Compositions and methods for expression of IL-12 and IL-1RA

Abstract

The present disclosure relates to the field of molecular virology, and in particular to nucleic acid molecules encoding viral genomes or self-replicating RNA (srRNA) constructs of modified alphaviruses, pharmaceutical compositions comprising the same, and the use of such nucleic acid molecules and compositions for the production of desired products in cell culture or in an organism. Methods of inducing a pharmacodynamic effect in a subject are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 17 / 537,211, filed November 29, 2021. The disclosures of the above-referenced applications are expressly incorporated by reference in their entirety, including any drawings.

[0002] Field The present disclosure relates to the fields of molecular virology and immunology, and in particular to the use of nucleic acid molecules encoding the viral genome and self-replicating RNA (srRNA) of modified alphaviruses, pharmaceutical compositions comprising the same, and the use of such nucleic acid molecules and compositions for the production of a desired product in cell culture or in vivo. Also provided are methods of inducing at least one pharmacodynamic effect in a subject.

[0003] Incorporating sequence tables The attached sequence listing material is incorporated into this application by reference. The attached sequence listing file (Name: 058462-508001WO_Sequence Listing_ST26.xml) was created on November 28, 2022 and is 35KB in size. [Background technology]

[0004] background Interleukin-12 (IL-12) is a pleiotropic proinflammatory cytokine produced in response to infection by various cells of the immune system, including phagocytes, B cells, and activated dendritic cells (Colombo and Trinchieri (2002), Cytokine & Growth Factor Reviews, 13: 155-168). IL-12 plays a key role in mediating the interactions of the innate and adaptive arms of the immune system, acting on T cells and natural killer (NK) cells to enhance the proliferation and activity of cytotoxic lymphocytes as well as the production of other proinflammatory cytokines, especially interferon-γ. IL-12 is a heterodimeric molecule consisting of an α chain (p35 subunit, IL-12A) and a β chain (p40 subunit, IL-12B) covalently linked by disulfide bonds to form a biologically active 74 kDa heterodimer.

[0005] The presence of endogenous IL-12 has also been shown to be necessary for immunological resistance to a wide range of pathogens as well as transplanted and chemically induced tumors (Gateley et al. (1998), Annu. Rev. Immunol., 16: 495-521). IL-12 has been shown to have potent antitumor activity based on the induction of IFN-γ and activation of effector cells such as CD8+ T cells and NK cells (Brunda ef al. (1993), J. Exp. Med., 178: 1223-30). In response to IL-12, high levels of IFN-γ are produced by T cells and NK cells (Kobayashi et al., 1989, J Exp Med; 170: 827-45), enhancing antigen presentation through paracrine upregulation of MHC class I and class II expression (Wallach et al., 1982 Nature 1982;299:833-69). Because of its proven antitumor activity, IL-12 has been tested in human clinical trials as an immunotherapeutic agent for the treatment of various cancers, including renal, colon, ovarian, melanoma, and T-cell lymphoma (Atkins et al. (1997), Clin. Cancer Res., 3: 409-17; Gollob et al. (2000), Clin. Cancer Res., 6: 1678-92; and Hurteau et al. (2001), Gynecol. Oncol., 82: 7-10), and as an adjuvant for cancer vaccines (Lee et al. (2001), J. Clin. Oncol. 19: 3836-47).

[0006] Systemic administration of IL-12 has shown efficacy, primarily against several solid tumors, but its therapeutic use is limited due to dose-limiting toxicities (Gollob et al., 2000, Clin. Cancer Res. 6:1678-1692). Although more than a decade has passed since its initial clinical development, documented cases of severe toxicity and overall low response rates to recombinant IL-12 have prevented it from undergoing clinical development to date.

[0007] Interleukin-1 is also important in immune responses, but it is the inhibition of this cytokine that has attracted much interest. The mode of action of IL-1 is mediated by the interleukin-1 receptor antagonist protein (IL-1ra; also known as "IRAP"). IL-1ra binds to the same receptor on the cell surface as IL-1, thus preventing IL-1 from sending signals to that cell. IL-1ra is secreted by white blood cells, such as monocytes, macrophages, neutrophils, polymorphonuclear cells (PMN), and other cells, and can regulate a variety of IL-1-associated immune and inflammatory responses, as described in Arend WP, ​​Malyak M, Guthridge CJ, Gabay C (1998) "Interleukin-1 receptor antagonist: role in biology" Annu Rev. Immunol. 16:27-55. The production of IL-1ra can be stimulated by several substances, including adhesive immunoglobulin G (IgG), other cytokines, and bacterial or viral components. IL-1ra is a naturally occurring anti-inflammatory protein important in arthritis, colitis, and granulomatous lung disease.

[0008] IL-1ra can be used to treat rheumatoid arthritis, an autoimmune disease in which IL-1 plays an important role, to reduce inflammation and cartilage deterioration associated with the disease. For example, Kineret™ (anakinra) is a recombinant, non-glycosylated form of IL-1ra (Amgen Manufacturing, Ltd., Thousand Oaks, Calif.). Various recombinant interleukin-1 inhibitors and methods of treatment are described in U.S. Patent No. 6,599,873, issued July 29, 2003 to Sommer et al.; U.S. Patent No. 5,075,222, issued December 24, 1991 to Hannum et al.; and U.S. Patent Application Publication No. 2005 / 0197293, published September 8, 2005 to Mellis et al. Additionally, methods for producing IL-1ra from body fluids, including the use of autologous fluids, are described in U.S. Pat. No. 6,623,472, issued Sep. 23, 2003 to Reineke et al.; U.S. Pat. No. 6,713,246, issued Mar. 30, 2004 to Reineke et al.; and U.S. Pat. No. 6,759,188, issued Jul. 6, 2004 to Reineke et al. Summary of the Invention [Problem to be solved by the invention]

[0009] As described in U.S. Patent No. 6,096,728, issued Aug. 1, 2000 to Collins et al., IL-1ra has been delivered as part of a composition with hyaluronic acid. However, many of these methods and compositions involve problems related to the stability and half-life of IL-1ra, as well as the amount and rate at which IL-1ra is provided. Thus, improved compositions and methods for delivering IL-1ra are desirable.

[0010] The disclosure provided herein provides solutions to problems existing in previous attempts to deliver IL-12 and IL-1RA, and may provide improved methods for the treatment of conditions including cancer and infectious diseases. [Means for solving the problem]

[0011] summary The present disclosure relates generally to the development of immunotherapeutics, such as recombinant nucleic acid constructs and pharmaceutical compositions comprising the same, for use in the prevention and management of various health conditions. In particular, as described in more detail below, some embodiments of the present disclosure provide nucleic acid constructs comprising a sequence encoding a modified alphavirus genome or a self-replicating RNA (srRNA), wherein at least a portion of the nucleic acid sequence encoding a viral structural protein of the modified alphavirus genome or srRNA has been replaced with a coding sequence for a polypeptide construct comprising a) a coding sequence for the p35 subunit of interleukin-12 (p35 or IL-12A) or a functional variant thereof, b) a coding sequence for the subunit 40 of interleukin-12 (p40 or IL-12B) or a functional variant thereof, and c) a coding sequence for an interleukin-1 receptor antagonist (IL-1RA) or a functional variant thereof. Also disclosed are recombinant cells engineered to contain one or more of the nucleic acid constructs disclosed herein, methods for producing molecules of interest, and pharmaceutical compositions comprising one or more of the following: (a) the nucleic acid constructs of the present disclosure, (b) the recombinant cells of the present disclosure, or (c) the pharmaceutical compositions of the present disclosure. Additionally, in certain aspects of the present disclosure, compositions and methods are provided for inducing at least one pharmacodynamic effect in a subject. The foregoing summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the exemplary embodiments and features described herein, further aspects, embodiments, objects, and features of the present disclosure will become fully apparent from the drawings, detailed description, and claims.

[0012] In one embodiment of the present disclosure, provided herein is a nucleic acid construct comprising a nucleic acid sequence encoding a modified alphavirus genome or srRNA, wherein at least a portion of the nucleic acid sequence encoding a viral structural protein of the modified alphavirus genome or srRNA has been replaced with a coding sequence for a polypeptide construct comprising a) a coding sequence for the p35 subunit of interleukin-12 (p35 or IL-12A) or a functional variant thereof, b) a coding sequence for the p40 subunit of interleukin-12 (p40 or IL-12B) or a functional variant thereof, and c) a coding sequence for interleukin-1 receptor antagonist (IL-1RA) or a functional variant thereof, wherein the coding sequences for IL-12A, IL-12B, and IL-1RA are operably linked to each other.

[0013] In some embodiments, the modified alphavirus genome or srRNA does not include nucleic acid sequences encoding viral structural proteins.

[0014] In some embodiments, the nucleic acid sequence encoding the modified alphavirus or srRNA is operably linked to a promoter sequence, hi some embodiments, the promoter sequence is a 26S subgenomic (sg) promoter.

[0015] In some embodiments, the coding sequences of (a)-(c) are operably linked to each other in a single open reading frame (ie, in a polycistronic ORF).

[0016] In some embodiments, the coding sequences of (a)-(c) are operably linked to one another by one or more connector sequences encoding an autoproteolytic peptide or an internal ribosome entry site (IRES). In some embodiments, the autoproteolytic peptide comprises one or more autoproteolytic cleavage sequences from calcium-dependent serine endoprotease (furin), porcine teschovirus 1 2A (P2A), foot and mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), Thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), Flacherie virus 2A (BmIFV2A), or a combination thereof. In some embodiments, the IRES is derived from Kaposi's sarcoma-associated herpesvirus (KSHV) IRES, Hepatitis virus IRES, Pestivirus IRES, Cripavirus IRES, Rhopalosiphum padivirus IRES, fibroblast growth factor IRES, platelet-derived growth factor IRES, vascular endothelial growth factor IRES, insulin-like growth factor IRES, picornavirus IRES, encephalomyocarditis virus (EMCV) IRES, Pim-1 IRES, p53 IRES, Apaf-1 IRES, TDP2 IRES, L-myc IRES, and c-myc IRES.

[0017] In some embodiments, the modified alphavirus genome or srRNA is of an alphavirus belonging to the VEEV / EEEV group, the SFV group, or the SINV group.

[0018] In some embodiments, the polypeptide construct comprises, from N-terminal to C-terminal direction, a) an IL-12A polypeptide, an IL-12B polypeptide, and an IL-1RA polypeptide, or b) an IL-1RA polypeptide, an IL-12B polypeptide, and an IL-12A polypeptide, wherein the IL-12A, IL-12B, and IL-1RA polypeptides are operably linked to each other by one or more autoproteolytic cleavage sequences or internal ribosome entry sites.

[0019] In some embodiments, the nucleic acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:10.

[0020] In one aspect, a recombinant cell is provided that comprises a nucleic acid construct disclosed herein. In some embodiments, the recombinant cell is a mammalian cell or an insect cell.

[0021] In yet another aspect, provided herein is a pharmaceutical composition comprising a pharma- ceutically acceptable excipient and a nucleic acid construct of the present disclosure.

[0022] In some embodiments, the composition is formulated in a delivery system with a delivery vehicle, the delivery system comprising a liposome, a viral replicon particle (VRP), a lipid-based nanoparticle (LNP), a polymeric nanoparticle, a physiological buffer, a microsphere, an immune stimulating complex (ISCOM), a conjugate of a bioactive ligand, or any combination thereof. In some embodiments, the LNP delivery system comprises a cationic lipid, an ionizable cationic lipid, an anionic lipid, or a neutral lipid. In some embodiments, the lipid is present in a lipid to RNA mass ratio of about 100:1 to about 4:1. In some embodiments, the lipid-based nanoparticle has an average diameter of about 25 nm to about 1000 nm. In some embodiments, the composition is formulated as a biotherapeutic.

[0023] In another aspect, provided herein is a method of inducing at least one pharmacodynamic effect in a subject. The method comprises administering to the subject a composition comprising a nucleic acid construct of the present disclosure. In some embodiments, the administered composition results in an immune response and induced production of one or more of mediators, such as interferon gamma (IFNγ). In some embodiments, the at least one pharmacodynamic effect comprises one or more of an immunogenic effect, a biomarker response, a therapeutic effect, a preventive effect, a desired effect, an undesirable effect, an adverse effect, and an effect in a disease model. In some embodiments, the administered composition enhances anti-tumor immunity in the tumor microenvironment. In some embodiments, the subject suffers from cancer, an immune disease, or a chronic infection. In some embodiments, the composition is administered to the subject individually as a monotherapy (monotherapy) or as a first therapy in combination with at least one additional therapy. [Brief description of the drawings]

[0024] The features and advantages of the present disclosure will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0025] [Figure 1A-1B] 1A-1B are graphical representations of in vitro protein expression from monogenic and multigenic VEE srRNA constructs. Protein expression was measured by ELISA using supernatants from BHK-21 cells transfected with each srRNA construct. FIG. 1A shows the results of an IL-12 ELISA. The x-axis indicates the various constructs tested. FIG. 1B shows the results of an IL-RA ELISA. The x-axis indicates the various constructs tested.

[0026] [Figure 2A-2B]2A-2B are graphical representations of in vitro protein bioactivity from mono- and multi-gene constructs in VEE. Supernatants from BHK-21 cells transfected with each srRNA construct were used to measure cytokine protein bioactivity on reporter cells expressing the cognate receptor. FIG. 2A shows the results of an IL-12 bioassay. The x-axis indicates the various constructs tested. FIG. 2B shows the results of an IL-1RA bioactivity assay. The x-axis indicates the various constructs tested.

[0027] [Diagram 3] FIG. 3 is a graphical representation of the combined IL-12 bioactivity and IL-1RA expression data from the srRNA constructs.

[0028] [Figure 4A-4B] Figures 4A-4B are graphical representations of protein expression from the two best multigene constructs cloned into six different srRNA vectors. Figure 4A shows the results of an IL-12 ELISA. The x-axis indicates the various constructs tested. Figure 4B shows the results of an IL-RA ELISA. The x-axis indicates the various constructs tested.

