Apobec-deficient cells for adenoviral vector production

Abstract

The present invention relates to a population of cells having abolished or reduced expression of at least one protein from the APOBEC family of proteins, and their use to produce adenoviral vectors.

Description

APOBEC-DEFICIENT CELLS FOR ADENOVIRAL VECTOR PRODUCTIONFIELD OF INVENTION

[0001] 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.BACKGROUND OF INVENTION

[0002] 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.

[0003] 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.

[0004] Therefore, the optimization of the manufacturing processes of adenoviral vectors is an important challenge, in order to maximize the yield of adenoviral vectors produced.

[0005] 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. However, all cells possess innate intracellular antiviral defense mechanisms, which interfere with the production of the viral vector, and result in decreased yields of production. In addition,some of these antiviral defenses can result in unwanted mutations in the nucleic acid molecule(s) carried by the vector.

[0006] It exists several different clones of HEK-293 cells that are deemed to be especially efficient for vector production (for example the 293F cells from Thermo Fisher Scientific, the AAV-293 Cells from Agilent or the AAVpro® 293T Cell Line from Takara). But none are specifically engineered to antagonize the intracellular antiviral response.

[0007] There is therefore an unmet need to provide a solution to circumvent the cell’s antiviral defenses for optimizing the production of adenoviral vectors.

[0008] 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. In particular, the present invention provides means for optimizing the production of adenoviral vector in cells by reducing the expression of APOBEC.SUMMARY

[0009] 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.

[0010] In some embodiments, the cells have abolished or reduced expression of at least two proteins from the APOBEC family of proteins.

[0011] In some embodiments, 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), reduced (knock-down, KD) or combinations thereof.

[0012] In some embodiments, 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).

[0013] In some embodiments, 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.

[0014] In some embodiments, the at least one protein from the APOBEC family of proteins is APOBEC3B.

[0015] In some embodiments, the cells are mammalian cells, preferably human cells.

[0016] In some embodiments, 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.

[0017] In some embodiments, the cells are HEK293 cells.

[0018] In some embodiments, 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.

[0019] The present invention further relates to the use of the cells or population of cells according to the invention, for producing an adenoviral vector.

[0020] In some embodiments, the adenoviral vector is a human adenoviral vector.

[0021] In some embodiments, the adenoviral vector is from a species selected from the group comprising or consisting of A, B, C, D, E, F and G.

[0022] In some embodiments, the adenoviral vector further comprises at least one transgene of interest.

[0023] 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.DEFINITIONS

[0024] In the present invention, the following terms have the following meanings:

[0025] The term “about”, when preceding a figure, means plus or less 10% of the value of said figure.

[0026] The term “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0027] The terms “comprising", "comprises" and "comprised of" are used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. These terms also encompass “consisting of’.

[0028] The expression “at least one” includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 250, 500, 750, 103,104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015or more.

[0029] The term “adenoviral vector” refers to a vector construct comprising nucleotide sequences derived from the genome of a virus of the Adenoviridae family and, optionally, one or more heterologous nucleic acid sequence of interest. Said heterologous nucleicacid sequence of interest, once within the host cell, encodes at least one protein or peptide of interest.

[0030] The term “in vitro method” refers to a method comprising steps performed in vitro (e.g., culture of cells or genome editing) or ex-vivo.

[0031] The terms “nucleic acid” or “polynucleotide” refer to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Nucleic acid” or “Polynucleotides” include, without limitation single-and doublestranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double- stranded RNA, and RNA that is a mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions. In addition, “Nucleic acid” or “polynucleotide” refers to triplestranded regions comprising RNA or DNA or both RNA and DNA. The term “nucleic acid” or “polynucleotide” also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications has been made to DNA and RNA; thus, “nucleic acid” or “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

[0032] The terms “protein”, “polypeptide” and “peptide” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified by, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, pegylation, or any other manipulation, such as conjugation with a labelling component. As used herein the term “amino acid” includes natural and / or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.

[0033] The term “vaccine” refers to any preparation comprising substance or group of substances meant to cause the immune system of a subject to respond to pathogens, such as bacteria or viruses, or to a tumor. Prophylactic vaccines are used to prevent a subject from ever having a particular disease or to only have a mild case of the disease. Such prophylactic vaccines usually comprise the pathogen responsible for the disease, either live and weakened or killed, or components thereof, purified or recombinant. Therapeutic vaccines are intended to treat specific diseases in a subject, in particular cancer. Such therapeutic anti-cancer vaccines comprise a tumor- ntigen or tumor-antigens, eliciting an immune response directed against the tumor cells.

[0034] The term “extract” or “lysate”, as used herein, means a lysed cell content. In one embodiment, such extract or lysate has not been further purified and thus contains the whole cell lysate content. In another embodiment, such extract refers to a protein extract, an RNA extract, a lipid, or a membrane vesicle. In practice, extracts include cellular and extracellular extracts. In one embodiment, extracts according to the present invention include metabolites from the cells. According to the present invention, lysate may include soluble fractions and / or insoluble fractions.

[0035] The terms “cell culture medium” or “culture medium” or “medium” refer to an aqueous liquid or gelatinous substance comprising nutrients which can be used for maintenance or growth of cells. Cell culture medium can contain serum or can be serum- free.DETAILED DESCRIPTION

[0036] The present invention relates to a cell or a population of cells, wherein the cells have abolished or reduced expression of at least one protein from the apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC) family of proteins. As used herein, the expression “at least one” means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0037] In one embodiment, the cells of the present invention are engineered cells, i.e., cells naturally expressing at least one APOBEC protein and engineered to abolish or1 reduce the expression of said protein, e.g., wherein the gene encoding said protein is KO or KD.

[0038] In some embodiments, said cells or populations of cells are adapted to the production of adenoviral vectors.

[0039] In some embodiments, the cells have abolished or reduced expression of at least two proteins from the APOB EC family of proteins. In some embodiments, the cells have abolished or reduced expression of at least three proteins from the APOB EC family of proteins. In some embodiments, the at least two or at least three proteins are distinct APOB EC protein family members.

[0040] In some embodiments, the cells have abolished or reduced expression of one, two or three proteins from the APOBEC family of proteins. In one embodiment, the cells have abolished or reduced expression of one protein from the APOBEC family of proteins. In another embodiment, the cells have abolished or reduced expression of two proteins from the APOBEC family of proteins. In yet another embodiment, the cells have abolished or reduced expression of three proteins from the APOBEC family of proteins. In some embodiments, the cells have abolished or reduced expression of all the proteins from the APOBEC family of proteins. In some embodiments, the two or three proteins are distinct APOBEC protein family members.

[0041] The apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC) is a family of cytidine deaminases, including APOBEC 1 to APOBEC4. The APOBEC family includes 11 members: APOBEC1, APOBEC2, APOBEC3A (A3A), APOBEC3B (A3B), APOBEC3C (A3C), APOBEC3D (A3D), APOBEC3F (A3F), APOBEC3G (A3G), APOBEC3H (A3H), APOBEC4 and Activation-induced cytidine deaminase (AID).

[0042] APOBEC3 (A3) enzymes act as antiviral effectors that catalyze the conversion of cytidine to uridine in the viral genome, rendering it nonfunctional. APOBEC3 (A3) enzymes are subdivided into APOBEC3A (A3A), APOBEC3B (A3B), APOBEC3C (A3C), APOBEC3D (A3D), APOBEC3F (A3F), APOBEC3G (A3G) and APOBEC3H (A3H).

[0043] In some embodiments, the at least one, at least two or at least three proteins from the APOB EC family of proteins are selected from the group comprising or consisting of APOBEC3B, APOBEC3A, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC1, APOBEC2 and APOBEC4.

[0044] In some embodiments, the at least one, at least two or at least three proteins from the APOB EC family of proteins are selected from the group compri sing or consisting of APOBEC3B, APOBEC3A, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC2, and APOBEC4. In some embodiments, the at least one, at least two or at least three proteins from the APOBEC family of proteins are selected from the group comprising or consisting of APOBEC3B, APOBEC3A, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOB EC 1, and APOBEC4. In some embodiments, the at least one, at least two or at least three proteins from the APOBEC family of proteins are selected from the group comprising or consisting of APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, and APOBEC3H, APOB EC 1, and APOBEC2.

[0045] In a preferred embodiment, the at least one, at least two or at least three proteins from the APOBEC family of proteins are selected from the group comprising or consisting of APOBEC3B, APOBEC3A, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G and APOBEC3H. In one embodiment, the at least one, at least two or at least three proteins from the APOBEC family of proteins are selected from the group comprising or consisting of APOBEC3B, APOBEC3A, APOBEC3C, APOBEC3D, APOBEC3F, and APOBEC3H. In another embodiment, the at least one, at least two or at least three proteins from the APOBEC family of proteins are selected from the group comprising or consisting of APOB EC 1, APOBEC2, and APOBEC4.

[0046] In one embodiment, the at least one protein from the APOBEC family of proteins is APOBEC3B. In one embodiment, the cells have abolished or reduced expression of at least APOBEC3B. In one embodiment, the cells have abolished or reduced expression of APOBEC3B and of at least another APOBEC family member.

[0047] In one embodiment, APOBEC3B is a protein having a sequence of SEQ ID NO: 46.

[0048] SEQ ID NO: 46MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTG VFRGQVYFKPQYHAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLA EFLSEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVTIMDYEEFAYCWE NFVYNEGQQFMPWYKFDENYAFLHRTLKEILRYLMDPDTFTFNFNNDPLVLRRRQTYLCYEVERLDNGTWVLMDQHMGFLCNEAKNLLCGFYGRHAELRFLDLVP SLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDP LYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQAL SGRLRAILQNQGN

[0049] In some embodiments, the at least one, at least two or at least three proteins from the APOBEC family of proteins are not selected from the group comprising or consisting of APOBEC3G, APOBEC 1, APOBEC2, and / or APOBEC4. In some embodiments, the at least one protein from the APOBEC family of protein is not APOBEC3G.

[0050] In some embodiments, the cells have abolished or reduced expression of APOBEC3B. In some embodiments, the cells have abolished or reduced expression of APOBEC3B and APOBEC3A.

