RNA replicon for somatic cell reprogramming

Abstract

The present invention comprises an RNA replicon that can be replicated by an alphaviral replicase and includes an open reading frame encoding a reprogramming factor. Such RNA replicons are useful for expressing a reprogramming factor in a cell, particularly a somatic cell. Cells genetically modified to express such reprogramming factors are useful in cell transplantation therapies.

Description

RNA replicon for somatic cell reprogramming Technical field The present invention encompasses an RNA replicon that can be replicated by an alphavirus-derived replicase and comprises an open reading frame encoding a reprogramming factor. Such RNA replicons are useful for producing cells with stem cell characteristics from somatic cells and, in particular, in methods for dedifferentiating somatic cells into cells with stem cell characteristics, particularly pluripotency, such as stem cell-like cells, without generating embryos or fetuses, by introducing one or more of the RNA replicons into somatic cells and culturing the somatic cells, allowing the cells to dedifferentiate. After dedifferentiation, the cells can be induced to redifferentiate into the same somatic cell type or into a different one, such as neuronal, hematopoietic, muscle, epithelial, and other cell types.The stem cell-like cells derived by the present invention have medical applications for the treatment of degenerative diseases by means of "cell therapy" and can be used in novel therapeutic strategies for the treatment of cardiac, neurological, endocrinological, vascular, retinal, dermatological, musculoskeletal and other diseases. Background Stem cells, also called progenitor cells, are cells with the capacity to self-renew, remain undifferentiated, and differentiate into one or more specialized cell types with mature phenotypes. Stem cells are not terminally differentiated, nor are they at the end of a differentiation pathway. Totipotent cells contain all the genetic information necessary to create every cell in the body, including placental cells. Human cells possess this totipotent capacity only during the first few divisions of a fertilized egg. After three or four divisions of totipotent cells, a series of stages occur in which the cells become increasingly specialized. The next stage of division results in pluripotent cells, which are highly versatile and can give rise to any cell type except placental cells or other supporting tissues of the uterus. In the following stage, the cells become multipotent, meaning they can give rise to several other cell types, but the number of those types is limited.At the end of the long chain of cell divisions that make up the embryo are the "terminally differentiated" cells, which are considered to be permanently committed to a specific function. There are three main groups of stem cells: (i) adult or somatic (postnatal) stem cells, which exist in all postnatal organisms, (ii) embryonic stem cells, which can be derived from a pre-embryonic or embryonic stage of development, and (iii) fetal (prenatal) stem cells, which can be isolated from the developing fetus. Stem cell technologies, which involve the isolation and use of human embryonic stem cells, have become a major area of ​​medical research. Human embryonic stem cells have the potential to differentiate into every cell type in the human body, including complex tissues. It is anticipated that many diseases resulting from cellular dysfunction may be treatable through the administration of human embryonic stem cells or cells derived from human embryonic stem cells. The ability of pluripotent embryonic stem cells to differentiate and give rise to a multitude of specialized mature cells reveals the potential application of these cells as a means to replace, restore, or supplement damaged or diseased cells, tissues, and organs.However, scientific and ethical considerations have slowed the progress of research using embryonic stem cells recovered from aborted embryos or embryos formed using in vitro fertilization techniques. Adult stem cells are present only at low frequencies and exhibit limited differentiation potential and poor growth. An additional problem associated with the use of adult stem cells is that these cells are not immunologically privileged, or may lose their immunological privilege after transplantation, where the term "immunologically privileged" denotes a state in which the recipient's immune system does not recognize the cells as foreign. Therefore, in most cases, when adult stem cells are used, only autologous transplants are possible. Most forms of stem cell therapy currently being considered are essentially personalized medical procedures, and thus the economic factors associated with such procedures limit their broad potential. Following fusion with embryonic stem cells, the restoration of the expression of at least some embryo-specific genes in somatic cells has been observed. However, the resulting cells are hybrid, often with a tetraploid genotype, and are therefore not suitable as normal or histocompatible cells for transplantation purposes. Somatic cell nuclear transfer (SCNT) has been shown to allow for the successful reprogramming of the nuclear contents of somatic cells to achieve pluripotency; however, it raises a number of concerns that extend beyond ethical considerations. The stresses placed on both the egg cell and the introduced nucleus are enormous, resulting in significant cell loss. Furthermore, the procedure must be performed manually under a microscope, making SCNT resource-intensive. Additionally, not all of the donor cell's genetic information is transferred, as the donor cell's mitochondria, containing their own mitochondrial DNA, remain in the cell. The resulting hybrid cells retain those mitochondrial structures that originally belonged to the egg cell. Consequently, the clones are not perfect copies of the nuclear donor. In 2006, Takahashi et al. took an important step toward obtaining patient-derived pluripotent stem cells. They demonstrated that overexpression of specific transcription factors (TFs) known to regulate and maintain stem cell pluripotency (Takahashi et al., 2006, Cell 126, 663-676; Schulz & Hoffmann, 2007, Epigenetics 2, 37-42) can induce a pluripotent state in murine somatic fibroblasts, termed induced pluripotent stem cells (iPSCs). In this study, the authors identified OCT3 / 4, SOX2, KLF4, and c-MYC as required for iPSC generation (Takahashi et al., 2006). In a subsequent study, the authors demonstrated that the same TFs are capable of reprogramming adult human fibroblasts (Takahasi et al., 2007, Cell 131, 861-872), while others attributed this activity to a modified TF cocktail composed of OCT3 / 4, SOX2, NANOG and LIN28 in relation to humans (Yu et al., 2007, Science 318, 1917) or murine fibroblasts (Wernig et al., 2007, Nature 448, 318-324). In both these initial studies and most subsequent studies, reprogramming transcription factors were overexpressed using retroviral or lentiviral vectors. Due to the silencing of viral promoters, these studies reproducibly show that the expression of exogenous TFs is deactivated during the reprogramming process (reviewed by Hotta and Ellis, 2008, J. Cell Biochem. 105, 940-948). Consequently, the pluripotent state is maintained by activated endogenous transcription factors. Furthermore, the silencing of virally expressed TFs is a prerequisite for the subsequent redifferentiation of iPS cells into tissue-specific precursors (Yu et al., 2007).A major drawback of viral administration is the stochastic reactivation of integrated retroviruses encoding potent oncogenes, which in the case of c-MYC led to tumor induction in chimeric mice (Okita et al., 2007, Nature 448, 313-317). Meanwhile, iPS cell generation has been shown to be possible in the absence of MYC (Nakagawa et al., 2008, Nat. Biotechnol., 26(1), 10-106). In general, only OCT4 and SOX2 have been reported as essential for reprogramming; oncogenes such as MYC and KLF4 appear to act as enhancers (McDevitt & Palecek, 2008, Curr. Opin. Biotechnol. 19, 527-33). Consequently, other transforming gene products such as the SV40 large T antigen or hTERT have been shown to improve the efficiency of iPS generation (Mali et al., 2008, Stem Cells 26, 1998-2005).Since epigenetic reprogramming involves chromatin remodeling, the addition of histone deacetylase (HDAC) inhibitors (such as valproic acid) or DNA methyltransferase inhibitors (such as 5'-azaC) greatly improves the efficiency of reprogramming (Huangfu et al., 2008, Nat. Biotechnol. 26, 795-797) and reduced the need for TF to OCT4 and SOX2 (Huangfu et al., 2008, Nat. Biotechnol. 26, 1269-1275). Another strategy to reduce the risk associated with retroviral integration into the host genome is the use of non-integrating adenoviral vectors, which mediate transient transgene expression sufficient for reprogramming (Stadtfeld et al., 2008, 322, 945-949). Transgene integration is also avoided by using conventional eukaryotic expression plasmids, resulting in transient gene expression. So far, using this strategy, MEFs have been successfully reprogrammed into iPS cells (Okita et al., 2008, Science 322, 949-53). In this study, genomic integration was not detected; however, stable genomic integration in a small fraction of the cells with transfected plasmid DNA cannot be completely ruled out. Adult human fibroblasts can be easily obtained from healthy donors or—in future clinical applications—from patients without the need for risky surgery. However, a recent study has shown that human keratinocytes are more easily and efficiently reprogrammed into iPS cells, and that, for example, hair follicle-derived keratinocytes could be the best source of choice for patient-derived iPS cells (Aasen et al., 2008, Nat. Biotechnol.26 (11), 1276-84). There remains a need for technologies to reprogram differentiated somatic cells in order to produce dedifferentiated or reprogrammed cells in large quantities and with good quality. Nucleic acid molecules that contain foreign genetic information and encode one or more polypeptides for prophylactic and therapeutic purposes have been studied in biomedical research for many years. Due to safety concerns associated with the use of deoxyribonucleic acid (DNA) molecules, ribonucleic acid (RNA) molecules have received increasing attention in recent years. Various approaches have been proposed, including the delivery of single-stranded or double-stranded RNA, either as naked RNA or in complex or packaged forms, for example, in non-viral or viral delivery vehicles. In viruses and viral delivery vehicles, the genetic information is typically encapsulated by proteins and / or lipids (viral particle).For example, genetically modified RNA virus particles derived from RNA viruses have been proposed as delivery vehicles for plant treatment (WO 2000 / 053780 A2) or for mammalian vaccination (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 subgrouped into single-stranded RNA viruses (smRNA) and double-stranded RNA viruses (dsRNA), and smRNA viruses can be further divided, generally, into positive-strand (+) and / or negative-strand (-) viruses. Positive-strand RNA viruses are prima facie attractive as a delivery system in biomedicine because their RNA can serve directly as a template for translation in the host cell. Alphaviruses are typical representatives of positive-strand RNA viruses. Hosts of alphaviruses include a wide range of organisms, including insects, fish, and mammals such as domestic animals and humans. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see José et al., Future Microbiol., 2009, vol. 4, pp. 837–856). The total genome length of many alphaviruses typically ranges from 11,000 to 12,000 nucleotides, and the genomic RNA typically has a 5' protectant and a poly(A) 3' tail. The alphavirus genome encodes nonstructural proteins (involved in the transcription, modification, and replication of viral RNA and in protein modification) and structural proteins (which form the viral particle). There are typically two open reading frames (ORFs) in the genome.The four non-structural proteins (nsP1-nsP4) are typically co-encoded by a first ORF that begins near the 5' end of the genome, while the structural proteins of alphaviruses are co-encoded by a second ORF that lies downstream of the first ORF and extends near the 3' end of the genome. Typically, the first ORF is larger than the second ORF, with a ratio of approximately 2:1. In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule resembling eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124). After infection, i.e., in the early stages of the viral life cycle, the (+) strand genomic RNA acts directly as a messenger RNA for the translation of the open reading frame encoding the non-structural polyprotein (nsP1234).In some alphaviruses, there is an opal stop codon between the coding sequences of nsP3 and nsP4: the P123 polyprotein, which contains nsP1, nsP2, and nsP3, is produced when translation terminates at the opal stop codon, and the P1234 polyprotein, which also contains nsP4, is produced after the reading of this opal codon (Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491–562; Rupp et al., 2015, J. Gen. Virology, vol. 96, pp. 2483–2500). nsP1234 is autoproteolytically cleaved into the nsP123 and nsP4 fragments. The nsP123 and nsP4 polypeptides associate to form the replicase complex of (-) strand that transcribes (-) strand RNA, using the (+) strand genomic RNA as a template. Typically, in later stages, the nsP123 fragment is completely cleaved into the individual proteins nsP1, nsP2, and nsP3 (Shirako & Strauss, 1994, J. Virol., vol.68, pp.1874-1885).All four proteins form the (+) strand replicase complex that synthesizes new (+) strand genomes using the (-) strand complement of the genomic RNA as a 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, both subgenomic and newly formed genomic RNA are provided with a 5-protector by nsP1 (Pettersson et al. 1980, Eur. J. Biochem. 105, 435-443; Rozanov et al., 1992, J. Gen. Virology, vol. 73, pp. 2129-2134), and are provided with a polyadenylate [poly(A)] tail by nsP4 (Rubach et al., Virology, 2009, vol. 384, pp. 201-208). Thus, both subgenomic and genomic RNA resemble messenger RNA (mRNA). The structural proteins of alphaviruses (nucleocapsid core protein C, envelope protein E2, and envelope protein E1, all constituents of the viral particle) are typically encoded by a single open reading frame under the control of a subgenomic promoter (Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491–562). The subgenomic promoter is recognized by cis-acting alphaviral nonstructural proteins. In particular, the alphavirus replicase synthesizes a (+) strand subgenomic transcript using the (-) strand complement of the genomic RNA as a template. The (+) strand subgenomic transcript encodes the structural proteins of the alphavirus (Kim et al., 2004, Virology, vol.323, pp.153-163, Vasiljeva et al., 2003, J. Biol. Chem. vol.278, pp.41636-41645).The subgenomic RNA transcript serves as a template for translation of the open reading frame that encodes the structural proteins as a polyprotein, and this polyprotein is cleaved to produce the structural proteins. At a late stage of alphavirus infection in a host cell, a packaging signal located within the coding sequence of nsP2 ensures the selective packaging of genomic RNA into budding virions, packaged by structural proteins (White et al., 1998, J. Virol., vol.72, pp.4320-4326). In infected cells, negative-strand RNA synthesis is typically observed only in the first 3–4 h after infection and is undetectable in later stages, at which point only positive-strand RNA synthesis (both genomic and subgenomic) is observed. According to Frolov et al., 2001, RNA, vol. 7, pp. 1638–1651, the prevailing model for the regulation of RNA synthesis suggests a dependence on non-structural polyprotein processing: the initial cleavage of the non-structural polyprotein nsP1234 produces nsP123 and nsP4; nsP4 acts as an RNA-dependent RNA polymerase (RdRp) that is active for negative-strand synthesis but inefficient for positive-strand RNA generation. Further processing of the nsP123 polyprotein, including cleavage at the nsP2 / nsP3 junction, changes the replicase template specificity to increase (+) strand RNA synthesis and decrease or terminate (-) strand RNA synthesis. Alphaviral RNA synthesis is also regulated by cis-active RNA elements, including four conserved sequence elements (CSes; Strauss & Strauss, Microbiol. Rev., 1994, vol.58, pp.491-562; and Frolov, 2001, RNA, vol.7, pp.1638-1651). In general, the 5' replication recognition sequence of the alphavirus genome is characterized by low overall homology among different alphaviruses, but it has a predicted conserved secondary structure. The 5' replication recognition sequence of the alphavirus genome not only participates in translation initiation, but also comprises two conserved sequence elements involved in viral RNA synthesis, CSE1 and CSE2. For the function of CSE1 and CSE2, the secondary structure is thought to be more important than the linear sequence (Strauss & Strauss, Microbiol. Rev., 1994, vol.58, pp.491-562). In contrast, the 3' terminal sequence of the alphavirus genome, i.e., the sequence immediately upstream of the poly(A) sequence, is characterized by a conserved primary structure, in particular by conserved sequence element 4 (CSE 4), also called the "19 nt conserved sequence", which is important for the initiation of the (-) chain synthesis. CSE3, also called the "junction sequence," is a conserved sequence element on the (+) strand of alphaviral genomic RNA, and the complement of CSE3 on the (-) strand acts as a promoter for transcription of subgenomic RNA (Strauss & Strauss, Microbiol. Rev., 1994, vol.58, pp.491-562; Frolov et al., 2001, RNA, vol.7, pp.1638-1651). The CSE3 region typically overlaps with the region encoding the C-terminal fragment of nsP4. In addition to alphavirus proteins, host cell factors, presumably proteins, can also bind to conserved sequence elements (Strauss & Strauss, supra). Alphavirus-derived vectors have been proposed for delivering foreign genetic information into target cells or organisms. In the simplest methodologies, the open reading frame encoding the structural proteins of alphaviruses is replaced with an open reading frame encoding a protein of interest. Alphavirus-based transreplication systems rely on alphavirus nucleotide sequence elements in two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase (typically as the nsP1234 polyprotein), and the other nucleic acid molecule is capable of being replicated in trans by that replicase (hence the designation transreplication system). Transreplication requires the presence of both nucleic acid molecules in a given host cell.The in trans must include certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase. (See WO2013177133, Yoshioka & Dowdy 2017, STEMCELL Technologies 2016, Yoshioka et al.) 2013, EMD Millipore Corporation 2016, and WO2017087763 have been identified in the Search Report established by the International Searching Authority as descriptive of RNA replicons comprising open reading frames encoding a reprogramming factor, methods for reprogramming somatic cells to acquire stem cell characteristics, and / or methods for differentiating such cells. As described herein, the aspects and realizations of this teaching address the need to provide technologies for reprogramming differentiated somatic cells to produce high-quality, high-quality dedifferentiated or reprogrammed cells. Summary of the invention The present invention is defined by the appended claims. In a first aspect, the present invention relates to an RNA replicon comprising an open reading frame encoding a reprogramming factor and a 5' replication recognition sequence, wherein the 5' replication recognition sequence is a functional variant of a native alphavirus 5' replication recognition sequence characterized in that it does not contain any start codon compared to the native alphavirus 5' replication recognition sequence. In one embodiment, the RNA replicon comprises at least one additional open reading frame that encodes a different reprogramming factor. In some embodiments, the reprogramming factor is selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG. In some embodiments, the RNA replicon does not comprise an open reading frame encoding a functional non-structural alphavirus protein, the nsP1234. In some embodiments, the RNA replicon comprises a first open reading frame that encodes a reprogramming factor. In some implementations, the first open read frame does not overlap with the 5' replication acknowledgment sequence. In some embodiments, the start codon of the first open reading frame is in the 5' to 3' direction of the RNA replicon, the first functional start codon. In a second aspect, the present invention relates to a set of RNA replicons, wherein each of the RNA replicons comprises at least one open reading frame encoding a reprogramming factor and the set of RNA replicons encodes a set of reprogramming factors; and wherein at least one RNA replicon of the set is an RNA replicon according to the first aspect and embodiments thereof. In one embodiment, the set of reprogramming factors is useful for reprogramming somatic cells into cells that have stem cell characteristics. In some embodiments, each RNA replicon in the ensemble is an RNA replicon according to the first aspect and its embodiments. In a third aspect, the present invention relates to a method for producing cells having stem cell characteristics comprising the step of introducing into somatic cells one or more RNA replicons according to the first aspect and its embodiments. In a fourth aspect, the present invention relates to a method for providing cells having stem cell characteristics comprising the following steps: (i) provide a cell population comprising somatic cells, (ii) providing one or more RNA replicons, wherein at least one of the one or more RNA replicons is an RNA replicon according to the first aspect and wherein each of the one or more RNA replicons comprises an open reading frame encoding the functional non-structural protein of alphavirus nsP1234, can be replicated by the functional non-structural protein of alphavirus nsP1234 and comprises at least one open reading frame encoding a reprogramming factor, (iii) introducing one or more RNA replicons into somatic cells, so that the cells express a set of reprogramming factors useful for reprogramming somatic cells into cells that have stem cell characteristics, and (iv) allow the development of cells that have stem cell characteristics. In a fifth aspect, the present invention relates to a method for providing cells having stem cell characteristics comprising the following steps: (i) provide a cell population comprising somatic cells, (ii) providing an RNA construct encoding the functional non-structural protein of alphavirus nsP1234, (iii) providing one or more RNA replicons, wherein at least one of the one or more replicons is an RNA replicon according to the first aspect and its realizations, wherein each of the one or more RNA replicons can be replicated by the functional non-structural protein of alphavirus nsP1234 and comprises at least one open reading frame encoding a reprogramming factor, (iv) introducing the RNA construct and one or more RNA replicons into somatic cells, so that the cells express a set of reprogramming factors useful for reprogramming somatic cells into cells that have stem cell characteristics, and (v) allow the development of cells that have stem cell characteristics. In one embodiment, the methods in the third through fifth aspects further comprise the introduction of miRNA into somatic cells, thereby enhancing the reprogramming of somatic cells to convert them into cells that have stem cell characteristics. In some embodiments of the fifth aspect method and its embodiments, at least one RNA replicon of the one or more RNA replicons is an RNA replicon according to the first aspect and its embodiments. In some embodiments of the methods of the third to fifth aspect and their embodiments, each RNA replicon of the one or more RNA replicons is an RNA replicon according to the first aspect and its embodiments. In some embodiments of the methods of the third to fifth aspect, the one or more RNA replicons comprise a set of RNA replicons according to the second aspect and its embodiments. In a sixth aspect, the present invention relates to cells comprising an RNA replicon according to the first aspect and embodiments thereof or a set of RNA replicons according to the second aspect and embodiments thereof. In a seventh aspect, the present invention relates to a method for providing differentiated cell types comprising the steps of (i) providing cells having stem cell characteristics using the method of any one of the third to fifth aspects, and (ii) growing the cells having stem cell characteristics under conditions that induce or direct partial or complete differentiation to a differentiated cell type. Teaching summary This disclosure provides insights that, in some respects, go beyond the disclosure of the invention itself, which is defined solely by the appended claims. These insights are provided to place the present invention in a broader technical context and to illustrate possible related technical developments. Such additional technical information that is not within the scope of the appended claims does not form part of the invention. In particular, the term "embodiment" should not be construed as necessarily referring to an embodiment of the invention unless the embodiment in question is within the scope of the claims. References to treatment methods in the description should be interpreted as references to the compounds, pharmaceutical compositions, and drugs of the present invention for use in a method for treating the human (or animal) body by therapy (or for diagnosis). This course provides technologies for producing reprogrammed cells using RNA replicons that encode reprogramming factors. These technologies utilize cells that are readily and inexpensively obtained in unlimited quantities and yield reprogrammed cells useful in cell therapy. The approach described here opens the possibility of reprogramming without altering the host genome. This teaching takes advantage of the fact that, when provided with the appropriate factors, the fate of a terminally differentiated cell can be redirected toward pluripotency. Specifically, this teaching provides the technology necessary to reprogram a differentiated animal somatic cell into a cell with stem cell properties. This method allows the dedifferentiation of a somatic cell type into stem cell-like pluripotent cells using a defined in vitro system. In one embodiment, the teaching method provides autologous (isogenic) cell types for cell transplantation in the same individual who donated the initial somatic cell sample. According to this teaching, one or more somatic cells are provided with one or more RNA replicons capable of expressing one or more factors that induce the reprogramming of somatic cells into cells with stem cell characteristics. The expression of these factors confers characteristics of an undifferentiated cell upon a somatic cell and facilitates its reprogramming. According to the teaching, different types of RNA replicons are useful. In one type of RNA replicon, the open reading frame of an alphavirus-derived RNA vector encoding alphavirus structural proteins is replaced by an open reading frame encoding a reprogramming factor. Figure 1 illustrates such a replicon as a "cis-replicon; WT-RRS". Other types of RNA replicons, according to the teaching, are related to alphavirus-based trans-replication systems. Figure 1 illustrates such a replicon as a "trans-replicon; WT-RRS". Such a replicon is associated with the advantage of allowing the amplification of an open reading frame encoding a reprogramming factor under the control of a subgenomic promoter. The open reading frame encoding nsP1234 typically overlaps with the 5' replication recognition sequence of the alphavirus genome (coding sequence for nsP1) and typically also with the subgenomic motor comprising CSE 3 (coding sequence for nsP4). Consequently, in such a "trans-replicon," the 5' replication recognition sequence required for RNA replication comprises an AUG start codon for nsP1 and thus overlaps with the coding sequence for the N-terminal fragment of the alphavirus nonstructural protein, and a replicon comprising the 5' replication recognition sequence will typically encode (at least) a portion of the alphavirus nonstructural protein, typically the N-terminal fragment of nsP1.This is disadvantageous in several respects: In the case of cis-replicons, this overlap limits, for example, the adaptation of the replicase ORF codon usage to different mammalian target cells (humans, mice, farm animals). It is conceivable that the secondary structure of the 5' replication recognition sequence, as found in viruses, may not be optimal in all target cells. However, the secondary structure cannot be freely altered, as the resulting amino acid changes in the replicase ORF must be considered and tested to determine their effect on replicase function. It is also not possible to replace the entire replicase ORF with replicas of heterologous origin, as this could result in an alteration of the 5' replication recognition sequence structure.In the case of trans-replicons, this overlap results in the synthesis of a fragment of the nsP1 protein, since the 5' replication recognition sequence must be conserved in trans-replicons. Typically, an nsP1 fragment is neither required nor desired: unwanted translation imposes an unnecessary burden on the host cell, and RNA replicons intended for therapeutic applications that encode both a pharmaceutically active protein and an nsP1 fragment may face regulatory challenges. For example, it will be necessary to demonstrate that the truncated nsP1 protein does not produce undesirable side effects.Furthermore, the presence of an AUG start codon for nsP1 within the 5' replication recognition sequence has prevented the design of trans-replicons encoding a heterologous gene of interest in such a way that the start codon for translation of the gene of interest is at the most accessible 5' position for ribosomal translation initiation. In turn, 5'-protector-dependent translation of transgenes from trans-replicon RNA using the above technique is challenging unless cloned as an in-frame fusion protein with the nsP1 start codon (such fusion constructs are described, for example, by Michel et al., 2007, Virology, vol. 362, pp. 475–487). Such fusion constructs result in the same unnecessary translation of the nsP1 fragment mentioned above, raising the same concerns as before.Furthermore, fusion proteins raise additional concerns, as they could alter the function or activity of the fused transgene of interest, or, when used as a vaccine vector, peptides spanning the fusion region could alter the immunogenicity of the fused antigen. Accordingly, this instruction provides an additional type of RNA replicon comprising sequence elements required for replicase replication, but these sequence elements do not encode any protein or protein fragment, such as an alphavirus non-structural protein or a fragment thereof. Thus, the sequence elements required for replicase replication and the protein-coding regions are uncoupled. Figure 1 illustrates a respective replicon as "trans-replicon; 5ATG-RRSSGP." The uncoupling is achieved by the removal of at least one start codon compared to a native alphavirus genomic RNA. The replicase may be encoded by the RNA replicon or by an independent nucleic acid molecule.In a particularly preferred embodiment, said replicon does not comprise a subgenomic promoter and the start codon for translation of the open reading frame encoding a reprogramming factor is at the 5' position that is accessible for initiation of ribosomal translation. In one aspect, the present teaching provides an RNA replicon comprising an open reading frame encoding a reprogramming factor. In one embodiment, the RNA replicon comprises one or more additional open reading frames encoding identical or different reprogramming factors. In one embodiment, the RNA replicon comprises open reading frames encoding a functional set of reprogramming factors, which is a set of reprogramming factors useful for reprogramming somatic cells into cells with stem cell characteristics; that is, the set of reprogramming factors, when expressed in a somatic cell, is sufficient to effect the reprogramming of the somatic cell into a cell with stem cell characteristics. In one embodiment, the reprogramming factor is selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG. In one embodiment, the RNA replicon is either a cis-replicon or a trans-replicon. In one embodiment, the RNA replicon comprises an open first reading frame that encodes a functional alphavirus nonstructural protein or a reprogramming factor. In one embodiment, the RNA replicon comprises a 5' replication recognition sequence, wherein the 5' replication recognition sequence is characterized in that it comprises the deletion of at least one start codon compared to a native alphavirus 5' replication recognition sequence. In one embodiment, the first open read frame does not overlap with the replication acknowledgment sequence 5'. In one embodiment, the start codon of the first open reading frame is in the 5' to 3' direction of the RNA replicon, being the first functional start codon. In one embodiment, particularly if the RNA replicon is a cis-replicon, the replicon comprises an open reading frame encoding a functional non-structural protein of the alphavirus. The RNA replicon may comprise one or more open reading frames encoding one or more reprogramming factors. In another embodiment, particularly if the RNA replicon is a trans-replicon, the RNA replicon does not comprise an open reading frame encoding a functional non-structural protein of the alphavirus. In this embodiment, the functional non-structural protein of the alphavirus may be provided for in-trans replicon replication as described herein. The RNA replicon may comprise one or more open reading frames encoding one or more reprogramming factors. Thus, in one embodiment, the RNA replicon may comprise only one open reading frame encoding a reprogramming factor.Several of these RNA replicons can form an RNA replicon set, such as a functional RNA replicon set as described herein. In one embodiment, the RNA replicon comprises a first open reading frame encoding a protein of interest, for example, a functional nonstructural protein of an alphavirus or a reprogramming factor. In one embodiment, the first open reading frame does not overlap with the 5' replication recognition sequence. If the RNA replicon is a cis-replicon, the first open read will generally be an open reading frame encoding a functional nonstructural protein of the alphavirus. In this embodiment, the RNA replicon generally comprises at least one additional open reading frame encoding a reprogramming factor that is under the control of a subgenomic promoter. If the RNA replicon is a transreplicon, the first open read will generally be an open reading frame encoding a reprogramming factor, and the RNA replicon preferably does not comprise any open reading frames encoding a functional nonstructural protein of the alphavirus. In one embodiment, the RNA replicon comprises a (modified) 5' replication recognition sequence and a first open reading frame encoding a protein of interest, e.g., a functional non-structural alphavirus protein or a reprogramming factor, located downstream of the 5' replication recognition sequence, wherein the 5' replication recognition 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 5' replication recognition sequence does not contain a functional start codon and, preferably, does not contain any start codon.Ideally, the start codon of the first open reading frame should be in the 5' to 3' direction of the RNA replicon, the first functional start codon, preferably the first start codon. In one embodiment, the first open reading frame, and preferably the entire RNA replicon, does not express non-functional alphavirus non-structural proteins, such as a fragment of an alphavirus non-structural protein, particularly a fragment of nsP1 and / or nsP4. In one embodiment, the functional alphavirus non-structural protein is heterologous to the 5' replication recognition sequence. In one embodiment, the first open reading frame is not under the control of a subgenomic promoter. In one embodiment, the first open reading frame encodes a functional nonstructural alphavirus protein, and the RNA replicon comprises at least one additional open reading frame encoding a reprogramming factor that is under the control of a subgenomic promoter. In one embodiment, the subgenomic promoter and the first open reading frame do not overlap. In another embodiment, the first open reading frame encodes a reprogramming factor, and the RNA replicon preferably does not comprise any open reading frame encoding a functional nonstructural alphavirus protein. The RNA replicon may comprise at least one additional open reading frame encoding one or more reprogramming factors (e.g., one or more reprogramming factors that, together with the reprogramming factor encoded by the first open reading frame, form a functional set of reprogramming factors) that is under the control of a subgenomic promoter. In one embodiment, the subgenomic promoter and the first open reading frame do not overlap. In a particularly preferred embodiment, the first open reading frame, located downstream of the 5' replication recognition sequence, encodes a reprogramming factor, the 5' replication recognition sequence and the first open reading frame do not overlap, the 5' replication recognition sequence does not contain a functional start codon and preferably does not contain any start codon, and the RNA replicon does not comprise an open reading frame encoding a functional nonstructural protein of the alphavirus.In this embodiment, the start codon of the first open reading frame is in the 5' to 3' direction of the RNA replicon, being the first functional start codon, preferably the first start codon such that the RNA replicon does not express a non-functional alphavirus non-structural protein, such as an alphavirus non-structural protein fragment, in particular a fragment of nsP1 and / or nsP4. The RNA replicon may comprise at least one additional open reading frame encoding one or more reprogramming factors (e.g., one or more reprogramming factors that, together with the reprogramming factor encoded by the first open reading frame, form a functional set of reprogramming factors) that is under the control of a subgenomic promoter. In one embodiment, the subgenomic promoter and the first open reading frame do not overlap. In one embodiment, the 5' replication recognition sequence of the RNA replicon, characterized by the deletion of at least one start codon, comprises a sequence homologous to approximately 250 nucleotides at the 5' end of an alphavirus. In a preferred embodiment, it comprises a sequence homologous to approximately 300 to 500 nucleotides at the 5' end of an alphavirus. In a further preferred embodiment, it comprises the 5' terminal sequence required for the efficient replication of the specific alphavirus species that is the parent of 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 conserved sequence element 2 (CSE 2) of an alphavirus. In a preferred embodiment, the RNA replicon comprises CSE2 and is further characterized in that it comprises a fragment of an open reading frame of a non-structural protein of an alphavirus. In a preferred embodiment, said fragment of an open reading frame of a non-structural protein does not comprise any start 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 of an alphavirus, wherein the sequence homologous to an open reading frame of a non-structural protein or a fragment thereof of an alphavirus is characterized in that it comprises the deletion of at least one start codon compared to the native sequence of the alphavirus. In a preferred embodiment, the sequence homologous to an open reading frame of a non-structural protein or a fragment thereof of an alphavirus is characterized in that it comprises the deletion of at least the native start codon of the open reading frame of a non-structural protein. In a preferred embodiment, the sequence homologous to an open reading frame of a nonstructural protein or a fragment thereof from an alphavirus is characterized in that it comprises the deletion of one or more initiation