[0029] [Figure 5A-5B] 5A-5B are graphical representations of protein bioactivity from the two best multigene constructs cloned into six different srRNA vectors. FIG. 5A shows the results of an IL-12 bioassay. The x-axis indicates the various constructs tested. FIG. 5B shows the results of an IL-RA bioassay. The x-axis indicates the various constructs tested.

[0030] [Figure 6] FIG. 6 is a graphical representation summarizing the IL-12 and IL-1RA bioactivity of the two best multigene constructs in six different srRNA vectors.

[0031] [Figure 7A-7B]7A-7B are graphical representations of in vivo protein expression in mouse serum by the two best multigene constructs in six different srRNA vectors. FIG. 7A shows the results of an IL-12 ELISA. The x-axis indicates the various constructs tested. FIG. 7B shows the results of an IL-1RA ELISA. The x-axis indicates the various constructs tested.

[0032] [Figure 8A-8B] 8A-8B are graphical representations of in vivo protein expression from various formulations of optimal srRNA constructs. FIG. 8A shows the results of an IL-12 ELISA. The x-axis indicates the various constructs and formulations tested. FIG. 8B shows the results of an IL-1RA ELISA. The x-axis indicates the various constructs and formulations tested. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Detailed Description of the Disclosure The present disclosure generally relates to nucleic acid constructs expressing the p35 subunit of interleukin-12 (p35 or IL-12A), the p40 subunit of interleukin-12 (p40 or IL-12B), and variants of interleukin-1 receptor antagonist (IL-1RA) for the treatment of human diseases such as cancer, immune disorders, and chronic infections. These constructs address the problem of therapeutic approaches such as recombinant cytokine administration due to previously proven dose-limiting toxicity issues. Provided herein are gene expression systems with enhanced expression capabilities suitable for expressing in recombinant cells, in particular, the coding sequence of the p35 subunit of interleukin-12 (p35 or IL-12A) or a functional variant thereof, the coding sequence of the p40 subunit of interleukin-12 (p40 or IL-12B) or a functional variant thereof, and the coding sequence of the interleukin-1 receptor antagonist (IL-1RA) or a functional variant thereof. For example, some embodiments of the present disclosure relate to nucleic acid constructs, such as: expression constructs and vectors comprising modified alphavirus genomes or srRNAs, in which at least a portion of the nucleic acid sequence encoding the viral structural proteins of the modified alphavirus genomes or srRNAs has been replaced with a coding sequence for the p35 subunit of interleukin-12 (p35 or IL-12A) or a functional variant thereof, a coding sequence for the p40 subunit of interleukin-12 (p40 or IL-12B) or a functional variant thereof, and a coding sequence for interleukin-1 receptor antagonist (IL-1RA) or a functional variant thereof. Additionally, recombinant cells genetically engineered to contain one or more of the nucleic acid molecules disclosed herein are provided. Biological materials and recombinant products obtained from such recombinant cells are also within the scope of the present application. Also provided are compositions and methods useful for eliciting pharmacodynamic effects of cancer in a subject in need thereof.

[0034] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0035] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may, for clarity, be described herein in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment.

[0036] definition Unless otherwise specified, all technical terms, notations, and other scientific or technical terms used herein are intended to have the meaning commonly understood by those of ordinary skill in the art to which this application pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein does not necessarily imply a significant difference from what is commonly understood in the art. Many of the techniques and operations described or referenced herein are well understood and commonly used by those of ordinary skill in the art using conventional methodology.

[0037] The singular forms "a," "an," and "the" include the plural forms unless the context clearly indicates otherwise. For example, the term "a cell" includes one or more cells, including mixtures thereof. "A and / or B" is used herein to include all of the following options: "A," "B," "A or B," and "A and B."

[0038] As used herein, the terms "administration" and "administering" refer to the delivery of a bioactive composition or formulation by a route of administration including, but not limited to, intranasal, transdermal, intravenous, intraarterial, intramuscular, intranodal, intraperitoneal, subcutaneous, intramuscular, oral, intravaginal, and topical administration, or a combination thereof. The term includes, but is not limited to, administration by a medical professional and self-administration.

[0039] The terms "cell," "cell culture," and "cell line" refer not only to a particular subject cell, cell culture, or cell line, but also to the progeny or potential progeny of such a cell, cell culture, or cell line, regardless of the number of transplants or passages in culture. It is understood that not all progeny are exactly identical to the parent cell. This is because certain modifications may occur in subsequent generations, either due to mutations (e.g., intentional or unintended mutations) or environmental influences (e.g., methylation or other epigenetic modifications), so that the progeny may not actually be identical to the parent cell, but are still within the scope of the term as used herein, so long as the progeny retains the same function as the original cell, cell culture, or cell line.

[0040] The term "construct" refers to a recombinant molecule, e.g., a recombinant nucleic acid or polypeptide, that includes one or more nucleic acid or amino acid sequences of heterologous origin. For example, a polypeptide construct can be a chimeric polypeptide molecule in which two or more amino acid sequences of different origins are operably linked to each other 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. Exemplary nucleic acid constructs include any recombinant nucleic acid molecule, linear or circular, single-stranded or double-stranded DNA or RNA nucleic acid molecule, capable of genomic integration or autonomous replication, from any source, such as plasmids, cosmids, viruses, autonomously replicating polynucleotide molecules, phages, etc., that includes one or more nucleic acid sequences operably linked thereto. The two or more nucleic acid constructs can be included in a single nucleic acid molecule, such as a single vector, or can be included in two or more separate nucleic acid molecules, such as two or more separate vectors.

[0041] The term "operably linked" as used herein refers to a physical or functional connection between two or more elements, e.g., polypeptide or polynucleotide sequences, that allows these elements to operate in an intended manner. For example, the term "operably linked" as used in the context of a nucleic acid molecule or coding and promoter sequences within a nucleic acid molecule described herein means that the coding and promoter sequences are in frame and in proper spatial and distance to allow binding of each by a transcription factor or RNA polymerase to affect transcription. It is understood that operably linked elements may be contiguous or non-contiguous (e.g., linked to each other via a linker). In the context of a polypeptide construct, "operably linked" refers to a physical connection (e.g., direct or indirect connection) between amino acid sequences (e.g., different segments, portions, regions, or domains) to provide the described activity of the construct. The operably linked segments, portions, regions, and domains of the polypeptide or nucleic acid molecules disclosed herein may be contiguous or non-contiguous (e.g., linked to each other via a linker).

[0042] The terms "effective amount", "therapeutically effective amount", or "pharmaceutical effective amount" of the disclosed compositions, e.g., nucleic acid constructs, srRNA, recombinant cells, and / or pharmaceutical compositions, generally refer to an amount sufficient for the composition to achieve a stated purpose (e.g., achieve the effect of being administered, stimulate an immune response, prevent or treat a disease, or alleviate one or more symptoms of a disease, disorder, infection, or condition) compared to the absence of the composition. An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or alleviation of one or more symptoms of a disease, also referred to as a "therapeutically effective amount". "Alleviation" of a symptom refers to a decrease in the severity or frequency of the symptom, or elimination of the symptom. The precise amount of a composition that comprises a "therapeutically effective amount" will depend on the purpose of the treatment, and will be ascertainable by one of ordinary skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0043] The term "portion" as used herein refers to a fraction. With respect to a particular structure, such as a polynucleotide sequence or an amino acid sequence or a protein, the term "portion" may refer to a contiguous or discontinuous portion of said structure. For example, a portion of an amino acid sequence includes at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% of the amino acids of said amino acid sequence. Additionally or alternatively, when a portion is a discontinuous portion, said discontinuous portion may be comprised of 2, 3, 4, 5, 6, 7, 8, or more portions of the structure (e.g., domains of a protein), each portion being a contiguous element of the structure. For example, the non-contiguous portions of an amino acid sequence can consist of 2, 3, 4, 5, 6, 7, 8 or more, such as up to 4 portions of said amino acid sequence, where 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.

[0044] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is included within the scope of the disclosure to the nearest tenth of the lower limit. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also included within the scope of the disclosure, subject to any specifically excluded limit in the stated range. Where one or both of the limits are included in the stated range, ranges excluding either or both of those included limits are also included in the disclosure.

[0045] In this specification, a specific range is provided with the term "about" before the numerical values. The term "about" is used herein to literally support the exact numerical value followed by the term, and a numerical value close to or approximately the numerical value followed by the term. In determining whether a numerical value is close to or approximately a specifically stated numerical value, a close or approximately unstated numerical value may be a numerical value that is substantially equivalent to the specifically stated numerical value in the context in which it is presented. If the degree of approximation is not clear from the context, "about" means a numerical value within plus or minus 10% of the provided value, or in all cases rounded to the nearest significant figure inclusive of the provided value. In some embodiments, the term "about" refers to the specified value ±10%, ±5%, or ±1%.

[0046] The term "percent identity" as used herein in the context of two or more nucleic acids or proteins refers to two or more sequences or subsequences that are the same or have a certain percentage of the same nucleotides or amino acids (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a particular region when compared and aligned for maximum correspondence over a comparison window or designated region) as determined using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters as described below, or by manual alignment and visual inspection. See, for example, the NCBI website ncbi.nlm.nih.gov / BLAST. Such sequences are then said to be "substantially identical". This definition can also refer to or apply to the complement of a sequence. This definition also includes sequences that contain deletions and / or additions, as well as substitutions. Sequence identity can be calculated using published procedures and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J Mol Biol 215:403, 1990). Sequence identity can be measured using sequence analysis software, such as the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, using default parameters.

[0047] The term "pharmaceutical acceptable excipient" as used herein refers to any suitable substance that provides a pharmaceutical acceptable carrier, additive, or diluent for administering a compound of interest to a subject. Thus, "pharmaceutical acceptable excipient" can include substances that are called pharmaceutical acceptable diluents, pharmaceutical acceptable additives, and pharmaceutical acceptable carriers. As used herein, the term "pharmaceutical acceptable carrier" includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics and additional therapeutic agents) can also be incorporated into the composition.

[0048] The term "recombinant" as used with respect to a cell, nucleic acid, protein, or vector indicates that the cell, nucleic acid, protein, or vector has been altered or produced by human intervention, e.g., modified by or is the result of a laboratory method. Thus, for example, recombinant proteins and nucleic acids include proteins and nucleic acids produced by laboratory methods. Recombinant proteins may include amino acid residues not found in the native (non-recombinant or wild-type) form of the protein or may include modified (e.g., labeled) amino acid residues. The term may include any modification to a peptide, protein, or nucleic acid sequence. Such modifications may include: any chemical modification of a peptide, protein, or nucleic acid sequence, including one or more amino acids, deoxyribonucleotides, or ribonucleotides; the addition, deletion, and / or substitution of one or more amino acids in a peptide or protein; the creation of a fusion protein (e.g., a fusion protein with an antibody fragment); and the addition, deletion, and / or substitution of one or more nucleic acids in a nucleic acid sequence. The term "recombinant" as used with respect to cells is not intended to include naturally occurring cells, but rather to encompass cells that have been engineered / modified to contain or express a polypeptide or nucleic acid that is not present in the cell unless engineered / modified.

[0049] As used herein, a "subject" or "individual" includes animals, such as humans (e.g., human individuals) and non-human animals. In some embodiments, a "subject" or "individual" is a patient under the care of a physician. Thus, a subject may be a human patient or individual who has, is at risk of, or is suspected of having a health condition of interest (e.g., cancer) and / or one or more symptoms of a health condition. A subject may also be an individual who has been diagnosed at or after diagnosis as being at risk for a health condition of interest. The term "non-human animal" includes all vertebrates, such as mammals, such as rodents, e.g., mice, non-human primates, and other mammals, such as sheep, dogs, cows, chickens, and non-mammals, such as amphibians, reptiles, and the like.

[0050] It is understood that aspects and embodiments of the disclosure described herein include aspects and embodiments that "comprise," "consist," or "consist essentially of." As used herein, "comprising" is synonymous with "comprise," "contain," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting" excludes elements, steps, or components 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. When the term "comprising" is recited herein, specifically in a description of a component of a composition or a description of a step of a method, it is understood to encompass compositions and methods that consist essentially of and consist of the recited components or steps.

[0051] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs of any species to which the compositions and methods disclosed herein are applicable. Thus, these terms include, but are not limited to, human and mouse genes and gene products. Where a gene or gene product of a particular species is disclosed, it is understood that this disclosure is intended to be illustrative only and should not be construed as limiting unless the context in which it appears clearly indicates so. Thus, for example, the genes or gene products disclosed herein relate in some embodiments to mammalian nucleic acid and amino acid sequences, but are intended to encompass homologous and / or orthologous genes and gene products from other animals, including, but not limited to, other mammals, fish, amphibians, reptiles, and birds. In some embodiments, the genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides, and proteins are human. The term "gene" is also intended to include variants thereof.

[0052] It is understood that certain features of the present disclosure that are described for clarity in the context of separate embodiments can also be provided in combination in a single embodiment. Conversely, various features of the disclosure that are described for brevity in the context of a single embodiment can also be provided separately or in any suitable subcombination. All combinations of the embodiments related to the disclosure are specifically embraced by the present disclosure and are disclosed herein as if each combination were individually and expressly disclosed herein. Moreover, all subcombinations of the various embodiments and elements thereof are specifically embraced by the present disclosure and are disclosed herein as if each such subcombination were individually and expressly disclosed herein. Alphaviruses

[0053] Alphaviruses are small enveloped RNA viruses with a single-stranded, positive-sense RNA genome. The Alphavirus genus includes, among others, Sindbis virus (SINV), Semliki Forest virus (SFV), Ross River virus (RRV), Venezuelan equine encephalitis virus (VEEV), and Eastern equine encephalitis virus (EEEV), all of which are closely related and can infect a variety of vertebrates, such as mammals, rodents, fish, birds, and larger mammals such as humans and horses, as well as invertebrates, such as insects. In particular, Sindbis virus and Semliki Forest virus have been extensively studied, and the life cycle, replication mode, etc. of these viruses have been well characterized.

