NZ785730A - RNA replicon for versatile and efficient gene expression - Google Patents

Abstract

The present invention embraces a RNA replicon that can be replicated by a replicase of alphavirus origin. The RNA replicon comprises sequence elements required for replication by the replicase, but these sequence elements do not encode any protein or fragment thereof, such as an alphavirus non-structural protein or fragment thereof. Thus, in the RNA replicon according to the invention, sequence elements required for replication by the replicase and protein-coding region(s) are uncoupled. According to the present invention the uncoupling is achieved by the removal of at least one initiation codon compared to a native alphavirus genomic RNA. In particular, the RNA replicon comprises a 5' replication recognition sequence, wherein the 5' replication recognition sequence is characterized in that it comprises the removal of at least one initiation codon compared to a native alphavirus 5' replication recognition sequence. The replicase of alphavirus origin may be encoded by an open reading frame on the RNA replicon or on a separate RNA molecule. The present invention enables efficient and safe expression of a protein of interest in a cell or organism, but is not associated with undesired production of fragments of alphavirus non-structural protein. Methods of protein production in vitro and in vivo, as well as medical uses, are provided herein.

Description

RNA replicon for versatile and efficient gene expression This is a divisional application of New Zealand Application 745739, the entire contents of which are incorporated herein by reference.

Technical Field of the ion The present invention embraces a RNA replicon that can be replicated by a replicase of alphavirus origin. The RNA replicon comprises sequence elements required for replication by the replicase, but these sequence elements do not encode any protein or fragment thereof, such as an alphavirus non-structural protein or fragment thereof. Thus, in the RNA replicon according to the invention, the sequence elements ed for replication by the replicase and protein-coding regions are uncoupled. According to the present invention the uncoupling is achieved by the removal of at least one initiation codon ed to a native alphavirus genomic RNA. The RNA replicon may comprise a gene ng a protein of interest, such as a pharmaceutically active protein. The replicase may be d by the RNA replicon or by a separate nucleic acid molecule.

Background of the Invention Nucleic acid molecules comprising foreign genetic information encoding one or more polypeptides for prophylactic and eutic purposes have been studied in biomedical research for many years. Prior art ches share the delivery of a nucleic acid molecule to a target cell or sm, but differ in the type of nucleic acid molecule and/or delivery system: influenced by safety concerns ated with the use of deoxyribonucleic acid (DNA) molecules, ribonucleic acid (RNA) molecules have received growing attention in recent years. Various approaches have been proposed, ing administration of single stranded or stranded RNA, in the form of naked RNA, or in complexed or packaged form, e.g. in non-viral or viral ry vehicles. In viruses and in viral delivery es, the genetic information is typically ulated by proteins and/or lipids (virus particle). For example, engineered RNA virus particles derived from RNA viruses have been proposed as delivery vehicle for treating plants ( or for vaccination of mammals (Tubulekas et al., 1997, Gene, vol. 190, pp. 191- 195). In general, RNA viruses are a diverse group of infectious particles with an RNA genome. RNA viruses can be sub-grouped into single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) viruses, and the ssRNA viruses can be further generally divided into [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg positive-stranded [(+) stranded] and/or negative-stranded [(-) ed] viruses.

Positive-stranded RNA viruses are prima facie attractive as a delivery system in biomedicine because their RNA may directly as template for translation in the host cell.

Alphaviruses are l representatives of positive-stranded RNA viruses. The hosts of alphaviruses include a wide range of organisms, comprising insects, fish and s, such as domesticated animals and humans. iruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see José et al., Future Microbiol., 2009, vol. 4, pp. 837—856). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5’-cap, and a '3 poiy(A) tail. The genome of alphaviruses encodes non—structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four ructural proteins (nsP1—nsP4) are typically encoded together by a first ORF ing near the 5' terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3' terminus of the genome.

Typically, the first ORF is larger than the second ORF, the ratio being roughly 2:1.

In cells infected by an irus, only the nucleic acid sequence encoding non- ural proteins is translated from the genomic RNA, while the genetic information encoding structural ns is translatable from a subgenomic ript, which is an RNA molecule that resembles otic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111—124). Following infection, i.e. at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural poly-protein (nsP1234). In some iruses, there is an opal stop codon between the coding sequences of nsP3 and nsP4: otein P123, containing nsP1, nsP2, and nsP3, is produced when translation terminates at the opal stop codon, and polyprotein P1234, containing in addition nsP4, is produced upon readthrough of this opal codon (StraD& 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 [Annotation] mpg None set by mpg ation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg ation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg the fragments nsP123 and nsP4. The polypeptides nsP123 and nsP4 associate to form the (-) strand replicase complex that transcribes (-) stranded RNA, using the (+) stranded genomic RNA as template. Typically at later stages, the nsP123 nt is completely cleaved into individual proteins nsP1, nsP2 and nsP3 (Shirako & Strauss, 1994, J. Virol., vol. 68, pp. 1874-1885). All four proteins form the (+) strand replicase complex that synthesizes new (+) stranded genomes, using the (-) stranded complement of genomic RNA as template (Kim et al., 2004, Virology, vol. 323, pp. 153-163, Vasiljeva et al., 2003, J. Biol. Chem. vol. 278, pp. 41636-41645).

In infected cells, omic RNA as well as new genomic RNA is provided with a ’-cap by nsP1 (Pettersson et al. 1980, Eur. J. Biochem. 105, ; Rozanov et al., 1992, J. Gen. Virology, vol. 73, pp. 2129-2134), and provided with a poly- adenylate [poly(A)] tail by nsP4 h et al., Virology, 2009, vol. 384, pp. 201- 208). Thus, both subgenomic RNA and genomic RNA resemble messenger RNA (mRNA).

Alphavirus structural ns (core nucleocapsid n C, envelope protein E2 and envelope protein E1, all tuents of the virus particle) are typically encoded by one single open reading frame under l of a subgenomic promoter (Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491-562). The subgenomic promoter is recognized by alphaviral non-structural proteins acting in cis. In particular, alphavirus replicase synthesizes a (+) stranded subgenomic transcript using the (-) stranded complement of genomic RNA as te. The (+) stranded subgenomic transcript encodes the alphavirus structural proteins (Kim et al., 2004, gy, vol. 323, pp. 153-163, Vasiljeva et al., 2003, J. Biol. Chem. vol. 278, pp. 41636-41645). The subgenomic RNA transcript serves as template for translation of the open reading frame encoding the structural proteins as one poly-protein, and the poly-protein is cleaved to yield the structural proteins. At a late stage of alphavirus infection in a host cell, a packaging signal which is located within the coding sequence of nsP2 ensures selective packaging of genomic RNA into budding virions, packaged by structural proteins (White et al., 1998, J. , vol. 72, pp. 4320-4326).

In inDed cells, (-) strand RNA synthesis is typically observed only in the first 3-4 h post infection, and is undetectable at late stages, at which time the synthesis of only [Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg (+) strand RNA (both genomic and subgenomic) is ed. ing to Frolov et al., 2001, RNA, vol. 7, pp. 651, the prevailing model for regulation of RNA synthesis suggests a dependence on the processing of the non-structural poly- protein: initial cleavage of the non-structural polyprotein nsP1234 yields nsP123 and nsP4; nsP4 acts as RNA-dependent RNA polymerase (RdRp) that is active for (-) strand synthesis, but inefficient for the generation of (+) strand RNAs. Further sing of the polyprotein , including cleavage at the sP3 junction, changes the template specificity of the replicase to increase synthesis of (+) strand RNA and to decrease or terminate synthesis of (-) strand RNA.

The synthesis of alphaviral RNA is also regulated ba IH sis-acting RNA ts, including four conserved ce elements (CSEs; Sr-o-reuss & Strauss, Microbiol.

Rev., 1994, vol. 58, pp. 491-562; and Frolov, 2001, RNA, vol. 7, pp. 1638-1651).

In general, the 5’ replication recognition sequence of the alphavirus genome is characterized by low overall homology between different alphaviruses, but has a conserved predicted secondary structure. The 5’ replication recognition sequence of the alphavirus genome is not only involved in translation initiation, but also comprises the 5’ replication recognition sequence comprising two conserved sequence elements involved in sis of viral RNA, CSE 1 and CSE 2. For the function of CSE 1 and 2, the secondary structure is believed to be more important than the linear ce (Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491-562). in contrast, the 3’ terminal sequence of the alphavirus genome, is. the sequence immediately upstream of the poly(A) sequence, is characterized by a conserved primary structure, ularly by conserved sequence element 4 (CSE 4), also termed "19-nt ved sequence", which is important for initiation of (-) strand synthesis.

CSE 3, also termed "junction sequence" is a conserved sequence element on the (+) strand of alphaviral genomic RNA, and the complement of CSE 3 on the (—) strand acts as promoter for subgenomic RNA transcription (Strauss & Strauss, Microbiol.

Rev.DQ4, vol. 58, pp. 491-562; Frolov et al., 2001, RNA, vol. 7, pp. 1638-1651).

CSE 3 typically ps with the region encoding the inal fragment of nsP4.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg In addition to alphavirus proteins, also host cell factors, presumably proteins, may bind to conserved sequence elements (Strauss & Strauss, supra).

Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms. In simple approaches, the open reading frame encoding alphaviral structural proteins is replaced by an open reading frame encoding a protein of interest. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase (typically as poly-protein nsP1234), and the other c acid moiecuie is capable of being replicated by said replicase in trans (hence the designation trans-replication system). trans-replication requires the presence of both these nucleic acid molecules in a given host cell. The nucleic acid moiecuie capabie of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase. A respective replicon is illustrated as "Template RNA WT-RRS" in Fig. 1. Such replicon is ated with the age of allowing for amplification of a gene of interest under control of a subgenomic er; however, more versatile vectors are difficult to develop because the open reading frame encoding 4 overlaps with the 5’ replication recognition sequence of the alphavirus genome g sequence for nsP1) and lly also with the subgenomic promoter comprising CSE 3 (coding sequence for nsP4).

For example, Michel et al. (2007, Virology, vol. 362, pp. 475—487) describe that the introduction of 95 silent ons (i.e. mutations which do not affect the encoded protein sequence) into the coding region of nsP1 of the alphavirus elan encephalitis virus (VEEV) tely abolished the capacity of VEEV to ate in cells, presumably because the silent ons destroyed the ary ure of the RNA. 1723-1727) describe helper RNA (i.e. trans-replicating RNA expressing VEEV capsid and envelope) which were modified such that the specific AUG base triplet that serves as start codon for nsP1 of VEEV found in nature can be removed (by convnon into a stop codon) to create modified constructs. According to Kamrud et al., the conversion of the nsP1 start codon into a stop codon conserved the [Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg replicative potential of the RNA and these ed helper RNAs yielded VEEV conversion of all AUGs found within the CSE1/2 region to stop codons resulted in helper RNA that replicated poorly in the presence of alphavirus replicase. The authors attribute the poor replication to a putative disruption of the underlying RNA secondary structure, A compromised ary structure is a quite likely explanation since the authors did not mention that they controlled correct RNA g of their ed helper RNA.

The fact that the 5’ replication ition sequence required for RNA replication comprises an AUG start codon for nsP1 and thus overiaps with the coding ce for the N-terminal fragment of the irus non-structural protein ents a serious bottle-neck for the engineering of alphavirus-based vectors because a replicon comprising the 5’ replication recognition sequence will typically encode (at least) a part of alphavirus non-structural protein, typically the N-terminal fragment of nsP1. This is disadvantageous in several aspects: In the case of cis-replicons this overlap limits for instance adaptation of codon usage of 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 as it is found in the viruses is not optimal in every target cell.

However, the secondary structure cannot be altered freely as possibly resulting amino acid changes in the replicase ORF have to be considered and tested for the effect on ase function. It is also not possible to exchange the te ase ORF for replicases from heterologous origin since this can results in disruption of the 5’ replication recognition sequence structure.

In the case of trans-replicons this overlap results in the synthesis of a fragment of nsP1 protein since the 5' replication recognition sequence needs to be retained in trans replicons. A fragment of nsP1 is typically not required and not desired: the undesired translation imposes an unnecessary burden on the host cell, and RNA replicons intended for therapeutic ations that encode, in addition to a pharmaceutically active n, a fragment of nsP1, may face regulatory concerns.

For instance, it will be necessary to trate that the truncated nsP1 does not creatDwanted side effects. In addition, the presence of an AUG start codon for nsP1 within the 5’ replication recognition sequence has prevented the design of ation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg trans-replicons encoding a heterologous gene of interest in a fashion wherein the start codon for translation of the gene of st is at the most 5’ position that is accessible for ribosomal translation initiation. In turn, —dependent translation of transgenes from prior art trans-replicon RNA is challenging, unless cloned as fusion protein in frame to the start codon of nsP1 (such fusion constructs are described e.g. by Michel et al., 200?, Virology, vol. 362, pp. 475-48?) Such fusion ucts lead to the same unnecessary translation of the nsP1 fragment mentioned above, raising the same concerns as above. Moreover, fusion proteins cause additional concerns as they might alter the function or activity of the fused transgene of interest, or when used as vaccine vector, peptides spanning the fusion region could alter immunogenicity of the fused antigen.

There is a need to overcome these disadvantages. For instance, there is a need to e improved replicons for expressing a nucleic acid encoding a n of interest, such as a pharmaceutically active n, in a safe and ent manner. As described herein, the aspects and embodiments of the present invention address this need.

Summary of the invention lmmunotherapeutic strategies represent promising options for the prevention and therapy of e.g. infectious diseases and cancer diseases. The identification of a growing number of pathogen- and tumor-associated antigens led to a broad collection of suitable targets for immunotherapy. The present invention embraces improved agents and methods suitable for efficient expression of antigens, suitable for immunotherapeutic ent for the prevention and therapy of diseases.

In a first aspect, the present invention provides a RNA replicon comprising a 5' replication recognition sequence, wherein the 5' ation recognition sequence is characterized in that it comprises the removal of at least one initiation codon ed to a native irus 5' replication recognition sequence.

In one embodiment, the RNA replicon of the invention comprises a (modified) 5' replicon recognition sequence and a first open reading frame encoding a n of interest, e.g. functional alphavirus non-structural protein or a transgene which is [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg [Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg ed set by mpg preferably not derived from an alphavirus, in particular an alphavirus non-structural protein, located ream from the 5' replication recognition sequence, wherein the 5' replication ition sequence and the first open reading frame encoding a 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 ' replication recognition sequence does not contain a functional initiation codon and preferably does not contain any initiation codon. Most preferably, the initiation codon of the first open reading frame encoding a protein of st is in the 5' —> 3' direction of the RNA replicon the first functional initiation codon, ably the first tion codon. In one embodiment, the first open reading frame ng a protein of interest encodes functional alphavirus non-structural protein. in one embodiment, the first open reading frame encoding a protein of interest and preferably the entire RNA replicon does not express non-functional alphavirus non-structural protein, such as a fragment of alphavirus non-structural protein, in particular a nt of nsP1 and/or nsP4. In one embodiment, the functional alphavirus ructural protein is heterologous to the 5' replication recognition ce. In one embodiment, the first open reading frame encoding a protein of interest is not under control of a subgenomic promotor. In one embodiment, the RNA replicon comprises at least one further open reading frame encoding a protein of interest which is under control of a subgenomic promotor. In one embodiment, the subgenomic promotor and the first open reading frame encoding a protein of interest do not overlap.

In one embodiment, the 5' replication recognition ce of the RNA on that is characterized by the removal of at least one initiation codon comprises a sequence homologous to about 250 nucleotides at the 5' end of an irus. In a preferred embodiment, it comprises a sequence homologous to about 300 to 500 nucleotides at the 5' end of an alphavirus. In a preferred embodiment it comprises the 5’-termina| sequence required for efficient replication of the specific alphavirus species that is al to the vector system.

In one embodiment, the 5' replication recognition sequence of the RNA replicon comprises sequences homologous to conserved sequence element 1 (CSE 1) and consGd sequence element 2 (CSE 2) of an alphavirus.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg In a red embodiment, the RNA replicon comprises CSE 2 and is further terized in that it comprises a nt of an open reading frame of a non- structural protein from an irus. In a more preferred embodiment, said fragment of an open reading frame of a non—structural protein does not comprise any initiation codon.

In one embodiment, the 5' replication recognition sequence comprises a sequence homologous to an open reading frame of a non-structural protein or a fragment thereof from an irus, wherein the sequence homologous to an open reading frame of a non-structural protein or a fragment thereof from an alphavirus is characterized in that it comprises the removal of at least one tion codon compared to the native alphavirus sequence.

In a preferred embodiment, the sequence homologous to an open reading frame of a non-structural protein or a fragment thereof from an alphavirus is terized in that it comprises the removal of at least the native start codon of the open reading frame of a non-structural protein.

In a red embodiment, the sequence gous to an open reading frame of a non-structural protein or a fragment thereof from an alphavirus is characterized in that it comprises the removal of one or more initiatic.. ccdons other than the native start codon of the open reading frame of a non-structural protein. In a more preferred embodiment, said nucleic acid sequence is additionally characterized by the removal of the native start codon of the open reading frame of a non-structural protein, preferably of nsP1.

In a preferred embodiment, the 5’ replication recognition sequence comprises one or more stem loops providing functionality of the 5’ replication recognition ce with respect to RNA replication. In a preferred embodiment, one or more stem loops of the 5’ replication recognition sequence are not d or disrupted. More preferably, one or more of stem loops 1, 3 and 5, preferably all stem loops 1, 3 and 4, or stem loops 3 and 4 are not deleted or disrupted. More preferably, none of the stem e 5’ ation recognition sequence is deleted or disrupted.

[Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg In a preferred ment, the RNA replicon comprises one or more nucleotide changes compensating for nucleotide pairing disruptions within one or more stem loops introduced by the removal of at least one initiation codon.

In one embodiment, the RNA replicon does not comprise an open reading frame encoding a ted alphavirus non—structural protein.

In one embodiment, the RNA replicon comprises a 3' replication ition sequence.

-A CD one Fl)rrnbodinsent, the RNA replicon comprises a first open reading frame encoding a protein of interest.

In one embodiment, the RNA replicon is characterized in that the protein of interest d by the first open reading frame can be expressed from the RNA on as a template.

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

In a preferred embodiment, the protein of interest encoded by the first open reading frame can be expressed from the RNA replicon as a te. in a more preferred embodiment, the protein of interest encoded by the first open reading frame can additionally be expressed from the subgenomic RNA.

In a preferred embodiment, the RNA replicon is further terized in that it comprises a subgenomic promoter controlling production of subgenomic RNA comprising a second open reading frame encoding a protein of interest. The protein of interest may be a second protein that is identical to or different from the protein of interest encoded by the first open reading frame. ation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg In a more red embodiment, the subgenomic promoter and the second open reading frame encoding a protein of interest are located ream from the first open reading frame encoding a protein of interest.

In one embodiment, the protein of interest encoded by the first and/or second open reading frame is functional irus non-structural protein.

In one embodiment, the RNA replicon comprises an open reading frame encoding functional alphavirus non-structural protein.

In one embodiment, the open reading frame encoding functional alphavirus non- structural protein does not overlap with the 5' replication recognition sequence.

In one embodiment, the RNA replicon that encodes functional alphavirus non- structural protein can be replicated by the functional alphavirus non-structural protein.

In one embodiment, the RNA replicon does not se an open reading frame ng functional alphavirus non-structural n. In this embodiment, the functional irus non-structural protein for replication of the replicon may be ed in trans as described herein.

In a second aspect, the present invention provides a system comprising: a RNA construct for expressing functional alphavirus non-structural protein, the RNA replicon according to the first aspect of the invention, which can be replicated by the onal alphavirus non-structural protein in trans. Preferably, the RNA replicon is further characterized in that it does not encode a functional alphavirus non-structural protein.

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

[Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg In a third , the present invention provides a DNA comprising a nucleic acid sequence encoding the RNA on according to the first aspect of the present invenfion.

In a fourth aspect, the present invention provides a method for producing a protein of interest in a cell comprising the steps of: (a) ing the RNA replicon according to the first aspect of the invention, which comprises an open reading frame encoding functional alphavirus non-structural protein, which can be replicated by the functional alphavirus non-structural protein and which further comprises an open reading frame encoding the protein of interest, (b) inoculating the RNA replicon into the cell.

In various embodiments of the , the RNA replicon is as defined above for the replicon of the invention.

In a fifth , the present invention provides a method for producing a n of interest in a cell comprising the steps of: (a) obtaining a RNA construct for sing functional alphavirus non—structural protein, (b) obtaining the RNA replicon according to the first aspect of the invention, which can be replicated by the functional alphavirus non-structural protein according to (a) in trans and which comprises an open reading frame encoding the protein of interest, (c) culating the RNA construct for expressing functional alphavirus non- structural protein and the RNA replicon into the cell.

In various embodiments of the method, the RNA construct for sing functional alphavirus non-structural protein and/or the RNA replicon are as defined above for the system of the invention. According to the fifth aspect, the RNA replicon does typicD not itself encode functional irus non-structural protein.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg ation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg In a sixth aspect, the invention provides a cell containing 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, 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 or by the method of the fifth aspect of the invention. In one embodiment, the cell is part of an organism.

In a seventh aspect, the present invention provides a method for ing a protein of interest in a subject comprising the steps of: (a) obtaining the RNA replicon ing to the first aspect of the invention, which comprises an open reading frame encoding functional aiphavirus non-structural protein, which can be replicated by the functional aiphavirus ructural protein and which further comprises an open reading frame encoding the protein of interest, and (b) administering the RNA replicon to the subject.

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

In an eighth aspect, the present invention provides a method for producing a protein of interest in a t comprising the steps of: (a) obtaining a RNA construct for expressing functional irus non—structural protein, (b) obtaining the RNA replicon according to the first aspect of the invention, which can be replicated by the functional aiphavirus non-structural protein according to (a) in trans and which comprises an open reading frame encoding the protein of interest, (c) administering the RNA uct for expressing functional aiphavirus non- ural protein and the RNA replicon to the subject.

In VEDJS embodiments of the method, the RNA construct for expressing onal irus non-structural protein and/or the RNA replicon are as defined above for [Annotation] mpg None set by mpg ation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg the system of the invention. According to the eighth aspect, the RNA replicon does typically not itself encode functional irus non-structural protein.

Brief description of the drawings Fig. 1: Schematic representation of RNA replicons comprising an unmodified or a modified 5’ replication recognition sequence Abbreviations: AAAA = Poly(A) tail; ATG = start codon/initiation codon (ATG on DNA level; AUG on RNA level); 5x ATG = nucleic acid sequence comprising all start 1O ccdcns in the nucleic acid sequence encoding nsP1* (in the case of the c acid n .. Annn in P.4* sequence emu ...g nl from Semliki Forest virus 5x ATG corresponds to five specific start codons, see Example 1); ASATG = nucleic acid sequence ponding to a nucleic acid sequence encoding nsP1*; r not comprising any start codons of the nucleic acid ce that encodes nsP1* in alphavirus found in nature (in the case of nsP1* d from Semliki Forest virus, "A5ATG" corresponds to the removal of five specific start codons compared to Semliki Forest virus found in nature, see Example 1); EcoRV = EcoRV restriction site; nsP = nucleic acid ce encoding an alphavirus non-structural protein (e.g. nsP1, nsP2, nsP3, nsP4); nsP1* = nucleic acid sequence encoding a fragment of nsP1, wherein the fragment does not comprise the C-terminal fragment of nsP1; *nsP4 = nucleic acid sequence encoding a fragment of nsP4, n the fragment does not comprise the N-terminal fragment of nsP4; RRS = 5' replication recognition sequence; Sall = Sall restriction site; SGP = subgenomic promoter; SL = stem loop (e.g. SL1, SL2, SL3, SL4); the positions of SL1-4 are graphically illustrated; UTR = untranslated region (e.g. 5’-UTR, 3’-UTR)9NT = wild type. cisReplicon WT-RRS: RNA replicon essentially corresponding to the genome of an alphavirus, except that the nucleic acid sequence encoding alphavirus structural proteins has been replaced by an open reading frame ng a gene of interest ("Transgene"). When "Replicon WT-RRS" is introduced into a cell, the translation product of the open g frame encoding replicase 34 or fragment(s) thereof) can drive ation of the RNA replicon in cis and drive synthesis of a nuclacid ce (the subgenomic transcript) downstream of the subgenomic promoter (SGP).

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg Trans-replicon or template RNA : RNA replicon essentially corresponding to "Replicon WT-RRS", except that most of the nucleic acid sequence encoding alphavirus non—structural proteins nsP1-4 has been removed. More specifically, the nucleic acid sequence encoding nsP2 and nsP3 has been removed completely; the nucleic acid sequence encoding nsP1 has been truncated so that the "Template RNA WT—RRS" encodes a fragment of nsP1, which fragment does not comprise the C- terminal fragment of nsP1 (but it comprises the inal fragment of nsP1; nsP1*); the nucleic acid sequence encoding nsP4 has been truncated so that the "Template RNA WT=RRS" encodes a fragment of nsP4, which fragment does not comprise the inal fragment "if nsP4 {but ft comprises the fnat nt cf "rsP4; *nsP4). This truncated nsP4 sequence overlaps partially with the fully active subgenomic promoter. The nucleic acid sequence ng nsP1* comprises all initiation codons of the nucleic acid sequence that encodes nsP1* in alphavirus found in nature (in the case of nsP1* from Semliki Forest virus, five ic initiation codons) A5ATG-RRS: RNA replicon essentially corresponding to "Template RNA WT-RRS", except that it does not comprise any initiation codons of the nucleic acid sequence that encodes nsP1* in alphavirus found in nature (in the case of Semliki Forest virus, "ASATG-RRS" ponds to the removal of five specific initiation codons ed to Semliki Forest virus found in ). All nucleotide s introduced to remove start codons were sated by additional nucleotide changes to conserve the predicted secondary structure of the RNA.

A5ATG-RRSASGP: RNA replicon essentially corresponding to -RRS", except that it does not comprise the subgenomic er (SGP) and does not comprise the nucleic acid sequence that encodes *nsP4. "Transgene 1" = a gene of interest.

A5ATG-RRS —- bicistronic: RNA replicon essentially corresponding to "A5ATG- RRS", except that it comprises a first open reading frame encoding a first gene of interfl‘Transgene 1") upstream of the subgenomic promoter, and a second open reading frame encoding a second gene of interest ("Transgene 2") downstream of the [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg ation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg subgenomic promoter. The localization of the second open g frame corresponds to the localization of the gene of interest ("Transgene") in the RNA replicon "A5ATG-RRS". cisReplicon A5ATG-RRS: RNA replicon essentially corresponding to "ASATG-RRS — bicistronic". except that the open reading frame encoding a first gene of st encodes functional alphavirus non—structural protein (typically one open reading frame encoding the poly-protein nsP1—nsP2-nsP3—nsP4, i.e. nsP1234). "Transgene" in "cisReplicon A5ATG-RRS" corresponds to "Transgene 2" in "A5ATG-RRS — bicistronic". The functional alphavirus non—structural protein is capable of recognizing the subgenomic promoter and of synthesizing subgenomic transcripts comprising the nucleic acid sequence encoding the gene of interest ("Transgene"). plicon A5ATG-RRS" encodes a functional alphavirus non-structural protein in cis as does "cisReplicon WT-RRS"; r, it is not required that the coding sequence for nsP’i d by plicon A5ATG-RRS" comprises the exact nucleic acid ce of "cisReplicon WT-RRS" including all stem loops.

Fig. 2: The removal of start codons within the 5’ replication recognition sequence does not affect replication of trans-replicon RNAs. A: illustration of c acid les used in Example 2. B: Measured luciferase expression of electroporated BHK21 cells. For details, see Example 2. Shown is the mean i SD of 2 independent experiments with 2 independently produced batches of RNA replicon ("template"); (N=4).

Fig. 3: The removal of start codons within the 5’ replication recognition sequence enables cap-dependent translation. Left: illustration of nucleic acid molecules (RNA replicons) used in Example 3. Right: Measured luciferase expression of electroporated human foreskin fibroblasts. For details, see Example 3.

Shown is the mean i SD of one experiment med in triplicates.

Fig. 4: Right after transfection, cap-dependent translation from trans-replicon characterized by the removal of start codons within the 5’ replication recontion sequence is stronger than translation from a omic transcript. Left: illustration of nucleic acid les. RNA replicons used in [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg Example 4 are illustrated as "ASATG-RRS" and "ASATG-RRSASGP". Right: Measured rase expression of electroporated BHK21 cells. For details, see Example 4. Shown is the mean of one experiment performed in triplicates.

Fig. 5: A capped trans-replicon characterized by the removal of start codons within the 5’ replication recognition sequence enables expression of a transgene at early stages. Left: illustration of the "A5ATG-RRSASGP" c acid molecule used in Example 5. Right: Measured luciferase expression of oporated BHK21 cells. For details, see Example 5. Shown is the mean i SD of one experiment performed in cates.

Fig. 6. Structures of cap dinucvleotides. Top: a natural cap dinucleotide, m7GpppG.

Bottom: Phosphorothioate cap analog beta-S—ARCA dinucleotide: There are two reomers of beta—S-ARCA due to the stereogenic P center, which are designated D1 and D2 according to their elution characteristics in reverse phase HPLC.

Fig. 7. Re-constructed cis-replicons with a ATG-deleted RRS are functional. The ORF of SFV replicase was inserted into "A5ATG-RRS" encoding firefly luciferase downstream of the omic promoter (SGP). Within the inserted replicase the regions ponding to CSE2 and the core SGP were disrupted by nucleotide exchanges (hashed boxes) to avoid duplication of these regulatory regions. This resulted in a structed cis—replicon. BHK21 cells were ctroporated with either 2,5ug "cis—replicon WT-RRS" or "cis-replicon A5ATG-RRS". 24h after electroporation luciferase expression was measured. MeaniSD of one experiment in triplicates.

