Method for improved AAV production

Abstract

The present invention relates to methods for producing preparations comprising recombinant adeno-associated virus (rAAV) particles. More particularly, the present invention relates to methods for producing a preparation comprising recombinant adeno-associated virus (rAAV) particles wherein an Open Reading Frame (ORF) is introduced to a host cell under suitable conditions, so that said ORF is overexpressed, and wherein said preparation comprises rAAV particles have improved and / or increased viral genome titer, capsid titer, and / or functional titer. The disclosure also provides preparations comprising recombinant adeno-associated virus (rAAV) particles having improved and / or increased viral genome titer, capsid titer, and / or functional titer.

Description

[0001] METHOD FOR IMPROVED AAV PRODUCTION

[0002] Field of the Invention

[0003] The present invention relates to methods for producing preparations comprising recombinant adeno-associated virus (rAAV) particles. More particularly, the present invention relates to methods for producing a preparation comprising recombinant adeno-associated virus (rAAV) particles wherein an Open Reading Frame (ORF) is introduced to a host cell under suitable conditions, so that said ORF is overexpressed, and wherein said preparation comprises rAAV particles have improved and / or increased viral genome titer, capsid titer, and / or potency. The disclosure also provides preparations comprising recombinant adeno-associated virus (rAAV) particles having improved and / or increased viral genome titer, and / or potency.

[0004] Background to the Invention

[0005] Adeno-associated virus (AAV) is a member of the Parvoviridae family. The AAV genome is composed of a linear single-stranded DNA molecule which contains approximately 4.7 kilobases (kb) and consists of two major open reading frames encoding the non-structural Rep (replication) and structural Cap (capsid) proteins. Flanking the AAV coding regions are two cis-acting inverted terminal repeat (ITR) sequences, approximately 145 nucleotides in length, with interrupted palindromic sequences that can fold into hairpin structures that function as primers during initiation of DNA replication. In addition to their role in DNA replication, the ITR sequences have been shown to be necessary for viral integration, rescue from the host genome, and encapsidation of viral nucleic acid into mature virions (Muzyczka; 1992; Curr. Top. Micro. Immunol.; 158:97-129).

[0006] Multiple serotypes of AAV exist and offer varied tissue tropism. Known serotypes include, for example, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11 .

[0007] Recombinant adeno-associated virus (rAAV) vectors are a leading gene delivery vehicle, have remarkable potential for gene therapy due to their promising safety profile and their ability to transduce many tissues in vivo, and several rAAV-mediated therapies have recently been approved (Ameri; 2018; J. Curr. Ophthalmol. 30, 1-2; Yla-Herttuala; 2012; Mol. Ther. 20:1831-1832). However, despite these advances in the clinic, rAAV vector manufacturing remains a challenge. As an example, a phase 1 / 2 trial for hemophilia B required over 400 ten-layer cell stacks to generate sufficient material for six patients (Allay et al; 2011 ; Hum. Gene Ther. 22: 595-604). Whilst the therapy was efficacious, the poor production efficiency limits commercial opportunities and highlights the need for new methods to improve vector generation. Increased production efficiency will reduce manufacturing costs, improve patient access, and make this emerging modality more feasible for large disease indications. Higher gene therapy vector loads are crucial to account for larger patient sizes, systemic diseases, or diseases in less accessible body sites. Thus, beyond the efforts in improving safety, alternative host cells, and alternative viral helpers, an additional area of rAAV research has been in scale-up, moving production from laboratory scale to industrial scale (Clement; 2016; Mol Ther Methods Clin Dev; 3:16002). An important focus of the AAV vector development field has been to fine-tune the manufacturing process to augment the vector yield, purity, or its potency so that dose of vectors required per patient is low. AAV transduction requires a reasonable multiplicity of infection of ~103to 105vector genomes (vg) per cell depending on cell type. However, for a clinical trial, an estimated 1012to 1014viral particles are needed to be efficacious during gene transfer. This high vector dose requirement in the clinical settings has underscored the need for optimizing vector production.

[0008] Taken together there remains a need to develop methods to increase yield in the manufacture of an AAV pharmaceutical product while retaining quality standards e.g. low empty capsids, low host ceil protein, and / or low contaminating DNA.

[0009] Summary of the Invention

[0010] The present inventors have identified human Open Reading Frames (ORFs) which when overexpressed increase the capacity of cells to produce AAV. The inventors used an arrayed targeted library for AAV screening (ATLAS) platform to perform a screen using a library of -18,000 human ORFs and identify enhancers of AAV production. The screen identified transcription factors, epigenetic regulators, DNA replication factors, RNA regulators, protein ubiquitination, and metabolic targets as primary enhancers of AAV9 production. Confirmatory studies indicated a dose-dependent increase in AAV9 production, up to 3-fold from baseline while no negative impact on the functional titer of AAV was observed.

[0011] 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 ORFs that increased rAAV9 production in a robust and dose-dependent manner. The inventors confirmed these findings using capsid titer, vector genome quantification and a cell-based (viral potent titer) assay.

[0012] 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).

[0013] Specifically, the present disclosure provides the following aspects, advantageous features and specific embodiments, respectively alone or in combination:

[0014] E1. A method for producing a preparation comprising recombinant adeno-associated virus (rAAV) particles, wherein said method comprises i)a step of introducing a human Open Reading Frame (ORF) to a host cell under suitable conditions, so that said ORF is overexpressed, ii)a step of culturing said host cell under suitable conditions to produce the preparation; and wherein said preparation comprises rAAV particles having improved and / or increased viral genome titer, and / or potency compared to a preparation produced by a corresponding method without introducing said same ORF.

[0015] E2. The method of E1 , wherein said method comprises i)a step of introducing a human ORF expressing vector to a host cell under suitable conditions, preferably by transfecting an ORF expressing vector to a host cell under suitable conditions, so that said ORF is overexpressed, ii)a step of culturing said host cell under suitable conditions to produce the preparation; and wherein said preparation comprises rAAV particles having improved and / or increased viral genome titer, and / or potency compared to a preparation produced by a corresponding method without introducing said same ORF expressing vector.

[0016] E3. The method of E1 or E2, wherein said method comprises i)a step of introducing a human ORF to a host cell or introducing an ORF expressing vector to a host cell, preferably by transfecting an ORF expressing vector to a host cell under suitable conditions, so that said ORF is overexpressed, wherein said host cell is preferably a mammalian cell, more preferably a human cell ii)a step of transfecting said host cell with a recombinant AAV production plasmid system, preferably a three plasmid rAAV production system or a two plasmid rAAV production system, or synthetic DNA molecules suitable for AAV production, iii)a step of culturing said cell under conditions suitable for recombinant AAV production iv)a step of obtaining recombinant AAV particles from said transfected cells, wherein said preparation comprises rAAV particles having increased viral genome titer, and / or potency compared to a preparation produced by a corresponding method without introducing said same ORF or introducing said same ORF expressing vector or transfecting said same ORF expressing vector.

[0017] E4. The method of any one of E1 to E3 wherein said ORF encodes a protein related to cell cycle regulation, cell fate and / or differentiation, DNA repair, epigenetic modulation, immune response, metabolism, protein homeostasis RNA processing, signal transduction or transcription.

[0018] E5. The method of any one of 1 to 4 wherein the viral genome titer is measured by qPCR or ddPCR; and / or potency is measured by a cell-based transduction assay.

[0019] E6. The method according of any one of E1 to E6, wherein said increase is at least 1 .05 fold, at least 1.1 fold, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1 .9, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold or at least 6 fold compared to a corresponding method without introducing said same ORF or same ORF expressing vector or without transfection with said same ORF expressing vector.

[0020] E7. A method for improving and / or increasing the viral genome titer, and / or potency of recombinant adeno-associated virus (rAAV) particles produced during recombinant AAV production, wherein said method comprises i)a step of introducing a human ORF to a host cell under suitable conditions so that said ORF molecule is overexpressed, ii)a step of culturing said host cell line under suitable conditions to produce said rAAV, and wherein said increase is at least 1 .05 fold, at least 1.1 fold, at least 1 .2, at least 1 .3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold or at least 6 fold compared to a corresponding method without introducing said same ORF.

[0021] E8. The method of E7, wherein said method comprises i)a step of introducing a human ORF or introducing a human ORF expressing vector, preferably by transfecting a host cell with a human ORF expressing vector, under suitable conditions so that said ORF is overexpressed ii)a step of transfecting said cell with a recombinant AAV production plasmid system, preferably a two -plasmid system rAAV production system or synthetic DNA molecules suitable for AAV production iii)a step of culturing said host cell under conditions suitable for recombinant AAV production iv)a step of obtaining recombinant AAV particles from said transfected cells, and wherein said increase is at least 1 .05 fold, at least 1.1 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold or at least 6 fold compared to a corresponding method without introducing said same ORF or introducing said same ORF expressing vector or transfection with said same ORF expressing vector.

