Replicon compositions for the treatment of diseases and methods of using the same

Abstract

The present invention encompasses a composition comprising at least two RNA replicons (self-amplifying RNA vectors (saRNA or rRNA)) that can be replicated by a replicase of a self-replicating virus, for example, a replicase of alphavirus origin. Among the at least two replicons, at least one optionally contains an open reading frame encoding an RNA-dependent RNA polymerase or replicase that can replicate each of the at least two replicons. Further, each replicon contains an open reading frame encoding different antigens of interest, for example, different antigens derived from the same or different pathogenic organisms, such as the glycoprotein and nucleoprotein of Ebola virus.

Description

Technical Field

[0001] The present invention encompasses a composition comprising at least two RNA replicons (self-amplifying RNA vectors (saRNAs)) that can be replicated by an RNA-dependent RNA polymerase (replicase) of a self-replicating virus, such as a replicase of alphavirus origin. Each replicon contains an open reading frame encoding a different antigen of interest, such as different antigens derived from the same or different pathogenic organisms, such as the glycoprotein and nucleoprotein of Ebola virus. The composition also includes a replicase capable of replicating each replicon. The replicase can be encoded by another open reading frame contained in one or both of the replicons and / or provided by another RNA molecule (non-replicating RNA) that has an open reading frame encoding the replicase but cannot be replicated by a replicase encoded by a third RNA molecule.

Background Art

[0002] Recently, mRNA-based vaccines have proven their immunogenicity in clinical trials to combat the COVID-19 pandemic. These RNA vaccines are highly effective and induce a very strong T cell immune response and high levels of neutralizing antibodies (Walsh et al., 2020, N Engl J Med 383:2439-2450; Sahin et al., 2020, Nature 586:594-599). These approved RNA vaccines require 30-100 μg of RNA per dose and two consecutive administrations at intervals of several weeks (prime-boost regimen). This results in 60-200 g of RNA required to immunize one million people. Thus, a dose reduction to less than 1 μg would have a major impact on the production time required to supply the population with vaccines against new pathogens.

[0003] A vaccine approach under test that promises to achieve a significant dose reduction is to use self-amplifying RNA (saRNA). SaRNA can be engineered from the alphavirus genome by replacing the alphavirus structural gene with an antigen for which an immune response is desired. SaRNA can encode the alphavirus replicase, which has all the enzymatic functions for replicating the saRNA molecule, thus resulting in amplification of the input vaccine dose.

[0004] Alphaviruses are typical representatives of plus-strand RNA viruses with an envelope. The hosts of alphaviruses include a wide range of organisms, including insects, fish, and mammals such as livestock and humans. Alphaviruses replicate in the cytoplasm of infected cells (see Jose et al., 2009, Future Microbiol. 4:837-856 for a review of the alphavirus life cycle). The total genome length of many alphaviruses typically ranges from 11,000 to 12,000 nucleotides, and the genomic RNA typically has a 5' cap and a 3' poly(A) tail. The alphavirus genome encodes non-structural proteins (involved in the transcription, modification, and replication of viral RNA and protein modification) and structural proteins (forming viral particles). Typically, there are two open reading frames (ORFs) in the genome. The four non-structural proteins (nsP1-nsP4) are typically encoded together by the first ORF, which starts near the 5' end of the genome, and the alphavirus structural proteins are found downstream of the first ORF and are encoded together by the second ORF, which extends near the 3' end of the genome. Typically, the first ORF is larger than the second ORF, with a ratio of approximately 2:1.

[0005] In cells infected with alphaviruses, only non-structural proteins are translated from the genomic RNA, and structural proteins can be translated from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNAs (mRNAs; Gould et al., 2010, Antiviral Res. 87:111-124). After infection, i.e., at the initial stage of the viral life cycle, the (+) strand genomic RNA acts directly like a messenger RNA for the translation of the open reading frame encoding the non-structural polyprotein (nsP1234). In some alphaviruses, an opal stop codon is present between the coding sequence of nsP3 and the coding sequence of nsP4: when translation terminates at the opal stop codon, the polyprotein P123, which includes nsP1, nsP2, and nsP3, is produced, and when the opal codon is read through, the polyprotein P1234, which also includes nsP4, is produced (Strauss & Strauss, 1994, Microbiol. Rev. 58:491-562; Rupp et al., 2015, J. Gen. Virology 96:2483-2500). nsP1234 is autocatalytically cleaved into nsP123 and nsP4 fragments. The polypeptides nsP123 and nsP4 associate to form a (-) strand replicase complex that uses the (+) strand genomic RNA as a template to transcribe (-) strand RNA. Typically, at a later stage, the nsP123 fragment is completely cleaved into the individual proteins nsP1, nsP2, and nsP3 (Shirako & Strauss, 1994, J. Virol. 68:1874-1885). All four proteins assemble to form a (+) strand replicase complex that uses the (-) strand complement of the genomic RNA as a template to synthesize new (+) strand genomic and subgenomic RNAs (Kim et al., 2004, Virology 323:153-163, Vasiljeva et al., 2003, J. Biol. Chem. 278:41636-41645).

[0006] In infected cells, nsP1 adds a 5' cap to subgenomic RNA and new genomic RNA (Pettersson et al., 1980, Eur. J. Biochem. 105:435-443; Rozanov et al., 1992, J. Gen. Virology 73:2129-2134), and nsP4 adds a polyadenylic acid [poly(A)] tail (Rubach et al., 2009, Virology 384:201-208). Thus, both subgenomic RNA and genomic RNA resemble messenger RNA (mRNA).

[0007] The synthesis of alphavirus RNA is also regulated by cis-acting RNA elements that contain four conserved sequence elements (CSEs; Strauss & Strauss, 1994, Microbiol. Rev. 58:491-562; and Frolov, 2001, RNA 7:1638-1651). The alphavirus genome contains four CSEs that are understood to be important for viral RNA replication in host cells. CSE 1, which is located at or near the 5’ end of the viral genome, is thought to function as a promoter for (+) strand synthesis from the (-) strand template. CSE 2, which is located downstream of CSE 1 but still near the 5’ end of the genome within the coding sequence of nsP1, is thought to act as a promoter for the initiation of (-) strand synthesis from the genomic RNA template (note that subgenomic RNA transcripts that do not contain CSE 2 do not function as templates for (-) strand synthesis). CSE 3 is located at the junction region between the coding sequences of the nonstructural and structural proteins and acts as a core promoter for efficient transcription of subgenomic transcripts. Finally, CSE 4, which is located immediately upstream of the poly(A) sequence in the 3’ untranslated region of the alphavirus genome, is understood to function as a core promoter for the initiation of (-) strand synthesis (Jose et al., 2009, Future Microbiol. 4:837-856). The CSE 4 and poly(A) tail of alphavirus are understood to function together for efficient (-) strand synthesis (Hardy & Rice, 2005, J. Virol. 79:4630-4639).

[0008] Alphavirus-derived vectors have been proposed for delivering foreign genetic information to target cells or target organisms. In a simple approach, the open reading frame encoding the alphavirus structural protein is replaced by an open reading frame encoding the protein of interest. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules (RNA molecules): one RNA molecule encodes the viral replicase (typically as the polyprotein nsP1234), and the other RNA molecule can be replicated in trans by said replicase (hence the name trans-replication system and / or nanotransreplicons). Trans-replication requires the presence of both of these nucleic acid molecules in a given host cell. The nucleic acid molecule that can be replicated in trans by the replicase must contain specific alphavirus sequence elements to enable recognition and RNA synthesis by the alphavirus replicase.

[0009] The Ebola virus (EBOV) outbreak in West Africa from 2014 to 2016, as well as the ongoing SARS-CoV-2 pandemic, have taught us that emerging and re-emerging viruses can suddenly lead to global disasters that may require millions of vaccine administrations within months. Due to increasing globalization and improved access to local areas, the incidence of emerging and re-emerging pathogens is on the rise (Murray et al., 2015, Proc. Natl. Acad. Sci. USA 112:12746-12751).

Prior Art Documents

Non-Patent Documents

[0010]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 16

Summary of the Invention

Problems to be Solved by the Invention

[0011] Therefore, there is still an urgent need for vaccines that can be rapidly developed and enable the treatment of diseases such as infectious diseases. The present invention meets such a need.

Means for Solving the Problems

[0012] The present invention generally relates to a composition comprising at least two replicable RNA molecules (replicons), each comprising a first open reading frame (ORF) encoding at least one peptide or protein comprising an antigen or epitope suitable for inducing an immune response against the antigen or epitope when administered to a subject, wherein at least one peptide or protein encoded by one of the replicable RNA molecules is different from at least one peptide or protein encoded by another replicable RNA molecule, and optionally, at least one of the replicable RNA molecules further comprises a second ORF encoding an RNA-dependent RNA polymerase (replicase) capable of replicating the replicable RNA molecules in the composition in cis or in trans. Optionally, the replicase capable of replicating the replicable RNA molecules in the composition in cis or in trans can be encoded by another (third) RNA molecule contained within the composition, which RNA molecule can be replicated by the encoded replicase, but preferably cannot be replicated by the encoded replicase.

[0013] The present invention is based, in part, on the observation that administration of a non-DNA-based vaccine composition of at least two antigens from a pathogenic organism encoded by at least two replicons can induce a specific antibody response to the antigens, as well as a CD4+ and / or CD8+ T cell response.

[0014] In one aspect, the present invention is a composition comprising at least two replicable RNA molecules, each comprising a first open reading frame (ORF) encoding at least one peptide or protein comprising an antigen or epitope suitable for inducing an immune response to the antigen or epitope when administered to a subject, wherein at least one peptide or protein encoded by one of the replicable RNA molecules is different from at least one peptide or protein encoded by another replicable RNA molecule, and optionally, at least one of the replicable RNA molecules further comprises a second ORF encoding an RNA-dependent RNA polymerase (replicase) capable of replicating the replicable RNA molecule in cis or in trans.

[0015] When used in the context of particular embodiments, the term "different", in the context of different peptides or proteins encoded by the first and second replicable RNA molecules, does not include the same peptides or proteins from different strains of a microorganism even if the amino acid sequences of the encoded peptides or proteins are different. Thus, in certain embodiments, the encoded peptides or proteins are not considered "different" due solely to differences in amino acid sequence. In one embodiment, different peptides or proteins are not the same peptides or proteins with different amino acid sequences. In one embodiment, different peptides or proteins have different functions or are derived from peptides or proteins having different functions, such as viral nucleoproteins and viral proteins expressed on the surface of a virus, such as glycoproteins. In one embodiment, different peptides or proteins are encoded by separate open reading frames in the genome of a pathogen, such as a virus.

[0016] The labeling of the first and second open reading frames does not necessarily indicate that the first open reading frame is 5' to the second open reading frame of the replicable RNA molecule. In one embodiment, the "second" open reading frame encoding a replicase within the replicable RNA molecule is 5' to an open reading frame encoding at least one peptide or protein.

[0017] In one embodiment, the composition can further comprise a third RNA molecule encoding a replicase capable of replicating the replicable RNA molecule and / or the third RNA molecule in cis or in trans. In one embodiment, the third RNA molecule is replicable. In one embodiment, the third RNA molecule is not replicable, i.e., is a non-replicating RNA molecule. In one embodiment, the composition comprises at least two replicable RNA molecules each not encoding a replicase and a third non-replicating RNA molecule encoding a replicase. In one embodiment, the non-replicating RNA is mRNA.

[0018] In one embodiment, the replicable RNA molecule in the composition can include an internal ribosome entry site (IRES) that controls the expression of a first ORF encoding a protein or peptide containing an antigen or epitope, and / or can include an internal ribosome entry site (IRES) that controls the expression of a second ORF encoding, for example, a replicase. In various embodiments, the IRES can be insensitive to cellular stress, and / or the IRES can be insensitive to interferon, preferably type I interferon, and / or the IRES can be a cellular or viral IRES, preferably a viral IRES. For example, the IRES can be derived from a virus selected from the group consisting of picornavirus, flavivirus, or dicistrovirus. In one embodiment, the IRES can be derived from picornavirus or dicistrovirus, preferably dicistrovirus. In one embodiment, the IRES can be a type IV IRES.

[0019] In one embodiment, the expression controlled by the IRES can be independent of the IRES trans-acting factor, and / or the expression controlled by the IRES can be independent of the cellular translation initiation factor. In one embodiment, the expression controlled by the IRES can be independent of the phosphorylation of eukaryotic initiation factor 2 (eIF2).

[0020] In one embodiment, both replicable RNA molecules can include a second ORF encoding a replicase. In one embodiment, neither of the replicable RNA molecules includes an ORF encoding a replicase.

[0021] In one embodiment, any of the RNA molecules, whether replicative or non-replicative, can include a 5' cap for driving the translation of the replicase and / or for driving the translation of a peptide or protein containing an antigen or epitope. The 5' cap can be a natural 5' cap or a 5' cap analog.

[0022] In one embodiment, at least one replicable RNA may comprise a 5' replication recognition sequence characterized in that at least one start codon is removed as compared to a natural alphavirus 5' replication recognition sequence. In one embodiment, all start codons except one are removed. In one embodiment, the 5' replication recognition sequence can comprise a sequence homologous to an open reading frame or a part thereof of a non-structural protein derived from a self-replicating virus, and the sequence homologous to an open reading frame or a part thereof of a non-structural protein derived from a self-replicating virus may be characterized in that it comprises the removal of at least one start codon as compared to a natural virus sequence. In one embodiment, the sequence homologous to an open reading frame or a part thereof of a non-structural protein derived from a self-replicating virus may be characterized in that it comprises the removal of at least the natural start codon of the open reading frame of the non-structural protein derived from the self-replicating virus. In one embodiment, the sequence homologous to an open reading frame or a part thereof of a non-structural protein derived from a self-replicating virus may be characterized in that it comprises the removal of at least one start codon other than the natural start codon of the open reading frame of the non-structural protein derived from the self-replicating virus. In one embodiment, the sequence homologous to an open reading frame or a part thereof of a non-structural protein derived from a self-replicating virus may be characterized in that it does not contain a start codon. In one embodiment, at least one nucleotide change can be introduced to compensate for nucleotide pairing disruption within at least one stem-loop introduced by the removal of at least one start codon.

[0023] In one embodiment, the open reading frame encoding a functional non-structural protein derived from a self-replicating virus (replicase) does not overlap with the 5' replication recognition sequence. In one embodiment, the first or second ORF may be present downstream of the 5' replication recognition sequence and upstream of the IRES.

[0024] In one embodiment, the replicable RNA may include a subgenomic promoter that controls the production of a subgenomic RNA encoding a protein or peptide. The subgenomic RNA may be a transcript of an RNA-dependent RNA polymerase (replicase) derived from a functional non-structural protein of a self-replicating virus. Optionally, the protein or peptide may be expressed from the subgenomic RNA as a template. In one embodiment, the first ORF encoding a protein or peptide controlled by the subgenomic promoter may be downstream of the second ORF encoding the replicase. In one embodiment, the subgenomic promoter may overlap with the second ORF.

[0025] In one embodiment, at least one of the replicable RNA molecules may include a 3' replication recognition sequence. In one embodiment, the 5' and / or 3' replication recognition sequences and the subgenomic promoter may be derived from a self-replicating virus, preferably the same self-replicating virus species.

[0026] In one embodiment, the replicable RNA molecule may be replicated by an RNA-dependent RNA polymerase derived from a functional non-structural protein of a self-replicating virus. The self-replicating virus may be an alphavirus selected from the group consisting of, for example, Venezuelan equine encephalitis complex virus, eastern equine encephalitis complex virus, western equine encephalitis complex virus, chikungunya virus, Semliki Forest virus complex virus, Sindbis virus, Barmah Forest virus, Middelburg virus and Ndumu virus.

[0027] In one embodiment, at least one of the RNA molecules may include a 3' poly(A) sequence.

[0028] In one embodiment, the first and / or second ORF may be adjacent to a 5' untranslated region (UTR) and / or a 3' UTR. In one embodiment, the 5' UTR and / or 3' UTR is not native to the alphavirus from which the replicase is derived. In one embodiment, the 5' UTR and / or 3' UTR is native to the alphavirus from which the replicase is derived.

[0029] In embodiments where two or more RNA molecules in the composition encode a replicase, the replicases can be from different alphaviruses, and thus the encoding replicase sequences in the RNA molecules are different. In embodiments where two or more RNA molecules in the composition encode a replicase, the replicases can be the same, and thus the encoding replicase sequences in the RNA molecules can be the same. In embodiments where two or more RNA molecules in the composition encode a replicase, the replicases can be variants from the same alphavirus. In one embodiment, the replicase is derived from Semliki Forest virus (SFV) or from Venezuelan equine encephalitis virus (VEEV). In one embodiment, the replicase comprises non-structural proteins nsp1, nsp2, nsp3, and nsp4.

[0030] In one embodiment, at least one, if not both, of the replicable RNA molecules does not contain an open reading frame of an intact alphavirus structural protein.

[0031] In one embodiment, the replicable RNA molecule comprises, in the 5’ to 3’ order, a 5’ cap, a 5’ UTR, an open reading frame encoding a replicase, an IRES, an open reading frame encoding an antigen, a 3’ UTR, and a polyA sequence. In this embodiment, both open reading frames can be directly translated, i.e., it is not necessary to generate daughter RNA to translate the open reading frame encoding the antigen. In one embodiment, the replicable RNA molecule comprises, in the 5’ to 3’ order, a 5’ cap, a 5’ UTR, an open reading frame encoding a replicase, a subgenomic promoter, an open reading frame encoding an antigen, a 3’ UTR, and a polyA sequence.

[0032] An exemplary polyA sequence is shown in SEQ ID NO: 78. In one embodiment, the polyA sequences useful for the RNA molecules described herein are those that are at least 75%, 80%, 85%, 90%, 95%, 98% or 99% homologous to SEQ ID NO: 78. Exemplary 5’ UTR sequences are shown in SEQ ID NO: 74 and 75. In one embodiment, the 5’ UTR sequences useful for the RNA molecules described herein are those that are at least 75%, 80%, 85%, 90%, 95%, 98% or 99% homologous to SEQ ID NO: 74 or 75. Exemplary 3’ UTR sequences are shown in SEQ ID NO: 76 and 77. In one embodiment, the 3’ UTR sequences useful for the RNA molecules described herein are those that are at least 75%, 80%, 85%, 90%, 95%, 98% or 99% homologous to SEQ ID NO: 76 or 77. An exemplary subgenomic promoter is shown in SEQ ID NO: 73. In one embodiment, the subgenomic promoters useful for the RNA molecules described herein are those that are at least 85%, 90%, 95%, 98% or 99% homologous to SEQ ID NO: 73. It is contemplated that the RNA molecules described herein can have any combination of functional 5’ UTR, 3’ UTR, polyA and subgenomic sequences. Thus, in certain embodiments, the RNA molecules described herein can have any combination of the exemplified 5’ UTR, 3’ UTR, polyA and subgenomic sequences.

[0033] In one embodiment, the antigen or epitope of the encoded protein or peptide is or is derived from an antigen of bacteria, virus, parasite or fungus. In one embodiment, the protein or peptide encoded by the first replicable RNA and the protein or peptide encoded by the second replicable RNA can both be obtained from or be derived from the same bacteria, virus, parasite or fungus. In one embodiment, the protein or peptide encoded by the first replicable RNA and the protein or peptide encoded by the second replicable RNA can each be obtained from or be derived from different strains of the same bacteria, virus, parasite or fungus, or can be obtained from or be derived from different pathogenic organisms, such as different viruses. In one embodiment, the peptide or protein encoded by the first replicable RNA and the peptide or protein encoded by the second replicable RNA are not the same peptide or protein derived from different strains of an organism. For example, the peptide or protein encoded by the first replicable RNA and the peptide or protein encoded by the second replicable RNA are not, for example, influenza hemagglutinin (HA) proteins from two different influenza strains. In one embodiment, the epitope of the encoded protein can be a T cell epitope.

[0034] In one embodiment, the protein or peptide encoded by the first replicable RNA is a surface-expressed protein or peptide, and the protein or peptide encoded by the second replicable RNA is not a surface-expressed protein or peptide. The surface-expressed protein or peptide and the non-surface-expressed protein or peptide can each be obtained from or derived from the same or different strains of the same bacterium, virus, parasite, or fungus, or can be obtained from or derived from different pathogenic organisms, such as different viruses. In one embodiment, the protein or peptide encoded by the first replicable RNA is not a surface-expressed protein or peptide, the protein or peptide encoded by the second replicable RNA is also not a surface-expressed protein or peptide, and is different from that encoded by the first replicable RNA. The different non-surface-expressed proteins or peptides can each be obtained from or derived from the same or different strains of the same bacterium, virus, parasite, or fungus, or can be obtained from or derived from different pathogenic organisms, such as different viruses. In one embodiment, the protein or peptide encoded by the first replicable RNA is a surface-expressed protein or peptide, the protein or peptide encoded by the second replicable RNA is also a surface-expressed protein or peptide, and is different from that encoded by the first replicable RNA. The surface-expressed proteins or peptides can each be obtained from or derived from the same or different strains of the same bacterium, virus, parasite, or fungus, or can be obtained from or derived from different pathogenic organisms, such as different viruses.

[0035] In one embodiment, the surface-expressed protein is expressed on the surface of the virus / viral particle, or if the virus is an enveloped virus, the surface-expressed protein is expressed on the surface of the viral envelope. For example, the surface-expressed protein is a viral capsid protein or a viral envelope or glycoprotein.

[0036] In one embodiment, a protein or peptide that is not a surface-expressed protein or peptide is a viral matrix protein, a viral nucleoprotein, or a viral capsid protein where the virus is an enveloped virus.

[0037] In one embodiment, the protein or peptide encoded by the first replicable RNA is a viral glycoprotein, and the protein or peptide encoded by the second replicable RNA is a viral nucleoprotein. The glycoprotein and the nucleoprotein can be obtained from, or be derived from, the same virus, optionally the same strain of the same virus.

[0038] In one embodiment, the surface-expressed protein or peptide can be the glycoprotein GP of Ebola virus. In one embodiment, the non-surface-expressed protein or peptide can be the matrix protein VP40 or the nucleoprotein NP of Ebola virus. The amino acid sequence of an exemplary Ebola virus GP protein is shown in SEQ ID NO: 89, and the amino acid sequence of an exemplary Ebola virus NP protein is shown in SEQ ID NO: 91.

[0039] In one embodiment, the surface-expressed protein or peptide can be the glycoprotein Gc of CCHFV virus. In one embodiment, the non-surface-expressed protein or peptide can be the nucleoprotein NP of CCHFV virus. The amino acid sequence of an exemplary CCHFV virus Gc protein is shown in SEQ ID NO: 93, and the amino acid sequence of an exemplary CCHFV virus NP protein is shown in SEQ ID NO: 95.

[0040] In one embodiment, the surface-expressed protein or peptide can be the spike (S) protein of the MERS CoV virus. In one embodiment, the non-surface-expressed protein or peptide can be the nucleoprotein NP of the MERS CoV virus. The amino acid sequence of an exemplary MERS CoV virus S protein is shown in SEQ ID NO: 97, and the amino acid sequence of an exemplary MERS CoV virus NP protein is shown in SEQ ID NO: 99.

[0041] In one embodiment, the amino acid sequence of the surface-expressed protein or peptide or the amino acid sequence of the non-surface-expressed protein or peptide can be at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the respective SEQ ID NOs above.

[0042] In one embodiment, the composition comprises a first replicable RNA molecule encoding an Ebola virus GP protein having the amino acid sequence shown in SEQ ID NO: 89 and a second replicable RNA molecule encoding an Ebola virus NP protein having the amino acid sequence shown in SEQ ID NO: 91. In one embodiment, the composition comprises a first replicable RNA molecule encoding a CCHFV virus Gc protein having the amino acid sequence shown in SEQ ID NO: 93 and a second replicable RNA molecule encoding a CCHFV NP protein having the amino acid sequence shown in SEQ ID NO: 95. In one embodiment, the composition comprises a first replicable RNA molecule encoding a MERS CoV S protein having the amino acid sequence shown in SEQ ID NO: 97 and a second replicable RNA molecule encoding a MERS CoV virus NP protein having the amino acid sequence shown in SEQ ID NO: 99. Optionally, the first or second replicable RNA molecule encodes a replicase. The composition may optionally be replicable or non-replicable and comprises a third RNA molecule encoding an RNA replicase. The third RNA molecule can be a non-replicable mRNA.

[0043] In a preferred embodiment, the first and second replicable RNA molecules in the composition do not encode a replicase, and the composition further comprises an mRNA encoding a replicase capable of replicating the first and second replicable RNA molecules in trans.

[0044] In one embodiment, the immune response induced against an antigen or epitope can be an antibody response against the antigen or epitope. In one embodiment, the immune response induced against an antigen or epitope can be an increase in the activity of CD4+ T cells and / or CD8+ T cells. In one embodiment, the immune response induced against an antigen or epitope can be an increase in the activity of both CD4+ and CD8+ T cells. In one embodiment, the immune response induced against an antigen or epitope can be an antibody response against the antigen or epitope, and an increase in the activity of both CD4+ T cells and CD8+ T cells.

[0045] In one embodiment, the protein or peptide encoded by the first replicable RNA can be an Ebola virus protein or a fragment thereof or an epitope of an Ebola virus protein, and the protein or peptide encoded by the second replicable RNA can be a different Ebola virus protein or a fragment thereof or an epitope of a different Ebola virus protein. The protein or peptide encoded by at least one replicable RNA molecule can be a structural Ebola virus protein selected from the group consisting of glycoprotein (GP), nucleoprotein (NP), polymerase cofactor (VP35), VP40, transcription factor (VP30), VP24, or RNA-dependent RNA polymerase (L), or a fragment thereof, or an epitope of an Ebola virus structural protein. The epitope can be a T cell epitope. In one embodiment, the protein or peptide encoded by the first replicable RNA can be a viral surface protein such as Ebola virus (EBOV) GP, CCHFV Gc-TM, or MERS-CoV S1, and the protein or peptide encoded by the second replicable RNA can be a protein derived from a nucleoprotein complex such as Ebola virus NP, CCHFV NP, or MERS-CoV NP. The protein or peptide can include a T cell epitope.

[0046] In one embodiment, the protein or peptide encoded by at least one replicable RNA molecule can be expressed as a fusion protein, for example, the protein or peptide can be fused to a targeting motif or a secretion motif.

[0047] In one embodiment, the first of at least two replicable RNA molecules included in the composition encodes a protein or an epitope thereof of a strain of a pathogen such as Ebola virus, and the second of at least two replicable RNA molecules included in the composition encodes the same or a different protein or an epitope thereof from a different strain of the pathogen. For example, one replicable RNA molecule encodes the GP protein or an epitope thereof of the Ebola virus Zaire subtype, and the second replicable molecule encodes the GP protein or an epitope thereof of the Ebola virus Sudan strain. In one embodiment, the first of at least two replicable RNA molecules included in the composition encodes several proteins or epitopes thereof from a strain of the pathogen, and the second of at least two RNA molecules included in the composition encodes several proteins or epitopes thereof from a different strain of the same pathogen. In one embodiment, the pathogen is Ebola virus or Crimean-Congo hemorrhagic fever virus (CCHFV). Exemplary Ebola virus strains include Zaire, Sudan, Tai Forest, and Bundibugyo. Exemplary Zaire strains include H.sapiens-wt / SLE / 2014 / Makona-EM095B (GenBank: KM034551.1), H.sapiens-wt / GIN / 2014 / Makona-Gueckedou-C07 (GenBank: KJ660347.2), H.sapiens-tc / COD / 1976 / Yambuku-Mayinga (GenBank NC_002549.1), Ebola virus strain EBO-0003 (GenBank: MZ605321.1), and Ebola virus strain EBO-0002 (GenBank: MZ605320.1). Other strains include those involved in the 2013-2016 EBOV epidemics in Guinea, Liberia, and Sierra Leone in West Africa, and their sequences are available in the European Nucleotide Archive under accession number PRJEB43650 and on github.com under PFHVG / EBOVsequencing.

[0048] In one embodiment, the protein or peptide encoded by at least one of the replicable RNA molecules can be the Ebola virus structural GP protein or a fragment thereof, or an epitope of the GP protein. The GP protein can be derived from or obtained from the Ebola virus Zaire subtype, virus strain H.sapiens-wt / SLE / 2014 / Makona-EM095B (GenBank: KM034551.1). In one embodiment, the protein or peptide encoded by at least one of the replicable RNA molecules can be the Ebola virus structural NP protein or a fragment thereof, or an epitope of the NP protein. The NP antigen can be derived from or obtained from the Ebola virus Zaire subtype, virus strain H.sapiens-wt / GIN / 2014 / Makona-EM096 (GenBank: KM034551.1). In one embodiment, the GP amino acid sequence of the Ebola virus sequence H.sapiens-wt / GIN / 2014 / Makona-Gueckedou-C07 (GenBank: KJ660347.2) can be mutated at position 82, for example, by substitution from alanine to valine (Ala82Val), compared to the C07 wild type.

[0049] In one embodiment, the protein or peptide encoded by at least one of the replicable RNA molecules can be the Ebola virus structural GP protein or a fragment thereof, or an epitope of the GP protein. The GP protein can be derived from or obtained from the Ebola virus sequence H.sapiens-wt / GIN / 2014 / Makona-Gueckedou-C07 (GenBank: KJ660347.2). In one embodiment, the protein or peptide encoded by at least one of the replicable RNA molecules can be the Ebola virus structural NP protein or a fragment thereof, or an epitope of the NP protein. The NP antigen can be derived from or obtained from the Ebola virus sequence H.sapiens-wt / GIN / 2014 / Makona-Gueckedou-C07 (GenBank: KJ660347.2). In one embodiment, the NP amino acid sequence of the Ebola virus Zaïre subtype, virus strain H.sapiens-wt / GIN / 2014 / Makona-EM096 (GenBank: KM034551.1) can be mutated at position 111, for example, by substitution from alanine to cysteine (Ala111Cys) compared to the EM096 wild type.

[0050] In one embodiment, the protein or peptide encoded by at least one of the replicable RNA molecules can be the Crimean-Congo hemorrhagic fever virus (CCHFV) structural GP protein or a fragment thereof, or an epitope of the GP protein. The GP protein can be derived from or obtained from the Crimean-Congo hemorrhagic fever virus strain Afg09-2990 sequence (GenBank: HM452306.1). In one embodiment, the protein or peptide encoded by at least one of the replicable RNA molecules can be the Crimean-Congo hemorrhagic fever virus (CCHFV) structural NP protein or a fragment thereof, or an epitope of the NP protein. The NP antigen can be derived from or obtained from the Crimean-Congo hemorrhagic fever virus (CCHFV) strain Afg09-2990 sequence (GenBank: HM452305.1).

[0051] In one embodiment, at least one, preferably both, of the replicable RNA molecules can be codon-optimized, for example, for human use, by replacing only the worst (suboptimal) triplets and optionally without increasing the overall GC content of the molecule.

[0052] In one embodiment, the protein or peptide encoded by the first replicable RNA molecule can be an Ebola virus structural GP protein or a fragment thereof, or an epitope of the GP protein, and the protein or peptide encoded by the second replicable RNA molecule can be an Ebola virus structural NP protein or a fragment thereof, or an epitope of the NP protein. In one embodiment, the amino acid sequence of an exemplary Ebola virus glycoprotein is shown in SEQ ID NO: 89. In one embodiment, the amino acid sequence of an exemplary Ebola virus nucleoprotein is shown in SEQ ID NO: 91. In one embodiment, the nucleotide sequence of a replicable RNA encoding an exemplary Ebola virus glycoprotein is shown in SEQ ID NO: 79. In one embodiment, the nucleotide sequence of a replicable RNA encoding an exemplary Ebola virus nucleoprotein is shown in SEQ ID NO: 80.

