Method for the production of replication-defective recombinant polyomaviral vector particles

Abstract

The present invention relates to a method for the production of replication-defective recombinant polyomaviral vector particles, the method comprising the steps of a) providing a mammalian cell line permissive or non-permissive for the wildtype polyomavirus, b) introducing into said cell line a recombinant polyomaviral first DNA construct comprising recombinant DNA and a polyomaviral intergenic region, wherein said recombinant polyomaviral first DNA construct not encoding functional polyomaviral T antigens, and wherein said intergenic region comprises the early and late promoters, the origin-of-replication and the packaging signal, c) culturing the cells of said cell line in a growth medium under conditions allowing the formation of said vector particles, and d) extracting the vector particles from said growth medium and / or said cells, wherein a gene encoding functional polyomaviral large T antigen is introduced into said cell line via a separate second DNA construct.

Description

[0001] Method for the production of replication-defective recombinant polyomaviral vector particles

[0002] Technical field

[0003] The present invention relates to a method for the production of replicationdefective recombinant polyomaviral vector particles.

[0004] Background

[0005] Over the last decades, much effort has been dedicated to the development of efficient gene or nucleic acid delivery technologies for introduction and proper expression of genes or nucleic acids in target cells. Therapeutic genes or nucleic acids can be used to restore malfunctioning genes to treat genetic disorders, to induce an immune response to treat cancer and infectious diseases or to suppress an immune response e.g., for inducing / restoring immune tolerance to prevent transplant rejection or to treat autoimmune diseases and allergies. The therapeutic genes or nucleic acids can be administered as naked molecules or as nucleic acids packaged in lipid and / or proteinaceous compounds.

[0006] Since viruses evolved to deliver and express their genetic information into host target cells, viral vectors are by far the most effective gene delivery vehicles to express self or foreign proteins in vivo. Among the viral vectors currently used for treating genetic disorders, cancer, autoimmune diseases and allergies, and for preventing transplant rejection, replication-defective lentiviral (LV) vectors derived from the human immunodeficiency virus type 1 and replication-defective adeno-associated viral (AAV) vectors derived from adeno-associated virus are the most popular. For both replication-defective vectors it has been shown that they are non-immunogenic or tolerogenic in hosts that are naive to the cognate virus. LV vectors permanently modify transduced target cells by integrating their viral genomes randomly in the host genome. Since the particles are highly instable LV vectors are mainly used for ex vivo gene replacement therapy to treat blood-related genetic disorders and cancer. The genomes of AAV vectors remain as stable episomes in the nuclei of transduced target cells and since the particles are highly stable AAV vectors are mainly used for in vivo gene therapies. AAV is a primarily human virus that co-replicates with adenoviruses: the causal agents of the common cold. The vast majority of the human population has been exposed to AAV and developed a strong immune memory for the viral capsid proteins. Numerous clinical studies using recombinant AAV vectors indeed confirmed that administration of vector particles elicits innate and adaptive immune responses against the viral and transgene-encoded proteins in the vast majority of treated patients. The immune responses lead to elimination of the transduced cells from the body and decreasing expression levels of the therapeutic transgenes over time, compromising re-administration of the vector. The few treated patients that showed long term transgene expression most likely have never been infected with AAV and thus were immunologically naive to the AAV vector used in the study. In these patients the treatment of a single dose of an AAV vector may lead to over-expression of the transgene resulting in the development of in some cases severe adverse effects. AAV’s immunogenicity and toxicity in humans, and as a result its clinical inefficacy, will remain the major challenges for the approval of new AAV vector-based interventions.

[0007] Replication-defective polyomaviral vectors are an attractive alternative to AAV vectors for clinical gene therapy. Polyomavirus strictly replicate in their natural host, where they cause chronic asymptomatic infections. Replication-defective polyomaviral vectors are non-immunogenic in hosts that are immunologically naive for the cognate polyomavirus. Simian virus 40 (SV40) is a polyomavirus that naturally and strictly infects macaques, where it causes chronic asymptomatic infections. SV40 particles enter infected cells via the caveolar-endosomal route, but in contrast with other viruses are able to avoid lysosomal degradation, thereby evading exposure to the host immune system. SV40 has a 5.25 kb long circular double-stranded DNA possessing two genes. The early gene encodes two non-structural replication-associated proteins Small T antigen (STag) and Large T antigen (LTag). The late gene codes for the structural viral proteins VP1 , VP2 and VP3.