[0054] The genome of alphaviruses is approximately 12 Kb long and consists of two open reading frames (ORFs): a 7 Kb frame that encodes the nonstructural proteins (nsPs) and a 4 Kb frame that encodes the structural polyprotein. The nonstructural polyprotein (nsP) is cleaved into four distinct proteins (nsP1, nsP2, nsP3, and nsP4) that are required for the transcription and translation of viral mRNA in the cytoplasm of the host cell.

[0055] The nsP1 protein is an mRNA capping enzyme with both guanine-7-methyltransferase (MTase) and guanyltransferase (GTase) activities that direct the methylation and capping of newly synthesized viral genomic and subgenomic RNAs. The MTase motif in the N-terminal domain of nsP1 catalyzes the transfer of a methyl group from S-adenosylmethionine (AdoMet) to the N7 position of a GTP molecule (m7Gppp). The GTase then binds to m7Gppp and forms a covalent bond with the catalytic histidine (m7Gp-GTase), releasing PPi. The GTase then transfers the m7Gp molecule to the 5'-diphosphate RNA to create m7GpppNp-RNA. The resulting cap structure is essential for viral mRNA translation and protects the mRNA from degradation by cellular 5' exonucleases. Following the N-terminal domain are features that allow the nsP1 protein to be attached to the cell membrane. The presence of the α-helical amphipathic loop and the palmitoylation site allows the nsP1 protein and nsP1-containing replication complexes to be anchored on the plasma membrane, presumably via interactions of nsP1 with anionic membrane phospholipids.

[0056] The nsP2 protein has multiple enzymatic activities and functional roles. Its N-terminal region contains a helicase domain with seven signature motifs of superfamily 1 (SF1) helicases. It functions as an RNA triphosphatase that executes the first step of the viral RNA capping reaction. It also functions as a nucleotide triphosphatase (NTPase) and promotes the RNA helicase activity. The C-terminal region of nsP2 contains a papain-like cysteine ​​protease, which is responsible for processing the viral nonstructural polyprotein. This protease recognizes conserved motifs within the polyprotein. This proteolytic function is highly regulated and is modulated by other domains of nsP2. Alphavirus nsP2 proteins have also been described as virulence factors involved in the inhibition of interferon (IFN)-mediated antiviral responses that contribute to the shutoff of transcription and translation in infected host cells and the control of the translation machinery by viral factors.

[0057] The exact role of alphavirus nsP3 protein in the replication complex is unclear. The nsP3 protein has three recognized domains: an N-terminal macrodomain with phosphatase activity and nucleic acid binding capacity, an alphavirus unique domain (AUD), and a C-terminal hypervariable domain. It has been demonstrated that deletion of this domain in SFV nsP3 reduces the pathogenicity of the virus, suggesting the importance of this domain in regulating viral RNA transcription.

[0058] The nsP4 polymerase is the most highly conserved protein in alphaviruses, differing most by its greater than 50% amino acid sequence identity when compared to other alphavirus nsP4s. nsP4 contains a core RNA-dependent RNA polymerase (RdRp) domain at the C-terminus and has been found to be solely responsible for the RNA synthesis properties of the viral replication complex. RdRp is involved in replicating genomic RNA via negative-strand RNA and transcribing 26S subgenomic RNA. Its N-terminal domain is specific to alphaviruses and may be structurally partially disordered.

[0059] The 5' two-thirds of the alphavirus genome encodes numerous nonstructural proteins (nsPs) required for viral RNA transcription and replication. These proteins are directly translated from RNA and, together with cellular proteins, generate the RNA-dependent RNA polymerase essential for viral genome replication and sgRNA transcription. Four nsPs (nsP1-4) are generated as a single polyprotein and constitute the viral replication machinery. Polyprotein processing is performed in a highly regulated manner, with cleavage at the P2 / 3 junction influencing the use of the RNA template during genome replication. This site is located at the bottom of a narrow groove and is not easily accessible. Once cleaved, nsP3 forms a ring structure to surround nsP2. These two proteins have an extensive interface. Mutations in nsP2 that produce noncytopathic viruses or temperature-sensitive phenotypes are concentrated in the P2 / P3 interface region. P3 mutations opposite the location of nsP2 noncytopathic mutations prevent efficient cleavage of P2 / 3. This in turn can affect RNA infectivity and alter the levels of viral RNA production.

[0060] The 3' third of the genome contains the sgRNA, which serves as a template for the translation of all structural proteins required for the formation of viral particles (core nucleocapsid protein C and envelope proteins P62 and E1, which assemble as a heterodimer). Surface glycoproteins anchored in the viral membrane are responsible for receptor recognition and entry into target cells by membrane fusion. The sgRNA is transcribed from the p26S subgenomic promoter located at the 3' end of the RNA sequence encoding the nsp4 protein. The proteolytic maturation of P62 to E2 and E3 results in changes to the viral surface. The glycoprotein "spikes" of E1, E2, and sometimes E3 form E1 / E2 dimers or E1 / E2 / E3 trimers, where E2 extends from the center to the apexes, E1 fills the space between the apexes, and E3, if present, resides at the distal end of the spike. When the virus is exposed to the acidity of the endosome, E1 dissociates from E2 to form the E1 homotrimer, which is required for the fusion step that drives the cellular and viral membranes together. The alphavirus glycoprotein E1 is a class II viral fusion protein, which differs in structure from the class I fusion proteins found in influenza viruses and HIV. The E2 glycoprotein functions to interact with the nucleocapsid through its cytoplasmic domain, and its ectodomain is responsible for binding to cellular receptors. Most alphaviruses have lost the peripheral protein E3, but in Semlikivirus, E3 remains associated with the viral surface.

[0061] It has been reported that alphavirus replication occurs on membranous surfaces within host cells. In the first step of the infection cycle, the 5' end of the genomic RNA is translated into polyproteins (nsP1-4) with RNA polymerase activity, which generate a minus strand complementary to the genomic RNA. The sequence at the 3' end of the genomic RNA plays a key role in initiating minus strand synthesis, where a minimum of adenylate residues has been confirmed to be essential for replication to occur. In particular, it has been previously reported that for alphavirus genome replication, there must be at least 11 residues in the poly(A) tail following the 3'UTR for efficient initiation of minus strand synthesis and thus replication to occur. It has also been previously reported that extending the poly(A) tail to 25 residues enhances replication, but no further enhancement of replication was observed when the poly(A) tail was further extended to 34 residues. Furthermore, our results suggest that replicon RNAs that do not contain only 3' adenylate residues following the 3' UTR are not beneficial for enzymatic poly(A) tailing, since internal non-A residues within poly(A) are most often detrimental to replication. It has been previously reported that RNA templates containing more than 25 adenylate residues in the poly(A) tail do not enhance minus-strand synthesis. In the second step of replication, the minus strand is used as a template, generating two RNAs each: (1) a positive genomic RNA corresponding to the genome of a secondary virus that produces other nsPs by translation and acts as the genome of the virus; and (2) an sgRNA that encodes a structural protein of the virus that generates the infectious particle. The positive genomic RNA / sgRNA ratio is regulated by proteolytic self-cleavage of the polyprotein into nsP1, nsP2, nsP3, and nsP4. In fact, viral gene expression occurs in two stages. In the first stage, the main synthesis of the positive genomic strand and the negative strand takes place. In the second stage, the synthesis of sgRNA is virtually exclusive, resulting in the production of large amounts of structural proteins.

[0062] self-replicating RNA As will be understood by those skilled in the art, the term "self-replicating RNA" refers to an RNA molecule that contains all the genetic information necessary to direct its own self-amplification or self-replication in permissive cells. To direct its own replication, srRNA generally (1) encodes a polymerase, replicase, or other protein that may interact with viral or host cell derived proteins, nucleic acids, or ribonucleoproteins to catalyze the RNA amplification process; (2) contains cis-acting RNA sequences necessary for the replication and transcription of the RNA encoded in the subgenomic replicon. These sequences may bind to self-encoded proteins, or non-self-encoded cell derived proteins, nucleic acids, or ribonucleoproteins, or complexes between any of these components during the replication process. In some embodiments of the present disclosure, an alphavirus srRNA construct generally contains the following elements: 5' viral or defective interfering RNA sequences required in cis for replication, sequences encoding biologically active alphavirus nonstructural proteins (e.g., nsP1, nsP2, nsP3, and nsP4), a subgenomic promoter (sg) for the subgenomic RNA (sgRNA), 3' viral sequences required in cis for replication, and optionally a polyadenylic acid moiety (poly(A)). Optionally, a subgenomic promoter (sg) directing expression of a heterologous sequence can be included in the srRNA construct of the present disclosure.

[0063] Additionally, the term srRNA generally refers to a molecule of positive polarity or "message" sense, and the srRNA may be of a length different from that of known naturally occurring alphaviruses. In some embodiments of the present disclosure, the srRNA does not include at least a portion of the coding sequence for one or more alphavirus structural proteins, and / or the sequence encoding the structural genes can be replaced with heterologous sequences. In these instances, when the srRNA is packaged into recombinant alphavirus particles, it may include one or more sequences, so-called packaging signals, that serve to initiate interactions with alphavirus structural proteins that lead to particle formation.

[0064] The srRNA constructs of the present disclosure generally have a length of at least about 2 kb. For example, the srRNA can have a length of at least about 2 kb, at least about 3 kb, at least about 4 kb, at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 11 kb, at least about 12 kb, or more than 12 kb. In some embodiments, the srRNA is about 4 kb to about 20 kb, about 4 kb to about 18 kb, about 5 kb to about 16 kb, about 6 kb to about 14 kb, about 7 kb to about 12 kb, about 8 kb to about 16 kb, about 9 kb to about 14 kb, about 10 kb to about 18 kb, about 11 kb to about 16 kb, about 5 kb to about 18 kb, about 6 kb to about 20 kb, about 5 kb to about 10 kb, about 5 kb to about 8 kb, about 5 kb to about 7 kb, about 5 kb to about 6 kb, b, about 6 kb to about 12 kb, about 6 kb to about 11 kb, about 6 Kb to about 10 kb, about 6 kb to about 9 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, about 7 kb to about 11 kb, about 7 kb to about 10 kb, about 7 kb to about 9 kb, about 7 kb to about 8 kb, about 8 kb to about 11 kb, about 8 kb to about 10 kb, about 8 kb to about 9 kb, about 9 kb to about 11 kb, about 9 kb to about 10 kb, or about 10 kb to about 11 kb. In 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. IL-12

[0065] IL-12 is a 70 kDa heterodimer consisting of a heavy chain subunit (p40) and a light chain subunit (p35), which are covalently linked by disulfide bonds. p40 is produced in large amounts by phagocytes, whereas p35 is ubiquitously and constitutively expressed at low levels, and co-expression of p40 is thought to be required for secretion of biologically active cytokines (Babik JM, Adams E, Tone Y, Fairchild PJ, Tone M, Waldmann H. Expression of murine IL-12 is regulated by translational control of the p35 subunit. JImmunol 1999; 162: 4069-4078).

[0066] IL-12 signals through the receptor complex IL-12Rβ1 and IL-12Rβ2 expressed on NK and T cells. Dimerization of the IL-12 receptor induces activation of receptor-associated Janus kinase (JAK) molecules, which phosphorylate each other and residues on the intracellular domain of IL-12Rβ2 that serve as docking sites for SH2-containing signal transducer and activator of transcription 4 (STAT4). The receptor-associated STAT4 protein is then phosphorylated and translocates to the nucleus, where it promotes expression of IFNγ and polarization of CD4+ T cells toward a T helper 1 (Th1) phenotype. Given the similarities between immunity to intracellular pathogens and cancer, therapeutic approaches to stimulate Th1 responses have been explored for cancer immunotherapy, either indirectly through vaccine adjuvants and epitope selection, or directly through administration of IL-12. Despite promising preclinical models, the therapeutic efficacy of IL-12 administration is limited due to the toxicity associated with NK cell-mediated production of IFNγ.

[0067] IL-1RA Interleukin-1 (IL-1) comprises a family of cytokines that stimulate lymphocytes and macrophages, activate phagocytes, increase prostaglandin production, contribute to bone joint degeneration, increase proliferation of bone marrow cells, and are involved in many chronic inflammatory conditions. IL-1α and IL-1β are produced by macrophages, monocytes, and dendritic cells and can be part of the inflammatory response to infection.

[0068] The mode of action of IL-1 cytokines is mediated by the interleukin-1 receptor antagonist protein (IL-1RA, also known as "IRAP"), which is encoded by the IL1RN gene. IL-1RA binds to the same cell surface receptor as IL-1α and IL-1β, thus preventing these cytokines from signaling to the cell. IL-1RA is secreted by white blood cells, including monocytes, macrophages, neutrophils, polymorphonuclear cells (PMNs), and other cells, and can regulate a variety of IL-1-associated immune and inflammatory responses, as described in Arend WP, ​​Malyak M, Guthridge CJ, Gabay C (1998) "Interleukin-1 receptor antagonist: role in biology" Annu. Rev. Immunol. 16: 27-55. Production of IL-1RA can be stimulated by several substances, including adhesion immunoglobulin G (IgG), other cytokines, and bacterial or viral components. IL-1RA is a naturally occurring anti-inflammatory protein important in arthritis, colitis, and granulomatous lung disease.

[0069] Compositions of the Disclosure As described in more detail below, one aspect of the disclosure relates to nucleic acid constructs comprising a sequence encoding a modified alphavirus genome or srRNA, wherein at least a portion of the nucleic acid sequence encoding the viral structural proteins of the modified alphavirus genome or srRNA has been replaced with a coding sequence for a polypeptide construct comprising a) a coding sequence for the p35 subunit of interleukin-12 (p35 or IL-12A) or a functional variant thereof, b) a coding sequence for the p40 subunit of interleukin-12 (p40 or IL-12B) or a functional variant thereof, and c) a coding sequence for interleukin-1 receptor antagonist (IL-1RA) or a functional variant thereof. Also provided are recombinant cells and cell cultures engineered to contain the nucleic acid constructs disclosed herein.