[0053] In one embodiment, the ratio (molar ratio) of the number of the first replicable RNA molecules to the number of the second replicable RNA molecules in the composition can vary from approximately equal numbers of molecules. In one embodiment, the ratio of the first replicable RNA molecule to the second replicable RNA molecule can be in the range of about 20:1 to about 1:20, about 15:1 to about 1:15, about 10:1 to about 1:10, or about 5:1 to about 1:5. In one embodiment, the ratio can be about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10.

[0054] In one embodiment, the composition further comprises a reagent capable of forming particles with the replicable RNA molecule, for example, the reagent can be a lipid or a polyalkyleneimine. In various embodiments, the lipid can comprise a cationic head group and / or the lipid can be a pH-responsive lipid and / or the lipid can be a PEGylated lipid. In one embodiment, the reagent can be conjugated to polysarcosine.

[0055] In one embodiment, the particles formed from the replicable RNA molecule and the reagent can be polymer-based polyplexes (PLX) or lipid nanoparticles (LNP), and the LNP is preferably a lipoplex (LPX) or a liposome. In one embodiment, the particles can further comprise at least one phosphatidylserine. In one embodiment, the particles are nanoparticles, wherein (i) the number of positive charges in the nanoparticles does not exceed the number of negative charges in the nanoparticles and / or (ii) the nanoparticles have a neutral or net negative charge and / or (iii) the charge ratio of positive to negative charges in the nanoparticles is 1.4:1 or less and / or (iv) the zeta potential of the nanoparticles is 0 or less. In one embodiment, the charge ratio of positive to negative charges in the nanoparticles can be from 1.4:1 to 1:8, preferably from 1.2:1 to 1:4.

[0056] In one embodiment, the nanoparticles can comprise at least one lipid, preferably at least one cationic lipid, and optionally, the positive charge is provided by at least one cationic lipid and the negative charge is provided by the replicable RNA molecule.

[0057] In one embodiment, the nanoparticles can further include at least one helper lipid. For example, the helper lipid can be a neutral lipid. In various embodiments, the at least one cationic lipid can include 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTMA), 1,2 - dioleyloxy - 3 - dimethylaminopropane (DODMA), and / or 1,2 - dioleoyl - 3 - trimethylammonium propane (DOTAP). In various embodiments, the at least one helper lipid can include 1,2 - di - (9Z - octadecenoyl) - sn - glycero - 3 - phosphoethanolamine (DOPE), cholesterol (Chol), 1,2 - dioleoyl - sn - glycero - 3 - phosphocholine (DOPC), and / or 1,2 - distearoyl - sn - glycero - 3 - phosphocholine (DSPC). In various embodiments, the molar ratio of the at least one cationic lipid to the at least one helper lipid can be from 10:0 to 3:7, preferably from 9:1 to 3:7, 4:1 to 1:2, 4:1 to 2:3, 7:3 to 1:1, or 2:1 to 1:1, preferably about 1:1.

[0058] In one embodiment, the nanoparticles can be lipoplexes containing DODMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and the charge ratio of the positive charge in DODMA to the negative charge in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2. In one embodiment, the nanoparticles can be lipoplexes containing DODMA and cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and the charge ratio of the positive charge in DODMA to the negative charge in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2. In one embodiment, the nanoparticles can be lipoplexes containing DODMA and DSPC in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and the charge ratio of the positive charge in DODMA to the negative charge in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2. In one embodiment, the nanoparticles are lipoplexes containing DODMA:cholesterol:DOPE:PEGcerC16 in a molar ratio of 40:48:10:2.

[0059] In one embodiment, the nanoparticles can be lipoplexes containing DOTMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and the charge ratio of the positive charges in DOTMA to the negative charges in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2. In one embodiment, the nanoparticles can be lipoplexes containing DOTMA and cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and the charge ratio of the positive charges in DOTMA to the negative charges in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2. In one embodiment, the nanoparticles can be lipoplexes containing DOTAP and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and the charge ratio of the positive charges in DOTMA to the negative charges in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2.

[0060] In one embodiment, the reagent can include a lipid, and the particles formed can be LNPs that form a complex with and / or encapsulate a replicable RNA molecule. In one embodiment, the reagent can include a lipid, and the particles formed can be vesicles that encapsulate a replicable RNA molecule, preferably unilamellar liposomes.

[0061] In one embodiment, the reagent is a polyalkyleneimine. For example, the molar ratio (N:P ratio) of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphorus atoms (P) in the replicable RNA molecule can be 2.0 to 15.0, preferably 6.0 to 12.0, and / or the ionic strength of the composition can be 50 mM or less, preferably the concentration of monovalent cationic ions can be 25 mM or less, and the concentration of divalent cationic ions can be 20 μM or less.

[0062] In one embodiment, the particles to be formed can be polyplexes.

[0063] In one embodiment, the polyalkyleneimine has the following general formula (I): [Chemical formula] (wherein, R is H, an acyl group or the following general formula (II): [Chemical formula] is a group containing, wherein, R 1 is H or the following general formula (III): [Chemical formula] is a group containing, n, m and l are independently selected from integers of 2 to 10; and p, q and r are integers, and the sum of p, q and r is such that the average molecular weight of the polymer is 1.5×10 2 ~10 7 Da, preferably 5000~10 5 Da, more preferably 10000~40000Da, still more preferably 15000~30000Da, and even more preferably 20000~25000Da) can be included. In one embodiment, n, m and l can be independently selected from 2, 3, 4 and 5, preferably 2 and 3, and / or R 1 can be H. In one embodiment, R can be H or an acyl group.

[0064] In one embodiment, the polyalkyleneimine can include polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. In one embodiment, at least 92% of the N atoms in the polyalkyleneimine can be protonatable.

[0065] In one embodiment, the composition can further comprise one or more peptide-based adjuvants, which optionally include immunomodulatory molecules such as cytokines, lymphokines, and / or costimulatory molecules. In one embodiment, the composition can further comprise one or more additives, which are optionally selected from the group consisting of buffering substances, saccharides, stabilizers, cryoprotectants, lyoprotectants, and chelating agents. The buffering substance can include at least one selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), acetic acid, acetate buffer and analogs, phosphoric acid and phosphate buffer, and citric acid and citrate buffer. The saccharides can include at least one selected from the group consisting of monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides, preferably glucose, trehalose, and sucrose. The cryoprotectant can include at least one selected from the group consisting of glycols such as ethylene glycol, propylene glycol, and glycerol. In one embodiment, the chelating agent can include EDTA.

[0066] In one embodiment, the replicable RNA molecules in the composition are present at a relative ratio of 6:1 to 1:6. In one embodiment, the replicable RNA molecules in the composition are present at a relative ratio of 3:1 to 1:3. In one embodiment, the replicable RNA molecules in the composition are present at a relative ratio of 2:1 to 1:2. In one embodiment, the replicable RNA molecules in the composition are present at a ratio of 1:1. In one embodiment, the ratio is determined by the molecular weight of the RNA molecule.

[0067] In one embodiment, the composition of the invention of at least two replicable RNA molecules can be a vaccine.

[0068] In one aspect, the present invention relates to a pharmaceutical composition comprising a composition of the present invention of at least two replicable RNA molecules and a pharmaceutically acceptable carrier. In various embodiments, the pharmaceutical composition can be formulated for intradermal, intranasal, intrapulmonary, subcutaneous, and / or intramuscular administration, such as by injection.

[0069] In one aspect, the present invention relates to the use of a composition of the present invention of at least two replicable RNA molecules in the induction of an immune response or in a treatment such as vaccination. In one aspect, the present invention relates to the use of a composition of the present invention of at least two replicable RNA molecules in a method of inducing an immune response specific for a protein or peptide encoded in a subject, preferably the subject is a mammal, more preferably the mammal is a human, and the method comprises administering a pharmaceutical composition of the present invention.

[0070] In one aspect, the present invention relates to a method of inducing an immune response specific for at least two antigens or epitopes in a subject, the method comprising administering a pharmaceutical composition of the present invention to the subject, preferably the subject is a mammal, more preferably the mammal is a human.

[0071] In one embodiment, the immune response can include activation of T cells and / or B cells, preferably the activated T cells include T helper cells and cytotoxic T cells. In one embodiment, the immune response can include activation of antigen-specific T helper cells, optionally the T helper cells proliferate, release T cell cytokines, and mediate the proliferation and / or activation of antigen-specific cytotoxic T cells. In one embodiment, the immune response can include activation of antigen-specific T helper cells, and the T helper cells stimulate B cell proliferation, antibody class switching, production and / or secretion of neutralizing antibodies.

[0072] In one aspect, the present invention relates to a method for producing at least two proteins or peptides of interest in a cell, comprising inoculating a cell with the pharmaceutical composition of the present invention. In one aspect, the present invention relates to a method for producing at least two proteins or peptides of interest in a subject, comprising administering the pharmaceutical composition of the present invention to the subject. In one embodiment, the method comprises administering to the subject a first replicable RNA molecule comprising the nucleotide sequence set forth in SEQ ID NO: 27 and a second replicable RNA molecule comprising the nucleotide sequence set forth in SEQ ID NO: 29. In one embodiment, the method comprises administering to the subject a replicable RNA molecule set forth in SEQ ID NO: 30 and a replicable RNA molecule set forth in SEQ ID NO: 31.

[0073] In one aspect, the present invention is a method for treating or preventing a bacterial, viral, parasitic or fungal infection in a subject, comprising administering to the subject a composition comprising at least two replicable RNA molecules, each comprising a first open reading frame (ORF) encoding at least one peptide or protein comprising an antigen or epitope suitable for inducing an immune response against a bacterium, virus, parasite or fungus, wherein at least one peptide or protein encoded by one of the replicable RNA molecules is different from at least one peptide or protein encoded by another of the replicable RNA molecules, and at least one of the replicable RNA molecules further comprises a second ORF encoding an RNA-dependent RNA polymerase (replicase) capable of replicating the replicable RNA molecule in cis or in trans. In one embodiment, the method comprises administering to the subject a first replicable RNA molecule comprising the nucleotide sequence set forth in SEQ ID NO: 27 and a second replicable RNA molecule comprising the nucleotide sequence set forth in SEQ ID NO: 29. In one embodiment, the method comprises administering to the subject a replicable RNA molecule set forth in SEQ ID NO: 30 and a replicable RNA molecule set forth in SEQ ID NO: 31.

[0074] In one embodiment, the immune response is a specific immune response to bacteria, viruses, parasites or fungi respectively, and / or the immune response reduces the severity of one or more symptoms of an infectious disease. In one embodiment, the infectious disease can be a viral infectious disease, and optionally, the infectious disease is an Ebola virus infectious disease.

[0075] In one embodiment, the treatment method includes only a single administration of the composition, or the treatment method includes multiple administrations of the composition. In one embodiment, the method may further include administering a booster dose of the pharmaceutical composition of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0076] The present invention will be described in detail below, but it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, and these may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention, and the scope of the present invention is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0077] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0078] The practice of the present invention uses conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the literature of the art (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989), unless otherwise indicated.

[0079] In the following, the elements of the present invention are described. It should be understood that these elements are listed with specific embodiments, but they may be combined in any manner and in any number to create additional embodiments. The examples and preferred embodiments described variously should not be construed as limiting the present invention to only the explicitly described embodiments. This description is to be understood as disclosing and encompassing embodiments that combine the explicitly described embodiments with any number of disclosed elements and / or preferred elements. Further, any rearrangement and combination of all the elements described in this application should be considered to be disclosed by this description, unless otherwise specifically indicated in the context.

[0080] The term "about" means approximately or nearly and, in the context of the numerical values or ranges described herein, preferably means + / - 10% of the recited or claimed numerical value or range.

[0081] The terms "a", "an", and "the" and similar references used in the context of describing the present invention (especially in the context of the claims) are to be construed to include both the singular and the plural unless specifically indicated otherwise herein or clearly contradicted by the context. The recitation of a range of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless specifically indicated otherwise herein, each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless specifically indicated otherwise herein or clearly contradicted by the context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is merely intended to better illustrate the invention and does not impose a limitation on the scope of the claimed invention. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0082] Unless otherwise specified, the term "comprising" is used in the context of this document to indicate that in addition to the members of the list introduced by "comprising", further members may optionally be present. However, the term "comprising" is contemplated as a specific embodiment of the present invention to encompass the possibility that no further members are present, i.e., for the purposes of this embodiment, "comprising" is to be understood to have the meaning of "consisting of".

[0083] An indication of the relative amount of a component characterized by a generic name means the total amount of all specific variants or members included by said generic name. When a specific component defined by a generic name is specified to be present in a particular relative amount and is further characterized as being a particular variant or member included by the generic name, it means that no other variants or members included by the generic name are additionally present such that the total relative amount of the component included by the generic name exceeds the specified relative amount, more preferably that no other variants or members included by the generic name are present at all.

[0084] Throughout the text of this specification, several references are cited. Each of the references cited in this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) is hereby incorporated by reference in its entirety, either above or below. Nothing in this specification shall be construed as an admission that the present invention has any right to antedate such disclosure.

[0085] As used herein, terms such as "reduce" or "inhibit" preferably mean the ability to cause an overall decrease at a level of 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more. The term "inhibit" or similar phrases includes complete or substantially complete inhibition, i.e., a decrease to zero or substantially zero.

[0086] Terms such as "increase" or "enhance" preferably relate to an increase or enhancement of at least about 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%.

[0087] The term "net charge" refers to the charge of an entire object such as a compound or a particle.

[0088] Ions having an overall net positive charge are cations, and ions having an overall net negative charge are anions. Thus, according to the present invention, an anion is an ion having more electrons than protons, giving a net negative charge, and a cation is an ion having fewer electrons than protons, giving a net positive charge.

[0089] For a given compound or particle, the terms "charged", "net charge", "negatively charged" or "positively charged" refer to the net charge of the given compound or particle when dissolved or suspended in water at pH 7.0.

[0090] As used herein, the term "nucleic acid" also includes chemical derivatizations of nucleic acids on nucleotide bases, sugars or phosphates, as well as nucleic acids containing unnatural nucleotides and nucleotide analogs. In some embodiments, the nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Generally, a nucleic acid molecule or nucleic acid sequence refers to a nucleic acid that is preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). According to the present invention, nucleic acids include genomic DNA, cDNA, mRNA, viral RNA, recombinantly prepared molecules and chemically synthesized molecules. According to the present invention, nucleic acids can be in the form of single-stranded or double-stranded linear molecules or covalently closed circular molecules.

[0091] As used herein, the term "nucleic acid sequence" refers to the sequence of nucleotides in a nucleic acid, such as ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). This term can refer to an entire nucleic acid molecule (such as a single strand of an entire nucleic acid molecule) or a portion thereof (such as a fragment).

[0092] As used herein, the term "RNA" or "RNA molecule" relates to a molecule that contains ribonucleotide residues and preferably consists entirely or substantially of ribonucleotide residues. The term "ribonucleotide" relates to a nucleotide having a hydroxyl group at the 2'-position of a β-D-ribofuranosyl group. The term "RNA" includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially or fully purified RNA, essentially pure RNA, synthetic RNA, and recombinantly produced RNA such as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or modification of one or more nucleotides. Such modifications can include, for example, the addition of non-nucleotide substances to the ends (one or both) or within the RNA, such as at one or more nucleotides of the RNA. The nucleotides in an RNA molecule can also include non-standard nucleotides such as unnatural nucleotides or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs can be called analogs, particularly analogs of naturally occurring RNA.

[0093] According to the present invention, the RNA can be single-stranded or double-stranded. In some embodiments of the present invention, single-stranded RNA is preferred. The term "single-stranded RNA" generally refers to an RNA molecule in which complementary nucleic acid molecules (typically complementary RNA molecules) are not associated. A single-stranded RNA can contain self-complementary sequences that allow a portion of the RNA to fold back and form secondary structure motifs including, but not limited to, base pairs, stems, stem-loops, and bulges. A single-stranded RNA can exist as a minus strand [(-) strand] or a plus strand [(+) strand]. The (+) strand is the strand that contains or encodes genetic information. The genetic information can be, for example, a polynucleotide sequence encoding a protein. When the (+) strand RNA encodes a protein, the (+) strand can function directly as a template for translation (protein synthesis). The (-) strand is the complement of the (+) strand. In the case of double-stranded RNA, the (+) strand and the (-) strand are two separate RNA molecules, and both of these RNA molecules associate with each other to form double-stranded RNA ("double-stranded RNA").

[0094] The term "stability" of RNA relates to the "half-life" of the RNA. "Half-life" relates to the period required to remove half of the activity, amount, or number of molecules. In the context of the present invention, the half-life of the RNA is an indicator of the stability of said RNA. The half-life of the RNA can affect the "duration of expression" of the RNA. An RNA having a long half-life can be expected to be expressed over a long period of time.

[0095] The term "translation efficiency" relates to the amount of translation product provided by an RNA molecule within a specific period of time.

[0096] "Fragment" relates to a nucleic acid sequence and refers to a sequence that is a part of the nucleic acid sequence, i.e., a nucleic acid sequence shortened at the 5'-end and / or 3'-end. Preferably, a fragment of a nucleic acid sequence contains at least 80%, preferably at least 90%, 95%, 96%, 97%, 98%, or 99% of the nucleotide residues from the said nucleic acid sequence. In the present invention, a fragment of an RNA molecule that retains the stability and / or translation efficiency of RNA is preferred.

[0097] "Fragment" relates to an amino acid sequence (peptide or protein) and refers to a sequence that is a part of the amino acid sequence, i.e., an amino acid sequence shortened at the N-terminus and / or C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame lacking the 3'-end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame lacking the 5'-end of the open reading frame as long as the truncated open reading frame contains a start codon that functions to initiate translation. A fragment of an amino acid sequence contains, for example, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from the amino acid sequence.

[0098] For example, the term "variant" with respect to nucleic acid and amino acid sequences according to the present invention includes any variant, particularly mutants, viral strain variants, splice variants, conformations, isoforms, allelic variants, species variants and species homologs, particularly those that occur naturally. Allelic variants are associated with changes in the normal sequence of a gene, and their significance is often unclear. Complete gene sequencing often identifies a number of allelic variants for a given gene. With respect to nucleic acid molecules, the term "variant" includes degenerate nucleic acid sequences, and degenerate nucleic acids according to the present invention are nucleic acids in which the codon sequence differs from the reference nucleic acid due to the degeneracy of the genetic code. Species homologs are nucleic acid or amino acid sequences that originate from a species different from that of a given nucleic acid or amino acid sequence. Viral homologs are nucleic acid or amino acid sequences that originate from a virus different from that of a given nucleic acid or amino acid sequence. For example, hemagglutinin from one influenza strain and hemagglutinin from another influenza strain having a different amino acid sequence are variants of each other. In another example, the same bacterial protein derived from two different bacterial strains and having different amino acid sequences are variants of each other.

[0099] Nucleic acid variants include deletions, additions, mutations, substitutions and / or insertions of single or multiple nucleotides as compared to the reference nucleic acid. Deletions include the removal of one or more nucleotides from the reference nucleic acid. Addition variants include the fusion of one or more nucleotides, such as 1, 2, 3, 5, 10, 20, 30, 50 or more nucleotides, at the 5' end and / or 3' end. In the case of substitutions, at least one nucleotide in the sequence is removed and at least one other nucleotide is inserted in its place (such as transversions and transitions). Mutations include abasic sites, cross-linked sites, and bases that have been chemically modified or altered. Insertions include the addition of at least one nucleotide to the reference nucleic acid.

[0100] According to the present invention, a "nucleotide change" can refer to a deletion, addition, mutation, substitution, and / or insertion of one or more nucleotides as compared to a reference nucleic acid. In some embodiments, a "nucleotide change" is selected from the group consisting of a single nucleotide deletion, a single nucleotide addition, a single nucleotide mutation, a single nucleotide substitution, and / or a single nucleotide insertion as compared to a reference nucleic acid. According to the present invention, a nucleic acid variant can contain one or more nucleotide changes as compared to a reference nucleic acid.

[0101] A variant of a particular nucleic acid sequence preferably has at least one functional property of the particular sequence and is preferably functionally equivalent to the particular sequence, for example, a nucleic acid sequence that exhibits the same or similar properties as the properties of the particular nucleic acid sequence.

[0102] As described below, some embodiments of the present invention are characterized, inter alia, by nucleic acid sequences that are homologous to other nucleic acid sequences. These homologous sequences are variants of other nucleic acid sequences.

[0103] Preferably, the degree of identity between a given nucleic acid sequence and a nucleic acid sequence that is a variant of the given nucleic acid sequence is at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, or most preferably at least 95%, 96%, 97%, 98% or 99%. The degree of identity is preferably given for a region of at least about 30, at least about 50, at least about 70, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, or at least about 400 nucleotides. In preferred embodiments, the degree of identity is given for the full length of the reference nucleic acid sequence.

[0104] "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two polypeptide or nucleic acid sequences indicates the percentage of amino acids or nucleotides that are identical between the sequences.

[0105] The term "percent identity" is specifically intended to refer to the percentage of nucleotides that are identical in the optimal alignment between two arrays being compared, said percentage being purely statistical and the differences between the two arrays may be randomly distributed over the entire length of the arrays, and the arrays being compared may include additions or deletions compared to a reference array in order to obtain the optimal alignment between the two arrays. The comparison of two arrays is usually done by comparing said arrays with respect to segments or "comparison windows" after optimal alignment in order to identify local regions of the corresponding arrays. The optimal alignment for comparison can be done manually or using local homology algorithms by Smith and Waterman, 1981, Ads App. Math. 2, 482, by Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, and by the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or by invoking computer programs that use said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA of the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0106] The percent identity is obtained by determining the number of identical positions where the arrays being compared match, dividing this number by the number of positions being compared, and multiplying the result by 100.

[0107] For example, the BLAST program "BLAST 2 sequences" available at the website http: / / www.ncbi.nlm.nih.gov / blast / bl2seq / wblast2.cgi may be used.

[0108] When two arrays are complementary to each other, a nucleic acid can "hybridize to" or "hybridize with" another nucleic acid. When two arrays can form a stable double strand with each other, the nucleic acid is "complementary" to another nucleic acid. According to the present invention, hybridization is preferably carried out under conditions (stringent conditions) that allow specific hybridization between polynucleotides. Stringent conditions are described, for example, in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Editors, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989 or Current Protocols in Molecular Biology, F.M. Ausubel et al., Editors, John Wiley & Sons, Inc., New York, and refer, for example, to hybridization in a hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH2PO4 (pH 7), 0.5% SDS, 2 mM EDTA) at 65°C. SSC is 0.15 M sodium chloride / 0.15 M sodium citrate, pH 7. After hybridization, the membrane on which the DNA has been transcribed is washed, for example, in 2×SSC at room temperature and then in 0.1 - 0.5×SSC / 0.1×SDS at a temperature up to 68°C.

[0109] The percentage of complementarity indicates the proportion of consecutive residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Fully complementary" or "wholly complementary" means that all consecutive residues of a nucleic acid sequence hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence. Preferably, the degree of complementarity according to the present invention is at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, or most preferably at least 95%, 96%, 97%, 98%, or 99%. Most preferably, the degree of complementarity according to the present invention is 100%.

[0110] The term "derivative" includes any chemical derivatization of a nucleic acid on a nucleotide base, sugar, or phosphate. The term "derivative" also includes nucleic acids containing nucleotides and nucleotide analogs that do not occur naturally. Preferably, the derivatization of the nucleic acid enhances its stability.

[0111] A "nucleic acid sequence derived from a nucleic acid sequence" refers to a nucleic acid that is a variant of the nucleic acid from which it is derived. Preferably, when substituting a specific sequence within an RNA molecule, the sequence that is a variant of the specific sequence retains the stability and / or translation efficiency of the RNA.

[0112] "nt" is an abbreviation for one nucleotide or for a plurality of nucleotides, preferably consecutive nucleotides in a nucleic acid molecule.

[0113] According to the present invention, the term "codon" refers to a base triplet in a coding nucleic acid that specifies which amino acid is to be added next during protein synthesis on a ribosome.

[0114] The terms "transcription" and "transcribe" relate to the process by which a nucleic acid molecule having a particular nucleic acid sequence (the "nucleic acid template") is read by RNA polymerase, resulting in the production by the RNA polymerase of a single-stranded RNA molecule. During transcription, the genetic information in the nucleic acid template is transcribed. The nucleic acid template may be DNA; however, in the case of transcription from, for example, an alphavirus nucleic acid template, the template is typically RNA. Subsequently, the transcribed RNA may be translated into protein. According to the present invention, the term "transcription" includes "in vitro transcription", and the term "in vitro transcription" relates to the process by which RNA, particularly mRNA, is synthesized in vitro in a cell-free system. Preferably, a cloning vector is applied for the production of the transcript. These cloning vectors are generally referred to as transcription vectors and are encompassed by the term "vector" according to the present invention. The cloning vector is preferably a plasmid. According to the present invention, the RNA is preferably in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0115] The single-stranded nucleic acid molecule produced during transcription typically has a nucleic acid sequence that is the complementary sequence of the template.

[0116] According to the present invention, the term "template" or "nucleic acid template" or "template nucleic acid" generally refers to a nucleic acid sequence that can be replicated or transcribed.

[0117] The terms "nucleic acid sequence transcribed from a nucleic acid sequence" and similar terms refer, where appropriate, to a nucleic acid sequence as part of a complete RNA molecule that is the transcription product of the template nucleic acid sequence. Typically, the transcribed nucleic acid sequence is a single-stranded RNA molecule.

[0118] According to the present invention, the "3'-end of a nucleic acid" refers to its end having a free hydroxy group. In a schematic diagram of a double-stranded nucleic acid, particularly DNA, the 3'-end is always on the right side. The "5'-end of a nucleic acid" refers to its end having a free phosphate group. In a schematic diagram of a double-stranded nucleic acid, particularly DNA, the 5'-end is always on the left side. 5'-end 5'- -P-NNNNNNN-OH-3' 3'-end 3'-HO-NNNNNNN-P- -5'

[0119] "Upstream" represents the relative position of a first element of a nucleic acid molecule with respect to a second element of that nucleic acid molecule, where both elements are contained in the same nucleic acid molecule and the first element is located closer to the 5'-end of the nucleic acid molecule than the second element of that nucleic acid molecule. In that case, the second element is said to be "downstream" of the first element of that nucleic acid molecule. An element located "upstream" of the second element can equivalently be referred to as being located on the "5'-side" of the second element. For a double-stranded nucleic acid molecule, designations such as "upstream" and "downstream" are given with respect to the "+" strand.

[0120] According to the present invention, "functionally linked" or "functionally connected" relates to a connection within a functional relationship. Nucleic acids are "functionally linked" when they are functionally related to another nucleic acid sequence. For example, a promoter is functionally linked to a coding sequence if it affects the transcription of the coding sequence. Functionally linked nucleic acids are typically adjacent to each other, but in appropriate cases are separated by additional nucleic acid sequences and, in certain embodiments, are transcribed by RNA polymerase to give a single RNA molecule (a co-transcript).

[0121] In certain embodiments, a nucleic acid is functionally linked, according to the present invention, to an expression control sequence that can be homologous or heterologous with respect to the nucleic acid.

[0122] According to the present invention, the term "expression control sequence" includes a promoter, a ribosome binding sequence, and other control elements that control the transcription of a gene or the translation of the induced RNA. In certain embodiments of the present invention, the expression control sequence can be regulated. The exact structure of the expression control sequence can vary depending on the species or cell type, but usually includes a 5' non-transcribed sequence and 5' and 3' untranslated sequences that are involved in the initiation of transcription and translation, respectively. More specifically, the 5' non-transcribed expression control sequence includes a promoter region that includes a promoter sequence for the transcriptional control of a functionally linked gene. The expression control sequence may also include an enhancer sequence or an upstream activation sequence. The expression control sequence of a DNA molecule usually includes 5' non-transcribed sequences such as a TATA box, a capping sequence, a CAAT sequence, and 5' and 3' untranslated sequences. The expression control sequence of an alphavirus RNA may include a subgenomic promoter and / or one or more conserved sequence elements. A specific expression control sequence according to the present invention is the subgenomic promoter of an alphavirus, as described herein.

[0123] The nucleic acid sequences identified herein, particularly the transcribable coding nucleic acid sequences, may be combined with any expression control sequence, particularly a promoter, that may be homologous or heterologous to the nucleic acid sequence. The term "homologous" refers to the fact that the nucleic acid sequence is naturally or functionally linked to the expression control sequence, and the term "heterologous" refers to the fact that the nucleic acid sequence is not naturally functionally linked to the expression control sequence.

[0124] A transcribable nucleic acid sequence, particularly a nucleic acid sequence encoding a peptide or protein, and an expression control sequence are "functionally" linked to each other when they are covalently bonded to each other such that the transcription or expression of the transcribable, particularly the coding nucleic acid sequence, is under the control or influence of the expression control sequence. When the nucleic acid sequence is translated into a functional peptide or protein, induction of the expression control sequence functionally linked to the coding sequence results in transcription of the coding sequence without causing a frameshift of the coding sequence or making it impossible for the coding sequence to be translated into the desired peptide or protein.

[0125] The term "promoter" or "promoter region" refers to a nucleic acid sequence that controls the synthesis of a transcript, such as a transcript containing a coding sequence, by providing a recognition and binding site for RNA polymerase. The promoter region may include additional recognition or binding sites for additional factors involved in the regulation of transcription of the gene. A promoter can control the transcription of a prokaryotic gene or a eukaryotic gene. A promoter can be "inducible", capable of initiating transcription in response to an inducer, or "constitutive" if transcription is not controlled by an inducer. An inducible promoter is expressed very little or not at all in the absence of the inducer. In the presence of the inducer, the gene is "switched on" or the level of transcription increases. This is usually mediated by the binding of a specific transcription factor. Specific promoters according to the present invention are, as described herein, for example, the subgenomic promoter of an alphavirus. Other specific promoters are, for example, the genomic plus-strand or minus-strand promoter of an alphavirus.

[0126] The term "core promoter" refers to the nucleic acid sequence contained within a promoter. The core promoter is typically the minimal part of the promoter necessary to properly initiate transcription. The core promoter typically includes the transcription start site and the binding site for RNA polymerase.

[0127] "Polymerase" generally refers to a molecular entity that can catalyze the synthesis of polymer molecules from monomer building blocks. "RNA polymerase" is a molecular entity that can catalyze the synthesis of RNA molecules from ribonucleotide building blocks. "DNA polymerase" is a molecular entity that can catalyze the synthesis of DNA molecules from deoxyribonucleotide building blocks. In the case of DNA polymerase and RNA polymerase, the molecular entity is typically a protein or an aggregate or complex of multiple proteins. Typically, DNA polymerase synthesizes DNA molecules based on a template nucleic acid, which is typically a DNA molecule. Typically, RNA polymerase synthesizes RNA molecules based on a template nucleic acid that is either a DNA molecule (in which case the RNA polymerase is a DNA-dependent RNA polymerase, DdRP) or an RNA molecule (in which case the RNA polymerase is an RNA-dependent RNA polymerase, RdRP).

[0128] "RNA-dependent RNA polymerase" or "RdRP" is an enzyme that catalyzes the transcription of RNA from an RNA template. In the case of alphavirus RNA-dependent RNA polymerase, RNA replication is brought about by the sequential synthesis of the (-) strand complement of the genomic RNA and the (+) strand genomic RNA. Thus, RNA-dependent RNA polymerase is synonymously referred to as "RNA replicase" or simply "replicase". In nature, RNA-dependent RNA polymerase is typically encoded by all RNA viruses except retroviruses. A typical representative of the viruses that encode RNA-dependent RNA polymerase is alphavirus.

