WO2025003977A2 - Procédé de transcription in vitro - Google Patents

Procédé de transcription in vitro Download PDF

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Publication number
WO2025003977A2
WO2025003977A2 PCT/IB2024/056309 IB2024056309W WO2025003977A2 WO 2025003977 A2 WO2025003977 A2 WO 2025003977A2 IB 2024056309 W IB2024056309 W IB 2024056309W WO 2025003977 A2 WO2025003977 A2 WO 2025003977A2
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rna molecule
subject
virus
rna
disorder
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WO2025003977A3 (fr
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Cheng Chang
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Seqirus Inc
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Seqirus Inc
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Priority to EP24831207.6A priority Critical patent/EP4735007A2/fr
Priority to CN202480043745.8A priority patent/CN121568700A/zh
Priority to AU2024309482A priority patent/AU2024309482A1/en
Publication of WO2025003977A2 publication Critical patent/WO2025003977A2/fr
Publication of WO2025003977A3 publication Critical patent/WO2025003977A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination

Definitions

  • the present disclosure relates generally to methods of producing RNA from a DNA template. More particularly, the method of the present disclosure comprises an in vitro transcription method for producing RNA from close-ended DNA (ceDNA) templates.
  • ceDNA close-ended DNA
  • Plasmid DNA is commonly used in IVT methods to produce an RNA of interest.
  • conventional bacterial fermentation techniques used to produce plasmid DNA are slow, expensive, limited by a lack of manufacturing capacity and may produce poor DNA quality owing to unstable or difficult sequences.
  • the production of template DNA of sufficient quality and quantity imposes a bottleneck in the process of mRNA synthesis.
  • IVT In vitro transcription
  • plasmid DNA template In vitro transcription
  • plasmid DNA template Close-ended DNA (ceDNA) templates are generated through a cell-free and enzymatically-based process which avoids the selective pressure often associated with plasmid instability, while also delivering rapid and reliable GMP DNA.
  • IVT reactions also generally require less ceDNA material as compared to when using plasmid DNA templates.
  • ceDNA templates can further support the production of difficult sequences, such as those encompassing long polyA tails or those that comprise large (>20kb) genes of interest, thereby allowing greater flexibility in supporting a range of genetic medicines.
  • the inventors have surprisingly found that certain parameters, and in particular pH, can play an important role in the yield or efficiency of RNA production from IVT based on a close-ended DNA template.
  • the present disclosure provides a method of producing an RNA molecule by in vitro transcription, said method including the steps of:
  • the reaction mixture has a pH of about 6.5 to about 7.5.
  • the reaction mixture has a pH of about 7.0.
  • the close-ended DNA template is formulated in a solution having a pH of about 6 to about 8.
  • the solution has a pH of about 7.0.
  • the solution comprises a buffer.
  • the solution comprises a Tris buffer.
  • the solution comprises the Tris buffer at a concentration of about 8 mM to about 12 mM.
  • the solution comprises the Tris buffer at a concentration of about 10 mM.
  • the close-ended DNA template is or comprises doggybone DNA (dbDNA).
  • dbDNA doggybone DNA
  • the close-ended DNA template has been linearized.
  • the method further includes the earlier step of linearizing the close-ended DNA template.
  • the RNA molecule encodes an immunogenic protein.
  • the present disclosure provides an isolated RNA molecule produced by the method described herein.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated RNA molecule described herein and optionally a pharmaceutically acceptable carrier, diluent or excipient.
  • the isolated RNA molecule is contained in or otherwise associated with a lipid-based carrier.
  • the lipid-based carrier is or comprises a lipid nanoparticle.
  • the present disclosure provides an isolated RNA molecule or a pharmaceutical composition as described herein for use in: (a) eliciting an immune response; and/or (b) treating a disease, disorder or condition in a subject.
  • the present disclosure provides a method of eliciting an immune response in a subject, said method including of step of administering a therapeutically effective amount of the isolated RNA molecule or the pharmaceutical composition as described herein to the subject to thereby elicit the immune response.
  • the present disclosure provides a method of preventing and/or treating a disease, disorder or condition in a subject, said method including of step of administering a therapeutically effective amount of the isolated RNA molecule or the pharmaceutical composition as described herein to the subject to thereby prevent and/or treat the disease, disorder or condition.
  • the present disclosure provides for the use of the isolated RNA molecule or the pharmaceutical composition as described herein in the manufacture of a medicament for eliciting an immune response in a subject.
  • the present disclosure provides for the use of the isolated RNA molecule or the pharmaceutical composition as described herein in the manufacture of a medicament for preventing and/or treating a disease, disorder or condition in a subject.
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (z.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
  • the present disclosure provides a method of producing an RNA molecule by in vitro transcription, said method including the step of:
  • the present disclosure relates to an isolated RNA molecule produced by the aforementioned method.
  • RNA molecules are synthesized from a DNA template in a cell-free system (i.e., in vitro).
  • a DNA template and more particularly a linearized DNA template (e.g., a linearized plasmid DNA template, a linearized ceDNA template, a linearized dbDNA template), can be used as a template for the generation of RNA transcripts.
  • a DNA template for RNA in vitro transcription may be obtained by cloning of a nucleic acid molecule, in particular a cDNA molecule corresponding to the respective RNA to be transcribed in vitro, and introducing it into an appropriate vector for RNA in vitro transcription.