[0051] In some embodiments, the expression of the at least one protein from the APOBEC family of proteins is abolished (knock-out, KO), reduced (knock-down, KD) or combinations thereof.

[0052] In a preferred embodiment, the expression of the at least one protein from the APOBEC family of proteins is abolished (knock-out, KO).

[0053] As used herein, the term “abolished”, or KO, means that the protein from the APOBEC family of proteins is either not produced by the cell, or a non-functional protein is produced, for example a truncated protein or a protein having a missing or altered functional domain.

[0054] Thus, in some embodiments, the enzymatic activity of the at least one protein from the APOBEC family of proteins is entirely suppressed. In other words, the enzymatic activity is 0% of that of a normal (wild-type) cell.

[0055] Gene KO is obtained by altering the nucleotide sequence of the gene, e.g., by introducing a mutation. In some embodiments, the gene(s) encoding the at least one protein from the APOBEC family of proteins comprise at least one mutation, wherein this mutation leads to the production of a non-functional protein.

[0056] In some embodiments, the mutation is selected from the group comprising or consisting of substitution, deletion, insertion, or inversion. In some embodiments, the mutation is a substitution. In some embodiments, the mutation is a deletion. In some embodiments, the mutation is an insertion. In some embodiments, the mutation is an inversion.

[0057] In some embodiments, the mutation introduces a premature stop codon in the sequence of the gene. In some embodiments, the mutation introduces a frameshift in the sequence reading frame.

[0058] In some embodiments, the mutation leads to the substitution of at least one amino acid in the amino acid sequence of the at least one protein from the APOBEC family of proteins, preferably the at least one substituted amino acid has different chemical properties from the original one (e.g., substitution of an acidic amino acid by a basic amino acid), even more preferably the substitution disrupts the tridimensional conformation of the amino acid.

[0059] In some embodiments, the mutation is not a silent mutation.

[0060] In some embodiments, the gene encoding the at least one protein from the APOBEC family of proteins is disrupted by the insertion of a nucleic acid sequence comprising more than one nucleotides (e.g., a cassette) in its sequence.

[0061] In some embodiments, more than one nucleotide of the sequence of the gene encoding the at least one protein from the APOBEC family of proteins is deleted, excised or removed.

[0062] Methods to knock-out a gene are well known in the art. Non-limitative examples include Cre-Lox recombinase, CRISPR-Cas (clustered regularly interspaced short palindromic repeats), TALEN (transcription-activator-like effector nuclease), ZFN (zinc finger nuclease), and integrative viral vector.

[0063] In some embodiments, the KO is a conditional KO, i.e., it is specific of one or more cell type or tissue.

[0064] In some embodiments, the KO is an activable KO, i.e., it can be differentially activated upon administration of a drug.

[0065] The KO of the at least one protein from the APOBEC family of proteins can be validated by any means known in the art. In some embodiments, the KO is validated at the enzymatic level, by testing the deaminase activity of the at least one protein from the APOBEC family of proteins. Illustratively a test of cytidine deaminase activity can be performed by incubating cells lysates with a fluorescent DNA molecule comprising a definite number of cytidine nucleotides, typically one cytidine, typically for 6 to 24 hours; upon cytidine deaminase activity, the cytidine nucleotide of the fluorescent DNA molecule is converted into uracil. Uracil DNA glycosylase is then added, which cuts the DNA molecule only if the deaminase activity was present, optionally upon heating conditions.

[0066] In some embodiments, the KO is validated at the protein level by, e.g., Western Blot, protein sequencing, liquid chromatography - mass spectrometry and the like. In some embodiments, the KO is validated at the RNA level by, e.g., RT-qPCR, RT-PCR, RNA sequencing, and the like. In some embodiments, the KO is validated at the DNA level by, e.g., qPCR, PCR, DNA sequencing, and the like.

[0067] In another embodiment, the expression of the at least one protein from the APOBEC family of proteins is knocked-down (KD), reduced, silenced or inhibited. As used herein, the terms knocked-down expression, reduced expression, silenced expression, and inhibited expression are used interchangeably. It is to be understood that, contrarily to KO, a KD does not entirely suppress the activity of the enzyme.

[0068] In one embodiment, the expression of the at least one protein from the APOB EC family of proteins is reduced in the cell at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, 10-fold or more, compared to a cell without KD. In another embodiment, the expression of the at least one protein from the APOB EC family of proteins is reduced in the cell at least 10-fold, 100-fold, 1,000-fold or more or more, compared to a cell without KD.

[0069] In one embodiment, the expression of the at least one protein from the APOB EC family of proteins is reduced so that the remaining enzymatic activity in the cell is at most 75%, 50%, 25%, 10% or less, compared to the normal enzymatic activity present in the cell. In another embodiment, the expression of the at least one protein from the APOB EC family of proteins is reduced so that the remaining enzymatic acti vity in the cell is at most 10%, 1%, 0.1%, 0.01% or less, compared to the normal enzymatic activity present in the cell. In some embodiments, the expression of the at least one protein from the APOB EC family of proteins is not reduced so that the remaining enzymatic activity in the cell is 0% of the normal enzymatic activity present in the cell. In some embodiments, the expression of the at least one protein from the APOB EC family of proteins is more than 0% of the normal enzymatic activity present in the cell. Methods for measuring the remaining enzymatic activity of a protein of the APOB EC family are well-known in the art and include, without limitation, the test of deaminase activity described herein.

[0070] Methods to knock-down, reduce or inhibit the expression of a gene are well known in the art and comprise, inter alia, RNA-based gene interference, DNA-based gene interference, CRISPR-mediated interference, and protein disruption.

[0071] In some embodiments, the KD is performed by the means of RNA-based gene interference (RNAi) or DNA-based gene interference. Typically, RNA-based and DNA- based gene interference techniques comprise the use of an interfering nucleic acid molecule. In some embodiments, the interfering nucleic acid molecule is a DNA molecule or a RNA molecule. In some embodiments, the interfering nucleic acid molecule is linear or circular. In some embodiments, the interfering nucleic acid molecule is delivered alone. In some embodiments, the interfering nucleic acid molecule is a naked molecule.

[0072] In some embodiments, the interfering nucleic acid molecule is comprised in a vector, e.g., a lentiviral vector or a plasmid. Typically, the interfering nucleic acid molecule is delivered to a cell, preferably the interfering nucleic acid molecule is delivered to the cytoplasm or to the nucleus of the cell.

[0073] In some embodiments, the interfering nucleic acid molecule is delivered on the form of a nucleic acid molecule encoding the interfering nucleic acid molecule.

[0074] In some embodiments, the interfering nucleic acid molecule is transfected in the cell by a technique selected from the group comprising or consisting of lipofection, electroporation, sonication, nanoparticles, microinjection, integrative viral vector (e.g., lentiviral vector) or non-integrative viral vector infection (i.e., transduction), preferably the interfering nucleic acid molecule is transfected by integrative viral vector (e.g., lentiviral vector) transduction, lipofection or electroporation, even more preferably the interfering nucleic acid molecule is transfected by integrative viral vector (e.g., lentiviral vector) transduction.

[0075] In one embodiment, the cells are stably transfected, typically by an integrative viral vector. In some embodiments, the cells stably express the interfering nucleic acid molecule, which in turn reduces the expression of its target as long as the interfering nucleic acid molecule is expressed. In another embodiment, the cells are transiently transfected, typically by a plasmid.

[0076] In some embodiments, the cells transfected with the interfering nucleic acid molecule are cultured in suitable conditions.

[0077] Methods to culture cells in vitro are well known in the art and are routine practice. Typically, cells are maintained in a controlled atmosphere (e.g., 37°C, 5 % CO2) and maintained in a suitable culture medium (e.g., Dulbecco’s Modified Eagle Medium, supplemented with 5% to 15% serum and 5 to 15 mM of a source of L-glutamine).

[0078] In some embodiments, the cells are cultured from 1 hour to 72 hours, from 2 hours to 72 hours, from 3 hours to 72 hours, from 4 hours to 72 hours, from 5 hours to 72 hours, from 6 hours to 72 hours, from 7 hours to 72 hours, from 8 hours to 72 hours, from9 hours to 72 hours, from 10 hours to 72 hours, from 11 hours to 72 hours, from 12 hours to 72 hours, from 24 hours to 72 hours, from 36 hours to 72 hours, from 48 hours to 72 hours. In some embodiments, the cells are cultured from 1 hour to 60 hours, from 1 hour to 48 hours, from 1 hour to 36 hours, from 1 hour to 24 hours, from 1 hour to 12 hours, from 1 hour to 11 hours, from 1 hour to 10 hours, from 1 hour to 9 hours, from 1 hour to 8 hours, from 1 hour to 7 hours, from 1 hour to 6 hours, from 1 hour to 5 hours, from 1 hour to 4 hours, from 1 hour to 3 hours, from 1 hour to 2 hours.

[0079] In one embodiment, the cells are transfected once with the interfering nucleic acid molecule. Typically, when cells stably express the interfering nucleic acid molecule, a second transfection is not required.

[0080] In another embodiment, the cells are transfected more than once, typically when cells transiently express the interfering nucleic acid molecule. It is to be understood that, for silencing proteins having a low turnover, several rounds of exposure to the interfering nucleic acid molecule may be necessary. In some embodiments, the cells are transfected again 12 hours, 24 hours, 36 hours, 48 hours or more after the previous transfection with the interfering nucleic acid molecule.

[0081] In some embodiments, the interfering nucleic acid molecule is complementary to a fragment of a nucleic acid sequence encoding a protein from the APOB EC family of proteins (e.g., a inRNA). As used herein, the term “complementary” means that the interfering nucleic acid molecule can hybridize with the nucleic acid molecule encoding a protein from the APOBEC family of proteins, or with a nucleic acid molecule comprising thereof. Typically, the interfering nucleic acid molecule may have 50%, 60%, 70%, 80%, 90%, 95% or 100% complementarity, preferably more than 80% complementarity .

