Patent Description:
Adeno-associated virus (AAV) is a non-pathogenic, replication-defective parvovirus. Recombinant AAV vectors (rAAV) have many unique features that make them attractive as vectors for gene therapy. In particular, rAAV vectors can deliver therapeutic genes to dividing and nondividing cells, and these genes can persist for extended periods without integrating into the genome of the targeted cell. Given the widespread therapeutic applications of rAAV, there exists an ongoing need for improved methods of rAAV vector production including methods to achieve high-titer rAAV vector yields. Previous attempts to improve the production of a variety of viral vectors have included the use of cell culture additives such as metals, trace supplements, salts, and others (See, e.g., <NPL>), <NPL>), <NPL>), and <CIT>). rAAV are often produced by means of a helper virus, such as adenovirus (AV). However, any helper virus is a contaminant that must be removed before the rAAV can be used in therapeutic applications. There is therefore also a need in the art to produce rAAV with as little contaminating helper virus as possible.

The invention is based, in part, upon the discovery that a host cell used in the production of recombinant adeno-associated virus vectors (rAAV) will produce increased amounts of rAAV and decreased amounts of helper virus when the osmolality of the cell culture media is increased via the addition of an ionic tonicifying agent, such as NaCl. The invention is also based, in part, on the discovery that a host cell used in the production of rAAV will produce decreased amount of helper virus when the osmolality of the cell culture media is increased via the addition of a non-ionic tonicifying agent such as sucrose. Described herein is a a method for producing a rAAV comprising incubating a host cell in a culture media with increase osmolality. Also described herein is a method for decreasing the amount of helper virus produced by a rAAV-producing host cell, comprising incubating the rAAV-producing host cell in cell culture media with increased osmolality due to supplementation with a non-ionic tonicifying agent such as sucrose. Described herein is a method for increasing the production of rAAV and decreasing the production of helper virus produced by a host cell, comprising incubating the host cell in cell culture media with increased osmolality due to supplementation with an ionic tonicifying agent such as NaCl. Also described herein are cell culture systems comprising a host cell capable of producing both rAAV and helper virus, and a cell culture media with increased osmolality due to supplementation with a tonicifying agent.

The present invention provides a cell culture system for use in increasing total rAAV production compared to production from a cell culture system comprising a cell culture medium having an osmolality of <NUM> mOsm/kg, the cell culture system comprising:.

Optionally, in the cell culture system of the present invention, the HeLa host cell comprises a genome, said genome comprising one or more AAV genes stably integrated therein; optionally wherein the HeLa host cell is capable of producing helper virus selected from the group consisting of adenovirus, herpes virus, baculovirus, and recombinant forms of any of the foregoing viruses. Optionally in the cell culture system of the present invention, the helper virus is an adenovirus (AV); optionally wherein the helper virus is Ad5.

Optionally in the cell culture system of the present invention, the cell culture medium of the present invention can have an osmolality of.

Optionally the cell culture system of the present invention can further comprise one or more ionic tonicifying agents; wherein the ionic tonicifying agent is selected from the group comprising: NaCl, KCI, NaNO<NUM>, Na<NUM>SO<NUM>, Na<NUM>HPO<NUM>, NaH<NUM>PO<NUM>, NaNO<NUM>, KNO<NUM>, K<NUM>SO<NUM>, K<NUM>HPO<NUM>, KH<NUM>PO<NUM>, and KNO<NUM>; optionally wherein the tonicifying agent is NaCl; further optionally wherein the concentration of NaCl in the cell culture medium is.

Optionally, in the cell culture system of the present invention, the non-ionic tonicifying agent is a sugar; optionally a disaccharide; further optionally a disaccharide selected from the group consisting of sucrose, fructose, glucose, galactose, mannose, maltose, and trehalose.

Optionally, in the cell culture system of the present invention, the non-ionic tonicifying agent is sucrose;
optionally wherein the concentration of sucrose in the cell culture medium is.

Optionally, in cell culture system of the present invention, the cell culture medium is a serum-free cell culture medium;.

Optionally, in the cell culture system of the present invention, the cell culture medium consists essentially of DMEM supplemented with one or more non-ionic tonicifying agents.

Optionally in the cell culture system of the present invention the HeLa host cell comprises a heterologous nucleotide sequence flanked by AAV inverted terminal repeats, rep and cap genes, and helper virus genes.

The present invention provides a method for producing a recombinant adeno-associated virus vector (rAAV) using a cell culture system of the present invention; optionally wherein the HeLa host cell is incubated in the cell culture medium for a period of at least <NUM> days; optionally at least <NUM> days; and further optionally about <NUM> days.

