Compositions and methods for producing polyomavirus vector particles

Abstract

The present invention relates to a composition for use in producing polyomavirus vector particles, the composition comprising a first DNA construct comprising recombinant DNA and a polyomavirus intergenic region, and a second DNA construct comprising a polyomavirus functional coding sequence encoding a functional polyomavirus capsid protein, wherein the first DNA construct does not comprise the polyomavirus functional coding sequence and a bacterial plasmid sequence, and the second DNA construct is not capable of being encapsulated into a polyomavirus vector particle. The present invention further relates to a method for producing polyomavirus vector particles using the first and second DNA constructs, and to polyomavirus vector particles for use as a pharmaceutical, preferably for the treatment of genetic and immune-related diseases.

Description

[Technical Field]

[0001] The present invention relates to compositions for use in producing polyomavirus vector particles and methods for producing polyomavirus vector particles. The present invention further relates to polyomavirus vector particles for use as pharmaceuticals, preferably for use in the treatment of genetic and immune-related diseases. [Background technology]

[0002] Over the past few decades, much effort has been devoted to developing efficient gene and nucleic acid delivery technologies for introducing genes and nucleic acids into target cells and ensuring their appropriate expression. Therapeutic genes and nucleic acids can be used to restore dysfunctional genes to treat genetic diseases, induce immune responses to treat cancer and infectious diseases, induce / restore immune tolerance to prevent transplant rejection, and suppress immune responses to treat autoimmune diseases and allergies. Therapeutic genes or nucleic acids can be administered as naked molecules or as nucleic acids packaged in lipid and / or proteinaceous compounds.

[0003] Because viruses have evolved to deliver and express genetic information in host target cells, viral vectors are the most effective gene delivery vehicles for expressing self or foreign proteins in vivo. Among the viral vectors currently used to treat genetic diseases, cancer, autoimmune diseases, allergies, and to prevent transplant rejection, replication-deficient lentiviral (LV) vectors derived from human immunodeficiency virus type 1 and replication-deficient adeno-associated viral (AAV) vectors derived from adeno-associated viruses are the most widely used. Both replication-deficient vectors have been shown to be non-immunogenic or tolerogenic in hosts naive to their cognate viruses. LV vectors permanently alter transformed target cells by randomly integrating their viral genome into the host genome. Because these particles are highly unstable, LV vectors are primarily used in ex vivo gene replacement therapy to treat blood-related genetic diseases and cancer. Because the AAV vector genome remains as a stable episome in the nucleus of transduced target cells, and the particles are highly stable, AAV vectors are primarily used in in vivo gene therapy. AAV is a primarily human virus that co-replicates with adenovirus, the causative agent of the common cold. The majority of humans have been exposed to AAV and have acquired strong immunological memory against the viral capsid protein. Numerous clinical studies using recombinant AAV vectors have confirmed that administration of vector particles induces innate and adaptive immune responses against the virus and transgene-encoded proteins in the majority of treated patients. This immune response eliminates transduced cells from the body, reducing the expression level of the therapeutic transgene over time and compromising vector re-administration. The few treated patients who demonstrated long-term transgene expression likely had no prior AAV infection and were therefore immunologically naive to the AAV vector used in the study. In such patients, a single administration of AAV vectors can lead to transgene overexpression, potentially resulting in severe side effects. The immunogenicity and toxicity of AAV in humans, and the resulting clinical inefficacy, remain major challenges to the approval of new AAV vector-based therapies.

[0004] Replication-deficient polyomavirus vectors are an attractive alternative to AAV vectors for clinical gene therapy. Polyomaviruses replicate strictly in their natural hosts, causing chronic, asymptomatic infections. Replication-deficient polyomavirus vectors are not immunogenic in immunologically naive hosts. Simian virus 40 (SV40) is a polyomavirus that naturally and strictly infects macaques, causing chronic, asymptomatic infections. SV40 particles enter infected cells via the caveolae-endosomal pathway, but, in contrast to other viruses, they avoid lysosomal degradation, avoiding exposure to the host immune system. SV40 contains a 5.25-kb circular double-stranded DNA molecule carrying two genes. The early genes encode two nonstructural replication-associated proteins, small T antigen (STag) and large T antigen (LTag). The late genes encode the structural viral proteins VP1, VP2, and VP3.

