Modified western equine encephalitis virus and uses thereof

Abstract

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

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 332,606, filed on April 19, 2022. The disclosures of the above-referenced applications are expressly incorporated herein by reference in their entirety, including any drawings. Incorporation of Sequence Listing

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

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

[0004] In recent years, several different animal virus groups have been genetically manipulated by homologous recombination or direct engineering of their genomes. The availability of reverse genetics systems for both DNA and RNA viruses has created new prospects 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 viral-based expression vectors have been developed for expressing 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 area include further development of techniques and systems for producing multi-subunit protein complexes and co-expressing protein modifying enzymes to improve heterologous protein production. Other recent advances in viral expression vector technology include many advanced genome engineering applications for controlling gene expression, preparing viral vectors, in vivo gene therapy applications, and producing vaccine delivery vectors.

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

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

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

[0009] In one aspect of the present disclosure, provided herein are nucleic acid constructs 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., 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 construct of the present disclosure may include one or more of the following features. In some embodiments, a modified viral genome or replicon (e.g., srRNA) lacks a large portion of a nucleic acid sequence encoding one or more viral structural proteins. In some embodiments, a 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, each of which comprises a promoter operably connected to a heterologous nucleic acid sequence. In some embodiments, at least one of the expression cassettes comprises a subgenomic (sg) promoter operably connected 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 constructs of the present disclosure further comprise one or more untranslated regions (UTRs). In some embodiments, at least one of the UTRs is a heterologous UTR.

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

[0014] In some embodiments, the nucleic acid 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 aspect, the present invention provides a recombinant cell comprising a nucleic acid construct as disclosed herein. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a vertebrate cell or an invertebrate cell. In some embodiments, the recombinant cell is an insect cell. In some embodiments, the recombinant cell is a mosquito cell. In some embodiments, the recombinant cell is a mammalian cell. In some embodiments, the recombinant cell is selected from monkey kidney CV1 cells (COS-7) transformed by SV40, human embryonic kidney cells (e.g., HEK 293 or HEK 293 cells), baby hamster kidney cells (BHK), mouse supporting cells (e.g., TM4 cells), monkey kidney cells (CV1), human cervical cancer cells (HeLa), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells, FS4 cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (Vero cells), human A549 cells, human cervical cells, human CHME5 cells, human PER.C6 cells, NS0 mouse myeloma cells, human epidermoid laryngeal cells, human fibroblasts, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocytes, human macrophages, human RAW264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells and rabbit kidney cells. In a related aspect, a cell culture is also provided, comprising at least one recombinant cell as disclosed herein and a culture medium.

[0016] In another aspect, transgenic animals are provided herein, comprising a nucleic acid construct as described herein. In some embodiments, the transgenic animal is a vertebrate or an invertebrate. In some embodiments, the transgenic animal is 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, a method for producing a polypeptide of interest is provided herein, wherein the method comprises (i) raising an animal as disclosed herein under certain conditions; or (ii) culturing a recombinant cell comprising a nucleic acid construct as disclosed herein under certain conditions, wherein the transgenic animal or recombinant cell produces a polypeptide encoded by the GOI under the conditions.

[0017] In another aspect, provided herein is a method for producing a polypeptide of interest in a subject, wherein the method comprises administering to the subject a nucleic acid construct as disclosed herein. In some embodiments, the subject is a vertebrate or an invertebrate. In some embodiments, the subject is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the subject is a mammalian subject. In some embodiments, the mammalian subject is a human subject. In yet another aspect, 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 pharmaceutically acceptable excipient and: a) a nucleic acid construct of the present disclosure; b) a recombinant cell of the present disclosure; and / or c) a recombinant polypeptide of the present disclosure.

[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, compositions are provided herein, comprising nucleic acid constructs as disclosed herein and pharmaceutically acceptable excipients. In some embodiments, compositions are provided herein, comprising recombinant cells as disclosed herein and pharmaceutically acceptable excipients. In some embodiments, compositions comprise recombinant polypeptides as disclosed herein and pharmaceutically acceptable excipients. In some embodiments, compositions are provided herein, and the compositions are formulated in liposomes, lipid-based nanoparticles (LNPs), polymer nanoparticles, polyplexes (polyplex), viral replicon particles (VRPs), microspheres, immunostimulatory complexes (ISCOMs), conjugates of bioactive ligands, or any combination thereof. In some embodiments, the compositions are immunogenic compositions. In some embodiments, immunogenic compositions are formulated as vaccines. In some embodiments, the immunogenic compositions are substantially non-immunogenic for subjects. In some embodiments, pharmaceutical compositions are formulated as adjuvants. In some embodiments, the pharmaceutical composition is formulated for one or more of intranasal administration, intrathecal administration, transdermal administration, intraperitoneal administration, intramuscular administration, intratracheal administration, intranodal administration, intratumoral administration, intraarticular administration, intravenous administration, subcutaneous administration, intravaginal administration, intraocular administration, rectal administration, and oral administration.

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

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

[0022] The non-limiting exemplary embodiments of the method of the present disclosure may include one or more of the following features. In some embodiments, the disease is a proliferative disorder or a microbial infection. In some embodiments, the subject suffers from or is suspected of suffering from a disease related to a proliferative disorder or a microbial infection. In some embodiments, the composition applied causes an increase in the production of interferon in the subject. In some embodiments, the composition is applied to the subject individually or as a first therapy in combination with at least one other therapy as a monotherapy (monotherapy). In some embodiments, the at least one other therapy is selected from chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy and surgery.

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

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

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

[0026] 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 nucleic acid sequences encoding viral structural proteins of the original virus have been completely deleted. The modified WEEV design depicted in the figure contains native 5'UTR and 3'UTR derived from WEEV strain Imperial, and further contains a heterologous gene of interest (GOI) placed under the control of a 26S subgenomic promoter. The coding sequences of nonstructural proteins nsP1, nsP2, nsP3, and nsP4 are shown.

[0027] Figure 2A-2B is a graphic representation of a non-limiting exemplary WEEV srRNA design according to some embodiments of the present disclosure, wherein a sequence encoding a modified WEEV genome from the Imperial strain is incorporated into a plasmid DNA vector ( Figure 2A), which also comprises the coding sequence of an exemplary target gene (GOI) (e.g., hemagglutinin precursor (HA) of influenza A virus H5N1) ( Figure 2B ). DETAILED DESCRIPTION

[0028] In particular, a viral expression system with excellent expression potential is provided herein, which is 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), which contain a modified genome or replicon (e.g., srRNA) of a Western equine encephalitis virus (WEEV), wherein at least some of the original viral sequences encoding structural proteins of the virus have been deleted. In some embodiments of the present disclosure, a virus-based expression vector is also provided, which contains one or more expression cassettes encoding heterologous polypeptides. Further provided are recombinant cells that are genetically engineered to contain one or more of the nucleic acid molecules disclosed herein. Biomaterials and recombinant products derived from such recombinant cells are also within the scope of the present application. Compositions and methods that can be used to cause pharmacodynamic effects in subjects in need and methods for preventing and / or treating a variety of health conditions are also provided.

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

[0030] Alphaviruses use motifs contained in their UTRs, structural regions, and nonstructural regions to influence their replication in host cells. These regions also contain mechanisms to evade host cell innate immunity. However, significant differences have been reported between alphavirus species. For example, New World and Old World alphaviruses have evolved different components to utilize intracellular stress granules, JAK-STAT signaling, FXR, and G3BP proteins to assemble viral replication complexes. Which part of the genome contains these components also varies between alphaviruses. For example, bypassing the activation of PKR and subsequent phosphorylation of EIF2α is accomplished via a downstream loop in some Old World alphaviruses (e.g., Sindbis), but bypassing this pathway in Chikungunya, which lacks a recognizable DLP, is thought to be accomplished via nsP4. In addition, in addition to variations between individual alphaviruses, there are often differences within alphavirus strains, which can explain changes in features such as virulence. As an example, sequence variations between North American and South American strains of the New World Eastern Equine Encephalitis Virus (EEEV) alter the ability to regulate the STAT1 pathway, leading to differential induction of type I interferons and resulting in changes in virulence. As another example, mouse studies conducted with a panel of North American WEEV isolates with less than 2.7% genomic sequence divergence had outcomes ranging from zero to complete lethality.

[0031] Given the differential presence of host cell attenuating factors in the nonstructural and structural regions of alphaviruses, deleting structural genes to allow heterologous gene expression in synthetic vectors will have different effects on individual vectors. Synthetic replicons with different host attenuating factors in the nonstructural region will have different advantages in inducing immune responses to the expressed heterologous genes. For example, while in Old World viruses, shutting down of host cell functions has been associated with nsP2, in New World viruses (e.g., EEEV, VEEV, and WEEV), this activity is primarily associated with the capsid protein (C), which is partially or completely deleted in synthetic vectors. The hypervariable domain (HVD) of the nsP3 protein has host interactions that are unique to each alphavirus. Among them, EEEV nsP3 has been shown to interact with cellular FXR and G3BP protein families, DDX3, S100A4, IKKβ, PGAM5, and cytoskeletal reorganization and vesicle trafficking proteins. Few studies have clearly detailed the WEEV nonstructural proteins, however, genomic sequencing of WEEV revealed that it is the result of recombination between the original species of EEEV and SINV. The amino acid identity of the WEEV nsP is reported to be 80%+ identical to that of the EEEV nsP, however, substitution of the EEEV nsP with the WEEV nsP produced attenuated chimeras, demonstrating that changes in nucleotide and coding sequences retain some activities essential to the viral life cycle, but confer significant differences in biological activity. The known and undescribed mechanisms by which the EEEV and WEEV nsPs contribute to their wide range of pathogenicity suggest that WEEV-based srRNA vectors could be unique and advantageous vehicles for the expression of heterologous proteins for vaccine and biotherapeutic applications. The advantages conferred by these previously undescribed srRNA vectors have been completely unexplored and unpredicted. definition

[0032] Unless otherwise defined, all technical terms, symbols and other scientific terms or expressions used herein are intended to have the meanings commonly understood by those skilled in the art to which this application belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ease of reference, and including such definitions herein should not necessarily be interpreted as representing substantial differences commonly understood in the art. Many of the techniques and procedures described or mentioned herein are well understood by those skilled in the art and are typically adopted by those skilled in the art using conventional methods.

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

[0034] As used herein, the terms "administration" and "administering" refer to the delivery of a biologically active composition or formulation by an administration route including, but not limited to, intranasal, transdermal, intravenous, intraarterial, intramuscular, intranodal, intraperitoneal, subcutaneous, intramuscular, oral, intravaginal, and topical administration or 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 specific subject cell, cell culture or cell line, but also to the progeny or potential progeny of such a cell, cell culture or cell line, without regard to the number of transfers or passages in culture. It should be understood that not all progeny are identical to the parental cell. This is because some modifications may occur in the offspring due to mutations (e.g., intentional or unintentional mutations) or environmental influences (e.g., methylation or other epigenetic modifications), so that the progeny may actually be different from the parental cell, but are still included in the scope of the term as used herein, as long as the progeny retains the same function as the original cell, cell culture or cell line.

[0036] The terms "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" of a composition (e.g., a nucleic acid construct (e.g., a replicon construct, such as an srRNA construct), a recombinant cell, a recombinant polypeptide, and / or a pharmaceutical composition) of the present disclosure generally refer to an amount sufficient to enable the composition to achieve its stated purpose (e.g., to achieve the effect for which it is administered, to stimulate an immune response, to prevent or treat a disease, or to alleviate one or more symptoms of a disease, disorder, infection, or health condition) relative to the absence of the composition. An example of an "effective amount" is an amount sufficient to facilitate the treatment, prevention, or alleviation of one or more symptoms of a disease, which may also be referred to as a "therapeutically effective amount." "Allowance" of a symptom means a reduction in the severity or frequency of one or more symptoms or the elimination of one or more symptoms. The exact amount of the composition (including a "therapeutically effective amount") will depend on the purpose of the treatment, and will be determined by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (Vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th edition, 2003, Gennaro ed., Lippincott, Williams & Wilkins).

