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WO2024133550A1 - Système d'auto-amplification eucaryote - Google Patents

Système d'auto-amplification eucaryote Download PDF

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WO2024133550A1
WO2024133550A1 PCT/EP2023/087081 EP2023087081W WO2024133550A1 WO 2024133550 A1 WO2024133550 A1 WO 2024133550A1 EP 2023087081 W EP2023087081 W EP 2023087081W WO 2024133550 A1 WO2024133550 A1 WO 2024133550A1
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virus
nucleic acid
acid sequence
promoter
utr
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Matthias BOZZA
Maysam MANSOURI
Philip OHLAND
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Vector Biopharma Ag
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Vector Biopharma Ag
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36141—Use of virus, viral particle or viral elements as a vector
    • C12N2770/36143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/40—Systems of functionally co-operating vectors

Definitions

  • the present invention provides a novel DNA-based expression system based on a self-amplifying system that makes use of alphavirus replicase.
  • the system is broadly applicable in mammalian cells and can be launched from minuscule DNA quantities. It can be used for the targeted and regulatable expression of genes and proteins of interest in mammalian cells for the prevention or treatment of diseases and disorders. Background
  • the introduction of foreign genetic information encoding one or more polypeptides for prophylactic and therapeutic purposes has been a goal of biomedical research for many years.
  • Nucleic acid molecules can be introduced in the form of deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules, either in unmodified or modified, typically stabilized, form.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • expression systems that harbour the coding sequence responsible for producing the protein of interest under the control of a promoter that guarantees its expression in target cells were generated and tested in animal models and, ultimately, humans.
  • DNA expression systems can be delivered as naked or formulated DNA complexes with molecular carriers. DNA can also be transferred to target cells through the generation of viral and viral-like particles.
  • a DNA-based expression system must reach the nucleus of target cells to be functional, where exploiting the cellular expression machinery can start the production of the gene of interest.
  • RNA viruses are a diverse group of infectious particles with an RNA genome.
  • RNA viruses can be sub-grouped into single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) viruses, and the ssRNA viruses can be further generally divided into positive-stranded [(+) stranded] and/or negative-stranded [(-) stranded] viruses.
  • RNA viruses are attractive as a delivery system in biomedicine because their RNA may serve directly as template for translation in the host cell.
  • Alphaviruses are representatives of positive-stranded RNA viruses.
  • Alphaviruses replicate in the cytoplasm of infected cells.
  • the total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genome typically has a 5'-cap, and 3' poly(A) tail.
  • the genome of alphaviruses encodes non- structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome.
  • the four non-structural proteins are typically encoded together by a first ORF beginning near the 5' terminus of the genome, while alphavirus structural proteins are encoded together in a second ORF which is found downstream of the first ORF and extends near the 3' terminus of the genome.
  • alphavirus structural proteins are encoded together in a second ORF which is found downstream of the first ORF and extends near the 3' terminus of the genome.
  • mRNA RNA molecule that resembles eukaryotic messenger RNA
  • the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the non- structural poly-protein (nsP1234).
  • nsP1234 is autoproteolytically cleaved into the fragments nsP123 and nsP4.
  • the polypeptides nsP123 and nsP4 associate to form the (-) strand replicase complex that transcribes (-) stranded RNA, using the (+) stranded genomic RNA as template.
  • nsP123 fragment is completely cleaved into individual proteins nsP1, nsP2, nsP3 and nsP4 (J Virol (1994) 68: 1874-85); these proteins assemble to form the (+) strand replicase complex that synthesizes new (+) stranded genomes, using the (-) stranded complement of genomic RNA as template (Virology (2004) 323: 153-63; J Biol. Chem (2003) 278: 41636- 45).
  • Alphavirus structural proteins (core nucleocapsid protein C, envelope protein P62 and envelope protein E1, all constituents of the virus particle) are typically encoded by one single open reading frame under control of a subgenomic promoter (Microbiol Rev (1994) 58: 491-562).
  • the subgenomic promoter is recognized by alphaviral non-structural proteins acting in cis.
  • alphavirus replicase synthesizes a (+) stranded subgenomic transcript using the (-) stranded complement of genomic RNA as template.
  • the (+) stranded subgenomic transcript encodes the alphavirus structural proteins.
  • the subgenomic RNA transcript serves as template for translation of the open reading frame encoding the structural proteins as one poly-protein, and the poly-protein is cleaved to yield the structural proteins.
  • a packaging signal which is located within the coding sequence of nsP2 ensures selective packaging of genomic RNA into budding virions, packaged by structural proteins (J Virol (1998) 72: 4320-6).
  • RNA messenger RNA
  • CSEs conserved sequence elements
  • CSE 1 found at or near the 5' end of the virus genome, is believed to function as a promoter for (+) strand synthesis from (-) strand templates.
  • CSE 2 located downstream of CSE1 but still close to the 5' end of the genome within the coding sequence for nsP1 is thought to act as a promoter for initiation of (-) strand synthesis from a genomic RNA template (the subgenomic RNA transcript, which does not comprise CSE 2, does not function as a template for (-) strand synthesis).
  • CSE 3 is located in the junction region between the coding sequence for the non-structural and structural proteins and acts as core promoter for the efficient transcription of the subgenomic transcript.
  • CSE 4 which is located just upstream of the poly(A) sequence in the 3' untranslated region of the alphavirus genome, is understood to function as a core promoter for initiation of (-) strand synthesis.
  • CSE 4 and the poly(A) tail of the alphavirus are understood to function together for efficient (-) strand synthesis.
  • the hosts of alphaviruses include a wide range of animals, comprising insects, fish and mammals, such as domesticated animals and humans; alphavirus-derived vectors have therefore been considered as a potential vector for delivery of foreign genetic information into a wide range of target organisms (Microbiol Rev (1994) 58: 491-562; Expert Rev Vaccines (2015) 14: 177-94).
  • Protein of interest may be encoded on respective vectors downstream of the subgenomic promoter.
  • the encoded replicase When introduced into a host cell, the encoded replicase is synthesized, forming replication complexes associated with membrane invaginationt, which may favor cis- replication (as exemplified for Rubella virus which has a similar genome organization as alphaviruses (Virology (2001) 282: 307-19).
  • replication is not cis-exclusive.
  • Alphavirus-based trans-replication systems comprise two nucleic acid molecules, wherein one nucleic acid molecule encodes a viral replicase and the other nucleic acid molecule is capable of being replicated by said replicase in trans. Such trans-replication systems require the presence of both nucleic acid molecules in a single host cell.
  • Viral RNA vectors have frequently been regarded as disadvantageous because of their potential to propagate in a treated individual by forming propagation competent virus particles. This can be associated with health risks, not only for the treated individual, but also for the general population: for example, some alphaviruses are pathogenic for humans (e.g. Chikungunya virus, Venezuelan equine encephalitis virus (VEEV).
  • VEEV Venezuelan equine encephalitis virus
  • it was proposed to introduce a non-viral trans-replication system into host cells Cellular Microbiol (2015) 17: 520-41; J Virol (2011) 85: 4739-51).
  • the trans-replication systems according to these references are based on the introduction of DNA vectors into host cells, wherein the vectors contain the bacteriophage T7 promoter and wherein the host cells are specialized engineered cells expressing the T7 polymerase (J Virol (1999) 73: 251-9).
  • the DNA constructs used therein encodes an internal ribosomal entry site (IRES) element downstream of the T7 promoter and describes the direct use of an RNA replicase construct (encoding nsP1 -4) downstream of an IRES; the RNA construct is prepared in vitro in the absence of the cap structure.
  • IRES internal ribosomal entry site
  • trans-replication systems are functional either as indirect DNA vectors with a bacteriophage promoter for synthesizing RNA in engineered host cells that express a bacteriophage RNA polymerase, or in the form of direct RNA systems that comprise an IRES for driving translation of the replicase.
  • bacteriophage promoter for synthesizing RNA in engineered host cells that express a bacteriophage RNA polymerase
  • IRES direct RNA systems that comprise an IRES for driving translation of the replicase.
  • WO2008/119827 relates to a DNA-based system which however differs from the system of the present disclosure in an important aspect.
  • the present invention hence addresses and solves several shortcomings.
  • a versatile system which enables a high level of transgene expression in eukaryotic cells.
  • said systems utilize indirect DNA vectors engineered to produce and stabilize RNA molecules derived from alphaviruses. More preferably, but not limited to, these DNA systems are transferred into the target cells in complexes with delivery reagents or packaged in viral particles.
  • the present disclosure relates to a DNA-based vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR.
  • said promoter is a eukaryotic or viral RNA Polymerase II promoter.
  • said eukaryotic or viral RNA Polymerase II promoter is selected from a CMV promoter, an EF1alpha promoter, a PGK promoter, an SSFV promoter, a CBA promoter, or a tissue-specific promoter.
  • said promoter is a CMV promoter.
  • said promoter comprises the nucleic acid sequence of SEQ ID No.18.
  • said first nucleic acid sequence further comprises a 5’UTR.
  • said 5’UTR is a eukaryotic 5’UTR.
  • said 5’UTR comprises a 5’ intronic sequence.
  • said intronic sequence contains a splicing donor, a branch and a splicing acceptor site.
  • said 5’ UTR comprises an alphavirus 5’UTR.
  • said alphavirus 5’UTR comprises conserved sequence elements.
  • said conserved sequence elements are selected from CSE 1, CSE 2, CSE 3 and/or CSE 4.
  • said 5’ UTR comprises the nucleic acid sequence of SEQ ID No.24.