[0005] The early and late genes are separated by the polyomavirus intergenic region, which contains the early and late promoters required for transcription of the early and late genes, the origin of replication 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 region of the early genes, leaving 2.7 kb of available space for cloning foreign DNA. When cells lacking the SV40 early genes were transduced with this vector, the absence of LTag prevented the production of all viral proteins. Because humans are considered naive to SV40, replication-defective SV40 vectors are not expected to be immunogenic in humans. This non-immunogenicity in humans makes SV40 vectors highly attractive for gene therapy to treat genetic diseases, cancer, autoimmune diseases, and allergies, as well as for preventing transplant rejection.

[0007] Replication-deficient SV40 vectors are produced in macaque packaging cell lines that express SV40 early genes, such as COS-1, COT18, and CMT4, or macaque cell lines that express SV40 early and late genes, such as COS-7. Packaging cell lines that express SV40 early and late genes can be used to produce replication-deficient SV40 vectors that lack the coding regions of the early and late genes. Such "gutless" vectors have the capacity to encode 4.8 kb of foreign DNA (Mueller C et al., Gene Therapy 17:227-237, 2010).

[0008] However, when the SV40 vector is passaged in the packaging cell line, wild-type SV40 particles emerge, likely through sequence homology-dependent recombination between chromosomally inserted and episomally replicated SV40-specific DNA sequences.

[0009] To prevent the generation of replication-competent virus particles in vector preparations, polyomavirus-based virus-like particle (VLP) vector systems have been developed. In vitro-produced VLPs consist of circular, double-stranded polyomavirus vector DNA encapsulated in polyomavirus VP1. Such VLPs lack VP2 and VP3 in the capsid and the histones that coat the encapsulated DNA molecule. While these particles exhibit high packaging capacity, the absence of VP2 / VP3 and histones in VLPs negatively impacts their transduction efficacy in vivo.

[0010] To overcome the risk of wild-type viral contamination during the production of replication-deficient SV40 vector particles, a safe and efficient Vero-based SV40 vector packaging cell line, named SuperVero, was engineered. SuperVero cells express only the viral LTag and accumulate fully replication-deficient vector particles at titers comparable to those obtained with conventional SV40 vector packaging cell lines (see, e.g., Toscano MG et al., Mol. Ther. Methods Clin. Dev. 6:124-134, 2017; International Patent Application WO 2010 / 122094 A1).

[0011] Currently, circular polyomavirus vector DNA, required to initiate vector particle production in packaging cell lines, is generated by releasing the vector DNA from the plasmid backbone by restriction enzyme digestion and then self-ligating the resulting linear vector DNA using T4 DNA ligase to generate a circular polyomavirus vector genome. A drawback of this method of generating circular vector DNA is the relaxed fidelity of the circular DNA molecule. Such relaxed circular vector DNA genomes are relatively poor substrates for SV40 LTag replication and packaging into SV40 vector particles by SV40 capsid proteins. To circumvent this issue, circular polyomavirus vector DNA with supercoiled fidelity can be generated by introducing two recognition sequences for tyrosine or serine DNA recombinase, such as the loxP sequences for the Cre recombinase from bacteriophage T1, flanking the vector DNA in the polyomavirus vector plasmid. Addition of tyrosine or serine DNA recombinase to polyomavirus vector plasmid DNA in vitro generates circular, supercoiled polyomavirus vector genomes. Alternatively, polyomavirus vector plasmid DNA containing a recognition sequence for tyrosine or serine DNA recombinase can be introduced into polyomavirus vector-packaging cells along with DNA encoding the cognate tyrosine or serine DNA recombinase, resulting in the generation of circular, supercoiled polyomavirus vector genomes in vivo (Shi X et al., Mol. Ther. Methods Clin. Dev. 9:225-233, 2018).

