Systems and compositions comprising trans-amplifying RNA vectors carrying miRNAs - Patent Application 201001229997

Abstract

The present invention encompasses systems, kits, and compositions comprising two RNA molecules, wherein the first RNA molecule comprises an open reading frame encoding a functional RNA-dependent RNA polymerase (replicase), and the second RNA molecule is a replicable RNA molecule comprising at least one miRNA sequence, which, when present in a cell, is excised from the second replicable RNA and is capable of regulating gene expression in the cell, and which replicable RNA molecule is capable of being replicated in trans by the replicase encoded by the first RNA molecule. The present invention further encompasses methods for treating or preventing cancer, infectious diseases, or other diseases and disorders using such systems and compositions, as well as the use of such systems and compositions in such treatment and prevention methods.

Description

[Technical Field]

[0001] Technical Field The present invention encompasses systems, kits, and compositions comprising two RNA molecules, wherein the first RNA molecule comprises an open reading frame encoding a functional RNA-dependent RNA polymerase (replicase), and the second RNA molecule is a replicable RNA molecule comprising at least one miRNA sequence, which, when present in a cell, is excised from the second replicable RNA and can regulate gene expression in the cell, and the replicable RNA molecule can be replicated in trans by the replicase encoded by the first RNA molecule. The present invention further encompasses methods for treating or preventing cancer, infectious diseases, or other diseases and disorders using such systems and compositions, as well as the use of such systems and compositions in such treatment and prevention methods. [Background technology]

[0002] background Alphaviruses are enveloped, positive-stranded RNA viruses belonging to the Togaviridae family. They infect insects, fish, and mammals, including livestock and humans. They replicate in the cytoplasm of infected cells (for an overview of the alphavirus life cycle, see Jose et al., 2009, Future Microbiol. 4:837-856). Alphavirus genomic RNAs are 5'-capped, 3'-polyadenylated, and 11-12 kilonucleotides in length (JH Strauss and EG Strauss, Microbiol. Rev., vol. 58, no. 3, pp. 491-562, 1994; JY-S. Leung, MM-L. Ng, and JJH Chu, Adv. Virol., vol. 2011, p. 249640, 2011). The genomic RNA contains two open reading frames (ORFs). The first ORF encodes the large polyprotein nsP1234, which forms the replication complex required for RNA transcription, modification, and replication. The second ORF, under the control of the subgenomic promoter (SGP), encodes the structural proteins required for viral particle assembly (JH Strauss and EG Strauss, Microbiol. Rev., vol. 58, no. 3, pp. 491-562, 1994; JY-S. Leung, MM-L. Ng, and JJH Chu, Adv. Virol., vol. 2011, pp. 249640, 2011). This bicistronic mRNA is flanked by conserved sequence elements (CSEs) that form the RNA structure required for subgenomic transcription and replication (JH Strauss and EG Strauss, Microbiol. Rev., vol. 58, no. 3, pp. 491-562, 1994).

[0003] During alphavirus infection, viral nonstructural proteins are translated directly from genomic RNA, while structural proteins are translated from subgenomic transcripts (Gould et al., Antiviral Res. 87:111-124, 2010). Early in infection, newly translated nsP1234 undergoes autolysis to cleave the short-lived alphaviral polyprotein intermediates nsP123 and nonstructural protein 4 (nsP4). nsP123 interacts with the nsP4 protein to form the core viral RNA-dependent RNA polymerase (MK Pietila, K. Hellstrom, and T. Ahola, Virus Res., 2017). Antisense RNA synthesis of the (+) genomic RNA is induced, generating at least one complementary (-) genomic copy as a template for plus-strand RNA synthesis. Shortly after generation of the antisense RNA template, nsP123 is sequentially processed by the viral nsP2 protease to nsP1 and nsP23, which are then converted to nsP2 and nsP3. Together with nsP4, these all form a stable replicase protein, or replication complex (L. Carrasco, M.A. Sanz, and E. Gonzalez-Almela, Viruses, vol. 10, no. 2, 2018). These replication complexes transcribe and amplify the positive-sense genomic and subgenomic RNAs (sgRNAs). Late in infection, only the sgRNAs are transcribed, which are structural proteins required for viral RNA encapsidation, final assembly, and virus release.

[0004] To generate alphavirus-based self-amplifying RNA vectors, or saRNA vectors, a heterologous gene of interest (GOI) replaces the structural genes within the genomic alphavirus RNA. The replicase polyprotein remains, allowing enhanced GOI expression with an increase in the number of newly synthesized saRNA copies. In this type of system, the absence of structural proteins inhibits virion formation and virus spread (JH Aberle, SW Aberle, RM Kofler, and CW Mandl, J. Virol., vol. 79, no. 24, pp. 15107-13, Dec. 2005). However, the RNA replication process of saRNA is identical to genome replication in alphavirus-infected cells. Furthermore, transient transfection with saRNA elicits a potent immune response because double-stranded RNA (dsRNA) replication intermediates activate the innate immune system. This is equivalent to the intrinsic self-adjuvanting activity of saRNA, which induces and enhances host immune responses (NP Restifo et al., Nat. Med., vol. 5, no. 7, pp. 823-827, July 1999; Perri et al., J. Virol., vol. 77, no. 19, pp. 10394-403, October 2003). This feature, combined with its properties as an antigen delivery vehicle, makes saRNA a suitable and attractive candidate for RNA vaccines (AJ Geall et al., Proc. Natl. Acad. Sci. USA, vol. 109, no. 36, pp. 14604-9, 2012; JB Ulmer and AJ Geall, Curr. Opin. Immunol., vol. 41, pp. 18-22, 2016).

[0005] Transamplifying RNA (taRNA) is a split vector system consisting of two alphavirus-based RNA molecules. One is a capped, replication-incompetent in vitro transcribed (IVT) mRNA encoding a replicase polyprotein. The IVT RNA encoding the target of infection (GOI) is flanked by the viral 5' and 3' CSEs, allowing it to be replicated in trans by the replicase proteins (termed the transreplicon (TR) and / or nano-transreplicon (NTR)) (JO Rayner, SA Dryga, and KI Kamrud, Reviews in Medical Virology, vol. 12, no. 5, pp. 279-296, 2002). When both RNA constructs are introduced into cells, the viral replicase proteins, using the mRNA as a template, recognize the 5' and 3' CSEs of the co-transfected TR / NTR and amplify them in trans.

[0006] Gene regulatory systems based on small non-coding RNAs (sncRNAs) are ubiquitous in biology. They are endogenous or exogenous single- or double-stranded RNA molecules less than 200 nucleotides (nt) in length and present in animals, plants, and viruses (RW Carthew and EJ Sontheimer, Cell, vol. 136, no. 4, pp. 642-55, Feb. 2009). By binding to specialized proteins, sncRNAs can silence the expression of invading genes (e.g., from viruses) or regulate the expression of the cell's own transcriptome. These mechanisms are generally collectively referred to as RNA interference (RNAi) (A. Fire, S. Xu, MK Montgomery, SA Kostas, SE Driver, and CC Mello, Nature, vol. 391, no. 6669, pp. 806–811, 1998; RC Wilson and JA Doudna, Annu. Rev. Biophys., vol. 42, no. 1, pp. 217–239, 2013.20). In bacteria, clusters of regularly interspaced short palindromic repeat RNAs are an important component of the endogenous immune defense system, protecting against invading foreign nucleic acids (LA Marraffini and EJ Sontheimer, Nat. Rev. Genet., vol. 11, no. 3, pp. 181–90, Mar. 2010). In invertebrates and plants, small interfering RNAs (siRNAs) constitute the host antiviral defense system. siRNAs are generated from endogenous, exogenous, or viral double-stranded RNAs and induce post-transcriptional silencing (PTS) of viral transcripts and transposons (S.-W. Ding, Nat. Rev. Immunol., vol. 10, no. 9, pp. 632-44, Sep. 2010).MicroRNAs (miRNAs), on the other hand, are a unique class of snRNAs conserved in eukaryotes and responsible for the PTS of endogenous mRNAs (M. Ghildiyal and PD Zamore, Nature Reviews Genetics, vol. 10, no. 2, pp. 94–108, 2009). Currently, 1,984 pre-miRNA sequences are known in humans (as of July 14, 2022, from the miRBase database, version 22.1). Each miRNA targets hundreds of genes, collectively affecting at least 50% of all human genes (RC Friedman, KKH Farh, CB Burge, and DP Bartel, Genome Res., vol. 19, no. 1, pp. 92–105, 2009). A single mRNA can be regulated by multiple miRNAs (DP Bartel, Cell, vol. 131, no. 4, pp. 11-29, 2007). Because so many protein-coding transcripts are regulated by miRNAs, miRNAs play a crucial role in almost every developmental and pathological process in animals. Therefore, defects or dysregulation of miRNAs are associated with many diseases, including neurological disorders, cancer, and cardiovascular diseases (MV Lorio and CM Croce, EMBO molecular medicine, vol. 4,3, pp. 143-59, 2012; C. Urbich, A. Kuehbacher, and S. Dimmeler, Cardiovascular Research, vol. 79, no. 4, pp. 581-588, 2008).

[0007] miRNAs act directly on target genes in a sequence-dependent manner (E. Huntzinger and E. Izaurralde, Nature Reviews Genetics, vol. 12, no. 2, pp. 99-110, 2011). Specifically, mature miRNAs associate with Argonaute (AGO) proteins, which are gene silencing effectors, to form the so-called RNA-induced silencing complex (RISC). RISC is a ribonucleoprotein complex that mediates post-transcriptional silencing (PTS) under miRNA guidance. The first 2–7 nucleotides of the 5′ end of the miRNA constitute the seed sequence. This section base-pairs with the 3′ UTR of the target mRNA, and depending on the number of base-pair matches, the active part of RISC, AGO, triggers cleavage, destabilization, or translational inhibition of the target mRNA (DP Bartel, Cell, vol. 136, no. 2, pp. 215–233, 2009). Most commonly, the level of PTS is low (~20%) because most miRNA target sites have only partial complementarity to their target mRNAs (D. Baek, J. Villen, C. Shin, FD Camargo, SP Gygi, and DP Bartel, Nature, vol. 455, no. 7209, pp. 64-71, Sep. 2008; H. Seitz, Curr. Biol., vol. 19, no. 10, pp. 870-873, May 2009).

[0008] Since its discovery, the RNAi machinery has been continuously exploited in basic and applied research and genome engineering. Its remarkable potential for loss-of-function studies in animals has led to the development of RNAi-based therapies for various genetic and viral diseases, such as Huntington's disease and viral hepatitis (S. Aguiar, B. van der Gaag, and F.A.B. Cortese, Translational Neurodegeneration, vol. 6, no. 1. 2017; D. Castanotto and J.J. Rossi, Nature, vol. 457, no. 7228, pp. 426–433, 2009).

[0009] siRNA and shRNA are commonly used as RNAi mediators for sequence-dependent cleavage and reduction of protein-coding transcripts. shRNAs are synthetic short hairpin RNAs that mimic miRNA precursors. Artificial miRNAs, or exogenous miRNAs, are also used for gene regulation. RNAi mediators are typically expressed transiently or stably from plasmid or viral DNA expression vectors. To achieve stable silencing of gene expression, significant efforts have been devoted to the construction and delivery of miRNA expression cassettes using viral vectors. Four popular and well-studied viral vector systems are commonly used to promote high levels of transgene and miRNA expression: adenovirus, adenovirus-associated virus, retrovirus, and subclass lentivirus. Each of the different viral vector systems has its own advantages and disadvantages. The main disadvantages of adenovirus-based vectors are the need for repeated administration and their relatively high immunogenicity. AAV-based vectors require a helper virus for replication. Furthermore, this vector system has a limited overall insert capacity (maximum 3–5 kb). A major known concern with retroviral and lentiviral vectors is the risk of insertional mutagenesis (for details, see E. Herrera-Carrillo, YP Liu, and B. Berkhout, Hum. Gene Ther. Methods, vol. 28, no. 4, 2017).

[0010] Therefore, there is an urgent need for systems and compositions that provide miRNA precursors in high copy numbers. Furthermore, there is a need for an efficient system that can easily deliver miRNAs together with protein-coding genes. The present invention fulfills these needs. Summary of the Invention

[0011] overview The present invention generally relates to a system comprising two RNA molecules, where a first RNA molecule comprises an open reading frame encoding a functional RNA-dependent RNA polymerase (replicase), and a second RNA molecule is a replicable RNA molecule comprising at least one miRNA sequence that, when present in a cell, is excised from the second replicable RNA and is capable of regulating gene expression in the cell, and where the replicable RNA molecule is capable of being replicated in trans by the replicase encoded by the first RNA molecule.

[0012] Without being bound by theory, in some embodiments, the second RNA molecule resembles a primary miRNA (pri-miRNA) from which miRNA is excised by enzymes present in the cell, such as Drosha and / or Dicer. According to the invention described herein, the second RNA molecule is processed within the cell to excise the miRNA sequence from the larger sequence of the second RNA molecule to generate a functional miRNA. The functional miRNA can form an RNA-induced silencing complex (RISC) with proteins from the host cell. Alternatively, the second RNA molecule can be processed within the cell to first excise the pre-miRNA molecule from the pri-miRNA sequence, which is then further processed within the cell to generate the functional miRNA. In some embodiments, the difference between a natural pri-miRNA and the second RNA molecule of the invention is that the second RNA molecule contains, in addition to the miRNA sequence, a sequence necessary for replication of the second RNA molecule by the replicase encoded by the first RNA molecule. Like the pri-miRNA sequence, the second RNA molecule contains a sequence necessary for excising the miRNA from the second RNA molecule. Such excision occurs intracellularly using cellular mechanisms, such as enzymes present in the cell, such as RNA cleavage enzymes, ribonucleases, and ribozymes, to excise functional miRNA sequences.

[0013] In one embodiment, the second RNA molecule comprises at least one pre-miRNA sequence. In this embodiment, the miRNA sequence is flanked by additional sequences that together with the miRNA sequence form the pre-miRNA sequence. Excision from the second RNA molecule typically occurs in cells that can excise the miRNA sequence from the second RNA molecule, such as cells that express Drosha and Dicer.

[0014] Without being bound by theory, the present invention is based in part on the observation that miRNAs can be introduced into cells without the need to introduce the pri-miRNA into the nucleus, where it is normally processed. The present invention is also based on the observation that it is beneficial to include miRNA sequences on replicable RNA to enhance the efficiency of the miRNA in regulating gene expression, for example, by inhibiting translation of mRNA molecules to which the miRNA binds.

[0015] In one embodiment, the first RNA molecule and / or the second RNA molecule, preferably the second RNA molecule, further comprises at least one open reading frame (ORF) encoding a protein of interest. The inventors surprisingly discovered that by combining an miRNA sequence with the coding sequence of the protein of interest on the same replicable RNA, the protein of interest and the miRNA can be provided to a subject simultaneously and within the same cell. For example, the protein of interest can be a dedifferentiation factor and the miRNA can inhibit the expression of a gene responsible for differentiation, or it can be a stem cell-specific miRNA (i.e., the miRNA is preferentially expressed in stem cells over differentiated cells). In another example, the protein of interest can be a tumor antigen and the miRNA can inhibit the expression of an oncogene expressed in tumors.

[0016] In one embodiment, the miRNA is a stem cell-specific miRNA (i.e., the miRNA is preferentially expressed in stem cells over differentiated cells), and the protein of interest can be a pluripotency inducer (e.g., OCT4). In one embodiment, the miRNA sequence targets an mRNA encoding a protein overexpressed in cancer cells, and the open reading frame encodes a protein useful in treating the cancer.

[0017] Described herein is a system comprising two RNA molecules, wherein the first RNA molecule comprises an open reading frame encoding a functional RNA-dependent RNA polymerase (replicase), and the second RNA molecule is a replicable RNA molecule comprising at least one non-coding RNA sequence that can be excised from the second replicable RNA molecule when present in a cell and can regulate gene expression in the cell, wherein the replicable RNA molecule can be replicated in trans by the replicase encoded by the first RNA molecule. In one embodiment, the second RNA molecule further comprises at least one open reading frame (ORF) encoding a protein of interest, as described herein. The length of each non-coding RNA sequence contained in the second RNA molecule is 10 to 500 nucleotides, optionally 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 50, 20 to 400, 20 to 300, 20 to 200, 20 to 100, 20 to 50, 10 to 40, 10 to 30, 20 to 40, or 20 to 30 nucleotides, and optionally 10 to 100 nucleotides, preferably 10 to 50 nucleotides. Examples of non-coding RNA sequences include miRNA, shRNA, siRNA, and antisense molecules, although those skilled in the art will recognize other non-coding RNA sequences capable of regulating gene expression in cells, which can also be incorporated into the second replicable RNA molecule. In one embodiment, the second RNA molecule can be an mRNA. In one embodiment, the second RNA molecule can be a replicable RNA molecule and an mRNA.

[0018] In one embodiment, the first RNA molecule can be a replicable RNA molecule that can be replicated by the replicase that it encodes. In one embodiment, the first RNA molecule is not a replicable RNA molecule. In one embodiment, the first RNA molecule can be an mRNA. In one embodiment, the first RNA molecule can be an mRNA, and the second RNA molecule can be an mRNA. In one embodiment, the first RNA molecule is an mRNA and is not a replicable RNA molecule, and the second RNA molecule is an mRNA and is a replicable RNA molecule. In one embodiment, the first RNA molecule is an mRNA and is a replicable RNA molecule, and the second RNA molecule is an mRNA and is a replicable RNA molecule.

[0019] In one embodiment, the replicase is derived from a functional nonstructural protein from an autonomously replicating virus. In one embodiment, the autonomously replicating virus is an autonomously replicating single-stranded RNA virus. In one embodiment, the autonomously replicating virus is a positive-sense single-stranded RNA virus (e.g., an alphavirus, a flavivirus, etc.). In one embodiment, the autonomously replicating virus is an alphavirus, preferably selected from the group consisting of Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, Chikungunya virus, Semliki Forest virus, Sindbis virus, Barmah Forest virus, Middelburg virus, and Ndumu virus. Preferably, the alphavirus is Venezuelan equine encephalitis virus or Semliki Forest virus.

[0020] In one embodiment, the second RNA molecule can contain at least 2, at least 3, at least 4, at least 5, or at least 10 miRNA sequences, preferably at least 5 miRNA sequences. In one embodiment, the second RNA molecule can contain 1 to 20, 1 to 10, 1 to 8, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 miRNA sequences, optionally 1 to 10, preferably 2 to 8 miRNA sequences. In one embodiment, the second RNA molecule can contain 1 to 20, 1 to 10, 1 to 8, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 different miRNA sequences, optionally 1 to 10, preferably 2 to 8 different miRNA sequences. In one embodiment, the second RNA molecule can comprise 1 to 20, 1 to 10, 1 to 8, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 copies of the same miRNA sequence, optionally 1 to 10 copies, preferably 2 to 8 copies of the same miRNA sequence.

[0021] In one embodiment, the sequence of at least one miRNA may be different from the sequence of at least one other miRNA, and preferably, the sequences of each miRNA may be different from each other. In one embodiment, the sequences of multiple miRNAs may be the same sequence.

[0022] In one embodiment, miRNAs target mRNAs and affect the translation of those mRNAs, thereby regulating the expression of genes encoding those mRNAs. For example, miRNAs bind to mRNAs and prevent them from being translated. In one embodiment, the same or different miRNAs can target the same mRNA. In one embodiment, different miRNAs target different mRNAs. In one embodiment, different miRNAs target 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different mRNAs, preferably 1 to 5 different mRNAs. In one embodiment, each miRNA sequence contained in the second RNA molecule can target a different mRNA. In one embodiment, different miRNAs can target different sites on the same mRNA, or different miRNAs can target different sites on two or more mRNAs.

[0023] In one embodiment, the miRNA sequence can be a naturally occurring miRNA sequence, preferably a human miRNA sequence. In one embodiment, the miRNA sequence can be an artificial miRNA sequence. In one embodiment, the miRNA can be a non-viral miRNA. In one embodiment, the miRNA can be a stem cell-specific miRNA.

[0024] In one embodiment, the miRNA can suppress the innate immune response in a cell, for example, by targeting the mRNA of a cytokine that contributes to the innate immune response, such as an interleukin.

[0025] In one embodiment, the target of miRNA can be the mRNA associated with the onset or progression of disease, preferably the mRNA of oncogene, mutated tumor suppressor gene, or virus, bacteria or fungus gene.In one embodiment, the target of miRNA can be mutated (non-functional) tumor suppressor gene.For example, the mutated tumor suppressor gene is mutated TP53.

[0026] In one embodiment, the miRNA target may be an interferon-stimulated gene, preferably RSAD2 (Viperin). These genes are upregulated by alphavirus infection, potentially leading to inhibition of the translational machinery. In one embodiment, the miRNA target may be retinoic acid-inducible gene I (RIG-I). In one embodiment, the miRNA target may be the eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2) gene, which encodes protein kinase R (PKR). Targeting RIG-I and / or PKR has the advantage, but not limitation, of being useful in suppressing innate immunity induced by transfection and resulting in inhibition of the intracellular translational machinery.

[0027] In one embodiment, the target of the miRNA may be DAZ-associated protein 2 (DAZAP2) and / or TGFβ receptor 2 (TGFβR2).

[0028] In one embodiment, the miRNA sequence may be flanked in the 5' and / or 3' directions by flanking and loop sequences derived from a natural miRNA, preferably mouse miR-155. These flanking and loop sequences are necessary to excise the miRNA sequence from the larger sequence of the second RNA molecule, as known in miRNA technology. In this embodiment, the miRNA is preferably an artificial miRNA, specifically an miRNA designed to perfectly bind to the target mRNA. In one embodiment, the miRNA sequence may be at least one of miR-30 and miR-124.

[0029] In one embodiment, the miRNA sequence may be at least one miRNA sequence of the miR-302 / 367 cluster. Preferably, the miRNA sequence is all miRNA sequences of the miR-302 / 367 cluster. Preferably, the miRNA sequence is the miR-302 / 367 cluster.

[0030] In one embodiment, the ORF may be flanked by a 5' untranslated region (UTR) and / or a 3' UTR.

[0031] Exemplary 5' UTR sequences are set forth in SEQ ID NOs: 47, 48, and 52. In one embodiment, 5' UTR sequences useful in the RNA molecules described herein are sequences that are at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% homologous to SEQ ID NO: 47, 48, or 52. Exemplary 3' UTR sequences are set forth in SEQ ID NOs: 49, 50, and 51. In one embodiment, 3' UTR sequences useful in the RNA molecules described herein are sequences that are at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% homologous to SEQ ID NO: 49, 50, or 51.

[0032] In some embodiments, the protein of interest may be a reporter protein, preferably GFP or a variant thereof. In some embodiments, the protein of interest may be a pharmacologically active peptide or protein, a pluripotency factor or differentiation factor, preferably a pluripotency factor. In some embodiments, the protein of interest may be an antigen or an epitope thereof, preferably a T cell epitope. In some embodiments, the protein of interest is a polyepitopic protein containing multiple antigenic epitopes. In some embodiments, the protein of interest comprises a signal sequence for extracellular expression and / or a sequence that enhances the expression or presentation of the protein, e.g., epitope, on the surface of a cell, such as an antigen-presenting cell. In some embodiments, the protein of interest further comprises an MHC class I transport signal (MITD) and / or an HLA-II helper epitope, e.g., the P2P16 amino acid sequence from tetanus toxoid of Clostridium tetanii. An exemplary MITD sequence is set forth in SEQ ID NO: 44. In one embodiment, MITD sequences useful in the RNA molecules described herein are sequences that are at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% homologous to SEQ ID NO: 44. An exemplary P2P16 sequence is set forth in SEQ ID NO: 45. In one embodiment, P2P16 sequences useful in the RNA molecules described herein are sequences that are at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% homologous to SEQ ID NO: 45.

[0033] In one embodiment, the antigen or epitope is or is derived from a bacterial, viral, parasitic, or fungal antigen. In one embodiment, the antigen or epitope is or is derived from a tumor antigen. Tumor antigens may be overexpressed in tumors, or preferably are expressed only in tumors / tumor tissue.

[0034] In one embodiment, the protein of interest can be an immune evasion protein of vaccinia virus (eg, E3 or B18).

[0035] In one embodiment, the miRNA sequence can be located anywhere within the second RNA molecule as long as its insertion does not inhibit translation or replication of the second RNA molecule. In one embodiment, the miRNA is not located in the 5' or 3' replication recognition sequence of the second RNA molecule. In one embodiment, the miRNA is not located within the ORF of the second RNA molecule. In one embodiment, the miRNA is not located in the poly(A) sequence of the second RNA molecule. In one embodiment, the miRNA sequence can be located in the 5' untranslated region (UTR) or 3' untranslated region (UTR) of the ORF of the second RNA molecule. In one embodiment, the miRNA sequence can be located in the 3' untranslated region (UTR) of the ORF of the second RNA molecule. In one embodiment, the 5' end of the miRNA sequence can be linked to the ORF by a linker sequence. In one embodiment, the 3' end of the miRNA sequence can be linked to the 3' UTR of the second RNA molecule by a linker sequence. In one embodiment, each miRNA sequence can be linked by a linker sequence. In one embodiment, each linker sequence comprises at least one cleavage site that can be cleaved when the second replicable RNA molecule is present in a cell. In one embodiment, the linker sequence can comprise 5 to 30 nucleotides.

[0036] In one embodiment, the first and / or second RNA molecule may be a modified RNA molecule or an unmodified RNA molecule. Preferably, the first and / or second RNA molecule is a modified RNA molecule.

[0037] In one embodiment, the first and / or second RNA molecule may be a modified RNA molecule containing at least one modified uridine. Preferably, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the uridines in the RNA molecule are pseudouridine (ψ), N1-methylpseudouridine (m1ψ), or 5-methyluridine (m5U), preferably N1-methylpseudouridine (m5U).

[0038] In one embodiment, the first and / or second RNA molecule can further comprise a 5' cap, a 5' regulatory region, a 5' replication recognition sequence, a 3' replication recognition sequence, and / or a poly(A) sequence. In one embodiment, the first and / or second RNA molecule can comprise a 5' cap that is a naturally occurring 5' cap or a 5' cap analog. In one embodiment, the 5' cap analog can be any one of ARCA, β-S-ARCA, β-S-ARCA(D1), β-S-ARCA(D2), CleanCap, Cap0, Cap1, or AU(Cap1).

[0039] In one embodiment, when at least one uridine in the first and / or second RNA molecule is a modified uridine and the RNA molecule includes a 5' cap, the 5' cap has the sequence NpppNU, where U in the 5' cap is an unmodified uridine. Preferably, the 5' cap has the sequence NpppAU, where U in the 5' cap is an unmodified uridine, and A can be a modified or unmodified adenosine nucleotide.

[0040] In one embodiment, the first and / or second RNA molecule comprises a 5' cap comprising Cap1 and a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA molecule, wherein: (i) Cap1 is m 7 G(5')ppp(5')(2'OMeN1)pN2, where N1 is the +1 position of the RNA molecule and N2 is the +2 position of the RNA molecule, and N1 and N2 are each independently selected from A, C, G, or U; and (ii) Cap-proximal sequences are N1 and N2 of Cap1, and: (a) a sequence selected from the group consisting of A3A4X5, C3A4X5, A3C4A5, and A3U4G5; or (b) Sequence containing X3Y4X5 wherein X3 or X5 is each independently selected from A, G, C, or U; and Y4 is not C.

[0041] In one embodiment, the first and / or second RNA molecule can comprise a modified 5' regulatory region of a self-replicating RNA virus, wherein the modified regulatory region comprises a point mutation at one or more of positions 67, 244, 245, 246, and 248 of the 5' regulatory region (SEQ ID NO: 1). Preferably, the self-replicating RNA virus is an alphavirus. Also preferably, the 5' regulatory region further comprises a point mutation at position 4 of the 5' regulatory region (SEQ ID NO: 1). Most preferably, the point mutation is G4A, A67C, G244A, C245A, G246A, or C248A.

[0042] In one embodiment, the first and / or second RNA molecule can comprise a 5' replication recognition sequence, characterized in that at least one start codon has been removed compared to the native 5' replication recognition sequence. In one embodiment, the 5' replication recognition sequence comprises a sequence homologous to an open reading frame or portion thereof of a nonstructural protein from an autonomously replicating virus, characterized in that the sequence homologous to an open reading frame or portion thereof of a nonstructural protein from an autonomously replicating virus has at least one start codon removed compared to the native viral sequence. In one embodiment, the sequence homologous to an open reading frame or portion thereof of a nonstructural protein from an autonomously replicating virus has at least one natural start codon of the open reading frame of the nonstructural protein from the autonomously replicating virus removed. In one embodiment, the sequence homologous to an open reading frame or portion thereof of a nonstructural protein from an autonomously replicating virus has at least one start codon removed other than the natural start codon of the open reading frame of the nonstructural protein from the autonomously replicating virus removed. In one embodiment, the sequence homologous to an open reading frame of a nonstructural protein from an autonomously replicating virus has no start codon. The sequence homologous to the nonstructural protein open reading frame or portion thereof further contains at least one nucleotide change that compensates for the disruption of nucleotide pairing within at least one stem-loop introduced by the removal of at least one start codon.

[0043] In one embodiment, the first and / or second RNA molecule can comprise a 3' replication recognition sequence. In one embodiment, the 5' replication recognition sequence and / or the 3' replication recognition sequence can be derived from a self-replicating virus, preferably from the same self-replicating virus species. In one embodiment, the 5' replication recognition sequence and / or the 3' replication recognition sequence can be derived from a self-replicating single-stranded RNA virus, such as a positive-sense single-stranded RNA virus (e.g., alphavirus, flavivirus, etc.), preferably from the same self-replicating virus species.

[0044] In one embodiment, the first and / or second RNA molecule may comprise an interrupted poly(A) sequence.

[0045] An exemplary poly(A) sequence is shown in SEQ ID NO: 42. In one embodiment, poly(A) sequences useful in the RNA molecules described herein are those that have at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% homology to SEQ ID NO: 42.

[0046] In one embodiment, the first and / or second RNA molecule does not comprise an open reading frame for an intact viral structural protein.

[0047] In one embodiment, the system may further include a third or more replicable RNA molecules that can be replicated by the replicase encoded by the first RNA molecule. All embodiments described herein relating to the second RNA molecule may also apply to the third or more replicable RNA molecules.

[0048] In one embodiment, the system may further include a reagent capable of forming particles with the RNA molecule. In one embodiment, the reagent is or can include a polyalkyleneimine or a lipid. In one embodiment, the reagent is or can include a lipid, preferably the lipid including a cationic head group. In one embodiment, the reagent is or can include a pH-responsive lipid. In one embodiment, the reagent is or can include a PEGylated lipid. In one embodiment, the reagent can be bound to polysarcosine (pSar), poly(oxazoline) (POX); poly(oxazine) (POZ), poly(vinylpyrrolidone) (PVP); poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA); poly(dehydroalanine) (pDha); poly(aminoethoxyethoxyacetic acid) (pAEEA), or poly(2-methylaminoethoxyethoxyacetic acid) (pmAEEA). Thus, the reagent may be or may include a "grafted" or "stealth" lipid, i.e., a lipid bound to a polymer selected from the group consisting of polyethylene glycol (PEG); poly(aminoethoxyethoxyacetic acid) (pAEEA); polysarcosine (pSar), poly(2-methylaminoethoxyethoxyacetic acid) (pmAEEA), poly(oxazoline) (POX); poly(oxazine) (POZ), poly(vinylpyrrolidone) (PVP); poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA); and poly(dehydroalanine) (pDha). The reagent may be or may include a lipid bound to pAEEA or pSar. In some cases, the reagent does not include a lipid bound to PEG.

[0049] In one embodiment, the particle formed from the RNA molecule and the reagent can be a lipid nanoparticle (LNP), a lipoplex (LPX), a liposome, or a polymer-based polyplex (PLX).

[0050] In one embodiment, the particles may further comprise at least one phosphatidylserine.