[0082] In a preferred embodiment, the KD is performed by the means of RNA-based gene interference (RNAi). Typically, RNA-based gene interference techniques comprise the use of an interfering RNA molecule. Thus, in some embodiments, the interfering nucleic acid molecule is an interfering RNA molecule.

[0083] In some embodiments, the RNA-based gene interference molecule is selected from the group comprising or consisting of small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), double-stranded RNA and piwi-interacting RNA (piRNA), preferably the RNA-based gene interference molecule is selected from the group comprising or consisting of siRNA, shRNA and miRNA, even more preferably the RNA-based gene interference molecule is siRNA or shRNA.

[0084] In a preferred embodiment, the RNA-based gene interference molecule is shRNA. In another embodiment, the RNA-based gene interference molecule is siRNA.

[0085] Examples of shRNA targeting the inRNA of APOBEC3B include, but are not limited to SEQ ID NO: 47(TCACATGGGCTTTCTATGCATTCAAGAGATGCATAGAAAGCCCATGTGTTT TTTC), SEQ ID NO: 48(TTGACCTACGATGAGTTTGATTCAAGAGATCAAACTCATCGTAGGTCATTTT TTC) and SEQ ID NO: 49(TGTGATTAATTGGCTCCATATTCAAGAGATATGGAGCCAATTAATCACTTTT TTC).

[0086] In another embodiment, the KD is performed by the means of DNA-based gene interference. Typically, DNA-based gene interference is mediated by a DNA antisense oligonucleotide. Thus, in some embodiments, the interfering nucleic acid molecule is an interfering DNA molecule.

[0087] In yet another embodiment, the KD is performed by the means of CRISPR- mediated interference.

[0088] In some embodiments, CRISPR-mediated interference is performed with a catalytically dead Cas9 (dCas9), i.e., a Cas9 devoid of nuclease activity or nickase activity.

[0089] In some embodiments, cells are contacted with a dCas9 associated with a guide RNA (gRNA) targeting a gene encoding a protein from the APOB EC family of proteins, so that expression of the gene is reduced.

[0090] In some embodiments, the dCas9 is further associated with a transcriptional repressor. Methods for associating two proteins are known in the art and comprise, inter alia, protein fusion.

[0091] In some embodiments, the CRISPR-mediated interference is performed with a Casl3 associated with a gRNA targeting a transcript from a gene encoding a protein from the APOBEC family of proteins, so that the transcript (e.g., messenger RNA) is cut by Casl3.

[0092] In some embodiments, the CRISPR-mediated interference is performed with any other suitable Cas protein.

[0093] It is to be understood that the Cas proteins described herein (Cas9, Cas 13 or other) may be delivered to the cell in protein form or in the form of a nucleic acid encoding thereof. Similarly, the gRNA may be delivered directly or encoded by a nucleic acid molecule.

[0094] In yet another embodiment, the KD is performed by the means of protein disruption.

[0095] Within the scope of the present invention, it is meant by “protein disruption” means by which either the protein itself is altered, degraded, sequestered or the like, or the activity of the protein is inhibited.

[0096] In some embodiments, the protein from the APOBEC family of proteins (or the activity of said protein) is inhibited by a molecule selected from the group comprising or consisting of a small molecule, a drug, an antibody or fragment thereof, and a peptide.

[0097] In some embodiments, the molecule covalently binds the protein from the APOBEC family of proteins. In some embodiments, the molecule alters the tridimensional structure of the protein from the APOBEC family of proteins, preferably the protein loses its function. In some embodiments, the molecule inhibits the catalytic site of the protein from the APOBEC family of proteins. In some embodiments, the protein from the APOBEC family of proteins is degraded, e.g., in the proteasome or the lysosome.

[0098] In some embodiments, at least two proteins from the APOB EC family of proteins have abolished or reduced expression. In some embodiments, at least one protein from the APOB EC family of proteins is KD, and at least one protein from the APOB EC family of proteins is KO. In some embodiments, more than one protein from the APOBEC family of proteins is KD, and more than one protein from the APOBEC family of proteins is KO.

[0099] In some embodiments, 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.

[0100] In some embodiments, the cells are further KO and / or KD for at least one gene encoding at least one protein having antiviral properties.

[0101] In some embodiments, the at least one gene encoding at least one protein having antiviral properties is selected from the group comprising or consisting of PKR, OAS2, BST2, HBXIP, ZAP, IFITM1, IFITM2, IFITM3, TRIM5a, TRIM19, TRIM79a, TRIM56, TRIM21, ISG15, ISG20, DDX60, SLAMF1, Viperin, IRF1, or combination or variants thereof.

[0102] In one embodiment, the further KO and / or KD for at least one gene encoding at least one protein having antiviral properties has synergistic effects with the KO and / or KD of the at least one, at least two or at least three protein(s) from the APOBEC family of proteins.

[0103] In some embodiments, the cells are mammalian cells, preferably human cells.

[0104] In some embodiments, the cells are primate cells.

[0105] In one embodiment, the cells according to the invention are suitable for adenovirus production and express at least one adenovirus attachment receptor and / or comprise in their genome sequences or domains useful for virus replication and / or production.

[0106] In some embodiments, the cells express at least one adenovirus attachment receptor, preferably the at least one 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. Thus, in some embodiments, the cells can be infected by an adenovirus, preferably the cells can be infected by a human adenovirus (HAd). In some embodiments, the cells express one adenovirus attachment receptor as defined herein.

[0107] In some embodiments, the adenovirus attachment receptor is endogenously expressed by the cells, or the cells are transformed, transfected, or transduced in order to express transiently or stably the at least one adenovirus attachment receptor. In a preferred embodiment, the cells endogenously express the at least one adenovirus attachment receptor.

[0108] In some embodiments, the cells further comprise in their genome any sequence or domain useful for adenovirus replication and / or production. In some embodiments, the genome of the cells comprises adenoviral early region 1 (El) and / or adenoviral early region 2 (E2) and / or adenoviral early region 3 (E3), or fragments or combinations thereof.

[0109] In some embodiments, the cells do not express a gene coding for a tetracyclin repressor.

[0110] In some embodiments, the cells are immortalized cells, typically cells from a cell line, or primary cells.

[0111] In a preferred embodiment, the cells are from a cell line.

[0112] In some embodiments, 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.

[0113] In some embodiments, 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, and A549.

[0114] In some embodiments, 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, and 293N3S.

[0115] In some embodiments, the cells are from a cell line selected from the group comprising or consisting of HEK293, PER.C6, HER, HeLa, and HEL299.

[0116] In some embodiments, the cells are from a cell line selected from the group comprising or consisting of HEK293, HBEC3-KT, and A549.

[0117] In a preferred embodiment, the cells are HEK293 cells.

[0118] In some embodiments, the cells are HEK293 variant cells selected from the group comprising or consisting of HEK293F, HEK293FT, HEK293T, HEK293S, HEK293FTM, HEK293SG, HEK293SGGD, HEK293H, HEK293E, HEK EBNA1-6E, HEK293MSR, HEK293A, or combinations thereof. In some embodiments, the HEK293 cells are any variant selected from the group comprising or consisting of HEK293F, HEK293FT, HEK293T, or combinations thereof.

[0119] In another embodiment, the cells are PER.C6 cells. In another embodiment, the cells are HEL299 cells. In another embodiment, the cells are HER cells. In another embodiment, the cells are HeLa cells. In another embodiment, the cells are LP-293 cells. In another embodiment, the cells are 911 cells. In another embodiment, the cells are AE1- 2A cells. In another embodiment, the cells are 293CRE4 cells. In another embodiment, the cells are E2T cells. In another embodiment, the cells are C7 cells. In another embodiment, the cells are 293N3S cells.

[0120] In certain embodiments, the cells are not HBEC3-KT cells nor A549 cells. In certain embodiments, the cells are not HBEC3-KT cells. In certain embodiments, the cells are not A549 cells.

[0121] In some embodiments, the cells form a cell population characterized in that it is substantially pure.

[0122] In some embodiments, the substantially pure cell population comprises less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001% or less of other contaminating cell type(s).

[0123] In some embodiments, the substantially pure cell population comprises 0% of other contaminating cell type(s). In some embodiments, the substantially pure cell population comprises no more than one cell type.

[0124] In some embodiments, the cells are helper cells. In some embodiments, the cells are packaging cells.

[0125] In some embodiments, the cells may be plated onto a substrate which allows adherence of cells thereto. In some embodiments, the substrate is plastic. In some embodiments, the plastic substrate is a vessel or container suitable for cell culture, typically a plastic flask or plastic dish.

[0126] In certain embodiments, the plastic substrate is treated with at least one agent that promotes cell adherence, survival and / or proliferation; illustratively, in some embodiments, the plastic substrate is treated with poly-D-lysine, gelatin and / or collagen. In some embodiments, the agent forms a layer or a matrix at the surface of the plastic substrate.

[0127] In some embodiments, the cells may be cultured in a culture medium sustaining their further proliferation, generally a liquid culture medium, which may contain serum or may be serum-free. These media, that can be further supplemented with appropriate mixture of organic or inorganic compounds may, besides providing nutrients and / or growth promoters, also promote the growth / adherence or the elimination / detachment of specific cell types.

[0128] In some embodiments, the culture medium comprises a basal medium formulation as known in the art. Many basal media formulations can be used to culture the primary cells herein, including but not limited to Eagle's Minimum Essential Medium (MEM), OPTI-MEM, Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), Iscove'sModified Dulbecco's Medium (IMDM), BGJb medium, F-12 Nutrient Mixture (Ham), Liebovitz L-15, DMEM / F-12, Essential Modified Eagle's Medium (EMEM), RPMI- 1640, Medium 199, Waymouth's MB 752 / 1 or Williams Medium E, and modifications and / or combinations thereof. Compositions of the above basal media are generally known in the art and it is within the skill of one in the art to modify or modulate concentrations of media and / or media supplements as necessary for the cells cultured.

[0129] In a preferred embodiment, the basal medium formulation may be one of DMEM, MEM, or variants thereof. In a more preferred embodiment, the basal medium is DMEM.

[0130] By means of illustration and not limitation, 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.

[0131] In some embodiments, 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. Further antioxidant supplements may be added, e.g., [3- mercaptoethanol. While many 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.