[0008] The early and late genes are separated by the polyomaviral intergenic region that contains the early and late promoters needed for transcription of the early and late gene, the origin-of-replication needed for polyomaviral DNA replication and the packaging signal needed for the formation of polyomavirus particles.

[0009] Replication-defective SV40 vectors have been generated by deleting the coding region of the early gene leaving 2.7 kb of available space to clone exogenous DNA. In cells lacking the SV40 early gene transduced with the vector the absence of LTag prevents the production of all viral proteins. Because humans can be considered naive to SV40, it is expected that replication-defective SV40 vectors are non-immunogenic when applied in humans. The non-immunogenicity in humans render SV40 vectors highly attractive for use in gene therapies to treat genetic disorders, cancer, autoimmune diseases and allergies, and to prevent transplant rejection.

[0010] Replication-defective SV40 vectors are produced in macaque packaging cell lines expressing the SV40 early gene such as COS-1 , COT18 and CMT4, or in macaque cell lines expressing the SV40 early and late genes, such as COS-7. Packaging cell lines expressing the SV40 early and late genes can be used to produce replication defective SV40 vectors lacking the coding regions of the early and late genes. Such “gutless” vectors have a coding capacity of 4.8 kb of exogenous DNA (Mueller C. et al., Gene Therapy 17: 227-237, 2010).

[0011] Passaging of SV40 vectors in said packaging cell lines however, results in the appearance of wild type SV40 particles. This most likely occurs by sequence homology-dependent recombination between the chromosomally inserted SV40- specific DNA sequences and episomally replicating SV40-specific DNA sequences.

[0012] To prevent the occurrence of replication-competent virus particles in the vector preparations, polyomavirus-based virus-like particle (VLP) vector systems have been developed. In vitro generated VLPs consisting of circular double-stranded polyomaviral vector DNA encapsidated with polyomaviral VP1. Such VLPs lack VP2 and VP3 in the capsids and histones covering the encapsidated DNA molecules. Although these particles display a higher packaging capacity, the absence of VP2 / VP3 and histones in the VLPs has a negative impact on their transduction efficacy in vivo.

[0013] In order to overcome the generation of wild type virus contaminants during the production of replication-defective SV40 vector particles a safe and efficient Vero- based SV40 vector packaging cell line, named SuperVero, was generated. SuperVero cells solely express the viral LTag and accumulate fully replication-defective vector particles at high titers, comparable to those obtained in the conventional SV40 vector packaging cell lines (see for example: Toscano M.G. et al., Mol. Ther. Methods Clin. Dev. 6: 124-134, 2017; International patent application published under number WO 2010 / 122094 A1).

[0014] Currently, circular polyomaviral vector DNA required for starting the production of vector particles in packaging cell lines is generated by releasing the vector DNA from a plasmid backbone by restriction enzyme digestion followed by self-ligation of the resulting linear vector DNA using T4 DNA ligase to generate the circular polyomaviral vector genomes. A disadvantage of this method to generate circular vector DNA is that the circular DNA molecules have a relaxed conformity. Such relaxed circular vector DNA genomes are relatively poor substrates for the SV40 LTag to become replicated and packaged by SV40 capsid proteins to SV40 vector particles.

[0015] Given the above, there is a constant need of further improving the method for the production of replication-defective recombinant polyomaviral vector particles.

[0016] Description of the invention

[0017] In order to provide such improved method, the present invention provides hereto a method for the production of replication-defective recombinant polyomaviral vector particles, wherein the method comprises the steps of: a) providing a mammalian cell line permissive or non-permissive for the wildtype polyomavirus; b) introducing into said mammalian cell line a recombinant polyomaviral first DNA construct comprising recombinant DNA and a polyomaviral intergenic region, wherein said recombinant polyomaviral first DNA construct not encoding functional polyomaviral T antigens, and wherein said intergenic region comprises the early and late promoters, the origin-of-replication and the packaging signal; c) culturing the cells of said mammalian cell line in a growth medium under conditions allowing the formation of replication-defective recombinant polyomaviral vector particles; and d) extracting the replication-defective recombinant polyomaviral vector particles from said growth medium and / or said cells.

