RNA replicons for versatile and efficient gene expression

Abstract

The present application relates to RNA replicons for multifunctional and efficient gene expression. The present invention includes RNA replicons that can be replicated by a replicase derived from an alphavirus. The RNA replicon contains sequence elements required for replication by the replicase, but these sequence elements do not encode any protein or fragment thereof. In the RNA replicon according to the present invention, the sequence elements required for replication by the replicase and the protein coding region are decoupled. According to the present invention, decoupling is achieved by removing at least one start codon compared to the natural alphavirus genomic RNA. In particular, the RNA replicon contains a 5' replication recognition sequence that contains at least one start codon removed compared to the natural alphavirus 5' replication recognition sequence. The alphavirus-derived replicase can be encoded by an open reading frame on the RNA replicon or on a separate RNA molecule. Methods for in vitro and in vivo protein production and medical uses are provided herein.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201780019155.1 and invention name “RNA replicon for multifunctional and efficient gene expression”. The original application is the PCT international application PCT / EP2017 / 055808 filed on March 13, 2017, which entered the Chinese national phase on September 21, 2018. Technical Field

[0002] The present invention includes RNA replicons that can be replicated by a replicase derived from an alphavirus. The RNA replicon contains sequence elements required for replication by the replicase, but these sequence elements do 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 are decoupled. According to the present invention, decoupling is achieved by removing at least one start codon compared to the natural alphavirus genomic RNA. The RNA replicon may contain a gene encoding a target protein (e.g., a pharmaceutically active protein). The replicase may be encoded by the RNA replicon or a separate nucleic acid molecule. Background Art

[0003] Nucleic acid molecules comprising genetic information encoding one or more polypeptides for prevention and treatment purposes have been studied in biomedical research for many years. The prior art methods are the same as the delivery of nucleic acid molecules to target cells or organisms, but the types of nucleic acid molecules and / or delivery systems are different: affected by the safety issues associated with the use of deoxyribonucleic acid (DNA) molecules, in recent years, ribonucleic acid (RNA) molecules have received increasing attention. Various methods have been proposed, including the use of naked RNA forms, or single-stranded or double-stranded RNA in a composite or packaged form (e.g., in a non-viral or viral delivery vector). In viruses and viral delivery vectors, genetic information is usually encapsulated by proteins and / or lipids (virions). For example, modified RNA virus particles derived from RNA viruses have been proposed as delivery vectors for treating plants (WO 2000 / 053780A2) or for mammalian vaccination (Tubulekas et al., 1997, Gene, Vol. 190, pp. 191-195). Generally, RNA viruses are a group of diverse infectious particles with RNA genomes. RNA viruses can be grouped into single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) viruses, and ssRNA viruses can be further generally divided into positive-strand [(+) strand] and / or negative-strand [(-) strand] viruses. Positive-strand RNA viruses are initially attractive as delivery systems in biomedicine because their RNA can be directly used as a template for translation in host cells.

[0004] Alphavirus is a typical representative of positive-strand RNA viruses. The host of alphavirus includes a variety of organisms, including insects, fish and mammals, such as domestic animals and humans. Alphavirus replicates in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see José et al., Future Microbiol., 2009, Vol. 4, pp. 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 generally two open reading frames (ORFs) in the genome. Four nonstructural proteins (nsP1-nsP4) are generally encoded together by the first ORF that starts near the 5' end of the genome, while alphavirus structural proteins are encoded together by the second ORF found downstream of the first ORF and extending near the 3' end of the genome. Typically, the first ORF outnumbers the second ORF, with a ratio of approximately 2:1.

[0005] In cells infected with alphaviruses, only nucleic acid sequences encoding nonstructural proteins are translated from genomic RNA, while genetic information encoding structural proteins can be translated from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., Vol. 87, pp. 111-124). After infection, i.e., in the early stages of the viral life cycle, the (+) strand genomic RNA, like messenger RNA, is directly used to translate the open reading frame encoding the nonstructural polyprotein (nsP1234). In some alphaviruses, an opal stop codon exists between the coding sequences for nsP3 and nsP4. When translation terminates at this opal stop codon, polyprotein P123 containing nsP1, nsP2, and nsP3 is produced, along with polyprotein P1234 containing nsP4, which is produced by reading through this opal stop codon (Strauss & Strauss, Microbiol. Rev., 1994, Vol. 58, pp. 491-562; Rupp et al., 2015, J. Gen. Virology, Vol. 96, pp. 2483-2500). nsP1234 is autoproteolytically cleaved into fragments nsP123 and nsP4. The polypeptides nsP123 and nsP4 associate to form the (-) strand replicase complex, which transcribes (-) strand RNA using the (+) strand genomic RNA as a template. Typically, at a later stage, the nsP123 fragment is completely cleaved into the individual proteins nsP1, nsP2, and nsP3 (Shirako & Strauss, 1994, J. Virol., Vol. 68, pp. 1874-1885). All four proteins form the (+)-strand replicase complex, which uses the (-)-strand complementary sequence of the genomic RNA as a template to synthesize a new (+)-strand genome (Kim et al., 2004, Virology, Vol. 323, pp. 153-163, Vasiljeva et al., 2003, J. Biol. Chem. Vol. 278, pp. 41636-41645).

[0006] In infected cells, subgenomic RNA and 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, Vol. 73, pp. 2129-2134), and a polyadenylate [poly(A)] tail by nsP4 (Rubach et al., Virology, 2009, Vol. 384, pp. 201-208). Therefore, both subgenomic and genomic RNAs are similar to messenger RNA (mRNA).

[0007] Alphavirus structural proteins (core nucleocapsid protein C, envelope protein E2 and envelope protein E1, all components of the virus particle) are typically encoded by a single open reading frame under the control of a subgenomic promoter (Strauss & Strauss, Microbiol. Rev., 1994, Vol. 58, pp. 491-562). The subgenomic promoter is recognized by cis-acting alphavirus non-structural proteins. In particular, the alphavirus replicase uses the (-) strand complementary sequence of the genomic RNA as a template to synthesize the (+) strand subgenomic transcript. The (+) strand subgenomic transcript encodes alphavirus structural proteins (Kim et al., 2004, Virology, Vol. 323, pp. 153-163, Vasiljeva et al., 2003, J. Biol. Chem. Vol. 278, pp. 41636-41645). The subgenomic RNA transcript serves as a template for translation of the open reading frame encoding the structural proteins into a polyprotein, and the polyprotein is cleaved to produce the structural proteins. During late stages of alphavirus infection in host cells, a packaging signal located within the nsP2 coding sequence ensures the selective packaging of the genomic RNA into budding virions that are packaged by the structural proteins (White et al., 1998, J. Virol., Vol. 72, pp. 4320-4326).

[0008] In infected cells, (-) strand RNA synthesis is typically observed only in the first 3 to 4 hours after infection and is undetectable in the late stages, when only (+) strand RNA (both genomic and subgenomic) synthesis is observed. According to Frolov et al., 2001, RNA, Vol. 7, pp. 1638-1651, the leading model for regulating RNA synthesis suggests dependence on processing by nonstructural polyproteins: initial cleavage of the nonstructural polyprotein nsP1234 produces nsP123 and nsP4; nsP4 acts as an RNA-dependent RNA polymerase (RdRp) that is active for (-) strand synthesis but inefficient for producing (+) strand RNA. Further processing of the polyprotein nsP123 (including cleavage at the nsP2 / nsP3 junction) alters the template specificity of the replicase to increase synthesis of (+) strand RNA and reduce or terminate synthesis of (-) strand RNA.

[0009] Alphavirus RNA synthesis is also regulated by cis-acting RNA elements, including four conserved sequence elements (CSEs; Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491-562; and Frolov, 2001, RNA, vol. 7, pp. 1638-1651).

[0010] Typically, the 5' replication recognition sequence of alphavirus genomes is characterized by low overall homology between different alphaviruses, but with a conserved predicted secondary structure. The 5' replication recognition sequence of the alphavirus genome is not only involved in translation initiation, but also contains a 5' replication recognition sequence that contains two conserved sequence elements involved in viral RNA synthesis, CSE1 and CSE2. For the function of CSE1 and 2, the secondary structure is believed to be more important than the linear sequence (Strauss & Strauss, Microbiol. Rev., 1994, Vol. 58, pp. 491-562).

[0011] In contrast, the 3' terminal sequence of the alphavirus genome, i.e., the sequence immediately upstream of the poly(A) sequence, is characterized by a conserved primary structure, in particular the conserved sequence element 4 (CSE4), also known as the "19-nt conserved sequence", which is important for the initiation of (-) strand synthesis.

[0012] CSE3, also known as the "junction sequence," is a conserved sequence element on the (+) strand of alphavirus genomic RNA. The complementary sequence of CSE3 on the (-) strand acts as a promoter for subgenomic RNA transcription (Strauss & Strauss, Microbiol. Rev., 1994, Vol. 58, pp. 491-562; Frolov et al., 2001, RNA, Vol. 7, pp. 1638-1651). CSE3 typically overlaps with the region encoding the C-terminal fragment of nsP4.

[0013] In addition to alphavirus proteins, host cell factors (presumably proteins) may also bind to conserved sequence elements (Strauss & Strauss, supra).

[0014] Alphavirus-derived vectors have been proposed for delivering exogenous genetic information into target cells or target organisms. In a simple approach, the open reading frame encoding the structural proteins of the alphavirus is replaced by an open reading frame encoding the protein of interest. The trans-replication system based on alphavirus relies on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes the viral replicase (usually the polyprotein nsP1234) and the other nucleic acid molecule is capable of being trans-replicated by the replicase (hence the name trans-replication system). Trans-replication requires the presence of both nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being trans-replicated by the replicase must contain certain alphavirus sequence elements to allow recognition and RNA synthesis by the alphavirus replicase. The respective replicons are Figure 1” is shown as “Template RNA WT-RRS” in the figure. Such replicons are associated with the advantage of allowing the amplification of the gene of interest under the control of a subgenomic promoter; however, since the open reading frame encoding nsP1234 overlaps with the 5′ replication recognition sequence of the alphavirus genome (coding sequence of nsP1) and often also with the subgenomic promoter containing CSE3 (coding sequence of nsP4), it is difficult to develop more functional vectors.

[0015] For example, Michel et al. (2007, Virology, Vol. 362, pp. 475-487) describe that the introduction of 95 silent mutations (i.e., mutations that do not affect the encoded protein sequence) into the coding region of the nsP1 of the alphavirus Venezuelan encephalitis virus (VEEV) completely abolished the ability of VEEV to replicate in cells, presumably because the silent mutations disrupted the secondary structure of the RNA. WO 2008 / 156829 A2 and Kamrud et al. (2010, J. Gen. Virol., Vol. 91, pp. 1723-1727) describe helper RNA (i.e., trans-replicating RNA that expresses the VEEV capsid and envelope) that was modified so that the specific AUG base triplet used as the start codon of nsP1 of VEEV found in nature can be removed (by conversion to a stop codon) to generate a modified construct. According to Kamrud et al., converting the nsP1 start codon to a stop codon preserves the RNA's replicative potential, and these modified helper RNAs produce VEEV particles with only slightly reduced titers. However, the authors observed that conversion of all AUGs found within the CSE1 / 2 region to stop codons resulted in poor helper RNA replication in the presence of the alphavirus replicase. The authors attributed this poor replication to a putative disruption of the underlying RNA secondary structure. Since the authors did not mention that they controlled for proper RNA folding of their modified helper RNAs, impaired secondary structure is a very likely explanation.

[0016] The fact that the 5' replication recognition sequence required for RNA replication contains the AUG start codon of nsP1 and thus overlaps with the coding sequence of the N-terminal fragment of the alphavirus nonstructural protein represents a significant bottleneck for engineering alphavirus-based vectors, since replicons containing the 5' replication recognition sequence usually encode (at least) a portion of the alphavirus nonstructural protein, usually the N-terminal fragment of nsP1. This is disadvantageous in several respects:

[0017] In the case of cis-replicons, this overlap limits the adaptability of the codon usage of, for example, the replicase ORF to different mammalian target cells (human, mouse, farm animals). It is conceivable that the secondary structure of the 5' replication recognition sequence found in viruses is not optimal in every target cell. However, the secondary structure cannot be freely changed, as the possible amino acid changes in the replicase ORF must be considered and their impact on replicase function must be tested. It is also not possible to exchange the complete replicase ORF for a replicase from a heterologous source, as this may lead to a disruption of the 5' replication recognition sequence structure. In the case of trans-replicons, this overlap leads to the synthesis of nsP1 protein fragments, as the 5' replication recognition sequence needs to be retained in the trans-replicons. Fragments of nsP1 are generally unnecessary and undesirable: unwanted translation puts an unnecessary burden on the host cell, and RNA replicons intended for therapeutic applications that encode fragments of nsP1 in addition to the pharmaceutically active protein may face regulatory problems. For example, it is necessary to demonstrate that truncated nsP1 does not produce unwanted side effects. Furthermore, the presence of the AUG start codon of nsP1 within the 5' replication recognition sequence has marginally prevented the design of transreplicons encoding heterologous genes of interest, in which the start codon for translation of the target gene is located at the most 5' position available for ribosomal translation initiation. Conversely, 5'-cap-dependent translation of transgenes from prior art transreplicon RNAs is challenging unless cloned as fusion proteins in frame with the start codon of nsP1 (such fusion constructs are described, for example, by Michel et al., 2007, Virology, Vol. 362, pp. 475-487). Such fusion constructs result in the same unwanted translation of the nsP1 fragments described above, raising the same issues as above. Furthermore, fusion proteins raise additional issues because they may alter the function or activity of the target fusion transgene, or, when used as vaccine vectors, peptides spanning the fusion region may alter the immunogenicity of the fusion antigen.

[0018] There is a need to overcome these shortcomings. For example, there is a need to provide improved replicons for expressing nucleic acids encoding target proteins (e.g., pharmaceutically active proteins) in a safe and effective manner. As described herein, aspects and embodiments of the present invention address this need. Summary of the Invention

[0019] Immunotherapeutic strategies represent a promising option for the prevention and treatment of diseases such as infectious diseases and cancer. The identification of an increasing number of pathogen- and tumor-associated antigens has led to a broad collection of suitable targets for immunotherapy. The present invention includes improved reagents and methods for the efficient expression of antigens, suitable for immunotherapy for the prevention and treatment of disease.

[0020] In a first aspect, the present invention provides an RNA replicon comprising 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 alphavirus 5' replication recognition sequence.

[0021] In one embodiment, the RNA replicon of the present invention comprises a (modified) 5' replication recognition sequence and a first open reading frame encoding a protein of interest, e.g., a functional alphavirus nonstructural protein or preferably a transgene that is not derived from an alphavirus, in particular an alphavirus nonstructural protein located downstream of the 5' replication recognition sequence, wherein the 5' replication recognition sequence and the first open reading frame encoding the protein of interest do not overlap, and preferably the 5' replication recognition sequence does not overlap with any open reading frame of the RNA replicon, e.g., the 5' replication recognition sequence does not contain a functional start codon, and preferably does not contain any start codon. Most preferably, the start codon of the first open reading frame encoding the protein of interest is in the 5'→3' direction of the first functional start codon, preferably the first start codon of the RNA replicon. In one embodiment, the first open reading frame encoding the protein of interest encodes a functional alphavirus nonstructural protein. In one embodiment, the first open reading frame encoding the protein of interest and preferably the entire RNA replicon do not express non-functional alphavirus nonstructural proteins, e.g., fragments of alphavirus nonstructural proteins, in particular fragments of nsP1 and / or nsP4. In one embodiment, the functional alphavirus nonstructural protein is heterologous to the 5' replication recognition sequence. In one embodiment, the first open reading frame encoding the protein of interest is not under the control of a subgenomic promoter. In one embodiment, the RNA replicon comprises at least one additional open reading frame encoding the protein of interest under the control of a subgenomic promoter. In one embodiment, the subgenomic promoter does not overlap with the first open reading frame encoding the protein of interest.

[0022] In one embodiment, the 5' replication recognition sequence of the RNA replicon characterized by removal of at least one start codon comprises a sequence homologous to approximately 250 nucleotides at the 5' terminus of an alphavirus. In a preferred embodiment, it comprises a sequence homologous to approximately 300 to 500 nucleotides at the 5' terminus of an alphavirus. In a preferred embodiment, it comprises a 5'-terminal sequence required for efficient replication of the specific alphavirus species from which the vector system is derived.

[0023] In one embodiment, the 5' replication recognition sequence of the RNA replicon comprises sequences homologous to conserved sequence element 1 (CSE 1) and conserved sequence element 2 (CSE 2) of alphaviruses.

[0024] In a preferred embodiment, the RNA replicon comprises CSE 2 and is further characterized in that it comprises a fragment of the open reading frame of a nonstructural protein from an alphavirus. In a more preferred embodiment, the fragment of the open reading frame of the nonstructural protein does not comprise any start codon.

[0025] 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 said 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.

[0026] In a preferred embodiment, the sequence homologous to the 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 the natural start codon of the open reading frame of the nonstructural protein.

[0027] In a preferred embodiment, the sequence homologous to the open reading frame of a nonstructural protein from an alphavirus or a fragment thereof is characterized in that it comprises the removal of one or more start codons other than the native start codon of the open reading frame of the nonstructural protein. In a more preferred embodiment, the nucleic acid sequence is further characterized by the removal of the native start codon of the open reading frame of the nonstructural protein, preferably nsP1.

[0028] In a preferred embodiment, the 5' replication recognition sequence comprises one or more stem loops, which provide the function of the 5' replication recognition sequence about RNA replication. In a preferred embodiment, the 5' replication recognition sequence comprises one or more stem loops ...

[0029] In a preferred embodiment, the RNA replicon comprises one or more nucleotide changes that compensate for the disruption of nucleotide pairing within one or more stem-loops introduced by removal of at least one start codon.

[0030] In one embodiment, the RNA replicon does not comprise an open reading frame encoding a truncated alphavirus nonstructural protein.

[0031] In one embodiment, the RNA replicon comprises a 3' replication recognition sequence.

[0032] In one embodiment, the RNA replicon comprises a first open reading frame encoding a protein of interest.

[0033] In one embodiment, the RNA replicon is characterized in that the protein of interest encoded by the first open reading frame can be expressed using the RNA replicon as a template.

[0034] In one embodiment, the RNA replicon is characterized in that it comprises a subgenomic promoter. Typically, the subgenomic promoter controls the production of a subgenomic RNA comprising an open reading frame encoding a protein of interest.

[0035] In a preferred embodiment, the protein of interest encoded by the first open reading frame can be expressed using an RNA replicon as a template. In a more preferred embodiment, the protein of interest encoded by the first open reading frame can additionally be expressed by a subgenomic RNA.

[0036] In a preferred embodiment, the RNA replicon is further characterized in that it comprises a subgenomic promoter that controls the production of a subgenomic RNA, wherein the subgenomic RNA comprises a second open reading frame encoding a protein of interest. The protein of interest can be a second protein that is the same as or different from the protein of interest encoded by the first open reading frame.

[0037] In a more preferred embodiment, the subgenomic promoter and the second open reading frame encoding the protein of interest are located downstream of the first open reading frame encoding the protein of interest.

[0038] In one embodiment, the protein of interest encoded by the first and / or second open reading frame is a functional alphavirus nonstructural protein.

[0039] In one embodiment, the RNA replicon comprises an open reading frame encoding a functional alphavirus nonstructural protein.

[0040] In one embodiment, the open reading frame encoding a functional alphavirus nonstructural protein does not overlap with the 5' replication recognition sequence.

[0041] In one embodiment, the RNA replicon encoding a functional alphavirus nonstructural protein can be replicated by the functional alphavirus nonstructural protein.

[0042] In one embodiment, the RNA replicon does not comprise an open reading frame encoding a functional alphavirus nonstructural protein. In this embodiment, the functional alphavirus nonstructural protein used to replicate the replicon can be provided in trans as described herein.

[0043] In a second aspect, the present invention provides a system comprising:

[0044] RNA constructs for expressing functional alphavirus nonstructural proteins,

[0045] The RNA replicon according to the first aspect of the present invention can replicate in trans via a functional alphavirus non-structural protein. Preferably, the RNA replicon is further characterized in that it does not encode a functional alphavirus non-structural protein.

[0046] In one embodiment the RNA replicon according to the first aspect or the system according to the second aspect is characterized in that said alphavirus is Semliki Forest Virus.

[0047] In a third aspect, the present invention provides a DNA comprising a nucleic acid sequence encoding an RNA replicon according to the first aspect of the invention.

[0048] In a fourth aspect, the present invention provides a method for producing a protein of interest in a cell, comprising the steps of:

[0049] (a) obtaining an RNA replicon according to the first aspect of the present invention comprising an open reading frame encoding a functional alphavirus non-structural protein, which can be replicated by the functional alphavirus non-structural protein and further comprising an open reading frame encoding a target protein, and

[0050] (b) inoculating the RNA replicon into cells.

[0051] In various embodiments of the method, the RNA replicon is as defined above for the replicon of the invention.

[0052] In a fifth aspect, the present invention provides a method for producing a protein of interest in a cell, comprising the steps of:

[0053] (a) obtaining an RNA construct for expressing a functional alphavirus nonstructural protein,

[0054] (b) obtaining an RNA replicon according to the first aspect of the invention, which can replicate in trans via the functional alphavirus nonstructural protein according to (a) and which comprises an open reading frame encoding a protein of interest, and

[0055] (c) The RNA replicon and an RNA construct for expressing functional alphavirus nonstructural proteins are co-inoculated into cells.

[0056] In various embodiments of the method, the RNA construct and / or RNA replicon for expressing a functional alphavirus nonstructural protein is as defined above for the system of the invention.According to the fifth aspect, the RNA replicon itself typically does not encode a functional alphavirus nonstructural protein.

[0057] In a sixth aspect, the present invention provides a cell comprising the replicon of the first aspect or the system of the second aspect. In one embodiment, the cell is inoculated according to the method of the fourth aspect of the invention or according to the method of the fifth aspect of the invention. In one embodiment, the cell is obtainable by the method of the fourth aspect of the invention or by the method of the fifth aspect of the invention. In one embodiment, the cell is part of an organism.

[0058] In a seventh aspect, the present invention provides a method for producing a protein of interest in a subject, comprising the steps of:

[0059] (a) obtaining an RNA replicon according to the first aspect of the present invention comprising an open reading frame encoding a functional alphavirus non-structural protein, which can be replicated by the functional alphavirus non-structural protein and further comprising an open reading frame encoding a target protein, and

[0060] (b) administering the RNA replicon to a subject.

[0061] In various embodiments of the method, the RNA replicon is as defined above for the replicon of the invention.

[0062] In an eighth aspect, the present invention provides a method for producing a protein of interest in a subject, comprising the steps of:

[0063] (a) obtaining an RNA construct for expressing a functional alphavirus nonstructural protein,

[0064] (b) obtaining an RNA replicon according to the first aspect of the invention, which can be replicated in trans by the functional alphavirus nonstructural protein according to (a) and comprises an open reading frame encoding a protein of interest, and

[0065] (c) administering to the subject an RNA replicon and an RNA construct for expressing a functional alphavirus nonstructural protein.

[0066] In various embodiments of the method, the RNA construct and / or RNA replicon for expressing a functional alphavirus nonstructural protein is as defined above for the system of the invention. According to the eighth aspect, the RNA replicon itself typically does not encode a functional alphavirus nonstructural protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 : Schematic diagram of RNA replicons containing unmodified or modified 5' replication recognition sequences

[0068] Abbreviations: AAAA = Poly(A) tail; ATG = start codon / start codon (ATG at the DNA level; AUG at the RNA level); 5x ATG = a nucleic acid sequence comprising all the start codons of the nucleic acid sequence encoding nsP1* (in the case of the nucleic acid sequence encoding nsP1* from Semliki Forest virus, 5x ATG corresponds to 5 specific start codons, see Example 1); Δ5ATG = a nucleic acid sequence corresponding to the nucleic acid sequence encoding nsP1*; however, not comprising any start codon of the nucleic acid sequence encoding nsP1* found in nature (in the case of nsP1* from Semliki Forest virus, “Δ5ATG” corresponds to the removal of 5 specific start codons compared to Semliki Forest virus found in nature, see Example 1); EcoRV = EcoRV restriction site; nsP = a nucleic acid sequence encoding a nonstructural protein of an alphavirus (e.g., nsP1, =nsP2, nsP3, nsP4); nsP1* = nucleic acid sequence encoding a fragment of nsP1, wherein the fragment does not include the C-terminal fragment of nsP1; *nsP4 = nucleic acid sequence encoding a fragment of nsP4, wherein the fragment does not include the N-terminal fragment of nsP4; RRS = 5' replication recognition sequence; SalI = SalI restriction site; SGP = subgenomic promoter; SL = stem-loop (e.g., SL1, SL2, SL3, SL4); the positions of SL1-4 are shown in the figure; UTR = untranslated region (e.g., 5'-UTR, 3'-UTR); WT = wild type.

[0069] Cis-replicon WT-RRS: The RNA replicon essentially corresponds to the alphavirus genome, except that the nucleic acid sequences encoding the alphavirus structural proteins have been replaced by an open reading frame encoding the gene of interest ("transgene"). When the "replicon WT-RRS" is introduced into cells, the translation product of the open reading frame encoding the replicase (nsP1234 or a fragment thereof) can drive cis-replication of the RNA replicon and drive the synthesis of nucleic acid sequences downstream of the subgenomic promoter (SGP) (subgenomic transcripts).

[0070] Trans-replicon or template RNA WT-RRS: The RNA replicon essentially corresponds to "Replicon WT-RRS," except that most of the nucleic acid sequences encoding the alphavirus nonstructural proteins nsP1-4 have been removed. More specifically, the nucleic acid sequences encoding nsP2 and nsP3 have been completely removed; the nucleic acid sequence encoding nsP1 has been truncated so that "Template RNA WT-RRS" encodes a fragment of nsP1 that does not include the C-terminal fragment of nsP1 (but does include the N-terminal fragment of nsP1; nsP1*); and the nucleic acid sequence encoding nsP4 has been truncated so that "Template RNA WT-RRS" encodes a fragment of nsP4 that does not include the N-terminal fragment of nsP4 (but does include the C-terminal fragment of nsP4; *nsP4). This truncated nsP4 sequence partially overlaps with the fully active subgenomic promoter. The nucleic acid sequence encoding nsP1* comprises all the start codons of nucleic acid sequences encoding nsP1* in alphaviruses found in nature (in the case of nsP1* from Semliki Forest virus, five specific start codons).

[0071] Δ5ATG-RRS: The RNA replicon essentially corresponds to "Template RNA WT-RRS," except that it does not contain any of the start codons of the nucleic acid sequence encoding nsP1* found in nature in alphaviruses (in the case of Semliki Forest virus, "Δ5ATG-RRS" corresponds to the removal of five specific start codons compared to the Semliki Forest virus found in nature). All nucleotide changes introduced to remove the start codons were compensated by additional nucleotide changes to preserve the predicted secondary structure of the RNA.

