Vesicular stomatitis virus rescue

Abstract

The present invention relates to a method for rescuing vesicular stomatitis virus (VSV) from DNA in a HEK293 cell line or a HEK293 cell line adapted to suspension growth in cell culture, comprising the steps of: (a) providing cells from a HEK293 cell line or a HEK293 cell line adapted to suspension growth in cell culture; (b) transfecting the cells with at least one plasmid, wherein the at least one plasmid comprises: (i) an expression cassette comprising a vesicular stomatitis virus (VSV) genomic cDNA; (ii) at least one expression cassette encoding a VSV nuclear (N) protein, a VSV phospho (P) protein, and a VSV large (L) protein; and (iii) an expression cassette encoding an SV40 large T antigen; (c) culturing the transfected cells; and (d) recovering a cell culture supernatant comprising the rescued VSV. Also provided is the use of a HEK293 cell line or a HEK293 cell line adapted to suspension growth for rescue of Vesicular Stomatitis Virus (VSV), or the use of a plasmid encoding SV40 large T antigen for rescue of Vesicular Stomatitis Virus (VSV) in a HEK293 cell line or a HEK293 cell line adapted to suspension growth of HEK293-F cells using transient transfection.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a method for rescuing vesicular stomatitis virus (VSV) from DNA in a HEK293 cell line or a HEK293 cell line adapted to suspension growth in cell culture, the method comprising the steps of: (a) providing cells from a HEK293 cell line or a HEK293 cell line adapted to suspension growth in cell culture; (b) transfecting the cells with at least one plasmid, wherein the at least one plasmid comprises: (i) an expression cassette comprising a vesicular stomatitis virus (VSV) genomic cDNA; (ii) at least one expression cassette encoding a VSV nuclear (N) protein, a VSV phospho (P) protein, and a VSV large (L) protein; and (iii) an expression cassette encoding an SV40 large T antigen; (c) culturing the transfected cells; and (d) recovering a cell culture supernatant containing the rescued VSV. Also provided is the use of a HEK293 cell line or a HEK293 cell line adapted to suspension growth for the rescue of vesicular stomatitis virus (VSV), or the use of a plasmid encoding SV40 large T antigen for the rescue of vesicular stomatitis virus (VSV) in a HEK293 cell line or a HEK293 cell line adapted to suspension growth using transient transfection.

[0002] Background of the Invention Vesicular stomatitis virus (VSV) is a negative-sense single-stranded RNA (ssRNA) virus of the order Mononegavirales, which belongs to the family Rhabdoviridae, just like rabies virus. The negative-sense viral RNA is complementary to mRNA and must be converted to positive-sense RNA by RNA-dependent RNA polymerase before translation. Therefore, purified negative-sense RNA is not infectious because it must first be transcribed, which requires the RNA-dependent RNA polymerase contained in the virus particle (virion).

[0003] The recovery of complete negative-strand RNA viruses from cloned cDNA was one of the most exciting breakthroughs in RNA virology in the 1990s because it opened the door to direct engineering of viral genomes. Therefore, the recovery of complete negative-strand RNA viruses from cloned cDNA is a prerequisite for the use of recombinant viruses such as VSV in clinical settings, e.g., for gene therapy or as oncolytic viruses. It allows VSV to be engineered to carry cargo or to modify viral proteins, e.g., glycoproteins, to alter tropism or immune evasion.

[0004] Schnell et al. discovered the key to the recovery of nonsegmented negative RNA viruses that had eluded the field for many years (Roberts & Rose, Virology, 1998, 247, 1-6; Schnell et al., EMBO J., 1994, 13(18), 4195-4203). The method they described was as follows: Plasmids encoding the viral nucleoprotein (N) and polymerase subunits (L and P) were transfected into cells previously infected with a recombinant vaccinia virus expressing the T7 polymerase protein (vTF7-3). In addition to these plasmids, a plasmid encoding the full-length antigenomic viral RNA under the control of a T7 promoter at the 5' end and a self-cleaving ribozyme at the 3' end was also transfected into the cells. After transcription of RNA from the T7 promoter and translation of the encoded protein, nucleoproteins are assembled around the antigenomic RNA, and polymerase proteins then replicate these ribonucleoproteins (RNPs) to form RNPs containing the genomic RNA. After transcription and translation of mRNA from the genomic RNP, infectious virus is assembled. This procedure has led to the successful recovery of recombinant rabies virus. In 1995, the first successful recovery of vesicular stomatitis virus (VSV) from cDNA was reported using a very similar strategy (Lawson et al., 1995, Proc. Natl., Acad. Sci, USA 92(10), 4477-4481; Whelan et al., 1995, Proc. Natl., Acad. Sci, USA 92(18), 8388-8392).

[0005] Only minor adaptations to this system have been performed recently, such as stable expression of T7 polymerase protein in BHK cells or cotransfection of a plasmid encoding T7 polymerase protein under the control of a strong promoter. However, although VSV normally has a broad tropism conferred by VSV G, successful recovery of VSV from cDNA has been limited to very few cell lines, including BHK and HEK293T cells.

[0006] High titers of VSV need to be produced for clinical use, which requires production in suspension culture. The present inventors have discovered that HEK293 cell lines adapted for suspension growth, which are not susceptible to VSV recovery from cDNA, can be made susceptible to VSV recovery by transient cotransfection with a plasmid encoding the SV40 large T antigen. This allows VSV to be rescued from the same cell line used for large-scale VSV production.

[0007] Summary of the Invention The present invention relates to a method for rescuing vesicular stomatitis virus (VSV) from DNA in a HEK293 cell line or a HEK293 cell line adapted to suspension growth in cell culture, the method comprising: (a) providing cells from a HEK293 cell line or a HEK293 cell line adapted to suspension growth in cell culture; (b) transfecting the cells with at least one plasmid, wherein the at least one plasmid comprises: (i) an expression cassette containing vesicular stomatitis virus (VSV) genomic cDNA; (ii) at least one expression cassette encoding a VSV nuclear (N) protein, a VSV phospho (P) protein, and a VSV large (L) protein; and (iii) an expression cassette encoding an SV40 large T antigen; (c) culturing the transfected cells; and (d) recovering a cell culture supernatant containing the rescued VSV. Preferably, the recovered cell culture supernatant contains infectious VSV. In certain embodiments, the method may further comprise step (e), which comprises transducing an HEK293 cell line or cells derived from an HEK293 cell line adapted to growth in suspension with the VSV obtained in step (d); and, optionally, step (f), which comprises producing VSV in the cells of step (e) in a large-scale suspension culture, preferably in a volume of more than 50 L. Preferably, the HEK293 cell line or HEK293 cell line adapted to growth in suspension described in step (e) is the same cell line as the HEK293 cell line or HEK293 cell line adapted to growth in suspension described in step (a).

[0008] In certain embodiments, (i) the cells are provided, transfected, and cultured as adherent cells; (ii) the cells are transiently transfected in step (b); (iii) transfection of the cells in step (b) comprises use of a chemical-based transfection agent, preferably wherein the chemical-based transfection agent is selected from lipofection, polyethyleneimine, or calcium phosphate; or any combination of (i), (ii), or (iii).

[0009] In certain embodiments, the cells of step (b) are further transfected or transduced with a plasmid or helper virus comprising an expression cassette encoding bacteriophage T7 RNA polymerase under the control of an RNA polymerase II-dependent promoter; and wherein the expression cassette comprising VSV genomic cDNA comprises VSV genomic cDNA under the control of a T7 promoter sequence and a T7 transcription termination sequence; and optionally wherein at least one expression cassette encoding a VSV nucleoprotein, a VSV phosphoprotein, and a VSV large protein comprises the VSV nucleoprotein, phosphoprotein, and / or large protein under the control of a promoter sequence and a transcription termination sequence. In a preferred embodiment, the above method is a helper virus-free method, wherein the cells of step (b) are transfected with a plasmid comprising an expression cassette encoding bacteriophage T7 RNA polymerase under the control of an RNA polymerase II-dependent promoter. In certain embodiments, the bacteriophage T7 RNA polymerase has the amino acid sequence of SEQ ID NO: 4 or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4. In certain alternative or additional embodiments, the nucleotide sequence encoding the bacteriophage T7 RNA polymerase is codon-optimized.

[0010] At least one expression cassette encoding a VSV nucleoprotein, a VSV phosphoprotein, and a VSV large protein can be transfected as one or more helper plasmids, such as (i) a first helper plasmid containing an expression cassette comprising a sequence encoding a VSV nucleoprotein, preferably under the control of a promoter and a transcription termination sequence; (ii) a second helper plasmid containing an expression cassette comprising a sequence encoding a VSV phosphoprotein, preferably under the control of a promoter and a transcription termination sequence; and (iii) a third helper plasmid containing an expression cassette comprising a sequence encoding a VSV large protein, preferably under the control of a promoter and a transcription termination sequence; and (iv) at least one additional helper plasmid, optionally containing an expression cassette comprising a sequence encoding a VSV glycoprotein (G) and / or an expression cassette comprising a sequence encoding a VSV matrix (M) protein, preferably under the control of a promoter and a transcription termination sequence.

[0011] The expression cassette encoding the SV40 large T antigen according to the methods of the present invention is transfected as a plasmid comprising the expression cassette encoding the SV40 large T antigen and / or comprises a nucleic acid sequence encoding the SV40 large T antigen under the control of a promoter, and further comprises a transcription termination sequence, preferably under the control of a strong RNA polymerase II-dependent promoter, more preferably the cauliflower mosaic virus promoter or the CAG promoter. In a specific embodiment, the expression cassette comprises a nucleic acid sequence encoding the SV40 large T antigen having the amino acid sequence of SEQ ID NO:5 or at least 95% sequence identity to the amino acid sequence of SEQ ID NO:5.