Fig. 8. Bicistronic replicons express both transgenes. Secretable Nano— rase (SNL) was cloned downstream of the subgenomic promoter (SGP) of a trans-replicon WT-RSS The position upstream of the SGP does not encode a transgene (-)SGP(SNL). in the lower construct, SNL was cloned downstream of AATG-RSS, and firefly luciferase (Luc) inserted downstream of the SGP (SNLD5P(Luc). BHK21 cells were co-electroporated with 0.9ug trans-replicating [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg ation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg RNA and 5ug SFV-replicase coding mRNA, 48h after electroporation SNL and Luc expression were measured. Data of one experiment.

Fig. 9. Sindbis Virus replicons lacking start codons in the replication recognition sequence replicate efficiently. Trans-replicons were engineered from Sindbis Virus genome by gene synthesis and GFP was ed downstream of the subgenomic promoter (SGP). In addition to this trans-replicon with unmodified replication recognition sequence (WT-RSS) two variants thereof were generated. In AATG-RRS the original start codon plus 4 further ATGs were deleted from the WT- RRS. satory nucleotide s to keep RNA secondary structure were also introduced as required. To generate AATG-RRSASGP, the region corresponding to the subgenomic promoter was deleted from the AATG-RRS resulting in a vector with GFP ly downstream of the ATG-deleted 5'RRS. BHK21 cells were co- electroporated with 0.1ug replicating RNA and 2.4ug SFV—replicase coding mRNA. 24h after electroporation GFP sion (transfection rate [%] and mean fluorescence intensity (MFI)) was assessed. Data of one experiment. ed description of the invention Although the present invention is described in detail below, it is to be understood that this invention is not d to the particular methodologies. protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the t invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B.

Nagei, and H. Kélbl, Eds., Helvetica Chimica Acta, CH-401O Basel, Switzerland, (1995).

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg The practice of the present invention will employ, unless otherwise indicated, tional methods of try, biochemistry, cell biology, immunology, and inant DNA techniques which are explained in the literature in the field (cf., e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create onal embodiments. The variously described examples and preferred embodiments should not be construed t" limit the present invention to only the explicitly describe embodiments. This description should be understood to disclose and encompass embodiments which combine the explicitly described ments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and ations of all described elements in this application should be Considered disclosed by this description unless the context indicates otherwise.

The term "about" means imately or , and in the context of a numerical value or range set forth herein preferably means +/- 10 % of the cal value or range recited or claimed.

The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the t of the claims) are to be construed to cover both the singular and the plural, unless othewvise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated , each individual value is incorporated into the specification as if it was individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otheIDe claimed. No language in the specification should be construed as indicating any non-claimed element essential to the ce of the invention.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg Unless expressly specified otherwise, the term "comprising" is used in the t of the present document to indicate that further s may optionally be present in addition to the members of the list uced by "comprising". It is, however, contemplated as a specific embodiment of the present invention that the term "comprising" encompasses the possibility of no further members being present, i.e. for the purpose of this embodiment "comprising" is to be understood as having the meaning of "consisting of". indications of ve amounts of a component characterized by a generic term are meant to refer to the total amount of aii specific ts or members covered by said generic term. If a certain component defined by a generic term is specified to be present in a certain relative amount, and if this component is further characterized to be a specific variant or member covered by the generic term, it is meant that no other variants or members covered by the generic term are onally present such that the total ve amount of components covered by the generic term exceeds the specified relative amount; more preferably no other variants or members covered by the generic term are present at all.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific ations, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their ty. Nothing herein is to be construed as an admission that the present invention was not entitled to antedate such disclosure.

Terms such as "reduce" or it" as used herein means the ability to cause an l decrease, preferably of 5% or greater, 10% or greater, 20% or greater, more preferably of 50% or greater, and most preferably 75% or greater, in the level. The term "inhibit" or similar phrases includes a complete or essentially complete inhibition, Le. a reduction to zero or ially to zero.

TernGuch as "increase" or ce" preferably relate to an increase or enhancement by about at least 10%, preferably at least 20%, preferably at least [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg %, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%.

The term "net " refers to the charge on a whole object, such as a nd or particle.

An ion having an overall net positive charge is a cation, while an ion having an overall net negative charge is an anion. Thus, according to the invention, an anion is an ion with more electrons than protons, giving it a net negative charge; and a cation 1O is an ion with fewer electrons than s, giving it a net positive charge.

Terms as "charged", "net charge", "negatively charged" or "positively charged", with nce to a given compound or particle, refer to the electric net charge of the given compound or particle when dissolved or suspended in water at pH 7.0.

The term "nucleic acid" according to the invention also comprises a chemical derivatization of a nucleic acid on a nucleotide base, on the sugar or on the phosphate, and nucleic acids containing non-natural tides and tide analogs. In some embodiments, the nucleic acid is a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). In general, a nucleic acid molecule or a nucleic acid sequence refers to a nucleic acid which is preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). According to the ion, nucleic acids comprise genomic DNA, cDNA, mRNA, viral RNA, recombinantly prepared and chemically synthesized molecules. According to the invention, a nucleic acid may be in the form of a single- ed or -stranded and linear or covalently closed circular molecule.

According to the invention "nucleic acid sequence" refers to the sequence of nucleotides in a nucleic acid, eg. a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). The term may refer to an entire nucleic acid molecule (such as to the single 3O strand of an entire nucleic acid molecule) or to a part (eg. a fragment) thereof.

According to the present invention, the term "RNA" or "RNA le" relates to a moleG which comprises ribonucleotide residues and which is preferably entirely or substantially composed of ribonucleotide residues. The term "ribonucleotide" relates [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg to a nucleotide with a hydroxyl group at the 2’-position of a B-D—ribofuranosyl group.

The term "RNA" comprises double-stranded RNA, single stranded RNA, isolated RNA such as partially or completely purified RNA, essentially pure RNA, synthetic RNA, and recombinantly generated RNA such as modified RNA which differs from naturally occurring RNA by addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of nan-nuciectide material, such as to the end(s) of a RNA or ally, for example at one or more nucleotides of the RNA. Nucleotides in RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring tides or ally synthesized 1O nucleotides or deoxynucleotides. These altered RNAs can be ed to as analogs, particularly s of naturally occurring RNAs. ing to the ion, RNA may be single-stranded or double-stranded. In some embodiments of the present invention, -stranded RNA is preferred. The term "single-stranded RNA" generally refers to an RNA molecule to which no complementary nucleic acid molecule ally no complementary RNA molecule) is associated. Single-stranded RNA may contain self-complementary sequences that allow parts of the RNA to fold back and to form secondary structure motifs ing without limitation base pairs, stems, stem loops and bulges. Single-stranded RNA can exist as minus strand [(-) strand} or as plus strand [(+) strand}. The (+) strand is the strand that comprises or encodes genetic information. The genetic information may be for example a polynucleotide sequence encoding a protein. When the (+) strand RNA encodes a protein, the (+) strand may serve directly as te for translation (protein synthesis). The (=) strand is the cempiement ef the (+) strand. in the case of double-stranded RNA, (+) strand and (-) strand are two separate RNA molecules, and both these RNA molecules associate with each other to form a double-stranded RNA ex RNA").

The term "stability" of RNA relates to the "half-life" of RNA. "Half-life" s to the period of time which is needed to eliminate half of the activity, amount, or number of molecules. In the context of the present invention, the half-life of an RNA is indicative for the stability of said RNA. The half-life of RNA may influence the "duration of exprDon" of the RNA. It can be expected that RNA having a long ife will be expressed for an extended time period.

[Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg The term "translation efficiency" relates to the amount of translation product provided by an RNA molecule within a particular period of time, ent", with reference to a nucleic acid ce, relates to a part of a nucleic acid sequence, Le. a sequence which represents the nucleic acid sequence shortened at the 5'- and/or 3'—end(s). Preferably, a fragment of a c acid sequence comprises at least 80%, preferably at least 90%, 95%, 96%, 97%, 98%, or 99% of the nucleotide residues from said c acid sequence. In the present invention those fragments of RNA molecules are preferred which retain RNA stability and/or translational efficiency.

"Fragment", with reference to an amino acid sequence (peptide or protein), relates to a part of an amino acid sequence, Le. a sequence which represents the amino acid sequence shortened at the N-terminus and/or C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) is obtainable e.g. by translation cf a truncated open reading frame that lacks the 3’-end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) is obtainable e.g. by translation of a truncated open reading frame that lacks the 5’-end of the open reading frame, as 2O long as the truncated open reading frame comprises a start codon that serves to initiate ation= A fragment at an amino acid sequence comprises e.g. at least 1 %, at least 2 %, at least 3 %, at least 4 %, at least 5 %, at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80%, at least 90% of the amino acid residues from an amino acid sequence.

The term "variant" with respect to, for example, nucleic acid and amino acid sequences, according to the invention includes any variants, in ular s, viral strain variants, splice variants, conformations, isoforms, c variants, species variants and species homologs, in particular those which are naturally present. An c t s to an alteration in the normal sequence of a gene, the icance of which is often unclear. Complete gene sequencing often identifies numerous allelic variants for a given gene. With respect to nucleic acid molecules, the ta "variant" includes degenerate nucleic acid sequences, wherein a degenerate nucleic acid according to the invention is a nucleic acid that differs from a reference [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg nucleic acid in codon sequence due to the degeneracy of the genetic code. A species homolog is a nucleic acid or amino acid ce with a different species of origin from that of a given nucleic acid or amino acid sequence. A virus homolog is a nucleic acid or amino acid sequence with a different virus of origin from that of a given nucleic acid or amino acid sequence.

According to the invention, nucleic acid variants include single or multiple nucleotide deletions, additions, mutations, substitutions and/or insertions in ison with the reference nucleic acid. Deletions include removal of one or more nucleotides from the _.\ CD reference nucleic acid. Addition variants comprise 5'- and/or 3'—terminal fusions of one or more nucleotides, such as 1, 2, 3, 5, 10, 20, 30, 50, or more nucleotides. in the case of substitutions, at least one tide in the sequence is removed and at least one other nucleotide is inserted in its place (such as transversions and transitions). Mutations include abasic sites, crosslinked sites, and chemically altered or modified bases. Insertions e the addition of at least one tide into the reference nucleic acid.

According to the invention, "nucleotide change" can refer to single or multiple nucleotide deletions, additions, mutations, substitutions and/or insertions in comparison with the reference nucleic acid. In some embodiments, a "nucleotide change is ed from the group consisting of a deletion of a single nucleotide, the addition of a single nucleotide, the mutation of a single nucleotide, the tution of a single nucleotide and/or the insertion of a single nucleotide, in comparison with the reference c acid. According to the invention, a nucleic acid variant can se one or more tide changes in comparison with the reference nucleic acid.

Variants of specific nucleic acid sequences preferably have at least one functional property of said specific ces and preferably are onally equivalent to said specific sequences, e.g. c acid sequences exhibiting properties identical or similar to those of the ic nucleic acid sequences.

As daibed below, some embodiments of the present invention are characterized inter alia by nucleic acid sequences that are homologous to nucleic acid sequences [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg of an alphavirus, such as an alphavirus found in nature. These homologous sequences are variants of nucleic acid sequences of an alphavirus, such as an alphavirus found in nature.

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

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

The term "% identical" is intended to refer, in particular, to a percentage of tides which are identical in an optimal alignment between two sequences to be compared, with said percentage being purely tical, and the ences 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 in comparison with the reference sequence, in order to obtain optimal alignment between two sequences. Comparisons of two sequences are usuaiiy carried out by comparing said sequences, after optimal ent, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences. The l alignment for a ison may be carried out manually or with the aid of the local homology thm by Smith and Waterman, 1981, Ads App.

Math. 2, 482, with the aid of the local homology algorithm by Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, and with the aid of the similarity search i by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444 or with the aid of computer ms using said algorithms (GAP, BESTFIT, FASTA, BLAST [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg ation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

Percentage identity is obtained by ining the number of identical positions in which the sequences to be compared correspond, dividing this number by the number of positions compared and multiplying this result by 100.

For example, the BLAST program "BLAST 2 sequences" which is available on the website http://www.ncbi.nlm.nih.gov/blast/bl23eq/wblast2.cgi may be used.

A c acid is "capable of hybridizing" or dizes" to r nucleic acid if the two sequences are complementary with one another. A nucleic acid is "complementary" to another nucleic acid if the two sequences are capable of forming a stable duplex with one another. According to the ion, hybridization is preferably carried out under conditions which allow specific ization between polynucleotides (stringent conditions). Stringent conditions are described, for example, in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Editors, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989 or Current Protocols in Molecular Biology, F.M. Ausubel et al., Editors, John Wiley & Sons, Inc., New York and refer, for example, to hybridization at 65°C in hybridization buffer (3.5 x SSC, 0.02% Ficoli, 0.02% poiyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH2PO4 (pH 7), 0.5% SDS, 2 mM 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 erred is washed, for example, in 2 x SSC at room temperature and then in 01-05 x 1 x SDS at temperatures of up to 68°C.

A percent complementarity indicates the percentage of contiguous 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 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" or "fully complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous es in a secoDucleic acid ce. Preferably, the degree of complementarity according to the invention is at least 70%, ably at least 75%, preferably at least 80%, [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg 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 ing to the invention is 100%.

The term "derivative" comprises any chemical derivatization of a nucleic acid on a nucleotide base, on the sugar or on the phosphate. The term "derivative" also comprises nucleic acids which contain nucleotides and nucleotide analogs not occurring naturally. Preferably, a derivatization of a nucleic acid increases its stability.

According to the invention, a "nucleic acid sequence which is derived from a nucleic acid ce" refers to a nucleic acid which is a variant of the nucleic acid from which it is derived. Preferably, a sequence which is a variant with respect to a ic ce. when it replaces the specific sequence in an RNA molecule retains RNA stability and/or ational ency. "nt" is an abbreviation for nucleotide; or for nucleotides, preferably consecutive nucleotides in a nucleic acid molecule.

According to the invention, the term " refers to a base triplet in a coding nucleic acid that specifies which amino acid will be added next during n sis at the ribosome.

The terms "transcription" and "transcribing" relate to a process during which a nucleic acid molecule with a particular nucleic acid ce (the "nucleic acid template") is read by an RNA polymerase so that the RNA polymerase produces a single-stranded RNA molecule. During transcription, the genetic information in a nucleic acid template is transcribed. The nucleic acid te may be DNA; r, eg. in the case of ription from an alphaviral nucleic acid template, the template is typically RNA.

Subsequently, the transcribed RNA may be translated into protein. According to the present invention, the term "transcription" comprises "in vitro transcription", wherein the term "in vitro transcription" relates to a process wherein RNA, in particular mRNA, is in vitro synthesized in a cell-free . Preferably, cloning vectors are applied for the gration of transcripts. These cloning vectors are generally designated as transcription vectors and are according to the present invention encompassed by the [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg term "vector". The cloning vectors are preferably plasmids. According to the present invention, RNA preferably is in vitro ribed RNA (lVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The er for controlling transcription can be any promoter for any RNA polymerase. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

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

According to the invention, the terms "template" or "nucleic acid template" or "template nucleic acid" generally refer to a nucleic acid sequence that may be replicated or transcribed.

"Nucleic acid sequence ribed from a nucleic acid sequence" and similar terms refer to a nucleic acid sequence, where appropriate as part of a complete RNA molecule, which is a transcription product of a template nucleic acid sequence.

Typically, the transcribed nucleic acid sequence is a single-stranded RNA molecule. "3' end of a nucleic acid" refers ing to the invention to that end which has a free hydroxy group. in a mmatic representation of double-stranded nucleic acids, in particular DNA, the 3' end is always on the right-hand side. "5' end of a nucleic acid" refers according to the invention to that end which has a free phosphate group. In a diagrammatic representation of -strand c acids, in particular DNA, the 5' end is always on the left-hand side. ' end 5 ' -—P—NNNNNNN—OH—3' 3 ' end 3 ' -HO—NNNNNNN-P--5' "Upstream" describes the relative oning of a first element of a nucleic acid le with respect to a second element of that c acid molecule, wherein both Dnents are comprised in the same nucleic acid molecule, and wherein the first element is located nearer to the 5’ end of the nucleic acid molecule than the second [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg t of that nucleic acid molecule. The second element is then said to be "downstream" of the first element of that nucleic acid molecule. An element that is located "upstream" of a second t can be synonymously referred to as being located "5’" of that second element. For a double-stranded nucleic acid molecule, indications like "upstream" and "downstream" are given with respect to the (+) .

According to the ion, ional linkage" or "functionally linked" relates to a connection within a functional relationship. A nucleic acid is "functionally linked" if it is functionally related to another nucleic acid ce. For example, a promoter is _.\ (D "hunctionaily linked to a coding sequence if it influences transcription of said coding sequence. Functionally tic-"ed nucleic acids are typically adjacent to one another, where appropriate separated by further nucleic acid sequences, and, in particular embodiments, are ribed by RNA polymerase to give a single RNA molecule (common ript).

In particular embodiments, a nucleic acid is functionally linked according to the invention to expression control sequences which may be homologous or heterologous with respect to the nucleic acid.

The term "expression l sequence" comprises according to the invention promoters, ribosome-binding sequences and other control elements which control transcription of a gene or translation of the derived RNA. In particular embodiments of the invention, the expression control sequences can be regulated. The precise structure of expression control sequences may vary depending on the s or cell type but y es 5'—untranscribed and 5'- and 3'-untranslated sequences involved in initiating ription and translation, respectively. More specifically, 5'- untranscribed expression control sequences include a promoter region which encompasses a promoter sequence for transcription control of the functionally linked gene. Expression control sequences may also include er ces or upstream activator sequences. An expression control sequence of a DNA molecule usually includes 5'—untranscribed and 5'— and 3'-untranslated sequences such as TATA box, g sequence, CAAT sequence and the like. An expression control sequDe of alphaviral RNA may include a subgenomic promoter and/or one or more conserved sequence element(s). A specific sion control sequence according [Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg to the present invention is a subgenomic promoter of an alphavirus, as described herein.

The nucleic acid sequences specified herein, in particular transcribable and coding nucleic acid sequences, may be combined with any expression control sequences, in ular promoters, which may be homologous or heterologous to said c acid sequences, with the term ogous" referring to the fact that a nucleic acid ce is also functionally linked naturally to the sion control sequence, and the term ologous" referring to the fact that a nucleic acid sequence is not naturally functionally linked to the expression l sequence.

A transcribable nucleic acid sequence, in particular a nucleic acid sequence coding for a peptide or protein, and an expression l sequence are "functionally" linked to one another, if they are covalently linked to one another in such a way that transcription or expression of the transcribable and in particular coding nucleic acid sequence is under the control or under the influence of the expression control sequence. If the nucleic acid ce is to be translated into a onal peptide or protein, ion of an expression control sequence functionally linked to the coding sequence results in transcription of said coding sequence, without causing a frame shift in the coding sequence or the coding sequence being unable to be translated into the desired peptide or protein.

The term "promoter" or "promoter region" refers to a nucleic acid sequence which controls synthesis of a transcript, e.g. a transcript comprising a coding sequence, by providing a recognition and binding site for RNA polymerase. The promoter region may include further recognition or binding sites for further s involved in regulating transcription of said gene. A promoter may control transcription of a prokaryotic or eukaryotic gene. A promoter may be "inducible" and initiate transcription in response to an inducer, or may be "constitutive" if transcription is not controlled by an inducer. An inducible promoter is sed only to a very small extent or not at all, if an inducer is absent. In the presence of the inducer, the gene is "switched on" or the level of transcription is increased. This is usually mediated by bindDof a specific transcription factor. A specific promoter ing to the present invention is a subgenomic promoter of an alphavirus, as described herein. Other [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg ation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg specific promoters are genomic plus-strand or negative-strand promoters of an alphavirus.

The term "core promoter" refers to a nucleic acid sequence that is comprised by the promoter. The core promoter is typically the minimal portion of the promoter required to properly initiate transcription. The core promoter typically includes the transcription start site and a binding site for RNA rase.

A "polymerase" generally refers to a molecular entity capable of zing the 1O synthesis of a polymeric molecule from ric ng blocks. A "RNA polymerase" is a lar entity capable of catalyzing the synthesis of a RNA molecule from ribonucleotide building blocks. A "DNA polymerase" is a molecular entity capable of catalyzing the synthesis of a DNA molecule from deoxy ribonucleotide building blocks. For 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 te nucleic acid, which is typiCally a DNA molecule. Typically, a RNA polymerase synthesizes a RNA le based on a template nucleic acid, which is either a DNA molecule (in that case the RNA polymerase is a DNA-dependent RNA rase, DdRP), or is a RNA molecule (in that case the RNA polymerase is a RNA-dependent RNA polymerase, RdRP).

A "RNA-dependent RNA polymerase" or "RdRP", is an enzyme that catalyzes the transcription of RNA from an RNA te. In the case of alphaviral pendent RNA polymerase, sequential synthesis of (-) strand complement of genomic RNA and of (+) strand genomic RNA leads to RNA replication. Alphaviral RNA—dependent RNA polymerase is thus synonymously referred to as "RNA replicase". In nature, RNA-dependent RNA polymerases are typically encoded by all RNA viruses except retroviruses. Typical representatives of viruses encoding a RNA-dependent RNA polymerase are alphaviruses.

According to the present ion, "RNA replication" lly refers to an RNA moleG sized based on the nucleotide sequence of a given RNA molecule (template RNA molecule). The RNA molecule that is synthesized may be 9.9.

[Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg cal or complementary to the template RNA molecule. In general, RNA replication may occur via synthesis of a DNA intermediate, or may occur directly by RNA-dependent RNA replication mediated by a RNA-dependent RNA polymerase (RdRP). In the case of alphaviruses, RNA replication does not occur via a DNA intermediate, but is mediated by a RNA-dependent RNA polymerase (RdRP): a template RNA strand (first RNA strand) — or a. part thereof — serves as template for the synthesis of a second RNA strand that is complementary to the first RNA strand or to a part thereof. The second RNA strand — or a part f — may in turn optionally serve as a template for synthesis of a third RNA strand that is complementary to the second RNA strand or to a part thereof. y, the third RNA strand is identical to the first RNA strand or to a part thereof. Thus, RNA- dependent RNA polymerase is capable of directly synthesizing a mentary RNA strand of a template, and of indirectly synthesizing an identical RNA strand (via a complementary intermediate strand).

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

According to the invention, the term "gene" refers to a particular nucleic acid sequence which is responsible for producing one or more ar products and/or for ing one or more intercellular or intracellular functions. More specifically, said term relates to a nucleic acid section (typically DNA; but RNA in the case of RNA viruses) which comprises a nucleic acid coding for a specific protein or a onal or structural RNA molecule.

An "isolated molecule" as used herein, is intended to refer to a molecule which is substantially free of other molecules such as other cellular material. The term "isolated nucleic acid" means according to the invention that the nucleic acid has been (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) inantly produced by cloning, (iii) purified, for example by cleavage and gelelectrophoretic onation, or (iv) synthesized, for example by chemical synthesis.

An isolated c acid is a nucleic acid ble to lation by recombinant techrDes.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg The term "vector" is used here in its most general meaning and comprises any intermediate vehicles for a nucleic acid which, for example, enable said nucleic acid to be introduced into prokaryotic and/or eukaryotic host cells and, where appropriate, to be ated into a genome. Such vectors are preferably replicated and/or expressed in the cell. s comprise plasmids, phagemids, virus genomes, and fractions thereof.

The term "recombinant" in the context of the present ion means "made through genetic engineering". Preferably, a "recombinant objec " such as a recombinant cell in the context of the present invention is not occurring naturally.

The term "naturally occurring" as used herein 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 viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring. The term "found in nature" means "present in nature" and includes known objects as well as s that have not yet been ered and/or isolated from nature, but that may be discovered and/or isolated in the future from a natural source.

According to the invention, the term "expression" is used in its most general meaning and comprises production of RNA, or of RNA and protein. It also comprises partial expression of nucleic acids. rmore, expression may be ent or stable. With t to RNA, the term "expression" or lation" relates to the process in the ribosomes of a cell by which a strand of coding RNA (eg. messenger RNA) directs the ly of a sequence of amino acids to make a peptide or protein.

According to the invention, the term "mRNA" means "messenger-RNA" and relates to a transcript which is lly generated by 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 may be generated by in vitro ription from a DNA template. The in vitro transcription methodology is known to the skilled person. For example, there is a variety of in vitro transcription kits commercially availa. According to the invention, mRNA may be modified by stabilizing modifications and capping.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg According to the ion, the terms "poly(A) sequence" or "poly(A) tail" refer to an uninterrupted or upted sequence of adenylate residues which is typically located at the 3' end of an RNA molecule. An uninterrupted sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical. While a poly(A) sequence is normally not encoded in eukaryotic DNA, but is attached during eukaryotic transcription in the cell nucleus to the free 3‘ end of the RNA by a te-independent RNA polymerase after transcription, the present invention encompasses poly(A) sequences encoded by DNA. _.\ CD According to the invention, the term "primary structure", with nce to a nucleic acid molecule, refers to the linear sequence of nucleotide monomers.

According to the invention, the term "secondary structure", with reference to a c acid molecule, refers to a two-dimensional representation of a nucleic acid le that reflects base pairings; so. in the case of a single-stranded RNA le particularly intramolecular base gs. Although each RNA molecule has only a single polynucleotide chain, the molecule is typically terized by regions of (intramolecular) base pairs. According to the invention, the term "secondary structure" comprises structural motifs including without limitation base pairs, stems, stem loops, bulges, loops such as interior loops and branch loops. The secondary structure of a nucleic acid molecule can be represented by a two- dimensional drawing (planar graph), showing base pairings (for further details on 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 certain RNA les is relevant in the context of the present ion. ing to the invention, secondary structure of a c acid molecule, particularly of a single-stranded RNA molecule, is determined by prediction using the web server for RNA secondary structure prediction (http://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1 .html).

Preferably, according to the invention, "secondary structure", with reference to a nuclenacid molecule, specifically refers to the secondary structure determined by [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg said prediction. The prediction may also be performed or confirmed using MFOLD structure prediction (http://unafold.rna.albany.edu/?q=mfold).

According to the invention, a "base pair" is a structural motif of a secondary structure n two nucleotide bases ate with each other through hydrogen bonds between donor and acceptor sites on the bases. The complementary bases, A:U and G:C, form stable base pairs h hydrogen bonds between donor and acceptor sites on the bases; the A:U and G:C base pairs are called Watson-Crick base pairs.

A weaker base pair (called Wobble base pair) is formed by the bases G and U (GzU).

The base pairs A:U and G:C are called canonical base pairs. Other base pairs like GiU (which occurs fairly eften in RNA) and other rare base-pairs (e.g. A:C; U:U) are called nonical base pairs.

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

According to the invention, the terms "stem loop" or "hairpin" or "hairpin loop", with reference to a nucleic acid molecule, all interchangeably refer to a particular secondary structure of a nucleic acid molecule, typically a single-stranded nucleic acid molecule, such as single-stranded RNA. The particular secondary structure ented by the stem loop consists of a consecutive nucleic acid sequence comprising a stem and a (terminal) loop, also called hairpin loop, wherein the stem is formed by two neighbored entirely or partially complementary sequence elements; which are separated by a short sequence (e.g. 3-10 tides), which forms the loop of the stem-loop structure. The two neighbored entirely or partially complementary sequences may be defined as e.g. stem loop elements stem 1 and stem 2. The stem loop is formed when these two neighbored ly or partially reverse complementary sequences, e.g. stem loop elements stem 1 and stem 2, form base-pairs with each other, leading to a double stranded nucleic acid sequence comprising an unpaired loop at its terminal ending formed by the short ce between stem loop elements stem 1 and stem 2. Thus, a stem loop comprises two stems (stem 1 and stem 2), which — at the level of secondary structure [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg of the nucleic acid molecule — form base pairs with each other, and which — at the level of the primary structure of the nucleic acid molecule — are separated by a short sequence that is not part of stem 1 or stem 2. For illustration, a two-dimensional representation of the stem loop resembles a lollipop-shaped structure. The formation of a stem-loop structure es the presence of a ce that can fold back on itself to form a paired double strand; the paired double strand is formed by stem 1 and stem 2. The stability of paired stem loop elements is typically determined by the length, the number of nucleotides of stem 1 that are capable of forming base pairs (preferably canonical base pairs, more preferably Watson-Crick base pairs) with nucleotides of stem 2, versus the number of nucleotides of stem 1 that are not capable of forming such base pairs with nucleotides of stem 2 tches or bulges). According to the present invention, the l loop length is 3-10 nucleotides, more preferably 4 to 7, nucleotides, such as 4 nucleotides, 5 nucleotides, 6 nucleotides or 7 tides. If a given nucleic acid sequence is characterized by a stem loop, the respective complementary nucleic acid sequence is typically also characterized by a stem loop. A stem loop is lly formed by single-stranded RNA molecules. For example, several stem loops are present in the ’ replication recognition sequence of alphaviral genomic RNA (illustrated in Fig. 1).

According to the invention, "disruption" or "disrupt", with reference to a specific secondary structure of a nucleic acid molecule (e.g. a stem loop) means that the specific ary structure is absent or altered. Typically, a secondary structure may be disrupted as a consequence of a change of at least one nucleotide that is part of the secondary ure. For example, a stem loop may be disrupted by change of one or more nucleotides that form the stem, so that nucleotide pairing is not le.