  • IVT In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530, 101-114; all of which are incorporated herein by reference).
  • IVT can be performed using a variety of commercially available kits including, but not limited to, RiboMax Large Scale RNA Production System (Promega), MegaScript Transcription kits (Life Technologies), mMESSAGE Transcription kits (Invitrogen), as well as with commercially available reagents including RNA polymerases (e.g., T7 RNA polymerase) and ribonucleotide triphosphates (rNTPs).
  • RNA polymerases e.g., T7 RNA polymerase
  • rNTPs ribonucleotide triphosphates
  • RNA is an abbreviation for ribonucleic acid.
  • These nucleic acid molecules usually comprise adenosine-monophosphate, uridine-monophosphate, guanosine-monophosphate and cytidine-monophosphate monomers or analogues or modified versions thereof, which are connected to each other along a so-called backbone.
  • the backbone is formed by phosphodiester bonds between the sugar (i.e., ribose) of a first monomer and a phosphate moiety of a second, adjacent monomer.
  • the specific order of the monomers i.e. the order of the bases linked to the sugar/phosphate-backbone, is called the RNA-sequence.
  • RNA may refer to a molecule or to a molecule species selected from the group consisting of long-chain RNA, coding RNA, non-coding RNA, single stranded RNA (ssRNA), double stranded RNA (dsRNA), linear RNA (linRNA), circular RNA (circRNA), messenger RNA (mRNA), RNA oligonucleotides, small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR/Cas9 guide RNAs, riboswitches, immuno stimulating RNA (isRNA), ribozymes, aptamers, ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA (vRNA), retroviral RNA or replicon RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), circular RNA (circRNA), and a Piwi-interacting RNA (p
  • the RNA molecule referred to herein is an mRNA molecule.
  • the RNA molecule is a non-replicating mRNA molecule or a self-amplifying mRNA molecule.
  • the RNA molecule is a non-replicating mRNA molecule.
  • the RNA molecule is a self-amplifying mRNA molecule.
  • double stranded DNA comprises two opposing strands in terms of the 5' to 3' direction of the two single strands present in the double strand, it is common to nevertheless refer to a 5' end and a 3' end of the double stranded DNA, namely if the DNA comprises a coding sequence element that introduces a direction of the transcription into the double stranded DNA (and accordingly also a direction of the translation).
  • DNA template refers to a polynucleotide template for an RNA polymerase.
  • a DNA template includes the sequence for a gene of interest operably linked to an RNA polymerase promoter sequence.
  • the DNA template is a double stranded DNA template.
  • a DNA template can be prepared for IVT from a number of sources with appropriate techniques which are well-known in the art (see, e.g., Linpinsel, J.L and Conn, G.L., General protocols for preparation of plasmid DNA template; and Bowman, J.C., Azizi, B., Lenz, T.K., Ray, P., and Williams, L.D. in RNA IVT and RNA purification by denaturing PAGE. In: Recombinant and in vitro RNA synthesis. Methods in Molecular Biology, v. 941 Conn G.L. (ed), New York, N.Y. Humana Press, 2012).
  • the DNA template comprises a double stranded DNA molecule.
  • Exemplary DNA templates may include plasmid DNA, a PCR product, ceDNA or the like. Referring to the present method, however, the DNA template is suitably a ceDNA template.
  • the DNA template typically comprises a suitable RNA polymerase promoter sequence, such as a T7, T3 or SP6 promoter, for IVT at or towards a 5’ end thereof, which is followed by the desired nucleotide sequence, such as a nucleotide sequence encoding a desired mRNA molecule to be prepared, and a 3 ’ terminator for IVT.
  • the desired nucleotide sequence generally includes, in a 5’ to 3’ direction, a 5’UTR, an open reading frame (ORF) encoding a protein of interest and a 3’ UTR.
  • the desired nucleotide sequence or a part thereof may be codon optimized.
  • the DNA template suitably is or comprises a close-ended or closed linear DNA template that encodes the RNA molecule of interest.
  • close-ended DNA e.g., a linear double stranded covalently closed DNA molecule
  • linear DNA molecule e.g., a double stranded linear DNA molecule
  • covalently closed linear DNA e.g., a linear double stranded covalently closed DNA molecule
  • hairpin loops suitably join the ends of complementary DNA strands.
  • the hairpin loops may themselves contain complementary sequences, particularly if the hairpin loops comprise part of a protelomerase target sequence. Because they have complementary internal sequences, such DNA molecules can assume a “dumbbell” shape. It is envisaged, however, that the ceDNA described herein may include additional secondary and tertiary nucleic acid structures, as are known in the art (e.g., one or more stem loop structures).
  • ceDNA may be generated from open or closed double stranded or single stranded DNA using conventional molecular biology techniques.
  • ceDNA can be generated by attaching hairpin DNA linkers to one or both ends of an open double- stranded or single stranded DNA molecule, such as under the action of a ligase (see, e.g., US6,451,563 for dsDNA and WO2019101596 for ssDNA).
  • ceDNA may be produced by the enzymatic activity of a recombinase or a protelomerase (see, e.g., W02010086626, W02012017210 and WO2016132129 for exemplary methods of producing ceDNA using a protelomerase).
  • the ceDNA template may comprise at least one processing enzyme target sequence, more particularly a recombinase target sequence or a protelomerase target sequence.
