Engineered cells for virus production

Abstract

The present disclosure relates to a genetically modified cell comprising at least one nucleic acid molecule encoding a B18R protein, or a functional fragment thereof, of a poxvirus, and / or an E3L protein, or a functional fragment thereof, of a poxvirus, and methods of using said genetically modified cell for producing a virus of interest, comprising culturing the virus of interest with said genetically modified cell.

Description

ENGINEERED CELLS FOR VIRUS PRODUCTION

[0001] CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 567,985 filed March 21, 2024, the contents of which are incorporated herein by reference.

[0003] FIELD

[0004] The present invention relates to genetically modified cells providing enhanced production of cultured viruses.

[0005] BACKGROUND

[0006] Viruses require host cells to be able to replicate. To produce virus in vitro, the virus of interest must be cultured with a host cell in which the virus of interest can replicate. The virus of interest infects the host cell and takes advantage of the host cell’s replication, transcription, and / or translation elements that are lacking in the virus genome. The virus of interest can then propagate in the cell culture until a maximum titer of virus in the culture medium is achieved. The virus of interest can then be isolated from the culture medium.

[0007] There are different factors that can limit the amount of virus produced in culture with host cells. One significant limitation is anti-viral responses induced in the host cells. Most cells have evolved mechanisms to detect the presence of virus and induce anti-viral responses that limit virus replication. For example, in response to viral infection, cells may produce cytokines known as interferons (IFN) that activate anti-viral mechanisms. These IFN responses inhibit viral propagation in host cells and therefore limit the amount of virus that can be produced. Furthermore, the presence of IFN responses in host cells acts as a selective pressure that can lead to unwanted selection for viral mutants that evade IFN responses, such as by mutations that modify the sequence or expression levels of genes encoded in the virus genome. This is undesirable when the goal is to produce a pure culture of a virus of interest without uncharacterized mutations.

[0008] SUMMARY

[0009] The present invention relates to genetically modified cells providing enhanced production of cultured viruses. By culturing a virus of interest with the genetically modified cells of the present invention, the virus of interest is propagated to high titers and without suppressing expression of genes encoded by the virus of interest.

[0010] In an embodiment, there is provided a genetically modified cell, wherein the cell comprises at least one nucleic acid molecule encoding: (i) a B18R protein, or a functional fragment thereof, of a poxvirus; and / or (ii) an E3L protein, or a functional fragment thereof, of a poxvirus.

[0011] In another embodiment, there is provided a method for producing a virus of interest, comprising culturing the virus of interest with the genetically modified cell as described herein.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Embodiments will be described, by way of example only, with reference to the accompanying figures.

[0014] Figure 1 shows virus titers as measured in plaque forming units per mL (PFU / mL) after a series of passages in culture with cells according to the present invention (Engineered) or control cells (Parental).

[0015] Figure 2 shows virus titers as measured in plaque forming units per mL (PFU / mL) after a series of amplification rounds in culture with cells according to the present invention (E3, B18, or both B18-E3 with Kozak sequence (B18-E3+) or without Kozak sequence (B18-E3-)) or control cells (Parental).

[0016] Figure 3 shows levels of IL-12 polypeptide produced in adherent cell culture with 10% FBS measured by ELISA (ng / mL). Culture with control cells (Parent) or with cells according to the present invention (E3, Bl 8, or both Bl 8 and E3 with Kozak sequence (Bl 8- E3L(+)) or without Kozak sequence (B18-E3L(-)) after a series of application rounds.

[0017] Figure 4 shows levels of IL-12 polypeptide produced in serum-free suspension cell culture measured by ELISA (ng / mL). Culture with control cells (Parental) or with cells according to the present invention (E3, Bl 8, or both Bl 8 and E3 with Kozak sequence(B18- E3(+) and E3-B18) after two rounds of culture.

[0018] DETAILED DESCRIPTION

[0019] The present inventor has developed genetically modified cells with enhanced properties for use in the production of viruses in vitro. The genetically modified cells of the present invention are engineered to express one or more poxvirus genes that inhibit anti-viral responses, leading to increased virus titers and improved expression of virus-encoded genes. As shown in Examples 1 and 2, using genetically modified cells of the present invention as hostcells for the production of a virus of interest resulted in higher viral titers compared to control cells. Furthermore, as shown in Example 2, the genetically modified cells of the present invention did not suppress the expression of a virus-encoded transgene, maintaining normal levels of transgene expression.

[0020] Genetic Modification

[0021] As used herein, the term “genetically modified” or “genetically engineered” refers to altering genetic material using molecular biology techniques known in the art such as, but not limited to, molecular cloning, recombinant DNA methods, and gene editing by known techniques such as with the use of restriction endonucleases, viral insertion, Gibson assembly, zinc finger nucleases, TALENs, and / or Crispr-Cas systems such as Crispr-Cas9. Genetic modification includes addition, deletion, substitution, modification, and / or mutation of genetic material. As used herein, the term “modified” may refer to a nucleic acid molecule or a polypeptide that contains one or more changes in the nucleotide or amino acid sequence compared to the un-modified wild-type version of said nucleic acid molecule or polypeptide.

[0022] As used herein, the term “wild-type” carries the ordinary meaning in the art of an organism, nucleic acid molecule, or polypeptide that can be found naturally occurring in the absence of a modification. A naturally occurring, wild-type nucleic acid molecule or polypeptide can be modified to differ in sequence, structure, and / or biological properties as compared to the un-modified wild-type version of the nucleic acid molecule or polypeptide.

[0023] As used herein, the term “nucleic acid molecule” may refer to a large sequence of nucleotides encoding multiple cassettes, operons, genes, and / or reading frames (e.g. a chromosome, a plasmid, an artificial chromosome, a viral genome) but may also refer to a smaller sequence of nucleotides encoding one or a small number (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of cassettes, operons, genes and / or reading frames (e.g. a transgene or a cassette) wherein the smaller sequence of nucleic acids may be isolated or may be comprised in a larger sequence of nucleotides (e.g. a chromosome, a plasmid, an artificial chromosome, a viral genome). Further, as used herein, the term "nucleic acid molecule" is intended to include unmodified DNA or RNA or modified DNA or RNA. The nucleic acid molecules of the disclosure may contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus "nucleic acid molecule" embraces chemically, enzymatically, or metabolically modified forms.