According to the invention, "compensates for secondary structure tion" 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 ce, which nucleic acid sequence also comprises one or more first nucleotide changes, terized as follows: while the one Gore first nucleotide changes, in the absence of the one or more second nucleotide changes, cause a disruption of the secondary structure of the nucleic acid [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg sequence, the co-occurrence of the one or more first nucleotide changes and the one or more second nucleotide changes does not cause the secondary ure of the nucleic acid to be ted. Co-occurrence means presence of both. the one or more first nucleotide changes and of the one or more second tide changes. lly, the one or more first nucleotide changes and the one or more second nucleotide changes are present er in the same nucleic acid molecule. In a specific embodiment, one or more nucleotide changes that compensate for secondary structure tion is/are one or more nucleotide changes that compensate for one or more nucleotide pairing disruptions. Thus, in one embodiment, "compensating for secondary structure disruption" means "compensating for nucleotide g disruptions", i.e. one or more nucleotide g tions, for example one or more nucleotide pairing disruptions within one or more stem loops. The one or more one or more nucleotide pairing disruptions may have been introduced by the removal of at least one initiation codon. Each of the one or more nucleotide changes that compensates for ary structure disruption is a nucleotide change, which can each be independently selected from a deletion, an addition, a substitution and/or an insertion of one or more nucleotides. In an illustrative example, when the nucleotide pairing A:U has been disrupted by substitution of A to C (C and U are not typically suitable to form a nucleotide pair); then a nucleotide change that compensates for nucleotide pairing disruption may be substitution of U by G, thereby enabling formation of the C:G nucleotide pairing. The tution of U by G thus compensates for the tide pairing disruption. In an alternative example, when the nucleotide pairing A:U has been disrupted by substitution of A to C; then a nucleotide change that compensates for nucleotide pairing disruption may be substitution of C by A, thereby ing formation of the original A:U nucleotide pairing. In general, in the present ion, those nucleotide changes compensating for secondary structure disruption are preferred which do neither restore the original nucleic acid sequence nor create novel AUG triplets. In the above set of examples, the U to G substitution is preferred over the C to A substitution.

According to the invention, the term ary structure", with reference to a nucleic acid Decule, refers to the three dimensional structure of a nucleic acid molecule, as defined by the atomic coordinates.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg According to the invention, a nucleic acid such as RNA, e.g. mRNA, may encode a peptide or protein. Accordingly, a transcribable nucleic acid sequence or a ript thereof may n an open reading frame (ORF) encoding a peptide or protein.

According to the invention, the term "nucleic acid ng a peptide or protein" means that the nucleic acid, if present in the appropriate environment, preferably within a cell, can direct the assembly of amino acids to produce the peptide or protein during the process of translation. Preferably, coding RNA ing to the invention is able to interact with the ar translation machinery allowing translation of the coding RNA to yieid a peptide or protein.

According to the invention, the term "peptide" comprises oligo- and polypeptides and refers to substances which comprise two or more, preferably 3 or more, ably 4 or more, preferably 6 or more, preferably 8 or more, preferably 10 or more, preferably 13 or more, preferably 16 or more, preferably 20 or more, and up to preferably 50, preferably 100 or preferably 150, consecutive amino acids linked to one another via peptide bonds. The term "protein" refers to large peptides, preferably peptides having at least 151 amino acids, but the terms "peptide" and "protein" are used herein usually as synonyms.

The terms "peptide" and "protein" comprise, according to the invention, substances which contain not only amino acid components but also non-amino acid components such as sugars and phosphate structures, and also comprise substances containing bonds such as ester, thioether or disulfide bonds.

According to the invention, the terms "initiation codon" and "start codon" synonymously refer to a codon (base triplet) of a RNA molecule that is potentially the first codon that is translated by a me. Such codon typically encodes the amino acid methionine in eukaryotes and a modified methionine in yotes. The most common initiation codon in eukaryotes and prokaryotes is AUG. Unless specifically stated herein that an tion codon other than AUG is meant, the terms "initiation codoDand "start codon", with nce to an RNA molecule, refer to the codon AUG. According to the invention, the terms "initiation codon" and "start codon" are [Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg also used to refer to a corresponding base triplet of a ibonucleic acid, namely the base triplet ng the tion codon of a RNA. If the initiation codon of messenger RNA is AUG, the base triplet encoding the AUG is ATG. According to the invention, the terms "initiation codon" and "start codon" preferably refer to a functional initiation codon or start codon, i.e. to an initiation codon or start codon that is used or would be used as a codon by a ribosome to start translation. There may be AUG codcns in an RNA molecule that are not used as codons by a ribosome to start translation, 9.9. due to a short distance of the codons to the cap. These codons are not encompassed by the term functional initiation codon or start codon. _.\ C: According to the invention, the terms "start codon of the open reading frame" or "initiation codon of the open reading frame" refer to the base triplet that serves as initiation codon for protein synthesis in a coding sequence, eg. in the coding sequence of a nucleic acid molecule found in nature. In an RNA le, the start codon of the open reading frame is often preceded by a 5' untranslated region (5’- UTR), although this is not strictly required.

According to the ion, the terms e start codon of the open reading frame" or "native initiation codon of the open reading frame" refer to the base triplet that serves as initiation codon for protein synthesis in a native coding sequence. A native coding sequence may be e.g. the coding senuenc of a nucleic acid molecule found in nature. In some embodiments, the present invention provides variants of c acid molecules found in nature, which are 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 molecule). ing to the invention, "first AUG" means the most upstream AUG base triplet of a messenger RNA molecule, preferably the most upstream AUG base triplet of a messenger RNA molecule that is used or would be used as a codon by a ribosome to start translation. Accordingly, "first ATG" refers to the ATG base triplet of a coding DNA sequence that s the first AUG. In some instances, the first AUG of a mRNA molecule is the start codon of an open reading frame, i.e. the codon that is usednstart codon during ribosomal n synthesis.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg According to the invention, the terms ises the removal" or "characterized by the removal" and similar terms, with reference to a certain element of a nucleic acid variant, mean that said certain element is not functional or not present in the nucleic acid variant, compared to a reference nucleic acid molecule. Without limitation, a removal can consist of deletion of all or part of the certain element, of substitution of all or part of the certain element, or of tion of the functional or structural properties of the certain element. The removai of a functional element of a c acid sequence requires that the function is not ted at the on of the nucleic acid variant comprising the removal. For example, a RNA variant characterized by -A (D the l of a certain initiation coden requires that ribosomal n synthesis is net initiated at the position of the RNA variant c .aractertzed by the removal. The removal of a structural element of a nucleic acid sequence requires that the structural element is not present at the position of the nucleic acid variant comprising the removal. For example, a RNA t terized by the removal of a certain AUG base triplet, i.e. of a AUG base triplet at a certain position, may be terized, e.g. by deletion of part or all of the certain AUG base triplet (e.g. AAUG), or by substitution of one or more nucleotides (A, U, G) of the certain AUG base triplet by any one or more different nucleotides, so that the resulting tide sequence of the variant does not comprise said AUG base triplet. Suitable substitutions of one nucleotide are those that convert the AUG base triplet into a GUG, CUG or UUG base triplet, or into a AAG, ACG or AGG base triplet, or into a AUA, AUC or AUU base triplet. Suitable substitutions of more nucleotides can be selected accordingly.

According to the invention, the term "alphavirus" is to be understood broadly and includes any virus particle that has teristics of alphaviruses. Characteristics of alphavirus include the presence of a (+) stranded RNA which encodes genetic information suitable for replication in a host cell, ing RNA polymerase activity.

Further characteristics of many alphaviruses are described e.g. in Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491-562. The term "alphavirus" includes alphavirus found in nature, as well as any variant or derivative thereof. In some embodiments, a t or derivative is not found in nature.

In orDambodiment, the alphavirus is an alphavirus found in nature. Typically, an alphavirus found in nature is infectious to any one or more otic organisms, [Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg such as an animal (including a vertebrate such as a human, and an arthropod such as an insect).

An alphavirus found in nature is preferably selected from the group consisting of the ing: Barmah Forest virus complex (comprising Barmah Forest virus); Eastern equine encephalitis complex {comprising seven antigenic types of Eastern equine encephalitis virus); Middelburg virus complex (comprising Middelburg virus); Ndumu virus complex ising Ndumu virus); Semliki Forest virus complex (comprising Bebaru virus, Chikungunya virus, Mayaro virus and its subtype Una virus, O’Nyong 1O Nyong virus, and its subtype lgbo-Ora virus, Ross River virus and its subtypes Bebaru virus, Getah virus, Sagiyama virus, Semliki Forest virus and its subtype Me Tri virus); Venezuelan equine encephalitis complex (comprising 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, elan equine encephalitis virus); Western equine encephalitis x (comprising Aura virus, Babanki virus, Kyzylagach virus, Sindbis virus, Ockelbo virus, Whataroa virus, Buggy Creek virus, Fort Morgan virus, Highlands J virus, Western equine encephalitis virus); and some unclassified viruses including Salmon pancreatic disease virus; Sleeping Disease virus; Southern elephant seal virus; Tonate virus.

More preferably, the alphavirus is selected from the group consisting of Semliki Forest virus complex (comprising the virus types as indicated above, including Semliki Forest , Western equine encephalitis x (comprising the virus types as indicated above, ing Sindbis virus), Eastern equine encephalitis virus (comprising the virus types as indicated above), Venezuelan equine encephalitis complex ising the virus types as indicated above, including Venezuelan equine encephalitis virus).

In a further preferred embodiment, the alphavirus is Semliki Forest virus. in an ative further red embodiment, the alphavirus is s virus. In an alternative further preferred embodiment, the alphavirus is Venezuelan equine encephalitis virus. ln son embodiments of the present invention, the alphavirus is not an alphavirus found in . Typically, an alphavirus not found in nature is a variant or derivative [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg of an alphavirus found in , that is distinguished from an alphavirus found in nature by at least one mutation in the nucleotide sequence, i.e. the genomic RNA.

The mutation in the nucleotide sequence may be selected from an insertion, a substitution or a deletion of one or more nucleotides, compared to an alphavirus found in nature. A mutation in the nucleotide sequence may or may not be associated with a on in a polypeptide or protei. encoded by the nucleotide sequence. For example, an alphavirus not found in nature may be an attenuated alphavirus. An attenuated alphavirus not found in nature is an alphavirus that typically has at least one mutation in its nucleotide ce by which it is 1O distinguished from an alphavirus found in , and that is either not infectious at all, or that is infectious but has a lower disease-producing ability or no diseaseproducing ability at all. As an illustrative example, T083 is an attenuated alphavirus that is distinguished 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). s of the alphavirus genus may also be classified based on their relative clinical features in : alphaviruses associated primarily with encephalitis, and alphaviruses associated primarily with fever, rash, and thritis.

The term "alphaviral" means found in an alphavirus, or originating from an alphavirus or derived from an alphavirus, eg. by genetic engineering.

According to the invention, "SFV" stands for Semliki Forest virus. According to the invention, "SIN" or "SINV" stands for s virus, According to the invention, "VEE" or "VEEV" stands for elan equine encephalitis virus.

According to the invention, the term "of an alphavirus" refers to an entity of origin from an alphavirus. For ration, a protein of an alphavirus may refer to a n that is found in alphavirus and/or to a protein that is encoded by alphavirus; and a nucleic acid ce of an alphavirus may refer to a nucleic acid sequence that is found in alphavirus and/or to a nucleic acid ce that is encoded by alphavirus.

Preferably, a nucleic acid sequence "of an alphavirus" refers to a nucleic acid sequae "of the genome of an alphavirus" and/or "of genomic RNA of an alphavirus".

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg According to the invention, the term "alphaviral RNA" refers to any one or more of alphaviral genomic RNA (Le (+) strand), complement of iral genomic RNA (Le. (-) strand), and the subgenomic transcript (i.e. (+) strand), or a fragment of any thereof.

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

According to the invention, the term "native alphavirus sequence" and. similar terms lly refer to a (e.g. nucleic acid) ce of a naturaiiy occurring alphavirus (alphavirus found in nature). in some embodiments, the term e alphavirus sequence" also includes a sequence of an attenuated alphavirus.

According to the invention, the term "5’ replication recognition ce" preferably refers to a continuous c acid sequence, preferably a ribonucleic acid sequence, that is identical or homologous to a 5’ fragment of the alphavirus genome. The "5’ replication recognition sequence" is a nucleic acid sequence that can be recognized by an alphaviral replicase. The term 5’ replication recognition sequence includes native 5’ replication recognition sequences as well as functional equivalents thereof, such as, e..,g functional variants at a 5’ replication recognition sequence of alphavirus found in nature. According to the ion, functional equivalents include derivatives of 5’ replication ition sequences characterized by the l of at least one initiation codon as described herein. The 5’ repiication recognition sequence is required for synthesis of the (-) strand complement of irus genomic RNA, and is required for synthesis of (+) strand viral genomic RNA based on a (-) strand template. A native 5’ replication recognition ce typically encodes at least the N-terminal fragment of nsP1; but does not comprise the entire open reading frame encoding nsP1234. In view of the fact that a native 5’ replication recognition sequence typically encodes at least the N-terminal nt of nsP1, a native 5’ replication ition sequence typically ses at least one initiation codon, typically AUG. In one embodiment, the 5’ replication recognition sequence comst conserved sequence element 1 of an alphavirus genome (CSE 1) or a variant f and conserved sequence element 2 of an alphavirus genome (CSE 2) [Annotation] mpg None set by mpg ation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg or a variant thereof. The 5’ replication recognition sequence is lly capable of forming four stem loops (SL), i.e. SL1, SL2, SL3, SL4. The numbering of these stem loops begins at the 5’ end of the 5’ replication recognition sequence.

According to the invention, the term "at the 5' end of an alphavirus" refers to the 5' end of the genome of an alphavirus. A nucleic acid sequence at the 5‘ end of an alphavirus encompasses the tide located at the 5’ terminus of alphavirus genomic RNA, plus optionally a consecutive sequence of further nucleotides. In one embodiment, a nucleic acid sequence at the 5’ end of an alphavirus is identical to the _; CD 5’ replication recognition sequence of the irus genome.

The term "conserved sequence element" or "CSE" refers to a nucleotide sequence found in alphavirus RNA. These sequence elements are termed "conserved" because orthologs are present in the genome of different alphaviruses, and orthologous CSEs of different alphaviruses preferably share a high percentage of sequence identity and/or a similar secondary or tertiary structure. The term CSE includes CSE 1, CSE 2, CSE 3 and CSE 4.

According to the invention, the terms "CSE 1" or "44-nt CSE" synonymously refer to a nucleotide sequence that is ed for (+) strand synthesis from a (-) strand template. The term "CSE 1" refers to a sequence on the (+) strand; and the complementary sequence of CSE 1 (on the (—) strand) ons as a promoter for (+) strand sis. Preferably, the term CSE 1 includes the most 5’ nucleotide of the alphavirus genome. CSE 1 typically forms a conserved stem-loop structure. Without wishing to be bound to a particular , it is believed that, for CSE 1, the secondary structure is more important than the primary structure, i.e. the linear sequence. in genomic RNA of the model alphavirus Sindbis virus, CSE 1 consists of a consecutive sequence of 44 nucleotides, which is formed by the most 5’ 44 tides of the c RNA (Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491-562).

According to the invention, the terms "CSE 2" or "51-nt CSE" synonymously refer to a nucleDe sequence that is required for (-) strand synthesis from a (+) strand template. The (+) strand te is typically alphavirus genomic RNA or an RNA [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg replicon (note that the subgenomic RNA transcript, which does not comprise CSE 2, does not function as a te for H strand synthesis). In irus genomic RNA, CSE 2 is typically localized within the coding sequence for nsP1. In genomic RNA of the model alphavirus Sindbis virus, the 51-nt CSE is located at nucleotide positions 155—205 of genomic RNA v et al., 2001, RNA, vol. 7, pp. 1638-1651). CSE 2 forms typically two conserved stem loop structures. These stem loop structures are designated as stem loop 3 (SL3) and stem loop 4 (SL4) because they are the third and fourth conserved stem loop, respectively, of alphavirus genomic RNA, counted from the 5’ end of alphavirus genomic RNA. Without wishing to be bound to a particular theory, it is ed that, for CSE 2, the secondary structure is more important than the primary structure, i.e. the linear sequence.

According to the ion, the terms "CSE 3" or "junction sequence" mously refer to a nucleotide sequence that is derived from iral genomic RNA and that comprises the start site of the omic RNA. The complement of this sequence in the (-) strand acts to promote subgenomic RNA transcription. In alphavirus genomic RNA, CSE 3 typically overlaps with the region encoding the C-terminal fragment of nsP4 and extends to a short non-coding region d upstream of the open reading frame encoding the structural proteins. According to Strauss & Strauss biol.

Rev., 1994, vol. 58, pp. 491-562), CSE 3 is characterized by the consensus sequence ACCUCUACGGCGGUCCUAAAUAGG (SEQ ID NO: 1; sus junction sequence (consensus CSE 3); underlined nucleotides represent the first five nucleotides of the subgenomic transcript) In one embodiment of the present ion, CSE 3 consists 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 °/o or more, or 99 % or more.

According to the invention, the terms "CSE 4" or "19-nt conserved sequence" or "19- nt CSDsynonymously refer to a nucleotide sequence from alphaviral genomic RNA, immediately upstream of the po|y(A) sequence in the 3' untranslated region of the [Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg ation] mpg ed set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg irus genome. CSE 4 lly consists of 19 consecutive nucleotides. Without wishing to be bound to a particular theory, CSE 4 is understood to function as a core promoter for initiation of (-) strand synthesis (José et al., Future Microbiol., 2009, vol. 4, pp. 6); 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).

According to Strauss & Strauss, CSE 4 is characterized by the conserved sequence AUUUUGUUUUUAAUAUUUC (SEQ ID NO: 2; 19 nt conserved ce) In one embodiment of the present invention, CSE 4 consists 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.

According to the invention, the term "subgenomic promoter" or "SGP" refers to a c acid ce upstream (5‘) of a nucleic acid sequence (e.g. coding sequence), which controls transcription of said nucleic acid sequence by providing a recognition and binding site for RNA polymerase, typically RNA-dependent RNA polymerase, in particular functional alphavirus ructural n. The SGP may include further recognition or binding sites for further factors. A subgenomic promoter is typically a genetic element of a positive strand RNA virus, such as an alphavirus. A subgenomic promoter of alphavirus is a nucleic acid sequence comprised in the viral genomic RNA= The subgenomic er is generally characterized in that it allow initiation of the transcription (RNA synthesis) in the presence of an RNA-dependent RNA polymerase, e.g. functional alphavirus non-structural protein. A RNA (-) strand, i.e. the complement of alphaviral genomic RNA, serves as a template for synthesis of a (+) strand omic transcript, and synthesis of the (+) strand subgenomic transcript is lly initiated at or near the subgenomic promoter. The term "subgenomic promoter" as used herein, is not confined to any particular localization in a nucleic acid comprising such subgenomic promoter. In some embodiments, the SGP is identical to CSE 3 or overlaps with CSE 3 or comprises CSE 3.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg The terms nomic transcript" or "subgenomic RNA" mously refer to a RNA molecule that is obtainable as a result of transcription using a RNA molecule as promoter that ls transcription of the subgenomic transcript. The subgenomic transcript is obtainable in the presence of an RNA-dependent RNA polymerase, in ar functional alphavirus non-structural protein. F-c-r instance, the term nomic transcript" may refer to the RNA transcript that is ed in a cell infected by an alphavirus, using the (-) strand ment of alphavirus genomic RNA as template. However, the term "subgenomic transcript", as used herein, is not 1O limited thereto and also includes transcripts obtainable by using heterologous RNA as template. For example, subgenomic transcripts are also obtainable by using the (- ) strand complement of SGP-containing ons according to the present ion as template. Thus, the term "subgenomic transcript" may refer to a RNA molecule that is obtainable by transcribing a fragment of alphavirus genomic RNA, as well as to a RNA le that is obtainable by transcribing a fragment of a replicon according to the present invention.

The term "autologous" is used to describe anything that is d from the same subject. For example, "autologous cell" refers to a cell derived from the same subject.

Introduction of autologous cells into a subject is advantageous because these cells overcome the immunological barrier which otherwise results in rejection.

The term "allogeneic" is used to describe anything that is derived from different individuals of the same s. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical.

The term "syngeneic" is used to describe anything that is derived from individuals or tissues having identical genotypes, i.e., identical twins or animals of the same inbred strain, or their tissues or cells.

The term "heterologous" is used to describe something ting of multiple different elements. As an example, the introduction of one individual's cell into a different Dl constitutes a heterologous transplant. A logous gene is a gene derived from a source other than the subject.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg The ing provides specific and/or preferred variants of the individual features of the invention. The present invention also contemplates as particularly preferred ments those embodiments, which are generated by ing two or more of the ic and/or preferred variants described for two or more of the es of the present invention.

RNA replicon In a first aspect, the present invention provides a replicon comprising a 5' ation recognition sequence, wherein the 5‘ replication recognition sequence is terized in that it com-prises the r‘moval of at least one initiation codon compared to a native alphavirus 5! replication recognition sequence. The replicon is ably a RNA replicon.

The 5' replication recognition sequence that is characterized in that it comprises the removal of at least one initiation codon compared to a native alphavirus 5' replication recognition sequence, ing to the present invention, can be referred to herein as "modified 5' replication recognition sequence" or "5’ replication ition sequence according to the invention". As described herein below, the 5’ replication recognition sequence according to the invention may optionally be characterized by the presence of one or more additional nucleotide changes.

A nucleic acid construct that is capable of being replicated by a replicase, preferably an ira! replicase, is termed replicon. According to the invention, the term "replicon" s a RNA le that can be replicated by RNA-dependent RNA polymerase, yielding - without DNA intermediate - one or multiple identical or essentially identical copies of the RNA replicon. "Without DNA intermediate" means that no deoxyribonucleic acid (DNA) copy or complement of the replicon is formed in the process of forming the copies of the RNA replicon, and/or that no deoxyribonucleic acid (DNA) molecule is used as a template in the process of forming the copies of the RNA replicon, or complement thereof. The ase function is typically provided by functional alphavirus non-structural protein.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg According to the invention, the terms "can be ated" and "capable of being ated" generally describe that one or more identical or essentially identical copies of a nucleic acid can be prepared. When used together with the term "replicase", such as in "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, eg. a RNA replicon, with respect to a replicase. These functional characteristics comprise at least one of (i) the replicase is e of recognizing the replicon and (ii) the replicase is e of acting as RNA-dependent RNA polymerase (RdRP). Preferably, the replicase is capable of both (i) recognizing -3 CD the on and (ii) acting as RNA-dependent RNA polymerase.

The expression "capable of recognizing" describes that the replicase is capable of physically associating with the replicon, and preferably, that the replicase is capable of binding to the replicon, typically non—covalently. The term "binding" can mean that the replicase has the capacity of binding to any one or more of a conserved sequence elemen. 1 (CSE 1) or complementary sequence thereof (if comprised by the replicon), conserved sequence element 2 (CSE 2) or complementary ce f (if comprised by the replicon), conserved sequence element 3 (CSE 3) or complementary sequence thereof (if comprised by the replicon), conserved sequence element 4 (CSE 4) or complementary sequence thereof (if comprised by the replicon). Preferably, the replicase is capable of binding to CSE 2 [Le to the (+) strand] and/or to CSE 4 [Le to the (+) strand], or of binding to the ment of CSE 1 [Le to the (-) strand] and/orto the complement of CSE 3 [Le to the (-) strand].

The expression "capable of acting as RdRP" means that the replicase is capable to catalyze the synthesis of the (-) strand complement of iral genomic (+) strand RNA, wherein the (+) strand RNA has te function, and/or that the replicase is capable to catalyze the synthesis of (+) strand alphaviral genomic RNA, wherein the (-) strand RNA has template function. In general, the expression "capable of acting as RdRP" can also include that the ase is capable to catalyze the synthesis of a (+) strand subgenomic ript wherein a (—) strand RNA has template function, and n sis of the (+) strand subgenomic transcript is typically initiated at an alphas subgenomic promoter.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg The sions le of binding" and "capable of acting as RdRP" refer to the capabiiity at normal physiological conditions. In particular, they refer to the conditions inside a cell, which expresses functional alphavirus non-structural protein or which has been transfected with a nucleic acid that codes for functional alphavirus non- structural protein. The cell is preferably a otic cell. The capability of binding and/or the capability of acting as RdRP can be experimentally tested, e.g. in a cell- free in vitro system or in a eukaryotic cell. aiiy, said eukaryotic cell is a cell from a species to which the particular alphavirus that represents the origin of the replicase is infectious. For example, when the alphavirus replicase from a particular -.\ CD aiphavirus is used that is infectious to humans, the normal physiologicai conditions are conditions in a huma": cell. More preferably, the eukaryotic cell (in one example human cell) is from the same tissue or organ to which the ular alphavirus that represents the origin of the replicase is infectious.

According to the invention, red 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 irus sequence.

In one embodiment, the RNA replicon comprises a 3‘ replication recognition sequence. A 3’ replication recognition sequence is a nucleic acid sequence that can be recognized by functional alphavirus non-structural n. in other words, functional alphavirus non-structural 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 on does not comprise a poly(A) tail), or immediately upstream of the poly(A) tail (if the replicon comprises a poly(A) tail). In one embodiment, the 3’ replication recognition sequence ts of or comprises CSE 4.

In one embodiment, the 5' replication recognition sequence and the 3' replication recognition sequence are capable of directing replication of the RNA replicon according to the present invention in the presence of functional alphavirus non- ural protein. Thus, when present alone or ably er, these recognition sequnes direct replication of the RNA replicon in the presence of functional irus non-structural protein.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg It is preferable that a functional irus non-structural n is provided in cis (encoded as protein of interest by an open reading frame on the replicon) or in trans ed as protein of interest by an open reading frame on a separate replicase construct as described in the second aspect), that is e of recognizing both the modified 5’ replication recognition sequence and the 3’ replication ition sequence of the replicon. In one embodiment, this is achieved when the 3’ replication recognition sequence is native to the alphavirus from which the functional alphavirus non-structural protein is derived, and when the modified 5’ replication recognition 1O sequence is a variant of the 5’ replication recognition sequence that is native to the alphavirus from which the onal irus non-structural protein is derived.

Native means that the natural origin 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 non-structural protein is derived, provided that the functional alphavirus non-structural protein is capable of izing both the modified 5’ replication recognition sequence and the 3' ation recognition sequence of the replicon. In other words, the functional alphavirus non-structural protein is compatible to the modified 5’ replication recognition sequence and the 3' replication ition sequence. When a non-native functional alphavirus non-structural protein is capable of recognizing a respective sequence or sequence element, the functional alphavirus non-structural protein is said to be compatible -virus compatibility). Any combination of (3’l5’) replication recognition sequences and CSEs, respectively, with functional alphavirus ruc.ural protein is possible as long as crcss—virus ibility exists. Cross-virus compatibility can readily be tested by the skilled person working the present invention by ting a functional alphavirus non- structural protein to be tested together with an RNA, wherein the RNA has 3’- and (optionally ed) 5’ replication recognition sequences to be tested, at conditions suitable for RNA replication, e.g. in a le host cell. If replication occurs, the (3’/5’) replication recognition sequences and the functional alphavirus non-structural n are determined to be compatible.

The Doval of at least one initiation codon provides several advantages over prior art trans-replicons (such as, e.g. represented by "Template RNA WT-RRS" of Fig. 1).

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg Absence of an initiation codon in the nucleic acid sequence encoding nsP1* will typically cause that nsP1* (N-terminal fragment of nsP1) is not ated. Further, ("Transgene") is the most upstream open g frame accessible to the ribosome; thus when the replicon is present in a cell, translation is initiated 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. 751): replicons according to Spuul et al. direct the sion of the inal portion of nsP1, a peptide of 74 amino acids. It is also 1O known from the prior art that construction of RNA replicons from full-length virus s is not a trivial matter, as certain mutations can render RNA incapable of being replicated ( that is ant for replication of alphavirus affects efficiency of replication (Kamrud et al., 2010, J. Gen. Virol., vol. 91, pp. 1723-1727).

The advantage over prior art cis-replicons is that removal of at least one initiation codon les the coding region for the alphaviral non-structural protein from the ’ replication recognition sequence. This enables a further engineering of cis- replicons e.g. by exchanging the native 5’ replication recognition sequence to an artificial sequence, a mutated sequence, or a heterologous sequence taken from another RNA virus. Such sequence manipulation in prior art cis—replicons are restricted by the amino acid sequence of nsP1. Any point on, er clusters of point mutations, would require experimental assessment whether replication is affected and small insertions or deletion leading to frame shift mutations are impossible due to their detrimental effect on the n.

The removal of at least one initiation codon ing to the present invention can be achieved by any le method known in the art. For example, a suitable DNA molecule encoding the replicon according to the invention, i.e. characterized by the removal of an initiation codon, can be designed in silica, and subsequently 3O synthesized in vitro (gene synthesis); alternatively, a le DNA molecule may be obtained by site-directed mutagenesis of a DNA sequence encoding a replicon. In any case, the respective DNA molecule may serve as template for in vitro transntion, thereby providing the replicon according to the invention.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg The removal of at least one initiation codon compared to a native alphavirus 5' replication recognition ce is not ularly limited and may be selected from any nucleotide cation, including substitution of one or more nucleotides (including, on DNA level, a substitution of A and/or T and/or G of the initiation codon); deletion of one or more nucleotides (including, on DNA level, a deletion of A and/or T and/or G of the initiation codon), and insertion of one or more nucleotides (including, on DNA level, an insertion of one or more tides between A and T and/or between T and G of the initiation codon). Irrespective of whether the nucleotide modification is a substitution, an insertion or a deletion, the nucleotide modification must not result in the ion of a new initiation codon (as an rative example: an insertion 9* "A love m II, UH. l‘l’"\ Lil, IIIC (D0-.- :5ct be an insertion of an ATG).

