WO2023031428A1 - Utilization of micro-rna for downregulation of cytotoxic transgene expression by modified vaccinia virus ankara (mva) - Google Patents

Utilization of micro-rna for downregulation of cytotoxic transgene expression by modified vaccinia virus ankara (mva) Download PDF

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Publication number
WO2023031428A1
WO2023031428A1 PCT/EP2022/074510 EP2022074510W WO2023031428A1 WO 2023031428 A1 WO2023031428 A1 WO 2023031428A1 EP 2022074510 W EP2022074510 W EP 2022074510W WO 2023031428 A1 WO2023031428 A1 WO 2023031428A1
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mva
mirblock
seq
rsv
mirna target
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French (fr)
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Jürgen HAUSMANN
Markus Kalla
Marc Schweneker
Matthias Habjan
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Bavarian Nordic AS
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Bavarian Nordic AS
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Priority to JP2024513977A priority Critical patent/JP2024535145A/ja
Priority to CA3230406A priority patent/CA3230406A1/en
Priority to EP22772951.4A priority patent/EP4396333A1/en
Priority to AU2022338199A priority patent/AU2022338199A1/en
Priority to KR1020247010053A priority patent/KR20240051214A/ko
Priority to US18/688,756 priority patent/US20240392257A1/en
Priority to MX2024002641A priority patent/MX2024002641A/es
Priority to CN202280073591.8A priority patent/CN118234850A/zh
Priority to IL311078A priority patent/IL311078A/en
Publication of WO2023031428A1 publication Critical patent/WO2023031428A1/en
Anticipated expiration legal-status Critical
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    • 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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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/275—Poxviridae, e.g. avipoxvirus
    • A61K39/285—Vaccinia virus or variola virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
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    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
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    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24151—Methods of production or purification of viral material
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    • C12N2760/00011—Details
    • C12N2760/00022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the mature miRNAs are loaded into the so-called RISC multi-protein complex that mediates the miRNA effects. Binding of miRNAs to their cognate target sequences within the coding sequence or the 5' or the 3'-untranslated region (UTR) of cellular mRNAs leads to either a reduction in their translatability when the match is imperfect or even to mRNA degradation when the match is perfect. In the latter process, the miRNAs mediating mRNA degradation are not degraded themselves and are retrieved by the microRNA effector machinery. Thus, they can initiate a new cycle of mRNA degradation upon recognition of their target sequences. Of note, downmodulation of cellular protein levels by miRNAs is usually lower than two-fold (6, 7), but these effects can be enhanced e.g. by perfect target matching and by tandem arrangement of target sequences (8).
  • the invention provides a transcriptional unit, preferably suitable for use in a recombinant MVA, comprising a nucleotide sequence comprising a transgene operably linked to a poxvirus promoter, the nucleotide sequence further comprising a miRNA target sequence that is linked to the transgene, or a series of miRNA target sequences arranged in a miRblock that is linked to the transgene, wherein the miRNA target sequence or each miRNA target sequence in the miRblock corresponds to a miRNA in a eukaryotic MVA producer cell.
  • the invention provides of a series of miRNA target sequences arranged in a miRblock, preferably suitable for use in a recombinant MVA, wherein each miRNA target sequence corresponds to a miRNA in a eukaryotic MVA producer cell.
  • the invention provides a plasmid comprising a nucleotide sequence comprising a transgene operably linked to a promoter which is active in a eukaryotic producer cell, the nucleotide sequence further comprising a miRNA target sequence that is linked to the transgene, or a series of miRNA target sequences arranged in a miRblock that is linked to the transgene, wherein the miRNA target sequence or each miRNA target sequence in the miRblock corresponds to a miRNA in the eukaryotic producer cell.
  • the invention provides a process for producing a recombinant MVA according to the invention, comprising the steps of:
  • the invention provides a use of a miRNA target sequence according to the invention for downregulation of the expression of an MVA encoded transgene in a eukaryotic MVA producer cell, particularly for industrial-scale production of a vaccine.
  • Figure 4 illustrates the design of recombinant MVA inserts encoding EGFP and containing miRNA target sequences arranged in hetero-oligomeric miRblocks in the EGFP 3'-UTR.
  • Figure 6 shows an analysis of miRblock effects on EGFP expression after infection with recombinant MVA at different MOIs.
  • GMFI of EGFP (top) and BFP (bottom) of BFP- positive cells (GM with geoSD) determined by flow cytometry on day 1 after transfection are shown.