  • said alphavirus replicase is or is derived from a replicase selected from the group of: Barmah Forest virus complex (comprising Barmah Forest virus); Eastern equine encephalitis complex (comprising seven antigenic types of Eastern equine encephalitis virus); Middelburg virus complex (comprising Middelburg virus); Ndumu virus complex (comprising Ndumu virus); Semliki Forest virus complex (comprising Bebaru virus, Chikungunya virus, Mayaro virus and its subtype Una virus, O'Nyong Nyong virus, and its subtype Igbo-Ora virus, Ross River virus and its subtypes Bebaru virus, Getah virus, Sagiyama virus, Semliki Forest virus and its subtype Me Tri virus); Venezuelan equine encephalitis complex (comprising Cabassou virus, Everglades virus, Mosso das Pedras virus, Mucambo virus, Paramana virus, Pix
  • said alphavirus replicase is or is derived from Venezuelan equine encephalitis virus.
  • said alphavirus replicase comprises nsP1, nsP2, nsP3 and/or nsP4.
  • said nucleic acid encodes nsP1, nsP2, nsP3 and/or nsP4 comprising SEQ ID Nos.2, 4, 6 and/or 8, respectively.
  • said nucleic acid encodes nsP1, nsP2, nsP3 and/or nsP4 which are at least 80% identical to SEQ ID Nos.2, 4, 6 and/or 8.
  • said subgenomic promoter is or is derived from a subgenomic promoter selected from the group of: Barmah Forest virus complex (comprising Barmah Forest virus); Eastern equine encephalitis complex (comprising seven antigenic types of Eastern equine encephalitis virus); Middelburg virus complex (comprising Middelburg virus); Ndumu virus complex (comprising Ndumu virus); Semliki Forest virus complex (comprising Bebaru virus, Chikungunya virus, Mayaro virus and its subtype Una virus, O'Nyong Nyong virus, and its subtype Igbo-Ora virus, Ross River virus and its subtypes Bebaru virus, Getah virus, Sagiyama virus, Semliki Forest virus and its subtype Me Tri virus); Venezuelan equine encephalitis complex (comprising Cabassou virus, Everglades virus, Mosso das Pedras virus, Mucambo virus, Paramana virus
  • said subgenomic promoter is or is derived from Venezuelan equine encephalitis virus.
  • said subgenomic promoter comprises the nucleic acid sequence of SEQ ID No.13 or 14.
  • said said subgenomic promoter has one nucleotide, two nucleotides nucleotides difference to the nucleic acid sequence of SEQ ID No.13 or 14.
  • said 3’ UTR comprises an alphavirus 3’UTR.
  • said alphavirus 3’UTR comprises a nucleic acid sequence encoding CSE5.
  • said said CSE5 comprises SEQ ID No.23.
  • said CSE5 comprises an amino acid sequence which is at least 80 percent identical to SEQ ID Nos.23.
  • said second nucleic acid sequence further comprises a ribozyme or a pseudoknot.
  • said ribozyme comprises the nucleic acid sequence of SEQ ID No. 11.
  • said pseudoknot comprises the nucleic acid sequence of SEQ ID No.12.
  • said second nucleic acid sequence further comprises one or more polyadenylation sequences.
  • said polyadenylation sequence is selected from an SV40 polyadenylation sequence, a bGH polyadeylation sequence and a hGH polyadeylation sequence.
  • said polyadenylation sequence comprises the nucleic acid sequence of SEQ ID No.26 or 27.
  • said first nucleic acid sequence and said second nucleic acid sequence are on the same nucleic acid molecule. In certain embodiments said first nucleic acid sequence and said second nucleic acid sequence are on different nucleic acid molecules.
  • said second nucleic acid sequence comprises a 5’UTR. In certain embodiments said 5’UTR on said second nucleic acid sequence is 5’ of the subgenomic promoter. In certain embodiments said first nucleic acid sequence and said second nucleic acid sequence are deoxyribonucleic acids. In certain embodiments, the present disclosure relates to a host cell comprising any one of aforementioned vector systems.
  • the present disclosure relates to a method for expressing a gene of interest in a host cell comprising a) introducing any one of aforementioned vector systems into a host call, and b) incubating said host cells at conditions suitable for the expression of said gene of interest.
  • the present disclosure relates to any one of aforementioned vector systems or aforementioned host cell for treating a disease or disorder.
  • Figure legends Figure 1 Schematic depiction of the esaRNA technology. The esaRNA system is launched from a DNA molecule that, to be transcribed, needs to reach to the nucleus.
  • the eukaryotic transcription machinery binds the promoter of the esaRNA construct and produces an RNA molecule that is translocated into the cellular cytoplasm.
  • the esaRNA transcript engages with the cellular translation machinery that produces the alphavirus Replicase (Nsp1-4) and the GOI.
  • the alphavirus replicase in the cytoplasm starts the replication process on the esaRNA transcript amplifying the GOI production.
  • Figure 2. esaRNAs with eukaryotic 3’ and 5’ UTRs provide higher expression levels.
  • Construct 1 (esaRNA), 2 (esaRNA_3’UTR) and 3(esaRNA_5’UTR) were tested in BHK-21 cells for the expression of the reporter gene nano Luciferase (nLuc) 72h post DNA delivery.
  • Figure 3 esaRNA launched from a different Pol II promoter provides a more robust transgene expression than a canonical Pol II-based one.
  • the constructs were used to make adenoviral particles in which the CMV promoter was either driving the expression of the fluorescent reporter gene GFP (A) or the esaRNA GFP-based system (B) and delivered at different Transducing Particles (TU) per cell.
  • Figure 5. esaRNA elicits a more robust transgene expression, starting with minuscule DNA quantities.
  • the efficiency of transgene expression of a construct where the CMV promoter was driving the expression of the reporter gene IgK-nLuc (1) was compared to the esaRNA IgK-nLuc system (2) launched from the same promoter.
  • the transgene expression was monitored daily for 196h in cells that were transfected with a total of 25ng (A), 10 ng (B) or 1 ng (C) and passaged once 144h post DNA delivery.
  • Figure 6. esaRNA expression in various cell lines. The esaRNA-GFP system was tested across multiple cell lines upon delivery with an Adenoviral particle: BHK-21 (1), A549 (2), HEK293 (3), MDA-MB231 (4) and Jurkat (5). While there was some variability, successful expression of the reporter construct could be measured in all cell lines tested.
  • Figure 7. esaRNA delivered on two different nucleic acids maintains functionality. The esaRNA components were placed on two different nucleic acids (DNA).
  • the first contains the Replicase (Nsp1-4) under the control of the CMV, and the second contains the sequences necessary for the replication (CSEs), the SGP and the reporter gene IgK-nLuc as well launched from the CVM promoter.50 ng of the construct containing the expression cassette for the Replicase was delivered upon DNA formulation together with 5 ng (1), 10 ng (2), 25 ng (3) or 50 ng (4) of the reporter construct and the reporter gene expression was measured 48 h post DNA delivery. As can be seen, the esaRNA system, also when used in trans, effectively produces the functional reporter gene.
  • the terms “reduce” or “inhibit” as used herein refer to the ability to cause an overall decrease, preferably of 5 percent or greater, 0 percent or greater, 20 percent or greater, more preferably of 50 percent or greater, and most preferably 75 percent or greater, in the level.
  • the term “inhibit” or similar phrases includes a complete or essentially complete inhibition, i.e. a reduction to zero or essentially to zero.
  • the terms “increase” or “enhance” as used herein refer to an increase or enhancement by about at least 10 percent, preferably at least 20 percent, preferably at least 30 percent, more preferably at least 40 percent, more preferably at least 50 percent, even more preferably at least 80 percent, and most preferably at least 100 percent.
  • nucleic acid as used herein is art recognized and includes deoxyribonucleic acids (DNA) and a ribonucleic acids (RNA). It also includes chemical derivatization of a nucleic acid on a nucleotide base, on the sugar or on the phosphate, and nucleic acids containing non-natural nucleotides and nucleotide analogs.
  • RNA or "RNA molecule” as used herein refers to a molecule which comprises ribonucleotide residues and which is preferably entirely or substantially composed of ribonucleotide residues.
  • Nucleotides in RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxy nucleotides. These altered RNAs can be referred to as analogs, particularly analogs of naturally occurring RNAs.
  • RNA may be single-stranded or double-stranded.
  • the term "single-stranded RNA” generally refers to embodiments wherein no complementary nucleic acid strand (typically no complementary RNA strand; i.e. no complementary RNA molecule) is associated with the RNA molecule.
  • Single-stranded RNA can exist as minus strand [(-) strand] or as plus strand [(+) strand].
  • the (+) strand is the strand that comprises or encodes genetic information.
  • the genetic information may be for example a polynucleotide sequence encoding a protein.
  • the (+) strand may serve directly as template for translation (protein synthesis).
  • the (-) strand is the complement of the (+) strand.
  • (+) strand and (-) strand are two separate RNA molecules, and both these RNA molecules associate with each other to form a double-stranded RNA ("duplex RNA").
  • duplex RNA double-stranded RNA
  • “Half-life” relates to the period of time which is needed to eliminate half of the activity, amount, or number of molecules.
  • the half-life of RNA is indicative for the stability of said RNA.
  • the half-life of RNA may influence the duration of expression of the RNA. It can be expected that RNA having a long half-life will be expressed for an extended time period.
  • DNA or “DNA molecule” as used herein refers to a molecule which comprises deoxyribonucleotide residues and is entirely or substantially composed of deoxyribonucleotide residues.