codons other than the native start codon of the open reading frame of a nonstructural protein. In a more preferred embodiment, said nucleic acid sequence is further characterized by the deletion of the native start codon of the open reading frame of a nonstructural protein, preferably nsP1. In a preferred embodiment, the 5' replication recognition sequence comprises one or more stem loops that provide the functionality of the 5' replication recognition sequence with respect to RNA replication. In a preferred embodiment, one or more stem loops of the 5' replication recognition sequence are not deleted or interrupted. More preferably, one or more of stem loops 1, 3, and 4, preferably all stem loops 1, 3, and 4, or stem loops 3 and 4 are not deleted or interrupted. Most preferably, none of the stem loops of the 5' replication recognition sequence are deleted or interrupted. In a preferred embodiment, the RNA replicon comprises the one or more nucleotide changes that compensate for the disruptions in nucleotide pairing within the one or more stem loops introduced by the deletion of at least one start codon. In one embodiment, the RNA replicon does not comprise an open reading frame encoding a truncated alphavirus nonstructural protein. In one embodiment, the RNA replicon comprises a 3' replication recognition sequence. In one embodiment, the RNA replicon is characterized in that the protein of interest encoded by the first open reading frame can be expressed from the RNA replicon as a template. In one embodiment, the RNA replicon comprises a subgenomic promoter that controls the production of subgenomic RNA comprising the first open reading frame. In one embodiment, the RNA replicon is characterized by comprising a subgenomic promoter. Typically, the subgenomic promoter controls the production of subgenomic RNA comprising an open reading frame that encodes a protein of interest. In one embodiment, the protein of interest encoded by the first open reading frame can be expressed from the RNA replicon as a template. In a more preferred embodiment, the protein of interest encoded by the first open reading frame can also be expressed from subgenomic RNA. In a preferred embodiment, the RNA replicon is further characterized in that it comprises a subgenomic promoter that controls the production of subgenomic RNA comprising a second open reading frame encoding a protein of interest. The protein of interest can be a second protein identical to or different from the protein of interest encoded by the first open reading frame. In a more preferred embodiment, the subgenomic promoter and the second open reading frame encoding a protein of interest are located downstream of the first open reading frame encoding a protein of interest. In one embodiment, the RNA replicon can be replicated by means of a functional non-structural protein of the alphavirus. In a second aspect, the present teaching provides a set of RNA replicons, i.e., a set comprising at least two, such as two, three, four, five, six, or even more RNA replicons, wherein each of the RNA replicons comprises at least one open reading frame encoding a reprogramming factor, and the set of RNA replicons encodes a set of reprogramming factors. In one embodiment, each of the RNA replicons comprises an open reading frame encoding a reprogramming factor. In one embodiment, the set of reprogramming factors is a functional set of reprogramming factors, i.e., it is useful and / or sufficient to reprogram somatic cells into cells that have stem cell characteristics. In one embodiment, the reprogramming factor set comprises OCT4 and SOX2. In one embodiment, the reprogramming factor set further comprises KLF4 and / or c-MYC. In one embodiment, the reprogramming factor set further comprises NANOG and / or LIN28. In one embodiment, the reprogramming factor set comprises OCT4, SOX2, KLF4, and c-MYC. In another embodiment, the reprogramming factor set further comprises LIN28 and, optionally, NANOG. In one embodiment, the reprogramming factor set comprises OCT4, SOX2, NANOG, and LIN28. In one embodiment, at least one RNA replicon in the set is a teachable RNA replicon. In one embodiment, each RNA replicon in the set is a teachable RNA replicon. In a third aspect, the present teaching provides a system that comprises: an RNA construct to express the functional non-structural protein of alphavirus, The RNA replicon, according to the first aspect of instruction, or the set of RNA replicons, according to the second aspect of instruction, which can be replicated by the functional alphavirus nonstructural protein in trans. Preferably, the RNA replicon or the set of RNA replicons is further characterized in that it does not encode a functional alphavirus nonstructural protein. In one embodiment, the RNA replicon according to the first aspect, the set of RNA replicons according to the second aspect, or the system according to the third aspect is characterized in that the alphavirus is the Venezuelan equine encephalitis virus. In a fourth aspect, the present teaching provides DNA (i.e., one or more DNA molecules) comprising a nucleic acid sequence encoding the RNA replicon according to the first aspect, the set of RNA replicons according to the second aspect, or the system according to the third aspect. In an additional aspect, the present teaching provides a method for producing cells that have stem cell characteristics, which comprises the step of introducing one or more RNA replicons into somatic cells, according to the teaching. In an additional aspect, the present teaching provides a method for providing cells that have stem cell characteristics, comprising the following steps: (i) provide a cell population comprising somatic cells, (ii) providing one or more RNA replicons, wherein each of the one or more RNA replicons comprises an open reading frame encoding a functional non-structural protein of the alphavirus, can be replicated by the functional non-structural protein of the alphavirus and comprises at least one open reading frame encoding a reprogramming factor, (iii) introducing one or more RNA replicons into somatic cells, so that the cells express a set of reprogramming factors useful for reprogramming somatic cells into cells that have stem cell characteristics, and (iv) allow the development of cells that have stem cell characteristics. In an additional aspect, this teaching provides a method for obtaining cells that have stem cell characteristics, comprising the following steps: (i) provide a cell population comprising somatic cells, (ii) providing an RNA construct for expressing the functional non-structural protein of the alphavirus, (iii) providing one or more RNA replicons, wherein each of the one or more RNA replicons can be replicated in trans by the functional non-structural protein of the alphavirus and comprises at least one open reading frame encoding a reprogramming factor, (iv) introducing the RNA construct and one or more RNA replicons into somatic cells, so that the cells express a set of reprogramming factors useful for reprogramming somatic cells into cells that have stem cell characteristics, and (v) allow the development of cells that have stem cell characteristics. According to the present teaching, the one or more RNA replicons and, optionally, the RNA construct to express the functional non-structural protein of the alphavirus are preferably introduced into somatic cells by electroporation or lipofection. In one embodiment, an RNA replicon comprises open reading frames encoding a functional set of reprogramming factors. In another embodiment, different RNA replicons comprise open reading frames encoding different reprogramming factors. In this latter embodiment, these different RNA replicons can be introduced simultaneously or at different times (optionally along with an RNA construct to express the functional alphavirus nonstructural protein) into cells to provide a functional set of reprogramming factors. In one embodiment, the cells express the reprogramming factors. In one embodiment, one or more RNA replicons encode a set of reprogramming factors. In one embodiment, each of the one or more RNA replicons comprises an open reading frame that encodes a reprogramming factor. In one embodiment, the set of reprogramming factors encoded by one or more RNA replicons is a functional set of reprogramming factors, i.e., a set of reprogramming factors useful and / or sufficient to reprogram somatic cells into cells that have stem cell characteristics. In one embodiment, the reprogramming factor set comprises OCT4 and SOX2. In one embodiment, the reprogramming factor set further comprises KLF4 and / or c-MYC. In one embodiment, the reprogramming factor set further comprises NANOG and / or LIN28. In one embodiment, the reprogramming factor set comprises OCT4, SOX2, KLF4, and c-MYC. In another embodiment, the reprogramming factor set further comprises LIN28 and, optionally, NANOG. In one embodiment, the set of reprogramming factors comprises OCT4, SOX2, NANOG, and LIN28. In another embodiment, the teaching method further comprises the introduction of miRNA into somatic cells, thereby enhancing the reprogramming of somatic cells into cells that have stem cell characteristics. In one embodiment, the teaching method further comprises culturing somatic cells in the presence of at least one histone deacetylase inhibitor. In one embodiment, at least one histone deacetylase inhibitor comprises valproic acid. In one embodiment, the step of enabling the development of cells having stem cell characteristics comprises culturing somatic cells under embryonic stem cell culture conditions, preferably conditions suitable for maintaining pluripotent stem cells in an undifferentiated state. In one embodiment, the stem cell characteristics comprise an embryonic stem cell morphology, where said embryonic stem cell morphology preferably comprises selected morphological criteria from the group consisting of compact colonies, a high nucleus-to-cytoplasm ratio, and prominent nucleoli. In certain embodiments, the cells that have stem cell characteristics have normal karyotypes, express telomerase activity, express cell surface markers characteristic of embryonic stem cells, and / or express genes characteristic of embryonic stem cells.Cell surface markers characteristic of embryonic stem cells can be selected from the group consisting of stage-3 specific embryonic antigen (SSEA-3), SSEA-4, tumor-related antigen-1-60 (TRA-1-60), TRA-1-81, and TRA-2-49 / 6E, and genes that are characteristic of embryonic stem cells can be selected from the group consisting of endogenous OCT4, endogenous NANOG, growth and differentiation factor 3 (GDF3), reduced expression 1 (REX1), fibroblast growth factor 4 (FGF4), embryo cell-specific gene 1 (ESG1), developmental pluripotency-associated 2 (DPPA2), DPPA4, and telomerase reverse transcriptase (TERT). In one embodiment, the cells having stem cell characteristics are dedifferentiated and / or reprogrammed somatic cells. Preferably, the cells having stem cell characteristics exhibit the essential characteristics of embryonic stem cells, such as the pluripotent state. Preferably, the cells having stem cell characteristics have the developmental potential to differentiate into advanced derivatives of the three primary germ layers. In one embodiment, the primary germ layer is the endoderm and the advanced derivative is an epithelial tissue similar to that of the gut. In another embodiment, the primary germ layer is the mesoderm and the advanced derivative is striated muscle and / or cartilage. In a further embodiment, the primary germ layer is the ectoderm and the advanced derivative is neural tissue and / or epidermal tissue.In a preferred embodiment, cells that have stem cell characteristics have the developmental potential to differentiate into neuronal cells and / or cardiac cells. In one embodiment, the somatic cells are somatic cells derived from embryonic stem cells with a mesenchymal phenotype. In a preferred embodiment, the somatic cells are fibroblasts, such as fetal fibroblasts or postnatal fibroblasts, or keratinocytes, preferably hair follicle-derived keratinocytes. In further embodiments, the fibroblasts are lung fibroblasts, foreskin fibroblasts, or dermal fibroblasts. In particular embodiments, the fibroblasts are fibroblasts deposited in the American Type Culture Collection (ATCC) under catalog number CCL-186 or deposited in the American Type Culture Collection (ATCC) under catalog number CRL-2097. In one embodiment, the fibroblasts are adult human dermal fibroblasts. Preferably, the somatic cells are human cells. According to the present teaching, the somatic cells may be genetically modified. In one embodiment, at least one RNA replicon of the one or more RNA replicons is an RNA replicon as taught. In one embodiment, each RNA replicon of the one or more RNA replicons is an RNA replicon as taught. In one embodiment, the one or more RNA replicons comprise a set of RNA replicons as taught. Some specific realizations of the methods described in this teaching also include one or more of the steps of culturing, propagating and cryopreservation of cells that have stem cell characteristics. In a further aspect, the present teachable provides cells having stem cell characteristics produced by the teachable method. In one embodiment, the cell is a recombinant cell. In a further aspect, the present teachable provides a cell expressing one or more reprogramming factors, preferably a functional set of reprogramming factors, comprising one or more RNA replicons of the teachable, which comprise an open reading frame encoding one or more reprogramming factors. The present teachable also provides a population of such cells. In an additional aspect, the present teaching provides a method for providing differentiated cell types comprising the steps of (i) providing cells having stem cell characteristics using the teaching method, and (ii) culturing the cells having stem cell characteristics under conditions that induce or direct partial or complete differentiation to a differentiated cell type. In an additional aspect, the present teaching refers to a method for obtaining differentiated cell types comprising the step of culturing the cells having stem cell characteristics of the present teaching under conditions that induce or direct partial or complete differentiation to a differentiated cell type. In one embodiment, the conditions that induce or direct partial or complete differentiation into a differentiated cell type comprise the presence of at least one differentiation factor. Preferably, the somatic cell type of the differentiated cells obtained according to this instruction is different from the somatic cell type of the somatic cells used for dedifferentiation. Preferably, the dedifferentiated cells are derived from fibroblasts, and the redifferentiated cell types are different from fibroblasts. In another embodiment, the dedifferentiated cells are derived from keratinocytes, and the redifferentiated cell types are different from keratinocytes. In a further aspect, this instruction relates to a kit for producing cells with stem cell characteristics, comprising the RNA replicon according to the first aspect, the set of RNA replicons according to the second aspect, or the system according to the third aspect. The kit may also include a culture medium for embryonic stem cells. In an additional aspect, the present teaching provides a pharmaceutical composition comprising cells having characteristics of teaching stem cells, for example, comprising a set of teaching RNA replicons, each RNA replicon encoding one of the reprogramming factors of a functional set of reprogramming factors. In yet another aspect, the present teaching refers to a pharmaceutical composition comprising the RNA replicon according to the first aspect, the set of RNA replicons according to the second aspect, or the system according to the third aspect. In an additional aspect, this teaching provides the pharmaceutical composition of the compound for its use as a medicine. In additional aspects, the present teaching refers to the use of cells or the pharmaceutical composition of the present teaching in medicine, particularly in transplant medicine. In an additional aspect, the present teaching provides a method for the treatment of a disease comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition of the teaching. In an additional aspect, the present teaching provides a method for treating a subject suffering from a disease, the method comprising administering to the subject cells that have characteristics of stem cells produced by the teaching method. In one embodiment, the cells can be autologous, allogeneic, or syngeneic to the subject. In one embodiment encompassing all aspects of the teaching method, the treatment method further comprises obtaining a sample of somatic cells from a subject and treating these cells using the teaching methods to obtain cells with stem cell characteristics. In one embodiment encompassing all aspects of the teaching method, the cells with stem cell characteristics are transiently transfected with nucleic acid encoding one or more reprogramming factors. Therefore, the nucleic acid encoding one or more reprogramming factors does not integrate into the cell's genome. In one embodiment of the teaching method, the somatic cells are derived from the subject to whom the cells with stem cell characteristics are administered. In another embodiment of the teaching method, the somatic cells are derived from a mammal different from the mammal to whom the cells with stem cell characteristics are administered. In a teaching context, a treatment includes cell therapy, such as cell transplantation therapy. In an additional aspect, the teaching provides the agents and compositions described herein for use in the methods described herein. Other features and advantages of current teaching will become evident in the following detailed description and in the claims. Brief description of the drawings Figure 1: Parental viral genome and cis and trans replication RNA vectors (A) General organization of alphavirus genomes. Two large open reading frames (ORFs) are separated by a subgenomic promoter (SGP). The 5' ORF encodes an enzyme complex for RNA amplification (replicase), while the 3' ORF encodes the viral structural genes (capsid and envelope glycoproteins). At the 5' end, two conserved sequence elements (CSEs) form the 5' replication recognition sequence (RRS), which partially overlaps with the replicase coding region. The 3' RRS is constructed from a CSE4 (19 nucleotides at the 3' terminal) and approximately 15 nucleotides of the poly-A tail (An). (B) Cis-replicon vectors conserve the wild-type (WT) sequence of the RRS and SGP but lack the ORF of the structural genes, which is replaced by genes of interest. (C) trans-replicon (TR) vector systems.The cis-replicon is cleaved into an mRNA that encodes the replicase but cannot replicate itself, and short RNAs amplified in trans by the replicase. These so-called trans-replicons have two different designs: one contains all the viral RRSs in the WT sequence identical to the cis-replicon. The other version contains a shortened 5'CSE mutated to remove any AUG codon that could serve as a translation start codon. The removal of 5'AUG ensures that translation begins exclusively with the start codon of the ORF of interest, which is inserted downstream of the mutated 5'CSE. The genes of interest in this teaching are six reprogramming transcription factors (rTFs) that promote pluripotency (OCT4, SOX2, MYC, KLF4, NANOG, LIN28) and three vaccinia virus interferon escape proteins (E3, K3, B18). Figure 2: RNA reprogramming using trans-replicon technology (WT-RRS) Figure 3: RNA reprogramming using trans-replicon technology (5ATG-RRSSGP) Figure 4: RNA-based reprogramming by transfection using trans-replicon technology (WT-RSS) Figure 5: Schematic representation of RNA replicons comprising an unmodified or modified 5' replication recognition sequence, useful according to teaching. Abbreviations: AAAA = Poly(A) tail; ATG = start codon / initiation codon (ATG at the DNA level; AUG at the RNA level); 5x ATG = nucleic acid sequence comprising all start codons in the nucleic acid sequence encoding nsP1* (in the case of the nucleic acid sequence encoding nsP1* from Semliki Forest virus, 5x ATG corresponds to five specific start codons); 5ATG = nucleic acid sequence corresponding to a nucleic acid sequence encoding nsP1*; however, it does not comprise any start codons from the nucleic acid sequence encoding nsP1* in the naturally occurring alphavirus (in the case of nsP1* derived from Semliki Forest virus, "5ATG" corresponds to the deletion of five specific start codons compared to the naturally occurring Semliki Forest virus); EcoRV = EcoRV Restriction Site;nsP = nucleic acid sequence encoding a non-structural alphavirus 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, wherein the fragment does not comprise the N-terminal fragment of nsP4; RRS = 5' replication recognition sequence; SalI = Sall restriction site; SGP = subgenomic promoter; SL = stem loop (e.g., SL1, SL2, SL3, SL4); SL1-4 positions are illustrated graphically; UTR = untranslated region (e.g., 5'-UTR, 3'-UTR); WT = wild type; Transgene preferably refers to an open reading frame that encodes a reprogramming factor. cisReplicon WT-RRS: An RNA replicon that essentially corresponds to the genome of an alphavirus, except that the nucleic acid sequence encoding the alphavirus's structural proteins has been replaced by an open reading frame encoding a gene of interest ("Transgene"). When the "WT-RRS Replicon" is introduced into a cell, the translation product of the open reading frame encoding the replicase (nsP1234 or fragments thereof) can drive replication of the cis RNA replicon and drive the synthesis of a nucleic acid sequence (the subgenomic transcript) downstream of the subgenomic promoter (SGP). WT-RRS trans-replicon or template RNA: An RNA replicon that essentially corresponds to the "WT-RRS Replicon", except that most of the nucleic acid sequence encoding the alphavirus non-structural proteins nsP1-4 has been deleted. More specifically, the nucleic acid sequence encoding nsP2 and nsP3 has been completely deleted; the nucleic acid sequence encoding nsP1 has been truncated such that the "WT-RRS template RNA" encodes a fragment of nsP1, which fragment does not comprise the C-terminal fragment of nsP1 (but does comprise the N-terminal fragment of nsP1; nsP1*); The nucleic acid sequence encoding nsP4 has been truncated such that the "WT-RRS template RNA" encodes a fragment of nsP4, which fragment does not comprise the N-terminal fragment of nsP4 (but does comprise the C-terminal fragment of nsP4; *nsP4). This truncated nsP4 sequence partially overlaps with the fully active subgenomic promoter.The nucleic acid sequence encoding nsP1* comprises all the initiation codons of the nucleic acid sequence encoding nsP1* in naturally occurring alphaviruses (in the case of nsP1* from Semliki forest virus, five specific initiation codons). 5ATG-RRS: An RNA replicon that essentially corresponds to the "WT-RRS template RNA", except that it does not comprise any start codons from the nucleic acid sequence encoding nsP1* in the naturally occurring alphavirus (in the case of Semliki Forest virus, "5ATG-RRS" corresponds to the deletion of five specific start codons compared to the naturally occurring Semliki Forest virus). All nucleotide changes introduced to remove the start codons were compensated for with additional nucleotide changes to preserve the intended secondary structure of the RNA. 5ATG-RRSSGP: RNA replicon that corresponds essentially to "5ATG-RRS", except that it does not comprise the subgenomic promoter (SGP) and does not comprise the nucleic acid sequence encoding *nsP4. "Transgene 1" = a gene of interest. 5ATG-RRS - bicistronic: An RNA replicon that corresponds essentially to "5ATG-RRS", except that it comprises a first open reading frame encoding a first gene of interest ("Transgene 1") upstream of the subgenomic promoter, and a second open reading frame encoding a second gene of interest ("Transgene 2") downstream of the subgenomic promoter. The location of the second open reading frame corresponds to the location of the gene of interest ("Transgene") in the "5ATG-RRS" RNA replicon. cisReplicon 5ATG-RRS: An RNA replicon that corresponds essentially to "5ATG-RRS - bicistronic", except that the open reading frame encoding a first gene of interest encodes a functional nonstructural protein of the alphavirus (typically an open reading frame encoding the nsP1-nsP2-nsP3-nsP4 polyprotein, i.e., nsP1234). "Transgene" in "cisReplicon 5ATG-RRS" corresponds to "Transgene 2" in "5ATG-RRS - bicistronic".The functional non-structural protein of the alphavirus is capable of recognizing the subgenomic promoter and synthesizing subgenomic transcripts comprising the nucleic acid sequence encoding the gene of interest ("Transgene"). "cisReplicon 5ATG-RRS" encodes a functional non-structural protein of the alphavirus in cis, as does "cisReplicon WT-RRS"; however, the coding sequence for nsP1 encoded by "cisReplicon 5ATG-RRS" does not necessarily comprise the exact nucleic acid sequence of "cisReplicon WT-RRS", including all stem loops. Figure 6. Structures of the protectant dinucleotides. Top: A natural protectant dinucleotide, m7GpppG. Bottom: Beta-S-ARCA dinucleotide analog of the phosphorothioate protectant: Due to the stereogenic P center, there are two diastereomers of beta-S-ARCA, which are named D1 and D2 according to their elution characteristics in reversed-phase HPLC. Detailed description Although the present teaching is described in detail below, it should be understood that this teaching is not limited to the particular methodologies, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein is solely for the purpose of describing particular embodiments and is not intended to limit the scope of this teaching, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as they are commonly understood by a person skilled in the art. Preferably, the terms used in this document are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", HGW Leuenberger, B. Nagel, and H. Kölbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995). The practice of the present teaching will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques explained in the literature of the field (cf., for example, Molecular Cloning: A Laboratory and Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory and Press, Cold Spring Harbor 1989). The elements of this teaching are described below. These elements are listed with specific realizations; however, it should be understood that they can be combined in any way and in any quantity to create additional realizations. The various examples described and preferred realizations should not be interpreted as limiting this teaching solely to the realizations explicitly described. This description should be understood as a disclosure encompassing realizations that combine the explicitly described realizations with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all the elements described in this application should be considered disclosed by this description unless the context indicates otherwise. The term "approximately" means approximately or nearly, and in the context of a numerical value or range stated herein, preferably means + / - 10% of the numerical value or range stated or claimed. The terms "a," "one," "the," and similar references used in the context of the teaching description (especially in the context of the claims) should be interpreted to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. The citation of value ranges herein is solely for the purpose of serving as a shorthand method for referring individually to each separate value within the range. Unless otherwise indicated herein, each individual value is incorporated into the descriptive report as if it were mentioned individually therein. All methods described herein may be performed in any appropriate order, unless otherwise indicated herein or clearly contradicted by the context.The use of any and all examples or exemplary language (e.g., "such as") provided in this document is solely for the purpose of better illustrating the teaching. Nothing in this descriptive memorandum should be construed as indicating any unclaimed element essential to the practice of teaching. Unless expressly stated otherwise, the term "comprising" is used in the context of this document to indicate that other members besides those in the list introduced by "comprising" may optionally be present. However, it is intended as a specific realization of this teaching that the term "comprising" encompasses the possibility that no other members may be present; that is, for the purposes of this realization, "comprising" is to be understood as having the meaning of "consisting of." Indications of relative quantities of a component characterized by a generic term refer to the total quantity of all variants or specific members covered by that generic term. If a particular component defined by a generic term is specified as being present in a certain relative quantity, and if this component is further characterized as a variant or specific member covered by the generic term, it is understood that no other variants or members covered by the generic term are present such that the total relative quantity of components covered by the generic term exceeds the specified relative quantity; more preferably, no other variants or members covered by the generic term are present at all. Terms such as "reduce" or "inhibit," as used herein, mean the ability to bring about an overall decrease, preferably of 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more, in the level. The term "inhibit" or similar expressions include complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero. Terms such as "increase" or "improve" preferably refer to an increase or improvement of at least 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%. The term "net charge" refers to the charge of a whole object, such as a compound or a particle. An ion with a net positive charge is a cation, while an ion with a net negative charge is an anion. Thus, according to this teaching, an anion is an ion with more electrons than protons, giving it a net negative charge; and a cation is an ion with fewer electrons than protons, giving it a net positive charge. The terms "charged", "net charge", "negatively charged" or "positively charged", with reference to a given compound or particle, refer to the net electrical charge of that compound or particle when it is dissolved or suspended in water at a pH of 7.0. According to this teaching, a nucleic acid is either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Generally, a nucleic acid molecule or nucleic acid sequence refers to a nucleic acid, preferably either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). According to this teaching, nucleic acids include genomic DNA, cDNA, mRNA, viral RNA, molecules prepared by recombination, and chemically synthesized molecules. According to this teaching, a nucleic acid can exist as a single-stranded or double-stranded molecule, linear or covalently closed circular. According to this teaching, the term "nucleic acid" also encompasses the chemical derivation of a nucleic acid based on a nucleotide, sugar, or phosphate, as well as nucleic acids containing non-natural nucleotides and nucleotide analogues. According to the teaching, "nucleic acid sequence" refers to the sequence of nucleotides in a nucleic acid, for example, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). The term can refer to an entire nucleic acid molecule (such as, for example, the single strand of a complete nucleic acid molecule) or to a part (for example, a fragment) of it. According to this teaching, the term "RNA" or "RNA molecule" refers to a molecule comprising ribonucleotide residues and preferably composed entirely or substantially of ribonucleotide residues. The term "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. The term "RNA" includes double-stranded RNA, single-stranded RNA, isolated RNA, such as partially or completely purified RNA, essentially pure RNA, synthetic RNA, and RNA generated by recombination, such as modified RNA, which differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide material, such as at the ends of an RNA molecule or internally, for example, within one or more nucleotides of the RNA.The nucleotides in RNA molecules can also include non-standard nucleotides, such as nucleotides that do not occur naturally or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs can be called analogs, particularly analogs of naturally occurring RNA. According to the teaching, RNA can be single-stranded or double-stranded. In some embodiments of the present teaching, single-stranded RNA is preferred. The term "single-stranded RNA" generally refers to an RNA molecule to which no complementary nucleic acid molecule (typically no complementary RNA molecule) is associated. Single-stranded RNA may contain self-complementary sequences that allow portions of the RNA to fold and form secondary structure motifs, including, without limitation, base pairs, stems, stem loops, and protrusions. Single-stranded RNA can exist as a negative strand [(-) strand] or as a positive strand [(+) strand]. The (+) strand is the strand that comprises or encodes the genetic information. The genetic information may be, for example, a polynucleotide sequence that codes for a protein.When the (+) strand of RNA codes for a protein, the (+) strand can directly serve as a template for translation (protein synthesis). The (-) strand is the complement of the (+) strand. In the case of double-stranded RNA, the (+) strand and the (-) strand are two separate RNA molecules, and both RNA molecules associate with each other to form a double-stranded RNA ("duplex RNA"). The term "stability" of RNA refers to the "half-life" of the RNA. Half-life refers to the period of time required to eliminate half of the activity, quantity, or number of molecules. In the context of this lesson, the half-life of an RNA molecule is an indicator of its stability. The half-life of an RNA molecule can influence the "duration of expression" of the RNA. RNA with a long half-life is expected to be expressed for a longer period of time. The term "translation efficiency" refers to the amount of translation product that an RNA molecule provides in a given period of time. "Fragment," with reference to a nucleic acid sequence, refers to a part of a nucleic acid sequence, that is, a sequence representing the nucleic acid sequence shortened at the 5' and / or 3' ends. Preferably, a fragment of a nucleic acid sequence comprises at least 80%, and preferably at least 90%, 95%, 96%, 97%, 98%, or 99% of the nucleotide residues of that nucleic acid sequence. For the purposes of this instruction, fragments of RNA molecules that preserve RNA stability and / or translation efficiency are preferred. "Fragment," with reference to an amino acid sequence (peptide or protein), refers to a portion of an amino acid sequence, that is, a sequence representing the amino acid sequence shortened at the N-terminus and / or the C-terminus. A C-terminus shortened fragment (N-terminus fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end. A C-terminus shortened fragment (C-terminus fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end, provided that such a truncated open reading frame contains a start codon to initiate translation.A fragment of an amino acid sequence comprises, for example, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues in an amino acid sequence. The term "variant," with respect to, for example, nucleic acid and amino acid sequences, as taught, includes any variant, particularly mutants, viral strain variants, splicing variants, conformations, isoforms, allelic variants, species variants, and species homologs, especially those that occur naturally. An allelic variant refers to an alteration in the normal sequence of a gene, the significance of which is often unclear. Complete sequencing of a gene often allows the identification of numerous allelic variants for a given gene. With respect to nucleic acid molecules, the term "variant" includes degenerate nucleic acid sequences, where a degenerate nucleic acid, as taught, is a nucleic acid that differs from a reference nucleic acid in its codon sequence due to the degeneracy of the genetic code.A species homolog is a nucleic acid or amino acid sequence with a different species of origin than a given nucleic acid or amino acid sequence. A viral homolog is a nucleic acid or amino acid sequence whose virus of origin is different from that of the sequence. According to the teaching, nucleic acid variants include deletions, additions, mutations, substitutions, and / or insertions of one or more nucleotides compared to the reference nucleic acid. Deletions involve the removal of one or more nucleotides from the 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 nucleotide is removed from the sequence and at least one other nucleotide is inserted in its place (such as transversions and transitions). Mutations include abasic sites, cross-linking sites, and chemically altered or modified bases. Insertions involve the addition of at least one nucleotide to the reference nucleic acid. According to the teaching, a "nucleotide change" can refer to deletions, additions, mutations, substitutions, and / or insertions of one or more nucleotides compared to the reference nucleic acid. In some embodiments, a "nucleotide change" is selected from the group consisting of a single nucleotide deletion, single nucleotide addition, single nucleotide mutation, single nucleotide substitution, and / or single nucleotide insertion, compared to the reference nucleic acid. According to the teaching, a nucleic acid variant can comprise one or more nucleotide changes compared to the reference nucleic acid. The variants of specific nucleic acid sequences preferably have at least one functional property of those specific sequences and, preferably, be functionally equivalent to those specific sequences; for example, nucleic acid sequences that have identical or similar properties to those of the specific nucleic acid sequences. As described below, some embodiments of the present teaching are characterized, inter alia, by nucleic acid sequences homologous to the nucleic acid sequences 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 nucleic acid sequence that is a variant of that given nucleic acid sequence shall 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 approximately 30, at least approximately 50, at least approximately 70, at least approximately 90, at least approximately 100, at least approximately 150, at least approximately 200, at least approximately 250, at least approximately 300, or at least approximately 400 nucleotides. In preferred embodiments, the degree of identity is specified for the entire length of the reference nucleic acid sequence. Sequence similarity indicates the percentage of amino acids that are identical or that represent conservative amino acid substitutions. Sequence identity between two polypeptide or nucleic acid sequences indicates the percentage of amino acids or nucleotides that are identical between the sequences. The term "% identical" refers, in particular, to a percentage of nucleotides that are identical in an optimal alignment between two sequences to be compared. This percentage is purely statistical, and the differences between the two sequences may be randomly distributed along the entire length of the sequence. The sequence to be compared may include additions or deletions compared to the reference sequence in order to obtain an optimal alignment between the two sequences.The comparison of two sequences is usually performed by comparing these sequences, after optimal alignment, with respect to a segment or "comparison window," in order to identify local regions of the corresponding sequences. Optimal alignment for a comparison can be performed manually or with the aid of the local homology algorithm 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 algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or with the aid of computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). The percentage of identity is obtained by determining the number of identical positions in which the sequences to be compared coincide, dividing this number by the number of positions compared and multiplying this result by 100. For example, you can use the "BLAST 2 sequences" program, which is available on the website http: / / www.ncbi.nlm.nih.gov / blast / bl2seq / wblast2.cgi. A nucleic acid is "capable of hybridizing" or "hybridizes" with another nucleic acid if the two sequences are complementary to each other. A nucleic acid is "complementary" to another nucleic acid if the two sequences are capable of forming a stable duplex with each other. According to the teaching, hybridization is preferably carried out under conditions that allow specific hybridization between polynucleotides (rigorous conditions). Strict conditions are described, for example, in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Editors, 2nd Edition, Cold Spring Harbor Laboratory and Press, Cold Spring Harbor, New York, 1989 or Current Protocols in Molecular Biology, FM 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% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH2PO4 (pH 7), 0.5% SDS, 2 mM EDTA). The SSC is 0.15 M sodium chloride / 0.15 M sodium citrate, pH 7. After hybridization, the membrane to which the DNA has been transferred is washed, for example, in 2 × SSC at room temperature and then in 0.1-0.5 × SSC / 0.1 × SDS at temperatures up to 68 °C. The percentage of 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 of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" or "fully complementary" means that all contiguous residues in one nucleic acid sequence will form hydrogen bonds with the same number of contiguous residues in a second nucleic acid sequence. Preferably, the degree of complementarity according to the teaching method is at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, or most preferably at least 95%, 96%, 97%, 98%, or 99%. Ideally, the degree of complementarity according to the teaching method should be 100%. The term "derivative" includes any chemical derivation of a nucleic acid at a nucleotide base, sugar, or phosphate. The term "derivative" also includes nucleic acids containing nucleotides and nucleotide analogues that are not naturally occurring. Preferably, the derivation of a nucleic acid increases its stability. According to the teaching, a "nucleic acid sequence derived from a nucleic acid sequence" refers to a nucleic acid that is a variant of the nucleic acid from which it is derived. Preferably, a sequence that is a variant with respect to a specific sequence, when it replaces the specific sequence in an RNA molecule, preserves RNA stability and / or translation efficiency. "nt" is an abbreviation for nucleotide; or nucleotides, preferably consecutive nucleotides in a nucleic acid molecule. According to the teaching, the term "codon" refers to a triplet of bases in a coding nucleic acid that specifies which amino acid will be added next during protein synthesis in the ribosome. The terms "transcription" and "transcribe" refer to a process during which a nucleic acid molecule with a particular nucleic acid sequence (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 contained in a nucleic acid template is transcribed. The nucleic acid template can be DNA; however, for example, in the case of transcription from an alphaviral nucleic acid template, the template is typically RNA. Subsequently, the transcribed RNA can be translated into protein. For the purposes of this teaching, the term "transcription" includes "in vitro transcription," where "in vitro transcription" refers to a process in which RNA, particularly mRNA, is synthesized in vitro in a cell-free system.Preferably, cloning vectors are used for generating transcripts. These cloning vectors are generally referred to as transcription vectors and, for the purposes of this instruction, are encompassed by the term "vector." The preferred cloning vectors are plasmids. For the purposes of this instruction, the RNA is preferably in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription from a suitable DNA template. The promoter controlling transcription can be any promoter for any RNA polymerase. A DNA template for in vitro transcription can be obtained by cloning a nucleic acid, in particular cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA. The single-stranded nucleic acid molecule that is produced during transcription typically has a nucleic acid sequence that is the complementary sequence to the template. According to the teaching, the terms "template", "nucleic acid template" or "nucleic acid template" generally refer to a nucleic acid sequence that can be replicated or transcribed. "Nucleic acid sequence transcribed from a nucleic acid sequence" and similar terms refer to a nucleic acid sequence, when appropriate as part of a complete RNA molecule, that is a transcription product of a template nucleic acid sequence. Typically, the transcribed nucleic acid sequence is a single-stranded RNA molecule. According to the teaching, the "3' end of a nucleic acid" refers to the end with a free hydroxyl group. In a schematic representation of double-stranded nucleic acids, particularly DNA, the 3' end is always on the right. According to the teaching, the "5' end of a nucleic acid" refers to the end with a free phosphate group. In a schematic representation of double-stranded nucleic acids, particularly DNA, the 5' end is always on the left. 5' end 5'--P-NNNNNNN-OH-3' 3' end 3'-HO-NNNNNNN-P--5' The term "upstream" describes the relative position of a first element of a nucleic acid molecule with respect to a second element of that same molecule, where both elements are contained within the same nucleic acid molecule, and where the first element is closer to the 5' end of the nucleic acid molecule than the second element. The second element is then said to be "downstream" of the first element of that nucleic acid molecule. An element that is "upstream" of a second element can be synonymously described as being located "5'" of that second element. For a double-stranded nucleic acid molecule, the designations "upstream" and "downstream" are given with respect to the (+) strand. According to the teaching, "functional linkage" or "functionally linked" refers to a connection within a functional relationship. A nucleic acid is "functionally linked" if it is functionally related to another nucleic acid sequence. For example, a promoter is functionally linked to a coding sequence if it influences the transcription of that coding sequence. Functionally linked nucleic acids are typically adjacent to each other, separated, where appropriate, by additional nucleic acid sequences, and, in certain embodiments, are transcribed by RNA polymerase to yield a single RNA molecule (common transcript). In particular embodiments, a nucleic acid is functionally linked, according to the methodology employed, to expression control sequences that may be homologous or heterologous with respect to the nucleic acid. The term "expression control sequence" encompasses, according to teaching promoters, ribosome-binding sequences and other control elements that regulate gene transcription or the translation of the resulting RNA. In specific teaching implementations, expression control sequences can be regulated. The precise structure of expression control sequences can vary depending on the species or cell type, but it usually includes 5' non-transcribed sequences and 5' and 3' non-translated sequences, involved in the initiation of transcription and translation, respectively. More specifically, 5' non-transcribed expression control sequences include a promoter region that encompasses a promoter sequence for the control of transcription of the functionally linked gene. Expression control sequences may also include upstream enhancer or activator sequences.A DNA expression control sequence typically includes 5' non-transcribed sequences and 5' and 3' non-translated sequences, such as the TATA box, the protection sequence, the CAAT sequence, and similar sequences. An alphaviral RNA expression control sequence may include a subgenomic promoter and / or one or more conserved sequence elements. For the purposes of this instruction, a specific expression control sequence is a subgenomic promoter of an alphavirus, as described herein. The nucleic acid sequences specified herein, in particular the transcribable and coding nucleic acid sequences, may be combined with any expression control sequence, particularly promoters, which may be homologous or heterologous to said nucleic acid sequences. The term "homologous" refers to the fact that a nucleic acid sequence is also naturally functionally linked to the expression control sequence, and the term "heterologous" refers to the fact that a nucleic acid sequence is not naturally functionally linked to the expression control sequence. A transcribable nucleic acid sequence, in particular a nucleic acid sequence encoding a peptide or protein, and an expression control sequence are "functionally" linked if they are covalently linked such that the transcription or expression of the transcribable nucleic acid sequence, and in particular the coding sequence, is under the control or influence of the expression control sequence. If the nucleic acid sequence is to be translated into a functional peptide or protein, the induction of an expression control sequence functionally linked to the coding sequence results in the transcription of that coding sequence without causing a frameshift in the coding sequence or preventing its translation into the desired peptide or protein. The term "promoter" or "promoter region" refers to a nucleic acid sequence that controls the synthesis of a transcript—for example, a transcript comprising a coding sequence—by providing a recognition and binding site for RNA polymerase. The promoter region may include additional recognition or binding sites for other factors involved in regulating the transcription of that gene. A promoter can control the transcription of a prokaryotic or eukaryotic gene. A promoter can be "inducible," initiating transcription in response to an inducer, or it can be "constitutive," meaning transcription is not controlled by an inducer. An inducible promoter is expressed only to a very small extent or not at all if there is no inducer. In the presence of an inducer, the gene is "activated," or the level of transcription increases. This is usually mediated by the binding of a specific transcription factor.According to this teaching, a specific promoter is a subgenomic promoter of an alphavirus, as described herein. Other specific promoters are the positive- or negative-strand genomic promoters of an alphavirus. The term "core promoter" refers to a nucleic acid sequence that comprises the promoter. The core promoter is typically the minimum portion of the promoter necessary to properly initiate transcription. It typically includes the transcription start site and an RNA polymerase binding site. A "polymerase" generally refers to a molecular entity capable of catalyzing the synthesis of a polymer molecule from monomeric building blocks. An "RNA polymerase" is a molecular entity capable of catalyzing the synthesis of an RNA molecule from ribonucleotide building blocks. A "DNA polymerase" is a molecular entity capable of catalyzing the synthesis of a DNA molecule from deoxyribonucleotide building blocks. In the case of DNA polymerases and RNA polymerases, the molecular entity is typically a protein or an assembly or complex of multiple proteins. Typically, a DNA polymerase synthesizes a DNA molecule from a template nucleic acid, which is typically a DNA molecule.Typically, an RNA polymerase synthesizes an RNA molecule from a template nucleic acid, which can be a DNA molecule (in which case the RNA polymerase is a DNA-dependent RNA polymerase, DdRP) or an RNA molecule (in which case the RNA polymerase is an RNA-dependent RNA polymerase, RdRP). An RNA-dependent RNA polymerase, or RdRP, is an enzyme that catalyzes the transcription of RNA from an RNA template. In the case of alphaviral RNA-dependent RNA polymerase, the sequential synthesis of the complement of the negative (-) strand of the genomic RNA and the positive (+) strand of the genomic RNA leads to RNA replication. Therefore, the RNA-dependent RNA polymerase of alphaviruses is sometimes referred to as an RNA replicase. In nature, RNA-dependent RNA polymerases are typically encoded by all RNA viruses except retroviruses. Typical representatives of viruses that encode an RNA-dependent RNA polymerase are alphaviruses. According to this teaching, "RNA replication" generally refers to an RNA molecule synthesized based on the nucleotide sequence of a given RNA molecule (template RNA molecule). The synthesized RNA molecule may be, for example, identical or complementary to the template RNA molecule. In general, RNA replication can occur through the synthesis of a DNA intermediate, or it can occur directly through RNA-dependent RNA replication mediated by an RNA-dependent RNA polymerase (RdRP). In the case of alphaviruses, RNA replication does not occur through a DNA intermediate, but is mediated by an RNA-dependent RNA polymerase (RdRP): a template RNA strand (first RNA strand)—or a portion thereof—serves as a template for the synthesis of a second RNA strand that is complementary to the first RNA strand or a portion thereof.The second RNA strand—or a portion of it—can optionally serve as a template for the synthesis of a third RNA strand that is complementary to the second RNA strand or a portion of it. Thus, the third RNA strand is identical to the first RNA strand or a portion of it. In this way, RNA-dependent RNA polymerase is able to directly synthesize a complementary RNA strand from a template and to indirectly synthesize an identical RNA strand (via a complementary intermediate strand). According to the teaching, the term "template RNA" refers to RNA that can be transcribed or replicated by an RNA-dependent RNA polymerase. According to the teaching, the term "gene" refers to a particular sequence of nucleic acid that is responsible for producing one or more cellular products and / or carrying out one or more intercellular or intracellular functions. More specifically, the term refers to a section of nucleic acid (typically DNA, but RNA in the case of RNA viruses) that comprises a nucleic acid encoding a specific protein or a functional or structural RNA molecule. In this document, "isolated molecule," as used herein, is intended to refer to a molecule that is substantially free of other molecules, such as other cellular material. The term "isolated nucleic acid" means, according to the teaching, that the nucleic acid has been (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, for example by gel electrophoretic cleavage and fractionation, or (iv) synthesized, for example by chemical synthesis. An isolated nucleic acid is a nucleic acid that can be manipulated by recombinant techniques. The term "vector" is used here in its broadest sense and includes any intermediate vehicle for a nucleic acid that, for example, allows the introduction of that nucleic acid into prokaryotic and / or eukaryotic host cells and, where appropriate, its integration into a genome. Preferably, these vectors are replicated and / or expressed within the cell. Vectors include plasmids, phagemids, viral genomes, and fragments thereof. The term "recombinant" in the context of this teaching means "created through genetic engineering." Preferably, a "recombinant object," such as a recombinant cell in the context of this teaching, does not occur 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 that can be isolated from a natural source, and that has not been intentionally modified by humans in a laboratory, is considered to be naturally occurring. The term "found in nature" means "present in nature" and includes known objects as well as objects that have not yet been discovered or isolated from nature but that could be discovered or isolated in the future from a natural source. According to the teaching, the term "expression" is used in its broadest sense and encompasses the production of RNA, or of RNA and protein. It also includes the partial expression of nucleic acids. Furthermore, expression can be transient or stable. With regard to RNA, the term "expression" or "translation" refers to the process that takes place in the ribosomes of a cell whereby a coding RNA strand (e.g., messenger RNA) directs the assembly of an amino acid sequence to form a peptide or a protein. According to the teaching, the term "mRNA" stands for "messenger RNA" and refers to a transcript that is typically generated using a DNA template and encodes a peptide or protein. Typically, mRNA comprises a 5-UTR, a protein-coding region, a 3-UTR, and a poly(A) sequence. mRNA can be generated by in vitro transcription from a DNA template. The methodology for in vitro transcription is well-known to experts. For example, a variety of in vitro transcription kits are commercially available. According to the teaching, mRNA can be modified by stabilizing agents and the addition of a protective coating. According to the teaching, the terms "poly(A) sequence" or "poly(A) tail" refer to an uninterrupted or interrupted sequence of adenylate residues typically found at the 3' end of an RNA molecule. An uninterrupted sequence is characterized by the presence of consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical. While a poly(A) sequence is not typically encoded in eukaryotic DNA but is attached during eukaryotic transcription in the cell nucleus to the free 3' end of RNA by a template-independent RNA polymerase after transcription, this teaching covers DNA-encoded poly(A) sequences. According to the teaching, the term "primary structure", with reference to a nucleic acid molecule, refers to the linear sequence of nucleotide monomers. According to the teaching, the term "secondary structure," with reference to a nucleic acid molecule, refers to a two-dimensional representation of a nucleic acid molecule that reflects base pairings; for example, in the case of a single-stranded RNA molecule, specifically intramolecular base pairings. Although each RNA molecule has only one polynucleotide chain, the molecule is typically characterized by (intramolecular) base-pairing regions. According to the teaching, the term "secondary structure" encompasses structural motifs that include, but are not limited to, base pairs, stems, stem loops, protrusions, loops such as internal loops, and multi-branched loops.The secondary structure of a nucleic acid molecule can be represented by a two-dimensional drawing (planar graph), which shows the base pairings (for more details on the secondary structure of RNA molecules, see Auber et al., J. Graph Algorithms Appl., 2006, vol.10, pp.329-351). As described herein, the secondary structure of certain RNA molecules is relevant in the context of this teaching. According to the instruction, the secondary structure of a nucleic acid molecule, particularly a single-stranded RNA molecule, is determined by prediction using the RNA Secondary Structure Prediction Web Server (http: / / rna.urmc.rochester.edu / RNAstructureWeb / Servers / Predict1 / Predict1.html). Preferably, according to the instruction, "secondary structure," with reference to a nucleic acid molecule, refers specifically to the secondary structure determined by this prediction. The prediction can also be made or confirmed using the MFOLD structure prediction tool (http: / / unafold.rna.albany.edu / ?q=mfold). According to the instruction, a "base pair" is a structural motif of a secondary structure in which two nucleotide bases are associated with each other by hydrogen bonds between the donor and acceptor sites of the bases.The complementary bases A:U and G:C form stable base pairs through hydrogen bonds between the donor and acceptor sites of the bases; the A:U and G:C base pairs are called Watson-Crick base pairs. The G and U bases (G:U) form a weaker base pair (called a wobble base pair). The A:U and G:C base pairs are called canonical base pairs. Other base pairs such as G:U (which occurs quite frequently in RNA) and other rare base pairs (e.g., A:C; U:U) are called non-canonical base pairs. According to the teaching, "nucleotide pairing" refers to two nucleotides associating with each other so that their bases form a base pair (canonical or non-canonical base pair, preferably canonical base pair, and most preferably Watson-Crick base pair). According to the teaching, the terms "stem loop," "hairpin," or "hairpin loop," with reference to a nucleic acid molecule, refer interchangeably 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 represented by the stem loop consists of a consecutive nucleic acid sequence comprising a stem and a (terminal) loop, also called a hairpin loop, where the stem is formed by two neighboring sequence elements, totally or partially complementary, which are separated by a short sequence (e.g., 3–10 nucleotides), forming the loop of the stem-loop structure. The two neighboring sequences, totally or partially complementary, can be defined, for example, as stem-loop elements, stem 1 and stem 2.A stem loop is formed when two complementary, wholly or partially inverse, and adjacent sequences—for example, the stem loop elements, stem 1 and stem 2—pair with each other, resulting in a double-stranded nucleic acid sequence that includes an unpaired loop at its terminal end formed by the short sequence located between the stem loop elements, stem 1 and stem 2. Thus, a stem loop comprises two stems (stem 1 and stem 2), which, at the level of the secondary structure of the nucleic acid molecule, pair 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 either stem 1 or stem 2. As an example, a two-dimensional representation of the stem loop resembles a lollipop-shaped structure.The formation of a stem-loop structure requires the presence of a sequence 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 usually determined by their length, that is, the number of nucleotides in stem 1 that are capable of forming base pairs (preferably canonical base pairs, and more preferably Watson-Crick base pairs) with the nucleotides in stem 2, compared to the number of nucleotides in stem 1 that are not capable of forming such base pairs with the nucleotides in stem 2 (non-matches or bulges). According to the present teaching, the optimum loop length is 3–10 nucleotides, preferably 4–7 nucleotides, such as 4, 5, 6, or 7 nucleotides.If a given nucleic acid sequence is characterized by a stem-loop structure, the corresponding complementary nucleic acid sequence is also usually characterized by a stem-loop structure. A hairpin loop is typically formed from single-stranded RNA molecules. For example, several stem loops are found in the 5' replication recognition sequence of alphavirus genomic RNA (illustrated in Figure 5). According to the teaching, "alteration" or "distort," with reference to a specific secondary structure of a nucleic acid molecule (e.g., a stem-loop), means that the specific secondary structure is absent or altered. Typically, a secondary structure can be altered as a result of a change in at least one nucleotide that is part of the secondary structure. For example, a stem-loop can be altered by changing one or more nucleotides that form the stem, so that nucleotide pairing is no longer possible. According to the teaching, "compensating for alteration of secondary structure" or "compensating for alteration of secondary structure" refers to one or more nucleotide changes in a nucleic acid sequence; more typically, it refers to one or more second nucleotide changes in a nucleic acid sequence, which also includes one or more first nucleotide changes, characterized as follows: while one or more first nucleotide changes, in the absence of one or more second nucleotide changes, cause an alteration of the secondary structure of the nucleic acid sequence, the co-occurrence of one or more first nucleotide changes and one or more second nucleotide changes does not cause the secondary structure of the nucleic acid to be altered. Co-occurrence means the presence of both one or more first nucleotide changes and one or more second nucleotide changes.Typically, one or more first nucleotide changes and one or more second nucleotide changes are present together in the same nucleic acid molecule. In a specific embodiment, one or more nucleotide changes that compensate for the alteration of the secondary structure are one or more nucleotide changes that compensate for one or more alterations in nucleotide pairing. Thus, in one embodiment, "compensating for the alteration of the secondary structure" means "compensating for alterations in nucleotide pairing," i.e., one or more alterations in nucleotide pairing, for example, one or more alterations in nucleotide pairing within one or more stem loops. The one or more alterations in nucleotide pairing may have been introduced by the deletion of at least one start codon.Each of the one or more nucleotide changes that compensate for the alteration of the secondary structure is a nucleotide change, which can 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 A:U nucleotide pairing has been altered by the substitution of A for C (C and U are not typically suitable to form a nucleotide pair), then a nucleotide change that compensates for the altered nucleotide pairing could be the substitution of U for G, allowing the formation of the C:G nucleotide pairing. The substitution of U for G thus compensates for the altered nucleotide pairing.In an alternative example, when the A:U nucleotide pairing has been altered by the substitution of A for C, then a nucleotide change that compensates for the altered nucleotide pairing could be the substitution of C for A, thus restoring the original A:U nucleotide pairing. In general, for the purposes of this instruction, nucleotide changes that compensate for the alteration of the secondary structure and do not restore the original nucleic acid sequence or create novel AUG triplets are preferred. In the preceding set of examples, the substitution of U for G is preferred to the substitution of C for A. According to the teaching, the term "tertiary structure", with reference to a nucleic acid molecule, refers to the three-dimensional structure of a nucleic acid molecule, as defined by atomic coordinates. According to the teaching, a nucleic acid such as RNA, for example mRNA, can encode a peptide or a protein. Consequently, a transcribable nucleic acid sequence or a transcript thereof can contain an open reading frame (ORF) that encodes a peptide or a protein. According to the teaching, the term "nucleic acid encoding 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, according to the teaching, the coding RNA is capable of interacting with the cellular translation machinery, allowing the translation of the coding RNA to produce a peptide or protein. According to the teaching, the term "peptide" comprises oligopeptides and polypeptides and refers to substances comprising two or more, preferably three or more, preferably four or more, preferably six or more, preferably eight or more, preferably ten or more, preferably thirteen or more, preferably sixteen or more, preferably twenty or more, and up to preferably fifty, preferably one hundred, or preferably one hundred and fifty, consecutive amino acids linked together by peptide bonds.The term "protein" refers to large peptides, preferably peptides that have at least 151 amino acids, but the terms "peptide" and "protein" are usually used here as synonyms. According to the teaching, the terms "peptide" and "protein" comprise substances that contain not only amino acid components, but also non-amino acid components such as sugars and phosphate structures, and also substances that contain bonds such as ester, thioether, or disulfide bonds. According to the teaching, the terms "initiation codon" and "start codon" refer interchangeably to a codon (triplet of bases) in an RNA molecule that is potentially the first codon translated by a ribosome. This codon typically codes for the amino acid methionine in eukaryotes and a modified methionine in prokaryotes. The most common initiation codon in eukaryotes and prokaryotes is AUG. Unless specifically stated herein as referring to an initiation codon other than AUG, the terms "initiation codon" and "start codon," with reference to an RNA molecule, refer to the AUG codon. According to the teaching, the terms "initiation codon" and "start codon" are also used to refer to a corresponding triplet of bases in a deoxyribonucleic acid, specifically the triplet of bases that codes for the initiation codon of an RNA. If the start codon of the messenger RNA is AUG, the triplet of bases that codes for AUG is ATG.According to the teaching, the terms "initiation codon" and "start codon" preferably refer to a functional initiation codon, that is, a codon that a ribosome uses or would use as the codon to initiate translation. In an RNA molecule, there may be AUG codons that are not used by a ribosome to initiate translation, for example, due to the short distance between the codons and the protectant. These codons are not included in the term functional initiation codon. According to the teaching, the terms "open reading frame start codon" or "open reading frame initiation codon" refer to the triplet of bases that serves as the start codon for protein synthesis in a coding sequence, for example, in the coding sequence of a nucleic acid molecule found in nature. In an RNA molecule, the open reading frame start codon is usually preceded by a 5' untranslated region (5'-UTR), although this is not strictly necessary. According to the teaching, the terms "native open reading frame start codon" or "native open reading frame initiation codon" refer to the triplet of bases that serves as the start codon for protein synthesis in a native coding sequence. A native coding sequence can be, for example, the coding sequence of a nucleic acid molecule found in nature. In some embodiments, the present teaching provides variants of naturally occurring nucleic acid molecules, which are characterized by the removal of the native start codon (present in the native coding sequence) (so that it is not present in the variant nucleic acid molecule). According to the teaching, "first AUG" means the upstream AUG base triplet of a messenger RNA molecule, preferably the upstream AUG base triplet of a messenger RNA molecule that is used or would be used as a codon by a ribosome to initiate translation. Consequently, "first ATG" refers to the ATG base triplet of a coding DNA sequence that encodes the first AUG. In some cases, the first AUG of an mRNA molecule is the open reading frame start codon, that is, the codon used as the start codon during ribosomal protein synthesis. According to the teaching, the terms "comprising deletion" or "characterized by deletion" and similar terms, with reference to a particular element of a nucleic acid variant, mean that the element is non-functional or absent in the nucleic acid variant compared to a reference nucleic acid molecule. Without limitation, a deletion may consist of the complete or partial removal of a particular element, the complete or partial substitution of a functional element, or the alteration of the functional or structural properties of the particular element. The deletion of a functional element from a nucleic acid sequence requires that the function is not manifested at the position in the nucleic acid variant comprising the deletion.For example, an RNA variant characterized by the deletion of a particular start codon requires that ribosomal protein synthesis not begin at the position of the RNA variant characterized by the deletion. Similarly, the deletion of a structural element from a nucleic acid sequence requires that the structural element not be present at the position of the nucleic acid variant containing the deletion.For example, an RNA variant characterized by the deletion of a particular AUG base triplet, i.e., an AUG base triplet at a specific position, can be characterized, for example, by deleting part or all of that AUG base triplet (e.g., AUG), or by substituting one or more nucleotides (A, U, G) of the particular AUG base triplet with any one or more different nucleotides, such that the resulting nucleotide sequence of the variant does not include that AUG base triplet. Suitable nucleotide substitutions are those that convert the AUG base triplet into a GUG, CUG, or UUG base triplet, or into an AAG, ACG, or AGG base triplet, or into an AUA, AUC, or AUU base triplet. Consequently, suitable substitutions of more nucleotides can be selected. According to the teaching, the term "alphavirus" should be understood broadly and includes any viral particle that has alphavirus characteristics. Alphavirus characteristics include the presence of a (+) stranded RNA that encodes genetic information suitable for replication in a host cell, including RNA polymerase activity. Additional features of many alphaviruses are described, for example, in Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491–562. The term "alphavirus" includes naturally occurring alphaviruses as well as any variants or derivatives thereof. In some embodiments, a variant or derivative is not found in nature. In one of its realizations, an alphavirus is an alphavirus found in nature. Typically, a naturally occurring alphavirus is infectious to one or more eukaryotic organisms, such as an animal (including vertebrates such as humans and arthropods such as insects). Preferably, an alphavirus found in nature is selected from the group consisting of the following: 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 (comprising Ndumu virus); Semliki Forest virus complex (comprising Bebaru virus, Chikungunya virus, Mayaro virus and its subtype Una virus, O'Nyong Nyong virus and its subtype Igbo-Ora virus, Ross River virus and its 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, Venezuelan equine encephalitis virus); Western equine encephalitis complex (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 sickness virus; southern elephant seal virus;the Tonate virus. More preferably, the alphavirus is selected from the group consisting of the Semliki forest virus complex (comprising the virus types indicated above, including the Semliki forest virus), the western equine encephalitis complex (comprising the virus types indicated above, including the Sindbis virus), the eastern equine encephalitis virus (comprising the virus types indicated above), and the Venezuelan equine encephalitis complex (comprising the virus types indicated above, including the Venezuelan equine encephalitis virus). In a further preferred embodiment, the alphavirus is the Semliki forest virus. In a preferred alternative embodiment, the alphavirus is the Sindbis virus. In a preferred alternative embodiment, the alphavirus is the Venezuelan equine encephalitis virus. In some versions of this teaching, an alphavirus is not a naturally occurring alphavirus. Typically, a non-natural alphavirus is a variant or derivative of a naturally occurring alphavirus, distinguished from the latter by at least one mutation in its nucleotide sequence, i.e., in its genomic RNA. The nucleotide sequence mutation may be selected by an insertion, substitution, or deletion of one or more nucleotides, compared to a naturally occurring alphavirus. A nucleotide sequence mutation may or may not be associated with a mutation in a polypeptide or protein encoded by that nucleotide sequence. For example, a non-natural alphavirus 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 sequence that distinguishes it from a naturally occurring alphavirus, and is either not infectious at all, or is infectious but has a reduced capacity to cause disease or no capacity to cause disease. As an illustrative example, TC83 is an attenuated alphavirus that is distinct from the naturally occurring Venezuelan equine encephalitis virus (VEEV) (McKinney et al., 1963, Am. J. Trop. Med. Hyg., 1963, vol.12; pp.597-603). Members of the genus Alphavirus can also be classified based on their relative clinical characteristics in humans: alphaviruses primarily associated with encephalitis and alphaviruses primarily associated with fever, rash, and polyarthritis. The term "alphaviral" means that it is found in an alphavirus, or that it originates from an alphavirus, or that it is derived from an alphavirus, for example, through genetic engineering. According to the teaching, "SFV" stands for Semliki Forest virus. According to the teaching, "SIN" or "SINV" stands for Sindbis virus. According to the teaching, "VEE" or "VEEV" stands for Venezuelan equine encephalitis virus. According to the teaching, the term "of an alphavirus" refers to an entity originating from an alphavirus. For illustration, a protein of an alphavirus may refer to a protein found in the alphavirus and / or a protein encoded by the alphavirus; and a nucleic acid sequence of an alphavirus may refer to a nucleic acid sequence found in the alphavirus and / or a nucleic acid sequence encoded by the alphavirus. Preferably, a nucleic acid sequence "of an alphavirus" refers to a nucleic acid sequence "from the genome of an alphavirus" and / or "from the genomic RNA of an alphavirus." According to the teaching, the term "alphaviral RNA" refers to one or more alphaviral genomic RNAs (i.e., the (+) strand), complements of alphaviral genomic RNA (i.e., the (-) strand), and the subgenomic transcript (i.e., the (+) strand), or a fragment of either. According to the teaching, "alphavirus genome" refers to the (+) RNA genomic chain of an alphavirus. According to the teaching, the term "native alphavirus sequence" and similar terms typically refer to a sequence (e.g., nucleic acid) of a naturally occurring alphavirus (an alphavirus found in nature). In some embodiments, the term "native alphavirus sequence" also includes a sequence of an attenuated alphavirus. According to the teaching, the term "5' replication recognition sequence" preferably refers to a continuous nucleic acid sequence, preferably a ribonucleic acid sequence, that is identical or homologous to 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 both native 5' replication recognition sequences and their functional equivalents, such as, for example, functional variants of a 5' alphavirus replication recognition sequence found in nature. According to the teaching, functional equivalents include derivatives of 5' replication recognition sequences characterized by the deletion of at least one start codon, as described herein.The 5' replication recognition sequence is required for the synthesis of the (-) strand complement of the alphavirus genomic RNA and is required for the synthesis of the (+) strand viral genomic RNA based on a (-) strand template. A native 5' replication recognition sequence typically encodes at least the N-terminal fragment of nsP1; however, it does not comprise the entire open reading frame encoding nsP1234. Since a native 5' replication recognition sequence typically encodes at least the N-terminal fragment of nsP1, it also typically comprises at least one start codon, typically AUG. In one embodiment, the 5' replication recognition sequence comprises conserved sequence element 1 of an alphavirus genome (CSE 1) or a variant thereof and conserved sequence element 2 of an alphavirus genome (CSE 2) or a variant thereof.The 5' replication recognition sequence is usually able to form four stem loops (SL), namely SL1, SL2, SL3, SL4. The numbering of these stem loops starts at the 5' end of the 5' replication recognition sequence. According to the teaching, the term "at the 5' end of an alphavirus" refers to the 5' end of the alphavirus genome. A nucleic acid sequence at the 5' end of an alphavirus comprises the nucleotide located at the 5' terminal of the alphavirus genomic RNA, plus optionally a consecutive sequence of additional nucleotides. In one embodiment, a nucleic acid sequence at the 5' end of an alphavirus is identical to the 5' replication recognition sequence of the alphavirus genome. The term "conserved sequence element" or "CSE" refers to a nucleotide sequence found in the RNA of alphaviruses. These sequence elements are called "conserved" because orthologs exist in the genomes of different alphaviruses, and the ortholog CSEs of different alphaviruses preferably share a high percentage of sequence identity and / or a similar secondary or tertiary structure. The term CSE includes CSE1, CSE2, CSE3, and CSE4. According to the teaching, the terms "CSE 1" or "44 nt CSE" refer interchangeably to a nucleotide sequence required for the synthesis of the (+) strand 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) functions as a promoter for the synthesis of the (+) strand. Preferably, the term CSE 1 includes the 5' end nucleotide of the alphavirus genome. CSE 1 typically forms a conserved stem-loop structure. Without adhering to any particular theory, it is believed that, for CSE 1, the secondary structure is more important than the primary structure, i.e., the linear sequence. In the genomic RNA of the model alphavirus Sindbis virus, CSE 1 consists of a consecutive sequence of 44 nucleotides, which is formed by the 44 nucleotides plus 5' of the genomic RNA (Strauss & Strauss, Microbiol. Rev., 1994, vol.58, pp.491-562). According to the teaching, the terms "CSE 2" or "51-nt CSE" refer interchangeably to a nucleotide sequence required for the synthesis of the (-) strand from a (+) strand template. The (+) strand template is typically alphavirus genomic RNA or an RNA replicon (note that the subgenomic RNA transcript, which does not comprise CSE 2, does not function as a template for (-) strand synthesis). In alphavirus genomic RNA, CSE 2 is typically located within the coding sequence of nsP1. In the genomic RNA of the model alphavirus Sindbis virus, the 51-nt CSE is found at nucleotide positions 155–205 of the genomic RNA (Frolov et al., 2001, RNA, vol. 7, pp. 1638–1651). CSE 2 typically forms two conserved stem-loop structures.These stem-loop structures are called stem-loop 3 (SL3) and stem-loop 4 (SL4) because they are the third and fourth conserved stem-loops, respectively, of the alphavirus genomic RNA, counting from the 5' end of the alphavirus genomic RNA. Without adhering to any particular theory, it is believed that, for CSE 2, the secondary structure is more important than the primary structure, i.e., the linear sequence. According to the teaching, the terms "CSE 3" or "binding sequence" refer interchangeably to a nucleotide sequence derived from alphaviral genomic RNA that comprises the subgenomic RNA start site. The complement of this sequence on the (-) strand acts to promote transcription of the subgenomic RNA. 