Methods for viral vector production with reduced host cell aggregation and methods for dissolving host cell aggregates

Abstract

The present invention relates to the field of cell culture and viral vector production. Specifically, the invention provides a method for producing a viral vector, such as AAV, wherein a HEK293 suspension culture comprises spermine or a salt thereof which increased the viral vector titer and reduced cell aggregation. The invention further provides a method for reducing host cell aggregation for production of a viral vector. The invention further provides a method for dissolving host cell aggregates. The invention further provides a composition for reducing host cell aggregation for production of a viral vector. The invention further provides a supplement composition for dissolving host cell aggregates. The invention further provides spermine or a salt thereof for different uses. The invention further provides polyamine or a salt thereof for use in a method for dissolving host cell aggregates.

Description

[0001] METHODS FOR VIRAL VECTOR PRODUCTION WITH REDUCED HOST CELL AGGREGATION AND METHODS FOR DISSOLVING HOST CELL AGGREGATES

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention relates to the field of cell culture and viral vector production. Specifically, the invention provides a method for producing a viral vector, such as AAV, wherein a HEK293 suspension culture comprises spermine or a salt thereof which increased the viral vector titer and reduced cell aggregation. The invention further provides a method for reducing host cell aggregation for production of a viral vector. The invention further provides a method for dissolving host cell aggregates. The invention further provides a composition for reducing host cell aggregation for production of a viral vector. The invention further provides a supplement composition for dissolving host cell aggregates. The invention further provides spermine or a salt thereof for different uses. The invention further provides polyamine or a salt thereof for use in a method for dissolving host cell aggregates.

[0004] BACKGROUND OF THE I NVENTION

[0005] Gene therapy allows for using nucleic acids to repair malfunctioning DNA sequences or to introduce a compensatory change that will restore the normal physiological functions of the cell. Viral vectors provide an efficient mean for modification of eukaryotic cells, and their use is commonplace in academic laboratories and industry for both research and clinical gene therapy applications. Gene therapy via gammaretroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAV) is attractive because of the natural ability of viruses to enter and deliver genetic material to cells. Recombinant AAV-based gene transfer vectors are generated by utilizing the capsid protein shell of the wildtype virus but replacing the ssDNA genome of the natural virus with a therapeutic transgene cassette, retaining only the viral inverted terminal repeats (ITRs). In the absence of the rep proteins, ITR-flanked transgenes encoded by rAAV can form circular concatemers that persist as episomes in the nucleus of transduced cells. Because recombinant episomal DNA does not integrate into the host genome, rAAV is ideal for gene therapy applications and AAV-vector-mediated gene delivery and was recently approved e.g., for the treatment of inherited blindness and spinal muscular atrophy. Long-term therapeutic effects have been achieved for other rare diseases, including hemophilia and Duchenne muscular dystrophy. Also, the European Medicines Agency approved HEMGENIX® by CSL Behring GmbH, Roctavian® by BioMarin International Limited and Upstaza by PTC Therapeutics International Limited as AAV based gene therapies, underscoring its increasing importance. As a vector, AAV has many advantages, such as a negligible rate of integration into the host cell genome, no association with any human disease, and the inability to replicate without a helper virus.

[0006] In most instances, advanced therapeutics such as AAV-derived vectors are produced in mammalian cell lines. A considerable advantage that especially the mammalian cell lines, e.g., such as those of human origin, share is their ability to confer certain post-translational modification (PTM) to the vector capsid. These PTMs can affect the stability, infectivity, and immunogenicity in vivo, thereby making them a crucial quality parameter.

[0007] To date, a cell line derived from human embryonic kidney (referred to as "HEK293" or "293") cells is most applied and well characterized for viral vector production, in particular for recombinant AAV vector production. HEK293 cells were originally developed in the 1970ies and first published by Graham et al. (J. Gen. Virol., 1977, Vol. 36, pp. 59-72). H EK293 cells were originally established by transfection of primary human embryonic kidney cells with fragments of mechanically sheared adenovirus 5 DNA inserted in human chromosome 19. It has been shown that HEK293 cells stably express the adenoviral E1A and ElB-55k proteins due to integration of a 4 kb adenoviral DNA fragment in chromosome 19. Hence, E1A and E1B do not need to be included in the genetic design for modifying such host cell (see Louis et al., Virol., 1997, Vol. 233, pp. 423-429). This is advantageous, as E1A and E1B are inter alia required for production of rAAV vectors and certain adenoviral vectors.

[0008] However, despite extensive research, low productivity of HEK293 cells is limiting a broader clinical use of viral vectors, such as rAAV vectors. In addition, like other epithelial cells, HEK293 cells readily form aggregates when cultured in suspension. In some serum-free media, the cell aggregates even reached up to 3 mm in diameter. Aggregation in suspension culture can be detrimental to a growing population since it restricts the cells inside of the aggregate from access to sufficient oxygen and necessary nutrients, leading to increased cell death and toxin accumulation. Moreover, in a transient production process, the transfection complex comprising the transfection reagent and complexed nucleic acids, e.g. plasmid DNA, cannot reach the cells on the inside of the aggregate. Hence, the aggregation lowers product yield in transient production processes. Aggregation can be related to the Ca2+and Mg2+content in the medium, as well as agitation in the bioreactor and overall hydrodynamic stress, e.g., in shake flask culture (see Seidel et al., Bioengineering, 2023, Vol. 10, 478). Also, dextran sulfate has been reported to reduce host cell aggregation (see WO 1998 / 008934 Al) but is also known for its interaction with viral capsids and to interfere with exogeneous nucleic acid delivery efficiency, e.g. in transient viral vector manufacture (see Williams-Fegredo et al., 2024).

[0009] Hence, there is a need to further increase the efficiency of viral vector production. In particular, there is a need to increase the production capacity of HEK293 cells for viral vectors, such as rAAV, and to reduce the aggregation of cells in suspension culture.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention addresses the above-described needs. The present invention provides a method for producing a viral vector, as well as a method for reducing host cell aggregation for production of a viral vector. Within these methods, a host cell selected from a HEK293 cell or a derivative thereof and a cell culture medium is provided in step (a) and contacted with each other to form a suspension culture in step (b). The suspension culture undergoes culturing in step (c) a nd production in step (d) to produce a viral vector by the host cell in the suspension culture. Importantly, the suspension culture of step (b), step (c), and / or step (d), preferably at least the suspension culture of step (d), comprises spermine or a salt thereof. The presence of spermine or a salt thereof advantageously results in an increase in the viral vector titer, including the genomic and the transducing vial vector titer. Spermine or a salt thereof further reduced the aggregation of the host cells which can be detrimental in culture, particularly for HEK293 cells, as the aggregation severely restricts the mass transfer to the cells within the aggregate, such that these do not receive sufficient oxygen and nutrients. As a result, cells within the aggregate may become apoptotic which can increase toxin levels within the suspension culture. Hence, the reduction in aggregation improves overall culture performance. At the same time, spermine or a salt thereof does not have a negative impact on (transient) viral vector production as other known compounds which can reduce aggregate formation, such as dextran sulfate, but interfere with viral vector production. Indeed, it was even found that spermine or a salt thereof can increase the viral vector production by being used as an enhancer within or shortly after transient transfection.

[0012] According to a first aspect, a method for producing a viral vector is provided, the method comprising:

[0013] (a) providing a host cell selected from a HEK293 cell or a derivative thereof, and a cell culture medium for culturing the host cell;

[0014] (b) contacting the host cell with the culture medium to form a suspension culture;

[0015] (c) culturing the suspension culture; and

[0016] (d) producing a viral vector by the host cell in the suspension culture, wherein the suspension culture of step (b), step (c), and / or step (d), preferably at least the suspension culture of step (d), comprises spermine or a salt thereof.

[0017] According to a second aspect, a method for reducing host cell aggregation for production of a viral vector is provided, the method comprising:

[0018] (a) providing a host cell selected from a HEK293 cell or a derivative thereof, and a cell culture medium for culturing the host cell;

[0019] (b) contacting the host cell with the culture medium to form a suspension culture;

[0020] (c) culturing the suspension culture; and

[0021] (d) producing a viral vector by the host cell in the suspension culture, wherein the suspension culture of step (b), step (c), and / or step (d), preferably at least the suspension culture of step (d), comprises spermine or a salt thereof and wherein host cell aggregation is reduced compared to a suspension culture without spermine or a salt thereof.

[0022] The methods according to the first and second aspects advantageously allow for producing consistently increased viral vector yields. This was demonstrated throughout the examples herein, wherein various HEK293 cell types, different transfection systems, and different culture processes and compositions were tested (see Figs. 1-4, 12, 13). Also, the methods allow for a remarkable reduction in cell aggregation of the host cells, improving the overall cell culture by enhancing oxygen and nutrient supply to the cells without negatively impacting viral vector production (see Figs. 5-8). The presence of spermine or a salt thereof in suspension culture of step (b), step (c), and / or step (d), preferably at least the suspension culture of step (d), indeed significantly improves the production of a viral vector, such as AAV, using a HEK293 host cell-based culturing system. Furthermore, by including spermine or a salt thereof during or shortly after transient transfection for producing a viral vector, such as AAV, improved viral vector production (see Figs. 11-13).

[0023] Based on the surprising finding of the inventors that spermine or a salt thereof significantly reduced host cell aggregation, it was further found that by contacting a suspension culture comprising host cells selected from a HEK293 cell or a derivative thereof with a polyamine such as spermine or a salt thereof, cell aggregates can be dissolved. Hence, according to a third aspect, a method for dissolving host cell aggregates is provided, the method comprising:

[0024] (a) providing a suspension culture comprising host cells selected from a HEK293 cell or a derivative thereof, wherein at least a fraction of the host cells is provided as cell aggregates, and a polyamine or a salt thereof, preferably spermine or a salt thereof; (b) contacting the suspension culture with the polyamine or the salt thereof, preferably spermine or a salt thereof; and

[0025] (c) optionally, culturing the suspension culture, wherein the polyamine or the salt thereof, preferably spermine or a salt thereof, dissolves host cell aggregates.

[0026] The method according to the third aspect advantageously dissolves cell aggregates, which can be detrimental in culture. As disclosed above, cell aggregates can lead to severe restrictions in mass transport limiting oxygen and nutrient supply to cells within such aggregates. This can result in cell death, toxin formation, hindrance of reliable cell counting, and lower culture performance, especially considering production of viral vectors. In addition, the restrictions in mass transport of cell aggregates also apply to transfection complexes delivered to the cells in a transient viral vector production process reducing productivity. As demonstrated in the examples, by contacting a suspension culture of HEK293 cells with a polyamine, such as spermine or a salt thereof, cell aggregates can be effectively dissolved, leading to higher fractions of single cells (see Figs. 9, 10).

[0027] According to a fourth aspect, a composition for reducing host cell aggregation for production of a viral vector is provided, wherein the host cell is selected from a HEK293 cell or a derivative thereof, the composition comprising:

[0028] (i) a cell culture medium for culturing the host cell; and

[0029] (ii) spermine or a salt thereof.

[0030] According to a fifth aspect, a supplement composition for reducing host cell aggregation for production of a viral vector is provided, wherein the host cell is selected from a HEK293 cell or a derivative thereof, the supplement composition comprising spermine or a salt thereof, preferably further comprising:

[0031] (i) a polyamine, preferably spermidine or a salt thereof; and / or a further polyamine, preferably putrescine or a salt thereof; optionally, a culture medium for culturing the host cell; or

[0032] (ii) a polyamine, preferably spermidine or a salt thereof; and / or a downregulating compound capable of downregulating conversion of spermine to spermidine and / or spermidine to putrescine, preferably an inhibitor of an enzyme involved in the spermine synthesis pathway, preferably an inhibitor of spermidine / spermine N 1-acetyltransferase 1 (SAT 1) or polyamine oxidase (PAO), more preferably an inhibitor of SAT 1, most preferably phenylbutyrate or a salt thereof; optionally, a liquid for dissolving the spermine or salt thereof; optionally, the supplement composition is substantially free of putrescine or a salt thereof.

[0033] According to a sixth aspect, a supplement composition for dissolving host cell aggregates is provided, wherein the host cells are selected from a HEK293 cell or a derivative thereof, the supplement composition comprising a polyamine or a salt thereof, preferably spermine or a salt thereof.

[0034] The compositions according to the fourth, fifth and sixth aspect allow for obtaining the advantages disclosed above for the methods according to the first, second and third aspect.

[0035] According to a seventh aspect, spermine or a salt thereof for use for producing a viral vector by a host cell is provided, wherein the host cell is selected from a HEK293 cell or a derivative thereof. As demonstrated in the examples, using spermine or a salt thereof for producing a viral vector by a host cell leads consistently to increased viral vector yields. Such increase was obtained for various HEK293 cell types, different transfection systems, and different culture processes and compositions were tested (see Figs. 1-4, 12, 13). The presence of spermine or a salt thereof in suspension culture significantly improved the production of viral vector, such as AAV, using a HEK293 host cell-based culturing system. Furthermore, by including spermine ora saltthereof during orshortly aftertransient transfection for producing a viral vector, such as AAV, improved viral vector production (see Figs. 11- 13).

[0036] According to an eighth aspect, spermine or a salt thereof for use for reducing host cell aggregation for production of a viral vector is provided, wherein the host cell is selected from a HEK293 cell or a derivative thereof. As demonstrated in the examples, spermine or a salt thereof for use for reducing host cell aggregation for production of a viral vector allowed for a remarkable reduction in cell aggregation of the host cells, improving the overall cell culture by enhancing oxygen and nutrient supply to the cells (see Figs. 5-8).

[0037] According to a ninth aspect, polyamine or a salt thereof, preferably spermine or a salt thereof, for use for dissolving host cell aggregates, preferably for production of a viral vector, wherein the host cell is selected from a HEK293 cell or a derivative thereof. As demonstrated in the examples, polyamine or a salt thereof, preferably spermine or a salt thereof, effectively dissolved host cell aggregates, leading to higher fractions of single cells and an optimized cell culture (see Figs. 9, 10). Further aspects of the invention are disclosed below. Other objects, features, advantages, and aspects of the present application will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples, while indicating preferred embodiments of the application, are given by way of illustration only.

[0038] BRIEF DESCRIPTION OF THE FIGURES

[0039] Some exemplary embodiments of the present disclosure will now be described with reference to the accompanying drawings.

[0040] Fig. 1 shows supplementation of spermine on HEKTF which increases AAV2 genomic titer.

[0041] The inhouse HEK cell line and Expi293F cells were adapted to HEK TF supplemented with 50, 100 and 500 pM spermine for five passages at 125 ml shake flask scale. Cells were transiently transfected for AAV2 production using a two- plasmid system and PEI-MAX. Lysis was performed at 72 h post transfection and genomic titer was measured by ddPCR.

[0042] Fig. 2 shows supplementation of spermine on internally developed HEK medium which increases AAV2 titer upon transfection with FectoVIR-AAV. Expi293F and VPC2.0 cells as well as the inhouse HEK clone were adapted to an internally developed HEK medium supplemented with 75 pM spermine for four to five passages at 125 ml shake flask scale. Cells were transiently transfected for AAV2 production using a two-plasmid system and FectoVIR-AAV. Lysis was performed at 72 h post transfection and genomic titer was measured by ddPCR. Depicted is the mean of duplicates ± standard deviation of one repetition.

[0043] Fig. 3 shows supplementation of spermine on internally developed HEK medium which increases AAV2 titer upon transfection with PEI-MAX. Expi293F and VPC2.0 cells as well as the inhouse HEK clone were adapted to an internally developed HEK medium supplemented with 75 pM spermine for four to five passages at 125 ml shake flask scale. Cells were transiently transfected for AAV2 production using a two-plasmid system and PEI-MAX. Lysis was performed at 72 h post transfection and genomic titer was measured by ddPCR. Depicted is the mean of duplicates ± standard deviation of one experiment.

[0044] Fig. 4 shows that spermine as part of a full weighing increases AAV2 titer in Ambr 15. Cells were adapted to a full weighing of the internally developed HEK medium with or without 72 pM spermine and cobalt chloride for three passages. Following inoculation, cells were cultured for one more passage in the Ambr 15 system at 630 rpm upstirring, pH 7.2, DO 40%, and 37°C. Transfection for AAV2 production was performed using a two-plasmid system and PEI-MAX. Cells were lysed at 72 h post transfection and genomic titers were analyzed by ddPCR.

[0045] Fig. 5 shows the supplementation of spermine which decreases cell aggregation in 3-day cultures. Expi293F and VPC2.0 cells as well as the inhouse H EK clone were adapted to an internally developed HEK medium supplemented with 75 pM spermine for three to four passages at 125 ml shake flask scale. Subsequently, cells were allowed to grow for three days. Cell cultures were imaged with help of an automated cell counter. A custom image analysis software (Fiji, ImageJ) script was used to quantify cell aggregation based on object size. Clusters of two cells were considered small aggregates, whereas large aggregates contained three or more cells. Values obtained for single cells, small and large aggregates were summed up for 20 images and are depicted as relatives of all cells counted per condition for one time point.

[0046] Fig. 6 shows that supplementation of spermine decreases cell aggregation in 4-day cultures. Expi293F and VPC2.0 cells as well as the inhouse HEK clone were adapted to an internally developed HEK medium supplemented with 75 pM spermine for three to four passages at 125 ml shake flask scale. Subsequently, cells were allowed to grow for four days. Cell cultures were imaged with help of an automated cell counter. A custom image analysis software (Fiji, ImageJ) script was used to quantify cell aggregation based on object size. Clusters of two cells were considered small aggregates, whereas large aggregates contained three or more cells. Values obtained for single cells, small and large aggregates were summed up for 20 images and are depicted as relatives of all cells counted per condition for one time point.

[0047] Fig. 7 shows that spermine as part of a full weighing medium reduces cell aggregation.

[0048] VPC2.0 cells were adapted to a full weighing with or without 72 pM spermine for two passages at 125 mL shake flask scale. Subsequently, cells were grown for four days in the respective culture medium and imaged with an automated cell counter. A custom image analysis software (Fiji, ImageJ) script was used to quantify cell aggregation based on object size. Clusters of two cells were considered small aggregates, whereas large aggregates contained three or more cells. Values obtained for single cells, small and large aggregates were summed up for 20 images and are depicted as relatives of all cells counted per condition for one time point.

[0049] Fig. 8 shows that spermine supplemented on HEK ViP NB reduces cell aggregation.

[0050] VPC2.0 cells were adapted to HEK ViP NB with or without 72 pM spermine for five passages at 125 mL shake flask scale. Cells were cultured for three days prior to imaging with an automated cell counter. A custom image analysis software (Fiji, ImageJ) script was used to quantify cell aggregation based on object size. Clusters of two cells were considered small aggregates, whereas large aggregates contained three or more cells. Values obtained for single cells, small and large aggregates were summed up for 20 images and are depicted as relatives of all cells counted per condition for one time point.

[0051] Fig. 9 shows that aggregation of VPC2.0 cells increases throughout cultivation. VPC2.0 cells were adapted to an internally developed HEK medium without spermine for three passages at 125 mL shake flask scale. Cells were inoculated at 2E6 VCD / mL and cultured for four days. Imaging with an automated cell counter was performed daily. A custom image analysis software (Fiji, ImageJ) script was used to quantify cell aggregation based on object size. Clusters of two cells were considered small aggregates, whereas large aggregates contained three or more cells. Values obtained for single cells, small and large aggregates were summed up for 20 images and are depicted as relatives of all cells counted per day.

[0052] Fig. 10 shows that addition of spermine dissolves cell aggregates within 4.6 h. VPC2.0 cells were adapted to an internally developed HEK medium without spermine for three passages at 125 mL shake flask scale. Cells were inoculated at 2E6 VCD / mL and cultured for four days. At day 4, 72 pM spermine were added to the culture and cells were imaged with an automated cell counter at 0.5, 2.6, 3.6 and 4.6 h post addition. A custom image analysis software (Fiji, ImageJ) script was used to quantify cell aggregation based on object size. Clusters of two cells were considered small aggregates, whereas large aggregates contained three or more cells. Values obtained for single cells, small and large aggregates were summed up for 20 images and are depicted as relatives of all cells counted per time point.

[0053] Fig. 11 shows spermine added during transfection for AAV2 production. The inhouse HEK clone was adapted to a full weighing of the internally developed HEK medium containing 72 pM spermine at 125 ml shake flask scale. Cells were transfected using a two-plasmid system and FectoVIR-AAV or PEI-MAX for AAV2 production. During transfection complex formation, additional 72 pM spermine was added. Lysis was performed at 72 h post transfection and genomic titer was measured by ddPCR.

[0054] Fig. 12 shows the genomic titer (A) and the fold-change (B) normalized to the P6 media

[0055] (inhouse HEK cell culture media) for spermine and / or M344 added at 4 to 6 h after transfection of VPC2.0 cells for AAV2 production. VPC2.0 cells were adapted to an internally developed HEK medium without spermine at 125 mL shake flask scale. Cells were transfected using a two-plasmid system and FectoVIR-AAV for AAV2 production. At 4 to 6 h after transfection, 72 pM Spermine, or 10 pM M344, or a combination thereof was added. Lysis was performed at 72 h post transfection and genomic titer was measured by ddPCR.

[0056] Fig. 13 shows the genomic titer (A) and the fold-change (B) normalized to the P6 media

[0057] (inhouse HEK cell culture media) for spermine and / or M344 added at 4 to 6 h after transfection of inhouse HEK clone 2.0 for AAV2 production. The inhouse HEK clone 2.0 was adapted to an internally developed HEK medium without spermine at 125 mL shake flask scale. Cells were transfected using a two-plasmid system and FectoVIR-AAV for AAV2 production. At 4 to 6 h after transfection, 72 pM spermine, or 10 pM M344, or a combination thereof was added. Lysis was performed at 72 h post transfection and genomic titer was measured by ddPCR.

[0058] Fig. 14 shows that supplementing spermine and spermidine at a physiological ratio combined with phenylbutyrate increases AAV2 genomic titer upon transfection with FectoVIR-AAV. The inhouse HEK clone was adapted to a full weighing of a HEK medium containing 72 pM spermine at 125 ml shake flask scale for three passages. The HEK medium was supplemented with spermidine, putrescine, and / or phenylbutyrate at the indicated concentrations. Cells were transfected using a two- plasmid system and FectoVIR-AAV for AAV2 production. 72 h post transfection cells underwent lysis and genomic titer was measured by ddPCR.