[0012] The HEK293 cell line or HEK293 cell line adapted to suspension growth, including but not limited to, may be selected from the group consisting of HEK293, HEK293-F, HEK-293-H, Expi293F cells, and freestyle HEK293-F. Preferably, the cells are derived from a HEK293 cell line adapted to suspension growth, more preferably, the cells are derived from a HEK293 cell line adapted to suspension growth selected from the group consisting of HEK293-F, HEK-293-H, Expi293F, and freestyle HEK293-F. Even more preferably, the cells are HEK293-F cells.

[0013] The VSV genomic cDNA used in the methods of the present invention is a full-length viral genomic cDNA or a modified viral genomic cDNA. In a specific embodiment, the VSV genomic cDNA is a modified viral genomic cDNA encoding a modified G protein. In a specific embodiment, the VSV genomic cDNA is a modified viral genomic cDNA encoding a modified G protein, wherein the gene encoding glycoprotein G in the VSV genomic cDNA is replaced with a gene encoding glycoprotein GP of lymphocytic choriomeningitis virus (LCMV); preferably, the glycoprotein GP comprises the amino acid sequence set forth in SEQ ID NO: 7 or a functional variant that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7.

[0014] In a particular embodiment, the SV40 large T antigen encoded by the expression cassette in the methods described herein has the amino acid sequence of SEQ ID NO:5 or has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:5.

[0015] In another aspect, the present invention relates to the use of HEK293 cell lines or HEK293 cell lines adapted to suspension growth for the rescue of vesicular stomatitis virus (VSV) using transient transfection of at least one plasmid containing (i) an expression cassette comprising VSV genomic cDNA, and (ii) at least one expression cassette encoding the VSV core (N) protein, the VSV phospho (P) protein, and the VSV large (L) protein, and an expression cassette encoding the SV40 large T antigen.

[0016] In yet another aspect, the present invention relates to the use of a plasmid encoding vesicular stomatitis virus (VSV) SV40 large T antigen for rescue of VSV in HEK293 cell lines or HEK293 cell lines adapted for suspension growth using transient co-transfection with at least one plasmid comprising (i) an expression cassette comprising VSV genomic cDNA and (ii) at least one expression cassette encoding VSV core (N) protein, VSV phospho (P) protein, and VSV large (L) protein. [Brief explanation of the drawings]

[0017] [Figure 1] Schematic overview of VSV rescue in HEK293-F cells. [Figure 2] Flow diagram of the VSV rescue and amplification process. [Figure 3]Expression of viral proteins after transfection in HEK293T cells (lanes 1-3) and HEK293-F cells (lanes 4-6) on Western blots stained with anti-P protein polyclonal antibody (top) or general serum against VSV (bottom). As a positive control, a preparation of purified VSV-GP virions (D-106-442) at 2.36 x 10 TCID (50% tissue culture infectious dose) / ml is shown at 1.0 μl and 0.2 μl in lanes 7 and 9, respectively. P, N, and P / M indicate the expected positions of the P protein, N protein, and mixed band containing the P and M proteins, respectively. NPL-Helper generally refers to the plasmids pCAG VSV-N, pCAG VSV-P, and pCAG VSV-L, T7-Pol refers to the plasmid pCAGGS T7-RNAP IRES Puro, and VSV-GP refers to the plasmid containing the VSV genomic cDNA of VSV-GP. [Figure 4A] Transient transfection of HEK293-F cells with or without SV40 large T antigen for VSV rescue. HEK293T and HEK293-F cells were transfected with the plasmids pVSV-LCMV GP (VSV-GP), pCAG VSV-N, pCAG VSV-P, and pCAG VSV-L (VSV-N, -P, -L), and pCAGGS T7-RNAP IRES Puro, with or without cotransfection of the plasmid pCAG SV40 large T (1 μg or 5 μg SV40 large T) (no SV40 large T). (A) Representative bright-field microscopy images are shown 4 h post-transfection (4 h pt, top row), and representative dark-field microscopy images are shown 48 h post-transfection (48 h pt, middle row), and 72 h post-transfection (72 h pt, bottom row). On the left-hand side, untransfected HEK293T cells (top) and HEK293-F cells (bottom) are shown as dark-field images, along with a bright-field image in the upper left corner for comparison. [Figure 4B]Transient transfection of HEK293-F cells with or without SV40 large T antigen for VSV rescue. HEK293T and HEK293-F cells were transfected with the plasmids pVSV-LCMV GP (VSV-GP), pCAG VSV-N, pCAG VSV-P, and pCAG VSV-L (VSV-N, -P, -L), and pCAGGS T7-RNAP IRES Puro, with or without cotransfection of the plasmid pCAG SV40 large T (1 μg or 5 μg of SV40 large T). (B) Supernatant passages from harvests of HEK293-F and HEK293T cells 48 h posttransfection and 72 h posttransfection were further analyzed for infection of BHK21C1.13 cells. Representative dark-field microscopy images 48 hours postinfection with the harvest supernatants from (A) above or with fresh medium as a negative control are shown (left-hand column). A (-) symbol below a column indicates no VSV rescue, and a (+) symbol below a column indicates successful VSV rescue. A clear cytopathic effect (CPE) was visible in BHK21C1.13 cells infected with 48- and 72-hour posttransfection harvest supernatants from HEK293T and HEK293-F cells transiently transfected with SV40 large T antigen, whereas no cytopathic effect was observed after infection with 48- and 72-hour posttransfection harvest supernatants from HEK293-F cells in the absence of SV40 large T antigen. [Figure 5A]Transient transfection of HEK293-F cells with or without SV40 large T antigen for VSV rescue, performed in an independent experiment. HEK293-F cells were transfected with the plasmids pVSV-LCMV GP (VSV-GP), pCAG VSV-N, pCAG VSV-P, and pCAG VSV-L (VSV-N, -P, -L), and pCAGGS T7-RNAP IRES Puro, with or without cotransfection of the plasmid pCAG SV40 large T (1 μg or 5 μg of SV40 large T). (A) Representative dark-field microscopy images at 4 h posttransfection (top row), 48 h posttransfection (middle row), and 72 h posttransfection (bottom row), as well as the respective bright-field images, are shown in the upper left corner. [Figure 5B]Independent experiments were performed on HEK293-F cells for VSV rescue using transient transfection of SV40 large T antigen + / -. HEK293-F cells were transfected with the plasmids pVSV-LCMV GP (VSV-GP), pCAG VSV-N, pCAG VSV-P, and pCAG VSV-L (VSV-N, -P, -L), and pCAGGS T7-RNAP IRES Puro, with or without cotransfection of the plasmid pCAG SV40 large T (1 μg or 5 μg of SV40 large T). (B) Passages of supernatants from harvests of HEK293-F cells at 4 h posttransfection (top row), 48 h posttransfection (middle row), and 72 h posttransfection (bottom row) were further analyzed for infection in adherent HEK293-F cells. Representative microscopy images are shown at 24 h postinfection (pi) using supernatants from harvests at 4 h posttransfection (top row), 48 h posttransfection (middle row) as dark-field images, and supernatants from harvests at 72 h posttransfection (bottom row) as bright-field images. A (-) symbol below a row indicates no VSV rescue, and a (+) symbol below a row indicates successful VSV rescue. Clear cytopathic effect (CPE) was visible in HEK293-F cells infected with supernatants from 48 and 72 h posttransfection harvests from HEK293-F cells transiently transfected with 5 μg of SV40 large T antigen, whereas no cytopathic effect was observed after infection with supernatants from 48 and 72 h posttransfection harvests from HEK293-F cells transiently transfected with 1 μg of SV40 large T antigen or in the absence of SV40 large T antigen. Dark-field images of HEK293-F negative control cells are shown on the left.

[0018] Detailed Description of the Invention The present invention relates to the rescue of vesicular stomatitis virus (VSV) or genetically modified forms thereof from cDNA using transfection (e.g., CaPO4-mediated transfection) of HEK293 cells or suspension-adapted HEK293 cells, and the generation of viral seed stocks following viral rescue.

[0019] The general embodiments "comprise" or "comprising" encompass the more specific embodiment "consisting of." Furthermore, the singular and plural forms are not used in a limiting sense. As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural, unless expressly stated to refer to the singular only.

[0020] The term "protein" is used synonymously with "amino acid sequence" or "polypeptide" and refers to a polymer of amino acids of any length. These terms also include proteins that have been post-translationally modified through reactions including, but not limited to, glycosylation, acetylation, phosphorylation, glycation, or proteolytic processing. Modifications and changes, such as amino acid sequence substitutions, deletions, or insertions, can be made in the structure of a polypeptide, yet the molecule maintains its biologically functional activity. For example, specific amino acid sequence substitutions can be made in a polypeptide or its underlying nucleic acid coding sequence to obtain a protein with the same properties.

[0021] Vesicular stomatitis virus (VSV) is a negative-sense, single-stranded RNA (ssRNA) virus of the order Mononegavirales, which belongs to the Rhabdoviridae family along with rabies virus. Negative-sense viral RNA is complementary to mRNA and must be converted to positive-sense RNA by RNA-dependent RNA polymerase before translation. Therefore, purified negative-sense RNA is not infectious because it must first be transcribed, which requires RNA-dependent RNA polymerase contained in the virus particle (virion). Because RNA sequences are reverse-transcribed for sequencing, sequences of recombinant RNA viruses are generally provided as cDNA sequences.

