Modified Vero Cells and Methods of Using Same for Virus Production - Patent application

Abstract

Disclosed herein are engineered cell lines that contain a modification in the ISG15 gene, where the modification in the ISG15 gene increases total viral particle production and / or infectious viral particle production compared to a control cell line that is identical to the engineered cell line except for the modification in the ISG15 gene. Also disclosed herein are methods for increasing viral particle production and methods for identifying genes to delete in a cell or cell line.

Description

[Technical field]

[0001] Disclosed herein are engineered cell lines for producing viral particles and methods of making the engineered cell lines. Also disclosed herein are methods of increasing viral particle production in the cell lines and methods of identifying genes to target for deletion in cell lines to enhance viral particle production. [Background technology]

[0002] Viruses (e.g., live attenuated and inactivated viruses) used in vaccines are often produced using host cells derived from cultured cell lines such as plants, yeast, or animal cell lines (e.g., insect or mammalian cell lines). Examples of mammalian cells include, but are not limited to, COS-7 cells, human embryonic kidney (HEK) cells, such as HEK293 cells; baby hamster kidney (BHK) cells; Chinese hamster ovary (CHO) cells; mouse Sertoli cells; African green monkey kidney (Vero) cells; human cervical carcinoma cells (e.g., HeLa); canine kidney cells (e.g., MDCK), and the like.

[0003] A variety of these cell culture-based platforms have been used to produce viral vaccines as an alternative to egg-based vaccine production methods. Thus, the ability to increase the rate of viral production may enhance the potential of cell lines for more widespread use.

[0004] Vero cells are a female African green monkey kidney-derived cell line that have been widely used for over 40 years in the production of viral vaccines, including against dengue fever (using vesicular stomatitis virus (VSV) recombinant virus (rVSV)), influenza, Japanese encephalitis, polio, rabies, rotavirus, smallpox, and Ebola. Additionally, Vero cells have been identified as a cell line that is highly susceptible to coronaviruses (e.g., MERS-CoV, SARS-CoV, and SARS-CoV-2) (Liu et al., A recombinant VSV-vectored MERS-CoV vaccine induces neutralizing antibody and T cell responses in rhesus monkeys after single dose immunization, Antiviral Res. 2018, 150:30-38; Hoffmann et al., SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor, Cell 2020, 181(2):271-280). Therefore, successful engineering of cell lines such as the Vero cell line to increase viral production of viruses could have a major impact on global health.

[0005] Recent advances in gene editing allow for the use of available genomic information to edit the genomes of cell lines, particularly those used for viral infection studies and those used for vaccine manufacturing for vaccine bioprocess enhancement purposes. Previously, attempts have been made to increase the rate of virus production in Vero cells by gene editing using genome-wide RNA interference screen datasets to select gene targets, but repeated experiments have not shown a significant increase. Van der Sanden, SM et al., Engineering Enhanced Vaccine Cell Lines To Eradicate Vaccine-Preventable Diseases: the Polio End Game, J. Virol. 2016, 90(4): 1694-704; Hoeksema F. et al., Enhancing viral vaccine production using engineered knockout vero cell lines-A second look, Vaccine 2018, 36(16): 2093-2103. Among the possible reasons cited by the authors to explain such results was the use of a genome other than the Vero cell genome and the fact that phenotypes induced by transcriptional suppression (RNAi-based knockdown) and gene deletion (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) knockout) may differ, such that the former but not the latter can increase virus production.

[0006] Thus, disclosed herein are methods for identifying valuable target genes for editing that do not rely on genome-wide screens, and engineered cell lines in which the desired gene target or targets are modified to enhance viral particle production. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure provides engineered cell lines that contain modifications in one or more genes that result in increased total viral particle production and / or infectious viral particle production compared to a control cell line that is identical to the engineered cell line except for the modification in the one or more genes.

[0008] In certain embodiments, disclosed herein are engineered cell lines that contain a modification in the ISG15 gene, which increases total and / or infectious viral particle production as compared to a control cell line that is identical to the engineered cell line except for the modification in the ISG15 gene. In certain embodiments, the engineered cell line contains a modification in one or more genes selected from APOA1, CCL2, CCL5, CYP19A1, CXCL8, ELF3, FOS, HERC3, HERC5, IFIT1, IFIT2, IFIT3, IRF7, ISG15, KRT15, ​​KRT19, MX1, NGFR, PTGS2, PTPN6, RET, ROS1, SFRP1, SOX2, SPP1, TNF, TNFRSF4, TRAF1, and VAV3.

[0009] In one embodiment of the disclosure, the increase in viral particle production is at least about 20% compared to the control cell line, e.g., at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%. In certain embodiments, the ratio of infectious viral particle production to total viral particle production is increased by at least about 3% compared to the control cell line, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60%.

[0010] In certain embodiments, a modification in one or more genes results in the deletion of the one or more genes from the engineered cell line, and in certain embodiments, the engineered cell line has reduced expression of the one or more genes compared to a control cell line. For example, in certain embodiments, a modification in the ISG15 gene results in the deletion of the ISG15 gene from the engineered cell line, and in certain embodiments, the engineered cell line has reduced expression of the ISG15 gene compared to a control cell line.

[0011] In certain embodiments, the engineered cell line is derived from monkey cells or mouse cells, hi certain embodiments, the engineered cell line is Vero cells.

[0012] In certain embodiments, one or more genes are modified or deleted from the engineered cell line using a CRISPR-associated (Cas) system. In one embodiment of the present disclosure, the ISG15 gene is modified or deleted from the engineered cell line using a CRISPR-Cas system.

[0013] In certain embodiments of any aspect of the present disclosure, the virus is selected from influenza virus, dengue virus, yellow fever virus, respiratory syncytial virus (RSV), herpes simplex virus, human immunodeficiency virus (HIV), hepatitis virus, coronavirus, or a virus from the Rhabdoviridae family, such as rabies virus or vesicular stomatitis virus (VSV).In certain aspects, the virus is an influenza virus, such as influenza A virus or influenza B virus.

[0014] Also provided herein is a method of increasing viral particle production comprising infecting an engineered cell line with a virus, incubating the engineered cell line under conditions suitable for production of virus by the engineered cell line, and recovering virus produced by the engineered cell line, wherein the engineered cell line comprises a modification in one or more genes, such as the ISG15 gene, wherein the modification in the one or more genes, such as the ISG15 gene, increases total viral particle production and / or infectious viral particle production compared to a control cell line identical to the engineered cell line except for the modification in the one or more genes, such as the ISG15 gene.

[0015] In certain embodiments of the methods disclosed herein, the engineered cell line increases viral particle production by at least about 20% compared to a control cell line, e.g., at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%. In certain embodiments, modifications in one or more genes, such as the ISG15 gene, result in the deletion of the one or more genes from the engineered cell line, and in certain embodiments, the engineered cell line has reduced expression of one or more genes, such as the ISG15 gene, from the engineered cell line. In certain embodiments of the methods disclosed herein, the engineered cell line is a Vero cell line, and in certain embodiments, the virus is selected from influenza virus, dengue virus, yellow fever virus, RSV, herpes simplex virus, HIV, hepatitis virus, coronavirus, or a virus from the Rhabdoviridae family, e.g., rabies virus or VSV.

[0016] Also disclosed herein is a method for identifying genes to be deleted in a cell or cell line, comprising: (1) infecting the cell or cell line with a virus; (2) detecting the expression level of a plurality of genes in the infected cell or cell line and comparing the expression level to the expression level of a plurality of genes in a control cell or cell line that is not infected with the virus; (3) identifying gene targets that are differentially expressed in the infected cell or cell line; and (4) analyzing the differentially expressed gene targets to identify one or more gene targets that are involved in a plurality of protein-protein networks, wherein the plurality of protein-protein networks are involved in a plurality of protein-protein networks. The method includes (5) analyzing the protein-protein network, which comprises at least two of the following: defense response, response to virus, viral genome replication, response to cytokines, response to type I interferon, regulation of viral genome replication, defense response to virus, cell death, viral life cycle, negative regulation of viral genome replication, and cellular response to cytokine stimulation; and (6) selecting at least one differentially expressed gene target to be deleted in the cell or cell line, wherein deletion of the at least one differentially expressed gene target increases viral viral particle production. In certain embodiments, the method further includes (6) using gene editing technology such as CRISPR-Cas system to delete the at least one differentially expressed gene target from the cell or cell line.

[0017] In certain embodiments of the method of identifying genes to be deleted in cells or cell lines, the cells or cell lines are Vero cells, Madin-Darby Canine (MDCK) cells, or human embryonic kidney (HEK) cells. In certain embodiments, the cells or cell lines are Vero cells. In certain embodiments of the method, the virus is selected from influenza virus, dengue virus, yellow fever virus, RSV, herpes simplex virus, HIV, hepatitis virus, coronavirus, or a virus from the Rhabdoviridae family, such as rabies virus or VSV. In certain embodiments, the virus is an influenza virus, such as influenza A virus or influenza B virus. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a graph showing normalized expression scores from gene set enrichment analysis (GSEA) for influenza-infected Vero cells at 24 hours post-infection, as described in Example 2. [Diagram 2] 1 is a graph showing normalized expression scores from GSEA for rVSV-GFP infected Vero cells at 6 hours post-infection, as described in Example 2. [Diagram 3] 1 is a sequence alignment comparing human ISG15 (hISG15) (SEQ ID NO: 15), mouse ISG15 (mISG15) (SEQ ID NO: 16), Vero cell ISG15 (vISG15) (SEQ ID NO: 14), and canine ISG15 (caISG15) (SEQ ID NO: 17), as described in Example 3. Amino acid residues known to interact with influenza NS1 protein, coronavirus PLP, and nairovirus OTU are shown below the sequence alignment. [Figure 4] FIG. 1 is a schematic diagram showing the positions of PCR primers sgRNA A and sgRNA B described in Example 4 for the detection of non-deletion and deletion bands, respectively. [Diagram 5]13 is an image of a PCR screen showing non-deleted and deleted bands in parental Vero cells (left) and ISG15− / − Vero cells (right), as described in Example 4. [Figure 6] FIG. 13 is a Western blot of parental and ISG15− / − Vero cells showing detection of ISG− / − Vero cells as indicated by the absence of a band at approximately 17 kDa and described in Example 4. [Figure 7] Graphs showing viral genomes / mL and TCID50 / mL for influenza virus A (IVA)-infected parental Vero cells, ISG15- / - Vero cells infected with IVA, rVSV-GFP-infected parental Vero cells, and rVSV-GFP-infected ISG15- / - Vero cells, as described in Example 5. [Figure 8] FIG. 13 is a graph showing growth rate analysis for ISG15− / − and control cell lines in serum-free medium as described in Example 6, where dt is doubling time and the line is plotted as a comparison of the fit to a non-linear fit. [Figure 9A] FIG. 13 is a graph showing the number of viable cells over time post-infection for ISG15− / − (p5+4), ISG15− / − (p17), and control cell lines in serum-free medium in a bioreactor, as described in Example 7. [Figure 9B] FIG. 13 is a graph showing cell viability over time post-infection for ISG15− / − (p5+4), ISG15− / − (p17), and control cell lines in serum-free medium in a bioreactor, as described in Example 7. [Figure 10A] Plots showing log titer (PFU / mL) over time post-infection (left) and titer (PFU / mL) over time post-infection (right) for ISG15- / -(p5+4), ISG15- / -(p17), and control cell lines in serum-free medium in bioreactors, as described in Example 8. [Figure 10B]FIG. 13 is a plot showing log titers per 106 cells at 3 days post-infection for ISG15- / -(p5+4), ISG15- / -(p17), and control cell lines in serum-free medium in bioreactors, as described in Example 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Disclosed herein is the use of cell lines (e.g., Vero cell lines) to identify various factors acting during viral infection and replication in host cells to enhance the production of viral particles (e.g., viral particles used in the manufacture of vaccine compositions). In certain embodiments, candidate antiviral genes for gene editing can be selected using any combination of methods including, for example, differential gene expression analysis, gene set enrichment analysis (GSEA), and / or protein-protein interaction analysis (e.g., network topology analysis). After selecting target genes for genome editing, sequences can be analyzed for gene editing. For example, sequences can be isolated for CRISPR guide RNA (gRNA) design and off-target prediction determination to further reduce potential gRNA candidates to those with the highest knockout efficiency scores. Then, engineered cell lines can be designed in which the target genes are modified to enhance the production of viral particles in the infected engineered cell lines.