[0037] The term "construct" refers to a recombinant molecule, such as a recombinant nucleic acid or polypeptide, comprising one or more isolated nucleic acid sequences or amino acid sequences from a heterologous source. 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 in a single polypeptide construct. Similarly, a nucleic acid construct can be a chimeric nucleic acid molecule in which two or more nucleic acid sequences of different origins are assembled into a single nucleic acid molecule. Thus, representative nucleic acid constructs include any construct containing: (1) a nucleic acid sequence including regulatory and coding sequences that are not found adjacent to each other in nature (e.g., at least one of the nucleotide sequences is heterologous relative to at least one of its other nucleotide sequences); or (2) a sequence encoding a portion of a functional RNA molecule or protein that is not naturally adjacent; or (3) a portion of a non-naturally adjacent promoter. Representative nucleic acid constructs can include any recombinant nucleic acid molecule (single-stranded or double-stranded DNA or RNA nucleic acid molecule) derived from any source (such as plasmid, cosmid, virus, self-replicating polynucleotide molecule, bacteriophage) that can carry out genome integration or autonomous replication, including nucleic acid molecules that one or more nucleotide sequences have been operably connected. The nucleic acid constructs of the present disclosure can include the necessary elements for guiding the expression of the target nucleic acid sequence also included in the construct. Such elements can include control elements (such as the promoter that is operably connected to the target nucleic acid sequence (so as to guide its transcription)), and 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 (e.g., a single vector), or can be incorporated (e.g., inserted) into two or more separate nucleic acid molecules (e.g., two or more separate vectors). The term "vector" is used herein to refer to a nucleic acid molecule or sequence that can transfer or transport another nucleic acid molecule. Therefore, the term "vector" encompasses both DNA-based vectors and RNA-based vectors. The term "vector" includes cloning vectors and expression vectors as well as viral vectors and integration vectors. "Expression vectors" are vectors that include regulatory regions so that DNA sequences and fragments can be expressed in vitro, ex vivo, and / or in vivo. In some embodiments, a vector may include a sequence that directs autonomous replication in a cell, such as a plasmid (DNA-based vector) or a self-replicating RNA vector. In some embodiments, a vector may include a sequence sufficient to allow integration into a host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. In some embodiments, the vector of the present disclosure may be a single-stranded vector (e.g., ssDNA or ssRNA). In some embodiments, the vector of the present disclosure can be a double-stranded vector (e.g., dsDNA or dsRNA). In some embodiments, the vector is a gene delivery vector. In some embodiments, the vector is used as a gene delivery vehicle to transfer the gene into a cell. 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 can also include, for example, one or more selective markers, one or more replication origins (such as prokaryotic and eukaryotic origins), at least one multiple cloning site and / or an element that promotes the stable integration of the construct into the genome of the cell. Two or more constructs can be incorporated into a single nucleic acid molecule (such as a single vector), or can be incorporated into two or more independent nucleic acid molecules (such as two or more independent vectors). An "expression construct" usually comprises at least a control sequence that is operably connected to a target nucleotide sequence. In this way, for example, a promoter that is operably connected to a nucleotide sequence to be expressed is provided in the expression construct for expression in a cell. For the practice of the present disclosure, compositions and methods for preparing and using constructs and cells are known to those skilled in the art.

[0040] As used herein, the term "operably connected" represents a physical or functional connection between two or more elements (e.g., a polypeptide sequence or a polynucleotide sequence) that allows them to operate in the manner they expect. For example, when used in the context of a coding sequence and a promoter sequence in a nucleic acid molecule or a nucleic acid molecule as described herein, the term "operably connected" means that the coding sequence and the promoter sequence are in frame, and in an appropriate space and distance, to allow the corresponding combination of a transcription factor or RNA polymerase to exert an influence on transcription. It should be understood that the elements that are operably connected can be continuous or discontinuous (e.g., connected to each other by a joint). In the context of a polypeptide construct, "operably connected" refers to a physical connection (e.g., directly or indirectly connected) between an amino acid sequence (e.g., different sections, parts, regions or domains) to provide the described activity of a construct. The sections, parts, regions and domains that are operably connected of a polypeptide or a nucleic acid molecule disclosed herein can be continuous or discontinuous (e.g., connected to each other by a joint).

[0041] As used herein, the term "portion" refers to a fraction. With respect to a particular structure (such as a polynucleotide sequence or an amino acid sequence or a protein), the term "portion" thereof may represent a continuous or discontinuous fraction of the structure. For example, a portion of an amino acid sequence comprises at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% and at least 90% of the amino acids of the amino acid sequence. Additionally or alternatively, if the portion is a discontinuous fraction, the discontinuous fraction is composed of 2, 3, 4, 5, 6, 7, 8 or more parts of a structure (e.g., a domain of a protein), each of which is a continuous element of the structure. For example, a discontinuous small portion of an amino acid sequence may consist of 2, 3, 4, 5, 6, 7, 8 or more (e.g., no more than 4) portions of the amino acid sequence, wherein each portion comprises 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] When used with respect to cells, nucleic acids, proteins or vectors, the term "recombinant" indicates that the cell, nucleic acid, protein or vector has been changed or produced by human intervention, such as having been modified by a laboratory method or being 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 contain amino acid residues that are not found in the natural (non-recombinant or wild-type) form of the protein, or may contain amino acid residues that have been modified (e.g., labeled). The term may include any modification to a peptide, protein or nucleic acid sequence. Such modifications may include the following: any chemical modification to a peptide, protein or nucleic acid sequence, including any chemical modification to one or more amino acids, deoxyribonucleotides or ribonucleotides; addition, deletion and / or substitution of one or more amino acids in a peptide or protein; production of fusion proteins (e.g., fusion proteins comprising antibody fragments); and addition, deletion and / or substitution of one or more nucleic acids in a nucleic acid sequence. When used with respect to cells, the term "recombinant" is not intended to include naturally occurring cells, but covers cells that have been engineered / modified to contain or express polypeptides or nucleic acids that are not present in cells when the cells are not engineered / modified.

[0043] In the context of two or more nucleic acids or proteins, the term "percent identity" as used herein refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides or amino acids (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned over a comparison window or specified region to obtain maximum correspondence), as measured using BLAST or BLAST 2.0 sequence comparison algorithms using the default parameters described below or by manual alignment and visual inspection. See, for example, the NCBI website ncbi.nlm.nih.gov / BLAST. Such sequences are then said to be "substantially identical". This definition also relates to or can be applied to the complement of a query sequence. This definition includes sequence comparisons performed by a BLAST algorithm, wherein the parameters of the algorithm are selected to give the maximum match between the respective sequences in the full length of the respective reference sequences. This definition also includes sequences with deletion and / or addition and those sequences with substitution.Sequence identity can be calculated in a region of at least about 20 amino acids or nucleotides in length, or in a region of 10-100 amino acids or nucleotides in length, or over the entire length of a given sequence.Sequence identity can be calculated using disclosed technology and widely available computer programs, such as GCS program packages (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 using its default parameters, such as the sequence analysis software package of the genetics computer group (Genetics Computer Group) of the University of Wisconsin Biotechnology Center (University of Wisconsin Biotechnology Center) (1710 University Avenue, Madison, Wisconsin, 53705). Additional methods that may be suitably used to determine the similarity or identity of amino acid sequences include those that rely on a position-specific structure scoring matrix (P3SM) that incorporates the structure prediction scores from Rosetta, and those based on length-normalized edit distance, as previously described in, for example, Setcliff et al., Cell Host & Microbe 23(6), May 2018.

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

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

[0046] It should be understood that aspects and embodiments of the present disclosure described herein include "comprising aspects and embodiments," "consisting of aspects and embodiments," and "consisting essentially of aspects and embodiments." As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of excludes any element, step, or ingredient not specified in the claimed composition or method. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. The term "comprising" in any recitation herein, particularly in the description of components of a composition or in the description of steps of a method, is understood to cover those compositions and methods consisting essentially of and consisting of the recited components or steps.

[0047] It should be understood that certain features of the disclosure described in the context of a separate embodiment for clarity may also be provided in combination in a single embodiment. On the contrary, various features of the disclosure described in the context of a single embodiment for brevity may also be provided individually or in any suitable sub-combination. All combinations of embodiments belonging to the disclosure are specifically covered by the disclosure and disclosed herein, as each combination is individually and clearly disclosed. In addition, all sub-combinations of various embodiments and elements thereof are also specifically covered by the disclosure and disclosed herein, as each such sub-combination is individually and clearly disclosed herein.

[0048] In the case of providing a range of values, it is understood that unless the context clearly specifies otherwise, each intermediate value (to one tenth of the unit of the lower limit) between the upper and lower limits of the range and any other stated or intermediate values ​​in the stated range are included in the present disclosure. These upper and lower limits of smaller ranges may be independently included in smaller ranges and are also included in the present disclosure, subject to any definite exclusion of limits in the stated range. In the case where the stated range includes one or two limits, the range excluding any one or two of those included limits is also included in the present disclosure.

[0049] Certain ranges are presented herein with numerical values ​​preceded by the term "about", as used herein, the term "about" has its approximately common meaning. The term "about" is used herein to provide textual support for the precise number thereafter and the number of the number close to or approximating the number after the term. When determining whether a number is close to or approximate to a specifically listed number, the close or approximate unlisted number can be a number that provides a basic equivalent form of the specifically listed number in the context in which it is presented. If the approximation is not clear from the context, "about" means within plus or minus 10% of the provided value, or rounded to the nearest significant figure, including the provided value in all cases. In some embodiments, the term "about" indicates a specified value ± at most 10%, at most ± 5%, or at most ± 1%.

[0050] In the case of providing a value range, it will be understood by those of ordinary skill in the art that all scopes disclosed herein encompass any and all possible sub-ranges and combinations of sub-ranges thereof.Any listed scope can be easily identified as fully describing the same scope and enabling the same scope to be decomposed into at least equal two equal parts, three equal parts, four equal parts, five equal parts, ten equal parts, etc. As a non-limiting example, each scope discussed herein can be easily decomposed into a lower third, a middle third, and an upper third, etc. As will also be understood by those skilled in the art, all languages ​​such as "at most", "at least", "greater than", "less than", etc. include the numbers listed, and refer to the scopes that can be subsequently decomposed into sub-ranges as discussed above. Finally, as will be understood by those skilled in the art, scope includes each individual member.Therefore, for example, a group with 1-3 articles refers to a group with 1, 2 or 3 articles. Similarly, a group with 1-5 articles refers to a group with 1, 2, 3, 4 or 5 articles, etc.

[0051] The headings (e.g., (a), (b), (i), etc.) are presented only to facilitate reading of the specification and claims. The use of headings in the specification or claims does not require that the steps or elements be performed in alphabetical or numerical order or in the order in which they are presented. Western equine encephalitis virus (WEEV)

[0052] Western equine encephalitis virus (WEEV) is a mosquito-borne virus belonging to the genus Alphavirus, which includes a group of Togaviridae viruses that are genetically, structurally and serologically related and is classified as a Group IV positive-sense single-stranded RNA virus. Currently, the genus Alphavirus includes, among others, Sindbis virus (SINV), Semliki Forest virus (SFV), Ross River virus (RRV), Venezuelan equine encephalitis virus (VEEV) and Eastern equine encephalitis virus (EEEV), all of which are closely related and can infect a variety of vertebrates (such as mammals, rodents, fish, bird species and large mammals (such as humans and horses)) and invertebrates (such as insects). An 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 genus Alphavirus (including WEEV and EEEV) has been extensively studied, and the life cycle, replication mode, etc. of these viruses are well characterized. More information in this regard can be found in, for example, Corrin T. et al., Vector-Borne and Zoonotic Diseases, Vol. 21, No. 5, 2021. In addition, it has been shown that alphaviruses can replicate very efficiently in animal cells, which makes them valuable as vectors for producing proteins and nucleic acids in such cells. Transmission between species and individuals is mainly carried out via mosquitoes, making alphaviruses a contributor to the collection of arboviruses or arthropod-borne viruses.

[0054] Each of these alphaviruses has a single-stranded RNA genome of positive polarity, which is encapsulated in a nucleocapsid surrounded by an envelope containing a viral spike protein. Alphavirus particles are enveloped, tend to be spherical (although slightly polymorphic), and have equidistant nucleocapsids. The alphavirus genome is a single-stranded RNA of positive polarity of about 11-12 kb in length, comprising a 5' cap, a 3' poly-A tail, and two open reading frames, wherein the first open reading frame encodes a non-structural protein with an enzymatic function and the second open reading frame encodes a viral structural protein (e.g., capsid protein CP, E1 glycoprotein, E2 glycoprotein, E3 protein, and 6K protein). For example, WEEV has a single-stranded positive RNA genome of about 11.5 kb, which comprises two open reading frames (ORFs), the ORFs are flanked by 5'- and 3'-untranslated regions (UTRs), and are capped at the 5' end and polyadenylated at the 3' end. WEEV is primarily transmitted in agricultural habitats by its primary mosquito vector, Culex (Culex) tarsalis. Mammals may participate in the secondary cycle. Humans and horses are considered the terminal hosts, however, some equids, such as donkeys and ponies, produce low to moderate levels of viremia (slightly less than 104 PFU / ml), which may make these hosts a contributor to epizootic spread. Symptoms of human infection with WEEV vary from no obvious symptoms (the vast majority of cases) to an influenza-like syndrome to life-threatening encephalitis and meningitis. Symptomatic infection usually involves an abrupt onset with fever, headache, nausea, vomiting, anorexia, and fatigue, followed by cognitive symptoms, weakness, and meningeal involvement.