[0018] In a first aspect of the present invention, the mammalian cell line used in the method of the present invention is preferably selected from a mammalian cell not encoding functional polyomaviral large T antigen. In other words, the mammalian cell line used in the method of the present invention does not comprise a gene encoding a functional polyomaviral large T antigen. Instead, a gene encoding functional polyomaviral large T antigen is introduced into the mammalian cell line via a separate second DNA construct, which separate second DNA construct is another DNA construct than said recombinant polyomaviral first DNA construct introduced into said mammalian cell line of step b). In the method of the present invention the separate second DNA construct comprises the gene encoding functional polyomaviral large T antigen. The separate second DNA construct may further comprise, but not necessarily, a gene encoding functional polyomaviral small T antigen. In a preferred embodiment the separate second DNA construct is substantially free of a gene encoding functional polyomaviral small T antigen.

[0019] It was found that by providing the method of the present invention in its first aspect, i.e., using a mammalian cell line not encoding large T antigen, replicationdefective recombinant polyomaviral vector particles can be efficiently and repeatedly produced. In fact, using the method of the present invention, replication-defective recombinant polyomaviral vector particles can be produced in any cell type, such as HEK293 cell lines.

[0020] In a preferred embodiment, the mammalian cell line as used in the method of the present invention does not comprise a gene encoding functional polyomaviral small T antigen and does not comprise gene encoding a functional polyomaviral large T antigen. It was found that by providing a recombinant polyomaviral first DNA construct not encoding polyomaviral functional proteins, and by providing a cell line not encoding functional polyomaviral T antigens, a reliable and safe production of polyomaviral vector particles is provided. In addition, it was also found that the method of the present invention provides in a high flexibility in designing the recombinant DNA to be used in the polyomaviral vector particles having an increased packaging capacity of the recombinant DNA compared to the currently used polyomaviral vector particles containing the polyomaviral late gene.

[0021] The recombinant polyomaviral first DNA construct of the present invention may be substantially free of (e.g., does not comprise any) polyomaviral functional coding sequences. In such embodiment of the present invention the recombination polyomaviral first DNA construct not encoding polyomaviral functional proteins, including functional polyomaviral capsid proteins (e.g., VP1 , VP2 and VP3), and functional polyomaviral T antigens, including small T antigen (Stag) and large T antigen (Ltag).

[0022] It is noted that the term ‘recombinant polyomaviral first DNA construct’ as used herein may refer to similar terms used in the field including gutless vector, gutless vector plasmid or gutless vector DNA. In a preferred embodiment of the present invention, the recombinant polyomaviral first DNA construct may be a circular double-stranded recombinant polyomaviral vector DNA molecule.

[0023] The actual size of the recombinant DNA comprised in the recombinant polyomaviral DNA construct may vary, but is selected such that the recombinant DNA can be encapsidated into the polyomaviral vector particles. By providing a recombinant polyomaviral first DNA construct not encoding functional polyomaviral T antigens, the packaging capacity of the recombinant polyomaviral first DNA construct in relation to the recombinant DNA is increased. The present invention now provides for a recombinant polyomaviral first DNA construct, wherein the recombinant DNA may have a size of at least 3.0 kb. Typically, the recombinant DNA comprised in the recombinant polyomaviral first DNA construct may have a size between 4.0 kb and 6.0 kb. In total, the size of the recombinant polyomaviral first DNA construct should preferably not exceed a size of 9.0 kb or 10 kb, as it is believed that a DNA construct having a size of not exceeding the size of 9.0 kb or 10 kb can still be encapsidated into the polyomaviral vector particles.

[0024] It is noted that the term ‘recombinant DNA’ as used herein may refer to similar terms used in the field including DNA insert, transgene or transgene construct. The recombinant DNA may encode one or multiple therapeutic proteins or RNA molecules.

[0025] Next to the recombinant DNA, the recombinant polyomaviral first DNA construct further comprises a polyomaviral intergenic region. As used herein, the term ‘polyomaviral intergenic region' refers to the region of the polyomavirus vector DNA comprising the early and late promoters needed for transcription of the early and late genes, the origin-of-replication needed for polyomaviral DNA replication and the packaging signal needed for the formation of polyomaviral vector particles.