[0022] E9. The method of any one of E1 to E8 wherein

[0023] (a) said ORF is exogenously or endogenously expressed in the host cell;

[0024] (b) the ORF is transiently or stably expressed in the host cell; and / or

[0025] (c) said ORF is transiently, conditionally, or permanently overexpressed in the host cell

[0026] E10. The method of any one of E1 to E9, wherein said ORF comprises or consists of the sequence of any one of SEQ ID NOs: 1 to 29

[0027] E11 . The method of any one of E1 to E10 wherein said 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;

[0028] (b) a HEK293 cell, a HEK293T cell, a HEK293SF cell or a HEK293-F cell;

[0029] (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;

[0030] (d) derived from a HEK293 cell, a HEK293T cell, a HEK293SF cell or a HEK293-F cell; or e) a sf9 cell, a sf21 cell or a High five cell

[0031] E12. The method of E11 , wherein said host cell is a HEK293 cell line, preferably a suspension HEK293 cell line.

[0032] E13. The method of anyone of E1 to E12 wherein the rAAV particles comprise a capsid protein of:

[0033] (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.rhW, 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;

[0034] (b) the AAV8 or AAV9 serotype;

[0035] (c) the AAV8 serotype;

[0036] (d) the AAV9 serotype;

[0037] (e) a non-human AAV serotype or

[0038] (f) an engineered AAV preferably an engineered serotype of any one of (a)-(e).

[0039] E14. A preparation comprising rAAV particles obtained or obtainable by the method of any one of E1 to E13

[0040] E15. A preparation according to E14 for use in a method of treating or preventing a disease.

[0041] E16. A cell line for use in a method according to any one E1 to E13, wherein said cell expresses an ORF, preferably said ORF comprising or consisting of the sequence of any one of SEQ ID NOs: 1 to 29, preferably the sequence of any one of SEQ ID NOs:1 to 5.

[0042] E17. Use of a preparation according to E14 or E15 or a cell line according to E16 for the manufacture of a medicament for treating or preventing a disease preferably said disease being a genetic disorder. Brief Description of the Figures

[0043] Figure 1 (A-C). Human ORF screen identifies pathways improving AAV9 production. Fig. 1A shows a schematic of the experimental design. The ORF expression screening vector includes an EF1 a promoter driving the expression of the ORF. Arrayed ORFs were co -transfected individually with the split plasmid system. AAV production was evaluated by quantifying assembled capsids and measuring viral functional titer. Fig. 1 B shows the results from the primary screen. Absolute capsid titer and functional AAV titer were normalized to the plate average and plotted. Hits were defined as having a 3-fold increase in both capsid and functional titers as measured by a cellbased functional titer assay, resulting in a hit rate of 0.8%. Fig. 2C is a pie chart showing that the majority of ORF hits from the primary screen can be clustered into 9 pathways or gene functions: cell cycle regulation, cell fate & differentiation, DNA repair, epigenetic modulation, immune response, metabolism, protein homeostasis RNA processing, signal transduction or transcription. The remainder of the ORFs cluster to the target class “other”.

[0044] Figure 2 (A-B). Secondary screening of ORFs identifies numerous ORFs that, when over-expressed, enhance AAV9 production. Fig. 2A: The top 140 ORFs from the primary screen were distributed across three 96-well plates and overexpressed during AAV production to assess their impact on AAV yield, with each ORF tested on eight independent plates. AAV9 production was measured using capsid ELISA (capsid titer) and qPCR (vector genome titer). Overexpression of 29 ORFs resulted in an increase in both capsid and vector genome titers compared to a group that contains on 3rd plasmid (no ORF). ORFs that had the most significant impact on AAV production include RARA, OSBPL5, SMARCAL1 , ZNF385B, and MAFB are highlighted. Fig. 2B: The top 11 ORFs identified from the secondary screen were arrayed and overexpressed during AAV production in 96-well suspension cultures. All tested ORFs resulted in a significant increase in AAV9 production compared to the GFP control vector. . Data are shown as means ± SEM.

[0045] Figure 3 (A-C). Overexpression of top ORFs during AAV2, AAV5 and AAV8 production increases yield. Figures 3A-C show that co-transfection of the top ORFs with the two-plasmid split system in 96-well suspensions increased AAV production in two three different AAV serotypes, assessed by vector genome concentration measured by qPCR. ZNF85B expression led to a modest, nonsignificant increase in AAV2 (Fig. 3A), AAV5 (Fig. 3B), and AAV8 (Fig. 3C) yields. Overexpression of SMARCAL1 significantly enhanced AAV5 and AAV8 production. RARA overexpression significantly increased AAV production across all tested serotypes, while OSBPL5 and MAFB overexpression led to significant increases in AAV5 and AAV8. Data are shown as means ± SEM. Statistical analysis was performed using One-Way ANOVA with each group compared to the GFP control. *P < 0.05, **P < 0.01 , ***P < 0.001 , and ****p < 0.0001.

[0046] Figure 4 (A-F). Overexpression of the top ORFs increase AAV9 production in a dosedependent manner. Figure 4A shows a schematic illustration of the minimal expression vector used, with ORF expression driven by a strong ubiquitous CMV promoter. Figures 4B-F illustrate the dosedependent effect of overexpression of the top ORFs on AAV9 production by varying plasmid DNA amounts from 0.13 pg / cell to 4pg / cell. MAFB (Fig. 4B), RARA (Fig. 4D), SMARCAL1 (Fig. 4E), and OSBPL5 (Fig. 4F) exhibited dose-dependent increases in yield up to 0.5 pg of plasmid per cell, while ZNF385B (Fig. 4C) reached maximal yield at 1 pg per cell. Vector genome data was normalized to the no ORF control and shown as means ± SEM. Statistical analysis was performed using One-Way ANOVA with each group compared to the no ORF group. *P < 0.05, **P < 0.01 , ***P < 0.001 , and 0.0001.

[0047] Figure 5. MAFB, ZNF385B, RARA, SMARCAL1, and OSBPL5 significantly enhance AAV9 production in shake flasks compared to a GFP control vector. Co-transfection of 2pg / cell of each ORF expression vector concurrently with the two-plasmid split system led to a significant increase in AAV9 titer as assessed by AAV9 capsid titer measured from crude lysate. Data are shown as means ± SEM. Statistical analysis was performed using One-Way ANOVA with each group compared to the GFP control. ****P < 0.0001 .

[0048] Figure 6 (A-C). Overexpression of MECP2 during AAV9 production in an Ambr15 microbioreactor leads to increased yields. HEK293 cells cultured in BalanCD medium supplemented with L-glutamine were co-transfected using PEIpro with the split two-plasmid system and either an CMV expression vector driving expression of GFP or MECP2. Expression of MECP2 resulted in a significant increase in (Fig, 6A) vector genome yield and (Fig. 6B) capsid yield, as well as a decrease in (Fig. 6C) host cell DNA impurities. Data are shown as means ± SD. Statistical analysis was performed using Student’s t test.

[0049] Detailed Description of the Invention

[0050] Since 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 deliver more efficiently to the desired tissue type and de-target away from non-desired tissues for improved safety.

[0051] 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. The inventors of the present disclosure have surprisingly observed, after extensive experimentation and utilizing ATLAS (Arrayed Targeted Library for AAV Screening) miniaturized screening platform which allows for overexpression of approximately 18,000 ORFs, that several human ORFs when overexpressed during the preparation of rAAVs, lead to increased viral genome titer, capsid titer, and potency.

[0052] Therefore, it is a subject of the present disclosure to provide methods for producing a preparation comprising recombinant adeno-associated virus (rAAV) particles wherein an Open Reading Frame (ORF) is introduced to a host cell under suitable conditions, so that said ORF is overexpressed, and wherein said preparation comprises rAAV particles have improved and / or increased viral genome titer, capsid titer, and / or potency. The disclosure also provides preparations comprising recombinant adeno-associated virus (rAAV) particles having improved and / or increased viral genome titer, capsid titer, and / or potency.

[0053] General Definitions

[0054] Unless 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.

[0055] In general, the term “comprising” is intended to mean including but not limited to. For example, the phrase a method is one “comprising” particular 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.

[0056] The terms “protein” and “polypeptide” are used interchangeably herein, and are intended to refer to a polymeric chain of amino acids of any length.

[0057] 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).

[0058] 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: 30, SEQ ID NO: 30 would be the reference sequence. To assess whether a sequence is at least 80% identical to SEQ ID NO: 30 (an example of a reference sequence), the skilled person would carry out an alignment over the length of SEQ ID NO: 30, and identify how many positions in the test sequence were identical to those of SEQ ID NO: 30. If at least 80% of the positions are identical, the test sequence is at least 80% identical to SEQ ID NO: 30. If the sequence is shorter than SEQ ID NO: 30, the gaps or missing positions should be considered to be non-identical positions.

[0059] 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.

[0060] 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.

[0061] 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 wherein said same ORF is not overexpressed” or “equivalent method wherein said same ORF is not overexpressed’ is a method which is identical to a method of the invention, except that the same ORF of the invention is not overexpessed.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] The singular forms “a”, “an", and “the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an Open Reading Frame" includes two or more instances or versions of an Open Reading Framer. "AAV" is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or "rAAV vector").

[0066] An "rAAV vector" as used herein refers to an AAV vector comprising a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a sequence of interest for the genetic transformation of a cell. In preferred vector constructs of this invention, the heterologous polynucleotide is flanked by at least one, preferably two AAV inverted terminal repeat sequences (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids.

[0067] An "AAV virus" or "AAV viral particle" refers to a viral particle composed of at least one AAV capsid protein (preferably by all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "rAAV vector particle" or simply an "rAAV vector".