[0055] The 5'-end two-thirds of the alphavirus genome encodes many nonstructural proteins necessary for viral RNA transcription and replication. These proteins are translated directly from RNA, and together with cellular proteins form RNA-dependent RNA polymerases that are essential for viral genome replication and subgenomic RNA transcription. Four nonstructural proteins (nsP1-4) are produced as a single polyprotein, constituting the viral replication machinery. The processing of the polyprotein is carried out in a highly regulated manner, wherein the cutting of the P2 / 3 junction affects the use of RNA templates during genome replication. This site is located at the bottom of a narrow cleft and is not easily accessible. Once cut, nsP3 produces a ring structure surrounding nsP2. These two proteins have a wide interface. Mutations in nsP2 that produce non-cytopathic viruses or temperature-sensitive phenotypes gather in the P2 / P3 interface region. P3 mutations relative to the position of nsP2 non-cytopathic mutations prevent the effective cutting of P2 / 3. This in turn may affect RNA infectivity and change the level of viral RNA production.

[0056] The 3' end third of the genome contains subgenomic RNA, which is used as a translation template for all structural proteins required to form viral particles (core nucleocapsid protein C and envelope proteins P62 and E1 associated as heterodimers). The surface glycoproteins anchored by the viral membrane are responsible for receptor recognition and enter the target cell through membrane fusion. Subgenomic RNA is transcribed from the p26S subgenomic promoter present at the 3' end of the RNA sequence encoding the nsP4 protein. P62 proteolytic maturation to E2 and E3 causes changes on the surface of the virus. E1, E2, and sometimes E3, glycoprotein "spikes" together form E1 / E2 dimers or E1 / E2 / E3 trimers, in which E2 extends from the center to the apex, E1 fills the space between the apex, and E3 (if present) is located at the distal end of the spike. When the virus is exposed to the acidity of the endosome, E1 dissociates from E2 to form an E1 homotrimer, which is necessary for driving the fusion step of the cell membrane with the viral membrane. The alphavirus glycoprotein E1 is a class II viral fusion protein that is structurally distinct from the class I fusion proteins found in influenza virus and HIV. The E2 glycoprotein functions through its cytoplasmic domain to interact with the nucleocapsid, while its extracellular domain is responsible for binding to cellular receptors. Most alphaviruses, including WEEV, have lost the peripheral protein E3, while in Semliki virus it remains associated with the viral surface.

[0057] Similar to most alphaviruses, WEEV replication occurs on the membrane surface within the host cell. In the first step of the infection cycle, the 5′ end of the genomic RNA is translated into a polyprotein (nsP1-4) with RNA polymerase activity, which produces a negative strand complementary to the genomic RNA. In the second step, the negative strand is used as a template for the production of the following two RNAs: (1) a positive genomic RNA corresponding to the genome of the secondary virus that produces other nsP proteins by translation and serves as the viral genome; and (2) a subgenomic RNA encoding the structural proteins of the virus that forms the infection particles. The positive genomic RNA / subgenomic RNA ratio is regulated by proteolytic self-cleavage of the polyprotein to nsP1, nsP2, nsP3 and nsP4. In fact, viral gene expression is carried out in two stages. In the first stage, the positive genomic strand and the negative strand are mainly synthesized. During the second stage, the synthesis of subgenomic RNA is actually exclusive, thus resulting in the production of a large amount of structural proteins. Self-replicating RNA

[0058] As will be appreciated by the skilled artisan, the term "self-replicating RNA" refers to an RNA molecule that contains all the genetic information required to direct its own amplification or self-replication within a permissive cell. Therefore, srRNA is sometimes also referred to as "self-amplifying RNA" (saRNA). In some embodiments, srRNA is a "replicon", which can be a linear or circular portion of DNA or RNA that is replicated sequentially as a unit. Non-limiting examples of replicons include "replicon RNA" or "RNA replicon". In order to guide its own replication, srRNA generally (1) encodes a polymerase, replicase, or other protein that can interact with a protein, nucleic acid, or ribonucleoprotein of viral or host cell origin to catalyze the RNA amplification process; and (2) contains cis-acting RNA sequences required for replication and transcription of subgenomic RNA. These sequences can be bound to proteins encoded by themselves or proteins, nucleic acids, or ribonucleoproteins of non-self-encoded cell origin 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, such as WEEV. In some embodiments of the present disclosure, an alphavirus srRNA construct (e.g., srRNA, saRNA, or replicon molecule) generally contains the following elements: one or more cis 5' viral or defective interfering RNA sequences required for replication, sequences encoding biologically active alphavirus nonstructural proteins (e.g., nsP1, nsP2, nsP3, and nsP4), a subgenomic promoter (sg) of a subgenomic RNA (sgRNA), a cis 3' viral sequence required for replication, and optionally a polyadenylic acid tract (poly (A)). In some cases, a subgenomic promoter (sg) directing expression of a heterologous sequence may be included in an srRNA construct of the present disclosure.

[0059] In addition, the term srRNA molecule (e.g., srRNA, saRNA, or replicon molecule) generally refers to a molecule of positive polarity or sense ("message" sense), and the length of the srRNA can be different from the length of any known naturally occurring alphavirus. In some embodiments of the present disclosure, the srRNA does not contain at least a portion of the coding sequence of one or more alphavirus structural proteins; and / or the sequence encoding the structural gene can be replaced by a heterologous sequence. In those cases, where the srRNA is to be packaged into recombinant alphavirus particles, it can contain one or more sequences for initiating interaction with alphavirus structural proteins, resulting in particle formation (so-called packaging signals).

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

[0061] As described in more detail below, one aspect of the disclosure relates to a nucleic acid construct comprising a nucleic acid sequence encoding a modified viral genome or srRNA, wherein the modified genome or srRNA lacks (e.g., does not contain) at least a portion of a nucleic acid sequence encoding one or more structural proteins of the corresponding unmodified viral genome or srRNA. Some embodiments of the disclosure provide a modified alphavirus genome or srRNA in which the coding sequence for the nonstructural proteins nsP1, nsP2, nsP3, and nsP4 is present, but at least a portion or the entire sequence encoding one or more structural proteins is absent. Recombinant cells and cell cultures are also provided that have been engineered to contain a nucleic acid construct as disclosed herein. A. Nucleic Acid Constructs

[0062] As described in more detail below, one aspect of the present disclosure relates to novel nucleic acid constructs comprising a nucleic acid sequence 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 comprise one or more deletions, one or more substitutions, and / or one or more insertions in one or more genomic regions of a parental alphavirus genome.

[0063] Non-limiting exemplary embodiments of nucleic acid constructs (e.g., replicon constructs, such as srRNA constructs) of the present disclosure may include one or more of the following features. In some embodiments, the nucleic acid construct comprises a nucleic acid sequence encoding a modified WEEV genome or srRNA, wherein the modified WEEV genome or srRNA lacks at least a portion of a nucleic acid sequence encoding one or more structural proteins of an unmodified WEEV genome or srRNA, such as a modified WEEV genome or srRNA that does not comprise at least a portion of a coding sequence for one or more of the WEEV structural proteins CP, E1, E2, E3, and 6K. Both virulent and avirulent WEEV strains are suitable. Non-limiting examples of WEEV strains suitable for use in 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. Additional examples of WEEV strains suitable for use in the compositions and methods of the present disclosure include 5614, 93A27, 93A30, 93A38, 93A79, B628 (C115), CBA87, CNTR34, CO921356, Fleming, Lake43, PV012357A, PV02808A, PV72102, R02PV001807A, R02PV002957B, R02PV003422B, R05PV003422B, R0PV003814A, and R0PV00384A. Additional suitable WEEV strains include, but are not limited to, those described in Bergren NA et al., J. Virol. 88(16):9260–9267, August 2014, and the Virus Pathogen Resource website (ViPR, which can be found at www.viprbrc.org / brc / vipr_genome_search.spg? method = SubmitForm & blockId = 868 & decorator = toga (publicly available) In some embodiments, the modified WEEV genome or srRNA is derived from the WEEV strain Imperial.

[0064] Non-limiting exemplary embodiments of nucleic acid constructs (e.g., replicon constructs, such as srRNA constructs) of the present disclosure may include one or more of the following features. In some embodiments, the modified viral genome or srRNA lacks at least a portion of the nucleic acid sequence encoding one or more of the viral structural proteins CP, E1, E2, E3, and 6K of the unmodified viral genome or srRNA. In some embodiments, the modified viral genome or srRNA lacks a portion or the entire sequence of the sequence encoding CP. In some embodiments, the modified viral genome or srRNA lacks a portion or the entire sequence of the sequence encoding E1. In some embodiments, the modified viral genome or srRNA lacks a portion or the entire sequence of the sequence encoding E2. In some embodiments, the modified viral genome or srRNA lacks a portion or the entire sequence of the sequence encoding E3. In some embodiments, the modified viral genome or srRNA lacks a portion or the entire sequence of the sequence encoding 6K. In some embodiments, the modified viral genome or srRNA lacks a portion or the entire sequence of the sequence encoding the combination of CP, E1, E2, E3, and 6K. Some embodiments of the present disclosure provide a modified WEEV genome or srRNA, wherein the coding sequences of the nonstructural proteins nsP1, nsP2, nsP3, and nsP4 of the unmodified WEEV genome or srRNA are present, however at least a portion or the entire sequence of the sequence encoding one or more structural proteins (e.g., CP, E1, E2, E3, and 6K) of the WEEV genome or srRNA is absent. Some embodiments of the present disclosure provide a modified WEEV genome or srRNA, wherein the coding sequences of the nonstructural proteins nsP1, nsP2, nsP3, and nsP4 of the unmodified WEEV genome or srRNA are present, however at least a portion or the entire sequence of the sequence encoding one or more structural proteins (e.g., CP, E1, E2, E3, and 6K) of the WEEV genome or srRNA is absent.

[0065] In some embodiments, the modified viral genome or srRNA lacks a large portion of the nucleic acid sequence encoding one or more viral structural proteins. It will be appreciated by a skilled person that a large portion of the nucleic acid sequence encoding a viral structural polypeptide may include sufficient nucleic acid sequence encoding a viral structural polypeptide to provide a putative identification of the polypeptide in the following manner: the sequence is manually evaluated by a person skilled in the art, or an algorithm such as BLAST (see, for example, "Basic Local Alignment Search Tool"; Altschul SF et al., J. Mol. Biol. 215: 403-410, 1993) is used for computer automatic sequence comparison and identification. Therefore, a large portion of the nucleotide sequence includes sufficient sequence to provide specific identification and / or separation of nucleic acid fragments comprising the sequence. For example, a large portion of the nucleic acid sequence may include at least about 20% of the full-length nucleic acid sequence, for example, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%. As described above, the disclosure provides nucleic acid molecules and constructs that lack part or complete nucleic acid sequences encoding one or more viral structural proteins. A skilled person benefits from sequences as disclosed herein and can easily use all or a large part of the disclosed sequences for the compositions and methods of the disclosure. Therefore, the application includes complete sequences as disclosed herein, such as those sequences shown in the attached sequence table, and a large part of those sequences as 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 comprise a nucleic acid sequence encoding a structural protein of the viral unmodified genome or srRNA.

[0067] In some embodiments, the nucleic acid construct of the present disclosure further comprises one or more expression cassettes. In principle, the nucleic acid construct disclosed herein can generally comprise any number of expression cassettes. In some embodiments, the nucleic acid construct disclosed herein can comprise at least two, at least three, at least four, at least five or at least six expression cassettes. It will be understood by those skilled in the art that the term "expression cassette" refers to the construct of genetic material, which contains a coding sequence and enough regulatory information to guide the coding sequence to correctly transcribe and / or translate in vivo and / or in vitro in cells. The expression cassette can be inserted into the vector for targeting the desired host cell and / or in a subject or individual. Therefore, in some embodiments, the term expression cassette can be used interchangeably with the term "expression construct". In some embodiments, the term "expression cassette" refers to such a nucleic acid construct, which comprises a gene or functional RNA of a protein encoding protein operably connected to a regulatory element (e.g., a promoter and / or termination signal, and optionally, any other nucleic acid sequence or combination of other nucleic acid sequences that affect gene transcription or translation).