  • Deoxyribonucleotide relates to a nucleotide which lacks a hydroxyl group at the 2'-position of a beta-D-ribofuranosyl group.
  • the terms "DNA” and “DNA molecule” comprise isolated DNA such as partially or completely purified DNA, essentially pure DNA. Synthetic DNA, and recombinantly generated DNA and includes modified DNA which differs from naturally occurring DNA by addition, deletion, substitution and/or alteration of one or more nucleotides.
  • “Fragment” or “fragment of a nucleic acid sequence” relates to a part of a nucleic acid sequence, i.e. a sequence which represents the nucleic acid sequence shortened at the 5'- and/or 3'-end(s).
  • a fragment of a nucleic acid sequence comprises at least 80 percent, preferably at least 90 percent, 95 percent, 96 percent, 97 percent, 98 percent, or 99 percent of the nucleotide residues from said nucleic acid sequence.
  • variant refers to any variants, in particular mutants, viral strains, splice variants, conformations, isoforms, allelic variants, species variants and species homologs. Complete gene sequencing often identifies numerous allelic variants for a given gene.
  • nucleic acid variants include single or multiple nucleotide deletions, additions, mutations and/or insertions in comparison with the reference nucleic acid.
  • Deletions include removal of one or more nucleotides from the reference nucleic acid.
  • Addition variants comprise 5'- and/or 3'-terminal fusions of one or more nucleotides, such as 1, 2, 3, 5, 10, 20, 30, 50, or more nucleotides.
  • Mutations can include but are not limited to substitutions, wherein at least one nucleotide in the sequence is removed and another nucleotide is inserted in its place (such as transversions and transitions), abasic sites, crosslinked sites, and chemically altered or modified bases.
  • Insertions include the addition of at least one nucleotide into the reference nucleic acid.
  • Variants of specific nucleic acid sequences preferably have at least one functional property of said specific sequences and preferably are functionally equivalent to said specific sequences, e.g. nucleic acid sequences exhibiting properties identical or similar to those of the specific nucleic acid sequences.
  • the degree of identity between a given nucleic acid sequence and a nucleic acid sequence which is a variant of said given nucleic acid sequence will be at least 70 percent, preferably at least 75 percent, preferably at least 80 percent, more preferably at least 85 percent, even more preferably at least 90 percent or most preferably at least 95 percent, 96 percent, 97 percent, 98 percent or 99 percent.
  • the degree of identity is preferably given for a region of at least about 30, at least about 50, at least about 70, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, or at least about 400 nucleotides. In preferred embodiments, the degree of identity is given for the entire length of the reference nucleic acid sequence.
  • sequence similarity'' refers to the percentage of amino acids that either are identical or that represent conservative amino acid substitutions.
  • Sequence identity between two polypeptide or nucleic acid sequences indicates the percentage of amino acids or nucleotides that are identical between the sequences.
  • vector refers to any intermediate vehicles for a nucleic acid which, for example, enables said nucleic acid to be introduced into prokaryotic and/or eukaryotic host cells and, where appropriate, to be integrated into a genome.
  • vectors are preferably replicated and/or expressed in the cell.
  • Vectors comprise plasmids, phagemids, free nucleic acid molecules, virus-like particles (VLPs), enveloped delivery vehicles (EDVs), lipid nanoparticles (LNPs), adeno-associated viruses (AAVs) and adenoviruses.
  • virus-like particle refers to a structure that in at least one attribute resembles a virus, but which has not been demonstrated to be infectious.
  • a VLP may be a nonreplicating, noninfectious viral shell that contains a viral capsid but lacks all or part of the viral genome, in particular, the replicative components of the viral genome.
  • VLPs are generally composed of one or more viral proteins, such as, but not limited to those proteins referred to as capsid, coat, shell, surface, and structural proteins (e.g., VPl, VP2).
  • a VLP may also resemble the structure of a bacteriophage, being non-replicative and noninfectious, and lacking at least the gene or genes coding for the replication machinery of the bacteriophage, and also lacking the gene or genes encoding the protein or proteins responsible for viral attachment to or entry into the host.
  • the ter virs- like particles includes "enveloped delivery vehicles” or “EDVs” in which viral glycoproteins are leveraged for cell- type-specific delivery of cargo into cells (Cell Rep (2021) 35: 109207, bioRxiv https://www.biorxiv.org/content/10.1101/2022.08.24.505004v1.full.pdf).
  • lipid nanoparticle refers to particles having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which include one or more specified lipids.
  • Lipid nanoparticles may be included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like).
  • a target site of interest e.g., cell, tissue, organ, tumor, and the like.
  • Lipid nanoparticles typically comprise a cationic lipid and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids.
  • An AAV can be an AAV derived from a naturally occurring "wild-type" virus, an AAV derived from a recombinant AAV (rAAV) genome packaged into a capsid derived from capsid proteins encoded by a naturally occurring cap gene and/or a rAAV genome packaged into a capsid derived from capsid proteins encoded by a non-natural capsid cap gene.
  • adenovirus refers to any adenovirus, i.e. to human and non-human serotypes. The human isolates are classified into subgroups A-G.
  • a preferred adenovirus of the present disclosure is adenovirus subtype 5 (“HAdV-C5”).
  • the nucleic acid template may be DNA; however, e.g. in the case of transcription from an alphaviral nucleic acid template, the template is typically RNA. Subsequently, the transcribed RNA may be translated into protein.
  • cloning vectors are applied for the generation of transcripts. These cloning vectors are generally designated as transcription vectors and are according to the present invention encompassed by the term "vector" as defined above.
  • the promoter for controlling transcription can be any promoter for any RNA polymerase.
  • a DNA template for transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for transcription.
  • the cDNA may be obtained by reverse transcription of RNA.
  • expression and “expressing” are used herein in their most general meaning and include the production of RNA and/or protein. Expression may be transient or stable.
  • expression or “translation” relates to the process in the ribosomes of a cell by which a strand of messenger RNA directs the assembly of a sequence of amino acids to make a peptide or protein.
  • alphavirus refers to any virus particle that has characteristics of alphaviruses. Characteristics of alphavirus include the presence of a (+) stranded RNA which encodes genetic information suitable for replication in a host cell, including RNA polymerase activity.
  • An alphavirus found in nature is preferably selected from the group consisting of the following: Barmah Forest virus complex (comprising Barmah Forest virus); Eastern equine encephalitis complex (comprising seven antigenic types of Eastern equine encephalitis virus); Middelburg virus complex (comprising Middelburg virus); Ndumu virus complex (comprising Ndumu virus); Semliki Forest virus complex (comprising Bebaru virus, Chikungunya virus, Mayaro virus and its subtype Una virus, O'Nyong Nyong virus, and its subtype Igbo-Ora virus, Ross River virus and its subtypes Bebaru virus, Getah virus, Sagiyama virus, Semliki Forest virus and its subtype Me Tri virus); Venezuelan equine encephalitis complex (comprising Cabassou virus, Everglades virus, Mosso das Pedras virus, Mucambo virus, Paramana virus, Pixuna virus, Rio Negro
  • an alphavirus not found in nature is a variant or derivative of an alphavirus found in nature, which is distinguished from an alphavirus found in nature by at least one mutation in the nucleotide sequence, i.e. the genomic RNA.
  • the mutation in the nucleotide sequence may be selected from an insertion, a substitution or a deletion of one or more nucleotides, compared to an alphavirus found in nature.
  • a mutation in the nucleotide sequence may or may not be associated with a mutation in a polypeptide or protein encoded by the nucleotide sequence.
  • an alphavirus not found in nature may be an attenuated alphavirus.
  • An attenuated alphavirus not found in nature is an alphavirus that typically has at least one mutation in its nucleotide sequence by which it is distinguished from an alphavirus found in nature, and that is either not infectious at all, or that is infectious but has a lower disease-producing ability or no disease- producing ability at all.
  • a "polymerase” as used herein refers to a molecular entity capable of catalyzing the synthesis of a polymeric molecule from monomeric building blocks.
  • a "RNA polymerase” is a molecular entity capable of catalyzing the synthesis of a RNA molecule from ribonucleotide building blocks.
  • a "DNA polymerase” is a molecular entity capable of catalyzing the synthesis of a DNA molecule from deoxy ribonucleotide building blocks.
  • the molecular entity is typically a protein or an assembly or complex of multiple proteins.
  • a DNA polymerase synthesizes a DNA molecule based on a template nucleic acid, which is typically a DNA molecule.
  • a RNA polymerase synthesizes a RNA molecule based on a template nucleic acid, which is either a DNA molecule (in that case the RNA polymerase is a DNA-dependent RNA polymerase, DdRP), or is RNA molecule (in that case the RNA polymerase is a RNA- dependent RNA polymerase, RdRP).
  • a "RNA dependent RNA polymerase” or an “RdRP” is an enzyme that catalyzes the transcription of RNA from an RNA template.
  • alphaviral RNA-dependent RNA polymerase sequential synthesis of (-) strand complement of genomic RNA and of (+) strand genomic RNA leads to RNA replication.
  • RNA-dependent RNA polymerase is thus synonymously referred to as "RNA replicase” or “alphavirus replicase”.
  • RNA- dependent RNA polymerases are typically encoded by all RNA viruses except retroviruses. Typical representatives of viruses encoding a RNA-dependent RNA polymerase are alphaviruses. Any RNA replicase of an alphavirus may be used withing the present disclosure.
  • RNA replicase of alphaviruses are consists of four individual proteins nsP1, nsP2, nsP3 and nsP4, which are autoproteolytically cleaved from a progenitor polypeptide, nsP1234.
