Production of adeno-associated viruses

Abstract

The present invention relates to a platform for production of adeno-associated viruses (AAVs). In particular, the present invention relates to a microcarrier comprising cellulose nanofibers upon which cells readily proliferate and act as hosts for efficient and scalable production of AAVs.

Description

[0001] Production of adeno-associated viruses

[0002] Technical field of the invention

[0003] The present invention relates to a platform for production of adeno-associated viruses (AAVs). In particular, the present invention relates to a microcarrier comprising cellulose nanofibers upon which cells readily proliferate and act as hosts for efficient and scalable production of AAVs.

[0004] Background of the invention

[0005] Adeno-associated viruses (AAVs) are small, non-enveloped viruses that have gained prominence in gene therapy due to their favorable characteristics. AAVs belong to the Parvoviridae family and consist of a protein capsid that encapsulates their single-stranded DNA genetic material. The capsid proteins play a crucial role in the virus's ability to deliver genes to target cells. Sustained therapeutic effects can be achieved as AAVs have the ability to establish long-term, stable integration of genetic material into the host cell's genome. Moreover, in contrast to other viruses, AAVs are considered non-pathogenic and do not cause human disease. This selection of desired properties together with their ability to infect a broad range of dividing and non-dividing cells makes them attractive candidates for gene therapy.

[0006] In gene therapy, the viral genome is replaced with therapeutic genes of interest, allowing for the targeted delivery of therapeutic cargo to specific cells. AAV-based gene therapy has shown promise in treating a variety of genetic disorders, including inherited diseases and conditions caused by a lack of specific proteins. Examples hereof include hemophilia and muscular dystrophy.

[0007] The production of adeno-associated viruses (AAVs) for gene therapy involves several key steps, including vector design, cell culture, transfection or infection, viral replication, virus harvest and purification. Design of the AAV vector may involve construction of a plasmid that contains the AAV genome with the therapeutic gene of interest, the AAV rep and cap genes (which encode replication and capsid proteins, respectively), and the adenoviral helper genes. A suitable cell line is chosen, such as Human embryonic kidney (HEK) 293 cells, and the AAV vector plasmid, along with helper plasmids encoding rep and cap genes, is introduced into the host cells. This process is known as transfection. Next, the AAV vector undergoes replication in the host cells to reach a sufficient level of AAV replicative intermediates followed by harvesting of the host cells. The harvested cells are subjected to lysis to release the AAV particles. Cell lysis methods may include freeze-thaw cycles, mechanical disruption, or other techniques to break open the cells and release the viral particles. Finally, the released AAVs are purified to remove cellular debris, host cell proteins, and other impurities.

[0008] The entire AAV production process must adhere to Good Manufacturing Practice (GMP) standards to ensure the safety, quality, and consistency of the vectors for use in clinical applications. Optimization of each step, from vector design to purification, is essential to achieve high yields, purity, and transduction efficiency, especially when scaling up production for clinical use.

[0009] The production of adeno-associated viruses (AAVs) for gene therapy faces several challenges, ranging from technical and biological hurdles to scale-up issues. Traditional AAV production methods do not easily translate to large-scale manufacturing which is required for clinical and commercial utility. Importantly, achieving high viral yields and productivity is essential for cost-effective production. Thus, optimization of the transfection of host cells and the resulting viral yield are key challenges to keep costs down. Furthermore, consistent and high quality of the produced AAVs across batches is critical for regulatory compliance and patient safety.

[0010] Thus, there is an unmet need for provision of a method for production of high quality adeno-associated viruses (AAVs) that can be used on industrial scale.

[0011] Hence, it would be advantageous to provide a method for culturing cells that ensures high transfection efficiency of host cells and high viral titers of AAVs.

[0012] Specifically, it would be advantageous to provide a cost-effective method to produce AAVs to reduce cost of gene therapies.

[0013] Summary of the invention

[0014] Herein are provided a method for producing adeno-associated virus (AAV) in an efficient and scalable manner. Host cells are grown on a microcarrier comprising cellulose nanofibers that offer a large surface area and low dead volume. Transfection efficiency of host cells is high and AAV particles are recovered at elevated virus titers. The method is simple and inexpensive as the microcarrier can be produced in bulk quantities.

[0015] Thus, an object of the present invention relates to the provision of a cost-effective and scalable method for production of adeno-associated virus (AAV). Another object of the present invention relates to provision of nanofibrous cellulose scaffold suitable for use in the production of adeno-associated virus (AAV).

[0016] Thus, an aspect of the present invention relates to a method for production of adeno- associated virus (AAV), said method comprising the steps of: providing a microcarrier;

[0017] - adding one or more adeno-associated virus (AAV) vector(s) to said microcarrier;

[0018] - seeding a host cell on said microcarrier;

[0019] - culturing said host cell; and

[0020] - collecting AAV particles produced by said host cell; wherein said microcarrier comprises cellulose nanofibers.

[0021] An embodiment of the present invention relates to the method for production of adeno- associated virus (AAV), wherein said microcarrier comprises a nanofibrous cellulose scaffold as described herein.

[0022] Another aspect of the present invention relates to a nanofibrous cellulose scaffold comprising a processed cellulose nanofiber material with a mean length of the cellulose nanofibers of less than about 250 pm, and wherein the processed cellulose nanofiber material is functionalized with a functional moiety.

[0023] Yet another aspect of the present invention relates to a method of preparing a nanofibrous cellulose scaffold, said method comprising the steps of:

[0024] (i) providing an initial cellulose nanofiber material,

[0025] (ii) dividing said initial cellulose nanofiber material into a processed cellulose nanofiber material,

[0026] (iii) functionalizing the processed cellulose nanofiber material by addition of a reagent comprising a functional moiety, and

[0027] (iv) drying the processed cellulose nanofiber material, thereby providing the nanofibrous cellulose scaffold.

[0028] A further aspect of the present invention relates to a nanofibrous cellulose scaffold obtainable from the method of preparing a nanofibrous cellulose scaffold as described herein.

[0029] A still further aspect of the present invention relates to use of a nanofibrous cellulose scaffold as described herein for production of adeno-associated virus (AAV). Brief description of the figures

[0030] Figure 1 shows (A) microscopy image of nanofibrous cellulose scaffold functionalized with quaternary ammonium (Cellevate QA). (B) Falcon tube comprising the nanofibrous cellulose scaffold in dry powder form. (C) Scanning electron microscopy (SEM) image of the nanofibrous cellulose scaffold demonstrating the spatial arrangement of individual nanofibers.

[0031] Figure 2 shows through microscopy the binding of HEK293T cells on the nanofibrous cellulose scaffold at (A) 6 hours after seeding (shown at two different magnifications) and (B) their growth in a 3D spheroid structure after culturing them with nanofibrous cellulose scaffold for 72 hours. (C) Quantification of proliferation and (D) viability of HEK293T cells cultured on the nanofibrous cellulose scaffold. Data are presented as mean ± SEM. Each data point represents a biological replicate from independently produced nanofibrous cellulose scaffold batches.

[0032] Figure 3 shows (A) microscope images of 3D spheroids formed from ZombieGreen- transfected HEK293T cells on nanofibrous cellulose scaffolds at 96 hours post-transfection. (B) Transfection efficiency of HEK293T cell spheroids on nanofibrous cellulose scaffolds as determined using flow cytometry. (C) HEK293T cell transduction efficiency using different AAV2 dilutions.

[0033] Figure 4 shows examples of fibers cut by laser. (A) The cellulose material cut by laser results in pieces of cellulose that trap air bubbles and float. (B) Electrospun cellulose material cut with a laser. The laser burns the cellulose sheets. (C) SEM images of cellulose nanofibers cut by laser. The cellulose nanofibers are melted and fused together.

[0034] Figure 5 shows determination of cellulose nanofiber diameters. (A-B) SEM micrographs of cellulose nanofibers after 1 hour dispersing at 18000 rpm. (C) Histogram depicting the cellulose nanofiber diameter distribution of five separate samples. Approximately 2500 individual cellulose nanofibers were measured using ImageJ software.

[0035] Figure 6 shows scanning electron microscopy (SEM) images of cellulose nanofibers blended (left) or dispersed (right). The cellulose nanofibers were mixed for different periods of time; 1 min (A-B), 5 min (C-D), 15 min (E-F) or 60 min (G-H).

[0036] Figure 7 shows histograms of size (length) distributions of cellulose nanofibers that have been divided by (A) blending or (B) dispersing. The histograms are for samples divided for 1, 5, 15, or 60 min (left to right). Nanofiber lengths are displayed as relative frequency of mean nanofiber lengths in bins of 200 pm.

[0037] Figure 8 shows scanning electron microscopy (SEM) images of electrospun nanofibers; (A) PCL, (B) PLA, (C) PLA / PCL, and (D) cellulose. All images are recorded at 600x magnification, scale bar is 50 pm.

[0038] Figure 9 shows representative photographs (left) and SEM images (right) of different electrospun nanofibers subsequent to 5 minutes of mixing in a blender. (A-B) PCL, (C-D) PLA, (E-F) PLA / PCL. Scale bar of SEM images is 200 pm.

[0039] Figure 10 shows representative photographs (left) and SEM images (right) of different electrospun nanofibers subsequent to 5 minutes of mixing in a disperser. (A-B) PCL, (C- D) PLA, (E-F) PLA / PCL. Scale bar of SEM images is 50 pm.

[0040] Figure 11 shows (A) cell density of HEK293T cells grown in spinner flasks of different culture volumes or a 2.4L stirred tank bioreactor over a 4 day period. Data are presented as mean. (B) Cell density of HEK293T cells upon passaging from spinner flask to bioreactor. Data are presented as mean ± SEM. (C) Cell density and viability of HEK293T cells grown in a spinner flask or a bioreactor over a 5 day period. (D) Glucose and lactate levels of HEK293T cells during culture in spinner flask or bioreactor. (E) Bright field microscope images (4x) showing the appearance of nanofibers in solution before starting the culture (left) and the absence of nanofibers in solution after cell lysis and depth filtration (right).

[0041] Figure 12 shows (A) HEK293T cells cultured on the nanofibrous cellulose scaffold (nanofiber microcarrier), with plasmid transfection efficiency (%) determined by flow cytometry for AAV2 (left) and fluorescence microscopy of ZombieGreenl-expressing transfected 3D spheroids (right). Data are presented as mean ± SEM. (B) AAV2 titer measured by qPCR from cells cultured on the nanofiber microcarrier. (C) Comparison of virus yield from cells cultured on the nanofiber microcarrier compared to cells cultured in 2D (culture flasks). Number of cells and culture volume were kept equal between 3D and 2D. (D) Percentage of full viral capsids produced from cells grown on nanofiber microcarrier. Full capsids are presented as capsid titer to viral genome titer ratio.

[0042] Figure 13 shows bright-field microscope images of spheroids cultured on nanofiber microcarriers at (A) the timepoint of transfection (24h after seeding) and (B) at the timepoint of virus harvest (72 h post-transfection). (C) Spheroid diameter of 110 spheroids at transfection and at harvest (Mean ± SEM). Fluorescence microscope images (4x) of ZombieGreenl expression 72h post-transfection from spheroids cultured on nanofiber microcarrier (D) or standard microcarrier (E). (F) Transfection efficiency for spheroids grown on nanofiber microcarrier or standard microcarrier. Quantification of AAV2 viral genome titer by qPCR presented as (G) viral genome titer per mL of culture and (H) viral genome titer per cell in culture.

[0043] Figure 14 shows transfection efficiency analysed by flow cytometry reported as (A) percentage of GFP-positive cells and (B) mean fluorescence intensity in arbitrary units (AU). (C) AAV9 titer in vg / mL. All data are presented as mean ± SEM.

[0044] Detailed description of the invention

[0045] Definitions

[0046] Prior to outlining the present invention in more details, a set of terms and conventions is first defined:

[0047] Host cell

[0048] In the present context, the term "host cell" refers to a living cell for virus production that is susceptible to transfection and / or viral infection and can support the replication and assembly of the virus. Thus, a host cell expresses necessary receptors for the virus to recognize and bind, and comprise the cellular machinery for replication of the viral genome, such as factors required transcription, translation, and replication of the viral genetic material.

[0049] It is preferred that the host cell is amenable to large scale cultivation, i.e. that the host cell is selected with factors such as growth rate, purity and adaptability to bioreactors are considered. Also, the host cell should be free from endogenous viruses that could potentially interfere with production of the virus.

[0050] Examples of suitable mammalian cells include, but are not limited to, HEK293, HEK293T, SF9, BHK21, SF21, CAP-T, Vero and CHO cells.

[0051] Vectors

[0052] In the present context, the term "vector" refers to a vehicle or carrier system designed to introduce foreign genetic material into a host cell. The vectors can be either viral or non- viral. Viral and non-viral vectors may be used in combination. Herein, viral vectors have the ability to infect cells and transfer their genetic payload, making them efficient tools for delivering genes into target cells.

[0053] Non-viral vectors are delivery systems that do not involve the use of viruses. They are typically synthetic or naturally occurring substances that can facilitate the transfer of genetic material into cells. A non-limiting example of a non-viral vector is a plasmid. A non-viral vector system may comprise more than one plasmid, such as supplementary plasmids that encode helper functions for production of viral particles.

[0054] The viral and non-viral vectors referred to herein carry a genetic payload suitable for production of adeno-associated virus (AAV) and are thus collectively termed AAV vectors. Host cells transfected and / or infected with AAV vectors gain the ability to produce AAV particles.

[0055] In the present context, auxiliary vectors are non-viral agents that assist the transfer of the genetic material of the AAV vectors into the host cells, i.e. transfection reagents. Auxiliary vectors may be used together with e.g. non-viral vectors, such as plasmids, to enhance transfer of negatively charged DNA into the host cells. A non-limiting example of auxiliary vectors are cationic polymers. Accordingly, the terms "auxiliary vectors" and "transfection reagents" may be used interchangeably herein.

[0056] Replication

[0057] In the present context, the term "replication" refers to the entire process of cellular production of viral particles from transfected viral vectors. Thus, replication includes uncoating of the viral genome, multiplication of the viral genome, transcription, translation, and assembly of viral particles.

[0058] Nanofiber

[0059] In the present context, the term "nanofiber" refers to fibers with diameters in the range of 10-2000 nm. The fibers may be generated from different types of polymers, such as cellulose.

[0060] Cellulose nanofiber material

[0061] In the present context, the term "cellulose nanofiber material" refers to an initial material prepared from cellulose. The cellulose nanofiber material may be prepared by any method suitable for preparing sheets of cellulose that can subsequently be processed as described herein to provide the nanofibrous cellulose scaffold. These methods include, but are not limited to, electrospinning, meltblowing, and drawing of cellulose nanofibers. Preferably, the cellulose nanofiber material is prepared by electrospinning of cellulose nanofibers. Electrospinning may be performed from a solution of cellulose acetate.

[0062] Processed cellulose nanofiber material

[0063] In the present context, the term "processed cellulose nanofiber material" refers to a cellulose nanofiber material that has been treated to reduce the length of the cellulose nanofibers.

[0064] Dispersing

[0065] In the present context, the term "dispersing" refers to the process of dividing the cellulose nanofiber material by means of a disperser. Dispersing is preferably performed in a liquid.

[0066] In the present context, a disperser is a high-speed mixing device that can comminute a solid, such as a cellulose sheet, into smaller fragments. The disperser comprises one or more heads which constitute the means of dividing the cellulose nanofiber material. The head may be in the form of a disc blade. The head of the disperser may comprise a blade with a propeller design. The disperser creates a turbulent flow and a vortex which ensures homogenous dividing of the cellulose nanofiber material into smaller fragments, i.e. cellulose nanofibers of reduced length compared to the initial cellulose nanofiber material.

[0067] Any type of disperser may be used, including, but not limited to, laboratory high-speed disperser, pilot-scale high-speed dispersers, and production-scale high-speed dispersers. The disperser can be chosen according to the batch size to be divided, and the diameter of the disperser blades are adjusted accordingly. The disperser blade may be raised and lowered during dispersing to eliminate stratification, such mechanism may be automatic. For large batches the disperser may be a floor-mounted or tank-mounted model and / or be a multishaft model.

[0068] Functional moiety

[0069] In the present context, the term "functional moiety" refers to a chemical or biological group or molecule positioned on the nanofibrous cellulose scaffold, and which interacts with the cells associated with scaffold. A functional moiety may interact with the cells via interactions including, but not limited to, electrostatic interactions, affinity, and hydrophobicity / hydrophilicity. The functional moiety may promote attachment / adhesion of cells to the nanofibrous cellulose scaffold, induce cell differentiation and proliferation, and / or assist maintenance of in vivo cellular functions. Chemical moieties may have one or more positive or negative charges to induce electrostatic interaction with the charged cell membranes. Examples hereof include, but are not limited to, quaternary ammonium (QA), carboxymethyl (CM), and diethylaminoethyl (DEAE). For many cells, the cell membrane would be negatively charged, and electrostatic interactions would be induced for nanofibrous cellulose scaffolds functionalized with positively charged functional moieties, such as QA or DEAE.

[0070] Biological moieties may be any type of biological molecule that can secure cell attachment to the nanofibrous cellulose scaffold, including but not limited to, lipid anchors, celladhesion molecules (CAMs), antigens, receptors, and antibodies. CAMs include integrins, immunoglobulins, cadherins, and selectins. Biological moieties may also assist cellular differentiation through receptor-ligand interactions, antigen-antibody interactions, protein interactions, and / or immune stimulatory signalling.

[0071] Adherent cell

[0072] In the present context, the term "adherent cells" refers to any cell that requires a surface or artificial substrate, such as a microcarrier, to form an adherent cell culture. Preferably, the adherent cell is derived from a solid tissue.

[0073] Adherent culture is to be distinguished from suspension culture in which cells are grown freely floating in suspension.