[0068] In some embodiments, at least one of the expression cassettes comprises a promoter operably connected to a heterologous nucleic acid sequence. Therefore, as provided herein, a nucleic acid construct can be used as, for example, an expression vector, which can affect the expression of a heterologous nucleic acid sequence when comprising a regulatory element (e.g., a promoter) operably connected to a heterologous nucleic acid sequence. In some embodiments, at least one of the expression cassettes comprises a subgenomic (sg) promoter operably connected to a heterologous nucleic acid sequence. In some embodiments, the sg promoter is a 26S subgenomic promoter. In some embodiments, at least one nonstructural protein (nsP) or a portion thereof of the modified WEEV genome or srRNA is heterologous relative 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 a portion thereof. In some embodiments, the nucleic acid molecule of the present disclosure further comprises one or more untranslated regions (UTRs). In some embodiments, at least one of the UTRs is a heterologous UTR.

[0069] In some embodiments, at least one of the expression cassettes comprises a coding sequence of a gene of interest (GOI). In some embodiments, the coding sequence of the GOI comprises a coding sequence of a polypeptide construct of interest, and the polypeptide construct of interest comprises a single polypeptide (e.g., a single gene PCI). In some embodiments, the coding sequence of the PCI comprises a plurality of polypeptides (e.g., a polygene (e.g., a double gene, a triple gene, or a quadruple gene, etc.) PCI) coding sequence. 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 in a single open reading frame (e.g., in a polycistronic ORF). In some embodiments, the coding sequence of the polycistronic ORF is operably linked to a promoter sequence. In some embodiments, at least one of the promoter sequences is a subgenomic (sg) promoter. In some embodiments, the sg promoter is a 26S genomic promoter.

[0070] In some embodiments, the plurality of polypeptides may be directly or indirectly connected to each other (e.g., via one or more linking sequences). For example, in some embodiments, the plurality of polypeptides may be directly connected to each other, e.g., connected adjacent to each other. In some embodiments, at least two (e.g., 2, 3, 4, or 5) of the plurality of polypeptides are operably connected to each other via one or more linking sequences. In some embodiments, the length and amino acid composition of the linking sequences may 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, the linking sequences in the plurality of linking sequences include one or more self-proteolytic peptide sequences. Non-limiting examples of autoproteolytic peptide sequences suitable for use in the methods and compositions of the present disclosure include autoproteolytic cleavage sequences derived from calcium-dependent serine endoprotease (Furin), porcine Teschovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis virus (ERAV) 2A (E2A), beta-tetrasomal virus of the thrush moth 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), and malarial disease virus 2A (BmIFV2A). In some embodiments, at least two of the plurality of polypeptides are operably linked to each other via the P2A autoproteolytic cleavage sequence.

[0071] In some embodiments, the coding sequences of multiple polypeptides are operably connected to each other through 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, cricket paralysis virus IRES, Rhopalosiphum padi virus IRES, fibroblast growth factor IRES, platelet-derived growth factor IRES, vascular endothelial growth factor IRES, insulin-like growth factor IRES, small RNA virus 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 on 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 use in 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, dedifferentiation factors, immune cell receptors or reporters, or any combination thereof.

[0073] In some embodiments, the coding sequence of the GOI is redesigned, reconstructed and / or optimized to obtain desired properties, such as increased stability, efficacy and expression (e.g., translation efficiency), which in turn can maximize the impact of producing, delivering and administering the biotherapeutic. 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%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% higher than the reference coding sequence). In some embodiments, the reference coding sequence is a wild-type non-optimized sequence. With regard to sequence optimization of nucleotide sequences, the degeneracy of the genetic code provides the possibility of replacing at least one base of a gene sequence encoding a protein with a different base without causing the amino acid sequence of the polypeptide produced by the gene to be changed. Therefore, the nucleic acid construct of the present disclosure may also have any base sequence that has been changed from any polynucleotide sequence disclosed herein by substitution according to the degeneracy of the genetic code. References describing codon usage are readily publicly available. In some embodiments, polynucleotide sequence variants may be generated for a variety of reasons, such as to optimize expression in a particular host (e.g., to change the codon usage in an alphavirus mRNA to those preferred by other organisms such as humans, non-human primates, hamsters, mice, or monkeys). Thus, in some embodiments, the coding sequence of the GOI is optimized for expression in a target host cell by using codons optimized for expression. Techniques for constructing a synthetic nucleic acid sequence encoding a GOI using preferred codons that are optimal for host cell expression can be determined by analyzing the universality of codon usage and computational methods of their relative abundance of native proteins encoding the host cell genome via techniques known in the art. Codon usage databases (e.g., http: / / www.kazusa.or.jp / codon) can all be used to generate codon-optimized sequences in a mammalian cell environment. In addition, a variety of software tools can be used to convert the sequence of an organism to the optimal codon usage for a different host organism, such as the JCat codon optimization tool (www.jcat.de), the Integrated DNA Technologies (IDT) codon optimization tool (https: / / www.idtdna.com / CodonOpt) or the Optimizer online codon optimization tool (http: / / genomes.urv.es / OPTIMIZER). Such synthetic sequences can be constructed by techniques known in the art for constructing synthetic nucleic acid molecules and can be obtained from multiple commercial suppliers. Therefore, in some embodiments, the coding sequence of the GOI is optimized for expression at a level higher than the expression level of a reference coding sequence (e.g., 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 an increase in expression level of at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold compared to a reference coding sequence that is not codon-optimized.

[0074] In some embodiments, the coding sequence of GOI is optimized for enhancing RNA stability and / or expression. The stability of RNA is generally related to the "half-life" of RNA. "Half-life" refers to the time period required to eliminate half of the activity, amount or quantity of a molecule. In the context of the present disclosure, the half-life of RNA indicates the stability of the RNA. The half-life of RNA can affect the "expression duration" of RNA. Additional information on the principles, strategies and methods used in enhancing RNA stability can be found in, for example, Leppek K. et al., Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics (Nature Communications. Vol. 13, Article No. 1536, March 2022).

[0075] The polypeptide encoded by the GOI can generally be any polypeptide, and can be, for example, a therapeutic polypeptide, a preventive 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 antibodies, antigens, immunomodulators, enzymes, signaling proteins, and cytokines. In some embodiments, the GOI can encode microbial proteins, viral proteins, bacterial proteins, fungal proteins, mammalian proteins, and any combination thereof. In some embodiments, the GOI encodes the hemagglutinin precursor (HA) of influenza A virus H5N1. Non-limiting examples of GOIs include interleukins and interacting proteins, including: G-CSF, GM-CSF, IL-1, IL-10, IL-10-like protein, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-18BP, IL-1-like protein, IL-1RA, IL-1α, IL-1β, IL-2, IL-20, IL-3, IL-4, IL-5, IL-6, IL-6-like protein, IL-7, IL-9, IL-21, IL-22, IL-33, IL-37, IL-38, LIF and OSM. Additional suitable GOIs include, but are not limited to, interferons (e.g., IFN-α, IFN-β, IFN-γ), 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 (e.g., XCL1, XCL2, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL 8, 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 use in the compositions and methods of the present disclosure include, but are not limited to, immunostimulatory gene products (e.g., CD27 / CD70, CD40, CD40L, B7.1, BTLA, MAVS, OX40, OX40L, RIG-I, and STING), drug resistance gene mutants / variants such as ABCB1, ABCC1, ABCG2, AKT1, ALK, BAFF, BCR-ABL, BRAF, CCND1, cMET, EGFR, ERBB2, ERBB3, ERK2, ESR1, GRB2, KRAS, MDR1, MRP1, NTRK1, PDC4, P-gp, PI3K, PTEN, RET, ROS1, RSK1, RSK2, SHIP, and STK11. Also suitable for use in the compositions and methods of the present disclosure include sequences encoding viral proteins, particularly spike proteins, fiber proteins, structural proteins, and adsorption proteins.

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

[0077] In some embodiments, the GOI may encode an enzyme whose deficiency or mutation is associated with a disease or health condition, such as, for example, agalsidase beta, agalsidase alfa, imiglucerase, taliglucerase alfa, velaglucerase alfa, arabinocerebrosidase, sebelipase alpha, laronidase, idosulfatase, elosulfase alpha, galactosidase, alglucerase alpha, and CTFR.

[0078] In some embodiments, the GOI may encode a polypeptide selected from the following: an antigenic molecule, a biotherapeutic molecule, or any combination thereof. In some embodiments, the GOI may encode a polypeptide selected from the following: a tumor-associated antigen, a tumor-specific antigen, a new antigen, and any combination thereof. As will be appreciated by a skilled artisan, TAAs include molecules, such as proteins, that are present on tumor cells and normal cells or on many normal cells but at concentrations much lower than those on tumor cells. In contrast, TSAs typically include molecules, such as proteins, that are present on tumor cells but not on normal cells. Tumor-associated antigens may be antigens associated with, for example, the following cancer cells: breast cancer cells, B-cell lymphomas, 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 also be expressed by non-cancerous cells. In some embodiments, the GOI may encode a polypeptide selected from the following: estrogen receptors, intracellular signal transduction enzymes, and human epidermal growth receptors. In some embodiments, the GOI may encode a biological therapeutic polypeptide selected from the following: immunomodulators, angiogenesis regulators, extracellular matrix regulators, metabolic regulators, neuromodulators, and any combination thereof. In some embodiments, the GOI may encode a cytokine selected from the following: chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. 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 may encode a biotherapeutic polypeptide selected from the group consisting of IL-12A, IL-12B, IL-1RA, and any combination thereof.

[0079] In some embodiments, the nucleic acid constructs of the present disclosure comprise 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, the nucleic acid constructs of the present disclosure comprise 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, the nucleic acid construct of the present 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 one, two, three, four, five or more nucleotides of the nucleic acid sequence may be substituted with different nucleotides. In some embodiments, the nucleic acid construct of the present 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 one, two, three, four, five or more nucleotides of the nucleic acid sequence may be substituted with different nucleotides.

[0081] Nucleic acid sequences having a high degree of sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the sequence of the modified WEEV of interest can be identified and / or isolated by genomic sequence analysis, hybridization, and / or PCR using degenerate primers or gene-specific primers from sequences identified in the corresponding WEEV genome using the sequences identified herein (e.g., SEQ ID NO: 1 or SEQ ID NO: 2), or any other sequence known in the art.

[0082] In some embodiments, the nucleic acid constructs of the present disclosure comprise one or more adapter sequences, such as cloning adapter sequences. In some embodiments, the one or more adapter sequences comprise the following sequence: 5'-CTGGAGACGTGGAGGAGAACCCTGGACCT-3' (SEQ ID NO: 3). In some embodiments, the one or more adapter 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 endonuclease cleavage sites, such as SapI and SpeI restriction endonuclease cleavage sites (see, e.g., Examples 1-3). For example, a unique restriction endonuclease cleavage site (SpeI, 5'-A'CTAG, T-3') can be incorporated in place of the coding sequence of the native WEEV structural gene (wherein the 5'A matches the position of the structural polyprotein ATG start codon, and the 3'T matches 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 subsequent Gibson Procedure (Gibson et al., Nat. Methods 6, 343-345, 2009). In some embodiments, a promoter sequence (e.g., a bacteriophage T7 RNA polymerase promoter) may be included upstream of the WEEV genomic sequence and may contain a poly(A) sequence downstream, followed by a restriction endonuclease cleavage site (e.g., Sapl), which cuts upstream of the recognition site. Immediately downstream of the restriction endonuclease cleavage site (SapI) may include a terminator sequence (e.g., a T7 terminator sequence), followed by a unique restriction endonuclease cleavage site (e.g., NotI').

[0084] In some experiments, the sequence encoding one or more nsPs was replaced by a heterologous nsP. In some other experiments, the sequence encoding one or more UTRs was replaced by a heterologous UTR.

[0085] The molecular techniques and methods of assembling and characterizing these new nucleic acid constructs are more fully described in the examples of the application. In some embodiments, nucleic acid molecules are recombinant nucleic acid molecules. As used herein, the term recombinant means any molecule (e.g., DNA, RNA, polypeptide) from human manipulation or produced from (however indirectly) human manipulation. As a non-limiting example, cDNA is a recombinant DNA molecule, such as any nucleic acid molecule produced by one or more in vitro polymerase reactions or attached to a joint or integrated into a carrier (e.g., a cloning vector or an expression vector). As non-limiting examples, a recombinant nucleic acid molecule: 1) has been synthesized or modified in vitro, e.g., using chemical or enzymatic techniques, e.g., by using chemical nucleic acid synthesis, or by using an enzyme to perform replication, polymerization, exonucleolytic digestion, endonucleolytic digestion, ligation, reverse transcription, transcription, base modification (including, e.g., methylation), or recombination (including homologous recombination and site-specific recombination) of nucleic acid molecules; 2) contains linked nucleotide sequences that are not linked in nature; 3) has been engineered using molecular cloning techniques such that it lacks one or more nucleotides relative to a naturally occurring nucleotide sequence; and / or 4) has been manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements relative to a naturally occurring nucleotide sequence.