[0051] In one embodiment, the particles may be nanoparticles, wherein: (i) the number of positive charges in the nanoparticles does not exceed the number of negative charges in the nanoparticles, and / or (ii) the nanoparticles have a neutral or net negative charge, and / or (iii) the charge ratio of positive to negative charges in the nanoparticles is 1.4:1 or less; and / or (iv) The zeta potential of the nanoparticles is 0 or less.

[0052] Preferably, the charge ratio of positive to negative charges in the nanoparticles is 1.4:1 to 1:8, preferably 1.2:1 to 1:4.

[0053] In one embodiment, nanoparticles can comprise at least one lipid, preferably at least one cationic lipid.In one embodiment, positive charge is provided by at least one cationic lipid, and negative charge is provided by RNA molecule.In one embodiment, nanoparticles can further comprise at least one helper lipid.Preferably, helper lipid is neutral lipid.

[0054] In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), and / or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP). In one embodiment, the at least one helper lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and / or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In one embodiment, the molar ratio of the at least one cationic lipid to the at least one helper lipid is 10:0 to 3:7, preferably 9:1 to 3:7, 4:1 to 1:2, 4:1 to 2:3, 7:3 to 1:1, or 2:1 to 1:1, preferably about 1:1.

[0055] In one embodiment, the nanoparticles are lipoplexes containing DODMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably 7:3 to 5:5, with a charge ratio of positive charges on DODMA to negative charges on RNA of 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2. In one embodiment, the nanoparticles are lipoplexes containing DODMA and cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and even more preferably 7:3 to 5:5, with a charge ratio of positive charges on DODMA to negative charges on RNA of 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2. In one embodiment, the nanoparticles are lipoplexes containing DODMA and DSPC in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably 7:3 to 5:5, and the charge ratio of the positive charges of DODMA to the negative charges of RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2. In one embodiment, the nanoparticles are lipoplexes containing DODMA:cholesterol:DOPE:PEGcerC16 in a molar ratio of 40:48:10:2.

[0056] In one embodiment, the nanoparticles are lipoplexes containing DOTMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably 7:3 to 5:5, with a charge ratio of positive charges on DOTMA to negative charges on RNA of 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2. In one embodiment, the nanoparticles are lipoplexes containing DOTMA and cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and even more preferably 7:3 to 5:5, with a charge ratio of positive charges on DOTMA to negative charges on RNA of 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2.

[0057] In one embodiment, the nanoparticles are lipoplexes containing DOTAP and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, and more preferably 7:3 to 5:5, and the charge ratio of the positive charges of DOTAP to the negative charges of RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:2.

[0058] In one embodiment, the reagent can include a lipid, and the particles formed are LNPs that are complexed with and / or encapsulate nucleic acid molecules (e.g., RNA molecules). In one embodiment, the reagent can include a lipid, and the particles formed are vesicles, optionally unilamellar liposomes, that encapsulate nucleic acid molecules (e.g., RNA molecules). In one embodiment, the composition comprising nucleic acid molecules is an LNP composition, such as an RNA-LNP composition. The reagent capable of forming particles with nucleic acid molecules can be or can include cationic ionizable lipids, neutral (e.g., helper) lipids, steroids (e.g., cholesterol), and polymer-bound lipids.

[0059] In one embodiment, the reagent may be or may include a polyalkyleneimine.

[0060] In one embodiment, the molar ratio (N:P ratio) of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphorus atoms (P) in the RNA molecule can be 2.0 to 15.0, preferably 6.0 to 12.0. In one embodiment, the molar ratio (N:P ratio) of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphorus atoms (P) in the RNA molecule can be at least about 48, and optionally about 48 to 300, about 60 to 200, or about 80 to 150.

[0061] In one embodiment, the ionic strength of the composition may be 50 mM or less, preferably with a monovalent cation concentration of 25 mM or less and a divalent cation concentration of 20 μM or less.

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

[0063] In one embodiment, the polyalkyleneimine has the following general formula (I): [ka] [In the formula, R is H, an acyl group, or a group represented by the following general formula (II): [ka] is a group comprising During the ceremony, R1 is H or the following general formula (III): [ka] is a group comprising n, m, and l are independently selected from integers from 2 to 10; and p, q, and r are integers, and the sum of p, q, and r is such that the average molecular weight of the polymer is 1.5 × 10 2 ~10 7 Da, preferably 5000 to 10 5 Da, more preferably 10,000 to 40,000 Da, even more preferably 15,000 to 30,000 Da, and even more preferably 20,000 to 25,000 Da. Preferably, n, m, and l are independently selected from 2, 3, 4, and 5, preferably 2 and 3. Preferably, R1 is H. Preferably, R is H or an acyl group. Includes:

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

[0065] In one embodiment, the system may further comprise one or more peptide-based adjuvants, which optionally include immunomodulatory molecules such as cytokines, lymphokines and / or costimulatory molecules.

[0066] In one embodiment, the system can further include one or more additives, optionally selected from the group consisting of buffers, sugars, stabilizers, cryoprotectants, cryoprotectants, and chelating agents. Preferably, the buffers include at least one selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), acetic acid, acetate buffers and analogs, phosphoric acid and phosphate buffers, and citric acid and citrate buffers. Preferably, the sugars include at least one selected from the group consisting of monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides, and preferably include glucose, trehalose, and sucrose. Preferably, the cryoprotectant includes at least one selected from the group consisting of glycols, such as ethylene glycol, propylene glycol, and glycerol. Preferably, the chelating agent includes EDTA.

[0067] The present invention also provides a kit comprising two RNA molecules, wherein the first RNA molecule comprises an open reading frame encoding a functional RNA-dependent RNA polymerase (replicase), and the second RNA molecule is a replicable RNA molecule comprising at least one miRNA sequence, which, when present in a cell, can be excised from the second replicable RNA and can regulate gene expression in the cell, and the replicable RNA molecule can be replicated in cis or trans by the replicase encoded by the first RNA molecule. In one embodiment, the two RNA molecules are contained in separate containers within the kit.

[0068] The present invention also provides a pharmaceutical composition comprising two RNA molecules, wherein a first RNA molecule comprises an open reading frame encoding a functional RNA-dependent RNA polymerase (replicase), and a second RNA molecule is a replicable RNA molecule comprising at least one miRNA sequence, which, when present in a cell, can be excised from the second replicable RNA and can regulate gene expression in the cell, and which replicable RNA molecule can be replicated in trans by the replicase encoded by the first RNA molecule, and a pharmaceutically acceptable carrier.

[0069] In one embodiment, the first and / or second RNA molecule in the composition, preferably the second RNA molecule, further comprises at least one open reading frame (ORF) encoding a protein of interest.

[0070] In one embodiment, the pharmaceutical composition can be formulated for intradermal, subcutaneous, and / or intramuscular administration, such as by injection. In one embodiment, the kit or pharmaceutical composition can be used for treatment. In one embodiment, the kit or pharmaceutical composition can be used in a method for treating or preventing a disease, preferably wherein the subject is a mammal, more preferably wherein the mammal is a human, and the method comprises administering a pharmaceutical composition according to the present invention to the subject. Preferably, administration of the kit or pharmaceutical composition comprises intradermal, subcutaneous, or intramuscular administration, such as intradermal, subcutaneous, or intramuscular injection. The injection uses a needle or a needleless injection device. Preferably, administration comprises administration by intramuscular injection, preferably using a needle. In one embodiment, the RNA molecules are administered separately, preferably by the same administration route.

[0071] In one embodiment, the disease is a bacterial, viral, parasitic, fungal infection, or cancer. The subject is preferably a human.

[0072] The present invention also provides a method for treating or preventing a bacterial, viral, parasitic, or fungal infection in a subject, comprising administering to the subject a composition, preferably a pharmaceutical composition, described herein.The present invention also provides a method for treating or preventing cancer in a subject, comprising administering to the subject a composition, preferably a pharmaceutical composition, described herein.

[0073] The present invention also provides a first RNA molecule and a second RNA molecule for use in treatment.The first RNA molecule comprises an open reading frame encoding a functional RNA-dependent RNA polymerase (replicase), and the second RNA molecule is a replicable RNA molecule comprising at least one miRNA sequence, which can be excised from the second replicable RNA when present in a cell and can regulate gene expression in the cell, and the replicable RNA molecule can be replicated in trans by the replicase encoded by the first RNA molecule.Preferably, the treatment is the treatment or prevention of cancer or infectious disease.

[0074] Detailed Description Although the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described herein, as they may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0075] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)," H.G. W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0076] The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology as described in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0077] The elements of the present invention are described below. While these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. The description should be understood to disclose and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered disclosed by the description, unless the context dictates otherwise.

[0078] The term "about" means approximately or near, and in the context of numerical values ​​or ranges described herein, preferably means ±10% of the stated or claimed numerical value or range.

[0079] As used in the context of describing the present invention (particularly in the context of the claims), the terms "a," "an," "the," and similar references are intended to include both the singular and the plural unless otherwise stated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended as a shorthand method of individually referring to each individual value falling within the range. If otherwise stated herein, each individual value is incorporated herein as if individually stated herein. All methods described herein can be performed in any suitable order unless otherwise stated herein or clearly contradicted by context. The use of any examples or exemplary language (e.g., "etc.") described herein is intended only to better illustrate the present invention and does not limit the scope of the present invention as set forth in the claims. No language in the specification should be construed as indicating any element essential to the practice of the invention not recited in the claims.

[0080] Unless otherwise indicated, the term "comprising" is used herein to indicate that additional members may optionally be present in addition to the members of the list introduced by "comprising." However, for specific embodiments of the present invention, it is envisioned that the term "comprising" encompasses the possibility that additional members are not present. That is, for the purposes of this embodiment, "comprising" is understood to have the meaning of "consisting of."

[0081] The indication of a relative amount of a component characterized by a generic term is meant to refer to the total amount of all specific variants or members encompassed by that generic term. When a specific component defined by a generic term is specified to be present in a specific relative amount, and this component is further characterized as a specific variant or member encompassed by that generic term, it means that no other variants or members encompassed by that generic term are additionally present, such that the total relative amount of components encompassed by that generic term does not exceed the specified relative amount. More preferably, no other variants or members encompassed by that generic term are present at all.

[0082] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure.

[0083] As used herein, terms such as "reduce" or "inhibit" refer to the ability to reduce levels overall, preferably by 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more. The term "inhibit" or similar phrases includes complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0084] Terms such as "increase" or "enhancement" preferably relate to an increase or enhancement of about 10% or more, preferably 20% or more, preferably 30% or more, more preferably 40% or more, more preferably 50% or more, even more preferably 80% or more, and most preferably 100% or more.

[0085] The term "net charge" refers to the overall charge of an object, such as a compound or particle.

[0086] Ions that have an overall net positive charge are cations, and ions that have an overall negative charge are anions. Thus, according to the present invention, anions are ions that have more electrons than protons and have a net negative charge, and cations are ions that have fewer electrons than protons and have a net positive charge.

[0087] The terms "charged," "net charge," "negatively charged," or "positively charged," with respect to a particular compound or particle, refer to the net charge of the particular compound or particle when dissolved or suspended in water at a pH of 7.0.

[0088] The term "nucleic acid" as used herein also includes chemical derivatives of nucleic acids based on nucleotide bases, sugars, or phosphates, as well as nucleic acids containing non-natural nucleotides and nucleotide analogs. In some embodiments, the nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Generally, a nucleic acid molecule or nucleic acid sequence refers to a nucleic acid, preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). According to the present invention, nucleic acids include genomic DNA, cDNA, mRNA, viral RNA, recombinantly prepared molecules, and chemically synthesized molecules. According to the present invention, nucleic acids can be in the form of single-stranded or double-stranded linear or covalently closed circular molecules.

[0089] According to the present invention, a "nucleic acid sequence" refers to the sequence of nucleotides in a nucleic acid, such as ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). The term can refer to an entire nucleic acid molecule (e.g., a single strand of an entire nucleic acid molecule) or a portion thereof (e.g., a fragment).

[0090] According to the present invention, the term "RNA" or "RNA molecule" refers to a molecule containing ribonucleotide residues, preferably consisting entirely or substantially of ribonucleotide residues. The term "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. The term "RNA" includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially or completely purified RNA, essentially pure RNA, synthetic RNA, and recombinantly produced RNA (e.g., modified RNA that differs from natural RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides). Such modifications can include the addition of non-nucleotide material to the end of the RNA or within the RNA, for example, to one or more nucleotides of the RNA. Nucleotides in an RNA molecule can also include non-natural nucleotides or non-standard nucleotides such as chemically synthesized nucleotides or deoxynucleotides. These modified RNAs are sometimes referred to as analogs, particularly analogs of natural RNA.

[0091] According to the present invention, RNA may be single-stranded or double-stranded. In some embodiments of the present invention, single-stranded RNA is preferred. The term "single-stranded RNA" generally refers to an RNA molecule that is not associated with a complementary nucleic acid molecule (typically a complementary RNA molecule). Single-stranded RNA can contain self-complementary sequences that allow portions of the RNA to fold and form secondary structural motifs, including, but not limited to, base pairs, stems, stem-loops, and bulges. Single-stranded RNA can exist as a minus strand ((-) strand) or a plus strand ((+) strand). The (+) strand is the strand that contains or encodes genetic information. Genetic information can be, for example, a polynucleotide sequence encoding a protein. When the (+) strand RNA encodes a protein, the (+) strand can directly serve as a template for translation (protein synthesis). The (-) strand is the complementary strand of the (+) strand. In the case of double-stranded RNA, the (+) strand and the (-) strand are two separate RNA molecules, and these two RNA molecules associate with each other to form double-stranded RNA ("duplex RNA").

[0092] The term "stability" of RNA relates to the "half-life" of RNA. "Half-life" is the time required for half of the activity, amount, or number of a molecule to disappear. In the present invention, the half-life of RNA is an indicator of the stability of the RNA. The half-life of RNA may affect the "expression period" of RNA. RNA with a longer half-life can be expected to be expressed for a longer period.

[0093] The term "translation efficiency" relates to the amount of translation product produced by an RNA molecule in a specific period of time.

[0094] With respect to a nucleic acid sequence, the term "fragment" refers to a portion of the nucleic acid sequence, i.e., a sequence representing a nucleic acid sequence truncated at the 5' and / or 3' end. Preferably, a fragment of a nucleic acid sequence comprises at least 80%, preferably at least 90%, 95%, 96%, 97%, 98%, or 99% of the nucleotide residues from said nucleic acid sequence. In the present invention, fragments of RNA molecules that maintain RNA stability and / or translation efficiency are preferred.

[0095] The term "fragment" in reference to an amino acid sequence (peptide or protein) refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. A C-terminally truncated fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. An N-terminally truncated fragment (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains an initiation codon useful for initiating translation. A fragment of an amino acid sequence can, for example, contain 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%, or at least 90% of the amino acid residues from the amino acid sequence.

[0096] In the present invention, the term "variant" with respect to, for example, nucleic acid and amino acid sequences, includes all variants, particularly mutants, virus strain variants, splice variants, conformations, isoforms, allelic variants, species variants, and species homologs, especially those that occur naturally. Allelic variants are associated with changes in the normal sequence of a gene, the significance of which is often unclear. Complete gene sequencing often identifies multiple allelic variants for a particular gene. With respect to nucleic acid molecules, the term "variant" includes degenerate nucleic acid sequences, which in the present invention are nucleic acids that differ in codon sequence from a reference nucleic acid due to the degeneracy of the genetic code. A species homolog is a nucleic acid or amino acid sequence originating from a different species than the specified nucleic acid or amino acid sequence. A viral homolog is a nucleic acid or amino acid sequence of a different viral origin than the specified nucleic acid or amino acid sequence.

[0097] Nucleic acid variants include deletions, additions, mutations, substitutions, and / or insertions of single or multiple nucleotides compared to a reference nucleic acid. Deletions include the removal of one or more nucleotides from the reference nucleic acid. Addition variants include 5'- and / or 3'-terminal fusions of one or more nucleotides, such as 1, 2, 3, 5, 10, 20, 30, 50, or more nucleotides. Substitutions involve the removal of at least one nucleotide in a sequence and the insertion of at least one other nucleotide (e.g., translocations and transitions). Mutations include abasic sites, cross-linked sites, and chemically altered or modified bases. Insertions include the addition of at least one nucleotide to the reference nucleic acid.

[0098] According to the present invention, a "nucleotide change" can refer to a deletion, addition, mutation, substitution, and / or insertion of a single or multiple nucleotides compared to a reference nucleic acid. In some embodiments, a "nucleotide change" is selected from the group consisting of a single nucleotide deletion, a single nucleotide addition, a single nucleotide mutation, a single nucleotide substitution, and / or a single nucleotide insertion compared to a reference nucleic acid. According to the present invention, a nucleic acid variant can contain one or more nucleotide changes compared to a reference nucleic acid.

[0099] A variant of a specific nucleic acid sequence preferably has at least one functional property of the specific sequence, and is preferably functionally equivalent to the specific sequence, e.g., a nucleic acid sequence that exhibits properties identical or similar to those of the specific nucleic acid sequence.

[0100] As described below, some embodiments of the present invention feature, inter alia, nucleic acid sequences that have homology to other nucleic acid sequences. These homologous sequences are variants of the other nucleic acid sequences.

[0101] Preferably, the degree of identity between a given nucleic acid sequence and a nucleic acid sequence that is a variant of the given nucleic acid sequence, or between a given amino acid sequence of a protein and an amino acid sequence that is a variant of the given amino acid sequence, is at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, or most preferably at least 95%, 96%, 97%, 98%, or 99%. The degree of identity is preferably shown over a region of at least about 30, at least about 50, at least about 70, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, or at least about 400 nucleotides. In a preferred embodiment, the degree of identity is shown over the entire length of the reference nucleic acid sequence.

[0102] "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid or nucleic acid sequences refers to the percentage of amino acids or nucleotides that are identical between the sequences.

[0103] The term "% identity" is intended to refer in particular to the percentage of amino acids or nucleotides that are identical in optimal alignment between two sequences being compared, said percentage being purely statistical; the differences between the two sequences may be randomly distributed over the entire length of the sequences, and the compared sequence may contain additions or deletions relative to the reference sequence in order to achieve optimal alignment between the two sequences. Comparison of two sequences is usually performed by comparing the sequences over segments or "comparison windows" in order to identify local regions of corresponding sequences after optimal alignment. Optimal alignment for comparison can be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2:482, or the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, and the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, or computer programs using the above algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0104] The percentage identity is obtained by determining the number of identical positions where the compared sequences correspond, dividing this number by the number of positions compared, and multiplying the result by 100.

[0105] For example, the BLAST program "BLAST 2 sequences" available at the website http: / / www.ncbi.nlm.nih.gov / blast / bl2seq / wblast2.cgi can be used.

[0106] A nucleic acid is "capable of hybridizing" or "hybridizes" to another nucleic acid if the two sequences are complementary to each other. A nucleic acid is "complementary" to another nucleic acid if the two sequences are capable of forming a stable duplex with each other. According to the present invention, hybridization is preferably performed under conditions that allow specific hybridization between polynucleotides (stringent conditions). Stringent conditions are described, for example, in *Molecular Cloning: A Laboratory Manual*, J. Sambrook et al., Editors, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or *Current Protocols in Molecular Biology*, F. M. Ausubel et al., Editors, John Wiley & Sons, Inc., New York, referring to hybridization in a hybridization buffer (3.5xSSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaHPO (pH 7), 0.5% SDS, 2 mM EDTA) at 65°C. SSC is 0.15 M sodium chloride / 0.15 M sodium citrate, pH 7. After hybridization, the membrane onto which the DNA has been transferred is washed, for example, with 2xSSC at room temperature, and then with 0.1-0.5xSSC / 0.1xSDS at temperatures up to 68°C.

[0107] Percent complementarity refers to the percentage of consecutive residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). "Fully complementary" or "fully complementary" means that all consecutive residues in a nucleic acid sequence will hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence. Preferably, the degree of complementarity according to the present invention is at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%, 96%, 97%, 98%, or 99%. Most preferably, the degree of complementarity according to the present invention is 100%.

[0108] The term "derivative" includes chemical derivatization of nucleic acids with nucleotide bases, sugars, or phosphates. The term "derivative" also includes nucleic acids containing non-naturally occurring nucleotides and nucleotide analogs. Preferably, derivatization of a nucleic acid increases its stability.

[0109] 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 variant with respect to a particular sequence maintains the stability and / or translation efficiency of the RNA when it replaces the particular sequence in an RNA molecule.

[0110] "nt" is an abbreviation for a nucleotide, or nucleotides, preferably consecutive nucleotides in a nucleic acid molecule.

[0111] According to the present invention, the term "codon" refers to a triplet of bases in a coding nucleic acid that specifies which amino acid is added next during protein synthesis in the ribosome.

[0112] The terms "transcription" and "transcribe" refer to the process in which a nucleic acid molecule ("nucleic acid template") having a specific nucleic acid sequence is read by an RNA polymerase, which generates a single-stranded RNA molecule. During transcription, the genetic information in the nucleic acid template is transcribed. The nucleic acid template may be DNA, but in the case of transcription from an alphavirus nucleic acid template, for example, the template is typically RNA. The transcribed RNA can then be translated into protein. According to the present invention, the term "transcription" includes "in vitro transcription," which refers to a process in which RNA, particularly mRNA, is synthesized in vitro in a cell-free system. Preferably, a cloning vector is used to generate the transcript. These cloning vectors are commonly called transcription vectors and are encompassed by the term "vector" in the present invention. The cloning vector is preferably a plasmid. According to the present invention, the RNA is preferably in vitro transcribed RNA (IVT-RNA), which can be obtained by in vitro transcription of an appropriate DNA template. The promoter for 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 a cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0113] The single-stranded nucleic acid molecule produced during transcription typically has a nucleic acid sequence that is the complement of the template.

[0114] According to the present invention, the term "template" or "nucleic acid template" or "template nucleic acid" generally refers to a nucleic acid sequence that can be replicated or transcribed.

[0115] "A nucleic acid sequence transcribed from a nucleic acid sequence" and similar terms refer, where appropriate, to a nucleic acid sequence as part of an entire RNA molecule that is the product of transcription of a template nucleic acid sequence. Typically, the transcribed nucleic acid sequence is a single-stranded RNA molecule.

[0116] In the present invention, the "3' end of a nucleic acid" refers to the end having a free hydroxyl group. In a diagrammatic representation of a double-stranded nucleic acid, particularly DNA, the 3' end is always located on the right side. In the present invention, the "5' end of a nucleic acid" refers to the end having a free phosphate group. In a diagrammatic representation of a double-stranded nucleic acid, particularly DNA, the 5' end is always located on the left side. [ka]

[0117] "Upstream" refers to the relative location of a first element of a nucleic acid molecule relative to a second element of the nucleic acid molecule, where both elements are contained in the same nucleic acid molecule and the first element is closer to the 5' end of the nucleic acid molecule than the second element of the nucleic acid molecule. In this case, the second element is said to be "downstream" of the first element of the nucleic acid molecule. An element located "upstream" of a second element can equivalently be said to be located "5'" of the second element. In the case of double-stranded nucleic acid molecules, designations such as "upstream" and "downstream" are used relative to the (+) strand.

[0118] According to the present invention, "operably linked" or "operably linked" refers to a connection in a functional relationship. A nucleic acid is said to be "operably linked" when it is functionally associated with another nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription of the coding sequence. Functionally linked nucleic acids are typically adjacent to each other, optionally separated by additional nucleic acid sequences, and in certain embodiments, are transcribed by RNA polymerase into a single RNA molecule (common transcript).

[0119] In a particular embodiment, a nucleic acid according to the present invention is operably linked to an expression control sequence, which may be homologous or heterologous to the nucleic acid.

[0120] The term "expression control sequence," according to the present invention, includes promoters, ribosomal binding sequences, and other control elements that control the transcription of a gene or the translation of a derived RNA. In certain embodiments of the present invention, expression control sequences can be regulated. The exact structure of an expression control sequence can vary depending on the species or cell type, but typically includes 5' non-transcribed sequences, involved in the initiation of transcription and translation, respectively, as well as 5' and 3' non-translated sequences. More specifically, 5' non-transcribed expression control sequences include a promoter region containing a promoter sequence for transcriptional control of an operably linked gene. Expression control sequences may also include enhancer sequences and upstream activating sequences. Expression control sequences for DNA molecules typically include 5' non-transcribed sequences, such as a TATA box, capping sequence, or CAAT sequence, as well as 5' and 3' non-translated sequences. Expression control sequences for alphavirus RNAs may include a subgenomic promoter and / or one or more conserved sequence elements. A specific expression control sequence according to the present invention is the subgenomic promoter of an alphavirus described herein.

[0121] The nucleic acid sequences described herein, in particular the transcribable and coding nucleic acid sequences, can be combined with any expression control sequence, in particular a promoter, which can be homologous or heterologous to said nucleic acid sequence, the term "homologous" indicating that the nucleic acid sequence is also naturally operably linked to an expression control sequence, and the term "heterologous" indicating that the nucleic acid sequence is not naturally operably linked to an expression control sequence.

[0122] A transcribable nucleic acid sequence, particularly a nucleic acid sequence encoding a peptide or protein, and an expression control sequence are "operably" linked to each other when they are covalently linked to each other such that transcription or expression of the transcribable nucleic acid sequence, particularly the coding nucleic acid sequence, is under the control or influence of the expression control sequence. When a nucleic acid sequence is to be translated into a functional peptide or protein, induction of an expression control sequence operably linked to a coding sequence results in transcription of the coding sequence without causing frameshifting of the coding sequence or preventing the coding sequence from being translated into the desired peptide or protein.

[0123] The term "promoter" or "promoter region" refers to a nucleic acid sequence that controls the synthesis of a transcript (e.g., a transcript containing a coding sequence) by providing recognition and binding sites for RNA polymerase. A promoter region may further include recognition or binding sites for additional factors involved in regulating the transcription of that gene. A promoter may control the transcription of a prokaryotic or eukaryotic gene. A promoter may be "inducible," meaning that it initiates transcription in response to an inducer, or "constitutive," meaning that transcription is not controlled by an inducer. An inducible promoter is expressed very little or not at all in the absence of an inducer. In the presence of an inducer, the gene is "switched on," or transcription levels increase, usually mediated by the binding of specific transcription factors. A particular promoter according to the present invention is, for example, an alphavirus subgenomic promoter, as described herein. An exemplary subgenomic promoter is set forth in SEQ ID NO: 46. In one embodiment, subgenomic promoters useful in the RNA molecules described herein are those that share at least 85%, 90%, 95%, 98%, or 99% homology with SEQ ID NO: 46. Other exemplary promoters include, for example, alphavirus genomic positive- or negative-strand promoters.

[0124] The term "core promoter" refers to the nucleic acid sequence that constitutes a promoter. A core promoter is typically the minimal portion of a promoter required to properly initiate transcription. A core promoter typically includes a transcription initiation site and an RNA polymerase binding site.

[0125] "Polymerase" generally refers to a molecular entity capable of catalyzing the synthesis of a polymer molecule from monomer building blocks. "RNA polymerase" is a molecular entity capable of catalyzing the synthesis of an RNA molecule from ribonucleotide building blocks. "DNA polymerase" is a molecular entity capable of catalyzing the synthesis of a DNA molecule from deoxyribonucleotide building blocks. In the case of DNA and RNA polymerases, the molecular entities are typically proteins or assemblies or complexes of multiple proteins. DNA polymerases typically synthesize DNA molecules based on a template nucleic acid (typically a DNA molecule). RNA polymerases typically synthesize RNA molecules based on a template nucleic acid (either a DNA molecule (in which case the RNA polymerase is a DNA-dependent RNA polymerase, DdRP)) or an RNA molecule (in which case the RNA polymerase is an RNA-dependent RNA polymerase, RdRP).

[0126] "RNA-dependent RNA polymerase" or "RdRP" is an enzyme that catalyzes the transcription of RNA from an RNA template. In the case of alphavirus RNA-dependent RNA polymerase, RNA replication occurs through the sequential synthesis of the (-) strand complementary strand of the genomic RNA and the (+) strand genomic RNA. Therefore, RNA-dependent RNA polymerase is synonymously referred to as "RNA replicase" or simply "replicase." In nature, RNA-dependent RNA polymerase is typically encoded by all RNA viruses except retroviruses. Representative viruses that encode RNA-dependent RNA polymerase are alphaviruses.

[0127] According to the present invention, "RNA replication" generally refers to an RNA molecule synthesized based on the nucleotide sequence of a predetermined RNA molecule (template RNA molecule). The synthesized RNA molecule can be, for example, identical to or complementary to the template RNA molecule. Generally, RNA replication can occur via the synthesis of a DNA intermediate or directly by RNA-dependent RNA replication mediated by RNA-dependent RNA polymerase (RdRP). In the case of alphaviruses, RNA replication is mediated by RNA-dependent RNA polymerase (RdRP) without a DNA intermediate. The template RNA strand (first RNA strand) or a portion thereof serves as a template for the synthesis of a second RNA strand complementary to the first RNA strand or a portion thereof. The second RNA strand or a portion thereof may optionally serve as a template for the synthesis of a third RNA strand complementary to the second RNA strand or a portion thereof. This results in the third RNA strand being identical to the first RNA strand or a portion thereof. Therefore, the RNA-dependent RNA polymerase can directly synthesize a complementary RNA strand of the template or indirectly synthesize an identical RNA strand via a complementary intermediate strand.

[0128] According to the present invention, the term "template RNA" refers to an RNA that can be transcribed or replicated by an RNA-dependent RNA polymerase.

[0129] According to the present invention, the term "gene" refers to a specific nucleic acid sequence that is responsible for producing one or more cellular products and / or performing one or more inter- or intracellular functions. More specifically, the term relates to a nucleic acid that encodes a specific protein, or a nucleic acid portion (typically DNA, or RNA in the case of RNA viruses) that comprises a functional or structural RNA molecule.

[0130] As used herein, an "isolated molecule" is intended to refer to a molecule that is substantially free of other molecules, such as other cellular material. In the present invention, the term "isolated nucleic acid" means that the nucleic acid has either been (i) amplified in a test tube, e.g., by polymerase chain reaction (PCR), (ii) produced recombinantly by cloning, (iii) purified, e.g., by cleavage and gel electrophoretic fractionation, or (iv) synthesized, e.g., by chemical synthesis. An isolated nucleic acid is a nucleic acid that is amenable to manipulation by recombinant techniques.

[0131] The term "vector" is used herein in its most general sense and includes, for example, any intermediate vehicle for a nucleic acid that allows the nucleic acid to be introduced into a prokaryotic and / or eukaryotic host cell and, if necessary, integrated into the genome. Such vectors are preferably replicated and / or expressed intracellularly. Vectors include plasmids, phagemids, viral genomes, and fractions thereof.

[0132] In the present invention, the term "recombinant" means "produced by genetic engineering." Preferably, a "recombinant entity," such as a recombinant cell, in the context of the present invention does not occur in nature.

[0133] The term "naturally occurring" used herein refers to the fact that an object is found in nature.For example, a peptide or nucleic acid that exists in living organisms (including viruses), can be isolated from natural sources, and has not been intentionally modified by humans in laboratories, is naturally occurring.The term "naturally occurring" means "existing in nature", and includes not only known objects, but also objects that have not yet been discovered and / or isolated from nature, but may be discovered and / or isolated from natural sources in the future.

[0134] According to the present invention, the term "expression" is used in its most general sense and includes the production of RNA and / or protein. It also includes partial expression of a nucleic acid. Furthermore, expression can be transient or stable. With respect to RNA, the terms "expression" or "translation" refer to the process by which a chain of coding RNA (e.g., messenger RNA) directs the assembly of an amino acid sequence in the ribosomes of a cell to produce a peptide or protein.

[0135] According to the present invention, the term "mRNA" refers to "messenger RNA" and refers to a transcript that encodes a peptide or protein. mRNA is translated to produce the encoded peptide or protein. In a broader sense, mRNA may also refer to transcripts that are not translated but still encode / provide functional nucleotide sequences, such as miRNA and other non-coding RNA species. Typically, mRNA contains a 5'-UTR, a protein-coding region, a 3'-UTR, and a poly(A) sequence. Replicable RNA molecules, such as self-amplifying RNA (saRNA), cis replicons, trans replicons (TR), and nano-trans replicons (NTR), can be considered types of mRNA, regardless of whether they are actually translated. mRNA can be generated from a DNA template by in vitro transcription. In vitro transcription techniques are known to those skilled in the art. For example, various in vitro transcription kits are commercially available. According to the present invention, mRNA can be modified by stabilizing modifications and capping.