The 5' replication recognition sequence of the RNA replicon that is characterized by the removal of at least one initiation codon (i.e. the modified 5' replication recognition sequence according to the present invention) is preferably a variant of a 5' replication recognition sequence of the genome of an alphavirus found in nature. In one embodiment, the modified 5' replication recognition sequence according to the t 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 ably 95 % or more, to the 5' replication recognition sequence of the genome of at least one alphavirus found in nature.

In one embodiment, the 5' replication recognition sequence of the RNA replicon that is characterized by the removal of at least one initiation codon comprises a ce homologous to about 250 nucleotides at the 5' end of an alphavirus, i.e. at the 5’ end of the alphaviral genome. In a preferred embodiment, it comprises a sequence homologous to about 250 to 500, preferably about 300 to 500 nucleotides at the 5' end of an alphavirus, i.e. at the 5’ end of the alphaviral genome. "At the 5’ end of the alphaviral genome" means a c acid sequence beginning at, and including, the most upstream nucleotide of the alphaviral genome. In other words, the most upstream nucleotide of the alphaviral genome is ated nucleotide no. 1, and eg. "250 nucleotides at the 5' end of the alphaviral genome" means nucleotides 1 to 250 aha alphaviral genome. In one ment, the 5‘ replication recognition ce of the RNA replicon that is characterized by the removal of at least one [Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg initiation codon is characterized by a degree of sequence identity of 80 % or more, preferably 85 % or more, more preferably 90 % or more, even more ably 95 % or more, to at least 250 nucleotides at the 5’ end of the genome of at least one alphavirus found in nature. At least 250 nucleotides es e.g. 250 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides.

The 5' replication recognition sequence of an alphavirus found in nature is typically characterized by at least one initiation codon and/or by conserved secondary structural motifs. For example, the native 5' replication ition sequence of _.\ CD CDemliki Forest virus (SFV) comprises five ic AUG base triplets, corresponding to ATG base triplets in the DNA of SEQ ID NO: 4 (see Example 1). According to Frolov et ai. (2001, RNA, vol. 7, pp. 1638-1651) analysis by MFOLD revealed that the native 5’ replication recognition ce of Semliki Forest virus is ted to form four stem loops (SL), termed stem loops 1 to 4 (SL1, SL2, SL3, SL4). ing to Frolov et al., analysis by MFOLD revealed that also the native 5' replication recognition sequence of a different alphavirus, Sindbis virus, is predicted to form four stem loops: SL1, SL2, SL3, SL4. ln Example 1 of the present invention, the predicted presence of four stem loops in the native 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) It is known that the 5’ end of the iral genome comprises sequence elements that enable replication of the alphaviral genome by functional alphavirus non- structural protein. ln one embodiment of the t invention, the 5‘ replication recognition sequence of the RNA replicon comprises a sequence homologous to conserved sequence element 1 (CSE 1) and/or a sequence homologous to conserved sequence element 2 (CSE 2) of an alphavirus.

Conserved sequence element 2 (CSE 2) of irus genomic RNA typically is represented by SL3 and SL4 which is preceded by SL2 comprising at least the native initiatncodon that encodes the first amino acid residue of alphavirus non-structural protein nsP1. In this description, however, in some embodiments, the conserved [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg ce t 2 (CSE 2) of alphavirus genomic RNA refers to a region spanning from SL2 to SL4 and comprising the native initiation codon that encodes the first amino acid residue of alphavirus non-structural protein nsP1. In a preferred embodiment, the RNA replicon comprises CSE 2 or a sequence homologous to CSE 2. In one embodiment, the RNA replicon comprises a sequence homologous to CSE 2 that is preferably characterized by a degree of sequence identity of 80 °/o or more, preferably 85 % or more, more preferably 90 % or more, even more preferably 95 % or more, to the sequence of CSE 2 of at least one alphavirus found in nature. _.\ CD I". a preferred embodiment, the 5 replication ition sequence comprises a sequence that is homologous to CSE 2 of an alphavirus. The CSE 2 of an alphavirus may comprise a fragment of an open reading frame of a non-structural protein from an alphavirus.

Thus, in a preferred embodiment, the RNA replicon is terized in that it ses a sequence homologous to an open reading frame of a non-structural protein or a fragment thereof from an alphavirus. The sequence homologous to an open reading frame of a non-structural protein or a fragment thereof is lly a variant of an open g frame of a non-structural protein or a fragment thereof of an alphavirus found in nature. In one embodiment, the sequence gous to an open reading frame of a non—structural n or a fragment thereof 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 an open g frame of a non-structural protein or a fragment thereof of at least one alphavirus found in nature.

In a more preferred embodiment, the sequence homologous to an open reading frame of a ructural protein that is comprised by the replicon of the present invention does not comprise the native initiation codon of a non-structural protein, and more preferably does not comprise any initiation codon of a non-structural protein. In a preferred embodiment, the sequence homologous to CSE 2 is characterized by the removal of all initiation codons compared to a native alphavirus CSEGequence. Thus, the sequence homologous to CSE 2 does preferably not comprise any initiation codon.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg ation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg When the sequence homologous to an open reading frame does not comprise any initiation codon, the sequence homologous to an open reading frame is not itself an open reading frame since it does not serve as a template for translation.

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

In a preferred ment, the sequence homologous to an open reading frame of a non-structural n or a fragment thereof from an alphavirus is characterized in that it comprises the removal of at least the native start codon of the open reading frame of a non-structural protein. Preferably, it is characterized in that it comprises the removal of at least the native start codon of the open reading frame encoding nsP1.

The native start codon is the AUG base triplet at which translation on ribosomes in a host cell begins when an RNA is present in a host cell. In other words, the native start codon is the first base triplet that is translated during ribosomal protein synthesis, e.g. in a host cell that has been inoculated with RNA comprising the native start codon. In one embodiment, the host cell is a. cell from a otio species that is a natural host of the specific alphavirus that comprises the native irus 5' replication recognition sequence. In a preferred embodiment, the host cell is a BHK21 cell from the cell line "BHK21 [C13] (ATCC® CCL10TM)", available from American Type Culture Collection, Manassas, Virginia, USA.

The s of many alphaviruses have been fully ced and are publically accessible, and the sequences of ructural proteins d by these genomes are publically ible as well. Such sequence information allows to determine the nativDart codon in silico.

[Annotation] mpg None set by mpg ation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg For illustration, in Example 1, the DNA base triplet corresponding to the native start codon of SFV nsP1 is described. in one embodiment, the native start codon is sed by a Kozak sequence or a functionally equivalent sequence. The Kozak sequence is a sequence initially described by Kozak (1987, Nucleic Acids Res, vol. 15, pp. 8125—8148). The Kozak sequence on an mRNA molecule is recognized by the me as the translational start site. According to this reference, the Kozak sequence comprises an AUG start codon, immediately followed by a highly conserved G nucleotide: MG (see also 1O initiation codon in the DNA sequence according to SEQ lD NO: 4 (Example 1)). in particular, it was bed by this reference that a Kozak sequence may be identified by (gcc)gccRcch (SEQ ID NO: 6), as follows: (i) lower case letters denote the most common base at a position where the base can nevertheless vary; (ii) upper case 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 significance; (v) the underlined AUG base t represents the start codon. in one embodiment of the present invention, the sequence homologous to an open reading frame of a ructural protein or a fragment thereof from an alphavirus is terized in that it comprises the removal of an initiation codon that is part of a Kozak ce. in one embodiment of the present invention, the 5' replication recognition sequence of the replicon is characterized by the removal of at least all those initiation codons, which, at RNA level, are part of an AUGG ce. in a preferred embodiment, the ce homologous to an open reading frame of a ructural protein or a fragment thereof from an alphavirus is characterized in that it comprises the l of one or more initiation codons other than the native start codon of the open reading frame of a non-structural protein. In a more preferred embodiment, said nucleic acid sequence is additionally characterized by the removal of the native start codon. For example, in addition to the removal of the native start codon, any one or two or three or four or more than four (e.g. five) initiation codons may Demoved.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg If the replicon is characterized by the l of the native start codon, and optionally by the removal of one or more initiation codons other than the native start codon, of the open reading frame of a non-structural protein, the sequence homologous to an open reading frame is not itself an open reading frame since it does not serve as a template for translation.

The one or more initiation codon other than the native start codon that is removed, preferably in addition to removal of the native start codon, is preferably selected from an AUG base triplet that has the potential to initiate translation. An AUG base triplet that has the potential to initiate translation may be referred to as "potential initiation . Whether a given AUG base t has the potential to initiate transiation can be determined in siiico or in a ceii-based in vitro assay.

In one ment, it is determined in silico whether a given AUG base triplet has the ial to initiate translation: in that embodiment, the nucleotide sequence is examined, and an AUG base triplet is determined to have the potential to initiate translation if it is part of an AUGG sequence, ably part of a Kozak sequence (SEQ lD NO: 6).

In one ment, it is determined in a cell-based in vitro assay r a given AUG base triplet has the potential to initiate translation: a RNA replicon characterized by the removal of the native start codon and comprising the given AUG base triplet downstream of the position of the l of the native start codon is introduced into a host cell. In one embodiment, the host cett is a cell from a eukaryotic species that is a natural host of the specific alphavirus that ses the native alphavirus 5' replication recognition sequence. In a preferred embodiment, the host cell is a BHK21 cell from the cell line "BHK21 [C13] (ATCC® CCL10TM)", available from American Type Culture Collection, Manassas, Virginia, USA. It is preferable that no further AUG base triplet is present between the position of the removal of the native start codon and the given AUG base triplet. If, following transfer of the RNA replicon - characterized by the removal of the native start codon and comprising the given AUG base t - into the host cell, translation is ted at the given AUG base triplet, the a 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 [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg ed set by mpg the art. For example, the replicon may , downstream of the given AUG base triplet and in-frame with the given AUG base triplet, a tag that facilitates detection of the translation t (if any), e.g. a myc-tag or a HA-tag; r or not an expression product having the encoded tag is present may be ined e.g. by Western Blot. In this embodiment, it is preferable that no further AUG base triplet is present between the given AUG base triplet and the nucleic acid sequence ng the tag. The cell-based in vitro assay can be performed individually for more than one given AUG base triplet: in each case, it is preferable that no further AUG base triplet is present between the position of the removal of the native start codon and the given -_\ (3 AUG base t. This can be achieved by removing all A' G base triplets (if any) between the position of the removal of the native start codon and the given AUG base triplet. y, the given AUG base triplet is the first AUG base triplet downstream of the position of the removal of the native start codon.

Preferably, the replicon according to the present invention is characterized by the removal of all potential initiation codons that are downstream of the position of the removal of the native start codon and that are located within the open reading frame of alphavirus non-structural protein or of a fragment thereof. Thus, according to the invention, the 5’ replication recognition sequence preferably does not comprise an open reading frame that can be translated to protein. in a preferred embodiment, the 5’ replication recognition ce of the RNA replicon according to the ion is characterized by a secondary structure that is equivalent to the secondary structure of the 5’ replication recognition sequence of alphaviral genomic RNA. In a preferred embodiment, the 5’ ation recognition sequence of the RNA replicon according to the invention is characterized by a predicted secondary structure that is equivalent to the predicted secondary ure of the 5’ replication recognition sequence of alphaviral genomic RNA. According to the present invention, the secondary structure of an RNA molecule is preferably predicted by the web server for RNA secondary structure prediction http://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1lPredict1 .html.

By cDaring the secondary structure or predicted secondary structure of a 5’ replication recognition sequence of an RNA replicon characterized by the removal of [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg at least one tion codon compared to the native alphavirus 5’ ation recognition sequence, the presence or absence of a nucleotide pairing tion can be fied. For example, at least one base pair may be absent at a given position, compared to a native alphavirus 5’ ation recognition sequence, e.g. a base pair within a stem loop, in particular the stem of the stem loop. in a red embodiment, one or more stem loops of the 5’ replication recognition sequence are not deleted or disrupted. More preferably, stem loops 3 and 4 are not deleted or disrupted. More preferably, none of the stem loops of the 5’ replication -_\ (3 ition sequence is deleted or disrupted.

In one embodiment, the removal of at least one initiation codon does not disrupt the secondary structure of the 5’ replication recognition sequence. In an alternative embodiment, the l of at least one initiation codon does disrupt the secondary structure of the 5’ replication recognition sequence. In this embodiment, the removal of at least one initiation codon may be causative for the absence of at least one base pair at a given position, e.g. a base pair within a stem loop, compared to a native alphavirus 5’ replication recognition sequence. If a base pair is absent within a stem loop, compared to a native alphavirus 5’ replication recognition sequence, the removal of at least one initiation codon is determined to introduce a nucleotide pairing disruption within the stem loop. A base pair within a stem loop is typically a base pair in the stem of the stem loop.

In a preferred embodiment, the RNA replicon comprises one or more tide changes compensating for nucleotide pairing tions within one or more stem loops introduced by the removal of at least one initiation codon.

If the removal of at least one tion codon introduces a nucleotide pairing disruption within a stem loop, ed to a native alphavirus 5’ replication ition sequence, one or more nucleotide changes may be introduced which are expected to compensate for the nucleotide pairing disruption, and the secondary structure or predicted secondary structure obtained thereby may be compared to a natithavirus 5’ replication recognition sequence.

[Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg Based on the common general knowledge and on the disclosure herein, certain nucleotide s can be expected by the skilled person to compensate for nucleotide pairing disruptions. For example, if a base pair is disrupted at a given position of the ary ure or predicted secondary ure of a given 5’ replication recognition sequence of an RNA replicon characterized by the l of at least one initiation codon, compared to the native alphavirus 5’ replication recognition sequence, a tide change that restores a base pair at that position, preferably without re-introducing an initiation codon, is expected to compensate for the nucleotide pairing disruption. In one example, the 5’ ation recognition 1O sequence of a on comprising one or more nucleotide changes compensating for nucleotide pairing disruptions within one or more stem loops introduced by the removal of at least one initiation codon is encoded by a DNA sequence as represented by SEQ ID NO: 5 (Example 1).

In a preferred embodiment, the 5’ replication recognition sequence of the replicon does not overlap with, or does not comprise, a translatable nucleic acid sequence, i.e. translatable into a peptide or protein, in particular a nsP, in particular nsP1, or a fragment of any thereof. For a nucleotide sequence to be "translatable", it requires the presence of an tion codon; the initiation codon s the most N—terminal amino acid residue of the peptide or protein. In one embodiment, the 5’ replication recognition sequence of the replicon does not overlap with, or does not comprise, a translatable c acid sequence encoding an N-terminal fragment of nsP1. v-vu- .14 at least one subgenomic promoter. In a preferred embodiment, the subgenomic promoter of the replicon does not overlap with, or does not comprise, a translatable nucleic acid sequence, i.e. translatable into a peptide or protein, in particular a nsP, in ular nsP4, or a fragment of any thereof. In one embodiment, the subgenomic promoter of the replicon does not overlap with, or does not comprise, a translatable nucleic acid sequence that encodes a C-terminal fragment of nsP4. A RNA replicon having a subgenomic promoter that does not overlap with, or does not comprise, a translatable nucleic acid ce, e.g. translatable into the inal fragment of nsP4Dy be generated by deleting part of the coding sequence for nsP4 (typically the part encoding the N-terminal part of nsP4), and/or by removing AUG base triplets [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg in the part of the coding sequence for nsP4 that has not been deleted. If AUG base triplets in the coding sequence for nsP4 or a part thereof are removed, the AUG base triplets that are d are preferably potential initiation codons. atively, if the subgenomic promoter does not overlap with a nucleic acid sequence that encodes nsP4, the entire nucleic acid sequence encoding nsP4 may be deleted.

In one embodiment, the RNA replicon does not comprise an open reading frame encoding a truncated alphavirus non-structural n. In the context of this ment, it is particularly preferable that the RNA on does not comprise an open reading frame encoding the N-terminal fragment of nsP1, and optionally does not comprise an open reading frame ng the C-terminal fragment of nsP4. The N-terminal fragment of nsP1 is a truncated alphavirus n; the C-terminal fragment of nsP4 is also a truncated alphavirus protein.

In some embodiments the replicon according to the present invention does not comprise stem loop 2 (SL2) of the 5’ terminus of the genome of an alphavirus.

According to Frolov et al., supra, stem loop 2 is a conserved ary structure found at the 5’ terminus of the genome of an alphavirus, upstream of CSE 2, but is dispensible for replication.

In one embodiment, the 5’ replication recognition sequence of the replicon does not p with a nucleic acid sequence that encodes alphavirus non-structural protein or a fragment thereof. Thus, the present invention encompasses replicons that are characterized, compared to genomic alphaviral RNA, by the removal of at least one initiation codon, as described herein, optionally combined with the deletion of the coding region for one or more alphavirus non-structural ns, or a part thereof.

For example, the coding region for nsP2 and nsP3 may be deleted, or the coding region for nsP2 and nsP3 may be deleted together with the deletion of the coding region for the C—terminal fragment of nsP1 and/or of the coding region for the N- terminal fragment of nsP4, and one or more remaining initiation codons, i.e. remaining after said removal, may be removed as bed herein.

Deletnof the coding region for one or more alphavirus non-structural proteins may be achieved by standard methods, e.g., at DNA level, excision by the help of [Annotation] mpg None set by mpg ation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg _ restriction enzymes, preferably restriction enzymes that recognize unique restriction sites in the open reading frame (for illustration: see e 1). Optionally, unique restriction sites may be introduced into an open reading frame by nesis, e.g. irected mutagenesis. The respective DNA may be used as template for in vitro transcription.

A restriction site is a nucleic acid sequence, e.g. DNA sequence, which is ary and ient to direct ction (cleavage) of the nucleic acid molecule, e.g. DNA molecule, in which the restriction site is contained, by a specific restriction . A restriction site is unique for a given nucleic acid moiecuie if one copy of the restriction site is present in the nucieic acid moiecuie.

A restriction enzyme is an endonuclease that cuts a nucleic acid molecule, e.g. DNA molecule, at or near the restriction site.

Alternatively, a nucleic acid sequence characterized by the deletion of part or all of the open reading frame may be obtained by synthetic methods.

The RNA replicon according to the present invention is preferably a single stranded RNA moiecuie. The RNA replicon according to the present ion is typically a (+) stranded RNA molecule. in one embodiment, the RNA replicon of the present invention is an isolated nucleic acid moiecuie.

At least one open reading frame comprised by the replicon In one embodiment, the RNA replicon according to the present ion comprises at least one open reading frame encoding a peptide of interest or a protein of interest. ably, the protein of interest is encoded by a heterologous nucleic acid sequence. The gene encoding the peptide or protein of interest is synonymously termed "gene of interest" or "transgene". in various embodiments, the peptide or protein of interest is encoded by a heterologous nucleic acid sequence. According to the present invention, the term "heterologous" refers to the fact that a nucleic acid sequence is not naturally onally or structurally linked to an alphavirus nucleic acid aence.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg The replicon according to the present invention may encode a single ptide or multiple polypeptides. Multiple polypeptides can be encoded as a single polypeptide (fusion polypeptide) or as te polypeptides. In some ments, the replicon ing to the present invention may comprise more than one open reading frames, each of which may independently be selected to be under the control of a subgenomic promoter or not. Alternatively, a poly-protein or fusion polypeptide comprises individual polypeptides ted by an optionally autocatalytic protease cleavage site (e.g. foot-and—mouth disease virus 2A protein), or an intein. _.\ C3 Proteins of interest may e.g. be selected frcrn the group consisting of reporter proteins, pharmaceutically active peptides or proteins, inhibitors of intracellular interferon (lFN) signaling, and onal aiphavirus non-structural protein.

Reporter protein In one ment, an open reading frame encodes a reporter protein. In that embodiment, the open reading frame comprises a reporter gene. Certain genes may be chosen as reporters because the characteristics they confer on cells or organisms expressing them may be readily identified and measured, or because they are selectable markers. Reporter genes are often used as an indication of whether a certain gene has been taken up by or expressed in the cell or sm tion.

Preferably, the expression t of the reporter gene is visually detectable.

Common visually detectable reporter proteins typically possess fluorescent or luminescent ns. Examples of specific reporter genes e the gene that encodes jellyfish green fluorescent protein (GFP), which causes cells that express it to glow green under blue light, the enzyme luciferase, which catalyzes a reaction with Iuciferin to produce light, and the red fluorescent protein (RFP). ts of any of these specific reporter genes are possible, as long as the variants possess visually detectable properties. For example, eGFP is a point mutant variant of GFP. The er protein embodiment is particularly suitable for testing expression, see e.g.

Examples 2 to 5.

Pharmaceutically active e or protein Accong to the invention, in one embodiment, RNA of the replicon comprises or consists of ceutically active RNA. A "pharmaceutically active RNA" may be [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg ation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg RNA that encodes a pharmaceutically active peptide or n. Preferably, the RNA replicon according to the present invention s a pharmaceutically active peptide or protein. Preferably, an open reading frame encodes a pharmaceutically active peptide or protein. Preferably, the RNA replicon comprises an open reading frame that encodes a pharmaceutically active peptide or protein, optionally under control of the subgenomic promoter.

A aceutically active peptide or protein" has a positive or advantageous effect on the condition or disease state of a subject when administered to the subject in a -_\ <3 therapeutically effective amount. Preferabt, a pharmaceutically active peptide Gr protein has curative er palliative properties and may be administered te ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder. A pharmaceutically active e or protein may have prophylactic properties and may be used to delay the onset of a disease or to lessen the severity of such disease or pathological condition. The term "pharmaceutically active peptide or protein" includes entire proteins or ptides, and can also refer to pharmaceutically active fragments thereof. It can also e pharmaceutically active analogs of a 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.

In one ment, the ceutically active peptide or protein is or comprises an immunologically active compound or an antigen or an epitope.

According to the ion, the term ologically active compound" relates to any compound ng an immune response, preferably by inducing and/or suppressing tion of immune cells, inducing and/or suppressing cytokine biosynthesis, and/or altering humoral immunity by stimulating antibody production by B cells. In one embodiment, the immune response involves ation of an antibody response (usually including immunoglobulin G (lgG)). Immunologically active compounds possess potent immunostimulating activity including, but not limited to, antivinand mor activity, and can also down-regulate other aspects of the immune response, for example shifting the immune response away from a TH2 [Annotation] mpg None set by mpg ation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg ation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg immune response, which is useful for treating a wide range of TH2 mediated diseases.

According to the invention, the term "antigen" or "immunogen" covers any substance that will elicit an immune response. In particular, an "antigen" relates to any substance that reacts specifically with antibodies or T-Iymphocytes {T—cells).

According to the present invention, the term "antigen" comprises any molecule which comprises at least one epitope. Preferably, an antigen in the context of the present invention is a molecule which, optionally after processing, induces an immune 1O reaction, which is preferably specific for the antigen. According to the present invention, any suitable n may be used, which is a ate for an immune on, wherein the immune reaction may be both a humoral as well as a cellular immune reaction. In the t of the embodiments of the t invention, the antigen is preferably presented by a cell, preferably by an antigen presenting cell, in the context of MHC molecules, which results in an immune reaction against the antigen. An antigen is preferably a product which corresponds to or is derived from a naturally occurring antigen. Such naturally occurring antigens may include or may be derived from allergens, viruses, bacteria, fungi, parasites and other ious agents and pathogens or an antigen may also be a tumor antigen. According to the present invention, an antigen may pond to a naturally occurring product, for example, a virai protein, or a part thereof. In preferred embodiments, the n is a surface polypeptide, Le. a polypeptide naturally displayed on the surface of a cell, a pathogen, a bacterium, a virus, a fungus, a parasite, an allergen, or a tumor. The antigen may elicit an immune response against a cell, a pathogen, a bacterium, a virus, a fungus, a parasite, an allergen, or a tumor.

The term "pathogen" refers to enic biological material capable of causing disease in an organism, preferably a vertebrate organism. ens include rganisms such as bacteria, unicellular eukaryotic organisms (protozoa), fungi, as well as viruses.

The terms "epitope", "antigen peptide", "antigen e", "immunogenic peptide" and "MHCD'Iding peptide" are used interchangeably herein and refer to an antigenic inant in a molecule such as an antigen, i.e., to a part in or fragment of an [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg immunologically active compound that is recognized by the immune system, for example, that is recognized by a T cell, in particular when presented in the context of MHC molecules. An epitope of a protein preferably comprises a continuous or discontinuous portion of said protein and is preferably between 5 and 100, preferably between 5 and 50, more preferably between 8 and 30, most preferably between 10 and 25 amino acids in length, for example, the epitope may be preferably 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

According to the invention an e may bind to MHC molecules such as MHC molecules on the e of a cell and thus, may be a "MHC binding e" or "antigen peptide". The term "major histocompatibility complex" and the abbreviation "MHC" include MHC class i and MHC class II les and relate to a complex of genes which is t in all vertebrates. MHC proteins or molecules are ant for signaling between lymphocytes and n presenting cells or diseased cells in immune reactions, wherein the MHC proteins or molecules bind peptides and present them for recognition by T cell receptors. The proteins encoded by the MHC are expressed on the surface of cells, and display both self-antigens (peptide fragments from the cell ) and non-self—antigens (e.g., fragments of ng microorganisms) to a T cell. Preferred such immunogenic portions bind to an MHC class I or class II molecule. As used herein, an immunogenic portion is said to "bind to" an MHC class I or class II molecule if such binding is detectable using any assay known in the art. The term "MHC- binding peptide" relates to a peptide which binds to an MHC class I and/or an MHC class II molecule. In the case of class I MHC/peptide complexes, the binding peptides are typically 8-10 amino acids long although longer or shorter peptides may be effective. In the case of class II MHC/peptide complexes, the binding peptides are typically 10-25 amino acids long and are in particular 13-18 amino acids long, whereas longer and shorter peptides may be effective.

In one embodiment, the n of interest according to the present invention comprises an epitope suitable for vaccination of a target organism. A person skilled in the art will know that one of the principles of immunobiology and ation is based on the fact that an immunoprotective reaction to a e is produced by immunizing an organism with an n, which is logically relevant with respao the disease to be treated. According to the present invention, an antigen is selected from the group comprising a self-antigen and non-self—antigen. A non-self- [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg ation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg n is preferably a bacterial antigen, a virus antigen, a fungus antigen, an allergen or a parasite antigen. It is preferred that the antigen comprises an epitope that is capable of eliciting an immune response in a target sm. For example, the epitope may elicit an immune response against a ium, a virus, a , a parasite, an allergen, or a tumor.

In some embodiments the non-self—antigen is a bacterial antigen. In some embodiments, the antigen elicits an immune response against a bacterium which infects animals, including birds, fish and mammals, including domesticated animals.

Preferably, the bacterium against which the immune response is elicited is a pathogenic ium.

In some embodiments the non-seIf-antigen is a virus antigen. A virus antigen may for example be a peptide from a virus surface protein, e.g. a capsid ptide or a spike polypeptide. In some embodiments, the antigen elicits an immune response against a virus which infects s, including birds, fish and mammals, including domesticated animals. Preferably, the virus against which the immune response is elicited is a pathogenic virus.

In some embodiments the non-seIf-antigen is a polypeptide or a protein from a fungus. In some embodiments, the antigen elicits an immune response against a fungus which infects s, including birds, fish and mammals, ing domesticated animals. Preferably, the fungus against which the immune response is elicited is a pathogenic .

In some embodiments the non-self—antigen is a polypeptide or protein from a unicellular eukaryotic parasite. In some embodiments, the antigen elicits an immune response against a unicellular eukaryotic parasite, preferably a pathogenic uniceIIuIar eukaryotic parasite. Pathogenic lular eukaryotic parasites may be e.g. from the genus Plasmodium, e.g. P. falciparum, P. vivax, P. malariae or P. ovale, from the genus Leishmania, or from the genus Trypanosoma, e.g. T. cruzi or T. brucei. ation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg In some embodiments the non-self—antigen is an allergenic polypeptide or an allergenic protein. An enic protein or enic polypeptide is suitable for allergen immunotherapy, also known as hypo-sensitization.

In some ments the antigen is a self-antigen, particularly a tumor antigen.

Tumor antigens and their ination are known to the skilled person. in the context of the present invention, the term "tumor antigen" or -associated antigen" relates to proteins that are under normal conditions specifically expressed in a limited number of tissues and/or organs or in specific developmental stages, for example, the tumor antigen may be under normal conditions specifically expressed in stomach tissue, preferably in the gastric mucosa, in reproductive organs, e.g., in testis, in trophoblastic tissue, e.g., in placenta, or in germ line cells, and are expressed or ntly expressed in one or more tumor or cancer tissues. In this context, "a limited number" preferably means not more than 3, more preferably not more than 2. The tumor ns in. the context of the t ion include, for example, entiation antigens, preferably cell type specific differentiation ns, i.e., proteins that are under normal conditions specifically expressed in a certain cell type at a certain differentiation stage, cancer/testis antigens, i.e., proteins that are under normal conditions specifically sed in testis and sometimes in placenta, and germ line specific ns. in the context of the present ion, the tumor antigen is preferably associated with the cell surface of a cancer cell and is preferably not or only rarely expressed in normal tissues. Preferably, the tumor antigen or the aberrant expression of the tumor antigen identifies cancer cells. In the context of the present invention, the tumor antigen that is expressed by a cancer cell in a subject, e.g., a patient suffering from a cancer disease, is preferably a self- protein in said subject. In preferred embodiments, the tumor antigen in the context of the present invention is expressed under normal conditions specifically in a tissue or organ that is non-essential, i.e., tissues or organs which when damaged by the immune system do not lead to death of the subject, or in organs or structures of the body which are not or only hardly accessible by the immune system. Preferably, the amino acid sequence of the tumor antigen is identical between the tumor antigen whicl'D expressed in normal tissues and the tumor antigen which is expressed in cancerfissues.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg Examples for tumor antigens that may be useful in the t invention are p53, ART-4, BAGE, beta-catenin/m, Bcr—abL CAMEL, CAP-1, CASP-8, CDCZ7/m, CDK4/m, CEA, the cell surface proteins of the claudin family, such as CLAUDlN-B, CLAUDlN-18.2 and CLAUDlN-12, c—MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2/neu, HPV—E7, HPV—E6, , hTE T (or hTRT), LAGE, LDLR/FUT, MAGE-A, preferably iviAGE-Af, MAGE—AZ, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, , MART-1/Melan-A, MC‘! R, Myosin/m, MUCl, MUM-1, -2, —3, NA88=A, NF1, NY-ESO-f, NY-BR-f, p190 minor BCR—abL, me/RARa, FRAME, prcteinase 3, PSA, PSM, RAGE, RU1 or RUZ, SAGE, SART-1 or SART-S, SCGBBAZ, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/lNT2, TPTE and WT. Particularly preferred tumor antigens include CLAUDlN-18.2 (CLDN18.2) and CLAUDlN-6 (CLDN6).