[0074] Mean diameter (of cellulose nanofiber)

[0075] In the present context, the term "mean diameter" refers to the average diameter of cellulose nanofibers in the nanofibrous cellulose scaffold. The mean diameter may be determined from SEM images of the nanofibrous cellulose scaffold. Preferably, the mean diameter is determined from measurement of at least 100 individual nanofibers within the sample, e.g. by use of image analysis software, such as Image!

[0076] The mean diameter of the cellulose nanofibers can be adjusted in the process of preparing the initial cellulose nanofiber material. This may be achieved by varying the parameters of e.g. the electrospinning process.

[0077] Preferably, the mean diameter of the cellulose nanofibers in the nanofibrous cellulose scaffold is from about 250 nm to about 750 nm, such as about 400 nm to about 600 nm.

[0078] Mean length (of cellulose nanofiber) In the present context, the term "mean length" refers to the average length of cellulose nanofibers in the nanofibrous cellulose scaffold. The mean length may be determined as the volume weighted mean value (D[4,3]) measured by light scattering, e.g. on a Malvern Mastersizer S. D[4,3] is also known as the De Brouckere mean value.

[0079] The mean fiber length in a sample may be determined using the following settings on a Malvern Mastersizer S:

[0080] Range lens: 300RF mm

[0081] Presentation: 3OHD

[0082] Analysis model: Polydisperse

[0083] Particle refractive index: (1.5295, 0.1000)

[0084] Dispersant refractive index: (1.33000)

[0085] Density: 1.5000 g / cm3

[0086] Preferably, the mean length of the cellulose nanofibers is from about 30 pm to about 250 pm, such as about 40 pm to about 200 pm, such as about 50 pm to about 150 pm, preferably about 60 pm to about 100 pm.

[0087] Surface area

[0088] In the present context, the term "surface area" refers to the Brunauer-Emmett-Teller (BET) surface area. The BET surface area may be determined by measurement of the physiosorption of a gas, usually nitrogen, to give a value of the sample. The BET method can accurately determine the surface area of the nanofibrous cellulose scaffold since the gas molecules can travel within the nanofibrous matrix to probe also interior surfaces.

[0089] The surface area is given as area per unit mass (e.g. cm2 / g) and can be measured according to ISO 9277:2022 - Determination of the specific surface area of solids by gas adsorption — BET method.

[0090] Degree of substitution (DS)

[0091] In the present context, the term "degree of substitution (DS)" refers to the average number of functional moieties attached per base unit of the condensation polymer cellulose. The base unit of cellulose is 0(1— >4) linked D-glucose, which comprise three hydroxyl groups that may be subjected to substitution. Accordingly, the theoretical maximum value of DS is 3.

[0092] Degree of substitution (DS) may be determined using the following formula:

[0093] DS = (162N / (1400-CAxN)) where 162 is the molecular weight of the anhydrous glucose unit (AGU); N is the percentage of nitrogen; CA is the molecular weight of the cationic reagent.

[0094] Substitution of the cellulose nanofibers can also be quantified as equivalent of charge per base unit mass of cellulose, and is given in units of meq / g. This value can be determined by potentiometric titration, zeta potential measurements, pH titration or electrokinetic chromatography.

[0095] Dead volume

[0096] In the present context, the term "dead volume" refers to the volume occupied by the microcarrier when culturing cells. Ideally, the dead volume is minimised to allow for more cell proliferation per volume in the container used for culturing cells.

[0097] Mercerization

[0098] In the present context, the term "mercerization" refers to a process comprising swelling of the cellulose nanofiber material in an aqueous or ethanolic NaOH solution to break internal hydrogen bonds of cellulose and increase the number of available hydroxyl groups (-OH).

[0099] Microcarrier

[0100] In the present context, the term "microcarrier" refers to any support matrix upon which adherent cells may grow in adherent culture.

[0101] The nanofibrous cellulose scaffold described herein is considered to be a microcarrier, and may be referred to as microcarrier or nanofiber microcarrier herein.

[0102] It is to be understood that the nanofibrous cellulose scaffold prepared herein is not a hydrogel, which amongst others are characterized by its ability to absorb and retain large amounts of water creating a highly viscous environment and are generally used for 3D cell culture in small scale or static conditions. In particular, hydrogels are not suited as microcarriers for large scale cell production, such as in a bioreactor. While hydrogels can be alternatively used as shear-thinning, the shear stress in stirred-tank or perfusion bioreactors may disrupt their structure.

[0103] Instead, the nanofibrous cellulose scaffold are lyophilised and grinded to provide a dry powder that are more robust and can be efficiently agitated in bioreactors, providing a surface for anchorage-dependent cells to attach and grow. Suspension based bioreactor

[0104] In the present context, the term "suspension based bioreactor" refers to a bioreactor wherein the microcarriers and cells adhere thereto are freely floating in the bioreactor. In a suspension based bioreactor, it is possible to add more microcarriers and / or medium during culturing. It is also possible to remove medium or have a constant perfusion mode.

[0105] Thus, the term "suspension based bioreactor" includes, but is not limited to, stirred tank bioreactors, fluidized bed bioreactors, airlift bioreactors, wave bioreactors and orbitally shaken bioreactors.

[0106] In contrast, a suspension based bioreactor is to be distinguished from a bioreactor wherein the microcarriers are fixed within the bioreactor, such as a packed bed bioreactor wherein the microcarriers are immobilised in a bed.

[0107] About

[0108] Wherever the term "about" is employed herein in the context of amounts, for example absolute amounts, such as numbers, purities, weights, concentrations, sizes, etc., or relative amounts (e.g. percentages, equivalents or ratios), timeframes, and parameters such as temperatures, pressure, etc., it will be appreciated that such variables are approximate and as such may vary by ±10%, for example ± 5% and preferably ± 2% (e.g. ± 1%) from the actual numbers specified. This is the case even if such numbers are presented as percentages in the first place (for example 'about 10%' may mean ±10% about the number 10, which is anything between 9% and 11%).

[0109] Method for production of adeno-associated virus (AAV)

[0110] AAV are widely used for gene therapy application because of their ability to deliver genetic material to both dividing and non-dividing cells, while having low immunogenicity and no etiologic association with any known diseases. Moreover, AAV provide efficient and longterm transgene expression in vivo in a wide selection of tissues without notably cellular immune responses or toxicity.

[0111] Naturally, the virus attaches to specific receptors on the surface of the host cell. This attachment is often mediated by viral surface proteins interacting with host cell receptors. Once attached, the virus enters the host cell. Entry mechanisms vary and may include direct fusion with the host cell membrane or endocytosis. After entry, the viral genome is released from its protective protein coat (capsid) in a process known as uncoating. This step exposes the viral genetic material to the host cell's machinery and initiates replication of the viral genome. The replicated viral genome is transcribed to produce messenger RNA (mRNA). The mRNA is then translated by the host cell's ribosomes to synthesize viral proteins. These proteins play roles in the formation of new viral particles. Newly synthesized viral proteins and replicated viral genomes are assembled into new viral particles. The assembled viral particles undergo maturation, where they acquire their final structure.

[0112] Production of AAV involves a series of steps that mostly resembles the natural cycle of the AAV, and include introduction of AAV genetic material into the host cells, uncoating of the viral genome, replication, transcription, translation, assembly of viral particles, and harvest of AAV particles.

[0113] While the complex production cycle is largely governed by the host cell's machinery, any optimization of the process that can e.g. reduce production time or increase virus yield is welcomed and can reduce the cost of the resulting gene therapy. Key parameters for AAV production methods include transfection efficiency, virus yield, and transduction efficiency.

[0114] Transfection efficiency is typically assessed by measuring the expression of a reporter gene (e.g. green fluorescent protein, luciferase) carried by the transfected genetic material. Techniques such as flow cytometry, fluorescence microscopy, or luciferase assays can be employed to quantify the percentage of cells successfully transfected. Transfection efficiency plays a crucial role in virus production processes and optimizing transfection conditions is essential for achieving successful and scalable virus production.

[0115] Transfection efficiency significantly affects the resulting virus yield of AAV production. However, virus yield may also be influenced by the choice of host cell, AAV vector design, hereunder use of supplementary plasmids, cell density and confluence at the time of transfection, timing of harvesting of cells, and purification methods.

[0116] Ideally the resulting AAV particles are highly infectious to enable gene therapy applications, where the successful delivery and expression of therapeutic genes are essential for the intended therapeutic effect. The infectivity may be quantified as the transduction efficiency and is a measure of the quality of the AAV particles.

[0117] Culturing of mammalian cells remains a cornerstone for biomanufacturing of biologies. Among the most utilised mammalian cell lines are adherent cells, such as human embryonic kidney (HEK) 293 cells. However, adherent mammalian cells are highly dependent on a suitable support matrix for cell proliferation, viability and productivity. Herein are described a method for cost-efficient and scalable production on adeno- associated virus (AAV). The method is based on growing the host cells on cellulose nanofibers which provides excellent transfection efficiency, virus yield, infectivity and enables industrial scale production.

[0118] Thus, an aspect of the present inventio relates to a method for production of adeno- associated virus (AAV), said method comprising the steps of: providing a microcarrier;

[0119] - adding one or more adeno-associated virus (AAV) vector(s) to said microcarrier;

[0120] - seeding a host cell on said microcarrier;

[0121] - culturing said host cell; and

[0122] - collecting AAV particles produced by said host cell; wherein said microcarrier comprises cellulose nanofibers.

[0123] The flexible nature of the cellulose nanofibers allows them to form a network of strands that efficiently exploit the space occupied and reduces the microcarrier dead volume that is not accessible to cells. Accordingly, more surface area can be packed into a smaller volume which is advantageous for use in cell culturing containers wherein only a finite volume is available.

[0124] Preferably, the cellulose nanofibers are short to limit entanglement and create a homogeneous matrix for the host cells to populate.

[0125] Therefore, an embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the cellulose nanofibers have a mean length of less than about 250 pm.

[0126] Another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the cellulose nanofibers have a mean length in the range of about 30 pm to about 250 pm, such as about 40 pm to about 200 pm, such as about 50 pm to about 175 pm, such as about 60 pm to about 150 pm, preferably about 70 pm to about 120 pm.

[0127] The mean diameter of the cellulose nanofibers can be guided during production of the nanofibers. This may be achieved by varying the parameters of e.g. the electrospinning or meltblowing process, such as the voltage or heat applied, the speed and type of injection, and / or the rotational speed of the collector drum. The mean diameter of the cellulose nanofibers may be varied depending on the application, e.g. the type of cells to be cultured. It has been found that for many applications, a mean diameter of about 400 nm to about 600 nm, such as about 500 nm, is advantageous.

[0128] Thus, an embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the cellulose nanofibers have a mean diameter in the range of about 10 nm to about 2000 nm, such as about 50 nm to about 1500 nm, such as about 100 nm to about 1000 nm, preferably about 250 nm to about 750 nm.

[0129] A preferred embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the cellulose nanofibers have a mean diameter in the range of about 400 nm to about 600 nm, preferably about 500 nm.

[0130] Another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the cellulose nanofibers are electrospun, meltblown or drawn, preferably electrospun.

[0131] The cellulose nanofibers may be functionalized with functional moieties such as ECM proteins, peptides, and / or charged groups to e.g. increase attachment levels of the cells to the scaffold, promote differentiation of the cells, or assist in the release and isolation of the cells from the scaffold.

[0132] The functional moieties attached to the cellulose nanofibers may be of either chemical or biological origin. In particular, positively charged groups are advantageous as they induce electrostatic interactions between the nanofibrous cellulose scaffold and cells with a negatively charged membrane, thereby increasing attachment of cells to the microcarrier. Biological moieties include proteins and peptides that are normally an integral part of the interaction between the cell and the extracellular environment. This interaction may further promote attachment of cells to the nanofibrous cellulose scaffold.

[0133] Therefore, an embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the cellulose nanofibers are functionalized with a functional moiety.

[0134] Another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the functional moiety is selected from chemical moieties or biological molecules. A further embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the chemical moieties are selected from the group consisting of negatively charged groups, positively charged groups, zwitterionic groups, hydrophobic groups, hydrophilic groups, amphiphilic groups, ligands, and combinations thereof.

[0135] Yet another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the chemical moieties are positively charged groups.

[0136] A still further embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the chemical moieties are selected from the group consisting of quaternary ammonium (QA), carboxymethyl (CM), diethylaminoethyl (DEAE), 4-(l-bromoethyl)benzoic acid, 4-(dimethylamino)pyridine, epoxide, and combinations thereof.

[0137] It is to be understood that the chemical moieties may be attached to the cellulose backbone using conventional chemistry. Thus reagents such as, but not limited to, 3- chloro-2-hydroxypropyl trimethyl ammonium chloride (CHPTAC), 2-chloro-N,N diethylethylamine hydrochloride (DAECH), and monochloro acetic acid (MCAA) may be used for attachment of QA, DEAE, and CM, respectively.

[0138] A preferred embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the chemical moiety is quaternary ammonium (QA).

[0139] Preferably, the microcarrier is provided as a dry material that is convenient and easy to handle for the end user. The dry material may be packed in a vessel suitable for direct addition to the cell culturing container. The microcarrier in dry powder form may also be reconstituted / rehydrated in aqueous solution, such as PBS and / or culture medium, prior to addition to the cell culturing container.

[0140] Accordingly, an embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the microcarrier is provided as a dry material. Another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the microcarrier is provided as a lyophilized material.

[0141] The microcarrier has a high surface area. Without being bound by theory, it is contemplated that the high surface area, amongst others, is caused by the homogenous distribution of cellulose nanofibers in the material, which ensures that large entanglements and clusters of nanofibers are avoided. Accordingly, the microcarrier provides a superior available surface area that is advantageous for culturing of adherent cells. It is also contemplated that the improved culturing conditions benefits the host cells' ability to produce AAV particles.

[0142] Therefore, an embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the nanofibrous cellulose scaffold has a BET surface area of at least about 40000 cm2 / g, such as at least about 50000 cm2 / g, such as at least about 55000 cm2 / g, such as at least about 60000 cm2 / g, such as at least about 70000 cm2 / g, such as at least about 80000 cm2 / g.

[0143] Another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein BET surface area is measured according to ISO 9277:2022 - Determination of the specific surface area of solids by gas adsorption — BET method.

[0144] The adeno-associated virus (AAV) vector(s) used for transfecting the host cells may be any conventional set of viral or non-viral vectors, such as a helper free virus plasmid system, usually used for production of AAVs. The AAV vectors may comprise regulatory elements such as promoters and enhancers to drive the expression of the introduced genetic material, and an origin of replication that allow the vectors to replicate within the host cell. The AAV vectors may also comprise a multiple cloning site (MCS) which is a region with multiple unique restriction enzyme recognition sites, facilitating the insertion of foreign DNA. The AAV vectors may include selectable markers (e.g. antibiotic resistance genes) to identify and select cells that have successfully taken up the foreign DNA, or reporter genes (e.g. GFP) that allow researchers to visually identify and study transfected cells. Selectable markers and reporter genes are particularly useful for optimization of transfection, whereas final production methods may produce AAV particles without these features. Importantly, the AAV vectors should comprise nucleic acids encoding any genes or proteins of interest that the produced AAV particles should introduce into target cells, i.e. for therapeutics applications. Adeno-associated viruses (AAVs) are derived from defective parvoviruses and have a single-stranded DNA genome which consists of approximately 4.7 kb. All characterized AAV serotypes share three key features, including two copies of AAV inverted terminal repeats (ITRs), one rep region and one cap region. The ITRs are capable of forming T- shape secondary structure and are the only cis elements that are required for AAV replication, packaging, integration, and rescue. The rep region encodes four overlapping proteins designated as Rep78, Rep68, Rep52, and Rep40, according to the apparent molecular mass of the protein. In addition to their well-defined roles in AAV replication, Rep proteins also regulate AAV packaging and site-specific integration. The cap region encodes three structural proteins, VP1, VP2, and VP3. It is to be understood that any serotype of AAV can be produced by the method described herein, e.g. AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9.

[0145] Therefore, an embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the AAV is of serotype AAV2 or AAV5, preferably AAV2.

[0146] Another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein said one or more AAV vector(s) comprises a nucleic acid sequence of interest and AAV rep and cap genes.

[0147] Yet another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein said one or more AAV vector(s) further comprises inverted terminal repeats (ITRs) flanking said nucleic acid sequence of interest.

[0148] A further embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein said nucleic acid sequence of interest encodes a therapeutic protein, a gene of interest, or a reporter gene.

[0149] A still further embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein said one or more AAV vector(s) are non-viral AAV vector(s).

[0150] An even further embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein said one or more AAV vector(s) are plasmids. For efficient viral replication and propagation, AAV relies on certain exogenous helper functions provided by other viruses, such as adenovirus or herpes virus. However, production of infective AAV particles can be achieved by providing the gene products encoding the helper functions on supplementary plasmids. Thus, the supplementary plasmids of such AAV helper-free systems are co-transfected into the host cells with AAV vector DNA. The supplementary plasmid may contain e.g. E2A, E4 and VA RIMA adenoviral genes, and thereby eliminate the need for a helper adenovirus.

[0151] Preferably, the rep and cap genes are removed from the AAV vector that contains AAV ITRs and are supplied in trans on a supplementary plasmid. The removal of the AAV rep and cap genes allows for insertion of a gene of interest in the viral genome.

[0152] Thus, an embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein said one or more AAV vector(s) comprises three plasmids.

[0153] Another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein said one or more adeno- associated virus (AAV) vector(s) comprises:

[0154] - a first plasmid comprising a nucleic acid sequence of interest and inverted terminal repeats (ITRs) flanking said nucleic acid sequence of interest;

[0155] - a second plasmid comprising AAV rep and cap genes; and

[0156] - a third plasmid comprising E2A, E4 and VA RNA genes.