[0024] The term “nucleic acid sequence” as used herein refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages and includes cDNA. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. The nucleic acid can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term "nucleic acid" includes the complementary nucleic acid sequences.

[0025] Nucleic acid sequences as described herein can be provided in nucleic acid molecules in different arrangements or combinations. As used herein with reference to nucleic acid molecules that encode a gene or polypeptide, the term “at least one nucleic acid molecule encoding” means that all encoded sequences are collectively comprised in one or more nucleic acid molecules. In some embodiments, all encoded sequences are comprised in separate nucleic acid molecules, all encoded sequences are comprised in the same nucleic acid molecule, or combinations where some encoded sequences are comprised in separate nucleic acid molecules and some encoded sequences are comprised together in the same nucleic acid molecule. For example, in one embodiment wherein a genetically modified cell comprises a gene encoding both a B18R protein and a gene encoding an E3L protein, or functional fragments thereof, the B18R gene and the E3L gene may be provided in the same nucleic acid molecule. In another embodiment wherein a genetically modified cell comprises a gene encoding both a B18R protein and a gene encoding an E3L protein, or functional fragments thereof, the B18R gene and the E3L gene may be provided in separate nucleic acid molecules. Where more than one sequence that encodes B18R and / or E3L, or functional fragments thereof, is provided in the same nucleic acid molecule, the sequences can be provided in separate expression cassettes, or together in the same expression cassette. Where two or more sequences are in the same expression cassette, they may form a polycistronic mRNA for subsequent translation, wherein each of the sequences has its own ribosomal binding site for translation. Where two or more sequences are in the same expression cassette, they can be provided in the same open reading frame so as to produce a fusion protein. Two or more sequences that encode a fusion protein can be separated by linker sequences that encode restriction nuclease recognition sites or self-cleaving peptide linkers. Nucleic acid molecules encoding genes or polypeptides can include, without limitation,plasmids, artificial chromosomes, episomes, and expression cassetes inserted into known vectors or inserted into chromosomes or plastid or mitochondrial DNA.

[0026] As used herein, the term “vector” or “nucleic acid vector” is a type of nucleic acid molecule, such as a plasmid, comprising regulatory elements and a site for introducing transgenic DNA, which is used to introduce said transgenic DNA into a microorganism. The transgenic DNA can encode a heterologous protein, which can be expressed in and isolated from a microorganism. The transgenic DNA can be integrated into nuclear, mitochondrial or chloroplastic genomes through homologous or non-homologous recombination. The transgenic DNA can also replicate without integrating into nuclear, mitochondrial or chloroplastic genomes in an extra-chromosomal vector. The vector can contain a single, operably linked set of regulatory elements that includes a promoter, a 5’ untranslated region (5’ UTR), an insertion site for transgenic DNA, a 3’ untranslated region (3’ UTR) and a terminator sequence. Vectors useful in the present methods are well known in the art. In some embodiments, the exogenous nucleic acid molecule is a vector comprising a nucleic acid sequence encoding one or both of B18R and E3L, or functional fragments thereof.

[0027] The term “operably linked”, as used herein, refers to an arrangement of two or more components, wherein the components so described are in a relationship permiting them to function in a coordinated manner. For example, a transcriptional regulatory sequence or a promoter is operably linked to a coding sequence if the transcriptional regulatory sequence or promoter facilitates aspects of the transcription of the coding sequence. The skilled person can readily recognize aspects of the transcription process, which include, but are not limited to, initiation, elongation, atenuation and termination. In general, an operably linked transcriptional regulatory sequence joined in cis with the coding sequence, but it is not necessarily directly adjacent to it.

[0028] The word “expression” as used herein refers to the translation of a polypeptide (for example B18R, E3L, or a functional fragment thereof) encoded by a nucleic acid (such as an exogenous nucleic acid molecule) and / or transcription of an RNA transcript (for example, mRNA or an interfering RNA, such as microRNA (miRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA) or Dicer-substrate small-interfering RNA (DsiRNA)) encoded by a nucleic acid (such as an exogenous nucleic acid molecule). As used herein, “increased expression” refers to an increase in the amount of translated polypeptide and / or RNA transcript encoded by a nucleic acid (such as an exogenous nucleic acid) in a cell. The increased expressionmay be relative to a wild-type organism or relative to a virus cultivated in a cell other than a genetically modified cell of the present invention.

[0029] As used herein, the term “exogenous” refers to an element that has been introduced into a nucleic acid molecule, such as a genome or chromosome. An exogenous nucleic acid molecule is introduced into a genome by a method of genetic modification. An exogenous nucleic acid molecule may code for the expression of an RNA and / or a protein. An exogenous nucleic acid molecule may have been derived from the same species (homologous) or from a different species (heterologous). An exogenous nucleic acid molecule may comprise a homologous sequence that is altered such that it is introduced into a genome in a form that is not naturally found in the genome. For example, an exogenous nucleic acid molecule that is homologous may contain mutations or be integrated into a different region of the genome, relative to the endogenous version of the nucleic acid molecule. An exogenous nucleic acid molecule may be incorporated into a chromosome of a genetically modified cell in one or more copies, into the plastid or mitochondrial DNA of the genetically modified cell, or be maintained as a separate nucleic acid molecule outside of the cell genome (e.g. an episome).

[0030] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two nucleic acid (polynucleotide) or two amino acid (polypeptide) sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions / total number of positions multiplied by 100%). The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin, S. and Altschul, S., PNAS (1990), 87(6):2264-2268, modified in Karlin, S. and Altschul, S., PNAS (1993), 90(12):5873-5877. Such an algorithm is incorporated into the BLAST programs. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a given nucleic acid molecule. BLAST protein searches can be performed with the XBLAST programparameters set, e.g., to score=50, wordlength=3 to obtain amino acid sequences homologous to a given polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. (1997), 25(17):3389-3402.Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., of XB LAST and NBLAST) can be used (see, e.g., the NCBI website). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers, E. and Miller, W., Bioinformatics (1988), 4(1): 11-17. Such an algorithm is incorporated in the ALIGN program which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0031] Cells

[0032] Numerous types of cells are known and available as host cells for the culture and production of virus of interest, and may be genetically modified as described herein to encode and express B18R and / or E3L protein, or functional fragments thereof, for use in the present invention.