[0136] According to the present invention, the term "poly(A) sequence" or "poly(A) tail" or "poly(A) structure" refers to a continuous or interrupted sequence of adenylate residues typically located at the 3' end of an RNA molecule. A continuous sequence is characterized by consecutive adenylate residues. Continuous poly(A) sequences are typical in nature. Poly(A) sequences are not usually encoded by eukaryotic DNA and are added to the free 3' end of RNA by template-independent RNA polymerase in the cell nucleus during eukaryotic transcription, but the present invention encompasses poly(A) sequences encoded by DNA. In a preferred embodiment, the RNA molecules described herein contain a continuous poly(A) sequence.

[0137] According to the present invention, with respect to a nucleic acid molecule, the term "primary structure" refers to the linear sequence of nucleotide monomers.

[0138] According to the present invention, the term "secondary structure" in reference to a nucleic acid molecule refers to the two-dimensional representation of the nucleic acid molecule that reflects base pairing, particularly in the case of a single-stranded RNA molecule, the intramolecular base pairing. Although each RNA molecule has only a single polynucleotide strand, the molecule is typically characterized by regions of (intramolecular) base pairing. According to the present invention, the term "secondary structure" includes structural motifs, including, but not limited to, base pairs, stems, stem-loops, bulges, and loops (e.g., internal loops and multi-branched loops). The secondary structure of a nucleic acid molecule can be represented by a two-dimensional diagram (planar graph) showing base pairing (for details on the secondary structure of RNA molecules, see Auber et al., 2006; J. Graph Algorithms Appl. 10:329-351). As described herein, the secondary structure of a particular RNA molecule is important in the context of the present invention.

[0139] According to the present invention, the secondary structure of nucleic acid molecules, particularly single-stranded RNA molecules, 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 present invention, the "secondary structure" of a nucleic acid molecule specifically refers to the secondary structure determined by the prediction. Predictions can also be performed or confirmed using MFOLD structure prediction (http: / / unafold.rna.albany.edu / ?q=mfold).

[0140] According to the present invention, a "base pair" is a structural motif in a secondary structure in which two nucleotide bases associate with each other through hydrogen bonds between the donor and acceptor sites on the bases. Complementary bases A:U and G:C form stable base pairs through hydrogen bonds between the donor and acceptor sites on the bases. The A:U and G:C base pair is called a Watson-Crick base pair. A weaker base pair (wobble base pair) is formed by G and U (G:U). The A:U and G:C base pair is called a canonical base pair. Other base pairs, such as G:U (relatively common in RNA) and other rare base pairs (e.g., A:C, U:U), are called non-canonical base pairs.

[0141] According to the present invention, "nucleotide pairing" refers to the association of two nucleotides with each other such that their bases form a base pair (canonical or non-canonical, preferably a canonical, most preferably a Watson-Crick base pair).

[0142] According to the present invention, the terms "stem-loop," "hairpin," and "hairpin loop" interchangeably refer to a specific secondary structure of a nucleic acid molecule, typically a single-stranded nucleic acid molecule, such as single-stranded RNA. The specific secondary structure represented by a stem-loop consists of a continuous nucleic acid sequence including a stem and a (terminal) loop (also called a hairpin loop), where the stem is formed by two adjacent fully or partially complementary sequence elements, separated by a short sequence (e.g., 3 to 10 nucleotides) that forms the loop of the stem-loop structure. The two adjacent fully or partially complementary sequences can be defined, for example, as stem-loop elements stem 1 and stem 2. A stem-loop is formed when these two adjacent fully or partially reverse-complementary sequences, for example, stem-loop elements stem 1 and stem 2, base-pair with each other, resulting in a double-stranded nucleic acid sequence containing an unpaired loop at its terminus formed by the short sequence located between stem-loop elements stem 1 and stem 2. Thus, a stem-loop comprises two stems (stem 1 and stem 2) that base-pair with each other at the secondary structure level of a nucleic acid molecule and are separated by a short sequence that is not part of stem 1 or stem 2 at the primary structure level of the nucleic acid molecule. For illustrative purposes, a two-dimensional representation of a stem-loop resembles a lollipop structure. Formation of the stem-loop structure requires the presence of a sequence that can fold back on itself to form a paired duplex. The paired duplex is formed by stem 1 and stem 2. The stability of a paired stem-loop element is typically determined by the number of nucleotides in stem 1 that can form base pairs (preferably canonical base pairs, more preferably Watson-Crick base pairs) with nucleotides in stem 2, i.e., their length, and the number of nucleotides in stem 1 that cannot form such base pairs with nucleotides in stem 2 (mismatches or bulges). According to the present invention, the optimal loop length is 3 to 10 nucleotides, more preferably 4 to 7 nucleotides, e.g., 4 nucleotides, 5 nucleotides, 6 nucleotides, or 7 nucleotides. If a given nucleic acid sequence is characterized by a stem-loop, then each complementary nucleic acid sequence will typically also be characterized by a stem-loop.Stem loops are typically formed by single-stranded RNA molecules, for example, the 5' replication recognition sequence of alphavirus genomic RNA has multiple stem loops.

[0143] According to the present invention, "disruption" or "disrupt" in relation to a specific secondary structure (e.g., stem loop) of a nucleic acid molecule means that the specific secondary structure does not exist or is changed.Typically, the secondary structure can be disrupted as a result of changing at least one nucleotide that is part of the secondary structure.For example, a stem loop can be disrupted by changing one or more nucleotides that form the stem, which can result in nucleotide pairing being impossible.

[0144] According to the present invention, "compensating for disruption of secondary structure" or "compensating for disruption of secondary structure" refers to one or more nucleotide changes in a nucleic acid sequence, more typically one or more second nucleotide changes in a nucleic acid sequence, which also contains one or more first nucleotide changes, and has the following characteristics: the one or more first nucleotide changes cause disruption of the secondary structure of the nucleic acid sequence in the absence of one or more second nucleotide changes, but the coexistence of one or more first nucleotide changes and one or more second nucleotide changes does not cause disruption of the secondary structure of the nucleic acid. Coexistence 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 coexist in the same nucleic acid molecule. In certain embodiments, the one or more nucleotide changes that compensate for disruption of secondary structure are one or more nucleotide changes that compensate for one or more nucleotide pairing disruptions. Therefore, in one embodiment, "compensating for a disruption of a secondary structure" means "compensating for a disruption of nucleotide pairing," i.e., one or more nucleotide pairing disruptions, for example, one or more nucleotide pairing disruptions in one or more stem-loops. The one or more nucleotide pairing disruptions may be introduced by removing at least one start codon. Each of the one or more nucleotide changes that compensate for the disruption of a secondary structure can be independently selected from the deletion, addition, substitution, and / or insertion of one or more nucleotides. In an illustrative example, if the nucleotide pairing A:U is disrupted by a substitution of A with C (C and U are usually not suitable for forming a nucleotide pair), the nucleotide change that compensates for the disruption of nucleotide pairing is to replace U with G, thereby allowing the formation of a C:G nucleotide pairing. In this way, the substitution of U with G compensates for the disruption of nucleotide pairing. As another example, if the nucleotide pairing A:U is disrupted by a substitution of A for C, a nucleotide change that compensates for the disrupted nucleotide pairing is to substitute C for A, thereby restoring formation of the original A:U nucleotide pairing.Generally, in the present invention, nucleotide changes that compensate for the disruption of secondary structure without restoring the original nucleic acid sequence or creating a new AUG triplet are preferred, e.g., in the above example, a U to G substitution is preferred over a C to A substitution.

[0145] According to the present invention, the term "tertiary structure" in relation to a nucleic acid molecule refers to the three-dimensional structure of the nucleic acid molecule as defined by its atomic coordinates.

[0146] According to the present invention, nucleic acids such as RNA (e.g., rRNA) can encode proteins. Thus, a transcribable nucleic acid sequence or its transcription product can comprise an open reading frame (ORF) that encodes a protein.

[0147] According to the present invention, the term "nucleic acid encoding a protein" means that the nucleic acid, when present in an appropriate environment, preferably a cell, is capable of directing the assembly of amino acids to produce a protein during the translation process. Preferably, the coding RNA according to the present invention is capable of interacting with the cellular translation machinery to translate the coding RNA to produce a protein.

[0148] In the present invention, the term "peptide" includes oligopeptides and polypeptides and refers to a substance containing two or more, preferably three or more, preferably four or more, preferably six or more, preferably eight or more, preferably ten or more, preferably thirteen or more, preferably six or more, preferably twenty or more, preferably fifty or less, preferably one hundred or less, and preferably one hundred and fifty consecutive amino acids linked together via peptide bonds. In this specification, the terms "peptide" and "protein" are generally used synonymously.

[0149] According to the present invention, the terms "peptide" and "protein" include substances that contain not only amino acid components but also non-amino acid components such as sugar and phosphate structures, as well as substances that contain bonds such as ester bonds, thioether bonds, disulfide bonds, etc.

[0150] According to the present invention, the term "polyprotein" refers to a single peptide containing the amino acid sequences of at least two, preferably at least three, and preferably at least four proteins, preferably as intermediates. This single peptide is cleaved by a protease to produce a single protein. The proteins contained in the polyprotein may already function within the polyprotein framework, or may acquire function by cleavage from the polyprotein. Furthermore, the function of a protein may change upon cleavage from the polyprotein. The protease that cleaves the polyprotein may be contained within the polyprotein itself, i.e., the polyprotein has autoproteolytic activity. Polyproteins are usually produced by translation of a single open reading frame of RNA.

[0151] According to the present invention, the terms "start codon" and "start codon" refer synonymously to the codon (base triplet) of an RNA molecule that is the first codon that can be translated by a ribosome. Such codons typically encode the amino acid methionine in eukaryotes and a modified methionine in prokaryotes. The most common start codon in eukaryotes and prokaryotes is AUG. Unless otherwise specified herein, the terms "start codon" and "start codon" with respect to an RNA molecule refer to the codon AUG. According to the present invention, the terms "start codon" and "start codon" are also used to refer to the corresponding base triplet of a deoxyribonucleic acid, i.e., the base triplet that encodes the start codon of an RNA. When the start codon of a messenger RNA is AUG, the base triplet that encodes AUG is ATG. According to the present invention, the terms "start codon" and "initiation codon" preferably refer to a functional initiation codon or initiation codon, i.e., an initiation codon or initiation codon that is or will be used as a codon by ribosomes to initiate translation. In RNA molecules, there may be AUG codons that ribosomes do not use as codons to initiate translation, for example, because the distance from the cap to the codon is short. These codons are not included in the term functional initiation codon or initiation codon.

[0152] According to the present invention, the term "start codon of an open reading frame" or "start codon of an open reading frame" refers to a triplet of bases that functions as the initiation codon for protein synthesis in a coding sequence, such as a coding sequence of a nucleic acid molecule found in nature. In RNA molecules, the start codon of an open reading frame is often preceded by a 5' untranslated region (5'-UTR), although this is not required.

[0153] According to the present invention, the term "native start codon of an open reading frame" or "native start codon of an open reading frame" refers to the base triplet that functions as the initiation codon for protein synthesis in a natural coding sequence. A natural coding sequence can be, for example, the coding sequence of a nucleic acid molecule found in nature. In some embodiments, the present invention provides variants of naturally occurring nucleic acid molecules, characterized in that the natural start codon (present in the natural coding sequence) has been removed (and is therefore absent in the variant nucleic acid molecule).

[0154] According to the present invention, the "first AUG" refers to the most upstream AUG base triplet of a messenger RNA molecule, preferably the most upstream AUG base triplet of a messenger RNA molecule that a ribosome uses or will use as a codon to initiate translation. Thus, the "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 start codon of an open reading frame, i.e., the codon used as the start codon during ribosomal protein synthesis.

[0155] According to the present invention, the terms "comprising a deletion" or "characterized by a deletion" and similar terms, when referring to a specific element of a nucleic acid variant, mean that the specific element is non-functional or absent in the nucleic acid variant compared to a reference nucleic acid molecule. Deletion can consist of, but is not limited to, the deletion of all or part of the specific element, the substitution of all or part of the specific element, or an alteration of the functional or structural properties of the specific element. Removal of a functional element in a nucleic acid sequence requires that the function is not exhibited at the position of the nucleic acid variant containing the deletion. For example, an RNA variant characterized by the deletion of a specific start codon requires that ribosomal protein synthesis does not initiate at the position of the RNA variant characterized by the deletion. Removal of a structural element in a nucleic acid sequence requires that the structural element is not present at the position of the nucleic acid variant containing the deletion. For example, RNA variants characterized by the removal of a specific AUG base triplet, i.e., an AUG base triplet at a specific position, can be characterized, for example, by deleting part or all of the specific AUG base triplet (e.g., ΔAUG), or by substituting one or more nucleotides (A, U, G) of the specific AUG base triplet with one or more different nucleotides, so that the resulting nucleotide sequence of the variant does not contain the AUG base triplet. Suitable substitutions of one nucleotide are those that convert the AUG base triplet to a GUG, CUG, or UUG base triplet, or to an AAG, ACG, or AGG base triplet, or to an AUA, AUC, or AUU base triplet. Suitable substitutions of more nucleotides can be selected accordingly.

[0156] According to the present invention, the term "self-replicating virus" includes RNA viruses that can replicate autonomously in host cells.Self-replicating viruses have single-stranded RNA (ssRNA) genomes, including alphaviruses, flaviviruses, measles viruses (MV), and rhabdoviruses.Alphaviruses and flaviviruses have positive-polarity genomes, while the genomes of measles viruses (MV) and rhabdoviruses are negative-polarity ssRNA.Typically, self-replicating viruses have a (+)-strand RNA genome that can be directly translated after infecting cells, and this translation provides RNA-dependent RNA polymerase, which produces both antisense and sense transcripts from the infected RNA.Hereinafter, the present invention will be described with reference to alphavirus-derived vectors as an example of self-replicating virus-derived vectors.However, it should be understood that the present invention is not limited to alphavirus-derived vectors.

[0157] In accordance with the present invention, the term "alphavirus" should be understood broadly and include any virus particle that has the characteristics of an alphavirus. Characteristics of an alphavirus include the presence of positive-strand RNA encoding genetic information suitable for replication in a host cell, including RNA polymerase activity. Additional characteristics of many alphaviruses are described, for example, in Strauss & Strauss, 1994, Microbiol. Rev. 58:491-562. The term "alphavirus" includes alphaviruses found in nature, as well as any mutants or derivatives thereof. In some embodiments, the mutants or derivatives are not found in nature.

[0158] In one embodiment, the alphavirus is an alphavirus found in nature. Typically, naturally occurring alphaviruses are infectious to any one or more eukaryotic organisms, such as animals (including vertebrates, such as humans, and arthropods, such as insects). The naturally occurring alphaviruses are preferably the following: Barmah Forest virus complex (including Barmah Forest virus); Eastern equine encephalitis virus complex (including seven serotypes of Eastern equine encephalitis virus); Middelburg virus complex (including Middelburg virus); Ndum virus complex (including Ndum virus); Semliki Forest virus complex (including Bebaru virus, Chikungunya virus, Mayaro virus and its subspecies Una virus, O'nyong-nyong virus and its subspecies Ibo-Ora virus, Ross River virus and its subspecies Bebaru virus, Getah virus, Sagiyama virus, Semliki Forest virus and its subspecies Metori virus); and Venezuelan equine encephalitis complex. (including Kabaso virus, Everglades virus, Mosso das Pedras virus, Mucambo virus, Paramana virus, Pixuna virus, Rio Negro virus, Trocara virus and its subspecies Bijou Bridge virus, and Venezuelan equine encephalitis virus); Western equine encephalitis complex (including Ora virus, Babanki virus, Kislagachi virus, Sindbis virus, Ockelbo virus, Wataroa virus, Boggy Creek virus, Fort Morgan virus, Highland J virus, and Western equine encephalitis virus); and unclassified viruses such as salmon pancreas disease virus, sleeping sickness virus, southern elephant seal virus, and Tonate virus. More preferably, the alphavirus is selected from the group consisting of the Semliki Forest virus complex (including the virus types listed above, including Semliki Forest virus), the Western equine encephalitis complex (including the virus types listed above, including Sindbis virus), the Eastern equine encephalitis virus (including the virus types listed above), and the Venezuelan equine encephalitis complex (including the virus types listed above, including Venezuelan equine encephalitis virus).

[0159] In a further preferred embodiment, the alphavirus is Semliki Forest virus. In another further preferred embodiment, the alphavirus is Sindbis virus. In another further preferred embodiment, the alphavirus is Venezuelan equine encephalitis virus.

[0160] In some embodiments of the present invention, the alphavirus is not an alphavirus found in nature. Typically, a non-naturally occurring alphavirus is a variant or derivative of a naturally occurring alphavirus and is distinguished from naturally occurring alphaviruses by at least one mutation in the nucleotide sequence, i.e., genomic RNA. The mutation in the nucleotide sequence can be selected from an insertion, substitution, or deletion of one or more nucleotides compared to an alphavirus found in nature. The mutation in the nucleotide sequence may or may not be associated with a mutation in the polypeptide or protein encoded by the nucleotide sequence. For example, the non-naturally occurring alphavirus can be an attenuated alphavirus. An attenuated alphavirus that does not occur in nature is typically an alphavirus that has at least one mutation in the nucleotide sequence that distinguishes it from naturally occurring alphaviruses and that is not infectious, infectious but with reduced pathogenicity, or not pathogenic at all. For example, TC83 is an attenuated alphavirus distinct from the naturally occurring Venezuelan equine encephalitis virus (VEEV) (McKinney et al., 1963, Am. J. Trop. Med. Hyg. 12:597-603).

[0161] Members of the alphavirus genus can also be classified based on their relative clinical characteristics in humans: those alphaviruses associated primarily with encephalitis and those associated primarily with fever, rash, and polyarthritis.

[0162] The term "alphaviral" means found in, derived from, or derived from an alphavirus, for example by genetic engineering.

[0163] According to the present invention, "SFV" stands for Semliki Forest virus. According to the present invention, "SIN" or "SINV" stands for Sindbis virus. According to the present invention, "VEE" or "VEEV" stands for Venezuelan equine encephalitis virus.

[0164] According to the present invention, the term "alphaviral" refers to an entity derived from an alphavirus. For example, an alphaviral protein can refer to a protein found in and / or encoded by an alphavirus, and an alphaviral nucleic acid sequence can refer to a nucleic acid sequence found in and / or encoded by an alphavirus. Preferably, an "alphaviral" nucleic acid sequence refers to a nucleic acid sequence "of the alphavirus genome" and / or a nucleic acid sequence "of the alphavirus genomic RNA."

[0165] According to the present invention, the term "alphavirus RNA" refers to any one or more of the alphavirus genomic RNA (i.e., the (+) strand), the complementary strand of the alphavirus genomic RNA (i.e., the (-) strand), and the subgenomic transcript (i.e., the (+) strand), or fragments of any of these.

[0166] According to the present invention, "alphavirus genome" refers to the genomic (+) strand RNA of an alphavirus.

[0167] In accordance with the present invention, the term "native alphavirus sequence" and similar terms typically refer to a (e.g., nucleic acid) sequence of a naturally occurring alphavirus (an alphavirus found in nature). In some embodiments, the term "native alphavirus sequence" also includes sequences of attenuated alphaviruses.

[0168] According to the present invention, the term "5' replication recognition sequence" preferably refers to a contiguous nucleic acid sequence, preferably a ribonucleic acid sequence, that is identical or homologous to the 5' segment of the genome of a self-replicating virus, such as an alphavirus genome. A "5' replication recognition sequence" is a nucleic acid sequence that can be recognized by a replicase, such as an alphavirus replicase. The term "5' replication recognition sequence" includes not only naturally occurring 5' replication recognition sequences, but also functional equivalents thereof, such as functional variants of the 5' replication recognition sequence of a naturally occurring self-replicating virus (e.g., a naturally occurring alphavirus). According to the present invention, functional equivalents include derivatives of 5' replication recognition sequences characterized by the removal of at least one initiation codon, as described herein. The 5' replication recognition sequence is required for the synthesis of the (-) strand complementary strand of alphavirus genomic RNA and is also required for the synthesis of (+) strand viral genomic RNA based on a (-) strand template. The natural 5' replication recognition sequence typically encodes at least the N-terminal fragment of nsP1, but does not include the entire open reading frame encoding nsP1234. Given the fact that the natural 5' replication recognition sequence typically encodes at least the N-terminal fragment of nsP1, the natural 5' replication recognition sequence typically includes at least one initiation codon (typically AUG). In one embodiment, the 5' replication recognition sequence includes conserved sequence element 1 (CSE1) of the alphavirus genome or a variant thereof, and conserved sequence element 2 (CSE2) of the alphavirus genome or a variant thereof. The 5' replication recognition sequence can usually form four stem loops (SL), designated SL1, SL2, SL3, and SL4. The numbering of these stem loops begins at the 5' end of the 5' replication recognition sequence.

[0169] The term "conserved sequence element" or "CSE" refers to a nucleotide sequence found in alphaviral RNA. These sequence elements are called "conserved" because homologous genes exist in the genomes of different alphaviruses, and homologous CSEs from different alphaviruses preferably share a high degree of sequence identity and / or similar secondary or tertiary structure. The term CSE includes CSE1, CSE2, CSE3, and CSE4.

[0170] According to the present invention, the terms "CSE1" and "44-nt CSE" refer synonymously to the nucleotide sequence required for (+)-strand synthesis from a (-)-strand template. The term "CSE1" refers to the sequence on the (+)-strand, and the complementary sequence of CSE1 (on the (-)-strand) functions as a promoter for (+)-strand synthesis. Preferably, the term CSE1 includes the 5'-most nucleotides of an alphavirus genome. CSE1 typically forms a conserved stem-loop structure. Without being bound by any particular theory, it is believed that the secondary structure of CSE1 is more important than the primary structure, i.e., the linear sequence. In the genomic RNA of the model alphavirus Sindbis virus, CSE1 consists of a 44-nucleotide continuous sequence, formed by the 5'-terminal 44 nucleotides of the genomic RNA (Strauss & Strauss, 1994, Microbiol. Rev. 58:491-562).

[0171] According to the present invention, the terms "CSE2" and "51-nucleotide CSE" refer synonymously to the nucleotide sequence required for negative-strand synthesis from a positive-strand template. The positive-strand template is typically an alphavirus genomic RNA or an RNA replicon (note that subgenomic RNA transcripts without CSE2 do not serve as templates for negative-strand synthesis). In alphavirus genomic RNA, CSE2 is typically located within the coding sequence of nsP1. In the genomic RNA of the model alphavirus, Sindbis virus, the 51-nucleotide CSE is located at nucleotides 155-205 of the genomic RNA (Frolov et al., 2001, RNA, vol. 7, pp. 1638-1651). CSE2 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 from the 5' end of the alphavirus genomic RNA. Without wishing to be bound by any particular theory, it is believed that in CSE2, secondary structure is more important than primary structure, i.e., linear sequence.

[0172] According to the present invention, the terms "CSE3" or "junction sequence" refer synonymously to a nucleotide sequence derived from an alphavirus genomic RNA and comprising the initiation site of the subgenomic RNA. The complementary sequence on the negative strand of this sequence acts to promote transcription of the subgenomic RNA. In alphavirus genomic RNAs, CSE3 typically overlaps with the region encoding the C-terminal fragment of nsP4 and extends into a short non-coding region located upstream of the open reading frame encoding the structural proteins.

[0173] According to the present invention, the terms "CSE4" or "19-nucleotide conserved sequence" or "19-nucleotide CSE" refer synonymously to a nucleotide sequence derived from alphavirus genomic RNA located immediately upstream of the poly(A) sequence in the 3' untranslated region of the alphavirus genome. CSE4 typically consists of 19 consecutive nucleotides. Without being bound by any particular theory, CSE4 is understood to function as a core promoter in the initiation of negative-strand synthesis (Jose et al., 2009, Future Microbiol. 4:837-856). Alternatively, CSE4 and the poly(A) tail of the alphavirus genomic RNA are understood to function together for efficient negative-strand synthesis (Hardy & Rice, 2005, J. Virol. 79:4630-4639).

[0174] According to the present invention, the term "subgenomic promoter" or "SGP" refers to a nucleic acid sequence upstream (5') of a nucleic acid sequence (e.g., a coding sequence) that controls transcription of said nucleic acid sequence by providing recognition and binding sites for an RNA polymerase, typically an RNA-dependent RNA polymerase, particularly a functional alphavirus nonstructural protein. SGPs may further contain recognition or binding sites for additional factors. Subgenomic promoters are typically genetic elements of positive-strand RNA viruses, such as alphaviruses. Alphavirus subgenomic promoters are nucleic acid sequences contained in the viral genomic RNA. Subgenomic promoters are generally characterized by allowing initiation of transcription (RNA synthesis) in the presence of an RNA-dependent RNA polymerase (e.g., a functional alphavirus nonstructural protein). The RNA (-) strand, i.e., the complementary strand of the alphavirus genomic RNA, serves as a template for synthesis of a (+)-strand subgenomic transcript, which typically initiates at or near the subgenomic promoter. As used herein, the term "subgenomic promoter" is not limited to a specific location within the nucleic acid containing such a subgenomic promoter. In some embodiments, the SGP is identical to, overlaps with, or comprises a CSE3.

[0175] The terms "subgenomic transcript" or "subgenomic RNA" refer synonymously to an RNA molecule obtained by transcribing an RNA molecule as a template ("template RNA"), where the template RNA includes a subgenomic promoter that controls transcription of the subgenomic transcript. Subgenomic transcripts are obtained in the presence of an RNA-dependent RNA polymerase, particularly functional alphavirus nonstructural proteins. For example, the term "subgenomic transcript" can refer to an RNA transcript prepared in an alphavirus-infected cell using the negative strand complement of an alphavirus genomic RNA as a template. However, the term "subgenomic transcript" as used herein is not limited thereto and also includes transcripts obtained by using heterologous RNA as a template. For example, a subgenomic transcript can also be obtained by using the negative strand complement of an SGP-containing replicon of the present invention as a template. Thus, the term "subgenomic transcript" can refer not only to an RNA molecule obtained by transcribing a fragment of an alphavirus genomic RNA, but also to an RNA molecule obtained by transcribing a fragment of a replicable RNA of the present invention.

[0176] The term "xenogeneic" is used to describe something that is made up of two or more different elements. For example, introducing cells from one individual into another constitutes xenotransplantation. A xenogeneic gene is a gene that originates from a source other than the subject.

[0177] The cell that can be used in the method for identifying sequence variation is any suitable cell that can replicate and / or translate RNA, regardless of whether nucleotide modification is present or not.Cell can be mammalian cell, for example, human cell.Cell can constitutively express replicase that recognizes and replicates the sequence present in replicable RNA, or can transiently express such replicase.

[0178] Below are presented specific and / or preferred variations of individual features of the invention. The present invention also contemplates, as particularly preferred embodiments, embodiments produced by combining two or more of the described specific and / or preferred variations of two or more features of the invention.

[0179] A system containing two RNA molecules According to the present invention, the system comprising two RNA molecules refers to a combination of physical entities, and these entities can be realized, for example, as separate compositions or as a single composition.In a preferred embodiment, this system is a composition comprising RNA molecules and additional components, such as lipids that form particles with RNA.This system can also be made by combining two different compositions, where the first composition comprises the first RNA, and the second composition comprises the second RNA.In another embodiment, the two RNAs can exist as separate compositions, and each composition can comprise lipids or polymers for complexing RNA.In this embodiment, each composition can be used separately to provide RNA to a subject, for example, by administration, etc.

[0180] In a preferred embodiment, the system can include one or more cells, where the two RNA molecules can be present in the same cell or in different cells, preferably the same cell. In a preferred embodiment, the cells can be present in a subject or administered to a subject.

[0181] RNA The RNA molecules of the present invention can optionally be characterized by additional features such as a 5' cap, a 5' UTR, a 3' UTR, a poly(A) sequence, and / or adaptation of codon usage for optimized translation and / or stabilization of the RNA molecule, as described in more detail below.

[0182] cap In some embodiments, an RNA molecule of the invention comprises a 5' cap.

[0183] The terms "5' cap," "cap," "5' cap structure," and "cap structure" are used interchangeably to refer to the dinucleotide found at the 5' end of some eukaryotic primary transcripts, such as precursor messenger RNA. A 5' cap is a structure in which an (optionally modified) guanosine is attached to the first nucleotide of an mRNA molecule via a 5'-5' triphosphate linkage (or, in the case of certain cap analogs, a modified triphosphate linkage). These terms may refer to a conventional cap or a cap analog.

[0184] "RNA containing a 5' cap" or "RNA provided with a 5' cap" or "RNA modified with a 5' cap" or "capped RNA" refers to RNA that includes a 5' cap. For example, providing an RNA with a 5' cap can be achieved by in vitro transcription of a DNA template in the presence of the 5' cap, where the 5' cap is incorporated into the generated RNA strand during transcription. Alternatively, the RNA can be generated, for example, by in vitro transcription, and the 5' cap can be added to the RNA post-transcriptionally using a capping enzyme, such as vaccinia virus capping enzyme. In capped RNA, the 3' position of the first base of the (capped) RNA molecule is linked to the 5' position of the next base (the "second base") of the RNA molecule via a phosphodiester bond.

[0185] In one embodiment, the RNA molecule comprises a 5' cap. In one embodiment, the RNA molecule does not comprise a 5' cap. In one embodiment, only one of the first or second RNA molecule comprises a 5' cap.

[0186] The term "conventional 5' cap" refers to a naturally occurring 5' cap, preferably a 7-methylguanosine cap, in which the guanosine of the cap is a modified guanosine consisting of a methylation at the 7 position.

[0187] As used herein, the term "5' cap analog" refers to a molecular structure similar to a conventional 5' cap, but preferably modified to have the ability to stabilize RNA when bound to RNA in vivo and / or in cells. A cap analog is not a conventional 5' cap.

[0188] In eukaryotic mRNA, the 5' cap is generally believed to be involved in efficient translation of mRNA. Generally, in eukaryotes, translation begins only at the 5' end of a messenger RNA (mRNA) molecule unless an internal ribosome entry site (IRES) is present. Eukaryotic cells can add a 5' cap to RNA during transcription in the nucleus. Newly synthesized mRNA is usually modified with a 5' cap structure, for example, when the transcript reaches a length of 20–30 nucleotides. First, the 5'-terminal nucleotide pppN (ppp represents a triphosphate and N represents any nucleoside) is converted to 5' GpppN in the cell by a capping enzyme with RNA 5'-triphosphatase and guanylyltransferase activities. GpppN is then methylated by a second enzyme with (guanine-7) methyltransferase activity in the cell, resulting in monomethylated m 7 A GpppN cap is formed. In one embodiment, the 5' cap used in the present invention is a natural 5' cap.

[0189] In the present invention, naturally occurring 5' cap dinucleotides typically include unmethylated cap dinucleotides (G(5')ppp(5')N; also referred to as GpppN) and methylated cap dinucleotides (m 7 G(5')ppp(5')N;m 7 m 7 GpppN (wherein N is G) is represented by the following formula: [ka] .

[0190] The capped RNA of the present invention can be prepared in vitro and is therefore independent of the capping machinery within the host cell. The most frequently used method for producing capped RNA in vitro is the synthesis of all four ribonucleoside triphosphates and m 7 G(5')ppp(5')G(m 7 The transcription of a DNA template using a bacterial or bacteriophage RNA polymerase in the presence of a cap dinucleotide such as GpppG. 7 The 3'-OH of the guanosine moiety of GpppG then initiates transcription by nucleophilic attack on the α-phosphate of the template nucleoside triphosphate (pppN), forming intermediate m 7 This produces GpppGpN (where N is the second base in the RNA molecule). The formation of the competing product pppGpN, a GTP-initiated product, is suppressed by setting the cap to GTP molar ratio between 5 and 10 during in vitro transcription.