  • a protelomerase target sequence comprises a palindromic sequence (i.e., a double-stranded DNA sequence having two-fold rotational symmetry, also known as an inverted repeat).
  • Suitable protelomerase target sequences are known in the art and are discussed in EP2,391,731, which is incorporated herein by reference.
  • a suitable protelomerase enzyme for the production of ceDNA may be TelN from the Escherichia coli phage N 15.
  • the ceDNA template provided herein is suitably an enzymatically-amplified ceDNA template or vector.
  • the ceDNA template has suitably been produced by a cell-free process and more particularly a bacterial cell-free process.
  • the ceDNA template is suitably free from any bacterial propagation elements and antibiotic resistance elements or markers.
  • the ceDNA template provided herein is suitably not or does not comprise plasmid DNA.
  • the ceDNA template provided herein is a DoggyboneTM DNA (dbDNA) template.
  • dbDNA is a minimal, closed linear DNA vector developed by Touchlight Genetics Ltd.
  • dbDNA which can be rapidly produced, is plasmid- free and is synthesized through an enzymatic process using a DNA polymerase, Phi29, and the protelomerase enzyme, TelN.
  • DNA polymerase Phi29
  • TelN protelomerase enzyme
  • the present inventors have surprisingly found that utilising the ceDNA template when included and/or previously stored in a solution (e.g., a storage solution) which is at a relatively neutral pH (e.g., a pH of about 6.0 to about 8.0) prior to its use in an IVT reaction can increase the efficiency of said reaction and improve RNA production.
  • a solution e.g., a storage solution
  • a relatively neutral pH e.g., a pH of about 6.0 to about 8.0
  • the close-ended DNA template is formulated in a solution having a buffer.
  • buffer denotes a weak acid or base used to maintain acidity/alkalinity (pH) of a solution near a chosen value after the addition of another acid or base.
  • pH acidity/alkalinity
  • Exemplary buffers that may be used herein include trisaminomethane (Tris) buffers (e.g., Tris HC1), phosphate buffers (e.g., PBS), citrate buffers, glutamate buffers, 4-(2- hydroxyethyl)-l -piperazineethanesulfonic acid (HEPES) and the like.
  • Tris trisaminomethane
  • PBS phosphate buffers
  • citrate buffers e.g., glutamate buffers
  • glutamate buffers e.g., 4-(2- hydroxyethyl)-l -piperazineethanesulfonic acid (HEPES) and the like.
  • the solution comprises a Tris buffer, such as Tris HC1.
  • the Tris buffer may be included in the solution at a concentration of about 8 mM to about 12 mM (e.g., about 8.0, 8.25, 8.5, 8.75, 9.0, 9.25, 9.5, 9.75, 10.0, 10.25, 10.5, 10.75, 11.0, 11.25, 11.5, 11.75, 12.0 mM or any range therein), more particularly about 8.5 mM to about 11.5 mM, even more particularly about 9 mM to about 11 mM or yet even more particularly about 9.5 mM to about 10.5 mM,.
  • the solution containing the ceDNA template comprises a Tris buffer at a concentration of about 10 mM.
  • the solution containing the ceDNA template has a pH of about 7.0. In other examples, the solution containing the ceDNA template has a pH of about 6.9. For some examples, the solution containing the ceDNA template has a pH of about 7.1.
  • the present method comprises the step of adjusting the pH of the solution containing the ceDNA template to a pH of about 7.1.
  • pH adjustment may be achieved by the addition of one or more suitable buffers, such as those provided herein (e.g., Tris HC1).
  • suitable buffers such as those provided herein (e.g., Tris HC1).
  • adjustment of the pH of the solution containing the ceDNA template may be performed, for example, after production of the ceDNA and more particularly prior to linearization thereof if required.
  • a pH adjustment or buffering step may be performed after linearization of the ceDNA template so that the solution has a pH in the range provided herein.
  • the DNA template, and more particularly the ceDNA template may be linearized prior to use in the IVT reaction. Accordingly, the present method may include the earlier or initial step of linearizing the DNA template.
  • linearized refers to a DNA molecule that comprises at least one free end (e.g., a free 5’ end and/or a free 3’ end) and more particularly two free ends.
  • the linearized ceDNA template includes a free 5’ end and a free 3’ end, which are not linked to each other. Accordingly, the linearized ceDNA template may no longer include hairpin loops at a 5’ end and/or a 3’ end thereof.
  • the linearized ceDNA template may be considered or referred to as being “deended”.
  • a linearized ceDNA template in the context of the present disclosure may be obtained by a restriction digest of a ceDNA. It is envisaged that the respective free ends of the linearized ceDNA template may be sticky and/or blunt depending on the restriction enzyme utilised.
  • the ceDNA template (e.g., the dbDNA template) may be linearized with a suitable restriction enzyme and optionally isolated or purified before it is subjected to IVT.
  • a suitable restriction enzyme e.g., the dbDNA template
  • the terms “restriction endonuclease” and “restriction enzyme” refer to a class of enzymes that cleave phosphodiester bonds in both strands of a DNA molecule within specific base sequences. Restriction enzymes recognize specific binding sites, referred to as recognition sequences, on a double-stranded DNA molecule. The sites at which said phosphodiester bonds in the DNA are cleaved by said enzymes are referred to as cleavage sites.