[0033] In some embodiments, the host cell for use in the present invention is an adherent cell. In some embodiments, the host cell for use in the present invention is a non-adherent cell.

[0034] In some embodiments, the host cell for use in the present invention is a cell line. It is routine in the art to select an appropriate cell line that is capable of being infected by the virus of interest that is to be grown in culture. Further, established cell lines can be engineered to express the viral entry receptor for the virus of interest to enable culture of the virus of interest in the modified cell line. In some embodiments, the host cell for use in the present invention is a cell line selected from CHO (Chinese hamster ovary), HEK239 (human embryonic kidney), A549 (human lung carcinoma; HSV, influenza, measles, mumps, parainfluenza, poliovirus, RSV, rotavirus, N N, MPV), BHK21 (Syrian hamster kidney; human adenovirus D, reovirus, vesicular stomatitis virus, dengue, influenza, rabies, foot and mouth, rubella, modified vaccinia ankara), CV-1 (African green monkey kidney fibroblast; RSV, measles, HSV, VZV), HeLa (human cervix adenocarcinoma; poliovirus, adenovirus, CMV, echovirus, HSV, rhinovirus, vesicular stomatitis virus, VZV), LLCMK2 (Rhesus monkey kidney; poliovirus, enterovirus,rhinovirus, poxvirus), McCoy (mouse fibroblast; HSV), MDCK (Madin-Darby canine kidney; influenza, adenovirus, reovirus), MRC-5 (human fetal lung; CMV, HSV, adenovirus, influenza, mumps, echovirus, poliovirus, rhinovirus, RSV, VZV), NCI-H292 (human lung mucoepidermoid carcinoma; vaccinia virus, HSV, adenovirus, measles, reovirus, BK polyomavirus, RSV, influenza, enterovirus, rhino virus), Vero (African green monkey kidney; coxsackie virus, HSV, measles, mumps, poliovirus, rotavirus, rubella), Vero76 (African green monkey kidney; coxsackie virus, HSV, West Nile virus), or WI 38 (human fetal lung; adenovirus, CMV, echovirus, HSV, mumps, influenza, rhinovirus, RSV, VZV). In some embodiments, the host cell is EB66 or AGE1 (duck cell lines; influenza, poxvirus).

[0035] Poxyirus

[0036] Poxviruses are viruses of the family Poxviridae, comprising a family of doublestranded DNA viruses. Poxviridae comprises the genera Avipoxvirus, Capripoxvirus, Centapoxvirus, Cervidpoxvirus, Crocody lipoxvirus, Leporipoxvirus, Macropopoxvirus, Molluscipoxvirus, Mustelpoxvirus , Orthopoxvirus, Oryzopoxvirus, Parapoxvirus, Pteropopoxvirus, Salmonpoxvirus, Sciuripoxvirus, Suipoxvirus, Vespertilionpoxvirus, Yatapoxvirus, Alphaentomopoxvirus, Betaentomopoxvirus, Deltaent omopoxvirus, Diachasmimorpha entomopoxvirus, and Gammaentomopoxvirus . Four genera of poxviruses are known to infect humans: Molluscipoxvirus, Orthopoxvirus, Parapoxvirus, and Yatapoxvirus . The Molluscipoxvirus comprises the molluscum contagiosum virus (MCV). The Orthopoxvirus comprises the variola virus, vaccinia virus, cowpox virus, buffalopox virus, horsepox virus, rabbitpox virus, taterapox virus, camelpox virus, ectromelia virus, alaskapox virus, skunkpox virus, racconpox virus, and monkeypox virus. The Parapoxvirus comprises the orf virus, pseudocowpox virus, and bovine popular stomatitis virus. The Yatapoxvirus comprises the tanapox virus, and yaba monkey tumor virus.

[0037] In some embodiments, the B18R protein, or a functional fragment thereof, of a poxvirus is of a poxvirus from the genus Orthopoxvirus. In some embodiments, the B18R protein, or a functional fragment thereof, of a poxvirus is from a poxvirus selected from vaccinia virus, cowpox virus, buffalopox virus, horsepox virus, and monkeypox virus. In some embodiments, the E3L protein, or a functional fragment thereof, of a poxvirus is of a poxvirus from the genus Orthopoxvirus. In some embodiments, the E3L protein, or a functional fragment thereof, of a poxvirus is from a poxvirus selected from variola virus, vaccinia virus, cowpox virus, buffalopox virus, horsepox virus, rabbitpox virus, taterapox virus, camelpox virus, ectrolmelia virus, alaskapox virus, skunkpox virus, raccoonpox virus, and monkeypox virus.

[0038] In some embodiments, the B18R protein, or a functional fragment thereof, of a poxvirus is of a vaccinia virus, including but not limited to the Western Reserve strain of vaccinia virus. In some embodiments, the E3L protein, or a functional fragment thereof, of a poxvirus is of a vaccinia virus, including but not limited to the modified vaccinia Ankara (MV A) strain of vaccinia virus. Vaccinia is a double-stranded DNA virus with a genome of about 190 kb and encoding for approximately 250 genes, including B18R and E3L. Vaccinia virus replicates only in the cytoplasm of the host cell, and the large genome codes for various enzymes and proteins needed for viral DNA replication (Greseth, M. and Traktman, P., Annu. Rev. Virol. (2022), 9:239-259). Many strains of vaccinia virus are known in the art and may be used to provide the B18R and / or E3L, or functional fragments thereof. Although some strains of vaccinia virus comprise deletions to the B18R and / or E3L genes, it is within the routine skill in the art to identify vaccinia strains comprising genes encoding B18R and / or E3L for use in the present invention.

[0039] B18R

[0040] In some embodiments, the genetically modified cell comprises a nucleic acid molecule encoding a B18R protein, or a functional fragment thereof, of a poxvirus. The B18R protein is encoded in the genome of numerous poxviruses. B18R binds to type I IFN and inhibits activation of interferon-mediated signal transduction (Colamonici et al., J. Biol. Chem. (1995), 270(27): 15974-15978). Without being bound by theory, it is believed that by binding to type I IFN with high affinity, B18R prevents IFN from binding to native IFN receptors in cells and inducing IFN-mediated antiviral responses.