[0022] The negative-strand ssRNA genome of VSV contains five open reading frames encoding, from 3' to 5', the N protein, P protein, M protein, G protein, and L protein. The nucleoprotein (N protein) is the main component of the nucleocapsid and is required for the initiation of genome synthesis. The large protein (L protein) is an RNA-dependent RNA polymerase that binds to the phosphorylated protein (P protein) to catalyze mRNA replication. The matrix protein (M protein) is associated with the inner surface of the viral membrane, and the glycoprotein (G protein) is a glycosylated transmembrane protein localized within the viral membrane that enables the virus to enter host cells.

[0023] The present invention relates to the rescue of vesicular stomatitis virus (VSV) from DNA in HEK293 cell lines or HEK293 cell lines adapted for suspension growth, which are typically used for large-scale virus production, e.g., for therapeutic applications. While there are several VSV serotypes, the best-characterized and therapeutically used VSV serotype is VSV Indiana (VSIV). All sequences disclosed and used herein are derived from VSIV. Because VSV Indiana is an RNA virus, several complete genomic nucleotide sequences are available, one example being the cDNA sequence of SEQ ID NO: 6 (GenBank Accession No. MH919398.1). The VSV N protein, P protein, or L protein is preferably VSIV, e.g., having the amino acid sequence of SEQ ID NO: 1, 2, or 3, respectively, or a sequence having at least 80%, 85%, 90%, or more preferably at least 95% sequence identity thereto.

[0024] As used herein, the term "VSV rescue" refers to the recovery of negative-strand RNA virus from DNA, such as plasmid DNA. The goal is to produce infectious VSV that can be used to transduce cells for analysis or large-scale production. As used herein, the term "infectious VSV" refers to VSV particles that infect cells susceptible to VSV infection, such as BHK cells or HEK293 cells or their derivatives. To confirm the production of infectious virus, a passage of the supernatant from the harvest after transfection (e.g., 48 or 72 hours post-transfection (pi)) is added to VSV-susceptible cells (also called seed stock infections, P0 infections) and analyzed microscopically for cytopathic effect (CPE) 48 hours post-infection (pi).

[0025] As used herein, the term "genomic RNA" refers to the inherited genetic information of an RNA virus. Those skilled in the art will understand that an RNA virus genome may be provided as a DNA sequence within a vector, such as a plasmid. In the context of the present invention, the VSV genome is provided as a "VSV genomic cDNA." This means that it is provided as a DNA sequence within a vector, such as a plasmid. The RNA genome is then produced within the host cell after transfection via transcription. Typically, the vector contains an expression cassette containing a VSV genomic cDNA under the control of a promoter and further containing at least one transcription termination sequence. Furthermore, the VSV genomic cDNA typically encodes the minus strand of the genome, which is transcribed in situ into VSV antigenome RNA ((+)strand RNA). Preferably, the VSV genomic cDNA is under the control of a T7 promoter and further contains a T7 transcription termination sequence. Complementary DNA (cDNA) is DNA synthesized from single-stranded RNA. In the case of VSV genomic cDNA, the cDNA is synthesized from VSV genomic RNA.

[0026] As used herein, the term "gene" refers to a DNA or RNA locus of an inherited genomic sequence that influences the characteristics of an organism by being expressed as a functional product or by regulating gene expression. Genes and polynucleotides can include introns and exons, as in a genomic sequence, or only coding sequences, such as an open reading frame (ORF), including a start codon (methionine codon) and a translation stop codon, as in cDNA. Genes and polynucleotides can also include regions that regulate their expression, such as transcription initiation, translation, and transcription termination. Thus, regulatory sequences, such as promoter sequences and transcription termination sequences, are also included.

[0027] As used herein, the terms "nucleic acid," "nucleotide," and "polynucleotide" are used synonymously and refer to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end, including double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA, negative and positive orientation), double-stranded RNA (dsRNA), genomic DNA, cDNA, cRNA, recombinant DNA, or recombinant RNA and derivatives thereof, such as those containing modified backbones.

[0028] As used herein, the terms "ribonucleic acid," "RNA," or "RNA oligonucleotide" refer to a molecule consisting of a nucleotide sequence, which is constructed from nucleic acid bases, ribose sugars, and phosphate groups. RNA is usually a single-stranded molecule and can perform a variety of functions. The term ribonucleic acid specifically includes messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA), each of which plays a specific role in biological cells. It also includes small non-coding RNAs, such as microRNA (miRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), and Piwi-interacting RNA (piRNA). The term "non-coding" means that the RNA molecule is not translated into an amino acid sequence. The term "coding" means that the RNA molecule is translated into an amino acid sequence.

[0029] The term "coding strand" or "plus strand" refers to the RNA strand that encodes the protein.

[0030] The terms "non-coding strand," "antisense strand," or "negative sense strand" or "negative strand" refer to the RNA strand that must be transcribed by an RNA-dependent RNA polymerase into positive-sense RNA before translation.

[0031] A "vector" is a nucleic acid that can be used to introduce a heterologous polynucleotide into a cell. One type of vector is a "plasmid," which refers to a linear or circular double-stranded DNA molecule into which additional nucleic acid segments can be ligated. Other types of vectors (e.g., retroviruses, adenoviruses, adeno-associated viruses, VSV, and MeV replication-defective or active forms) allow additional DNA or RNA segments to be introduced into the viral genome. Introduction of nucleic acid is commonly referred to as transfection, while introduction of nucleic acid via viral infection is commonly referred to as transduction.

[0032] The terms "encode" and "encoding" broadly refer to any process using information within a polymeric macromolecule to direct the production of a second molecule that differs from the first. The second molecule may have a chemical structure that differs from the chemical makeup of the first molecule. For example, the term "encode" refers to the semi-conservative DNA replication process in which one strand of a double-stranded DNA molecule is used as a template to encode a newly synthesized complementary sister strand by a DNA-dependent DNA polymerase. Furthermore, a DNA molecule can encode an RNA molecule (e.g., by using a DNA-dependent RNA polymerase), or an RNA molecule (minus strand) can encode an RNA molecule (plus strand) (e.g., by using an RNA-dependent RNA polymerase). An RNA molecule (plus strand) can also encode a polypeptide, as in the process of translation. When used to describe the translation process, the term "encode" also extends to triplet codons that encode amino acids. An RNA molecule can also encode a DNA molecule, for example, by the process of reverse transcription using an RNA-dependent DNA polymerase. When referring to a DNA molecule encoding a polypeptide, the processes of transcription and translation are referred to.

[0033] As used herein, the term "expression" refers to the transcription and / or translation of a (heterologous) nucleic acid sequence in a host cell. The expression level of a gene product of interest in a host cell can be determined based on either the amount of corresponding mRNA (or positive-strand RNA) present in the cell or the amount of a polypeptide encoded by a selected sequence. For example, RNA transcribed from a selected sequence can be quantified by Northern blot hybridization, ribonuclease RNA protection, in situ hybridization to intracellular RNA, or PCR, e.g., quantitative PCR. The protein encoded by a selected sequence can be quantified by various methods, such as ELISA, Western blot, radioimmunoassay, immunoprecipitation, assaying for protein biological activity, FACS analysis after immunostaining of the protein, or homogeneous time-resolved fluorescence (HTRF) assay. The product of interest encoded by a heterologous nucleic acid sequence can also be a non-coding RNA. The expression level of non-coding RNA, such as miRNA, siRNA, or shRNA, can be quantified by PCR, e.g., quantitative PCR.

[0034] As used herein, the term "expression cassette" refers to the individual components of a DNA molecule involved in RNA synthesis, including a sequence to be expressed and regulatory sequences, typically at least a promoter sequence and a transcription termination sequence. If the final product is a protein, the sequence to be expressed includes an open reading frame encoding the protein. According to the present invention, the expression cassette encodes one or more proteins or a VSV genomic cDNA. The expression cassette is typically part of a vector, such as a plasmid or viral vector.

[0035] The term "gene product" refers to both mRNA polynucleotides and polypeptides encoded by a gene or DNA polynucleotide.

[0036] As used herein, the term "HEK293 cell line" refers to an adherent human cell line that originated from human embryonic kidney and was first immortalized in 1973 by integration of a 4-kbp adenovirus 5 (ad5) genomic fragment containing the E1A and E1B genes of chromosome 19 (Graham et al., J. Gen. Virol. (1977) 36: 59-72; Malm et al., Nature research, Scientific Reports (220) 10:18996). This cell line can be obtained, for example, from ATCC and DSMZ (ATCC-CRL-1573; DSMZ No: ACC305; RRID:CVCL_0045). This cell line may also be referred to as the parent HEK293 cell line or parent HEK293 cell line. Those skilled in the art will understand that the term HEK293 cell line, as used herein, includes subclones thereof. The term "HEK293 cell line adapted to suspension growth" refers to a cell line clonally derived from a parent HEK293 cell line adapted to high-density suspension growth in serum-free medium, allowing for large-scale cultivation and biological production of therapeutic proteins or viruses in bioreactors. These include, but are not limited to, industrially relevant suspension cell lines HEK293-F cells, HEK293-H cells, and Style-Free HEK293-F cells. Free-Style HEK293-F cells are adapted to suspension culture in Free-Style™ 293 Expression Medium, which can be obtained, for example, from Thermo Fisher Scientific (R79007; RRID:CVCL_D603). HEK293-F cells and HEK293-H cells were prepared by clonal selection from HEK293 cells due to their rapid growth in serum-free medium (SFM), excellent transfection efficiency, and high-level protein expression, and can be obtained, for example, from Thermo Fisher Scientific (HEK293-F: 11625019, RRID:CVCL_6642; HEK293-H: 11631017, RRID:CVCL_6643).The HEK293-H line is a variant that, when grown in serum-supplemented medium, demonstrates better adhesion in monolayer cultures and amenability for plaque assays and other anchorage-dependent applications. HEK293-F and HEK-293-H cells are provided in Gibco® CD293 medium. Other HEK293 cell lines adapted to suspension growth in cell culture include, but are not limited to, HEK293.2sus (ATCC CRL-1573.3), HEK293-SF-3F6 (ATCC CRL-12585; RRID: CVCL_4V94), Expi293F (ThermoFisher A14527 / A14528 / 100044202 (cGMP banked); RRID: CVCL_D615), and HEK293-S (Ximbio154155; RRID: CVCL_A784). HEK293 cell lines adapted to suspension growth may also be referred to as "293 cells adapted to serum-free medium."