[0086] In some embodiments, nucleic acid molecules disclosed herein are produced using recombinant DNA techniques (e.g., polymerase chain reaction (PCR) amplification, cloning, etc.) or chemical synthesis. Nucleic acid molecules as disclosed herein include natural nucleic acid molecules and homologs thereof, including but not limited to natural allelic variants and modified nucleic acid molecules in which one or more nucleotide residues have been inserted, deleted and / or substituted in such a manner that such modifications provide the desired properties for achieving biological activity as described herein.

[0087] Nucleic acid molecules (including variants of naturally occurring nucleic acid sequences) can be produced using a variety of methods known to those skilled in the art (see, e.g., Sambrook et al., in: Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1989)). The sequence of a nucleic acid molecule can be modified relative to the naturally occurring sequence from which it is derived using a variety of techniques, including, but not limited to, classical mutagenesis techniques and recombinant DNA techniques, such as, 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, including chemical synthesis of oligonucleotide mixtures and ligation of mixed sets to "build" a mixture of nucleic acid molecules; and combinations thereof. Nucleic acid molecule homologs can be selected from a mixture of modified nucleic acid molecules by screening for the function of a protein or srRNA encoded by the nucleic acid molecule, and / or hybridization with a wild-type gene or fragment thereof, or PCR using primers having homology to a target or wild-type nucleic acid molecule or sequence. B. Recombinant Cells

[0088] The nucleic acid construct of the present disclosure can be introduced into a host cell to produce a recombinant cell containing a nucleic acid molecule. Therefore, a prokaryotic cell or eukaryotic cell containing a nucleic acid construct encoding a modified WEEV genome as described herein is also a feature of the present disclosure. In related aspects, some embodiments disclosed herein relate to a method for transforming a cell, comprising introducing a nucleic acid construct as provided herein into a host cell (such as an animal cell), and then selecting or screening the transformed cell. The introduction of the nucleic acid construct of the present disclosure into a cell can be achieved by methods known to those skilled in the art, such as viral infection, transfection, conjugation, protoplast fusion, liposome transfection, electroporation, nuclear transfection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, direct microinjection, nanoparticle-mediated nucleic acid delivery, etc.

[0089] In one aspect, some embodiments of the present disclosure relate to recombinant cells comprising nucleic acid constructs as described herein, such as recombinant animal cells. The nucleic acid construct can be stably integrated into the host genome, or can be replicated as an episome, or present in a recombinant host cell as a mini-ring expression vector for stable or transient expression. Therefore, in some embodiments of the present disclosure, the nucleic acid construct is maintained and replicated as an episomal unit in a recombinant host cell. In some embodiments, the nucleic acid construct is stably integrated into the genome of the recombinant cell. Stable integration can be completed using classical random genome recombination technology or with more accurate genome editing technology (such as CRISPR / Cas9 or TALEN genome editing guided by guide RNA). In some embodiments, the nucleic acid construct is present in a recombinant host cell as a mini-ring expression vector for stable or transient expression.

[0090] The host cell may be an untransformed cell or a cell that has been transfected with at least one nucleic acid molecule. Thus, in some embodiments, the host cell may be genetically engineered (eg, transduced or transformed or transfected) with at least one nucleic acid molecule.

[0091] Suitable host cells for cloning or expressing polypeptides of interest as described herein include prokaryotic or eukaryotic cells as described herein. Therefore, in some embodiments, recombinant cells are prokaryotic cells, such as bacterial Escherichia coli (E. coli); or eukaryotic cells, such as insect cells (e.g., mosquito cells or Sf21 cells) or mammalian cells (e.g., COS cells, NIH 3T3 cells or HeLa cells). In some embodiments, cells are in vivo, such as recombinant cells in living bodies, such as cells of transgenic subjects. In some embodiments, subjects are vertebrates or invertebrates. In some embodiments, subjects are insects. In some embodiments, subjects are mammalian subjects. In some embodiments, cells are ex vivo, such as having been extracted from living bodies or organisms as separate cells or as part of an organ or tissue for treatment or procedure, and then returned to living bodies or organisms. In some embodiments, cells are in vitro, such as 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 selected from monkey kidney CV1 cells (e.g., COS-7) transformed by SV40, human embryonic kidney cells (e.g., HEK293 or HEK 293 cells), baby hamster kidney cells (BHK), mouse supporting cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1), human cervical cancer cells (e.g., HeLa), canine kidney cells (e.g., MDCK), buffalo rat liver cells (e.g., BRL 3A), human lung cells (e.g., W138), human liver cells (e.g., HepG2), mouse breast tumor cells (e.g., MMT 060562), TRI cells, FS4 cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (e.g., Vero cells), human A549 cells, human cervical cells, human CHME5 cells, human PER.C6 cells, NS0 mouse myeloma cells, human epidermoid laryngeal cells, human fibroblasts, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocytes, human macrophages, human RAW 264.7 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 African green monkey kidney cells (Vero cells), baby hamster kidney (BHK) cells, Chinese hamster ovary cells (CHO cells), human A549 cells, human cervical cells, human CHME5 cells, human epidermoid laryngeal cells, human fibroblasts, human HEK-293 cells, human HeLa cells, human HepG2 cells, human HUH-7 cells, human MRC-5 cells, human muscle cells, mouse 3T3 cells, mouse connective tissue cells, mouse muscle cells and rabbit kidney cells. In some embodiments, the recombinant cell is a cell derived from the cell described above (i.e., a derivative cell of the original cell described herein), such as amplified from a clone of the original cell (engineered form of the original cell) or as a reclassified cell of the original cell after having undergone extensive passage or having been passaged by another host.

[0094] In some embodiments, the recombinant cell is an insect cell, such as a cell of an insect cell line. In some embodiments, the recombinant cell is an Sf21 cell. Other suitable insect cell lines include, but are not limited to, cell lines established from the insect orders Diptera, Lepidoptera, and Hemiptera, and may 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 with about 50 lines per decade. More information about available lepidopteran insect cell lines can be found in, for example, Lynn D.E., Available lepidopteran insect cell lines. Methods Mol Biol. 2007; 388: 117-38, which is incorporated herein by reference. In some embodiments, the recombinant cell is a mosquito cell, such as a cell of a mosquito species within the genera Anopheles (An.), Culex (Cx.), and Aedes (subgenus Stegomyia) (Ae.). Exemplary mosquito cell lines suitable for use in the compositions and methods described herein include cell lines from the following mosquito species: Aedes aegypti, Aedes albopictus, Aedes pseudoscutellaris, Aedes triseriatus, Aedes vexans, Anopheles gambiae, Anopheles stephensi, Anopheles albimanus, Culex quinquefasciatus, Culex theileri, Culextritae niorhynchus, Culex bitae niorhynchus, and Toxorhynchites amboinensis. Suitable mosquito cell lines include, but are not limited to, CCL-125, Aag-2, RML-12, C6 / 26, C6 / 36, C7-10, AP-61, AtGRIP-1, AtGRIP-2, UM-AVE1, Mos.55, Sua1B, 4a-3B, Mos.43, MSQ43, and LSB-AA695BB. In some embodiments, the mosquito cell is a cell of the C6 / 26 cell line.

[0095] In another aspect, cell cultures are provided herein, comprising at least one recombinant cell as disclosed herein and a culture medium. Typically, the culture medium can be any suitable culture medium for culturing cells as described herein. Techniques for transforming various host cells and species mentioned above are known in the art and are described in the technical and scientific literature. Therefore, cell cultures comprising at least one recombinant cell as disclosed herein are also within the scope of the present application. Methods and systems suitable for producing and maintaining cell cultures are known in the art. C. Transgenic Animals

[0096] In another aspect, transgenic animals are also provided, the transgenic animals comprising nucleic acid constructs as described herein (e.g., vectors, replicons, or srRNA molecules). In some embodiments, transgenic animals are vertebrates or invertebrates. In some embodiments, transgenic animals are insects. In some embodiments, insects are mosquitoes. In some embodiments, transgenic animals are mammals. In some embodiments, transgenic mammals are non-human mammals. Generally, the transgenic animals of the present disclosure can be any non-human animals known in the art. In some embodiments, the non-human animals of the present disclosure are non-human primates. Other animal species suitable for the compositions and methods of the present disclosure include animals that (i) are suitable for transgenic effects, and (ii) can rearrange immunoglobulin gene segments to produce antibody responses. Examples of such species include, but are not limited to, mice, rats, hamsters, rabbits, chickens, goats, pigs, sheep, and cattle. Additional examples of non-human animals suitable for use with the compositions and methods of the present disclosure can include, but are not limited to, laboratory animals (e.g., mice, rats, hamsters, gerbils, guinea pigs, etc.), livestock (e.g., horses, cows, pigs, sheep, goats, ducks, geese, chickens, etc.), domestic animals and pets (e.g., cats, dogs, etc.), non-human primates (e.g., apes, chimpanzees, orangutans, monkeys, etc.), fish, amphibians (e.g., frogs, salamanders, etc.), reptiles (e.g., snakes, lizards, etc.), and other animals (e.g., foxes, weasels, rabbits, minks, beavers, ferrets, otters, sables, seals, coyotes, chinchillas, deer, muskrats, opossums, etc.).

[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 with such germ cells and somatic cells, and the germ cells and somatic cells contain one or more (for example, one or more, two or more, three or more, four or more, etc.) nucleic acid constructs of the present disclosure. In some embodiments, one or more nucleic acid constructs are stably integrated into the genome of the transgenic animal. In some embodiments, the genome of the transgenic animal of the present disclosure can include any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more copies of one or more nucleic acid constructs of the present disclosure.

[0098] Means and methods for preparing transgenic non-human animals are known in the art. Exemplary methods include pronuclear microinjection, DNA microinjection, lentiviral vector-mediated transfer of DNA into early embryos and sperm-mediated transgenic effects, adenovirus-mediated introduction of DNA into animal sperm (e.g., in pigs), retroviral vectors (e.g., avian species), somatic cell nuclear transfer (e.g., in goats). The state of the art in the preparation of transgenic livestock farm animals is reviewed in Niemann, H. et al. (2005) Rev. Sci. Tech. 24: 285-298. In some embodiments, standard methods known in the art for introducing exogenous nucleic acids into the genome of non-human animals are used to prepare the transgenic non-human host animals of the present disclosure. In some embodiments, the transgenic animals of the present disclosure can be produced using classical random genome recombination techniques or using more precise techniques, such as CRISPR / Cas genome editing guided by guide RNA, 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 prepared using transgenic microinjection technology, and do not require the use of homologous recombination technology, and are therefore considered to be easier to prepare and select than methods using homologous recombination. In some embodiments, transgenic animals produce a protein 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 (i) are suitable for transgenic effects, and (ii) are capable of rearranging immunoglobulin gene segments to produce antibody responses. Examples of such species include, but are not limited to, mice, rats, hamsters, rabbits, chickens, goats, pigs, sheep and cattle. The modes 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 to early embryos and sperm-mediated transgenic effects, adenovirus-mediated DNA introduction into animal sperm (e.g., in pigs), retroviral vectors (e.g., avian species), somatic cell nuclear transfer (e.g., in goats). The state of the art in the preparation of transgenic livestock farm animals is reviewed in Niemann, H. et al. (2005) Rev. Sci. Tech. 24: 285-298.

