Method for producing recombinant adeno-associated virus particles

Abstract

The invention relates to methods for producing preparations comprising recombinant adeno-associated virus (rAAV) particles. More particularly, the present invention relates to methods comprising a step of contacting a host cell with a polo like kinase (PLK) modulator, a cell cycle modulator preferably an Aurora kinase modulator or a FOXM1 modulator, a microtubule modulator, a histone deacetylase (HDAC) modulator, a G protein-coupled receptor (GPCR) modulator or a JAK modulator.

Description

METHODField of the inventionThe present invention relates to methods for producing preparations comprising recombinant adeno- associated virus (rAAV) particles. More particularly, the present invention relates to methods comprising a step of contacting a host cell with a polo-like kinase (PLK) modulator, a cell cycle modulator preferably an Aurora kinase modulator or a Forkhead Box M1 (FOXM1) modulator, a microtubule modulator, a histone deacetylase (HDAC) modulator, a G protein-coupled receptor (GPCR) modulator or a Janus kinase (JAK) modulator.Background to the InventionSince 2014 there has been a rapid growth and interest in the gene therapy field. The use of recombinant adeno-associated virus (rAAV) as a vector for gene delivery has recently become widespread, with over 900 pre-clinical and clinical programs underway. rAAV vectors have considerable potential for gene therapy due to their well-established safety profile, their ability to transduce many tissues in vivo and the nonpathogenic and non-integrating nature of the adeno- associated virus (AAV). In addition, capsid engineering efforts allow AAVs to more efficiency deliver to the desired tissue type and de-target away from non-desired tissues for improved safety.However, it remains difficult to obtain high quality rAAV at high yield.Production of rAAV is still quite difficult and scale-up of production to an industrial scale has been accomplished only to a limited degree. One obstacle that limits the widespread use of rAAVs in gene therapy is the high cost of these products. Inefficient manufacturing methods result in high costs of these medicines, limiting the wide-spread availability of gene therapies. Therefore, there is a need to provide improved processes that can produce higher quantities of rAAV than currently available.Summary of the InventionThe present inventors have identified compounds that increase the capacity of cells to produce AAV using a high-throughput small molecule screening strategy. The inventors used an arrayed targeted library for AAV screening (ATLAS) platform to perform a screen using a library of -3300 small molecules and identify enhancers of AAV production.Target enhancers identified clustered into 5 pathways or target classes: cell cycle modulators, GPCR modulators, HDAC modulators, JAK modulators and metabolic modulators. Targets identified clustered into 5 pathways or target classes: cell cycle modulators, GPCR modulators, HDAC modulators, JAK modulators and metabolic modulators.The top performing enhancers were re-evaluated in a dose-response manner in a suspension HEK293 cell line. After a series of studies in small, large-scale shake flasks and suspension cells in the Ambr®15 microbioreactor, the inventors identified PLK inhibitors such as HMN-214, HMN-176 and Rigosertib) Aurora kinase inhibitors such asSNS-314, SP-96 and PHA-680632) and FOXM1 inhibitors such as FDI-6 and RCM- that increased rAAV9 production in a and dose-dependent manner. The inventorsconfirmed these findings using capsid titre, vector genome quantification and a cell-based potency (viral potent titre) assay.Accordingly, in a first aspect of the invention, there is provided a method for producing a preparation comprising recombinant adeno-associated virus (rAAV) particles, wherein the method comprises a step of contacting a host cell with a polo- like kinase (PLK) modulator, a cell cycle modulator preferably an Aurora kinase modulator or a FOXM1 modulator, a microtubule modulator, a histone deacetylase (HDAC) modulator, a G protein-coupled receptor (GPCR) modulator or a JAK modulator; and a step of culturing the host cell under suitable conditions to produce the preparation.In a second aspect of the invention, there is provided a method for increasing the viral genome titre, capsid titre and / or potency of recombinant adeno-associated virus (rAAV) in a preparation, wherein the method comprises a step of contacting a host cell with a polo- like kinase (PLK) modulator, a cell cycle modulator preferably an Aurora kinase modulator or a FOXM1 modulator, a microtubule modulator, a histone deacetylase (HDAC) modulator, a G protein-coupled receptor (GPCR) modulator or a JAK modulator; and a step of culturing the host cell under suitable conditions to produce the preparation.In a third aspect of the invention, there is provided preparation comprising rAAV obtained or obtainable by the method of any one of the first or second aspects.In a fourth aspect of the invention, there is provided a method for the treatment or prevention of a disease, comprising administering the preparation of the third aspect to a subject in need thereof.In a fifth aspect of the invention, there is provided a preparation according to the third aspect, for use in a method of treating or preventing a disease.In a sixth aspect of the invention, there is provided a use of a preparation according to the third aspect for the manufacture of a medicament for treating or preventing a diseaseDescription of the FiguresFigure 1 (A-D). Compound screen identifies pathways improving AAV9 production. Fig 1 A shows aschematic overview of the high-throughput compound study to screen for increased AAV9 production. Compounds were added at the point of triple transfection using pHelper, RepCap9 and an ITR containing reporter vector. Fig. 1 B showsa small molecule screen of bioactive compounds which was performed at a 1 pM dose on HEK293 cells. AAV9 capsid titre was measured using ELISA, 3 days post triple transfection and normalized to DMSO. Top performing compounds (3o above mean DMSO controls) increasing AAV9 capsid titre yields can be clustered into 5 pathways or target classes: cell cycle modulators, GPCR, HDAC, JAK inhibitors and metabolic targets, as indicated in a clockwise direction starting from the top right of the pie chart. The top left of the pie chart indicates compounds clustering in the target class “other”. Fig. 1CTop performing AAV9 potentiators from the primary screen were examined in a confirmation study at 4 doses in HEK293 cells and data is plotted as a heatmap. Addition of HMN-214 (PLK1 inhibitor) and four microtubule inhibitors (Vindesine sulfate, Combretastatin A4, Monomethyl auristatin E (MMAE) and Ansamitocin p-3) at the time of triple transfection showed an enhancement effect on AAV9 production. Fig. 1 D shows relative AAV9 potenttitre which was also measured using the cell-based potency assay and data were plotted against capsid titre. The top performing compounds that improved both capsid titre and potent titre were HMN-214 and Combretastatin A4.Figure 2 (A-l). HMN-214, HMN-176 and Rigosertib increase AAV9 capsid titre, potency and vector genome titre in a dose dependent manner in suspension HEK293. Cells. Fig. 2A, 2B and 2C show that addition of HMN-214 (PLK1 inhibitor) at the point of triple transfection exhibited a dosedependent improvement in AAV9 production using suspension HEK293 cells which was measured as capsid titre depicted in Fig.2A, as potency depicted in Fig. 2B and vector genome titre depicted in Fig 2C. HMN-176 (an active metabolite of HMN-214 and inhibitor of PLK1) as shown in Fig. 2D, 2E and 2F which depict measurement of capsid titre, potency and vector genome respectively, and Rigosertib (a structurally different PLK1 inhibitor) as shown in Fig. 2G, 2H, 2I which depict measurement of capsid titre, potency and vector genome respectively, also, show activity in increasing AAV9 production in suspension HEK293 cells. AAV9 production was measured using 3 assays: capsid titre, cell-based potency and vector genome titre. Data are shown as means ± SD.Figure 3 (A-Y). HMN-214 increases AAV9 titre in multiple HEK293 cells and AAV serotypes. HMN-214 (1 pM) significantly improved AAV9 production in three polyclonal HEK293 cells: 293F (ThermoFisher) shown in Fig 3A, AC001 (CLS) shown in Fig. 3B and AC018 (DSMZ) shown in Fig. 3C. Two additional clonally derived cell lines from AC001 (AC001.230 depicted in Fig. 3D and AC015.55 depicted in Fig.3E) also showed approximately a 4-6-fold increase in AAV9 production. Fig. 3F to Fig. 30 HMN-214 (1 pM) significantly increased AAV genome in AAV1 , AW2, AAV23, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and Rh10 respectively relative to DMSO control. n=1-3 independent studies, n>3 technical replicates per study. Fig. 3P to Fig. 3Y. HMN-214 (1 pM) significantly increased AAV potent titre in AAV1 , AVV2, AAV23, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and Rh10 respectively and potent titre relative to DMSO control. n=1-3 independent studies, n>3 technical replicates per study. Data are shown as means ± SD. Statistical analysis was performed using Student’s t test. *P < 0.05, **P < 0.01 , ***P < 0.001 , and ****P < 0.0001 .Figure 4(A-G). HMN-214 increases AAV9 titre using a split plasmid system in Ambr15 microbioreactor. Fig.4A. Schematic overview of confirmatory studies in Ambr15 microbioreactor system for AAV9 production. A split plasmid system (one carrying ITR containing reporter with Cap9 and the second carrying Helper elements and Rep2) was introduced into HEK293 cells using PEIPro and cultured in BalanCD+L-Glutamine. HMN-214 was added at multiple times duri ng AAV9 production. A dose-dependent increase in AAV9 capsid titre as shown in Fig. 4B and vector genome titre as shown in Fig. 4C was observed up to 1 pM and titres dropped at higher doses. Fig. 4D Vg:Cap ratios remained constant up to 1 pM and then a declined at higher doses. A dose-dependent increase in host cell DNA as shown in Fig. 4E, Cap as shown in Fig. 4F and KanR as shown in Fig. 4G impurities were observed with increasing doses of HMN-214. Data are shown as means ± SD.Figure 5 (A-F). HMN-214 blunts growth rate of HEK293 cells by blocking cell cycle progression from M to G1 phase. Fig. 5A. HEK293 cells were triple transfected with a pHelper, RepCap9 and ITR containing reporter vector. Samples were treated with either DMSO or HMN-214 (1 pM) at the time oftransfection, and cell count was monitored over time. The DMSO treated condition shows a slight dip in cell count post transfection, but cells continue to grow after day 3, while the HMN-214 treated cells show a slow decline in cell number over time. Fig. 5B AAV9 capsid titre was measured from DMSO or HMN-214 treated samples on day 3 post triple transfection and plotted against cell growth rate. An anticorrelation effect was observed between the AAV9 capsid titre and cell growth rate (DMSO point is in the bottom right of the graph). A fluorescent ubiquitination-based cell cycle indicator (FUCCI) reporter cell line was used to analyze cell proliferation and cell cycle states in various treatment groups. Fig. 5C. Group triple transfected and treated with HMN-214. Fig. 5D. Group treated with HMN-214 but not transfected. Fig. 5E. Group triple transfected, but not treated with HMN-214. Fig. 5F. Group not transfected and not HMN-214 treated. Groups treated with 1 pM of HMN-214 show a reduction of cells in the M to G1 phase and increase in the G1 to S and S / G2 / M phase. Data are shown as means ± SD.Figure 6(A-F). High-throughput screening optimization and potency assay development. Cell seeding density and transfection optimization using a single vector luciferase expression cassette in Fig. 6A. suspension and Fig. 6B adherent HEK293F cells (at 3.00 cells (M / mL). The top curve corresponds to 4.0 pg / mL DNA, the second from top curve corresponds to 2.0 pg / mL DNA, the second from bottom curve corresponds to 1 .0 pg / mL DNA and the bottom curve corresponds to 0.5 pg / mL DNA). Fig. 6C Triple transfection optimization using PEIPro and TransIT-VirusGEN (TransIT). Ratios indicate molar ratios of pHelper plasmid: RepCap9 plasmid: ITR containing reporter vector. Plasmid Ratio (to each other) was a 1 :1 :1 molar ratio. Transfection Ratio (M cells: pg DNA: pL Reagent) was 1 : 1.2: 2.4 (75M cells : 90pg DNA : 180pL TransIT). Fig. 6D. A reporter stable cell line (AC006) containing a flipped mCherry, NanoLuciferase (NLuc) flanked by loxP sites was developed to measure AAV transduction potency. Fig, 6E. Upon introduction of an ITR-containing GFP-Cre expressing vector (pAX130) the mCherry-NanoLuciferase cassette is flipped and both the mCherry and NIuc signals were detected as a measure of AAV potent titre (i), compared to a non-transduced control (ii). Fig. 6F. Luminescence signal in relation to AAV capsid titre. Scale bar, 100 pm. Data are shown as means ± SD.Figure 7 (A-.F). Microtubule inhibitors increase AAV9 capsid titre and potency. Addition of HMN- 214 at the point of transfection (t=0 hr) had the most significant boost on AAV9 genome titre as shown in Fig. 7A and potent titre as shown in Fig. 7B. No significant effect of HMN-214 was observed at 4 hr and 12 hr post transfection. At 24 hr post transfection there was ~2-fold increase in AAV9 genome titre (non-significant), while a significant reduction in potent titre was observed. Pre-dosing HMN-214 prior to transfection lead to a significant reduction in AAV9 genome titre while not affecting the potent titre. Using a single transfection reporter system, addition of HMN-214 showed a dose-dependent decrease in the mCherry reporter signal with either TransIT-VirusGEN shown in Fig. 7C(i) and PEIpro shown in Fig. 7C(ii) as transfection agents, suggesting that the mechanism of AAV9 production is not enhancement of transfection efficiency. Addition of two microtubule inhibitors Combretastatin A4 showin in Fig 7D(i-ii) and Monomethyl auristatin E (MMAE) shown in Fig. 7E(iii) at the point of triple transfection exhibited a dose-dependent response in AAV9 production using suspension HEK293 cells. AAV9 production was measured using capsid titre (Fig. 7D(i), Fig. 7E(i)) and cell-based potency (Fig. 7D(ii), Fig. 7E(ii)) assays. Fig. 7F Co-dosing HMN-214 (1 pM) with a dose range of Combretastatin A4did not increase AAV9 capsid titre, suggesting no additive increase in AAV9 production can be achieved. Data are shown as means ± SD. For pairwise analysis, statistical analysis was performed using Student’s t test. Multiple comparisons were made using one-way ANOVA. *P < 0.05, **P < 0.01 , ***P < 0.001 , and ****P < 0.0001 .Figure 8(A-I). HEK293 clonal selection with improved AAV9 yields. Fig. 8A The Solentim VIPS™ single cell seeder was used to isolate clonal HEK293 cells. A two-step clonality verification was performed to identify 274 single-cell derived clones. Top performing colonies were selected based on transfection efficiency and AAV9 production using a cell-based reporter gene assay. Fig. 8B The top 12 performing clones for AAV9 production were further validated in 96 well plates. The top performing clonal cell line (AC00.1230) showed a 3-4-fold increase in AAV9 production compared to HEK293F cells as measuring using (Fig. 8C and Fig. 8D) potent and (Fig. 8E and Fig. 8f) vector genome-titre. (G, H, I) Growth rate of 293F cells was measured post thaw recovery and compared to AC001 polyclone and AC001 .230 clonal cell line. Data are shown as means ± SD.Figure 9. HMN-214 increases AAV9 titre using split two-plasmid transfection in Ambr15 microbioreactor. Fig. 9A Addition of HMN-214, 4 hr post transfection had a minimal effect on AAV9 production expressed as capsid titre shown in Fig. 9A(i), vector genome titre shown in Fig .9A(ii) and Vg:cap ratio shown in Fig. 9A(iii), while increasing host cell derived (HCD) shown in Fig.9A(iv), Cap shown in Fig. 9A(v) and KanR shown in Fig. 9b(vi) impurities. Additional redosing with HMN-214 at 24 hr shown in Fig. 9B (i to vi) and 48 hr, shown in Fig. 9C (i to vi) post transfection did not further improve AAV9 yields compared to addition at transfection only condition. Data are shown as means ± SD.Figure 10 (A-E). FUCCI reporter cell line development to study effects of HMN-214. Fig. 10A Schematic diagram of the FUCCI cell line reporter. Fluorescent ubiquitination-based cell cycle indicator (FUCCI) reporter cell line was developed by integrating the FUCCI reporter in HEK293 cells. A CBh promoter drives the expression of mK02 florescent reporter fused to Cdt1 followed by T2A and mAzami-Green florescent reporter fused to Gem1. Fig. 10B. Florescent image of FUCCI cell line showing expression of mAzami-Green and mK02 (FUCCI cell line expresses GFP and mCherry). Fig. 10C Phases of the cell cycle. Fig. 10D Flow cytometry plots showing HMN-214 reduced the fraction of cells in M / G1 while increases fraction of cells in the G1 , G1 / S and S / G2 / M phase. More specifically, a fluorescent ubiquitination-based cell cycle indicator (FUCCI) reporter cell line was used to analyze cell proliferation and cell cycle states in various treatment groups. Groups treated with 1 pM of HMN-214 show a reduction of cells in the M to G1 phase and increase in the G1 to S and S / G2 / M phase. Fig. 10E Addition of HMN-214 (in either alone or in combination with triple transfection) showed a timedependent reduction in the cells in the M / G1 phase. Data are shown as means ± SD. Bars for 0 hr on the left of each set of four bars; bars for 72 hr on the right of each set of four bars.Figure 11(A-B). PLK inhibitors increase AAV9 production. Inhibition of PLK enhances AAV9 production in adherent HEK293 cells. Fig. 11 A shows the relative AAV9 capsid titre and Fig. 11 B shows vector genome titres, which are displayed as a heatmap normalized to the DMSO control. Numbers in heatmap represent fold over the DMSO control.Figure 12 (A-B). Aurora kinase and FOXM1 inhibitors increase AAV9 production. Inhibition of Aurora kinase and F0XM1 enhanced AAV9 production in adherent HEK293 cells. Fig. 12A shows relative AAV9 capsid titre and Fig. 12B shows vector genome titres, which are displayed as a heatmap normalized to the DMSO control. Numbers in heatmap represent fold over the DMSO control.Figure 13 (A-D). Aurora kinase inhibitors increase AAV9 production. Aurora kinase inhibitors PHA- 680632 (50 nM), SP-96 (500 nM), and SNS-314 (50 nM increase AAV9 production in adherent HEK293 cells. AAV production was measured a relative capsid titre which is shown in Fig. 13A and vector genome titre which is shown in Fig. 13B, over DMSO control. Adding SNS-314 (Aurora kinase inhibitor) at the point of transfection led to a dose-dependent increase in AAV9 production in adherent HEK293 cells. AAV9 production measured as relative capsid titre which is shown in Fig. 13C and vector genome titre which is shown in Fig. 13D, over DMSO control. Data are shown as mean ± SD.Figure 14 (A-D). HMN-214 and SNS-314 increase AAV9 production. HMN-214 (at 1 pM) and SNS- 314 (50 nM) increased AAV9 production in two different HEK293 cells lines (AC230 and AC18P). AAV9 production was measured as capsid titre which is shown in Fig. 14A, and as vector genome titre which is shown in Fig. 14.B. Fig. 14C shows vector genome to capsid (Vg:Cap) ratios measured in the different treatment groups. Fig. 14D shows that HMN-214 and SNS-314 halt cells in the G2 / M phase and prevent entry to G1 phase. Data are shown as mean ± SD.Figure 15 (A-C). SNS-314, SP-96 and HMN-214 increase AAV9 in the Ambr15 microbioreactor. SNS-314 (at 50 nM), SP-96 (at 500 nM) and HMN-214 (at 1 pM) increased AAV9 production in the Ambr15 microbioreactor system. AAV9 production was measured as capsid titre shown in Fig. 15A, and as vector genome titre shown in Fig. 15B. Fig. 15C shows vector genome to capsid (Vg:Cap) ratios. Data are shown as mean ± SD.Figure 16 (A-D). SNS-314 increases AAV1 , AAV2, and AAV6 titres using split two-plasmid transfection in Ambrl 5 microbioreactor. Addition of SNS-314 (at 50 nM) increased AAV1 production as shown in Fig. 16A, AAV2 production as shown in Fig. 16B, AAV6 as shown in Fig. 16C, and AAV9 production as shown in Fig. 16D, in the Ambrl 5 microbioreactor system. AAV production for all four tested AAV serotypes was measured as vector genome titre. Data are shown as mean ± SD.Detailed Description of the InventionGeneral DefinitionsUnless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.In general, the term “comprising” is intended to mean including but not limited to. For example, the phrase a method is one “comprising” steps, the method should be interpreted to mean that the method includes those steps, but the method may comprise further steps. In some embodiments, “comprising” may be replaced by “consisting of’. The term “consisting of is intended to be limiting.The terms “protein” and “polypeptide” are used interchangeably herein and are intended to refer to a polymeric chain of amino acids of any length.The terms “nucleic acid’ “nucleic acid sequence”, “polynucleotide” and “nucleotide sequence” are used interchangeably herein, and are intended to refer to a polymeric chain of nucleotides of any length e.g. deoxyribonucleotides, ribonucleotides, or analogues thereof. For example, the polynucleotide may comprise DNA (deoxyribonucleotides) or RNA (ribonucleotides). The polynucleotide may consist of DNA. The polynucleotide may be mRNA. Since the polynucleotide may comprise RNA or DNA, all references to T (thymine) nucleotides may be replaced with U (uracil).For the purpose of this invention, in order to determine the percent identity of two sequences (such as two polynucleotide or two polypeptide sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in a first sequence for optimal alignment with a second sequence). The nucleotides or amino acids at each position are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, then the amino acids or nucleotides are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e. , % identity = number of identical positions / total number of positions in the reference sequence x 100).Typically the sequence comparison is carried out over the length of the reference sequence. For example, if the user wished to determine whether a given (“test”) sequence is 95% identical to SEQ ID NO: 1 , SEQ ID NO: 1 would be the reference sequence. To assess whether a sequence is at least 80% identical to SEQ ID NO: 1 (an example of a reference sequence), the skilled person would carry out an alignment over the length of SEQ ID NO: 1 , and identify how many positions in the test sequence were identical to those of SEQ ID NO: 1 . If at least 80% of the positions are identical, the test sequence is at least 80% identical to SEQ ID NO: 1 . If the sequence is shorter than SEQ ID NO: 1 , the gaps or missing positions should be considered to be non-identical positions.The skilled person is aware of different computer programs that are available to determine the homology or identity between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In an embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the EMBOSS Needle Pairwise Sequence Alignment tool.Herein, the term “plasmid’ is intended to refer to a nucleic acid molecule that can replicate independently of a cell chromosome. The term “plasmid’ is intended to cover circular nucleic acid molecules and linear nucleic acid molecules. Furthermore, the term “plasmid’ is intended to cover bacterial plasmids, but also cosmids, minicircles (Nehlsen, K., Broil S., Bode, J. (2006), Gene Ther. Mol. Biol., 10: 233-244; Kay, M.A., He, C.-Y, Chen, Z.