[0072] Δ5ATG-RRSΔSGP: RNA replicon essentially corresponds to "Δ5ATG-RRS", except that it does not contain the subgenomic promoter (SGP) and does not contain the nucleic acid sequence encoding *nsP4. "Transgene 1" = gene of interest.

[0073] Δ5ATG-RRS-bicistronic: The RNA replicon essentially corresponds to "Δ5ATG-RRS," except that it contains a first open reading frame encoding a first gene of interest ("transgene 1") upstream of a subgenomic promoter and a second open reading frame encoding a second gene of interest ("transgene 2") downstream of a subgenomic promoter. The positioning of the second open reading frame corresponds to the positioning of the gene of interest ("transgene") in the RNA replicon "Δ5ATG-RRS."

[0074] Cis-replicon Δ5ATG-RRS: The RNA replicon essentially corresponds to "Δ5ATG-RRS-bicistronic," except that the open reading frame encoding the first gene of interest encodes a functional alphavirus nonstructural protein (typically an open reading frame encoding the polyprotein nsP1-nsP2-nsP3-nsP4, i.e., nsP1234). The "transgene" in "cis-replicon Δ5ATG-RRS" corresponds to the "transgene 2" in "Δ5ATG-RRS-bicistronic." The functional alphavirus nonstructural protein is capable of recognizing a subgenomic promoter and synthesizing a subgenomic transcript comprising a nucleic acid sequence encoding the gene of interest ("transgene"). "Cis-replicon Δ5ATG-RRS" encodes a functional alphavirus nonstructural protein in cis, just like "cis-replicon WT-RRS"; however, the coding sequence of nsP1 encoded by "cis-replicon Δ5ATG-RRS" is not required to contain the exact nucleic acid sequence of "cis-replicon WT-RRS," including all stem-loops.

[0075] Figure 2 : Removal of the start codon within the 5' replication recognition sequence does not affect replication of the trans-acting replicon RNA. A: Description of the nucleic acid molecules used in Example 2. B: Luciferase expression measured in electroporated BHK21 cells. For details, see Example 2. Shown are the mean ± SD of two independent experiments with two independently generated RNA replicon batches ("templates"); (N = 4).

[0076] Figure 3 : Removal of the start codon within the 5' replication recognition sequence enables cap-dependent translation. Left: Schematic representation of the nucleic acid molecule (RNA replicon) used in Example 3. Right: Luciferase expression measured in electroporated human foreskin fibroblasts. For details, see Example 3. Shown are the mean ± SD of one experiment performed in triplicate.

[0077] Figure 4 : Immediately after transfection, cap-dependent translation of a trans-replicon characterized by removal of the start codon within the 5' replication recognition sequence is stronger than translation from a subgenomic transcript. Left: Schematic representation of nucleic acid molecules. The RNA replicons used in Example 4 are shown as "Δ5ATG-RRS" and "Δ5ATG-RRSΔSGP." Right: Luciferase expression measured in electroporated BHK21 cells. See Example 4 for details. Shown are the average values ​​of one experiment performed in triplicate.

[0078] Figure 5: A capped trans-replicon characterized by removal of the start codon within the 5' replication recognition sequence, enabling early-stage transgene expression. Left: Schematic representation of the "Δ5ATG-RRSΔSGP" nucleic acid molecule used in Example 5. Right: Luciferase expression measured in electroporated BHK21 cells. For details, see Example 5. Shown are the mean ± SD of one experiment performed in triplicate.

[0079] Figure 6 .Structure of cap dinucleotide. Top: Natural cap dinucleotide, m 7 GpppG. Bottom: Phosphorothioate cap analog β-S-ARCA dinucleotide: Based on its elution profile in reverse phase HPLC, there are two diastereomers of β-S-ARCA, designated D1 and D2, due to the stereogenic P center.

[0080] Figure 7 .The reconstructed cis-replicon with ATG-deleted RRS is functional. The ORF of SFV replicase was inserted into the "Δ5ATG-RRS" encoding firefly luciferase downstream of the subgenomic promoter (SGP). Within the inserted replicase, the regions corresponding to CSE2 and core SGP were destroyed by nucleotide exchange (dashed box) to avoid duplication of these regulatory regions. This resulted in a reconstructed cis-replicon. BHK21 cells were co-electroporated with 2.5 μg of "cis-replicon WT-RRS" or "cis-replicon Δ5ATG-RRS". Luciferase expression was measured 24 hours after electroporation. Mean ± SD of a triplicate experiment.

[0081] Figure 8 .Bicistronic replicon expressing two transgenes. Secretable nanoluciferase (SNL) was cloned downstream of the subgenomic promoter (SGP) of the transreplicon WT-RSS. The position upstream of SGP does not encode the transgene (-)SGP(SNL). In the following construct, SNL was cloned downstream of ΔATG-RSS, and firefly luciferase (Luc) was inserted downstream of SGP(SNL)SGP(Luc). BHK21 cells were co-electroporated with 0.9 μg of transreplicated RNA and 5 μg of SFV-replicase encoding mRNA, and SNL and Luc expression was measured 48 hours after electroporation. Data from one experiment.

[0082] Figure 9.A Sindbis virus transreplicon lacking a start codon in its replication recognition sequence replicates efficiently. The transreplicon was engineered from the Sindbis virus genome by gene synthesis, and GFP was inserted downstream of the subgenomic promoter (SGP). In addition to this transreplicon with an unmodified replication recognition sequence (WT-RSS), two variants were also generated. In ΔATG-RRS, the original start codon plus four additional ATGs were deleted from WT-RRS. Compensatory nucleotide changes were also introduced as needed to maintain RNA secondary structure. To generate ΔATG-RRSΔSGP, the region corresponding to the subgenomic promoter was deleted from ΔATG-RRS, resulting in a vector with GFP directly downstream of the 5' RRS with the ATG deletion. BHK21 cells were co-electroporated with 0.1 μg of transreplicon RNA and 2.4 μg of SFV-replicase encoding mRNA. GFP expression (transfection efficiency [%] and mean fluorescence intensity (MFI)) was assessed 24 hours after electroporation. Data from one experiment. DETAILED DESCRIPTION

[0083] 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 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 defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art.

[0084] 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 Chimica Acta, CH-4010 Basel, Switzerland, ed., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0085] 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, for example, Molecular Cloning: A Laboratory Manual, 2nd ed., J. Sambrook et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0086] Hereinafter, the elements of the present invention will be described. These elements are listed together with specific embodiments, however, it should be understood that they can be combined in any way and in any quantity to produce another embodiment. The embodiments and preferred embodiments of the 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 embodiments that combine the embodiments clearly described with any number of disclosed and / or preferred elements. In addition, unless the context indicates otherwise, any arrangement and combination of all described elements in this application should be considered to be disclosed by this specification.

[0087] 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.

[0088] Unless otherwise stated herein or clearly contradictory with context, otherwise the terms "one" and "a / kind" and "said" and similar references used in the context of describing the present invention (particularly in the context of claims) should be interpreted as covering both the singular and the plural. The description of numerical ranges herein is only intended to be used as a shorthand method for referring to each individual value falling within the range individually. Unless otherwise stated herein, each individual value is incorporated into this specification as if it were quoted separately in this article. Unless otherwise stated herein or clearly 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., "such as") provided herein is only intended to better illustrate the present invention, rather than limiting the scope of the present invention, unless otherwise stated. Any language in the specification should not be interpreted as representing any unclaimed element essential for the practice of the present invention.

[0089] Unless expressly 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 no other members are present, i.e., for the purposes of this embodiment, "comprising" should be understood to have the meaning of "consisting of."

[0090] The indication of the relative amount of a component characterized by a general term refers to the total amount of all specific variants or members encompassed by the general term. If a 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 encompassed by the general term, it means that no other variants or members encompassed by the general term are present such that the total relative amount of the component encompassed by the general term exceeds the specified relative amount; more preferably, no other variants or members encompassed by the general term are present at all.

[0091] 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 disclosure.

[0092] As used herein, terms such as "reduce / lower" or "inhibit" refer to the ability to cause an overall decrease / reduction 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., a reduction to zero or substantially to zero.

[0093] Terms such as "increase / enhance" or "enhance" preferably relate to an increase / enhancement 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%, most preferably at least 100%.

[0094] The term "net charge" refers to the charge on an entire object (eg, a compound or particle).

[0095] 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; a cation is an ion that has fewer electrons than protons, giving it a net positive charge.

[0096] With respect to a given compound or particle, the terms "charged," "net charge," "negatively charged," or "positively charged" refer to the net charge of the given compound or particle when dissolved or suspended in water at pH 7.0.

[0097] Term " nucleic acid " according to the present invention also includes the chemical derivatization of nucleic acid on nucleotide bases, on sugar or on phosphate, and the nucleic acid containing non-natural nucleotides and nucleotide analogs.In some embodiments, nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).Usually, nucleic acid molecule or nucleic acid sequence refer to the nucleic acid that is preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).According to the present invention, nucleic acid includes genomic DNA, cDNA, mRNA, viral RNA, recombinant preparation and chemically synthesized 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.

[0098] According to the present invention, "nucleic acid sequence" refers to a sequence of nucleotides in a nucleic acid, such as ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). The term can refer to an entire nucleic acid molecule (e.g., a single strand of an entire nucleic acid molecule) or a portion thereof (e.g., a fragment).

[0099] According to the present invention, the term "RNA" or "RNA molecule" refers to a molecule comprising ribonucleotide residues and preferably consisting entirely or essentially of ribonucleotide residues. The term "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of the β-D-ribofuranosyl group. The term "RNA" includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially or completely purified RNA, essentially pure RNA, synthetic RNA, and recombinantly produced RNA, such as modified RNA that differs from naturally occurring RNA by addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide material, such as at one or more nucleotides of the RNA, such as at the ends or within the 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.

[0100] 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 is not associated with any complementary nucleic acid molecule (usually without a complementary RNA molecule). Single-stranded RNA can contain self-complementary sequences, which allow part of the RNA to fold back and form secondary structure motifs, including but not limited to base pairs, stems, stem loops, and protrusions. Single-stranded RNA can exist as a minus strand [(-) strand] or as a plus strand [(+) strand]. The (+) strand is a strand that contains or encodes genetic information. Genetic information can be, for example, a polynucleotide sequence that encodes a protein. When the (+) 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 these two RNA molecules bind to each other to form double-stranded RNA ("double-stranded RNA").

[0101] The term "stability" of RNA relates to the "half-life" of the RNA. "Half-life" refers to the time required to eliminate half of the activity, amount, or number of molecules. In the context of the present invention, the half-life of an RNA represents the stability of the RNA. The half-life of an RNA may affect the "duration of expression" of the RNA. It can be expected that RNA with a long half-life will be expressed for a long time.

[0102] The term "translation efficiency" relates to the amount of translation product provided by an RNA molecule within a specific period of time.

[0103] When referring to a nucleic acid sequence, a "fragment" refers to a portion of a nucleic acid sequence, i.e., a sequence that represents a shortened nucleic acid sequence at its 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 the nucleic acid sequence. In the present invention, fragments of RNA molecules that retain RNA stability and / or translation efficiency are preferred.

[0104] When referring 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 truncated at the N-terminus and / or C-terminus. A fragment truncated at the C-terminus (N-terminal fragment) can be obtained by, for example, translating a truncated open reading frame at the 3' end of the open reading frame. A fragment truncated at the N-terminus (C-terminal fragment) can be obtained by, for example, translating a truncated open reading frame at the 5' end of the open reading frame, as long as the truncated open reading frame comprises a start codon for initiating 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% amino acid residues from the amino acid sequence.

[0105] With regard to, for example, nucleic acid and amino acid sequences, the term "variant" according to the present invention includes any variant, particularly mutants, virus strain variants, splice variants, conformations, isoforms, allelic variants, species variants and species homologs, particularly naturally occurring ones. 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 different from the reference nucleic acid in the codon sequence due to the degeneracy of the genetic code. Species homologs are nucleic acids or amino acid sequences having species of origin different from a given nucleic acid or amino acid sequence. Viral homologs are nucleic acids or amino acid sequences having viruses of origin different from a given nucleic acid or amino acid sequence.

[0106] According to the present invention, nucleic acid variants include single or multiple nucleotide deletions, additions, mutations, replacements and / or insertions compared to a reference nucleic acid. Deletion includes removing one or more nucleotides from a reference nucleic acid. Addition variants include one or more nucleotides, such as 1, 2, 3, 5, 10, 20, 30, 50 or more nucleotides 5'- and / or 3'-terminal fusions. In the case of replacement, at least one nucleotide in the sequence is removed and at least one other nucleotide (such as transversion and conversion) is inserted in its place. Mutation includes abasic sites, crosslinking sites and chemically altered or modified bases. Insertion includes adding at least one nucleotide in a reference nucleic acid.

[0107] According to the present invention, "nucleotide changes" can refer to single or multiple nucleotide deletions, additions, mutations, substitutions and / or insertions compared to a reference nucleic acid. In some embodiments, "nucleotide changes" are selected from the group consisting of single nucleotide deletions, single nucleotide additions, single nucleotide mutations, single nucleotide substitutions and / or single nucleotide insertions compared to a reference nucleic acid. According to the present invention, nucleic acid variants can comprise one or more nucleotide changes compared to a reference nucleic acid.

[0108] Variants of a specific nucleic acid sequence preferably have at least one functional property of the specific sequence and are preferably functionally equivalent to the specific sequence, eg, nucleic acid sequences that exhibit the same or similar properties as those of the specific nucleic acid sequence.

[0109] As described below, some embodiments of the present invention are characterized, inter alia, by nucleic acid sequences homologous to nucleic acid sequences of alphaviruses (e.g., naturally occurring alphaviruses). These homologous sequences are variants of nucleic acid sequences of alphaviruses (e.g., naturally occurring alphaviruses).

[0110] Preferably, the degree of identity between a given nucleic acid sequence and a nucleic acid sequence that is a variant of the given nucleic acid sequence will be at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, or most preferably at least 95%, 96%, 97%, 98% or 99%. Preferably, the degree of identity is given for a region of at least about 30, at least about 50, at least about 70, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, or at least about 400 nucleotides. In a preferred embodiment, the degree of identity is given for the entire length of the reference nucleic acid sequence.

[0111] "Sequence similarity" refers to the percentage of amino acids that are identical or 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.

[0112] The term "% identity" means the percentage of identical nucleotides, particularly in an optimal alignment between two sequences to be compared, said 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 in order to obtain an optimal alignment between the two sequences. The comparison of two sequences is usually performed by comparing the sequences after optimal alignment over a segment 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, BLASTP, BLASTN, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0113] The percent identity is obtained by determining the number of corresponding identical positions in the sequences being compared, dividing that number by the number of positions compared and multiplying the result by 100.

[0114] For example, the BLAST program "BLAST2 sequences" available at the website http: / / www.ncbi.nlm.nih.gov / blast / bl2seq / wblast2.cgi can be used.

[0115] A nucleic acid is "capable of hybridizing" or "hybridizing" to another nucleic acid if the two sequences are complementary to each other. A nucleic acid is "complementary" to another nucleic acid if the two sequences are capable of forming a stable duplex with each other. 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., ed., 2nd ed., Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989, or in Molecular Biology, FM Ausubel et al., ed., Current Protocols by John Wiley & Sons, Inc., New York, for example, referring to hybridization at 65°C 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). SSC is 0.15 M sodium chloride / 0.15 M sodium citrate, pH 7. After hybridization, the membrane to which the DNA has been transferred is washed, for example, in 2×SSC at room temperature and then in 0.1-0.5×SSC / 0.1×SDS at a temperature up to 68°C.

[0116] 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 will hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence. Preferably, the degree of complementarity according to the present invention is at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90% or most preferably at least 95%, 96%, 97%, 98% or 99%. Most preferably, the degree of complementarity according to the present invention is 100%.

[0117] 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 the nucleic acid increases its stability.

[0118] According to the present invention, a "nucleic acid sequence derived from a nucleic acid sequence" refers to a nucleic acid that is a variant of the nucleic acid from which it is derived. Preferably, when it replaces a specific sequence in an RNA molecule, the sequence that is a variant of the specific sequence retains RNA stability and / or translation efficiency.

[0119] "nt" is an abbreviation for nucleotide; or an abbreviation for nucleotides, preferably consecutive nucleotides in a nucleic acid molecule.

[0120] According to the present invention, the term "codon" refers to a base triplet in a coding nucleic acid that specifies which amino acid will be added next during protein synthesis in the ribosome.

[0121] The term "transcription" refers to the process by which an RNA polymerase reads a nucleic acid molecule ("nucleic acid template") having a specific nucleic acid sequence, 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 can be DNA; however, in the case of transcription from an alphavirus nucleic acid template, for example, the template is typically RNA. The transcribed RNA can then be translated into protein. According to the present invention, the term "transcription" includes "in vitro transcription," wherein the term "in vitro transcription" refers to methods in which RNA, particularly mRNA, is synthesized in vitro in a cell-free system. Preferably, a cloning vector is used to produce the transcript. These cloning vectors are generally referred to as transcription vectors and, according to the present invention, are encompassed by the term "vector." The cloning vector is preferably a plasmid. According to the present invention, the RNA is preferably in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter used to control transcription can be any promoter for any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0122] The single-stranded nucleic acid molecule produced during transcription typically has a nucleic acid sequence that is complementary to the template.

[0123] 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.

[0124] "Nucleic acid sequence transcribed from a nucleic acid sequence" and similar terms refer to a nucleic acid sequence that is part of a complete RNA molecule, where appropriate, that is the transcription product of a template nucleic acid sequence. Typically, the transcribed nucleic acid sequence is a single-stranded RNA molecule.

[0125] According to the present invention, the term "3' end of a nucleic acid" refers to the end with a free hydroxyl group. In diagrammatic representations of double-stranded nucleic acids, particularly DNA, the 3' end is always on the right. According to the present invention, the term "5' end of a nucleic acid" refers to the end with a free phosphate group. In diagrammatic representations of double-stranded nucleic acids, particularly DNA, the 5' end is always on the left.

[0126] 5' end 5'--P-NNNNNNN-OH-3' 3' end

[0127] 3'-HO-NNNNNNN-P-5'

[0128] "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 located closer to the 5' end of the nucleic acid molecule than the second element of the nucleic acid molecule. The second element is then referred to as "downstream" of the first element of the nucleic acid molecule. An element located "upstream" of a second element can be synonymously referred to as being located "5'" of the second element. For double-stranded nucleic acid molecules, the indications "upstream" and "downstream" are given, for example, with respect to the (+) strand.

[0129] According to the present invention, "functional linkage" or "functionally linked" refers to a linkage within 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 it influences the transcription of the coding sequence. Functionally linked nucleic acids are generally adjacent to each other, separated by additional nucleic acid sequences where appropriate, and in a specific embodiment, transcribed by RNA polymerase to produce a single RNA molecule (co-transcript).

[0130] In a particular embodiment, according to the invention, the nucleic acid is functionally linked to an expression control sequence, which may be homologous or heterologous to the nucleic acid.

[0131] According to the present invention, the term "expression control sequence" includes promoters, ribosome binding sequences, and other control elements that control gene transcription or translation of derived RNA. In some specific embodiments of the present invention, the expression control sequence can be regulated. The precise structure of the expression control sequence can vary depending on the species or cell type, but generally includes 5'-non-transcribed and 5'- and 3'-non-translated sequences that are involved in initiating transcription and translation, respectively. More specifically, the 5'-non-transcribed expression control sequence includes a promoter region that contains promoter sequences for transcriptional control of functionally linked genes. 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 5'-non-transcribed and 5'- and 3'-non-translated sequences, such as a TATA box, a capping sequence, a CAAT sequence, and the like. 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.

[0132] The nucleic acid sequences indicated herein, in particular transcribable and coding nucleic acid sequences, may be combined with any expression control sequences, in particular promoters, which may be homologous or heterologous to the nucleic acid sequence, wherein the term "homologous" refers to the fact that the nucleic acid sequence is also functionally linked to an expression control sequence in nature, and the term "heterologous" refers to the fact that the nucleic acid sequence is not functionally linked to an expression control sequence in nature.

[0133] A transcribable nucleic acid sequence, in particular a nucleic acid sequence encoding a peptide or protein, and an expression control sequence are "functionally" linked to one another if they are covalently linked to one another in such a way that the transcription or expression of the transcribable and in particular coding nucleic acid sequence is controlled or influenced by the expression control sequence. If the nucleic acid sequence is to be translated into a functional peptide or protein, induction of the expression control sequence functionally linked to the coding sequence results in the transcription of the coding sequence without causing a frame shift of the coding sequence or preventing translation into the desired peptide or protein.

[0134] The term "promoter" or "promoter region" refers to a nucleic acid sequence that controls the synthesis of transcripts (e.g., transcripts comprising a coding sequence) by providing recognition and binding sites for RNA polymerase. The promoter region may include additional recognition or binding sites for other factors involved in regulating the transcription of the gene. A promoter can control the transcription of prokaryotic or eukaryotic genes. 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 very small extent or not at all. In the presence of an inducer, the gene is "switched on" or the transcription level increases. This is typically mediated by the combination of a specific transcription factor. As described herein, a specific promoter according to the present invention is a subgenomic promoter of an alphavirus. Other specific promoters are genomic positive or negative chain promoters of an alphavirus.

[0135] The term "core promoter" refers to the nucleic acid sequence contained in a promoter. A core promoter is typically the smallest portion of a promoter required to correctly initiate transcription. A core promoter typically includes a transcription start site and a binding site for RNA polymerase.

[0136] "Polymerase" generally refers to a molecular entity that can catalyze the synthesis of a polymer molecule from monomeric building blocks. "RNA polymerase" is a molecular entity that can catalyze the synthesis of an RNA molecule from ribonucleotide building blocks. "DNA polymerase" is a molecular entity that can catalyze the synthesis of a DNA molecule from deoxyribonucleotide building blocks. In the case of DNA polymerases and RNA polymerases, the molecular entity is typically a protein or an assembly or complex of multiple proteins. Typically, a DNA polymerase synthesizes a DNA molecule based on a template nucleic acid, which is typically a DNA molecule. Typically, an RNA polymerase synthesizes an RNA molecule based on a template nucleic acid, which is either a DNA molecule (in which case the RNA polymerase is a DNA-dependent RNA polymerase, DdRP) or an RNA molecule (in which case the RNA polymerase is an RNA-dependent RNA polymerase, RdRP).

[0137] "RNA-dependent RNA polymerase" or "RdRP" is an enzyme that catalyzes the transcription of RNA from an RNA template. In the case of alphavirus RNA-dependent RNA polymerase, the sequential synthesis of the (-) strand complementary sequence and the (+) strand genomic RNA leads to RNA replication. Therefore, alphavirus RNA-dependent RNA polymerase is synonymously referred to as "RNA replicase." In nature, RNA-dependent RNA polymerases are generally encoded by all RNA viruses except retroviruses. Alphaviruses are a typical example of viruses encoding RNA-dependent RNA polymerases.

[0138] According to the present invention, "RNA replication" generally refers to an RNA molecule synthesized based on the nucleotide sequence of a given RNA molecule (template RNA molecule). The synthesized RNA molecule can, for example, be identical or complementary to the template RNA molecule. Generally, RNA replication can occur through the synthesis of a DNA intermediate, or can occur directly through RNA-dependent RNA polymerase (RdRP)-mediated RNA-dependent RNA replication. In the case of alphaviruses, RNA replication does not occur through a DNA intermediate, but is mediated by RNA-dependent RNA polymerase (RdRP): a template RNA chain (a first RNA chain)-or a portion thereof-is used 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-can then optionally be used 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 (through a complementary intermediate chain).

[0139] According to the present invention, the term "template RNA" refers to an RNA that can be transcribed or replicated by an RNA-dependent RNA polymerase.

[0140] According to the present invention, the term "gene" refers to a specific nucleic acid sequence that is responsible for producing one or more cellular products and / or for carrying out one or more intercellular or intracellular functions. More specifically, the term relates to a nucleic acid segment (usually DNA; but in the case of RNA viruses, RNA) that contains a nucleic acid that encodes a specific protein or functional or structural RNA molecule.

[0141] As used herein, "isolated molecule" means a molecule that is substantially free of other molecules, such as other cellular material. The term "isolated nucleic acid" means, according to the present invention, a nucleic acid that has been (i) amplified in vitro, such as by polymerase chain reaction (PCR), (ii) produced recombinantly by cloning, (iii) purified, such as by cleavage and gel electrophoresis fractionation, or (iv) synthesized, such as by chemical synthesis. An isolated nucleic acid is a nucleic acid that can be manipulated by recombinant techniques.

[0142] The term "vector" is used herein in its most general sense and includes any intermediate carrier for a nucleic acid that, for example, enables the nucleic acid to be introduced into a prokaryotic and / or eukaryotic host cell and, where appropriate, integrated into the genome. Such vectors are preferably replicated and / or expressed in the cell. Vectors include plasmids, phagemids, viral genomes, and portions thereof.

[0143] In the context of the present invention, the term "recombinant" means "produced by genetic engineering." Preferably, in the context of the present invention, a "recombinant subject," such as a recombinant cell, does not exist naturally.

[0144] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including a virus) and can be isolated from a source in nature and has not been intentionally modified by man in the laboratory is naturally occurring. The term "found in nature" means "occurring 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.

[0145] According to the present invention, the term "expression" is used in its most general sense and includes the production of RNA, or the production of RNA and protein. It also includes partial expression of nucleic acids. Furthermore, expression can be transient or stable. With respect to RNA, the term "expression" or "translation" refers to the process in the ribosomes of the cell by which a chain of coding RNA (e.g., messenger RNA) directs the assembly of an amino acid sequence to produce a peptide or protein.

[0146] According to the present invention, the term "mRNA" means "messenger RNA" and refers to a transcript that is typically produced using a DNA template and encodes a peptide or protein. Typically, mRNA comprises a 5'-UTR, a protein coding region, a 3'-UTR, and a poly(A) sequence. mRNA can be produced by in vitro transcription from a DNA template. In vitro transcription methods are known to those skilled in the art. For example, a variety of in vitro transcription kits are commercially available. According to the present invention, mRNA can be modified by stabilization modification and capping.

[0147] According to the present invention, the term "poly(A) sequence" or "poly(A) tail" refers to an uninterrupted or interrupted sequence of adenylate residues that is typically located at the 3' end of an RNA molecule. An uninterrupted sequence is characterized by a continuous series of adenylate residues. In nature, uninterrupted poly(A) sequences are typical. Although poly(A) sequences are not typically encoded in eukaryotic DNA, they are post-transcriptionally attached to the free 3' end of RNA by template-independent RNA polymerases during eukaryotic transcription in the cell nucleus, and the present invention encompasses poly(A) sequences encoded by DNA.