[0132] In some embodiments, the culture medium is further supplemented with antibiotic and / or antimycotic compounds, such as, typically, mixtures of penicillin and streptomycin, and / or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, zeocin, and combinations thereof.

[0133] In some embodiments, the culture medium is further supplemented with hormones selected from the group comprising or consisting of D-aldosterone, diethylstilbestrol (DES), dexamethasone, estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin / human growth hormone (HGH), thyrotropin, thyroxine, L- thyronine, epithelial growth factor (EGF), human recombinant epidermal growth factor, and combinations thereof. The final concentration of hormones may range from 0.01 to 1000 ng / mL, preferably from 0.1 to 100 ng / mL, more preferably from 1 to 10 ng / mL.

[0134] In some embodiments, the culture medium is further supplemented with bovine pituitary extract. In some embodiments, the bovine pituitary extract is present at a final concentration of about 50 g / mL in the culture medium.

[0135] In some embodiments, the culture medium is further supplemented with lipids and lipid carriers selected from the group comprising or consisting of cyclodextrin, cholesterol, linoleic acid conjugated to albumin, linoleic acid and oleic acid conjugated to albumin, unconjugated linoleic acid, linoleic-oleic-arachidonic acid conjugated to albumin, oleic acid unconjugated and conjugated to albumin, and the like, and combinations thereof. Albumin can similarly be used in fatty-acid free formulations.

[0136] In some embodiments, the culture medium is further supplemented with mammalian plasma or serum. In some embodiments, the concentration of the plasma or serum in the culture medium is from 0.01% to 20%, preferably from 0.1% to 10%, more preferably from 1% to 5%. In some embodiments, the concentration of the plasma or serum in the culture medium is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.

[0137] The term "serum", as conventionally defined, is obtained from a sample of whole blood by first allowing clotting to take place in the sample and subsequently separating the so formed clot and cellular components of the blood sample from the liquid component (serum) by an appropriate technique, typically by centrifugation. An inert catalyst, e.g., glass beads or powder, can facilitate clotting. Advantageously, serum can be prepared using serum- separating vessels (SST), which contain the inert catalyst tomammals. Plasma or serum often contains cellular factors and components that are necessary for viability and expansion.

[0138] In some embodiments, the plasma or serum for use in the culture medium as described herein may include human plasma or serum; or plasma or serum derived from non-human animals, preferably non-human mammals, such as, e.g., non-human primates (e.g., lemurs, monkeys, apes), fetal or adult bovine, horse, porcine, lamb, goat, dog, rabbit, mouse or rat serum or plasma, etc. In a preferred embodiment, the serum is fetal bovine or calf serum. In another embodiment, the any combination of the above plasma and / or serum may be used in the cell medium.

[0139] In some embodiments, the culture medium is further supplemented with a serum replacement or analogue.

[0140] In some embodiments, when passaged, the cultured cells are detached and dissociated from the culture substrate and from each other. Detachment and dissociation of the cells can be carried out as generally known in the art, e.g., by enzymatic treatment with proteolytic enzymes (e.g., chosen from trypsin, collagenase, e.g., type I, II, III or IV, dispase, pronase, papain, etc.), treatment with bivalent ion chelators (e.g., EDTA or EGTA) or mechanical treatment (e.g., repeated pipetting through a small-bore pipette or pipette tip), or any combination of these treatments.

[0141] A suitable method of cell detachment and dispersion should ensure a desired degree of cell detachment and dispersion, while preserving a majority of cells in the culture.

[0142] In a preferred embodiment, the detachment and dissociation of the cultured cells yields a substantial proportion of cells as single, viable cells (e.g., at least 50 %, 70 %, 80 %, 90 % of the cells or more). The remaining cells may be present in cell clusters, each containing a relatively small number of cells (e.g., on average, between 1 and 100 cells).

[0143] In some embodiments, the detached and dissociated cells (typically as a cell suspension in an isotonic buffer or a medium) may be re-plated onto a substrate whichallows the adherence of cells thereto, and are subsequently cultured in a culture medium as described hereinabove.

[0144] As appreciated by those skilled in the art, the cells may be counted in order to facilitate subsequent plating of the cells at a desired density.

[0145] In some embodiments, the cells are cultured by re-plating them at a density of between 10 and 105cells / cm2, and at a splitting ratio between about 1 / 100 and Yi. As used herein, the splitting ratio denotes the fraction of the passaged cells that is seeded into an empty (typically a new) culture vessel of the same surface area as the vessel from which the cells were obtained.

[0146] The type of culture vessel, as well as of surface allowing cell adherence into the culture vessel and the cell culture media, can be the same as initially used and as described hereinabove, or may be different.

[0147] In some embodiments, the cells are devoid of contamination by bacteria, fungi, protozoa, archaea, or other microbial species.

[0148] In some embodiments, the cells are stored for future use.

[0149] In some embodiments, cells are cryopreserved for storage. In some embodiments, cells are stored at a temperature from -20°C to -200°C, preferably from -80°C to -200°C. In some embodiments, the cells are stored at about -196°C, typically cells are stored in liquid nitrogen. In some embodiments, cells are “flash-frozen” in liquid nitrogen prior to storage.

[0150] In some embodiments, the cells are contacted with at least one cryoprotective agent prior to cryopreservation. Cryoprotective agent are known in the art and include, e.g., glycerol, sucrose and the like.

[0151] In some embodiments, the cell population is producing viral vectors. In one embodiment, the cell population is producing adenoviral vectors.

[0152] The present invention further relates to the use of the cells according to the invention, for producing an adenoviral vector. In one embodiment, said use is an in vitro use. In one embodiment, said use is an ex vivo use.

[0153] Adenoviral vectors are useful in many applications, from genome editing in vitro to therapeutic applications such as vaccination, anti-cancer treatment and gene therapy. There is therefore a need to improve the production of these vectors and make it more efficient. The present invention provides a solution to this need.

[0154] In some embodiments, the adenoviral vector derives or is obtained from a mammalian adenovirus (Ad). In some embodiments, the adenoviral vector is a human adenoviral vector or a non-human adenoviral vector. In some embodiments, the adenoviral vector is a human adenoviral vector. In one embodiment, the adenoviral vector is obtained from any human Ad (HAd) serotype.

[0155] Human adenoviruses (HAd) comprise more than 60 identified serotypes, distributed among 7 species, namely HAd-A to HAd-G. In some embodiments, the adenoviral vector is from a species selected from the group comprising or consisting of A, B, C, D, E, F and G. In some embodiments, the adenoviral vector is from a species selected from the group comprising or consisting of A, B, D, F and G.

[0156] In some embodiments, the adenoviral vector is of any human adenovirus serotype selected from the group comprising or consisting of Ad serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 and 75.

[0157] In some embodiments, the adenoviral vector is of a serotype selected from the group comprising or consisting of Ad serotypes 3, 4, 5, 6, 11, 26, 28 and 35 e.g., HAd3, Had4, HAd 5, HAd 6, HAd 11, HAd 26, Had 28 and HAd 35). In some embodiments, the adenoviral vector is obtained from any one of human Ad serotypes. In some embodiments, the adenoviral vector is obtained from the Ad serotype selected from the group comprising or consisting of HAd3, HAd4, HAd 5, HAd 6, HAd 11, HAd 26, Had 28 and HAd 35. In some preferred embodiments, the adenoviral vector is obtained fromthe Ad serotype selected from the group comprising or consisting of HAd 5, HAd6 and HAd 26.

[0158] In another embodiment, the adenoviral vector is obtained from any non-human Ad serotype.

[0159] Non-human adenoviruses are also used for adenoviral vector design. Non limitative examples of non-human adenovirus serotypes used for vector design include bovine Ad serotype 3 (BAd3), canine Ad serotype 2 (CAd2), chimpanzee Ad serotypes 1, 2, 3, 5, 6, 7, 25 and 68 (ChAdl, ChAd2, ChAd3, ChAd5, ChAd6, ChAd7, ChAd25, ChAd68), ovine Ad serotype 7 (OAd7), porcine Ad serotype 3 and 5 (PAd3, PAd5) and fowl Ad serotypes 1, 4, 8, 9, and 10 (FAdl, Fad4, FAd8, FAd9, FAdlO). In some embodiments, 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. In some embodiments, 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.

[0160] In some embodiments, the adenoviral vector is a recombinant adenoviral vector. As used herein, “recombinant adenoviral vector” means that the adenoviral vector comprises at least one mutation compared to an unmodified adenoviral vector.

[0161] In one embodiment, the adenoviral vector is replication-defective. Such vectors are preferred for gene delivery and vaccination purposes.

[0162] In one embodiment, the adenoviral vector is replication competent. Such vectors are preferred for their oncolytic properties, in particular in cancer therapy.

[0163] In some embodiments, the adenoviral vector is a helper-dependent adenoviral vector.

[0164] In some embodiments, the adenoviral vector further comprises at least one transgene of interest.

[0165] In some embodiments, the at least one transgene of interest comprises at least one gene. In some embodiment, the at least one gene is absent from the genome of the cell. In some embodiment, the at least one gene is an allele of a gene of the genome of the cell, wherein this allele is not endogenous to the genome of the cell. In some embodiments, the transgene of interest comprises at least one gene from the same species than the cell. In another embodiment, the transgene of interest comprises at least one gene from a different species than the cell.

[0166] In some embodiments, the at least one transgene of interest encodes at least one protein of interest or at least one nucleic acid of interest.

[0167] In some embodiments, the at least one transgene of interest has a length of at least 500 base pairs (bp). As use herein, at least 500 means 500, 600, 700, 800, 900, 1,000, 10,000, 20,000 bp or more.

[0168] In some embodiments, the at least one transgene of interest encodes at least one protein of interest.