[0020] definition In order that this disclosure may be more readily understood, certain terms are first defined below. Throughout the specification, additional definitions of the following terms and other terms may be set forth. In the event that a definition of a term set forth below conflicts with a definition in an application or patent incorporated by reference, the meaning of the term shall be understood using the definition set forth in this application.

[0021] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. So, for example, reference to a "method" includes one or more methods and / or steps of the type described herein and / or that will be apparent to those skilled in the art upon reading this disclosure etc.

[0022] The use of ordinal terms such as "first," "second," "third," etc. in the claims to modify claim elements does not, in and of itself, imply any importance, priority, or order of one claim element relative to another claim element, or the temporal order in which operations of a method are performed, but is merely used as a label (but for the purposes of the use of ordinal terms) to distinguish one claim element having a certain name from another element having the same name, in order to distinguish between the claim elements.

[0023] Approximately: As used herein, the term "approximately" or "about" when applied to one or more subject values ​​refers to a value similar to a stated reference value. In some embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the stated reference value (except where such number exceeds 100% of possible values), unless otherwise specified or clear from the context.

[0024] CRISPR-Cas system: As used herein, "CRISPR-associated (Cas) system" or "CRISPR-Cas system" refers to transcripts and other elements (e.g., sequences encoding Cas genes) involved in directing the expression or activity of Cas genes, such as Cas9. Generally, CRISPR systems include elements (e.g., protospacers) that promote the formation of a CRISPR complex at the site of a target sequence. A guide sequence is designed to have complementarity with the target sequence, where hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. A target sequence can include any polynucleotide, such as a DNA or RNA polynucleotide.

[0025] sgRNA: As used herein, the term "guide RNA", "single guide RNA", or "sgRNA" refers to any polynucleotide sequence (e.g., DNA or RNA polynucleotides) that has sufficient complementarity with a target sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity is at least about 50%, e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or about 100%, when optimally aligned using a suitable alignment algorithm. Optimal alignment can be determined by use of any suitable algorithm for aligning sequences, including, for example, the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, Burrows Wheeler Aligner, ClustalW, Clustal X, BLAST, Novoalign, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0026] Gene: As used herein, the term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises a coding sequence for the production of a polypeptide or precursor (e.g., protein). The polypeptide can be encoded by a full-length coding sequence or any portion of the coding sequence, so long as the desired activity or functional property of the full-length or fragment is retained. The term "gene" encompasses both cDNA and genomic forms of a gene.

[0027] Gene expression: The term "gene expression" refers to the expression level of a gene in a sample. As understood in the art, the expression level of a gene can be analyzed by measuring the expression of a nucleic acid (e.g., mRNA or cDNA) or a polypeptide encoded by the nucleic acid. Gene expression can be upregulated, indicating that the expression level of a gene in a sample is increased when compared to a normalized gene expression, or downregulated, indicating that the expression level of a gene in a sample is decreased when compared to a normalized gene expression.

[0028] Normalized Gene Expression: The term "normalized gene expression" refers to the average gene expression level of a given gene in a disease- or virus-free sample or pool of samples.

[0029] Pharmaceutically acceptable carrier: The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art. In certain embodiments, the pharmaceutically acceptable carrier or excipient is not naturally occurring.

[0030] Polypeptide: The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acids.

[0031] Primer: The term "primer" refers to a polynucleotide capable of binding to a region of a target nucleic acid or its complement and facilitating nucleic acid amplification of the target nucleic acid. Generally, a primer has a free 3' end that can be extended by a nucleic acid polymerase. A primer also generally comprises a base sequence that can hybridize with at least one strand of a target nucleic acid via direct complementary base interactions or with a strand complementary to the target sequence. A primer may comprise a target-specific sequence and, optionally, other sequences that are non-complementary to the target sequence. The non-complementary sequence may comprise, for example, a promoter sequence or a restriction endonuclease recognition site.

[0032] Subject: The term "subject" refers to any animal (e.g., a mammal, e.g., a human, a non-human primate, a rodent, etc.) that will be the recipient of a particular treatment. In some embodiments, "subject" refers to a human. In some embodiments, "subject" refers to a non-human animal.

[0033] Vaccine: As used herein, the term "vaccine" refers to a composition administered to induce or stimulate an immune response against a particular disease, such as influenza infection. The term vaccine includes prophylactic and therapeutic vaccines. Prophylactic vaccines are designed to prevent a subject from acquiring a particular disease, such as influenza infection, or to have only mild disease. Therapeutic vaccines aim to improve the immune response against a particular disease or to alleviate the symptoms of the disease.

[0034] Viral infection: As used herein, the term "viral infection" describes a disease state in which a virus invades a healthy cell, grows or replicates using the cell's replication machinery, and eventually lyses the cell, resulting in cell death, release of viral particles, and infection of other cells with the newly produced progeny virus. Latent infection with certain viruses (e.g., HIV-1) can also occur as a result of viral infection.

[0035] Viral particle: As used herein, a "viral particle" is a virion that replicates inside a living cell. A viral particle comprises genetic material (i.e., DNA or RNA), a protein coat or capsid that surrounds the genetic material, and an optional lipid envelope.

[0036] Infectious viral particles: As used herein, an "infectious viral particle" is a viral particle that has a cytopathic effect on a host cell. Infectious viral particles can be counted, for example, at a 50% Median Tissue Culture Infectious Dose (TCID ) as described herein. 50 ) assay and / or plaque assay.

[0037] Disclosed herein are various genes.Table 1 below lists the gene name, species, NCBI reference sequence number, and description of the genes discussed herein.This list does not constitute a complete list of genes that may be considered and are considered within the scope of this disclosure.

[0038] [Table 1]

[0039] [Table 2]

[0040] [Table 3]

[0041] Presented herein is an analysis of the virus-host interactions that play out during viral infection, as host cells attempt to minimize the impact of viral infection and viruses attempt to evade host cell immune responses.By using a multidisciplinary approach that combines functional genomics and cell biology, disclosed herein is a novel strategy of more efficient targeted gene editing to enhance viral particle production in host cells, thereby opening up new possibilities for pandemic-ready host cells (e.g., Vero cells) that are high throughout the vaccine production platform.

[0042] A newly assembled and annotated Vero genome has recently been published. Sene, M.-A. et al., Haplotype-resolved de novo assembly of the Vero cell line genome, NPJ Vaccine 2021, 6(1):106. Based on this information and using functional genomics, it is possible to better control and monitor the effects of gene editing in Vero cells, which allows for a deeper understanding of potential gene targets before selection and a better understanding of the mechanisms at work during infection. Deletion of entire genomic regions, such as coding regions (coding DNA sequences, or CDS regions), may increase the chances of obtaining biallelic deletions compared to gene knockdown using single guide RNA-based cleavage. This increases the chances that the deletion will lead to the desired loss of function of the targeted gene product and also simplifies validation of gene knockouts, ensuring rapid and high-throughput gene editing protocols.

[0043] As disclosed herein, deletion of ISG15 in Vero cells resulted in an overall increase in total virus particle production, and an increase in infectious virus particle production, and an increase in the ratio of infectious to total virus particles.

[0044] ISG15 is a 17 kDa antiviral protein (15 kDa after maturation by N-terminal Met excision and removal of C-terminal peptide) that protects host cells from viral infection through inhibition of viral replication in a conjugation-dependent manner. Pattyn E. et al., HyperISGylation of Old World monkey ISG15 in human cells, PLoS One 2008;3(6):e2427. ISG15 has been associated with antiviral responses to various viruses, including SARS, influenza, HIV, and hepatitis, but functional diversity of ISG15 exists across species, as ISG15-deficient human patients do not show increased susceptibility to viral infection compared to ISG15-deficient mice, which are susceptible to viral infection. Perng, YC et al., ISG15 in antiviral immunity and beyond, Nat Rev Microbiol. 2018,16(7):423-439. Therefore, it is desirable to consider diversity between cell types to ensure that gene editing results in the desired phenotypic modification with respect to viral infection. Thus, protein sequences can be compared between species of interest, including those derived from cell types used in vaccine production such as Vero, HEK293, and MDCK.

[0045] Thus, disclosed herein are engineered cell lines that contain modifications in one or more genes (e.g., modifications in the ISG15 gene of the Vero cell line) that increase total and / or infectious viral particle production compared to a control cell line. Also disclosed are methods of making the engineered cell lines and methods of using the engineered cell lines to increase viral particle production. Additionally, disclosed herein are methods of identifying target genes to modify (e.g., delete) in a cell or cell line.

[0046] Engineered cell lines In certain embodiments, disclosed herein are engineered cell lines that contain a modification in one or more genes that increases total and / or infectious viral particle production compared to a control cell line that is identical to the engineered cell line except for the modification. As used herein, a control cell line that is identical to the engineered cell line except for the modification can be a parent cell line (i.e., derived from the same cell culture) of the engineered cell line. Thus, in certain embodiments, the control cell line is a parent cell line. In certain embodiments, the engineered cell line is a Vero cell line, and in certain embodiments, the modification is a modification of the ISG15 gene, such as a deletion of the ISG15 gene.

[0047] In some embodiments, the engineered cell line comprises a modification of one or more of the following genes: APOA1, CCL2, CCL5, CYP19A1, CXCL8, ELF3, FOS, HERC3, HERC5, IFIT1, IFIT2, IFIT3, IRF7, ISG15, KRT15, ​​KRT19, MX1, NGFR, PTGS2, PTPN6, RET, ROS1, SFRP1, SOX2, SPP1, TNF, TNFRSF4, TRAF1, and VAV3, compared to a control cell. In some embodiments, the engineered cell line comprises a modification of one or more of the following genes: CCL2, CCL5, CXCL8, HERC5, IFIT1, IFIT2, IFIT3, and ISG15, compared to a control cell line. In certain embodiments, the engineered cell line comprises a modification of the ISG15 gene.

[0048] As used herein, modification refers to any unnatural rearrangement of the genome of a cell line that affects the expression of a gene or gene in the cell line.For example, modification to a gene can increase the expression of this gene, or in certain embodiments, modification can decrease the expression of this gene.In certain embodiments, modification can include the deletion of a part of a gene or the entire gene from the genome of a cell line, including the deletion of a CDS region.

[0049] Decreased expression refers to a decrease in transcription of the coding region of a gene, a decrease in translation of the mRNA encoded by this coding region, or a decrease in the activity of the resulting protein encoded by this coding region. Increased expression refers to an increase in transcription of the coding region of a gene, an increase in translation of the mRNA encoded by this coding region, or an increase in the activity of the resulting protein encoded by this coding region.