[0129] According to the present invention, "RNA replication" generally refers to an RNA molecule synthesized based on the nucleotide sequence of a given RNA molecule (template RNA molecule). The synthesized RNA molecule can be, for example, identical or complementary to the template RNA molecule. Generally, RNA replication can occur via the synthesis of a DNA intermediate or directly by RNA-dependent RNA replication mediated by an RNA-dependent RNA polymerase (RdRP). In the case of alphaviruses, RNA replication occurs not via a DNA intermediate but is mediated by an RNA-dependent RNA polymerase (RdRP): a template RNA strand (the first RNA strand) - or a part thereof - serves as a template for the synthesis of a second RNA strand complementary to the first RNA strand or a part thereof. The second RNA strand - or a part thereof - then optionally serves as a template for the synthesis of a third RNA strand complementary to the second RNA strand or a part thereof. Thereby, the third RNA strand is identical to the first RNA strand or a part thereof. Thus, an RNA-dependent RNA polymerase can directly synthesize a complementary RNA strand of the template and can indirectly synthesize an identical RNA strand (via a complementary intermediate strand).

[0130] According to the present invention, the term "template RNA" refers to an RNA that can be transcribed or replicated by an RNA-dependent RNA polymerase.

[0131] According to the present invention, the term "gene" refers to a specific nucleic acid sequence responsible for the production of one or more cellular products and / or the accomplishment of one or more intercellular or intracellular functions. More specifically, the term relates to a nucleic acid moiety (typically DNA; however, RNA in the case of RNA viruses) that includes a nucleic acid encoding a specific protein or a functional or structural RNA molecule.

[0132] As used herein, "isolated molecule" is intended to refer to a molecule that is substantially free of other molecules such as other cellular materials. The term "isolated nucleic acid", according to the present invention, means that the nucleic acid has been (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, for example by cleavage and gel electrophoresis fractionation, or (iv) synthesized, for example by chemical synthesis. An isolated nucleic acid is a nucleic acid that is available for manipulation by recombinant techniques.

[0133] The term "vector" is used herein in its broadest sense and includes any intermediate vehicle for a nucleic acid that enables, for example, the introduction of the nucleic acid into prokaryotic and / or eukaryotic host cells and, where appropriate, its integration into the genome. Such vectors are preferably replicated and / or expressed intracellularly. Vectors include plasmids, phagemids, viral genomes, and fractions thereof.

[0134] The term "recombinant" in the context of the present invention means "produced through genetic manipulation". Preferably, a "recombinant", such as a recombinant cell, in the context of the present invention does not occur naturally.

[0135] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that exists in an organism (including a virus), can be isolated from a natural source, and has not been intentionally modified by a person in the laboratory is naturally occurring. The term "found in nature" means "existing in nature" and includes known objects as well as objects that have not yet been discovered and / or isolated from natural sources but may be discovered and / or isolated from natural sources in the future.

[0136] According to the present invention, the term "expression" is used in its most general sense and includes the production of RNA and / or protein. This term also includes partial expression of nucleic acids. Furthermore, expression can be transient or stable. With respect to RNA, the terms "expression" or "translation" relate to the process in the ribosomes of a cell in which the strand of coding RNA (e.g., messenger RNA) instructs the assembly of an amino acid sequence to produce a peptide or protein.

[0137] According to the present invention, the term "mRNA" means "messenger RNA" and relates to a transcript typically produced by using a DNA template and encoding a peptide or protein. Typically, mRNA includes a 5'-UTR, a protein-coding region, a 3'-UTR, and a poly(A) sequence. mRNA can be produced by in vitro transcription from a DNA template. Methods of in vitro transcription are known to those skilled in the art. For example, various in vitro transcription kits are commercially available. According to the present invention, mRNA can be modified by stabilization modification and capping.

[0138] According to the present invention, the term "poly(A) sequence" or "poly(A) tail" typically refers to a continuous or intermittent sequence of adenylate residues located at the 3'-end of an RNA molecule. A continuous sequence is characterized by consecutive adenylate residues. In nature, a continuous poly(A) sequence is typical. The poly(A) sequence is usually not encoded in eukaryotic DNA, but during eukaryotic transcription in the cell nucleus, it binds to the free 3'-end of the RNA by a post-transcriptional template-independent RNA polymerase, and the present invention includes poly(A) sequences encoded by DNA.

[0139] According to the present invention, with respect to a nucleic acid molecule, the term "primary structure" refers to the linear sequence of nucleotide monomers.

[0140] According to the present invention, with respect to a nucleic acid molecule, the term "secondary structure" refers to a two-dimensional representation of the nucleic acid molecule that reflects base pairing, for example, in the case of a single-stranded RNA molecule, particularly intramolecular base pairing. Each RNA molecule has only a single polynucleotide chain, but the molecule is typically characterized by regions of (intramolecular) base pairs. According to the present invention, the term "secondary structure" includes structural motifs including, but not limited to, loops such as base pairs, stems, stem-loops, bulges, internal loops, and multi-branched loops. The secondary structure of a nucleic acid molecule can be represented by a two-dimensional drawing (planar graph) showing base pairing (for further details regarding the secondary structure of RNA molecules, see Auber et al., 2006; J. Graph Algorithms Appl. 10:329-351). As described herein, the secondary structure of a particular RNA molecule is relevant in the context of the present invention.

[0141] According to the present invention, the secondary structure of a nucleic acid molecule, particularly a single-stranded RNA molecule, is determined by prediction using a web server for RNA secondary structure prediction (http: / / rna.urmc.rochester.edu / RNAstructureWeb / Servers / Predict1 / Predict1.html). Preferably, according to the present invention, with respect to a nucleic acid molecule, "secondary structure" specifically refers to the secondary structure determined by said prediction. The prediction can also be performed or confirmed using MFOLD structure prediction (http: / / unafold.rna.albany.edu / ?q=mfold).

[0142] According to the present invention, a "base pair" is a structural motif of a secondary structure in which two nucleotide bases associate with each other via a hydrogen bond between a donor site and an acceptor site on the base. Complementary bases A:U and G:C form stable base pairs via hydrogen bonds between a donor site and an acceptor site on the base; A:U base pairs and G:C base pairs are called Watson-Crick base pairs. Weaker base pairs (called wobble base pairs) are formed by bases G and U (G:U). Base pairs A:U and G:C are called canonical base pairs. Other base pairs such as G:U (which are quite frequent in RNA) and other rare base pairs (for example A:C; U:U) are called non-canonical base pairs.

[0143] According to the present invention, "nucleotide pairing" refers to two nucleotides in which the bases of the two nucleotides associate with each other to form a base pair (canonical or non-canonical base pair, preferably a canonical base pair, most preferably a Watson-Crick base pair).

[0144] According to the present invention, the terms "stem-loop", "hairpin", or "hairpin loop" with respect to a nucleic acid molecule all interchangeably refer to a specific secondary structure of a nucleic acid molecule, typically a single-stranded nucleic acid molecule such as single-stranded RNA. The specific secondary structure represented by a stem-loop consists of a continuous nucleic acid sequence including a (terminal) loop also called a stem and a hairpin loop, and the stem is formed by two adjacent completely or partially complementary sequence elements separated by a short sequence (e.g., 3 to 10 nucleotides) that forms the loop of the stem-loop structure. The two adjacent completely or partially complementary sequences can be defined, for example, as stem 1 and stem 2 of the stem-loop element. A stem-loop is formed when these two adjacent completely or partially reverse-complementary sequences, e.g., stem 1 and stem 2 of the stem-loop element, form base pairs with each other, resulting in a double-stranded nucleic acid sequence containing an unpaired loop at its end formed by the short sequence located between stem 1 and stem 2 of the stem-loop element. Thus, a stem-loop contains two stems (stem 1 and stem 2) which, at the level of the secondary structure of the nucleic acid molecule, form base pairs with each other and, at the level of the primary structure of the nucleic acid molecule, are separated by a short sequence that is not part of stem 1 or stem 2. For illustrative purposes, the two-dimensional representation of a stem-loop resembles a lollipop-shaped structure. The formation of a stem-loop structure requires the presence of a sequence that can itself fold back and form a paired double-strand; the paired double-strand is formed by stem 1 and stem 2. The stability of the paired stem-loop element is typically determined by the length, i.e., the number of nucleotides in stem 2 that can form base pairs (preferably canonical base pairs, more preferably Watson-Crick base pairs) with the nucleotides in stem 1, relative to the number of nucleotides in stem 1 that cannot form such base pairs with the nucleotides in stem 2 (mismatches or bulges). According to the present invention, the optimal loop length is 3 to 10 nucleotides, more preferably 4 to 7 nucleotides, such as 4 nucleotides, 5 nucleotides, 6 nucleotides, or 7 nucleotides.If a given nucleic acid sequence is characterized by a stem-loop, each complementary nucleic acid sequence will also typically be characterized by a stem-loop. Stem-loops are typically formed by single-stranded RNA molecules. For example, the 5' replication recognition sequence of alphavirus genomic RNA contains several stem-loops.

[0145] According to the present invention, with respect to a particular secondary structure (e.g., a stem-loop) of a nucleic acid molecule, "disrupt" or "disrupting" means that the particular secondary structure is absent or modified. Typically, the secondary structure can be disrupted as a result of a change in at least one nucleotide that is part of the secondary structure. For example, a stem-loop can be disrupted by a change in one or more nucleotides forming the stem, such that nucleotide pairing is not possible.

[0146] According to the present invention, "compensating for secondary structure disruption" or "compensation for secondary structure disruption" refers to one or more nucleotide changes in a nucleic acid sequence; more typically, this is one or more second nucleotide changes in a nucleic acid sequence, where one or more first nucleotide changes cause disruption of the secondary structure of the nucleic acid sequence in the absence of one or more second nucleotide changes, but the co-occurrence of one or more first nucleotide changes and one or more second nucleotide changes does not cause disruption of the secondary structure of the nucleic acid, and refers to one or more second nucleotide changes in the nucleic acid sequence, including one or more first nucleotide changes. Co-occurrence means the presence of both one or more first nucleotide changes and one or more second nucleotide changes. Typically, one or more first nucleotide changes and one or more second nucleotide changes are present together within the same nucleic acid molecule. In certain embodiments, the one or more nucleotide changes that compensate for secondary structure disruption are one or more nucleotide changes that compensate for one or more nucleotide pairing disruptions. Thus, in one embodiment, "compensation for secondary structure disruption" means "compensation for nucleotide pairing disruption", i.e., compensation for one or more nucleotide pairing disruptions, such as one or more nucleotide pairing disruptions within one or more stem-loops. The one or more nucleotide pairing disruptions may be introduced by removal of at least one start codon. Each of the one or more nucleotide changes that compensate for secondary structure disruption can be independently selected from one or more nucleotide deletions, additions, substitutions and / or insertions. In an exemplary example, if the nucleotide pairing A:U is disrupted by a substitution of A to C (C and U are typically not suitable for forming a nucleotide pair), the nucleotide change that compensates for the nucleotide pairing disruption can be a substitution of U with G, thereby allowing the formation of a C:G nucleotide pairing. Thus, the substitution of U with G compensates for the nucleotide pairing disruption. In an alternative example, if the nucleotide pairing A:U is disrupted by a substitution of A to C, the nucleotide change that compensates for the nucleotide pairing disruption can be a substitution of C with A, thereby restoring the formation of the original A:U nucleotide pairing.In general, in the present invention, nucleotide changes that compensate for secondary structure disruption without restoring the original nucleic acid sequence and without creating a new AUG triplet are preferred. In the above series of examples, the substitution from U to G is more preferred than the substitution from C to A.

[0147] According to the present invention, with respect to a nucleic acid molecule, the term "tertiary structure" refers to the three-dimensional structure of the nucleic acid molecule defined by atomic coordinates.

[0148] According to the present invention, a nucleic acid such as RNA, for example rRNA, can encode a peptide or a protein. Thus, a transcribable nucleic acid sequence or its transcript may contain an open reading frame (ORF) that encodes a peptide or a protein.

[0149] According to the present invention, the term "nucleic acid encoding a peptide or a protein" means that the nucleic acid can direct the assembly of amino acids to produce a peptide or a protein during the translation process when the nucleic acid is present in a suitable environment, preferably within a cell. Preferably, the coding RNA according to the present invention can interact with a cellular translation machinery that enables translation of the coding RNA to produce a peptide or a protein.

[0150] According to the present invention, the term "peptide" includes oligopeptides and polypeptides and refers to a substance containing two or more, preferably three or more, preferably four or more, preferably six or more, preferably eight or more, preferably ten or more, preferably thirteen or more, preferably sixteen or more, preferably twenty or more, and up to a maximum of preferably fifty, preferably one hundred or preferably one hundred and fifty consecutive amino acids linked to each other via peptide bonds. The term "protein" refers to a large peptide, preferably a peptide having at least 151 amino acids, but the terms "peptide" and "protein" are generally used as synonyms herein.

[0151] According to the present invention, the terms "peptide" and "protein" include substances containing not only amino acid components but also non-amino acid components such as sugars and phosphate structures, and substances containing bonds such as ester, thioether or disulfide bonds.

[0152] According to the present invention, the terms "start codon" and "initiation codon" synonymously refer to the codon (base triplet) of an RNA molecule that may be the first codon translated by a ribosome. Such a codon typically codes for the amino acid methionine in eukaryotes and modified methionine in prokaryotes. The most common start codon in both eukaryotes and prokaryotes is AUG. Unless otherwise specified herein to mean a start codon other than AUG, the terms "start codon" and "initiation codon" with respect to an RNA molecule refer to the codon AUG. According to the present invention, the terms "start codon" and "initiation codon" are also used to refer to the corresponding base triplet of deoxyribonucleic acid, i.e., the base triplet that codes for the start codon of RNA. When the start codon of messenger RNA is AUG, the base triplet that codes for AUG is ATG. According to the present invention, the terms "start codon" and "initiation codon" preferably refer to a functional start codon or initiation codon, i.e., a start codon or initiation codon that is or will be used as a codon by a ribosome to initiate translation. For example, an AUG codon that is not used as a codon by a ribosome to initiate translation may be present in an RNA molecule because the distance from the codon to the cap is short. These codons are not included in the term functional start codon or initiation codon.

[0153] According to the present invention, the term "open reading frame start codon" or "open reading frame initiation codon" refers to a base triplet that functions as an initiation codon for protein synthesis in a coding sequence, such as the coding sequence of a nucleic acid molecule found in nature. In RNA molecules, a 5' untranslated region (5'-UTR) often exists before the start codon of the open reading frame, but this is not strictly necessary.

[0154] According to the present invention, the term "natural open reading frame start codon" or "natural open reading frame initiation codon" refers to a base triplet that functions as an initiation codon for protein synthesis in a natural coding sequence. The natural coding sequence can be, for example, the coding sequence of a nucleic acid molecule found in nature. In some embodiments, the present invention provides a variant of a nucleic acid molecule found in nature, characterized in that the natural start codon (present in the natural coding sequence) has been removed (and thus is not present in the variant nucleic acid molecule).

[0155] According to the present invention, "the first AUG" means the most upstream AUG base triplet of a messenger RNA molecule, preferably the most upstream AUG base triplet of a messenger RNA molecule that is used or will be used as a codon by a ribosome to initiate translation. Thus, "the first ATG" refers to the ATG base triplet of the coding DNA sequence that encodes the first AUG. In some cases, the first AUG of an mRNA molecule is the start codon of the open reading frame, i.e., the codon that is used as the start codon during ribosomal protein synthesis.

[0156] According to the present invention, the terms "including removal" or "characterized by removal" and similar terms with respect to a particular element of a nucleic acid variant mean that the particular element is non-functional or absent in the nucleic acid variant as compared to a reference nucleic acid molecule. Without limitation, removal can consist of deletion of all or part of a particular element, substitution of all or part of a particular element, or modification of the functional or structural properties of a particular element. Removal of a functional element of a nucleic acid sequence requires that the function is not exerted at the position of the nucleic acid variant including the removal. For example, an RNA variant characterized by removal of a particular start codon requires that ribosomal protein synthesis does not start at the position of the RNA variant characterized by the removal. Removal of a structural element of a nucleic acid sequence requires that the structural element is not present at the position of the nucleic acid variant including the removal. For example, an RNA variant characterized by removal of a particular AUG triplet, i.e., the AUG triplet at a particular position, can be characterized by deletion of some or all of the particular AUG triplet (e.g., ΔAUG), or substitution of one or more nucleotides (A, U, G) of the particular AUG triplet by any one or more different nucleotides, such that the resulting nucleotide sequence of the variant does not contain the AUG triplet. A suitable substitution of one nucleotide is one that converts the AUG triplet to a GUG, CUG or UUG triplet, or to an AAG, ACG or AGG triplet, or to an AUA, AUC or AUU triplet. Accordingly, suitable substitutions of more nucleotides can be selected.

[0157] According to the present invention, the term "self-replicating virus" includes RNA viruses that can replicate autonomously within a host cell. Self-replicating viruses can have a single-stranded RNA (ssRNA) genome and include alphaviruses, flaviviruses, measles virus (MV), and rhabdoviruses. Alphaviruses and flaviviruses have a positive-polarity genome, while the genomes of measles virus (MV) and rhabdoviruses are minus-strand ssRNA. Typically, a self-replicating virus is a virus having a (+) strand RNA genome that can be directly translated after cell infection, and this translation provides an RNA-dependent RNA polymerase that produces both antisense and sense transcripts from the infecting RNA. Hereinafter, the present invention will be described by referring to an alphavirus-derived vector as an example of a self-replicating virus-derived vector. However, it should be understood that the present invention is not limited to alphavirus-derived vectors.

[0158] According to the present invention, the term "alphavirus" should be understood broadly and includes any viral particle having the characteristics of an alphavirus. The characteristics of an alphavirus include the presence of a (+) strand RNA encoding genetic information suitable for replication in a host cell, including RNA polymerase activity. Further characteristics of many alphaviruses are described, for example, in Strauss & Strauss, 1994, Microbiol. Rev. 58:491-562. The term "alphavirus" includes alphaviruses found in nature, and any variants or derivatives thereof. In some embodiments, the variants or derivatives are not found in nature.

[0159] In one embodiment, the alphavirus is an alphavirus found in nature. Typically, alphaviruses found in nature are infectious to any one or more eukaryotes such as animals (including vertebrates such as humans and arthropods such as insects). Alphaviruses found in nature are preferably selected from the group consisting of: Barmah Forest virus complex (including Barmah Forest virus); Eastern equine encephalitis complex (including seven antigenic types of Eastern equine encephalitis virus); Middelburg virus complex (including Middelburg virus); Ndumu virus complex (including Ndumu virus); Semliki Forest virus complex (including Bebaru virus, Chikungunya virus, Mayaro virus and its subtype Una virus, O'nyong'nyong virus and its subtype Igbo-Ora virus, Ross River virus and its subtype Bebaru virus, Getah virus, Sagiyama virus, Semliki Forest virus and its subtype Metavir); Venezuelan equine encephalitis complex (including Cabassou virus, Everglades virus, Mossuril virus, Mucambo virus, Paraná virus, Pixuna virus, Rio Negro virus, Trocará virus and its subtype Bijou Bridge virus, Venezuelan equine encephalitis virus); Western equine encephalitis complex (including Aura virus, Babanki virus, Kyzylagach virus, Sindbis virus, Ockelbo virus, Wataroa virus, Baggie Creek virus, Fort Morgan virus, Highlands J virus, Western equine encephalitis virus); and several unclassified viruses including Salmon pancreas disease virus; Sleeping sickness virus; Southern sea otter virus; Tonate virus. More preferably, the alphavirus is selected from the group consisting of: Semliki Forest virus complex (including Semliki Forest virus, including the virus types shown above); Western equine encephalitis complex (including Sindbis virus, including the virus types shown above); Eastern equine encephalitis virus (including the virus types shown above); Venezuelan equine encephalitis complex (including Venezuelan equine encephalitis virus, including the virus types shown above).

[0160] In a further preferred embodiment, the alphavirus is Semliki Forest virus. In an alternative further preferred embodiment, the alphavirus is Sindbis virus. In an alternative further preferred embodiment, the alphavirus is Venezuelan equine encephalitis virus.

[0161] In some embodiments of the invention, the alphavirus is not an alphavirus found in nature. Typically, an alphavirus not found in nature is a variant or derivative of an alphavirus found in nature that is distinguished from the alphavirus found in nature by at least one mutation in the nucleotide sequence, i.e., the genomic RNA. Mutations in the nucleotide sequence can be selected from insertions, substitutions, or deletions of one or more nucleotides compared to the alphavirus found in nature. Mutations in the nucleotide sequence may or may not be associated with mutations in the polypeptide or protein encoded by the nucleotide sequence. For example, an alphavirus not found in nature can be an attenuated alphavirus. An attenuated alphavirus not found in nature typically has at least one mutation in its nucleotide sequence, thereby distinguishing it from the alphavirus found in nature, and is an alphavirus that is either not infectious at all, or is infectious but has a lower ability to cause disease or no ability to cause disease at all. As an illustrative example, TC83 is an attenuated alphavirus that is distinguished from Venezuelan equine encephalitis virus (VEEV) found in nature (McKinney et al., 1963, Am. J. Trop. Med. Hyg. 12:597-603).

[0162] Members of the alphavirus genus can also be classified, based on their relative clinical characteristics in humans, into alphaviruses mainly associated with encephalitis and alphaviruses mainly associated with fever, rash, and polyarthritis.

[0163] The term "alphavirus-like" means found in, derived from, or for example, derivable from an alphavirus by genetic manipulation.

[0164] According to the present invention, "SFV" represents Semliki Forest virus. According to the present invention, "SIN" or "SINV" represents Sindbis virus. According to the present invention, "VEE" or "VEEV" represents Venezuelan equine encephalitis virus.

[0165] According to the present invention, the term "alphavirus-derived" refers to an entity originating from an alphavirus. For the purposes of explanation, an alphavirus-derived protein may refer to a protein found in an alphavirus and / or a protein encoded by an alphavirus, and an alphavirus-derived nucleic acid sequence may refer to a nucleic acid sequence found in an alphavirus and / or a nucleic acid sequence encoded by an alphavirus. Preferably, an "alphavirus-derived" nucleic acid sequence refers to a nucleic acid sequence of "alphavirus genome" and / or "alphavirus genomic RNA".

[0166] According to the present invention, the term "alphavirus RNA" refers to any one or more of alphavirus genomic RNA (i.e., (+) strand), the complement of alphavirus genomic RNA (i.e., (-) strand), and subgenomic transcripts (i.e., (+) strand), or any fragment thereof.

[0167] According to the present invention, "alphavirus genome" refers to the genomic (+) strand RNA of an alphavirus.

[0168] According to the present invention, the term "native alphavirus sequence" and similar terms typically refer to the (e.g., nucleic acid) sequence of a naturally occurring alphavirus (an alphavirus found in nature). In some embodiments, the term "native alphavirus sequence" also includes the sequence of an attenuated alphavirus.

[0169] According to the present invention, the term "5' replication recognition sequence" preferably refers to a continuous nucleic acid sequence, preferably a ribonucleic acid sequence, that is identical or homologous to the 5' fragment of the genome of a self-replicating virus such as an alphavirus genome. The "5' replication recognition sequence" is a nucleic acid sequence that can be recognized by a replicase such as an alphavirus replicase. The term "5' replication recognition sequence" includes natural 5' replication recognition sequences as well as functional equivalents thereof, such as functional variants of 5' replication recognition sequences of self-replicating viruses found in nature, such as alphaviruses found in nature. According to the present invention, functional equivalents include derivatives of the 5' replication recognition sequence characterized by the removal of at least one start codon described herein. The 5' replication recognition sequence is required for the synthesis of the (-) strand complement of the alphavirus genomic RNA and for the synthesis of the (+) strand viral genomic RNA based on the (-) strand template. Natural 5' replication recognition sequences typically encode at least the N-terminal fragment of nsP1 but do not include the entire open reading frame encoding nsP1234. Considering the fact that natural 5' replication recognition sequences typically encode at least the N-terminal fragment of nsP1, natural 5' replication recognition sequences typically include at least one start codon, typically AUG. In one embodiment, the 5' replication recognition sequence includes the conserved sequence element 1 (CSE 1) of the alphavirus genome or a variant thereof, and the conserved sequence element 2 (CSE 2) of the alphavirus genome or a variant thereof. The 5' replication recognition sequence can typically form four stem-loops (SLs), namely SL1, SL2, SL3, and SL4. The numbering of these stem-loops starts from the 5' end of the 5' replication recognition sequence.

[0170] The term "conserved sequence element" or "CSE" refers to nucleotide sequences found in alphavirus RNA. These sequence elements are present in the genomes of alphaviruses with different orthologs, and the orthologous CSEs of different alphaviruses are called "conserved" because they preferably share a high percentage of sequence identity and / or similar secondary or tertiary structures. The term CSE includes CSE 1, CSE 2, CSE 3, and CSE 4.

[0171] According to the present invention, the term "CSE 1" or "44-nt CSE" synonymously refers to the nucleotide sequence required for (+) strand synthesis from a (-) strand template. The term "CSE 1" refers to the sequence on the (+) strand, and the complementary sequence of CSE 1 (on the (-) strand) functions as a promoter for (+) strand synthesis. Preferably, the term CSE 1 includes the most 5'-terminal nucleotides of the alphavirus genome. CSE 1 typically forms a conserved stem-loop structure. Without wishing to be bound by a particular theory, in the case of CSE 1, the secondary structure is considered to be more important than the primary structure, i.e., the linear sequence. In the genomic RNA of Sindbis virus, a model alphavirus, CSE 1 consists of a continuous sequence of 44 nucleotides formed by the most 5'-terminal 44 nucleotides of the genomic RNA (Strauss & Strauss, 1994, Microbiol. Rev. 58:491-562).

[0172] According to the present invention, the terms "CSE 2" or "51-nt CSE" are used synonymously to refer to the nucleotide sequence required for (-) strand synthesis from a (+) strand template. The (+) strand template is typically an alphavirus genomic RNA or an RNA replicon (note that a subgenomic RNA transcript that does not contain CSE 2 does not function as a template for (-) strand synthesis). In the genomic RNA of alphaviruses, CSE 2 is typically located within the coding sequence of nsP1. In the genomic RNA of the model alphavirus Sindbis virus, the 51-nt CSE is located at nucleotide positions 155-205 of the genomic RNA (Frolov et al., 2001, RNA, vol. 7, pp. 1638-1651). CSE 2 typically forms two conserved stem-loop structures. These stem-loop structures are the third and fourth conserved stem-loops of the alphavirus genomic RNA, respectively, when counted from the 5' end of the alphavirus genomic RNA, and are therefore called stem-loop 3 (SL3) and stem-loop 4 (SL4). Without wishing to be bound by any particular theory, in the case of CSE 2, the secondary structure is thought to be more important than the primary structure, i.e., the linear sequence.

[0173] According to the present invention, the terms "CSE 3" or "junction sequence" are used synonymously to refer to a nucleotide sequence derived from alphavirus genomic RNA and containing the start site of subgenomic RNA. The complement of this sequence in the (-) strand acts to promote subgenomic RNA transcription. In alphavirus genomic RNA, CSE 3 typically overlaps with the region encoding the C-terminal fragment of nsP4 and extends into a short non-coding region located upstream of the open reading frame encoding the structural proteins.

[0174] According to the present invention, the terms "CSE 4" or "19-nt conserved sequence" or "19-nt CSE" are used synonymously to refer to the nucleotide sequence from alphavirus genomic RNA immediately upstream of the poly(A) sequence within the 3' untranslated region of the alphavirus genome. CSE 4 typically consists of 19 consecutive nucleotides. Without wishing to be bound by a particular theory, CSE 4 is understood to function as a core promoter for the initiation of (-) strand synthesis (Jose et al., 2009, Future Microbiol. 4:837-856), and / or CSE 4 and the poly(A) tail of alphavirus genomic RNA are understood to function together for efficient (-) strand synthesis (Hardy & Rice, 2005, J. Virol. 79:4630-4639).

[0175] According to the present invention, the term "subgenomic promoter" or "SGP" refers to a nucleic acid sequence upstream (5' side) of a nucleic acid sequence (e.g., a coding sequence) that controls the transcription of said nucleic acid sequence by providing a recognition and binding site for an RNA polymerase, typically an RNA-dependent RNA polymerase, in particular a functional alphavirus non-structural protein. The SGP may contain additional recognition or binding sites for additional factors. The subgenomic promoter is typically a genetic element of a plus-strand RNA virus such as an alphavirus. The subgenomic promoter of an alphavirus is a nucleic acid sequence contained in the viral genomic RNA. The subgenomic promoter is generally characterized by enabling the initiation of transcription (RNA synthesis) in the presence of an RNA-dependent RNA polymerase, such as a functional alphavirus non-structural protein. The RNA (-) strand, i.e., the complement of the alphavirus genomic RNA, serves as a template for the synthesis of the (+) strand subgenomic transcript, and the synthesis of the (+) strand subgenomic transcript typically starts at or near the subgenomic promoter. The term "subgenomic promoter" as used herein is not limited to a specific localization within a nucleic acid containing such a subgenomic promoter. In some embodiments, the SGP is identical to, overlaps with, or contains CSE 3.

[0176] The terms "subgenomic transcript" or "subgenomic RNA" are used synonymously to refer to an RNA molecule that is the result of transcription using an RNA molecule as a template ("template RNA"), where the template RNA contains a subgenomic promoter that controls the transcription of the subgenomic transcript. Subgenomic transcripts can be obtained in the presence of an RNA-dependent RNA polymerase, particularly a functional alphavirus nonstructural protein. For example, the term "subgenomic transcript" can refer to an RNA transcript prepared in an alphavirus-infected cell using the (-) strand complement of the alphavirus genomic RNA as a template. However, the term "subgenomic transcript" as used herein is not limited thereto and also includes transcripts obtained by using a heterologous RNA as a template. For example, subgenomic transcripts can also be obtained by using the (-) strand complement of an SGP-containing replicon according to the present invention as a template. Thus, the term "subgenomic transcript" can refer to an RNA molecule obtained by transcribing a fragment of an alphavirus genomic RNA, as well as an RNA molecule obtained by transcribing a fragment of a replicon according to the present invention.

[0177] The term "autologous" is used to denote something that is derived from the same subject. For example, "autologous cells" refer to cells derived from the same subject. Introducing autologous cells into a subject is advantageous because these cells overcome the immunological barriers that would otherwise result in rejection.

[0178] The term "allogeneic" is used to denote something that is derived from different individuals of the same species. Two or more individuals are said to be allogeneic to each other if the genes at one or more loci are not identical.

[0179] The term "syngeneic" is used to denote something that is derived from an individual or tissue having the same genotype, i.e., identical twins or animals of the same inbred strain, or their tissues or cells.

[0180] The term "heterologous" is used to denote something consisting of multiple different elements. As an example, introducing the cells of one individual into a different individual constitutes a xenotransplant. A heterologous gene is a gene that is derived from a source other than the subject.

[0181] The cells that can be used in a method for identifying sequence changes are any suitable cells in which replicable RNA can be replicated and / or translated, regardless of the presence or absence of nucleotide modifications. The cells can be mammalian cells, such as human cells. The cells can constitutively express a replicase that recognizes the sequences present in the replicable RNA for replication, or can transiently express such a replicase.

[0182] The following provides specific and / or preferred variations of the individual features of the present invention. The present invention also contemplates, as particularly preferred embodiments, embodiments generated by combining two or more of the specific and / or preferred variations described for two or more of the features of the present invention.

[0183] RNA replicon A replicable RNA molecule (rRNA) is an RNA that can be replicated by an RNA-dependent RNA polymerase (replicase) by including a nucleotide sequence that can be recognized by the replicase such that the RNA is replicated. Since the rRNA does not necessarily encode the replicase, the rRNA can be replicated in cis (by the encoded replicase) or in trans (by a separately provided replicase, such as a separate replicase encoding a nucleic acid such as mRNA). The terms "RNA replicon", "replicon" and "replicable RNA molecule" can be used interchangeably.

