Artificial alphavirus-derived RNA replicon expression system

Abstract

A synthetic alphavirus-derived replicon expression system is described that includes a nucleic acid sequence encoding at least one modified nonstructural protein and a synthetic nucleic acid sequence encoding at least one heterologous protein. Methods of producing at least one heterologous protein in a cell or inducing an immune response in a subject by administering and / or expressing the synthetic alphavirus-derived replicon expression system are provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 163,475, filed March 19, 2021, which is incorporated by reference as if set forth in full.

[0002] The sequence listing submitted electronically with this application entitled "Sequence Listing", which was created on March 18, 2022, and has a size of 413,759 bytes, is hereby incorporated by reference as if set forth in full.

[0003] FIELD OF THEINVENTION This disclosure relates to self-replicating RNA molecules derived from the genome of an alphavirus and methods for improving the safety and gene expression capabilities of those self-replicating RNA molecules by mutating or otherwise editing the primary nucleotide sequence. This disclosure further relates to methods for producing and using such engineered RNA molecules for the treatment of and prophylaxis against disease in humans or animals. [Background technology]

[0004] Self-replicating RNA or "replicon" RNA has been used as a vector for transgene expression in vitro and in vivo. Viruses of the alphavirus genus are used as templates for the design of replicon RNA because their genome layout is consistent across species and amenable to molecular biological manipulation. Alphavirus-based replicons derived from many different species of alphaviruses (e.g., Venezuelan equine encephalitis virus, Sindbis virus, Semliki Forest virus) have been used as vectors for transgene expression both in vitro and in vivo. These vectors rely on the activity of nonstructural viral proteins encoded therein to mediate the expression of exogenous proteins encoded in place of structural gene open reading frames (ORFs) that are translated from subgenomic (SG) mRNAs synthesized late in the replicative life cycle.

[0005] Replicons are potent inducers of the innate immune response, making them useful for vaccines and other immunomodulatory applications. Alphavirus replicon RNAs are often based on common virus species, such as Venezuelan equine encephalitis virus (VEEV), which is naturally occurring and endemic in much of the world. The risk of recombination with wild-type virus is greater when the sequence of the replicon vector is identical to the wild-type genome. The primary sequence of RNA viruses is sensitive to changes due to the conservation of secondary structures and non-protein-coding functions required for genome replication, translation, and packaging into virions.

[0006] If only the nonstructural polyproteins (nsPs) are expressed at effective intracellular concentrations, it is difficult to modulate the immune response to the replicon RNA, since the initial interferon response is initiated at an early stage of the viral life cycle. Modifications to incorporate additional regulatory gene products into the replicon must currently be done by insertion into the SG ORF, which results in expression only at a later stage of the viral replication cycle, disrupting any other transgene sequences encoded in that ORF. RNA replicons would be much more useful if transgenes could be expressed in cis from and in addition to the nsP ORF, ensuring their expression early in the replication life cycle if important biological processes such as the induction of pattern recognition receptors (PRRs) or post-translational processing of proteins could be deliberately regulated by delivery of exogenous transgenes encoded in that nsP ORF. Furthermore, RNA replicons would be safer for use in the field if the coding sequences of the nsP regions contained less sequence identity with the naturally occurring viral genome. Summary of the Invention [Means for solving the problem]

[0007] In one aspect, the present invention relates to a synthetic alphavirus-derived self-replicating or replicon nucleic acid (RNA) molecule comprising a first nucleic acid encoding alphavirus nonstructural proteins nsP1, nsP2, nsP3, and nsP4 and including at least one silent mutation introduced anywhere within the regions nt503 to nt658, nt658 to nt1620, nt1620 to nt2560, nt2560 to nt3954, nt3954 to nt4120, nt6381 to nt7083, and nt6966 to nt7526 of the alphavirus genome sequence shown in SEQ ID NO: 17. The synthetic alphavirus-derived replicon nucleic acid molecule also comprises a second nucleic acid comprising a subgenomic promoter (SGP) and a modified subgenomic open reading frame (SG ORF).

[0008] In one aspect, the invention relates to an alphavirus-derived RNA replicon expression system comprising any one of said synthetic alphavirus-derived replicon nucleic acid molecules in which the C-terminus of nonstructural protein 4 (nsP4) is fused to a heterologous protein via a non-cleavable, self-cleavable, or proteolytically cleavable peptide linker sequence. The synthetic alphavirus-derived replicon nucleic acid molecule also encodes a functional SGP sequence downstream (i.e., 3') of the nsP4 fusion protein to drive expression of a modified SG ORF. In one aspect, there is at least one silent mutation in the nsP4 coding sequence that renders the SGP-homologous sequence contained in the nsP4 coding sequence non-functional and non-identical to the functional SGP encoded downstream of the nsP4 fusion protein ORF.

[0009] In one aspect, the invention relates to an alphavirus-derived RNA replicon expression system comprising any one of the synthetic alphavirus-derived replicon nucleic acid molecules described herein encapsulated or formulated for delivery to cells of an organism in the form of a virus, virus-like particle, liposome, or lipid, polymer-based or dendrimer-based nanoparticle.

[0010] In one aspect, the invention relates to a vaccine comprising any one of the synthetic alphavirus-derived replicon nucleic acid molecules described herein.

[0011] In one aspect, the invention relates to a therapeutic agent or medicament, the therapeutic agent or medicament comprising any one of the synthetic alphavirus-derived replicon nucleic acid molecules described herein.

[0012] In one aspect, the invention relates to a method of producing at least one heterologous protein in a cell, the method comprising expressing in the cell any one of the synthetic alphavirus-derived replicon nucleic acid molecules described herein.

[0013] In one aspect, the invention relates to a method of preventing, inhibiting, or treating a symptom of a disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of any one of the synthetic alphavirus-derived replicon nucleic acid molecules described herein. [Brief description of the drawings]

[0014] The following detailed description of the preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, specific embodiments are shown in the drawings, it being understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown in the drawings.

[0015] [Figure 1] FIG. 1 is a schematic diagram showing the steps of gene expression and post-translational processing of the wild-type alphavirus genome nonstructural polyproteins (nsP1-nsP2-nsP3-nsP4). [Diagram 2] Figure 2 is a schematic diagram showing the gene expression process of an alphavirus genome nonstructural polyprotein (nsP1-nsP2-nsP3-nsP4-xP) engineered to express an additional exogenous protein ("xP") by in-frame addition with nsP4. [Diagram 3] FIG. 3 is a schematic diagram showing the steps for cloning the modifiable nsP4 open reading frame (ORF) while maintaining the extended subgenomic promoter (SGP, overlapping region, striped box) intact. [Figure 4] Figure 4 is a schematic diagram showing the optimized steps for cloning the modifiable nsP4 open reading frame (ORF) while maintaining an intact extended subgenomic promoter (SGP), in which the grey boxes indicate the synthetic nsP4 C-terminal and xP regions, and the striped box indicates the original nsP4 C-terminal CDS (untranslated). [Diagram 5]5 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 8 (C08). Transcription of this plasmid from the T7 promoter produces the C08 replicon RNA. The 5'UTR, nsP1-4 ORF, and SGP of C08 are shown in SEQ ID NO:27. [Figure 6] Figure 6 is a schematic diagram depicting an exemplary process used to select regions of the alphavirus genome that can be altered by applying silent mutations that reduce homology to the wild-type virus without reducing the replicative function of the replicon. In this diagram, nucleobases that can be altered without disrupting the secondary structure amino acid coding sequence are circled. Such modification of nucleobases to generate silent mutations is referred to as the process of codon adaptation. [Figure 7] Figure 7 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 2 (C02). In this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codons at genomic nucleotide positions 503-658 (grey box) have been altered to reduce homology to wild-type virus. Transcription of this plasmid from the T7 promoter produces the C02 replicon RNA. The 5'UTR, nsP1-4 ORFs, and SGPs of C02 are shown in SEQ ID NO:21. [Figure 8] Figure 8 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 6 (C06). In this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codon at genomic nucleotide positions 658-1620 (grey box) has been altered to reduce homology to wild-type virus. Transcription of this plasmid from the T7 promoter produces the C06 replicon RNA. The 5'UTR, nsP1-4 ORFs, and SGPs of C06 are shown in SEQ ID NO:25. [Figure 9]Figure 9 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 5 (C05). In this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codons at genomic nucleotide positions 1620-2560 (grey box) have been altered to reduce homology to wild-type virus. Transcription of this plasmid from the T7 promoter produces the C05 replicon RNA. The 5'UTR, nsP1-4 ORFs, and SGPs of C05 are shown in SEQ ID NO:24. [Figure 10] Figure 10 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 3 (C03). In this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codon at genomic nucleotide positions 3694-3954 (grey box) has been altered to reduce homology to wild-type virus. Transcription of this plasmid from the T7 promoter produces the C03 replicon RNA. The 5'UTR, nsP1-4 ORFs, and SGPs of C03 are shown in SEQ ID NO:22. [Figure 11] Figure 11 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 7 (C07). In this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codons at genomic nucleotide positions 2560-3954 (grey box) have been altered to reduce homology to wild-type virus. Transcription of this plasmid from the T7 promoter produces the C07 replicon RNA. The 5'UTR, nsP1-4 ORFs, and SGPs of C07 are shown in SEQ ID NO:26. [Figure 12]Figure 12 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 4 (C04). In this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codon at genomic nucleotide positions 3954-4120 (grey box) has been altered to reduce homology to wild-type virus. Transcription of this plasmid from the T7 promoter produces the C04 replicon RNA. The 5'UTR, nsP1-4 ORFs, and SGPs of C04 are shown in SEQ ID NO:23. [Figure 13] Figure 13 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 1 (C01). In this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codons at genomic nucleotide positions 6381-7083 (grey box) have been altered to reduce homology to wild-type virus. Transcription of this plasmid from the T7 promoter produces the C01 replicon RNA. The 5'UTR, nsP1-4 ORFs, and SGPs of C01 are shown in SEQ ID NO:20. [Figure 14] Figure 14 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 9 (C09). In this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codons at genomic nucleotide positions 658-1620 and 2560-3954 (grey box) have been altered to reduce homology to wild-type virus. Transcription of this plasmid from the T7 promoter produces the C09 replicon RNA. The 5'UTR, nsP1-4 ORFs, and SGPs of C09 are shown in SEQ ID NO:28. [Figure 15]Figure 15 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 10 (C10). In this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codons at genomic nucleotide positions 3694-3954 and 6381-7083 (grey box) have been altered to reduce homology to wild-type virus. Transcription of this plasmid from the T7 promoter produces the C10 replicon RNA. The 5'UTR, nsP1-4 ORFs, and SGPs of C10 are shown in SEQ ID NO:29. [Figure 16] Figure 16 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 11 (C11). In this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codons at genomic nucleotide positions 503-658, 658-1620, 1620-2560, 2560-3954, 3954-4120, and 6381-7083 (grey boxes) have been altered to reduce homology to wild-type virus. Transcription of this plasmid from the T7 promoter produces the C11 replicon RNA. The 5'UTR, nsP1-4 ORFs, and SGPs of C11 are shown in SEQ ID NO:30. [Figure 17A] Figures 17A-17D show the results of a SEAP expression study to demonstrate the functionality of the SG ORF encoding SEAP present in the codon-adapted replicon RNA and to quantify SEAP concentration by colorimetric assay. Figure 17A is a bar graph showing the results of a SEAP expression study for replicon RNAs C01, C02, C03, C04, C05, C06, C07, and C08 compared to wild type (WT, a non-codon-adapted replicon RNA that also encodes SEAP), performed by sampling conditioned supernatants of transfected BHK cells for the indicated times: 4 hours (hr) (left bar), 16 hours (middle bar), and 24 hours (right bar). [Figure 17B]Figures 17A-17D show the results of a SEAP expression test to demonstrate the functionality of the SG ORF encoding SEAP present in the codon-adapted replicon RNA and to quantify SEAP concentration by colorimetric assay. Figure 17B is a bar graph showing the results of a SEAP expression test by sampling conditioned supernatants of BHK cells transfected for 16 h with the indicated replicon RNA (C09) and quantifying SEAP expression by measuring absorbance at 650 nm in a colorimetric assay. The codon-adapted construct was observed to express more strongly than the wild-type (WT) control. [Figure 17C] Figures 17A-17D show the results of a SEAP expression test to demonstrate the functionality of the SG ORF encoding SEAP present in the codon-adapted replicon RNA and to quantify SEAP concentration by colorimetric assay. Figure 17C is a bar graph showing the results of a SEAP expression test by sampling conditioned supernatants of BHK cells transfected for 16 h with the indicated replicon RNA (C10) and quantifying SEAP expression by measuring absorbance at 650 nm in a colorimetric assay. The codon-adapted construct was observed to express more strongly than the wild-type (WT) control. [Figure 17D] Figures 17A-17D show the results of a SEAP expression study to demonstrate the functionality of the SG ORF encoding SEAP present in the codon-adapted replicon RNA and to quantify SEAP concentrations by colorimetric assay. Figure 17D is a column scatter plot showing the results of SEAP expression quantified by luminescence assay (RLU) for constructs C03, C05, and C06 compared to a control wild-type RNA replicon (VEEVrep-SEAP; WT) and untreated (untransfected) mice. For treatment, four mice per group were injected with 1 μg of the indicated RNA and serum was collected the next day. Serum SEAP concentrations were measured in relative luminescence units (RLU) by luminescence assay. Bars: mean RLU of the group with error bars = SD. [Figure 18]FIG. 18 is a scale diagram of the VEEV genome, with regions of the nonstructural polyprotein that were determined to be enriched in nonstructured regions susceptible to silent mutations by RNA folding analysis and the experiments described herein marked as grey boxes. [Figure 19] Figure 19 is a schematic diagram showing an example of the nsP4 replicon sequence shown in Figure 4 modified for an experiment, where the exogenous protein (xP) added to the nsP4 protein is green fluorescent protein (GFP; grey box). In this figure, the structural polyprotein in the SG ORF is replaced with the reporter gene SEAP. This replicon RNA is called VEEVrep-nsP4[GFP]-SEAP, and the RNA sequence is as shown in SEQ ID NO:31. [Figure 20A] Figures 20A-20C show gene expression in cells in vitro mediated by engineered nsP4 modified VEEV replicons. BHK cells were transfected with an experimental nsP4 modified replicon (VEEVrep-nsP4[GFP]-SEAP) encoding GFP in the nonstructural protein ORF and SEAP in the SG ORF. Figure 20A shows the results of fluorescence microscopy demonstrating multiple GFP positive cells when transfection was performed with nsP4(GFP) modified VEEV replicon RNA (VEEVrep-nsP4[GFP]-SEAP) and the absence of GFP expression in untransfected control cells. [Figure 20B]Figures 20A-20C show gene expression in cells in vitro mediated by engineered nsP4 modified VEEV replicons. BHK cells were transfected with an experimental nsP4 modified replicon (VEEVrep-nsP4[GFP]-SEAP) encoding GFP in the nonstructural protein ORF and SEAP in the SG ORF. Figure 20B is a photograph of an immunoblot of transfected BHK cell lysates with a GFP-specific monoclonal antibody, with GAPDFI detection serving as a loading control showing confirmation of GFP expression by two independently isolated clones of VEEVrep-nsP4[GFP]-SEAP. [Figure 20C] Figures 20A-20C show gene expression in cells in vitro mediated by engineered nsP4 modified VEEV replicons. BHK cells were transfected with an experimental nsP4 modified replicon (VEEVrep-nsP4[GFP]-SEAP) encoding GFP in the nonstructural protein ORF and SEAP in the SG ORF. Figure 20C is a bar graph showing the functionality of the SG ORF encoding SEAP, which was tested by sampling conditioned supernatants of transfected BHK cells and quantifying SEAP expression by a colorimetric assay. [Figure 21A] Figures 21A-21B show gene expression mediated by an engineered nsP4 modified VEEV replicon, which combines codon adaptation and nsP4 C-terminal modifications as described in embodiments herein. Figure 21A shows the results of fluorescence microscopy revealing multiple GFP positive cells (upper panel, right) when transfected with codon adapted nsP4(GFP) modified VEEV-SEAP replicon RNA (C13) and the absence of GFP expression in untransfected control cells (lower panel, right) compared to bright field (BF) microscopy (left panel). [Figure 21B]21A-21B show gene expression mediated by an engineered nsP4 modified VEEV replicon. This replicon RNA combines codon adaptation and nsP4 C-terminal modifications as described in embodiments herein. FIG. 21B is a chart showing the results of a luminescence assay (RLU) to quantify the concentration of SEAP in serum collected from mice injected with wild-type (WT) VEEV-SEAP replicon RNA or with nsP4(GFP) modified (modified) VEEV replicon RNA with optimized codons in the nonstructural region of the nonstructural polyprotein gene compared to serum collected from uninjected control mice. Serum was collected on days 1, 3, and 5 after injection. [Figure 22A] Figures 22A-22B show gene expression from an alphavirus replicon RNA modified to encode the 3Cpro protease required for picornavirus P1 structural polyprotein (encoded in the SG ORF). Figure 22A is a schematic diagram showing a construct with modification of the nsP4 coding sequence of an alphavirus replicon to encode the 3Cpro protease required for picornavirus P1 structural polyprotein (O1 Manisa P1 polyprotein), designated VEEVrep-nsP4[3C]-P1. The sequences of the 5'UTR, nsP1-4, 3Cpro and SGP regions of this RNA are shown in SEQ ID NO:32. The amino acid sequence of the P1 polyprotein encoded by this SG ORF is shown in SEQ ID NO:40. [Figure 22B]Figures 22A-22B show gene expression from an alphavirus replicon RNA modified to encode the 3Cpro protease required for the picornavirus P1 structural polyprotein (encoded in the SG ORF). Figure 22B is a photograph of an immunoblot performed on lysates of BHK cells transfected with the following RNAs: lane 1, VEEVrep-nsP4[3C]-P1; lane 2, a replicon RNA similar to that in lane 1 but with an insertion mutation that introduces a premature stop codon and eliminates expression of the 3Cpro polypeptide as a negative control (VEEVrep-nsP4[3Cmut]-P1); lane 3, VEEVrep-P1 (similar to VEEVrep-nsP4[3C]-P1 but lacking the nsP4-fused 3Cpro); lane 4, VEEVrep-P1 transfected in combination with 1 / 40 of the mass of the mRNA encoding 3Cpro as a positive control. As shown in this figure, successful proteolytic processing of the ∼100 kDa P1 polyprotein into the immunogenic ∼35 kDa VP0 capsid fragment was mediated to completion only by VEEVrep-nsP4[3C]-P1. An intermediate incompletely cleaved product representing intact VP4+VP2+VP3 can be seen proceeding with an apparent molecular weight of ∼55 kDa. A VP2-specific antibody was used to probe the blot. M, molecular weight marker; C, untreated negative control cells. [Figure 23A] Figures 23A-23D show gene expression data from an alphavirus replicon RNA modified to encode a human STING protein mutated to be constitutively active for IFN-stimulating activity and to encode the reporter gene SEAP in the SG ORF. Figure 23A is a schematic diagram of the construct VEEVrep-nsP4[STING]-SEAP, a replicon RNA containing modifications of the nsP4 coding sequence to encode a human STING protein mutated to be constitutively active for IFN-stimulating activity, and the reporter gene SEAP encoded in the SG ORF. The sequences of the 5'UTR, nsP1-4, STING and SGP regions of this RNA are shown in SEQ ID NO:34. [Figure 23B] Figures 23A-23D show gene expression data from an alphavirus replicon RNA modified to encode a human STING protein mutated to be constitutively active for IFN stimulatory activity and to encode a reporter gene, SEAP, in the SG ORF. Figure 23B is a photograph of an immunoblot performed on HEK-Lucia™ Null cells transfected with the following RNAs: lane 1, VEEVrep-SEAP; lane 2, VEEVrep-nsP4[STING]-SEAP; lane 3, no transfection control. [Figure 23C] Figures 23A-23D show gene expression data from alphavirus replicon RNAs engineered to encode human STING protein mutated to be constitutively active for IFN stimulatory activity and to encode the reporter gene SEAP in the SG ORF. Figure 23C is a bar graph showing expression of the SEAP reporter gene encoded in the SG ORF in VEEVrep-SEAP and VEEVrep-nsP4[STING]-SEAP replicon RNAs (as measured by absorbance at 650 nm in a colorimetric assay) compared to controls (untransfected cells). [Figure 23D] Figures 23A-23D show gene expression data from an alphavirus replicon RNA modified to encode a human STING protein mutated to be constitutively active for IFN stimulatory activity and to encode a reporter gene, SEAP, in the SG ORF. Figure 23D is a bar graph showing surrogate measures of IFN signaling activity induced by VEEVrep-SEAP SEAP and VEEVrep-nsP4[STING]-SEAP replicon RNAs compared to control (untransfected cells). [Figure 24A]Figures 24A-24D show gene expression in B16 melanoma from an alphavirus replicon RNA modified to encode a human STING protein mutated to be constitutively active for IFN stimulatory activity and to encode a reporter gene SEAP in the SG ORF. Figure 24A is a photograph showing GFP expression approximately 1 day after transfection in B16 melanoma cells cultured in 12-well dishes after transfection with 1 μg of VEEVrep-nsP4[GFP]-SEAP replicon RNA compared to untransfected control cells. [Figure 24B] Figures 24A-24D show gene expression in B16 melanoma from an alphavirus replicon RNA modified to encode a human STING protein mutated to be constitutively active for IFN stimulatory activity and to encode a reporter gene SEAP in the SG ORF. Figure 24B is a bar graph showing SEAP expression in B16 melanoma cells cultured in 12-well dishes following transfection with 1 μg of the indicated replicon RNA, VEEVrep-nsP4[STING]-SEAP, VEEVrep-nsP4[GFP]-SEAP, and VEEVrep-SEAP, compared to untransfected control cells. [Figure 24C] Figures 24A-24D show gene expression in B16 melanoma from alphavirus replicon RNAs engineered to encode human STING protein mutated to be constitutively active for IFN stimulatory activity and to encode the reporter gene SEAP in the SG ORF. Figure 24C is a bar graph showing measurement of IFN-β secretion (measured by sandwich ELISA on culture supernatants) by B16 melanoma cells approximately 1 day after transfection with 1 μg of the indicated replicon RNAs, VEEVrep-nsP4[STING]-SEAP and VEEVrep-SEAP, compared to untransfected control cells. [Figure 24D]Figures 24A-24D show gene expression in B16 melanoma from an alphavirus replicon RNA modified to encode a human STING protein mutated to be constitutively active for IFN stimulatory activity and to encode a reporter gene, SEAP, in the SG ORF. Figure 24D is a photograph of light microscopy of B16 cells 1 day after transfection with 2 μg of VEEVrep-nsP4[GFP]-SEAP or VEEVrep-nsP4[STING]-SEAP compared to healthy untransfected control cells (leftmost panel). [Figure 25A] Figures 25A-25D show gene expression in TC-1 cancer cells from an alphavirus replicon RNA modified to encode a human STING protein mutated to be constitutively active for IFN stimulatory activity and to encode a reporter gene, SEAP, in the SG ORF. Figure 25A is a bar graph showing SEAP expression in TC-1 cancer cells cultured in 12-well dishes 2 days after transfection with 2 μg of the indicated replicon RNA, VEEVrep-nsP4[GFP]-SEAP, VEEVrep-nsP4[STING]-SEAP, compared to untransfected control cells. In this experiment, SEAP expression was quantified by a colorimetric assay in the culture medium, indicating successful expression of the SEAP gene encoded in the SG ORF. [Figure 25B] Figures 25A-25D show gene expression in TC-1 cancer cells from an alphavirus replicon RNA modified to encode a human STING protein mutated to be constitutively active for IFN stimulatory activity and to encode a reporter gene, SEAP, in the SG ORF. Figure 25B is a bar graph showing measurement of IFN-β secretion (measured by sandwich ELISA on culture supernatants) by TC-1 cancer cells 1 day after transfection with 2 μg of the indicated replicon RNA, VEEVrep-nsP4[GFP]-SEAP, or VEEVrep-nsP4[STING]-SEAP, compared to untransfected control cells. [Figure 25C] Figures 25A-25D show gene expression in TC-1 cancer cells from an alphavirus replicon RNA modified to encode human STING protein mutated to be constitutively active for IFN stimulatory activity and to encode the reporter gene SEAP in the SG ORF. Figure 25C is a photograph showing GFP expression in TC-1 cancer cell aggregates after transfection with 1 μg of VEEVrep-nsP4[GFP]-SEAP, approximately 3 days after transfection. As shown in this figure, widespread GFP expression was observed throughout the multicellular structure driven by the encoding of a fluorescent protein at the nsP4 C-terminus of the construct (upper panel, right), compared to no GFP expression in untreated control cells (lower panel, right). [Figure 25D] Figures 25A-25D show gene expression in TC-1 cancer cells from an alphavirus replicon RNA modified to encode a human STING protein mutated to be constitutively active for IFN stimulatory activity and to encode a reporter gene SEAP in the SG ORF. Figure 25D is a photograph showing the results of light microscopy of TC-1 cancer cells 1 day after transfection with 4 μg of the indicated replicon RNA VEEVrep-nsP4[STING]-SEAP compared to untreated control cells. As shown in this figure, untreated cells (control) were observed to grow and fill the dish, while growth inhibition and cell death were observed after transfection with VEEVrep-nsP4[STING]-SEAP. [Figure 26A]Figures 26A-26G show VEEV replicon RNAs modified with reduced homology to wild-type RNA and expression data from these constructs. Figure 26A is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 12 (VEEVrepHK-SEAP). As shown in this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codon at genomic nucleotide positions 705-3406 (grey box) was altered to reduce homology to wild-type virus, regardless of the possible presence of any secondary structure elements. [Figure 26B] Figures 26A-26G show VEEV replicon RNAs modified with reduced homology to wild-type RNA and expression data from these constructs. Figure 26B is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 13 (VEEVrepHK(ΔU)-SEAP). As shown in this figure, the DNA plasmid is similar to that shown in Figure 58, except that only the codons at genomic nucleotide positions 658-3359 (grey box) were altered to reduce homology to wild-type virus, with codons that avoid uracil bases being preferentially selected, regardless of the possible presence of any secondary structure elements. [Figure 26C] Figures 26A-26G show VEEV replicon RNAs modified with reduced homology to wild-type RNA and expression data from these constructs. Figure 26C is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 14 (VEEVrepES-SEAP). As shown in this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codon at genomic nucleotide positions 4120-6965 (grey box) was altered to reduce homology to wild-type virus, regardless of the possible presence of any secondary structure elements. [Figure 26D]Figures 26A-26G show VEEV replicon RNAs modified with reduced homology to wild-type RNA and expression data from these constructs. Figure 26D is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 15 (VEEVrepES(ΔU)-SEAP). As shown in this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codons at genomic nucleotide positions 4120-6965 (grey box) were altered to reduce homology to wild-type virus, with codons that avoided uracil bases being preferentially selected, regardless of the possible presence of any secondary structure elements. [Figure 26E] Figures 26A-26G show VEEV replicon RNAs modified with reduced homology to wild-type RNA and expression data from these constructs. Figure 26E is a bar graph showing SEAP expression in BHK cells cultured in 12-well dishes 16 hours after transfection with 1 μg of VEEVrep-SEAP, VEEVrepHK-SEAP, or VEEVrepHK(ΔU)-SEAP RNA compared to untransfected control cells. In this experiment, SEAP expression was quantified by a colorimetric assay in the culture medium, indicating successful expression of the SEAP gene encoded by the SG ORF. [Figure 26F] Figures 26A-26G show VEEV replicon RNAs modified with reduced homology to wild-type RNA and expression data from these constructs. Figure 26F is a bar graph showing SEAP expression in BHK cells cultured in 12-well dishes 16 hours after transfection with 1 μg of VEEVrep-SEAP, VEEVrepES-SEAP, or VEEVrepES(ΔU)-SEAP RNA compared to untransfected controls. [Figure 26G]Figures 26A-26G show VEEV replicon RNAs modified with reduced homology to wild-type RNA and expression data from these constructs. Figure 26G is a bar graph showing serum SEAP expression measured in vivo (by chemiluminescence assay) following injection of 2 μg of LNP-formulated VEEVrep-SEAP, VEEVrepHK-SEAP, or VEEVrepHK(ΔU)-SEAP compared to control (serum from untreated mice). [Figure 27A] Figures 27A-27D show VEEV replicon RNA expression in normal HFF cells. Figure 27A is a bar graph showing SEAP expression in normal HFF cells cultured in 12-well dishes 1 day after transfection with 2 μg or 4 μg of the indicated replicon RNA, VEEVrep-nsP4[STING]-SEAP, VEEVrep-SEAP, compared to untransfected control cells. In this experiment, SEAP expression was quantified by a colorimetric assay in the culture medium, indicating successful expression of the SEAP gene encoded by the SG ORF. [Figure 27B] Figures 27A-27D show VEEV replicon RNA expression in normal HFF cells. Figure 27B is a photograph of GFP expression in trypsin-dissociated normal human HFF cells after transfection with 4 μg of VEEVrep-nsP4[GFP]-SEAP approximately 4 days after transfection, demonstrating the lack of efficient gene expression from the GFP protein encoded at the C-terminus of nsP4 in these healthy, non-cancerous cells. [Figure 27C]Figures 27A-27D show VEEV replicon RNA expression in normal HFF cells. Figure 27C is a bar graph showing measurements of IFN-β activity from transfected HFFs. Conditioned media from HFFs in 12-well dishes was collected 1 day after transfection with the indicated RNAs, VEEVrep-nsP4[STING]-SEAP, VEEVrep-nsP4[GFP]-SEAP, VEEVrep-SEAP, compared to control untransfected HFF cells and no media controls, and doses were applied to HEK-Lucia NULL cells to measure response to type I IFN in the media. In this experiment, the IFN-responsive reporter gene Lucia was assayed in the supernatants the next day by chemiluminescence assay. [Figure 27D] Figures 27A-27D show VEEV replicon RNA expression in normal HFF cells. Figure 27D shows light microscopy of HFF cells 4 days after transfection with 4 μg of the indicated replicon RNA, VEEVrep-nsP4[GFP]-SEAP and VEEVrep-nsP4[STING]-SEAP, compared to untransfected controls. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The terms "a" and "one," as used in the claims and corresponding parts of the specification, are defined as including one or more of the referenced items, unless otherwise specified. This terminology includes the words specifically mentioned above, derivatives thereof, and words of similar import. The phrase "at least one" followed by a list of two or more items, such as "A, B, or C," means any individual one of A, B, or C, and any combination thereof.