-H. (2010), Nature Biotechnology, 28: 1287- 1289) and ministrings (Nafissi N, Alqawlaq S, Lee EA, Foldvari M, Spagnuolo PA, Slavcev RA. (2014), Mol Ther Nucleic 15 Acids, 3:e165). Optionally, the plasmid is a circular nucleic acid molecule. Optionally, the plasmid is a nucleic acid molecule that is of bacterial origin.The term “corresponding method’ or “equivalent method’ refers to a method that is identical to a different method, but for one feature. For example, a “corresponding method without a modulator” or “equivalent method without a modulator1’ is a method which is identical to a method of the invention, except that the same modulator is not present.The term “about’ or “around’ when referring to a value refers to that value but within a reasonable degree of scientific error. Optionally, a value is “about x” or “around x” if it is within 10%, within 5%, or within 1 % of x.In addition, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the content clearly dictates otherwise.The term “between” in relation to a pair of reference numerical values and its grammatical equivalents as used herein can include the numerical values themselves and the range of values between the reference numerical values. For example, the term “density between 2.5 - 5 M / mL” may refer to a density of 2.5 M / mL, 5M / mL, or any value falling within the range 2.5 to 5 M / mL.The singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a PLK inhibitor1’ includes two or more instances or versions of a PLK inhibitor or “an Aurora kinase inhibitor” includes two or more instances or versions of an Aurora kinase inhibitor.All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.Preparation comprising recombinant AAVA “preparation” is a solution produced by any of the methods of the present invention. Optionally, a preparation may comprise recombinant AAV. “Recombinant A A V" or “rAAV" refers to AAV particles, i.e. particles comprising an AAV genome (such as a vector genome) and an AAV capsid. The rAAV may be of human, porcine, canine, porcine, canine bovine, primate or any other animal origin. The rAAV may be preferably human. The rAAV may be of any serotype. Optionally, the rAAV may comprise a genome of one serotype and a capsid of another serotype. The AAV capsid may comprise proteins from more than one serotype, otherwise known as a pseudotyped capsid. Optionally, the rAAV may be an engineered rAAV. An engineered rAAV may be an engineered version of any rAAV serotype. Optionally, an engineered rAAV in the context of the current disclosure shall also include a vector in which a recombinant adeno-associated virus genome e.g.AAV 2 is pseudopackaged into another suitable human virus capsid such as bocavirus 1 (HBoV1) capsid. The preparation may comprise further components such as pharmaceutically acceptable excipients as discussed in more details below.A preparation comprising recombinant AAV may be obtained by any method of the present invention. In addition, or alternatively, a preparation comprising recombinant AAV may be obtainable by any method of the present invention.AAV production assayAn AAV production assay may be used to test whether certain features of the methods of the invention allow for suitable AAV production.The user provides a “reference” host (e.g. mammalian host) cell that comprises sufficient genetic material to produce recombinant AAV when cultured under conditions suitable for the production of rAAV.For example, the user provides a reference host cell comprising wild type Adenovirus 5 helper genes encoding E2A, E4 and VA RNA I and II, i.e. the adenovirus helper genes comprised within SEQ ID NO: 2. Details of the nucleic acid positions in SEQ ID NO: 2 which encode these genes are set out in more detail below under the heading “helper genes”. The reference host cell also comprises a wild type rep gene encoding Rep 40, Rep 52, Rep 68 and Rep 78 and the rep promoters p5, p19 and p40, i.e. the sequences comprised within nucleotides 200-2252 of SEQ ID NO: 1.The reference host cell also comprises a wild type cap gene operably linked to a wild type cap gene promoter comprising p5, p19 and p40, i.e. the cap gene comprised within SEQ ID NO: 1 (nucleotides 5961-8171 of SEQ ID NO: 1). The host cell further comprises a transgene flanked by two AAV2 ITRs, i.e. the ITRs comprised within nucleotides 1-145 and 4535-4679 of SEQ ID NO: 1.The user then provides a “test” host cell that is based on the reference host cell, but has a single change relating to a characteristic that the user wishes to test. For example, if the user wishes to see whether a given Rep protein was functional, the user could swap out the rep gene of the “reference host cell” and replace it with the test rep protein to provide a “test host cell”. If the user wishes to see whether a particular modulator improves viral genome titre, capsid titre and / or potency in a preparation comprising rAAV, the user could compare the viral genome titre, capsid titre and / or potency of an rAAV produced from a host cell that has not been contacted with the modulator (i.e. the “reference host cell") and a host cell that has been contacted with the same modulator (i.e. the “test host cell”).In other words, the user compares the ability of the reference host cell and the test host cell to allow for production of rAAV. To do this, the user can incubate the reference host cells and test host cells for a period of time suitable for the rAAV production to occur. The yield of rAAV produced from the reference host cell and the test host cell may then be harvested and measured using qPCR to quantify the number of vector genomes. For example, qPCR may be used to determine the number of instances of nucleic acid molecules comprising a component of the vector genome, such as a promoter sequence, that are produced in the test host cells compared to the reference host cells. Alternatively, the comparative yield of particles may be determined, for example by an anti-capsid ELISA.Host cellsThe present invention relates to methods for producing a preparation comprising recombinant adeno- associated virus (rAAV) particles, wherein a host cell is contacted with a modulator. The method may result in improved viral genome titre, capsid titre and / or potency, compared to a preparation comprising rAAV using a corresponding method without the modulator.AAV are viruses that are useful in applications such as gene therapy, and replicate in human cells. It is typical to employ a “host” cell for rAAV vector replication and packaging. Optionally, a host cell is a producer cell. The host cell may suitably be a mammalian host cell. The host cell generally comprises or is modified to comprise several different types of components for rAAV production. Thus, mammalian cells may be used to produce AAV in quantities suitable for harvesting the AAV (in a preparation comprising recombinant AAV). Cells that are suitable for production of AAV may be referred to as “host cells”. The host cells used in the methods of the invention comprise recombinant AAV. For example, the host cells may comprise AAV because they comprise sufficient genetic material for AAV to propagate and / or because they have been cultured under conditions suitable for the production of rAAV. The skilled person can easily determine whether a given cell is suitable for the production of AAV using the assay described under the heading AAV production assay by using the given cell as a “test” host cell.In one embodiment of the present invention, the host cell is a human cell. Optionally, the host cells are human kidney cells. Host cells are human cells or human kidney cells if they are derived from human cells or human kidney cells, for example the HEK293 immortalised human kidney cells should be considered both human cells and kidney cells.Optionally, the host cells are HEK293 cells, HEK293T cells, HEK293SF cells, HEK293-F cells, HEK293EBNA cells, HEK293-derived cells, CHO cells, HeLa cells, HeLa S3 cells, A549 cells, PerC6 cells, CAP cells, CAPT cells, EB66 cells, AGE1 .OR cells, C139 cells, BHK cells, COS cells, Vero cells, or other cells derived from any of these cells. In one embodiment of the present invention, the host cells are selected from the group consisting of HEK293 cells, HEK293T cells, HEK293SF cells, HEK293-F cells, HEK293EBNA cells, HEK293-derived cells, CHO cells, HeLa cells, HeLa S3 cells, A549 cells, PerC6 cells, CAP cells, CAPT cells, EB66 cells, AGE1.CR cells, C139 cells, BHK cells, COS cells, and Vero cells. In one embodiment of the present invention, the host cells are HEK293 cells, HEK293T cells, HEK293SF cells, HEK293-F cells, HEK293EBNA cells, HEK293-derived cells, CHO cells, HeLa cells, HeLa S3 cells, A549 cells, PerC6 cells, CAP cells, CAPT cells, EB66 cells, AGE1 .CR cells, C139 cells, BHK cells, COS cells, or Vero cells. According to a method of the invention, the host cell may be:(a) a HEK293 cell, a HEK293T cell, a HEK293SF cell, a HEK293-F cell, a CHO cell, a HeLa cell, a HeLa S3 cell, a HEK293EBNA cell, a CAP cell, a CAP-T cell, an AGE1 .CR cell, a PerC6 cell, a C139 cell, an EB66 cell, a BHK cell, a COS cell, a Vero cell, an A549 cell, or a K562 cell;(b) a HEK293 cell, a HEK293T cell, a HEK293SF cell or a HEK293-F cell;(c) derived from a HEK293 cell, a HEK293T cell, a HEK293SF cell, a HEK293-F cell, a CHO cell, a HeLa cell, a HeLa S3 cell, a HEK293EBNA cell, a CAP cell, a CAP-T cell, an AGE1 .CR cell, a PerC6 cell, a C139 cell, an EB66 cell, a BHK cell, a COS cell, a Vero cell or an A549 cell; or(d) derived from a HEK293 cell, a HEK293T cell, a HEK293SF cell or a HEK293-F cell.Optionally, the host cells are of a cell type that is suited to suspension or adherent cell culture or were cultured in suspension or adherent cell culture. Optionally, the host cells are of a cell type that is suited to suspension cell culture. Optionally, the host cells were cultured in suspension culture. As set out in more detail below under the heading “Cell culture” cells that are cultured in suspension culture tend to have a different morphology compared to cells that are cultured in adherent culture. In particular, cells cultured in adherent culture tend to be flatter and less rounded that cells that are cultured in suspension culture.ContactingBy “contacting” , it is to be understood that the host cell may interact with the at least one of the specified modulators in a way that still allows the host cell to produce a preparation comprising rAAV when the host cell is cultured.For example, the interaction may be a direct or indirect interaction with the host cell, such as a host cell surface component such as a receptor, a cytoplasmic component of the host cell and / or a nuclear component of the host cell such as chromatin. The interaction may alter gene expression of the cell being contacted and in doing so allows a stem cell to be obtained. The interaction with the modulator and host cell may be transient, for example in the order of minutes and / or if the effect of the modulator is irreversible. Such a transient interaction could be achieved by any suitable means, such as pulsing a host cell culture or an immobilised host cell with the modulator. Alternatively, or in addition, the interaction with the modulator may be more long term, for example in the order of hours or days, for example by longer term culture of cells in the presence of the modulator and / or if the effect of the modulator is reversible.Where two molecules are used, the molecules may be conveniently referred to as a “first” and “second” molecule. However, the use of the terms “first” and “second” does not necessarily imply an order in which a cell is contacted with each molecule. For example, a host cell may be contacted with one modulator and then the first modulator replaced with the other modulator, such that there is no overlap between the first and second modulators contacting the host cells. Alternatively, the host cell may be contacted with both modulators at the same time. Here, contact of the host cell with each of the modulators may be stopped at different times or at the same time. Therefore, by way of example, the host cell may be contacted with the second modulator and then contact with the second modulator replaced by contact with the first modulator. By way of a further example, the host cell may be contacted with the first and second modulator at the same time, and then after a suitable period of time the first modulator removed such that contact between the host cell and first modulator ceases but contact between the host cell and second modulator continues.When the host cell has been transfected with one or more polynucleotide, such as a polynucleotide comprising: (i)at least one AAV cap gene and / or at least one AAV rep gene, at least one inverted terminal repeat (ITR) and / or at least one helper gene; and / or (ii) expression cassette, wherein the expression cassette comprises a transgene between two ITRs, the host cell may be contacted with a modulator at the time of transfection, substantially at the time of transfection, or at any point after transfection. In other words, the modulator may be added when one or more of the at least one AAV cap gene and / or at least one AAV rep gene, at least one inverted terminal repeat (ITR) and / or at least one helper gene is transfected into the host cell. The host cell may be contacted with the modulator once, twice, three times, four times, five times, or six times or more. Contacting the host cell with the modulator ideally is carried out in such a way as to not prevent or unduly affect host cell replication prior to rAAV production. For example, contacting the host cell with a modulator may only take place after the host cell has been subjected to a suitable growth phase.ModulatorsThe invention relates to a method for producing a preparation comprising recombinant adeno- associated virus (rAAV) particles, wherein the method comprises a step of contacting a host cell with a modulator, and a step of culturing the host cell under suitable conditions to produce the preparation.The modulator may be any suitable molecule that alters a specified function. For example, the modulator may be any molecule that increases, decreases, delays the onset of, speeds up the onset of, shortens the effect or lengthens the effect of a specified function. In all instances where it is stated that the modulator alters a specified function, it is intended that the modulator is also capable of altering that function. By way of example, a modulator of a polo- like kinase (PLK) may increase, decrease, delay the onset of, speed up the onset of, shorten the effect or lengthen the canonical biological effect of a PLK (i.e. the modulator may be capable of increasing decreasing, delaying the onset of, speeding up the onset of, shortening the effect or lengthening the canonical biological effect of a PLK). In one preferred embodiment, the modulator may be a PLK inhibitor (PLKi). By way of another example, a modulator of cell cycle may increase, decrease, delay the onset of, speed up the onset of, shorten the effect or lengthen the canonical biological effect of cell cycle. In one preferred embodiment, the cell cycle modulator may be an Aurora kinase inhibitor or a FOXM1 inhibitor.The modulator may be a compound selected from Table 1 or Table 2 or Table 3.The modulator may function by altering the activity of its target directly, for example by binding directly to its target or altering gene expression of the target. Alternatively or additionally, the modulator may function by altering the activity of its target indirectly, for example by acting on a molecule that is upstream or downstream of its target in a signalling pathway.The modulator may comprise an organic or inorganic molecule, such as a small molecule. The modulator may comprise a CRISPR-associated protein (Cas), a nucleic acid-binding protein, a RNA- and / or DNA-binding protein, a nucleic acid, a nucleic acid analogue, gRNA, miRNA, shRNA, siRNA, piRNA or DNA. Alternatively, the modulator may comprise an antibody and / or antigen-binding variantor fragment thereof, a single chain variable fragment (scFv), VHH, a nanobody, an unnatural nucleic acid, a phosphorodiamidate morpholino oligomer (PMO) or a small molecule.In one embodiment of the invention, the modulator may alter the function of a polo- like kinase (PLK), the cell cycle such as an Aurora kinase modulator, or FOXM1 modulator a microtubule, a histone deacetylase (HDAC), a G protein-coupled receptor (GPCR) or a JAK. More preferably, the modulator may alter the function of a polo- like kinase (PLK), the cell cycle such as an Aurora kinase modulator or FOXM1 modulator, a microtubule or a histone deacetylase (HDAC). Even more preferably, the modulator may alter the function of a polo- like kinase (PLK) or a cell cycle such as an Aurora kinase modulator or FOXM1 modulator. Thus, the host cell may be contacted with a polo- like kinase (PLK) modulator, a cell cycle modulator such as an Aurora kinase modulator or FOXM1 modulator, a microtubule modulator, a histone deacetylase (HDAC) modulator, a G protein-coupled receptor (GPCR) modulator or a Janus kinase (JAK) modulator.Where the modulator increases a specified function (i.e. is an activator), the specified function may be increased by at least 1.1 times, at least 1 .5 times, at least 2 times, at least 5 times, at least 10 times, at least 25 times, at least 50 times or at least 100 times, compared to when the modulator is not present. For example, a modulator that is a PLK activator may increase kinase activity of the PLK or Aurora kinase or FOXM1 by at least 1.1 times, at least 1 .5 times, at least 2 times, at least 5 times, at least 10 times, at least 25 times, at least 50 times or at least 100 times compared to when the activator is not present.Where the modulator decreases a specified function (i.e. is an inhibitor), the specified function may be decreased to a level of at least 90% thereof, at least 80% thereof, at least 70% thereof, at least 60% thereof, at least 50% thereof, at least 40% thereof, at least 30% thereof, at least 20% thereof at least 10% thereof, at least 5% thereof, at least 1 % thereof, or substantially 0% thereof, compared to when the modulator is not present. Where the modulator decreases a specified function to substantially 0% thereof, the specified function is substantially eliminated compared to when the modulator is not present. For example, a modulator may be a PLK inhibitor. The PLK inhibitor may decrease kinase activity of the PLK to a level of at least 90% thereof, at least 80% thereof, at least 70% thereof, at least 60% thereof, at least 50% thereof, at least 40% thereof, at least 30% thereof, at least 20% thereof at least 10% thereof, at least 5% thereof, at least 1 % thereof, or substantially 0% thereof, compared to when the inhibitor is not present. As another example, a modulator may be an Aurora kinase inhibitor. The Aurora kinase inhibitor may decrease kinase activity of the Aurora kinase to a level of at least 90% thereof, at least 80% thereof, at least 70% thereof, at least 60% thereof, at least 50% thereof, at least 40% thereof, at least 30% thereof, at least 20% thereof or at least 10% , compared to when the inhibitor is not present. In an even further example, a modulator may be an FOXM1 inhibitor. The FOXM1 inhibitor may decrease kinase activity of the FOXM1 to a level of at least 90% thereof, at least 80% thereof, at least 70% thereof, at least 60% thereof, at least 50% thereof, at least 40% thereof, at least 30% thereof, at least 20% thereof or at least 10% thereof, compared to when the inhibitor is not presentWhere the modulator delays the onset of a specified function, the specified function may be delayed by at least 1.1 times, at least 1 .5 times, at least 2 times, at least 5 times, at least 10 times, at least 25times, at least 50 times or at least 100 times, compared to when the modulator is not present. Where the modulator speeds up the onset of a specified function, the specified function may be sped up by at least 1 .1 times, at least 1 .5 times, at least 2 times, at least 5 times, at least 10 times, at least 25 times, at least 50 times or at least 100 times, compared to when the modulator is not present.Where the modulator shortens the effect of a specified function, the effect may be shortened by at least 1 .1 times, at least 1 .5 times, at least 2 times, at least 5 times, at least 10 times, at least 25 times, at least 50 times or at least 100 times, compared to when the modulator is not present. Where the modulator lengthens the effect of a specified function, the effect may be lengthened by at least 1.1 times, at least 1 .5 times, at least 2 times, at least 5 times, at least 10 times, at least 25 times, at least 50 times or at least 100 times, compared to when the modulator is not present.The skilled person would readily be able to measure the specified function using assays known in the art. For example, the skilled person would be able to make use of a PLK kinase enzyme system using an ADP colorimetric assay, such as the ADP-Glo™ assay (Promega Corporation), to measure ADP formation from the PLK-catalysed kinase reaction. Using this system, ADP is converted into ATP, which is converted into light by a Luciferase such as the Ultra-Gio™ Luciferase. The luminescent signal positively correlates with ADP amount and kinase activity. Such a kinase assay is well suited for measuring the effects chemical compounds have on the activity of a broad range of kinases, including PLK and Aurora kinases, making it ideal for both primary screening as well as kinase selectivity profiling. Such an assay can be used to monitor the activity of virtually any ADP-generating enzyme (e.g., kinase or ATPase) using up to 1 mM ATP. Specifically, the skilled person could dilute enzyme, substrate, ATP, and the PLK modulator such as a PLK inhibitor or an Aurora kinase inhibitor to be tested (or a suitable control such as 5% DMSO), in a suitable kinase buffer to create a mixture that may be added to a microwell plate, such as a 96-well or 384-well plate. The mixture can then be incubated at room temperature for about 1 hour after which the luciferase reagent may be added. After further incubation for about 40 minutes, the kinase detection reagent (e.g. comprising a compatible luciferin) may be added, followed by a further incubation step of about 30 minutes in the dark. Luminescence may then be read, for example on a plate reader set to an integration time of 0.5-1 second. If various suitable concentrations of the inhibitor to be tested are used, a dose-dependent curve can be generated and ICso and EC50 values calculated accordingly. Additionally, an indicative assay to measure the function of FOXM1 is described by Lee et al in https: / / pubmed.ncbi.nlm.nih.gov / 34602556 / An IC50 value refers to a concentration value having half maximal inhibitory concentration. Thus, it is a measure of the potency of a substance in inhibiting a specific biological or biochemical function. IC50 is a quantitative measure that indicates how much of a particular inhibitory substance (e.g. inhibitory modulator) is needed to inhibit, in vitro, a given biological process or biological component by 50%.An EC50 value refers to a concentration value having half maximal effective concentration. Thus, it is a measure of the concentration of a substance (such as an activating modulator) which induces a biological response halfway between the baseline and maximum after a specified exposure time.Appropriate assays to determine the activity of other modulators, such as modulators of the cell cycle such as Aurora kinase modulators or FOXM1 modulators, a microtubule, a histone deacetylase (HDAC), a G protein-coupled receptor (GPCR) or a JAK would also be known to the skilled person. For example, a flow cytometry-based cell cycle analysis assay, an in vitro tubulin polymerization assay kit, a HDAC fluorometric assay kit, a GPCR signalling assay, or a JAK kinase assay by be selected as appropriate.The modulator may have an EC50 of 0.05nM or less, 0.1 nM or less, 1 nM or less, 5nM or less, 10nM or less, 25nM or less, 50nM or less, 100nM or less, 0.5pM or less, 1 pM or less, 10pM or less, or 50pM or less. Alternatively, or in addition, the modulator may have an IC50 of 0.05nM or less, 0.1 nM or less, 1 nM or less, 5nM or less, 10nM or less, 25nM or less, 50nM or less, 100nM or less, 0.5pM or less, 1 pM or less, 10pM or less, or 50pM or less.In one preferable embodiment, the modulator may have an EC50 of 25nM or less, 50nM or less, 100nM or less, 0.5pM or less, or 1 pM or less; and / or an IC50 of 25nM or less, 50nM or less, 100nM or less, 0.5pM or less, or 1 pM or less.When the modulator is HMN-214, the modulator may have an ICso of 0.5pM or less, preferably 150nM or less, and more preferably about 120nM.When the modulator is HMN-176, the modulator may have an IC50 of 0.5pM or less, preferably 150nM or less, and more preferably about 118nM.When the modulator is Rigosertib, the modulator may have an ICso of 150nM or less, preferably 25nM or less, and more preferably about 9nM.When the modulator is PHA-680632 the modulator may have an IC50 of 0.5pM or less, preferably 150nMWhen the modulator is SP96 the modulator may have an ICso of 0.5pM or less, preferably 0.5 nM or less,When the modulator is SNS-314 the modulator may have an ICso of 0.5pM or less, preferably 35 nM or less,When the modulator is FDI-6 the modulator may have an IC50 of 25pM or less.The concentration of the modulator used in the method of the invention is not particularly limiting and the skilled person would be able to select a suitable concentration, for example based on their common general knowledge, the prior art, and the known IC50 and / or EC50 values. By way of example, suitable concentrations for the modulator may be 1-10nM, 1-100nM, 1-500nM, 1-1000nM, 10-100nM, 10- 500nM, 10-1000nM, 100-500nM, 100-1000nM or 500-1 OOOnM. Optionally, the concentration is sufficient to modulate the specified function by least 1.1 times, at least 1.5 times, at least 2 times, at least 5 times, at least 10 times, at least 25 times, at least 50 times or at least 100 times, compared to when the modulator is not present. Optionally, the concentration is sufficient to decrease a specified function to a level of at least 90% thereof, at least 80% thereof, at least 70% thereof, at least 60%thereof, at least 50% thereof, at least 40% thereof, at least 30% thereof, at least 20% thereof at least 10% thereof, at least 5% thereof, at least 1 % thereof, or substantially 0% thereof, compared to when the modulator is not present. Where the modulator decreases a specified function to substantially 0% thereof, the specified function is substantially eliminated compared to when the modulator is not present.Where the modulator is a cell cycle modulator, the modulator may increase, decrease, delay the onset of, speed up the onset of, shorten or lengthen the G1 (Gap 1 , cell growth) to S (DNA synthesis) transition, the S to G2 (Gap 2, Growth and preparation for mitosis) transition, the G2 to M (Mitosis phase) transition, and / or the M to G1 transition. The cell cycle modulator may be selected from a cyclin family member, a cyclin dependent kinase (cdk), a cdk inhibitor, an Aurora kinase inhibitor, FOXM1 inhibitor or a pocket protein retinoblastoma (pRB) family member. In one embodiment, the cell cycle modulator may be selected from one or more of Adavosertib, Cobimetinib, Irbinitinib, Olmutinib, Venetoclax, SNS-314, PHA-680632, SP-96, FDI-6 or RCM-1.In one preferred embodiment, the modulator is a microtubule modulator. The modulator may increase, decrease, delay the onset of, speed up the onset of, shorten or lengthen the polymerisation of tubulin. The microtubule modulator may also stabilise or destabilise microtubules. In a more preferred embodiment, the microtubule modulator may be a microtubule inhibitor. The microtubule modulator may be selected from one or more of Ansamitocin p-3, Combretastatin A4, Monomethyl auristatin E (MMAE), Vinblastine sulfate, Vindesine sulfate, Docetaxel, Paclitaxel, Colchicine (NSC 757), Nocodazole, T-1101 tosylate, Trastuzumab Emtansine (T-DM1), Monomethylauristatin F (MMAF), Indibulin, Myoseverin, ABT-751 (E7010), Epothilone A, Patupilone (Epothilone B), Albendazole (SKF-62979), Albendazole Oxide, Lexibulin (CYT997), 4-Demethylepipodophyllotoxin (NSC-122819, VM-26, DMEP), Colchicine (NSC 757), Estramustine phosphate sodium, Parbendazole (SKF 29044), Ansamitocin p-3 (Maytansinol isobutyrate, NSC292222), Cabazitaxel (XRP6258), 5a- Pregnane-3, 20-dione, Trigonelline, ELR-510444, Vindoline, Griseofulvin, Triclabendazole, Vinorelbine detartrate, Vinblastine (NSC-49842) sulfate, Methylene Blue, Benproperine phosphate, KFIS101 hydrochloride, VcMMAE, Mertansine, CK-869 (CK 0157869), Fosbretabulin (Combretastatin A4 Phosphate, CA4P, CA4DP) Disodium, CW069, INH1 (IBT13131), INH6, TAI-1 , CK-636 (CK-0944636), Monomethyl auristatin E (MMAE, SGD-1010), TRx 0237 mesylate (LMTX, Hydromethylthionine), Combretastatin A4, Docetaxel (RP56976, NSC 628503), Ixabepilone (BMS-247550), SSE15206, Eribulin Mesylate (E7389), Vincristine, iHAPI, RMC-4630, EGFR Inhibitor, Plinabulin (NPI-2358), 2- Methoxyestradiol (2-MeOE2), Verubulin hydrochloride, PLK1 , PLK2, PLK3, PLK4 or PLK5. The microtubule modulator may also be selected from US 2023 / 0263783 A1 , which is hereby incorporated by reference, and in particular paragraph