[0068] "Packaging" refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle.

[0069] AAV "rep" and "cap" genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus. They have been found in all AAV serotypes examined and are in the art. AAV rep and cap are referred to herein as AAV "packaging genes".

[0070] A "helper virus" for AAV refers to a virus that allows AAV (e.g. wild-type AAV) to be replicated and packaged by a mammalian cell. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV): which are also available from depositories such as ATCC.

[0071] 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, Algawlaa S. Lee EA, Foldvari M, Spagnuolo PA, Slavcev RA. (2014), Mol Ther Nucleic 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 “ helped is not intended to be limiting. Accordingly, a “ helper plasmid’ is any plasmid that comprises at least one rep gene encoding at least one functional Rep protein and may or may not comprise a cap gene encoding a functional set of Cap proteins.

[0072] The term “three or triple plasmid AAV production system" refers to a rAAV production system which involves the transfection of host cells with three separate plasmids, namely a rAAV vector plasmid which contains a promoter, a gene of interest (GOI) flanked by inverted terminal repeats (ITRs), which are essential for AAV replication and packaging, a rAAV helper plasmid carrying the Rep and Cap genes, which encode the replication and capsid proteins of AAV, respectively and an adenovirus helper plasmid (Ad helper) containing the necessary adenoviral genes, such as, E2a, E4orf6, and VA RNA, which are required to support AAV replication and packaging. Currently, there are available several variations of the above-mentioned system in the art, which are herein incorporated by reference.

[0073] The term “two-plasmid AAV production system” in the context of the current disclosure refers to a system that comprises only two plasmids and can be used without the need for additional plasmids to produce rAAV. Optionally, the two-plasmid system can be used to produce rAAV without the need for helper virus such as adenovirus. Optionally, the two plasmid system can be used to produce rAAV without the need for genetic material originating from a host cell, optionally with the exception of a gene encoding E1A / B. However, the system may comprise additional non-plasmid components. Optionally, the two plasmid system does not comprise a helper virus. Optionally, the two plasmid system of the invention comprises all the necessary genetic information for the production of rAAV. For example, the two plasmid system of the invention may comprise at least one rep gene, at least one cap gene and at least one helper gene. Optionally, the two plasmid system of the invention comprises all the necessary genetic information required for the production of rAAV suitable for use in gene therapy. For example, the two plasmid system of the invention may comprise at least one rep gene, at least one cap gene, at least one helper gene and an expression cassette comprising a transgene operably linked to at least one regulatory control element. However, in embodiments the two plasmid system of the invention may lack a functional cap gene (required for the production of rAAV) and / or an expression cassette comprising a transgene operably linked to at least one regulatory control element (required for the production of rAAV suitable for use in gene therapy). Suitably, a two plasmid system of the invention comprises a helper plasmid comprising at least one AAV rep gene encoding at least one functional AAV Rep protein and at least one helper virus gene, and which does not comprise a cap gene encoding a functional set of Cap proteins and a vector plasmid comprising (a) an AAV cap gene encoding at least one functional AAV Cap protein; or (b) at least one AAV cap gene promoter, a cloning site operably linked to the AAV cap gene promoter, and an expression cassette flanked on at least one side by an inverted terminal repeat (ITR); wherein the vector plasmid does not comprise a rep gene encoding a functional Rep protein and the expression cassette comprises a transgene operably linked to at least one regulatory control element. Suitable two plasmid systems are described in WO 2020 / 208379 A, EP3722434 B1 and WO 2022 / 079429 A1 (incorporated by reference). It is an advantage of the present invention that a cap gene and / or a transgene in the vector plasmid may be exchanged with another in order to treat different genetic disorders. Optionally, therefore, the two plasmid system of the invention may comprise all the necessary genetic information for the production of rAAV except a functional cap gene, and in such embodiments the two plasmid system of the invention may comprise a site suitable for cloning in a cap gene. Such a site may comprise a cloning site adjacent to a cap gene promoter. The site suitable for cloning in a cap gene will be present on the vector plasmid. Optionally, the two plasmid system of the invention comprises all the necessary genetic information for the production of rAAV suitable for use in gene therapy except a functional cap gene and an expression cassette comprising a transgene and a regulatory control element,

[0074] The term “nucleic acid molecule" refers to a polymeric form of nucleotides of any length. The nucleotides may be deoxyribonucleotides, ribonucleotides or analogs thereof. Preferably, the plasmid is made up of deoxyribonucleotides or ribonucleotides. Even more preferably, the plasmid is made up of deoxyribonucleotides, i.e. the plasmid is a DNA molecule. In all instances herein, the term “nucleic acid sequence" may be replaced by the term “polynucleotide".

[0075] The terms “potency”, “potent titer” or “functional titer” may be used interchangeably in the context of the present disclosure. 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).

[0076] The terms “wild type" and “native" are synonymous and refer to genes present in the genome of a strain / serotype of AAV or adenovirus, or to proteins encoded by genes present in the genome of a strain / serotype of AAV or adenovirus.

[0077] The helper plasmid may be useful for producing rAAV. Optionally, the helper plasmid is suitable for use in producing rAAV. Optionally, the helper plasmid is for producing rAAV. Optionally, the helper plasmid is suitable for producing rAAV suitable for use in gene therapy. Optionally, the helper plasmid is for producing rAAV for use in gene therapy.

[0078] Transcription regulatory elements are nucleotide sequences which effect the level of expression of a gene, and include, for example, promoters, enhancers, introns, untranslated regions, and transcriptional terminators. Some transcription regulatory elements promote greater levels of transcription compared to others (stronger transcription regulatory elements). For example, some promoters are known to promote transcription at a higher level than others. Generally, a promoter will be a stronger promoter if it comprises a sequence that allows for strong binding to the transcription complex. Promoters which are known to be generally strong promoters in human cells include viral promoters. Promoters that are generally believed to be strong promoters in human cells include the EF1A, CMV, CAG and SV40 promoters. Promoters that are generally believed to be weak promoters in human cells include UBC and PGK promoters. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0079] Preparation comprising recombinant AA V

[0080] A “preparation" is a solution produced by any of the methods of the present invention. Optionally, a preparation may comprise recombinant AAV. “ Recombinant AAV” or “rAA V” 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.

[0081] 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.

[0082] AA V production assay

[0083] An AAV production assay may be used to test whether certain features of the methods of the invention allow for suitable AAV production.

[0084] 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.

[0085] 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: 31. Details of the nucleic acid positions in SEQ ID NO: 31 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: 30.

[0086] The reference host cell also comprises a wildtype cap gene operably linked to a wildtype cap gene promoter comprising p5, p19 and p40, i.e. the cap gene comprised within SEQ ID NO: 30 (nucleotides 5961 -8171 of SEQ ID NO: 30). 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: 30.

[0087] 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 ORF improves viral genome titer, and / or potency in a preparation comprising rAAV, the user could compare the viral genome titer, capsid titer and / or potency of an rAAV produced from a host cell wherein said ORF is not overexpessed (i.e. the “reference host cell") and a host cell that overexpresses thesame modulator (i.e. the “test host cell”).

[0088] 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.

[0089] Host cells

[0090] The present invention relates to methods for producing a preparation comprising recombinant adeno-associated virus (rAAV) particles, wherein a host cell overexpresses an ORF. The method may result in improved viral genome titer, capsid titer and / or potency, compared to a preparation comprising rAAV using a corresponding method without the modulator.

[0091] AAV are viruses that are useful in applications such as gene therapy and can 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.

[0092] 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.

[0093] 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.CR 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:

[0094] (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;

[0095] (b) a HEK293 cell, a HEK293T cell, a HEK293SF cell or a HEK293-F cell;

[0096] (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

[0097] (d) derived from a HEK293 cell, a HEK293T cell, a HEK293SF cell or a HEK293-F cell.

[0098] (e) a sf9 cell, a sf21 cell or a High five cell

[0099] 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.

[0100] Open Reading Frames

[0101] The invention relates to a method for producing a preparation comprising recombinant adeno-associated virus (rAAV) particles, wherein the method comprises a step of introducing an Open Reading Frame (ORF) to a host cell under suitable conditions, so that said ORF is overexpressed, a step of culturing said host cell under suitable conditions to produce the preparation; and wherein said preparation comprises rAAV particles having improved and / or increased viral genome titer, capsid titer, and / or potency compared to a preparation produced by a corresponding method without introducing said same ORF.

[0102] An open reading frame (ORF) refers to the part of a gene or polynucleotide that has the potential to be transcribed and / or translated. ORFs span intron / exon regions, which in some embodiments can be spliced together after transcription of the ORF to yield a final mRNA for protein translation. Thus, ORFs include both introns and exons, when applicable. In some embodiments, an ORF is a continuous stretch of codons that contain a start codon and a stop codon. In some embodiments, the transcription termination site is located after the ORF, beyond the translation stop codon. In some embodiments an ORF is a continuous segment of DNA beginning with an initiation codon, methionine ATG, and ending with one of the three termination codons, TAA, TAG, or TGA, that is coded into a polypeptide chain or a protein. An ORF contains the coding sequence of a gene (CDS) and lacks both the 5' and 3' UTRs. ORFs are inserted into expression vectors (containing an artificial stop codon) and their expression may be modulated (overexpressed) to understand the corresponding gene / protein function. ORFs also play an important role in RNA interference and CRISPR rescue experiments, where gene expression is restored (gain-of-function) for gene validation. For example, the Mission® TRC3 arrayed ORF library (Human LentiORFs) is a collection of sequenced and pre-cloned human open reading frames which allows for stable integration, enrichment of cells, and long-term gene expression indifficult-to-transfect cell lines utilizing lentiviral vectors. This Human Lentiviral ORF Pool consists of plasmid-based, puromycin resistant ORF constructs expressing over 14,000 human genes. On average, there are 1 .2 ORFs for each gene in the ORFeome pool. The ORF plasmids are further processed into lentiviral particles to facilitate stable gene expression in both dividing and quiescent cells.