[0148] According to the present invention, with reference to a nucleic acid molecule, the term "primary structure" refers to the linear sequence of nucleotide monomers.

[0149] According to the present invention, referring to nucleic acid molecules, the term "secondary structure" refers to a two-dimensional diagram of the nucleic acid molecules reflecting base pairing; For example, in the case of single-stranded RNA molecules, particularly intramolecular base pairing. Although each RNA molecule has only a single polynucleotide chain, the molecule is generally characterized by the region of (intramolecular) base pairs. According to the present invention, the term "secondary structure" includes structural motifs, including but not limited to base pairs, stems, stem loops, protrusions, rings such as inner loops and multi-branched loops. The secondary structure of nucleic acid molecules can be represented by a two-dimensional diagram (plan view), showing base pairing (for further details about the secondary structure of RNA molecules, see Auber et al., J.Graph Algorithms Appl., 2006, Vol. 10, pp. 329–351). As described herein, the secondary structure of some RNA molecules is relevant to the context of the present invention.

[0150] 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, when referring to a nucleic acid molecule, "secondary structure" specifically refers to the secondary structure determined by the prediction. Predictions can also be performed or confirmed using MFOLD structure prediction (http: / / unafold.rna.albany.edu / ?q=mfold).

[0151] According to the present invention, a "base pair" is a structural motif of the 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). The base pairs A:U and G:C are called classical base pairs. Other base pairs like G:U (which occurs frequently in RNA) and other rare base pairs (e.g. A:C; U:U) are called non-classical base pairs.

[0152] According to the present invention, "nucleotide pairing" refers to two nucleotides associating with each other such that their bases form a base pair (classical or non-classical base pair, preferably a classical base pair, most preferably a Watson-Crick base pair).

[0153] According to the present invention, with reference to nucleic acid molecules, the terms "stem-loop" or "hairpin" or "hairpin loop" all refer interchangeably to a specific secondary structure of a nucleic acid molecule (typically a single-stranded nucleic acid molecule, such as single-stranded RNA). The specific secondary structure represented by 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; they are separated by a short sequence (e.g. 3 to 10 nucleotides) that forms the loop of the stem-loop structure. Two adjacent fully or partially complementary sequences can be defined, for example, as stem-loop elements stem 1 and stem 2. A stem-loop is formed 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, which results in a double-stranded nucleic acid sequence comprising an unpaired loop formed at its end by a short sequence located between the stem-loop elements stem 1 and stem 2. Thus, a stem-loop comprises two stems (stem 1 and stem 2) that form base pairs with each other at the secondary structure level of the nucleic acid molecule and, at the primary structure level of the nucleic acid molecule, are separated by a short sequence that is not part of stem 1 or stem 2. For illustration, a two-dimensional representation of a stem-loop resembles a lollipop-shaped structure. The formation of a stem-loop structure requires the presence of a sequence that can fold on itself 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 generally determined by the length of stem 1, the number of nucleotides that can form base pairs (preferably classical base pairs, more preferably Watson base pairs) with the nucleotides of stem 2, and the number of nucleotides of stem 1 that cannot form such base pairs (mismatches or bulges) 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, for example 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 generally characterized by a stem-loop. Stem-loops are generally formed by single-stranded RNA molecules. For example, several stem-loops are present in the 5' replication recognition sequence of alphavirus genomic RNA (e.g. Figure 1 ).

[0154] According to the present invention, with reference to a specific secondary structure (e.g., a stem-loop) of a nucleic acid molecule, "disruption" or "disrupt" means that the specific secondary structure is absent or altered. Typically, a secondary structure may be disrupted by a change in at least one nucleotide that is part of the secondary structure. For example, a stem-loop may be disrupted by changing one or more nucleotides that form the stem so that nucleotide pairing is impossible.

[0155] According to the present invention, "compensating for secondary structure disruption" or "compensating for secondary structure disruption" refers to one or more nucleotide changes in a nucleic acid sequence; more typically, it refers to one or more second nucleotide changes in a nucleic acid sequence that further comprises one or more first nucleotide changes, characterized in that when the one or more first nucleotide changes occur, in the absence of the one or more second nucleotide changes, the secondary structure of the nucleic acid sequence is disrupted, and the co-occurrence of the one or more first nucleotide changes and the one or more second nucleotide changes does not result in the secondary structure of the nucleic acid being disrupted. Co-occurrence means the presence of both one or more first nucleotide changes and one or more second nucleotide changes. Typically, the one or more first nucleotide changes and the 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 disruption are one or more nucleotide changes that compensate for one or more nucleotide pairing disruptions. Therefore, in one embodiment, "compensating for secondary structure disruption" means "compensating for nucleotide pairing disruptions", i.e., one or more nucleotide pairing disruptions, such as one or more nucleotide pairing disruptions within one or more stem loops. One or more nucleotide pairing disruptions can be introduced by removing at least one start codon. Each of the one or more nucleotide changes that compensate for secondary structure disruption is a nucleotide change, each of which can be independently selected from the deletion, addition, substitution and / or insertion of one or more nucleotides. In an illustrative example, when the nucleotide pairing A:U (C and U are generally not suitable for forming nucleotide pairs) is destroyed by replacing A with C; the nucleotide change that compensates for nucleotide pairing disruption can replace U with G, thereby enabling formation of C:G nucleotide pairing. Therefore, replacing U with G compensates for nucleotide pairing disruption. In an alternative example, when the nucleotide pairing A:U is destroyed by replacing A with C; the nucleotide change that compensates for nucleotide pairing disruption can replace C with A, thereby restoring the formation of original A:U nucleotide pairing. Generally, in the present invention, it is preferred that those nucleotide changes that compensate for secondary structure disruption neither restore the original nucleic acid sequence nor produce new AUG triplets. In the above-mentioned group of examples, the replacement of U to G is better than the replacement of C to A.

[0156] According to the present invention, with reference to nucleic acid molecules, the term "tertiary structure" refers to the three-dimensional structure of a nucleic acid molecule, as defined by atomic coordinates.

[0157] According to the present invention, nucleic acids such as RNA, for example RNA. mRNA can encode a peptide or protein. Therefore, a transcribable nucleic acid sequence or its transcript can contain an open reading frame (ORF) encoding a peptide or protein.

[0158] According to the present invention, the term "nucleic acid encoding a peptide or protein" means a nucleic acid that, if present in an appropriate environment, preferably within a cell, can direct the assembly of amino acids to produce a peptide or protein during translation. Preferably, the coding RNA according to the present invention is capable of interacting with the cellular translation machinery, thereby allowing translation of the coding RNA to produce a peptide or protein.

[0159] According to the present invention, the term "peptide" includes oligopeptides and polypeptides and refers to a substance comprising two or more, preferably three or more, preferably four or more, preferably six or more, preferably eight or more, preferably ten or more, preferably 13 or more, preferably 16 or more, preferably 20 or more, and preferably up to 50, preferably 100 or preferably 150 consecutive amino acids linked to each other by peptide bonds. The term "protein" refers to large peptides, preferably peptides having at least 151 amino acids, but the terms "peptide" and "protein" are often used synonymously herein.

[0160] According to the present invention, the terms "peptide" and "protein" include substances containing not only amino acid components but also non-amino acid components such as sugars and phosphate structures, and also substances containing bonds such as ester, thioether or disulfide bonds.

[0161] According to the present invention, the terms "initiation codon" and "start codon" refer synonymously to a codon (base triplet) of an RNA molecule, which may be the first codon translated by a ribosome. Such a codon typically encodes the amino acid methionine in eukaryotes and modified methionine in prokaryotes. The most common start codon in eukaryotes and prokaryotes is AUG. Unless otherwise specified herein to mean a start codon other than AUG, when referring to an RNA molecule, the terms "start codon" and "start codon" 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 encoding the RNA start codon. If the start codon of a messenger RNA is AUG, the basic triplet encoding AUG is ATG. According to the present invention, the terms "start codon" and "start codon" preferably refer to a functional start codon or initiation codon, i.e., a start codon or initiation codon that is used or will be used as a codon by the ribosome to initiate translation. In RNA molecules, there may be AUG codons that are not used as a codon by the ribosome to initiate translation, for example, because the distance from the codon to the cap is very short. These codons are not included in the term functional start codon or initiation codon.

[0162] According to the present invention, the term "start codon of an open reading frame" or "start codon of an open reading frame" refers to the basic triplet that serves as the start codon for protein synthesis in a coding sequence (e.g., in the coding sequence of a nucleic acid molecule found in nature). In RNA molecules, the start codon of the open reading frame is usually preceded by the 5' untranslated region (5'-UTR), although this is not strictly required.

[0163] According to the present invention, the term "native start codon of an open reading frame" or "native start codon of an open reading frame" refers to the basic triplet that serves as the start codon for protein synthesis in a native coding sequence. A native coding sequence can be, for example, the coding sequence of a nucleic acid molecule found in nature. In some embodiments, the present invention provides variants of nucleic acid molecules found in nature, characterized in that the native start codon (which is present in the native coding sequence) has been removed (so that it is not present in the variant nucleic acid).

[0164] According to the present invention, "first AUG" means the upstreammost AUG base triplet of a messenger RNA molecule, preferably the upstreammost AUG base triplet of a messenger RNA molecule that is used or will be used as a codon by the ribosome to initiate translation. Thus, "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., the codon that is used as a start codon during ribosomal protein synthesis.

[0165] According to the present invention, referring to certain elements of nucleic acid variants, the term "comprising removal" or "characterized by removal" and similar terms means that the certain elements are inactive or absent in the nucleic acid variant compared to the reference nucleic acid molecule. Without limitation, removal can include deleting all or part of a particular element, replacing all or part of a particular element, or changing the functional or structural properties of a particular element. Removing the functional elements of a nucleic acid sequence requires that the function is not exerted at the position of the nucleic acid variant comprising the removal. For example, an RNA variant characterized in that certain start codons are removed requires that ribosomal protein synthesis does not start at the position of the RNA variant characterized by the removal. Removing the structural elements of a nucleic acid sequence requires that the structural elements are not present at the position of the nucleic acid variant comprising the removal. For example, an RNA variant characterized in that certain AUG base triplets are removed, i.e., an AUG base triplet at a certain position may be characterized by, for example, by deleting part or all of certain AUG base triplets (e.g., ΔAUG), or by replacing one or more nucleotides (A, U, G) of a certain AUG base triplet with any one or more different nucleotides so that the nucleotide sequence of the variant obtained does not comprise the AUG base triplet. Suitable substitutions of one nucleotide are those that convert an AUG base triplet into a GUG, CUG or UUG base triplet, or into an AAG, ACG or AGG base triplet, or into an AUA, AUC or AUU base triplet. Thus, suitable substitutions of more nucleotides can be selected.

[0166] According to the present invention, the term "alphavirus" should be understood broadly and includes any virus particle having alphavirus characteristics. Characteristics of alphaviruses include the presence of (+) strand RNA that encodes genetic information suitable for replication in a host cell, including RNA polymerase activity. Further features of many alphaviruses are described, for example, in Strauss & Strauss, Microbiol. Rev., 1994, Vol. 58, pp. 491-562. The term "alphavirus" includes alphaviruses found in nature, as well as any variants or derivatives thereof. In some embodiments, variants or derivatives are not found in nature.

[0167] In one embodiment, the alphavirus is an alphavirus found in nature. Typically, an alphavirus found in nature is infectious to any one or more eukaryotic organisms, such as animals (including vertebrates such as humans, and arthropods such as insects).

[0168] The alphavirus found in nature is preferably selected from the group consisting of: Bama Forest virus complex (including Bama Forest virus); Eastern equine encephalitis complex (including seven antigenic types of Eastern equine encephalitis viruses); Middleburg virus complex (including Middleburg virus); Ndumu virus complex (including Ndumu virus); Semliki Forest virus complex (including Bebaru virus, Chikungunya virus, Mayaro virus and its subtype Una virus, O'Nyong Nyong virus and its subtype Igbo-Ora virus, Ross River virus and its subtype Bebaru virus, Getah virus, Sagiyama virus, Semliki Forest virus and its subtype Me Tri virus); Venezuelan equine encephalitis complex (including Cabassou virus, Everglades virus, Mosso das Pedras virus, Mucambo virus, Paramana virus, Pixuna virus, Rio Negro virus, Trocara virus and its subtype Bijou Bridge virus, Venezuelan equine encephalitis virus); Western equine encephalitis complex (including Aura virus, Babanki virus, Kyzylagach virus, Sindbis virus, Ockelbo virus, Whataroa virus, BuggyCreek virus, Fort Morgan virus, Highlands J virus, Western equine encephalitis virus); and some unclassified viruses, including salmon pancreatic virus; sleeping sickness virus; Southern elephant seal virus; Tonate virus. More preferably, the alphavirus is selected from the Semliki Forest virus complex (including the virus types described above, including Semliki Forest virus), Western equine encephalitis complex (including the virus types described above, including Sindbis virus), Eastern equine encephalitis virus (including the virus types described above), Venezuelan equine encephalitis complex (including the virus types described above, including Venezuelan equine encephalitis virus).

[0169] In another preferred embodiment, the alphavirus is Semliki Forest virus. In an alternative preferred embodiment, the alphavirus is Sindbis virus. In an alternative preferred embodiment, the alphavirus is Venezuelan equine encephalitis virus.

[0170] In some embodiments of the present invention, the alphavirus is not an alphavirus found in nature. Typically, an alphavirus not found in nature is a variant or derivative of an alphavirus found in nature that is distinguished from an alphavirus found in nature by at least one mutation in the nucleotide sequence (i.e., genomic RNA). The mutation in the nucleotide sequence can be selected from the insertion, substitution, or deletion of one or more nucleotides compared to an alphavirus found in nature. The mutation in the nucleotide sequence can be associated with or unassociated with a mutation in a polypeptide or protein encoded by the nucleotide sequence. For example, an alphavirus not found in nature can be an attenuated alphavirus. An attenuated alphavirus not found in nature is an alphavirus that typically has at least one mutation in its nucleotide sequence that distinguishes it from an alphavirus found in nature and that is not infective at all, or is infective but has a lower ability to cause disease or no ability to cause disease at all. As an illustrative example, TC83 is an attenuated alphavirus distinct from the Venezuelan equine encephalitis virus (VEEV) found in nature (McKinney et al., 1963, Am. J. Trop. Med. Hyg., 1963, Vol. 12; pp. 597-603).

[0171] Members of the alphavirus genus can also be classified based on their relative clinical characteristics in humans: alphaviruses primarily associated with encephalitis and alphaviruses primarily associated with fever, rash, and polyarthritis.

[0172] The term "alphaviral" means found in an alphavirus, or originating from or derived from an alphavirus, such as by genetic engineering.

[0173] 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.

[0174] According to the present invention, the term "alphavirus" refers to an entity derived from an alphavirus. For illustrative purposes, an alphavirus protein may refer to a protein found in an alphavirus and / or a protein encoded by an alphavirus; 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 an "alphavirus genome" and / or a nucleic acid sequence of an "alphavirus genomic RNA."

[0175] According to the present invention, the term "alphavirus RNA" refers to any one or more of the alphavirus genomic RNA (i.e., the (+) chain), the complementary sequence of the alphavirus genomic RNA (i.e., the (-) chain), and the subgenomic transcript (i.e., the (+) chain), or any fragment thereof.

[0176] According to the present invention, "alphavirus genome" refers to the genomic (+) strand RNA of an alphavirus.

[0177] 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 found in nature). In some embodiments, the term "natural alphavirus sequence" also includes the sequence of an attenuated alphavirus.

[0178] According to the present invention, the term "5' replication recognition sequence" preferably refers to a continuous nucleic acid sequence, preferably a ribonucleic acid sequence, which is identical or homologous to the 5' segment of the alphavirus genome. The "5' replication recognition sequence" is a nucleic acid sequence that can be recognized by the alphavirus replicase. The term 5' replication recognition sequence includes natural 5' replication recognition sequences and functional equivalents thereof, such as functional variants of the 5' replication recognition sequences of alphaviruses found in nature. According to the present invention, functional equivalents include derivatives of the 5' replication recognition sequence characterized by the removal of at least one start codon as described herein. The 5' replication recognition sequence is required for the synthesis of the (-) strand complementary sequence of the alphavirus genomic RNA and is required 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 segment of nsP1; but does not include the entire open reading frame encoding nsP1234. In view of the fact that the natural 5' replication recognition sequence usually encodes at least the N-terminal segment of nsP1, the natural 5' replication recognition sequence usually contains 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 typically capable of forming four stem-loops (SLs), namely SL1, SL2, SL3, and SL4. The numbering of these stem-loops begins at the 5' end of the 5' replication recognition sequence.

[0179] According to the present invention, the term "at the 5' end of an alphavirus" refers to the 5' end of the alphavirus genome. The nucleic acid sequence at the 5' end of an alphavirus includes the nucleotides located at the 5' end of the alphavirus genomic RNA, plus an optional contiguous sequence of other nucleotides. In one embodiment, the nucleic acid sequence at the 5' end of an alphavirus is identical to the 5' replication recognition sequence of the alphavirus genome.

[0180] The term "conserved sequence element" or "CSE" refers to a nucleotide sequence found in alphavirus RNA. These sequence elements are called "conserved" because orthologs exist in the genomes of different alphaviruses, and orthologous CSEs from different alphaviruses preferably share a high percentage of sequence identity and / or similar secondary or tertiary structure. The term CSE includes CSE 1, 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 (+) strand synthesis from a (-) strand template. The term "CSE 1" refers to the sequence on the (+) strand; the complementary sequence of CSE 1 (on the (-) strand) acts as a promoter for (+) strand synthesis. Preferably, the term CSE 1 includes the 5'-most nucleotides of the alphavirus genome. CSE 1 typically forms a conserved stem-loop structure. Without wishing to be bound by a particular theory, it is believed that for CSE 1, secondary structure is more important than primary structure, i.e., linear sequence. In the genomic RNA of the model alphavirus Sindbis virus, CSE 1 consists of a contiguous sequence of 44 nucleotides, formed by the 5'-most 44 nucleotides of the genomic RNA (Strauss & Strauss, Microbiol. Rev., 1994, Vol. 58, pp. 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 (note that subgenomic RNA transcripts that do not contain CSE 2 do not serve as templates for (-) strand synthesis). In 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 nucleotides 155-205 of the genomic RNA (Frolov et al., 2001, RNA, Vol. 7, pp. 1638-1651). CSE 2 typically forms two conserved stem-loop structures. These stem-loop structures are referred to as 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, secondary structure is more important than primary structure, i.e., linear sequence.

[0183] According to the present invention, the term "CSE 3" or "linker sequence" refers synonymously to a nucleotide sequence derived from the alphavirus genomic RNA and comprising the start site of the subgenomic RNA. The complement of this sequence in the (-) strand serves to promote transcription of the subgenomic RNA. In the alphavirus genomic RNA, CSE 3 typically overlaps with the region encoding the C-terminal fragment of nsP4 and extends to a short noncoding region upstream of the open reading frame encoding the structural proteins. According to Strauss & Strauss (Microbiol. Rev., 1994, Vol. 58, pp. 491-562), CSE 3 is characterized by the consensus sequence (SEQ ID NO: 1; consensus junction sequence (consensus sequence CSE 3); underlined nucleotides represent the first five nucleotides of the subgenomic transcript).

[0184] In one embodiment of the invention, CSE 3 consists of or comprises SEQ ID NO: 1 or a variant thereof, wherein the variant is preferably characterized by a degree of sequence identity to SEQ ID NO: 1 of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more.

[0185] 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 the alphavirus genomic RNA immediately upstream of the poly(A) sequence in the 3' untranslated region of the alphavirus genome. CSE 4 typically consists of 19 consecutive nucleotides. Without wishing to be bound by a particular theory, CSE 4 is understood to function as a core promoter for initiating (-) strand synthesis (José et al., Future Microbiol., 2009, Vol. 4, pp. 837-856); and / or CSE 4 and the poly(A) tail of the alphavirus genomic RNA are understood to function together for efficient (-) strand synthesis (Hardy & Rice, J. Virol., 2005, Vol. 79, pp. 4630-4639).

[0186] According to Strauss & Strauss, CSE 4 is characterized by a conserved sequence (SEQ ID NO: 2; 19 nt conserved sequence).

[0187] In one embodiment of the invention, CSE 4 consists of or comprises SEQ ID NO: 2 or a variant thereof, wherein the variant is preferably characterized by a degree of sequence identity to SEQ ID NO: 2 of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more.

[0188] 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 binding sites for an RNA polymerase (typically an RNA-dependent RNA polymerase, particularly a functional alphavirus nonstructural protein). The SGP may include other recognition or binding sites for additional factors. A subgenomic promoter is typically a genetic element of a positive-strand RNA virus (e.g., an alphavirus). The subgenomic promoter of an alphavirus is a nucleic acid sequence contained in the viral genomic RNA. A subgenomic promoter is typically characterized in that it allows initiation of transcription (RNA synthesis) in the presence of an RNA-dependent RNA polymerase (e.g., a functional alphavirus nonstructural protein). The RNA (-) chain, i.e., the complementary sequence of the alphavirus genomic RNA, is used as a template for the synthesis of (+) chain subgenomic transcripts, and the synthesis of (+) chain subgenomic transcripts typically begins at or near the subgenomic promoter. The term "subgenomic promoter" as used herein is not limited to any specific location in the nucleic acid comprising such a subgenomic promoter. In some embodiments, the SGP is the same as, overlaps with, or comprises, CSE 3.

[0189] The term "subgenomic transcript" or "subgenomic RNA" refers synonymously to an RNA molecule obtained 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. Subgenomic transcripts can be obtained in the presence of an RNA-dependent RNA polymerase, in particular a functional alphavirus nonstructural protein. For example, the term "subgenomic transcript" can refer to an RNA transcript prepared in an alphavirus-infected cell using the (-) strand complementary sequence 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 using heterologous RNA as a template. For example, subgenomic transcripts can also be obtained by using the (-) strand complementary sequence of the SGP-containing replicon according to the present invention as a template. Therefore, the term "subgenomic transcript" can refer to an RNA molecule obtained by transcribing an alphavirus genomic RNA fragment, as well as an RNA molecule obtainable by transcribing a replicon fragment according to the present invention.

[0190] The term "autologous" is used to describe anything that originates from the same subject. For example, "autologous cells" refer to cells derived from the same subject. Introducing autologous cells into a subject is advantageous because these cells overcome immune barriers that would otherwise lead to rejection.

[0191] The term "allogeneic" is used to describe any material derived from different individuals of the same species. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical.

[0192] The term "homologous" is used to describe any material derived from individuals or tissues having the same genotype, ie, identical twins or animals of the same inbred strain, or tissues or cells thereof.

[0193] The term "heterologous" is used to describe something that is composed of multiple different elements. For example, introducing cells from one individual into a different individual constitutes a xenograft. Heterologous genes are genes that originate from a source other than the subject.

[0194] Specific and / or preferred variations of individual features of the invention are provided below. The present invention also contemplates as particularly preferred embodiments those resulting from combining two or more specific and / or preferred variations described for two or more features of the invention.

[0195] RNA replicon

[0196] In a first aspect, the present invention provides a replicon comprising 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 alphavirus 5' replication recognition sequence. The replicon is preferably an RNA replicon.

[0197] According to the present invention, a 5' replication recognition sequence characterized in that it comprises the removal of at least one start codon compared to a native alphavirus 5' replication recognition sequence may be referred to herein as a "modified 5' replication recognition sequence" or a "5' replication recognition sequence according to the present invention". As described below, the 5' replication recognition sequence according to the present invention may optionally be characterized by the presence of one or more additional nucleotide changes.

[0198] A nucleic acid construct that can be replicated by a replicase (preferably an alphavirus replicase) is referred to as a replicon. According to the present invention, the term "replicon" defines an RNA molecule that can be replicated by an RNA-dependent RNA polymerase that 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 complementary sequence 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 sequence. The replicase function is usually provided by a functional alphavirus nonstructural protein.

[0199] According to the present invention, the terms "can be replicated" 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 with the term "replicase," for example, "capable of being replicated by a replicase," the terms "can be replicated" and "capable of being replicated" describe 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 capable of recognizing the replicon and (ii) the replicase is capable of functioning as an RNA-dependent RNA polymerase (RdRP). Preferably, the replicase is capable of both (i) recognizing the replicon and (ii) functioning as an RNA-dependent RNA polymerase.

[0200] The expression "capable of recognizing" describes that the replicase is capable of physically associating with the replicon, and preferably, the replicase is capable of binding to the replicon, typically non-covalently. The term "binding" may mean that the replicase has the ability to bind to any one or more of conserved sequence element 1 (CSE1) or its complement (if contained by the replicon), conserved sequence element 2 (CSE2) or its complement (if contained by the replicon), conserved sequence element 3 (CSE3) or its complement (if contained by the replicon), and conserved sequence element 4 (CSE4) or its complement (if contained by the replicon). Preferably, the replicase is capable of binding to CSE2 [i.e., to the (+) strand] and / or to CSE4 [i.e., to the (+) strand], or to the complement of CSE1 [i.e., to the (-) strand] and / or to the complement of CSE3 [i.e., to the (-) strand].

[0201] The expression "capable of serving as an RdRP" means that the replicase is capable of catalyzing the synthesis of the (-) strand complementary sequence of the (+) strand RNA of the alphavirus genome, wherein the (+) strand RNA has a template function, and / or the replicase is capable of catalyzing the synthesis of the (+) strand alphavirus genome RNA, wherein the (-) strand RNA has a template function. Generally, the expression "capable of serving as an RdRP" may also include the replicase being 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 is typically initiated at an alphavirus subgenomic promoter.

[0202] The expressions "capable of binding" and "capable of serving as an RdRP" refer to the ability under normal physiological conditions. In particular, they refer to the conditions within a cell that expresses a functional alphavirus nonstructural protein or has been transfected with a nucleic acid encoding a functional alphavirus nonstructural protein. The cell is preferably a eukaryotic cell. The binding ability and / or the ability to serve as an RdRP can be tested experimentally, for example, in a cell-free in vitro system or in eukaryotic cells. Optionally, the eukaryotic cell is a cell from a species in which the specific alphavirus representing the origin of the replicase is infectious. For example, when using an alphavirus replicase from a specific alphavirus that is infectious for humans, normal physiological conditions are the conditions in human cells. More preferably, the eukaryotic cell (in one example, a human cell) is from the same tissue or organ as the specific alphavirus representing the origin of the replicase is infectious.

[0203] According to the present invention, "compared to a native alphavirus sequence" and similar terms refer to a sequence that is a variant of a native alphavirus sequence. The variant is typically not itself a native alphavirus sequence.

[0204] 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 alphavirus nonstructural protein. In other words, the functional alphavirus nonstructural protein is capable of recognizing 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.

[0205] 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 functional alphavirus nonstructural proteins. Thus, when present alone or preferably together, these recognition sequences direct the replication of the RNA replicon in the presence of functional alphavirus nonstructural proteins.