[0184] In one embodiment, the replicable RNA (rRNA) molecule comprises a modified regulatory region of a self-replicating single-stranded positive-sense virus that contains sequence changes compared to a reference modified regulatory region, and these sequence changes restore or improve the function of the rRNA molecule that includes at least one modified nucleotide. These changes can be identified by the methods described herein for identifying such sequence changes. In one embodiment, the modified regulatory region is an alphavirus regulatory region, such as a 5' or 3' regulatory region. In one embodiment, the 5' regulatory region is the VEEV alphavirus 5' regulatory region.

[0185] In one embodiment, the RNA replicon can comprise an internal ribosome entry site (IRES) and an open reading frame encoding a functional non-structural protein from a self-replicating virus, and the IRES controls the expression of the functional non-structural protein, such as a replicase. Preferably, the RNA replicon comprises a sequence element that enables replication by the functional non-structural protein. In one embodiment, the self-replicating virus is an alphavirus and the sequence element that enables replication by the functional non-structural protein is derived from an alphavirus.

[0186] Alphavirus replicase has a capping enzyme function, and typically, genomic and subgenomic (+) strand RNAs are capped. The 5’ cap protects the mRNA from degradation, recruits ribosomal subunits and cellular factors to the mRNA to form a ribonucleoprotein complex on the mRNA, and then functions to initiate translation from the nearby start codon. This complex process is well described in the literature (Jackson et al., 2010, Nat Rev Mol Biol; Vol 10; 113 - 127). Despite the very elaborate and efficient mechanism of cap-dependent translation, cells have means to initiate translation either completely or partially independently of the 5’ cap (Thompson 2012; Trends in Microbiology 20:558 - 566). Thereby, in situations of cellular stress that result in overall downregulation of cap-dependent translation, cells can often still selectively express selected genes, often with the help of IRES.

[0187] Viruses have also evolved different means to utilize the cellular machinery for the translation of viral genes. Since viral infection is often sensed by cells that trigger an antiviral response in the cell (interferon response; stress response), many viruses also utilize cap-independent translation, especially RNA viruses. Cap-independent translation ensures the advantages for viral RNA translation during the cellular stress response and gives the viruses the opportunity to complete their life cycle and be released from infected cells.

[0188] Internal ribosome entry sites (IRESs) are RNA sequences that form an appropriate secondary structure to attract pre-initiation complexes in the vicinity of the translation start codon, such as AUG. Four classes of IRESs sharing common features have been described in the literature. The prototype IRESs are the poliovirus IRES (type I), encephalomyocarditis virus (EMCV) IRES (type II), hepatitis C virus (HCV) IRES (type III), and the IRES found in the intergenic region of dicistroviruses (type IV) (Thompson, 2012; Trends in Microbiology 20:558-566; Lozano et al. 2018; Open Biology 8:180155).

[0189] Types I-III IRESs have in common that they initiate translation at the AUG start codon, while type IV IRESs initiate at non-AUG codons (e.g., GCU). Thereby, types I-III require the initiator tRNA that delivers methionine with the help of eIF2 / GTP (eIF2 / GTP / Met-tRNAiMet). Activation of eIF2 kinases under stress phosphorylates the α subunit of eIF2, which inhibits translation initiating at AUG. Thereby, translation directed by type IV IRESs is not inhibited by eIF2 phosphorylation.

[0190] The term "internal ribosome entry site", abbreviated as "IRES", relates to an RNA element that recruits ribosomes to an internal region of an mRNA and initiates translation in a cap-independent manner. IRESs are generally located in the 5'-UTR of RNA viruses. However, mRNAs of viruses from the dicistroviridae family have two open reading frames (ORFs), and each translation is directed by two different IRESs. Also, some mammalian cell mRNAs have been suggested to have IRESs. These cellular IRES elements are thought to be located in eukaryotic mRNAs encoding genes involved in other processes important for stress survival and survival. The location of the IRES element is often in the 5'-UTR but can also be present in other places of the mRNA.

[0191] The term "internal ribosome entry site" includes IRESs present in viruses of the Picornaviridae family such as poliovirus (PV) and encephalomyocarditis virus, as well as pathogenic viruses including human immunodeficiency virus, hepatitis C virus (HCV), and foot-and-mouth disease virus. These viral IRESs contain diverse sequences, but many of them have similar secondary structures and initiate translation via similar mechanisms. Furthermore, the activity of IRESs often requires assistance from other factors known as IRES trans-acting factors (ITAFs). Based on the structures and requirements of translation initiation factors (IF) and ITAFs, viral IRESs are classified into four types described herein. Any of these IRES types are useful according to the present invention, and type IV IRESs are particularly preferred.

[0192] Two groups of viral IRESs, type I and type II, cannot directly bind to the 40S small ribosomal subunit. Instead, they recruit the 40S small ribosomal subunit via different ITAFs and require canonical IFs in cap-dependent translation (i.e., eIF2, eIF3, eIF4A, eIF4B, and eIF4G). The main difference between type I and type II IRESs is the requirement for 40S ribosome scanning, and 40S ribosome scanning is not required for type II IRESs. Examples of type I IRESs include the IRESs found in poliovirus (PV) and rhinovirus. Examples of type II IRESs include the IRESs found in encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), and Theiler's murine encephalomyelitis virus (TMEV).

[0193] Type III IRESs can interact directly with the 40S small ribosomal subunit, which has a special RNA structure, but their activities usually require the assistance of several IFs including eIF2 and eIF3 and the initiator Met-tRNAi. Examples include the IRESs found in hepatitis C virus (HCV), classical swine fever virus (CSFV), and porcine teschovirus (PTV).

[0194] Type IV viral IRESs generally have strong activities and can initiate translation from non-AUG start codons without the need for additional ITAFs or even the eIF2 / Met-tRNAi / GTP ternary complex. These IRESs are folded into a compact structure that interacts directly with the 40S small ribosomal subunit. Examples include the IRESs found in dicistroviruses such as cricket paralysis virus (CrPV), Plautia stali intestine virus (PSIV), and Taura syndrome virus (TSV).

[0195] The term "internal ribosome entry site" also includes IRESs found in cellular mRNAs, many of which encode proteins required for the stress response in conditions such as apoptosis, mitosis, hypoxia, and nutrient limitation. Cellular IRESs can be broadly classified into two types based on the mechanism of ribosome recruitment: Type I IRESs interact with ribosomes via cis-elements such as ITAFs bound to RNA-binding motifs and N-6-methyladenosine (m6A) modifications, while Type II IRESs contain short cis-elements that pair with 18S rRNA to recruit ribosomes.

[0196] In one embodiment, the rRNAs described herein may have modified nucleotides / nucleosides / backbone modifications. As used herein, the term "RNA modification" may refer to chemical modifications including backbone modifications as well as sugar or base modifications.

[0197] In this regard, the modified rRNA molecules defined herein may include nucleotide analogs / modifications, such as backbone modifications, sugar modifications or base modifications. Backbone modifications related to the present invention are modifications in which the phosphate of the backbone of the nucleotides contained in the rRNA molecules defined herein is chemically modified. Sugar modifications related to the present invention are chemical modifications of the sugars of the nucleotides of the rRNA molecules defined herein. Further, base modifications related to the present invention are chemical modifications of the base portions of the nucleotides of the rRNA molecules. In this regard, the nucleotide analogs or modifications are preferably selected from nucleotide analogs applicable to transcription and / or translation.

[0198] Sugar modifications: The modified nucleosides and nucleotides that can be incorporated into the modified rRNA molecules described herein can be modified at the sugar moiety. For example, the 2'-hydroxyl group (OH) can be modified or replaced with several different "oxy" or "deoxy" substituents. Examples of "oxy"-2'-hydroxyl group modifications include alkoxy or aryloxy (-OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethylene glycol (PEG), -O(CH2CH2O)nCH2CH2OR; "locked" nucleic acids (LNA) where the 2'-hydroxyl is attached to the 4'-carbon of the same ribose sugar, e.g., by a methylene bridge; and amino groups (-O-amino, where the amino group, e.g., NRR can be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxy, but are not limited thereto. "Deoxy" modifications include hydrogen, amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino or amino acid), or the amino group can be attached to the sugar via a linker, which contains one or more of the atoms C, N and O. The sugar group can also contain one or more carbons having a stereochemical configuration opposite to that of the corresponding carbon in ribose. Thus, the modified RNA molecule can contain, for example, nucleotides containing arabinose as the sugar.

[0199] Skeletal Modification: The phosphate backbone can be further modified with modified nucleosides and nucleotides that can be incorporated into the modified RNA molecules described herein. The phosphate groups of the backbone can be modified by substituting one or more of the oxygen atoms with different substituents. Further, the modified nucleosides and nucleotides can include complete substitution of the unmodified phosphate moiety with modified phosphates described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphorodithioate, phosphorodiselenoate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphoramidate, alkyl phosphonate or aryl phosphonate, and phosphotriester. In phosphorodithioate, both non-bridging oxygens are replaced by sulfur. The phosphate linker can also be modified by substituting the bridging oxygen with nitrogen (bridging phosphoramidate), sulfur (bridging phosphorothioate), and carbon (bridging methylene phosphonate).

[0200] Base Modification: The modified nucleosides and nucleotides that can be incorporated into the modified rRNA molecules described herein can be further modified at the nucleobase moiety. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. For example, the nucleosides and nucleotides described herein can be chemically modified on the major groove face. In some embodiments, the major groove chemical modification can include an amino group, a thiol group, an alkyl group, or a halo group.

[0201] In certain embodiments of the present invention, the nucleotide analog / modification is preferably 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine-riboside-5'-triphosphate, 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methylinosine-5'-triphosphate, 4-thio-uridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, N6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, or puromycin-5'-triphosphate, xanthosine-5'-triphosphate. Nucleotides for base modification selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate are particularly preferred.In some embodiments, modified nucleosides include pyridin-4-one ribonucleoside, 5-azauridine, 2-thio-5-azauridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thiouridine, l-taurinomethyl-4-thiouridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydro-pseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thiopseudouridine.

[0202] In some embodiments, modified nucleosides include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-l-methyl-pseudoisocytidine.

[0203] In other embodiments, modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diamino-purine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methyl-thio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In other embodiments, modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0204] In some embodiments, the nucleotide can be modified on the major groove face and can include substituting the hydrogen on C-5 of uracil with a methyl group or a halo group. In certain embodiments, the modified nucleoside is 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine, 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine or 5'-O-(1-thiophosphate)-pseudouridine.

[0205] In a further embodiment, the modified rRNA may include nucleoside modifications selected from 6-aza-cytidine, 2-thio-cytidine, a-thio-cytidine, pseudoiso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydro-uridine, a-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxythymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, a-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudoiso-cytidine, 6-chloro-purine, N6-methyl-adenosine, a-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine.

[0206] In certain preferred embodiments, the rRNA includes modified nucleosides in place of at least one (e.g., all) uridines.

[0207] As used herein, the term "uracil" refers to one of the nucleobases that can be present in the nucleic acids of RNA. The structure of uracil is:

Chem.

[0208] As used herein, the term "uridine" refers to one of the nucleosides that can be present in RNA. The structure of uridine is:

Chem.

[0209] UTP (uridine 5'-triphosphate) has the following structure:

Chem.

[0210] Pseudo-UTP (pseudouridine 5'-triphosphate) has the following structure:

Chem.

[0211] “Pseudouridine” is an example of a modified nucleoside that is an isomer of uridine, in which uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

[0212] Another exemplary modified nucleoside is N1-methyl-pseudouridine (m1Ψ), which has the structure:

Chem.

[0213] N1-methyl-pseudo-UTP has the following structure:

Chem.

[0214] Another exemplary modified nucleoside is 5-methyl-uridine (m5U), which has the structure:

Chem.

[0215] In certain preferred embodiments, one or more uridines in the rRNA described herein are replaced with modified nucleosides. In some embodiments, the modified nucleoside is a modified uridine.

[0216] In certain preferred embodiments, the RNA contains modified nucleosides instead of at least one uridine. In some embodiments, the RNA contains modified nucleosides instead of each uridine.

[0217] In certain preferred embodiments, the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleoside comprises 5-methyl-uridine (m5U). In some embodiments, the RNA may comprise two or more modified nucleosides, and the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ) and N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleoside comprises pseudouridine (ψ) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0218] In certain preferred embodiments, the modified nucleoside that replaces one or more, such as all, of the uridines in rRNA is 3-methyluridine (m 3 U), 5-methoxyuridine (mo 5 U), 5-azauridine, 6-azauridine, 2-thio-5-azauridine, 2-thiouridine (s 2 U), 4-thiouridine (s 4 U), 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine (ho 5 U), 5-aminoallyluridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), uridine 5-oxyacetic acid (cmo 5 U), methyl ester of uridine 5-oxyacetic acid (mcmo 5 U), 5-carboxymethyluridine (cm5 U), 1-carboxymethylpseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thiouridine (nm 5 s 2 U), 5-methylaminomethyluridine (mnm 5 U), 1-ethylpseudouridine, 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-selenouridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyluridine, 1-propynylpseudouridine, 5-taurinomethyluridine (τm 5 U), 1-taurinomethylpseudouridine, 5-taurinomethyl-2-thiouridine (τm5s2U), 1-taurinomethyl-4-thiopseudouridine), 5-methyl-2-thiouridine (m 5 s 2 U), 1-methyl-4-thiopseudouridine (m 1 s 4 Ψ), 4-thio-1-methylpseudouridine, 3-methylpseudouridine (m 3 Ψ), 2-thio-1-methylpseudouridine, 1-methyl-1-deazapseudouridine, 2-thio-1-methyl-1-deazapseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m 5D), 2-thiodihydrouridine, 2-thiodihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxypseudouridine, 4-methoxy-2-thiopseudouridine, N1-methylpseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 Ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thiouridine, 2'-O-methyluridine (Um), 5,2'-O-dimethyluridine (m 5 Um), 2'-O-methylpseudouridine (Ψm), 2-thio-2'-O-methyluridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm 5 Um), 3,2'-O-dimethyluridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm 5 Um), 1-thiouridine, deoxythymidine, 2'-F-arabouridine, 2'-F-uridine, 2'-OH-arabouridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any one or more of any other modified uridine known in the art may be used.

[0219] In one embodiment, the rRNA contains other modified nucleosides or further modified nucleosides, such as modified cytidines as described above. For example, in one embodiment, in the rRNA, cytidine is partially or completely, preferably completely, replaced with 5-methylcytidine. In one embodiment, the rRNA contains 5-methylcytidine and one or more selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In one embodiment, the rRNA contains 5-methylcytidine and N1-methyl-pseudouridine (m1ψ). In some embodiments, the rRNA contains 5-methylcytidine in place of each cytidine and N1-methyl-pseudouridine (m1ψ) in place of each uridine.

[0220] Functional non-structural protein The term "non-structural protein" relates to proteins encoded by a virus that are not part of the virus particle. This term typically includes various enzymes and transcription factors used by the virus to replicate itself, such as RNA replicase or other template-directed polymerases. The term "non-structural protein" includes all co-translational or post-translational modification forms, including glycosylation and lipid modification forms of non-structural proteins, and preferably relates to "alphavirus non-structural proteins".

[0221] In some embodiments, the term "alphavirus nonstructural protein" refers to any one or more of the individual nonstructural proteins (nsP1, nsP2, nsP3, nsP4) of alphavirus origin, or a polyprotein comprising the polypeptide sequences of multiple nonstructural proteins of alphavirus origin. In some embodiments, "alphavirus nonstructural protein" refers to nsP123 and / or nsP4. In other embodiments, "alphavirus nonstructural protein" refers to nsP1234. In one embodiment, the protein of interest encoded by the open reading frame consists of all of nsP1, nsP2, nsP3, and nsP4 as a single, optionally cleavable polyprotein: nsP1234. In one embodiment, the protein of interest encoded by the open reading frame consists of nsP1, nsP2, and nsP3 as a single, optionally cleavable polyprotein: nsP123. In that embodiment, nsP4 can be a protein of further interest and can be encoded by a further open reading frame.

[0222] In some embodiments, the nonstructural proteins can form complexes or associations, for example, in a host cell. In some embodiments, "alphavirus nonstructural protein" refers to a complex or association of nsP123 (synonymously P123) and nsP4. In some embodiments, "alphavirus nonstructural protein" refers to a complex or association of nsP1, nsP2, and nsP3. In some embodiments, "alphavirus nonstructural protein" refers to a complex or association of nsP1, nsP2, nsP3, and nsP4. In some embodiments, "alphavirus nonstructural protein" refers to any one or more complexes or associations selected from the group consisting of nsP1, nsP2, nsP3, and nsP4. In some embodiments, the alphavirus nonstructural protein comprises at least nsP4.

[0223] The term "complex" or "assembly" refers to two or more identical or different protein molecules that are spatially proximal. The proteins of the complex preferably are in physical or physicochemical contact with each other, either directly or indirectly. A complex or assembly can be composed of multiple different proteins (heteromultimer) and / or multiple copies of one particular protein (homomultimer). In the context of alphavirus nonstructural proteins, the term "complex or assembly" refers to at least two protein molecules, a number of which at least one is an alphavirus nonstructural protein. A complex or assembly can be composed of multiple copies of one particular protein (homomultimer) and / or multiple copies of multiple different proteins (heteromultimer). In the context of multimer, "multi" means more than one, such as two, three, four, five, six, seven, eight, nine, ten or more than ten.

[0224] The term "functional non-structural protein" includes non-structural proteins having replicase function. Thus, "functional non-structural proteins" include alphavirus replicases. "Replicase function" includes the function of an enzyme, RNA-dependent RNA polymerase (RdRP), that can catalyze the synthesis of (-) strand RNA based on a (+) strand RNA template and / or catalyze the synthesis of (+) strand RNA based on a (-) strand RNA template. Thus, the term "functional non-structural protein" can refer to a protein or complex that synthesizes (-) strand RNA using (+) strand (e.g., genomic) RNA as a template, a protein or complex that synthesizes new (+) strand RNA using the (-) strand complement of genomic RNA as a template, and / or a protein or complex that synthesizes a subgenomic transcript using a fragment of the (-) strand complement of genomic RNA as a template. Functional non-structural proteins can further have one or more additional functions, such as protease (for self-cleavage), helicase, terminal adenylyl transferase (for addition of the poly(A) tail), methyltransferase and guanylyl transferase (for providing a 5' cap to the nucleic acid), nuclear localization site, triphosphatase, etc. (Gould et al., 2010, Antiviral Res. 87:111-124; Rupp et al., 2015, J. Gen. Virol. 96:2483-500).

[0225] The term "replicase" includes RNA-dependent RNA polymerase. According to the present invention, the term "replicase" includes "alphavirus replicase" including RNA-dependent RNA polymerase derived from a naturally occurring alphavirus (alphavirus found in nature) and RNA-dependent RNA polymerase derived from a variant or derivative of an alphavirus such as a live-attenuated alphavirus.

[0226] The term "replicase" includes all variants of alphavirus replicase, particularly post-translationally modified variants, conformations, isoforms and homologs, that are expressed by alphavirus-infected cells or by cells transfected with a nucleic acid encoding alphavirus replicase. Further, the term "replicase" includes all forms of replicase that are produced by and can be produced by recombinant means. For example, a replicase that includes a tag, such as a myc tag, HA tag or oligo-histidine tag (His tag), to facilitate detection and / or purification of the replicase in the laboratory can be produced by recombinant means.

[0227] Optionally, alphavirus replicase is further functionally defined by its ability to bind to any one or more of the alphavirus conserved sequence element 1 (CSE 1) or its complementary sequence, conserved sequence element 2 (CSE 2) or its complementary sequence, conserved sequence element 3 (CSE 3) or its complementary sequence, conserved sequence element 4 (CSE 4) or its complementary sequence. Preferably, the replicase can bind to CSE 2 [i.e., the (+) strand] and / or CSE 4 [i.e., the (+) strand], or to the complement of CSE 1 [i.e., the (-) strand] and / or the complement of CSE 3 [i.e., the (-) strand].

[0228] The origin of the alphavirus replicase is not limited to a particular alphavirus. In preferred embodiments, the alphavirus replicase comprises non-structural proteins derived from Semliki Forest virus, including variants or derivatives of Semliki Forest virus such as naturally occurring Semliki Forest virus and attenuated Semliki Forest virus. In alternative preferred embodiments, the alphavirus replicase comprises non-structural proteins derived from Sindbis virus, including variants or derivatives of Sindbis virus such as naturally occurring Sindbis virus and attenuated Sindbis virus. In alternative preferred embodiments, the alphavirus replicase comprises non-structural proteins derived from Venezuelan equine encephalitis virus (VEEV), including variants or derivatives of VEEV such as naturally occurring VEEV and attenuated VEEV. In alternative preferred embodiments, the alphavirus replicase comprises non-structural proteins derived from Chikungunya virus (CHIKV), including variants or derivatives of CHIKV such as naturally occurring CHIKV and attenuated CHIKV.

[0229] The replicase may also comprise non-structural proteins from multiple viruses, e.g., multiple alphaviruses. Thus, heteromeric complexes or associations that contain alphavirus non-structural proteins and have replicase function are also encompassed by the present invention. For illustrative purposes only, the replicase may comprise one or more non-structural proteins (e.g., nsP1, nsP2) from a first alphavirus, and one or more non-structural proteins (nsP3, nsP4) from a second alphavirus. Non-structural proteins from multiple different alphaviruses may be encoded by separate open reading frames or may be encoded by a single open reading frame as a polyprotein, e.g., nsP1234.

[0230] In some embodiments, the functional non-structural protein is capable of forming a membrane replication complex and / or a vacuole within the cell in which the functional non-structural protein is expressed.

[0231] When a functional non-structural protein, i.e., a non-structural protein having replicase function, is encoded by a nucleic acid molecule according to the present invention, the sub-genomic promoter of the replicon, if present, is preferably compatible with said replicase. Being compatible in this context means that the replicase can recognize the sub-genomic promoter when the sub-genomic promoter is present. In one embodiment, this is achieved when the sub-genomic promoter is native to the virus from which the replicase is derived, i.e., the natural origin of these sequences is the same virus. In an alternative embodiment, the sub-genomic promoter is not native to the virus from which the viral replicase is derived as long as the viral replicase can recognize the sub-genomic promoter. In other words, the replicase is compatible with the sub-genomic promoter (cross-virus compatibility). Examples of cross-virus compatibility for sub-genomic promoters and replicases derived from different alphaviruses are known in the art. As long as cross-virus compatibility exists, any combination of sub-genomic promoter and replicase is possible. Cross-virus compatibility can be readily tested by those skilled in the art of practicing the present invention by incubating the replicase being tested with RNA having the sub-genomic promoter being tested under conditions suitable for RNA synthesis from the sub-genomic promoter. When a sub-genomic transcript is produced, it is determined that the sub-genomic promoter and the replicase are compatible. Various examples of cross-virus compatibility are known.

[0232] A replicon can preferably be replicated by a functional non-structural protein. In particular, an RNA replicon encoding a functional non-structural protein can be replicated by the functional non-structural protein encoded by the replicon. In a preferred embodiment, the RNA replicon contains an open reading frame encoding a functional alphavirus non-structural protein. In one embodiment, the replicon contains an additional open reading frame encoding a protein of interest. This embodiment is particularly suitable for several methods for generating a protein of interest according to the present invention. In one embodiment, the additional open reading frame encoding the protein of interest is downstream of the 5' replication recognition sequence and upstream of the IRES (and upstream of the open reading frame encoding the functional non-structural protein derived from the self-replicating virus) and / or downstream of the open reading frame encoding the functional non-structural protein derived from the self-replicating virus. An additional open reading frame encoding a protein of interest that is downstream of the 5' replication recognition sequence and upstream of the IRES (and upstream of the open reading frame encoding the functional non-structural protein derived from the self-replicating virus) can be expressed as a fusion protein with the sequence encoded by the 5' replication recognition sequence. An additional open reading frame encoding a protein of interest that is downstream of the 5' replication recognition sequence and upstream of the IRES (and upstream of the open reading frame encoding the functional non-structural protein derived from the self-replicating virus) may or may not be controlled by a subgenomic promoter. One or more additional open reading frames encoding one or more proteins of interest that are downstream of the open reading frame encoding the functional non-structural protein derived from the self-replicating virus are generally controlled by one or more subgenomic promoters.

[0233] The open reading frame encoding the functional non-structural protein preferably does not overlap with the 5' replication recognition sequence. In one embodiment, the open reading frame encoding the functional non-structural protein does not overlap with the subgenomic promoter when the subgenomic promoter is present. That embodiment is disclosed in International Publication No. WO 2017 / 162460, which is incorporated herein by reference.

[0234] Decoupling of sequence elements required for replication and protein coding regions The open reading frame encoding nsP1234 overlaps with the 5' replication recognition sequence (the coding sequence of nsP1) of the alphavirus genome and typically also overlaps with the subgenomic promoter (the coding sequence of nsP4) including CSE 3, so it is difficult to develop a multi-purpose alphavirus-derived vector.

[0235] The RNA replicons described herein generally include sequence elements required for replication by the replicase, particularly the 5' replication recognition sequence. In one embodiment, the coding sequence of the non-structural protein is under the control of an IRES, and thus the IRES is located upstream of the coding sequence of the non-structural protein. Thus, in one embodiment, the 5' replication recognition sequence, which normally overlaps with the coding sequence of the N-terminal fragment of the alphavirus non-structural protein, is located upstream of the IRES and does not overlap with the coding sequence of the non-structural protein.

[0236] In one embodiment, the coding sequence of the 5' replication recognition sequence, such as the nsP1 coding sequence, is fused in-frame to the gene of interest located upstream of the IRES.

[0237] In one embodiment, the 5' replication recognition sequence does not encode a protein or a fragment thereof, such as an alphavirus nonstructural protein or a fragment thereof. Thus, in the RNA replicon according to the present invention, the sequence elements necessary for replication by the replicase and the protein coding region may be separated. The separation can be achieved by removal of at least one start codon in the 5' replication recognition sequence as compared to the natural viral genomic RNA, for example, the natural alphavirus genomic RNA.

[0238] Thus, the rRNA may contain a 5' replication recognition sequence, which is characterized by including the removal of at least one start codon as compared to the natural viral 5' replication recognition sequence, for example, the natural alphavirus 5' replication recognition sequence.

[0239] The 5' replication recognition sequence characterized by including the removal of at least one start codon as compared to the natural viral 5' replication recognition sequence may be referred to herein as a "modified 5' replication recognition sequence" or a "5' replication recognition sequence according to the present invention". As described below herein, the 5' replication recognition sequence according to the present invention may optionally be characterized by the presence of one or more additional nucleotide changes, such as those detected by the method of the present invention.

[0240] Nucleic acid constructs that can be replicated by a replicase, preferably an alphavirus replicase, are called replicable RNAs or replicons. According to the present invention, the term "replicon" defines an RNA molecule that can be replicated by an RNA-dependent RNA polymerase and that can generate one or more identical or substantially identical copies of the RNA replicon without a DNA intermediate. "Without a DNA intermediate" means that no copy of deoxyribonucleic acid (DNA) or a complement of the replicon is formed during the process of forming a copy of the RNA replicon, and / or that no deoxyribonucleic acid (DNA) molecule is used as a template during the process of forming a copy of the RNA replicon or its complement. The function of the replicase is typically provided by a functional non-structural protein, such as a functional alphavirus non-structural protein.

[0241] According to the present invention, the terms "can be replicated" and "can be replicated" generally mean that one or more identical or substantially identical copies of a nucleic acid can be generated. When used with the term "replicase", for example, in "can be replicated by a replicase", the terms "can be replicated" and "can be replicated" represent functional characteristics of a nucleic acid molecule with respect to the replicase, such as an RNA replicon. These functional characteristics include at least one of (i) the ability of the replicase to recognize the replicon and (ii) the ability of the replicase to act as an RNA-dependent RNA polymerase (RdRP). Preferably, the replicase is capable of both (i) recognizing the replicon and (ii) acting as an RNA-dependent RNA polymerase.

[0242] The expression "capable of recognizing" means that the replicase can physically bind to the replicon and preferably can bind to the replicon, typically non-covalently. The term "bind" means that the replicase has the ability to bind to any one or more of conserved sequence element 1 (CSE 1) or its complementary sequence (if included in the replicon), conserved sequence element 2 (CSE 2) or its complementary sequence (if included in the replicon), conserved sequence element 3 (CSE 3) or its complementary sequence (if included in the replicon), conserved sequence element 4 (CSE 4) or its complementary sequence (if included in the replicon). Preferably, the replicase can bind to CSE 2 [i.e., the (+) strand] and / or CSE 4 [i.e., the (+) strand], or can bind to the complement of CSE 1 [i.e., the (-) strand] and / or the complement of CSE 3 [i.e., the (-) strand].

[0243] In one embodiment, the expression "capable of acting as an RdRP" means that the replicase can catalyze the synthesis of the (-) strand complement of the viral genomic (+) strand RNA, the (+) strand RNA has a template function, and / or the replicase can catalyze the synthesis of the (+) strand viral genomic RNA, the (-) strand RNA has a template function. Generally, the expression "capable of acting as an RdRP" also includes that the replicase can catalyze the synthesis of the (+) strand subgenomic transcript, the (-) strand RNA has a template function, and the synthesis of the (+) strand subgenomic transcript is typically initiated at a subgenomic promoter. In one embodiment, the virus is an alphavirus.

[0244] The expressions "capable of binding" and "capable of acting as an RdRP" refer to the ability under normal physiological conditions. In particular, they refer to the state within a cell that expresses a functional non-structural protein or is transfected with a nucleic acid encoding a functional non-structural protein. The cell is preferably a eukaryotic cell. The ability to bind and / or the ability to act as an RdRP can be experimentally tested, for example, in a cell-free in vitro system or in eukaryotic cells. Optionally, the eukaryotic cell is a cell derived from a species in which a particular virus that is the origin of the replicase is infectious. For example, when a viral replicase derived from a particular virus that is infectious to humans is used, the normal physiological conditions are the conditions within human cells. More preferably, the eukaryotic cell (in one example, a human cell) is derived from the same tissue or organ in which a particular virus that is the origin of the replicase is infectious.

[0245] According to the present invention, "compared to a native alphavirus sequence" and similar terms refer to a sequence that is a variant of a native alphavirus sequence. The variant is typically not itself a native alphavirus sequence.

[0246] In one embodiment, the RNA replicon comprises a 3' replication recognition sequence. The 3' replication recognition sequence is a nucleic acid sequence that can be recognized by a functional non-structural protein. In other words, the functional non-structural protein can recognize the 3' replication recognition sequence. Preferably, the 3' replication recognition sequence is located at the 3' end of the replicon (when the replicon does not contain a poly(A) tail) or immediately upstream of the poly(A) tail (when the replicon contains a poly(A) tail). In one embodiment, the 3' replication recognition sequence consists of or comprises CSE 4.

[0247] In one embodiment, the 5' replication recognition sequence and the 3' replication recognition sequence can direct the replication of the RNA replicon according to the present invention in the presence of a functional non-structural protein. Thus, these recognition sequences, when present alone or preferably together, direct the replication of the RNA replicon in the presence of a functional non-structural protein.

[0248] It is preferred that a functional non-structural protein be provided that can recognize both the 5' replication recognition sequence and the 3' replication recognition sequence of the replicon. In one embodiment, this is achieved when the 3' replication recognition sequence is native to the alphavirus from which the functional alphavirus non-structural protein is derived, and the 5' replication recognition sequence is native to the alphavirus from which the functional alphavirus non-structural protein is derived or is a variant of the 5' replication recognition sequence native to the alphavirus from which the functional alphavirus non-structural protein is derived. By native, it is meant that the natural origin of these sequences is the same alphavirus. In an alternative embodiment, the 5' replication recognition sequence and / or the 3' replication recognition sequence are not native to the alphavirus from which the functional alphavirus non-structural protein is derived when the functional alphavirus non-structural protein can recognize both the 5' replication recognition sequence and the 3' replication recognition sequence of the replicon. In other words, the functional alphavirus non-structural protein is compatible with the 5' replication recognition sequence and the 3' replication recognition sequence. A functional alphavirus non-structural protein is said to be compatible (cross-virus compatibility) when it can recognize the respective sequence or sequence element. Any combination of the (3' / 5') replication recognition sequence and CSE with the functional alphavirus non-structural protein is possible as long as cross-virus compatibility exists. Cross-virus compatibility can be readily tested by those skilled in the art of practicing the present invention by incubating the functional alphavirus non-structural protein to be tested with RNA having the 3' and 5' replication recognition sequences to be tested under conditions suitable for RNA replication, for example, in a suitable host cell. If replication occurs, it is determined that the (3' / 5') replication recognition sequence and the functional alphavirus non-structural protein are compatible.