Optionally the method of the present invention further comprises the steps of harvesting and purifying the rAAV.

Optionally the method of the present invention produces at least a <NUM>% increase in total rAAV production compared to production from a cell culture system comprising a cell culture medium having an osmolality of <NUM> mOsm/kg.

Optionally, the method of the present invention produces at least a <NUM>% reduction in total helper virus production compared to production from a cell culture system comprising a cell culture medium having an osmolality of <NUM> mOsm/kg.

The present invention provides a method for producing a recombinant adeno-associated virus vector (rAAV) using a helper virus, the method comprising incubating a HeLa host cell capable of producing rAAV in the presence of helper virus for an incubation period of at least <NUM> days in a cell culture medium containing helper virus and a non-ionic tonicifying agent and having an osmolality of <NUM> mOsm/kg or higher at the start of the incubation period, and wherein the method produces at least a <NUM>% increase in total rAAV production and a <NUM>% reduction in total helper virus production compared to a host cell incubated in a medium with an osmolality of <NUM> mOsm/kg.

In one aspect of the invention, the tonicifying agent is ionic. It is contemplated that the tonicifying agent may be any ionic tonicifying agent compatible with mammalian or insect cell culture media. Exemplary ionic tonicifying agents include NaCl, KCI, NaNO<NUM>, NaHCO<NUM>, Na<NUM>SO<NUM>, Na<NUM>HPO<NUM>, NaH<NUM>PO<NUM>, NaNO<NUM>, KNO<NUM>, K<NUM>SO<NUM>, K<NUM>HPO<NUM>, KH<NUM>PO<NUM>, or KNO<NUM>. In one embodiment, the tonicifying agent is NaCl. It is contemplated that, where the tonicifying agent is NaCl, it may be present at a concentration of at least <NUM>/L (<NUM>). It is further contemplated that NaCl may be present at a concentration of at least <NUM>/L (<NUM>), or at least <NUM>/L (<NUM>). It will be understood that other ionic tonicifying agents can be substituted for NaCl by substituting an equal osmolar amount of the alternative salt.

In another aspect of the invention, the tonicifying agent is non-ionic. It is contemplated that the tonicifying agent may be any non-ionic tonicifying agent compatible with mammalian or insect cell culture media. For example, the non-ionic tonicifying agents may be a sugar, including disaccharides and monosaccharides, such as sucrose, fructose, glucose, galactose, mannose, maltose, and trehalose. In one embodiment, the tonicifying agent is sucrose. It is contemplated that, where the tonicifying agent is sucrose, it may be present at a concentration of at least <NUM>/L (<NUM>). It is further contemplated that sucrose may be present at a concentration of at least <NUM>/L (<NUM>), <NUM>/L (<NUM>), or <NUM>/L (<NUM>). It will be understood that other non-ionic tonicifying agents can be substituted for sucrose by substituting an equal molar amount of the alternative non-ionic tonicifying agent.

It is contemplated that the osmolality of the culture media, when measured at the start of incubation of the host cell, will be <NUM> mOsm/kg or higher, <NUM> mOsm/kg or higher, or <NUM> mOsm/kg or higher. In one embodiment, the osmolality of the culture medium is sufficient to produce at least a <NUM>% reduction in total helper virus production, relative to that produced by a host cell in a cell culture medium at <NUM> mOsm/kg. It is further contemplated that the reduction in total helper virus production may be <NUM>%, <NUM>%, or <NUM>%. In another embodiment, the concentration of ionic tonicifying agent is sufficient to produce at least a <NUM>% increase in total rAAV production, relative to that produced by a host cell in a cell culture medium at <NUM> mOsm/kg. It is further contemplated that the increase in total rAAV production may be <NUM>%, <NUM>%, or <NUM>%. In one embodiment, the period of time the host cell incubated in the cell culture medium with increased osmolality is for at least <NUM> days. It is further contemplated that the incubation may be at least <NUM> days or about <NUM> days.

It is contemplated that the host cell may be a mammalian cell, for example, a HeLa, HEK293, COS, A549, BHK, or Vero cell. It is also contemplated that the host cell may be an insect cell, for example, a Sf9, Sf-<NUM>, Tn-<NUM>, or BTI-Tn-5B1-<NUM> (High-Five) cell. In the present invention , the host cell is a HeLa cell. It is contemplated that the host cell may comprise a heterologous nucleotide sequence flanked by AAV inverted terminal repeats, rep and cap genes, or helper virus genes. In one embodiment, the host cell comprises a heterologous nucleotide sequence flanked by AAV inverted terminal repeats, rep and cap genes, and helper virus genes. In one embodiment, the host cell comprises one or more AAV genes stably integrated into the host cell's genome.

