Compositions and methods of making polyomavirus vector particles

Abstract

The present invention relates to a composition for preparing polyomavirus vector particles, said composition comprising said first DNA construct comprising recombinant DNA and a polyomavirus intergene region wherein said first DNA construct does not comprise a polyomavirus functional coding sequence and a bacterial plasmid sequence, and a second DNA construct comprising recombinant DNA and a polyomavirus intergene region wherein said first DNA construct does not comprise a bacterial plasmid sequence and a polyomavirus functional coding sequence wherein said second DNA construct does not comprise a bacterial plasmid sequence. Comprising a polyomavirus functional coding sequence encoding a functional polyomavirus capsid protein wherein the second DNA construct cannot be wrapped in polyomavirus vector particles. The invention also relates to a method for preparing polyomavirus vector particles using the first and second DNA constructs, as well as polyomavirus vector particles for use as medicaments, preferably for the treatment of genetic and immune-related diseases.

Description

Technical Field

[0001] The present invention relates to a composition for preparing polyomavirus vector particles and a method for preparing polyomavirus vector particles. The present invention also relates to polyomavirus vector particles for use as a medicament, preferably for treating genetic diseases and immune-related diseases. Background Art

[0002] In the past several decades, many efforts have been devoted to developing effective gene or nucleic acid delivery technologies for introducing and correctly expressing genes or nucleic acids in target cells. Therapeutic genes or nucleic acids can be used to restore malfunctioning genes to treat hereditary diseases, induce immune responses to treat cancer and infectious diseases or suppress immune responses, for example, to induce / restore immune tolerance to prevent transplant rejection or to treat autoimmune diseases and allergies. Therapeutic genes or nucleic acids can be used as naked molecules or as nucleic acids packaged in lipids and / or protein compounds.

[0003] Since viruses have evolved to deliver and express their genetic information into host target cells, viral vectors are by far the most effective gene delivery vectors for expressing self or foreign proteins in vivo. Among the viral vectors currently used to treat genetic diseases, cancer, autoimmune diseases and allergies, and for preventing transplant rejection, replication-defective lentiviral (LV) vectors derived from human immunodeficiency virus type 1 and replication-defective adeno-associated virus (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 tolerant in hosts that are negative for the homologous virus. LV vectors permanently modify transduced target cells by randomly integrating their viral genomes into the host genome. Since the particles are highly unstable LV vectors, they are mainly used in ex vivo gene replacement therapy to treat blood-related genetic diseases and cancer. The genome of AAV vectors remains a stable episome in the nucleus of the transduced target cells, and since the particles are highly stable AAV vectors are mainly used for in vivo gene therapy. AAV is primarily a human virus that replicates with adenovirus: the pathogenic agent of the common cold. Most of the human population has been exposed to AAV and has developed a strong immune memory of the viral capsid protein. Many clinical studies using recombinant AAV vectors have indeed confirmed that administration of vector particles triggers innate and adaptive immune responses against proteins encoded by the virus and transgene in most treated patients. The immune response leads to elimination of transduced cells from the body and reduces the expression level of the therapeutic transgene over time, impairing re-administration of the vector. The few treated patients who show long-term transgene expression are most likely never infected with AAV and are therefore immunologically negative for the AAV vectors used in the study. In these patients, treatment with a single dose of AAV vectors may result in overexpression of the transgene, leading to severe side effects in some cases. The immunogenicity and toxicity of AAV in humans, as well as its clinical ineffectiveness, will remain a major challenge for the approval of new AAV vector-based interventions.

[0004] Replication-deficient polyomavirus vectors are attractive alternatives to AAV vectors for clinical gene therapy. Polyomaviruses replicate strictly in their natural hosts, where they cause chronic asymptomatic infections. Replication-deficient polyomavirus vectors are non-immunogenic in hosts that are immunologically negative for homologous polyomaviruses. Simian virus 40 (SV40) is a polyomavirus that naturally and strictly infects rhesus monkeys, where it causes chronic asymptomatic infections. SV40 particles enter infected cells via the plasma membrane microvesicle-endosome pathway, but compared to other viruses, are able to avoid lysosomal degradation, thereby avoiding exposure to the host immune system. SV40 has a 5.25 kb long circular double-stranded DNA with two genes. The early gene encodes two nonstructural replication-related proteins, small T antigen (STag) and large T antigen (LTag). The late gene encodes the structural viral proteins VP1, VP2, and VP3.