[0012] A drawback of replication-deficient polyomavirus vectors with deleted early gene coding regions is that the space for cloning foreign DNA is relatively limited to 2.7 kb. Summary of the Invention

[0013] In a first aspect, the present invention relates to a composition for use in producing polyomavirus vector particles, the composition comprising: a first DNA construct comprising recombinant DNA and a polyomavirus intergenic region; a second DNA construct comprising a polyomavirus functional coding sequence encoding a functional polyomavirus capsid protein; the first DNA construct does not contain polyomavirus functional coding sequences and bacterial plasmid sequences; The second DNA construct cannot be encapsulated into a polyomavirus vector particle. [Brief explanation of the drawings]

[0014] [Figure 1] 1A to 1D show vector maps of plasmids pAM467, pAM615, pAM560, and pAM486, respectively. [Figure 2] This figure shows the results of SuperVero cells transduced with MaxVec particles encoding GFP (MaxVGFP). MaxVGFP particles were produced in SuperVero cells using pAM486 as a helper plasmid. Three days after transfection, the supernatant was collected and added to fresh SuperVero cells. GFP+ signals indicate transduced cells. A: SVGFP (control), B: MaxVGFP (4.7 kB), C: negative cell control. Image 1 shows the cells, and image 2 shows the fluorescent signal. Three days after transduction, the cells were observed under a fluorescence microscope (509 nm) at 100x magnification. [Figure 3] Figure 1 shows the results of SuperVero cells transduced with MaxVLuc. MaxVLuc particles were produced in SuperVero cells using pAM486 as a helper plasmid. Firefly luciferase luminescence was measured 3 days after transfection in triplicates. Bars represent a single transfection. The SVLuc control is based on the SVec vector carrying the SV40 late gene. [Figure 4]This figure shows firefly luciferase expression in pSVLuc (control) and MaxVLuc vectors with different genome lengths. SuperVero cells were transfected with different vector DNAs with or without a BGH polyadenylation sequence downstream of the SV40 late promoter, with genome lengths ranging from 2.5 to 6.0 kb. Supernatants from transfected cells were collected and added to fresh SuperVero cells in triplicate (n=3). Firefly luciferase was measured 3 days after transduction. TRF = firefly luciferase signal after transfection into SuperVero cells as an indicator of transfection efficiency; TRD = firefly luciferase signal after transfection into SuperVero cells as an indicator of MaxVec titer. MaxVec vectors with genome lengths of 4.0 to 6.0 kb were produced at the highest titers. [Figure 5] Figure 1 shows serial passage of SVLuc versus MaxVLuc in SuperVero cells. SuperVero cells were used to produce MaxVLuc and SVLuc particles after transfection (TRF) of cre recombinant plasmid DNA. Cell supernatants were collected and added to fresh SuperVero cells and serially passaged for three passages. Luminescence was measured after the fourth transduction (TRD) cycle. Measurements showed that SuperVero cells transduced with SVLuc still produced vector particles. However, MaxVLuc showed a decrease in luminescence after the first passage, indicating that MaxVec particles were produced after transfection of SuperVero with cre recombinant plasmid DNA, but not after transduction of SuperVero cells. DETAILED DESCRIPTION OF THE INVENTION

[0015] As used herein, the term "polyomavirus intergenic region" refers to a region of polyomavirus vector DNA that contains the early and late promoters required for transcription of the early and late genes, the origin of replication required for polyomavirus DNA replication, and the packaging signal required for formation of polyomavirus vector particles.

[0016] It has been found that by providing a first DNA construct that does not encode a functional polyomavirus protein or a bacterial plasmid sequence and a second DNA construct that encodes a functional polyomavirus capsid protein, not only is the second DNA construct unable to be encapsulated into polyomavirus vector particles, but also reliable and safe production of polyomavirus vector particles is provided. Furthermore, it has been found that the composition of the present invention has improved recombinant DNA packaging ability compared to currently used polyomavirus vector particles containing polyomavirus late genes, allowing greater flexibility in designing the recombinant DNA used in polyomavirus vector particles.