[0012] of US 2023 / 0263783 A1 . Preferably, the microtubule modulator may be selected from one or more of Ansamitocin p-3, Combretastatin A4, Monomethyl auristatin E (MMAE), Vinblastine sulfate, Vindesine sulfate, Docetaxel, Paclitaxel, Colchicine (NSC 757), Nocodazole.Where the modulator is a HDAC modulator, the modulator may increase, decrease, delay the onset of, speed up the onset of, shorten or lengthen the activity of a HDAC. In one preferred embodiment, theHDAC modulator may be a HDAC inhibitor. The HDAC modulator, such as HDAC inhibitor, may modulate (and in the case of an inhibitor inhibit) one or more HDAC selected from HDAC1 , HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10 and HDAC11. In one embodiment, the HDAC modulator is selected from one or more of Abexinostat, Belinostat, CUDC- 907, Givinostat, GSK3117391 , Panobinostat, Pracinostat, Quisinostat 2HCI and Ricolinostat.Where the modulator is a GPCR modulator, the modulator may increase, decrease, delay the onset of, speed up the onset of, shorten the effect or lengthen the effect of a GPCR. The GPCR modulator may be selected from a modulator of a class A GPCR, a class B GPCR, a class C GPCR or a class F GPCR. In one embodiment, the GPCR modulator is selected from one or more of Cetirizine DiHCI, Desipramine Hydrochloride, Dexamethasone, Dexmedetomidine HCI, Dronedarone HCI, Pimavanserin, Sotagliflozin, Venlafaxine HCI and Adiphenine HCI.Where the modulator is a JAK modulator, the modulator may increase, decrease, delay the onset of, speed up the onset of, shorten the effect or lengthen the effect of a JAK. The JAK modulator may be selected from a modulator of JAK1 , JAK2, JAK3 or tyrosine kinase 2. In one embodiment, the JAK modulator is selected from one or more of Momelotinib, Oclacitinib maleate and PF-06700841 .In one preferred embodiment, the modulator is a PLK modulator, and in particular a PLK inhibitor. The PLK modulator may increase, decrease, delay the onset of, speed up the onset of, shorten or lengthen the activity of a PLK. The PLK modulator may be a modulator or inhibitor of PLK1 , PLK2, PLK3, PLK4 or PLK5; more preferably a modulator or inhibitor of PLK1 , PLK2 or PLK5; and even more preferably a modulator or inhibitor of PLK1 . The PLK modulator may be selected from one or more of HMN-214, HMN-176, Rigosertib, Onvansertib (NMS-P937), BI-2536, GSK461364, Volasertib (BI6727), Centrinone (LCR-263), (1 E)-CFI-400437 dihydrochloride, CFI-400945, Cu-CPT22, GW843682X, MLN0905, ON1231320, Poloxin, Ro3280, SBE 13 HCI and TAK-960, BI2536, TAK-960, Wortmannin, MLN0905, Poloxin, Onvansertib (NMS-P937) and CFI-400945. The PLK modulator may also be selected from US 2023 / 0263783 A1 , which is hereby incorporated by reference, and in particular paragraphs

[0013] ,

[0099] and

[0103] of US 2023 / 0263783 A1. The PLK modulator may also be selected from WO 2023 / 091932 A1 which is hereby incorporated by reference, and in particular paragraph