[0037] The present invention relates to a method for rescuing vesicular stomatitis virus (VSV) from DNA in a HEK293 cell line or a HEK293 cell line adapted to suspension growth in cell culture, comprising the steps of: (a) providing cells from a HEK293 cell line or a HEK293 cell line adapted to suspension growth in cell culture; (b) transfecting the cells with at least one plasmid, wherein the at least one plasmid comprises: (i) an expression cassette comprising a VSV genomic cDNA; (ii) at least one expression cassette encoding a VSV nuclear (N) protein, a VSV phospho (P) protein, and a VSV large (L) protein; and (iii) an expression cassette encoding an SV40 large T antigen; (c) culturing the transfected cells; and (d) recovering a cell culture supernatant containing the rescued VSV. The cell culture supernatant can be harvested at any time after transfection, preferably 24 to 96 hours after transfection, more preferably 48 to 72 hours after transfection. The cells prepared in cell culture medium are selected from the HEK293 cell line or an HEK293 cell line adapted to suspension growth. Preferably, the cells prepared in cell culture medium are an HEK293 cell line adapted to suspension growth. The HEK293 cell line can be any parent HEK293 cell line that has been adapted to grow efficiently in suspension, typically in the absence of serum, and maintains a high virus production capacity (e.g., specific cell productivity) comparable to that of the parent HEK293 cell line. Suitable HEK293 cell lines adapted to suspension growth include, but are not limited to, HEK293-F cells, HEK293-H cells, freestyle HEK293-F cells, HEK293-SF-3F6 cells, Expi293F cells, HEK293.2sus cells, and HEK293-S cells. Preferred HEK293 cell lines adapted to suspension growth in the context of the present invention are HEK293-F cells, HEK293-H cells, freestyle HEK293-F cells, and Expi293F cells, more preferably HEK293-F cells or Expi293F cells, and even more preferably HEK293-F cells. In a preferred embodiment, the harvested cell culture supernatant contains infectious VSV.Infectious particles can be determined by adding the collected cell culture supernatant to cells susceptible to VSV infection, such as BHK cells or HEK293 cells or their derivatives. Cells susceptible to VSV infection include the HEK293 cell lines described herein and HEK293 cell lines adapted for suspension growth, as well as other HEK293 derivatives, including, but not limited to, HEK293T cells and HEK293E cells. Suitable BHK cells include, but are not limited to, BHK-21C1.13 cells (ATCC CCL-10; RRID:CVCL_1915). Infectious virus is detected microscopically approximately 48 hours postinfection for cytopathic effect (CPE). Thus, in certain embodiments, infectious particles are determined by passaging virions, in which (i) harvested cell culture supernatant is added to cells susceptible to VSV infection, preferably BHK21 cells, e.g., BHK-21C1.13 cells or HEK293-F cells, and (ii) infectious virus is detected microscopically 48 hours post-infection for cytopathic effects.

[0038] An expression cassette containing a VSV genomic cDNA typically encodes the VSV genome in reverse orientation and further comprises a promoter sequence and a transcription termination sequence. At least one expression cassette encoding a VSV nucleoprotein (N), VSV phosphoprotein (P), and VSV large (L) protein comprises sequences encoding the VSV nucleoprotein, phosphoprotein, and / or large protein and further comprises a promoter sequence and a transcription termination sequence. As used herein, the term "at least one expression cassette" indicates that the nucleoprotein, VSV phosphoprotein, and large protein may be encoded by sequences within the same expression cassette, or by separate expression cassettes or a combination thereof. When more than one protein is encoded by an expression cassette, the sequences encoding the more than one protein are linked by sequences that allow for cap-independent translation initiation, such as an internal ribosome entry site (IRES). Furthermore, when the nuclear protein, phosphoprotein, or large protein are encoded by separate expression cassettes, i.e., three or more expression cassettes, the expression cassettes may be present on the same plasmid and / or on separate plasmids. The expression cassette encoding the SV40 large T antigen comprises a sequence encoding the SV40 large T antigen as well as a promoter sequence and a transcription termination sequence. (i) An expression cassette comprising a VSV genomic cDNA, (ii) at least one expression cassette encoding the nuclear protein, phosphoprotein, and large protein, and (iii) an expression cassette comprising the SV40 large T antigen may be present on one, two, three, four, or five plasmids. Preferably, the expression cassette (i), at least one expression cassette (ii), and expression cassette (iii) are present on separate plasmids. More preferably, the at least one expression cassette (ii) comprises at least three expression cassettes, i.e., a first expression cassette encoding the nuclear protein, a second expression cassette encoding the phosphoprotein, and a third expression cassette encoding the large protein.The first, second, and third expression cassettes may be present on one plasmid, or on three separate plasmids, or on two plasmids (one containing two of the expression cassettes and the other containing one of the expression cassettes), which may be derived from the same plasmid or from different plasmids.

[0039] In certain embodiments, the nucleoprotein, phosphoprotein, and large protein are derived from VSV Indiana (VSIV). In certain preferred embodiments, the nucleoprotein has the amino acid sequence of SEQ ID NO: 1 or a sequence having at least 95% sequence identity thereto, the phosphoprotein has the amino acid sequence of SEQ ID NO: 2 or a sequence having at least 95% sequence identity thereto, and / or the large protein has the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 95% sequence identity thereto. Those skilled in the art will understand that a protein having at least 95% sequence identity to the recited sequence of a particular protein is a functional homolog of that particular protein. For example, a nucleoprotein comprising a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1 is a functional homolog of the nucleoprotein having the amino acid sequence of SEQ ID NO: 1. Thus, homologous proteins further exhibit the same or similar protein activity as the original sequence.

[0040] In a specific embodiment, the method according to the present invention further comprises a step (e) comprising transducing cells derived from an HEK293 cell line or an HEK293 cell line adapted to suspension growth in suspension with the VSV obtained in step (d); and a step (f) comprising producing VSV in the cells of step (e), optionally on a large scale, preferably in a suspension culture of more than 50 L. Preferably, the HEK293 cell line or HEK293 cell line adapted to suspension growth described in step (e) is the same cell line as the HEK293 cell line or HEK293 cell line adapted to suspension growth described in step (a). Although VSV can be produced in adherent HEK293 cells, virus production is typically more efficient in suspension, particularly in large-scale culture, due to the higher cell density, which also allows virus production in the absence of serum. Preferably, the HEK293 cell line or HEK293 cell line adapted to suspension growth described in steps (e) and (a) is selected from the group consisting of HEK293-F cells, HEK293-H cells, freestyle HEK293-F cells, and Expi293F cells; more preferably, the HEK293 cell line or HEK293 cell line adapted to suspension growth described in steps (e) and (a) is HEK293-F cells or Expi293F cells, even more preferably HEK293-F cells. As used herein, the term "large scale" refers to a culture volume of more than 5 L, preferably more than 10 L, more preferably more than 25 L, and even more preferably more than 50 L.

[0041] Transfection can be performed in adherent cells or in suspension, but is typically performed in adherent cells for higher transfection efficiency. In certain embodiments, the cells in step (a) are provided, transfected, and cultured as adherent cells. HEK293 cell lines adapted to suspension growth can be made to adhere by adding fetal calf serum (FCS) to the culture medium in the range of 1 to 12%, preferably 3 to 10%, more preferably 5 to 10% (v / v). Typically, transient transfection is more efficient in adherent cells than in suspension cells. In certain embodiments, the cells are transiently transfected in step (b), and preferably, adherent cells are transiently transfected in step (b). Transfection of cells in step (b) can involve the use of a chemical reaction-based transfection agent, such as lipofection (lipid transfection), polyethyleneimine (PEI), DEAE-dextran, or calcium phosphate transfection, preferably calcium phosphate transfection. The cells are preferably transiently transfected using a chemical-based transfection agent, more preferably, adherent cells are transiently transfected using a chemical-based transfection agent.