[0017] As described above, the first DNA construct of the present invention does not contain polyomavirus functional coding sequences and bacterial plasmid sequences. In other words, the first DNA construct of the present invention is substantially free of (e.g., does not contain any) polyomavirus functional coding sequences, i.e., does not encode polyomavirus functional proteins including functional polyomavirus capsid proteins (e.g., VP1, VP2, and VP3), replication-associated proteins small T antigen (STag) and large T antigen (LTag), agnoprotein, and bacterial plasmid sequences.

[0018] It should be noted that the term "first DNA construct" as used herein may refer to a gutless vector, a gutless vector plasmid, or similar terms used in the art, including gutless vector DNA.

[0019] The actual size of the recombinant DNA contained in the first DNA construct may vary, but is selected so that the recombinant DNA can be encapsulated into polyomavirus vector particles. Providing a first DNA construct that does not contain sequences encoding functional polyomavirus proteins increases the packaging capacity of the first DNA construct for the recombinant DNA. The present invention provides first DNA constructs in which the recombinant DNA can have a size of at least 3.0 kb. Typically, the recombinant DNA contained in the first DNA construct can have a size of 4.0 kb to 6.0 kb.

[0020] It should be noted that the term "recombinant DNA" as used herein may refer to similar terms used in the art, including a DNA insert, a transgene, or a transgene construct. The recombinant DNA can encode one or more therapeutic proteins or RNA molecules.

[0021] As described above, the second DNA construct of the present invention encoding a functional polyomavirus capsid protein cannot be encapsulated into a polyomavirus vector particle. Preferably, the size of the second DNA construct is selected so that the second DNA construct cannot be encapsulated into a polyomavirus vector particle. The size of the second DNA construct can vary, but preferably, the second DNA construct has a size of at least 10 kb, at least 11 kb, or more preferably at least 12 kb.

[0022] As used herein, the term "second DNA construct" may refer to a support vector genome or support DNA vector, i.e., a DNA construct or vector that includes DNA sequences, such as sequences encoding functional polyomavirus capsid proteins, and that facilitates the formation of polyomavirus vector particles.

[0023] The second DNA construct comprises a polyomavirus functional coding sequence encoding a functional polyomavirus capsid protein (preferably selected from the group consisting of VP1, VP2, and VP3). Additionally, the second DNA construct may also comprise 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, production of polyomavirus vector particles is no longer dependent on the additional functionality of a polyomavirus-permissive cell into which the DNA construct of the present invention is introduced.

[0024] The polyomavirus intergenic regions and polyomavirus functional coding sequences used in the constructs of the present invention are preferably derived from a primate polyomavirus, preferably a simian polyomavirus. Preferably, the polyomavirus intergenic region and the polyomavirus functional coding sequence are selected from the group consisting of Simian Virus 40, Macaca fascicularis polyomavirus 1, Pan troglodytes verus polyomavirus 1a, Western chimpanzee polyomavirus 2a, Western chimpanzee polyomavirus 3, Western chimpanzee polyomavirus 4, Western chimpanzee polyomavirus 8, Pan troglodytes schweinfurthii polyomavirus 2, chimpanzee polyomavirus, Bornean orangutan polyomavirus, Sumatran orangutan polyomavirus, or Western lowland gorilla (Gorilla gorilla gorilla) polyomavirus 1, Yellow Baboon Polyomavirus 1, Yellow Baboon Polyomavirus 2, Vervet Monkey Polyomavirus The polyomavirus is derived from a polyomavirus selected from the group consisting of Polyomavirus 1, Vervet Monkey Polyomavirus 2, Vervet Monkey Polyomavirus 3, and Red-eared Mongolian (Cercopithecus erythrotis) Polyomavirus 1. In a preferred embodiment, the polyomavirus intergenic region and the polyomavirus functional coding sequence are derived from Simian Virus 40 (SV40), a macaque polyomavirus.