  • CSE1, CSE2, CSE3 and CSE4 may be partly or fully located on the nucleic acid sequence encoding nSP1.
  • nSP1 exemplified herein is encoded my the following nucleic acid: atgagagaagcccagaccaattacctacccaaaatggagaaagttcacgttgacatcgaggaagac agcccattcctcagagcttttgcagcggagcttcccgcagtttgaggtagaagccaagcaggtcact gataatgaccatgctaatgccagagcgttttcgcatctggcttcaaaactgatcgaaacggaggtg gacccatccgacacgatccttgacattggaagtgcgcccgcgcagaatgtattctaagcacaag tatcattgt
  • nSP1 exemplified herein is comprises the following amino acid sequence: MREAQTNYLPKMEKVHVDIEEDSPFLRALQRSFPQFEVEAKQVTDNDHANARAFSHLASKLIETEV DPSDTILDIGSAPARRMYSKHKYHCICPMRCAEDPDRLYKYATKLKKNCKEITDKELDKKMKELAA VMSDPDLETETMCLHDDESCRYEGQVAVYQDVYAVDGPTSLYHQANKGVRVAYWIGFDTTPFMFKN LAGAYPSYSTNWADETVLTARNIGLCSSDVMERSRRGMSILRKKYLKPSNNVLFSVGSTIYHEKRD LLRSWHLPSVFHLRGKQNYTCRCETIVSCDGYVVKRIAISPGLYGKPSGYAATMHREGFLCCKVTD TLNGERVSFPVCTYVPATLCDQMTGILATDVSADDAQKLLVGLNQRIVVNGRTQRNTNTMKNYLLP VVAQAFARWAKEYK
  • nSP2 exemplified herein is encoded my the following nucleic acid: ggctcagtggagacacctcgtggcttgataaaggttaccagctacgatggcgaggacaagatcggc tcttacgctgtgctttctcgcaggctgtactcaagagtgaaaaattatcttgcatccaccctctc gctgaacaagtcatagtgataacacactctggccgaaaagggcgttatgccgtggaaccataccat ggtaaagtagtggtgccagagggacatgcaatacccgtccaggacttttcaagctctgagtgaaagt gccaccaccattgtgtacaacgtgtgtgaaagt gccaccattgtgt
  • nSP2 exemplified herein is comprises the following amino acid sequence: GSVETPRGLIKVTSYDGEDKIGSYAVLSPQAVLKSEKLSCIHPLAEQVIVITHSGRKGRYAVEPYH GKVVVPEGHAIPVQDFQALSESATIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEY LYDIDRKQCVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVYGVPGSGKSGIIKS AVTKKDLVVSAKKENCAEIIRDVKKMKGLDVNARTVDSVLLNGCKHPVETLYIDEAFACHAGTLRA LIAIIRPKKAVLCGDPKQCGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFYDKKMR TTNPKETKIVIDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAAASQGLTRKGVYAVRYKV N
  • nSP3 exemplified herein is encoded my the following nucleic acid: gcaccctcatatcatgtggtgcgaggggatattgccacggccaccgaaggagtgattataaatgct gctaacagcaaaggacaacctggcggaggggtgtgcggagcgctgtataagaaattcccggaaagc ttcgatttacagccgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaacatatcatt catgccgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaacagttggcagaggct tatgagtccatcgctaagattgtcaacgataacaattacaagtcagtagcgattccactgttttg
  • nSP3 exemplified herein is comprises the following amino acid sequence: APSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGAAKHII HAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGIFSGNKDRLTQSLNHLLTAL DTTDADVAIYCRDKKWEMTLKEAVARREAVEEICISDDSSVTEPDAELVRVHPKSSLAGRKGYSTS DGKTFSYLEGTKFHQAAKDIAEINAMWPVATEANEQVCMYILGESMSSIRSKCPVEESEASTPPST LPCLCIHAMTPERVQRLKASRPEQITVCSSFPLPKYRITGVQKIQCSQPILFSPKVPAYIHPRKYL VETPPVDETPEPSAENQSTEGTPEQPPLITEDETRTRTPEPIIIEEEEEDSISLLSDGPTHQVLQV EADIHGPPSVSSSSWSIPHA
  • nSP4 exemplified herein is encoded my the following nucleic acid: TACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACAACAAAAATCAGTAAGGCAAACGGTGCTA TCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTATGCCCCGCGCCTCGACCAAGAAAAA GAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTGCTAACAGAAGCAGATACCAGTCC AGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTCTGCAAGGCCTAGGGCATTATTTG AAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTGTTCCTTTGTATTCATCTAGTGTG AACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCCATGTTGAAAGAACTTT CCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTGACGGAGCT TCATGCTGCTTAGAC
  • nSP4 exemplified herein is comprises the following amino acid sequence: YIFSSDTGQGHLQQKSVRQTVLSEVVLERTELEISYAPRLDQEKEELLRKKLQLNPTPANRSRYQS RKVENMKAITARRILQGLGHYLKAEGKVECYRTLHPVPLYSSSVNRAFSSPKVAVEACNAMLKENF PTVASYCIIPEYDAYLDMVDGASCCLDTASFCPAKLRSFPKKHSYLEPTIRSAVPSAIQNTLQNVL AAATKRNCNVTQMRELPVLDSAAFNVECFKKYACNNEYWETFKENPIRLTEENVVNYITKLKGPKA AALFAKTHNLNMLQDIPMDRFVMDLKRDVKVTPGTKHTEERPKVQVIQAADPLATAYLCGIHRELV RRLNAVLLPNIHTLFDMSAEDFDAIIAEHFQPGDCVLETDIASFDKSEDDAMALTALMILEDLGVD AELLTLIEAAFGEISSIHL
  • RNA replication does not occur via a DNA intermediate, but is mediated by a RNA-dependent RNA polymerase (RdRP): a template RNA strand (first RNA strand) serves as template for the synthesis of a second RNA strand that is complementary to the first RNA strand or to a part thereof.
  • the second RNA strand may in turn optionally serve as a template for synthesis of a third RNA strand that is complementary to the second RNA strand or to a part thereof.
  • the third RNA strand is identical to the first RNA strand or to a part thereof.
  • gene of interest and “protein of interest” as used herein refer to a gene or protein, as applicable, that is to be transcribed or expressed with the vectors, nucleic acid molecules and methods of the present disclosure.
  • the gene or protein of interest is Igk-Luc, the protein product of which can be analyzed and quantified by measuring luciferase activity.
  • the secretion signal of the Immunoglobulin kappa was fused to the reporter gene Luciferase. This synthetic fusion allows the secretion of the reporter gene into the cell-culturing medium.
  • reporter gene One such gene of interest or protein of interest which serves to analyze and quantify the functionality of a test system may also be referred to as “reporter gene” or “reporter protein”.
  • the gene of interest exemplified herein has the following nucleic acid sequence: ATGACTAGTGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGT GACGCTAGTggtggttctggtATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACA GCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGG GTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCAT GTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTG GTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCA
  • T he protein of interest exemplified herein has the following nucleic acid sequence: MTSETDTLLLWVLLLWVPGSTGDASGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLG VSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDG VTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERI LAASSGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHT AQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIP PPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKL
  • mRNA as used herein means "messenger RNA” and relates to a transcript which is typically generated by using a DNA template and encodes a peptide or protein.
  • mRNA typically comprises a 5’cap, a 5’ UTR, a protein coding region, a 3’ UTR, and a poly(A) sequence.
  • mRNA coding for a single protein is referred to as a “monocistronic mRNA”.
  • a mRNA may also encode for more than one protein or polypeptide (cistrons), such mRNA is referred to as “polycistronic mRNA”.
  • the mRNA may contain additional regulatory elements, such as a ribozyme for stabilization.
  • ribozyme is typically encoded on the 3’ of the mRNA.
  • regulatory elements may also be located between said proteins or polypeptides encoded on the mRNA.
  • Such regulatory elements may for a example be a promoter, such as a subgenomic promoter or an internal ribosome entry sequence (IRES).
  • IRS internal ribosome entry sequence
  • ribozyme refers to an RNA molecule capable of acting as an enzyme.
  • some ribozymes are capable of cleaving RNA molecules.
  • RNA cleaving ribozymes typically consist at least of a catalytic domain and a recognition sequence that is recognized by the catalytic domain.
  • the catalytic domain can be a part of the same RNA molecule as the recognition sequence, and thus mediate cis-cleavage.
  • the catalytic domain can be a separate RNA molecule from the RNA molecule comprising the recognition sequence, and thus mediate trans-cleavage.
  • the ribozyme exemplified herein has the following nucleic acid sequence: gggtcggcatggcatctccacctcctcgcggtccgacctgggcatccgaaggaggacgcacgtcca ctcggatggctaagggag (SEQ ID no.
  • the term "pseudoknot” as used herein refers to a nucleic acid secondary structure containing at least two stem-loop structures in which half of one stem is intercalated between the two halves of another stem. It includes, but is not limited to, hairpins, multiloops, kissing loops, coaxial stacking, triplexes, pseudoknot-like structures, pseudoknotted hairpins and/or a decoy pseudoknotted hairpins or other RNA structural motifs.
  • the pseudoknot exemplified herein has the following nucleic acid sequence: CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID no.
  • promoter refers to a nucleic acid sequence which controls synthesis of a transcript, e.g. a transcript comprising a coding sequence, by providing a recognition and binding site for RNA polymerase.
  • the promoter region may include further recognition or binding sites for further factors involved in regulating transcription of said gene.
  • a promoter may control transcription of a prokaryotic or eukaryotic gene.