[0206] Preferably, the functional alphavirus nonstructural protein is provided in cis (encoded as the protein of interest by an open reading frame on the replicon) or in trans (encoded as the protein of interest by an open reading frame on a separate replicase construct described in the second aspect) that is capable of recognizing both the modified 5' replication recognition sequence and the 3' replication recognition sequence of the replicon. In one embodiment, this is achieved when the 3' replication recognition sequence is native to the alphavirus from which the functional alphavirus nonstructural protein is derived, and when the modified 5' replication recognition sequence is a variant of the 5' replication recognition sequence. The variant of the 5' replication recognition sequence is native to the alphavirus from which the functional alphavirus nonstructural protein was derived. Native means that the natural source of these sequences is the same alphavirus. In an alternative embodiment, the modified 5' replication recognition sequence and / or the 3' replication recognition sequence are not native to the alphavirus from which the functional alphavirus nonstructural protein is derived, provided that the functional alphavirus nonstructural protein is capable of recognizing both the modified 5' replication recognition sequence and the 3' replication recognition sequence of the replicon. In other words, the functional alphavirus nonstructural protein is compatible with the modified 5' replication recognition sequence and the 3' replication recognition sequence. When the non-natural functional alphavirus nonstructural protein is able to recognize each sequence or sequence element, the functional alphavirus nonstructural protein is considered to be compatible (cross-virus compatibility). Any combination of (3' / 5') replication recognition sequences and CSEs with functional alphavirus nonstructural proteins is possible, as long as there is cross-virus compatibility. By incubating the functional alphavirus nonstructural protein to be tested with RNA, wherein the RNA has the 3'- and (optionally modified) 5' replication recognition sequences to be tested, those skilled in the art who perform the present invention can easily test cross-virus compatibility under conditions suitable for RNA replication (e.g., in a suitable host cell). If replication occurs, it is determined that the (3' / 5') replication recognition sequence and the functional alphavirus nonstructural protein are compatible.

[0207] Removal of at least one start codon provides advantages over prior art trans-replicons (e.g., Figure 1The invention relates to a method for expressing a transgenic protein of interest in a cell, wherein the replicon is expressed as a template RNA WT-RRS (denoted by "Template RNA WT-RRS"). The absence of a start codon in the nucleic acid sequence encoding nsP1* generally results in nsP1* (the N-terminal fragment of nsP1) not being translated. Furthermore, because nsP1* is not translated, the open reading frame encoding the protein of interest ("transgene") is the most upstream open reading frame accessible to ribosomes; thus, when the replicon is present in the cell, translation begins at the first AUG of the open reading frame (RNA) encoding the gene of interest. This represents an advantage over prior art trans-replicons, such as those described by Spuul et al. (J. Virol., 2011, Vol. 85, pp. 4739-4751): the replicon according to Spuul et al. directs expression of the N-terminal portion of nsP1 (a 74-amino acid peptide). It is also known from the prior art that the construction of RNA replicons from full-length viral genomes is not a trivial matter, since certain mutations can render the RNA incapable of replication (WO 2000 / 053780 A2), and the removal of parts of the 5' structure that are important for alphavirus replication affects the replication efficiency (Kamrud et al., 2010, J. Gen. Virol., Vol. 91, pp. 1723-1727).

[0208] An advantage over prior art cis-replicons is that removal of at least one start codon decouples the coding region for the alphavirus nonstructural protein from the 5' replication recognition sequence. This enables further engineering of the cis-replicons, for example by exchanging the native 5' replication recognition sequence for an artificial sequence, a mutant sequence, or a heterologous sequence from another RNA virus. Such sequence manipulation in prior art cis-replicons is limited by the amino acid sequence of nsP1. Any point mutation or cluster of point mutations requires experimental evaluation to determine whether replication is affected, and small insertions or deletions that result in frameshift mutations are not possible due to their adverse effects on the protein.

[0209] Removal of at least one start codon according to the present invention can be achieved by any suitable method known in the art. For example, a suitable DNA molecule encoding the replicon of the present invention, i.e., characterized by the removal of the start codon, can be designed in silico and subsequently 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 respective DNA molecule can serve as a template for in vitro transcription, thereby providing the replicon according to the present invention.

[0210] Compared to the natural alphavirus 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 example: at the DNA level, the insertion must not be an ATG insertion).

[0211] 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 by 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 the 5' replication recognition sequence of the genome of at least one alphavirus occurring in nature.

[0212] In one embodiment, the 5' replication recognition sequence of an RNA replicon characterized by the removal of at least one start codon comprises a sequence homologous to approximately 250 nucleotides from the 5' terminus of an alphavirus, i.e., at the 5' terminus of the alphavirus genome. In a preferred embodiment, it comprises a sequence homologous to approximately 250 to 500, preferably approximately 300 to 500, nucleotides from the 5' terminus of an alphavirus, i.e., at the 5' terminus of the alphavirus genome. "At the 5' terminus of the alphavirus genome" means the nucleic acid sequence starting from and including the most upstream nucleotide of the alphavirus genome. In other words, the most upstream nucleotide of the alphavirus genome is designated as nucleotide number 1, e.g., "the 250 nucleotides from the 5' terminus of the alphavirus genome" refers to nucleotides 1 to 250 of the alphavirus genome. In one embodiment, the 5' replication recognition sequence of an RNA replicon characterized by the removal of at least one start codon is characterized by a degree of sequence identity of 80% or greater, preferably 85% or greater, more preferably 90% or greater, and even more preferably 95% or greater, to at least 250 nucleotides from the 5' terminus of the genome of at least one alphavirus found in nature. At least 250 nucleotides includes, for example, 250 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides.

[0213] The 5' replication recognition sequence of alphaviruses found 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) comprises five specific AUG base triplets, corresponding to the ATG base triplets in the DNA of SEQ ID NO: 4 (see Example 1). According to Frolov et al. (2001, RNA, Vol. 7, pp. 1638-1651), MFOLD analysis showed that the natural 5' replication recognition sequence of Semliki Forest virus is predicted to form four stem loops (SL), referred to as stem loops 1 to 4 (SL1, SL2, SL3, SL4). According to Frolov et al., MFOLD analysis showed that the natural 5' replication recognition sequences of different alphaviruses and Sindbis virus are also predicted to form four stem loops: SL1, SL2, SL3, SL4. In Example 1 of the present invention, the presence of four predicted stem-loops in the natural 5' replication recognition sequence of Semliki Forest virus was confirmed by two web servers for RNA secondary structure prediction (http: / / rna.urmc.rochester.edu / RNAstructureWeb / Servers / Predict1 / Predict1.html) (http: / / unafold.rna.albany.edu / ?q=mfold).

[0214] It is known that the 5' end of the alphavirus genome contains sequence elements that enable replication of the alphavirus genome via 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) and / or a sequence homologous to the conserved sequence element 2 (CSE 2) of the alphavirus.

[0215] The conserved sequence element 2 (CSE 2) of the alphavirus genomic RNA is typically represented by SL3 and SL4, and is preceded by SL2, which contains the natural start codon encoding the first amino acid residue of the alphavirus nonstructural protein nsP1. However, in some embodiments herein, the conserved sequence element 2 (CSE 2) of the alphavirus genomic RNA refers to the region spanning from SL2 to SL4 and containing the natural start codon encoding the first amino acid residue of the alphavirus nonstructural protein nsP1. In a preferred embodiment, the RNA replicon contains CSE 2 or a sequence homologous to CSE 2. In one embodiment, the RNA replicon contains a sequence homologous to CSE 2, preferably characterized by a degree of sequence identity of 80% or greater, 85% or greater, more preferably 90% or greater, and even more preferably 95% or greater to the CSE 2 sequence of at least one alphavirus found in nature.

[0216] In a preferred embodiment, the 5' replication recognition sequence comprises a sequence homologous to the CSE 2 of an alphavirus. The CSE 2 of an alphavirus may comprise a fragment of the open reading frame from a nonstructural protein of an alphavirus.

[0217] Thus, in a preferred embodiment, the RNA replicon is characterized in that it comprises a sequence homologous to an open reading frame of a nonstructural protein from an alphavirus or a fragment thereof. Sequences homologous to an open reading frame of a nonstructural protein or a fragment thereof are typically variants of a naturally occurring open reading frame of a nonstructural protein from an alphavirus or a fragment thereof. In one embodiment, the sequence homologous to an open reading frame of a nonstructural protein or a fragment thereof is preferably characterized in that the degree of sequence identity to an open reading frame of a nonstructural protein from at least one naturally occurring alphavirus or a fragment thereof is 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more.

[0218] In a more preferred embodiment, the sequence homologous to the open reading frame of the nonstructural protein contained in the replicon of the present invention does not contain the natural start codon of the nonstructural protein, and more preferably does not contain any natural start codon of the nonstructural protein. In a preferred embodiment, the sequence homologous to CSE 2 is characterized by the removal of all start codons compared to the natural alphavirus CSE2 sequence. Therefore, the sequence homologous to CSE 2 preferably does not contain any start codon.

[0219] When the sequence homologous to the open reading frame does not contain any start codon, the sequence homologous to the open reading frame is not itself an open reading frame because it does not serve as a template for translation.

[0220] 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 includes the removal of at least one start codon compared to the native alphavirus sequence.

[0221] In a preferred embodiment, the sequence homologous to the open reading frame of a nonstructural protein from an alphavirus or a fragment thereof is characterized in that it comprises at least the removal of the natural start codon of the open reading frame of the nonstructural protein. Preferably, it is characterized in that it comprises at least the removal of the natural start codon of the open reading frame encoding nsP1.

[0222] The natural start codon is the AUG base triplet at which translation begins by ribosomes in a host cell 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, such as 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 a preferred embodiment, the host cell is from the cell line "BHK21[C13]( CCL10 TM )" BHK21 cells, available from American Type Culture Collection, Manassas, Virginia, USA.

[0223] 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 determination of the natural start codon in silico.

[0224] For illustration, in Example 1, the DNA base triplet corresponding to the natural start codon of SFV nsP1 is described.

[0225] In one embodiment, the natural start codon is comprised of a Kozak sequence or a functionally equivalent sequence. The Kozak sequence is a sequence originally described by Kozak (1987, Nucleic Acids Res., Vol. 15, pp. 8125–8148). The Kozak sequence on an mRNA molecule is recognized by the ribosome as a translation start site. According to this reference, the Kozak sequence comprises the AUG start codon, followed by the highly conserved G nucleotide: AUG G (see also the start codon in the DNA sequence according to SEQ ID NO: 4 (Example 1)). In particular, the reference describes that the Kozak sequence can be represented by (gcc)gccRcc AUG G (SEQ ID NO: 6) as follows: (i) lowercase letters indicate the most common base at that position, however, the base can vary; (ii) uppercase letters indicate highly conserved bases (e.g., 'AUGG'); (iii) 'R' indicates a purine (adenine or guanine); (iv) the sequence in brackets ((gcc)) is of uncertain importance; (v) the underlined AUG base triplet represents the start codon. In one embodiment of the present invention, the sequence homologous to the open reading frame of the nonstructural protein from an alphavirus or a fragment thereof is characterized in that it includes the removal of the start codon that is part of the Kozak sequence.

[0226] In one embodiment of the invention, the 5' replication recognition sequence of the replicon is characterized, at least at the RNA level, by AUG Removal of those start codons that are part of the G sequence.

[0227] In a preferred embodiment, the sequence homologous to the open reading frame of the nonstructural protein from an alphavirus or a fragment thereof is characterized in that it includes the removal of one or more start codons other than the natural start codon of the open reading frame of the nonstructural protein. In a more preferred embodiment, the nucleic acid sequence is further characterized in that the natural start codon is removed. For example, in addition to the removal of the natural start codon, any one, two, three, four, or more than four (e.g., five) start codons may be removed.

[0228] If the replicon is characterized by the removal of the natural start codon of the open reading frame of the nonstructural protein, and optionally one or more start codons in addition to the natural start codon, the sequence homologous to the open reading frame is not itself an open reading frame since it does not serve as a template for translation.

[0229] Preferably, in addition to the removal of the natural start codon, one or more start codons are removed in addition to the natural start codon and are preferably selected from AUG base triplets with the potential to start translation. The 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 in silico or in a cell-based in vitro assay.

[0230] In one embodiment, it is determined in silico whether a given AUG base triplet has the potential to initiate translation: in this embodiment, the nucleotide sequence is examined, and if it is AUG A portion of a G sequence, preferably a portion of a Kozak sequence (SEQ ID NO: 6), determines that the AUG base triplet has the potential to initiate translation.

[0231] In one embodiment, whether a given AUG base triplet has the potential to initiate translation is determined in a cell-based in vitro assay by introducing an RNA replicon into a host cell, the RNA replicon being characterized by the removal of the natural start codon and comprising the given AUG base triplet downstream of the position where the natural start codon was removed. In one embodiment, the host cell is a cell from a eukaryotic species that is a natural host of a particular alphavirus comprising a natural alphavirus 5' replication recognition sequence. In a preferred embodiment, the host cell is a cell from the cell line "BHK21[C13]( CCL10 TM)" BHK21 cells, available from American Type Culture Collection, Manassas, Virginia, USA. Preferably, no additional AUG base triplet exists between the natural start codon removal position and the given AUG base triplet. If, after an RNA replicon characterized by the removal of the natural start codon and comprising a given AUG base triplet is transferred into a host cell, translation is initiated at the given AUG base triplet, then the given AUG base triplet is determined to have the potential to initiate translation. Whether translation is initiated can be determined by any suitable method known in the art. For example, the replicon can encode a tag that facilitates detection of the translation product (if any), such as a myc-tag or an H-tag, downstream of the given AUG base triplet and in the same frame as the given AUG base triplet. A-tag; It can be determined, for example, by Western blot whether the expression product with the encoded tag exists. In this embodiment, preferably there is no additional AUG base triplet between the given AUG base triplet and the nucleic acid sequence encoding the tag. For more than one given AUG base triplet, a cell-based in vitro assay can be performed respectively: in each case, preferably there is no additional AUG base triplet between the natural start codon removal position and the given AUG base triplet. This can be achieved by removing all AUG base triplets (if any) between the natural start codon removal position and the given AUG base triplet. Therefore, a given AUG base triplet is the first AUG base triplet downstream of the natural start codon removal position.

[0232] Preferably, the replicon according to the invention is characterized by the removal of all potential start codons downstream of the removal position of the natural start codon and located within the open reading frame of the alphavirus nonstructural protein or its fragment. Thus, according to the invention, the 5' replication recognition sequence preferably does not contain an open reading frame that can be translated into protein.

[0233] In a preferred embodiment, the 5' replication recognition sequence of the RNA replicon according to the present invention is characterized by a secondary structure identical to the secondary structure of the 5' replication recognition sequence of the genomic RNA of an alphavirus. In a preferred embodiment, the 5' replication recognition sequence of the RNA replicon according to the present invention is characterized by a predicted secondary structure identical to the predicted secondary structure of the 5' replication recognition sequence of the genomic RNA of an alphavirus. According to the present invention, the secondary structure of the RNA molecule is preferably predicted using a web server for RNA secondary structure prediction: http: / / rna.urmc.rochester.edu / RNAstructureWeb / Servers / Predict1 / Predict1.html.

[0234] 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 be absent at a given position, such as within a stem-loop, and particularly within the stem of the stem-loop, compared to a native alphavirus 5' replication recognition sequence.

[0235] In a preferred 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. More preferably, no stem loop is deleted or disrupted in the 5' replication recognition sequence.

[0236] 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 really destroyed the secondary structure of 5 ' replication recognition sequence.In this embodiment, the removal of at least one start codon may be the reason that at least one base pair (for example base pair in the stem-loop) of a given position is not present compared with natural alphavirus 5 ' replication recognition sequence.If the given position base pair is not present compared with natural alphavirus 5 ' replication recognition sequence, then determine that the removal of at least one start codon has introduced nucleotide pairing destruction in the stem-loop.The base pair in the stem-loop is normally the base pair in the stem of the stem-loop.

[0237] In a preferred embodiment, the RNA replicon comprises one or more nucleotide changes that compensate for the disruption of nucleotide pairing within one or more stem-loops introduced by the removal of at least one start codon.

[0238] If removal of at least one start codon introduces a disruption in nucleotide pairing within the stem-loop compared to the native alphavirus 5' replication recognition sequence, one or more nucleotide changes can be introduced that are expected to compensate for the disruption in nucleotide pairing. The resulting or predicted secondary structure can then be compared to the native alphavirus 5' replication recognition sequence.

[0239] Based on common general knowledge and the disclosure of this article, a skilled person can anticipate certain nucleotide changes to compensate for nucleotide pairing disruption. For example, if the 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 compared to a natural alphavirus 5' replication recognition sequence is disrupted, it is expected that the base pair at that position will be restored and preferably the nucleotide change of the start codon will not be reintroduced to compensate for the nucleotide pairing disruption. In one example, the 5' replication recognition sequence of a replicon comprising one or more nucleotide changes that compensate for the nucleotide pairing disruption introduced within one or more stem loops by the removal of at least one start codon is encoded by the DNA sequence represented by SEQ ID NO:5 (Example 1).

[0240] In a preferred 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 an nsP, particularly nsP1, or a fragment thereof). For a nucleotide sequence to be "translatable," the presence of a start codon is required; the start codon encodes the majority of the N-terminal amino acid residues of the peptide or protein. In one embodiment, the 5' replication recognition sequence of the replicon does not overlap with or contain a translatable nucleic acid sequence encoding the N-terminal fragment of nsP1.

[0241] In some cases described in detail below, 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 nsP1, or a fragment thereof). In one embodiment, the subgenomic promoter of the replicon does not overlap with or contain a translatable nucleic acid sequence encoding the C-terminal fragment of nsP4. An RNA replicon having a subgenomic promoter that does not overlap with or contain a translatable nucleic acid sequence (e.g., translated into the C-terminal fragment of nsP4) can be generated by deleting a portion of the coding sequence of nsP4 (typically a portion encoding the N-terminal portion of nsP4) and / or by removing an AUG base triplet from the non-deleted portion of the coding sequence of nsP4. If an AUG base triplet is removed from the coding sequence of nsP4 or a portion thereof, 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.

[0242] In one embodiment, the RNA replicon does not contain an open reading frame encoding a truncated alphavirus nonstructural protein. In the context of this embodiment, it is particularly preferred that the RNA replicon does not contain an open reading frame encoding the N-terminal fragment of nsP1, and optionally does not contain an open reading frame encoding the C-terminal fragment of nsP4. The N-terminal fragment of nsP1 is a truncated alphavirus protein; the C-terminal fragment of nsP4 is also a truncated alphavirus protein.

[0243] In some embodiments, the replicon according to the invention does not comprise the stem-loop 2 (SL2) at the 5' end of the alphavirus genome. According to Frolov et al. (supra), the stem-loop 2 is a conserved secondary structure found at the 5' end of the alphavirus genome, upstream of CSE 2, but not essential for replication.

[0244] In one embodiment, the 5' replication recognition sequence of the replicon does not overlap with a nucleic acid sequence encoding an alphavirus nonstructural protein or a fragment thereof. Thus, the present invention includes replicons characterized by the removal of at least one start codon as described herein, and optionally also by the deletion of the coding region for one or more alphavirus nonstructural proteins, or a portion thereof, compared to genomic alphavirus RNA. For example, the coding regions for nsP2 and nsP3 can be deleted, or the coding regions for nsP2 and nsP3 can be deleted and the coding region for the C-terminal fragment of nsP1 and / or the coding region for the N-terminal fragment of nsP4 can be deleted, and one or more remaining (i.e., remaining after said removal) start codons can be removed as described herein.

[0245] Deletion of the coding region for one or more alphavirus nonstructural proteins can be achieved by standard methods, for example, at the DNA level, by excision with the aid of restriction enzymes, preferably restriction enzymes that recognize unique restriction sites within the open reading frame (e.g., see Example 1). Optionally, unique restriction sites can be introduced into the open reading frame by mutagenesis (e.g., site-directed mutagenesis). The corresponding DNA can be used as a template for in vitro transcription.

[0246] A restriction site is a nucleic acid sequence, such as a DNA sequence, that is necessary and sufficient to direct a specific restriction enzyme to restrict (cut) a nucleic acid molecule (such as a DNA molecule) that contains the restriction site. A restriction site is unique to a given nucleic acid molecule if there is one copy of the restriction site in the nucleic acid molecule.

[0247] Restriction enzymes are endonucleases that cleave nucleic acid molecules (eg, DNA molecules) at or near restriction sites.

[0248] Alternatively, nucleic acid sequences characterized by deletion of part or all of the open reading frame can be obtained synthetically.

[0249] The RNA replicon according to the present invention is preferably a single-stranded RNA molecule. The RNA replicon according to the present invention is typically a (+)-stranded RNA molecule. In one embodiment, the RNA replicon according to the present invention is an isolated nucleic acid molecule.

[0250] The replicon contains at least one open reading frame

[0251] In one embodiment, the RNA replicon according to the present invention comprises at least one open reading frame encoding a peptide or protein of interest. Preferably, the protein of interest is encoded by a heterologous nucleic acid sequence. The gene encoding the peptide or protein of interest is synonymously referred to as a "gene of interest" or a "transgene." In various embodiments, the peptide or protein of interest is encoded by a heterologous nucleic acid sequence. According to the present invention, the term "heterologous" refers to the fact that the nucleic acid sequence is not naturally functionally or structurally linked to an alphavirus nucleic acid sequence.

[0252] The replicons according to the present 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 replicons according to the present invention may comprise more than one open reading frame, each open reading frame being independently selectable as being under the control of a subgenomic promoter. Alternatively, the polyprotein or fusion polypeptide comprises individual polypeptides separated by an optional autocatalytic protease cleavage site (e.g., foot-and-mouth disease virus 2A protein) or intein.

[0253] The protein of interest can be, for example, selected from the group consisting of a reporter protein, a pharmaceutically active peptide or protein, an inhibitor of intracellular interferon (IFN) signaling, and a functional alphavirus nonstructural protein.

[0254] Reporter protein

[0255] In one embodiment, the open reading frame encodes a reporter protein. In this embodiment, the open reading frame comprises a reporter gene. Certain genes can be selected as reporter genes because the characteristics of the cells or organisms that express them can be easily identified and measured, or because they are selectable markers. Reporter genes are generally used to indicate whether a certain gene is absorbed or expressed by a cell or biological population. Preferably, the expression product of the reporter gene is visually detectable. Common visually detectable reporter proteins generally have fluorescence or luminescent proteins. Examples of specific reporter genes include genes encoding: jellyfish green fluorescent protein (GFP), which causes cells expressing it to emit green light under blue light; enzyme luciferase, which catalyzes and reacts with luciferin to produce light; and red fluorescent protein (RFP). Any variant of these specific reporter genes is possible, as long as the variant has visually detectable characteristics. For example, eGFP is a point mutant variant of GFP. The reporter protein embodiment is particularly suitable for testing expression, referring to, for example, Examples 2 to 5.

[0256] Pharmaceutically active peptides or proteins

[0257] According to the present invention, in one embodiment, the RNA of the replicon comprises or consists of a pharmaceutically active RNA. "Pharmaceutically active RNA" may be an RNA encoding a pharmaceutically active peptide or protein. Preferably, the RNA replicon according to the present invention encodes a pharmaceutically active peptide or protein. Preferably, the open reading frame encodes a pharmaceutically active peptide or protein. Preferably, the RNA replicon comprises an open reading frame encoding a pharmaceutically active peptide or protein, optionally under the control of a subgenomic promoter.

[0258] When administered to a subject in a therapeutically effective amount, a "pharmaceutically active peptide or protein" has a positive or beneficial effect on the subject's condition or disease state. Preferably, the pharmaceutically active peptide or protein has therapeutic or palliative properties and can be administered to improve, alleviate, relieve, reverse one or more symptoms of a disease or condition, delay its onset or reduce its severity. Pharmaceutically active peptides or proteins may have prophylactic properties 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 protein" includes intact proteins or polypeptides and may also refer to pharmaceutically active fragments thereof. It may also include pharmaceutically active analogs of the peptide or protein. The term "pharmaceutically active peptide or protein" includes peptides and proteins that are antigens, i.e., the peptide or protein elicits an immune response in a subject, which may be therapeutic or partially or fully protective.

[0259] In one embodiment, the pharmaceutically active peptide or protein is or comprises an immunologically active compound or antigen or epitope.

[0260] According to the present invention, the term "immunologically active compound" relates to any compound that alters the immune response, preferably by inducing and / or inhibiting the maturation of immune cells, inducing and / or inhibiting cytokine biosynthesis, and / or altering humoral immunity by stimulating antibody production by B cells. In one embodiment, the immune response involves stimulating an antibody response (typically including immunoglobulin G (IgG)). Immunologically active compounds have potent immunostimulatory activity, including but not limited to antiviral and antitumor activity, and may also downregulate other aspects of the immune response, for example, shifting the immune response away from a TH2 immune response, which is useful for treating a number of TH2-mediated diseases.

[0261] According to the present invention, the term "antigen" or "immunogen" encompasses any substance that triggers an immune response. In particular, "antigen" refers to any substance that specifically reacts with antibodies or T lymphocytes (T cells). According to the present invention, the term "antigen" includes any molecule that comprises at least one epitope. Preferably, the antigen in the context of the present invention is a molecule that induces an immune response, optionally after processing, and the immune response is preferably antigen-specific. 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 a humoral immune response or a cellular immune response. In the context of an embodiment of the present invention, in the case of an MHC molecule, the antigen is preferably presented by a cell, preferably an antigen-presenting cell, which results in an immune response against the antigen. Antigens are preferably products corresponding to or derived from naturally occurring antigens. These naturally occurring antigens can include or can be derived from allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens, or the antigen can also be a tumor antigen. According to the present invention, antigens can correspond to naturally occurring products, such as viral proteins or parts thereof. In a preferred embodiment, the antigen is a surface polypeptide, i.e., an antigen that is naturally displayed on the surface of a cell, pathogen, bacterium, virus, fungus, parasite, allergen, or tumor. The antigen can elicit an immune response against the cell, pathogen, bacterium, virus, fungus, parasite, allergen, or tumor.

[0262] The term "pathogen" refers to disease-causing biological material that is capable of causing disease in an organism, preferably a vertebrate organism. Pathogens include microorganisms such as bacteria, single-cell eukaryotes (protozoa), fungi, and viruses.