[0060] of WO 2023 / 091932 A1 .Preferably, the PLK modulator is selected from one or more of HMN-214, HMN-176, Rigosertib, Onvansertib (NMS-P937), BI-2536, GSK461364, Volasertib (BI6727), Centrinone (LCR-263), (1 E)- CFI-400437 dihydrochloride, CFI-400945, Cu-CPT22, GW843682X, MLN0905, ON1231320, Poloxin, Ro3280, SBE 13 HCI and TAK-960. Even more preferably, the PLK modulator is selected from one or more of:HMN-214In one preferred embodiment, the modulator is a cell cycle modulator, and in particular an Aurora kinase modulator. The Aurora kinase modulator may increase, decrease, delay the onset of, speed up the onset of, shorten or lengthen the activity of an Aurora kinase. The Aurora kinase modulator may be a modulator or inhibitor of Aurora kinase A, Aurora kinase B or Aurora kinase C; more preferably an inhibitor of Aurora kinase A, Aurora kinase B or Autora kinase C. The Aurora kinase modulator may be selected from one or more of SP600125, Alisertib, Barasertib, Tozasertib (VX-680), ZM 447439, MLN8054, Danusertib (PHA-739358), Hesperadin, MK-5108, TCS7010, AT9283, JNJ-7706621 , AMG 900, PHA-680632, KW-2449, Reversine, SNS-314, ENMD-2076, GSK1070916, CCT137690,CYC116, TAK-901 , BI-847325, CCT129202, SNS-314 Mesylate, LY3295668, SP-96, MK-8745,ENMD-2076 L-(+)-Tartaric acid, TAS-119, AKI-603, Chiauranib, H-1152 dihydrochloride, CD532, XL228, Tinengotinib,The Aurora kinase modulator may also be selected from WO 05 / 111039 A1 , which is hereby incorporated by reference. More preferably, the Aurora kinase modulator is selected from one or more of:PHA-680632In one preferred embodiment, the modulator is a cell cycle modulator, and in particular a FOXM1 modulator. The FOXM1 modulator may increase, decrease, delay the onset of, speed up the onset of,shorten or lengthen the activity of a F0XM1. The F0XM1 kinase modulator may be a modulator or inhibitor of F0XM1 ; more preferably an inhibitor of F0XM1 .More preferably, the FOXM1 modulator is selected from one or more of:FDI-6The modulator may be exogenously and / or endogenously expressed in the host cell. When endogenously expressed, the host cell may already stably or transiently express the modulator. For example, the genetic material encoding the modulator may already be present in the genome or episomally in the host cell. Alternatively, or in addition, the modulator may be exogenously loaded into the host cell, for example using any suitable exogenous loading system such as: electroporation, transfection with transfection reagents such a cationic transfection agents, lipofectamine, conjugation of modulator to a membrane anchoring moiety such as a lipid or cholesterol tail or loading by means of a cell penetrating peptide (CPP), either in the form of a CPP-cargo conjugate or in the form of a CPP-cargo non-covalent complex or any combination of these methods. Exogenous loading may result in stable or transient expression of the modulator in the host cell. Thus, by way of example, the modulator, such as one or more of PLK1 , PLK2, PLK3, PLK4, PLK5, Aurora kinase A, Aurora kinase B, Aurora kinase C or FOXM1 may be transiently, conditionally, or permanently knocked down, knocked out or underexpressed in the host cell.Cell cultureThe methods of the invention may comprise a step of culturing the host cells in cell culture medium. Optionally, the host cells are cultured under conditions suitable for rAAV production. Culturing the host cells under conditions suitable for rAAV production refers to culturing the host cell under conditions at which AAV can replicate. For example, the host cell may be cultured at a temperature between 32°C and 40°C, between 34°C and 38°C, between 35°C and 38°C, or around 37°C. Optionally, the host cell may be cultured in the presence of a complete cell culture medium such as Dulbecco’s Modified Eagle’s Medium (DMEM). A complete cell culture medium is a medium that provides all the essential nutrients required to support the host cell. Optionally, the complete cell culture medium is supplemented with serum, such as foetal bovine serum or bovine serum albumin. Optionally, the complete cell culture medium is serum-free.The term “suspension culture” refers to growing cells (such as host cells) in a system suitable for suspension cell culture, i.e. a system which allows cells to grow free-floating in culture medium. Cells in a suspension system may form aggregates or may be suspended in medium as single cells. The term “adherent culture” refers to growing cells (such as host cells) in a system suitable for adherent cell culture, i.e. for cells to be cultured whilst anchored to a substrate. Optionally, an adherent system refers to a flask or fermenter which forms a container to which cells can bind, and optionally is specifically treated to allow cell adhesion and spreading. Alternatively, an adherent system may be a “carrier system”, in which the container contains an additional carrier such as a bead or a fibre to which the cells can adhere. In such “carrier1’ adherent systems, the cells tend to adhere less tightly and to have a morphology that is more similar to the morphology of cells grown using suspension systems compared to cells grown in conventional adherent systems, for example, cells grown in a suspension system may have a more rounded morphology than adherent cells, which tend to be flatter.In another embodiment, the host cells have been cultured in suspension culture. In another embodiment, the host cells have been cultured in adherent culture. In another embodiment, the host cells have been cultured in adherent culture in a carrier system. In another embodiment, host cells have been cultured in both suspension and adherent culture.In one embodiment, the host cell is cultured for up to 2 days, up to 3 days, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days or for 2-10 days.Host cell capable of propagating AAVAs discussed above, the host cells are cells that are suitable for producing AAV. For cells to be able to propagate AAV, they should comprise genetic material encoding an AAV genome, and other genes required for AAV production and packaging to occur. The genetic material may be supplied, for example, by transfecting the host cells with genetic material comprising the AAV genome and other required genes, for example in the form of plasmids. Alternatively, the host cells may be propagated from a cell line that comprises some or all of the genetic material required, e.g. because the genetic material is comprised in the host cell genome or the genes may be supplied by infecting the host cellswith a virus. For example, the HEK293T cell line comprises an ElA gene which is an adenoviral gene that may be required for AAV production. Other methods of introducing genetic material, such as viral infection, may also be used.As set out in more detail below, the genetic material required for propagating AAV may comprise rep genes (such as AAV rep genes), cap genes (such as AAV cap genes), helper genes (such as adenoviral helper genes), and a viral genome (optionally a polynucleotide comprising two inverted terminal repeats (ITRs) and an expression cassette between the two ITRs). Optionally, the host cells comprise sufficient genetic material for the recombinant AAV to propagate. Optionally, the host cells comprise:(i) a rep 52 gene;(ii) a rep 40 gene;(iii) a rep 68 gene;(iv) a cap gene;(v) a viral associated (VA) nucleic acid;(vi) an E2a gene;(vii) an E4 gene;(viii) an ElA gene; and / or(ix) a polynucleotide comprising an expression cassette comprising a transgene between two ITRs.Accordingly, the methods of the present invention may comprise a step of transfecting the host cells with one or more plasmids or synthetic DNA molecules. In one embodiment of the present invention, the host cells have been transfected with one or more plasmids or synthetic DNA molecules. Optionally the one or more plasmids or synthetic DNA molecules comprise an AAV cap gene and / or AAV rep genes and optionally adenoviral helper genes and at least one inverted terminal repeat. In some embodiments, the helper genes may be from another AAV helper virus known in the art, e.g. HSVIn some embodiments, the synthetic DNA molecule is a doggybone or a DNA minicircle.Transfecting host cells with one or more plasmids or synthetic DNA molecules may comprise exposing the host cells to the one or more plasmids in conditions suitable for transfection. For example, the user of the method may add a transfection agent (addition of a transfection agent would be considered to be a condition suitable for transfection), such as Polyethylenimine (PEI). Alternatively, calcium phosphate transfection, electroporation or cationic liposomes could be used. Optionally, the step of transfecting the host cells takes place when the host cells have grown to confluence.Transfection may be stable or transient, i.e. cells transfected with a plasmid may stably express the genes comprised on the plasmid or may only transiently express the genes comprised on the plasmid. Furthermore, a cell may transiently express one plasmid and stably express another, e.g. a cell may be stably transfected with a plasmid comprising AAV rep and cap genes, but only transiently transfectedwith a plasmid comprising a transgene. By way of further example, the cell may be stably transfected with one or more plasmids comprising requisite genes of the helper virus (for example as discussed below in the section “Helper genes”), but only transiently transfected with a plasmid comprising a transgene.The methods of the present invention may comprise a step of infecting the host cells with one or more viruses. In one embodiment of the present invention, the host cells have been infected with one or more viruses. Optionally the one or more viruses comprise an AAV cap gene and / or AAV rep genes and optionally adenoviral helper genes and at least one inverted terminal repeat. In some embodiments, the helper genes may be from another AAV helper virus known in the art, e.g. HSVInfecting host cells with one or more viruses may comprise exposing the host cells to the one or more viruses in conditions suitable for infection.Infection may be stable or transient, i.e. cells transfected with a virus may stably express the genes comprised by the virus or may only transiently express the genes comprised by the virus. Furthermore, a cell may transiently express one gene and stably express another, e.g. a cell may stably express AAV rep and cap genes, but only transiently express a transgene.Expression cassetteAn “expression cassette” is a nucleotide sequence comprising a transgene operably linked to a transcription regulatory element (TRE). Thus, the transgene may comprise a TRE operatively linked to a polynucleotide encoding a protein.The term “transcription regulatory element’, or simply “regulatory element’ refers to a polynucleotide which can regulate the transcription of a gene to which it is operably linked. A TRE may comprise one or more promoter and / or enhancer elements. Suitable transcription regulatory elements include those disclosed in GB2109231.7, WO2021 / 084277, and WO16 / 181122, which are herein incorporated by reference.The transgene may be any suitable gene. The transgene may encode a protein or a non-translated RNA which may be, for example, an siRNA or miRNA or a snRNA or an antisense RNA. The transgene may encode a protein or a non-translated RNA which is associated with a genetic disorder. The transgene may be longer than 4,000 (4k) nucleotides, or 4,000 base pairs (4kbp). The transgene may be longer than 4.2k nucleotides. The transgene may be shorter than 4.4k nucleotides.If the preparation comprising recombinant AAV is for use in gene therapy, the transgene may be any gene that comprises or encodes a protein or nucleotide sequence that can be used to treat a disease. For example, the transgene may encode an enzyme, a metabolic protein, a signalling protein, an antibody, an antibody fragment, an antibody-like protein, an antigen, or a non-translated RNA such as a miRNA, siRNA, snRNA, or antisense RNA.At least one inverted terminal repeatIn one embodiment of the present invention, the one or more plasmids and / or the host cells comprise at least one inverted terminal repeat (ITR). Thus, optionally, the one or more plasmids and / or the host cells comprise at least one ITR, but, more typically, two ITRs (generally with one either end of the expression cassette, i.e. one at the 5’ end and one at the 3’ end). Optionally, the at least one ITR is an AAV ITR. Optionally, the at least one ITR is an AAV-derived ITR. There may be intervening sequences between the expression cassette and one or more of the ITRs. The expression cassette may be incorporated into a viral particle located between two regular ITRs or located on either side of an ITR engineered with two D regions. Optionally, the vector plasmid comprises ITR sequences which are derived from AAV1 , AAV2, AAV4 and / or AAV6.Helper genesAAV can only propagate in the presence of a helper virus, which encodes proteins that aid in AAV production. However, growing AAV in the presence of a helper virus is not advantageous as helper viruses can be lytic to cells, including host cells used to grow AAV. Furthermore, if helper viruses are used in the production of rAAV products, such as rAAV for use in gene therapy, the helper virus may contaminate the product. As an alternative to co-infecting with helper virus such as adenovirus, the requisite genes of the helper virus can be provided in the one or more plasmids and / or the host cells. Current understanding suggests that the following (adenoviral) helper genes are important for AAV replication: a (adenoviral) viral associated (VA) nucleic acid, an (adenoviral) E2a gene, an (adenoviral) E4 gene, and an (adenoviral) E1 A gene. Accordingly, the host cells and / or the one or more plasmids may comprise one or more of a viral associated (VA) nucleic acid, an E2a gene, an E4 gene and / or an E1A. One of more of these genes may be stably expressed in the cell type from which the host cells are derived. For example, if the host cells are HEK293T cells, then they will stably express the E1A gene. Suitably, the helper genes are genes corresponding to the (adenoviral) viral associated (VA) nucleic acid, the (adenoviral) E2a gene, the (adenoviral) E4 gene, and / or the (adenoviral) ElA gene from adenovirus 5. For host cells expressing the adenoviral E1 A / B genes (such as HEK293T cells) the remaining required adenoviral helper genes encode E4, E2A and VA RNA I and II.Optionally, the at least one helper virus gene is an adenovirus gene. Adenovirus is a virus which is known to aid production of AAV. Optionally, the at least one helper virus gene is an Adenovirus 5 gene or an Adenovirus 2 gene. The genome of Adenovirus 5 is set out in SEQ ID NO: 2, and the genome of Adenovirus 2 is set out in SEQ ID NO: 3. Accordingly, the helper genes may comprise a stretch of nucleotides present in SEQ ID NO: 2 or SEQ ID NO: 3, or a corresponding stretch of nucleotides in another serotype of adenovirus.The helper genes of adenoviruses encode E1 A, E1 B, E4, E2A and VA RNA I and II.E1A is encoded by nucleotides 560-1545 of the Adenovirus 5 genome (for example the genome of SEQ ID NO: 2). Nucleotides 560-1545 contain an intron, from nucleotide 1113 to nucleotide 1228. This intron is not essential, and so an ElA gene comprising nucleotides 560-1112 and 1229-1545 of SEQ ID NO: 2 would encode a functional E1 A protein.E1 B is actually two proteins E1 B 19K and E1 B 55K, which work together to block apoptosis in adenovirus-infected cells. E1 B is encoded by nucleotides 1714-2244 (E1 B 19K), and by nucleotides 2019-3509 (E1 B 55 K) of the Adenovirus 5 genome (for example the genome of SEQ ID NO: 2).E4 is encoded by a number of different open reading frames (ORFs) of the Adenovirus 5 genome (for example the genome of SEQ ID NO: 2). E4 ORF 6 / 7 is encoded by nucleotides 32914- 34077, which comprises an intron between nucleotides 33193 and 33903. This intron is not essential, and so an E4 ORF 6 / 7 comprising nucleotides 32914-33192 and 33904-34077 of SEQ ID NO: 2 is sufficient. E4 34K is encoded by nucleotides 33193-34077 of the Adenovirus 5 genome (for example the genome of SEQ ID NO: 2). E4 ORF 4 is encoded by nucleotides 33998-34342 of the Adenovirus 5 genome (for example the genome of SEQ ID NO: 2). E4 ORF 3 is encoded by nucleotides 34353- 34703 of the Adenovirus 5 genome (for example the genome of SEQ ID NO: 2). E4 ORF B is encoded by nucleotides 34700 to 35092 of the Adenovirus 5 genome (for example the genome of SEQ ID NO: 2). E4 ORF 1 is encoded by nucleotides 35140-35526 of the Adenovirus 5 genome (for example the genome of SEQ ID NO: 2).A functional E4 protein may only comprise amino acids encoded by ORFs 6 and 7, as only the amino acids encoded by ORFs 6 and 7 are required for activity. Optionally, therefore, the functional E4 protein comprises a polypeptide sequence encoded by all or a significant portion of ORFs 6 and 7. Optionally, the functional E4 protein does not comprise polypeptide sequence encoded by all or a portion of ORFs 1-4 and 34K. However, the amino acids encoded by ORFs 1-3 and 34K do improve the activity of the E4 protein, and so in some embodiments the functional E4 protein comprises amino acids encoded by ORFs 1-7.The E2 (E2A) gene is encoded by nucleotides 22443-24032 of the Adenovirus 5 genome (for example the genome of SEQ ID NO: 2).The VA RNA I and II is encoded by nucleotides 10589-11044 of the Adenovirus 5 genome (for example the genome of SEQ ID NO: 2).Rep genesThe one or more plasmids and / or host cells may comprise AAV rep genes. The host cells may comprise (i) a rep 52 gene, (ii) a rep 40 gene, and / or (iii) a rep 68 gene. AAV comprises a rep gene region which encodes four Rep proteins (Rep 78, Rep 68, Rep 52 and Rep 40). The rep gene region is under the control of the p5 and p19 promoters. When the p5 promoter is used, a gene that encodes Rep 78 and Rep 68 is transcribed. Rep 78 and Rep 68 are two alternative splice variants (Rep 78 comprises an intron that is excised in Rep 68). Similarly, when the p19 promoter is used, a gene that encodes Rep 52 and Rep 40 is transcribed. Rep 52 and Rep 40 are alternative splice variants (Rep 52 comprises an intron that is excised in Rep 40).The four Rep proteins are known to be involved in replication and packaging of the viral genome, and are, therefore, useful in rAAV production.It is not necessary for all four Rep proteins to be present. Optionally, however, the at least one rep gene encodes a large Rep protein (Rep 78 or Rep 68) and a small Rep protein (Rep 52 or Rep 40). Accordingly, the one or more plasmids and / or the host cells may comprise at least one rep gene encoding:(a) a functional (AAV) Rep 52 protein, i.e. a rep 52 gene;(b) a functional (AAV) Rep 40 protein, i.e. a rep 40 gene; and / or(c) a functional (AAV) Rep 68 protein, i.e. a rep 68 gene.For example, the one or more plasmids and / or the host cells may comprise a (AAV) rep 52 gene, a (AAV) rep 40 gene, and / or a (AAV) rep 68 gene. Optionally, the one or more plasmids and / or the host cells comprises a (AAV) rep 78 gene.A “functional" Rep protein is one which allows for production of AAV particles. In particular, Rep 78 or Rep 68 (the large Rep proteins) are believed to be involved in replication of the AAV genome, and Rep 52 and Rep 40 (the small Rep proteins) are believed to be involved in packaging of the AAV genome into a capsid. It is within the abilities of the skilled person to determine whether a given Rep protein is functional. The skilled person merely needs to determine whether the Rep protein supports AAV production using an AAV production assay for example using a “test” host cell which is identical to the “reference” host cell except that it comprises a gene encoding the Rep protein whose function is to be tested in place of the reference rep gene.In general, a Rep protein will only be able to support rAAV production if it is compatible with the ITR(s) surrounding the genome of the AAV to be packaged. Some Rep proteins may only be able to package genomic material (such as an expression cassette) when it is flanked by ITR(s) of the same serotype as the Rep protein. Other Rep proteins are cross-compatible, meaning that they can package genomic material that is flanked by ITR(s) of a different serotype. For example, it is preferred that the Rep protein is able to support replication and packaging of an expression cassette comprised within the one or more plasmids, and such a Rep protein will be compatible with the at least one ITR flanking the expression cassette (i.e. able to replicate and package the expression cassette flanked on at least one side by an ITR).Optionally, the one or more plasmids and / or the host cells comprise a gene encoding a functional (AAV) Rep 52 protein (a rep 52 gene), at least one gene encoding a functional (AAV) Rep 40 protein (a rep 40 gene), and a gene encoding a functional (AAV) Rep 68 protein (a rep 68 gene).The one or more plasmids and / or host cells may comprise two genes encoding a functional (AAV) Rep 40 protein. For example, the one or more plasmids and / or host cells may comprise two rep genes that are separated on the plasmid.SEQ ID NO: 1 provides the sequence of the genome of wild type AAV2, and nucleotides 321-2252 of SEQ ID NO: 1 encode the four Rep proteins. The full-length rep gene (nucleotides 321-2252) encodes all four Rep proteins (Rep 78 and Rep 68 from the p5 promoter and Rep 52 and Rep 40 from the p19 promoter). A shorter stretch of the rep gene downstream of the p19 promoter (nucleotides 993-2252)encodes Rep 52 and Rep 40 only ( / .e. this stretch of the rep gene reaches from the end of the p19 promoter to the end of the gene). Nucleotides 1907-2227 of SEQ ID NO: 1 correspond to an intron. Rep 78 and Rep 52 comprise amino acids encoded by the intron, but Rep 68 and Rep 40 are alternative splice variants that do not comprise amino acids encoded by the intron.Optionally, the one or more plasmids and / or the host cells comprise a gene encoding a functional Rep 52 protein (a rep 52 gene), and the rep 52 gene comprises a nucleic acid sequence having at least 95%, at least 98%, at least 99%, or 100% identity to the full length or a fragment of at least 800, at least 900, at least 1000, or at least 1100 nucleotides in length of nucleotides 993-2186 of SEQ ID NO: 1 , or to a corresponding stretch of nucleotides in a different serotype of AAV.It is within the abilities of the person skilled in the art to determine whether a particular (test) stretch of nucleotides is a “corresponding stretch of nucleotides in a different serotype of“ AAV. All that is required is that the person skilled in the art align the test stretch of nucleotides with the genome of the reference serotype ( / .e. SEQ ID NO: 1). If the test stretch of nucleotides has greater than 90% identity with a contiguous stretch of nucleotides of the same length in SEQ ID NO: 1 , the contiguous stretch is a corresponding stretch of nucleotides in a different serotype of AAV. The same applies in the case of adenovirus sequences (except here the reference serotype is SEQ ID NO: 2).Optionally, the one or more plasmids and / or the host cells comprise a gene encoding a functional Rep 40 protein (a rep 40 gene), and the rep 40 gene comprises a nucleic acid sequence having at least 95%, at least 98%, at least 99%, or 100% identity to the full length or to a fragment of at least 600, at least 700, at least 800, or at least 900 nucleotides in length of a stretch of nucleotides corresponding to nucleotides 993-2252 minus nucleotides 1907-2227 of SEQ ID NO: 1 , or to corresponding stretches of nucleotides in a different serotype of AAV. Hence, such a rep 40 gene has at least the abovespecified identity to a notional stretch of nucleotides consisting of nucleotides 993-1906 of SEQ ID NO: 1 immediately juxtaposed with nucleotides 2228-2252 of SEQ ID NO: 1 (5’-[993-1906]-[2228-2252]-3’) or to a notional stretch of nucleotides from a different AAV serotype.Optionally, the one or more plasmids and / or the host cells comprises at least one gene encoding a functional Rep 40 protein (i.e. a rep 40 gene), and the rep 40 gene comprises a nucleic acid sequence having at least 95%, at least 98%, at least 99%, or 100% identity to the full-length or to a fragment of at least 900, at least 1000, at least 1100, or at least 1200 nucleotides in length of nucleotides 993-2252 of SEQ ID NO: 1 , or to a corresponding stretch of nucleotides in a different serotype of AAV.Optionally, the one or more plasmids and / or the host cells comprise a gene encoding a functional Rep 68 protein (i.e. a rep 68 gene), and the rep 68 gene comprises a nucleic acid sequence having at least 95%, at least 98%, at least 99%, or 100% identity to the full-length or to a fragment of at least 1000, at least 1400, at least 1500, or at least 1600 nucleotides in length of a stretch of nucleotides corresponding to nucleotides 321-2252 minus nucleotides 1907-2227 of SEQ ID NO: 1 , or to corresponding stretches of nucleotides in a different serotype of AAV. Hence, such a rep 68 gene has at least the above-specified identity to a notional stretch of nucleotides consisting of nucleotides 321- 1906 of SEQ ID NO: 1 immediately juxtaposed with nucleotides 2228-2252 of SEQ ID NO: 1 (5’-[321 - 1906]-[2228-2252]-3’), or to a notional stretch of nucleotides from a different AAV serotype.Optionally, the one or more plasmids and / or host cells comprise a gene encoding functional Rep 68 and Rep 40 proteins ( / .e. a rep 68 gene and a rep 40 gene), wherein said gene comprises a nucleic acid having at least 95%, at least 98%, at least 99%, or 100% identity to the full length or to a fragment of at least 1400, 1500, 1600 or 1700 nucleotides in length of the following stretches of native AAV2 sequence (SEQ ID NO: 1) positioned in immediate juxtaposition from 5’ to 3’: 200-1906; 2228-2309, or to corresponding juxtaposed stretches of nucleotides from a different serotype of AAV.Optionally, the one or more plasmids and / or the host cells comprise a gene encoding functional Rep 52 and Rep 40 proteins, wherein said gene comprises a nucleic acid having at least 95%, at least 98%, at least 99%, or 100% identity to the full length or to a fragment of at least 1300, 1400, 1500 or 1600 nucleotides in length of the following stretch of native AAV2 sequence (SEQ ID NO: 1): 658-2300, or to a corresponding stretch of nucleotides from a different serotype of AAV.Optionally, the one or more plasmids and / or the host cells comprise a stretch of nucleotides encoding functional Rep 68, Rep 52 and Rep 40 proteins, wherein said stretch comprises a nucleic acid having at least 95%, at least 98%, at least 99%, or 100% identity to the full length or to a fragment of at least 3000, 3200, 3300 or 3400 nucleotides in length of the following stretches of native AAV2 sequence (SEQ ID NO: 1) positioned in immediate juxtaposition from 5’ to 3’: 200-1906; 2228-2309; 658-2300, or to corresponding juxtaposed stretches of nucleotides from a different serotype of AAV.Optionally, the one or more plasmids and / or the host cells do not comprise a contiguous sequence of at least 1700, at least 1800, or 1866 nucleotides corresponding to a contiguous stretch of nucleotides of equivalent length comprised within nucleotides 321-2186 of SEQ ID NO: 1 , orwithin a corresponding stretch of nucleotides in a different serotype of AAV. The contiguous stretch of nucleotides comprised within nucleotides 321-2186 corresponds to Rep 78.Cap geneThe one or more plasmids and / or the host cells may comprise a (AAV) cap gene. The cap gene encodes a functional Cap protein. The cap gene may encode a functional set of Cap proteins. AAV generally comprise three Cap proteins, VP1 , VP2 and VP3. These three proteins form a capsid into which the AAV genome is inserted, and allow the transfer of the AAV genome into a host cell. All of VP1 , VP2 and VP3 are encoded in native AAV by a single gene, the cap gene. The amino acid sequence of VP1 comprises the sequence of VP2. The portion of VP1 which does not form part of VP2 is referred to as VP1 unique or VP1 U. The amino acid sequence of VP2 comprises the sequence of VP3. The portion of VP2 which does not form part of VP3 is referred to as VP2unique or VP2U.A “functionaP set of Cap proteins is one which allows for encapsidation of AAV. It is within the abilities of the skilled person to determine whether a given Cap protein is or a set of Cap proteins are functional. The skilled person merely needs to determine whether the encoded Cap protein(s) support AAV production using an AAV production assay. The Cap