[0017] Alphaviruses As used herein, "alphavirus" refers to an enveloped, single-stranded, positive-sense RNA virus of the Togaviridae family. The Alphavirus genus includes about 30 members that can infect humans and other animals. Alphavirus particles typically have a diameter of 70 nm, tend to be spherical or slightly pleomorphic, and have an isometric nucleocapsid of 40 nm.

[0018] The alphavirus genome is a ssRNA molecule approximately 12000 nt long that encodes two open reading frames (ORFs): nonstructural (ns) and structural (also referred to herein as subgenomic, or SG, ORF). The ns ORF begins near the 5' end of the genome and represents the first 2 / 3 of the total genome length. It encodes the nonstructural proteins (nsPs), namely nsP1, nsP2, nsP3, and nsP4, which are produced as a single polyprotein precursor and cleaved into mature proteins by proteolytic processing. The nsPs are expressed by a typical cap-dependent translation in the cytoplasm. After infection, the alphavirus genomic RNA is translated to produce the nsP polyprotein, which is cleaved into four proteins, nsP1, nsP2, nsP3, and nsP4 (Figure 1), which collectively act to catalyze genome replication and transcription / translation of the second alphavirus ORF. nsP1 can be approximately 60 kDa in size, may have methyltransferase activity, and may be involved in the viral capping reaction. nsP2 can be approximately 90 kDa in size and may have helicase and protease activity, while nsP3 is approximately 60 kDa and contains three domains: a macrodomain, a central (or alphavirus-specific) domain, and a hypervariable domain (HVD). nsP4 is approximately 70 kDa in size and contains the core RNA-dependent RNA polymerase (RdRp) catalytic domain.

[0019] The structural SG ORF occupies approximately one third of the genome length and naturally encodes the virion structural proteins: the core nucleocapsid protein C and the envelope proteins P62 and E1 which assemble as a heterodimer. This viral membrane-anchored surface glycoprotein is involved in receptor recognition and entry into target cells via membrane fusion.

[0020] Translation of this SG ORF does not occur until the nsP products mediate RNA-dependent RNA transcription of the genome to generate a complementary negative-strand (-RNA) template. Starting from the 3' end of the -RNA template, the nsP products complete full-length transcription to recreate the original genome sequence, thus completing a cycle of genome copying. The initiation of genome replication represents a transition from the "early" to the "late" phase of the viral replication life cycle. As genome replication occurs via the generation of dsRNA intermediates that constitute pathogen-associated molecular patterns (PAMPs), a strong innate immune response is elicited by intracellular pattern recognition receptors (PRRs). From the -RNA template, the nsP products also mediate transcription from an internal RNA-dependent RNA polymerase promoter ("subgenomic promoter" or SGP) that maps to a genic region immediately upstream of the second ORF (when read in terms of the original sense-strand RNA genome) to generate a smaller or "subgenomic" (SG) mRNA that only codes for the second ORF. Transcription of the SG mRNA is favored over transcription of the full-length genome, resulting in high copy numbers of the SG mRNA in infected cells and, therefore, high levels of production of the structural genes encoded therein.

[0021] As used herein, an RNA replicon refers to a nucleic acid molecule derived in sequence from the alphavirus (Togaviridae) genome that can self-copy (replicate) when introduced into the appropriate intracellular environment. Because RNA replicons are self-copying, they may substantially amplify the production of the encoded protein, resulting in sustained translation of the desired protein in vivo. The terms "replicon RNA" and "RNA replicon" are used interchangeably herein to refer to these RNA molecules.

[0022] In one embodiment, the RNA replicon may be an RNA replicon derived from an alphavirus species. Alphavirus species include Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semliki Forest virus (SFV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), Onion-onion virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), and Una virus (Una virus). In some embodiments, the virus may include, but is not limited to, U.S. Pat. No. 6,313,411, Sindbis virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu (NDUV), and Buggy Creek virus.

[0023] In one embodiment, the RNA replicon may be a replicon derived from the genome of an RNA virus produced by natural selection.

[0024] In one embodiment, the RNA replicon disclosed herein may be an engineered RNA replicon, a synthetic RNA replicon, or a recombinant RNA replicon. The RNA replicon may be synthesized or modified in vitro. The modification may include, but is not limited to, the following techniques: performing chemical or enzymatic techniques, for example, by using chemical nucleic acid synthesis, enzymes for replication, polymerization, exonuclease digestion, endonuclease digestion, ligation, reverse transcription, transcription, base modification (e.g., methylation), or recombination of nucleic acid molecules. The RNA replicon may be engineered by using rational design approaches that selectively maintain important sequence elements, additional functions, and reduced homology to dangerous circulating viruses. The RNA replicon may combine nucleotide sequences that are not combined in nature. The RNA replicon may be engineered using molecular cloning techniques to have one or more sequence changes or rearrangements with respect to naturally occurring nucleotide sequences.

[0025] Alphavirus-based RNA replicons may be used as vectors for gene expression in vitro and in vivo. Alphavirus structural genes are encoded in a single subgenomic (SG) open reading frame (ORF) that may be replaced with the desired recombinant protein. The nonstructural (nsP) coding sequence in a separate preceding ORF has the necessary biochemical functionality to mediate replicon RNA replication in cells and translation of the SG ORF. If the nsP coding sequence is nearly identical in sequence to a circulating alphavirus, it creates the possibility of recombination with pathogenic viral genomes in situ, thus presenting a safety risk. The nsP sequence may also be modified to prevent recombination with sequences of pathogenic viruses. Modification of nsPs is difficult because modifications of the primary amino acid sequence may affect biochemical function, and changes in the primary nucleotide sequence may disrupt important secondary structures required for translation and transcription of the replicon.

[0026] Synthetic Alphavirus-Derived Replicon Nucleic Acid Molecules In one embodiment, a synthetic alphavirus-derived replicon nucleic acid molecule is provided. The synthetic alphavirus-derived replicon nucleic acid molecule may comprise a first nucleic acid encoding alphavirus nonstructural proteins nsP1, nsP2, nsP3, and nsP4. The first nucleic acid may comprise a sequence ranging from the beginning of the 5' untranslated region (UTR) to the end of the subgenomic promoter (SGP). The first nucleic acid may comprise at least one silent mutation introduced anywhere within the following regions of nt503 to nt658, nt658 to nt1620, nt1620 to nt2560, nt2560 to nt3954, nt3954 to nt4120, nt6381 to nt7083, and nt6966 to nt7526 in the sequence of the alphavirus genome shown in SEQ ID NO: 17. The synthetic alphavirus-derived replicon nucleic acid molecule may also comprise a second nucleic acid sequence comprising a modified subgenomic (SG) open reading frame (ORF). The modified SG ORF may include a sequence encoding a first heterologous protein. The first nucleic acid sequence may include a second heterologous protein encoded as a fusion to the C-terminus of nsP4. The nsP4 coding sequence may contain at least one silent mutation introduced anywhere within the region from nt 6966 to nt 7526.

[0027] As used herein, the term "heterologous protein" refers to any protein that does not naturally occur in an organism.

[0028] As used herein, the term "silent mutation" refers to a change in the sequence of nucleotide bases that does not change the amino acid sequence of the encoded protein.For example, silent mutation may be a point mutation that changes a first codon to a second codon that codes for the same amino acid.Silent mutation may include a change in one or more nucleotides.

[0029] As used herein, the term "codon optimization" refers to a process used to improve gene expression and increase the translation efficiency of a gene of interest by adapting to the codon bias of the host organism.

[0030] As used herein, the term "codon adaptation" refers to a process used to alter codons by silent mutations such that the alterations are selected so as not to affect secondary structural motifs determined by bioinformatics analysis of RNA folding.

[0031] In one embodiment, the first nucleic acid may include at least one silent mutation introduced anywhere within the region of the alphavirus genome from nt 503 to nt 658. As used herein, all genomic locations given refer to the Trinidad donkey strain of Venezuelan equine encephalitis virus, complete genome reference sequence L01442 in the NCBI GenBank database (SEQ ID NO: 17; Kinney, RM, Johnson, BJ, Brown, VL and Trent, DW 1986, Nucleotide sequence of the 26 S mRNA of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and deduced sequence of the encoded structural proteins, Virology 152(2), 400-413, which is incorporated herein by reference as if set forth in full).

[0032] The first nucleic acid having this silent mutation may comprise a polynucleotide that encodes nsP1 and comprises a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO:21.

[0033] In one embodiment, the first nucleic acid may comprise a sequence having at least one silent mutation introduced anywhere within the region from nt 658 to nt 1620 of the sequence of the alphavirus genome set forth in SEQ ID NO: 17. The first nucleic acid having the silent mutation may comprise a polynucleotide comprising a sequence encoding nsP1 and having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO:25.

[0034] In one embodiment, the first nucleic acid may comprise a sequence having at least one silent mutation introduced anywhere within the region of nt 1620 to nt 2560 of the alphavirus genome sequence (SEQ ID NO: 17). The first nucleic acid comprising this silent mutation may comprise a polynucleotide comprising a sequence encoding the junction (border) of nsP1 and nsP2 and having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO:24.

[0035] In one embodiment, the first nucleic acid may comprise a sequence having at least one silent mutation introduced anywhere within the region of nt 3694 to nt 3954 of the alphavirus genome sequence (SEQ ID NO: 17). The first nucleic acid comprising this silent mutation may comprise a polynucleotide comprising a sequence encoding the junction of nsP1 and nsP2 and having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO:22.

[0036] In one embodiment, the first nucleic acid may comprise a sequence having at least one silent mutation introduced anywhere within the region of nt 2560 to nt 3954 of the alphavirus genome sequence (SEQ ID NO: 17). The nucleic acid comprising the silent mutation may comprise a polynucleotide comprising a sequence encoding the junction of nsP1 and nsP2 and having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO:26.

[0037] In one embodiment, the first nucleic acid may comprise a sequence having at least one silent mutation introduced anywhere within the region of nt 3954 to nt 4120 of the alphavirus genome sequence (SEQ ID NO: 17). The first nucleic acid comprising this silent mutation may comprise a polynucleotide comprising a sequence encoding the junction of nsP2 and nsP3 and having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO:23.

[0038] In one embodiment, the first nucleic acid may comprise a sequence having at least one silent mutation introduced anywhere within the region from nt 6381 to nt 7083 of the alphavirus genome sequence (SEQ ID NO: 17). The first nucleic acid comprising the silent mutation may comprise a polynucleotide comprising a sequence encoding nsP4 and having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO:20.

[0039] In one embodiment, the first nucleic acid sequence may comprise a sequence having at least one silent mutation introduced anywhere within the region from nt 6966 to nt 7526 of the alphavirus genome sequence (SEQ ID NO: 17). The first nucleic acid comprising this silent mutation may comprise a polynucleotide comprising a sequence encoding the C-terminal region of nsP4 and having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO: 18 or SEQ ID NO: 19.

[0040] In one embodiment, the first nucleic acid may comprise a sequence with silent mutations in multiple regions of the genome. The nucleic acid may comprise multiple silent mutations introduced at any position within the region from nt 658 to nt 1620 and within the region from nt 2560 to nt 3954 of the sequence of the alphavirus genome (SEQ ID NO: 17). The first nucleic acid comprising silent mutations may comprise a polynucleotide encoding nsP1 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO: 25, and a polynucleotide encoding nsP2 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO: 26.

[0041] In one embodiment, the first nucleic acid may comprise a sequence having multiple silent mutations introduced anywhere within the region from nt 3694 to nt 3954 and within the region from nt 6381 to nt 7083 of the sequence of the alphavirus genome (SEQ ID NO: 17). The first nucleic acid comprising these silent mutations may comprise a polynucleotide comprising a sequence encoding nsP2 and having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO: 22, and a polynucleotide comprising a sequence encoding nsP4 and having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO: 20.

[0042] In one embodiment, the first nucleic acid may comprise a sequence having multiple silent mutations introduced at any position within the region from nt503 to nt658, the region from nt658 to nt1620, the region from nt1620 to nt2560, the region from nt2560 to nt3954, the region from nt3954 to nt4120, and the region from nt6381 to nt7083 of the alphavirus genome sequence (SEQ ID NO: 17). The first nucleic acid containing these silent mutations is a polynucleotide encoding nsP1 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO:21; a polynucleotide encoding nsP1 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO:25; a polynucleotide encoding the junction of nsP1 and nsP2 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO:24. The polynucleotides may include a polynucleotide encoding nsP2 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO:26, a polynucleotide encoding the junction of nsP2 and nsP3 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO:23; and a polynucleotide encoding nsP4 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO:20. The first nucleic acid containing these silent mutations may comprise a polynucleotide encoding the nonstructural protein and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO:30.

[0043] Determining the percent identity of two amino acid sequences or two nucleic acid sequences may involve aligning and comparing the amino acid residues or nucleotides at corresponding positions in the two sequences. If all positions in two sequences are occupied by the same amino acid residue or nucleotide, the sequences are said to be 100% identical. The percent identity is measured by the Smith Waterman algorithm (Smith TF, Waterman MS 1981, "Identification of Common Molecular Subsequences", J Mol Biol 147:195-197, which is incorporated herein by reference as if set forth in full).

