Replicon compositions and methods of treating diseases using same

Abstract

The present invention comprises compositions comprising at least two RNA replicons (self-amplifying RNA vector (saRNA or rRNA)) that can be replicated by a replicase of a self-replicating virus (e.g., a replicase of alphavirus origin). In the at least two replicons, at least one of the replicons optionally comprises an open reading frame encoding an RNA-dependent RNA polymerase or replicase, the RNA-dependent RNA polymerase or replicase being capable of replicating each of the at least two replicons. Furthermore, each replicon comprises an open reading frame encoding a different antigen of interest, such as a different antigen derived from the same or different pathogenic organism, such as glycoproteins and nucleoproteins of Ebola virus.

Description

Technical Field

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

[0002] Recently, mRNA-based vaccines have demonstrated their immunogenicity in clinical studies to combat the Covid-19 epidemic. These RNA vaccines are very effective and induce very strong T cell immune responses and high levels of neutralizing antibodies (Walshet al., 2020, N Engl J Med 383: 2439-2450; Sahin et al., 2020, Nature 586: 594-599). These approved RNA vaccines require 30 to 100 μg RNA per dose and two consecutive doses at intervals of several weeks (primary immunity-boosting regimen). This ultimately requires 60 to 200 g RNA to immunize 1 million people. Therefore, a dose reduction of less than 1 μg will have a huge impact on the production time required to provide a vaccine against a new pathogen to a population.

[0003] A vaccine approach under investigation that promises to achieve significant dose reduction is the use of self-amplifying RNA (saRNA). The saRNA from the alphavirus genome can be engineered by replacing the alphavirus structural genes with the antigens to which the desired immune response is directed. The saRNA can encode the alphavirus replicase, which has all the enzymatic functions to replicate the saRNA molecule, thus resulting in amplification of the amount of input vaccine.

[0004] Alphavirus is a typical representative of enveloped positive-strand RNA virus. The host of alphavirus includes a wide variety of organisms, including insects, fish and mammals, such as domesticated animals and humans. Alphavirus replicates in the cytoplasm of infected cells (for a review of the life cycle of alphavirus, see José et al., 2009, Future Microbiol. 4: 837-856). The total genome length of many alphaviruses is generally 11,000 to 12,000 nucleotides, and the genomic RNA generally has a 5' cap and a 3' poly (A) tail. The genome of alphavirus encodes nonstructural proteins (participating in the transcription, modification and replication of viral RNA and protein modification) and structural proteins (forming viral particles). There are usually two open reading frames (open reading frames, ORF) in the genome. Four nonstructural proteins (nsP1 to nsP4) are usually encoded together by the first ORF starting near the 5' end of the genome, while the alphavirus structural proteins are encoded together by the second ORF present downstream of the first ORF and extending to the 3' end of the genome. Generally speaking, the first ORF is more than the second ORF, with a ratio of about 2:1.

[0005] In cells infected with alphaviruses, only nonstructural proteins are translated from genomic RNA, while structural proteins can be translated from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNA (messenger RNA, mRNA; Gould et al., 2010, Antiviral Res. 87: 111-124). After infection, i.e., in the early stages of the viral life cycle, the (+) strand genomic RNA directly serves as messenger RNA for translation of the open reading frame encoding the nonstructural polyprotein (nsP1234). In some alphaviruses, an opal stop codon is present between the coding sequences of nsP3 and nsP4: when translation is terminated at the opal stop codon, a polyprotein P123 comprising nsP1, nsP2 and nsP3 is produced, and a polyprotein P1234 further comprising nsP4 is produced by reading through the opal codon (Strauss & Strauss, 1994, Microbiol. Rev. 58: 491-562; Rupp et al., 2015, J. Gen. Virology 96: 2483-2500). nsP1234 is cleaved by autoproteolysis into fragments nsP123 and nsP4. Polypeptides nsP123 and nsP4 associate to form a (-) chain replicase complex, which uses the (+) chain genomic RNA as a template to transcribe the (-) chain RNA. Usually at the late 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 the (+) strand replicase complex, which uses the (-) strand complementary sequence 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, subgenomic RNA as well as new genomic RNA are provided with a 5' cap by nsP1 (Pettersson et al., 1980, Eur. J. Biochem. 105:435-443; Rozanov et al., 1992, J. Gen. Virology 73:2129-2134), and a polyadenylic acid [poly (A)] tail by nsP4 (Rubach et al., 2009, Virology 384:201-208). Therefore, both subgenomic RNA and genomic RNA are similar to messenger RNA (mRNA).

[0007] The synthesis of alphavirus RNA is also regulated by cis-acting RNA elements, including four conserved sequence elements (conserved sequence element, CSE; Strauss & Strauss, 1994, Microbiol. Rev. 58: 491-562; and Frolov, 2001, RNA 7: 1638-1651). The alphavirus genome contains four CSEs, which are considered to be important for viral RNA replication in host cells. CSE 1, which is present at or near the 5' end of the viral genome, is considered to act as a promoter for synthesizing the (+) strand from the (-) strand template. CSE2, located downstream of CSE 1 but still close to the 5' end of the genome in the nsP1 coding sequence, is considered to act as a promoter for initiating (-) strand synthesis from the genomic RNA template (note that subgenomic RNA transcripts that do not contain CSE 2 do not serve as templates for (-) strand synthesis). CSE 3 is located in the junction region between the coding sequences for nonstructural and structural proteins and acts as a core promoter for efficient transcription of subgenomic transcripts. Finally, CSE 4, located just upstream of the poly(A) sequence in the 3' untranslated region of the alphavirus genome, is understood to serve as a core promoter for initiating (-) strand synthesis (José et al., 2009, Future Microbiol. 4: 837-856). CSE 4 and the poly(A) tail of alphavirus are understood to work together for efficient (-) strand synthesis (Hardy & Rice, 2005, J. Virol. 79: 4630-4639). .

[0008] It has been proposed that the vector of alphavirus origin is used to deliver foreign genetic information to target cells or target organisms. In a simple method, the open reading frame encoding the structural protein of alphavirus is replaced with the open reading frame encoding the protein of interest. The trans-replication system based on alphavirus depends on the alphavirus nucleotide sequence elements on two separated nucleic acid molecules (RNA molecules): an RNA molecule encoding viral replicase (usually polyprotein nsP1234), and another RNA molecule can be trans-replicated by the replicase (therefore referred to as trans-replication system and / or nano trans-replicator system). Trans-replication requires the presence of these two nucleic acid molecules in a given host cell. The nucleic acid molecules that can be trans-replicated by the replicase must include some alphavirus sequence elements to allow identification and RNA synthesis by the alphavirus replicase.

[0009] The Ebola virus (EBOV) outbreak in West Africa from 2014 to 2016 and the ongoing SARS-CoV-2 pandemic have taught us that emerging and re-emerging viruses can suddenly become a global disaster in a few months, requiring millions of doses of vaccines. The incidence of emerging and re-emerging pathogens is increasing due to increasing globalization and access to rural areas (Murray et al., 2015, Proc. Natl. Acad. Sci. USA 112:12746-12751).

[0010] Therefore, there remains a pressing need for vaccines that can be rapidly developed and allow for use in the treatment of diseases, such as infectious diseases. The present invention meets this need. Summary of the invention

[0011] The present invention generally relates to a composition comprising at least two replicable RNA molecules (replicons), each replicon 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 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 the other replicable RNA molecule, and optionally, wherein at least one of the replicable RNA molecules further comprises a second ORF encoding an RNA-dependent RNA polymerase (replicase) capable of cis- or trans-replicating the replicable RNA molecules in the composition. Optionally, the replicase capable of cis- or trans-replicating the replicable RNA molecules in the composition may be encoded by another (third) RNA molecule contained in the composition, which RNA molecule can be replicated by the encoded replicase, but preferably cannot be replicated by the encoded replicase.

[0012] 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 specific antibody responses, as well as CD4+ and / or CD8+ T cell responses to the antigens.

[0013] In one aspect, the present invention relates to a composition comprising at least two replicable RNA molecules, each RNA molecule 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 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 the other replicable RNA molecule, and optionally, wherein at least one of the replicable RNA molecules further comprises a second ORF encoding an RNA-dependent RNA polymerase (replicase) that is capable of replicating the replicable RNA molecule in cis or in trans.

[0014] The term "different" used in certain embodiments, in the context of different peptides or proteins encoded by the first and second replicable RNA molecules, does not encompass the same peptides or proteins derived from strains of different microorganisms, even if the amino acid sequences of the encoded peptides or proteins may be different. Therefore, in certain embodiments, the encoded peptides or proteins will not be considered "different" simply because of the amino acid sequence differences. In one embodiment, different peptides or proteins are not the same peptides or proteins with amino acid sequence differences. In one embodiment, different peptides or proteins have different functions, or are derived from peptides or proteins with different functions, such as viral nucleoproteins and viral proteins expressed on the surface of the virus, such as glycoproteins. In one embodiment, different peptides or proteins are encoded by different open reading frames in the genome of a pathogen (e.g., a virus).

[0015] The labeling of the first and second open reading frames does not necessarily mean that the first open reading frame is 5' to the second open reading frame in the replicable RNA molecule. In one embodiment, the "second" open reading frame encoding the replicase in the replicable RNA molecule is located 5' to the open reading frame encoding at least one peptide or protein.

[0016] In one embodiment, the composition can also include a third RNA molecule, which encodes a replicase that can cis or trans-replicate a replicable RNA molecule and / or a third RNA molecule. In one embodiment, the third RNA molecule is replicable. In one embodiment, the third RNA molecule is non-replicable, i.e., a non-replicating RNA molecule. In one embodiment, the composition includes at least two replicable RNA molecules (each of which does not encode a replicase), and the third non-replicating RNA molecule encoding a replicase. In one embodiment, the non-replicating RNA is an mRNA.

[0017] In one embodiment, the replicable RNA molecule in the composition may include an internal ribosome entry site (IRES) for the expression of a first ORF that controls the protein or peptide encoding an antigen or epitope, and / or may include an internal ribosome entry site (IRES) for the expression of a second ORF (e.g., encoding a replicase). In a number of embodiments, IRES may be insensitive to cell stress and / or IRES may be insensitive to interferon (preferably type I interferon) and / or IRES may be a cell IRES or a virus IRES, preferably a virus IRES, such as an IRES that may be derived from a virus selected from a picornavirus, a flavivirus, or a dicistrovirus. In one embodiment, IRES may be derived from a picornavirus or a dicistrovirus, preferably a dicistrovirus. In one embodiment, IRES may be a type IV IRES.

[0018] In one embodiment, expression controlled by IRES can be independent of IRES trans-acting factors, and / or expression controlled by IRES can be independent of cellular translation initiation factors. In one embodiment, expression controlled by IRES can be independent of phosphorylation of eukaryotic initiation factor 2 (eIF2).

[0019] In one embodiment, both replicable RNA molecules may comprise a second ORF encoding a replicase. In one embodiment, neither replicable RNA molecule comprises an ORF encoding a replicase.

[0020] In one embodiment, any of the RNA molecules, whether replicating or non-replicating, may comprise a 5' cap for driving translation of a replicase and / or for driving translation of a peptide or protein comprising an antigen or epitope. The 5' cap may be a natural 5' cap or a 5' cap analog.

[0021] 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 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 may comprise a sequence homologous to an open reading frame of a non-structural protein or a portion thereof from a self-replicating virus, wherein the sequence homologous to an open reading frame of a non-structural protein or a portion thereof from a self-replicating virus may be characterized in that, compared to a natural viral sequence, it comprises the removal of at least one start codon. In one embodiment, the sequence homologous to an open reading frame of a non-structural protein or a portion thereof from a self-replicating virus may be characterized in that it comprises the removal of at least one natural start codon of an open reading frame of a non-structural protein from a self-replicating virus. In one embodiment, the sequence homologous to an open reading frame of a non-structural protein or a portion thereof 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 an open reading frame of a non-structural protein from a self-replicating virus. In one embodiment, the sequence homologous to the open reading frame of a nonstructural protein or a portion thereof 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 may be introduced that compensates for the disruption of nucleotide pairing within at least one stem loop introduced by the removal of at least one start codon.

[0022] In one embodiment, the open reading frame encoding a functional nonstructural protein (replicase) from a self-replicating virus does not overlap with the 5' replication recognition sequence. In one embodiment, the first or second ORF may be located downstream of the 5' replication recognition sequence and upstream of the IRES.

[0023] In one embodiment, replicable RNA may include a subgenomic promoter that controls the production of subgenomic RNA, and the subgenomic RNA includes the first ORF encoding a protein or peptide. Subgenomic RNA can be a transcription product of an RNA-dependent RNA polymerase (replicase) derived from a functional nonstructural protein from a self-replicating virus. Optionally, a protein or peptide can be expressed from a subgenomic RNA as a template. In one embodiment, the first ORF encoding a protein or peptide controlled by a subgenomic promoter can be located downstream of a second ORF encoding a replicase. In one embodiment, the subgenomic promoter can overlap with the second ORF.

[0024] In one embodiment, the at least one replicable RNA molecule may comprise a 3' replication recognition sequence. In one embodiment, the 5' and / or 3' replication recognition sequence and the subgenomic promoter may be derived from a self-replicating virus, preferably the same self-replicating virus species.

[0025] In one embodiment, the replicable RNA molecule can be replicated by an RNA-dependent RNA polymerase derived from a functional nonstructural protein from a self-replicating virus. The self-replicating virus can be an alphavirus, for example, selected from 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, Middelburgvirus, and Ndumu virus.

[0026] In one embodiment, at least one RNA molecule may comprise a 3' poly(A) sequence.

[0027] In one embodiment, the first and / or second ORF may be flanked by a 5' untranslated region (UTR) and / or a 3'UTR. In one embodiment, the 5'UTR and / or the 3'UTR are not native to the alphavirus from which the replicase is derived. In one embodiment, the 5'UTR and / or the 3'UTR are native to the alphavirus from which the replicase is derived.

[0028] In the embodiment of more than one RNA molecule encoding replicase in the composition, the replicase can be derived from different alphaviruses, and therefore the sequence encoding the replicase in the RNA molecule is different. In the embodiment of more than one RNA molecule encoding replicase in the composition, the replicase can be the same, and therefore the sequence encoding the replicase in the RNA molecule can be the same. In one embodiment, when more than one RNA molecule encoding replicase in the composition, the replicase can be a variant derived 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.

[0029] In one embodiment, at least one (if not both) of the replicable RNA molecules does not contain an open reading frame for a complete alphavirus structural protein.

[0030] In one embodiment, the replicable RNA molecule comprises a 5' cap, a 5' UTR, an open reading frame encoding a replicase, an IRES, an open reading encoding an antigen, a 3' UTR, and a poly-A sequence in a 5' to 3' order. In this embodiment, both open reading frames can be directly translated, i.e., there is no need to generate progeny RNA to translate the open reading frame encoding the antigen. In one embodiment, the replicable RNA molecule comprises a 5' cap, a 5' UTR, an open reading frame encoding a replicase, a subgenomic promoter, an open reading encoding an antigen, a 3' UTR, and a poly-A sequence in a 5' to 3' order.

[0031] An exemplary poly-A sequence is set forth in SEQ ID NO:78. In one embodiment, a poly-A sequence useful in the RNA molecules described herein is a poly-A sequence having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% homology to SEQ ID NO:78. Exemplary 5'UTR sequences are set forth in SEQ ID NOs:74 and 75. In one embodiment, a 5'UTR sequence useful in the RNA molecules described herein is a 5'UTR sequence having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% homology to SEQ ID NO:74 or 75. Exemplary 3'UTR sequences are set forth in SEQ ID Nos:76 and 77. In one embodiment, a 3'UTR sequence useful in the RNA molecules described herein is a 3'UTR having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% homology to SEQ ID NO:76 or 77. An exemplary subgenomic promoter is shown in SEQ ID NO: 73. In one embodiment, a subgenomic promoter useful for the RNA molecules described herein is a subgenomic promoter having at least 85%, 90%, 95%, 98% or 99% homology to SEQ ID NO: 73. It is contemplated that the RNA molecules described herein may have any combination of functional 5'UTR, 3'UTR, poly-A and subgenomic sequences. Thus, in certain embodiments, the RNA molecules described herein may have any combination of the exemplified 5'UTR, 3'UTR, poly-A and subgenomic sequences.

[0032] In one embodiment, the antigen or epitope of the encoded protein or peptide is the following or is derived from the following: a bacterial, viral, parasitic or fungal antigen. 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 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 be obtained from or derived from different strains of the same bacteria, virus, parasite or fungus, respectively, or can be obtained from or 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 each, for example, influenza hemagglutinin (influenzahemagglutinin, HA) proteins from two different strains of influenza. In one embodiment, the epitope of the encoded protein can be a T cell epitope.

[0033] In one embodiment, the protein or peptide encoded by the first replicable RNA is a surface-expressed protein or peptide, while the protein or peptide encoded by the second replicable RNA is not a surface-expressed protein or peptide. Surface-expressed and non-surface-expressed proteins or peptides can be obtained from or derived from the same or different strains of the same bacteria, virus, parasite or fungus, respectively, 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, while the protein or peptide encoded by the second replicable RNA is not a surface-expressed protein or peptide and is different from the protein or peptide encoded by the first replicable RNA. These different non-surface-expressed proteins or peptides can be obtained from or derived from the same or different strains of the same bacteria, virus, parasite or fungus, respectively, 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, while the protein or peptide encoded by the second replicable RNA is also a surface-expressed protein or peptide and is different from the protein or peptide encoded by the first replicable RNA. These surface expressed proteins or peptides may be obtained or derived from the same or different strains of the same bacteria, virus, parasite or fungus, respectively, or may be obtained or derived from different pathogenic organisms, such as different viruses.

[0034] In one embodiment, the surface expressed protein is expressed on the surface of the virus / viral particle, or wherein, in the case where the virus is an enveloped virus, the surface expressed protein is expressed on the surface of the viral envelope, e.g., the surface expressed protein is a viral capsid protein or a viral envelope or glycoprotein.

[0035] In one embodiment, 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, wherein the virus is an enveloped virus.

[0036] 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, wherein the glycoprotein and nucleoprotein are obtainable or derived from the same virus, optionally obtained or derived from the same strain of the same virus.

[0037] 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 nucleoprotein NP of Ebola virus. The amino acid sequence of exemplary Ebola virus GP protein is shown in SEQ ID NO:89, and the amino acid sequence of exemplary Ebola virus NP protein is shown in SEQ ID NO:91.

[0038] In one embodiment, the surface expressed protein or peptide may be the glycoprotein Gc of the CCHFV virus. In one embodiment, the non-surface expressed protein or peptide may be the nucleoprotein NP of the 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.

[0039] 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.

[0040] In one embodiment, the amino acid sequence of a surface-expressed protein or peptide or the amino acid sequence of a non-surface-expressed protein or peptide can be an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the corresponding SEQ ID NO above.

[0041] In one embodiment, the composition comprises a first replicable RNA molecule encoding an Ebola virus GP protein having an amino acid sequence shown in SEQ ID NO:89 and a second replicable RNA molecule encoding an Ebola virus NP protein having an 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 an amino acid sequence shown in SEQ ID NO:93 and a second replicable RNA molecule encoding a CCHFV NP protein having an 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 an amino acid sequence shown in SEQ ID NO:97 and a second replicable RNA molecule encoding a MERS CoV virus NP protein having an amino acid sequence shown in SEQ ID NO:99. Optionally, the first or second replicable RNA molecule encodes a replicase. The composition optionally comprises a third RNA molecule, which may be replicable or non-replicable, and encodes an RNA replicase. The third RNA molecule may be a non-replicable mRNA.

[0042] 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.

[0043] In one embodiment, the induced immune response to an antigen or epitope can be an antibody response to an antigen or epitope. In one embodiment, the induced immune response to an antigen or epitope can be an increase in the activity of CD4+T cells and / or CD8+T cells. In one embodiment, the induced immune response to an antigen or epitope can be an increase in the activity of both CD4+T cells and CD8+T cells. In one embodiment, the induced immune response to an antigen or epitope can be an antibody response to an antigen or epitope and an increase in the activity of both CD4+T cells and CD8+T cells.

[0044] 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 following, or an epitope of a fragment thereof or an Ebola virus structural protein: glycoprotein (GP), nucleoprotein (NP), polymerase cofactor (VP35), VP40, transcription factor (VP30), VP24 or RNA-dependent RNA polymerase (L). 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 from a nucleoprotein complex, such as Ebola virus NP, CCHFV NP or MERS-CoV NP. The protein or peptide can contain a T cell epitope.

[0045] In one embodiment, the protein or peptide encoded by the 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 or secretion motif.

[0046] In one embodiment, the first of at least two replicable RNA molecules included in the composition encodes a protein or an epitope of a strain of a pathogen (e.g., Ebola virus), and the second of at least two replicable RNA molecules included in the composition encodes the same or different proteins or epitopes from different strains of the pathogen. For example, a replicable RNA molecule encodes the GP protein or an epitope of Ebola virus subtype Zaire, and the second replicable molecule encodes the GP protein or an epitope of Ebola virus strain Sudan. In one embodiment, the first of at least two replicable RNA molecules included in the composition encodes several proteins or epitopes from strains of pathogens, and the second of at least two RNA molecules included in the composition encodes several proteins or epitopes from different strains 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.

[0047] Exemplary Zaire strains include Homo sapiens -wt / SLE / 2014 / Makona-EM095B (GenBank: KM034551.1), Homo sapiens -wt / GIN / 2014 / Makona-Gueckedou-C07 (GenBank: KJ660347.2), Homo sapiens -tc / COD / 1976 / Yambuku-Mayinga (GenBank NC_002549.1), Zaire Ebola virus strain EBO-0003 (GenBank: MZ605321.1) and Zaire Ebola virus strain EBO-0002 (GenBank: MZ605320.1). Other strains include those implicated in the 2013-2016 EBOV epidemic in Guinea, Liberia, and Sierra Leone in West Africa, sequences of which are available at the European Nucleotide Archive under accession number PRJEB43650 and at github.com as PFHVG / EBOVsequencing.

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

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

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

[0051] In one embodiment, at least one, preferably both, replicable RNA molecules may be codon optimized, for example, by exchanging only the worst (least favorable) triplets for human usage, 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 may be an Ebola virus structural GP protein or a fragment thereof, or an epitope of a GP protein, and the protein or peptide encoded by the second replicable RNA molecule may be an Ebola virus structural NP protein or a fragment thereof, or an epitope of an 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 first replicable RNA molecules to the number of second replicable RNA molecules in the composition can be varied from approximately equal numbers of molecules. In one embodiment, the ratio of the first and second replicable RNA molecules can be 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 an agent capable of forming particles with the replicable RNA molecule, for example, the agent can be a lipid or a polyalkylene imine. 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 agent can be conjugated to polysarcosine.

[0055] In one embodiment, the particles formed by the replicable RNA molecule and the agent can be a polymer-based polyplex (PLX) or a lipid nanoparticle (LNP), wherein the LNP is preferably a lipid complex (LPX) or a liposome. In one embodiment, the particles may also contain at least one phosphatidylserine. In one embodiment, the particles may be 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 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. In one embodiment, the charge ratio of positive charges to negative charges in the nanoparticles may be 1.4:1 to 1:8, preferably 1.2:1 to 1:4.

[0056] In one embodiment, the nanoparticle may comprise at least one lipid, preferably at least one cationic lipid, optionally wherein the positive charge is contributed by the at least one cationic lipid and the negative charge is contributed by the replicable RNA molecule.

[0057] In one embodiment, the nanoparticles may further comprise at least one auxiliary lipid, wherein, for example, the auxiliary lipid may be a neutral lipid. In various embodiments, the at least one cationic lipid may comprise 1,2-di-O-octadecenyl-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 auxiliary lipid may comprise 1,2-di-(9Z-octadecanoyl)-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 10:0 to 3:7, preferably 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 nanoparticle may be a lipoplex comprising DODMA and DOPE, wherein the molar ratio of DODMA to DOPE is from 10:0 to 1:9, preferably from 8:2 to 3:7, and more preferably from 7:3 to 5:5, and wherein the charge ratio of positive charges in DODMA to negative charges in 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. In one embodiment, the nanoparticle may be a lipoplex comprising DODMA and cholesterol, wherein the molar ratio of DODMA to cholesterol is from 10:0 to 1:9, preferably from 8:2 to 3:7, and more preferably from 7:3 to 5:5, and wherein the charge ratio of positive charges in DODMA to negative charges in 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. In one embodiment, the nanoparticle can be a lipid complex comprising DODMA and DSPC, wherein the molar ratio of DODMA to DSPC is 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably 7:3 to 5:5, and wherein 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 nanoparticle is a lipid complex comprising DODMA:cholesterol:DOPE:PEGcerC16 in a molar ratio of 40:48:10:2.

[0059] In one embodiment, the nanoparticle may be a lipoplex comprising DOTMA and DOPE, wherein the molar ratio of DOTMA to DOPE is 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably 7:3 to 5:5, and wherein the charge ratio of positive charges in DOTMA to 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 nanoparticle may be a lipoplex comprising DOTMA and cholesterol, wherein the molar ratio of DOTMA to cholesterol is 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably 7:3 to 5:5, and wherein the charge ratio of positive charges in DOTMA to 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 nanoparticle can be a lipoplex comprising DOTAP and DOPE, wherein the molar ratio of DOTAP to DOPE is from 10:0 to 1:9, preferably from 8:2 to 3:7, and more preferably from 7:3 to 5:5, and wherein the charge ratio of positive charges in DOTMA to negative charges in 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.

[0060] In one embodiment, the reagent may include lipids, and the particles formed may be LNPs that are complexed with and / or encapsulate replicable RNA molecules. In one embodiment, the reagent may include lipids, and the particles formed may be vesicles, preferably unilamellar liposomes, that encapsulate replicable RNA molecules.

[0061] In one embodiment, the agent is a polyalkyleneimine, and, for example, the molar ratio of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphorus atoms (P) in the replicable RNA molecule (N:P ratio) 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 wherein the concentration of monovalent cations can be 25 mM or less and the concentration of divalent cations can be 20 μM or less.

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

[0063] In one embodiment, the polyalkylene imine may comprise the following general formula (I):

[0064]

[0065] in

[0066] R is H, acyl or a group comprising the following general formula (II):

[0067]

[0068] Wherein R1 is H or a group comprising the following general formula (III):

[0069]

[0070] n, m and l are independently selected from integers from 2 to 10; and

[0071] p, q and r are integers, wherein the sum of p, q and r is such that the average molecular weight of the polymer is 1.5×10 2 Up to 10 7 Da, preferably 5000 to 10 5 Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da. In one embodiment, n, m and l can be independently selected from 2, 3, 4 and 5, preferably selected from 2 and 3; and / or R1 can be H. In one embodiment, R can be H or acyl.

[0072] In one embodiment, the polyalkyleneimine may comprise polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. In one embodiment, at least 92% of the N atoms in the polyalkyleneimine may be protonatable.

[0073] In one embodiment, the composition may also include one or more peptide-based adjuvants, wherein the peptide-based adjuvant optionally includes immunomodulatory molecules, such as cytokines, lymphokines and / or co-stimulatory molecules. In one embodiment, the composition may also include one or more additives, wherein the additive is optionally selected from buffer substances, sugar, stabilizers, cryoprotectants, lyophilization protectants and chelating agents. Buffer substances may include at least one selected from the following: 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. Sugar may include at least one selected from monosaccharides, disaccharides, trisaccharides, oligosaccharides and polysaccharides, preferably including at least one selected from glucose, trehalose and sucrose. Cryoprotectants may include at least one selected from glycols such as ethylene glycol, propylene glycol, and glycerol. In one embodiment, chelating agents may include EDTA.