[0249] Removal of at least one start codon within the 5’ replication recognition array provides several advantages. Absence of a start codon in the nucleic acid sequence encoding nsP1* (the N-terminal fragment of nsP1) typically causes nsP1* not to be translated. Further, since nsP1* is not translated, the open reading frame encoding the protein of interest (“GOI 2”) is the most upstream open reading frame accessible to ribosomes; thus, when the replicon is present within a cell, translation is initiated at the first AUG of the open reading frame (RNA) encoding the gene of interest.

[0250] Removal of at least one start codon can be achieved by any suitable method known in the art. For example, a suitable DNA molecule encoding a replicon according to the invention, i.e., characterized by removal of a start codon, can be designed in silico and then synthesized in vitro (gene synthesis); alternatively, a suitable DNA molecule can be obtained by site-directed mutagenesis of a DNA sequence encoding a replicon. In either case, each DNA molecule can serve as a template for in vitro transcription, thereby providing a replicon according to the invention.

[0251] Removal of at least one start codon compared to the native 5’ replication recognition array is not particularly limited and can be selected from any nucleotide modification, including substitution of one or more nucleotides (including substitution of A and / or T and / or G of the start codon at the DNA level), deletion of one or more nucleotides (including deletion of A and / or T and / or G of the start codon at the DNA level), and insertion of one or more nucleotides (including insertion of one or more nucleotides between A and T and / or between T and G of the start codon at the DNA level). Regardless of whether the nucleotide modification is a substitution, insertion or deletion, the nucleotide modification must not result in the formation of a new start codon (exemplary example: insertion at the DNA level must not be an insertion of ATG).

[0252] The 5' replication recognition sequence of the RNA replicon, which is characterized by the removal of at least one start codon (i.e., the modified 5' replication recognition sequence according to the present invention), is preferably a variant of the 5' replication recognition sequence of the alphavirus genome found in nature. In one embodiment, the modified 5' replication recognition sequence according to the present invention preferably has a sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more with the 5' replication recognition sequence of the genome of at least one alphavirus found in nature.

[0253] In one embodiment, the 5' replication recognition sequence of the RNA replicon, which may be characterized by the removal of at least one start codon, comprises a sequence homologous to approximately 250 nucleotides at the 5' end of the alphavirus, i.e., the 5' end of the alphavirus genome. In a preferred embodiment, this comprises a sequence homologous to approximately 250 to 500, preferably approximately 300 to 500 nucleotides at the 5' end of the alphavirus, i.e., the 5' end of the alphavirus genome. The "5' end of the alphavirus genome" means the nucleic acid sequence starting from and including the most upstream nucleotide of the alphavirus genome. In other words, the most upstream nucleotide of the alphavirus genome is referred to as nucleotide number 1, and for example, the "250 nucleotides at the 5' end of the alphavirus genome" means nucleotides 1 to 250 of the alphavirus genome. In one embodiment, the 5' replication recognition sequence of the RNA replicon is characterized by a sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more with at least 250 nucleotides at the 5' end of the genome of at least one alphavirus found in nature. The at least 250 nucleotides include, for example, 250 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides.

[0254] The 5’ replication recognition sequences of alphaviruses found in nature typically feature at least one start codon and / or conserved secondary structure motifs. For example, the native 5’ replication recognition sequence of Semliki Forest virus (SFV) contains five specific AUG base triplets. According to Frolov et al., 2001, RNA 7:1638-1651, analysis by MFOLD revealed that the native 5’ replication recognition sequence of Semliki Forest virus is predicted to form four stem loops (SLs) called stem loops 1-4 (SL1, SL2, SL3, SL4). According to Frolov et al., analysis by MFOLD also revealed that the native 5’ replication recognition sequence of Sindbis virus, a different alphavirus, is predicted to form four stem loops: SL1, SL2, SL3, SL4.

[0255] It is known that the 5’ end of the alphavirus genome contains sequence elements that enable replication of the alphavirus genome by functional alphavirus non-structural proteins. In one embodiment of the present invention, the 5’ replication recognition sequence of the RNA replicon contains a sequence homologous to the conserved sequence element 1 (CSE 1) of alphaviruses and / or a sequence homologous to the conserved sequence element 2 (CSE 2).

[0256] The conserved sequence element 2 (CSE 2) of alphavirus genomic RNA is typically represented by SL3 and SL4 preceded by SL2, which includes at least the natural start codon encoding the first amino acid residue of alphavirus nonstructural protein nsP1. However, in this description, in some embodiments, the conserved sequence element 2 (CSE 2) of alphavirus genomic RNA refers to the region extending from SL2 to SL4 and including the natural start codon encoding the first amino acid residue of alphavirus nonstructural protein nsP1. In a preferred embodiment, the RNA replicon includes CSE 2 or a sequence homologous to CSE 2. In one embodiment, the RNA replicon preferably has a degree of sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more with the sequence of CSE 2 of at least one alphavirus found in nature, and includes a sequence homologous to CSE 2.

[0257] In one embodiment, the 5’ replication recognition sequence includes a sequence homologous to CSE 2 of alphavirus. CSE 2 of alphavirus may include a fragment of the open reading frame of the nonstructural protein derived from alphavirus.

[0258] Accordingly, in one embodiment, the RNA replicon is characterized by including a sequence homologous to the open reading frame of the nonstructural protein derived from alphavirus or a fragment thereof. The sequence homologous to the open reading frame of the nonstructural protein or a fragment thereof is typically a variant of the open reading frame of the nonstructural protein of alphavirus found in nature or a fragment thereof. In one embodiment, the sequence homologous to the open reading frame of the nonstructural protein or a fragment thereof preferably has a degree of sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more with the open reading frame of the nonstructural protein of at least one alphavirus found in nature or a fragment thereof.

[0259] In one embodiment, the sequence homologous to the open reading frame of the non-structural protein contained in the replicon of the present invention does not contain the natural start codon of the non-structural protein, and more preferably does not contain any start codon of the non-structural protein. In one embodiment, the sequence homologous to CSE 2 is characterized by the removal of all start codons as compared to the natural alphavirus CSE 2 sequence. Thus, the sequence homologous to CSE 2 preferably does not contain any start codon.

[0260] When the sequence homologous to the open reading frame does not contain any start codon, the sequence homologous to the open reading frame does not function as a template for translation and thus is not an open reading frame itself.

[0261] In one embodiment, the 5' replication recognition sequence contains a sequence homologous to the open reading frame of the non-structural protein derived from alphavirus or a fragment thereof, and the sequence homologous to the open reading frame of the non-structural protein derived from alphavirus or a fragment thereof is characterized by including the removal of at least one start codon as compared to the natural alphavirus sequence.

[0262] In one embodiment, the sequence homologous to the open reading frame of the non-structural protein derived from alphavirus or a fragment thereof is characterized by including the removal of at least the natural start codon of the open reading frame of the non-structural protein. Preferably, the sequence is characterized by including the removal of at least the natural start codon of the open reading frame encoding nsP1.

[0263] The natural start codon is the AUG triplet at which translation on ribosomes in the host cell begins when the RNA is present in the host cell. In other words, the natural start codon is the first triplet of bases that is translated during ribosomal protein synthesis, for example, in a host cell inoculated with RNA containing the natural start codon. In one embodiment, the host cell is a cell derived from a eukaryotic species that is the natural host of a particular alphavirus containing the natural alphavirus 5′ replication recognition sequence. In one embodiment, the host cell is BHK21 cells derived from the cell line “BHK21[C13] (ATCC® CCL10™)” available from the American Type Culture Collection, Manassas, Virginia, USA.

[0264] The genomes of many alphaviruses have been fully sequenced and are publicly accessible, and the sequences of the nonstructural proteins encoded by these genomes are also publicly accessible. Such sequence information enables the in silico determination of the natural start codon.

[0265] In one embodiment, a sequence homologous to the open reading frame or a fragment thereof of an alphavirus-derived nonstructural protein is characterized by the removal of one or more start codons other than the natural start codon of the open reading frame of the nonstructural protein. In one embodiment, the nucleic acid sequence is further characterized by the removal of the natural start codon. For example, in addition to the removal of the natural start codon, any one or two or three or four or more than four (e.g., five) start codons may be removed.

[0266] When the replicon is characterized by the removal of the natural start codon of the open reading frame of the nonstructural protein and optionally one or more start codons other than the natural start codon, the sequence homologous to the open reading frame does not function as a template for translation and thus is not itself an open reading frame.

[0267] One or more start codons other than the natural start codon to be removed are preferably selected from AUG triplets that preferably have the potential to initiate translation, in addition to the removal of the natural start codon. An AUG triplet that has the potential to initiate translation may be referred to as a "potential start codon." Whether a given AUG triplet has the potential to initiate translation can be determined by in silico or cell-based in vitro assays.

[0268] In one embodiment, whether a given AUG triplet has the potential to initiate translation is determined in silico: in that embodiment, the nucleotide sequence is examined and if the AUG triplet is part of an AUGG sequence, preferably part of a Kozak sequence, the AUG triplet is determined to have the potential to initiate translation.

[0269] In one embodiment, whether a given AUG triplet has the potential to initiate translation is determined in a cell-based in vitro assay: An RNA replicon that features the removal of the native start codon and contains a given AUG triplet downstream of the position where the native start codon was removed is introduced into a host cell. In one embodiment, the host cell is a cell derived from a eukaryotic species that is the natural host of a particular alphavirus containing the native alphavirus 5’ replication recognition sequence. In a preferred embodiment, the host cell is a BHK21 cell derived from the cell line “BHK21[C13] (ATCC® CCL10™)” available from the American Type Culture Collection, Manassas, Virginia, USA. It is preferred that there are no additional AUG triplets between the position of the removal of the native start codon and the given AUG triplet. If, after introduction of an RNA replicon that features the removal of the native start codon and contains a given AUG triplet into a host cell, translation is initiated at the given AUG triplet, then the given AUG triplet is determined to have the potential to initiate translation. Whether translation is initiated can be determined by any suitable method known in the art. For example, the replicon can encode, in-frame with the given AUG triplet, a tag, such as a myc tag or an HA tag, downstream of the given AUG triplet that facilitates detection of the translation product (if any); whether an expression product having the encoded tag is present can be determined, for example, by Western blot. In this embodiment, it is preferred that there are no additional AUG triplets between the given AUG triplet and the nucleic acid sequence encoding the tag. The cell-based in vitro assay can be performed individually for a plurality of given AUG triplets: In each case, it is preferred that there are no additional AUG triplets between the position of the removal of the native start codon and the given AUG triplet. This can be achieved by removing all AUG triplets (if any) between the position of the removal of the native start codon and the given AUG triplet.Thereby, a given AUG triplet is the first AUG triplet downstream of the removal position of the natural start codon.

[0270] Preferably, the 5' replication recognition sequence of the RNA replicon according to the invention is characterized by the removal of all potential start codons. Thus, according to the invention, the 5' replication recognition sequence preferably does not contain an open reading frame that can be translated into a protein.

[0271] In one embodiment, the 5' replication recognition sequence of the RNA replicon according to the invention is characterized by a secondary structure equal to the (predicted) secondary structure of the 5' replication recognition sequence of the viral genomic RNA. For this purpose, the RNA replicon may contain one or more nucleotide changes that compensate for nucleotide pair disruptions within one or more stem-loops introduced by the removal of at least one start codon.

[0272] In one embodiment, the 5' replication recognition sequence of the RNA replicon according to the invention is characterized by a secondary structure equal to the secondary structure of the 5' replication recognition sequence of the alphavirus genomic RNA. In a preferred embodiment, the 5' replication recognition sequence of the RNA replicon according to the invention is characterized by a predicted secondary structure equal to the predicted secondary structure of the 5' replication recognition sequence of the alphavirus genomic RNA. According to the invention, the secondary structure of the RNA molecule is preferably predicted by the web server for RNA secondary structure prediction, http: / / rna.urmc.rochester.edu / RNAstructureWeb / Servers / Predict1 / Predict1.html.

[0273] The presence or absence of nucleotide pair disruptions can be identified by comparing the secondary structure or predicted secondary structure of the 5' replication recognition sequence of an RNA replicon characterized by the removal of at least one start codon compared to the natural alphavirus 5' replication recognition sequence. For example, compared to the base pairs within the natural alphavirus 5' replication recognition sequence, such as within a stem-loop, especially within the stem of the stem-loop, there may be a case where at least one base pair is absent at a given position.

[0274] In one embodiment, one or more stem-loops of the 5' replication recognition sequence are not deleted or disrupted. More preferably, stem-loops 3 and 4 are not deleted or disrupted. Preferably, none of the stem-loops of the 5' replication recognition sequence are deleted or disrupted.

[0275] In one embodiment, the removal of at least one start codon does not disrupt the secondary structure of the 5' replication recognition sequence. In an alternative embodiment, the removal of at least one start codon disrupts the secondary structure of the 5' replication recognition sequence. In this embodiment, the removal of at least one start codon can cause the absence of at least one base pair, such as a base pair within a stem-loop, at a given position compared to the native 5' replication recognition sequence. If there is no base pair within the stem-loop compared to the native 5' replication recognition sequence, it is determined that the removal of at least one start codon introduces a nucleotide pairing disruption within the stem-loop. The base pair within the stem-loop is typically a base pair within the stem of the stem-loop.

[0276] In one embodiment, the RNA replicon comprises one or more nucleotide changes that compensate for the nucleotide pairing disruption within one or more stem-loops introduced by the removal of at least one start codon.

[0277] If the removal of at least one start codon introduces a nucleotide pairing disruption within the stem-loop, one or more nucleotide changes that are expected to compensate for the nucleotide pairing disruption can be introduced, and the resulting secondary structure or predicted secondary structure can be compared to the native 5' replication recognition sequence.

[0278] Based on common general knowledge and the disclosure of this specification, certain nucleotide changes can be expected by those skilled in the art to compensate for nucleotide pairing disruptions. For example, compared to the native 5' replication recognition sequence, if the base pair is disrupted at a given position in the secondary structure or predicted secondary structure of a given 5' replication recognition sequence of an RNA replicon characterized by the removal of at least one start codon, a nucleotide change that restores the base pair at that position without preferably reintroducing a start codon is expected to compensate for the nucleotide pairing disruption.

[0279] In one embodiment, the 5' replication recognition sequence of the replicon does not overlap with or contain a nucleic acid sequence that is translatable, i.e., a peptide or protein, particularly nsP, particularly nsP1, or a fragment of any of them. For a nucleotide sequence to be "translatable", the presence of a start codon is required; the start codon encodes the most N-terminal amino acid residue of a peptide or protein. In one embodiment, the 5' replication recognition sequence of the replicon does not overlap with or contain a translatable nucleic acid sequence encoding the N-terminal fragment of nsP1.

[0280] In some situations, the RNA replicon comprises at least one subgenomic promoter. In a preferred embodiment, the subgenomic promoter of the replicon does not overlap with or contain a nucleic acid sequence that is translatable into a peptide or protein, i.e., a translatable nucleic acid sequence, particularly nsP, particularly nsP4, or a fragment of any of them. In one embodiment, the subgenomic promoter of the replicon does not overlap with or contain a nucleic acid sequence that is translatable into the C-terminal fragment of nsP4. An RNA replicon having a subgenomic promoter that does not overlap with or contain a nucleic acid sequence that is translatable into, for example, the C-terminal fragment of nsP4 can be generated by deleting a part of the coding sequence of nsP4 (typically the part encoding the N-terminal portion of nsP4) and / or by removing the AUG triplet of the part of the coding sequence of nsP4 that has not been deleted. When the AUG triplet of the coding sequence of nsP4 or a part thereof is removed, the AUG triplet to be removed is preferably a potential start codon. Alternatively, if the subgenomic promoter does not overlap with the nucleic acid sequence encoding nsP4, the entire nucleic acid sequence encoding nsP4 may be deleted.

[0281] In one embodiment, the RNA replicon does not contain an open reading frame encoding a truncated non-structural protein, such as a truncated alphavirus non-structural protein. In the context of this embodiment, it is particularly preferred that the RNA replicon does not contain an open reading frame encoding the N-terminal fragment of nsP1 and optionally does not contain an open reading frame encoding the C-terminal fragment of nsP4. The N-terminal fragment of nsP1 is a truncated alphavirus protein; the C-terminal fragment of nsP4 is also a truncated alphavirus protein.

[0282] In some embodiments, the replicon according to the invention does not include stem-loop 2 (SL2) at the 5' end of the alphavirus genome. According to the above Frolov et al., stem-loop 2 is a conserved secondary structure found at the 5' end of the alphavirus genome, upstream of CSE 2, but is not essential for replication.

[0283] The RNA replicon according to the invention is preferably a single-stranded RNA molecule. The RNA replicon according to the invention is typically a (+) strand RNA molecule. In one embodiment, the RNA replicon of the invention is an isolated nucleic acid molecule. The RNA replicon according to the invention contains at least one modified nucleotide and preferably contains one or more sequence changes, in particular sequence changes detected by the methods disclosed herein for identifying sequence changes that restore or improve the function of rRNA containing at least one modified nucleotide.

[0284] At least one open reading frame encoding at least one gene product of interest In one embodiment, the RNA replicon according to the invention includes at least one open reading frame encoding a gene product of interest, such as a peptide or protein of interest. Preferably, the protein of interest is encoded by a heterologous nucleic acid sequence. The gene encoding the peptide or protein of interest is synonymously referred to as the "gene of interest" or "transgene". In various embodiments, the peptide or protein of interest is encoded by a heterologous nucleic acid sequence. According to the invention, the term "heterologous" refers to the fact that the nucleic acid sequence is not functionally or structurally linked in nature to a viral nucleic acid sequence, such as an alphavirus nucleic acid sequence.

[0285] The replicon according to the present invention can encode a single polypeptide or a plurality of polypeptides. The plurality of polypeptides can be encoded as a single polypeptide (fusion polypeptide) or as separate polypeptides. In some embodiments, the replicon according to the present invention can include a plurality of open reading frames, each of which can be optionally under the control of a subgenomic promoter. Alternatively, the polyprotein or fusion polypeptide can include individual polypeptides separated by a 2A self-cleaving peptide (e.g., derived from the foot-and-mouth disease virus 2A protein) or a protease cleavage site or an intein.

[0286] The protein of interest can be selected from the group consisting of, for example, a reporter protein, a pharmaceutically active peptide or protein, and an inhibitor of intracellular interferon (IFN) signaling. According to the present invention, the protein of interest preferably does not include a functional non-structural protein derived from a self-replicating virus, such as a functional alphavirus non-structural protein.

[0287] Reporter protein In one embodiment, the open reading frame encodes a reporter protein, such as a cell surface-expressed protein like CD90. In that embodiment, the open reading frame contains a reporter gene. A particular gene can be selected as a reporter because the characteristics it confers on the cells or organisms that express it can be readily identified and measured, or because they are selectable markers. Reporter genes are often used as indicators of whether a particular gene has been taken up by or expressed in a population of cells or organisms. Preferably, the expression product of the reporter gene is visually detectable. Common visually detectable reporter proteins typically have a fluorescent or luminescent protein. Examples of specific reporter genes include the jellyfish green fluorescent protein (GFP) that causes the cells expressing it to fluoresce green under blue light, the enzyme luciferase (Luc) that catalyzes a reaction with luciferin to produce light, and genes encoding red fluorescent protein (RFP). Variants of any of these specific reporter genes are possible as long as they have visually detectable properties. For example, eGFP is a point mutant of GFP. Embodiments of reporter proteins are particularly suitable for testing expression.

[0288] Pharmaceutically active gene products such as peptides or proteins or nucleic acids According to the present invention, in one embodiment, the rRNA comprises or consists of pharmaceutically active rRNA. "Pharmaceutically active RNA" can be RNA that encodes a pharmaceutically active peptide or protein. Preferably, the RNA replicon according to the present invention encodes a pharmaceutically active peptide or protein or other gene product. Preferably, the open reading frame encodes a pharmaceutically active peptide or protein. Preferably, the RNA replicon optionally contains an open reading frame encoding a pharmaceutically active peptide or protein under the control of a subgenomic promoter.

[0289] A "pharmaceutically active peptide or protein" has a positive or beneficial effect on the condition or disease state of a subject when administered to the subject in a therapeutically effective amount. Preferably, the pharmaceutically active peptide or protein has curative or palliative properties and can be administered to improve, alleviate, relieve, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or disorder. A pharmaceutically active peptide or protein can have prophylactic properties and can be used to delay the onset of a disease or to reduce the severity of such a disease or pathological condition. The term "pharmaceutically active peptide or protein" includes the entire protein or polypeptide and can also refer to its pharmaceutically active fragments. This term can also include pharmaceutically active analogs of the peptide or protein. The term "pharmaceutically active peptide or protein" includes peptides and proteins that are antigens, i.e., the peptide or protein induces an immune response in a subject that can be therapeutic or partially or fully protective.

[0290] In one embodiment, the pharmaceutically active peptide or protein is or comprises an immunologically active compound or antigen or epitope.

[0291] According to the present invention, the term "immunologically active compound" relates to any compound that modifies the immune response, preferably by inducing and / or suppressing the maturation of immune cells, inducing and / or suppressing cytokine biosynthesis, and / or altering humoral immunity by stimulating antibody production by B cells. In one embodiment, the immune response includes stimulation of an antibody response (usually including immunoglobulin G (IgG)). Immunologically active compounds have potent immunostimulatory activity including, but not limited to, antiviral and antitumor activity, and can also downregulate other aspects of the immune response, for example, shift the immune response from a Th2 immune response, which is useful for treating a wide range of Th2-mediated diseases.

[0292] According to the present invention, the term "antigen" or "immunogen" encompasses any substance that can induce an immune response. In particular, an "antigen" relates to any substance that specifically reacts with an antibody or a T lymphocyte (T cell). According to the present invention, the term "antigen" includes any molecule containing at least one epitope. Preferably, an antigen in the context of the present invention is a molecule that, optionally after processing, preferably induces an immune reaction specific to the antigen. According to the present invention, any suitable antigen that is a candidate for an immune reaction can be used, and the immune reaction can be both a humoral and a cellular immune reaction. In the context of embodiments of the present invention, an antigen is preferably presented in association with MHC molecules by a cell, preferably an antigen-presenting cell, resulting in an immune reaction against the antigen. An antigen is preferably a product corresponding to or derived from a naturally occurring antigen. Such naturally occurring antigens may include, or be derived from, allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, or the antigen may be a tumor antigen. According to the present invention, an antigen can correspond to a naturally occurring product, such as a viral protein, or a part thereof. In a preferred embodiment, the antigen is a surface polypeptide, i.e., a polypeptide that is naturally presented on the surface of a cell, pathogen, bacterium, virus, fungus, parasite, allergen, or tumor. An antigen can induce an immune response against a cell, pathogen, bacterium, virus, fungus, parasite, allergen, or tumor.

[0293] The term "pathogen" refers to a pathogenic biological substance that can cause disease in an organism, preferably a vertebrate. Pathogens include microorganisms such as bacteria, single-celled eukaryotes (protozoa), fungi, and viruses.

[0294] The terms "epitope", "antigenic peptide", "antigenic epitope", "immunogenic peptide" and "MHC-binding peptide" are used interchangeably herein and refer to an antigenic determinant in a molecule such as an antigen, i.e., a part or fragment of an immunologically active compound that is recognized by the immune system, for example, recognized by T cells when presented in association with MHC molecules, in particular. An epitope of a protein preferably comprises a continuous or discontinuous portion of said protein and is preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, and most preferably 10 to 25 amino acids in length. For example, an epitope can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. According to the present invention, an epitope can bind to MHC molecules such as MHC molecules on the surface of cells and can thus be a "MHC-binding peptide" or an "antigenic peptide". The term "major histocompatibility complex" and the abbreviation "MHC" relate to a complex of genes that includes MHC class I and MHC class II molecules and is present in all vertebrates. MHC proteins or molecules are important for signal transduction between lymphocytes and antigen-presenting cells or diseased cells in an immune response. MHC proteins or molecules bind to peptides and present them for recognition by T cell receptors. Proteins encoded by MHC are expressed on the surface of cells and present both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. Preferred such immunogenic moieties bind to MHC class I or class II molecules. As used herein, an immunogenic moiety is said to "bind" to MHC class I or class II molecules if such binding is detectable using any assay known in the art. The term "MHC-binding peptide" relates to peptides that bind to MHC class I and / or MHC class II molecules. In the case of class I MHC / peptide complexes, the binding peptide is typically 8 to 10 amino acids in length, although longer or shorter peptides can be effective.In the case of a class II MHC / peptide complex, the binding peptide is typically 10 to 25 amino acids in length, particularly 13 to 18 amino acids in length, although longer and shorter peptides can be effective.

[0295] In one embodiment, the protein of interest according to the invention comprises an epitope suitable for vaccination of a target organism. Those skilled in the art will understand that one of the principles of immunobiology and vaccination is based on the fact that an immune protective response against a disease is generated by immunizing an organism with an antigen immunologically relevant to the disease to be treated. The antigen is selected from the group comprising self-antigens and non-self-antigens. The non-self-antigen is preferably a bacterial antigen, a viral antigen, a fungal antigen, an allergen or a parasite antigen. The antigen preferably comprises an epitope capable of inducing an immune response in the target organism. For example, the epitope can induce an immune response, such as a cytotoxic T cell response, against bacteria, viruses, fungi, parasites, allergens, or tumors.

[0296] In some embodiments, the non-self-antigen is a bacterial antigen. In some embodiments, the antigen induces an immune response against bacteria that infect animals including mammals including birds, fish, and livestock. Preferably, the bacteria against which the immune response is induced are pathogenic bacteria.

[0297] In some embodiments, the non-self-antigen is a viral antigen. The viral antigen can be, for example, a peptide derived from a viral surface protein, such as a capsid polypeptide or a spike polypeptide, such as a peptide derived from the genus Coronavirus. In some embodiments, the antigen induces an immune response against viruses that infect animals including mammals including birds, fish, and livestock. Preferably, the virus against which the immune response is induced is a pathogenic virus such as the Ebola virus.

[0298] In some embodiments, the non-self antigen is a polypeptide or protein derived from a fungus. In some embodiments, the antigen elicits an immune response against a fungus that infects animals, including mammals such as birds, fish, and livestock. Preferably, the fungus against which the immune response is elicited is a pathogenic fungus.

[0299] In some embodiments, the non-self antigen is a polypeptide or protein derived from a single-celled eukaryotic parasite. In some embodiments, the antigen elicits an immune response against a single-celled eukaryotic parasite, preferably a pathogenic single-celled eukaryotic parasite. Pathogenic single-celled eukaryotic parasites can be derived from, for example, the genus Plasmodium, such as Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium ovale, the genus Leishmania, or the genus Trypanosoma, such as Trypanosoma cruzi or Trypanosoma brucei.

[0300] In some embodiments, the pharmaceutically active peptide or protein need not be an antigen that elicits an immune response. Suitable pharmaceutically active proteins or peptides include cytokines and immune system proteins, such as immunologically active compounds (e.g., interleukins, colony stimulating factors (CSF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, selectins, homing receptors, T cell receptors, chimeric antigen receptors (CAR), immunoglobulins), hormones (insulin, thyroid hormones, catecholamines, gonadotropins, trophic hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptin, etc.), growth hormones (e.g., human growth hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factors, etc.), growth factor receptors, enzymes (tissue plasminogen activator, streptokinase, cholesterol biosynthetic or degradative enzymes, steroidogenic enzymes, kinases, phosphodiesterases, methylases, demethylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatase, cytochrome, adenylate or guanylate cyclase, neuraminidase, etc.), receptors (steroid hormone receptors, peptide receptors), binding proteins (e.g., growth hormone or growth factor binding proteins), transcription and translation factors, tumor growth inhibitory proteins (e.g., proteins that inhibit angiogenesis), structural proteins (collagen, fibroin, fibrinogen, elastin, tubulin, actin, myosin, etc.), blood proteins (thrombin, serum albumin, factor VII, factor VIII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin granulocyte colony stimulating factor (GCSF) or modified factor VIII, anticoagulant factors, etc.) and may be selected from the group consisting of.In one embodiment, the pharmaceutically active protein according to the present invention is a cytokine involved in the regulation of lymphatic homeostasis, preferably a cytokine involved in the generation, priming, expansion, differentiation and / or survival of T cells, preferably inducing or enhancing them. In one embodiment, the cytokine is an interleukin, such as IL-2, IL-7, IL-12, IL-15, or IL-21.

[0301] Position of at least one open reading frame in the rRNA molecule The rRNA replicon is suitable for the expression of one or more genes encoding a peptide or protein of interest, optionally under the control of a subgenomic promoter. Various embodiments are possible. One or more open reading frames, each encoding a peptide or protein of interest, may be present on the RNA replicon. The most upstream open reading frame of the RNA replicon is referred to as the "first open reading frame". In one embodiment, the first open reading frame encoding the protein of interest is located downstream of the 5' replication recognition sequence and upstream of the IRES (and the open reading frame encoding the functional non-structural protein derived from the self-replicating virus). In some embodiments, the "first open reading frame" is the only open reading frame of the RNA replicon. Optionally, one or more additional open reading frames may be present downstream of the first open reading frame. One or more additional open reading frames downstream of the first open reading frame may be referred to as the "second open reading frame", "third open reading frame", etc. in the order in which they are present downstream of the first open reading frame (from 5' to 3'). In one embodiment, one or more additional open reading frames encoding one or more proteins of interest are located downstream of the open reading frame encoding the functional non-structural protein derived from the self-replicating virus and are preferably controlled by a subgenomic promoter. Preferably, each open reading frame contains a start codon (base triplet), typically AUG in the RNA molecule corresponding to ATG in each DNA molecule.

[0302] If the replicon contains a 3' replication recognition sequence, it is preferred that all open reading frames are located upstream of the 3' replication recognition sequence.

[0303] In some embodiments, at least one open reading frame of the replicon is under the control of a subgenomic promoter, preferably an alphavirus subgenomic promoter. The alphavirus subgenomic promoter is very efficient and thus suitable for high-level heterologous gene expression. Preferably, the subgenomic promoter is a promoter for subgenomic transcripts in an alphavirus. This means that the subgenomic promoter is native to the alphavirus and preferably controls the transcription of an open reading frame encoding one or more structural proteins in said alphavirus. Alternatively, the subgenomic promoter is a variant of the alphavirus subgenomic promoter, and any variant that functions as a promoter for subgenomic RNA transcription in a host cell is suitable. When the replicon contains a subgenomic promoter, the replicon preferably contains a conserved sequence element 3 (CSE 3) or a variant thereof.

[0304] Preferably, at least one open reading frame under the control of the subgenomic promoter is located downstream of the subgenomic promoter. Preferably, the subgenomic promoter controls the production of subgenomic RNA containing the transcript of the open reading frame.

[0305] In some embodiments, the first open reading frame is under the control of a subgenomic promoter. In one embodiment, when the first open reading frame is under the control of a subgenomic promoter, the gene encoded by the first open reading frame can be expressed from both the replicon and its subgenomic transcript (the latter in the presence of functional alpha-viral non-structural proteins). One or more additional open reading frames, each under the control of a subgenomic promoter, can be present downstream of the first open reading frame, which can be under the control of a subgenomic promoter. The gene encoded by one or more additional open reading frames, such as by a second open reading frame, can be translated from one or more subgenomic transcripts, each under the control of a subgenomic promoter. For example, an RNA replicon can include a subgenomic promoter that controls the production of a transcript encoding a second protein of interest.

