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WO2024126795A1 - Cellules déficientes en apobec pour la production de vecteurs adénoviraux - Google Patents

Cellules déficientes en apobec pour la production de vecteurs adénoviraux Download PDF

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Publication number
WO2024126795A1
WO2024126795A1 PCT/EP2023/086066 EP2023086066W WO2024126795A1 WO 2024126795 A1 WO2024126795 A1 WO 2024126795A1 EP 2023086066 W EP2023086066 W EP 2023086066W WO 2024126795 A1 WO2024126795 A1 WO 2024126795A1
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cells
proteins
protein
adenoviral vector
population
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WO2024126795A8 (fr
Inventor
Nicolas Gillet
Noémie LEJEUNE
Zoé BLOCKX
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Facultes Universitaires Notre Dame de la Paix
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Facultes Universitaires Notre Dame de la Paix
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N2710/10351—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N2710/10351—Methods of production or purification of viral material
    • C12N2710/10352—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)

Definitions

  • the present invention refers to vector production. Particularly, the present invention refers to cells with abolished or reduced expression of at least one protein from the APOBEC family of proteins for vector production.
  • Adenoviral vectors are particularly useful tools for transgene expression, as they allow efficient nucleic acid delivery into cells. Moreover, unlike lentiviral vectors, they generally do not involve the insertion of the transgene into the genome of the host cell, thereby preventing unwanted mutations.
  • Adenoviral vectors are commonly used in therapeutic applications, such as gene therapy, cancer therapy, and vaccination. They can be used for delivering a foreign antigen (vaccination), an antitumor protein or a protein defective in a genetic disease (gene therapy); some replication-competent adenoviral- vectors can also be used for their oncolytic properties in cancer therapy. These vectors offer many advantages, including their flexibility and adaptability. For example, AstraZeneca’s vaccine against SARS- CoV-2 (Vaxzevria) is adenoviral vector-based.
  • Cell lines such as, e.g., HEK293 cells or PER.C6 cells, are cultured in vitro, and transfected, transformed, or infected for expressing these vectors.
  • all cells possess innate intracellular antiviral defense mechanisms, which interfere with the production of the viral vector, and result in decreased yields of production.
  • some of these antiviral defenses can result in unwanted mutations in the nucleic acid molecule(s) carried by the vector.
  • the present invention provides a solution to this problem by identifying apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC), a conserved family of cytidine deaminases, as an actor of innate antiviral defenses.
  • APOBEC catalytic polypeptide
  • the present invention provides means for optimizing the production of adenoviral vector in cells by reducing the expression of APOBEC.
  • This invention thus relates to a cell or a population of cells, wherein said cell(s) have abolished or reduced expression of at least one protein from the apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC) family of proteins.
  • APOBEC catalytic polypeptide
  • the expression of said at least one protein or said at least two proteins from the APOBEC family of proteins is abolished (knock-out, KO).
  • the at least one protein or the at least two proteins from the APOBEC family of proteins is selected from the group comprising or consisting of APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H and APOBEC4.
  • the at least one protein from the APOBEC family of proteins is APOBEC3B.
  • the cells are mammalian cells, preferably human cells.
  • the cells are from a cell line selected from the group comprising or consisting of HEK293, PER.C6, HER, HeLa, HEL299, LP-293, 911, AE1- 2A, 293CRE4, E2T, C7, 293N3S, HBEC3-KT, A549, COS, CHO, VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0, and SP2/0-Agl4.
  • the cells express an adenovirus attachment receptor, preferably the adenovirus attachment receptor is selected from the group comprising or consisting of the coxsackievirus and adenovirus receptor (CAR), CD46, CD80, CD86, sialic acid and integrins.
  • CAR coxsackievirus and adenovirus receptor
  • the present invention further relates to the use of the cells or population of cells according to the invention, for producing an adenoviral vector.