[0157] Alternatively, the AAV may be produced with the assistance of a helper virus to provide exogenous helper functions. The helper virus may be an adenovirus or a herpes virus.

[0158] Thus, an embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein a helper virus is provided together with said one or more AAV vector(s).

[0159] A further embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein said one or more adeno- associated virus (AAV) vector(s) are viral vectors.

[0160] The host cells may lend their cellular machinery to support the replication and assembly of AAV particles. Thus, the host cell is capable of supporting AAV replication. This may include suitable transcription and translation machinery to express the Rep and Cap proteins, as well as any gene of interest inserted into the AAV vector. It can also include the ability to transport AAV genome and proteins into and out of the nucleus, and an endoplasmic reticulum (ER) and Golgi apparatus suited for assembling and packaging the AAV particles. In variants of the method, the host cells may stably express the adenovirus El gene, which further enhances AAV production efficiency.

[0161] Therefore, an embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein said host cell is capable of supporting AAV replication.

[0162] Another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein said host cell comprises the machinery for supporting AAV replication.

[0163] The microcarrier is particular advantageous for culturing of adherent cells which grow while adhering to microcarriers in the culture vessel. Ideally, cultured cells are cultured in a manner that reflects the conditions under which they exist in the living organism. Adherent cells are cells which adhere to extracellular matrices or surfaces to form tissues under physiological conditions.

[0164] Therefore, an embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the host cell is an adherent cell.

[0165] Another, an embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein said host cell is selected from the group consisting of mammalian cells, insect cells, and bacterial cells.

[0166] For the purpose of biomanufacturing, hereunder AAV production, some cell lines are preferred due to characteristics such as ease of handling and ability to propagate human viruses. An example hereof is HEK293 cells, which may be used for packaging and amplification of recombinant adenovirus.

[0167] Accordingly, an embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the host cell is selected from the group consisting of HEK293, HEK293T, SF9, BHK21, SF21, CAP-T, Vero and CHO cells. A preferred embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the host cell is a HEK 293 cell line.

[0168] The host cells are transfected with the AAV vector(s) to facilitate the production of AAV particles. Transfection may be achieved by any conventional means. Thus, the host cells may be seeded onto the microcarrier either before or after addition of the AAV vectors to the microcarrier. The microcarrier used in the present method has a large surface area and low dead volume which allow host cells to be seeded at high seeding density. It is preferred to provide host cells at high seeding density because proliferation after transfection typically is halted. If the seeding density is not sufficiently high at the time of seeding to provide acceptable virus titers, then the host cells must be grown for a period of time prior to transfection to reach an acceptable cell density. Any incubation time required to reach an acceptable cell density is unfavourable because it increases the overall processing time, and therefore reduces the total virus titer production capacity over time.

[0169] It is contemplated herein, that the production method described herein is favourable because it reduces the required processing time needed to obtain a high virus titer. This is amongst others because the microcarrier supports seeding of host cells at a high seeding density.

[0170] Therefore, an embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the seeding density of host cells on the microcarrier is in the range of about IxlO5to about IxlO7cells / ml culturing volume, such as 5xl05to about 5xl06cells / ml culturing volume, such as 8xl05to about 2xl06cells / ml culturing volume, preferably about IxlO6cells / ml culturing volume.

[0171] If the amount of microcarrier per cell culture volume becomes too high, the microcarrier may create a "carpet" like structure hindering cell proliferation and the creation of 3D cell structures. Thus, an embodiment of the present disclosure relates to the method as described herein applying at least 0.01 mg microcarrier per mL culture volume, such as at least 0.02 mg / mL, such as 0.05 mg / mL, such as 0.06 mg / mL, such as 0.075 mg / mL, such as 0.1 mg / mL, such as 0.2 mg / mL and preferably 0.06 mg / ml to 0.08 mg / ml and most preferably 0.075 mg / mL.

[0172] Another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the seeding density of host cells on the microcarrier is in the range of about IxlO5to about IxlO7cells / mg microcarrier, such as 5xl05to about 5xl06cells / mg microcarrier, such as 8xl05to about 2xl06cells / mg microcarrier, preferably about IxlO6cells / mg microcarrier.

[0173] Yet another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the cell density of host cells seeded or grown on the microcarrier is in the range of about 1.4xl06to about 1.4xl08cells / mg microcarrier, such as 7xl06to about 7xl08cells / mg microcarrier, such as 11.2xl05to about 11.2xl08cells / mg microcarrier, preferably about 1.4xl07cells / mg microcarrier.

[0174] A further embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, said host cells are seeded on said microcarrier before addition of said one or more adeno-associated virus (AAV) vector(s).

[0175] An still further embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the time between seeding said host cells and addition of said one or more adeno-associated virus (AAV) vector(s) is less than about 24 hours, such as less than about 20 hours, such as less than about 18 hours, such as less than about 16 hours, such as less than about 14 hours, such as less than about 12 hours, such as less than about 10 hours, such as less than about 8 hours, such as less than about 6 hours, such as less than about 4 hours, such as less than about 2 hours, such as less than about 1 hour, such as less than about 45 minutes, such as less than about 30 minutes, such as less than about 20 minutes, such as less than about 15 minutes, such as less than about 10 minutes, such as less than about 5 minutes.

[0176] A process wherein the host cells are seeded onto the microcarrier after or simultaneously with provision of the AAV vectors is known as reverse transfection. Interestingly, it has been found herein that the transfection efficiency is surprisingly high when transfection is performed as reverse transfection. Reverse transfection is preferred from a processing perspective because there is no lag in processing time after seeding of the host cells, which leads to a more cost-effective production method.

[0177] Therefore, a preferred embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein said one or more AAV vector(s) are added to said microcarrier before or simultaneous with seeding of the host cell on the microcarrier, preferably before seeding of the host cell on the microcarrier. Another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein there is a lag time between adding said one or more AAV vector(s) to said microcarrier and seeding of the host cell on the microcarrier, wherein said lag time is at least 1 min, such as at least 2 min, such as at least 3 min, such as at least 4 min, such as at least 5 min.

[0178] A further embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, said method comprising the following steps in the following order: i. providing a microcarrier; ii. adding one or more adeno-associated virus (AAV) vector(s) to said microcarrier; iii. seeding a host cell on said microcarrier; iv. culturing said host cell; and v. collecting AAV particles produced by said host cell; wherein said microcarrier comprises cellulose nanofibers.

[0179] Alternatively, conventional transient transfection of the host cells may be carried out to produce the adeno-associated virus (AAV).

[0180] Accordingly, an embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, said method comprising the following steps in the following order: i. providing a microcarrier; ii. seeding a host cell on said microcarrier; iii. culturing said host cell; iv. transfecting said host cell of step (iii) with one or more adeno-associated virus (AAV) vector(s); v. culturing said transfected host cell of step (iv); and vi. collecting AAV particles produced by said host cell; wherein said microcarrier comprises cellulose nanofibers.

[0181] Another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein said host cell of step (iii) is cultured for at least 24 hours prior to step (iv).

[0182] The method has been developed with the aim of achieving a high transfection efficiency in order to increase virus titer. Thus, auxiliary vectors (or transfection reagents), such as cationic polymers, may be added in conjunction with AAV vector(s) to improve transfection. The negatively charged DNA binds to the polycations and form a complex which is more easily taken up by the cell via endocytosis. Alternatively, physical treatment of the host cells can temporarily increase porosity of the cell membrane and increase transfection efficiency.

[0183] Therefore, an embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein transfection of the host cell with the one or more AAV vector(s) is facilitated by a chemical treatment or a physical treatment.

[0184] A further embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein said chemical treatment comprises providing one or more transfection reagents together with said one or more AAV vector(s).

[0185] A still further embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein said transfection reagents are selected from the group consisting of polymeric transfection reagents, lipid-based transfection reagents, and calcium phosphate, preferably polymeric transfection reagents.

[0186] An even further embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the polymeric transfection reagents are cationic polymers.

[0187] Different cationic polymers may be suitable to form complexes with the negatively charged DNA. PEI is a synthetic polymer with an exceptionally high positive charge density in pH- neutral solutions, and is therefore preferred.

[0188] Accordingly, an embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the cationic polymers are selected from the group consisting of polyethyleneimine (PEI), poly-L-lysine and DEAE- dextran, preferably PEI.

[0189] A further embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the cationic polymers are selected from the group consisting of polyethyleneimine (PEI), poly-L-lysine (PLL) and Poly(amidoamine) (PAMAM) dendrimers, Chitosan, and Poly(beta-amino esters) (PBAEs), preferably PEI.

[0190] Another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the ratio of PEI to total DNA comprised in the one or more AAV vector(s) are in the range of about 1:2 to about 5: 1 wt% / wt%, such as about 1: 1 to about 3: 1 wt% / wt%, preferably about 2: 1 wt% / wt%.

[0191] An alternative embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the physical treatment is selected from the group consisting of electroporation, sonication, and magnetofection.

[0192] Host cells may be cultured under conventional conditions for production of AAV. This includes culturing with or without agitation, but preferably with agitation.

[0193] Accordingly, an embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein culturing said host cell is performed under conditions conducive to AAV replication, expression of a nucleic acid sequence of interest, and production of AAV particles.

[0194] Another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein culturing said host cell is conducted under controlled conditions of temperature, pH, and nutrient supply.

[0195] Yet another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein culturing said host cell is performed under agitation.

[0196] A further embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein agitation is selected from the group consisting of stirring, shaking, rocking, waving, and bubbling, preferably stirring.

[0197] A still further embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein culturing said host cell is performed at about 30 rpm to about 100 rpm, such as at about 50 rpm to about 80 rpm, such as about 70 rpm. An even further embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein culturing said host cell is performed at 37°C.

[0198] The produced AAV particles may be collected after about 48 hours to about 96 hours, such as about 72 hours to about 96 hours, of culturing using any conventional method of harvesting. AAV particles are present in both intact cells and the culture medium, and therefore AAV particles may be collected from both. Preferably, collection of AAV particles includes lysing the host cells to release the AAV particles. Lysis may be achieved by cycling the host cells between a dry ice / ethanol bath and a water bath of 37°C and / or by addition of detergents that permeabilise cellular and nuclear membranes. Resulting lysed cells and cell debris can be removed by centrifugation and / or filtering.

[0199] Therefore, an embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein colleting said AAV particles comprises harvesting AAV particles produced by said host cell.

[0200] Another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein harvesting AAV particles comprises lysis of the host cell to produce a cell lysate.

[0201] Still another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein lysis of the host cell is achieved by a method selected from the group consisting of detergent lysis, freeze-thaw cycles, osmotic shock, and mechanical agitation, and combinations thereof.

[0202] A further embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein harvesting AAV particles comprises filtering of AAV particles from cell debris and dead cells.

[0203] A further embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein harvesting AAV particles includes comprises AAV particles contained in the supernatant and / or pellet of cell lysate.

[0204] Harvested AAV particles are purified to obtain an efficient and safe AAV product, which does not cause immunological or toxicological adverse effects. The purification may involve filtration, ultracentrifugation and / or high-performance liquid chromatography (HPLC). An embodiment of the present invention relates to the method for production of adeno- associated virus (AAV) as described herein further comprising a step of purifying said collected AAVs.

[0205] Another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein purifying said collected AAVs comprises chromatographic separation, ultracentrifugation, or a combination thereof.

[0206] The method for production of AAVs is preferably performed in a vessel suitable for culturing of cells. As such, the container may be loaded with the microcarrier prior to addition of cells. The microcarrier may be used in any traditional scale up step for cell culturing, such as adding microcarrier and cells to a small container (e.g. a flask), or performing the method in an industrial scale bioreactor.

[0207] Therefore, an embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein said microcarrier is provided in a container.

[0208] Another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein said container is selected from the group consisting of a bioreactor, a cell culturing plate, a cell culture bottle, a spinner flask, a shaker flask, and a roller bottle, a petri dish, and a tube, preferably a bioreactor.

[0209] Bioreactors are of particular importance to large scale industrial production. They may hold larger volumes than laboratory- or pilot study equipment and are used in upstream processes to expand and scale cell culture for production. For the purpose of AAV production it is important that the microcarrier in the bioreactor offers a controlled microenvironment and nutrient delivery to regulate host cell growth, improving standardization and reproducibility of viral yield. The microcarrier comprising cellulose nanofibers as described herein is particularly advantageous at the production scale where other microcarriers may be too expensive or otherwise unsuitable for use.

[0210] Suspension based bioreactors are preferred for many practical applications because they allow addition of more microcarriers and medium during the culturing process, which gives flexibility and control over the process. In the suspension based bioreactor, the microcarriers are freely floating in the bioreactor but provides the necessary support matrix for adherent to attach and proliferate. Important parameters for the microcarrier include the available surface area they provide and the small dead volume they occupy. This means that more volume is available for cells, and therefore that more viruses can be produced in a limited volume. Furthermore, the porous nature of the microcarrier ensures that the material stays in solution without precipitation. Accordingly, only light agitation is needed for distribution of nutrients, and undesired collisions and shear forces caused by heavy agitation can be avoided. Together, these effects make the present method advantageous for production of AAVs in bioreactors, such as suspension based bioreactors.

[0211] Thus, an embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the container is a bioreactor, preferably a suspension based bioreactor.

[0212] Another embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein the container is a bioreactor with a volume of at least about 500 mL, such as at least about IL, such as at least about 5L, such as at least about 10L, such as at least about 25L, such as at least about 50L, such as at least about 100L, such as at least about 250L, such as at least about 500L, such as at least about 1000L, such as at least about 5000L.

[0213] The cellulose nanofibers of the microcarrier may be processed to yield a nanofibrous cellulose scaffold which mimics the collagen and elastin fiber structures that make up the human extracellular matrix (ECM) and has a high surface area due to the homogeneous distribution of cellulose nanofibers. The ECM is important for survival, proliferation, differentiation and migration of the cells, and microcarriers mimicking the properties of the ECM is therefore considered a step in the direction of in v / vo-like cell culturing. Without being bound by theory, it is contemplated herein that host cells grown on the microcarrier presented herein adopt physiological characteristics which in turn results in a recovery or maintenance of in vivo functions that will positively affect the production of AAV particles.

[0214] Thus, a preferred embodiment of the present invention relates to the method for production of adeno-associated virus (AAV) as described herein, wherein said microcarrier comprises a nanofibrous cellulose scaffold as described herein.

[0215] The preparation of the nanofibrous cellulose scaffold will be described in the following. It is to be understood that the resulting product from this method may be used as a microcarrier in the method for production of AAV. The production of the nanofibrous cellulose scaffold can easily be scaled for industrial use without cost and applicability of the resulting microcarrier when used at high volumes being a hindrance. Accordingly, an aspect of the present invention relates to a method of preparing a nanofibrous cellulose scaffold, said method comprising the steps of:

[0216] (i) providing an initial cellulose nanofiber material,

[0217] (ii) dividing said initial cellulose nanofiber material into a processed cellulose nanofiber material,

[0218] (iii) functionalizing the processed cellulose nanofiber material by addition of a reagent comprising a functional moiety, and

[0219] (iv) drying the processed cellulose nanofiber material, thereby providing the nanofibrous cellulose scaffold.

[0220] Dividing of the initial cellulose nanofiber material is advantageous as it ensures a homogeneous distribution of the cellulose nanofibers in the final scaffold. The shorter cellulose nanofibers also significantly reduce the risk of entanglement of the nanofibers and formation of clusters of nanofibers, which leads to less available surface area exposed to the cells and increased risk of blocking the impeller of a bioreactor. As part of the upstream biomanufacturing process, pipetting or tapping (sampling) from the bioreactor is continuously performed in order to perform cell counting, viability and yield results. Therefore, a microcarrier with decreased risk of clogging is favoured.

[0221] It has been found that dispersing the initial cellulose nanofiber material results in a homogeneous material that is suitable for use as a microcarrier. Without being bound by theory, it is contemplated that dispersing the initial cellulose nanofiber material results in a distribution which are spaced so as to allow multiple attachment points for the cells leading to improved proliferation.

[0222] Therefore, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein dividing said initial cellulose nanofiber material is achieved by dispersing.

[0223] Another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein dispersing is performed with a high-speed disperser.

[0224] A further embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold, wherein dividing said initial nanofiber material comprises a step of cutting the initial nanofiber material with a disperser. Increasing the dispersing time reduces the mean length of the cellulose nanofibers in the nanofibrous cellulose scaffold. In particular, it is advantageous to disperse the material for at least a couple of minutes to reduce nanofiber entanglement and cluster formation. Also, without being bound by theory, it is contemplated that longer cellulose nanofibers are not as easily substituted on the hydroxyl groups, thereby leading to a lower degree of substitution (DS) of functional moieties.

[0225] Thus, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein dispersing is performed for at least 2 min, such as at least 5 min, such as at least 10 min, such as at least 15 min, such as at least 20 min, such as at least 30 min, such as at least 40 min, such as at least 50 min, such as at least 60 min, such as at least 90 min, such as at least 120 min.

[0226] Another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein dispersing is performed for a period of time in the range of 2 min to 120 min, such as 2 min to 90 min, such as 5 min to 60 min, such as 10 min to 60 min, such as 15 min to 60 min.

[0227] A preferred embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein dispersing is performed for at least 5 min.

[0228] Another preferred embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein dispersing is performed for at least 15 min.

[0229] A still further preferred embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein dispersing is performed for at least 60 min.

[0230] Dispersing of the initial cellulose nanofiber material for at least 15 min, and even at least 60 min, can be advantageous as it reduces entanglement and cluster formation of the cellulose nanofibers.