[0070] nucleic acid construct 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 alphavirus genome or srRNA, wherein at least a portion of the nucleic acid sequence encoding the viral structural proteins of the modified alphavirus genome or srRNA has been replaced with a coding sequence for a polypeptide construct comprising a) a coding sequence for the p35 subunit of interleukin-12 (p35 or IL-12A) or a functional variant thereof, b) a coding sequence for the p40 subunit of interleukin-12 (p40 or IL-12B) or a functional variant thereof, and c) a coding sequence for interleukin-1 receptor antagonist (IL-1RA) or a functional variant thereof. In some embodiments, the coding sequence for the srRNA construct can be operably linked, e.g., placed under the control of elements necessary for expression (e.g., promoter sequences), which allows for expression of the srRNA construct in a host cell, a subject, or an in vitro cell-free expression system.

[0071] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to both RNA and DNA molecules, including nucleic acid molecules including cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules including nucleic acid analogs. Nucleic acid molecules can be double-stranded or single-stranded (e.g., sense or antisense strands). Nucleic acid molecules can include unconventional or modified nucleotides. As used herein, the terms "polynucleotide sequence" and "nucleic acid sequence" are used interchangeably and refer to the sequence of a polynucleotide molecule. The nomenclature of nucleotide bases as set forth in 37 CFR § 1.822 is used herein.

[0072] Nucleic acid molecules of the present disclosure may be of any length, including, for example, about 1.5 Kb to about 50 Kb, about 5 Kb to about 40 Kb, about 5 Kb to about 30 Kb, about 5 Kb to about 20 Kb, or about 10 Kb to about 50 Kb, e.g., about 15 Kb to 30 Kb, about 20 Kb to about 50 Kb, about 20 Kb to about 40 Kb, about 5 Kb to about 25 Kb, or about 30 Kb to about 50 Kb.

[0073] Non-limiting exemplary embodiments of the disclosed methods may include one or more of the following features: In some embodiments, the alphavirus srRNA vector 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 alphavirus srRNA vector. In some embodiments, the alphavirus srRNA vector lacks some or all of the sequence encoding CP. In some embodiments, the alphavirus srRNA vector lacks some or all of the sequence encoding E1. In some embodiments, the alphavirus srRNA vector lacks some or all of the sequence encoding E2. In some embodiments, the alphavirus srRNA vector lacks some or all of the sequence encoding E3. In some embodiments, the alphavirus srRNA vector lacks some or all of the sequence encoding 6K. In some embodiments, the alphavirus srRNA vector lacks some or all of the sequence encoding a combination of CP, E1, E2, E3, and 6K. In some embodiments of the present disclosure, the coding sequences for the nonstructural proteins nsP1, nsP2, nsP3, and nsP4 of the alphavirus srRNA vector are present, but at least some or all of the sequences encoding one or more structural proteins (e.g., CP, E1, E2, E3, and 6K) of the alphavirus srRNA vector are absent.

[0074] In some embodiments, the alphavirus srRNA vector lacks a substantial portion of the nucleic acid sequence encoding one or more viral structural proteins. One skilled in the art will appreciate that a substantial portion of a nucleic acid sequence encoding a viral structural polypeptide may include sufficient nucleic acid sequence encoding a viral structural polypeptide to allow for putative identification of the polypeptide, either by manual evaluation of the sequence by one skilled in the art or by computer-automated sequence comparison and identification using algorithms such as BLAST (see, e.g., "Basic Local Alignment Search Tool"; Altschul SF et al., J. Mol. Biol. 215:403-410, 1993). Thus, a substantial portion of a nucleotide sequence includes sufficient sequence to allow for specific identification and / or isolation of a nucleic acid fragment comprising the sequence. For example, a substantial portion of a nucleic acid sequence may include at least about 20%, e.g., about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% of the full length nucleic acid sequence.

[0075] In some embodiments, the alphavirus srRNA vector lacks the entire sequence encoding a viral structural protein, eg, the alphavirus srRNA vector does not include nucleic acid sequences encoding viral structural proteins.

[0076] The nucleic acid construct of the present disclosure further comprises a coding sequence for a polypeptide construct that replaces at least a portion of the nucleic acid sequence encoding the viral structural protein of the modified alphavirus genome or srRNA. In principle, the nucleic acid construct disclosed herein can generally comprise any number of coding sequences for a polypeptide construct. In some embodiments, the nucleic acid construct disclosed herein can comprise at least one, at least two, at least three, at least four, at least five, or at least six coding sequences for a polypeptide construct. A coding sequence for a polypeptide construct can be a construct of genetic material that includes a coding sequence and sufficient regulatory information to direct the proper transcription and / or translation of the coding sequence in a cell, in vivo, and / or ex vivo. A coding sequence for a polypeptide construct can be inserted into a vector for targeting to a desired host cell and / or subject. Thus, in some embodiments, the term "coding sequence for a polypeptide construct" can be used interchangeably with the term "expression construct." In some embodiments, the coding sequence of a polypeptide construct may be a nucleic acid construct comprising any or a combination of a gene encoding a protein or functional RNA operably linked to regulatory elements, such as, for example, a promoter and / or a termination signal, and, optionally, other nucleic acid sequences that affect transcription or translation of the gene.

[0077] The nucleic acid constructs described herein include coding sequences for the p35 subunit of interleukin-12 (p35 or IL-12A) or a functional variant thereof, the p40 subunit of interleukin-12 (p40 or IL-12B) or a functional variant thereof, and the interleukin-1 receptor antagonist (IL-1RA) or a functional variant thereof, which encode polypeptides capable of eliciting a pharmacodynamic effect in a subject. Functional variants of IL-12A, IL-12B, and IL-1RA can include coding sequences for polypeptides having the same or essentially the same amino acid sequence as a reference protein (e.g., IL-12A, IL-12B, and IL-1RA), except that they contain at least one modified (e.g., deleted, inserted, or substituted, respectively) amino acid. The amino acid substitutions may be conservative amino acid substitutions, preferably at non-essential amino acid residues in the protein. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are known in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). A variant of a protein may have an amino acid sequence that is at least about 80%, 90%, 95%, or 99%, preferably at least about 90%, more preferably at least about 95% identical to the amino acid sequence of the protein. Preferably, a variant is a functional variant of a protein that retains the same function as the protein. The term "variant" when used in reference to nucleic acid sequences refers to a nucleic acid sequence that differs in one or more nucleotides from another, normally related nucleic acid sequence.The term "variant" can thus refer to a change in one or more nucleotides of the reference nucleic acid, including the insertion of one or more new nucleotides, the deletion of one or more nucleotides, and the substitution of one or more existing nucleotides. Variants can also include point mutations, multiple mutations, single nucleotide polymorphisms (SNPs), deletions, insertions, and translocations. Thus, variants of the coding sequences described herein include nucleic acids that encode polypeptides that can be, for example, full-length, mutated, truncated, inactivated peptides / epitopes of IL-12A, IL-12B, and IL-1RA, or combinations thereof.

[0078] The full length amino acid sequence of the human IL-12p35 subunit is set forth in SEQ ID NO:1 as follows: [ka]

[0079] Alternatively, the full length amino acid sequence of the human IL-12p35 subunit together with its signal sequence is set forth in SEQ ID NO:2 as follows: [ka]

[0080] In some embodiments, the coding sequence for the IL-12p35 subunit in the nucleic acid construct described herein encodes the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the nucleic acid construct of the present disclosure comprises a nucleic acid sequence encoding an IL-12p35 subunit 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 amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the coding sequence for the IL-12p35 subunit encodes smaller portions of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. These smaller portions may include at least 8, 10, 12, 14, 16, 18, 20, 30, or more amino acids of SEQ ID NO:1 or SEQ ID NO:2.

[0081] In some embodiments, the coding sequence of the polypeptide construct in the nucleic acid described herein encodes a functional variant of the IL-12p35 subunit.

[0082] As mentioned above, the nucleic acid constructs described herein also include a coding sequence for the IL-12p40 subunit or a functional variant thereof.

[0083] The full length amino acid sequence of the human IL-12p40 subunit is set forth in SEQ ID NO:3 as follows: [ka]

[0084] Alternatively, the full length amino acid sequence of the human IL-12p40 subunit together with its signal sequence is set forth in SEQ ID NO:4 as follows: [ka]

[0085] In some embodiments, the coding sequence for the human IL-12p40 subunit in the nucleic acid construct described herein encodes the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the nucleic acid construct of the present disclosure comprises a nucleic acid sequence encoding a human IL-12p40 subunit 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 amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the coding sequence for the human IL-12p40 subunit encodes smaller portions of the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. These smaller portions may include at least 8, 10, 12, 14, 16, 18, 20, or more amino acids of SEQ ID NO:3 or SEQ ID NO:4.

[0086] In some embodiments, the coding sequence of the polypeptide construct in the nucleic acid described herein encodes a functional variant of the human IL-12p40 subunit.

[0087] As stated above, the nucleic acid constructs described herein also include a coding sequence for IL-RA or a functional variant thereof.

[0088] In some embodiments, the coding sequence of the polypeptide construct in the nucleic acid described herein encodes one or more regions of IL-1RA transcript 1, 2, 3, or 4, intracellular IL-1RA (icIL-1Ra), or their corresponding polypeptide isoforms. Alternatively, the composition comprises the entire IL-1RA transcript 1, 2, 3, or 4, intracellular IL-1RA (icIL-1Ra), or their corresponding polypeptide isoforms. Compositions comprising any form of human IL-1Ra or a fragment thereof inhibit the function of IL-1R1.

[0089] The amino acid sequence of human IL-1RA encoded by IL1RN transcript 1, and set forth in SEQ ID NO:5, is as follows: [ka]

[0090] In some embodiments, a nucleic acid construct of the present disclosure comprises a nucleic acid sequence encoding an IL-1RA variant 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 amino acid sequence of SEQ ID NO:5.

[0091] The amino acid sequence of human IL-1RA encoded by IL1RN transcript 2 and set forth in SEQ ID NO:6 is as follows (NCBI accession no. NM_173841): [ka]

[0092] In some embodiments, a nucleic acid construct of the disclosure comprises a nucleic acid sequence encoding an IL-1RA variant 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 amino acid sequence of SEQ ID NO:6.

[0093] The amino acid sequence of human IL-1RA encoded by IL1RN transcript 3 and set forth in SEQ ID NO:7 is as follows (NCBI accession no. NM_000577): [ka]

[0094] In some embodiments, a nucleic acid construct of the present disclosure comprises a nucleic acid sequence encoding an IL-1RA variant 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 amino acid sequence of SEQ ID NO:7.

[0095] The amino acid sequence of human IL-1RA encoded by IL1RN transcript 4 and set forth in SEQ ID NO:8 is as follows (NCBI accession no. NM_173843): [ka]

[0096] In some embodiments, a nucleic acid construct of the disclosure comprises a nucleic acid sequence encoding an IL-1RA variant 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 amino acid sequence of SEQ ID NO:8.

[0097] The amino acid sequence of human intracellular IL-1RA, icIL-1Ra, set forth in SEQ ID NO:9, is as follows (NCBI Accession No. M55646): [ka]

[0098] In some embodiments, a nucleic acid construct of the disclosure comprises a nucleic acid sequence encoding an IL-1RA variant 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 amino acid sequence of SEQ ID NO:9.

[0099] In some embodiments, the coding sequence of the IL-12p35 subunit or a functional variant thereof, the IL-12p40 subunit or a functional variant thereof, and the IL-1RA or a functional variant thereof comprises a coding sequence of a single polypeptide (e.g., a single gene). In some embodiments, the coding sequence of the IL-12p35 subunit or a functional variant thereof, the IL-12p40 subunit or a functional variant thereof, and the IL-1RA or a functional variant thereof comprises a coding sequence of multiple polypeptides, for example, a coding sequence of a multigene (e.g., a bigene or trigene). In some embodiments, each of the coding sequences of the IL-12p35 subunit or a functional variant thereof, the IL-12p40 subunit or a functional variant thereof, and the IL-1RA or a functional variant thereof is operably linked to a separate promoter sequence. In some embodiments, the coding sequences of IL-12p35 subunit or functional variant thereof, IL-12p40 subunit or functional variant thereof, and IL-1RA or functional variant thereof are operably linked to each other in a single open reading frame (e.g., in a polycistronic ORF). In some embodiments, the coding sequences of the polycistronic ORF are 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.

[0100] In some embodiments, the coding sequences of the IL-12p35 subunit or functional variant thereof, the IL-12p40 subunit or functional variant thereof, and the IL-1RA or functional variant thereof can be directly or indirectly linked to each other (e.g., via one or more connector sequences). For example, in some embodiments, the coding sequences can be directly linked to each other, e.g., adjacent to each other. In some embodiments, at least two (e.g., 2, 3, 4, or 5) of the coding sequences are operably linked to each other by one or more connector sequences. In some embodiments, the length and amino acid composition of the connector sequences can be optimized to vary the orientation, flexibility, and / or proximity of the polypeptides relative to each other to achieve a desired activity or property of the encoded protein. In some embodiments, a connector sequence of the multiple connector sequences comprises one or more coding sequences of an autoproteolytic peptide sequence. In general, any proteolytic cleavage site known in the art can be incorporated into the nucleic acid molecules of the present disclosure, e.g., a proteolytic cleavage sequence that is cleaved after production by a protease. Further suitable proteolytic cleavage sites also include proteolytic cleavage sequences that can be cleaved after the addition of an external protease. The term "autoproteolytic peptide" as used herein refers to a "self-cleaving" peptide that has autoproteolytic activity and can cleave itself from a larger polypeptide moiety. First identified in the Foot and Mouth Disease Virus (FMDV), a member of the picornavirus group, several autoproteolytic peptides have since been identified, such as the "2A-like" peptide from Equine Rhinitis A Virus (E2A), Porcine Teschovirus-1 (P2A), and Thosea asigna Virus (T2A), and their activity in proteolytic cleavage has been shown in various ex in vitro, in vitro, ex vivo, and in vivo eukaryotic systems. Thus, the concept of an autoproteolytic peptide is available to those skilled in the art, and many naturally occurring autoprotease systems have been identified.Well-studied autoprotease systems include, for example, viral proteases, developmental proteins (e.g., HetR, hedgehog proteins), RumA autoprotease domain, UmuD, etc. Non-limiting examples of autoproteolytic peptides suitable for the compositions and methods of the present disclosure include one or more autoproteolytic cleavage sequences from calcium-dependent serine endoprotease (furin), porcine teschovirus-12A (P2A), foot and mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), tosea asignavirus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), bursaphelenchus disease virus 2A (BmIFV2A), or combinations thereof.