[0191] In preferred embodiments of the present invention, the 5'-cap (if present) is a 5'-cap analog. These embodiments are particularly suitable when the RNA is obtained by in vitro transcription, e.g., in vitro transcribed RNA (IVT-RNA). Cap analogs were first described to facilitate large-scale synthesis of RNA transcripts by in vitro transcription.

[0192] Several cap analogs (synthetic caps) have been generally described for messenger RNA, all of which can be used in the present invention. Ideally, a cap analog associated with higher translation efficiency and / or improved resistance to in vivo degradation and / or improved resistance to in vitro degradation will be selected.

[0193] Preferably, a cap analog is used that can be incorporated into an RNA strand in only one direction. Pasquinelli et al. (1995, RNA J. 1:957-967) demonstrated that bacteriophage RNA polymerase uses a 7-methylguanosine unit for transcription initiation in in vitro transcription. As a result, approximately 40-50% of capped transcripts have a reverse cap dinucleotide (i.e., the initial reaction product is Gpppm). 7 It has been shown that RNA with a reverse cap does not function in the translation of nucleic acid sequences into proteins, compared to RNA with a correct cap. Therefore, incorporating the cap in the correct orientation, i.e., m 7 It is desirable to obtain RNA with a structure essentially corresponding to GpppGpN, etc. Reverse incorporation of cap dinucleotides has been shown to be inhibited by substitution of either the 2'- or 3'-OH group of the methylated guanosine unit (Stepinski et al., 2001, RNA J. 7:1486-1495; Peng et al., 2002, Org. Lett. 24:161-164). RNA synthesized in the presence of such "anti-reverse cap analogs" is not susceptible to the traditional 5'-cap m 7 In the presence of GpppG, it is translated more efficiently than in vitro transcribed RNA. For this purpose, a cap analog in which the 3'OH group of the methylated guanosine unit is replaced with OCH3 is described, for example, by Holtkamp et al., 2006, Blood 108:4009-4017 (7-methyl(3'-O-methyl)GpppG; anti-reverse cap analog (ARCA)). ARCA is a suitable cap dinucleotide for the present invention. [ka]

[0194] In one embodiment, the RNA of the present invention is essentially decapping-resistant. This is important because the amount of protein produced from synthetic mRNA introduced into cultured mammalian cells is generally limited by spontaneous degradation of the mRNA. One in vivo pathway of mRNA degradation begins with the removal of the mRNA cap. This removal is catalyzed by a heterodimeric pyrophosphatase comprising a regulatory subunit (Dcp1) and a catalytic subunit (Dcp2). The catalytic subunit cleaves between the α and β phosphates of the triphosphate bridge. The present invention allows for the selection or presence of cap analogs that are not or are less susceptible to this type of cleavage. A suitable cap analog for this purpose is represented by the formula (I): [ka] [In the formula, R 1 is selected from the group consisting of optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl; R 2 and R 3 is independently selected from the group consisting of H, halo, OH, and optionally substituted alkoxy, or R 2 and R 3 together form OXO, where X is selected from the group consisting of optionally substituted CH, CHCH, CHCHCH, CHCH(CH), and C(CH); or R 2 is R 2 to form -O-CH2- or -CH2-O-, R 5 is selected from the group consisting of S, Se, and BH3; R 4 and R 6 are independently selected from the group consisting of O, S, Se, and BH3. The cap dinucleotide can be selected from the following:

[0195] n is 1, 2, or 3.

[0196] R 1 , R 2 , R 3 , R 4 , R 5 , R 6 Preferred embodiments of the above are disclosed in WO2011 / 015347A1 and can be selected accordingly in the present invention.

[0197] For example, in one embodiment, the RNA of the invention comprises a phosphorothioate cap analog, which has one of the three non-bridging O atoms in the triphosphate chain replaced with an S atom, i.e., R in formula (I): 4 , R 5 , or R 6 and R is S. Phosphorothioate cap analogs have been described by Kowalska et al., 2008, RNA, 14:1119-1131, as a solution to the undesired decapping process and thus increasing RNA stability in vivo. In particular, the replacement of the sulfur atom in the β-phosphate group of the 5' cap with an oxygen atom provides stabilization against Dcp2. In a preferred embodiment of the present invention, R in formula (I) is S. and R 4 and R 6 is O.

[0198] In a further embodiment, the RNA of the present invention comprises a phosphorothioate cap analog in which a phosphorothioate modification of the RNA 5' cap is combined with an "anti-reverse cap analog" (ARCA) modification. ARCA phosphorothioate cap analogs are described in WO2008 / 157688A2, all of which can be used in the RNA of the present invention. In this embodiment, R in formula (I) 2Or at least one of R3 is not OH, preferably R 2 and R 3 One of the groups is methoxy (OCH3), and preferably R 2 and R 3 In a preferred embodiment, the sulfur atom of the β-phosphate group is replaced by an oxygen atom (thus, R in formula (I) is S and R 4 and R 6 (The symbol O represents O.) The phosphorothioate modification of ARCA is thought to ensure that the α, β, and γ phosphorothioate groups are correctly positioned within the active sites of cap-binding proteins in both the translational and decapping machinery. At least some of these analogs are inherently resistant to pyrophosphatases Dcp1 / Dcp2. Phosphorothioate-modified ARCA has been reported to have significantly higher affinity for eIF4E than the corresponding ARCA lacking the phosphorothioate groups.

[0199] A particularly preferred cap analog in the present invention, m2'7,2'-OGppspG, is called β-S-ARCA (WO 2008 / 157688 A2; Kuhn et al., 2010, Gene Ther. 17:961-971). Thus, in one embodiment of the present invention, the RNA of the present invention is modified with β-S-ARCA. β-S-ARCA is represented by the following structure: [ka] .

[0200] Generally, substitution of the oxygen atom for the sulfur atom in the bridging phosphate group results in phosphorothioate diastereomers designated D1 and D2 based on their elution patterns in HPLC. Briefly, the "D1 diastereomer of β-S-ARCA" or "β-S-ARCA(D1)" is the diastereomer of β-S-ARCA that elutes first on an HPLC column compared to the D2 diastereomer of β-S-ARCA (β-S-ARCA(D2)), thus resulting in a shorter retention time. Determination of stereochemical configuration by HPLC is described in WO 2011 / 015347 A1.

[0201] In a particularly preferred first embodiment of the present invention, the RNA of the present invention is modified with the β-S-ARCA (D2) diastereomer. The two diastereomers of β-S-ARCA differ in their susceptibility to nucleases. RNAs bearing the D2 diastereomer of β-S-ARCA are almost completely resistant to Dcp2 cleavage (only 6% cleavage rate compared to RNA synthesized in the presence of an unmodified ARCA 5'-cap), whereas RNAs bearing the β-S-ARCA (D1) 5'-cap are moderately susceptible to Dcp2 cleavage (71% cleavage rate). Furthermore, improved stability against Dcp2 cleavage has been shown to correlate with increased protein expression in mammalian cells. In particular, RNAs bearing the β-S-ARCA (D2) cap have been shown to be translated more efficiently in mammalian cells than RNAs bearing the β-S-ARCA (D1) cap. Thus, in one embodiment of the present invention, the RNA of the present invention is modified with a cap analog of formula (I), characterized in that the stereochemical configuration at the P atom containing the substituent R5 of formula (I) corresponds to the stereochemical configuration at the Pβ atom of the D2 diastereomer of β-S-ARCA. 5 is S and R 4 and R 6 is O. Furthermore, R in formula (I) 2 or R 3 At least one of is preferably not OH, and preferably R 2 and R 3One of the groups is methoxy (OCH3), and R 2 and R 3 The other of is preferably OH.

[0202] In a particularly preferred second embodiment, the RNA of the present invention is modified with a β-S-ARCA (D1) diastereomer. This embodiment is particularly suitable for introducing capped RNA into immature antigen-presenting cells, such as for vaccination purposes. It has been demonstrated that the β-S-ARCA (D1) diastereomers are particularly suitable for improving RNA stability, increasing RNA translation efficiency, extending RNA translation time, increasing total protein expression of the RNA, and / or enhancing the immune response to the antigen or antigenic peptide encoded by the RNA when the capped RNA is introduced into immature antigen-presenting cells (Kuhn et al., 2010, Gene Ther. 17:961-971). Therefore, in another embodiment of the present invention, the RNA of the present invention is modified with a cap analog of formula (I), characterized in that the stereochemical configuration at the P atom containing the substituent R5 of formula (I) corresponds to the stereochemical configuration at the Pβ atom of the D1 diastereomer of β-S-ARCA. Respective cap analogs and their embodiments are described in WO 2011 / 015347 A1 and Kuhn et al., 2010, Gene Ther. 17:961-971. Any cap analog described in WO 2011 / 015347 A1, in which the stereochemical configuration at the P atom containing the substituent R5 corresponds to the stereochemical configuration at the Pβ atom of the D1 diastereomer of β-S-ARCA, can be used in the present invention. Preferably, R in formula (I) 5 is S and R 4 and R 6 is O. Furthermore, preferably, R in formula (I) 2 or R 3 At least one of is not OH, and preferably R 2 and R 3 One of the groups is methoxy (OCH3), and R 2 and R 3 The other of these is OH.

[0203] In one embodiment, the RNA of the present invention is modified with a 5' cap structure shown in Formula (I), in which any one phosphate group is replaced with a boranophosphate group or a phosphoroselenoate group. Such caps have improved stability both in vitro and in vivo. Optionally, each compound has a 2'-O- or 3'-O-alkyl group (wherein the alkyl group is preferably a methyl group), and the respective cap analogs are referred to as BH3-ARCA and Se-ARCA. Compounds particularly suitable for mRNA capping include β-BH3-ARCA and β-Se-ARCA, as described in WO 2009 / 149253 A2. Preferably, in these compounds, the stereochemical configuration at the P atom containing the substituent R5 in Formula (I) corresponds to the stereochemical configuration at the Pβ atom of the D1 diastereomer of β-S-ARCA.

[0204] In one embodiment, the 5' cap has the following structure, supplied by Trilink Biotechnologies, San Diego, CA: [ka] It can be a CleanCap with

[0205] In one embodiment, the 5' cap has the following structure, supplied by Trilink Biotechnologies, San Diego, CA: [ka] It can be a CleanCap with

[0206] In one embodiment, a modified RNA molecule comprises a 5' cap, wherein at least one of the uridines in the molecule is a modified uridine, preferably N1-methyl-pseudouridine (Imψ), and the molecule comprises a 5' cap with the sequence NpppNU, where U in the 5' cap is an unmodified uridine. In one embodiment, the 5' cap has the sequence NpppAU, where A represents a modified or unmodified adenosine nucleotide. For example, a modified nucleotide N or A at the 3' position of the triphosphate linkage has a modified ribose structure, such as 2'-O-methylated ribose (Nm or Am), resulting in a so-called "Cap 1." In contrast, a cap comprising a nucleotide N or A at the 3' position of the triphosphate linkage with an unmethylated ribose is typically referred to as "Cap 0."

[0207] In one embodiment, the modified adenosine is selected from the group consisting of 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, and N6-threonylcarbamoyladenosine. 2-methylthio-N6-threonylcarbamoyl adenosine, N6,N6-dimethyl adenosine, 7-methyladenine, 2-methylthioadenine, and 2-methoxyadenine.

[0208] UTR The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or the corresponding region in an RNA molecule, such as an mRNA molecule. Untranslated regions (UTRs) can be located 5' (upstream) of an open reading frame (5'-UTR) and / or 3' (downstream) of an open reading frame (3'-UTR).

[0209] A 3'-UTR, if present, is located at the 3' end of a gene, downstream of the stop codon of the protein-coding region, although the term "3'-UTR" preferably does not include the poly(A) tail. Thus, a 3'-UTR is upstream of, e.g., immediately adjacent to, the poly(A) tail (if present).

[0210] When present, a 5'-UTR is located at the 5' end of a gene, upstream of the start codon of the protein-coding region. The 5'-UTR is downstream of the 5'-cap (if present), e.g., immediately adjacent to the 5'-cap.

[0211] According to the present invention, 5'- and / or 3'-untranslated regions can be operably linked to an open reading frame such that these regions are associated with the open reading frame in a manner that enhances the stability and / or translation efficiency of RNA containing the open reading frame.

[0212] In some embodiments, an RNA molecule of the present invention comprises a 5'-UTR and / or a 3'-UTR, and in some embodiments, at least one miRNA sequence described herein is located or contained within the 3'-UTR of a second RNA molecule.

[0213] UTRs are involved in RNA stability and translation efficiency. In addition to the structural modifications of the 5' cap and / or 3' poly(A) tail described herein, both can be improved by selecting specific 5' and / or 3' untranslated regions (UTRs). It is understood that sequence elements within UTRs generally affect translation efficiency (mainly 5'-UTR) and RNA stability (mainly 3'-UTR). Preferably, an active 5'-UTR is present to increase the translation efficiency and / or stability of an RNA molecule. Also, preferably, an active 3'-UTR is present to increase the translation efficiency and / or stability of an RNA molecule, independently or in addition.

[0214] The terms "active to increase translation efficiency" and / or "active to increase stability," with respect to a first nucleic acid sequence (e.g., a UTR), mean that the first nucleic acid sequence can be modified in a manner that, in a co-transcription product with a second nucleic acid sequence, increases the translation efficiency and / or stability of the second nucleic acid sequence compared to the translation efficiency and / or stability of the second nucleic acid sequence in the absence of the first nucleic acid sequence.

[0215] In one embodiment, the RNA molecules of the present invention comprise heterologous or non-native 5'-UTRs and / or 3'-UTRs relative to the alphavirus from which the functional alphavirus replicase is derived. This allows the untranslated regions to be designed according to the desired translation efficiency and RNA stability. Thus, heterologous or non-native UTRs allow for a high degree of flexibility, which is advantageous compared to native alphavirus UTRs.

[0216] Preferably, the RNA molecules of the present invention comprise a 5'-UTR and / or a 3'-UTR that are not derived from a virus, particularly an alphavirus. In one embodiment, the RNA molecule comprises a 5'-UTR derived from a eukaryotic 5'-UTR and / or a 3'-UTR derived from a eukaryotic 3'-UTR.

[0217] The 5'-UTRs of the present invention can comprise any combination of two or more nucleic acid sequences, optionally separated by a linker. The 3'-UTRs of the present invention can comprise any combination of two or more nucleic acid sequences, optionally separated by a linker.

[0218] In the present invention, the term "linker" refers to a nucleic acid sequence that is added between two nucleic acid sequences and links the two nucleic acid sequences. There are no particular limitations on the linker sequence.

[0219] The length of the 3'-UTR is typically 200 to 2000 nucleotides, e.g., 500 to 1500 nucleotides. The 3'-untranslated regions of immunoglobulin mRNAs are relatively short (less than about 300 nucleotides), while the 3'-untranslated regions of other genes are relatively long. For example, the 3'-untranslated regions of tPA are about 800 nucleotides, factor VIII about 1800 nucleotides, and erythropoietin about 560 nucleotides. In some embodiments, the 3'-UTR of the second RNA molecule further comprises at least one miRNA sequence described herein. The length of each miRNA sequence is 10 to 200 nucleotides, optionally 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, or 20 to 30 nucleotides, optionally 10 to 50 nucleotides, preferably 10 to 30 nucleotides.

[0220] The 3' untranslated region of mammalian mRNAs typically contains a homologous region known as the AAUAAA hexanucleotide sequence. This sequence is likely a poly(A) addition signal and is often located 10-30 bases upstream of the poly(A) addition site. The 3' untranslated region may contain one or more inverted repeats that fold to form stem-loop structures and may act as a barrier to exonucleases or interact with proteins known to increase RNA stability (e.g., RNA-binding proteins).

[0221] Two consecutive identical copies of the human β-globin 3'-UTR, particularly the human β-globin 3'-UTR, contribute to high transcript stability and translation efficiency (Holtkamp et al., 2006, Blood 108:4009-4017). Therefore, in one embodiment, the RNA molecule of the present invention comprises two consecutive identical copies of the human β-globin 3'-UTR. Thus, in the 5' to 3' direction, it comprises: (a) an optional 5'-UTR, (b) an open reading frame, and (c) a 3'-UTR. The 3'-UTR comprises two consecutive identical copies of the human β-globin 3'-UTR, a fragment thereof, or a variant of the human β-globin 3'-UTR or a fragment thereof.

[0222] In one embodiment, an RNA molecule of the present invention comprises a 3'-UTR that is active to increase translation efficiency and / or stability, but is not the human β-globin 3'-UTR, a fragment thereof, or a variant of the human β-globin 3'-UTR or a fragment thereof. An exemplary human β-globin 3'-UTR sequence is set forth in SEQ ID NO: 51. In one embodiment, a human β-globin 3'-UTR sequence useful in the RNA molecules described herein is one that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 51.

[0223] In one embodiment, an RNA molecule according to the invention comprises an active 5'-UTR to increase translation efficiency and / or stability.

[0224] In some embodiments, the RNA molecule can contain a 3'-UTR sequence. This is a combination of two sequence elements (FI elements) derived from the "amino-terminal enhancer (AES) mRNA" (termed F) and the mitochondrially encoded 12S ribosomal RNA (termed I), located between the coding sequence and the poly(A) tail, ensuring higher maximum protein levels and extended mRNA duration. These were identified through an ex vivo selection process for sequences that confer RNA stability and enhance total protein expression (see WO 2017 / 060314, incorporated herein by reference). An exemplary FI element sequence is shown in SEQ ID NO: 43. In one embodiment, FI element sequences useful in the RNA molecules described herein are sequences that are at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% homologous to SEQ ID NO: 43.

[0225] Poly(A) sequence In some embodiments, the first and / or second RNA molecule according to the present invention comprises a poly(A) sequence. When the RNA molecule comprises conserved sequence element 4 (CSE4), the poly(A) sequence of the RNA molecule is preferably downstream of the CSE4, and most preferably immediately adjacent to the CSE4. In some embodiments, the poly(A) sequence is a 3' poly(A) sequence.

[0226] According to the present invention, in one embodiment, the poly(A) sequence comprises, consists essentially of, or consists of at least 20, preferably at least 26, preferably at least 40, preferably at least 80, preferably at least 100, preferably at most 500, preferably at most 400, preferably at most 300, preferably at most 200, particularly at most 150 A nucleotides, in particular about 120 A nucleotides. In this context, "consisting essentially of" means that the majority of the nucleotides in the poly(A) sequence, typically at least 50% and preferably at least 75% of the nucleotides in the "poly(A) sequence," are A nucleotides (adenylic acid), although the remaining nucleotides may be nucleotides other than A nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), and C nucleotides (cytidylic acid). In this context, "consisting of" means that all nucleotides in the poly(A) sequence, i.e., 100% of the nucleotides in the poly(A) sequence, are A nucleotides. The term "A nucleotide" or "A" refers to adenylic acid.

[0227] Indeed, it has been demonstrated that a 3' poly(A) sequence of approximately 120 A nucleotides has a beneficial effect on RNA levels in transfected eukaryotic cells, as well as on the levels of proteins translated from open reading frames located 5' upstream of the 3' poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0228] In alphaviruses, a 3' poly(A) sequence consisting of at least 11 consecutive adenylate residues or at least 25 consecutive adenylate residues is thought to be important for efficient minus-strand synthesis. In particular, in alphaviruses, a 3' poly(A) sequence consisting of at least 25 consecutive adenylate residues has been shown to promote (-)strand synthesis in cooperation with conserved sequence element 4 (CSE4) (Hardy & Rice, 2005, J. Virol. 79:4630-4639).

[0229] The present invention provides a 3' poly(A) sequence that is added during RNA transcription, i.e., during the preparation of in vitro transcribed RNA, based on a DNA template containing repetitive dT nucleotides (deoxythymidylic acid) in the strand complementary to the coding strand. The DNA sequence encoding the poly(A) sequence (coding strand) is called a poly(A) cassette.

[0230] The first and / or second RNA molecule may contain an interrupted 3' poly(A) sequence. In a preferred embodiment of the present invention, the 3' poly(A) cassette present in the coding strand of the DNA consists essentially of dA nucleotides, but is interrupted by random sequences of evenly distributed four nucleotides (dA, dC, dG, dT). Such random sequences may be 5 to 50 nucleotides in length, preferably 10 to 30 nucleotides, and more preferably 10 to 20 nucleotides in length. Such cassettes are disclosed in WO2016 / 005004A1. Any of the poly(A) cassettes disclosed in WO2016 / 005004A1 can be used in the present invention. Poly(A) cassettes composed essentially of dA nucleotides but interrupted by random sequences with an equal distribution of the four nucleotides (dA, dC, dG, dT) and a length of, for example, 5–50 nucleotides, at the DNA level show a constant propagation of plasmid DNA in E. coli and at the RNA level are still associated with the beneficial properties of supporting RNA stability and translation efficiency.

[0231] Thus, in a preferred embodiment of the present invention, the 3' poly(A) sequence contained in the RNA molecules described herein consists essentially of A nucleotides, but is interrupted by random sequences in which the four nucleotides (A, C, G, and U) are evenly distributed. Such random sequences may be 5 to 50 nucleotides in length, preferably 10 to 30 nucleotides, and more preferably 10 to 20 nucleotides in length. In some embodiments, the first and / or second RNA molecules contain an interrupted 3' poly(A) sequence consisting of A30-L-A70, and the linker (L) is 10 nucleotides in length.

[0232] Codon usage Generally, the degeneracy of the genetic code allows certain codons (base triplets encoding amino acids) present in an RNA sequence to be replaced with other codons (base triplets), while maintaining the same coding capacity (the substituted codon encodes the same amino acid as the substituted codon). In some embodiments of the present invention, at least one codon of an open reading frame contained in an RNA molecule differs from the corresponding codon in each open reading frame of the species from which the open reading frame is derived. In this embodiment, the coding sequence of the open reading frame is said to be "adapted" or "modified." The coding sequence of the open reading frame contained in the first RNA and / or the second RNA can be adapted.

[0233] For example, when the coding sequence of an open reading frame is adapted, frequently used codons can be selected.WO2009 / 024567A1 describes the adaptation of the coding sequence of a nucleic acid molecule, including replacing rare codons with more frequently used codons.Because the frequency of codon usage depends on host cells or host organisms, this type of adaptation is suitable for adapting nucleic acid sequences to the expression in specific host cells or host organisms.Generally, in host cells or host organisms, more frequently used codons are often translated more efficiently, but it is not necessary to adapt all the codons of an open reading frame.

[0234] For example, when adapting the coding sequence of an open reading frame, the content of G (guanylic acid) and C (cytidylic acid) residues can be altered by selecting the codon with the highest GC-rich content for each amino acid. It has been reported that RNA molecules with GC-rich open reading frames may suppress immune activation and improve RNA translation and half-life (Thess et al., 2015, Mol. Ther. 23:1457-1465).

[0235] In particular, the coding sequences of the nonstructural proteins can be adapted as needed because the open reading frames encoding the nonstructural proteins do not overlap with the 5' replication recognition sequence of the replicon.

[0236] RNA modification In one embodiment, the first RNA and / or second RNA described herein may have modified nucleotide / nucleoside / backbone modifications. As used herein, the term "RNA modification" may refer to chemical modifications, including backbone modifications, as well as sugar or base modifications.

[0237] In this context, modified RNA molecules as defined herein may contain nucleotide analogs / modifications, such as backbone modifications, sugar modifications, or base modifications. A backbone modification in the context of the present invention is a modification in which the phosphate of the backbone of a nucleotide contained in an RNA molecule as defined herein is chemically modified. A sugar modification in the context of the present invention is a chemical modification of the sugar of a nucleotide of an RNA molecule as defined herein. Furthermore, a base modification in the context of the present invention is a chemical modification of the base portion of a nucleotide of an RNA molecule. In this context, the nucleotide analog or modification is preferably selected from nucleotide analogs applicable to transcription and / or translation.

[0238] Sugar Modification: Modified nucleosides and nucleotides that can be incorporated into the modified RNA molecules described herein can have modified sugar moieties. For example, the 2' hydroxyl group (OH) can be modified or replaced with various "oxy" or "deoxy" substituents. Examples of "oxy"-2' hydroxyl group modifications include, but are not limited to, alkoxy or aryloxy (-OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), polyethylene glycol (PEG), -O(CH2CH20)nCH2CH2OR, "locked" nucleic acids (LNAs) in which the 2' hydroxyl is linked to the 4' carbon of the same ribose sugar, e.g., by a methylene bridge, and amino groups (-O-amino, where the amino group, e.g., NRR, can be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxy. A "deoxy" modification includes hydrogen, amino (e.g., NH, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid), or an amino group attached to the sugar via a linker, the linker containing one or more of C, N, and O atoms. The sugar group can also contain one or more carbons that have the opposite stereochemical configuration as the corresponding carbon in ribose. Thus, modified RNA molecules can include, for example, nucleotides containing arabinose as the sugar.

[0239] Backbone modification: In modified nucleosides and nucleotides, the phosphate backbone can be further modified. These can be incorporated into modified RNA molecules as described herein. The phosphate group of the backbone can be modified by replacing one or more oxygen atoms with different substituents. Furthermore, modified nucleosides and nucleotides can have all unmodified phosphate groups replaced with modified phosphate groups as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphate groups, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. In phosphorodithioates, non-linked oxygens are replaced with sulfur. Phosphate linkers can also be modified by replacing linking oxygens with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene phosphonates).

[0240] Base modification: The modified nucleosides and nucleotides that can be incorporated into modified RNA molecules as described herein can be further modified at the nucleobase portion. Examples of nucleobases contained in RNA include, but are not limited to, adenine, guanine, cytosine, uracil, etc. For example, the nucleosides and nucleotides described herein can be chemically modified on the major groove surface. In some embodiments, the chemical modification of the major groove can include an amino group, a thiol group, an alkyl group, or a halo group.

[0241] In certain embodiments of the invention, the nucleotide analogue / modification is a base modification, preferably 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine riboside-5'-triphosphate, 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methylinosine-5'-triphosphate Acid, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate Phosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine 5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, N6-methylguanosine-5'-triphosphate, pseudouridine 5'-triphosphate, puromycin-5'-triphosphate, xanthosine-5'-triphosphate. Particularly preferred is a base-modifying nucleotide selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine 5'-triphosphate.In some embodiments, modified nucleosides include pyridin-4-one ribonucleosides, 5-azauridine, 2-thio-5-azauridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethylpseudouridine, 5-propynyluridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethylpseudouridine, 5-taurinomethyl-2-thiouridine, 1-taurinomethyl 4-thiouridine, 5-methyluridine, 1-methylpseudouridine, 4-thio-1-methylpseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydro-pseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

[0242] In some embodiments, the modified nucleoside is 5-azacytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methylpseudoisocytidine, pyrrolopseudoisocytidine, pyrrolopseudoisocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thiopseudoisocytidine, 4-thio-1-methylcytidine, 5-hydroxymethylcytidine, 1-methylpseudoisocytidine, pyrrolopseudoiso ... 2-methyl-5-methylcytidine, 4-hydroxymethylcytidine, 1-methyl-5-methylcytidine, 4-hydroxymethylcytidine, 1-methyl-5-methylcytidine, 4-hydroxymethylcytidine, 1-methyl-5-methylcytidine, 4-hydroxymethylcytidine, 1-methyl-5-methylcytidine, 4-hydroxymethylcytidine, 1-methyl-5-methylcytidine, 4-hydroxymethylcytidine, 1-methyl-5-methylcytidine, 4-hydroxymethylcyt These include rupsoidoisocytidine, 4-thio-1-methyl-1-deazapseudoisocytidine, 1-methyl-1-deazapseudoisocytidine, zebularine, 5-azazebularine, 5-methylzebularine, 5-aza-2-thiozebularine, 2-thiozebularine, 2-methoxycytidine, 2-methoxy-5-methylcytidine, 4-methoxypseudoisocytidine, and 4-methoxy-1-methylpseudoisocytidine.

[0243] In other embodiments, the modified nucleoside is 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyl adenosine N6-(cis-hydroxyisopentenyl)adenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthioadenine, and 2-methoxyadenine. In other embodiments, modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thioguanosine.

[0244] In some embodiments, the nucleotide is modified on the major groove face and may include substituting the hydrogen at C-5 of uracil with a methyl or halo group. In specific embodiments, the modified nucleoside is 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine, 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, or 5'-O-(1-thiophosphate)-pseudouridine.

[0245] In further embodiments, the modified RNA may comprise a nucleoside modification selected from 6-azacytidine, 2-thiocytidine, α-thiocytidine, pseudoisocytidine, 5-aminoallyluridine, 5-iodouridine, N1-methylpseudouridine, 5,6-dihydrouridine, α-thiouridine, 4-thiouridine, 6-azauridine, 5-hydroxyuridine, deoxythymidine, 5-methyluridine, pyrrolocytidine, inosine, α-thioguanosine, 6-methylguanosine, 5-methylcytidine, 8-oxoguanosine, 7-deazaguanosine, N1-methyladenosine, 2-amino-6-chloropurine, N6-methyl-2-aminopurine, pseudoisocytidine, 6-chloropurine, N6-methyladenosine, α-thioadenosine, 8-azidoadenosine, 7-deazaadenosine.

[0246] In certain preferred embodiments, the RNA includes a modified nucleoside in place of at least one (eg, all) uridines.

[0247] The term "uracil" as used herein refers to one of the nucleobases that can occur in RNA nucleic acids. The structure of uracil is as follows: [ka] As stated above.

[0248] As used herein, the term "uridine" refers to one of the nucleosides present in RNA. The structure of uridine is: [ka] As stated above.

[0249] The structure of UTP (uridine 5'-triphosphate) is: [ka] As stated above.

[0250] The structure of pseudo-UTP (pseudouridine 5'-triphosphate) is: [ka] As stated above.

[0251] "Pseudouridine" is an example of a modified nucleoside that is an isomer of uridine in which uracil is attached to the pentose ring via a carbon-carbon bond rather than a nitrogen-carbon glycosidic bond.

[0252] Another example of a modified nucleoside is N1-methyl-pseudouridine (m1ψ), whose structure is: [ka] As stated above.

[0253] N1-methylpseudoUTP is: [ka] It has the following structure.

[0254] Another exemplary modified nucleoside is 5-methyluridine (m5U), which is: [ka] It has the following structure.

[0255] In certain preferred embodiments, one or more uridines in the RNA described herein are replaced with a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[0256] In certain preferred embodiments, the RNA comprises a modified nucleoside in place of at least one uridine, hi some embodiments, the RNA comprises a modified nucleoside in place of each uridine.

[0257] In certain preferred embodiments, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleoside comprises 5-methyl-uridine (m5U). In some embodiments, the RNA may comprise two or more modified nucleosides, wherein the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (ψ) and N1-methyl-pseudouridine (m1ψ). In some embodiments, modified nucleosides include pseudouridine (ψ) and 5-methyl-uridine (m5U). In some embodiments, modified nucleosides include N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U). In some embodiments, modified nucleosides include pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0258] In certain preferred embodiments, the modified nucleoside that replaces one or more, e.g., all, uridines in the RNA is 3-methyluridine (m 3 U), 5-methoxyuridine (mo5U), 5-azauridine, 6-azauridine, 2-thio-5-azauridine, 2-thiouridine (s 2 U), 4-thiouridine (s 4 U), 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine (ho 5 U), 5-aminoallyl uridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5U), 5-carboxymethyluridine (cm 5 U), 1-carboxymethylpseudouridine, 5-carboxyhydroxymethyluridine (chm 5 U), 5-carboxyhydroxymethyluridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyluridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-aminomethyl-2-thiouridine (nm 5 s 2 U), 5-methylaminomethyluridine (mnm 5 U), 1-ethylpseudouridine, 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-selenouridine (mnm 5 se 2 U), 5-carbamoylmethyluridine (ncm 5 U), 5-carboxymethylaminomethyluridine (cmnm 5 U) 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyluridine, 1-propynylpseudouridine, 5-taurinomethyluridine (τm5U), 1-taurinomethylpseudouridine, 5-taurinomethyl-2-thiouridine (τm 5 s 2 U), 1-taurinomethyl-4-thiopseudouridine), 5-methyl-2-thiouridine (m 5 s 2 U), 1-methyl-4-thiopseudouridine (m 1 s 4 ψ), 4-thio-1-methylpseudouridine, 3-methylpseudouridine (m 3 ψ), 2-thio-1-methylpseudouridine, 1-methyl-1-deazapseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m 5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U) 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thiouridine, 2'-O-methyluridine (Um), 5,2'-O-dimethyluridine (m 5 Um), 2'-O-methylpseudouridine (ψm), 2-thio-2'-O-methyluridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm 5 Um), 3,2'-O-dimethyluridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm 5The modified uridine may be any one or more of: uridine (Um), 1-thiouridine, deoxythymidine, 2'-F-aruridine, 2'-F-uridine, 2'-OH-aruridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art. In some embodiments, the first and second RNA molecules comprise a modified nucleoside in place of at least one uridine, preferably in place of each uridine; preferably, the modified nucleosides are independently selected from pseudouridine (ψ), N-methyl-pseudouridine (mψ), and 5-methyl-uridine (mU). In some embodiments, the first RNA molecule comprises a modified nucleoside in place of at least one uridine, preferably in place of each uridine, but the second RNA molecule does not. Preferably, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U). In some embodiments, the second RNA molecule is not the first RNA molecule and comprises a modified nucleoside in place of at least one uridine, preferably in place of each uridine. Preferably, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U).