[0044] In one embodiment, the first nucleic acid may comprise the sequence of the nsP region of one of the alphaviruses listed in Table 1, and multiple silent mutations in the nsP1 coding sequence are made after amino acid position 85 such that 10-30% of the nucleotides differ from the wild-type nsP1 coding sequence.

[0045] [Table 1(1)] [Table 1(2)] [Table 1(3)]

[0046] In one embodiment, the first nucleic acid may comprise the sequence of the nsP region of one of the alphaviruses listed in Table 1, and multiple silent mutations in the nsP2 coding sequence are made such that 10-30% of the nucleotides differ from the wild-type nsP2 coding sequence.

[0047] In one embodiment, the first nucleic acid may comprise the sequence of the nsP region of one of the alphaviruses listed in Table 1, and multiple silent mutations in the nsP3 coding sequence are made such that 10-30% of the nucleotides differ from the wild-type nsP3 coding sequence.

[0048] In one embodiment, the first nucleic acid may comprise the sequence of the nsP region of one of the alphaviruses listed in Table 1, and multiple silent mutations in the nsP4 coding sequence are made at positions preceding the last 30 amino acids of nsP such that 10-30% of the nucleotides differ from the wild-type nsP4 coding sequence.

[0049] In one embodiment, the first nucleic acid may comprise the sequence of the nsP region of one of the alphaviruses listed in Table 1, and multiple silent mutations in the nsP4 coding sequence are made at positions within the last 30 amino acids of the nsP such that 10-30% of the nucleotides differ from the wild-type nsP4 coding sequence, reducing homology to the endogenous SGP.

[0050] In one embodiment, an alphavirus-derived replicon nucleic acid molecule based on one of the species in Table 1 may include a second nucleic acid that includes a polynucleotide encoding a first heterologous protein. This first heterologous protein may be a protein that replaces an alphavirus structural protein encoded by an alphavirus subgenomic (SG) ORF.

[0051] In one embodiment, the alphavirus-derived replicon nucleic acid molecule may comprise a polynucleotide encoding a second heterologous protein, which may be a protein inserted into an altered nsP1, nsP2, nsP3, or nsP4 protein.

[0052] In one embodiment, the modified nsP protein may be a modified nsP4 protein. The modified nsP4 may include a second heterologous protein fused to its C-terminal region. The polynucleotide sequence encoding this heterologous C-terminal region may include a sequence encoding an alphavirus subgenomic promoter at its 3' end. The sequence may be inserted downstream (i.e., 3') of the nsP4 coding sequence region and may be translatable into a protein. Alternatively, the inserted sequence may not be translated into a protein. The non-translated sequence inserted into this region may be a small hairpin RNA (shRNA).

[0053] In one embodiment, the modified nsP4 may comprise a GFP protein as a second heterologous protein. The modified nsP4 may comprise an amino acid sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO: 12 or SEQ ID NO: 41. The modified nsP4 may be encoded by a polynucleotide sequence shown in SEQ ID NO: 31. The modified nsP4 may comprise an amino acid sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO: 15.

[0054] The first heterologous protein (in the SG ORF) or the second heterologous protein (in the nsP ORF) may be any antigenic protein isolated or derived from a viral pathogen, including but not limited to Adenoviridae, Papovaviridae, Parvoviridae, Herpesviridae, Poxviridae, Anelloviridae, Pleolipoviridae, Reoviridae, Picornaviridae, Caliciviridae, Togaviridae, Arenaviridae, Flaviviridae, Orthomyxoviridae, or any of the following: The virus may be selected from the families Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Rhabdoviridae, Filoviridae, Coronaviridae, Astroviridae, Bornaviridae, Arteriviridae, Hepeviridae, Retroviridae, Caulimoviridae, or Hepadnaviridae. For example, the viral pathogen may be an adenovirus, a rhinovirus, a rotavirus, a West Nile virus, a Zika virus, herpes, a filovirus, or a coronavirus (CoV). The coronavirus may be one of MERS CoV, SARS-CoV, and SARS-CoV-2.

[0055] As used herein, the term "antigenic protein" refers to a protein that contains one or more antigenic determinants that elicit an immune response. The immune response may include either antibody production or activation of specific immunologically active cells, or both. An antigenic protein may be a structural component of a pathogen or a cancer cell. An antigenic protein may be synthesized in the host, recombinantly produced, or derived from a biological sample, including, but not limited to, a tissue sample, a cell, or a biological fluid.

[0056] The antigenic protein may be, but is not limited to, a parasitic antigenic protein, a bacterial antigenic protein, a tumor antigenic protein, an environmental antigenic protein, a therapeutic antigenic protein, or an allergen. The antigenic protein may also be a protein that contains one or more antigenic determinants of a viral pathogen described herein.

[0057] In one embodiment, the first heterologous protein or the second heterologous protein may be an enzyme. The enzyme may be of mammalian origin and may modulate cell metabolism or cell signaling. The enzyme may be a cellular protease or a viral protease. The viral protease may be picornavirus 3C protease (3Cpro). As used herein, the term "3C pro ", "3C protease", "3Cpro" or "3CP" refers to a cysteine ​​protease found in picornavirus species, which typically contains a conserved cysteine-histidine-aspartic acid / glutamic acid catalytic triad or cysteine-histidine dyad within its active site. This catalytic triad or dyad typically forms a charge relay network that polarizes and activates a nucleophile (typically a cysteine) that attacks a substrate to form a covalent intermediate that is then hydrolyzed to regenerate the free enzyme. 3C properforms maturation cleavage in structural and non-structural regions of the polyprotein, with pronounced substrate preference for glutamine-glycine / serine / alanine / valine / histidine / arginine and glutamic acid-serine / glycine / arginine / methionine. 3Cpro protease may be derived from foot-and-mouth disease virus (FMDV).

[0058] FMDV 3C protease is a cysteine ​​protease with a molecular weight of approximately 23.1 kDa and containing 213 amino acids. The cysteine-histidine-aspartic acid catalytic triad in the active site of FMDV 3C protease is formed by residues H46, D84 and C163.

[0059] In one embodiment, the modified nsP4 may comprise a second heterologous protein that is a 3C protease. The modified nsP4 may comprise an amino acid sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO: 40. The modified nsP4 comprising the 3C protease may be encoded by a polynucleotide sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO: 32.

[0060] In one embodiment, the synthetic alphavirus-derived replicon nucleic acid molecule may further comprise a first heterologous protein that is an FMDV P1 precursor polypeptide or other polypeptide of interest. The FMDV P1 may be derived from FMDV serotype O1 Manisa, and is referred to herein as the O1 Manisa P1 polyprotein. The O1 Manisa P1 polyprotein may be encoded by a polynucleotide having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the reference sequence set forth in SEQ ID NO:33.

[0061] In one embodiment, the first heterologous protein can be an FMDV P1 precursor polypeptide (O1 Manisa P1 polyprotein) and the second heterologous protein can be a3C protease.

[0062] The first or second heterologous protein may be a viral or mammalian cell derived regulator of cellular metabolic, immune, or signal transduction function, and expression of the heterologous protein may affect the cellular antiviral innate immune response to enhance replication of the alphavirus replicon RNA molecule.

[0063] In one embodiment, the second heterologous protein may be a pattern recognition receptor (PRR) protein. The PRR protein may be a STING (stimulator of interferon genes) protein. The STING protein may be a wild-type STING protein. The modified nsP4 containing STING protein may comprise an amino acid sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO: 42. The modified nsP4 containing STING protein may be encoded by a polynucleotide sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO: 34. The first heterologous protein may comprise an antigen protein, and the second heterologous protein may comprise a STING protein.

[0064] The first or second heterologous protein may be any immunogenic polypeptide suitable for protecting a subject against a disease, which may be cancer or a disease caused by a microbial, bacterial, protozoan, parasitic, or viral pathogen.

[0065] The first heterologous protein may be the same protein as the second heterologous protein.

[0066] In one embodiment, the synthetic alphavirus-derived replicon nucleic acid molecule may include a translation interrupter sequence, such as a 2A sequence. The translation interrupter sequence allows for interruption of protein translation and efficient cleavage at sites not necessarily recognized by proteases, such as sites in the modified nsP4 protein. The 2A sequence may be inserted into the nucleic acid sequence encoding the modified nsP4 before the sequence encoding the second heterologous protein. Insertion of the 2A sequence may result in interruption of translation by 2A and production of a polypeptide without an N-terminal Met residue. The 2A sequence may be a 2A amino acid sequence derived from, but not limited to, Thosea asigna virus (SEQ ID NO: 43), porcine teschovirus-1 (SEQ ID NO: 44), foot and mouth disease virus (SEQ ID NO: 45), equine rhinitis A virus (SEQ ID NO: 46), or other Picornaviridae or Permutotetraviridae viruses.

[0067] In one embodiment, the synthetic alphavirus-derived replicon nucleic acid molecule may comprise a subgenomic (SGP) promoter that directs expression of a sequence encoding a first heterologous protein and / or a second heterologous protein. The subgenomic promoter may be a 26S subgenomic promoter derived from an alphavirus species (see Table 1). The heterologous protein-encoding sequence may be under the control of an internal ribosome entry site (IRES).

[0068] The synthetic alphavirus-derived replicon nucleic acid molecules described herein may contain one or more modified nucleotides, which may be 5-methyluridine, 5-methylcytidine, pseudouridine, or N6-methyladenosine.

[0069] The synthetic alphavirus derived replicon nucleic acid molecule may have a 3' polyA tail.

[0070] In one embodiment, the RNA sequence of the synthetic alphavirus-derived replicon nucleic acid molecule may be codon-optimized to improve translation efficiency. The RNA molecule may be modified by any method known in the art to enhance stability and / or translation. The RNA molecule may be modified, for example, by adding a poly-A tail of at least 30 adenosine residues. The RNA molecule may be modified by capping the 5' end with a modified ribonucleotide, for example a 7-methylguanosine cap, which may be incorporated during RNA synthesis or enzymatically engineered after RNA transcription.

[0071] In one embodiment, a nucleic acid is provided that comprises a DNA sequence encoding a synthetic alphavirus-derived replicon nucleic acid molecule. The nucleic acid may be, for example, a DNA plasmid or a fragment of a linearized DNA plasmid. The nucleic acid may further comprise a promoter, such as a T7 promoter, operably linked to the 5' end of the DNA sequence. The nucleic acid may be used for the production of an RNA replicon of the present application using methods known in the art in view of the present disclosure. For example, a synthetic alphavirus-derived replicon nucleic acid molecule may be obtained by in vivo or in vitro transcription of the nucleic acid.

[0072] In one embodiment, the DNA sequence may be a DNA plasmid, which may comprise a polynucleotide encoding nsP1 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO:2.

[0073] The DNA plasmid may comprise a polynucleotide encoding nsP1 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO:6.

[0074] The DNA plasmid may comprise a polynucleotide encoding the junction of nsP1 and nsP2 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO:5.

[0075] The DNA plasmid may comprise a polynucleotide encoding the junction of nsP1 and nsP2 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO:3.

[0076] The DNA plasmid may comprise a polynucleotide encoding the junction of nsP1 and nsP2 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO:7.

[0077] The DNA plasmid may comprise a polynucleotide encoding the junction of nsP2 and nsP3 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO:4.

[0078] The DNA plasmid may comprise a polynucleotide encoding nsP4 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO:1.

[0079] The DNA plasmid may comprise a polynucleotide encoding nsP1 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO:6, and a polynucleotide encoding nsP2 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO:7.

[0080] The DNA plasmid may comprise a polynucleotide encoding nsP2 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO:3, and a polynucleotide encoding nsP4 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO:1.

[0081] The DNA plasmids include a polynucleotide encoding nsP1 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO:2; a polynucleotide encoding nsP1 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO:6; a polynucleotide encoding the junction of nsP1 and nsP2 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence shown in SEQ ID NO:5. , a polynucleotide encoding nsP2 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO:7, a polynucleotide encoding the junction of nsP2 and nsP3 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO:4, and a polynucleotide encoding nsP4 and comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO:1. The first nucleic acid containing these silent mutations may comprise a polynucleotide encoding a nonstructural protein comprising a sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the reference sequence set forth in SEQ ID NO:11.

[0082] In one embodiment, the nucleic acid comprising a DNA sequence encoding an alphavirus-derived replicon nucleic acid molecule may comprise a second nucleic acid comprising a polynucleotide encoding a first heterologous protein, which may be a protein that replaces an alphavirus structural protein encoded by an alphavirus SG ORF.

[0083] In one embodiment, the nucleic acid may comprise a polynucleotide encoding a second heterologous protein, which may be a protein inserted into the modified nsP1, nsP2, nsP3, or nsP4 protein.

[0084] In one embodiment, the modified nsP protein may be a modified nsP4 protein. The modified nsP4 may comprise a second heterologous protein. The polynucleotide encoding the modified nsP4 may comprise a polynucleotide sequence encoding a first C-terminal region and a polynucleotide sequence encoding a second C-terminal region. The polynucleotide sequence encoding the second C-terminal region may comprise a sequence encoding an alphavirus subgenomic promoter. The sequence encoding the subgenomic promoter may have at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the sequence shown in SEQ ID NO:14.

[0085] In one embodiment, the modified nsP4 may comprise a GFP protein as a second heterologous protein. The modified nsP4 may comprise an amino acid sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with the reference sequence shown in SEQ ID NO: 12. The modified nsP4 may be encoded by a polynucleotide sequence shown in SEQ ID NO: 13. The modified nsP4 may comprise an amino acid sequence having at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with the reference sequence shown in SEQ ID NO: 15. The modified nsP4 may be encoded by a polynucleotide sequence shown in SEQ ID NO: 16.

[0086] Alphavirus-derived RNA replicon expression system In one embodiment, an alphavirus-derived RNA replicon expression system is provided, which may comprise any one of the synthetic alphavirus-derived replicon nucleic acid molecules described herein and a host cell(s).

[0087] The host cells may be, but are not limited to, hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines such as HEK293 cells, PER.C6 cells. The host cells may be yeast, fungi, insect cells, animal or plant cells. Primary non-transformed normal cells from humans or any mammalian species may be used, such as fibroblasts, stem cells, or cells of lymphoid or myeloid lineage.

[0088] In one embodiment, production of a first heterologous protein and / or a second heterologous protein in a host cell comprises introducing a synthetic alphavirus-derived replicon nucleic acid molecule comprising a nucleic acid sequence encoding the first heterologous protein and / or the second heterologous protein to be expressed in the host cell, culturing the host cell under conditions suitable for expression of the nucleic acid molecule, and allowing expression of the first heterologous protein and / or the second heterologous protein in the host cell.

[0089] For expression in a host cell, the synthetic alphavirus-derived replicon nucleic acid molecule containing a nucleic acid sequence encoding a first heterologous protein and / or a second heterologous protein may be included in an expression cassette that includes regulatory sequences required for or facilitating expression of the nucleic acid sequence. The regulatory sequences may include promoters, enhancers, promoters, and / or polyadenylation signals, and the like. A variety of promoters known in the art may be used to express the nucleic acids disclosed herein in a host cell. The promoter may be a constitutive or inducible promoter. The promoter may be derived from a prokaryotic or eukaryotic organism, or may be artificially designed. The host cell may be cultured in an appropriate culture medium that is commercially available for culturing cells to express the first heterologous protein and / or the second heterologous protein.

[0090] One embodiment provides a host cell containing a synthetic alphavirus-derived replicon nucleic acid molecule that includes a nucleic acid sequence encoding a 3C protease, and optionally also contains one or more polynucleotide constructs encoding an FMDV P1 precursor polypeptide or other polypeptide of interest.

[0091] Method for the production of at least one heterologous protein In one embodiment, a method of producing at least one heterologous protein in a cell is provided, which may comprise expressing in the cell any one of the synthetic alphavirus-derived replicon nucleic acid molecules described herein.

[0092] It is described herein that the nsP gene region of an alphavirus replicon has been successfully edited to reduce its homology with wild-type virus and to incorporate novel biological functions at the early stage of self-amplification of such artificial replicons, which precedes translation of a subgenomically encoded transgene. This may be achieved by identifying critical stretches of nucleotides in the nsP region that can be safely edited without disrupting conserved structural elements, and by adding additional sequences to the native ends of the nsPs. The present disclosure reveals how such modifications can be made without compromising the self-amplification function of the replicon. Thus, the present disclosure provides a method for generating safer replicons with additional functionality for modifying cellular and immunological parameters of host cells.

[0093] In one embodiment, the method may include culturing a host cell using a suitable medium and expressing the 3C protease in the host cell. The method may also include expressing and / or processing the FMDV P1 precursor polypeptide (or other polypeptide of interest) into FMDV viral proteins. The method may include culturing the host cell and recovering viral proteins VP0, VP1, VP2, VP3, or VP4, or other cleavage products of the 3C protease.

[0094] In one embodiment, the method may include culturing a host cell and expressing a synthetic alphavirus-derived replicon nucleic acid molecule containing a nucleic acid sequence encoding the O1 Manisa P1 polyprotein as a first heterologous protein and the 3C protease as a second heterologous protein.

[0095] In one embodiment, the method may include culturing a synthetic alphavirus-derived replicon nucleic acid molecule containing a translation interrupter sequence, such as a 2A sequence. The translation interrupter sequence allows for interruption of protein translation and efficient cleavage at sites not necessarily recognized by the 3C protease, such as sites in the modified nsP4 protein. The 2A sequence may be inserted into the nucleic acid sequence encoding the modified nsP4 prior to the sequence encoding the second heterologous protein. Insertion of the 2A sequence may result in interruption of translation by 2A and production of a polypeptide without an N-terminal Met residue.

[0096] composition In one embodiment, the synthetic alphavirus-derived replicon nucleic acid molecules or heterologous proteins described herein may be administered as a composition.

[0097] In one embodiment, the composition may be a therapeutic for an autoimmune or allergic disease, which may comprise any one of the alphavirus-derived replicon nucleic acid molecules containing a heterologous protein described herein that expresses an agent that inhibits or otherwise reduces the adaptive immune response to a desired target antigen.

[0098] In one embodiment, the composition may include a synthetic alphavirus-derived replicon nucleic acid molecule for expressing a target specific antigen, which may be a tumor antigen. The cancer specific antigen may be derived from any of the following: total mRNA isolated from a target cell(s), one or more specific target mRNA molecules, a protein lysate of a target cell(s), a specific protein from a target cell(s), or a synthetic target specific peptide or protein and synthetic mRNA or DNA encoding the target specific antigen or a peptide derived therefrom. Non-limiting examples are the following cancer or tumor specific antigens: WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2 / neu, idiotype, MAGE A3, p53 (non-mutant), NY-ESO-1, PSMA, GD2, CEA, MelanA / MART1, Ras mutant, gp100, p53 mutant, proteinase 3 (PR1), Bcr-abl, tyrosinase, survivin, PSA, hTERT, Sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, GD3, Fucosyl GM1, mesothelin, PSCA, MAGE A1, sLe(a), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, Tie2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-β, MAD-CT-2, Fos-related antigen 1.

[0099] In one embodiment, the composition may be a cancer therapeutic, which may comprise any one of the alphavirus-derived replicon nucleic acid molecules containing a heterologous protein described herein that expresses an agent that inhibits or otherwise reduces the growth or proliferation of tumor cells in the human or animal body.

[0100] In one embodiment, the composition may comprise a 3C protease. Such a composition may be used to induce or detect an immune response to the 3C protease, such as a humoral or cellular immune response to the 3C protease. The composition may further comprise a precursor polypeptide, such as an FMDV P1 precursor, that contains a site recognized by the modified FMDV 3C protease. The composition may be in a form useful for processing the precursor polypeptide in vitro.

[0101] In one embodiment, the composition may be a vaccine. The vaccine may comprise any one of the alphavirus-derived replicon nucleic acid molecules comprising antigenic proteins described herein. The term "vaccine" refers to a drug or composition containing active ingredients effective to induce some degree of immunity in a subject against a pathogen or disease, the induced immunity resulting in at least a reduction in the severity, duration, or other symptoms of, or elimination of, symptoms associated with infection or disease caused by the pathogen.

[0102] In one embodiment, the synthetic alphavirus-derived replicon nucleic acid molecules disclosed in any one of the embodiments herein may be encapsulated in a carrier system for in vivo administration. The carrier may be, but is not limited to, an anionic liposome, a cationic liposome, or a dendrimer. The term "anionic liposome" refers to a liposome that includes lipids that include anionic groups. Anionic liposomes may be formed by anionic phospholipids. The term "cationic liposome" refers to a liposome that is composed entirely or partially of positively charged lipids, or more specifically, lipids that include both cationic groups and lipophilic moieties. The positively charged portion of the cationic lipids used in cationic liposomes provides advantageous structural features. For example, the lipophilic portion of the cationic lipid is hydrophobic and therefore may orient itself away from the aqueous interior of the liposome and associate with other non-polar, hydrophobic species, or conversely, the cationic portion may associate with polar molecules and species that can form complexes with the cationic portion in the aqueous interior of the cationic liposome. Positively charged liposomes may interact with negatively charged nucleic acid molecules to form stable complexes.

[0103] The liposomes may comprise zwitterionic lipids. As used herein, the term "zwitterionic" refers to a molecule that contains both positive and negative charges but has a net neutral charge.

[0104] Liposomes may be formed from a single lipid or a mixture of lipids. The hydrophilic portion of the lipid can be PEGylated, i.e., modified by covalent attachment of polyethylene glycol, to increase the stability of the liposome and prevent non-specific adsorption (Heyes et al., (2005) J Controlled Release 107:276-87, which is incorporated herein by reference as if set forth in full).