[0059] Fig. 15 shows that supplementing spermine and spermidine at a physiological ratio combined with phenylbutyrate increases AAV2 genomic titer upon transfection with PEI-MAX. The internal HEK clone was adapted to a full weighing of a HEK medium containing 72 pM spermine at 125 ml shake flask scale for three passages. The medium was supplemented with spermidine, putrescine, and / or phenylbutyrate at the indicated concentrations. Cells were transfected using a two-plasmid system and PEI-MAX for AAV2 production. 72 h post transfection cells underwent lysis and genomic titer was measured by ddPCR.

[0060] Fig. 16 shows AAV2 production with FectoVIR-AAV after supplementation of spermine and spermidine at a physiological ratio combined with phenylbutyrate which increases AAV2 transducing titer. The inhouse HEK clone was adapted to a full weighing of the internally developed HEK medium containing 72 pM spermine at 125 ml shake flask scale for three passages. In a screening approach, the medium was supplemented with spermidine, or spermidine and phenylbutyrate at the indicated concentrations. Cells were transfected using a two-plasmid system and FectoVIR-AAV for AAV2 production. Dilution of harvested AAV2 samples were added to adherent HEK293 cells and transducing titers were calculated based on the number of GFP+ cells 72h after transduction measured by flow cytometry.

[0061] Fig. 17 shows AAV2 production with PEI MAX after supplementation of spermine and spermidine at a physiological ratio combined with phenylbutyrate which increases AAV2 transducing titer. The inhouse HEK clone was adapted to a full weighing of the internally developed HEK medium containing 72 pM spermine at 125 ml shake flask scale for three passages. In a screening approach, the medium was supplemented with spermidine, or spermidine and phenylbutyrate at the indicated concentrations. Cells were transfected using a two-plasmid system and PEI MAX for AAV2 production. Dilution of harvested AAV2 samples were added to adherent HEK293 cells and transducing titers were calculated based on the number of GFP+ cells 72h after transduction measured by flow cytometry.

[0062] DETAILED DESCRIPTION

[0063] The following description serves to deepen the understanding of the present disclosure and shall be understood to complement and be read together with the description of exemplary embodiments of the present disclosure as provided in the above section of this description. It is to be understood that this invention is not limited to the particular embodiments, methodologies, protocols and reagents described herein as these may vary within the scope set by the claims. It is also to be understood that terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which is defined by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0064] In the following description, certain elements of the present invention will be described. These elements may be discussed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples, features and particular embodiments should not be construed to limit the present invention to only the explicitly described embodiments or to the explicitly described combination of features. This description should be understood to disclose and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by this description unless the context indicates otherwise. As explained in the summary of the invention, the different aspects and embodiments of the invention disclosed herein make important contributions to the art by providing improved viral vector production and cell culture systems.

[0065] Methods according to the first and second aspects of the invention

[0066] According to a first aspect, a method for producing a viral vector is provided, the method comprising:

[0067] (a) providing a host cell selected from a HEK293 cell or a derivative thereof, and a cell culture medium for culturing the host cell;

[0068] (b) contacting the host cell with the culture medium to form a suspension culture;

[0069] (c) culturing the suspension culture; and

[0070] (d) producing a viral vector by the host cell in the suspension culture, wherein the suspension culture of step (b), step (c), and / or step (d), preferably at least the suspension culture of step (d), comprises spermine or a salt thereof.

[0071] According to a second aspect, a method for reducing host cell aggregation for production of a viral vector is provided, the method comprising:

[0072] (a) providing a host cell selected from a HEK293 cell or a derivative thereof, and a cell culture medium for culturing the host cell;

[0073] (b) contacting the host cell with the culture medium to form a suspension culture;

[0074] (c) culturing the suspension culture; and

[0075] (d) producing a viral vector by the host cell in the suspension culture, wherein the suspension culture of step (b), step (c), and / or step (d), preferably at least the suspension culture of step (d), comprises spermine or a salt thereof and wherein host cell aggregation is reduced compared to a suspension culture without spermine or a salt thereof.

[0076] The methods according to the first and second aspects advantageously allow for producing consistently increased viral vector yields. This was demonstrated throughout the examples herein, wherein various HEK293 cell types, different transfection systems, and different culture processes and compositions were tested (see Figs. 1-4, 12, 13). Also, the methods allow for a remarkable reduction in cell aggregation of the host cells, improving the overall cell culture by enhancing oxygen and nutrient supply to the cells (see Figs. 5-8), as well as increasing accessibility of the cells for transfection. The presence of spermine or a salt thereof in suspension culture of step (b), step (c), and / or step (d), preferably at least the suspension culture of step (d), significantly improves the production of viral vector, such as AAV, using a HEK293 host cell-based culturing system. Furthermore, by including spermine or a salt thereof during or shortly after transient transfection for producing a viral vector, such as AAV, improved viral vector production (see Figs. 11-13).

[0077] The individual steps and preferred embodiments of the method according to the first aspect will now be described in detail.

[0078] The viral vector

[0079] The viral vector according to the present disclosure is any viral material suitable for delivering genetic material into cells (e.g., alone or in conjunction with further viruses or biochemical cues). In particular, as used herein, the terms "virus (vector)," "viral vector," and "gene delivery vector" refer to a virus particle that functions as a nucleic acid delivery vehicle, and which comprises a nucleic acid molecule packaged within the viral particle. A viral vector may be suitable for application in gene therapy, i.e. allows for using nucleic acids to repair malfunctioning DNA sequences or to introduce a compensatory change that will restore the normal physiological functions of the cell. Such gene delivery is also referred to as transduction. In general, a viral vector for gene therapy is produced recombinantly such that it contains one or more target genes, also referred to as "transgenes". Hence, according to a preferred embodiment, the viral vector is a recombinant viral vector. The target genes can be transduced into a cell, e.g. patient cells, autologous cells, or allogenic cells, which can happen in vivo or in vitro or ex vivo. The one or more target genes are then either directly available for protein expression in the cell as non-integrative vectors which degrade naturally over time or are integrated into the nuclear DNA of the cell. As used herein, a "transgene" or "target gene" is a nucleic acid that is introduced into the genome, including but not limited to genes or nucleic acid having sequences which are not normally present in the viral vector genes, such as an AAV or Adenovirus, which are present but not normally transcribed and translated ("expressed") in the viral vector genome, such as AAV or Adenovirus genome, or any other gene or nucleic acid which one desires to position between the viral vector repeat sequences, such as AAV's ITR sequences. A transgene may include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid. A transgene can be as few as a couple of nucleotides long, but can preferably be at least about 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1.000, 1.100, 1.200, 1.300, 1.400, 1.500, 1.600, 1.700, 1.800, 1.900, 2.000, 2.100, 2.200, 2.300, 2.400, 2.500, 2.600, 2.700, 2.800, 2.900, 3.000, 3.100, 3.00, 3.300, 3.400, 3.500, 3.600, 3.700, 3.800, 3.900, 4.000, 4.100, 4.200, 4.300, 4.400, 4.500, or 4.600 nucleotides (nt) long. A transgene can comprise coding or non-coding sequences. Importantly, the methods disclosed herein are believed to be applicable for a range of viral vectors and shall not be limited to a particular type. A number of viruses have been suitable as viral vectors for gene therapy, which are known by the skilled person, see e.g. Roldao et al., 2017, Comprehensive Biotechnology, Vol. 1, pp. 633-656.

[0080] According to a preferred embodiment, the viral vector is a an enveloped or a non-enveloped viral vector, preferably a non-enveloped viral vector. An adeno-associated virus (AAV) is a typical example for a non-enveloped viral vector and provides a suitable model for other non-enveloped viral vector types, such that the skilled person would extrapolate the results disclosed herein also to other nonenveloped viral vector types. According to a preferred embodiment, the viral vector is a DNA viral vector or an RNA viral vector, preferably a DNA viral vector. An adeno-associated virus (AAV) is a typical example for a DNA viral vector and provides a suitable model for other DNA viral vector types, such that the skilled person would extrapolate the results disclosed herein also to other DNA viral vector types. As disclosed herein, it is hypothesized that the spermine has a particular effect on the DNA level, supporting the advantages for production of DNA viruses, such as an adeno-associated virus (AAV). According to a particularly preferred embodiment, the viral vector is a non-enveloped, DNA viral vector, such as an adeno-associated virus (AAV).

[0081] According to a further embodiment, the viral vector is suitable for gene therapy and preferably selected from the group consisting of retrovirus, such as lentivirus, or adenovirus, or adeno- associated virus (AAV), or hybrids thereof. A viral vector may be selected from retroviruses, such as lentivirus, adenoviruses, herpes simplex, vaccinia, and adeno-associated virus (AAV). According to preferred embodiments, the viral vector is an adeno-associated virus (AAV). As demonstrated in the examples below, the method according to the present disclosure improves production of AAV (see e.g., Figs. 1-4, 12, 13). The term "adeno-associated virus" or "AAV" is interchangeable used with the terms recombinant AAV, rAAV, rAAVs, adeno-associated viruses, or AAV vector, all of which refer to a viral vector which is based on a native adeno-associated virus, but which was produced recombinantly. AAV is a small virus capable of infecting humans but not known to cause any disease. AAV can infect both dividing and non-dividing cells but mostly stays episomal, performing long and stable expression. These features make AAV a very attractive candidate for creating viral vectors for gene therapy.

[0082] As used herein, the term "adeno-associated virus" or "AAV", includes but is not limited to, AAV type 1 (e.g., AAV of serotype 1, also referred to as AAV1), AAV type2 (e.g, AAV2), AAV type 3 (e.g, AAV3, including types 3A and 3B, AAV3A and AAV3B), AAV type 4 (e.g, AAV4), AAV type 5 (e.g, AAV5), AAV type 6 (e.g, AAV6), AAV type 7 (e.g, AAV7), AAV type 8 (e.g, AAV8), AAV type 9 (e.g, AAV9), AAV type 10 (e.g, AAV10), AAV type 11 (e.g, AAV11), AAV type 12 (e.g, AAV12), AAV type 13 (e.g, AAV13), AAV type rh32.33 (e.g, AAVrh32.33), AAV type rh8 (e.g, AAVrh8), AAV type rhIO (e.g, AAVrhIO), AAV type rh74 (e.g, AAVrh74), AAV type hu.68 (e.g, AAVhu.68), avian AAV (e.g, AAAV), bovine AAV (e.g, BAAV), canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAV now known or later discovered. According to some embodiments, the AAV is AAV type 2.

[0083] According to preferred embodiments, the host cell is modified to be configured to produce a viral vector. In other embodiments, it may also be started directly with the host cell without any active modification step by purchasing a host cell that is capable of producing the desired viral vector, e.g., a so-called packaging cell or stable cell which contains all genetic elements required for expressing the viral vector, typically without the target gene(s). In some embodiments, the applied host cell may from the beginning on also be itself capable of expressing one or more genes which are required for producing the viral vector, e.g., HEK293 cells are known to express E1A and E1B which are required for producing AAV.

[0084] According to a preferred embodiment, the host cell is modified by transiently transfecting the host cell with one or more plasmids for viral vector production allowing for transient viral vector production. Transiently transfected host cells allow essentially for a single run of viral vector manufacturing, such that for each manufacturing run a transfection has to be performed. According to an alternative preferred embodiment, the host cell is modified by genome editing the host cell with one or more nucleic acid molecules for viral vector production allowing for stable viral vector production. The stable integration allows for using a stock, e.g., cell bank or master cell bank, of modified host cells multiple times without the need to genetically modifying the host cells in advance of each manufacturing run (compared to the transient transfection). This has the advantage that an established process can be repeated multiple times without any necessity to perform transient transfection in advance, simplifying the process and rendering it more efficient. On the other hand, the transient transfection is more flexible, as for each run a different set of genetic elements can be transfected into the cells without complicated genome editing of the host cells in advance. Both ways of modifying the host cells for producing a viral vector are applicable in frame of the method according to the present disclosure and shall not be limiting in any way. Indeed, it is also in scope of the present disclosure to combine transient transfection and stable integration, e.g. by providing some genetic elements stably integrated and other genetic elements via transient transfection. For instance, the transgene gene cassette may be provided by transient transfection, whereas the remaining genetic elements may be stably integrated. According to a preferred embodiment, transiently transfecting comprises (x) combining the host cell with the one or more plasmids for viral vector production and a transfection reagent; and (y) incubating the host cell, the one or more plasmids for viral vector production and the transfection reagent to generate a transiently transfected cell. Preferably, spermine or a salt thereof is present in incubation step (y) and / or is added after incubation step (y). In preferred embodiments, wherein the spermine or salt thereof is present during transient transfection and / or is added after transient transfection. In such embodiments, spermine advantageously enhances the viral vector in that the production of the viral vector is increased (see e.g., Figs. 11-13). In some embodiments, the spermine or salt thereof is also combined in step (x) with the host cell with the one or more plasmids for viral vector production and a transfection reagent. For instance, the host cell may be provided in a cell culture medium comprising spermine or the salt thereof, such that it is automatically present in step (x). In other embodiments, spermine or a salt thereof may be provided separately, e.g., in form of a supplement composition comprising spermine or a salt thereof as disclosed herein. In some embodiments, step (x) comprises combining the host cell with the one or more plasmids for viral vector production and a transfection reagent and further with a supplement composition and / or a cell culture medium, wherein the supplement and / orthe cell culture medium comprises spermine or a salt thereof. It may be advantageous to have an early and possibly increased effect of the spermine or salt thereof on the host cell.

[0085] For the step of combining the host cell with the one or more plasmids for viral vector production and a transfection reagent (see step (x)) different procedures may be followed, including: combining a cell culture medium with the one or more plasm ids for viral vector production and the transfection reagent to form a transfection composition; optionally, mixing the transfection composition of (i); optionally, incubating the transfection composition of (i) or (ii); combining the transfection composition of (i), (ii) or (iii) and the host cell; and optionally, the transfection composition of (i), (ii), or (iii) or the composition of (iv) further comprises the spermine or salt thereof; or alternatively, adding the one or more plasmids for viral vector production and the transfection reagent to the host cell present in a cell culture medium, optionally further comprising the spermine or salt thereof.

[0086] Both methods are suitable for transfecting the host cells with the one or more plasmidsfor viral vector production. In some embodiments, the spermine or salt thereof as disclosed herein is combined with the transfection composition, such that the spermine or salt thereof is preferably added at the same time as the one or more plasmids for viral vector production and the transfection reagent to the host cell present in a cell culture medium. In alternative preferred embodiments, the spermine or salt thereof may be added subsequently to the transfection composition, which may be advantageous to adapt the concentration or content of spermine or salt thereof.

[0087] For the step of incubating the host cell, the one or more plasmids for viral vector production and the transfection reagent to generate a transiently transfected cell according to the prior embodiment (see step (y)), different incubation times are possible. In one embodiment, the incubation step comprises incubating for at least 5 min, at least 10 min, at least 15 min, at least 30 min, at least 45 min, at least 1 hour, at least 2 hours, at least 3 hours, preferably at least 4 hours. It may be advantageous to have longer incubation times such as at least 2 hours, at least 3 hours, at least 4 hours in order to ensure that as much plasmid has been introduced into the host cell as possible. Accordingly in some embodiments, it is incubated for a range of 1 to 48 hours, 2 to 36 hours, 3 to 24 hours, preferably 4 to 12 hours, more preferably 4 to 8 hours or 4 to 6 hours. According to a specific embodiment, it is incubated for 1 to 8 hours. Incubations times of e.g., 1 to 12 hours, preferably 2 to 8 hours or 4 to 6 hours, are advantageous for the method according to the present disclosure to make sure that the one or more plasmids have been introduced into the host cell. However, according to some embodiments, the spermine or salt thereof may be added before or during transiently transecting the host cell. For instance, during step (x) or during step (y) as disclosed herein spermine or salt thereof may be added, such that the host cell is contacted with the one or more plasmids for viral vector production and a transfection reagent and the spermine or salt thereof. As a result, incubation would take place in presence of the spermine or salt thereof. Afterwards, the host cell may be further contacted with the spermine or salt thereof and / or the host cell may be directly cultured according to step (c).

[0088] According to preferred embodiments, the method further comprises having M344 or a derivative thereof present during transient transfection and / or M344 or a derivative thereof is added after transient transfection. According to preferred embodiments, the method further comprises having M344 or a derivative thereof present in incubation step (y) and / or is added after incubation step (y). As is shown in the examples below, M344 is highly advantageous in that it increases the viral vector titer. M344, which is also referred to as 4-dimethylamino-N-(6-hydroxycarbamoylhexyl)-benzamide is an inhibitor of histone deacetylase (HDAC). HDACs remove acetyl moieties from lysine residues located on histones and cause changes in gene expression. This has been shown to reduce cancer growth (see Knoche et al., 2022, pLoS ONE 17(9): e0273518). HDAC inhibitors such as sodium butyrate and sodium valproate have been shown in the past to influence rAAV production, at high concentrations in the millimolar range (see WO 2020 / 033842 Al). The inventors surprisingly found that the HDAC inhibitor M344, particularly in combination with spermine or a salt thereof, is very potent in improving the viral vector production, as is demonstrated in the examples below, especially for AAV (see e.g., Figs. 13, 14). A "derivative" may be any compound chemically considered a derivative, e.g., Trichostatin A.

[0089] M344 or a derivative thereof may be added at any timepoint, e.g., before, during and / or after transient transfection. Considering the embodiments above, M344 may be present in the transfection composition of step (x) and / or during incubation in step (y) and / or after incubation in step (y). It may be preferred to add M344, optionally together with spermine or a salt thereof, after incubation step (y), e.g., after incubating for at least 5 min, at least 10 min, at least 15 min, at least 30 min, at least 45 min, at least 1 hour, at least 2 hours, at least 3 hours, preferably at least 4 hours, since M344 also can impact cell viability, such that it may be advantageous to add it after incubation.

[0090] According to a preferred embodiment, the transfection reagent is polymer-based or lipid-based, preferably being cationic, such as polyethylene imine or a derivative thereof. In a particular example (also shown below), the transfection reagent may comprise polyethyleneimine. However, the skilled person is well-aware of different transfection reagents and will readily understand that different transfection reagents are applicable in frame of the method according to the present disclosure. The other methods or techniques for transient transfection may be used as is readily known by the person skilled in the art. For instance, electroporation or calcium phosphate precipitation may be used for transfecting the plasmids forviral vector production. In such embodiments, spermine or a salt thereof, optionally also M344 or a derivative thereof, may be added before, during and / or after transfection, preferably after transfection, to enhance viral vector production.

[0091] In some embodiments, the one or more transfection reagent used in the methods disclosed herein is selected from commercially available transfection reagents such as Lipofectamine' 2000 & 3000 (ThermoFisher), TransIT reagents'(MirusBio), FuGene' (Promega), XtremeGene' (Roche), jetPRIME' (Polyplus-transfection), ViaFect' (Promega), FectoVIR' reagents (Polyplus-transfection), FectoVIR' AAV (Polyplus-transfection), PEI MAX® (Polysciences) or PEIpro' (Polyplus-transfection).

[0092] In some embodiments, the one or more transfection reagent used in the methods of the present disclosure is selected from any of the transfection reagents disclosed in WO 2021 / 023798 Al, which is hereby incorporated by reference in its entirety. In some embodiments, the one or more transfection reagent used in the methods of the present disclosure is selected from any of the transfection reagents disclosed in WO 2023 / 161409 Al, which is hereby incorporated by reference in its entirety. In some preferred embodiments, the transfection reagent used in the methods of the present disclosure is FectoVIR'or FectoVIR' AAV. In some embodiments, the transfection reagent comprises (i) at least one compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or an acceptable salt thereof, and (ii) an acceptable excipient, buffering agent, cell culture medium, or transfection medium: wherein:

[0093] - Z1represents H, XI-R3-X2-P+, XI-R3-P+, Xj-X2-P+, R3-X2-P+, Xj-P+, R3-P+, or X2-P+; or Z1is absent;

[0094] Z2represents H, a linear or branched, saturated or unsaturated Ci- Cis alkyl, Cs-Cis aryl, a linear or branched, saturated or unsaturated Cs-Cis aryl-Ci-Cis alkyl, a linear or branched, saturated or unsaturated C2-Cis heteroalkyl, C5-C10 heteroaryl, halogen, OH, a linear or branched, saturated or unsaturated Ci-Ci8alkylamine, a Ci-Ci2alkoxy, a linear or branched, saturated or unsaturated Ci- Cis alkyl-Ci-Ci2alkoxy, XI-R3-X2-P+, XI-R3-P+, Xi-X2-P+, R3-X2- P+, Xi-P+, R3-P+, orX2-P+; or Z2is absent;

[0095] Z3represents H, a linear or branched, saturated or unsaturated Ci-Cis alkyl, Cs-Cis aryl, a linear or branched, saturated or unsaturated C6-Ci8aryl-Ci-Cis alkyl, C5-C10 heteroaryl, a linear or branched, saturated or unsaturated C2-Cis heteroalkyl, C2-Cis alkylidene, OH, guanidine, halogen, XI-R3-X2-P+, XI-R3-P+, Xi-X2-P+, R3-X2-P+, Xi-P+, R3-P+, or X2-P+; or Z 3 is absent;

[0096] Xi and X2, which may be identical or different, represent CO or CH2;

[0097] - R3represents (CH2)m, (CH2)m-CHCH3-(CH2)n-, (CH2)m-C(CH3)2-(CH2)n-, (CH2)m-O-(CH2)n- (CH2)m-S-(CH2)n-, (CH2)m-CH2-O-, with m representing an integer between 1 and 3 and n representing an integer between 1 and 3;

[0098] P+represents a graft cationic polymer, which is a polyamine comprising secondary amines, tertiary amines, a mixture of primary and secondary amines, a mixture of primary and tertiary amines, a mixture of secondary and tertiary amines, or a mixture of primary, secondary and tertiary amines; R or V represents H, a linear or branched, saturated or unsaturated Ci-Cis alkyl or cycloalkyl, a C6-Ci8aryl, a linear or branched, saturated or unsaturated C6-Ci8aryl-Ci-Ci8alkyl, a linear or branched, saturated or unsaturated C2-Ci8heteroalkyl, a linear or branched, saturated or unsaturated C1-C24 ester, a C5-C10 heterocyclyl, a C5-C10 heteroaryl, a linear or branched, saturated or unsaturated Ci-Cis alkyl-Cs-Cio heteroaryl, XI-R8-X2-P+, XI-R8-P+, X1-X2-PT R3- X2-P+, Xi-P+, R3-P+, or X2-P+; with the provisos that: o at least one of Zi, Z2 or Z8is present; and o only one of Zi, Z2, Z8, R or V represents XI-R8-X2-P+, XI-R8-P+, X1-X2-P* R3-X2-P* Xi- P+, R3-P+, or X2-P+.

[0099] The compound of general formula (I) may be prepared according to various methods well known in the art, for example as disclosed in the patent applications WO2021 / 023796 and WO2021 / 023798.