[0005] The early and late genes are separated by the polyomavirus intergenic region, which includes the early and late promoters required for transcription of the early and late genes, the replication initiation site required for polyomavirus DNA replication, and the packaging signal required for the formation of polyomavirus particles.

[0006] Replication-defective SV40 vectors were generated by deleting the coding regions of the early genes, leaving 2.7 kb of available space to clone foreign DNA. In cells lacking the SV40 early genes transduced with the vector, the absence of LTag prevents the production of all viral proteins. Because humans can be considered negative for SV40, replication-defective SV40 vectors are expected to be non-immunogenic when applied to humans. Non-immunogenicity in humans makes SV40 vectors highly attractive for use in gene therapy for the treatment of genetic diseases, cancer, autoimmune diseases and allergies, and to prevent transplant rejection.

[0007] Replication-deficient SV40 vectors are produced in rhesus monkey packaging cell lines expressing the SV40 early genes, such as COS-1, COT18, and CMT4, or in rhesus monkey 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-deficient SV40 vectors lacking the coding regions of the early and late genes. Such "gutless" vectors have the coding capacity of 4.8 kb of foreign DNA (Mueller C et al ., GeneTherapy 17: 227-237, 2010).

[0008] However, passage of the SV40 vector in the packaging cell line results in the appearance of wild-type SV40 particles. This most likely occurs through sequence homology-dependent recombination between chromosomally inserted SV40-specific DNA sequences and episomal replicated SV40-specific DNA sequences.

[0009] In order to prevent the presence of replication-competent viral particles in vector preparations, polyomavirus-based virus-like particle (VLP) vector systems have been developed. VLPs produced in vitro consist of circular double-stranded polyomavirus vector DNA packaged with polyomavirus VV1. Such VLPs lack VP2 and VP3 in the capsid and histones covering the packaged DNA molecule. Although these particles show a higher packaging capacity, the absence of VP2 / VP3 and histones in VLPs has a negative impact on their transduction efficacy in vivo.

[0010] To overcome the generation of wild-type viral contaminants during the production of replication-defective SV40 vector particles, a safe and efficient Vero-based SV40 vector packaging cell line, termed SuperVero, was generated. SuperVero cells express only the viral LTag and accumulate fully replication-defective vector particles at high titers comparable to those obtained in conventional SV40 vector packaging cell lines (see example: Toscano MG et al ., Mol. Ther. Methods Clin. Dev. 6: 124-134, 2017; International patent application published under number WO 2010 / 122094 A1).

[0011] At present, the carrier DNA is released from the plasmid backbone by restriction enzyme digestion, and then the obtained linear carrier DNA is self-ligated with T4 DNA ligase to produce a circular polyomavirus vector genome, thereby producing the circular polyomavirus vector DNA required for starting to produce vector particles in the packaging cell line. The shortcoming of this method for producing circular carrier DNA is that the circular DNA molecule has a relaxed consistency. This relaxed circular carrier DNA genome is a relatively poor substrate for SV40 LTag to be replicated and packaged into SV40 vector particles by SV40 capsid protein. In order to avoid this problem, the carrier DNA in the polyomavirus vector plasmid can be flanked by the loxP sequence of the Cre recombinase of phage T1, to produce a circular polyomavirus vector DNA with supercoil consistency. In vitro, tyrosine or serine DNA recombinase is added to the polyomavirus vector plasmid DNA to cause the generation of a circular supercoil polyomavirus vector genome. Alternatively, introduction of polyomavirus vector plasmid DNA containing a recognition sequence for a tyrosine or serine DNA recombinase together with DNA encoding a cognate tyrosine or serine DNA recombinase into polyomavirus vector packaging cells results in the production of circular supercoiled polyomavirus vector genomes in vivo (Shi X et al ., Mol. Ther. Methods Clin. Dev. 9: 225-233, 2018).