[0105] The term "dendrimer" refers to a highly branched macromolecule having a globular shape. The surface of the dendrimer molecule may be modified in many ways, and many of the properties of the resulting construct may be determined by the surface. Dendrimers may be modified to have a positive surface charge, i.e., to become cationic dendrimers. Cationic dendrimers may transiently associate with nucleic acids. Once the dendrimer-nucleic acid complex has reached its destination, it may then be taken up into the cell via endocytosis.

[0106] Exemplary sizes of single dendrimer-nucleic acid complexes, also referred to herein as modified dendrimer nanoparticles (MDNPs), may range from 30 nm to 1,000 nm in the longest dimension. MDNPs may have an average size of 30 nm to 450 nm, inclusive, 50 nm to 300 nm, inclusive, or even 60 nm to 250 nm, inclusive. MDNPs may be alkyl-modified dendrimer nanoparticles. Nanoparticle size may be influenced by the length of the alkyl chains substituting the core dendrimer. Methods for making and formulating modified dendrimer nanoparticles are described in International Publication No. WO 2021 207020, published October 14, 2021; U.S. Patent Application Publication No. 20210330600, published October 28, 2021, and U.S. Patent Application Publication No. 20210338789, published November 4, 2021. all of which are incorporated by reference herein as if fully set forth.

[0107] In one embodiment, the composition may further comprise one or more adjuvants. The term "adjuvant" refers to one or more substances that cause stimulation of the immune system. Adjuvants may be used to enhance the immune response to heterologous proteins used to immunize a subject against a disease. The one or more adjuvants may include aluminum, for example as aluminum hydroxide and / or aluminum phosphate; oil-emulsion compositions or oil-in-water compositions, such as squalene-water emulsions, e.g., MF59; saponin preparations, e.g., QS21 and immune stimulating complexes (ISCOMs); bacterial or microbial derivatives, such as monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG motif-containing oligonucleotides, ADP-ribosylating bacterial toxins or mutants thereof, e.g., E. coli heat labile enterotoxin LT, cholera toxin CT, nucleic acids and other TLR agonists, such as poly(I:C); eukaryotic proteins, such as antibodies or fragments thereof, and ligands for receptors which stimulate an immune response upon interaction with recipient cells.

[0108] In one embodiment, the compositions described herein may be utilized without an adjuvant.

[0109] In one embodiment, the composition may be administered in a pharma- ceutically acceptable carrier or excipient. As used herein, the term "pharma-ceutically acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium stearate, or zinc stearate, or stearic acid), or solvent encapsulating material, that is involved in carrying or transporting the subject compound from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can serve as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol. (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances used in pharmaceutical formulations.Wetting agents, coloring agents, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives and antioxidants may also be present in the formulation. The terms "excipient", "carrier", "pharmaceutical acceptable carrier" and the like are used interchangeably herein. Pharmaceutically acceptable carriers and excipients are known in the art (Remington's Pharmaceutical Sciences, 18th Edition, AR Gennaro, Mack Publishing Company, 1990; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Taylor & Francis, 2000; Handbook of Pharmaceutical Excipients, 3rd Edition, A. Kibbe, Pharmaceutical Press, 2000). The synthetic alphavirus-derived replicon nucleic acid molecules described herein may be formulated and administered as sterile and / or lyophilized solutions. Sterile solutions may be prepared by sterile filtration or by any other known method. The solution may then be lyophilized or filled into pharmaceutical dosage containers. The pH of the solution may generally range from pH 3.0 to 9.5, for example, pH 5.0 to 7.5. In one embodiment, a stabilizer such as albumin or additional sugar (e.g., sucrose) may be added. In one embodiment, a surfactant may be added. In one embodiment, a nucleic acid mixture containing any of the replicon RNAs described herein may be formulated into an injectable preparation.

[0110] One embodiment provides for the use of a preparation of replicon RNA produced by the methods disclosed herein in the manufacture of a vaccine capable of eliciting an immune response against cancer in a patient in need thereof.

[0111] In one embodiment, a method of inducing an immune response in a subject is provided, which may comprise administering to the subject a therapeutically effective amount of a vaccine comprising any one of the synthetic alphavirus-derived replicon nucleic acid molecules described herein.

[0112] In one embodiment, a method of preventing and / or treating a subject against a disease or condition is provided. The method may comprise utilizing any one of the compositions and vaccines described herein. The method may comprise administering to a subject in need thereof a therapeutically effective amount of a composition or vaccine comprising a synthetic alphavirus-derived replicon nucleic acid molecule as described herein above.

[0113] Administration of the compositions or vaccines described herein may be performed using known routes of administration. The compositions or vaccines may be administered using intradermal, intramuscular, subcutaneous, transdermal, or mucosal administration, such as intranasal, oral, and other parenteral administration. In one embodiment, the composition is administered by intramuscular injection. As used herein, the term "administering" refers to placement of a composition in a subject by a method or route that results in at least partial localization of the composition at a desired site to produce a desired effect. The compounds or compositions described herein may be administered by any suitable route known in the art, including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, or topical (including oral and sublingual) administration.

[0114] Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In one embodiment, the composition may be administered by intravenous infusion or injection.

[0115] The vaccines described herein may be administered by any known route to induce an immune response to the antigen(s) in the vaccine.

[0116] In one embodiment, the method of administration or delivery may not be limited to those described above, and any means for intracellular delivery may be used.

[0117] A therapeutically effective amount refers to an amount of a protein or nucleic acid molecule effective to prevent, ameliorate and / or treat a disease or condition resulting from infection by a pathogen. Prevention encompasses inhibiting or reducing the spread of a pathogen, or inhibiting or reducing the onset, development or progression of one or more symptoms associated with infection by a pathogen. As used herein, amelioration refers to a reduction in visible or perceptible disease symptoms, viremia, or any other measurable sign of infection induced by a pathogen.

[0118] In relation to the treatment of cancer, a "therapeutically effective amount" is an amount effective to prevent further development of cancer or transformed growth, and even to cause regression of cancer or solid tumors.

[0119] Determination of a therapeutically effective amount is generally well within the capabilities of one of ordinary skill in the art. Generally, a therapeutically effective amount may vary depending on the subject's medical history, age, condition, sex, and the severity and type of medical condition in the subject, and the administration of other agents to ameliorate the disease or disorder being treated.

[0120] Toxicity and therapeutic efficacy may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example to determine LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compositions that exhibit large therapeutic indices are preferred. As used herein, the term ED stands for effective dose and is used in conjunction with animal models. The term EC stands for effective concentration and is used in conjunction with in vitro models.

[0121] Data obtained from cell culture assays and animal studies may be used in formulating a range of dosages for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosages may vary within this range depending on the dosage form employed and the route of administration utilized.

[0122] A therapeutically effective dose may be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the therapeutic agent that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Levels in plasma may be measured, for example, by high performance liquid chromatography. The effect of any particular dosage may be monitored by a suitable bioassay.

[0123] Dosage will be determined by the physician and may be adjusted, if necessary, to suit the observed therapeutic effect. In general, the compositions may be administered such that the active agent is given in a dose of 1 μg / kg to 150 mg / kg, 1 μg / kg to 100 mg / kg, 1 μg / kg to 50 mg / kg, 1 μg / kg to 20 mg / kg, 1 μg / kg to 10 mg / kg, 1 μg / kg to 1 mg / kg, 100 μg / kg to 100 mg / kg, 100 μg / kg to 50 mg / kg, 100 μg / kg to 20 mg / kg, 100 μg / kg to 10 mg / kg, 100 μg / kg to 1 mg / kg, 1 mg / kg to 100 mg / kg, 1 mg / kg to 50 mg / kg, 1 mg / kg to 20 mg / kg, 1 mg / kg to 10 mg / kg, 10 mg / kg to 100 mg / kg, 10 mg / kg to 50 mg / kg, or 10 mg / kg to 20 mg / kg. It will be understood that the ranges given herein include all intermediate ranges, for example, a range of 1tmg / kg to 10mg / kg includes 1mg / kg to 2mg / kg, 1mg / kg to 3mg / kg, 1mg / kg to 4mg / kg, 1mg / kg to 5mg / kg, 1mg / kg to 6mg / kg, 1mg / kg to 7mg / kg, 1mg / kg to 8mg / kg, 1mg / kg to 9mg / kg, 2mg / kg to 10mg / kg, 3mg / kg to 10mg / kg, 4mg / kg to 10mg / kg, 5mg / kg to 10mg / kg, 6mg / kg to 10mg / kg, 7mg / kg to 10mg / kg, 8mg / kg to 10mg / kg, 9mg / kg to 10mg / kg, etc. It should be further understood that intermediate ranges within the above ranges, for example, the range of 1 mg / kg to 10 mg / kg, dosage ranges such as 2 mg / kg to 8 mg / kg, 3 mg / kg to 7 mg / kg, 4 mg / kg to 6 mg / kg, etc., are also within the scope of the present invention.

[0124] In one embodiment, the composition may be administered at a dosage such that the active agent has an in vivo concentration of less than 500 nM, less than 400 nM, less than 300 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 25 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM, less than 0.05, less than 0.01 nM, less than 0.005 nM, less than 0.001 nM 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours or more after administration.

[0125] With regard to duration and frequency of treatment, a skilled clinician will typically monitor the subject to determine when the treatment is providing therapeutic benefit, as well as to determine whether to increase or decrease the dosage, increase or decrease the frequency of administration, discontinue treatment, resume treatment, or make other changes to the treatment regimen. The administration schedule may vary from once a week to daily, depending on several clinical factors, such as the subject's sensitivity to the polypeptide. The desired dose may be administered daily, or every 3, 4, 5, or 6 days. The desired dose may be administered all at once, or may be divided into partial doses, e.g., 2-4 partial doses, and administered over a period of time, e.g., at suitable intervals throughout the day, or on other suitable schedules. Such partial doses may be administered as unit dosage forms. In one embodiment, administration may be chronic, e.g., one or more doses daily over a period of weeks or months. Examples of dosing schedules may include daily, twice daily, three times daily, or four times daily or more dosing for a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or longer.

[0126] In one embodiment, a method of preparing a vaccine or therapeutic composition against a disease or condition is provided. The method may comprise providing a vaccine or therapeutic composition as described herein and formulating it into a pharma- ceutically acceptable composition. The vaccine composition may comprise an effective amount of a heterologous immunogenic protein and / or a nucleic acid molecule encoding the protein that provides an immune response against the disease.

[0127] In one embodiment, the disease may be caused by a viral pathogen. The pathogen may be a viral pathogen. The viral pathogen may be an adenovirus, a rhinovirus, a rotavirus, a West Nile virus, a Zika virus, a herpes virus, or a coronavirus (CoV). The coronavirus may be one of the following viruses: MERS CoV, SARS-CoV, and SARS-CoV-2.

[0128] In one embodiment, the disease may be foot and mouth disease (FMD). FMD is an acute and highly contagious viral disease of domestic and wild cloven-hoofed animals, including domestic and wild cattle and pigs. Exemplary susceptible animals include cattle, buffalo, sheep, goats, pigs, antelope, deer and bison, hedgehogs and elephants; llamas and alpacas. In laboratory experiments, mice, rats, and chickens have been successfully infected by artificial means, but are not thought to be susceptible to the disease under natural conditions. Humans are very rarely infected. Symptoms of FMD include a high fever for about 2-6 days, followed by blisters in the mouth and on the feet that can rupture and cause lameness. FMD has a significant impact on livestock production because it is highly infectious and can be spread by infected animals through aerosols, through contact with contaminated agricultural equipment, vehicles, clothing, or feed, as well as by domesticated and wild predators. In particular, symptoms in cattle and pigs caused by FMDV infection. Economic losses from FMD outbreaks are among the highest of all livestock diseases. The causative agent, foot and mouth disease virus (FMDV), is a member of the Picornaviridae family, and thus the viral pathogen is likely a picornavirus. As used herein, the term "picornavirus" refers to non-enveloped viruses that represent a family of small, cytoplasmic, positive-stranded RNA (7.0-8.5 kb) viruses with icosahedral capsids. Picornaviruses are a large viral family that includes multiple genera containing important human and animal pathogens such as enteroviruses (human polioviruses), hepatoviruses (hepatitis A virus), and aphthoviruses (foot and mouth disease viruses, aphthoviruses), with FMDV as the prototypic member.FMDV exists as seven different serotypes (Euroasiatic serotypes A, O, C, and Asia 1 and South African Territory [SAT] serotypes SAT1, SAT2, and SAT3) as well as multiple subtypes reflecting the remarkable genetic variability of the genus. Genera within this family include Aphthovirus, Aquamavirus, Avihepatovirus, Cardiovirus, Cosavirus, Dicipivirus, Enterovirus, Erbovirus, Hepatovirus, Kobuvirus, Megrivirus, Parechovirus, Piscevirus, Salivirus, Sapelovirus, Senecavirus, Teschovirus, and Tremovirus. The viral genome of picornaviruses generally contains one open reading frame encoding a single polyprotein that includes the structural protein region P1 and the nonstructural protein regions P2 and P3. Release of mature functional proteins from the polyprotein is primarily mediated by viral proteinases, including the 3C protease.

[0129] Providing vaccine compositions and immunization regimes The "vaccine" described herein may be provided as a pharmaceutical composition. The composition may include a pharma-ceutically acceptable diluent, carrier or excipient. The composition may include additional active ingredients. Administration may be provided in a single dose or in a prime-boost setting. A prime-boost setting may include a "priming" and "boost" immunization regime in which the immune response induced by a priming (prime) vaccine may be boosted by a booster (boost) vaccine. For example, after priming (at least once) with any one of the polynucleotides encoding a first heterologous protein, a booster vaccine comprising an effective amount of a second heterologous antigenic protein or a subunit thereof may be administered to boost the immune response in the primed host.

[0130] The compositions described herein may be administered to a subject, for example, a human subject. The total dose of heterologous immunogenic protein in the composition for a single administration may be, for example, about 0.01 μg to about 10 mg, for example, 1 μg to 1 mg, for example, 10 μg to 100 μg. Determining the recommended dose may be performed by experimentation, which is routine for those skilled in the art.

[0131] The terms "subject" and "individual" are used interchangeably herein and refer to a human or an animal. Typically, an animal is a vertebrate, such as a primate, a rodent, a domestic or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. A patient or subject includes any subset of the above, such as all of the above, excluding one or more groups or species, such as humans, primates, or rodents. In one embodiment, the subject may be a mammal, such as a primate, such as a human. The terms "patient" and "subject" are used interchangeably herein. The terms "patient" and "subject" are used interchangeably herein. Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, non-human primate, mouse, rat, dog, cat, horse, or cow.

[0132] The following list includes certain embodiments of the invention, but the list is not limiting and does not exclude alternative embodiments or embodiments otherwise described herein. The percent identity listed in the following list of embodiments refers to the identity of the listed sequence along the entire length of the reference sequence.

[0133] Cancer Treatment Methods One embodiment provides a method of treating cancer. The method may include administering to a subject in need thereof a therapeutically effective amount of a composition comprising any one of the immunogenic proteins described herein. The immunogenic protein may include a pattern recognition receptor or an effector protein that stimulates an innate immune response. The immunogenic protein may be an extracellular signaling molecule such as a cytokine, a membrane surface receptor, a membrane channel, or an integrin, or other glycoprotein that affects immune cell recognition. The immunogenic protein may include a STING protein. Stimulator of interferon genes (STING) plays a central role in innate immunity during infection and cancer. STING is endogenously activated by 2',3'-cyclic-GMP-AMP (cGAMP), a cyclic dinucleotide synthesized by cGAMP synthase (cGAS) in response to cytoplasmic DNA as a danger signal. Activation of STING mediates a pleiotropic type I interferon (IFN-I) response that promotes the maturation and migration of dendritic cells and primes cytotoxic T lymphocytes and natural killer cells for spontaneous immune responses.

[0134] In one embodiment, the method of treating the cancer may comprise administering a therapeutically effective amount of any one of the immunogenic proteins described herein to a subject in need thereof.

[0135] In one embodiment, the method may further include analyzing inhibition of tumor growth. The analyzing step may include observing greater than about 60%, greater than 70%, greater than 80%, or greater than about 90% inhibition of tumor growth in the subject.

[0136] A variety of known controlled- or extended-release dosage forms, formulations, and devices may be adapted for use with the immunogenic compositions of the embodiments of the present disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1, all of which are incorporated by reference herein as if fully set forth. These dosage forms may be used to provide a sustained or controlled release of one or more active ingredients using, for example, hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif., USA)), or combinations thereof to provide desired release profiles at various rates.

[0137] As used herein, the term "cancer" refers to the uncontrolled proliferation of cells that may interfere with the normal function of the body's organs and systems. Cancer may be either primary or metastatic cancer, or both. Cancer that migrates from its original location and disseminates to vital organs can ultimately lead to the death of the subject through the functional deterioration of the affected organ. Metastasis is a cancer cell or group of cancer cells distinct from the primary tumor location that results from the dissemination of cancer cells from the primary tumor to other parts of the body. Upon diagnosis of the primary tumor mass, the subject may be monitored for the presence of migration metastasis, e.g., cancer cells in the dissemination process.

[0138] As used herein, the term "cancer" includes, but is not limited to, solid tumors and blood-borne tumors. The term cancer refers to diseases of the skin, tissues, organs, bone, cartilage, blood and blood vessels. The term "cancer" includes malignant epithelial tumors (from the lung, stomach, duodenum, colon, rectum, breast, uterus, prostate, bladder, etc.).

[0139] The term "cancer" further encompasses primary and metastatic cancers. Examples of cancers that can be treated by the methods of the present invention include solid tumors; melanoma; brain cancers, including but not limited to glioma, glioblastoma, glioblastoma multiforme (GBM), oligodendroglioma, primitive neuroectodermal tumor, low-grade, intermediate-grade and high-grade astrocytoma, ependymoma (e.g., myxopapillary ependymoma, papillary ependymoma, subependymoma, anaplastic ependymoma), oligodendroglioma, medulloblastoma, meningioma, pituitary adenoma, neuroblastoma and craniopharyngioma; intraductal carcinoma in situ, invasive (or infiltrating) ductal carcinoma, invasive (or infiltrating) lobular carcinoma, adenoid cystic carcinoma (or adenoid cystic carcinoma), and cystic carcinoma (or adenoid cystic carcinoma). carcinoma), low-grade adenosquamous carcinoma, medullary carcinoma, mucinous carcinoma (or colloid carcinoma), papillary carcinoma, tubular carcinoma, inflammatory breast cancer, Paget's disease of the nipple, phyllodes tumor, triple-negative breast cancer, metastatic breast cancer; cancers including bladder cancer, breast cancer, colon cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, cervical cancer, thyroid cancer, and skin cancer, including squamous cell carcinoma; other tumors including seminoma, teratocarcinoma; tumors of the central and peripheral nervous system; and other tumors including, but not limited to, xeroderma pigmentosum, keratoacanthoma, follicular thyroid carcinoma, and teratocarcinoma.

[0140] The methods disclosed herein are useful for treating patients who have been previously treated for cancer, as well as patients who have not been previously treated for cancer, and indeed, the methods and compositions described herein may be used in first and second line cancer treatments.

[0141] As used herein, the term "precancerous condition" has its usual meaning, i.e., unregulated growth without metastasis, and includes various forms of hyperplasia and benign hypertrophy. Thus, a "precancerous condition" is a disease, syndrome, or finding that can lead to cancer if left untreated. It is a common condition associated with a significant increase in the risk of cancer. A premalignant lesion is a morphologically altered tissue that is more likely to develop cancer than its apparently normal counterpart. Examples of premalignant conditions include, but are not limited to, oral leukoplakia, actinic keratosis (solar keratosis), Barrett's esophagus, atrophic gastritis, benign hyperplasia of the prostate, precancerous polyps of the colon or rectum, gastric epithelial dysplasia, adenomatous dysplasia, hereditary nonpolyposis colon cancer syndrome (HNPCC), Barrett's esophagus, bladder dysplasia, precancerous cervical conditions, and cervical dysplasia.

[0142] In one embodiment, the cancer may be selected from the group consisting of: breast cancer; ovarian cancer; brain cancer; gastrointestinal cancer; prostate cancer; carcinoma, lung carcinoma, hepatocellular carcinoma, testicular cancer; cervical cancer; endometrial cancer; bladder cancer; head and neck cancer; lung cancer; gastroesophageal cancer, and gynecological cancer.

[0143] In one embodiment, the cancer may be breast cancer, including but not limited to intraductal carcinoma in situ, invasive (or infiltrating) ductal carcinoma, invasive (or infiltrating) lobular carcinoma, adenoid cystic carcinoma (or adenocystic carcinoma), low-grade adenosquamous carcinoma, medullary carcinoma, mucinous carcinoma (or colloid carcinoma), papillary carcinoma, tubular carcinoma, inflammatory breast cancer, Paget's disease of the nipple, phyllodes tumor, triple-negative breast cancer, metastatic breast cancer.

[0144] In one embodiment, the methods described herein may relate to treating a subject who has cancer or has been diagnosed with cancer. A subject who has cancer may be identified by a physician using current methods of diagnosing cancer. Symptoms and / or complications of cancer that characterize these conditions and aid in diagnosis are well known in the art and may include, but are not limited to, tumor growth, dysfunction of organs or tissues that have cancer cells, and the like. For example, tests that may aid in the diagnosis of cancer include, but are not limited to, tissue biopsy and histological examination. Family history of cancer or exposure to risk factors for cancer (e.g., tobacco products, radiation, etc.) may also aid in determining whether a subject is likely to have cancer or in making a diagnosis of cancer.

