNA electroporation and the effect on vector replication and protein expression is measured using the assays described above.

[0182] In some experiments, BHK-21 cells are transformed with WEEV srRNA constructs. WEEV srRNA without the target GOI is transformed by electroporation, and 20 hours after transformation, transformed cells are fixed, permeabilized, and stained using a PE-conjugated anti-dsRNA mouse monoclonal antibody (J2, Scicons) to quantitate the frequency of dsRNA+ cells by fluorescent flow cytometry. Additionally, WEEV srRNA containing the coding sequence of the target GOI is similarly transformed into BHK-21 cells, and in addition to dsRNA detection, transgene expression is detected using an APC-conjugated anti-GOI monoclonal antibody. Positive staining of individual cells with both anti-dsRNA and anti-GOI antibodies demonstrates that the modified WEEV design described herein is an effective synthetic srRNA capable of RNA replication and transgene expression.

[0183] Example 3 In vivo evaluation of modified WEEV vectors This example describes in vivo experiments performed to evaluate immune responses following vaccination with the synthetic WEEV srRNA constructs described in Examples 1 and 2 above (e.g., both unformulated and LNP-formulated vectors).

[0184] In these experiments, synthetic srRNA constructs derived from WEEV strain Imperial are designed and then evaluated.

[0185] Mice and injectionsFemale C57BL / 6 or BALB / c mice are purchased from Envigo, Charles River Labs or Jackson Laboratories. On the day of dosing, 0.1-10 μg of material is injected intramuscularly, split into both quadriceps. Vectors are administered either unformulated, LNP-formulated, or polymer-formulated in saline. Animals are monitored for weight and other general observations throughout the study. For immunogenicity studies, animals are dosed on days 0 and 21. Spleens are collected on day 35 and serum is isolated on days 14 and 35. For protein expression studies, animals are dosed on day 0 and protein expression or bioluminescence is assessed on days 1, 3, and / or 7. In some experiments where the WEEV srRNA construct encodes a reporter protein such as luciferase, in vivo imaging of luciferase activity is performed using the IVIS system at designated time points. In some experiments where the WEEV srRNA construct encodes a secreted protein, systemic levels will be assayed by serum ELISA.

[0186] LNP formulation In some experiments, srRNA is formulated into lipid nanoparticles using a microfluidic mixer and analyzed for particle size, polydispersity using dynamic light scattering, and encapsulation efficiency. In these experiments, a wide range of lipid molar ratios are used in formulating the LNP particles. Exemplary lipid molar ratios used in these experiments are 35% C12-200, 46.5% cholesterol, 2.5% PEG-2K, and 16% DOPE.

[0187] ELISpot To measure the magnitude of HA-specific T cell responses, IFNγ ELISpot analysis is performed using the Mouse IFNγ ELISpot PLUS Kit (HRP) (MabTech) according to the manufacturer's instructions. In these experiments, spleen cells are isolated and cultured at 100x10 ng / ml, e.g., 5x10 ng / ml, in medium containing a peptide representing a T cell epitope of the protein of interest encoded by the WEEV srRNA construct. 6Resuspend to an appropriate concentration, such as cells / mL. These experiments also include one or more positive controls, such as PMA / ionomycin, and DMSO used as a mock stimulus.

[0188] antibody Antibody responses to measure total HA-specific IgG are measured using an ELISA kit from Alpha Diagnostic International according to the manufacturer's instructions. While certain alternatives of the present disclosure are disclosed, it is understood that various modifications and combinations are possible and are considered to be within the true spirit and scope of the appended claims. Accordingly, there is no intention to be limited to the precise summary and disclosure presented herein.