  • Exemplary restriction enzymes include EcoRI, Xbal, PvuII, SapI, Ecil, Bpil, Aarl, Alol, Bael, BbvCI, Ppil, PsrI, BsrDI , BtsI, Earl, BmrI, Bsal, BsmBI, Faul, BbsI, BciVI, BfuAI, BspI, BseRI, Ecil, BtgZI, BpuEI, Bsgl, Mmel, CspCI, Bael, BsaMI, Mval269I, PctI, Bse3DI, BseMI, Bst6I, EamllO4I, Ksp632I, Bfil, Bso31I, BspTNI, BspQI, SapI, Eco31I, Esp3I, Bful, Acc36I, Aarl, Eco57I, Eco57MI, Gsul, Alol, Hin4I
  • the ceDNA template has been linearized with SapI, BspQI or an isoschizomer thereof.
  • the present method may include the earlier or initial step of linearizing the ceDNA template with SapI, BspQI or an isoschizomer thereof.
  • Reagents used in IVT typically comprise: the DNA template (e.g., a linearized ceDNA template) comprising a RNA polymerase promoter sequence; ribonucleotide triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil); optionally, a cap analogue (e.g., m 7 G(5')ppp(5')G(m 7 G) or other suitable cap analogues known in the art), a DNA- dependent RNA polymerase capable of binding to the promoter sequence within the DNA template; optionally, a ribonuclease (RNase) inhibitor to inactivate any potentially contaminating RNase; optionally, a pyrophosphatase to degrade pyrophosphate (an inhibitor of RNA synthesis); MgCh, which supplies Mg 2+ ions as a co-factor for the polymerase; optionally a reaction buffer, such as to maintain a suitable pH value, which
  • the reaction mixture includes a reaction buffer.
  • the reaction buffer is the same as the buffer included in the solution containing the ceDNA template. It is contemplated, however, that the reaction buffer and the buffer of the solution containing the ceDNA template may differ provided they impart suitable buffering to the reaction mixture.
  • the reaction buffer may be that as hereinbefore described for the solution containing the ceDNA template.
  • the reaction buffer comprises a Tris buffer (e.g., Tris HC1).
  • the Tris buffer may be included in the reaction mixture at a concentration of about 8 mM to about 12 mM (e.g., about 8.0, 8.25, 8.5, 8.75, 9.0, 9.25, 9.5, 9.75, 10.0, 10.25, 10.5, 10.75, 11.0, 11.25, 11.5, 11.75, 12.0 mM or any range therein), more particularly about 8.5 mM to about 11.5 mM, even more particularly about 9 mM to about 11 mM or yet even more particularly about 9.5 mM to about 10.5 mM.
  • the reaction buffer comprises a Tris buffer, such as Tris HC1, at a concentration of about 10 mM.
  • the reaction mixture suitably has a pH in the range of about 6.0 to about 8.0 (e.g., a pH of about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0 or any range therein), more particularly about 6.2 to about 7.8, even more particularly about 6.5 to about 7.5, yet even more particularly about 6.6 to about 7.4, still even more particularly about 6.7 to about 7.3, yet still even more particularly about 6.8 to about 7.2 or further more particularly about 6.9 to about 7.1.
  • the reaction mixture for IVT has a pH of about 7.0.
  • RNA molecules that are produced by IVT are typically capped in order for the mRNA to be translated.
  • mature mRNA molecules bear a “cap” structure at their 5 ’-termini, which plays an important role in translation and stability.
  • the 5’ cap plays a pivotal role in mRNA metabolism, and is required to varying degrees for processing and maturation of an RNA transcript in the nucleus, transport of mRNA from the nucleus to the cytoplasm, mRNA stability, and efficient translation of the mRNA to protein.
  • the 5’ cap comprises a 7-methyl guanosine (m 7 G) that is linked via a 5 ’-5 ’-triphosphate bridge to the 5'-end of the first transcribed nucleotide, resulting in a dinucleotide cap of m 7 GpppN, where N is any nucleoside (e.g. G, C, A or U) and is the first transcribed nucleotide. This is often referred to as capO.
  • capl m 7 GpppNmpN
  • cap2 m 7 GpppNmpNm
  • the in vitro transcribed mRNA may be further processed, for example by the addition of a poly(A) tail.
  • the poly(A) tail may be included in the ceDNA template, added via PCR, or added post-transcriptionally by enzymatic polyadenylation.
  • the ceDNA template may include a polyadenylation signal sequence.
  • the poly(A) tail is introduced by including a poly(dT) stretch at the end of the ceDNA template.
  • the poly(A) tail is added following IVT.
  • a 3’ poly(A) tail is added following IVT through the addition of ATP in conjunction with a poly(A) polymerase.
  • the DNA template may be removed using any technique known to the person skilled in the art.
  • the DNA template is removed by treatment with a DNase.
  • the DNase is DNAse I.
  • RNA transcripts may then be purified or isolated from the undesired components of the transcription or associated reactions.
  • Techniques for the isolation of RNA transcripts are well known in the art and include phenol/chloroform extraction or precipitation with either alcohol in the presence of monovalent cations or lithium chloride (LiCl).
  • the RNA molecule can be purified by LiCl precipitation, phenol: chloroform extraction followed by ethanol precipitation, precipitation with ether alcohol in the presence of monovalent cations or by using a spin column based method.
  • the RNA molecule is purified using tangential flow filtration (TFF), such as diafiltration.
  • the purification step comprises diafiltration into a suitable buffer.