[0042] In certain embodiments, the cells of step (b) are further transfected or transduced with a plasmid or helper virus comprising an expression cassette encoding bacteriophage T7 RNA polymerase under the control of an RNA polymerase II-dependent promoter, wherein the expression cassette comprising VSV genomic cDNA comprises VSV genomic cDNA under the control of a T7 promoter sequence and a T7 transcription termination sequence, and optionally, at least one expression cassette encoding a VSV nucleoprotein, a VSV phosphoprotein, and a VSV large protein comprises a sequence encoding the VSV nucleoprotein, phosphoprotein, and / or large protein under the control of a promoter sequence and a transcription termination sequence. The RNA polymerase II-dependent promoter in the expression cassette encoding bacteriophage T7 RNA polymerase is preferably a strong promoter, such as a cauliflower mosaic virus promoter or a CAG promoter. The CAG promoter contains the cytomegalovirus (CMV) early enhancer sequence (C), the promoter, first exon and first intron (A) of the chicken β-actin gene, and the splice acceptor (G) of the rabbit β-globin gene (Niwa H et al., (1991) Gene 108(2): 193-9). The term "T7 transcription termination sequence" may also include more than one T7 transcription termination sequence, for example, two or three T7 transcription termination sequences, preferably two T7 transcription termination sequences. The expression cassette further contains a transcription termination sequence and, optionally, a marker gene separated by an IRES sequence. Typical helper viruses used for the expression of bacteriophage T7 RNA polymerase include, but are not limited to, vaccinia virus. In a preferred embodiment, the method is a helper virus-free method, wherein the cells in step (b) are transfected with a plasmid containing an expression cassette encoding bacteriophage T7 RNA polymerase under the control of an RNA polymerase II-dependent promoter.In certain embodiments, the bacteriophage T7 RNA polymerase has the amino acid sequence of SEQ ID NO: 4 or has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4. In certain alternative or additional embodiments, the nucleotide sequence encoding the bacteriophage T7 RNA polymerase is codon-optimized.

[0043] At least one expression cassette encoding a VSV nucleoprotein, a VSV phosphoprotein, and a VSV large protein can be transfected as one or more helper plasmids, such as (i) a first helper plasmid containing an expression cassette comprising a sequence encoding a VSV nucleoprotein, preferably under the control of a promoter and a transcription termination sequence; (ii) a second helper plasmid containing an expression cassette comprising a sequence encoding a VSV phosphoprotein, preferably under the control of a promoter and a transcription termination sequence; and (iii) a third helper plasmid containing an expression cassette comprising a sequence encoding a VSV large protein, preferably under the control of a promoter and a transcription termination sequence; and (iv) optionally, at least one additional helper plasmid containing an expression cassette comprising a sequence encoding a VSV glycoprotein (G) and / or an expression cassette comprising a sequence encoding a VSV matrix (M) protein, preferably under the control of a promoter and a transcription termination sequence. Suitable promoters for expression cassettes containing sequences encoding VSV-N, -P, -L, G and / or M are strong promoters, preferably strong RNA polymerase II-dependent promoters, such as CMV or CAG.

[0044] The expression cassette encoding the SV40 large T antigen described in the methods of the present invention is transfected as a plasmid containing the expression cassette encoding the SV40 large T antigen. The expression cassette comprises a sequence encoding the SV40 large T antigen under the control of a promoter and further comprises a transcription termination sequence, preferably under the control of a strong RNA polymerase II-dependent promoter, more preferably a CMV promoter or a CAG promoter. In a specific embodiment, the SV40 large T antigen encoded by the expression cassette in the methods of the present invention has the amino acid sequence of SEQ ID NO:5 or has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:5. Thus, in a specific embodiment, the expression cassette comprises a nucleic acid sequence encoding the SV40 large T antigen having the amino acid sequence of SEQ ID NO:5 or having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:5.

[0045] The HEK293 cell line or HEK293 cell line adapted to suspension growth may be selected from the group consisting of, but not limited to, HEK293, HEK293-F, HEK-293-H, Expi293F, and freestyle HEK293-F. Preferably, the cells are derived from an HEK293 cell line adapted to suspension growth, and more preferably, the cells are derived from an HEK293 cell line adapted to suspension growth selected from the group consisting of HEK293-F, HEK-293-H, Expi293F, and freestyle HEK293-F. Even more preferably, the HEK293 cell line adapted to suspension growth is HEK293-F cells. An advantage of using HEK293, particularly an HEK293 cell line adapted to suspension growth, is that the same cell line can be used for VSV rescue from VSV genomic cDNA (typically performed using adherent cells) and for VSV production in suspension cell culture. This allows the entire production process to be carried out in a single cell line, which simplifies regulatory approval.

[0046] The VSV genomic cDNA described in the methods of the present invention may be a full-length viral genome cDNA or a modified viral genome cDNA. A full-length viral genome cDNA provides a wild-type virus, such as VSV Indiana. The VSV genomic cDNA may also be a modified viral genome cDNA. For example, the glycoprotein (G) may be replaced with a glycoprotein from a different (heterologous) virus, such as a glycoprotein from lymphocytic choriomeningitis virus (LCMV). Glycoprotein replacement may alter the tropism of the virus as well as other characteristics of the virus, such as avoiding neuroinflammation or immunogenicity associated with wild-type viruses. A VSV containing an LCMV glycoprotein may also be referred to as VSV-GP. In a specific embodiment, the VSV genomic cDNA described in the methods of the present invention is a modified viral genome cDNA (modified VSV genomic cDNA). Modified VSV genomic cDNAs include, but are not limited to, VSV genomic cDNAs in which the gene encoding the G protein has been replaced with another viral receptor, such as the glycoprotein GP of lymphocytic choriomeningitis virus (LCMV), the glycoprotein of Dandenong virus (DANDV) or Mopeia virus (MOPV) (as described in detail in WO 2020 / 104694), or a glycoprotein of an arenavirus. In a preferred embodiment, the glycoprotein G in the VSV genomic cDNA is replaced with the glycoprotein GP of lymphocytic choriomeningitis virus (LCMV), preferably from the WE-HPI strain. Such a VSV is described, for example, in WO 2010 / 040526 and is designated VSV-GP. The glycoprotein GP of lymphocytic choriomeningitis virus (LCMV) can be GP1 or GP2, but can also include glycoproteins from different LCMV strains. In particular, LCMV-GP can be derived from LCMV wild type or the LCMV strains LCMV-WE, LCMV-WE-HPI, LCMV-WE-HPI opt.In a preferred embodiment, the gene encoding the glycoprotein GP of LCMV encodes a protein having the amino acid sequence of SEQ ID NO:7 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:7, while maintaining the functional properties of VSV, including the glycoprotein GP, ​​which encodes the amino acid sequence as set forth in SEQ ID NO:7.

[0047] Furthermore, the modified viral genome cDNA can contain additional genes, for example, in intergenic loci of the genome cDNA. The additional genes are preferably heterologous genes encoding heterologous proteins. Thus, in one embodiment, the VSV genome cDNA further encodes at least one heterologous protein, such as a therapeutic protein, and an antigen, such as a tumor-specific or tumor-associated antigen, or a reporter gene.

[0048] As used herein, the term "heterologous polypeptide" or "heterologous protein" refers to a protein derived from a different organism or species from the recipient, i.e., an RNA virus, such as VSV. In the context of the present invention, those skilled in the art will understand that it refers to a protein that is not naturally expressed by VSV. The term "heterologous" when used in reference to a portion of a protein can also indicate that the protein comprises two or more amino acid sequences that are not found in the same relationship to each other in nature. In the context of the present invention, this is typically a therapeutic protein, an antigen, such as a tumor-specific or tumor-associated antigen, or a reporter (e.g., luciferase or a fluorescent protein). A heterologous polypeptide is encoded by a heterologous nucleic acid sequence or gene.

[0049] The term "therapeutic protein" refers to proteins that can be used in the medical treatment of humans and / or animals, including, but not limited to, antibodies, growth factors, blood clotting factors, cytokines such as interferons and interleukins, chemokines and hormones, preferably growth factors, cytokines, chemokines and antibodies.

[0050] The term "cytokine" refers to small proteins released by cells that act as intracellular mediators, for example, influencing the behavior of cells surrounding the secreting cell. Cytokines can be secreted by immune cells or other cells, such as T cells, B cells, natural killer cells, and macrophages. Cytokines can be involved in intracellular signaling events, such as autocrine signaling, paracrine signaling, and endocrine signaling. They can mediate a range of biological processes, including, but not limited to, immunity, inflammation, and hematopoiesis. Cytokines can be chemokines, interferons, interleukins, lymphokines, or tumor necrosis factors.

[0051] As used herein, "growth factor" refers to a protein or polypeptide that is capable of stimulating cell proliferation.

[0052] As used herein, a "reporter gene" is a polynucleotide that encodes a reporter protein, i.e., a "reporter" that can be easily detected and quantified. Thus, measuring the expression level of a reporter is typically an indicator of the transcription and / or translation level. A gene that encodes a reporter is a reporter gene. For example, a reporter gene can encode a reporter, e.g., an enzyme whose activity can be quantified, such as alkaline phosphatase (AP) (e.g., secreted embryonic alkaline phosphatase), chloramphenicol acetyltransferase (CAT), Cypridina, Gaussia, or firefly luciferase protein(s). Reporters also include fluorescent proteins, such as green fluorescent protein (GFP) or any recombinant variant of GFP (including enhanced GFP (EGFP)), blue fluorescent protein (BFP and other derivatives), cyan fluorescent protein (CFP and other derivatives), yellow fluorescent protein (YFP and other derivatives), and red fluorescent protein (RFP and other derivatives), or other fluorescent proteins, such as mCherry and mWasabi.