[0100] In some embodiments, the transfection reagent used in the methods of the present disclosure is PEI MAX® or PEIpro'.

[0101] According to preferred embodiments, genome editing the host cell comprises stably integrating the one or more nucleic acid molecules into the chromosome of the host cell to be capable of stably producing a viral vector. The stable integration is advantageous in that it allows using the same stock of genome edited cells multiple times without the need to genetically modifying the cells in advance of culture. The integration can be performed by providing at least one site-directed endonuclease, preferably being selected from a meganuclease, a ZFN, a TALEN, a CRISPR-nuclease, or a nickase or nuclease-dead variant therefrom, or a nucleic acid molecule encoding the same, and optionally in case of a CRISPR-nuclease: providing at least one suitable, functional guide RNA molecule, or a nucleic acid molecule encoding the same. In addition, the genetic elements required for producing the viral vector may be provided in form of a template to be integrated into the genome which may be cut by the at least one site-directed endonuclease, e.g., a CRISPR-nuclease. The way of integrating the genetic elements into the genome of the host cell shall not be limited and different techniques are available to the skilled person. According to particular embodiments, however, the one or more genetic elements required forviral vector production may be integrated using a CRISPR- nuclease, e.g., Casl2a or Cas9.

[0102] According to a preferred embodiment of modifying the host cell by genome editing with one or more nucleic acid molecules for viral vector production allowing for stable viral vector production, the one or more nucleic acid molecules comprise an induction system, such as in particular one or more inducible promoters. Thereby, the expression of viral vector genes can be regulated. This has the advantage that the viral vector genes are not uncontrollably expressed, which can have a negative impact on the host cell viability, as is e.g., known for some Rep proteins of AAV. It is preferred that one or more inducible promotors are used which can be induced by the same or different triggers. For instance, it may be desired to induce all promotors at the same time to start expression of the viral vector genes stably integrated into the genome of the host call. Alternatively, it may be desired to orchestrate the expression of the viral vector genes stably integrated into the genome of the host cell, e.g., by providing sequentially different triggers for inducing the promotor to start protein expression. The type and sequence of inducible system shall not be limiting in frame of the present disclosure, and the skilled person is well-aware of different suitable induction systems.

[0103] According to a preferred embodiment, the one or more plasmids or the one or more nucleic acid molecules for viral vector production encode at least part of an adeno-associated virus (AAV). In some cases, not all of the virus's genetic elements that are natively present need to be present in the one or more plasmids or the one or more nucleic acid molecules for viral vector production, e.g., if part of the genetic elements is already present in the host cells. For instance, HEK293 cells which are commonly used for producing a viral vector were originally established by transfection of primary human embryonic kidney cells with sheared adenovirus 5 DNA, and it has been shown that HEK293 cells stably express the adenoviral ElAand ElB-55k proteins due to integration of a 4 kbp adenoviral DNA fragment in chromosome 19. Hence, E1A and E1B do not need to be including in the genetic design for modifying such host cell. In addition, for gene therapy, the produced viral vector generally contains one or more genes to be delivered to a patient or another cell. Such one or more genes may be referred to as "target gene(s)" or"gene of interest" (GOI). In the final product, these then typically replace the viral vector replication machinery, which is packaged into the plasmid, as it is typically not desired to replicate the viral vector in vivo, i.e., in a potential patient. Therefore, not the native and full viral vector genetic elements may be delivered but only those genetic elements to produce the viral vector including the target gene(s). In some embodiments, the target genes are therapeutic nucleic acids, such as therapeutic DNA or RNA. In other embodiments, the target genes include a reporter gene. In preferred embodiments, the reporter gene can be detected by antibody-based assays. In further preferred embodiments, the reporter gene is a fluorescent molecule. Exemplary fluorescent molecules suitable as reporter gene are GFP, eGFP, mGFP, eYFP, citrine, eCFP, mCFP, Cerulean, dtTomato, mCherry, RFP, mRFP, and any variants thereof. In some embodiments, the reporter gene is a beta-galactosidase, luciferase or glutathione S-transferase, or any variant thereof. In particular embodiments, the target gene is suitable for screening assays or markers, e.g., fluorescence proteins, such as green fluorescent protein (GFP) or a derivative thereof, which are used for visualizing transduction of the viral vector. According to preferred embodiments, the one or more plasmids or the one or more nucleic acid molecules for viral vector production encode one or more of the group comprising Rep78, Rep68, Rep52, Rep40, VP1, VP2, VP3, ITR, AAP, MAAP, X Gene, VA RNA, E4orf6, and E2A, preferably all of the aforementioned, for producing an AAV. According to another embodiment, the one or more plasmids or the one or more nucleic acid molecules for viral vector production encode one or more of the group comprising Rep78, Rep68, Rep52, Rep40, VP1, VP2, VP3, ITR, AAP, MAAP, X Gene, VA RNA, E4orf6, E1A, E1B, and E2A, preferably all of the aforementioned, for producing an AAV. In any of these embodiments, the host cell may already encode one or more of the genetic elements for producing the AAV. For instance, E1A and E1B may already be produced by the host cell, e.g. HEK293, wherein a plasmid or nucleic acid molecule can support the cellular expression by additional copies of E1A or E1B or the cellular expression may be considered sufficient. The encoded genes may be provided on a single or multiple plasmids or a single or multiple nucleic acid molecules. According to one embodiment, more than one plasmid is provided for producing the AAV. For instance, two, three, four, five, six, seven, eight, nine or ten plasmids may be provided. In such case, the plasmids encode the same or different genetic elements required for producing the AAV. According to a specific, non-limiting embodiment, following plasmid system is used:

[0104] One expression plasmid comprising the target gene, such as a therapeutic nucleic acid (e.g., therapeutic RNA or DN A) or a reporter gene, in particular a fluorescent protein (e.g., GFP or a derivative thereof),

[0105] One packaging plasmids comprising: a plasmid encoding at least Rep78, Rep68, Rep52, Rep40, VP1, VP2, VP3, AAP, MAAP, X Gene, VA RNA, E4orf6, and E2A, optionally also E1A and E1B.

[0106] However, any distribution of genetic elements on the one or more (e.g., two, three, four or five) plasmids for producing the AAV is possible for use in a method according to the present disclosure. According to one embodiment, the sequences may also be split, such that only transduction with two or more rAAVs leads to assembled transgenes. According to a one embodiment of the methods disclosed herein, the viral vector is AAV, and two or three plasmids are transfected into the cell for producing the AAV.

[0107] The host cell

[0108] The method comprises providing a host cell selected from a HEK293 cell or a derivative thereof. HEK293 is particularly advantageous as it is well-characterized, widely used and very susceptible to transfection. The HEK293 cell was established by transforming human embryonic kidney cells with sheared adenovirus type 5 DNA. While HEK293 cells and derivatives thereof are widely used for viral vector production, they are - like other epithelial cells - also prone to aggregation. In some serum- free media, the cell aggregates even reached up to 3 mm in diameter. Aggregation in suspension culture can be detrimental to a growing population since it restricts the cells inside of the clump from access to sufficient oxygen and necessary nutrients, leading to increased cell death and toxin accumulation. Aggregation can be related to the Ca2+and Mg2+content in the medium, as well as the agitation in the bioreactor and the overall hydrodynamic stress, e.g., during shake flask culture (see Seidel et al., "Improvement of HEK293 Cell Growth by Adapting Hydrodynamic Stress and Predicting Cell Aggregate Size Distribution", Bioengineering, 2023, Vol. 10, 478).

[0109] As disclosed herein, the inventors surprisingly found that spermine or a salt thereof achieve a reduction on HEK293 aggregation and even allow dissolving existing aggregates (see example, e.g., Figs.5-10). Therefore, according to a second aspect of the invention, a method for reducing host cell aggregation for production of a viral vector is provided. "Reducing host cell aggregation" according to the present disclosure refers to reduced host cell aggregation compared to a suspension culture without spermine or a salt thereof. This may refer to a reduction in large aggregate and / or small aggregates. This may also refer to an increase in single cells. It may also be possible that over culture of the suspension culture comprising the spermine or salt thereof, the proliferating cells remain to a greater extent single cells compared to a suspension culture without spermine or a salt thereof, such that the formation of host cell aggregates is reduced. Such reduction can occur throughout culturing of the suspension culture and / or throughout the production of the viral vector. In some embodiments, the reduction may be achieved prior to production of the viral vector throughout the cell culture, e.g., throughout the upscaling of the cells, also referred to as a "seed train process". In some embodiments or in addition, the reduction may be achieved during production of the viral vector.

[0110] Without being bound to theory, it is hypothesized that the spermine interacts with DNA and other charged molecules in a manner that such interaction is less possible with the host cells, thereby "shielding" the host cells from interacting. Based on evidence in literature describing that spermine condensates DNA (see Katz et al., "Spermine Condenses DNA, but not RNA Duplexes", Biophysical Journal, 2017, Vol. E112(l), pp. 22-30), it is hypothesized that the spermine interacts with DNA in a manner that promotes the production of viral genomes. In addition, the examples disclosed herein show an increase in the genomic titer, i.e., on DNA level, whereas the capsid titer, i.e., on protein level, remains essentially constant (data not shown). Hence, spermine appears not to significantly alter protein production but has effects on a nucleic acid, particularly DNA, level.

[0111] According to preferred embodiments, the fraction of single cells is increased compared to a method wherein the suspension culture does not comprise spermine or a salt, preferably the fraction of single cells is increased by at least 2%, more preferably at least 4%, related to the total number of counted cells and aggregates. According to preferred embodiments, the fraction of cell aggregates is reduced compared to a method wherein the suspension culture does not comprise spermine or a salt, preferably the fraction of cell aggregates is reduced by at least 2%, more preferably at least 4%, related to the total number of counted cells and aggregates. According to preferred embodiments, the fraction of single cells is increased compared to a method wherein the suspension culture does not comprise spermine ora salt, preferably the fraction of single cells is increased by at least 2%, more preferably at least 4%, related to the total number of counted cells and aggregates; and the fraction of cell aggregates is reduced compared to a method wherein the suspension culture does not comprise spermine or a salt, preferably the fraction of cell aggregates is reduced by at least 2%, more preferably at least 4%, related to the total number of counted cells and aggregates. Such increase in single cells and decrease in aggregates has been shown in the example and is associated with overall beneficial effects on the cell culture, particularly by improving oxygen and nutrient access to the cells. Also, such parameters are associated with an increase in the viral vector titer, i.e., improved production of viral vectors, such as AAV. According to preferred embodiments, the fraction of single cells and the fraction of aggregates was measured by imaging a cell suspension and counting single cells and aggregates, preferably by automated cell counting using an image analysis software. The skilled person is also aware of other methods or techniques for cell and aggregate counting, such as automatic cell counters acquiring images of cell cultures which are either automatically analyzed by the device or can be downloaded and analyzed with image analysis software may also be used. For instance, Cedex® cell counters already provide an aggregation rate as a standard parameter for each measurement. Those are less accurate compared to values derived from the image analysis script but are often used as an aggregation readout. Also, the skilled person is aware of manual counting methods.

[0112] "HEK293" cells are readily known in the art and a well-known term for a host cell that was originally developed in the 1970ies and first published by Graham et al. (J. Gen. Virol., 1977, Vol. 36, pp. 59- 72). HEK293 were originally established by transfection of primary human embryonic kidney cells with fragments of mechanically sheared adenovirus 5 DNA inserted in human chromosome 19. It has been shown that HEK293 cells stably express the adenoviral E1A and ElB-55k proteins due to integration of a 4 kbp adenoviral DNAfragment in chromosome 19. Hence, ElA and E1B do not need to be including in the genetic design for modifying such host cell (see also Louis et al., Virol., 1997, Vol. 233, pp.423-429). "HEK293 cell" may herein be interchangeably used herein with the terms "HEK cell", "HEK", "293 cell", "293" or other terminology commonly used in the art for such cells. While "HEK293" are established in the field for viral vector production, since their creation in the 1970ies various derivatives thereof have been generated, which are also well suited for viral vector production. In general, according to preferred embodiments, the derivative of the HEK293 cell is characterized by being cell that roots back to HEK293 and / or was originally a HEK293 cell that was adapted, modified, differentiated, reprogrammed, transformed, transduced, transfected and / or mutated. The skilled person readily can identify a HEK293 cell as known in the art, as it typically contains the adenovirus 5 DNA inserted in human chromosome 19. While it is not excluded that such adenovirus 5 DNA insert has been modified, it is believed that certain similarities will always be existent in order to identify a HEK293 cell or a respective derivative thereof. According to preferred embodiments, the derivative of the HEK293 cell is selected from one or more of the group consisting of 293, 293T, 293T / 17, ANJOU 65, 293H, 293E, 293-6E, EBNA1-6E, 293F, 293FT, 293Flp-IN T- REx, 293FTM, 293S, 293SG, 293SGGD, 293MSR, 293A, or any modified variants thereof. Such derivatives may be advantageous for viral vector production. Some of these derivatives may have been particularly adapted in the past in order to enhance viral vector production.

[0113] According to preferred embodiments, the host cell is adapted for suspension culture. HEK293 cells are originally adherent cells but have been adapted in the past and present for suspension culture. Techniques to adapt the host cell are known in the art. Suspension culture cells are particularly suitable for large-scale culture of the host cells, as these are independent of potentially limiting surface areas. Also, no additional compounds such as microcarriers need to be provided in culture. HEK293 cells adapted for suspension culture are commercially available (e.g., Viral Production Cells2.0 ("VPC 2.0") or FreeStyle™ 293-F-cells from Thermo Fisher Scientific) or can be generated (see below examples for inhouse cell clone and "Clone 2.0").

[0114] Step (a)

[0115] The methods according to the first and second aspect comprise step (a) providing a host cell selected from a HEK293 cell or a derivative thereof, and a cell culture medium for culturing the host cell. Providing step (a) can be performed in any order, i.e., first the cells may be provided and then the cell culture medium or vice versa or simultaneously. The particular order shall not be limiting. The host cell and the cell culture medium may also be provided as separate components or as a single combined component, e.g., the host cells suspended in the cell culture medium.

[0116] The host cell can be provided in any manner known by the person skilled in the art. For instance, the host cell can be provided in form of a cryopreserved cell bank which is redispersed in suitable cell culture medium. Alternatively, the host cell can undergo a scale up or seed train process, wherein the host cell is cultured in cell culture medium to a desired cell number followed by transfer into a larger cell culture vessel or dish or bioreactor and the dilution with further cell culture medium. The host cell can also be provided as a mixture comprising the host cells and a cell culture medium suitable for culturing such host cell. In a particular embodiment, the host cell, which is a HEK293 cell or a derivative thereof, is provided in form of a suspension with cell culture medium suitable for culturing the HEK293 cell or derivative thereof.

[0117] A "cell culture medium for culturing the host cell" refers to a composition that is suitable for culturing the host cell, i.e., suitable for culturing the HEK293 cell or derivative thereof. Such cell culture media are well-known to the skilled person and shall not limit the scope of the present disclosure. Typically, such cell culture medium is in liquid form. However, the cell culture medium may also be provided in solid form, e.g., in form of a powder, and then dissolved in a liquid prior to contacting the host cell with the cell culture medium in step (b). Specific examples include the cell culture media used in the examples below. Specific examples can include in house designed cell culture medium (see examples below, e.g., "P6"), as well as commercially available cell culture media, see e.g., HEK ViP NB and HEK TF (Sartorius Xell GmbH), and DMEM (Pan-Biotech).

[0118] According to preferred embodiments, the cell culture medium of step (a) comprises spermine or a salt thereof. According to preferred embodiments, the cell culture medium of step (a) comprises spermine or a salt thereof and / or the method further comprises providing a supplement composition comprising spermine or a salt thereof. Providing the spermine or salt thereof in form of the cell culture medium has the advantage that no further compositions need to be added, such that the process is simplified. It may also be preferred to include the spermine or salt thereof in the cell culture medium in order to exactly control the content of the spermine in the medium and thus also in the later suspension culture. The examples below show that spermine as a full weighed in compound of the cell culture medium advantageously leads to higher viral vector titers and reduced host cell aggregation (see e.g., Figs. 4, 7). Providing the spermine or salt thereof in form of a supplement composition has the advantage that the spermine can be added to any kind of cell culture medium to be used, i.e. allows for being flexibly applied and in desired concentrations. As demonstrated in the examples below, providing a supplement composition comprising the spermine or salt thereof can advantageously be applied in scope of the methods disclosed herein, leading to higher viral vector titers, as well as reduced aggregation of host cells (see e.g., Figs. 1-3, 5, 6)

[0119] Step (b)

[0120] The methods according to the first and second aspect comprise step (b) contacting the host cell with the culture medium to form a suspension culture. Contacting may comprise adding the host cells to the cell culture medium or vice versa or joining both simultaneously. According to one embodiment, the host cell, e.g., in form of a concentrated suspension, is added to the cell culture medium, which is for instance advantageous in case the cell culture medium is present in the culture vessel, e.g., bioreactor. According to another embodiment, the cell culture medium, preferably in liquid form, is added to the host cells, which can be provided in form of a suspension or cell pellet in a container. The particular mode of contacting according to step (b) shall not be limiting in scope of the present disclosure.

[0121] According to preferred embodiment, the suspension culture of step (b) comprises spermine or a salt thereof. Such spermine or salt thereof may be present as part of the cell culture medium, i.e. may be provided by providing the cell culture medium. Such spermine may alternatively or in addition, be provided in contacting step such, such that the host cell, the cell culture medium, or the mixture of both is contacted with the spermine or salt thereof. This may preferably be achieved with a supplement composition as disclosed here, e.g., in form of a composition comprising spermine or a salt thereof, which is added to the suspension culture.

[0122] Step (c)

[0123] The methods according to the first and second aspect comprise step (c) culturing the suspension culture. Culturing may be understood as cultivating the host cell under conditions suitable for proliferation of the host cell. Such culture can take place in any format or scale, e.g., in a bioreactor, shake flask, well plate, etc. As is demonstrated in the examples, the method according to the present disclosure is independent of scale and thus applicable for any type of cultivation method. Culturing according to step (c) may comprise performing any mode suitable. For instance, culturing may comprise performing a batch, fed batch, or perfusion culture, or any combination thereof.

[0124] According to a preferred embodiment, the suspension culture of step (c) comprises spermine or a salt thereof. Such spermine or salt thereof may be present as part of the cell culture medium, i.e. may be provided by providing the cell culture medium, such that it is present throughout steps (a) (in the culture medium), step (b) (in the suspension culture) and step (c). Such spermine may alternatively or in addition, be provided in contacting step (c) such, such that the suspension culture of step (b) is contacted with the spermine or salt thereof. This may preferably be achieved with a supplement composition as disclosed here, e.g., in form of a composition comprising spermine or a salt thereof, which is added to the suspension culture. This may also be achieved by adding a cell culture medium comprising spermine or a salt thereof to the suspension culture. Adding the spermine to the suspension culture can be conducted prior to step (c) of within step (c). Step (d)

[0125] The methods according to the first and second aspect comprise step (d) producing a viral vector by the host cell in the suspension culture. Producing the viral vector may preferably be performed simultaneous to culturing the suspension culture. Hence, steps (c) and (d) can be performed simultaneously. However, it may also be desirable to first culture the cells to increase the cell number before starting to produce the viral vector. For instance, in some embodiments, the cells are first cultured in one or more steps to increase the cell number and scale up the cells, followed by production step (d). Nevertheless, culturing step (c) may continue throughout production step (d).

[0126] To start the production according to step (d) various methods and techniques are known in the art and shall not be limiting in scope of the present disclosure. As disclosed herein, production may be started by a step of transient transfection, wherein the host cells are modified for producing the viral vector, e.g., AAV. As also disclosed herein, the host cell may be modified in order to stably produce the viral vector. Hence, the viral vector may already be produced in any of steps (a), (b), (c), and / or (d). Nevertheless, it may be desired to include an induction system, such that the viral vector production of the host cell can be induced in order to initiate the viral vector production. Such systems are readily known in the art.

[0127] According to preferred embodiments, when producing in step (d), culturing of the cells is continued, which may comprise performing any culturing mode suitable. For instance, culturing during production may comprise performing a batch, fed batch, or perfusion culture, or any combination thereof. For instance, it may be desirable to produce the viral vector in batch or fed -batch mode when using a host cell that is configured to produce a viral vector by transient transfection. In another example, it may be desirable to produce the viral vector in fed batch or perfusion culture when using a host cell that is configured to produce a viral vector by genome editing (referred to as stable cell line). Also, combinations of culture modes are within the scope of the present disclosure, e.g., by first culturing in batch mode, followed by perfusion orfed-batch culture.

[0128] According to a preferred embodiment, step (d) comprises producing the viral vector for at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, preferably at least 48 hours or at least 72 hours. Producing the viral vector for such time advantageously leads to increased viral vector production. Indeed, production durations of at least 48 hours lead to high numbers of viral vectors. In the examples below a production duration of about 72 hours was performed showing superior viral vector yields and quality, however, the present disclosure shall not be limited to such specific duration. According to preferred embodiments, the suspension culture of step (d) comprises spermine or a salt thereof. According to preferred embodiments, the host cell in step (d) is present in a suspension culture comprising spermine ora salt thereof. Such embodiments are advantageous as demonstrated in the examples because presence of spermine or a salt thereof advantageously increases the viral vector titer, e.g., of AAV. Also, spermine reduced host cell aggregation.

[0129] In preferred embodiments, the suspension culture comprises spermine or a salt thereof in steps (b), (c), and / or (d), preferably at least in steps (c) and / or (d), more preferably at least in steps (c) and (d). In some embodiments, the suspension culture of step (b), (c), and (d) comprises spermine or a salt thereof.

[0130] According to preferred embodiments, the method comprises transferring the suspension culture one or more times after step (c) and before step (d). Such transfer may be performed in order change the culture container, e.g., from shake flask to microbioreactor, or from shake flask to bioreactor, or from small scale bioreactor to large scale bioreactor. Typically, such transfer method further comprises transferring, diluting, and culturing the suspension culture one or more times after step (c) and before step (d). Such embodiments, refer to a "seed train process", wherein the host cell undergoes one or more culturing steps in order to increase the cell number for seeding higher cell numbers into larger and larger culturing containers. For instance, one may use a cryo-preserved host cell vial and culture these combined with a cell culture medium in a shake flask. After a certain culture duration, the cells have proliferated and thus increased in number. Then, the cells may be transferred to another, typically larger container, e.g., a bioreactor or larger shake flask, with a higher volume, such that the cell suspension is diluted with fresh cell culture medium. In general, such scale up processes are well- known in the art. According to preferred embodiments, the spermine or salt thereof, is present throughout such culturing and transfer steps. For instance, it can be desirable to culture the suspension culture in presence of spermine or a salt thereof and then perform a transfer step, typically including a dilution in fresh medium. Such fresh medium may already comprise the spermine or salt thereof and / or spermine may be added as a supplement composition as disclosed herein. As a result, the host cell is continuously provided and contacted with spermine or a salt thereof, such that the spermine can continuously provide its beneficial effect, particularly in that the host cell aggregation is reduced and later viral vector production can be improved. According to preferred embodiments, the suspension culture of the production step (d) comprises spermine or a salt thereof, as well as one or more upfront cultivation steps, optionally including a transferand dilution step, may also comprise spermine or a salt thereof.