[0231] Preferably dispersing is performed at high speed, such as at about 18000 rpm. The speed may be adjusted depending on the type of disperser. Any type of disperser may be used, including, but not limited to, laboratory high-speed disperser, pilot-scale high-speed dispersers, and production-scale high-speed dispersers. The speed may be adjusted to produce a good vortex in the solution, and can depend on volume and viscosity of the solution.

[0232] Therefore, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein dispersing is performed at a speed in the range of about 10000 rpm to about 30000 rpm, such as at about 12000 rpm to about 25000 rpm, such as about 15000 rpm to about 20000 rpm.

[0233] During the dividing step, the cellulose nanofibers are reduced in length. It is advantageous that the cellulose nanofibers are not too long as it can cause entanglement of the nanofibers and cluster formation. Cells are not able to penetrate and migrate into these tight clusters of entangled fibers and thereby a portion of the large surface area of the nanofibers are lost.

[0234] Accordingly, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the mean length of the cellulose nanofibers in said processed cellulose nanofiber material is shorter than the mean length of the cellulose nanofibers in said initial cellulose nanofiber material.

[0235] Another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the mean length of the cellulose nanofibers in said processed cellulose nanofiber material is in the range of about 30 pm to about 250 pm, such as about 40 pm to about 200 pm, such as about 50 pm to about 175 pm, such as about 60 pm to about 150 pm, preferably about 70 pm to about 120 pm.

[0236] The dividing step may be performed under cooling to lower the ductility of the cellulose nanofibers, making them more brittle and easier to divide. The cooling may occur before or during the dividing of the initial cellulose nanofiber material. Cooling may comprise cooling of the container in which the initial cellulose nanofiber material is held during the dividing step or cooling the initial cellulose nanofiber material by exposure to a coolant, such as liquid hydrogen, liquid helium and / or liquid nitrogen, or by keeping the initial cellulose nanofiber material in a fridge or freezer immediately before the dividing step.

[0237] Thus, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the initial cellulose nanofiber material is cooled before or during the dividing step. Cellulose sheets, if prepared by electrospinning, may be highly static and difficult to handle. Therefore, the initial cellulose nanofiber material may conveniently be provided as a liquid sample that is ready for processing, e.g. by dispersing.

[0238] Thus, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the initial cellulose nanofiber material is provided as a liquid sample.

[0239] Another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the solvent of the liquid sample comprises water and / or ethanol.

[0240] Yet another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the solvent of the liquid sample comprises ethanol.

[0241] A further embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is about 0.1 wt% to about 10 wt%, such as about 0.5 wt% to about 5 wt%, such as about 0.75 wt% to about 4 wt%, preferably about 1 wt% to about 3 wt%, with respect to the total weight of the liquid sample.

[0242] A common source of cellulose nanofibers is pulp, which may be mechanically defibrillated to generate cellulosic nanofibers. Mechanical treatment may include high-pressure homogenization, grinding and / or microfluidization. Mechanical defibrillation is commonly utilised in the pulp and paper industry, as well as the textile industry. Unfortunately, it can be difficult to precisely control cellulose nanofiber dimensions and properties when the starting material is raw pulp (that can vary in composition depending on source) which is subsequently exposed to the harsh mechanical treatment. Thus, it is preferred to avoid cellulose material derived directly from a raw plant source (e.g. without any treatment to extract and / or purify the cellulose nanofibers) as the source of the cellulose nanofibers. Raw plant sources for cellulose material may include wood, such as softwood (e.g. spruce, pine, fir, larch, or hemlock) or hardwood (e.g. birch, aspen, poplar, alder, eucalyptus or acacia), or non-wood sources such as agricultural residues, grasses or other plant substances (e.g. straw, leaves, bark, seeds, hulls, flowers, vegetables or fruits from cotton, corn, wheat, oat, rye, barley, rice, flax, hemp, manilla hemp, sisal hemp, jute, ramie, kenaf, bagasse, bamboo or reed). Accordingly, it is advantageous to use initial cellulose nanofiber material derived from a sources, e.g. plant source, that has been treated to purify the cellulose nanofibers to ensure high quality and consistency of the cellulose nanofibers utilised for preparing the nanofibrous cellulose scaffold.

[0243] Therefore, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the initial cellulose nanofiber material is not derived from a raw plant source.

[0244] Another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the initial cellulose nanofiber material is not prepared by mechanical treatment, such as mechanical defibrillation, such as mechanical disintegration.

[0245] It is to be understood that a raw plant source is a source which has not been treated to extract or purify the cellulose nanofibers. Extraction may include chemical treatment of a cellulose source, such as a raw plant source. The chemical treatment can comprise treating the raw plant source with acetic acid and acetic anhydride in the presence of a catalyst, such as sulfuric acid. The chemical treatment can produce a cellulose acetate solution which can be further processed, e.g. by electrospinning, to yield cellulose nanofibers with highly controllable dimensions and properties.

[0246] Thus, the cellulose nanofibers may advantageously be obtained from electrospinning to allow precise control over nanofiber dimensions and quality. Electrospinning involves dissolving cellulose or its derivatives in a suitable solvent and then spinning it into nanofibers using an electric field. Electrospun nanofibers are spun as monofibers that can be aligned on a drum roll to yield a cellulose sheet that can subsequently be further processed.

[0247] Therefore, an embodiment of the resent invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the initial cellulose nanofiber material comprises monofibers.

[0248] Another embodiment of the resent invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein said nanofibrous cellulose scaffold comprises monofibers. The initial cellulose nanofiber material may be prepared from a cellulose acetate solution, e.g. by electrospinning of a cellulose acetate solution. However, the resulting cellulose acetate sheets are preferably regenerated to cellulose sheets in a sodium hydroxide bath before any further processing. This treatment opens up hydroxyl groups that may subsequently be used for binding of functional moieties.

[0249] Therefore, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the initial cellulose nanofiber material is prepared by electrospinning of a cellulose acetate solution to cellulose acetate sheets.

[0250] Another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein said initial cellulose nanofiber material is regenerated in a regeneration solution comprising sodium hydroxide.

[0251] Yet another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the regeneration solution comprises sodium hydroxide in the range of about 0.1 M to about 1 M, such as about 0.2 M to about 0.8 M, such as about 0.3 M to about 0.7 M, such as about 0.4 M to about 0.6 M, preferably about 0.5 M.

[0252] By regenerating the initial cellulose nanofiber material, e.g. by alkali treatment, the cellulose obtains a monoclinic crystal structure known as cellulose II. In contrast, native cellulose found in plants has the crystal structure cellulose I. Without being bound by theory, it is contemplated that the crystal structure cellulose II is advantageous for use as the initial cellulose nanofiber material, e.g. because it is more thermodynamically stable than other polymorphs such as cellulose I. This may be due to the antiparallel polymer chain orientation and different hydrogen bonding of cellulose II.

[0253] Accordingly, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein said initial cellulose nanofiber material comprises regenerated cellulose.

[0254] Another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the crystal structure of said initial cellulose nanofiber material is cellulose II. Regeneration of cellulose sheets is preferably performed in an ethanol solution comprising sodium hydroxide. It is possible to use varying amounts of ethanol, such as from 5% vol / vol to 99% vol / vol.

[0255] Accordingly, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the regeneration solution is an ethanol solution comprising about 5% vol / vol to about 99% vol / vol ethanol, such as about 10% vol / vol to about 95% vol / vol ethanol, such as about 20% vol / vol to about 90% vol / vol ethanol, such as about 30% vol / vol to about 80% vol / vol ethanol, such as about 40% vol / vol to about 70% vol / vol ethanol.

[0256] The content of ethanol in the regeneration solution may influence the mechanical properties of the resulting nanofibrous cellulose scaffold. Mechanical properties that can be impacted by the content of ethanol includes elasticity and brittleness. Without being bound by theory, it is contemplated that a more elastic and less stiff material is beneficial for interaction with the cells and to promote proliferation.

[0257] Thus, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the regeneration solution is an ethanol solution comprising at least about 50% vol / vol ethanol, such as at least 60% vol / vol ethanol, such as at least 70% vol / vol ethanol, such as at least 80% vol / vol ethanol, such as at least 90% vol / vol ethanol, preferably about 90% vol / vol ethanol.

[0258] Subsequent to dividing of the initial cellulose nanofiber material, the resulting processed material is preferably handled to make it ready for functionalization. Part of the treatment can include filtration, washing and re-suspension of the processed cellulose nanofiber material. Filtration ensures that excess water is removed from the processed cellulose nanofiber material. Washing assists in removal of any acetate ions still present after the previous treatment. When re-suspending the processed cellulose nanofiber material, the concentration of cellulose nanofibers may be adjusted if desired.

[0259] Accordingly, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the dividing step (ii) is followed by a step comprising filtration, washing and suspending the processed cellulose nanofiber material.

[0260] Prior to functionalization, the cellulose nanofibers are preferably mercerized to improve the substitution of functional moieties onto the cellulose nanofibers. Mercerization is a process in which the cellulose nanofibers are swollen by immersion in a concentrated aqueous NaOH solution. During the mercerization process, the crystal structure of the cellulose nanofiber is transformed from cellulose I to cellulose II. Under the action of concentrated alkaline solutions chemical, physicochemical and structural modifications of cellulose occur. Upon washing and neutralisation cellulose II is formed. As a result of the penetration of the base into the lattice, internal hydrogen bonds are broken and the number of available hydroxyl groups (-OH) in the cellulose nanofiber is increased. It is therefore contemplated that mercerization improves the degree of substitution (DS). The mercerization step may be performed before or after the dividing step.

[0261] Therefore, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein further comprising a step of mercerization of said processed cellulose nanofiber material.

[0262] Another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the mercerization step is immediately before or after the dividing step (ii).

[0263] A further embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein said mercerization step comprises addition of NaOH in a concentration of about 0.1 M to about 2 M, such as about 0.5 M to about 1.8 M, such as about 1 M to about 1.7 M, preferably about 1.4 M to about 1.6 M.

[0264] After functionalization of the processed cellulose nanofiber scaffold, the material is dried to yield the nanofibrous cellulose scaffold in its final form. Drying may be performed in two steps, such as freezing followed by lyophilization, or in a single step, such as by lyophilization. A lyophilizer performs a water removal process that can extend shelf life and / or make the material more convenient for transport. Lyophilizers work by freezing the material, then reducing the pressure and adding heat to allow the frozen water in the material to sublimate.

[0265] Accordingly, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the drying step (iv) comprises freezing and / or lyophilization of the processed cellulose nanofiber material.

[0266] Another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the drying step (iv) comprises freezing of processed cellulose nanofiber material followed by lyophilization of the frozen processed cellulose nanofiber material.

[0267] The dried nanofibrous cellulose scaffold may be further processed to provide a dry powder. This may be accomplished by grinding the dried product.

[0268] The method described herein provides a nanofibrous cellulose scaffold with large surface area and low dead volume that may advantageously be utilised as a microcarrier for culturing of host cells in the production of AAV. The microcarrier product may be in the form of a dry powder that is added to the cell culturing container, such as a bioreactor, to form a support matrix upon which cells may attach, proliferate and produce AAV particles.

[0269] Thus, an aspect of the present invention relates to a nanofibrous cellulose scaffold obtainable from the method of preparing a nanofibrous cellulose scaffold as described herein.

[0270] Another aspect of the present invention relates to use of a microcarrier comprising cellulose nanofibers for production of adeno-associated virus (AAV).

[0271] An embodiment of the present invention relates to the use of a microcarrier as described herein, wherein said microcarrier comprises a nanofibrous cellulose scaffold as described herein.

[0272] The listing or discussion of an apparently prior published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0273] Preferences, options and embodiments for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences, options and embodiments for all other aspects, features and parameters of the invention. This is especially true for the description of the method for production of adeno-associated virus (AAV), and all its features, which may readily be part of the corresponding use of the nanofibrous cellulose scaffold for production of AAV. Embodiments and features of the present invention are also outlined in the following items.

[0274] Items XI. A method of preparing a nanofibrous cellulose scaffold, said method comprising the steps of:

[0275] (i) providing an initial cellulose nanofiber material,

[0276] (ii) dividing said initial cellulose nanofiber material into a processed cellulose nanofiber material,

[0277] (iii) functionalizing the processed cellulose nanofiber material by addition of a reagent comprising a functional moiety, and

[0278] (iv) drying the processed cellulose nanofiber material, thereby providing the nanofibrous cellulose scaffold.

[0279] X2. The method according to item XI, wherein dividing said initial cellulose nanofiber material is achieved by dispersing.

[0280] X3. The method according to item X2, wherein dispersing is performed with a high-speed disperser.

[0281] X4. The method according to any one of items X2 or X3, wherein dispersing is performed for at least 2 min, such as at least 5 min, such as at least 10 min, such as at least 15 min, such as at least 20 min, such as at least 30 min, such as at least 40 min, such as at least 50 min, such as at least 60 min, such as at least 90 min, such as at least 120 min.

[0282] X5. The method according to any one of items X2-X4, wherein dispersing is performed at a speed in the range of about 10000 rpm to about 30000 rpm, such as at about 12000 rpm to about 25000 rpm, such as about 15000 rpm to about 20000 rpm.

[0283] X6. The method according to any one of the preceding items, wherein the mean length of the cellulose nanofibers in said processed cellulose nanofiber material is shorter than the mean length of the cellulose nanofibers in said initial cellulose nanofiber material.

[0284] X7. The method according to any one of the preceding items, wherein the mean length of the cellulose nanofibers in said processed cellulose nanofiber material is less than 250 pm, such as less than about 200 pm, such as less than about 150 pm, such as less than about 120 pm, such as less than about 100 pm, such as less than about 80 pm.

[0285] X8. The method according to any one of the preceding items, wherein the mean length of the cellulose nanofibers in said processed cellulose nanofiber material is in the range of about 30 pm to about 250 pm, such as about 40 pm to about 200 pm, such as about 50 pm to about 175 pm, such as about 60 pm to about 150 pm, preferably about 70 pm to about 120 pm.

[0286] X9. The method according to any one of the preceding items, wherein the initial cellulose nanofiber material is provided as a liquid sample.

[0287] X10. The method according to item X9, wherein the solvent of the liquid sample comprises water and / or ethanol.

[0288] XI 1. The method according to any one of the preceding items, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is about 0.1 wt% to about 10 wt%, such as about 0.5 wt% to about 5 wt%, such as about 0.75 wt% to about 4 wt%, preferably about 1 wt% to about 3 wt%, with respect to the total weight of the liquid sample.

[0289] X12. The method according to any one of the preceding items, wherein the mean diameter of the cellulose nanofibers in said initial cellulose nanofiber material and / or processed cellulose nanofiber material is in the range of about 10 nm to about 2000 nm, such as about 50 nm to about 1500 nm, such as about 100 nm to about 1000 nm, preferably about 250 nm to about 750 nm.

[0290] X13. The method according to any one of the preceding items, wherein the initial cellulose nanofiber material is prepared by a process selected from the group consisting of electrospinning, meltblowing, drawing, self-assembly, template synthesis, and thermal- induced phase separation.

[0291] X14. The method according to any one of the preceding items, wherein the initial cellulose nanofiber material is prepared by electrospinning of a cellulose acetate solution to cellulose acetate sheets.

[0292] X15. The method according to any one of the preceding items, wherein said initial cellulose nanofiber material is regenerated in a regeneration solution comprising sodium hydroxide.

[0293] X16. The method according to item X15, wherein the regeneration solution is an ethanol solution comprising at least about 50% vol / vol ethanol, such as at least 60% vol / vol ethanol, such as at least 70% vol / vol ethanol, such as at least 80% vol / vol ethanol, such as at least 90% vol / vol ethanol, preferably about 90% vol / vol ethanol. X17. The method according to any one of the preceding items further comprising a step of mercerization of said processed cellulose nanofiber material.

[0294] X18. The method according to item X17, wherein the mercerization step is immediately before or after the dividing step (ii).

[0295] X19. The method according to any one of the preceding items, wherein the functional moiety is selected from chemical moieties or biological molecules.

[0296] X20. The method according to item X19, wherein the chemical moieties are selected from the group consisting of negatively charged groups, positively charged groups, zwitterionic groups, hydrophobic groups, hydrophilic groups, amphiphilic groups, ligands, and combinations thereof.

[0297] X21. The method according to any one of items X19 or X20, wherein the chemical moieties are selected from the group consisting of quaternary ammonium (QA), carboxymethyl (CM), diethylaminoethyl (DEAE), 4-(l-bromoethyl)benzoic acid, 4- (dimethylamino)pyridine, epoxide, and combinations thereof.

[0298] X22. The method according to any one of items X19-X21, wherein the chemical moiety is quaternary ammonium (QA).

[0299] X23. The method according to any one of the preceding items, wherein the dividing step (ii) is followed by a step comprising filtration, washing and suspending the processed cellulose nanofiber material.

[0300] X24. The method according to any one of the preceding items, wherein the drying step (iv) comprises freezing and / or lyophilization of the processed cellulose nanofiber material.

[0301] X25. The method according to any one of the preceding items, wherein the drying step (iv) comprises freezing of processed cellulose nanofiber material followed by lyophilization of the frozen processed cellulose nanofiber material.

[0302] X26. The method according to any one of the preceding items, wherein the initial cellulose nanofiber material is not derived from a raw plant source. X27. The method according to any one of the preceding items, wherein the initial cellulose nanofiber material is not prepared by mechanical treatment, such as mechanical defibrillation, such as mechanical disintegration.

[0303] X28. The method according to any one of the preceding items, wherein said initial cellulose nanofiber material comprises regenerated cellulose.

[0304] X29. The method according to any one of the preceding items, wherein the crystal structure of said initial cellulose nanofiber material is cellulose II.