  • the purification step comprises a spin column based method or column chromatography.
  • the RNA molecule can be resuspended in, for example, nuclease-free water or a suitable buffer.
  • isolated or “purified” is meant material, such as protein and nucleic acid molecules, that has been removed from its natural state or otherwise been subjected to human manipulation.
  • Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state.
  • Isolated material may be in native, chemical synthetic or recombinant form. A chemically synthesized nucleic acid or protein or one synthesized using IVT/translation is considered “isolated”.
  • the RNA product of the method of IVT provided herein is analysed to assess reaction yield and quality.
  • Various methods for analysing mRNA will be apparent to the skilled person. Such methods may include, but are not limited to, mass spectrometry, gel electrophoresis, liquid chromatography, spectrometry (e.g., Nanodrop) or a combination thereof.
  • the RNA molecule described herein is or comprises a non-replicating mRNA molecule or a self-amplifying mRNA (sa-mRNA) molecule.
  • the ceDNA template suitably includes a nucleotide sequence that encodes a nonreplicating mRNA molecule or a sa-mRNA molecule.
  • the RNA molecule referred to herein is a non-replicating mRNA molecule.
  • non-replicating or non-replicative mRNA molecules are non-self-replicating and typically comprise, in order from 5’ to 3’ : a 5 ’cap structure, a 5’- UTR, a nucleotide sequence encoding a protein of interest, a 3’-UTR and a tailing sequence e.g. a polyadenylation signal or poly-A tail).
  • the non-replicating mRNA may further comprise a translation internal ribosome entry site e.g. Kozak consensus sequence or IRES), a chain terminating nucleotide and/or a stem loop.
  • IRES Kozak consensus sequence
  • non- replicating mRNA is suitably unable to make additional copies of the mRNA encoding a protein of interest.
  • the RNA molecule provided herein is a sa-mRNA molecule.
  • Selfamplifying mRNA sa-mRNA; also referred to as self-replicating mRNA
  • sa-mRNA Selfamplifying mRNA
  • dsRNA double-stranded RNA
  • sa-mRNA vector systems may be particularly suited for vaccine development, as they provide high transient transgene expression and inherent adjuvant effects.
  • self-amplifying mRNA or “sa-mRNA” refers to a construct based on an RNA virus that has been engineered to allow expression of heterologous mRNA and proteins.
  • Self-amplifying mRNA can also be referred to as a replicon.
  • Selfamplifying mRNA can amplify in host cells leading to expression of the desired gene product in the host cell.
  • the sa-mRNA of the present disclosure suitably comprises one or more features of a mRNA (e.g., a nucleotide sequence encoding a protein of interest), but further comprises nucleotide sequences encoding non- structural proteins (NSPs), which enable the sa-mRNA to direct its self-amplification.
  • NSPs non- structural proteins
  • Non-structural proteins can include a viral replicase (or viral polymerase), a viral protease, a viral helicase and optionally other non-structural viral proteins.
  • self-amplifying mRNA can be based on the genomic RNA of an RNA virus.
  • the viral RNA is typically positive (+)- stranded so that it can be directly translated after delivery to a cell without the need for intervening replication steps (e.g., reverse transcription). Translation of the viral RNA results in the production of non-structural proteins (NSPs) which combine to form a replicase complex (i.e., an RNA-dependent RNA polymerase).
  • NSPs non-structural proteins
  • the replicase complex is the component of the sa-mRNA which amplifies the mRNA molecule of interest producing both antisense and sense transcripts, resulting in production of multiple daughter mRNA molecules, and subsequently the encoded protein of interest.
  • the sa-mRNA comprises a viral replicase.
  • the sa-mRNA comprises NSPs derived from (or based on) an alphavirus.
  • alphaviruses include, but are not limited to, Venezuelan equine encephalitis virus (VEEV; e.g., Trinidad donkey, TC83CR), Semliki Forest virus (SFV), Sindbis virus (SIN), Ross River virus, Western equine encephalitis virus, Eastern equine encephalitis virus, Chikungunya virus, S.A.
  • VEEV Venezuelan equine encephalitis virus
  • SFV Semliki Forest virus
  • SIN Sindbis virus
  • Ross River virus Western equine encephalitis virus
  • Western equine encephalitis virus e.g., Eastern equine encephalitis virus
  • Chikungunya virus S.A.
  • alphavirus may also include chimeric alphaviruses (e.g., as described by Perri et al., (2003) J. Virol. 77(19): 10394-403) that contain genome sequences from more than one alphavirus.
  • the self-replicating RNA is derived from or based on a virus other than an alphavirus, for example, a positive-stranded RNA virus.
  • a positive-stranded RNA virus for use in the present disclosure can include, for example, a picomavirus, a flavivirus, a rubivirus, a pestivirus, a hepacivirus, a calicivirus, or a coronavirus.
  • subgenomic promoter refers to sequences that constitute a functional element required for the production of subgenomic RNA species.
  • a subgenomic promoter is generally necessary to drive the expression of genes using RNA as the template nucleic acid.
  • the subgenomic promoter can be recognized by an RNA-dependent RNA polymerase, which may be a viral RNA replicase.
  • the promoter itself may be a composite of segments derived from more than one source, naturally occurring or synthetic.
  • RNA molecule inclusive of non-replicating mRNA and sa-mRNA, of the present disclosure typically comprises a nucleotide or nucleic acid sequence encoding a peptide, polypeptide or protein of interest.