[0041] In some embodiments, the B18R protein is derived from a poxvirus, such as a virus of the genus Orthopoxvirus, including but not limited to vaccinia virus, cowpox virus, buffalopox virus, horsepox virus, and monkeypox virus. An exemplary B18R amino acid sequence from vaccinia virus Western Reserve strain is set forth as SEQ ID NO: 1 and is publicly available under GenBank accession number BAA00826.1. An exemplary B18R encoding nucleic acid sequence from vaccinia virus Western Reserve strain is set forth as SEQ ID NO: 2 and is publicly available under GenBank accession number D01019.1. Although there may be variations in the amino acid sequences of various poxviruses, or of strains thereof, the homologous B18R proteins for use in the present invention will share the functional characteristic of being able to bind to at least one type I IFN protein (e.g. IFN alpha, IFN beta, IFN kappa, IFN delta, IFN epsilon, IFN tau, IFN omega, IFN zeta). In some embodiments, the genetically modified cell comprises a nucleic acid molecule that encodes a functional fragmentof the B18R protein instead of the entire B18R protein. As used herein, the term “functional fragment” means a fragment of a protein that retains all or most of the biological function of the entire protein from which it is derived. In some embodiments, a functional fragment of B18R will exhibit at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the binding to one or more type I IFN protein exhibited by the wild-type B 18R protein from which the functional fragment is derived.

[0042] In some embodiments, the nucleic acid molecule encoding a B18R protein, or a functional fragment thereof, is operably linked to one or more regulatory elements. In some embodiments, the nucleic acid molecule is operably linked to a promoter, including but not limited to a CMV promoter or an EFla promoter. In some embodiments, the nucleic acid molecule encoding a B18R protein, or a functional fragment thereof, is operably linked to a KOZAK sequence.

[0043] In some embodiments, the sequence encoding the B18R protein, or functional fragment thereof, is encoded by a nucleic acid sequence identical or substantially similar to the sequence in the virus genome from which it is derived, such as at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the sequence in the virus genome. In some embodiments, the sequence encoding the B18R protein, or functional fragment thereof, is encoded by a nucleic acid sequence that is codon-optimized for expression in the desired host cell to be genetically modified. Codon optimization is a technique known in the art to improve protein expression in a host organism by replacing the codon coding for a particular amino acid with another codon which is better expressed in the host organism. This effect arises due to different organisms showing preferences for different codons. The process of altering the sequence of a nucleic acid to achieve better expression based on codon preference is called codon optimization. Many codon optimization tools are available, such as the IDT codon optimization tool and the VectorBuilder codon optimization tool.

[0044] E3L

[0045] In some embodiments, the genetically modified cell comprises a nucleic acid molecule encoding an E3L protein, or a functional fragment thereof, of a poxvirus. The E3L protein is encoded in the genome of numerous poxviruses. E3L is an intracellular protein that binds to double stranded RNA and blocks host defense factors from participating in the induction and action of IFN, namely blocking the activation of interferon regulatory factor 3 (IRF3), RNase L, and Protein Kinase R (PKR) (Li et al., Am. J. Biomed. Res. (2020), 10(3):343-350).Without being bound by theory, by binding double stranded RNA and blocking the activation of host defense factors, E3L inhibits cellular antiviral responses.

[0046] In some embodiments, the E3L protein is derived from a poxvirus, such as a virus of the genus Orthopoxvirus, including but not limited to variola virus, vaccinia virus, cowpox virus, buffalopox virus, horsepox virus, rabbitpox virus, taterapox virus, camelpox virus, ectrolmelia virus, alaskapox virus, skunkpox virus, raccoonpox virus, and monkeypox virus. An exemplary E3L amino acid sequence from vaccinia virus Western Reserve strain is set forth as SEQ ID NO: 3 and is publicly available as NCBI Reference Sequence YP 232941.1. An exemplary E3L encoding nucleic acid sequence from vaccinia virus Western Reserve strain is set forth as SEQ ID NO: 4 and is publicly available as NCBI Reference Sequence NC_006998.1. Another exemplary E3L amino acid sequence from vaccinia virus MV A strain is set forth as SEQ ID NO: 5 and is publicly available under GenBank accession number AAB96428.1. Another exemplary E3L encoding nucleic acid sequence from vaccinia virus MVA strain is set forth as SEQ ID NO: 6 and is publicly available under GenBank accession number U94848.1, (positions 42697-43269). Although there may be variations in the amino acid sequences of various poxviruses, or of strains thereof, the homologous E3L proteins for use in the present invention will share the functional characteristic of being able to bind double stranded RNA, inhibiting or blocking activation of IRF3, inhibiting or blocking activation of RNase L, and / or inhibiting or blocking activation of PKR. In some embodiments, the genetically modified cell comprises a nucleic acid molecule that encodes a functional fragment of the E3L protein instead of the entire E3L protein. As used herein, the term “functional fragment” means a fragment of a protein that retains all or most of the biological function of the entire protein from which it is derived. In some embodiments, a functional fragment of E3L will exhibit at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the binding to double stranded RNA exhibited by the wild-type E3L protein from which the functional fragment is derived. In some embodiments, a functional fragment of E3L will exhibit at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the blocking of activation of IRF3 exhibited by the wild-type E3L protein from which the functional fragment is derived. In some embodiments, a functional fragment of E3L will exhibit at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the blocking of activation of RNase L exhibited by the wild-type E3L protein from which the functional fragment is derived. In some embodiments, a functional fragment of E3L will exhibit at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the blocking of activation of PKR exhibited by the wild-type E3L protein from which the functional fragment is derived.

[0047] In some embodiments, the nucleic acid molecule encoding an E3L protein, or a functional fragment thereof, is operably linked to one or more regulatory elements. In some embodiments, the nucleic acid molecule is operably linked to a promoter, including but not limited to a CMV promoter or an EFla promoter. In some embodiments, the nucleic acid molecule encoding an E3L protein, or a functional fragment thereof, is operably linked to a KOZAK sequence.