[0131] According to preferred embodiments, the method further comprises one or more of the following: (i) prior to step (b) the cell culture medium is contacted with the supplement composition;

[0132] (ii) step (b) further comprises contacting the host cell with the culture medium and the supplement composition to form a suspension culture;

[0133] (iii) prior to step (c) or (d) the suspension culture is contacted with the supplement composition; and / or

[0134] (iv) in step (c) and / or step (d) the suspension culture is contacted with the supplement composition.

[0135] The supplement composition of such embodiment refers toa a supplement composition comprising the spermine or salt thereof. As demonstrated in the examples, contacting the suspension culture with the supplement composition increased the viral vector titer and reduced host cell aggregation. In some embodiments, such supplement composition does not comprise putrescine or a salt thereof.

[0136] According to preferred embodiments, the spermine or salt thereof in the supplement composition is present in a concentration of 100 mM or less. According to further embodiments, the supplement composition comprises spermine at a concentration of less than 100 mM, less than 90 mM, or less than 80 mM, preferably less than 70 mM or less than 60 mM, such as about 50 mM. In general, a high concentration in the supplement leads to less dilution of the suspension culture and a more exact control of the final spermine concentration. On the other hand, having a too high concentration may lead to a very high concentration at certain positions within the suspension culture when adding the supplement. Hence, a suitable but non-limiting example of a supplement composition comprises spermine or a salt thereof in a concentration of about 50 mM.

[0137] Spermine or salt thereof

[0138] The methods according to the first and second aspect of the present invention define that the suspension culture of step (b), step (c), and / or step (d), preferably at least the suspension culture of step (d), comprises spermine or a salt thereof. As disclosed above, there are various ways of including the spermine or salt thereof in any of the suspension cultures. For instance, the spermine or salt thereof may be present in the culture medium of step (a), such that the suspension cultures of steps (b), (c) and (d) can comprise the spermine or salt thereof. Furthermore, or in addition thereto, the method may further comprise providing a supplement composition comprising the spermine or salt thereof, which can be added to the suspension culture of steps (b), (c), and / or (d) and / or to the culture medium in step (a). The beneficial effects of having spermine in the suspension culture will be valid for all these embodiments, particularly by reducing the host cell aggregation. Nevertheless, it may be particularly advantageous for at least the suspension culture of step (d) to comprise the spermine or salt thereof for improving the viral vector production. Also, from practical point of view, it is advantageous to include the spermine and salt thereof throughout culture in order to avoid a change of cell culture media composition throughout steps (c) and (d), such that according to preferred embodiments, the spermine or salt thereof is present in the suspension cultures of steps (c) and (d), optionally also step (b).

[0139] As indicated above, it is hypothesized that the spermine interacts with DNA and other charged molecules in a manner that such interaction is less possible with the host cells, thereby "shielding" the host cells from interacting. In addition, the examples disclosed herein show an increase in the genomic titer, i.e., on DNA level, whereas the capsid titer, i.e., on protein level, remains essentially constant (data not shown). Hence, spermine appears not to significantly alter protein production but has effects on a nucleic acid, particularly DNA, level.

[0140] "Spermine" is a well-known polyamine, which refers to organic compounds containing two or more amino groups. Examples of particular preferred polyamines herein are spermine, spermidine, norspermine, norspermidine, homospermine, homospermidine, cadaverine, putrescine, agmatine and ornithine. Polyamines or salts thereof include hydrated or dehydrated forms, various salt forms or combinations of one or more polyamines. "Spermine" as disclosed herein refers to a compound comprising spermine or a derivative thereof, including thermospermine, norspermine, or homospermine. Such derivative of spermine, however, does not encompass spermidine or putrescine. Such compound may be provided in an ionic form such that one or more counterions are also provided, which would constitute a salt of spermine. "Spermine or a salt thereof" preferably is selected from one or more of spermine, spermine tetrahydrochloride, spermine dihydrate, thermospermine, and thermospermine tetrahydrochloride, as well as norspermine or homospermine.

[0141] According to preferred embodiments, the suspension culture comprises the spermine or salt thereof in a concentration of at least 1 pM, preferably at least 5 pM, at least 10 pM, at least 15 pM, or at least 25 pM, more preferably at least 30 pM. According to preferred embodiments, the suspension culture comprises the spermine or salt thereof in a concentration of less than 1.000 pm, preferably less than 800 pM, less than 700 pM, less than 600 pM, more preferably less than 500 pM or less than 400 pM. According to preferred embodiments, the suspension culture comprises spermine or saltthereof in a concentration of the range selected from 1 pM to 1.000 pM, preferably 5 pM to 800 pM, 10 pM to 700 pM, 15 pM to 600 pM, 25 pM to 500 pM, more preferably 30 pM to 400 pM or 40 pM to 250 pM. As disclosed in the examples, different concentrations of spermine or salt thereof can be applied in orderto achieve the beneficial effects disclosed herein (see e.g., Figs. 1-17). While for a particular cell model, very high concentrations were found to have a slightly negative effect (see Fig. Fig. 1, 500 pM spermine), it is not excluded that higher concentrations are beneficial for other cell models. Nevertheless, according to preferred embodiments, the suspension culture comprises the spermine or salt thereof in a concentration of less than 500 pM or less than 400 pM.

[0142] Polyamine(s)

[0143] According to preferred embodiments, the suspension culture further comprises a polyamine, preferably a spermine precursor, such as spermidine or a salt thereof or cadaverine or a salt thereof, more preferably spermidine or a salt thereof. A "polyamine" in scope of the present disclosure is preferably a spermine precursor. As is known in the art, cellular spermine synthesis is enzymatically controlled and tightly regulated. Compounds such as putrescine and spermidine serve as precursors for spermine synthesis. Consequently, supplementing a culture medium with a polyamine which is a spermine precursor, particularly spermidine ora salt thereof, can increase cellular spermine levelsand thus improve viral vector production. As is shown in the examples, addition of a further polyamine which is a spermine precursor, particularly spermidine or a salt thereof, increases the genomic and transducing titer of the viral vector, here AAV (see e.g., Figs. 14-17).

[0144] According to preferred embodiments, the suspension culture further comprises spermidine or a salt thereof. "Spermidine" is a well-known polyamine. "Spermidine" as disclosed herein refers to a compound comprising spermidine or a derivative thereof, including norspermidine, or homospermidine. Such derivative of spermidine, however, does not encompass spermine or putrescine. Such compound may be provided in an ionic form such that one or more counterions are also provided, which would constitute a salt of spermidine. "Spermidine or a salt thereof" preferably is selected from one or more of spermidine, norspermidine, homospermidine, spermidine trihydrochloride, spermidine phosphate.

[0145] According to preferred embodiments, the cell culture medium of step (a) comprises the polyamine, preferably a spermine precursor, such as spermidine or a salt thereof. In such embodiments, it may be particularly advantageous that the cell culture medium comprises spermine or a salt thereof and the polyamine or salt thereof, preferably a spermine precursor, such as spermidine or a salt thereof. This simplifies the process, as no addition compounds need to be added throughout the process and both compounds are already present in the suspension culture of step (b) and, preferably, continue to be present in steps (c) and (d).

[0146] According to preferred embodiments, the method further comprises providing a supplement composition comprising the polyamine, preferably a spermine precursor, such as spermidine or a salt thereof. It is preferred that such supplement composition further comprises the spermine or salt thereof, such that both compounds are already present in the desired concentration and not further supplement composition is needed. Nevertheless, in some embodiments, different supplement compositions may be provided, one comprises the spermine or salt thereof, the other comprising the polyamine or salt thereof, preferably a spermine precursor, such as spermidine or a salt thereof. Then each compound can be adjusted freely for optimizing the culturing conditions. The polyamine, preferably a spermine precursor, such as spermidine or a salt thereof, in the supplement composition is preferably present in a concentration of 200 mM or less. In some embodiments, the polyamine is present in a concentration of less than 200 mM, less than 150 mM, less than 100 mM, preferably less than 80 mM, less than 60 mM, or less than 50 mM. In some embodiments, such supplement composition does not comprise putrescine or a salt thereof.

[0147] According to preferred embodiments, the suspension culture comprises the polyamine, preferably a spermine precursor, such as spermidine or a salt thereof, in steps ( b), (c), and / or (d ), preferably at least in steps (c) and / or (d), more preferably at least in steps (c) and (d). As discussed above for spermine or a salt thereof, there are various ways of including the polyamine in any of the suspension cultures. For instance, the polyamine may be present in the culture medium of step (a), such that the suspension cultures of steps (b), (c) and (d) can comprise the polyamine. Furthermore, or in addition thereto, the method may further comprise providing a supplement composition comprising the polyamine, which can be added to the suspension culture of steps (b), (c), and / or (d) and / or to the culture medium in step (a). The beneficial effects of having the polyamine, preferably a spermine precursor, such as spermidine or a salt thereof, in the suspension culture will be valid for all these embodiments, particularly by reducing the host cell aggregation. Nevertheless, it may be particularly advantageous for at least the suspension culture of step (d) to comprise the polyamine, particularly the spermidine or salt thereof, for further improving the viral vector production. Also, from practical point of view, it is advantageous to include the polyamine throughout culture in order to avoid a change of cell culture media composition throughout steps (c) and (d), such that according to preferred embodiments, the polyamine, preferably spermidine or salt thereof, is present in the suspension cultures of steps (c) and (d), optionally also step (b).

[0148] According to preferred embodiments, the suspension culture comprises the polyamine, preferably a spermine precursor, such as spermidine or a salt thereof, in a concentration of at least 1 pM, preferably at least 5 pM, at least 10 pM, at least 15 pM, or at least 20 pM, more preferably at least 25 pM. Such concentrations have been found advantageous in the examples below (see e.g., Figs. 14- 17). In some embodiments, the concentration of the polyamine, preferably a spermine precursor, such as spermidine or a salt thereof, may be less than 1 mM, preferably less than 500 pM. In some embodiments, the concentration of the polyamine, preferably a spermine precursor, such as spermidine or a salt thereof, may be selected from the range of 1 pM to 1 mM, preferably 5 pM to 500 pM or 10 pM to 250 pM.

[0149] According to one embodiment, the suspension culture further comprises a further polyamine, preferably the further polyamine is a spermine precursor, such as putrescine or a salt thereof. Such further polyamine would be provided in addition to the spermine or salt thereof, preferably also in addition to the aforementioned polyamine, preferably spermidine or salt thereof. For instance, the suspension culture may comprise spermine or a salt thereof, spermidine or a salt thereof and a further polyamine, preferably putrescine or a salt thereof. While putrescine has been shown to have less or a negative effect in the examples when provided at a concentration of 1 mM (see Figs. 14, 15), it is believed that lower concentration of putrescine may be advantageous. Therefore, the suspension culture may comprise the further polyamine, preferably a spermine precursor, such as putrescine or salt thereof, in a concentration of less than 1 mM, less than 750 pM, or less than 500 pM. In some embodiments, the suspension culture comprises the further polyamine, preferably putrescine or salt thereof, in a concentration of at least 10 pM, at least 50 pM, at least 100 pM, at least 250 pM, or at least 500 pM. The suspension culture may thus further comprise putrescine or a salt thereof. "Putrescine" is a well-known polyamine. "Putrescine" as disclosed herein refers to a compound comprising putrescine or a derivative thereof. Such derivative of putrescine, however, does not encompass spermine or spermidine. Such compound may be provided in an ionic form such that one or more counterions are also provided, which would constitute a salt of putrescine. "Putrescine or a salt thereof" preferably is selected from one or more of putrescine or putrescine dihydrochloride.

[0150] According to some embodiments, the method has one or more of the following characteristics:

[0151] (i) the cell culture medium of step (a) comprises the further polyamine, preferably putrescine or salt thereof;

[0152] (ii) the method further comprises providing a supplement composition comprising the further polyamine, preferably putrescine or salt thereof, preferably wherein the supplement composition further comprises the spermine or salt thereof;

[0153] (iii) the polyamine, preferably putrescine or salt thereof, in the supplement composition is present in a concentration of 620 mM or less; and / or

[0154] (iv) the suspension culture comprises the further polyamine, preferably putrescine or salt thereof, in steps (b), (c), and / or (d), preferably at least in steps (c) and / or (d), more preferably at least in steps (c) and (d). Downregulating compound

[0155] As discussed above for the polyamine(s), which are preferably spermine precursor(s) it can be advantageous for increasing the spermine in the suspension culture and thus increase viral vector production. In addition to using spermine precursor(s), spermine can be converted back to spermidine by an enzyme termed spermidine / spermine-N 1-acetyltransferase (SSAT) (Pegg, 2008, American Journal of Physiology. Endocrinology and Metabolism, E. 294(6), pp. E995-1010.). Hence, downregulation of SSAT, e.g., by supplementation of phenylbutyrate, can increase spermine levels and thereby viral vector titer. Thus, according to preferred embodiments, the suspension culture further comprises a downregulating compound capable of downregulating conversion of spermine to spermidine and / or spermidine to putrescine. Such addition of a downregulating compound leads to increased production of viral vector, which is also demonstrated in the examples below showing increased genomic and transducing AAV titers (see Figs. 14-17).

[0156] According to preferred embodiments, the downregulating compound is an inhibitor of an enzyme involved in the spermine synthesis pathway, preferably the enzyme being spermidine / spermine N 1- acetyltransferase 1 (SAT1) or polyamine oxidase (PAO), more preferably an inhibitor of SAT1. Such downregulating compound is preferably phenylbutyrate or a salt thereof (such as sodium phenylbutyrate), or diminazene aceturate, preferably phenylbutyrate or a salt thereof, more preferably sodium phenylbutyrate. As is demonstrated in the examples, an enzyme inhibitor, such as phenylbutyrate significantly increases the viral vector yield.

[0157] According to other embodiments, the downregulating compound is a compound interfering with the expression of an enzyme involved in the spermine synthesis pathway, preferably the enzyme being spermidine / spermine N 1-acetyltransferase 1 (SAT1) or polyamine oxidase (PAO), more preferably an inhibitor of SAT1. Such downregulating compound is preferably an expression interfering nucleic acid, preferably a small interfering RNA (siRNA) or a microRNA (miRNA). As is readily understood by the skilled person, a compound interfering with the expression of an enzyme involved in the spermine synthesis pathway is a suitable alternative or addition to the inhibitor of an enzyme involved in the spermine synthesis pathway disclosed herein.

[0158] According to preferred embodiments, the method has one or more of the following characteristics:

[0159] (i) the cell culture medium of step (a) comprises the downregulating compound;

[0160] (ii) the method further comprises providing a supplement composition comprising the downregulating compound, preferably wherein the supplement composition further comprises the spermine or salt thereof; (iii) the downregulating compound, preferably an inhibitor of an enzyme involved in the spermine synthesis pathway, such as phenylbutyrate or a salt thereof, in the supplement composition is present in a concentration of 200 mM or less;

[0161] (iv) the suspension culture comprises spermine or a salt thereof in steps (b), (c), and / or (d), preferably at least in steps (c) and / or (d), more preferably at least in steps (c) and (d); and / or

[0162] (v) the downregulating compound is present in the suspension culture at least in step (d).

[0163] In some embodiments, such supplement composition does not comprise putrescine or a salt thereof.

[0164] According to preferred embodiments, the suspension culture comprises the downregulating compound, preferably an inhibitor of an enzyme involved in the spermine synthesis pathway, such as phenylbutyrate or a salt thereof, in a concentration of at least 0.1 pM, preferably at least 0.5 pM, at least 1 pM, at least 2.5 pM, or at least 5 pM, more preferably at least 10 pM. Such concentrations have been found advantageous in the examples (see e.g., Figs. 14-17).

[0165] According to preferred embodiments, the suspension culture further comprises a polyamine, preferably a spermine precursor, and a downregulating compound capable of downregulating conversion of spermine to spermidine and / or spermidine to putrescine, preferably an inhibitor of spermidine / spermine N l-acetyltransferase 1 (SAT1). As shown in the examples such combination was found highly advantageous and capable of synergistically increasing the viral vector production (see e.g. Figs. 14-17). Preferably, the polyamine is spermidine or a salt thereof in such an embodiment. In addition, it is preferred that the suspension culture does not comprise putrescine or a salt thereof originating from the cell culture medium or a supplement.

[0166] Further embodiments

[0167] According to preferred embodiments, the genomic viral vector titer and / or transducing viral vector titer is increased compared to a method wherein the suspension culture does not comprise spermine or a salt. According to particular embodiments, the genomic viral vector titer is increased by at least 10%, preferably at least 25%, compared to a method wherein the suspension culture does not comprise spermine or a salt. According to particular embodiments, the transducing viral vector titer is increased by at least 10%, preferably at least 25%, compared to a method wherein the suspension culture does not comprise spermine or a salt. Such increase in genomic and / or transducing titer has been shown in the examples below and is advantageous for improved production of viral vectors such as AAV.

[0168] According to preferred embodiments, the method further comprises step (e) harvesting the viral vector. Harvesting is typically understood as obtaining the viral vector produced by the host cell after or throughout culture. Hence, harvesting can take place at the end of the production, e.g., by stopping the culture and obtaining the viral vector. Alternatively, harvesting can be performed throughout culture by intermittently or continuously obtaining the viral vector, e.g., by obtaining the cell suspension or a permeate which could be obtained in perfusion culture. Harvesting of the viral vector can encompass obtaining the viral vector from the host cell (intracellular viral vector) and / or the surrounding cell culture medium, also referred to as supernatant (extracellular viral vector). Some viruses, e.g., AAV8, secrete directly into the medium, such that the viral vector can be directly obtained from the cell culture medium or supernatant (see e.g., WO 2007 / 127264 A2). Other viral vectors are mostly present within the host cell, such that the cells need to be disrupted or lysed in order to obtain the viral vector. It may be advantageous to use both the supernatant and the cells in order to harvest the viral vector to obtain a high yield of viral vector. According to one embodiment, step (e) comprises harvesting the viral vector by chemically lysing the host cell after step (d). Forthis purpose, the host cell may be separated from the cell culture medium or provided together with the cell culture medium. For chemical lysis, the host cell is contacted with a chemical lysis reagent or composition. In particular, a detergent-based chemical lysis reagent or composition may be used for lysing the host cell. Such detergent-based lysis composition is advantageously used in the examples, demonstrating efficient lysis, and thus harvesting of the viral vector. However, other methods of disrupting the host cell may be used, e.g., sonication and high-pressure homogenization, physical methods like freeze-thaw, and chemical methods, other mechanical lysis methods. The skilled person is well-aware of different methods of disrupting a cell in order to harvest the viral vector.

[0169] According to preferred embodiments, the method further comprises step (f) purifying the harvested viral vector. After harvesting, it may be required to purify the viral vector for subsequent steps, e.g., analytics, formulation, etc. Purifying the harvested viral vector may be performed by any method applicable and known by the skilled person. Indeed, various protocols and methods are available in the art for purifying a viral vector, typically including multiple-step processes (also referred to as downstream processing). For example, the harvested viral vector may undergo filtration, centrifugation, chromatography, dialysis, etc. Chromatographic purification may encompass anionic, cationic and / or affinity chromatography.

[0170] According to preferred embodiments, the method further comprises step (g) formulating the viral vector for gene therapy. Such step may advantageously be performed after the purification of the viral vector. Formulating the viral vector is particularly useful when applying the viral vector for gene therapy but also for storing the viral vector. According to a particular embodiment, the method according to the second or first aspect has following combination of characteristics:

[0171] (i) the viral vector is an adeno-associated virus (AAV);

[0172] (ii) the cell culture medium of step (a) comprises spermine or a salt thereof, preferably wherein the suspension culture of steps (b), (c), and (d) comprises spermine or a salt thereof; and

[0173] (iii) the suspension culture comprises spermine or salt thereof in a concentration of the range selected from 1 pM to 1.000 pM, preferably 5 pM to 800 pM, 10 pM to 700 pM, 15 pM to 600 pM, 25 pM to 500 pM, more preferably 30 pM to 400 pM or 40 pM to 250 pM.

[0174] Additionally, the particular embodiment may comprise transferring the suspension culture one or more times after step (c) and before step (d), optionally comprising transferring, diluting, and culturing the suspension culture one or more times after step (c) and before step (d). Within the particular embodiment, the cell culture medium of step (a) may further comprise a polyamine, preferably a spermine precursor, such as spermidine or a salt thereof. Within the particular embodiment, the cell culture medium of step (a) may further comprise a downregulating compound capable of downregulating conversion of spermine to spermidine and / or spermidine to putrescine, preferably an inhibitor of an enzyme involved in the spermine synthesis pathway or a compound interfering with the expression of an enzyme involved in the spermine synthesis pathway, more preferably an inhibitor of spermidine / spermine N l-acetyltransferase 1 (SAT1) or polyamine oxidase (PAO), most preferably an inhibitor of SAT1, such as phenylbutyrate or a salt thereof. Also, a combination of the polyamine and downregulation compound can be included. In such particular embodiment, preferably no supplement composition is provided that comprises putrescine or a salt thereof.

[0175] According to a particular embodiment, the method according to the second or first aspect has following combination of characteristics:

[0176] (i) the viral vector is an adeno-associated virus (AAV);

[0177] (ii) the method further comprises providing a supplement composition comprising spermine or a salt thereof, preferably wherein the spermine or salt thereof in the supplement composition is present in a concentration of 100 mM or less;

[0178] (iii) the suspension culture of step (d) comprises spermine or a salt thereof, optionally wherein also the suspension culture of steps (b) and (d) comprises spermine or a salt thereof; and

[0179] (iv) the suspension culture at least of step (d) comprises spermine orsalt thereof in a concentration of the range selected from 1 pM to 1.000 pM, preferably 5 pM to 800 pM, 10 pM to 700 pM, 15 pM to 600 pM, 25 pM to 500 pM, more preferably 30 pM to 400 pM or 40 pM to 250 pM.