  • protein is meant an amino acid polymer.
  • the amino acids may be natural or non-natural amino acids, D- or L- amino acids as are well understood in the art.
  • a “peptide” is generally considered a protein having no more than fifty (50) amino acids.
  • a “polypeptide” is generally considered a protein having more than fifty (50) amino acids.
  • the nucleotide sequence of the RNA molecule may encode any protein known to the person skilled in the art, including any naturally or non-naturally occurring or otherwise modified protein.
  • a protein encoded by the RNA molecule described herein may be of any size and may have any secondary structure or activity.
  • a protein encoded by the RNA molecule may have a therapeutic effect when expressed in a cell.
  • the nucleotide sequence of the RNA molecule encodes an immunogen or an antigen (e.g., a pathogenic antigen).
  • the antigen can induce or elicit an immune response in the subject.
  • the RNA molecule comprises a nucleotide sequence that encodes an antigen, such as those provided herein, expressed by, derived from or otherwise associated with a pathogen (e.g., a virus, a bacteria, a fungi, a protozoa, etc.) and more particularly an infectious pathogen, such as those described below.
  • a pathogen e.g., a virus, a bacteria, a fungi, a protozoa, etc.
  • infectious pathogen e.g., infectious pathogen, such as those described below.
  • the RNA molecule of the present disclosure comprises a nucleotide sequence that encodes an antigen from a virus.
  • pharmaceutically-acceptable carrier diluent or excipient
  • a solid or liquid filler diluent or encapsulating substance that may be safely used in systemic administration.
  • a variety of carriers well known in the art may be used.
  • These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, liposomes and other lipid-based carriers, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates and pyrogen-free water.
  • a useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington’s Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991), which is incorporated herein by reference.
  • any safe route of administration may be employed for providing a patient with the composition of the present disclosure.
  • oral, rectal, parenteral, sublingual, buccal, intravenous, intra- articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed.
  • Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.
  • compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is pharmaceutically-effective.
  • the dose administered to a patient should be sufficient to effect a beneficial response in a patient over an appropriate period of time.
  • the quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner.
  • the isolated RNA molecule of the pharmaceutical composition is contained in, encapsulated by or otherwise associated with (e.g., bound to, absorbed/adsorbed on) a lipid-based carrier, such as a cationic lipid, a lipid nanoparticle, a liposome, a cochleate, a virosome, an immune- stimulating complex, a microparticle, a microsphere, a nanosphere, a unilamellar vesicle, a multilamellar vesicle, an oil-in-water emulsion, a water-in-oil emulsion, an emulsome, and a polycationic peptide, a cationic nano-emulsion or combinations thereof.
  • a lipid-based carrier such as a cationic lipid, a lipid nanoparticle, a liposome, a cochleate, a virosome, an immune- stimulating complex, a microparticle, a microsphere,
  • the lipid-based carrier suitably comprises any lipid or mixture of lipids capable of forming a lipid bilayer structure.
  • lipids capable of forming a lipid bilayer structure.
  • phospholipids include phosphatidylcholine (PC) (lecithin), phosphatidic acid, phosphatidylethanolamine (PE) (cephalin), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI) and sphingomyelin (SM) or natural or synthetic derivatives thereof, as are known in the art.
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PG phosphatidylglycerol
  • PS phosphatidylserine
  • PI phosphatidylinositol
  • SM sphingomyelin
  • the pharmaceutical composition is formulated as a lipid nanoparticle (LNP) (i.e., the lipid-based carrier is or comprises a lipid nanoparticle).
  • LNP lipid nanoparticle
  • lipid nanoparticle refers to lipid-based particles having at least one dimension in the order of nanometers (e.g., 1-1,000 nm) which contain one or more lipids (e.g., neutral lipids, anionic lipids, cationic lipids, steroids, polymer-conjugated lipids).
  • lipid nanoparticles have a single phospholipid outer layer that encapsulates an inner space thereof, which may or may not be aqueous.
  • Formulation of LNPs to be administered may vary according to the route of administration and formulation (e.g., solution, emulsion, capsule) selected.
  • An appropriate pharmaceutical composition comprising an LNP to be administered can be prepared in a physiologically acceptable carrier.
  • suitable carriers include, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • aqueous carriers include water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution and glycine.
  • Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980).
  • the compositions can optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents and toxicity adjusting agents, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate.
  • the LNPs can be stored in the liquid stage or can be lyophilized for storage and reconstituted in a suitable carrier prior to use according to art-known lyophilization and reconstitution techniques.
  • the buffer suitable for injection purposes may contain salts selected from sodium chloride (NaCl), calcium chloride (CaCh) and optionally potassium chloride (KC1), wherein further anions may be present additional to the chlorides.
  • the salts in the injection buffer are present in a concentration of at least about 50 mM sodium chloride (NaCl), at least about 3mM potassium chloride (KCI) and at least about O.OlmM calcium chloride (CaCh).
  • the injection buffer may be hypertonic, isotonic or hypotonic with reference to the specific reference medium.
  • one or more compatible solid or liquid fillers or diluents or encapsulating compounds may be employed which are suitable for administration to a person.
  • Pharmaceutically acceptable carriers, fillers and diluents will have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to a subject.