[0012] The disadvantage of replication-defective polyomavirus vectors with early gene deletion coding regions is that the space for cloning foreign DNA is relatively limited, only 2.7 kb. Summary of the invention

[0013] In a first aspect, the present invention relates to a composition for preparing polyomavirus vector particles, the composition comprising: - a first DNA construct comprising recombinant DNA and a polyomavirus intergenic region, wherein the first DNA construct does not include a polyomavirus functional coding sequence and a bacterial plasmid sequence; and - A second DNA construct comprising a polyomavirus functional coding sequence encoding a functional polyomavirus capsid protein, wherein the second DNA construct is not capable of being packaged into a polyomavirus vector particle.

[0014] As used herein, the term "polyomavirus intergenic region" refers to a region of polyomavirus vector DNA that includes early and late promoters required for early and late gene transcription, a replication initiation site required for polyomavirus DNA replication, and a packaging signal required for the formation of polyomavirus vector particles.

[0015] It has been found that by providing a first DNA construct that does not encode polyomavirus functional proteins and bacterial plasmid sequences, and by providing a second DNA construct that encodes functional polyomavirus capsid proteins, which second DNA construct cannot be packaged in polyomavirus vector particles, a reliable and safe production of polyomavirus vector particles is provided. It has also been found that the composition of the present invention provides high flexibility in designing the recombinant DNA for the polyomavirus vector particles, which has an increased packaging capacity compared to the currently used polyomavirus vector particles containing polyomavirus late genes.

[0016] As described above, the first DNA construct of the present invention does not include polyoma virus functional coding sequences and bacterial plasmid sequences. In other words, the first DNA construct of the present invention does not substantially contain (e.g., does not contain any) polyoma virus functional coding sequences, i.e., does not encode polyoma virus functional proteins, including functional polyoma virus capsid proteins (e.g., VP1, VP2, and VP3), replication-related proteins small T antigen (STag) and large T antigen (LTag), unknown protein (agnoprotein), and bacterial plasmid sequences.

[0017] It should be noted that the term "first DNA construct" as used herein may refer to similar terms used in the art, including gutless vector, gutless vector plasmid, or gutless vector DNA.

[0018] The actual size of the recombinant DNA included in the first DNA construct can vary, but is selected so that the recombinant DNA can be packaged in polyomavirus vector particles. By providing a first DNA construct that does not include sequences encoding functional polyomavirus proteins, the packaging capacity of the first DNA construct relative to the recombinant DNA is increased. The present invention now provides a first DNA construct, wherein the recombinant DNA can have a size of at least 3.0 kb. Typically, the size of the recombinant DNA included in the first DNA construct can be between 4.0 kb and 6.0 kb.

[0019] It should be noted that the term "recombinant DNA" used herein may refer to similar terms used in the art, including DNA insert, transgene or transgenic construct. The recombinant DNA may encode one or more therapeutic proteins or RNA molecules.

[0020] As described above, the second DNA construct encoding a functional polyomavirus capsid protein of the present invention cannot be encapsulated in a polyomavirus vector particle. Preferably, the size of the second DNA construct is selected so that the second DNA construct cannot be encapsulated in a polyomavirus vector particle. Although the size of the second DNA construct can vary, preferably, the second DNA construct can have a size of at least 10 kb, at least 11 kb, or more preferably at least 12 kb.

[0021] As used herein, the term "second DNA construct" can refer to a supporting vector genome or a supporting DNA vector, i.e., a DNA construct or a vector comprising a DNA sequence, such as a sequence encoding a functional polyomavirus capsid protein, thereby facilitating the formation of polyomavirus vector particles.

[0022] Although the second DNA construct includes a polyomavirus functional coding sequence encoding a functional polyomavirus capsid protein (preferably selected from the group consisting of VP1, VP2 and VP3), in addition, the second DNA construct may also include a polyomavirus functional coding sequence encoding a functional polyomavirus LTag. It has been found that by providing a second DNA construct encoding a functional polyomavirus capsid protein and a functional polyomavirus LTag, the production of polyomavirus vector particles is no longer dependent on the additional functionality of the polyomavirus-permissive cells into which the DNA construct of the present invention is introduced.