  • a promoter may be "inducible” and initiate transcription in response to an inducer, or may be “constitutive” if transcription is not controlled by an inducer. An inducible promoter is expressed only to a very small extent or not at all, if an inducer is absent.
  • a specific promoter according to the present invention is a subgenomic promoter of an alphavirus, as described herein.
  • Other specific promoters are genomic plus-strand or negative-strand promoters of an alphavirus.
  • the term "subgenomic promoter” or “SGP” as used herein refers to a nucleic acid sequence upstream (5') of a nucleic acid sequence (e.g. coding sequence), which controls transcription of said nucleic acid sequence by providing a recognition and binding site for RNA polymerase, typically RNA-dependent RNA polymerase.
  • the SGP may include further recognition or binding sites for further factors.
  • a subgenomic promoter is typically a genetic element of a positive strand RNA virus, such as an alphavirus.
  • a subgenomic promoter of alphavirus is a nucleic acid sequence comprised in the viral genomic RNA.
  • the subgenomic promoter is generally characterized in that it allows initiation of the transcription (RNA synthesis) in the presence of an RNA-dependent RNA polymerase, e.g. alphavirus replicase.
  • a RNA (-) strand i.e. the complement of alphaviral genomic RNA, serves as a template for synthesis of a (+) strand subgenomic RNA molecule, and subgenomic (+) strand synthesis is typically initiated at or near the subgenomic promoter.
  • a subgenomic promoter exemplified herein has the following nucleic acid sequence: atggactacgacata (SEQ ID no. 13).
  • Another subgenomic promoter exemplified herein has the following nucleic acid sequence: [SGP(30)] atggactacgacatagtctagtccgccaag (SEQ ID no. 14).
  • the term “Kozak sequence” as used herein refers to a short nucleotide sequence that facilitates efficient initiation of translation of mRNA.
  • a typical consensus Kozak sequence is GCCRCC (SEQ ID No. 15) where R is a purine (A or G). Typically the Kozak sequence is located directly upstream of the translation start codon.
  • the Kozak sequence exemplified herein has the following nucleic acid sequence: GCCACC (SEQ ID no. 16).
  • the Kozak sequence may also be considered as part of a promoter, e.g. the subgenomic promoter.
  • the subgenomic promoter has the following nucleic acid sequence: atggactacgacatagccacc (SEQ ID no. 17).
  • the term "conserved sequence element" or "CSE" as used herein refers to a nucleotide sequence found in alphavirus RNA.
  • CSE includes CSE 1, CSE 2, CSE 3 and CSE 4 (Future Microbiol (2009) 4: 837-56).
  • CSE1 exemplified herein comprises the following nucleic acid sequence: ggcggcgcatgagagaagcccag (SEQ ID no. 19).
  • CSE2 exemplified herein comprises the following nucleic acid sequence: ctacccaaaatggagaaagttcacgttgacatcgaggaagacagcccattcctcagagctttgcag cggagcttccgcagtttgaggtag (SEQ ID no. 20).
  • CSE3 exemplified herein comprises the following nucleic acid sequence: agcaggtcactgataatgaccatgct (SEQ ID no. 21).
  • CSE4 exemplified herein comprises the following nucleic acid sequence: gccagagcgttttcgcatctggc (SEQ ID no. 22).
  • CSE5 exemplified herein comprises the following nucleic acid sequence: attttgtttttaatatttc (SEQ ID no. 23).
  • the term "untranslated region” or “UTR” as used herein refers to a region which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule.
  • a 3'-UTR if present, is located at the 3' end of a gene, downstream of the termination codon of a protein-encoding region, but the term “3'-UTR" or “3’ untranslated region” does preferably not include the poly(A) tail.
  • the 3'-UTR is upstream of the poly(A) tail (if present), e.g. directly adjacent to the poly(A) tail.
  • a “5'-UTR” or “3’ untranslated region”, if present, is located at the 5' end of a gene, upstream of the start codon of a protein-encoding region.
  • a 5'-UTR is downstream of the 5'-cap (if present), e.g. directly adjacent to the 5'- cap.
  • 5'- and/or 3'-untranslated regions may be functionally linked to an open reading frame, so as for these regions to be associated with the open reading frame in such a way that the stability and/or translation efficiency of the RNA comprising said open reading frame are increased.
  • an untranslated region can be present 5' (upstream) of the open reading frame encoding the replicase (5'-UTR) and/or 3' (downstream) of the open reading frame encoding the replicase (3'-UTR).
  • the RNA construct for expressing alphavirus replicase comprises a 5' UTR, an open reading frame encoding alphavirus replicase, and a 3' UTR.
  • UTRs are implicated in stability and translation efficiency of RNA. Both can be improved, besides structural modifications concerning the 5'-cap and/or the 3' poly(A)-tail, by selecting specific 5' and/or 3' untranslated regions (UTRs).
  • Sequence elements within the UTRs are generally understood to influence translational efficiency (mainly 5'-UTR) and RNA stability (mainly 3 -UTR). It is preferable that a 5’-UTR is present that is active in order to increase the transcription efficiency and the nuclear export.
  • the 5’ UTR is preferably an intron-like sequence flanked by a 5’ splicing donor and a 3’ splicing acceptor site. Independently or additionally, it is preferable that a 3’-UTR is present that is active in order to increase the translation efficiency and/or stability of a nucleic acid sequence.
  • the 5’ UTR exemplified herein has the following nucleic acid sequence: GTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGGCCAATAGAAACTGGGCTTGTCGAGACAGAAGATTC TTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAG (SEQ ID No. 24)
  • the 3’ UTR exemplified herein has the following nucleic acid sequence: ATACAGCAGCAATTGGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTT TTATTTTTTCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTC (SEQ ID no. 25).
  • the RNA construct for expressing alphavirus replicase may comprise a 5'-cap.
  • the terms "5'-cap”, “cap”, “5'- cap structure” and “cap structure” are used synonymously to refer to a dinucleotide that is found on the 5' end of some eukaryotic primary transcripts such as precursor messenger RNA.
  • a 5'-cap is a structure wherein a (optionally modified) guanosine is bonded to the first nucleotide of an mRNA molecule via a 5' to 5' triphosphate linkage (or modified triphosphate linkage in the case of certain cap analogs).
  • the terms can refer to a conventional cap or to a cap analog.
  • nucleic acid sequence which is active in order to increase the translation efficiency and/or stability of a nucleic acid sequence with reference to a first nucleic acid sequence (e.g. a UTR), means that the first nucleic acid sequence is capable of modifying, in a common transcript with the second nucleic acid sequence, the translation efficiency and/or stability of said second nucleic acid sequence in such a way that said translation efficiency and/or stability is increased in comparison with the translation efficiency and/or stability of said second nucleic acid sequence in the absence of said first nucleic acid sequence.
  • transcription efficiency relates to the amount of translation product provided by an RNA molecule within a particular period of time
  • stability relates to the half-life of an RNA molecule.
  • expression control sequence includes promoters, ribosome-binding sequences and other control elements which control transcription of a gene or translation of the derived RNA.
  • the precise structure of expression control sequences may vary depending on the species or cell type but usually includes 5'- untranscribed and 5'- and 3'-untranslated sequences involved in initiating transcription and translation, respectively. More specifically, 5'- untranscribed expression control sequences include a promoter region which encompasses a promoter sequence for transcription control of the functionally linked gene.
  • Expression control sequences may also include enhancer sequences or upstream activator sequences.
  • An expression control sequence of a DNA molecule usually includes 5'-untranscribed and 5'- and 3 -untranslated sequences such as TATA box, capping sequence, CAAT sequence and the like.
  • An expression control sequence of alphaviral RNA may include a subgenomic promoter and/or one or more conserved sequence element(s).
  • a specific expression control sequence according to the present invention is a subgenomic promoter of an alphavirus.
  • the terms "poly(A) sequence”, “poly(A) tail” and “polyadenylation sequence” as used herein refer to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3' end of an RNA molecule.
  • An uninterrupted sequence is characterized by consecutive adenylate residues.
  • an uninterrupted poly(A) sequence is typical.
  • a poly(A) sequence is normally not encoded in eukaryotic DNA but is attached during eukaryotic transcription in the cell nucleus to the free 3' end of the RNA by a template- independent RNA polymerase after transcription.
  • the present disclosure makes use of poly(A) sequences encoded by DNA.
  • the length of the poly(A) tail may vary.
  • the poly(A) tail exemplified herein consists of 26 adenylate residues.
  • the constructs of the present disclosure may comprise one or more polyadenylation signals.
  • the polyadenylation signal may be a polyadenylation signal of SV 40 with the following nucleic acid sequence: AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAA GCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAAGAT (SEQ ID no. 26).
  • the polyadenylation signal may also be the polyadenylation signal of human hormone gene HGH with the following nucleic acid sequence: CCCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTG CCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAAT ATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGC GGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCC TGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGG CTCAGCTAATTTTTGTTTTTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGCTCTCCAAC TCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAG
  • nucleic acid sequences specified herein, in particular transcribable and coding nucleic acid sequences may be combined with any expression control sequences, in particular promoters, which may be homologous or heterologous to said nucleic acid sequences, with the term “homologous” referring to the fact that a nucleic acid sequence is also functionally linked naturally to the expression control sequence, and the term “heterologous” referring to the fact that a nucleic acid sequence is not naturally functionally linked to the expression control sequence.