[0180] Within the particular embodiment, the cell culture medium of step (a) may further comprise a polyamine, preferably a spermine precursor, such as spermidine or a salt thereof and / or the method may further comprise providing a supplement composition comprising the polyamine, preferably a spermine precursor, such as spermidine or a salt thereof. Within the particular embodiment, the cell culture medium of step (a) may further comprise a downregulating compound capable of downregulating conversion of spermine to spermidine and / or spermidine to putrescine, preferably an inhibitor of an enzyme involved in the spermine synthesis pathway or a compound interfering with the expression of an enzyme involved in the spermine synthesis pathway, more preferably an inhibitor of spermidine / spermine N 1-acetyltransferase 1 (SAT1) or polyamine oxidase (PAO), most preferably an inhibitor of SAT1, such as phenylbutyrate or a salt thereof; and / or the method may further comprise providing a supplement composition comprising the downregulating compound capable of downregulating conversion of spermine to spermidine and / or spermidine to putrescine, preferably an inhibitor of an enzyme involved in the spermine synthesis pathway or a compound interfering with the expression of an enzyme involved in the spermine synthesis pathway, more preferably an inhibitor of spermidine / spermine N 1-acetyltransferase 1 (SAT1) or polyamine oxidase (PAO), most preferably an inhibitor of SAT 1, such as phenylbutyrate or a salt thereof. It may be particularly preferred to have one supplement composition comprising the spermine or salt thereof, the polyamine or salt thereof, preferably spermidine or salt thereof, and the downregulating compound capable of downregulating conversion of spermine to spermidine and / or spermidine to putrescine, preferably an inhibitor of an enzyme involved in the spermine synthesis pathway or a compound interfering with the expression of an enzyme involved in the spermine synthesis pathway, more preferably an inhibitor of spermidine / spermine N 1-acetyltransferase 1 (SAT1) or polyamine oxidase (PAO), most preferably an inhibitor of SAT1, such as phenylbutyrate or a salt thereof. However, these compounds can also be provided as two or three separate supplement compositions. In such particular embodiment, preferably no supplement composition is provided that comprises putrescine or a salt thereof.

[0181] According to a particular embodiment, the method according to the second or first aspect has following combination of characteristics:

[0182] (i) the viral vector is an adeno-associated virus (AAV);

[0183] (ii) the host cell is modified to be configured to produce a viral vector by transiently transfecting the host cell with one or more plasmids for viral vector production allowing for transient viral vector production, preferably wherein transiently transfecting comprises (x) combining the host cell with the one or more plasmids for viral vector production and a transfection reagent; and (y) incubating the host cell, the one or more plasmids for viral vector production and the transfection reagent to generate a transiently transfected cell; and

[0184] (iii) the spermine or salt thereof is present during transient transfection and / or is added after transient transfection, preferably after transient transfection. Preferably, the particular embodiment further comprises having M34 or a derivative thereof present during transient transfection and / or M344 or a derivative thereof is added after transient transfection, preferably M344 or a derivative thereof is after transient transfection further; and / or further M344 or a derivative thereof is present in incubation step (y) and / or is added after incubation step (y).

[0185] Method according to the third aspect of the invention

[0186] According to a third aspect, a method for dissolving host cell aggregates is provided, the method comprising:

[0187] (a) providing a suspension culture comprising host cells selected from a HEK293 cell or a derivative thereof, wherein at least a fraction of the host cells is provided as cell aggregates, and a polyamine or a salt thereof;

[0188] (b) contacting the suspension culture with the polyamine orthe salt thereof; and

[0189] (c) optionally, culturing the suspension culture, wherein the polyamine or the salt thereof dissolves host cell aggregates.

[0190] The method according to the third aspect advantageously dissolves cell aggregates, which can be detrimental in culture. As disclosed above, cell aggregates can lead to severe restrictions in mass transport limiting oxygen and nutrient supply to cells within such aggregates. This can result in cell death, toxin formation, hindrance of reliable cell counting and thus process control, as well as lower culture performance, especially considering production of viral vectors. As demonstrated in the examples, by contacting a suspension culture of HEK293 cells with a polyamine, such as spermine or a salt thereof, cell aggregates can be effectively dissolved, leading to higher fractions of single cells (see Figs. 9, 10). At the same time, the polyamine, such as spermine or a salt thereof, does not negatively affect viral vector production, e.g. transient viral vector production, known for conventional anti-clumping compounds, such as dextran sulfate, which are less suitable for (transient) viral vector production. Indeed, consistently increased viral vector production is achieved

[0191] (see e.g., Figs. 1-4, 11-17)

[0192] The individual steps and preferred embodiments of the method according to the third aspect mostly correspond to the individual steps and embodiments of the methods according to the first and second aspect. Therefore, it is referred to the above disclosure which shall equally be applicable for the method according to the second aspect. This particularly but not exclusively includes the host cell and aggregates thereof, the suspension culture comprising host cells selected from a HEK293 cell or a derivative thereof, the culture medium for culturing the host cell, the polyamine or salt thereof, particularly spermine or salt thereof, as well as culturing step (c). Furtherfeatures will now be described in detail.

[0193] According to preferred embodiments, the polyamine or the salt thereof is provided as a composition or suspension, preferably as a cell culture medium comprising the polyamine or the salt thereof and / or as a supplement composition comprising the polyamine or the salt thereof. A cell culture medium comprising the polyamine has the advantage that it is highly cell compatible. A supplement composition can be flexibly applied to any cell culture medium for culturing the host cell and simply added at any point throughout culture, or potential later production steps. According to one embodiment, the cell culture medium and / or the supplement composition is essentially free of dextran sulfate, particularly when applying the method according to the third aspect for producing a viral vector.

[0194] According to preferred embodiments, the method is for dissolving host cell aggregates for production of a viral vector, preferably, wherein the method further comprises step (d) producing a viral vector by the host cell in the suspension culture. Such embodiments make use of the benefits disclosed above for the method according to the first and second aspect of the invention, particularly by achieving consistently increased viral vector production (see e.g., Figs. 1-4, 11-17). The host cell aggregates may be dissolved by the method during the culturing step (c) and / or production step (d). Such production of a viral vector has been in detail described above for the methods according to the first and second aspect of the invention, which shall also be applicable for such embodiments, wherein the method is for dissolving host cell aggregates for production of a viral vector.

[0195] In other embodiments, the method is for dissolving host cell aggregates for production of a recombinant protein or recombinant RNA molecule of interest. In such embodiments, the method preferably further comprises step (d) producing a recombinant protein or recombinant RNA molecule by the host cell in the suspension culture. Preferably, the host cell accordingly comprises at least one recombinant gene encoding at least one recombinant protein of interest and / or encoding at least one recombinant RNA molecule of interest. The at least one recombinant protein of interest may be selected from the group consisting of therapeutic proteins, monoclonal antibodies, bispecific antibodies, fusion proteins, peptibodies, and peptides. The at least one recombinant RNA molecule of interest may be selected from the group consisting of mRNA therapeutics including mRNA vaccines, non-coding single-stranded RNA species, such as e.g. antisense RNAs and MicroRNAs (also called miRNAs), interfering RNAs (RNAi), such as e.g. small interfering RNA (siRNA) or micro RNA (miRNA), and RNA aptamers. According to preferred embodiments, the polyamine is one or more of the group selected from spermine, spermidine, cadaverine, and putrescine, preferably spermine, or spermidine, more the polyamine comprises spermine or a salt thereof. Other suitable polyamines may be norspermine, norspermidine, homospermine, homospermidine, agmatine and ornithine. Polyamines, such as spermine or a salt thereof, have been found advantageous in order to dissolve host cell aggregates (see e.g., Figs. 9, 10).

[0196] According to preferred embodiments, the polyamine is spermine or a salt thereof in a supplement composition present in a concentration of 100 mM or less. According to further embodiments, the supplement composition comprises spermine at a concentration of less than 100 mM, less than 90 mM, or less than 80 mM, preferably less than 70 mM or less than 60 mM, such as about 50 mM. In general, such high concentrations in the supplement lead to less dilution of the suspension culture and a more exact control of the final polyamine concentration.

[0197] According to preferred embodiments, after contacting the suspension culture with the polyamine or a salt thereof, preferably spermine or a salt thereof, the suspension culture comprises the polyamine or the salt thereof in a concentration of at least 1 pM, preferably at least 5 pM, at least 10 pM, at least 15 pM, or at least 25 pM, more preferably at least 30 pM. According to some embodiments, the suspension culture comprises the polyamine or a salt thereof, preferably spermine or a salt thereof, in a concentration of less than 1.000 pm, preferably less than 800 pM, less than 700 pM, less than 600 pM, more preferably less than 500 pM or less than 400 pM. According to preferred embodiments, after contacting the suspension culture with the polyamine or the salt thereof, preferably spermine or a salt thereof, the suspension culture comprises the polyamine or the salt thereof in a concentration of the range selected from 1 pM to 1.000 pM, preferably 5 pM to 800 pM, 10 pM to 700 pM, 15 pM to 600 pM, 25 pM to 500 pM, more preferably 30 pM to 400 pM or 40 pM to 250 pM. As disclosed in the examples, different concentrations of polyamine or a salt thereof, preferably spermine or a salt thereof, can be applied in order to achieve the beneficial effects disclosed herein (see e.g., Figs. 1-17).

[0198] According to preferred embodiments, the polyamine or salt thereof is spermine or salt thereof, preferably selected from one or more of spermine, spermine tetra hydrochloride, spermine di hydrate, thermospermine, and thermospermine tetrahydrochloride, as well as norspermine or homospermine.

[0199] According to preferred embodiments, contacting the suspension culture with the polyamine or the salt thereof, preferably spermine or a salt thereof, results in an increase of at least 2% of the fraction of single cells, preferably an increase of at least 4%, related to the total number of counted cells and aggregates. According to preferred embodiments, contacting the suspension culture with the polyamine or the salt thereof, preferably spermine or a salt thereof, results in a reduction of at least 2% of the fraction of aggregates, preferably a reduction of at least 4%, related to the total number of counted cells and aggregates. According to preferred embodiments, contacting the suspension culture with the polyamine or the salt thereof, preferably spermine or a salt thereof, results in an increase of at least 2% of the fraction of single cells, preferably an increase of at least 4%, related to the total number of counted cells and aggregates, and results in a reduction of at least 2% of the fraction of aggregates, preferably a reduction of at least 4%, related to the total number of counted cells and aggregates. Such increase in single cells and decrease in aggregates has been shown in the examples and is associated with overall beneficial effects on the cell culture, particularly by improving oxygen and nutrient access to the cells. Also, such parameters are associated with an increase in the viral vector titer, i.e. improved production of viral vectors, such as AAV. According to preferred embodiments, the fraction of single cells and the fraction of aggregates was measured by imaging a cell suspension and counting single cells and aggregates, preferably by automated cell counting using an image analysis software. The skilled person is also aware of other methods or techniques for cell and aggregate counting, such as automatic cell counters acquiring images of cell cultures which are either automatically analyzed by the device or can be downloaded and analyzed with image analysis software may also be used. For instance, Cedex® cell counters already provide an aggregation rate as a standard parameter for each measurement. Those are less accurate compared to values derived from the image analysis script but are often used as an aggregation readout. Also, the skilled person is aware of manual counting methods.

[0200] Composition according to the fourth aspect

[0201] According to a fourth aspect, a composition for reducing host cell aggregation for production of a viral vector is provided, wherein the host cell is selected from a HEK293 cell or a derivative thereof, the composition comprising:

[0202] (i) a cell culture medium for culturing the host cell; and

[0203] (ii) spermine or a salt thereof.

[0204] The composition according to the fourth aspect advantageously allows for reducing host cell aggregation for production of a viral vector. Furthermore, it improves the viral vector production by increasing the viral vector titer, as is shown in the examples. Such composition can be advantageously applied in scope of the methods disclosed here. Hence, the above disclosed advantages disclosed for the methods according to the first, second and third aspect of the invention can also be found for the composition according to the fourth aspect.

[0205] The individual and preferred embodiments of the composition according to the fourth aspect correspond to the embodiments of the methods according to the first, second, and third aspect. Therefore, it is referred to the above disclosure which shall equally be applicable for the composition according to the fourth aspect. This particularly but not exclusively includes the host cell and aggregates thereof, the viral vector, the suspension culture comprising host cells selected from a HEK293 cell or a derivative thereof, the culture medium for culturing the host cell, the spermine or salt thereof. Further features will now be described in detail.

[0206] According to preferred embodiments, the composition further comprises: a polyamine, preferably a spermine precursor, such as spermidine or a salt thereof or cadaverine or a salt thereof, preferably spermidine or a salt thereof; and / or a further polyamine, preferably a spermine precursor, such as putrescine or a salt thereof.

[0207] As disclosed above combining the spermine or salt thereof with a further polyamine, which is preferably a spermine precursor improves the viral vector production by increasing the genomic and transducing viral vector titer (see Figs. 14-17). Hence, the above disclosed advantages for such embodiment of the methods according to the first and second aspects apply as well. For further details of the polyamine(s) it is referred to the above disclosure.

[0208] According to preferred embodiments, the composition further comprises: a polyamine, preferably a spermine precursor, such as spermidine or a salt thereof; and / or a downregulating compound capable of downregulating conversion of spermine to spermidine and / or spermidine to putrescine, preferably an inhibitor of an enzyme involved in the spermine synthesis pathway or a compound interfering with the expression of an enzyme involved in the spermine synthesis pathway, more preferably an inhibitor of spermidine / spermine N l-acetyltransferase 1 (SAT1) or polyamine oxidase (PAO), most preferably an inhibitor of SAT1, such as phenylbutyrate or a salt thereof.

[0209] As disclosed above combining the spermine or salt thereof with a further polyamine, which is preferably a spermine precursor improves the viral vector production by increasing the genomic and transducing viral vector titer. Furthermore, as disclosed above, including a downregulating compound can further improve viral vector production up to a synergistic effect (see Figs. 14-17). Hence, the above disclosed advantages for such embodiment of the methods according to the first and second aspects apply as well. For further details of the polyamine and downregulating compound it is referred to the above disclosure. In such embodiment, the composition optionally is substantially free of putrescine or a salt thereof. In such embodiment, the composition optionally is substantially free of dextran sulfate.

[0210] According to preferred embodiments, the composition is for use in a method for producing a viral vector and / or for reducing host cell aggregation for production of a viral vector and / or for dissolving host cell aggregates, preferably for use in a method according to the invention (e.g. method according to the first aspect, method according to the second aspect, method according to the third aspect).

[0211] Supplement composition according to the fifth and sixth aspect

[0212] According to a fifth aspect, a supplement composition for reducing host cell aggregation for production of a viral vector is provided, wherein the host cell is selected from a HEK293 cell or a derivative thereof, the supplement composition comprising spermine or a salt thereof, preferably further comprising:

[0213] (i) a polyamine, preferably a spermine precursor, such as spermidine or a salt thereof; and / or a further polyamine, preferably a spermine precursor, such as putrescine or a salt thereof; optionally, a liquid for dissolving the spermine or salt thereof; or

[0214] (ii) a polyamine, preferably a spermine precursor, such as spermidine or a salt thereof; and / or a downregulating compound capable of downregulating conversion of spermine to spermidine and / or spermidine to putrescine, preferably an inhibitor of an enzyme involved in the spermine synthesis pathway or a compound interfering with the expression of an enzyme involved in the spermine synthesis pathway, more preferably an inhibitor of spermidine / spermine N l-acetyltransferase 1 (SAT1) or polyamine oxidase (PAO), most preferably an inhibitor of SAT1, such as phenylbutyrate or a salt thereof; optionally, a liquid for dissolving the spermine or salt thereof; optionally, the supplement composition is substantially free of putrescine or a salt thereof. The supplement composition according to the fifth aspects advantageously allow for a remarkable reduction in cell aggregation of the host cells, improving the overall cell culture by enhancing oxygen and nutrient supply to the cells (see Figs. 5-8). Furthermore, consistently increased viral vector yields were obtained (see Figs. 1-4, 12, 13).

[0215] According to a sixth aspect, a supplement composition for dissolving host cell aggregates is provided, wherein the host cells are selected from a HEK293 cell or a derivative thereof, the supplement composition comprising a polyamine or a salt thereof, preferably spermine or a salt thereof.

[0216] The supplement composition according to the sixth aspect, advantageously dissolves cell aggregates, which can be detrimental in culture. As disclosed above, cell aggregates can lead to severe restrictions in mass transport limiting oxygen and nutrient supply to cells within such aggregates. This can result in cell death, toxin formation and lower culture performance, especially considering production of viral vectors. As demonstrated in the examples, by contacting a suspension culture of HEK293 cells with a polyamine, such as spermine or a salt thereof, cell aggregates can be effectively dissolved, leading to higher fractions of single cells (see Figs. 9, 10).

[0217] Such supplement compositions according to the fifth and sixth aspect can be advantageous applied in scope of the methods disclosed here. Hence, the above disclosed advantages disclosed for the methods according to the first, second and third aspect of the invention can also be found for the supplement composition according to the fifth and sixth aspect. The individual preferred embodiments of the supplement compositions according to the fifth and sixth aspect correspond to the individual embodiments of the methods according to the first, second, and aspects, as well as of the composition of the fourth aspect. Therefore, it is referred to the above disclosure which shall equally be applicable. This particularly but not exclusively includes the host cell and aggregates thereof, the suspension culture comprising host cells selected from a HEK293 cell or a derivative thereof, the viral vector, the culture medium for culturing the host cell, the polyamine(s) or salt thereof, particularly spermine or salt thereof but also the other disclosed polyamines, and the downregulating compound. Further features will now be described in detail.

[0218] The term "substantially free of putrescine or a salt thereof" as disclosed herein means that such (supplement) composition does not contain putrescine or a salt thereof as defined herein or that the putrescine or a salt thereof are present in trace amounts or minor amounts of less than 1 pM, preferably less than 0.5 pM.

[0219] The supplement composition according to the sixth aspect, optionally further comprises: (i) a polyamine, preferably a spermine precursor, such as spermidine or a salt thereof or cadaverine or a salt thereof, preferably spermidine or a salt thereof; and / or a further polyamine, preferably a spermine precursor, such as putrescine or a salt thereof; optionally, a liquid for dissolving the polyamine or salt thereof; or

[0220] (ii) a polyamine, preferably a spermine precursor, such as spermidine or a salt thereof; and / or a downregulating compound capable of downregulating conversion of spermine to spermidine and / or spermidine to putrescine, preferably an inhibitor of an enzyme involved in the spermine synthesis pathway or a compound interfering with the expression of an enzyme involved in the spermine synthesis pathway, more preferably an inhibitor of spermidine / spermine N l-acetyltransferase 1 (SAT1) or polyamine oxidase (PAO), most preferably an inhibitor of SAT1, such as phenylbutyrate or a salt thereof; optionally, a liquid for dissolving the polyamine or salt thereof; optionally, it is substantially free of putrescine or a salt thereof.

[0221] The "liquid for dissolving the spermine or salt thereof" refers to a liquid that can dissolve the spermine or salt thereof and the polyamine or salt thereof, respectively. Such liquid is compatible with the host cell in that it does not significantly negatively affect the host cells. Such liquid may be water-based, such as demineralized water. In one embodiment, the liquid may be cell culture media. In another embodiment, the liquid may be water, such as demineralized water.

[0222] According to preferred embodiments, the supplement composition is for use in a method for producing a viral vector and / or for reducing host cell aggregation for production of a viral vector and / or for dissolving host cell aggregates, preferably for use in a method according to the invention (e.g. method according to the first aspect, method according to the second aspect, method according to the third aspect).

[0223] Uses according to the seventh, eighth and nineth aspect

[0224] According to a seventh aspect, spermine or a salt thereof for use for producing a viral vector by a host cell is provided, wherein the host cell is selected from a HEK293 cell or a derivative thereof. Spermine or a salt thereof is preferably for use in a method for producing a viral vector by a host cell, wherein the host cell is selected from a HEK293 cell or a derivative thereof. As demonstrated in the examples, using spermine or a salt thereof for producing a viral vector by a host cell leads consistently to increased viral vector yields. Such increase was obtained for various HEK293 cell types, different transfection systems, and different culture processes and compositions were tested (see Figs. 1-4, 12, 13). The presence of spermine or a salt thereof in suspension culture significantly improves the production of viral vector, such as AAV, using a HEK293 host cell-based culturing system. Furthermore, by including spermine or a salt thereof during or shortly after transient transfection for producing a viral vector, such as AAV, improved viral vector production (see Figs. 11-13).

[0225] According to an eighth aspect, spermine or a salt thereof for use for reducing host cell aggregation for production of a viral vector is provided, wherein the host cell is selected from a HEK293 cell or a derivative thereof. Spermine or a salt thereof is preferably for use in a method for reducing host cell aggregation for production of a viral vector, wherein the host cell is selected from a HEK293 cell or a derivative thereof. As demonstrated in the examples, spermine or a salt thereof for use for reducing host cell aggregation for production of a viral vector allows for a remarkable reduction in cell aggregation of the host cells, improving the overall cell culture by enhancing oxygen and nutrient supply to the cells (see Figs. 5-8).

[0226] According to a ninth aspect, polyamine or a salt thereof, preferably spermine or a salt thereof, for use for dissolving host cell aggregates, preferably for production of a viral vector, wherein the host cell is selected from a HEK293 cell or a derivative thereof. The polyamine or a salt thereof, preferably spermine or a salt thereof, is preferably for use in a method for dissolving host cell aggregates, preferably for production of a viral vector, wherein the host cell is selected from a HEK293 cell or a derivative thereof. As demonstrated in the examples, polyamine or a salt thereof, preferably spermine or a salt thereof, effectively dissolves host cell aggregates, leading to higher fractions of single cells and an optimized cell culture (see Figs. 9, 10).

[0227] The individual preferred embodiments of the uses correspond to the individual embodiments of the methods according to the first, second, and aspects, as well as of the compositions of the fourth, fifth and sixth aspects. Therefore, it is referred to the above disclosure which shall equally be applicable. This particularly but not exclusively includes the host cell and aggregates thereof, the suspension culture comprising host cells selected from a HEK293 cell or a derivative thereof, the viral vector, the culture medium for culturing the host cell, the polyamine(s) or salt thereof, particularly spermine or salt thereof but also the other disclosed polyamines, and the downregulating compound. Further features will now be described in detail. According to preferred embodiments, the host cell is adapted for suspension culture. HEK293 cells are originally adherent cells but have been adapted in the past and present for suspension culture. Techniques to adapt the host cell are known in the art. Suspension culture cells are particularly suitable for large-scale culture of the host cells, as these are independent of potentially limiting surface areas. Also, no additional compounds such as microcarriers need to be provided in culture. HEK293 cells adapted for suspension culture are commercially available (e.g., Viral Production Cells2.0 ("VPC 2.0") or FreeStyle™ 293-F-cells from Thermo Fisher Scientific) or can be generated (see below examples for inhouse cell clone and "Clone 2.0").