[0023] The polyomavirus intergenic regions and the polyomavirus functional coding sequences used in the constructs of the present invention are preferably derived from primate polyomaviruses, preferably simian polyomaviruses. Preferably, the polyomavirus intergenic region and the polyomavirus functional coding sequence are derived from a virus selected from simian virus 40, Macaca fascicularis polyomavirus 1, Senegal wild chimpanzee polyomavirus 1a, Senegal wild chimpanzee polyomavirus 2a, Senegal wild chimpanzee polyomavirus 3, Senegal wild chimpanzee polyomavirus 4, Senegal wild chimpanzee polyomavirus 8, Pan troglodytes schweinfurthii polyomavirus 2, chimpanzee polyomavirus, Bornean orang utan polyomavirus, Sumatran orang utan polyomavirus or West African lowland gorilla polyomavirus 1, yellow baboon polyomavirus 2a, Senegal wild chimpanzee polyomavirus 3, Senegal wild chimpanzee polyomavirus 4, Senegal wild chimpanzee polyomavirus 8, Pan troglodytes schweinfurthii polyomavirus 2, chimpanzee polyomavirus, Bornean orang utan polyomavirus, Sumatran orang utan polyomavirus or West African lowland gorilla polyomavirus 1, yellow baboon polyomavirus 2a, Senegal wild chimpanzee polyomavirus 3, Senegal wild chimpanzee polyomavirus 4, Senegal wild chimpanzee polyomavirus 8, baboon polyomavirus 1, yellow baboon polyomavirus 2, Vervet monkey polyomavirus 1, Vervet monkey polyomavirus 2, Vervetmonkey polyomavirus 3 and red-eared vervet monkey (Cercopithecus erythrotis) polyomavirus 1. In a preferred embodiment, the polyomavirus intergenic region and the polyomavirus functional coding sequence are derived from rhesus monkey polyomavirus simian virus 40 (SV40).

[0024] Composition of the present invention can be a solution, such as an aqueous solution, preferably a physiological solution. Composition of the present invention can include the first DNA construct as a first vector and the second DNA construct as a different independent second vector. Preferably, the first DNA construct and the second DNA constructed are included in the composition as the first plasmid and the second plasmid respectively. In addition, it should be noted that in the case where two DNA constructs are provided as independent constructs, i.e., independent vectors or plasmids, it is preferred to provide a composition comprising an excessive first DNA construct and a limited amount of the second DNA construct.

[0025] Alternatively, the first DNA construct and the second DNA construct of compositions of the present invention may be included in the same circular DNA. In other words, the compositions include a certain amount of, preferably excessive circular DNA, wherein each of the circular DNA is formed by the first DNA construct and the second DNA construct. In a preferred embodiment, the second DNA construct included in the circular DNA may further include a coding recombinase, for example a sequence of the Cre recombinase.

[0026] The second DNA construct can be inserted into the chromosomal DNA in the cells used to produce the polyomavirus vector particles of the present invention.

[0027] In a second aspect, the present invention relates to a method for preparing polyomavirus vector particles, the method comprising the following steps: a) providing a first DNA construct comprising recombinant DNA and a polyomavirus intergenic region, wherein the first DNA construct does not include a polyomavirus functional coding sequence and a bacterial plasmid sequence; b) provide a cell line that is permissive for wild-type polyomavirus; c) introducing the first DNA construct of step a) into the cell line of step b); d) culturing said cell line obtained in step c) in a growth medium under conditions allowing the formation of polyomavirus vector particles; and e) harvesting said polyomavirus vector particles from said cell culture obtained in step d), The method further comprises the step of introducing in step c) a second DNA construct capable of expressing a functional polyomavirus capsid protein, and wherein the second DNA construct cannot be encapsidated in the polyomavirus vector particle.

[0028] The second DNA construct includes a polyomavirus functional coding sequence encoding a functional polyomavirus capsid protein and optionally encoding a functional polyomavirus large T antigen.

[0029] The cell line permissive for said wild-type polyomavirus as used in the method of the invention and provided in step b) is preferably capable of expressing a functional polyomavirus large T antigen. Preferably, the cell line permissive for said wild-type polyomavirus is selected from the group consisting of Vero, CV1 or BSC-1 cells or derivatives thereof.

[0030] Furthermore, it should be noted that said first DNA construct comprising the recombinant DNA and said polyomavirus intergenic region is capable of replicating in said cell line permissive for said wild-type polyomavirus.