[0101] In some embodiments, the coding sequences of the IL-12p35 subunit or a functional variant thereof, the IL-12p40 subunit or a functional variant thereof, and the IL-1RA or a functional variant thereof are operably linked to each other by one or more coding sequences of internal ribosome entry sites (IRES). An IRES or "internal ribosome entry site" is a sequence located between polycistronic genes that allows the generation of an expression product from a second gene by internal initiation of translation of a bicistronic mRNA. This promotes direct internal ribosome entry to the start codon (e.g., ATG) of the cistron (protein coding region), thus resulting in cap-independent translation of the gene. See, for example, Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. In some embodiments, the IRES can be a viral IRES, a cellular IRES, or an artificial IRES. Examples of IRES commonly used by those skilled in the art include those described in U.S. Patent No. 6,692,736. In some embodiments, the IRES is selected from Kaposi's sarcoma-associated herpesvirus (KSHV) IRES, Hepatitis virus IRES, Pestivirus IRES, Cripavirus IRES, Rhopalosiphum padi virus IRES, fibroblast growth factor IRES, platelet-derived growth factor IRES, vascular endothelial growth factor IRES, insulin-like growth factor IRES, picornavirus IRES, encephalomyocarditis virus (EMCV) IRES, Pim-1 IRES, p53 IRES, Apaf-1 IRES, TDP2 IRES, L-myc IRES, and c-myc IRES. In some embodiments, the IRES is obtained from EMCV.

[0102] One skilled in the art will understand that different configurations of the coding sequences of the IL-12p35 subunit or functional variants thereof, the IL-12p40 subunit or functional variants thereof, and the IL-1RA or functional variants thereof, the sequence encoding the autoproteolytic peptide, or the IRES can be used, so long as the expression of the IL-12p35 subunit or functional variants thereof, the IL-12p40 subunit or functional variants thereof, and the IL-1RA or functional variants thereof is appropriately maintained. These sequences will typically be configured such that the polypeptide encoded by the gene of interest is released from the protease and other sequences after cleavage by the autoprotease.

[0103] The term "operably linked" as used herein means a functional linkage between two or more sequences. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional linkage that allows expression of the polynucleotide of interest. In this sense, the term "operably linked" refers to the arrangement of a regulatory region with a coding sequence to be transcribed such that the regulatory region is effective to regulate the transcription or translation of the coding sequence of interest. In some embodiments disclosed herein, the term "operably linked" refers to a configuration in which the regulatory sequence is appropriately positioned relative to the sequence encoding a polypeptide or functional RNA such that the control sequence directs or regulates the expression or cellular localization of the mRNA encoding the polypeptide, the polypeptide, and / or the functional RNA. Thus, a promoter is operably linked to a nucleic acid sequence if it is capable of mediating the transcription of the nucleic acid sequence. Operably linked elements may be contiguous or non-contiguous.

[0104] Basic techniques for operably linking two or more DNA sequences are well known to those of skill in the art, and such methods are described in many standard molecular biology texts (see, e.g., Maniatis et al., "Molecular Cloning: A Laboratory Manual" 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Gibson et al., Nature Methods 6:343-45, 2009).

[0105] As shown in Figures 1A-1B and 2A-2B, IL-12 and IL-1RA protein expression and biological activity were measured from different two-gene constructs with different gene orders. Exemplary configurations of the nucleic acid constructs described herein are shown in Table 1 below. [Table 1]

[0106] In some embodiments, the order of the nucleic acid construct is selected from the group consisting of pRB-299 and pRB-306.

[0107] In some embodiments, the polypeptide construct comprises, from N-terminal to C-terminal direction, a) an IL-12A polypeptide, an IL-12B polypeptide, and an IL-1RA polypeptide, or b) an IL-1RA polypeptide, an IL-12B polypeptide, and an IL-12A polypeptide, wherein the IL-12A, IL-12B, and IL-1RA polypeptides are operably linked to each other by one or more autoproteolytic cleavage sequences or internal ribosome entry sites.

[0108] In some embodiments, the nucleic acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:10.

[0109] In some embodiments, the coding sequences of IL-12p35, IL-12p40, and IL-1RA are redesigned and / or optimized for desired properties, such as improved stability, potency, and expression (e.g., translation efficiency), so that the biotherapeutic production, delivery, and administration can be maximized. For example, in some embodiments, the coding sequences are optimized for expression at a level higher than that of the reference coding sequence. With respect to sequence optimization of nucleotide sequences, the degeneracy of the genetic code provides the possibility to replace at least one base of the protein coding sequence of a gene with a different base without changing the amino acid sequence of the polypeptide produced from the gene. Thus, the nucleic acid constructs of the present disclosure may have any base sequence that is altered from any polynucleotide sequence disclosed herein by substitution according to the degeneracy of the genetic code. References describing codon usage are readily available to the public. In some embodiments, polynucleotide sequence variants can be generated for a variety of reasons, such as optimizing expression for a particular host (e.g., changing the codon usage in alphavirus mRNA to that preferred by other organisms, such as humans, non-human primates, hamsters, mice, or monkeys). Thus, in some embodiments, the coding sequence is optimized for expression in the target host cell by using codons optimized for expression. Techniques for constructing synthetic nucleic acid sequences encoding genes using optimal preferred codons for host cell expression can be determined by techniques well known in the art, computational methods that analyze the commonality and relative abundance of codon usage encoding native proteins in the host cell genome. Codon usage databases can be used to generate codon-optimized sequences in a mammalian cell environment. Additionally, various software tools are available for converting sequences from one organism to the optimal codon usage of another host organism, such as the JCat Codon Optimization Tool (www.jcat.de), the Integrated DNA Technologies (IDT) codon optimization tool, or the Optimizer online codon optimization tool.Such synthetic sequences can be constructed by techniques well known in the art for the construction of synthetic nucleic acid molecules, and are available from a variety of commercial sources.

[0110] In some embodiments, the coding sequence is optimized for enhancing RNA stability and / or expression. RNA stability is generally related to the "half-life" of the RNA. "Half-life" refers to the period required to remove half of the activity, amount, or number of a molecule. In the context of the present disclosure, the half-life of an RNA refers to the stability of said RNA. The half-life of an RNA can affect the "expression period" of the RNA. Additional information on the principles, strategies, and methods for use in enhancing RNA stability can be found, for example, in Leppek K. et al., Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics. bioRxiv. (preprint). Mar 30, 2021. doi: 10.1101 / 2021.03.29.437587.

[0111] Recombinant cells The nucleic acid constructs of the present disclosure can be introduced into a host cell to produce a recombinant cell comprising the nucleic acid molecule. Thus, prokaryotic or eukaryotic cells comprising a nucleic acid construct encoding a modified alphavirus genome as described herein are also a feature of the present disclosure. In a related aspect, some embodiments disclosed herein relate to a method of transforming a cell, comprising introducing a nucleic acid construct provided herein into a host cell, such as an animal cell, and then selecting or screening for transformed cells. Introduction of the nucleic acid constructs of the present invention into a cell can be accomplished by methods known to those of skill in the art, such as viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0112] In one aspect, some embodiments of the present disclosure relate to recombinant cells, such as recombinant animal cells, comprising the nucleic acid constructs described herein. The nucleic acid constructs can be stably integrated into the host genome, or can be episomally replicated, or can exist within the recombinant host cell as a minicircle expression vector for stable or transient expression. Thus, in some embodiments of the present disclosure, the nucleic acid constructs are maintained and replicated within the recombinant host cell as episomal units. In some embodiments, the nucleic acid constructs are stably integrated into the genome of the recombinant cell. Stable integration can be accomplished using classical random genome recombination techniques, or using more precise genome editing techniques such as guide RNA-guided CRISPR / Cas9 or TALEN genome editing. In some embodiments, the nucleic acid constructs can exist within the recombinant host cell as a minicircle expression vector for stable or transient expression.

[0113] In some embodiments, the recombinant cell is a prokaryotic cell, such as a bacterium, E. coli, or a eukaryotic cell, such as an insect cell (e.g., a mosquito cell or an Sf21 cell) or a mammalian cell (e.g., a COS cell, an NIH3T3 cell, or a HeLa cell). In some embodiments, the cell is in vivo. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vitro. 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 the group consisting of SV40 transformed monkey kidney CV1 cells (COS-7), human embryonic kidney cells (e.g., HEK293 or HEK293 cells), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells), monkey kidney cells (CV1), human cervical carcinoma cells (HeLa), canine kidney cells (MDCK), buffalo rat hepatocytes (BRL3A), human lung cells (W138), human hepatocytes (HepG2), mouse mammary tumor (MMT060562), TRI cells, FS4 cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (Vero cells), human A549 cells, human cervical cells, human CHME5 cells, human PER.C6 cells, NS0 mouse myeloma cells, human epidermoid laryngeal cells, human fibroblast cells, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocytes, human macrophage cells, human RAW264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells.

[0114] In some embodiments, the recombinant cell is an insect cell, for example, a cell of an insect cell line. In some embodiments, the recombinant cell is an Sf21 cell. Additional suitable insect cell lines include, but are not limited to, cell lines established from insects of the orders Diptera, Lepidoptera, and Hemiptera, and can be derived from different tissue sources. In some embodiments, the recombinant cell is a cell of a lepidopteran insect cell line. Over the past few decades, the availability of lepidopteran insect cell lines has increased by about 50 lines per decade. Further information on available lepidopteran insect cell lines can be found, for example, in Lynn DE, Available lepidopteran insect cell lines. Methods Mol Biol. 2007;388:117-38, which is incorporated herein by reference. In some embodiments, the recombinant cell is a mosquito cell, such as a cell of a mosquito species in the genera Anopheles (An.), Culex (Cx.), and Aedes: tegomyia (Ae.). Exemplary mosquito cell lines suitable for the compositions and methods described herein include cell lines from the following mosquito species: Aedes aegypti, Aedes albopictus, Aedes pseudoscutellaris, Aedes triseriatus, Aedes vexans, Anopheles gambiae, Anopheles stephensi, Anopheles albimanus, Culex quinquefasciatus, Culex theileri, Culex tritaeniorhynchus, Culex bitaeniorhynchus, and Toxorhynchites amboinensis.Suitable mosquito cell lines include, but are not limited to, CCL-125, Aag-2, RML-12, C6 / 26, C6 / 36, C7-10, AP-61, 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.

[0115] In another aspect, a cell culture is provided that comprises at least one recombinant cell disclosed herein and a culture medium. In general, the culture medium can be any suitable culture medium for culturing the cells described herein. Transformation techniques for a wide variety of the above host cells and species are known in the art and described in the technical and scientific literature. Thus, a cell culture that comprises at least one recombinant cell disclosed herein is also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.

[0116] Recombinant polypeptides produced by the methods disclosed herein are also within the scope of the disclosure.

[0117] Non-limiting exemplary embodiments of the disclosed methods for producing a recombinant polypeptide may include one or more of the following features: In some embodiments, the methods for producing a recombinant polypeptide of the present disclosure further include isolating and / or purifying the produced polypeptide. In some embodiments, the methods for producing a polypeptide of the present disclosure further include modifying the structure of the produced polypeptide to extend its half-life.

[0118] Pharmaceutical Compositions The nucleic acid constructs, recombinant cells, recombinant polypeptides of the present disclosure can be incorporated into compositions, including pharmaceutical compositions. Such compositions generally include one or more of the nucleic acid constructs, recombinant cells, recombinant polypeptides described and provided herein, and a pharma- ceutically acceptable excipient, such as a carrier. In some embodiments, the compositions of the present disclosure are formulated for the prevention, treatment, or management of a health condition, such as cancer, an immune disorder, or a chronic infectious disease. For example, the compositions of the present disclosure can be formulated as a prophylactic composition, a therapeutic composition, or a pharmaceutical composition with a pharma-ceutically acceptable excipient, or a mixture thereof. In some embodiments, the compositions of the present disclosure are formulated for use as a biotherapeutic. In some embodiments, the compositions of the present application are formulated for use as an adjuvant.

[0119] Accordingly, in one aspect, provided herein is a pharmaceutical composition comprising a pharma- ceutical acceptable excipient and a) a nucleic acid construct of the present disclosure, b) a recombinant cell of the present disclosure, and / or c) a recombinant polypeptide of the present disclosure.

[0120] Non-limiting exemplary embodiments of the pharmaceutical composition of the present disclosure may include one or more of the following features: The nucleic acid construct of the present disclosure may be used in a naked form or formulated with a delivery vehicle. An exemplary route is to use in a free form, for example, inserted into a nucleic acid (e.g., a vector). For example, as described in more detail below, the nucleic acid construct described herein may be used as a vaccine.

[0121] For use in the pharmaceutical compositions of the present disclosure, the nucleic acids or recombinant cells described herein can be formulated in or with a delivery vehicle. Examples of delivery vehicles suitable for the compositions and methods of the present disclosure include, but are not limited to, liposomes (e.g., neutral or anionic liposomes), microspheres, immune stimulating complexes (ISCOMs), lipid-based nanoparticles (LNPs), polymeric nanoparticles, viral replicon particles (VRPs), or those conjugated with bioactive ligands that can facilitate delivery and / or enhance immune responses. These compounds are readily available to those skilled in the art. See, for example, Liposomes: A Practical Approach, RCP New Ed, IRL press (1990). Other adjuvants besides liposomes, such as those used, are known in the art. Adjuvants can sequester antigens (e.g., srRNA constructs) in localized deposits, thereby preventing the antigen from dispersing rapidly, or can include substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. A person skilled in the art can make an appropriate selection, for example, from those described below.