[0306] In other embodiments, the first open reading frame is not under the control of a subgenomic promoter. In one embodiment, when the first open reading frame is not under the control of a subgenomic promoter, the gene encoded by the first open reading frame can be expressed from the replicon. One or more additional open reading frames, each under the control of a subgenomic promoter, can be present downstream of the first open reading frame. The gene encoded by one or more additional open reading frames can be expressed from a subgenomic transcript.

[0307] In a cell containing a replicon according to the invention, the replicon can be amplified by a functional non-structural protein. Further, when the replicon includes one or more open reading frames under the control of a subgenomic promoter, one or more subgenomic transcripts are expected to be produced by a functional non-structural protein.

[0308] When the replicon contains multiple open reading frames encoding the protein of interest, each open reading frame preferably encodes a different protein. For example, the protein encoded by the second open reading frame is different from the protein encoded by the first open reading frame.

[0309] Other features of the replicable RNA molecule according to the present invention The RNA molecule according to the present invention may optionally be characterized by further features, such as a 5' cap, 5'-UTR, 3'-UTR, poly(A) sequence, and / or adaptation of the codon usage frequency for optimized translation and / or stabilization of the RNA molecule, as detailed below.

[0310] Cap In some embodiments, the replicon according to the present invention includes a 5' cap.

[0311] The terms "5' cap", "cap", "5' cap structure", and "cap structure" are used synonymously to refer to the dinucleotide found at the 5' end of some eukaryotic primary transcripts, such as precursor messenger RNA. The 5' cap is a structure in which (optionally modified) guanosine is linked to the first nucleotide of the mRNA molecule via a 5'-5' triphosphate bond (or a modified triphosphate bond in the case of certain cap analogs). These terms can refer to conventional caps or cap analogs.

[0312] "RNA containing a 5' cap", "RNA having a 5' cap", "RNA modified with a 5' cap", or "capped RNA" refers to RNA containing a 5' cap. For example, providing a 5' cap to RNA can be achieved by in vitro transcription of a DNA template in the presence of said 5' cap, said 5' cap being co-transcriptionally incorporated into the resulting RNA strand, or RNA can be produced, for example, by in vitro transcription, and the 5' cap can be ligated to the RNA post-transcriptionally using a capping enzyme, such as the capping enzyme of vaccinia virus. In capped RNA, the 3' position of the first base of the (capped) RNA molecule is linked via a phosphodiester bond to the 5' position of the subsequent base of the RNA molecule ("the second base").

[0313] In one embodiment, the RNA replicon contains a 5' cap. In one embodiment, the RNA replicon does not contain a 5' cap.

[0314] The term "conventional 5' cap" refers to a naturally occurring 5' cap, preferably a 7-methylguanosine cap. In a 7-methylguanosine cap, the guanosine of the cap is a modified guanosine, and the modification consists of methylation at the 7 position.

[0315] In the context of the present invention, the term "5' cap analog" refers to a molecular structure that is similar to a conventional 5' cap but is modified to have the ability to stabilize RNA when bound to RNA in vivo and / or intracellularly. A cap analog is not a conventional 5' cap.

[0316] In the case of eukaryotic mRNA, the 5' cap is generally described as being involved in efficient translation of the mRNA: Generally, in eukaryotes, translation starts only at the 5' end of the messenger RNA (mRNA) molecule unless an internal ribosome entry site (IRES) is present. Eukaryotic cells can provide an RNA with a 5' cap during transcription in the nucleus: Newly synthesized mRNA is usually modified with a 5' cap structure when the transcript reaches a length of, for example, 20 to 30 nucleotides. First, the 5' terminal nucleotide pppN (where ppp represents triphosphate; N represents any nucleoside) is converted intracellularly to 5'GpppN by a capping enzyme having RNA 5'-triphosphatase and guanylyltransferase activities. GpppN is then methylated intracellularly by a second enzyme having (guanine-7)-methyltransferase activity to form a monomethylated m 7 GpppN cap. In one embodiment, the 5' cap used in the present invention is a natural 5' cap.

[0317] In the present invention, the natural 5' cap dinucleotide typically consists of an unmethylated cap dinucleotide (G(5')ppp(5')N; also called GpppN) and a methylated cap dinucleotide ((m 7 G(5')ppp(5')N; m 7 GpppN; also called). m 7 GpppN (where N is G) is represented by the following formula:

Chemical formula

[0318] The capped RNA of the present invention can be prepared in vitro and thus does not depend on the capping mechanism in host cells. The most frequently used method for producing capped RNA in vitro is to use all four ribonucleoside triphosphates and m 7 G(5')ppp(5')G(m 7In the presence of a capped dinucleotide (also called GpppG), it is to transcribe a DNA template with either a bacterial or bacteriophage RNA polymerase. The RNA polymerase adds an m to the α-phosphate of the next template nucleoside triphosphate (pppN). 7 Transcription is initiated by a nucleophilic attack by the 3'-OH of the guanosine moiety of GpppG, resulting in an intermediate m 7 GpppGpN (where N is the second base of the RNA molecule). The formation of the competing GTP-initiated product pppGpN is suppressed by setting the molar ratio of cap to GTP during in vitro transcription to 5-10.

[0319] In a preferred embodiment of the invention, the 5' cap (when present) is a 5' cap analog. These embodiments are particularly suitable when the RNA is obtained by in vitro transcription, for example, in vitro transcribed RNA (IVT-RNA). Cap analogs have first been described as promoting the large-scale synthesis of RNA transcripts by in vitro transcription.

[0320] For messenger RNA, several cap analogs (synthetic caps) have been generally described so far, and all of them can be used in the context of the present invention. Ideally, a cap analog is selected that is related to higher translation efficiency and / or increased resistance to in vivo degradation and / or increased resistance to in vitro degradation.

[0321] Preferably, a cap analog that can be incorporated into the RNA strand in only one orientation is used. Pasquinelli et al. (1995, RNA J. 1:957-967) showed that during in vitro transcription, bacteriophage RNA polymerase uses a 7-methylguanosine unit for the initiation of transcription, such that approximately 40-50% of the transcripts with a cap have the cap dinucleotide in the reverse orientation (i.e., the initial reaction product is Gpppm 7It was demonstrated that it is GpN. Compared with RNA having a correct cap, RNA having a reverse cap is not functional with respect to the translation of a nucleic acid sequence into a protein. Therefore, incorporating the cap in the correct orientation, i.e., m 7 It is desirable to obtain RNA having a structure essentially corresponding to GpppGpN or the like. Reverse incorporation of the cap dinucleotide has been shown to be inhibited by substitution of either the 2'-OH group or the 3'-OH group of the methylated guanosine unit (Stepinski et al., 2001, RNA J. 7:1486-1495; Peng et al., 2002, Org. Lett. 24:161-164). RNA synthesized in the presence of such an "anti-reverse cap analog" is translated more efficiently than RNA transcribed in vitro in the presence of the conventional 5' cap m 7 GpppG. For this purpose, one cap analog in which the 3'OH group of the methylated guanosine unit is replaced by OCH 3 is described, for example, by Holtkamp et al., 2006, Blood 108:4009-4017 (7-methyl(3'-O-methyl)GpppG; anti-reverse cap analog (ARCA)). ARCA is an appropriate cap dinucleotide according to the present invention. [Chemical formula]

[0322] In one embodiment, the RNA of the present invention is substantially resistant to cap removal. This is important because generally, the amount of protein produced from synthetic mRNA introduced into cultured mammalian cells is limited by the natural degradation of the mRNA. One in vivo pathway of mRNA degradation begins with the removal of the mRNA cap. This removal is catalyzed by a heterodimeric pyrophosphatase that includes a regulatory subunit (Dcp1) and a catalytic subunit (Dcp2). The catalytic subunit cleaves between the α and β phosphate groups of the triphosphate bridge. In the present invention, a cap analog that is less susceptible or more resistant to this type of cleavage can be selected or can be present. Suitable cap analogs for this purpose are of formula (I): [Chemical formula] Formula (I) (wherein R 1 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, R 2 and R 3 are independently selected from the group consisting of H, halo, OH, and optionally substituted alkoxy, or R 2 and R 3 together form O-X-O (where X is optionally substituted CH 2 , CH 2 CH 2 , CH 2 CH 2 CH 2 , CH 2 CH(CH 3 ), and C(CH 3 ) 2 selected from the group consisting of), or R 2 is bonded to the hydrogen atom at the 4'-position of the ring to which R 2 is attached to form -O-CH 2 - or -CH 2 -O-, R 5 is selected from the group consisting of S, Se, and BH 3 and, R 4 and R 6 are independently selected from the group consisting of O, S, Se, and BH 3 (). It may be selected from cap dinucleotides according to

[0323] n is 1, 2, or 3.

[0324] R 1 , R 2 , R3, R 4 , R 5 , R 6 Preferred embodiments of are disclosed in International Publication No. WO 2011 / 015347 A1 and may be selected accordingly in the present invention.

[0325] For example, in one embodiment, the RNA of the present invention comprises a phosphorothioate cap analog. The phosphorothioate cap analog is one in which one of the three non-bridging O atoms in the triphosphate chain is replaced by an S atom, i.e., R of formula (I) 4 , R 5 or R 6 is a specific cap analog in which one of them is S. The phosphorothioate cap analog is described by J. Kowalska et al., 2008, RNA, 14: 1119-1131 as a solution to the unwanted cap removal process and thus as a solution to enhance the stability of RNA in vivo. In particular, substituting a sulfur atom for an oxygen atom at the β-phosphate group of the 5' cap results in stabilization against Dcp2. In a preferred embodiment thereof in the present invention, R of formula (I) 5 is S, and R 4 and R 6 are O.

[0326] In a further embodiment, the RNA of the invention comprises a phosphorothioate cap analog in which the phosphorothioate modification of the RNA 5' cap is combined with an "anti-reverse cap analog" (ARCA) modification. Each ARCA-phosphorothioate cap analog is described in WO 2008 / 157688 A2, and all of them can be used for the RNA of the invention. In that embodiment, R 2 or R 3 of at least one is not OH, preferably one of R 2 and R 3 is methoxy (OCH 3 ), and the other of R 2 and R 3 is preferably OH. In a preferred embodiment, the oxygen atom is replaced by a sulfur atom at the β-phosphate group (thus, R 5 of formula (I) is S, and R 4 and R 6 are O). The phosphorothioate modification of ARCA is thought to ensure that the α, β, and γ phosphorothioate groups are correctly positioned within the active site of the cap-binding protein in both the translation machinery and the cap-removing machinery. At least some of these analogs are essentially resistant to pyrophosphatase Dcp1 / Dcp2. The phosphorothioate-modified ARCA has been described as having a much higher affinity for eIF4E than the corresponding ARCA lacking the phosphorothioate group.

[0327] Each cap analog particularly preferred in the present invention, namely m 2’ 7,2’-O Gpp s pG, is called β-S-ARCA (WO 2008 / 157688 A2; Kuhn et al., 2010, Gene Ther. 17:961-971). Thus, in one embodiment of the invention, the RNA of the invention is modified with β-S-ARCA. β-S-ARCA is represented by the following structure:

Chemical formula

[0328] Generally, replacing the sulfur atom with an oxygen atom in the cross-linked phosphate results in phosphorothioate diastereomers called D1 and D2, based on their elution patterns in HPLC. Briefly, the "D1 diastereomer of β-S-ARCA" or "β-S-ARCA (D1)" elutes first on the HPLC column compared to the D2 diastereomer of β-S-ARCA (β-S-ARCA (D2)) and thus exhibits a shorter retention time. The determination of the stereochemical configuration by HPLC is described in International Publication No. WO 2011 / 015347 A1.

[0329] In a first particularly preferred embodiment of the present invention, the RNA of the present invention is modified with the β-S-ARCA (D2) diastereomer. The two diastereomers of β-S-ARCA differ in their sensitivity to nucleases. RNA carrying the D2 diastereomer of β-S-ARCA is almost completely resistant to Dcp2 cleavage (only 6% cleavage compared to RNA synthesized in the presence of the unmodified ARCA 5'-cap), while RNA having the β-S-ARCA (D1) 5'-cap has been shown to exhibit moderate sensitivity to Dcp2 cleavage (71% cleavage). It has further been shown that the increased stability to Dcp2 cleavage correlates with an increase in protein expression in mammalian cells. In particular, RNA having the β-S-ARCA (D2) cap has been shown to be translated more efficiently in mammalian cells than RNA having the β-S-ARCA (D1) cap. Thus, in one embodiment of the present invention, the RNA of the present invention has a substituent R of formula (I) corresponding to the stereochemical configuration at the P β atom of the D2 diastereomer of β-S-ARCA 5 and is modified with a cap analog according to formula (I) characterized by the stereochemical configuration at the P atom containing the same. In that embodiment, R 5 of formula (I) is S, and R 4 and R 6 are O. Further, R 2 or R 3At least one of them is preferably not OH, preferably R 2 and R 3 One of them is methoxy (OCH3), and R 2 and R 3 The other is preferably OH.

[0330] In a second particularly preferred embodiment, the RNA of the invention is modified with a β-S-ARCA(D1) diastereomer. This embodiment is particularly suitable for the transfer of capped RNA into immature antigen-presenting cells, for example for vaccination purposes. The β-S-ARCA(D1) diastereomers have been shown to enhance the stability of RNA, enhance the translation efficiency of RNA, extend the translation of RNA, increase the total protein expression of RNA, and / or increase the immune response to the antigen or antigen peptide encoded by said RNA when each transferred the capped RNA into immature antigen-presenting cells (Kuhn et al., 2010, Gene Ther. 17:961-971). Thus, in an alternative embodiment of the invention, the RNA of the invention is modified with a cap analog according to formula (I) having a stereochemical configuration at the P β atom corresponding to the stereochemical configuration of the D1 diastereomer of β-S-ARCA at the substituent R 5 of the P atom containing it. Each cap analog and its embodiments are described in International Publication No. WO 2011 / 015347 A1 and Kuhn et al., 2010, Gene Ther. 17:961-971. Any cap analog described in International Publication No. WO 2011 / 015347 A1 having a stereochemical configuration at the P 5 atom containing the substituent R β corresponding to the stereochemical configuration of the D1 diastereomer of β-S-ARCA at the P 5 atom can be used in the present invention. Preferably, R 4 and R 6 are O. Further, at least one of R 2 or R 3 of formula (I) is preferably not OH, preferably R2 and R 3 One of them is methoxy (OCH3), and R 2 and R 3 The other is preferably OH.

[0331] In one embodiment, the RNA of the present invention is modified with a 5' cap structure according to formula (I) in which any one phosphate group is substituted by a boranophosphate group or a phosphoselenoate group. Such caps have increased stability both in vitro and in vivo. Optionally, each compound has a 2'-O- or 3'-O-alkyl group (alkyl is preferably methyl); each cap analog is BH 3 -ARCA or Se-ARCA. Compounds particularly suitable for capping of mRNA include β-BH 3 -ARCA and β-Se-ARCA as described in International Publication No. WO 2009 / 149253 A2. For these compounds, the stereochemical configuration at the P β atom of the D1 diastereomer of β-S-ARCA corresponds to the substituent R 5 of formula (I) at the P atom containing it, which is preferred.

[0332] In one embodiment, the 5' cap can be CleanCap supplied by Trilink Biotechnologies, San Diego, CA, having the following structure:

Chemical formula

[0333] UTR The term "untranslated region" or "UTR" relates to a region within a DNA molecule that is transcribed but not translated into an amino acid sequence, or the corresponding region within an RNA molecule such as an mRNA molecule. The untranslated region (UTR) can be present on the 5' side (upstream) (5'-UTR) and / or the 3' side (downstream) (3'-UTR) of the open reading frame.

[0334] The 3’-UTR, when present, is located at the 3’ end of the gene, downstream of the stop codon of the protein coding region, although the term “3’-UTR” preferably does not include the poly(A) tail. Thus, the 3’-UTR is upstream of the poly(A) tail (when present), for example, immediately adjacent to the poly(A) tail.

[0335] The 5’-UTR, when present, is located at the 5’ end of the gene, upstream of the start codon of the protein coding region. The 5’-UTR is downstream of the 5’ cap (when present), for example, immediately adjacent to the 5’ cap.

[0336] According to the present invention, the 5’ and / or 3’ untranslated regions can be operably linked to the open reading frame such that these regions associate with the open reading frame in a manner that enhances the stability and / or translation efficiency of the RNA comprising the open reading frame.

[0337] In some embodiments, the RNA replicons according to the present invention comprise a 5’-UTR and / or a 3’-UTR.

[0338] UTRs are involved in the stability and translation efficiency of RNA. In addition to the structural modifications to the 5’ cap and / or 3’ poly(A) tail described herein, both can be improved by selecting specific 5’ and / or 3’ untranslated regions (UTRs). Sequence elements within the UTRs are generally understood to affect translation efficiency (mainly 5’-UTR) and RNA stability (mainly 3’-UTR). It is preferred that an active 5’-UTR is present to enhance the translation efficiency and / or stability of the RNA replicon. Independently or additionally, it is preferred that an active 3’-UTR is present to enhance the translation efficiency and / or stability of the RNA replicon.

[0339] With respect to the first nucleic acid sequence (e.g., UTR), the terms "active to enhance translation efficiency" and / or "active to enhance stability" mean that the first nucleic acid sequence can modify the translation efficiency and / or stability of the second nucleic acid sequence in a transcript common with the second nucleic acid sequence, such that the translation efficiency and / or stability is increased as compared to the translation efficiency and / or stability of the second nucleic acid sequence in the absence of the first nucleic acid sequence.

[0340] In one embodiment, the RNA replicon according to the invention comprises a 5'-UTR and / or a 3'-UTR that is heterologous or non-native to the alpha virus from which the functional alpha virus non-structural protein is derived. This allows for the design of untranslated regions according to the desired translation efficiency and RNA stability. Thus, heterologous or non-native UTRs allow for a high degree of flexibility, which is advantageous compared to native alpha virus UTRs.

[0341] Preferably, the RNA replicon according to the invention comprises a 5'-UTR and / or a 3'-UTR that is not of viral origin, in particular not of alpha virus origin. In one embodiment, the RNA replicon comprises a 5'-UTR derived from a eukaryotic 5'-UTR and / or a 3'-UTR derived from a eukaryotic 3'-UTR.

[0342] The 5'-UTR according to the invention can comprise any combination of a plurality of nucleic acid sequences, optionally separated by a linker. The 3'-UTR according to the invention can comprise any combination of a plurality of nucleic acid sequences, optionally separated by a linker.

[0343] The term "linker" according to the invention relates to a nucleic acid sequence that is added between two nucleic acid sequences to ligate the two nucleic acid sequences. There are no particular restrictions on the linker sequence.

[0344] The 3'-UTR typically has a length of 200 to 2000 nucleotides, for example 500 to 1500 nucleotides. The 3' untranslated region of immunoglobulin mRNA is relatively short (less than about 300 nucleotides), while the 3' untranslated regions of other genes are relatively long. For example, the 3'-UTR of tPA is about 800 nucleotides long, the 3'-UTR of factor VIII is about 1800 nucleotides long, and the 3'-UTR of erythropoietin is about 560 nucleotides long. The 3' untranslated region of mammalian mRNA typically has a homologous region known as the AAUAAA hexanucleotide sequence. This sequence is probably the poly(A) addition signal and is often located 10 to 30 bases upstream of the poly(A) addition site. The 3'-untranslated region can contain one or more inverted repeat sequences that can fold to provide a stem-loop structure that acts as a barrier to exoribonucleases or interacts with proteins (such as RNA-binding proteins) known to enhance RNA stability.

[0345] Two consecutive identical copies of the human β-globin 3'-UTR, particularly the human β-globin 3'-UTR, contribute to high transcript stability and translation efficiency (Holtkamp et al., 2006, Blood 108:4009-4017). Thus, in one embodiment, the RNA replicon according to the present invention comprises two consecutive identical copies of the human β-globin 3'-UTR. Thus, this comprises, in the 5'→3' direction: (a) optionally a 5'-UTR; (b) an open reading frame; (c) a 3'-UTR, wherein the 3'-UTR comprises two consecutive identical copies of the human β-globin 3'-UTR, a fragment thereof, or a variant of the human β-globin 3'-UTR or a fragment thereof.

[0346] In one embodiment, the RNA replicon according to the present invention comprises a 3'-UTR that is active in enhancing translation efficiency and / or stability but is not the human β-globin 3'-UTR, a fragment thereof, or a variant of the human β-globin 3'-UTR or a fragment thereof.

[0347] In one embodiment, the RNA replicon according to the present invention includes an active 5'-UTR to enhance translation efficiency and / or stability.

[0348] Poly(A) sequence In some embodiments, the replicon according to the present invention includes a 3'-poly(A) sequence. When the replicon includes the conserved sequence element 4 (CSE 4), the 3'-poly(A) sequence of the replicon is preferably present downstream of CSE 4 and most preferably directly adjacent to CSE 4.

[0349] According to the present invention, in one embodiment, the poly(A) sequence comprises, consists essentially of, or consists of at least 20, preferably at least 26, preferably at least 40, preferably at least 80, preferably at least 100, and preferably at most 500, preferably at most 400, preferably at most 300, preferably at most 200, particularly at most 150, particularly at most about 120 A nucleotides. In this context, "consists essentially of" means that most of the nucleotides in the poly(A) sequence, typically at least 50%, preferably at least 75% of the number of nucleotides in the "poly(A) sequence", are A nucleotides (adenylic acid), but the remaining nucleotides are allowed to be nucleotides other than A nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), C nucleotides (cytidylic acid). In this context, "consists of" means that all of the nucleotides in the poly(A) sequence, i.e., 100% of the number of nucleotides in the poly(A) sequence, are A nucleotides. The term "A nucleotide" or "A" refers to adenylic acid.

[0350] Indeed, a 3' poly(A) sequence of approximately 120 A nucleotides has been demonstrated to have a beneficial effect on the level of RNA in transfected eukaryotic cells and on the level of the protein translated from the open reading frame present upstream (5' side) of the 3' poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0351] In alphaviruses, a 3' poly(A) sequence of at least 11 consecutive adenylate residues, or at least 25 consecutive adenylate residues, is thought to be important for efficient synthesis of the minus strand. In particular, in alphaviruses, a 3' poly(A) sequence of at least 25 consecutive adenylate residues is understood to function with the conserved sequence element 4 (CSE 4) to promote (-) strand synthesis (Hardy & Rice, 2005, J. Virol. 79:4630-4639).

[0352] The present invention provides a 3' poly(A) sequence that is added during transcription of RNA, i.e., during generation of in vitro transcribed RNA, based on a DNA template that includes repetitive dT nucleotides (deoxythymidylate) within the strand complementary to the coding strand. The DNA sequence (coding strand) encoding the poly(A) sequence is referred to as a poly(A) cassette.

[0353] In a preferred embodiment of the present invention, the 3' poly(A) cassette present in the coding strand of the DNA consists essentially of dA nucleotides but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT). Such a random sequence can be 5 to 50, preferably 10 to 30, more preferably 10 to 20 nucleotides in length. Such cassettes are disclosed in International Publication No. WO 2016 / 005004 A1. Any poly(A) cassette disclosed in International Publication No. WO 2016 / 005004 A1 can be used in the present invention. A poly(A) cassette consisting essentially of dA nucleotides but having an equal distribution of the four nucleotides (dA, dC, dG, dT) and interrupted by a random sequence having a length of, for example, 5 to 50 nucleotides shows sustained growth of plasmid DNA in Escherichia coli at the DNA level and is still associated with beneficial properties related to the support of RNA stability and translation efficiency at the RNA level.

[0354] As a result, in a preferred embodiment of the present invention, the 3' poly(A) sequence contained in the RNA molecule described herein consists essentially of A nucleotides but is interrupted by a random sequence having an equal distribution of the four nucleotides (A, C, G, U). Such a random sequence can be 5 to 50, preferably 10 to 30, more preferably 10 to 20 nucleotides in length.

[0355] Codon usage frequency Generally, codon degeneracy in the genetic code allows for the substitution of a particular codon (a base triplet encoding an amino acid) present in an RNA sequence with another codon (a base triplet) while maintaining the same coding ability (such that the substituting codon encodes the same amino acid as the codon being substituted). In some embodiments of the present invention, at least one codon of an open reading frame contained in an RNA (rRNA) molecule is different from each codon within each open reading frame of the species from which the open reading frame is derived. In such embodiments, the coding sequence of the open reading frame is said to be “adapted” or “modified”. The coding sequence of an open reading frame contained in a replicon can be adapted.

[0356] For example, when adapting the coding sequence of an open reading frame, codons that are frequently used can be selected: WO 2009 / 024567 A1 describes the adaptation of the coding sequence of a nucleic acid molecule that includes the substitution of rare codons with more frequently used codons. Since codon usage frequency depends on the host cell or host organism, this type of adaptation is suitable for adapting a nucleic acid sequence for expression in a particular host cell or host organism. Generally speaking, more frequently used codons are typically translated more efficiently in a host cell or host organism, but adaptation of all codons in an open reading frame is not necessarily required.

[0357] For example, when adapting the coding sequence of an open reading frame, the content of G (guanine) residues and C (cytidylic acid) residues can be altered by selecting the codon having the highest GC-rich content for each amino acid. RNA molecules having GC-rich open reading frames have been reported to have the potential to reduce immune activation and improve the translation and half-life of the RNA (Thess et al., 2015, Mol. Ther. 23, 1457-1465).

[0358] In particular, the coding sequences of non-structural proteins can be adapted as desired. This degree of freedom is possible because the open reading frames encoding non-structural proteins do not overlap with the 5' replication recognition sequences of the replicon.

[0359] Safety features of embodiments of the present invention The following features are preferred in the present invention, either alone or in any suitable combination: The replicon of the present invention is not particle-forming. This means that after inoculation of host cells with the replicon of the present invention, the host cells do not produce virus particles such as progeny virus particles. In one embodiment, the RNA replicon according to the present invention does not contain any genetic information encoding viral structural proteins, such as alphavirus structural proteins such as core nucleocapsid protein C, envelope protein P62, and / or envelope protein E1. Preferably, the replicon according to the present invention does not contain a viral packaging signal, such as an alphavirus packaging signal. For example, the alphavirus packaging signal contained in the coding region of nsP2 of SFV (White et al. 1998, J. Virol. 72:4320-4326) can be removed, for example, by deletion or mutation. Suitable methods for removing the alphavirus packaging signal include adapting the codon usage frequency of the coding region of nsP2. The degeneracy of the genetic code can make it possible to abolish the function of the packaging signal without affecting the amino acid sequence of the encoded nsP2.

[0360] DNA The present invention also provides DNA comprising a nucleic acid sequence encoding an RNA replicon according to the present invention.

[0361] Preferably, the DNA is double-stranded.

[0362] In a preferred embodiment, the DNA is a plasmid. As used herein, the term "plasmid" generally refers to an extrachromosomal genetic construct that can replicate independently of chromosomal DNA, usually a circular DNA double strand.

[0363] The DNA of the present invention may contain a promoter that can be recognized by a DNA-dependent RNA polymerase. This enables the in vivo or in vitro transcription of the encoded RNA, for example, the RNA of the present invention. The IVT vector can be used in a standardized manner as a template for in vitro transcription. Examples of preferred promoters according to the present invention are the promoters of SP6, T3 or T7 polymerase.

[0364] In one embodiment, the DNA of the present invention is an isolated nucleic acid molecule.

[0365] Method for preparing RNA The RNA molecule according to the present invention can be obtained by in vitro transcription. In vitro transcribed RNA (IVT-RNA) is of particular interest in the present invention. IVT-RNA can be obtained by transcription from a nucleic acid molecule (especially a DNA molecule). The DNA molecule(s) of the present invention is / are suitable for such purposes, especially when it / they contain a promoter that can be recognized by a DNA-dependent RNA polymerase.

[0366] The rRNA according to the present invention can be synthesized in vitro. This enables the addition of a cap analog to the in vitro transcription reaction. Typically, the poly(A) tail is encoded by a poly(dT) sequence on the DNA template. Alternatively, capping and the addition of the poly(A) tail can be achieved enzymatically after transcription.

[0367] Methods of in vitro transcription are known to those skilled in the art. For example, various in vitro transcription kits are commercially available as described in International Publication No. WO 2011 / 015347 A1.

[0368] Kit The present invention also provides a kit comprising at least two RNA replicons according to the present invention.

[0369] In one embodiment, the components of the kit exist as separate entities. For example, one component of the kit may be present in one entity and another component of the kit may be present in another entity. For example, an open container or a closed container is a suitable entity. A closed container is preferred. The container used should preferably be RNase-free or substantially RNase-free.

[0370] In one embodiment, the kit of the present invention contains RNA for inoculation of cells and / or administration to human or animal subjects.

[0371] The kit according to the present invention optionally contains a label or other form of information element, such as an electronic data carrier. The label or information element preferably contains instructions, such as printed written instructions, or optionally printable electronic form instructions. The instructions may refer to at least one appropriate and possible use of the kit.

[0372] Pharmaceutical composition The RNA replicons described herein may exist in the form of a pharmaceutical composition. The pharmaceutical composition according to the present invention may contain at least one nucleic acid molecule according to the present invention. The pharmaceutical composition according to the present invention contains a pharmaceutically acceptable diluent and / or a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable vehicle. The choice of pharmaceutically acceptable carrier, vehicle, excipient or diluent is not particularly limited. Any suitable pharmaceutically acceptable carrier, vehicle, excipient or diluent known in the art may be used.

[0373] In one embodiment of the present invention, the pharmaceutical composition can further contain a solvent such as an aqueous solvent or any solvent that enables preservation of the integrity of rRNA. In a preferred embodiment, the pharmaceutical composition is an aqueous solution containing RNA. The aqueous solution may optionally contain a solute, such as a salt.

[0374] In one embodiment of the present invention, the pharmaceutical composition is in the form of a lyophilized composition. The lyophilized composition can be obtained by lyophilizing each aqueous composition.

[0375] In one embodiment, the pharmaceutical composition comprises at least one cationic entity. Generally, cationic lipids, cationic polymers, and other substances with a positive charge can form complexes with negatively charged nucleic acids. The RNA according to the present invention can be stabilized by complexation with a cationic compound, preferably a polycationic compound, such as a cationic or polycationic peptide or protein. In one embodiment, the pharmaceutical composition according to the present invention comprises at least one cationic molecule selected from the group consisting of protamine, polyethyleneimine, poly-L-lysine, poly-L-arginine, histone, or a cationic lipid.

[0376] According to the present invention, the cationic lipid is a cationic amphiphilic molecule, for example, a molecule containing at least one hydrophilic and lipophilic moiety. The cationic lipid can be monocationic or polycationic. The cationic lipid typically has a lipophilic moiety such as a sterol chain, acyl chain or diacyl chain and has a net positive charge overall. The head group of the lipid typically bears a positive charge. The cationic lipid preferably has a positive charge of 1 to 10 valences, more preferably 1 to 3 valences, and even more preferably 1 valence. Examples of cationic lipids include 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), dimethyldioctadecylammonium (DDAB), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-diacyloxy-3-dimethylammonium propane, 1,2-dialkyloxy-3-dimethylammonium propane, dioctadecyldimethylammonium chloride (DODAC), 1,2-dimyristoyloxypropyl-1,3-dimethylhydroxyethylammonium (DMRIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), but are not limited thereto. Cationic lipids also include lipids having a tertiary amine group, including 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA). Cationic lipids are suitable for formulating RNA in lipid formulations described herein such as liposomes, emulsions and lipoplexes. Typically, at least one cationic lipid contributes to the positive charge and RNA contributes to the negative charge. In one embodiment, the pharmaceutical composition includes at least one helper lipid in addition to the cationic lipid. The helper lipid can be a neutral or anionic lipid. The helper lipid can be a natural lipid such as a phospholipid, or an analog of a natural lipid, or a fully synthetic lipid, or a lipid-like molecule having no similarity to a natural lipid.When the pharmaceutical composition contains both a cationic lipid and a helper lipid, the molar ratio of the cationic lipid to the neutral lipid can be appropriately determined in consideration of the stability of the formulation and the like.