In some embodiments, it is not ed that the pharmaceutically active peptide or protein is an antigen that elicits an immune response. Suitable pharmaceutically active proteins or peptides may be selected from the group consisting of cytokines and immune system proteins such as immunologically active nds (e.g., eukins, colony stimulating factor (CSF), granulocyte colony stimulating factor (G- CSF), granulocyte-macrophage colony stimulating factor F), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, seletins, homing receptors, T cell receptors, immunoglobulins), hormones (insulin, thyroid hormone, catecholamines, gonadotrophines, trophic hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptins and the like), growth hormones (e.g., human grown hormone), growth factors (e.g., epidermal growth , nerve growth factor, n- like growth factor and the like), growth factor receptors, enzymes (tissue plasminogen activator, streptokinase, cholesterol biosynthetic or degradative, steriodogenic s, kinases, phosphodiesterases, methylases, de-methylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, ases, cytochromes, adenylate or guanylaste cyclases, neuramidases and the like), receptors (steroid hormone receptors, peptide ors), binding ns (growth hormD or growth factor binding proteins and the like), transcription and translation factors, tumor growth ssing proteins (e.g., proteins which inhibit [Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg angiogenesis), structural proteins (such as en, 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 Wilebrand factor, antithrombin lll, glucocerebrosidase, erythropoietin ocyte colony stimulating factor (GCSF) or modified Factor VIII, anticoagulants and the like.

In one embodiment, the pharmaceutically active protein according to the invention is a cytokine which is involved in regulating lymphoid homeostasis, preferably a cytokine which is involved in and preferably induces or enhances development, priming, expansion, differentiation and/or survival of T cells. In one embodiment, the cytokine is an interleukin, e.g. lL-2, lL-7, lL-12, lL-15, or lL-21. tor of interferon (IFN) signaling A r suitable protein of interest encoded by an open reading frame is an inhibitor of interferon (IFN) signaling. While it has been reported that ity of cells in which RNA has been introduced for sion can be reduced, in particular, if cells are transfected multiple times with RNA, IFN inhibiting agents were found to e the viability of cells in which RNA is to be expressed ( the inhibitor is an inhibitor of IFN type | signaling. Preventing engagement of IFN receptor by extracellular IFN and inhibiting intracellular lFN signaling in the cells allows stable expression of RNA in the cells. Alternatively or additionally, preventing engagement of lFN receptor by extracellular IFN and inhibiting intracellular IFN signaling enhances survival of the cells, in particular, if cells are transfected repetitively with RNA. Without wishing to be bound by theory, it is envisaged that intracellular lFN signalling can result in inhibition of translation and/or RNA degradation. This can be sed by inhibiting one or more lFN-inducible antivirally active effector proteins. The ducible antivirally active effector protein can be selected from the group consisting of RNA-dependent n kinase (PKR), 2',5'—oligoadenylate synthetase (OAS) and RNaseL. Inhibiting intracellular lFN signalling may comprise inhibiting the pendent pathway and/or the OAS- 3O ent pathway. A le protein of interest is a protein that is capable of inhibiting the pendent pathway and/or the OAS-dependent y. inhibiting the PKR-dependent pathway may comprise inhibiting eIF2-alpha phosDrylation. Inhibiting PKR may comprise treating the cell with at least one PKR inhibitor. The PKR inhibitor may be a viral inhibitor of PKR. The preferred viral [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg inhibitor of PKR is vaccinia virus E3. If a peptide or protein (e.g. E3, K3) is to inhibit intracellular lFN signaling, intracellular expression of the peptide or protein is preferred. Vaccinia virus E3 is a 25 kDa dsRNA-binding protein (encoded by gene E3L) that binds and sequesters dsRNA to prevent the tion of PKR and OAS. E3 can bind directly to PKR and inhibits its activity, resulting in reduced phosphorylation of elFZ-alpha. Other suitable inhibitors of IFN signaling are Herpes simplex virus ICP34.5, a virus NSs, Bombyx mori nucleopolyhedrovirus PK2, and HCV N834A. 1O The inhibitor of lFN ing may be provided to the cell in the form of a nucleic acid sequence (e.g. RNA) encoding the inhibitor of lFN signaling.

In one embodiment, the inhibitor of intracellular or extracellular lFN signaling is encoded by an mRNA molecule. That mRNA molecule may se a non- polypeptide-sequence modifying modification as described herein, e.g. cap, 5’-UTR, 3’-UTR, poly(A) sequence, adaptation of the codon usage. Respective embodiments are illustrated e.g. in Example 3.

In an alternative embodiment, the inhibitor of intracellular or extracellular lFN signaling is encoded by a replicon, preferably a trans-replicon or a trans-replicon as described herein. The on comprises nucleic acid sequence ts that. allow replication by alphavirus replicase, typically CSE 1, CSE 2 and CSE 4; and preferably also nucleic acid sequence elements that allow production of a subgenomic transcript, Le. a subgenomic promoter, typically sing CSE 3. The replicon may additionally se one or more non-polypeptide-sequence ing modifications as described herein, e.g. cap, poly(A) sequence, adaptation of the codon usage. If multiple open reading frames are t on the replicon, then an inhibitor of ellular IFN signaling may be encoded by any one of them, optionally under control of a subgenomic promoter or not. In a preferred embodiment, the inhibitor of intracellular lFN signaling is encoded by the most upstream open reading frame of the RNA replicon. When an inhibitor of intracellular lFN signaling is encoded by the most upstream open reading frame of the RNA replicon, the genetic inforrrnnn encoding the inhibitor of ellular lFN signaling will be translated early [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg after introduction of the RNA on into a host cell, and the resulting protein may subsequently inhibit intracellular IFN signaling.

Functional alphavirus non-structural protein A r suitable protein of interest encoded by an open reading frame is functional alphavirus non-structural protein. The term "alphavirus non-structural protein" includes each and every 00- or post-translationally modified form, including carbohydrate-modified (such as glycosylated) and lipid-modified forms of irus non-structural protein. _A CD in some embodiments, the term "alphavirus non-strwtural protein" refers to any one or more of dual non-structural proteins of irus origin (nsP1, nsP2, nsP3, nsP4), or to a poly-protein comprising the polypeptide sequence of more than one non-structural protein of alphavirus origin. In some embodiments, "alphavirus non- structural protein" refers to nsP123 and/or to nsP4. In other embodiments, "alphavirus non-structural protein" refers to nsP1234. In one embodiment, the protein of interest encoded by an open reading frame ts of all of nsP1, nsP2, nsP3 and nsP4 as one single, ally cleavable poly-protein: nsP1234. In one embodiment, the protein of interest encoded by an open reading frame consists of nsP1, nsP2 and nsP3 as one single, optionally cleavable polyprotein: nsP123. In that embodiment, nsP4 may be a further protein of interest and may be d by a further open reading frame.

I". some embodiments, alphavirus non-structural protein is capable of forming a complex or association, e.g. in a host cell. In some embodiments, "alphavirus non- structural protein" refers to a complex or association of nsP123 (synonymously P123) and nsP4. In some embodiments, "alphavirus non-structural protein" refers to a complex or association of nsP1, nsP2, and nsP3. In some embodiments, "alphavirus ructural protein" refers to a complex or association of nsP1, nsP2, nsP3 and nsP4. In some embodiments, virus non-structural protein" refers to a complex or ation of any one or more selected from the group consisting of nsP1, nsP2, nsP3 and nsP4. In some embodiments, the alphavirus non-structural protein comns at least nsP4.

[Annotation] mpg None set by mpg ation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg ation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg The terms "complex" or "association" refer to two or more same or different protein molecules that are in spatial proximity. Proteins of a complex are preferably in direct or indirect physical or physicochemical contact with each other. A complex or ation can t of multiple different proteins (heteromultimer) and/or of multiple copies of one particular n (homomultimer). In the context of alphavirus non-structural protein, the term "complex or association" describes a multitude of at least two protein molecules, of which at least one is an irus non-structural protein. The complex or association can consist of multiple copies of one particular protein (homomultimer) and/or of le different proteins (heteromultimer). In the context of a er, "multi means more than one, such as two, thr‘e, four, five, six, seven, eight, nine, ten, or more tha :i 1-.-(D -3 The term "functional alphavirus non-structural protein" includes alphavirus non- structural protein that has replicase function. Thus, "functional alphavirus non- structural protein" includes alphavirus replicase. "Replicase on" comprises the function of an RNA-dependent RNA polymerase (RdRP), i.e. an enzyme which i capable to catalyze the synthesis of (-) strand RNA based on a (+) strand RNA template, and/or which is capable to catalyze the synthesis of (+) strand RNA based on a (-) strand RNA template. Thus, the term "functional alphavirus non-structural protein" can refer to a protein or complex that synthesizes (-) stranded RNA, using the (+) stranded (e.g. genomic) RNA as te, to a protein or complex that sizes new (+) stranded RNA, using the (-) stranded complement of genomic RNA as template, and/or to a protein or complex that synthesizes a omic transcript, using a fragment of the (-) stranded complement of genomic RNA as template. The functional alphavirus non-structural protein may additionally have one or more additional functions, such as e.g. a protease (for auto-cleavage), helicase, terminal adenylyltransferase (for poly(A) tail addition), methyltransferase and guanylyltransferase (for ing a nucleic acid with a 5’-cap), nuclear localization sites, triphosphatase (Gould et al., 2010, Antiviral Res., vol. 87 pp. 111—124; Rupp et al., 2015, J. Gen. Virol., vol. 96, pp. 2483-500).

According to the invention, the term "alphavirus replicase" refers to alphaviral RNA- depeDnt RNA polymerase, including a RNA-dependent RNA polymerase from a naturally occurring irus (alphavirus found in nature) and a RNA-dependent [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg RNA polymerase from a variant or derivative of an alphavirus, such as from an attenuated alphavirus. In the context of the present invention, the terms "replicase" and "alphavirus replicase" are used interchangeably, unless the context dictates that any particular ase is not an alphavirus ase.

The term "replicase" comprises all variants, in particular post—translationally modified variants, conformations, isoforms and homologs of alphavirus replicase, which are expressed by alphavirus-infected cells or which are expressed by cells that have been transfected with a nucleic acid that codes for alphavirus replicase. er, 1.0 the term "replicase" comprises all forms of replicase that have been produced and can be ed by recombinant methods. For example, a replicase compristng a tag that facilitates detection and/or purification of the replicase in the laboratory, e.g. a myc-tag, a HA-tag or an oligohistidine tag (His-tag) may be produced by recombinant methods.

Optionally, the alphavirus replicase is onally functionally defined by the capacity of binding to any one or more of alphavirus conserved ce element 1 (CSE 1) or complementary sequence f, conserved sequence element 2 (CSE 2) or complementary ce thereof, conserved sequence element 3 (CSE 3) or complementary sequence thereof, ved sequence element 4 (CSE 4) or complementary ce thereof. Preferably, the replicase is capable of g to CSE 2 [Le to the (+) strand] and/or to CSE 4 [Le to the (+) strand], or of binding to the complement of CSE 1 [Le to the (-) strand] and/or to the complement of CSE 3 [i.e. to the (-) strand].

The origin of the replicase is not limited to any particular alphavirus. In a preferred embodiment, the alphavirus ase comprises non-structural protein from Semliki Forest virus, including a lly occurring Semliki Forest virus and a variant or derivative of Semliki Forest virus, such as an attenuated Semliki Forest virus. In an alternative preferred ment, the alphavirus replicase comprises non-structural protein from s virus, including a naturally occurring Sindbis virus and a t or derivative of Sindbis virus, such as an attenuated Sindbis virus. In an alternative prefen embodiment, the alphavirus replicase comprises non-structural protein from Venezuelan equine encephalitis virus (VEEV), including a naturally occurring VEEV [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg and a t or derivative of VEEV, such as an attenuated VEEV. In an ative preferred embodiment, the alphavirus replicase comprises non-structural protein from gunya virus (CHIKV), including a naturally occurring CHIKV and a variant or derivative of CHIKV, such as an attenuated CHIKV.

A replicase can also comprise non-structural ns from more than one alphavirus.

Thus, heterologous complexes or associations comprising alphavirus non-structural protein and having replicase function are y comprised by the present invention.

Merely for illustrative purposes, replicase may comprise one or more ructural 1O proteins (e.g. nsP1, nsP2) from a first alphavirus, and one or more non-structural ns (nsP3, nsP4) from a second alphavirus. Non-structural proteins from more than one different irus may be encoded by te open reading frames, or may be encoded by a single open reading frame as poly-protein, e.g. nsP1234.

In some embodiments, functional alphavirus non-structural protein is capable of forming membranous replication complexes and/or vacuoles in cells in which the functional alphavirus non-structural protein is sed. lf functional alphavirus non-structural protein, i.e. alphavirus non-structural protein with replicase function, is encoded by a nucleic acid molecule according to the present invention, it is preferable that the subgenomic er of the replicon, if present, is compatible with said replicase. ible in this context means that the alphavirus replicase is capable of izing the subgenomic promoter, if present.

In one embodiment, this is achieved when the omic promoter is native to the alphavirus from which the replicase is derived, i.e. the natural origin of these sequences is the same alphavirus. In an ative embodiment, the subgenomic promoter is not native to the alphavirus from which the alphavirus replicase is derived, provided that the alphavirus replicase is capable of recognizing the subgenomic promoter. In other words, the replicase is compatible with the subgenomic promoter (cross-virus compatibility). Examples of cross-virus compatibility concerning subgenomic promoter and replicase originating from different alphaviruses are known in the art. Any combination of subgenomic promoter and Dicase is possible as long as cross-virus compatibility exists. Cross-virus compatibility can readily be tested by the skilled person working the present invention [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg ation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg by incubating a replicase to be tested together with an RNA, wherein the RNA has a subgenomic promoter to be tested, at conditions suitable for RNA synthesis from the a subgenomic promoter. If a subgenomic transcript is prepared, the subgenomic promoter and the replicase are determined to be compatible. Various examples of cross-virus compatibility are known (reviewed by Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491-562).

In one embodiment, alphavirus non-structural protein is not encoded as fusion protein with a logous protein, e.g. ubiquitin.

In the present invention, an open reading frame encoding functional alphavirus non— structural n can be provided on the RNA on, or alternatively, can be provided as separate nucleic acid molecule, e.g. mRNA le. A separate mRNA molecule may optionally comprise e.g. cap, 5’-UTR, 3’-UTR, poly(A) sequence, and/or adaptation of the codon usage. The separate mRNA molecule may be provided in trans, as bed herein for the system of the present invention.

When an open reading frame encoding functional alphavirus non-structural protein is ed on the RNA replicon, the replicon can preferably be replicated by the onal alphavirus non-structural n. In particular, the RNA on that encodes onal alphavirus non-structural protein can be replicated by the functional alphavirus non—structural protein encoded by the replicon. This embodiment is strongly preferred when no nucleic acid le encoding functional alphavirus non—structural n is provided in trans. In this embodiment, cis- replication of the replicon is aimed at. In a preferred embodiment, the RNA replicon comprises an open reading frame encoding functional alphavirus non-structural protein as well as a further open reading frame encoding a n of interest, and can be ated by the functional alphavirus non-structural protein. This embodiment is particularly suitable in some methods for ing a protein of interest according to the present invention. An example of a respective replicon is illustrated in Fig. 1 ("cisReplicon A5ATG-RRS").

If theDalicon comprises an open reading frame encoding functional alphavirus non- structural protein, it is preferable that the open reading frame encoding functional [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg irus non-structural protein does not overlap with the 5' replication recognition sequence. In one embodiment, the open reading frame ng functional irus non-structural protein does not overlap with the subgenomic promoter, if present. An example of a tive replicon is illustrated in Fig. 1 ("cisReplicon A5ATG—RRS").

If multiple open reading frames are present on the replicon. then the functional alphavirus non-structural protein may be encoded by any one of them, ally under control of a subgenomic promoter or not, preferably not under control of a -A CD subgenomic promoter. In a preferred embodiment, the functional atphavirus non- structural protein is encoded by the most upstream open reading frame of the RNA replicon. When the functional alphavirus non-structural protein is encoded by the most upstream open reading frame of the RNA replicon, the genetic information encoding onal irus non-structural protein will be translated early after introduction of the RNA replicon into a host cell, and the resulting protein can uently drive replication, and optionally production of a subgenomic transcript, in the host cell. An example of a respective replicon is illustrated in Fig. 1 ("cisReplicon A5ATG-RRS").

Presence of an open g frame encoding functional alphavirus non-structural protein, either comprised by the replicon or comprised by a separate nucleic acid molecule that is provided in trans, allows that the replicon is replicated, and consequently, that a gene of interest encoded by the on, optionally under control of a subgenomic promoter, is expressed at high ievels. This is associated with a cost advantage compared to other ene expression s. For example, in the case of animal vaccination, the cost of a e is key to its success in. the nary and farming community. Since the replicon of the present invention can be replicated in the presence of functional alphavirus non-structural protein, eg. in a cell of a vaccinated animal, high levels of expression of a gene of interest may be achieved even if relatively low amounts replicon RNA are administered. The low amounts of replicon RNA positively influence the costs of vaccine per subject.

[Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg ation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg Position of the at least one open reading frame in the RNA replicon The RNA replicon is suitable for expression of one or more genes encoding a peptide of st or a protein of interest, optionally under control of a subgenomic promoter.

Various embodiments are possible. One or more open reading frames, each encoding a e of interest or a protein of interest, can be present on the RNA replicon. The most upstream open reading frame of the RNA replicon is referred to as "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 further open reading frames can be present downstream of the first open reading frame.

One or more further open reading frames downstream of the first open reading frame may be referred to as "second open reading frame", "third open reading frame" and so on, in the order (5’ to 3’) in which they are t downstream of the first open reading frame. Preferably, each open reading frame ses a start codon (base triplet), typically AUG (in the RNA molecule), corresponding to ATG (in a respective DNA le).

If the replicon comprises a 3’ replication recognition sequence, it is preferred that all open reading frames are localized upstream of the 3’ replication ition sequence.

When the RNA replicon comprising one or more open reading frames is introduced into a host cell, translation is preferably not initiated at any position am of the first open reading frame, owing to the removal of at least one initiation codon from the 5' replication recognition sequence. Therefore, the replicon may serve directly as template for translation of the first open reading frame. Preferably, the on comprises a 5’—cap. This is helpful for expression of the gene encoded by the first open reading frame ly from the replicon.

In some ments, at least one open reading frame of the replicon is under the control of a subgenomic promoter, preferably an alphavirus subgenomic promoter.

The alphavirus subgenomic promoter is very efficient, and is therefore suitable for heterologous gene expression at high levels. ably, the subgenomic promoter is a water for a subgenomic transcript in an alphavirus. This means that the subgenomic promoter is one which is native to an alphavirus and which preferably ation] mpg None set by mpg ation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg controls transcription of the open reading frame encoding one or more structural proteins in said alphavirus. Alternatively, th (D subgenomic promoter is a variant of a subgenomic promoter of an alphavirus; any variant which functions as promoter for subgenomic RNA ription in a host cell is suitable. If the on ses a omic promoter, it is preferred that the replicon comprises a conserved sequence element 3 (CSE 3) or a variant thereof.

Preferably, the at least one open reading frame under control of a subgenomic promoter is zed downstream of the subgenomic promoter. Preferably, the subgenomic promoter controls production of subgenomic RNA comprising a ript of the open reading frame In some embodiments the first open reading frame is under control of a subgenomic promoter. When the first open reading frame is under control of a subgenomic promoter, its localization resembles the localization of the open reading frame encoding structural proteins in the genome of an alphavirus. When the first open reading frame is under control of the subgenomic promoter, the gene encoded by the first open reading frame can be expressed both from the replicon as well as from a subgenomic ript thereof (the latter in the presence of functional alphavirus non- structural protein). A respective embodiment is exemplified by the replicon "A5ATG- RRS" in Fig. 1. Preferably "ASATG-RRS" does not se any initiation codon in the nucleic acid sequence encoding the C-terminal fragment of nsP4 (*nsP4). One or more further open reading frames, each under control of a subgenomic promoter, may be present downstream of the first open reading frame that is under control of a subgenomic promoter (not illustrated in Fig. 1). The genes encoded by the one or more further open reading frames, e.g. by the second open reading frame, may be translated from one or more subgenomic transcripts, each under l of a subgenomic promoter. For e, the RNA replicon may comprise a subgenomic promoter controlling production of a transcript that encodes a second protein of interest.

In other embodiments the first open reading frame is not under control of a subganic promoter. When the first open reading frame is not under control of a omic promoter, the gene encoded by the first open reading frame can be ation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg expressed from the replicon. A tive embodiment is exemplified by the replicon "A5ATG-RRSASGP" in Fig. 1. One or more further open reading frames, each under control of a subgenomic promoter, may be present downstream of the first open reading frame (for illustration of two exemplary embodiments, see "A5ATG-RRS - ronic" and "cisReplicon A5ATG-RRS" in Fig. 1). The genes encoded by the one or more further open reading frames may be expressed from subgenomic transcripts.

In a cell which comprises the replicon according to the present invention, the replicon may be amplified by functional alphavirus non-structural protein. Additionally, if the replicon comprises one or more open reading frames under controi of a subgenomic promoter, one or more subgenomic transcripts are expected to be prepared by functionat alphavirus non-structural protein. Functional irus non-structural protein may be ed in trans, or may be d by an open g frame of the replicon.

If a replicon comprises more than one open reading frame encoding a protein of interest, it is preferable that each open reading frame encodes a different protein. For e, the protein encoded by the second open reading frame is different from the protein encoded by the first open reading frame.

In some embodiments, the protein of interest encoded by the first and/or a further open g frame, preferably by the first open reading frame, is functional alphavirus non-structural protein or an inhibitor of lFN signaling, e.g. E3. In some embodiments, the protein of interest encoded (3'y the :41iret’ anal Inv- {I Ir-I-lnnr lot HI I" UlI I uI u IUI Open reading frame, e.g. by the second open reading frame, is a pharmaceutically active peptide or protein, or a reporter protein. in one embodiment, the protein of interest encoded by the first open reading frame is functional alphavirus non—structural protein. In that embodiment the replicon ably comprises a 5’-cap. Particularly when the protein of interest encoded by the first open reading frame is functional alphavirus non-structural n, and ably when the replicon comprises a 5’-cap, the nucleic acid sequence encoding functiDl alphavirus non-structural protein can be efficiently ated from the replicon, and the resulting protein can subsequently drive replication of the replicon [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg and drive synthesis of subgenomic transcript(s). This embodiment may be preferred when no additional nucleic acid le encoding functional alphavirus non- structural protein is used or present together with the replicon. In this embodiment, cis-replication of the replicon is aimed at.

One embodiment wherein the first ope . reading frame encodes functional alphavirus non-structural protein is illustrated by "cisReplicon A5ATG-RRS" in Fig. 1. Following translation of the c acid sequence encoding nsP1234, the translation product (nsP1234 or nt(s) thereof) can act as replicase and drive RNA synthesis, i.e. 1O replication of the on and synthesis of a subgenomic transcript comprising the second open reading frame ("Transgene" in Fig. 1). replication system In a second aspect, the present invention provides a system comprising: a RNA construct for sing functional alphavirus non-structural n, the RNA replicon according to the first aspect of the invention, which can be replicated by the functional alphavirus ructural protein in trans.

In the second aspect it is preferred that the RNA replicon does not comprise an open reading frame ng functional alphavirus non-structural protein.

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

The functional alphavirus ructural protein is as defined above and is typically encoded by an open reading frame comprised by the ase construct. The functional alphavirus non-structural protein encoded by the replicase construct may be any functional alphavirus non-structural protein that is capable of replicating the replica [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg ation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg When the system of the present invention is introduced into a cell, preferably a otic cell, the open reading frame ng functional alphavirus non-structural protein can be translated. After translation, the functional alphavirus non-structural n is capable of replicating a separate RNA molecule (RNA replicon) in trans.

Thus, the present invention provides a system for replicating RNA in trans.

Consequently, the system of the present invention is a trans-replication system.

According to the second aspect, the replicon is a trans-replicon.

Herein, trans (e.g. in the context of trans-acting, regulatory), in general, means 1.0 "acting from a ent molecule" (i.e., intermolecular). it is the opposite of cis (e.g. in .he contex. of sis-acting, sis-regulatory), which, in general 'neans "acting from the same molecule" (i.e., intr’moiecular). In the context of RNA synthesis (including transcription and RNA replication), a trans-acting element includes a nucleic acid sequence that contains a gene encoding an enzyme capable of RNA synthesis (RNA polymerase). The RNA polymerase uses a second nucleic acid molecule, Le. a c acid molecule other than the one by which it is encoded, as template for the synthesis of RNA. Both the RNA polymerase and the c acid sequence that contains a gene encoding the RNA polymerase are said to "act in trans" on the second nucleic acid molecule. In the context of the t invention, the RNA polymerase encoded by the trans-acting RNA is functional irus non-structural protein. The functional alphavirus non-structural protein is capable of using a second c acid molecule, which is an RNA replicon, as template for the synthesis or RNA, including replication of the RNA replicon. The RNA replicon that can be replicated by the replicase .in trans according to the present invention is synonymously ed to herein as "trans-replicon".

In the system of the present invention, the role of the functional alphavirus non- structural protein is to amplify the replicon, and to prepare a subgenomic transcript, if a subgenomic promoter is present on the replicon. If the replicon encodes a gene of st for expression, the expression levels of the gene of interest and/or the duration of sion may be regulated in trans by modifying the levels of the functional alphavirus non-structural protein.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg ation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg The fact that alphaviral replicase is generally able to recognize and replicate a template RNA in trans was initially discovered in the 1980s, but the potential of trans- replication for ical applications was not recognized, inter alia because trans- replicated RNA was considered to inhibit efficient replication: it was discovered in the case of defective interfering (DI) RNA that co-replicates with alphaviral genomes in infected cells (Barrett et al., 1984, J. Gen. Virol., vol. 65 ( Pt 8), pp. 1273-1283; Lehtovaara et al., 1981, Proc. Natl. Acad. Sci. U. S. A, vol. .78, pp. 5353-5357; Pettersson, 1981, Proc. Natl. Acad. Sci. U. S. A, vol. 78, pp. 115-119). DI RNAs are trans-replicons that may occur quasi-naturally during infections of cell lines with high 1.0 virus load. DI elements co—replicate so efficiently that they reduce the virulence of the parental virus and thereby act as inhibitory tic RNA (Barrett et al., 1984, J.

Gen. , vol. 6-5 (Pt 11), pp. 1909-1920). Although the potential for biomedical ations was not recognized, the phenomenon of trans-replication was used in several basic studies aiming to elucidate isms of replication, without requiring to express the replicase from the same molecule in cis; further, the separation of ase and replicon also allows functional studies ing mutants of viral ns, even if respective s were Ioss—of—function s (Lemm et al., 1994, EMBO J., vol. 13, pp. 2925-2934). These loss-of function studies and DI RNA did not suggest that trans-activation systems based on alphaviral elements may eventually become available to suit therapeutic purposes.

The system of the present invention comprises at least two nucleic acid molecules.

Thus, it may 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 molecules, the replicon and the replicase construct. ln alternative preferred embodiments, the system comprises more than one replicon, each preferably encoding at least one protein of interest, and also ses the replicase construct. In these embodiments, the functional alphavirus non-structural protein encoded by the ase construct can act on each replicon to drive replication and tion of subgenomic transcripts, respectively. For example, each replicon may encode a pharmaceutically active peptide or protein.

This Dadvantageous e.g. if ation of a subject against several different antigens is desired. ation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg Preferably, the replicase construct lacks at least one conserved sequence element (CSE) that is required for (-) strand synthesis based on a (+) strand te, and/or for (+) strand synthesis based on a (-) strand te. More preferably, the replicase construct does not comprise any alphaviral conserved sequence elements (CSEs). In particular, among the four CSEs of alphavirus (Strauss & s, Microbiol. Rev., 1994, vol. 58, pp. 491-562; José et ai., Future Microbiol., 2009, vol. 4, pp. 837—856), any one or more of the following CSEs are preferably not present on the replicase construct: CSE 1; CSE 2; CSE 3; CSE 4. Particularly in the absence of any one or 1Q more alphaviral CSE, the replic{D (I)e construct of the present ion res mbles(I) typical eukaryotic rnRNA much more than it resembles alphaviral genomic RNA.