  • Figure 11 illustrates the design of recombinant MVA inserts expressing EGFP and containing selected miRblock-2 derived miRNA target sequences arranged in homo-oligomeric miRblocks.
  • Figure 20 shows an analysis of RSV-specific IgG antibody titers and RSV plaque reduction neutralization titers in blood serum from mice.
  • the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or”, a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.”
  • At least one miRNA target sequence in a miRblock is selected from the group consisting of nucleotide sequences as depicted in SEQ ID NO: 1 (corresponding to miR- 17-5p), SEQ ID NO: 2 (miR-20a-5p), SEQ ID NO: 3 (miR-21-5p), SEQ ID NO: 4 (miR-221 a- 3p), SEQ ID NO: 6 (miR-19a-3p), and SEQ ID NO: 7 (miR-199-3p).
  • the miRNA target sequences in a hetero-oligomeric miRblock are selected from the group consisting of nucleotide sequences as depicted in SEQ ID NO: 1 (corresponding to miR-17-5p), SEQ ID NO: 2 (miR-20a-5p), SEQ ID NO: 3 (miR-21 -5p), SEQ ID NO: 4 (miR-221 a-3p), SEQ ID NO: 5 (miR-18a-5p), SEQ ID NO: 6 (miR-19a-3p), SEQ ID NO: 7 (miR-199-3p), SEQ ID NO: 8 (miR-33-5p), SEQ ID NO: 9 (miR-218b-5p).
  • the miRblock comprises nucleotide sequences as depicted in SE NO: 1 (corresponding to miR-17-5p in miRblock-39), SEQ ID NO: 5 (miR-18a-5p - miRblock-39), SEQ ID and SEQ ID NO: 6 (miR-19a-3p - miRblock-39), and SEQ ID NO: 7 (miR-199-3p - miRblock-39).
  • the miRblock comprises nucleotide sequences as depicted in SEQ ID NO: 1 (corresponding to miR-17-5p in miRblock-41 ), and SEQ ID NO: 4 (miR-221 a-3p - miRblock-41 ), SEQ ID NO: 8 (miR-33-5p - miRblock-41 ), and SEQ ID NO: 9 (miR-218b-5p - miRblock-41 ).
  • the transgene encodes a tumor specific antigen (TSA) or a tumor associated antigen (TAA), or an antigenic part thereof.
  • TSA tumor specific antigen
  • TAA tumor associated antigen
  • the miRNA in a eukaryotic MVA producer cell is present or detectable or expressed in the eukaryotic MVA producer cell.
  • the miRNA is encoded by, preferably expressed by, a heterologous nucleotide sequence in a transgenic cell line.
  • the recombinant MVA comprises a first, second and third, or a first to fourth, or a first to fifth, or a first to sixth, or more transcriptional units.
  • not capable of reproductive replication in human cell lines in vitro as described above is, for example, described in WO 02/42480, which also teaches how to obtain MVA having the desired properties as mentioned above.
  • the term applies to a virus that has a virus amplification ratio in vitro at 4 days after infection of less than 1 using the assays described in WO 02/42480 or US 6,761 ,893.
  • Recombinant MVAs containing miRblock-17 and -18 (under the control of the PrS promoter) and the recombinants expressing EGFP-miRblock-2 and EGFP-scrbl2 under the control of the P r 13.5long immediate-early promoter were generated by standard homologous recombination in CEF cells using transfer plasmids with flanking homology regions targeting the transgenes into IGR MVA044L/MVA045L like in the BAC-derived recombinant MVAs described above. The resulting recombinants were purified by three rounds of plaque purification on CEF cells.
  • Plasmids were constructed with the objective to assess the potential of miRNA target sequences to mediate downregulation of transgene expression in chicken cells. Inserts of such plasmid constructs are illustrated in Fig. 1.
  • BFP blue fluorescent protein
  • miRblock-3 mediated a moderate EGFP downregulation
  • miRblock-5 to -10 showed no significant effect on EGFP expression (Fig. 2 top).
  • EGFP-miRb-1 EGFP-miRb-2
  • TTS poxviral early transcription termination signal
  • the RFP gene had not been modified by insertion of miRNA target sequences and should therefore not be regulated by the miRNA machinery of the infected cell. It was inserted to serve as a marker to monitor and compare infection levels produced by the different MVA constructs . Both EGFP and RFP were under control of the early/late PrS promoter in the recombinant MVA constructs (Fig. 4).
  • EGFP expression mediated by miRblock-1 and -2 was detectable at 6 hours p.i. at all MOIs tested.
  • EGFP expression is depicted as % of control (i.e., EGFP expression reference, “no miRb”) (Fig. 6 bottom) it becomes apparent that the downregulating effect of miRblock-1 and -2 is independent of the MOI used.
  • a higher EGFP expression level in an infected cell culture due to a higher MOI did not affect downregulation of EGFP expression.
  • vaccinia virus D8 protein used as an endogenous expression control was comparable in cells infected with MVA-BN-RSV-miRb1/2 or MVA-BN-RSV-miRb39/41 (Fig. 16).
  • the D8 signal was slightly stronger in lysates from cells infected with wildtype, i.e. non-recombinant MVA-BN (Fig. 16) as compared to the three recombinants.
  • This result might indicate that the cytotoxic effect of simultaneously expressed RSV derived transgenes without any miRNA-mediated downregulation affected the expression of autochthonous MVA genes such as D8, while on the other hand, miRNA-targeting of the RSV transgenes did not fully restore normal D8 expression.
  • RSV-specific neutralizing antibody titers were determined in sera of immunized mice at day 34 post prime (i.e., day 13 post boost) by a plaque reduction neutralization test (PRNT).
  • PRNT plaque reduction neutralization test