  • a transcribable nucleic acid sequence in particular a nucleic acid sequence coding for a peptide or protein, and an expression control sequence are "functionally” linked to one another, if they are covalently linked to one another in such a way that transcription or expression of the transcribable and in particular coding nucleic acid sequence is under the control or under the influence of the expression control sequence. If the nucleic acid sequence is to be translated into a functional peptide or protein, induction of an expression control sequence functionally linked to the coding sequence results in transcription of said coding sequence, without causing a frame shift in the coding sequence or the coding sequence being unable to be translated into the desired peptide or protein.
  • the single-stranded nucleic acid molecule produced during transcription typically has a nucleic acid sequence that is the complementary sequence of the template.
  • the terms “template” or “nucleic acid template” or “template nucleic acid” generally refer to a nucleic acid sequence that may be replicated or transcribed.
  • the term “nucleic acid sequence transcribed from a nucleic acid sequence” as used herein refers to a nucleic acid sequence, where appropriate as part of a complete RNA molecule, which is a transcription product of a template nucleic acid sequence.
  • the transcribed nucleic acid sequence is a single-stranded RNA molecule.
  • nucleic acid refers according to the invention to that end which has a free phosphate group.
  • 3' end of a nucleic acid refers to that end which has a free hydroxy group.
  • An element that is located "upstream” of a second element can be synonymously referred to as being located 5’ of that second element.
  • An element that is located “downstream” of a second element can be synonymously referred to as being located 3’ of that second element.
  • a nucleic acid is “functionally linked” if it is functionally related to another nucleic acid sequence.
  • a promoter is functionally linked to a coding sequence if it influences transcription of said coding sequence.
  • Functionally linked nucleic acids are typically adjacent to one another, where appropriate separated by further nucleic acid sequences, and, in particular embodiments, are transcribed by RNA polymerase to give a single RNA molecule (common transcript).
  • a nucleic acid is functionally linked according to the invention to expression control sequences which may be homologous or heterologous with respect to the nucleic acid.
  • an "isolated molecule” as used herein is intended to refer to a molecule which is substantially free of other molecules such as other cellular material.
  • the term "recombinant” as used herein refers to an entity or molecules which is made through genetic engineering. Recombinant entities or moieties are not occurring naturally.
  • the term “naturally occurring” as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.
  • the term “peptide” as used herein refers to as used herein refers to a molecule consisting of one or more chains of multiple, i.e.
  • polypeptide refers to a molecule consisting of one or more chains of multiple, i.e. two or more, amino acids linked via peptide bonds. A polypeptide typically consists of more than twenty amino acids linked via peptide bonds.
  • heterologous in the context of a gene, a nucleic acid, a polypeptide or a cell refers to such gene, nucleic acid, polypeptide or cell that is derived from a source other than the subject.
  • autologous refers to anything that is derived from the same subject.
  • autologous cell refers to a cell derived from the same subject.
  • introduction of autologous cells into a subject is advantageous because these cells overcome the immunological barrier which otherwise results in rejection.
  • allogeneic refers anything that is derived from different individuals of the same species. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical.
  • sergeneic is used to describe anything that is derived from individuals or tissues having identical genotypes, i.e., identical twins or animals of the same inbred strain, or their tissues or cells.
  • the vector system employed by the present disclosure makes use of two nucleic acid sequences.
  • nucleic acid sequences may be comprised on the same nucleic acid molecule (in cis) or on different nucleic molecules (in trans).
  • the present disclosure relates to a vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR.
  • said first nucleic acid sequence and said second nucleic acid sequence are deoxyribonucleic acids
  • the present disclosure relates to a DNA-based vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR.
  • the first nucleic acid sequence of the vector system of the present disclosure comprises a promoter.
  • Said promoter may be a eukaryotic promoter or a viral RNA Polymerase II promoter.
  • said eukaryotic or viral RNA Polymerase II promoter is selected from a CMV promoter, an EF1alpha promoter, a PGK promoter, an SSFV promoter, a CBA promoter, or a tissue-specific promoter.
  • said promoter is a CMV promoter.
  • said promoter is a miniCMV promoter.
  • said promoter comprises the nucleic acid sequence of GTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCC ATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCA TTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT (SEQ ID No.18). In certain embodiments said promoter consists of the amino acid sequence of SEQ ID No.18.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a eukaryotic promoter or a viral RNA Polymerase II promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a CMV promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a miniCMV promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said promoter of the first nucleic acid sequence comprises the amino acid sequence of SEQ ID No.18.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said promoter of the first nucleic acid sequence consists of the nucleic acid sequence of SEQ ID No.18.
  • the first nucleic acid sequence of the vector system of the present disclosure comprises an open reading frame encoding alphavirus replicase.
  • Said alphavirus replicase is or is derived from a replicase selected from the group of: Barmah Forest virus complex (comprising Barmah Forest virus); Eastern equine encephalitis complex (comprising seven antigenic types of Eastern equine encephalitis virus); Middelburg virus complex (comprising Middelburg virus); Ndumu virus complex (comprising Ndumu virus); Semliki Forest virus complex (comprising Bebaru virus, Chikungunya virus, Mayaro virus and its subtype Una virus, O'Nyong Nyong virus, and its subtype Igbo-Ora virus, Ross River virus and its subtypes Bebaru virus, Getah virus, Sagiyama virus, Semliki Forest virus and its subtype Me Tri virus); Venezuelan equine encephalitis complex (comprising Cabassou virus, Everglades virus, Mosso das Pedras virus, Mucambo virus, Paramana virus, Pixuna
  • said alphavirus replicase is or is derived from Venezuelan equine encephalitis virus.
  • said alphavirus replicase comprises nsP1, nsP2, nsP3 and/or nsP4.
  • said alphavirus replicase comprises nsP1, nsP2, nsP3 and nsP4.
  • said nsP1, nsP2, nsP3 and/or nsP4 comprise SEQ ID Nos.2, 4, 6 and/or 8, respectively.
  • said nsP1, nsP2, nsP3 and nsP4 comprise SEQ ID Nos.2, 4, 6 and 8, respectively.
  • said nsP1, nsP2, nsP3 and nsP4 comprise an amino acid sequence which is at least 80 percent, at least 90 percent, at least 95 percent, at least 97 percent, at least 98 percent or at least 99 percent identical to SEQ ID Nos.2, 4, 6 and 8.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said alphavirus replicase is or is derived from Venezuelan equine encephalitis virus.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said alphavirus replicase comprises nsP1, nsP2, nsP3 and/or nsP4.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said alphavirus replicase comprises nsP1, nsP2, nsP3 and nsP4.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said alphavirus replicase encoded by the first nucleic acid comprises SEQ ID Nos.2, 4, 6 and/or 8.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said alphavirus replicase encoded by the first nucleic acid comprises SEQ ID Nos.2, 4, 6 and 8.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said alphavirus replicase encoded by the first nucleic acid comprises SEQ ID Nos.2, 4, 6 and 8. wherein said first nucleic acid encodes an alphavirus replicase comprising nsP1, nsP2, nsP3 and nsP4 which are at least 80% identical to SEQ ID Nos.2, 4, 6 and/or 8.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a CMV promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said alphavirus replicase encoded by the first nucleic acid comprises SEQ ID Nos.2, 4, 6 and 8.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said alphavirus replicase encoded by the first nucleic acid comprises SEQ ID Nos.2, 4, 6 and 8, and wherein said promoter of the first nucleic acid sequence comprises the amino acid sequence of SEQ ID No.18.
  • the second nucleic acid sequence of the vector system of the present disclosure comprises one or more subgenomic promoters.
  • said second nucleic acid sequence of the vector system of the comprises one subgenomic promoters. In certain embodiments said second nucleic acid sequence of the vector system of the comprises two subgenomic promoters. In certain embodiments said second nucleic acid sequence of the vector system of the comprises more than two subgenomic promoters. If said second nucleic acid sequence comprises more than one subgenomic promoter, then said subgenomic promoter may be arranged in tandem.
  • said one or more subgenomic promoter is or is derived from a subgenomic promoter selected from the group of: Barmah Forest virus complex (comprising Barmah Forest virus); Eastern equine encephalitis complex (comprising seven antigenic types of Eastern equine encephalitis virus); Middelburg virus complex (comprising Middelburg virus); Ndumu virus complex (comprising Ndumu virus); Semliki Forest virus complex (comprising Bebaru virus, Chikungunya virus, Mayaro virus and its subtype Una virus, O'Nyong Nyong virus, and its subtype Igbo-Ora virus, Ross River virus and its subtypes Bebaru virus, Getah virus, Sagiyama virus, Semliki Forest virus and its subtype Me Tri virus); Venezuelan equine encephalitis complex (comprising Cabassou virus, Everglades virus, Mosso das Pedras virus, Muca
  • said subgenomic promoter is or is derived from Venezuelan equine encephalitis virus. In certain embodiments said subgenomic promoter comprises SEQ ID No.13 or 14. In certain embodiments said subgenomic promoter comprises SEQ ID No.13. In certain embodiments said subgenomic promoter comprises SEQ ID No. 14. In certain embodiments said subgenomic promoter consists of SEQ ID No. 13 or 14. In certain embodiments said subgenomic promoter consists of SEQ ID No.13. In certain embodiments said subgenomic promoter consists of SEQ ID No.14. In certain embodiments.
  • said subgenomic promoter has one nucleotide, two nucleotides or three nucleotides difference to the nucleic acid sequence of SEQ ID No. 13. In certain embodiments. In certain embodiments said subgenomic promoter has one nucleotide, two nucleotides or three nucleotides difference to the nucleic acid sequence of SEQ ID No.14.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said subgenomic promoter is or is derived from Venezuelan equine encephalitis virus.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said subgenomic promoter comprises the nucleic acid sequence of SEQ ID No.13 or 14.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said subgenomic promoter comprises the nucleic acid sequence of SEQ ID No 13.