[0228] According to preferred embodiments, the viral vector is an adeno-associated virus (AAV). As demonstrated in the examples below, the uses of spermine or a salt thereof according to the present disclosure improves production of AAV (see e.g. Figs. 1-4, 12, 13). According to preferred embodiments, the spermine or salt thereof has a concentration of at least 1 pM, preferably at least 5 pM, at least 10 pM, at least 15 pM, or at least 25 pM, more preferably at least 30 pM. As disclosed in the examples, different concentrations of spermine or salt thereof can be applied in order to achieve the beneficial effects disclosed herein (see e.g. Figs. 1-17). Such concentration corresponds particularly to the concentration when being used, i.e. when being in contact with the host cell, such as within a suspension culture. When being provided, e.g., in form of a supplement, the spermine or salt thereof may be higher concentrated as disclosed herein (see methods according to the first and second aspect).

[0229] According to preferred embodiments, in addition to the spermine or salt thereof or polyamine or salt thereof following is further provided for the respective use:

[0230] (i) a polyamine, preferably a spermine precursor, such as spermidine or a salt thereof; and / or a further polyamine, preferably a spermine precursor, such as putrescine or a salt thereof; optionally, a liquid for dissolving the spermine or salt thereof; or

[0231] (ii) a polyamine, preferably a spermine precursor, such as spermidine or a salt thereof; and / or a downregulating compound capable of downregulating conversion of spermine to spermidine and / or spermidine to putrescine, preferably an inhibitor of an enzyme involved in the spermine synthesis pathway or a compound interfering with the expression of an enzyme involved in the spermine synthesis pathway, more preferably an inhibitor of spermidine / spermine N l-acetyltransferase 1 (SAT1) or polyamine oxidase (PAO), most preferably an inhibitor of SAT1, such as phenylbutyrate or a salt thereof; optionally, a liquid for dissolving the spermine or salt thereof; optionally, it is substantially free of putrescine or a salt thereof.

[0232] Items according to the present disclosure

[0233] The following items provide further advantageous embodiments of the present disclosure:

[0234] 1. A method for producing a viral vector, the method comprising:

[0235] (a) providing a host cell selected from a HEK293 cell or a derivative thereof, and a cell culture medium for culturing the host cell;

[0236] (b) contacting the host cell with the culture medium to form a suspension culture;

[0237] (c) culturing the suspension culture; and

[0238] (d) producing a viral vector by the host cell in the suspension culture, wherein the suspension culture of step (b), step (c), and / or step (d) comprises spermine or a salt thereof.

[0239] 2. A method for reducing host cell aggregation for production of a viral vector, the method comprising:

[0240] (a) providing a host cell selected from a HEK293 cell or a derivative thereof, and a cell culture medium for culturing the host cell;

[0241] (b) contacting the host cell with the culture medium to form a suspension culture;

[0242] (c) culturing the suspension culture; and

[0243] (d) producing a viral vector by the host cell in the suspension culture, wherein the suspension culture of step (b), step (c), and / or step (d) comprises spermine or a salt thereof and wherein host cell aggregation is reduced compared to a suspension culture without spermine or a salt thereof.

[0244] 3. The method according to item 1 or 2, wherein the method has one or more of the following characteristics:

[0245] (i) cell culture medium of step (a) comprises spermine or a salt thereof;

[0246] (ii) the suspension culture of step (b) comprises spermine or a salt thereof;

[0247] (iii) the suspension culture of step (c) comprises spermine or a salt thereof; (iv) the suspension culture of step (d) comprises spermine or a salt thereof;

[0248] (v) the host cell in step (d) is present in a suspension culture comprising spermine or a salt thereof;

[0249] (vi) the suspension culture comprises spermine or a salt thereof in steps (b), (c), and / or (d), preferably at least in steps (c) and / or (d), more preferably at least in steps (c) and (d); and / or

[0250] (vii) the suspension culture of step (b), (c), and (d) comprises spermine or a salt thereof.

[0251] 4. The method according to one or more of items 1 to 3, wherein the method comprises transferring the suspension culture one or more times after step (c) and before step (d), optionally, the method further comprises transferring, diluting, and culturing the suspension culture one or more times after step (c) and before step (d).

[0252] 5. The method according to one or more of items 1 to 4, wherein the cell culture medium of step (a) comprises spermine or a salt thereof and / or the method further comprises providing a supplement composition comprising spermine or a salt thereof.

[0253] 6. The method according to item 5, wherein the method further comprises one or more of the following:

[0254] (i) prior to step (b) the cell culture medium is contacted with the supplement composition;

[0255] (ii) step (b) further comprises contacting the host cell with the culture medium and the supplement composition to form a suspension culture;

[0256] (iii) prior to step (c) or (d) the suspension culture is contacted with the supplement composition; and / or

[0257] (iv) in step (c) and / or step (d) the suspension culture is contacted with the supplement composition.

[0258] 7. The method according to item 5 or 6, wherein the spermine or salt thereof in the supplement composition is present in a concentration of 100 mM or less.

[0259] 8. The method according to one or more of items 1 to 7, wherein the suspension culture comprises the spermine or salt thereof in a concentration of at least 1 pM, preferably at least 5 pM, at least 10 pM, at least 15 pM, or at least 25 pM, more preferably at least 30 pM; and / or less than 1.000 pm, preferably less than 800 pM, less than 700 pM, less than 600 pM, more preferably less than 500 pM or less than 400 pM.

[0260] 9. The method according to one or more of items 1 to 8, wherein the suspension culture comprises spermine or salt thereof in a concentration of the range selected from 1 pM to 1.000 pM, preferably 5 pM to 800 pM, 10 pM to 700 pM, 15 pM to 600 pM, 25 pM to 500 pM, more preferably 30 pM to 400 pM or 40 pM to 250 pM. The method according to one or more of items 1 to 9, wherein the spermine or salt thereof is selected from one or more of spermine, spermine tetrahydrochloride, spermine dihydrate, thermospermine, and thermospermine tetrahydrochloride. The method according to one or more of items 1 to 10, wherein the suspension culture further comprises a polyamine, preferably a spermine precursor, such as spermidine or a salt thereof or cadaverine or a salt thereof, more preferably spermidine or a salt thereof. The method according to item 11, wherein the method has one or more of the following characteristics:

[0261] (i) the cell culture medium of step (a) comprises the polyamine, preferably spermidine or a salt thereof;

[0262] (ii) the method further comprises providing a supplement composition comprising the polyamine, preferably spermidine or a salt thereof, preferably wherein the supplement composition further comprises the spermine or salt thereof;

[0263] (iii) the polyamine, preferably spermidine or a salt thereof, in the supplement composition is present in a concentration of 200 mM or less; and / or

[0264] (iv) the suspension culture comprises the polyamine, preferably spermidine or a salt thereof, in steps (b), (c), and / or (d), preferably at least in steps (c) and / or (d), more preferably at least in steps (c) and (d). The method according to item 11 or 12, wherein the suspension culture comprises the polyamine, preferably a spermine precursor, such as spermidine or a salt thereof, in a concentration of at least 1 pM, preferably at least 5 pM, at least 10 pM, at least 15 pM, or at least 20 pM, more preferably at least 25 pM. The method according to one or more of items 11 to 13, wherein the suspension culture further comprises a further polyamine, preferably a further spermine precursor, such as putrescine or a salt thereof. The method according to item 14, wherein the method has one or more of the following characteristics:

[0265] (i) the cell culture medium of step (a) comprises the further polyamine, preferably putrescine or salt thereof;

[0266] (ii) the method further comprises providing a supplement composition comprising the further polyamine, preferably putrescine or salt thereof, preferably wherein the supplement composition further comprises the spermine or salt thereof;

[0267] (iii) the polyamine, preferably putrescine or salt thereof, in the supplement composition is present in a concentration of 620 mM or less; and / or (iv) the suspension culture comprises the further polyamine, preferably putrescine or salt thereof, in steps (b), (c), and / or (d), preferably at least in steps (c) and / or (d), more preferably at least in steps (c) and (d). The method according to item 14 or 15, wherein the suspension culture comprises the further polyamine, preferably a spermine precursor, such as putrescine or salt thereof, in a concentration of at least 10 pM, at least 50 pM, at least 100 pM, at least 250 pM, at least 500 pM, or at least 750 pM. The method according to one or more of items 1 to 16, wherein the suspension culture further comprises a downregulating compound capable of downregulating conversion of spermine to spermidine and / or spermidine to putrescine. The method according to item 17, wherein the downregulating compound is an inhibitor of an enzyme involved in the spermine synthesis pathway or a compound interfering with the expression of an enzyme involved in the spermine synthesis pathway, preferably the enzyme being spermidine / spermine N ^acetyltransferase 1 (SAT1) or polyamine oxidase (PAO), more preferably an inhibitor of SAT1. The method according to item 17 or 18, wherein the downregulating compound is phenylbutyrate or a salt thereof (such as sodium phenylbutyrate), or diminazene aceturate, preferably phenylbutyrate or a salt thereof, more preferably sodium phenyl butyrate, or the downregulating compound is an expression interfering nucleic acid, preferably a small interfering RNA (siRNA) or a microRNA (miRNA). The method according to one or more of items 17 to 19, wherein the method has one or more of the following characteristics:

[0268] (i) the cell culture medium of step (a) comprises the downregulating compound;

[0269] (ii) the method further comprises providing a supplement composition comprising the downregulating compound, preferably wherein the supplement composition further comprises the spermine or salt thereof;

[0270] (iii) the downregulating compound, preferably an inhibitor of an enzyme involved in the spermine synthesis pathway, such as phenylbutyrate or a salt thereof, in the supplement composition is present in a concentration of 200 mM or less;

[0271] (iv) the suspension culture comprises the downregulating compound in steps (b), (c), and / or (d), preferably at least in steps (c) and / or (d), more preferably at least in steps (c) and (d); and / or

[0272] (v) the downregulating compound is present in the suspension culture at least in step (d). The method according to one or more of items 17 to 20, wherein the suspension culture comprises the downregulating compound, preferably an inhibitor of an enzyme involved in the spermine synthesis pathway, such as phenylbutyrate or a salt thereof, in a concentration of at least 0.1 pM, preferably at least 0.5 pM, at least 1 pM, at least 2.5 pM, or at least 5 pM, more preferably at least 10 pM.

[0273] 22. The method according to one or more of items 1 to 16, wherein the suspension culture further comprises a polyamine and a downregulating compound capable of downregulating conversion of spermine to spermidine and / or spermidine to putrescine, preferably an inhibitor of spermidine / spermine N ^acetyltransferase 1 (SAT1).

[0274] 23. The method according to item 19, wherein the polyamine is spermidine or a salt thereof.

[0275] 24. The method according to item 19 or 20, wherein the suspension culture does not comprise putrescine or a salt thereof originating from the cell culture medium or a supplement.

[0276] 25. The method according to one or more of items 1 to 24, wherein the genomic viral vector titer and / or transducing viral vector titer is increased compared to a method wherein the suspension culture does not comprise spermine or a salt.

[0277] 26. The method according to item 25, wherein the genomic viral vector titer is increased by at least 10%, preferably at least 25%, compared to a method wherein the suspension culture does not comprise spermine or a salt.

[0278] 27. The method according to item 25 or 26, wherein the transducing viral vector titer is increased by at least 10%, preferably at least 25%, compared to a method wherein the suspension culture does not comprise spermine or a salt.

[0279] 28. The method according to one or more of items 1 to 27, wherein the fraction of single cells is increased compared to a method wherein the suspension culture does not comprise spermine or a salt, preferably the fraction of single cells is increased by at least 2%, more preferably at least 4%, related to the total number of counted cells and aggregates.

[0280] 29. The method according to one or more of items 1 to 28, wherein the fraction of cell aggregates is reduced compared to a method wherein the suspension culture does not comprise spermine or a salt, preferably the fraction of cell aggregates is reduced by at least 2%, more preferably at least 4%, related to the total number of counted cells and aggregates.

[0281] 30. The method according to item 28 or 29, wherein the fraction of single cells and the fraction of aggregates was measured by imaging a cell suspension and counting single cells and aggregates, preferably by automated cell counting using an image analysis software.

[0282] 31. The method according to one or more of items 1 to 30, wherein the host cell is adapted for suspension culture.

[0283] 32. The method according to one or more of items 1 to 31, wherein the viral vector is a nonenveloped viral vector and / or DNA viral vector, preferably a nonenveloped, DN A viral vector, such as an adeno-associated virus (AAV).

[0284] 33. The method according to one or more of items 1 to 32, wherein the derivative of the HEK293 cell is characterized by being cell that roots backto HEK293 and / or was originally a HEK293 cell that was adapted, modified, differentiated, reprogrammed, transformed, transduced, transfected and / or mutated. The method according to one or more of items 1 to 33, wherein the derivative of the HEK293 cell is selected from one or more of the group consisting of 293, 293T, 293T / 17, ANJOU 65, 293H, 293E, 293-6E, EBNA1-6E, 293F, 293FT, 293Flp-IN T-REx, 293FTM, 293S, 293SG, 293SGGD, 293MSR, 293A, or any modified variants thereof. The method according to one or more of items 1 to 34, wherein the host cell is modified to be configured to produce a viral vector. The method according to item 35, wherein the host cell is modified by:

[0285] (i) transiently transfecting the host cell with one or more plasmids for viral vector production allowing for transient viral vector production, optionally wherein the spermine or salt thereof is present during transient transfection and / or is added after transient transfection.; and / or

[0286] (ii) genome editing the host cell with one or more nucleic acid molecules for viral vector production allowing for stable viral vector production. The method according to item 36, wherein transiently transfecting comprises:

[0287] (x) combining the host cell with the one or more plasmids for viral vector production and a transfection reagent; and

[0288] (y) incubating the host cell with the one or more plasmids for viral vector production and the transfection reagent, to generate a transiently transfected cell; optionally, wherein spermine or a salt thereof is present in incubation step (y) and / or is added after incubation step (y). The method according to item 37, wherein step (x) comprises one of the following:

[0289] (x.l) i. combining the cell culture medium with the one or more plasmids for viral vector production and the transfection reagent to form a transfection composition; ii. optionally, mixing the transfection composition of (i); iii. optionally, incubating the transfection composition of (i) or (ii); iv. combining the transfection composition of (i), (ii) or (iii) and the host cell; and v. optionally, the transfection composition of (i), (ii), or (iii) or the composition of (iv) further comprises the spermine or salt thereof; or

[0290] (x.2) i. adding the one or more plasmids for viral vector production and the transfection reagent to the host cell present in the cell culture medium, optionally further comprising the spermine or salt thereof. The method according to one or more of items 36 to 38, wherein: the method further comprises having M344 or a derivative thereof present during transient transfection and / or M344 or a derivative thereof is added after transient transfection, preferably M344 or a derivative thereof is after transient transfection further; and / or further M344 ora derivative thereof is present in incubation step (y) and / or is added after incubation step (y).

[0291] 40. The method according to one or more of items 37 to 39, wherein step (y) comprises incubating for at least 1 hour, at least 2 hours, at least 3 hours, preferably at least 4 hours.

[0292] 41. The method according to one or more of items 37 to 40, wherein step (y) comprises incubating for 1 to 48 hours, 2 to 36 hours, 3 to 24 hours, preferably 4 to 12 hours, more preferably 4 to 8 hours or 4 to 6 hours.

[0293] 42. The method according to one or more of items 37 to 41, wherein the transfection reagent comprises a compound that is polymer-based or lipid-based, preferably being cationic, such as polyethylene imine or a derivative thereof.

[0294] 43. The method according to one or more of items 37 to 42, wherein genome editing the host cell comprises stably integrating the one or more nucleic acid molecules into the chromosome of the host cell to be capable of stably producing a viral vector.

[0295] 44. The method according to one or more of items 37 to 43, wherein the one or more plasmids or the one or more nucleic acid molecules for viral vector production encode at least part of an adeno-associated virus (AAV).

[0296] 45. The method according to item 44, wherein the one or more plasmids or the one or more nucleic acid molecules for viral vector production encode one or more of the group comprising Rep78, Rep68, Rep52, Rep40, VP1, VP2, VP3, ITR, AAP, MAAP, X Gene, VA RNA, E4orf6, and E2A, preferably all of the aforementioned, for producing an AAV.

[0297] 46. The method according to one or more of items 1 to 45, wherein the method further comprises step (e) harvesting the viral vector.

[0298] 47. The method according to item 46, wherein harvesting comprises lysing the host cell.

[0299] 48. The method according to one or more of items 1 to 47, wherein the method further comprises step (f) purifying the harvested viral vector.

[0300] 49. The method according to item 48, wherein the method further comprises step (g) formulating the viral vector for gene therapy.

[0301] 50. A method for dissolving host cell aggregates, the method comprising:

[0302] (a) providing a suspension culture comprising host cells selected from a HEK293 cell or a derivative thereof, wherein at least a fraction of the host cells is provided as cell aggregates, and a polyamine or a salt thereof;

[0303] (b) contacting the suspension culture with the polyamine or the salt thereof; and

[0304] (c) optionally, culturing the suspension culture, wherein the polyamine or the salt thereof dissolves host cell aggregates.

[0305] 51. The method according to item 50, wherein the polyamine orthe salt thereof is provided as a composition or suspension, preferably as a cell culture medium comprising the polyamine or the salt thereof and / or as a supplement composition comprising the polyamine or the salt thereof.

[0306] 52. The method according to item 50 or 51, wherein the method is for dissolving host cell aggregates for production of a viral vector, preferably, wherein the method further comprises step (d) producing a viral vector by the host cell in the suspension culture.

[0307] 53. The method according to one or more of items 50 to 52, wherein the polyamine is one or more of the group selected from spermine, spermidine, cadaverine, and putrescine, preferably spermine, or spermidine, more preferably spermine.

[0308] 54. The method according to one or more of items 50 to 53, wherein the polyamine is spermine or a salt thereof in a supplement composition present in a concentration of 100 mM or less.

[0309] 55. The method according to one or more of items 50 to 54, wherein, after contacting the suspension culture with the polyamine or a salt thereof, preferably spermine or a salt thereof, the suspension culture comprises the polyamine or the salt thereof in a concentration of at least 1 pM, preferably at least 5 pM, at least 10 pM, at least 15 pM, or at least 25 pM, more preferably at least 30 pM.

[0310] 56. The method according to one or more of items 50 to 55, wherein, after contacting the suspension culture with the polyamine or the salt thereof, preferably spermine or a salt thereof, the suspension culture comprises the polyamine or the salt thereof in a concentration of the range selected from 1 pM to 1.000 pM, preferably 5 pM to 800 pM, 10 pM to 700 pM, 15 pM to 600 pM, 25 pM to 500 pM, more preferably 30 pM to 400 pM or 40 pM to 250 pM.

[0311] 57. The method according to one or more of items 50 to 56, wherein the polyamine or salt thereof is spermine or salt thereof, preferably selected from one or more of spermine, spermine tetrahydrochloride, spermine dihydrate, thermospermine, and thermospermine tetra hydrochloride.

[0312] 58. The method according to one or more of items 50 to 57, wherein contacting the suspension culture with the polyamine or the salt thereof, preferably spermine or a salt thereof, results in an increase of at least 2% of the fraction of single cells, preferably an increase of at least 4%, related to the total number of counted cells and aggregates. 59. The method according to one or more of items 50 to 58, wherein contacting the suspension culture with the polyamine or the salt thereof, preferably spermine or a salt thereof, results in a reduction of at least 2% of the fraction of aggregates, preferably a reduction of at least 4%, related to the total number of counted cells and aggregates.

[0313] 60. A composition for reducing host cell aggregation for production of a viral vector, wherein the host cell is selected from a HEK293 cell or a derivative thereof, the composition comprising:

[0314] (i) a cell culture medium for culturing the host cell; and

[0315] (ii) spermine or a salt thereof.

[0316] 61. The composition according to item 60, wherein the composition further comprises: a polyamine, preferably a spermine precursor, such as spermidine or a salt thereof or cadaverine or a salt thereof, preferably spermidine or a salt thereof; and / or a further polyamine, preferably a spermine precursor, such as putrescine or a salt thereof.

[0317] 62. The composition according to item 60, wherein the composition further comprises: a polyamine, preferably a spermine precursor, such as spermidine or a salt thereof; and / or a downregulating compound capable of downregulating conversion of spermine to spermidine and / or spermidine to putrescine, preferably an inhibitor of an enzyme involved in the spermine synthesis pathway or a compound interfering with the expression of an enzyme involved in the spermine synthesis pathway, more preferably an inhibitor of spermidine / spermine N 1-acetyltransferase 1 (SAT1) or polyamine oxidase (PAO), most preferably an inhibitor of SAT1, such as phenylbutyrate or a salt thereof.

[0318] 63. The composition according to one or more of items 60 to 62, wherein the composition is for use in a method for producing a viral vector and / or for reducing host cell aggregation for production of a viral vector and / or for dissolving host cell aggregates, preferably for use in a method according to one or more of items 1 to 53.

[0319] 64. A supplement composition for reducing host cell aggregation for production of a viral vector, wherein the host cell is selected from a HEK293 cell or a derivative thereof, the supplement composition comprising spermine or a salt thereof, preferably further comprising:

[0320] (i) a polyamine, preferably a spermine precursor, such as spermidine or a salt thereof; and / or a further polyamine, preferably a spermine precursor, such as putrescine or a salt thereof; optionally, a liquid for dissolving the spermine or salt thereof; or (ii) a polyamine, preferably a spermine precursor, such as spermidine or a salt thereof; and / or a downregulating compound capable of downregulating conversion of spermine to spermidine and / or spermidine to putrescine, preferably an inhibitor of an enzyme involved in the spermine synthesis pathway, preferably an inhibitor of spermidine / spermine N ^acetyltransferase 1 (SAT1) or polyamine oxidase (PAO), more preferably an inhibitor of SAT1, most preferably phenylbutyrate or a salt thereof; optionally, a liquid for dissolving the spermine or salt thereof; optionally, the supplement composition is substantially free of putrescine or a salt thereof. A supplement composition for dissolving host cell aggregates, wherein the host cells are selected from a HEK293 cell ora derivative thereof, the supplement composition comprising a polyamine or a salt thereof, preferably spermine or a salt thereof, optionally further comprising:

[0321] (i) a polyamine, preferably a spermine precursor, such as spermidine or a salt thereof or cadaverine or a salt thereof, preferably spermidine or a salt thereof; and / or a further polyamine, preferably a spermine precursor, such as putrescine or a salt thereof; optionally, a liquid for dissolving the polyamine or salt thereof; or

[0322] (ii) a polyamine, preferably a spermine precursor, such as spermidine or a salt thereof; and / or a downregulating compound capable of downregulating conversion of spermine to spermidine and / or spermidine to putrescine, preferably an inhibitor of an enzyme involved in the spermine synthesis pathway or a compound interfering with the expression of an enzyme involved in the spermine synthesis pathway, more preferably an inhibitor of spermidine / spermine N 1-acetyltransferase 1 (SAT1) or polyamine oxidase (PAO), most preferably an inhibitor of SAT1, such as phenylbutyrate or a salt thereof; optionally, a liquid for dissolving the polyamine or salt thereof; optionally, the supplement composition is substantially free of putrescine or a salt thereof. 66. The supplement composition according to item 64 or 65, wherein the supplement composition is for use in a method for producing a viral vector and / or for reducing host cell aggregation for production of a viral vector and / or for dissolving host cell aggregates, preferably for use in a method according to one or more of items 1 to 59.