  • the adenoviral vector further comprises at least one transgene of interest.
  • the present invention further relates to a method of producing an adenoviral vector, comprising the steps of: a. culturing the cells or the population of cells according to the invention; b. transfecting the cells with at least one vector encoding the adenoviral vector, or fragments thereof; c. incubating the transfected cells for a duration from 1 day to one week; d. obtaining the adenoviral vector from the culture supernatant and/or from the cells; and e. optionally, purifying the adenoviral vector.
  • the KO is an activable KO, i.e., it can be differentially activated upon administration of a drug.
  • shRNA targeting the inRNA of APOBEC3B include, but are not limited to SEQ ID NO: 47
  • CRISPR-mediated interference is performed with a catalytically dead Cas9 (dCas9), i.e., a Cas9 devoid of nuclease activity or nickase activity.
  • dCas9 catalytically dead Cas9
  • protein disruption means by which either the protein itself is altered, degraded, sequestered or the like, or the activity of the protein is inhibited.
  • the cell of the present invention overexpresses a nonfunctional form of the at least one, at least two or at least three proteins of the APOBEC family.
  • the non-functional mutant may thus act as a dominant negative and effectively decreases the activity of the at least one, at least two or at least three protein(s) from the APOBEC family.
  • the cells are from a cell line selected from the group comprising or consisting of HEK293, HBEC3-KT, and A549.
  • the culture medium may further include inorganic salts (in particular salts containing Na, K, Mg, Ca, Cl, P and possibly Cu, Fe, Se and Zn), physiological buffers (e.g., HEPES, bicarbonate), nucleotides, nucleosides and/or nucleic acid bases, ribose, deoxyribose, amino acids, vitamins, antioxidants (e.g., glutathione) and sources of carbon (e.g., glucose, pyruvate, e.g., sodium pyruvate, acetate, e.g., sodium acetate), etc. It will also be apparent that many media are available as low-glucose formulations with or without sodium pyruvate.
  • physiological buffers e.g., HEPES, bicarbonate
  • nucleotides e.g., nucleosides and/or nucleic acid bases
  • ribose e.g., deoxyribose
  • amino acids
  • basal media are supplemented with one or more further components selected from the group comprising or consisting of transferrin, selenium salts, amino acids, sugar, and combinations thereof.
  • these components can be included in a salt solution such as, but not limited to, Hanks' Balanced Salt Solution (HBSS), Earle's Salt Solution.
  • HBSS Hanks' Balanced Salt Solution
  • Further antioxidant supplements may be added, e.g., [3- mercaptoethanol.
  • basal media already contain amino acids, some amino acids may be supplemented later, e.g., L-glutamine, which is known to be less stable when in solution.
  • the cells may be counted in order to facilitate subsequent plating of the cells at a desired density.
  • the adenoviral vector is obtained from the Ad serotype selected from the group comprising or consisting of BAd3, CAd2, ChAdl, ChAd2, ChAd3, ChAd5, ChAd6, ChAd7, ChAd25, ChAd68, OAd7, PAd3, PAd5, FAdl, Fad4, FAd8, FAd9 and FAdlO.
  • the adenoviral vector is obtained from the Ad serotype selected from the group comprising or consisting of ChAdl, ChAd2, ChAd3, ChAd5, ChAd6, ChAd7, ChAd25 and ChAd68.
  • the adenoviral vector is a helper-dependent adenoviral vector.
  • the at least one transgene of interest encodes at least one protein of interest.
  • the at least one nucleic acid of interest is a messenger RNA (mRNA) encoding the at least one protein of interest as described herein.
  • mRNA messenger RNA
  • the cells are transfected by the means of adenovirus infection.
  • the ratio of the number of adenoviruses to the number of cells is MOI 0.00001 to MOI 10 3 , preferably MOI 0.0001 to MOI 100, more preferably MOI 0.001 to MOI 10, even more preferably MOI 0.03 to MOI 3.
  • the MOI is about 0.03.
  • the MOI is about 0.3. In some embodiments, the MOI is about 3.
  • step a) comprises culturing exactly one cell type.
  • the adenoviral vectors are obtained by means according to the present invention, preferably the adenoviral vectors are obtained from the culture supernatant.
  • Adenovirus A12 (HAdV-12, ATCC VR-863) was purchased at the American Type Culture Collection.
  • HAdV-B3 and -C2 viral stocks were produced in A549 cells and HAdV-A12 viral stock in PKR-deficient A549 cells.