[0259] In one embodiment, the RNA contains other modified nucleosides or further modified nucleosides, such as modified cytidines as described above. For example, in one embodiment, cytidine is partially or completely, preferably completely, replaced by 5-methylcytidine in the RNA. In one embodiment, the RNA contains 5-methylcytidine and one or more selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U). In one embodiment, the RNA contains 5-methylcytidine and N1-methylpseudouridine (m1ψ). In some embodiments, the RNA contains 5-methylcytidine in place of each cytidine and N1-methylpseudouridine (m1ψ) in place of each uridine.

[0260] First RNA molecule The first RNA molecule comprises an open reading frame encoding a functional RNA-dependent RNA polymerase (replicase). In one embodiment, the first RNA molecule is a replicon, which can be replicated by the replicase it encodes. In this embodiment, the first RNA molecule comprises a nucleotide sequence that can be recognized by the replicase so that the RNA is replicated. The first RNA molecule may further comprise other features.

[0261] In one embodiment, the first RNA molecule is not replicated by its encoded replicase, preferably not by any replicase from a self-replicating virus, In this embodiment, the first RNA molecule may lack sequences normally required for replication, as described herein.

[0262] In one embodiment, the first RNA is an mRNA, preferably including additional features of typical eukaryotic mRNA, such as a 5' cap or poly(A) tail, as described herein.

[0263] In one embodiment, the first RNA molecule comprises an open reading frame encoding a functional replicase and a further open reading frame encoding a protein of interest.

[0264] Functional replicase The term "nonstructural proteins" refers to proteins that are encoded by the virus but are not part of the virus particle. This term typically includes various enzymes and transcription factors that viruses use to replicate themselves, such as RNA replicase and other template-specific polymerases. The term "nonstructural proteins" includes all co- or post-translationally modified forms of nonstructural proteins, including carbohydrate-modified (e.g., glycosylated) and lipid-modified forms, and preferably refers to "alphavirus nonstructural proteins."

[0265] In some embodiments, the term "alphavirus nonstructural proteins" refers to any one or more of the individual nonstructural proteins (nsP1, nsP2, nsP3, nsP4) from an alphavirus, or to a polyprotein comprising the polypeptide sequences of multiple nonstructural proteins from an alphavirus. In some embodiments, "alphavirus nonstructural proteins" refers to nsP123 and / or nsP4. In other embodiments, "alphavirus nonstructural proteins" refers to nsP1234. In one embodiment, the protein of interest encoded by the open reading frame includes nsP1, nsP2, nsP3, and nsP4 all as a single, optionally cleavable polyprotein, i.e., nsP1234. In one embodiment, the protein of interest encoded by the open reading frame is a single, optionally cleavable polyprotein composed of nsP1, nsP2, and nsP3, i.e., nsP123. In this embodiment, nsP4 is an additional protein of interest and may be encoded by an additional open reading frame.

[0266] In some embodiments, the nonstructural proteins are capable of forming complexes or associations, for example, within a host cell. In some embodiments, "alphavirus nonstructural proteins" refers to a complex or association of nsP123 (synonym: P123) and nsP4. In some embodiments, "alphavirus nonstructural proteins" refers to a complex or association of nsP1, nsP2, and nsP3. In some embodiments, "alphavirus nonstructural proteins" refers to a complex or association of nsP1, nsP2, nsP3, and nsP4. In some embodiments, "alphavirus nonstructural proteins" refers to a complex or association of one or more selected from the group consisting of nsP1, nsP2, nsP3, and nsP4. In some embodiments, the alphavirus nonstructural proteins include at least nsP4.

[0267] The term "complex" or "association" refers to two or more identical or different protein molecules in spatial proximity. The proteins that make up a complex are preferably in direct or indirect physical or physicochemical contact. A complex or association can be composed of multiple different proteins (heteromultimers) and / or multiple copies of a particular protein (homomultimers). In the context of alphavirus nonstructural proteins, the term "complex or association" refers to an assembly of at least two protein molecules, at least one of which is an alphavirus nonstructural protein. A complex or association can be composed of multiple copies of a particular protein (homomultimers) and / or multiple different proteins (heteromultimers). In the context of multimers, "multiple" means one or more, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10.

[0268] The term "functional nonstructural protein" includes nonstructural proteins with replicase function. Thus, "functional nonstructural protein" includes alphavirus replicases. "Replicase function" includes the function of an RNA-dependent RNA polymerase (RdRP), i.e., an enzyme capable of catalyzing the synthesis of (-)-strand RNA based on a (+)-strand RNA template, and / or the function of an enzyme capable of catalyzing the synthesis of (+)-strand RNA based on a (-)-strand RNA template. Thus, the term "functional nonstructural protein" can refer to a protein or complex that synthesizes (-)-strand RNA using (+)-strand (e.g., genomic) RNA as a template, a protein or complex that synthesizes new (+)-strand RNA using the (-)-strand complement of genomic RNA as a template, and / or a protein or complex that synthesizes a subgenomic transcript using a fragment of the (-)-strand complement of genomic RNA as a template. Functional nonstructural proteins may further possess one or more additional functions, such as, for example, a protease (for self-cleavage), a helicase, a terminal adenyltransferase (for poly(A) tail addition), a methyltransferase and a guanylyltransferase (to add a 5' cap to nucleic acids), a nuclear localization site, a triphosphatase (Gould et al., 2010, Antiviral Res. 87:111-124; Rupp et al., 2015, J. Gen. Virol. 96:2483-500), etc.

[0269] In some embodiments, the term "functional nonstructural protein" is synonymous with "functional replicase."

[0270] The term "replicase" includes RNA-dependent RNA polymerases. According to the present invention, the term "replicase" includes "alphavirus replicases," which include RNA-dependent RNA polymerases from native alphaviruses (alphaviruses occurring in nature) and RNA-dependent RNA polymerases from mutants or derivatives of alphaviruses, such as attenuated alphaviruses. The term "replicase" also includes RNA-dependent RNA polymerases from other self-replicating viruses, such as autonomously replicating single-stranded RNA viruses, and optionally positive-sense single-stranded RNA viruses (e.g., alphaviruses, flaviviruses, etc.).

[0271] The term "replicase" includes all variants, particularly post-translationally modified variants, conformations, isoforms, and homologs, of alphavirus replicases expressed by cells infected with alphaviruses or by cells into which nucleic acid encoding alphavirus replicases has been introduced. Furthermore, the term "replicase" includes all forms of replicase that have been produced or can be produced by recombinant methods. For example, replicases that contain tags that facilitate detection and / or purification of the replicase in the laboratory, such as myc tags, HA tags, or oligohistidine tags (His tags), can be produced by recombinant methods.

[0272] Optionally, the alphavirus replicase is further functionally defined by its ability to bind to any one or more of alphavirus conserved sequence element 1 (CSE1) or its complement, conserved sequence element 2 (CSE2) or its complement, conserved sequence element 3 (CSE3) or its complement, and conserved sequence element 4 (CSE4) or its complement. Preferably, the replicase is capable of binding to CSE2 (i.e., the (+) strand) and / or CSE4 (i.e., the (+) strand), or the complement of CSE1 (i.e., the (-) strand) and / or the complement of CSE3 (i.e., the (-) strand).

[0273] The source of the alphavirus replicase is not limited to a particular alphavirus. In a preferred embodiment, the alphavirus replicase comprises nonstructural proteins from Semliki Forest virus, including naturally occurring Semliki Forest virus and mutants or derivatives of Semliki Forest virus, such as attenuated Semliki Forest virus. In another preferred embodiment, the alphavirus replicase comprises nonstructural proteins from Sindbis virus, including naturally occurring Sindbis virus and mutants or derivatives of Sindbis virus, such as attenuated Sindbis virus. In another preferred embodiment, the alphavirus replicase comprises nonstructural proteins from Venezuelan equine encephalitis virus (VEEV), including naturally occurring VEEV and mutants or derivatives of VEEV, such as attenuated VEEV. In another preferred embodiment, the alphavirus replicase comprises nonstructural proteins from Chikungunya virus (CHIKV), including naturally occurring CHIKV and mutants or derivatives of CHIKV, such as attenuated CHIKV.

[0274] A replicase can also comprise nonstructural proteins from multiple viruses, e.g., multiple alphaviruses. Thus, heterologous complexes or aggregates comprising alphavirus nonstructural proteins and having replicase function are also encompassed by the present invention. By way of example only, a replicase can comprise one or more nonstructural proteins (e.g., nsP1, nsP2) from a first alphavirus and one or more nonstructural proteins (nsP3, nsP4) from a second alphavirus. The nonstructural proteins from multiple different alphaviruses can be encoded by separate open reading frames or can be encoded by a single open reading frame as a polyprotein (e.g., nsP1234).

[0275] In some embodiments, the functional nonstructural proteins are capable of forming membranous replication complexes and / or vacuoles in cells in which the functional nonstructural proteins are expressed.

[0276] When a functional nonstructural protein, i.e., a nonstructural protein with replicase function, is encoded by a nucleic acid molecule of the present invention, the subgenomic promoter of the replicon, if present, is preferably compatible with the replicase. Compatibility here means that the replicase can recognize the subgenomic promoter. In one embodiment, compatibility is achieved when the subgenomic promoter is native to the virus from which the replicase is derived, i.e., when these sequences naturally originate from the same virus. In another embodiment, the subgenomic promoter is not native to the virus from which the viral replicase is derived, but only if the viral replicase can recognize the subgenomic promoter. In other words, the replicase is compatible with the subgenomic promoter (interviral compatibility). Examples of interviral compatibility involving subgenomic promoters and replicases from different alphaviruses are known in the art. Any combination of subgenomic promoter and replicase is possible as long as interviral compatibility exists. Interviral compatibility can be easily tested by one skilled in the art practicing this invention by incubating the replicase to be tested with RNA bearing the subgenomic promoter to be tested under conditions suitable for RNA synthesis from the subgenomic promoter. If a subgenomic transcript is prepared, the subgenomic promoter and replicase are determined to be compatible. Various examples of compatibility between viruses are known.

[0277] The replicon is preferably capable of replication by functional nonstructural proteins. In particular, an RNA replicon encoding a functional nonstructural protein can be replicated by the functional nonstructural protein encoded by the replicon. In a preferred embodiment, the second RNA molecule comprises an miRNA and an open reading frame encoding a protein of interest. This embodiment is particularly suitable for some methods of producing a protein of interest together with an miRNA according to the present invention. The additional open reading frame encoding the protein of interest is preferably located downstream of the 5' replication recognition sequence and upstream of the miRNA. In one embodiment, the additional open reading frame is located downstream of the miRNA. In one embodiment, the second RNA molecule comprises one or more open reading frames encoding one or more proteins of interest.

[0278] One or more additional open reading frames encoding one or more proteins of interest are typically controlled by (a) a subgenomic promoter.

[0279] replicable RNA A replicable RNA molecule or replicable RNA (rRNA) is RNA that can be replicated by an RNA-dependent RNA polymerase (replicase) by containing a nucleotide sequence that is recognized and replicated by the RNA-dependent RNA polymerase. Replication of rRNA produces one or more identical or essentially identical copies of the rRNA without a DNA intermediate. "Without a DNA intermediate" means that a deoxyribonucleic acid (DNA) copy or complementary strand of the rRNA is not formed during the process of forming the rRNA copy, and / or a deoxyribonucleic acid (DNA) molecule is not used as a template during the process of forming the rRNA copy or its complementary strand. Replicase function is typically provided by functional nonstructural proteins, such as functional alphavirus nonstructural proteins.

[0280] According to the present invention, at least the second RNA molecule is a replicable RNA molecule.The second RNA molecule according to the present invention is preferably replicated in trans, for example, by the functional replicase encoded by the first RNA molecule, not by the replicase not encoded by the second RNA molecule.Preferably, the second RNA molecule does not contain a functional replicase.The first RNA molecule may also be a replicable RNA molecule.Preferably, any additional RNA molecule, for example, a third RNA molecule, is a replicable RNA molecule.

[0281] The terms "RNA replicon," "replicon," "replicable RNA molecule," and "replicable RNA" can be used interchangeably.

[0282] According to the present invention, the terms "replicable" and "replicable" generally refer to the ability to prepare one or more identical or essentially identical copies of a nucleic acid. When used in conjunction with the term "replicase" (e.g., "capable of being replicated by a replicase"), the terms "capable of being replicated" and "capable of being replicated" refer to the functional properties of a nucleic acid molecule (e.g., an RNA replicon) for a replicase. These functional properties include at least one of (i) the ability of the replicase to recognize the replicon and (ii) the ability of the replicase to act as an RNA-dependent RNA polymerase (RdRP). Preferably, the replicase (i) recognizes the replicon and (ii) can act as an RNA-dependent RNA polymerase. In a preferred embodiment, the term "replicable" means that the RNA contains one or more sequences that can be recognized or bound by a functional replicase, such as 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 / or conserved sequence element 4 (CSE4) or its complementary sequence.

[0283] The term "capable of recognizing" means that the replicase is capable of physically associating with the replicon, preferably, that the replicase is capable of binding to the replicon, typically non-covalently. The term "binding" can mean that the replicase has the ability to bind to any one or more of conserved sequence element 1 (CSE1) or its complementary sequence (if contained in the replicon), conserved sequence element 2 (CSE2) or its complementary sequence (if contained in the replicon), conserved sequence element 3 (CSE3) or its complementary sequence (if contained in the replicon), and conserved sequence element 4 (CSE4) or its complementary sequence (if contained in the replicon). Preferably, the replicase is capable of binding to CSE2 (i.e., the (+) strand) and / or CSE4 (i.e., the (+) strand), or to the complementary strand of CSE1 (i.e., the (-) strand) and / or the complementary strand of CSE3 (i.e., the (-) strand).

[0284] In one embodiment, the phrase "capable of acting as an RdRP" means that the replicase can catalyze the synthesis of a (-) strand complementary to the viral genomic (+) strand RNA, where the (+) strand RNA serves as a template, and / or that the replicase can catalyze the synthesis of a (+) strand of the viral genomic (-) strand RNA, where the (-) strand RNA serves as a template. In general, the phrase "capable of acting as an RdRP" also includes that the replicase can catalyze the synthesis of a (+) strand subgenomic transcript for which the (-) strand RNA serves as a template, where synthesis of the (+) strand subgenomic transcript typically initiates at a subgenomic promoter. In one embodiment, the virus is an alphavirus.

[0285] The terms "capable of binding" and "acting as an RdRP" refer to the ability under normal physiological conditions. In particular, they refer to the conditions in cells expressing nonstructural proteins or cells into which nucleic acids encoding functional nonstructural proteins have been introduced. The cells are preferably eukaryotic cells. The ability to bind and / or act as an RdRP can be experimentally tested, for example, in a cell-free in vitro system or in eukaryotic cells. Optionally, the eukaryotic cells are cells of a species infected by the specific virus from which the replicase originates. For example, when a viral replicase derived from a specific virus that infects humans is used, the normal physiological conditions are the conditions in human cells. More preferably, the eukaryotic cells (e.g., human cells) are derived from the same tissue or organ as that infected by the specific virus from which the replicase originates.

[0286] Separation of replication-required sequence elements and protein-coding regions In one embodiment, the first and / or second replicable RNA (rRNA) comprises a modified regulatory region of a self-replicating, single-stranded, positive-sense virus that contains a sequence change compared to a reference modified regulatory region, which sequence change restores or improves the function of the rRNA molecule containing at least one modified nucleotide. These changes can be identified by the methods described herein for identifying such sequence changes. In one embodiment, the modified regulatory region is an alphavirus regulatory region, e.g., a 5' or 3' regulatory region. In one embodiment, the 5' regulatory region is a VEEV alphavirus 5' regulatory region.

[0287] The open reading frame encoding nsP1234 overlaps with the 5' replication recognition sequence of the alphavirus genome (the coding sequence for nsP1) and also overlaps with the subgenomic promoter that typically contains CSE3 (the coding sequence for nsP4), making the development of versatile alphavirus-derived vectors challenging.

[0288] The rRNAs described herein generally contain sequence elements necessary for replication by a replicase, particularly a 5' replication recognition sequence. In one embodiment, the coding sequence for one or more nonstructural proteins is under the control of an IRES, and thus the IRES is located upstream of the coding sequence for the nonstructural proteins. Thus, in one embodiment, the 5' replication recognition sequence, which normally overlaps with the coding sequence for the N-terminal fragment of an alphavirus nonstructural protein, is located upstream of the IRES and does not overlap with the coding sequence for one or more nonstructural proteins.

[0289] In one embodiment, the coding sequence for a 5' replication recognition sequence, such as the nsP1 coding sequence, is fused in frame to the gene of interest upstream of the IRES.

[0290] In one embodiment, the 5' replication recognition sequence does not encode a protein or fragment thereof, such as an alphavirus nonstructural protein or fragment thereof. Thus, in the rRNA according to the present invention, the sequence elements necessary for replication by the replicase and the protein coding region may be separated. This separation may be achieved by removing at least one start codon in the 5' replication recognition sequence compared to the native viral genomic RNA, e.g., the native alphavirus genomic RNA.

[0291] Thus, the rRNA can comprise a 5' replication recognition sequence, which is characterized by the removal of at least one start codon compared to a native viral 5' replication recognition sequence, e.g., a native alphavirus 5' replication recognition sequence.

[0292] A 5' replication recognition sequence characterized by the removal of at least one initiation codon compared to the 5' replication recognition sequence of a native virus may be referred to herein as a "modified 5' replication recognition sequence" or a "5' replication recognition sequence according to the invention." As described herein below, a 5' replication recognition sequence according to the invention may optionally be characterized by the presence of one or more additional nucleotide changes, such as those detected by the methods of the invention.

[0293] In one embodiment, the rRNA comprises a 3' replication recognition sequence. The 3' replication recognition sequence is a nucleic acid sequence that can be recognized by a functional replicase. In other words, a functional replicase can recognize the 3' replication recognition sequence. Preferably, the 3' replication recognition sequence is located at the 3' end of the replicon (if the replicon does not contain a poly(A) tail) or immediately upstream of the poly(A) tail (if the replicon contains a poly(A) tail). In one embodiment, the 3' replication recognition sequence consists of or comprises CSE4.

[0294] In one embodiment, the 5' and 3' replication recognition sequences are capable of directing replication of rRNA according to the present invention in the presence of a functional replicase. Thus, these recognition sequences, alone or preferably together, direct replication of rRNA in the presence of a functional replicase.

[0295] Preferably, the first rRNA provides a functional replicase capable of recognizing both the 5' and 3' replication recognition sequences of each rRNA. In one embodiment, this is achieved when the 3' replication recognition sequence is native to the alphavirus from which the functional alphavirus replicase is derived, and the 5' replication recognition sequence is native to the alphavirus from which the functional alphavirus replicase is derived, or is a variant of the 5' replication recognition sequence native to the alphavirus from which the functional alphavirus replicase is derived. "Native" means that the natural origin of these sequences is the same alphavirus. In another embodiment, the 5' replication recognition sequence and / or the 3' replication recognition sequence is not native to the alphavirus from which the functional alphavirus replicase is derived, provided that the functional alphavirus replicase is capable of recognizing both the 5' and 3' replication recognition sequences of each rRNA. In other words, the functional alphavirus replicase is compatible with the 5' and 3' replication recognition sequences. A functional alphavirus replicase is said to be compatible (inter-virus compatible) if the non-naturally occurring functional alphavirus replicase can recognize the respective sequences or sequence elements. Any combination of functional alphavirus replicase, (3' / 5') replication recognition sequence, and CSE is possible, as long as inter-virus compatibility exists. Inter-virus compatibility can be easily tested by one skilled in the art practicing the present invention by incubating the functional alphavirus replicase to be tested with RNA having the 3' and 5' replication recognition sequences to be tested under conditions suitable for RNA replication, e.g., in a suitable host cell. If replication occurs, the (3' / 5') replication recognition sequence and the functional alphavirus replicase are determined to be compatible.In some cases, the replicase may be derived from a self-replicating single-stranded RNA virus, such as a positive-sense single-stranded RNA virus (e.g., alphavirus, flavivirus, etc.), in which case the 5' replication recognition sequence and 3' replication recognition sequence of each rRNA may also be derived from the same self-replicating single-stranded RNA virus, such as the same positive-sense single-stranded RNA virus (e.g., alphavirus, flavivirus, etc.).

[0296] Removal of at least one initiation codon within the 5' replication recognition sequence provides several advantages. * If there is no initiation codon in the nucleic acid sequence encoding the N-terminal fragment of nsP1, * is not translated. * Since the open reading frame encoding the protein of interest ("GOI 2") is not translated, the open reading frame encoding the protein of interest ("GOI 2") is the most upstream open reading frame accessible to ribosomes. Therefore, if rRNA is present in the cell, translation will begin at the first AUG of the open reading frame (RNA) encoding the protein of interest.

[0297] Removal of at least one start codon can be achieved by any suitable method known in the art. For example, a suitable DNA molecule encoding an rRNA characterized by a removed start codon can be designed in silico and then synthesized in vitro (gene synthesis). Alternatively, a suitable DNA molecule can be obtained by site-directed mutagenesis of the DNA sequence encoding the rRNA. In either case, the respective DNA molecule serves as a template for in vitro transcription, thereby obtaining the rRNA according to the present invention.

[0298] The removal of at least one start codon compared to the native 5' replication recognition sequence is not particularly limited and may be selected from any nucleotide modification, including a substitution of one or more nucleotides (at the DNA level, including substitution of A and / or T and / or G from the start codon), a deletion of one or more nucleotides (at the DNA level, including deletion of A and / or T and / or G from the start codon), and an insertion of one or more nucleotides (at the DNA level, including insertion of one or more nucleotides between A and T and / or T and G from the start codon). Regardless of whether the nucleotide modification is a substitution, insertion, or deletion, the nucleotide modification must not result in the formation of a new start codon (for example, an insertion at the DNA level must not be an insertion of ATG).

[0299] The 5' replication recognition sequence of an rRNA characterized by the removal of at least one initiation codon (i.e., the modified 5' replication recognition sequence of the present invention) is preferably a variant of the 5' replication recognition sequence of an alphavirus genome found in nature. In one embodiment, the modified 5' replication recognition sequence of the present invention is preferably characterized by having at least 80% sequence identity, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% sequence identity to the 5' replication recognition sequence of at least one naturally occurring alphavirus genome.

[0300] In one embodiment, the 5' replication recognition sequence of an rRNA, which may be characterized by the removal of at least one start codon, comprises a sequence homologous to the 5' end of an alphavirus, i.e., approximately 250 nucleotides of the 5' end of the alphaviral genome. In a preferred embodiment, it comprises a sequence homologous to the 5' end of an alphavirus, i.e., approximately 250-500, preferably approximately 300-500, nucleotides of the 5' end of the alphaviral genome. By "5' end of the alphaviral genome" is meant a nucleic acid sequence beginning with and including the most upstream nucleotide of the alphaviral genome. In other words, the most upstream nucleotide of the alphaviral genome is nucleotide number 1; for example, "250 nucleotides of the 5' end of the alphaviral genome" means nucleotides 1 through 250 of the alphaviral genome. In one embodiment, the 5' rRNA replication recognition sequence is characterized by having at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% sequence identity to at least 250 nucleotides of the 5' terminus of at least one naturally occurring alphavirus genome, including, for example, 250, 300, 400, or 500 nucleotides.

[0301] The 5' replication recognition sequences of naturally occurring alphaviruses are typically characterized by at least one initiation codon and / or conserved secondary structure motifs. For example, the native 5' replication recognition sequence of Semliki Forest virus (SFV) is composed of five specific AUG triplets. According to Frolov et al., 2001, RNA 7:1638-1651, MFOLD analysis revealed that the native 5' replication recognition sequence of Semliki Forest virus is predicted to form four stem-loops (SLs), designated stem-loops 1 through 4 (SL1, SL2, SL3, and SL4). According to Frolov et al., MFOLD analysis also revealed that the native 5' replication recognition sequence of another alphavirus, Sindbis virus, is predicted to form four stem-loops: SL1, SL2, SL3, and SL4.

[0302] The 5' end of the alphaviral genome is known to contain sequence elements that allow replication of the alphaviral genome by a functional alphaviral replicase. In one embodiment of the present invention, the 5' rRNA replication recognition sequence contains a sequence homologous to alphavirus conserved sequence element 1 (CSE1) and / or a sequence homologous to alphavirus conserved sequence element 2 (CSE2).

[0303] Conserved sequence element 2 (CSE2) of alphavirus genomic RNA is typically represented by SL3 and SL4, preceded by SL2, which contains the native initiation codon encoding at least the first amino acid residue of the alphavirus nonstructural protein nsP1. However, as used herein, in some embodiments, conserved sequence element 2 (CSE2) of alphavirus genomic RNA refers to the region spanning from SL2 to SL4 and including the native initiation codon encoding the first amino acid residue of the alphavirus nonstructural protein nsP1. In preferred embodiments, rRNAs according to the present invention comprise CSE2 or a sequence homologous to CSE2. In one embodiment, rRNAs according to the present invention comprise a sequence homologous to CSE2, which sequence is preferably characterized by 80% or greater, preferably 85% or greater, more preferably 90% or greater, and even more preferably 95% or greater sequence identity to the sequence of CSE2 of at least one alphavirus found in nature.

[0304] In one embodiment, the 5' replication recognition sequence comprises a sequence homologous to the CSE2 of an alphavirus. The CSE2 of an alphavirus may comprise a fragment of the open reading frame of a nonstructural protein from an alphavirus.

[0305] Thus, in one embodiment, the rRNA of the present invention is characterized in that it contains a sequence homologous to a nonstructural protein open reading frame or fragment thereof from an alphavirus. The sequence homologous to a nonstructural protein open reading frame or fragment thereof is typically a variant of a naturally occurring alphavirus nonstructural protein open reading frame or fragment thereof. In one embodiment, the sequence homologous to a nonstructural protein open reading frame or fragment thereof is characterized in that it has at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% sequence identity to at least one naturally occurring nonstructural protein open reading frame or fragment thereof from an alphavirus.

[0306] In one embodiment, the sequence homologous to the open reading frame of a nonstructural protein contained in the rRNA of the present invention does not contain the native start codon of the nonstructural protein, and more preferably does not contain any start codons for the nonstructural protein. In one embodiment, the sequence homologous to CSE2 is characterized in that all start codons have been removed compared to the native alphavirus CSE2 sequence. Thus, the sequence homologous to CSE2 preferably does not contain a start codon.

[0307] If a sequence homologous to an open reading frame does not contain an initiation codon, the sequence homologous to an open reading frame is not itself an open reading frame because it cannot function as a translation template.

[0308] In one embodiment, the 5' replication recognition sequence comprises a sequence homologous to an open reading frame or a fragment thereof of a nonstructural protein derived from an alphavirus, wherein the sequence homologous to an open reading frame or a fragment thereof of a nonstructural protein derived from an alphavirus is characterized by having at least one start codon removed compared to the native alphavirus sequence.

[0309] In one embodiment, the sequence homologous to an alphavirus-derived nonstructural protein open reading frame or fragment thereof is characterized in that at least the native start codon of the nonstructural protein open reading frame has been removed, preferably including the removal of at least the native start codon of the open reading frame encoding nsP1.

[0310] The native start codon is the AUG base triplet that initiates translation on a ribosome within a host cell when the RNA is present in the host cell. 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 derived from a eukaryotic species that is the natural host for a particular alphavirus containing the native alphavirus 5' replication recognition sequence. In one embodiment, the host cell is a BHK21 cell derived from cell line "BHK21[C13] (ATCC® CCL10®)" available from the American Type Culture Collection, Manassas, Virginia, USA.

[0311] The genomes of many alphaviruses have been completely sequenced and are publicly available, as are the sequences of the nonstructural proteins encoded by these genomes. This sequence information allows the natural start codon to be determined in silico.

[0312] In one embodiment, the sequence homologous to the nonstructural protein open reading frame or fragment thereof from an alphavirus is characterized in that one or more start codons other than the native start codon of the nonstructural protein open reading frame have been removed. In one embodiment, the nucleic acid sequence is characterized in that the native start codon has been removed. For example, in addition to the removal of the native start codon, any one, two, three, four, or more than four (e.g., five) start codons may be removed.

[0313] When the rRNA of the present invention is characterized by the removal of the native start codon, and optionally one or more start codons other than the native start codon, from the open reading frame of a nonstructural protein, the sequence homologous to the open reading frame is not itself an open reading frame, since it does not function as a template for translation.

[0314] The one or more initiation codons other than the natural initiation codon that are removed in addition to the natural initiation codon are preferably selected from AUG base triplets that have the potential to initiate translation. AUG base triplets that have the potential to initiate translation are sometimes referred to as "cryptic initiation codons." Whether a given AUG base triplet has the potential to initiate translation can be determined in silico or in cell-based in vitro assays.

[0315] In one embodiment, the ability of a given AUG base triplet to initiate translation is determined in silico by examining a nucleotide sequence and determining that the AUG base triplet has the ability to initiate translation if it is part of an AUGG sequence, preferably a Kozak sequence.

[0316] In one embodiment, the ability of a given AUG base triplet to initiate translation is determined in a cell-based in vitro assay. That is, the native start codon is removed, and rRNA containing the given AUG base triplet downstream of the removal of the native start codon is introduced into a host cell. In one embodiment, the host cell is a cell derived from a eukaryotic species that is the natural host for a particular alphavirus containing the native alphavirus 5' replication recognition sequence. In a preferred embodiment, the host cell is a BHK21 cell derived from cell line "BHK21[C13] (ATCC® CCL10®)" available from the American Type Culture Collection, Manassas, Virginia, USA. Preferably, no additional AUG base triplets are present between the removal of the native start codon and the given AUG base triplet. A given AUG base triplet is determined to be capable of initiating translation if translation is initiated at the given AUG base triplet after removal of the native start codon and introduction of rRNA containing the given AUG base triplet into a host cell. Whether translation has been initiated can be determined by any suitable method known in the art. For example, the rRNA may encode a tag downstream of the predetermined AUG base triplet, in frame with the predetermined AUG base triplet, such as a myc tag or an HA tag, which facilitates detection of the translation product (if present). The presence of an expression product bearing the encoded tag can be determined, for example, by Western blotting. In this embodiment, it is preferable that no additional AUG base triplets exist between the predetermined AUG base triplet and the nucleic acid sequence encoding the tag. Cell-based in vitro assays can be performed separately for multiple predetermined AUG base triplets. In either case, it is preferable that no additional AUG base triplets exist between the deletion position of the natural start codon and the predetermined AUG base triplet. This can be achieved by removing all AUG base triplets (if present) between the deletion position of the natural start codon and the predetermined AUG base triplet.This ensures that the given AUG base triplet is the first AUG base triplet downstream of the removal position of the natural start codon.

[0317] Preferably, the 5' replication recognition sequence of the rRNA according to the present invention is characterized in that all potential initiation codons have been removed. Thus, according to the present invention, the 5' replication recognition sequence preferably does not contain an open reading frame that can be translated into a protein.