[0031] Both DNA constructs may be introduced into said cell line in step c) as separate DNA constructs, wherein said first DNA construct provided in step a) is comprised in a circular DNA and / or wherein said second DNA construct is comprised in a circular DNA.

[0032] Alternatively, the two DNA constructs may be provided as a single circular DNA comprising the first DNA construct and the second DNA construct. In such embodiments, the single circular DNA is introduced into the cell line in step c).

[0033] In a third aspect, the present invention relates to a composition comprising polyomavirus vector particles obtainable by the method described in the present invention. It should be noted that the polyomavirus vector particles obtainable by the method described in the present invention are polyomavirus vector particles that do not encode functional polyomavirus proteins, such as functional polyomavirus capsid proteins and functional polyomavirus replication-associated proteins and bacterial plasmid sequences. It should also be noted that the polyomavirus vector particles obtainable by the method described in the present invention cannot replicate in cells that allow the wild-type polyomavirus. In particular, the present invention relates to a composition that does not contain a single polyomavirus particle, and the polyomavirus particle is able to replicate in cells that allow the wild-type polyomavirus, wherein the cells do not express functional polyomavirus large T antigen and functional polyomavirus capsid protein.

[0034] In a preferred embodiment, the present invention relates to a composition comprising more than one million polyomavirus vector particles obtainable by the method of the present invention.

[0035] In a fourth aspect, the present invention relates to polyomavirus vector particles obtainable by the method of the present invention for use as a medicament. Preferably, the present invention relates to polyomavirus vector particles obtainable by the method of the present invention for use in the treatment of genetic diseases and immune-related diseases, including degenerative diseases, inflammatory diseases, autoimmune diseases, allergies, cancer and transplant rejection. DETAILED DESCRIPTION

[0036] Example Construction of the double gutless MaxVEC vector system The MaxVec dual-replicon gut-free vector system was constructed using two previously described plasmids: pSVac (Toscano M et al ., 2017), which is a plasmid encoding the SV40 intergenic region and late region; and pHY359, which is a pBluescript-based plasmid encoding SV40 LTag under the transcriptional control of the EIF1a promoter.

[0037] The MaxVec vector plasmid pMaxVec (pAM467; SEQ ID NO: 1; see also: Figure 1A ) was constructed by removing the late region from pSVac. Subsequently, a gateway recombination DNA cassette was inserted to facilitate cloning of transgenes by Gateway recombination (Thermo Fisher). A multiple cloning site was added downstream of the SV40 late promoter to allow for the addition of transgenic DNA, in this case "stuffer" DNA (pAM615; SEQ ID NO: 2; see also: Figure 1B ).

[0038] Note here that if a transgene with a size that does not result in vector particle formation is used, the addition of filler DNA is important.

[0039] Furthermore, two LoxP recombination sites flanking the viral genes within the plasmid were added. This enabled the bacterial backbone containing the ampicillin resistance gene and the bacterial origin of replication to be excluded from the viral vector using the enzyme Cre-recombinase.

[0040] Because MaxVec vector particles cannot be produced in cells lacking SV40 LTag and capsid proteins, an SV40MaxVec helper plasmid (pAM560; SEQ ID NO: 3; see also: Figure 1C ). This helper plasmid was constructed by adding the blasticidin resistance gene behind the SV40 early promoter and the SV40 LTag gene under the transcriptional control of the EF1α promoter from pHY359. The EF1-α-Ltag sequence was inserted downstream of the blasticidin resistance gene. The SV40 late region remained under the transcriptional control of the SV40 late promoter.

[0041] Testing of the Double Gutless MaxVEC Vector System Test the newly constructed MaxVec dual vector system. SuperVero cells were co-cultured with the helper plasmid pAM560 and the vector encoding firefly luciferase (MaxV Luc ) or hrGFPII reporter gene (MaxV GFP ) was co-transfected with the MaxVec plasmid containing Cre-recombinase (New England Biolabs) as a transgene. Here, cells were cultured at 10,000 cells / cm 1 day before transfection. 2 After transfection with polyethyleneimine (PEI) at a PEI:DNA weight ratio of 4:1, the cells were incubated overnight at 37°C and washed with optipro medium the next day. Three days after transfection, the supernatant and cells were collected separately. The cells containing MaxV were lysed according to the manufacturer's instructions (Promega). LucThe cells were cultured and the luminescence of firefly luciferase (Glomax, Promega) was measured. Luc The supernatant was plated at 10,000 cells / cm 2 Three days later, transduced cells were examined by fluorescence microscopy for the presence of GFP.