[0305] Yl. A nanofibrous cellulose scaffold obtainable from a method according to any one of items X1-X29.

[0306] Zl. A nanofibrous cellulose scaffold comprising cellulose nanofibers with a mean length of the cellulose nanofibers of less than about 250 pm, and wherein the cellulose nanofibers are functionalized with a functional moiety.

[0307] Z2. The nanofibrous cellulose scaffold according to item Zl, wherein the cellulose nanofibers have a mean length in the range of about 30 pm to about 250 pm, such as about 40 pm to about 200 pm, such as about 50 pm to about 175 pm, such as about 60 pm to about 150 pm, preferably about 70 pm to about 120 pm.

[0308] Z3. The nanofibrous cellulose scaffold according to any one of items Zl or Z2, wherein the cellulose nanofibers have a mean diameter in the range of about 10 nm to about 2000 nm, such as about 50 nm to about 1500 nm, such as about 100 nm to about 1000 nm, preferably about 250 nm to about 750 nm.

[0309] Z4. The nanofibrous cellulose scaffold according to any one of items Z1-Z3, wherein the cellulose nanofibers are electrospun, meltblown or drawn, preferably electrospun.

[0310] Z5. The nanofibrous cellulose scaffold according to any one of items Z1-Z4, wherein the functional moiety is selected from chemical moieties or biological molecules.

[0311] Z6. The nanofibrous cellulose scaffold according to item Z5, wherein the chemical moieties are selected from the group consisting of negatively charged groups, positively charged groups, zwitterionic groups, hydrophobic groups, hydrophilic groups, amphiphilic groups, ligands, and combinations thereof. Z7. The nanofibrous cellulose scaffold according to any one of items Z5 or Z6, wherein the chemical moieties are positively charged groups.

[0312] Z8. The nanofibrous cellulose scaffold according to any one of items Z5-Z7, wherein the chemical moieties are selected from the group consisting of quaternary ammonium (QA), carboxymethyl (CM), diethylaminoethyl (DEAE), 4-(l-bromoethyl)benzoic acid, 4- (dimethylamino)pyridine, epoxide, and combinations thereof.

[0313] Z9. The nanofibrous cellulose scaffold according to any one of items Z5-Z8, wherein the chemical moiety is quaternary ammonium (QA).

[0314] Z10. The nanofibrous cellulose scaffold according to any one of items Z1-Z9, wherein the nanofibrous cellulose scaffold is provided as a dry material.

[0315] Zll. The nanofibrous cellulose scaffold according to any one of items Z1-Z10, wherein the nanofibrous cellulose scaffold is provided as a lyophilized material.

[0316] Z12. The nanofibrous cellulose scaffold according to any one of items Zl-Zll, wherein the nanofibrous cellulose scaffold has a BET surface area of at least about 40000 cm2 / g, such as at least about 50000 cm2 / g, such as at least about 55000 cm2 / g, such as at least about 60000 cm2 / g, such as at least about 70000 cm2 / g, such as at least about 80000 cm2 / g.

[0317] Z13. The nanofibrous cellulose scaffold according to any one of items Z1-Z12, wherein the cellulose nanofibers comprise monofibers.

[0318] Z14. The nanofibrous cellulose scaffold according to any one of items Z1-Z13, wherein the cellulose nanofibers are derived from regenerated cellulose.

[0319] Z15. The nanofibrous cellulose scaffold according to any one of items Z1-Z14, wherein the cellulose nanofibers have the cellulose II crystal structure.

[0320] Z16. The nanofibrous cellulose scaffold according to any one of items Z1-Z15, wherein the cellulose nanofibers are not derived from a raw plant source.

[0321] Z17. The nanofibrous cellulose scaffold according to any one of items Z1-Z16, wherein the cellulose nanofibers are not obtained by mechanical treatment, such as mechanical defibrillation, such as mechanical disintegration. Z18. The nanofibrous cellulose scaffold according to any one of items Z1-Z17, wherein said nanofibrous cellulose scaffold is not a hydrogel.

[0322] QI. A method for production of adeno-associated virus (AAV), said method comprising the steps of: providing a microcarrier;

[0323] - adding one or more adeno-associated virus (AAV) vector(s) to said microcarrier;

[0324] - seeding a host cell on said microcarrier;

[0325] - culturing said host cell; and

[0326] - collecting AAV particles produced by said host cell; wherein said microcarrier comprises cellulose nanofibers.

[0327] Q2. The method according to item QI, wherein said microcarrier comprises a nanofibrous cellulose scaffold according to items Y1 or Z1-Z18.

[0328] Q3. The method according to any one of items QI or Q2, wherein said one or more AAV vector(s) comprises a nucleic acid sequence of interest and AAV rep and cap genes.

[0329] Q4. The method according to item Q3, wherein said one or more AAV vector(s) further comprises inverted terminal repeats (ITRs) flanking said nucleic acid sequence of interest.

[0330] Q5. The method according to any one of items Q3 or Q4, wherein said nucleic acid sequence of interest encodes a therapeutic protein, a gene of interest, or a reporter gene.

[0331] Q6 The method according to any one of items Q1-Q5, wherein said one or more AAV vector(s) are non-viral AAV vector(s).

[0332] Q7 The method according to any one of items Q1-Q6, wherein said one or more AAV vector(s) are plasmids.

[0333] Q8. The method according to any one of items Q1-Q7, wherein said one or more AAV vector(s) comprises three plasmids.

[0334] Q9. The method according to any one of items Q1-Q8, wherein said one or more adeno- associated virus (AAV) vector(s) comprises:

[0335] - a first plasmid comprising a nucleic acid sequence of interest and inverted terminal repeats (ITRs) flanking said nucleic acid sequence of interest;

[0336] - a second plasmid comprising AAV rep and cap genes; and a third plasmid comprising E2A, E4 and VA RIMA genes.

[0337] Q10. The method according to any one of items Q1-Q9, wherein said host cell comprises the machinery for supporting AAV replication.

[0338] Qll. The method according to any one of items Q1-Q10, wherein said host cell is selected from the group consisting of mammalian cells, insect cells, and bacterial cells.

[0339] Q12. The method according to any one of items Ql-Qll, wherein the host cell is selected from the group consisting of HEK293, HEK293T, SF9, BHK21, SF21, CAP-T, Vero and CHO cells.

[0340] Q13. The method according to any one of items Q1-Q12, wherein said one or more AAV vector(s) are added to said microcarrier before or simultaneous with seeding of the host cell on the microcarrier, preferably before seeding of the host cell on the microcarrier.

[0341] Q14. The method according to item Q13, wherein there is a lag time between adding said one or more AAV vector(s) to said microcarrier and seeding of the host cell on the microcarrier, wherein said lag time is at least 1 min, such as at least 2 min, such as at least 3 min, such as at least 4 min, such as at least 5 min.

[0342] Q15. The method according to any one of items Q1-Q14, wherein transfection of the host cell with the one or more AAV vector(s) is facilitated by a chemical treatment or a physical treatment.

[0343] Q16. The method according to item Q15, wherein said chemical treatment comprises providing one or more transfection reagents together with said one or more AAV vector(s).

[0344] Q17. The method according to item Q16, wherein said transfection reagents are selected from the group consisting of polymeric transfection reagents, lipid-based transfection reagents, and calcium phosphate, preferably polymeric transfection reagents.

[0345] Q18. The method according to item Q17, wherein the polymeric transfection reagents are cationic polymers. Q19. The method according to item Q18, wherein the cationic polymers are selected from the group consisting of polyethyleneimine (PEI), poly-L-lysine and DEAE-dextran, preferably PEI.

[0346] Q20. The method according to item Q19, wherein the ratio of PEI to total DNA comprised in the one or more AAV vector(s) are in the range of about 1:2 to about 5: 1 wt% / wt%, such as about 1: 1 to about 3: 1 wt% / wt%, preferably about 2: 1 wt% / wt%.

[0347] Q21. The method according to any one of items Q15-Q20, wherein the physical treatment is selected from the group consisting of electroporation, sonication, and magnetofection.

[0348] Q22. The method according to any one of items Q1-Q21, wherein the cell density of host cells seeded or grown on the microcarrier is in the range of about 1.4xl06to about 1.4xl08cells / mg microcarrier, such as 7xl06to about 7xl08cells / mg microcarrier, such as 11.2xl05to about 11.2xl08cells / mg microcarrier, preferably about 1.4xl07cells / mg microcarrier.

[0349] Q23. The method according to any one of items Q1-Q22, wherein culturing said host cell is performed under conditions conducive to AAV replication, expression of a nucleic acid sequence of interest, and production of AAV particles.

[0350] Q24. The method according to any one of items Q1-Q23, wherein culturing said host cell is conducted under controlled conditions of temperature, pH, and nutrient supply.

[0351] Q25. The method according to any one of items Q1-Q24, wherein colleting said AAV particles comprises harvesting AAV particles produced by said host cell.

[0352] Q26. The method according to item Q25, wherein harvesting AAV particles comprises lysis of the host cell to produce a cell lysate.

[0353] Q27. The method according to item Q26, wherein lysis of the host cell is achieved by a method selected from the group consisting of detergent lysis, freeze-thaw cycles, osmotic shock, and mechanical agitation, and combinations thereof.

[0354] Q28. The method according to any one of items Q25-Q27, wherein harvesting AAV particles comprises filtering of AAV particles from cell debris and dead cells. Q29. The method according to any one of items Q25-Q28, wherein harvesting AAV particles includes comprises AAV particles contained in the supernatant and / or pellet of lysate.

[0355] Q30. The method according to any one of items Q1-Q29 further comprising a step of purifying said collected AAVs.

[0356] Q31. The method according to item Q30, wherein purifying said collected AAVs comprises filtration, chromatographic separation, ultracentrifugation, or a combination thereof.

[0357] Q32. The method according to any one of items Q1-Q31, wherein said microcarrier is provided in a container.

[0358] Q33. The method according to any one of items Q32, wherein said container is selected from the group consisting of a bioreactor, a cell culturing plate, a cell culture bottle, a spinner flask, a shaker flask, and a roller bottle, a petri dish, and a tube, preferably a bioreactor.

[0359] Q34. The method according to any one of items Q32 or Q33, wherein the container is a bioreactor, preferably a suspension based bioreactor.

[0360] Q35. The method according to any one of items Q32-Q34, wherein the container is a bioreactor with a volume of at least about 500 mL, such as at least about IL, such as at least about 5L, such as at least about 10L, such as at least about 25L, such as at least about 50L, such as at least about 100L, such as at least about 250L, such as at least about 500L, such as at least about 1000L, such as at least about 5000L.

[0361] Q36. The method according to any one of items Q1-Q35, wherein the AAV is of serotype AAV2, AAV5, or AAV9, preferably AAV2.

[0362] Ul. Use of a microcarrier comprising cellulose nanofibers for production of adeno- associated virus (AAV).

[0363] U2. The use according to item Ul, wherein said microcarrier comprises a nanofibrous cellulose scaffold according to items Y1 or Z1-Z18.

[0364] The invention will now be described in further details in the following non-limiting examples. Examples

[0365] Example 1: Preparation of nanofibrous cellulose scaffold

[0366] In this example is given a non-limiting demonstration of how the nanofibrous cellulose scaffold can be prepared. The core properties of the material, such as surface area and degree of substitution, was characterized.

[0367] Method

[0368] Preparation of initial cellulose nanofiber material

[0369] A solution of 19% cellulose acetate was prepared by addition of cellulose acetate (6.38 g) to 12 ml of acetone in an Erlenmeyer flask, followed by addition of 12 ml DMF and 6 ml 96% ethanol. The solution was stirred using a magnetic stirrer overnight at room temperature.

[0370] The 19% cellulose acetate solution was electrospun to prepare cellulose acetate nanofiber sheets. The cellulose nanofibers were electrospun (Fluidnatek LE50) using a drum speed of 200 rpm and a flow of 10 ml / hour at 18 kV-i- (emitter) and 10 kV- (collector). The cellulose acetate nanofibers were collected on an aluminium substrate, with temperature being 23°C and at a relative humidity of 63%. After the electrospinning process completed, the cellulose acetate sheet comprising cellulose nanofibers was removed from the drum.

[0371] The cellulose acetate sheets were regenerated to cellulose by submergence in a 0.5 M NaOH in 95% ethanol solution. The cellulose acetate sheets were left in the solution for 24 hours at room temperature followed by filtering through a Buchner filter and washing with copious volumes of distilled water. Filtering and washing was repeated until the conductivity of the final washing step was 0 pS / cm to make sure there were no residual NaOH, or acetate left. The washed sheets were put in the oven at 80°C for 12 hours to provide dry cellulose sheets. The cellulose sheets were weighed.

[0372] Processing of initial cellulose nanofiber material

[0373] The dry cellulose sheets were cut into rough pieces of approximately 2x2 cm squares using scissors. The size of the pieces does not have to be exact, but larger pieces should be avoided as they may hamper the dividing step. The rough pieces of cellulose were fully submerged in water and dispersed using a high-speed disperser (IKA T25 digital Ultra Turrax) at 18,000 rpm for 60 min (with intermittent stops to cool the disperser). The processed cellulose nanofiber material was transferred to a sieve, washed with water to remove any remaining acetate ions, and drained to remove excess water. Cellulose nanofiber material was transferred to a flask and fresh water was added to provide a cellulose concentration of 2 wt%.

[0374] Functionalization of processed cellulose nanofiber material

[0375] The processed cellulose nanofiber material was functionalized with quaternary ammonium (QA).

[0376] Briefly, 1 g of regenerated cellulose nanofiber material was resuspended in 50 ml of 1.5 M NaOH solution and mercerised for 2 hours under stirring at room temperature.

[0377] After the mercerization step, the temperature of the cellulose suspension was increased to 80°C and 2.5 ml of 3-chloro-2-hydroxypropyltrimethyl ammonium chloride (CHPTAC) was added dropwise. The reaction was continued at 80°C for 4 hours. After the reaction, the reaction mixture was cooling down to room temperature and filtered through a Buchner filter, immersed in 200 mL db O for 1 minute and filtered again. This process was repeated until the conductivity of the final washing step was 0 pS / cm to make sure there were no residual CHPTAC or NaOH left. Finally, the filtered processed and functionalized cellulose nanofiber material were resuspended in water to a 2% solution and freeze dried for further experimentation.

[0378] Drying

[0379] The functionalized cellulose nanofiber material was transferred to a -85°C freezer and left overnight. The frozen cellulose nanofiber material was then transferred to a lyophilizer and processed for 48 hours to yield a dry product. The dry product was grinded to provide a dry powder of nanofibrous cellulose scaffold.

[0380] Microscopy

[0381] Samples of nanofibrous cellulose scaffold (diluted to 0.2 wt% cellulose) were added to a glass slide. Images of the network of cellulose nanofibers in the sample were captured on a light microscope (Leica) using 40x magnification.

[0382] Scanning electron microscopy (SEM)

[0383] Morphological studies of the nanofibrous cellulose scaffolds were carried out using scanning electron microscopy (SEM). The samples were dried and sputter coated with gold before carrying out the analysis. The micrographs were obtained in secondary electron (SE) imaging mode on a Hitachi SU3500 at an accelerating voltage of 5 kV and a working distance of 7 mm at varying magnifications. Surface area measurements

[0384] The Brunauer-Emmett-Teller (BET) model was employed to measure the specific surface areas using a molecule of nitrogen bearing molecular cross-sectional area of 0.162 nm2. Dried samples were degassed under vacuum for 6 hours before the BET surface area was measured. Nitrogen adsorption and desorption isotherms were acquired on an ASAP 2020 M analyzer (Micromeritics) at 77.3 K.

[0385] Elemental analysis and Degree of substitution

[0386] Elemental analysis of the nanofibrous cellulose scaffold was performed on an elemental analyzer FlashEA 1112 (Thermo Fischer Scientific) using 5 mg of sample. The samples were well dried before the analysis to remove any adsorbed moisture.

[0387] Degree of substitution (DS) of QA functionalized cellulose was calculated using the following formula:

[0388] DS = (162N / (1400-CAxN)) where 162 is the molecular weight of the anhydrous glucose unit (AGU); N is the percentage of nitrogen; CA is the molecular weight of the cationic reagent. For the present nanofibrous cellulose scaffold the cationic reagent is CHPTAC with a molecular weight of 188.1.

[0389] Results

[0390] Nanofibrous cellulose scaffolds with QA functional moieties were prepared and the dry powder product (Figure IB) was visualized by light microscopy (Figure 1A) and SEM (Figure 1C). The resulting nanofibrous cellulose material is highly homogenous in the sense that individual nanofibers are clearly visible without any large degree of entanglement or clusters. The homogenous distribution of nanofibers in the material ensures optimal exposure of the surface area for cell attachment and interaction.

[0391] Elemental composition and degree of substitution (DS) was assessed for the nanofibrous cellulose scaffold functionalized with QA. The content of carbon (C) was 37.71%, the content of hydrogen (H) was 6.59%, and the content of nitrogen (N) was 1.28%. Based on the content of N, the DS was calculated to 0.18. The amount of substituted QA may influence the cell growth as the positive charge facilitates electrostatic interactions with negatively charged cell membranes.

[0392] The surface area of the nanofibrous cellulose scaffold can be approximated in a theoretical calculation using the following equation:

[0393] Theoretical surface area per weight (cm2 / g) = 2 / (r x 6) wherein m is the mass of nanofibrous cellulose material, r is the radius of the cellulose nanofibers, and 6 is the density of cellulose sheets.

[0394] The nanofibrous cellulose scaffold offers an increased surface area compared to the market standard microcarrier. The accuracy of the theoretical calculation was supported by a measurement of the BET surface area of a 0.5% cellulose sample, which gave a BET surface area of 58000 cm2 / g.