  • Some examples of compounds which can be used as pharmaceutically acceptable carriers, fillers or constituents thereof are sugars, such as lactose, glucose, trehalose and sucrose; starches, such as corn starch or potato starch; dextrose; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants, such as stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as groundnut oil, cottonseed oil, sesame oil, olive oil, com oil and oil from theobroma; polyols, such as polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; and alginic acid.
  • sugars such as lactose, glucose, trehalose and sucrose
  • starches such as corn starch or potato starch
  • dextrose such as sodium carb
  • the pharmaceutical composition such as one formulated as an LNP
  • the pharmaceutical composition may further comprise one or more pharmaceutically acceptable adjuvants to enhance the immunostimulatory properties of the pharmaceutical composition.
  • the adjuvant may be any compound, which is suitable to support administration and delivery of the pharmaceutical composition and which may initiate or increase an immune response of the innate immune system, (i.e., a non-specific immune response).
  • an adjuvant may be selected from any adjuvant known to a skilled person and suitable for the particular nature of the vaccine or immunogenic composition (i.e., for the induction of a suitable immune response in a mammal).
  • the adjuvant may be selected from the group consisting of: MF59® (squalene-water emulsion), TDM, MDP, muramyl dipeptide, pluronics, alum solution, aluminium hydroxide, ADJUMERTM (polyphosphazene); aluminium phosphate gel; glucans from algae; algammulin; aluminium hydroxide gel (alum); highly protein-adsorbing aluminium hydroxide gel; low viscosity aluminium hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%), Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4); AVRIDINETM (propanediamine); BAY R1005TM
  • the concentration of the RNA molecule in the pharmaceutical composition can vary and will be selected based on fluid volumes, viscosities, body weight, type of mRNA molecule (non-replicating vs self-amplifying) and other considerations in accordance with the particular mode of administration.
  • the concentration of the RNA molecule in the pharmaceutical composition will suitably be effective for prevention or treatment of a disease, disorder or condition, either in a single dose or as part of a series of doses.
  • the amount may vary depending upon the health, physical condition, age and taxonomic group of the individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to react to the encoded antigenic protein or peptide, the condition to be treated and other relevant factors.
  • RNA molecules and pharmaceutical compositions disclosed herein may be for use in therapy, such as in methods of: eliciting an immune response; and/or treating a disease, disorder or condition in a subject.
  • the immune response described herein includes one or more elements of the immune system, such as T lymphocytes, B lymphocytes, antibodies, neutrophils, dendritic cells inclusive of plasmacytoid dendritic cells, cytokines and/or chemokines.
  • cytokines include pro-inflammatory cytokines such as TNF-a, IL-2, IL-6, IL-8, IL-17A and IL-1 (e.g., IL-ip).
  • a non-limiting example of a chemokine is the neutrophil chemo -attractant IL-8.
  • the immune response that is elicited by the vaccine compositions described herein is protective.
  • RNA molecules or pharmaceutical compositions of the present disclosure may include, but are not limited to, rare diseases, infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and reno-vascular diseases, and metabolic diseases.
  • the disease, disorder or condition is at least partly caused or mediated by a respiratory virus, such an influenza virus, a parainfluenza virus (e.g., parainfluenza virus type 3; PIV3), a rhinovirus, an avian influenza virus, a coronavirus (e.g., a SARS virus, such as SARS-CoV-1 and SARS-CoV-2), a me tapneumo virus (e.g., human metapneumovirus; hMPV) or a respiratory syncytial virus (RSV).
  • a respiratory virus such an influenza virus, a parainfluenza virus (e.g., parainfluenza virus type 3; PIV3), a rhinovirus, an avian influenza virus, a coronavirus (e.g., a SARS virus, such as SARS-CoV-1 and SARS-CoV-2), a me tapneumo virus (e.g., human metapneumovirus; hMPV) or a respiratory
  • the disease, disorder or condition can be at least partly caused or mediated by SARS-CoV-2, an influenza A virus, an influenza B virus, RSV, PIV3 or hMPV.
  • the disease, disorder or condition is at least partly caused or mediated by an influenza virus.
  • a “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement of a particular disease, disorder or condition.
  • a therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the RNA molecule of the present disclosure to elicit a desired response, such as an immune response, in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the RNA molecule are outweighed by the therapeutically beneficial effects thereof.
  • the therapeutically effective amount may vary according to the disease, disorder or condition to be treated or factor to be altered and also according to the weight, age, racial background, sex, health and/or physical condition and other factors relevant to the subject being treated.
  • the therapeutically effective amount will fall within a relatively broad range (e.g., a “dosage” range) that can be determined through routine trial and experimentation by a medical practitioner. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity, such as weight or number of RNA molecules.
  • the therapeutically effective amount can be administered in a single dose or in a dose repeated once or several times over a treatment period.
  • RNA molecule described herein can be delivered as naked RNA (e.g., merely as an aqueous solution of RNA).
  • the RNA molecule is administered in combination with a lipid-based carrier, such as a liposome or lipid nanoparticle, as described herein.
  • RNA molecule or pharmaceutical composition of the present disclosure may be administered to a subject in the form of one or more dosage units, where for example, a tablet or injectable liquid volume may be a single dosage unit.
  • a dosage unit where for example, a tablet or injectable liquid volume may be a single dosage unit.
  • the RNA molecule or pharmaceutical composition is administered within a pre-defined timespan. Such a timespan may be a week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks up until one year.
  • Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art (see, e.g., Remington: The Science and Practice of Pharmacy, 20th Edition, Philadelphia College of Pharmacy and Science, 2000).
  • RNA molecules described herein include a nucleotide sequence encoding an antigenic or immunogenic protein, or a fragment, variant or derivative thereof.
  • the antigenic protein may be a pathogen antigen, a tumour antigen, an allergenic antigen or an autoimmune self-antigen.
  • pathogen antigens may be those derived from pathogenic organisms, in particular bacterial, viral, fungal or protozoal pathogenic organisms, which evoke an immunological reaction in a mammalian subject, such as a human.
  • Pathogen antigens may be surface antigens, for example proteins or fragments thereof, located at the surface of, for example, the virus, bacteria, fungus or protozoa.
  • Pathogen antigens may include those derived from one or more of: Acinetobacter baumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae family, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocysts hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei, Calicivi
  • relevant antigens or immunogenic proteins may be derived from the pathogens selected from: Severe Acute Respiratory Syndrome (SARS), Severe Acute Respiratory Syndrome Coronavirus and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-1 and SARS-CoV-2), Influenza virus (inclusive of influenza A and B), respiratory syncytial virus (RSV), Herpes simplex virus (HSV), human Papilloma virus (HPV), Human immunodeficiency virus (HIV), Plasmodium, Staphylococcus aureus, Dengue virus, Chlamydia trachomatis, Cytomegalovirus (CMV), Hepatitis B virus (HBV), Mycobacterium tuberculosis, Rabies virus, Hendra virus, Parainfluenza virus (e.g., Parainfluenza virus type 3 or PIV3), human Metapneumo virus (hMPV) and Yellow Fever Virus.
  • SARS Severe Acute Respiratory
  • the antigen may be derived from SARS-CoV-2, an influenza A virus, an influenza B virus, RSV, PIV3, hMPV, Hendra virus or CMV.
  • the antigen or immunogenic protein is derived from SARS-CoV-2, an influenza A virus, an influenza B virus, RSV, PIV3 or hMPV.
  • the antigen or immunogenic protein is derived from an influenza virus.
  • the mRNA may have a nucleotide sequence encoding at least one antigenic protein derived from hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (Ml), matrix protein 2 (M2), non-structural protein 1 (NS1), non-structural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB 1, PB 1-F2, or polymerase basic protein 2 (PB2) of an influenza virus or a fragment, derivative or variant thereof.
  • HA hemagglutinin
  • NA nucleoprotein
  • NP nucleoprotein
  • Ml matrix protein 1
  • M2 matrix protein 2
  • NEP nuclear export protein
  • PA polymerase acidic protein
  • PB 1F2 polymerase basic protein 2
  • PB2 polymerase basic protein 2
  • the nucleotide sequence encodes at least one antigenic protein derived from hemagglutinin (HA) and/or neuraminidase (NA) of an influenza virus or a fragment, derivative or variant thereof.
  • HA and/or NA may, independently, be derived from an influenza A virus or an influenza B virus or a fragment, variant or derivative (e.g., chimeric versions) of either.
  • the antigen or immunogenic protein is from a coronavirus, such as a SARS-CoV-1 or SARS-CoV-2.
  • the mRNA may have a nucleotide sequence encoding at least one antigenic protein derived from the Spike (S) protein or a fragment, derivative or variant thereof.
  • a method of producing an RNA molecule by in vitro transcription including the steps of:
  • reaction mixture has a pH of about 6.5 to about 7.5.
  • RNA molecule is or comprises a non-replicating mRNA molecule or a self-amplifying mRNA molecule.
  • RNA molecule encodes an immunogenic protein
  • a pharmaceutical composition comprising the isolated RNA molecule of claim 16 and optionally a pharmaceutically acceptable carrier, diluent or excipient.
  • RNA molecule is contained in or otherwise associated with a lipid-based carrier.
  • RNA molecule of claim 16 or the pharmaceutical composition of any one of claims 17 to 19 for use in: (a) eliciting an immune response; and/or (b) treating a disease, disorder or condition in a subject.
  • a method of eliciting an immune response in a subject including of step of administering a therapeutically effective amount of the isolated RNA molecule of claim 16 or claim 20 or the pharmaceutical composition of any one of claims 17 to 20 to the subject to thereby elicit the immune response.
  • a method of preventing and/or treating a disease, disorder or condition in a subject including of step of administering a therapeutically effective amount of the isolated RNA molecule of claim 16 or claim 20 or the pharmaceutical composition of any one of claims 17 to 20 to the subject to thereby prevent and/or treat the disease, disorder or condition.
  • RNA molecule of claim 16 or claim 20 or the pharmaceutical composition of any one of claims 17 to 20 in the manufacture of a medicament for eliciting an immune response in a subject.
  • RNA molecule of claim 16 or claim 20 or the pharmaceutical composition of any one of claims 17 to 20 in the manufacture of a medicament for preventing and/or treating a disease, disorder or condition in a subject.

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Abstract

La présente divulgation concerne de manière générale des procédés de production d'ARN à partir d'une matrice d'ADN. Plus particulièrement, le procédé de la présente divulgation comprend un procédé de transcription in vitro pour produire de l'ARN à partir de matrices d'ADN à extrémité fermée (ADNce).
PCT/IB2024/056309 2023-06-29 2024-06-28 Procédé de transcription in vitro Ceased WO2025003977A2 (fr)

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