[0025] The composition of the present invention can be a solution, for example, an aqueous solution, preferably a physiological solution.The composition of the present invention can comprise a first DNA construct as a first vector and a second DNA construct as a different second vector.Preferably, the first DNA construct and the second DNA construct are comprised in the composition as a first plasmid and a second plasmid, respectively.Furthermore, when both DNA constructs are provided as separate constructs, i.e., separate vectors or plasmids, it is noted that it is preferable to provide a composition comprising an excess amount of the first DNA construct and a limited amount of the second DNA construct.

[0026] Alternatively, the first DNA construct and the second DNA construct of the composition of the present invention may be contained in the same circular DNA.In other words, the composition contains a certain amount, preferably an excess amount, of circular DNA, and each circular DNA is formed from the first DNA construct and the second DNA construct.In a preferred embodiment, the second DNA construct contained on the circular DNA may further contain a sequence encoding a recombinase enzyme, such as Cre recombinase.

[0027] The second DNA construct may be inserted into the chromosomal DNA of the cells used to produce the polyomavirus vector particles of the invention.

[0028] In a second aspect, the present invention relates to a method for producing polyomavirus vector particles, the method comprising: a) providing a first DNA construct comprising recombinant DNA and a polyomavirus intergenic region; b) providing a cell line permissive to wild-type polyomavirus; c) introducing the first DNA construct of step a) into the cell line of step b); d) culturing the cell line obtained in step c) in a growth medium under conditions that allow the formation of polyomavirus vector particles; e) harvesting polyomavirus vector particles from the cell culture obtained in step d); Including, In step c), the method further includes introducing a second DNA construct capable of expressing a functional Polyomavirus capsid protein, wherein the first DNA construct does not contain functional Polyomavirus coding sequences and bacterial plasmid sequences, and the second DNA construct is incapable of being encapsulated into a Polyomavirus vector particle.

[0029] The second DNA construct contains a polyomavirus functional coding sequence that encodes a functional polyomavirus capsid protein and, optionally, encodes a functional polyomavirus large T antigen.

[0030] The cell line permissive to wild-type polyomavirus 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 to wild-type polyomavirus is selected from the group consisting of Vero, CV1 or BSC-1 cells or derivatives thereof.

[0031] Furthermore, it should be noted that the first DNA construct comprising the recombinant DNA and the polyomavirus intergenic region is capable of replicating in a cell line permissive for wild-type polyomavirus.

[0032] Both DNA constructs can be introduced into the cell line as separate DNA constructs in step c), wherein the first DNA construct provided in step a) is comprised in a circular DNA and / or the second DNA construct is comprised in a circular DNA.

[0033] Alternatively, both DNA constructs can be provided as a single circular DNA comprising the first and second DNA constructs, in such an embodiment, the single circular DNA is introduced into the cell line in step c).

[0034] In a third aspect, the present invention relates to a composition comprising polyomavirus vector particles obtainable by the method of the present invention. It should be noted that the polyomavirus vector particles obtainable by the method of 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 obtained by the method of the present invention are unable to replicate in cells permissive to wild-type poliomavirus. In particular, the present invention relates to a composition that does not comprise a single polyomavirus particle capable of replicating in cells permissive to wild-type polyomavirus, wherein the cells do not express a functional polyomavirus large T antigen or a functional polyomavirus capsid protein.

[0035] In a preferred embodiment, the present invention relates to a composition comprising more than one million polyomavirus vector particles obtained by the methods of the present invention.