[0395] Conclusion

[0396] This example demonstrates that it is possible to produce a nanofibrous cellulose scaffold in a simple manner which is readily scalable for industrial usage. The nanofibrous cellulose scaffold has a large surface area which is available to the cells and does not comprise nanofibers collapsed in clusters or entangled in a fashion that exclude cells from gaining access to the surface.

[0397] Example 2: Growth of host cells on nanofibrous cellulose scaffold and evaluation of AAV2 production performance

[0398] In this example the ability of host cells to grow on the nanofibrous cellulose scaffolds was assessed. Transfection with non-viral AAV vectors at different time points to identify best conditions for obtaining high transfection efficiency. Virus titer was the determined and the transduction efficiency was assessed.

[0399] Method

[0400] Preparation of nanofibrous cellulose scaffold

[0401] The nanofibrous cellulose scaffold (nanofiber microcarrier) was prepared as described in Example 1.

[0402] Seeding and transfection of cells

[0403] HEK293T cells were seeded onto the nanofibrous cellulose scaffold (nanofiber microcarrier) and cultured at a starting density of 1.5xl05cells / ml. Addition of AAV2 vectors to the samples was performed either before seeding of the cells (reverse transfection) or after seeding of the cells (conventional transfection).

[0404] Reverse transfection protocol

[0405] First, the transfection (DNA: PEI) mix was prepared to be 60000 molecules of each plasmid per cell by adding equimolar ratio 1 : 1: 1 of the three plasmids included in AAV-vector helper free system and pAAV-ZsGreenl Vector (adeno-associated virus (AAV) vector that contains the ZsGreenl gene) (Takara Bio) to 1 ml of Opti-MEM (Thermo Fisher) followed by dropwise administration of polyethyleneimine (PEIpro, VWR) in 1.5 ml Opti-MEM (Total DNA:PEI 1:2 wt / wt ratio) and then vortexing. The DNA:PEI mix was incubated for 10 min in room temperature, to generate cloudy solution (formation of DNA / PEI complex). Then, 2.5 ml DNA:PEI mix was added into the 125 ml E-flask containing 0.3 ml (3 mg) nanofiber microcarriers, mixed by pipetting and incubated for 5 min. 3 ml of DMEM+10% FBS cell suspension containing 3xl06HEK293T cells was added to the E-flask to initiate transfection in a total cell culture transfection volume of 5.8 ml (500000 cells / ml). Cells were incubated at 37°C, 5% CO2 at 35 rpm. The next day (after 18 hours), fresh cell media (DMEM+10% FBS) was added up to 20 ml total cell culture volume (150000 cells / ml). Cells were further incubated at 37°C, 5% CO2 at 70 rpm for 72 hours.

[0406] In experiments for determination of virus titer (e.g. benchmarking against commercial microcarrier) the reverse transfection protocol was used with a cell density of IxlO6cells / ml in 20 ml culture volume at inoculum to give 20xl06total cells. The final cell density was 400000 cells / ml in a total cell culture volume of 50 ml.

[0407] Conventional transfection protocol

[0408] For this protocol, 0.3 ml (3 mg) of nanofiber microcarrier was added to 125 ml E-flasks. HEK293T cells were seeded onto the nanofiber microcarrier as described above, but without prior addition of the DNA:PEI mix. The cells were cultured on the nanofiber microcarrier (24 hours or 48 hours) and then carefully spun down followed by removal of medium. 5 ml new medium (DMEM + 10% FBS) was added, followed by addition of the DNA:PEI mix (as described above), and the suspension was incubated for approximately 5 hours before additional medium (DMEM+10% FBS) was added up to a total volume of 20 ml. Cells were further incubated at 37°C, 5% CO2 at 70 rpm for 72 hours.

[0409] Following cell culturing, transfection efficiency, virus titer, and transduction efficiency were assessed.

[0410] Transfection efficiency

[0411] The transfected cells were imaged by fluorescence microscopy (EVOS M7000, Thermo Fisher) and the transfection efficiency was measured using flow cytometry (Attune NXT, Thermo Fisher). Imaging was performed after 72 hours and 96 hours of culturing.

[0412] For fluorescence microscopy imaging, 2 ml of each sample was transferred to a 12-well plate. The samples were analysed with 4x magnification, the GFP and TRANS channels was used to image the samples. For flow cytometry analysis 2 ml of each sample was transferred to a 15 ml tube. The tube was centrifuged for 5 min at 350xg, the supernatant was removed and the sample was resuspended in 3 ml DPBS and washed again. The supernatant was removed and 1 ml TrypLE was added to the tube, and the content was mixed. The sample was then incubated for 5 min at 37°C after which 4 ml of DPBS with 1% hiFBS was added. The sample was then centrifuged for 5 min at 350xg, the supernatant was removed and the sample was resuspended and mixed in 2 ml DPBS with 1% hiFBS. The sample was then filtered through a 0.35 pm filter into a FACS tube. The sample was analysed for the expression of ZombieGreen in the BL-1 channel. Cells were separated from debris using FSC-A and SSC- A, and duplicate cells was removed using FSC-A and FSC-H. A transfected sample was used as a negative control to set the positive gate.

[0413] Harvesting AAV2

[0414] After 72 hours of culturing, the samples comprising transfected cells were centrifuged at 1500 rpm for 4 minutes, whereafter the supernatant containing secreted virus was collected, and filtered through 0.2 pm filters to remove dead cells and residual nanofiber microcarrier.

[0415] The cell pellets were washed twice in PBS, resuspended in 3 pellet volume of sterile milli- Q water (osmotic shock) and vortexed (maximum speed) for 5 min to break cells and release virus particles. The cell lysate was further centrifuged at 14000 rpm, 5min, 4°C to pellet cell debris and fibers. Harvested AAV2 viruses (from both supernatant or cell lysate) were kept at 4°C for further analysis.

[0416] Virus titer measurements

[0417] The virus titer of total viral genomes (vg) / ml was determined by quantitative PCR (qPCR) using ITR primers and protocols as described previously by Aurnhammer etal. (2012) with no further modifications.

[0418] Infectivity assay

[0419] Produced (harvested) AAV2 in limiting dilutions was used to transduce HEK293T cells for 72 hours, and the transduction efficiency was estimated using flow cytometry. In brief, a 96-well plate was seeded with 10000 HEK293T cells in 100 pl of DMEM + 10% FBS and incubated at 37°C and 5% CO2. After 4 hours, a serial dilution in DMEM of cell lysates and supernatants containing produced AAV2 particles was added. Following 72 hours of incubation the cells were disassociated and the expression of ZombieGreen was then analyzed using flow cytometry (Attune NXT). The fraction of ZombieGreen positive cells was used to evaluate the infectivity rates and if the produced viruses were infectious. Results

[0420] The cells attached and adhered to the nanofibrous cellulose scaffold (nanofiber microcarrier) as single cells quickly following seeding (Figure 2A). The cells grow as spheroids on the nanofibrous cellulose scaffold (Figure 2B). Cells proliferated well and reached high cell densities of 1.2-1.5 M cells / ml after 72 hours of culturing (Figure 2C), maintaining high viability (Figure 2D).

[0421] Cells were readily transfected by the reverse transfection protocol (Figure 3A) and performed better than cells transfected after seeding of the inoculum on the nanofibrous cellulose scaffold. Cells transfected with the reverse transfection protocol reached a transfection efficiency of approx. 95% (Figure 3B) and resulted in a 5 times higher virus titer than cells transfected 24 hours after seeding and 1000 times higher virus titer than cells transfected 48 hours after seeding.

[0422] The virus titer was determined as total viral genomes (vg) / ml by qPCR. The total virus titer (lysate + supernatant) for the reverse transfected cells was determined to be 1.25X1011vg / ml in the final cell culture volume of 50 ml. Virus titer numbers were derived from 6 replicate experiments.

[0423] The virus titer numbers were benchmarked against publicly available data on a commercially available microcarrier as published by the distributor (Cytiva). Because the experimental conditions in the experiments are slightly different, the yield expressed in vg / ml was also normalized to the total number of cells at the point of transfection, considering the difference in both the cell culture volume and the cell density at the point of transfection. Thus, the normalised virus titer is an indication of the virus titer produced per "input" cell. A summary of the data is provided in table 1, and show that the output AAV2 titer per cell for cells cultured on the nanofibrous cellulose scaffold is about 2 times higher than when cells are cultured on a commercially available microcarrier. scaffold as microcarrier or a commercially available microcarrier.

[0424] The infectivity test confirmed the virulence of the produced AAV2, and the vast majority of HEK293T cells exposed to the produced AAV2 were after 72 hours of incubation ZombieGreen positive cells as determined by subsequent cytometric analysis. At lowest dilution factor of AAV2 the transduction efficiency was as high as 95% (Figure 3C).

[0425] Conclusion

[0426] This example demonstrates that host cells grow efficiently as spheroids on the nanofibrous cellulose scaffold and are readily transfected with vectors for AAV2 production. This is particularly true when the AAV2 vectors are introduced by reverse transfection. The virus titer produced per cell by cells cultured on the nanofibrous cellulose scaffold is improved over a commercially available microcarrier, and the resulting AAV2 are of high quality as measured by their virulence. Therefore, the present method of producing AAV2 is advantageous in that high virus titers of virulent AAV2 are achieved, which may potentially reduce production costs significantly.

[0427] Example 3: Processing of the initial cellulose nanofiber material

[0428] In this example various methods of dividing the initial cellulose nanofiber material were tested and their influence on the nanofibrous cellulose scaffold was assessed.

[0429] Method

[0430] Preparation of the initial cellulose nanofiber material was performed as described in example 1, with the exception that different means of dividing the fibers were tested.

[0431] Processinq / dividinq protocols

[0432] Different methods of dividing the initial cellulose nanofiber material were evaluated. Thus, samples were prepared according to the following:

[0433] Mechanical cutting (by scissor):

[0434] 2.5 g Cellulose sheets were cut up in 10x10 mm pieces with a pair of scissors and assayed directly as fragmented cellulose sheets.

[0435] Laser cutting: Cellulose sheets were cut with a laser cutter (Epilog laser, Zing 24) in three different sizes (0.75x0.75mm, 1.5x1.5mm and 3x3mm). The cut sheets were assayed as is.

[0436] Blending:

[0437] 2.5 g cellulose sheets were cut up in 20x20 mm pieces with a pair of scissors and added together with 250 ml water to a lab blender (LB20, Waring Laboratory). The cellulose nanofiber material was processed at 7000 rpm for 1 (Bl), 5 (B5), 15 (B15) or 60 (B60) minutes.

[0438] Dispersing:

[0439] 2.5 g cellulose sheets were cut up in 20x20 mm pieces with a pair of scissors and added together with 250 ml water to an Erlenmeyer flask. The sample was processed using a disperser (IKA T25 digital Ultra-Turrax with S25 NB - 25 G disperser tool) at 18000 rpm for 1 (DI), 5 (D5), 15 (D15) or 60 (D60) minutes.

[0440] Fiber length measurements

[0441] Fiber length of the cellulose nanofibers was determined either by SEM (Hitachi SU3500) or by light scattering (Malvern Mastersizer S).

[0442] Samples of processed cellulose nanofiber material were diluted 1000-10000X in water and a droplet of sample is applied to a SEM fixture. The samples were dried and sputter coated with gold before capturing images on a Hitachi SU3500. Images were captured at different magnifications.

[0443] SEM images were evaluated manually by visual inspection to ensure that all measured fibers had both endings visible. The fiber lengths were determined by use of ImageJ software. Fiber lengths from several SEM images were determined to get a larger dataset.

[0444] Fiber lengths were also determined using light scattering. Briefly, 1 ml of sample was added in water to the Malvern Mastersizer S and measurements were performed with the settings described under the definition of "mean length". Sample was added to the sample container until the obscuration value was between 15-20%. From each sample a fiber length histogram displaying the fiber length distribution was generated. The statistics of the distribution are calculated from the results using the derived diameters D[m,n] - an internationally agreed method of defining the mean and other moments of particle size. D(v, 0.5), D(v, 0.1) and D(v, 0.9) are standard "percentile" readings from the analysis. D(v, 0.5) is the fiber length at which 50% of the sample is smaller and 50% is larger than this length. This value is also known as the Mass median diameter (MMD) when used for particles. D(v, 0.1) is the fiber length for which 10% of the sample is below this length. D(v, 0.9) gives a fiber length for which 90% of the sample is below this length. The volume weighted mean fiber length D[4,3] was also determined.

[0445] Pipettinq / floatinq test

[0446] Samples of processed cellulose nanofiber material were tested for their ability to be pipetted. 1 ml of sample was pipetted out of the sample container and subsequently expelled from the pipette tip into a tube with water. The ease of pipetting was evaluated, including the propensity for processed cellulose nanofiber material to clog the pipette.

[0447] Processed cellulose nanofiber material transferred to a tube containing water was then assessed for its propensity to float. Samples were vigorously shaken and it was observed by visual inspection whether the material floated immediately after and 24 hours after shaking.

[0448] Results

[0449] The pipetting test of scissor cut and laser cut cellulose sheets showed that these cut pieces are too large to pipette as they will clog the pipette tip. Also, cellulose nanofiber material processed in this manner generates pieces of dimensions that trap air bubbles and float in solution (Figure 4A). Furthermore, cutting with a laser makes the cellulose nanofibers melt, burn and stick together and is therefore not a suitable operation for dividing the cellulose nanofibers (Figure 4B-C).

[0450] None of the cellulose nanofiber materials processed by dispersing trapped air bubbles or had any tendency to float in solution. Pipetting of these samples was least challenging for samples that had been dispersed for longer durations of time. As such it was preferred to disperse for at least 10 min to improve flow through the pipette.

[0451] SEM measurements were used to determine cellulose nanofiber diameter (Figure 5A-B) and length (Figure 6A-H + Figure 7).

[0452] More than 2000 individual cellulose nanofibers were measured using ImageJ software and gave a mean cellulose nanofiber diameter of 500 nm (Figure 5C).

[0453] It is clear from the SEM images (Figure 6A-H) that the disperser quickly provides a homogenous nanofiber population with relatively few long nanofibers and without any significant entanglement. While large chunks of uncut nanofibers are present in the samples after only 1 min of dividing (Figure 6A-B), the disperser presents a finer population of nanofibers with only few smaller chunks of entangled nanofibers already after 5 min of dispersing (Figure 6D). After 15 and 60 min of dispersing the presence of entangled nanofibers is almost completely eliminated (Figure 6F and 6H). In contrast, the blended samples comprise large chunks of entangled fibers even after 60 min of blending (Figure 6G).

[0454] The data are summarised for each of the samples in Figure 7A-B. It is clear that the frequency of long nanofibers is higher in the blended samples compared to the dispersed samples. Furthermore, there is a tendency towards longer processing times producing fewer long nanofibers.

[0455] This relative trend is supported by measurements of the nanofibers using light diffraction. These data are summarised in Table 2. Shorter fiber lengths were obtained for longer dispersing times.

[0456] Table 2. Fiber length measurements of cellulose nanofibers prepared by blending (Bl, B5, B15, B60) and dispersing (DI, D5, D15, D60). Measurements were performed on a Malvern Mastersizer S.

[0457] Overall, it is desired that the mean length of the cellulose nanofibers is reduced. It is also preferred that the population of nanofibers does not contain many long nanofibers as these may act as nucleus for larger clusters of nanofibers and entanglements. These clusters or entanglements of nanofibers are undesirable in the nanofibrous scaffold as large fractions of the surface area remains inaccessible to the cells seeded thereupon and increase the risk of clogging process equipment. Moreover, an inhomogeneous population of nanofibers comprising clusters and entanglements are undesirable because the variability between batches of the nanofibrous scaffold becomes inconsistent and therefore unreliable for cell growth. Conclusion

[0458] This example demonstrates that not every method for dividing the initial cellulose nanofiber material is equally effective and suitable for preparation of the nanofibrous cellulose scaffold. In particular, dispersing is advantageous because it rapidly removes longer fibers and yields a homogeneous population of cellulose nanofiber material without generation of entanglements or clusters.

[0459] Example 4: Processing nanofibers of material different from cellulose

[0460] In this example the processing of a variety of nanofibers different from cellulose were assessed with the aim of identifying other nanofibers suitable for preparation of nanofibrous scaffolds for growing cell cultures on. The electrospun nanofibers were characterized by scanning electron microscopy (SEM) and by visual inspection (following dividing of the nanofibers).

[0461] Method

[0462] Electrospinning

[0463] Nanofibers of polycaprolactone (PCL), poly-L-lactic acid (PLA), a mixture of PCL and PLA (PLA / PCL) and cellulose were electrospun. Electrospinning was performed on a Fluidnatek LE50 apparatus as described in Example 1.

[0464] PCL fibers were obtained by dissolving 8% Polycaprolactone pellets (Sigma Aldrich, MW 80000), in Chloroform: Methanol, 1 : 1 solution. Needle to collector distance was set to 20 cm, flow rate to 3ml / h, and voltage to 18kV.

[0465] PLA fibers were obtained by dissolving 8% PLLA pellets (Goodfellow, MFR:24), in Chloroform: Methanol, 3:2. Needle to collector distance was set to 24cm, flow rate to 4.5 ml / h and voltage to 35kV.

[0466] PLA / PCL fibers were obtained by dissolving PLA pellets (Goodfellow, MFR: 65) and PCL pellets (Sigma Aldrich, MW 80000) 1:2 to an 8% polymer solution in Chloroform: Methanol, 3:2. Needle to collector distance was set to 20cm, flow rate to 3 ml / h and voltage to 18kV

[0467] 1 ml of polymer solution was spun for each sheet of fibers.

[0468] Scanning electron microscopy The electrospun material of PCL, PLA, mixed PCL / PLA and cellulose were imaged on a Hitachi SU3500 as described in Example 1. Images of the nanofiber materials were obtained for both undivided and divided nanofibers.