[0145] The following list includes certain embodiments of the invention, but the list is not limiting and does not exclude alternative embodiments or embodiments otherwise described herein. The percent identity listed in the following list of embodiments refers to the identity of the listed sequence along the entire length of the reference sequence.

[0146] Embodiment 1. A synthetic alphavirus-derived replicon nucleic acid molecule comprising: (i) a first nucleic acid encoding alphavirus nonstructural proteins nsP1, nsP2, nsP3, and nsP4 and comprising at least one silent mutation introduced at any position within the regions nt503 to nt658, nt658 to nt1620, nt1620 to nt2560, nt2560 to nt3954, nt3954 to nt4120, nt6381 to nt7083, and nt6966 to nt7526 in the alphavirus genome sequence set forth in SEQ ID NO:17; and (ii) a second nucleic acid comprising a modified subgenomic open reading frame (ORF). 2. A synthetic alphavirus-derived replicon nucleic acid molecule as described in embodiment 1, wherein said modified subgenomic ORF comprises a sequence encoding a first heterologous protein. 3. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or both of embodiment 1 and embodiment 2, wherein the first nucleic acid comprises at least one silent mutation introduced at any position within the nt503 to nt658 region of the alphavirus genome. 4. A synthetic alphavirus-derived replicon nucleic acid molecule described in any one or more of embodiments 1 to 3, wherein the first nucleic acid comprises a polynucleotide encoding nsP1 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:21. 5. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 4, wherein the first nucleic acid comprises at least one silent mutation introduced at any position within the region from nt 658 to nt 1620 of the alphavirus genome. 6. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 5, wherein the first nucleic acid comprises a polynucleotide encoding nsP1 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:25. 7. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 6, wherein the first nucleic acid comprises at least one silent mutation introduced at any position within the region from nt 1620 to nt 2560 of the alphavirus genome. 8. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 7, wherein the first nucleic acid comprises a polynucleotide encoding the junction of nsP1 and nsP2 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:24. 9. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 8, wherein the first nucleic acid comprises at least one silent mutation introduced at any position within the region from nt3694 to nt3954 of the alphavirus genome. 10. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 9, wherein the first nucleic acid comprises a polynucleotide encoding the junction of nsP1 and nsP2 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 22. 11. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 10, wherein the first nucleic acid comprises at least one silent mutation introduced at any position within the region from nt 2560 to nt 3954 of the alphavirus genome. 12. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 11, wherein the first nucleic acid comprises a polynucleotide encoding the junction of nsP1 and nsP2 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:26. 13. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 12, wherein the first nucleic acid comprises at least one silent mutation introduced at any position within the region from nt3954 to nt4120 of the alphavirus genome. 14. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 13, wherein the first nucleic acid comprises a polynucleotide encoding the junction of nsP2 and nsP3 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 23. 15. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 15, wherein the first nucleic acid comprises at least one silent mutation introduced at any position within the region from nt6381 to nt7083 of the alphavirus genome. 16. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 15, wherein the first nucleic acid comprises a polynucleotide encoding nsP4 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:20. 17. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 16, wherein the first nucleic acid comprises at least one silent mutation introduced at any position within the regions nt658 to nt1620 and nt2560 to nt3954 of the alphavirus genome. 18. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 17, wherein the first nucleic acid comprises a polynucleotide encoding nsP1 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 25, and a polynucleotide encoding nsP2 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 26. 19. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 18, wherein the first nucleic acid comprises at least one silent mutation introduced at any position within the regions nt3694 to nt3954 and nt6381 to nt7083 of the alphavirus genome. 20. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 19, wherein the first nucleic acid comprises a polynucleotide encoding nsP2 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 22, and a polynucleotide encoding nsP4 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 20. 21. The synthetic alphavirus-derived replicon nucleic acid molecule according to one or more of embodiments 1 to 20, wherein the first nucleic acid comprises a polynucleotide encoding nsP1 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:21, a polynucleotide encoding nsP1 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:25, a polynucleotide encoding a junction of nsP1 and nsP2 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:24, a polynucleotide encoding nsP2 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:26, a polynucleotide encoding a junction of nsP2 and nsP3 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:23; and a polynucleotide encoding nsP4 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:20. 22. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 21, wherein the first nucleic acid comprises a polynucleotide encoding the nonstructural protein and comprising a polynucleotide sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 30. 23. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 22, wherein the first nucleic acid molecule comprises at least one silent mutation introduced at any position within the region from nt6966 to nt7523 of the alphavirus genome. 24. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 23, wherein the first nucleic acid molecule comprises a polynucleotide sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 18. 25. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 24, wherein the first nucleic acid comprises at least one silent mutation introduced into the polynucleotide encoding the nsP4 protein. 26. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 25, wherein the first nucleic acid molecule comprises a polynucleotide sequence having at least 90% identity to the reference sequence set forth in SEQ ID NO:19. 27. A synthetic alphavirus-derived replicon nucleic acid molecule according to one or more of embodiments 1 to 26, wherein the first nucleic acid comprises a polynucleotide encoding a modified nsP4 comprising a second heterologous protein. 28. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 27, wherein the polynucleotide encoding the modified nsP4 comprises a polynucleotide sequence encoding a first C-terminal region and a polynucleotide sequence encoding a second C-terminal region. 29. A synthetic alphavirus-derived replicon nucleic acid molecule of one or more of embodiments 1 to 28, wherein said second C-terminal region is translatable into a protein. 30. A synthetic alphavirus-derived replicon nucleic acid molecule of one or more of embodiments 1 to 28, wherein said second C-terminal region is not translatable into a protein. 31. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 28, wherein the polynucleotide sequence encoding the first C-terminal region is different from the polynucleotide sequence encoding the second C-terminal region. 32. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 31, wherein the second heterologous protein comprises a GFP protein and the modified nsP4 comprises an amino acid sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 41. 33. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 32, wherein the modified nsP4 is encoded by a polynucleotide sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:31. 34. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 33, wherein the first heterologous protein or the second heterologous protein comprises a viral or mammalian cell-derived regulator of a cellular metabolic, immune, or signal transduction function. 35. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 34, wherein the first heterologous protein or the second heterologous protein affects a cellular antiviral innate immune response and enhances the replication of the alphavirus replicon RNA molecule. 36. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 35, wherein the first heterologous protein or the second heterologous protein comprises an antigenic protein isolated from or derived from a viral pathogen. 37. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 36, wherein the viral pathogen is selected from the group consisting of picornavirus, adenovirus, rhinovirus, rotavirus, West Nile virus, Zika virus, herpes, or coronavirus (CoV). 38. The synthetic alphavirus-derived replicon nucleic acid molecule of one or more of embodiments 1 to 37, wherein said viral pathogen is a picornavirus. 39. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 35, wherein the second heterologous protein comprises a 3C protease protein and the modified nsP4 comprises an amino acid sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 40. 40. A synthetic alphavirus-derived replicon nucleic acid molecule described in one or more of embodiments 1 to 35 and embodiment 39, wherein the modified nsP4 is encoded by a polynucleotide sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 32. 41. A synthetic alphavirus-derived replicon nucleic acid molecule of a molecule according to one or more of embodiments 1 to 35 and embodiments 39 to 40, wherein said first heterologous protein comprises an O1 Manisa P1 protein. 42. A synthetic alphavirus-derived replicon nucleic acid molecule of a molecule described in one or more of embodiments 1 to 35 and embodiments 39 to 41, wherein the first heterologous protein comprises O1 Manisa P1 protein and the second heterologous protein comprises 3C protease. 43. A synthetic alphavirus-derived replicon nucleic acid molecule of a molecule described in one or more of embodiments 1 to 35, wherein the second heterologous protein comprises a STING protein and the modified nsP4 comprises an amino acid sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 42. 44. A synthetic alphavirus-derived replicon nucleic acid molecule of a molecule described in one or more of embodiments 1 to 35 and embodiment 44, wherein the first heterologous protein comprises an antigen protein and the second heterologous protein comprises a STING protein. 45. A nucleic acid comprising a DNA sequence encoding a synthetic alphavirus-derived replicon nucleic acid molecule comprising: (i) a first nucleic acid encoding alphavirus nonstructural proteins nsP1, nsP2, nsP3, and nsP4 and comprising at least one silent mutation introduced at any position within the regions nt503 to nt658, nt658 to nt1620, nt1620 to nt2560, nt2560 to nt3954, nt3954 to nt4120, nt6381 to nt7083, and nt6966 to nt7526 in the sequence of the alphavirus genome set forth in SEQ ID NO:17; and (ii) a second nucleic acid comprising a modified subgenomic open reading frame (ORF). 46. ​​The nucleic acid of embodiment 45, wherein the modified subgenomic ORF comprises a sequence encoding a first heterologous protein. 47. A nucleic acid described in one or both of embodiments 45 and 46, wherein the first nucleic acid comprises at least one silent mutation introduced at any position within the region from nt503 to nt658 of the alphavirus genome. 48. A nucleic acid described in any one or more of embodiments 45 to 47, wherein the first nucleic acid comprises a polynucleotide encoding nsP1 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:2. 49. A nucleic acid described in any one or more of embodiments 45 to 48, wherein the first nucleic acid comprises at least one silent mutation introduced at any position within the region from nt 658 to nt 1620 of the alphavirus genome. 50. A nucleic acid described in any one or more of embodiments 45 to 49, wherein the first nucleic acid comprises a polynucleotide encoding nsP1 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 6. 51. A nucleic acid described in any one or more of embodiments 45 to 50, wherein the first nucleic acid comprises at least one silent mutation introduced at any position within the region from nt 1620 to nt 2560 of the alphavirus genome. 52. A nucleic acid described in any one or more of embodiments 45 to 51, wherein the first nucleic acid comprises a polynucleotide encoding the junction of nsP1 and nsP2 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:5. 53. A nucleic acid described in any one or more of embodiments 45 to 52, wherein the first nucleic acid comprises at least one silent mutation introduced at any position within the region from nt 3694 to nt 3954 of the alphavirus genome. 54. A nucleic acid described in any one or more of embodiments 45 to 53, wherein the first nucleic acid comprises a polynucleotide encoding the junction of nsP1 and nsP2 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 3. 55. A nucleic acid described in any one or more of embodiments 45 to 54, wherein the first nucleic acid comprises at least one silent mutation introduced at any position within the region from nt 2560 to nt 3954 of the alphavirus genome. 56. A nucleic acid described in any one or more of embodiments 45 to 55, wherein the first nucleic acid comprises a polynucleotide encoding the junction of nsP1 and nsP2 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 7. 57. A nucleic acid described in any one or more of embodiments 45 to 57, wherein the first nucleic acid comprises at least one silent mutation introduced at any position within the region from nt3954 to nt4120 of the alphavirus genome. 58. A nucleic acid described in any one or more of embodiments 45 to 57, wherein the first nucleic acid comprises a polynucleotide encoding the junction of nsP2 and nsP3 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 4. 59. A nucleic acid described in any one or more of embodiments 45 to 58, wherein the first nucleic acid comprises at least one silent mutation introduced at any position within the region from nt6381 to nt7083 of the alphavirus genome. 60. A nucleic acid described in any one or more of embodiments 45 to 59, wherein the first nucleic acid comprises a polynucleotide encoding nsP4 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:1. 61. A nucleic acid described in any one or more of embodiments 45 to 60, wherein the first nucleic acid comprises at least one silent mutation introduced at any position within the regions of nt 658 to nt 1620 and nt 2560 to nt 3954 of the alphavirus genome. 62. The nucleic acid according to any one or more of embodiments 45 to 61, wherein the first nucleic acid comprises a polynucleotide encoding nsP1 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 6, and a polynucleotide encoding nsP2 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 7. 63. A nucleic acid described in any one or more of embodiments 45 to 63, wherein the first nucleic acid comprises at least one silent mutation introduced at any position within the regions nt3694 to nt3954 and nt6381 to nt7083 of the alphavirus genome. 64. The nucleic acid according to any one or more of embodiments 45 to 63, wherein the first nucleic acid comprises a polynucleotide encoding nsP2 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 3, and a polynucleotide encoding nsP4 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 1. 65. The nucleic acid according to any one or more of embodiments 45 to 64, wherein the first nucleic acid comprises a polynucleotide encoding nsP1 and comprising a sequence having at least 90% identity with the reference sequence shown in SEQ ID NO: 2, a polynucleotide encoding nsP1 and comprising a sequence having at least 90% identity with the reference sequence shown in SEQ ID NO: 6, a polynucleotide encoding a junction of nsP1 and nsP2 and comprising a sequence having at least 90% identity with the reference sequence shown in SEQ ID NO: 5, a polynucleotide encoding nsP2 and comprising a sequence having at least 90% identity with the reference sequence shown in SEQ ID NO: 7, a polynucleotide encoding a junction of nsP2 and nsP3 and comprising a sequence having at least 90% identity with the reference sequence shown in SEQ ID NO: 4, and a polynucleotide encoding nsP4 and comprising a sequence having at least 90% identity with the reference sequence shown in SEQ ID NO: 1. 66. A nucleic acid described in any one or more of embodiments 45 to 65, wherein the first nucleic acid comprises a polynucleotide encoding the nonstructural protein and comprising a polynucleotide sequence having at least 90% identity to the reference sequence set forth in SEQ ID NO:11. 67. A nucleic acid described in any one or more of embodiments 45 to 66, wherein the first nucleic acid comprises a polynucleotide encoding a modified nsP4 comprising a second heterologous protein. 68. A nucleic acid described in any one or more of embodiments 45 to 67, wherein the polynucleotide encoding the modified nsP4 comprises a polynucleotide sequence encoding a first C-terminal region and a polynucleotide sequence encoding a second C-terminal region. 69. The nucleic acid of any one or more of embodiments 45 to 68, wherein the second C-terminal region comprises a subgenomic promoter. 70. A nucleic acid described in any one or more of embodiments 45 to 69, wherein the subgenomic promoter is encoded by a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 14. 71. The nucleic acid of any one or more of embodiments 45 to 70, wherein the second C-terminal region is translatable into a protein. 72. The nucleic acid of any one or more of embodiments 45 to 70, wherein the second C-terminal region is not translatable into a protein. 73. The nucleic acid of any one or more of embodiments 45 to 70, wherein the polynucleotide sequence encoding the first C-terminal region is different from the polynucleotide sequence encoding the second C-terminal region. 74. A nucleic acid described in any one or more of embodiments 45 to 73, wherein the second heterologous protein comprises a GFP protein and the modified nsP4 comprises an amino acid sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 12. 75. A nucleic acid described in any one or more of embodiments 45 to 74, wherein the modified nsP4 is encoded by the polynucleotide sequence shown in SEQ ID NO: 13. 76. A nucleic acid described in any one or more of embodiments 45 to 75, wherein the second heterologous protein comprises a GFP protein and the modified nsP4 comprises an amino acid sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 15. 77. A nucleic acid described in any one or more of embodiments 45 to 76, wherein the modified nsP4 is encoded by the polynucleotide sequence shown in SEQ ID NO: 16. 78. The nucleic acid of any one or more of embodiments 45 to 73, wherein the first heterologous protein or the second heterologous protein comprises a viral or mammalian cell-derived regulator of a cellular metabolic, immune, or signal transduction function. 79. A nucleic acid described in any one or more of embodiments 45 to 73, wherein the first heterologous protein or the second heterologous protein affects a cellular antiviral innate immune response and enhances the replication of an alphavirus replicon RNA molecule. 80. The nucleic acid of any one or more of embodiments 45 to 73, wherein the first heterologous protein and / or the second heterologous protein comprises an antigenic protein isolated from or derived from a viral pathogen. 81. The nucleic acid of any one or more of embodiments 45 to 73 and 80, wherein the viral pathogen is selected from the group consisting of picornavirus, adenovirus, rhinovirus, rotavirus, West Nile virus, Zika virus, herpes, or coronavirus (CoV). 82. The nucleic acid of any one or more of embodiments 45 to 73 and embodiments 80 to 81, wherein the viral pathogen is a picornavirus. 83. The nucleic acid described in any one or more of embodiments 45 to 73 and embodiments 80 to 82, wherein the first heterologous protein comprises O1 Manisa P1 protein and / or the second heterologous protein comprises 3C protease. 84. A nucleic acid described in any one or more of embodiments 45 to 73, wherein the first heterologous protein and / or the second heterologous protein comprises a STING protein, and the first heterologous protein comprises an antigen protein. 85. An alphavirus-derived RNA replicon expression system comprising a synthetic alphavirus-derived replicon nucleic acid molecule described in any one or more of embodiments 1 to 44, and a host cell. 86. A pharma- ceutically acceptable composition comprising an alphavirus-derived replicon described in any one or more of embodiments 1 to 44, and a pharma- ceutically acceptable carrier or excipient. 87. A method for producing at least one heterologous protein in a cell, comprising expressing in a host cell a synthetic alphavirus-derived replicon nucleic acid molecule described in any one or more of embodiments 1 to 44. 88. A method for preventing, inhibiting, or treating a symptom of a disease or condition in a subject, comprising: (i) a first nucleic acid encoding alphavirus nonstructural proteins nsP1, nsP2, nsP3, and nsP4, the first nucleic acid comprising at least one silent mutation introduced at any position within the regions nt503 to nt658, nt658 to nt1620, nt1620 to nt2560, nt2560 to nt3954, nt3954 to nt4120, nt6381 to nt7083, and nt6966 to nt7526 in the sequence of the alphavirus genome set forth in SEQ ID NO:17; and (ii) a second nucleic acid comprising a modified subgenomic open reading frame (ORF); providing a synthetic alphavirus-derived replicon nucleic acid molecule comprising: administering to a subject a therapeutically effective amount of said synthetic alphavirus-derived replicon nucleic acid molecule; A method comprising: 89. The method of embodiment 88, wherein the modified subgenomic ORF comprises a sequence encoding a first heterologous protein. 90. A method according to one or both of embodiments 88 and 89, wherein the first nucleic acid comprises at least one sequence having at least 90% identity to a reference sequence selected from the group consisting of SEQ ID NO: 20 to SEQ ID NO: 30. 91. The method of any one or more of embodiments 88 to 90, wherein the first nucleic acid comprises a polynucleotide encoding a modified nsP4 that comprises a second heterologous protein. 92. A method according to any one or more of embodiments 88 to 91, wherein the polynucleotide encoding the modified nsP4 comprises a polynucleotide sequence encoding a first C-terminal region and a polynucleotide sequence encoding a second C-terminal region. 93. The method of any one or more of embodiments 88 to 92, wherein the second C-terminal region is translatable into a protein. 94. The method of any one or more of embodiments 88 to 92, wherein the second C-terminal region is not translatable into a protein. 95. A method according to any one or more of embodiments 88 to 92, wherein the polynucleotide sequence encoding the first C-terminal region is different from the polynucleotide sequence encoding the second C-terminal region. 96. The method of any one or more of embodiments 88 to 95, wherein the first heterologous protein or the second heterologous protein comprises a viral or mammalian cell-derived regulator of a cellular metabolic, immune, or signal transduction function. 97. A method according to any one or more of embodiments 88 to 95, wherein the first heterologous protein or the second heterologous protein affects a cellular antiviral innate immune response and enhances the replication of an alphavirus replicon RNA molecule. 98. The method of any one or more of embodiments 88 to 95, wherein the first heterologous protein or the second heterologous protein comprises an antigenic protein isolated from or derived from a viral pathogen. 99. The method of any one or more of embodiments 88 to 95 and embodiment 98, wherein the viral pathogen is selected from the group consisting of picornavirus, adenovirus, rhinovirus, rotavirus, West Nile virus, Zika virus, herpes, or coronavirus (CoV). 100. The method of any one or more of embodiments 88 to 95 and embodiments 98 to 99, wherein the viral pathogen is a picornavirus. 101. The method of any one or more of embodiments 88 to 95 and embodiments 98 to 100, wherein the first heterologous protein comprises O1 Manisa P1 protein and the second heterologous protein comprises 3C protease. 102. A method according to any one or more of embodiments 88 to 95, wherein the first heterologous protein or the second heterologous protein comprises a STING protein. 103. A method according to any one or more of embodiments 88 to 95 and embodiment 102, wherein the second heterologous protein comprises a STING protein and the first heterologous protein comprises an antigen protein. 104. A method according to any one or more of embodiments 88 to 104, wherein the administering step results in preventing the disease, treating the disease, reducing the severity of the disease, or slowing the progression of the disease in the subject. 105. The method according to any one or more of embodiments 88 to 95 and embodiments 103 to 104, wherein the disease is cancer. 106. The method of any one or more of embodiments 88 to 95 and embodiments 103 to 105, wherein the cancer is melanoma or an epithelial tumor. 107. A method according to any one or more of embodiments 88 to 95 and embodiments 103 to 106, wherein the method further comprises co-administering one or more additional anti-cancer therapies to the subject. 108. The method according to any one or more of embodiments 88 to 95 and embodiments 103 to 107, wherein the additional anticancer therapy is selected from the group consisting of surgery, chemotherapy, radiation therapy, hyperthermia, immunotherapy, hormonal therapy, laser therapy, antiangiogenic therapy, and any combination thereof. 109. The method according to any one or more of embodiments 88 to 95 and embodiments 98 to 101, wherein the disease is FMD. 110. The method of any one or more of embodiments 88 to 109, wherein the subject is a mammal. 111. The method of any one or more of embodiments 88 to 110, wherein the mammal is an even-toed animal or a human. 112. The method of any one or more of embodiments 88 to 111, wherein the cloven-hoofed animal is selected from the group consisting of cows, buffaloes, sheep, goats, pigs, antelopes, deer, bisons, hedgehogs, elephants, llamas, and alpacas.

[0147] Further embodiments herein may be formed by supplementing an embodiment with one or more elements from any one or more other embodiments herein and / or by substituting one or more elements from an embodiment with one or more elements from one or more other embodiments. EXAMPLES

[0148] The following non-limiting examples are provided to illustrate certain embodiments. Throughout, an embodiment may be supplemented with one or more details from one or more of the following examples, and / or one or more elements from an embodiment may be substituted with one or more details from one or more of the following examples.