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 located upstream of the open reading frame that encodes the structural proteins. According to the teaching, the terms "CSE 4," "19-nt conserved sequence," or "19-nt CSE" refer interchangeably to a nucleotide sequence in the genomic RNA of alphaviruses, immediately upstream of the poly(A) tail in the 3' untranslated region of the alphavirus genome. The CSE 4 sequence typically consists of 19 consecutive nucleotides. Without adhering to any particular theory, CSE 4 is understood to function as a central promoter for the initiation of (-) strand synthesis (José et al., Future Microbiol., 2009, vol. 4, pp. 837–856); and / or CSE 4 and the poly(A) tail of the alphavirus genomic RNA are understood to work together for efficient (-) strand synthesis (Hardy & Rice, J. Virol., 2005, vol. 79, pp. 4630-4639) . According to the teaching, the term "subgenomic promoter" or "SGP" refers to a nucleic acid sequence located upstream (5') of another nucleic acid sequence (e.g., a coding sequence) that controls the transcription of that sequence by providing a recognition and binding site for RNA polymerase, typically RNA-dependent RNA polymerase, specifically the functional non-structural protein of the alphavirus. The SGP may include additional recognition or binding sites for other factors. A subgenomic promoter is typically a genetic element of a positive-strand RNA virus, such as an alphavirus. An alphavirus subgenomic promoter is a nucleic acid sequence that is part of the viral genomic RNA.The subgenomic promoter is generally characterized by allowing the initiation of transcription (RNA synthesis) in the presence of an RNA-dependent RNA polymerase, for example, a functional nonstructural protein of the alphavirus. A negative RNA strand, i.e., the complement of the alphaviral genomic RNA, serves as a template for the synthesis of a positive strand subgenomic transcript, and the synthesis of the positive strand subgenomic transcript typically begins at or near the subgenomic promoter. The term "subgenomic promoter," as used herein, is not limited to any particular location on a nucleic acid containing such a subgenomic promoter. In some embodiments, the SGP is identical to, overlaps with, or comprises CSE3. The terms "subgenomic transcript" or "subgenomic RNA" refer interchangeably to an RNA molecule that can be obtained as a result of transcription using an RNA molecule as a template ("template RNA"), where the template RNA comprises a subgenomic promoter that controls the transcription of the subgenomic transcript. The subgenomic transcript can be obtained in the presence of an RNA-dependent RNA polymerase, in particular the functional nonstructural protein of the alphavirus. For example, the term "subgenomic transcript" may refer to the RNA transcript prepared in a cell infected by an alphavirus, using the complement of the (-) strand of the alphavirus genomic RNA as a template. However, the term "subgenomic transcript," as used herein, is not limited to this; it also includes transcripts that can be obtained using heterologous RNA as a template.For example, subgenomic transcripts can also be obtained using the minus strand complement of SGP-containing replicons as a template, as described herein. Therefore, the term "subgenomic transcript" can refer either to an RNA molecule obtained by transcribing a fragment of alphavirus genomic RNA or to an RNA molecule obtained by transcribing a fragment of a replicon as described herein. The term "autologous" is used to describe anything derived from the same individual. For example, an "autologous cell" refers to a cell derived from the same individual. Introducing autologous cells into a subject is advantageous because these cells overcome the immune barrier that would otherwise cause rejection. The term "allogeneic" is used to describe anything derived from different individuals of the same species. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. The term "syngenic" is used to describe anything derived from individuals or tissues that have 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 that consists of multiple different elements. For example, introducing cells from one individual into another constitutes a heterologous transplant. A heterologous gene is a gene derived from a source other than the subject. The following are specific and / or preferred variations of the individual teaching features. This teaching also considers as particularly preferred realizations those generated by combining two or more of the specific and / or preferred variations described for two or more of the features of this teaching. RNA replicon A nucleic acid construct capable of being replicated by a replicase, preferably an alphaviral replicase, is called a replicon. According to the teaching, the term "replicon" defines an RNA molecule that can be replicated by RNA-dependent RNA polymerase, producing—without a DNA intermediate—one or more identical or essentially identical copies of the RNA replicon. "Without a DNA intermediate" means that no deoxyribonucleic acid (DNA) copy or complement of the replicon is formed in the process of forming the RNA replicon copies, and / or that no DNA molecule is used as a template in the process of forming the RNA replicon copies or its complement. The replicase function is typically provided by a functional nonstructural protein of the alphavirus. According to the teaching, the terms "can be replicated" and "capable of being replicated" generally describe that one or more identical or essentially identical copies of a nucleic acid can be prepared. When used in conjunction with the term "replicase," as in "capable of being replicated by a replicase," the terms "can be replicated" and "capable of being replicated" describe the functional characteristics of a nucleic acid molecule, for example, an RNA replicon, with respect to a replicase. These functional characteristics comprise at least one of the following: (i) the replicase is capable of recognizing the replicon, and (ii) the replicase is capable of acting as an RNA-dependent RNA polymerase (RdRP). Preferably, the replicase is capable of (i) recognizing the replicon and (ii) acting as an RNA-dependent RNA polymerase. The expression "recognizable" describes that the replicase is able to physically associate with the replicon and, preferably, that the replicase is able to bind to the replicon, typically in a non-covalent manner. The term "binding" may mean that the replicase has the ability to bind to one or more of the following elements: conserved sequence element 1 (CSE 1) or its complement sequence (if included in the replicon), conserved sequence element 2 (CSE 2) or its complement sequence (if included in the replicon), conserved sequence element 3 (CSE 3) or its complement sequence (if included in the replicon), conserved sequence element 4 (CSE 4) or its complement sequence (if included in the replicon).Preferably, the replicase is able to bind to CSE 2 [i.e., the (+) strand] and / or to CSE 4 [i.e., the (+) strand], or to bind to the complement of CSE 1 [i.e., the (-) strand] and / or to the complement of CSE 3 [i.e., the (-) strand]. The expression "capable of acting as RdRP" means that the replicase is capable of catalyzing the synthesis of the (-) strand complement of the (+) strand alphaviral genomic RNA, where the (+) strand RNA serves as a template, and / or that the replicase is capable of catalyzing the synthesis of the (+) strand alphaviral genomic RNA, where the (-) strand RNA serves as a template. In general, the expression "capable of acting as RdRP" may also include that the replicase is capable of catalyzing the synthesis of a (+) strand subgenomic transcript where a (-) strand RNA serves as a template, and where the synthesis of the (+) strand subgenomic transcript is typically initiated at an alphaviral subgenomic promoter. The terms "capable of binding" and "capable of acting as RdRP" refer to the capacity under normal physiological conditions. Specifically, they refer to the conditions within a cell that expresses the functional alphavirus non-structural protein or that has been transfected with a nucleic acid encoding the functional alphavirus non-structural protein. The cell is preferably a eukaryotic cell. The binding capacity and / or the capacity to act as RdRP can be tested experimentally, for example, in a cell-free in vitro system or in a eukaryotic cell. Optionally, such a eukaryotic cell is a cell of a species to which the particular alphavirus representing the origin of the replicase is infectious. For example, when using the replicase from a particular alphavirus that is infectious to humans, the normal physiological conditions are those found in a human cell.More preferably, the eukaryotic cell (in an example human cell) is from the same tissue or organ to which the particular alphavirus representing the origin of the replicase is infectious. According to the teaching, "compared to a native alphavirus sequence" and similar terms refer to a sequence that is a variant of a native alphavirus sequence. The variant is typically not itself a native alphavirus sequence. The teaching RNA replicon comprises a 5' replication recognition sequence. A 5' replication recognition sequence is a nucleic acid sequence that can be recognized by the alphavirus's functional nonstructural protein. In other words, the alphavirus's functional nonstructural protein is capable of recognizing the 5' replication recognition sequence. In one embodiment, the teaching RNA replicon comprises a 5' replication recognition sequence, wherein the 5' replication recognition sequence is characterized in that it comprises the deletion of at least one start codon compared to a native alphavirus 5' replication recognition sequence. The 5' replication recognition sequence characterized by the deletion of at least one start codon compared to a native alphavirus 5' replication recognition sequence, as described herein, may be referred to herein as a "modified 5' replication recognition sequence" or a "5' replication recognition sequence as described herein." As described below, the 5' replication recognition sequence as described herein may optionally be characterized by the presence of one or more additional nucleotide changes. 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 the functional nonstructural protein of the alphavirus. In other words, the functional nonstructural protein of the alphavirus is capable of recognizing the 3' replication recognition sequence. Preferably, the 3' replication recognition sequence is located at the 3' end of the replicon (if the replicon does not 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 consists of or comprises CSE 4.In one embodiment, the 5' replication recognition sequence and the 3' replication recognition sequence are capable of directing RNA replicon replication according to the present instruction in the presence of the functional alphavirus nonstructural protein. Thus, when present alone or, preferably, together, these recognition sequences direct RNA replicon replication in the presence of the functional alphavirus nonstructural protein. It is preferable to provide a functional non-structural alphavirus protein in cis (encoded as a protein of interest by an open reading frame in the replicon) or in trans (encoded as a protein of interest by an open reading frame in a separate replicase construct as described in the second aspect), which is capable of recognizing both the optionally modified 5' replication recognition sequence and the 3' replication recognition sequence of the replicon.In one embodiment, this is achieved when the 5' replication recognition sequence and the 3' replication recognition sequence are unique to the alphavirus from which the functional nonstructural protein of the alphavirus is derived, or when the 3' replication recognition sequence is unique to the alphavirus from which the functional nonstructural protein of the alphavirus is derived and the modified 5' replication recognition sequence is a variant of the 5' replication recognition sequence that is unique to the alphavirus from which the functional nonstructural protein of the alphavirus is derived. Native means that the natural origin of these sequences is the same alphavirus.In an alternative embodiment, the 5' (modified) replication recognition sequence and / or the 3' replication recognition sequence are not native to the alphavirus from which the functional alphavirus nonstructural protein is derived, provided that the functional alphavirus nonstructural protein is capable of recognizing both the 5' (modified) replication recognition sequence and the 3' replication recognition sequence of the replicon. In other words, the functional alphavirus nonstructural protein is compatible with both the 5' (modified) replication recognition sequence and the 3' replication recognition sequence. When a functional nonstructural protein of a nonnative alphavirus is capable of recognizing a specific sequence or sequence element, that functional alphavirus nonstructural protein is said to be compatible (virus compatibility).Any combination of replication recognition sequences (3' / 5') and CSE, respectively, with functional alphavirus nonstructural protein is possible, provided there is virus compatibility. Virus compatibility can be easily verified by the qualified instructor by incubating the functional alphavirus nonstructural protein to be analyzed with an RNA molecule containing 3' and 5' replication recognition sequences (optionally modified) to be analyzed, under conditions suitable for RNA replication, for example, in a suitable host cell. If replication occurs, the replication recognition sequences (3' / 5') and the functional alphavirus nonstructural protein are determined to be compatible. The removal of at least one start codon provides several advantages. The absence of a start codon in the nucleic acid sequence encoding nsP1* will typically result in nsP1* (the N-terminal fragment of nsP1) not being translated. Furthermore, since nsP1* is not translated, the open reading frame encoding the protein of interest ("transgene") is the most upstream open reading frame accessible to the ribosome; therefore, 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 transreplicons of the previous technique, such as those described by Spuul et al. (J. Virol., 2011, vol.85, pp.4739-4751): According to Spuul et al., replicons direct the expression of the N-terminal portion of nsP1, a peptide of 74 amino acids.It is also known from the above technique that the construction of RNA replicons from full-length viral genomes is not a trivial matter, since certain mutations can make the RNA unable to replicate (WO 2000 / 053780 A2), and the removal of some parts of the 5' structure that is important for alphavirus replication affects the efficiency of replication (Kamrud et al., 2010, J. Gen. Virol., vol.91, pp.1723-1727). The advantage over conventional cis-replicons is that deleting at least one start codon uncouples the coding region of the alphaviral nonstructural protein from the 5' replication recognition sequence. This allows for greater engineering of cis-replicons, for example, by replacing the native 5' replication recognition sequence with an artificial sequence, a mutated sequence, or a heterologous sequence taken from another RNA virus. Such sequence manipulations in conventional cis-replicons are restricted by the amino acid sequence of nsP1. Any point mutation, or clusters of point mutations, would require experimental evaluation to determine whether replication is affected, and small insertions or deletions that cause frameshift mutations are impossible due to their detrimental effect on the protein. The deletion of at least one start codon, according to the present instruction, can be achieved by any suitable method known to the art. For example, a suitable DNA molecule encoding the replicon according to the instruction, i.e., characterized by the deletion of a start codon, can be designed in silico and subsequently synthesized in vitro (gene synthesis). Alternatively, a suitable DNA molecule can be obtained by mutagenesis directed at a specific site in a DNA sequence encoding a replicon. In either case, the corresponding DNA molecule can serve as a template for transcription in vitro, thereby providing the replicon according to the instruction. The deletion of at least one start codon compared to a native 5' alphavirus replication recognition sequence is not particularly restricted and can be selected from any nucleotide modification, including the substitution of one or more nucleotides (including, at the DNA level, a substitution of A and / or T and / or G of the start codon); the deletion of one or more nucleotides (including, at the DNA level, a deletion of A and / or T and / or G of the start codon); and the insertion of one or more nucleotides (including, at the DNA level, an insertion of one or more nucleotides between A and T and / or between T and G of the start codon). Regardless of whether the nucleotide modification is a substitution, an insertion, or a deletion, such modification must not result in the formation of a new start codon (as an illustrative example: an insertion, at the DNA level, must not be an insertion of an ATG). The 5' replication recognition sequence of the RNA replicon characterized by the deletion of at least one start codon (i.e., the 5' replication recognition sequence modified according to the present instruction) is preferably a variant of a 5' replication recognition sequence from the genome of a naturally occurring alphavirus. In one embodiment, the 5' replication recognition sequence modified according to the present instruction is preferably characterized by a degree of sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, with the 5' replication recognition sequence from the genome of at least one naturally occurring alphavirus. In one embodiment, the 5' replication recognition sequence of the RNA replicon, characterized by the deletion of at least one start codon, comprises a sequence homologous to approximately 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 approximately 250 to 500, preferably approximately 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 nucleic acid sequence that begins at, and includes, the upstream nucleotide of the alphaviral genome. In other words, the most northerly nucleotide at the top of the alphaviral genome is designated as nucleotide no. 1, and, for example, "250 nucleotides at the 5' end of the alphaviral genome" means nucleotides 1 through 250 of the alphaviral genome.In one embodiment, the 5' replication recognition sequence of the RNA replicon, characterized by the deletion of at least one start codon, is characterized by a degree of sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, with at least 250 nucleotides at the 5' end of the genome of at least one naturally occurring alphavirus. The "at least 250 nucleotides" includes, for example, 250 nucleotides, 300 nucleotides, 400 nucleotides, or 500 nucleotides. The 5' replication recognition sequence of a naturally occurring alphavirus is typically characterized by at least one start codon and / or conserved secondary structural motifs. For example, the native 5' replication recognition sequence of Semliki forest virus (SFV) comprises five specific AUG base triplets. According to Frolov et al. (2001, RNA, vol.7, pp. 1638-1651) The analysis performed by MFOLD revealed that the native 5' replication recognition sequence of the Semliki forest virus is predicted to form four stem loops (SL), designated stem loops 1 to 4 (SL1, SL2, SL3, SL4). According to Frolov et al., the analysis performed by MFOLD also revealed that the native 5' replication recognition sequence of another alphavirus, the Sindbis virus, is predicted to form four stem loops: SL1, SL2, SL3, SL4. The 5' end of the alphaviral genome is known to comprise sequence elements that enable replication of the alphaviral genome by the functional nonstructural protein of the alphavirus. In one embodiment of the present teaching, 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. The conserved sequence element 2 (CSE 2) of the alphavirus genomic RNA is typically represented by SL3 and SL4, preceded by SL2, which comprises at least the native start codon encoding the first amino acid residue of the alphavirus nonstructural protein nsP1. In this description, however, in some embodiments, the conserved sequence element 2 (CSE 2) of the alphavirus genomic RNA refers to a region spanning SL2 to SL4 and comprising the native start codon encoding the first amino acid residue of the alphavirus nonstructural protein nsP1. In a preferred embodiment, the RNA replicon comprises CSE 2 or a sequence homologous to CSE 2.In one embodiment, the RNA replicon comprises a sequence homologous to CSE 2 that 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, with the CSE 2 sequence of at least one naturally occurring alphavirus. In a preferred embodiment, the 5' replication recognition sequence comprises a sequence homologous to CSE2 of an alphavirus. CSE2 of an alphavirus may comprise a fragment of an open reading frame of a non-structural protein of an alphavirus. Thus, in a preferred embodiment, the RNA replicon is characterized by comprising a sequence homologous to an open reading frame of a nonstructural protein or a fragment thereof from an alphavirus. The sequence homologous to an open reading frame of a nonstructural protein or a fragment thereof is typically a variant of an open reading frame of a nonstructural protein or a fragment thereof from a naturally occurring alphavirus. In one embodiment, the sequence homologous to an open reading frame of a nonstructural protein 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, and even more preferably 95% or more, with an open reading frame of a nonstructural protein or a fragment thereof from at least one naturally occurring alphavirus. In a more preferred embodiment, the open reading frame homologue sequence of a nonstructural protein comprising the replicon of the present instruction does not comprise the native start codon of a nonstructural protein, and more preferably does not comprise any start codon of a nonstructural protein. In a preferred embodiment, the CSE2 homologue sequence is characterized by the deletion of all start codons compared to a native alphavirus CSE2 sequence. Therefore, the CSE2 homologue sequence preferably contains no start codon. When a sequence homologous to an open reading frame does not comprise any start codon, such a sequence 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 deletion of at least one start codon compared to the native sequence of the alphavirus. In a preferred embodiment, the sequence homologous to an open reading frame of a nonstructural protein or a fragment thereof from an alphavirus is characterized in that it comprises the deletion of at least the native start codon of the open reading frame of a nonstructural protein. Preferably, it is characterized in that it comprises the deletion of at least the native start codon of the open reading frame encoding nsP1. The native start codon is the AUG base triplet, where translation begins in the ribosomes of a host cell when RNA is present. In other words, the native start codon is the first base triplet translated during ribosomal protein synthesis, for example, in a host cell inoculated with RNA containing the native start codon. In one embodiment, the host cell is a cell of a eukaryotic species that is a natural host of the specific alphavirus comprising the alphavirus's native 5' replication recognition sequence. In a preferred embodiment, the host cell is a BHK21 cell of the "BHK21 [C13] (ATCC® CCL10™)" cell line, available from the American Type Culture Collection, Manassas, Virginia, USA. The genomes of many alphaviruses have been fully sequenced and are publicly available, as are the sequences of the non-structural proteins encoded by these genomes. This sequence information allows for the in silico determination of the native start codon. In one embodiment, the native start codon is composed of 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 in an mRNA molecule is recognized by the ribosome as the translation start site. According to this reference, the Kozak sequence comprises a start codon AUG, immediately followed by a highly conserved G nucleotide: AUGG. In one embodiment of the present teaching, the sequence homologous to an open reading frame of a nonstructural protein or a fragment thereof from an alphavirus is characterized by the deletion of a start codon that is part of a Kozak sequence. In one embodiment of the present teaching, the 5' replication recognition sequence of the replicon is characterized by the removal of at least all those initiation codons that, at the RNA level, are part of an AUGG sequence. In a preferred embodiment, the sequence homologous to an open reading frame of a nonstructural protein or a fragment thereof from an alphavirus is characterized in that it comprises the deletion of one or more start codons other than the native start codon of the open reading frame of a nonstructural protein. In a preferred embodiment, said nucleic acid sequence is further characterized by the deletion of the native start codon. For example, in addition to the deletion of the native start codon, any one, two, three, four, or more of four (for example, five) start codons may be deleted.If the replicon is characterized by the removal of the native start codon, and optionally by the removal of one or more initiation codons other than the native start codon, from 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 codons other than the native start codon that are deleted, preferably in addition to the deletion of the native start codon, are 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 a "potential start codon." Whether a given AUG base triplet has the potential to initiate translation can be determined in silico or in a cell-based in vitro assay. In one embodiment, it is determined in silico whether a given AUG base triplet has the potential to initiate translation: in that embodiment, the nucleotide sequence is examined and it is determined that an AUG base triplet has the potential to initiate translation if it is part of an AUGG sequence, preferably part of a Kozak sequence. In one embodiment, it is determined in an in vitro cell-based assay whether a given AUG base triplet has the potential to initiate translation: an RNA replicon characterized by the deletion of the native start codon and comprising the given AUG base triplet downstream of the native start codon deletion position is introduced into a host cell. In one embodiment, the host cell is a cell of a eukaryotic species that is a natural host of the specific alphavirus comprising the alphavirus's native 5' replication recognition sequence. In a preferred embodiment, the host cell is a BHK21 cell of the "BHK21 [C13] (ATCC® CCL10™)" cell line, available from the American Type Culture Collection, Manassas, Virginia, USA. Preferably, no other AUG base triplet is present between the native start codon deletion position and the given AUG base triplet.If, after the transfer of the RNA replicon, characterized by the deletion of the native start codon and comprising the given AUG base triplet, to the host cell, translation is initiated at said AUG base triplet, then said AUG base triplet is determined to have the potential to initiate translation. The initiation or continuation of translation can be determined by any suitable method known to the art. For example, the replicon may encode, downstream of the given AUG base triplet and framed within the given AUG base triplet, a tag that facilitates the detection of the translation product (if any), for example, a myc tag or an HA tag; the presence or absence of an expression product containing the encoded tag can be determined, for example, by Western blot.In this embodiment, it is preferable that there be no other AUG base triplets between the given AUG base triplet and the nucleic acid sequence encoding 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 there be no other AUG base triplets between the native start codon deletion position and the given AUG base triplet. This can be achieved by removing all AUG base triplets (if any) between the native start codon deletion position and the given AUG base triplet. Thus, the given AUG base triplet is the first AUG base triplet downstream of the native start codon deletion position.Preferably, the replicon according to the present instruction is characterized by the deletion of all potential initiation codons downstream of the deletion position of the native start codon and located within the open reading frame of the alphavirus nonstructural protein or a fragment thereof. Therefore, according to the instruction, the 5' replication recognition sequence preferably does not comprise an open reading frame that can be translated into protein. In a preferred embodiment, the 5' replication recognition sequence of the RNA replicon, as taught, is characterized by a secondary structure equivalent to the secondary structure of the 5' replication recognition sequence of alphaviral genomic RNA. In a preferred embodiment, the 5' replication recognition sequence of the RNA replicon, as taught, is characterized by a predicted secondary structure that is equivalent to the predicted secondary structure of the 5' replication recognition sequence of alphaviral genomic RNA. According to the present teaching, the secondary structure of an RNA molecule is preferably predicted using the RNA secondary structure prediction web server http: / / rna.urmc.rochester.edu / RNAstructureWeb / Servers / Predict1 / Predict1.html. By comparing the secondary structure or predicted secondary structure of a 5' replication recognition sequence of an RNA replicon characterized by the deletion of at least one start codon with the native 5' replication recognition sequence of the alphavirus, the presence or absence of a nucleotide pairing disruption can be identified. For example, at least one base pair may be missing at a specific position compared to a native 5' replication recognition sequence of the alphavirus—for instance, a base pair within a stem loop, particularly the stem of the stem loop. In a preferred embodiment, one or more stem loops of the 5' replication recognition sequence are not deleted or interrupted. Preferably, stem loops 3 and 4 are not deleted or altered. Preferably, none of the stem loops of the 5' replication recognition sequence are deleted or interrupted. In one embodiment, the deletion of at least one start codon does not alter the secondary structure of the 5' replication recognition sequence. In an alternative embodiment, the deletion of at least one start codon does alter the secondary structure of the 5' replication recognition sequence. In this embodiment, the deletion of at least one start codon can cause the absence of at least one base pair at a given position, for example, a base pair within a stem loop, compared to a native 5' replication recognition sequence of the alphavirus. If a base pair is missing within a stem loop, compared to a native 5' replication recognition sequence of the alphavirus, the deletion of at least one start codon is determined to introduce a disruption in nucleotide pairing within the stem loop.A base pair within a stem loop is typically a base pair on the stem of the stem loop. In a preferred embodiment, the RNA replicon comprises the one or more nucleotide changes that compensate for the disruptions in nucleotide pairing within the one or more stem loops introduced by the deletion of at least one start codon. If the deletion of at least one start codon introduces a nucleotide pairing alteration within a stem loop, compared to a native 5' alphavirus replication recognition sequence, one or more nucleotide changes can be introduced that are expected to compensate for the nucleotide pairing alteration, and the secondary structure or predicted secondary structure obtained in this way can be compared to a native 5' alphavirus replication recognition sequence. Based on common knowledge and the information disclosed here, a person skilled in the art can predict that certain nucleotide changes will compensate for the nucleotide pairing alterations.For example, if a base pair is interrupted at a given position in the secondary structure or intended secondary structure of a given 5' replication recognition sequence of an RNA replicon characterized by the deletion of at least one start codon, compared to the native 5' replication recognition sequence of the alphavirus, a nucleotide change that restores a base pair at that position, preferably without reintroducing an start codon, is expected to compensate for the disruption of nucleotide pairing. In a preferred embodiment, the 5' replication recognition sequence of the replicon does not overlap with, nor comprise, a translatable nucleic acid sequence, i.e., a sequence translatable into a peptide or protein, in particular an nsP, specifically nsP1, or a fragment thereof. For a nucleotide sequence to be "translatable," it requires the presence of a start codon; the start codon encodes 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 comprise, a translatable nucleic acid sequence encoding an N-terminal fragment of nsP1. In some cases, described in detail below, the RNA replicon comprises at least one subgenomic promoter. In a preferred embodiment, the replicon's subgenomic promoter does not overlap with, nor comprise, a translatable nucleic acid sequence, i.e., translatable to a peptide or protein, in particular a nsP, specifically nsP4, or a fragment thereof. In one embodiment, the replicon's subgenomic promoter does not overlap with, nor comprise, a translatable nucleic acid sequence encoding a C-terminal fragment of nsP4.An RNA replicon having a subgenomic promoter that does not overlap with, or comprise, a translatable nucleic acid sequence, for example, translatable to the C-terminal fragment of nsP4, can be generated by deleting part of the nsP4 coding sequence (typically the portion encoding the N-terminal part of nsP4) and / or by deleting AUG base triplets in the portion of the nsP4 coding sequence that has not been deleted. If AUG base triplets are deleted in the nsP4 coding sequence or part of it, the deleted AUG base triplets are preferably potential start codons. Alternatively, if the subgenomic promoter does not overlap with a nucleic acid sequence encoding nsP4, the entire nucleic acid sequence encoding nsP4 can be deleted. In one embodiment, the RNA replicon does not comprise an open reading frame encoding a truncated alphavirus nonstructural protein. In the context of this embodiment, it is particularly preferable that the RNA replicon does not comprise an open reading frame encoding the N-terminal fragment of nsP1 and, optionally, does not comprise an open reading frame encoding the C-terminal fragment of nsP4. The N-terminal fragment of nsP1 is a truncated alphavirus protein; the C-terminal fragment of nsP4 is also a truncated alphavirus protein. In some embodiments, the replicon according to the present teaching does not include the 5' end stem loop 2 (SL2) of an alphavirus genome. According to Frolov et al., supra, stem loop 2 is a conserved secondary structure located at the 5' end of an alphavirus genome, upstream of CSE 2, but is dispensable for replication. In one embodiment, the 5' replication recognition sequence of the replicon does not overlap with a nucleic acid sequence encoding the alphavirus nonstructural protein or a fragment thereof. Therefore, the present teaching covers replicons characterized, in comparison with genomic alphaviral RNA, by the deletion of at least one start codon, as described herein, optionally combined with the deletion of the coding region of one or more alphavirus nonstructural proteins, or a portion thereof.For example, the coding region of nsP2 and nsP3 can be removed, or the coding region of nsP2 and nsP3 can be removed together with the coding region of the C-terminal fragment of nsP1 and / or the coding region of the N-terminal fragment of nsP4, and one or more remaining start codons, i.e., those that remain after such removal, can be removed, as described herein. Deletion of the coding region of one or more alphavirus nonstructural proteins can be achieved using standard methods, for example, at the DNA level, by excision with restriction enzymes, preferably restriction enzymes that recognize unique restriction sites in the open reading frame. Alternatively, unique restriction sites can be introduced into an open reading frame by mutagenesis, for example, site-directed mutagenesis. The resulting DNA can then be used as a template for in vitro transcription. A restriction site is a nucleic acid sequence, such as a DNA sequence, that is necessary and sufficient to direct the restriction (cleavage) of the nucleic acid molecule, such as a DNA molecule, in which the restriction site is contained, by a specific restriction enzyme. A restriction site is unique to a given nucleic acid molecule if there is only one copy of that restriction site in the nucleic acid molecule. A restriction enzyme is an endonuclease that cuts a nucleic acid molecule, for example a DNA molecule, at or near the restriction site. Alternatively, a nucleic acid sequence characterized by the removal of part or all of the open reading frame can be obtained using synthetic methods. According to the present teaching, the RNA replicon is preferably a single-stranded RNA molecule. According to the present teaching, the RNA replicon is typically a (+)-stranded RNA molecule. In one embodiment, the RNA replicon of the present teaching is an isolated nucleic acid molecule. Cell reprogramming and reprogramming factors This teaching provides the technology to transform one type of highly specialized somatic cell, for example fibroblasts or keratinocytes, into another type, for example neuronal cells, through a pluripotent cell intermediate. Specifically, by providing a differentiated somatic cell with reprogramming factors that are preferably present in pluripotent cell types, preferably stem cells, and most preferably embryonic stem cells, this teaching method restores the cell's epigenetic memory to a pluripotent stem cell-like state. With this teaching method, it is not necessary to use, create, or destroy embryos to generate cells with stem cell characteristics, particularly pluripotency, thus eliminating ethical concerns. Furthermore, this teaching method does not require the use of vectors that integrate into the genome, such as viral vectors, which could introduce mutations at the insertion site. The somatic cells used according to this teaching have a significant advantage over oocytes as a means of inducing reprogramming, as they can be readily increased in number in vitro. Furthermore, this teaching allows the use of patient-specific somatic cells and thus largely eliminates