[0469] Processing of PCL, PLA and PCL / PLA nanofiber material

[0470] Electrospun sheets of PCL, PLA and mixed PLA / PCL were cut into smaller lOxlOmm pieces and mixed with two different mixing tools for 5 minutes. The cut sheets were mixed either with a blender at 7000 rpm (LB20E Laboratory blender, Waring) or with a disperser at 18000 rpm (IKA T25 digital Ultra Turrax).

[0471] Results

[0472] Nanofibers could be electrospun from all materials (PCL, PLA, PLA / PCL, and cellulose) and formed nanofibrous sheets (see Figure 8A-D).

[0473] The sheets made from nanofibers different from cellulose were subjected to two individual modes of dividing the nanofibers, namely blending (Figure 9A-F) and dispersing (Figure 10A-F). The tests showed that it was not possible to uniformly blend or disperse either PCL, PLA, or PLA / PCL nanofibers into a homogenous mixture of short strand nanofibers.

[0474] Both the PCL nanofibers (Figure 9A-B and Figure 10A-B) and the PLA / PCL nanofibers (Figure 9E-F and Figure 10E-F) melted or deformed during the process. In particular, the nanofibers melted together to form either large pieces of solid polymer or large entangled clusters of nanofibers with semi-melted nanofibers.

[0475] The PLA nanofibers (Figure 9C-D and Figure 10C-D) did not melt in the same way as the PCL fibers, but all materials did easily get stuck in the mixing tool or got entangled on the blades of the blender and halted the processing.

[0476] Conclusion

[0477] This example demonstrates that it is not possible to uniformly blend or disperse nanofibers of all materials. Accordingly, not all nanofiber materials can be easily transformed into shorter strands followed by formation of a nanofibrous scaffold as described herein. Thus, it is preferred to use cellulose nanofibers for the preparation of the nanofibrous scaffolds.

[0478] Example 5: Scalability of cell production method

[0479] In this example the ability to scale-up production was assessed by inoculating microcarrier cultures and performing passaging in a series of containers of different volume. Cell density and viability was tested and extraction of the cell product from the nanofibrous cellulose scaffold was evaluated.

[0480] Method

[0481] Cell line, maintenance and expansion in 2D

[0482] For routine maintenance of adherent HEK293T cells (ATCC, CRL-3216), the cells were seeded in culture flasks at a density of 2xl05cells / cm2 in Dulbecco's Modified Eagle Medium (DMEM high glucose, glutaMAX and pyruvate; Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco). The cells were incubated at 37°C, 5% CO2 and passaged every two to three days when reaching a maximum confluency of 80- 90%. The cells were detached by removing the cell medium and adding TrypLE Select (Gibco) for 5 min at 37°C. An equal amount of culture medium was then added before centrifugation at 300xg for 5 min. The cells were resuspended in culture media, loaded into a Via2-cassette and counted using the Count&Viability protocol on the NucleoCounter NC-202 (ChemoMetec).

[0483] Preparation of nanofiber microcarrier

[0484] The nanofibrous cellulose scaffold (nanofiber microcarrier) was prepared as described in Example 1 with the following adjustments:

[0485] - The cellulose acetate sheets were regenerated to cellulose in a 0.75 M NaOH in 95% ethanol solution for 3 hours.

[0486] - The cellulose nanofiber material was functionalized in a 0.75 M NaOH solution without the mercerization step for 1 hour at 45°C.

[0487] - The functionalized cellulose nanofiber material (100 mg) was freeze dried in autoclavable plastic vials.

[0488] 4g of nanofiber microcarriers were washed and prepared according to the following protocol. Aliquots of 100 mg of nanofiber microcarriers were hydrated with 20 mL DPBS (Ca2+, Mg2+free) and then transferred into centrifuge tube with additional 30 mL DPBS. The hydrated nanofiber microcarriers were centrifuged at 2000xg for 5 min. Supernatant was removed and the nanofiber microcarriers were resuspended in 50 mL DPBS and centrifugated an additional two times before sanitization in 50 mL of 70% ethanol for 2h. The nanofiber microcarriers were spun down at 2000xg for 5 min and washed three time with 50 mL of DPBS. The nanofiber microcarriers were stored in 20 mL of DPBS (stock solution of 5 mg / mL) at 5°C. Prior to seeding, a desired amount of nanofiber microcarriers was spun down and resuspended in culture medium at a concentration of 5 mg / mL.

[0489] Cell culture and evaluation Two overall sets of data were collected; First, cell culture in spinner flasks and small bioreactor. Here, cells were grown in various container volumes and the cell density assessed. Second, cell culture and passage in a 10L bioreactor. Here, not only cell density was assessed but also metabolite profile and purification of the cell product was evaluated.

[0490] Cell culture in spinner flask and small bioreactor

[0491] HEK293T cells were seeded on the nanofiber microcarrier in spinner flasks or Applikon AppliFlex 3L single-use (SU) stirred bioreactor with an inoculum density of 1.5xl05cells / mL in DMEM+10% FBS. Cell growth studies were performed for 96h. The spinner flasks were kept at 37°C with 5% CO2 and the bioreactor at 37°C, 40% DO and pH 7.2.

[0492] To monitor cell growth and viability, 1 mL of spheroid suspension was sampled every 24 hours from each culture vessel, following the NC-202 NucleoCounter microcarrier protocol. Samples were centrifuged at 300xg for 5 minutes, supernatants removed, and 1 mL of TrypLE Select added for cell dissociation at 37°C, 5% CO2 for 5 minutes. Cells were resuspended by pipetting, an equivalent volume of culture medium was added, followed by a second centrifugation and removal of supernatant, leaving 100 pL. The volume was then adjusted to 1 mL with culture medium, and pipetted for uniformity. Cell suspensions were transferred to 1.5 mL microcentrifuge tubes, with 500 pL moved to new tubes and mixed with 500 pL Lysis Buffer 2 for 5 minutes. Lysed and non-lysed samples were loaded into separate NC-202 cassettes for analysis.

[0493] Cell culture and passage in 10L bioreactor

[0494] HEK293T cells were seeded on the nanofiber microcarrier (0.075 mg / mL) in spinner flasks with an inoculum density of 1.5xl05cells / mL in culture medium that was prepared by aseptically adding 10% heat-inactivated fetal bovine serum (FBS), 1% penicillinstreptomycin, 1% GlutaMAX Supplement and 1% Sodium Pyruvate to a bottle of Dulbecco's Modified Eagle Medium (DMEM) (high glucose, no glutamine, no phenol red). The spinner flasks were kept at 37°C with 5% CO2 for 72h and then a volume of spheroid suspension (corresponding to 1500xl06cells) was passaged into an Xcellerex XDR-10 SU stirred bioreactor with an inoculum density of 1.5xl05cells / mL in culture medium and with additional nanofiber microcarrier (750 mg). In parallel, a volume of spheroid suspension (corresponding to 150xl06cells) was also passaged into a IL spinner flask with an inoculum density of 1.5xl05cells / mL in culture medium and with additional nanofiber microcarrier (75 mg). The bioreactor was kept at 37°C, 40% DO and pH 7.2 and the spinner flask was kept at 37°C with 5% CO2. Both the bioreactor and the control spinner flask were sampled every 24h up to 120h for cell counting, metabolite analysis and microscopy. Two consecutive bioreactor runs were preformed. To monitor cell growth and viability, samples were collected every 24 hours, and the NC- 202 NucleoCounter microcarrier protocol was used. From spinner flasks, 1 mL samples were taken while maintaining cell suspension motion using a magnetic stirrer in the biosafety cabinet. Bioreactor sampling involved cleaning the port, discarding 7 mL of waste, and collecting a fresh 7 mL sample. From this, 1 mL was transferred into a 15 mL centrifuge tube. Cells were prepared and analyzed as described above.

[0495] Metabolites (glucose and lactate) were measured using the Cedex Bio Analyzer throughout the study. Culture medium sampling was conducted every 24h across each cell culture system. A 1 mL sample was collected, centrifuged to remove residual cells, and the resulting supernatant was used for metabolite analysis.

[0496] A mock-harvest of AAV-viruses was performed to investigate the purity of the lysate regarding cell debris and nanofibers after depth filtrations. 4L of the 3D cell culture from the bioreactor was collected in a 5 L bottle using the drain tube on the bioreactor. The spheroid suspension was centrifuged at 300xg for 5 min. The pellets from two 500 mL (1 L in total) centrifugation buckets were resuspended in DPBS to a volume of 900 mL in a 2.8L shake flask and 100 mL AAV-max lysis buffer (10X) was added. This was repeated to make four 2.8 L shake flasks in total, each one containing a IL of cell suspension in diluted lysis buffer. The four shake flasks were placed in an orbital shaking incubator (50 mm orbit) set at 90 rpm for 3h.

[0497] The cell lysate was filtered using two different sizes of depth filters, Supracap 50 Seitz™ HP-series depth filter and the Mini Profile capsule depth filter, without any pre-processing prior to filtration. The filters were prepared with a 1 / 8" x 1 / 4" inch tube that was fitted to the pump head on the inlet side of the filter. The other end of the tube was connected to a port on the lysate flask containing 1 L cell lysate. The outlet of the filter was fitted with a 1 / 8" x 1 / 4" inch tube and connected to a collection flask. Using a peristaltic pump connected to the inlet tube, the lysate was pumped through the filter. The filtrate was stored in 500 mL Nalgene storage flasks at 4°C for further analysis.

[0498] The 500 mL Nalgene bottles containing the filtrates were shaken to mix the content in case of sedimentation. 1 mL was sampled from the filtrates of both the Supracap 50 and the Mini Profile and placed in two 1.5 mL centrifuge tubes. The samples were centrifuged at 17000xg for 10 min. Then 700 pl of the supernatant from each sample was removed without disturbing the pellet. The pellet was resuspended in the remaining 300 pl supernatant for all three tubes and placed in the 24-well plate for microscopy. Results

[0499] Scale-up of cell cultivation was successfully demonstrated from spinner flasks to a small bioreactor, with cell densities reaching 3xl06cells / mL within 96h of culture (figure 11A). Moreover, cells grown as spheroids could be passaged to a larger bioreactor (figure 11B) and yielded reproducible cell growth curves and high cell viability (consistently above 92%) comparable to spheroids passaged in spinner flasks in parallel (figure 11C).

[0500] Metabolite profiling of glucose and lactate revealed consistent profiles between the bioreactors and spinner flasks in batch mode (figure 11D).

[0501] Following cell lysis, depth filtration was able to effectively remove the nanofibers, leaving only residual cell debris in the upstream feed, as confirmed by microscopy (figure HE). The efficient purification of the cell product presents the nanofibrous cellulose scaffold as a viable scalable microcarrier for cell production.

[0502] Conclusion

[0503] This example demonstrates that the methodology described herein is geared for upscaling to industrial production. The AAV producing cells grow viably on the nanofiber microcarrier in larger culture vessels and can be efficiently separate from the nanofiber microcarrier upon harvesting.

[0504] Example 6: Assessment of AAV production using transient reverse transfection

[0505] In this example the AAV production from host cells cultured on the nanofibrous cellulose scaffold (nanofiber microcarrier) was assessed based on transient transfection efficiency, volumetric productivity, and percentage of full capsids.

[0506] Method

[0507] Preparation of nanofiber microcarrier

[0508] The nanofibrous cellulose scaffold (nanofiber microcarrier) was prepared as described in Example 1.

[0509] 100 mg of nanofiber microcarriers were hydrated with 20 mL DPBS (Ca2+, Mg2+free) and then transferred into centrifuge tube with additional 30 mL DPBS. The hydrated nanofiber microcarriers were centrifuged at lOOOxg for 5 min. Supernatant was removed and the nanofiber microcarriers were resuspended in 50 mL DPBS and centrifugated an additional two times before sanitization. The rehydrated nanofiber microcarriers were sanitized in 50 mL of 70% ethanol for 2h. The nanofiber microcarriers were spun down at lOOOxg for 5 min and washed three time with 50 mL of DPBS. The nanofiber microcarriers were stored in DPBS at 5°C. Prior to seeding, an amount of nanofiber microcarriers corresponding to 10 mg of nanofiber microcarriers per flask were spun down and resuspended in Opti-MEM medium at a concentration of 5 mg / mL.

[0510] Cell culture

[0511] Adherent HEK293T cells (ATCC) were maintained in DMEM growth medium supplemented with 10% FBS and grown at 37°C in humidified incubator at 5% CO2. HEK293T cells were inoculated with the nanofiber microcarriers during reverse transfection in shake flasks (125 mL) with a working volume of 20 mL and placed on an orbital shaker (19 mm) at 32- 64 rpm. For parallel 2D cultures, HEK293T cells were grown in tissue-treated culture flasks (175 cm2) for 24h prior plasmid transfection in a volume of 20 mL.

[0512] Viral vector production

[0513] Plasmids encoding helper components (pHelper plasmid), AAV2 envelope (AAV-pRC2 plasmid), and an AAV transgene plasmid encoding ZombieGreenl (AAV-ZombieGreenl plasmid) were utilized. DNA and PEIpro were pre-diluted in Opti-MEM medium. After 1 min vortex, the DNA / PEIpro mixture was incubated for 10 min at room temperature, then the complex was immediately added to the cells. For all conditions, 20xl06cells were transfected with a mixture of plasmids, at a ratio of 1: 1 : 1 of transgene:helper:envelope (i.e., ensuring 60,000 copies of each plasmid per cell), and PEIpro, at a 2: 1 ratio of PEIpro:totalDNA (w / w).

[0514] For the 2D culture flask, on the day of transfection, viable cells were counted from an additional parallel flask grown for 24h post-seeding. The culture medium in the remaining flask for transfection was exchanged with 17.5 mL of Opti-MEM prior transfection and the cells were transfected with 2.5 mL of transfection mixture which was added dropwise to cells.

[0515] For nanofiber microcarrier cultures, 10 mg of nanofiber microcarriers (in 2 mL of Opti- MEM) were added into the shake flasks and incubated with the DNA / PEIpro mixture (i.e. 2.5 mL per shake flask) for 5 min. After incubation, viable cells were also added (in 5 mL of DMEM+10%FBS) and the shake flask placed on an orbital shaker (19 mm) at 35 rpm. At 18-24h post-transfection, the culture volume was increased with Opti-MEM medium to 20 mL, and shaking speed increased to 75 rpm. Viruses from all conditions were harvested at 72h post-transfection.

[0516] Viral vector harvest AAV vectors were harvested at 72h post-transfection according to the below processes.

[0517] For 2D cultures, the cells were manually scraped from the flask, transferred to 50 ml tubes, and spun at 300xg for 5 min. The supernatant was removed and stored at 5°C until analysis and the cell pellet resuspended in 9mL of DPBS and ImL of AAV-MAX lysis buffer was added. The 10 mL mixture was transferred to a shake flask (125mL) and incubated at 37°C on an orbital shaker (19mm) at 124rpm.

[0518] Each replicate of the nanofiber microcarrier culture was transferred to a 50 mL tube (saving the shake flasks for a later step), and spun at 300xg for 5 min, then the supernatant was removed and stored at 5°C until analysis. The pellets (composed of cells and nanofibers) were resuspended in 9mL of DPBS and ImL of AAV-MAX lysis buffer was added. The 10 mL mixtures were transferred back to the shake flasks and incubated at 37°C on an orbital shaker (19mm) at 124rpm.

[0519] After 3h in lysis buffer, the cell lysates from both 2D and 3D cultures were transferred into 50 mL tubes and centrifuged at 2000xg for 10 min at 4°C to pellet nanofiber microcarriers and / or cell debris. The supernatants containing viral vectors from cell lysates were removed and stored at 5°C until analysis.

[0520] Qualitative assessment of transfection efficiency

[0521] The 3D spheroids cultures were microscopically analyzed for ZoombieGreenl transgene expression in the GFP channel using EVOS M7000 (Thermo Fisher). Sample of 0.5-1 mL from the cultures were transferred to a 24-well plate for imaging and analyzed with 4x magnification using the GFP and TRANS channels.

[0522] Flow cytometry assessment of transfection efficiency

[0523] From the nanofiber microcarrier cultures, 1-2 mL of spheroid suspension was removed, spun at 300-350xg for 5 min and the supernatant was removed. The pellet was washed in 1-3 mL of DPBS with 1% FBS to remove residual viruses and then resuspended in 1 mL of TrypLE and incubated for 5 min, and vortexed. Then 1 mL of DPBS with 1% FBS was added, the sample was spun at 300-350xg for 5 min and the supernatant was removed. The pellet was resuspended in 1 mL DPBS with 1% FBS and the cell suspension was passed through a 35-40 pm cell strainer prior to analysis, to remove nanofibers. Flow cytometry analysis was performed using Attune NXT (Thermo Fisher) flow cytometer. Cells were gated by forward scatter and side scatter to identify monocellular viable cells and by excitation with 488 nm laser to detect ZombieGreenl expression. A non-transfected sample was used as a negative control to set the positive gate. AAV titration

[0524] Vector genome titers were quantified by qPCR targeting the AAV ITR. A serial dilution of each vector preparation was loaded into the plate and analyzed according to AAVpro Titration Kit (Takara). AAV2 capsid protein was detected using an AAV2 specific ELISA kit (Progen).

[0525] Results

[0526] Transient reverse transfection of cells arranging into spheroids with the nanofiber microcarrier achieved a very high transfection efficiency of 94% for AAV2 as quantified by flow cytometry and visually demonstrated by fluorescence microscopy of spheroids transfected with plasmids carrying ZombieGreenl (figure 12A).

[0527] Likewise, high volumetric productivity of AAV2 vectors was achieved, reaching 1011viral genomes / mL (or 1014viral genomes / L) of cell culture (figure 12B). Compared to 2D cultures, the cell cultures cultured on the nanofiber microcarrier achieved 1.5-fold higher product yield (figure 12C).