[0048] In some embodiments, the sequence encoding the E3L protein, or functional fragment thereof, is encoded by a nucleic acid sequence identical or substantially similar to the sequence in the virus genome from which it is derived, such as at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the sequence in the virus genome. In some embodiments, the sequence encoding the E3L protein, or functional fragment thereof, is encoded by a nucleic acid sequence that is codon-optimized for expression in the desired host cell to be genetically modified.

[0049] Methods

[0050] In some embodiments, the present invention provides a method of producing a virus of interest, comprising culturing the virus of interest with the genetically modified cell as described herein.

[0051] Techniques for the culture of cells are well known in the art (ATCC Animal CellCulture Guide, 2014), including culture of cells for the purpose of virus production (ATCC Virology Guide, 2022). It is routine in the art to select appropriate cell culture conditions (media, supplements, temperature, CO2) for the selected host cell type. Examples of known recommended culture conditions for various cell lines include but are not limited to: CHO, Dulbecco’s Modified Eagle Medium (DMEM) + 10% FBS, 5% CO2, 37°C; HEK293. DMEM + 10% FBS, 5% CO2, 37°C; A549. Ham's F12K + 2mM Glutamine + 10% Fetal BovineSerum (FBS), 5% CO2, 37°C; BHK 21. Glasgow’s Minimal Essential medium (GMEM) + 2mM Glutamine + 5% Tryptose Phosphate Broth + 5-10% FBS, 5% CO2, 37°C; CV-1, DMEM + 2 mM L-Glutamine + 10% FBS, 5% CO2, 37°C; HeLa, Eagle’s Minimum Essential Medium (EMEM) + 2mM Glutamine + 1% Non Essential Amino Acids + 10% FBS, 5% CO2, 37°C; LLCMK2, EMEM + 2 mM L-Glutamine + 1% Non Essential Amino Acids + 5% FBS, 5% CO2, 37°C; McCoy. EMEM + 2mM Glutamine + 1% Non Essential Amino Acids + 10% FBS, 5% CO2, 37°C; MDCK. EMEM + 2mM Glutamine + 1% Non Essential Amino Acids + 10% FBS, 5% CO2, 37°C; NCI-H292. Roswell Park Memorial Institute Medium (RPMI) 1640 + 2mM Glutamine + 10% FBS, 5% CO2, 37°C; Vero76. DMEM + 2 mM L-Glutamine + 10% FBS, 5%CO2, 37°C; WI 38. EMEM + 2mM Glutamine + 1% Non Essential Amino Acids + 10% FBS, 5% CO2, 37°C; EB66 and AGE 1. 7.5% CO2, 37°C in either DMEM / F12 + 10% FBS, or specially formulated serum-free chemically defined medium.

[0052] In some embodiments, wherein when the genetically modified cell is cultured with a virus of interest, the culture produces a titer of the virus of interest that is at least 100 fold higher after 5 replication rounds compared to when the virus of interest is cultured with a parental cell lacking the B18R protein, or the functional fragment thereof and / or the E3L protein, or the functional fragment thereof. Methods of producing a virus of interest using the genetically modified cells of the present invention can achieve higher viral titers as compared to culture using other cell lines. Numerous methods of measuring viral titers are known in the art (ATCC Virology Guide, 2022). For example, viral titers may be measured by plaque assay in a unit called Plaque Forming Units (PFU). Briefly, the basis of this assay is to measure the ability of a single infectious virus to form a plaque on a cell culture monolayer. A plaque is developed as part of the viral infection cycle, where following viral replication the host cell dies and the necrotic debris forms a plaque. Host cells are grown to a monolayer and inoculated with medium containing the virus of interest, and the average number of plaques per culture is multiplied by the dilution of the viral media to obtain the PFU per volume of viral media. Viral titers may also be measured by tissue culture infectious dose (TCID). Briefly, this calculation is obtained through an endpoint dilution assay in cell culture. The most reproducible endpoint of the dilution assay is the dilution of the virus that will produce a pathological change in 50% of the cell cultures inoculated. This number is expressed as 50% of the infectious dose, or TCID50. Host cells are grown in culture to an optimal density and inoculated with dilution series of the viral media; measuring a titer as a dilution that results in infection of 50% of cells / cultures.Alternatively, viral titers may be measured by qPCR. Briefly, primers specific for a region of the virus genome are used to amplify viral DNA and measure the copy number of virus genomes in the virus media, measured as copy number per volume of media or mass (nanogram or picogram) per volume of media. As used herein, the number of “replication rounds” or “amplification rounds” in a viral cell culture refers to the number of times that the virus of interest is passaged. For example, in some embodiments, host cells are harvested 3 days after infection, the host cells are lysed to release virus, and then a portion of the virus (e.g. l / 20th to l / 200th of the harvested virus) is used for next round of infection on a fresh culture of host cells.

[0053] In some embodiments, wherein when the genetically modified cell is cultured with a virus of interest, the culture produces replicated virus of interest exhibiting higher expression of a virus-encoded gene in infected cells compared to replicated virus produced byculture with a parental cell lacking the B18R protein, or the functional fragment thereof and / or the E3L protein, or the functional fragment thereof. In some embodiments, the virus-encoded gene exhibits at least 2, 3, 4, 5, 10, or 15 fold higher expression after 2, 3, or 4 replication rounds. As used herein, “virus-encoded gene” refers to a nucleic acid sequence in the genome of the virus of interest that encodes for a polypeptide or RNA transcript. The virus-encoded gene will be expressed as a polypeptide and / or RNA transcript in the host cell. In some embodiments, the virus-encoded gene is an endogenous gene such as, for example but not limited to, a viral enzyme, glycoprotein, or antigen. In some embodiments, the virus-encoded gene is an exogenous gene. An exogenous gene can comprise, for example but not limited to, a transgene inserted into the virus genome by known techniques. In some embodiments, the virus-encoded gene encodes a reporter (for example Green Fluorescent Protein), an antigen (for example a viral, bacterial, or tumor antigen), a cytokine (for example an interleukin), an enzyme (for example a DNA- modifying enzyme), a receptor (for example a chemokine receptor), a ligand (for example a chemokine ligand), or an antibody (for example an anti-CTLA4, anti-PD-1, or anti-PD-Ll antibody). Many techniques are known for measuring the expression of a gene. For example, the expression of a gene may be measured at the transcript level by qPCR, which measures the amount of RNA transcripts. Alternatively, the expression of a gene may be measured at the translation level by ELISA, which measures the amount of translated polypeptide in solution. These techniques for measuring the expression of a gene are routine in the art and may easily be adapted for use in measuring gene expression of a virus-encoded gene in the present invention.