[0050] For example, in certain embodiments, the modification comprises the deletion of one or more of the following genes from the engineered cell line: APOA1, CCL2, CCL5, CYP19A1, CXCL8, ELF3, FOS, HERC3, HERC5, IFIT1, IFIT2, IFIT3, IRF7, ISG15, KRT15, ​​KRT19, MX1, NGFR, PTGS2, PTPN6, RET, ROS1, SFRP1, SOX2, SPP1, TNF, TNFRSF4, TRAF1, and VAV3, and in certain embodiments, the modification comprises the deletion of one or more of the following genes from the engineered cell line: CCL2, CCL5, CXCL8, HERC5, IFIT1, IFIT2, IFIT3, and ISG15. In certain embodiments, the modification comprises the deletion of the ISG15 gene from the engineered cell line.

[0051] As known in the art, a cell line is a clonal cell culture developed from a single cell, in which the cells continue to divide over an extended period of time without undergoing senescence in culture. A control cell line differs from an engineered cell line in that the control cell line is derived from the same or a similar clonal cell culture and is therefore genetically similar to the engineered cell line, but the control cell line has not been engineered to modify the expression of one or more target genes. In certain embodiments, the engineered cell line and the control cell line are selected from primate cells, such as monkey cells or human cells, mouse cells, or dog cells. For example, in certain embodiments, the engineered cell line and the control cell line can be Vero cells, Madin-Darby Canine (MDCK), or human embryonic kidney (HEK) cells. In certain embodiments, the engineered cell line and the control cell line are Vero cells.

[0052] The engineered cell line can be designed to alter the expression of one or more genes by any method known in the art. In certain embodiments, the engineered cell line is modified by editing the cell genome, for example, by using CRISPR technology.

[0053] In certain embodiments, the CRISPR system comprises Cas9 endonuclease and single guide RNA ("sgRNA") to create engineered cell lines with one or more genes knocked out or deleted. CRISPR stands for clustered regularly interspaced short palindromic repeats and is a system for genome engineering that can be used to knock out specific genes. The sgRNA is a short guide RNA that contains a Cas9 endonuclease binder of about 20 nucleotides that can be used as a target sequence. In general, "CRISPR system" refers collectively to the transcripts and other elements involved in directing the expression or activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g., tracrRNA, or active partial tracrRNA), tracr-mate sequences (including "direct repeats" and tracrRNA-processed partial direct repeats associated with endogenous CRISPR systems), guide sequences (also referred to as "spacers" associated with endogenous CRISPR systems), or other sequences and transcripts from a CRISPR locus. "Target sequence" refers to a sequence to which the guide sequence is designed to have complementarity, where hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. The target sequence may include any polynucleotide, such as a DNA polynucleotide or an RNA polynucleotide. The target sequence may be altered to repress or activate specific genes to generate customized knockout cell lines. The Cas9 / sgRNA complex acts by binding to cellular DNA, cleaving at a designated target spot, and then repairing the double-stranded break after cleavage. Shalem et al., Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells, Science, 343; 6166, 84-87 (2014). In this way, specific gene knockouts can be created.

[0054] In some embodiments, expression of any of the selected target genes is modified using a method comprising introducing a CRISPR / Cas endonuclease (Cas) 9 system into a cell using a CRISPR / Cas guide RNA, where the guide RNA targets the gene or a fragment thereof. Thus, in one aspect, disclosed herein is a method of genetically engineering a cell line (e.g., a Vero cell line) comprising: a) obtaining an sgRNA specific for a target DNA sequence in the cell; and b) introducing into the target cell (e.g., by electroporation) a CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA that hybridizes to the target sequence in the genomic DNA of the cell.

[0055] "Guide RNA", "single guide RNA", and "synthetic guide RNA" are used interchangeably and refer to a polynucleotide sequence that includes a guide sequence, a tracr sequence, and a tracr mate sequence. The term "guide sequence" refers to an approximately 20 bp sequence within a guide RNA that identifies a target site and may be used interchangeably with the terms "guide" or "spacer". In some embodiments, the gRNA includes a sequence of ACCAGCATTCGAGCAAGATCAAGG (SEQ ID NO: 33), and in some embodiments, the gRNA includes a sequence of GGAAACCGAAACTTGGCCACCGG (SEQ ID NO: 34). In some embodiments, the CRISPR / Cas system includes a first guide sequence of ACCAGCATTCGAGCAAGATCAAGG (SEQ ID NO: 33) and a second guide sequence of GGAAACCGAAACTTGGCCACCGG (SEQ ID NO: 34).

[0056] In certain embodiments disclosed herein, one or more genes are deleted from the engineered cell line using the CRISPR-Cas system. For example, in certain embodiments, one or more of the following genes have been engineered and deleted from the cell line using the CRISPR-Cas system: APOA1, CCL2, CCL5, CYP19A1, CXCL8, ELF3, FOS, HERC3, HERC5, IFIT1, IFIT2, IFIT3, IRF7, ISG15, KRT15, ​​KRT19, MX1, NGFR, PTGS2, PTPN6, RET, ROS1, SFRP1, SOX2, SPP1, TNF, TNFRSF4, TRAF1, and VAV3, and in certain embodiments, one or more of the following genes have been deleted from the engineered cell line using the CRISPR-Cas system: CCL2, CCL5, CXCL8, HERC5, IFIT1, IFIT2, IFIT3, and ISG15. In certain embodiments, the ISG15 gene is deleted from the engineered cell line using the CRISPR-Cas system.

[0057] The engineered cell lines disclosed herein provide increased total viral particle production compared to a control cell line. In certain embodiments, the increase in viral particle production is at least about 10% compared to a control cell line, e.g., at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%. In certain embodiments, the engineered cell line may increase viral particle production by up to about 10% compared to a control cell line, e.g., up to about 15%, up to about 20%, up to about 25%, up to about 30%, up to about 35%, up to about 40%, up to about 45%, up to about 50%, up to about 55%, up to about 60%, up to about 65%, up to about 70%, up to about 75%, up to about 80%, up to about 85%, up to about 90%, up to about 95%, up to about 100%, up to about 150%, up to about 200%, up to about 250%, or up to about 300%. In certain embodiments, the engineered cell line may increase viral particle production by at least 0.3 logs compared to a control cell line, e.g., at least 0.4 logs, at least 0.5 logs, at least 0.6 logs, at least 0.7 logs, at least 0.8 logs, at least 0.9 logs, at least 1.0 logs, at least 1.1 logs, at least 1.2 logs, at least 1.3 logs, at least 1.4 logs, or at least 1.5 logs. In certain embodiments, the engineered cell line may increase viral particle production by up to 0.3 logs compared to a control cell line, e.g., up to 0.4 logs, up to 0.5 logs, up to 0.6 logs, up to 0.7 logs, up to 0.8 logs, up to 0.9 logs, up to 1.0 logs, up to 1.1 logs, up to 1.2 logs, up to 1.3 logs, up to 1.4 logs, or up to 1.5 logs.In certain embodiments, the engineered cell line may provide a 0.5 to 1.5 log increase in viral particle production compared to a control cell line, or a 1.0 to 1.5 log increase, or about a 1.5 log increase.

[0058] In certain embodiments, the ratio of infectious viral particle release to total viral particle production is increased compared to a control cell line. In certain embodiments, the increase in the ratio of infectious viral particle production to total viral particle production is at least about 1% compared to a control cell line, e.g., at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, or at least about 75%.

[0059] Viral particles can be derived from any virus known to use cellular replication machinery to replicate in living cells.In certain embodiments, the virus is selected from influenza virus, dengue virus, yellow fever virus, RSV, herpes simplex virus, HIV, hepatitis virus, coronavirus, or virus from the Rhabdoviridae family, such as rabies virus or VSV.For example, in certain aspects, the virus is influenza virus, such as influenza A virus or influenza B virus.

[0060] All nomenclatures used to classify influenza viruses are those commonly used by those skilled in the art. Thus, influenza virus types or groups refer to three major types of influenza: influenza A, influenza B, or influenza C that infect humans. Influenza A and B cause significant morbidity and mortality annually. It is understood by those skilled in the art that the designation of a virus as a particular type is related to sequence differences in the respective M1 (matrix) protein or P (nucleoprotein). Influenza A viruses are further classified into group 1 and group 2. These groups are further classified into subtypes, which refer to classification of the virus based on the sequences of two proteins on the surface of the virus, hemagglutinin (HA) and neuraminidase (NA). Currently, there are 18 recognized HA subtypes (H1-H18) and 11 recognized NA subtypes (N1-N11). Group 1 includes N1, N4, N5, and N8, and H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18. Group 2 includes N2, N3, N6, N7, and N9, and H3, H4, H7, H10, H14, and H15. There are potentially 198 different influenza A subtype combinations, but only about 131 subtypes have been detected in nature. Current subtypes of influenza A viruses that are commonly circulating in human populations and cause seasonal pandemics include A(H1N1) and A(H3N2). Influenza B subtypes can include any subtype known in the art, including, for example, influenza virus strains from the B / Victoria lineage or influenza virus strains from the B / Yamagata lineage.

[0061] Thus, the engineered cell lines disclosed herein can include Vero cells or Vero cell lines that contain a modification of the ISG15 gene, such as a deletion of the ISG15 gene, which results in increased total influenza virus particle production and / or infectious influenza virus particle production compared to a control Vero cell line.

[0062] Identification of target genes In certain embodiments, disclosed herein is a method for identifying a target gene to be deleted in a host cell genome, wherein the deletion of the target gene enhances the production of viral particles and / or infectious viral particles. In certain embodiments, the host cell is a Vero cell. The target gene can be identified by any method or combination of methods known in the art.

[0063] In certain embodiments, disclosed herein is a method for identifying genes to be deleted in a cell or cell line, comprising: (1) infecting a cell or cell line with a virus; (2) detecting the expression level of a plurality of genes in the infected cell or cell line and comparing the expression level with the expression level of a plurality of genes in a control cell or cell line not infected with the virus; and (3) identifying target genes that are differentially expressed in the infected cell or cell line. In certain embodiments, the method further comprises analyzing the differentially expressed target genes to identify one or more gene targets involved in a plurality of protein-protein networks, and selecting at least one differentially expressed gene target to be deleted in the cell or cell line, where deletion of the at least one differentially expressed gene target increases viral particle production of the virus.

[0064] In certain embodiments, the target genes are identified by at least one of differential gene expression analysis, GSEA, and / or network topology analysis to identify protein-protein networks. In certain embodiments, all three of differential gene expression analysis, GSEA, and protein-protein interaction analysis may be used to identify the target genes.

[0065] Differential gene expression analysis: Gene expression differences, such as up-regulation or down-regulation, can be evaluated between virus-infected host cells and control host cells that are not infected with the virus. To determine gene expression differences between infected and uninfected cells, RNA sequencing data from both genomes can be obtained using methods known in the art, such as DESeq2 analysis. Love MI, et al., Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2, Genome Biology 2014. From the RNA sequencing data, genes that are differentially expressed across multiple groups of samples are identified. Thus, in certain embodiments, RNA sequencing data from virus-infected host cells can be obtained and compared with RNA sequencing data from uninfected host cells, or with known normalized gene expression data. In certain embodiments, RNA sequencing data from infected cells may be obtained at any time period post-infection, for example, at about 30 minutes post-infection, about 1 hour post-infection (hpi), about 2 hpi, about 4 hpi, about 6 hpi, about 8 hpi, about 10 hpi, about 12 hpi, about 16 hpi, about 20 hpi, about 24 hpi, about 48 hpi, or about 72 hpi.

[0066] As used herein, measuring or detecting the expression of any of the aforementioned genes or nucleic acids includes measuring or detecting any nucleic acid transcript (e.g., mRNA, cDNA, or genomic DNA) corresponding to the gene of interest or the protein encoded thereby. In cases where a gene is associated with multiple mRNA transcripts or isoforms, expression of the gene may be measured or detected by measuring or detecting one or more mRNA transcripts of the gene, or all of the mRNA transcripts associated with the gene.