[0169] In some embodiments, the at least one protein of interest is for vaccine purposes. In some embodiments, the protein of interest is recognized by the immune system of a subject, preferably a human subject; preferably the protein of interest elicits an immune response. As used herein, vaccine refers to any preparation comprising a substance or a group of substances meant to cause the immune system of an organism to respond to pathogens, such as bacteria or viruses, or to a tumor. Prophylactic vaccines are used to prevent a subject from ever having a particular disease or to only have a mild case of the disease. Such prophylactic vaccines usually comprise the pathogen responsible for the disease, either live and weakened, inactivated, or killed, or components thereof, purified or recombinant. Therapeutic vaccines are intended to treat specific diseases in a subject, in particular cancer. Such therapeutic anti-cancer vaccines comprise a tumor-antigen or tumor- antigens, eliciting an immune response directed against the tumor cells.

[0170] Therefore, in some embodiments, the at least one protein of interest is a protein from a pathogen species (e.g., virus, bacteria, fungi and the like) responsible for one or more infectious disease. Non-limitative examples of infectious diseases includeAnaplasmosis; Anthrax; Babesiosis; Botulism; Brucellosis; Burkliolderia mallei infection (glanders); Burkliolderia pseudomallei infection (melioidosis); Campylobacteriosis; Carbapenemresistant Enterobacteriaceae infection (CRE); Chancroid; Chikungunya infection; Chlamydia infection; Ciguatera; Clostridium difficile infection; Clostridium perfringens infection (Epsilon Toxin); Coccidioidomycosis fungal infection (Valley fever); Creutzfeldt-Jacob Disease, transmissible spongiform (CJD); Cryptosporidiosis; Cyclosporiasis; Dengue Fever; Diphtheria; E. Coli infection; Eastern Equine Encephalitis (EEE); Ebola Hemorrhagic Fever (Ebola); Ehrlichiosis; Arboviral or parainfectious encephalitis; Non-polio enterovirus infection; D68 enterovirus infection, (EV-D68); Giardiasis; Gonococcal infection (Gonorrhea); Granuloma inguinale; Type B Haemophilus influenza disease, (Hib or H-flu); Flu; Hantavirus pulmonary syndrome (HPS); Hemolytic uremic syndrome (HUS); Hepatitis A (Hep A); Hepatitis B (Hep B); Hepatitis C (Hep C); Hepatitis D (Hep D); Hepatitis E (Hep E); Herpes; Herpes zoster, zoster VZV (Shingles); Histoplasmosis; Human Immunodeficiency Virus / AIDS (HIV / AIDS); Human Papillomavirus (HPV); Influenza (Flu); Lead poisoning; Legionellosis (Legionnaires Disease); Leprosy (Hansens Disease); Leptospirosis; Listeriosis; Lyme Disease; Lymphogranuloma venereum infection (LVG); Malaria; Measles; Viral meningitis; Meningococcal disease; Middle East respiratory syndrome coronavirus 20 (MERS-CoV); Mumps; Norovirus; Paralytic shellfish poisoning; Pediculosis (lice, head and body lice); Pelvic inflammatory disease (PID); Pertussis; Bubonic, septicemic or pneumonic plague,; Pneumococcal disease; Poliomyelitis (Polio); Psittacosis; Pthiriasis (crabs; pubic lice infestation); Pustular rash diseases (small pox, monkeypox, cowpox); Q-Fever; Rabies; Ricin poisoning; Rickettsiosis (Rocky Mountain Spotted Fever); Rubella, including congenital rubella (German Measles); Salmonellosis gastroenteritis infection; Scabies infestation; Scombroid; Severe acute respiratory syndrome (SARS); Shigellosis gastroenteritis infection; Smallpox; Methicillin-resistant Staphylococcal infection (MRSA); Staphylococcal food poisoning; Vancomycin intermediate Staphylococcal infection (VISA); Vancomycin resistant Staphylococcal infection (VRSA); Streptococcal disease, Group A; Streptococcal disease, Group B; Streptococcal toxic-shock syndrome (STSS); Primary, secondary, early latent, late latent or congenital syphilis; Tetanus infection (Lock Jaw); Trichinosis; Tuberculosis (TB); Latent tuberculosis (LTBI); Tularemia (rabbit fever); Typhoid fever, Group D; Typhus;Vaginosis; Varicella (chickenpox); Vibrio cholerae infection (Cholera); Vibriosis (Vibrio); Viral hemorrhagic fever (Ebola, Lassa, Marburg); West Nile virus infection; Yellow Fever; Yersinia infection; SARS-CoV infection; SARS-CoV-2 infection and Zika virus infection.

[0171] In some embodiments, the at least one protein of interest is a protein from a tumor or cancer. Non-1 imitative examples of cancer include carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, vulvar cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.

[0172] In a preferred embodiment, the at least one protein from a tumor or cancer comprises or consists of a neoantigen, e.g., an antigen normally found intracellularly, but aberrantly expressed extracell ularly or secreted by a cancer or tumor cell.

[0173] In some embodiments, the at least one protein of interest is for gene therapy purposes. As used herein, “gene therapy” refers to the exogenous expression of at least one gene encoding at least one protein in a cell having reduced, abnormal, low or no expression of this at least one protein, or reduced activity of this protein, or combinations thereof. Gene therapy is useful for the treatment of diseases, in particular genetic diseases and cancer. In a preferred embodiment, the at least one protein of interest is expressed in, or delivered to, the cell having reduced, abnormal, low or no expression of this at least one protein, or reduced activity of this protein, or combinations thereof, in a cell- specific or tissue specific manner, i.e., the at least one protein of interest is not expressed in, nor delivered to, normal cells.

[0174] In some embodiments, the disease is a genetic disease. In some embodiments, the genetic disease is characterized in that at least one gene is partially or completely silenced, inactivated or malfunctional. In some embodiments, the genetic disease is characterized in that at least one gene in a cell of the subject in need thereof encodes at least one non-functional protein. In some embodiments, the genetic disease is characterized in that at least one gene in a cell of the subject in need thereof encodes at least one protein or polypeptide having deleterious effects in the cell.

[0175] Non-limitative examples of genetic diseases include sickle cell anemia, cystic fibrosis, Huntington disease, congenital muscular dystrophy, Duchenne muscular dystrophy, Fabry disease, Marfan syndrome, thalassemia, cystinosis, familial hypercholesterolemia, hemochromatosis and the like.

[0176] Thus, in some embodiments, the at least one protein of interest is the at least one protein whose expression is reduced, abnormal, low, or absent in the genetic disease. The protein altered or absent in the genetic disease will be apparent to those skilled in the art.

[0177] In some embodiments, the disease is cancer. Examples of cancer have been described hereinabove.

[0178] Cancer or tumor cells typically comprise mutations inducing decreased or abolished expression of anti-tumor genes (e.g., p53), and / or increased expression of proto-oncogenes (e.g., Ras).

[0179] Thus, in some embodiments, the at least one protein of interest is an anti-tumor protein (also named tumor suppressor protein). Non-limitative examples of tumor suppressor protein include p53, APC, MADR2, Bcl-2, CDKN2A, BCRA1-2, and the like.

[0180] In some embodiments, the at least one protein of interest is not a protein having therapeutic properties.

[0181] In some embodiments, the at least one transgene of interest encodes at least one nucleic acid of interest.

[0182] In some embodiments, the at least one nucleic acid of interest is a messenger RNA (mRNA) encoding the at least one protein of interest as described herein.

[0183] In some embodiments, the at least one nucleic acid of interest is an interfering nucleic acid molecule. In some embodiments, the interfering nucleic acid molecule isselected from the group comprising or consisting of DNA antisense oligonucleotides, small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), double- stranded RNA and piwi-inter cting RNA (piRNA).

[0184] In some embodiments, the interfering nucleic acid molecule effectively decreases at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold the expression of at least one deleterious protein.

[0185] It is to be understood that “deleterious protein” refers to both abnormal proteins being inherently dangerous for the cell (e.g., a mutant protein) and proteins that are abnormally overexpressed (e.g., proto-oncogenes in cancer). In some embodiments, the deleterious is a proto-oncogene. Non-limitative examples of proto-oncogenes include HER-2, c-Kit, Ras, beta-Catenin, Cyclin E, B-Raf, BCR-ABL, EGF, VEGF, EGFR, c- Myc, and the like.

[0186] It will be apparent that the handling and production of viral vector requires safety measures that are known to the skilled person. Typically, experiments on such vectors will be performed in a laboratory or culture platform having a biosafety index equal to or greater than 2.

[0187] Culture conditions for the cells are described hereinabove. For the particular purpose of producing adenoviral vectors, any suitable medium can be used.

[0188] In some embodiments, the cells are transfected with at least one vector encoding the adenoviral vector as described herein. In some embodiments, the at least one vector further comprises the transgene of interest. In some embodiments, the at least one vector encoding the adenoviral vector and the at least one vector comprising the transgene of interest are distinct.

[0189] Cells may be transfected by methods known in the art; non-limitative examples include lipofection, electroporation, sonoporation, gene gun, microinjection, transduction with a viral vector or chemical-based such as calcium phosphate.

[0190] In some embodiments, the cells are transfected by the means of adenovirus infection. In some embodiments, the ratio of the number of adenoviruses to the numberof cells (multiplicity of infection, MOI) is MOI 0.00001 to MOI 103, preferably MOI 0.0001 to MOI 100, more preferably MOI 0.001 to MOI 10, even more preferably MOI 0.03 to MOI 3. In some embodiments, the MOI is about 0.03. In some embodiments, the MOI is about 0.3. In some embodiments, the MOI is about 3.

[0191] In some embodiments, the transfected cells are incubated as described hereinabove, for a duration from 12 hours to one week, from 1 day to one week, from 2 days to one week, from 3 days to one week, from 4 days to one week, from 5 days to one week, from 6 days to one week after transfection. In some embodiments, the transfected cells are incubated for a duration of about 5 days after transfection. In some embodiments, the transfected cells are incubated for a duration of about 7 days after transfection.

[0192] In some embodiments, the transfected cells are incubated as described hereinabove, until 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100% of the cells show cytopathic effects. In some embodiments, the transfected cells are incubated until about 80% of the cells show cytopathic effects. Examples of cytopathic effects include, but are not limited to, a change in the shape of the cell (that may, for example, become spherical), or detachment from the culture support.