[0149] Example 1. Design of nsP4 engineered replicons FIG. 1 shows the steps of gene expression of the alphavirus genome nonstructural polyproteins (nsPs; nsP1-nsP2-nsP3-nsP4). The figure shows that expression of the nonstructural polyproteins is a prerequisite for genome synthesis and subsequent expression of the subgenomic ORFs (SG ORFs) that code for the structural polyproteins in the native virus. In the figure, nsPs are shown to contain an early stop codon present in wild-type VEEV, which results in the termination of translation of the nsP-1-2-3 polyproteins. nsP4 is translated by low-frequency translational read-through of an amber stop codon (UGA) at genomic position 5682, incorporating arginine, cysteine ​​or tryptophan. Subsequent proteolysis of the polyprotein occurs, which results in the separated nsP1, nsP2, nsP3 and nsP4 proteins.

[0150] The "early stage" of viral infection refers to the period after infection characterized primarily by expression of the nsP polyproteins before genome replication occurs, whereas the "late stage" is defined by the gene expression pattern after genome replication has occurred and expression of the subgenomic polyproteins becomes the predominant viral gene product.

[0151] It is reported herein that the nsP gene region of an alphavirus replicon has been successfully edited to reduce its homology with the wild-type virus and to incorporate novel biological functions at the early stage of self-amplification of such artificial replicons, which precedes the translation of a subgenomically encoded transgene. This is achieved by identifying key stretches of nucleotides in the nsP region that can be safely edited without disrupting conserved structural elements, and by adding additional sequences to the native ends of the nsPs. The present disclosure shows how such modifications can be made without compromising the self-amplification function of the replicon. Thus, the present disclosure provides a new method for generating safer replicons with additional functionality for modifying cellular and immunological parameters of host cells.

[0152] Alphavirus replicons are currently used primarily as vaccine vectors, where an antigen of interest is encoded in a subgenomic ORF to replace the native alphavirus structural proteins that would normally constitute the virion particle. Currently, if an auxiliary biological function is desired in the form of an additional polypeptide (e.g., a reporter gene, an immune regulator, or other host cell function regulator), the auxiliary factor must be (i) encoded in the same subgenomic ORF as the first transgene by fusion of the coding sequence, (ii) encoded under the control of a separate RNA transcription / translation promoter such as a duplicate subgenomic promoter (SGP) or an internal ribosome entry site (IRES), or (iii) provided in trans separately from the replicon by mixing additional nucleic acid or protein species with the replicon RNA product. Method (i) requires modification of the functional polypeptide, which is not always possible for a particular antigen without modifying its primary sequence. Method (ii) requires the addition of extensive extra nucleotide sequences to generate the SGP or IRES in addition to the desired auxiliary ORF. Both the use of additional SGPs as approaches in methods (i) and (ii) only allow the expression of auxiliary factors simultaneously with the subgenomic ORFs at later stages of the viral replication cycle, but cannot affect replicon function at earlier stages. Method (iii) requires the production of an entirely additional biological product, whose co-delivery with the replicon RNA must be addressed by some means. Currently, replicon nsP sequences essentially identical to circulating endemic alphaviruses are used, which increases the risk of recombination when host cells are also co-infected with another closely related alphavirus genome, e.g., a circulating pathogenic wild-type virus strain. Such recombination poses the threat of generating viable replicating viruses and raises environmental safety concerns. A replicon with reduced homology to wild-type native virus strains would have the advantage of being more safe.

[0153] The challenge of (a) sequence homology with the native virus and (b) means of including auxiliary early biological functions is addressed by specifically editing the native coding sequence of the alphavirus genome such that important primary and secondary structural elements are purposely left intact. By analyzing the pre-edited region to detect conserved primary and secondary sequence elements predicted to have specific functionality (e.g., as shown in Figure 6), the native codons in the nsP coding region can be altered to result in silent mutations that result in the same protein product and do not impair the functionality of the replicon. Extending this technology, the C-terminus of the nsP polyprotein can be edited to allow the addition of a new transgene x by fusion to the nsP coding sequence, again without impairing the functionality of the replicon. Duplication of necessary non-coding elements in the 3' region of the nsP ORF is performed to recreate the SGP to ensure SG ORF expression, and silent codon changes are made in the upstream copy to create novel nsP-x fusion proteins and to eliminate spurious homologies between the replication sites that would inhibit propagation of DNA templates for such constructs in standard bacterial production strains.

[0154] Figure 2 shows the genetic process of an alphavirus genome nonstructural polyprotein (nsP1-nsP2-nsP3-nsP4-xP) engineered to express an additional exogenous protein (commonly "xP") by in-frame addition with nsP4. The diagram shows that the exogenous protein xP is introduced into the gene encoding the nsP4 segment of the nsP polyprotein, which results in the translation and proteolysis of the nsP4 and xP proteins joined by the junction. This nsP4-xP junction may contain a peptide linker sequence that is non-cleavable, autocleavable (e.g., 2A peptide), or proteolytically cleavable (e.g., nsP2 protease or furin recognition site).

[0155] Figure 3 is a schematic depicting the steps that can be taken to genetically modify an alphavirus replicon RNA sequence to generate a construct such as that described in Figure 2. As shown in Figure 3, the desired insertion location is within the subgenomic promoter (SGP) region, and step 1 involves duplication of a portion of the SGP region. Step 2 involves insertion of a sequence encoding the xP protein within the duplicated region. This method results in proximal identical sequences due to duplication of the SGP, which makes the construct genetically unstable. A solution to this problem is found in implementing silent codon changes in the upstream duplicated element that eliminates the problematic homology.

[0156] Figure 4 shows the optimal steps for cloning the modifiable nsP4 open reading frame (ORF) while maintaining an intact extended subgenomic promoter. As shown in Figure 4, step 1 involves inserting the synthetic nsP4 terminal region into the SGP region (gray box) using different codon-optimized selections and incorporating terminal restriction sites. Step 2 involves inserting the sequence encoding the xP protein between the synthetic nsP4 terminal region and the original nsP4 C-terminal coding sequence (non-translated coding sequence CDS, striped box). This method does not result in any proximal identical sequence stretches and generates a genetically stable construct. This approach eliminates problematic repetitive sequence elements and the DNA template can be easily propagated and produced in common bacterial strains. This modification of nsP4 to add transgene x can be performed in combination with other silent mutations in the nsP region described herein to create further improved replicon RNAs.

[0157] Example 2. VEEV replicon RNA modified to have reduced homology at nsP1 within nucleotide positions 45-260 of the genome To demonstrate the sensitivity of alphavirus nsPs to arbitrary nucleotide sequence alterations, a series of silent mutations were substituted across the beginning of the nsP1 protein, corresponding to genomic nucleotide positions 45 to 260, as shown in Figure 5. Figure 5 is a schematic diagram of the DNA plasmid encoding the replicon sequence template for construct 8 (C08).

[0158] In this diagram, the replicon sequence is derived from the Venezuelan Equine Encephalitis Virus (VEEV) species of alphavirus. The DNA encodes an ampicillin resistance gene (AmpR) to facilitate specific production in bacterial cell culture, an origin of replication (ori) to allow growth in bacterial cells, and a DNA template for an engineered VEEV replicon (containing the viral genomic 5'UTR, nsPs, subgenomic promoter and 5'UTR, gene of interest in the subgenomic ORF, genomic 3'UTR, and poly(A) tail) that can be transcribed into RNA by run-off transcription initiated at the T7 promoter. Codons at genomic nucleotide positions 45-260 (grey box) were altered to reduce homology with wild-type virus. This 45-260 nt coding region contains the alphavirus conserved sequence element (CSE) required for RNA replication in cells. The CSE, known to be required for replicon function, has been disrupted at the secondary structure level by silent mutations (codon adaptation) in this construct. Therefore, this construct served as a negative control. In total, the silent mutations reduced the sequence identity of the nsP1 CDS to the wild type by 19.04%. Transcription of this plasmid from the T7 promoter produces the C08 replicon RNA. The 5'UTR, nsP1-4 ORFs, and SGPs of C08 are shown in SEQ ID NO:27.

[0159] This C08 region contains a well-established alphavirus conserved sequence element (CSE), a 51 nt nucleotide span that folds into a regular stem-loop. This secondary structure motif is required for genome replication and therefore transgene expression from the subgenomic ORF. Transfection of BHK cells in vitro confirmed that this RNA (C08) was unable to mediate expression of the reporter gene, secreted placental alkaline phosphatase, encoded in the subgenomic ORF (shown in Figure 17A).

[0160] Example 3. VEEV replicon RNA modified to have reduced homology at nsP1 within nucleotide positions 503-658 of the genome In contrast to construct C08 described in Example 1, a different nsP1 ORF modification was made in the region downstream of the CSE, where a bioinformatics examination of local RNA folding based on thermodynamic parameters was used to select a region of unstructured nucleotides, as shown in Figure 6, and thus a series of silent mutations were substituted across that region to generate the construct called C02, as shown in Figure 7.

[0161] Figure 6 is a schematic diagram depicting an exemplary process used to select regions of an alphavirus genome that can be altered by applying silent mutations that reduce homology to the wild-type virus without reducing the replicative function of the replicon. In this figure, the RNA secondary structure of the region spanning the BsiWI to EcoNI restriction fragment of the wild-type VEEV genome (reference sequence L01442 in the NCBI GenBank database) is shown as predicted by calculation of the minimum free energy of hybridization at 77°C. Boxed in the figure are polynucleotide stretches involved in local (i.e., within a window of approximately 100 nt) hairpin structures that are left intact and unmutated to preserve the overall secondary structure of the genome. Examples of nucleic acid positions that can be altered to create silent mutations while preserving the disordered nature of the local sequence are circled in the figure.

[0162] Figure 7 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 2 (C02). In this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codon at genomic nucleotide positions 503-658 (gray box) was altered to reduce homology to wild-type virus. In total, this silent mutation reduced the sequence identity of the nsP1 CDS to wild-type by 14.09%. Transcription of this plasmid from the T7 promoter produces the C02 replicon RNA. The 5'UTR, nsP1-4 ORFs, and SGPs of C02 are shown in SEQ ID NO:21.

[0163] It was observed that transfection of BHK cells in vitro confirmed that the RNA of the C02 construct was capable of mediating expression of the reporter gene, secreted placental alkaline phosphatase, encoded in the subgenomic ORF (FIG. 17A).

[0164] Example 4. VEEV replicon RNA modified to have reduced homology at nsP1 within nucleotide positions 658-1620 of the genome Using bioinformatics testing of local RNA folding based on thermodynamic parameters, as described in Example 2 for construct C02, a region of unstructured nucleotides was selected and a series of silent mutations were substituted across that region accordingly to generate a construct designated C06. Figure 8 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 6 (C06). In this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codon at genomic nucleotide positions 658-1620 (grey box) was altered to reduce homology to the wild-type virus. In total, the silent mutations reduced the sequence identity of the nsP1 CDS to wild-type by 8.96%. Transcription of this plasmid from the T7 promoter produces the C06 replicon RNA. The 5'UTR, nsP1-4 ORFs, and SGPs of C06 are shown in SEQ ID NO:25.

[0165] It was observed that transfection of BHK cells in vitro confirmed that this RNA (construct C06) was capable of mediating expression of the reporter gene, secreted placental alkaline phosphatase, encoded in the subgenomic ORF (FIG. 17A). Mice injected with this construct showed mean SEAP serum expression that was comparable to or greater than that of mice injected with wild-type VEEV-SEAP (FIG. 17D).

[0166] Example 5. VEEV replicon RNA modified to have reduced homology at the nsP1-nsP2 junction region within nucleotide positions 1620-2560 of the genome Using a bioinformatics test of local RNA folding based on thermodynamic parameters, as described in Example 2 for construct C02, a region of unstructured nucleotides spanning the junction of nsP1 and nsP2 was selected, and a series of silent mutations were substituted across that region accordingly to generate a construct designated C05. Figure 9 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 5 (C05). In this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codon at genomic nucleotide positions 1620-2560 (grey box) was altered to reduce homology to the wild-type virus. In total, the silent mutations reduced the sequence identity of the nsP1 CDS to wild-type by 15.94% and the sequence identity of the nsP2 CDS by 0.8%.

[0167] Transcription of this plasmid from the T7 promoter generates the C05 replicon RNA. The 5'UTR, nsP1-4 ORFs, and SGPs of C05 are shown in SEQ ID NO:24.

[0168] Transfection of BHK cells in vitro confirmed that this RNA (C05) could mediate expression of the reporter gene, secreted placental alkaline phosphatase, encoded in the subgenomic ORF (Figure 17A). Mice injected with this construct showed mean SEAP serum expression that was similar to or greater than that of mice injected with wild-type VEEV-SEAP (Figure 17D).

[0169] Example 6. VEEV replicon RNA modified to have reduced homology in nsP2 within nucleotide positions 3694-3954 of the genome Using a bioinformatics test of local RNA folding based on thermodynamic parameters as described in Example 2 for construct C02, a region of unstructured nucleotides within the nsP2 coding sequence was selected and a series of silent mutations were substituted across that region accordingly to generate a construct designated C03. Figure 10 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 3 (C03). In this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codon at genomic nucleotide positions 3694-3954 (grey box) was altered to reduce homology to the wild-type virus. In total, the silent mutations reduced the sequence identity of the nsP2 CDS to the wild-type by 3.19%.

[0170] Transcription of this plasmid from the T7 promoter generates the C03 replicon RNA. The 5'UTR, nsP1-4 ORFs, and SGPs of C03 are shown in SEQ ID NO:22.

[0171] Transfection of BHK cells in vitro confirmed that this RNA was capable of mediating expression of the reporter gene, secreted placental alkaline phosphatase, encoded in the subgenomic ORF (FIG. 17A). Mice injected with construct C03 were observed to exhibit mean SEAP serum expression that was comparable to or greater than that of mice injected with wild-type VEEV-SEAP (FIG. 17D).

[0172] Example 7. VEEV replicon RNA modified to have reduced homology in nsP2 within nucleotide positions 2560-3954 of the genome Given the success observed in Example 5, the homology reduction region was further extended in the 5' direction. A bioinformatics examination of local RNA folding based on thermodynamic parameters spanning genomic nucleotide positions 2560-3954 was again performed and the region of unstructured nucleotides was correspondingly replaced with silent mutations to generate a construct designated C07. Figure 11 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 7 (C07). In this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codon at genomic nucleotide positions 2560-3954 (grey box) was altered to reduce homology to the wild-type virus. In total, the silent mutations reduced the sequence identity of the nsP2 CDS to the wild-type by 15.97%. Transcription of this plasmid from the T7 promoter produces the C07 replicon RNA. The 5'UTR, nsP1-4 ORFs, and SGPs of C07 are shown in SEQ ID NO:26.

[0173] Transfection of BHK cells in vitro confirmed that this RNA (C07) could mediate expression of a reporter gene, secreted placental alkaline phosphatase, encoded in a subgenomic ORF (FIG. 17A).

[0174] Example 8. VEEV replicon RNA modified to have reduced homology at the nsP2-nsP3 junction region within nucleotide positions 3954-4120 of the genome Using a bioinformatics test of local RNA folding based on thermodynamic parameters, as described in Example 2 for construct C02, a region of unstructured nucleotides spanning the junction of nsP2 and nsP3 was selected and a series of silent mutations were substituted across that region accordingly to generate a construct designated C04. Figure 12 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 4 (C04). In this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codon at genomic nucleotide positions 3954-4120 (grey box) was altered to reduce homology to wild-type virus. In total, the silent mutations reduced the sequence identity of the nsP2 CDS to wild-type by 4.61% and the sequence identity of the nsP3 CDS by 0.12%. Transcription of this plasmid from the T7 promoter produces the C04 replicon RNA. The 5'UTR, nsP1-4 ORFs, and SGPs of C04 are shown in SEQ ID NO:23.

[0175] Transfection of BHK cells in vitro confirmed that this RNA was able to mediate expression of a reporter gene, secreted placental alkaline phosphatase, encoded in a subgenomic ORF (FIG. 17A).

[0176] Example 9. VEEV replicon RNA modified to have reduced homology in nsP4 within nucleotide positions 6381-7083 of the genome Using bioinformatics testing of local RNA folding based on thermodynamic parameters, as described in Example 2 for construct C02, a region of unstructured nucleotides within the nsP4 coding sequence was selected and a series of silent mutations were substituted across that region accordingly to generate a construct designated C01. Figure 13 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 1 (C01). In this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codon at genomic nucleotide positions 6381-7083 (grey box) was altered to reduce homology to the wild-type virus. In total, the silent mutations reduced the sequence identity of the nsP4 CDS to the wild-type by 4.69%. Transcription of this plasmid from the T7 promoter produces the C01 replicon RNA. The 5'UTR, nsP1-4 ORFs, and SGPs of C01 are shown in SEQ ID NO:20.

[0177] Transfection of BHK cells in vitro confirmed that this RNA (C01) was able to mediate expression of a reporter gene, secreted placental alkaline phosphatase, encoded in a subgenomic ORF (FIG. 17A).

[0178] Example 10. Single VEEV replicon RNA modified with nsP1 homology reduction in C06 and nsP2 homology reduction in C07 Given the success observed in Examples 4 and 7, the regions of reduced homology were combined in a single clone to generate a construct designated C09. Figure 14 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 9 (C09). In this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codons at genomic nucleotide positions 658-1620 and 2560-3954 (grey boxes) were altered to reduce homology to the wild-type virus. In total, the silent mutations reduced the sequence identity of the nsP1 CDS to wild-type by 8.96% and the sequence identity of the nsP2 CDS by 15.97%. Transcription of this plasmid from the T7 promoter produces the C09 replicon RNA. The 5'UTR, nsP1-4 ORFs, and SGPs of C09 are shown in SEQ ID NO:28.

[0179] FIG. 17B is a bar graph showing the results of SEAP expression testing by sampling conditioned supernatants of BHK cells transfected for 16 hours with the indicated replicon RNAs and quantifying SEAP expression by measuring absorbance at 650 nm in a colorimetric assay.

[0180] As shown in FIG. 17B, transfection of BHK cells in vitro confirmed that this RNA (C09) could mediate expression of the reporter gene, secreted placental alkaline phosphatase, encoded in the subgenomic ORF.

[0181] Example 11. Single VEEV replicon RNA modified with nsP2 homology reduction in C03 and nsP4 homology reduction in C01 Given the success observed in Examples 6 and 9, the regions of reduced homology were combined in a single clone to generate a construct designated C10. Figure 15 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 10 (C10). In this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codons at genomic nucleotide positions 3694-3954 and 6381-7083 (grey boxes) were altered to reduce homology to the wild-type virus. In total, the silent mutations reduced the sequence identity of the nsP2 CDS to wild-type by 3.19% and the sequence identity of the nsP4 CDS by 4.69%. Transcription of this plasmid from the T7 promoter produces the C10 replicon RNA. The 5'UTR, nsP1-4 ORFs, and SGPs of C10 are shown in SEQ ID NO:29.

[0182] FIG. 17C is a bar graph showing the results of SEAP expression testing by sampling conditioned supernatants of BHK cells transfected for 16 hours with the indicated replicon RNAs and quantifying SEAP expression by measuring absorbance at 650 nm in a colorimetric assay.

[0183] As shown in FIG. 17C, transfection of BHK cells in vitro confirmed that this RNA (C10) could mediate expression of the reporter gene, secreted placental alkaline phosphatase, encoded in a subgenomic ORF.

[0184] Example 12. A single VEEV replicon RNA modified with reduced homology expressed in a region spanning all nsPs Given the generalizable success of the approach described in the previous examples, the previously analyzed regions with reduced homology were reanalyzed en bloc and silently mutated across the determined unstructured nucleotide stretches to generate constructs C01, C02, C04, C05, C06, and C07, generating a construct designated C11. Figure 16 is a schematic diagram of a DNA plasmid encoding the replicon sequence template for construct 11 (C11). In this figure, the DNA plasmid is similar to that shown in Figure 5, except that only codons at genomic nucleotide positions 503-658, 658-1620, 1620-2560, 2560-3954, 3954-4120, and 6381-7083 (grey boxes) were altered to reduce homology to the wild-type virus. In this example, the silent mutations applied resulted in an overall reduction in nsP CDS homology comparable to the sum of all previously mutated regions combined compared to wild-type virus. Transcription of this plasmid from the T7 promoter produces C11 replicon RNA. The C11 5'UTR, nsP1-4 ORFs, and SGP are shown in SEQ ID NO:30.

[0185] Figures 17A-17D show the results of a SEAP expression study to demonstrate the functionality of the subgenomic ORF encoding SEAP present in the codon-adapted replicon RNA. Figure 17A is a bar graph showing the results of a SEAP expression study by sampling conditioned supernatants of BHK cells transfected for the indicated times, 4 hours (left bar), 16 hours (middle bar) and 24 hours (right bar), compared to control, wild type (WT) and untransfected (No Transfect) cells. Figures 17B and 17C are bar graphs showing the results of a SEAP expression study by sampling conditioned supernatants of BHK cells transfected for 16 hours with the indicated replicon RNAs (Figures 17B-C09 and Figures 17C-C10) and quantifying SEAP expression by measuring absorbance at 650 nm in a colorimetric assay. As demonstrated by this data, modification of nsP codons in the region identified by bioinformatics analysis does not negatively affect the expression of transgenes encoded in subgenomic ORFs in vitro. Figure 17D shows the results of SEAP expression in mice. In these experiments, 4 mice per group were administered 1 μg of the indicated codon-compatible replicon by intramuscular injection. Serum was collected and the relative expression of SEAP was quantified by luminescence assay. As shown in Figure 17D, the average RLU of each animal, error bars for each group, and standard deviation are shown. Two non-injected mice (labeled "untransfected") were used as negative controls to show background signals. As demonstrated by this data, modification of nsP codons in the region identified by bioinformatics analysis does not negatively affect the expression of transgenes encoded in subgenomic ORFs in vivo.