[0036] In a fourth aspect, the present invention relates to polyomavirus vector particles obtainable by the method of the invention for use as a medicament. Preferably, the present invention relates to polyomavirus vector particles obtainable by the method of the invention for use in the treatment of genetic and immune-related diseases, including degenerative diseases, inflammatory diseases, autoimmune diseases, allergies, cancer, and in the treatment of transplant rejection. [Example]

[0037] Construction of a dual gutless MaxVec vector system The MaxVec dual replicon gutless vector system was constructed using two previously described plasmids: pSVac, a plasmid encoding the SV40 intergenic and late regions (Toscano et al., 2017); and pHY359, a pBluescript-based plasmid encoding the SV40 LTag under the transcriptional control of the EIF1a promoter.

[0038] The MaxVec vector plasmid pMaxVec (pAM467; SEQ ID NO: 1; see also Figure 1A) was constructed by removing the late region from pSVac. A Gateway recombination DNA cassette was then inserted to facilitate transgene cloning by Gateway recombination (ThermoFisher Scientific). Multiple cloning sites were added downstream of the SV40 late promoter to allow for the addition of transgene DNA, in this case, "filler" DNA (pAM615; SEQ ID NO: 2; see also Figure 1B).

[0039] Here, it was noted that the addition of filler DNA becomes important when using a transgene of a size that does not allow vector particles to be formed.

[0040] Additionally, two LoxP recombination sites were added to flank the viral genes in the plasmid, allowing the bacterial backbone, including the ampicillin resistance gene and bacterial replication origin, to be removed from the viral vector using the enzyme Cre recombinase.

[0041] Because MaxVec vector particles cannot be produced in cells lacking the SV40 LTag and capsid proteins, an SV40 MaxVec helper plasmid (pAM560; SEQ ID NO: 3; see also Figure 1C) was constructed. This helper plasmid was constructed by adding a blasticidin resistance gene behind the SV40 early promoter and SV40 LTag gene under the transcriptional control of the EF1-α promoter from pHY359. The EF1-α-Ltag sequence was inserted downstream of the blasticidin resistance gene. The SV40 late region was placed under the transcriptional control of the SV40 late promoter.

[0042] Testing the Dual Gutless MaxVec Vector System To test the newly constructed MaxVec dual vector system, the helper plasmid pAM560 and Cre-modified (New England Biolabs) MaxVec plasmids encoding firefly luciferase (MaxVLuc) or the hrGFPII reporter gene (MaxVGFP) as transgenes were cotransfected into SuperVero cells at a 1:1 ratio, where cells were grown at 10,000 cells / cm the day before transfection. 2 Cells were seeded at a density of 10,000 cells / cm. After transfection with polyethyleneimine (PEI) at a PEI:DNA mass ratio of 4:1, the cells were incubated overnight at 37°C and washed the next day with Optipro medium. Three days after transfection, the supernatant and cells were harvested separately. MaxVLuc-containing cells were lysed according to the manufacturer's instructions (Promega), and firefly luciferase luminescence was measured (Glomax, Promega). MaxVLuc-containing supernatant was cultured at a density of 10,000 cells / cm. 2 The cells were incubated with fresh SuperVero cells seeded at a density of 1000 μg / ml. After 3 days, the transduced cells were examined for the presence of GFP by fluorescence microscopy.

[0043] Transfection experiments demonstrated that the SV40 early promoter remained active and drove transgene expression (see Figures 2 and 3). Furthermore, luminescence in transduced cells was detected using the supernatant of transfected cells, demonstrating the production of MaxVec vectors in transfected SuperVero cells. These results demonstrate that the MaxVec dual replicon vector system provides potent MaxVec vector particles capable of transducing cells and expressing transgenes in transduced cells.

[0044] Optimization of MaxVec particle manufacturing To identify the optimal genome size for producing MaxVec particles in SuperVero cells, different transgene sequence lengths were tested. Multiple MaxVec constructs were generated, each encoding firefly luciferase and different lengths of non-coding "filler" DNA. The resulting MaxVec plasmids were cre-recombined to generate circular vector DNA molecules. SuperVero cells were co-transfected with the MaxVec filler DNA variants and a helper plasmid. Supernatants were harvested 3 days post-transfection. To confirm the transduction efficiency / capacity of various MaxVec particles, 5x10 supernatants were used. 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.