protein(s) will be considered to be “functionaP if it / they support(s) rAAV production at a level at least 25%, at least 40%, at least 50%, at least 70%, at least 80%, at least 90% or at least 95% of the level supported by the wild type cap gene product, i.e.if the yield of rAAV produced is at least 25%, at least 40%, at least 50%, at least 70%, at least 80%, at least 90% or at least 95% of the yield of rAAV produced using the reference two-plasmid system. Preferably, the Cap protein(s) will be considered to be “functional’ if it / they support(s) rAAV production at a level at least 70%, at least 80%, at least 90% or at least 95% of the level supported by the wildtype cap gene product.Optionally, VP2 and / or VP3 proteins are “functional" if an AAV comprising the VP2 and / or the VP3 proteins is able to transduce host cell susceptible to infection with the AAV, e.g. Huh7 cells at a level at least 25%, at least 40%, at least 50%, at least 70%, at least 80%, at least 90% or at least 95% of that of an equivalent AAV comprising a wild type VP2 and / or VP3 protein. The ability of an AAV particle to transduce host cells, e.g. Huh7 cells can be tested by adding a reporter protein such as green fluorescent protein (GFP) to the AAV particle, mixing the AAV particle with Huh7 cells, and measuring the fluorescence produced.Optionally, the one or more plasmids and / or the host cells comprise a cap gene that encodes a VP1 , a VP2 and / or a VP3 protein. Optionally, the VP1 , VP2 and VP3 proteins are expressed from more than one cap gene. Optionally, the one or more plasmids and / or the host cells comprise a cap gene that encodes a VP1 , a VP2 and a VP3 protein. Optionally the one or more plasmids and / or the host cells comprise a cap gene encoding a functional VP1 , i.e. a VP1 protein capable of assembling with other Cap proteins to encapsidate a viral genome.Different serotypes of AAV have Cap proteins having different amino acid sequences. A cap gene encoding any (set of) Cap protein(s) is suitable for use in connection with the present invention. The Cap protein can be a native Cap protein expressed in AAV of a certain serotype. Alternatively, the Cap protein can be a non-natural, for example an engineered, Cap protein, which is designed to comprise a sequence different to that of a native AAV Cap protein. Genes encoding non-natural Cap proteins are particularly advantageous, as in the context of gene therapy applications it is possible that fewer potential patients have levels of antibodies that prevent transduction by AAV comprising non-natural Cap proteins, relative to native capsids.Optionally, the cap gene encodes a Cap protein from a serotype selected from the group consisting of serotypes 1 , 2, 3A, 3B, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13. Optionally, the cap gene encodes a Cap protein selected from the group consisting of LK03, rh74, rh10 and Mut C (WO 2016 / 181123; WO 2013 / 029030; WO 2017 / 096164, which are hereby incorporated by reference).In one embodiment of the invention, the rAAV particles comprise a capsid protein of:(a) the AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11 , AAV 12, AAV13, AAV 14, AAV 15, AAV 16, AAV.rh8, AAV.rhI O, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1 , AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1 , AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11 , AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15 or AAV.HSC16 serotype;(b) the AAV8 or AAV9 serotype;(c) the AAV8 serotype;(d) the AAV9 serotype;(e) a non-human AAV serotype or(f) an engineered AAV preferably an engineered serotype of any one of (a)-(d).Sufficient genetic materialThe minimum required genetic material for a host cell to produce rAAV, or for AAV to propagate in the host cells, when cultured under conditions suitable for rAAV production. Sufficient genetic material may comprise:(i) a rep 52 gene;(ii) a rep 40 gene;(iii) a rep 68 gene;(iv) a cap gene;(v) a VA nucleic acid;(vi) an E2a gene;(vii) an E4 gene;(viii) an ElA gene; and(ix) a polynucleotide comprising an expression cassette comprising a transgene between the two ITRs.The skilled person can easily determine if a host cell comprises sufficient genetic material for the production of rAAV by culturing the host cell under conditions suitable for the production of rAAV, e.g. as described under the heading Cell culture, and assaying for the presence of rAAV, for example by detecting the viral genome titre using qPCR, for example, as described under the heading Viral genome titre assay or detecting the capsid titre using an ELISA, for example, as described under the heading Capsid titre assay. Alternatively, a suitable AAV production assay is described under the heading AAV production assay. A host cell comprises sufficient genetic material for the production of rAAV if rAAV is detected in an AAV production assay after the host cell has been cultured under conditions suitable for the production of rAAV.The genetic material may be comprised in one or more plasmids. The genetic material may be comprised within the genome of the host cell. For example, the genome of the host cell may comprise an ElA gene.In one embodiment of the present invention, the host cells have been transfected with one or more plasmids comprising an AAV cap gene and / or AAV rep genes, and optionally adenoviral helper genes and at least one inverted terminal repeat (ITR).In another embodiment, the method or use further comprises a step of transfecting the host cells with one or more plasmids comprising an AAV cap gene and / or AAV rep genes, and optionally adenoviral helper genes and at least one inverted terminal repeat (ITR).Increased viral genome titreThe methods of the invention may result in a preparation that comprises recombinant AAV at an improved, increased or higher viral genome titre. Viral genome titre is the concentration of viral genome particles present in a preparation. If the preparation comprises rAAV, the viral genome particles will be AAV viral genome particles. Viral genome titre can be used as a measure of the yield of rAAV. A preparation comprising recombinant AAV produced by the methods of the invention may have an improved, increased or higher viral genome titre when compared to a preparation comprising recombinant AAV produced by a corresponding method, such as a corresponding method in which the host cell is not contacted with a modulator. In one embodiment, the method according to the invention produces a preparation comprising recombinant AAV having an improved, increased or higher viral genome titre. The preparation comprising recombinant AAV is the preparation that is produced by the methods of the invention. Accordingly, to determine whether a method produces a preparation comprising recombinant AAV having or an improved, increased or higher viral genome titre, the viral genome titre of the preparation should be measured once the method been completed, i.e. after contact of a host cell with a modulator and if the method or use comprises further subsequent steps once those further subsequent steps have been completed, and compared to the viral genome titre of a preparation made using an equivalent method in which the host cell is not contacted with the same modulator.In another embodiment, the method according to the invention produces a preparation comprising recombinant AAV having at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% improved, increased or higher viral genome titre when compared to a preparation comprising recombinant AAV produced by a corresponding method in which the host cell is not contacted with the relevant modulator.In another embodiment, the method according to the invention is a method for improving or increasing the viral genome titre of a preparation comprising recombinant AAV.In another embodiment, the method according to the invention is a method for improving or increasing the viral genome titre of a preparation comprising recombinant AAV by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%, at least 70%, or at least 80%, when compared to a preparation comprising recombinant AAV produced by a corresponding method in which the host cell is not contacted with the relevant modulator.Viral genome titre assayThe skilled person will understand that there are many suitable methods for determining the viral genome titre of a preparation comprising recombinant AAV. Viral genome titre may be measured using digital droplet polymerase chain reaction (ddPCR). Viral genome titre may be measured by thequantitative polymerase chain reaction (qPCR). qPCR or ddPCR may be carried out with primers specific to the viral genome. For example, if the viral genome is the AAV genome, primers specific to the AAV genome will be used. Viral genome titre may be measured using photometric quantification.The AAV viral genome assay may be based on a quantitative polymerase chain reaction (qPCR) specific for the promoter sequence of the rAAV expression cassette. In principle, the qPCR primers can be designed to bind any part of the recombinant AAV genome which is not common to wild type AAV genomes, but it is recommended against using primer template sequences very close to the ITRs as doing so can lead to an exaggerated vector genome titre measurement. Suitably, qPCR is carried out using a pair of primers that are able to amplify at least a region of the promoter of the expression cassette. Optionally, at least one of the primers is specific for (reverse and complementary to or identical to depending on whether the primer is a forward primer or a reverse primer) a region of at least 12, at least 14, at least 16, or at least 18 nucleotides of the promoter of the expression cassette. Optionally, one primer is specific for the start of the promoter (the first at least 12 nucleotides of the promoter) and the other primer is specific for a region of the expression cassette that is 150 base pairs from the binding site of the first primer. The qPCR may be performed using SYBR green or another intercalating dye that can be used for detection of the amplification product. Alternatively, the qPCR product may be detected using Taqman™ assay or similar.Cell lysate test samples may be subjected to a nuclease treatment procedure in order to remove nonpacked vector genomes prior to performing qPCR or ddPCR.“Droplet digital PCR” (ddPCR) refers to a digital PCR assay that measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions that support PCR amplification ( / .e., a plurality of such compartments). Typically, a “droplet” refers to water-in-oil droplet ( / .e., an oil droplet that may be generated by emulsifying a sample with droplet generator oil); an individual partition of the droplet digital PCR assay. A droplet supports PCR amplification of template molecule(s) using homogenous assay chemistries and workflows similar to those widely used for real-time PCR applications (Hinson et al (2011) Anal. Chem. 83:8604-8610; Pinheiro et al (2012) Anal. Chem. 84:1003-1011). A single ddPCR reaction may typically be comprised of at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 12,000, at least 14,000, at least 16,000, at least 18,000 or at least 20,000 compartments.Droplet digital PCR may be performed using any platform that performs a digital PCR assay that measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions that support PCR amplification. The strategy for droplet digital PCR may be summarized as follows: a sample is diluted and partitioned into thousands to millions of separate reaction chambers (water-in-oil droplets) so that each contains one or no copies of the nucleic acid molecule of interest. The number of positive droplets detected, which contain the target amplicon (i.e., nucleic acid molecule of interest), versus the number of negative droplets, which do not contain the target amplicon (i.e., nucleic acid molecule of interest), may be used to determine the number of copies of the nucleic acid molecule of interest that were in the original sample. Examplesof droplet digital PCR systems include the QX100™ Droplet Digital PCR System by Bio-Rad, which partitions samples containing nucleic acid template into 20,000 nanolitre-sized droplets.Droplet digital PCR may thus be used to detect a single target in a sample, for example using a single primer pair. However, ddPCR may also be used to detect two different targets in a sample, for example using two primer pairs, each primer pair hybridising to a different target, i.e., duplexing of targets. Duplexing may be extended to look at more targets in a sample, such as at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or at least 12 different targets in a sample, i.e., multiplexing of targets. Duplexing and multiplexing with ddPCR allows for improved sensitivity and precision, increased low level detection, and also the inference of the size of a nucleic acid. ddPCR may be quantitative.Increased capsid titreCapsid titre is the concentration of capsid particles present in a preparation. For example, if the preparation comprises rAAV, the capsid titre is the concentration of AAV capsid particles in the preparation. The preparation comprising recombinant AAV produced when a host cell is contacted with a modulator may have increased or increased capsid titre when compared to a preparation comprising recombinant AAV produced by a corresponding method in which the host cell is not contacted with the same modulator.In one embodiment, the method according to the invention produces a preparation comprising recombinant AAV having increased or improved capsid titre. The preparation comprising recombinant AAV is the preparation that is produced by the methods.In another embodiment, the method or use according to the invention produces a preparation comprising recombinant AAV having at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% increased or improved capsid titre when compared to a preparation comprising recombinant AAV produced by a corresponding method in which the host cell is not contacted with a modulator.In another embodiment, the method or use according to the invention is a method or use for increasing the capsid titre of a preparation comprising recombinant AAV.In another embodiment, the method or use according to the invention is a method or use for increasing the capsid titre of a preparation comprising recombinant AAV by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%, when compared to a preparation comprising recombinant AAV produced by a corresponding method in which the host cell is not contacted with the relevant modulator.In the context of the present disclosure an “improved and / or increased capsid titre” is used as an indication of “improved and / or increased genome titre”.Capsid titre assayThe skilled person will understand that there are many suitable methods for determining the capsid titre of a preparation comprising recombinant AAV. Capsid titre may be measured by an enzyme-linked immunosorbent assay (ELISA). For example, the capsid-specific ELISA may comprise exposing the rAAV preparation to an antibody that binds to the capsid protein. If, for example, the vector plasmid comprises a cap gene that encodes a capsid from an AAV2 serotype, the antibody may be an antibody that binds to the AAV2 capsid. For example, the user may coat a plate with an antibody that is specific for the capsid. The user may then pass the rAAV preparation over the surface of the plate. The capsids will bind to the antibody and be immobilised on the plate. The plate may then be washed to remove contaminants. The amount of capsids present can then be detected by addition of a detection antibody that can bind to the capsid and is conjugated to a detection agent such as streptavidin peroxidase. The amount of capsids present will be proportional to the colour change obtained when the streptavidin peroxidase is exposed to the chromogenic substrate TMB (tetramethylbenzidine). In one aspect of the present invention, the capsid titre is measured by ELISA.Increasing the potency of recombinant AAV produced during recombinant AAV productionThe present invention provides a method for increasing the potency of recombinant AAV produced during recombinant AAV production.The term “potency" refers to the ability of the recombinant AAV to transduce cells and deliver a transgene. The “potency” may be measured by transducing cells with recombinant AAV produced using the methods of the invention which comprise a transgene and determining the activity of a polypeptide encoded by the transgene (i.e. a cell-based transduction assay). For example, a user may determine the potency of recombinant AAV produced by a particular method by carrying out the particular method of the invention comprising a transgene whose activity can be measured, for example a chromogenic / fluorogenic protein such as green fluorescent protein (GFP) or a blood clotting factor such as Factor VIII. The user may then measure the potency of the recombinant AAV by transducing host cells, such as Huh7 cells, with the recombinant AAV (for example at a pre-selected multiplicity of infection) and culturing the host cell under conditions suitable for expression of the transgene to occur. The user may then isolate the protein encoded by the transgene, and test its activity. The potency assay may be an absolute potency assay.For example, a suitable chromogenic assay is as follows. The Factor VIII polypeptide is mixed with human Factor X polypeptide and Factor IXa polypeptide, thrombin, phospholipids and calcium. The thrombin activates the Factor VIII polypeptide to form Factor Villa polypeptide. The thrombin-activated Factor VIII polypeptide forms an enzymatic complex with Factor IXa polypeptide, phospholipids and calcium, which enzymatic complex can catalyse the conversion of Factor X polypeptide to Factor Xa polypeptide. The activity of the Factor Xa polypeptide can catalyse cleavage of a chromogenic substrate (e.g. SXa-11) to produce pNA. The level of pNA generated can be measured by determining colour development at 405 nm (e.g. measured by absorbance). Factor X polypeptide, and therefore Factor Xa polypeptide, is provided in excess. Therefore, the limiting factor is Factor Villa polypeptide. Thus, the level of pNA generated is proportional to the amount of the Factor Xa polypeptide generated by Factor FVIIIa polypeptide in the sample, which is proportional to the activity of Factor FVIIIapolypeptide in the sample. The activity of Factor FVIIIa polypeptide in the sample is a measure of the cofactor activity of the Factor FVIII polypeptide in the sample.For example, a suitable chromogenic assay is the BIOPHEN FVIII:C assay (Ref: 221406) manufactured by HYPHEN BioMed. The activity of the Factor VIII polypeptide may be measured using the BIOPHEN FVII I :C assay. Optionally, FVIII activity (potency) is expressed as a percentage of normal FVIII activity (e.g. as a percentage of the FVIII activity in a control which comprises human plasma from a person without haemophilia).Optionally, “potency" of recombinant AAV is measured by transducing host cells with the recombinant AAV, culturing the host cells and measuring the activity of a polypeptide produced by the host cells, wherein the recombinant AAV comprise a transgene and the polypeptide is encoded by the transgene. Optionally, potency of recombinant AAV is measured by transducing Huh7 cells with the recombinant AAV, culturing the Huh7 cells and measuring the activity of a polypeptide produced by the host cells, wherein the recombinant AAV comprise a transgene and the polypeptide is encoded by the transgene. Optionally, the polypeptide whose activity is measured is Factor VIII, and the activity of the Factor VIII is measured using a chromogenic assay.The present invention also provides a method for producing a recombinant AAV preparation having higher potency than the potency obtained using an equivalent method in which the host cell is not contacted with a modulator.Optionally, the higher potency is at least 1.1 fold, at least 1.15 fold, between 1.1 fold and 5 fold, or between 1 .15 fold and 3 fold higher than the potency of recombinant AAV obtained using said equivalent method. Optionally, the higher potency is between 1.15 fold and 3 fold higher than the potency of recombinant AAV obtained using said equivalent method. Optionally, the higher potency is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80% higher than the potency of recombinant AAV obtained using said equivalent method.Endonuclease treatmentAn endonuclease is an enzyme capable of breaking down polynucleotides in a preparation. Endonuclease treatment may be used to remove unpackaged viral genomes or host cell DNA impurities. Endonuclease treatment therefore improves the purity of a preparation comprising rAAV. The skilled person would understand that there are many suitable endonuclease enzymes which could be used in a step of endonuclease treatment. Suitably, the endonuclease enzyme is denarase™. Suitably, the endonuclease enzyme is benzonase™. Suitably, the endonuclease enzyme is Turbonuclease™. The skilled person would understand that there are many suitable methods of performing endonuclease treatment.Optionally, a preparation comprising recombinant AAV may be treated with endonuclease at 20 units / ml (U / ml). Optionally, the treatment comprises the step of incubating the preparation comprising recombinant AAV with the endonuclease. Optionally, the step of incubation lasts for around 1 hour, around 2 hours, around 3 hours, around 4 hours, around 5 hours, around 6 hours, around 7 hours, around 8 hours, around 9 hours, around 10 hours, around 11 hours, around 12 hours, around 13 hours,around 14 hours, around 15 hours, around 16 hours, around 17 hours, around 18 hours, around 19 hours, around 20 hours, around 21 hours, around 22 hours, around 23 hours, around 24 hours or overnight. Suitably, the endonuclease incubation step is performed at room temperature, e.g. a temperature of around 18°C to around 25°C.The methods according to the present invention may further comprise a step of endonuclease treatment.Depth filtrationThe methods according to the present invention may further comprise a step of depth filtration. Depth filtration comprises passing a preparation through a depth filter at a particular flux in order to remove impurities such as host cell debris. Removing impurities may increase the concentration of rAAV in a preparation, and thus increase the viral genome and capsid titres. In addition to removing impurities depth filtration of the preparation will improve the purity / quality of the preparation.The skilled person would understand that there are many commercially available depth filters. Depth filters have a micron rating, which describes the range of particle sizes which may pass through the depth filter and will therefore be retained in the preparation after performing the step of depth filtration. For example, a depth filter may have a micron rating of 0.2 - 1.1 pm, meaning that particles of a size between 0.2 and 1.1 pm will pass through the filter, and larger particles will not. Depth filters may comprise an organic filter and an inorganic filter aid. For example, the organic filter may comprise cellulose fibres, and the inorganic filter aid may comprise a perlite or resin or diatomaceous earth. A suitable depth filter is a COHC filter. Suitably, the depth filtration step is performed at room temperature, e.g. a temperature of around 18°C to around 25°C.The “flux” at which depth filtration occurs is a measure of the speed at which the preparation passes through the depth filter. Higher flux means that the preparation is passing through the depth filter at an increased rate. The skilled person can easily determine the flux at which depth filtration is performed according to the following equation:Volume (L)Flux (LMH) = - — - - - - rTime (h) * Surface area (cm2) wherein the volume is the total preparation volume and the surface area is the surface area of the filter.The depth filtration may be performed at a flux of between 100 and 600 LMH, between 200 and 400 LMH, between 250 and 350 LMH, or around 300 LMH.The step of depth filtration may use a filter which is an organic filter. Optionally, the filter comprises cellulose fibres and an inorganic filter aid. Optionally, the filter used in the depth filtration has a micron rating falling within the range of 0.1 to 10pm. Optionally, the filter has a micron rating of 0.2 to 1.1 m. Optionally, the filter is a COHC filter.The step of depth filtration may increase the viral genome titre of the preparation comprising recombinant AAV.Viral genome / capsid ratioThe viral genome / capsid ratio (as measured as a percentage of the total number of particles that are full particles) may be determined using qPCR to determine the number of vector genomes (as discussed in the section headed Viral genome titre assay), and using a capsid-specific ELISA to measure the total number of particles (as discussed in the section headed Capsid titre assay). Optionally, the viral genome / capsid ratio may be calculated by dividing the viral genome titre as measured by qPCR by the capsid titre as measured by ELISA and multiplying the result by 100%, wherein the units, in which the viral genome titre and the capsid titre are expressed, are the same.Methods of the present invention may increase capsid titre more than the viral genome titre, resulting in a reduced viral genome / capsid ratio. In one embodiment, the methods of the present invention do not significantly reduce the viral genome / capsid ratio of the preparation comprising recombinant AAV when compared to a corresponding in which the host cell is not contacted with a modulator.In some cases the methods of the present invention may reduce the viral genome / capsid ratio of the preparation comprising recombinant AAV by no more than 10% when compared to a corresponding method in which the host cell is not contacted with a modulator.In some cases, the methods of the present invention may reduce the viral genome / capsid ratio of the preparation comprising recombinant AAV by no more than 15% when compared to a corresponding method in which the host cell is not contacted with a modulator.The step of depth filtration may increase the viral genome / capsid ratio of the preparation comprising recombinant AAV.The preparation comprising recombinant AAV may have an increased viral genome / capsid ratio when compared to a preparation comprising recombinant AAV produced by a corresponding method or use not comprising a step of performing depth filtration.The step of depth filtration may increase the viral genome / capsid ratio of the preparation comprising recombinant AAV by at least 1 %, at least 2%, at least 3%, or at least 4%.In one embodiment of the invention, the viral genome to capsid ratio of the preparation comprising rAAV may be increased by at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20% or at least 25%.Reduced turbidityTurbidity is a measure of the opacity of a solution. Impurities, such as cell debris or precipitated macromolecules, may increase turbidity by more than correctly folded proteins in solution e.g. AAV capsids. The turbidity of a preparation may therefore be used as an indirect measure of the impuritiespresent in a preparation comprising recombinant AAV. It is often advantageous to reduce the turbidity of a preparation comprising recombinant AAV. The skilled person would understand that there are many methods to measure the turbidity of a preparation. For example, turbidity may be measured using the TL2360 (Hach) device. A suitable method for measuring the turbidity of a preparation comprising recombinant AAV is set out in the Examples.The preparation of the invention comprising recombinant AAV may have reduced turbidity, for example compared to a preparation comprising recombinant AAV produced by a method not comprising the step of depth filtration. The preparation of the invention comprising recombinant AAV may have a turbidity of less than 50 Nephelometric Turbidity Units (NTU), less than 40 NTU, less than 30 NTU, or less than 20 NTU.Sterile filtrationThe methods of the invention may further comprise a step of sterile filtration. Sterile filtration comprises the step of passing a preparation through a sterile filter, optionally a bottle-top filter. A filter used in sterile filter has a micron rating, meaning that only particles which are smaller than said rating may pass through the filter, while larger particles will be retained. Optionally, the sterile filtration is performed using a 0.22pm filter. Optionally, the step of sterile filtration reduces the turbidity of the preparation