[0067] Typically, gene expression can be detected or measured based on mRNA or cDNA levels, but protein levels can also be used if appropriate. Any quantitative or qualitative method for measuring mRNA, cDNA, or protein levels can be used. Suitable methods for detecting or measuring mRNA or cDNA levels include, for example, Northern blotting, microarray analysis, or nucleic acid amplification procedures, such as reverse transcription PCR (RT-PCR), real-time RT-PCR, also known as quantitative RT-PCR (qRT-PCR), and / or digital droplet PCR (ddPCR). Such methods are known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 th Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 2012. Other techniques include digital multiplexed analysis of gene expression (e.g., nCounter® (NanoString Technologies, Seattle, WA) gene expression assays), which are further described in U.S. Patent Application Publication Nos. 2010 / 0112710 and 2010 / 0047924.

[0068] Detection of a nucleic acid of interest generally involves hybridization between a target (e.g., mRNA, cDNA, or genomic DNA) and a probe. The sequences of many genes are readily known. Thus, one of skill in the art can readily design hybridization probes to detect these genes. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 thEd., Cold Spring Harbor Press, Cold Spring Harbor, NY, 2012. Each probe can be substantially specific for its target to avoid any cross-hybridization and false positives. An alternative to using specific probes is to use specific reagents when obtaining material from transcripts (e.g., using target-specific primers during cDNA production or amplification). In both cases, specificity can be achieved by hybridization of the probe to a portion of the target that is substantially unique within the gene group analyzed; hybridization to, for example, the polyA tail would not provide specificity. If the target has multiple splice variants, it is possible to design hybridization reagents that recognize a region common to each variant and / or to use multiple reagents, each of which can recognize one or more variants.

[0069] In certain embodiments, microarray analysis or PCR-based methods are used. In this regard, measuring the expression of the aforementioned nucleic acid may include, for example, contacting the sample with a polynucleotide probe specific to the gene of interest, or with primers designed to amplify a portion of the gene of interest, and detecting binding of the probe to the nucleic acid target or amplification of the nucleic acid, respectively. Detailed protocols for designing PCR primers are known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 th Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 2012. Similarly, detailed protocols for preparing and using microarrays to analyze gene expression are known in the art and described herein.

[0070] Alternatively, or in addition, the expression level of a gene may be determined at the protein level, which means measuring the level of a protein encoded by a gene discussed herein. Several methods and devices are known for determining levels of proteins, such as immunoassays described in, for example, U.S. Patent Nos. 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; 5,458,852; and 5,480,792 (each of which is incorporated herein by reference in its entirety). These assays may include various sandwich, competitive, or non-competitive assay formats to generate a signal related to the presence or amount of the protein of interest. Any suitable immunoassay (e.g., lateral flow, enzyme-linked immunoassay (ELISA), radioimmunoassay (RIA), competitive binding assay, etc.) may be utilized. Numerous formats for antibody arrays have been described. Such arrays may include various antibodies with specificity for the various proteins intended to be detected. For example, at least 100 different antibodies are used to detect 100 different protein targets, each antibody specific for one target. Other ligands with specificity for a particular protein target may also be used, such as the synthetic antibodies disclosed in WO 2008 / 048970, which is incorporated herein by reference in its entirety. Other compounds with the desired binding specificity may be selected from random libraries of peptides or small molecules. U.S. Pat. No. 5,922,615, which is incorporated herein by reference in its entirety, describes a device that uses multiple discrete zones of antibodies immobilized on a membrane to detect multiple target antigens in an array. Using microtiter plates or automation, large numbers of different proteins can be easily detected.

[0071] Immunoassays have been used to identify and quantify proteins, but recent advances in mass spectrometry (MS) technology have led to the development of highly sensitive and high-throughput MS protein analysis. MS methods can be used to detect low-abundance proteins in complex biological samples. For example, targeted MS can be performed by fractionating biological samples prior to MS analysis. Common techniques for performing such fractionation prior to MS analysis include, for example, two-dimensional electrophoresis, liquid chromatography, and capillary electrophoresis.

[0072] Western blotting allows the identification of specific proteins (native or denatured) from extracts made from cells or tissues, before or after any purification steps. Proteins are generally separated by size using gel electrophoresis, and then transferred to a synthetic membrane (typically nitrocellulose or PVDF) by dry, semi-dry, or wet blotting methods. The membrane can then be probed using antibodies, using methods similar to immunohistochemistry, but without the need for fixation. Detection is typically performed using peroxidase-conjugated antibodies to catalyze a chemiluminescent reaction. Western blotting is a routine molecular biology method that can be used to semi-quantitatively or quantitatively compare protein levels between extracts. Protein molecular weights can be measured by size separation prior to blotting, relative to known molecular weight markers. Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. Gel electrophoresis is used to separate proteins by polypeptide length (denaturing conditions) or by the three-dimensional structure of the protein (native / non-denaturing conditions).

[0073] Gene set enrichment analysis: Gene set enrichment analysis (GSEA) may be used to further interpret differential gene expression data. GSEA focuses on gene sets or groups of genes that share common biological functions, chromosomal location, and / or regulation. Subramanian, A. et al., Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles, Proc. Nat'l Acad. Sci. USA 2005, 102(43):15545-15550. In certain embodiments of all aspects of the present disclosure, GSEA may be used to identify classes of genes that may be associated with increased viral particle production and / or infectious viral particle production in cells or cell lines. GSEA allows gene expression data to be interpreted by focusing on gene sets or groups of genes that share common functions, locations, or regulations. Gene sets are available in a searchable format, for example, in the electronic Molecular Signatures Database (MSigDB). Genes from the samples can be ranked by differential gene expression, comparing the amount of viral particles produced in infected and uninfected cells, and used to screen enriched gene sets in MSigDB.

[0074] In certain embodiments, GSEA may include calculation of an enrichment score ES, which represents the extent to which the gene set is over-represented among all of these various expressed genes (due to either over- or under-expression of the various expressed genes). The statistical significance (p-value) of the ES, and a normalized enrichment score value (NES) may also be calculated, as well as a false discovery rate (FDR) that corresponds to the proportion of false positives for any given NES. Subramanian 2005. The calculated NES for each gene set may reflect the extent to which the gene set correlates with increased viral particle production. The NES of GSEA may be calculated by any means known in the art, such as using Reactome (Croft, D et al., The Reactome pathway knowledgebase, Nucleic Acids Res. 2014, 42:D472-D477) and / or WebGestalt (WEB-based Gene SeT AnaLysis Toolkit) (Liao, Y. et al., WebGestalt 2019:gene set analysis toolkit with revamped UIs and APIs, Nucleic Acids Res. 2019, 47(W1):W199-W205). In certain embodiments, the NES for a gene set may be a positive value indicating upregulated expression of genes in the gene set and may be greater than about 0.5, such as greater than about 1.0, greater than about 1.5, greater than about 2.0, or greater than about 2.5. In certain embodiments, the NES for a gene set may be a negative value indicating downregulated expression of genes in the gene set, and may be less than about -0.5, such as less than about -1.0, less than about -1.5, less than about -2.0, or less than about -2.5.

[0075] In certain embodiments, characteristic gene pathways may be identified by the use of GSEA disclosed herein. In certain embodiments, at least one of the following gene pathways may be upregulated in cells or cell lines following infection with influenza virus: G-protein coupled receptor (GPCR) ligand binding, signaling by GPCR, class A / 1 (rhodopsin-like receptor), GPCR downstream signaling, G alpha(i) signaling events, interferon alpha / beta signaling, DNA synthesis, visual phototransduction, peptide ligand binding receptor, DNA replication, removal of Orc1 from chromatin, assembly of pre-replicative complexes, pre-initiation of DNA replication, metaphase and anaphase, anaphase, G2 / M checkpoint, origin switch to post-replicative state, antiviral mechanisms by IFN-stimulated genes, separation of sister chromatids, and cross-presentation of soluble foreign antigens (endosomes).

[0076] In certain embodiments, at least one of the following genetic pathways may be upregulated in a cell or cell line following infection with the VSV virus: interferon alpha / beta signaling, interferon signaling, GPCR ligand binding, antiviral mechanisms by IFN stimulated genes, class A / 1 (rhodopsin-like receptors), peptide ligand binding receptors, interleukin-10 signaling, signaling by GPCRs, chemokine receptors bind chemokines, cytokine signaling in the immune system, interferon gamma signaling, GPCR downstream signaling, ISG15 antiviral mechanisms, G alpha(i) signaling events, class B / 2 (secretin family receptors), OAS antiviral response, SLC transporter impairment, cholesterol biosynthesis, DDX58 / IFIH1 mediated induction of interferon-alpha / beta, and activation of matrix metalloproteinases.

[0077] In certain embodiments, at least one of the following genetic pathways: GPCR ligand binding, GPCR signaling, class A / 1 (rhodopsin-like receptors), GPCR downstream signaling, G alpha(i) signaling events, interferon alpha / beta signaling, peptide ligand binding receptors, and IFN-stimulated gene antiviral mechanisms may be upregulated in infected cells following infection with a virus.

[0078] In certain embodiments, at least one of the following genetic pathways may be downregulated in a cell or cell line following infection with an influenza virus: selenocysteine ​​synthesis, major pathway of rRNA processing in the nucleolus and cytosol, eukaryotic translation termination, EJC-independent NMD, peptide chain elongation, rRNA processing, EJC-enhanced NMD, NMD, eukaryotic translation elongation, formation of a pool of free 40S subunits, L13a-mediated translational silencing of ceruloplasmin expression, GTP hydrolysis and binding of 60S ribosomal subunits, signaling by non-receptor tyrosine kinases, signaling by PTK6, cap-dependent translation initiation, eukaryotic translation initiation, signaling by NTRK2 (TRKB), viral mRNA translation, signaling by ERBB2, and selenoamino acid metabolism.

[0079] In certain embodiments, at least one of the following genetic pathways may be downregulated in cells or cell lines following infection with VSV virus: rRNA processing in the nucleus and cytosol, eukaryotic translation elongation, the major pathway of rRNA processing in the nucleolus and cytosol, rRNA processing, EJC-independent NMD, peptide chain elongation, L13a-mediated translational silencing of ceruloplasmin expression, cap-dependent translation initiation, eukaryotic translation initiation, eukaryotic translation termination, selenocysteine ​​synthesis, GTP hydrolysis and binding of 60S ribosomal subunits, formation of a pool of free 40S subunits, EJC-enhanced NMD, NMD, viral mRNA translation, selenoamino acid metabolism, activation of mRNA by binding of cap-binding complex and eIF, formation of translation initiation complex, and influenza virus RNA transcription and replication.

[0080] In certain embodiments, at least one of the following genetic pathways may be downregulated in infected cells following infection with a virus: selenocysteine ​​synthesis, major pathway of rRNA processing in the nucleolus and cytosol, eukaryotic translation termination, EJC-independent NMD, peptide chain elongation, rRNA processing, EJC-enhanced NMD, NMD, eukaryotic translation elongation, formation of a pool of free 40S subunits, L13a-mediated translational silencing of ceruloplasmin expression, GTP hydrolysis and binding of 60S ribosomal subunits, cap-dependent translation initiation, eukaryotic translation initiation, viral mRNA translation, and selenoamino acid metabolism.

[0081] In certain embodiments disclosed herein, the method of identifying genes to be modified in a cell or cell line includes analyzing genes identified by differential expression and GSEA, and does not include analyzing gene targets involved in multiple protein-protein interactions. However, in certain embodiments, potential target genes can be further identified by combining differential expression and / or GSEA results with network topology analysis (e.g., analyzing multiple protein-protein interactions to identify target genes to be modified in a cell), and modification of the target genes increases viral particle production.