[0186] Overall, extensive regions of nsPs were found through this analysis to contain unstructured nucleotide stretches that accept silent mutations that do not affect gene expression. Figure 18 is a scale diagram of the VEEV genome, in which regions of the unstructured polyprotein that are enriched in unstructured regions that accept silent mutations, as determined by the RNA folding analysis and experiments described herein, are marked as grey boxes. Embodiments herein disclose silent mutations across these regions such that the resulting sequences diverge (deviation) from naturally occurring VEEV-type genome nsP coding regions by up to 10% non-identity by Basic Local Alignment Search (BLAST).

[0187] Example 13. VEEV replicon RNA with nsP4 C-terminal coding region duplicated to introduce C-terminal fusion sites and reconstruct the subgenomic promoter (SGP) Direct insertion of any additional protein ("xP") by conventional cloning to fuse the nsP4 and xP ORFs would disrupt the primary nucleotide sequence elements that constitute the SGP. Since the SGP is required to mediate transcription of the subgenomic RNA and thus the later translation of the subgenomic ORF, such an approach to fuse xP to the nsP4 C-terminus would destroy the ability of the replicon to mediate transgene expression from the subgenomic locus. Figure 3 shows a process to circumvent this problem by duplicating the SGP region including the coding region of the nsP4 C-terminus. The upstream duplicated region can then be used as a target for in-frame insertion of xP by incorporation of nucleotides that generate a restriction site for conventional cloning. Figure 3 shows an example of a simple step that can be followed to clone a modifiable nsP4 open reading frame (ORF) while maintaining an intact extended subgenomic promoter (SGP, striped box). However, this oversimplified approach leaves two regions of identical sequence that result in genetic instability when propagating this DNA template sequence in the bacterial strain required for production.

[0188] Indeed, when this construct was cloned as a DNA template, identical sequences representing the codons encoding the C-terminus of nsP4 were present, and a standard bacterial strain (DH5-alpha) was therefore unable to maintain the repeated sequences intact. All clones isolated from DNA templates constructed using the method outlined in Figure 3 contained a deletion that eliminated the nsP4 C-terminal duplication.

[0189] Example 14. VEEV replicon RNA with nsP4 C-terminal coding region duplicated and reduced in homology to introduce C-terminal fusion sites and thus reconstruct the subgenomic promoter (SGP) and eliminate repetitive sequence elements FIG. 4 shows an example of optimal steps that can be followed to clone a modifiable nsP4 open reading frame (ORF) while maintaining an intact extended subgenomic promoter (SGP, striped box). This approach eliminates problematic repetitive sequence elements, and the DNA template can be easily propagated and produced in common bacterial strains. FIG. 4 shows an optimal process for introducing an xP fusion to the nsP4 C-terminus while maintaining an intact SGP region. The upstream duplicated sequence is first modified to remove its codon homology with the downstream duplicated sequence, which maintains the wild-type sequence to ensure an intact SGP in close proximity to the subgenomic ORF. The sequence of the downstream SGP located 3' of any xP is shown as SEQ ID NO: 14. The upstream duplicated region of reduced homology (exemplified in the sequence shown as SEQ ID NO: 18) can then be fused to any xP inserted in frame at the end of nsP4 by conventional molecular cloning. An example of the final nsP4 coding RNA sequence is shown in SEQ ID NO: 19. FIG. 19 is a schematic diagram illustrating an nsP4 replicon sequence modified for the experiment, where the exogenous protein (xP) added to the nsP4 protein is green fluorescent protein (GFP). In this figure, the structural polyprotein in the SG ORF is replaced with the reporter gene SEAP. In this figure, the nsP contains a gene encoding GFP preceded by a self-cleaving 2A peptide as the xP, and the subgenomic ORF contains a secreted placental alkaline phosphatase (SEAP) reporter gene. The nsP1-nsP2-nsP3-nsP4-2A fusion polyprotein amino acid sequence resulting from these modifications is shown as SEQ ID NO: 12, to which the xP of interest (e.g., GFP) can be further fused. An exemplary DNA sequence encoding this engineered nsP-2A ORF is shown as SEQ ID NO: 13. The nsP1-nsP2-nsP3-nsP4-2A-GFP amino acid sequence is shown in SEQ ID NO: 15, and an exemplary DNA sequence encoding this engineered nsP-2A-GFP ORF is shown in SEQ ID NO: 16. The entire nsP4-2A-GFP modified replicon RNA construct is designated C16.This replicon RNA is called VEEVrep-nsP4[GFP]-SEAP, and the corresponding RNA sequences of the 5′UTR, nsP1 to 4-GFP fusion ORF, and SGP are shown in SEQ ID NO:31.

[0190] Figures 20A-C show gene expression in cells in vitro mediated by engineered nsP4 modified VEEV replicons. BHK cells were transfected with an experimental nsP4 modified replicon (VEEVrep-nsP4[GFP]-SEAP) encoding GFP in the nonstructural protein ORF and SEAP in the subgenomic ORF. Figure 20A shows the results of fluorescence microscopy demonstrating multiple GFP positive cells (top right) when transfected with nsP4(GFP) modified VEEV-SEAP replicon RNA, and the absence of GFP expression in untransfected control cells (bottom right). Figure 20B shows an immunoblot of lysates of transfected BHK cells with a GFP specific monoclonal antibody (top band) and GAPDH detection serves as a loading control (bottom band) showing confirmation of GFP expression by two independently isolated clones (clone 1 and clone 2) of nsP4(GFP) modified VEEV replicon RNA compared to the untransfected control (NoTx). FIG. 20C is a bar graph showing the functionality of the subgenomic ORF encoding SEAP, which was tested by sampling conditioned supernatants of transfected BHK cells and quantifying SEAP expression by a colorimetric assay. As shown in this figure, both clone 1 and clone 2 show high SEAP expression compared to the untransfected control, where only assay background absorbance was observed. As shown in FIG. 20A-C, this RNA simultaneously drove both GFP expression (FIG. 20A and FIG. 29B) and SEAP expression (FIG. 20C) when transfected into BHK cells. This confirmed that both nsP4 function-mediated genome replication transcription and SGP-mediated translation were maintained, but an additional accessory function protein (GFP) was successfully produced from an early-translating locus independent of the subgenomic ORF.

[0191] Example 15. A single VEEV replicon RNA incorporating both nsP4 C-terminal xP fusions and reduced homology nsPs Building on the success of Examples 9 and 12, a VEEV replicon was created that combines the reduced homology region of C09 and the nsP4-2A-GFP modifications of C16 to generate a new construct designated C13. Figures 21A-B show gene expression mediated by this engineered nsP4 modified VEEV replicon. This replicon RNA combines the codon adaptation and nsP4 C-terminal modifications as described in this application.

[0192] FIG. 21A is a photograph showing the results of fluorescence microscopy demonstrating multiple GFP-positive cells upon transfection with codon-compatible nsP4(GFP)-modified VEEV-SEAP replicon RNA (C13) (upper panel, right) and the absence of GFP expression in untransfected control cells (lower panel, right).

[0193] FIG. 21B is a chart showing the results of a luminescence assay (RLU) to quantify the concentration of SEAP in serum collected from mice injected with wild-type (WT) VEEVrep-SEAP replicon RNA or nsP4(GFP)-modified (modified) VEEV replicon RNA with optimized codons in the nonstructural region of the nonstructural polyprotein gene (similar to VEEVrep-nsP4[GFP]-SEAP) compared to serum collected from non-injected control mice. Serum was collected on days 1, 3, and 5 after injection. In these experiments, mice (3 per group) were administered an equal dose of standard VEEV-SEAP replicon RNA or nsP4(GFP)-modified VEEV replicon RNA with optimized codons in the nonstructural region of the nonstructural polyprotein gene by intramuscular injection. Serum was collected at the indicated times and the concentration of SEAP was quantified by luminescence assay. The average RLU of mice in each group is shown with error bars, standard deviation. Two non-injected mice were used as negative controls to show background signal. This data demonstrates that modification of the nsP4 gene by the methods described herein does not negatively affect expression of the transgene encoded in the subgenomic ORF in vivo.

[0194] It was observed that this RNA was capable of expressing GFP in transfected cells in vitro, as shown in Figure 21 A. It was also observed that injection of C13 in mice resulted in SEAP serum concentrations comparable to the parental VEEV-SEAP replicon RNA, as shown in Figure 21 B, indicating that the combination of reduced homology to wild-type virus and the additional transgene payload incorporated by fusion to nsP4 did not interfere with the efficacy of gene expression in vivo.

[0195] Example 16. Replicon RNA encoding a functional enzyme fused to the nsP4 C-terminus and a separate transgene in the SG ORF An enzyme may be introduced at the C-terminus of nsP4 (i.e. xP may be a functional enzyme) according to the design described in Example 14. As outlined above and shown in SEQ ID NO: 18 and SEQ ID NO: 14, the SGP may be maintained by duplicating and placing the wild-type SGP sequence downstream of the xP fusion (in this example, downstream of the enzyme), and the upstream duplicated sequence in the C-terminal coding region of nsP4 is altered to maintain the correct amino acid sequence while reducing homology with the wild-type SGP. This ensures genetic stability of the construct and avoids homology-driven recombination that would otherwise result in the deletion of xP (which would occur if no codon changes were made, as exemplified in Example 13).

[0196] 22A-B show gene expression from an alphavirus replicon RNA engineered to encode the 3Cpro protease required for proteolytic processing of the picornavirus P1 structural polyprotein (encoded by the SG ORF).

[0197] 22A is a schematic diagram showing a construct with modification of the nsP4 coding sequence of an alphavirus replicon to encode the 3Cpro protease required for the picornavirus P1 structural polyprotein (O1 Manisa P1 polyprotein), designated VEEVrep-nsP4[3C]-P1. The sequences of the 5'UTR, nsP1-4, 3Cpro and SGP regions of this RNA are shown in SEQ ID NO:32. The RNA sequence encoding the P1 structural polyprotein in the SG ORF is shown in SEQ ID NO:33. The amino acid sequence of the nsP4-2A-3Cpro fusion polyprotein encoded by the nsP ORF is shown in SEQ ID NO:40.

[0198] This figure shows an exemplary VEEV replicon RNA design in which the C-terminus is fused to a viral protease via a 2A self-cleaving peptide using the approach described herein. The viral protease in this example is 3Cpro from the picornavirus, foot and mouth disease virus (FMDV), strain A12. The 3Cpro enzyme is an example of a good candidate for insertion into the NPS4 modified region of the replicon. 3Cpro is a highly processive enzyme and requires a relatively low molecular copy number compared to its substrate when delivered exogenously to cells. 3Cpro activity is required for post-translational processing of the picornavirus capsid polyprotein (called the P1 polyprotein). In addition to being fully functional at low expression levels, the low copy number of 3Cpro is ideal due to the inherent toxicity of the protease. When high levels of 3Cpro are expressed in vitro, cellular metabolism shuts down and viability decreases. Attempts have been made to create an FMDV vaccine in which the replicon RNA encoding the P1 polyprotein and the mRNA encoding 3Cpro are co-formulated with the aim of delivering both molecules to the same cells and allowing processing of P1 (trans-complementation). This trans-complementation vaccine design requires a ratio of P1-2A to 3Cpro of approximately 30-40:1 to minimize the toxicity of 3Cpro. Although delivery platforms exist that allow for the co-encapsulation and delivery of multiple RNA molecules to the same cells, it is unlikely that 100% of the nanoparticles will contain at least one copy of the mRNA encoding 3Cpro due to the requirements of these ratios. Therefore, the trans-complementation approach minimizes the potential efficacy of the vaccine product, since individual nanoparticles without at least one copy of both RNA molecules will not be effective in generating processed capsids and therefore will not contribute to the humoral immune response.

[0199] Given that the 3Cpro enzyme is highly cytotoxic and must be maintained at a low steady-state concentration to avoid eventual disruption of cellular processes through its proteolytic activity, the 3Cpro enzyme was encoded in the nsP4 C-terminal region to mediate expression as part of the FMDV vaccine construct. The P1 polyprotein contains VP0, VP2, and VP3 subunits, which must be cleaved into separate polypeptide chains by 3Cpro to allow assembly into capsid structures. To generate a functional picornavirus vaccine, these capsid structures must assemble in order to be recognized by the immune system and thus drive neutralizing antibody production. The VP0 fragment of the P1 polyprotein further contains the VP4 and VP2 proteins, which undergo cleavage by a 3Cpro-independent process and are not required for capsid assembly. To generate a useful FMDV vaccine, the P1 polyprotein of FMDV strain O1 Manisa was encoded in the SG ORF of a VEEV-based replicon. The replicon, designated VEEVrep-nsP4[3C]-P1, provides 3Cpro activity in cis by encoding it at the nsP4 C-terminus, as shown in Figure 22A. An additional replicon, VEEVrep-nsP4[3C mut ]-P1 contains an insertion mutation that abolishes translation of the 3Cpro sequence and serves as a parallel control. As an additional control, a replicon (VEEVrep-P1) was generated with wild-type nsP4 and SGP regions only encoding the P1 polyprotein in the SG ORF. These RNAs were synthesized by in vitro transcription and post-transcriptional capping. BHK cells in 12-well dishes were transfected with 1 μg of each P1-encoding replicon per well, and cell lysates were prepared for analysis by immunoblot approximately 1 day later.

[0200] FIG. 22B is a photograph of an immunoblot performed on lysates of BHK cells transfected with the following RNAs: lane 1, VEEVrep-nsP4[3C]-P1; lane 2, a replicon RNA similar to that in lane 1, but with an insertion mutation (VEEVrep-nsP4[3Cmut]-P1) that introduced a premature stop codon and eliminated expression of the 3C polypeptide as a negative control; lane 3, VEEVrep-P1 (similar to VEEVrep-nsP4[3C]-P1, but lacking the nsP4-fused 3C); lane 4, VEEVrep-P1 transfected in combination with 1 / 40 of the mass of the mRNA encoding 3C as a positive control. As shown in this figure, successful proteolytic processing of the ∼100 kDa P1 polyprotein into the immunogenic ∼35 kDa VP0 capsid fragment was mediated to completion only by VEEVrep-nsP4[3C]-P1 (lane 1). An intermediate incomplete cleavage product, representing intact VP4+VP2+VP3, can be seen proceeding with an apparent molecular weight of approximately 55 kDa (lane 4). A VP2-specific antibody was used to probe the blot. M, molecular weight marker; C, untreated negative control cells. This figure shows the results of an immunoblot on samples after probing with an FMDV VP2-specific monoclonal antibody. VEEVrep-nsP4[3C mut]-P1 and VEEVrep-P1 produced only the full-length intact P1 polypeptide seen as a band at approximately 100 kDa. VEEVrep-nsP4[3C]-P1 successfully produced the desired fully processed VP0 fragment, indicating that capsid processing occurred as desired. Of note, processing was superior to the positive control (lane 4) in which VEEVrep-P1 was transfected in combination with a separate 3Cpro encoded in trans in a conventional mRNA molecule at a mass ratio of 40:1, a ratio known to generate protective antibody titers in pigs when administered as a vaccine. This superior processing efficiency is also evident in the absence of processing of the intermediate fragment VP4+2+3. This demonstrates the utility of encoding a functional protein, in this particular example an enzyme, in the C-terminal region of nsP4 using the methods described in this disclosure to induce a desired biochemical outcome.

[0201] Example 17. Replicon RNA encoding an immune regulator fused to the nsP4 C-terminus and a separate transgene in the SG ORF To achieve a desired result, it is useful to increase or decrease the immune response induced by replicon RNA activity in cells. For example, it is possible to treat cancer by inducing a strong natural immune response in cancer cells.

[0202] Figures 23A-D show gene expression data from an alphavirus replicon RNA modified to encode a human STING protein mutated to be constitutively active for IFN stimulatory activity and to encode the reporter gene SEAP in the SG ORF.

[0203] FIG. 23A is a schematic diagram of construct VEEVrep-nsP4[STING]-SEAP, a replicon RNA containing modification of the nsP4 coding sequence of an alphavirus replicon to encode a human STING protein mutated to be constitutively active for IFN stimulatory activity, and a reporter gene SEAP encoded in the SG ORF. The sequences of the 5'UTR, nsP1-4, STING and SGP regions of this RNA are shown in SEQ ID NO: 34. The resulting translated amino acid sequence of the nsP4-2A-STING fusion polypeptide is shown in SEQ ID NO: 42. This diagram shows a replicon RNA construct in which the potent immune-modulating protein STING (mutated to be constitutively active) is encoded by fusion to the nsP4 C-terminus. The SG ORF is where tumor-associated antigens or additional immune-modulating factors may be encoded to drive specific cellular immunity against the desired cancer target in the case of cancer therapy. To illustrate the function of STING-modified nsP4, in this example, the SG ORF encodes a reporter gene SEAP that can be easily quantified from cultures of tumor cells. To demonstrate that a STING- and SEAP-encoding replicon of this design, termed VEEVrep-nsP4[STING]-SEAP, functions in human cells, HEK-Lucia™ Null cells were transfected in 12-well dishes with 1 μg of VEEVrep-nsP4[STING]-SEAP or non-nsP4 modified VEEVrep-SEAP as a control, using a commercial transfection reagent (TransIT-mRNA, Mirus). After approximately 1 day, the supernatant culture medium was sampled, cells were lysed and STING content was analyzed by immunoblot. Figure 23B is a photograph of an immunoblot performed on HEK-Lucia™ Null cells transfected with the following RNAs: lane 1, VEEVrep-SEAP; lane 2, VEEVrep-nsP4[STING]-SEAP; lane 3, no transfection control. In this figure, common background bands are marked with an asterisk ( *We marked the blot with nsP4 (presumably endogenous wild-type STING) and probed the blot with a human STING-specific antibody. We observed that only VEEVrep-nsP4[STING]-SEAP displayed an additional band representing the constitutively active STING protein encoded in the C-terminal region of nsP4.

[0204] FIG. 23C is a bar graph showing the expression of the SEAP reporter gene encoded by the SG ORF in VEEVrep-SEAP and VEEVrep-nsP4[STING]-SEAP replicon RNAs (measured by absorbance at 650 nm in a colorimetric assay) compared to the control (untransfected cells). It was observed that the VEEVrep-nsP4[STING]-SEAP subgenomic promoter activity was retained and transgene expression was retained despite the novel nsP4 C-terminal modification with an immune-modulating polypeptide. As shown in this figure, a colorimetric assay to quantify SEAP in the culture supernatant showed that the gene was expressed by both constructs, proving that the VEEVrep-nsP4[STING]-SEAP construct successfully replicated and expressed the transgene driven by SGP. The lower SEAP expression is likely due to the reduced viability of HEK cells induced by the strong innate immune response elicited by STING protein. Type I IFN signaling activity can be measured from HEK-Lucia™ Null cells by the supernatant medium concentration of secreted luciferase, which in these cells is encoded under the control of an IFN-inducible promoter that includes the IFN-stimulated gene (ISG) 54 promoter enhanced by multimeric IFN-stimulated response elements (ISREs). Figure 23D is a bar graph showing surrogate measurements of IFN signaling activity induced by VEEVrep-SEAP and VEEVrep-nsP4[STING]-SEAP replicon RNAs compared to control (untransfected cells). HEK-Lucia™ Null cells encode a secreted luciferase reporter gene under the control of an IFN-inducible promoter that includes the IFN-stimulated gene (ISG) 54 promoter enhanced by multimeric IFN-stimulated response elements (ISREs). We observed that VEEVrep-nsP4[STING]-SEAP RNA, but not unmodified VEEVrep-SEAP RNA, induced potent IFN signaling.

[0205] As shown in Figure 23D, in a luminescence-based assay for secreted luciferase, only VEEVrep-nsP4[STING]-SEAP showed IFN type I signal activation, demonstrating that the active STING protein was functional and conferred the desired effect on transfected human cells. STING represents an immunomodulatory protein that has both immunostimulatory and immunosuppressive properties via the IFN signaling cascade, depending on the tissue and cell type context of administration. Therefore, this approach of embedding STING or other immunomodulatory proteins represents a way to either induce or attenuate adaptive immune responses, making the engineered replicon useful not only in infectious diseases and cancer, but also in the treatment of autoimmune diseases and allergies by inducing immune tolerance when necessary.

[0206] Example 18. Treatment of melanoma Replicons engineered to express accessory factors from the nsP4 coding region using the methods described herein may be used to treat cancers such as melanoma. Figures 24A-D show gene expression and biological effects in B16 melanoma from alphavirus replicon RNA modified to encode GFP or human STING protein (mutated to be constitutively active for IFN stimulatory activity) in the nsP ORF and the reporter gene SEAP in the SG ORF.

[0207] Figure 24A is a photograph showing GFP expression in B16 melanoma cells cultured in 12-well dishes after transfection with 1 μg VEEVrep-nsP4[GFP]-SEAP, compared to untransfected control cells, about 1 day after transfection. Widespread GFP expression was observed, driven by the encoding of fluorescent protein at the nsP4 C-terminus of the construct described herein. Figure 24A shows the successful GFP expression in B16 melanoma cells mediated by RNA VEEVrep-nsP4[GFP]-SEAP, demonstrating the ability of nsP4 fusion protein to express efficiently in tumor cell lines.