[0377] In one embodiment, the pharmaceutical composition according to the present invention contains protamine. According to the present invention, protamine is useful as a cationic carrier agent. The term "protamine" refers to any of a variety of relatively low molecular weight, highly basic proteins that are rich in arginine and are found associated with DNA in place of somatic histones, particularly in the sperm cells of animals such as fish. In particular, the term "protamine" refers to a protein found in fish sperm that is highly basic, soluble in water, not coagulated by heat, and contains multiple arginine monomers. According to the present invention, the term "protamine" as used herein is intended to include any protamine amino acid sequence obtained from or derived from a natural or biological source, including fragments thereof, and multimeric forms of said amino acid sequence or fragments thereof. Furthermore, the term includes artificial, specially designed polypeptides for a particular purpose that cannot be isolated from a natural or biological source (synthetic).

[0378] In some embodiments, the compositions of the invention may include one or more adjuvants. Adjuvants may be added to vaccines to stimulate the immune system's response; adjuvants typically do not provide immunity themselves. Exemplary adjuvants include, but are not limited to, the following: inorganic compounds (such as alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide); mineral oils (such as paraffin oil), cytokines (such as IL-1, IL-2, IL-12); immunostimulatory polynucleotides (RNA or DNA; such as CpG-containing oligonucleotides, etc.); saponins (such as plant saponins from Quillaja, soybean, Polygala senega); oil emulsions or liposomes; polyoxyethylene ether and polyoxyethylene ester formulations; polyphosphazenes (PCPP); muramyl peptides; imidazoquinolone compounds; thiosemicarbazone compounds; Flt3 ligand (International Publication No. WO 2010 / 066418 A1); or any other adjuvant known to those skilled in the art. A preferred adjuvant for the administration of RNA according to the invention is Flt3 ligand (International Publication No. WO 2010 / 066418 A1). When Flt3 ligand is administered together with RNA encoding an antigen, a strong increase in antigen-specific CD8+ T cells can be observed.

[0379] The pharmaceutical compositions according to the invention can be buffered (e.g., using acetate buffer, citrate buffer, succinate buffer, Tris buffer, phosphate buffer).

[0380] RNA-containing particles In some embodiments, due to the instability of unprotected RNA, it is advantageous to provide the RNA molecules of the invention in a complexed or encapsulated form. Each pharmaceutical composition is provided in the present invention. In particular, in some embodiments, the pharmaceutical composition of the present invention comprises nucleic acid-containing particles, preferably RNA-containing particles. Each pharmaceutical composition is referred to as a particle formulation. In the particle formulation according to the present invention, the particles comprise a nucleic acid according to the present invention and a pharmaceutically acceptable carrier or pharmaceutically acceptable vehicle suitable for delivery of the nucleic acid. The nucleic acid-containing particles can be, for example, in the form of proteinaceous particles or lipid-containing particles. Suitable proteins or lipids are referred to as particle-forming agents. Proteinaceous particles and lipid-containing particles have previously been described as being suitable for delivery of alphavirus RNA in particulate form (e.g. Strauss & Strauss, 1994, Microbiol. Rev. 58:491-562). In particular, alphavirus structural proteins (e.g. provided by a helper virus) are suitable carriers for delivering RNA in the form of proteinaceous particles.

[0381] In one embodiment, the particle formulation of the present invention is a nanoparticle formulation. In that embodiment, the composition according to the present invention comprises a nucleic acid according to the present invention in the form of nanoparticles. Nanoparticle formulations can be obtained by various protocols and using various complexing compounds. Lipids, polymers, oligomers, or amphiphilic substances are typical components of nanoparticle formulations.

[0382] As used herein, the term "nanoparticle" refers to any particle having a diameter that renders the particle suitable, particularly for systemic administration of nucleic acids, particularly parenteral administration, typically a diameter of 1000 nanometers (nm) or less. In one embodiment, the nanoparticles have an average diameter in the range of about 50 nm to about 1000 nm, preferably about 50 nm to about 400 nm, preferably about 100 nm to about 300 nm, such as about 150 nm to about 200 nm. In one embodiment, the nanoparticles have a diameter in the range of about 200 to about 700 nm, about 200 to about 600 nm, preferably about 250 to about 550 nm, particularly about 300 to about 500 nm or about 200 to about 400 nm. In one embodiment, the average diameter is about 50 to 150 nm, preferably about 60 to 120 nm. In one embodiment, the average diameter is less than 50 nm. In one embodiment, the polydispersity index (PI) of the nanoparticles described herein is 0.5 or less, preferably 0.4 or less, or even more preferably 0.3 or less when measured by dynamic light scattering. The "polydispersity index" (PI) is a measure of the uniform or non-uniform size distribution of the individual particles (such as liposomes) in a particle mixture and indicates the width of the particle distribution in the mixture. The PI can be determined, for example, as described in WO 2013 / 143555 A1.

[0383] As used herein, the term "nanoparticle formulation" or similar terms refers to any particulate formulation containing at least one nanoparticle. In some embodiments, the nanoparticle composition is a uniform aggregate of nanoparticles. In some embodiments, the nanoparticle composition is a lipid-containing pharmaceutical formulation such as a liposome formulation or an emulsion.

[0384] Lipid-containing pharmaceutical composition In one embodiment, the pharmaceutical composition of the present invention contains at least one lipid. Preferably, the at least one lipid is a cationic lipid. The lipid-containing pharmaceutical composition contains the nucleic acid according to the present invention. In one embodiment, the pharmaceutical composition according to the present invention contains RNA encapsulated in vesicles, such as liposomes. In one embodiment, the pharmaceutical composition according to the present invention contains RNA in the form of an emulsion. In one embodiment, the pharmaceutical composition according to the present invention contains rRNA in a complex with a cationic compound, thereby forming, for example, a so-called lipoplex or polyplex. Encapsulation of RNA into vesicles such as liposomes is different, for example, from lipid / RNA complexes. Lipid / RNA complexes can be obtained, for example, when RNA is mixed with pre-formed liposomes.

[0385] In one embodiment, the pharmaceutical composition according to the present invention contains rRNA encapsulated in vesicles. Such a formulation is a specific particle formulation according to the present invention. A vesicle is a lipid bilayer wrapped around a spherical shell, enclosing a small space and separating that space from the space outside the vesicle. Typically, the space inside the vesicle is an aqueous space, i.e., it contains water. Typically, the space outside the vesicle is an aqueous space, i.e., it contains water. The lipid bilayer is formed by one or more lipids (vesicle-forming lipids). The membrane surrounding the vesicle is in a lamellar phase similar to the plasma membrane. The vesicles according to the present invention can be multilamellar vesicles, unilamellar vesicles, or a mixture thereof. When encapsulated in vesicles, rRNA is typically separated from the external medium. Thus, it exists in a protected form that is functionally equivalent to the protected form of native alphavirus. Suitable vesicles are the particles described herein, particularly nanoparticles.

[0386] For example, RNA (rRNA) can be encapsulated in liposomes. In that embodiment, the pharmaceutical composition is a liposome formulation or contains a liposome formulation. Encapsulation into liposomes typically protects RNA from RNase digestion. Liposomes can contain some external RNA (e.g., on their surface), but at least half (ideally all) of the RNA is encapsulated within the core of the liposome.

[0387] Liposomes are microscopic lipid vesicles that often have one or more bilayers of vesicle-forming lipids such as phospholipids and can encapsulate drugs, such as RNA. Various types of liposomes can be used in connection with the present invention, including, but not limited to, multilamellar vesicles (MLV), small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), sterically stabilized liposomes (SSL), multi-vesicular vesicles (MV) and large multi-vesicular vesicles (LMV), and other bilayer forms known in the art. The size and lamellarity of liposomes depend on the method of preparation. There are several other morphological forms of supramolecular structures in which lipids can exist in an aqueous medium, including lamellar phase, hexagonal and inverse hexagonal phases, cubic phase, micelles, and inverse micelles consisting of a single layer. These phases may be obtained in combination with DNA or RNA, and the interaction with RNA and DNA can substantially affect the phase state. Such phases can be present in the nanoparticle RNA formulations of the present invention.

[0388] Liposomes can be formed using standard methods known to those skilled in the art. Each method includes reverse evaporation, ethanol injection, dehydration-rehydration, sonication or other suitable methods. After liposome formation, the liposomes can be sized to obtain a population of liposomes having a substantially uniform size range.

[0389] In a preferred embodiment of the present invention, the rRNA is present in liposomes comprising at least one cationic lipid. Each liposome can be formed from a single lipid or a mixture of lipids, provided that at least one cationic lipid is used. Preferred cationic lipids have a nitrogen atom that can be protonated, and preferably such cationic lipids are lipids having a tertiary amine group. A particularly suitable lipid having a tertiary amine group is 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA). In one embodiment, the RNA according to the present invention is present in a liposome formulation as described in International Publication No. WO 2012 / 006378 A1, the liposome having a lipid bilayer encapsulating an aqueous core containing the RNA, the lipid bilayer preferably containing a lipid having a pKa in the range of 5.0 to 7.6 and having a tertiary amine group. Preferred cationic lipids having a tertiary amine group include DLinDMA (pKa 5.8), which is generally described in International Publication No. WO 2012 / 031046 A2. According to WO 2012 / 031046 A2, liposomes containing each compound are particularly suitable for encapsulation of RNA and thus for liposomal delivery of RNA. In one embodiment, the RNA according to the present invention is present in a liposome formulation, the liposome containing at least one cationic lipid whose head group contains at least one nitrogen atom (N) that can be protonated, and the liposome and the RNA having an N:P ratio of 1:1 to 20:1. According to the present invention, the "N:P ratio" refers to the molar ratio of nitrogen atoms (N) in the cationic lipid to phosphate atoms (P) in the RNA contained in the lipid-containing particles (e.g., liposomes), as described in International Publication No. WO 2013 / 006825 A1. The N:P ratio of 1:1 to 20:1 is related to the net charge of the liposome and the delivery efficiency of the RNA to vertebrate cells.

[0390] In one embodiment, the rRNA according to the present invention is present in a liposome formulation containing at least one lipid comprising a polyethylene glycol (PEG) moiety, and the RNA is encapsulated within PEGylated liposomes such that the PEG moiety is present on the outer side of the liposomes, as described in WO 2012 / 031043 A1 and WO 2013 / 033563 A1.

[0391] In one embodiment, the rRNA according to the present invention is present in a liposome formulation in which the liposomes have a diameter in the range of 60 to 180 nm, as described in WO 2012 / 030901 A1.

[0392] In one embodiment, the rRNA according to the present invention is present in a liposome formulation in which the rRNA-containing liposomes have a net charge close to zero or negative, as disclosed in WO 2013 / 143555 A1.

[0393] In other embodiments, the rRNA according to the present invention exists in the form of an emulsion. Emulsions have been previously described as being used to deliver nucleic acid molecules, such as rRNA molecules, to cells. Herein, an oil-in-water emulsion is preferred. Each emulsion particle contains an oil core and a cationic lipid. A cationic oil-in-water emulsion in which the RNA according to the present invention is complexed to the emulsion particles is more preferred. The emulsion particles contain an oil core and a cationic lipid. The cationic lipid can interact with the negatively charged rRNA, thereby immobilizing the rRNA to the emulsion particles. In an oil-in-water emulsion, the emulsion particles are dispersed in an aqueous continuous phase. For example, the average diameter of the emulsion particles can typically be about 80 nm to 180 nm. In one embodiment, the pharmaceutical composition of the present invention is a cationic oil-in-water emulsion in which the emulsion particles contain an oil core and a cationic lipid, as described in International Publication No. WO 2012 / 006380 A2. The rRNA according to the present invention can exist in the form of an emulsion containing a cationic lipid, wherein the N:P ratio of the emulsion is at least 4:1, as described in International Publication No. WO 2013 / 006834 A1. The rRNA according to the present invention can exist in the form of a cationic lipid emulsion, as described in International Publication No. WO 2013 / 006837 A1. In particular, the composition may contain rRNA complexed to the particles of a cationic oil-in-water emulsion, and the oil / lipid ratio is at least about 8:1 (molar:molar).

[0394] In other embodiments, the pharmaceutical composition according to the invention comprises RNA in the form of a lipoplex. The term "lipoplex" or "RNA lipoplex" refers to a complex of a lipid and a nucleic acid such as RNA. A lipoplex can be formed from cationic (positively charged) liposomes and anionic (negatively charged) nucleic acids. The cationic liposomes can also include neutral "helper" lipids. In the simplest case, lipoplexes are spontaneously formed by mixing nucleic acids with liposomes according to a specific mixing protocol, although various other protocols can also be applied. It is understood that the electrostatic interaction between positively charged liposomes and negatively charged nucleic acids is the driving force for lipoplex formation (WO 2013 / 143555 A1). In one embodiment of the invention, the net charge of the RNA lipoplex particles is near zero or negative. Electrically neutral or negatively charged lipoplexes of RNA and liposomes result in substantial RNA expression in splenic dendritic cells (DCs) after systemic administration and are not associated with the increased toxicity reported for positively charged liposomes and lipoplexes (see WO 2013 / 143555 A1). Thus, in one embodiment of the invention, the pharmaceutical composition according to the invention comprises RNA in the form of nanoparticles, preferably lipoplex nanoparticles, wherein (i) the number of positive charges in the nanoparticles does not exceed the number of negative charges in the nanoparticles, and / or (ii) the nanoparticles have a neutral or net negative charge, and / or (iii) the charge ratio of positive to negative charges in the nanoparticles is 1.4:1 or less, and / or (iv) the zeta potential of the nanoparticles is 0 or less. As described in WO 2013 / 143555 A1, the zeta potential is the scientific term for the electrokinetic potential at the interface in a colloidal system. In the present invention, both (a) the zeta potential and (b) the charge ratio of the cationic lipid to the RNA in the nanoparticles can be calculated as disclosed in WO 2013 / 143555 A1. In summary, as disclosed in WO 2013 / 143555 A1, a pharmaceutical composition that is a nanoparticle lipoplex formulation having a defined particle size and a net charge of the particles near zero or negative is a preferred pharmaceutical composition in the context of the present invention.

[0395] In one embodiment, nucleic acids such as rRNA described herein are administered in the form of lipid nanoparticles (LNPs). The LNP can comprise any lipid capable of forming particles to which one or more nucleic acid molecules are bound or encapsulated.

[0396] In one embodiment, the LNP comprises one or more cationic lipids and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.

[0397] In one embodiment, the LNP comprises a cationic lipid, a neutral lipid, a steroid, a polymer-conjugated lipid, and RNA encapsulated within or associated with the lipid nanoparticle.

[0398] In one embodiment, the LNP comprises 40 - 55 mol%, 40 - 50 mol%, 41 - 49 mol%, 41 - 48 mol%, 42 - 48 mol%, 43 - 48 mol%, 44 - 48 mol%, 45 - 48 mol%, 46 - 48 mol%, 47 - 48 mol%, or 47.2 - 47.8 mol% of a cationic lipid. In one embodiment, the LNP comprises about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9 or 48.0 mol% of a cationic lipid.

[0399] In one embodiment, the neutral lipid is present at a concentration in the range of 5 - 15 mol%, 7 - 13 mol%, or 9 - 11 mol%. In one embodiment, the neutral lipid is present at a concentration of about 9.5, 10 or 10.5 mol%.

[0400] In one embodiment, the steroid is present at a concentration in the range of 30 - 50 mol%, 35 - 45 mol% or 38 - 43 mol%. In one embodiment, the steroid is present at a concentration of about 40, 41, 42, 43, 44, 45 or 46 mol%.

[0401] In one embodiment, the LNP contains 1 to 10 mol%, 1 to 5 mol%, or 1 to 2.5 mol% of polymer-conjugated lipid.

[0402] In one embodiment, the LNP contains 40 to 50 mol% of cationic lipid; 5 to 15 mol% of neutral lipid; 35 to 45 mol% of steroid; 1 to 10 mol% of polymer-conjugated lipid; and RNA encapsulated within or associated with the lipid nanoparticle.

[0403] In one embodiment, the mole percent is determined based on the total moles of lipid present in the lipid nanoparticle.

[0404] In one embodiment, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In one embodiment, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In one embodiment, the neutral lipid is DSPC.

[0405] In one embodiment, the steroid is cholesterol.

[0406] In one embodiment, the polymer-conjugated lipid is a pegylated lipid. In one embodiment, the pegylated lipid has the following structure: [Chemical formula] (where R 12 and R 13 are each independently a straight-chain or branched saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, where the alkyl chain may be interrupted by one or more ester bonds, and w has an average value in the range of 30 to 60) or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. In one embodiment, R 12 and R 13is, independently of each other, a straight-chain saturated alkyl chain containing 12 to 16 carbon atoms. In one embodiment, w has an average value in the range of 40 to 55. In one embodiment, the average w is about 45. In one embodiment, R 12 and R 13 are, independently of each other, straight-chain saturated alkyl chains containing about 14 carbon atoms, and w has an average value of about 45.

[0407] In one embodiment, the pegylated lipid is DMG-PEG 2000, which has, for example, the following structure:

Chemical formula

[0408] In some embodiments, the cationic lipid component of the LNP has the structure of formula (III):

Chemical formula

[0409] In some of the foregoing embodiments of formula (III), the lipid has the following structure (IIIA) or (IIIB):

Chemical formula

[0410] In some of the foregoing embodiments of formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0411] In other embodiments of formula (III), the lipid is of the following structure (IIIC) or (IIID):

Chemical formula

[0412] In any of the foregoing embodiments of formula (III), one of L 1 or L 2 is -O(C=O)-. For example, in some embodiments, each of L 1 and L 2 is -O(C=O)-. In some different embodiments of any of the foregoing, each of L 1 and L 2 is independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, each of L 1 and L 2 is -(C=O)O-.

[0413] In some different embodiments of formula (III), the lipid has one of the following structures (IIIE) or (IIIF):

Chemical formula

Chemical formula

[0414] In some of the foregoing embodiments of formula (III), n is an integer in the range of 2 to 12, such as 2 to 8 or 2 to 4. For example, in some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0415] In some other embodiments of the foregoing embodiments of formula (III), y and z are each independently an integer in the range of 2 to 10. For example, in some embodiments, y and z are each independently an integer in the range of 4 to 9 or 4 to 6.

[0416] In some of the foregoing embodiments of formula (III), R 6 is H. In other embodiments of the foregoing embodiments, R 6 is C 1 -C 24 alkyl. In other embodiments, R 6 is OH.

[0417] In some embodiments of formula (III), G 3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G 3 is linear C 1 -C 24 alkylene or linear C 1 -C 24 alkenylene.

[0418] In some of the other foregoing embodiments of formula (III), R 1 or R 2 , or both of them, are C 6 -C24 is alkenyl. For example, in some embodiments, R 1 and R 2 each independently has the following structure: [Chemical formula] (where R 7a and R 7b are each independently, in each occurrence, H or C 1 -C 12 alkyl; and a is an integer from 2 to 12, where R 7a , R 7b and a are each selected such that R 1 and R 2 each independently contain 6 to 20 carbon atoms) has. For example, in some embodiments, a is an integer in the range of 5 to 9 or 8 to 12.

[0419] In some of the aforementioned embodiments of formula (III), at least one occurrence of R 7a is H. For example, in some embodiments, R 7a is H in each occurrence. In other different embodiments of the aforementioned embodiments, at least one occurrence of R 7b is C 1 -C 8 alkyl. For example, in some embodiments, C 1 -C 8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.

[0420] In different embodiments of formula (III), R 1 or R 2 , or both, have one of the following structures: [Chemical formula]

[0421] In some of the foregoing embodiments of formula (III), R 3 is OH, CN, -C(=O)OR 4 , -OC(=O)R 4 or -NHC(=O)R 4 . In some embodiments, R 4 is methyl or ethyl.

[0422] In various different embodiments, the cationic lipid of formula (III) has one of the structures shown in the following table.

[0423] Representative compounds of formula (III).

Chemical formula

[0424]

Chemical formula

[0425]

Chemical formula

[0426]

Chemical formula

[0427]

Chemical formula

[0428] In some embodiments, the LNP comprises a lipid of formula (III), RNA, a neutral lipid, a steroid and a pegylated lipid. In some embodiments, the lipid of formula (III) is compound III-3. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the pegylated lipid is ALC-0159.

[0429] In some embodiments, the cationic lipid is present in the LNP in an amount of about 40 to about 50 mol%. In one embodiment, the neutral lipid is present in the LNP in an amount of about 5 to about 15 mol%. In one embodiment, the steroid is present in the LNP in an amount of about 35 to about 45 mol%. In one embodiment, the pegylated lipid is present in the LNP in an amount of about 1 to about 10 mol%.

[0430] In some embodiments, the LNP comprises compound III-3 in an amount of about 40 to about 50 mol%, DSPC in an amount of about 5 to about 15 mol%, cholesterol in an amount of about 35 to about 45 mol%, and ALC-0159 in an amount of about 1 to about 10 mol%.

[0431] In some embodiments, the LNP comprises compound III-3 in an amount of about 47.5 mol%, DSPC in an amount of about 10 mol%, cholesterol in an amount of about 40.7 mol%, and ALC-0159 in an amount of about 1.8 mol%.

[0432] In various different embodiments, the cationic lipid has one of the structures shown in the following table.

Chemical formula

[0433] In some embodiments, the LNP comprises the cationic lipid shown in the above table, such as the cationic lipid of formula (B) or formula (D), particularly the cationic lipid of formula (D), RNA, neutral lipid, steroid and pegylated lipid. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the pegylated lipid is DMG-PEG 2000.

[0434] In one embodiment, the LNP comprises a cationic lipid that is an ionizable lipid-like substance (lipidoid). In one embodiment, the cationic lipid has the following structure:

Chemical formula

[0435] The N / P value is preferably at least about 4. In some embodiments, the N / P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In one embodiment, the N / P value is about 6.

[0436] In one embodiment, the LNPs described herein can have an average diameter in the range of about 30 nm to about 200 nm, or about 60 nm to about 120 nm.

[0437] RNA targeting Some aspects of the present disclosure include the targeted delivery of rRNA (e.g., RNA encoding a vaccine antigen and / or an immune activator) disclosed herein.

[0438] In one embodiment, the present disclosure includes targeting the lung. When the RNA to be administered is RNA encoding a vaccine antigen, targeting the lung is particularly preferred. The RNA can be delivered to the lung by inhalation of an RNA formulated, for example, as a particle as described herein, such as a lipid particle.

[0439] In one embodiment, the present disclosure includes targeting the lymphatic system, particularly secondary lymphoid organs, more specifically the spleen. When the RNA to be administered is RNA encoding a vaccine antigen, targeting the lymphatic system, particularly secondary lymphoid organs, more specifically the spleen, is particularly preferred.

[0440] In one embodiment, the target cells are spleen cells. In one embodiment, the target cells are antigen-presenting cells such as professional antigen-presenting cells in the spleen. In one embodiment, the target cells are dendritic cells in the spleen.

[0441] The "lymphatic system" is a part of the circulatory system and an important part of the immune system that includes a network of lymphatic vessels that carry lymph. The lymphatic system consists of lymphoid organs, a conductive network of lymphatic vessels, and circulating lymph. Primary or central lymphoid organs generate lymphocytes from immature progenitor cells. The thymus and bone marrow constitute the primary lymphoid organs. Secondary or peripheral lymphoid organs, including lymph nodes and the spleen, maintain mature naive lymphocytes and initiate an adaptive immune response.

[0442] RNA can be delivered to the spleen by so-called lipoplex formulations, in which the RNA binds to liposomes containing cationic lipids and optionally additional lipids or helper lipids to form an injectable nanoparticle formulation. Liposomes can be obtained by injecting an ethanol solution of the lipid into water or a suitable aqueous phase. RNA lipoplex particles can be prepared by mixing liposomes with RNA. RNA lipoplex particles targeting the spleen are described in International Publication No. WO 2013 / 143683, which is incorporated herein by reference. It has been found that RNA lipoplex particles having a net negative charge can be used to selectively target spleen tissue or spleen cells, such as antigen-presenting cells, particularly dendritic cells. Thus, RNA accumulation and / or RNA expression occurs in the spleen after administration of the RNA lipoplex particles. Accordingly, the RNA lipoplex particles of the present disclosure can be used to express RNA in the spleen. In one embodiment, after administration of the RNA lipoplex particles, no or essentially no RNA accumulation and / or RNA expression occurs in the lungs and / or liver. In one embodiment, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression occurs in antigen-presenting cells, such as professional antigen-presenting cells, in the spleen. Accordingly, the RNA lipoplex particles of the present disclosure can be used to express RNA in such antigen-presenting cells. In one embodiment, the antigen-presenting cells are dendritic cells and / or macrophages.

[0443] The charge of the RNA lipoplex particles of the present disclosure is the sum of the charge present in at least one cationic lipid and the charge present in the RNA. The charge ratio is the ratio of the positive charge present in at least one cationic lipid to the negative charge present in the RNA. The charge ratio of the positive charge present in at least one cationic lipid to the negative charge present in the RNA is calculated by the following formula: Charge ratio = [(concentration of cationic lipid (mol)) * (total number of positive charges in the cationic lipid)] / [(concentration of RNA (mol)) * (total number of negative charges in the RNA)].

[0444] The spleen-targeting RNA lipoplex particles described herein at physiological pH preferably have a net negative charge, such as a charge ratio of positive to negative charges of about 1.9:2 to about 1:2, or about 1.6:2 to about 1:2, or about 1.6:2 to about 1.1:2. In certain embodiments, the charge ratio of positive to negative charges in the RNA lipoplex particles at physiological pH is about 1.9:2.0, about 1.8:2.0, about 1.7:2.0, about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about 1:2.0.

[0445] An immunostimulant can be provided to a subject by administering to the subject an RNA encoding the immunostimulant in a formulation for the selective delivery of the RNA to the liver or liver tissue. Delivery of such RNA to a target organ or tissue is particularly preferred when it is desirable to express a large amount of the immunostimulant and / or when the systemic presence of the immunostimulant, particularly in a significant amount, is desirable or required.

[0446] The RNA delivery system has selectivity specific to the liver. This is related to lipid nanoparticles such as lipid-based particles, cationic and neutral nanoparticles, particularly liposomes, nanomicelles and lipophilic ligands in bioconjugates. Liver accumulation is caused by the discontinuous nature of the hepatic vasculature or lipid metabolism (liposomes and lipid or cholesterol conjugates).

[0447] For in vivo delivery of RNA to the liver, a drug delivery system can be used to transport the RNA to the liver by preventing its degradation. For example, polyplex nanomicelles consisting of a poly(ethylene glycol) (PEG)-coated surface and an mRNA-containing core are a useful system because the nanomicelles provide excellent in vivo stability of the RNA under physiological conditions. Furthermore, the stealth properties provided by the polyplex nanomicelle surface composed of high-density PEG paricles effectively avoid the host's immune defense.

[0448] Examples of suitable immunostimulants for targeting the liver are cytokines involved in T cell proliferation and / or maintenance. Examples of suitable cytokines include IL2 or IL7, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended PK cytokines.

[0449] In another embodiment, the RNA encoding the immunostimulant can be administered in a formulation for the selective delivery of RNA to the lymphatic system, particularly secondary lymphoid organs, more specifically the spleen. Delivery of an immunostimulant to such target tissues is particularly desirable when the presence of the immunostimulant in this organ or tissue is desired (e.g., to induce an immune response, particularly during T cell priming or when an immunostimulant such as a cytokine is required for activation of resident immune cells), but it is not desirable for the immunostimulant to be present systemically, particularly in a significant amount (e.g., because the immunostimulant has systemic toxicity).

[0450] Examples of suitable immunostimulants are cytokines involved in T cell priming. Examples of suitable cytokines include IL12, IL15, IFN-α, or IFN-β, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended PK cytokines.

[0451] Method for producing a protein The present invention also relates to a method for producing a protein of interest in a cell, comprising (a) Obtaining an RNA replicon according to the present invention comprising an open reading frame encoding a protein of interest, and (b) Inoculating the RNA replicon into a cell A method comprising the steps is provided.

[0452] In various embodiments of the method, the RNA replicon comprises an open reading frame encoding a protein of interest and optionally an open reading frame encoding a functional non-structural protein, and is as defined above for the RNA replicon of the present invention as long as it can be replicated by the functional non-structural protein. The rRNA can comprise at least one modified nucleotide and one or more point mutations in regulatory sequences that restore or improve the function of the modified rRNA.

[0453] A cell into which one or more nucleic acid molecules can be inoculated can be referred to as a "host cell". According to the present invention, the term "host cell" refers to any cell that can be transformed or transfected with an exogenous nucleic acid molecule. The term "cell" preferably refers to an intact cell, i.e., a cell with an intact membrane that has not released its normal intracellular components such as enzymes, organelles, or genetic material. The intact cell is preferably a viable cell, i.e., a living cell that can perform its normal metabolic functions. The term "host cell" according to the present invention includes prokaryotic cells (e.g., Escherichia coli) or eukaryotic cells (e.g., human and animal cells, plant cells, yeast cells, and insect cells). Mammalian cells, including domestic animals such as humans, mice, hamsters, pigs, horses, cows, sheep, and goats, as well as cells derived from primates, are particularly preferred. The cells can be derived from a number of tissue types and can include primary cells and cell lines. Specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells, and embryonic stem cells. In other embodiments, the host cell is an antigen-presenting cell, particularly a dendritic cell, monocyte, or macrophage. The nucleic acid can be present in the host cell in a single or several copies, and in one embodiment, is expressed in the host cell.

[0454] The cell can be a prokaryotic cell or a eukaryotic cell. Prokaryotic cells are, for example, suitable for the propagation of DNA according to the present invention in this specification, and eukaryotic cells are, for example, suitable for the expression of the open reading frame of the replicon in this specification.

[0455] In the method of the present invention, any one of the RNA replicon according to the present invention, the kit according to the present invention, or the pharmaceutical composition according to the present invention can be used. RNA can be used in the form of a pharmaceutical composition or as naked RNA, for example, for electroporation.

[0456] In a method for producing a protein intracellularly according to the present invention, the cell can be an antigen-presenting cell, and this method can be used to express RNA encoding an antigen. For this purpose, the present invention may include introducing RNA encoding an antigen into an antigen-presenting cell such as a dendritic cell. A pharmaceutical composition containing RNA encoding an antigen can be used for the transfection of an antigen-presenting cell such as a dendritic cell.

[0457] In one embodiment, the method for producing a protein intracellularly is an in vitro method. In one embodiment, the method for protein production intracellularly does not include the removal of cells from a human or animal subject by surgery or treatment.

[0458] In this embodiment, the cells inoculated according to the present invention can produce a protein in the subject and can be administered to the subject to provide the protein to the subject. The cells can be autologous, syngeneic, allogeneic or xenogeneic with respect to the subject.

[0459] In other embodiments, the cells in the method for producing a protein intracellularly can be present in a subject such as a patient. In these embodiments, the method for producing a protein intracellularly is an in vivo method that includes administering an RNA molecule to the subject.

[0460] In this regard, the present invention also provides a method for producing a protein of interest in a subject, comprising (a) Obtaining an RNA replicon according to the present invention that contains an open reading frame encoding a protein of interest, and (b) Administering the RNA replicon to a subject A method comprising is provided.

[0461] In various embodiments of the present method, the RNA replicon contains an open reading frame encoding a protein of interest and optionally an open reading frame encoding a functional non-structural protein, and is as defined above for the RNA replicon of the present invention as long as it can be replicated by the functional non-structural protein. The rRNA can contain at least one modified nucleotide and one or more point mutations in a regulatory sequence that restores or improves the function of the modified rRNA.