[0193] In some embodiments, adenoviral vectors are collected from the cell culture. In some embodiments, adenoviral vectors are collected from the cell culture supernatant and / or from the cells after a step of cell lysis. In some embodiments, cell lysis is performed by any suitable technique known in the art, comprising or consisting of freeze / thaw cycles, typically from 1 to 10 cycles, or about 3 cycles; mechanical lysis, for example with beads or influx / reflux in a syringe; enzymatic lysis, for example with a protease; perforating agent, for example perforin or digitonin; osmotic choc; chemical lysis; or combination thereof.

[0194] In some embodiments, the adenoviral vectors are purified from the culture medium of the cells. Purification of adenoviral vectors to enhance the concentration can be accomplished by any suitable method, such as by density gradient purification e.g., cesium chloride (CsCl)), by chromatography techniques (e.g., column or batch chromatography), or by ultracentrifugation. For example, the adenoviral vector can besubjected to two or three CsCl density gradient purification steps. The adenoviral vector is desirably purified from transfected cells using a method that comprises lysing cells, applying the lysate to a chromatography resin, eluting the adenoviral vector from the chromatography resin, and collecting a fraction containing the adenoviral vector.

[0195] In some embodiments, the cell culture supernatant is treated with a nuclease for 1 minute to 60 minutes, preferably for about 30 minutes, in order to degrade the unpackaged nucleic acids. In some embodiments, the nuclease is an endonuclease, typically Benzonase®. In some embodiments, the nuclease is an exonuclease. In some embodiments, the nuclease is administered at a dose of about lU / mL or according to the manufacturer’ s recommendations.

[0196] In some embodiments, the supernatant is filtered one or more times. In some embodiments, the filter size is from 0.10 m to 1 pm, preferably from 0.15 pm to 0.30 pm, more preferably about 0.22 pm. In some embodiments, the filter size is from 10 to 1,000 kDa, preferably about 100 kDa.

[0197] In some embodiments, the adenoviral particles are stored for future use.

[0198] In some embodiments, the adenoviral vectors are stored in a suitable biological buffer. Suitable biological buffers for adenoviral vectors are known in the art.

[0199] In some embodiments, the biological buffer is devoid of protease, lipase, detergent and / or ion chelator.

[0200] In some embodiments, the adenoviral vectors are cryopreserved for storage. In some embodiments, adenoviral vectors are stored at a temperature from -20°C to -200°C. In some embodiments, the adenoviral vectors are stored at about -80°C. In some embodiments, the adenoviral vectors are stored at about -196°C, typically adenoviral vectors are stored in liquid nitrogen. In a less preferred embodiment, the adenoviral vectors are stored at about -20°C In some embodiments, adenoviral vectors are “flash- frozen” in liquid nitrogen prior to storage.

[0201] In some embodiments, the adenoviral vectors are contacted with at least one cryoprotective agent prior to cryopreservation. Cryoprotective agent are known in the art and include, e.g., glycerol, sucrose and the like.

[0202] The present invention further relates to a method of producing an adenoviral vector, comprising the steps of: a) culturing cells according to the invention; b) transfecting the cells obtained in step a) with at least one vector encoding the adenoviral vector according to the invention, or fragments thereof; c) incubating the transfected cells obtained in step b) for a duration from 1 day to one week; d) obtaining the adenoviral vector from the culture supernatant and / or from the cells obtained in step c); and e) optionally, purifying the adenoviral vector obtained in step ad.

[0203] The cells and means to culture the cells at step a) are described herein.

[0204] In some embodiments, the cells are helper cells. In some embodiments, the cells are packaging cells.

[0205] In one embodiment, step a) comprises culturing exactly one cell type.

[0206] In another embodiment, step a) comprises culturing more than one cell type, typically wherein another cell population of helper cells or packaging cells is cultured in addition to the cell population of the present invention. Such cell lines are known to the skilled in the art.

[0207] In some embodiments, the at least one vector at step b) is selected from the group comprising or consisting of a plasmid, a messenger RNA (mRNA), a naked DNA, a cosmid, a fosmid, a prokaryotic chromosome (e.g., bacterial artificial chromosome), a eukaryotic chromosome (e.g., yeast artificial chromosome or human artificialchromosome), a viral vector (e.g., lentiviral vector or adeno-associated viral vector), or combinations thereof.

[0208] In one embodiment, step b) comprises transfecting the cells with one vector as described hereinabove, wherein the one vector is sufficient to induce the production of the complete adenoviral vector in the cells. In other words, the vector comprises all the adenoviral genes and optional regulatory elements to enable the production of the adenoviral vectors by the cells.

[0209] In another embodiment, step b) comprises transfecting the cells with at least two vectors as described hereinabove, wherein only the co-transfection with the at least two vectors is sufficient to induce the production of the complete adenoviral vector in the cells. In other words, each vector (e.g., 1, 2, 3 or more vectors) comprises at least one adenoviral gene or optional regulatory element distinct from the other vector(s), and the combination of all vectors is necessary and sufficient to enable the production of the adenoviral vectors by the cells.

[0210] In a preferred embodiment, the at least one vector at step b) further comprises the at least one transgene of interest.

[0211] In another embodiments, the transgene is comprised in another vector, wherein the other vector is transfected before step b), concomitantly with step b), or after step b).

[0212] In some embodiments, the transfected cells at step c) are incubated for a duration from 12 hours to one week, from 1 day to one week, from 2 days to one week, from 3 days to one week, from 4 days to one week, from 5 days to one week, from 6 days to one week after transfection. In some embodiments, the transfected cells are incubated for a duration of about 5 days after transfection. In some embodiments, the transfected cells are incubated for a duration of about 7 days after transfection.

[0213] In some embodiments, the transfected cells at step c) are incubated until 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100% of the cells show cytopathic effects. In some embodiments, the transfected cells are incubated until about 80% of the cells show cytopathic effects.

[0214] In some embodiments, the transfected cells at step c) are incubated at about 37°C, under about 5% CO2.

[0215] In some embodiments, the transfected cells at step c) are incubated in a culture medium as described in the present invention.

[0216] In some embodiments, the method further comprises a step c’), occurring after step c) and before step d), comprising or consisting of quantifying the number of transfected cells (z.e., cells producing at least one adenoviral vector). In some embodiments, cells are quantified using flow cytometry, FACS, fluorescence microscopy, Western Blot, nested PCR, RNA-seq, or combination thereof, preferably flow cytometry and / or RNA-seq.

[0217] In some embodiments, at step d), the adenoviral vectors are obtained by means according to the present invention, preferably the adenoviral vectors are obtained from the culture supernatant.

[0218] In some embodiments, at step e), the adenoviral vectors are purified by means described herein, such as, for example, centrifugation, ultracentrifugation, chromatography, density gradient purification, affinity purification, filtration, and / or combination thereof.

[0219] In some embodiments, the purification step results in a 1.5-fold, 2-fold, 5-fold, 10-fold or more enrichment of adenoviral vectors in the purified fraction compared to the culture supernatant and / or the cell lysate.

[0220] In some embodiments, the purified fraction comprises less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001% or less contaminants. In some embodiments, contaminants are any molecule that are not the adenoviral vector or the biological buffer. In some embodiments, the contaminants are cell components, fragments or fractions, typically proteins, fragments of membranes, organelles and the like. In some embodiments, the contaminants are components of the culture medium, e.g., antibacterial agents.

[0221] In a preferred embodiment, the purified fraction is devoid of naked (or unpackaged) nucleic acid. The present disclosure teaches means of eliminating naked nucleic acids during the obtention of adenoviral vector, e.g., by nuclease treatment.

[0222] In some embodiments, the method further comprises a step of sequencing the adenoviral vectors produced, by means of DNA sequencing. In some embodiments, the method further comprises a step of quantifying and / or counting the adenoviral vectors. In some embodiments, the adenoviral vectors may be quantified and / or counted in the supernatant and / or intracellularly.

[0223] In some embodiments, the method further comprises a step of storage of the adenoviral vector.

[0224] In some embodiments, the adenoviral vectors are cryopreserved for storage. In some embodiments, adenoviral vectors are stored at a temperature from -20°C to -200°C. In some embodiments, the adenoviral vectors are stored at about -80°C. In some embodiments, the adenoviral vectors are stored at about -196°C, typically adenoviral vectors are stored in liquid nitrogen. In a less preferred embodiment, the adenoviral vectors are stored at about -20°C In some embodiments, adenoviral vectors are “flash- frozen” in liquid nitrogen prior to storage. In some embodiments, the adenoviral vectors are contacted with at least one cryoprotective agent prior to cryopreservation. Cryoprotective agent are known in the art and include, e.g., glycerol, sucrose and the like.

[0225] In some embodiments, the adenoviral vectors are comprised in a buffer suitable for long term storage. Illustratively, GTS buffer (2.5% glycerol, 20 mM Tris pH 8, 25 mM NaCl) may be used.

[0226] The present invention further relates to a kit comprising the population of cells according to the invention, and means to culture the cells.

[0227] In some embodiments, the kit further comprises means to produce at least one adenoviral vector as described herein. In some embodiments, the kit further comprises at least one nucleic acid molecule encoding at least one adenoviral vector as described herein.

[0228] The present invention further relates to a composition comprising at least one vector encoding the adenoviral vector produced by the method according to the invention, or fragments thereof.

[0229] In some embodiments, the at least one vector is selected from the group comprising or consisting of a plasmid, a messenger RNA (mRNA), a naked DNA, a cosmid, a fosmid, a prokaryotic chromosome (e.g., bacterial artificial chromosome), a eukaryotic chromosome (e.g., yeast artificial chromosome or human artificial chromosome), a viral vector (e.g., lentiviral vector or adeno-associated viral vector), or combinations thereof.

[0230] The present invention further relates to a method for in vitro culturing cells as described herein.

[0231] In some embodiments, the method is a method to in vitro culture adenoviral vector-producing cells.

[0232] The present invention further relates to an adenoviral vector produced by the method according to the invention.

[0233] The present invention further relates to a composition comprising an adenoviral vector produced by the method according to the invention.

[0234] The present invention further relates to a pharmaceutical composition comprising an adenoviral vector produced by the method according to the invention, and at least one pharmaceutically acceptable excipient.