[0528] The percentage of full capsids (figure 12D) was within the expected range (5-15%) for AAV2 in the upstream feed, indicating the suitability of the method for large scale production of AAV.

[0529] Conclusion

[0530] This example demonstrates that the present AAV production method presents a significant upgrade over 2D culturing systems.

[0531] Example 7: Conventional transient transfection of host cells grown on microcarrier

[0532] In this example transfection efficiency and virus yield were assessed for host cells transfected only after seeding on the microcarrier (i.e. conventional transfection). The nanofibrous cellulose scaffold (nanofiber microcarrier) was compared to a commercially available standard (bead-based) microcarrier. Culture media and transfection parameters were kept the same, and transfection and analytics were run in parallel.

[0533] Method

[0534] Microcarrier preparation

[0535] The nanofibrous cellulose scaffold (nanofiber microcarrier) was prepared as described in Example 5. The nanofiber microcarriers were hydrated in DPBS, washed twice and either sanitized for 2h in 70% ethanol and washed again 2 times, then stored in PBS a concentration of 5 mg / mL at 5°C until use or autoclaved at a concentration of 5 mg / mL at 121°C for 15 min, then stored at 5°C until use.

[0536] Standard microcarriers (Cytodexl, Cytiva) were hydrated in DPBS, washed twice and autoclaved at a concentration of 20 mg / mL according to the manufacturer's instructions then stored at 5°C until use.

[0537] Cell culture

[0538] Adherent HEK293T cells (ATCC, CRL-3216) were maintained in DMEM growth medium supplemented with 10% FBS and grown at 37°C in humidified incubator at 5% CO2. HEK293T cells were inoculated with 0.075 mg of nanofiber microcarriers per mL of culture in shake flasks with a working volume of 25 mL (density of 0.2xl06live cells / mL) and placed on an orbital shaker (19 mm) at 85 rpm (after initial inoculum at 45 rpm for a period of 3h). HEK293T cells were inoculated with 3 mg of standard bead-based microcarriers per mL of culture in spinner flasks with a working volume of 50 mL (density of 0.2xl06live cells / mL) and placed on a magnetic stirrer at 50 rpm (after initial intermittent inoculum of 15 min static conditions and 2 min agitation for a period of 3h). Cells were grown for 24h prior to plasmid transfection.

[0539] Viral vector production using conventional transfection

[0540] Plasmids encoding helper components (pHelper plasmid), AAV2 envelope (AAV-pRC2 plasmid), and an AAV transgene plasmid encoding ZombieGreenl (AAV-ZombieGreenl plasmid) were utilized. DNA and PEIpro were pre-diluted in Opti-MEM medium. After 1 min vortex, the DNA / PEIpro mixture was incubated for 10 min at room temperature, then the complex was immediately added to the cultures where 50% of the working volume was removed and the agitation speed lowered to 65 rpm and 25 rpm for shake and spinner flasks respectively.

[0541] Spheroids formed with nanofiber microcarriers and cells grown on standard microcarriers were transfected with a mixture of plasmids, at a ratio of 1 : 1: 1 of transgene:helper:envelope (i.e., ensuring 60,000 copies of each plasmid per cell), and PEIpro, at a 2: 1 ratio of PEIpro:totalDNA (w / w). At 3h post transfection, a final working volume of 25 mL and 50 mL for shake and spinner flasks respectively was reached by addition of Opti-MEM. Viruses from all conditions were harvested at 72h post-transfection.

[0542] Cell counts at transfection

[0543] To assess cell density at transfection, samples were collected at 24h post seeding and the NC-202 NucleoCounter microcarrier protocol was used.

[0544] From shake flasks (nanofiber microcarrier cultures), 1 mL samples were taken and cells were prepared and analysed as described in Example 4. From spinner flasks (bead-based microcarrier cultures), 1 mL samples were taken and transferred to 1.5 mL microcentrifuge tubes, and 500 pL was mixed with 500 pL Lysis Buffer 2 for 2 minutes. Lysed and nonlysed samples were loaded into separate NC-202 cassettes for analysis.

[0545] For cells grown on nanofiber microcarriers, transfection was performed at 24h postseeding with 0.3xl06live cells / mL.

[0546] For cells grown on standard microcarriers, transfection performed at 24h post-seeding with 0.4xl06live cells / mL.

[0547] Spheroid diameter analysis

[0548] The diameter of the spheroids formed on the nanofiber microcarriers was measured using the imaging software of the Olympus CKX53 Inverted microscope system from brightfield images (4x) of 0.4 mL samples taken from the cultures both at the timepoints of transfection and virus harvest and placed in 24-well plates for imaging. 110 spheroids were measured for each timepoint of analysis.

[0549] Viral vector harvest

[0550] AAV vectors were harvested at 72h post-transfection according to the below processes. All the microcarrier cultures were transferred to separates 50 mL tubes and spun at 300xg for 5 min, then the supernatants were removed and stored at 5°C until analysis. The pellets (composed of cells and microcarriers) were resuspended in 9-18 mL of DPBS and 1-2 mL of AAV-MAX lysis buffer was added to each tube (for shake and spinner flasks respectively). The 10-20 mL mixtures were transferred into shake flasks and incubated at 37°C on an orbital shaker (19mm) at 124rpm. After 3h in lysis buffer, the cell lysates were transferred into 50 mL tubes and centrifuged at 1000-2000xg for 10 min at 4°C to pellet cell debris and microcarriers. The supernatants containing viral vectors from cell lysates were removed and stored at 5°C until analysis. Qualitative assessment of transfection efficiency

[0551] The 3D spheroids cultures were microscopically analyzed for ZoombieGreenl transgene expression as described in Example 5.

[0552] AAV titration

[0553] Vector genome titers were quantified by qPCR targeting the AAV ITR. A dilution of 1: 1000 for cell lysates and 1 : 100 for supernatants was loaded into the plate and viral genome (vg) titer per mL of culture analyzed according to AAVpro Titration Kit (Takara) and presented as the sum of vg / mL from cell lysates and supernatants. To calculate the total vg in the culture, the vg / mL obtained from the measurements was multiplied by the respective volumes of the supernatants and cell lysates. The total vg was then divided by the total number of cells in the culture at the moment of transfection to obtain the vg per cell.

[0554] Results

[0555] Cells were seeded on the nanofibrous cellulose scaffold (nanofiber microcarrier) and formed small spheroids which were visibly present at the time of transfection, corresponding to 24 hours after seeding (figure 13A). Continued proliferation of the cells, leading to the formation of larger spheroids by the harvest timepoint (72h posttransfection), as show by microscopy (figure 13B) and quantified by spheroid diameter measurements (figure 13C, increasing from an average of 91 to 167 pm), may contribute to a reduction in transfection efficiency due transient (non-integrative) nature of the plasmids.

[0556] Comparing the transfection efficiency 72 hours post transfection, there was very significant difference between transfection of cells cultured on nanofiber microcarriers (82.2%) and cells cultured on standard microcarriers (12.8%), as visually demonstrated by fluorescence microscopy (figure 13D-E) and quantified by flow cytometry (figure F). This difference also translated to virus yield achieved upon viral vector harvest, with the nanofiber microcarrier being the superior support material for achieving high yields of AAV (figures 13G-H).

[0557] Conclusions

[0558] This example demonstrates that the nanofibrous cellulose scaffold is suitable for use as a microcarrier in the production of AAV when cells are transfected after seeding (i.e. conventional transfection). Improved virus yield was achieved when benchmarking against a commercially available standard microcarrier. Example 8: Production of AAV9

[0559] In this example production of AAV9 on the nanofibrous cellulose scaffold (nanofiber microcarrier) was evaluated and compared to a standard microcarrier. The reverse transfection protocol was used for the nanofiber microcarrier and the conventional transfection protocol was used for the standard microcarrier. Cell density at transfection, culture volume, culture media and transfection parameters were kept equal.

[0560] Method

[0561] Microcarrier preparation

[0562] The nanofibrous cellulose scaffold (nanofiber microcarrier) was prepared as described in Example 1.

[0563] Nanofiber microcarriers were hydrated in PBS, washed twice, sanitized for 2h in 70% ethanol and washed again 2 times, then stored in PBS at 5°C until use.

[0564] Standard microcarriers (Cytodexl, Cytiva) were hydrated in PBS, washed twice and autoclaved according to the manufacturer's instructions then stored at 5°C until use.

[0565] Cell culture

[0566] Adherent HEK293T cells (AAVpro 293T Cell Line, # 632273, Takara Bio) were maintained in DMEM growth medium supplemented with 10% FBS and grown at 37°C in humidified incubator at 5% CO2. HEK293T cells were inoculated with nanofiber microcarriers during reverse transfection in shake flasks (125 mL) with a working volume of 20 mL and placed on an orbital shaker (19 mm) at 32-64 rpm.

[0567] For standard microcarrier cultures, cells were grown for 24h prior plasmid transfection. To establish standard microcarrier cultures, 12xl06viable cells were seeded in spinner flasks (125 mL, Corning) along with microcarriers (at a final concentration of 3 g / L, according to manufacturer's protocol), in a 20 mL volume and cultures were placed on a magnetic stirrer set to 60 rpm (after intermitted inoculum as of Cytiva 's instructions).

[0568] Viral vector production

[0569] Plasmids encoding helper components (pHelper; Charles River Laboratories), AAV9 RepCap, and an AAV transgene plasmid encoding GFP from a CAG promoter were utilized. DNA and PEIpro were pre-diluted in Opti-MEM medium. After a brief vortex, the DNA / PEIpro mixture was incubated for 15 min at room temperature, then the complex was immediately added to the cells. For all conditions, 20xl06cells were transfected with 30 pg plasmid mixture, at a ratio of 3: 1: 1 of target: backcone:envelope, and 1 pL PEIpro per 1 pg DNA and cells were harvested 72h post-transfection.

[0570] For nanofiber microcarrier cultures, 10 mg of microcarriers (in 2 mL volume, concentration of 5 mg / mL in Opti-MEM) was added into the shake flask and incubated with the DNA / PEI mixture (i.e. 2.5 mL per shake flask) for 5 min. After incubation, 20xl06viable cells were also added (in 5 mL of culture medium) and the shake flask placed on an orbital shaker (19 mm) at 32 rpm. At 24h post-transfection, the culture volume was increased with DMEM growth medium with 10% FBS to 20 mL, and shaking speed increased to 64 rpm.

[0571] For standard microcarrier cultures, on the day of transfection, the viable cell density was measured to IxlO6cells / mL per flask in 20 mL working volume (based on sampling and counting from the spinner flask). DNA: PEIpro mixture (i.e. 2 mL per spinner flask) was added to cells that had grown on microcarriers for 24h prior transfection, ensuring a final volume of 20 mL. Temperature, agitation, and CO2 levels remained constant (i.e. 37°C, 60 rpm, 5%).

[0572] Viral vector harvest

[0573] Each replicate nanofiber microcarrier culture was transferred to a 50 mL tube, and spun at 500xg for 5 min, then the supernatant was removed and stored at 5°C. The pellet (composed of cells and nanofibers) was resuspended in 1 mL of in-house produced lysis buffer.

[0574] Each replicate standard microcarrier culture was also transferred to a 50 mL tube and allowed to settle by gravity, then the supernatant removed and stored at 5°C. The pellet (composed of cells and beads) was resuspended in 1 mL of lysis buffer.

[0575] Once in 1 mL lysis buffer, cells from all conditions were transferred to microcentrifuge tubes and subjected to five freeze-thaw cycles to lysate the cells. The resulting AAV particles were passed through 0.45 pm filters to remove cell debris and nanofibres / beads, prior to titration / ELISA analysis. All viruses from cell lysates were stored at 5°C until analysis.

[0576] Flow cytometry assessment of transfection efficiency

[0577] For nanofiber microcarrier cultures, 1 mL of spheroid suspension was removed, spun at 500xg for 5 min and the supernatant was removed. The pellet was resuspended in 500 pL of TrypLE and incubated for 10 min, then 500 pL of culture medium was added. Cell suspension was passed through a 40 pm cell strainer prior to analysis, to remove nanofibers.

[0578] For standard microcarrier cultures, 1 mL of beads was removed, and the beads left settling by gravity, then the medium was removed. The beads were resuspended in 500 pL of TrypLE and incubated for 10 min, then 500 pL of culture medium was added. The beads were allowed to settle by gravity, then the cell suspension was passed through a 40 pm cell strainer prior to analysis, to remove residual beads.

[0579] Flow cytometry analysis of 1 mL of cell suspension from all conditions was performed on a Cytoflex flow cytometer (Beckman Coulter Ltd). Cells were gated by forward scatter and side scatter to identify monocellular viable cells and by excitation with 488 nm laser to detect GFP expression. Analysis was performed using FlowJo software to quantify the number of GFP-positive cells in each population and the mean fluorescence intensity in GFP-positive cells.

[0580] AAV titration

[0581] Vector genome titers were quantified by digital droplet PCR (ddPCR) using a QX ONE machine (BioRad), targeting the AAV ITR. A serial dilution of each vector preparation was loaded into the plate and the median values were utilized for reporting of titers. AAV9 capsid protein was detected using an AAV9 specific ELISA kit (Progen) and following manufacturer's instructions.

[0582] AAV capsid ELISA data were analyzed using Matlab R2023b software with titers extrapolated via linear regression. Only statistically significant differences calculated using GraphPad Prism Software are annotated on graphs. Statistical significance was inferred by Mann-Whitney test (* p<0.05, ** p<0.01).

[0583] Results

[0584] Host cells were efficiently transfected with viral vectors, with cells cultured on the nanofibrous cellulose scaffold (nanofiber microcarrier) yielding the highest transfection efficiency both in terms of cells transfected and the amount of GFP protein expressed per cell (figure 14A-B). Further, a 3.1-fold increase of AAV9 titer was obtained when the host cells were cultured on the nanofiber microcarriers compared to the standard microcarrier (figure 14C).

[0585] Conclusions This example demonstrates that AAV9 can be efficiently manufactured when cultured on a microcarrier comprising cellulose nanofibers. The method is scalable as demonstrated in example 4.

[0586] References

[0587] - Aurnhammer et al. (2012), Hum Gene Ther Methods, 23(1), 18-28

Claims

Claims1. A method for production of adeno-associated virus (AAV), said method comprising the steps of: providing a microcarrier;- adding one or more adeno-associated virus (AAV) vector(s) to said microcarrier;- seeding a host cell on said microcarrier;- culturing said host cell; and- collecting AAV particles produced by said host cell; wherein said microcarrier comprises cellulose nanofibers.

2. The method according to claim 1, wherein the cellulose nanofibers have a mean length of less than about 250 pm.

3. The method according to any one of claims 1 or 2, wherein the cellulose nanofibers have a mean length in the range of about 30 pm to about 250 pm, such as about 40 pm to about 200 pm, such as about 50 pm to about 175 pm, such as about 60 pm to about 150 pm, preferably about 70 pm t o about 120 pm.

4. The method according to any one of the preceding claims, wherein the cellulose nanofibers are electrospun cellulose nanofibers.

5. The method according to any one of the preceding claims, wherein the cellulose nanofibers are monofibers.

6. The method according to any one of the preceding claims, wherein the cellulose nanofibers are derived from regenerated cellulose.

7. The method according to any one of the preceding claims, wherein the cellulose nanofibers have the cellulose II crystal structure.

8. The method according to any one of the preceding claims, wherein the cellulose nanofibers are functionalised with a functional moiety.

9. The method according to claim 8, wherein the functional moiety is selected from chemical moieties or biological molecules.

10. The method according to claim 9, wherein the chemical moieties are positively charged groups.

11. The method according to any one of claims 7-10, wherein the functional moiety is selected from the group consisting of quaternary ammonium (QA), carboxymethyl (CM), diethylaminoethyl (DEAE), 4-(l-bromoethyl)benzoic acid, 4-(dimethylamino)pyridine, epoxide, and combinations thereof, preferably quaternary ammonium (QA).

12. The method according to any one of the preceding claims, wherein the microcarrier is provided as a dry material.

13. The method according to any one of the preceding claims, wherein said one or more AAV vector(s) comprises a nucleic acid sequence of interest and AAV rep and cap genes.

14. The method according to claim 13, wherein said nucleic acid sequence of interest encodes a therapeutic protein, a gene of interest, or a reporter gene.

15. The method according to any one of the preceding claims, wherein said host cell wherein said host cell comprises the machinery for supporting AAV replication.

16. The method according to any one of the preceding claims, wherein said one or more AAV vector(s) are added to said microcarrier before or simultaneous with seeding of the host cell on the microcarrier.

17. The method according to any one of the preceding claims, wherein said one or more AAV vector(s) are added to said microcarrier before seeding of the host cell on the microcarrier.

18. The method according to any one of the preceding claims, wherein one or more transfection reagents are added together with said one or more AAV vector(s), wherein said transfection reagents are selected from the group consisting of polymeric transfection reagents, lipid-based transfection reagents, and calcium phosphate, preferably polymeric transfection reagents.

19. The method according to claim 18, wherein the polymeric transfection reagents are cationic polymers selected from the group consisting of polyethyleneimine (PEI), poly-L- lysine (PLL) and Poly(amidoamine) (PAMAM) dendrimers, Chitosan, and Poly(beta-amino esters) (PBAEs) , preferably PEI.

20. The method according to any one of the preceding claims, wherein colleting said AAV particles comprises lysis of the host cell to produce a cell lysate.

21. The method according to any one of the preceding claims further comprising a step of purifying said collected AAVs.

22. The method according to claim 21, wherein purifying said collected AAVs comprises filtration, chromatographic separation, ultracentrifugation, or a combination thereof.

23. The method according to any one of the preceding claims, wherein said microcarrier is provided in a container, preferably a bioreactor.

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