[0122] Thus, in some embodiments, the compositions of the present disclosure may comprise one or more of the following: a physiological buffer, a liposome, a lipid-based nanoparticle (LNP), a polymeric nanoparticle, a viral replicon particle (VRP), a microsphere, an immune stimulating complex (ISCOM), a conjugate of a biologically active ligand, or any combination thereof.

[0123] In some embodiments, the nucleic acid construct of the present disclosure can be delivered to cells or subjects by lipid-based nanoparticles (LNPs).LNPs are generally less immunogenic than viral particles.Many people have pre-existing immunity to viral particles, but no pre-existing immunity to LNPs.In addition, adaptive immune responses to LNPs are unlikely to occur, so LNPs can be administered repeatedly.

[0124] Lipids suitable for the compositions and methods described herein can be cationic lipids, ionizable cationic lipids, anionic lipids, or neutral lipids.

[0125] In some embodiments, the LNPs of the present disclosure may include one or more ionizable lipids. The term "ionizable lipid" as used herein refers to a lipid that is cationic or ionizable (protonated) when the pH is lower than the pKa of the ionizable group of the lipid, but is more neutral at higher pH values. At pH values ​​lower than the pKa, the lipid can bind to negatively charged nucleic acids (e.g., oligonucleotides). The term "ionizable lipid" as used herein includes lipids that become positively charged as the pH decreases from physiological pH, and any of a number of lipid species that carry a net positive charge at a selective pH, such as physiological pH. Permanently cationic lipids, such as DOTMA, have proven too toxic for clinical use. The ionizable lipid may be present in the lipid formulation according to other embodiments in a proportion of preferably about 30 to about 70 mol%, in some embodiments about 30 mol%, in other embodiments about 40 mol%, in other embodiments about 45 mol%, in other embodiments about 47.5 mol%, in yet other embodiments about 50 mol%, and in yet other embodiments about 60 mol% ("mol %" refers to the percentage of the total moles of a particular component). The term "about" in this paragraph refers to a range of plus or minus 5 mol%. DODMA, i.e. 1,2-dioleyloxy-3-dimethylaminopropane, is an ionizable lipid, as is DLin-MC3-DMA or 0-(Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N-dimethylamino) ("MC3").

[0126] Exemplary ionizable lipids suitable for the compositions and methods of the present disclosure include those described in PCT Publication Nos. WO2020252589A1 and WO2021000041A1, U.S. Patent Nos. 8,450,298 and 10,844,028, and Love KT et al., Proc Natl Acad Sci USA, Feb. 2, 2010 107 (5) 1864-1869, all of which are incorporated herein by reference in their entirety. Thus, in some embodiments, the LNPs of the present disclosure comprise one or more lipid compounds, such as C16-96, C14-110, and C12-200, as described in Love KT et al., 2010 (supra). In some embodiments, the LNP comprises an ionizable cationic lipid selected from the group consisting of ALC-0315, C12-200, LN16, MC3, MD1, SM-102, and any combination thereof. In some embodiments, the LNP of the present disclosure comprises C12-200. The structure of C12-200 lipids is known in the art and is described, for example, in U.S. Patent Nos. 8,450,298 and 10,844,028, which are incorporated herein by reference in their entireties. In some embodiments, C12-200 is combined with cholesterol, C14-PEG2000, and DOPE. In some embodiments, C12-200 is combined with DSPC and DMG-PEG2000.

[0127] In some embodiments, the LNPs of the present disclosure comprise one or more cationic lipids. Suitable cationic lipids include, but are not limited to, 98N12-5, C12-200, C14-PEG2000, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. In some embodiments, the LNPs of the present disclosure comprise one or more neutral lipids. Non-limiting neutral lipids suitable for the compositions and methods of the present disclosure include DPSC, DPPC, POPC, DOPE, and SM. In some embodiments, the LNPs of the present disclosure comprise one or more ionized lipid compounds described in PCT Publications WO2020252589A1 and WO2021000041A1, which are incorporated herein by reference in their entirety.

[0128] Many other lipids or lipid combinations known in the art can be used to generate LNPs. Non-limiting examples of lipids suitable for use in generating LNPs include DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Non-limiting examples of cationic lipids include 98N12-5, C12-200, C14-PEG2000, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, 7C1, and any combination thereof. Non-limiting examples of neutral lipids include DPSC, DPPC, POPC, DOPE, and SM. Non-limiting examples of PEG-modified lipids include PEG-DMG, PEG-CerC14, and PEG-CerC20.

[0129] In some embodiments, the LNPs of the 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). In some embodiments, C12-200 is combined with cholesterol, C14-PEG2000, and DOPE. In some embodiments, C12-200 is combined with DSPC and DMG-PEG2000.

[0130] In some embodiments, the mass ratio of lipids to nucleic acids 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 lipids to nucleic acids in the LNP delivery system is about 16:1 to 4:1. In some embodiments, the mass ratio of lipids to nucleic acids in the LNP delivery system is about 20:1. In some embodiments, the mass ratio of lipids to nucleic acids in the LNP delivery system is about 8:1. In some embodiments, the average diameter of the lipid-based nanoparticles is less than about 1000 nm, about 500 nm, about 250 nm, about 200 nm, about 150 nm, about 100 nm, about 75 nm, about 50 nm, or about 25 nm. In some embodiments, the average diameter of the LNPs is in the range of about 70 nm to 100 nm. In some embodiments, the average diameter of the LNPs is in the range of about 88 nm to about 92 nm, 82 nm to about 86 nm, or about 80 nm to about 95 nm.

[0131] In some embodiments, the compositions of the present disclosure are formulated in liposomes. In some embodiments, the compositions of the present disclosure are formulated in lipid-based nanoparticles (LNPs). In some embodiments, the compositions of the present disclosure are formulated in polymeric nanoparticles.

[0132] As mentioned above, neurolipids, also known as "structured lipids" or "helper lipids", can also be incorporated into lipid formulations, and in some embodiments, lipid particles. Lipid formulations and lipid particles can include one or more structured lipids at about 10-40 mole % of the composition. Suitable structured lipids support particle formation during manufacture. Structured lipids refer to any of a number of lipid species that exist in anionic, uncharged, or neutral zwitterionic form at physiological pH. Exemplary structured lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, diacylphosphatidylglycerol, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0133] 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-dielideyl-sn-glycero-3-phosphoethanolamine (trans DOPE).

[0134] In another embodiment, the structured lipid can be any lipid that is negatively charged at physiological pH.These lipids include phosphatidylglycerols, such as dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, and other anionic modification groups attached to neutral lipids.Other suitable structured lipids include glycolipids (such as monosialoganglioside GM1).

[0135] An embodiment of the lipid formulation may include a stabilizer to ensure the integrity of the mixture. Stabilizers are a class of molecules that disrupt or aid in the formation of hydrophobic-hydrophilic interactions between molecules. Suitable stabilizers include, but are not limited to, polysorbate 80 (also known as Tween 80, IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl octadec-9-enoate), Myrj52 (polyoxyethylene(40) stearate), and Brij™ S10 (polyoxyethylene(10) stearyl ether). Polyethylene glycol-linked lipids may also be used. Stabilizers may be used alone or in combination with each other.

[0136] In some embodiments, the stabilizer comprises about 1-3 mol% of the total lipid mixture. In some embodiments, the stabilizer comprises about 0.5-2.5 mol% of the total lipid mixture. In some embodiments, the stabilizer is present at greater than 2.5 mol%. In some embodiments, the stabilizer is present at 5 mol%. In some embodiments, the stabilizer is present at 10 mol%. In some embodiments, the stabilizer is present at about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 mol%, etc. In other embodiments, the stabilizer is present at 2.6-10 mol% of the lipid mixture. In other embodiments, the stabilizer is present at greater than 10 mol% of the lipid mixture.

[0137] For certain applications, the lipid composition may also include steroids, and the lipid particles produced therefrom may include sterols, such as cholesterol or plant sterols.

[0138] 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 to prevent or treat a subject suffering from, suspected of suffering from, or who may be at high risk for developing cancer.

[0139] 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. In some embodiments, the immunogenic composition is formulated as a biotherapeutic agent, e.g., as a vehicle for gene delivery of different molecules with biological activity. Non-limiting examples of biotherapeutic agents include cytokines, chemokines, 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.

[0140] In some embodiments, the immunogenic composition is substantially non-immunogenic or minimally immunogenic (e.g., a composition that minimally stimulates an immune response in a subject). In some embodiments, the non-immunogenic or minimally immunogenic composition is formulated as a biotherapeutic. In some embodiments, the pharmaceutical composition is formulated for one or more of intranasal, transdermal, intraperitoneal, intramuscular, intranodal, intratumoral, intraarticular, intravenous, subcutaneous, intravaginal, and oral administration.

[0141] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, sterile water, Cremophor EK™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS), Tris (tromethamine), and HEPES. In these cases, the composition should be sterile and fluid to the extent that easy syringability exists. It is stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants, for example, sodium dodecyl sulfate. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is common to include isotonic agents, such as sugars, polyalcohols (e.g., mannitol, sorbitol), sucrose, trehalose, and / or sodium chloride in the composition. In some embodiments, the composition includes tris and sucrose. The absorption of the injectable composition can be prolonged by including an agent that delays absorption, such as aluminum monostearate and gelatin, in the composition.

[0142] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent containing one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0143] In some embodiments, the composition is formulated for one or more of intranasal, transdermal, intramuscular, intratumoral, intranodal, intravenous, intraperitoneal, oral, intravaginal, or intracranial administration.

[0144] Method of Disclosure Administration of any one of the therapeutic compositions described herein, such as a nucleic acid construct, a recombinant cell, a recombinant polypeptide, and / or a pharmaceutical composition, can be used to induce at least one pharmacodynamic effect in a subject. In some embodiments, the nucleic acid construct, the recombinant cell, the recombinant polypeptide, and / or the pharmaceutical composition described herein can result in an immune response and induced production of one or more mediators, such as interferon gamma (IFNγ). In some embodiments, the administered composition described herein enhances tumor immunity in the tumor microenvironment. In some embodiments, the subject may have, be suspected of having, or be at high risk of developing one or more associated health conditions or diseases. In some embodiments, the subject has cancer, an immune disease, or a chronic infection. In some embodiments, the subject is a patient receiving treatment from a physician.

[0145] Examples of immune disorders suitable for the methods of the present disclosure include, but are not limited to, rheumatoid arthritis, osteoarthritis, Still's disease, familial Mediterranean fever, systemic sclerosis, multiple sclerosis, ankylosing spondylitis, Hashimoto's thyroiditis, systemic lupus erythematosus, Sjogren's syndrome, diabetic retinopathy, diabetic vasculopathy, diabetic neuralgia, insulitis, psoriasis, alopecia major, warm-cold autoimmune hemolytic anemia (AIHA), pernicious anemia, acute inflammatory disease, autoimmune adrenalitis, chronic inflammatory demyelinating polyneuropathy (CIDP), Lambert-Eaton syndrome, lichen sclerosis, Lyme disease, Graves' disease, Behcet's disease, Meniere's disease, reactive arthritis (Reiter's syndrome), Churg-Strauss syndrome, Cogan's syndrome, CREST syndrome, pemphigus vulgaris and pemphigus foliaceus, bullous pemphigoid, Lyme's syndrome, Polymyalgia umatica, polymyositis, primary biliary cirrhosis, pancreatitis, peritonitis, psoriatic arthritis, rheumatic fever, sarcoidosis, Sjögrensen's syndrome, scleroderma, celiac disease, stiff man syndrome, Takayasu's arteritis, transient gluten intolerance, autoimmune uveitis, vitiligo, polychondritis, dermatitis herpetiformis (DH) or Duhring's disease, fibromyalgia, Goodpasture's syndrome, Guillain-Barré syndrome, Barre syndrome, Hashimoto's thyroiditis, autoimmune hepatitis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, myasthenia gravis, immune complex disease, glomerulonephritis, polyarteritis nodosa, antiphospholipid syndrome, autoimmune polyglandular syndrome, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), urticaria, autoimmune infertility, juvenile rheumatoid arthritis, sarcoidosis, and autoimmune cardiomyopathy.

[0146] Non-limiting examples of infections suitable for the methods of the present disclosure include infections with viruses such as, for example, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus (CMV), respiratory syncytial virus (RSV), human papillomavirus (HPV), Epstein-Barr virus (EBV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome (MERS), influenza virus, Ebola virus, etc. Additional infections suitable for the methods of the present disclosure include infections with intracellular parasites such as, for example, Leishmania, Rickettsia, Chlamydia, Coxiella, Plasmodium, Brucella, Mycobacteria, Listeria, Toxoplasma, and Trypanosoma.

[0147] In some embodiments, the nucleic acid constructs, recombinant cells, recombinant RNA molecules, recombinant polypeptides, and / or pharmaceutical compositions may be useful for the treatment and / or prevention of the following diseases: immune diseases, autoimmune diseases, or inflammatory diseases, such as glomerulonephritis, inflammatory bowel disease, nephritis, peritonitis, psoriatic arthritis, osteoarthritis, Still's disease, familial Mediterranean fever, systemic sclerosis and sclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, acute lung injury, meningitis, encephalitis, uveitis, multiple myeloma, glomerulonephritis, nephritis, asthma, atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud's syndrome, Sjogren's syndrome, juvenile onset diabetes, Reiter's disease, Behce's disease, and / or other diseases. thyroiditis, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, immune-mediated thrombocytopenia, hemolytic anemia, myasthenia gravis, lupus nephritis, lupus erythematosus, rheumatoid arthritis (RA), ankylosing spondylitis, pemphigus, Graves' disease, Hashimoto's thyroiditis, small vessel vasculitis, Omens' syndrome, chronic renal failure, autoimmune thyroid disease, acute infectious mononucleosis, HIV, herpes virus-related diseases, human viral infections, coronaviruses, other enteroviruses, herpes viruses, influenza viruses, parainfluenza viruses, respiratory syncytial virus or adenovirus infections, bacterial pneumonia, wounds, sepsis, stroke / cerebral edema, ischemia-reperfusion injury, and hepatitis C.