  • said subgenomic promoter has one nucleotide, two nucleotides or three nucleotides difference to the nucleic acid sequence of SEQ ID No.13.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said subgenomic promoter comprises the nucleic acid sequence of SEQ ID No.14.
  • said subgenomic promoter has one nucleotide, two nucleotides or three nucleotides difference to the nucleic acid sequence of SEQ ID No.14.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said alphavirus replicase encoded by the first nucleic acid comprises SEQ ID Nos.2, 4, 6 and 8, and wherein said subgenomic promoter comprises SEQ ID No.14.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said subgenomic promoter comprises the nucleic acid sequence of SEQ ID No.14, and wherein said promoter of the first nucleic acid sequence comprises the nucleic acid sequence of SEQ ID No.18.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said alphavirus replicase encoded by the first nucleic acid comprises SEQ ID Nos.2, 4, 6 and 8, wherein said promoter of the first nucleic acid sequence comprises the nucleic acid sequence of SEQ ID No.18, and wherein said subgenomic promoter comprises the nucleic acid sequence of SEQ ID No.14.
  • the second nucleic acid sequence of the vector system of the present disclosure comprises a 3’UTR.
  • said 3’UTR is or is derived from an alphavirus.
  • said 3’UTR comprises a nucleic acid sequence encoding CSE5.
  • said CSE5 comprises the amino acid sequence of SEQ ID No.23.
  • said CSE5 consists of the amino acid sequence of SEQ ID No.23.
  • said CSE5 comprises an amino acid sequence which is at least 80 percent, at least 90 percent, at least 95 percent, at least 97 percent, at least 98 percent or at least 99 percent identical to SEQ ID Nos.23.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said 3’UTR is or is derived from an alphavirus.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said 3’UTR comprises CSE5.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said 3’UTR comprises the nucleic acid sequence of SEQ ID No.23.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said 3’UTR comprises a nucleic acid sequence which is at least 80 percent, at least 90 percent, at least 95 percent, at least 97 percent, at least 98 percent or at least 99 percent identical to SEQ ID Nos. 23.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said promoter comprises the nucleic acid sequence of SEQ ID No.18, and wherein said 3’UTR comprises the nucleic acid sequence of SEQ ID No.23.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said alphavirus replicase encoded by the first nucleic acid comprises SEQ ID Nos.2, 4, 6 and 8, wherein said 3’UTR comprises the nucleic acid sequence of SEQ ID No.23.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said subgenomic promoter comprises the nucleic acid sequence of SEQ ID No.13 or 14,and wherein said 3’UTR comprises the nucleic acid sequence of SEQ ID No.23.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said promoter comprises the nucleic acid sequence of SEQ ID No.18, wherein said alphavirus replicase encoded by the first nucleic acid comprises SEQ ID Nos.2, 4, 6 and 8, wherein said subgenomic promoter comprises the nucleic acid sequence of SEQ ID No.13 or 14,and wherein said 3’UTR comprises the nucleic acid sequence of SEQ ID No.23.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR.
  • the first nucleic acid sequence of the vector system of the present disclosure may further comprise a 5’UTR.
  • said 5’UTR is a eukaryotic 5’UTR.
  • said 5’UTR comprises a 5’ intronic sequence.
  • intronic sequence contains a splicing donor, a branch and a splicing acceptor site.
  • RNA splicing A newly-made precursor messenger RNA (pre-mRNA) transcript is transformed into a mature messenger RNA (mRNA) by removing non-coding sequences.
  • pre-mRNA precursor messenger RNA
  • mRNA mature messenger RNA
  • splicing occurs in the nucleus either during or immediately after transcription, and it is needed to create an mRNA molecule that can be translated into protein.
  • the RNA splicing is catalyzed by the nuclear complex spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs) that facilitate the removal of the sequences comprised through a splice donor and a splice acceptor site.
  • snRNPs small nuclear ribonucleoproteins
  • said 5’UTR is an alphavirus 5’UTR.
  • said alphavirus 5’UTR comprises conserved sequence elements.
  • said conserved sequence elements are selected from CSE 1, CSE 2, CSE 3 and/or CSE 4.
  • said 5’ UTR comprises the amino acid sequence of SEQ ID No.24.
  • said 5’ UTR consists of the amino acid sequence of SEQ ID No.24.
  • said 3’UTR comprises an nucleic acid sequence which is at least 80 percent, at least 90 percent, at least 95 percent, at least 97 percent, at least 98 percent or at least 99 percent identical to SEQ ID No.24. Therefore, in certain embodiments the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a 5’UTR promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a eukaryotic 5’UTR promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a eukaryotic 5’UTR promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR wherein said 5’UTR comprises a 5’ intronic sequence.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a eukaryotic 5’UTR promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR wherein said 5’UTR comprises a 5’ intronic sequence and wherein said 5’ intronic sequence contains a splicing donor, a branch and a splicing acceptor site.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising an alphavirus 5’UTR promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising an alphavirus 5’UTR promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said alphavirus 5’UTR comprises conserved sequence elements selected from CSE 1, CSE 2, CSE 3 and/or CSE 4.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising an alphavirus 5’UTR promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said alphavirus 5’UTR comprises the nucleic acid sequence of SEQ ID No.24.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising an alphavirus 5’UTR promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said alphavirus 5’UTR consists of the nucleic acid sequence of SEQ ID No.24.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising an alphavirus 5’UTR promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said alphavirus 5’UTR comprises an nucleic acid sequence which is at least 80 percent, at least 90 percent, at least 95 percent, at least 97 percent, at least 98 percent or at least 99 percent identical to SEQ ID Nos.24.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said alphavirus 5’UTR comprises the nucleic acid sequence of SEQ ID No.24,.
  • said promoter comprises the nucleic acid sequence of SEQ ID No.18, wherein said alphavirus replicase encoded by the first nucleic acid comprises SEQ ID Nos.2, 4, 6 and 8, wherein said subgenomic promoter comprises the nucleic acid sequence of SEQ ID No.13 or 14,and wherein said 3’UTR comprises the nucleic acid sequence of SEQ ID No.23.
  • the second nucleic acid sequence of the vector system of the present disclosure may further comprise a ribozyme or a pseudoknot where the presence of such sequences catalyzed a self-cleavage reaction that aims to remove the eukaryotic 3’UTR, beneficial for the nuclear export and RNA stability during transcription, but not for the Replicase activity.
  • said second nucleic acid sequence of the vector system of the present disclosure comprises a ribozyme.
  • said ribozyme comprises SEQ ID No.11.
  • said ribozyme consists of the nucleic acid sequence of SEQ ID No.11.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said second nucleic acid sequence of the vector system of the present disclosure comprises a ribozyme.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said second nucleic acid sequence of the vector system of the present disclosure comprises a ribozyme comprising the nucleic acid sequence of SEQ ID No.11.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said alphavirus 5’UTR comprises the nucleic acid sequence of SEQ ID No.24,.
  • said promoter comprises the nucleic acid sequence of SEQ ID No.18, wherein said alphavirus replicase encoded by the first nucleic acid comprises SEQ ID Nos.2, 4, 6 and 8, wherein said subgenomic promoter comprises the nucleic acid sequence of SEQ ID No.13 or 14, wherein said 3’UTR comprises the nucleic acid sequence of SEQ ID No.23, and wherein said second nucleic acid sequence of the vector system of the present disclosure comprises a ribozyme comprising the nucleic acid sequence of SEQ ID No.11.
  • said second nucleic acid sequence of the vector system of the present disclosure comprises a pseudoknot.
  • said pseudoknot comprises the nucleic acid sequence of SEQ ID No.12.
  • said pseudoknot consists of the nucleic acid sequence of SEQ ID No.12. Therefore, in certain embodiments the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said second nucleic acid sequence of the vector system of the present disclosure comprises a pseudoknot.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said second nucleic acid sequence of the vector system of the present disclosure comprises a pseudoknot comprising the nucleic acid sequence of SEQ ID No.12.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said alphavirus 5’UTR comprises the nucleic acid sequence of SEQ ID No.24, wherein said promoter comprises the nucleic acid sequence of SEQ ID No.18, wherein said alphavirus replicase encoded by the first nucleic acid comprises SEQ ID Nos.2, 4, 6 and 8, wherein said subgenomic promoter comprises the nucleic acid sequence of SEQ ID No.13 or 14, wherein said 3’UTR comprises the nucleic acid sequence of SEQ ID No.23, and wherein said second nucleic acid sequence of the vector system of the present disclosure comprises a pseudoknot comprising the nucleic acid sequence of SEQ ID No.12.
  • the second nucleic acid sequence of the vector system of the present disclosure may further comprise one or more polyadenylation sequences.
  • said polyadenylation sequence is selected from an SV40 polyadenylation sequence, a bGH polyadeylation sequence and a hGH polyadeylation sequence.
  • said polyadenylation comprises the nucleic acid sequence of SEQ ID No. 26 or 27.
  • said polyadenylation comprises the nucleic acid sequence of SEQ ID No. 26.
  • said polyadenylation comprises the nucleic acid sequence of SEQ ID No. 27.
  • said polyadenylation consists of the nucleic acid sequence of SEQ ID No. 26.