[0263] The terms "epitope", "antigenic peptide", "antigenic epitope", "immunogenic peptide" and "MHC binding peptide" are used interchangeably herein and refer to an antigenic determinant in a molecule (such as an antigen), i.e., a part or fragment of an immunologically active compound that is recognized by the immune system, e.g., 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 said protein and preferably has a length of 5 to 100, preferably 5 to 50, more preferably 8 to 30, and most preferably 10 to 25 amino acids, e.g., 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. According to the present invention, an epitope may bind to an MHC molecule, e.g., an MHC molecule on the surface of a cell, and may therefore be an "MHC binding peptide" or "antigenic peptide." The term "major histocompatibility complex" and the abbreviation "MHC" include MHC class I and MHC class II molecules and refer to gene complexes 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, where 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 self-antigens (peptide fragments from the cell itself) and non-self antigens (e.g., fragments of invading microorganisms) to T cells. Preferred such immunogenic portions bind to MHC class I or class II molecules. As used herein, if such binding is detected using any assay known in the art, the immunogenic portion is said to be "bound" to an MHC class I or class II molecule. The term "MHC binding peptide" refers to a peptide that binds to an MHC class I and / or MHC class II molecule. In the case of class I MHC / peptide complexes, the length of the binding peptide is typically 8-10 amino acids, although longer or shorter peptides may also be effective. In the case of MHC class II / peptide complexes, binding peptides are typically 10-25 amino acids in length, particularly 13-18 amino acids in length, although longer and shorter peptides may also be effective.

[0264] In one embodiment, the protein of interest according to the present invention comprises an epitope suitable for vaccination of the target organism. It is known to those skilled in the art that one of the principles of immunobiology and vaccination is based on the fact that an immunoprotective response is generated by immunizing an organism with an antigen that is immunologically related to the disease to be treated. According to the present invention, the antigen is selected from self-antigens and non-self-antigens. The non-self-antigen is preferably a bacterial antigen, a viral antigen, a fungal antigen, an allergen or a parasite antigen. Preferably, the antigen comprises an epitope that can elicit an immune response in the target organism. For example, the epitope can elicit an immune response against bacteria, viruses, fungi, parasites, allergens or tumors.

[0265] 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 domestic animals. Preferably, the bacteria against which the immune response is elicited are pathogenic bacteria.

[0266] In some embodiments, the non-self antigen is a viral antigen. The viral antigen can be, for example, a peptide derived from a viral surface protein, such as a capsid polypeptide or a spike polypeptide. In some embodiments, the antigen elicits an immune response against a virus that infects an animal, including birds, fish, and mammals, including domestic animals. Preferably, the virus against which the immune response is elicited is a pathogenic virus.

[0267] 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 domestic animals. Preferably, the fungus against which the immune response is elicited is a pathogenic fungus.

[0268] 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 against a pathogenic unicellular eukaryotic parasite. Pathogenic unicellular eukaryotic parasites 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 Trypanosoma cruzi or T. brucei.

[0269] In some embodiments, the non-self antigen is an allergen polypeptide or an allergen protein.The allergen protein or allergen polypeptide is suitable for allergen immunotherapy (also known as desensitization).

[0270] In some embodiments, the antigen is a self-antigen, particularly a tumor antigen.Tumor antigens and their determination are known to those skilled in the art.

[0271] In the context of the present invention, the term "tumor antigen" or "tumor-associated antigen" relates to proteins that are specifically expressed in a limited number of tissues and / or organs or in a specific developmental stage under normal conditions. For example, a tumor antigen may be specifically expressed in gastric tissue (preferably in the gastric mucosa), in reproductive organs (e.g., in the testes), in trophoblastic tissue (e.g., in the placenta), or in germline cells under normal conditions, and is expressed or abnormally expressed in one or more tumor or cancer tissues. In this context, "a limited number" preferably means no more than 3, more preferably no more than 2. Tumor antigens in the context of the present invention include, for example: differentiation antigens, preferably cell type-specific differentiation antigens, i.e., proteins that are specifically expressed in a specific cell type at a specific differentiation stage under normal conditions; cancer / testicular antigens, i.e., proteins that are specifically expressed in the testes and sometimes in the placenta under normal conditions; and germline-specific antigens. In the context of the present invention, tumor antigens are preferably associated with the cell surface of cancer cells and are preferably not expressed or only rarely expressed in normal tissues. Preferably, tumor antigens or abnormal expression of tumor antigens identify cancer cells. In the context of the present invention, a tumor antigen expressed by a cancer cell in a subject (e.g., a patient suffering from a cancer disease) is preferably a self-protein in the subject. In some preferred embodiments, a tumor antigen in the context of the present invention is specifically expressed under normal conditions in non-essential tissues or organs (i.e., tissues or organs that would not lead to death in the subject if damaged by the immune system) or in body organs or structures that are inaccessible or nearly inaccessible to the immune system. Preferably, the amino acid sequence of the tumor antigen is identical between the tumor antigen expressed in normal tissue and the tumor antigen expressed in cancer tissue.

[0272] Examples of tumor antigens that can be used in the present invention are p53, ART-4, BAGE, β-catenin / m, Bcr-abLCAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, cell surface proteins of the occludin family, such as CLAUDIN-6, CLAUDIN-18.2 and CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2 , hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE -A10, MAGE-A11 or MAGE-A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, Myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 Minor BCR-abL, Pm1 / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART- or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, and WT. Particularly preferred tumor antigens include CLAUDIN-18.2 (CLDN18.2) and CLAUDIN-6 (CLDN6).

[0273] In some embodiments, it is not required that the pharmaceutically active peptide or protein is an antigen that elicits an immune response. Suitable pharmaceutically active proteins or peptides can be selected from the following: cytokines and immune system proteins, such as immunoactive compounds (e.g., interleukins, colony stimulating factor (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, immunoglobulins), hormones (insulin, thyroid hormones, catecholamines, gonadotropins, nutritional hormones, prolactin, oxytocin, dopamine, bovine growth hormone, 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, leptin, etc.), activators, streptokinase, cholesterol biosynthesis or degradation, steroid hormone synthase, kinase, phosphodiesterase, methylase, demethylase, dehydrogenase, cellulase, protease, lipase, phospholipase, aromatase, cytochrome, adenylate or guanylate cyclase, ceramidase, etc.), receptors (steroid hormone receptors, peptide receptors), binding proteins (growth hormone or growth factor binding protein, etc.), transcription and translation factors, proteins that inhibit tumor growth (such as proteins that inhibit angiogenesis), structural proteins (such as 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 Hippelmann-Stokes protein), 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, priming, 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.

[0274] Inhibitors of interferon (IFN) signaling

[0275] Another suitable target protein encoded by the open reading frame is an inhibitor of interferon (IFN) signal transduction. Although it has been reported that the viability of cells into which RNA has been introduced for expression can be reduced, particularly if cells are transfected multiple times with RNA, it has been found that IFN inhibitors enhance the viability of cells in which RNA is expressed (WO 2014 / 071963 A1). Preferably, the inhibitor is an inhibitor of IFN type I signal transduction. By preventing IFN receptors from being bound by extracellular IFN and inhibiting intracellular IFN signal transduction in the cell, RNA can be stably expressed in the cell. Alternatively or in addition, preventing IFN receptors from being bound by extracellular IFN and inhibiting intracellular IFN signal transduction enhances cell survival, particularly if cells are repeatedly transfected with RNA. Without wishing to be bound by theory, it is envisioned that intracellular IFN signal transduction can lead to inhibition of translation and / or RNA degradation. This can be solved by inhibiting one or more IFN-inducible antiviral activity effector proteins. IFN-inducible antiviral activity effector proteins can be selected from RNA-dependent protein kinase (PKR), 2', 5'-oligoadenylate synthetase (OAS) and RNaseL. Inhibiting intracellular IFN signaling may include inhibiting PKR-dependent pathways and / or OAS-dependent pathways. Suitable target proteins are proteins capable of inhibiting PKR-dependent pathways and / or OAS-dependent pathways. Inhibiting PKR-dependent pathways may include inhibiting eIF2-α phosphorylation. Inhibiting PKR may include treating cells with at least one PKR inhibitor. The PKR inhibitor may be a viral inhibitor of PKR. A preferred viral inhibitor of PKR is vaccinia virus E3. If a peptide or protein (e.g., E3, K3) is used to inhibit intracellular IFN signaling, intracellular expression of the peptide or protein is preferred. Vaccinia virus E3 is a 25 kDa dsRNA binding protein (encoded by the gene E3L) that binds to and sequesters dsRNA to prevent activation of PKR and OAS. E3 can directly bind to PKR and inhibit its activity, resulting in reduced phosphorylation of eIF2-α. Other suitable IFN signaling inhibitors include herpes simplex virus ICP34.5, Tuscany virus NSs, Bombyx mori nuclear polyhedrosis virus PK2, and HCV NS34A.

[0276] The inhibitor of IFN signaling can be provided to the cell in the form of a nucleic acid sequence (eg, RNA) encoding the inhibitor of IFN signaling.

[0277] In one embodiment, an inhibitor of intracellular or extracellular IFN signaling is encoded by an mRNA molecule. The mRNA molecule may include modifications other than polypeptide sequence modifications as described herein, such as a cap, 5'-UTR, 3'-UTR, poly(A) sequence, or adjustments to codon usage. Representative embodiments are described, for example, in Example 3.

[0278] In another embodiment, the inhibitor of intracellular or extracellular IFN signaling is encoded by a replicon, preferably a trans-replicon or trans-replicon as described herein. The replicon comprises nucleic acid sequence elements that allow replication by the alphavirus replicase, typically CSE1, CSE2, and CSE4; preferably, it also comprises nucleic acid sequence elements that allow production of subgenomic transcripts, i.e., a subgenomic promoter, typically comprising CSE 3. The replicon may also comprise one or more modifications of non-polypeptide sequence modifications as described herein, such as caps, poly (A) sequences, and adjustments to codon usage. If multiple open reading frames are present on the replicon, the inhibitor of intracellular IFN signaling can be encoded by any one of them, optionally under the control of a subgenomic promoter or not under its control. In a preferred embodiment, the inhibitor of intracellular IFN signaling is encoded by the most upstream open reading frame of the RNA replicon. When the inhibitor of intracellular IFN signaling is encoded by the most upstream open reading frame of the RNA replicon, the genetic information encoding the inhibitor of intracellular IFN signaling will be translated early after the RNA replicon is introduced into the host cell, and the resulting protein can then inhibit intracellular IFN signaling.

[0279] Functional alphavirus nonstructural protein

[0280] Another suitable protein of interest encoded by the open reading frame is a functional alphavirus nonstructural protein. The term "alphavirus nonstructural protein" includes each and every co-translationally or post-translationally modified form, including carbohydrate-modified (e.g., glycosylated) and lipid-modified forms of alphavirus nonstructural protein.

[0281] In some embodiments, the term "alphavirus nonstructural protein" refers to any one or more of the individual nonstructural proteins of alphavirus origin (nsP1, nsP2, nsP3, nsP4), or 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 an additional protein of interest and may be encoded by an additional open reading frame.

[0282] In some embodiments, alphavirus nonstructural proteins are capable of forming complexes or associates, for example, in host cells. In some embodiments, "alphavirus nonstructural proteins" refer to a complex or associate of nsP123 (synonymous P123) and nsP4. In some embodiments, "alphavirus nonstructural proteins" refer to a complex or associate of nsP1, nsP2, and nsP3. In some embodiments, "alphavirus nonstructural proteins" refer to a complex or associate of nsP1, nsP2, nsP3, and nsP4. In some embodiments, "alphavirus nonstructural proteins" refer to a complex or associate of any one or more selected from nsP1, nsP2, nsP3, and nsP4. In some embodiments, alphavirus nonstructural proteins comprise at least nsP4.

[0283] The term "complex" or "associate" refers to two or more identical or different protein molecules that are close in space. The proteins of the complex are preferably in direct or indirect physical or physicochemical contact with each other. The complex or associate can be composed of a variety of different proteins (heteromultimers) and / or multiple copies of a specific protein (homomultimers). In the case of alphavirus nonstructural proteins, the term "complex or associate" describes a variety of at least two protein molecules, at least one of which is an alphavirus nonstructural protein. The complex or associate can be composed of multiple copies of a specific protein (homomultimers) and / or a variety of different proteins (heteromultimers). In the case of multimers, "multi" means more than one, for example two, three, four, five, six, seven, eight, nine, ten or more than ten.

[0284] The term "functional alphavirus nonstructural protein" includes alphavirus nonstructural proteins that have replicase function. Therefore, "functional alphavirus nonstructural protein" includes alphavirus replicase. "Replicase function" includes the function of RNA-dependent RNA polymerase (RdRP), that is, an enzyme that can catalyze the synthesis of (-) strand RNA based on a (+) strand RNA template and / or can catalyze the synthesis of (+) strand RNA based on a (-) strand RNA template. Therefore, the term "functional alphavirus nonstructural protein" can 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 alphavirus nonstructural proteins may additionally have one or more additional functions, for example, a protease (for self-cleavage), a helicase, a terminal adenylyltransferase (for poly(A) tail addition), a methyltransferase and a guanylyltransferase (for providing nucleic acids with a 5' cap), a nuclear localization site, a triphosphatase (Gould et al., 2010, Antiviral Res., Vol. 87, pp. 111-124; Rupp et al., 2015, J. Gen. Virol., Vol. 96, pp. 2483-500).

[0285] According to the present invention, the term "alphavirus replicase" refers to an alphavirus RNA-dependent RNA polymerase, including RNA-dependent RNA polymerases from naturally occurring alphaviruses (alphaviruses found in nature) and RNA-dependent RNA polymerases from variants or derivatives of alphaviruses (e.g., attenuated alphaviruses). In the context of the present invention, the terms "replicase" and "alphavirus replicase" are used interchangeably unless the context dictates that any particular replicase is not an alphavirus replicase.

[0286] 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, particularly post-translationally modified variants, conformations, isoforms and homologs. In addition, the term "replicase" includes all forms of replicase produced or producible by recombinant methods. For example, replicase comprising a tag (e.g., a myc tag, HA tag, or oligohistidine tag (His tag)) that facilitates detection and / or purification of the replicase in the laboratory can be produced by recombinant methods.

[0287] 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., to the (-) strand] and / or to the complementary strand of CSE 3 [i.e., to the (-) strand].

[0288] The source of the replicase is not limited to any particular alphavirus. In a preferred embodiment, the alphavirus replicase comprises a nonstructural protein from Semliki Forest virus, including naturally occurring Semliki Forest virus and variants or derivatives of Semliki Forest virus, such as an attenuated Semliki Forest virus. In another preferred embodiment, the alphavirus replicase comprises a nonstructural protein from Sindbis virus, including naturally occurring Sindbis virus and variants or derivatives of Sindbis virus, such as an attenuated Sindbis virus. In another preferred embodiment, the alphavirus replicase comprises a nonstructural protein from Venezuelan equine encephalitis virus (VEEV), including naturally occurring VEEV and variants or derivatives of VEEV, such as an attenuated VEEV. In another preferred embodiment, the alphavirus replicase comprises a nonstructural protein from Chikungunya virus (CHIKV), including naturally occurring CHIKV and variants or derivatives of CHIKV, such as an attenuated CHIKV.

[0289] The replicase may also comprise nonstructural proteins from more than one alphavirus. Thus, heterocomplexes 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 (e.g., nsP1, nsP2) from a first alphavirus and one or more nonstructural proteins (nsP3, nsP4) from a second alphavirus. Nonstructural proteins from more than one different alphavirus may be encoded by separate open reading frames, or may be encoded as a polyprotein by a single open reading frame, such as nsP1234.

[0290] In some embodiments, the functional alphavirus nonstructural protein is capable of forming membrane replication complexes and / or vacuoles in a cell in which the functional alphavirus nonstructural protein is expressed.

[0291] If a functional alphavirus nonstructural protein, i.e. an alphavirus nonstructural protein with replicase function, is encoded by a nucleic acid molecule according to the present invention, then preferably the subgenomic promoter of the replicon (if present) is compatible with the replicase. Compatible in this context means that the alphavirus replicase is able to recognize the subgenomic promoter (if present). In one embodiment, this is achieved when the subgenomic promoter is native to the alphavirus from which the replicase is derived, i.e. the natural source of these sequences is the same alphavirus. In an alternative embodiment, the subgenomic promoter is not native to the alphavirus from which the alphavirus replicase is derived, provided that the alphavirus replicase is able to recognize the subgenomic promoter. In other words, the replicase is compatible with the subgenomic promoter (cross-virus compatibility). Examples of cross-virus compatibility involving subgenomic promoters and replicases derived from different alphaviruses are known in the art. As long as there is cross-virus compatibility, any combination of subgenomic promoters and replicase is possible. One skilled in the art can easily test for cross-viral compatibility by incubating the replicase to be tested with RNA under conditions suitable for synthesizing RNA from a subgenomic promoter, wherein the RNA has the subgenomic promoter to be tested. If a subgenomic transcript is prepared, the subgenomic promoter and replicase are determined to be compatible. Several examples of cross-viral compatibility are known (reviewed by Strauss & Strauss, Microbiol. Rev., 1994, Vol. 58, pp. 491-562).

[0292] In one embodiment, the alphavirus nonstructural protein is not encoded as a fusion protein with a heterologous protein, eg, ubiquitin.

[0293] In the present invention, an open reading frame encoding a functional alphavirus nonstructural protein can be provided on an RNA replicon or can be provided as an independent nucleic acid molecule (e.g., an mRNA molecule). The independent mRNA molecule can optionally include, for example, a cap, a 5'-UTR, a 3'-UTR, a poly(A) sequence, and / or adjustments to codon usage. As described herein for the system of the present invention, the independent mRNA molecule can be provided in trans.

[0294] When an open reading frame encoding a functional alphavirus nonstructural protein is provided on the RNA replicon, the replicon can preferably be replicated by the functional alphavirus nonstructural protein. In particular, the RNA replicon encoding a functional alphavirus nonstructural protein can be replicated by the functional alphavirus nonstructural protein encoded by the replicon. This embodiment is very preferred when no nucleic acid molecule encoding a functional alphavirus nonstructural protein is provided in trans. In this embodiment, cis replication of the replicon is intended to be achieved. In a preferred embodiment, the RNA replicon comprises an open reading frame encoding a functional alphavirus nonstructural protein and a further open reading frame encoding a protein of interest, and can be replicated by the functional alphavirus nonstructural protein. This embodiment is particularly suitable for some methods of producing a protein of interest according to the present invention. Examples of individual replicons are Figure 1 As shown ("cis replicon Δ5ATG-RRS").

[0295] If the replicon comprises an open reading frame encoding a functional alphavirus nonstructural protein, then preferably the open reading frame encoding the functional alphavirus nonstructural protein does not overlap with the 5' replication recognition sequence. In one embodiment, the open reading frame encoding the functional alphavirus nonstructural protein does not overlap with the subgenomic promoter (if present). Examples of various replicons are Figure 1 As shown ("cis replicon Δ5ATG-RRS").

[0296] If multiple open reading frames are present on the replicon, the functional alphavirus nonstructural protein can be encoded by any of them, optionally under the control of a subgenomic promoter or not under the control of a subgenomic promoter, preferably not under the control of a subgenomic promoter. In a preferred embodiment, the functional alphavirus nonstructural protein is encoded by the upstream-most open reading frame of the RNA replicon. When the functional alphavirus nonstructural protein is encoded by the upstream-most open reading frame of the RNA replicon, the genetic information encoding the functional alphavirus nonstructural protein will be translated early after the RNA replicon is introduced into the host cell, and the resulting protein can then drive replication in the host cell and optionally produce subgenomic transcripts. Examples of individual replicons are Figure 1 As shown ("cis replicon Δ5ATG-RRS").

[0297] The presence of an open reading frame encoding a functional alphavirus nonstructural protein, contained by the replicon or contained by a separate nucleic acid molecule provided in trans, allows the replicon to be replicated and, therefore, to express the gene of interest encoded by the replicon at high levels, optionally under the control of a subgenomic promoter. This is associated with a cost advantage compared to other transgenic expression systems. For example, in the case of animal vaccination, the cost of the vaccine is key to its success in the veterinary and agricultural communities. Since the replicon of the present invention can replicate in the presence of functional alphavirus nonstructural proteins, for example, in the cells of the vaccinated animal, high levels of expression of the gene of interest can be achieved even with the administration of relatively low amounts of replicon RNA. Small amounts of replicon RNA have a positive impact on the cost of the vaccine per subject.

[0298] Position of at least one open reading frame in the RNA replicon

[0299] RNA replicon is suitable for expressing one or more genes encoding target peptide or target protein, optionally under the control of a subgenomic promoter. Various embodiments are possible. One or more open reading frames may be present on the RNA replicon, each open reading frame encoding 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 some embodiments, the "first open reading frame" is the only open reading frame of the RNA replicon. Optionally, one or more other open reading frames may be present in the downstream of the first open reading frame. The one or more other open reading frames downstream of the first open reading frame may be referred to as the "second open reading frame", "third open reading frame" etc. according to the order (5' to 3') in which they exist in the downstream of the first open reading frame. Preferably, each open reading frame comprises a start codon (base triplet), typically AUG (in RNA molecules), corresponding to ATG (in corresponding DNA molecules).

[0300] If the replicon comprises a 3' replication recognition sequence, preferably all open reading frames are located upstream of the 3' replication recognition sequence.

[0301] When an RNA replicon comprising one or more open reading frames is introduced into a host cell, translation preferably does not start anywhere upstream of the first open reading frame due to the removal of at least one start codon from the 5' replication recognition. Thus, the replicon can be used directly as a translation template for the first open reading frame. Preferably, the replicon comprises a 5'-cap. This facilitates expression of the gene encoded by the first open reading frame directly from the replicon.

[0302] 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-level 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 native to the alphavirus and preferably controls the transcription of an open reading frame encoding one or more structural proteins in the alphavirus. Alternatively, the subgenomic promoter is a variant of an alphavirus subgenomic promoter; any variant that functions as a promoter of subgenomic RNA transcription in the host cell is suitable. If the replicon comprises a subgenomic promoter, it is preferred that the replicon comprises conserved sequence element 3 (CSE3) or a variant thereof.

[0303] Preferably, at least one open reading frame under the control of the subgenomic promoter is located downstream of the subgenomic promoter. Preferably, the subgenomic promoter controls the production of subgenomic RNA containing transcripts of the open reading frame.

[0304] In some embodiments, the first open reading frame is under the control of a subgenomic promoter. When the first open reading frame is under the control of a subgenomic promoter, its positioning is similar to the positioning of the open reading frame encoding the structural protein in the alphavirus genome. When the first open reading frame is under the control of a subgenomic promoter, the gene encoded by the first open reading frame can be expressed from both the replicon and its subgenomic transcript (the latter of which contains functional alphavirus non-structural proteins). Figure 1 The replicon "Δ5ATG-RRS" in is shown. Preferably, "Δ5ATG-RRS" does not contain any start codon in the nucleic acid sequence encoding the C-terminal fragment of nsP4 (*nsP4). Downstream of the first open reading frame under the control of the subgenomic promoter, there may be one or more additional open reading frames, each under the control of the subgenomic promoter ( Figure 1 ). A gene encoded by one or more additional open reading frames (e.g., by a second open reading frame) can be translated from one or more subgenomic transcripts, each under the control of a subgenomic promoter. For example, an RNA replicon can include a subgenomic promoter that controls the production of a transcript encoding a second protein of interest.

[0305] In other embodiments, the first open reading frame is not under the control of a subgenomic promoter. When the first open reading frame is not under the control of a subgenomic promoter, the gene encoded by the first open reading frame can be expressed from the replicon. Figure 1The replicon "Δ5ATG-RRSΔSGP" in FIG. 1 is shown. Downstream of the first open reading frame, one or more additional open reading frames may be present, each under the control of a subgenomic promoter (for illustration of two exemplary embodiments, see Figure 1 "Δ5ATG-RRS-bicistronic" and "cis-replicon Δ5ATG-RRS" in . Genes encoded by one or more additional open reading frames can be expressed from the subgenomic transcripts.

[0306] In cells containing the replicon according to the present invention, the replicon can be amplified by functional alphavirus nonstructural proteins. In addition, if the replicon contains one or more open reading frames under the control of a subgenomic promoter, it is expected that one or more subgenomic transcripts will be produced by functional alphavirus nonstructural proteins. Functional alphavirus nonstructural proteins can be provided in trans or can be encoded by the open reading frame of the replicon.

[0307] If the replicon comprises more than one open reading frame encoding a protein of interest, it is preferred that 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.

[0308] In some embodiments, the protein of interest encoded by the first and / or additional open reading frame, preferably the first open reading frame, is a functional alphavirus nonstructural protein or an inhibitor of IFN signaling, e.g., E3. In some embodiments, the protein of interest encoded by the first and / or additional open reading frame, e.g., the second open reading frame, is a pharmaceutically active peptide or protein, or a reporter protein.

[0309] In one embodiment, the protein of interest encoded by the first open reading frame is a functional alphavirus nonstructural protein. In this embodiment, the replicon preferably comprises a 5'-cap. In particular, when the protein of interest encoded by the first open reading frame is a functional alphavirus nonstructural protein, and preferably when the replicon comprises a 5'-cap, the nucleic acid sequence encoding the functional alphavirus nonstructural protein can be effectively translated from the replicon, and the resulting protein can then drive the replication of the replicon and drive the synthesis of subgenomic transcripts. This embodiment may be preferred when no additional nucleic acid molecule encoding a functional alphavirus nonstructural protein is used or is not present with the replicon. In this embodiment, cis-replication of the replicon is intended to be achieved.

[0310] One embodiment wherein the first open reading frame encodes a functional alphavirus nonstructural protein is Figure 1After translation of the nucleic acid sequence encoding nsP1234, the translation product (nsP1234 or a fragment thereof) can act as a replicase and drive RNA synthesis, i.e., replication of the replicon and synthesis of a subgenomic transcript containing a second open reading frame ( Figure 1 “Genetically modified” in the text).

[0311] trans-replication system

[0312] In a second aspect, the present invention provides a system comprising:

[0313] RNA constructs for expressing functional alphavirus nonstructural proteins,

[0314] The RNA replicon according to the first aspect of the present invention is capable of trans replication via a functional alphavirus non-structural protein.

[0315] In the second aspect, preferably the RNA replicon does not comprise an open reading frame encoding a functional alphavirus non-structural protein.

[0316] Thus, the present invention provides a system comprising two nucleic acid molecules: a first RNA construct for expressing a functional alphavirus nonstructural protein (i.e., encoding a functional alphavirus nonstructural protein); and a second RNA molecule, an RNA replicon. The RNA construct for expressing a functional alphavirus nonstructural protein is herein synonymously referred to as an "RNA construct for expressing a functional alphavirus nonstructural protein" or a "replicase construct."

[0317] Functional alphavirus nonstructural proteins are as defined above and are typically encoded by the open reading frame comprised by the replicase construct.The functional alphavirus nonstructural protein encoded by the replicase construct may be any functional alphavirus nonstructural protein capable of replicating the replicon.

[0318] When the system of the present invention is introduced into a cell, preferably a eukaryotic cell, an open reading frame encoding a functional alphavirus nonstructural protein can be translated. After translation, the functional alphavirus nonstructural protein can replicate an independent RNA molecule (RNA replicon) in trans. Therefore, the present invention provides a system for replicating RNA in trans. Therefore, the system of the present invention is a trans-replicon. According to a second aspect, the replicon is a trans-replicon.