[0053] The term xenogeneic refers to an RNA virus rather than a host or patient infected with the virus, and therefore specifically encompasses eukaryotic proteins, particularly human proteins. A heterologous protein is a protein derived from a different organism or species from the recipient, i.e., the RNA virus VSV. At least one heterologous protein encoded by the RNA virus described in the present invention can be a therapeutic protein, a reporter, or a tumor antigen. Preferably, the at least one heterologous protein is a therapeutic protein with immunomodulatory or cell death-regulating function, preferably selected from the group consisting of cytokines, chemokines, growth factors, and antibodies. The therapeutic protein can also be a membrane-associated protein or can be membrane-bound by fusion of a transmembrane domain, such as the transmembrane domain of CD4, to the heterologous protein, preferably linked via a linker. The therapeutic protein may also encode a suicide gene. Alternatively or additionally, the at least one heterologous protein is a tumor antigen (including tumor-specific and / or tumor-associated antigens), such as a lineage antigen, neoantigen, testis antigen, and oncovirus antigen. The term "tumor-specific antigen" refers to an antigen that is exclusively expressed in tumor cells and not in any other tissue of an organism. The term "tumor-associated antigen" refers to an antigen that is overexpressed, i.e., expressed at a higher level, in tumor cells compared to other tissues in an organism. A tumor antigen may also be a neoantigen or a group of neoantigens. Neoantigens are newly formed antigens resulting from tumor somatic mutations. Those skilled in the art will know how to detect and determine neoantigens in patients. In another embodiment, the heterologous protein is a reporter protein, such as green fluorescent protein, red fluorescent protein, mCherry, or mWasabi. Heterologous proteins, particularly for therapeutic purposes, are preferably therapeutic proteins or tumor antigens with immunomodulatory or cell death-regulating functions.

[0054] Those skilled in the art will appreciate that while the methods of the present invention are exemplified for the rescue of vesicular stomatitis virus (VSV) from DNA in HEK293 cell lines or HEK293 cell lines adapted to suspension growth, they can be readily adapted to other negative-strand RNA viruses, such as Mononegavirales, Orthomyxovirales, Bunyavirales, and / or Arenavirales, particularly Mononegavirales. Thus, in another embodiment, a method for rescuing a negative-strand RNA virus includes: (a) providing cells from a HEK293 cell line or a HEK293 cell line adapted to suspension growth in cell culture; (b) transfecting the cells with at least one plasmid, wherein the at least one plasmid includes: (i) an expression cassette containing a negative-strand RNA virus genome cDNA; (ii) at least one expression cassette encoding a negative-strand RNA virus protein, such as a nucleocapsid protein (e.g., a nucleoprotein (N / NP) protein, a phosphoprotein (P) protein, and a large (L) protein of a mononegavirus, particularly a rhabdovirus, a paramyxovirus, a filovirus, and a bornavirus); and (iii) an expression cassette encoding an SV40 large T antigen; (c) culturing the transfected cells; and (d) recovering the cell culture supernatant, which contains the rescued negative-strand RNA virus. In a preferred embodiment, the recovered cell culture supernatant contains infectious negative-strand RNA virus. Further embodiments and aspects exemplified herein for VSV also apply to other negative-strand RNA viruses.

[0055] In another aspect, the present invention relates to the use of HEK293 cell lines or HEK293 cell lines adapted for suspension growth for the rescue of vesicular stomatitis virus (VSV) using transient transfection of at least one plasmid containing (i) an expression cassette containing VSV genomic cDNA and (ii) at least one expression cassette encoding the VSV core (N) protein, VSV phospho (P) protein, and VSV large (L) protein, as well as an expression cassette encoding the SV40 large T antigen. The embodiments and detailed descriptions relating to the methods described herein also apply to the use of HEK293 cell lines or HEK293 cell lines adapted for suspension growth for the rescue of vesicular stomatitis virus (VSV) described herein. Accordingly, the present invention also relates to the use of HEK293 cell lines or HEK293 cell lines adapted for suspension growth in the method for rescuing vesicular stomatitis virus (VSV) from DNA described herein. In a particular embodiment of the use of an HEK293 cell line or an HEK293 cell line adapted to suspension growth according to the present invention, the HEK293 cell line or the HEK293 cell line adapted to suspension growth is preferably an HEK293 cell line adapted to suspension growth selected from the group consisting of HEK293-F, HEK-293-H, Expi293F, and Freestyle HEK293-F, more preferably the HEK293 cell line adapted to suspension growth is an HEK293-F cell.

[0056] In yet another aspect, the present invention relates to the use of a plasmid encoding SV40 large T antigen for the rescue of vesicular stomatitis virus (VSV) in HEK293 cell lines or HEK293 cell lines adapted for suspension growth using transient cotransfection with at least one plasmid comprising (i) an expression cassette containing VSV genomic cDNA and (ii) at least one expression cassette encoding the VSV core (N) protein, VSV phospho (P) protein, and VSV large (L) protein. The embodiments and detailed descriptions relating to the methods described in the present invention also apply to the use of a plasmid encoding SV40 large T antigen for the rescue of vesicular stomatitis virus (VSV) in HEK293 cell lines or HEK293 cell lines adapted for suspension growth described in the present invention. Accordingly, the present invention also relates to the use of a plasmid encoding SV40 large T antigen in the method for rescuing vesicular stomatitis virus (VSV) from DNA described in the present invention. In a particular embodiment of the use of a plasmid encoding SV40 large T according to the present invention, the HEK293 cell line or the HEK293 cell line adapted to suspension growth is preferably an HEK293 cell line adapted to suspension growth selected from the group consisting of HEK293-F, HEK-293-H, Expi293F and Freestyle HEK293-F, more preferably the HEK293 cell line adapted to suspension growth is an HEK293-F cell.

[0057] Example For helper virus-free VSV rescue from plasmids using transient transfection, cells were transfected with at least five plasmids: an RNA polymerase II-dependent vector encoding bacteriophage T7 RNA polymerase; three separate plasmids encoding viral proteins N, P, or L under the control of a CAG promoter (pCAG VSV-N, pCAG VSV-P, and pCAG VSV-L plasmids); and a plasmid encoding a recombinant vesicular stomatitis virus cDNA clone (in which the coding sequence for VSV glycoprotein G was replaced by LCMV-GP). The viral genome could be transcribed from the plasmid by bacteriophage T7 RNA polymerase, generating a full-length positive-strand RNA complementary to the VSV genome. The resulting virus from the cDNA was designated VSV-LCMV-GP. A schematic diagram of the process is demonstrated in Figure 1. T7 RNA polymerase transcribes DNA encoding the VSV genome into viral RNA with a sense strand orientation. Expression of this RNA in cells that also express nucleoprotein N and the two polymerase subunits P and L leads to the production of VSV ribonucleoproteins, which are subsequently packaged into VSV-LCMV-GP virions and released from the cell by budding.

[0058] material kit Calcium Phosphate Transfection Kit The kit contains: 5 ml of 2.5 M CaCl2 25 ml 2x HEPES-buffered saline ·25ml molecular biology grade water

[0059] Chemicals, media, and buffers Chloroquine diphosphate ·Complete DMEM (cDMEM): 500 ml of DMEM (Dulbecco's Modified Eagle's Medium (DMEM), high glucose, glutamine-free) 50 ml of fetal bovine serum (FBS), heat-inactivated 10ml of CTS™ Glutamine™-I Supplement TrypLE™ Select Enzyme (1x), Phenol Red Free PBS-1×water-in-oil type, calcium, magnesium Plasmid-free, animal component-free, and quality control tested: 1. pCAG VSV-N plasmid 2. pCAG VSV-P plasmid 3. pCAG VSV-M Plasmid (Optional) 4. pCAG VSV-G plasmid (optional) 5. pCAG VSV-L plasmid 6. pCAG SV40 Large T 7. pCAGGS T7-RNAP IRES Puro 8. pVSV-LCMV GP (viral cDNA, also referred to as "VSV-GP").

[0060] The helper plasmids pCAG VSV-N, pCAG VSV-P, and pCAG VSV-L encode viral transactivation proteins required for RNP assembly, transcription, and replication, including nucleoprotein (SEQ ID NO: 1), phosphoprotein (SEQ ID NO: 2), and large protein (SEQ ID NO: 3). Additional expression of matrix (M) protein and viral glycoprotein (G) protein can further enhance viral rescue. Plasmids pCAG VSV-N, pCAG VSV-P, pCAG VSV-M, pCAG VSV-G, pCAG VSV-L, and pCAG SV40 large T were generated from the pSF-CAG_AMP background vector (Sigma-Aldrich, product number OGS504), which contains the ampicillin resistance gene and encodes the VSV-N, -P, -M, -G, and -L proteins under the control of the CAG promoter. VSV N, P, M, G, and L were derived from the Indiana serotype genomic cDNA clone (Lawson et al., 1995). A Kozak consensus sequence was included 5' of the start codon to provide an optimal sequence context for translation. The RNA polymerase II-dependent vector pCAGGS T7-RNAP IRES puro was originally derived from the pCAGGS background vector (Niwa H et al., (1991) Gene 108(2):193-9) and contains an ampicillin resistance gene, SV40 onset, and pBR322 onset, and encodes a codon-optimized T7 polymerase (SEQ ID NO:4) under the control of the CAG promoter and linked to the puromycin coding sequence via an IRES sequence. Plasmid pVSV-LCMV GP encodes the full-length viral genome cDNA, in which the gene encoding the G protein has been replaced by the gene encoding the GP protein from lymphocytic choriomeningitis virus (LCMV) (SEQ ID NO: 7), generating VSV-GP. The SV40 large T antigen (SEQ ID NO: 5) is under the control of the CMV promoter.A T7 RNAP promoter at the 5' end directs the synthesis of a positive genomic transcript before transcription is terminated by the phage T7 transcription termination sequence, and the plasmid contains the pBR322 ori and ampicillin resistance gene.