[0026] Preferably, the polyomaviral intergenic region is derived from a primate polyomavirus, preferably a simian polyomavirus. Preferably the polyomaviral intergenic region as used in the recombinant polyomaviral first DNA construct is derived from polyomaviruses selected from the group consisting of Simian virus 40, Macaca fascicularis polyomavirus 1 , Pan troglodytes verus polyomavirus 1a, Pan troglodytes verus polyomavirus 2a, Pan troglodytes verus polyomavirus 3, Pan troglodytes verus polyomavirus 4, Pan troglodytes verus polyomavirus 8, Pan troglodytes schweinfurthii polyomavirus 2, Chimpanzee polyomavirus, Bornean orang utan polyomavirus, Sumatran orang utan polyomavirus or Gorilla gorilla gorilla polyomavirus 1 , yellow baboon polyomavirus 1 , yellow baboon polyomavirus 2, Vervet monkey polyomavirus 1 , Vervet monkey polyomavirus 2, Vervet monkey polyomavirus 3 and Cercopithecus erythrotis polyomavirus 1. In a preferred embodiment, the polyomaviral intergenic region is derived from macaque polyomavirus Simian Virus 40 (SV40).

[0027] Similarly, the term ‘polyomaviral functional coding sequences’ refers to sequences derived from a primate polyomavirus, preferably a simian polyomavirus. Preferably the term ‘polyomaviral functional coding sequences’ refers to sequences derived from polyomaviruses selected from the group consisting of Simian virus 40, Macaca fascicularis polyomavirus 1 , Pan troglodytes verus polyomavirus 1a, Pan troglodytes verus polyomavirus 2a, Pan troglodytes verus polyomavirus 3, Pan troglodytes verus polyomavirus 4, Pan troglodytes verus polyomavirus 8, Pan troglodytes schweinfurthii polyomavirus 2, Chimpanzee polyomavirus, Bornean orang utan polyomavirus, Sumatran orang utan polyomavirus or Gorilla gorilla gorilla polyomavirus 1 , yellow baboon polyomavirus 1 , yellow baboon polyomavirus 2, Vervet monkey polyomavirus 1 , Vervet monkey polyomavirus 2, Vervet monkey polyomavirus 3 and Cercopithecus erythrotis polyomavirus 1. In a preferred embodiment, the term ‘polyomaviral functional coding sequences’ refers to sequences derived from macaque polyomavirus Simian Virus 40 (SV40).

[0028] It is further noted that the term ‘polyomaviral functional coding sequences’ refers to sequences coding functional polyomaviral capsid proteins (e.g., VP1 , VP2 and VP3). Further the term ‘polyomaviral functional coding sequences’ refers to sequences coding functional polyomaviral T antigens, including small T antigen (STag) and large T antigen (LTag).

[0029] The mammalian cell line as used in the method of the present invention is preferably selected from the group consisting of a human cell line, a non-human primate cell line, a bovine cell line or a rodent cell line. Preferably, the mammalian cell line may be selected from the group consisting of HEK293 cell lines, PER.C6 cell lines, Vero cell lines, CV1 cell lines, BSC-1 cell lines, BHK cell lines, CHO cell lines, or derivatives of these cell lines.

[0030] Although any cell line may be selected for use in the present method, it is noted that the cell line is preferably selected such that the recombinant polyomaviral first DNA construct comprising recombinant DNA and a polyomaviral intergenic region is capable of replication in said mammalian cell line.

[0031] As stated above, the mammalian cell line as used in the method of the present invention is preferably selected from the group consisting of cell lines permissive for wildtype polyomavirus and cell lines non-permissive for wildtype polyomavirus. The polyomavirus may be selected from the group consisting of Simian virus 40, Macaca fascicularis polyomavirus 1 , Pan troglodytes verus polyomavirus 1a, Pan troglodytes verus polyomavirus 2a, Pan troglodytes verus polyomavirus 3, Pan troglodytes verus polyomavirus 4, Pan troglodytes verus polyomavirus 8, Pan troglodytes schweinfurthii polyomavirus 2, Chimpanzee polyomavirus, Bornean orang utan polyomavirus, Sumatran orang utan polyomavirus or Gorilla gorilla gorilla polyomavirus 1 , yellow baboon polyomavirus 1 , yellow baboon polyomavirus 2, Vervet monkey polyomavirus 1 , Vervet monkey polyomavirus 2, Vervet monkey polyomavirus 3 and Cercopithecus erythrotis polyomavirus 1. In a preferred embodiment, the polyomavirus is selected from a primate polyomavirus, preferably a simian polyomavirus, such as macaque polyomavirus Simian Virus 40 (SV40).