[0100] In some embodiments, the animal is a vertebrate or an invertebrate. In some embodiments, the animal is an insect. In some embodiments, the insect is a mosquito. In some embodiments, the animal is a mammalian subject. In some embodiments, the mammal is a non-human animal. In some embodiments, the mammal is a non-human primate. In some embodiments, the transgenic animal of the present disclosure can be prepared using classical random genome recombination technology or with more precise technology, such as CRISPR / Cas genome editing guided by guide RNA, or endonuclease genome editing guided by DNA of NgAgo (Gray's halophilic alkaline bacillus Argonaute), or TALEN genome editing (transcription activator-like effector nuclease). In some embodiments, the transgenic animal of the present disclosure can be prepared using transgenic microinjection technology, and does not need to use homologous recombination technology, and is therefore considered to be easier to prepare and select than the method using homologous recombination. In another aspect, provided herein is a method for producing a polypeptide of interest, wherein the method comprises (i) raising an animal as disclosed herein under certain conditions; or (ii) culturing a recombinant cell comprising a nucleic acid construct as disclosed herein under certain conditions, under 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, wherein the method comprises administering a nucleic acid construct as disclosed herein to the subject. 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. Therefore, 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 recombinant polypeptides may include one or more of the following features. In some embodiments, the method for producing a recombinant polypeptide of the present disclosure further comprises isolating and / or purifying the produced polypeptide. In some embodiments, the method for producing a polypeptide of the present disclosure further comprises structurally modifying the produced polypeptide to increase half-life. D. Pharmaceutical Composition

[0102] The nucleic acid construct, recombinant cell, and recombinant polypeptide of the present disclosure can be incorporated into a composition (including a pharmaceutical composition). Such compositions generally include one or more of the nucleic acid constructs, recombinant cells, and recombinant polypeptides described and provided herein and a pharmaceutically acceptable excipient (e.g., carrier). In some embodiments, the compositions of the present disclosure are formulated for preventing, treating, or managing health conditions, such as immune diseases or microbial infections (e.g., viral infections, microfungal infections, or bacterial infections). For example, the compositions of the present disclosure can be formulated as preventive compositions, therapeutic compositions, or pharmaceutical compositions or mixtures thereof comprising pharmaceutically acceptable excipients. In some embodiments, the compositions of the present disclosure are formulated for use as vaccines. In some embodiments, the compositions of the present application are formulated for use as adjuvants.

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

[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, compositions are provided herein, comprising nucleic acid constructs as disclosed herein and pharmaceutically acceptable excipients. In some embodiments, compositions are provided herein, comprising recombinant cells as disclosed herein and pharmaceutically acceptable excipients. In some embodiments, compositions comprise recombinant polypeptides as disclosed herein and pharmaceutically acceptable excipients.

[0105] In some embodiments, the nucleic acid construct of the present disclosure (e.g., carrier or srRNA molecule) can be used in naked form or formulated with a delivery vehicle. Exemplary delivery vehicles suitable for the compositions and methods of the present disclosure include, but are not limited to, liposomes (e.g., neutral or anionic liposomes), microspheres, immunostimulatory complexes (ISCOMs), lipid-based nanoparticles (LNPs), solid lipid nanoparticles (SLNs), polyplexes, polymer nanoparticles, viral replicon particles (VRPs), or conjugates with bioactive ligands, which can promote delivery and / or enhance immune responses. These compounds are readily available to those skilled in the art; for example, see Liposomes: A Practical Approach, RCP New Ed, IRL press (1990). Adjuvants other than liposomes, etc. are also used, and the adjuvants are known in the art. Adjuvants can protect antigens (e.g., nucleic acid constructs, carriers, srRNA molecules) from rapid diffusion by isolating them in local deposits, or they can contain substances that stimulate the host to secrete factors that are chemotactic to macrophages and other components of the immune system.

[0106] The compositions of the present disclosure may be formulated in a form compatible with its intended route of administration, such as liposomes, lipid-based nanoparticles (LNPs), polymer nanoparticles, polyplexes, viral replicon particles (VRPs), microspheres, immunostimulatory complexes (ISCOMs), conjugates of biologically active 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 in lipid-based nanoparticles (LNPs).Exemplary types of lipids suitable for use in the delivery systems described herein include cationic lipids, ionizable cationic lipids, anionic lipids, neutral lipids, and combinations thereof.

[0108] In some embodiments, the LNP of the present disclosure may 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 WO 2020252589A1 and WO 2021000041A1 and Love KT et al., Proc Natl Acad Sci USA, February 2, 2010, 107(5)1864-1869, which are incorporated herein by reference in their entirety.

[0109] Therefore, in some embodiments, the LNP of the present disclosure comprises 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 LNP comprises an ionizable cationic lipid selected from the following: ALC-0315, C12-200, LN16, MC3, MD1, SM-102 and any combination thereof. In some embodiments, the LNP of the present disclosure comprises C12-200.

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

[0111] In some embodiments, the LNP of the present disclosure comprises one or more neutral lipids. As described above, in some embodiments, neutral lipids (also referred to as "structural lipids" or "helper lipids") can also be incorporated into lipid formulations and lipid particles. Lipid formulations and lipid particles can include one or more structural lipids of about 10Mol% to 40Mol% of the composition. Suitable structural lipids support the formation of particles during manufacture. Structural lipids refer to any of the many lipid species that exist in the form of anions, uncharged or neutral zwitterions at physiological pH. Representative structural lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, diacylphosphatidylglycerol, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin and cerebrosides.

[0112] Exemplary structured lipids include zwitterionic lipids such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-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-dielaidoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE).

[0113] In another embodiment, the structural lipid can be any lipid with negative charge at physiological pH. These lipids include phosphatidylglycerol, such as dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid and other anionic modifying groups connected to neutral lipids. Other suitable structural lipids include glycolipids (e.g., monosialoganglioside GM1).

[0114] Non-limiting neutral lipids suitable for use in 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 WO 2021000041A1, which are incorporated herein by reference in their entirety.

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

[0116] In some embodiments in which the delivery system described herein comprises 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 average diameter of lipid-based nanoparticles (LNP) is less than about 1000nm, about 500nm, about 250nm, about 200nm, about 150nm, about 100nm, about 75nm, about 50nm or about 25nm. In some embodiments, the range of the average diameter of LNP is about 70nm to 100nm. In some embodiments, the range of the average diameter of LNP is about 88nm to about 92nm, 82nm to about 86nm or about 80nm to about 95nm.

[0117] Stabilizers can be included in lipid formulation embodiments to ensure the integrity of the mixture. Stabilizers are molecules that destroy or help form 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)oxolane-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl octadecane-9-enoate), Myrj52 (polyoxyethylene (40) stearate), and Brij TM S10 (polyoxyethylene (10) stearyl ether). Polyethylene glycol-conjugated lipids may also be used. Stabilizers may be used alone or in combination with one another.

[0118] In some embodiments, the stabilizer accounts for about .1Mol% to 3Mol% of the overall lipid mixture. In some embodiments, the stabilizer accounts for about 0.5Mol% to 2.5Mol% of the overall lipid mixture. In some embodiments, the stabilizer is present at more than 2.5Mol%. In some embodiments, the stabilizer is present at 5Mol%. In some embodiments, the stabilizer is present at 10Mol%. In some embodiments, the stabilizer is about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, etc. In other embodiments, the stabilizer is 2.6Mol%-10Mol% of the lipid mixture. In other embodiments, the stabilizer is present at more than 10Mol% of the lipid mixture.

[0119] Steroids may also be included in lipid compositions for certain applications, and lipid particles prepared therefrom include sterols such as cholesterol and phytosterols.

[0120] In some embodiments, the composition of the present disclosure is formulated in polymer nanoparticles. Examples of polymers suitable for the composition and method of the present disclosure can be found in Jiang X et al. (Polymeric nanoparticlesfor RNA delivery.Encyclopedia of Nanomaterials, 2021), which are incorporated herein by reference. Exemplary polymers suitable for the composition and method of the present disclosure include cationic polymers, non-cationic polymers and combinations thereof. In some embodiments, the polymer nanoparticles of the present disclosure may include cationic polymers of natural origin. In some embodiments, cationic polymers of natural origin may include chitosan, gelatin, dextran, cellulose, cyclodextrin or a combination thereof. In some embodiments, cationic polymers may be synthetic cationic polymers. In some embodiments, the synthetic cationic polymer may include polyethyleneimine (PEI), poly-L-lysine (PLL), poly(amino acid) (PAA), poly(amidoamine) (PAMAM), poly(amino-co-ester) (PAE), poly(2-N,N-dimethylaminoethyl methacrylate, poly(β-amino ester) (PBAE), imidazole-containing polymers, tertiary amine-containing polymers, poly(2-(dimethylamino)ethyl methacrylate), poly-N-(2-hydroxy-propyl)methacrylamide, polyamidoamine dendrimers, cationic sugar-containing polymers, or derivatives thereof.

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

[0122] In some embodiments, the polymer may be water soluble and / or biodegradable. In some embodiments, the polymer 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), polyalkylcyanoacrylate (PACA), polyanhydrides, polyhydroxy acids, polypropyl fumarate, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, [N-(2-hydroxypropyl) methacrylamide] (HPMA) copolymers, polyvinyl alcohol, polyurethanes, polyphosphazenes, polyacrylates, polyureas, polyamines, poly-ε-caprolactone (PCL) and copolymers thereof.

[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 biotherapeutic, such as a vehicle for gene delivery of different molecules with biological activity. Non-limiting examples of biotherapeutics include cytokines, chemokines and other soluble immunomodulators, enzymes, peptide and protein agonists, peptide and protein antagonists, hormones, receptors, antibodies and antibody derivatives, growth factors, transcription factors, and gene silencing / editing molecules. In some embodiments, the pharmaceutical 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 administration, transdermal administration, intrathecal administration, intraperitoneal administration, intramuscular administration, intratracheal administration, intranodal administration, intratumoral administration, intraarticular administration, intravenous administration, subcutaneous administration, intravaginal administration, intraocular administration, rectal administration, and oral administration.

[0126] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, New Jersey) or phosphate buffered saline (PBS). In these cases, the composition should be sterile and should be fluid to the extent that it is easy to inject. It can be stable under the conditions of manufacture and storage and can be preserved against the contaminating effects of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and a suitable mixture thereof. For example, appropriate fluidity can be maintained by using a coating (such as lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant (e.g., sodium lauryl sulfate). Preventing the effects of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, isotonic agents such as sugars, polyols (such as mannitol, sorbitol) and / or sodium chloride will generally be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

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

[0128] In some embodiments, the pharmaceutical compositions of the present disclosure are formulated for inhalation, such as aerosols, sprays, mists, liquids or powders. Administration by inhalation can be in the form of dry powder or aerosol formulations, which are inhaled by a subject (e.g., a patient) using an inhalation device (e.g., a microsprayer, a pressure metered dose inhaler or a nebulizer).

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

[0130] Administration of any of the therapeutic compositions described herein (e.g., nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions) can be used to treat related health conditions, such as proliferative disorders (e.g., cancer), infectious diseases (e.g., acute infections, chronic infections, or viral infections), rare diseases, and / or autoimmune diseases, and / or inflammatory diseases. In some embodiments, nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions as described herein can be used to modulate (e.g., cause or inhibit) a pharmacodynamic effect in a subject in need thereof. In some embodiments, the pharmacodynamic effect includes causing 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, side effects, and effects in disease models. Thus, one aspect of the present disclosure relates to a method for modulating a pharmacodynamic effect in a subject in need thereof, the method comprising administering to the subject a composition comprising one or more of the following: (a) a nucleic acid construct as described herein; (b) a recombinant cell as described herein; (c) a recombinant polypeptide as described herein; and (d) a pharmaceutical composition as described herein. In some embodiments, the pharmacodynamic effect includes one or more of: an immunogenic effect, a biomarker response, a therapeutic effect, a prophylactic effect, a desired effect, an undesired effect, a side effect, and an effect in a disease model.

[0131] In some embodiments, the nucleic acid constructs, recombinant cells, recombinant polypeptides and / or pharmaceutical compositions as described herein can be incorporated into therapeutic agents for use in methods for treating subjects who have, are suspected of having, or may be at high risk of developing one or more related health conditions or diseases. Therefore, in another aspect, provided herein is a method for preventing or treating a health condition of a subject, the method comprising prophylactically or therapeutically administering to the subject a composition comprising one or more of the following: (a) a self-replicating RNA construct as described herein; (b) a nucleic acid as described herein; (c) a recombinant cell as described herein; and (d) a pharmaceutical composition as described herein. In some embodiments, the composition administered causes an immune response in the subject. In some embodiments, the composition administered induces the production of one or more pro-inflammatory molecules in the subject. In some embodiments, one or more pro-inflammatory molecules include interferon gamma (IFNγ), cytokines, TNF-α, GM-CSF, and MIP1α, granzyme B, granzyme A, perforin, or any combination thereof. In some embodiments, the subject has previously been treated with one or more therapies and has developed at least partial resistance to the one or more therapies.