[0462] Any of the RNA replicon according to the present invention, or the kit according to the present invention, or the pharmaceutical composition according to the present invention can be used in a method for producing a protein in a subject according to the present invention. For example, in the method of the present invention, the RNA can be used in the form of a pharmaceutical composition as described herein, for example, or as naked RNA.

[0463] In consideration of the ability to be administered to a subject, each of the RNA replicon according to the present invention, or the kit according to the present invention, or the pharmaceutical composition according to the present invention can be referred to as a "pharmaceutical product" or the like. The present invention anticipates that the RNA replicon, kit, and pharmaceutical composition of the present invention are provided for use as a pharmaceutical product. The pharmaceutical product can be used to treat a subject. "Treating" means administering a compound or composition or other entity described herein to a subject. This term includes methods for the treatment of the body of a human or animal by therapy.

[0464] The above-mentioned pharmaceutical product typically does not contain DNA and thus has additional safety features compared to the DNA vaccines described in the prior art (for example, WO 2008 / 119827 A1).

[0465] Alternative medical uses according to the present invention include methods for producing proteins intracellularly according to the present invention, wherein the cells can be antigen-presenting cells such as dendritic cells, and subsequently introducing said cells into a subject. For example, RNA encoding a pharmaceutically active protein such as an antigen may be introduced (transfected) ex vivo into antigen-presenting cells, such as antigen-presenting cells collected from a subject, and optionally the antigen-presenting cells clonally expanded ex vivo may be reintroduced into the same or a different subject. The transfected cells can be reintroduced into the subject using any means known in the art.

[0466] The pharmaceutical according to the present invention can be administered to a subject in need thereof. The pharmaceutical of the present invention can be used in prophylactic and therapeutic methods for the treatment of a subject.

[0467] The pharmaceutical according to the present invention is administered in an effective amount. "Effective amount" relates to an amount sufficient to produce a response or a desired effect, alone or in combination with other dosages. In the case of the treatment of a particular disease or a particular condition in a subject, the desired effect is inhibition of disease progression. This includes deceleration of disease progression, particularly interruption of disease progression. The desired effect in the treatment of a disease or condition can also be delay or inhibition of the onset of the disease.

[0468] The effective amount depends on individual parameters of the patient including the condition being treated, the severity of the disease, age, physiological state, size and weight, the duration of the treatment, the type of concomitant treatment (if any), the particular mode of administration and other factors.

[0469] Vaccination The terms "immunization" or "vaccination" generally refer to the process of treating a subject for therapeutic or prophylactic reasons. Treatment, particularly prophylactic treatment, is preferably a treatment or includes a treatment aimed at inducing or enhancing the subject's immune response to, for example, one or more antigens. According to the present invention, when it is desired to induce or enhance an immune response by using the rRNA described herein, the immune response can be induced or enhanced by the rRNA. In one embodiment, the present invention provides a prophylactic treatment that preferably is or includes vaccination of a subject. Embodiments of the invention in which the replicon encodes a pharmaceutically active peptide or protein that is an immunologically active compound or antigen as the protein of interest are particularly useful for vaccination.

[0470] RNA has been previously described for vaccination against foreign agents including pathogens or cancer (recently reviewed by Ulmer et al., 2012, Vaccine 30:4414-4418). In contrast to the general approach of the prior art, the replicon according to the present invention is a particularly suitable element for efficient vaccination due to its ability to be replicated by the functional alphavirus nonstructural proteins described herein. The vaccination according to the present invention can be used, for example, to induce an immune response against a poorly immunogenic protein. In the case of the RNA vaccine according to the present invention, the protein antigen is never exposed to serum antibodies and is produced by the transfected cells themselves after translation of the RNA. Thus, anaphylaxis should not be a problem. Thus, the present invention enables repeated immunization of patients without the risk of allergic reactions.

[0471] In a method comprising vaccination according to the present invention, the pharmaceutical of the present invention is administered to a subject when it is desired to treat a subject having a disease involving an antigen or at risk of developing a disease involving an antigen.

[0472] In the method involving vaccination according to the present invention, the protein of interest encoded by the replicon according to the present invention encodes, for example, a bacterial antigen to which an immune response is directed, or a viral antigen to which an immune response is directed, or a cancer antigen to which an immune response is directed, or an antigen of a unicellular organism to which an immune response is directed. The effectiveness of vaccination can be evaluated by known standard methods such as measurement of antigen-specific IgG antibodies derived from an organism. In the method involving allergen-specific immunotherapy according to the present invention, the protein of interest encoded by the replicon according to the present invention encodes an antigen associated with allergy. Allergen-specific immunotherapy (also known as desensitization) is defined as administering to an organism having one or more allergies, preferably in increasing doses of an allergen vaccine, in order to achieve a state in which symptoms associated with subsequent exposure to the causative allergen are alleviated. The effectiveness of allergen-specific immunotherapy can be evaluated by known standard methods such as measurement of allergen-specific IgG and IgE antibodies derived from an organism.

[0473] The pharmaceutical of the present invention can be administered to a subject for the treatment of the subject, including, for example, vaccination of the subject.

[0474] The term "subject" relates to vertebrates, particularly mammals. For example, mammals in the context of the present invention are humans, non-human primates, dogs, cats, sheep, cows, goats, pigs, horses and other domestic animals, mice, rats, rabbits, guinea pigs and other laboratory animals, and captive animals such as zoo animals. The term "subject" also relates to non-mammalian vertebrates such as birds (particularly poultry such as chickens, ducks, geese, turkeys) and fish (particularly farmed fish such as salmon or catfish). The term "animal" as used herein includes humans.

[0475] In some embodiments, administration to livestock such as dogs, cats, rabbits, guinea pigs, hamsters, sheep, cows, goats, pigs, horses, chickens, ducks, geese, turkeys, or to wild animals such as foxes is preferred. For example, prophylactic vaccination according to the present invention may be suitable for vaccinating animal populations or wild animal populations in, for example, agriculture. Other captive animal populations such as pet animals or zoo animals can be vaccinated.

[0476] In one embodiment, the pharmaceutical can be administered multiple times. The multiple doses can be administered such that the individual doses can be administered at different intervals. For example, one dose can be administered 14 to 35 days after the previous dose has been administered. In one embodiment, one dose is administered 21 days after the previous dose. In one embodiment, one dose is administered 35 days after the previous dose.

[0477] In one embodiment, when administered to a subject, the replicon used as a pharmaceutical preferably does not contain sequences from viruses of the type that are infectious for the species or genus to which the subject being treated belongs, for example alphaviruses. Preferably, in that case, the replicon does not contain nucleotide sequences from alphaviruses that can infect each species or genus. This embodiment has the advantage that recombination with infectious (e.g., fully functional or wild-type) alphaviruses is not possible even if the subject to which the RNA is administered is (accidentally) infected with an infectious alphavirus. As an illustrative example, for the treatment of pigs, the replicon used does not contain nucleotide sequences from alphaviruses that can infect pigs.

[0478] Route of administration The pharmaceutical according to the present invention can be applied to a subject by any suitable route.

[0479] For example, the pharmaceutical can be administered systemically, for example, by intravenous (i.v.), intramuscular (i.m.), subcutaneous (s.c.), intradermal (i.d.) or inhalation.

[0480] In one embodiment, the pharmaceutical according to the present invention is administered to muscle tissue such as skeletal muscle, or to the skin, for example subcutaneously. It is generally understood t...

Claims

**Claim 1** A composition comprising at least two replicable RNA molecules, each comprising a first open reading frame (ORF) encoding at least one peptide or protein comprising said antigen or epitope suitable for inducing an immune response against said antigen or epitope when administered to a subject ; wherein at least one peptide or protein encoded by one of said replicable RNA molecules is different from at least one peptide or protein encoded by another replicable RNA molecule optionally, at least one of said replicable RNA molecules further comprises a second ORF encoding an RNA-dependent RNA polymerase (replicase) capable of replicating said replicable RNA molecule in cis or in trans a composition. **Claim 2** The composition according to claim 1, wherein one or both of said at least two replicable RNA molecules comprise a second ORF encoding an RNA-dependent RNA polymerase (replicase) capable of replicating said replicable RNA molecule in cis or in trans. **Claim 3** The composition according to claim 1 or 2, further comprising a third RNA molecule encoding a replicase capable of replicating said replicable RNA molecule and / or said third RNA molecule in cis or in trans. **Claim 4** The composition according to claim 3, wherein said third RNA molecule is replicable. **Claim 5** The composition according to claim 3, wherein said third RNA molecule is a non-replicating RNA molecule. **Claim 6** The composition according to any one of claims 1 to 5, comprising at least two replicable RNA molecules each not encoding said replicase and a third non-replicating RNA molecule encoding said replicase. **Claim 7** The composition according to claim 5 or 6, wherein said non-replicating RNA is mRNA. **Claim 8** The composition according to any one of claims 1 to 7, wherein said replicable RNA molecule comprises an internal ribosome entry site (IRES) controlling the expression of said first ORF encoding said protein or peptide comprising an antigen or epitope. **Claim 9** The composition according to any one of claims 1 to 8, wherein said replicable RNA molecule comprises an internal ribosome entry site (IRES) controlling the expression of said second ORF encoding said replicase. **Claim 10** The composition according to claim 8 or 9, wherein the IRES is insensitive to cellular stress.

11. The composition according to any one of claims 8 to 10, wherein the IRES is insensitive to interferon, preferably type I interferon.

12. The composition according to any one of claims 8 to 11, wherein the IRES is a cellular or viral IRES, preferably a viral IRES.

13. The composition according to any one of claims 8 to 12, wherein the IRES is derived from a virus selected from the group consisting of picornavirus, flavivirus or dicistrovirus.

14. The composition according to any one of claims 8 to 13, wherein the IRES is derived from picornavirus or dicistrovirus, preferably dicistrovirus.

15. The composition according to any one of claims 8 to 14, wherein the IRES is a type IV IRES.

16. The composition according to any one of claims 8 to 15, wherein the expression controlled by the IRES is independent of the IRES trans - acting factor.

17. The composition according to any one of claims 8 to 16, wherein the expression controlled by the IRES is independent of the cellular translation initiation factor.

18. The composition according to any one of claims 8 to 17, wherein the expression controlled by the IRES is independent of the phosphorylation of eukaryotic initiation factor 2 (eIF2).

19. The composition according to any one of claims 1 to 18, wherein both replicable RNA molecules contain a second ORF encoding the replicase.

20. The composition according to any one of claims 1 to 19, wherein at least one of the replicable RNA molecules contains a 5' cap for driving the translation of the replicase or for driving the translation of the peptide or protein containing the antigen or epitope.

21. The composition according to claim 20, wherein the 5' cap is a natural 5' cap or a 5' cap analog.

22. The composition according to any one of claims 1 to 21, wherein at least one replicable RNA contains a 5' replication recognition sequence characterized in that at least one start codon is removed as compared to the natural alphavirus 5' replication recognition sequence.

23. The composition according to claim 22, wherein the 5′ replication recognition sequence contains a sequence homologous to an open reading frame of a non-structural protein derived from a self-replicating virus or a part thereof, and the sequence homologous to the open reading frame of the non-structural protein derived from a self-replicating virus or a part thereof includes removal of at least one start codon as compared with the natural virus sequence.

24. The composition according to claim 23, wherein the sequence homologous to the open reading frame of the non-structural protein derived from a self-replicating virus or a part thereof includes removal of at least the natural start codon of the open reading frame of the non-structural protein derived from a self-replicating virus.

25. The composition according to claim 23 or 24, wherein the sequence homologous to the open reading frame of the non-structural protein derived from a self-replicating virus or a part thereof includes removal of at least one start codon other than the natural start codon of the open reading frame of the non-structural protein derived from a self-replicating virus.

26. The composition according to any one of claims 22 to 25, wherein the sequence homologous to the open reading frame of the non-structural protein derived from a self-replicating virus or a part thereof does not contain a start codon.

27. The composition according to any one of claims 22 to 26, including at least one nucleotide change that compensates for nucleotide pairing disruption within at least one stem-loop introduced by removal of at least one start codon.

28. The composition according to any one of claims 22 to 27, wherein the open reading frame encoding the functional non-structural protein derived from a self-replicating virus does not overlap with the 5′ replication recognition sequence.

29. The composition according to any one of claims 8 to 28, wherein the first ORF is downstream of the 5′ replication recognition sequence and upstream of the IRES.

30. The composition according to any one of claims 1 to 29, wherein the replicable RNA includes a subgenomic promoter that controls the production of a subgenomic RNA containing the first ORF encoding the protein or peptide.

31. The composition according to claim 30, wherein the subgenomic RNA is a transcription product of an RNA-dependent RNA polymerase derived from the functional non-structural protein derived from a self-replicating virus.

32. The composition according to claim 30 or 31, wherein the protein or peptide can be expressed from the subgenomic RNA as a template.

33. The composition according to any one of claims 30 to 32, wherein the first ORF encoding the protein or peptide controlled by the subgenomic promoter is downstream of the second ORF encoding the replicase.

34. The composition according to claim 33, wherein the subgenomic promoter overlaps with the second ORF.

35. The composition according to any one of claims 1 to 34, wherein at least one of the replicable RNA molecules contains a 3' replication recognition sequence.

36. The composition according to claim 35, wherein the second ORF encoding the replicase, the 5' and / or 3' replication recognition sequences, and the subgenomic promoter are derived from a self-replicating virus, preferably the same self-replicating virus species.

37. The composition according to any one of claims 1 to 36, wherein the replicable RNA molecule can be replicated by an RNA-dependent RNA polymerase derived from the functional non-structural protein derived from a self-replicating virus.

38. The composition according to claim 37, wherein the self-replicating virus is preferably an alphavirus selected from the group consisting of Venezuelan equine encephalitis complex virus, Eastern equine encephalitis complex virus, Western equine encephalitis complex virus, chikungunya virus, Semliki Forest virus complex virus, Sindbis virus, Barmah Forest virus, Middelburg virus, and Ndumu virus.

39. The composition according to claim 37 or 38, wherein the alphavirus is Venezuelan equine encephalitis virus or Semliki Forest virus.

40. The composition according to any one of claims 1 to 39, wherein at least one of the replicable RNA molecules contains a 3' poly(A) sequence.

41. The composition according to any one of claims 1 to 40, wherein the antigen or epitope of the encoded protein or peptide is an antigen of a bacterium, virus, parasite, or fungus, or is derived therefrom.

42. The composition according to any one of claims 1 to 41, wherein the protein or peptide encoded by the first replicable RNA and the protein or peptide encoded by the second replicable RNA are both obtained from or derived from the same bacterium, virus, parasite or fungus.

43. The composition according to any one of claims 1 to 42, wherein the protein or peptide encoded by the first replicable RNA and the protein or peptide encoded by the second replicable RNA are each obtained from or derived from different strains of the same bacterium, virus, parasite or fungus, or are obtained from or derived from different pathogenic organisms, such as different viruses.

44. The composition according to any one of claims 1 to 43, wherein the protein or peptide encoded by the first replicable RNA is a surface-expressed protein or peptide, the protein or peptide encoded by the second replicable RNA is not a surface-expressed protein or peptide, and the surface-expressed and non-surface-expressed proteins or peptides are each obtained from or derived from the same or different strains of the same bacterium, virus, parasite or fungus, or are obtained from or derived from different pathogenic organisms, such as different viruses.

45. The composition according to any one of claims 1 to 43, wherein the protein or peptide encoded by the first replicable RNA is not a surface-expressed protein or peptide, the protein or peptide encoded by the second replicable RNA is not a surface-expressed protein or peptide, and, different from that encoded by the first replicable RNA, the different non-surface-expressed proteins or peptides are each obtained from or derived from the same or different strains of the same bacterium, virus, parasite or fungus, or are obtained from or derived from different pathogenic organisms, such as different viruses.

46. The protein or peptide encoded by the first replicable RNA is a surface-expressed protein or peptide, the protein or peptide encoded by the second replicable RNA is a surface-expressed protein or peptide, and, different from that encoded by the first replicable RNA, the surface-expressed protein or peptide is obtained from or derived from the same or different strains of the same bacterium, virus, parasite or fungus, or is obtained from or derived from different pathogenic organisms, such as different viruses, the composition according to any one of claims 1 to 43.

47. The composition according to claim 44 or 46, wherein the surface-expressed protein is expressed on the surface of the virus / viral particle, or, when the virus is an enveloped virus, the surface-expressed protein is expressed on the surface of the viral envelope.

48. The composition according to claim 47, wherein the surface-expressed protein is a viral capsid protein or a viral envelope or glycoprotein.

49. The composition according to claim 44 or 45, wherein the protein or peptide that is not a surface-expressed protein or peptide is a viral matrix protein, a viral nucleoprotein, or a viral capsid protein, and the virus is an enveloped virus.

50. The protein or peptide encoded by the first replicable RNA is a viral glycoprotein, the protein or peptide encoded by the second replicable RNA is a viral nucleoprotein, and the glycoprotein and the nucleoprotein are obtained from or derived from the same virus, optionally the same strain of the same virus, the composition according to any one of claims 1 to 49.

51. The composition according to any one of claims 1 to 50, wherein the induced immune response against the antigen or epitope is an increase in the activity of CD4+ T cells and / or CD8+ T cells, preferably an increase in the activity of both CD4+ and CD8+ T cells.

52. The protein or peptide encoded by the first replicable RNA is an Ebola virus protein or a fragment thereof, or an epitope of the Ebola virus protein, and the protein or peptide encoded by the second replicable RNA is a different Ebola virus protein or a fragment thereof, or an epitope of the different Ebola virus protein. The composition according to any one of claims 1 to 51.

53. The protein or peptide encoded by at least one replicable RNA molecule is a structural Ebola virus protein selected from the group consisting of glycoprotein (GP), nucleoprotein (NP), polymerase cofactor (VP35), VP40, transcription factor (VP30), VP24, or RNA-dependent RNA polymerase (L), or a fragment thereof, or an epitope of the Ebola structural protein. The composition according to any one of claims 1 to 52.

54. The protein or peptide encoded by at least one replicable RNA molecule is expressed as a fusion protein. The composition according to any one of claims 1 to 53.

55. The protein or peptide is fused to a targeting motif or a secretion motif. The composition according to claim 54.

56. The protein or peptide encoded by at least one of the replicable RNA molecules is an Ebola structural GP protein or a fragment thereof, or an epitope of the GP protein. The composition according to any one of claims 1 to 55.

57. The GP protein is derived from or obtained from the Ebola Zaire subtype, virus strain H. sapiens-wt / SLE / 2014 / Makona-EM095B. The composition according to claim 56.

58. The protein or peptide encoded by at least one of the replicable RNA molecules is an Ebola structural NP protein or a fragment thereof, or an epitope of the NP protein. The composition according to any one of claims 1 to 57.

59. The NP antigen is derived from or obtained from the Ebola Zaire subtype, virus strain H. sapiens-wt / GIN / 2014 / Makona-EM096. The composition according to claim 58.

60. The protein or peptide encoded by the first replicable RNA molecule is the Ebola structural GP protein or a fragment thereof, or an epitope of the GP protein, and the protein or peptide encoded by the second replicable RNA molecule is the Ebola structural NP protein or a fragment thereof, or an epitope of the NP protein. The composition according to any one of claims 1 to 59.

61. The protein or peptide encoded by the first replicable RNA is the CCHFV virus protein or a fragment thereof, or an epitope of the CCHFV virus protein, and the protein or peptide encoded by the second replicable RNA is a different CCHFV virus protein or a fragment thereof, or an epitope of the different CCHFV virus protein. The composition according to any one of claims 1 to 51.

62. The protein or peptide encoded by the first replicable RNA is the MERS-CoV virus protein or a fragment thereof, or an epitope of the MERS-CoV virus protein, and the protein or peptide encoded by the second replicable RNA is a different MERS-CoV virus protein or a fragment thereof, or an epitope of the different MERS-CoV virus protein. The composition according to any one of claims 1 to 51.

63. The first and / or second ORF is adjacent to the 5' untranslated region (UTR) and / or 3' UTR, and preferably the 5' UTR and / or 3' UTR is not native to the alphavirus from which the replicase is derived. The composition according to any one of claims 1 to 62.

64. At least one of the replicable RNA molecules does not contain an open reading frame of an intact alphavirus structural protein. The composition according to any one of claims 1 to 63.

65. The composition according to any one of claims 1 to 64, further comprising a reagent capable of forming particles with the replicable RNA molecule.

66. The composition according to claim 65, wherein the reagent is a lipid or a polyalkyleneimine.

67. The composition according to claim 65 or 66, wherein the reagent is a lipid containing a cationic head group.

68. The composition according to any one of claims 65 to 67, wherein the reagent is a pH-responsive lipid.

69. The composition according to any one of claims 65 to 68, wherein the reagent is a PEGylated lipid.

70. The composition according to any one of claims 65 to 69, wherein the reagent is conjugated to polysarcosine.

71. The composition according to any one of claims 65 to 70, wherein the particles formed from the replicable RNA molecule and the reagent are polymer-based polyplexes (PLX) or lipid nanoparticles (LNP), and the LNP is preferably a lipoplex (LPX) or a liposome.

72. The composition according to any one of claims 65 to 71, wherein the particles further comprise at least one phosphatidylserine.

73. The particles are nanoparticles, (i) the number of positive charges in the nanoparticles does not exceed the number of negative charges in the nanoparticles, and / or (ii) the nanoparticles have a neutral or net negative charge, and / or (iii) the charge ratio of positive charges to negative charges in the nanoparticles is 1.4:1 or less, and / or (iv) the zeta potential of the nanoparticles is 0 or less, The composition according to any one of claims 65 to 72.

74. The composition according to claim 73, wherein the charge ratio of positive charges to negative charges in the nanoparticles is 1.4:1 to 1:8, preferably 1.2:1 to 1:

4.

75. The composition according to claim 73 or 74, wherein the nanoparticles comprise at least one lipid, preferably at least one cationic lipid.

76. The composition according to claim 75, wherein the at least one cationic lipid contributes to the positive charge and the replicable RNA molecule contributes to the negative charge.

77. The composition according to claim 75 or 76, wherein the nanoparticles further comprise at least one helper lipid.

78. The composition according to claim 77, wherein the helper lipid is a neutral lipid.

79. The composition according to any one of claims 75 to 78, wherein the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), and / or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP).

80. The composition according to any one of claims 77 to 79, wherein the at least one helper lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and / or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

81. The composition according to any one of claims 77 to 80, wherein the molar ratio of the at least one cationic lipid to the at least one helper lipid is from 10:0 to 3:7, preferably from 9:1 to 3:7, from 4:1 to 1:2, from 4:1 to 2:3, from 7:3 to 1:1, or from 2:1 to 1:1, preferably about 1:

1.

82. The composition according to any one of claims 71 to 81, wherein the nanoparticles are lipoplexes comprising DODMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and the charge ratio of the positive charge in DOTMA to the negative charge in the RNA is from 1.8:2 to 0.8:2, more preferably from 1.6:2 to 1:2, even more preferably from 1.4:2 to 1.1:2, and even more preferably about 1.2:

2.

83. The composition according to any one of claims 71 to 81, wherein the nanoparticles are lipoplexes comprising DODMA and cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and the charge ratio of the positive charge in DOTMA to the negative charge in the RNA is from 1.8:2 to 0.8:2, more preferably from 1.6:2 to 1:2, even more preferably from 1.4:2 to 1.1:2, and even more preferably about 1.2:

2.

84. The nanoparticle is a lipoplex containing DO-DMA and DSPC in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and the charge ratio of the positive charge in DOTMA to the negative charge in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:

2. The composition according to any one of claims 71 to 81.

85. The nanoparticle is a lipoplex containing DO-DMA:cholesterol:DOPE:PEGcerC16 in a molar ratio of 40:48:10:

2. The composition according to any one of claims 71 to 81.

86. The nanoparticle is a lipoplex containing DOTMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and the charge ratio of the positive charge in DOTMA to the negative charge in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:

2. The composition according to any one of claims 71 to 81.

87. The nanoparticle is a lipoplex containing DOTMA and cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and the charge ratio of the positive charge in DOTMA to the negative charge in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:

2. The composition according to any one of claims 71 to 81.

88. The nanoparticle is a lipoplex containing DOTAP and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and the charge ratio of the positive charge in DOTMA to the negative charge in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:

2. The composition according to any one of claims 71 to 81.

89. The reagent contains a lipid, and the formed particle is an LNP that forms a complex with and / or encapsulates the replicable RNA molecule. The composition according to any one of claims 65 to 88.

90. The composition according to any one of claims 65 to 88, wherein the reagent contains a lipid, and the formed particles are vesicles encapsulating the replicable RNA molecule, preferably monolayer liposomes.

91. The composition according to claim 65 or 66, wherein the reagent is a polyalkyleneimine.

92. The composition according to claim 91, wherein the molar ratio (N:P ratio) of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphorus atoms (P) in the replicable RNA molecule is 2.0 to 15.0, preferably 6.0 to 12.

0.

93. The composition according to claim 91 or 92, wherein the ionic strength of the composition is 50 mM or less, preferably the concentration of monovalent cationic ions is 25 mM or less, and the concentration of divalent cationic ions is 20 μM or less.

94. The composition according to any one of claims 91 to 93, wherein the formed particles are polyplexes.

95. The polyalkyleneimine is represented by the following general formula (I): 【Chemical 1】 (wherein, R is H, an acyl group or a group containing the following general formula (II): [Chemical 2] is a group containing, In the formula, R １ is H or the following general formula (III): [Chemical Formula 3] is a group containing, n, m and l are independently selected from integers of 2 to 10; and p, q, and r are integers, and the sum of p, q, and r is such that the average molecular weight of the polymer is 1.5×10 ２ to 10 ７ Da, preferably 5000 to 10 ５ Da, more preferably 10000 to 40000 Da, even more preferably 15000 to 30000 Da, and still even more preferably 20000 to 25000 Da) The composition according to any one of claims 91 to 94, including.

96. The composition according to claim 95, wherein n, m and l are independently selected from 2, 3, 4 and 5, preferably 2 and 3.

97. R １ The composition according to claim 95 or 96, wherein R is H.

98. The composition according to any one of claims 95 to 97, wherein R is H or an acyl group.

99. The composition according to any one of claims 95 to 98, wherein the polyalkyleneimine contains polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine.

100. The composition according to any one of claims 95 to 99, wherein at least 92% of the N atoms in the polyalkyleneimine are protonatable.

101. The composition according to any one of claims 1 to 100, further comprising one or more peptide-based adjuvants, and the peptide-based adjuvant optionally contains immunomodulatory molecules such as cytokines, lymphokines and / or costimulatory molecules.

102. The composition according to any one of claims 1 to 101, further comprising one or more additives, wherein the additives are optionally selected from the group consisting of a buffering substance, a saccharide, a stabilizer, a cryoprotectant, a lyoprotectant, and a chelating agent.

103. The composition according to claim 102, wherein the buffering substance comprises at least one selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), acetic acid, acetate buffer and analogs, phosphoric acid and phosphate buffer, and citric acid and citrate buffer.

104. The composition according to claim 102 or 103, wherein the saccharide comprises at least one selected from the group consisting of monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides, preferably glucose, trehalose, and sucrose.

105. The composition according to any one of claims 102 to 104, wherein the cryoprotectant comprises at least one selected from the group consisting of glycols such as ethylene glycol, propylene glycol, and glycerol.

106. The composition according to any one of claims 102 to 105, wherein the chelating agent comprises EDTA.

107. The composition according to any one of claims 1 to 106, which is a vaccine.

108. A pharmaceutical composition comprising the molecule according to any one of claims 1 to 107 and a pharmaceutically acceptable carrier.

109. The pharmaceutical composition according to claim 108, formulated for intradermal, subcutaneous and / or intramuscular administration by injection or the like.

110. The pharmaceutical composition according to claim 108 or 109, for use in inducing an immune response or in a treatment such as vaccination.

111. The pharmaceutical composition according to claim 108 or 109, for use in a method for inducing a specific immune response against the encoded protein or peptide in a subject, preferably wherein the subject is a mammal, more preferably wherein the mammal is a human, and the method comprises administering the pharmaceutical composition according to claim 93 or 94 to the subject.

112. The pharmaceutical composition for use according to claim 111, wherein administration of the pharmaceutical composition comprises intradermal, subcutaneous or intramuscular administration, such as by intradermal, subcutaneous or intramuscular injection.

113. The pharmaceutical composition for use according to claim 112, wherein the injection is by use of a needle or by use of a needleless injection device.

114. The pharmaceutical composition for use according to any one of claims 111 to 113, wherein the administration preferably comprises intramuscular administration using a needle.

115. A method of inducing an immune response specific for at least two antigens or epitopes in a subject, the method comprising administering to the subject the pharmaceutical composition according to claim 108 or 109, preferably wherein the subject is a mammal, more preferably wherein the mammal is a human.

116. The pharmaceutical composition for use according to claim 114 or the method according to claim 115, wherein the immune response comprises activation of T cells and / or B cells, preferably wherein the activated T cells comprise T helper cells and cytotoxic T cells.

117. The pharmaceutical composition for use according to claim 114 or the method according to claim 115, wherein the immune response comprises activation of antigen-specific T helper cells, optionally wherein the T helper cells proliferate, release T cell cytokines, and mediate proliferation and / or activation of antigen-specific cytotoxic T cells.

118. The pharmaceutical composition for use according to claim 114 or the method according to claim 115, wherein the immune response comprises activation of antigen-specific T helper cells, and the T helper cells stimulate B cell proliferation, antibody class switching, production and / or secretion of neutralizing antibodies.

119. A method of producing at least two proteins or peptides of interest in the cells, the method comprising inoculating the cells with the pharmaceutical composition according to claim 108 or 109.

120. A method of producing at least two proteins or peptides of interest in the subject, the method comprising administering to the subject the pharmaceutical composition according to claim 108 or 109.

121. A method of treating or preventing a bacterial, viral, parasitic or fungal infection in a subject, the method comprising administering to the subject a composition comprising at least two replicable RNA molecules, each comprising a first open reading frame (ORF) encoding at least one peptide or protein comprising an antigen or epitope suitable for inducing an immune response against bacteria, viruses, parasites or fungi, respectively, wherein the at least one peptide or protein encoded by one of the replicable RNA molecules is different from the at least one peptide or protein encoded by another replicable RNA molecule, optionally, wherein at least one of the replicable RNA molecules further comprises a second ORF encoding an RNA-dependent RNA polymerase (replicase) capable of replicating the replicable RNA molecule in cis or in trans, a method. **Claim 122** The method according to claim 121, wherein the composition further comprises a third RNA molecule encoding the replicase capable of replicating the replicable RNA molecule and / or the third RNA molecule in cis or in trans. **Claim 123** The method according to claim 121, wherein the immune response is a specific immune response against bacteria, viruses, parasites or fungi, respectively. **Claim 124** The method according to claim 121 or 123, wherein the immune response reduces the severity of one or more symptoms of the infection. **Claim 125** The method according to any one of claims 121 to 124, comprising only a single administration of the composition. **Claim 126** The method according to any one of claims 121 to 124, comprising multiple administrations of the composition. **Claim 127** The method according to any one of claims 121 to 126, further comprising administering a booster dose of the composition. **Claim 128** The method according to any one of claims 121 to 127, wherein the infection is a viral infection, and optionally, the infection is an Ebola virus infection. **Claim 129** The method according to any one of claims 115 and 120 to 128, wherein administering the composition comprises intradermal, subcutaneous or intramuscular administration, such as by intradermal, subcutaneous or intramuscular injection. **Claim 130** The method according to claim 129, wherein the injection is by use of a needle or by use of a needle-free injection device. **Claim 131** The method according to any one of claims 115 and 120 to 128, wherein the administration preferably comprises intramuscular injection using a needle.

Images