[0074] In one embodiment, the replicable RNA molecules in the composition are present in a relative ratio of 6:1 to 1:6. In one embodiment, the replicable RNA molecules in the composition are present in a relative ratio of 3:1 to 1:3. In one embodiment, the replicable RNA molecules in the composition are present in a relative ratio of 2:1 to 1:2. In one embodiment, the replicable RNA molecules in the composition are present in a ratio of 1:1. In one embodiment, the ratio is determined by the molecular weight of the RNA molecules.

[0075] In one embodiment, the composition of the invention having at least two replicable RNA molecules may be a vaccine.

[0076] 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, for example, by injection.

[0077] In one aspect, the present invention relates to the use of a composition of the present invention having at least two replicable RNA molecules in treatment, such as inducing an immune response or vaccination. In one aspect, the present invention relates to the use of a composition of the present invention having at least two replicable RNA molecules in a method for inducing an immune response specific for an encoded protein or peptide in a subject, preferably wherein the subject is a mammal, more preferably wherein the mammal is a human, the method comprising administering a pharmaceutical composition of the present invention.

[0078] In one aspect, the invention relates to a method for inducing an immune response specific for at least two antigens or epitopes in a subject, comprising administering to the subject a pharmaceutical composition of the invention, preferably wherein the subject is a mammal, more preferably wherein the mammal is a human.

[0079] In one embodiment, the immune response may include activation of T cells and / or B cells, preferably wherein the activated T cells include T helper cells and cytotoxic T cells. In one embodiment, the immune response may include activation of antigen-specific T helper cells, optionally wherein T helper cells proliferate, release T cell cytokines, mediate growth and / or activation of antigen-specific cytotoxic T cells. In one embodiment, the immune response may include activation of antigen-specific T helper cells, wherein T helper cells stimulate B cell proliferation, antibody class switching, the production and / or secretion of neutralizing antibodies.

[0080] 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 the pharmaceutical composition of the present invention into the cell. In one aspect, the present invention relates to a method for producing at least two proteins or peptides of interest in an object, comprising administering a pharmaceutical composition of the present invention to the object. In one embodiment, the method comprises administering to the object a first replicable RNA molecule comprising the nucleotide sequence shown in SEQ ID NO:27 and a second replicable RNA molecule comprising the nucleotide sequence shown in SEQ IN NO:29. In one embodiment, the method comprises administering to the object a replicable RNA molecule shown in SEQ ID NO:30 and a replicable RNA molecule shown in SEQ ID NO:31.

[0081] In one aspect, the present invention relates to a method for 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 replicable RNA molecule comprising a first open reading frame (ORF) encoding at least one peptide or protein, the 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 the other replicable RNA molecule, and wherein at least one of the replicable RNA molecules further comprises a second ORF encoding an RNA-dependent RNA polymerase (replicase) capable of cis- or trans-replicating the replicable RNA molecule. In one embodiment, the method comprises administering to the subject a first replicable RNA molecule comprising the nucleotide sequence shown in SEQ ID NO: 27 and a second replicable RNA molecule comprising the nucleotide sequence shown in SEQIN NO: 29. In one embodiment, the method comprises administering to the subject a replicable RNA molecule shown in SEQ ID NO: 30 and a replicable RNA molecule shown in SEQ ID NO: 31.

[0082] In one embodiment, the immune response is a specific immune response against bacteria, viruses, parasites or fungi, respectively, and / or the immune response reduces the severity of one or more symptoms of the infection. In one embodiment, the infection may be a viral infection, optionally wherein the infection is an Ebola virus infection.

[0083] In one embodiment, the method of treatment involves only a single administration of the composition or the method of treatment comprises multiple administrations of the composition. In one embodiment, the method may further comprise administering a booster dose of a pharmaceutical composition of the invention. DETAILED DESCRIPTION

[0084] Although the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only used for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art.

[0085] Preferably, the terms used herein are as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", HGW Leuenberger, B. Nagel, and H. Definitions as described in Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0086] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology and recombinant DNA techniques, which are explained in the literature in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0087] Hereinafter, some elements of the present invention will be described. These elements are listed together with some specific embodiments, however, it should be understood that they can be combined in any way and in any quantity to produce other embodiments. The embodiments and preferred embodiments of multiple descriptions should not be interpreted as limiting the present invention to only the embodiments clearly described. This description should be understood to disclose and encompass the embodiments of the embodiments clearly described and any number of disclosed and / or preferred element combinations. In addition, unless otherwise indicated by the context, any arrangement and combination of all described elements in this application should be considered to be disclosed by this specification.

[0088] The term "about" means approximately or close to, and in the context of numerical values ​​or ranges recited herein, preferably means + / - 10% of the recited or claimed numerical value or range.

[0089] Unless otherwise specified herein or obviously contradictory with context, otherwise the term of undefined number used in the context of describing the present invention (particularly in the context of claim) and similar references should be interpreted as covering both singular and plural. The description of numerical range herein is only intended to be used as a shorthand method for referring to each individual value falling within the range separately. Unless otherwise specified herein, each individual value is incorporated into this specification as if it is quoted separately in this article. Unless otherwise specified herein or obviously contradictory with context, all methods described herein can be carried out in any suitable order. The use of any and all embodiments or exemplary language (e.g., "for example") provided herein is only intended to better illustrate the present invention, rather than to limit the scope of the present invention, unless otherwise stated. Any language in the specification should not be interpreted as representing any unclaimed element essential to the practice of the present invention.

[0090] Unless explicitly stated otherwise, the term "comprising" is used in the context of this document to indicate that other members may optionally be present in addition to the members of the list introduced by "comprising". However, it is contemplated that as a specific embodiment of the present invention, the term "comprising" includes the possibility that other members are not present, that is, for the purpose of this embodiment, "comprising" should be understood to have the meaning of "consisting of..."

[0091] The indication of the relative amount of a component characterized by a general term means the total amount of all specific variants or members covered by the general term. If a certain component defined by a general term is specified to be present in a certain relative amount, and if the component is further characterized as a specific variant or member covered by the general term, it means that no other variants or members covered by the general term are additionally present, such that the total relative amount of the component covered by the general term exceeds the specified relative amount; more preferably, no other variants or members covered by the general term are present at all.

[0092] Several documents are cited throughout this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether above or below, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosures.

[0093] As used herein, terms such as "reduce" or "inhibit" mean the ability to cause an overall decrease in levels of preferably 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 include complete or substantially complete inhibition, i.e., reduction to zero or substantially to zero.

[0094] Terms such as "increase" or "enhance" preferably relate to an increase or enhancement of about at least 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%.

[0095] The term "net charge" refers to the charge on an entire object (eg, a compound or particle).

[0096] An ion with an overall net positive charge is a cation, while an ion with an overall net negative charge is an anion. Thus, according to the present invention, an anion is an ion that has more electrons than protons, giving it a net negative charge, and a cation is an ion that has fewer electrons than protons, giving it a net positive charge.

[0097] With respect to a given compound or particle, terms such as "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.

[0098] Term " nucleic acid " according to the present invention also includes the chemical derivatization of nucleic acid on nucleotide base, sugar or phosphate, and the nucleic acid containing non-natural nucleotide and nucleotide analog.In some embodiments, nucleic acid is deoxyribonucleic acid (deoxyribonucleic acid, DNA) or ribonucleic acid (ribonucleic acid, RNA).In general, nucleic acid molecule or nucleic acid sequence refers to the nucleic acid preferably of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).According to the present invention, nucleic acid includes genomic DNA, cDNA, mRNA, viral RNA, recombinant preparation and chemical synthesis molecule.According to the present invention, nucleic acid can be the form of single-stranded or double-stranded and linear or covalently closed circular molecule.

[0099] According to the present invention, "nucleic acid sequence" refers to the sequence of nucleotides in a nucleic acid, such as ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). The term can refer to the entire nucleic acid molecule (e.g., a single strand of the entire nucleic acid molecule) or a portion thereof (e.g., a fragment).

[0100] According to the present invention, the term "RNA" or "RNA molecule" refers to a molecule comprising ribonucleotide residues and preferably consisting entirely or substantially of ribonucleotide residues. The term "ribonucleotide" refers 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 completely purified RNA, substantially pure RNA, synthetic RNA, and recombinantly produced RNA, such as modified RNA that is different from naturally occurring RNA by adding, deleting, replacing and / or changing one or more nucleotides. Such changes may include adding non-nucleotide substances to, for example, the end or interior of RNA, such as at one or more nucleotides of RNA. The nucleotides in the RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs, particularly analogs of naturally occurring RNA.

[0101] According to the present invention, 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 that does not have a complementary nucleic acid molecule (generally without a complementary RNA molecule) associated with it. Single-stranded RNA may include a self-complementary sequence that allows a portion of RNA to fold back and form a secondary structure motif, including but not limited to base pairs, stems, stem loops, and protrusions. Single-stranded RNA may exist as a negative strand [(-) strand] or as a positive strand [(+) strand]. (+) strand is a strand that contains or encodes genetic information. Genetic information may be, for example, a polynucleotide sequence that encodes a protein. When (+) strand RNA encodes a protein, the (+) strand can be directly used as a template for translation (protein synthesis). The (-) strand is the complementary strand of the (+) strand. In the case of double-stranded RNA, the (+) strand and the (-) strand are two independent RNA molecules, and the two RNA molecules associate with each other to form a double-stranded RNA ("duplex RNA").

[0102] The term "stability" of RNA relates to the "half-life" of the RNA. "Half-life" relates to the period of time required to eliminate half of the activity, amount or number of a molecule. In the context of the present invention, the half-life of an RNA indicates the stability of the RNA. The half-life of an RNA may affect the "duration of expression" of the RNA. It is expected that an RNA with a long half-life will be expressed over an extended period of time.

[0103] The term "translation efficiency" relates to the amount of translation product provided by an RNA molecule within a specific period of time.

[0104] With respect to nucleic acid sequences, a "fragment" relates to a part of a nucleic acid sequence, i.e. a sequence representing a nucleic acid sequence shortened at the 5' and / or 3' end. Preferably, a fragment of a nucleic acid sequence comprises at least 80%, preferably at least 90%, 95%, 96%, 97%, 98% or 99% of the nucleotide residues from said nucleic acid sequence. In the present invention, preference is given to those fragments of RNA molecules which retain RNA stability and / or translation efficiency.

[0105] With respect to an amino acid sequence (peptide or protein), a "fragment" refers to a portion of an amino acid sequence, i.e., a sequence representing 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 translating 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 translating 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 for starting translation. A fragment of an amino acid sequence comprises, 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.

[0106] With regard to nucleic acid and amino acid sequences, for example, according to the present invention, the term "variant" includes any variant, particularly mutants, virus strain variants, splice variants, conformations, isotypes, allelic variants, species variants and species homologs, particularly those that occur naturally. Allelic variants relate to changes in the normal sequence of a gene, the importance of which is generally unclear. Complete gene sequencing generally identifies many allelic variants of a given gene. With regard to nucleic acid molecules, the term "variant" includes degenerate nucleic acid sequences, wherein the degenerate nucleic acid according to the present invention is a nucleic acid that is different from a reference nucleic acid in codon sequence due to the degeneracy of the genetic code. Species homologs are nucleic acids or amino acid sequences having different origin species from a given nucleic acid or amino acid sequence. Viral homologs are nucleic acids or amino acid sequences having different origin viruses from a given nucleic acid or amino acid sequence. For example, a hemagglutinin derived from an influenza strain and a hemagglutinin derived from another influenza strain (which does not have the same amino acid sequence) are variants of each other. In another example, the same bacterial proteins derived from two different bacterial strains and not having the same amino acid sequence are variants of each other.

[0107] Compared to a reference nucleic acid, nucleic acid variants include deletions, additions, mutations, substitutions and / or insertions of single or multiple nucleotides. Deletions include removing one or more nucleotides from a reference nucleic acid. Addition variants include 5' and / or 3' end fusions of one or more nucleotides, such as 1, 2, 3, 5, 10, 20, 30, 50 or more nucleotides. In the case of substitution, at least one nucleotide in the sequence is removed, and at least one other nucleotide is inserted into its position (e.g., transversion and conversion). Mutations include abasic sites, crosslinking sites, and chemically altered or modified bases. Insertions include adding at least one nucleotide to a reference nucleic acid.

[0108] According to the present invention, "nucleotide changes" may refer to the deletion, addition, mutation, substitution and / or insertion of a single or more nucleotides compared to a reference nucleic acid. In some embodiments, "nucleotide changes" are selected from the deletion of a single nucleotide, the addition of a single nucleotide, the mutation of a single nucleotide, the substitution of a single nucleotide and / or the insertion of a single nucleotide compared to a reference nucleic acid. According to the present invention, a nucleic acid variant may comprise one or more nucleotide changes compared to a reference nucleic acid.

[0109] A variant of a specific nucleic acid sequence preferably has at least one functional property of the specific sequence, and is preferably functionally equivalent to the specific sequence, e.g., a nucleic acid sequence having the same or similar properties as the specific nucleic acid sequence.

[0110] As described below, some embodiments of the present invention are characterized in particular by nucleic acid sequences that are homologous to other nucleic acid sequences. These homologous sequences are variants of other nucleic acid sequences.

[0111] 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 preferably 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%. Preferably, the degree of identity is given over 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 some preferred embodiments, the degree of identity over the entire length of the reference nucleic acid sequence is given.

[0112] "Sequence similarity" refers to the percentage of amino acids that are identical or that represent conservative amino acid substitutions. "Sequence identity" between two polypeptide or nucleic acid sequences refers to the percentage of amino acids or nucleotides that are identical between the sequences.

[0113] The term "% identity" is particularly intended to refer to the percentage of identical nucleotides in an optimal alignment between two sequences to be compared, the percentage being purely statistical, and the differences between the two sequences may be randomly distributed over the entire length of the sequence, and the sequence to be compared may comprise additions or deletions compared to the reference sequence to obtain an optimal alignment between the two sequences. The comparison of two sequences is usually performed by comparing the sequences after optimal alignment with respect to a fragment or "comparison window" to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually, or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2:482, with the aid of the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, and with the aid of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85:2444, or with the aid of computer programs that use such algorithms (GAP, BESTFIT, FASTA, BLAST P, BLASTN, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0114] The percent identity is calculated by determining the number of identical positions in the sequences being compared, dividing that number by the number of positions being compared, and multiplying the result by 100.

[0115] For example, the BLAST program "BLAST 2 sequences" can be used, which is available at the website http: / / www.ncbi.nlm.nih.gov / blast / N2seq / wblast2.cgi.

[0116] If a nucleic acid is complementary to another nucleic acid, then two sequences are "capable of hybridizing" or "hybridizing". If a nucleic acid is complementary to another nucleic acid and is capable of forming a stable duplex with each other, then the two sequences are "complementary". According to the present invention, hybridization is preferably carried out under conditions that allow specific hybridization between polynucleotides (stringent conditions). 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, FM Ausubel ct al., Editors, John Wiley & Sons, Inc., New York, and refer, for example, to hybridization in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5mM NaH2PO4 (pH 7), 0.5% SDS, 2mM EDTA) at 65°C. SSC is 0.15 M sodium chloride / 0.15 M sodium citrate, pH 7. After hybridization, the membrane to which the DNA was transferred is washed, for example, in 2X SSC at room temperature and then in 0.1 to 0.5X SSC / 0.1X SDS at temperatures up to 68°C.

[0117] Percent complementarity represents the percentage 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, 10 out of 10 are 50%, 60%, 70%, 80%, 90% and 100% complementary). "Perfect complementarity" or "complete complementarity" means that all consecutive residues of a nucleic acid sequence are hydrogen bonded to 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%.

[0118] The term "derivative" includes any chemical derivatization of a nucleic acid on a nucleotide base, a sugar or a phosphate. The term "derivative" also includes nucleic acids containing non-naturally occurring nucleotides and nucleotide analogs. Preferably, the derivatization of a nucleic acid improves its stability.

[0119] "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 the sequence of the variant with respect to a specific sequence replaces the specific sequence in an RNA molecule, it retains RNA stability and / or translation efficiency.

[0120] "nt" is an abbreviation for a nucleotide, or for nucleotides, preferably consecutive nucleotides, in a nucleic acid molecule.

[0121] According to the present invention, the term "codon" refers to a base triplet in a coding nucleic acid that indicates which amino acid is to be added next during protein synthesis in the ribosome.

[0122] The term "transcription" and its variations relate to a process in which a nucleic acid molecule having a specific nucleic acid sequence ("nucleic acid template") is read by an RNA polymerase, causing the RNA polymerase to produce 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 an alphavirus nucleic acid template, for example, the template is typically RNA. The transcribed RNA may subsequently be translated into a protein. According to the present invention, the term "transcription" includes "in vitro transcription", wherein the term "in vitro transcription" relates to a process in which RNA, in particular 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 covered 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 an in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter used to control transcription may be any promoter for any RNA polymerase. The DNA template used for in vitro transcription may be obtained by cloning nucleic acids, in particular c DNA and introduce it into a suitable vector for in vitro transcription. c DNA can be obtained by reverse transcription of RNA.

[0123] The single-stranded nucleic acid molecule produced during transcription generally has a nucleic acid sequence that is complementary to the sequence of the template.

[0124] 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.

[0125] "Nucleic acid sequence transcribed from a nucleic acid sequence" and similar terms refer to a nucleic acid sequence that is a transcription product of a template nucleic acid sequence, where appropriate, as part of a complete RNA molecule. Typically, the transcribed nucleic acid sequence is a single-stranded RNA molecule.

[0126] According to the present invention, the "3' end of a nucleic acid" refers to the end with a free hydroxyl group. In schematic diagrams of double-stranded nucleic acids (especially DNA), the 3' end is always on the right. According to the present invention, the "5' end of a nucleic acid" refers to the end with a free phosphate group. In schematic diagrams of double-stranded nucleic acids (especially DNA), the 5' end is always on the left.

[0127] 5' end 5'--P-NNNNNNN-OH-3' 3' end

[0128] 3'-HO-NNNNNNN-P--5'

[0129] "Upstream" describes the relative positioning of a first element of a nucleic acid molecule relative to a second element of the nucleic acid molecule, wherein both elements are contained in the same nucleic acid molecule and wherein the first element is closer to the 5' end of the nucleic acid molecule than the second element of the nucleic acid molecule. The second element is then said to be "downstream" of the first element of the nucleic acid molecule. An element that is "upstream" of a second element may be synonymously referred to as being "5'" of the second element. For double-stranded nucleic acid molecules, indications such as "upstream" and "downstream" are given with respect to the (+) strand.

[0130] According to the invention, "functionally linked" or "functionally linked" relates to a connection that is in a functional relationship. A nucleic acid is "functionally linked" if it is functionally related to another nucleic acid sequence. For example, a promoter is functionally linked to a coding sequence if a promoter influences the transcription of said coding sequence. Functionally linked nucleic acids are usually adjacent to each other, separated by further nucleic acid sequences where appropriate, and in a specific embodiment, transcribed by RNA polymerase to give a single RNA molecule (co-transcript).

[0131] In some specific embodiments, the nucleic acid according to the invention is functionally linked to an expression control sequence, which may be homologous or heterologous to the nucleic acid.

[0132] According to the present invention, the term "expression control sequence" includes promoters, ribosome binding sequences and other control elements for the transcription of genes or the translation of source RNA. In a specific embodiment of the present invention, the expression control sequence can be adjusted. The precise structure of the expression control sequence can vary according to the species or cell type, but generally includes 5'-non-transcribed sequences and 5'- and 3'-non-translated sequences that participate in the initiation of transcription and translation, respectively. More specifically, the 5'-non-transcribed expression control sequence includes a promoter region, which contains a promoter sequence for the transcriptional control of the gene to be functionally connected. The expression control sequence may also include an enhancer sequence or an upstream activator sequence. The expression control sequence of a DNA molecule generally includes a 5'-non-transcribed sequence and a 5'- and 3'-non-translated sequence, such as a TATA box, a capping sequence, a CAAT sequence, etc. The expression control sequence of an alphavirus RNA may include a subgenomic promoter and / or one or more conserved sequence elements. As described herein, a specific expression control sequence according to the present invention is a subgenomic promoter of an alphavirus.

[0133] The nucleic acid sequences specified herein (particularly transcribable and encoding nucleic acid sequences) may be combined with any expression control sequences (particularly promoters), which may be homologous or heterologous to the nucleic acid sequences, the term "homologous" referring to the fact that the nucleic acid sequence is also naturally functionally linked to the expression control sequence, and the term "heterologous" referring to the fact that the nucleic acid sequence is not naturally functionally linked to the expression control sequence.

[0134] If a transcribable nucleic acid sequence, in particular a nucleic acid sequence encoding a peptide or protein, and an expression control sequence are covalently linked to each other in such a way that the transcription or expression of the transcribable nucleic acid sequence, and in particular the coding nucleic acid sequence, is under the control or influence of the expression control sequence, then they are "functionally" linked to each other. If the nucleic acid sequence is to be translated into a functional peptide or protein, then the functional linkage of the inducible expression control sequence to the coding sequence results in the transcription of the coding sequence without causing a frameshift in the coding sequence or the inability of the coding sequence to be translated into the desired peptide or protein.

[0135] The term "promoter" or "promoter region" refers to a nucleic acid sequence that controls the synthesis of a transcript (e.g., a transcript comprising a coding sequence) by providing recognition and binding sites for RNA polymerase. The promoter region may contain additional recognition or binding sites for additional factors involved in the regulation of gene transcription. A promoter can control the transcription of a prokaryotic or eukaryotic gene. A promoter can be "inducible" and initiate transcription in response to an inducer, or can be "constitutive" if transcription is not controlled by an inducer. If there is no inducer, an inducible promoter is expressed only to a small extent or not at all. In the presence of an inducer, a gene is "turned on" or the transcription level is increased. This is typically mediated by the binding of a specific transcription factor. A specific promoter according to the present invention is a subgenomic promoter of, for example, an alphavirus as described herein. Other specific promoters are, for example, alphavirus genome positive or negative chain promoters.

[0136] The term "core promoter" refers to the nucleic acid sequence contained in the promoter. The core promoter is usually the smallest part of the promoter required to correctly initiate transcription. The core promoter usually contains a transcription start site and a binding site for RNA polymerase.

[0137] "Polymerase" generally refers to a molecular entity that can catalyze the synthesis of polymer molecules by monomeric structural units. "RNA polymerase" is a molecular entity that can catalyze the synthesis of RNA molecules by ribonucleotide structural units. "DNA polymerase" is a molecular entity that can catalyze the synthesis of DNA molecules by deoxyribonucleotide structural units. For the case of DNA polymerase and RNA polymerase, the molecular entity is generally an assembly or complex of proteins or multiple proteins. Generally speaking, DNA polymerase synthesizes DNA molecules based on template nucleic acid, and the template nucleic acid is generally a DNA molecule. Generally speaking, RNA polymerase synthesizes RNA molecules based on template nucleic acid, and the template molecule is a DNA molecule (in this case, RNA polymerase is a DNA-dependent RNA polymerase, DdRP), or an RNA molecule (in this case, RNA polymerase is an RNA-dependent RNA polymerase, RdRP).

[0138] "RNA-dependent RNA polymerase" or "RdRP" is an enzyme that catalyzes RNA transcription from an RNA template. In the case of alphavirus RNA-dependent RNA polymerase, the sequential synthesis of the (-) strand complementary strand of the genomic RNA and the (+) strand genomic RNA results in RNA replication. Therefore, RNA-dependent RNA polymerases are synonymously referred to as "RNA replicases" or simply "replicases". In fact, RNA-dependent RNA polymerases are generally encoded by all RNA viruses except retroviruses. The typical representative of viruses encoding RNA-dependent RNA polymerases is the alphavirus.

[0139] 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 may be, for example, identical or complementary to the template RNA molecule. Generally speaking, RNA replication may occur by the synthesis of a DNA intermediate, or may occur directly by RNA-dependent RNA replication mediated by RNA-dependent RNA polymerase (RdRP). In the case of alphavirus, RNA replication does not occur by a DNA intermediate, but is mediated by RNA-dependent RNA polymerase (RdRP): a template RNA chain (the first RNA chain) or a portion thereof serves as a template for synthesizing a second RNA chain complementary to the first RNA chain or a portion thereof. The second RNA chain or a portion thereof may then optionally serve as a template for synthesizing a third RNA chain complementary to the second RNA chain or a portion thereof. Therefore, the third RNA chain is identical to the first RNA chain or a portion thereof. Therefore, RNA-dependent RNA polymerase is capable of directly synthesizing the complementary RNA chain of the template, and is capable of indirectly synthesizing the same RNA chain (by a complementary intermediate chain).

[0140] According to the present invention, the term "template RNA" refers to RNA that can be transcribed or replicated by an RNA-dependent RNA polymerase.

[0141] According to the present invention, the term "gene" refers to a specific nucleic acid sequence responsible for producing one or more cell products and / or achieving one or more intercellular or intracellular functions. More specifically, the term relates to a nucleic acid portion (usually DNA; but RNA in the case of RNA viruses) comprising a nucleic acid encoding a specific protein or a functional or structural RNA molecule.

[0142] As used herein, "isolated molecules" are intended to refer to molecules that are substantially free of other molecules (e.g., other cellular materials). According to the present invention, the term "isolated nucleic acid" means that the nucleic acid has been (i) amplified in vitro, such as by polymerase chain reaction (PCR), (ii) produced by cloning recombination, (iii) purified, such as by cutting and gel electrophoresis fractionation, or (iv) synthesized, such as by chemical synthesis. An isolated nucleic acid is a nucleic acid that can be used for manipulation by recombinant techniques.

[0143] The term "vector" is used herein in its most general sense and includes any intermediate carrier for a nucleic acid, which is capable, for example, of introducing the nucleic acid into a prokaryotic and / or eukaryotic host cell and, where appropriate, of integrating into the genome. Such vectors are preferably replicated and / or expressed in the cell. Vectors include plasmids, phagemids, viral genomes, and fractions thereof.

[0144] In the context of the present invention, the term "recombinant" means "produced by genetic modification." Preferably, in the context of the present invention, a "recombinant subject" (eg, a recombinant cell) does not occur naturally.

[0145] As used herein, the term "naturally occurring" refers to the fact that an object can exist in nature. For example, a peptide or nucleic acid that exists in an organism (including a virus) and can be isolated from a natural source and has not been intentionally artificially modified in a laboratory is naturally occurring. The term "existing in nature" means "existing in nature" and includes known objects as well as objects that have not yet been discovered and / or isolated from nature but may be discovered and / or isolated from natural sources in the future.

[0146] According to the present invention, the term "expression" is used in its most general sense and includes the production of RNA and / or protein. It also includes partial expression of nucleic acids. In addition, expression can be transient or stable. For RNA, the term "expression" or "translation" refers to the process in the ribosomes of the cell by which a coding RNA (e.g., messenger RNA) chain directs the assembly of an amino acid sequence to produce a peptide or protein.

[0147] According to the present invention, the term "mRNA" means "messenger RNA" and refers to a transcript that is usually produced by using a DNA template and encodes a peptide or protein. Generally speaking, mRNA contains a 5'-UTR, a protein coding region, a 3'-UTR and a poly (A) sequence. mRNA can be produced from a DNA template by in vitro transcription. In vitro transcription methods are known to the skilled person. For example, there are many commercially available in vitro transcription kits. According to the present invention, mRNA can be modified by stabilization modification and capping.

[0148] According to the present invention, the term "poly (A) sequence" or "poly (A) tail" refers to an uninterrupted or interrupted sequence of adenylic acid residues usually located at the 3' end of an RNA molecule. An uninterrupted sequence is characterized by consecutive adenylic acid residues. In fact, an uninterrupted poly (A) sequence is typical. Although poly (A) sequences are not usually encoded in eukaryotic DNA, but are attached to the free 3' end of RNA by a template-independent RNA polymerase after transcription during eukaryotic transcription in the nucleus, the present invention covers poly (A) sequences encoded by DNA.