[0208] Figure 24B is a bar graph showing SEAP expression in B16 melanoma cells cultured in 12-well dishes after transfection with 1 μg of the indicated replicon RNA, VEEVrep-nsP4[STING]-SEAP, VEEVrep-nsP4[GFP]-SEAP, and VEEVrep-SEAP, compared to untransfected control cells. In this experiment, SEAP expression was quantified by colorimetric assay in the culture medium one day after transfection, indicating successful expression of the SEAP gene encoded by the SG ORF. As shown in Figure 24B, the reporter gene (SEAP) encoded by the SG ORF (representing the transgene location of the antigen or other cancer therapeutic gene sequence) is efficiently expressed in the same cells, regardless of the nature of the nsP4 fusion protein.

[0209] Figure 24C is a bar graph showing measurement of IFN-β secretion (measured by sandwich ELISA on culture supernatants) by B16 melanoma cells approximately 1 day after transfection with 1 μg of the indicated replicon RNA, VEEVrep-nsP4[STING]-SEAP and VEEVrep-SEAP, compared to untransfected control cells. As shown in this figure, VEEVrep-nsP4[STING]-SEAP RNA was observed to successfully induce IFN-β secretion in these cancer cells at approximately 73% higher levels than VEEVrep-SEAP. This indicates that STING protein expressed by fusion to nsP4 successfully induced an innate immune response in cancer cells, which correlates with successful tumor clearance in a clinical setting.

[0210] FIG. 24D is a photograph of light microscopy of B16 cells 1 day after transfection with 2 μg of VEEVrep-nsP4[GFP]-SEAP or VEEVrep-nsP4[STING]-SEAP compared to healthy untransfected control cells (Control; leftmost panel). As shown in this figure, untransfected control cells proliferated and filled the dish. Moderate growth defects and cytopathology were observed after transfection with VEEVrep-nsP4[GFP]-SEAP. Maximal growth inhibition and cell death were observed after transfection with VEEVrep-nsP4[STING]-SEAP.

[0211] As shown in FIG. 24D, when B16 cells were examined by bright field microscopy one day after transfection, untreated cells (control) grew and filled the dish as expected. In contrast, a moderate growth defect and cytopathology were observed upon transfection with VEEVrep-nsP4[GFP]-SEAP, and an even higher degree of growth inhibition and cell death was observed upon transfection with VEEVrep-nsP4[STING]-SEAP. Altogether, these data indicate that the nsP4 modified replicon can replicate in melanoma cells, drive gene expression from both the nsP4 C-terminal encoded ORF and the SG ORF, and activate a higher innate immune response and inhibit melanoma cell growth and survival when an immunomodulatory protein is fused to nsP4. In all B16 melanoma cell experiments described in this example, cells were grown in 12-well dishes in DMEM+10% inactivated fetal bovine serum (IFS). For analysis of gene expression (GFP, SEAP) or immune activation (IFN-β), cells were transfected with 1 μg of the indicated RNA and analyzed after 1 day. For measurement of cell proliferation and viability by microscopy, cells were transfected with 2 μg of RNA and images were captured after 1 day.

[0212] Example 19. Treatment of HPV-induced epithelial tumors To demonstrate the universal applicability of the engineered replicons as disclosed in this application, the functionality of the nsP4 modified replicon RNA was demonstrated in an additional tumor cell type, TC-1 epithelial tumor cells, which exhibit malignant growth driven by HPV E6 and E7 oncogene transformation.

[0213] Figures 25A-D show gene expression and biological effects in TC-1 cancer cells from alphavirus replicon RNA modified to encode GFP or human STING protein (mutated to be constitutively active for IFN stimulatory activity) in the nsP ORF and the reporter gene SEAP in the SG ORF.

[0214] Figure 25A is a bar graph showing SEAP expression in TC-1 cancer cells cultured in 12-well dishes 2 days after transfection with 2 μg of the indicated replicon RNA, VEEVrep-nsP4[GFP]-SEAP, VEEVrep-nsP4[STING]-SEAP, compared to untransfected control cells. In this experiment, SEAP expression was quantified by a colorimetric assay in the culture medium, indicating successful expression of the SEAP gene encoded by the SG ORF. As shown in Figure 25A, as seen in B16 melanoma (Example 18), SEAP expression confirmed the activity of the SG ORF for both VEEVrep-nsP4[STING]-SEAP and VEEVrep-nsP4[GFP]-SEAP RNAs in this unrelated cancer type.

[0215] Figure 25B is a bar graph showing measurement of IFN-β secretion (measured by sandwich ELISA on culture supernatants) by TC-1 cancer cells 1 day after transfection with 2 μg of the indicated replicon RNA, VEEVrep-nsP4[GFP]-SEAP, or VEEVrep-nsP4[STING]-SEAP, compared to untransfected control cells. As shown in Figure 25B, the constitutively active STING fusion protein encoded by nsP4 induced greater IFN-β secretion than the non-STING-containing replicon.

[0216] Figure 25C is a photograph showing GFP expression in TC-1 cancer cell aggregates after transfection with 1 μg of VEEVrep-nsP4[GFP]-SEAP, approximately 3 days after transfection. As shown in this figure, widespread GFP expression was observed throughout the multicellular structures driven by the fluorescent protein encoding at the nsP4 C-terminus of the construct (upper panel, right), compared to no GFP expression in untreated control cells (lower panel, right). As shown in Figure 25C, in cell aggregates isolated from TC-1 cultures, GFP expression by VEEVrep-nsP4[GFP]-SEAP was confirmed to be widespread throughout the structures.

[0217] Figure 25D is a photograph showing the results of light microscopy of TC-1 cancer cells 1 day after transfection with 4 μg of the indicated replicon RNA VEEVrep-nsP4[STING]-SEAP compared to untransfected control cells. As shown in this figure, untreated cells (control) were observed to grow and fill the dish, whereas growth inhibition and cell death were observed after transfection with VEEVrep-nsP4[STING]-SEAP.

[0218] As shown in Figure 25D, as observed for B16 melanoma cells, proliferation and viability were significantly inhibited by treatment with VEEVrep-nsP4[STING]-SEAP.

[0219] These data support that nsP4 modified replicons have broad applicability in the treatment of diverse cancer types. In all TC-1 cancer cell experiments described in this example, cells were grown in 12-well dishes in DMEM + 10% IFS. For analysis of GFP expression by microscopy, cells were transfected with 1 μg of RNA, scraped to release clumps, and imaged after 3 days. To measure immune activation by IFN-β ELISA, cells were transfected with 2 μg of the indicated RNA, and culture medium was analyzed after 1 day. To measure cell proliferation and viability by microscopy, cells were transfected with 4 μg of RNA, and images were captured after 1 day. SEAP expression was measured by a colorimetric assay performed on culture medium 2 days after transfection with 2 μg of the indicated RNA.

[0220] Example 20. VEEV replicon RNA modified to have reduced homology without bioinformatics testing Four additional reduced homology replicon RNAs were generated. Figures 26A-G show VEEV replicon RNAs modified to have reduced homology to wild-type RNA and expression data from these constructs. VEEVrepHK-SEAP was generated by applying a series of silent mutations across the junction of nsP1 and nsP2 at nucleotide positions 658-3359 (Figure 26A). This nucleotide position references the VEEV genome sequence SEQ ID NO:17. Figure 26A is a schematic diagram of construct 12 (C12; VEEVrepHK-SEAP). As shown in this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codon at genomic nucleotide positions 658-3359 (grey box) was altered to reduce homology to wild-type virus, regardless of the possible presence of any secondary structure elements. The 5'UTR, nsP1-4 coding region, and SGP of the RNA produced by transcription of the C12 plasmid are shown in SEQ ID NO:35.

[0221] VEEVrepHK(ΔU)-SEAP was similarly mutated over the same region, but by selecting only codons that lacked a uridine base when possible (Figure 26B). Figure 26B is a schematic diagram of construct 13 (C13; VEEVrepHK(ΔU)-SEAP). As shown in this figure, the DNA plasmid is similar to that shown in Figure 5, except that only codons at genomic nucleotide positions 658-3359 (grey box) were altered to reduce homology to wild-type virus, regardless of the possible presence of any secondary structural elements, and codons that avoided uracil bases were preferentially selected. The 5'UTR, nsP1-4 coding regions, and SGPs of the RNA produced by transcription of the C13 plasmid are shown in SEQ ID NO:36.

[0222] VEEVrepES-SEAP was generated by applying a series of silent mutations across the junction of nsP3 and nsP4 at nucleotide positions 4120-6965. Figure 26C is a schematic diagram of construct 14 (C14; VEEVrepES-SEAP). As shown in this figure, the DNA plasmid is similar to that shown in Figure 5, except that only the codon at genomic nucleotide positions 4120-6965 (gray box) was altered to reduce homology to wild-type virus, regardless of the possible presence of any secondary structure elements. The 5'UTR, nsP1-4 coding region, and SGP of the RNA produced by transcription of the C14 plasmid are shown in SEQ ID NO:37.

[0223] VEEVrepES(ΔU)-SEAP was similarly mutated over the same region, but by selecting only codons that lacked a uridine base when possible (Figure 26D). Figure 26D is a schematic diagram of construct 15 (C15; VEEVrepES(ΔU)-SEAP). As shown in this figure, the DNA plasmid is similar to that shown in Figure 5, except that only codons at genomic nucleotide positions 4120-6965 (grey box) were altered to reduce homology to wild-type virus, and codons that avoided uracil bases were preferentially selected, regardless of the possible presence of any secondary structure elements. The 5'UTR, nsP1-4 coding regions, and SGPs of the RNA produced by transcription of the C15 plasmid are shown in SEQ ID NO:38.

[0224] These constructs were made without considering the secondary structure elements present in the sequence in order to evaluate the performance of a typical non-bioinformatics approach to reduce homology with the wild-type virus.

[0225] 26E is a bar graph showing SEAP expression in BHK cells cultured in 12-well dishes 16 hours after transfection with 1 μg of VEEVrep-SEAP, VEEVrepHK-SEAP, or VEEVrepHK(ΔU)-SEAP RNA compared to untransfected control cells. In this experiment, SEAP expression was quantified by a colorimetric assay in the culture medium, indicating successful expression of the SEAP gene encoded by the SG ORF.

[0226] 26F is a bar graph showing SEAP expression in BHK cells cultured in 12-well dishes 16 hours after transfection with 1 μg of VEEVrep-SEAP, VEEVrepES-SEAP, or VEEVrepES(ΔU)-SEAP RNA compared to untransfected controls. In this experiment, SEAP expression was quantified by a colorimetric assay in the culture medium, which showed no expression of the SEAP gene encoded by the SG ORF for the VEEVrepES-SEAP or VEEVrepES(ΔU)-SEAP constructs.

[0227] Transfection of BHK cells in vitro confirmed that VEEVrepHK-SEAP and VEEVrepHK(ΔU)-SEAP could mediate expression of the reporter gene, secreted placental alkaline phosphatase, encoded in the subgenomic ORF (Figure 26E), but not VEEVrepES-SEAP and VEEVrepES(ΔU)-SEAP (Figure 26F). This indicated that silent mutations of arbitrary coding segments cannot reliably yield functional replicons without examination of the primary RNA sequence to identify secondary structural elements. VEEVrepHK-SEAP and VEEVrepHK(ΔU)-SEAP appeared to represent a serendipitous finding indicating that this naive approach could spontaneously yield a working replicon; however, further characterization revealed that both of these constructs lost SEAP expression activity in vivo, as measured by serum SEAP concentrations in mice (n=5) 1 day after intramuscular injection of 2 μg of LNP-formulated RNA by chemiluminescence assay. FIG. 26G is a bar graph showing serum SEAP expression measured in vivo (by chemiluminescence assay) after injection of 2 μg LNP-formulated VEEVrep-SEAP, VEEVrepHK-SEAP, or VEEVrepHK(ΔU)-SEAP compared to control (serum from untreated mice). In this experiment, mice (5 per group) were administered the indicated RNA by intramuscular (im) injection, serum was sampled the next day, and SEAP was quantified by chemiluminescence assay. The mean RLU for each group of mice is plotted with error bars and standard deviation. Serum from two untreated mice (n=2) was used as a negative control group.

[0228] In vivo performance was confirmed, but with less than average levels of expression, highlighting the superiority of the bioinformatics methods described in this disclosure (see Example 15) which resulted in comparable or superior gene expression in vivo compared to wild-type replicons.

[0229] Example 21. Engineered replicons are safe and do not harm healthy human cells To demonstrate the safety of the engineered replicons as disclosed in this application, the functionality of nsP4 modified replicon RNA was confirmed to be low in non-cancerous human cells. Human foreskin fibroblasts (HFF) are a commonly studied normal cell type that represents healthy non-transformed cell biology, yet still serves as a host for efficient transfection and infection for a number of nucleic acids and viral species. Figures 27A-D show the expression and effect of VEEV replicon RNA in normal HFF cells. Figure 27A is a bar graph showing SEAP expression in normal HFF cells cultured in 12-well dishes 1 day after transfection with 2 μg or 4 μg of the indicated replicon RNA, VEEVrep-nsP4[STING]-SEAP, or VEEVrep-SEAP, compared to untransfected control cells. In this experiment, SEAP expression was quantified by a colorimetric assay in the culture medium.

[0230] As shown in this figure, transfection of HFFs in 12-well dishes with 2 μg or 4 μg of a non-engineered replicon encoding a SEAP reporter gene in the SG ORF (VEEVrep-SEAP) resulted in low levels of SEAP expression as measured by a colorimetric assay performed on culture medium 1 day after transfection. In contrast, a replicon containing a constitutively active STING fusion protein encoded by nsP4 (VEEVrep-nsP4[STING]-SEAP) did not induce SEAP secretion, indicating that replication and transgene expression in healthy cells are suppressed for this engineered replicon.

[0231] Figure 27B is a photograph of GFP expression in trypsin-dissociated normal human HFF cells after transfection with 4 μg of VEEVrep-nsP4[GFP]-SEAP approximately 4 days after transfection, demonstrating the lack of efficient gene expression from the GFP protein encoded at the C-terminus of nsP4 in these healthy, non-cancerous cells. As shown in this figure, unlike the cancer cell types tested above (see Examples 18 and 19), no GFP expression was observed in HFF cells transfected with VEEVrep-nsP4[GFP]-SEAP.

[0232] FIG. 27C is a bar graph showing measurements of IFN-β activity from transfected HFFs. Conditioned medium from HFFs in 12-well dishes was collected one day after transfection with the indicated RNAs, VEEVrep-nsP4[STING]-SEAP, VEEVrep-nsP4[GFP]-SEAP, VEEVrep-SEAP at doses of 1 μg, 2 μg, and 4 μg. Medium was collected from untransfected cells (control HFFs) as a negative control. The collected medium was fed to HEK-Lucia Null cells to measure the response to type I IFN in the medium. Untreated HEK-LuciaNull cells were included as an additional negative control (no medium control). In this experiment, the IFN-responsive reporter gene Lucia was assayed in the supernatant of HEK-Lucia Null cells the next day by chemiluminescence assay. The engineered replicons VEEVrep-nsP4[GFP]-SEAP and VEEVrep-nsP4[STING]-SEAP, engineered as described in this disclosure, did not stimulate type I IFN innate immune responses in healthy HFF cell types. Based on the results of treating IFN-β-sensitive HEK-Lucia NULL cells with conditioned HFF culture medium, as shown in Figure 27C, the engineered replicons VEEVrep-nsP4[STING]-SEAP and VEEVrep-nsP4[GFP]-SEAP also did not drive a spike in type I IFN signaling in these normal cells.

[0233] FIG. 27D shows photographs of light microscopy of HFF cells 4 days after transfection with 4 μg of the indicated replicon RNAs, VEEVrep-nsP4[GFP]-SEAP and VEEVrep-nsP4[STING]-SEAP, compared to untransfected controls. Untreated cells (control) were observed to grow and fill the dish, exhibiting healthy fiber morphology. RNA-transfected cells showed equal growth and healthy morphology. As shown in FIG. 27D, no adverse effects on cell growth, morphology, or viability were observed. To measure cell growth and assess health by microscopy, cells were transfected with 2 μg or 4 μg of RNA and images were captured 3 days later. Cells under all conditions grew and remained intact with the expected fiber morphology even when transfected with the high 4 μg dose of RNA (FIG. 27D), suggesting that these RNAs are safe to administer in the context of healthy cells and exhibit negligible off-target immunogenicity to non-cancerous bystander cells. This is in contrast to the effects of the RNAs on cancer cells (see Examples 18 and 19), where both the nsP4 fusion protein and the SG ORF were strongly expressed and were able to disrupt cancer cell proliferation and induce an innate immune response.

[0234] References US Patent Application Publication No. 20140079734A1 - Modifications to an alphavirus replicon that duplicate elements from the 5' end of the genome into a subgenomic region to affect replication. WO2014170493A2 - Different modifications to the replicon, referring to mutations in the 3' region of nsP4 to modulate SGP. J Virol. 2006 Oct;80(20):9962-9969 - describes the function of nsP4 and the C-terminal tagging of this protein, suggesting that structure may be maintained by modifying this end.

Claims

1. 1. A synthetic alphavirus-derived replicon nucleic acid molecule comprising: (i) a first nucleic acid encoding alphavirus nonstructural proteins nsP1, nsP2, nsP3, and nsP4 and comprising at least one silent mutation introduced anywhere within the regions of nt 503 to nt 658, nt 658 to nt 1620, nt 1620 to nt 2560, nt 2560 to nt 3954, nt 3954 to nt 4120, nt 6381 to nt 7083, and nt 6966 to nt 7526 in the alphavirus genome sequence set forth in SEQ ID NO:17; and (ii) a second nucleic acid comprising a modified subgenomic open reading frame (ORF).

2. 2. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 1, wherein said modified subgenomic ORF comprises a sequence encoding a first heterologous protein.

3. 3. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 2, wherein said first nucleic acid comprises a polynucleotide encoding a modified nsP4 that comprises a second heterologous protein.

4. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 1, wherein the first nucleic acid comprises a polynucleotide encoding nsP1 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:21 or SEQ ID NO:

25.

5. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 1, wherein the first nucleic acid comprises a polynucleotide encoding the junction of nsP1 and nsP2 and comprising a sequence having at least 90% identity to a reference sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24 and SEQ ID NO:

26.

6. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 1, wherein the first nucleic acid comprises a polynucleotide encoding the junction of nsP2 and nsP3 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:

23.

7. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 1, wherein the first nucleic acid comprises a polynucleotide encoding nsP4 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:19 or SEQ ID NO:

20.

8. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 1, wherein the first nucleic acid comprises a polynucleotide encoding nsP1 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:25, and a polynucleotide encoding nsP2 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:

26.

9. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 1, wherein the first nucleic acid comprises a polynucleotide encoding nsP2 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO: 22, and a polynucleotide encoding nsP4 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:

20.

10. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 1, wherein the first nucleic acid comprises a polynucleotide encoding nsP1 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:21, a polynucleotide encoding nsP1 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:25, a polynucleotide encoding the junction of nsP1 and nsP2 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:24, a polynucleotide encoding nsP2 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:26, a polynucleotide encoding the junction of nsP2 and nsP3 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:23, and a polynucleotide encoding nsP4 and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:

20.

11. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 1, wherein the first nucleic acid comprises a polynucleotide encoding the nonstructural protein and comprising a polynucleotide sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:

30.

12. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 1, wherein the first nucleic acid molecule comprises a polynucleotide sequence having at least 90% identity to the reference sequence set forth in SEQ ID NO:

18.

13. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 3, wherein the first heterologous protein or the second heterologous protein is selected from the group consisting of proteins including viral or mammalian cell-derived regulators of cellular metabolic, immune, or signal transduction functions, antigenic proteins isolated from or derived from viral pathogens, and proteins that affect cellular antiviral innate immune responses and enhance replication of alphavirus replicon RNA molecules.

14. 4. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 3, wherein the first heterologous protein or the second heterologous protein comprises an antigenic protein isolated from or derived from a viral pathogen.

15. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 13, wherein said viral pathogen is a picornavirus.

16. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 3, wherein the second heterologous protein comprises a 3C protease protein and the modified nsP4 comprises an amino acid sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:

40.

17. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 16, wherein the modified nsP4 is encoded by a polynucleotide sequence having at least 90% identity to the reference sequence set forth in SEQ ID NO:

32.

18. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 3, wherein the second nucleic acid comprises a polynucleotide encoding the O1 Manisa P1 protein and comprising a sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:

33.

19. 4. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 3, wherein said first heterologous protein comprises an O1 Manisa P1 protein and said second heterologous protein comprises a 3C protease.

20. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 3 , wherein the second nucleic acid comprises a polynucleotide encoding an antigenic protein.

21. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 3, wherein the second heterologous protein comprises a STING protein and the modified nsP4 comprises an amino acid sequence having at least 90% identity to the reference sequence shown in SEQ ID NO:

42.

22. 4. The synthetic alphavirus-derived replicon nucleic acid molecule of claim 3, wherein the first heterologous protein comprises an antigen protein and the second heterologous protein comprises a STING protein.

23. 10. An alphavirus-derived RNA replicon expression system comprising the synthetic alphavirus-derived replicon nucleic acid molecule of claim 1 and a host cell.

24. A pharma- ceutically acceptable composition comprising the alphavirus-derived replicon of claim 1 and a pharma- ceutically acceptable carrier or excipient.

25. 25. Use of a pharma- ceutically acceptable composition according to claim 24 for the manufacture of a medicament for preventing, inhibiting, or treating a symptom of a disease or condition in a subject.

26. 26. The use of claim 25, wherein the treatment results in a reduction in the severity of the disease or a slowing of the progression of the disease in the subject.

27. 26. The use according to claim 25, wherein the disease is cancer.

28. 28. The use according to claim 27, wherein the cancer is melanoma or an epithelial tumor.

29. The use according to claim 25, wherein the disease is FMD.

30. 26. The use according to claim 25, wherein the subject is a cloven-hoofed animal or a human.

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