[0318] In one embodiment, the 5' replication recognition sequence of the rRNA of the present invention is characterized by a secondary structure equivalent to the (predicted) secondary structure of the 5' replication recognition sequence of the viral genomic RNA, and thus the rRNA may contain one or more nucleotide changes that compensate for the disruption of nucleotide pairing within one or more stem-loops introduced by the removal of at least one start codon.

[0319] In one embodiment, the 5' replication recognition sequence of an rRNA of the present invention is characterized by a secondary structure equivalent to that of the 5' replication recognition sequence of an alphavirus genomic RNA. In a preferred embodiment, the 5' replication recognition sequence of an rRNA of the present invention is characterized by a predicted secondary structure equivalent to that of the 5' replication recognition sequence of an alphavirus genomic RNA. In accordance with the present invention, the secondary structure of an RNA molecule is preferably predicted by the RNA secondary structure prediction web server http: / / rna.urmc.rochester.edu / RNAstructureWeb / Servers / Predict1 / Predict1.html.

[0320] The presence or absence of nucleotide pairing inhibition can be identified by comparing the secondary structure or predicted secondary structure of a 5' rRNA replication recognition sequence, characterized by the removal of at least one initiation codon, with a native alphavirus 5' replication recognition sequence. For example, at least one base pair may be missing at a specific position, e.g., within a stem-loop, particularly in the stem of the stem-loop, compared to the native alphavirus 5' replication recognition sequence.

[0321] In one embodiment, one or more stem loops of the 5' replication recognition sequence are not deleted or disrupted. More preferably, stem loops 3 and 4 are not deleted or disrupted. Preferably, none of the stem loops of the 5' replication recognition sequence are deleted or disrupted.

[0322] In one embodiment, the removal of at least one start codon does not disrupt the secondary structure of the 5' replication recognition sequence. In another embodiment, the removal of at least one start codon disrupts the secondary structure of the 5' replication recognition sequence. In this embodiment, the removal of at least one start codon can cause the absence of at least one base pair at a predetermined position, for example, a base pair within a stem loop, compared to the natural 5' replication recognition sequence. If there is no base pair within the stem loop, the removal of at least one start codon is considered to introduce disruption of nucleotide pairing within the stem loop, compared to the natural 5' replication recognition sequence. The base pair within the stem loop is typically a base pair within the stem of the stem loop.

[0323] In one embodiment, an rRNA of the present invention comprises one or more nucleotide changes that compensate for the disruption of nucleotide pairing within one or more stem-loops introduced by the removal of at least one start codon.

[0324] If removal of at least one start codon introduces a disruption in nucleotide pairing within the stem-loop compared to the natural 5' replication recognition sequence, one or more nucleotide changes that are predicted to compensate for the disruption in nucleotide pairing can be introduced, and the resulting or predicted secondary structure can be compared to the natural 5' replication recognition sequence.

[0325] Based on general knowledge and the disclosures herein, those skilled in the art can predict that a specific nucleotide change will compensate for the disruption of nucleotide pairing. For example, if a base pair is disrupted at a specific position in the secondary structure or predicted secondary structure of a 5' replication recognition sequence of an rRNA characterized by the removal of at least one start codon, a nucleotide change that restores the base pair at that position (preferably without reintroducing a start codon) compared to the natural 5' replication recognition sequence is predicted to compensate for the disruption of nucleotide pairing.

[0326] In one embodiment, the 5' replication recognition sequence of the rRNA of the present invention does not overlap with or include a translatable nucleic acid sequence, i.e., a nucleic acid sequence translatable into a peptide or protein, particularly an nsP, particularly nsP1, or any fragment thereof. For a nucleotide sequence to be "translatable," the presence of an initiation codon is required. The initiation codon encodes the N-terminal amino acid residue of the peptide or protein. In one embodiment, the 5' replication recognition sequence of the rRNA of the present invention does not overlap with or include a translatable nucleic acid sequence encoding the N-terminal fragment of nsP1.

[0327] In some scenarios, the rRNA contains at least one subgenomic promoter. In a preferred embodiment, the subgenomic promoter of the rRNA does not overlap or contain a translatable nucleic acid sequence, i.e., a nucleic acid sequence translatable into a peptide or protein, particularly an nsP, particularly nsP4, or any fragment thereof. In one embodiment, the subgenomic promoter of the rRNA does not overlap or contain a translatable nucleic acid sequence encoding the C-terminal fragment of nsP4. For example, an rRNA having a subgenomic promoter that does not overlap or contain a translatable nucleic acid sequence translatable into the C-terminal fragment of nsP4 can be generated by deleting a portion of the nsP4 coding sequence (typically the portion encoding the N-terminal portion of nsP4) and / or removing an AUG base triplet from the non-deleted portion of the nsP4 coding sequence. When an AUG base triplet is removed from the nsP4 coding sequence or a portion thereof, the removed AUG base triplet is preferably a cryptic start codon. Alternatively, if the subgenomic promoter does not overlap with the nucleic acid sequence encoding nsP4, the entire nucleic acid sequence encoding nsP4 can be deleted.

[0328] In one embodiment, the rRNA of the present invention does not contain an open reading frame encoding only the N-terminal fragment of nsP1, and optionally does not contain an open reading frame encoding only the C-terminal fragment of nsP4.

[0329] In some embodiments, the rRNA of the present invention does not contain stem-loop 2 (SL2) at the 5' end of the alphavirus genome. According to Frolov et al. (supra), stem-loop 2 is a conserved secondary structure found at the 5' end of the alphavirus genome, upstream of CSE2, but is not essential for replication.

[0330] The rRNA of the present invention is preferably a single-stranded RNA molecule. The rRNA of the present invention is typically a (+)-strand RNA molecule. In one embodiment, the rRNA of the present invention is an isolated nucleic acid molecule. The rRNA of the present invention contains at least one modified nucleotide, and preferably contains one or more sequence changes, particularly sequence changes that are detected by the method disclosed herein for identifying sequence changes that restore or improve the function of rRNA containing at least one modified nucleotide.

[0331] In one embodiment, the rRNA comprises a modified 5' regulatory region of the self-replicating RNA virus of SEQ ID NO: 1, preferably a modified version of the 5' regulatory region of VEEV Trinidad donkey strain (Accession No. L01442), which contains point mutations at one or more of positions 67, 244, 245, 246, and 248 of the 5' regulatory region (SEQ ID NO: 1). Preferably, the 5' regulatory region also contains a point mutation at position 4 of the 5' regulatory region (SEQ ID NO: 1). The point mutation is preferably G4A, A67C, G244A, C245A, G246A, or C248A.

[0332] Safety Features of Embodiments of the Invention In the present invention, the following features are preferred either alone or in any suitable combination.

[0333] The replicons of the present invention are not particle-forming. This means that after inoculation of the replicon of the present invention into host cells, the host cells do not produce virus particles, such as next-generation virus particles. In one embodiment, the RNA replicon of the present invention does not contain any genetic information encoding viral structural proteins, such as alphavirus structural proteins (e.g., core nucleocapsid protein C, envelope protein P62, and / or envelope protein E1). Preferably, the replicon of the present invention does not contain a viral packaging signal, such as an alphavirus packaging signal. For example, the alphavirus packaging signal contained in the coding region of nsP2 of SFV (White et al., 1998, J. Virol. 72:4320-4326) can be removed, for example, by deletion or mutation. A suitable method for removing the alphavirus packaging signal includes adjusting the codon usage in the coding region of nsP2. Due to the degeneracy of the genetic code, it may be possible to eliminate the function of the packaging signal without affecting the amino acid sequence of the encoded nsP2.

[0334] miRNA The second RNA molecule of the present invention comprises, and optionally encodes, at least one miRNA sequence. The miRNA sequence, when present in a cell, can be excised from the second replicable RNA molecule and can regulate gene expression in the cell. The second RNA molecule of the present invention comprises, and optionally encodes, at least one non-coding RNA sequence. The non-coding RNA sequence, when present in a cell, can be excised from the second replicable RNA molecule and can regulate gene expression in the cell. Preferably, the cell is a eukaryotic cell, preferably a mammalian cell, and preferably a human cell. The cell in which the second RNA is present for excision must typically have the ability to excise the miRNA sequence from the second RNA molecule. For example, it must possess the necessary enzymes, such as Drosha and Dicer. The cell may endogenously (i.e., naturally) express factors (typically enzymes) necessary for excising the non-coding RNA sequence, preferably the miRNA sequence, from the second RNA molecule, or may be modified to express factors (typically enzymes) necessary for excising the non-coding RNA sequence, preferably the miRNA sequence, from the second RNA molecule. Such agents (typically enzymes) can excise a sequence comprising the miRNA sequence from the second RNA molecule and, if necessary, further process that sequence to provide a functional miRNA sequence.

[0335] The miRNA that can be excised from the second RNA molecule in cells usually has adjacent sequences upstream and / or downstream of miRNA.These adjacent sequences function as or contain the recognition sequence for excising miRNA from the second RNA molecule.Therefore, the above-mentioned factor or enzyme can target the recognition sequence in the adjacent sequence and excise miRNA from the second RNA molecule.

[0336] In one embodiment, the upstream and / or downstream flanking sequences of at least one miRNA sequence are naturally occurring flanking sequences, such as sequences flanking a naturally occurring miRNA derived from mouse miR-155. When the miRNA is a naturally occurring miRNA, the flanking sequences may be flanking sequences that naturally flank the miRNA sequence, or flanking sequences that do not naturally flank the miRNA, such as flanking sequences that flank another miRNA sequence. The flanking sequences may be derived from the same organism as the miRNA sequence, or from a different organism.

[0337] In one embodiment, the upstream and / or downstream flanking sequences of at least one miRNA sequence are artificial flanking sequences.

[0338] The term "can regulate gene expression" means that miRNA affects the expression level of a specific gene product, such as a protein encoded by the gene, thereby regulating the level of the protein. Regulation can be complete cessation of gene expression (also called silencing), or attenuation, meaning that gene expression is reduced, or enhancement of expression. Preferably, regulation is achieved by targeting mRNA and inhibiting its translation.

[0339] The target of miRNA is not particularly limited.Preferably, the target is particularly interested in the onset or progression of disease or disorder, and its regulation is useful for the treatment or prevention of these diseases or disorders.The target can also be related to the induction of pluripotency.

[0340] In the present invention, the term "targeting" means binding of miRNA to an at least partially complementary sequence, preferably to the sequence of mRNA, and regulating expression from the mRNA.

[0341] The origin of miRNA sequence can be natural or artificial.Natural miRNA sequence is preferably derived from the same organism as the organism into which the RNA molecule of the present invention is introduced.For example, when the system of the present invention is intended to be introduced into human cells, miRNA is preferably derived from human.

[0342] The artificial pre-miRNA sequence can also comprise a naturally occurring mature miRNA sequence. In this embodiment, for example, the sequence of a naturally occurring mature miRNA is included in the artificial pre-miRNA, with flanking and loop sequences not naturally associated with the mature miRNA.

[0343] The miRNA sequence can also be designed to be at least partially complementary to, e.g., capable of binding to, a particular mRNA of interest, i.e., a target mRNA. Thus, the second RNA molecule comprises an miRNA sequence that is at least partially complementary to (i.e., targets) the mRNA of interest, and can optionally further comprise flanking sequences as described herein.

[0344] The terms "mature miRNA" or "functional miRNA" are used interchangeably in this application and refer to approximately 22-nucleotide miRNAs that can directly regulate gene expression by binding to a target, e.g., a target mRNA, via protein binding.

[0345] In some embodiments, the miRNA sequence contained in the second RNA molecule may be 10 to 200 nucleotides in length, optionally 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, or 20 to 30 nucleotides in length, optionally 10 to 50, preferably 10 to 30 nucleotides in length.

[0346] At least one open reading frame encoding at least one gene product of interest In one embodiment, the first RNA and / or second RNA, preferably the second RNA molecule, of the present invention comprises at least one open reading frame encoding a gene product of interest, such as a protein of interest. Preferably, the protein of interest is encoded by a heterologous nucleic acid sequence. A gene encoding a protein of interest is synonymously referred to as a "gene of interest" or a "transgene." In various embodiments, the protein of interest is encoded by a heterologous nucleic acid sequence. In accordance with the present invention, the term "heterologous" preferably refers to a nucleic acid sequence that is not naturally functionally or structurally associated with a viral nucleic acid sequence, e.g., an alphavirus nucleic acid sequence.

[0347] In some embodiments, the first and / or second RNAs of the present invention may contain multiple open reading frames encoding proteins of interest, each of which may be independently selected to be under the control of a subgenomic promoter or not. Alternatively, the polyprotein or fusion polypeptide may contain individual polypeptides separated by a 2A self-cleaving peptide (e.g., from the foot-and-mouth disease virus 2A protein), a protease cleavage site, or an intein.

[0348] Location of at least one open reading frame encoding a protein of interest The first and second RNAs are suitable for expressing one or more genes encoding proteins of interest, optionally under the control of a subgenomic promoter. Various embodiments are possible. One or more open reading frames encoding each protein of interest can be present on the first and / or second RNA, preferably the second RNA. The most upstream open reading frame of each RNA is referred to as the "first open reading frame." In one embodiment, on the first RNA, one or more open reading frames encoding proteins of interest are located downstream of an open reading frame encoding a functional nonstructural protein. In one embodiment, the first open reading frame encoding a protein of interest is located downstream of the 5' replication recognition sequence, and in the case of the first RNA, optionally downstream of an open reading frame encoding one or more nonstructural proteins from a self-replicating virus. In one embodiment, the first open reading frame encoding a protein of interest is located downstream of the 5' replication recognition sequence, and in the case of the first RNA, upstream of an IRES and, optionally, upstream of an open reading frame encoding one or more nonstructural proteins from a self-replicating virus. In some embodiments, one or more additional open reading frames may be present downstream of the first open reading frame. The one or more additional open reading frames downstream of the first open reading frame may be referred to as a "second open reading frame," a "third open reading frame," etc., in the order in which they are present downstream of the first open reading frame (5' to 3'). In one embodiment, one or more additional open reading frames encoding one or more proteins of interest are located downstream of an open reading frame encoding one or more nonstructural proteins from a self-replicating virus on the first RNA, and are preferably controlled by a subgenomic promoter. Preferably, each open reading frame encoding a protein of interest is controlled by a subgenomic promoter.Preferably, each open reading frame includes a start codon (base triplet), typically AUG in an RNA molecule corresponding to ATG in the corresponding DNA molecule.

[0349] When the replicon contains a 3' replication recognition sequence, it is preferred that all open reading frames are located upstream of the 3' replication recognition sequence.

[0350] In some embodiments, at least one open reading frame of the first RNA and / or second RNA is under the control of a subgenomic promoter, preferably an alphavirus subgenomic promoter. Alphavirus subgenomic promoters are highly efficient and therefore suitable for high-level expression of heterologous genes. Preferably, the subgenomic promoter is a promoter of an alphavirus subgenomic transcript. This means that the subgenomic promoter is a promoter native to the alphavirus and preferably controls the transcription of an open reading frame encoding one or more structural proteins in the alphavirus. Alternatively, the subgenomic promoter is a variant of an alphavirus subgenomic promoter, and any variant that functions as a promoter of subgenomic RNA transcription in a host cell is suitable. When the first and / or second RNA comprises a subgenomic promoter, the first and / or second RNA preferably comprises conserved sequence element 3 (CSE3) or a variant thereof.

[0351] 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 a subgenomic RNA comprising a transcription product of the open reading frame.

[0352] In some embodiments, the first open reading frame is under the control of a subgenomic promoter. In one embodiment, when the first open reading frame is under the control of a subgenomic promoter, the gene encoded by the first open reading frame can be expressed from both the RNA and its subgenomic transcript (the latter in the presence of a functional alphavirus replicase). Downstream of the first open reading frame, which may be under the control of a subgenomic promoter, there may be one or more additional open reading frames, each under the control of a subgenomic promoter. Proteins encoded by one or more additional open reading frames, such as a second open reading frame, can be translated from one or more subgenomic transcripts, each under the control of a subgenomic promoter. For example, the first RNA may contain a subgenomic promoter that controls the production of a transcript encoding a third protein of interest.

[0353] In other embodiments, the first open reading frame is not under the control of a subgenomic promoter. In one embodiment, when the first open reading frame is not under the control of a subgenomic promoter, the protein encoded by the first open reading frame can be expressed from RNA. One or more additional open reading frames, each under the control of a subgenomic promoter, can be present downstream of the first open reading frame. The proteins encoded by one or more additional open reading frames can be expressed from the subgenomic transcript.

[0354] In cells containing the first and second RNAs of the present invention, the second RNA and optionally the first RNA can be amplified by a functional replicase. Furthermore, if the first RNA and / or the second RNA contains one or more open reading frames under the control of a subgenomic promoter, the functional replicase will produce one or more subgenomic transcripts.

[0355] When the first RNA and / or the second RNA comprises multiple open reading frames encoding target proteins, it is preferred that each open reading frame encodes a different protein.For example, the protein encoded by the second open reading frame encoding the target protein is different from the protein encoded by the first open reading frame encoding the target protein.

[0356] IRES In one embodiment, the first RNA comprises an internal ribosome entry site (IRES) and an open reading frame encoding one or more nonstructural proteins derived from a self-replicating virus, where the IRES controls expression of the one or more nonstructural proteins, such as nsp1234. Preferably, the first and / or second rRNA comprises sequence elements that allow replication by a functional replicase. In one embodiment, the self-replicating virus is an alphavirus, and the sequence elements that allow replication by a functional replicase are derived from an alphavirus.

[0357] Alphavirus replicases possess capping enzyme functions, typically capping not only the genomic strand RNA but also the subgenomic strand RNA (+). The 5' cap protects mRNA from degradation and also attracts ribosomal subunits and cellular factors to the mRNA, allowing it to form a ribonucleoprotein complex on the mRNA and initiate translation from the nearby start codon. This complex process has been described in detail in the literature (Jackson et al., 2010, Nat Rev Mol Biol; Vol 10:113-127). Despite the sophisticated and efficient mechanism of cap-dependent translation, cells possess the means to initiate translation completely or partially independent of the 5' cap (Thompson 2012; Trends in Microbiology 20:558-566). Therefore, even under cellular stress that leads to a global downregulation of cap-dependent translation, cells may continue to preferentially express selected genes, often with the aid of an IRES.

[0358] Viruses have also evolved various means to exploit the cellular viral gene translation machinery. Because viral infection is sensed by cells and often leads to cellular antiviral responses (interferon response, stress response), many viruses, especially RNA viruses, also utilize cap-independent translation. Cap-independent translation favors viral RNA translation during cellular stress responses, allowing the virus the opportunity to complete its life cycle and be released from the infected cell.

[0359] Internal ribosome entry sites (IRES) are RNA sequences that form the appropriate secondary structure to attract the preinitiation complex to the vicinity of the translation start codon, e.g., AUG. Four classes of IRES with common characteristics have been described in the literature. Typical IRES include the poliovirus IRES (type I), the encephalomyocarditis virus (EMCV) IRES (type II), the hepatitis C virus (HCV) IRES (type III), and the IRES found in the intergenic regions of dicistroviruses (type IV) (Thompson, 2012; Trends in Microbiology 20:558-566; Lozano et al., 2018; Open Biology 8:180155).

[0360] Types I to III IRESs have one thing in common: they initiate translation at an AUG start codon, whereas type IV IRESs initiate translation at a codon other than AUG (e.g., GCU). Therefore, types I to III require an initiator tRNA to deliver methionine via eIF2 / GTP (eIF2 / GTP / Met-tRNAiMet). Activation of eIF2 kinase under stress conditions phosphorylates the α subunit of eIF2, inhibiting AUG-initiated translation. Therefore, type IV IRES-induced translation is not inhibited by eIF2 phosphorylation.

[0361] An "internal ribosome entry site" (abbreviated "IRES") is an RNA element that recruits ribosomes to an internal region of an mRNA and initiates translation in a cap-independent manner. IRESs are often found in the 5'-UTR of RNA viruses. However, the mRNAs of Dicistroviridae viruses contain two open reading frames (ORFs), the translation of which is controlled by two different IRESs. IRESs have also been suggested to exist in some mammalian cellular mRNAs. These intracellular IRES elements are thought to be present in eukaryotic mRNAs encoding genes involved in stress resistance and other processes essential for survival. IRES elements are often located in the 5'-UTR, but can also be found elsewhere in mRNAs.

[0362] The term "internal ribosome entry site (IRES)" includes IRESs found in picornaviruses, such as poliovirus (PV) and encephalomyocarditis virus, as well as pathogenic viruses such as human immunodeficiency virus, hepatitis C virus (HCV), and foot-and-mouth disease virus. While the IRESs of these viruses contain diverse sequences, many share similar secondary structures and initiate translation through similar mechanisms. Furthermore, IRES activity often requires the assistance of other factors known as IRES-transacting factors (ITAFs). Based on their structure and the requirement for translation initiation factors (IFs) and ITAFs, viral IRESs are classified into four types, as described herein. While all of these IRES types are useful in the present invention, type IV IRESs are particularly preferred.

[0363] There are two groups of viral IRES: type I and type II. These cannot directly bind to the 40S small ribosomal subunit. Instead, they recruit the 40S small ribosomal subunit through distinct ITAFs and require canonical IFs (i.e., eIF2, eIF3, eIF4A, eIF4B, and eIF4G) for cap-dependent translation. The main difference between type I and type II IRES is that 40S ribosomal scanning is required for IRES, whereas type II IRES do not. Examples of type I IRES include those found in poliovirus (PV) and rhinovirus. Examples of type II IRES include those found in encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), and Theiler's murine encephalomyelitis virus (TMEV).

[0364] Type III IRESs, which have a specialized RNA structure, can directly interact with the 40S small ribosomal subunit, but their activity typically requires the assistance of multiple IFs, including eIF2, eIF3, and the initiation factor Met-tRNAi. Examples include the IRESs found in hepatitis C virus (HCV), classical swine fever virus (CSFV), and porcine tescovirus (PTV).

[0365] Type IV viral IRESs generally have strong activity and can initiate translation from start codons other than AUG without the need for additional ITAFs or the eIF2 / Met-tRNAi / GTP ternary complex. These IRESs fold into compact structures that directly interact with the 40S small ribosomal subunit. Examples include the IRESs found in dicistroviruses such as cricket paralysis virus (CrPV), Plautia stari enteric virus (PSIV), and taura syndrome virus (TSV).

[0366] The term "internal ribosome entry site" also includes IRESs found on cellular mRNAs. Many of these encode proteins required for stress responses, such as apoptosis, mitosis, hypoxia, and nutrient limitation. Cellular IRESs can be broadly classified into two types based on the mechanism of ribosome recruitment: type I IRESs interact with ribosomes via ITAFs, which bind to cis-elements such as RNA-binding motifs and N-6-methyladenosine (m6A) modifications. Type II IRESs, on the other hand, have short cis-elements that pair with 18S rRNA to recruit ribosomes.

[0367] Protein of interest The protein of interest may be selected from the group consisting of, for example, a reporter protein, a pharmacologically active peptide or protein, an intracellular interferon (IFN) signaling inhibitor, a pluripotency factor, a differentiation factor, a vaccinia virus immune evasion protein, or an antigen or epitope thereof. According to the present invention, the protein of interest preferably does not include a functional nonstructural protein from an autonomously replicating virus, such as a functional alphavirus nonstructural protein.

[0368] Reporter proteins In one embodiment, the open reading frame encodes a reporter protein, e.g., a cell surface-expressed protein such as CD90. In this embodiment, the open reading frame comprises a reporter gene. Certain genes can be chosen as reporters because they confer easily identifiable and measurable properties on the expressing cell or organism, or because they are selectable markers. Reporter genes are often used as indicators of whether a particular gene has been incorporated or expressed in a cell or population of organisms. Preferably, the expression product of the reporter gene is visually detectable. Common visually detectable reporter proteins typically contain fluorescent or luminescent proteins. Examples of specific reporter genes include the gene encoding jellyfish green fluorescent protein (GFP), which causes expressing cells to glow green under blue light; the enzyme luciferase (Luc), which catalyzes a reaction with luciferin to produce light; and red fluorescent protein (RFP). Any variant of these specific reporter genes is possible, as long as it has a visually detectable property. For example, eGFP is a point mutant of GFP. Reporter protein embodiments are particularly suited for testing expression.

[0369] Pharmaceutically active peptides or proteins According to the present invention, in one embodiment, the first RNA and / or the second RNA comprises or consists of a pharmaceutically active RNA. A "pharmaceutically active RNA" can be an RNA encoding a pharmaceutically active peptide or protein. Preferably, the RNA according to the present invention encodes a pharmaceutically active peptide or protein. Preferably, the RNA according to the present invention comprises a pharmaceutically active miRNA. In some embodiments, the system according to the present invention encodes a pharmaceutically active peptide or protein and a pharmaceutically active miRNA. Preferably, the first RNA molecule encodes a replicase described herein, and a second replicable RNA molecule capable of being replicated in trans by the replicase encoded by the first RNA molecule encodes a pharmaceutically active peptide or protein and a pharmaceutically active miRNA. Preferably, the open reading frame encodes a pharmaceutically active peptide or protein. Preferably, the RNA comprises an open reading frame encoding a pharmaceutically active peptide or protein, optionally under the control of a subgenomic promoter.

[0370] A "pharmaceutically active peptide or protein" or "pharmaceutically active miRNA" has a positive or beneficial effect on a subject's condition or pathology when administered to a subject in a therapeutically effective amount. Preferably, the pharmaceutically active peptide or protein or pharmaceutically active miRNA has a curative or palliative effect and can be administered to improve, alleviate, relieve, reverse, delay the onset, or reduce the severity of one or more symptoms of a disease or disorder. The pharmaceutically active peptide or protein or pharmaceutically active miRNA can have a prophylactic effect and can be used to delay the onset of a disease or reduce the severity of such a disease or pathology. The term "pharmaceutically active peptide or protein" includes whole proteins or polypeptides and can also refer to pharmaceutically active fragments thereof. It also includes pharmaceutically active analogs of peptides or proteins. The term "pharmaceutically active peptide or protein" includes peptides and proteins that are antigens. That is, the peptide or protein elicits an immune response in a subject that has a therapeutic, partial, or complete protective effect.

[0371] In one embodiment, the pharmaceutically active peptide or protein is or comprises an immunologically active compound, antigen, or epitope.

[0372] According to the present invention, the term "immunologically active compound" relates to any compound that alters the immune response, preferably by inducing and / or suppressing immune cell maturation, inducing and / or suppressing cytokine biosynthesis, and / or altering humoral immunity by stimulating antibody production by B cells. In one embodiment, the immune response includes stimulation of an antibody response (usually involving immunoglobulin G (IgG)). Immunoactive compounds have potent immunostimulatory activity, including, but not limited to, antiviral and antitumor activity, and can also downregulate other aspects of the immune response, e.g., by shifting the immune response away from a Th2-type immune response, making them useful for the treatment of a wide range of Th2-mediated diseases.

[0373] According to the present invention, the term "antigen" or "immunogen" encompasses any substance that elicits an immune response. In particular, "antigen" refers to any substance that specifically reacts with antibodies or T lymphocytes (T cells). According to the present invention, the term "antigen" includes any molecule containing at least one epitope. Preferably, an antigen in the present invention is a molecule that, after optional processing, induces an immune response, preferably specific to the antigen. According to the present invention, any suitable antigen that is a candidate for an immune response can be used, and the immune response can be both a humoral immune response and a cellular immune response. In an embodiment of the present invention, the antigen is preferably presented via an MHC molecule by a cell, preferably an antigen-presenting cell, resulting in an immune response against the antigen. The antigen is preferably a product corresponding to a naturally occurring antigen or derived from a naturally occurring antigen. Such naturally occurring antigens may include or be derived from allergens, viruses, bacteria, fungi, parasites, other infectious agents and pathogens, or may be tumor antigens. According to the present invention, the antigen may correspond to a naturally occurring product, for example, a viral protein or a portion thereof. In a preferred embodiment, the antigen is a surface polypeptide, i.e., a polypeptide that is naturally displayed on the surface of a cell, pathogen, bacterium, virus, fungus, parasite, allergen, or tumor, and is capable of eliciting an immune response against the cell, pathogen, bacterium, virus, fungus, parasite, allergen, or tumor.

[0374] The term "pathogen" refers to a pathogenic biological agent capable of causing disease in an organism, preferably a vertebrate. Pathogens include microorganisms such as bacteria, unicellular eukaryotes (protozoa), fungi, parasites, and viruses.

[0375] The terms "epitope," "antigenic peptide," "antigenic epitope," "immunogenic peptide," and "MHC-binding peptide" are used interchangeably herein and refer to an antigenic determinant in a molecule, such as an antigen, i.e., a portion or fragment of an immunologically active compound that is recognized by the immune system, e.g., by T cells, particularly when presented in the context of an MHC molecule. A protein epitope preferably consists of a continuous or discontinuous portion of the protein and is preferably 5 to 100 amino acids, preferably 5 to 50 amino acids, more preferably 8 to 30 amino acids, and most preferably 10 to 25 amino acids in length. For example, an epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. According to the present invention, an epitope is capable of binding to an MHC molecule, such as an MHC molecule on a cell surface, and thus may be an "MHC-binding peptide" or an "antigenic peptide." The term "major histocompatibility complex" and abbreviation "MHC" refer to a genetic complex that contains MHC class I and MHC class II molecules and is present in all vertebrates. MHC proteins or molecules are important in signaling between lymphocytes and antigen-presenting or diseased cells during the immune response; they bind peptides and present them for recognition by T cell receptors. Proteins encoded by MHC are expressed on the cell surface and present both self-antigens (peptide fragments of the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. Preferred such immunogenic moieties bind to MHC class I or class II molecules. As used herein, an immunogenic moiety is said to "bind" to an MHC class I or class II molecule if such binding is detectable using any assay known in the art. The term "MHC-binding peptide" refers to a peptide that binds to an MHC class I and / or class II molecule. For class I MHC / peptide complexes, the binding peptide is typically 8-10 amino acids in length, although longer or shorter peptides may also be effective.For class II MHC / peptide complexes, the binding peptides are typically 10-25 amino acids in length, particularly 13-18 amino acids in length, although longer and shorter peptides are also effective.

[0376] In one embodiment, the protein of interest of the present invention comprises an epitope suitable for vaccination of a target organism. Those skilled in the art will appreciate that one of the principles of immunobiology and vaccination is based on the fact that immunizing an organism with an antigen immunologically relevant to the disease being treated generates an immune protective response against the disease. The antigen is selected from the group consisting of self-antigens and non-self-antigens. Preferably, the non-self-antigen is a bacterial antigen, a viral antigen, a fungal antigen, an allergen, or a parasitic antigen. Preferably, the antigen comprises an epitope capable of eliciting an immune response in the target organism. For example, the epitope may elicit an immune response (e.g., a cytotoxic T cell response) against bacteria, viruses, fungi, parasites, allergens, or tumors.

[0377] In some embodiments, the non-self antigen is a bacterial antigen. In some embodiments, the antigen induces an immune response against bacteria that infect animals, including mammals, including birds, fish, and livestock. Preferably, the bacteria against which the immune response is induced are pathogenic bacteria.

[0378] In some embodiments, the non-self antigen is a viral antigen. The viral antigen can be, for example, a peptide derived from a viral surface protein, such as a capsid polypeptide or spike polypeptide derived from coronavirus. In some embodiments, the antigen induces an immune response against a virus that infects animals, including birds, fish, and mammals, including livestock. Preferably, the virus that induces an immune response is a pathogenic virus, such as Ebola virus.

[0379] In some embodiments, the non-self antigen is a polypeptide or protein derived from a fungus. In some embodiments, the antigen induces an immune response against a fungus that infects animals, including mammals, including birds, fish, and livestock. Preferably, the fungus against which the immune response is induced is a pathogenic fungus.

[0380] In some embodiments, the non-self antigen is a polypeptide or protein derived from a unicellular eukaryotic parasite. In some embodiments, the antigen induces an immune response against a unicellular eukaryotic parasite, preferably a pathogenic unicellular eukaryotic parasite. The pathogenic unicellular eukaryotic parasite can be, for example, a Plasmodium genus, such as Plasmodium falciparum, Plasmodium malariae, or Plasmodium ovale, a Leishmania genus, or a Trypanosoma genus, such as Trypanosoma cruzi or Trypanosoma brucei.