comprising recombinant AAV to 5 NTU or lower. Optionally, the step of sterile filtration reduces the turbidity of the preparation comprising recombinant AAV to 3 NTU or lower. Optionally, the step of sterile filtration reduces the turbidity of the preparation comprising recombinant AAV to 1 NTU or lower.Purifying the recombinant AAVThe method of the invention may further comprise a step of isolating and / or purifying the rAAV. In general, a step of isolating and / or purifying the rAAV will involve increasing the concentration of the rAAV compared to other components of the preparation. Optionally, the step of isolating and / or purifying the rAAV results in a concentrated rAAV preparation. Optionally, the step of purifying the rAAV results in an isolated rAAV, or substantially isolated rAAV.Any suitable isolation or purification method may be used. Optionally, the step of purifying the rAAV is carried out using a technique selected from the group consisting of gradient density centrifugation (such as CsCI or lodixanol gradient density centrifugation), filtration, ion exchange chromatography, size exclusion chromatography, affinity chromatography and hydrophobic interaction chromatography or a combination of some or all of these techniques. Optionally, the method comprises further concentrating the rAAV using ultracentrifugation, tangential flow filtration, or gel filtration.In one embodiment, the method or use further comprises a step of purifying and / or isolating the rAAV. Purification and / or isolation may take place after the step of endonuclease treatment, depth filtration, reducing turbidity and / or sterile filtration may be used to increase the purity of the rAAV. Alternatively, endonuclease treatment, depth filtration, reducing turbidity and / or sterile filtration may itself be used to increase the purity of the rAAV.Pharmaceutical compositionsThe methods of the invention may further comprise the step of formulating the preparation comprising recombinant AAV with a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipients may comprise carriers, diluents and / or other medicinal agents, pharmaceutical agents or adjuvants, etc. Optionally, the pharmaceutically acceptable excipients comprise saline solution. Optionally, the pharmaceutically acceptable excipients comprise human serum albumin. Optionally, the preparation is suitable for human administration.In one aspect of the present invention, the method further comprises a step of formulating the preparation comprising recombinant AAV with a pharmaceutically acceptable excipient.In one aspect, the present invention provides a pharmaceutical composition or preparation comprising recombinant AAV obtained or obtainable by the methods of the present invention.Methods of treatmentFurther provided is a method for the treatment or prevention of a disease, comprising administering the preparation or pharmaceutical composition of the invention to a subject in need thereof. Further provided is the preparation or the pharmaceutical composition of the invention for use in a method of treating or preventing a disease. Further provided is the preparation or the pharmaceutical composition of the invention for use in the manufacture of a medicament for use in a method of treating or preventing a disease.Optionally, the method of treating or preventing a disease comprises administering an effective amount of the preparation or pharmaceutical composition of the invention to a patient. Optionally, the patient suffers from a genetic disorder. Optionally, the disease is a genetic disorder. For the purposes of the present invention, a genetic disorder is any disorder associated with a mutation in a gene. Optionally, the genetic disorder is a genetic disorder that can be treated by gene therapy, for example using AAV as a gene therapy vector.Based on the disclosure provided herein, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following embodiments (E).Specifically, the present disclosure provides the following aspects, advantageous features and specific embodiments, respectively alone or in combination:E1 . A method for producing a preparation comprising recombinant adeno-associated virus (rAAV) particles, wherein the method comprises a step of contacting a host cell with a polo- like kinase (PLK) modulator, a cell cycle modulator preferably an Aurora kinase modulator or a Forkhead box M1 (FOXM1) modulator, a microtubule modulator, a histone deacetylase (HDAC) modulator, a G protein- coupled receptor (GPCR) modulator or a JAK modulator; and a step of culturing the host cell under suitable conditions to produce the preparation.E2. A method according to E1 for producing a preparation comprising recombinant adeno-associated virus (rAAV) particles, wherein the method comprises a step of contacting a host cell with a polo- like kinase (PLK) modulator and a step of culturing the host cell under suitable conditions to produce the preparation.E3. A method according to E1 for producing a preparation comprising recombinant adeno-associated virus (rAAV) particles, wherein the method comprises a step of contacting a host cell with a cell cycle modulator preferably an Aurora kinase modulator or a Forkhead box M1 (FOXM1) modulator; and a step of culturing the host cell under suitable conditions to produce the preparation.E4. A method according to E1 for producing a preparation comprising recombinant adeno-associated virus (rAAV) particles, wherein the method comprises a step of contacting a host cell with an Aurora kinase modulator; and a step of culturing the host cell under suitable conditions to produce the preparation.E5. A method according to E1 for producing a preparation comprising recombinant adeno-associated virus (rAAV) particles, wherein the method comprises a step of contacting a host cell with a Forkhead box M1 (FOXM1) modulator; and a step of culturing the host cell under suitable conditions to produce the preparation.E6. A method according to E1 for producing a preparation comprising recombinant adeno-associated virus (rAAV) particles, wherein the method comprises a step of contacting a host cell with a microtubule modulator, and a step of culturing the host cell under suitable conditions to produce the preparation.E7. A method according to E1 to E6 for producing a preparation comprising recombinant adeno- associated virus (rAAV) particles, wherein the method comprises a step of contacting a host cell with a polo- like kinase (PLK) modulator and / or a cell cycle modulator preferably an Aurora kinase modulator or a Forkhead box M1 (FOXM1) modulator and / or a microtubule modulator, a histone deacetylase (HDAC) modulator, a G protein-coupled receptor (GPCR) modulator or a JAK modulator, preferably contacting a host cell with a polo- like kinase (PLK) modulator and / or a cell cycle modulator preferably an Aurora kinase modulator or a Forkhead box M1 (FOXM1) modulator and / or a microtubule modulator; and a step of culturing the host cell under suitable conditions to produce the preparation.E8. A method according to any one of E1 to E7, wherein the method is a method for producing a preparation comprising rAAV having improved viral genome titre, capsid titre and / or potency compared to an rAAV using a corresponding method without the modulator.E9. A method for increasing the viral genome titre, capsid titre and / or potency of recombinant adeno- associated virus (rAAV) in a preparation, wherein the method comprises a step of contacting a host cell with a polo- like kinase (PLK) modulator, a cell cycle modulator preferably an Aurora kinase modulator or a FOXM1 modulator, a microtubule modulator, a histone deacetylase (HDAC) modulator, a G protein-coupled receptor (GPCR) modulator or a JAK modulator; and a step of culturing the host cell under suitable conditions to produce the preparation.E10. A method according to E9 for increasing the viral genome titre, capsid titre and / or potency of recombinant adeno-associated virus (rAAV) in a preparation, wherein the method comprises a step of contacting a host cell with a polo- like kinase (PLK) modulator; and a step of culturing the host cell under suitable conditions to produce the preparation.E11. A method according to E9 for increasing the viral genome titre, capsid titre and / or potency of recombinant adeno-associated virus (rAAV) in a preparation, wherein the method comprises a step of contacting a host cell with a cell cycle modulator preferably an Aurora kinase modulator or a FOXM1 modulator; and a step of culturing the host cell under suitable conditions to produce the preparation.E12. A method according to E9 for increasing the viral genome titre, capsid titre and / or potency of recombinant adeno-associated virus (rAAV) in a preparation, wherein the method comprises a step of contacting a host cell with an Aurora kinase modulator; and a step of culturing the host cell under suitable conditions to produce the preparation.E13. A method according to E9 for increasing the viral genome titre, capsid titre and / or potency of recombinant adeno-associated virus (rAAV) in a preparation, wherein the method comprises a step of contacting a host cell with a FOXM1 modulator; and a step of culturing the host cell under suitable conditions to produce the preparation.E14. A method according to E9 for increasing the viral genome titre, capsid titre and / or potency of recombinant adeno-associated virus (rAAV) in a preparation, wherein the method comprises a step of contacting a host cell with a microtubule modulator; and a step of culturing the host cell under suitable conditions to produce the preparation.E15. A method according to E9 for increasing the viral genome titre, capsid titre and / or potency of recombinant adeno-associated virus (rAAV) in a preparation, wherein the method comprises a step of contacting a host cell with a polo- like kinase (PLK) modulator, and / or a cell cycle modulator preferably an Aurora kinase modulator or a FOXM1 modulator, and / or a microtubule modulator, and / or a histone deacetylase (HDAC) modulator, and / or a G protein-coupled receptor (GPCR) modulator and / or a JAK modulator, preferably contacting a host cell with a polo- like kinase (PLK) modulator, and / or a cell cycle modulator preferably an Aurora kinase modulator or a FOXM1 modulator, and / or a microtubule modulator; and a step of culturing the host cell under suitable conditions to produce the preparation.E16. The method of any one of E9 to E15, wherein the viral genome titre, capsid titre and / or potency is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or at least 60%.E17. The method of any one of E9 to E16, wherein the viral genome titre is measured by qPCR or ddPCR; and / or the capsid titre is measured by an immunoassay preferably ELISA or a sandwich immunometric assay; and / or the potency is measured by a cell-based transduction assay.E18. The method of any one of E1 to E17, wherein the method comprises a step of contacting a host cell with a polo- like kinase (PLK) modulator, a cell cycle modulator preferably an Aurora kinase modulator or a FOXM1 modulator, a microtubule modulator or a histone deacetylase (HDAC) modulator.E19. The method of any one of E1 to E18, wherein the method comprises a step of contacting a host cell with a polo- like kinase (PLK) modulator, a cell cycle modulator preferably an Aurora kinase modulator or a FOXM1 modulator, or a microtubule modulator.E20. The method of any one of E1 to E19, wherein the method comprises a step of contacting a host cell with a polo- like kinase (PLK) modulator, and / or a cell cycle modulator preferably an Aurora kinase modulator and / or a FOXM1 modulator, and / or a microtubule modulator.E21 . The method of any one of E1 to E19, wherein the method comprises a step of contacting the host cell with a PLK modulator and / or an Aurora kinase modulator and / or a FOXM1 modulator.E22. The method of any one of E1 to E21 , wherein:(a) the PLK modulator is a PLK inhibitor;(b) the cell cycle modulator is a modulator and / or inhibitor of the G1 (Gap 1 , cell growth) to S (DNA synthesis) transition, the S to G2 (Gap 2, Growth and preparation for mitosis) transition, the G2 to M (Mitosis phase) transition, and / or the M to G1 transition, preferably an Aurora kinase inhibitor or FOXM1 inhibitor;(c) the microtubule modulator is a microtubule inhibitor; or(d) the HDAC modulator is a HDAC inhibitor.E23. The method of any one of E1 to E22, wherein:(a) the PLK modulator is a PLK inhibitor;(b) the cell cycle modulator is a modulator and / or inhibitor of the G1 (Gap 1 , cell growth) to S (DNA synthesis) transition, the S to G2 (Gap 2, Growth and preparation for mitosis) transition, the G2 to M (Mitosis phase) transition, and / or the M to G1 transition, preferably an Aurora kinase inhibitor or FOXM1 inhibitor; or(c) the microtubule modulator is a microtubule inhibitor;E24. The method of any one of E1 to E23, wherein the modulator:(a) has an EC50 of 0.05nM or less, 0.1 nM or less, 1 nM or less, 5nM or less, 10nM or less, 25nM or less, 50nM or less, 100nM or less, 0.5pM or less, 1 pM or less, 10pM or less, or 50pM or less; and / or(b) has an IC50 of 0.05nM or less, 0.1 nM or less, 1 nM or less, 5nM or less, 10nM or less, 25nM or less, 50nM or less, 10OnM or less, 0.5pM or less, 1 pM or less, 1 OpM or less, or 50pM or less.E25. The method of any one of E1 to E24, wherein the modulator:(a) has an EC50 of 25nM or less, 50nM or less, 100nM or less, 0.5pM or less, or 1 pM or less; and / or(b) has an IC50 of 25nM or less, 50nM or less, 10OnM or less, 0.5pM or less, or 1 pM or less.E26. The method of any one of E1 to E20, wherein:(a) the PLK modulator is selected from a modulator of PLK1 , PLK2, PLK3, PLK4 or PLK5; a modulator of PLK1 , PLK2 or PLK5; or a modulator of PLK1 ;(b) the cell cycle modulator is selected from a modulator of a cyclin family, a cyclin dependent kinase (cdk), a cdk inhibitor, an Aurora kinase A, an Aurora kinase B, an Aurora kinase C FOXM1 or a pocket protein retinoblastoma (pRB) family;(c) the HDAC modulator is selected from a modulator of HDAC1-11 ;(d) the GPCR modulator is selected from a modulator of a class A GPCR, a class B GPCR, a class C GPCR or a class F GPCR; or(e) the JAK modulator is selected from a modulator of JAK1 , JAK2, JAK3 or tyrosine kinase 2.E27. The method of any one of E1 to E26, wherein the modulator is selected from:(a) a CRISPR-associated protein (Cas), a nucleic acid-binding protein, a RNA- and / or DNA-binding protein, a nucleic acid, a nucleic acid analogue, gRNA, miRNA, shRNA, siRNA, piRNA or DNA; or(b) an antibody and / or antigen-binding variant or fragment thereof, a single chain variable fragment (scFv), VHH, a nanobody, an unnatural nucleic acid, a PMO or a small molecule.E28. The method of any one of E1 to E27, wherein:(a) the modulator is exogenously or endogenously expressed in the host cell;(b) the modulator is transiently or stably expressed in the host cell; and / or(c) one or more of PLK1 , PLK2, PLK3, PLK4 and PLK5 is transiently, conditionally, or permanently knocked down, knocked out or underexpressed in the host cell or one or more of Aurora kinase A, Aurora kinase B, Aurora kinase C or FOXM1 is transiently, conditionally, or permanently knocked down, knocked out or underexpressed in the host cellE29. The method of any one of E1 to E28 wherein:(a) the PLK modulator is selected from HMN-214, HMN-176, Rigosertib, Onvansertib (NMS-P937), Bl- 2536, GSK461364, Volasertib (BI6727), Centrinone (LCR-263), (1 E)-CFI-400437 dihydrochloride, CFI- 400945, CU-CPT22, GW843682X, MLN0905, ON1231320, Poloxin, Ro3280, SBE 13 HCI and TAK- 960;(b) the cell cycle modulator is selected from Adavosertib, Cobimetinib, Irbinitinib, Olmutinib Venetoclax, SNS-314, SP-96, PHA-680632, RCM-1 , and FDI-6;(c) the microtubule modulator is selected from Ansamitocin p-3, Combretastatin A4, Monomethyl auristatin E (MMAE), Vinblastine sulfate, Vindesine sulfate, Docetaxel, Paclitaxel, Colchicine (NSC 757) and Nocodazole;(d) the HDAC modulator is selected from Abexinostat, Belinostat, CUDC-907, Givinostat, GSK3117391 , Panobinostat, Pracinostat, Quisinostat 2HCI and Ricolinostat;(e) the GPCR modulator is selected from Cetirizine DiHCI, Desipramine Hydrochloride, Dexamethasone, Dexmedetomidine HCI, Dronedarone HCI, Pimavanserin, Sotagliflozin, Venlafaxine HCI and Adiphenine HCI; or(f) the JAK modulator is selected from Momelotinib, Oclacitinib maleate and PF-06700841 .E30. The method of any one of E1 to E29, wherein the rAAV particles comprise a capsid protein of:(a) the AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11 , AAV 12, AAV13, AAV 14, AAV 15, AAV 16, AAVrh8, AAV.rh , AAVrh20, AAVrh39, AAV.Rh74, AAVRHM4-1 , AAVhu37, AAVAnc80, AAVAnc80L65, AAV7m8, AAVPHP.B, AAV2.5, AAV2tYF, AAV3B, AAVLK03, AAVHSC1 , AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10 , AAVHSC11 , AAVHSC12, AAVHSC13, AAVHSC14, AAVHSC15 or AAVHSC16 serotype;(b) the AAV8 or AAV9 serotype;(c) the AAV8 serotype;(d) the AAV9 serotype;(e) a non-human AAV serotype;or(f) an engineered AAV preferably an engineered serotype of any one of (a)-(d).E31 . The method of any one of E1 to E30, wherein the host cell has been transfected with one or more polynucleotide comprising at least one AAV cap gene and / or at least one AAV rep gene, at least one inverted terminal repeat (ITR) and / or at least one helper gene.E32. The method of any one of E1 to E31 wherein the host cell has been transfected with a polynucleotide comprising an expression cassette, wherein the expression cassette comprises a transgene between two ITRs.E33. The method of E31 or E32, wherein the modulator is added before transfection or substantially at the time of transfection or after transfection.E34. The method of any one of E31 to E33, wherein the transgene comprises a regulatory element operatively linked to a polynucleotide encoding a polypeptide.E35. The method of any one of the previous claims, wherein the host cell comprises:(i) a rep 52 gene;(ii) a rep 40 gene;(iii) a rep 68 gene;(iv) a cap gene;(v) a viral associated (VA) nucleic acid;(vi) an E2A gene;(vii) an E4 gene;(viii) an ElA gene; and / or(ix) a polynucleotide comprising an expression cassette comprising a transgene between two ITRs.E36. The method of any one of E1 to E35, wherein the host cell is a mammalian cell.E37. The method according to any one of E1 to E36, wherein the host cell is:(a) a HEK293 cell, a HEK293T cell, a HEK293SF cell, a HEK293-F cell, a CHO cell, a HeLa cell, a HeLa S3 cell, a HEK293EBNA cell, a CAP cell, a CAP-T cell, an AGE1 .CR cell, a PerC6 cell, a C139 cell, an EB66 cell, a BHK cell, a COS cell, a Vero cell, an A549 cell, or a K562 cell;(b) a HEK293 cell, a HEK293T cell, a HEK293SF cell or a HEK293-F cell;(c) derived from a HEK293 cell, a HEK293T cell, a HEK293SF cell, a HEK293-F cell, a CHO cell, a HeLa cell, a HeLa S3 cell, a HEK293EBNA cell, a CAP cell, a CAP-T cell, an AGE1 .CR cell, a PerC6 cell, a C139 cell, an EB66 cell, a BHK cell, a COS cell, a Vero cell or an A549 cell; or(d) derived from a HEK293 cell, a HEK293T cell, a HEK293SF cell or a HEK293-F cell.E38. The method according to any one of E1 to E37, wherein the culturing step comprises culturing the host cell in suspension culture and / or adherent culture.E39. The method according to any one of E1 to E38, wherein the culturing step comprises culturing the host cell for up to 2 days, up to 3 days, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days or for 2-10 days.E40. The method according to any one of E1 to E39, wherein the viral genome to capsid ratio of the preparation is increased by at least 10%, at least 20%, at least 30%, or at least 40%.E41. The method according to any one of E1 to E40, further comprising a step of formulating the preparation with a pharmaceutically acceptable excipient.E42. The method according to any one of E1 to E41 , further comprising a step of isolating and / or purifying the rAAV.E43. A preparation comprising rAAV obtained or obtainable by the method of any one of E1 to E42.E44. A method for the treatment or prevention of a disease, comprising administering the preparation of E43 to a subject in need thereof.E45. A preparation according to E43, for use in a method of treating or preventing a disease.E46. Use of a preparation according to E45 for the manufacture of a medicament for treating or preventing a disease.E47. The method of E44, preparation for use according to E45, or use of E46, wherein the disease is a genetic disorder.The following examples illustrate the invention.Example 1 - Methods and MaterialsAdherent cell culture and suspension adaptation of HEK293 cellsAdherent HEK293 cells were expanded in DMEM, high glucose, GlutaMAX™ Supplement, pyruvate supplemented with 10% Fetal Bovine Serum at 37°C, 5% CO2 incubator. Once cells had grown to -80% confluency, the media was carefully aspirated and replaced with Expi293™ expression medium or BalanCD HEK293 supplemented with 4mM L-Glutamine and incubated for 48 hours in the adherent format at 37°C, 5% CO2. After 48 hours of media adaption, the cells were resuspended in 2mL of fresh Expi293™ expression medium or BalanCD HEK293 supplemented with 4mM L-Glutamine and placed on an orbital shaker (125 rpm,19 mm orbital throw) at 37°C, 5% CO2. Cell viability and density were monitored following suspension adaption. Cells were maintained at 0.5 - 5 M / mL.Culturing suspension HEK293 cellsExpi293F™ HEK cells (“293F” for short) (Thermo Fisher Scientific; Catalog number: A14527) were maintained in suspension in Expi293™ expression medium at 37°C, 5% CO2 incubator on an orbital shaker (125 rpm,19 mm orbital throw). Cell growth and viability was monitored for 7-10 days until cells had reached a density between 2.5 - 5 M / mL and >95% viability prior to transfection. Cells were then centrifuged at 500g for 5 minutes. Spent media was aspirated and the cell pellet was resuspended in the appropriate volume of Expi293™ expression medium to achieve a density of 2.5M / mL in a 30mL shake flask.Plasmids and ITR containing reporter vectorThe pHelper plasmid contains Ad5 E4 and Ad5 E2A and Ad5 VA-RNA. The RepCap9 plasmid expresses AAV2-derived Rep78 and Rep68 under the control of the P5 promoter, Rep52 and Rep40 under the control of the P19 promoter and VP1 ,VP2 and VP3 derived from AAV9 under the control of the P40 promoter. The ITR-containing reporter vector expresses mCherry, P2A, nano luciferase (nluc) under a CMV promoter.Compound library screenpHelper plasmid, RepCap9 plasmid, and the ITR containing reporter vector were prepared in a 1 :1 :1 molar ratio. The DNA mix was then diluted in PBS to a volume of 10% of the total culture volume to give a final concentration of 3.0 pg / mL. Next, room temperature TransIT-VirusGEN (Mirus) transfection reagent was added to the diluted DNA at 2x the volume of the predilution DNA mix (2:1 mixture of transfection reagent to total DNA) and incubated for 15 minutes at room temperature. After incubation, TransIT-VirusGEN: DNA complex was added to a shake flask containing HEK293F cells at a density of 2.5 M / mL and incubated for 4 hr. Next, transfected cells were diluted 1 :5 with Expi293™ expression medium to obtain a cell density of 0.5 M / mL. Using a multichannel liquid handler, 90 pL of cells were seeded per well of a 96-well plate to obtain a final cell density of 50 K cells / well. A compound library consisting of bioactive drugs was diluted to working library at the concentration of 10 pM. Using a multichannel liquid handler, 10 pL of 10 pM drug mixture was added into each well and mixed 5 times to obtain a final drug concentration of 1 pM per well. Plates were then incubated at 37°C and 5% CO2 for 72 hr with no media change prior to sample harvest. 3 days post transfection and compound addition, samples were harvested and AAV9 capsid titre was determined using ELISA. rAAV production rAAV was produced in an Ambr™ 15 Generation 2 Cell Culture 24 Bioreactor System (Sartorius Stedim Biotech GmbH, Gottingen, Germany). Ambr™ 15cc bioreactors with sparge were equilibrated with 12 mL of BalanCD HEK293 media (FUJIFILM Irvine Scientific, Santa Ana, California, USA) supplemented with 4 mM L-glutamine (Thermo Fisher Scientific, Waltham, Massachusetts, USA)) at 37 °C, 40% DO and pH set-point 7.4. 20 pl EX-CELL™ Antifoam (Merck, Darmstadt, Germany) was added to each bioreactor every 12 hours starting directly post media fill. 1 molar sodium bicarbonate solution was used to adjust pH levels. HEK293 suspension cells were seeded into Ambr™ 15cc bioreactors at a final concentration of 2.5E+06 viable cells per mL. Cells were transfected with a 4:3 molar mixture of helper and vector plasmid using the linear polyethylenimine transfection reagent PEIpro™ (Polyplus / Sartorius Stedim Biotech GmbH, Gottingen, Germany) according to the manufacturer’s manual. Per viable cell, a total plasmid DNA amount of 0.52 pg was applied. The PEI-DNA complexes were prepared in BalanCD HEK293 media supplemented with 4 mM L-glutamine. Where required, compound such as HMN-214 was added to respective bioreactors by addition of a 100X stock solution in BalanCD HEK293 media containing 0.1 % DMSO. Cells were cultured until day 3 post-transfection, followed by harvest and lysis by three freeze-thaw cycles (-80°C and 37°C). Cell debris was removed by centrifugation at 3,700 x g for 30 min at 4 °C. Cell lysates were subjected to a nuclease treatment procedure to remove extra-particulate DNA. To that aim, the samples were treated by addition of Denarase™ endonuclease (c-LEcta GmbH., Leipzig, Germany) at a final activity of 0.02 U / pL and incubation at room temperature for at least 12 hours. Subsequently, samples were centrifugated and supernatants were retained to remove any precipitate arising from the treatment.AA V harvest and purificationThree days post transfection plasmid transfection, BD Pharmigen Cell Lysis Buffer (BDB559759) and Benzonase (70846-3) were added to the cell culture and incubated for 1 hour at 37°C. To remove cell debris, the mixture was centrifuged for 3 minutes at 4000 rpm and the supernatant was transferred toa fresh tube. POROS AAV9 Affinity Resin (A27354) was added to the supernatant and incubated on a shaker for 30 minutes at room temperature. The affinity resin was then transferred through a Pierce Centrifuge Columns (PI89868) for collection and washed 3x times with PBS. Virus was eluted from the column with 0.2M Glycine (J67094) and quenched with 1 M Tris-HCL (50-843-270). The sample was then concentrated in an Amicon-Ultra Centrifugal Filter Unit (UFC510096).Alternatively, or additionally, rAAV particles were purified from the Denarase™ treated cell lysates by a spin-column based affinity chromatography approach applying POROS™ CaptureSelect™ AAVX Affinity Resin (Thermo Fisher Scientific, Waltham, Massachusetts, USA). 150 pl of resuspended affinity resin was used per cell lysate. Prior to use, the affinity resin was washed 3 times with 300 pl of equilibration / wash buffer (20 mM Tris, 100 mM NaCI, pH 7.5). Between wash steps, the affinity resin suspension was centrifugated at 1000 g for 1 minute, and supernatants were discarded. After the final centrifugation step, the affinity resin was resuspended in 300 pl of equilibration / wash buffer and transferred to the cell lysate. The mixture was incubated at room temperature for 90 minutes in an overhead shaker. After incubation, the affinity resin containing captured rAAV was recovered by centrifugation at 1000 g for 1 minute and discarding of the supernatant. The affinity resin containing captured rAAV particles was transferred to Pierce™ cellulose acetate filter spin cups (Thermo Fisher Scientific, Waltham, Massachusetts, USA). 