[0149] According to the present invention, the term "primary structure" with respect to a nucleic acid molecule refers to the linear sequence of nucleotide monomers.

[0150] According to the present invention, the term "secondary structure" with respect to nucleic acid molecules refers to a two-dimensional representation of nucleic acid molecules that reflects base pairing; for example, in the case of single-stranded RNA molecules, in particular intramolecular base pairing. Although each RNA molecule has only one polynucleotide chain, the molecule is generally characterized by an (intramolecular) base pair region. According to the present invention, the term "secondary structure" includes structural motifs, which include but are not limited to base pairs, stems, stem loops, protrusions, loops (e.g., internal loops and multi-branched loops). The secondary structure of a nucleic acid molecule can be represented by a two-dimensional graph (plan view) showing base pairing (for more detailed information on the secondary structure of RNA molecules, see Auber ct al., 2006; J. Graph Algorithms App1.10: 329-351). As described herein, the secondary structure of certain RNA molecules is relevant in the context of the present invention.

[0151] According to the present invention, the secondary structure of a nucleic acid molecule, in particular 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, the "secondary structure" of a nucleic acid molecule refers in particular to the secondary structure determined by the prediction. The prediction can also be performed or confirmed using MFOLD structure prediction (http: / / unafold.rna.albany.edu / ?q=mfold).

[0152] 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 through hydrogen bonds between donor and acceptor sites on the bases. Complementary bases A:U and G:C form stable base pairs through hydrogen bonds between donor and acceptor sites on the bases; A:U 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 (frequently found in RNA) and other rare base pairs (e.g. A:C; U:U) are called non-canonical base pairs.

[0153] According to the present invention, "nucleotide pairing" refers to two nucleotides that associate with each other so that their bases form a base pair (canonical or non-canonical base pair, preferably a canonical base pair, most preferably a Watson-Crick base pair).

[0154] According to the present invention, the terms "stem-loop" or "hairpin" or "hairpin loop" with respect to nucleic acid molecules all interchangeably refer to a specific secondary structure of a nucleic acid molecule (typically a single-stranded nucleic acid molecule, such as a single-stranded RNA). The specific secondary structure represented by the stem-loop consists of a continuous nucleic acid sequence comprising a stem and a (terminal) loop (also referred to as a hairpin loop), wherein the stem is formed by two adjacent fully or partially complementary sequence elements, which are separated by a short sequence (e.g. 3 to 10 nucleotides) forming a loop of the stem-loop structure. Two adjacent fully or partially complementary sequences can be defined as, for example, stem-loop elements stem 1 and stem 2. When these two adjacent fully or partially reverse complementary sequences (e.g. stem-loop elements stem 1 and stem 2) form base pairs with each other, a stem-loop is formed, which results in a double-stranded nucleic acid sequence comprising an unpaired loop at its terminal end formed by a short sequence between the stem-loop elements stem 1 and stem 2. Therefore, the stem loop comprises two stems (stem 1 and stem 2), which form base pairs with each other at the level of the secondary structure of the nucleic acid molecule and are separated by a short sequence that is not part of stem 1 or stem 2 at the level of the primary structure of the nucleic acid molecule. For illustration, the two-dimensional representation of the stem loop is similar to a lollipop-shaped structure. The formation of the stem loop structure requires the presence of a sequence that can be folded back to 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 usually determined by the length, the number of nucleotides of stem 1 that can form base pairs (preferably canonical base pairs, more preferably Watson-Crick base pairs) with the nucleotides of stem 2 compared to the number of nucleotides of stem 1 that cannot form such base pairs (mismatches or protrusions) with the nucleotides of stem 2. 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, the corresponding complementary nucleic acid sequence is also usually characterized by a stem loop. Stem-loops are usually formed by single-stranded RNA molecules. For example, there are several stem-loops in the 5' replication recognition sequence of the alphavirus genomic RNA.

[0155] According to the present invention, "destruction" or its variation on a specific secondary structure (e.g., stem loop) of a nucleic acid molecule means that the specific secondary structure does not exist or is changed. Generally speaking, the secondary structure can be destroyed due to the change of at least one nucleotide of a part of the secondary structure. For example, the stem loop can be destroyed by changing one or more nucleotides forming the stem so that nucleotide pairing cannot be performed.

[0156] According to the present invention, "compensation for secondary structure destruction" or its variations refers to one or more nucleotide changes in a nucleic acid sequence; more generally refers to one or more second nucleotide changes in a nucleic acid sequence, the nucleic acid sequence also comprising one or more first nucleotide changes, characterized in that: although one or more first nucleotide changes cause nucleic acid secondary structure destruction in the absence of one or more second nucleotide changes, the coexistence of one or more first nucleotide changes and one or more second nucleotide changes does not cause nucleic acid secondary structure destruction. Coexistence means the presence of both one or more first nucleotide changes and one or more second nucleotide changes. Generally speaking, one or more first nucleotide changes and one or more second nucleotide changes are present together in the same nucleic acid molecule. In a specific embodiment, the one or more nucleotide changes that compensate for secondary structure destruction are one or more nucleotide changes that compensate for one or more nucleotide pairing destruction. Therefore, in one embodiment, "compensation for secondary structure destruction" means "compensation for nucleotide pairing destruction", i.e., one or more nucleotide pairing destruction, such as one or more nucleotide pairing destruction in one or more stem loops. One or more one or more nucleotide pairing destructions can be introduced by removing at least one start codon. Each in one or more nucleotide changes that compensate for secondary structure destruction is such nucleotide change, and it can be selected from the disappearance, interpolation, replacement and / or insertion of one or more nucleotide independently of one another.In an illustrative example, when nucleotide pairing A:U is destroyed by the replacement of A to C (C and U are usually not suitable for forming nucleotide pair), then the nucleotide change that compensates for nucleotide pairing destruction can be that U is replaced by G, thereby can form C:G nucleotide pairing.Therefore U is replaced by G and compensates for nucleotide pairing destruction.In an alternative example, when nucleotide pairing A:U is destroyed by the replacement of A to C, then the nucleotide change that compensates for nucleotide pairing destruction can be that C is replaced by A, thereby recovers the formation of original A:U nucleotide pairing.Generally speaking, in the present invention, those nucleotide changes that compensate for secondary structure destruction preferably neither recover the original nucleic acid sequence, nor produce new AUG triplet.In the above example concentration, relative to the replacement of C to A, the replacement of U to G is preferred.

[0157] According to the present invention, the term "tertiary structure" with respect to a nucleic acid molecule refers to the three-dimensional structure of a nucleic acid molecule defined by atomic coordinates.

[0158] According to the present invention, nucleic acid (such as RNA, such as rRNA) can encode a peptide or protein. Therefore, the transcribable nucleic acid sequence or its transcript can contain an open reading frame (ORF) encoding a peptide or protein.

[0159] According to the present invention, the term "nucleic acid encoding a peptide or protein" means a nucleic acid that, if present in a suitable environment, preferably within a cell, can direct the assembly of amino acids during the translation process to produce a peptide or protein. Preferably, the coding RNA according to the present invention is capable of interacting with the cellular translation machinery, thereby allowing the translation of the coding RNA to produce a peptide or protein.

[0160] According to the present invention, the term "peptide" includes oligopeptides and polypeptides, and refers to a substance comprising two or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 10 or more, preferably 13 or more, preferably 16 or more, preferably 20 or more, and up to preferably 50, preferably 100 or preferably 150 consecutive amino acids linked to each other by peptide bonds. The term "protein" refers to a large peptide, preferably a peptide with at least 151 amino acids, but the terms "peptide" and "protein" are often used as synonyms herein.

[0161] 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 sugar and phosphate structures), and also include substances containing bonds (such as ester, thioether or disulfide bonds).

[0162] According to the present invention, the terms "initiation codon" and "start codon" refer synonymously to the codons (base triplet) of RNA molecules, which are the potential first codons translated by ribosomes. Such codons usually encode the amino acid methionine in eukaryotes and encode modified methionine in prokaryotes. The most common start codon in eukaryotes and prokaryotes is AUG. Unless specifically indicated herein to refer to a start codon other than AUG, the terms "start codon" and "start codon" for RNA molecules refer to the codon AUG. According to the present invention, the terms "start codon" and "start codon" are also used to refer to the corresponding base triplet of deoxyribonucleic acid, i.e., the base triplet of the start codon encoding RNA. If the start codon of messenger RNA is AUG, the base triplet encoding AUG is ATG. According to the present invention, the terms "start codon" and "start codon" preferably refer to a functional start codon, i.e., a start codon that is used or will be used by the ribosome as a codon for starting translation. AUG codons may be present in the RNA molecule that are not used by the ribosome as a codon for starting translation, for example due to the short distance between the codon and the cap. The term functional start codon does not include these codons.

[0163] According to the present invention, the terms "start codon of the open reading frame" and "initiation codon of the open reading frame" refer to the base triplet used as the start codon for protein synthesis in a coding sequence (e.g., a coding sequence of a nucleic acid molecule occurring in nature). In RNA molecules, the start codon of the open reading frame is usually preceded by a 5' untranslated region (5'-UTR), although this is not a strict requirement.

[0164] According to the present invention, the terms "native start codon of the open reading frame" and "native initiation codon of the open reading frame" refer to the base triplet used as the start codon for protein synthesis in a native coding sequence. A native coding sequence can be, for example, a coding sequence of a nucleic acid molecule existing in nature. In some embodiments, the present invention provides variants of nucleic acid molecules existing in nature, characterized in that the native start codon (present in the native coding sequence) has been removed (so that it is not present in the variant nucleic acid molecule).

[0165] According to the present invention, "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 used or to be used by the ribosome as a codon to start translation. Accordingly, "first ATG" refers to the ATG base triplet of a coding DNA sequence encoding the first AUG. In some cases, the first AUG of an mRNA molecule is the start codon of an open reading frame, i.e., a codon used as a start codon during ribosomal protein synthesis.

[0166] According to the present invention, the term "comprising the removal ..." or "characterized by the removal ..." and similar terms with respect to an element of a nucleic acid variant means that the element is not functional or does not exist in the nucleic acid variant compared to a reference nucleic acid molecule. Without limitation, the removal may consist of the deletion of all or part of an element, the replacement of all or part of an element, or the change of the functional or structural properties of an element. The removal of a functional element of a nucleic acid sequence requires that the nucleic acid variant comprising the removal does not exhibit a function at that position. For example, an RNA variant characterized by the removal of a start codon requires that the RNA variant characterized by the removal does not initiate ribosomal protein synthesis at that position. The removal of a structural element of a nucleic acid sequence requires that the nucleic acid variant comprising the removal does not have the structural element at that position. For example, the RNA variant characterized by removing a certain AUG base triplet (i.e., an AUG base triplet at a certain position) may be characterized by, for example, a partial or complete deletion of the certain AUG base triplet (e.g., ΔAUG), or one or more nucleotides (A, U, G) of the certain AUG base triplet are replaced by any one or more different nucleotides, so that the nucleotide sequence of the resulting variant does not include the AUG base triplet. A suitable replacement of a nucleotide is to convert the AUG base triplet into a GUG, CUG or UUG base triplet, or into an AAG, ACG or AGG base triplet, or into those of an AUA, AUC or AUU base triplet. Suitable replacements of more nucleotides may be selected accordingly.

[0167] According to the present invention, the term "self-replicating virus" includes RNA viruses that can replicate autonomously in host cells. Self-replicating viruses may have a single-stranded RNA (ssRNA) genome, and include alphaviruses, flaviviruses, measles viruses (MV) and rhabdoviruses. Alphaviruses and flaviviruses have positive polarity genomes, while the genomes of measles viruses (MV) and rhabdoviruses are negative strand ssRNA. Generally speaking, self-replicating viruses are viruses with (+) strand RNA genomes that are directly translated after infecting cells, and this translation provides RNA-dependent RNA polymerases, which then produce both antisense and sense transcripts from the infected RNA. In the following, the present invention is illustrated by reference to an alphavirus-derived vector as an example of a vector derived from a self-replicating virus. However, it should be understood that the present invention is not limited to alphavirus-derived vectors.

[0168] According to the present invention, the term "alphavirus" should be understood broadly and includes any virus particles having alphavirus characteristics. The characteristics of alphaviruses include the presence of (+) strand RNA, which encodes genetic information suitable for replication in host cells, including RNA polymerase activity. Many additional features of alphaviruses are described in Strauss & Strauss, 1994, Microbiol. Rev. 58: 491-562. The term "alphavirus" includes alphaviruses existing in nature and any variants or derivatives thereof. In some embodiments, variants or derivatives do not exist in nature.

[0169] In one embodiment, the alphavirus is an alphavirus found in nature. Generally speaking, an alphavirus found in nature can infect any one or more eukaryotic organisms, such as animals, including vertebrates (such as humans) and arthropods (such as insects). The alphaviruses occurring in nature are preferably selected from the following: Bama Forest virus complex (comprising Bama Forest virus); Eastern equine encephalitis complex (comprising seven antigenic types of Eastern equine encephalitis viruses); Middelburg virus complex (comprising Middelburg virus); Ndumu virus complex (comprising Ndumu virus); Semliki Forest virus complex (comprising Bebaru virus, Chikungunya virus, Mayaro virus and its subtypes Una virus, O'Nyong Nyongvirus and its subtypes Igbo-Ora virus, Ross River virus and its subtypes Bebaru virus, Getah virus, Sagiyama virus, Semliki Forest virus and its subtypes Me Tri virus); Venezuelan equine encephalitis complex (comprising Cabassou virus, Everglades virus, virus), Mosso das Pedras virus, Mucambo virus, Paramana virus, Pixuna virus, Rio Negro virus, Trocaravirus and its subtypes, Bijou Bridge virus, Venezuelan equine encephalitis virus); Western equine encephalitis complex (including Aura virus, Babanki virus, Kyzylagach virus, Sindbis virus, Ockelbo virus, Whataroa virus, Buggy Creek virus, Fort Morgan virus, Highlands J virus, Western equine encephalitis virus);And some unclassified viruses, including salmon pancreatic disease virus; Sleeping Disease virus; Southern elephant seal virus; Tonate virus. More preferably, the alphavirus is selected from the following: Semliki Forest virus complex (including the virus types as described above, including Semliki Forest virus), Western equine encephalitis complex (including the virus types as described above, including Sindbis virus), Eastern equine encephalitis virus (including the virus types as described above), Venezuelan equine encephalitis complex (including the virus types as described above, including Venezuelan equine encephalitis virus). ;

[0170] In another preferred embodiment, the alphavirus is Semliki Forest virus.In another preferred embodiment, the alphavirus is Sindbis virus.In another alternative preferred embodiment, the alphavirus is Venezuelan equine encephalitis virus.

[0171] In some embodiments of the present invention, the alphavirus is not an alphavirus that exists in nature. Generally speaking, an alphavirus that does not exist in nature is a variant or derivative of an alphavirus that exists in nature, which is distinguished from an alphavirus that exists in nature by at least one mutation in the nucleotide sequence (i.e., genomic RNA). Compared with the alphavirus that exists in nature, the mutation in the nucleotide sequence can be selected from the insertion, substitution or deletion of one or more nucleotides. The mutation in the nucleotide sequence may be associated or unrelated to the mutation in the polypeptide or protein encoded by the nucleotide sequence. For example, the alphavirus that does not exist in nature may be an attenuated alphavirus. An attenuated alphavirus that does not exist in nature is an alphavirus that generally has at least one mutation in its nucleotide sequence, by which it is distinguished from an alphavirus that exists in nature, and it is not completely infective, or it is infective but has a lower disease-producing ability or is completely infective. As an illustrative example, TC83 is an attenuated alphavirus that is distinct from the naturally occurring Venezuelan equine encephalitis virus (VEEV) (McKinney et al., 1963, Am. J. Trop. Med. Hyg. 12:597-603).

[0172] Members of the genus Alphavirus can also be classified according to their relative clinical characteristics in humans: alphaviruses primarily associated with encephalitis and alphaviruses primarily associated with fever, rash, and polyarthritis.

[0173] The term "alphaviral" means present in, derived from, or derived from an alphavirus (eg, by genetic modification) an alphavirus.

[0174] According to the present invention, "SFV" stands for Semliki Forest virus. According to the present invention, "SIN" or "SINV" stands for Sindbis virus. According to the present invention, "VEE" or "VEEV" stands for Venezuelan equine encephalitis virus.

[0175] According to the present invention, the term "alphavirus" refers to an entity derived from an alphavirus. For illustration, an alphavirus protein may refer to a protein present in an alphavirus and / or a protein encoded by an alphavirus; and an alphavirus nucleic acid sequence may refer to a nucleic acid sequence present in an alphavirus and / or a nucleic acid sequence encoded by an alphavirus. Preferably, an "alphavirus" nucleic acid sequence refers to a nucleic acid sequence "of the genome of an alphavirus" and / or "of the genomic RNA of an alphavirus".

[0176] According to the present invention, the term "alphavirus RNA" refers to any one or more of the following: alphavirus genomic RNA (i.e., the (+) strand), the complementary strand of the alphavirus genomic RNA (i.e., the (-) strand), and subgenomic transcripts (i.e., the (+) strand) or any fragment thereof.

[0177] According to the present invention, "alphavirus genome" refers to the genomic (+) strand RNA of an alphavirus.

[0178] According to the present invention, the term "natural alphavirus sequence" and similar terms generally refer to the (e.g., nucleic acid) sequence of a naturally occurring alphavirus (an alphavirus occurring in nature). In some embodiments, the term "natural alphavirus sequence" also includes the sequence of an attenuated alphavirus.

[0179] 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 self-replicating virus genome (e.g., the alphavirus genome). "5' replication recognition sequence" is a nucleic acid sequence that can be recognized by a replicase (e.g., an alphavirus replicase). The term 5' replication recognition sequence includes natural 5' replication recognition sequences and functional equivalents thereof, for example, functional variants of the 5' replication recognition sequence of a self-replicating virus (e.g., an alphavirus) existing 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 as described herein. The 5' replication recognition sequence is necessary for the synthesis of the (-) strand complementary strand of the alphavirus genomic RNA and is necessary for the synthesis of the (+) strand viral genomic RNA based on the (-) strand template. The natural 5' replication recognition sequence usually encodes at least the N-terminal fragment of nsP1; but does not include the entire open reading frame encoding nsP1234. Taking into account the fact that the natural 5' replication recognition sequence usually encodes at least the N-terminal fragment of nsP1, the natural 5' replication recognition sequence usually comprises at least one start codon, usually AUG. In one embodiment, the 5' replication recognition sequence comprises conserved sequence element 1 (CSE 1) of the alphavirus genome or a variant thereof and conserved sequence element 2 (CSE 2) of the alphavirus genome or a variant thereof. The 5' replication recognition sequence is usually able to form four stem loops (SL), namely SL1, SL2, SL3, and SL4. The numbering of these stem loops starts from the 5' end of the 5' replication recognition sequence.

[0180] The term "conserved sequence element" or "CSE" refers to a nucleotide sequence present in alphavirus RNA. These sequence elements are called "conserved" because there are orthologs in the genomes of different alphaviruses, and orthologous CSEs of different alphaviruses preferably have a high percentage of sequence identity and / or similar secondary or tertiary structure. The term CSE includes CSE1, CSE 2, CSE 3 and CSE 4.

[0181] According to the present invention, the term "CSE 1" or "44-nt CSE" refers synonymously to the nucleotide sequence required for synthesizing the (+) strand from the (-) strand template. The term "CSE 1" refers to the sequence on the (+) strand; and the complementary sequence of CSE 1 (on the (-) strand) serves as a promoter for (+) strand synthesis. Preferably, the term CSE 1 includes the 5'-most nucleotides of the alphavirus genome. CSE 1 generally forms a conserved stem-loop structure. Without wishing to be bound by a particular theory, it is believed that for CSE 1, the secondary structure is more important than the primary structure (i.e., the linear sequence). In the genomic RNA of the model alphavirus Sindbis virus, CSE 1 consists of a continuous sequence of 44 nucleotides, which is formed by the 5'-most 44 nucleotides of the genomic RNA (Strauss & Strauss, 1994, Microbiol. Rev. 58: 491-562).

[0182] According to the present invention, the term "CSE 2" or "51-nt CSE" refers synonymously to the nucleotide sequence required for synthesizing the (-) strand from the (+) strand template. The (+) strand template is typically an alphavirus genomic RNA or RNA replicon (it should be noted that subgenomic RNA transcripts that do not contain CSE 2 are not used as templates for (-) strand synthesis). In the alphavirus genomic RNA, 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 to 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 named stem-loop 3 (SL3) and stem-loop 4 (SL4) because they are the third and fourth conserved stem-loops of the alphavirus genomic RNA, respectively, counting from the 5' end of the alphavirus genomic RNA. Without wishing to be bound by a particular theory, it is believed that for CSE 2, the secondary structure is more important than the primary structure (ie, linear sequence).

[0183] According to the present invention, the term "CSE 3" or "junction sequence" refers synonymously to a nucleotide sequence derived from the alphavirus genomic RNA and comprising the start site of the subgenomic RNA. The complementary sequence of this sequence in the (-) strand functions to promote transcription of the subgenomic RNA. In the alphavirus genomic RNA, CSE 3 generally overlaps with the region encoding the C-terminal fragment of nsP4 and extends to a short non-coding region located upstream of the open reading frame encoding the structural proteins.

[0184] According to the present invention, the term "CSE 4" or "19-nt conserved sequence" or "19-nt CSE" refers synonymously to a nucleotide sequence from an alphavirus genomic RNA that is immediately upstream of the poly(A) sequence in the 3' untranslated region of the alphavirus genome. CSE 4 is generally composed of 19 consecutive nucleotides. Without wishing to be bound by a particular theory, CSE 4 is understood to act as a core promoter that initiates (-) strand synthesis (José et al., 2009, Future Microbiol. 4: 837-856); and / or CSE 4 and the poly(A) tail of the alphavirus genomic RNA are understood to act together to efficiently perform (-) strand synthesis (Hardy & Rice, 2005, J. Virol. 79: 4630-4639).

[0185] According to the present invention, the term "subgenomic promoter" or "SGP" refers to a nucleic acid sequence upstream (5') of a nucleic acid sequence (e.g., a coding sequence) that controls the transcription of the nucleic acid sequence by providing recognition and structural sites for RNA polymerases, typically RNA-dependent RNA polymerases, particularly functional alphavirus nonstructural proteins. The SGP may contain additional recognition or binding sites for additional factors. Subgenomic promoters are typically genetic elements of positive-strand RNA viruses (e.g., alphaviruses). The subgenomic promoter of an alphavirus is a nucleic acid sequence contained in the viral genomic RNA. The general feature of a subgenomic promoter is that it allows transcription (RNA synthesis) to begin in the presence of an RNA-dependent RNA polymerase (e.g., a functional alphavirus nonstructural protein). The RNA (-) strand (i.e., the complementary strand of the alphavirus genomic RNA) serves as a template for synthesizing (+) strand subgenomic transcripts, and the synthesis of (+) strand subgenomic transcripts typically begins at or near the subgenomic promoter. The term "subgenomic promoter" as used herein is not limited to any specific positioning in a nucleic acid comprising such a subgenomic promoter. In some embodiments, the SGP is the same as or overlaps with or comprises CSE 3.

[0186] The term "subgenomic transcript" or "subgenomic RNA" refers synonymously to an RNA molecule obtainable by transcription using an RNA molecule as a template ("template RNA"), wherein the template RNA comprises a subgenomic promoter that controls the transcription of the subgenomic transcript. The subgenomic transcript can be obtained in the presence of an RNA-dependent RNA polymerase, in particular a functional alphavirus non-structural protein. For example, the term "subgenomic transcript" may refer to an RNA transcript prepared in an alphavirus-infected cell using the (-) strand complementary strand of the alphavirus genomic RNA as a template. However, the term "subgenomic transcript" as used herein is not limited thereto, and also includes transcripts obtainable by using heterologous RNA as a template. For example, subgenomic transcripts can also be obtained by using the (-) strand complementary strand of the replicon containing SGP according to the present invention as a template. Therefore, the term "subgenomic transcript" may refer to an RNA molecule obtainable by transcribing a fragment of the alphavirus genomic RNA, as well as an RNA molecule obtainable by transcribing a fragment of the replicon according to the present invention.

[0187] The term "autologous" is used to describe anything that originates from the same subject. For example, "autologous cells" refer to cells that originate from the same subject. Introducing autologous cells into a subject is advantageous because these cells overcome immunological barriers that would otherwise lead to rejection.

[0188] The term "allogeneic" is used to describe anything that originates from different individuals of the same species. Two or more individuals are considered allogeneic when the genes at one or more loci are not identical.

[0189] The term "isogenic" is used to describe anything derived from individuals or tissues having the same genotype (ie, identical twins or the same inbred strain of animals, or tissues or cells thereof).

[0190] The term "heterologous" is used to describe something that is composed of multiple different elements. As an example, introducing cells from one individual into a different individual constitutes a heterologous transplant. Heterologous genes are genes that originate from a source other than the subject.

[0191] The cell that can be used in the method for identifying sequence changes is any suitable cell in which a replicable RNA with or without any nucleotide modification can be replicated and / or translated. The cell can be a mammalian cell, such as a human cell. The cell can constitutively express a replicase that recognizes a sequence present in the replicable RNA for replication, or can transiently express such a replicase.

[0192] Specific and / or preferred variations of the various features of the invention are provided below. The invention also contemplates those embodiments produced by combining two or more specific and / or preferred variations described for two or more features of the invention as some particularly preferred embodiments.

[0193] RNA replicon

[0194] A replicable RNA (rRNA) molecule is an RNA that can be replicated by an RNA-dependent RNA polymerase (replicase), which allows the RNA to be replicated by virtue of containing a nucleotide sequence that can be recognized by the replicase. The rRNA does not necessarily encode a replicase, so that the rRNA can be replicated in cis (by an encoded replicase) or in trans (by a replicase provided in another way, such as a nucleic acid encoding a separate replicase, such as mRNA). The terms "RNA replicon", "replicon" and "replicable RNA molecule" are used interchangeably.

[0195] In one embodiment, the replicable RNA (rRNA) molecule comprises a modified regulatory region of a self-replicating single-stranded positive-sense virus, comprising sequence changes compared to a reference modified regulatory region, which restore or improve the function of the rRNA molecule comprising 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 5' regulatory region of a VEEV alphavirus.

[0196] In one embodiment, the RNA replicon may comprise an internal ribosome entry site (IRES) and an open reading frame encoding a functional nonstructural protein from a self-replicating virus, wherein the IRES controls the expression of the functional nonstructural protein, such as a replicase. Preferably, the RNA replicon comprises sequence elements that allow replication by the functional nonstructural protein. In one embodiment, the self-replicating virus is an alphavirus, and the sequence elements that allow replication by the functional nonstructural protein are derived from the alphavirus.

[0197] Alphavirus replicase has a capping enzyme function, and generally speaking, genome and subgenomic (+) strand RNA are capped. 5' cap is used to protect mRNA from degradation, and ribosomal subunits and cytokines are directed to mRNA to form a ribonucleoprotein complex on mRNA, and then translation can be started from the nearby start codon. This complex process is widely described in the literature (Jackson et al., 2010, Nat Rev Mol Biol; Vol 10: 113-127). Although the mechanism of cap-dependent translation is very complex and effective, cells have a way of initiating translation that is completely or partially independent of 5' caps (Thompson 2012; Trends in Microbiology 20: 558-566). Therefore, in the case of cell stress that causes the overall downregulation of cap-dependent translation, cells can still preferentially express selected genes, usually with the help of IRES.