The replicase construct of the present invention is preferably distinguished from alphaviral genomic RNA at least in that it is not capable of self-replication and/or that it does not comprise an open g frame under the control of a sub-genomic er. When unable to self-replicate, the replicase construct may also be termed "suicide construct".

The trans-replication system is associated with the following advantages: First and foremost, the versatility of the replication system allows that replicon and ase construct can be designed and/or ed at different times and/or at different sites. In one embodiment, the replicase construct is prepared at a first point in time, and the replicon is ed at a later point in time. For example, following its preparation, the replicase construct may be stored for use at a later point in time. The present invention provides increased flexibility compared to plicons: when a new pathogen emerges, the system of the present invention may be designed for vaccination, by g into the replicon a nucleic acid encoding a polypeptide that elicits an immune response against the new pathogen. A previously prepared replicase construct may be recovered from storage. Thus, it is not required that, at the time the replicase construct is designed and prepared, the nature of a ular pathogen, or of the antigen(s) of a particular pathogen, is known. Consequently, it is not rDired that, at the time the replicase construct is designed and prepared, a replicon encoding a polypeptide that elicits an immune response against a particular [Annotation] mpg None set by mpg ation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg new pathogen is available. In other words, the replicase construct can be designed and prepared independently of any particular replicon. This allows to rapidly react to the emergence of new pathogens, or to ens characterized by expression of at least one new antigen, because preparation of the replicon devoid of replicase es less effort and resources than the preparation of cis-replicons. History tells that a system allowing for rapid reaction to pathogens is needed: this is rated e.g. by the occurrence of pathogens causing severe acute respiratory me (SARS), Ebola and various influenza virus subtypes in recent years.

Second, the trans-replicon according to the present invention is typically a shorter nucleic acid molecule than a typical cis-replicon. This enables faster cloning of a on encoding a protein of st, e.g. an immunogenic polypeptide, and provides high yields of the protein of interest.

Further ages of the system of the present invention include the independence from nuclear transcription and the presence of key genetic information on two separate RNA molecules, which es unprecedented design freedom. In view of its versatile elements, which are combinable with each other, the present ion allows to optimize replicase expression for a desired level of RNA amplification, for a desired target organism, for a desired level of production of a protein of interest, etc.

The system according to the invention allows to nsfect varying amounts or ratios of replicon and replicase construct for any given cell type — resting or cycling, in vitro or in vivo. The trans-replication system of the present invention is suitable for inoculation of a host cell, and for expression of a gene of interest in a host cell (see e.g. Examples 2, 3 and 5).

The replicase construct according to the present invention is preferably a single stranded RNA molecule. The replicase construct according to the present invention is typically a (+) stranded RNA molecule. In one ment, the replicase construct of the present invention is an isolated nucleic acid molecule.

[Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg Preferred features of RNA molecules according to the invention RNA molecules according to the invention may optionally be characterized by further features, e.g. by a 5’-cap, a 5’-UTR, a 3’-UTR, a ) sequence, and/or adaptation of the codon usage. Details are described in the following.

In some embodiments, the replicon according to the t invention comprises a '-cap. 1O In some embodiments, the replicase construct according to the present invention comprises a 5’-cap.

The terms "5’-cap", "cap", "5’-cap structure", "cap structure" are used mously to refer to a dinucleotide that is found on the 5’ end of some eukaryotic y transcripts such as precursor messenger RNA. A 5’-cap is a structure wherein a (optionally modified) ine is bonded to the first nucleotide of an mRNA molecule via a 5' to 5' triphosphate linkage (or modified triphosphate linkage in the case of certain cap analogs). The terms can refer to a conventional cap or to a cap analog. For illustration, some particular cap dinucleotides (including cap analog dinucleotides) are shown in Fig. 6.

"RNA which comprises a 5’-cap" or "RNA which is provided with a 5'-cap" or "RNA which is modified with a 5'-cap" or "capped RNA" refers to RNA which comprises a ’-cap. For example, providing an RNA with a 5‘-cap may be ed by in vitro transcription of a DNA template in presence of said 5'-cap, n said 5'—cap is co- transcriptionally incorporated into the generated RNA strand, or the RNA may be generated, for example, by in vitro transcription, and the 5'-cap may be attached to the RNA post-transcriptionally using capping enzymes, for e, g enzymes of ia virus. In capped RNA, the 3’ position of the first base of a (capped) RNA molecule is linked to the 5’ position of the subsequent base of the RNA molecule ("second base") via a phosphodiester bond.

Presae of a cap on an RNA molecule is ly preferred if translation of a nucleic acid sequence encoding a protein at early stages after introduction of the respective [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg RNA into host cells or into a host organism is desired. For e, as shown in Example 4, presence of a cap allows that a gene of interest encoded by RNA replicon is translated efficiently at early stages after introduction of the respective RNA into host cells. "Early stages" typically means within the first 1 hour, or within the first two hours, or within the first three hours after introduction of the RNA.

Presence of a cap on an RNA molecule is also preferred if it is desired that translation occurs in the absence of functional ase, or when only minor levels of replicase are present in a host cell. For example, even if a nucleic acid molecule encoding replicase is introduced into a host cell, at early stages after introduction the levels of replicase will typically be minor.

In the system according to the invention, it is preferred that the RNA construct for ma A:HI‘ ‘Ilnn'flnnnl tirlln nan n'hnln Ilrnl +run nnmnnnnnn n 5’ Ann eprcSanly IuIIULIUIIal alpnavnuo IIUII'auuuLUIaI pr0tcm buulplloca a 'bdp.

In ular when the RNA replicon according to the present invention is not used or provided together with a second nucleic acid le (e.g. mRNA) that encodes functional alphavirus non-structural protein, it is preferred that the RNA replicon comprises a . Independently, the RNA replicon may also comprise a 5’—cap even when it is used or provided er with a second c acid molecule that encodes functional irus non-structural protein.

The term "conventional 5’-cap" refers to a naturally occurring 5’-cap, preferably to the 7-methylguanosine cap. In the 7-methylguanosine cap, the guanosine of the cap is a modified guanosine wherein the modification consists of a methylation at the 7- position (top of Fig. 6).

In the context of the present invention, the term "5’-cap analog" refers to a molecular structure that resembles a conventional 5’-cap, but is modified to possess the ability to stabilize RNA if attached thereto, preferably in vivo and/or in a cell. A cap analog is not a tional .

For tDcase of eukaryotic mRNA, the 5’-cap has been generally described to be involved in efficient translation of mRNA: in general, in otes, translation is [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg initiated only at the 5' end of a messenger RNA (mRNA) molecule, unless an internal ribosomal entry site (IRES) is present. Eukaryotic cells are capable of providing an RNA with a 5’-cap during transcription in the nucleus: newly synthesized mRNAs are usually modified with a 5’-cap structure, e.g. when the transcript reaches a length of to 30 nucleotides. First, the 5' terminal tide pppN (ppp representing triphosphate; N representing any nucleoside) is converted in the cell to 5' GpppN by a capping enzyme having RNA 5‘-triphosphatase and guanylyltransferase activities.

The GpppN may uently be methylated in the cell by a second enzyme with (guanine-7)-methyltransferase activity to form the ethylated m7GpppN cap. ln ' ! I I one embodiment, the 5 -capused'In the presentInve.two. :8 a natural 5 -cap.’ in he presentiImention, a natural 5’-cap dinucieotide is typically selected from the group consisting of a non-methylated cap dinucieotide (G(5')ppp(5')N; also termed GpppN) and a ated cap dinucieotide ((m7G(5‘)ppp(5')N; also termed N). m7GpppN (wherein N is G) is represented by the following formula: C? CH: m7Gp'ppG O " CII3 (ll) Cl)! N IN/J\N ‘2 Hz (5H P~o—P—o—P-o—VDZ\ [Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg In preferred embodiments of the present invention, the 5’-cap (if present) is a 5’-cap analog. These ments are ularly suitable if the RNA is obtained by in vitro transcription, e.g. is an in vitro transcribed RNA (lVT-RNA). Cap analogs have been initially described to facilitate large scale synthesis of RNA transcripts by means of in vitro ription.

For messenger RNA, some cap s (synthetic caps) have been generally described to date, and they can all be used in the context of the present invention.

Ideally, a cap analog is ed that is associated with higher translation efficiency andler increased resistance to in viva degradation and/or increased resistance to in vitro degradation.

Preferably, a cap analog is used that can only be incorporated into an RNA chain in one orientation. Pasquinelli et al. (1995, RNA J., vol., 1, pp. 957-967) demonstrated that during in vitro transcription, bacteriophage RNA polymerases use the 7- methylguanosine unit for tion of transcription, whereby around 40-50% of the transcripts with cap s the cap dinucleotide in a reverse orientation (i.e., the initial reaction product is Gpppm7GpN). Compared to the RNAs with a correct RNAs with a reverse cap are not functional with respect to translation of a nucleic acid sequence into protein. Thus, it is desirable to incorporate the cap in the correct orientation, i.e., ing in an RNA with a structure essentially corresponding to m7GpppGpN etc. It has been shown that the reverse integration of the cap- dinucleotide is inhibited by the substitution of either the 2'- or the 3'-OH group 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). RNAs which are synthesized in ce of such "anti reverse cap analogs" are translated more efficiently than RNAs which are in vitro transcribed in presence of the conventional 5’-cap m7GpppG.

To that end, one cap analog in which the 3’ OH group of the methylated guanosine unit is replaced by OCH3 is bed e.g. by Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017 (7-methyl(3’-O-methyl)GpppG; anti-reverse cap analog (ARCA)).

ARCA is a suitable cap dinucleotide according to the present invention.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg MW m; 3'-OGPPPG (ARCA) {NJKNH .r 7 try" I ‘\ {N/jkN/KNHz OH OH In a preferred embodiment of the present ion, the RNA of the present invention is essentially not susceptible to decapping. This is important because, in l, the (J1 amount of n ed from synthetic mRNAs introduced into cultured mammalian cells is d by the natural degradation of mRNA. One in vivo pathway for mRNA degradation begins with the removal of the mRNA cap. This removal is zed by a heterodimeric pyrophosphatase, which contains a regulatory subunit (Dcp1) and a catalytic subunit (Dcp2). The catalytic subunit cleaves between the a and B phosphate groups of the triphosphate bridge. In the present invention, a cap analog may be selected or present that is not susceptible, or less susceptible, to that type of cleavage. A le cap analog for this purpose may be selected from a cap dinucleotide according to formula (I): R‘°’..R3 0 2 3' NH R4 R5 [R6 l Preferred embodiments for R1, R2, R3, R4, R5, R6 are disclosed in A1 and may be selected ingly in the present invention.

For example, in a preferred embodiment of the present invention, the RNA of the present ion comprises a phosphorothioate-cap-analog. Phosphorothioate—cap- analogs are specific cap analogs in which one of the three idging O atoms in the triphosphate chain is replaced with an 8 atom, Le: one of R4, R5 or R6 in Formuia (I) is S. Phosphorothioate—cap-analogs have been described by J. Kowalska et al., 2008, RNA, vol. 14, pp. 1119-1131, as a solution to the undesired ing process, and thus to increase the stability of RNA in vivo. In particular, the substitution of an oxygen atom for a sulphur atom at the beta-phosphate group of the '—cap results in ization against Dcp2. In that embodiment, which is preferred in the present invention, R5 in Formula (I) is S; and R4 and R6 are 0.

In a further 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 " (ARCA) modification. tive ARCA—phosphorothioate—cap—analogs are described in WC 2008D688 A2, and they can all be used in the RNA of the present invention. In that embodiment, at least one of R2 or R3 in Formula (I) is not OH, preferably one [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg among R2 and R3 is methoxy (OCH3), and the other one among R2 and R3 is preferably OH. In a preferred embodiment, an oxygen atom is substituted for a sulphur atom at the beta-phosphate group (so that R5 in Formula (I) is S; and R4 and R6 are 0). It is believed that the phosphorothioate modification of the ARCA ensures that the or, [3, and y phosphorothioate groups are precisely positioned within the machinery. At least some of these analogs are essentially ant to pyrophosphatase Dcp1/Dcp2. Phosphorothioate—modified ARCAs were described to have a much higher ty for elF4E than the corresponding ARCAs lacking a phosphorothioate group. ’ A respective cap analog that is ularly preferred in the present invention, i.e., m2v7’2"°GppspG, is termed beta-S—ARCA (WO 57688 A2; Kuhn et al., Gene Ther., 2010, vol. 17, pp. 961-971). Thus, in one embodiment of the present invention, the RNA of the present invention is modified with beta-S-ARCA. beta—S-ARCA is represented by the following structure: J\ > 3? =3 3; HN/ N k!2' 3‘{j fiho-fi" C O 0' 3HCC13 0 DH 3' 2' OH. OH in general, the replacement of an oxygen atom for a sulphur atom at a bridging phosphate s in phosphorothioate diastereomers which are designated D1 and D2, based on their elution pattern in HPLC. Briefly, the D1 diastereomer of beta-S- ARCA" or "beta-S—ARCA(D1)" is the diastereomer of beta-S—ARCA which elutes first on an HPLC column compared to the D2 diastereomer of beta-S-ARCA (beta-S- 2)) and thus exhibits a shorter retention time. ination of the sterefiemical configuration by HPLC is described in [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg In a first particularly preferred embodiment of the present invention, RNA of the present invention is modified with the beta-S-ARCA(D2) diastereomer. The two diastereomers of beta-S-ARCA differ in sensitivity t nucleases. It has been shown that RNA carrying the D2 diastereomer of beta-S-ARCA is almost fully ant against Dcp2 ge (only 6% cleavage compared to RNA which has been synthesized in presence of the unmodified ARCA 5'-cap), whereas RNA with the beta-S—ARCA(D1) 5'—cap exhibits an intermediary sensitivity to Dcp2 cleavage (71% cleavage). It has further been shown that the increased stability against Dcp2 cleavage correlates with increased protein expression in mammalian cells. In -.\ CD particular, it has been shown that RNAe carrying the beta-S-ARCA(DZ) cap are mereIIIU cap. Therefore, in one embodiment of the present invention, RNA of the present invention is modified with a cap analog according to Formula (I), characterized by a chemical configuration at the P atom comprising the substituent R5 in Formula (I) that corresponds to that at the PB atom of the D2 reomer of beta-S-ARCA. In that embodiment, R5 in Formula (I) is S; and R4 and R6 are 0. Additionally, at least one of R7- or R3 in Formula (I) is preferably not OH, preferably one among R2 and R3 is methoxy , and the other one among R2 and R3 is preferably OH.

In a second particularly preferred embodiment, RNA of the present invention is modified with the beta-S—ARC-A(D1) diastereomer. This embodiment is particularly suitable for transfer of capped RNA into immature antigen presenting cells, such as for vaccination purposes. It has been trated that the beta-S-ARCA(D1) reomer, upon transfer of respectively capped RNA into immature antigen presenting cells, is ularly suitable for increasing the stability of the RNA, increasing translation efficiency of the RNA, prolonging translation of the RNA, increasing total protein expression of the RNA, and/or increasing the immune se against an antigen or n peptide encoded by said RNA (Kuhn et al., 2010, Gene Ther., vol. 17, pp. 961-971). Therefore, in an ative embodiment of the present ion, RNA of the present invention is modified with a cap analog according to Formula (I), characterized by a stereochemical configuration at the P atom comprising the substituent R5 in Formula (I) that corresponds to that at the P5 atomn the D1 diastereomer of beta-S-ARCA. Respective cap analogs and embodiments thereof are described in [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg Gene Ther., vol. 17, pp. 961-971. Any cap analog described in WO 15347 A1, wherein the stereochemical configuration at the P atom comprising the substituent R5 corresponds to that at the P5 atom of the D1 diastereomer of beta-S-ARCA, may be used in the present invention. Preferably, R5 in Formula (I) is S; and R4 and R6 are 0.

Additionally, at least one of R2 or R3 in Formula (I) is preferably not OH, preferably one among R2 and R3 is methoxy (OCH3), and the other one among R2 and R3 is preferably OH.

In one embodiment, RNA of the present invention is modified with a 5'-cap structure according to Formula (l) n any one phosphate group is replaced by a boranophosphate group or a orcselenoate group. Such caps have increased stability both in vitro and in vivo. Optionally, the respective compound has a 2'-O- or 3'-O-alkyl group in alkyl is ably methyl); respective cap analogs are termed BH3-ARCAs or Se-ARCAs. Compounds that are particularly suitable for capping of mRNA include the ARCAs and B-Se-ARCAS, as described in WO 2009/149253 A2. For these compounds, at stereochemical configuration at the P atom comprising the substituent R5 in Formula (I) that corresponds to that at the Pt; atom of the D1 reomer of beta-S-ARCA is preferred.

UTR The term "untranslated region" or "UTR" relates to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be present 5' eam) of an open reading frame (5’—UTR) and/or 3’ (downstream) of an open g frame (3’-UTR).

A , if present, is located at the 3' end of a gene, downstream of the termination codon of a protein-encoding region, but the term "3’-UTR" does preferably not e the poly(A) tail. Thus, the 3’-UTR is upstream of the poly(A) tail (if present), e.g. directly adjacent to the poly(A) tail.

A 5’-UTR, if present, is located at the 5‘ end of a gene, upstream of the start codon of a punt-encoding region. A 5’-UTR is downstream of the 5’-cap (if present), e.g. directly adjacent to the 5’—cap.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg '— and/or 3'—untranslated regions may, according to the invention, be functionally linked to an open reading frame, so as for these regions to be associated with the open reading frame in such a way that the stability and/or ation efficiency of the RNA comprising said open reading frame are increased.

In some embodiments, the ase construct according to the present invention comprises a 5’-UTR and/or a .

In a preferred embodiment, the replicase construct according to the present invention comprises (1) a 5’-UTR, (2) an open reading frame, and (3) a 3’-UTR.

UTRs are implicated in stability and translation efficiency of RNA. Both can be improved, besides structural modifications concerning the 5’-cap and/or the 3’ poly(A)-tail as described herein, by selecting specific 5’ and/or 3’ untranslated regions (UTRs). Sequence ts within the UTRs are generally understood to influence translational efficiency (mainly 5’-UTR) and RNA stability (mainly 3’-UTR). It is preferable that a 5’-UTR is present that is active in order to increase the translation ency and/or stability of the replicase construct. Independently or additionally, it is preferable that a 3’-UTR is present that is active in order to increase the translation efficiency and/or ity of the replicase construct.

The terms e in order to increase the translation efficiency" and/or "active in order to increase the stability", with reference to a first nucleic acid sequence (eg. a UTR), means that the first nucleic acid sequence is capable of modifying, in a common transcript with a second nucleic acid sequence, the ation efficiency and/or stability of said second nucleic acid sequence in such a way that said translation efficiency and/or stability is increased in comparison with the translation efficiny and/or stability of said second nucleic acid sequence in the e of said first c acid sequence.

[Annotation] mpg None set by mpg ation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg In one embodiment, the replicase construct according to the present invention comprises a 5'—UTR and/or a 3'-UTR which is heterologous or non-native to the alphavirus from which the onal alphavirus non-structural protein is derived. This allows the slated regions to be designed according to the desired ation efficiency and RNA stability, Thus, heterologous or non-native UTRs allow for a high degree of ility, and this flexibility is advantageous compared to native alphaviral UTRs. In particular, while it is known that alphaviral (native) RNA also comprises a 5’- UTR and/or a 3’-UTR, alphaviral UTRs fulfil a dual function, i.e. (i) to drive RNA 1O replication as well as (ii) to drive translation. While alphaviral UTRs were reported to be inefficient for translation (Berben-Bloemheuvel et al., 1992, Eur. J. Biochem., vol. 208, pp. 581-587), they can typically not readily be replaced by more efficient UTRs because of their dual function. In the t invention, however, a 5’-UTR and/or a 3’-UTR comprised in a replicase construct for replication in trans can be selected ndent of their potential influence on RNA replication.

Preferably, the ase construct according to the present invention comprises a 5'- UTR and/or a 3’-UTR that is not of virus origin; particularly 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 d from a eukaryotic 3’-UTR.

A 5’-UTR according to the present invention can comprise any ation of more than one nucleic acid sequence, ally separated by a linker. A 3’-UTR according to the present invention can comprise any combination of more than one nucleic acid sequence, optionally separated by a linker.

The term "linker" according to the invention relates to a nucleic acid sequence added between two c acid sequences to connect said two nucleic acid sequences.

There is no particular limitation regarding the linker sequence.

A 3’-UTR typically has a length of 200 to 2000 nucleotides, eg. 500 to 1500 nucleotides. The 3'—untranslated regions of globulin mRNAs are relatively shortD/ver than about 300 tides), while the 3'-untranslated regions of other genes are relatively long. For example, the 3'-untranslated region of tPA is about 800 [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg ation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg nucleotides in length, that of factor VIII is about 1800 nucleotides in length and that of erythropoietin is about 560 nucleotides in length. The 3'—untranslated regions of mammalian mRNA typically have a homology region known as the AAUAAA cleotide sequence. This ce is presumably the ) attachment signal and is frequently located from 10 to 30 bases upstream of the poly(A) attachment site. 3'—untranslated regions may contain one or more inverted repeats which can fold to give stem-loop structures which act as barriers for exoribonucleases or interact with proteins known to increase RNA ity (e.g. RNA- g proteins).

The human beta-globin 3’-UTR, particularly two consecutive identical copies of the human beta-globin 3’-UTR, contributes to high transcript stability and translational efficiency (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017). Thus, in one embodiment, the replicase construct according to the present ion comprises two consecutive identical copies of the human beta-globin 3’-UTR. Thus, it comprises in the 5‘ —> 3' direction: (a) optionally a 5’-UTR; (b) an open reading frame; (c) a 3’- UTR; said 3'-UTR comprising two consecutive cal copies of the human beta- globin 3’-UTR, a nt thereof, or a variant of the human beta-globin 3’-UTR or fragment thereof.

In one embodiment, the replicase construct according to the present invention comprises a 3’-UTR which is active in order to increase translation efficiency and/or stability, but which is not the human beta-globin 3’-UTR, a fragment thereof, or a variant of the human beta-globin 3’-UTR or fragment thereof.

In one embodiment, the replicase uct according to the present inventionIIV comprises a 5’-UTR which is active in order to increase translation efficiency and/or stability.

A UTR-containing replicase construct according to the invention can be prepared by in vitro transcription. This may be ed by genetically ing a template c acid molecule (e.g. DNA) in such a way that it allows transcription of RNA with SDRS and/or 3’-UTRs.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg As illustrated in Fig. 1, also the replicon can be characterized by a 5’-UTR and/or a . The UTRs of the replicon are typically alphaviral UTRs or variants thereof.

Poly(A) sequence In some embodiments, the replicon ing to the present invention comprises a 3’-po|y(A) sequence. If the replicon comprises ved 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. _.\ (D I." some ments, the replicase construct according to the present invention comprises a 3 -poiy(A) sequence.

According to the invention, in one embodiment, a poly(A) sequence comprises or ially consists 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 up to 500, preferably up to 400, preferably up to 300, preferably up to 200, and in particular up to 150, A nucleotides, and in particular about 120 A nucleotides. In this context "essentially consists of" means that most nucleotides in the poly(A) ce, typically at least 50 %, and preferably at least 75 % by number of nucleotides in the "poly(A) sequence", are A nucleotides (adenylate), but permits that ing nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G tides (guanylate), C nucleotides (cytidylate). In this context "consists of" means that all nucleotides in the poly(A) sequence, i.e. 100 % by number of nucleotides in the poly(A) sequence, are A tides. The term "A nucleotide" or "A" refers to adenylate.

Indeed, it has been demonstrated that a 3' poly(A) ce of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5’) of the 3' poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

In alpDiruses, a 3' poly(A) sequence of at least 11 consecutive adenylate es, or at least 25 utive adenylate residues, is thought to be important for efficient ation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg synthesis of the minus strand. In particular, in alphaviruses, a 3' poly(A) sequence of at least 25 consecutive adenylate residues is understood to function together with conserved sequence element 4 (CSE 4) to promote synthesis of the (-) strand (Hardy & Rice, J. Virol., 2005, vol. 79, pp. 4630-4639).

The present invention provides for a 3’ poly(A) sequence to be attached during RNA transcription, i.e. during preparation of in vitrc transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence ng a poly(A) 1O sequence (coding strand) is referred to as poly(A) cassette. in a preferred embodiment of the t ion, the 3' poly(A) cassette t in the coding strand of DNA essentially consists of dA tides, but is upted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT). Such random sequence may be 5 to 50, preferably 10 to 30, more preferably 10 to 20 nucleotides in length. Such a cassette is disclosed in poly(A) cassette disclosed in ion. A poly(A) te that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal bution of the four nucleotides (dA, dC, dG, dT) and having a length of eg. 5 to 50 nucleotides shows, on DNA level, constant propagation of d DNA in E.coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency.

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

Codon usage In general, the degeneracy of the genetic code will allow the substitution of certain codorDase triplets coding for an amino acid) that are t in an RNA sequence by other codons (base triplets), while maintaining the same coding capacity (so that [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg ation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg the ing codon encodes the same amino acid as the replaced codon). In some embodiments of the present ion, at least one codon of an open reading frame comprised by a RNA molecule differs from the respective codon in the respective open reading frame in the species from which the open reading frame ates. in that embodiment, the coding sequence of the open reading frame is said to be "adapted" or "modified". The coding sequence of an open reading frame comprised by the replicon may be adapted. Alternatively or additionally, the coding sequence for functional alphavirus non-structural protein comprised by the replicase construct may be adapted.

For example, when the coding sequence of an open reading frame is adapted, frequently used codons may be selected: WO 2009/024567 A’l describes the adaptation of a coding ce of a nucleic acid molecule, involving the substitution of rare codons by more frequently used codons. Since the frequency of codon usage depends on the host cell or host organism, that type of adaptation is suitable to fit a nucleic acid sequence to expression in a particular host cell or host organism.

Generally, speaking, more frequently used codons are typically translated more efficiently in a host cell or host organism, although adaptation of all codons of an open reading frame is not always required.

For example, when the coding sequence of an open reading frame is d, the content of G (guanylate) residues and C (cytidylate) residues may be altered by ing codons with the t GC-rich content for each amino acid. RNA molecules with GC-rich open reading frames were reported to have the potential to reduce immune tion and to improve translation and half-life of RNA (Thess et al., 2015, Mol. Ther. 23, 1457-1465).

When the replicon according to the present invention encodes irus non- structural n, the coding sequence for alphavirus ructural protein can be adapted as desired. This m is possible because the open reading frame encoding alphavirus non-structural protein does not overlap with the 5’ replication recognition sequence of the replicon.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg Safety features of ments of the present invention The following features are preferred in the present invention, alone or in any suitable ation: Preferably, the replicon or the system of the present invention is not particle-forming.

This means that, following inoculation of a host cell by the replicon or the system of the t invention, the host cell does not produce virus particles, such as next generation virus particles. In one ment, all RNA molecules according to the invention are completely free of genetic information ng any alphavirus structural protein, such as core capsid protein C, envelope protein P62, andlor envelope protein E1. This aspect of the present invention provides an added value in terms of safety over prior art systems wherein structural proteins are encoded on trans-replicating helper RNA (e.g. Bredenbeek eta/., J. Virol, 1993, vol. 67, pp. 6439- 6446).

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

Preferably, the replicon and the replicase construct of the system of the present invention are non-identical to each other. In one embodiment, the replicon does not encode functional alphavirus non-structural protein. in one embodiment, the replicase construct lacks at least one sequence element (preferably at least one CSE) that is required for (-) strand synthesis based on a (+) strand template, and/or for (+) strand synthesis based on a (-) strand template. In one ment, the replicase construct does not comprise CSE 1 and/or CSE 4. ably, neither the replicon according to the present ion nor the replicase construct according to the present invention ses an irus packaging signal. For example, the alphavirus packaging signal comprised in the coding region of nsP2 of SFV (White et al. 1998, J. Virol., vol. 72, pp. 4320-4326) may be removed, e.g. by deletion or mutation. A le way of removing the alphavirus packaging signancludes adaptation of the codon usage of the coding region of nsP2. The [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg degeneration of the genetic code may allow to delete the function of the ing signal without affecting the amino acid sequence of the encoded nsP2.

In one embodiment, the system of the present invention is an ed system. In that embodiment, the system is not present inside a cell, such as inside a mammalian cell, or is not present inside a virus capsid, such as inside a coat comprising alphavirus structural proteins. In one embodiment, the system of the present invention is present in vitro. 1O DNA in a third aspect, the present invention provides a DNA comprising a nucleic acid ce encoding the RNA repiicon ing to the first aspect of the present invenfion.

Preferably, the DNA is -stranded.

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

The DNA of the present invention may comprise a promoter that can be recognized by a DNA-dependent lymerase. This allows for transcription of the encoded RNA in vivo or in vitro, e.g. of the RNA of the present invention. IVT vectors may be used in a standardized manner as template for in vitro transcription. Examples of ers preferred according to the invention are promoters for SP6, T3 or T7 polymerase.

In one ment, the DNA of the present invention is an isolated nucleic acid molecule.

Methods of preparing RNA Any DA molecule according to the present invention, be it part of the system of the present invention or not, may be obtainable by in vitro transcription. In vitro- ation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg transcribed RNA (lVT-RNA) is of particular interest in the present invention. lVT-RNA is obtainable by transcription from a c acid molecule (particularly a DNA molecule). The DNA molecule(s) of the third aspect of the present invention are suitable for such purposes, particularly if cOmprising a promoter that can be recognized by a DNA-dependent RNA-polymerase.

RNA according to the present invention can be synthesized in vitro. This allows to add cap-analogs to the in vitro transcription reaction. Typically, the poly(A) tail is encoded by a poly-(dT) sequence on the DNA template. Alternatively, capping and _.\ C3 poly(A) tail addition can be achieved enzymatically after ription.