[0319] Herein, trans (e.g., in the case of trans-action, trans-regulation) generally means "action from different molecules" (i.e., intermolecular). It is opposite to cis (e.g., in the case of cis-action, cis-regulation), which generally means "action from the same molecule" (i.e., intramolecular). In the context of RNA synthesis (including transcription and RNA replication), trans-acting elements include nucleic acid sequences containing genes encoding enzymes (RNA polymerases) capable of RNA synthesis. RNA polymerase uses a second nucleic acid molecule (i.e., a nucleic acid molecule other than the nucleic acid molecule encoding it) as a template for RNA synthesis. RNA polymerase and nucleic acid sequences containing genes encoding RNA polymerases are both referred to as "trans" acting on a second nucleic acid molecule. In the context of the present invention, the RNA polymerase encoded by trans-acting RNA is a functional alphavirus nonstructural protein. Functional alphavirus nonstructural proteins can use a second nucleic acid molecule (which is an RNA replicon) as a synthesis template or RNA, including the replication of an RNA replicon. The RNA replicon that can be replicated by the trans-replicon according to the present invention is synonymously referred to as a "trans-replicon" herein.

[0320] In the system of the present invention, the role of the functional alphavirus nonstructural protein is to amplify the replicon and, if a subgenomic promoter is present on the replicon, to produce subgenomic transcripts. If the replicon encodes a gene of interest for expression, the expression level and / or duration of the gene of interest can be trans-regulated by altering the level of the functional alphavirus nonstructural protein.

[0321] The fact that alphavirus replicase is often able to recognize and replicate template RNA in trans was first discovered in the 1980s, but the potential of trans-replication for biomedical applications has not yet been recognized, especially because trans-replicating RNA is believed to inhibit efficient replication: in infected cells, defective interfering (DI) RNA is found to co-replicate with the alphavirus genome (Barrett et al., 1984, J. Gen. Virol., Vol. 65 (Pt 8), pp. 1273-1283; Lehtovaara et al., 1981, Proc. Natl. Acad. Sci. USA, Vol. 78, pp. 5353-5357; Pettersson, 1981, Proc. Natl. Acad. Sci. USA, Vol. 78, pp. 115-119). DI RNA is a trans-replicon that can occur quasi-naturally during infection of cell lines with high viral loads. The DI element is so effective in co-replicating that it reduces the virulence of the parent virus, thereby acting as an inhibitory parasitic RNA (Barrett et al., 1984, J. Gen. Virol., Vol. 65 (Pt 11), pp. 1909-1920). Although the potential for biomedical applications has not been recognized, the phenomenon of trans-replication has been used in some basic studies aimed at elucidating the replication mechanism, without the need to express the replicase in cis from the same molecule; in addition, the separation of the replicase and the replicon also allows functional studies involving viral protein mutants, even if the corresponding mutants are loss-of-function mutants (Lemm et al., 1994, EMBO J., Vol. 13, pp. 2925-2934). These loss-of-function studies and DI RNA do not indicate that the transactivation system based on alphavirus elements can ultimately be used for therapeutic purposes.

[0322] The system of the present invention comprises at least two nucleic acid molecules. Therefore, it can comprise two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more nucleic acid molecules, which are preferably RNA molecules. In a preferred embodiment, the system consists of exactly two RNA molecule replicons and replicase constructs. In an alternative preferred embodiment, the system comprises more than one replicon, each replicon preferably encoding at least one target protein, and further comprising a replicase construct. In these embodiments, the functional alphavirus nonstructural protein encoded by the replicase construct can act on each replicon to drive the replication and production of subgenomic transcripts, respectively. For example, each replicon can encode a pharmaceutically active peptide or protein. This is advantageous, for example, if it is necessary to vaccinate a subject against a variety of different antigens.

[0323] Preferably, the replicase construct lacks at least one conserved sequence element (CSE) required for (-) strand synthesis based on a (+) strand template and / or (+) strand synthesis based on a (-) strand template. More preferably, the replicase construct does not contain any alphavirus conserved sequence element (CSE). In particular, of the four alphavirus CSEs (Strauss & Strauss, Microbiol. Rev., 1994, Vol. 58, pp. 491-562; José et al., Future Microbiol., 2009, Vol. 4, pp. 837-856), any one or more of the following CSEs are preferably absent from the replicase construct: CSE 1; CSE 2; CSE 3; CSE 4. In particular, in the absence of any one or more alphavirus CSEs, the replicase construct of the present invention is more similar to a typical eukaryotic mRNA than to an alphavirus genomic RNA.

[0324] The replicase construct of the present invention is preferably distinguished from the alphavirus genomic RNA in that it is at least incapable of self-replication and / or it does not contain an open reading frame under the control of a subgenomic promoter. When it is incapable of self-replication, the replicase construct may also be referred to as a "suicide construct."

[0325] The trans-system has the following advantages:

[0326] First, the versatility of the trans-replication system allows replicon and replicase constructs to be designed and / or prepared at different times and / or different sites. In one embodiment, the replicase construct is prepared at a first time point, and the replicon is prepared at a later time point. For example, after its preparation, the replicase construct can be stored for later use. Compared to cis-replicons, the present invention provides increased flexibility: when a new pathogen emerges, the system of the present invention can be designed for vaccination by cloning a nucleic acid encoding a polypeptide that elicits an immune response against the new pathogen into the replicon. Previously prepared replicase constructs can be recovered from storage. Therefore, when designing and preparing the replicase construct, it is not necessary to know the properties of a specific pathogen or the antigens of a specific pathogen. Therefore, when designing and preparing the replicase construct, it is not necessary to have a replicon encoding a polypeptide that elicits an immune response against a specific new pathogen available. In other words, the replicase construct can be designed and prepared independently of any specific replicon. This allows for a rapid response to the emergence of a new pathogen or a pathogen characterized by the expression of at least one new antigen, as the preparation of a replicase-deficient replicon requires less effort and resources than the preparation of a cis-replicon. History teaches us that a system is needed that allows a rapid response to pathogens: this is illustrated for example by the emergence in recent years of the pathogen causing Severe Acute Respiratory Syndrome (SARS), Ebola virus and various influenza virus subtypes.

[0327] Secondly, the trans-replicons according to the present invention are generally shorter nucleic acid molecules than typical cis-replicons. This enables faster cloning of replicons encoding target proteins (e.g., immunogenic polypeptides) and provides high yields of target proteins.

[0328] Other advantages of the system of the present invention include independence from nuclear transcription and the presence of key genetic information on two separate RNA molecules, which provides unprecedented design freedom. In view of its multifunctional elements that can be combined with each other, the present invention allows optimization of replicase expression to obtain the desired level of RNA amplification for the desired target organism, for the production level of the desired protein of interest, etc. The system according to the present invention allows co-transfection of different amounts or ratios of replicon and replicase constructs for any given cell type - resting or circulating, in vitro or in vivo. The trans-replication system of the present invention is suitable for inoculating host cells, and for expressing target genes in host cells (see, for example, Examples 2, 3 and 5).

[0329] The replicase construct according to the invention is preferably a single-stranded RNA molecule. The replicase construct according to the invention is typically a (+)-stranded RNA molecule. In one embodiment, the replicase construct of the invention is an isolated nucleic acid molecule.

[0330] Preferred features of the RNA molecules according to the invention

[0331] The RNA molecules according to the present invention may optionally be characterized by other features, such as, for example, 5'-cap, 5'-UTR, 3'-UTR, poly(A) sequence and / or modification of codon usage. Details are described below.

[0332] cap

[0333] In some embodiments, a replicon according to the invention comprises a 5'-cap.

[0334] In some embodiments, the replicase constructs according to the invention comprise a 5'-cap.

[0335] The terms "5'-cap", "cap", "5'-cap structure", and "cap structure" are used synonymously to refer to a dinucleotide found at the 5' end of some eukaryotic primary transcripts, such as pre-messenger RNA. The 5'-cap is a structure in which an (optionally modified) guanosine is bonded to the first nucleotide of an mRNA molecule via a 5' to 5' triphosphate linkage (or a modified triphosphate linkage in the case of certain cap analogs). These terms can refer to traditional caps or cap analogs. For illustration, some specific cap dinucleotides (including cap analog dinucleotides) are shown below. Figure 6 middle.

[0336] "RNA comprising a 5'-cap" or "RNA having 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 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., vaccinia virus capping enzyme). 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 (the "second base") of the RNA molecule via a phosphodiester bond.

[0337] If it is desired to translate the protein-encoding nucleic acid sequence at an early stage after the respective RNA is introduced into the host cell or host organism, the presence of a cap on the RNA molecule is highly preferred. For example, as shown in Example 4, the presence of a cap allows efficient translation of the gene of interest encoded by the RNA replicon at an early stage after the respective RNA is introduced into the host cell. "Early stage" generally means within the first hour, or within the first two hours, or within the first three hours after the introduction of the RNA.

[0338] The presence of a cap on the RNA molecule is also preferred if translation is desired to occur in the absence of a functional replicase, or when only a small amount of replicase is present in the host cell. For example, even if a nucleic acid molecule encoding a replicase is introduced into a host cell, the level of replicase is generally minimal in the early stages after introduction.

[0339] In the system according to the present invention, it is preferred that the RNA construct for expressing a functional alphavirus nonstructural protein comprises a 5'-cap.

[0340] In particular, when the RNA replicon according to the present invention is not used or provided with a second nucleic acid molecule (e.g., mRNA) encoding a functional alphavirus nonstructural protein, it is preferred that the RNA replicon comprises a 5'-cap. Alternatively, the RNA replicon may comprise a 5'-cap even if it is used or provided with a second nucleic acid molecule encoding a functional alphavirus nonstructural protein.

[0341] 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 position 7 ( Figure 6 (top of the ).

[0342] 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 when attached thereto, preferably in vivo and / or the cap analogue is not a conventional 5'-cap.

[0343] In the case of eukaryotic mRNA, the 5'-cap is often described as being involved in the efficient translation of mRNA: Normally, in eukaryotes, translation only begins 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, such as a 5'-cap. 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 guanyltransferase activity. GpppN can then be methylated in the cell by a second enzyme with (guanine-7)-methyltransferase activity to form a monomethylated m 7 GpppN-cap. In one embodiment, the 5'-cap used in the present invention is a natural 5'-cap.

[0344] In the present invention, the natural 5'-cap dinucleotide is generally selected from the group consisting of 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 (wherein N is G) is represented by the following formula:

[0345]

[0346] 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 common method for preparing capped RNA in vitro is to combine all four ribonucleoside triphosphates and a cap dinucleotide such as mRNA. 7 G(5')ppp(5')G(also known as m 7 GpppG) is used to transcribe the DNA template using bacterial or phage RNA polymerase. RNA polymerase is transcribed by m 7 The nucleophilic attack of the 3'-OH of the guanosine moiety of GpppG on the α-phosphate of the next template nucleoside triphosphate (pppN) initiates transcription, producing the intermediate m 7 GpppGpN (where N is the second base of the RNA molecule). The formation of the product pppGpN, which is a competitive GTP-primed product, is inhibited by setting the molar ratio of cap to GTP between 5 and 10 during in vitro transcription.

[0347] In some preferred embodiments of the present 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, for example, in vitro transcribed RNA (IVT-RNA). Cap analogs have been initially described to facilitate large-scale synthesis of RNA transcripts by in vitro transcription.

[0348] For messenger RNA, a number of cap analogs (synthetic caps) have been generally described to date, all of which can be used in the context of the present invention. Ideally, cap analogs are selected that are associated with higher translation efficiency and / or increased resistance to degradation in vivo and / or increased resistance to degradation in vitro.

[0349] Preferably, a cap analog is used that can be incorporated into the RNA chain in only one direction. Pasquinelli et al. (1995, RNA J., Vol. 1, pp. 957-967) demonstrated that during in vitro transcription, bacteriophage RNA polymerase uses a 7-methylguanosine unit to initiate transcription, with approximately 40-50% of capped transcripts having the cap dinucleotide in the reverse direction (i.e., the initial reaction product is Gpppm). 7 GpN). RNA with an inverted cap is less effective in translating the nucleic acid sequence into protein than RNA with a correct cap. Therefore, it is desirable to introduce the cap in the correct orientation, i.e., to produce an RNA with a cap substantially corresponding to m 7 GpppGpN etc. structure RNA. It has been shown that the reverse integration of cap dinucleotides is suppressed by replacing any one of the 2'- or 3'-OH groups of the methylated guanosine unit (Stepinski et al., 2001; RNA J., Vol. 7, pp. 1486-1495; Peng et al., 2002; Org. Lett., Vol. 24, pp. 161-164). RNA synthesized in the presence of this "anti-reverse cap analogue" is more efficiently translated than RNA transcribed in vitro in the presence of conventional 5'-cap m7GpppG. Therefore, for example, Holtkamp et al., 2006, Blood, Vol. 108, pp. 4009-4017 describe a cap analogue in which the 3'OH group of the methylated guanosine unit is replaced by OCH3 (7-methyl (3'-O-methyl) GpppG; anti-reverse cap analogue (ARCA)). ARCA is a suitable cap dinucleotide according to the present invention.

[0350]

[0351] In a preferred embodiment of the present invention, the RNA of the present invention is not easy to remove the cap substantially. This is important because, in general, the amount of protein produced by the synthetic mRNA introduced into cultured mammalian cells is limited by the natural degradation of the mRNA. An in vivo pathway for mRNA degradation begins with the removal of the mRNA cap. This removal is catalyzed by a heterodimeric pyrophosphatase, which contains 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 not easily affected by this type of cutting can be selected or present. Suitable cap analogues for this purpose can be selected from cap dinucleotides according to formula (I):

[0352]

[0353] where R 1 is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl and optionally substituted heteroaryl,

[0354] 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

[0355] C(CH3)2 or R 2 With R 2 The hydrogen atom at the 4' position of the connected ring combines to form -O-CH2- or -CH2-O-,

[0356] R 5 selected from S, Se and BH3,

[0357] R 4 and R 6 Independently selected from O, S, Se and BH3.

[0358] n is 1, 2, or 3.

[0359] R 1 、R 2 , R3, R 4 、R 5 、R 6 Preferred embodiments are disclosed in WO 2011 / 015347 A1 and can be selected accordingly in the present invention.

[0360] For example, in a preferred embodiment of the present invention, the RNA of the present 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 are described by J. Kowalska et al., 2008, RNA, Vol. 14, pp. 1119-1131 as a solution to the undesirable decapping process, thereby improving the stability of RNA in vivo. In particular, the replacement of the sulfur atom on the β-phosphate group of the 5'-cap with an oxygen atom leads to stabilization of Dcp2. In this preferred embodiment of the invention, R in formula (I) 5 is S; and R 4 and R 6 It's O.

[0361] In another preferred embodiment of the present invention, the RNA of the present 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. Various ARCA-phosphorothioate-cap-analogs are described in WO 2008 / 157688 A2, and they can all be used in the RNA of the present 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 replaces the sulfur atom on the β-phosphate group (so that R in formula (I) 5 is S; and R 4 and R 6 (O). It is believed that the phosphorothioate modification of ARCA ensures that the α, β, and γ phosphorothioate groups are precisely positioned within the active site of the cap-binding protein during translation and decapping machinery. At least some of these analogs are substantially resistant to the pyrophosphatase Dcp1 / Dcp2. Phosphorothioate-modified ARCA is described to have a much higher affinity for eIF4E than the corresponding ARCA lacking the phosphorothioate group.

[0362] The corresponding cap analogs (ie, m 2’ 7,2’-O Gpp spG) is known as β-S-ARCA (WO2008 / 157688 A2; Kuhn et al., Gene Ther., 2010, Vol. 17, pp. 961-971). Therefore, in one embodiment of the present invention, β-S-ARCA is used to modify the RNA of the present invention. β-S-ARCA is represented by the following structure:

[0363]

[0364] Typically, thiophosphate diastereomers are obtained by replacing oxygen atoms with sulfur atoms at the bridged phosphate esters, which are named D1 and D2 based on their elution patterns in HPLC. In short, the D1 diastereomer of β-S-ARCA or "β-S-ARCA (D1)" is a 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)). Therefore, it exhibits a shorter retention time. Stereochemical configurations determined by HPLC are described in WO2011 / 015347A1.

[0365] In a first particularly preferred embodiment of the present invention, the RNA of the present invention is modified with the β-S-ARCA (D2) diastereomer. The two diastereomers of β-S-ARCA differ in their sensitivity to nucleases. It has been shown that RNA carrying the D2 diastereomer of β-S-ARCA is almost completely resistant to Dcp2 cleavage (only 6% cleavage compared to RNA synthesized in the presence of the unmodified ARCA 5'-cap), while RNA with a β-S-ARCA (D1) 5'-cap exhibits moderate sensitivity to Dcp2 cleavage (71% cleavage). It has further been shown that increased stability to Dcp2 cleavage is associated with increased protein expression in mammalian cells. In particular, it has been shown that RNA carrying a β-S-ARCA (D2) cap is more efficiently translated in mammalian cells than RNA carrying 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 of formula (I), characterized in that formula (I) comprises a substituent R 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 them is preferably not OH, preferably R 2 and R 3One of them is a methoxy group (OCH3), and R 2 and R 3 Another preferred one is OH.

[0366] In a second particularly preferred embodiment, the RNA of the present invention is modified with β-S-ARCA (D1) diastereomers. This embodiment is particularly suitable for transferring capped RNA into immature antigen-presenting cells, for example, for vaccination purposes. It has been shown that β-S-ARCA (D1) diastereomers are particularly suitable for improving the stability of RNA, improving the translation efficiency of RNA, extending 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., Vol. 17, pp. 961-971). Therefore, in an alternative embodiment of the present invention, the RNA of the present invention is modified with a cap analog of 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 Atom. Various cap analogs and embodiments thereof are described in WO2011 / 015347 A1 and Kuhn et al., 2010, Gene Ther., Vol. 17, pp. 961-971. Any cap analog described in WO2011 / 015347 A1 can be used in the present invention, wherein the cap analogs comprising the 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 Preferably, R in formula (I) 5 is S; In addition, R in formula (I) 2 or R 3 At least one of them is preferably not OH, preferably R 2 and R 3 One of them is methoxy (OCH3), R 2 and R 3 Another preferred one is OH.

[0367] In one embodiment, the RNA of the present invention is modified with a 5'-cap structure according to formula (I), wherein any phosphate group is replaced by a boronate phosphate 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 analogs are referred to as β-BH3-ARCAs or β-Se-ARCAs. Compounds particularly suitable for mRNA capping include β-BH3-ARCAs and β-Se-ARCAs, as described in WO2009 / 149253A2. For these compounds, it is preferred to include the substituent R in formula (I) 5 The stereochemical configuration of the P atom corresponds to the P of the D1 diastereomer of β-S-ARCA. β atom.

[0368] UTR

[0369] The term "untranslated region" or "UTR" refers to a region of a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region in an RNA molecule (e.g., an mRNA molecule). An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5'-UTR) and / or 3' (downstream) of an open reading frame (3'-UTR).

[0370] 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, directly adjacent to the poly(A) tail.

[0371] If present, the 5'-UTR 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, directly adjacent to the 5'-cap.

[0372] According to the present invention, the 5' and / or 3' untranslated regions may be functionally linked to the open reading frame so that these regions are associated with the open reading frame such that the stability and / or translation efficiency of the RNA comprising said open reading frame is increased.

[0373] In some embodiments, a replicase construct according to the invention comprises a 5'-UTR and / or a 3'-UTR.

[0374] In a preferred embodiment, the replicase construct according to the present invention comprises

[0375] (1) 5'-UTR,

[0376] (2) open reading frame, and

[0377] (3) 3'-UTR.

[0378] In some embodiments, the present invention relates to the stability and translation efficiency of RNA. In addition to the structural modification of 5'-cap and / or 3'poly (A) tail as described herein, both can be improved by selecting specific 5' and / or 3' untranslated region (UTR). The sequence elements in UTR are generally understood to affect translation efficiency (mainly 5'-UTR) and RNA stability (mainly 3'-UTR). Preferably, there is 5'-UTR, which is active to improve translation efficiency and / or stability of replicase construct. Independently or additionally, preferably, there is 3'-UTR, which is active to improve translation efficiency and / or stability of replicase construct.

[0379] The terms "active to increase translation efficiency" and / or "active to increase stability" with reference to a first nucleic acid sequence (e.g., UTR) mean that in a co-transcript with a second nucleic acid sequence, the first nucleic acid sequence is capable of modifying the translation efficiency and / or stability of the second nucleic acid sequence such 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.

[0380] In one embodiment, the replicase construct according to the present invention comprises 5'-UTR and / or 3'-UTR, which is heterologous or non-natural for the alphavirus that obtains functional alphavirus nonstructural protein. This allows the design of non-translational region according to required translation efficiency and RNA stability. Therefore, heterologous or non-natural UTR allows high flexibility, and compared with natural alphavirus UTR, this flexibility is advantageous. In particular, although it is known that alphavirus (natural) RNA also comprises 5'-UTR and / or 3'-UTR, alphavirus UTR has dual function, i.e. (i) drives RNA replication and (ii) drives translation. Although it is reported that alphavirus UTR is inefficient for translation (Berben-Bloemheuvel et al., 1992, Eur.J.Biochem., Vol. 208, pp. 581-587), they are usually not easily replaced by more efficient UTR because of their dual function. However, in the present invention, the 5'-UTR and / or 3'-UTR included in the replicase construct for trans replication can be selected regardless of their potential effects on RNA replication.

[0381] Preferably, the replicase construct according to the present invention comprises a 5'-UTR and / or a 3'-UTR of non-viral origin; in particular not of alphavirus origin. In one embodiment, the replicase construct comprises a 5'-UTR derived from a eukaryotic 5'-UTR and / or a 3'-UTR derived from a eukaryotic 3'-UTR.

[0382] The 5'-UTR according to the invention may comprise any combination of more than one nucleic acid sequences, optionally separated by linkers.The 3'-UTR according to the invention may comprise any combination of more than one nucleic acid sequences, optionally separated by linkers.

[0383] 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.

[0384] 3'-UTR generally has a length of 200 to 2000 nucleotides, for example, 500 to 1500 nucleotides. The 3'-untranslated region of immunoglobulin mRNA is relatively short (less than about 300 nucleotides), while the 3'-untranslated region of other genes is relatively long. For example, the 3'-untranslated region of tPA is about 800 nucleotides in length, the 3'-untranslated region of factor VIII is about 1800 nucleotides in length, and the 3'-untranslated region of erythropoietin is about 560 nucleotides in length. The 3'-untranslated region of mammalian mRNA generally has a homology region called the AAUAAA hexanucleotide sequence. This sequence may 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 contain one or more inverted repeats that can fold to produce 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.

[0385] The human β-globin 3'-UTR, in particular two consecutive identical copies of the human β-globin 3'-UTR, contributes to high transcriptional stability and translation efficiency (Holtkamp et al., 2006, Blood, Vol. 108, pp. 4009-4017). Therefore, in one embodiment, the replicase construct according to the present invention comprises two consecutive identical copies of the human β-myosin 3'-UTR. Thus, it comprises, in the 5'→3' direction: (a) optionally a 5'-UTR; (b) an open reading frame; (c) a 3'-UTR; the 3'-UTR comprising two consecutive identical copies of the human β-globin 3'-UTR, a fragment thereof, or a variant of the human β-globin 3'-UTR or a fragment thereof.

[0386] In one embodiment, the replicase construct according to the invention comprises a 3'-UTR that has activity to increase translation efficiency and / or stability, but is not human β-globin 3'-UTR, a fragment thereof, or a variant of human β-globin 3'-UTR or a fragment thereof.

[0387] In one embodiment, the replicase construct according to the present invention comprises a 5'-UTR active to increase translation efficiency and / or stability.

[0388] The replicase construct containing UTRs according to the present invention can be prepared, for example, by in vitro transcription. This can be achieved by modifying the template nucleic acid molecule (eg, DNA) in a manner that allows transcription of RNA having a 5'-UTR and / or a 3'-UTR.

[0389] like Figure 1 As shown, the replicon can also be characterized by a 5'-UTR and / or a 3'-UTR. The UTR of the replicon is typically an alphavirus UTR or a variant thereof.

[0390] poly(A) sequence

[0391] In some embodiments, the replicon according to the present 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 directly adjacent to CSE 4.

[0392] In some embodiments, the replicase construct according to the invention comprises a 3'-poly(A) sequence.

[0393] 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 particularly at most 150 A nucleotides, particularly about 120 A nucleotides. In this context, "essentially consisting of" means that the majority of the nucleotides in the poly(A) sequence, typically at least 50%, preferably at least 75% of the nucleotides in the "poly(A) sequence" are A nucleotides (adenylic acid), but the remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), and C nucleotides (cytidylic acid). In this context, "consisting of" means that all nucleotides in the poly(A) sequence, i.e., 100% of the nucleotides in the poly(A) sequence are A nucleotides. The term "A nucleotide" or "A" refers to adenosine.

[0394] Indeed, it has been demonstrated that a 3' poly(A) sequence of approximately 120 A nucleotides has a beneficial effect on RNA levels in transfected eukaryotic cells as well as on the levels of proteins translated from open reading frames present upstream (5') of the 3' poly(A) sequence (Holtkamp et al., 2006, Blood, Vol. 108, pp. 4009-4017).

[0395] In alphaviruses, a 3' poly(A) sequence of at least 11 consecutive adenylate residues or at least 25 consecutive adenylate residues is believed to be important for efficient synthesis of the negative strand. In particular, in alphaviruses, a 3' poly(A) sequence of at least 25 consecutive adenylate residues is understood to function together with conserved sequence element 4 (CSE 4) to promote (-) strand synthesis (Hardy & Rice, J. Virol., 2005, Vol. 79, pp. 4630-4639).

[0396] The present invention provides a 3' poly(A) sequence that is ligated to a DNA template during RNA transcription, i.e., during the preparation of in vitro transcribed RNA, based on repeated dT nucleotides (deoxythymidylate) contained in a strand complementary to the coding strand. The DNA sequence encoding the poly(A) sequence (coding strand) is referred to as a poly(A) cassette.

[0397] In a preferred embodiment of the present invention, the 3' poly (A) box present in the DNA coding chain is essentially composed of dA nucleotides, but is interrupted by a random sequence of four nucleotides (dA, dC, dG, dT) with equal distribution. The length of this 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. A poly (A) box essentially composed of dA nucleotides but interrupted by a random sequence of four nucleotides (dA, dC, dG, dT) with equal distribution and having a length of, for example, 5 to 50 nucleotides shows constant proliferation of plasmid DNA in Escherichia coli at the DNA level, and is still associated with the beneficial properties of supporting RNA stability and translation efficiency at the RNA level.