concerns about immune rejection and the problems associated with patient immunosuppression. The use of cells generated according to this teaching for autologous cell transplantation is unlikely to induce adverse side effects or resistance. If necessary, repeated cell transplants can be performed. However, since this teaching will significantly reduce the need for patient immunosuppression to decrease acute and hyperacute rejection, it will also alleviate the need for repeated transplant procedures, reducing the cost of treating the disease. In this document, the term "cells having stem cell characteristics" is used to designate cells that, while derived from differentiated non-stem somatic cells, exhibit one or more characteristics typical of stem cells, particularly embryonic stem cells. These characteristics include embryonic stem cell morphology, such as compact colonies, a high nucleus-to-cytoplasm ratio and prominent nucleoli, normal karyotypes, expression of telomerase activity, expression of cell surface markers characteristic of embryonic stem cells, and / or expression of genes characteristic of embryonic stem cells.Cell surface markers characteristic of embryonic stem cells are selected, for example, from the group consisting of stage-3 embryonic antigen (SSEA-3), SSEA-4, tumor-related antigen-1-60 (TRA-1-60), TRA-1-81, and TRA-2-49 / 6E. Genes characteristic of embryonic stem cells are selected, for example, from the group consisting of endogenous OCT4, endogenous NANOG, growth and differentiation factor 3 (GDF3), reduced expression 1 (REX1), fibroblast growth factor 4 (FGF4), embryonic cell-specific gene 1 (ESG1), developmental pluripotency-associated gene 2 (DPPA2), DPPA4, and telomerase reverse transcriptase (TERT). In one embodiment, one or more typical stem cell characteristics include pluripotency. In one embodiment, cells that have stem cell characteristics exhibit a pluripotent state.In one embodiment, cells with stem cell characteristics have the developmental potential to differentiate into advanced derivatives of the three primary germ layers. In one embodiment, the primary germ layer is the endoderm, and the advanced derivative is an epithelial tissue similar to that of the gut. In another embodiment, the primary germ layer is the mesoderm, and the advanced derivative is striated muscle and / or cartilage. In a further advanced embodiment, the primary germ layer is the ectoderm, and the advanced derivative is neural tissue and / or epidermal tissue. In a preferred embodiment, cells with stem cell characteristics have the developmental potential to differentiate into neuronal cells and / or cardiac cells.According to the teaching, in general, the terms "cells that have stem cell characteristics", "cells that have stem cell properties", "stem cell-like cells", "reprogrammed cells" and "dedifferentiated cells" or similar terms have similar meanings and are used interchangeably in this document. A "stem cell" is a cell with the capacity to self-renew, remain undifferentiated, and differentiate. A stem cell can divide indefinitely, at least during the lifespan of the animal in which it naturally resides. A stem cell is not terminally differentiated; it is not at the final stage of a differentiation pathway. When a stem cell divides, each daughter cell can either remain a stem cell or embark on a path that leads to terminal differentiation. Totipotent stem cells are cells that possess totipotent differentiation properties and are capable of developing into a complete organism. These cells possess this property up to the 8-cell stage after fertilization of the oocyte by the sperm. When these cells are isolated and transplanted into the uterus, they can develop into a complete organism. Pluripotent stem cells are cells capable of developing into various cell types and tissues derived from the ectodermal, mesodermal, and endodermal layers. Pluripotent stem cells, which originate from the inner cell mass located within the blastocyst and are generated 4-5 days after fertilization, are called "embryonic stem cells" and can differentiate into various cells of other tissues, but they cannot form new living organisms. Multipotent stem cells are stem cells that typically differentiate only into cell types specific to their tissue and organ of origin. Multipotent stem cells participate not only in the growth and development of various tissues and organs during the fetal, neonatal, and adult periods, but also in maintaining homeostasis in adult tissues and in inducing regeneration after tissue damage. Tissue-specific multipotent cells are collectively referred to as "adult stem cells." An "embryonic stem cell" is a stem cell that is present in an embryo or isolated from an embryo. It can be pluripotent, having the capacity to differentiate into every cell type present in the organism, or multipotent, with the capacity to differentiate into more than one cell type. As used herein, "embryo" refers to an animal in the early stages of its development. These stages are characterized by implantation and gastrulation, where the three germ layers are defined and established, and by the differentiation of the germ layers into the respective organs and organ systems. The three germ layers are the endoderm, the ectoderm, and the mesoderm. A blastocyst is an embryo at an early stage of development where the fertilized egg has undergone cleavage and a spherical layer of cells is forming, or has already formed, surrounding a fluid-filled cavity. This spherical layer of cells is the trophectoderm. Inside the trophectoderm is a group of cells called the inner cell mass (ICM). The trophectoderm is the precursor to the placenta, and the ICM is the precursor to the embryo. An adult stem cell, also called a somatic stem cell, is a stem cell found in an adult. An adult stem cell resides in differentiated tissue, can self-renew, and can differentiate, with some limitations, to generate specialized cell types from its tissue of origin. Examples include mesenchymal stem cells, hematopoietic stem cells, and neural stem cells. A "differentiated cell" is a mature cell that has undergone progressive developmental changes toward a more specialized form or function. Cell differentiation is the process a cell goes through as it matures into a clearly specialized cell type. Differentiated cells possess distinctive characteristics, perform specific functions, and are less likely to divide than their less differentiated counterparts. An "undifferentiated" cell, for example, an immature, embryonic, or primitive cell, typically has a nonspecific appearance, can perform multiple nonspecific activities, and may perform poorly, or not at all, in the functions typically performed by differentiated cells. "Somatic cell" refers to any and all differentiated cells, and does not include stem cells, germ cells, or gametes. Preferably, the term "somatic cell," as used herein, refers to a terminally differentiated cell. In one embodiment, somatic cells are fibroblasts, such as lung fibroblasts, foreskin fibroblasts, or dermal fibroblasts, or keratinocytes. In one embodiment, the somatic cells are somatic cells derived from embryonic stem cells with a mesenchymal phenotype. In a preferred embodiment, the somatic cells are fibroblasts, such as fetal fibroblasts or postnatal fibroblasts, or keratinocytes, preferably hair follicle-derived keratinocytes. In other embodiments, the fibroblasts are lung fibroblasts, foreskin fibroblasts, or dermal fibroblasts. In particular embodiments, the fibroblasts are fibroblasts deposited in the American Type Culture Collection (ATCC) under catalog number CCL-186, deposited in the American Type Culture Collection (ATCC) under catalog number CRL-2097, or deposited in the American Type Culture Collection (ATCC) under catalog number CRL-2522, distributed by SBI System Biosciences under catalog number PC501A-HFF, or distributed by Innoprot under catalog number P10857.In one embodiment, fibroblasts are adult human dermal fibroblasts. Preferably, somatic cells are human cells. According to the present teaching, the somatic cells can be genetically modified. As used herein, "committed" refers to cells that are considered permanently dedicated to a specific function. Committed cells are also called "terminally differentiated cells." As used herein, "differentiation" refers to the adaptation of cells for a particular form or function. In cells, differentiation results in a cell more committed to a specific purpose. As used herein, "dedifferentiation" refers to the loss of specialization in form or function. In cells, dedifferentiation results in a cell less committed to its differentiation. As used herein, "reprogramming" refers to the resetting of a cell's genetic program. A reprogrammed cell preferably exhibits pluripotency. In this document, the terms "dedifferentiated" and "reprogrammed" or similar terms are used interchangeably to refer to somatic cell-derived cells that have stem cell characteristics. However, these terms are not intended to limit the scope of this document for mechanistic or functional reasons. As used herein, "germ cell" refers to a reproductive cell, such as a spermatocyte or an oocyte, or a cell that will develop into a reproductive cell. As used herein, "pluripotent" refers to cells that can give rise to any type of cell, except for placental cells or other supporting cells of the uterus. According to the teaching, it is preferred that the introduction of RNA replicons capable of expressing reprogramming factors, as described herein, into somatic cells results in the expression of these factors for a period of time sufficient to complete the reprogramming process and the development of cells with stem cell characteristics. Preferably, the introduction of RNA capable of expressing certain factors, as described herein, into somatic cells results in the expression of these factors for an extended period, preferably for at least 10 days, preferably for at least 11 days, and most preferably for at least 12 days. To achieve long-term expression, it is preferred that the RNA be introduced into the cells periodically more than once, preferably by electroporation or lipofection.Preferably, the RNA is introduced into the cells at least twice, more preferably at least three times, more preferably at least four times, even more preferably at least five times, up to preferably six times, more preferably up to seven times, or even up to eight, nine, or ten times, preferably over a period of at least ten days, preferably for at least eleven days, and more preferably for at least twelve days to ensure the expression of one or more factors over a prolonged period. Preferably, the time intervals between repeated introductions of the RNA are from 24 to 120 hours, preferably from 48 to 96 hours. In one embodiment, the time intervals between repeated introductions of the RNA are not longer than 72 hours, preferably not longer than 48 hours or 36 hours.In one embodiment, before the next electroporation, the cells are allowed to recover from the previous electroporation. In this embodiment, the time intervals between repeated RNA introductions are at least 72 hours, preferably at least 96 hours, and more preferably at least 120 hours. In any case, the conditions must be selected so that the factors are expressed in the cells in quantities and for periods of time that favor the reprogramming process. However, in one embodiment, a single introduction of RNA replicons may be sufficient for the reprogramming process and for the development of cells with stem cell characteristics. Preferably at least 0.01 µg, preferably at least 0.05 µg, preferably at least 0.1 µg, preferably at least 0.2 µg, more preferably at least 0.3 µg and preferably up to 5 µg, more preferably up to 2 µg, more preferably up to 1 µg, more preferably up to 0.5 µg, preferably from 0.05 to 1 µg, even more preferably from 0.05 to 0.7 µg, or from 0.05 to 0.5 µg of RNA replicon are used for each factor per transfection. Preferably, the step that enables the development of cells having stem cell characteristics comprises the culture of somatic cells under embryonic stem cell culture conditions, preferably conditions suitable for maintaining pluripotent stem cells in an undifferentiated state. Preferably, to enable the development of cells with stem cell characteristics, the cells are cultured in the presence of one or more DNA methyltransferase inhibitors and / or one or more histone deacetylase inhibitors. Preferred compounds are selected from the group consisting of 5'-azacytidine (5'-azaC), suberoylanilidone hydroxamic acid (SAHA), dexamethasone, trichostatin A (TSA), sodium butyrate (NaBu), Scriptaid, and valproic acid (VPA). Preferably, the cells are cultured in the presence of valproic acid (VPA), preferably at a concentration of between 0.5 and 10 mM, more preferably between 1 and 5 mM, and most preferably at a concentration of approximately 2 mM. In a preferred embodiment of the present instruction, the RNA is introduced into somatic cells by repeated electroporations. Preferably, if loss of cell viability occurs, previously electroporated cells are added as carrier cells. Preferably, the previously electroporated cells are added before, during, or after one or more of the 4th and, preferably, the 5th and subsequent electroporations, such as before, during, or after the 4th and 6th electroporations. Preferably, the previously electroporated cells are added before, during, or after the 4th or 5th and each subsequent electroporation. Preferably, the previously electroporated cells are the same cells into which the RNA is introduced. Preferably, the introduction of RNA replicons capable of expressing one or more reprogramming factors into a cell causes the expression of said reprogramming factors in the cell. The term "reprogramming factor," as taught, includes proteins and peptides, as well as their derivatives and variants, which, optionally in conjunction with other agents such as other reprogramming factors, induce the reprogramming of somatic cells into cells with stem cell characteristics. For example, the term "reprogramming factor" encompasses OCT4, SOX2, NANOG, LIN28, KLF4, and c-MYC. Reprogramming factors can be of any animal species; for example, mammals and rodents. Examples of mammals include, but are not limited to, human and non-human primates. Primates include, but are not limited to, humans, chimpanzees, baboons, cynomolgus monkeys, and any other New or Old World monkeys. Rodents include, but are not limited to, mice, rats, guinea pigs, hamsters, and gerbils. In one embodiment of the present teaching, the reprogramming factors capable of enabling the reprogramming of somatic cells into cells having stem cell characteristics comprise a set of factors selected from the group consisting of (i) OCT4 and SOX2, (ii) OCT4, SOX2 and one or both of NANOG and LIN28, (iii) OCT4, SOX2 and one or both of KLF4 and c-MYC. In one embodiment, said reprogramming factors comprise OCT4, SOX2, NANOG and LIN28, OCT4, SOX2, KLF4 and c-MYC, or OCT4, SOX2, KLF4, c-MYC, NANOG and LIN28. OCT4 is a transcription factor of the eukaryotic POU transcription factors and an indicator of embryonic stem cell pluripotency. It is a maternally expressed octamer-binding protein. It has been observed in oocytes, the inner cell mass of blastocysts, and primordial germ cells. The POU5F1 gene encodes the OCT4 protein. Synonyms for the gene name include OCT3, OCT4, OTF3, and MGC22487. The presence of OCT4 at specific concentrations is necessary for embryonic stem cells to remain undifferentiated. Preferably, "OCT4 protein" or simply "OCT4" refers to human OCT4. Sox2 belongs to the Sox (SRY-related HMG box) gene family, which encodes transcription factors with a single HMG DNA-binding domain. SOX2 has been found to control neuronal progenitor cells by inhibiting their ability to differentiate. Repression of this factor results in delamination of the ventricular zone, followed by cell cycle exit. These cells also begin to lose their progenitor character due to the loss of progenitor cell markers and early neuronal differentiation markers. Preferably, "SOX2 protein" or simply "SOX2" refers to the human SOX2 protein. NANOG is an NK-2 type homeodomain gene and has been proposed to play a key role in maintaining stem cell pluripotency, presumably by regulating the expression of genes critical for embryonic stem cell renewal and differentiation. NANOG behaves as a transcription activator, with two unusually strong activation domains embedded in its C-terminus. Reduction in NANOG expression induces embryonic stem cell differentiation. Preferably, "NANOG protein" or simply "NANOG" refers to the human NANOG protein. LIN28 is a conserved cytoplasmic protein with an unusual pairing of RNA-binding motifs: a cold shock domain and a retroviral-like CCHC zinc finger pair. In mammals, it is abundant in various undifferentiated cell types.In mammalian pluripotent cells, LIN28 is observed in RNase-sensitive complexes with poly(A)-binding protein and in polysomal fractions of sucrose gradients, suggesting its association with mRNA translation. Preferably, "LIN28 protein" or simply "LIN28" refers to the human LIN28 protein. Krueppel-like factor 4 (KLF4) is a zinc-finger transcription factor that is strongly expressed in post-mitotic epithelial cells of various tissues, such as the colon, stomach, and skin. KLF4 is essential for the terminal differentiation of these cells and is involved in cell cycle regulation. Preferably, "KLF4 protein" or simply "KLF4" refers to human KLF4. MYC (cMYC) is a proto-oncogene that is overexpressed in a wide range of human cancers. When it undergoes a specific mutation or is overexpressed, it increases cell proliferation and functions as an oncogene. The MYC gene encodes a transcription factor that regulates the expression of 15% of all genes by binding to Enhancer Box (E-box) sequences and recruiting histone acetyltransferases (HATs). MYC belongs to the MYC family of transcription factors, which also includes the N-MYC and L-MYC genes. MYC family transcription factors contain the bHLH / LZ (basic helix-loop-helix with leucine zipper) domain. Preferably, "cMYC protein" or simply "cMYC" refers to the human cMYC protein. The term "miRNA" (microRNA) refers to non-coding RNAs 21–23 nucleotides long present in eukaryotic cells that, by inducing the degradation or preventing the translation of target mRNAs, modulate various cellular functions, including those related to the self-renewal / differentiation of embryonic stem cells and cell cycle progression. miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNAs (mRNAs), usually resulting in translation repression, target degradation, and gene silencing. In the appropriate combination, miRNAs have been found to induce direct cellular reprogramming of somatic cells into cells with stem cell-like characteristics in vitro. For example, the 302–367 miRNA group (e.g., group 302a–d / 367) has been observed to enhance somatic cell reprogramming. Any reference in this document to specific factors such as OCT4, SOX2, NANOG, LIN28, KLF4, or c-MYC should be understood to include all variants of these factors. In particular, it should be understood to include all splicing variants, post-translational modified variants, conformations, isoforms, and species homologs of these factors that are naturally expressed by cells. For the purposes of this teaching, "variants" of a protein or peptide or of an amino acid sequence comprise amino acid insertion variants, amino acid deletion variants and / or amino acid substitution variants. Amino acid insertion variants comprise amino- and / or carboxy-terminal fusions, as well as insertions of one, two, or more amino acids into a given amino acid sequence. In the case of amino acid sequence variants with an insertion, one or more amino acid residues are inserted at a particular site in the sequence, although random insertion is also possible with appropriate screening of the resulting product. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence. Amino acid substitution variants are characterized by the removal of at least one residue from the sequence and the insertion of another in its place. Preference is given to modifications that occur at positions in the amino acid sequence that are not conserved between homologous proteins or peptides and / or to the substitution of amino acids with others that have similar properties. "Conservative substitutions" can be made, for example, based on the similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved. For example: (a) Nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; (b) neutral polar amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positively charged (basic) amino acids include arginine, lysine, and histidine; and (d) negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Typically, substitutions can be made within groups (a)–(d). In addition, glycine and proline can be substituted for each other based on their ability to disrupt helices. Some preferred substitutions can be made between the following groups: (i) S and T; (ii) P and G; and (iii) A, V, L and I.Given the known genetic code and recombinant and synthetic DNA techniques, the skilled scientist can easily construct DNA that codes for the conserved amino acid variants. Preferably, the degree of similarity, preferably identity, between a specific amino acid sequence described herein and an amino acid sequence that is a variant of that specific amino acid sequence shall be at least 70%, preferably at least 80%, preferably at least 85%, even more preferably at least 90%, or most preferably at least 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given for a region of at least approximately 20, at least approximately 40, at least approximately 60, at least approximately 80, at least approximately 100, at least approximately 120, at least approximately 140, at least approximately 160, at least approximately 200, or 250 amino acids. In preferred embodiments, the degree of similarity or identity is stated for the entire length of the reference amino acid sequence. According to the teaching, a variant of a protein or peptide preferably possesses a functional property of the protein or peptide from which it was derived. Such functional properties are described above for OCT4, SOX2, NANOG, LIN28, KLF4, and c-MYC, respectively. Preferably, a variant of a protein or peptide should have the same property of reprogramming a differentiated animal cell as the protein or peptide from which it was derived. Preferably, the variant induces or enhances the reprogramming of a differentiated animal cell. The methods described herein can be used to effect the dedifferentiation of any type of somatic cell. The cells that can be used include cells that can be dedifferentiated or reprogrammed by the methods described herein, particularly cells that are fully or partially differentiated, preferably terminally differentiated. Preferably, the somatic cell is a diploid cell derived from pre-embryonic, embryonic, fetal, and postnatal multicellular organisms.Examples of cells that may be used include, but are not limited to, fibroblasts, such as fetal and neonatal fibroblasts or adult fibroblasts, keratinocytes, particularly primary keratinocytes, preferably those derived from hair, B cells, T cells, dendritic cells, adipose cells, epithelial cells, epidermal cells, chondrocytes, cumulus cells, neuronal cells, glial cells, astrocytes, cardiac cells, esophageal cells, muscle cells, melanocytes, hematopoietic cells, osteocytes, macrophages, monocytes, and mononuclear cells. Examples of cells that may be used include blood-derived endothelial cells and endothelial progenitor cells, as well as urine-derived epithelial cells. The cells that can be used with the teaching methods can be from any animal species; for example, mammals and rodents. Examples of mammalian cells that can be dedifferentiated and redifferentiated using this teaching method include, but are not limited to, human cells and cells from non-human primates. Primate cells that can be used for teaching include, but are not limited to, cells from humans, chimpanzees, baboons, cynomolgus monkeys, and any other New or Old World monkeys. Rodent cells that can be used for teaching include, but are not limited to, cells from mice, rats, guinea pigs, hamsters, and gerbils. According to this teaching, the term "organism" refers to any biological unit capable of multiplying or transmitting genetic material, and includes plants and animals, as well as microorganisms such as bacteria, yeasts, fungi, and viruses. The term "organism" includes, but is not limited to, a human being, a non-human primate, or another animal, particularly a mammal such as a cow, horse, pig, sheep, goat, dog, cat, or a rodent such as a mouse or rat. In a particularly preferred embodiment, the organism is a human being. It is expected that dedifferentiated cells prepared according to this instruction will exhibit many of the same requirements as pluripotent stem cells and will be able to expand and be maintained under the conditions used for embryonic stem cells, for example, ES cell culture medium or any medium that favors embryonic cell growth. Embryonic stem cells retain their pluripotency in vitro when maintained in culture on inactivated fetal fibroblasts, such as irradiated mouse embryonic fibroblasts or human fibroblasts (for example, human foreskin fibroblasts, human skin fibroblasts, human endometrial fibroblasts, human oviductal fibroblasts). In one embodiment, the human support cells can be autologous support cells derived from the same culture of cells reprogrammed by direct differentiation. Furthermore, human embryonic stem cells can be successfully cultured in Matrigel in a medium conditioned with mouse fetal fibroblasts. Human stem cells can be cultured for an extended period and remain undifferentiated under specific culture conditions. In certain embodiments, cell culture conditions may include contact of the cells with factors that can inhibit differentiation or enhance dedifferentiation of the cells, for example, preventing differentiation of the cells into cells other than embryonic stem cells, trophectoderm, or other cell types. Dedifferentiated cells prepared according to this instruction can be evaluated using methods that include monitoring changes in cell phenotype and characterizing gene and protein expression. Gene expression can be determined by RT-PCR, and translation products can be determined by immunocytochemistry and Western blot. In particular, dedifferentiated cells can be characterized to determine their gene expression pattern and whether reprogrammed cells exhibit a gene expression pattern similar to that expected in undifferentiated pluripotent control cells, such as embryonic stem cells, using well-established techniques, including transcriptomics. In this regard, the expression of the following genes of dedifferentiated cells can be evaluated: OCT4, NANOG, LIN28, growth and differentiation factor 3 (GDF3), reduced expression 1 (REX1), fibroblast growth factor 4 (FGF4), embryonic cell-specific gene 1 (ESG1), developmental pluripotency-associated 2 (DPPA2), DPPA4, telomerase reverse transcriptase (TERT), embryonic antigen-3 (SSEA-3), SSEA-4, tumor-related antigen-1-60 (TRA-1-60), TRA-1-81 and TRA-2-49 / 6E. The embryonic or undifferentiated stem cells to which the reprogrammed cells can be compared may belong to the same species as the differentiated somatic cells. Alternatively, the embryonic or undifferentiated stem cells to which the reprogrammed cells can be compared may belong to a different species than the differentiated somatic cells. In some embodiments, there is a similarity in the gene expression pattern between a reprogrammed cell and an undifferentiated cell, such as an embryonic stem cell, if certain genes specifically expressed in an undifferentiated cell are also expressed in the reprogrammed cell. For example, certain genes, such as telomerase, which are typically undetectable in differentiated somatic cells, can be used to monitor the degree of reprogramming. Likewise, for certain genes, the absence of expression can be used to assess the degree of reprogramming. The capacity for self-renewal, marked by the induction of telomerase activity, is another characteristic of stem cells that can be observed in dedifferentiated cells. Karyotypic analysis can be performed using chromosomal preparations of mitotic cells, spectral karyotyping, telomere length assays, total genomic hybridization, or other well-known techniques in the field. At least one open reading frame understood by the replicant. The RNA replicon, according to the present teaching, comprises an open reading frame encoding a peptide or protein of interest, an open reading frame encoding a reprogramming factor, and may comprise one or more additional open reading frames encoding a peptide or protein of interest, such as one or more additional reprogramming factors, which together with the first reprogramming factor form a functional set of reprogramming factors. Preferably, the protein of interest is encoded by a heterologous nucleic acid sequence. The gene encoding the peptide or protein of interest is referred to interchangeably as the "gene of interest" or "transgene." In various embodiments, the peptide or protein of interest is encoded by a heterologous nucleic acid sequence.According to this teaching, the term "heterologous" refers to the fact that a nucleic acid sequence is not naturally linked, either functionally or structurally, to a nucleic acid sequence of an alphavirus. According to this teaching, the replicon may encode a single polypeptide, i.e., a reprogramming factor, or multiple polypeptides, such as several reprogramming factors or a reprogramming factor and another polypeptide. Multiple polypeptides may be encoded as a single polypeptide (fusion polypeptide) or as separate polypeptides. In some embodiments, the replicon according to this teaching may comprise more than one open reading frame, each of which may be independently selected to be under the control of a subgenomic promoter or not.Alternatively, a fusion polyprotein or polypeptide comprises individual polypeptides separated by an optionally autocatalytic protease cleavage site (e.g., foot-and-mouth disease virus protein 2A) or an intein. The proteins of interest can be selected, for example, from the group consisting of reporter proteins, pharmaceutically active peptides or proteins, inhibitors of IFN receptor interaction with extracellular IFN, inhibitors of intracellular interferon (IFN) signaling, and functional alphavirus nonstructural proteins. Interferon (IFN) signaling inhibitor Another suitable protein of interest, encoded by an open reading frame, is an interferon (IFN) signaling inhibitor. While it has been reported that the viability of cells into which RNA has been introduced for expression can be reduced, particularly if the cells are transfected multiple times with RNA, IFN-inhibiting agents have been found to improve the viability of cells in which RNA is to be expressed (WO 2014 / 071963 A1). Preferably, the inhibitor should be a type I IFN signaling inhibitor. By preventing the interaction of the IFN receptor with extracellular IFN and / or by inhibiting intracellular IFN signaling in the cells, stable RNA expression in the cells is achieved.As an alternative or complement, preventing the interaction of extracellular IFN with the IFN receptor and / or inhibiting intracellular IFN signaling improves cell survival, particularly if cells are repeatedly transfected with RNA. In one embodiment, preventing the interaction of IFN with the IFN receptor comprises providing a binding agent for extracellular IFN, such as a viral binding agent for extracellular IFN. In one embodiment, the viral binding agent for extracellular IFN is a viral interferon receptor. In one embodiment, the viral binding agent for extracellular IFN is vaccinia virus B18R. The B18R protein is a type I interferon receptor encoded by vaccinia virus, specific for type I interferons from mouse, human, rabbit, pig, rat, and bovine sources, and possessing potent neutralizing activity. The B18R protein is encoded by the B18R gene of the Western Reserve strain of vaccinia virus. The 60-65 kD glycoprotein is related to interleukin-1 receptors and belongs to the immunoglobulin superfamily, unlike other type I IFN receptors, which belong to the class II cytokine receptor family.The B18R protein has a high affinity (KD, 174 pM) for human IFN-alpha. Among modifiers of the host viral response, the B18R protein is unique because it exists as a soluble extracellular protein as well as a cell surface protein, allowing it to block both autocrine and paracrine IFN functions. Without wishing to be limited to any single theory, it is anticipated that intracellular IFN signaling may lead to the inhibition of RNA translation and / or degradation. This can be addressed by inhibiting one or more IFN-inducible antiviral effector proteins. The IFN-inducible antiviral effector protein can be selected from the group consisting of RNA-dependent protein kinase (PKR), 2',5'-oligoadenylate synthase (OAS), and RNase L. Inhibition of intracellular IFN signaling may involve inhibition of the PKR-dependent pathway and / or the OAS-dependent pathway. A suitable protein of interest is one capable of inhibiting either the PKR-dependent pathway or the OAS-dependent pathway. Inhibition of the PKR-dependent pathway may involve inhibition of eIF2-alpha phosphorylation. Inhibition of PKR may involve treating the cell with at least one PKR inhibitor. The PKR inhibitor could be a viral inhibitor of PKR.The preferred viral inhibitor of PKR is the vaccinia virus E3 protein. If a peptide or protein (e.g., E3, K3) is intended to inhibit intracellular IFN signaling, intracellular expression of the peptide or protein is preferred. The vaccinia virus E3 protein is a 25 kDa double-stranded RNA-binding protein (encoded by the E3L gene) that binds to and sequesters double-stranded RNA to prevent PKR and OAS activation. E3 can bind directly to PKR and inhibit its activity, resulting in reduced eIF2-alpha phosphorylation. The vaccinia virus K3L gene encodes a 10.5 kDa homolog of the eIF2-alpha subunit that acts as a non-phosphorylatable pseudosubstrate of PKR and competitively inhibits eIF2-alpha phosphorylation. Other suitable inhibitors of IFN signaling include herpes simplex virus ICP34.5, Toscana NSs virus, Bombyx mori nucleopolyhedrovirus PK2, and HCV NS34A. In one embodiment, the present instruction may comprise supplying cells with vaccinia virus B18R or a functional variant thereof and vaccinia virus E3 or a functional variant thereof, or vaccinia virus K3 or a functional variant thereof, or both. The viral escape protein NSs(N) from Toscana virus is a potent inhibitor of the IFN response and can be used to replace EKB(E3, K3, B18R) for successful RNA-based reprogramming. The IFN signaling inhibitor can be provided to the cell in the form of a nucleic acid sequence (e.g., RNA) that encodes the IFN signaling inhibitor. In one embodiment, the inhibitor of intracellular or extracellular IFN signaling is encoded by an mRNA molecule. That mRNA molecule may comprise a modification that does not alter the polypeptide sequence as described here, e.g., protectant, 5'-UTR, 3'-UTR, poly(A) sequence, codon usage adaptation. In an alternative embodiment, the inhibitor of intracellular or extracellular IFN signaling is encoded by a replicon, preferably a trans-replicon or a trans-replicon as described herein. The replicon comprises nucleic acid sequence elements that allow replication by the alphavirus replicase, typically CSE1, CSE2, and CSE4; and preferably also nucleic acid sequence elements that allow the production of a subgenomic transcript, i.e., a subgenomic promoter, typically comprising CSE3. The replicon may further comprise one or more modifications that do not alter the polypeptide sequence, as described herein, e.g., a protectant, a poly(A) sequence, or codon usage adaptation.If multiple open reading frames exist in the replicon, any of them can encode an IFN signaling inhibitor, optionally under the control of a subgenomic promoter. In a preferred embodiment, the IFN signaling inhibitor is encoded by the upstream open reading frame of the RNA replicon. When an IFN signaling inhibitor is encoded by the upstream open reading frame of the RNA replicon, the genetic information encoding the IFN signaling inhibitor will be translated shortly after the RNA replicon is introduced into a host cell, and the resulting protein can subsequently inhibit IFN signaling. Functional non-structural protein of the alphavirus An additional suitable protein of interest, encoded by an open reading frame, is the functional alphavirus nonstructural protein. The term "alphavirus nonstructural protein" includes any and all co-translational or post-translational modified forms, including carbohydrate-modified (such as glycosylated) and lipid-modified forms of the alphavirus nonstructural protein. In some embodiments, the term "alphavirus nonstructural protein" refers to one or more individual alphavirus-derived nonstructural proteins (nsP1, nsP2, nsP3, nsP4), or to a polyprotein comprising the polypeptide sequence of more than one alphavirus-derived nonstructural protein. In some embodiments, "alphavirus nonstructural protein" refers to nsP123 and / or nsP4. In other embodiments, "alphavirus nonstructural protein" refers to nsP1234.In one embodiment, the protein of interest encoded by an open reading frame consists of nsP1, nsP2, nsP3, and nsP4 as a single optionally cleavable polyprotein: nsP1234. In another embodiment, the protein of interest encoded by an open reading frame consists of nsP1, nsP2, and nsP3 as a single optionally cleavable polyprotein: nsP123. In that embodiment, nsP4 may be an additional protein of interest and may be encoded by an additional open reading frame. In some embodiments, the alphavirus nonstructural protein is capable of forming a complex or association, for example, in a host cell. In some embodiments, "alphavirus nonstructural protein" refers to a complex or association of nsP123 (synonym P123) and nsP4. In some embodiments, "alphavirus nonstructural protein" refers to a complex or association of nsP1, nsP2, and nsP3. In some embodiments, "alphavirus nonstructural protein" refers to a complex or association of nsP1, nsP2, nsP3, and nsP4. In some embodiments, "alphavirus nonstructural protein" refers to a complex or association of one or more proteins selected from the group consisting of nsP1, nsP2, nsP3, and nsP4. In some embodiments, the alphavirus nonstructural protein comprises at least nsP4. The terms "complex" or "association" refer to two or more protein molecules, identical or different, that are in close proximity. The proteins in a complex are preferably in direct or indirect physical or physicochemical contact with each other. A complex or association may consist of multiple different proteins (heteromultimer) and / or multiple copies of a particular protein (homomultimer). In the context of alphavirus nonstructural proteins, the term "complex or association" describes a group of at least two protein molecules, at least one of which is an alphavirus nonstructural protein. The complex or association may consist of multiple copies of a particular protein (homomultimer) and / or multiple different proteins (heteromultimer). In the context of a multimer, "multi" means more than one, such as two, three, four, five, six, seven, eight, nine, ten, or more than ten. The term "functional non-structural protein of alphavirus" includes the alphavirus non-structural protein that has replicase function. Therefore, the "functional non-structural protein of alphavirus" includes the alphavirus replicase. "Replicase function" encompasses the function of an RNA-dependent RNA polymerase (RdRP), that is, an enzyme capable of catalyzing the synthesis of negative-strand RNA based on a positive-strand RNA template, and / or capable of catalyzing the synthesis of positive-strand RNA based on a negative-strand RNA template.Thus, the term "functional non-structural alphavirus protein" may refer to a protein or complex that synthesizes (-) strand RNA, using (+) strand RNA (e.g., genomic) as a template, to a protein or complex that synthesizes new (+) strand RNA, using the (-) strand complement of the genomic RNA as a template, and / or to a protein or complex that synthesizes a subgenomic transcript, using a fragment of the (-) strand complement of the genomic RNA as a template. The functional nonstructural protein of the alphavirus may also have one or more additional functions, such as a protease (for self-cleavage), helicase, terminal