[0198] Viruses have also evolved different ways to exploit cellular machinery to translate viral genes. Since viral infection is often sensed by cells, leading to cellular antiviral responses (interferon responses; stress responses), many viruses also utilize cap-independent translation, especially RNA viruses. Cap-independent translation ensures the advantage of viral RNA translation during cellular stress responses, giving the virus a chance to complete its life cycle and be released from infected cells.

[0199] Internal ribosome entry site (IRES) is an RNA sequence that forms a suitable secondary structure, which attracts a pre-initiation complex near a translation start codon (AUG or other). Four types of IRES with common characteristics are described in the literature. Prototype IRES is poliovirus IRES (type I), encephalomyocarditis virus (EMCV) IRES (type II), hepatitis C virus (hepathitis C virus, HCV) IRES (type III) and IRES (type IV) present in the intergenic region of dicistroviruses (dicistroviruses) (Thompson, 2012; Trends in Microbiology 20: 558-566; Lozano et al., 2018; Open Biology 8: 180155).

[0200] The commonality of type I to type III IRES is that they initiate translation at the AUG start codon, while type IV IRES initiates at a non-AUG codon (e.g., GCU). Therefore, type I to type III require an initiator tRNA that delivers methionine (eIF2 / GTP / Met-tRNAiMet) with the help of eIF2 / GTP. Under stress, activation of the eIF2 kinase phosphorylates the α subunit of eIF2, inhibiting translation initiated at AUG. Therefore, translation directed by type IV IRES is not inhibited by eIF2 phosphorylation.

[0201] The term "internal ribosome entry site", abbreviated as "IRES", refers to an RNA element that recruits ribosomes to the internal region of mRNA to start translation in a cap-independent manner. IRES is usually located in the 5'-UTR of RNA viruses. However, the mRNA of viruses from the dicistroviridae family has two open reading frames (ORFs), and the translation of each is guided by two different IRESs. It has also been shown that some mammalian cell mRNAs also have IRES. These cellular IRES elements are believed to be located in eukaryotic mRNAs encoding genes that are involved in stress survival and other processes that are critical for survival. The location of the IRES element is usually in the 5'-UTR, but it can also appear at other positions in the mRNA.

[0202] The term "internal ribosome entry site" includes IRES 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. Although these viral IRESs contain different sequences, many of them have similar secondary structures and initiate translation by similar mechanisms. In addition, the activity of IRESs generally requires the help of other factors called IRES-transacting factors (ITAFs). Based on the structure and requirements of translation initiation factors (IFs) and ITAFs, viral IRESs are divided into four types, as described herein. Any of these IRES types are useful according to the present invention, with type IV IRES being particularly preferred.

[0203] Two groups of viral IRES, type I and type II, cannot bind directly to the 40S small ribosomal subunit. Instead, it recruits the 40S small ribosomal subunit through different ITAFs and requires typical IFs (i.e., eIF2, eIF3, eIF4A, eIF4B, and eIF4G) in cap-dependent translation. The main difference between type I and type II IRES is that 40S ribosome scanning is required, where 40S ribosome scanning is unnecessary for type II IRES. Some examples of type I IRES include IRES present in poliovirus (PV) and rhinovirus. Some examples of type II IRES include IRES present in encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), and Theiler's murineencephalomyelitis virus (TMEV).

[0204] Type III IRES can directly interact with the 40S small ribosomal subunit with a specialized RNA structure, but its activity usually requires the assistance of several IFs, including eIF2 and eIF3 and the initiator Met-tRNAi. Some examples include IRES present in hepatitis C virus (HCV), classical swine fever virus (CSFV) and porcine teschovirus (PTV).

[0205] Type IV viral IRESs are generally highly active and can initiate translation from non-AUG start codons in the absence of additional ITAFs or even eIF2 / Met-tRNAi / GTP ternary complexes. These IRESs are folded into compact structures that interact directly with the 40S small ribosomal subunit. Some examples include IRESs present in dicistroviruses such as cricket paralysis virus (CrPV), soybean looper virus (PSIV) and Taura syndrome virus (TSV).

[0206] The term "internal ribosome entry site", also includes IRES present in cellular mRNAs, many of which encode proteins required in stress responses such as apoptosis, mitosis, hypoxia and nutrient limitation. Based on the mechanism of ribosome recruitment, cellular IRES can be roughly divided into two types: type I IRES interacts with ribosomes through ITAFs such as RNA binding motifs and N-6-methyladenosine (m6A) modifications on cis elements, while type II IRES contains short cis elements that pair with 18S rRNA to recruit ribosomes.

[0207] In one embodiment, the rRNA described herein may have modified nucleotides / nucleosides / backbone modifications. The term "RNA modification" as used herein may refer to chemical modifications including backbone modifications as well as sugar modifications or base modifications.

[0208] In the present context, the modified rRNA molecule as defined herein may comprise nucleotide analogs / modifications, such as backbone modifications, sugar modifications or base modifications. The backbone modification associated with the present invention is a modification in which the phosphate of the nucleotide backbone included in the rRNA molecule as defined herein is chemically modified. The sugar modification associated with the present invention is a chemical modification of the nucleotide sugar of the rRNA molecule as defined herein. In addition, the base modification associated with the present invention is a chemical modification of the nucleotide base portion of the rRNA molecule. In the present context, nucleotide analogs or modifications are preferably selected from nucleotide analogs suitable for transcription and / or translation.

[0209] Sugar modifications: Modified nucleosides and nucleotides can be modified in the sugar moiety, which can be incorporated into modified rRNA molecules as described herein. For example, the 2' hydroxyl (OH) can be modified or replaced with many different "oxy" or "deoxy" substituents. Some examples of "oxy"-2' hydroxyl modifications include, but are not limited to, alkoxy or aryloxy (-OR, such as R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethylene glycol (PEG), -0 (CH2CH2O) nCH2CH2 OR; "locked" nucleic acid (LNA), in which the 2' hydroxyl group is connected to the 4' carbon of the same ribose sugar, for example, by a methylene bridge; and amino (-O-amino, where the amino group, for example, NRR, can be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxy. "Deoxy" modifications include hydrogen, amino (for example, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino or amino acid); or the amino group can be connected to the sugar through a linker, wherein the linker comprises one or more atoms C, N and O. The sugar group can also contain one or more carbons having a stereochemical configuration opposite to the corresponding carbon in ribose. Thus, the modified RNA molecule can contain nucleotides containing, for example, arabinose as a sugar.

[0210] Backbone modification: The phosphate backbone can be further modified in modified nucleosides and nucleotides, which can be incorporated into modified RNA molecules as described herein. The phosphate group of the backbone can be modified by replacing one or more oxygen atoms with different substituents. In addition, modified nucleosides and nucleotides can include completely replacing the unmodified phosphate moiety with modified phosphates as described herein. Some examples of modified phosphate groups include but are not limited to phosphorothioates, selenophosphates, borane phosphates (borano phosphate), boron phosphate esters (borano phosphate esters), hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates and phosphotriesters. Phosphorodithioates have two non-connected oxygens replaced by sulfur. Phosphate joints can also be modified by replacing connected oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates) and carbon (bridged methylene phosphonates).

[0211] Base modification: modified nucleosides and nucleotides can also be modified in the nucleobase portion, which can be incorporated into modified rRNA molecules as described herein. Some examples of nucleobases present 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 surface of the major groove. In some embodiments, the major groove chemical modification can include an amino group, a thiol group, an alkyl group, or a halogen group.

[0212] In a specific embodiment of the invention, the nucleotide analogs / modifications are selected from base modifications, which are preferably selected from 2-amino-6-chloropurine nucleoside-5'-triphosphate, 2-aminopurine-nucleoside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxy-cytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-0-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 nucleoside-5'-triphosphate, 7-deaza-adenosine-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, 06-methylguanosine-5'-triphosphate, N6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, or puromycin-5'-triphosphate, xanthosine-5'-triphosphate. The particularly preferred nucleotides considered for base modification are selected from the group of base-modified nucleotides composed of: 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate.In some embodiments, modified nucleosides include pyridin-4-one ribonucleosides, 5-aza-uridine, 2-thio-5-aza-uridine, 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-taurine methyl uridine, 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thiouridine, 1-taurine methyl- 4-thio-uridine, 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-thio-pseudouridine.

[0213] 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-1-methyl-pseudoisocytidine.

[0214] In other embodiments, the 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-isopentyl 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, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0215] In some embodiments, the nucleotides may be modified on the surface of the major groove and may include replacing the hydrogen on C-5 of uracil with a methyl or halogen group. In specific embodiments, the modified nucleoside is 5'-0-(1-phosphorothioate)-adenosine, 5'-0-(1-phosphorothioate)-cytidine, 5'-0-(1-phosphorothioate)-guanosine, 5'-0-(1-phosphorothioate)-uridine or 5'-0-(1-phosphorothioate)-pseudouridine.

[0216] In other embodiments, the modified rRNA may comprise a nucleoside modification selected from the group consisting of 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudoisocytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine, α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine (cytdine), 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudoisocytidine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine.

[0217] In certain preferred embodiments, the rRNA comprises a modified nucleoside replacing at least one (eg, each) uridine.

[0218] The term "uracil" as used herein describes one of the nucleobases that can be present in RNA nucleic acids. The structure of uracil is:

[0219]

[0220] The term "uridine" as used herein describes one of the nucleosides that can be present in RNA. The structure of uridine is:

[0221]

[0222] UTP (uridine 5'-triphosphate) has the following structure:

[0223]

[0224] Pseudo-UTP (pseudouridine 5'-triphosphate) has the following structure:

[0225]

[0226] "Pseudouridine" is an example of a modified nucleoside and is an isomer of uridine in which uracil is linked to the pentose ring by a carbon-carbon bond rather than a nitrogen-carbon glycosidic bond.

[0227] Another exemplary modified nucleoside is N1-methyl-pseudouridine (m1ψ), which has the following structure:

[0228]

[0229] N1-methyl-pseudoUTP has the following structure:

[0230]

[0231] Another exemplary modified nucleoside is 5-methyl-uridine (m5U), which has the following structure:

[0232]

[0233] In certain preferred embodiments, one or more uridines in the rRNA described herein are replaced by modified nucleosides. In some embodiments, the modified nucleosides are modified uridines.

[0234] In certain preferred embodiments, the RNA comprises a modified nucleoside replacing at least one uridine. In some embodiments, the RNA comprises a modified nucleoside replacing each uridine.

[0235] In certain preferred embodiments, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (ψ). In some embodiments, the modified nucleosides comprise N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleosides comprise 5-methyl-uridine (m5U). In some embodiments, the RNA may comprise more than one type of 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 nucleosides comprise pseudouridine (ψ) and N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleosides comprise 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).

[0236] In certain preferred embodiments, the modified nucleosides replacing one or more (eg, all) uridines in the rRNA may be one or more of the following: 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-hydroxyacetic acid (cmo 5U), uridine 5-hydroxyacetate (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 s 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurine methyl-uridine (τm 5 U), 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thio-uridine (τm5s2U), 1-taurine methyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl) pseudouridine (acp3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-arabino-uridine, 2'-F-uridine, 2'-OH-arabino-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0237] In one embodiment, rRNA comprises other modified nucleosides or comprises other modified nucleosides, such as those modified cytidines as described above. For example, in one embodiment, 5-methylcytidine is partially or completely replaced by cytidine in rRNA, preferably completely replaced. In one embodiment, rRNA comprises 5-methylcytidine and one or more selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In one embodiment, rRNA comprises 5-methylcytidine and N1-methyl-pseudouridine (m1ψ). In some embodiments, rRNA comprises 5-methylcytidine replacing each cytidine and N1-methyl-pseudouridine (m1ψ) replacing each uridine.

[0238] Functional nonstructural proteins

[0239] The term "nonstructural protein" relates to proteins encoded by the virus but not part of the viral particle. The term generally includes various enzymes and transcription factors used by the virus for its own replication, such as RNA replicase or other template-directed polymerases. The term "nonstructural protein" includes each and every co-translationally or post-translationally modified form, including carbohydrate-modified (e.g., glycosylated) and lipid-modified forms of nonstructural proteins, and preferably relates to "alphavirus nonstructural proteins".

[0240] In some embodiments, the term "alphavirus nonstructural protein" refers to any one or more individual nonstructural proteins (nsP1, nsP2, nsP3, nsP4) of alphavirus origin, or refers to a polyprotein comprising the polypeptide sequences of more than one nonstructural protein 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 this embodiment, nsP4 may be another protein of interest and may be encoded by another open reading frame.

[0241] In some embodiments, the nonstructural protein is capable of forming a complex or association, for example, in a host cell. In some embodiments, an "alphavirus nonstructural protein" refers to a complex or association of nsP123 (synonymous P123) and nsP4. In some embodiments, an "alphavirus nonstructural protein" refers to a complex or association of nsP1, nsP2, and nsP3. In some embodiments, an "alphavirus nonstructural protein" refers to a complex or association of nsP1, nsP2, nsP3, and nsP4. In some embodiments, an "alphavirus nonstructural protein" refers to a complex or association of any one or more selected from nsP1, nsP2, nsP3, and nsP4. In some embodiments, an alphavirus nonstructural protein comprises at least nsP4.

[0242] The term "complex" or "associate" refers to two or more identical or different protein molecules that are adjacent in space. The proteins of the complex are preferably in direct or indirect physical or physicochemical contact with each other. The complex or associate may be composed of a variety of different proteins (heteromultimers) and / or multiple copies of a specific protein (homomultimers). In the context of alphavirus nonstructural proteins, the term "complex or associate" describes a large number of at least two protein molecules, at least one of which is an alphavirus nonstructural protein. The complex or associate may be composed of multiple copies of a specific protein (homomultimers) and / or a variety of different proteins (heteromultimers). In the case of a polymer, "multi" means more than one, for example two, three, four, five, six, seven, eight, nine, ten or more than ten.

[0243] The term "functional nonstructural protein" includes nonstructural proteins with replicase function. Therefore, "functional nonstructural protein" includes alphavirus replicase. "Replicase function" includes the function of RNA-dependent RNA polymerase (RdRP), i.e., an enzyme that can catalyze the synthesis of (-) strand RNA based on a (+) strand RNA template and / or an enzyme that can catalyze the synthesis of (+) strand RNA based on a (-) strand RNA template. Therefore, the term "functional nonstructural protein" may refer to a protein or complex that uses a (+) strand (e.g., genomic) RNA as a template to synthesize (-) strand RNA, a protein or complex that uses the (-) strand complementary strand of genomic RNA as a template to synthesize new (+) strand RNA, and / or a protein or complex that uses a fragment of the (-) strand complementary strand of genomic RNA as a template to synthesize subgenomic transcripts. Functional nonstructural proteins may additionally have one or more additional functions, such as, for example, proteases (for self-cleavage), helicases, terminal adenosyltransferases (for poly(A) tail addition), methyltransferases and guanylyltransferases (for providing nucleic acids with 5' caps), nuclear localization sites, triphosphatases (Gould et al., 2010, Antiviral Res. 87: 111-124; Rupp et al., 2015, J. Gen. Virol. 96: 2483-500).

[0244] The term "replicase" includes RNA-dependent RNA polymerases. According to the present invention, the term "replicase" includes "alphavirus replicase", including RNA-dependent RNA polymerases from naturally occurring alphaviruses (alphaviruses present in nature) and RNA-dependent RNA polymerases from alphavirus variants or derivatives (e.g., from attenuated alphaviruses).

[0245] The term "replicase" includes all variants of alphavirus replicase expressed by cells infected with alphavirus or expressed by cells transfected with nucleic acids encoding alphavirus replicase, in particular variants, conformations, isoforms and homologs that are modified post-translationally. In addition, the term "replicase" includes all forms of replicase that have been produced and can be produced by recombinant methods. For example, a replicase containing a tag that facilitates detection and / or purification of the replicase in the laboratory, such as a myc tag, an HA tag or an oligohistidine tag (His tag) can be produced by recombinant methods.

[0246] Optionally, the alphavirus replicase is further functionally defined by the ability to bind to any one or more of the following: alphavirus conserved sequence element 1 (CSE 1) or its complement, conserved sequence element 2 (CSE 2) or its complement, conserved sequence element 3 (CSE 3) or its complement, conserved sequence element 4 (CSE 4) or its complement. Preferably, the replicase is capable of binding to CSE 2 [i.e., the (+) strand] and / or CSE 4 [i.e., the (+) strand] or to the complementary strand of CSE 1 [i.e., the (-) strand] and / or the complementary strand of CSE 3 [i.e., the (-) strand].

[0247] The source of the alphavirus replicase is not limited to any particular alphavirus. In a preferred embodiment, the alphavirus replicase comprises a nonstructural protein from a Semliki Forest virus, including a naturally occurring Semliki Forest virus and a variant or derivative of a Semliki Forest virus, such as an attenuated Semliki Forest virus. In an alternative preferred embodiment, the alphavirus replicase comprises a nonstructural protein from a Sindbis virus, including a naturally occurring Sindbis virus and a variant or derivative of a Sindbis virus, such as an attenuated Sindbis virus. In an alternative preferred embodiment, the alphavirus replicase comprises a nonstructural protein from a Venezuelan equine encephalitis virus (VEEV), including a naturally occurring VEEV and a variant or derivative of VEEV, such as an attenuated VEEV. In an alternative preferred embodiment, the alphavirus replicase comprises a nonstructural protein from a Chikungunya virus (CHIKV), including a naturally occurring CHIKV and a variant or derivative of CHIKV, such as an attenuated CHIKV.

[0248] The replicase may also comprise nonstructural proteins from more than one virus (e.g., from more than one alphavirus). Thus, heterologous complexes or associations comprising alphavirus nonstructural proteins and having replicase function are also encompassed by the present invention. For illustrative purposes only, a replicase may comprise one or more nonstructural proteins from a first alphavirus (e.g., nsP1, nsP2) and one or more nonstructural proteins from a second alphavirus (nsP3, nsP4). Nonstructural proteins from more than one different alphavirus may be encoded by separate open reading frames or may be encoded as polyproteins by a single open reading frame, e.g., nsP1234.

[0249] In some embodiments, the functional nonstructural protein is capable of forming membrane replication complexes and / or vesicles in a cell in which the functional nonstructural protein is expressed.

[0250] If by the functional nonstructural protein encoded by the nucleic acid molecule according to the present invention, i.e. the nonstructural protein with replicase function, it is preferred that the subgenomic promoter of the replicon (if present) is compatible with the replicase. Compatible in this case means that the replicase can recognize the subgenomic promoter (if present). In one embodiment, when the subgenomic promoter is natural to the virus in the replicase source, i.e. the natural source of these sequences is the same virus, this is achieved. In an alternative embodiment, the subgenomic promoter is not natural for the virus in the viral replicase source, provided that the viral replicase can recognize the subgenomic promoter. In other words, the replicase is compatible with the subgenomic promoter (cross-virus compatibility). The example of the cross-virus compatibility of the subgenomic promoter and the replicase derived from different alphaviruses is known in the art. Any combination of subgenomic promoters and replicase is possible, as long as there is cross-virus compatibility. Cross-virus compatibility can be easily tested by the technician engaged in the work of the present invention under the conditions suitable for RNA synthesis from the subgenomic promoter by incubating the replicase to be tested with RNA, wherein RNA has the subgenomic promoter to be tested. If subgenomic transcripts are made, make sure the subgenomic promoters and replicases are compatible. Various examples of cross-viral compatibility are known.

[0251] Replicon can be preferably replicated by functional nonstructural protein. In particular, RNA replicon encoding functional nonstructural protein can be replicated by the functional nonstructural protein encoded by the replicon. In a preferred embodiment, RNA replicon comprises an open reading frame encoding functional alphavirus nonstructural protein. In one embodiment, replicon comprises another open reading frame encoding target protein. This embodiment is particularly suitable for producing some methods of target protein according to the present invention. In one embodiment, another open reading frame encoding target protein is located downstream of 5' replication recognition sequence and upstream of IRES (and upstream of the open reading frame encoding the functional nonstructural protein from self-replicating virus) and / or is located downstream of the open reading frame encoding the functional nonstructural protein from self-replicating virus. Another open reading frame encoding the target protein located downstream of 5' replication recognition sequence and upstream of IRES (and upstream of the open reading frame encoding the functional nonstructural protein from self-replicating virus) can be expressed as a fusion protein with a sequence encoded by 5' replication recognition sequence. Another open reading frame encoding a protein of interest located downstream of the 5' replication recognition sequence and upstream of the IRES (and upstream of the open reading frame encoding a functional nonstructural protein from a 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 located downstream of the open reading frame encoding a functional nonstructural protein from a self-replicating virus are typically controlled by a subgenomic promoter.

[0252] It is preferred that the open reading frame encoding the functional nonstructural protein does not overlap with the 5' replication recognition sequence. In one embodiment, the open reading frame encoding the functional nonstructural protein does not overlap with the subgenomic promoter (if present). Its embodiments are disclosed in WO 2017 / 162460, which is incorporated herein by reference.

[0253] Uncoupling of sequence elements required for replication and protein coding regions

[0254] Vectors derived from various alphaviruses are difficult to develop because the open reading frame encoding nsP1234 overlaps with the 5' replication recognition sequence of the alphavirus genome (coding sequence of nsP1) and often also overlaps with the subgenomic promoter containing CSE 3 (coding sequence of nsP4).

[0255] The RNA replicons described herein typically contain sequence elements required for replication by a replicase, particularly a 5' replication recognition sequence. In one embodiment, the coding sequence of the nonstructural protein is under the control of an IRES, and thus the IRES is located upstream of the coding sequence of the nonstructural protein. Thus, in one embodiment, the 5' replication recognition sequence, which typically overlaps with the coding sequence of the N-terminal fragment of the alphavirus nonstructural protein, is located upstream of the IRES and does not overlap with the coding sequence of the nonstructural protein.

[0256] In one embodiment, the coding sequence of the 5' replication recognition sequence (eg, the nsP1 coding sequence) is fused in frame to the gene of interest upstream of the IRES.

[0257] In one embodiment, the 5' replication recognition sequence does not encode any protein or fragment thereof, such as an alphavirus nonstructural protein or fragment thereof. Therefore, in the RNA replicon according to the present invention, the sequence elements required for replication by the replicase and the protein coding region may not be coupled. Compared to a natural viral genomic RNA (e.g., a natural alphavirus genomic RNA), decoupling can be achieved by removing at least one start codon in the 5' replication recognition sequence.

[0258] Thus, the rRNA may comprise a 5' replication recognition sequence, wherein the 5' replication recognition sequence is characterized in that it comprises the removal of at least one start codon compared to a native viral 5' replication recognition sequence (eg, a native alphavirus 5' replication recognition sequence).

[0259] A 5' replication recognition sequence is characterized in that it comprises the removal of at least one start codon compared to a native viral 5' replication recognition sequence and may be referred to herein as a "modified 5' replication recognition sequence" or a "5' replication recognition sequence according to the invention". As described below, a 5' replication recognition sequence according to the invention may optionally be characterized by the presence of one or more additional nucleotide changes, such as those detected by the methods of the invention.

[0260] A nucleic acid construct that can be replicated by a replicase (preferably an alphavirus replicase) is referred to as a replicable RNA or replicon. According to the present invention, the term "replicon" defines an RNA molecule that can be replicated by an RNA-dependent RNA polymerase, which produces one or more identical or substantially identical copies of an RNA replicon in the absence of a DNA intermediate. "Without a DNA intermediate" means that a deoxyribonucleic acid (DNA) copy or a complementary strand of the replicon is not formed in the process of forming a copy of the RNA replicon, and / or a deoxyribonucleic acid (DNA) molecule is not used as a template in the process of forming a copy of the RNA replicon or its complementary strand. The replicase function is usually provided by a functional nonstructural protein such as a functional alphavirus nonstructural protein.

[0261] According to the present invention, the terms "replicable" and "capable of being replicated" generally describe that one or more identical or substantially identical copies of a nucleic acid can be prepared. When used together with the term "replicase", for example in "capable of being replicated by a replicase", the terms "replicable" and "capable of being replicated" describe the functional characteristics of a nucleic acid molecule (e.g., an RNA replicon) with respect to the replicase. These functional characteristics include at least one of the following: (i) the replicase is able to recognize the replicon and (ii) the replicase is able to act as an RNA-dependent RNA polymerase (RdRP). Preferably, the replicase is able to both (i) recognize the replicon and (ii) act as an RNA-dependent RNA polymerase.

[0262] The expression "capable of recognizing" describes that the replicase is able to physically associate with the replicon, and preferably, the replicase is able to bind to the replicon (usually non-covalently). The term "binding" may mean that the replicase has the ability to bind to any one or more of the following: conserved sequence element 1 (CSE 1) or its complementary sequence (if contained by the replicon), conserved sequence element 2 (CSE 2) or its complementary sequence (if contained by the replicon), conserved sequence element 3 (CSE 3) or its complementary sequence (if contained by the replicon), conserved sequence element 4 (CSE 4) or its complementary sequence (if contained by the replicon). Preferably, the replicase is able to bind to CSE 2 [i.e., the (+) strand] and / or CSE 4 [i.e., the (+) strand], or to the complementary sequence of CSE 1 [i.e., the (-) strand) and / or the complementary sequence of CSE 3 [i.e., the (-) strand].

[0263] In one embodiment, the expression "capable of acting as an RdRP" means that the replicase is capable of catalyzing the synthesis of the (-) strand complementary strand of the viral genomic (+) strand RNA, wherein the (+) strand RNA has a template function, and / or the replicase is capable of catalyzing the synthesis of the (+) strand viral genomic RNA, wherein the (-) strand RNA has a template function. Generally speaking, the expression "capable of acting as an RdRP" may also include that the replicase is capable of catalyzing the synthesis of the (+) strand subgenomic transcript, wherein the (-) strand RNA has a template function, and wherein the synthesis of the (+) strand subgenomic transcript generally starts at a subgenomic promoter. In one embodiment, the virus is an alphavirus.

[0264] The expressions "capable of binding" and "capable of acting as an RdRP" refer to the ability under normal physiological conditions. In particular, it refers to the conditions inside a cell that expresses a functional non-structural protein or has been 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 tested experimentally, for example in a cell-free in vitro system or in a eukaryotic cell. Optionally, the eukaryotic cell is a cell from a species for which a specific virus representing a source of the replicase is infectious. For example, when a viral replicase from a specific virus that is infectious to humans is used, normal physiological conditions are conditions in human cells. More preferably, the eukaryotic cell (in one example, a human cell) is from the same tissue or organ to which the specific virus representing the source of the replicase is infectious.

[0265] 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 itself is typically not a native alphavirus sequence.

[0266] 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 is able to recognize the 3' replication recognition sequence. Preferably, the 3' replication recognition sequence is located at the 3' end of the replicon (if the replicon does not contain a poly(A) tail), or immediately upstream of the poly(A) tail (if the replicon contains a poly(A) tail). In one embodiment, the 3' replication recognition sequence consists of or comprises CSE 4.

[0267] In one embodiment, the 5' replication recognition sequence and the 3' replication recognition sequence are capable of directing the replication of the RNA replicon according to the invention in the presence of a functional nonstructural protein. Thus, when present alone or preferably together, these recognition sequences direct the replication of the RNA replicon in the presence of a functional nonstructural protein.