[0103] ORFs in the context of the present disclosure may refer to gene functions such as cell cycle regulation, cell fate & differentiation, DNA repair, epigenetic modulation, immune response, metabolism, protein homeostasis RNA processing, signal transduction or transcription.

[0104] ORFs can be used to “turn on” or “overexpress” particular genes e.g. flipping the switches on genes one at a time can help reveal the functions of individual genes, such as those that play a role in cancer. Overexpression of genes and proteins is widely used in functional genomics, proteomics, and system biology studies. Table 1 , in Example 2 below, shows particular examples of Open Reading Frames.

[0105] Open reading frame (ORF) overexpression is the artificial increase in expression of a gene’s full coding sequence relative to its native (endogenous) expression level in the same organism or cell type. This is typically achieved by placing the ORF under the control of a stronger or inducible promoter, resulting in substantially higher mRNA and protein abundance than would occur under physiological conditions. The ORF(s) of the invention may be exogenously and / or endogenously expressed in the host cell. When endogenously expressed, the host cell may already stably or transiently express the ORF. For example, the genetic material encoding the ORF may already be present in the genome or episomally in the host cell. Alternatively, or in addition, the ORF may be exogenously loaded into the host cell, for example using any suitable exogenous loading system such as: electroporation, transfection with transfection reagents etc. Exogenous loading may result in stable or transient expression of the ORF in the host cell. Thus, by way of example, the ORF may be transiently, conditionally, or permanently overexpressed in the host cell.

[0106] Cell culture

[0107] The 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.

[0108] 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 “carrier 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.

[0109] 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.

[0110] 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.

[0111] Host cell capable of propagating AA V

[0112] As 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 cells with a virus. For example, the HEK293T cell line comprises an E1A 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.

[0113] 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:

[0114] (i) a rep 52 gene;

[0115] (ii) a rep 40 gene;

[0116] (iii) a rep 68 gene;

[0117] (iv) a cap gene;

[0118] (v) a viral associated (VA) nucleic acid;

[0119] (vi) an E2a gene;

[0120] (vii) an E4 gene;

[0121] (viii) an ElA gene; and / or

[0122] (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. HSV.

[0123] In some embodiments, the synthetic DNA molecule may be a doggybone or a DNA minicircle.

[0124] 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 transfected with 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.

[0125] 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. HSV.

[0126] Infecting host cells with one or more viruses may comprise exposing the host cells to the one or more viruses in conditions suitable for infection.

[0127] 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.

[0128] Expression cassette

[0129] An “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.

[0130] 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.

[0131] The transgene may be any suitable gene. The transgene may encode a protein or a nontranslated 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.

[0132] 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 repeat

[0133] In 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.

[0134] Helper genes

[0135] AAV 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) ElA 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) E1 A 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.

[0136] 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: 31 , and the genome of Adenovirus 2 is set out in SEQ ID NO: 32. Accordingly, the helper genes may comprise a stretch of nucleotides present in SEQ ID NO: 31 or SEQ ID NO: 32, or a corresponding stretch of nucleotides in another serotype of adenovirus.

[0137] The helper genes of adenoviruses encode E1A, E1 B, E4, E2A and VA RNA I and II.

[0138] E1A is encoded by nucleotides 560-1545 of the Adenovirus 5 genome (for example the genome of SEQ ID NO: 31 ). Nucleotides 560-1545 contain an intron, from nucleotide 1113 to nucleotide 1228. This intron is not essential, and so an E1A gene comprising nucleotides 560-1112 and 1229-1545 of SEQ ID NO: 2 would encode a functional E1A protein.

[0139] 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: 31 ).

[0140] 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: 31 ). 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: 31 ). E4 ORF 4 is encoded by nucleotides 33998-34342 of the Adenovirus 5 genome (for example the genome of SEQ ID NO: 31 ). E4 ORF 3 is encoded by nucleotides 34353- 34703 of the Adenovirus 5 genome (for example the genome of SEQ ID NO: 31 ). E4 ORF B is encoded by nucleotides 34700 to 35092 of the Adenovirus 5 genome (for example the genome of SEQ ID NO: 31 ). E4 ORF 1 is encoded by nucleotides 35140-35526 of the Adenovirus 5 genome (for example the genome of SEQ ID NO: 31 ).

[0141] 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.

[0142] The E2 (E2A) gene is encoded by nucleotides 22443-24032 of the Adenovirus 5 genome (for example the genome of SEQ ID NO: 31 ).

[0143] 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: 31 ).

[0144] Rep genes

[0145] The 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.

[0146] 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:

[0147] (a) a functional (AAV) Rep 52 protein, i.e. a rep 52 gene;

[0148] (b) a functional (AAV) Rep 40 protein, i.e. a rep 40 gene; and / or

[0149] (c) a functional (AAV) Rep 68 protein, i.e. a rep 68 gene.

[0150] 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.

[0151] 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.

[0152] 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).

[0153] 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).

[0154] 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.

[0155] SEQ ID NO: 30 provides the sequence of the genome of wild type AAV2, and nucleotides 321 -2252 of SEQ ID NO: 30 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 (i.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: 30 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.

[0156] 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: 30, or to a corresponding stretch of nucleotides in a different serotype of AAV.

[0157] 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 (i.e. SEQ ID NO: 30). 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: 30, 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: 31 ). 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: 30, or to corresponding stretches of nucleotides in a different serotype of AAV. Hence, such a rep 40 gene has at least the above-specified identity to a notional stretch of nucleotides consisting of nucleotides 993-1906 of SEQ ID NO: 30 immediately juxtaposed with nucleotides 2228-2252 of SEQ ID NO: 30 (5’-[993-1906]-[2228-2252]-3’) or to a notional stretch of nucleotides from a different AAV serotype.

[0158] 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: 30, or to a corresponding stretch of nucleotides in a different serotype of AAV.

[0159] 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: 30, 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: 30 immediately juxtaposed with nucleotides 2228-2252 of SEQ ID NO: 30 (5’-[321 -1906]-[2228-2252]-3’), or to a notional stretch of nucleotides from a different AAV serotype.

[0160] Optionally, the one or more plasmids and / or host cells comprise a gene encoding functional Rep 68 and Rep 40 proteins (i.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: 30) 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.

[0161] 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: 30): 658-2300, or to a corresponding stretch of nucleotides from a different serotype of AAV.

[0162] 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 afragment of at least 3000, 3200, 3300 or 3400 nucleotides in length of the following stretches of native AAV2 sequence (SEQ ID NO: 30) 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.

[0163] 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: 30, or within 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.

[0164] Cap gene

[0165] The 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.

[0166] A “functionaF 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 “functional" 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 wild-type cap gene product.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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).

[0171] In one embodiment of the invention, the rAAV particles comprise a capsid protein of:

[0172] (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.rhW, 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;

[0173] (c) the AAV8 serotype;

[0174] (d) the AAV9 serotype;

[0175] (e) a non-human AAV serotype

[0176] (or

[0177] (f) an engineered AAV preferably an engineered serotype of any one of (a)-(e).

[0178] Sufficient genetic material

[0179] The 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:

[0180] (i) a rep 52 gene;

[0181] (ii) a rep 40 gene;

[0182] (iii) a rep 68 gene;

[0183] (iv) a cap gene;

[0184] (v) a VA nucleic acid;

[0185] (vi) an E2a gene;

[0186] (vii) an E4 gene;

[0187] (viii) an E1A gene; and

[0188] (ix) a polynucleotide comprising an expression cassette comprising a transgene between the two ITRs.

[0189] 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 titer using qPCR, for example, as described under the heading Viral genome titer assay or detecting the capsid titer using an ELISA, for example, as described under the heading Capsid titer 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.

[0190] 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 E1 A gene.

[0191] 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).

[0192] 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 titer

[0193] The methods of the invention may result in a preparation that comprises recombinant AAV at an improved, increased or higher viral genome titer. Viral genome titer 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 titer 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 titer when compared to a preparation comprising recombinant AAV produced by a corresponding method, such as a corresponding method in which an ORF of the invention is not overexpressed. In one embodiment, the method according to the invention produces a preparation comprising recombinant AAV having an improved, increased or higher viral genome titer. 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 titer, the viral genome titer of the preparation should be measured once the method been completed, i.e. after overexpression of an ORF and if the method or use comprises further subsequent steps once those further subsequent steps have been completed, and compared to the viral genome titer of a preparation made using an equivalent method in which an ORF of the invention is not overexpressed

[0194] 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 titer when compared to a preparation comprising recombinant AAV produced by a corresponding method in which an ORF of the invention is not overexpressed

[0195] In another embodiment, the method according to the invention is a method for improving or increasing the viral genome titer of a preparation comprising recombinant AAV.