[0054] Any culturable virus can be grown as a virus of interest for use in the present invention. It is routine in the art to select an appropriate host cell that can be infected by the virus of interest, or to engineer a host cell to express the entry receptor for a virus of interest, to be genetically modified as described herein for use in the present invention. In some embodiments, the virus of interest is a DNA virus. In some embodiments, the virus of interest is an RNA virus. In some embodiments, the virus of interest is selected from influenza, measles, mumps, parainfluenza, poliovirus, respiratory syncytial virus (RSV), rotavirus, poxvirus, vaccinia virus, varicella zoster virus (VZV), metapneumovirus (MPV), reovirus, vesivular stomatitis virus (VSV), dengue, rabies, foot and mouth, rubella, adenovirus, cytomegalovirus (CMV), rhinovirus, enterovirus, echovirus, herpes simplex virus (HSV), human immunodeficiency virus (HIV), BK polyomavirus, coxsackie virus, coronavirus, West Nile virus. In some embodiments, the virus is a vaccinia virus, including but not limited to a LISTER, modified virus Ankara (MV A), Copenhagen, Wyeth, Western Reserve, or Tian Tan strain of vaccinia virus.

[0055] Particular embodiments of the disclosure include, without limitation, the following:1. A genetically modified cell, wherein the cell comprises at least one nucleic acid molecule encoding: (i) a B18R protein, or a functional fragment thereof, of a poxvirus; and / or (ii) an E3L protein, or a functional fragment thereof, of a poxvirus.2. The genetically modified cell of embodiment 1, wherein the at least one nucleic acid molecule encodes the B18R protein, or the functional fragment thereof.3. The genetically modified cell of embodiment 1 or 2, wherein the B18R protein, or the functional fragment thereof, is of a poxvirus from the genus Orthopoxvirus.4. The genetically modified cell of embodiment 3, wherein the B18R protein, or the functional fragment thereof, is of a vaccinia virus, cowpox virus, buffalopox virus, horsepox virus, or monkeypox virus.5. The genetically modified cell of embodiment 4, wherein the B18R protein, or the functional fragment thereof, is of a vaccinia virus.6. The genetically modified cell of embodiment 5, wherein the vaccinia virus is the Western Reserve strain of vaccinia virus.7. The genetically modified cell of embodiment 1 or 2, wherein the B18R protein, or the functional fragment thereof, comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 1.8. The genetically modified cell of any one of embodiments 1 to 7, wherein the B18R protein, or the functional fragment thereof, exhibits at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the binding to Type I Interferon exhibited by the wild-type B18R protein.9. The genetically modified cell of any one of embodiments 1 to 8, wherein the B18R protein is encoded by a nucleic acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 2, or is encoded by a codon-optimized sequence of SEQ ID NO: 2.10. The genetically modified cell of any one of embodiments 1 to 9, wherein the at least one nucleic acid molecule encoding the B18R protein, or the functional fragment thereof, is operably linked to a promoter.11. The genetically modified cell of embodiment 10, wherein the promoter is a CMV promoter or an EFla promoter.12. The genetically modified cell of any one of embodiments 1 to 11, wherein the at least one nucleic acid molecule encoding the B18R protein, or the functional fragment thereof, is operably linked to a KOZAK sequence.13. The genetically modified cell of any one of embodiments 1 to 12, wherein the at least one nucleic acid molecule encodes the E3L protein, or the functional fragment thereof.14. The genetically modified cell of any one of embodiments 1 to 13, wherein the E3L protein, or the functional fragment thereof, is of a poxvirus from the genus Orthopoxvirus.15. The genetically modified cell of embodiment 14, wherein the E3L protein, or the functional fragment thereof, is of a variola virus, vaccinia virus, cowpox virus, buffalopox virus, horsepox virus, rabbitpox virus, taterapox virus, camelpox virus, ectrolmelia virus, alaskapox virus, skunkpox virus, racconpox virus, or monkeypox virus.16. The genetically modified cell of embodiment 15, wherein the E3L protein, or the functional fragment thereof, is of a vaccinia virus.17. The genetically modified cell of embodiment 16, wherein the vaccinia virus is the Western Reserve strain of vaccinia virus.18. The genetically modified cell of any one of embodiments 1 to 17, wherein the E3L protein, or the functional fragment thereof, comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 3.19. The genetically modified cell of any one of embodiments 1 to 18, wherein the E3L protein, or the functional fragment thereof, exhibits: (i) at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the binding to double stranded RNA exhibited by the wild-type E3L protein; (ii) at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the blocking of activation of Interferon Regulatory Factor 3 (IRF3) exhibited by the wild-type E3L protein; and / or (iii) at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the blocking of activation of RNase L exhibited by the wild-type E3L protein; and / or (iv) at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the blocking of activation of Protein Kinase R (PKR) exhibited by the wild-type E3L protein.20. The genetically modified cell of any one of embodiments 1 to 19, wherein the E3L protein is encoded by a nucleic acid sequence with at least 70%, 75%, 80%, 85%, 90%,95%, 99%, or 100% sequence identity to SEQ ID NO: 4, or is encoded by a codon-optimized sequence of SEQ ID NO: 4.21. The genetically modified cell of any one of embodiments 1 to 20, wherein the at least one nucleic acid molecule encoding the E3L protein, or the functional fragment thereof, is operably linked to a promoter.22. The genetically modified cell of embodiment 21, wherein the promoter is a CMV promoter or an EFla promoter.23. The genetically modified cell of any one of embodiments 1 to 22, wherein the at least one nucleic acid molecule encoding the E3L protein, or the functional fragment thereof, is operably linked to a KOZAK sequence.24. The genetically modified cell of any one of embodiments 1 to 23, which is an adherent cell.25. The genetically modified cell of any one of embodiments 1 to 23, which is anon- adherent cell.26. The genetically modified cell of any one of embodiments 1 to 25, which is a mammalian cell.27. The genetically modified cell of any one of embodiments 1 to 25, wherein the cell is a CHO cell, HEK239 cell, A549 cell, BHK 21 cell, CV 1 cell, HeLa cell, LLCMK2 cell, McCoy cell, MDCK cell, MRC-5 cell, NCI-H292 cell, Vero cell, Vero76 cell, or WI 38 cell.28. The genetically modified cell of any one of embodiments 1 to 27, wherein when the genetically modified cell is cultured with a virus of interest, the culture produces a titer of the virus of interest that is at least 100 fold higher after 5 replication rounds compared to when the virus of interest is cultured with a parental cell lacking the B18R protein, or the functional fragment thereof and / or the E3L protein, or the functional fragment thereof.29. The genetically modified cell of any one of embodiments 1 to 27, wherein when the genetically modified cell is cultured with a virus of interest, the culture produces replicated virus of interest exhibiting higher expression of a virus-encoded gene in infected cells compared to replicated virus produced by culture with a parental cell lacking the B18R protein, or the functional fragment thereof and / or the E3L protein, or the functional fragment thereof.30. The genetically modified cell of embodiment 29, wherein the virus-encoded gene exhibits at least 2, 3, 4, or 5 fold higher expression after 2 replication rounds.31. The genetically modified cell of embodiment 29, wherein the virus-encoded gene exhibits at least 2, 3, 4, 5, 10, or 15 fold higher expression after 3 replication rounds.32. The genetically modified cell of embodiment 29, wherein the virus-encoded gene exhibits at least 2, 3, 4, 5, 10, or 15 fold higher expression after 4 replication rounds.33. The genetically modified cell of any one of embodiments 29-32, wherein the virus-encoded gene comprises an endogenous gene.34. The genetically modified cell of any one of embodiments 29-33, wherein the virus-encoded gene comprises an exogenous gene.35. The genetically modified cell of embodiment 34, wherein the exogenous gene encodes a reporter, an antigen, a cytokine, an enzyme, a receptor, a ligand, or an antibody.36. A method for producing a virus of interest, comprising culturing the virus of interest with the genetically modified cell of any one of embodiments 1 to 35.37. The method of embodiment 36, wherein the virus of interest is a DNA virus.38. The method of embodiment 36, wherein the virus of interest is an RNA virus.39. The method of embodiment 36, wherein the virus of interest is a virus selected from influenza, measles, mumps, parainfluenza, poliovirus, respiratory syncytial virus (RSV), rotavirus, poxvirus, vaccinia virus, varicella zoster virus (VZV), metapneumovirus (MPV), reovirus, vesivular stomatitis virus (VSV), dengue, rabies, foot and mouth, rubella, adenovirus, cytomegalovirus (CMV), rhinovirus, enterovirus, echovirus, herpes simplex virus (HSV), human immunodeficiency virus (HIV), BK polyomavirus, coxsackie virus, coronavirus, and West Nile virus.40. The method of embodiment 36, wherein the virus of interest is a vaccinia virus.41. The method of embodiment 40, wherein the virus of interest is a LISTER, modified virus Ankara (MV A), Copenhagen, Wyeth, Western Reserve, or Tian Tan strain of vaccinia virus.