[0132] Exemplary health conditions or diseases may include, but are not limited to, cancer, immune diseases, autoimmune diseases, inflammatory diseases, gene therapy, gene replacement, cardiovascular disease, 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 use with 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 tyroidism), systemic lupus erythematosus, Sjögren's syndrome, diabetic retinopathy, diabetic vasculopathy, diabetic neuropathy, insulitis, psoriasis, alopecia areata, warm and cold autoimmune hemolytic anemia (AIHA), pernicious anemia, acute inflammatory diseases, autoimmune adrenalitis, chronic inflammatory demyelinating polyneuropathy (CIDP), Lambert-Eaton syndrome, lichen sclerosus, Lyme disease, Graves' disease, Behçet's disease, Meniere's disease, reactive arthritis (Reiter's syndrome), Schüll-Strauss syndrome, Cogan syndrome, CREST syndrome, pemphigus vulgaris and pemphigus foliaceus, bullous pemphigoid, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, pancreatitis, peritonitis, psoriatic arthritis, rheumatic fever, sarcoidosis, Schergensen syndrome ( syndrome, scleroderma, celiac disease, stiff-person syndrome, Takayasu arteritis, transient gluten intolerance, autoimmune uveitis, vitiligo, polychondritis, dermatitis herpetiformis (DH) or Duling disease, fibromyalgia, Goodpasture's syndrome, Guillain-Barré syndrome, Hashimoto's thyroiditis, autoimmune hepatitis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, myasthenia gravis, immune complex disorders, glomerulonephritis, polyarteritis nodosa, antiphospholipid syndrome, polyglandular autoimmune syndrome, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), urticaria, autoimmune infertility, juvenile rheumatoid arthritis, sarcoidosis, and autoimmune cardiomyopathy.

[0134] Non-limiting examples of infections suitable for the methods of the present disclosure include infections with viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis B virus (HCV), cytomegalovirus (CMV), respiratory syncytial virus (RSV), human papillomavirus (HPV), Epstein-Barr virus (EBV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome (MERS), influenza virus, and Ebola virus. Additional infections suitable for the methods of the present disclosure include infections with intracellular parasites such as Leishmania, Rickettsia, Chlamydia, Coxiella, Plasmodium, Brucella, Mycobacterium, Listeria, Toxoplasma, and Trypanosoma. In some embodiments, the replicon constructs, srRNA constructs, nucleic acid constructs, recombinant cells and / or pharmaceutical compositions can be used to treat and / or prevent immune, autoimmune or inflammatory diseases, such as, for example, glomerulonephritis, inflammatory bowel disease, nephritis, peritonitis, psoriatic arthritis, osteoarthritis, Still's disease, familial Mediterranean fever, systemic scleroderma and sclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, acute lung injury, meningitis, encephalitis, uveitis, multiple myeloma, glomerulonephritis, nephritis, asthma, atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud's syndrome, Sjögren's syndrome, juvenile diabetes, Reiter's disease, Behcet's disease, immune complex nephropathy, inflammation, IgA nephropathy, IgM polyneuropathy, immune-mediated thrombocytopenia, hemolytic anemia, myasthenia gravis, lupus nephritis, lupus erythematosus, rheumatoid arthritis (RA), ankylosing spondylitis, pemphigus, Graves' disease, Hashimoto's thyroiditis, small vessel vasculitis, Omen's syndrome, chronic renal failure, autoimmune thyroid disease, acute infectious mononucleosis, HIV, herpes virus-related diseases, human viral infections, coronaviruses, other enteroviruses, herpes viruses, influenza virus, parainfluenza virus, respiratory syncytial virus or adenovirus infections, bacterial pneumonia, trauma, sepsis, stroke / cerebral edema, ischemia-reperfusion injury, and hepatitis C.

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

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

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

[0138] In some embodiments, the disclosed compositions are formulated to be compatible with its expected route of administration. For example, the nucleic acid constructs (for example, replicon constructs, for example srRNA constructs), recombinant cells, recombinant polypeptides and / or pharmaceutical compositions of the present disclosure can be administered orally or by inhalation, but more likely, they will be administered by parenteral routes. The example of parenteral route of administration includes, 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 for parenteral use may include the following components: sterile diluents, such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, phosphates, tris, sucrose; and agents for adjusting tonicity, such as sodium chloride or dextrose. pH may be adjusted with acid or alkali (such as sodium dihydrogen phosphate and / or sodium hydrogen phosphate, hydrochloric acid or sodium hydroxide) (e.g., to a pH of about 7.2-7.8, e.g., 7.5). Parenteral preparations may be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0139] The LD50 (the dose lethal to 50% of the population) and ED can be determined in cell cultures or experimental animals, for example. 50 The dosage, toxicity, and therapeutic efficacy of such subject nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure are determined by standard pharmaceutical procedures (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxic effects and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD50 / ED 50 Compounds that exhibit high therapeutic indices are generally suitable. Although compounds that exhibit toxic side effects may be used, care should be taken to design delivery systems that target such compounds to the affected tissue site to minimize potential damage to uninfected cells and thereby reduce side effects.

[0140] For example, data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. Dosages of such compounds are generally in the range of ED 50 The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods of the present disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve an IC value including that determined in cell culture. 50 The invention also provides a method for determining the circulating plasma concentration range of the test compound (e.g., the concentration of the test compound that achieves 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 described herein, such as nucleic acid constructs (e.g., srRNA constructs), recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions, may be administered from one or more times per day to one or more times per week (including administration every other day). The skilled artisan will appreciate that certain factors may affect the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the disease, previous treatment, the general health and / or age of the subject, and the presence of other diseases. In addition, treating a subject with a therapeutically effective amount of the subject multivalent polypeptides and multivalent antibodies of the present disclosure may include a single treatment, or may include a series of treatments. In some embodiments, the composition is administered every 8 hours for five days, followed by a rest period of 2 to 14 days (e.g., 9 days), followed by administration every 8 hours for another five days. With respect to nucleic acid constructs (e.g., replicon constructs, such as srRNA constructs) and recombinant polypeptides, the therapeutically effective amount (e.g., effective dose) of the nucleic acid construct or recombinant polypeptide of the present disclosure depends on the selected nucleic acid construct or recombinant polypeptide. For example, a single dose amount in the range of about 0.001 to 0.1 mg / kg patient weight may be administered. In some embodiments, about 0.005, 0.01, 0.05 mg / kg can be administered. In some embodiments, a single dose amount in the range of about 0.03 μg to 300 μg / kg patient weight can be administered. In some embodiments, a single dose amount in the range of about 0.3 mg to 3 mg / kg patient weight can be administered.

[0142] As discussed above, a therapeutically effective amount includes an amount of a therapeutic composition sufficient to promote a specific effect when administered to a subject, such as a subject suffering from, suspected of suffering from, or at risk of suffering from a health condition (e.g., a disease or infection). In some embodiments, an effective amount includes an amount sufficient to prevent or delay the development of symptoms of a disease or infection, to change the course of symptoms of a disease or infection (e.g., but not limited to, to slow the progression of symptoms of a disease or infection), or to reverse the symptoms of a disease or infection. It should be understood that for any given case, one of ordinary skill in the art can determine an appropriate effective amount using routine experiments.

[0143] The efficacy of a treatment comprising the disclosed therapeutic compositions for treating a disease or infection can be determined by a skilled clinician. However, if at least any one or all of the signs or symptoms of the disease or infection are improved or ameliorated, the treatment is considered to be an effective treatment. Efficacy can also be measured by failure of individual deterioration (e.g., cessation or at least slowing of disease or infection progression) as assessed by hospitalization or the need for medical intervention. Methods for measuring these indicators are known to those skilled 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, such as stopping or slowing the progression of symptoms; or (2) alleviating the disease or infection, such as causing regression of symptoms; and (3) preventing the development of symptoms or reducing their likelihood.

[0144] In some embodiments, the nucleic acid constructs (e.g., replicon constructs, such as srRNA constructs), recombinant cells, recombinant polypeptides and / or pharmaceutical compositions of the present disclosure can be administered to a subject in a composition with a pharmaceutically acceptable carrier and in an amount effective to stimulate an immune response. Typically, a subject can be immunized by an initial series of injections (or administered by one of the other routes described below), followed by a booster to increase the protection provided by the initial series of administrations. The initial series of injections and subsequent boosters are administered at a dose necessary to stimulate the subject's immune response and over a period of time necessary to stimulate the subject's immune response. In some embodiments, the composition administered results in an increase in the production of interferon in the subject. In some embodiments of the disclosed method, the subject is a mammal. In some embodiments, the mammal is a human subject.

[0145] As described above, pharmaceutically acceptable carriers suitable for injection include sterile aqueous solutions (in the case of water-soluble) or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. In these cases, the composition must be sterile and must be a fluid of a degree that is easy to inject. The composition must also be stable under manufacturing and storage conditions, and must be preserved against the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium, and the solvent or dispersion medium contains, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. For example, appropriate fluidity can be maintained by using a coating (such as lecithin), by maintaining the required particle size in the case of a dispersion, and by using a surfactant. Preventing the effect of microorganisms can be achieved by various antibacterial and antifungal agents (for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.).

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

[0147] When nucleic acid constructs, recombinant cells, recombinant polypeptides and / or pharmaceutical compositions are appropriately protected, as described above, they can be administered orally, for example, with an inert diluent or an assimilable edible carrier. Nucleic acid constructs, recombinant cells, recombinant polypeptides and / or pharmaceutical compositions and other ingredients can also be encapsulated in hard or soft shell gelatin capsules, compressed into tablets, or directly incorporated into the diet of an individual. For oral therapeutic administration, the active compound can be combined with an excipient and used in the form of an ingestible tablet, buccal tablet, lozenge, capsule, elixir, suspension, syrup, wafer, etc.

[0148] In some embodiments, the nucleic acid constructs and recombinant polypeptides of the present disclosure can be delivered to cells or subjects via lipid-based nanoparticles (LNPs). The immunogenicity of LNPs is generally lower than that of viral particles. Although many people have pre-existing immunity to viral particles, they do not have pre-existing immunity to LNPs. In addition, adaptive immune responses to LNPs are unlikely to occur, which enables 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 in particular. For example, in one type of LNP, the GalNAc moiety is attached to the outside of the LNP and acts as a ligand to be absorbed into the liver via the asialoglycoprotein receptor. Any of these cationic lipids can be used to prepare LNPs to deliver the nucleic acid constructs and recombinant polypeptides of the present disclosure to the liver.

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

[0151] LNP can be made of cation, anion or neutral lipid.Neutral lipid (such as fusogenic phospholipid DOPE or membrane component cholesterol) can be included in LNP as "helper lipid" to enhance transfection activity and nanoparticle stability.The limitation of cationic lipid includes low efficacy caused by poor stability and rapid removal, and produces inflammatory response or anti-inflammatory response.LNP can also have hydrophobic lipid, hydrophilic lipid or both hydrophobic and hydrophilic lipid.

[0152] Many lipids known in the art or the combination of lipids can be used for producing LNP.The non-limiting example of the lipid suitable for producing LNP comprises DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE and GL67A-DOPE-DMPE-polyethylene glycol (PEG).The non-limiting example of cationic lipid comprises 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1 and 7C1.The non-limiting example of neutral lipid comprises DPSC, DPPC, POPC, DOPE and SM.The non-limiting example of PEG-modified lipid comprises PEG-DMG, PEG-CerC14 and PEG-CerC20.

[0153] In some embodiments, lipids can be combined in any number of molar ratios to produce LNPs. In addition, one or more polynucleotides can be combined in a wide range of molar ratios with one or more lipids 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 having, suspected of having, or who may be at high risk of developing cancer, an autoimmune disease, and / or an infection.

[0155] 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 for preventing or treating subjects who have, are suspected of having, or may be at high risk of developing a microbial infection. In some embodiments, the microbial infection is a bacterial infection. In some embodiments, the microbial infection is a fungal infection. In some embodiments, the microbial infection is a viral infection. Other treatments

[0156] In some embodiments, the composition according to the present disclosure is administered to the subject as a monotherapy (monotherapy) alone or as a first therapy in combination with at least one other therapy (e.g., a second therapy). In some embodiments, the second therapy is selected from chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery. In some embodiments, the second therapy is selected from chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, or surgery. In some embodiments, the first therapy and the second therapy are administered concomitantly. In some embodiments, the first therapy and the second therapy are administered simultaneously. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy is administered before the second therapy. In some embodiments, the first therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and / or after the second therapy. In some embodiments, the first therapy and the second therapy are administered in turn. In some embodiments, the first therapy and the second therapy are administered together with a single formulation. Reagent test kit

[0157] Also provided herein are various kits for practicing the methods described herein and written instructions for preparing and using the kits. Specifically, some embodiments of the present disclosure provide kits for regulating pharmacodynamic effects. Some embodiments of the present disclosure provide kits for causing an immune response in a subject. Some other embodiments relate to kits for preventing the health conditions of a subject in need. Some other embodiments relate to kits for treating methods for the health conditions of a subject in need. For example, in some embodiments, provided herein are kits comprising one or more of the nucleic acid constructs, recombinant cells, recombinant polypeptides and / or pharmaceutical compositions as provided and described herein and written instructions for preparing and using them.