[0323] 67. Spermine or a salt thereof for use for producing a viral vector by a host cell, wherein the host cell is selected from a HEK293 cell or a derivative thereof.

[0324] 68. Spermine or a salt thereof for use for reducing host cell aggregation for production of a viral vector, wherein the host cell is selected from a HEK293 cell or a derivative thereof.

[0325] 69. Polyamine or a salt thereof, preferably spermine or a salt thereof, for use for dissolving host cell aggregates, preferably for production of a viral vector, wherein the host cell is selected from a HEK293 cell or a derivative thereof.

[0326] 70. Spermine or a salt thereof for use according to item 67 or 68 or polyamine or salt thereof for use according to item 69, wherein the host cell is adapted for suspension culture.

[0327] 71. Spermine or a salt thereof or polyamine or a salt thereof for use according to one or more of items 67 to 70, wherein the viral vector is an adeno-associated virus (AAV).

[0328] 72. Spermine or a salt thereof for use according to one or more of items 67 to 71, wherein the spermine or salt thereof has a concentration of at least 1 pM, preferably at least 5 pM, at least 10 pM, at least 15 pM, or at least 25 pM, more preferably at least 30 pM.

[0329] 73. Spermine or a salt thereof or polyamine or a salt thereof for use according to one or more of items 67 to 72, wherein in addition to the spermine or salt thereof or polyamine or salt thereof following is further provided for the respective use:

[0330] (i) a polyamine, preferably a spermine precursor, such as spermidine or a salt thereof; and / or a further polyamine, preferably a spermine precursor, such as putrescine or a salt thereof; optionally, a liquid for dissolving the spermine or salt thereof; or

[0331] (ii) a polyamine, preferably a spermine precursor, such as spermidine or a salt thereof; and / or a downregulating compound capable of downregulating conversion of spermine to spermidine and / or spermidine to putrescine, preferably an inhibitor of an enzyme involved in the spermine synthesis pathway or a compound interfering with the expression of an enzyme involved in the spermine synthesis pathway, more preferably an inhibitor of spermidine / spermine N 1-acetyltransferase 1 (SAT1) or polyamine oxidase (PAO), most preferably an inhibitor of SAT1, such as phenylbutyrate or a salt thereof; optionally, a liquid for dissolving the spermine or salt thereof; optionally, it is substantially free of putrescine or a salt thereof.

[0332] Throughout the description, where methods, compositions or uses are described as having, including, or comprising specific components or steps, it is contemplated that, additionally, there are methods, compositions or uses of the present invention that consist essentially of, or consist of, the recited components or steps.

[0333] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0334] Terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0335] The use of the term "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including grammatical equivalents thereof, should be understood generally as open- ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0336] Where the use of the term "about" or "approximately" is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a ±10% variation from the nominal value unless otherwise indicated or inferred.

[0337] Also as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or"). The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0338] All citations are hereby incorporated by reference. All individual embodiments and aspects as disclosed herein can be combined with each other within the framework and context of the present disclosure. It will be understood that the embodiments disclosed herein are only exemplary, and that any feature presented for a particular exemplary embodiment may be used with the present disclosure on its own or in combination with any feature presented for the same or another particular exemplary embodiment and / or in combination with any other feature not mentioned. It will further be understood that any feature presented for an example embodiment in a particular category may also be used in a corresponding manner in an example embodiment of any other category.

[0339] EXAMPLES

[0340] It should be understood that the following examples are for illustrative purpose only and are not to be construed as limiting this invention in any manner. The following examples demonstrate the advantageous improved viral vector production method by providing spermine ora salt thereof in the suspension culture. It is shown that spermine increases the viral vector titer consistently for different HEK293-based host cells, different culture systems, different transfection system, as well as a supplement and full-weighed in media component. Furthermore, the examples show that spermine is beneficial in reducing host cell aggregation for HEK293 based cells culture systems, which is beneficial for culture viability, particularly by enhancing cell oxygen transfer and nutrition. In another example, spermine was used for dissolving host cell aggregates of a suspension culture, further highlighting the beneficial effects of spermine, especially as supplement throughout various steps of suspension culture. Spermine was also used as an enhancer compound for transient transfection in a viral vector production method, either alone or in combination with another enhancing molecule (M344), showing improvements in viral vector production. Finally, spermine was also tested together with other spermine pathway compounds and / or regulator or the spermine pathway, showing superior viral vector production. Overall, the examples demonstrate the suitability of spermine for viral vector production and HEK293-based host cell culture, as well as beneficial combinations of spermine with other compounds for viral vector production.

[0341] Materials and Methods

[0342] Equipment and material

[0343] The used equipment and material are listed below in Tables 1 - 4: Table 1: Technical equipment

[0344] Table 2: HEK293 cell lines Table 3: Chemicals

[0345] Table 4: Cell culture media and feeds

[0346] Routine cultivation of HEK293 cells:

[0347] Expi293F and VPC2.0 cells as well as the internally developed H EK clones ("inhouse H EK clone" and "Clone 2.0") were thawed and cultivated in HEK TF, HEK ViP NB, or the internally developed HEK medium. Cells were sub-cultured to a VCD of 0.3-0.4E6 cel Is / mL every three to four days. Cells were cultivated in plain bottom shake flasks with a vented cap at a culture volume of 30 mL, 37°C, 5% CO 2, 185 rpm and 50 mm orbit. Cells were adapted to culture media with indicated supplementations for three to five passages prior to AAV production. rAAV production at shake flask scale

[0348] One day before transfection, cells were inoculated at a VCD of 2.0E6 cells / mL in 22 mL culture medium. For transfection, 1 pg DNA was used per 1E6 cells at a final cell density of 3E6 cells / mL. A two-plasmid system consisting of pAAV-ssGFP and pDG (both plasmid factory) was employed at a molar ratio of 1:1.3, respectively. As transfection reagents, PEI-MAX or FectoVIR-AAV were used at a 2.7:1 or 1:1 ratio with the DNA. Pre-complexation of DNA and transfection reagent was performed in the indicated culture medium using 10%, i.e., 3 mL of the final culture volume. Mixing the components at an overhead shaker for 20 seconds at 15 rpm was followed by incubation of 15 or 30 min at room temperature for FectoVIR-AAV or PEI-MAX, respectively. Subsequently, the transfection mixture was added to the culture, followed by addition of 6 mL fresh culture medium. If applicable, enhancers (including spermine and / or M344) were added at 4 to 6 h post transfection at the indicated concentrations.

[0349] VCD and viability were measured daily using an automated cell counter. At 72 h post transfection, cells were lysed using a lOx tergitol lysis buffer at lx working concentration. After buffer addition, cells were incubated on a shaking platform at 37°C, 130 rpm, and 50 mm orbit for 2 h. Cell debris was pelleted by centrifugation at 2000 x g for 15 min. Supernatants were collected and stored at -80°C until further analysis. rAAV production in Ambr 15

[0350] For rAAV production at Ambr 15 scale, the indicated culture medium was filled in microbioreactors and equilibrated to culture conditions (37°C, 40% DO, pH 7.2) for one day. Subsequently, microbioreactors were inoculated with the indicated cells at a VCD of 0.3-0.5E6 cells / mL in 14.5 mL final volume per vessel. Cultures were stirred at 630 rpm (upstirring). After three days, cells were passaged to a VCD of 2.0E6 cells / mL in 11 mL final volume. The next day, transfection for rAAV production was performed using the same parameters as described for shake flask experiments. Based on the smallerfinal volume, 1.1 mL transfection mixture and 2.2 mLfresh medium were added. To respect incubation times of transfection reagents, transfection was performed in sets of 6 microbioreactors. VCD and viability were measured daily using an automated cell counter. Cells were lysed by addition of lOxtergitol buffer (lx final concentration) using the liquid handler. Subsequently, cells were incubated for 2 h at 630 rpm upstirring and 37°C for 2 h. Cell lysates were extracted from microbioreactors and processed as described for shake flask experiments.

[0351] Quantification of rAAV genomes

[0352] As a first step, free DNA was digested by mixing 5 pL cell lysate with 35 pL ddPCR dilution buffer, 5 pL DNase I and 5 pL DNase I reaction buffer followed by incubation at 37°C for 1 h. As preparation for DNase I inactivation, 20 pL PCR grade water, 8 pL EDTA (50 mM), and 1 pLSDS (10%) were mixed. Subsequently, 29 pL of this mixture were added to the digested cell lysate (50 pL). Next, 14 pL PCR grade water, 2 pL Proteinase K and 5 pL Proteinase K buffer (20x) were mixed and 21 pL were added to the DNase I digested cell lysate. To lyse viral capsids, the sample was then incubated at 50°C for 1 h. Proteinase K was inactivated at 95°C for 15 minutes. The fully prepared rAAV genomes were quantified on a QX200 ddPCR system following the manufacturer's instructions using an eGFP- specific primer / probe assay.

[0353] Transduction assay

[0354] For the determination of the transducing titer of AAV-containing samples, a transduction assay was performed. Briefly, on the day of transduction, 100,000 cells in 1 mL of DMEM (+10 % FCS, + 2 mM L-glutamine) of adherent HEK293 (ATCC) were seeded in 12-well plates. Serial dilution of AAV- containing samples was performed in PBS. Subsequently, 10 pL of either the undiluted or diluted sample was added to each well and cells were incubated for 72 h at 37 °C and 5 % CO2.

[0355] For the quantification of GFP+ cells, cells were washed once with PBS and trypsinated with 100 pL TrypLE Select per well. Trypsination was stopped by the addition of 900 pL DMEM (+10 % FCS, + 2 mM L-glutamine). Flow cytometry analysis was performed on an iQue® 3 flow cytometer system (Sartorius AG). Positive cells were gated above the 99 % interval of the negative (untransduced) control. For the calculation of the transducing titer, dilutions with equal or less than 25 % positive cells were used. The calculations were based on the following formula:

[0356] TU fraction of GFP + cells x 100.000 cells x 1E6 |1L / L

[0357] T" 10 |1L Analysis of cell aggregation with Fiji

[0358] Cell aggregation analysis was performed based on images acquired with automated cell counters, as is well-known in the art (see manual for ImageJ / Fiji). Initially, a threshold for cell segmentation was applied with Fiji. As a next step, the "Analyze Particles" function was executed for automated cell detection and measurement of object size for 20 images per condition. All steps were performed with a custom automated script. As a last step, objects detected by Fiji were categorized based on cell size using Excel to distinguish between single cells, two-cell aggregates and aggregates that contain three or more cells. Importantly, for each cell line, the size of single cells and aggregates of two and three cells was assessed on example images. Results are depicted as a fraction of all objects.

[0359] Example 1: Supplementation of spermine increases AAV titer

[0360] In example 1, spermine was supplemented to a cell culture medium for HEK cell culture (see 1A, IB, and 1C) or used as full-weighing media compound (see ID). For supplementing, spermine tetrahydrochloride was dissolved in water at a concentration of about 50 mM. Afterwards, the supplement composition was sterile filtered and added to the cell culture medium to adjust the spermine concentration to the indicated ranges. Different HEK cell lines were cultured in presence of spermine, which was tested at shake flask scale, as well as in Amr 15 microbioreactors. Furthermore, different transfection systems were tested. Example 1 demonstrates that the presence of spermine in the HEK suspension culture significantly increases the AAV genomic titer. Such increase was consistently observed for the different culture systems, transfection systems, and cell lines.

[0361] Example 1A: Supplementation of different spermine concentrations increases AAV2 genomic titer To assess the effect of spermine supplementation on AAV titer, Expi293F cells and an suspension- adapted HEK293 clone (referred to as "inhouse HEK cell line") were cultured in HEK TF medium or HEK TF medium supplemented with spermine at shake flask scale. The inhouse HEK cell line and Expi293F cells were adapted to HEK TF supplemented with 50, 100 and 500 pM spermine for five passages at 125 ml shake flask scale. For AAV2 production, transient transfection was carried out as described above using a two-plasmid system and PEI-MAX. Lysis was performed at 72 h post transfection and genomic titer was measured by ddPCR as described above. The results of these measurements are shown in Fig. 1.

[0362] Fig. 1 shows that compared to HEK TF control medium, the supplementation of 50 and 100 pM spermine led to a 1.8 to 6.2-fold increase in AAV2 genomic titer for the inhouse HEK cell line and Expi293F cells, respectively. Notably, addition of 500 pM spermine was detrimental for AAV2 production by the inhouse HEK cell line and resulted in aggregation with trypan blue at concentrations used in automated cell counting devices. However, for some other HEK cell lines which are more robust to spermine, high concentrations of 500 pM or more may still result in titer increases.

[0363] Example IB: Supplementation of spermine increases AAV2 titer for different HEK cell lines transfected with FectoVIR-AAV

[0364] To test whet her supplementation of spermine was also beneficial in the context of other HEK culture media and for a FectoVIR-AAV based transfection, it was tested on an internally developed HEK medium using Expi293F and VPC2.0 cells, as well as the inhouse HEK cel I line. Expi293F and VPC2.0 cells as well as the inhouse HEK clone were adapted to an internally developed HEK medium supplemented with 75 pM spermine for four to five passages at 125 ml shake flask scale. Cells were transiently transfected for AAV2 production using a two-plasmid system and FectoVIR-AAV. Lysis was performed at 72 h post transfection and genomic titer was measured by ddPCR as described above. The results are shown in Fig. 2.

[0365] Fig. 2 shows that for cultures transfected with FectoVIR-AAV, the AAV2 titer was increased 1.7 and 2.4-fold for Expi293 and VPC2.0 cells, respectively, in cultures supplemented with 75 pM spermine compared to unsupplemented controls. For the inhouse HEK clone, no benefit of spermine supplementation was observed. Overall, supplementation of spermine increases the AAV2 titer for a FectoVIR based transfection using the inhouse developed cell culture media.

[0366] Example 1C: Supplementation of spermine increases AAV2 titer for different HEK cell lines transfected with PEI-MAX

[0367] Furthermore, it was also tested whether supplementation of spermine was also beneficial in the context of other HEK culture media and for a PEI-MAX based transfection. Again, the internally developed HEK medium using Expi293F and VPC2.0 cells was tested, as well as the inhouse HEK cell line. Expi293F and VPC2.0 cells as well as the inhouse HEK clone were adapted to an internally developed HEK medium supplemented with 75 pM spermine for four to five passages at 125 ml shake flask scale. Cells were transiently transfected for AAV2 production using a two-plasmid system and PEI-MAX. Lysis was performed at 72 h post transfection and genomic titer was measured by ddPCRas described above. The results are shown in Fig. 3.

[0368] As shown in Fig. 3, for cultures transfected with PEI-MAX, AAV2 titer was increased 2.6-fold for

[0369] Expi293F cells, and 3.3-fold for VPC2.0 cells and the inhouse HEK cell line in presence of 75 pM spermine. In conclusion, supplementation of spermine also on other HE Keel I culture media increases

[0370] AAV2 titer for PEI-MAX based transfection.

[0371] Example ID: Spermine a full weighing media component increases AAV2 titer

[0372] Since the experiments outlined above indicated that supplementation of spermine was beneficial for AAV production, 72 pM spermine was included in a full weighing of our internally developed HEK medium. Such full weighing media does not require supplementation of spermine, as spermine is present from the beginning on. This full weighing was used for AAV2 production in the Ambr 15 and compared to a full weighing without spermine. Cells were adapted to a full weighing of the internally developed HEK medium with or without 72 pM spermine and cobalt chloride for three passages. Following inoculation, cells were cultured for one more passage in the Ambr 15 system at 630 rpm upstirring, pH 7.2, DO 40%, and 37°C. Transfection for AAV2 production was performed using a two- plasmid system and PEI-MAX. Cells were lysed at 72 h post transfection and genomic titers were analyzed by ddPCR as described above. Notably, in this experiment, the full weighing with spermine also contained cobalt chloride, which was identified of having no or even negative impact in later experiments. The results of the genomic titer are shown in Fig. 4.

[0373] Fig. 4 highlights that the AAV2 titer was increased by a factor of 2.0, 1.3, and 3.3-fold for Expi293F cells, VPC2.0 cells, and the inhouse HEK clone, respectively. Thus, spermine as part of a full weighing used in a regulated system such as the Ambrl5 increases AAV2 titer for all cell lines tested.

[0374] Conclusions

[0375] Example 1 demonstrates that spermine is beneficial as supplement and compound of a cell culture medium for culturing HEK cells in suspension culture. It was shown that spermine consistently increases the genomic titer using different HEK cell lines (Expi293F, VPC2.0, inhouse HEK clone), different cell culture media (HEK TF, inhouse developed media), different transfection systems (FectoVIR, PEI-MAX), as well as different culturing processes (shake flasks, microbioreactors). Furthermore, various concentrations of spermine (50 pM, 72 pM, 75 pM and 100 pM) showed the beneficial impact of spermine on the AAV genomic titer. Based on evidence in literature describing that spermine condensates DNA (see Katz et al., "Spermine Condenses DNA, but not RNA Duplexes", Biophysical Journal, 2017, Vol. E112(l), pp. 22-30), it is hypothesized that the spermine interacts with DNA in a manner that promotes the production of viral genomes. The increase in the genomic titer, i.e., on DNA level, of Example 1 supports this hypothesis (whereas the capsid titer, i.e., on protein level, remains essentially constant (data not shown)). Hence, spermine appears not to significantly alter protein production but has effects on a nucleic acid, particularly DNA, level. Overall, presence of spermine in suspension culture is thus highly advantageous for producing a viral vector, such as AAV, using a host cell selected from a HEK293 cell or a derivative thereof.

[0376] Example 2: Spermine decreases cell aggregation in H EK suspension culture

[0377] It was surprisingly observed by the inventors in example 1 above, that the presence of spermine led to a reduction in cell aggregation. A reduction in aggregation is highly beneficial. Specifically, due to restricted access to cells inside the aggregate, cell aggregation reduces transfectability which decreases product yields in transient production processes. Moreover, cell aggregation is detrimental for cell monitoring and control of the cellular environment, and might impairtransport of nutrients to, and products from the cell. Cell aggregates are also restricted from access to oxygen and nutrients increasing cell death and toxin accumulation. Hence, reducing aggregation addresses the challenges of cell aggregation, which is also oftentimes observed for HEK cells (see Seidel et al., Bioengineering, 2023, Vol. 10, 478).

[0378] In example 2, the surprising finding that spermine decreases cell aggregation in HEK suspension culture is further assessed by quantifying the presence of cell aggregates using an image-based analysis (ImageJ script) as described above. Note that the skilled person is also aware of other methods or techniques for cell and aggregate counting, such as automatic cell counters acquiring images of cell cultures which are either automatically analyzed by the device or can be downloaded and analyzed with image analysis software may also be used. For instance, Cedex® cell counters already provide an aggregation rate as a standard parameter for each measurement. Those are less accurate compared to values derived from the image analysis script but are often used as an aggregation readout. Also, the skilled person is aware of manual counting methods.

[0379] Example 2 demonstrates that spermine as supplement and component of full-weighing medium consistently reduces cell aggregation of HEK suspension culture, resulting in higher fractions of single cells. This significantly reduces aggregation-based side effects which are undesirable for viral vector production, such as oxygen or nutrient shortages and toxin formation but also access to the transfection complexes. Without being bound to theory, it is believed that spermine binds to DNA and other charged molecules to neutralize these and thereby shields the cells from interacting via DNA or other charged molecules with each other. In addition, as demonstrated in example 1 above, spermine increases the genomic titer, i.e., on DNA level, whereas the capsid titer, i.e., on protein level, remains essentially constant (data not shown). Hence, spermine appears not to significantly alter protein production but has effects on a nucleic acid, particularly DNA, level. Example 2A: Supplementation of spermine decreases cell aggregation

[0380] To quantify the improvement, cells cultured for 3 or 4 days in an internally developed HEK medium with and without spermine were imaged in an automated cell counter. A custom image analysis software (ImageJ) script was used to analyze cell aggregation based on object size, as described above. The results are shown in Figs. 5 and 6.

[0381] Figs. 5 and 6 show the supplementation of spermine which decreases cell aggregation in 3-day cultures (Fig. 5) and 4-days cultured (Fig.6). Expi293F and VPC2.0 cells as well as the inhouse HEK clone were adapted to an internally developed HEK medium with and without supplemented 75 pM spermine for three to four passages at 125 ml shake flask scale. Subsequently, cells were allowed to grow for three days and 4 days, respectively. Cell cultures were imaged with help of an automated cell counter. A custom image analysis software (Fiji) script was used to quantify cell aggregation based on object size as described above. Clusters of two cells were considered small aggregates, whereas large aggregates contained three or more cells.

[0382] As shown in Figs. 5, in 3-day cultures, the fraction of large (>3 cells) aggregates was reduced by 2.3, 1.8, and 1.6-fold for Expi293F cells, the inhouse HEK clone, and VPC2.0 cells, respectively, in presence of spermine. For these cells, a 2.6, 1.5 and 1.1-fold decrease in large aggregates was observed in 4-day cultures, respectively (Fig.6). In all cases, the percentage of single cells increased by 2.4-14% in presence of spermine compared to the control (Figs. 5 and 6). This indicated that supplementation of spermine consistently led to the reduction of cell aggregation for different HEK cell lines (Expi293F, VPC 2.0 and the inhouse HEK clone) cultured in suspension culture for different culture durations (3 days and 4 days).

[0383] Example 2B: Spermine decreases aggregation as part of a full weighing medium

[0384] Next, it was tested whether spermine also reduced the aggregation of VPC2.0 cells as part of a full weighing medium. VPC2.0 cells were adapted to a full weighing with or without 72 pM spermine for two passages at 125 mL shake flask scale. Subsequently, cells were grown for four days in the respective culture medium and imaged with an automated cell counter. A custom image analysis software (ImageJ) script was used to quantify cell aggregation based on object size as described above. Clusters of two cells were considered small aggregates, whereas large aggregates contained three or more cells. The results are shown in Fig. 7.