[0268] Preferably, a functional non-structural protein capable of recognizing both the 5' replication recognition sequence and the 3' replication recognition sequence of the replicon is provided. In one embodiment, this is achieved when the 3' replication recognition sequence is natural for the alphavirus from which the functional alphavirus non-structural protein is derived and when the 5' replication recognition sequence is natural for the alphavirus from which the functional alphavirus non-structural protein is derived or is a variant of the natural 5' replication recognition sequence for the alphavirus from which the functional alphavirus non-structural protein is derived. Natural means that the natural source 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 natural for the alphavirus from which the functional alphavirus non-structural protein is derived, provided that the functional alphavirus non-structural protein is capable of recognizing 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 both the 5' replication recognition sequence and the 3' replication recognition sequence. When the non-natural functional alphavirus non-structural protein is capable of recognizing the corresponding sequence or sequence element, the functional alphavirus non-structural protein is considered to be compatible (cross-virus compatibility). Any combination of a (3' / 5') replication recognition sequence and a CSE, respectively, with a functional alphavirus nonstructural protein is possible, as long as cross-viral compatibility exists. Cross-viral compatibility can be easily tested by a technician working with the present invention by incubating the functional alphavirus nonstructural protein to be tested with RNA under conditions suitable for RNA replication, such as in a suitable host cell, wherein the RNA has the 3' and 5' replication recognition sequences to be tested. If replication occurs, it is determined that the (3' / 5') replication recognition sequence is compatible with the functional alphavirus nonstructural protein.

[0269] Removal of at least one start codon within the 5' replication recognition sequence provides several advantages. The absence of a start codon in the nucleic acid sequence encoding nsP1* (the N-terminal fragment of nsP1) generally results in nsP1* not being translated. In addition, 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 the ribosome; therefore, when the replicon is present in the cell, translation starts at the first AUG of the open reading frame (RNA) encoding the gene of interest.

[0270] The 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 the replicon according to the invention (i.e., characterized by the removal of the start codon) can be designed on a computer (in silico) and then synthesized in vitro (gene synthesis); alternatively, a suitable DNA molecule can be obtained by site-directed mutagenesis of the DNA sequence encoding the replicon. In any case, the corresponding DNA molecule can be used as a template for in vitro transcription, thereby providing a replicon according to the invention.

[0271] Compared with the natural 5' replication recognition sequence, the removal of at least one start codon 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 substitution, insertion or deletion, the nucleotide modification must not result in the formation of a new start codon (as an illustrative example: at the DNA level, the insertion must not be an insertion of ATG).

[0272] The 5' replication recognition sequence of the RNA replicon 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 genome of an alphavirus occurring in nature. In one embodiment, the modified 5' replication recognition sequence according to the present invention is preferably characterized in that it has a degree of 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 occurring in nature.

[0273] 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 about 250 nucleotides of the 5' end of an alphavirus (i.e., the 5' end of the alphavirus genome). In a preferred embodiment, it comprises a sequence homologous to about 250 to 500, preferably about 300 to 500 nucleotides of the 5' end of an alphavirus (i.e., the 5' end of the alphavirus genome). "The 5' end of the alphavirus genome" means a 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 designated as nucleotide number 1, and, for example, "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 having 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 at least 250 nucleotides of the 5' end of the genome of at least one alphavirus present in nature. At least 250 nucleotides includes, for example, 250 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides.

[0274] The 5' replication recognition sequence of alphaviruses present in nature is generally characterized by at least one start codon and / or a conserved secondary structure motif. For example, the natural 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, the analysis by MFOLD shows that the natural 5' replication recognition sequence of Semliki Forest virus is expected to form four stem loops (SL), referred to as stem loops 1 to 4 (SL1, SL2, SL3, SL4). According to the analysis by MFOLD by Frolov et al., the natural 5' replication recognition sequence of different alphaviruses Sindbis virus is also expected to form four stem loops: SL1, SL2, SL3, SL4.

[0275] It is known that the 5' end of the alphavirus genome contains sequence elements that enable replication of the alphavirus genome through functional alphavirus nonstructural proteins. In one embodiment of the present invention, the 5' replication recognition sequence of the RNA replicon comprises a sequence homologous to the conserved sequence element 1 (CSE 1) of the alphavirus and / or a sequence homologous to the conserved sequence element 2 (CSE 2) of the alphavirus.

[0276] The conserved sequence element 2 (CSE 2) of the alphavirus genomic RNA is generally represented by SL3 and SL4, which is preceded by SL2 comprising at least the natural start codon encoding the first amino acid residue of the alphavirus nonstructural protein nsP1. However, in the present specification, in some embodiments, the conserved sequence element 2 (CSE 2) of the alphavirus genomic RNA refers to a region spanning from SL2 to SL4 and comprising the natural start codon encoding the first amino acid residue of the alphavirus nonstructural protein nsP1. In a preferred embodiment, the RNA replicon comprises CSE 2 or a sequence homologous to CSE 2. In one embodiment, the RNA replicon comprises a sequence homologous to CSE 2, the sequence preferably being characterized in having a degree of sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more with the sequence of CSE 2 of at least one alphavirus present in nature.

[0277] In one embodiment, the 5' replication recognition sequence comprises a sequence homologous to CSE 2 of an alphavirus. CSE 2 of an alphavirus may comprise a fragment of an open reading frame from a nonstructural protein of an alphavirus.

[0278] Thus, in one embodiment, the RNA replicon is characterized in that it comprises a sequence homologous to an open reading frame of a nonstructural protein or a fragment thereof from an alphavirus. Sequences homologous to an open reading frame of a nonstructural protein or a fragment thereof are typically variants of an open reading frame of a nonstructural protein or a fragment thereof of an alphavirus occurring in nature. In one embodiment, the sequence homologous to an open reading frame of a nonstructural protein or a fragment thereof is preferably characterized in that it has a degree of sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more to an open reading frame of a nonstructural protein or a fragment thereof of at least one alphavirus occurring in nature.

[0279] In one embodiment, the sequence homologous to the open reading frame of the nonstructural protein comprised by the replicon of the present invention does not contain the natural start codon of the nonstructural protein, and more preferably, does not contain any start codon of the nonstructural protein. In one embodiment, the sequence homologous to CSE 2 is characterized in that all start codons are removed compared to the natural alphavirus CSE 2 sequence. Therefore, the sequence homologous to CSE 2 preferably does not contain any start codon.

[0280] When the sequence homologous to the open reading frame does not contain any start codons, the sequence homologous to the open reading frame itself is not an open reading frame because it cannot serve as a template for translation.

[0281] In one embodiment, the 5' replication recognition sequence comprises a sequence homologous to an open reading frame of a nonstructural protein from an alphavirus or a fragment thereof, wherein the sequence homologous to an open reading frame of a nonstructural protein from an alphavirus or a fragment thereof is characterized in that it comprises the removal of at least one start codon compared to the native alphavirus sequence.

[0282] In one embodiment, the sequence homologous to the open reading frame of a nonstructural protein or a fragment thereof from an alphavirus is characterized in that it comprises the removal of at least the native start codon of the open reading frame of the nonstructural protein. Preferably, it is characterized in that it comprises the removal of at least the native start codon of the open reading frame encoding nsP1.

[0283] The natural start codon is the AUG base triplet at which translation on the ribosome in the host cell begins when RNA is present in the host cell. In other words, the natural start codon is the first base triplet translated during ribosomal protein synthesis in a host cell that has been inoculated with RNA containing the natural start codon. In one embodiment, the host cell is a cell from a eukaryotic species that is a natural host of a particular alphavirus that contains a natural alphavirus 5' replication recognition sequence. In one embodiment, the host cell is from the cell line "BHK21[C13]( CCL10TM )" BHK21 cells can be obtained from the American Type Culture Collection, Manassas, Virginia, USA.

[0284] The genomes of many alphaviruses have been completely sequenced and are publicly available, and the sequences of the nonstructural proteins encoded by these genomes are also publicly available. Such sequence information allows the natural start codon to be determined in silico.

[0285] In one embodiment, the sequence homologous to the open reading frame of the nonstructural protein or fragment thereof from an alphavirus is characterized in that it comprises 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 removing the natural start codon, any one or two or three or four or more than four (e.g., five) start codons may be removed.

[0286] If the replicon is characterized by the removal of the native start codon of the open reading frame of the non-structural protein and optionally one or more start codons other than the native start codon, then the sequence homologous to the open reading frame is not itself an open reading frame since it cannot serve as a template for translation.

[0287] Preferably, in addition to removing the natural start codon, one or more start codons removed except the natural start codon are preferably selected from AUG base triplets with the potential to start translation. AUG base triplets with the potential to start translation can be referred to as "potential start codons". Whether a given AUG base triplet has the potential to start translation can be determined on a computer or in a cell-based in vitro assay.

[0288] In one embodiment, whether a given AUG base triplet has the potential to initiate translation is determined in computer: In this embodiment, the nucleotide sequence is examined and if the AUG base triplet is part of an AUGG sequence, preferably part of a Kozak sequence, it is determined to have the potential to initiate translation.

[0289] In one embodiment, whether a given AUG base triplet has the potential to initiate translation is determined in a cell-based in vitro assay: an RNA replicon characterized by the removal of a natural start codon and comprising a given AUG base triplet downstream of the removal position of the natural start codon is introduced into a host cell. In one embodiment, the host cell is a cell from a eukaryotic species that is a natural host of a specific alphavirus that contains a natural alphavirus 5' replication recognition sequence. In a preferred embodiment, the host cell is from the cell line "BHK21[C13]( CCL10 TM )" BHK21 cells, available from the American Type Culture Collection in Manassas, Virginia, USA. Preferably, no additional AUG base triplet exists between the removal position of the natural start codon and the given AUG base triplet. If translation is initiated at the given AUG base triplet after an RNA replicon characterized by the removal of the natural start codon and comprising the given AUG base triplet is transferred into a host cell, it is determined that the given AUG base triplet has the potential to initiate translation. Whether translation has been initiated can be determined by any suitable method known in the art. For example, the replicon can encode a tag that facilitates detection of the translation product (if any), such as my, downstream of the given AUG base triplet and in the same frame with the given AUG base triplet. c-tag or HA-tag; Whether there is an expression product with an encoded tag can be determined, for example, by Western blotting. In this embodiment, preferably no other AUG base triplets are present between a given AUG base triplet and the nucleic acid sequence encoding the tag. Cell-based in vitro assays can be performed separately for more than one given AUG base triplet: in each case, preferably no other AUG base triplets are present between the removal position of the natural start codon and the given AUG base triplet. This can be achieved by removing all AUG base triplets (if any) between the removal position of the natural start codon and the given AUG base triplet. Therefore, a given AUG base triplet is the first AUG base triplet downstream of the removal position of the natural start codon.

[0290] Preferably, the 5' replication recognition sequence of the RNA replicon according to the invention is characterized in that all potential start codons are removed. Therefore, according to the invention, the 5' replication recognition sequence preferably does not contain an open reading frame that can be translated into protein.

[0291] In one embodiment, the 5' replication recognition sequence of the RNA replicon according to the invention is characterized by a secondary structure that is equivalent to the (predicted) secondary structure of the 5' replication recognition sequence of the viral genomic RNA. To this end, the RNA replicon may comprise one or more nucleotide changes to compensate for the disruption of nucleotide pairing within one or more stem loops introduced by the removal of at least one start codon.

[0292] In one embodiment, the 5' replication recognition sequence of the RNA replicon according to the invention is characterized by a secondary structure equivalent 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 equivalent 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 a web server for RNA secondary structure prediction, http: / / rna.urmc.rochester.edu / RNAstructureWeb / Servers / Predictl / Predictl.html.

[0293] The presence or absence of nucleotide pairing disruption can be determined by comparing the secondary structure or predicted secondary structure of a 5' replication recognition sequence of an RNA replicon characterized by the removal of at least one start codon compared to a native alphavirus 5' replication recognition sequence. For example, at least one base pair may not be present at a given position, such as a base pair in a stem-loop, particularly in the stem of a stem-loop, compared to a native alphavirus 5' replication recognition sequence.

[0294] In one embodiment, one or more of the 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.

[0295] In one embodiment, the removal of at least one start codon does not destroy the secondary structure of 5' replication recognition sequence. In an alternative embodiment, the removal of at least one start codon has destroyed the secondary structure of 5' replication recognition sequence. In this embodiment, compared with natural 5' replication recognition sequence, the removal of at least one start codon can be the reason that at least one base pair (for example base pair in stem loop) at a given position does not exist. Compared with natural 5' replication recognition sequence, if the base pair in stem loop does not exist, the removal of at least one start codon is determined as having introduced nucleotide pairing destruction in stem loop. The base pair in stem loop is normally the base pair in the stem of stem loop.

[0296] In one embodiment, the RNA replicon comprises one or more nucleotide changes to compensate for disruption of nucleotide pairing within one or more stem-loops introduced by removal of at least one start codon.

[0297] If removal of at least one start codon introduces a disruption in nucleotide pairing within the stem-loop compared to the native 5' replication recognition sequence, one or more nucleotide changes expected to compensate for the disruption in nucleotide pairing can be introduced, and the secondary structure or predicted secondary structure thereby obtained can be compared to the native 5' replication recognition sequence.

[0298] Based on common knowledge and the disclosure herein, a skilled artisan may anticipate certain nucleotide changes to compensate for nucleotide pairing disruptions. For example, if a base pair at a given position of 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 is disrupted compared to the native 5' replication recognition sequence, a nucleotide change that restores the base pair at that position, preferably without reintroducing the start codon, is expected to compensate for the nucleotide pairing disruption.

[0299] In one embodiment, the 5' replication recognition sequence of the replicon does not overlap with or contain a translatable nucleic acid sequence (i.e., translatable into a peptide or protein, particularly nsP, particularly nsP1, or a fragment of any thereof). For a nucleotide sequence to be "translatable", it requires the presence of a start codon; the start codon encodes the N-terminal-most 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 an N-terminal fragment of nsP1.

[0300] In some cases, 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 translatable nucleic acid sequence (i.e., translatable into a peptide or protein, particularly nsP, particularly nsP4, or a fragment of any thereof). In one embodiment, the subgenomic promoter of the replicon does not overlap with or contain a translatable nucleic acid sequence encoding a C-terminal fragment of nsP4. By deleting a portion of the coding sequence of nsP4 (typically a portion encoding the N-terminal portion of nsP4) and / or by removing the AUG base triplet in the non-deleted portion of the nsP4 coding sequence, an RNA replicon with a subgenomic promoter can be generated, the subgenomic promoter does not overlap with or contain a translatable nucleic acid sequence, e.g., translatable into a C-terminal fragment of nsP4. If the AUG base triplet in the coding sequence of nsP4 or a portion thereof is removed, the removed AUG base triplet 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.

[0301] In one embodiment, the RNA replicon does not comprise an open reading frame encoding a truncated nonstructural protein, such as a truncated alphavirus nonstructural protein. In the context of this embodiment, it is particularly preferred that the RNA replicon does not comprise an open reading frame encoding the N-terminal fragment of nsP1, and optionally does not comprise 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.

[0302] In some embodiments, the replicon according to the invention does not comprise stem-loop 2 (SL2) at the 5' end of the alphavirus genome. According to Frolov et al. (supra), stem-loop 2 is a conserved secondary structure present at the 5' end of the alphavirus genome upstream of CSE 2, but is not essential for replication.

[0303] 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 according to the invention is an isolated nucleic acid molecule. The RNA replicon according to the invention comprises at least one modified nucleotide, and preferably comprises one or more sequence changes, in particular sequence changes detected by the methods disclosed herein, which are used to identify sequence changes that restore or improve the function of rRNA comprising at least one modified nucleotide.

[0304] At least one open reading frame encoding at least one gene product of interest

[0305] In one embodiment, the RNA replicon according to the present invention comprises at least one open reading frame encoding a target gene product (e.g., a target peptide or a target protein). Preferably, the target protein is encoded by a heterologous nucleic acid sequence. Genes encoding target peptides or target proteins are synonymously referred to as "target genes" or "transgenes". In a plurality of embodiments, target peptides or target proteins are encoded by heterologous nucleic acid sequences. According to the present invention, the term "heterologous" refers to the fact that a nucleic acid sequence is not naturally functionally or structurally connected to a viral nucleic acid sequence, such as an alphavirus nucleic acid sequence.

[0306] The replicon according to the invention may encode a single polypeptide or multiple polypeptides. Multiple polypeptides may be encoded as a single polypeptide (fusion polypeptide) or separate polypeptides. In some embodiments, the replicon according to the invention may comprise more than one open reading frame, each open reading frame being independently selected to be under the control of a subgenomic promoter or not under its control. Alternatively, the polyprotein or fusion polypeptide comprises separate polypeptides separated by a 2A self-cleaving peptide (e.g., from the 2A protein of foot-and-mouth disease virus) or a protease cleavage site or an intein.

[0307] The target protein can be selected from reporter proteins, pharmaceutically active peptides or pharmaceutically active proteins, inhibitors of intracellular interferon (IFN) signaling. According to the present invention, the target protein preferably does not include functional non-structural proteins from self-replicating viruses, such as functional non-structural proteins of alphaviruses.

[0308] Reporter protein

[0309] In one embodiment, the open reading frame encodes a reporter protein, such as a cell surface expressed protein such as CD90. In this embodiment, the open reading frame comprises a reporter gene. Certain genes can be selected as reporters because the characteristics of the cells or organisms expressing them can be easily identified and measured, or because these genes are selectable markers. Reporter genes are generally used to indicate whether a gene has been absorbed by a cell or organism population or expressed in a cell or organism population. Preferably, the expression product of the reporter gene is visually detectable. Common visually detectable reporter proteins generally have fluorescent or luminescent proteins. Examples of specific reporter genes include genes encoding jellyfish green fluorescent protein (green fluorescent protein, GFP), luciferase (Luc) and red fluorescent protein (red fluorescent protein, RFP), the jellyfish green fluorescent protein causes the cells expressing it to emit green light under blue light, and the luciferase catalyzes the reaction with luciferin to produce light. Variants of any of these specific reporter genes are possible, as long as the variant has visually detectable properties. For example, eGFP is a point mutant variant of GFP. Reporter protein embodiments are particularly suitable for testing expression.

[0310] Pharmaceutically active gene products, such as peptides or proteins or nucleic acids

[0311] According to the present invention, in one embodiment, the rRNA comprises or consists of a pharmaceutically active rRNA. "Pharmaceutically active RNA" may be an RNA encoding a pharmaceutically active peptide or a pharmaceutically active protein. Preferably, the RNA replicon according to the present invention encodes a pharmaceutically active peptide or a pharmaceutically active protein or other gene products. Preferably, the open reading frame encodes a pharmaceutically active peptide or a pharmaceutically active protein. Preferably, the RNA replicon comprises an open reading frame encoding a pharmaceutically active peptide or a pharmaceutically active protein, which is optionally under the control of a subgenomic promoter.

[0312] When a "pharmaceutically active peptide or pharmaceutically active protein" is administered to a subject in a therapeutically effective amount, it has a positive or beneficial effect on the subject's condition or disease state. Preferably, the pharmaceutically active peptide or pharmaceutically active protein has a curative or palliative property and can be administered to improve, alleviate, relieve, reverse one or more symptoms of a disease or condition, delay the onset of one or more symptoms of a disease or condition, or reduce its severity. A pharmaceutically active peptide or pharmaceutically active protein may have a preventive property and may be used to delay the onset of a disease or reduce the severity of such a disease or pathological condition. The term "pharmaceutically active peptide or pharmaceutically active protein" includes complete proteins or polypeptides, and may also refer to pharmaceutically active fragments thereof. It may also include pharmaceutically active analogs of peptides or proteins. The term "pharmaceutically active peptide or pharmaceutically active protein" includes peptides and proteins that are antigens, i.e., the peptide or protein elicits an immune response in a subject, which may be therapeutic or partially or completely preventive.

[0313] In one embodiment, the pharmaceutically active peptide or pharmaceutically active protein is or comprises an immunologically active compound or an antigen or epitope.

[0314] According to the present invention, the term "immunologically active compound" relates to any compound that preferably alters the immune response by inducing and / or inhibiting the maturation of immune cells, inducing and / or inhibiting cytokine biosynthesis, and / or alters humoral immunity by stimulating B cells to produce antibodies. In one embodiment, the immune response involves stimulating an antibody response (usually including immunoglobulin G (IgG)). The immunologically active compound has potent immunostimulatory activity, including but not limited to antiviral and antitumor activity, and may also downregulate other aspects of the immune response, such as shifting the immune response from a Th2 immune response, which is useful for treating a wide range of Th2-mediated diseases.

[0315] According to the present invention, the term "antigen" or "immunogen" encompasses any substance that will trigger an immune response. In particular, "antigen" relates to any substance that specifically reacts with antibodies or T lymphocytes (T cells). According to the present invention, the term "antigen" comprises any molecule containing at least one epitope. Preferably, an antigen in the context of the present invention is a molecule that induces an immune response, optionally after processing, which is preferably specific for the antigen. According to the present invention, any suitable antigen can be used, which is a candidate for an immune response, wherein the immune response can be both a humoral immune response and a cellular immune response. In the context of an embodiment of the present invention, in the context of an MHC molecule, the antigen is preferably presented by a cell, preferably by an antigen presenting cell, which results in an immune response against the antigen. The antigen is preferably a product corresponding to or derived from a naturally occurring antigen. Such naturally occurring antigens may include or may be derived from allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens, or the antigen may also be a tumor antigen. According to the present invention, the antigen may correspond to a naturally occurring product, such as a viral protein, or a portion thereof. In some preferred embodiments, the antigen is a surface polypeptide, i.e. a polypeptide naturally displayed on the surface of a cell, pathogen, bacterium, virus, fungus, parasite, allergen or tumor. The antigen can induce an immune response against a cell, pathogen, bacterium, virus, fungus, parasite, allergen or tumor.

[0316] The term "pathogen" refers to pathogenic biological material that is capable of causing disease in an organism, preferably a vertebrate organism. Pathogens include microorganisms such as bacteria, unicellular eukaryotes (protozoa), fungi, and viruses.

[0317] 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 immunoactive compound that is recognized by the immune system, such as by T cells, particularly when presented in the context of an MHC molecule. An epitope of a protein preferably comprises a continuous or discontinuous portion of the protein and is preferably 5 to 100 amino acids in length, preferably 5 to 50, more preferably 8 to 30, most preferably 10 to 25 amino acids in length, for example, an epitope may 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 may bind to an MHC molecule, such as an MHC molecule on the surface of a cell, and may therefore be an "MHC binding peptide" or an "antigenic peptide". The term "major histocompatibility complex" and the abbreviation "MHC" include MHC class I and MHC class II molecules, and refer to a complex of genes present in all vertebrates. MHC proteins or molecules are important for signal transduction between lymphocytes and antigen presenting cells or diseased cells in immune responses, wherein MHC proteins or molecules bind peptides and present them for recognition by T cell receptors. Proteins encoded by MHC are expressed on the cell surface and display both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. Preferably, such immunogenic moieties are bound to MHC class I or class II molecules. As used herein, if such binding can be detected using any assay known in the art, the immunogenic moiety is said to be "bound to" MHC class I or class II molecules. The term "MHC binding peptide" refers to a peptide that is bound to MHC class I and / or MHC class II molecules. In the case of class I MHC / peptide complexes, although longer or shorter peptides may be effective, the binding peptide is typically 8 to 10 amino acids long. In the case of MHC class II / peptide complexes, binding peptides are typically 10 to 25 amino acids long, and particularly 13 to 18 amino acids long, although longer and shorter peptides may be effective.

[0318] In one embodiment, the protein of interest according to the present invention comprises an epitope suitable for vaccination of a target organism. It will be appreciated by those skilled in the art that one of the principles of immunobiology and vaccination is based on the fact that an immunoprotective response to a disease is produced by immunizing an organism with an antigen, which has an immunological correlation with the disease to be treated. Antigens are selected from self-antigens and non-self-antigens. Non-self-antigens are preferably bacterial antigens, viral antigens, fungal antigens, allergens or parasite antigens. It is preferred that the antigen comprises an epitope that can induce an immune response in the target organism. For example, an epitope can induce an immune response, such as a cytotoxic T cell response, to a bacterium, a virus, a fungus, a parasite, an allergen or a tumor.

[0319] In some embodiments, the non-self antigen is a bacterial antigen. In some embodiments, the antigen elicits an immune response against bacteria that infect animals (including birds, fish, and mammals, including domesticated animals). Preferably, the bacteria against which the immune response is elicited are pathogenic bacteria.

[0320] In some embodiments, the non-self antigen is a viral antigen. The viral antigen can be, for example, a peptide from a viral surface protein, such as a capsid polypeptide or a spike polypeptide from a coronavirus. In some embodiments, the antigen elicits an immune response to a virus that infects an animal (including birds, fish, and mammals, including domesticated animals). Preferably, the virus that elicits an immune response thereto is a pathogenic virus, such as Ebola virus.

[0321] In some embodiments, the non-self antigen is a polypeptide or protein from a fungus. In some embodiments, the antigen elicits an immune response against a fungus that infects an animal (including birds, fish, and mammals, including domesticated animals). Preferably, the fungus against which the immune response is elicited is a pathogenic fungus.

[0322] In some embodiments, the non-self antigen is a polypeptide or protein from a unicellular eukaryotic parasite. In some embodiments, the antigen elicits an immune response against a unicellular eukaryotic parasite, preferably a pathogenic unicellular eukaryotic parasite. The pathogenic unicellular eukaryotic parasite can be, for example, from the genus Plasmodium, such as P. falciparum, P. vivax, P. malariae, or P. ovale, from the genus Leishmania, or from the genus Trypanosoma, such as T. cruzi or T. brucei.

[0323] In some embodiments, it is not required that the pharmaceutically active peptide or pharmaceutically active protein is an antigen that triggers an immune response. Suitable pharmaceutically active proteins or pharmaceutically active peptides can be selected from cytokines and immune system proteins, such as immunoactive 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 (chimeric antigen receptors), receptor, CAR), immunoglobulins), hormones (insulin, thyroid hormone, catecholamines, gonadotropins, troponins, 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 factor, etc.), growth factor receptors, enzymes (tissue plasminogen activator, streptokinase, cholesterol biosynthetic enzyme or degrading enzyme, steroidogenic enzyme, kinase, phosphodiesterase, methylase, demethylase, dehydrogenase, cellulase, protease, lipase, phospholipases, aromatases, cytochromes, adenylate or guanylate cyclases, ceramidase, etc.), receptors (steroid hormone receptors, peptide receptors), binding proteins (growth hormone or growth factor binding proteins, etc.), transcription and translation factors, tumor growth inhibitory proteins (e.g., proteins that inhibit angiogenesis), structural proteins (e.g., collagen, fibroin, fibrinogen, elastin, tubulin, actin and myosin), blood proteins (thrombin, serum albumin, factor VII, factor VIII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von Hippel-Lindau, 1994), Wilebrand factor, antithrombin III, glucocerebrosidase, erythropoietin, granulocyte colony stimulating factor (GCSF) or modified factor VIII, anticoagulants, etc. In one embodiment, the pharmaceutically active protein according to the present invention is a cytokine involved in regulating lymphoid homeostasis, preferably a cytokine that participates in and preferably induces or enhances the development, sensitization, expansion, differentiation and / or survival of T cells. In one embodiment, the cytokine is an interleukin, such as IL-2, IL-7, IL-12, IL-15 or IL-21.

[0324] The location of at least one open reading frame in an rRNA molecule

[0325] The rRNA replicon is suitable for expressing one or more genes encoding a target peptide or target protein, optionally under the control of a subgenomic promoter. A plurality of embodiments are possible. One or more open reading frames may be present on the RNA replicon, each open reading frame encoding a target peptide or target protein. 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 target protein is located downstream of the 5' replication recognition sequence and upstream of the IRES (and the open reading frame encodes a functional non-structural protein from a 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", "the third open reading frame", and so on, in the order (5' to 3') of their presence downstream of the first open reading frame. In one embodiment, one or more additional open reading frames encoding one or more target proteins are located downstream of the open reading frame encoding a functional non-structural protein from a self-replicating virus, and are preferably controlled by a subgenomic promoter. Preferably, each open reading frame comprises a start codon (base triplet), usually AUG (in RNA molecules), corresponding to ATG (in the respective DNA molecules).