Claims

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

2. (a) The modified viral genome or srRNA lacks a substantial portion of a nucleic acid sequence encoding one or more viral structural proteins, (b) The modified viral genome or srRNA does not contain a nucleic acid sequence encoding a viral structural protein, (c) The nucleic acid construct further comprises one or more expression cassettes, each of which comprises a promoter operably linked to a heterologous nucleic acid sequence, (d) The nucleic acid construct further comprises one or more expression cassettes, each of which comprises a promoter operably linked to a heterogeneous nucleic acid sequence, and at least one of the expression cassettes comprises a subgenome (sg) promoter operably linked to a heterogeneous nucleic acid sequence, (e) The nucleic acid construct further comprises one or more expression cassettes, each of which comprises a promoter operably linked to a heteronucleotide sequence, and at least one of the expression cassettes comprises a subgenome (sg) promoter operably linked to a heteronucleotide sequence, and the sg promoter is a 26S subgenome promoter. (f) At least one non-structural protein (nsP) or a portion thereof of the modified WEEV genome or srRNA is heterogeneous to the rest of the modified WEEV genome or srRNA, (g) The nucleic acid construct further comprises a nucleic acid sequence encoding a heterogeneous nsP or a part thereof, (h) The nucleic acid construct further comprises one or more untranslated regions (UTRs), (i) The nucleic acid construct further comprises one or more untranslated regions (UTRs), and at least one of the UTRs is a heterogeneous UTR, (j) The nucleic acid construct further comprises one or more expression cassettes, each of which comprises a promoter operably linked to a heterogeneous nucleic acid sequence, and at least one of the expression cassettes comprises the coding sequence of a target gene (GOI), (k) The nucleic acid construct further comprises one or more expression cassettes, each of which comprises a promoter operably linked to a heterogeneous nucleic acid sequence, at least one of which comprises a coding sequence for a target gene (GOI), and the GOI codes for a polypeptide selected from the group consisting of therapeutic polypeptides, prophylactic polypeptides, diagnostic polypeptides, dietary supplement polypeptides, industrial enzymes, and reporter polypeptides. (l) The nucleic acid construct further comprises one or more expression cassettes, each of which comprises a promoter operably linked to a heterogeneous nucleic acid sequence, at least one of which comprises a coding sequence for a target gene (GOI), and the GOI encodes a polypeptide selected from the group consisting of antibodies, antigens, immunomodulators, enzymes, signaling proteins, and cytokines. (m) The nucleic acid construct further comprises one or more expression cassettes, each of which comprises a promoter operably linked to a heterogeneous nucleic acid sequence, at least one of which comprises a coding sequence for a target gene (GOI), and the coding sequence for the GOI is optimized to be expressed at a higher level than the expression level of a reference coding sequence. (n) The nucleic acid construct further comprises one or more expression cassettes, each of which comprises a promoter operably linked to a heterogeneous nucleic acid sequence, at least one of which comprises a coding sequence for a target gene (GOI), and the coding sequence for the GOI is optimized to enhance RNA stability, and / or (o) 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 Sequence ID No. 1 or Sequence ID No.

2. The nucleic acid construct according to claim 1.

3. Recombinant cells comprising the nucleic acid construct according to claim 1.

4. (a) The recombinant cell is a eukaryotic cell, (b) The recombinant cells are animal cells, (c) The recombinant cells are vertebrate cells or invertebrate cells, (d) The recombinant cells are insect cells, (e) The recombinant cells are mosquito cells, (f) The recombinant cells are mammalian cells, or (g) The recombinant cells are vertebrate cells, and the recombinant cells are SV40-transformed monkey kidney CV1 cells (COS-7), human fetal kidney cells (e.g., HEK293 or HEK293 cells), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells), monkey kidney cells (CV1), human cervical cancer cells (HeLa), canine kidney cells (MDCK), buffalo rat hepatocytes (BRL3A), human lung cells (W138), human hepatocytes (HepG2), mouse mammary tumor cells (MMT060562), TRI cells, FS4 cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (Vero cells), human A549 cells, human cervical cells, human CHME5 cells, human PER. Selected from the group consisting of C6 cells, NS0 mouse myeloma cells, human epithelial-like laryngeal cells, human fibroblasts, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocytes, human macrophage cells, human RAW264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells. Recombinant cells according to claim 3.