[0148] Non-limiting examples of inflammatory diseases suitable for the methods of the present disclosure include inflammatory diseases such as asthma, inflammatory bowel disease (IBD), chronic colitis, splenomegaly, and rheumatoid arthritis. In one aspect, a method of inducing a pharmacodynamic effect in a subject is provided herein, 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, and (b) a pharmaceutical composition as described herein. In some embodiments, the pharmacodynamic effect comprises eliciting an immune response in the subject.

[0149] Non-limiting exemplary embodiments of the method of inducing a pharmacodynamic effect may include one or more of the following features. In some embodiments, the administered composition induces production of one or more pro-inflammatory molecules in the subject. In some embodiments, the one or more pro-inflammatory molecules include interferon gamma (IFNγ), cytokines, TNF-α, GM-CSF, and MIP1α, granzyme B, granzyme A, perforin, or any combination thereof. In some embodiments, the administered composition inhibits production of one or both of the IL-1α or IL-1β signaling pathways. In some embodiments, the administered composition induces changes in IL-6 or VEGF expression levels, angiogenesis, tumor metastasis, immunosuppression, or tissue remodeling. In some embodiments, the subject has been previously treated with one or more therapies and has developed at least partial resistance to said one or more therapies. In some embodiments, at least one of said one or more therapies includes a small molecule. In some embodiments, the subject is suffering from cancer, an immune disorder, or a chronic infection.

[0150] As mentioned above, administration of any of the therapeutic compositions described herein, such as nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions, can be used to induce at least one pharmacodynamic effect in a subject. In some embodiments, the analysis of the ability of the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions described herein to impart at least one pharmacodynamic effect is performed in vivo or ex vivo. Examples of pharmacodynamic effects that can be analyzed include immunogenic effects (e.g., eliciting an in vivo immune response), biomarker responses, therapeutic effects, preventive effects, desired effects, undesirable effects, adverse effects, and effects in disease models. In some embodiments, the evaluation of the pharmacodynamic effect includes evaluation of the induction of an in vivo immune response. In some embodiments, the evaluation of the pharmacodynamic effect includes evaluation of the induction of cytokine pathways that can enhance immune responses and prevent angiogenesis and metastasis.

[0151] In some embodiments, the disclosed compositions are formulated to be compatible with the intended route of administration. For example, the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure may be administered by oral, inhalation, or parenteral routes. Examples of parenteral administration routes include, for example, intramuscular, intratumoral, intraocular, intravenous, intranodal, intradermal, subcutaneous, transdermal (topical), transmucosal, intravaginal, and rectal administration. In some embodiments, the composition is administered intramuscularly. In some embodiments, the composition is administered intratumorally. Solutions or suspensions used for parenteral administration may include the following components: a sterile diluent, such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, phosphates, Tris, sucrose, and tonicity adjusters, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as monobasic and / or dibasic sodium phosphate, hydrochloric acid, or sodium hydroxide (e.g., to a pH of about 7.2 to 7.8, e.g., 7.5). Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0152] The therapeutic compositions, e.g., nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions described herein, can be administered from once or more per day to once or more per week, including once every other day. Treatment of a subject with a therapeutically effective amount of a subject nucleic acid construct, recombinant cell, recombinant polypeptide, and / or pharmaceutical composition of the present disclosure can include a single treatment, or a series of treatments. In some embodiments, the compositions are administered at weekly intervals, e.g., 1 to 2, 2 to 3, or 3 to 4 doses at 1 to 2 week, 2 to 3, or 3 to 4 week intervals. This may be followed by additional doses every month, 2 months, 3 months, or 4 months. In some embodiments, three doses can be administered intramuscularly at 3 to 4 week intervals, followed by intramuscular administration every 3 months. Alternatively, the compositions can be administered at shorter intervals, e.g., every 8 hours for 5 days, followed by a rest period of 2 to 14 days, e.g., 9 days, followed by every 8 hours for another 5 days. With regard to nucleic acid constructs and recombinant polypeptides, a therapeutically effective amount (eg, an effective dosage) of a nucleic acid construct or recombinant polypeptide of the present disclosure will depend on the nucleic acid construct or recombinant polypeptide selected.

[0153] As discussed above, a therapeutically effective amount includes an amount of a therapeutic composition sufficient to promote a particular effect when administered to a subject suffering from, suspected of suffering from, or at risk of suffering from a health condition such as cancer. In some embodiments, an effective amount includes an amount sufficient to prevent or delay the onset of a disease symptom, alter the course of a disease symptom (such as, but not limited to, slow the progression of a disease symptom), or reverse a disease symptom.

[0154] A treatment is considered effective if at least any one or all of the signs or symptoms of the disease are improved or alleviated. Efficacy can also be measured by the lack of worsening of an individual's condition (e.g., progression of the disease is stopped or at least slowed) as assessed by the need for hospitalization or medical intervention. Methods for measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease in a subject or animal (some non-limiting examples include humans or mammals), including (1) suppression of the disease, e.g., stopping or slowing the progression of the symptoms, or (2) alleviation of the disease, e.g., regression of the symptoms, and (3) prevention or reduction in the likelihood of onset of the symptoms.

[0155] In some embodiments, the nucleic acid construct, recombinant cell, recombinant polypeptide, and / or pharmaceutical composition of the present disclosure can be administered to a subject in a composition comprising a pharma- ceutically acceptable carrier in an amount effective to stimulate an immune response. In general, a subject is immunized with an initial series of injections (or administered by one of the other routes described below), and then a booster can be administered to enhance the protection provided by the initial series. The initial series of injections and subsequent boosters are administered at a dose and for a period of time necessary to stimulate an immune response in the subject. In some embodiments of the disclosed methods, the subject is a mammal. In some embodiments, the mammal is a human subject.

[0156] As mentioned above, pharma- ceutically acceptable carriers suitable for injection include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In these cases, the compositions must be sterile and must be fluid to the extent that easy syringability exists. The compositions must further be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.

[0157] Sterile injectable solutions can be prepared by incorporating the nucleic acid constructs, recombinant cells, and / or recombinant polypeptides in the required amount in an appropriate solvent containing one or a combination of components enumerated above, as required, followed by filtered sterilization.

[0158] When the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions are appropriately protected as described above, they can be orally administered, for example, with an inert diluent or an assimilable edible carrier. The nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions and other components can also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into an individual's diet. For oral therapeutic administration, the active compounds can be incorporated into excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0159] Additional Treatments In some embodiments, the compositions according to the present disclosure are administered to a subject individually as a single therapy (monotherapy) or as a first therapy in combination with at least one additional therapy (e.g., a second therapy). In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiation therapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery. In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiation therapy, immunotherapy, hormone therapy, toxin therapy, or surgery. In some embodiments, the first therapy and the second therapy are administered simultaneously. In some embodiments, the first therapy is administered simultaneously with the second therapy. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy is administered before the second therapy. In some embodiments, the first therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and / or after the second therapy. In some embodiments, the first and second therapies are administered alternately. In some embodiments, the first and second therapies are administered together in a single formulation.

[0160] kit Also provided herein are various kits for carrying out the methods described herein, as well as instructions for making and using the same. In particular, some embodiments of the present disclosure provide kits for inducing a pharmacodynamic effect in a subject. Some other embodiments relate to kits for treating cancer in a subject in need thereof. For example, in some embodiments, provided herein are kits that include one or more of the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions provided and described herein, as well as instructions for making and using the same.

[0161] In some embodiments, the kit of the present disclosure further comprises one or more means useful for administering any one of the provided nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions to a subject. For example, in some embodiments, the kit of the present disclosure further comprises one or more syringes (including pre-filled syringes) and / or catheters (including pre-filled catheters) used to administer any one of the provided nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions to a subject. In some embodiments, the kit may have one or more additional therapeutic agents that can be administered simultaneously or sequentially with other kit components for a desired purpose, such as diagnosis, prevention, or treatment of a condition in a subject in need thereof.

[0162] Any of the above kits may further comprise one or more additional reagents, where such additional reagents may be selected from the following: dilution buffers; reconstitution solutions, wash buffers, control reagents, control expression vectors, negative controls, positive controls, reagents suitable for in vitro production of the provided nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure.

[0163] In some embodiments, the components of the kit may be in separate containers. In some other embodiments, the components of the kit may be combined in a single container. Thus, in some embodiments of the present disclosure, the kit includes one or more of the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions provided and described herein in one container (e.g., a sterile glass or plastic vial) and another therapeutic agent in another container (e.g., a sterile glass or plastic vial).

[0164] In another embodiment, the kit includes a combination of compositions as described herein, in which one or more of the nucleic acid constructs, recombinant cells, and / or recombinant polypeptides of the present disclosure are contained in a single common container, optionally in combination with one or more additional therapeutic agents formulated together in a pharmaceutical composition.

[0165] Where the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit may include a device for performing such administration (e.g., an injection device or catheter). For example, the kit may include one or more hypodermic needles or other injection devices described above that contain one or more nucleic acid constructs, recombinant cells, and / or recombinant polypeptides of the present disclosure.

[0166] In some embodiments, the kit may further include instructions for using the components of the kit to carry out the methods disclosed herein. For example, the kit may include a package insert containing information about the pharmaceutical compositions and dosage forms in the kit. In general, such information helps patients and physicians to effectively and safely use the enclosed pharmaceutical compositions and dosage forms. For example, the following information about the combination of the disclosed contents may be included in the package insert: pharmacokinetics, pharmacodynamics, clinical trials, efficacy parameters, indications and usage, contraindications, warnings, precautions, side effects, overdosage, appropriate dosage and administration method, method of supply, appropriate storage conditions, references, manufacturer / distributor information, and intellectual property information.

[0167] The instructions for carrying out the method are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. The instructions can be present as a kit insert, on a label on the kit's container or a component thereof (e.g., in connection with a package or subpackage), or the like. The instructions can be present as an electronic storage data file present on a suitable computer-readable storage medium, such as a CD-ROM, disk, flash drive, or the like. In some cases, the actual instructions are not present in the kit, but a means is provided for retrieving the instructions from a remote source (e.g., via the Internet). An example of this embodiment is a kit that includes a web address where the instructions can be viewed or from which the instructions can be downloaded. As with the instructions, this means for retrieving the instructions can be recorded on a suitable substrate.

[0168] 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.

[0169] No admission is made that any reference cited herein constitutes prior art. The discussion of a reference states what its author asserts, and applicants reserve the right to challenge the accuracy and pertinence of the cited documents. Although a number of sources of information, including scientific journal articles, patent documents, and textbooks, have been referenced herein, this reference is expressly not an admission that any of these documents constitutes part of the common general knowledge in the art.

[0170] The general method descriptions provided herein are for illustrative purposes only: other alternative methods and means will be apparent to those of skill in the art upon review of this disclosure, and are intended to be within the spirit and scope of this application.

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

[0172] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those of skill in the art. Such techniques are described in, e.g., Sambrook, J., & Russell, DW (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russell, DW (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (together 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, Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, EA (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, SL et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley (including supplements through 2014); and Makrides, SC (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences BV, the disclosures of which are incorporated herein by reference.

[0173] Further embodiments are disclosed in more detail in the following examples, which are provided by way of illustration and are not intended to limit the scope of the disclosure or claims in any way.

[0174] Example 1 Alphavirus vector construction This example describes experiments performed to construct a basic alphavirus vector (e.g., a vector that does not contain a heterologous gene) and then use this vector to construct vectors that express one or more genes of interest (e.g., IL-12p35 subunit or a functional variant thereof, IL-12p40 subunit or a functional variant thereof, and IL-1RA or a functional variant thereof).

[0175] EEEV Basic Vector The basic EEEV vector (i.e., without the heterologous gene of interest) was constructed as follows: The basic EEEV vector was synthesized de novo (Twist Bioscience) from four approximately 4 kb segments of the reference sequence (Genbank EF151502) with some modifications. Silent mutations G301A, A3550C, G4516A, G5725A, and G7399A were introduced to remove restriction sites. A unique restriction site (SpeI, 5'-A'CTAG,T-3') was introduced in place of the coding sequence of the original EEEV structural gene (where 5'A corresponds to the position of the ATG start codon of the structural polyprotein and 3'T corresponds to the position of the TAA stop codon of the structural polyprotein). A 5' adaptor sequence (5'-CTGGAGACGTGGAGGAGAACCCTGGACCT-3'; SEQ ID NO:11) was inserted upstream of the SpeI site and a 3' adaptor sequence (5'-GACCGCTACGCCCCAATGACCCGACCAGC-3'; SEQ ID NO:12) was inserted downstream of the SpeI site for subsequent Gibson Assembly® manipulations (Gibson et al., Nat. Methods 6, 343-345, 2009). A bacteriophage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO:13) was included upstream of the EEEV genomic sequence and downstream of a poly(A) sequence followed by a SapI site, which cleaves upstream of the recognition site. Immediately downstream of the SapI site is a T7 terminator sequence (5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO: 14) followed by a unique restriction site (NotI, 5'-GC'GGCC,GC-3'). These portions were combined into a five-piece Gibson Assembly® reaction (linearized pYL backbone and four synthetic fragments) to generate the EEEV base vector.