[0235] In some embodiments, pharmaceutical composition further comprises at least one other therapeutic agent.

[0236] The present invention further relates to the adenoviral vector produced by the method according to the invention or the pharmaceutical composition according to the invention, for use for treating a disease.

[0237] In some embodiments, the disease is selected from the group comprising or consisting of genetic disease, cancer, and infectious disease. In some embodiments, the disease is selected from the group comprising or consisting of genetic disease, and cancer.

[0238] The present invention further relates to the adenoviral vector produced by the method according to the invention or the pharmaceutical composition according to the invention, for use as a vaccine.

[0239] In some embodiments, the vaccine is for immunization against a pathogen selected from the group comprising or consisting of virus, bacteria, fungi, archaea, and protozoa, preferably selected from the group comprising or consisting of virus, bacteria, and fungi, more preferably selected from the group comprising or consisting of virus and bacteria.

[0240] The present invention further relates to the adenoviral vector produced by the method according to the invention or the pharmaceutical composition according to the invention, for use as a vaccine adjuvant.

[0241] The present invention further relates to a method for treating a disease as described herein comprising administering to a subject in need thereof a therapeutically effective amount of the adenoviral vector produced by the method according to the invention or the pharmaceutical composition according to the invention.

[0242] The present invention further relates to a cell or population of cells according to the invention, the adenoviral vector produced by the method according to the invention, or the pharmaceutical composition according to the invention for the manufacture of a medicament for treating a disease as described herein, preferably selected from the group comprising or consisting of genetic disease, cancer and infectious disease.

[0243] The present invention further relates to an adenoviral vector produced by the method according to the invention, for use in an in vitro method.

[0244] In some embodiments, the in vitro method is a transfection method or a transduction method.BRIEF DESCRIPTION OF THE DRAWINGS

[0245] Figure 1A-1C is a set of graphs showing that A3B slows down adenovirus propagation by a deaminase-dependent mechanism. HBEC-WT, -GFP, -A3B and -A3B- DD were infected with HAdV-A12 (Fig. 1A), -B3 (Fig. IB) or -C2 (Fig. 1C) at a MOI=0.03. The percentage of infected cells were quantified by flow cytometry at different time points post infection. The results of 2 biological replicates are shown separately. P-values were calculated by ratio t- tests. Only significant differences are shown.

[0246] Figure 2A-2C is a set of dot plots showing the establishment of A3B knockdown lung cancer cells. A549 lung cancer cells were transduced to stably express APOBEC3B- targeting shRNAs (shA3B) or a nontargeting control shRNA (scramble). A3B mRNA levels were quantified by RTqPCR and expressed relative to GAPDH mRNA (Fig. 2A). A3B protein levels were assessed by western blot, quantified by densitometry and expressed relative to HSP90 (Fig. 2B). Deamination activities of the cell lysates were expressed as the percentage of substrate converted into product and compared to the deamination activity of the WT cells (Fig. 2C). P-values were calculated by t-tests.

[0247] Figure 3A-3C is a set of graphs showing that A3B knockdown promotes replication of adenoviruses. A549-WT, -scramble and -shA3B were infected with HAdV- A12 (Fig. 3A), -B3 (Fig. 3B) or -C2 (Fig. 3C) at a MOI=0.03. The percentage of infected cells were quantified by flow cytometry at 2-, 4-, 7-days post infection (dpi). The results for 2 replicates are depicted.

[0248] Figure 4 is a set of graphs showing that A3B knockdown promotes replication and secretion of adenovirus of strain A12. A549-WT, -scramble and -shA3B were infected with HAdV-A12, at a MOI=0.03. Extracellular viral DNA levels were quantified by qPCR at 2-, 4- and 7-days post infection. The results for 2 replicates are depicted.

[0249] Figure 5A-5B is a combination of a photograph and a scheme showing that A3B knockdown reduces the presence of hypermutated viral genomes. A549-WT, -scrambleand -shA3B were infected with HAdV-A12, -B3 or -C2 and the intracellular DNA was extracted 48 hours post infection. 3DPCR reactions were conducted on the E1B, L3 and E4 genes. Fig. 5A shows agarose gels illustrating 3DPCR specific for the L3 gene of the A12 strain. The dotted white line indicates the threshold between mutated and unmutated 3DPCR products. Low denaturation temperature amplicons were recovered in WT and scramble A549 cells. Fig. 5B is a graphical representation of the 3DPCR results obtained for the 3 viral genes (E1B, L3 and E4), for the 3 viral strains (A12, B3 and C2) and for the 3 cell lines (WT, scramble and shA3B). The lowest denaturation temperatures allowing the production of the expected amplicons were represented on the gradients by black, grey, and white circles.

[0250] Figure 6A-6B is a set of photographs showing the knockdown of constitutively expressed A3B in HEK293 cells. HEK293 cells were transduced with lentiviral vectors encoding shRNA targeting the A3B transcript. The HEK293 cells lost detectable A3B protein expression (Fig. 6A) and deaminase activity (Fig. 6B).EXAMPLES

[0251] The present invention is further illustrated by the following examples.Example 1: APOBEC3B (A3B) limits the propagation of adenovirusesMaterials and MethodsCell lines, transduction and knock-down:

[0252] HBEC3-KT cells are normal human bronchial epithelial cells immortalized with the human TERT and mouse Cdk4 genes. HBEC3-KT were cultured in keratinocyte-SFM medium supplemented with bovine pituitary extract (50 pg / mL) and human recombinant epidermal growth factor (5 ng / mL) (Gibco) on gelatin-coated flasks. HBEC3-KT constitutively expressing GFP (HBEC-GFP), A3B (HBEC-A3B) or A3B Deaminase Dead (HBEC-A3B-DD) were established by transduction with lentiviral vectors encoding for a GFP, A3B or A3B-DD protein (pLenti4-A3B and pLenti4-A3B-E68Q-E255Qencoding respectively the catalytically active and inactive A3B). Of note, the puromycin markers originally present in the pSicoR-M2 and pLenti4-A3B plasmids have been replaced by a blasticidin resistance gene prior to HBEC3-KT transduction. Blasticidin was used to select for a polyclonal population of effectively transduced cells.

[0253] The human lung carcinoma cell line A549 (American Type Culture Collection) and PKR-deficient A549 cell line were cultured in Dulbecco’s Modified Eagle’s medium (Gibco) supplemented with 10% fetal calf serum (Gibco) and 10 mM of L-glutamine. A549 cells knock-down for A3B (A549 A3B shRNAs) were established by transduction with lentiviral vectors encoding A3B-targeting shRNAs (pSicoR-MS2 plasmids). A549 cells were also transduced with a lentiviral vector encoding non-targeting shRNA (A549 NTC shRNA) and used as control.

[0254] Of note, the puromycin markers originally present in the pSicoR-M2 and pLenti4-A3B plasmids have been replaced by a blasticidin resistance gene prior to A549 or HBEC3-KT transduction. Blasticidin was used to select for a polyclonal population of effectively transduced cells. All cell lines were incubated at 37°C and 5% CO2.Virus preparation and infection procedure:

[0255] Handling of human adenoviruses was done in a biosafety level 2 laboratory. 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. During viral stock production, A549 cells and 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 residualcell 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.

[0256] HBEC3-KT cells were infected with the HAdV-A12, -B3 or -C2 strain at a multiplicity of infection (MOI) of 0.03 or 3 in keratinocyte-SFM medium supplemented with bovine pituitary extract (50 pg / mL) and human recombinant epidermal growth factor (5 ng / mL) (Gibco) on gelatin-coated flasks.

[0257] A549 cells were infected with the HAdV-A12, -B3 or -C2 strain at a multiplicity of infection (MOI) of 0.03 or 3 in Dulbecco’s Modified Eagle’s medium (Gibco) supplemented with 2% fetal calf serum (Gibco) and 10 mM of L-glutamine.Percentage of infected cells by flow cytometry:

[0258] Two-, four- or seven-days post infection, cells were collected and washed in PBS. Cells were fixed and permeabilized using the eBiosciencesTM Foxp3 / Transcription Factor Staining Buffer set (InvitrogenTM) according manufacturer’s instructions. Cells were then incubated 30 minutes at 4°C with anti-HAdV antibody (MAB 8051, Merck Millipore) used at a dilution of 1:200 in permeabilization buffer supplemented with 2% of goat serum (Gibco). After 2 washes in permeabilization buffer supplemented with goat serum, cells were incubated 30 minutes with an anti-mouse antibody coupled with phycoerythrin (Miltenyi Biotec). Cells were washed two times with permeabilization buffer supplemented with 2% of goat serum and then resuspended in PBS. Percentage of HAdV-infected cells was quantified using BD FACSVerse Flow Cytometer and BD FACSuite vl.0.6 software (BD Biosciences).Statistics and data representation:

[0259] P-values were calculated according to the indicated test. Error bars show standard deviations. ND for stands for Not Detected, NS for Not Significant, * p< 0.05, ** p< 0.01, *** p< 0.001, **** p<0.0001. The experiments were replicated at least three times and representative images are shown.Results

[0260] 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).

[0261] HBEC-WT, HBEC-A3B, HBEC-A3B-DD and HBEC-GFP cells were infected with the different adenoviral strains, A12, B3 and C2 at a low MOI (0.03), and viral replication was monitored in the 4 cell lines. Figure 1A-C shows the percentage of infected cells measured by flow cytometry at different time points post infection. Of note, the propagation of the A 12 strain is consistently slower than that of the B3 and C2 strains; thus, the follow-up of the A12 infection was extended up to 10 days post infection (dpi), whereas it was stopped after 7 days for the B3 and C2 strains.

[0262] As seen on Figure 1A-C, viral propagation is repeatedly slower in HBEC-A3B cells than in HBEC-WT, HBEC-GFP or HBEC-A3B-DD. Most of the cells were lysed at 7 days post-infection, precluding FACS and intracellular DNA qPCR analysis for this time point.

[0263] 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.