[0082] Network Topology Analysis: In certain embodiments, the differentially expressed genes and gene sets identified by GSEA can be further refined through the use of network topology analysis to identify target genes involved in a certain protein-protein interaction (PPI) network. In certain embodiments, the list of target genes can be created and / or filtered based on the Network Retrieval and Prioritization construction method described in, for example, Wang, J. et al., Proteome Profiling Outperforms Transcriptome Profiling for Coexpression Based Gene Function Prediction, Mol Cell Proteomics 2017, 16(1):121-134. For example, in certain embodiments, random walk analysis can be used to calculate the random walk probability of a given gene (known as a seed). The relationships between these seeds can then be identified in the selected network to arrive at a search sub-network, and genes with high levels of random walk probability can be selected. Network topology analysis may use random walk-based network propagation by identifying genes that are potentially biologically significant, and each gene in the PPI network may be assigned a score, and the statistical significance of the score may be calculated using two p-values: a global p-value (this significance is a result of non-random associations between genes in the PPI network and the input seeds) and a local p-value (this significance indicates that the gene did not gain significant association with the input seeds due to network topology alone).

[0083] Enrichment analysis of the retrieved sub-networks can then be performed using any means known in the art, such as, for example, the PPI BIOGRID database (Stark C. et al., BioGRID: a general repository for interaction datasets, Nucleic Acids Res. 2006, 34: D535-539) and Gene Ontology (GO) Biology Process terms (Harris, MA et al., The Gene Ontology (GO) database and informatics resource, Nucleic Acids Res. 2004, 32: D258-D261). As known in the art, GO terms provide a systematic language for the description of genes and gene products in three separate domains shared by all organisms: molecular function, biological process, and cellular component. GO terms can be used to glean functional and biological significance from large datasets, such as those that may result from differential gene expression analysis and GSEA.

[0084] In certain embodiments disclosed herein, gene targets, such as those identified by differential gene expression and GSEA, may be further analyzed to identify one or more gene targets involved in multiple protein-protein networks. For example, in certain embodiments, a single gene target may share at least two GO pathways. In certain embodiments, the plurality of protein-protein networks includes at least two of the following: defense response (GO:0006952), response to viruses (GO:0009615), viral genome replication (GO:0019079), response to cytokines (GO:0034097), response to type I interferon (GO:0034340), regulation of viral genome replication (GO:0045069), defense response to viruses (GO:0051607), cell death (GO:0008219), viral life cycle (GO:0019058), negative regulation of viral genome replication (GO:0045071), and cellular response to cytokine stimulation (GO:0071345). In certain embodiments, the plurality of protein-protein networks comprises at least two of the following: defense response, response to virus, viral genome replication, response to cytokines, response to type 1 interferon, regulation of viral genome replication, defense response to virus, negative regulation of viral genome replication, and cellular response to cytokine stimulation. In certain embodiments, the plurality of protein-protein networks comprises at least two of the following: response to virus, viral genome replication, response to type 1 interferon, and defense response to virus.

[0085] In certain embodiments of the methods of identifying genes to be deleted disclosed herein, the cell or cell line can be any host cell or host cell line known for growing viral particles, for example, for the production of viral vaccines. In certain embodiments, the cell or cell line is a primate, such as monkey (e.g., Vero cell line), or human, dog, cow, pig, cat, mouse, hamster, or rabbit. In certain embodiments, the cell or cell line is a Vero cell line. In certain embodiments, the cell or cell line is a Madin-Darby Canine (MDCK) cell, and in certain embodiments, the cell or cell line is a human embryonic kidney (HEK) cell.

[0086] In certain embodiments of the method of identifying genes to be deleted disclosed herein, the virus can be any virus known to replicate in cells or cell lines, including but not limited to Vero cell lines. For example, in certain embodiments, the virus is selected from influenza virus (e.g., influenza A virus or influenza B virus), dengue virus, yellow fever virus, RSV, herpes simplex virus, HIV, hepatitis virus, coronavirus, or a virus from the Rhabdoviridae family, such as rabies virus or VSV.

[0087] After identifying the gene to be modified in the cell or cell line, the method disclosed herein may further comprise modifying or deleting said gene, or reducing the expression of said gene, by any method known in the art. In certain embodiments, the identified gene is deleted from the cell or cell line using the CRISPR-Case system, as disclosed above.

[0088] Methods for using engineered cell lines The engineered cell line disclosed herein can be used to, for example, increase viral particle production. In certain embodiments, disclosed herein is a method for increasing viral particle production. In the method disclosed herein, the engineered cell line can be infected with a virus and incubated under conditions suitable for the production of the virus by the engineered cell line, and then the virus produced by the engineered cell line can be harvested. The harvested viral particles can then be used, for example, for further research and / or for the manufacture of vaccine compositions. In certain embodiments, the engineered cell line comprises at least one genetic modification, resulting in increased total viral particle production and / or infectious viral particle production compared to a control cell line identical to the engineered cell line except for this modification.

[0089] In certain embodiments, the modification is present in at least one of the following genes: APOA1, CCL2, CCL5, CYP19A1, CXCL8, ELF3, FOS, HERC3, HERC5, IFIT1, IFIT2, IFIT3, IRF7, ISG15, KRT15, ​​KRT19, MX1, NGFR, PTGS2, PTPN6, RET, ROS1, SFRP1, SOX2, SPP1, TNF, TNFRSF4, TRAF1, and VAV3. In certain embodiments, the modification is present in at least one of the following genes: CCL2, CCL5, CXCL8, HERC5, IFIT1, IFIT2, IFIT3, and ISG15. In certain embodiments, the modification is present in the ISG15 gene. The modification can be a modification to reduce the expression of at least one gene. For example, in certain embodiments, the modification results in a deletion of at least one gene, such as deletion by a CRISPR-Cas system.

[0090] In certain embodiments, the engineered cell line may increase viral particle production by at least about 20% compared to a control cell line, e.g., at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%. In certain embodiments, the engineered cell line may increase viral particle production by about 20% to about 300%, by about 50% to about 300%, or by about 100% to about 300% compared to a control cell line. In certain embodiments, the engineered cell line may increase viral particle production by up to 1.5 logs compared to a control cell line (e.g., an increase of 0.5-1.5 logs, or 1.0-1.5 logs, or about 1.5 logs). Viral particle production may be measured by any means known in the art for counting viral genomes. For example, in certain embodiments, viral particle production is measured at 50% tissue culture infectious dose (TCID 50 ) assay, hemagglutination assay, or PCR (e.g., ddPCR or qRT-PCR).

[0091] The number of infectious viral particles can also be quantified by any means known in the art. In certain embodiments, the number of infectious viral particles is determined by the TCID 50 Quantitatively determine the TCID using the TCID assay and / or plaque assay. 50Both the plaque assay and the TCID of the virus sample are added to cells, for example in a 96-well plate format. Cell types are specifically selected to show cytopathic effect (CPE) (i.e., morphological changes due to viral infection or cell death). After an incubation period, the cells are examined for CPE or cell death, and each well is classified as infected or uninfected. Colorimetric or fluorometric readouts are also possible, which may increase the sensitivity of the assay. The dilution at which 50% of the wells show CPE is used to determine the TCID of the virus sample. 50 This calculation can generally be performed by various mathematical approaches, for example by the Spearman-Karber method or the Reed-Muench method. The viral titer is calculated as TCID 50 In the case of plaque assay quantification, the number of plaques formed by the virus at various dilutions can be quantified per well, i.e. the number of plaque forming units (PFU) can be quantified per well. Log titers are expressed as the logarithm of PFU / mL. TCID 50 A comparison of the assay with plaque assays is discussed, for example, in Smither, SJ et al., Comparison of the plaque assay and 50% tissue culture infectious dose assay as methods for measuring filovirus infectivity, J. Virological Methods 2013, 193(2):565-71.

[0092] The hemagglutinin assay employs the process of hemagglutination, in which sialic acid receptors on the surface of red blood cells (RBCs) bind to the hemagglutinin glycoprotein found on the surface of viruses, such as influenza viruses, creating a network or lattice structure of interconnected RBCs and virus particles (termed hemagglutination) that occurs in a concentration-dependent manner in virus particles. One goal of the hemagglutinin assay can be to characterize the concentration of virus particles relative to their ability to induce hemagglutination in this assay.

[0093] PCR technology can also be used to amplify and quantify viral genomes (i.e., DNA or RNA). In certain embodiments, quantification by PCR involves multiple serial dilutions of samples of unknown concentration in parallel with samples of known concentration for reference and calibration. Quantification can be achieved, for example, using a wide variety of known fluorescent detection strategies. One method of PCR includes ddPCR, a form of digital PCR that relies on water-oil emulsion droplet technology. In certain embodiments, a sample can be fractionated into thousands of droplets, so that PCR amplification of target nucleic acid occurs within each individual droplet. Viral particles can then be quantified, for example, in units of Vg / mL.

[0094] As is known in the art, PCR amplifies all target nucleic acid material (e.g., nucleic acid from both intact infectious and defective viral particles, as well as free nucleic acid). Thus, PCR results, which may be expressed in units of viral genomes (Vg) / mL, are often expressed as viral titers (e.g., TCID 50 / mL). Therefore, in addition to measuring viral particle production, for example by PCR, 50Infectious viral particle production by the engineered cell line may also be measured, and the ratio between these two values ​​may then be compared. In certain embodiments of the engineered cell lines and methods disclosed herein, the ratio of infectious viral particle production to total viral particle production is at least about 3%, e.g., at least about 5%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, or at least about 70%.

[0095] Vaccine composition: Also disclosed herein are methods of using engineered cell lines to produce viral particles for use in vaccine compositions. For example, in certain embodiments, after harvesting the virus produced by the engineered cell lines, the virus, which may be live, live attenuated, or inactivated, may be added to the vaccine composition. For example, certain known approved influenza vaccine compositions are inactivated vaccines that contain whole virions or virions treated with agents that dissolve lipids ("split" vaccines), purified glycoproteins expressed in cell culture ("subunit vaccines"), or live attenuated virus vaccines.

[0096] In certain embodiments, disclosed herein are vaccine compositions comprising viral particles recovered from an engineered cell line that contains modifications in one or more genes (e.g., ISG15), where the modifications in the one or more genes increase total viral particle production and / or infectious viral particle production compared to a control cell line that is identical to the engineered cell line except for the modifications in the one or more genes.

[0097] The vaccine composition may also further comprise an adjuvant. As used herein, the term "adjuvant" refers to a substance or vehicle that non-specifically enhances the immune response to an antigen. Adjuvants include inorganic substances to which antigens are adsorbed (alum, aluminum salts (e.g., aluminum hydroxide / aluminum oxyhydroxide (AlOOH), aluminum phosphate (AlPO 4 Adjuvants may include suspensions of glycerol, ... Exemplary biological adjuvants include AS04 (Didierlaurent, AM et al, AS04, an Aluminum Salt- and TLR4 Agonist-Based Adjuvant System, Induces a Transient Localized Innate Immune Response Leading to Enhanced Adaptive Immunity, J. Immunol. 2009, 183:6186-6197), IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, and 41 BBL.

[0098] In addition to the viral particles and optional adjuvants, the vaccine composition may also further comprise one or more pharma- ceutically acceptable excipients. In general, the nature of the excipient will depend on the particular mode of administration used. For example, parenteral formulations usually contain an injectable fluid, including pharma- ceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, and the like, as a vehicle. In the case of solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the vaccine composition to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pharma- ceutically acceptable salts for adjusting osmotic pressure, preservatives, stabilizers, buffers, sugars, amino acids, pH buffers, and the like (e.g., sodium acetate or sorbitan monolaurate).