[0398] Thus, in a preferred embodiment of the present invention, the 3' poly (A) sequence contained in the RNA molecules described herein consists essentially of A nucleotides, but is interrupted by a random sequence having four nucleotides (dA, dC, dG, dT) with equal distribution. The length of this random sequence may be 5 to 50, preferably 10 to 30, more preferably 10 to 20 nucleotides.

[0399] Codon usage

[0400] 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 with other codons (base triplets) while maintaining the same coding capacity (making the replacement codon encode the same amino acid as the replaced codon). In some embodiments of the present invention, at least one codon of the open reading frame comprised by the RNA molecule is different from the corresponding codon in the corresponding open reading frame in the species from which the open reading frame originates. In this embodiment, the coding sequence of the open reading frame is referred to as "adjustment" or "modification". The coding sequence of the open reading frame comprised by the replicon can be adjusted. Alternatively or in addition, the coding sequence of the functional alphavirus non-structural protein comprised by the replicase construct can be modified.

[0401] For example, when adjusting the coding sequence of an open reading frame, frequently used codons can be selected: WO 2009 / 024567 A1 describes the adjustment of the coding sequence of a nucleic acid molecule, involving the replacement of rare codons with more frequently used codons. Since the frequency of codon usage depends on the host cell or host organism, this type of adjustment is suitable for adapting the nucleic acid sequence to expression in a specific host cell or host organism. In general, more frequently used codons are usually more efficiently translated in the host cell or host organism, although it is not always necessary to adjust all codons of the open reading frame.

[0402] For example, when adjusting the coding sequence of the open reading frame, the content of G (guanylate) residues and C (cytidylate) residues can be changed by selecting codons with the highest GC content for each amino acid. It has been reported that RNA molecules with GC-rich open reading frames have the potential to reduce immune activation and improve RNA translation and half-life (Thess et al., 2015, Mol. Ther. 23, 1457-1465).

[0403] When the replicon according to the present invention encodes an alphavirus nonstructural protein, the coding sequence of the alphavirus nonstructural protein can be adjusted as needed. This freedom is possible because the open reading frame encoding the alphavirus nonstructural protein does not overlap with the 5' replication recognition sequence of the replicon.

[0404] Security features of embodiments of the present invention

[0405] The following features are preferred in the present invention, alone or in any suitable combination:

[0406] Preferably, the replicon or system of the present invention is not particle-forming. This means that, after inoculation of a host cell with the replicon or system of the present invention, the host cell does not produce viral particles, e.g., next generation viral particles. In one embodiment, all RNA molecules according to the present invention are completely free of genetic information encoding any alphavirus structural proteins (e.g., core nucleocapsid protein C, envelope protein P62 and / or envelope protein E1). This aspect of the present invention provides additional value in terms of safety over prior art systems in which structural proteins are encoded on trans-replicating helper RNAs (e.g., Bredenbeek et al., J. Virol, 1993, Vol. 67, pp. 6439-6446).

[0407] Preferably, the system of the present invention does not comprise any alphavirus structural proteins, such as core nucleocapsid protein C, envelope protein P62 and / or envelope protein E1.

[0408] Preferably, the replicon and replicase constructs of the system of the present invention are distinct from one another. In one embodiment, the replicon does not encode functional alphavirus nonstructural proteins. In one embodiment, the replicase construct lacks at least one sequence element (preferably at least one CSE) required for (-) strand synthesis based on a (+) strand template and / or (+) strand synthesis based on a (-) strand template. In one embodiment, the replicase construct does not contain CSE1 and / or CSE4.

[0409] Preferably, neither the replicon according to the invention nor the replicase construct according to the invention comprises an alphavirus packaging signal. For example, the alphavirus packaging signal contained in the nsP2 coding region of SFV (White et al. 1998, J. Virol., Vol. 72, pp. 4320-4326) can be removed, for example, by deletion or mutation. Suitable means of removing the alphavirus packaging signal include adjusting the codon usage of the nsP2 coding region. The degeneracy of the genetic code allows for the deletion of the packaging signal function without affecting the amino acid sequence of the encoded nsP2.

[0410] In one embodiment, the system of the present invention is an isolated system. In this embodiment, the system is not present in a cell, such as a mammalian cell, or in a viral capsid, such as an outer shell containing alphavirus structural proteins. In one embodiment, the system of the present invention is present in vitro.

[0411] DNA

[0412] In a third aspect, the present invention provides a DNA comprising a nucleic acid sequence encoding an RNA replicon according to the first aspect of the invention.

[0413] Preferably, the DNA is double-stranded.

[0414] In a preferred embodiment, the DNA according to the third aspect of the invention is a plasmid. As used herein, the term "plasmid" generally relates to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of chromosomal DNA.

[0415] The DNA of the present invention may comprise a promoter that can be recognized by a DNA-dependent RNA polymerase. This allows the encoded RNA (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.

[0416] In one embodiment, the DNA of the present invention is an isolated nucleic acid molecule.

[0417] Methods for preparing RNA

[0418] Any RNA molecule according to the present invention, whether or not part of the system of the present invention, can be obtained by in vitro transcription. In vitro transcribed RNA (IVT-RNA) is of particular interest in the present invention. IVT-RNA can be obtained by transcription from a nucleic acid molecule, particularly a DNA molecule. The DNA molecules of the third aspect of the invention are suitable for this purpose, particularly if they contain a promoter that can be recognized by a DNA-dependent RNA polymerase.

[0419] RNA according to the present invention can be synthesized in vitro. This allows the addition of cap-analogs to in vitro transcription reactions. Typically, the poly(A) tail is encoded by a poly(dT) sequence on the DNA template. Alternatively, capping and poly(A) tail addition can be achieved enzymatically after transcription.

[0420] In vitro transcription methodology is known to those skilled in the art. For example, various in vitro transcription kits are commercially available as described in WO2011 / 015347A1.

[0421] Reagent test kit

[0422] The present invention also provides a kit comprising the RNA replicon according to the first aspect of the invention or the system according to the second aspect of the invention.

[0423] In one embodiment, the components of the test kit exist as separate entities. For example, one nucleic acid molecule of the test kit can be present in one entity, and another nucleic acid of the test kit can be present in a separate entity. For example, an open or closed container is a suitable entity. A sealed container is preferred. The container used should preferably be free of RNase or substantially free of RNase.

[0424] In one embodiment, the kits of the invention comprise RNA for inoculating cells and / or for administration to a human or animal subject.

[0425] The kit according to the present invention optionally comprises a label or other form of information element, for example, an electronic data carrier. The label or information element preferably comprises instructions, for example, printed written instructions or instructions in an optional printable electronic form. The instructions may relate to at least one suitable possible use of the kit.

[0426] Pharmaceutical composition

[0427] The replicase constructs and / or replicons described herein can be present in the form of pharmaceutical compositions. Pharmaceutical compositions according to the present invention can comprise at least one nucleic acid molecule according to the present invention. Pharmaceutical compositions according to the present invention can comprise a pharmaceutically acceptable diluent and / or a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable vehicle. The choice of pharmaceutically acceptable carrier, vehicle, excipient, or diluent is not particularly limited. Any suitable pharmaceutically acceptable carrier, vehicle, excipient, or diluent known in the art can be used.

[0428] In one embodiment of the invention, the pharmaceutical composition may further comprise a solvent, such as an aqueous solvent or any solvent capable of maintaining RNA integrity. 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.

[0429] 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 freeze-drying the respective aqueous composition.

[0430] In one embodiment, pharmaceutical composition comprises at least one cationic entity.Usually, cationic lipids, cationic polymers and other materials with positive charge can form complex with negatively charged nucleic acid.By with cationic compounds, preferably with polycationic compounds (such as cations or polycationic peptides or proteins), RNA according to the present invention can be stabilised.In one embodiment, pharmaceutical composition according to the present invention comprises at least one cationic molecule selected from protamine, polyethyleneimine, poly-L-lysine, poly-L-arginine, histone or cationic lipid.

[0431] According to the present invention, cationic lipid is a cationic amphiphilic molecule, for example, a molecule comprising at least one hydrophilic and lipophilic portion. Cationic lipid can be monocationic or polycationic. Cationic lipid usually has a lipophilic portion, for example a sterol, an acyl group or a diacyl chain, and has a total net positive charge. The head group of lipid usually carries a positive charge. Cationic lipid preferably has a positive charge of 1 to 10 valences, more preferably a positive charge of 1 to 3 valences, more preferably a positive charge of 1 valence. Examples of cationic lipids include, but are not limited to, 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA); dimethyldioctadecyl ammonium (DDAB); 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkoxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride (DODAC), 1,2-dimyristoyloxypropyl-1,3-dimethylhydroxyethylammonium (DMRIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propylamine trifluoroacetate (DOSPA). Cationic lipids also include lipids with tertiary amine groups, including 1,2-divinyloxy-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 RNA. In one embodiment, in addition to the cationic lipid, the pharmaceutical composition also includes at least one helper 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 lipid-like molecule that has no similarity to a natural lipid. When the pharmaceutical composition includes 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.

[0432] 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 strongly basic proteins with relatively low molecular weight, which is rich in arginine and is particularly associated with DNA to replace the somatic histones in the sperm cells of animals such as fish. In particular, the term "protamine" refers to the protein found in fish sperm, which is strongly alkaline, soluble in water, does not coagulate when heated, and comprises multiple arginine monomers. According to the present invention, as used herein, the term "protamine" means any protamine amino acid sequence obtained or derived from natural or biological sources, including its fragment and the polymer form of the amino acid sequence or its fragment. In addition, the term includes (synthetic) polypeptide, which is artificial and is specially designed for a specific purpose, and can not be separated from natural or biological sources.

[0433] In some embodiments, the compositions of the present invention may include one or more adjuvants. Adjuvants can be added to vaccines to stimulate the response of the immune system; adjuvants generally do not provide immunity by themselves. Exemplary adjuvants include, but are not limited to, the following: inorganic compounds (e.g., alum, aluminum hydroxide, aluminum phosphate, calcium hydrogen phosphate); mineral oils (e.g., paraffin oil), cytokines (e.g., IL-1, IL-2, IL-12); immunostimulatory polynucleotides (e.g., RNA or DNA; e.g., CpG-containing oligonucleotides); saponins (e.g., plant saponins from Quillaja saponaria, soybeans, and Polygala senega); oil emulsions or liposomes; polyoxyethylene ether and polyoxyethylene ester preparations; polyphosphazenes (PCPP); muramyl peptides; imidazoquinolones; thiosemicarbazone compounds; Flt3 ligand (WO2010 / 066418A1); or any other adjuvant known to those skilled in the art. A preferred adjuvant for administering RNA according to the present invention is Flt3 ligand (WO2010 / 066418A1). When Flt3 ligand is administered together with RNA encoding the antigen, a robust increase in antigen-specific CD8+ T cells is observed.

[0434] The pharmaceutical composition according to the invention may be buffered (eg with acetate buffer, citrate buffer, succinate buffer, Tris buffer, phosphate buffer).

[0435] RNA-containing particles

[0436] 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. Various pharmaceutical compositions are provided in the present invention. In particular, in some embodiments, the pharmaceutical compositions of the present invention comprise particles containing nucleic acids, preferably particles containing RNA. Each pharmaceutical composition is referred to as a granular formulation. In the granular formulation according to the present invention, the particles comprise nucleic acids according to the present invention and a pharmaceutically acceptable carrier or pharmaceutically acceptable carrier suitable for delivering nucleic acids. The particles containing nucleic acids can be, for example, in the form of protein particles or in the form of lipid-containing particles. Suitable proteins or lipids are referred to as particle-forming agents. Protein particles and lipid-containing particles suitable for delivering alphavirus RNA in granular form have been previously described (e.g., Strauss & Strauss, Microbiol. Rev., 1994, Vol. 58, pp. 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.

[0437] When formulated as a granule formulation according to the system of the present invention, each RNA species (e.g., replicon, replicase construct, and optionally other RNA species, e.g., RNA encoding a protein suitable for inhibiting IFN) can be formulated separately as a separate granule formulation. In that case, each separate granule formulation comprises one RNA species. The separate granule formulations can exist as separate entities, e.g., in separate containers. Such formulations are provided by separately providing each RNA species (usually each in the form of an RNA-containing solution) and a granule-forming agent, thereby allowing granules to form. Each granule will contain only the specific RNA species (separate granule formulation) provided when the granule is formed.

[0438] In one embodiment, a pharmaceutical composition according to the present invention comprises more than one individual granule formulation. Each pharmaceutical composition is referred to as a mixed granule formulation. The mixed granule formulation according to the present invention can be obtained by separately forming the individual granule formulations as described above and then mixing the individual granule formulations. By the mixing step, a single formulation comprising a mixed population of RNA-containing particles can be obtained (for example, a first particle population can comprise a replicon according to the present invention, and a second particle formulation can comprise a replicase construct according to the present invention). The individual particle populations can be together in one container containing the mixed individual particle formulation populations.

[0439] Alternatively, all RNA species of a pharmaceutical composition (e.g., replicons, replicase constructs, and optionally other species, such as RNA encoding a protein suitable for inhibiting IFN) can be formulated together into a combined granule formulation. Such a formulation can be obtained by providing a combined formulation (typically a combined solution) of all RNA species and a granule-forming agent, thereby allowing granules to form. In contrast to mixed granule formulations, combined granule formulations typically contain granules containing more than one RNA species. In a combined granule composition, different RNA species are typically present together in a single granule.

[0440] 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 protocols and various complex compounds. Lipids, polymers, oligomers or amphiphiles are typical components of nanoparticle formulations.

[0441] As used herein, the term "nanoparticle" refers to any particle having a diameter that makes the particle suitable for systemic, particularly parenteral, administration, particularly of nucleic acids, typically having a diameter of 1000 nanometers (nm) or less. In one embodiment, the nanoparticles have an average diameter of about 50 nm to about 1000 nm, preferably about 50 nm to about 400 nm, preferably about 100 nm to about 300 nm, for example about 150 nm to about 200 nm. In one embodiment, the nanoparticles have a diameter of about 200 to about 700 nm, about 200 to about 600 nm, preferably about 250 to about 550 nm, particularly about 300 to about 500 nm or about 200 to about 400 nm.

[0442] 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. The "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 represents the width of the distribution of particles in the mixture. For example, the PI can be determined as described in WO2013 / 143555A1.

[0443] As used herein, the term "nanoparticle formulation" or similar terms refers to any particle formulation containing at least one nanoparticle. In some embodiments, the nanoparticle composition is a uniform collection of nanoparticles. In some embodiments, the nanoparticle composition is a lipid-containing pharmaceutical formulation, such as a liposomal formulation or an emulsion.

[0444] Pharmaceutical compositions containing lipids

[0445] 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 lipid 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 RNA that forms a complex with a cationic compound, thereby forming, for example, so-called lipid complexes (lipoplexes) or polymer complexes (polyplexes). The encapsulation of RNA in vesicles (e.g., liposomes) is different from, for example, lipid / RNA complexes. Lipid / RNA complexes can be obtained, for example, when RNA is mixed with preformed liposomes.

[0446] In one embodiment, the pharmaceutical composition according to the present invention comprises RNA encapsulated in a vesicle. This formulation is a specific particle formulation according to the present invention. A vesicle is a lipid bilayer rolled into a spherical shell that surrounds a small space and separates the space from the space outside the vesicle. Typically, the space inside the vesicle is an aqueous space, i.e., it contains water. Typically, the space outside the vesicle is an aqueous space, i.e., it contains water. The lipid bilayer is formed by one or more lipids (the lipids that form the vesicle). The membrane surrounding the vesicle is a lamellar phase, similar to 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, the RNA is typically separated from any external culture medium. Therefore, it exists in a protected form, functionally equivalent to the protected form in natural alphaviruses. Suitable vesicles are particles as described herein, particularly nanoparticles.

[0447] For example, RNA can be encapsulated in liposomes. In this embodiment, the pharmaceutical composition is or comprises a liposome formulation. Encapsulation within the liposome will generally 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 the RNA) is encapsulated within the core of the liposome.

[0448] Liposome is microscopic lipid vesicle, usually has the double layer of one or more lipids forming vesicle, such as phospholipid, and can encapsulate drug, such as RNA. Different types of liposomes can be used in the context of the present invention, including but not limited to multilamellar vesicle (multilamellar vesicle, MLV), small unilamellar vesicle (small unilamellar vesicle, SUV), large unilamellar vesicle (large unilamellar vesicle, LUV), sterically stabilized liposome (sterically stabilized liposome, SSL), multivesicular vesicle (multivesicular vesicle, MV) and most mutivesicular vesicle (large mutivesicular vesicle, LMV) and other double-layer forms known in the art. The size of liposome and layer matter (lamellarity) will depend on preparation mode. There is multiple other forms of supramolecular organization, wherein lipid can be present in aqueous medium, including lamellar phase, hexagonal phase (hexagonal phase) and anti-hexagonal phase, cubic phase, micelle, the reverse micelle being composed of single layer. These phases can also be obtained in combination with DNA or RNA, and the interaction with RNA and DNA can significantly affect the phase state. These phases can be present in the nanoparticle RNA formulations of the present invention.

[0449] Liposomes can be formed using standard methods known to those skilled in the art. Various methods include reverse evaporation, ethanol infusion, dehydration-rehydration, ultrasonic treatment, or other suitable methods. After liposome formation, the size of the liposomes can be adjusted to obtain a liposome population with a substantially uniform size range.

[0450] In a preferred embodiment of the present invention, the RNA is present in liposomes, and the liposomes include at least one cationic lipid. The corresponding liposomes can be formed by a single lipid or 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-divinyloxy-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 WO2012 / 006378A1: a liposome having a lipid bilayer encapsulating an aqueous core containing 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 WO2012 / 031046A2. According to WO2012 / 031046A2, liposomes comprising the corresponding compounds are particularly suitable for encapsulating RNA and are therefore suitable for liposomal delivery of RNA. In one embodiment, the RNA according to the present invention is present in a liposomal formulation, wherein the liposome comprises at least one cationic lipid, whose head group comprises at least one nitrogen atom (N) that can be protonated, wherein the N:P ratio of the liposome and the 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 to the phosphate atom (P) in the RNA contained in the lipid-containing particle (e.g., liposome), as described in WO2013 / 006825A1. The N:P ratio of 1:1 to 20:1 is relevant to the net charge of the liposome and the delivery efficiency of the RNA to vertebrate cells.

[0451] In one embodiment, the RNA 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 within the PEGylated liposomes such that the PEG moiety is present on the exterior of the liposomes, as described in WO 2012 / 031043 A1 and WO 2013 / 033563 A1.

[0452] In one embodiment, the RNA according to the invention is present in a liposomal formulation, wherein the liposomes have a diameter of 60-180 nm, as described in WO 2012 / 030901 A1.

[0453] In one embodiment, the RNA according to the present invention is present in a liposomal formulation, wherein the RNA-containing liposomes have a net charge close to zero or negative, as disclosed in WO 2013 / 143555 A1.

[0454] In other embodiments, the RNA according to the present invention is present in the form of an emulsion. Emulsions have been previously described for delivering nucleic acid molecules (e.g., RNA molecules) to cells. Preferred herein are oil-in-water emulsions. Each emulsion particle comprises an oil core and a cationic lipid. More preferred are cationic oil-in-water emulsions, 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 RNA, thereby anchoring the RNA 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 from about 80 nm to 180 nm. 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 WO2012 / 006380A2. As described in WO2013 / 006834A1, the RNA 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 / 006837A1, the RNA according to the present invention may be present in the form of a cationic lipid emulsion. In particular, the composition may comprise RNA complexed with cationic oil-in-water emulsion particles, wherein the oil / lipid ratio is at least about 8:1 (mol:mol).

[0455] In other embodiments, pharmaceutical compositions according to the present invention comprise RNA in the form of a lipoplex. The term "lipoplex" or "RNA lipoplex" refers to a complex of a lipid and a nucleic acid (e.g., RNA). Lipoplexes can be formed from cationic (positively charged) liposomes and anionic (negatively charged) nucleic acids. Cationic liposomes may also include a neutral "helper" lipid. In the simplest case, lipoplexes spontaneously form by mixing the nucleic acid with liposomes using certain mixing protocols, although various other protocols may be employed. It is understood that electrostatic interactions between the positively charged liposomes and the negatively charged nucleic acids are the driving force for lipoplex formation (WO2013 / 143555A1). In one embodiment of the present invention, the net charge of the RNA lipoplex particle is close to zero or negative. Neutral or negatively charged lipoplexes of RNA and liposomes are known to result in substantial RNA expression in splenic dendritic cells (DCs) following systemic administration, and are unrelated to the reported increased toxicity of positively charged liposomes and lipoplexes (see WO2013 / 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 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 WO2013 / 143555A1, zeta potential is the scientific term for the electromotive force in a colloidal system. In the present invention, (a) the zeta potential and (b) the charge ratio of the cationic lipid to the RNA in the nanoparticles can be calculated as disclosed in WO2013 / 143555A1. In summary, a preferred pharmaceutical composition in the context of the present invention is a pharmaceutical composition of a nanoparticle lipoplex formulation of a defined particle size as disclosed in WO2013 / 143555A1, wherein the net charge of the particles is close to zero or negative.

[0456] Methods of producing proteins

[0457] In a fourth aspect, the present invention provides a method for producing a target protein in a cell, comprising the following steps:

[0458] (a) obtaining an RNA replicon according to the first aspect of the present invention, comprising an open reading frame encoding a functional alphavirus nonstructural protein, which can be replicated by the functional alphavirus nonstructural protein, and further comprising an open reading frame encoding a protein of interest, and

[0459] (b) RNA replicons are inoculated into cells.

[0460] 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 functional alphavirus nonstructural protein and an open reading frame encoding a protein of interest and can be replicated by the functional alphavirus nonstructural protein.

[0461] In a fifth aspect, the present invention provides a method for producing a protein of interest in a cell, comprising the steps of:

[0462] (a) obtaining an RNA construct for expressing a functional alphavirus nonstructural protein,

[0463] (b) obtaining an RNA replicon according to the first aspect of the present invention, which is capable of replicating in trans via the functional alphavirus nonstructural protein according to (a) and comprises an open reading frame encoding a protein of interest, and

[0464] (c) RNA replicons and RNA constructs expressing functional alphavirus nonstructural proteins are co-inoculated into cells.

[0465] In a plurality of embodiments of the method, the RNA construct and / or RNA replicon for expressing a functional alphavirus nonstructural protein is as defined above for the system of the present invention, as long as the RNA replicon can be trans-replicated by the functional alphavirus nonstructural protein and comprises an open reading frame encoding the protein of interest. The RNA construct and RNA replicon for expressing a functional alphavirus nonstructural protein can be inoculated at the same time point, or alternatively, at different time points. In the second case, the RNA construct for expressing a functional alphavirus nonstructural protein is usually inoculated at the first time point, and the RNA replicon is usually inoculated at a subsequent second time point. In this case, it is envisioned that the replicon will replicate immediately because the replicase has been synthesized in the cell. The second time point is usually shortly after the first time point, for example, 1 minute to 24 hours after the first time point.

[0466] The cell into which one or more nucleic acid molecules are inoculated is referred to as a "host cell". According to the present invention, the term "host cell" refers to any cell that can be transformed or transfected with an exogenous nucleic acid molecule. The term "cell" is preferably a complete cell, i.e., a cell with an intact membrane that does not release its normal intracellular components, such as enzymes, organelles, or genetic material. Complete cells are preferably living cells, i.e., living cells that can exert their normal metabolic functions. According to the present invention, the term "host cell" includes prokaryotes (e.g., Escherichia coli) or eukaryotic cells (e.g., human and animal cells, plant cells, yeast cells, and insect cells). Mammalian cells are particularly preferred, for example, from humans, mice, hamsters, pigs, domesticated animals, including horses, cattle, sheep, and goats, as well as cells from primates. Cells can be derived from a variety of tissue types and include primary cells and cell lines. Specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells, and embryonic stem cells. In other embodiments, the host cell is an antigen presenting cell, particularly a dendritic cell, a monocyte, or a macrophage. The nucleic acid may be present in the host cell in single or multiple copies and, in one embodiment, is expressed in the host cell.

[0467] The cell may be a prokaryotic cell or a eukaryotic cell. Prokaryotic cells are suitable herein, for example, for propagating the DNA according to the invention, and eukaryotic cells are suitable herein, for example, for expressing the open reading frame of a replicon.

[0468] In the method of the present invention, any system according to the present invention, or a kit according to the present invention, or a pharmaceutical composition according to the present invention can be used. The RNA can be used in the form of a pharmaceutical composition, or as naked RNA, for example for electroporation.

[0469] According to the method of the present invention, efficient expression of target genes in host cells can be achieved (see, for example, Examples 2 to 5).

[0470] In one embodiment, the cells can be inoculated with additional RNA molecules, preferably mRNA molecules. Optionally, the additional RNA molecules encode proteins suitable for inhibiting IFN, such as E3 as described herein. Optionally, the additional RNA molecules can be inoculated prior to inoculation with the replicon or replicase construct or system according to the invention.

[0471] In the method for producing a protein in a cell according to the present invention, the cell may be an antigen-presenting cell, and the method may be used to express RNA encoding an antigen. To this end, the present invention may involve introducing RNA encoding an antigen into an antigen-presenting cell, such as a dendritic cell. For transfection of antigen-presenting cells, such as dendritic cells, a pharmaceutical composition comprising RNA encoding an antigen may be used.

[0472] 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.

[0473] In this embodiment, cells seeded according to the fourth aspect of the invention may be administered to a subject to produce and provide the protein to the subject. The cells may be autologous, syngeneic, allogeneic or heterologous relative to the subject.

[0474] In other embodiments, the cell in the method of producing a protein in a cell can be present in a subject, such as a patient. In these embodiments, the method for producing a protein in a cell is an in vivo method, which comprises administering an RNA molecule to the subject.

[0475] In this aspect, the present invention also provides a method for producing a protein of interest in a subject, comprising the steps of:

[0476] (a) obtaining an RNA replicon according to the first aspect of the present invention, comprising an open reading frame encoding a functional alphavirus nonstructural protein, which can be replicated by the functional alphavirus nonstructural protein, and further comprising an open reading frame encoding a protein of interest, and

[0477] (b) Administering the RNA replicon to a subject.

[0478] 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 functional alphavirus non-structural protein and an open reading frame encoding a protein of interest, and can be replicated by the functional alphavirus non-structural protein.

[0479] The present invention further provides a method for producing a protein of interest in a subject, comprising the steps of:

[0480] (a) obtaining an RNA construct for expressing a functional alphavirus nonstructural protein,

[0481] (b) obtaining an RNA replicon according to the first aspect of the present invention, which is capable of replicating in trans via the functional alphavirus nonstructural protein according to (a) and comprises an open reading frame encoding a protein of interest, and

[0482] (c) administering to the subject an RNA replicon and an RNA construct for expressing a functional alphavirus nonstructural protein.