[0176] CHIKV basic vector The basic CHIKV S27 vector was de novo synthesized in four approximately 4 kb pieces (Twist Bioscience, Thermo Fisher GeneArt) from a reference sequence (Genbank AF369024) with a silent A5366G mutation and a unique restriction enzyme cleavage site (SpeI, 5'-A'CTAG,T-3') in place of the coding sequence of the CHIKV structural gene (wherein 5'A corresponds to the position of the ATG start codon of the structural polyprotein and 3'T corresponds to the position of the TAA stop codon of the structural polyprotein). A 5' adapter sequence (5'-CTGGAGACGTGGAGGAGAACCCTGGACCT-3'; SEQ ID NO: 11) was inserted upstream of the SpeI site, and a 3' adapter sequence (5'-GACCGCTACGCCCCAATGACCCGACCAGC-3'; SEQ ID NO: 12) was inserted downstream of the SpeI site for the subsequent Gibson Assembly® manipulation. A bacteriophage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO: 13) is included upstream of the CHIKV genomic sequence and downstream of a poly(A) sequence followed by a SapI site, which cleaves upstream of the recognition site. Immediately downstream of the SapI site is the T7 terminator sequence (5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO: 14), followed by a unique restriction enzyme cleavage site (NotI, 5'-GC'GGCC,GC-3'). These parts were combined into a five-piece Gibson Assembly® reaction (linearized pYL backbone and four synthetic fragments) to generate the CHIKV S27 base vector.

[0177] The CHIKV DRDE base vector was similarly constructed from the reference sequence (Genbank EF210157), but the S27 3′UTR was used instead of the DRDE 3′UTR.

[0178] SINV basic vector The basic SINV Girdwood vector was synthesized de novo in four approximately 4 kb portions (Twist Bioscience, Thermo Fisher GeneArt) from the Girdwood strain reference sequence (Genbank MF459683) with a unique restriction enzyme cleavage site (SpeI, 5'-A'CTAG,T-3') in place of the coding sequence of the SINV structural genes (where 5'A is the next nucleotide of the P2A sequence following nucleotide 93 of the structural polyprotein gene, and 3'T coincides with the position of the stop codon TGA of the structural polyprotein). A bacteriophage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO: 13) was included upstream of the SINV genomic sequence and downstream of the poly(A) sequence followed by a SapI site, which cleaves upstream of the recognition site. Immediately downstream of the SapI site is a T7 terminator sequence (5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO: 14), followed by a unique restriction site (NotI, 5'-GC'GGCC,GC-3'). These sections were combined into a five-piece Gibson Assembly® reaction (e.g., linearized pYL backbone and four synthetic fragments) to generate the SINV Girdwood base vector.

[0179] The basic SINV AR86 vector was similarly constructed from the reference sequence (Genbank U38305), with the nsP2 coding sequence obtained from the Girdwood reference sequence.

[0180] VEE Basic Vector The basic VEE vector was de novo synthesized in four approximately 4 kb portions (Twist Bioscience, Thermo Fisher GeneArt) from the TC-83 strain reference sequence (Genbank L01443) with a silent A2087G mutation and a unique restriction enzyme cleavage site (SpeI, 5'-A'CTAG,T-3') in place of the coding sequence of the VEE structural gene (where 5'A is the next nucleotide of the P2A sequence following nucleotide 93 of the structural polyprotein gene and 3'T corresponds to the position of the stop codon TGA of the structural polyprotein). A 5' adaptor sequence (5'-CTGGAGACGTGGAGGAGAACCCTGGACCT-3'; SEQ ID NO:11) was inserted upstream of the SpeI site and a 3' adaptor sequence (5'-GACCGCTACGCCCCAATGACCCGACCAGC-3'; SEQ ID NO:12) was inserted downstream of the SpeI site for the subsequent Gibson Assembly® manipulation. A bacteriophage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO:13) is included upstream of the VEE genomic sequence and downstream of a poly(A) sequence followed by a SapI site, which cleaves upstream of the recognition site. Immediately downstream of the SapI site is the T7 terminator sequence (5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO:14), followed by a unique restriction enzyme cleavage site (NotI, 5'-GC'GGCC,GC-3'). These parts were combined into a five-piece Gibson Assembly® reaction (linearized pYL backbone and four synthetic fragments) to generate the VEE base vector.

[0181] Final Vector Construction of vectors containing heterologous genes was performed as follows: empty base vector was linearized by SpeI digestion. IL-12A, IL12-B, IL-1RN genes were codon optimized / refactored in silico for human expression and synthesized de novo with EMCV IRES (IDT). The synthesis products were amplified using primers that add 5' or 3' adapter sequences to the ends of the genes or P2A and / or homologous sequences to the adjacent gene inserts. Digestion and PCR products were combined by Gibson Assembly® procedure to obtain the final vectors.

[0182] Example 2 In vitro evaluation of modified alphavirus vectors This example describes the results of in vitro experiments performed to assess the expression levels of the synthetic srRNA constructs described in Example 1 above and to examine their differential behavior (e.g., replication and protein expression).

[0183] In vitro transcription : RNA was prepared by in vitro transcription from SapI-linearized plasmid templates using 5'ARCA-capped bacteriophage T7 polymerase (HiScribe™ T7 ARCA mRNA Kit, NEB) or by uncapped transcription (HiScribe™ T7 High Yield RNA Synthesis Kit, NEB) followed by addition of a 5' Cap 1 (Vaccinia Capping System, mRNA Cap 2'-O-Methyltransferase, NEB). RNA was then purified using phenol / chloroform extraction or column purification (Monarch® RNA Cleanup Kit, NEB). RNA concentration was measured by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific).

[0184] ReproductionRNA was transfected by electroporation into BHK-21 or Vero cells (e.g., 4D-Nucleofector™, Lonza). 15-22 hours after transfection, cells were fixed, permeabilized (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen), and stained using a PE-conjugated anti-dsRNA mouse monoclonal antibody (J2, Scicons), and the frequency of dsRNA+ cells and the mean fluorescence intensity (MFI) of dsRNA in individual cells were quantified by fluorescent flow cytometry.

[0185] Protein expression by ELISA Human IL-12p70 and IL-1RA were detected from electroporated BHK-21 cells using 500ng of srRNA monogene or multigene constructs. Supernatants were harvested approximately 24 and 48 hours post-transfection and assayed with RnD Systems' Human IL-12p70 (Cat. No. DY1270) and RnD Systems' Human IL-1ra / IL-1F3 DuoSet ELISA (Cat. No. DY280).

[0186] Bioactivity assays Human IL-12p70 and IL-1RA were detected using 500ng of srRNA mono- or multi-gene constructs from electroporated BHK-21 cells. Supernatants were harvested approximately 24 and 48 hours post-transfection and assayed for IL-12 using Promega's GloMax bioassay (catalog no. JA2601). IL-1RA was assayed using supernatants from transfected BHK cells and IL-1β reporter HEK293 cells (Invivogen, hkb-il1bv2) pre-incubated with 4ng / mL IL1β (final concentration 1ng / mL) according to the manufacturer's protocol.

[0187] Assessment of gene number and order The results of the mouse IL-12 and IL-1RA detection ELISA assay measured from transfected BHK-21 cells are shown in Figures 1A, 1B, 4A, and 4B. Various constructs in the form of monogenes or multigenes with different orders of IL-12 subunit p35, IL-12 subunit p40, and IL-1RA were tested to determine which genetic configuration within the construct would result in the most robust expression of IL-12 and IL-1RA. The Y-axis shows the concentration of IL-12 or IL-1RA in ng / mL. Figure 3 shows the corresponding IL-12 and IL-1RA concentrations measured from each construct tested.

[0188] Murine IL-12 and IL-1RA detection bioactivity was measured from the supernatants of BHK-21 cells transfected with different srRNA constructs tested in reporter cells expressing different cytokine receptors. The results are shown in Figures 2A, 2B, 5A, and 5B. Figure 6 shows the corresponding IL-12 and IL-1RA bioactivity measured from each construct tested.

[0189] Example 3 In vivo evaluation of modified alphavirus vectors This example describes the results of in vivo experiments performed to evaluate the srRNA constructs described herein (e.g., both unformulated and LNP-formulated vectors).

[0190] In these experiments, synthetic srRNA constructs derived from various alphavirus strains were designed and then evaluated.

[0191] Mice and injections BALB / c mice were purchased from Charles River Labs, Envigo, or Jackson Laboratories. On the day of dosing, 0.01-40 μg of material was injected intramuscularly in split doses into one or both quadriceps muscles. Vectors were administered unformulated or LNP-formulated in saline. Mice were followed for weight and other general observations throughout the study period. For pharmacokinetic studies, mice were dosed only on day 0.

[0192] LNP formulation Replicon RNA was formulated into lipid nanoparticles using a microfluidic mixer and analyzed for particle size, polydispersity using dynamic light scattering, and for encapsulation efficiency. Lipids were suspended in ethanol. For L1, RNA was suspended in 10 mM citrate buffer (pH 5.0) at a concentration of 172 μg / ml and mixed at a flow rate of 3:1 (aqueous:organic). For L2, RNA was suspended in 250 mM NaOAc (pH 4.0) at a concentration of 82 μg / ml and mixed at a flow rate of 3:1 (aqueous:organic).

[0193] ELISA To measure serum concentrations of IL-12 and IL-1RA, ELISA analysis was performed using human IL-12 p70 DuoSet ELISA (RnD Systems, Cat. No. DY1270) and human IL-1ra ELISA kits (Abcam, ab211650) according to the manufacturer's protocols.

[0194] Evaluation of gene and lipid composition order The two best multigene constructs obtained from the in vitro assay were then tested in vivo with six different srRNA vectors for protein expression in mouse serum. The results are shown in Figures 7A and 7B. Note that in some cases IL-1RA was not detected, likely due to the short half-life of the protein.

[0195] The multigene construct was then tested in different formulations and analyzed for protein expression in vivo by ELISA, and the results are shown in Figures 8A and 8B.

[0196] While certain alternative embodiments of the present disclosure have been disclosed, it is to be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. Accordingly, there is no intention to be limited to the precise content of the summary and disclosure presented herein.

Claims

1. 1. A nucleic acid construct comprising a nucleic acid sequence encoding a modified alphavirus genome or a self-replicating RNA (srRNA), wherein at least a portion of the nucleic acid sequence encoding a viral structural protein of said modified alphavirus genome or srRNA comprises: a) a coding sequence for the p35 subunit of interleukin-12 (p35 or IL-12A) or a functional variant thereof; b) a coding sequence for the p40 subunit of interleukin-12 (p40 or IL-12B) or a functional variant thereof; and c) a coding sequence for an interleukin-1 receptor antagonist (IL-1RA) or a functional variant thereof, A nucleic acid construct wherein the coding sequences for IL-12A, IL-12B, and IL-1RA are operably linked to each other.

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

3. 2. The nucleic acid construct of claim 1, wherein the coding sequences of (a) to (c) are operably linked to each other within a single open reading frame (i.e., within a polycistronic ORF).

4. The nucleic acid construct of claim 1 , wherein the nucleic acid sequence encoding the polypeptide construct is operably linked to a promoter sequence.

5. The nucleic acid construct of claim 4, wherein the promoter sequence is a 26S subgenomic (sg) promoter.

6. 2. The nucleic acid construct of claim 1, wherein the coding sequences of (a) to (c) are operably linked to each other by one or more connector sequences encoding an autoproteolytic peptide or an internal ribosome entry site (IRES).

7. 7. The nucleic acid construct of claim 6, wherein the autoproteolytic peptide comprises one or more autoproteolytic cleavage sequences from calcium-dependent serine endoprotease (furin), porcine teschovirus 1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), tosea asignavirus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), flacherie virus 2A (BmIFV2A), or a combination thereof.

8. 7. The nucleic acid construct of claim 6, wherein the internal ribosome entry site (IRES) is derived from Kaposi's sarcoma-associated herpesvirus (KSHV) IRES, hepatitis virus IRES, pestivirus IRES, crispa virus IRES, Rhopalosiphum padivirus IRES, fibroblast growth factor IRES, platelet-derived growth factor IRES, vascular endothelial growth factor IRES, insulin-like growth factor IRES, picornavirus IRES, encephalomyocarditis virus (EMCV) IRES, Pim-1 IRES, p53 IRES, Apaf-1 IRES, TDP2 IRES, L-myc IRES, or c-myc IRES.

9. 2. The nucleic acid construct of claim 1, wherein the modified alphavirus genome or srRNA is from an alphavirus belonging to the VEEV / EEEV group, the SFV group, or the SINV group.

10. 10. The nucleic acid construct of claim 9, wherein the alphavirus is Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Chikungunya virus (CHIKV), or Sindbis virus (SINV).

11. The polypeptide construct comprises, in N-terminal to C-terminal direction: a) an IL-12A polypeptide, an IL-12B polypeptide, and an IL-1RA polypeptide; or b) comprising an IL-1RA polypeptide, an IL-12B polypeptide, and an IL-12A polypeptide; 2. The nucleic acid construct of claim 1, wherein the IL-12A, IL-12B, and IL-1RA polypeptides are operably linked to each other by one or more autoproteolytic cleavage sequences or internal ribosome entry sites.

12. 2. The nucleic acid construct of claim 1, wherein the nucleic acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:

10.

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

14. The recombinant cell of claim 13 , wherein the recombinant cell is a mammalian cell or an insect cell.

15. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the nucleic acid construct of claim 1.

16. 16. The pharmaceutical composition of claim 15, wherein the composition is formulated in a delivery system together with a delivery vehicle, wherein the delivery system comprises a liposome, a viral replicon particle (VRP), a lipid-based nanoparticle (LNP), a polymeric nanoparticle, a physiological buffer, a microsphere, an immunostimulating complex (ISCOM), a conjugate of a biologically active ligand, or any combination thereof.

17. 17. The pharmaceutical composition of claim 16, wherein the LNP delivery system comprises a cationic lipid, an ionizable cationic lipid, an anionic lipid, or a neutral lipid.

18. 17. The pharmaceutical composition of claim 16, wherein the lipids are present in a lipid to RNA mass ratio of 100:1 to 4:

1.

19. 17. The pharmaceutical composition of claim 16, wherein the lipid-based nanoparticles have an average diameter of 25 nm to 1000 nm.

20. 17. The pharmaceutical composition of claim 16, wherein the composition is formulated as a biotherapeutic.

21. 16. The pharmaceutical composition of claim 15 for treating cancer, immune disorders, or chronic infections.

22. 16. The pharmaceutical composition of claim 15, wherein the composition is administered individually to the subject as a monotherapy or as a first therapy in combination with at least one additional therapy.

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