[0099] Typically, the vaccine composition is a sterile liquid solution formulated for parenteral administration, such as intravenous, subcutaneous, intraperitoneal, intradermal, or intramuscular. The vaccine composition may also be formulated for intranasal or inhalation administration. The vaccine composition may also be formulated for any other intended route of administration.

[0100] In some embodiments, the vaccine composition is formulated for intradermal, intranasal, or intramuscular injection. In some embodiments, the injectables are prepared in conventional forms, either as liquid solutions or suspensions, as solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. In some embodiments, the injectable solutions and suspensions are prepared from sterile powders or granules. General considerations in the formulation and manufacture of pharmaceuticals for administration by these routes are discussed, for example, in Remington's Pharmaceutical Sciences, 1999, ed. ... thed., Mack Publishing Co., Easton, PA, 1995, incorporated herein by reference. Currently, oral or nasal spray or aerosol routes (e.g., by inhalation) are most commonly used to deliver therapeutics directly to the lungs and respiratory system. In some embodiments, the vaccine composition is administered using a device that delivers a metered amount of the vaccine composition. Suitable devices for use in delivering the intradermal pharmaceutical compositions described herein include short needle devices, such as those described in U.S. Pat. Nos. 4,886,499, 5,190,521, 5,328,483, 5,527,288, 4,270,537, 5,015,235, 5,141,496, and 5,417,662, all of which are incorporated herein by reference. Intradermal compositions may also be administered by devices that limit the effective penetration length of a needle into the skin, such as those described in WO 1999 / 34850, which is incorporated herein by reference, and functional equivalents thereof. Also suitable are jet injection devices that deliver liquid vaccines to the dermis via a liquid jet injector or via a needle that pierces the stratum corneum and produces a jet that reaches the dermis.Jet injection devices are described, for example, in U.S. Pat. Nos. 5,480,381, 5,599,302, 5,334,144, 5,993,412, 5,649,912, 5,569,189, 5,704,911, 5,383,851, 5,893,397, 5,466,220, 5,339,163, and 5,31 The ballistic powder / particle delivery device is described in WO 2,335, WO 5,503,627, WO 5,064,413, WO 5,520,639, WO 4,596,556, WO 4,790,824, WO 4,941,880, WO 4,940,460, WO 1997 / 37705, and WO 1997 / 13537 (all of which are incorporated herein by reference). Also suitable is a ballistic powder / particle delivery device that uses compressed gas to accelerate the powder form of the vaccine through the outer layer of the skin to the dermis.In addition, a conventional syringe can be used in the classical Mantoux method of intradermal administration.

[0101] Preparations for parenteral administration typically include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions (e.g., saline and buffered media). Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose, and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0102] The present disclosure will be better understood with reference to the following examples. EXAMPLES

[0103] The following examples should be considered as illustrative and not limiting the scope of the disclosure above.

[0104] Cell lines and culture media: The Vero WHO cell line disclosed in the examples below was at passage 138. The cell line was derived from a vial of Vero ATCC CCL-81 at passage 124 sent to the WHO for analysis and establishment of a Vero WHO master cell bank approved for vaccine production. The cells were cultured at 37°C and 5% CO in a humidified incubator (Infors HT, Switzerland). 2 The cells were grown in static culture at 37 °C for 24 h. Cells were passaged twice weekly using TrypLE® Express (Thermo Fisher Scientific) as the dissociation reagent. A serum-free adapted sub-cell line grown in OptiPRO® medium (Thermo Fisher Scientific) supplemented with 4 mM GlutaMAX® (Thermo Fisher Scientific) was cryopreserved at passage number 151 in OptiPRO® medium supplemented with 4 mM GlutaMAX® and 10% DMSO (Sigma, USA).

[0105] The ISG15- / - Vero cell line was received and thawed for five passages before cryopreservation. The cells were then thawed again and continued passage for four more passages ("p5+4") or serially passaged from the first thaw for 17 passages ("p17"). The cell line was derived from a vial of Vero ATCC CCL-81 at the 124th passage sent to the WHO for analysis and establishment of a Vero WHO Master Cell Bank approved for vaccine production. The ISG15- / - Vero cells, and control cell lines, were cultured in serum-free, ultra-low protein medium (VP-SFM AGT™ from Thermo Fisher Scientific) without proteins, peptides, or other components of animal or human origin using pharmaceutical grade reagents and equipment.

[0106] Example 1 - Cell kinetic analysis and infection First, quality control kinetic experiments were performed to quantitate virus production rates and cell viability over time in which Vero cells were infected with influenza virus A (IVA) Puerto Rico 8 strain or rVSV-GFP at a multiplicity of infection (MOI) of 10. The results were used to estimate the optimal time to harvest samples for RNA sequencing (e.g., maximum viability before induction of cell death).

[0107] Supernatants were collected at several time points to monitor cell viability. TCID 50 and hemagglutination assays were used to quantitate viral particles, as well as infectious viral particles (for IVA) and infectious viral particles (for rVSV-GFP).

[0108] Optimal harvest time points for RNA sequencing were selected for IVA and rVSV based on the level of infectious viral particle production and cell viability. Therefore, for IVA, the selected time points were determined to be relatively early in infection (4 hours post-infection (hpi)) and the peak of infectious viral particle production (24 hpi). For rVSV-GFP, given the appearance of cytopathic effects at an early stage of infection (8 hpi), the selected time points were 2 hpi and 6 hpi to ensure that pathways such as cell death were not falsely enriched due to sample quality issues.

[0109] Based on the results of the kinetic analysis, Vero WHO cells at passage 153 were infected with either IVA Puerto Rico 8 or rVSV-GFP at an MOI of 10 for further transcriptome analysis. IVA-infected cells were harvested at 4 hpi and 24 hpi, and rVSV-GFP-infected cells were harvested at 2 hpi and 6 hpi. These samples were harvested using TrypLE® Express and centrifuged at 300×g for 5 min. Cell pellets of approximately 6 million cells were lysed, quickly frozen in a mixture of dry ice / ethanol, and stored at −80° C. until further analysis. Samples from non-infected cells were also prepared and sent for sequencing as a control batch. All samples were generated in triplicate.

[0110] Example 2 - Functional genomic analysis and selection of target genes Identification of differentially expressed genes: Total RNA sequencing (TrueSeq) was performed using Illumina NovaSeq6000 Sprime v1.5, PE100. Following standard quality control, reads were first aligned to the Vero cell genome published in Sene, M.-A. et al., Haplotype-resolved de novo assembly of the Vero cell line genome, NPJ Vaccine 2021, 6(1):106 using STAR alignment as described in Dobin, A. et al., STAR: ultrafast universal RNA-seq aligner, Bioinformatics 2013, 29(1):15-21. The resulting BAM files were classified by name using SAMtools (Li, H. et al., The Sequence Alignment / Map format and SAMtools, Bioinformatics 2009, 25(16):2078-2079) prior to read counting. Transcripts were quantified using featureCounts (Liao, Y. et al., featureCounts: an efficient general purpose program for assigning sequence reads to genomic features, Bioinformatics 2014, 30(7)923-930). Differential expression analysis of raw read counts was performed using DESeq2 and quality control graphs were generated using DESeq2 and the R package described in Love, MI et al., Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2, Genome Biology 2014,15:550. The resulting differentially expressed (DE) gene list was filtered with a p-value cutoff of 0.0001.

[0111] Gene set enrichment analysis: DE genes were then ranked based on their log2 fold change. WebGestalt (WEB-based GEne SeT AnaLysis Toolkit) was used for gene set enrichment analysis with the Reactome gene set collection (Croft, 2014). To find differential expression pathways of genes between adherent and suspension cell lines, gene sets were filtered and the top 20 gene sets with adjusted p-values ​​less than 0.05 were considered significantly changed. The results are shown in Table 2 and Figure 1 below for 24hpi IVA and Table 3 and Figure 2 for rVSV-GFP.

[0112] [Table 4]

[0113] [Table 5]

[0114] As shown in Figure 1, for IVA infection at 24 hpi, gene set enrichment analysis (GSEA) using Reactome as the gene set showed downregulation of key RNA processing gene sets, such as L13a-mediated translational silencing of ceruloplasmin expression, correlating with a viral evasion strategy via cap snatching and the host cell's attempt to address this evasion. Selenium-related pathways, such as selenoamino acid metabolism and selenocysteine ​​synthesis, were also downregulated. See Figure 1. Indeed, Guillin, OM et al., Selenium, Selenoproteins and Viral Infection, Nutrients 2019, 11(9):2101, previously showed that selenium and selenoprotein deficiency leads to increased host susceptibility to viral infection. Meanwhile, key immune response-related pathways, such as interferon signaling, were significantly upregulated. Upregulation of IFN-stimulated genes was also observed.

[0115] Similar to IVA, rVSV-GFP interaction with Vero cells at 6 hpi showed downregulation of one of the key quality control mechanisms of RNA processing: nonsense-mediated decay (NMD), which promotes viral replication concomitantly with downregulation of eukaryotic translation elongation (Figure 2). Furthermore, previously identified antiviral pathways related to interferon were also upregulated in the case of rVSV-GFP infection, in particular the antiviral mechanisms by IFN-stimulated genes such as ISG15.

[0116] Network topology analysis: To identify antiviral genes involved in protein-protein interaction (PPI) networks across gene sets and pathways, network topology analysis was performed on previously identified significantly up-regulated genes, including 130 genes on IVA 4 hpi, 264 genes on IVA 24 hpi, and 235 genes on rVSV-GFP 6 hpi.

[0117] Random walk analysis was first used to calculate the random walk probability of the input gene IDs (seeds), and then the upregulated portion of the gene list produced by DESeq2 was filtered by identifying the relationships between the seeds in the selected network and returning the search sub-networks, considering genes with |log2 fold change|>2 for network search and prioritization construction method based network topology analysis (NTA), highlighting the top 20 genes with the highest random walk probability. Indeed, assuming a close association between mechanistically important genes and the random distribution of other genes on the network, NTA uses random walk-based network propagation by identifying genes that are potentially biologically important. Using the input gene IDs (already filtered upregulated genes) as seeds, a score was imputed to each gene in the PPI network based on their overall proximity (quantified by random walk similarity) to the input seeds. The statistical significance of these scores was then calculated by two p-values: a global p-value (this significance is a result of non-random associations between genes in the PPI network and the input seeds) and a local p-value (this significance ensures that genes did not acquire significant associations with the input seeds due to network topology alone).

[0118] Finally, an enrichment analysis of the retrieved sub-networks was performed using the protein-protein interaction (PPI) BIOGRID database and Gene Ontology (GO) Biology Process terms (Stark C. et al., BioGRID: a general repository for interaction datasets, Nucleic Acids Res. 2006, 34:D535-539; Harris, MA et al., The Gene Ontology (GO) database and informatics resource, Nucleic Acids Res. 2004, 32:D258-D261). GO terms were first ranked based on their adjusted p-values, and the top 10 highly significant terms were considered, with an adjusted p-value cutoff of 0.01. The identified pathways and top-ranked genes are shown in Table 4 below.

[0119] [Table 6]

[0120] [Table 7]

[0121] In all three cases (IVA 4 hpi, IVA 24 hpi, and rVSAV 6 hpi), ISG15 plays a central role in one of the identified pathways (e.g., defense response, viral life cycle, response to cytokines, interferon, negative regulation of viral genome regulation, among others) and therefore emerges as an attractive candidate for CRISPR / Cas9-mediated knockout.