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PCT/EP2022/074510 2021-09-03 2022-09-02 Utilization of micro-rna for downregulation of cytotoxic transgene expression by modified vaccinia virus ankara (mva) Ceased WO2023031428A1 (en)

Priority Applications (9)

Application Number Priority Date Filing Date Title
JP2024513977A JP2024535145A (ja) 2021-09-03 2022-09-02 改変ワクシニアウイルスアンカラ（ｍｖａ）による細胞傷害性導入遺伝子発現を下方調節するためのマイクロｒｎａの利用
CA3230406A CA3230406A1 (en) 2021-09-03 2022-09-02 Utilization of micro-rna for downregulation of cytotoxic transgene expression by modified vaccinia virus ankara (mva)
EP22772951.4A EP4396333A1 (en) 2021-09-03 2022-09-02 Utilization of micro-rna for downregulation of cytotoxic transgene expression by modified vaccinia virus ankara (mva)
AU2022338199A AU2022338199A1 (en) 2021-09-03 2022-09-02 Utilization of micro-rna for downregulation of cytotoxic transgene expression by modified vaccinia virus ankara (mva)
KR1020247010053A KR20240051214A (ko) 2021-09-03 2022-09-02 변형 백시니아 바이러스 앙카라(mva)에 의한 세포독성 전이유전자 발현의 하향조절을 위한 마이크로rna의 활용
US18/688,756 US20240392257A1 (en) 2021-09-03 2022-09-02 Utilization of Micro-RNA for Downregulation of Cytotoxic Transgene Expression by Modified Vaccinia Virus Ankara (MVA)
MX2024002641A MX2024002641A (es) 2021-09-03 2022-09-02 Uso de micro ácido ribonucleico (arn) para la regulación por disminución de la expresión de transgenes citotóxicos mediante el virus vaccinia ankara modificado (mva).
CN202280073591.8A CN118234850A (zh) 2021-09-03 2022-09-02 利用微rna来下调经修饰的安卡拉痘苗病毒(mva)的细胞毒性转基因的表达
IL311078A IL311078A (en) 2021-09-03 2022-09-02 Use of microRNA to downregulate expression of cytotoxic transgenes by modified vicinia ankara virus (mva)

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EP21194940.9 2021-09-03

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Publication number Priority date Publication date Assignee Title
WO2024188801A1 (en) * 2023-03-10 2024-09-19 Bavarian Nordic A/S Use of quail cell lines for poxvirus production
WO2024245722A3 (en) * 2023-05-26 2025-03-13 Probiogen Ag Rapid selection system for the generation of recombinant enveloped viruses
WO2025120141A1 (en) * 2023-12-08 2025-06-12 Bavarian Nordic A/S Downregulation of yield-reducing transgenes expressed by poxvirus

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025153852A1 (en) * 2024-01-17 2025-07-24 1. Revvity Gene Delivery Gmbh Production of viral vectors

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WO2002042480A2 (en) 2000-11-23 2002-05-30 Bavarian Nordic A/S Modified vaccinia ankara virus variant
WO2003048184A2 (en) 2001-12-04 2003-06-12 Bavarian Nordic A/S Flavivirus ns1 subunit vaccine
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WO2010112847A2 (en) * 2009-03-30 2010-10-07 Lotus Cars Limited A reheated gas turbine system, in particular such a system having a fuel cell
US20130280215A1 (en) * 2011-10-18 2013-10-24 Geovax, Inc. Mva vectors expressing polypeptides and having high level production in certain cell lines
WO2014019718A1 (en) 2012-08-01 2014-02-06 Bavarian Nordic A/S Recombinant modified vaccinia virus ankara (mva) respiratory syncytial virus (rsv) vaccine
WO2014063832A1 (en) 2012-10-28 2014-05-01 Bavarian Nordig A/S Pr13.5 promoter for robust t-cell and antibody responses

Patent Citations (9)

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Publication number Priority date Publication date Assignee Title
WO2002042480A2 (en) 2000-11-23 2002-05-30 Bavarian Nordic A/S Modified vaccinia ankara virus variant
US6761893B2 (en) 2000-11-23 2004-07-13 Bavarian Nordic A/S Modified vaccinia ankara virus variant
WO2003048184A2 (en) 2001-12-04 2003-06-12 Bavarian Nordic A/S Flavivirus ns1 subunit vaccine
US20100029003A1 (en) * 2004-12-23 2010-02-04 Whitehead Institute For Biomedical Research System and methods for identifying miRNA targets and for altering miRNA and target expression
WO2010112847A2 (en) * 2009-03-30 2010-10-07 Lotus Cars Limited A reheated gas turbine system, in particular such a system having a fuel cell
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