[0056] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude thepresence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps.

[0057] As used herein, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. In embodiments comprising an “additional” or “second” component, the second component as used herein is different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

[0058] All publications and patents cited herein are incorporated by reference in their entirety as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. In the case of any conflict between a definition of a term in the present disclosure and a definition in a cited publication or patent, the definition provided in the present disclosure is to be used in describing the present invention.

[0059] The present invention will now be described by way of non-limiting examples having regard to the appended drawings.

[0060] EXAMPLE 1

[0061] A virus of interest was grown in cell culture to compare the viral titers produced using genetically modified cells of the present invention that contain a gene for E3L to the parental cells that do not contain a gene encoding E3L. Unmodified control AGE1.CR cells or AGE1.CR cells modified to encode the E3L protein from vaccinia MVA strain were grown in cell culture and inoculated with an MVA strain of vaccinia virus.

[0062] Equal amount of MVA vaccinia virus was inoculated at the beginning (Passage 0) in control (Parental) or E3L-expressing (Engineered) cells. Virus was harvested at day 3 post infection. l / 20thof the harvested virus was used for the next passage.

[0063] As shown in Figure 1, genetically modified cells of the present invention comprising a gene encoding E3L (Engineered) maintained high titers of MVA vaccinia virusafter repeated passages. By contrast, cultures of the control cells that do not have an E3L gene (Parental) exhibited a gradual decrease in viral titers. These results show that the present invention achieves enhanced production of virus in vitro.

[0064] EXAMPLE 2

[0065] A virus of interest encoding human IL-12 was grown in adherent cell culture (10% FBS, DMEM / F12 regular medium) to compare the viral titers produced using genetically modified cells of the present invention to the unmodified parental cells. Unmodified control AGE1.CR cells or AGE1.CR cells modified to encode the B18R and / or E3L protein from vaccinia Western Reserve and MVA strains, respectively, were grown in cell culture and inoculated with an MVA strain of vaccinia virus. The MVA strain of vaccinia virus is modified to include a transgene for human IL- 12.

[0066] Equal amount of MVA vaccinia virus was inoculated at the beginning (Passage 0) in control (Parental) or genetically modified cells of the present invention (E3, Bl 8, or both Bl 8- E3 with Kozak sequence (+) or without Kozak sequence (-). Viruses were harvested at day 3 post infection. 1 / 50thof the harvested virus was used for the next passage.

[0067] Figure 2 shows a comparison between cells with genes for both Bl 8R and E3L, B18R only, E3L only, and unmodified parental cells. As well, cells encoding both B18R and E3L, linked by a cleavable 2A linker, were compared with the genes being operably linked to a KOZAK sequence (+) or in the absence of a KOZAK sequence (-). As shown in Figure 2, titers of MVA vaccinia virus remain low in culture with the control cells (Parental) even after 4 amplification rounds. By contrast, genetically modified cells expressing B18R alone or E3L alone achieved significantly higher titers of virus. Similar titers were achieved with cells expressing both B18R and E3L, which was further enhanced when the genes were operably linked to a KOZAK sequence. These results show that the present invention achieves enhanced production of virus in vitro.