[0032] The mammalian cell line as used in the method of the present invention may comprise a gene encoding functional polyomaviral capsid proteins. Functional polyomaviral capsid proteins are preferably selected from the group consisting of VP1 , VP2 and VP3. In addition, it is noted that the gene encoding functional polyomaviral capsid proteins may be stably integrated in the genome of the mammalian cell line used in the method of the present invention. Alternatively, a gene encoding functional polyomaviral capsid proteins may be introduced into said cell line via the recombinant polyomaviral first DNA construct of the present invention, wherein the recombinant polyomaviral first DNA construct comprises a gene encoding functional polyomaviral capsid proteins. Further alternatively, a gene encoding functional polyomaviral capsid proteins may be introduced into said cell line via the separate second DNA construct or a separate third DNA construct, wherein the separate third DNA construct is another DNA construct than the recombinant polyomaviral first DNA construct and the separate second DNA construct. Preferably, the separate second or third DNA construct comprises a gene encoding functional polyomaviral capsid proteins, and wherein the separate second or third DNA construct cannot be encapsidated into the polyomaviral vector particles. It is noted that the separate DNA construct for introducing into said cell line a gene encoding functional polyomaviral capsid proteins and the separate DNA construct for introducing into said cell line a gene encoding functional polyomaviral large T antigen as used in the second aspect of the method of the present invention may be the same DNA construct when used in combination. However, both separate DNA constructs may also be different (separated) DNA constructs when used in combination. Preferably, the functional polyomaviral capsid proteins and functional polyomaviral large T antigen encoded by the separate DNA construct(s) are expressed in a cell under transcriptional control of promoters other than polyomaviral early and late promoters. In a preferred embodiment the functional polyomaviral capsid proteins and functional polyomaviral large T antigen encoded by the separate DNA construct(s) are expressed in a cell under transcriptional control of polyomaviral early and late promoters. The size of such separate DNA construct(s) may be selected such that the separate DNA construct(s) cannot be encapsidated into the recombinant polyomaviral vector particles to be produced. To this end, although the size of the separate DNA construct(s) may vary, preferably the separate DNA construct(s) may have a size of at least 10 kb, at least 11 kb, or more preferably a size of at least 12 kb.

[0033] Preferably, the separate DNA construct (second and third DNA constructs), other than the recombinant polyomaviral first DNA construct, introduced into the mammalian cell line do not comprise the polyomaviral intergenic region.

[0034] As used herein, the term ‘separate DNA construct’ may refer to a supporting vector genome or supporting DNA vector, i.e. , a DNA construct or vector comprising DNA sequences, such as sequences encoding functional polyomaviral capsid proteins and / or sequences encoding functional polyomaviral large T antigen, facilitating the formation of polyomaviral vector particles.

[0035] Examples

[0036] Example 1 : Construction of the SV40 derived gene delivery vector

[0037] The following oligonucleotides were designed: where:

[0038] WdV101 containing a Xhol and a Notl restriction site;

[0039] WdV102 containing a Kpnl restriction site;

[0040] WdV103 containing from 5' to 3' subsequently a Notl sticky restriction site, a Padl, Sbfl, Pmel and an Asci intact restriction site and a Clal sticky restriction site;

[0041] WdV104 containing from 3' to 5' subsequently a Notl sticky restriction site, a Padl, Sbfl, Pmel and an Asci intact restriction site and a Clal sticky restriction site;

[0042] WdV105 containing a BamHI restriction site; and

[0043] WdV106 containing a Notl restriction site.

[0044] Purified plasmid DNA of the SV40 vector pSL-PL (De La Luna, S. et al., Journal of General Virology 74: 535-539, 1993) was subjected to PCR using oligonucleotides WdV105 and WdV106. The resulting amplified DNA fragment comprised the SV40- polyadenylation signal flanked by a BamHI restriction site at the 5' end and a Notl restriction site at the 3' end. This SV40 polyadenylation signal fragment was digested with BamHI and Notl and the resulting 150 bp long DNA fragment was isolated from an agarose gel and cloned into a likewise digested pBluescript SK- plasmid (Promega), yielding pAM002.