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

[0159] Any of the kits described above may further comprise one or more additional reagents, wherein such additional reagents may be selected from: dilution buffer, reconstitution solution, wash buffer, control reagent, control expression vector, negative control, positive control, 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 can be in separate containers. In some other embodiments, the components of the kit can be combined in a single container. Therefore, in some embodiments of the present disclosure, the kit is included in one container (e.g., in a sterile glass or plastic vial) as provided and described herein nucleic acid constructs, recombinant cells, recombinant polypeptides and / or pharmaceutical compositions One or more and another therapeutic agent in another container (e.g., in a sterile glass or plastic vial).

[0161] In another embodiment, the kit comprises a combination of a composition described herein (comprising one or more nucleic acid constructs, recombinant cells and / or recombinant polypeptides of the present disclosure) and one or more other therapeutic agents, optionally formulated together as a pharmaceutical composition, in a single common container.

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

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

[0164] In some embodiments, the kit may further include instructions for practicing the methods disclosed herein using the components of the kit. For example, the kit may include a package insert containing information about the pharmaceutical composition and dosage form in the kit. Typically, such information helps patients and doctors to effectively and safely use the encapsulated pharmaceutical composition and dosage form. For example, the following information about the combination of the present disclosure can be provided in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdose, correct dosage and administration, supply specifications, correct storage conditions, references, manufacturer / distributor information, and intellectual property information.

[0165] The instructions for practicing the method are usually recorded on a suitable recording medium. For example, the instructions can be printed on a substrate (such as paper or plastic, etc.). The instructions can be present in the test kit as a package insert, can be present in the label of the container of the test kit or its component (for example, related to packaging or sub-packaging), etc. The instructions can exist as an electronic storage data file present in a suitable computer-readable storage medium (for example, CD-ROM, floppy disk, flash drive, etc.). In some cases, the real instructions are not present in the test kit, but can provide a means for obtaining the instructions from a remote source (for example, via the Internet). The example of this embodiment is a test kit comprising a website, in which the instructions can be viewed and / or can be downloaded from it. Like 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 any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that although a number of information sources are referred to herein, including scientific journal articles, patent documents and textbooks; this reference does not constitute an admission that any of these documents form part of the common general knowledge in the field.

[0168] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be clear to those skilled in the art after reviewing this disclosure and are to be included within the spirit and scope of this application.

[0169] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration only and are not intended to limit the scope of the disclosure or the claims in any way. Example

[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 fully explained in the literature, such as Sambrook, J., & Russell, DW (2012). Molecular Cloning: A Laboratory Manual (4th Edition). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J. and Russel, DW (2001). Molecular Cloning: A Laboratory Manual (3rd Edition). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (collectively referred to herein as "Sambrook"); Ausubel, FM (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (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, Ferré, F. and Gibbs, R. (1994). PCR: The PolymeraseChain Reaction. Boston: Birkhauser Publisher; Greenfield, EA(2014). Antibodies: A Laboratory Manual (2nd Edition). 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 only and are not intended to limit the scope of the disclosure or the claims in any way. Example 1 Construction of WEEV vector

[0172] This example describes experiments performed to construct a basic WEEV vector (eg, without heterologous genes), which was subsequently used to construct a WEEV vector expressing a gene of interest (eg, the hemagglutinin precursor HA of influenza A virus H5N1).

[0173] The basic WEEV vector (ie, without a heterologous gene of interest) was constructed as follows: the basic WEEV vector was synthesized de novo in four approximately 4 kb parts from a reference sequence (Genbank GQ287641) with several modifications (see, e.g., Figure 2A). Ambiguous nucleotide assignments in the reference sequence were assigned to nucleotides that matched synonymous codons for the encoded residues. Silent mutations were incorporated to eliminate SapI and SpeI restriction endonuclease cleavage sites. A unique restriction endonuclease cleavage site (SpeI, 5'-A'CTAG, T-3') can be incorporated in place of the coding sequence of the native WEEV structural gene (wherein the 5'A matches the position of the structural polyprotein ATG start codon and the 3'T matches the position of the structural polyprotein stop codon TAA). A 5' adapter sequence (5'-CTGGAGACGTGGAGGAGAACCCTGGACCT-3'; SEQ ID NO: 3) was inserted upstream of the SpeI site, and a 3' adapter sequence (5'-GACCGCTACGCCCCAATGACCCGACCAGC-3'; SEQ ID NO: 4) was inserted downstream of the SpeI site for subsequent Gibson Procedure (Gibson et al., Nat. Methods 6, 343-345, 2009). The bacteriophage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO: 5) is included upstream of the WEEV genomic sequence and may contain a poly(A) sequence downstream, followed by a SapI site that cuts upstream of the recognition site. Immediately downstream of the SapI site is the T7 terminator sequence (5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO: 6), followed by a unique restriction endonuclease cleavage site (NotI, 5'-GC'GGCC, GC-3'). These parts are assembled in a five-piece Gibson The pYL backbone and four synthetic fragments were combined in the reaction to generate the WEEV base vector.

[0174] In some experiments, the sequence encoding one or more nsPs was replaced by a heterologous nsP. In some other experiments, the sequence encoding one or more UTRs was replaced by a heterologous UTR.

[0175] The construction of WEEV vectors containing heterologous genes was performed as follows: Figure 2A The empty WEEV vector was linearized to construct Figure 2B The WEEV vector described in . The hemagglutinin (HA) gene from influenza virus (Genbank AY651334) was codon optimized / reconstructed for human expression in silico and synthesized de novo (IDT). The synthetic product was amplified using primers that added 5' and 3' adapter sequences to the ends of the HA gene. The digestion product and PCR product were separated by Gibson The programs are combined to produce the final vector. Example 2 In vitro evaluation of modified WEEV vectors

[0176] This example describes in vitro experiments performed to evaluate the expression levels of the synthetic WEEV srRNA constructs described in Example 1 above and to investigate any differential behavior thereof (eg, replication and protein expression).

[0177] In these experiments, synthetic srRNA constructs derived from the WEEV strain Imperial were designed and subsequently evaluated.

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

[0179] copy: Transform RNA into BHK-21 or Vero cells by electroporation (e.g., 4D-Nucleofector TM , Lonza). 15-20 hours after transformation, cells were fixed and permeabilized (eBioscience TM The cells were stained with Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen) followed by staining with PE-conjugated anti-dsRNA mouse monoclonal antibody (J2, Scicons) to quantify the frequency of dsRNA+ cells and the mean fluorescence intensity (MFI) of dsRNA in individual cells by fluorescent flow cytometry.

[0180] Protein expression : Transform RNA into BHK-21 or Vero cells by electroporation (e.g., 4D-Nucleofector TM , Lonza). 15-20 hours after transformation, cells were fixed and permeabilized (eBioscience TMFoxp3 / Transcription Factor Staining Buffer Set, Invitrogen) and stained with APC-conjugated anti-HA mouse monoclonal antibody (2B7, Abcam) to quantify the frequency of cells expressing HA protein and the mean fluorescence intensity (MFI) of HA protein in individual cells by fluorescence flow cytometry.

[0181] Other experiments: Prior to RNA electroporation, BHK-21 or Vero cells were pretreated with a titration curve of recombinant interferon (IFN), and the effects on vector replication and protein expression were measured using the assays described above.

[0182] In some experiments, BHK-21 cells were transformed with WEEV srRNA constructs. WEEV srRNA without the target GOI was transformed by electroporation, and 20 hours after transformation, the transformed cells were fixed, permeabilized, and stained with PE-conjugated anti-dsRNA mouse monoclonal antibodies (J2, Scicons) to quantify the frequency of dsRNA+ cells by fluorescent flow cytometry. In addition, WEEV srRNA containing the coding sequence of the target GOI was similarly transformed into BHK-21 cells, and in addition to dsRNA detection, transgenic expression was detected using APC-conjugated anti-GOI monoclonal antibodies. Positive staining of individual cells with both anti-dsRNA and anti-GOI antibodies demonstrated that the modified WEEV designs described herein are viable synthetic srRNAs and are capable of undergoing RNA replication and expressing transgenes. Example 3 In vivo evaluation of modified WEEV vectors

[0183] This example describes in vivo experiments conducted to evaluate immune responses following vaccination with the synthetic WEEV srRNA constructs described in Examples 1 and 2 above (eg, both unformulated vectors and LNP formulated vectors).

[0184] In these experiments, synthetic srRNA constructs derived from the WEEV strain Imperial were designed and subsequently evaluated.

[0185] Mice and injections. Female C57BL / 6 or BALB / c mice were purchased from Envigo, Charles RiverLabs or Jackson Laboratories. On the day of dosing, between 0.1-10 μg of material was injected intramuscularly into both quadriceps muscles. The carrier was administered in an unformulated form in saline, in an LNP-formulated form or in a polymer-formulated form. Throughout the study, the animals were monitored for weight and other comprehensive observations. For immunogenicity studies, animals were dosed on days 0 and 21. Spleens were collected on day 35, and serum was separated on days 14 and 35. For protein expression studies, animals were dosed on day 0, and protein expression or bioluminescence was assessed on days 1, 3 and / or 7. In some experiments where the WEEV srRNA construct encoded a reporter protein (such as luciferase), luciferase activity was imaged in vivo using an IVIS system at specified time points. In some experiments where the WEEV srRNA construct encoded a secreted protein, systemic levels were determined by serum ELISA.

[0186] LNP Formulations In some experiments, srRNA was formulated in lipid nanoparticles using a microfluidic mixer, and the particle size, polydispersity (using dynamic light scattering), and encapsulation efficiency were analyzed. In these experiments, a wide range of lipid molar ratios were used when formulating the LNP particles. An exemplary molar ratio of lipids used in these experiments can be 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 was performed using the Mouse IFNγ ELISpot PLUS Kit (HRP) (MabTech) according to the manufacturer's instructions. In these experiments, splenocytes were isolated and resuspended in culture medium to an appropriate concentration (e.g., 5×10 6 Cells / mL) containing peptides representing T cell epitopes of the protein of interest encoded by the WEEV srRNA construct. One or more positive controls (such as, for example, PMA / ionomycin) and DMSO used as a mock stimulus were also included in these experiments.

[0188] Antibody. Antibody responses for measuring total HA-specific IgG were measured using an ELISA kit from Alpha Diagnostic International according to the manufacturer's instructions. Although specific alternatives to the present disclosure have been disclosed, it should be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. Therefore, there is no intention to be limited to the exact abstract and disclosure presented herein.

Claims

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

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

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

4. according to the nucleic acid construct described in any one among the claims 1-3, described nucleic acid construct further comprises one or more expression cassettes, each in wherein said expression cassette comprises the promoter that is operably connected to the heterologous nucleic acid sequence.

5. The nucleic acid construct of claim 4, wherein at least one of the expression cassettes comprises a subgenomic (sg) promoter operably linked to a heterologous nucleic acid sequence. The nucleic acid construct according to claim 5 , wherein the sg promoter is a 26S subgenomic promoter.

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

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

9. according to the nucleic acid construct described in any one among claim 1-8, described nucleic acid construct further comprises one or more untranslated regions (UTR).

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

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

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

13. The nucleic acid construct according to any one of claims 11-12, wherein the GOI encodes a polypeptide selected from the group consisting of an antibody, an antigen, an immunomodulator, an enzyme, a signaling protein, and a cytokine.

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

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

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

2.

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

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

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

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

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

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

24. The recombinant cell of claim 20, wherein the recombinant cell is selected from monkey kidney CV1 cells (COS-7) transformed by SV40, human embryonic kidney cells (e.g., HEK 293 or HEK 293 cells), baby hamster kidney cells (BHK), mouse supporting cells (e.g., TM4 cells), monkey kidney cells (CV1), human cervical cancer cells (HeLa), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse breast tumor cells (MMT 060562), TRI cells, FS4 cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (Vero cells), human A549 cells, human cervical cells, human CHME5 cells, human PER.C6 cells, NS0 mouse myeloma cells, human epidermoid laryngeal cells, human fibroblasts, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocytes, human macrophages, human RAW264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells and rabbit kidney cells.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

42. A pharmaceutical composition according to any one of claims 38-41, wherein the composition is formulated in liposomes, lipid-based nanoparticles (LNPs), polymer nanoparticles, polyplexes, viral replicon particles (VRPs), microspheres, immunostimulatory complexes (ISCOMs), conjugates of biologically active ligands, or any combination thereof.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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