The in vitro transcription methodology is known to the skilled person. For e, as mentioned in commercially available.

The present invention also provides a kit comprising an RNA replicon according to the first aspect of the invention or a system according to the second aspect of the invention. !n one embodiment, the constituents of the kit are present as separate entities. For example, one nucleic acid molecule of the kit may be present in one entity, and the r nucleic acid of the kit may be present in a te . For example, an open or closed ner is a suitable entity. A closed container is preferred. The container used should preferably be RNAse-free or essentially RNAse-free.

In one ment, the kit of the present invention ses RNA for inoculation with a cell and/or for administration to a human or animal subject.

The kit according to the present invention optionally comprises a label or other form of information element, eg. an electronic data carrier. The label or information element preferably comprises ctions, e.g. printed written ctions or instrLDns in electronic form that are optionally printable. The instructions may refer to at least one suitable possible use of the kit.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg ation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg Pharmaceutical composition The ase construct and/or the replicon described herein may be present in the form of a pharmaceutical composition. A pharmaceutical composition according to (J1 the ion may comprise at least one nucleic acid molecule according to the present invention. A pharmaceutical composition according to the invention comprises a pharmaceutically acceptable diluent and/or a pharmaceutically able excipient and/or a pharmaceutically acceptable carrier and/or a pharmaceutically able e. The choice of pharmaceutically acceptable .A CD carrier, vehicle, ent or diluent is not particularly limited. Any suitable pharmaceutically acceptable carrier, vehicle, excipient or diluent known in the art may be used.

In one ment of the present invention, a pharmaceutical composition can further comprise a solvent such as an aqueous solvent or any solvent that makes it possible to preserve the integrity of the RNA. In a preferred embodiment, the pharmaceutical composition is an aqueous solution comprising RNA. The aqueous solution may optionally comprise solutes, e.g. salts.

In one ment of the present invention, the pharmaceutical composition is in the form of a freeze-dried composition. A freeze-dried composition is obtainable by freeze-drying a respective aqueous composition.

In one embodiment, the pharmaceutical composition comprises at least one cationic entity. In l, ic lipids, cationic polymers and other substances with positive charges may form complexes with negatively charged nucleic acids. It is possible to stabilize the RNA according to the invention by complexation with cationic compounds, preferably polycationic compounds such as for example a cationic or polycationic peptide or protein. In one embodiment, the pharmaceutical composition ing to the present invention comprises at least one cationic molecule selected from the group ting ine, polyethylene imine, a poly-L—Iysine, a poly-L- arginine, a histone or a cationic lipid.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg According to the present invention, a cationic lipid is a ic amphiphilic molecule, e.g., a molecule which comprises at least one hydrophilic and lipophilic moiety. The cationic lipid can be monocationic or polycationic. Cationic lipids typically have a lipophilic moiety, such as a sterol, an acyl or diacyl chain, and have an overall net positive . The head group of the lipid typically carries the ve charge. The cationic lipid preferably has a positive charge of 1 to 10 valences, more preferably a positive charge of 1 to 3 valences, and more preferably a positive charge of 1 valence. Examples of cationic lipids include, but are not limited to 1,2-di-O- octadecenyItrimethylammonium propane (DOTMA); dimethyldioctadecylammonium (DDAB); 1,2-dioleoyltrimethylammonium-propane (DOTAP); 1,2-dioleoyl—3-dimethylammonium-propane (DODAP); acyloxy—3- dimethylammonium propanes; aikyloxy—S-dimethyiammonium propanes; decyldimethyl ammonium chloride (DODAC), 1,2-dimyristoyloxypropyl-1,3- dimethylhydroxyethyl ammonium (DMRIE), and 2,3-dioleoyloxy—N-[2(spermine carboxamide)ethyl]-N,N-dimethylpropanamium trifluoroacetate (DOSPA). Cationic lipids also include lipids with a tertiary amine group, including linoleyloxy—N,N- dimethylaminopropane (DLinDMA). Cationic lipids are suitable for formulating RNA in lipid formulations as described herein, such as liposomes, emulsions and lipoplexes. Typically positive charges are contributed by at least one cationic lipid and negative charges are contributed by the RNA. In one embodiment, the pharmaceutical composition comprises at least one helper lipid, in addition to a ic lipid. The helper lipid may be a l or an anionic lipid. The helper lipid may be a natural lipid, such as a phospholipid, or an ue of a natural lipid, or a fully synthetic lipid, or lipid-like molecule, with no similarities with natural lipids. In the case where a pharmaceutical composition includes both a cationic lipid and a helper lipid, the molar ratio of the cationic lipid to the neutral lipid can be appropriately determined in view of ity of the formulation and the like.

In one embodiment, the pharmaceutical composition according to the t invention comprises protamine. According to the invention, protamine is useful as cationic carrier agent. The term "protamine" refers to any of s strongly basic proteins of relatively low molecular weight that are rich in arginine and are found assoned especially with DNA in place of somatic histones in the sperm cells of animals such as fish. In particular, the term mine" refers to proteins found in ation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and se multiple arginine monomers. According to the ion, the term "protamine" as used herein is meant to comprise any ine amino acid sequence obtained or derived from native or ical sources including fragments thereof and multimeric forms of said amino acid sequence or fragment thereof.

Furthermore, the term encompasses (synthesized) polypeptides which are artificial and specifically ed for specific purposes and cannot be isolated from native ical sources. 1O In some embodiments, the compositions of the invention may comprise one or more adjuvants. Adjuvants may be added to es to stimulate the immune system‘s response; adjuvants do not typically provide immunity themselves. Exemplary adjuvants include without limitation the following: Inorganic compounds (eg. alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide); mineral oil (e.g. paraffin oil), cytokines (e.g. lL-1, lL-2, |L-12); stimulatory polynucleotide (such as RNA or DNA; e.g., CpG-containing oligonucleotides); saponins (e.g. plant saponins from Quillaja, Soybean, Polygala senega); oil emulsions or liposomes; polyoxy ethylene ether and poly oxy ne ester formulations; polyphosphazene (PCPP); l peptides; imidazoquinolone compounds; thiosemicarbazone compounds; the Flt3 ligand ( known by a person skilled in the art. A preferred nt for administration of RNA according to the present invention is the Flt3 ligand ( Flt3 ligand is administered er with RNA that codes for an antigen, a strong increase in n-specific coa+ T cells may be observed.

The pharmaceutical composition ing to the invention can be buffered, (e.g., with an acetate buffer, a citrate buffer, a succinate buffer, a Tris buffer, a phosphate buffer).

RNA-containing particles In some embodiments, owing to the instability of non-protected RNA, it is advantageous to provide the RNA molecules of the present invention in complexed or enDsulated form. Respective pharmaceutical compositions are provided in the present invention. In particular, in some embodiments, the pharmaceutical [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg composition of the t invention comprises nucleic acid-containing particles, preferably RNA-containing particles. Respective pharmaceutical compositions are referred to as particulate formulations. In particulate formulations according to the present invention, a particle ses nucleic acid according to the invention and a pharmaceutically acceptable carrier or a ceutically acceptable vehicle that is suitable for delivery of the nucleic acid. The nucleic acid-containing particles may be, for example, in the form of proteinaceous particles or in the form of lipid-containing particles. Suitable proteins or lipids are referred to as particle forming agents.

Proteinaceous particles and containing particles have been described previously to be suitable for delivery of ira! RNA in particulate form (e.g. Strauss & s, Microbiol. Rev., 1994, vol. 58, pp. 491-562). In ular, alpha‘v‘irus structurat proteins (provided e.g. by a helper virus) are a le carrier for delivery of RNA in the form of proteinaceous les.

When the system according to the present invention is formulated as a particulate ation, it is possible that each RNA species (e.g. replicon, replicase construct, and optional onal RNA species such as an RNA encoding a protein suitable for inhibiting IFN) is separately formulated as an individual particulate formulation. In that case, each individual particulate formulation will comprise one RNA species. The dual particulate formulations may be present as separate entities, e.g. in separate containers. Such formulations are able by providing each RNA species separately (typically each in the form of an RNA-containing solution) together with a particle-forming agent, y allowing the formation of particles. Respective particles will contain exclusively the specific RNA species that is being provided when the particles are formed (individual particulate formulations).

In one embodiment, a pharmaceutical composition according to the invention comprises more than one individual particle formulation. Respective ceutical compositions are referred to as mixed ulate formulations. Mixed particulate formulations according to the invention are obtainable by forming, separately, individual particulate formulations, as described above, followed by a step of mixing of the individual particulate formulations. By the step of mixing, one formulation compatg a mixed population of RNA-containing particles is obtainable (for illustration: e.g. a first population of particles may contain replicon according to the [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg ation] mpg None set by mpg ation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg invention, and a second formulation of particles may contain replicase construct ing to the invention). Individual particulate populations may be together in one container, comprising a mixed tion of individual particulate formulations.

Alternatively, it is possible that all RNA species of the ceutical composition (e.g. repliccn, replicase construct, and optional additional species such as RNA encoding a protein le for inhibiting lFN) are formulated together as a combined particulate formulation. Such formulations are obtainable by providing a combined formulation (typically combined solution) of all RNA species together with a particle- —}l C) forming agent, thereby allowing the formation of particles. As opposed to a mixed particulate formulation, a combined ulate formulation will typically se particles which comprise more than one RNA species. In a ed particulate composition different RNA species are typically present together in a single particle.

In one embodiment, the particulate formulation of the present invention is a nanoparticulate formulation. in that embodiment, the composition according to the present invention comprises nucleic acid according to the invention in the form of nanoparticles. Nanoparticulate formulations can be obtained by various protocols and with various complexing compounds. Lipids, polymers, oligomers, or amphipiles are typical constituents of nanoparticulate formulations.

As used herein, the term "nanoparticle" refers to any particle having a diameter making the particle suitable for systemic, in ular parenteral, administration, of, in particular, nucleic acids, typically a diameter of 1000 nanometers (nm) or less. In one embodiment, the nanoparticles have an average diameter in the range of from about 50 nm to about 1000 nm, preferably from about 50 nm to about 400 nm, preferably about 100 nm to about 300 nm such as about 150 nm to about 200 nm. In one embodiment, the rticles have a diameter in the range of about 200 to about 700 nm, about 200 to about 600 nm, preferably about 250 to about 550 nm, in particular about 300 to about 500 nm or about 200 to about 400 nm. in one embodiment, the polydispersity index (PI) of the nanoparticles described s measured by c light ring, is 0.5 or less, preferably 0.4 or less or even more preferably 0.3 or less. The "polydispersity index" (PI) is a measurement [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg of homogeneous or heterogeneous size distribution of the dual particles (such as liposomes) in a partrcle mrxture and indicates the breadth of the p9) rttcle distribution in a mixture. The PI can be determined, for example, as described in WO 2013/143555 A1.

As used herein, the term "nanoparticulate formulation" or similar terms refer to any particulate formulation that contains at least one nanoparticle. In some ments, a nanoparticulate composition is a uniform collection of nanoparticles. In some ments, a nanoparticulate composition is a lipid-containing pharmaceutical _k C3 formulation, such as a tiposome formulation or an emulsion.

Lipid-containing pharmaceutical compositions In one embodiment, the pharmaceutical ition of the present invention comprises at least one lipid. Preferably, at least one lipid is a cationic lipid. Said lipid- containing pharmaceutical composition ses nucleic acid according to the present ion. In one embodiment, the pharmaceutical composition according to the invention comprises RNA encapsulated in a vesicle, e.g. in a liposome. In one embodiment, the pharmaceutical composition according to the invention comprises RNA in the form of an emulsion. In one embodiment, the pharmaceutical composition according to the invention comprises RNA in a complex with a cationic compound, thereby g e.g. so-called exes or polyplexes. Encapsulation of RNA within vesicles such as liposomes is distinct from, for instance, lipid/RNA complexes.

RNA complexes are able e.g. when RNA is e.g. mixed with pre-formed liposomes.

In one embodiment, the pharmaceutical composition according to the invention comprises RNA encapsulated in a vesicle. Such formulation is a particular particulate formulation according to the invention. A vesicle is a lipid bilayer rolled up into a cal shell, enclosing a small space and ting that space from the space outside the vesicle. Typically, the space inside the vesicle is an aqueous space, i.e. comprises water. Typically, the space outside the vesicle is an aqueous space, i.e. comprises water. The lipid bilayer is formed by one or more lipids (vesicle-forming lipidsate membrane enclosing the vesicle is a lamellar phase, similar to that of the plasma membrane. The e according to the present invention may be a [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg multilamellar e, a unilamellar vesicle, or a e thereof. When encapsulated in a vesicle, the RNA is typically separated from any external medium. Thus it is present in protected form, functionally equivalent to the protected form in a natural alphavirus. Suitable vesicles are particles, particularly nanoparticles, as described For example, RNA may be encapsulated in a Iiposome. In that embodiment, the pharmaceutical composition is or comprises a liposome formulation. Encapsulation within a liposome will typically protect RNA from RNase digestion. It is possible that 1O the liposomes include some external RNA (e.g. on their e), but at least half of the RNA (and ideally all of it) is encapsulated within the core of the liposome.

Liposomes are microscopic lipidic vesicles often having one or more bilayers of a vesicle-forming lipid, such as a phospholipid, and are capable of encapsulating a drug, e.g. RNA. Different types of liposomes may be ed in the context of the present invention, including, without being limited thereto, multilamellar vesicles (MLV), small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), sterically stabilized liposomes (SSL), multivesicular vesicles (MV), and large multivesicular vesicles (LMV) as well as other red forms known in the art. The size and 2O lamellarity of the me will depend on the manner of preparation. There are several other forms of supramolecular organization in which lipids may be t in an aqueous medium, comprising lamellar phases, hexagonal and inverse hexagonal phases, cubic phases, micelles, reverse es composed of monolayers. These phases may also be obtained in .he combination with DNA or RNA, and the ction with RNA and DNA may substantially affect the phase state. Such phases may be present in rticulate RNA formulations of the present invention.

Liposomes may be formed using rd methods known to the skilled person.

Respective methods include the reverse evaporation method, the ethanol injection method, the dehydration-rehydration method, sonication or other le methods.

Following liposome formation, the liposomes can be sized to obtain a population of liposomes having a substantially homogeneous size range.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg In a red embodiment of the present invention, the RNA is present in a liposome which includes at least one cationic lipid. Respective liposomes can be formed from a single lipid or from a mixture of lipids, provided that at least one cationic lipid is used. red cationic lipids have a nitrogen atom which is capable of being ated; preferably, such cationic lipids are lipids with a tertiary amine group. A particularly suitable lipid with a tertiary amine group is 1,2-dilinoleyloxy—N,N-dimethyI aminopropane (DLinDMA). In one embodiment, the RNA according to the present invention is t in a liposome ation as described in liposome having a lipid bilayer encapsulating an s core including RNA, wherein the lipid bilayer comprises a lipid with a pKa in the range of 5.0 to 7.6, which preferably has a tertiary amine group. Preferred cationic lipids with a tertiary amine group include DLinDMA (pKa 5.8) and are generally described in A2. According to are particularly suitable for encapsulation of RNA and thus mal delivery of RNA. In one embodiment, the RNA according to the present ion is present in a liposome formulation, wherein the liposome includes at least one cationic lipid whose head group includes at least one nitrogen atom (N) which is capable of being protonated, wherein the liposome and the RNA have a N:P ratio of between 1:1 and :1. ing to the present invention, "N:P ratio" refers to the molar ratio of nitrogen atoms (N) in the cationic lipid to phosphate atoms (P) in the RNA comprised in a lipid containing particle (eg. liposome), as described in The N:P ratio of between 1:1 and 20:1 is implicated in the net charge of the liposome and in efficiency of delivery of RNA to a vertebrate cell.

In one embodiment, the RNA ing to the present invention is present in a me formulation that comprises at least one lipid which es a polyethylene glycol (PEG) moiety, wherein RNA is encapsulated within a PEGylated liposome such that the PEG moiety is present on the liposome’s exterior, as described in WO 2012/031043 A1 and In one embodiment, the RNA according to the present invention is present in a liposome formulation, n the liposome has a diameter in the range of 60-180 nm, anescribed in wo 2012/030901 A1.

[Annotation] mpg None set by mpg ation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg In one embodiment, the RNA according to the present invention is present in a Iiposome formulation, wherein the RNA-containing Iiposomes have a net charge close to zero or negative, as disclosed in WC) 201311. 43555 A1 In other embodiments, the RNA according to the present invention is present in the form of an emulsion. Emulsions have been previously described to be used for delivery of nucleic acid molecules, such as RNA les, to cells. Preferred herein are oil-in-water emulsions. The respective emulsion particles comprise an oil core and a cationic lipid. More preferred are cationic oil-in-water emulsions in which the RNA according to the present invention is complexed to the emulsion particles. The emulsion particles comprise an oil core and a cationic iipid. The ic lipid can interact with the negatively d RNA, y anchoring the RNA to the emulsion particles. In an oil-in-water emulsion, emulsion particles are dispersed in an aqueous continuous phase. For example, the average diameter of the emulsion les 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 WO 2012/006380 A2. The RNA ing to the present ion may be present in the form of an emulsion comprising a cationic lipid wherein the NP ratio of the emulsion is at least 4:1, as described in present invention may be present in the form of a cationic lipid emulsion, as bed in complexed with a particle of a cationic oil-in-water emulsion, wherein the ratio of pid is at least about 8:1 mole).

In other embodiments, the pharmaceutical ition according to the invention comprises RNA in the format of a lipoplex. The term, "lipoplex" or "RNA lipoplex" refers to a complex of lipids and nucleic acids such as RNA. Lipoplexes can be formed of cationic (positively charged) Iiposomes and the anionic (negatively d) nucleic acid. The cationic Iiposomes can also include a neutral "helper" lipid. In the simplest case, the lipoplexes form spontaneously by mixing the nucleic acid with the Iiposomes with a certain mixing protocol, however s other proton may be applied. It is understood that ostatic interactions between positively charged Iiposomes and negatively charged nucleic acid are the driving [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg force for the lipoplex formation ( present invention, the net charge of the RNA lipoplex particles is close'to zero or ve. It is known that electro-neutral or negatively charged Iipoplexes of RNA and Iiposomes lead to ntial RNA expression in spleen dendritic cells (DCs) after systemic stration and are not associated with the elevated toxicity that has been reported for positively charged Iiposomes and Iipoplexes (cf.

A1). Therefore, in one embodiment of the present invention, the pharmaceutical composition according to the invention comprises RNA in the format of nanoparticles, preferably lipoplex nanoparticles, in which (i) the number of positive charges in the -3. CD nanoparticles does not exceed the number of negative charges in the nanoparticles and/or (ii) the nanoparticles have a neutral or net negative charge and/or (iii) the charge ratio of positive charges to negative charges in the nanoparticles is 1.4:1 or less and/or (iv) the zeta potential of the nanoparticles is O or less. As described in colloidal systems. In the present ion, (a) the zeta ial and (b) the charge ratio of the cationic lipid to the RNA in the nanoparticles can both be calculated as disclosed in are rticulate lipoplex formulations with a defined particle size, wherein the net charge of the les is close to zero or negative, as disclosed in WO 43555 A1, are red pharmaceutical compositions in the context of the present invention.

Methods for ing a protein in a fourth aspect, the present ion provides a method for producing a n of interest in a cell comprising the steps of: (a) obtaining the RNA replicon according to the first aspect of the invention, which comprises an open reading frame encoding functional alphavirus non-structural protein, which can be replicated by the functional alphavirus ructural protein and which further comprises an open reading frame encoding the protein of interest, (b) inoculating the RNA replicon into the cell.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg In various embodiments of the method, the RNA replicon is as defined above for the RNA replicon of the invention, as long as the RNA rep icon comprises an open reading frame encoding functional alphavirus non-structural protein and an open reading frame encoding the protein of interest, and can be replicated by the functional alphavirus non-structural protein.

In a fifth aspect, the present invention provides a method for producing a protein of interest in a cell sing the steps of: -3 CD (a) obtaining a RNA construct fcr expressing functional alphavirus non-structural protein, (b) obtaining the RNA replicon according to the first aspect of the ion, which can be replicated by the onal alphavirus non-structural protein according to (a) in trans and which comprises an open reading frame encoding the n of interest, and (c) co-inoculating the RNA construct for expressing functional alphavirus non- structural protein and the RNA replicon into the cell.

In various embodiments of the method, the RNA construct for expressing functional alphavirus non-structural protein and/or the RNA replicon are as defined above for the system of the ion, as long as the RNA replicon can be replicated by the functional alphavirus non-structural protein in trans and comprises an open reading frame encoding the protein of interest. The RNA construct for sing functional alphavirus non-structural protein and the RNA on may either be inoculated at the same point in time, or may alternatively be ated at different points in time.

In the second case, the RNA construct for expressing functional alphavirus non- structural protein is typically inoculated at a first point in time, and the replicon is lly ated at a , later, point in time. In that case, it is envisaged that the replicon will be immediately replicated since replicase will already have been synthesized in the cell. The second point in time is typically shortly after the first point in time, e.g. 1 minute to 24 hours he first point in time.

The D into which one or more nucleic molecule can be inoculated can be referred to as "host cell". According to the invention, the term "host cell" refers to any cell [Annotation] mpg None set by mpg [Annotation] mpg ionNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg which can be transformed or transfected with an exogenous nucleic acid molecule.

The term "cell" preferably is an intact cell, Le. a cell with an intact membrane that has not released its normal intracellular components such as enzymes, organelles, or genetic material. An intact cell preferably is a viable cell, Le. a living cell capable of carrying out its normal lic functions. The term "host cell" ses, according to the invention, prokaryotic (e.g. Eco/i} or eukaryotic cells (e.g. human and animal cells, plant cells, yeast cells and insect cells). Particular preference is given to mammalian cells such as cells from humans, mice, hamsters, pigs, domesticated animals including horses, cows, sheep and goats, as well as primates. The cells may be derived from a multiplicity of tissue types and comprise primary cells and cell lines. Specific examples include keratinocytes, peripheral blood leukocytes. bone marrow stem cells and embryonic stem cells. In other ments, the host cell is an antigen-presenting cell, in ular a dendritic cell, a monocyte or a macrophage.

A nucleic acid may be present in the host cell in a single or in several copies and, in one embodiment is expressed in the host cell.

The cell may be a prokaryotic cell or a eukaryotic cell. yotic cells are suitable herein e.g. for ation of DNA according to the invention, and eukaryotic cells are suitable herein e.g. for expression of the open g frame of the replicon.

In the method of the present ion, any of the system according to the invention, or the kit according to the invention, or the pharmaceutical composition according to the invention, can be used. RNA can be used in the form of a pharmaceutical composition, or as naked RNA e.g. for electroporation.

According to the method of the present invention, efficient expression of a gene of st in a host cell can be achieved (see e.g. Examples 2 to 5).

In one embodiment, an onal RNA molecule, preferably an mRNA molecule, may be inoculated with the cell. Optionally, the additional RNA molecule encodes a protein suitable for inhibiting IFN, such as E3, as described herein. Optionally, the additional RNA molecule may be inoculated prior to inoculation of the replicon or of the rnsase construct or of the system according to the invention.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg 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 for expressing the RNA encoding the antigen. To this end, the invention may involve introduction of RNA encoding antigen into antigen presenting cells such as dendritic cells. For transfection of antigen presenting cells such as dendritic cells a ceutical composition sing RNA encoding the antigen may be used.

In one embodiment, a method for producing a protein in a cell is an in vitro method.

In one embodiment, a method for production of a protein in a cell does not comprise the removal of a cell from a human or animal subject by surgery or therapy.

In this ment, the cell inoculated according to the fourth aspect of the invention may be administered to a subject so as to produce the n in the subject and to provide the subject with the protein. The cell may be autologous, syngenic, allogenic or heterologous with respect to the subject.

In other embodiments, the cell in a method for producing a protein in a cell may 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 administration of RNA molecules to the subject.

In this respect, the invention also provides a method for producing a protein of interest in a subject comprising the steps of: (a) obtaining the RNA replicon according to the first aspect of the invention, which comprises an open g frame encoding onal alphavirus non-structural protein, which can be replicated by the onal alphavirus ructural protein and which further comprises an open reading frame encoding the protein of interest, (b) administering the RNA replicon to the subject.

In various embodiments of the method, the RNA replicon is as defined above for the RNAnillcon of the invention, as long as the RNA replicon ses an open reading frame encoding onal alphavirus ructural protein and an open [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg reading frame encoding the protein of interest, and can be replicated by the functional alphavirus ructural protein.

The invention further provides a method for producing a protein of interest in a t comprising the steps of: (a) obtaining a RNA construct for expressing functional alphavirus ructural protein, (b) obtaining the RNA replicon according to the first aspect of the invention, which can be replicated by the functional alphavirus non-structural protein according to (a) in trans- and which comprise" an open reading frame encoding the protein of interest, (c) administering the RNA construct for expressing functional alphavirus non- structural protein and the RNA on to the subject.

In various embodiments of the method, the RNA construct for expressing functional alphavirus non-structural n and/or the RNA replicon are as defined above for the system of the invention, as long as the RNA replicon can be replicated by the functional alphavirus ructural n in trans and comprises an open reading frame encoding the protein of interest. The RNA construct for expressing functional alphavirus non-structural protein and the RNA replicon may either be administered at the same point in time, or may alternatively be stered at different points in time. In the second case, the RNA construct for expressing functional alphavirus non- structural protein is typically administered at a first point in time, and the RNA replicon is typically administered at a second, later, point in time. In that case, it is envisaged that the on will be immediately replicated since ase will already have been synthesized in the cell. The second point in time is typically shortly after the first point in time, eg. 1 minute to 24 hours after the first point in time. Preferably the stration of the RNA replicon is performed at the same site and via the same route of administration as the administration of the RNA construct for expressing functional alphavirus non-structural protein, in order to increase the prospects that the RNA replicon and the RNA construct for expressing functional alphas non-structural protein reach the same target tissue or cell. "Site" refers to [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg ed set by mpg the position of a t’s body. Suitable sites are for example, the left arm, right arm, In one embodiment, an additional RNA molecule, preferably an mRNA le, may be administered to the subject. Optionally, the additional RNA molecule encodes a protein suitable for inhibiting lFN, such as E3, as described . Optionally, the additional RNA molecule may be administered prior to administration of the on or of the replicase construct or of the system according to the invention.

...\ CD Any of the RNA replicon according to the invention, the system ing to the invention, or the kit according to the ion, or the pharmaceutical ition according to the invention can be used in the method for producing a protein in a subject ing to the invention. For example, in the method of the invention, RNA can be used in the format of a pharmaceutical composition, e.g. as described herein, or as naked RNA.

In view of the capacity to be administered to a subject, each of the RNA replicon according to the invention, the system according to the invention, or the kit according to the invention, or the pharmaceutical composition according to the invention, may be referred to as "medicament", or the like. The present invention foresees that the RNA replicon, the system, the kit, the pharmaceutical ition of the present invention are provided for use as a medicament. The medicament can be used to treat a subject. By "treat" is meant to administer a compound or composition or other entity as described herein to a subject. The term es methods for treatment of the human or animal body by therapy.

The above described medicament does typically not comprise a DNA, and is thus associated with additional safety features compared to DNA vaccines described in the prior art (e.g.

An ative medical use according to the present invention comprises a method for producing a protein in a cell according to the fourth aspect of the present invention, whernthe cell may be an antigen presenting cell such as a dendritic cell, followed by the introduction of said cell to a subject. For example, RNA encoding a [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg pharmaceutically active protein, such as an antigen, may be introduced (transfected) into antigen-presenting cells ex vivo, e.g. antigen-presenting cells taken from a subject, and the antigen-presenting cells, optionally clonally propagated ex vivo, may be oduced into the same or a different subject. Transfected cells may be reintroduced into the subject using any means known in the art.

The medicament according to the present ion may be stered to a subject in need f. The medicament of the present invention can be used in prophylactic as well as in therapeutic methods of treatment of a subject. _; C3 The medicament according to the invention is administered in an effective amount.

An "effective amount" concerns an amount that is sufficient, alone or together with other doses, to cause a reaction or a desired effect. In the case of treatment of a certain disease or a certain condition in a subject, the d effect is the inhibition of disease ssion. This es the deceleration of disease progression, in particular the interruption of disease progression. The desired effect in the treatment of a e or a condition can also be a delay of disease outbreak or the tion of disease outbreak.

The effective amount will depend on the condition being treated, the ty of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, duration of the treatment, type of accompanying y (if any), the specific mode of administration and other factors.

Vaccination The term "immunization" or "vaccination" generally refers to a process of treating a t for therapeutic or prophylactic reasons. A treatment, particularly a prophylactic treatment, is or comprises ably a treatment aiming to induce or enhance an immune response of a subject, e.g. against one or more antigens. If, according to the present invention, it is desired to induce or enhance an immune se by using RNA as described herein, the immune response may be triggered or enhanced by the RNA. In one embodiment, the invention provides a prophylactic treatrDit which is or comprises ably the vaccination of a subject. An embodiment of the present invention wherein the replicon encodes, as a protein of [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg interest, a pharmaceutically active peptide or protein which is an immunologically active compound or an antigen is ularly useful for vaccination.

RNA has been previously bed for vaccination against foreign agents including pathogens or cancer (reviewed ly by Ulmer et al., 2012, Vaccine, vol. 30, pp. 4414-4418). In contrast to common approaches in the prior art, the replicon according to the present invention is a particularly suitable element for efficient vaccination because of the ability to be ated by functional alphavirus non- structural protein as described herein. The vaccination according to the present invention can be used for example for induction of an immune response to weakly genic proteins. In the case of the RNA vaccines according to the ion, the protein antigen is never exposed to serum antibodies, but is produced by transfected cells themselves after translation of the RNA. Therefore anaphylaxis should not be a problem. The invention therefore permits the repeated immunization of a patient without risk of allergic reactions.