[0326] If the replicon comprises a 3' replication recognition sequence, preferably all open reading frames are located upstream of the 3' replication recognition sequence.

[0327] 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. Alphavirus subgenomic promoters are very efficient and therefore suitable for high levels of heterologous gene expression. Preferably, the subgenomic promoter is a promoter of a subgenomic transcript in an alphavirus. This means that the subgenomic promoter is a promoter that is natural to the alphavirus and preferably controls the transcription of an open reading frame in the alphavirus, the open reading frame encoding one or more structural proteins. Alternatively, the subgenomic promoter is a variant of a subgenomic promoter of an alphavirus; any variant that acts as a promoter for transcription of subgenomic RNA in a host cell is suitable. If the replicon comprises a subgenomic promoter, preferably the replicon comprises a conserved sequence element 3 (CSE 3) or a variant thereof.

[0328] 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 a subgenomic RNA comprising a transcript of the open reading frame.

[0329] 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 a replicon or from its subgenomic transcript (the latter in the presence of a functional alphavirus nonstructural protein). One or more additional open reading frames that are respectively under the control of a subgenomic promoter may be present downstream of the first open reading frame that may be under the control of a subgenomic promoter. Genes encoded by one or more additional open reading frames (e.g., by a second open reading frame) may be translated from one or more subgenomic transcripts that are respectively under the control of a subgenomic promoter. For example, an RNA replicon may include a subgenomic promoter that controls the generation of a transcript encoding a second protein of interest.

[0330] In other embodiments, the first open reading frame is not controlled by the subgenomic promoter. In one embodiment, when the first open reading frame is not controlled by the subgenomic promoter, the gene encoded by the first open reading frame can be expressed from the replicon. One or more additional open reading frames respectively under the control of the subgenomic promoter can be present downstream of the first open reading frame. Genes encoded by one or more additional open reading frames can be expressed from subgenomic transcripts.

[0331] In cells comprising the replicon according to the invention, the replicon can be amplified by functional nonstructural proteins. In addition, if the replicon comprises one or more open reading frames under the control of a subgenomic promoter, it is expected that one or more subgenomic transcripts will be prepared by functional nonstructural proteins.

[0332] If the replicon comprises more than one open reading frame encoding a protein of interest, preferably each open reading frame 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.

[0333] Other features of the replicable RNA molecules according to the invention

[0334] The RNA molecules according to the invention may optionally be characterized by further features, such as a 5' cap, a 5'-UTR, a 3'-UTR, a poly(A) sequence and / or adaptations in codon usage for optimizing translation and / or stabilization of the RNA molecule, as described in detail below.

[0335] cap

[0336] In some embodiments, a replicon according to the invention comprises a 5' cap.

[0337] The terms "5' cap", "cap", "5' cap structure", "cap structure" are used synonymously to refer to a dinucleotide present at the 5' end of certain eukaryotic primary transcripts (e.g., precursor messenger RNA). A 5' cap is a structure in which a (optionally modified) guanosine is bound to the first nucleotide of an mRNA molecule via a 5' to 5' triphosphate bond (or a modified triphosphate bond in the case of certain cap analogs). The term may refer to a conventional cap or a cap analog.

[0338] "RNA comprising a 5' cap" or "RNA provided with a 5' cap" or "RNA modified with a 5' cap" or "capped RNA" refers to RNA comprising a 5' cap. For example, providing RNA with a 5' cap can be achieved by in vitro transcription of a DNA template in the presence of the 5' cap, wherein the 5' cap is co-transcriptionally incorporated into the resulting RNA strand, or the RNA can be produced, for example, by in vitro transcription, and the 5' cap can be attached to the RNA post-transcriptionally using a capping enzyme (e.g., a capping enzyme of vaccinia virus). In capped RNA, the 3' position of the first base of the (capped) RNA molecule is linked to the 5' position of the subsequent base ("second base") of the RNA molecule via a phosphodiester bond.

[0339] In one embodiment, the RNA replicon comprises a 5' cap. In one embodiment, the RNA replicon does not comprise a 5' cap.

[0340] 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, wherein the modification consists of a methylation at the 7-position.

[0341] In the context of the present invention, the term "5' cap analogue" refers to a molecular structure similar to a conventional 5' cap but modified to have the ability to stabilize RNA if attached thereto, preferably in vivo or in a cell. A cap analogue is not a conventional 5' cap.

[0342] In the case of eukaryotic mRNA, the 5' cap has been generally described as being involved in the efficient translation of the mRNA: Generally speaking, in eukaryotes, translation only starts at the 5' end of a messenger RNA (mRNA) molecule, unless an internal ribosome entry site (IRES) is present. Eukaryotic cells are able to provide RNA with a 5' cap during transcription in the nucleus: Newly synthesized mRNA is often modified with a 5' cap structure, for example, when the transcript reaches a length of 20 to 30 nucleotides. First, the 5' terminal nucleotides pppN (ppp stands for triphosphate; N stands for any nucleoside) are converted in the cell to 5'GpppN by a capping enzyme with RNA5'-triphosphatase and guanylyltransferase activities. GpppN can then be methylated in the cell by a second enzyme with (guanine-7)-methyltransferase activity to form a monomethylated m7 GpppN-cap. In one embodiment, the 5'-cap used in the present invention is a natural 5'-cap.

[0343] In the present invention, the natural 5' cap dinucleotide is generally selected from a non-methylated cap dinucleotide (G(5')ppp(5')N; also referred to as GpppN) and a methylated cap dinucleotide ((m 7 G(5')ppp(5')N; also known as m 7 GpppN).m 7 GpppN (where N is G) is represented by the following formula:

[0344]

[0345] The capped RNA of the present invention can be prepared in vitro and therefore does not rely on the capping machinery in the host cell. The most commonly used method for preparing capped RNA in vitro is to prepare the capped RNA in the presence of all four ribonucleoside triphosphates and a cap dinucleotide (e.g., m 7 G(5')ppp(5')G(also known as m 7 GpppG)), the DNA template is transcribed using either bacterial or bacteriophage RNA polymerase. RNA polymerase is transcribed by m 7 The 3'-OH of the guanosine portion of GpppG initiates transcription by nucleophilic attack on the α-phosphate of the next template nucleoside triphosphate (pppN), producing the intermediate m 7 GpppGpN (where N is the second base of the RNA molecule). By setting the molar ratio of cap to GTP to 5 to 10 during in vitro transcription, the formation of the competing GTP initiation product pppGpN can be inhibited.

[0346] In some preferred embodiments of the invention, the 5' cap (if present) is a 5' cap analog. These embodiments are particularly suitable if the RNA is obtained by in vitro transcription, such as in vitro transcribed RNA (IVT-RNA). Cap analogs have been initially described to facilitate large-scale synthesis of RNA transcripts by in vitro transcription.

[0347] For messenger RNA, several cap analogs (synthetic caps) have been described so far in general, and all of them can be used in the context of the present invention. Ideally, a cap analog is selected that is associated with higher translation efficiency and / or increased resistance to in vitro degradation and / or increased resistance to in vivo degradation.

[0348] Preferably, a cap analog is used that can be incorporated into the RNA chain in only one orientation. Pasquinelli et al., 1995, RNA J., 1: 957-967) showed that during in vitro transcription, bacteriophage RNA polymerase uses a 7-methylguanosine unit to initiate transcription, with about 40% to 50% of the capped transcripts having the cap dinucleotide in the opposite orientation (i.e., the initial reaction product is Gpppm 7 GpN). RNA with an inverted cap is ineffective in translating the nucleic acid sequence into protein compared to RNA with a correct cap. Therefore, it is desirable to incorporate the cap in the correct orientation, i.e., to produce an RNA with a cap substantially corresponding to m 7 GpppGpN and other structures. It has been shown that reverse integration of the cap-dinucleotide is inhibited by substitution of either the 2'- or 3'-OH group of the guanosine unit with a methylated guanosine (Stepinski et al., 2001, RNA J. 7: 1486-1495; Peng et al., 2002, Org. Lett. 24: 161-164). 7 RNA synthesized in the presence of such an "anti-reverse cap analogue" is more efficiently translated than RNA transcribed in vitro in the presence of GpppG. For this purpose, a cap analogue is described in which the 3'OH group of the methylated guanosine unit is replaced by OCH3, for example Holtkamp et al., 2006, Blood, 108: 4009-4017 (7-methyl (3'-O-methyl) GpppG; anti-reverse cap analog (anti-reverse cap analog, ARCA)). ARCA is a suitable cap dinucleotide according to the present invention.

[0349]

[0350] In one embodiment, the RNA of the present invention is substantially not easy to remove the cap. This is important because, in general, the amount of protein produced from the synthetic mRNA introduced into the cultured mammalian cells is limited by the natural degradation of the mRNA. An in vivo approach for mRNA degradation begins with the removal of the mRNA cap. This removal is catalyzed by a heterodimeric pyrophosphatase containing a regulatory subunit (Dcp1) and a catalytic subunit (Dcp2). The catalytic subunit cuts between the α and β phosphate groups of the triphosphate bridge. In the present invention, a cap analogue that is insensitive or less sensitive to this type of cutting may be selected or present. For this purpose, a suitable cap analogue may be selected from a cap dinucleotide according to formula (I):

[0351]

[0352] Where R 1is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl,

[0353] R 2 and R 3 are independently selected from H, halogen, OH and optionally substituted alkoxy, or R 2 and R 3 Together they form OXO, wherein X is selected from optionally substituted CH2, CH2CH2, CH2CH2CH2, CH2CH(CH3), and

[0354] C(CH3)2, or R 2 With R 2 The hydrogen atom at the 4' position of the ring connected to is combined to form -O-CH2- or -CH2-O-,

[0355] R 5 Selected from S, Se and BH3,

[0356] R 4 and R 6 Independently selected from O, S, Se and BH3.

[0357] n is 1, 2 or 3.

[0358] R 1 , R 2 , R 3 , R 4 , R 5 , R 6 Some preferred embodiments of are disclosed in WO 2011 / 015347 A1 and can be selected accordingly in the present invention.

[0359] For example, in one embodiment, the RNA of the invention comprises a phosphorothioate cap analog. A phosphorothioate-cap analog is a specific cap analog in which one of the three non-bridging O atoms in the triphosphate chain is replaced by an S atom, i.e., R in formula (I) 4 , R 5 or R 6 One of them is S. Phosphorothioate-cap analogs have been described by Kowalska et al., 2008, RNA, 14: 1119-1131 as a solution to the undesirable decapping process and thus improve the stability of RNA in vivo. In particular, the replacement of the oxygen atom with a sulfur atom on the β-phosphate group of the 5' cap leads to stabilization against Dcp2. In this preferred embodiment of the invention, R in formula (I) 5 is S; and R 4 and R 6is O.

[0360] In another embodiment, the RNA of the invention comprises a phosphorothioate-cap analog, wherein the phosphorothioate modification of the RNA 5' cap is combined with an "anti-reverse cap analog" (ARCA) modification. Corresponding ARCA-phosphorothioate-cap analogs are described in WO 2008 / 157688 A2, and they can all be used in the RNA of the invention. In this embodiment, R in formula (I) 2 or R 3 At least one of them is not OH, preferably R 2 and R 3 One of them is a methoxy group (OCH3), and R 2 and R 3 In a preferred embodiment, the oxygen atom on the β-phosphate group is replaced by a sulfur atom (so that R in formula (I) 5 is S; and R 4 and R 6 ARCA is described as having a much higher affinity for eIF4E than the corresponding ARCA lacking the phosphorothioate group.

[0361] The corresponding cap analogs (i.e., 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). Therefore, in one embodiment of the present invention, the RNA of the present invention is modified with β-S-ARCA. β-S-ARCA is represented by the following structure:

[0362]

[0363] In general, replacing the oxygen atom with a sulfur atom at the bridged phosphate gives thiophosphate diastereomers, which are referred to as D1 and D2 based on their elution patterns on HPLC. In short, "D1 diastereomer of β-S-ARCA" or "β-S-ARCA (D1)" is the diastereomer of β-S-ARCA that elutes first on the HPLC column compared to the D2 diastereomer of β-S-ARCA (β-S-ARCA (D2)), and therefore exhibits a shorter retention time. Determination of stereochemical configuration by HPLC is described in WO 2011 / 015347 A1.

[0364] In a first particularly preferred embodiment of the present invention, the RNA of the present invention is modified with a β-S-ARCA (D2) diastereomer. The two diastereomers of β-S-ARCA differ in their sensitivity to nucleases. It has been shown that RNA with 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 an unmodified ARCA 5' cap), while RNA with a β-S-ARCA (D1) 5' cap exhibits moderate sensitivity to Dcp2 cleavage (71% cleavage). It has further been shown that increased stability against Dcp2 cleavage is associated with increased protein expression in mammalian cells. In particular, it has been shown that RNA with a β-S-ARCA (D2) cap is more efficiently translated in mammalian cells than RNA with a β-S-ARCA (D1) cap. Therefore, in one embodiment of the present invention, the RNA of the present invention is modified with a cap analog according to formula (I), characterized in that the substituent R is contained in formula (I) 5 The stereochemical configuration at the P atom of β-S-ARCA corresponds to the P of the D2 diastereomer of β-S-ARCA. β In this embodiment, R in formula (I) 5 is S; and R 4 and R 6 is O. In addition, R in formula (I) 2 or R 3 At least one of is preferably not OH, preferably R 2 and R 3 One of them is a methoxy group (OCH3), and R 2 and R 3 The other one of is preferably OH.

[0365] In a second particularly preferred embodiment, the RNA of the present invention is modified with a β-S-ARCA (D1) diastereomer. This embodiment is particularly suitable for transferring capped RNA into immature antigen-presenting cells, for example for vaccination purposes. It has been shown that when the corresponding capped RNA is transferred into immature antigen-presenting cells, the β-S-ARCA (D1) diastereomer is particularly suitable for improving the stability of RNA, improving the translation efficiency of RNA, prolonging the translation of RNA, increasing the total protein expression of RNA, and / or increasing the immune response to the antigen or antigenic peptide encoded by the RNA (Kuhn et al., 2010, Gene Ther. 17: 961-971). Therefore, in an alternative embodiment of the present invention, the RNA of the present invention is modified with a cap analog according to formula (I), characterized in that the formula (I) contains a substituent R 5 The stereochemical configuration at the P atom of β-S-ARCA corresponds to the P of the D1 diastereomer of β-S-ARCA. β The stereochemical configuration at the atom. Corresponding cap analogs and embodiments thereof are described in WO 2011 / 015347 A1 and Kuhn et al., 2010, Gene Ther. 17:961-971. Any cap analog described in WO 2011 / 015347 A1 can be used in the present invention, wherein the substituent R 5 The stereochemical configuration at the P atom of β-S-ARCA corresponds to the P of the D1 diastereomer β Preferably, R in formula (I) 5 is S; and R 4 and R 6 is O. In addition, R in formula (I) 2 or R 3 At least one of is preferably not OH, preferably R 2 and R 3 One of them is a methoxy group (OCH3), and R 2 and R 3 The other one of is preferably OH.

[0366] In one embodiment, the RNA of the present invention is modified with a 5' cap structure according to formula (I), wherein any one of the phosphate groups is replaced by a borophosphate group or a selenophosphate group. Such a cap has improved stability both in vitro and in vivo. Optionally, the corresponding compound has a 2'-O- or 3'-O-alkyl group (wherein the alkyl group is preferably a methyl group); the corresponding cap analog is called BH3-ARCA or Se-ARCA. As described in WO 2009 / 149253 A2, compounds particularly suitable for mRNA capping include β-BH3-ARCA and β-Se-ARCA. For these compounds, preferably, the substituent R is included in formula (I). 5 The stereochemical configuration at the P atom of β-S-ARCA corresponds to the P of the D1 diastereomer β The stereochemical configuration at an atom.

[0367] In one embodiment, the 5' cap can be a CleanCap supplied by Trilink Biotechnologies, San Diego, CA, which has the following structure:

[0368]

[0369] UTR

[0370] The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or to a corresponding region in an RNA molecule (e.g., an mRNA molecule). An untranslated region (UTR) may be located 5' (upstream) of an open reading frame (5'-UTR) and / or 3' (downstream) of an open reading frame (3'-UTR).

[0371] If present, the 3'-UTR is located at the 3' end of the gene, downstream of the stop codon of the protein coding region, but the term "3'-UTR" preferably does not include the poly(A) tail. Thus, the 3'-UTR is located upstream of the poly(A) tail (if present), for example, immediately adjacent to the poly(A) tail.

[0372] The 5'-UTR, if present, is located at the 5' end of the gene, upstream of the start codon of the protein coding region. The 5'-UTR is located downstream of the 5' cap (if present), for example, immediately adjacent to the 5' cap.

[0373] According to the invention, the 5' and / or 3' untranslated regions may be functionally linked to the open reading frame in such a way that these regions are associated with the open reading frame: the stability and / or the translation efficiency of the RNA comprising the open reading frame is increased.

[0374] In some embodiments, the RNA replicon according to the invention comprises a 5'-UTR and / or a 3'-UTR.

[0375] UTR is related to the stability and translation efficiency of RNA. In addition to the structural modifications involving the 5' cap and / or 3' poly (A) tail as described herein, these two can be improved by selecting specific 5' and / or 3' untranslated regions (untranslated region, UTR). It is generally believed that the sequence elements in UTR will affect the translation efficiency (mainly 5'-UTR) and RNA stability (mainly 3'-UTR). Preferably, there is an active 5'-UTR to improve the translation efficiency and / or stability of the RNA replicon. Independently or additionally, it is preferred that there is an active 3'-UTR to improve the translation efficiency and / or stability of the RNA replicon.

[0376] The terms "active to increase translation efficiency" and / or "active to increase stability" with respect to a first nucleic acid sequence (e.g., UTR) mean that the first nucleic acid sequence is capable of altering the translation efficiency and / or stability of a second nucleic acid sequence in a co-transcript with the second nucleic acid sequence in such a way that the translation efficiency and / or stability is increased compared to the translation efficiency and / or stability of the second nucleic acid sequence in the absence of the first nucleic acid sequence.

[0377] 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 alphavirus from which the functional alphavirus non-structural proteins are derived. This allows the non-translated regions to be designed according to the desired translation efficiency and RNA stability. Thus, heterologous or non-native UTRs allow a high degree of flexibility, and this flexibility is advantageous compared to native alphavirus UTRs.

[0378] Preferably, the RNA replicon according to the invention comprises a 5'-UTR and / or a 3'-UTR which is not of viral origin, in particular not of alphavirus 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.

[0379] The 5'-UTR according to the invention may comprise any combination of more than one nucleic acid sequence, optionally separated by a linker.The 3'-UTR according to the invention may comprise any combination of more than one nucleic acid sequence, optionally separated by a linker.

[0380] The term "linker" according to the present invention relates to a nucleic acid sequence added between two nucleic acid sequences to connect the two nucleic acid sequences. There is no particular limitation on the linker sequence.

[0381] 3'-UTR generally has a length of 200 to 2000 nucleotides, such as 500 to 1500 nucleotides. The 3' untranslated region of immunoglobulin mRNA is relatively short (less than about 300 nucleotides), while the 3' untranslated region of other genes is relatively long. For example, the length of the 3'-untranslated region of tPA is about 800 nucleotides, the length of the 3'-untranslated region of factor VIII is about 1800 nucleotides, and the length of the 3'-untranslated region of erythropoietin is about 560 nucleotides. The 3' untranslated region of mammalian mRNA generally has a homology region known as the AAUAAA hexanucleotide sequence. This sequence can be a poly (A) attachment signal, and is generally located 10 to 30 bases upstream of the poly (A) attachment site. The 3'-untranslated region may include one or more inverted repeats, which may fold to form a stem-loop structure that acts as a barrier to exoribonucleases or interacts with proteins (such as RNA binding proteins) known to improve RNA stability.

[0382] Human β-globin 3'-UTR, in particular two consecutive identical copies of human β-globin 3'-UTR contribute to high transcript stability and translation efficiency (Holtkamp et al., 2006, Blood 108: 4009-4017). Therefore, in one embodiment, the RNA replicon according to the present invention comprises two consecutive identical copies of human β-globin 3'-UTR. Therefore, it comprises in the 5'→3' direction: (a) an optional 5'-UTR; (b) an open reading frame; (c) a 3'-UTR; the 3'-UTR comprises two consecutive identical copies of human β-globin 3'-UTR, a fragment thereof, or a variant of human β-globin 3'-UTR or a fragment thereof.

[0383] In one embodiment, the RNA replicon according to the present invention comprises a 3'-UTR having activity to improve translation efficiency and / or stability, but the 3'-UTR is not human β-globin 3'-UTR, a fragment thereof, or a variant of human β-globin 3'-UTR or a fragment thereof.

[0384] In one embodiment, the RNA replicon according to the present invention comprises a 5'-UTR which is active to increase translation efficiency and / or stability.

[0385] Poly(A) sequence

[0386] In some embodiments, the replicon according to the invention comprises a 3'-poly(A) sequence. If the replicon comprises conserved sequence element 4 (CSE 4), the 3'-poly(A) sequence of the replicon is preferably present downstream of CSE 4, most preferably immediately adjacent to CSE 4.

[0387] According to the present invention, in one embodiment, the poly(A) sequence comprises or 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, and in particular at most 150 A nucleotides, and in particular about 120 A nucleotides. In the present context, "consisting essentially of" means that most of the nucleotides in the poly(A) sequence, usually at least 50%, and preferably at least 75% by 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 the present context, "consisting of" means all nucleotides in the poly(A) sequence, i.e. 100% by number of nucleotides in the poly(A) sequence are A nucleotides. The term "A nucleotide" or "A" refers to adenylic acid.

[0388] Indeed, it has been shown that a 3' poly(A) sequence of approximately 120 A nucleotides has a beneficial effect on the levels of RNA in transfected eukaryotic cells as well as the levels of protein translated from the open reading frame present upstream (5') of the 3' poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0389] In alphaviruses, a 3'poly(A) sequence of at least 11 consecutive adenylic acid residues or at least 25 consecutive adenylic acid residues is considered important for efficient synthesis of the negative strand. In particular, in alphaviruses, a 3'poly(A) sequence of at least 25 consecutive adenylic acid residues is understood to work together with conserved sequence element 4 (CSE 4) to promote synthesis of the (-) strand (Hardy & Rice, 2005, J. Virol. 79: 4630-4639).

[0390] The present invention provides a 3' poly(A) sequence to be ligated during RNA transcription (i.e., during the preparation of in vitro transcribed RNA), the process being based on a DNA template containing repeated dT nucleotides (deoxythymidylic acid) in a strand complementary to the coding strand. The DNA sequence encoding the poly(A) sequence (coding strand) is called a poly(A) box.

[0391] In a preferred embodiment of the present invention, the 3'poly (A) box present in the coding strand of the DNA is essentially composed of dA nucleotides, but is interrupted by a random sequence with an equal distribution of four nucleotides (dA, dC, dG, dT). The length of such a random sequence can be 5 to 50, preferably 10 to 30, more preferably 10 to 20 nucleotides. Such a box is disclosed in WO 2016 / 005004A1. Any poly (A) box disclosed in WO 2016 / 005004 A1 can be used in the present invention. The poly (A) box essentially composed of dA nucleotides but interrupted by a random sequence shows constant propagation of plasmid DNA in Escherichia coli (E. coli) at the DNA level, and is still associated with beneficial properties regarding supporting RNA stability and translation efficiency at the RNA level, wherein the random sequence has an equal distribution of four nucleotides (dA, dC, dG, dT) and a length of, for example, 5 to 50 nucleotides.

[0392] Therefore, in a preferred embodiment of the present invention, the 3'poly (A) sequence contained in the RNA molecule described herein is essentially composed of A nucleotides, but is interrupted by a random sequence having an equal distribution of four nucleotides (A, C, G, U). The length of such a random sequence can be 5 to 50, preferably 10 to 30, more preferably 10 to 20 nucleotides.

[0393] Codon usage

[0394] In general, the degeneracy of the genetic code will allow certain codons (base triplets encoding amino acids) present in the RNA sequence to be replaced by other codons (base triplets) while maintaining the same coding capacity (so that the replacement codon encodes the same amino acid as the replaced codon). In some embodiments of the invention, at least one codon of the open reading frame comprised by the RNA (rRNA) molecule is different from the corresponding codon in the corresponding open reading frame of the species from which the open reading frame originates. In this embodiment, the coding sequence of the open reading frame is referred to as being "adapted" or "modified". The coding sequence of the open reading frame comprised by the replicon can be adapted.

[0395] For example, when adapting the coding sequence of an open reading frame, commonly used codons may be selected: WO 2009 / 024567 A1 describes the adaptation of the coding sequence of a nucleic acid molecule, involving the replacement of rare codons with more commonly used codons. Since the frequency of codon usage depends on the host cell or host organism, this type of adaptation is suitable for making the nucleic acid sequence suitable for expression in a specific host cell or host organism. In general, although it is not always necessary to adapt all codons of an open reading frame, the more commonly used codons are usually more efficiently translated in a host cell or host organism.

[0396] For example, when adapting the coding sequence of the open reading frame, the content of G (guanylic acid) residues and C (cytidylic acid) residues can be changed by selecting the codon with the highest GC-rich content for each amino acid. RNA molecules with GC-rich open reading frames have been reported to have the potential to reduce immune activation and improve RNA translation and half-life (Thess et al., 2015, Mol. Ther. 23: 1457-1465).

[0397] In particular, the coding sequences of the nonstructural proteins can be adapted as desired. This freedom is possible due to the fact that the open reading frames encoding the nonstructural proteins do not overlap with the 5' replication recognition sequence of the replicon.

[0398] Safety features of embodiments of the present invention

[0399] The following features, alone or in any suitable combination, are preferred in the present invention:

[0400] The replicon of the present invention is not particle-forming. This means that after the host cell is inoculated with the replicon of the present invention, the host cell does not produce virus particles, such as the next generation of virus particles. In one embodiment, the RNA replicon according to the present invention is completely free of genetic information encoding any viral structural protein, such as alphavirus structural protein (e.g. 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. The alphavirus packaging signal (White et al., 1998, J. Virol. 72: 4320-4326) contained in the coding region of nsP2 of SFV can be removed, for example, by deletion or mutation. Suitable methods for removing the alphavirus packaging signal include adaptation of the codon usage of the nsP2 coding region. The degeneracy of the genetic code allows the function of the packaging signal to be deleted without affecting the amino acid sequence of the encoded nsP2.

[0401] DNA

[0402] The present invention also provides a DNA comprising a nucleic acid sequence encoding the RNA replicon according to the present invention.

[0403] Preferably, the DNA is double-stranded.

[0404] In a preferred embodiment, the DNA is a plasmid.The term "plasmid" as used herein generally relates to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of the chromosomal DNA.

[0405] DNA of the present invention may comprise a promoter that can be recognized by a DNA-dependent RNA polymerase. This allows the RNA of the coding (e.g., RNA of the present invention) to be transcribed in vivo or in vitro. IVT vectors can be used as templates for in vitro transcription in a standardized manner. Examples of preferred promoters according to the present invention are promoters of SP6, T3 or T7 polymerases.

[0406] In one embodiment, the DNA of the present invention is an isolated nucleic acid molecule.

[0407] Methods for preparing RNA

[0408] The RNA molecules according to the 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, in particular a DNA molecule. The DNA molecules of the invention are suitable for such purposes, in particular if they contain a promoter that can be recognized by a DNA-dependent RNA polymerase.