[0045] The results (see Figure 4) demonstrated that vector sizes up to 6.0 kb can be packaged. Genome lengths of 4.0 kb to 6 kb were shown to produce particles that could most efficiently transduce SuperVero cells.

[0046] MaxVec production generates replication-defective particles MaxVec particles encoding firefly luciferase (MaxVLuc) were produced in SuperVero cells using the pAM486 helper plasmid (SEQ ID NO: 4; see also Figure 1D). Particles were collected from the supernatant on days 3 and 6 posttransfection, measured, and pooled. SuperVero cells were seeded, and MaxVLuc particles were added to the cells. The supernatant from the transduced SuperVero cells was then collected, and the cells were isolated on day 7 posttransduction and measured for luminescence. Luminescence was measured using a dual luciferase assay (Promega). The supernatant was then added back to fresh SuperVero cells, and this procedure was repeated for two more rounds. Neither the initially transfected nor the transduced cells exhibited luminescence. SuperVero cells transduced with supernatant collected from the initial or subsequent transductions did not exhibit luminescence (see Figure 5). These data demonstrate that MaxVec particles cannot replicate in cells lacking SV40 LTag and capsid proteins. Therefore, MaxVec particles cannot be produced in SuperVero cells that lack SV40 capsid protein.

Claims

1. 1. A composition for use in producing polyomavirus vector particles, comprising: a first DNA construct comprising recombinant DNA and a polyomavirus intergenic region; a second DNA construct comprising a polyomavirus functional coding sequence encoding a functional polyomavirus capsid protein; Including, the first DNA construct does not contain polyomavirus functional coding sequences and bacterial plasmid sequences; The composition, wherein said second DNA construct is incapable of being encapsulated into the produced polyomavirus vector particle.

2. 2. The composition of claim 1, wherein the size of the recombinant DNA is selected so that the recombinant DNA can be encapsulated into the polyomavirus vector particle, and is preferably at least 3.0 kb, more preferably 4.0 kb to 6.0 kb.

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

4. The composition of any one of claims 1 to 3, wherein the second DNA construct encodes a functional polyomavirus large T antigen.

5. 5. The composition of any one of claims 1 to 4, wherein the polyomavirus intergenic region and the polyomavirus functional coding sequence are derived from a simian polyomavirus, such as a primate polyomavirus, preferably the macaque polyomavirus Simian Virus 40 (SV40).

6. The composition of any one of claims 1 to 5, 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 contained in the same circular DNA.

8. The composition of claim 7 , wherein the second DNA construct contained on the circular DNA further comprises a sequence encoding a recombinase enzyme, such as Cre recombinase.

9. a) providing a first DNA construct comprising recombinant DNA and a polyomavirus intergenic region; b) providing a cell line permissive to wild-type polyomavirus; c) introducing the first DNA construct of step a) into the cell line of step b); d) culturing the cell line obtained in step c) in a growth medium under conditions that allow the formation of polyomavirus vector particles; e) recovering polyomavirus vector particles from the cell culture obtained in step d); Equipped with In step c), the method further comprises introducing a second DNA construct capable of expressing a functional polyomavirus capsid protein; said first DNA construct is free of polyomavirus functional coding sequences and bacterial plasmid sequences; The method for producing a polyomavirus vector particle, wherein the second DNA construct is incapable of being encapsulated into the polyomavirus vector particle.

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

11. 11. The method of claim 9 or 10, wherein the first DNA construct provided in step a) is contained in a circular DNA, said circular DNA further comprising a second DNA construct comprising a polyomavirus functional coding sequence encoding a functional polyomavirus capsid protein and, optionally, a functional polyomavirus large T antigen.

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

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

14. 12. A polymeric viral vector particle obtainable by the method of any one of claims 9 to 11 for use in the treatment of genetic and immune-related diseases, including degenerative diseases, inflammatory diseases, autoimmune diseases, allergies, cancer and transplant rejection.

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