[0264] The percentage of B3-infected cells in A3B -expressing cells is significantly lower compared to the percentage measured in the WT cells at 2 days post infection but no significant difference could be observed later (Figure IB). Finally, the percentage of C2-infected cells in A3B -expressing cells is significantly lower compared to thepercentage measured in the WT cells at 2- and 4- days post infection but no significant difference could be observed at 7 dpi (Figure 1C). Thus, A3B also limits the propagation of B3 and C2, but with less efficiency than against A12 strain.

[0265] Overall, these results show that A3B restricts the replication of these different adenoviral strains, that the deaminase activity of the protein is required, and that the magnitude of restriction is influenced by the strain of virus, the most sensiti ve strain being the A12.Example 2; A3B knock-down promotes adenoviral replicationMaterials and MethodsCell lines, transduction and knock-down: see example 1.Virus preparation and infection procedure: see example 1.Statistics and data, representation: see example 1.Viral and cellular mRNA quantification by RT-qPCR

[0266] One or two-days post infection, cells were collected, washed in PBS and resuspended in 1ml of TRIzol reagent (Invitrogen) for RNA extraction. Two hundred (200) pl of chloroform were added and samples were centrifugated at 4°C at 12 000g for 15 minutes. Five hundred (500) pL from the aqueous phase was collected and added to 450 pL of isopropanol. Samples were centrifuged at 12,000g for 15 minutes. Pellets were then washed into 900 pL of 75% ethanol, centrifuged at 12,000g for 10 minutes and resuspended into 30 pL of RNAse free water. cDNA was obtained by reverse transcription of 1 pg of RNA using iScript cDNA Synthesis Kit (Bio-rad) following manufacturer’s instructions. The cDNA was subjected to qPCR using Takyon™ No Rox SYBR 2X MasterMix blue dTTP (Eurogentec) and CFX96TM lOOOTouch Real-Time PCR System (Biorad). The primers used to titrate A3B were designed by Refsland and colleagues (Refsland et al., 2010).

[0267] The A3B mRNA level was expressed relatively to the abundance of GAPDH.Primers used for mRNA quantification by RTqPCR are listed in Table 1.

[0268] Table 1: Primers for cellular and viral mRNAs quantificationViral copy number quantification by qPCR:

[0269] DNA was extracted from infected cells using Nucleospin Tissue (MachereyNagels) according manufacturer’s instructions and subjected to PCR. In parallel, supernatant containing virions was inactivated 15 minutes at 70°C and viral copies were directly quantified. A fragment of the penton gene was amplified using TakyonTM No Rox SYBR 2X MasterMix blue DTTP (Eurogentec) and CFX96TM lOOOTouch Real- Time PCR System (Biorad). Serial dilutions of plasmids containing the target sequences were used as calibration curves. Primers used for DNA quantification by qPCR are listed in Table 2.

[0270] Table 2: Primers for adenovirus DNA quantificationA3B protein detection by immunoblotting:

[0271] One or two-days post infection, cells were collected and washed in PBS. Cellswere 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). Twenty-five pg of proteins were loaded on a 10% SDS-PAGE gel and transferred actively to PVDF membrane (GE healthcare Life Sciences). Membrane was blocked in TBS (Tris Buffered Saline) supplemented with 0.1% Tween 20 and 5% bovine serum albumin (BSA). A3B protein was detected with anti-Human APOBEC3B monoclonal antibody (5210-87-13, cat# 12397) used at a dilution of 1:1000 in TBS supplemented with 0.1% Tween 20 and 5% BSA. Hsp90 was used as loading control and was detected with an anti-HSP90AB 1 antibody (Sigma- Aldrich) at a dilution of 1:1000 in TBS supplemented with 0.1% Tween 20 and 5% BSA. An HRP-coupled anti-rabbit IgG secondary antibody (Dako) was used at a dilution of 1:2000 in TBS with 0.1% Tween 20 and 5% BSA or 4% milk. Membranes were incubated with the chemoluminescent SuperSignal TM West Femto Maximun Sensitivity Substrate (ThermoFisher Scientific) and chemoluminescence was read using an ImageQuant LAS4000 (GE Healthare Life Sciences). Densitometry analysis was done using ImageJ software.Deamination assay:

[0272] 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 Pierce TM BCA Protein Assay Kit (ThermoFisher Scientific). Forty pg of proteins were incubated overnight at 37°C with 1 pmol of a fluorescent oligo substrate (5’- ATTATTATTATTCAAATGGATTTATTTATTTATTTATTTATTT-Cy5-3’) (SEQ ID NO: 45), ImM ZnC12, 0.025U uracil DNA glycosylase (NEB), 2 pL lOx UDG buffer(NEB) and 100 pg / mL RNAse A (Thermofisher Scientific). Reaction mixture was treated with 50 mM NaOH and heated to 95°C for 10 minutes to cleave DNA probes at the abasic site. Reaction mixture was then neutralized with 50 mM HC1 and mixed with 1.25x formamide buffer. Substrates (43 bases-long) were separated from products (30 bases- long) on a 15% tris-borate-EDTA (TBE)-urea gel. The Cy5-labeled substrates and deamination products were detected using ImageQuant LAS4000 mini (GE Healthcare Life Sciences). Densitometry analysis was done using ImageJ software.Results

[0273] It was next assessed whether knock-down (KD) of A3B could facilitate adenovirus replication. 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 and Figure 2C shows that A549-shA3B does not display deaminase activity, contrary to the WT or scramble controls.

[0274] Similarly with the previous experiment in HBEC3-KT cells, the A549-WT, - scramble and -shA3B cells were infected with the 3 HAdVs strains at a low MOI (0.03) and the viral replication was monitored during several days post infection. Figure 3A-B shows the percentage of infected cells measured by flow cytometry (2 replicates).

[0275] The percentage of A12-infected cells is increased in the A549-shA3B cells compared to A549-WT cells at 4- and 7-days post infection (Figure 3A), and to a lesser extent with the B3 and C2 strains (Figures 3B and 3C).

[0276] Figure 4 shows the amount of viral DNA in the supernatant. The viral load in the supernatant of the A549-shA3B cells infected with the A12 strain, used illustratively herein, is increased at 7-days post infection.

[0277] To summarize, the down regulation of A3B speeds up the propagation of adenovirus, in particular that of the A12 strain, in cell culture, increasing its DNAreplication and virion production. In other words, an endogenously-expressed A3B protein effectively restricts the propagation of adenoviruses, notably adenoviruses of the A 12 strain. Example 3: A3B knock-down reduces the presence of hypermutated viral genomesMaterials and MethodsCell lines, transduction and knock-down: see example 1.Virus preparation and infection procedure: see example 1.Statistics and data representation: see example 1. Selective amplification of hypermutated viral sequences by 3DPCR:

[0278] Total DNA from infected cells was extracted using the MasterPure complete DNA and RNA purification kit (Epicentre) and resuspended in 50 pL sterile water. All amplifications were performed using first-round standard PCR followed by nested 3DPCR. PCR was performed with 1U Taq DNA polymerase (Bioline) per reaction. After purification, PCR products were cloned into TOPO 2.1 vector (Life Technologies) and sequencing was outsourced to Eurofins Genomics. Primers used for classic PCR and 3DPCR are listed in Table 3.

[0279] Table 3: Primers and cycling conditions for 3DPCRResults

[0280] It was then assessed whether the A3B knockdown would prevent the generation of hypermutated viruses. 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.

[0281] 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.

[0282] This demonstrates that an endogenously expressed A3B can hypermutate adenovirus genome.Example 4: A3B knock-down generation on HEK293 cellsMaterials and Methods

[0283] 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: seeExample 2.Results

[0285] The HEK293 cells, widely used as helper cells for the production of viral vectors, express an active APOBEC3B protein (Figure 6A).

[0286] 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.

[0287] These results indicate that knock-down or knock-out of the APOBEC3B gene in HEK293 cells greatly improves their potency as viral vector producer cells, in particular adenoviral vector producer cells.

Claims

CLAIMS1. A population of cells, wherein said cells have abolished or reduced expression of at least one protein from the apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOB EC) family of proteins.

2. The population of cells according to claim 1, wherein said cells have abolished or reduced expression of at least two proteins from the APOBEC family of proteins.

3. The population of cells according to any one of claims 1 to 2, wherein 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), reduced (knock-down, KD) or combinations thereof.

4. The population of cells according to any one of claims 1 to 3, wherein 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).

5. The population of cells according to any one of claims 1 to 4, wherein said at least one protein or said at least two proteins from the APOBEC family of proteins is selected from the group comprising or consisting of APOBEC3B, APOBEC3A, APOBEC3C, APOBEC3D, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC 1, APOBEC2, and APOBEC4.

6. The population of cells according to any one of claims 1 to 5, wherein said at least one protein from the APOBEC family of proteins is APOBEC3B.

7. The population of cells according to any one of claims 1 to 6, wherein said cells are mammalian cells, preferably human cells.

8. The population of cells according to any one of claims 1 to 7, wherein said cells are from a cell line selected from the group comprising HEK293, PER.C6, HER, HeLa, HEL299, LP-293, 911, AE1-2A, 293CRE4, E2T, C7, 293N3S, HBEC3-KT, A549, COS, CHO, VERO, MDCK, WI38, V79, B 14AF28-G3, BHK, HaK, NSO, and SP2 / 0-Agl4.The population of cells according to any one of claims 1 to 8, wherein said cells are HEK293 cells. The population of cells according to any one of claims 1 to 9, wherein said cells express an adenovirus attachment receptor, preferably said adenovirus attachment receptor is selected from the group comprising the coxsackievirus and adenovirus receptor (CAR), CD46, CD80, CD86, sialic acid and integrins. Use of the population of cells according to any one of claims 1 to 10, for producing an adenoviral vector. The use according to claim 11, wherein said adenoviral vector is a human adenoviral vector. The use according to claim 11 or 12, wherein said adenoviral vector is from a species selected from the group comprising or consisting of A, B, C, D, E, F and G. The use according to any one of claims 11 to 13, wherein said adenoviral vector further comprises at least one transgene of interest. A method of producing an adenoviral vector, comprising the steps of: a) culturing a cell population according to any one of claims 1 to 10; b) transfecting said 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 said cells; and e) optionally, purifying the adenoviral vector.

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