[0381] In some embodiments, a pharmaceutically active peptide or protein need not be an antigen to elicit an immune response. Suitable pharmacologically active proteins or peptides include cytokines and immune system proteins, such as immunoactive compounds (e.g., interleukins, colony-stimulating factors (CSFs), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, ceretins, homing receptors, T-cell receptors, chimeric antigen receptors (CARs), immunoglobulins), hormones (e.g., insulin, thyroid hormones, catecholamines, gonadotropins, trophic hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptin, etc.), growth hormones (e.g., human growth hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, etc., insulin-like growth factor, etc.), growth factor receptors, enzymes (e.g., tissue plasminogen activator, streptokinase, cholesterol biosynthetic or degradative enzymes, steroidogenic enzymes, kinases, phosphodiesterases, etc.), and the like. enzymes, methylases, demethylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatase, cytochromes, adenylate or guanylate cyclase, neuraminidase, etc.), receptors (steroid hormone receptors, peptide receptors), binding proteins (growth hormone or growth factor binding proteins, etc.), transcription and translation factors, tumor growth suppressor proteins (e.g., proteins that inhibit angiogenesis), structural proteins (collagen, fibroin, fibrinogen, elastin, tubulin, actin, myosin, etc.), blood proteins (thrombin, serum albumin, factor VII, factor VIII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin, granulocyte colony-stimulating factor (GCSF) or modified factor VIII, anticoagulants, etc.In one embodiment, the pharmacologically active protein of the present invention is a cytokine involved in regulating lymphatic homeostasis, preferably a cytokine that is involved in, and preferably induces or promotes, the development, priming, proliferation, differentiation and / or survival of T cells. In one embodiment, the cytokine is an interleukin, such as IL-2, IL-7, IL-12, IL-15, or IL-21.

[0382] Another suitable target protein encoded by the open reading frame is an inhibitor of interferon (IFN) signaling. It has been reported that the viability of cells into which RNA has been introduced for expression decreases, especially when the cells are transfected multiple times with the RNA; however, IFN inhibitors have been found to increase the viability of cells expressing the RNA (WO 2014 / 071963 A1). Preferably, the inhibitor is an inhibitor of IFN type I signaling. Preventing extracellular IFN from binding to the IFN receptor and inhibiting intracellular IFN signaling allows stable expression of the RNA in cells. Alternatively or additionally, preventing extracellular IFN from binding to the IFN receptor and inhibiting intracellular IFN signaling improves cell viability, especially when the cells are repeatedly transfected with the RNA. Without being bound by theory, it is believed that intracellular IFN signaling may result in the inhibition of translation and / or RNA degradation. This can be addressed by inhibiting one or more IFN-induced antiviral effector proteins. The IFN-induced antiviral activity effector protein can be selected from the group consisting of RNA-dependent protein kinase (PKR), 2',5'-oligoadenylate synthetase (OAS), and RNase L. Inhibition of intracellular IFN signaling can include inhibition of the PKR-dependent pathway and / or the OAS-dependent pathway. Suitable proteins of interest are proteins that can inhibit the PKR-dependent pathway and / or the OAS-dependent pathway. Inhibition of the PKR-dependent pathway can include inhibition of eIF2-α phosphorylation. Inhibition of PKR can include treating cells with at least one PKR inhibitor. The PKR inhibitor can be a viral inhibitor of PKR. A preferred viral inhibitor of PKR is vaccinia virus E3. When a peptide or protein (e.g., E3, K3) inhibits intracellular IFN signaling, intracellular expression of the peptide or protein is preferred.Vaccinia virus E3 is a 25 kDa dsRNA-binding protein (encoded by the gene E3L) that binds to and sequesters dsRNA, preventing the activation of PKR and OAS. E3 directly binds to PKR, inhibiting its activity and reducing the phosphorylation of eIF2-α. A more preferred viral inhibitor is vaccinia virus B18, particularly B18R. Vaccinia virus B18 is a soluble inhibitor of IFN-α with a molecular weight of 41 kDa. Other suitable inhibitors of IFN signaling include herpes simplex virus ICP34.5, Tuscany virus NSs, silkworm nucleopolyhedrovirus PK2, and HCV NS34A.

[0383] pluripotency factor The term "pluripotency factor" or "reprogramming transcription factor" refers to a molecule, particularly a peptide or protein, that, when expressed in a somatic cell, optionally together with other factors such as additional reprogramming factors, reprograms or dedifferentiates the somatic cell into a cell with stem cell properties, particularly pluripotency. Specific examples of reprogramming factors include OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG.

[0384] differentiation factor The target protein encoded by the RNA molecule may preferably be a differentiation factor. This factor can be used for transdifferentiation, which means that the factor is introduced into, preferably, an already differentiated cell, thereby (re)programming the cell into a (different) specific cell type. Transdifferentiation particularly means that a pluripotent state has not been established to reprogram cells from one cell type to another. An example of such a protein is MYOD1, which can also be used as a transdifferentiation factor to reprogram fibroblasts into muscle cells.

[0385] RNA preparation method The RNA molecules of the present invention can be obtained by in vitro transcription. In the present invention, in vitro transcribed RNA (IVT-RNA) is particularly interesting. IVT-RNA can be obtained by transcription from a nucleic acid molecule (especially a DNA molecule). The DNA molecules of the present invention are suitable for such purposes, especially if they contain a promoter recognized by a DNA-dependent RNA polymerase.

[0386] The RNA of the present invention can be synthesized in vitro. This allows the addition of a cap analog during the in vitro transcription reaction. Typically, the poly(A) tail is encoded by a poly(dT) sequence on the DNA template. Alternatively, cap formation and addition of the poly(A) tail can be achieved enzymatically after transcription.

[0387] In vitro transcription methods are known to those skilled in the art, and various in vitro transcription kits are commercially available, for example as described in WO 2011 / 015347 A1.

[0388] DNA The present invention also provides DNA comprising a nucleic acid sequence encoding the RNA of the present invention.

[0389] Preferably, the DNA is double-stranded.

[0390] In a preferred embodiment, the DNA is a plasmid. As used herein, the term "plasmid" generally refers to a construct of extrachromosomal genetic material, usually a circular DNA duplex, that can replicate independently of chromosomal DNA.

[0391] The DNA of the present invention may contain a promoter that can be recognized by DNA-dependent RNA polymerase. This allows in vivo or in vitro transcription of the encoded RNA, such as the RNA of the present invention. The IVT vector can be used as a template for in vitro transcription using standardized methods. Examples of promoters preferred for the present invention include the promoters of SP6, T3, or T7 polymerase.

[0392] In one embodiment, the DNA of the present invention is an isolated nucleic acid molecule.

[0393] Further components of the system The system described herein can be in the form of a composition or two separate compositions.The system can include additional components.The following embodiments of the system are applicable to the embodiment where the system is a composition or separate compositions, for example, where only one of the RNAs is present.

[0394] In one embodiment of the present invention, the system can further comprise a solvent such as an aqueous solvent, or any solvent that can maintain the integrity of RNA.In a preferred embodiment, the system is an aqueous solution that contains RNA.The aqueous solution can optionally contain a solute, such as a salt.

[0395] In one embodiment of the invention, the system is in the form of a lyophilized composition or at least two lyophilized compositions obtained by lyophilizing each aqueous composition.

[0396] In some embodiments, the systems described herein may further include a reagent capable of forming particles with the RNA molecule.

[0397] The systems described herein may further include salts, buffers, or other components, as further described below.

[0398] In some embodiments, the salt used in the systems described herein comprises sodium chloride. Without wishing to be bound by theory, sodium chloride functions as an ionic osmotic agent to pretreat the RNA before mixing with lipids. In some embodiments, the systems described herein may include alternative organic or inorganic salts. Alternative salts include, but are not limited to, potassium chloride, dipotassium phosphate, monopotassium phosphate, potassium acetate, potassium bicarbonate, potassium sulfate, disodium phosphate, monosodium phosphate, sodium acetate, sodium bicarbonate, sodium sulfate, lithium chloride, magnesium chloride, magnesium phosphate, calcium chloride, and the sodium salt of ethylenediaminetetraacetic acid (EDTA).

[0399] Generally, systems or compositions for freezing RNA particles contain a low sodium chloride concentration or a low ionic strength. In some embodiments, the sodium chloride concentration is 0 mM to about 50 mM, 0 mM to about 40 mM, or about 10 mM to about 50 mM.

[0400] According to the present disclosure, the system described herein has a pH suitable for the stability of RNA particles, particularly the stability of RNA. Without wishing to be bound by theory, the pH of the particle composition described herein is maintained during the preparation, storage, and use of the composition by using a buffer system. In some embodiments of the present disclosure, the buffer system can include a solvent (particularly water, such as deionized water, particularly water for injection) and a buffer substance. The buffer substance can be selected from 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris), acetic acid, and histidine. A preferred buffer substance is HEPES.

[0401] The systems described herein may include cryoprotectants and / or surfactants as stabilizers, e.g., to reduce or prevent aggregation, particle collapse, RNA degradation, and / or other types of damage, to avoid significant loss of product quality, particularly RNA activity, during storage, freezing, spray drying, and / or lyophilization.

[0402] In one embodiment, the cryoprotectant is a carbohydrate. As used herein, the term "carbohydrate" refers to and includes monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides.

[0403] In one embodiment, the cryoprotectant is a monosaccharide. As used herein, the term "monosaccharide" refers to a single carbohydrate unit (e.g., a monosaccharide) that is not hydrolyzed into simpler carbohydrate units. Exemplary monosaccharide cryoprotectants include glucose, fructose, galactose, xylose, ribose, etc.

[0404] In one embodiment, the cryoprotectant is a disaccharide. As used herein, the term "disaccharide" refers to a compound or chemical moiety formed from two monosaccharide units linked via a glycosidic bond, such as a 1-4 bond or a 1-6 bond. A disaccharide can be hydrolyzed into two monosaccharides. Exemplary disaccharide cryoprotectants include sucrose, trehalose, lactose, maltose, etc.

[0405] The term "trisaccharide" means three sugars joined together to form one molecule. Examples of trisaccharides include raffinose and melezitose.

[0406] In one embodiment, the cryoprotectant is an oligosaccharide. As used herein, the term "oligosaccharide" refers to a compound or chemical moiety consisting of 3 to about 15, e.g., 3 to about 10, monosaccharide units linked by glycosidic bonds (e.g., 1 to 4 or 1 to 6) to form a linear, branched, or cyclic structure. Exemplary oligosaccharide cryoprotectants include cyclodextrin, raffinose, melezitose, maltotriose, stachyose, acarbose, and the like. Oligosaccharides can be oxidized or reduced.

[0407] In one embodiment, the cryoprotectant is a cyclic oligosaccharide. As used herein, the term "cyclic oligosaccharide" refers to a compound or chemical moiety consisting of 3 to about 15, e.g., 6, 7, 8, 9, or 10, monosaccharide units joined by glycosidic bonds (e.g., 1-4 or 1-6 bonds) to form a ring structure. Examples of cyclic oligosaccharide cryoprotectants include cyclic oligosaccharides that are independent compounds, such as α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.

[0408] Other exemplary cyclic oligosaccharide cryoprotectants include compounds containing a cyclodextrin moiety within a larger molecular structure, such as a polymer containing a cyclic oligosaccharide moiety. The cyclic oligosaccharide can be oxidized or reduced, for example, to a dicarbonyl form. As used herein, the term "cyclodextrin moiety" refers to a cyclodextrin (e.g., α-, β-, or γ-cyclodextrin) radical that is incorporated into or forms part of a larger molecular structure, such as a polymer. The cyclodextrin moiety can be directly bonded to one or more other moieties or can be bonded via an optional linker. The cyclodextrin moiety can be oxidized or reduced, for example, to a dicarbonyl form.

[0409] The carbohydrate-based cryoprotectant, e.g., a cyclic oligosaccharide-based cryoprotectant, can be a derivatized carbohydrate. For example, in one embodiment, the cryoprotectant is a derivatized cyclic oligosaccharide, e.g., a derivatized cyclodextrin, e.g., 2-hydroxypropyl-β-cyclodextrin, e.g., a partially etherified cyclodextrin (e.g., a partially etherified β-cyclodextrin).

[0410] An exemplary cryoprotectant is a polysaccharide. As used herein, the term "polysaccharide" refers to a compound or chemical moiety in which at least 16 monosaccharide units are linked via glycosidic bonds (e.g., 1-4 or 1-6 bonds) to form a linear, branched, or cyclic structure, including polymers that contain polysaccharides as part of their backbone structure. In the backbone, the polysaccharides can be linear or cyclic. Exemplary polysaccharide cryoprotectants include glycogen, amylase, cellulose, dextran, maltodextrin, and the like.

[0411] In some embodiments, the system may include sucrose. Without wishing to be bound by theory, sucrose has the function of promoting cryoprotection, thereby preventing aggregation of RNA (especially rRNA) particles and maintaining the chemical and physical stability of the composition. In some embodiments, the system may include a cryoprotectant that replaces sucrose. Alternative stabilizers include, but are not limited to, trehalose and glucose. In a specific embodiment, the alternative stabilizer to sucrose is trehalose or a mixture of sucrose and trehalose.

[0412] Preferred cryoprotectants are selected from the group consisting of sucrose, trehalose, glucose, and combinations thereof (e.g., a combination of sucrose and trehalose). In a preferred embodiment, the cryoprotectant is sucrose.

[0413] In some embodiments of the present disclosure, a chelating agent is contemplated for use in the systems described herein. A chelating agent refers to a compound capable of forming at least two coordinate covalent bonds with a metal ion, thereby forming a stable, water-soluble complex. Without being bound by theory, the chelating agent reduces the concentration of free divalent ions, which may cause accelerated RNA degradation in the present disclosure. Examples of suitable chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), salts of EDTA, desferrioxamine B, deferoxamine, dithiocarbamate sodium, penicillamine, calcium pentetate, sodium pentetate, succinimer, trientine, nitrilotriacetic acid, trans-diaminocyclohexanetetraacetic acid (DCTA), diethylenetriaminepentaacetic acid (DTPA), and bis(aminoethyl)glycolether-N,N,N',N'-tetraacetic acid. In some embodiments, the chelating agent is EDTA or a salt of EDTA. In an exemplary embodiment, the chelating agent is disodium EDTA dihydrate. In some embodiments, the concentration of EDTA is about 0.05 mM to about 5 mM, about 0.1 mM to about 2.5 mM, or about 0.25 mM to about 1 mM.

[0414] In another embodiment, the systems described herein are chelating agent-free.

[0415] As used herein, terms such as "stability" or "desired storage stability" may refer to the physicochemical stability of a product (e.g., a Tris / sucrose finished product) when stored in an unopened thawed vial at 30° C. for up to 24 hours, and in a syringe at 2-8° C. for up to 24 hours and 12 hours at 30° C. Such terms may refer to a product having a shelf life of 6 months or more when stored at -90 to -60° C.

[0416] In some embodiments, the systems of the present invention may include one or more adjuvants. Adjuvants can be added to vaccines to stimulate immune system responses, but adjuvants themselves typically do not provide immunity. Exemplary adjuvants include, but are not limited to, inorganic compounds (e.g., alum, aluminum hydroxide, aluminum phosphate, calcium hydroxide phosphate), mineral oils (e.g., paraffin oil), cytokines (e.g., IL-1, IL-2, IL-12), immunostimulatory polynucleotides (e.g., RNA or DNA, e.g., CpG-containing oligonucleotides), saponins (e.g., plant saponins from Quillaja, soybean, and Quercus serrata), oil emulsions or liposomes, polyoxyethylene ether and polyoxyethylene ester formulations, polyphosphazene (PCPP), muramyl peptides, imidazoquinolone compounds, thiosemicarbazone compounds, Flt3 ligand (WO 2010 / 066418 A1), or other adjuvants known to those skilled in the art. A preferred adjuvant for RNA administration according to the present invention is Flt3-ligand (WO2010 / 066418A1). When Flt3-ligand is administered together with RNA encoding an antigen, a significant increase in antigen-specific CD8+ T cells can be observed.

[0417] The system according to the invention can be buffered (for example with acetate buffer, citrate buffer, succinate buffer, Tris buffer, phosphate buffer).

[0418] RNA-containing particles In some embodiments, due to the instability of unprotected RNA, it is advantageous to provide the RNA molecules of the present invention in a complexed or encapsulated form. The present invention provides respective systems, particularly compositions. In particular, in some embodiments, the system of the present invention comprises nucleic acid-containing particles, preferably RNA-containing particles. The nucleic acid-containing particles may be in the form of, for example, proteinaceous particles or lipid-containing particles. Suitable proteins or lipids are referred to as particle-forming agents. Proteinaceous particles and lipid-containing particles have previously been reported to be suitable for delivering alphavirus RNA in particulate form (e.g., Strauss & Strauss, 1994, Microbiol. Rev. 58:491-562). In particular, alphavirus structural proteins (e.g., provided by helper viruses) are suitable carriers for delivering RNA in the form of proteinaceous particles. The system may comprise a first composition comprising a first RNA molecule, a second composition comprising a second RNA molecule, and, optionally, one or more additional compositions comprising any additional RNA molecules (e.g., a third RNA molecule). The system may include a composition comprising a first RNA molecule, a second RNA molecule, and optionally, any additional RNA molecule (e.g., a third RNA molecule). The system may include a composition comprising a particle comprising the first RNA molecule and a particle comprising the second RNA molecule. The system may also include a composition comprising a particle comprising a mixture of the first RNA molecule and the second RNA molecule.

[0419] In one embodiment, the system according to the present invention comprises the nucleic acid according to the present invention in the form of nanoparticles. Nanoparticle preparations can be obtained using various protocols and various complex compounds. Lipids, polymers, oligomers, or amphiphiles are typical components of nanoparticle preparations.

[0420] As used herein, the term "nanoparticle" refers to particles having a diameter suitable for systemic administration, particularly parenteral administration, of nucleic acids, and typically has a diameter of 1000 nanometers (nm) or less. In one embodiment, the average diameter of the nanoparticles is about 50 nm to about 1000 nm, preferably about 50 nm to about 400 nm, preferably about 100 nm to about 300 nm, for example, about 150 nm to about 200 nm. In one embodiment, the diameter of the nanoparticles is in the range of about 200 to about 700 nm, about 200 to about 600 nm, preferably about 250 to about 550 nm, particularly about 300 to about 500 nm or about 200 to about 400 nm. In one embodiment, the average diameter is about 50 to 150 nm, preferably about 60 to 120 nm. In one embodiment, the average diameter is less than 50 nm.

[0421] In one embodiment, the polydispersity index (PI) of the nanoparticles described herein is 0.5 or less, preferably 0.4 or less, and more preferably 0.3 or less, as measured by dynamic light scattering. The "polydispersity index" (PI) is a measure of the uniform or non-uniform size distribution of individual particles (e.g., liposomes) in a particle mixture and indicates the breadth of particle distribution in the mixture. PI can be measured, for example, as described in WO 2013 / 143555 A1.

[0422] As used herein, the term "nanoparticle formulation" or "nanoparticle system," or similar terms, refers to any system, particularly a composition, that includes at least one nanoparticle. In some embodiments, the nanoparticle system is a homogeneous collection of nanoparticles. In some embodiments, the nanoparticle system is a lipid-containing system, such as a liposome formulation or an emulsion.

[0423] Lipid-containing systems In one embodiment, the system of the present invention comprises at least one lipid. Preferably, the at least one lipid is a cationic lipid. The lipid-containing system comprises a nucleic acid according to the present invention. In one embodiment, the system of the present invention comprises RNA encapsulated in a vesicle, such as a liposome. In one embodiment, the system of the present invention comprises RNA in emulsion form. In one embodiment, the system of the present invention comprises RNA complexed with a cationic compound, thereby forming, for example, a so-called lipoplex. The encapsulation of RNA in a vesicle, such as a liposome, is different from, for example, a lipid / RNA complex. The lipid / RNA complex can be obtained, for example, by mixing RNA with preformed liposomes.

[0424] In one embodiment, the system according to the present invention comprises RNA encapsulated in a vesicle. Such a formulation is a specific system according to the present invention. A vesicle is a lipid bilayer rolled up into a spherical shell, enclosing a small space and separating it from the space outside the vesicle. Typically, the space inside the vesicle is aqueous, i.e., contains water. Typically, the space outside the vesicle also contains aqueous, i.e., contains water. The lipid bilayer is formed by one or more lipids (vesicle-forming lipids). The membrane surrounding the vesicle is a lamellar phase similar to that of a cell membrane. Vesicles according to the present invention may be multilamellar vesicles, unilamellar vesicles, or a mixture thereof. The RNA encapsulated in the vesicles is typically isolated from the external medium. Thus, the RNA is present in a protected form, functionally equivalent to the protected form in natural alphaviruses. Suitable vesicles are the particles, particularly nanoparticles, described herein.

[0425] For example, RNA can be encapsulated in liposome.In this embodiment, the system is or includes liposome preparation.The encapsulation in liposome typically protects RNA from degradation by RNase.Although liposome can contain RNA on the outside (for example, on the surface), at least half (ideally all) of RNA is encapsulated in the core of liposome.

[0426] Liposomes are tiny lipid vesicles, often containing one or more bilayers of vesicle-forming lipids such as phospholipids, that can encapsulate drugs such as RNA. Various types of liposomes can be used in the present invention, including, but not limited to, multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), sterically stabilized liposomes (SSLs), multivesicles (MVs), large multivesicles (LMVs), and other bilayer structures known in the art. The size and degree of lamellae of liposomes depend on the preparation method. There are several other supramolecular structures in which lipids exist in aqueous media, including lamellar phases, hexagonal and reverse hexagonal phases, cubic phases, micelles, and reverse micelles consisting of a single layer. These phases can also be obtained in combination with DNA or RNA, and interactions with RNA and DNA can significantly affect the phase state. Such phases can be present in the nanoparticle RNA formulations of the present invention.

[0427] Liposomes can be formed using standard methods known to those skilled in the art, including back-evaporation, ethanol injection, dehydration and rehydration, sonication, or other suitable methods. After liposome formation, the liposomes can be sized to obtain a population of liposomes with a substantially uniform size range.

[0428] In a preferred embodiment of the present invention, RNA is present in liposomes containing at least one cationic lipid. Each liposome can be formed from a single lipid or a mixture of lipids, so long as at least one cationic lipid is used. Preferred cationic lipids have a protonatable nitrogen atom. Preferably, such cationic lipids are lipids containing tertiary amine groups. A particularly suitable lipid containing tertiary amine groups is 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA). In one embodiment, the RNA of the present invention is present in a liposome formulation such as that described in WO 2012 / 006378 A1. The liposomes have a lipid bilayer encapsulating an aqueous core containing RNA, and the lipid bilayer contains lipids with a pKa in the range of 5.0 to 7.6, preferably containing tertiary amine groups. A preferred cationic lipid containing tertiary amine groups is DLinDMA (pKa 5.8), which is generally described in WO 2012 / 031046 A2. According to WO 2012 / 031046 A2, liposomes containing each compound are particularly suitable for encapsulating RNA and thus for liposomal delivery of RNA. In one embodiment, the RNA of the present invention is present in a liposomal formulation, and the liposome contains at least one cationic lipid containing at least one protonatable nitrogen atom (N) in its head group, with the N:P ratio between the liposome and the RNA being 1:1 to 20:1. In the present invention, "N:P ratio" refers to the molar ratio of nitrogen atoms (N) in the cationic lipid to phosphate atoms (P) in the RNA contained in the lipid-containing particle (e.g., liposome), as described in WO 2013 / 006825 A1. An N:P ratio of 1:1 to 20:1 is related to the net charge of the liposome and the efficiency of RNA delivery to vertebrate cells.

[0429] In one embodiment, the RNA of the invention is present in a liposomal formulation comprising at least one lipid comprising a polyethylene glycol (PEG) moiety, and the RNA is encapsulated within a PEGylated liposome such that the PEG moiety is present on the exterior of the liposome, as described in WO2012 / 031043A1 and WO2013 / 033563A1.

[0430] In one embodiment, the RNA of the invention is not present in a liposomal formulation comprising at least one lipid comprising a polyethylene glycol (PEG) moiety.

[0431] In one embodiment, the RNA of the present invention is present in a liposome preparation in which the liposome diameter is in the range of 60 to 180 nm, as described in WO2012 / 030901A1.

[0432] In one embodiment, the RNA of the invention is present in a liposome formulation in which the net charge of the RNA-containing liposomes is near zero or negative, as described in WO2013 / 143555A1.

[0433] In another embodiment, the system of the present invention comprises RNA in the form of an emulsion. Emulsions have previously been described for use in delivering nucleic acid molecules, such as RNA molecules, to cells. Oil-in-water emulsions are preferred herein. Each emulsion particle comprises an oily core and a cationic lipid. More preferred are cationic oil-in-water emulsions in which the RNA of the present invention is complexed with emulsion particles. The emulsion particles comprise an oily core and a cationic lipid. The cationic lipid can interact with the negatively charged RNA and immobilize the RNA in the emulsion particles. In oil-in-water emulsions, the emulsion particles are dispersed in an aqueous continuous phase. For example, the average particle size of the emulsion particles can typically be about 80 nm to 180 nm. In one embodiment, the system of the present invention is a cationic oil-in-water emulsion, as described in WO 2012 / 006380, in which the emulsion particles comprise an oily core and a cationic lipid. The RNA of the present invention can be present in the form of an emulsion containing cationic lipids, as described in WO 2013 / 006834, where the N:P ratio of the emulsion is at least 4:1. The RNA of the present invention can be present in the form of a cationic lipid emulsion, as described in WO 2013 / 006837. In particular, the composition comprises RNA complexed with particles of a cationic oil-in-water emulsion, where the oil / lipid ratio is at least about 8:1 (mol:mol).

[0434] In another embodiment, the system of the present invention includes RNA in the form of a lipoplex. The term "lipoplex" or "RNA lipoplex" refers to a complex of lipids and nucleic acids, such as RNA. Lipoplexes can be formed from cationic (positively charged) liposomes and anionic (negatively charged) nucleic acids. Cationic liposomes can also contain neutral "helper" lipids. In the simplest case, lipoplexes form spontaneously by mixing nucleic acids and liposomes using a specific mixing protocol, although various other protocols can also be applied. It is understood that electrostatic interactions between positively charged liposomes and negatively charged nucleic acids are the driving force behind lipoplex formation (WO 2013 / 143555 A1). In one embodiment of the present invention, the net charge of the RNA lipoplex particles is close to zero or negative. Neutral or negatively charged lipoplexes composed of RNA and liposomes are known to induce significant RNA expression in splenic dendritic cells (DCs) after systemic administration without the increased toxicity reported for positively charged liposomes and lipoplexes (see WO 2013 / 143555 A1). Thus, in one embodiment of the present invention, the system comprises RNA in the form of nanoparticles, preferably lipoplex nanoparticles, where (i) the number of positive charges in the nanoparticles does not exceed the number of negative charges in the nanoparticles, and / or (ii) the nanoparticles have a neutral or net negative charge, and / or (iii) the charge ratio of positive to negative charges in the nanoparticles is 1.4:1 or less, and / or (iv) the nanoparticles have a zeta potential of 0 or less. As described in WO 2013 / 143555 A1, zeta potential is a scientific term that describes the electrokinetic potential in a colloidal system. In the present invention, (a) the zeta potential and (b) the charge ratio of the cationic lipid to the RNA in the nanoparticles can both be calculated as disclosed in WO2013 / 143555A1. In summary, as disclosed in WO2013 / 143555A1, nanoparticulate lipoplex formulations having defined particle sizes in which the net charge of the particles is close to zero or negative are preferred systems in the context of the present invention.

[0435] In another embodiment, lipoplexes are obtained according to the method disclosed in WO2019 / 077053A1. According to WO2019 / 077053A1, lipoplexes are obtained by adding a solution containing RNA to a liposome colloid. According to WO2019 / 077053A1, liposome colloids are obtained by a method comprising injecting a lipid solution in ethanol into an aqueous phase to produce a liposome colloid, wherein the concentration of at least one lipid in the lipid solution is equal to or higher than the equilibrium solubility of the at least one lipid in ethanol. A particularly preferred method for producing liposome colloids comprises injecting a lipid solution containing DOTMA and DOPE in an approximately 2:1 molar ratio in ethanol into water stirred at a stirring speed of approximately 150 rpm to produce a liposome colloid, wherein the concentration of DOTMA and DOPE in the lipid solution is approximately 330 mM.

[0436] In another embodiment, the lipoplex is an RNA lipoplex particle described in WO 2020 / 069632 A1, comprising RNA, at least one cationic lipid, at least one additional lipid, sodium chloride at a concentration of about 10 mM or less, a stabilizer at a concentration of more than about 10% weight / volume percent (% w / v) and less than about 15% weight / volume percent (% w / v), and a buffer. Preferably, the lipoplex according to the present invention is an RNA lipoplex particle as described in WO 2020 / 069632 A1, comprising DOTMA and DOPE in a molar ratio of about 2:1, the ratio of positive charges to negative charges in the composition being about 1.3:2.0, the sodium chloride concentration being about 8.2 mM, the sucrose concentration being about 13% (w / v), the HEPES concentration being about 5 mM (pH about 6.7), and the EDTA concentration being about 2.5 mM.

[0437] In one embodiment, the nucleic acids, such as RNA, described herein are in the form of lipid nanoparticles (LNPs). LNPs can include any lipid capable of forming particles to which one or more nucleic acid molecules are bound or encapsulated.

[0438] In one embodiment, the LNP comprises one or more cationic lipids and one or more stabilizing lipids, including neutral lipids and PEGylated lipids.

[0439] In one embodiment, the LNP does not comprise a PEGylated lipid.

[0440] In one embodiment, the LNP comprises a cationic lipid, a neutral lipid, a steroid, a polymer-conjugated lipid, and RNA encapsulated or associated within the lipid nanoparticle.

[0441] In one embodiment, the LNP comprises 40-55 mole percent, 40-50 mole percent, 41-49 mole percent, 41-48 mole percent, 42-48 mole percent, 43-48 mole percent, 44-48 mole percent, 45-48 mole percent, 46-48 mole percent, 47-48 mole percent, or 47.2-47.8 mole percent cationic lipid. In one embodiment, the LNP comprises about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, or 48.0 mole percent cationic lipid.

[0442] In one embodiment, the neutral lipid is present at a concentration of 5-15 mol%, 7-13 mol%, or 9-11 mol%. In one embodiment, the neutral lipid is present at a concentration of about 9.5, 10, or 10.5 mol%.

[0443] In one embodiment, the steroid is present in a concentration of 30-50 mol%, 35-45 mol%, or 38-43 mol%. In one embodiment, the steroid is present in a concentration of about 40, 41, 42, 43, 44, 45, or 46 mol%.

[0444] In one embodiment, the LNP comprises 1-10 mol%, 1-5 mol%, or 1-2.5 mol% of polymer-bound lipid.

[0445] In one embodiment, the LNP comprises 40-50 mol% cationic lipid, 5-15 mol% neutral lipid, 35-45 mol% steroid, 1-10 mol% polymer-conjugated lipid, and RNA encapsulated or associated within the lipid nanoparticle.

[0446] In one embodiment, the mole percentage is determined based on the total number of moles of lipid present in the lipid nanoparticle.

[0447] In one embodiment, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In one embodiment, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In one embodiment, the neutral lipid is DSPC.

[0448] In one embodiment, the steroid is cholesterol.

[0449] In one embodiment, the polymer-bound lipid is a PEGylated lipid. In one embodiment, the PEGylated lipid is: [ka] [In the formula, R 12 and R 13 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, the alkyl chain optionally being interrupted by one or more ester bonds; and the average value of w is in the range of 30 to 60. or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. In one embodiment, R 12 and R 13 are each independently a linear saturated alkyl chain containing 12 to 16 carbon atoms. In one embodiment, the average value of w is about 40 to 55. In one embodiment, the average value of w is about 45. In one embodiment, R 12 and R13 are each independently a linear saturated alkyl chain containing about 14 carbon atoms, and w has an average value of about 45.