[0061] Transfection medium 500 ml of DMEM (Dulbecco's Modified Eagle's Medium (DMEM), High Glucose) (Glutamine-free) 10ml of CTS™ Glutamine™-I Supplement

[0062] Samples and cell lines HEK293-F (Thermo Fisher, serial number: 11625-019) HEK293T (provided by EUFETS GmbH / BioNTech IMFS) BHK21Cl.13 (Cell line services (CLS) serial number: 603126)

[0063] Preparation of adherent HEK293-F cell cultures For virus recovery, HEK293-F suspension cell cultures were transformed into adherent cell cultures at least one passage prior to transfection. HEK293-F suspension cells were transformed by changing cell culture conditions from balanced CD® HEK293 medium (Fujifilm) to fully supplemented cDMEM (10% fetal bovine serum, 2% CTS™ Glutamine™-I supplement). Cells were counted using a Nucleocounter NC-200 instrument (Chemometec) and counted at 2.0 x 10 cells per T175 cell culture flask in 30 ml of cDMEM. 6 Cells were seeded at 1000 x 1000 cells. The cells were cultured at 37°C, 6% carbon dioxide, and 95% humidity for 2-3 days. Adherent HEK293-F cells are required not only for transient transfection but, more importantly, for subsequent plaque purification to obtain clonal viruses.

[0064] Transfection of HEK293-F or HEK293T cells with plasmid DNA Cells were plated at 5 × 10 in 10 ml of cDMEM in a 10 cm dish one day before transfection (d-1). 6 HEK293-F cells or HEK293T cells were seeded onto the plates and cultured for 16 to 24 hours in an incubator at 37°C. The cells were 80% confluent on the day of transfection.

[0065] On the day of transfection (d0), the plasmids were mixed in the following amounts per 10 cm dish: 10.0 μg of viral cDNA (e.g., pVSV-LCMV GP) 2.4 μg pCAG VSV-N plasmid 1.8 μg pCAG VSV-P plasmid 0.6 μg pCAG VSV-L plasmid 10.0μg pCAGGS T7-RNAP IRES Puro 1.0-5.0 μg of pCAG SV40 large T (optional)

[0066] Although not added in the present experiments, the plasmids pCAG VSV-M and pCAG VSV-G can be added to the mixture at 1 μg each.

[0067] One hour before transfection, the cells were carefully washed with 5 ml of pre-warmed transfection medium (DMEM with glutamine), and 8 ml of pre-warmed transfection medium was carefully added to the cells without disturbing the cell layer and incubated in an incubator for 1 hour. Meanwhile, a DNA master mix was prepared in a 1.5 ml reaction tube for transfection using calcium phosphate precipitation, mixed by rocking the tube, followed by rapid spinning and settling.

[0068] For each rescue, tubes containing sterile cell culture-grade water were prepared to provide a final total volume (including the DNA mixture) of at least 450 μl. The specific amount of viral cDNA and / or pCAG SV40 large T was added to each tube and mixed by pipetting up and down. The DNA-master mix was then added, the tube was mixed by rocking, and quickly spun down. 50.0 μl of 2.5 M CaCl2 (kept on ice until use) was added to each tube and mixed by pipetting up and down. The DNA / CaCl2 mixture was then incubated at 4°C for exactly 5 minutes.

[0069] For each rescue, 500 μl of 2× HEPES buffer (Sigma) was pipetted into a 15 ml reaction tube, placed on a vortex mixer, and the DNA / CaCl mixture was added dropwise using a 1000 μl microtiter pipette over approximately 30 seconds while vortexing vigorously, followed by an additional 30 seconds of vortexing. The mixture was incubated at room temperature for a total of 20 minutes to allow the calcium phosphate-DNA precipitate to form without further mixing.

[0070] Two ml of transfection medium was mixed with 10 μl of 25 mM chloroquine stock solution and carefully added to a 10 cm tissue culture dish containing 80% confluent cells using a serological pipette. The dish was gently agitated to mix (total volume in dish: 10 ml, final concentration: 25 μM chloroquine), and the cells were returned to the incubator for the remainder of the 20-minute incubation. The transfection mixture was added dropwise to different areas of the cell layer using a 1000 μl microtiter pipette, and the dish was gently agitated to ensure uniform distribution of the calcium phosphate-DNA complexes. Cells were incubated in a 37°C incubator, and the chloroquine-containing medium was replaced with 10 ml of pre-warmed cDMEM after 4 hours, after which the cells were washed once with 5 ml of cDMEM and cultured as indicated. 50% of the medium was replaced every 2 days, if necessary, to prevent the medium from becoming acidic.

[0071] Rescue Recovery At the designated time points post-transfection, the supernatant from the 10 cm dish was collected and transferred to a 15 ml tube. The tube was centrifuged at 300 rcf for 4 minutes. The supernatant was filtered using a 0.20 μm filter, and 1 ml aliquots (rescue supernatant) were transferred to 6 x 1.5 ml tubes. The tubes were frozen at -80°C until further processing, or stored at 4°C if virion passaging was performed on the same day as rescue harvest.

[0072] Passaging of infectious virions HEK293-F cells or BHK21Cl.13 cells were cultured at 5 x 10 cells per 10 cm dish. 6 1 × 10 cells per 10 cm dish in 10 ml of cDMEM one day before infection (d-1). 7 Cells were seeded on the morning of transfection (d0) and cultured in an incubator at 37° C. Cells were approximately 80% confluent on the day of infection with rescue supernatant.

[0073] One ml of rescue supernatant (stored at 4°C until use or thawed for approximately 30 minutes before infection) was added dropwise to each plate without disturbing the cell layer. The culture vessel was gently rocked back and forth side to side to evenly distribute the supernatant, and the cells were incubated in a 37°C incubator for 24 to 48 hours. As a negative control, medium may be added to one dish.

[0074] Harvesting virus seed stock After 24-48 hours, virus seed stocks were harvested when clear cytopathic effect (CPE) was visible. The supernatant was removed from each dish and transferred to a 15 ml tube. The tubes were centrifuged at 300 rcf for 4 minutes. The supernatant was filtered using a 0.20 μm filter, and 0.6 ml was transferred to a 1.5 ml tube in 1 ml aliquots (rescue supernatant). The tubes were frozen at -80°C until plaque purification.

[0075] A generalized process overview is provided graphically in Figure 2.

[0076] Example 1 HEK293-F cells are frequently used as a suspension-producer cell line for VSV or recombinant VSV (rVSV, e.g., containing genetically modified glycoproteins) for clinical use as a vaccine or gene therapy. For regulatory reasons, it is advantageous to also use a suspension-producer cell line for rescue of helper virus-free VSV or rVSV from a plasmid using transient transfection. However, the present inventors observed that VSV or rVSV was not efficiently rescued using transient transfection in HEK293-F cells. HEK293T cells, a related cell line known to be easily transfected and frequently used for virus production in laboratory settings, on the other hand, have been shown to be an efficient cell line for VSV rescue.

[0077] To optimize VSV rescue in HEK293-F cells, viral protein expression was analyzed in HEK293-F and HEK293T cells after transfection. HEK293-F and HEK293T cells were transiently transfected as adherent cells using 10 μg of pVSV-LCMV GP, 2.4 μg of pCAG VSV-N plasmid, 1.8 μg of pCAG VSV-P plasmid, 0.6 μg of pCAG VSV-L plasmid, and 10 μg of pCAGGS T7-RNAP IRES Puro, as described above. In some cases, 1 μg of pCAG VSV-GP plasmid was added to the DNA mixture for transfection, or all NPL-helper plasmids (pCAG VSV-N, pCAG VSV-P, and pCAG VSV-L) were omitted as negative controls, as shown in Figure 3.

[0078] Cell lysates were analyzed by SDS page and Western blot using a polyclonal antibody against the P protein (Figure 3, upper panel) and a general antibody against VSV proteins that recognizes at least the N, P, and M proteins (Figure 3, lower panel). Because the P and M proteins migrate as a single band, they cannot be distinguished using this serum. As a positive control, 2.36 x 10 VSV purified by cation exchange chromatography (CEX) and purified by tangential flow filtration was used. 10 VSV-GP virus concentrated to TCID50 / ml (D-106-442) was used at 1 μl and 0.2 μl in separate lanes. No clear differences in P and N protein expression were observed in HEK293T and HEK293-F cells (Fig. 3, lanes 1 and 4). T7 RNA polymerase expression was not directly detected. Because expression of the P and N proteins is not T7 RNA polymerase-dependent, the data suggest that overall differences in transfection efficiency and expression of at least the P and N proteins cannot explain the observed differences in VSV rescue in HEK293-F and HEK293T cells.

[0079] Example 2 To analyze whether SV40 large T antigen can support VSV rescue in HEK293-F cells, HEK293-F cells were transfected with five plasmids for VSV rescue as described above, using two different amounts of plasmid (1 μg and 5 μg) in the absence or presence of SV40 large T antigen. HEK293-F cells were cultured as adherent cells and transiently transfected with 10 μg of pVSV-LCMV GP, 2.4 μg of pCAG VSV-N plasmid, 1.8 μg of pCAG VSV-P plasmid, 0.6 μg of pCAG VSV-L plasmid, and 10 μg of pCAGGS T7-RNAP IRES Puro, with or without the pCAG SV40 large T plasmid (1 μg or 5 μg). As a control, HEK293T cells were transfected in the same manner without co-transfection of the plasmid pCAG SV40 large T. Representative photographs at various time points are provided in Figure 4A.