[0196] In another embodiment, the method according to the invention is a method for improving or increasing the viral genome titer 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 which an ORF of the invention is not overexpressed

[0197] Viral genome titer assay

[0198] The skilled person will understand that there are many suitable methods for determining the viral genome titer of a preparation comprising recombinant AAV. Viral genome titer may be measured using digital droplet polymerase chain reaction (ddPCR). Viral genome titer may be measured by the quantitative 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 titer 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 titer 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.

[0199] Cell lysate test samples may be subjected to a nuclease treatment procedure in order to remove non-packed vector genomes prior to performing qPCR or ddPCR.

[0200] “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 (i.e., a plurality of such compartments). Typically, a “droplet” refers to water-in-oil droplet (i.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.

[0201] 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. Examples of droplet digital PCR systems include the QX100™ Droplet Digital PCR System by Bio-Rad, which partitions samples containing nucleic acid template into 20,000 nanolitresized 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.

[0202] Increased capsid titer

[0203] Capsid titer is the concentration of capsid particles present in a preparation. For example, if the preparation comprises rAAV, the capsid titer is the concentration of AAV capsid particles in the preparation. The preparation comprising recombinant AAV produced when a host cell is engineered so that an ORF of the invention is overexpressed have increased or increased capsid titer when compared to a preparation comprising recombinant AAV produced by a corresponding method in which the same ORF is not overexpressed.

[0204] In one embodiment, the method according to the invention produces a preparation comprising recombinant AAV having increased or improved capsid titer. The preparation comprising recombinant AAV is the preparation that is produced by the methods.

[0205] 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 titer when compared to a preparation comprising recombinant AAV produced by a corresponding method in which an ORF of the invention is not overexpressed.

[0206] In another embodiment, the method or use according to the invention is a method or use for increasing the capsid titer of a preparation comprising recombinant AAV.

[0207] In another embodiment, the method or use according to the invention is a method or use for increasing the capsid titer 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 an ORF of the invention is not overexpressed.

[0208] In the context of the present disclosure an “improved and / or increased capsid titer” is used as an indication of “improved and / or increased genome titer”.

[0209] Capsid titer assay

[0210] The skilled person will understand that there are many suitable methods for determining the capsid titer of a preparation comprising recombinant AAV. Capsid titer 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 titer is measured by ELISA.

[0211] Increasing the potency of recombinant AA V produced during recombinant AA V production

[0212] The present invention provides a method for increasing the potency of recombinant AAV produced during recombinant AAV production.

[0213] 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 preselected 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.

[0214] 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 FVIIIa polypeptide 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).

[0215] 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.

[0216] 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 an ORF of the invention is not overexpressed.

[0217] 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.

[0218] Endonuclease treatment

[0219] An 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.

[0220] 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.

[0221] The methods according to the present invention may further comprise a step of endonuclease treatment.

[0222] Depth filtration

[0223] The 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 titers. In addition to removing impurities depth filtration of the preparation will improve the purity / quality of the preparation.

[0224] 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.

[0225] 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:

[0226] Volume (LI

[0227] Flux (LMH) = - — - - - - r

[0228] Time (h) * Surface area (cm2) wherein the volume is the total preparation volume and the surface area is the surface area of the filter.

[0229] 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.

[0230] 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 pm. Optionally, the filter is a COHC filter. The step of depth filtration may increase the viral genome titer of the preparation comprising recombinant AAV.

[0231] Viral genome / capsid ratio

[0232] The 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 titer assay), and using a capsid-specific ELISA to measure the total number of particles (as discussed in the section headed Capsid titer assay). Optionally, the viral genome / capsid ratio may be calculated by dividing the viral genome titer as measured by qPCR by the capsid titer as measured by ELISA and multiplying the result by 100%, wherein the units, in which the viral genome titer and the capsid titer are expressed, are the same.

[0233] Methods of the present invention may increase capsid titer more than the viral genome titer, 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 an ORF of the invention is not overexpressed.

[0234] 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 an ORF of the invention is not overexpressed.

[0235] 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 an ORF of the invention is not overexpressed.

[0236] The step of depth filtration may increase the viral genome / capsid ratio of the preparation comprising recombinant AAV.

[0237] 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.

[0238] 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%.

[0239] 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%.

[0240] Reduced turbidity

[0241] Turbidity 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 impurities present 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.

[0242] 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.

[0243] Sterile filtration

[0244] The 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.

[0245] Purifying the recombinant AAV

[0246] The 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.

[0247] 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.

[0248] 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 compositions

[0249] The 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.

[0250] 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.

[0251] 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.

[0252] Methods of treatment

[0253] Further 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.

[0254] 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.

[0255] 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).

[0256] Specifically, the present disclosure provides the following aspects, advantageous features and specific embodiments, respectively alone or in combination:

[0257] E1. A method for producing a preparation comprising recombinant adeno-associated virus (rAAV) particles, wherein said method comprises i) a step of expressing Open Reading Frame (ORF) to a host cell under suitable conditions, so that said ORF is overexpressed, ii)a step of culturing said host cell under suitable conditions to produce the preparation; E1 a. The method of E1 for producing a preparation comprising recombinant adeno-associated virus (rAAV) particles, wherein said method comprises i)a step of introducing a human Open Reading Frame (ORF) to a host cell under suitable conditions, so that said ORF is overexpressed, ii)a step of culturing said host cell under suitable conditions to produce the preparation;

[0258] E2. The method of E1 or E1 a wherein said method comprises i)a step of introducing a human Open Reading Frame (ORF) to a host cell under suitable conditions, so that said ORF is overexpressed, ii)a step of culturing said host cell under suitable conditions to produce the preparation; and wherein said preparation comprises rAAV particles having improved and / or increased viral genome titer and / or potency compared to a preparation produced by a corresponding method without introducing said same ORF.

[0259] E3. The method of E1 to E2, wherein said method comprises i)a step of introducing a human ORF expressing vector to a host cell under suitable conditions, so that said ORF is overexpressed, ii)a step of culturing said host cell under suitable conditions to produce the preparation; and wherein said preparation comprises rAAV particles having improved and / or increased viral genome titer, and / or potency compared to a preparation produced by a corresponding method without introducing said same ORF expressing vector.

[0260] E4. The method of any one of E1 or E3, wherein said method comprises i)a step of transfecting a host cell with an ORF expressing vector under suitable conditions, so that said ORF is overexpressed, ii)a step of culturing said host cell under suitable conditions to produce the preparation; and wherein said preparation comprises rAAV particles having improved and / or increased viral genome titer, and / or potency compared to a preparation produced by a corresponding method without introducing said same ORF expressing vector.

[0261] E5. The method of any one of E1 to E4, wherein said method comprises the steps of i)introduci ng a human Open Reading Frame (ORF) to a host cell under suitable conditions, so that said ORF is overexpressed or introducing an Open Reading Frame (ORF) expressing vector to a host cell under suitable conditions, so that said ORF is overexpressed or transfecting a host cell with an ORF expressing vector under suitable conditions, so that said ORF is overexpressed, wherein said host cell is preferably a mammalian cell, more preferably a human cell. ii)transfecting said host cell with a recombinant AAV production plasmid system, preferably a three plasmid rAAV production system or a two plasmid rAAV production system, or synthetic DNA molecules suitable for AAV production iii)culturing said cell line under conditions suitable for recombinant AAV production iv)obtai ning recombinant AAV particles from said transfected cells, wherein said preparation comprises rAAV particles having increased viral genome titer and / or potency compared to a preparation produced by a corresponding method without introducing said same ORF, or without introducing said same ORF expressing vector or without transfection with said same ORF expressing vector.

[0262] E6. The method of any one of E1 to E5, wherein said host cell is transfected with a recombinant AAV production plasmid system, suitably a three plasmid rAAV production system

[0263] E7. The method of any one of E1 to E5, wherein said host cell is transfected with a recombinant AAV production plasmid system, suitably a two plasmid rAAV production system

[0264] E7. The method of any one of E1 to E5, wherein said host cell is transfected with synthetic DNA molecules suitable for AAV production

[0265] E8. The method of any one E1 to E7 wherein said ORF preferably encodes a protein related to cell cycle regulation, cell fate and / or differentiation, DNA repair, epigenetic modulation, immune response, metabolism, protein homeostasis RNA processing, signal transduction or transcription.

[0266] E9. The method of E8 wherein said ORF encodes a protein selected from the group consisting of MAFB, OSBPL5, RARA, ZNF385B, SMARCAL1 , HIF1A, PARVG, PAN3, TSSK2, POLD3, SPANXA2, SSX2, SMARCAL1 , H2AB2, DAXX, MECP2, NR1 D2, HOXC10, DHX29, NR1 H2, RARG, WDR55, USP10, NUP88, AKR1 C1 , BZW2, AK5, FNTA, and DCLK3

[0267] E10. The method of E9 wherein said ORF encodes a protein selected from the group consisting of RARA, OSBPL5, MAFB, SMARCAL1 and ZNF385B.