adenylyltransferase (for poly(A) tail addition), methyltransferase and guanylyltransferase (to provide a nucleic acid with a 5-protectant), 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 teaching, the term "alphavirus replicase" refers to alphavirus RNA-dependent RNA polymerase, including an RNA-dependent RNA polymerase from a naturally occurring alphavirus (an alphavirus found in nature) and an RNA-dependent RNA polymerase from a variant or derivative of an alphavirus, such as an attenuated alphavirus. In the context of this teaching, the terms "replicase" and "alphavirus replicase" are used interchangeably unless the context indicates that a particular replicase is not an alphavirus replicase. The term "replicase" encompasses all variants, particularly post-translational modified variants, conformations, isoforms, and homologs of alphavirus replicase that are expressed in alphavirus-infected cells or in cells transfected with nucleic acid encoding alphavirus replicase. Furthermore, the term "replicase" includes all replicase forms that have been and can be produced by recombinant methods. For example, a replicase can be produced by recombinant methods containing a tag that facilitates the detection and / or purification of the replicase in the laboratory, such as a myc tag, an HA tag, or an oligohistidine tag (His tag). Optionally, the alphavirus replicase is further functionally defined by its ability to bind to any one or more of the alphavirus conserved sequence element 1 (CSE1) or its complementary sequence, conserved sequence element 2 (CSE2) or its complementary sequence, conserved sequence element 3 (CSE3) or its complementary sequence, and conserved sequence element 4 (CSE4) or its complementary sequence. Preferably, the replicase is capable of binding to CSE2 (i.e., the (+) strand) and / or CSE4 (i.e., the (+) strand), or of binding to the complement of CSE1 (i.e., the (-) strand) and / or the complement of CSE3 (i.e., the (-) strand). The origin of the replicase is not limited to any particular alphavirus. In a preferred embodiment, the alphavirus replicase comprises a nonstructural protein from Semliki Forest virus, including a naturally occurring Semliki Forest virus and a variant or derivative of Semliki Forest virus, such as an attenuated Semliki Forest virus. In an alternative preferred embodiment, the alphavirus replicase comprises a nonstructural protein from Sindbis virus, including a naturally occurring Sindbis virus and a variant or derivative of Sindbis virus, such as an attenuated Sindbis virus. In an alternative preferred embodiment, the alphavirus replicase comprises a nonstructural protein from Venezuelan equine encephalitis virus (VEEV), including a naturally occurring VEEV and a variant or derivative of VEEV, such as an attenuated VEEV.In an alternative preferred embodiment, the alphavirus replicase comprises a non-structural protein of the chikungunya virus (CHIKV), which includes a naturally occurring CHIKV and a variant or derivative of CHIKV, such as an attenuated CHIKV. A replicase can also be composed of nonstructural proteins from more than one alphavirus. Therefore, heterologous complexes or associations comprising alphavirus nonstructural proteins that function as replicas are also covered in this teaching. For example, the replicase may be composed of one or more nonstructural proteins (e.g., nsP1, nsP2) from a first alphavirus and one or more nonstructural proteins (nsP3, nsP4) from a second alphavirus. The nonstructural proteins from more than one different alphavirus may be encoded by separate open reading frames, or they may be encoded by a single open reading frame as a polyprotein, e.g., nsP1234. In some embodiments, the functional non-structural protein of the alphavirus is able to form membranous replication complexes and / or vacuoles in cells in which the functional non-structural protein of the alphavirus is expressed. If the functional non-structural protein of the alphavirus, i.e., the alphavirus non-structural protein with replicase function, is encoded by a nucleic acid molecule according to the present teaching, it is preferable that the replicon's subgenomic promoter, if present, be compatible with that replicase. In this context, compatible means that the alphavirus replicase is capable of recognizing the subgenomic promoter, if present. In one embodiment, this is achieved when the subgenomic promoter is specific to the alphavirus from which the replicase is derived; that is, the natural origin of these sequences is the same alphavirus. In an alternative embodiment, the subgenomic promoter is not specific 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 (virus compatibility).Examples of compatibility between viruses with respect to the subgenomic promoter and replicase originating from different alphaviruses are known in the literature. Any combination of subgenomic promoter and replicase is possible, provided there is compatibility between viruses. Compatibility between viruses can be easily verified by a qualified instructor by incubating the replicase to be analyzed with an RNA sample containing the subgenomic promoter to be analyzed, under conditions suitable for RNA synthesis from a subgenomic promoter. If a subgenomic transcript is produced, it is determined that the subgenomic promoter and replicase are compatible. Several examples of compatibility between viruses are known (reviewed by Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491–562). In one embodiment, the alphavirus nonstructural protein is not encoded as a fusion protein with a heterologous protein, e.g., ubiquitin. In this instruction, an open reading frame encoding a functional non-structural alphavirus protein in the RNA replicon may be provided, or it may be provided as a separate nucleic acid molecule, e.g., an mRNA molecule. A separate mRNA molecule may optionally comprise, e.g., a protectant, 5'-UTR, 3'-UTR, poly(A) sequence, and / or an adaptation of codon usage. The separate in-trans mRNA molecule may be provided as described here for the system in this instruction. When an open reading frame encoding a functional alphavirus nonstructural protein is provided in the RNA replicon, the replicon can be preferentially replicated by the functional alphavirus nonstructural protein. In particular, the RNA replicon encoding the functional alphavirus nonstructural protein can be replicated by the functional alphavirus nonstructural protein encoded by the replicon. This embodiment is highly preferred when no nucleic acid molecule encoding a functional alphavirus nonstructural protein is provided in trans. In this embodiment, the goal is cisreplication of the replicon. In a preferred embodiment, the RNA replicon comprises an open reading frame encoding a functional alphavirus nonstructural protein, as well as another open reading frame encoding a protein of interest, and can be replicated by the functional alphavirus nonstructural protein.This embodiment is particularly suitable for some methods for producing a protein of interest according to this instruction. Figure 5 shows an example of the corresponding replicon ("cisReplicon 5ATG-RRS"). If the replicon comprises an open reading frame encoding a functional non-structural protein of the alphavirus, it is preferable that the open reading frame encoding a functional non-structural protein of the alphavirus not overlap with the 5' replication recognition sequence. In one embodiment, the open reading frame encoding the functional non-structural protein of the alphavirus not overlap with the subgenomic promoter, if present. Figure 5 shows an example of the corresponding replicon ("cisReplicon 5ATG-RRS"). If multiple open reading frames exist in the replicon, then the functional nonstructural protein of the alphavirus can be encoded by any of them, optionally under the control of a subgenomic promoter or not, preferably without the control of a subgenomic promoter. In a preferred embodiment, the functional nonstructural protein of the alphavirus is encoded by the open reading frame most upstream of the RNA replicon. When the functional nonstructural protein of the alphavirus is encoded by the open reading frame most upstream of the RNA replicon, the genetic information encoding the functional nonstructural protein of the alphavirus will be translated shortly after the introduction of the RNA replicon into a host cell, and the resulting protein can subsequently drive replication and, optionally, the production of a subgenomic transcript in the host cell.Figure 5 shows an example of the corresponding replicon ("cisReplicon 5ATG-RRS"). The presence of an open reading frame encoding a functional alphavirus non-structural protein, whether composed of the replicon or a separate nucleic acid molecule provided in trans, allows the replicon to replicate and, consequently, enables a gene of interest encoded by the replicon, optionally under the control of a subgenomic promoter, to be expressed at high levels. This is associated with a cost advantage compared to other transgene expression systems. Since the replicon in this teaching can replicate in the presence of the functional alphavirus non-structural protein, high levels of expression of a gene of interest can be achieved even with relatively low amounts of replicon RNA. The low amounts of replicon RNA positively impact costs. Position of at least one open reading frame in the RNA replicon The RNA replicon is suitable for the expression of one or more genes encoding a peptide or protein of interest, optionally under the control of a subgenomic promoter. Several embodiments are possible. The RNA replicon may have one or more open reading frames, each encoding a peptide or protein of interest. The open reading frame most upstream of the RNA replicon is called the "first open reading frame." In some embodiments, the "first open reading frame" is the only open reading frame of the RNA replicon. Optionally, one or more additional open reading frames may exist downstream of the first open reading frame.One or more additional open reading frames downstream of the first open reading frame may be designated "second open reading frame," "third open reading frame," and so on, in the order (5' to 3') in which they are present downstream of the first open reading frame. Preferably, each open reading frame comprises a start codon (triplet of bases), typically AUG (in the RNA molecule), which corresponds to ATG (in a respective DNA molecule). If the replicon comprises a 3' replication recognition sequence, it is preferable that all open reading frames be located upstream of the 3' replication recognition sequence. When the RNA replicon comprising one or more open reading frames is introduced into a host cell, translation preferably does not initiate at any position upstream of the first open reading frame, due to the deletion of at least one start codon from the 5' replication recognition sequence. Therefore, the replicon can serve directly as a template for translation of the first open reading frame. Preferably, the replicon comprises a 5' protectant. This is useful for the expression of the gene encoded by the first open reading frame directly from the replicon. In some embodiments, at least one open reading frame of the replicon is under the control of a subgenomic promoter, preferably an alphavirus subgenomic promoter. The alphavirus subgenomic promoter is highly efficient and thus well-suited for the expression of heterologous genes at high levels. Preferably, the subgenomic promoter is a promoter for a subgenomic transcript in an alphavirus. This means that the subgenomic promoter is specific to an alphavirus and, preferably, controls the transcription of the open reading frame encoding one or more structural proteins in that alphavirus. Alternatively, the subgenomic promoter can be a variant of an alphavirus subgenomic promoter; any variant that functions as a promoter for the transcription of subgenomic RNA in a host cell is suitable.If the replicon comprises a subgenomic promoter, it is preferable that the replicon comprises a conserved sequence element 3 (CSE 3) or a variant thereof. Preferably, at least one open reading frame under the control of a subgenomic promoter is located downstream of the subgenomic promoter. Preferably, the subgenomic promoter controls the production of subgenomic RNA comprising a transcript of the open reading frame. In some embodiments, the first open reading frame is under the control of a subgenomic promoter. When the first open reading frame is under the control of a subgenomic promoter, its location resembles the location of the open reading frame encoding the structural proteins in the alphavirus genome. When the first open reading frame is under the control of the subgenomic promoter, the gene encoded by the first open reading frame can be expressed from either the replicon or a subgenomic transcript of it (the latter in the presence of the functional non-structural protein of the alphavirus). An example of this embodiment is shown in Figure 5 using the replicon "5ATG-RRS". Preferably, "5ATG-RRS" does not include any start codon in the nucleic acid sequence encoding the C-terminal fragment of nsP4 (*nsP4).There may be one or more additional open reading frames, each under the control of a subgenomic promoter, upstream of the first open reading frame that is under the control of a subgenomic promoter (not illustrated in Fig. 5). Genes encoded by one or more additional open reading frames, for example, by the second open reading frame, may be translated from one or more subgenomic transcripts, each under the control of a subgenomic promoter. For example, the RNA replicon may comprise a subgenomic promoter that controls the production of a transcript encoding a second protein of interest. In other embodiments, the first open reading frame is not under the control of a subgenomic promoter. When the first open reading frame is not under the control of a subgenomic promoter, the gene encoded by the first open reading frame may be expressed from the replicon.One such embodiment is exemplified in Figure 5 by the "5ATG-RRSSGP" replicon. There may be one or more additional open reading frames, each under the control of a subgenomic promoter, downstream of the first open reading frame (to illustrate two exemplary embodiments, see "5ATG-RRS-bistronic" and "cisReplicon 5ATG-RRS" in Fig. 5). Genes encoded by one or more additional open reading frames may be expressed from subgenomic transcripts. In a cell comprising the replicon as described herein, the replicon can be amplified by the functional nonstructural protein of the alphavirus. Furthermore, if the replicon comprises one or more open reading frames under the control of a subgenomic promoter, one or more subgenomic transcripts are expected to be produced by a functional nonstructural protein of the alphavirus. This functional nonstructural protein of the alphavirus may be provided in trans, or it may be encoded by an open reading 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 encode a different protein. For example, the protein encoded by the second open reading frame is different from the protein encoded by the first open reading frame. In some embodiments, the protein of interest encoded by the first open reading frame and / or by an additional open reading frame, preferably the first open reading frame, is a functional nonstructural protein of the alphavirus. In other embodiments, the protein of interest encoded by the first open reading frame and / or by an additional open reading frame, for example, the second open reading frame, is a reprogramming factor. In one embodiment, the protein of interest encoded by the first open reading frame is a functional non-structural protein of the alphavirus. In that embodiment, the replicon preferably comprises a 5' protectant. Particularly when the protein of interest encoded by the first open reading frame is a functional non-structural protein of the alphavirus, and preferably when the replicon comprises a 5' protectant, the nucleic acid sequence encoding the functional non-structural protein of the alphavirus can be efficiently translated from the replicon, and the resulting protein can subsequently drive replicon replication and the synthesis of subgenomic transcripts. This embodiment may be preferable when no additional nucleic acid molecule encoding a functional non-structural protein of the alphavirus is used or present alongside the replicon. In this embodiment, the objective is cis-replication of the replicon.Figure 5 illustrates an embodiment in which the first open reading frame encodes a functional non-structural alphavirus protein using the "cisReplicon 5ATG-RRS". Following translation of the nucleic acid sequence encoding nsP1234, the translation product (nsP1234 or fragments thereof) can act as a replicase and drive RNA synthesis, i.e., replication of the replicon and synthesis of a subgenomic transcript comprising the second open reading frame ("Transgene" in Fig. 5). Trans-replication system In an additional aspect, the present teaching provides a system that comprises: an RNA construct to express the functional non-structural protein of alphavirus, the RNA replicon according to the first aspect of teaching, which can be replicated by the functional non-structural in-trans protein of the alphavirus. In this respect, it is preferred that the RNA replicon does not comprise an open reading frame that encodes a functional non-structural protein of the alphavirus. Thus, the present instruction provides a system comprising two nucleic acid molecules: a first RNA construct for expressing the functional non-structural protein of alphavirus (i.e., encoding the functional non-structural protein of alphavirus); and a second RNA molecule, the RNA replicon. In this document, the RNA construct for the expression of the functional non-structural protein of alphavirus is referred to interchangeably as the "RNA construct for the expression of the functional non-structural protein of alphavirus" or the "replicase construct." The functional nonstructural protein of the alphavirus is the one defined above and is typically encoded by an open reading frame comprising the replicase construct. The functional nonstructural protein of the alphavirus encoded by the replicase construct can be any functional nonstructural protein of the alphavirus capable of replicating the replicon. When the system described herein is introduced into a cell, preferably a eukaryotic cell, the open reading frame encoding the functional non-structural protein of the alphavirus can be translated. After translation, the functional non-structural protein of the alphavirus is able to replicate an independent RNA molecule (RNA replicon) in trans. Therefore, the system described herein provides a system for replicating RNA in trans. Consequently, the system described herein is a transreplication system. Based on the second aspect, the replicon is a trans-replicon. In this document, trans (e.g., in the context of trans-action, trans-regulation) generally means "acting from a different molecule" (i.e., intermolecular). It is the opposite of cis (e.g., in the context of cis-action, cis-regulation), which generally means "acting from the same molecule" (i.e., intramolecular). In the context of RNA synthesis (which includes RNA transcription and replication), a trans-action element comprises a nucleic acid sequence containing a gene that encodes an enzyme capable of synthesizing RNA (RNA polymerase). The RNA polymerase uses a second nucleic acid molecule—that is, a nucleic acid molecule other than the one that encodes it—as a template for RNA synthesis. Both the RNA polymerase and the nucleic acid sequence containing a gene that encodes the RNA polymerase are said to "act in trans" on the second nucleic acid molecule.In the context of this instruction, the trans-RNA-encoded RNA polymerase is a functional non-structural protein of the alphavirus. This functional non-structural protein is capable of using a second nucleic acid molecule, an RNA replicon, as a template for RNA synthesis, including replication of the RNA replicon. The RNA replicon that can be replicated by the trans-RNA replicase, as described in this instruction, is referred to herein as a synonym for "trans-replicon." In the system described here, the function of the functional alphavirus non-structural protein is to amplify the replicon and prepare a subgenomic transcript, if a subgenomic promoter is present on the replicon. If the replicon encodes a gene of interest for expression, the expression levels of the gene of interest and / or the duration of expression can be regulated in trans by modifying the levels of the functional alphavirus non-structural protein. The fact that alphaviral replicase is generally able to recognize and replicate an in trans template RNA was initially discovered in the 1980s, but the potential of trans-replication for biomedical applications was not recognized, inter alia because trans-replicated RNA was thought to inhibit efficient replication: it was discovered in the case of defective interfering RNA (DI) 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. USA, vol.78, pp.5353-5357; Pettersson, 1981, Proc. Natl. Acad. Sci. USA, vol.78, pp.115-119) DI RNAs are trans-replicons that can appear almost naturally during infections of cell lines with a high viral load.The DI elements replicate together so efficiently that they reduce the virulence of the parental virus and thus act as inhibitory parasitic RNA (Barrett et al., 1984, J. Gen. Virol., vol.65 (Pt 11), pp.1909-1920). Although its potential for biomedical applications was not recognized, the trans-replication phenomenon was used in several basic studies aimed at elucidating replication mechanisms, without the need to express the replicase from the same in cis molecule; moreover, the separation of the replicase and the replicon also allows for functional studies involving viral protein mutants, even if the respective mutants were loss-of-function mutants (Lemm et al., 1994, EMBO J., vol.13, pp.2925-2934). These loss-of-function studies and RNA DI do not suggest that alphaviral element-based transactivation systems may become available for therapeutic purposes. The system described herein comprises at least two nucleic acid molecules. Therefore, 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. In other preferred embodiments, the system comprises more than one replicon, each of which preferably encodes at least one protein of interest, and also comprises the replicase construct. In these embodiments, the functional alphavirus nonstructural protein encoded by the replicase construct can act on each replicon to drive replication and the production of subgenomic transcripts, respectively. For example, each replicon may encode a reprogramming factor.This is advantageous, for example, if the expression of more than one reprogramming factor is desired in a cell to form a functional set of reprogramming factors. Preferably, the replicase construct lacks at least one conserved sequence element (CSE) required for the synthesis of the (-) strand based on a (+) strand template, and / or for the synthesis of the (+) strand based on a (-) strand template. Preferably, the replicase construct does not comprise any alphaviral conserved sequence elements (CSEs). In particular, among the four alphavirus CSEs (Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491-562; José et al., Future Microbiol., 2009, vol. 4, pp. 837-856), preferably one or more of the following CSEs should not be present in the replicase construct: CSE 1; CSE 2; CSE 3; CSE 4. Particularly in the absence of one or more alphaviral CSEs, the replicase construct of the present teaching is much more like typical eukaryotic mRNA than alphaviral genomic RNA. The replicase construct discussed here is preferably distinguished from alphaviral genomic RNA at least insofar as it is not capable of self-replication and / or does not comprise an open reading frame under the control of a subgenomic promoter. When it cannot self-replicate, the replicase construct may also be referred to as a "suicide construct." The trans-replication system is associated with the following advantages: First and foremost, the versatility of the trans-replication system allows the replicon and replicase constructs to be designed and / or prepared at different times and / or locations. In one embodiment, the replicase construct is prepared first, and the replicon is prepared later. For example, once prepared, the replicase construct can be stored for later use. The present teaching offers greater flexibility compared to cis-replicons: the system in the present teaching can be designed for processing by cloning a nucleic acid encoding a novel reprogramming factor into the replicon. A previously prepared replicase construct can then be retrieved from storage. In other words, the replicase construct can be designed and prepared independently of any particular replicon. Secondly, according to the present teaching, a trans-replicon is typically a shorter nucleic acid molecule than a typical cis-replicon. This allows for faster cloning of a replicon encoding a protein of interest and provides high yields of the protein of interest. Other advantages of the system described herein include independence from nuclear transcription and the presence of key genetic information in two separate RNA molecules, providing unprecedented design freedom. Given its versatile and combinable elements, this system allows for the optimization of replicase expression for a desired level of RNA amplification, a desired target organism, a desired level of protein production, and so on. The system, as described herein, allows for the cotransfection of varying amounts or ratios of the replicon and replicase construct into any given cell type—whether at rest or during cycling, in vitro or in vivo. According to this teaching, the replicase construct is preferably a single-stranded RNA molecule. The replicase construct, according to this teaching, is typically a positive-stranded RNA molecule. In one embodiment, the replicase construct of this teaching is an isolated nucleic acid molecule. Preferred characteristics of RNA molecules according to teaching According to the teaching, RNA molecules can optionally be characterized by other features, for example, a 5'-protector, a 5'-UTR, a 3'-UTR, a poly(A) sequence, and / or codon usage adaptation. Details are described below. Protective In some realizations, the replicant according to the present teaching comprises a 5'-protector. In some embodiments, the replicase construct as presented here includes a 5' protectant. The terms "5' protectant," "protector," "5' protectant structure," and "protector structure" are used synonymously to refer to a dinucleotide found at the 5' end of some primary eukaryotic transcripts, such as precursor messenger RNA. A 5' protectant is a structure in which a guanosine (optionally modified) is attached to the first nucleotide of an mRNA molecule by a 5'-5' triphosphate bond (or a modified triphosphate bond in the case of certain protectant analogues). The terms may refer to a conventional protectant or a protectant analogue. As an example, Figure 6 shows some particular protectant dinucleotides (including protectant analogue dinucleotides). "RNA comprising a 5'-protector" or "RNA provided with a 5'-protector" or "RNA modified with a 5'-protector" or "protected RNA" refers to RNA comprising a 5'-protector. For example, RNA with a 5'-protector can be provided by in vitro transcription of a DNA template in the presence of said 5'-protector, wherein said 5'-protector is co-transcriptionally incorporated into the generated RNA strand, or the RNA can be generated, for example, by in vitro transcription, and the 5'-protector can be attached to the RNA post-transcriptionally using protecting enzymes, for example, vaccinia virus protecting enzymes. In protected RNA, the 3' position of the first base of an RNA molecule (protected) is linked to the 5' position of the subsequent base of the RNA molecule ("second base") by a phosphodiester bond. The presence of a protectant on an RNA molecule is highly preferable if the translation of a nucleic acid sequence encoding a protein is desired in the early stages after the introduction of the corresponding RNA into host cells or a host organism. For example, the presence of a protectant allows a gene of interest encoded by an RNA replicon to be efficiently translated in the early stages after the introduction of the corresponding RNA into host cells. "Early stages" typically refers to the period within one hour, or within the first two hours, or within the first three hours after RNA introduction. The presence of a protectant on an RNA molecule is also preferable if translation is to occur in the absence of functional replicase, or when only minimal 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, replicase levels are typically minimal in the early stages following introduction. In the system according to the teaching, it is preferred that the RNA construct to express the functional non-structural protein of the alphavirus include a 5-protector. In particular, when the RNA replicon according to the present instruction is not used or provided together with a second nucleic acid molecule (e.g., mRNA) encoding a functional nonstructural protein of the alphavirus, it is preferable that the RNA replicon include a 5'-protector. Separately, the RNA replicon may also comprise a 5'-protector even when it is used or provided together with a second nucleic acid molecule encoding a functional nonstructural protein of the alphavirus. The term "conventional 5'-protector" refers to a naturally occurring 5'-protector, preferably the 7-methylguanosine protectant. In the 7-methylguanosine protectant, the guanosine of the protectant is a modified guanosine where the modification consists of methylation at position 7 (top of Fig. 6). In the context of this instruction, the term "5' protectant analogue" refers to a molecular structure that resembles a conventional 5' protectant but is modified to stabilize RNA when it binds to it, preferably in vivo and / or in a cell. A 5' protectant analogue is not a conventional 5' protectant. In the case of eukaryotic mRNA, the 5' protectant has generally been described as participating in efficient mRNA translation: in general, in eukaryotes, translation is initiated only at the 5' end of a messenger RNA (mRNA) molecule, unless an internal ribosomal entry site (IRES) is present. Eukaryotic cells are able to provide a 5' protectant to the RNA during transcription in the nucleus: newly synthesized mRNAs are often modified with a 5' protectant structure, for example, when the transcript reaches a length of 20 to 30 nucleotides. First, the terminal nucleotide 5' pppN (where ppp represents the triphosphate and N represents any nucleoside) is converted in the cell to 5' GpppN by a protecting enzyme that has RNA 5'-triphosphatase and guanylyltransferase activities.Subsequently, GpppN can be methylated in the cell by a second enzyme with (guanine-7)-methyltransferase activity to form the monomethylated m7GpppN protectant. In one embodiment, the 5-protectant used in this instruction is a naturally occurring 5-protectant. In the present teaching, a natural 5'-protecting dinucleotide is typically selected from the group consisting of an unmethylated protecting dinucleotide (G(5')ppp(5')N; also referred to as GpppN) and a methylated protecting dinucleotide (m7G(5')ppp(5')N; also referred to as m7GpppN). m7GpppN (where N is G) is represented by the following formula: The protected RNA discussed here can be prepared in vitro and, therefore, does not depend on a protection mechanism in a host cell. The most common method for producing protected RNA in vitro involves transcribing a DNA template with a bacterial or bacteriophage RNA polymerase in the presence of the four ribonucleoside triphosphates and a protecting dinucleotide such as m7G(5')ppp(5')G (also called m7GpppG). The RNA polymerase initiates transcription with a nucleophilic attack by the 3'-OH of the guanosine moiety of m7GpppG on the -phosphate of the next nucleoside triphosphate (pppN) in the template, resulting in the intermediate m7GpppGpN (where N is the second base of the RNA molecule). The formation of the GTP-initiated product pppGpN, which competes with the protected RNA, is suppressed by adjusting the molar ratio of the protecting RNA to GTP between 5 and 10 during in vitro transcription. In the preferred embodiments of this instruction, the 5' protectant (if present) is a 5' protectant analogue. These embodiments are particularly suitable if the RNA is obtained by in vitro transcription, for example, if it is an in vitro transcribed RNA (IVT-RNA). Protectant analogues were initially described to facilitate the large-scale synthesis of RNA transcripts by in vitro transcription. In the case of messenger RNA, several protectant analogues (synthetic protectants) have been described to date, and all of them can be used in the context of this instruction. Ideally, a protectant analogue is selected that is associated with higher translation efficiency and / or greater resistance to degradation in vivo and / or greater resistance to degradation in vitro. Preferably, a protectant analogue is used that can only be incorporated into an RNA strand in a specific 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 subunit to initiate transcription, so approximately 40–50% of protected transcripts possess the protecting dinucleotide in the reverse orientation (i.e., the initial reaction product is Gpppm7GpN). Compared to RNAs with a correct protectant, RNAs with an inverted protectant are nonfunctional with respect to the translation of a nucleic acid sequence into protein. Therefore, it is desirable to incorporate the protectant in the correct orientation, resulting in an RNA with a structure that essentially corresponds to m7GpppGpN, etc.It has been shown that the reverse integration of the protectant dinucleotide is inhibited by substitution of the 2'- or 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 synthesized in the presence of such "anti-reverse protectant analogues" are translated more efficiently than RNAs transcribed in vitro in the presence of the conventional 5'-protector m7GpppG. To that end, a protectant analogue is described in which the 3' OH group of the methylated guanosine unit is replaced by OCH3, for example, by Holtkamp et al., 2006, Blood, vol.108, pp.4009-4017 (7-methyl (3'-O-methyl) GpppG; anti-reverse protectant analogue (ARCA)). ARCA is a suitable protectant dinucleotide according to the present teaching. In a preferred embodiment of the present instruction, the RNA of the present instruction is not essentially susceptible to removal of the protectant. This is important because, in general, the amount of protein produced from synthetic mRNAs introduced into cultured mammalian cells is limited by natural mRNA degradation. One in vivo mRNA degradation pathway begins with the removal of the mRNA protectant. This removal is catalyzed by a heterodimeric pyrophosphatase, which contains a regulatory subunit (Dcp1) and a catalytic subunit (Dcp2). The catalytic subunit performs the cleavage between the phosphate and β groups of the triphosphate bridge. In the present instruction, a protectant analogue that is not susceptible, or is less susceptible, to this type of cleavage may be selected or presented. A suitable protectant analogue for this purpose may be selected from a protectant dinucleotide according to Formula (I): wherein R1 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, R2 and R3 are independently selected from the group consisting of H, halogen, OH and optionally substituted alkoxy, or R2 and R3 together form OXO, wherein X is selected from the group consisting of optionally substituted CH2, CH2CH2, CH2CH2CH2, CH2CH (CH3), and C(CH3)2, or R2 combines with the hydrogen atom in the 4' position of the ring to which R2 is attached to form -O-CH2- or -CH2-O-, R5 is selected from the group consisting of S, Se and BH3, R4 and R6 are selected independently from the group consisting of O, S, Se and BH3. n is 1, 2 or 3. Preferred realizations for R1, R2, R3, R4, R5, R6 are disclosed in WO 2011 / 015347 A1 and may be selected accordingly in the present teaching. For example, in a preferred embodiment of the present teaching, the RNA of the present teaching comprises a phosphorothioate protectant analogue. Phosphorothioate protectant analogues are specific protectant analogues in which one of the three non-bridging O atoms in the triphosphate chain is replaced by a S atom, i.e., one of R4, R5, or R6 in Formula (I) is S. Phosphorothioate protectant analogues have been described by J. Kowalska et al., 2008, RNA, vol. 14, pp. 1119–1131, as a solution to the undesirable deprotection process and thus to increase the stability of RNA in vivo. In particular, the substitution of an oxygen atom for a sulfur atom in the beta-phosphate group of the 5'-protectant results in stabilization against Dcp2. In that embodiment, which is preferred in the present teaching, R5 in Formula (I) is S; and R4 and R6 are O. In a further preferred embodiment of the present instruction, the RNA of the present instruction comprises a phosphorothioate protectant analogue wherein the phosphorothioate modification of the 5-protectant of the RNA is combined with an "anti-reverse protectant analogue" (ARCA) modification. The respective ARCA phosphorothioate protectant analogues are described in WO 2008 / 157688 A2, and all of them may be used in the RNA of the present instruction. In that embodiment, at least one of R2 or R3 in Formula (I) is not OH, preferably one between R2 and R3 is methoxy (OCH3), and the other between R2 and R3 is preferably OH. In a preferred embodiment, an oxygen atom is substituted for a sulfur atom in the beta-phosphate group (so that R5 in Formula (I) is S; and R4 and R6 are O).The phosphorothioate modification of ARCA is believed to ensure that the phosphorothioate, β, and β groups are precisely positioned within the active sites of the shield-binding proteins, in both the translation and unshielding machinery. At least some of these analogs are essentially resistant to the Dcp1 / Dcp2 pyrophosphatase. Phosphorthioate-modified ARCAs have been described as having a much higher affinity for elF4E than the corresponding ARCAs lacking a phosphorothioate group. A respective protective analogue that is particularly preferred in the present teaching, namely m2'7, 2'-OGppspG, is designated beta-S-ARCA (WO 2008 / 157688 A2; Kuhn et al., Gene Ther., 2010, vol. 17, pp. 961–971). Thus, in one embodiment of the present teaching, the RNA of the present teaching is modified with beta-S-ARCA. The beta-S-ARCA is represented by the following structure: In general, the substitution of an oxygen atom for a sulfur atom in a bridging phosphate results in phosphorothioate diastereomers designated D1 and D2, based on their elution pattern in HPLC. In summary, the D1 diastereomer of beta-S-ARCA, or beta-S-ARCA (D1), is the beta-S-ARCA diastereomer that elutes first on an HPLC column compared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA (D2)) and therefore exhibits a shorter retention time. The determination of the stereochemical configuration by HPLC is described in WO 2011 / 015347 A1. In a particularly preferred first embodiment of the present instruction, the RNA of the present instruction is modified with the beta-S-ARCA (D2) diastereomer. The two beta-S-ARCA diastereomers differ in their sensitivity to nucleases. RNA containing the beta-S-ARCA D2 diastereomer has been shown to be almost completely resistant to Dcp2 cleavage (only 6% cleavage compared to RNA synthesized in the presence of the unmodified ARCA 5' protectant), whereas RNA with the beta-S-ARCA (D1) 5' protectant shows intermediate sensitivity to Dcp2 cleavage (71% cleavage). Furthermore, greater stability against Dcp2 cleavage has been shown to correlate with increased protein expression in mammalian cells.In particular, it has been shown that RNAs carrying the beta-S-ARCA (D2) protectant are translated more efficiently in mammalian cells than RNAs carrying the beta-S-ARCA (D1) protectant. Therefore, in one embodiment of the present instruction, the RNA of the present instruction is modified with an analog of the protectant according to Formula (I), characterized by a stereochemical configuration at the P atom comprising the substituent R5 in Formula (I) corresponding to that of the Pβ atom of the beta-S-ARCA D2 diastereomer. In that embodiment, R5 in Formula (I) is S; and R4 and R6 are O. Furthermore, at least one of R2 or R3 in Formula (I) is preferably not OH, preferably one of R2 and R3 is