[0042] Transfection experiments showed that the SV40 early promoter remained active and drove transgene expression (see: Figure 2 and Figure 3 ). In addition, luminescence could be detected in cells transduced using the supernatant of transfected cells, indicating that the MaxVec vector was produced in the transfected SuperVero cells. These results indicate that the MaxVec dual-replicon vector system provides potent MaxVec vector particles capable of transducing cells and expressing transgenes in transduced cells.

[0043] Optimization of MaxVEC Granule Production Different transgene sequence lengths were tested to identify the optimal genome size for producing MaxVec particles in SuperVero cells. Multiple MaxVec constructs were prepared, each encoding firefly luciferase with different lengths of non-coding "stuffer" DNA. The resulting MaxVec plasmids were cre-recombined to generate circular vector DNA molecules. SuperVero cells were co-transfected with MaxVec stuffer DNA variants and helper plasmids. Supernatants were harvested 3 days after transfection. To determine the transduction efficiency / capacity of the various MaxVec particles, supernatants were then added to 5x10 3 Cells / cm 2 The transfected cells were incubated for 3 days and the number of vector particles produced in the transfected cells was measured by luminescence using a dual luciferase assay.

[0044] The results show (see: Figure 4 ) can package vectors up to 6.0 kb in size. Genome lengths between 4.0 kb and 6 kb have been shown to produce particles that are most efficient in transducing SuperVero cells.

[0045] MaxVEC production produces replication-defective particles The pAM486 helper plasmid (SEQ ID NO: 4; see also: Figure 1D ) in SuperVero cells to produce a protein encoding firefly luciferase (MaxV Luc ) of MaxVec particles. Particles were collected from the supernatant on days 3 and 6 after transfection, measured, and pooled. SuperVero cells were inoculated and MaxV Luc The particles were added to the cells. Subsequently, the supernatant from the transduced SuperVero cells was collected and the luminescence was measured by isolating the cells 7 days after transduction. The luminescence was measured using a dual luciferase assay (Promega). Thereafter, the supernatant was added again to fresh SuperVero cells and the process was repeated 2 more times. The initially transfected or transduced cells showed luminescence. SuperVero cells transduced with supernatant collected from the first or subsequent transductions did not show luminescence (see: Figure 5 ). These data indicate that MaxVec particles cannot replicate in cells that lack SV40 LTag and capsid proteins. Therefore, MaxVec particles cannot be produced in SuperVero cells that lack SV40 capsid proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1A -D depict the vector maps of plasmids pAM467, pAM615, pAM560, and pAM486, respectively.

[0047] Figure 2 Displayed with GFP (MaxV GFP pAM486 was used as a helper plasmid to produce MaxVec in SuperVero cells. GFP Pellets. Three days after transfection, the supernatant was harvested and added to fresh SuperVero cells. GFP+ signal indicates transduced cells. A: SV GFP (Control), B:MaxV GFP (4.7kB) and C: negative cell control. Image 1 shows cells and image 2 shows fluorescent signal. Observed under a fluorescent microscope (509nm) 3 days after transduction at 100x magnification.

[0048] Figure 3 Shows MaxV Luc Results of transduced SuperVero cells. Production of MaxV in SuperVero cells using pAM486 as a helper plasmid Luc Pellets. Luminescence of firefly luciferase was measured 3 days after transduction in triplicate. Bar graphs represent a single transduction. SV Luc The control was based on the SVec vector with the SV40 late genes.

[0049] Figure 4 pSV is shown as a control Luc and MaxV with different genome lengths LucFirefly luciferase expression of vectors. SuperVero cells were transfected with different vector DNAs with genome lengths ranging from 2.5 to 6.0 kb with or without BGH polyadenylation sequences downstream of the SV40 late promoter. The supernatant of the transfected cells was collected and then added to fresh SuperVero cells, repeated three times (n=3). Three days after transduction, firefly luciferase was measured. TRF=firefly luciferase signal after transfection of SuperVero cells was used as a measure of transfection efficiency, and TRD=firefly luciferase signal after transduction of SuperVero cells was used as a measure of MaxVec effectiveness. MaxVec vectors with genome lengths of 4.0 to 6.0 kb were produced at the highest titer.