  • said polyadenylation consists of the nucleic acid sequence of SEQ ID No.27. Therefore, in certain embodiments the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said second nucleic acid sequence further comprise one or more polyadenylation sequences.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said second nucleic acid sequence further comprise one or more polyadenylation sequences comprising the nucleic acid sequence of SEQ ID No.26.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said second nucleic acid sequence further comprise one or more polyadenylation sequences comprising the nucleic acid sequence of SEQ ID No.27.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said alphavirus 5’UTR comprises the nucleic acid sequence of SEQ ID No.24, wherein said promoter comprises the nucleic acid sequence of SEQ ID No.18, wherein said alphavirus replicase encoded by the first nucleic acid comprises SEQ ID Nos.2, 4, 6 and 8, wherein said subgenomic promoter comprises the nucleic acid sequence of SEQ ID No.13 or 14, wherein said 3’UTR comprises the nucleic acid sequence of SEQ ID No.23, wherein said second nucleic acid sequence comprises a pseudoknot comprising the nucleic acid sequence of SEQ ID No.12, and wherein said second nucleic acid sequence comprises
  • the present disclosure relates to any one of aforementioned vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said first nucleic acid sequence and said second nucleic acid sequence are on the same nucleic acid molecule.
  • the present disclosure relates to any one of aforementioned vector system comprising a) a first nucleic acid sequence comprising a promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR, wherein said first nucleic acid sequence and said second nucleic acid sequence are on different nucleic acid molecule.
  • the second nucleic acid molecule may comprise a 5’UTR, in addition to the 5’UTR which is located on the first nucleic acid molecule.
  • the present disclosure relates to vector system comprising a) a first nucleic acid sequence comprising a 5’UTR promoter, and an open reading frame encoding alphavirus replicase, and b) a second nucleic acid sequence comprising a 5’UTR, one or more subgenomic promoters, one or more open reading frames encoding a gene of interest and a 3’ UTR.
  • the present disclosure relates to a host cell comprising the vector system of the present invention.
  • said host cell is a mammalian cell.
  • said host cell is a human cell.
  • Table 1 Construct Gene architecture 1 CMV(nsp1-4)-SGP-lgk-nLuc 2 CMV(nsp1-4)-SGP(15)Kozak-lgk-nLuc 3 CMV(nsp1-4)-SGP(15)Kozak-lgk-nLuc-3’UTR 4 CMV-5’UTR-(nsp1-4)-SGP(15)Kozak-lgk-nLuc-3’UTR 5 CMV-(nsp1-4, GDD>GAA)-SGP-lgk-nLuc 6 CMV-(nsp1-4, GDD>GAA)-SGP-lgk-nLuc-3’UTR 7 miniCMV-(nsp1-4)-SGP-IgK-nLuc 8 miniCMV-(nsp1-4)-SGP(15)Kozak-IgK-nLuc-3’UTR 9 miniCMV-IgK-nLuc CMV: CMV promoter miniCMV: minimal version of the CMV promoter ns
  • Naked DNA can also be used if an electric field is applied, for example, using the Lonza 4D Nucleofector, following manufacture guidelines.
  • An Infinite 200 Pro (Tecan) plate reader with a wavelength of 460nm was used to quantify the Luciferase expression.
  • the experiments were carried out in a 96-well format (unless otherwise stated in the figure legend), and the transfection was performed following the manufacturer's guidelines. Briefly, 24h post DNA delivery, the medium was replaced to eliminate the transfection complexes and 72 hours post DNA delivery, an aliquot of the culturing medium (e.a 10 ⁇ l) was taken out, mixed with the nano Luciferase substrate (Promega) and its activity was measured in an Infinite 100 Pro (Tecan).
  • the reporter gene was the fluorescent protein GFP
  • the cells were trypsinized, washed twice in PBS containing 1% FBS (Fetal Bovine Serum), resuspended in 100 ⁇ l of PBS + 1% FBS and analyzed by Fluorescent Activated Cell Sorting (FACS).
  • FACS Fluorescent Activated Cell Sorting
  • the GFP fluorescence was also measured in real-time assays using the Incucyte (Sartorius) device, taking measures every 6 hours.
  • Example 2 Comparative study for reporter gene expression from different versions of the esaRNA The esaRNA system described in the present disclosure was iteratively developed starting from known systems in the prior art.
  • Construct 1 was generated by introducing the DNA from the alphavirus VEE (Venezuelan Equine Encephalitis) under the control of the Pol II viral promoter isolated from the Cytomegalovirus (CMV).
  • CMV Cytomegalovirus
  • construct 2 the SGP from the VEE alphavirus was reduced from 30 to 15 bp, and the Kozak sequence was introduced to promote the efficient translation of the gene of interest.
  • the eukaryotic 3’UTR was introduced after the CSE5 sequence from the VEE alphavirus (construct 3) and in the last iteration (construct 4), a eukaryotic 5’UTR was introduced between the Pol II promoter and the nsp1 coding sequence.
  • the constructs’ functionality was tested in the Baby Hamster Kidney 21 (BHK-21) cell lines using the common transgene nano Luciferase as a reporter. Results are shown in Figure 2. As can be seen, esaRNAs expressed from constructs with eukaryotic 3’ and 5’ UTRs provide higher expression levels.
  • Example 3 The esaRNA can be launched from different Pol II promoters The system's versatility was demonstrated by launching the esaRNA system from a different Pol II promoter.
  • constructs 6 (esaRNA_miniCMV_dead), 9 (miniCMV_IgK-nLuc), 7 (esaRNA_miniCMV_IgK- nLuc 1st generation), and 8 (esaRNA_miniCMV_Igk-nLuc 2nd generation)) were generated by swapping the full- length Cytomegalovirus enhancer-promoter with its minimal version (miniCMV) and their functionality was assessed with the aforementioned Luciferase assay. Constructs were transfected into BHK-21 cells, and the expression of the reporter gene nLuc was measured 48h post DNA delivery. Results are shown in Figure 3.
  • esaRNA launched from a different Pol II promoter provides a more robust transgene expression than a canonical Pol II-based one.
  • Example 4 The esaRNA can be launched from Adenoviral particles In the esaRNA, the reporter gene IgK-nLuc was swapped for the Green Fluorescent Protein (GFP), and the construct was introduced into the backbone scaffold necessary for producing Helper-dependent or Gutless Adenovirus. Viral particles were produced, and the system's functionality was compared to control particles harbouring a similar expression cassette where the CMV promoter drew the expression of the reporter gene GFP. The expression of the reporter gene was measured every 6 hours in the Incucyte (Sartorius). Results are shown in Figure 4.
  • Example 5 The esaRNA system produces functional proteins starting from minuscule DNA quantities and sustains a more robust transgene expression over time.
  • the esaRNA system launched from plasmid DNA was tested using a minuscule quantity of DNA as starting material (1 ng).
  • the cells were cultured in DMEM (Gibco) supplemented with 10% final concentration of FBS and passaged once, following standard techniques. Results are shown in Figure 5.
  • esaRNA showed a significantly higher expression level demonstrating its ability to amplify the transgene expression once delivered into a target cell compared to a canonical Pol II-driven transgenic expression. Moreover, the transgene expression was proven to be more robust over time when launched from the esaRNA system in mitotically active and rapidly growing BHK-21 cells.
  • Example 6 The esaRNA functionality is not restricted by the cell type. The esaRNA harbouring the expression of the reporter gene GFP was tested for its functionality and effectiveness in several human-derived cell lines. The following cell lines were tested: the lung-derived human cancer cell line A549 (ATCC No. CCL-185), the breast-derived human cancer cell line MDA-MB231 (ATCC No.
  • Example 7 The esaRNA can be launched from two different nucleic acids In this experiment it was tested if the system is not only functional when launched from a single nucleic acid molecule (in cis), but also when it is divided onto two nucleic acids (in trans).
  • the first nucleic acid molecule contains the elements for the production of the Nsp1-4 complex, the second nucleic acid molecule the elements necessary for the replication (CSE1-4 and CSE5), the SGP promoter and the Gene Of Interest (GOI). Results are shown in Figure 7. As can be seen, the esaRNA system, also when used in trans, effectively produces the functional reporter gene.

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Abstract

La présente invention concerne un nouveau système d'expression fondé sur l'ADN, reposant sur un système d'auto-amplification qui utilise la réplicase d'alphavirus. Le système est largement applicable aux cellules de mammifères et peut être lancé à partir de quantités minuscules d'ADN. Il peut être utilisé pour l'expression ciblée et régulable de gènes et de protéines d'intérêt dans des cellules de mammifères pour la prévention ou le traitement de maladies et de troubles.
PCT/EP2023/087081 2022-12-22 2023-12-20 Système d'auto-amplification eucaryote Ceased WO2024133550A1 (fr)

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WO2008119827A1 (fr) 2007-04-02 2008-10-09 Fit Biotech Oy Produits de synthèse transréplicases
EP3433368A1 (fr) 2016-03-21 2019-01-30 BioNTech RNA Pharmaceuticals GmbH Arn à réplication trans
WO2022165789A1 (fr) 2021-02-03 2022-08-11 郑州大学 Construction d'arn de réplicon cis pour exprimer efficacement une protéine cible

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WO2005026316A2 (fr) * 2003-09-15 2005-03-24 Bioption Ab Vaccins contre les arbovirus
WO2008119827A1 (fr) 2007-04-02 2008-10-09 Fit Biotech Oy Produits de synthèse transréplicases
EP3433368A1 (fr) 2016-03-21 2019-01-30 BioNTech RNA Pharmaceuticals GmbH Arn à réplication trans
WO2022165789A1 (fr) 2021-02-03 2022-08-11 郑州大学 Construction d'arn de réplicon cis pour exprimer efficacement une protéine cible

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