[0409] The rRNA according to the present invention can be synthesized in vitro. This allows the addition of cap analogs in an in vitro transcription reaction. Typically, the poly(A) tail is encoded by a poly-(dT) sequence on a DNA template. Alternatively, capping and poly(A) tail addition can be achieved enzymatically after transcription.

[0410] In vitro transcription methods are known to the skilled person. Various in vitro transcription kits are commercially available, for example as described in WO 2011 / 015347 A1.

[0411] Reagent test kit

[0412] The invention also provides a kit comprising at least two RNA replicons according to the invention.

[0413] In one embodiment, the components of the test kit are present as separate entities. For example, one component of the test kit may be present in one entity, and another component of the test kit may be present in a separate entity. For example, an open or closed container is a suitable entity. A closed container is preferred. The container used should preferably be free of RNase or substantially free of RNase.

[0414] In one embodiment, the kits of the invention comprise RNA for cell inoculation and / or administration to a human or animal subject.

[0415] The kit according to the invention optionally comprises a label or other form of information element, such as an electronic data carrier. The label or information element preferably comprises instructions, such as printed written instructions or instructions in electronic form, which are optionally printable. The instructions may relate to at least one suitable possible use of the kit.

[0416] Pharmaceutical composition

[0417] The RNA replicon composition described herein can be present in the form of a pharmaceutical composition. The pharmaceutical composition according to the present invention may comprise at least one nucleic acid molecule according to the present invention. The pharmaceutical composition according to the present invention comprises a pharmaceutically acceptable diluent and / or a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable carrier. The selection of pharmaceutically acceptable carriers, carriers, excipients or diluents is not particularly limited. Any suitable pharmaceutically acceptable carrier, carrier, excipient or diluent known in the art can be used.

[0418] In one embodiment of the invention, the pharmaceutical composition may further comprise a solvent, such as an aqueous solvent or any solvent that allows the integrity of the rRNA to be retained. In a preferred embodiment, the pharmaceutical composition is an aqueous solution comprising RNA. The aqueous solution may optionally comprise a solute, such as a salt.

[0419] 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 the corresponding aqueous composition.

[0420] In one embodiment, the pharmaceutical composition comprises at least one cationic entity. Generally speaking, cationic lipids, cationic polymers and other positively charged substances can form complexes with negatively charged nucleic acids. By compounding with cationic compounds, preferably polycationic compounds, such as cations or polycationic peptides or proteins, the RNA according to the present invention can be stabilized. In one embodiment, the pharmaceutical composition according to the present invention comprises at least one cationic molecule selected from protamine, polyethyleneimine, poly-L-lysine, poly-L-arginine, histones or cationic lipids.

[0421] According to the present invention, cationic lipids are cationic amphiphilic molecules, such as molecules comprising at least one hydrophilic and lipophilic part. Cationic lipids can be monocationic or polycationic. Cationic lipids generally have a lipophilic part, such as a sterol, an acyl group or a diacyl chain, and have a total net positive charge. The head group of the lipid generally carries a positive charge. Cationic lipids preferably have a positive charge of 1 to 10 valencies, more preferably a positive charge of 1 to 3 valencies, and more preferably a positive charge of 1 valency. Examples of cationic lipids include, but are not limited to, 1,2-di-O-octadecenyl-3-trimethylammonium propane (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-dialkoxy-3-dimethylammonium propane; dioctadecyldimethyl ammonium chloride (dioctadecyldimethyl Cationic lipids include 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane, DLinDMA). Cationic lipids are suitable for preparing RNA in lipid formulations as described herein, such as liposomes, emulsions and lipid complexes. Typically, the positive charge is contributed by at least one cationic lipid and the negative charge is contributed by the RNA.In one embodiment, the pharmaceutical composition comprises 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 lipidoid molecule that has no similarity to a natural lipid. In the case where the pharmaceutical composition comprises both a cationic lipid and a helper lipid, the molar ratio of the cationic lipid to the neutral lipid can be appropriately determined taking into account the stability of the preparation, etc.

[0422] In one embodiment, the pharmaceutical composition according to the present invention comprises protamine. According to the present invention, protamine can be used as a cationic carrier agent. The term "protamine" refers to any of a variety of relatively low molecular weight strongly basic proteins, which are rich in arginine, and are found to be particularly associated with DNA, to replace the somatic histones in the sperm cells of animals (e.g., fish). In particular, the term "protamine" refers to a protein present in fish semen, which is strongly alkaline, soluble in water, not heat-condensed and comprising multiple arginine monomers. According to the present invention, as used herein, the term "protamine" means any protamine amino acid sequence obtained or sourced from natural or biological sources, the amino acid sequence including its fragments and the polymeric form of the amino acid sequence or its fragments. In addition, the term encompasses (synthetic) polypeptides, which are artificial and are specially designed for a specific purpose, and can not be separated from natural or biological sources.

[0423] In some embodiments, the compositions of the present invention may include one or more adjuvants. Adjuvants may be added to vaccines to stimulate the response of the immune system; adjuvants themselves generally do not provide immunity. Exemplary adjuvants include, but are not limited to, the following: inorganic compounds (e.g., alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide); mineral oils (e.g., paraffin oil), cytokines (e.g., IL-1, IL-2, IL-12); immunostimulatory polynucleotides (e.g., RNA or DNA; e.g., oligonucleotides containing CpG); saponins (e.g., plant saponins from Quillaja, soybean, Polygala senega); oil emulsions or liposomes; polyoxyethylene ether and polyoxyethylene ester preparations; polyphosphazene (PCPP); muramyl peptides; imidazoquinolone compounds; thiosemicarbazide compounds; Flt3 ligands (WO 2010 / 066418 A1); or any other adjuvants known to those skilled in the art. A preferred adjuvant for administering RNA according to the invention is Flt3 ligand (WO 2010 / 066418A1).When Flt3 ligand is administered together with RNA encoding an antigen, a strong increase in antigen-specific CD8+ T cells can be observed.

[0424] The pharmaceutical composition according to the invention may be buffered (eg with acetate buffer, citrate buffer, succinate buffer, Tris buffer, phosphate buffer).

[0425] RNA-containing particles

[0426] In some embodiments, due to the instability of unprotected RNA, it is advantageous to provide the RNA molecules of the present invention in a composite or encapsulated form. The present invention provides corresponding pharmaceutical compositions. In particular, in some embodiments, the pharmaceutical compositions of the present invention comprise particles containing nucleic acids, preferably particles containing RNA. The corresponding pharmaceutical compositions are referred to as granule preparations. In the granule preparations according to the present invention, the particles comprise nucleic acids according to the present invention and pharmaceutically acceptable carriers or pharmaceutically acceptable carriers suitable for delivering nucleic acids. The particles containing nucleic acids may be, for example, in the form of protein particles or in the form of particles containing lipids. Suitable proteins or lipids are referred to as particle forming agents. It has been previously described that protein particles and particles containing lipids are suitable for delivering alphavirus RNA in the form of particles (e.g., Strauss & Strauss, 1994, Microbiol. Rev. 58: 491-562). In particular, alphavirus structural proteins (e.g., provided by helper viruses) are suitable carriers for delivering RNA in the form of protein particles.

[0427] In one embodiment, the particle formulation of the present invention is a nanoparticle formulation. In this embodiment, the composition according to the present invention comprises the nucleic acid according to the present invention in the form of nanoparticles. Nanoparticle formulations can be obtained by various schemes and with various complexing compounds. Lipids, polymers, oligomers or amphiphilic substances are typical components of nanoparticle formulations.

[0428] As used herein, the term "nano-particle" refers to any particle with a diameter that makes the particle suitable for whole body, particularly parenteral administration, particularly a diameter that is generally 1000 nanometers (nm) or smaller nucleic acids. In one embodiment, the mean diameter of the nano-particle is from about 50nm to about 1000nm, preferably from about 50nm to about 400nm, preferably from about 100nm to about 300nm, for example from about 150nm to about 200nm. In one embodiment, the diameter of the nano-particle is from about 200 to about 700nm, from about 200 to about 600nm, preferably from about 250 to about 550nm, particularly from about 300 to about 500nm or from about 200 to about 400nm. In one embodiment, the mean diameter is from about 50 to 150nm, preferably from about 60 to 120nm. In one embodiment, the mean diameter is less than 50nm.

[0429] In one embodiment, the polydispersity index (PI) of the nanoparticles described herein as measured by dynamic light scattering is 0.5 or less, preferably 0.4 or less, or even more preferably 0.3 or less. "Polydispersity index" (PI) is a measure of the uniform or heterogeneous size distribution of individual particles (e.g., liposomes) in a mixture of particles, and indicates the width of the distribution of particles in the mixture. For example, PI can be determined as described in WO 2013 / 143555 A1.

[0430] As used herein, the term "nanoparticle formulation" or similar terms refers to any particle formulation comprising at least one nanoparticle. In some embodiments, the nanoparticle composition is a uniform collection of nanoparticles. In some embodiments, the nanoparticle composition is a pharmaceutical formulation containing lipids, such as a liposomal formulation or an emulsion.

[0431] Pharmaceutical compositions containing lipids

[0432] In one embodiment, the pharmaceutical composition of the present invention comprises at least one lipid. Preferably, at least one lipid is a cationic lipid. The pharmaceutical composition containing lipids comprises a nucleic acid according to the present invention. In one embodiment, the pharmaceutical composition according to the present invention comprises RNA encapsulated in a vesicle (e.g., liposome). In one embodiment, the pharmaceutical composition according to the present invention comprises RNA in the form of an emulsion. In one embodiment, the pharmaceutical composition according to the present invention comprises rRNA complexed with a cationic compound, thereby forming, for example, a so-called lipid complex or a polycomplex. RNA encapsulation in a vesicle (e.g., liposome) is different from, for example, a lipid / RNA complex. Lipid / RNA complexes can be obtained, for example, when RNA is mixed with preformed liposomes.

[0433] In one embodiment, the pharmaceutical composition according to the present invention comprises rRNA encapsulated in a vesicle. Such a preparation is a specific particle preparation according to the present invention. Vesicles are lipid bilayers rolled into a spherical shell, which enclose a small space and separate the space from the space outside the vesicle. Generally speaking, the space inside the vesicle is an aqueous space, i.e., it contains water. Generally speaking, the space outside the vesicle is an aqueous space, i.e., it contains water. The lipid bilayer is formed by one or more lipids (lipids forming vesicles). The membrane surrounding the vesicle is a lamellar phase, similar to the lamellar phase of the plasma membrane. The vesicle according to the present invention can be a multilamellar vesicle, a unilamellar vesicle, or a mixture thereof. When encapsulated in a vesicle, rRNA is usually separated from any external medium. Therefore, it exists in a protected form, which is functionally equivalent to the protected form in natural alphaviruses. Suitable vesicles are particles as described herein, particularly nanoparticles.

[0434] For example, RNA (rRNA) can be encapsulated in liposomes. In this embodiment, the pharmaceutical composition is or comprises a liposome formulation. Encapsulation in the liposome will usually protect the RNA from RNase digestion. The liposome may contain some external RNA (e.g., on its surface), but at least half of the RNA (and ideally all of it) is encapsulated in the core of the liposome.

[0435] Liposome is a microscopic lipid vesicle, usually with one or more double layers of the lipid (e.g., phospholipid) forming a vesicle, and can encapsulate drugs, such as RNA. Different types of liposomes can be used in the context of the present invention, including but not limited to multilamellar vesicles (MLV), small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), sterically stabilized liposomes (SSL), multivesicular vesicles (MV) and large multivesicular vesicles (LMV) and other double-layer forms known in the art. The size and lamellarity of liposomes will depend on the preparation method. There are multiple other forms of supramolecular organization, in which lipids can be present in aqueous media, including lamellar phases, hexagonal phases and anti-hexagonal phases, cubic phases, micelles, reverse micelles consisting of single layers. These phases can also be obtained in combination with DNA or RNA, and the interaction with RNA and DNA may significantly affect the phase state. Such phases can be present in the nanoparticle RNA formulations of the present invention.

[0436] Liposomes can be formed using standard methods known to the skilled person. Appropriate methods include reverse evaporation, ethanol infusion, dehydration-rehydration, ultrasonic treatment or other suitable methods. After liposome formation, the liposomes can be sized to obtain a liposome population with a substantially uniform size range.

[0437] In a preferred embodiment of the invention, rRNA is present in liposomes comprising at least one cationic lipid. The corresponding liposomes can be formed by a single lipid or by a lipid mixture, provided that at least one cationic lipid is used. Preferred cationic lipids have nitrogen atoms that can be protonated; preferably, such cationic lipids are lipids with tertiary amine groups. A particularly suitable lipid with 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 WO 2012 / 006378 A1: a liposome having a lipid bilayer, the lipid bilayer encapsulating an aqueous core comprising RNA, wherein the lipid bilayer comprises a lipid with a pKa of 5.0 to 7.6, preferably with a tertiary amine group. Preferred cationic lipids with tertiary amine groups include DLinDMA (pKa 5.8), and are generally described in WO 2012 / 031046 A2. According to WO 2012 / 031046 A2, liposomes comprising corresponding compounds are particularly suitable for the encapsulation of RNA, and are therefore suitable for the liposome delivery of RNA. In one embodiment, RNA according to the present invention is present in a liposome formulation, wherein the liposome comprises at least one cationic lipid, the head group of the cationic lipid comprises at least one nitrogen atom (N) that can be protonated, wherein the N:P ratio of liposome and RNA is 1:1 to 20:1. According to the present invention, "N:P ratio" refers to the molar ratio of the nitrogen atom (N) in the cationic lipid included in the particle (e.g., liposome) comprising lipid to the phosphate atom (P) in the RNA, as described in WO 2013 / 006825A1. 1:1 to 20:1 N:P ratios are relevant to the net charge of the liposome and the delivery efficiency of the RNA to vertebrate cells.

[0438] In one embodiment, the rRNA according to the invention is present in a liposomal formulation comprising at least one lipid comprising a polyethylene glycol (PEG) moiety, wherein the RNA is encapsulated in the PEGylated liposomes such that the PEG moiety is present outside the liposomes, as described in WO 2012 / 031043 A1 and WO 2013 / 033563 A1.

[0439] In one embodiment, the rRNA according to the invention is present in a liposomal formulation, wherein the diameter of the liposomes is between 60 and 180 nm, as described in WO 2012 / 030901 A1.

[0440] In one embodiment, the rRNA according to the invention is present in a liposomal formulation, wherein the rRNA-containing liposomes have a net charge close to zero or negative, as disclosed in WO 2013 / 143555 A1.

[0441] In other embodiments, the rRNA according to the present invention is present in the form of an emulsion. Emulsions for delivering nucleic acid molecules (e.g., rRNA molecules) to cells have been described previously. Preferred herein are oil-in-water emulsions. The corresponding emulsion particles comprise an oil core and a cationic lipid. More preferably, a cationic oil-in-water emulsion is present, wherein the RNA according to the present invention is complexed with the emulsion particles. The emulsion particles comprise an oil core and a cationic lipid. The cationic lipid can interact with the negatively charged rRNA, thereby anchoring 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 80nm to 180nm. In one embodiment, the pharmaceutical composition of the present invention is a cationic oil-in-water emulsion, wherein the emulsion particles comprise an oil core and a cationic lipid, as described in WO 2012 / 006380 A2. The rRNA according to the present invention can be present in the form of an emulsion comprising a cationic lipid, wherein the N:P ratio of the emulsion is at least 4:1, as described in WO2013 / 006834 A1. The rRNA according to the invention may be present in the form of a cationic lipid emulsion, as described in WO2013 / 006837 A1. In particular, the composition may comprise rRNA complexed with cationic oil-in-water emulsion particles, wherein the oil / lipid ratio is at least about 8:1 (mol:mol).

[0442] In other embodiments, the pharmaceutical composition according to the present invention comprises RNA in the form of a lipid complex. The term "lipid complex" or "RNA lipid complex" refers to a complex of lipids and nucleic acids (e.g., RNA). Lipid complexes can be formed by cationic (positively charged) liposomes and anionic (negatively charged) nucleic acids. Cationic liposomes may also include neutral "helper" lipids. In the simplest case, lipid complexes are spontaneously formed by mixing nucleic acids with liposomes in a certain mixing scheme, but a variety of other schemes can be applied. It should be understood that the electrostatic interaction between positively charged liposomes and negatively charged nucleic acids is the driving force for the formation of lipid complexes (WO 2013 / 143555A1). In one embodiment of the present invention, the net charge of the RNA lipid complex particles is close to zero or negative. It is known that electrically neutral or negatively charged lipid complexes of RNA and liposomes result in a large amount of RNA expression in splenic dendritic cells (dendritic cells, DC) after systemic administration, and are not related to the increased toxicity reported for positively charged liposomes and lipid complexes (see WO 2013 / 143555A1). Thus, in one embodiment of the present invention, the pharmaceutical composition according to the present invention comprises RNA in the form of nanoparticles, preferably RNA in the form of 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, zeta potential is a scientific term for the electromotive force in a colloidal system. In the present invention, both (a) zeta potential and (b) charge ratio of cationic lipid to RNA in the nanoparticles can be calculated as disclosed in WO 2013 / 143555 A1. In summary, the pharmaceutical composition of the nanoparticle lipoplex formulation with a defined particle size as disclosed in WO 2013 / 143555 A1, wherein the net charge of the particles is close to zero or negative, is a preferred pharmaceutical composition in the context of the present invention.

[0443] In one embodiment, the nucleic acids described herein, such as rRNA, are administered in the form of lipid nanoparticles (LNPs). LNPs may comprise any lipid capable of forming particles that are linked to or encapsulate one or more nucleic acid molecules.

[0444] 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.

[0445] In one embodiment, the LNP comprises a cationic lipid, a neutral lipid, a steroid, a polymer-conjugated lipid; and the RNA encapsulated within or associated with the lipid nanoparticle.

[0446] In one embodiment, LNP comprises 40 to 55 mol percent, 40 to 50 mol percent, 41 to 49 mol percent, 41 to 48 mol percent, 42 to 48 mol percent, 43 to 48 mol percent, 44 to 48 mol percent, 45 to 48 mol percent, 46 to 48 mol percent, 47 to 48 mol percent or 47.2 to 47.8 mol percent of cationic lipid. In one embodiment, 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 percent of cationic lipid.

[0447] In one embodiment, the neutral lipid is present in a concentration range of 5 to 15 mol percent, 7 to 13 mol percent, or 9 to 11 mol percent. In one embodiment, the neutral lipid is present in a concentration of about 9.5, 10, or 10.5 mol percent.

[0448] In one embodiment, the steroid is present in a concentration range of 30 to 50 mol percent, 35 to 45 mol percent, or 38 to 43 mol percent. In one embodiment, the steroid is present in a concentration of about 40, 41, 42, 43, 44, 45, or 46 mol percent.

[0449] In one embodiment, the LNP comprises 1 to 10 mol percent, 1 to 5 mol percent, or 1 to 2.5 mol percent of polymer-conjugated lipid.

[0450] In one embodiment, the LNP comprises 40 to 50 mol percent of a cationic lipid; 5 to 15 mol percent of a neutral lipid; 35 to 45 mol percent of a steroid; 1 to 10 mol percent of a polymer-conjugated lipid; and RNA encapsulated within or associated with the lipid nanoparticle.

[0451] In one embodiment, the mole percentage is determined based on the total moles of lipid present in the lipid nanoparticle.

[0452] In one embodiment, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE and SM. In one embodiment, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In one embodiment, the neutral lipid is DSPC.

[0453] In one embodiment, the steroid is cholesterol.

[0454] In one embodiment, the polymer-conjugated lipid is a pegylated lipid. In one embodiment, the pegylated lipid has the following structure.

[0455]

[0456] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0457] R 12 and R 13 Each is independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has an average value ranging from 30 to 60. In one embodiment, R 12 and R 13 Each independently is a straight chain saturated alkyl chain containing 12 to 16 carbon atoms. In one embodiment, the average value of w ranges from 40 to 55. In one embodiment, the average w is about 45. In one embodiment, R 12 and R 13 Each is independently a straight saturated alkyl chain containing about 14 carbon atoms, and w has an average value of about 45.

[0458] In one embodiment, the PEGylated lipid is DMG-PEG 2000, for example having the following structure:

[0459]

[0460] In some embodiments, the cationic lipid component of the LNP has the structure of Formula (III):

[0461]

[0462] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

[0463] L 1 or L 2One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-,-C(=O)NR a -,NR a C(=O)NR a -,-OC(=O)NR a -or-NR a C(=O)O-, and L 1 or L 2 The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-,-C(=O)NR a -,NR a C(=O)NR a -,-OC(=O)NR a -or-NR a C(=O)O- or direct bond;

[0464] G 1 and G 2 are each independently unsubstituted C1-C 12 Alkylene or C1-C 12 alkenylene;

[0465] G 3 It is C1-C 24 Alkylene, C1-C 24 Alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;

[0466] R a Is H or C1-C 12 alkyl;

[0467] R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 alkenyl;

[0468] R 3 Yes H, QR 5 ,CN,-C(=O)OR 4 ,-OC(=O)R 4 or -NR 5 C(=O)R 4 ;

[0469] R4 It is C1-C 12 alkyl;

[0470] R 5 is H or C1-C6 alkyl; and

[0471] x is 0, 1, or 2.

[0472] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIA) or (IIIB):

[0473]

[0474] in:

[0475] A is a 3- to 8-membered cycloalkyl or cycloalkylene ring;

[0476] R 6 is independently H, OH or C1-C 24 alkyl;

[0477] n is an integer of 1-15.

[0478] In some of the foregoing embodiments of Formula (III), the lipid has structure (IIIA), while in other embodiments, the lipid has structure (IIIB).

[0479] In other embodiments of Formula (III), the lipid has one of the following structures (IIIC) or (IIID):

[0480]

[0481] wherein y and z are each independently an integer from 1 to 12.

[0482] In any of the foregoing embodiments of formula (III), L 1 or L 2 One of them is -O(C=O)-. For example, in some embodiments, L 1 and L 2 In some different embodiments of any of the foregoing, L 1 and L 2 Each is independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of is -(C=O)O-.

[0483] In some different embodiments of Formula (III), the lipid has one of the following structures (IIIE) or (IIIF):

[0485]

[0486] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ):

[0487]

[0488] In some of the foregoing embodiments of Formula (III), n is an integer from 2 to 12, such as from 2 to 8 or from 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.

[0489] In other of the foregoing embodiments of Formula (III), y and z are each independently an integer from 2 to 10. For example, in some embodiments, y and z are each independently an integer from 4 to 9 or 4 to 6.

[0490] In some of the foregoing embodiments of Formula (III), R 6 is H. In other of the foregoing embodiments, R 6 It is C1-C 24 In other embodiments, R 6 It's OH.

[0491] In some embodiments of Formula (III), G 3 In other embodiments, G 3 In various embodiments, G 3 It is a straight chain C1-C 24 Alkylene or straight chain C1-C 24 Alkenylene.

[0492] In other of the foregoing embodiments of Formula (III), R 1 or R 2 or both are C6-C 24 For example, in some embodiments, R 1 and R 2 Each independently has the following structure:

[0493]

[0494] in:

[0495] R 7a and R 7b is independently H or C1-C at each occurrence 12 Alkyl; and

[0496] a is an integer from 2 to 12,

[0497] Where R 7a , R 7b and a are each chosen so that R 1 and R 2 Each independently contains 6 to 20 carbon atoms. For example, in some embodiments, a is an integer from 5 to 9 or from 8 to 12.

[0498] In some of the foregoing embodiments of Formula (III), R 7a At least one occurrence of is H. For example, in some embodiments, R 7a In each occurrence, R is H. In yet other different embodiments of the foregoing, 7b At least one occurrence of is C1-C8 alkyl. For example, in some embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0499] In various embodiments of formula (III), R 1 or R 2 or both have one of the following structures:

[0500]

[0501] In some of the foregoing embodiments of Formula (III), R 3 OH, CN, -C(=O)OR 4 、-OC(=O)R 4 or -NHC(=O)R 4 In some embodiments, R 4 It is methyl or ethyl.

[0502] In various embodiments, the cationic lipid of formula (III) has one of the structures listed in the table below.

[0503] Representative compounds of formula (III).

[0504]

[0505]

[0506]

[0507]

[0508]

[0509] 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.

[0510] In some embodiments, cationic lipid is present in LNP with the amount of about 40 to about 50 molar percent. In one embodiment, neutral lipid is present in LNP with the amount of about 5 to about 15 molar percent. In one embodiment, steroid is present in LNP with the amount of about 35 to about 45 molar percent. In one embodiment, pegylated lipid is present in LNP with the amount of about 1 to about 10 molar percent.

[0511] In some embodiments, the LNP comprises compound III-3 in an amount of about 40 to about 50 mole percent, DSPC in an amount of about 5 to about 15 mole percent, cholesterol in an amount of about 35 to about 45 mole percent, and ALC-0159 in an amount of about 1 to about 10 mole percent.

[0512] In some embodiments, the LNP comprises Compound III-3 in an amount of about 47.5 mole percent, DSPC in an amount of about 10 mole percent, cholesterol in an amount of about 40.7 mole percent, and ALC-0159 in an amount of about 1.8 mole percent.

[0513] In various embodiments, the cationic lipid has one of the structures listed in the table below.

[0514]

[0515] In some embodiments, LNP comprises the cationic lipid shown in the above table, for example the cationic lipid of formula (B) or formula (D), in particular 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.

[0516] In one embodiment, LNP comprises the cationic lipid that is ionizable lipidoid substance (lipidoid).In one embodiment, cationic lipid has following structure:

[0517]

[0518] 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.

[0519] The LNPs described herein may have an average diameter ranging from about 30 nm to about 200 nm, or from about 60 nm to about 120 nm in one embodiment.

[0520] RNA targeting

[0521] Some aspects of the present disclosure relate to targeted delivery of rRNA disclosed herein (eg, RNA encoding vaccine antigens and / or immunostimulatory agents).

[0522] In one embodiment, the disclosure relates to targeting the lung. If the RNA administered is an RNA encoding a vaccine antigen, targeting the lung is particularly preferred. The RNA can be delivered to the lung, for example, by inhalation administration of RNA that can be formulated as particles (e.g., lipid particles) as described herein.

[0523] In one embodiment, the present disclosure relates to targeting the lymphatic system, in particular secondary lymphoid organs, more particularly the spleen. If the administered RNA is RNA encoding a vaccine antigen, targeting the lymphatic system, in particular secondary lymphoid organs, more particularly the spleen is particularly preferred.

[0524] In one embodiment, the target cell is a spleen cell. In one embodiment, the target cell is an antigen presenting cell, such as a professional antigen presenting cell in the spleen. In one embodiment, the target cell is a dendritic cell in the spleen.

[0525] The "lymphatic system" is part of the circulatory system and is an important part of the immune system, comprising a network of lymphatic vessels that transport lymph. The lymphatic system consists of lymphatic organs, a conducting network of lymphatic vessels, and circulating lymph. The primary or central lymphoid organs produce lymphocytes from immature progenitor cells. The thymus and bone marrow constitute the primary lymphoid organs. Secondary or peripheral lymphoid organs (including lymph nodes and spleen) maintain mature initial lymphocytes and initiate adaptive immune responses.