  • PKR-deficient A549 cells were cultured in OPTI-MEM®! Reduced-Serum Medium without phenol red (ThermoFisher Scientific). When 80% of the cells showed cytopathic effects, cells were scraped, collected with the culture medium and centrifuged at 3,500g for 15 minutes.
  • the supernatant was collected and set aside.
  • the cell pellet was resuspended in 2 mL of culture medium, submitted to 3 freeze/thaw cycles and centrifuged at 3,500g for 15 minutes.
  • the supernatant was collected, pooled with the previous supernatant and treated with the endonuclease Benzonase® (1 U/mL) (Sigma- Aldrich) at 37°C for 30 minutes to degrade the unpackaged nucleic acids.
  • Supernatant was then filtered on a 0.22 m Steriflip® Filters (Merck Millipore) to remove residual cell debris. Eluate was collected and further filtered through Amicon® Ultra- 15 Centrifugal Filter Unit 100 KDa (Merck Millipore). Virions were retained on the filter, resuspended in PBS and stored at -80°C. Titration of the viral stocks were done by fluorescent forming unit (FFU) assay and by qPCR quantification of genome copies.
  • the human epithelial bronchial cells (HBEC3-KT, thereafter named HBEC-WT for Wild Type) were transduced by lentiviral vectors in order to induce a constitutive expression of A3B.
  • Three different cell lines were established: the HBEC-A3B cells (expressing an enzymatically active A3B protein), the HBEC-A3B-DD cells (producing a Deaminase Dead A3B mutant) and the HBEC-GFP cells as control.
  • the levels of A3B mRNA in the HBEC-A3B and HBEC-A3B-DD cells are increased by more than 100 folds compared to WT (data not shown).
  • A3B-expressing cells The percentage of A12-infected cells in A3B-expressing cells is significantly lower compared to the percentage measured in the WT cells and that from 2 days post infection and up to 10 days (Figure 1A). On the contrary, the percentage of A12-infected cells in GFP or A3B-DD cells is not different to the percentage measured in the WT cells. Thus, it appears that A3B restricts the propagation of the A12 virus by a deaminasedependent mechanism.
  • A3B protein detection by immunoblotting [0271] One or two-days post infection, cells were collected and washed in PBS. Cells were resuspended in HED buffer (20mM HEPES pH 7.4, 5mM EDTA, ImM DTT, 10% glycerol) supplemented with cOmpleteTM Protease Inhibitor Cocktail (Roche). Cells were then submitted to one freeze/thaw cycle and sonicated 15 cycles of 30 sec ON / 30 sec OFF using Bioruptor Pico device (Diagenode) at 4°C. Cell lysates were spun down at 14,000 rpm for 15 minutes to remove cell debris. Proteins were quantified using PierceTM BCA Protein Assay Kit (ThermoFisher Scientific).
  • the A549 lung cancer cell line was used because it constitutively expresses A3B (Figure 2A).
  • the A549 cells were transduced with lentiviral vectors expressing shRNAs against A3B (shA3B) or scramble control (scramble).
  • Figure 2A demonstrates that A549-shA3B produces 26 times less mRNA that the WT or scramble control.
  • Figure 2B shows that the A549-shA3B barely express the A3B protein
  • Figure 2C shows that A549-shA3B does not display deaminase activity, contrary to the WT or scramble controls.
  • Virus preparation and infection procedure see example 1.
  • Figure 5A illustrates the L3-specific 3DPCR amplicons obtained during HAdV-A12 infection of WT, scramble, and shA3B cells.
  • Low denaturation temperature amplicons Figure 5A were observed in the 3DPCR reactions performed on the DNA extracted from WT and scramble cells, but not from shA3B cells, indicating that A3B is necessary for the generation of hypermutated viruses.
  • Figure 5B reports the 3DPCR results for the 3 viral genes (E1B, L3 and E4), the 3 viral strains (A12, B3 and C2) and the 3 cell lines (WT, scramble and shA3B).
  • the dotted lines arbitrarily separating mutated from unmutated amplicons were placed based on the results obtained on the HBEC-WT cells which do not constitutively express A3B.
  • Amplicons from WT and scramble cells generally display a lower denaturation temperature than the amplicons generated in A3B-knockdown cells.
  • Knock-down (KD) of A3B by A3B shRNA was performed on HEK293 cells, as described hereinabove. [0284] For A3B protein detection by immunoblotting and deamination assay: see
  • the HEK293 cells widely used as helper cells for the production of viral vectors, express an active APOBEC3B protein (Figure 6A).
  • the KD of A3B in HEK293 cells ( Figure 6A) effectively abolishes A3B deaminase activity ( Figure 6B) that is required for its effect on virus replication.