[0268] E11.The method of any one of E1 to E10 wherein the viral genome titer is measured by qPCR or ddPCR; and / or the functional titer is measured by a cell-based transduction assay.

[0269] E12. The method of any one of E2 to E11 , wherein said increase is at least 1.05 fold, at least 1.1 fold, at least 1 .2. fold, at least 1 .3 fold, at least 1 .4 fold, at least 1 .5 fold at, at least 1 .6 fold, at least 1 .7 fold, at least 1 .8 fold, at least 19 fold, at least 1 .9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold or at least 6 fold compared to a corresponding method without introducing said same ORF or same ORF expressing vector or without transfection with said same ORF expressing vector.

[0270] E13. The method of E12, wherein said increase is at least 1.05 fold, at least 1.1 fold, at least 1.2. fold, at least 1 .3 fold, at least 1 .4 fold, at least 1 .5 fold at, at least 1 .6 fold, at least 1 .7 fold, at least 1 .8 fold, at least 19 fold, at least 1 .9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold or at least 6 fold compared to a corresponding method without introducing said same ORF.

[0271] E14. The method of E12, wherein said increase is at least 1.05 fold, at least 1.1 fold, a at least 1.2. fold, at least 1 .3 fold, at least 1 .4 fold, at least 1 .5 fold at, at least 1 .6 fold, at least 1 .7 fold, at least 1 .8 fold, at least 19 fold, at least 1 .9 fold,t least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold or at least 6 fold compared to a corresponding method without introducing said same ORF expressing vector.

[0272] E15. The method of E12 wherein said increase is at least 1.05 fold, at least 1.1 fold, at least 1.2. fold, at least 1 .3 fold, at least 1 .4 fold, at least 1 .5 fold at, at least 1 .6 fold, at least 1 .7 fold, at least 1 .8 fold, at least 19 fold, at least 1 .9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold or at least 6 fold compared to a corresponding method without transfection with said same ORF expressing vector.

[0273] E16. A method for improving and / or increasing the viral genome titer and / or potency of recombinant adeno-associated virus (rAAV) particles produced during recombinant AAV production, wherein said method comprises i)a step of introducing a human ORF to a host cell under suitable conditions so that said ORF molecule is overexpressed, ii)a step of culturing said host cell line under suitable conditions to produce said rAAV, and wherein said increase is at least 1 .05 fold, at least 1.1 fold, at least 1 .2. fold, at least 1 .3 fold, at least 1 .4 fold, at least 1 .5 fold at, at least 1 .6 fold, at least 1 .7 fold, at least 1 .8 fold, at least 19 fold, at least 1 .9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold or at least 6 fold compared to a corresponding method without introducing said same ORF.

[0274] E17. The method of E16, wherein said method comprises i)a step of introducing a human ORF expressing vector to a host cell under suitable conditions so that said ORF molecule is overexpressed, ii)a step of culturing said host cell line under suitable conditions to produce said rAAV, and wherein said increase is at least 1 .05 fold, at least 1.1 fold, at least 1 .2. fold, at least 1 .3 fold, at least 1 .4 fold, at least 1 .5 fold at, at least 1 .6 fold, at least 1 .7 fold, at least 1 .8 fold, at least 19 fold, at least 1 .9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold or at least 6 fold compared to a corresponding method without introducing said same ORF expressing vector.

[0275] E18. The method of E16, wherein said method comprises i)a step of transfecting a human ORF expressing vector to a host cell under suitable conditions so that said ORF molecule is overexpressed, ii)a step of culturing said host cell line under suitable conditions to produce said rAAV, and wherein said increase is at least 1 .05 fold, at least 1.1 fold, at least 1 .2. fold, at least 1 .3 fold, at least 1 .4 fold, at least 1 .5 fold at, at least 1 .6 fold, at least 1 .7 fold, at least 1 .8 fold, at least 19 fold, at least 1 .9 fold,, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold or at least 6 fold compared to a corresponding method without introducing said same ORF expressing vector.

[0276] E19. The method of E16 wherein said method comprises i)a step of introducing a human ORF to a host cell under suitable conditions so that said ORF molecule is overexpressed, ii)a step of transfecting said cell with a recombinant AAV production plasmid system or synthetic DNA molecules iii)a step of culturing said host cell under conditions suitable for recombinant AAV production iv)a step of obtaining recombinant AAV particles from said transfected cells, and wherein said increase is at least 1 .05 fold, at least 1.1 fold, at least 1 .2. fold, at least 1 .3 fold, at least 1 .4 fold, at least 1 .5 fold at, at least 1 .6 fold, at least 1 .7 fold, at least 1 .8 fold, at least 19 fold, at least 1 .9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold or at least 6 fold compared to a corresponding method without introducing said same ORF

[0277] E20. The method of E16 wherein said method comprises i)a step of introducing a human ORF expressing vector to a host cell under suitable conditions so that said ORF molecule is overexpressed, ii)a step of transfecting said cell with a recombinant AAV production plasmid system or synthetic DNA molecules iii)a step of culturing said host cell under conditions suitable for recombinant AAV production iv)a step of obtaining recombinant AAV particles from said transfected cells, and wherein said increase is at least 1 .05 fold, at least 1.1 fold, at least 1 .2. fold, at least 1 .3 fold, at least 1 .4 fold, at least 1 .5 fold at, at least 1 .6 fold, at least 1 .7 fold, at least 1 .8 fold, at least 19 fold, at least 1 .9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold or at least 6 fold compared to a corresponding method without introducing said same ORF expressing vector. E21 . The method of E16 wherein said method comprises i)a step of transfecting a host cell with a human ORF expressing vector under suitable conditions so that said ORF molecule is overexpressed, ii)a step of transfecting said cell with a recombinant AAV production plasmid system or synthetic DNA molecules iii)a step of culturing said host cell under conditions suitable for recombinant AAV production iv)a step of obtaining recombinant AAV particles from said transfected cells, and wherein said increase is at least 1 .05 fold, at least 1.1 fold, at least 1 .2. fold, at least 1 .3 fold, at least 1 .4 fold, at least 1 .5 fold at, at least 1 .6 fold, at least 1 .7 fold, at least 1 .8 fold, at least 19 fold, at least 1 .9 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold or at least 6 fold compared to a corresponding method without transfection with said ORF expressing vector.

[0278] E22. The method of any one of E19 to E21 , wherein said recombinant AAV production plasmid system is a three plasmid AAV production system.

[0279] E23. The method of any one of E19 to E21 , wherein said recombinant AAV production plasmid system is a two plasmid AAV production system

[0280] E24. The method of E19 to E21 wherein said recombinant AAV production plasmid system is a two plasmid AAV production system and wherein said two plasmid system comprises helper plasmid and a vector plasmid, wherein the helper plasmid comprises at least one rep gene encoding at least one functional Rep protein and does not comprise a cap gene encoding a functional set of Cap proteins. E25. The method of any one of E19 to E24, wherein said 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.

[0281] E26. The method of E25, wherein said helper genes may be from another AAV helper virus known in the art preferably HSV

[0282] E27. The method of E25 or E26 wherein said synthetic DNA molecules are optionally doggybone DNA or minicircle DNA

[0283] E28. The method of any one of E1 to E27 wherein

[0284] (a) said ORF is exogenously or endogenously expressed in the host cell;

[0285] (b) the ORF is transiently or stably expressed in the host cell; and / or

[0286] (c) said ORF is transiently, conditionally, or permanently overexpressed in the host cell E29. The method according to any one of E1 to E28, wherein said ORF comprises or consists of the sequence of any one of SEQ ID NOs: 1 to 29, preferably any one of SEQ ID NOs: 1 to 5.

[0287] E30. The method according to any one of E1 to E28, wherein said ORF comprises the sequence of any one of SEQ ID NOs: 1 to 5

[0288] E31 . The method according to any one of E1 to E28, wherein said ORF consists of the sequence of any one of SEQ ID NOs: 1 to 5

[0289] E32. The method of any one of 1 to 31 wherein said 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;

[0290] (b) a HEK293 cell, a HEK293T cell, a HEK293SF cell or a HEK293-F cell;

[0291] (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;

[0292] (d) derived from a HEK293 cell, a HEK293T cell, a HEK293SF cell or a HEK293-F cell<

[0293] (e ) a sf9 cell or a sf21 cell or a high five cell

[0294] E33. The method of E32, wherein said host cell line is a HEK293 cell line, preferably a suspension HEK293 cell line.

[0295] E36. The method according to any one of E1 to E35 wherein wherein the rAAV particles comprise a capsid protein of:

[0296] (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.rhW, 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;

[0297] (b) the AAV8 or AAV9 serotype;

[0298] (c) the AAV8 serotype;

[0299] (d) the AAV9 serotype;

[0300] (e) a non-human AAV serotype or

[0301] (f) an engineered AAV preferably an engineered serotype of any one of (a)-(e).

[0302] E34. A preparation comprising rAAV obtained or obtainable by the method of any one of E1 to E33. E35 A preparation according to E34 for use in a method of treating or preventing a disease.

[0303] E36. A cell line for use in a method according to any one of E1 to E33 E37. The cell line of E36, wherein said cell line encodes an ORF, preferably said ORF comprising or consisting of the sequence of any one of SEQ ID NOs: 1 to 29, preferably any one of SEQ ID NOs: 1 to 5.