[0526] RNA can be delivered to the spleen by so-called lipoplex formulations, in which RNA is combined with liposomes comprising cationic lipids and optionally additional lipids or auxiliary lipids to form injectable nanoparticle formulations. Liposomes can be obtained by injecting a solution of lipids in ethanol 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 WO 2013 / 143683, which is incorporated herein by reference. It has been found that RNA lipoplex particles with a net negative charge can be used to preferentially target spleen tissue or spleen cells, such as antigen presenting cells, particularly dendritic cells. Therefore, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression occur in the spleen. Therefore, 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, RNA accumulation and / or RNA expression do not occur or substantially do not occur in the lungs and / or liver. In one embodiment, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression occur in antigen presenting cells in the spleen, such as professional antigen presenting cells. Thus, the RNA lipid complex 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.

[0527] The charge of the RNA lipid complex particles of the present disclosure is the sum of the charge present in the at least one cationic lipid and the charge present in the RNA. The charge ratio is the ratio of the positive charge present in the at least one cationic lipid to the negative charge present in the RNA. The charge ratio of the positive charge present in the at least one cationic lipid to the negative charge present in the RNA is calculated by the following equation: Charge ratio = [(cationic lipid concentration (mol)) * (total number of positive charges in the cationic lipid)] / [(RNA concentration (mol)) * (total number of negative charges in the RNA)].

[0528] The spleen-targeted RNA lipoplex particles described herein preferably have a net negative charge at physiological pH, for example, a charge ratio of positive charge to negative charge 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 some specific embodiments, the charge ratio of positive charge to negative charge 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.

[0529] Immunostimulants can be provided to a subject by administering to the subject an RNA encoding the immunostimulant in a formulation for preferentially delivering the RNA to the liver or liver tissue. RNA is preferably delivered to such a target organ or tissue, particularly if it is desired to express a large amount of the immunostimulant and / or if it is desired or required that the immunostimulant be present systemically, particularly in significant amounts.

[0530] RNA delivery systems have an inherent preference for the liver. This involves lipid-based particles, cationic and neutral nanoparticles, particularly lipid nanoparticles, such as liposomes, nanomicelles, and lipophilic ligands, which are in bioconjugates. Liver accumulation is caused by the discontinuous nature of the liver vasculature or lipid metabolism (liposomes and lipid or cholesterol conjugates).

[0531] In order to deliver RNA to the liver in vivo, a drug delivery system can be used to transport RNA to the liver by preventing RNA degradation. For example, the surface coated with poly (ethylene glycol) (PEG) and the complex (polyplex) nano micelles containing the core of mRNA are available systems because nano micelles provide excellent RNA stability in vivo under physiological conditions. In addition, the stealth (stealth) characteristics provided by the complex nano micelle surface consisting of dense PEG fences effectively circumvent host immune defense.

[0532] Some examples of suitable immunostimulants for targeting the liver are cytokines involved in T cell proliferation and / or maintenance. Some examples of suitable cytokines include IL2 or IL7, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as cytokines with extended PK.

[0533] In another embodiment, the RNA encoding the immunostimulatory agent can be used for preferentially delivering RNA to the lymphatic system, particularly secondary lymphoid organs, more particularly the spleen in a preparation for administration. Preferably, the immunostimulatory agent is delivered to such a target tissue, particularly if it is desired that the immunostimulatory agent exists in the organ or tissue (e.g., for inducing an immune response, particularly during T cell sensitization or for activating resident immune cells and requiring an immunostimulatory agent such as a cytokine), while it is not desired that the immunostimulatory agent exists systemically, particularly in significant amounts (e.g., because the immunostimulatory agent has systemic toxicity).

[0534] Some examples of suitable immunostimulants are cytokines involved in T cell sensitization. Some 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 cytokines with extended PK.

[0535] Methods for producing proteins

[0536] The present invention also provides a method for producing a target protein in a cell, comprising the following steps:

[0537] (a) obtaining an RNA replicon composition according to the present invention, which comprises an open reading frame encoding a target protein, and

[0538] (b) Inoculation of RNA replicons into cells.

[0539] In various embodiments of the method, the RNA replicon is as defined above for the RNA replicon of the present invention, as long as the RNA replicon comprises an open reading frame encoding a protein of interest, optionally an open reading frame encoding a functional non-structural protein and can be replicated by a functional non-structural protein, wherein the rRNA may comprise at least one modified nucleotide and one or more point mutations in the regulatory sequence, which point mutations restore or improve the function of the modified rRNA.

[0540] Cells that can be inoculated with one or more nucleic acid molecules can be referred to as "host cells". According to the present invention, the term "host cell" refers to any cell that can be transformed or transfected with exogenous nucleic acid molecules. The term "cell" is preferably a complete cell, i.e., a cell with a complete membrane that does not release its normal intracellular components, such as enzymes, organelles, or genetic material. Complete cells are preferably viable cells, i.e., living cells that can perform their normal metabolic functions. The term "host cell" includes prokaryotic cells (e.g., Escherichia coli (E.coli)) or eukaryotic cells (e.g., human and animal cells, plant cells, yeast cells, and insect cells) according to the present invention. Particularly preferred are mammalian cells, such as cells from humans, mice, hamsters, pigs, domesticated animals (including horses, cattle, sheep, and goats), and primates. Cells can be derived from a variety of tissue types and include primary cells and cell lines. Some 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, a monocyte, or a macrophage. The nucleic acid may be present in a host cell in a single copy or in multiple copies, and in one embodiment is expressed in the host cell.

[0541] The cell may be a prokaryotic cell or a eukaryotic cell. Prokaryotic cells are suitable herein, for example, for the propagation of the DNA according to the invention, and eukaryotic cells are suitable herein, for example, for the expression of the open reading frame of a replicon.

[0542] In the method of the invention, any RNA replicon composition according to the invention or a kit according to the invention or a pharmaceutical composition according to the invention can be used. The RNA can be used in the form of a pharmaceutical composition or as naked RNA, for example for electroporation.

[0543] In the method for producing protein in cells according to the present invention, the cell can be an antigen presenting cell, and the method can be used to express the RNA encoding the antigen. For this reason, the present invention may relate to introducing the RNA encoding the antigen into the antigen presenting cell (e.g., dendritic cell). In order to transfect the antigen presenting cell (e.g., dendritic cell), a pharmaceutical composition comprising the RNA encoding the antigen can be used.

[0544] In one embodiment, the method for producing a protein in a cell is an in vitro method. In one embodiment, the method for producing a protein in a cell does not comprise removing the cell from a human or animal subject by surgery or therapy.

[0545] In this embodiment, cells seeded according to the invention can be administered to a subject to produce a protein in the subject and provide the protein to the subject.The cells can be autologous, syngeneic, allogeneic or xenogeneic with respect to the subject.

[0546] In other embodiments, the cell in the method for producing a protein in a cell may be present in an object, such as a patient. In these embodiments, the method for producing a protein in a cell is an in vivo method, which includes administering an RNA molecule to the object.

[0547] In this aspect, the present invention also provides a method for producing a protein of interest in a subject, comprising the following steps:

[0548] (a) obtaining an RNA replicon according to the present invention, which comprises an open reading frame encoding a protein of interest, and

[0549] (b) Administering the RNA replicon to a subject.

[0550] In various embodiments of the method, the RNA replicon is as defined above for the RNA replicon of the present invention, as long as the RNA replicon comprises an open reading frame encoding a protein of interest, optionally an open reading frame encoding a functional non-structural protein and can be replicated by a functional non-structural protein, wherein the rRNA may comprise at least one modified nucleotide and one or more point mutations in the regulatory sequence, which point mutations restore or improve the function of the modified rRNA.

[0551] Any RNA replicon according to the invention or a kit according to the invention or a pharmaceutical composition according to the invention can be used in the method according to the invention for producing a protein in a subject. For example, in the method of the invention, the RNA can be used in the form of a pharmaceutical composition, for example as described herein, or as naked RNA.

[0552] In view of the ability to be administered to a subject, each RNA replicon according to the invention or the kit according to the invention or the pharmaceutical composition according to the invention may be referred to as a "drug", etc. The present invention contemplates that the RNA replicon, kit and pharmaceutical composition of the invention are provided for use as a drug. The drug can be used to treat a subject. "Treatment" means administering a compound or composition as described herein or other entity to a subject. The term includes methods of treating the human or animal body by therapy.

[0553] The abovementioned medicaments generally do not contain DNA and therefore have additional safety features compared to the DNA vaccines described in the prior art (eg WO 2008 / 119827 A1).

[0554] Alternative medical uses according to the present invention include methods for producing proteins in cells according to the present invention, wherein the cells may be antigen presenting cells, such as dendritic cells, and then the cells are introduced into the subject. For example, RNA encoding a pharmaceutically active protein (such as an antigen) may be introduced (transfected) into antigen presenting cells in vitro, such as those taken from the subject, and the antigen presenting cells optionally cloned and propagated in vitro may be reintroduced into the same or different subject. Any method known in the art may be used to reintroduce the transfected cells into the subject.

[0555] The medicament according to the present invention can be administered to a subject in need thereof. The medicament according to the present invention can be used in a prophylactic as well as a therapeutic method for treating a subject.

[0556] The medicament according to the invention is administered in an effective amount. An "effective amount" relates to an amount sufficient to cause a response or desired effect alone or together with other agents (dose). In the case of a certain disease or a certain condition in the treatment subject, the desired effect is the inhibition of disease progression. This includes the deceleration of disease progression, in particular the interruption of disease progression. The desired effect of treating a disease or condition can also be the delay of disease outbreak or the inhibition of disease outbreak.

[0557] The effective amount will depend on the condition being treated, the severity of the disease, the individual parameters of the patient including age, physical condition, size and weight, the duration of treatment, type of concomitant treatment (if any), the particular mode of administration and other factors.

[0558] Vaccination

[0559] The term "immunization" or "vaccination" generally refers to the process of treating a subject for therapeutic or prophylactic reasons. Treatment, in particular prophylactic treatment, preferably is or includes treatment intended to elicit or enhance an immune response in a subject, for example against one or more antigens. According to the present invention, if it is desired to induce or enhance an immune response by using rRNA as described herein, the immune response can be elicited or enhanced by rRNA. In one embodiment, the present invention provides a prophylactic treatment, which preferably is or includes vaccination of a subject. One embodiment of the present invention is particularly suitable for vaccination, wherein the replicon encodes a pharmaceutically active peptide or a pharmaceutically active protein as a protein of interest, the pharmaceutically active peptide or the pharmaceutically active protein being an immunologically active compound or an antigen.

[0560] RNA has been previously described for vaccination against foreign substances including pathogens or cancer (recently reviewed by Ulmer et al., 2012, Vaccine 30: 4414-4418). Compared to conventional methods in the prior art, the replicon according to the present invention is a particularly suitable element for effective vaccination because it can be replicated by functional alphavirus non-structural proteins as described herein. Vaccination according to the present invention can be used, for example, to elicit an immune response to weakly immunogenic proteins. In the case of RNA vaccines according to the present invention, the protein antigens are never exposed to serum antibodies, but are produced by the transfected cells themselves after RNA translation. Therefore, allergic reactions should not be a problem. Therefore, the present invention allows patients to be repeatedly immunized without the risk of allergic reactions.

[0561] In a method involving vaccination according to the invention, a medicament of the invention is administered to a subject, particularly if it is desired to treat a subject suffering from or at risk of contracting a disease involving the antigen.

[0562] In the method involving vaccination according to the invention, the protein of interest encoded by the replicon according to the invention encodes, for example, a bacterial antigen to be directed by an immune response, a viral antigen to be directed by an immune response, or a cancer antigen to be directed by an immune response, or an antigen of a unicellular organism to be directed by an immune response. The efficacy of the vaccination can be assessed by known standard methods, for example by measuring antigen-specific IgG antibodies from the organism. In the method involving allergen-specific immunotherapy according to the invention, the protein of interest encoded by the replicon according to the invention encodes an antigen associated with an allergy. Allergen-specific immunotherapy (also called desensitization) is defined as the administration of an allergen vaccine, preferably in increasing doses, to an organism with one or more allergies in order to achieve a state of reduced symptoms associated with subsequent exposure to the pathogenic allergen. The efficacy of the allergen-specific immunotherapy can be assessed by known standard methods, for example by measuring allergen-specific IgG and IgE antibodies from the organism.

[0563] The medicament of the present invention can be administered to a subject, for example, for treatment of the subject, including vaccination of the subject.

[0564] The term "subject" relates to vertebrates, in particular mammals. For example, mammals in the context of the present invention are humans, non-human primates, domesticated mammals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., and captive animals such as zoo animals. The term "subject" also relates to non-mammalian vertebrates, such as birds (in particular poultry, such as chickens, ducks, geese, turkeys) and fish (in particular farmed fish, such as salmon or catfish). The term "animal" as used herein also includes humans.

[0565] In some embodiments, administration to domesticated animals such as dogs, cats, rabbits, guinea pigs, hamsters, sheep, cattle, goats, pigs, horses, chickens, ducks, geese, turkeys or wild animals such as foxes is preferred. For example, the preventive vaccination according to the present invention may be suitable for vaccinating animal groups, for example, in agriculture, or wild animal groups. Other animal groups in captivity such as pets or animals in zoos can be vaccinated.

[0566] In one embodiment, the drug can be administered more than once. Multiple doses can be administered so that a single dose can be administered at different time intervals. For example, it can be administered 14 to 35 days after the previous dose was administered. In one embodiment, the dose is administered 21 days after the previous dose. In one embodiment, the dose is administered 35 days after the previous dose.

[0567] In one embodiment, when administered to a subject, the replicon used as a medicament preferably does not contain sequences from a virus type (e.g., an alphavirus) that is infectious to the species or genus to which the subject being treated belongs. Preferably, in this case, the replicon does not contain any nucleotide sequences from an alphavirus that can infect the corresponding species or genus. An advantage of this embodiment is that even if the subject to whom the RNA is administered is infected (e.g., accidentally) with an infectious alphavirus, there is no possibility of recombination with an infectious (e.g., fully functional or wild-type) alphavirus. As an illustrative example, for the treatment of pigs, the replicon used does not contain any nucleotide sequences from an alphavirus that can infect pigs.

[0568] Mode of administration

[0569] The medicament according to the present invention may be administered to a subject by any suitable route.

[0570] For example, the drug can be administered systemically, such as intravenously (iv), intramuscularly (im), subcutaneously (sc), intradermally (id), or by inhalation.

[0571] In one embodiment, the drug according to the present invention is administered subcutaneously to muscle tissue, such as skeletal muscle or skin. It is generally understood that RNA transfer to skin or muscle leads to high and sustained local expression, while strongly inducing humoral and cellular immune responses (Johansson et al., 2012, PLoS. One. 7: e29732; Geall et al., 2012, Proc. Natl. Acad. Sci. USA 109: 14604-14609).

[0572] Alternatives for administration to muscle tissue or skin include, but are not limited to, intradermal, intranasal, intraocular, intraperitoneal, intravenous, interstitial, buccal, transdermal, or sublingual administration. Intradermal and intramuscular administration are two preferred routes.

[0573] Administration can be achieved in a variety of ways. In one embodiment, the medicament according to the present invention is administered by injection. In a preferred embodiment, it is injected by needle. Needle-free injection can be used as an alternative.

[0574] The present invention is described and illustrated in detail by the drawings and examples, which are only for illustrative purposes and are not meant to be limiting. According to the description and examples, the technician can obtain other embodiments that are also included in the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0575] Figure 1 :On days 0 and 21 or on days 0 and 35, C57Bl / 6IFNAR- / - mice were injected intramuscularly with saRNA encoding Ebola virus GP or NP formulated as polyplexes (PLX) or buffer controls. Serum samples were collected before immunization (day -1) before booster immunization (d20 or d34) and at specified times. 1A) Schematic overview of the experimental setup for testing different primary-boost intervals. 1B) Average PLX size and size distribution were determined by: Dynamic light scattering characterization was performed on a DynaPro PlateReader II, and dynamic light scattering (DLS) was used to calculate the hydrodynamic size of nanoparticles on the Wyatt device. LNP samples were diluted in PBS and measured in duplicate. Ten data points were recorded per well, each for 10 seconds. Dyamicsv.7.8.1 (Wyatt Technology) was used to analyze the average size (Z aver...

Claims

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 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 the other replicable RNA molecule, 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.

2. The composition of claim 1, wherein one or both of the at least two replicable RNA molecules comprise a second ORF encoding an RNA-dependent RNA polymerase (replicase) capable of replicating the replicable RNA molecule in cis or in trans.

3. A composition according to claim 1 or 2, wherein the composition further comprises a third RNA molecule encoding a replicase capable of cis or trans replicating the replicable RNA molecule and / or the third RNA molecule.

4. The composition of claim 3, wherein the third RNA molecule is replicable.

5. The composition of claim 3, wherein the third RNA molecule is a non-replicating RNA molecule.

6. The composition of any one of claims 1 to 5, wherein the composition comprises the at least two replicable RNA molecules, each of which does not encode a replicase, and a third non-replicating RNA molecule encoding a replicase.

7. The composition of claim 5 or 6, wherein the non-replicating RNA is mRNA.

8. The composition of any one of claims 1 to 7, wherein the replicable RNA molecule comprises an internal ribosome entry site (IRES) that controls expression of a first ORF encoding a protein or peptide containing an antigen or epitope.

9. The composition of any one of claims 1 to 8, wherein the replicable RNA molecule comprises an internal ribosome entry site (IRES) that controls expression of a second ORF encoding a replicase.

10. The composition of 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 insensitive to 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 a picornavirus, a flavivirus or a dicistrovirus.

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

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

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

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

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

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

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

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

22. The composition of any one of claims 1 to 21, wherein at least one replicable RNA comprises a 5' replication recognition sequence characterized by the removal of at least one start codon compared to a native alphavirus 5' replication recognition sequence.

23. The composition of claim 22, wherein the 5' replication recognition sequence comprises a sequence homologous to an open reading frame of a non-structural protein or a portion thereof from a self-replicating virus, wherein the sequence homologous to an open reading frame of a non-structural protein or a portion thereof from a self-replicating virus is characterized in that it comprises the removal of at least one start codon compared to the native viral sequence.

24. The composition according to claim 23, wherein the sequence homologous to an open reading frame of a non-structural protein from a self-replicating virus or a portion thereof is characterized in that it comprises the removal of at least the native start codon of the open reading frame of a non-structural protein from a self-replicating virus.

25. The composition according to claim 23 or 24, wherein the sequence homologous to the open reading frame of a non-structural protein from a self-replicating virus or a part thereof is characterized in that it comprises the removal of at least one start codon other than the native start codon of the open reading frame of the non-structural protein from a self-replicating virus.

26. The composition according to any one of claims 22 to 25, wherein the sequence homologous to an open reading frame of a nonstructural protein from a self-replicating virus or a portion thereof is characterized in that it does not contain a start codon.

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

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

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

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

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

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

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

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

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

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

37. A 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 a functional non-structural protein from a self-replicating virus.

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

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

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

41. A composition according to any one of claims 1 to 40, wherein the antigen or epitope of the encoded protein or peptide is or is derived from a bacterial, viral, parasitic or fungal antigen.

42. according to the composition described in 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. A 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 obtained or derived from different strains of the same bacteria, virus, parasite or fungus, respectively, or are obtained or derived from different pathogenic organisms, such as different viruses.

44. A 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, and wherein 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 obtained or derived from the same or different strains of the same bacteria, virus, parasite or fungus, respectively, or are obtained or derived from different pathogenic organisms, such as different viruses.

45. A 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, and wherein the protein or peptide encoded by the second replicable RNA is not a surface-expressed protein or peptide and is different from the protein or peptide encoded by the first replicable RNA, the different non-surface-expressed proteins or peptides being obtained or derived from the same or different strains of the same bacteria, virus, parasite or fungus, or from different pathogenic organisms, such as different viruses, respectively.

46. ​​A 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, and wherein the protein or peptide encoded by the second replicable RNA is a surface-expressed protein or peptide and is different from the protein or peptide encoded by the first replicable RNA, and the surface-expressed proteins or peptides are obtained or derived from the same or different strains of the same bacteria, virus, parasite or fungus, or are obtained or derived from different pathogenic organisms, such as different viruses.

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

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

49. The composition of 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, wherein the virus is an enveloped virus.

50. according to the composition described in any one of claims 1 to 49, wherein 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, wherein the glycoprotein and the nucleoprotein are obtained from or derived from the same virus, optionally obtained from or derived from the same strain of the same virus.

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

52. The composition of any one of claims 1 to 51, wherein 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.

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

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

55. The composition of claim 54, wherein the protein or peptide is fused to a targeting motif or a secretion motif.

56. A composition according to any one of claims 1 to 55, wherein 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.

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

58. The composition of any one of claims 1 to 57, wherein 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.

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

60. The composition of any one of claims 1 to 59, wherein the protein or peptide encoded by the first replicable RNA molecule is an 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.

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

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

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

64. The composition of any one of claims 1 to 63, wherein at least one of the replicable RNA molecules does not comprise an open reading frame of a complete alphavirus structural protein.

65. according to the composition described in any one in claim 1 to 64, it also comprises the reagent that can form particle with described replicable RNA molecule.

66. The composition of claim 65, wherein the agent is a lipid or a polyalkylene imine.

67. A composition according to claim 65 or 66, wherein the agent is a lipid comprising a cationic head group.

68. A composition according to any one of claims 65 to 67, wherein the agent is a pH responsive lipid.

69. A composition according to any one of claims 65 to 68, wherein the agent is a pegylated lipid.

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

71. A composition according to any one of claims 65 to 70, wherein the particle formed by the replicable RNA molecule and the agent is a polymer-based polyplex (PLX) or a lipid nanoparticle (LNP), wherein the LNP is preferably a lipid complex (LPX) or a liposome.

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

73. The composition of any one of claims 65 to 72, wherein 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 charge or a net negative charge, and / or (iii) the charge ratio of positive charge to negative charge in the nanoparticle is 1.4:1 or less, and / or (iv) The zeta potential of the nanoparticles is 0 or less.

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

4.

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

76. The composition of claim 75, wherein the positive charge is contributed by the at least one cationic lipid and the negative charge is contributed by the replicable RNA molecule.

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

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

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

80. The composition of any one of claims 77 to 79, wherein the at least one auxiliary lipid comprises 1,2-di-(9Z-octadecanoyl)-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 of 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 10:0 to 3:7, preferably 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.

82. The composition of 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, wherein the molar ratio of DODMA to DOPE is preferably 8:2 to 3:7 and more preferably 7:3 to 5:5, and wherein the charge ratio of positive charges in DOTMA to 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.

83. The composition of any one of claims 71 to 81, wherein the nanoparticles are lipid complexes comprising DODMA and cholesterol in a molar ratio of 10:0 to 1:9, wherein the molar ratio of DODMA to cholesterol is preferably 8:2 to 3:7 and more preferably 7:3 to 5:5, and wherein the charge ratio of positive charges in DOTMA to 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.

84. The composition of any one of claims 71 to 81, wherein the nanoparticles are lipid complexes comprising DODMA and DSPC in a molar ratio of 10:0 to 1:9, wherein the molar ratio of DODMA to DSPC is preferably 8:2 to 3:7 and more preferably 7:3 to 5:5, and wherein the charge ratio of positive charges in DOTMA to 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.

85. The composition of any one of claims 71 to 81, wherein the nanoparticles are lipid complexes comprising DODMA:cholesterol:DOPE:PEGcerC16 in a molar ratio of 40:48:10:

2.

86. The composition of any one of claims 71 to 81, wherein the nanoparticles are lipoplexes comprising DOTMA and DOPE in a molar ratio of 10:0 to 1:9, wherein the molar ratio of DOTMA to DOPE is preferably 8:2 to 3:7 and more preferably 7:3 to 5:5, and wherein the charge ratio of positive charges in DOTMA to 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.

87. The composition of any one of claims 71 to 81, wherein the nanoparticle is a lipid complex comprising DOTMA and cholesterol in a molar ratio of 10:0 to 1:9, wherein the molar ratio of DOTMA to cholesterol is preferably 8:2 to 3:7 and more preferably 7:3 to 5:5, and wherein the charge ratio of positive charges in DOTMA to 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.

88. The composition of any one of claims 71 to 81, wherein the nanoparticles are lipoplexes comprising DOTAP and DOPE in a molar ratio of 10:0 to 1:9, wherein the molar ratio of DOTAP to DOPE is preferably 8:2 to 3:7 and more preferably 7:3 to 5:5, and wherein the charge ratio of positive charges in DOTMA to 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.

89. according to the composition described in any one in claim 65 to 88, wherein said reagent comprises lipid, and the particle formed is the LNP that is compounded with said replicable RNA molecule and / or encapsulates said replicable RNA molecule.

90. The composition of any one of claims 65 to 88, wherein the agent comprises a lipid and the particles formed are vesicles, preferably unilamellar liposomes, encapsulating the replicable RNA molecule.

91. The composition of claim 65 or 66, wherein the agent is a polyalkyleneimine.

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

0.

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

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

95. The composition of any one of claims 91 to 94, wherein the polyalkylene imine comprises the following general formula (I): in: R is H, acyl or a group comprising the following general formula (II): Wherein R1 is H or a group comprising the following general formula (III): n, m and l are independently integers selected from 2 to 10; and p, q and r are integers, wherein the sum of p, q and r is such that the average molecular weight of the polymer is 1.5×10 2 Up to 10 7 Da, preferably 5000 to 10 5 Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da.

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

97. A composition according to claim 95 or 96, wherein R1 is H.

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

99. The composition according to any one of claims 95 to 98, wherein the polyalkylene imine comprises polyethylene imine and / or polypropylene imine, preferably polyethylene imine.

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

101. A composition according to any one of claims 1 to 100, further comprising one or more peptide-based adjuvants, wherein the peptide-based adjuvant optionally comprises an immunomodulatory molecule, such as a cytokine, a lymphokine and / or a co-stimulatory molecule.

102. The composition of any one of claims 1 to 101, further comprising one or more additives, wherein the additives are optionally selected from buffer substances, sugars, stabilizers, cryoprotectants, lyoprotectants and chelating agents.

103. The composition of claim 102, wherein the buffer 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 buffers and the like, phosphoric acid and phosphate buffers, and citric acid and citrate buffers.

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

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

107. The composition of any one of claims 1 to 106, wherein the composition is a vaccine.

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

109. The pharmaceutical composition according to claim 108, which is formulated for intradermal, subcutaneous and / or intramuscular administration, such as by injection.

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

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

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

113. The pharmaceutical composition for use according to claim 112, wherein the injection is performed by using a needle or by using a needle-free injection device.

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

115. A method for inducing an immune response specific for at least two antigens or epitopes in a subject, comprising administering to the subject a 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 activation of B cells, preferably wherein the activated T cells comprise T helper cells and cytotoxic T cells.

117. A pharmaceutical composition for use according to claim 114 or a 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 growth 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, wherein the T helper cells stimulate B cell proliferation, antibody class switching, production and / or secretion of neutralizing antibodies.

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

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

121. A method for 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 said bacterium, virus, parasite or fungus, respectively; 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 the other replicable RNA molecule, 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.

122. The method of claim 121, wherein the composition further comprises 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.

123. The method of claim 121, wherein the immune response is a specific immune response against the bacteria, virus, parasite or fungus, respectively.

124. The method of claim 121 or 123, wherein the immune response reduces the severity of one or more symptoms of infection.

125. The method of any one of claims 121 to 124, wherein the method involves only a single administration of the composition.

126. The method of any one of claims 121 to 124, wherein the method comprises multiple administrations of the composition.

127. The method of any one of claims 121 to 126, further comprising administering the composition as a booster.

128. The method of any one of claims 121 to 127, wherein the infection is a viral infection, optionally wherein the infection is an Ebola virus infection.

129. The method of 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.

130. The method of claim 129, wherein the injecting is performed by using a needle or by using a needle-free injection device.

131. The method of any one of claims 115 and 120 to 128, wherein administering comprises administering by intramuscular injection, preferably comprises administering by intramuscular injection using a needle.

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