[0045] Purified pEF5 / FRT / 5-DEST (Invitrogen) plasmid DNA was subjected to PCR using oligonucleotides WdV101 and WdV102. The resulting amplified DNA fragment comprising the bovine growth hormone (BGH) polyadenylation signal flanked by subsequently a Xhol and a Notl restriction site at the 5' end and an Kpnl restriction site at the 3' end. This BGH polyadenylation signal fragment was digested with Kpnl and Notl, and the resulting 250 bp long DNA fragment was isolated from an agarose gel and ligated into the likewise digested pAM002 plasmid. Transformation with this ligation mixture was performed in a methylation insensitive E. coli strain. This resulted in plasmid pAM003.

[0046] The two complementary oligonucleotides WdV103 and WdV104 were annealed by incubating them in a water bath that was cooling down autonomously from boiling temperature to room temperature, yielding a DNA linker containing subsequently a Notl sticky restriction site, a Padl, Sbfl, Pmel and a Asci intact restriction site and a Clal sticky restriction site. This linker was ligated into the pAM002 plasmid that was digested with Notl and Clal and isolated from an agarose gel. The ligation mixture was subsequently used to transform a methylation insensitive E. coli strain, yielding pAM004.

[0047] Purified plasmid DNA of the SV40 vector pSL-PL was digested with Clal and BamHI. The resulting 2.6 kb DNA fragment that contains the SV40 origin and the SV40 late region was purified from agarose and cloned into likewise digested pAM004. This resulted in the new SV40 vector plasmid pAM005.

[0048] Example 2: Molecular cloning of SV40 vectors encoding luciferase and the SV40 large T antigen

[0049] The expression plasmid pGL3 (Promega) was used as template for cloning of the firefly luciferase using PCR. The following oligonucleotides were designed: where:

[0050] WdV407 contains respectively restriction sites Asci and Pad; and WdV442 contains respectively BamHI and Notl restriction site.

[0051] The PCR amplified luciferase fragment was subsequently Asci and Pad digested and ligated into SV40 vector plasmid pAM005, resulting in an SV40 vector encoding luciferase named pAM006 or pSVLt / c. The pSL-PL vector was used as template for cloning of the large T antigen trailer sequence using PCR. The resulting PCR fragment was digested with BamHI and Notl and cloned into the BamHI and Notl (partially digested) pAM006, resulting in expression plasmid pAM020 encoding the SV40 large T antigen.

[0052] Example 3: Production of SV40 vector particles encoding luciferase (SVLt / c) in HEK293 suspension cells

[0053] HEK293 suspension cells are transfected with pSVLt / c DNA or with pSVLt / c DNA and different amounts of pAM020 DNA. Up to six days after transfection the number of SVLt / c infectious units (lUs) were determined in SuperVero producer cells (Toscano M.G. et al., Mol. Ther. Methods Clin. Dev. 6: 124-134, 2017). The assay to measure I Us was performed by seeding M96 wells plates with SuperVero cells containing the gene encoding the SV40 Large T antigen on the genomic DNA, in such that the cell cultures were 30% confluent the next day. Four replicates of serial dilutions of the test sample were added to infect these 30% confluent cell cultures, which were subsequently incubated at 37°C with 5% CO2. Four days post-infection the mRNAs were isolated from the infected cells and used as templates for reverse transcriptase (RT)-qPCR reactions with primers and a probe specific for a SV40 VP2 coding sequence. The cycle threshold (Ct) values obtained from these RT-qPCR reactions were used to score the M96 wells plate to be either positive or negative for an infection event. All wells with a Ct value less than 36 were scored positive and wells with a CT more than 35 were scored negative. These scores were subsequently used to enter in the spearman Karber formula to calculate the lU / mL of the test sample.

[0054] It was found that the number of SVLt / c lUs produced in HEK293 cells transfected with pSVLt / c DNA in the absence of the SV40 large T antigen was 100 times lower than that produced in HEK293 cells transfected with pSVLt / c DNA in the presence of the SV40 large T antigen.