[0304] E38. Use of a preparation according to E34 or E35 or a cell line according to E36 or E37 for the manufacture of a medicament for treating or preventing a disease preferably said disease being a genetic disorder.

[0305] The following examples illustrate the invention.

[0306] Example 1 - Methods and Materials

[0307] Culturing suspension HEK293 cells

[0308] HEK293 cells were cultured in suspension using BalanCD HEK293 media (FUJIFILM Irvine Scientific) supplemented with 4 mM L-glutamine (Thermo Fisher Scientific). The cells were maintained at 37°C in a 5% CO2 incubator on an orbital shaker set to 125 rpm with a 19 mm orbital throw. The cells were observed for 7-10 days to ensure cell density reached between 2.5 and 5 million cells per mL and viability exceeded 95%, prior to initiating AAV production.

[0309] Split two-plasmid system

[0310] The two-plasmid (“split” plasmid system) system for recombinant AAV production consists of the first plasmid which contains the human Ad5 E4, E2A, and VA RNA genes, along with the AAV2 Rep gene that has a mutation to inactivate the P40 promoter and the second plasmid contains an expression cassette flanked by ITRs, which includes a CMV enhancer, a CMV promoter, b-Globin intron, inverted mCherry and nano luciferase reporter transgenes, a WPRE post-transcriptional regulatory element, and a bGH polyadenylation signal. Additionally, the second plasmid houses the AAV cap gene (e.g., AAV9), with its expression controlled by a mutant tandem promoter comprising the AAV2 P5 (with an ATG deletion corresponding to AAV2 nucleotides 321-323), P19 (with an ATG to ATT conversion corresponding to AAV2 nt 766-768, an ATG deletion at AAV2 nt 955-957, an ATG deletion at AAV2 nt 993-995, and a GTG deletion at AAV2 nt 1014-1016), and an unmodified P40 promoter.

[0311] AA V production in pseudo-adherent 96-well format

[0312] HEK293 cells were maintained in culture as previously described. 24 hours prior to transfection, 96- well flat bottom plates (Thermo Fisher) were coated with Fibronectin (Thermo Fisher) diluted 1 :1000 in PBS and incubated at 37°C in 5% CO2 for 30 minutes. After coating the plate, 50,000 cells per well were seeded in BalanCD HEK293 media and left to adhere overnight. On the day of transfection, 60 ng / well of pAX413 and 40 ng / well of pAX391 were combined in BalanCD HEK293 media. For groups requiring a third plasmid, an additional 50 ng / well of DNA from the third plasmid was included. The plasmid DNA mixture was then complexed with TransIT-VirusGEN® (Mirus; Catalog Number MIR6704) in a 2:1 ratio as per the manufacturer's instructions. After incubating the transfection complex at room temperature for 15 minutes, it was added to the 96-well plate and cells were further cultured at 37°C with 5% CO2 for 72 hours. Following incubation, the virus was released from the cells by subjecting the 96-well plate to three cycles of freezing at -80°C and thawing at 37°C.

[0313] Human Open Reading Frames (OTFs) library screen

[0314] One day prior to transfection, HEK293 cells were seeded and cultured in 96-well well format as previously described. On the day of transfection, 2.5uL (~50ng) of each well of the Mission® TRC3 arrayed ORF library (purchased by Sigma Aldrich) was complexed with 60 ng / well of the first and 40 ng / well of the second plasmid of the two plasmid rAAV production system in BalanCD HEK293 media. The plasmid DNA mixture was then complexed with TransIT-VirusGEN® in a 2:1 ratio as per the manufacturer's instructions. After incubating the transfection complex at room temperature for 15 minutes, it was added to the 96-well plate and cells were further cultured at 37°C with 5% COzfor 72 hours. Following incubation, the virus was released from the cells by subjecting the 96-well plate to three cycles of freezing at -80°C and thawing at 37°C. AAV production was quantified by measuring AAV9 capsid titer and functional titer (potency) using a cell-based assay. rAAV production in suspension cells ell 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 BalanCD HEK293 media to achieve a density of 2.5M / mL in a 30mL shake flask. The three plasmids were prepared in a 4:3:3 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 2.5M / mL. rAA 1 / production in bioreactor 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 4:3:3 molar mixture of helper, vector and ORF expression plasmid using the linear polyethylenimine transfection reagent PEIpro™ (Polyplus I 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. 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.

[0315] AA V harvest and purification

[0316] Three 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 to a 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).

[0317] 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 titer using ELISA

[0318] A Streptavidin coated high-capacity plate (Thermo Fisher Scientific; Catalog No. PI15500) 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 ELISA substrate (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 (Promega).

[0319] Determination of vector genome titer using qPCR

[0320] Purified 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 real-time PCR system (Thermo Fisher Scientific). To generate a standard curve, a six-point dilution series ranging from 2x1011cp / mL to 2x106cp / mL was prepared by performing 10- fold dilutions of plasmid DNA. The AAV titer was determined by interpolating the quantification data from the standard curve.

[0321] 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 titer 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 titer was interpolated from the standard curve.

[0322] Immunoassay quantification of rAA V 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 Titer 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 Titer Sample Dilution Buffer. All samples and controls were tested in duplicate. Data were analyzed using the Gyrolab™ Evaluator Software Version 3.7.1 .252.

[0323] Vector genome to total particle ratio

[0324] The ratio of vector genomes to total AAV particles is expressed as a percentage. This is based on the vector genome titer (determined by qPCR, as described above) and the number of total AAV particles (determined by the capsid immunoassay, as described above).

[0325] Cell-based AA V functional titer assay

[0326] A cell line suitable for functional titer assessment was developed by randomly integrating an expression construct encoding AAVR and Cre recombinase into a HEK293F cell line. Single-cell seeding was subsequently performed, and the cell line was subsequently identified for further use. Twenty-four hours before transduction, the developed cells were seeded in a 96-well plate at a density of 50,000 cells per well. On the day of transduction, 5 pL of virus-containing cell lysate, which harbored an inverted mCherry-P2A-NLuc reporter cassette, was added to each well. 72 hours posttransduction, nano luciferase transgene expression was measured using the Nano-Gio Luciferase Assay System (Promega) according to the manufacturer’s instructions.

[0327] Quantification of plasmid-derived impurities Prokaryotic 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.

[0328] Split plasmid system

[0329] The split plasmid system used herein has been described in detail elsewhere. See, for example, EP3722434A1 , which is incorporated herein by reference in its entirety.

[0330] Statistical analysis

[0331] The number of independent studies (biological replicates) and technical replicates are indicated in the figure legends. Unless otherwise specified, One-Way ANOVA 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 error of the mean (SEM).

[0332] Example 2 - Screening

[0333] A high-throughput screening platformed for optimizing AA V production

[0334] To identify candidate Open Reading Frames (ORFs) which when overexpressed improve AAV production, an arrayed targeted library for AAV screening (ATLAS), in particular screening of Mission® TRC3 arrayed ORF library (Sigma Aldrich), was conducted. First, the cell seeding density and transfection conditions were optimised using an inverted tandem mCherry and luciferase expression vector in suspension and adherent HEK293F cells. Next, a cell-based functional assay was developed to enable high-throughput measurement of functional AAV particles in microwell plates. To overcome the low in vitro AAV9 transduction efficiency in cells, a reporter cell line containing a cassette overexpressing AAVR and Cre recombinase was generated. Upon transduction of an rAAV vector containing inverted mCherry-nano Luciferase vector genome is flipped and both the mCherry and nLuc signals can be detected.

[0335] Human ORF screen identified several ORFs putative potentiators of AA V9 production Next, an unbiased high-throughput screen using a library of -18,000 was conducted to screen for AAV9 potentiators. Twenty-four hours prior to transfection, HEK293F cells were seeded at a density of 50,000 cells per well into a fibronectin-coated 96-well plate. On the day of transfection, the cells were triple-transfected with the split two-plasmid system and the ORF screening vector. AAV9 capsid ELISA and the cell-based functional titer assay was used to assess AAV9 production. Results from the primary screen were normalized to the the plate average and plotted and are shown in Figure 1 B. The majority of ORF hits from the primary screen can be clustered into 9 pathways or gene functions: cell cycle regulation, cell fate & differentiation, DNA repair, epigenetic modulation, immune response, metabolism, protein homeostasis RNA processing, signal transduction or transcription. The remainder of the ORFs cluster to the target class “other”. (Figure 1 C). The top 140 ORFs from the primary screen were distributed across three 96-well plates and over-expressed during AAV production to assess their impact on AAV yield, with each ORF tested on eight independent plates. AAV9 production was measured using capsid ELISA (capsid titer) and qPCR (vector genome titer). Over-expression of 29 ORFs resulted in an increase in both capsid and vector genome titers compared to a group that contains on 3rd plasmid (no ORF).As shown in Figure 2, theORFs that had the largest impact on AAV production include RARA, OSBPL5, SMARCAL1 , ZNF385B, and MAFB are highlighted .

[0336] A list of ORFs identified in the secondary screen (n=29) is summarized in Table 1 .