5 wash steps with equilibration / wash buffer were performed with intermittent centrifugation at 1000 g. Finally, elution of the bound rAAV particles was performed in three rounds, each consisting of addition of 100 pl elution buffer (20 mM Sodium citrate, 98 mM NaCI, pH 2.5) followed by neutralization of the eluate with neutralization buffer (2 M Tris).Determination of capsid titre using ELISAA Streptavidin coated high-capacity plate (Thermo Fisher Scientific; Catalog No. P115500) was coated for 1 hr at room temperature with the CaptureSelect™ Biotin anti-AAV9 conjugate (Thermo Fisher Scientific; Catalog No.7103332500), at a dilution of 1 :10,000 in PBST. After incubation, the capture antibody solution was aspirated from each well of the plate, and each well was washed 3 times with 150 pL of PBST. 100 pL of either standard or sample was added to the plate. A 5-7-point standard was used in all studies using the AAV9 empty capsid standard (Progen; Catalog No. 66V090). The plate was covered with foil sealing film and incubated on a shaker for 1 hr at room temperature. After incubation, the entire sample was aspirated off, and the plate was washed 3 times with 150 pL of PBST. CaptureSelect™ HRP anti-AAV9 conjugate (Thermo Fisher Scientific; Catalog No. 7303332100) was diluted 1 :50,000 in PBST and 100 pL of the diluted detection antibody was added to each well. The plate was covered with foil sealing film and incubated on a shaker for 1 hr at room temperature. After incubation, the HRP anti-AAV9 conjugate solution was aspirated and washed 3 times with 150 pL of PBST. 100 pL of TMB ELISAsubstrate (highest sensitivity) (Abeam; Catalog No. ab171522) was added per well and incubated for 2-5 minutes before adding 100 pL the ELISA stop solution (Thermo Fisher Scientific; Catalog No. SS04). Absorbance was measured on the Varioskan microplate reader at 450 nm (Thermo Fisher Scientific).Determination of vector genome titre using qPCRPurified or crude viral supernatant was DNase I treated for 90 minutes at 37°C according to manufacturer’s instructions (New England BioLabs; Catalog Number M0303S). qPCR was performed using a custom FAM probe against Nano Luciferase (Thermo Fisher Scientific) and TaqMan fast advanced master mix (Thermo Fisher Scientific; Catalog No. 4444557) on a QuantStudio 6 Flex realtime PCR system (Thermo Fisher Scientific). Plasmid DNA was used from a concentration of 1 ng / pL to 0.0001 ng / pL to generate a standard curve.The AAV vector genome assay is based on a quantitative polymerase chain reaction (qPCR) specific for a sequence of the rAAV expression cassette. Per sample, 10.0 pl iTaq™ Universal SYBR™ Green Supermix (Bio-Rad Laboratories Inc., Hercules, California) were mixed with 0.80 pl of qPCR primer working stock solution (containing 10pM of each primer) and filled up to a volume of 15 pl with nuclease-free water. 5 pl of Denarase™ treated cell lysate or purified virus test sample were added to the mix (total reaction volume 20 pl, final primer concentration in the reaction 400 nM each) and qPCR was performed in a CFX 96 Touch Real Time PCR cycler (Bio-Rad Laboratories Inc., Hercules, USA) with following program steps: 95°C 5 min; 39 cycles (95°C 5 s, 60°C 30 s, plate read); 95°C 10 sec; 60-95°C (+0.5°C / step), 10 sec; plate read. To control for the quality of the qPCR, a trending control with known AAV vector genome titre was measured in parallel. To check for contaminations, a no template control (NTC, 5 pl H2O) was also included. Standard row, test samples and controls were measured in triplicates for each dilution. Denarase™ treated cell lysates, purified virus test samples and trending control were generally measured in 3 different dilutions in EB buffer (10 mM Tris-CI, pH 8.5). Data were analyzed using the CFX ManagerTM Software 3.1 (Bio-Rad Laboratories Inc.). Melting curve analysis confirmed the presence of only one amplicon. Amplification results in nascent double stranded DNA amplicons detected with the fluorescent intercalator SYBR Green to monitor the PCR reaction in real time. Known quantities of the expression cassette genetic material, in the form of a linearized plasmid, were serially diluted to create a standard curve and sample vector genome titre was interpolated from the standard curve.Immunoassay quantification of rAAV particles (capsids) rAAV capsids were quantified using the automated immunoassay platform Gyrolab xPlore™ (Gyros Protein Technologies AB, Uppsala, Sweden) in conjunction with the Gyrolab™ AAV9 Titre Kit. The sandwich immunometric technique used to capture and detect AAV9 capsids is based on the Thermo Scientific™ CaptureSelect™ Biotin Anti-AAV9 Conjugate and Thermo Scientific CaptureSelect Alexa Fluor™ 647 Anti-AAV9 Conjugate from Thermo Fisher Scientific (Waltham, Massachusetts, USA). A serial dilution of AAV9 empty capsid control (Progen, Heidelberg, Germany) was applied to interpolate AAV9 concentrations in the test samples. A negative control was included to check for contamination. Denarase™ treated cell lysates, purified virus test samples and AAV9 control serial dilution were diluted in Gyrolab™ AAV9 Titre Sample Dilution Buffer. All samples and controls were tested in duplicate. Data were analyzed using the Gyrolab™ Evaluator Software Version 3.7.1 .252.Vector genome to total particle ratioThe ratio of vector genomes to total AAV particles is expressed as a percentage. This is based on the vector genome titre (determined by qPCR, as described above) and the number of total AAV particles (determined by the capsid immunoassay, as described above).Quantification of plasmid-derived impuritiesProkaryotic DNA sequences, such as antibiotic resistance genes or parts of them originating from the bacterial backbone of the producer plasmids, can be packaged into the rAAV particles, constituting product-related impurities. Plasmid-derived impurity quantification is based on experiments using qPCR techniques specific for defined sequences of the kanamycin resistance gene (kanR), present on both helper and vector plasmid, and the AAV cap gene, which is present on the vector plasmid. Plasmid-derived impurity qPCRs were performed on the purified material. The plasmid-derived impurity qPCRs were performed according to the method for quantification of rAAV vector genomes outlined above with appropriate modifications to reflect the impurity templates, such as the primer pairs, standards and controls. Specifically, modifications were made to the impurity specific primer pairs, the linearized plasmid standards, the trending controls and the annealing temperatures in the qPCR setup.Quantification of host cell-derived impuritiesDNA sequences, such as nucleic acid material from the genome of host cells in which AAV are produced, can be packaged into rAAV particles, constituting product-related impurities. Host cell- derived impurity quantification was carried out using droplet digital polymerase chain reaction (ddPCR) specific for defined sequences of the 18S rRNA gene locus of the 45S ORF present in HEK293 cells. Host cell-derived impurity ddPCR was performed on the purified material. The rAAV vector samples were diluted in EB (10mM Tris-CI, pH 8.5) in a range of 1 :5 to 1 :40. The reaction mixtures were prepared using ddPCR Supermix for Probes (1863026, Bio-Rad) with 0.9 pM primers specific for the 18S rRNA gene locus and 0.25 pM probe specifically binding to the 18S rRNA gene locus and 8 pL sample dilution in a final volume of 20 pL as described in the manufacturer's protocol. Samples were partitioned with droplet generator oil for probes (Bio-Rad) using a QX-200 droplet generator (Bio-Rad) according to manufacturer's instructions. PCR amplification of the droplets was performed using a thermal cycler with the following parameters: 95°C for 10 min, followed by 39 cycles of 94°C for 30 s, 59°C for 1 min 20 followed by a final 98°C heat treatment for 10 min. Droplet read-out of the PCR plate wells was carried out on the QX200 droplet reader (Bio-Rad) and data were analyzed with QuantaSoft software (Bio-Rad). To control for the quality of the ddPCR, a trending control was measured in parallel. To check for contaminations, a no template control (NTC, H2O) was also included. Test samples and controls were measured in triplicates for each dilution.Confirmation studies in suspension HEK293 cellsConfirmation studies were performed in suspension using Expi293FTM HEK cells (Thermo Fisher Scientific; Catalog number: A14527) and 3 clonally derived suspension HEK293 cell lines (AC001 .230, AC010.68, AC015.55). Cells were maintained in suspension in Expi293™ expression medium at 37°C, 5% CO2 incubator on an orbital shaker (125 rpm,19 mm orbital throw). Cell growth and viability was monitored for 7-10 days until cells had reached a density between 2.5 - 5 M / mL and >95% viabilityprior to transfection. Cells were then centrifuged at 500g for 5 minutes. Spent media was aspirated and the cell pellet was resuspended in the appropriate volume of Expi293™ expression medium to achieve a density of 2.5 M / mL in a 30mL shake flask. pHelper plasmid, RepCap9 plasmid, and the ITR containing reporter vector were prepared in a 1 :1 :1 ratio at a final concentration of 3.0 pg / pL. The DNA mix was then diluted in PBS (67:1 PBS to DNA ratio). Next, room temperature TransIT-VirusGEN (MirusBio) transfection regent was added to the diluted DNA at 2x the volume of the predilution DNA mix (2:1 mixture of transfection reagent to total DNA) and incubated for 15 minutes. After incubation, TransIT-VirusGEN: DNA complex was added to the shake flask with cells at a density of 2.5 M / mL. Compounds were solubilized in a 1000x concentration in DMSO and added to the shake flask at the point of transfection. Shake flasks were then incubated at 37°C and 5% CO2 for 72 hr with no media change prior to sample harvest.Generation of FUCCI reporterA plasmid containing the fluorescent ubiquitination-based cell cycle indicator (FUCCI) reporter cassette along with a CMV-driven puromycin selectable marker was synthesized. A CBh promoter drives the expression of mK02 florescent reporter fused to Cdt1 followed by T2A and mAzami-Green florescent reporter fused to Gem1 . 1 pg of the FUCCI plasmid was nucleofected into 1 M HEK293 cells using the 4D-nucleofector transfection system using the SF cell line nucleofector solution (Lonza).5 days post nucleofection, cells were selected using 5 pM puromycin (Thermo Fisher Scientific). After the 10 days of selection, the polyclonal population was single cell seeded using VIPS single cell seeder (Advanced Instruments). Clones were isolated and verified using microscopy and flow cytometry. A single clone based on expression levels and growth rate was chosen for further studies.Cell lines were used to analyze cell proliferation and cell cycle states in various treatment groups.Flow cytometrySuspension HEK293 cells were collected and pelleted at 500 g for 5 minutes. For each sample 10,000 live events were captured on the Attune flow cytometer (ThermoFisher) and analyzed using AttuneTM software.Split plasmid systemThe split plasmid system (two- plasmid system for AAV production) used has been described elsewhere, see, for example, EP3722434A1 , which is incorporated herein by reference in its entirety.Statistical analysisThe number of independent studies (biological replicates) and technical replicates are indicated in the figure legends. Unless otherwise specified, Student’s t-test was used for statistical analysis, with significant differences defines as *P <0.05, “ P <0.01 , “* P<0.001 , *“* P<0.0001 . Error bars indicate standard deviation (SD).Example 2 - ScreeningA high-throughput screening platformed for optimizing AAV productionTo identify candidate targets and pathways that improve AAV production, an arrayed targeted library for AAV screening (ATLAS) was conducted. This is a high-throughput screen amenable to gain and loss-of-function screens. First, the cell seeding density and transfection conditions were optimised using a luciferase expression vector in suspension and adherent HEK293F cells. An optimal transfection efficiency of 2.5 M / ml and 3 pg / mL of DNA was chosen for further studies (Figure 1 and 6A). Next, a cell-based functional assay was developed to enable high-throughput measurement of potent AAV particles in microwell plates. To overcome the low in vitro AAV9 transduction efficiency in cells, a reporter cell line containing a flipped mCherry, NanoLuciferase (NLuc) flanked by loxP sites was developed. Upon introduction of an ITR-containing GFP-Cre expressing vector the mCherry- NanoLuciferase cassette is flipped and both the mCherry and NIuc signals can be detected (Figure 1 ; and Figure 6, in particular Figure 6B - D).Compound screen identified cell cycle modulators, GPCR, HDAC, JAK and metabolic targets as putative potentiators of AAV9 productionNext, an unbiased high-throughput screen using a library of -3300 bioactive compounds was conducted to screen for AAV9 potentiators. HEK293F cells were triple transfected with a RepCap9 plasmid, Helper plasmid and an ITR-containing reporter vector (for example as referenced in the section “Compound library screen” in Example 1 above). Transfected cells were seeded onto 96-well plates and treated with the bioactive compound library (Figure 1A). AAV9 capsid ELISA was used to determine the capsid titre and screen to potentiators of AAV9 production. Results from the primary screen were normalized to the DMSO mean across all plates and rank ordered (Figure 1 B). Top performing compounds that increased AAV9 capsid titre 3o above mean DMSO controls were clustered into 5 pathways or target classes: cell cycle modulators, GPCR, HDAC, JAK inhibitors and metabolic targets. A list of compounds identified in the primary screen (n=46) is summarized in Table 1.Table 1. List of compounds identified in primary screen.Example 3 - Confirmation and Validation StudiesConfirmation study showed cell cycle regulation using PLK and microtubule inhibitors increased AAV9 capsid titre and potency in HEK293 cells Top hits from the primary screen were examined in a confirmation study at 4 different doses in HEK293 cells and measured using AAV9 capsid ELISA (for example, as referenced in the section “Compound library screen”, in Example 1 above). HMN-214 (PLK1 inhibitor) and microtubule inhibitors (Vindesine sulfate, Combretastatin A4, Monomethyl auristatin E (MMAE) and Ansamitocin p-3) showed an increase in AAV9 capsid titre (Figure 1 C). Relative AAV9 potent titre was also measured using the cell- based potency assay described above and data was plotted against capsid titre (Figure 1 D). Top 2 performing compounds that improved both capsid titre and potent titre were HMN-214 and Combretastatin A4.HMN-214, HMN-176 and Rigosertib increased AAV9 capsid titre, potency and vector genome titre in a dose dependent manner in suspension HEK293 cellsNext, three PLK1 inhibitors were investigated in suspension HEK293 cells. Addition of HMN-214 (PLK1 inhibitor), HMN-176 (an active metabolite of HMN-214 and inhibitor of PLK1) and Rigosertib (a structurally different PLK1 inhibitor) at the point of triple transfection exhibited a dose-dependent response in AAV9 production using suspension HEK293 cells. AAV9 production was measured using 3 assays: capsid titre, cell-based potency and vector genome titre (Figure 2A - C respectively).Further studies on HMN-214 were conducted to identify the most optimal time of compound addition and found that the most significant effect was observed at the point of transfection (t=0 hr), with no significant effect of HMN-214 at 4 hr and 12 hr post transfection. At 24 hr post transfection there was ~2-fold increase in AAV9 production (non-significant). Pre-dosing HMN-214 prior to transfection lead to a significant reduction in AAV9 production (Figure 2; and Figures 7A and 7B). Using a single transfection reporter system, addition of HMN-214 showed a dose-dependent decrease in the mCherry reporter signal, suggesting that the mechanism of AAV9 production is not enhancement of transfection efficiency (Figure 2; and Figure 7C).Combination of HMN-214 and microtubule inhibitors did not show an additive effect on AAV9 productionTo test if co-administration of HMN-214 and microtubule inhibitors further boost AAV9 production, two microtubule inhibitors (Combretastatin A4 and MMEA) were first tested as a single agent in suspension HEK293 cells. Both Combretastatin A4 and MMEA showed a dose dependent increase in AAV9 production (Figure 2; and Figure 7D, E). However, combination of Combretastatin A4 and HMN-214 did not show an increase in AAV9 production (Figure 2; and Figure 7F).HMN-214 increases AAV9 production in multiple clonal HEK293 cellsTo ensure the effects of HMN-214 was not specific to HEK293F cells, polyclonal HEK293 cells were obtained from two difference cell line suppliers (DSMZ and CLS). Clonal cell lines were also developed using the Solentim VIPS™ (Verified in-situ plate seeding) single cell seeder. A double imaging system (once at droplet dispense and whole well imaging) is used for clonality assurance. 274 clonally -derived HEK293 cells were developed screened for transfection efficiency and AAV9 production (Figure 3; and Figure 8A). Top 12 performing clones were first tested in adherent format and later suspension-adapted and further tested for AAV9 production. Several of the clonal cell lines showed improved AAV9 production compared to their polyclonal parental lines and HEK293F cells (Figure 3; and Figure 8B). The top AAV9 producer clonal cell line (AC001 .230) was further validated in 125mL and 1 L shake flasks and production capacity was shown to be approximately 3 times higher compared to 293F cells using the potency and vector genome titre (Figure 3; and Figure 8, in particular Figures 8C-F). The most efficacious dose of HMN-214 from previous studies (1 pM) was used in these cell lines and both polyclonal and clonal cells lines showed a significant increase in AAV9 production showing the effect of HMN-214 was broadly applicable to other HEK293 cells (Figure 3A).Additionally, the effects of PLK inhibition on AAV production was largely consistent across capsid serotypes, suggesting that production benefits are capsid independent.HMN-214 increases AAV9 titre using a split plasmid system in Ambr15 microbioreactorTo further confirm the enhanced AAV9 production effects of HMN-214, a split AAV9 plasmid transfection system was used. One plasmid carries the ITR containing reporter with Cap9 and the second plasmid carries the Helper elements and Rep2. The split plasmids were transfected into HEK293 cells (AC018) using polyethyleneimine (PEIpro (Polyplus) and cultured in BalanCD supplemented with L-Glutamine. HMN-214 was added at multiple times during AAV9 production (Figure 4A). A dose-dependent increase in AAV9 capsid titre and vector genome titre was observed up to 1 pM and titres dropped at higher doses (Figure 4B, C). Vg:Cap ratios remained constant up to 1 pM and then a declined at higher doses. (Figure 4D). A dose-dependent increase in host cell DNA, Cap and KanRimpurities were observed with increasing doses of HMN-214 (Figure 4E-G). Addition of HMN-214, 4 hr post transfection had a minimal effect on AAV9 production, while increasing host cell derived (HCD), Cap and KanRimpurities, suggesting the most ideal timing for PLK1 inhibition is at the time of transfection (Figure 4; and Figure 9A). Additional redosing with HMN-214 at 24 hr and 48 hr post transfection did not further improve AAV9 yields compared to addition at transfection only condition (Figure 4; and Figure 9B, C).HMN-214 blunts growth rate of HEK293 cells by blocking cell cycle progression from M to G1 phase and a FUCCI reporter cell line developed to study effects of HMN-214HEK293 cells were triple transfected with a pHelper, RepCap9 and ITR containing reporter vector. Samples were treated with either DMSO or HMN-214 (1 pM) at the time of transfection, and cell count was monitored over time. The DMSO treated condition showed a slight dip in cell count post transfection, but cells continue to grow after day 3, while the HMN-214 treated cells show a slow decline in cell number over time. AAV9 capsid titre was also measured from DMSO or HMN-214 treated samples on day 3 post triple transfection and plotted against cell growth rate. An anticorrelation effect was observed between the AAV9 capsid titre and cell growth rate. Further, a fluorescent ubiquitination- based cell cycle indicator (FUCCI) reporter cell line was used to analyze cell proliferation and cell cycle states in various treatment groups. Groups treated with 1 pM of HMN-214 show a reduction of cells in the M to G1 phase and increase in the G1 to S and S / G2 / M phase. See Figure 5In addition, a fluorescent ubiquitination-based cell cycle indicator (FUCCI) reporter cell line was developed by integrating the FUCCI reporter in HEK293 cells. A CBh promoter drives the expression of mK02 florescent reporter fused to Cdt1 followed by T2A and mAzami-Green florescent reporter fused to Gem1 . Flow cytometry plots showing HMN-214 demonstrated reduction in the fraction of cells in M / G1 along with an increase in the fraction of cells in the G1 , G1 / S and S / G2 / M phase. More specifically, a fluorescent ubiquitination-based cell cycle indicator (FUCCI) reporter cell line was used to analyze cell proliferation and cell cycle states in various treatment groups. Groups treated with 1 pM of HMN-214 show a reduction of cells in the M to G1 phase and increase in the G1 to S and S / G2 / M phase. Addition of HMN-214 (in either alone or in combination with triple transfection) showed a timedependent reduction in the cells in the M / G1 phase. See Figure 10.Example 4 - Confirmation Studies with Additional Cell Cycle ModulatorsCell cycle regulation using several PLK inhibitors increased AAV9 capsid titre and vector genome titre in HEK293 cellsAdditional PLK inhibitors, identified in a follow up screen performed as described in Example 2, were tested in HEK293 cells and AAV production was measured using capsid and vector genome titre using the appropriate assays described in Example 1. As it can be seen in Figure 11 (A-B), several of the selected PLK inhibitors enhanced AAV9 production in adherent HEK293 cells. A list of additional PLK inhibitors tested for along with their maximal effect on AAV9 yield, measured as capsid and vector genome titres respectively, are disclosed in Table 2.Table 2. List of additional PLK inhibitors tested for their effect on AAV productionExample 5- Cell cycle regulation using selected Aurora kinase and FOXM1 inhibitors and assessment of their effect on recombinant AAV9 production in HEK293 cell culture.It is known that PLK1 is activated by its upstream regulator Aurora Kinase (A or B) . In addition, PLK1 dependent phosphorylation of FOXM1 regulates a transcriptional program required for mitotic progression . Therefore, based on these knowledge, inventors of the current disclosure explored the potential impact of cell modulators involved in the above-mentioned pathways. To this end, a number of Aurora kinase inhibitors and FOXM1 inhibitors were selected and tested for their effect on AAV9 production. The selected compounds were added at the point of transfection with a two plasmid system. As it is shown in Figure 12 (A-B), several Aurora kinase and FOXM1 inhibitors enhanced AAV9 production in adherent HEK293 cells. A list of inhibitors selected and tested along with their maximal effect on AAV yield measured as capsid and vector genome titres respectively are disclosed in Table 3.Table 3. List of additional cell cycle modulators tested for AAV productionAurora kinase inhibitors PHA-680632, SP96, and SNS-314 increase AAV9 capsid titre and vector genome titre in suspension HEK293 cellsThree selected Aurora kinase inhibitors were further tested for their effect on AAV9 production in adherent HEK293 cells. For this purpose, PHA-680632 (Aurora kinase A / B / C inhibitor), SP96 (Aurora B inhibitor) and SNS-314 (Aurora kinase A / B / C inhibitor) were added to HEK293 cells at the time of transfection with the two -plasmid system. AAV9 production was measured using two assays, measuring capsid titre and vector genome titre respectively, as described above in detail. As it is shown in Figure 13 (A-B), all three Aurora kinase inhibitors (PHA-680632 at 50 nM, SP-96 at 500 nM and SNS-314 at 50 nM) increased AAV9 production in adherent HEK293 cells over the DMSO control. Moreover, an additional experiment was carried out using a confirmatory dose ranging for the selected Aurora kinase inhibitor SNS-314. As it is shown in Fig. 13 (C-D), adding SNS-314 at the point of transfection, led to a dose-dependent increase in AAV9 production in adherent HEK293 cells. AAV9 production was measured as relative capsid titre (Fig. 13C) and vector genome titre (Fig. 13D) over DMSO control.HMN-214 and SNS-314 increase AAV9 production in suspension HEK293 cells by blocking cell cycle progression from M to G1 phaseIn order to assess the effect of a combination of a PLK inhibitor and an Aurora kinase inhibitor in AAV9 production, two selected compounds described in previous experiments, were added in suspension HEK293 cells at the point of transfection with the two plasmid system. The experiment has shown that HMN-214 (PLK inhibitor, 1 pM) and SNS-314 (Aurora kinase inhibitor, 50 nM) increased AAV9 production in suspension using two different HEK293 cells lines (AC230 and AC18P). Figure 14 (A-B) shows the effect of adding these two compounds on AAV9 production, measured as capsid titre and vector genome titre. Also, vector genome to capsid (Vg:Cap) ratios were measured in the different treatment groups as depicted in Figure 14C. To measure the effects of PLK and Aurora kinase inhibition on AAV9 producing cells, Click-iT EdU and FxCycle Violet were used for the flow cytometry stain assay (as described below, in the following paragraph). This assay allows to quantify the percentage of proliferating cells and DNA content per cell in different treatment conditions. HMN-214 and SNS-314 halt cells in the G2 / M phase and prevent entry to G1 phase as depicted in as shown in Figure 14D. As it can be seen HMN-214 and SNS-314 treatment reduced the percentage of proliferating cells (EdU- positive) and increased the fraction of cells in the G2 / M phase (EdU-negative, double-DNA content).Click-iT EdU and FxCycle Violet for the flow cytometry stain assayDNA replication in proliferating cells was analyzed with the Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit according to manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA). Briefly, cells were incubated with 50 pM EdU for 1 hr, washed once with 1 % bovine serum albumin in PBS, fixed with Click-iT fixative for 15 min, washed once with 1 % bovine serum albumin in PBS, and permeabilized by resuspending in 1X Click-iT saponin-based permeabilization and wash reagent. Then, cells were incubated in Click-iT detection cocktail for 30 min and washed once with 1X Click-iT saponin-based permeabilization and wash reagent. Next, DNA content in fixed cells was measured using FxCycle Violet stain according to manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA). Two drops of FxCycle Violet Ready Flow Reagent were added to EdU-stained and fixed cells, incubated for 30 min, and protected from light using aluminum foil. Samples were analyzed on the Attune flow cytometer (Thermo Fisher Scientific, Waltham, MA) using the VL1 and RL1 channels.Aurora kinase inhibitors SNS-314 and SP-96 increase AAV9 titre in Ambr15 microbioreactorTo further confirm the enhanced AAV9 production effects of Aurora kinase inhibitors in a small-scale bioreactor format, two Aurora kinase inhibitors, SNS-314 and SP-96) were tested along with HMN-214, used as a positive control, in an Ambr15 microbioreactor system. A split AAV9 plasmid transfection system was used, as described in Example 1. One plasmid carries the ITR containing reporter gene with Cap9 and the second plasmid carries the Helper elements and Rep2. The two plasmids were transfected into HEK293 cells (AC018) using polyethyleneimine (PEIpro (Polyplus) and cultured in BalanCD supplemented with L-Glutamine. SNS-314 (50 nM), SP-96 (500 nM) and HMN-214 (1 pM) were added at the time of transfection. An 2 fold increase approximately in AAV9 capsid titre and vector genome titre was observed with all three compounds compared to the no treatment and DMSO controls, as shown in Figure 15A and 15B respectively. Viral genome to capsid ratios for all treatment groups remained constant compared to the no treatment and DMSO controls, as shown in Figure 15C.SNS-314 increased AAV1, AAV2, and AAV6 titres in Ambr15 microbioreactorIn a further experimental setup, the selected Aurora kinase inhibitor SNS-314 was tested for its effect on the production of other AAV serotypes in a small-scale bioreactor format (Ambr15 microbioreactor). For this purpose, SNS-314 (50 nM) was tested in suspension HEK293 cells with three additional AAV serotypes, namely AAV1 , AAV2 and AAV6, in addition to AAV9 which is in this case was used as the positive control. Cells were treated with SNS-314 at a concentration of 50 nM at the time of transfection. A split-plasmid system, as described with the previous experiments, was introduced into HEK293 cells using PEIpro and cultured in BalanCD HEK293 media with 4 mM L-glutamine. One of the two plasmid carries an ITR-containing reporter with the corresponding Cap serotype sequence. The second plasmid carries the Helper elements and Rep2. As it can been seen in Figure 16 (A-D), SNS-314 increases vector genome titer across AAV1 , AAV2 and AAV6 (in addition to AAV9 as reported previously)