Claims

CLAIMS1. Method for the production of replication-defective recombinant polyomaviral vector particles, the method comprising the steps of: a) providing a mammalian cell line permissive or non-permissive for the wildtype polyomavirus; b) introducing into said mammalian cell line a recombinant polyomaviral first DNA construct comprising recombinant DNA and a polyomaviral intergenic region, wherein: said recombinant polyomaviral first DNA construct not encoding functional polyomaviral T antigens; and said intergenic region comprises the early and late promoters, the origin-of-replication and the packaging signal; c) culturing the cells of said mammalian cell line in a growth medium under conditions allowing the formation of replication-defective recombinant polyomaviral vector particles; and d) extracting the replication-defective recombinant polyomaviral vector particles from said growth medium and / or said cells, characterised in that a gene encoding functional polyomaviral large T antigen is introduced into said mammalian cell line via a separate second DNA construct other than said recombinant polyomaviral first DNA construct introduced into said mammalian cell line of step b).

2. Method according to claim 1 , wherein the mammalian cell line is selected from the group consisting of a human cell line, a non-human primate cell line, a bovine cell line or a rodent cell line.

3. Method according to claim 1 or 2, wherein the mammalian cell line is selected from the group consisting of HEK293 cell lines, PER.C6 cell lines, Vero cell lines, CV1 cell lines, BSC-1 cells, BHK cell lines, CHO cell lines, or derivatives of these cell lines.

4. Method according to any of the preceding claims, wherein the polyomavirus is selected from the group consisting of Simian virus 40, Macaca fascicularispolyomavirus 1 , Pan troglodytes verus polyomavirus 1a, Pan troglodytes verus polyomavirus 2a, Pan troglodytes verus polyomavirus 3, Pan troglodytes verus polyomavirus 4, Pan troglodytes verus polyomavirus 8, Pan troglodytes schweinfurthii polyomavirus 2, Chimpanzee polyomavirus, Bornean orang utan polyomavirus, Sumatran orang utan polyomavirus or Gorilla gorilla gorilla polyomavirus 1 , yellow baboon polyomavirus 1 , yellow baboon polyomavirus 2, Vervet monkey polyomavirus 1 , Vervet monkey polyomavirus 2, Vervet monkey polyomavirus 3 and Cercopithecus erythrotis polyomavirus 1 , preferably wherein the polyomavirus is a primate polyomavirus, preferably a simian polyomavirus, such as macaque polyomavirus Simian Virus 40 (SV40).

5. Method according to any of the preceding claims, wherein the recombinant polyomaviral first DNA construct is a circular double-stranded recombinant polyomaviral vector DNA molecule.

6. Method according to any of the preceding claims, wherein a gene encoding functional polyomaviral capsid proteins is introduced into said mammalian cell line the separate second DNA construct or via a separate third DNA construct other than the recombinant polyomaviral first DNA construct and other than the separate second DNA construct.

7. Method according to any of the preceding claims, wherein the separate second and third DNA constructs, other than the recombinant polyomaviral first DNA construct, introduced into the mammalian cell line cannot be encapsidated into recombinant polyomaviral vector particles.

8. Method according to any of the preceding claims, wherein the separate second and third DNA constructs, other than the recombinant polyomaviral first DNA construct, introduced into the mammalian cell line do not comprise the polyomaviral intergenic region.

9. Method according to any of the preceding claims, wherein the polyomaviral intergenic region and the functional polyomaviral T antigens, such as large T antigen,are derived from a polyomavirus selected from the group consisting of Simian virus 40, Macaca fascicularis polyomavirus 1 , Pan troglodytes verus polyomavirus 1a, Pan troglodytes verus polyomavirus 2a, Pan troglodytes verus polyomavirus 3, Pan troglodytes verus polyomavirus 4, Pan troglodytes verus polyomavirus 8, Pan troglodytes schweinfurthii polyomavirus 2, Chimpanzee polyomavirus, Bornean orang utan polyomavirus, Sumatran orang utan polyomavirus or Gorilla gorilla gorilla polyomavirus 1 , yellow baboon polyomavirus 1 , yellow baboon polyomavirus 2, Vervet monkey polyomavirus 1 , Vervet monkey polyomavirus 2, Vervet monkey polyomavirus 3 and Cercopithecus erythrotis polyomavirus 1 , preferably wherein the polyomaviral intergenic region and the functional polyomaviral large T antigen are derived from a primate polyomavirus, preferably a simian polyomavirus, such as macaque polyomavirus Simian Virus 40 (SV40).

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