[0385] As obtainable from Fig. 7, supplementation of spermine led to a 1.3-fold decrease in large aggregates, whereas a 1.8-fold reduction was observed for the full weighing medium containing spermine. The percentage of single cells increased by 4.4 and 8.3% for the supplemented and full weighing medium, respectively. In conclusion, cell aggregation is reduced upon supplementation of spermine or its integration into a full weighing HEK medium. Again, the reduction in aggregation was observed for different HEK cells (Expi293F, VPC 2.0 and the internal HEK clone).

[0386] Example 2C: Supplementation of spermine decreases cell aggregation for further HEK medium Since a reduction in cell aggregation was observed in presence of spermine as part of an internally developed HEK medium, the effect of spermine on the aggregation of VPC2.0 cells grown in the commercial medium HEK ViP NB was tested. VPC2.0 cells were adapted to HEK ViP NB with or without 72 pM spermine for five passages at 125 mL shake flask scale. Cells were cultured for three days prior to imaging with an automated cell counter. A custom image analysis software (Fiji) script was used to quantify cell aggregation based on object size as described above. Clusters of two cells were considered small aggregates, whereas large aggregates contained three or more cells. The results are shown in Fig.8.

[0387] Fig.8 shows a 2-fold reduction in large aggregates and an increase of the percentage of single cells by 15.3%. Thus, spermine also reduces cell aggregation when added to other HEK media. Again, this highlights the consistency of the effect of spermine to reduce aggregation of the HEK cells present in suspension culture.

[0388] Conclusions

[0389] Example 2 demonstrates that spermine as supplement for different cell culture media or compound of a full-weighed cell culture medium leads to a reduction in host cell aggregation. Specifically, it was shown for different types of HEK cells that the presence of spermine throughout culture in suspension leads to an increase in single cells, as well as reduction in small and large cell aggregates. As explained above, the reduction in aggregation is highly beneficial in order to enhance oxygen and nutrient transfer to the cells but also transfection complexes, which can be restricted in aggregates, particularly in larger aggregates. The inventors hypothesize that the positive impact of spermine relies on its positive charges leading to binding of DNA and other charged molecules, thereby possibly neutralizing or shielding these. As a result, the cells are shielded from interaction triggered by the DNA or charged molecules. Other or additional affects may also play a role for the reduction in aggregation.

[0390] Example 3: Spermine dissolved cell aggregates in HEK suspension culture

[0391] Based on the surprising result that spermine reduced cell aggregation (see example 2), the inventors further tested whether spermine also has the ability to dissolve host cell aggregates of a HEK cell suspension culture in example 3. Such application is highly advantageous, as supplementing media with spermine or using media containing spermine can be used to dissolve existing cell aggregates and improve culture by increasing the accessibility of such cells of the aggregate to oxygen and nutrients. In transient production processes, dissolving existing aggregates by addition of spermine can be used to increase the accessibility of cells to transfection complexes. Hence, dissolving aggregates also has the advantage that spermine can be used as a booster to H EK suspension culture in order to improve or revitalize cells in culture present in aggregates. The results of example 3 below demonstrate that spermine can be used to dissolve aggregates of host cells of a HEK suspension culture.

[0392] To address whether an addition of spermine leads to dissociation of existing cell aggregates, VPC2.0 cells were adapted to an internally developed HEK medium without spermine for three passages at 125 mL shake flask scale. Shake flasks containing the internally developed HEK medium without spermine were inoculated with VPC2.0 cells at 2E6 VCD / mL. Initially, cells were cultured for four days, and aggregation was monitored daily. Imaging with an automated cell counter was performed daily. A custom image analysis software (ImageJ) script was used to quantify cell aggregation based on object size as described above. Clusters of two cells were considered small aggregates, whereas large aggregates contained three or more cells. Results are shown in Fig.9.

[0393] VCDs of 4.4, 7.8, 9.8, and 12.0 E6 VCD / mL were reached at days one, two, three, and four, respectively. The fraction of single cells decreased from 73.2% to 53% at day four (Fig.9). Conversely, the fraction of large aggregates more than doubled, whereas the fraction of small aggregates increased by 11.7% (Fig. 9). In conclusion, cell aggregation of the HEK cells increases throughout cultivation.

[0394] To test whether supplementation of spermine is capable of decreasing cell aggregation, 72 pM spermine were added as supplement to the culture at day four. Subsequently, cells were imaged with an automated cell counter at 0.5, 2.6, 3.6 and 4.6 h post addition. Clusters of two cells were considered small aggregates, whereas large aggregates contained three or more cells. The results are shown in Fig. 10.

[0395] Notably, the VCD was comparable for all timepoints. At 4.6 h post spermine addition, the fraction of single cells had increased by 8.4% (Fig. 10). Conversely, the fraction of large aggregates had decreased by a factor of 1.6, whereas the fraction of small aggregates had slightly decreased by 3.7% (Fig. 10). Overall, addition of spermine dissolves a fraction of cell aggregates withing 4.6 h. In example 3 it is shown that spermine not only reduces aggregation when present throughout suspension culture but that adding spermine at anytime point throughout the culturing process, e.g. cell scale-up, transfection, production, etc., aggregates of host cells can be dissolved. As a results, it is believed that the cells can be "recovered" or "revitalized" from their aggregate condition by dissolving the aggregating and again providing sufficient oxygen and nutrition to the cells, which can also reduce toxin formation. This is highly advantageous for any type of host cell culture, particularly also for improving the viral vector production.

[0396] Example 4: Use of spermine as an enhancer during transfection

[0397] In example 4, it was further tested whether spermine has positive effect when being present in a viral production method. Specifically, the use of auxiliary compounds, for example in the form of small molecule enhancers, has already been proven to be effective in increasing recombinant protein yield. One enhancer is sodium butyrate, which is involved in the modulation of condensation of specific regions of DNA in the host cell genome, thereby influencing global gene expression. Other examples of enhancers of recombinant protein expression encompass valproic acid and dimethyl sulfoxide (DMSO). The present inventors further investigate whet her spermine also has a positive impact when being added as such "enhancer" during transient transfection and / or shortly afterwards due to the believed interaction with DNA. Thus, it was tested in example 4 whether spermine was beneficial for transfection with FectoVIR-AAV or PEI-MAX during AAV2 production with the inhouse HEK clone. The cells were grown in an internally developed HEK medium containing 72 pM spermine. During transfection, additional 72 pM spermine was added in the complexation step. Analysis of the AAV2 genomic titer at 72 h post transfection revealed that an addition of spermine during transfection was neither beneficial nor detrimental for AAV2 production, independent of the transfection reagent used (Fig. 11). A slight improvement may be seen for PEI-MAX.

[0398] The experiment was repeated for two further host cells selected from HEK293 (VPC 2.0 and another internal HEK cell clone referred to as "clone 2.0") and using the inhouse developed HEK cell culture medium. For transient transfection FectoVIR-AAV was used and as a transfection enhancer, spermine, M344, as well as the combination of spermine and M344 were tested as a supplement added 4 to 6 hours after transfection. Analysis of the AAV2 genomic titer was done at 72 h post transfection by lysing the cells and measuring the genomic titer as described above. The results are shown in Figs. 12 (for VPC 2.0) and 13 (for the clone 2.0).

[0399] Fig. 12 shows the measured genomic titer (see A) and the fold change (see B) when adding spermine, M344 or a combination of both 4 to 6 hours after adding the transfection reagents to the VPC 2.0 HEK cells. Spermine clearly increases the genomic titer, indicating that spermine functions as an enhancer for transient transfection. Furthermore, also M344, as well as the combination of spermine and M344 enhances viral vector production significantly with a factor of 3.4 and 3.3, respectively. Hence, the combination of spermine and M344 lead to a similar enhancement as M344 alone, which is likely reasoned in other limitations of the system, such as for instance the used plasmid system.

[0400] Fig. 13 shows the measured genomic titer (see A) and the fold change (see B) when adding spermine, M344 or a combination of both 4 to 6 hours after adding the transfection reagents to the internal HEK cell clone 2.0. Fig. 13 shows that the genomic titer is slightly increased when adding spermine alone and increased by a factor of 1.7 when adding M344. The combination of spermine and M344 appeared to synergistically enhance the viral vector production by leading to a factor of 2.8.

[0401] Overall, the results of example 4 substantiate that spermine alone can have beneficial effects on the viral vector production when being added as an enhancer, particularly as additive during or shortly after transient transfection. While the effect is higher for some host cells (here VPC 2.0) a small increase is also observed for other cells (inhouse HEK clone and clone 2.0). M344 also enhances the viral vector production, whereas the combination of spermine and M344 can even further increase viral vector production due to synergistic effects (see Fig. 13).

[0402] Example 5: Components of the spermine synthesis pathway further increase AAV production

[0403] The cellular spermine synthesis is enzymatically controlled and tightly regulated. Putrescine and spermidine serve as precursors for spermine synthesis. Consequently, supplementing a culture medium with these components may increase cellular spermine levels and thus enhance viral vector production, e.g. by increasing the AAV titer. In addition, spermine can be converted back to spermidine by an enzyme termed spermidine / spermine-N 1-acetyltransferase (SSAT) (see Pegg, 2008, American Journal of Physiology. Endocrinology and Metabolism, E. 294(6), pp. E995-1010.). Downregulation of SSAT, e.g., by supplementation of phenylbutyrate may increase spermine levels and thereby AAV titer (see Tao et al., 2022, JCI Insight 7(13)).

[0404] To test these hypotheses, a full weighing of an internally developed HEK medium including spermine was supplemented with spermidine, phenylbutyrate, and / or additional putrescine in example 5. The inhouse HEK clone was adapted to the respective media containing 72 pM spermine at 125 ml shake flask scale for three passages. In a screening approach, the medium was supplemented with spermidine (42 pM final concentration), putrescine (1 mM final concentration), and / or phenylbutyrate (25 pM final concentration) and transfected with FectoVIR-AAV or PEI-MAX for AAV2 production. Notably, a physiological spermine:spermidine ratio was obtained by addition of 42 pM spermidine (Pegg, 2014, International Union of Biochemistry and Molecular Biology, E. 66). Lysis was performed at 72 h post transfection and genomic titer was measured by ddPCR. The results are shows in Figs. 14 (for FectoVIR-AAV) and 15 (for PEI-MAX).

[0405] As shown in Figs. 14 and 15, supplementation of 42 pM spermidine increased AAV2 titer by 1.6 or 1.9-fold compared to the control containing spermine only. Compared to supplementation of spermidine alone, combination with 1 mM putrescine did not further increase AAV2 titer. Thus, spermidine and putrescine are not interchangeable. As discussed herein, it is hypothesized that spermine has an impact on the nucleic acid, particularly DNA, level, whereas putrescine is described in the literature to possibly influence protein production (see, e.g., WO 2012 / 078270A2). The biggest increase in AAV2 titer was achieved when spermidine was combined with 25 pM phenylbutyrate. A 3- or 2-fold increase was achieved for transfection with FectoVIR-AAV and PEI- MAX, respectively (Figs. 14 and 15). Importantly, at the concentration used, phenylbutyrate was not detrimental for cell growth (data not shown). In conclusion, addition of spermine and spermidine at a physiological ratio combined with phenylbutyrate increases AAV2 genomic titer synergistically.

[0406] In conclusion, example 5 demonstrates that the addition of further compounds of the spermine synthesis pathway or compounds effecting the pathway advantageously can improve the viral vector production. Specifically, the addition of spermidine or phenylbutyrate as SSAT downregulator (in addition to spermine) significantly increases the genomic AAV titer. The combination of spermine, spermidine and phenylbutyrate appears to have a synergistic effect on viral vector production. On the other hand, 1 mM putrescine decreased the viral vector titer, which may be due to the high concentration of putrescine. It is hypothesized that such high concentration leads to an overaccumulation of positive charges which can have negative effects. Nevertheless, it is possible that also putrescine has a beneficial effect, e.g., if used at lower concentration. Without being bound to theory, based on evidence in literature describing that spermine condensates DNA (see Katz et al., "Spermine Condenses DNA, but not RNA Duplexes", Biophysical Journal, 2017, Vol. E112(l), pp. 22- 30), it is hypothesized that the spermine interacts with DNA in a manner that promotes the production of viral genomes.

[0407] Example 6: Supplementation of Phenyl butyrate and Spermidine increases AAV2 transducing titer

[0408] To test whether the observed increase in genomic titer in presence of spermine, spermidine, and phenylbutyrate also transfers to an increase in functional viral particles, a transducing titer assay was performed in example 6. Therefore, the inhouse HEK clone was adapted to a full weighing of the internally developed HEK medium containing 72 pM spermine at 125 ml shake flask scale for three passages. In a screening approach, the medium was supplemented with spermidine, or spermidine and phenylbutyrate at the indicated concentrations. Cells were transfected using a two-plasmid system and FectoVIR-AAV or PEI-MAX for AAV2 production. AAV2 was harvested from the inhouse HEK clone cells after production as described above. Adherent growing HEK293 cells, which are highly susceptible for infection by AAV2, were seeded and transduced with dilutions of the AAV2 containing harvest. Transduced cells were harvested at 72 h post transduction, and the transducing titer was calculated based on the number of cells expressing the transgene (GFP) measured by flow cytometry. The results are shown in Figs. 16 (for FectoVIR-AAV) and 17 (for PEI-MAX).

[0409] As shown in Figs. 16 and 17 and in line with the genomic titer data in example 5, the transducing titer was increased by the addition of 42 pM spermidine and further increased when spermine was combined with 25 pM phenylbutyrate, especially for FectoVIR-AAV-based transfection. Therefore, the results confirm the observations made in example 5 in that spermine pathway compounds and / or compounds effecting the pathway advantageously can improve the viral vector production. Combined with the results of example 5, the beneficial effect of spermine with spermidine and / or phenylbutyrate on viral vector production (genomic titer and transducing titer) for various HEK- based host cells and culture systems is demonstrated. Again, the combination of spermine, spermidine and phenylbutyrate even led to synergistic improvements of viral vector production.

Claims

CLAIMS1. A method for producing a viral vector, the method comprising:(a) providing a host cell selected from a HEK293 cell or a derivative thereof, and a cell culture medium for culturing the host cell;(b) contacting the host cell with the culture medium to form a suspension culture;(c) culturing the suspension culture; and(d) producing a viral vector by the host cell in the suspension culture, wherein the suspension culture of step (b), step (c), and / or step (d), preferably at least the suspension culture of step (d), comprises spermine or a salt thereof.

2. A method for reducing host cell aggregation for production of a viral vector, the method comprising:(a) providing a host cell selected from a HEK293 cell or a derivative thereof, and a cell culture medium for culturing the host cell;(b) contacting the host cell with the culture medium to form a suspension culture;(c) culturing the suspension culture; and(d) producing a viral vector by the host cell in the suspension culture, wherein the suspension culture of step (b), step (c), and / or step (d), preferably at least the suspension culture of step (d), comprises spermine or a salt thereof and wherein host cell aggregation is reduced compared to a suspension culture without spermine or a salt thereof.

3. The method according to claim 1 or 2, wherein the cell culture medium of step (a) comprises spermine or a salt thereof and / or the method further comprises providing a supplement composition comprising spermine or a salt thereof.

4. The method according to claim 3, wherein the method further comprises one or more of the following:(i) prior to step (b) the cell culture medium is contacted with the supplement composition;(ii) step (b) further comprises contacting the host cell with the culture medium and the supplement composition to form a suspension culture;(iii) prior to step (c) or (d) the suspension culture is contacted with the supplement composition; and / or(iv) in step (c) and / or step (d) the suspension culture is contacted with the supplement composition.

5. The method according to one or more of claims 1 to 4, wherein the suspension culture comprises the spermine or salt thereof: in a concentration of at least 1 pM, preferably at least 5 pM, at least 10 pM, at least 15 pM, or at least 25 pM, more preferably at least 30 pM; in a concentration of less than 1.000 pm, preferably less than 800 pM, less than 700 pM, less than 600 pM, more preferably less than 500 pM or less than 400 pM; and / or in a concentration of the range selected from 1 pM to 1.000 pM, preferably 5 pM to 800 pM, 10 pM to 700 pM, 15 pM to 600 pM, 25 pM to 500 pM, more preferably 30 pM to 400 pM or 40 pM to 250 pM.

6. The method according to one or more of claims 1 to 5, wherein the suspension culture further comprises a polyamine, preferably spermidine or a salt or cadaverine or a salt thereof, more preferably spermidine or a salt thereof, optionally, wherein the suspension culture comprises the polyamine in a concentration of at least 1 pM, preferably at least 5 pM, at least 10 pM, at least 15 pM, or at least 20 pM, more preferably at least 25 pM.

7. The method according to one or more of claims 1 to 6, wherein the suspension culture further comprises a downregulating compound capable of downregulating conversion of spermine to spermidine and / or spermidine to putrescine, preferably the downregulating compound is an inhibitor of an enzyme involved in the spermine synthesis pathway or a compound interfering with the expression of an enzyme involved in the spermine synthesis pathway, optionally the enzyme being spermidine / spermine N ^acetyltransferase 1 (SAT1) or polyamine oxidase (PAO), more preferably an inhibitor of SAT1.

8. The method according to claim 7, wherein the downregulating compound is phenylbutyrate or a salt thereof (such as sodium phenylbutyrate), or diminazene aceturate, preferably phenylbutyrate or a salt thereof, more preferably sodium phenylbutyrate, or the downregulating compound is an expression interfering nucleic acid, preferably a small interfering (siRNA) or a microRNA (miRNA), optionally, wherein the suspension culture comprises the downregulating compound, preferably an inhibitor of an enzyme involved in the spermine synthesis pathway, such as phenylbutyrate or a salt thereof, in a concentration of at least 0.1 pM, preferably at least 0.5 pM, at least 1 pM, at least 2.5 pM, or at least 5 pM, more preferably at least 10 pM.

9. The method according to one or more of claims 1 to 8, wherein the viral vector is a nonenveloped viral vector and / or a DNA viral vector, such as an adeno-associated virus (AAV).

10. The method according to one or more of claims 1 to 9, wherein the method has the following characteristics: the viral vector is a non-enveloped, DNA viral vector, preferably an adeno-associated virus (AAV);the suspension culture comprises spermine or salt thereof in a concentration of less than 500 pM, such as in a concentration of the range selected from 30 pM to 400 pM; and the host cell is modified to be configured to produce a viral vector by transiently transfecting the host cell with one or more plasm ids for viral vector production allowing for transient viral vector production; optionally, wherein the suspension culture is substantially free of dextran sulfate; optionally, no supplement composition is provided that is not substantially free of putrescine or a salt thereof.

11. A method for dissolving host cell aggregates, the method comprising:(a) providing a suspension culture comprising host cells selected from a HEK293 cell or a derivative thereof, wherein at least a fraction of the host cells is provided as cell aggregates, and a polyamine or a salt thereof, preferably spermine or a salt thereof;(b) contacting the suspension culture with the polyamine or the salt thereof, preferably spermine or a salt thereof; and(c) optionally, culturing the suspension culture, wherein the polyamine or the salt, preferably spermine or a salt thereof, thereof dissolves host cell aggregates.

12. The method according to claim 11, wherein, after contacting the suspension culture with the polyamine or a salt thereof, preferably spermine or a salt thereof, the suspension culture comprises the polyamine or the salt thereof in a concentration of at least 1 pM, preferably at least 5 pM, at least 10 pM, at least 15 pM, or at least 25 pM, more preferably at least 30 pM.

13. The method according to one or more of claims 1 to 12, wherein the host cell is modified to be configured to produce a viral vector by transiently transfecting the host cell with one or more plasmids for viral vector production allowing for transient viral vector production, optionally wherein the spermine or salt thereof is present during transient transfection and / or is added after transient transfection, preferably the transient transfection comprising:(x) combining the host cell with the one or more plasmids for viral vector production and a transfection reagent; and(y) incubating the host cell with the one or more plasmids for viral vector production and the transfection reagent, to generate a transiently transfected cell; optionally, wherein spermine or a salt thereof is present in incubation step (y) and / or is added after incubation step (y).

14. The method according to claim 13, whereinthe method further comprises having M344 or a derivative thereof present during transient transfection and / or M344 or a derivative thereof is added after transient transfection, preferably M344 or a derivative thereof is after transient transfection further; and / or further M344 ora derivative thereof is present in incubation step (y) and / or is added after incubation step (y).

15. A composition for reducing host cell aggregation for production of a viral vector, wherein the host cell is selected from a HEK293 cell or a derivative thereof, the composition comprising:(i) a cell culture medium for culturing the host cell; and(ii) spermine or a salt thereof, optionally further comprising:(iii) a polyamine, preferably spermidine or a salt thereof or cadaverine or a salt thereof, preferably spermidine or a salt thereof; and / or(iv) a downregulating compound capable of downregulating conversion of spermine to spermidine and / or spermidine to putrescine, preferably an inhibitor of an enzyme involved in the spermine synthesis pathway or a compound interfering with the expression of an enzyme involved in the spermine synthesis pathway, more preferably an inhibitor of spermidine / spermine N ^acetyltransferase 1 (SAT1) or polyamine oxidase (PAO), most preferably an inhibitor of SAT1, such as phenylbutyrate or a salt thereof.

16. A supplement composition for reducing host cell aggregation for production of a viral vector, wherein the host cells are selected from a HEK293 cell or a derivative thereof, the supplement composition comprising a polyamine or a salt thereof, preferably spermine or a salt thereof, optionally further comprising:(i) a polyamine, preferably spermidine or a salt thereof or cadaverine or a salt thereof, preferably spermidine or a salt thereof; and / or optionally, a culture medium for culturing the host cells; or(ii) a polyamine, preferably spermidine or a salt thereof; and / or a downregulating compound capable of downregulating conversion of spermine to spermidine and / or spermidine to putrescine, preferably an inhibitor of an enzyme involved in the spermine synthesis pathway, preferably an inhibitor of spermidine / spermine N ^acetyltransferase 1 (SAT1) or polyamine oxidase (PAO), more preferably an inhibitor of SAT1, most preferably phenylbutyrate or a salt thereof; optionally, a culture medium for culturing the host cells;optionally, the supplement composition is substantially free of putrescine or a salt thereof.

17. A supplement composition for dissolving host cell aggregates, wherein the host cells are selected from a HEK293 cell or a derivative thereof, the supplement composition comprising a polyamine or a salt thereof, preferably spermine or a salt thereof.

18. Spermine or a salt thereof for use for producing a viral vector by a host cell and / or for reducing host cell aggregation for production of a viral vector, wherein the host cell is selected from a HEK293 cell or a derivative thereof.

19. Polyamine or a salt thereof, preferably spermine or a salt thereof, for use for dissolving host cell aggregates, preferably for production of a viral vector, wherein the host cell is selected from aHEK293 cell or a derivative thereof.

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