[0483] In various embodiments of this method, the RNA construct and / or RNA replicon for expressing a functional alphavirus nonstructural protein is as defined above for the system of the present invention, provided that the RNA replicon is trans-replicable by the functional alphavirus nonstructural protein and comprises an open reading frame encoding the protein of interest. The RNA construct for expressing a functional alphavirus nonstructural protein and the RNA replicon can be administered at the same time point, or alternatively, at different time points. In the second embodiment, the RNA construct for expressing a functional alphavirus nonstructural protein is typically administered at a first time point, and the RNA replicon is typically administered at a subsequent second time point. In this case, it is assumed that the replicon will replicate immediately because the replicase has already been synthesized in the cell. The second time point is typically shortly after the first time point, for example, 1 minute to 24 hours after the first time point. Preferably, the RNA replicon is administered at the same site and via the same route of administration as the RNA construct for expressing a functional alphavirus nonstructural protein, to increase the likelihood that the RNA replicon and the RNA construct for expressing a functional alphavirus nonstructural protein reach the same target tissue or cell. "Site" refers to a location in the subject's body. Suitable sites are, for example, the left arm, the right arm, etc.

[0484] In one embodiment, an additional RNA molecule, preferably an mRNA molecule, can be administered to a subject. Optionally, the additional RNA molecule encodes a protein suitable for inhibiting IFN, such as E3, as described herein. Optionally, the additional RNA molecule can be administered prior to administration of a replicon or replicase construct or system according to the invention.

[0485] Any of the RNA replicon according to the invention, the system according to the invention, the kit according to the invention, or the pharmaceutical composition according to the invention can be used in a 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, e.g., as described herein, or as naked RNA.

[0486] In view of the ability to be administered to a subject, each of the RNA replicons of the present invention, the systems of the present invention, the kits of the present invention, or the pharmaceutical compositions of the present invention may be referred to as "drugs," etc. The present invention contemplates providing the RNA replicons, systems, kits, and pharmaceutical compositions of the present invention for use as medicaments. The medicaments can be used to treat a subject. "Treatment" refers to administering a compound or composition described herein or other entity to a subject. The term includes methods of treating the human or animal body by therapy.

[0487] The above-mentioned medicaments generally do not contain DNA and are therefore associated with additional safety features compared to the DNA vaccines described in the prior art (eg WO 2008 / 119827 A1).

[0488] The alternative medical uses according to the present invention include methods for producing proteins in cells according to the fourth aspect of the present invention, wherein the cells can be antigen-presenting cells, such as dendritic cells, and then the cells are introduced into a subject. For example, RNA encoding a pharmaceutically active protein (e.g., an antigen) can be introduced (transfected) into ex vivo antigen-presenting cells, e.g., antigen-presenting cells taken from a subject and, optionally, ex vivo cloned antigen-presenting cells can be reintroduced into the same or different subject. The transfected cells can be reintroduced into the subject using any method known in the art.

[0489] 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 preventive as well as therapeutic methods of treating a subject.

[0490] The medicaments according to the present invention are administered in an effective amount. An "effective amount" refers to an amount sufficient, alone or in combination with other doses, to elicit a response or desired effect. In the case of treating a disease or condition in a subject, the desired effect is to inhibit disease progression. This includes slowing disease progression, and in particular, interrupting disease progression. The desired effect of treating a disease or condition may also be delaying disease onset or inhibiting disease onset.

[0491] 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, duration of treatment, type of concomitant treatment (if any), the particular mode of administration and other factors.

[0492] Vaccination

[0493] 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 is intended to include treatment intended to induce or enhance an immune response to one or more antigens in a subject. According to the present invention, if it is desired to induce or enhance an immune response by using RNA as described herein, the immune response can be elicited or enhanced by RNA. 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 protein as the protein of interest, which is an immunologically active compound or antigen.

[0494] RNA has been previously described for vaccination against foreign substances including pathogens or cancer (recently reviewed by Ulmer et al., 2012, Vaccine, Vol. 30, pp. 4414-4418). In contrast 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 nonstructural proteins as described herein. Vaccination according to the present invention can be used, for example, to induce an immune response to weakly immunogenic proteins. In the case of the RNA vaccine according to the present invention, the protein antigen is never exposed to serum antibodies, but is 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.

[0495] In a method involving vaccination according to the invention, the medicament of the invention is administered to a subject, in particular a subject having or at risk of a disease involving the antigen if treatment is required.

[0496] In the method involving vaccination according to the invention, the protein of interest encoded by the replicon according to the invention encodes a bacterial antigen to be directed by the immune response, a viral antigen to be directed by the immune response, or a cancer antigen to be directed by the immune response. The efficacy of 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 allergenicity. Allergen-specific immunotherapy (also known as desensitization) is defined as the administration of preferably increasing doses of an allergen vaccine to an organism having one or more allergens in order to achieve a state of reduced symptoms associated with subsequent exposure to the pathogenic allergen. The efficacy of allergen-specific immunotherapy can be assessed by known standard methods, for example by measuring allergen-specific IgG and IgE antibodies from the organism.

[0497] The medicaments of the present invention can be administered to a subject, for example, for treatment of the subject, including vaccination of the subject.

[0498] The term "subject" refers 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, cows, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., as well as captive animals, such as zoo animals. The term "subject" also refers 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). As used herein, the term "animal" also includes humans.

[0499] In some embodiments, administration to domestic 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 prophylactic vaccination according to the present invention can be applied to vaccinating animal populations, for example, in agriculture, or wild animal populations. Other animal populations kept in captivity, such as pets or animals in zoos, can be vaccinated.

[0500] When administered to a subject, the replicon and / or replicase construct used as a medicament preferably does not contain sequences from an alphavirus type that is infectious for the species or genus to which the subject is being treated. Preferably, in that case, the replicon and / or replicase construct does not contain any nucleotide sequences from an alphavirus that can infect the corresponding species or genus. This embodiment has the advantage that even if the subject to whom the RNA is administered is (e.g., accidentally) exposed to an infectious alphavirus, recombination with an infectious (e.g., fully functional or wild-type) alphavirus is unlikely. As an illustrative example, for the treatment of pigs, the replicon and / or replicase construct used does not contain any nucleotide sequences from an alphavirus that can infect pigs.

[0501] Administration

[0502] The medicament according to the present invention may be administered to a subject by any suitable route.

[0503] For example, the drug can be administered systemically, such as intravenously (iv), subcutaneously (sc), intradermally (id), or by inhalation.

[0504] 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 into skin or muscle results in 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, Vol. 109, pp. 14604-14609).

[0505] Alternatives to administration to muscle tissue or skin include, but are not limited to, intradermal, intranasal, intraocular, intraperitoneal, intravenous, interstitial, oral, transdermal, or sublingual administration. Intradermal and intramuscular administration are two preferred routes.

[0506] 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 administered by needle injection. Needle-free injection can be used as an alternative.

[0507] The present invention is described and explained in detail by the accompanying drawings and examples, which are only for illustrative purposes and not limiting. According to the description and examples, the skilled person can obtain other embodiments that are also included in the present invention.

[0508] Example

[0509] Materials and methods:

[0510] The following materials and methods were used in the examples described below.

[0511] Plasmid cloning, in vitro transcription, RNA purification:

[0512] Plasmids were cloned using standard techniques. Details regarding the cloning of each plasmid used in the examples of the present invention are described in Example 1. In vitro transcription was performed using the plasmids described in Example 1 and T7 RNA polymerase, and RNA purification was performed as previously described (Holtkamp et al., 2006, Blood, Vol. 108, pp. 4009-4017; Kuhn et al., 2010, Gene Ther., Vol. 17, pp. 961-971).

[0513] The quality of the purified RNA was assessed spectrophotometrically and analyzed on a 2100 BioAnalyzer (Agilent, Santa Clara, USA).All RNA transfected into the cells in the examples was in vitro transcribed RNA (IVT-RNA).

[0514] RNA transfection:

[0515] For electroporation, RNA was electroporated into mammalian cells at room temperature using a square wave electroporation device (BTX ECM 830, Harvard Apparatus, Holliston, MA, USA) using the following settings: for BHK21 cells: 750 V / cm, 16 millisecond pulse; for human foreskin fibroblasts: 500 V / cm, 24 millisecond pulse. Mixtures of different RNA species were prepared in RNase-free tubes and kept on ice until transfection. For electroporation, the RNA or RNA mixture was resuspended in a final volume of 62.5 μl / mm cuvette gap size.

[0516] For lipofection, cells were plated at approximately 20,000 cells / cm 2 The growth areas were plated and the cells were stained with a total of 260 ng / cm according to the manufacturer's instructions (Life Technologies, Darmstadt, Germany). 2 RNA and 1 μl / cm 2MessengerMax reagent for transfection.

[0517] Cell culture:

[0518] All growth media, fetal calf serum (FCS), antibiotics, and other supplements were provided by Life Technologies / Gibco unless otherwise stated. Human foreskin fibroblasts obtained from System Bioscience (HFF, neonatal) or ATCC (CCD-1079Sk) were cultured in minimal essential medium (MEM) containing 15% FCS, 1% nonessential amino acids, and 1 mM sodium pyruvate at 37°C. Cells were grown at 37°C in a humidified atmosphere equilibrated to 5% CO2. The cell line "BHK21[C13]( CCL10 TM BHK21 cells were grown in Eagle's minimal essential medium supplemented with 10% FCS.

[0519] Flow cytometry:

[0520] The expression of RNA encoding GFP was measured by flow cytometry using a FACS Canto II flow cytometer (BD Bioscience, Heidelberg, Germany), and the obtained data were analyzed by the corresponding Diva software or FlowJo software (Tree Star Inc., Ashland, OR, USA).

[0521] Luciferase assay:

[0522] To evaluate the expression of firefly luciferase, transfected cells were plated into 96-well black microplates and supernatants from Nanoluc transfected cells were transferred to 96-well black microplates (Nunc, Langenselbold, Germany). Firefly luciferase expression was measured using the Bright-Glo luciferase assay system, and Nanoluc expression was measured using the Nano-Glo luciferase assay system (both Promega, Madison, WI, USA) according to the manufacturer's instructions. Luminescence was measured using a microplate reader Infinite M200 (Tecan Group, Bioluminescence was measured by luciferase-negative cells in Switzerland. Data are expressed as relative light units [RLU], and luciferase-negative cells were used to subtract background signal.

[0523] Example 1: Plasmid cloning

[0524] A. A plasmid encoding a replicon based on Semliki Forest virus (SFV) was obtained using a PCR-based seamless cloning technique. Seamless cloning means a cloning technique in which the fragments generated by PCR are recombined into a linearized vector based on the use of homologous sequence segments. Therefore, the DNA sequence encoding the replicon corresponding to the SFV genome except for the lack of an open reading frame encoding viral structural genes was transferred from pSFV-gen-GFP (Ehrengruber & Lundstrom, 1999, Proc. Natl. Acad. Sci. USA, Vol. 96, pp. 7041-7046; Lundstrom, 2001, Histochem. Cell Biol., Vol. 115, pp. 83-91) to a pST1 plasmid backbone (Holtkamp et al., 2006, Blood, Vol. 108, pp. 4009-4017), which is immediately downstream of the T7 phage RNA polymerase promoter. A plasmid encoding a poly(A) cassette of 120 adenylate residues (Holtkamp et al., supra) or a modified poly(A) cassette consisting of 30 and 70 adenylate residues separated by a 10-nucleotide random sequence (WO 2016 / 005004 A1) was added immediately downstream of the last nucleotide of the SFV 3'-UTR. A SapI restriction site was placed immediately downstream of the poly(A) cassette or the modified poly(A) cassette. In addition, the coding sequence encoding the myc-tag was inserted into the XhoI site found in the coding region of the SFV nsP3 variable region. Insertion into the nsP3 variable region did not affect the activity of the replicase polyprotein (Spuul et al., 2010, J. Virol, Vol. 85, pp. 7543–7557). The resulting plasmid contained a DNA sequence encoding the 5' replication recognition sequence of SFV under the control of the T7 polymerase promoter. The DNA sequence encoding the 5' replication recognition sequence of SFV is represented by SEQ ID NO: 4:

[0525]

[0526] In the above representation of SEQ ID NO:4, the first underlined ATG serves as the start codon for the synthesis of the N-terminal fragment of nsP1; the bases translated into protein are indicated in bold. Other ATGs within the nsP1 coding region are also underlined. During in vitro transcription of a plasmid containing the 5' replication recognition sequence of SFV (represented by SEQ ID NO:4) by T7 polymerase, a transcript containing an RNA sequence corresponding to SEQ ID NO:3 was obtained: the 5'-terminal G corresponds to the first nucleotide transcribed by T7 polymerase. This G precedes the alphavirus sequence and is required for efficient transcription by T7 polymerase.

[0527]

[0528] In the above representation of SEQ ID NO: 3, five specific ATG base triplets are underlined. The unique EcoRV restriction site (GATATC) in the SFV replicase coding region is highlighted in bold.

[0529] As a result, plasmid A was obtained. Plasmid A contains the open reading frame of a functional alphavirus nonstructural protein.

[0530] B. A plasmid encoding a trans-replicon with an unmodified 5' replication recognition sequence was obtained by removing the nonstructural protein coding sequence from EcoRV to SalI from plasmid A (see above). Thus, the major portion of the open reading frame encoding nsP1234 was removed, i.e., the sequence extending from the unique EcoRV restriction site to the unique SalI restriction site. For example: GATATC (the 3'most six nucleotides of SEQ ID NO:3, in bold in the above representation) corresponds to the unique EcoRV restriction site of the coding sequence for the SFV replicase. After removal of the major portion of the open reading frame encoding nsP1234, the 5' replication recognition sequence and the subgenomic promoter remained. Due to the removal of the major portion of the open reading frame encoding nsP1234, the RNA encoded by the corresponding plasmid cannot drive replication in cis when present in a host cell, but replication requires the presence of functional alphavirus nonstructural proteins.

[0531] The open reading frame encoding firefly luciferase (transgene) was inserted downstream of the subgenomic promoter (SGP). Thus, plasmid B was obtained. The RNA replicon encoded by the corresponding plasmid was expressed in Figure 1 It is shown as “Template RNA WT-RRS” in the figure.

[0532] The RNA secondary structure prediction web server (http: / / rna.urmc.rochester.edu / RNAstructureWeb / Servers / Predict1 / Predict1.html) confirmed that the unmodified 5' replication recognition sequence (RNA) was predicted to fold into four stem-loops, indistinguishable from the parental RNA and consistent with the literature (Frolov, 2001, RNA, vol. 7, pp. 1638-1651).

[0533] C-1. Starting from plasmid B, a plasmid encoding a trans-replicon with a modified 5' replication recognition sequence (including the removal of the ATG triplet encoding the first amino acid residue of nsP1 and the removal of four additional ATG triplets within the 5' replication recognition sequence) was generated by replacing one base of each ATG triplet with a different base (i.e., replacing A, T, or G). In other words, the ATG triplets of SEQ ID NO: 3 underlined above were removed by a single nucleotide change (replacement).

[0534] The fold of the trans-acting replicon RNA encoded by the plasmid was predicted using the RNA Secondary Structure Prediction Web Server (http: / / rna.urmc.rochester.edu / RNAstructureWeb / Servers / Predict1 / Predict1.html) and Mfold (http: / / unafold.rna.albany.edu / ?q=mfold).

[0535] The predicted folding of the modified 5' replication recognition sequence characterized by ATG removal was compared with the predicted folding of each unmodified 5' replication recognition sequence. If the prediction showed that the secondary structure of the modified 5' replication recognition sequence was different from the secondary structure of the unmodified 5' replication recognition sequence, one or more additional nucleotide changes (replacements) were made in a trial-and-error approach until the RNA folding of the 5' replication recognition sequence without ATG (Δ5ATG-RRS) was predicted to be identical to the RNA folding of the unmodified 5' replication recognition sequence encoded by plasmid B. As a result, a DNA sequence according to SEQ ID NO: 5 was obtained:

[0536]

[0537] The plasmid encoding a modified 5' replication recognition sequence characterized by the same predicted fold (ie, the plasmid comprising SEQ ID NO: 5) is referred to herein as C-1.

[0538] Depend on Figure 1 The RNA replicon encoded by each plasmid shown in is named "Δ5ATG-RRS".

[0539] It is understood that removal of the five specific ATGs will prevent the synthesis of nsP1 or fragments thereof, as exemplified by SEQ ID NO: 5. Due to the removal of the native start codon of nsP1, it is understood that protein synthesis begins at the first start codon downstream of the subgenomic promoter (SGP), resulting in transcription of the open reading frame (transgene) encoding firefly luciferase.

[0540] C-2. Starting from C-1, SGP was removed by digestion with EcoRV and SmaI and religation. The corresponding RNA replicon is schematically shown in Figure 1 If the corresponding RNA replicon contains a 5' cap, the transgene encoding luciferase can be placed under the direct translational control of the 5'-cap.

[0541] C-3. Starting from C-1, the open reading frame encoding the SFV replicase was inserted using the EcoRV and SalI restriction sites. Thus, a plasmid encoding a self-replicating RNA was obtained, wherein the replicase ORF did not overlap with the 5' replication recognition sequence nor with the subgenomic promoter. Each RNA replicon was Figure 1 It is schematically depicted in FIG, and is referred to as the “cis-replicon Δ5ATG-RRS”.

[0542] D. To enable translation of the replicase-encoding nucleic acid sequence from non-replicating mRNA, the open reading frame encoding the SFV replicase was cloned into a plasmid containing the human α-globin 5'-UTR, a synthetic 3'-UTR, and a plasmid-encoded poly(A) tail of 30 plus 70 adenylate residues separated by a stable 10 nucleotide (10 nt) linker (WO 2016 / 005324 A1). The open reading frame encoding the SFV replicase was cloned downstream of the human α-globin 5'-UTR. The plasmid contained a T7 promoter for transcription of the open reading frame encoding the SFV replicase.

[0543] E. To enable translation of the nucleic acid sequence encoding the vaccinia virus protein kinase R inhibitor E3 from non-replicating mRNA, the open reading frame encoding the vaccinia virus protein kinase R inhibitor E3 ("E3") was cloned into a plasmid containing the human α-globin 5'-UTR, a synthetic 3'-UTR, and a plasmid-encoded poly(A) tail of 30 plus 70 adenylate residues separated by a stable 10 nucleotide (10 nt) linker (WO 2016 / 005324 A1). The open reading frame encoding E3 was cloned downstream of the human α-globin 5'-UTR. The plasmid contained a T7 promoter for transcription of the open reading frame encoding E3.

[0544] Example 2: Removal of the start codon within the 5' replication recognition sequence does not affect the replication of the replicon RNA in trans.

[0545] BHK21 cells were co-electroporated with: (i) 5 μg of mRNA encoding SFV replicase (encoded by plasmid D of Example 1 ) and (ii) varying amounts of trans -replicon RNA (encoded by plasmid B or C-1 of Example 1 ; Figure 2The trans-replicon encodes firefly luciferase; the trans-replicon contains the wild-type 5' replication recognition sequence ( Figure 2 : "WT-RRS"; encoded by plasmid B); or a 5' replication recognition sequence characterized by removal of all start codons ( Figure 2 : "Δ5ATG-RRS"; encoded by plasmid C-1). The trans-replicon RNA was not capped (uncapped). 5000 electroporated BHK21 cells were plated in each well of a 96-well plate to measure luciferase expression 24 hours after electroporation. The results are shown in Figure 2 In B.

[0546] Example 3: Removal of the start codon within the 5' replication recognition sequence enables translation of the transgene under the control of the cap

[0547] A trans-replicon characterized by the removal of all start codons and containing a subgenomic promoter ( Figure 3 "Δ5ATG-RRS"; encoded by plasmid C-1 of Example 1), a trans-replicon characterized by the removal of all start codons but containing no subgenomic promoter ( Figure 3 "Δ5ATG-RRSΔSGP"; encoded by plasmid C-2 of Example 1), and a trans-replicon with all start codons and containing a subgenomic promoter ( Figure 3 Human foreskin fibroblasts were co-electroporated with: (i) 0.45 μg of Figure 3 Each trans-replicon RNA as shown, and either (ii-a) 2.5 μg of mRNA encoding vaccinia virus E3 protein (encoded by plasmid E of Example 1, in Figure 3 (ii-b): 2.5 μg mRNA encoding replicase (encoded by plasmid D of Example 1, in Figure 3 2.5 μg of mRNA encoding vaccinia virus E3 protein (encoded by plasmid E in Example 1) was added to the 50 μg vaccinia virus E3 protein mRNA to inhibit protein kinase R activation, thereby promoting the expression of luciferase and replicase. Figure 3 : “uncapped”) or co-transcriptionally capped with a β-S-ARCA (D2) cap analog ( Figure 3 : "D2-cap"). Luciferase expression was assessed 24 hours later. The results are shown in Figure 3 Middle (right picture).

[0548] This example demonstrates that removal of the start codon from the 5' replication recognition sequence enables efficient translation of the transgene directly from the capped trans-replicon RNA.

[0549] Example 4: Cap-dependent translation is stronger from a trans-replicon characterized by removal of the start codon within the 5' replication recognition sequence than from subgenomic RNA at early stages after transfection.

[0550] A trans-replicon characterized by the removal of all start codons and containing a subgenomic promoter ( Figure 4 : “Δ5ATG-RRS”; encoded by plasmid C-1 of Example 1), and a trans-replicon characterized by the removal of all start codons but containing no subgenomic promoter ( Figure 4 In the example, "Δ5ATG-RRSΔSGP"; encoded by plasmid C-2 of Example 1). BHK21 cells were co-electroporated with: (i) 0.45 μg of each trans-replicon RNA and (ii) 2.5 μg of mRNA encoding the replicase (encoded by plasmid D of Example 1). The trans-replicon RNA was not capped ( Figure 4 : “uncapped”) or co-transcriptionally capped with a β-S-ARCA (D2) cap analog ( Figure 4 : "D2-cap"). Luciferase expression was assessed over time. Results are shown in Figure 4 Middle (right picture).

[0551] Example 5: Cap-dependent translation from a trans-replicon characterized by removal of the start codon within the 5' replication recognition sequence enables transgene expression at an early stage, independent of prior expression of the replicase.

[0552] Using a trans-replicon characterized by the removal of all start codons but without a subgenomic promoter ( Figure 5 "Δ5ATG-RRSΔSGP"; encoded by plasmid C-2 of Example 1). The trans-replicon RNA is not capped ( Figure 5 : “uncapped”) or co-transcriptionally capped with a β-S-ARCA (D2) cap analog ( Figure 5 BHK21 cells were electroporated with (i) 0.45 μg of each trans-replicon RNA and (ii) 0.45 μg of each tra...

Claims

1. A pharmaceutical composition comprising an RNA replicon comprising a modified 5' replication recognition sequence of an alphavirus, wherein the modified 5' replication recognition sequence is characterized in that all start codons have been removed from the conserved sequence element 2 (CSE2) of the native alphavirus 5' replication recognition sequence, and wherein the modified 5' replication recognition sequence comprises one or more additional nucleotide changes that compensate for the disruption of nucleotide pairing within one or more stem-loops (SLs) introduced by the removal of the start codon, wherein said CSE 2 spans from SL2 to SL4 and comprises the natural start codon encoding the first amino acid residue of the alphavirus nonstructural protein nsP1, and wherein the modified 5' replication recognition sequence is characterized by a predicted secondary structure identical to the predicted secondary structure of the 5' replication recognition sequence of the alphavirus genomic RNA, The RNA replicon comprises a first open reading frame encoding a target protein.

2. The pharmaceutical composition according to claim 1, wherein the RNA replicon comprises a 3' replication recognition sequence.

3. The pharmaceutical composition according to claim 1, wherein the target protein can be expressed using the RNA replicon as a template.

4. The pharmaceutical composition of claim 1, wherein the RNA replicon comprises a subgenomic promoter that controls the production of subgenomic RNA, and the subgenomic RNA comprises the first open reading frame encoding the protein of interest.

5. The pharmaceutical composition according to claim 4, wherein the protein of interest encoded by the first open reading frame can be expressed by the RNA replicon and subgenomic RNA.

6. The pharmaceutical composition of claim 1, wherein the RNA replicon comprises a subgenomic promoter that controls the production of a subgenomic RNA, and the subgenomic RNA comprises a second open reading frame encoding a protein of interest.

7. The pharmaceutical composition according to claim 6, wherein the subgenomic promoter and the second open reading frame encoding the target protein are located downstream of the first open reading frame encoding the target protein.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the protein of interest encoded by the first and / or second open reading frame is a functional alphavirus non-structural protein.

9. The pharmaceutical composition according to claim 8, wherein the RNA replicon is capable of replicating through the functional alphavirus non-structural protein.

10. The pharmaceutical composition of claim 1, wherein the RNA replicon does not contain an open reading frame encoding a functional alphavirus nonstructural protein.

11. The pharmaceutical composition according to any one of claims 1 to 7 and 10, further comprising: RNA constructs for expressing functional alphavirus nonstructural proteins, The RNA replicon is capable of trans replication through the functional alphavirus non-structural protein.

12. The pharmaceutical composition according to claim 1, wherein the alphavirus is Semliki Forest virus.

13. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises a pharmaceutically acceptable diluent and / or a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable carrier.

14. The pharmaceutical composition according to claim 1, comprising a particle preparation of the RNA replicon, wherein the particle is a protein particle or a lipid-containing particle.

15. The pharmaceutical composition of claim 14, wherein the lipid-containing particles comprise at least one cationic lipid.

16. The pharmaceutical composition of claim 15, wherein the cationic lipid is 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA); dimethyldioctadecyl ammonium (DDAB); 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkoxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride (DODAC), 1,2-dimyristoyloxypropyl-1,3-dimethylhydroxyethylammonium (DMRIE) and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propylamine trifluoroacetate (DOSPA).

17. The pharmaceutical composition of claim 15, wherein the cationic lipid comprises a lipid having a tertiary amine group, including 1,2-divinyloxy-N,N-dimethyl-3-aminopropane (DLinDMA).

18. The pharmaceutical composition of claim 1, wherein the RNA replicon is contained in a liposome.

19. The pharmaceutical composition of claim 14, wherein the lipid-containing particle is a lipid / RNA complex.

20. The pharmaceutical composition of claim 14, wherein the lipid-containing particles are lipid complexes.

21. The pharmaceutical composition of claim 14, wherein the lipid-containing particle is a polyplex, wherein the polyplex is a complex of RNA and a cationic compound.

22. The pharmaceutical composition of claim 20, wherein the lipid complex is formed by cationic liposomes.

23. The pharmaceutical composition of claim 22, wherein the lipid complex comprises a neutral helper lipid.

24. Use of the pharmaceutical composition according to claim 1 in the preparation of a medicament for treatment, wherein the RNA replicon encodes a pharmaceutically active peptide or protein.

25. Use according to claim 24, wherein the pharmaceutical composition is suitable for intravenous (iv), subcutaneous (sc), intradermal (id), by inhalation, intranasal, intraocular, intraperitoneal, interstitial, oral, transdermal or sublingual administration.

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