Claims

CLAIMS1 . A method for producing a preparation comprising recombinant adeno-associated virus (rAAV) particles, wherein the method comprises a step of contacting a host cell with a polo- like kinase (PLK) modulator, a cell cycle modulator preferably an Aurora kinase modulator or a FOXM1 modulator, a microtubule modulator, or a G protein-coupled receptor (GPCR) modulator and a step of culturing the host cell under suitable conditions to produce the preparation.

2. A method according to claim 1 , wherein the method is a method for producing a preparation comprising rAAV having improved viral genome titre, capsid titre and / or potency compared to an rAAV using a corresponding method without the modulator.

3. A method for increasing the viral genome titre, capsid titre and / or potency of recombinant adeno-associated virus (rAAV) in a preparation, wherein the method commprises a step of contacting a host cell with a polo- like kinase (PLK) modulator, a cell cycle modulator preferably an Aurora kinase modulator or a FOXM1 modulator, a microtubule modulator, modulator, or a G protein-coupled receptor (GPCR) modulator; and a step of culturing the host cell under suitable conditions to produce the preparation.

4. The method of claim 2 or 3, wherein the viral genome titre, capsid titre and / or potency is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or at least 60%.

5. The method of any one of claims 2-4, wherein the viral genome titre is measured by qPCR or ddPCR; and / or the capsid titre is measured by an immunoassay preferably by ELISA or a sandwich immunometric assay; and / or the potency is measured by a cell-based transduction assay.

6. The method of any one of the previous claims, wherein the method comprises a step of contacting a host cell with a polo- like kinase (PLK) modulator, a cell cycle modulator preferably an Aurora kinase modulator and / or a FOXM1 modulator, and / or a microtubule modulator.

7. The method of any one of the previous claims, wherein the method comprises a step of contacting the host cell with a PLK modulator and / or an Aurora kinase modulator and / or a FOXM1 modulator.

8. The method of any one of the previous claims, wherein:(a) the PLK modulator is a PLK inhibitor;(b) the cell cycle modulator is a modulator and / or inhibitor of the G1 (Gap 1 , cell growth) to S (DNA synthesis) transition, the S to G2 (Gap 2, Growth and preparation for mitosis) transition, the G2 to M (Mitosis phase) transition, and / or the M to G1 transition, preferably an Aurora kinase inhibitor or a FOXM1 inhibitor; or(c) the microtubule modulator is a microtubule inhibitor9. The method of any one of the previous claims, wherein:(a) the PLK modulator is selected from a modulator of PLK1 , PLK2, PLK3, PLK4 or PLK5; a modulator of PLK1 , PLK2 or PLK5; or a modulator of PLK1 ;(b) the cell cycle modulator is selected from a modulator of a cyclin family, a cyclin dependent kinase (cdk), a cdk inhibitor, an Aurora kinase A inhibitor, an Aurora kinase B inhibitor, an Aurora kinase C inhibitor, FOXM1 inhibitor or a pocket protein retinoblastoma (pRB) family;(c) the GPCR modulator is selected from a modulator of a class A GPCR, a class B GPCR, a class C GPCR or a class F GPCR10. The method of any one of claims 1-9, wherein:(a) the modulator is exogenously or endogenously expressed in the host cell;(b) the modulator is transiently or stably expressed in the host cell; and / or(c) one or more of PLK1 , PLK2, PLK3, PLK4 and PLK5 is transiently, conditionally, or permanently knocked down, knocked out or underexpressed in the host cell orr one or more of Aurora kinase A, Aurora kinase B, Aurora kinase C or FOXM1 is transiently, conditionally, or permanently knocked down, knocked out or underexpressed in the host cell11. The method of any one of claims 1-10, wherein:(a) the PLK modulator is selected from HMN-214, HMN-176, Rigosertib, Onvansertib (NMS-P937), Bl- 2536, GSK461364, Volasertib (BI6727), Centrinone (LCR-263), (1 E)-CFI-400437 dihydrochloride, CFI- 400945, CU-CPT22, GW843682X, MLN0905, ON1231320, Poloxin, Ro3280, SBE 13 HCI and TAK- 960, preferably selected from HMN-214, HMN-176, Rigosertib;(b) the cell cycle modulator is selected from Adavosertib, Cobimetinib, Irbinitinib, Olmutinib Venetoclax, SNS-314, SP-96, PHA-680632, RCM-1 , and FDI-6, preferably selected from SNS-314, SP-96, PHA- 680632, RCM-1 , and FDI-6;(c) the microtubule modulator is selected from Ansamitocin p-3, Combretastatin A4, Monomethyl auristatin E (MMAE), Vinblastine sulfate, Vindesine sulfate, Docetaxel, Paclitaxel preferably Combretastatin;(d) the GPCR modulator is selected from Cetirizine DiHCI, Desipramine Hydrochloride, Dexamethasone, Dexmedetomidine HCI, Dronedarone HCI, Pimavanserin, Sotagliflozin, Venlafaxine HCI and Adiphenine HCI; or12. The method of any one of the previous claims, wherein the rAAV particles comprise a capsid protein of:(a) the AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11 , AAV 12, AAV13, AAV 14, AAV 15, AAV 16, AAV.rh8, AAV.rhI O, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1 , AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1 ,AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11 , AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15 or AAV.HSC16 serotype;(b) the AAV8 or AAV9 serotype;(c) the AAV8 serotype;(d) the AAV9 serotype;(e) a non-human AAV serotype, or(f) an engineered AAV preferably an engineered serotype of any one of (a)-(e).

13. The method of any one of the previous claims, wherein the host cell has been transfected with one or more polynucleotide comprising at least one AAV cap gene and / or at least one AAV rep gene, at least one inverted terminal repeat (ITR) and / or at least one helper gene.

14. The method of any one of the previous claims, wherein the host cell has been transfected with a polynucleotide comprising an expression cassette, wherein the expression cassette comprises a transgene between two ITRs.

15. The method of claim 13 or 14, wherein the modulator is added before transfection, or substantially at the time of transfection or after transfection.

16. The method of any one of claims 13-15, wherein the transgene comprises a regulatory element operatively linked to a polynucleotide encoding a polypeptide.

17. The method of any one of the previous claims, wherein the host cell comprises:(i) a rep 52 gene;(ii) a rep 40 gene;(iii) a rep 68 gene;(iv) a cap gene;(v) a viral associated (VA) nucleic acid;(vi) an E2A gene;(vii) an E4 gene;(viii) an ElA gene; and / or(ix) a polynucleotide comprising an expression cassette comprising a transgene between two ITRs.

18. The method according to any one of the previous claims, wherein the host cell is:(a) a HEK293 cell, a HEK293T cell, a HEK293SF cell, a HEK293-F cell, a CHO cell, a HeLa cell, a HeLa S3 cell, a HEK293EBNA cell, a CAP cell, a CAP-T cell, an AGE1 .CR cell, a PerC6 cell, a C139 cell, an EB66 cell, a BHK cell, a COS cell, a Vero cell, an A549 cell, or a K562 cell;(b) a HEK293 cell, a HEK293T cell, a HEK293SF cell or a HEK293-F cell; (c) derived from a HEK293 cell, a HEK293T cell, a HEK293SF cell, a HEK293-F cell, a CHO cell, aHeLa cell, a HeLa S3 cell, a HEK293EBNA cell, a CAP cell, a CAP-T cell, an AGE1 .CR cell, a PerC6 cell, a C139 cell, an EB66 cell, a BHK cell, a COS cell, a Vero cell or an A549 cell; or(d) derived from a HEK293 cell, a HEK293T cell, a HEK293SF cell or a HEK293-F cell.

19. The method according to any one of the previous claims, wherein the viral genome to capsid ratio of the preparation is increased by at least 10%, at least 20%, at least 30%, or at least 40%.

20. A preparation comprising rAAV obtained or obtainable by the method of any one of the previous claims.21 . A preparation according to claim 20, for use in a method of treating or preventing a disease.

22. Use of a preparation according to claim 20 for the manufacture of a medicament for treating or preventing a disease preferably said disease being a genetic disorder.

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