[0050] Figure 5 SV in SuperVero cells Luc MaxV Luc MaxV was produced using SuperVero cells after transfection (TRF) of cre-recombinant plasmid DNA. Luc and SV Luc The supernatant from the cells was collected and added to fresh SuperVero cells and serially passaged into fresh SuperVero cells for 3 generations. Luminescence was measured after the fourth transduction (TRD) cycle. The results showed that the SV Luc Transduced SuperVero cells still produced vector particles. However, luminescence was reduced at MaxV Luc The expression of cre-recombinant plasmid DNA decreased after the first passage, indicating that MaxVec particles were generated after transfection of SuperVero cells with cre-recombinant plasmid DNA rather than after transduction of SuperVero cells.

Claims

1. A composition for preparing polyomavirus vector particles, the composition comprising: - a first DNA construct comprising recombinant DNA and a polyomavirus intergenic region, wherein the first DNA construct does not comprise a polyomavirus functional coding sequence and a bacterial plasmid sequence; and - A second DNA construct comprising a polyomavirus functional coding sequence encoding a functional polyomavirus capsid protein, wherein the second DNA construct cannot be packaged into the polyomavirus vector particle to be prepared.

2. The composition according to claim 1, wherein The size of the recombinant DNA is selected so that it can be packaged in polyomavirus vector particles, preferably having a size of at least 3.0 kb, more preferably having a size of 4.0 kb to 6.0 kb.

3. A composition according to claim 1 or 2, wherein The size of the second DNA construct is selected such that the second DNA construct cannot be encapsulated within the polyomavirus vector particle.

4. A composition according to any one of the preceding claims, wherein The second DNA construct encodes a functional polyomavirus large T antigen.

5. A composition according to any one of the preceding claims, wherein The polyomavirus intergenic region and the polyomavirus functional coding sequence are derived from primate polyomavirus, preferably simian polyomavirus, for example, rhesus polyomavirus simian virus 40 (SV40).

6. A composition according to any one of the preceding claims, wherein The first DNA construct is a first vector and the second DNA construct is a second vector, preferably a second bacterial plasmid.

7. The composition according to any one of claims 1 to 5, wherein The first DNA construct and the second DNA construct are included in the same circular DNA.

8. The composition according to claim 7, wherein The second DNA construct included in the circular DNA also includes a sequence encoding a recombinase, such as Cre recombinase.

9. A method for preparing polyomavirus vector particles, the method comprising the following steps: a) providing a first DNA construct comprising recombinant DNA and a polyomavirus intergenic region, wherein the first DNA construct does not include a polyomavirus functional coding sequence and a bacterial plasmid sequence; b) provide a cell line that is permissive for wild-type polyomavirus; c) introducing the first DNA construct of step a) into the cell line of step b); d) culturing said cell line obtained in step c) in a growth medium under conditions allowing the formation of polyomavirus vector particles; and e) harvesting said polyomavirus vector particles from said cell culture obtained in step d), The method further comprises the step of introducing in step c) a second DNA construct capable of expressing a functional polyomavirus capsid protein, and wherein the second DNA construct is not encapsulated in the polyomavirus vector particle.

10. The method according to claim 9, wherein: Said wild-type polyomavirus permissive cell line provided in step b) is capable of expressing functional polyomavirus large T antigen.

11. The method according to claim 9 or 10, wherein: The first DNA construct provided in step a) is included in a circular DNA, wherein the circular DNA further includes the second DNA construct, which includes a polyoma functional coding sequence encoding a functional polyoma capsid protein and optionally encoding a functional polyoma large T antigen.

12. A composition comprising polyomavirus vector particles obtained by the method of any one of claims 9 to 11.

13. Polyomavirus vector particles obtainable by the method of any one of claims 9 to 11 for use as a medicament.

14. Polyomavirus vector particles obtainable by the method of any one of claims 9 to 11 for use in treating genetic diseases and immune-related diseases, including degenerative diseases, inflammatory diseases, autoimmune diseases, allergies, cancer and transplant rejection.

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