methoxy (OCH3), and the other of R2 and R3 is preferably OH. In a second particularly preferred embodiment, the RNA of the present instruction is modified with the beta-S-ARCA (D1) diastereomer. The beta-S-ARCA (D1) diastereomer, following the transfer of the corresponding protected RNA to immature antigen-presenting cells, has been shown to be particularly suitable for increasing RNA stability, increasing RNA translation efficiency, prolonging RNA translation, increasing total protein expression of the RNA, and / or increasing the immune response against an antigen or antigenic peptide encoded by said RNA (Kuhn et al., 2010, Gene Ther., vol. 17, pp. 961–971). Therefore, in an alternative embodiment of the present teaching, the RNA of the present teaching is modified with a protectant analogue according to Formula (I), characterized by a stereochemical configuration at the P atom comprising the R5 substituent in Formula (I) corresponding to that of the Pβ atom of the D1 diastereomer of beta-S-ARCA. The respective protectant analogues and their embodiments are described in WO 2011 / 015347 A1 and Kuhn et al., 2010, Gene Ther., vol. 17, pp. 961–971. Any protectant analogue described in WO 2011 / 015347 A1, wherein the stereochemical configuration at the P atom comprising the R5 substituent corresponds to that of the Pβ atom of the D1 diastereomer of beta-S-ARCA may be used in the present teaching. Preferably, R5 of Formula (I) is S; and R4 and R6 are O.Furthermore, at least one of R2 or R3 in Formula (I) is preferably not OH, preferably one between R2 and R3 is methoxy (OCH3), and the other between R2 and R3 is preferably OH. In one embodiment, the RNA of the present instruction is modified with a 5-protector structure according to Formula (I), wherein any phosphate group is replaced by a boranophosphate or phosphoroselenote group. These protectants have increased stability both in vitro and in vivo. Optionally, the corresponding compound has a 2'-O- or 3'-O-alkyl group (where the alkyl group is preferably methyl); the corresponding protectant analogues are designated BH3-ARCA or Se-ARCA. Compounds that are particularly suitable for mRNA protection include β-BH3-ARCA and β-Se-ARCA, as described in WO 2009 / 149253 A2. For these compounds, a stereochemical configuration at the P atom comprising the R5 substituent in Formula (I) corresponding to that of the Pβ atom of the D1 diastereomer of β-S-ARCA is preferred. UTR The term "untranslated region" or "UTR" refers to a region of a DNA molecule that is transcribed but not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be present at the 5' (upstream) end of an open reading frame (5'-UTR) and / or the 3' (downstream) end of an open reading frame (3'-UTR). A 3'-UTR, if present, is located at the 3' end of a gene, downstream of the stop codon of a protein-coding region, but the term "3'-UTR" preferably does not include the poly(A) tail. Therefore, the 3'-UTR is upstream of the poly(A) tail (if present), for example, 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 protein-coding region. A 5'-UTR is downstream of the 5'-protector (if present), for example, directly adjacent to the 5'-protector. According to the teaching, the 5' and / or 3' untranslated regions can be functionally linked to an open reading frame, such that these regions associate with the open reading frame in a way that increases the stability and / or efficiency of the translation of the RNA comprising that open reading frame. In some embodiments, the replicase construct according to the present teaching comprises a 5'-UTR and / or a 3'-UTR. In a preferred embodiment, the replicase construct according to the present teaching comprises (1) a 5'-UTR, (2) an open reading framework, and (3) a 3'-UTR. Untranslated translation regions (UTRs) are involved in the stability and efficiency of RNA translation. Both can be improved, in addition to the structural modifications related to the 5' protectant and / or the 3' poly(A) tail described herein, by selecting specific 5' and / or 3' untranslated regions (UTRs). It is generally understood that sequence elements within UTRs influence translation efficiency (primarily the 5'-UTR) and RNA stability (primarily the 3'-UTR). The presence of an active 5'-UTR is preferable to increase translation efficiency and / or the stability of the replicase construct. Independently or additionally, the presence of an active 3'-UTR is also preferable to increase translation efficiency and / or the stability of the replicase construct. The terms "translation efficiency-enhancing" and / or "stability-enhancing," with reference to a first nucleic acid sequence (e.g., a UTR), mean that the first nucleic acid sequence is capable of modifying, in a common transcript with a second nucleic acid sequence, the translation efficiency and / or stability of that second nucleic acid sequence in such a way that that translation efficiency and / or stability is increased compared to the translation efficiency and / or stability of that second nucleic acid sequence in the absence of that first nucleic acid sequence. In one embodiment, the replicase construct according to the present teaching comprises a 5'-UTR and / or a 3'-UTR that is heterologous or non-native to the alphavirus from which the functional alphavirus non-structural protein is derived. This allows the untranslated regions to be designed according to the desired translation efficiency and RNA stability. Therefore, the heterologous or non-native UTRs allow for a high degree of flexibility, and this flexibility is advantageous compared to native alphaviral UTRs. In particular, while it is known that (native) alphaviral RNA also comprises a 5'-UTR and / or a 3'-UTR, the alphaviral UTRs serve a dual function, namely, (i) driving RNA replication and (ii) driving translation. Although alphaviral UTRs were reported to be inefficient for translation (Berben-Bloemheuvel et al., 1992, Eur. J. Biochem., vol.208, pp.581-587), typically cannot be easily replaced by more efficient UTRs due to their dual function. In the present teaching, however, a 5'-UTR and / or a 3'-UTR included in a replicase construct for in trans replication can be selected independently of their potential influence on RNA replication. Preferably, the replicase construct according to the present instruction comprises a 5'-UTR and / or a 3'-UTR that is not of viral origin; in particular, not of alphavirus origin. In one embodiment, the replicase construct comprises a 5'-UTR derived from a eukaryotic 5'-UTR and / or a 3'-UTR derived from a eukaryotic 3'-UTR. According to this teaching, a 5'-UTR may comprise any combination of more than one nucleic acid sequence, optionally separated by a linker. According to this teaching, a 3'-UTR may comprise any combination of more than one nucleic acid sequence, optionally separated by a linker. According to the teaching, the term "linker" refers to a nucleic acid sequence that is added between two nucleic acid sequences to connect them. There are no particular limitations regarding the linker sequence. A 3'-UTR typically ranges from 200 to 2000 nucleotides in length, for example, 500 to 1500 nucleotides. The 3' untranslated regions of immunoglobulin mRNAs are relatively short (less than approximately 300 nucleotides), whereas the 3' untranslated regions of other genes are relatively long. For example, the 3' untranslated region of tPA is approximately 800 nucleotides long, that of factor VIII is approximately 1800 nucleotides long, and that of erythropoietin is approximately 560 nucleotides long. The 3' untranslated regions of mammalian mRNA typically have a homology region known as the hexanucleotide sequence AAUAAA. This sequence is presumed to be the poly(A) binding signal and is frequently found 10 to 30 bases upstream of the poly(A) binding site.The 3' untranslated regions may contain one or more inverted repeats that can fold to form stem-loop structures that act as barriers to exoribonucleases or interact with proteins known to increase RNA stability (e.g., RNA-binding proteins). The 3'-UTR of human beta-globin, in particular two consecutive identical copies of the 3'-UTR of human beta-globin, contributes to high transcript stability and translation efficiency (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009–4017). Thus, in one embodiment, the replicase construct according to the present teaching comprises two consecutive identical copies of the 3'-UTR of human beta-globin. Therefore, it comprises, in the 5' to 3' direction: (a) optionally a 5'-UTR; (b) an open reading frame; (c) a 3'-UTR; said 3'-UTR comprising two consecutive identical copies of the 3'-UTR of human beta-globin, a fragment thereof, or a variant of the 3'-UTR of human beta-globin or a fragment thereof.In one embodiment, the replicase construct according to the present teaching comprises a 3'-UTR that is active to increase the efficiency and / or stability of translation, but is not the 3'-UTR of human beta-globin, a fragment thereof, or a variant of the 3'-UTR of human beta-globin or a fragment thereof. In one embodiment, the replicase structure described in the present teaching comprises a 5-UTR that is active to increase the efficiency and / or stability of translation. A replicase construct containing UTRs can be prepared according to the teaching method, for example, by in vitro transcription. This can be achieved by genetically modifying a template nucleic acid molecule (e.g., DNA) to allow the transcription of RNA with the 5'-UTR and / or 3'-UTR. As illustrated in Fig. 5, the replicon can also be characterized by a 5-UTR and / or a 3-UTR. The replicon's UTRs are typically alphavirus UTRs or variants thereof. Poly sequence (A) In some embodiments, the replicon according to the present teaching comprises a 3'-poly(A) sequence. If the replicon comprises the conserved sequence element 4 (CSE 4), the 3'-poly(A) sequence of the replicon is preferably present downstream of CSE 4, and more preferably directly adjacent to CSE 4. In some realizations, the replicase construct according to the present teaching comprises a 3'-poly (A) sequence. According to the teaching, in one embodiment, a poly(A) sequence comprises or essentially 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 approximately 120 A nucleotides. In this context, "essentially consists of" means that the majority of the nucleotides in the poly(A) sequence, typically at least 50%, and preferably at least 75% by number of nucleotides in the "poly(A) sequence", are A (adenylate) nucleotides, but allows the remaining nucleotides to be nucleotides other than A nucleotides, such as U (uridylate) nucleotides, G (guanylate) nucleotides, and 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 nucleotides. The term "A nucleotide" or "A" refers to adenylate. In fact, a poly(A) 3' sequence of approximately 120 A nucleotides has been shown to have a beneficial influence on RNA levels in transfected eukaryotic cells, as well as on protein levels that are translated from an open reading frame that is present upstream (5') of the poly(A) 3' sequence (Holtkamp et al., 2006, Blood, vol.108, pp.4009-4017). In alphaviruses, a poly(A)3' sequence of at least 11 consecutive adenylate residues, or at least 25 consecutive adenylate residues, is thought to be important for efficient negative-strand synthesis. In particular, in alphaviruses, a poly(A)3' sequence of at least 25 consecutive adenylate residues is understood to work in conjunction with conserved sequence element 4 (CSE 4) to promote (-)-strand synthesis (Hardy & Rice, J. Virol., 2005, vol.79, pp.4630-4639). This instruction anticipates the addition of a 3' poly(A) sequence during RNA transcription, i.e., during the preparation of in vitro transcribed RNA based on a DNA template comprising repeated dT (deoxythymidylate) nucleotides on the strand complementary to the coding strand. The DNA sequence encoding a poly(A) sequence (coding strand) is called a poly(A) cassette. In a preferred embodiment of the present teaching, the 3' poly(A) cassette present in the DNA coding strand consists essentially of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT). Such a random sequence may be 5 to 50 nucleotides long, preferably 10 to 30, and more preferably 10 to 20 nucleotides. Such a cassette is disclosed in WO 2016 / 005004 A1. Any poly(A) cassette disclosed in WO 2016 / 005004 A1 may be used in the present teaching. A poly(A) cassette consisting essentially of dA nucleotides, but interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of, for example, 5 to 50 nucleotides, shows, at the DNA level, a constant spread of plasmid DNA in E.coli and remains associated, at the RNA level, with beneficial properties regarding support for RNA stability and translation efficiency. Accordingly, in a preferred embodiment of the present teaching, the poly(A) 3' sequence contained in an RNA molecule described herein consists essentially of A nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (A, C, G, U). Such a random sequence may be 5 to 50 nucleotides long, preferably 10 to 30, and more preferably 10 to 20 nucleotides. Use of codons In general, the degeneracy of the genetic code allows the substitution of certain codons (triplets of bases that code for an amino acid) present in an RNA sequence by other codons (triplets of bases), maintaining the same coding capacity (so that the replacement codon codes for the same amino acid as the replaced codon). In some embodiments of this teaching, at least one codon in an open reading frame composed of an RNA molecule differs from the corresponding codon in the corresponding open reading frame in the species from which the open reading frame originates. In such an embodiment, the coding sequence of the open reading frame is said to be "adapted" or "modified." The coding sequence of an open reading frame comprising the replicon can be adapted.As an alternative or complement, the coding sequence of the functional non-structural protein of the alphavirus comprising the replicase construct can be adapted. For example, when adapting the coding sequence of an open reading frame, frequently used codons can be selected: WO 2009 / 024567 A1 describes the adaptation of a nucleic acid molecule's coding sequence, which involves replacing infrequent codons with more frequently used ones. Since codon frequency depends on the host cell or host organism, this type of adaptation is suitable for adjusting a nucleic acid sequence for expression in a particular host cell or host organism. Generally speaking, more frequently used codons are usually translated more efficiently in a host cell or host organism, although it is not always necessary to adapt all codons in an open reading frame.For example, when adapting the coding sequence of an open reading frame, the content of G (guanylate) and C (cytidylate) residues can be modified by selecting codons with the highest GC-rich content for each amino acid. RNA molecules with GC-rich open reading frames have been reported to have the potential to reduce immune activation and improve RNA translation and half-life (Thess et al., 2015, Mol. Ther.23, 1457-1465). When the replicon, according to the present teaching, encodes the alphavirus nonstructural protein, the coding sequence for the alphavirus nonstructural protein can be adapted as desired. This freedom is possible because the open reading frame encoding the alphavirus nonstructural protein does not overlap with the replicon's 5' replication recognition sequence. Safety characteristics of the achievements of this teaching The following characteristics are preferred in the present teaching, either individually or in any suitable combination: Preferably, the replicon or system of the present instruction is non-particle-forming. This means that, after inoculation of a host cell by the replicon or system described herein, the host cell does not produce viral particles, such as next-generation viral particles. In one embodiment, all RNA molecules, according to the instruction, are completely free of genetic information encoding any alphavirus structural protein, such as the central nucleocapsid C protein, the envelope protein P62, and / or the envelope protein E1. This aspect of the present instruction provides added value in terms of safety compared to prior art systems where the structural proteins are encoded in trans-replicating helper RNA (e.g., Bredenbeek et al., J. Virol, 1993, vol. 67, pp. 6439–6446). Preferably, the system of the present teaching does not comprise any alphavirus structural proteins, such as the core nucleocapsid C protein, the P62 envelope protein and / or the E1 envelope protein. Preferably, the replicon and replicase structure of the system described herein are not identical to each other. In one embodiment, the replicon does not encode the functional nonstructural protein of the alphavirus. In one embodiment, the replicase construct lacks at least one sequence element (preferably at least one CSE) that is required for the synthesis of the (-) strand based on a (+) strand template, and / or for the synthesis of the (+) strand based on a (-) strand template. In one embodiment, the replicase structure does not comprise CSE 1 and / or CSE 4. Preferably, neither the replicon as taught here nor the replicase construct as taught here includes an alphavirus packaging signal. For example, the alphavirus packaging signal contained in the nsP2 coding region of SFV (White et al. 1998, J. Virol., vol. 72, pp. 4320-4326) can be eliminated, for example, by deletion or mutation. A suitable way to eliminate the alphavirus packaging signal is to adapt the codon usage of the nsP2 coding region. Degeneracy of the genetic code may allow the elimination of the packaging signal function without affecting the amino acid sequence of the encoded nsP2. In one embodiment, the system described herein is an isolated system. In that embodiment, the system is not present within a cell, such as, for example, within a mammalian cell, nor is it present within a viral capsid, such as within a shell comprising alphavirus structural proteins. In one embodiment, the system described herein is present in vitro. DNA In a further aspect, the present instruction provides DNA comprising a nucleic acid sequence encoding the RNA replicon, the set of RNA replicons, or the system as described herein. According to the instruction, instead of using RNA, for example, for cell transfection, DNA encoding RNA can be used, for example, for cell transfection such that the RNA is produced within the cells. Preferably, DNA is double-stranded. In a preferred embodiment, the DNA is a plasmid. The term "plasmid," as used herein, generally refers to a construct of extrachromosomal genetic material, usually a circular DNA duplex, that can replicate independently of chromosomal DNA. The DNA in this instruction may include a promoter that can be recognized by a DNA-dependent RNA polymerase. This allows for the transcription of the encoded RNA in vivo or in vitro, for example, from the RNA in this instruction. IVT vectors can be used in a standardized manner as a template for in vitro transcription. Examples of preferred promoters, depending on the instruction, are the promoters for the SP6, T3, or T7 polymerases. In one embodiment, the DNA of the present teaching is an isolated nucleic acid molecule. RNA preparation methods According to this teaching, any RNA molecule, whether or not it is part of the system described herein, can be obtained by in vitro transcription. In vitro transcribed RNA (IVT-RNA) is of particular interest herein. IVT-RNA can be obtained by transcription from a nucleic acid molecule (in particular, a DNA molecule). The DNA molecules presented herein are suitable for this purpose, particularly if they comprise a promoter that can be recognized by a DNA-dependent RNA polymerase. According to this teaching, RNA can be synthesized in vitro. This allows for the addition of protectant analogs to the in vitro transcription reaction. Typically, the poly(A) tail is encoded by a poly-(dT) sequence in the DNA template. Alternatively, the addition of the protectant and the poly(A) tail can be performed enzymatically after transcription. The methodology of in vitro transcription is known to the expert. For example, as mentioned in WO 2011 / 015347 A1, a variety of in vitro transcription kits are commercially available. Methods for producing cells and the cells produced thus. In additional aspects, the present teaching provides a method for producing cells that have stem cell characteristics comprising the transduction of somatic cells with one or more RNA replicons from the teaching and, optionally, an RNA construct to express the functional non-structural alphavirus protein. In one embodiment, the present teaching provides a method for obtaining cells that have stem cell characteristics comprising the following steps: (i) provide a cell population comprising somatic cells, (ii) providing one or more RNA replicons, wherein each of the RNA replicons comprises an open reading frame encoding a functional non-structural protein of the alphavirus, can be replicated by the functional non-structural protein of the alphavirus, and comprises at least one open reading frame encoding a reprogramming factor, (iii) introducing the one or more RNA replicons into somatic cells, such that the cells express a set of reprogramming factors useful for reprogramming somatic cells into cells having stem cell characteristics, and (iv) allow the development of cells that have stem cell characteristics. In a further embodiment, the present teaching provides a method for obtaining cells that have stem cell characteristics comprising the steps of: (i) provide a cell population comprising somatic cells, (ii) providing an RNA construct for expressing the functional non-structural protein of the alphavirus, (iii) providing one or more RNA replicons, wherein each of the one or more RNA replicons can be replicated in trans by the functional non-structural protein of the alphavirus and comprises at least one open reading frame encoding a reprogramming factor, (iv) introducing the RNA construct and one or more RNA replicons into somatic cells, so that the cells express a set of reprogramming factors useful for reprogramming somatic cells into cells that have stem cell characteristics, and (v) allow the development of cells that have stem cell characteristics. According to the teaching, the term "introduction of RNA into somatic cells" includes and preferably refers to embodiments in which RNA is introduced into only a portion of the cells, and another portion of the cells remains untransfected. If more than one RNA molecule is to be introduced into the cells—for example, more than one RNA replicon or an RNA construct to express a functional non-structural alphavirus protein and one or more RNA replicons—the term includes and preferably relates to embodiments in which all the RNA molecules are introduced into only a portion of the cells, and another portion of the cells remains untransfected or remains transfected by not all of those RNA molecules.In any case, if according to the teaching more than one RNA molecule is to be introduced into the cells, for example, more than one RNA replicon that expresses a functional set of reprogramming factors or an RNA construct to express a functional non-structural alphavirus protein and one or more RNA replicons, the objective is to introduce all the RNA molecules into a cell, for example, to express a functional set of reprogramming factors within a single cell. In one embodiment, transfected cells express the functional alphavirus nonstructural protein encoded by one or more transfected replicons and / or by a transfected replicase construct and / or reprogramming factors encoded by one or more transfected replicons. The different reprogramming factors may be encoded by different open reading frames residing on the same RNA replicon or on different RNA replicons. In this latter embodiment, the different replicons are preferably transfected together into a single cell. In various embodiments of the method, the RNA construct for expressing the functional alphavirus nonstructural protein and / or the RNA replicon are those defined above for the teaching system, provided that the RNA replicon can be replicated in trans by the functional alphavirus nonstructural protein and comprises an open reading frame encoding a reprogramming factor. The RNA construct for expressing the functional alphavirus nonstructural protein and one or more RNA replicons can be inoculated at the same time or, alternatively, at different time points. In the latter case, the RNA construct for expressing the functional alphavirus nonstructural protein is typically inoculated first, and the one or more replicons are typically inoculated at a later time point. In that case, it is anticipated that one or more replicons will replicate immediately, since the replicase will have already been synthesized in the cell.The second point in time is typically shorter after the first point in time, for example, 1 minute to 24 hours after the first point in time. The cell into which one or more nucleic acid molecules can be inoculated or transfected is called a "host cell." According to the teaching, the term "host cell" refers to any cell that can be transformed or transfected with an exogenous nucleic acid molecule. The term "cell" preferably refers to an intact cell, that is, a cell with an intact membrane that has not released its normal intracellular components, such as enzymes, organelles, or genetic material. An intact cell is preferably a viable cell, that is, a living cell capable of carrying out its normal metabolic functions. The term "host cell" includes, according to the teaching, prokaryotes (e.g., E. coli) or eukaryotic cells (e.g., human and animal cells, plant cells, yeast cells, and insect cells).Mammalian cells are preferred, such as those from humans, mice, hamsters, pigs, domestic animals including horses, cows, sheep, and goats, as well as primates. The cells can be derived from multiple 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 embodiments, the host cell is an antigen-presenting cell, particularly a dendritic cell, monocyte, or macrophage. A nucleic acid may be present in the host cell in a single copy or in multiple copies and, in one embodiment, is expressed within the host cell. The cell can be prokaryotic or eukaryotic. Prokaryotic cells are suitable in this case, for example, for DNA propagation as taught, and eukaryotic cells are suitable in this case, for example, for the expression of the replicon's open reading frame. For the purposes of this instruction, terms such as "transduction" or "transfection" refer to the introduction or incorporation of a nucleic acid into a cell or the uptake of a nucleic acid by a cell in vitro or in vivo. According to this instruction, a cell may be present for the transfection of a nucleic acid described herein in vitro or in vivo. For example, the cell may be part of an organ, tissue, and / or organism of a patient. According to this instruction, transfection may be transient or stable. For some transfection applications, it is sufficient for the transfected genetic material to be expressed only transiently. Since the nucleic acid introduced in the transfection process generally does not integrate into the nuclear genome, the foreign nucleic acid will be diluted by mitosis or degraded.Cells that allow episomal amplification of nucleic acids considerably reduce the dilution rate. If the transfected nucleic acid is to remain in the genome of the cell and its daughter cells, a stable transfection must occur. RNA can be transfected into cells to transiently express its encoded protein. According to this teaching, any useful technique may be used to introduce—that is, incorporate, transfer, or transfect—nucleic acids into cells. Preferably, the nucleic acid, such as RNA, is transfected into cells using standard techniques. These techniques include electroporation, lipofection, and microinjection. In a particularly preferred embodiment of this teaching, the RNA is introduced into cells by electroporation. Electroporation, or electropermeabilization, refers to a significant increase in the electrical conductivity and permeability of the cell's plasma membrane caused by an externally applied electric field. It is commonly used in molecular biology as a method for introducing a substance into a cell. According to this teaching, it is preferred that the introduction of nucleic acid encoding a protein or peptide into cells results in the expression of that protein or peptide.A pharmaceutical composition containing nucleic acid can be used for in vivo cell transfection. A delivery vehicle that directs the nucleic acid to a specific cell can be administered to a patient, resulting in an in vivo transfection. In one embodiment, a method for producing cells is an in vitro method. In one embodiment, a method for producing cells may or may not include the removal of cells from a human or animal subject by surgery or therapy. In this embodiment, the cells produced according to the instructions can be administered to a subject. The cell can be autologous, syngeneic, allogeneic, or heterologous with respect to the subject. The cells can be (re)introduced into a subject using any means known to the technique. In other embodiments, somatic cells may be present in a subject, such as a patient. In these embodiments, the method for producing cells with stem cell characteristics is an in vivo method involving the administration of RNA and / or DNA molecules to the subject. In this sense, the teaching also provides a method for producing cells that have stem cell characteristics in a subject that includes the following steps: (i) providing one or more RNA replicons, wherein each of the one or more RNA replicons comprises an open reading frame encoding a functional non-structural protein of the alphavirus, can be replicated by the functional non-structural protein of the alphavirus and comprises at least one open reading frame encoding a reprogramming factor, (ii) administer one or more RNA replicons to the subject, and (iii) allow the development of cells that have stem cell characteristics. In several embodiments of the method, the RNA replicon is as defined above for the teaching RNA replicon, provided that the RNA replicon comprises an open reading frame encoding a functional non-structural protein of the alphavirus and an open reading frame encoding a reprogramming factor, and can be replicated by the functional non-structural protein of the alphavirus. The course also provides a method for producing cells that have stem cell characteristics in a subject that comprises the following steps: (i) providing an RNA construct for expressing the functional non-structural protein of the alphavirus, (ii) providing one or more RNA replicons, wherein each of the one or more RNA replicons can be replicated in trans by the functional non-structural protein of the alphavirus and comprises at least one open reading frame encoding a reprogramming factor, (iii) administer the RNA construct and one or more RNA replicons to the subject, and (v) allow the development of cells that have stem cell characteristics. In various embodiments of the method, the RNA construct for expressing the functional alphavirus nonstructural protein and / or the RNA replicon are those defined above for the teaching system, provided that the RNA replicon can be replicated in trans by the functional alphavirus nonstructural protein and comprises an open reading frame encoding a reprogramming factor. The RNA construct for expressing the functional alphavirus nonstructural protein and the one or more RNA replicons can be administered simultaneously or, alternatively, at different time points. In the latter case, the RNA construct for expressing the functional alphavirus nonstructural protein is typically administered at a first time point, and the one or more RNA replicons are typically administered at a later time point.In this case, one or more replicons are expected to replicate immediately, as the replicase will already have been synthesized in the cell. The second time point is usually shortly after the first time point, for example, 1 minute to 24 hours after the first time point. Preferably, the administration of the one or more RNA replicons is performed at the same site and via the same route of administration as the administration of the RNA construct for the expression of the functional alphavirus non-structural protein, in order to increase the likelihood that the one or more RNA replicons and the RNA construct for the expression of the functional alphavirus non-structural protein will reach the same target tissue or cell. "Site" refers to a position on a subject's body. Suitable sites are, for example, the left arm, the right arm, etc. In one embodiment, an additional RNA molecule, preferably an mRNA molecule, may be administered to the subject. Optionally, the additional RNA molecule encodes a protein suitable for inhibiting IFN, such as E3. Optionally, the additional RNA molecule may be administered prior to the administration of one or more replicons, the replicase construct, or the system, as per the teaching instructions. Any of the RNA replicons, as taught, the assembly, the system, the kit, or the pharmaceutical composition may be used in the method to produce cells in a subject. For example, in the teaching method, the RNA may be used in the form of a pharmaceutical composition, as described herein, or as unaltered RNA. In one embodiment of the teaching, a functional set of reprogramming factors may comprise more than one reprogramming factor, all of which must be present within a cell to achieve reprogramming. Accordingly, the present teaching involves the materialization of a set of RNA replicons encoding the different reprogramming factors. For example, different RNA replicons may comprise different open reading frames encoding different reprogramming factors. These different RNA replicons, i.e., a set of RNA replicons, can be co-inoculated (optionally along with an RNA construct to express the functional non-structural alphavirus protein) into a cell to provide a functional set of reprogramming factors. Kit The present instruction also provides a kit comprising an RNA replicon according to the instruction, an assembly according to the instruction, or a system according to the instruction. In one embodiment, the kit components are presented as separate entities. For example, one nucleic acid molecule from the kit may be present in one entity, and another nucleic acid molecule from the kit may be present in a different entity. For example, an open or closed container is a suitable entity. A closed container is preferred. Preferably, the container used should be RNase-free or essentially RNase-free. In one embodiment, the present teaching kit comprises RNA for inoculation with a cell and / or for administration to a human or animal subject. The kit, as per this instruction, may optionally include a label or other information item, such as an electronic data carrier. The label or information item may preferably include instructions, such as printed written instructions or electronic instructions that can be optionally printed. The instructions may refer at least to one possible appropriate use of the kit. Uses of reprogrammed cells Using this method, RNA replicons encoding the appropriate factors are incorporated into one or more somatic cells, for example, by electroporation or lipofection. After incorporation, the cells are preferably cultured under conditions that favor the maintenance of dedifferentiated cells (i.e., stem cell culture conditions). The dedifferentiated cells can then be administered to a subject, for example, in cell therapy, or expanded and induced to differentiate back into somatic cells that can then be administered to a subject, for example, in cell therapy. Thus, dedifferentiated cells obtained according to this method can be induced to differentiate into one or more desired somatic cell types, both in vitro and in vivo. The term "cell therapy" (also called cytotherapy) refers to a therapy in which cellular material, usually live and intact cells, is provided to, and preferably administered to, a patient. The term includes allogeneic cell therapy, where the donor is a different person than the cell recipient, and autologous cell therapy. Cell therapy addresses numerous clinical indications in multiple organs and uses various cell delivery methods. Consequently, the specific mechanisms of action involved in these therapies are highly diverse. However, there are two main principles by which cells facilitate therapeutic action: (1) cells that replace damaged tissue and thus facilitate improved organ or tissue function. An example of this is the use of cells to replace cardiomyocytes after a myocardial infarction.(2) Cells that have the capacity to release soluble factors such as cytokines, chemokines, and growth factors that act in a paracrine or endocrine manner. These factors facilitate the self-healing of the affected organ or region. Some examples of this are cells that secrete factors that promote angiogenesis, anti-inflammation, and anti-apoptosis. Preferably, the undifferentiated cells obtained according to the present instruction can give rise to cells of any of the three embryonic germ layers, namely, endoderm, mesoderm, and ectoderm. For example, the undifferentiated cells can differentiate into skeletal muscle, skeleton, dermis of the skin, connective tissue, urogenital system, heart, blood (lymphatic cells), and spleen (mesoderm); stomach, colon, liver, pancreas, urinary bladder; lining of the urethra, epithelial parts of the trachea, lungs, pharynx, thyroid, parathyroid, intestine (endoderm); or central nervous system, retina and lens, cranial and sensory system, ganglia and nerves, pigment cells, connective tissue of the head, epidermis, hair, and mammary glands (ectoderm). The dedifferentiated cells obtained according to the present teaching can be redifferentiated in vitro or in vivo using techniques known in the art. In one embodiment of this instruction, the reprogrammed cells resulting from the methods described herein are used to produce differentiated offspring. Thus, in one aspect, this instruction provides a method for producing differentiated cells, comprising: (i) obtaining reprogrammed cells using the methods described herein; and (ii) inducing the differentiation of the reprogrammed cells to produce differentiated cells. Step (ii) can be performed in vivo or in vitro. Furthermore, differentiation can be induced by the presence of appropriate differentiation factors, which can be added or be present in situ, for example, in a body, organ, or tissue into which the reprogrammed cells have been introduced. The differentiated cells can be used to obtain cells, tissues, and / or organs that are advantageously used in the field of cell, tissue, and / or organ transplantation.If desired, genetic modifications can be introduced, for example, into somatic cells before reprogramming. The differentiated cells described herein preferably do not possess the pluripotency of an embryonic stem cell or an embryonic germ cell, and are, in essence, partially or fully differentiated cells specific to a tissue. An advantage of the methods described herein is that the reprogrammed cells obtained can be differentiated without prior selection, purification, or establishment of a cell line. Consequently, in certain embodiments, a heterogeneous cell population composed of reprogrammed cells is differentiated into the desired cell type. In one embodiment, a mixture of cells obtained using the methods described herein is exposed to one or more differentiation factors and cultured in vitro. Methods for differentiating reprogrammed cells obtained using the methods described herein may include a permeabilization step. For example, cells generated using the reprogramming techniques described herein, or alternatively a heterogeneous cell mixture comprising reprogrammed cells, may be permeabilized prior to exposure to one or more differentiation factors, cell extract, or other preparation containing differentiation factors. For example, differentiated cells can be obtained by culturing undifferentiated reprogrammed cells in the presence of at least one differentiation factor and selecting the differentiated cells from the culture. The selection of differentiated cells can be based on phenotype, such as the expression of certain cellular markers present in the differentiated cells, or on functional assays (e.g., the ability to perform one or more functions of a particular type of differentiated cell). In another embodiment, the cells reprogrammed acco...