[0068] Furthermore, the amount of IL-12 polypeptide expressed from the viral-encoded IL-12 transgene was measured in each culture by ELISA. Supernatants from producing cells were collected at the time of viral harvest. Human IL-12 was quantified by ELISA using R&D Systems Human IL- 12 p70 DuoSet ELISA kit (Cat# DY1270). As shown in Figure 3, in the culture with the control cells (Parental) IL-12 expression was high after the amplification round (Round 1), but the levels of IL-12 dropped rapidly afterwards. By contrast, in the cultures with the genetically modified cells of the present invention, IL-12 levels remained high or increasedup to Round 4. These results show that the present invention maintains normal levels of virus- encoded genes.

[0069] Without being bound by theory, it is believed that the IL-12 encoded by the virus may stimulate anti-viral mechanisms in the host cells. This may create a selective pressure for mutants of the cultured virus that express lower levels of IL-12. By contrast, use of the genetically modified cells of the present invention may remove this selection pressure, allowing continued high expression of the virus-encoded transgene.

[0070] EXAMPLE 3

[0071] A virus of interest encoding human IL- 12 was grown in suspension cell culture (serum-free chemically defined medium) in a shaker to compare the viral titers produced using genetically modified cells of the present invention to the unmodified parental cells. Unmodified control AGE1.CR cells or AGE1.CR cells modified to encode the B18R and / or E3L protein from vaccinia Western Reserve and MVA strains, respectively, were grown in suspension cell culture and inoculated with an MVA strain of vaccinia virus. The MVA strain of vaccinia virus is modified to include a transgene for human IL- 12.

[0072] Equal amount of MVA vaccinia virus was inoculated at the beginning (Passage 0) in control (Parental) or genetically modified cells of the present invention (E3, Bl 8, or both B18and E3 with Kozak sequence wherein the B18R gene is placed upstream of the E3L gene (B18-E3(+)) or wherein the E3L gene is placed upstream of the B18R gene (E3-B18).

[0073] Figure 4 shows a comparison between cells with genes for both Bl 8R and E3L, B18R only, E3L only, and unmodified parental cells. The amount of IL-12 polypeptide expressed from the viral-encoded IL-12 transgene was measured in each culture by ELISA essentially as described in Example 2.

[0074] As shown in Figure 4, in the culture with the control cells (Parental) IL- 12 expression was insignificant. By contrast, in the cultures with the genetically modified cells of the present invention, IL-12 levels were much higher. These results show that the present invention improves expression of virus-encoded genes.

Claims

CLAIMS:

1. A genetically modified cell, wherein the cell comprises at least one nucleic acid molecule encoding:(i) a B18R protein, or a functional fragment thereof, of a poxvirus; and / or(ii) an E3L protein, or a functional fragment thereof, of a poxvirus.

2. The genetically modified cell of claim 1, wherein the at least one nucleic acid molecule encodes the B18R protein, or the functional fragment thereof.

3. The genetically modified cell of claim 1 or 2, wherein the B18R protein, or the functional fragment thereof, is of a poxvirus from the genus Orthopoxvirus .

4. The genetically modified cell of claim 3, wherein the B18R protein, or the functional fragment thereof, is of a vaccinia virus.

5. The genetically modified cell of claim 4, wherein the vaccinia virus is the Western Reserve strain of vaccinia virus.

6. The genetically modified cell of claim 1 or 2, wherein the B18R protein, or the functional fragment thereof, comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 1.

7. The genetically modified cell of any one of claims 1 to 6, wherein the B18R protein, or the functional fragment thereof, exhibits at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the binding to Type I Interferon exhibited by the wild-type B18R protein.

8. The genetically modified cell of any one of claims 1 to 7, wherein the at least one nucleic acid molecule encoding the B18R protein, or the functional fragment thereof, is operably linked to a KOZAK sequence.

9. The genetically modified cell of any one of claims 1 to 8, wherein the at least one nucleic acid molecule encodes the E3L protein, or the functional fragment thereof.

10. The genetically modified cell of any one of claims 1 to 9, wherein the E3L protein, or the functional fragment thereof, is of a poxvirus from the genus Orthopoxvirus.

11. The genetically modified cell of claim 10, wherein the E3L protein, or the functional fragment thereof, is of a vaccinia virus.

12. The genetically modified cell of claim 11, wherein the vaccinia virus is the Western Reserve strain of vaccinia virus.

13. The genetically modified cell of any one of claims 1 to 12, wherein the E3L protein, or the functional fragment thereof, comprises an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 3.

14. The genetically modified cell of any one of claims 1 to 13, wherein the E3L protein, or the functional fragment thereof, exhibits:(i) at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the binding to double stranded RNA exhibited by the wild-type E3L protein;(ii) at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the blocking of activation of Interferon Regulatory Factor 3 (IRF3) exhibited by the wild-type E3L protein; and / or(iii) at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the blocking of activation of RNase L exhibited by the wild-type E3L protein; and / or(iv) at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the blocking of activation of Protein Kinase R (PKR) exhibited by the wild-type E3L protein.

15. The genetically modified cell of any one of claims 1 to 14, wherein the at least one nucleic acid molecule encoding the E3L protein, or the functional fragment thereof, is operably linked to a KOZAK sequence.

16. The genetically modified cell of any one of claims 1 to 15, which is a mammalian cell.

17. The genetically modified cell of any one of claims 1 to 15, wherein the cell is a CHO cell, HEK239 cell, A549 cell, BHK 21 cell, CV 1 cell, HeLa cell, LLCMK2 cell, McCoy cell, MDCK cell, MRC-5 cell, NCI-H292 cell, Vero cell, Vero76 cell, WI 38 cell, RK 13 cell, EB66 cell or AGE1.CR cell.

18. A method for producing a virus of interest, comprising culturing the virus of interest with the genetically modified cell of any one of claims 1 to 17.

19. The method of claim 18, wherein the virus of interest is a vaccinia virus.

20. The method of claim 19, wherein the virus of interest is a LISTER, modified virus Ankara (MV A), Copenhagen, Wyeth, Western Reserve, or Tian Tan strain of vaccinia virus.

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