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Abstract

La présente invention concerne une population de cellules dont l'expression d'au moins une protéine de la famille des protéines APOBEC est abolie ou réduite, et leur utilisation pour produire des vecteurs adénoviraux.
PCT/EP2023/086066 2022-12-15 2023-12-15 Cellules déficientes en apobec pour la production de vecteurs adénoviraux Ceased WO2024126795A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118480512A (zh) * 2024-07-11 2024-08-13 广东省农业科学院动物卫生研究所 A3z2基因敲除的st细胞株及其构建方法和用途

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019081673A1 (fr) * 2017-10-25 2019-05-02 Nouscom Ag Lignée de cellules eucaryotes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019081673A1 (fr) * 2017-10-25 2019-05-02 Nouscom Ag Lignée de cellules eucaryotes

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
JIN ZHE ET AL: "The role of APOBEC3B in chondrosarcoma", ONCOLOGY REPORTS, vol. 32, no. 5, 22 August 2014 (2014-08-22), pages 1867 - 1872, XP093048841, ISSN: 1021-335X, DOI: 10.3892/or.2014.3437 *
LEJEUNE NOÉMIE ET AL: "Infection of Bronchial Epithelial Cells by the Human Adenoviruses A12, B3, and C2 Differently Regulates the Innate Antiviral Effector APOBEC3B", vol. 95, no. 13, 10 June 2021 (2021-06-10), US, XP093021378, ISSN: 0022-538X, Retrieved from the Internet <URL:https://journals.asm.org/doi/pdf/10.1128/JVI.02413-20> DOI: 10.1128/JVI.02413-20 *
LEJEUNE NOÉMIE ET AL: "The APOBEC3B cytidine deaminase is an adenovirus restriction factor", PLOS PATHOGENS, vol. 19, no. 2, 6 February 2023 (2023-02-06), pages e1011156, XP093048847, DOI: 10.1371/journal.ppat.1011156 *
MACIEJOWSKI JOHN ET AL: "APOBEC3-dependent kataegis and TREX1-driven chromothripsis during telomere crisis", NATURE GENETICS, NATURE PUBLISHING GROUP US, NEW YORK, vol. 52, no. 9, 27 July 2020 (2020-07-27), pages 884 - 890, XP037235613, ISSN: 1061-4036, [retrieved on 20200727], DOI: 10.1038/S41588-020-0667-5 *
POULAIN FLORIAN ET AL: "Footprint of the host restriction factors APOBEC3 on the genome of human viruses", PLOS PATHOGENS, vol. 16, no. 8, 14 August 2020 (2020-08-14), pages e1008718, XP093021336, DOI: 10.1371/journal.ppat.1008718 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118480512A (zh) * 2024-07-11 2024-08-13 广东省农业科学院动物卫生研究所 A3z2基因敲除的st细胞株及其构建方法和用途

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