[0450] In one embodiment, the pegylated lipid is DMG-PEG 2000, for example, the following structure: [ka] It has.

[0451] In some embodiments, the polymer-conjugated lipid is not a pegylated lipid.

[0452] In some embodiments, the cationic lipid component of the LNP has formula (III): [ka] [In formula: L 1 or one of L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa, -OC(=O)NRa-, or -NRaC(=O)O-; and L 1 or L 2 the other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa, -OC(=O)NRa-, or -NRaC(=O)O- or a direct bond; G 1 and G 2 are each independently an unsubstituted C1-C 12 Alkylene or C1-C 12 alkenylene; G3 is C1-C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R ais H or C1-C 12 is alkyl; R 1 and R 2 are independently C6-C 24 Alkyl or C6-C 24 is alkenyl; R 3 is H, OR 5 , CN, C(=O)OR 4 , OC(=O)R 4 , or -NR 5 C(=O)R 4 and; R 4 is C1-C 12 is alkyl; R 5 is H or C1-C6 alkyl; and x is 0, 1, or 2] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0453] In some of the foregoing embodiments of formula (III), the lipid has the following structure (IIIA) or (IIIB): [ka] [In the formula, A is a 3-8 membered cycloalkyl or cycloalkylene ring; R 6 is, in each occurrence, independently H, OH, or C-C 24 is alkyl; n is an integer ranging from 1 to 15]. The device has one of the following:

[0454] In some of the foregoing embodiments of formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0455] In other embodiments of formula (III), the lipid has the following structure (IIIC) or (IIID): [ka] [In the formula, y and z are each independently an integer ranging from 1 to 12. The device has one of the following:

[0456] In any of the foregoing embodiments of formula (III), L 1 or L 2 is O(C=O). For example, in some embodiments, L 1 and L 2 Each of the following is O(C=O). In any of the foregoing different embodiments, L 1 and L 2 are each independently (C=O)O or O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of is (C=O)O.

[0457] In some different embodiments of formula (III), the lipid has the following structure (IIIE) or (IIIF): [ka] The device has one of the following:

[0458] In some of the foregoing embodiments of formula (III), the lipid has the following structure (IIIG), (IIIH), (IIII), or (IIIJ): [ka] [ka] The device has one of the following:

[0459] In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, e.g., from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0460] In some other embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0461] In some of the foregoing embodiments of Formula (III), R6 is H. In other embodiments, R6 is C1-C24 alkyl. In other embodiments, R6 is OH.

[0462] In some embodiments of Formula (III), G3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G 3 is a straight chain C1-C 24 Alkylene or straight chain C1-C 24 It is alkenylene.

[0463] In some other embodiments of Formula (III), R 1 or R 2 , or both are C6-C 24 For example, in some embodiments, R 1 and R 2 are each independently of the following structure: [ka] [In the formula, R 7a and R 7b is, in each occurrence, independently H or C1-C 12 is alkyl; and a is an integer between 2 and 12, During the ceremony, R 7a , R 7b and a are R 1 and R 2 are each independently selected to contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or from 8 to 12. It has.

[0464] In some of the foregoing embodiments of formula (III), R 7a At least one occurrence of is H. For example, in some embodiments, R 7a are each H. In other different embodiments of the foregoing, R 7b is C1-C8 alkyl. For example, in some embodiments, the C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0465] In different embodiments of formula (III), R 1 or R 2 , or both, have the following structure: [ka] It has one of the following.

[0466] In some of the foregoing embodiments of formula (III), R 3 OH, CN, C(=O)OR 4 , OC(=O)R 4 , or -NHC(=O)R 4 In some embodiments, R 4 is methyl or ethyl.

[0467] In various different embodiments, the cationic lipid of formula (III) has any of the structures shown in the table below.

[0468] Representative compounds of formula (III).

[0469] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0470] In some embodiments, the LNP comprises a lipid of Formula (III), RNA, a neutral lipid, a steroid, and a PEGylated lipid. In some embodiments, the lipid of Formula (III) is compound III-3. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the PEGylated lipid is ALC-0159.

[0471] In some embodiments, the cationic lipid is present in the LNP in an amount of about 40 to about 50 mole percent. In one embodiment, the neutral lipid is present in the LNP in an amount of about 5 to about 15 mole percent. In one embodiment, the steroid is present in the LNP in an amount of about 35 to about 45 mole percent. In one embodiment, the pegylated lipid is present in the LNP in an amount of about 1 to about 10 mole percent.

[0472] In some embodiments, the LNP comprises about 40 to about 50 mole percent compound III-3, about 5 to about 15 mole percent DSPC, about 35 to about 45 mole percent cholesterol, and about 1 to about 10 mole percent ALC-0159.

[0473] In some embodiments, the LNPs comprise about 47.5 mole percent compound III-3, about 10 mole percent DSPC, about 40.7 mole percent cholesterol, and about 1.8 mole percent ALC-0159.

[0474] In various different embodiments, the cationic lipid has one of the structures shown in the table below.

[0475] [Table 2]

[0476] In some embodiments, the LNP comprises a cationic lipid shown in the table above, e.g., a cationic lipid of formula (B) or formula (D), particularly a cationic lipid of formula (D), RNA, a neutral lipid, a steroid, and a PEGylated lipid. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the PEGylated lipid is DMG-PEG 2000.

[0477] In one embodiment, the LNP comprises a cationic lipid that is an ionizable lipid-like substance (lipidoid). In one embodiment, the cationic lipid has the following structure: [ka] It has.

[0478] The N / P value is preferably at least about 4. In some embodiments, the N / P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In one embodiment, the N / P value is about 6.

[0479] The LNPs described herein, in one embodiment, can have an average diameter ranging from about 30 nm to about 200 nm, or from about 60 nm to about 120 nm.

[0480] RNA targeting Some aspects of the present disclosure include targeted delivery of the RNAs disclosed herein (eg, RNAs encoding vaccine antigens and / or immunostimulatory agents).

[0481] In one embodiment, the present disclosure is targeted to lung.When the RNA that is administered is the RNA that encodes vaccine antigen or the miRNA that is related to the treatment of infectious diseases in lung, it is particularly preferred to target lung.For example, RNA can be delivered to lung by inhalation administration, and can be formulated as particle as described herein, for example, lipid particle.

[0482] In one embodiment, the present invention includes targeting the lymphatic system, particularly secondary lymphoid organs, more particularly the spleen. When the administered RNA is RNA encoding a vaccine antigen, it is particularly preferred to target the lymphatic system, particularly secondary lymphoid organs, more particularly the spleen.

[0483] In one embodiment, the target cell is a spleen cell. In one embodiment, the target cell is an antigen-presenting cell, such as a professional antigen-presenting cell in the spleen. In one embodiment, the target cell is a dendritic cell in the spleen.

[0484] The "lymphatic system" is part of the circulatory system and an important part of the immune system, and includes a network of lymphatic vessels that transport lymph. The lymphatic system consists of lymphoid organs, a conducting network of lymphatic vessels, and circulating lymph. Primary, or central, lymphoid organs generate lymphocytes from immature precursor cells. The thymus and bone marrow comprise the primary lymphoid organs. Secondary, or peripheral, lymphoid organs (including lymph nodes and the spleen) maintain mature, naive lymphocytes and initiate adaptive immune responses.

[0485] RNA can be delivered to the spleen using so-called lipoplex formulations. In lipoplex formulations, RNA is bound to liposomes containing cationic lipids and, optionally, additional lipids or helper lipids, to form injectable nanoparticle formulations. Liposomes are obtained by injecting a lipid solution in ethanol into water or a suitable aqueous phase. RNA lipoplex particles can be prepared by mixing liposomes with RNA. Spleen-targeting RNA lipoplex particles are described in WO 2013 / 143683, which is incorporated herein by reference. It has been found that RNA lipoplex particles with a net negative charge can be used to preferentially target spleen tissue or spleen cells, such as antigen-presenting cells, particularly dendritic cells. Thus, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression occurs in the spleen. Therefore, the RNA lipoplex particles of the present disclosure can be used for RNA expression in the spleen. In one embodiment, after administration of the RNA lipoplex particles, no or substantially no RNA accumulation and / or RNA expression occurs in the lungs and / or liver. In one embodiment, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression occurs in antigen-presenting cells, such as professional antigen-presenting cells in the spleen. Therefore, the RNA lipoplex particles of the present disclosure can be used for RNA expression in such antigen-presenting cells. In one embodiment, the antigen-presenting cells are dendritic cells and / or macrophages.

[0486] The charge of the RNA lipoplex particles of the present disclosure is the sum of the charge present in at least one cationic lipid and the charge present in RNA. The charge ratio is the ratio of the positive charge present in at least one cationic lipid to the negative charge present in RNA. The charge ratio of the positive charge present in at least one cationic lipid to the negative charge present in RNA is calculated by the following formula: charge ratio = [(cationic lipid concentration (molar)) x (total number of positive charges in cationic lipid)] / [(RNA concentration (molar)) x (total number of negative charges in RNA)].

[0487] The spleen-targeted RNA lipoplex particles described herein preferably have a net negative charge at physiological pH, such as a positive to negative charge ratio of about 1.9:2 to about 1:2, or about 1.6:2 to about 1:2, or about 1.6:2 to about 1.1:2. In specific embodiments, the positive to negative charge ratio in the RNA lipoplex particles at physiological pH is about 1.9:2.0, about 1.8:2.0, about 1.7:2.0, about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about 1:2.0.

[0488] The immunostimulant can be provided to the subject by administering to the subject the RNA encoding the immunostimulant in a formulation for preferential delivery of the RNA to the liver or liver tissue.The delivery of RNA to such target organ or tissue is preferred, particularly when it is desired to express a large amount of the immunostimulant, and / or when it is desired or required that a significant amount of the immunostimulant is present systemically.

[0489] RNA delivery systems are inherently liver-selective. This applies to lipid-based particles, cationic and neutral nanoparticles, especially lipid nanoparticles such as liposomes, nanomicelles, and lipophilic ligands in bioconjugates. Liver accumulation can be caused by discontinuities in the hepatic vasculature or by lipid metabolism (liposomes, lipid, or cholesterol conjugates).

[0490] To deliver RNA to the liver, a drug delivery system can be used to prevent RNA degradation and transport it to the liver. For example, polyplex nanomicelles, which consist of a polyethylene glycol (PEG)-coated surface and an mRNA-containing core, are useful systems because they provide excellent in vivo stability of RNA under physiological conditions. Furthermore, the stealth properties of the polyplex nanomicelle surface, which consists of a dense PEG palisade structure, effectively evade host immune defenses.

[0491] Examples of suitable immunostimulants that target the liver include cytokines that are involved in the proliferation and / or maintenance of T cells.Examples of suitable cytokines include IL2 or IL7, their fragments and variants, and fusion proteins of these cytokines, fragments and variants (for example, PK-extended cytokines).

[0492] In another embodiment, RNA encoding an immunostimulatory agent may be administered in a formulation for preferential delivery of the RNA to the lymphatic system, particularly secondary lymphoid organs, more particularly the spleen. Delivery of an immunostimulatory agent to such a target tissue is preferred, particularly when the presence of the immunostimulatory agent in that organ or tissue is desirable (e.g., when an immunostimulatory agent such as a cytokine is needed to induce an immune response, particularly during T cell priming or for activation of resident immune cells), but when systemic presence of the immunostimulatory agent, especially in significant amounts, is undesirable (e.g., when the immunostimulatory agent has systemic toxicity).

[0493] Examples of suitable immunostimulants include cytokines involved in T cell priming. Examples of suitable cytokines include IL12, IL15, IFN-α, or IFN-β, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants (e.g., extended PK cytokines).

[0494] Polymer-Based Systems In one embodiment, the system of the present invention comprises at least one polymer, preferably a polyalkyleneimine.

[0495] In some embodiments, particles formed from RNA and a polymer, preferably a polyalkyleneimine, are polymer-based polyplexes.

[0496] Polymers are commonly used materials for nanoparticle-based delivery due to their high chemical flexibility. Cationic polymers are typically used to electrostatically condense negatively charged nucleic acids into nanoparticles. These positively charged groups are often composed of amines whose protonation state changes between pH 5.5 and 7.5, which is thought to cause ionic imbalance and lead to endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine, and polyethyleneimine, as well as natural polymers such as chitosan, have all been applied to nucleic acid delivery and are suitable as cationic polymers in the present invention. Furthermore, some researchers have reported polymers synthesized specifically for nucleic acid delivery. In particular, poly(β-amino esters) are widely used in nucleic acid delivery due to their ease of synthesis and high biodegradability. These synthetic polymers are also suitable as cationic polymers in the present invention.

[0497] As used herein, the term "polymer" has its conventional meaning: a molecular structure containing one or more repeating units (monomers) linked by covalent bonds. The repeating units may all be identical, or in some cases, there may be more than one type of repeating unit present within a polymer. The polymer may also be of biological origin, i.e., a biopolymer such as a protein. Additional moieties, such as targeting moieties, may also be present within the polymer.

[0498] ...

Claims

1. A system comprising two RNA molecules, the first RNA molecule comprises an open reading frame encoding a functional RNA-dependent RNA polymerase (replicase); and The second RNA molecule is a replicable RNA molecule comprising at least one miRNA sequence, which can be excised from the second replicable RNA molecule when present in a cell and can regulate gene expression in the cell, and the replicable RNA molecule can be replicated in trans by a replicase encoded by the first RNA molecule.

2. The system of claim 1 , wherein the second RNA molecule comprises at least one pre-miRNA sequence.

3. The system of claim 1 or 2, wherein the first and / or second RNA molecule further comprises at least one open reading frame (ORF) encoding a protein of interest.

4. The system according to any one of claims 1 to 3, wherein the first RNA molecule is a replicable RNA molecule that can be replicated by a replicase that it encodes.

5. The system according to any one of claims 1 to 3, wherein the first RNA molecule is not a replicable RNA molecule.

6. The system of any one of claims 1 to 3 or 5, wherein the first RNA molecule is mRNA.

7. The system according to any one of claims 1 to 6, wherein the replicase is derived from a functional non-structural protein from a self-replicating virus.

8. 8. The system of claim 7, wherein the self-replicating virus is an alphavirus, preferably selected from the group consisting of Venezuelan equine encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus, chikungunya virus, Semliki Forest virus, Sindbis virus, Barma Forest virus, Middelburg virus, and Ndum virus.

9. 9. The system of claim 8, wherein the alphavirus is Venezuelan equine encephalitis virus or Semliki Forest virus.

10. The system of any one of claims 1 to 9, wherein the second RNA molecule comprises at least 2, at least 3, at least 4, at least 5, or at least 10 miRNA sequences.

11. The system of claim 10, wherein the sequence of at least one miRNA sequence differs from the sequences of other miRNAs, and preferably the sequences of each miRNA differ from each other.

12. The system of claim 10 , wherein the sequences of the miRNAs are identical.

13. The system of any one of claims 10 to 12, wherein the miRNAs target the same mRNA.

14. The system of any one of claims 10 to 12, wherein the miRNAs target different mRNAs.

15. The system of any one of claims 10 to 12, wherein the miRNAs target different sites on the same mRNA, or the miRNAs target different sites on two or more mRNAs.

16. The system according to any one of claims 1 to 15, wherein the miRNA sequence is a naturally occurring miRNA sequence, preferably a human miRNA sequence.

17. The system according to any one of claims 1 to 15, wherein the miRNA sequence is an artificial miRNA sequence.

18. The system of any one of claims 1 to 17, wherein the miRNA is a non-viral miRNA.

19. The system according to any one of claims 1 to 18, wherein the miRNA is a stem cell-specific miRNA.

20. The system of any one of claims 1 to 18, wherein the miRNA suppresses an innate immune response.

21. The system according to any one of claims 1 to 18, wherein the target of the miRNA is an mRNA associated with the onset or progression of a disease, preferably an mRNA of an oncogene, a mutated tumor suppressor gene, or a viral, bacterial or fungal gene.

22. The system of claim 21 , wherein the target of the miRNA is a mutated tumor suppressor gene.

23. The system of claim 22, wherein the mutated tumor suppressor gene is TP53.

24. The system according to any one of claims 1 to 21, wherein the target of the miRNA is an interferon stimulated gene, preferably RSAD2 (Viperin).

25. The system according to any one of claims 1 to 21, wherein the target of the miRNA is retinoic acid-inducible gene I (RIG-I).

26. The system of any one of claims 1 to 21, wherein the target of the miRNA is the eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2) gene, which encodes protein kinase R (PKR).

27. The system according to any one of claims 1 to 21, wherein the target of the miRNA is DAZ-associated protein 2 (DAZAP2) and / or TGFβ receptor 2 (TGFβR2).

28. The system of any one of claims 1 to 27, wherein the sequence of the miRNA comprises flanking and loop sequences derived from a naturally occurring miRNA, preferably mouse miR-155.

29. The system according to any one of claims 1 to 19, wherein the miRNA sequence is at least one miRNA sequence of the miR-302 / 367 cluster.

30. The system according to any one of claims 3 to 29, wherein the ORF is flanked by a 5' untranslated region (UTR) and / or a 3' UTR.

31. The system according to any one of claims 3 to 30, wherein the protein of interest is a reporter protein, preferably GFP or a mutant thereof.

32. The system according to any one of claims 3 to 30, wherein the protein of interest is a pluripotency factor or a differentiation factor.

33. The system according to any one of claims 3 to 30, wherein the protein of interest is an antigen or an epitope thereof, preferably a T-cell epitope.

34. 34. The system of claim 33, wherein the antigen or epitope is or is derived from a bacterial, viral, parasitic, or fungal antigen.

35. The system according to any one of claims 3 to 30, wherein the target protein is an immune evasion protein of vaccinia virus.

36. The system of claim 35, wherein the protein of interest is E3 or B18.

37. The system of any one of claims 3 to 36, wherein the sequence of the miRNA is located in the 3' untranslated region (UTR) of at least one ORF of the second RNA molecule.

38. The system of any one of claims 3 to 38, wherein the 5' end of the miRNA sequence is connected to the ORF by a linker sequence and / or the 3' end of the miRNA sequence is connected to a 3' conserved sequence element of the 3' UTR of a second RNA molecule by a linker sequence.

39. The system of any one of claims 3 to 39, wherein each of the miRNA sequences is connected by a linker sequence.

40. 40. The system of claim 38 or 39, wherein the linker sequence comprises 5 to 30 nucleotides.

41. The system of any one of claims 1 to 40, wherein the first and / or second RNA molecule is a modified RNA molecule.

42. 42. The system of claim 41, wherein the first and / or second RNA molecule is a modified RNA molecule comprising at least one modified uridine.

43. 43. The system of claim 42, wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the uridines in the RNA molecule are pseudouridine (ψ), N1-methylpseudouridine (m1ψ), or 5-methyluridine (m5U), preferably N1-methylpseudouridine (lmψ).

44. 44. The system of any one of claims 1 to 43, wherein the first and / or second RNA molecule further comprises a 5' cap, a 5' regulatory region, a 5' replication recognition sequence, a 3' replication recognition sequence, and / or a poly(A) sequence.

45. 45. The system of any one of claims 1 to 44, wherein the first and / or second RNA molecule comprises a 5' cap that is a naturally occurring 5' cap or a 5' cap analog.

46. The system of claim 45, wherein the 5' cap analog is any one of ARCA, β-S-ARCA, β-S-ARCA (D1), β-S-ARCA (D2), CleanCap, Cap0, Cap1, or AU(Cap1).

47. 47. The system of any one of claims 1 to 46, wherein the first and / or second RNA molecule comprises at least one modified uridine and the RNA molecule comprises a 5' cap having the sequence NpppNU, wherein the U in the 5' cap is an unmodified uridine.

48. 48. The system of claim 47, wherein the 5' cap has the sequence NpppAU, where A represents a modified or unmodified adenosine nucleotide.

49. the first and / or second RNA molecule comprises a 5' cap comprising Cap1 and a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA molecule, wherein: (i) Cap1 is m ７ G(5')ppp(5')(2'OMeN １ ) pN ２ where N １ is the +1 position of the RNA molecule, and N ２ is the +2 position of the RNA molecule, and N １ and N ２ are each independently selected from A, C, G, or U; and (ii) The cap-proximal sequence is N of Cap1 １ and N ２ , and: (a) A ３ A ４ X ５ , C ３ A ４ X ５ , A ３ C ４ A ５ , and A ３ U ４ G ５ or a sequence selected from the group consisting of (b) X ３ Y ４ X ５ an array containing where X ３ or X ５ are each independently selected from A, G, C, or U; Y ４ The system of any one of claims 1 to 48, wherein is not C.

50. 50. The system of any one of claims 1 to 49, wherein the first and / or second RNA molecule comprises a modified 5' regulatory region of a self-replicating RNA virus, and the modified regulatory region comprises a point mutation at one or more of positions 67, 244, 245, 246, 248 of the 5' regulatory region (SEQ ID NO: 1).

51. 51. The system of claim 50, wherein the self-replicating RNA virus is an alphavirus.

52. 52. The system of claim 50 or 51, wherein the 5' regulatory region further comprises a point mutation at position 4 of the 5' regulatory region (SEQ ID NO: 1).

53. The system of any one of claims 50 to 52, wherein the point mutation is G4A, A67C, G244A, C245A, G246A, or C248A.

54. 54. The system of any one of claims 1 to 53, wherein the first and / or second RNA molecule comprises a 5' replication recognition sequence characterized in that at least one start codon has been removed compared to the natural 5' replication recognition sequence.

55. The system described in claim 54, characterized in that the 5' replication recognition sequence comprises a sequence homologous to an open reading frame or a portion thereof of a nonstructural protein derived from an autonomously replicating virus, and the sequence homologous to an open reading frame or a portion thereof of a nonstructural protein derived from an autonomously replicating virus comprises the removal of at least one start codon compared to the native viral sequence.

56. The system described in claim 55, characterized in that the sequence homologous to the open reading frame or part thereof of a nonstructural protein from an autonomously replicating virus includes removal of at least the natural start codon of the open reading frame of the nonstructural protein from an autonomously replicating virus.

57. The system described in claim 55 or 56, characterized in that the sequence homologous to the open reading frame or part thereof of a nonstructural protein from an autonomously replicating virus includes the removal of at least one start codon other than the natural start codon of the open reading frame of the nonstructural protein from an autonomously replicating virus.

58. The system according to any one of claims 55 to 57, characterized in that the sequence homologous to the open reading frame or a part thereof of a nonstructural protein derived from an autonomously replicating virus does not contain an initiation codon.

59. 59. The system of any one of claims 55 to 58, wherein the first and / or second RNA molecule comprises at least one nucleotide change that compensates for the disruption of nucleotide pairing in at least one stem loop introduced by removal of at least one start codon.

60. 60. The system of any one of claims 1 to 59, wherein the first and / or second RNA molecule comprises a 3' replication recognition sequence.

61. 61. The system according to any one of claims 1 to 60, wherein the 5' and / or 3' replication recognition sequences are derived from an autonomously replicating virus, preferably from the same autonomously replicating virus species.

62. 62. The system of any one of claims 1 to 61, wherein the first and / or second RNA molecule comprises a poly(A) sequence comprising about 80 to about 150 A residues, or an interrupted poly(A) sequence.

63. 63. The system of any one of claims 1 to 62, wherein the first and / or second RNA molecule does not comprise an open reading frame for an intact viral structural protein.

64. 64. The system of any one of claims 1 to 63, further comprising a third or more replicable RNA molecules capable of being replicated by the replicase encoded by the first RNA molecule.

65. 65. The system of any one of claims 1 to 64, further comprising a reagent capable of forming a particle with at least one of the RNA molecules.

66. 66. The system of claim 65, wherein the reagent is or comprises a polyalkyleneimine or a lipid.

67. 67. A system according to claim 65 or 66, wherein the reagent is a lipid or a system comprising a lipid, preferably the lipid comprising a cationic head group.

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

69. 69. The system of any one of claims 65 to 68, wherein the reagent is or comprises a PEGylated lipid.

70. 70. The system of any one of claims 65 to 69, wherein the reagent is conjugated to polysarcosine, and optionally the reagent comprises a lipid conjugated to the polysarcosine.

71. 71. The system of any one of claims 65 to 70, wherein the particle formed from at least one of the RNA molecules and the reagent is a polymer-based polyplex (PLX), a lipid nanoparticle (LNP), a lipoplex (LPX), or a liposome.

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

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

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

4.

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

76. 76. The system of claim 75, wherein the positive charge is imparted by at least one cationic lipid and the negative charge is imparted by an RNA molecule.

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

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

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

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

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

1.

82. 82. The system of any one of claims 73 to 81, wherein the nanoparticles are lipoplexes comprising DODMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and wherein the charge ratio of positive charges in DODMA to negative charges in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, even more preferably about 1.2:

2.

83. 82. The system of any one of claims 73 to 81, wherein the nanoparticles are lipoplexes comprising DODMA and cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and wherein the charge ratio of positive charges in DODMA to negative charges in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, even more preferably about 1.2:

2.

84. 82. The system of any one of claims 73 to 81, wherein the nanoparticles are lipoplexes comprising DODMA and DSPC in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and wherein the charge ratio of the positive charges of DODMA to the negative charges of RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:

2.

85. 82. The system of any one of claims 73 to 81, wherein the nanoparticles are lipoplexes comprising DODMA:cholesterol:DOPE:PEGcerC16 in a molar ratio of 40:48:10:

2.

86. 82. The system of any one of claims 73 to 81, wherein the nanoparticles are lipoplexes comprising DOTMA and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and wherein the charge ratio of positive charges in DOTMA to negative charges in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, even more preferably about 1.2:

2.

87. 82. The system of any one of claims 73 to 81, wherein the nanoparticles are lipoplexes comprising DOTMA and cholesterol in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and wherein the charge ratio of positive charges in DOTMA to negative charges in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, even more preferably about 1.2:

2.

88. 82. The system of any one of claims 73 to 81, wherein the nanoparticles are lipoplexes comprising DOTAP and DOPE in a molar ratio of 10:0 to 1:9, preferably 8:2 to 3:7, more preferably 7:3 to 5:5, and wherein the charge ratio of positive charges in DOTAP to negative charges in RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1:2, even more preferably 1.4:2 to 1.1:2, and even more preferably about 1.2:

2.

89. 89. The system of any one of claims 65 to 88, wherein the reagent comprises a lipid and the particles formed are LNPs that are complexed with and / or encapsulate RNA molecules.

90. 90. A system according to any one of claims 65 to 89, wherein the reagent comprises a lipid and the particles formed are vesicles, preferably unilamellar liposomes, that encapsulate the RNA molecules.

91. 67. The system of claim 65 or 66, wherein the reagent is or comprises a polyalkyleneimine.

92. The system of claim 90, wherein (a) the molar ratio (N:P ratio) of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphorus atoms (P) in the RNA molecule is 2.0 to 15.0, preferably 6.0 to 12.0, or (b) the molar ratio (N:P ratio) of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphorus atoms (P) in the RNA molecule is at least about 48, optionally about 48 to 300, about 60 to 200, or about 80 to 150.

93. 92. The system of claim 90 or 91, wherein the ionic strength of the composition is 50 mM or less, preferably the concentration of monovalent cations is 25 mM or less and the concentration of divalent cations is 20 μM or less.

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

95. The polyalkyleneimine is represented by the following general formula (I): 【Chemical 1】 [In the formula, R is H, an acyl group, or a group represented by the following general formula (II): 【Chemistry 2】 is a group comprising During the ceremony, R １ is H or the following general formula (III): 【Chemistry 3】 is a group comprising n, m, and l are independently selected from integers from 2 to 10; and p, q, and r are integers, and the sum of p, q, and r is such that the average molecular weight of the polymer is 1.5×10 ２ ~10 ７ Da, preferably 5000 to 10 ５ Da, more preferably 10,000 to 40,000 Da, more preferably 15,000 to 30,000 Da, and even more preferably 20,000 to 25,000 Da] 95. The system of any one of claims 91 to 94, comprising:

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

97. R １ 97. The system of claim 95 or 96, wherein is H.

98. R is H or an acyl group.

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

100. 100. The system of any one of claims 98 to 99, wherein at least 92% of the N atoms in the polyalkyleneimine are protonatable.

101. 101. The system of any one of claims 1 to 100, further comprising one or more peptide-based adjuvants, which optionally include immunomodulatory molecules such as cytokines, lymphokines and / or co-stimulatory molecules.

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

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

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

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

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

107. A kit comprising two RNA molecules, wherein the first RNA molecule comprises an open reading frame encoding a functional RNA-dependent RNA polymerase (replicase); and A kit, wherein the second RNA molecule is a replicable RNA molecule comprising at least one miRNA sequence, which can be excised from the second replicable RNA when present in a cell and can regulate gene expression in the cell, and the replicable RNA molecule can be replicated in trans by a replicase encoded by the first RNA molecule.

108. 108. The kit of claim 107, wherein the first and / or second RNA molecule, preferably the second RNA molecule, further comprises at least one open reading frame (ORF) encoding a protein of interest.

109. 109. The kit of claim 107 or 108, wherein the two RNA molecules are contained in separate containers.

110. 1. A pharmaceutical composition comprising two RNA molecules, wherein the first RNA molecule comprises an open reading frame encoding a functional RNA-dependent RNA polymerase (replicase); and A pharmaceutical composition comprising a pharmaceutically acceptable carrier, wherein the second RNA molecule is a replicable RNA molecule comprising at least one miRNA sequence, which can be excised from the second replicable RNA when present in a cell and can regulate gene expression in the cell, and the replicable RNA molecule can be replicated in trans by a replicase encoded by the first RNA molecule.

111. 111. The pharmaceutical composition of claim 110, wherein the first and / or second RNA molecule, preferably the second RNA molecule, further comprises at least one open reading frame (ORF) encoding a protein of interest.

112. 112. The pharmaceutical composition of claim 110 or 111, formulated for intradermal, subcutaneous, and / or intramuscular administration, such as by injection.

113. 113. A kit or pharmaceutical composition according to any one of claims 107 to 112 for use in therapy.

114. 114. A kit or pharmaceutical composition according to any one of claims 107 to 113 for use in a method of treating or preventing a disease, preferably wherein the subject is a mammal, more preferably wherein the mammal is a human, said method comprising administering to the subject the kit or pharmaceutical composition according to any one of claims 107 to 112.

115. 115. The kit or pharmaceutical composition for use according to claim 114, wherein administration of the pharmaceutical composition comprises intradermal, subcutaneous, or intramuscular administration, such as intradermal, subcutaneous, or intramuscular injection.

116. 115. The kit or pharmaceutical composition for use according to claim 114, wherein injection is by use of a needle or by use of a needle-free injection device.

117. 117. The kit or pharmaceutical composition for use according to any one of claims 114 to 116, wherein administration comprises administration by intramuscular injection, preferably with a needle.

118. The kit or pharmaceutical composition for use according to any one of claims 114 to 117, wherein the RNA molecules are administered separately, preferably by the same route of administration.

119. 118. The kit or pharmaceutical composition of any one of claims 114 to 117, wherein the disease is a bacterial, viral, parasitic or fungal infection, cardiovascular disease, or cancer in a subject.

120. 120. A method for treating or preventing a bacterial, viral, parasitic or fungal infection in a subject, comprising administering to the subject a kit or composition according to any one of claims 107 to 119.

121. A method for treating or preventing cancer in a subject, comprising administering to the subject the kit or composition of any one of claims 107 to 119.

122. 1. A first RNA molecule and a second RNA molecule for use in therapy, wherein the first RNA molecule comprises an open reading frame encoding a functional RNA-dependent RNA polymerase (replicase), and the second RNA molecule is a replicable RNA molecule comprising at least one miRNA sequence, which can be excised from the second replicable RNA when present in a cell and which can regulate gene expression in the cell, and which replicable RNA molecule can be replicated in trans by the replicase encoded by the first RNA molecule, and wherein optionally either the first or the second RNA molecule, preferably the second RNA molecule, further comprises at least one open reading frame (ORF) encoding a protein of interest.

123. 123. The first and second RNA molecules for use according to claim 122, wherein the therapy is the treatment or prevention of cancer or an infectious disease.

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