[0337] Table 1 : Selected ORFs of the secondary library screen that boost AAV production

[0338] Example 3 - Confirmation and Validation Studies

[0339] Selected hits from the ORF screen described in Example 2 were examined in a confirmation study for their impact on different AAV serotypes, namely AAV2, AAV5 and AAV8 in HEK293 cells and measured using qPCR (for example, as referenced in Example 1 above). Vector genome titer was normalized to the no ORF expressing control group which did not receive a 3rdplasmid during transfection. As shown in Figure 3 (A-C), among checked ORFs, RARA (SEQ ID NO: 3), OSBPL5 (SEQ ID NO: 2), SMARCAL1 (SEQ ID NO: 5), ZNF385B (SEQ ID NO: 4), and MAFB (SEQ ID NO:

[0340] 1 ) exhibit enhanced AAV production across all three serotypes when compared to the GFP control group.

[0341] To determine the impact on AAV9 production, ORF hits evaluated in Confirmation Study I were examined by transfecting varying amounts of plasmid into HEK293 cells. RARA (SEQ ID NO:

[0342] 3), OSBPL5 (SEQ ID NO: 2), SMARCAL1 (SEQ ID NO:), ZNF385B (SEQ ID NO: 4), and MAFB (SEQ ID NO: 1 ) ORFs were cloned into an expression vector wherein ORF expression was driven by a CMV promoter (as shown in Figure 4A). AAV9 yield was assessed using vector genome titer and normalized to the no ORF control. As shown in Figure 4 (B-F), AAV9 yield increased in a dose dependent manner to a maximal level of 0.5pg / cell for RARA (SEQ ID NO: 3), OSBPL5 (SEQ ID NO:

[0343] 2), SMARCAL1 (SEQ ID NO: 5), and MAFB (SEQ ID NO: 1 ) or 1 pg / cell for ZNF385B (SEQ ID NO:

[0344] 4). Confirmation study III demonstrated that overexpression of selected human ORFs increased AA V9 capsid yield in HEK293 cells cultured in 125mL suspension shake flasks

[0345] The top-performing ORFs— RARA (SEQ ID NO: 3), OSBPL5 (SEQ ID NO: 2), SMARCAL1 (SEQ ID NO: 5), ZNF385B (SEQ ID NO: 4), and MAFB (SEQ ID NO: 1 )— were evaluated in HEK293 cells using 125 mL shake flasks. Control groups were transfected with either the split plasmid system alone or the split two-plasmid system along with a CMV-GFP plasmid to account for effects of adding a third plasmid. Experimental groups received the split two-plasmid system plus an ORF expression vector, as shown in Figure 4A, at a concentration of 1 pg / cell. AAV9 yield was measured by capsid ELISA, revealing a significant increase for all tested ORFs. Notably, MAFB overexpression resulted in a 9-fold higher yield compared to the GFP control group, as illustrated in Figure 5.

[0346] Confirmation study IV demonstrated that overexpression of MECP2 in HEK293 cells cultured in Ambr15 bioreactors during results in increased rAAV9 quality and yield

[0347] The impact of MECP2 (SEQ ID NO: 16) overexpression on vector yield and quality during rAAV9 production was evaluated in the Ambrl 5 system to simulate conditions of larger bioreactors. For this, cells were transfected with the split two-plasmid system alongside either a control vector (CMV-GFP) or CMV-MECP2. MECP2 overexpression resulted in a significant increase in vector genome yield (as shown in Figure 6A) and capsid titer (as illustrated in Figure 6B), as well as a marked reduction in host-cell DNA impurities (as illustrated in Figure 6C). These findings highlight the beneficial effect of MECP2 overexpression during rAAV9 production in a scaled-down bioreactor model.

Claims

CLAIMS1 . A method for producing a preparation comprising recombinant adeno-associated virus (rAAV) particles, wherein said method comprises i)a step of introducing a human Open Reading Frame (ORF) to a host cell under suitable conditions, so that said ORF is overexpressed, ii)a step of culturing said host cell under suitable conditions to produce the preparation; and wherein said preparation comprises rAAV particles having improved and / or increased viral genome titre and / or potency compared to a preparation produced by a corresponding method without introducing said same ORF.

2. A method according to claim 1 , wherein said method comprises i)a step of introducing a human ORF expressing vector to a host cell under suitable conditions, preferably by transfecting an ORF expressing vector to a host cell under suitable conditions, so that said ORF is overexpressed, ii)a step of culturing said host cell under suitable conditions to produce the preparation; and wherein said preparation comprises rAAV particles having improved and / or increased viral genome titre and / or potency compared to a preparation produced by a corresponding method without introducing said same ORF expressing vector.

3. A method according to claim 1 or 2, wherein said method comprises i) a step of introducing a human ORF to a host cell or introducing an ORF expressing vector to a host cell, preferably by transfecting an ORF expressing vector to a host cell under suitable conditions, so that said ORF is overexpressed, wherein said host cell is preferably a mammalian cell, more preferably a human cell ii)a step of transfecting said host cell with a recombinant AAV production plasmid system, preferably a three plasmid rAAV production system or a two plasmid rAAV production system, or synthetic DNA molecules suitable for AAV production, iii)a step of culturing said cell under conditions suitable for recombinant AAV production iv)a step of obtaining recombinant AAV particles from said transfected cells, wherein said preparation comprises rAAV particles having increased viral genome titre and / or potency compared to a preparation produced by a corresponding method without introducing said same ORF or introducing said same ORF expressing vector or transfecting said same ORF expressing vector.

4. A method according to any one of 1 to 3 wherein said ORF encodes a protein related to cell cycle regulation, cell fate and / or differentiation, DNA repair, epigenetic modulation, immuneresponse, metabolism, protein homeostasis RNA processing, signal transduction or transcription.

5. A method according to any one of 1 to 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. A method according to any one of claimsl to 5, wherein said increase is at least 1 .05 fold, at least 1 .1 fold, at least 1 .2, at least, 1 .3, at least 1 .4, at least 1 .5, at least 1 .6, at least 1 .5, at least 1 .6, at least 1 .7, at least 1 .

8. at least 1 .9, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold or at least 6 fold compared to a corresponding method without introducing said same ORF or same ORF expressing vector or without transfection with said same ORF expressing vector.

7. A method for improving and / or increasing the viral genome titre and / or potency of recombinant adeno-associated virus (rAAV) particles produced during recombinant AAV production, wherein said method comprises i)a step of introducing an ORF to a host cell under suitable conditions so that said ORF molecule is overexpressed, ii)a step of culturing said host cell line under suitable conditions to produce said rAAV, and wherein said increase is at least 1 .05 fold, at least, 1 .1 fold, at least 1 .2, at least, 1 .3, at least 1 .4, at least 1 .5, at least 1 .6, at least 1 .5, at least 1 .6, at least 1 .7, at least 1 .

8. at least 1 .9, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold or at least 6 fold compared to a corresponding method without introducing said same ORF expressing vector.

8. The method of claim 7, wherein said method comprises i)a step of introducing an ORF or introducing an ORF expressing vector, preferably by transfecting a host cell with an ORF expressing vector, under suitable conditions so that said ORF is overexpressed ii)a step of transfecting said cell with a recombinant AAV production plasmid system, preferably a two -plasmid system rAAV production system or synthetic DNA molecules suitable for AAV production iii)a step of culturing said host cell under conditions suitable for recombinant AAV production iv)a step of obtaining recombinant AAV particles from said transfected cells, and wherein said increase is at least 1 .05 fold, at least 1 .1 fold, at least, 1 .1 fold, at least 1 .2, at least, 1 .3, at least 1 .4, at least 1 .5, at least 1 .6, at least 1 .5, at least 1 .6, at least 1 .7, at least 1 .

8. at least 1 .9, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold or at least 6 fold compared to a corresponding method without introducing said same ORF or introducing said same ORF expressing vector or transfection with said same ORF expressing vector.

9. A method according to any one of cla8ms 1 to 8 wherein(a) said ORF is exogenously or endogenously expressed in the host cell;(b) the ORF is transiently or stably expressed in the host cell; and / or(c) said ORF is transiently, conditionally, or permanently overexpressed in the host cell10. A method according to any one of claims 1 to 9, wherein said ORF comprises or consists of the sequence of any one of SEQ ID NOs: 1 to 29, preferably any one of SEQ ID NOs: 1 to 5.1 1 . A method according to any one of 1 to 10 wherein said 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;(d) derived from a HEK293 cell, a HEK293T cell, a HEK293SF cell or a HEK293-F cell; or (e ) a sf9 cell or a sf21 cell or a high five cell12. A method to claim 1 1 , wherein said host cell is a HEK293 cell line, preferably a suspension HEK293 cell line.

13. A method according to anyone of claims 1 to 12 wherein the rAAV particles comprise a capsid protein of:(a) the AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV1 1 , AAV 12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhW, 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.HSC1 1 , 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).

14. A preparation comprising rAAV particles obtained or obtainable by the method of any one of the previous claims.

15. A preparation according to claim 14 for use in a method of treating or preventing a disease.

16. A cell line for use in a method according to any one of claims 1 to 15, wherein said cell expresses an ORF, preferably said ORF comprising or consisting of the sequence of any one of SEQ ID NOs: 1 to 29.

17. Use of a preparation according to claim 14 or 15 or a cell line according to claim 16 for the manufacture of a medicament for treating or preventing a disease preferably said disease being a genetic disorder.

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