5. A cell culture comprising at least one recombinant cell and a culture medium as described in claim 3.

6. A transgenic animal comprising the nucleic acid construct described in claim 1.

7. (a) The animal is a vertebrate or an invertebrate, (b) If the animal is an insect, (c) The animal is a mammal, or (d) The animal is a non-human mammal, The transgenic animal according to claim 6.

8. A method for producing a target polypeptide, wherein (a) (i) (ii) Raising the transgenic animals described in claim 6 or 7 The method comprises culturing recombinant cells containing a nucleic acid construct according to any one of claims 2(k) to (o) under conditions in which the transgenic animal or recombinant cells produce polypeptides encoded by the GOI, or (b) comprising administering the nucleic acid construct according to any one of claims 2(k) to (o) to a subject, method.

9. comprising administering the nucleic acid construct according to any one of claims (k) to (o) of claim 2 to a subject, (a) The subject is a vertebrate or an invertebrate, (b) The subject is an insect, (c) The subject is a mammalian subject, or (d) The subject is a human subject, The method according to claim 8.

10. Recombinant polypeptide produced by the method described in claim 8.

11. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the following: a) A nucleic acid construct according to claim 1 or 2, b) Recombinant cells according to claim 3 or 4, and / or c) The recombinant polypeptide according to claim 10.

12. (a) The pharmaceutical composition is incorporated in liposomes, lipid-based nanoparticles (LNPs), polymer nanoparticles, polyplexes, viral replicon particles (VRPs), microspheres, immunostimulatory complexes (ISCOMs), bioactive ligand conjugates, or any combination thereof (b) The pharmaceutical composition is an immunogenic composition, (c) The pharmaceutical composition is an immunogenic composition, and the immunogenic composition is formulated as a vaccine, (d) The composition is substantially non-immunogenic to the subject, (e) The pharmaceutical composition is formulated as an adjuvant, and / or (f) The pharmaceutical composition is formulated for one or more of the following administration methods: intranasal administration, intrathecal administration, transdermal administration, intraperitoneal administration, intramuscular administration, intratracheal administration, intranodal administration, intratumoral administration, intraarticular administration, intravenous administration, subcutaneous administration, vaginal administration, intraocular administration, rectal administration, and oral administration. The pharmaceutical composition according to claim 11.

13. A pharmaceutical composition for use in a method for inducing a pharmacodynamic effect in a subject requiring such effect, wherein the method comprises administering the pharmaceutical composition to the subject, according to claim 11.

14. The pharmacodynamic effect includes one or more of the following: immunogenicity, biomarker response, therapeutic effect, preventive effect, desired effect, undesirable effect, adverse effect, and effect in a disease model, and / or The pharmacodynamic effect includes inducing an immune response in the subject, The pharmaceutical composition according to claim 13.

15. A pharmaceutical composition for use in a method of preventing and / or treating a health condition of a subject requiring such treatment, wherein the method comprises administering the pharmaceutical composition to the subject prophylactically or therapeutically. The pharmaceutical composition according to claim 11.

16. (a) The administered pharmaceutical composition induces an immune response in the subject, (b) The condition is a proliferative disorder or a microbial infection, (c) The subject has or is suspected to have a condition related to proliferative disorders or microbial infections, (d) The administered pharmaceutical composition results in an increase in interferon production in the subject, (e) The pharmaceutical composition is administered to the subject individually as a single treatment (monotherapy) or as a first treatment in combination with at least one additional treatment, and / or (f) The pharmaceutical composition is administered individually as a first treatment in combination with at least one additional treatment, wherein the at least one additional treatment is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery. The pharmaceutical composition according to claim 15.

17. A kit containing the following for inducing pharmacodynamic effects, for inducing immune responses, and / or for the prevention and / or treatment of health conditions or microbial infections: a) The nucleic acid construct according to claim 1; b) Recombinant cells according to claim 3; c) The recombinant polypeptide according to claim 10; and / or d) The pharmaceutical composition according to claim 11, And instructions.

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