[0122] Example 3 - Comparison of ISG15 protein sequences across species To verify that gene editing of ISG15 would result in phenotypic modification with respect to viral infection, ISG15 protein sequences were compared across species of interest (i.e., species from which cell lines used in vaccine production are derived), such as human, mouse, Vero, and dog. ISG15 protein sequences were read from RefSeq for Vero cells (XP_007979280.1), human (NP_005092.1), mouse (NP_056598.2), and dog (XP_003639101.1), and are described herein as SEQ ID NO: 14 (Vero); SEQ ID NO: 15 (human); SEQ ID NO: 16 (mouse); and SEQ ID NO: 17 (dog). These sequences were aligned using T-Coffee and exported to the ESPript server for sequence alignment graphic design. See Figure 3. Regions known to interact with viruses were also highlighted.

[0123] As shown in Figure 3, the mutations between human and mouse ISG15 are similar to those between human and Vero ISG15, especially at position 89, which has previously been highlighted as playing an important role in the ability of Old World monkey ISG15 (including Vero cells) to efficiently ISGylate proteins compared to human ISG15, thus providing some indication of the desired effect of ISG15 deletion in Vero cells. Pattyn E. et al., HyperISGylation of Old World monkey ISG15 in human cells, PLoS One 2008,3(6):e2427. In Figure 3, residues of ISG15 known to interact with influenza NS1 protein, coronavirus PLP, and nairovirus OTU are also shown below the sequence alignment. Dzimianski, J. et al., ISG15: it's Complicated, J. Mol Biol. 2019,431(21):4203-4216.

[0124] Example 4 - Genomic deletion of ISG15 using CRISPR / Cas9 and validation of the deletion at the genomic / proteomic level The strategy used for the genomic deletion protocol relied on the cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double-strand breaks (DSBs) at the gene locus to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. This method has been used to delete genes of 1-10 kb in length (Bauer, DE et al., Generation of genomic deletions in mammalian cell lines via CRISPR / Cas9, J Vis Exp. 2015, 95:e52118), and was applied herein to the deletion of the CDS region of the ISG15 gene. In certain cases, genomic deletions may be advantageous for homology-directed repair (HDR) or single-site small-scale indel production. The high frequency of deletions limits the number of clones that need to be screened to find the clone of interest, and monoallelic and biallelic deletions can be easily identified by PCR, thus avoiding more laborious methods. In addition, assuming a substantial portion of the gene of interest is deleted, reliable loss-of-function alleles can be obtained.

[0125] A pair of guide RNAs was designed using the freely available online tools CRISPOR and EuPaGDT, which already included the Vero cell genome in their list of custom genomes. These tools helped to identify guide sequences that minimized identical or similar genome matches, reducing the risk of cleavage from the target site (off-target effects). The guide sequences contained a 20-mer ("protospacer sequence") upstream of a "NGG" sequence ("protospacer adjacent motif" or PAM) at the genome recognition site. The plasmid construct pX458 (Addgene plasmid ID 48138) purchased from GenScript contained GFP as a selectable marker and one of the two designed gRNAs guide A or guide B, where guide A was ACCAGCATTCGAGCAAGATCAAGG (SEQ ID NO: 33) and guide B was GGAAACCGAAACTTGGCCACCGG (SEQ ID NO: 34).

[0126] Delivery of CRISPR / Cas9 plasmids was performed by electroporation. For transfection, 2.6 × 10 cells were added in 90 uL of growth medium. 6 Four vials containing 1000 cells each were prepared. The cells were washed twice with ice-cold phosphate-buffered saline (PBS), resuspended, and transferred to a 4 mm gap cuvette. Four tubes containing 10 mL of growth medium were prepared and placed in an incubator for 10 minutes. 5 μg of each CRISPR / Cas9 construct containing guide A and guide B were mixed with the Vero cell suspension, and the samples were immediately pulsed using an electroporator at 250 volts square wave for 20 ms. The cells were then diluted with 10 mL of previously prepared and pre-warmed complete growth medium and transferred to a T75cm 2 After seeding in flasks, the cells were incubated at 37°C, 5% CO for 48 hours. 2 For all tests, non-transfected cells were included as a negative control.

[0127] Following CRISPR / Cas9-based genomic deletion of the ISG15 CDS region, several validation steps were designed to confirm plasmid delivery using a GFP reporter and cell sorting, the intended deletion using PCR, protein deletion using Western blot, and the deletion phenotypic effect by viral infection and virus production quantification.

[0128] Approximately the top 3% of GFP-positive cells were sorted using fluorescence-activated cell sorting (FACS) to enrich for cells that received high levels of CRISPR / Cas9 constructs. Sorted cells were individually plated into 96-well plates with 100 μl cell culture medium per well using a FACS sorter. Clones were incubated at 37°C for 3 weeks. Resulting monoclonal colonies were passaged and split for further validation steps.

[0129] PCR was used to verify the intended genomic deletion of the ISG15 CDS region. To confirm the genomic deletion, two pairs of PCR primers were designed as shown in Figure 4. The first primer sgRNA A (non-deleted band) flanked the inside of the deleted region, and the second primer sgRNA B (deleted band) flanked the outside of the deleted region (these allow screening of the deleted and non-deleted bands). sgRNA A had the forward primer GTCCCAGCTCTGCAGACATTA (SEQ ID NO: 35) and the reverse primer GAGCTCGGCCAGGTTCTAAG (SEQ ID NO: 36). sgRNA B had the forward primer CCTCGAGGCTGTAACTGCAA (SEQ ID NO: 37) and the reverse primer ACCATAGGGGTGTTTTCCGT (SEQ ID NO: 38).

[0130] In the absence of deletions, the deletion band is often too large to amplify efficiently. A primer at least 100 bp from the predicted cleavage site was used to ensure that detection was not affected by small indels at the sgRNA target site. Genomic DNA was extracted from each clone using the Invitrogen PureLink Genomic DNA Mini Kit, and DNA concentration was measured. Each clone was screened for both non-deletion and deletion band detection using the following PCR protocol: for each detection, 12.5 μL master mix, 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), 100 ng gDNA, and H. 2 o PCR reactions containing up to 25 μL were run in a thermocycler (98°C for 30 sec, 35 cycles of (98°C for 10 sec, 60°C for 30 sec, 72°C for 1 min), and 72°C for 2 min). PCR products were then run on a 2% agarose gel at 10 V / cm using 1× Tris-Acetate-EDTA (TAE) buffer. Samples were checked for detection of non-deleted and deleted bands using a Chemidoc (Biorad) and clones with biallelic deletions were passaged and split for cell banking and further validation analysis. This validation was repeated after 1 week for quality control.

[0131] Biallelic clones are demonstrated by the absence of non-deleted bands and the presence of deleted bands in the ISG15 CDS region, as shown in Figure 5. Among 100 clones screened, 6 were identified with biallelic deletions and good fitness (by monitoring clonal doubling time).

[0132] At the protein level, Western blot analysis was performed to further confirm ISG15 deletion. In this protocol, 20 μL of each cell lysate sample was mixed with SDS loading buffer, separated on an SDS-PAGE gel (BioRad Criterion TGX Precast gel), and transferred to a polyvinylidene difluoride (PVDF) membrane. Immunoblotting was performed using the relevant antibody (anti-ISG15, Invitrogen). Horseradish peroxidase-conjugated secondary antibody was detected with BioRad Clarity Western ECL substrate. The resulting signal was imaged with Chemidoc (BioRad) and analyzed with ImageJ. Western blot analysis showed that none of the previously selected clones had the bands at 15-17 kDa seen in parental or wild-type Vero cells. See Figure 6 showing the absence of the ISG15 band in ISG- / - cells but the presence of a band at approximately 17 kDa in parental Vero cells.

[0133] Example 5 - ISG- / - Vero Cell Virus Production Quantification To quantify the viral production of IVA and rVSV-GFP in parental and ISG- / - Vero cells, cells were cultured in triplicate and infected with either IVA Puerto Rico 8 or rVSV-GFP at an MOI of 10. Supernatants from each sample were harvested 24 hours post-infection and viral production was quantified by ddPCR (viral genome) and TCID50 (infectious viral particles). Infection with engineered clones showed a significant increase in both total viral particle production and infectious viral particles. Indeed, with IVA infection, a 70.3-fold increase in total viral particles was observed, while with rVSV-GFP, an 87-fold increase was shown. Interestingly, the infectious / total viral particle ratio also increased significantly, from 0.0316 to 0.653 for IVA and from 0.0542 to 0.679 for rVSV-GFP. The results are shown in Table 5 below and illustrated in FIG. 7, where STD is calculated as the standard deviation across the population.

[0134] [Table 8]

[0135] These results demonstrate that deletion of the CDS region of ISG15 from Vero cells can increase viral particle production and infectious viral particle production of IVA and rVSV.

[0136] Example 6 - Growth kinetics of ISG15- / - and control cell lines The pharmaceutical industrialization process for vaccine production in large-scale bioreactors begins with thawing frozen vials of master cell bank seed into cell culture treated flasks or cell factories such as polystyrene Corning® CellSTACK® chambers (Corning).

[0137] First, quality control experiments were performed in which ISG15- / -v and control cell lines were cultured in serum-free, very low protein medium (VP-SFM AGT™ Medium (Thermo Fisher Scientific)) containing no proteins, peptides, or other components of animal or human origin using pharmaceutical grade reagents and equipment. Cells were cultured at 0.44 × 10 5 Cells / cm 2 or 0.24 x 10 5 Cells / cm 2 The cells were grown every 3-4 days at a seeding density of 100 μg / ml. The cells were maintained at 37°C and 5% CO in a humidified incubator (Sanyo). 2 Cells were grown in static culture at 37 °C for 24 h. Cells were passaged twice weekly using trypsin (Roche) as the dissociation reagent and inhibited with trypsin inhibitor (Sigma) in sodium citrate buffer. The passaging process for ISG15- / - included optimization from standard industry procedures. This optimization included the addition of a washing step with sodium citrate buffer at 37 °C prior to trypsinization and incubation in a humidified incubator with 5% CO. 2The trypsinization was performed at 37° C. during incubation. However, the trypsin exposure period and trypsin concentration were not altered and remained consistent with the cell culture of the control cell line.

[0138] As shown in Figure 8, the growth rate of ISG15- / - cells was significantly lower compared to the control cell line. Figure 8 shows that the ISG15- / - cell line had a doubling time of 1.390 days compared to 1.147 days for the control cell line (P<0.0001). The growth rates (k) of the ISG15- / - and control cell lines were 0.4987 and 0.6045 days, respectively. The goodness of fit to the nonlinear regression for the ISG15- / - and control cell lines was 0.9997 and 1.000, respectively. However, there was only a two-fold difference in yield within two weeks of culture, confirming that both the ISG15- / - and control cell lines could be grown sufficiently in static culture to generate material to inoculate the bioreactor.

[0139] Example 7 - Kinetic analysis of cell performance in bioreactors The pharmaceutical industry process for adherent Vero cells in vaccine production employs the use of microcarriers to grow adherent cells in bioreactors. Quality control experiments were performed to characterize the growth of ISG15- / - cells using microcarriers in bioreactors.

[0140] Cells from two different passages of the ISG15- / - cell line (p5+4 and p17, as indicated above), along with control cells, were harvested from cell culture treated flasks and cultured at a final density of 1 g / L (Cytiva) and 20,000 cells / cm. 2The cells were seeded on Cytodex® 1 microcarriers at a seeding density of 1000 x g / ml. The cells were cultured in a bioreactor at 37°C with 30% dissolved oxygen and a pH of 7.2. The cells were cultured in a 2-way single-use bioreactor system (AMBR250, Sartorius Stedim) in 0.2 L of VP-SFM AGT™ medium (Thermo Fisher Scientific) using pharmaceutical grade reagents and equipment at CO 2 and sodium bicarbonate was added to adjust the pH.