In methods involving vaccination according to the present invention, the medicament of the present invention is administered to a subject, in ular if treating a subject having a disease involving the antigen or at risk of g ill with the disease involving the antigen is d.

In methods involving vaccination ing to the present ion, the protein of interest encoded by the replicon according to the present invention codes for example for a bacteria! antigen, against which an immune response is to be directed, or for a viral n, against which an immune response is to be directed, or for a cancer antigen, against which an immune response is to be directed, or for an antigen of a unicellular organism, against which an immune response is to be directed. The efficacy of vaccination can be assessed by known standard s such as by measurement of antigen-specific lgG antibodies from the organism. ln methods ing allergen-specific immunotherapy according to the present invention, the protein of interest encoded by the replicon according to the present invention codes for an antigen relevant to an allergy. Allergen-specific immLDherapy (also known as hypo-sensitization) is defined as the administration of preferably increasing doses of an allergen e to an organism with one or more [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg ation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg allergies, in order to achieve a state in which the symptoms that are associated with a subsequent re to the causative allergen are alleviated. The efficacy of an allergen-specific immunotherapy can be assessed by known standard methods such as by ement of allergen-specific lgG and lgE antibodies from the sm.

The medicament of the t invention can be administered to a subject, e.g. for treatment of the subject, ing vaccination of the subject.

The term "subjec " relates to vertebrates, ularly mammals. For example, 1O mammals in the context of the present invention are humans, nonahuman primates, icated mammals such as dogs, cats, sheep, cattle, goats, pigs, horses etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc. as well as animals in captivity such as animals of zoos. The term "subject" also relates to non-mammalian vertebrates such as birds (particularly domesticated birds such as chicken, ducks, geese, turkeys) and to fish (particularly farmed fish, e.g. salmon or h). The term "animal" as used herein also includes humans.

The administration to domesticated animals such as dogs, cats, rabbits, guinea pigs, hamsters, sheep, cattle, goats, pigs, horses, chicken, ducks, geese, turkeys, or wild animals, e.g. foxes, is preferred in some embodiments. For example, a prophylactic vaccination according to the present invention may be suitable to vaccinate an animal population, e.g. in the g industry, or a wild animal population. Other animal populations in captivity, such as pets, or animals of zoos, may be vaccinated.

When administered to a subject, the replicon and/or the replicase construct used as a medicament do preferably not comprise sequences from a type of alphavirus that is infectious to the species or genus to which the treated subject belongs. Preferably, in that case, the replicon and/or the replicase construct do not comprise any nucleotide ce from an alphavirus that can infect the respective species or genus. This 3O embodiment bears the advantage that no recombination with ious (e.g. fully onal or wild-type) alphavirus is possible, even if the subject to which the RNA is administered is (e.g. accidentally) affected by ious alphavirus. As an illustrative exarra, for treatment of pigs, the on and/or the replicase construct used do not comprise any nucleotide sequence from an alphavirus that can infect pigs.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg Mode of administration The medicament according to the t invention can be applied to a subject in any suitable route.

For example, the medicament may be administered sys ly, for example intravenously (i.v.), subcutaneously (s.c.), intradermally (id) or by inhalation.

In one embodiment, the medicament according to the present invention is administered to muscle tissue, such as skeletal muscle, or skin, e.g. subcutaneously. it is generaiiy understood that er of RNA into the skin or muscles leads to high and sustained local expression, paralleled by a strong induction of humoral and cellular immune responses (Johansson et al. 2012, PLoS. One., 7, e29732; Geall et al., 2012, Proc. Natl. Acad. Sci. U. S. A, vol. 109, pp. 14604-14609).

Alternatives to stration to muscle tissue or skin include, but are not limited to: intradermal, intranasal, intraocular, intraperitoneal, intravenous, titial, buccal, transdermal, or gual administration. lntradermal and intramuscular administration are two preferred routes.

Administration can be achieved in various ways. In one embodiment, the medicament ing to the present invention is administered by injection. In a preferred embodiment, injection is via a needle. Needle-free injection may be used as an alternative.

The present invention is described in detail and is illustrated by the figures and examples, which are used only for illustration purposes and are not meant to be limiting. Owing to the description and the examples, r embodiments which are likewise included in the invention are accessible to the skilled worker.

Examples Material and Methods: The waing materials and s were used in the examples that are described below.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg Cloning of plasmids, In vitro transcription, RNA purification: Plasmids were cloned using rd technology. The details on the cloning of individual plasmids used in the examples of this invention are described in Example 1. In vitro transcription, using the plasmids described in Example 1 and T7 RNA- polymerase, and purification of RNA. were performed as previously described (Hoitkamp et ai., 2006, Blood, vol. 108, pp. 4009-4017; Kuhn et al., 2010, Gene Ther., vol. 17, pp. 961-971). y of purified RNA was assessed by spectrophotometr ', and analysis on the 2100 lyzer (Agilent, Santa Clara, USA). All RNA transfected into cells in in the examples was in vitro ribed RNA (IVT-RNA).

RNA transfection: For electroporation, RNA was oporated 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, 1 pulse of 16 ms; for human foreskin fibroblasts: 500V/cm, 1 pulse of 24 ms.

Mixtures of different RNA species were ed in RNAse-free tubes and kept on ice until transfection. For electroporation, RNA or RNA mixtures were resuspended in a final volume of 62.5 pl/mm cuvette gap size.

For lipofection, cells were plated at approximately 20,000 cells/cm2 growth area, and transfected with a total amount of 260 ng/cm2 RNA and 1 pl/cm2 MessengerMax t following the manufacturer’s instructions (Life Technologies, Darmstadt, Germany).

Cell culture: All growth media, fetal calf serum (FCS), antibiotics and other ments were supplied by Life Technologies/Gibco, except when stated otherwise. Human foreskin fibroblasts obtained from System Bioscience (HFF, al) or ATCC (CCD- ) were cultivated in minimum essential media (MEM) containing 15% FCS, 1% rDessential amino acids, 1 mM sodium pyruvate at 37°C. Cells were grown at 37°C in humidified atmosphere equilibrated to 5% 002. BHK21 cells from the cell line ation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg "BHK21 [C13] (ATCC® CCL10TM)", available from American Type Culture Collection, Manassas, Virginia, USA, were grown in Eanle’sany V Minimum Essential medium supplemented with 10% FCS.

Flow cytometry: The expression of RNA encoding GFP was measured by flow cytornetry using a FACS Canto l| flow cytometer (BD Bioscience, Heidelberg, Germany), and acquired data were analyzed by the corresponding Diva software or FlowJo software (Tree Star lnc., Ashland, OR, USA).

Luciferase Assays: To assess 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). y Luciferase expression was measured using with the Bright-Glo Luciferase Assay System, c sion was measured using the Nano-Glo Luciferase assay system (both Promega, Madison, WI, USA) according to the manufacturer’s instructions. Bioluminescence was measured using a microplate luminescence reader Infinite M200 (Tecan Group, Ménnedorf, Switzerland). Data are represented in relative light units [RLU], luciferase—negative cells were used to subtract the background signal. e 1: g of ds A. Plasmids encoding on based on Semliki forest virus (SFV) were obtained using sed seamless cloning techniques. Seamless cloning meaning cloning techniques based on recombination of PCR generated fragment into linearized vectors using homologous sequence stretches. Thereby a DNA sequence encoding a replicon that corresponds to the SFV genome, except for the absence of an open reading frame ng viral structural genes, was transferred from pSFV—gen-GFP (Ehrengruber & Lundstrom, 1999, Proc. Natl. Acad. Sci. U. S. A, vol. 96, pp. 7041- 7046; Lundstrom, 2001, Histochem. Cell Biol., vol. 115, pp. 83-91) into pST1 plasmid backbone (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017) immediately dowrnaam of a T7 phage RNA-polymerase promoter. A d-encoded poly(A) cassette of either 120 adenylate residues (Holtkamp et al., supra) - or a modified [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg poly(A) cassette consisting of 30 and 70 adenylate residues, separated by a 10 nucleotide random ce (WO 2016/005004 A1), was added immediately downstream of the very last nucleotide of the SFV 3'-UTR. A Sapl restriction site was placed immediately ream of the poly(A) cassette or the modified poly(A) cassette. Furthermore a coding ce encoding the myc—tag was inserted into the Xhol site that is found in the coding region for the variable region of SF‘V’ nsP3.

Insertions into the nsP3 variable region do not affect the activity of the replicase polyprotein (Spuul et al., 2010, J. Virol, vol. 85, pp. 7543—7557). The resulting plasmid comprises a DNA sequence encoding the 5’ replication recognition 1O sequence of SFV under the control of a promoter for T7 polymerase. The DNA ce encoding the 5’ replication recognition sequence of SFV is represented by SEQ ID NO: 4: ATGGCGGATGTGTGACATACACGACGCCAAAAGATTTTGTTCCAGCTCCTGCCACCTCCG CTACGCGAGAGATTAACCACCCACcgggcccecCAAAGTGcgggTTGATATTGAGGcTGA ATTCATCAAGTCTTTGCAGAAGGCATTTCCGTCGTTCGAGGTGGAGTCATTGCA GGTCACACCAAAEEACCéEECAAéEECCAGAGCATTTTCGCACCTGGCTACCAAATTGA (SEQ ID NO: 4) In the above representation of SEQ ID NO: 4, the first underlined ATG serves as tion codon for synthesis of the N-terminal fragment of nsP1; bases translated into protein are represented in bold face. Further ATGs within nsP1 coding region are underlined, too. During in vitro transcription of the plasmid comprising the 5’ replication recognition se(1| u ICe of SFV (represented by SEQ lD NO: 4) b" T7 polymerase, a transcript sing an RNA sequence corresponding to SEQ ID NO: 3 is obtained: the 5' terminal G corresponds to the first nucleotide that is transcribed by T7 polymerase. This G precedes the alphaviral sequence and is required for ent transcription by T7 polymerase. 3O GATGGCGGATGTGTGACATACACGACGCCAAAAGATTTTGTTCCAGCTCCTGCCACCTCCGC TACGCGAGAGATTAACCACCCACGgggGCCGCCAAAGTGcgggTTGATATTGAGGCTGACAG CCCATTCATCAAGTCTTTGCAGAAGGCATTTCCGTCGTTCGAGGTGGAGTCATTGCAGGTCA CACCQEQACCEQCAAJLTQCCAGAGCATTTTCGCACCTGGCTACCAAATTGATCGAGCAG GAGACTGACAAAGACACACTCATCTTGGATATC (SEQ ID NO: 3) [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg In the above representation of SEQ ID NO: 3, five specific ATG base triplets are underlined. A unique EcoRV restriction site in the coding region for SFV replicase (GATATC) is highlighted in bold face.

As a result, plasmid A was obtained. Plasmid A comprises an open reading frnmnIlul I IV or functional alphavirus non-structural protein.

B. A plasmid encoding a trans-replicon with non-modified 5’ replication recognition (I)eq Ience was obtained by removing ructural protein coding ce from EcoRV to Sell in plasmid A (see above). Thereby, the major part of the open reading frame encoding nsP1234, i.e. the sequence extending from the unique EcoRV restriction site to the unique Sal! restriction site, was removed. For illustration: GATATC (the most 3’ six nucleotides of SEQ ID NO: 3, bold in the above representation) corresponds to the unique EcoRV restriction site of the coding sequence of SFV replicase. After removal of the major part of the open reading frame encoding nsP1234, the 5’ replication recognition sequence and the subgenomic promoter are still present. Owing to the removal of the major part of the open reading frame encoding nsP1234, the RNA encoded by the respective plasmid, when present in a host cell, is not e to drive replication in cis, but requires for replication the presence of functional alphavirus non—structural protein.

An open reading frame encoding y rase (transgene) was inserted downstream of the omic promoter (SGP). Thereby, plasmid B was ed.

An RNA replicon encoded by a respective plasmid is illustrated as "Template RNA " in Fig. 1.

It was confirmed using a web server for RNA secondary ure prediction (http://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1 .html) that the non-modified 5’ replication ition sequence (RNA) is predicted to fold into four stem loops, indistinguishable from the parental RNA and in accordance with the literature v, 2001, RNA, vol. 7, pp. 1638-1651).

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg C-1. Starting from plasmid B, a plasmid encoding a trans-replicon with a modification of the 5’ replication ition sequence (comprising the removal of the ATG base triplet that encodes the first amino acid residue of nsP1, as well as the l of four additional ATG base ts within the 5’ replication recognition sequence) was generated by single-nucleotide changes, i.e. substitution of one base of the respective ATG base triplet (i.e. substitution of either A or T or G) by a different base.

In other words, those ATG base triplets of SEQ ID NO: 3 that are underlined above were removed, each by change of a single-nucleotide (substitution), respectively.

The folding of the trans-replicon RNA encoded by the piasmid was predicted using the web server for RNA secondary structure prediction (http://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1 .html) and Mfold (http://unafold.rna.a|bany.edu/?q=mfold).

The predicted folding of the modified 5’ replication recognition ce characterized by ATG removal was compared to the predicted folding of the respective fied 5’ replication recognition sequence. In case the prediction revealed that the secondary ure of the modified 5’ ation recognition sequence differed from the ary structure of the unmodified 5’ replication recognition sequence, one or more additional nucleotide changes (substitutions) were carried out in a trial-and-error approach until the RNA folding of the ATG-free 5' replication recognition sequence (A5ATG-RRS) was predicted to be identical to the RNA folding of the non-modified 5’ replication recognition sequence encoded by piasmid B. As a , the DNA sequence according to SEQ ID NO: 5 was ed: GATGGCGGATGTGTGACATACACGACGCCAAAHGATTTTGTTCCAGCTCCTGCCACCTCC-CC7 AGAGATTAACCACCCACGAEGGCCGCCAAAGTGCETGTTGATATTGAGGCTGACAG CCCATTCATCAAGTCTTQGCAGAAGGCATTTCCGTCGTTCGAGGTGGAGTCATTGEAGGTEA CACCAAATEACCATECAAATECCAGAGCATTTTCGCACCTGGETACCAAATTGATCGAGCAG GAGACTGACAAAGACACACTCATCTTGGATATC (SEQ ID NO: 5) The plasmid encoding the modified 5’ replication recognition sequence characterized by prDted identical folding, is. the d comprising SEQ ID NO: 5, is termed herein C-1.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg An RNA replicon encoded by a respective plasmid is illustrated in Fig. 1, designated "A5ATG-RRS".

It is understood that removal of five specific ATGs, as exemplified in SEQ ID NO: 5, will prevent the synthesis of nsP1. or a fragment f. Owing to removal of the native initiation codon for nsPt, it is understood that protein synthesis is initiated at the first initiation codon ream of the subgenomic er (SGP), resulting in transcription of the open reading frame encoding y luciferase (transgene). (2-2. Starting from C-1, the SGP was removed by digestion with EcoRV and Smal and re-ligation. A respective RNA replicon is schematically depicted in Fig. 1, designated "A5ATG-RRSASGP". If a respective RNA replicon comprises a 5’ cap, the transgene encoding luciferase can placed under direct translational control of the 5’-cap. 6-3. Starting from C-1, an open g frame ng SFV replicase was inserted using the EcoRV and Sell restriction sites. Thereby, a plasmid encoding RNA capable of self-replication was obtained, wherein the replicase ORF does neither overlap with the 5’ replication recognition ce nor with the subgenomic er. A respective RNA replicon is schematically depicted in Fig. ’l, ated "cisReplicon A5ATG-RRS".

D. In order to enable translation of a nucleic acid sequence encoding replicase from a non-replicating mRNA, an open reading frame ng SFV replicase was cloned into a plasmid containing a human alpha-globin 5’-UTR, a synthetic 3’-UTR and a d-encoded poly(A) tail of 30 plus 70 adenylate residues, separated by a stabilizing 10 nucleotide (10 nt) linker ( frame encoding SFV replicase was cloned downstream of the human alpha-globin 5’- UTR. The plasmid contained a T7 promoter for transcription of the open g frame encoding SFV replicase.

E. Inner to enable translation of a nucleic acid sequence encoding ia virus protein kinase R inhibitor E3 from a non-replicating mRNA, an open reading frame ation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg encoding Vaccinia virus protein kinase R inhibitor E3 ("E3") was cloned into a d containing a human alpha-globin 5’—UTR, a synthetic 3’=UTR and a plasmid- encoded poly(A) tail of 30 plus 70 adenylate residues, separated by a stabilizing 10 nucleotide (10 nt) linker ( U] was cloned downstream of the human alpha-globin 5’—UTR. The plasmid contained a T7 promoter for transcription of the open reading frame encoding E3.

Example 2: The removal of start codons within the 5’ replication recognition sequence does not affect replication of trans-replicon RNAs.

BHK21 cells were co-electroporated with (i) 5 pg mRNA encoding SFV replicase (encoded by plasmid D of Example 1) and (ii) varying amounts of trans-repiioon RNA (encoded by plasmids B or C-1 of Example 1; in Fig. 2: "template"). trans-replicon encodes y rase; trans-replicon either contains a wild type 5' replication recognition ce (Fig. 2: "WT-RRS"; encoded by plasmid B); or a 5’ replication ition sequence characterized by the removal of all initiation codons (Fig. 2: "A5ATG-RRS"; encoded by plasmid C-1). trans-replicon RNA was uncapped (no cap). 5000 oporated BHK21 cells were plated into each well of 96-well-plates to measure luciferase expression 24h after electroporation. Results are shown in Fig.

Example 3: The removal of start codons within the 5’ replication recognition ce enables translation of a ene under control of the cap trans-replicon characterized by the removal of all initiation codons, and comprising a subgenomic promoter (Fig. 3: "A5ATG-RRS"; encoded by plasmid C-1 of Example 1), trans-replicon characterized by the removal of all tion codons, but not comprising a subgenomic er (Fig. 3: "A5ATG-RRSASGP"; encoded by plasmid C-2 of Example 1), and replicon having all initiation codons and comprising a omic promoter (Fig. 3 ate RNA WT-RRS") were used.

Human foreskin fibroblasts were co-electroporated with (i) 0.45 pg of the respective trans-replicon RNA, as indicated in Fig. 3, and either (ii-a) 2.5 pg mRNA encoding Vaccinia virus E3 protein (encoded by plasmid E of Example 1, in Fig. 3: "E3"), or (ii— b) 2.5 pg mRNA encoding replicase (encoded by plasmid D of Example 1, in Fig. 3: "repliG") plus 2.5 pg mRNA encoding Vaccinia virus E3 protein (encoded by d E of Example 1). mRNA encoding Vaccinia virus E3 protein was added in [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg order to inhibit protein kinase R activation and to thereby promote expression of luciferase and replicase. trans—replicon RNA was either uncapped (Fig. 3: "no cap") or co-transcriptionally capped with a beta-S-ARCA(D2) cap analog (Fig. 3: "DZ-cap").

Luciferase sion was assessed after 24h. Results are shown in Fig. 3 (right panel).

This example demonstrates that removal of tion codons from the 5’ replication ition sequence enables a transgene to be efficiently translated ly from capped trans-replicon RNA.

Example 4: At early stages after transfection the cap-dependent translation from trans-replicon characterized by the l of start codons within the 5’ replication recognition sequence is stronger than from subgenomic RNA. trans-replicon characterized by the removal of all initiation codons, and comprising a subgenomic promoter (Fig. 4: "A5ATG-RRS"; encoded by plasmid C-1 of Example 1), and trans-replicon characterized by the removal of all initiation codons, but not comprising a subgenomic promoter (in Fig. 4: "A5ATG-RRSASGP"; d by plasmid C—2 of Example 1), were used. BHK21 cells were co-electroporated with (i) 0.45 pg of the respective trans-replicon RNA and with (ii) 2.5 pg mRNA encoding replicase (encoded by plasmid D of Example 1). The trans-replicon RNA was either uncapped (Fig. 4: "no Cap") or co-transcriptionally capped with a beta-S—ARCA(D2) cap analog (Fig. 4: "DZ-cap"). Luciferase sion was assessed over time.

Results are shown in Fig. 4 (right panel).

Example 5: Cap-dependent translation from trans-replicon characterized by the removal of start codons within the 5’ replication recognition sequence s transgene sion at early stages, without being dependent on prior expression of replicase. trans-replicon characterized by the removal of all initiation codons, but not comprising a omic promoter (Fig. 5: -RRSASGP"; encoded by plasmid C-2 of Example 1), was used. The trans-replicon RNA was either uncapped (Fig. 5: "no cap") or co-transcriptionally capped with a beta-S—ARCA(D2) cap analog (Fig. 5: "D2- cap")D-IK21 cells were electroporated with (i) 0.45 pg of the respective trans- on RNA, and, where indicated ("replicase" in Fig. 5), additionally with (ii) 2.5 pg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg replicase coding mRNA (encoded by plasmid D of Example 1). Luciferase expression was assessed over time. Results are shown in Fig. 5 (right .

Example 6: Re-constructed cis-replicons with a ATG-deleted replication ition sequence are functional. The ORF of SFV replicase was inserted into "ASATG-RRS" (Fig. 7A: "A5ATC-RRS"; encoded by plasmid C-1. of Example 1.) resulting in plasmid C-3 of example 1 "cisReplicon A5ATG-RRS" and encoding firefly luciferase downstream of the subgenomic promoter (SGP). Within the inserted replicase the regions corresponding to CSE2 and the core SGP were disrupted by nucleotide exchanges (hashed boxes) to avoid duplication of these regulatory regions. This resulted in a re-constructed cis—repiicon. BHK21 cells were co- electroporated with either 2.5ug "cis—replicon WT-RRS" or eplicon A5ATG-RRS". 24h after oporation luciferase sion was measured and trates that this re-constructed licon is functional (Fig. 78).

Example 7: Bicistronic trans-replicons express bcth transgenes. Secretable uciferase (SNL) was cloned downstream of the subgenomic promoter (SGP) of a trans-replicon WT-RSS (plasmid B of example 1). The position upstream of the SGP does not encode a transgene since it is not ible for translation (Fig. 8A).

In a second construct, SNL was cloned downstream of AATG—RSS (plasmid C-1 of example 1‘, and y luciferase (Luc) inserted ream of the SGP. BHK21 cells were co-electroporated with 0.9pg trans-replicating RNA and 5ug SFV-replicase coding mRNA. 48h after electroporation SNL and Luc expression were measured (Fig. SB). This experiment provides ce that transgens are expressed from both positions within "A5ATG-RRS — bicistronic" trans-replicons. e 8: Sindbis virus trans-replicons lacking start codons in the replication recognition sequence replicate efficiently. replicons were engineered from Sindbis virus genome by gene synthesis similarly to the constructs described in 3O example 1 for SFV. Besides a trans-replicon with unmodified replication recognition sequence (WT-RSS) two variants were generated. First one (AATG-RRS) contains deletions of the original start codon plus 4 further ATGs and corresponding comrnatory nucleotide changes to preserve RNA secondary structure. In the next step, the region corresponding to the subgenomic promoter was additionally deleted [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg ation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg ed set by mpg to obtain AATG-RRSASGP. GFP was inserted into the trans-replicon RNA directly downstream of the ATG-deleted 5’RRS in A5ATG-RRSASGP-vectors, or downstream of the subgenomic promoter (SGP) in A5ATG-RRS and WT-RRS vectors (Fig. 9A). BHK21 cells were co-electroporated with 0.1pg trans-replicating RNA and 2.4ug SFV-replicase coding mRNA and 24h later GFP expression (transfection rate [°/o] and mean fluorescence intensity (MFl) was assessed (Fig. 93)). This experiment shows that the same principle of sequence modification that was applied to SFV engineered replicons can be d to Sindbis virus engineered replicons.

[Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg ation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg

Claims (1)

Claims 1. A 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 initiation codon compared to a native alphavirus 5' replication ition sequence. 2. The RNA replicon according to claim 1, wherein the 5' replication recognition sequence comprises a sequence homologous to about 250 nucleotides at the 5' end ..x CD of an alphavirus, and preferably comprises a sequence homologous to about 300 to about 500 tides at the 5' end of an alphavirus. 3. The RNA replicon according to claim 1 or 2, n the 5' replication recognition sequence comprises sequences homologous to conserved sequence 15 element 1 and conserved sequence t 2 of an irus. 4. The RNA replicon according to claim 3, wherein conserved sequence element 2 comprises a fragment of an open reading frame of a non-structural protein from an alphavirus. 5. The RNA replicon according to any one of claims 1 to 4, wherein the 5' replication recognition sequence comprises a sequence gous to an open reading frame of a non-structural protein or a fragment thereof from an alphavirus, wherein the sequence homologous to an open g frame of a non-structural 25 protein or a fragment thereof from an alphavirus is terized in that it ses the removal of at least one initiation codon compared to the native irus sequence. 6. The RNA replicon according to claim 5, wherein the ce homologous to 30 an open reading frame of a non-structural protein or a fragment thereof from an alphavirus is characterized in that it comprises the removal of at least the native start codon of the open reading frame of a non-structural protein. [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg 7. The RNA replicon ing to claim 5 or 6, wherein the sequence homologous to an open reading frame of a hen-structural protein or a fragment thereof from an alphavirus is characterized in that it comprises the removal of one or more initiation codons other than the native start codon of the open reading frame of a non—structural protein. 8. The RNA replicon according to any one of claims 1 to 7, which comprises one or more nucleotide s compensating for nucleotide pairing disruptions within one or more stem loops introduced by the l of at least one initiation codon. -A (D 9. The RNA replicon according to any one of claims 1 to 8, which does not se an open reading frame encoding a truncated alphavirus non-structural protein. 15 10. The RNA replicon according to any one of claims 1 to 9, which comprises a 3' replication recognition sequence. 11. The RNA replicon according to any one of claims 1 to 10, which ses a first open reading frame encoding a protein of interest. 12. The RNA replicon according to claim 11, wherein the protein of interest encoded by the first open reading frame can be expressed from the RNA replicon as a template. 25 13. The RNA on according to claim 11 or 12, which comprises a subgenomic promoter controlling production of subgenomic RNA comprising the first open reading frame encoding a protein of st. 14. The RNA replicon according to claim 13, wherein the protein of interest 3O encoded by the first open g frame can be expressed from the RNA replicon and the subgenomic RNA. [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg ation] mpg ed set by mpg 15. The RNA replicon according to claim 11 or 12, which comprises a subgenomic promoter controlling production of subgenomic RNA comprising a second open reading frame encoding a protein of interest. 16. The RNA replicon according to claim 15, wherein the subgenomic promoter and the second open reading frame encoding a n of interest are located downstream from the first open reading frame encoding a protein of interest. 17. The RNA replicon ing to any one of claims 11 to 16, wherein the protein 10 of interest encoded by the first and/or second open reading frame is functional alphavirus non-structural protein. 18. The RNA replicon according to any one of claims 1 to 17, which comprises an open reading frame encoding functional alphavirus non-structural protein. 19. The RNA replicon according to claim 17 or 18, wherein the open reading frame encoding functional alphavirus non-structural protein does not overlap with the 5' replication recognition sequence. 20 20. The RNA replicon according to any one of claims 17 to 19, which can be replicated by the functional alphavirus non-structural protein. 21. The RNA replicon according to any one of claims 1 to 16, which does not comprise an open reading frame encoding functional alphavirus non-structural 25 protein. 22. A system comprising: a RNA construct for expressing functional alphavirus non-structural n, the RNA replicon ing to any one of claims 1 to 16 and 21 which can be 30 ated by the functional alphavirus non-structural protein in trans. 23. The RNA on according to any one of claims 1 to 21 or the system accorng to claim 22, wherein the alphavirus is Semliki Forest Virus. [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg 24. A DNA comprising a nucleic acid sequence encoding the RNA replicon of one of claims 1 to 21 and 23. 25. A method for ing a protein of interest in a cell comprising the steps of: (a) obtaining the RNA replicon according to any one of claims 1 to 20 and 23 which comprises an open g frame encoding functional alphavirus non-structural protein, can be replicated by the functional alphavirus non-structural n and comprises an open reading frame encoding the protein of interest, and (b) inoculating the RNA replicon into the cell. 26. A method for producing a protein of interest in a cell comprising the steps of: (a) obtaining a RNA construct for sing functional irus ructural (b) obtaining the RNA replicon according to any one of claims 1 to 16, 21 and 23 15 which can be replicated by the onal alphavirus non-structural protein in trans and comprises an open reading frame encoding the protein of interest, and (c) co-inoculating the RNA construct for expressing functional alphavirus non- structural protein and the RNA replicon into the cell. 20 27. A cell inoculated according to the method of claim 25 or 26. 28. A method for producing a protein of interest in a subject comprising the steps (a) obtaining the RNA replicon according to any one of claims 1 to 20 and 23 which 25 comprises an open reading frame encoding functional alphavirus non-structural protein, can be ated by the functional alphavirus non-structural n and comprises an open reading frame encoding the protein of interest, and (b) stering the RNA replicon to the subject. 30 29. A method for producing a protein of interest in a subject comprising the steps (a) obtaining a RNA construct for expressing functional alphavirus non-structural proteD [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg [Annotation] mpg None set by mpg [Annotation] mpg MigrationNone set by mpg [Annotation] mpg Unmarked set by mpg (b) obtaining the RNA replicon ing to any one of claims 1 to 16, 21 and 23 which can be replicated by the onal alphavirus non-structural protein in trans and comprises an open reading frame encoding the protein of interest, and (c) stering the RNA construct for expressing functional alphavirus non- structural protein and the RNA replicon to the subject. 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