Claims

1. An RNA replicon comprising an open reading frame encoding a reprogramming factor and a 5' replication recognition sequence, wherein the 5' replication recognition sequence is a functional variant of a native alphavirus 5' replication recognition sequence, characterized in that it does not contain any start codon compared to the native alphavirus 5' replication recognition sequence.

2. The RNA replicon according to claim 1, comprising at least one additional open reading frame encoding a different reprogramming factor.

3. The RNA replicon according to claim 1 or 2, wherein the reprogramming factor is selected from the group consisting of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

4. The RNA replicon according to any one of claims 1 to 3,5. The RNA replicon according to any one of claims 1 to 4, comprising a first open reading frame encoding a functional non-structural alphavirus nsP1234 protein.

6. The RNA replicon according to any one of claims 1 to 5, wherein the first open reading frame does not overlap with the 5' replication recognition sequence.

7. The RNA replicon according to any one of claims 1 to 6, wherein the start codon of the first open reading frame is in the 5' to 3' direction of the RNA replicon, the first functional start codon.

8. An array of RNA replicons,wherein each of the RNA replicons comprises at least one open reading frame encoding a reprogramming factor and the RNA replicon assembly encodes a set of reprogramming factors; and wherein at least one RNA replicon of the assembly is an RNA replicon according to any one of claims 1 to 7.

9. The RNA replicon assembly according to claim 8, wherein the set of reprogramming factors is useful for reprogramming somatic cells into cells having stem cell characteristics.

10. The RNA replicon assembly according to claim 8 or 9,wherein each RNA replicon of the assembly is an RNA replicon according to any one of claims 1 to 7.

11. A method for producing cells having stem cell characteristics comprising the step of introducing into somatic cells one or more RNA replicons according to any one of claims 1 to 7.

12. A method for providing cells having stem cell characteristics comprising the steps of: (i) providing a cell population comprising somatic cells, (ii) providing one or more RNA replicons, wherein at least one RNA replicon of the one or more RNA replicons is an RNA replicon according to any one of claims 1 to 3 and 5 to 7 and wherein each of the one or more RNA replicons comprises an open reading frame encoding the functional nonstructural protein of alphavirus nsP1234,can be replicated by the functional non-structural protein of alphavirus nsP1234 and comprises at least one open reading frame encoding a reprogramming factor, (iii) introducing the one or more RNA replicons into somatic cells, such that the cells express a set of reprogramming factors useful for reprogramming somatic cells to cells having stem cell characteristics, and (iv) enabling the development of cells having stem cell characteristics.

13. A method for providing cells having stem cell characteristics comprising the steps of: (i) providing a cell population comprising somatic cells, (ii) providing an RNA construct encoding the functional non-structural protein of alphavirus nsP1234, (iii) providing one or more RNA replicons, wherein at least one RNA replicon of the one or more replicons is an RNA replicon according to any one of claims 1 to 7,wherein each of the one or more RNA replicons can be replicated by the functional non-structural protein of the alphavirus nsP1234 and comprises at least one open reading frame encoding a reprogramming factor, (iv) introducing the RNA construct and the one or more RNA replicons into somatic cells, such that the cells express a set of reprogramming factors useful for reprogramming somatic cells to cells having stem cell characteristics, and (v) enabling the development of cells having stem cell characteristics.

14. The method according to any one of claims 11 to 13, further comprising introducing into somatic cells miRNA that enhances the reprogramming of somatic cells to cells having stem cell characteristics.

15. The method according to any one of claims 11 to 15,wherein each RNA replicon of the one or more RNA replicons is an RNA replicon according to any one of claims 1 to 7.

16. The method according to any one of claims 11 to 14, wherein the one or more RNA replicons comprise a set of RNA replicons according to any one of claims 8 to 10.

17. Cells comprising an RNA replicon according to any one of claims 1 to 7 or a set of RNA replicons according to any one of claims 8 to 10.

18. A method for providing differentiated cell types comprising the steps of (i) providing cells having stem cell characteristics using the method of any one of claims 11 to 14, and (ii) culturing the cells having stem cell characteristics under conditions that induce or direct partial or complete differentiation to a differentiated cell type.