[0141] All cells were grown in the same bioreactor. After 72 hours from seeding the bioreactor, the cells were dissociated from the microcarriers using 12.5 U / mL trypsin (Roche) and mechanical agitation using the bioreactor impeller for 10 minutes. The dissociation process was inhibited using trypsin inhibitor (Sigma) in sodium citrate buffer. Visual inspection of the cell solution confirmed cell dissociation from the microcarriers. The total Cytodex® 1 microcarriers (Cytiva) concentration was increased to 4 g / L and the culture was continued for another 72 hours.

[0142] Medium exchange was performed 24 hours after each cell passage. Medium exchange was performed by temporarily stopping the impeller agitation to allow the cells attached to the microcarriers to settle by gravity before removing 80% of the total bioreactor volume from the surface of the solution in the bioreactor. Three and six independent bioreactors were tested for each control and ISG15- / - cell line (three shown for each passage), respectively. Consistent with static cell culture in flasks, the total cell number from the bioreactor containing ISG15- / - cells was lower compared to the bioreactor containing control cells. As shown in Figure 9A and Figure 9B, two-tailed ANOVA, Dunnett's multiple comparison test showed that the total cell number and cell viability were significantly lower in ISG15- / - cells compared to the control cells.

[0143] Example 8 - Kinetic analysis of virus production in bioreactors As discussed in Example 7 above, during characterization of the growth of ISG15- / - cells using microcarriers in bioreactors, all bioreactors were infected with RSV at a multiplicity of infection (MOI) of 0.01, representative of pharmaceutical industrial bioprocesses.

[0144] RSV infection was performed by temporarily stopping impeller agitation to allow the cells attached to the microcarriers to settle by gravity before removing 80% of the total bioreactor volume from the surface of the solution in the bioreactor. Fresh medium supplemented with 0.1% (vol / vol) SyntheChol® (Sigma Aldrich) was added to a final volume of 0.2 L. The virus stock was then added at an MOI of 0.01 and adjusted to the total viable cell count of each individual bioreactor, and impeller agitation was then resumed. During the infection period, the bioreactor temperature was reduced to 34° C. and the pH was increased to 7.3. Cultures were maintained for 4 days post-infection and daily sample analysis was performed, including total and viable cell counts. Infectious titers were tested by plaque assay at 72 hours post-infection. As shown in Figures 9A and 9B, control cells had significantly reduced viability compared to ISG15- / - cells at 72 hours post-infection, the standard time point for crude harvest following virus production.

[0145] Samples were tested for log infectious virus titer using a plaque assay in parallel from samples stored frozen in 1×HSG and at −80° C. until testing. Results are shown in FIG. 10A based on an unpaired two-tailed T-test. As shown in FIG. 10A, the log titer (PFU / mL) was significantly higher from the bioreactor using ISG15− / − cells compared to the control line cells after 72 hours. At 72 hours post-infection, the increase in virus particle production was 0.6967 log difference (P=0.0248) in infectious titer per liter of culture in crude harvest material for vaccine production. These results demonstrate that deletion of the CDS region of ISG15 from Vero cells can increase virus particle production and infectious virus particle production of RSV using pharmaceutical industrialization processes in bioreactors using microcarriers. The increase in virus particle production was approximately 300% difference in infectious titer per million cells in crude harvest material for vaccine production.

[0146] Due to the significant difference in total cell density during infection and virus production, an unpaired two-tailed T-test was used to determine the total virus production per million cells, as shown in Figure 10B. Considering that during virus production, the total cells in the bioreactors containing ISG15- / - were on average 32% less than those containing the control cell line, this virus productivity translated into an increase in virus particle production that was about 1.5 log difference in the infectious titer per million cells in the crude harvest material for vaccine production. See Figure 10B. The increase in virus particle production was about 1000% difference in the infectious titer per million cells in the crude harvest material for vaccine production.

[0147] Also, it should be noted that, as used in this disclosure and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Optional or optionally means that the subsequently described event or circumstance may or may not occur, and the description is meant to include the event or circumstance occurring or not occurring. For example, the phrase "a composition may optionally include a combination" means that the composition may or may not include a combination of different molecules, such that the description includes both the combination and the absence of the combination (i.e., the individual members of the combination). Ranges may be expressed herein as from about one particular value and / or to about another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent, it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges have meaning both in relation to the other endpoint, and independently of the other endpoint. All references cited in this disclosure are incorporated herein by reference in their entirety.

[0148] array Chlorocebus sabaeus APOA1: [ka]

[0149] Chlorocebus sabaeus CCL2: MKVSAALLCLLLIAATFSPQGLAQPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQIQTPKP (SEQ ID NO: 2)

[0150] Chlorocebus sabaeus CCL5: MKVSVAALAVILVATALCAPASASPYASDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEMS (SEQ ID NO: 3)

[0151] Chlorocebus sabaeus CYP19A1: [ka]

[0152] Chlorocebus sabaeus CXCL8: MTSKLAVALLAAFLLSAALCEGAVLPRSAKELRCQCIKTYSKPIHPKFIKELRVIESGPHCVNTEIIVKLSDGRELCLDPKVPWVSRVVEKFLKRAESQNS (SEQ ID NO: 5)

[0153] Chlorocebus sabaeus ELF3: [ka]

[0154] Chlorocebus sabaeus FOS: [ka]

[0155] Chlorocebus sabaeus HERC3: [ka]

[0156] Chlorocebus sabaeus HERC5: [ka]

[0157] Chlorocebus sabaeus IFIFT1: [ka]

[0158] Chlorocebus sabaeus IFIT2: [ka]

[0159] Chlorocebus sabaeus IFIFIT3: [ka]

[0160] Chlorocebus sabaeus IRF7: [ka]

[0161] Chlorocebus sabaeus ISG15: [ka]

[0162] Homo sapiens ISG15: [ka]

[0163] Mus musculus ISG15: [ka]

[0164] Canine ISG15: [ka]

[0165] Chlorocebus sabaeus KRT15: [ka]

[0166] Chlorocebus sabaeus KRT19: [ka]

[0167] Chlorocebus sabaeus MX1: [ka]

[0168] Chlorocebus sabaeus NGFR: [ka]

[0169] Chlorocebus sabaeus PTGS2: [ka]

[0170] Chlorocebus sabaeus PTPN6: [ka]

[0171] Chlorocebus sabaeus RET: [ka]

[0172] Chlorocebus sabaeus ROS1: [ka] [ka]

[0173] Chlorocebus sabaeus SFRP1: [ka]

[0174] Chlorocebus sabaeus SOX2: [ka]

[0175] Chlorocebus sabaeus SPPP1: [ka]

[0176] Chlorocebus sabaeus TNF: [ka]

[0177] Chlorocebus sabaeus TNFRSF4: [ka]

[0178] Chlorocebus sabaeus TRAF1: [ka]

[0179] Chlorocebus sabaeus VAV3: [ka]

[0180] Guide A: ACCAGCATTCGAGCAAGATCAAGG (SEQ ID NO: 33)

[0181] Guide B: GGAAACCGAAACTTGGCCACCGG (SEQ ID NO: 34)

[0182] sgRNA A forward primer GTCCCAGCTCTGCAGACATTA (SEQ ID NO: 35)

[0183] sgRNA A reverse primer GAGCTCGGCCAGGTTCTAAG (SEQ ID NO: 36)

[0184] sgRNA B forward primer CCTCGAGGCTGTAACTGCAA (SEQ ID NO: 37)

[0185] sgRNA B reverse primer ACCATAGGGGTGTTTTCCGT (SEQ ID NO: 38)

Claims

1. 1. An engineered cell line comprising a modification in the ISG15 gene, An engineered cell line, wherein the modification in the ISG15 gene increases total viral particle production and / or infectious viral particle production compared to a control cell line that is identical to the engineered cell line except for the modification in the ISG15 gene.

2. the increase in viral particle production is at least about 20% compared to the control cell line, e.g., at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%; 2. The engineered cell line of claim 1, wherein the ratio of infectious viral particle production to total viral particle production is increased by at least about 3%, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least 60%, compared to the control cell line.

3. 2. The engineered cell line of claim 1, wherein the modification in the ISG15 gene results in the ISG15 gene being deleted from the engineered cell line or the engineered cell line having reduced expression of the ISG15 gene compared to the control cell line.

4. The engineered cell line of any one of claims 1 to 3, wherein the engineered cell line is derived from monkey cells, such as Vero cells, or mouse cells.

5. 4. The engineered cell line of claim 3, wherein the ISG15 gene is deleted from the engineered cell line using a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system.

6. The virus may be an influenza virus, such as an influenza A virus or an influenza B virus; a dengue virus; a yellow fever virus; Respiratory syncytial virus (RSV); Herpes simplex virus; Human immunodeficiency virus (HIV); Hepatitis virus; Ko 6. The engineered cell line of any one of claims 1 to 3 and 5, wherein the host cell is selected from a virus selected from the group consisting of: a rabies virus, a rabies virus, a vesicular stomatitis virus (VSV), ...rabies virus, a vesicular stomatitis virus (VSV), a rab

7. 1. A method for increasing viral particle production, comprising: infecting an engineered cell line with a virus, said engineered cell line comprising a modification in an ISG15 gene, wherein said modification in said ISG15 gene increases total viral particle production and / or infectious viral particle production compared to a control cell line identical to said engineered cell line except for said modification in said ISG15 gene; incubating the engineered cell line under conditions suitable for production of the virus by the engineered cell line; and recovering the virus produced by the engineered cell line. A method comprising:

8. 8. The method of claim 7, wherein the engineered cell line increases viral particle production by at least about 20%, e.g., at least about 40%, at least about 50%, at least about 60%, or at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%, compared to the control cell line.

9. 8. The method of claim 7, wherein the modification in the ISG15 gene results in the deletion of the ISG15 gene from the engineered cell line or the engineered cell line has reduced expression of the ISG15 gene compared to the control cell line.

10. The method of any one of claims 7 to 9, wherein the engineered cell line is a Vero cell line.

11. 10. The method of any one of claims 7 to 9, wherein the virus is selected from influenza virus, dengue virus, yellow fever virus, RSV, herpes simplex virus, HIV, hepatitis virus, coronavirus, or a virus from the family Rhabdoviridae, such as rabies virus or VSV.

12. 1. A method for identifying genes to delete in a cell or cell line, comprising: infecting said cells or cell lines with a virus; detecting the expression level of a plurality of genes in said infected cell or cell line and comparing said expression level with the expression level of said plurality of genes in a control cell or cell line not infected with said virus; identifying gene targets that are differentially expressed in said infected cells or cell lines; analyzing the differentially expressed gene targets to identify one or more gene targets involved in a plurality of protein-protein networks, the plurality of protein-protein networks including at least two of a defense response, a response to a virus, a viral genome replication, a response to a cytokine, a response to type I interferon, regulation of viral genome replication, a defense response to a virus, cell death, a viral life cycle, negative regulation of viral genome replication, and a cellular response to a cytokine stimulus; and selecting at least one differentially expressed gene target to be deleted in said cell or cell line; Including, The method, wherein deletion of said at least one differentially expressed gene target increases viral particle production of said virus.

13. 13. The method of claim 12, further comprising deleting the at least one differentially expressed gene target from the cell or cell line using a clustered regularly interspaced short palindromic repeats (CRISPR) associated (Cas) system.

14. 14. The method of claim 12 or 13, wherein the cells or cell lines are Vero cells, Madin-Darby Canine (MDCK) cells, or human embryonic kidney (HEK) cells.

15. 14. The method of claim 12 or 13, wherein the virus is selected from an influenza virus, such as influenza A virus or influenza B virus; a dengue virus; a yellow fever virus; an RSV; a herpes simplex virus; an HIV; a hepatitis virus; a coronavirus; or a virus from the Rhabdoviridae family, such as a rabies virus or a VSV.

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