[0080] To confirm the production of infectious virus in HEK293-F and HEK293T cells, passages of supernatants from HEK293-F and HEK293T cell harvests at 48 and 72 hours posttransfection were added to BHK21C1.13 cells and analyzed microscopically for cytopathic effect (CPE) at 48 hours postinfection (pi). Surprisingly, we found that transient transfection of HEK293-F cells with SV40 large T antigen allowed the rescue of VSV or rVSV. Distinct cytopathic effects were visible in BHK21C1.13 cells infected with 48- and 72-h posttransfection supernatants from HEK293T and HEK293-F cells transiently transfected with SV40 large T antigen, whereas no cytopathic effects were observed after infection with 48- and 72-h posttransfection supernatants from HEK293-F cells in the absence of SV40 large T antigen (Fig. 4B). After 48 h of infection, infection of BHK21C1.13 cells with 48- and 72-h posttransfection supernatants from HEK293-F cells in the absence of SV40 large T antigen was indistinguishable from BHK21C1.13 cells incubated with medium instead of harvest supernatant as a negative control. It can therefore be concluded that the presence of T antigen is required for infectious virus in HEK293-F cells.

[0081] Example 3 The previous experiment was independently repeated using HEK293-F cells for passage of infectious virions to confirm successful rescue of VSV. HEK293-F cells were transfected with the plasmids pVSV-LCMV GP (VSV-GP), pCAG VSV-N, pCAG VSV-P, and pCAG VSV-L (VSV-N, -P, L), and pCAGGS T7-RNAP IRES Puro, with or without cotransfection of the plasmid pCAG SV40 large T (1 μg or 5 μg). Representative photographs at various time points are provided in Figure 5A. To confirm the production of infectious virus in HEK293-F cells, supernatants from HEK293-F cell harvests 48 h posttransfection and 72 h posttransfection were added to 80% confluent, adherent HEK293-F cells and analyzed microscopically for cytopathic effect (CPE) 24 h postinfection (pi). At this earlier time point using HEK293-F cells, clear cytopathic effect (CPE) was visible in HEK293-F cells infected with 48- and 72-hour posttransfection harvest supernatants from HEK293-F cells transiently transfected with 5 μg of SV40 large T antigen, whereas no cytopathic effect was observed after infection with 48- and 72-hour posttransfection harvest supernatants from HEK293-F cells transiently transfected with 1 μg of SV40 large T antigen or in the absence of SV40 large T antigen (Fig. 5B).

[0082] Example 4 The presence of large T antigen was associated with increased amplification of vectors containing the SV40 onori. The only plasmid used for VSV rescue containing the SV40 onori is the plasmid encoding T7-RNAP (pCAGGS T7-RNAP IRES Puro). Because expression of viral proteins N, P, and L from the corresponding pCAG vector is driven by an artificial CAG promoter and does not depend on the presence of T polymerase, differences in viral protein levels do not explain the difference in virus recovery observed in HEK293T cells compared to HEK293-F cells (Figure 3). However, we cannot exclude that T7 polymerase-dependent primary transcription of VSV-GP genomic RNA in the early steps after plasmid transfection affects virus recovery in various cell lines.

[0083] To analyze the effect of the SV40 ori on VSV rescue in HEK293-F cells in the presence of T7 RNA polymerase, we repeated VSV rescue in HEK293-F cells using a different vector that did not contain the SV40 ori for expression of T7 RNA polymerase. Therefore, an expression cassette encoding T7 RNA polymerase from the plasmid pCAGGS T7-RNAP IRES Puro was cloned into the pCAG plasmid used for expression of VSV P, N, and L proteins. Although the efficiency was generally lower compared to pCAGGS T7-RNA polymerase IRES Puro, large T antigen-dependent VSV rescue was still observed using a plasmid lacking the SV40 ori for expression of T7 RNA polymerase. Again, VSV rescue was observed only in the presence of large T antigen in HEK293-F cells.

[0084] Sequence Listing SEQ ID NO: 1 (Vesicular stomatitis Indiana virus, VSV-N): [ka]

[0085] SEQ ID NO: 2 (Vesicular Stomatitis Indiana Virus, VSV-P): [ka]

[0086] SEQ ID NO: 3 (Vesicular stomatitis Indiana virus, VSV-L): [ka]

[0087] SEQ ID NO: 4 (T7 RNA polymerase): [ka]

[0088] SEQ ID NO: 5 (simian virus 40, SV40 large T antigen): [ka]

[0089] SEQ ID NO: 6 (Vesicular stomatitis Indiana virus strain T1026R1 complete sequence): [ka] TIFF0007762791000007.tif244161 TIFF0007762791000008.tif191161

[0090] SEQ ID NO: 7 (Lymphocytic choriomeningitis virus, LCMV GP): [ka]

Claims

1. (a) providing cells from a HEK293 cell line or a HEK293 cell line adapted to suspension growth in cell culture; (b) transfecting the cells with at least one plasmid, wherein the at least one plasmid is (i) an expression cassette containing vesicular stomatitis virus (VSV) genomic cDNA; (ii) at least one expression cassette encoding a VSV nucleo (N) protein, a VSV phospho (P) protein, and a VSV large (L) protein; and (iii) an expression cassette encoding the SV40 large T antigen; Includes; (c) culturing the transfected cells; and (d) recovering the cell culture supernatant containing the rescued VSV Including, Rescue of vesicular stomatitis virus (VSV) from DNA in HEK293 cell lines or HEK293 cell lines adapted to suspension growth.

2. The method of claim 1, wherein the harvested cell culture supernatant contains infectious vesicular stomatitis virus.

3. (i) the cells are provided, transfected, and cultured as adherent cells; (ii) the cells are transiently transfected in step (b); (iii) the transfection of the cells in step (b) comprises the use of a chemical reaction-based transfection agent; and / or (iv) The method of claim 1 or 2, wherein the transfection of the cells in step (b) comprises the use of a chemical reaction-based transfection agent selected from lipofection, polyethyleneimine, or calcium phosphate.

4. The method of claim 1, wherein the cells in step (b) are further transfected or transduced with a plasmid or helper virus containing an expression cassette encoding bacteriophage T7 RNA polymerase under the control of an RNA polymerase II-dependent promoter; and the expression cassette containing the vesicular stomatitis virus genomic cDNA contains VSV genomic cDNA under the control of a T7 promoter sequence and a T7 transcription termination sequence.

5. 5. The method of claim 4, wherein the method is a helper virus-free method and the cells in step (b) are transfected with a plasmid containing an expression cassette encoding bacteriophage T7 RNA polymerase under the control of an RNA polymerase II-dependent promoter.

6. (a) the nucleotide sequence encoding the bacteriophage T7 RNA polymerase is codon-optimized; and / or 6. The method of claim 4 or 5, wherein (b) the bacteriophage T7 RNA polymerase has the amino acid sequence of SEQ ID NO:4 or has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

4.

7. 2. The method of claim 1, wherein the at least one expression cassette encoding a VSV phosphoprotein, a VSV nucleoprotein, and a VSV large protein is transfected as one or more helper plasmids.

8. The one or more helper plasmids (i) a first helper plasmid containing an expression cassette comprising a sequence encoding a VSV nucleoprotein under the control of a promoter sequence and a transcription termination sequence; (ii) a second helper plasmid containing an expression cassette comprising a sequence encoding a VSV phosphoprotein under the control of a promoter sequence and a transcription termination sequence; and (iii) a third helper plasmid containing an expression cassette comprising a sequence encoding a VSV large protein under the control of a promoter sequence and a transcription termination sequence.

8. The method of claim 7, comprising:

9. The expression cassette encoding the SV40 large T antigen is (a) transfected as a plasmid containing an expression cassette encoding the SV40 large T antigen; and / or (b) a nucleic acid sequence encoding an SV40 large T antigen under the control of a promoter, and further comprising a transcription termination sequence; (c) a nucleic acid sequence encoding an SV40 large T antigen having the amino acid sequence of SEQ ID NO:5 or having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

5.

10. 2. The method of claim 1, wherein the HEK293 cell line or the HEK293 cell line adapted to suspension growth is selected from the group consisting of HEK293, HEK293-F, HEK-293-H, Expi293F, and Freestyle HEK293-F.

11. The method of claim 1, wherein the cells are HEK293-F cells.

12. The method according to claim 1, wherein the VSV genomic cDNA is a full-length viral genomic cDNA or a modified viral genomic cDNA.

13. 2. The method of claim 1, wherein the VSV genomic cDNA is a modified viral genomic cDNA encoding a modified G protein.

14. The method according to claim 12 or 13, wherein the gene encoding glycoprotein G in the VSV genome cDNA is replaced by the gene encoding glycoprotein GP of lymphocytic choriomeningitis virus (LCMV).

15. (e) transducing the HEK293 cell line or cells derived from the HEK293 cell line adapted to growth in suspension with the VSV obtained in step (d). The method of claim 1 further comprising:

16. Use of a HEK293 cell line or a HEK293 cell line adapted to suspension growth for rescue of vesicular stomatitis virus (VSV) using transient transfection of at least one plasmid containing (i) an expression cassette containing VSV genomic cDNA, and (ii) at least one expression cassette encoding VSV core (N) protein, VSV phospho (P) protein, and VSV large (L) protein, and an expression cassette encoding SV40 large T antigen.

17. Use of a plasmid encoding SV40 large T antigen for rescue of vesicular stomatitis virus (VSV) in HEK293 cell lines or HEK293 cell lines adapted for suspension growth using transient co-transfection with at least one plasmid comprising (i) an expression cassette comprising VSV genomic cDNA, and (ii) at least one expression cassette encoding VSV core (N) protein, VSV phospho (P) protein, and VSV large (L) protein.

18. The one or more helper plasmids at least one additional helper plasmid containing an expression cassette comprising a sequence encoding a VSV glycoprotein (G) and / or an expression cassette comprising a sequence encoding a VSV matrix (M) protein; 9. The method of claim 8.

19. The method of claim 19, further comprising: (f) producing VSV in the cells of step (e) in large-scale suspension culture. Including, 16. The method of claim 15.

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