It is contemplated that the helper virus may be any virus capable of allowing AAV contained in a host cell to enter the infections phase, for example adenovirus, herpes simplex virus, papilloma virus, or baculovirus. In one embodiment, the helper virus is adenovirus subtype <NUM> (Ad5).

In certain embodiments, the host cell will be capable of producing both rAAV and helper virus. A host cell is capable of producing rAAV or AV if, in the absence of intervention and given appropriate culture conditions, the cell will produce viral particles, whether or not the particles are released into the cell culture media. A host cell may be capable of producing a virus because it was infected by a live virus, or because it was transfected with viral genes that may exist in the cell transiently, for example, on a plasmid or other extrachromosomal body, or be permanently integrated into the host cell genome. It is contemplated host cells transiently transfected with one or more plasmids containing AAV inverted terminal repeats, rep and cap genes, and infected by live helper virus may be considered capable of producing both rAAV and helper virus. It is further contemplated that host cells containing AAV inverted terminal repeats, rep and cap genes integrated in the host cell's chromosomes and infected with a live helper virus will be considered capable of producing both rAAV and helper virus.

In other embodiments, the host cell is inoculated with non-replicating helper virus. In this situation, the host cell is capable of producing rAAV but not capable of producing helper virus, and thus the beneficial effects of increased osmolality on helper virus production will not occur, however the beneficial effect of increased rAAV production will still be realized.

In other aspects, this disclosure provides a rAAV produced by any of the contemplated methods, a composition comprising a rAAV produced by any of the contemplated methods, or a cell culture system comprising a host cell capable of producing both rAAV and helper virus and cell culture media with an osmolality of <NUM> mOsm/kg or higher.

These and other aspects and features of the invention are described in the following detailed description and claims.

The foregoing and other objects, features and advantages of the invention will become apparent from the following description of preferred embodiments, as illustrated in the accompanying drawings. Like referenced elements identify common features in the corresponding drawings.

The invention is based, in part, upon the discovery that the production of rAAV and helper virus in a host cell can be optimized by increasing the osmolality of the culture media through the use of a tonicifying agent, such as NaCl or sucrose. In one aspect, the invention provides a method for producing rAAV using a helper virus, the method comprising incubating a host cell capable of producing both rAAV and helper virus in a cell culture medium containing one or more tonicifying agents and having an osmolality of <NUM> mOsm/kg or higher at the start of the incubation period. In another aspect, the invention provides a method for decreasing the amount of helper virus produced during the production of rAAV by incubating a host cell in a cell culture medium containing one or more tonicifying agents and having an osmolality of <NUM> mOsm/kg or higher at the start of the incubation period. In yet another aspect, the invention provides a method for increasing the amount of rAAV produced by a host cell while simultaneously decreasing the amount of helper virus produced by a host cell, by incubating a host cell in a cell culture medium containing one or more tonicifying agents and having an osmolality of <NUM> mOsm/kg or higher at the start of the incubation period.

Adeno-associated virus (AAV) is a small, nonenveloped icosahedral virus of the genus Dependoparvovirus and family Parvovirus. AAV has a single-stranded linear DNA genome of approximately <NUM> kb. AAV includes numerous serologically distinguishable types including serotypes AAV-<NUM> to AAV-<NUM>, as well as more than <NUM> serotypes from nonhuman primates (See, e.g., <NPL>), and <NPL>)). Any AAV type may be used in the methods of the present invention. AAV is capable of infecting both dividing and quiescent cells of several tissue types, with different AAV serotypes exhibiting different tissue tropism. AAV is non-autonomously replicating, and has a life cycle with a latent phase and an infectious phase. In the latent phase, after a cell is infected with an AAV, the AAV site-specifically integrates into the host's genome as a provirus. The infectious phase does not occur unless the cell is also infected with a helper virus (for example, adenovirus (AV) or herpes simplex virus (HSV)), which allows the AAV to replicate.

The wild-type AAV genome contains two <NUM> nucleotide inverted terminal repeats (ITRs), which contain signal sequences directing AAV replication, genome encapsidation and integration. In addition to the ITRs, three AAV promoters, p5, p19, and p40, drive expression of two open reading frames encoding rep and cap genes. Two rep promoters, coupled with differential splicing of the single AAV intron, result in the production of four rep proteins (Rep <NUM>, Rep <NUM>, Rep <NUM>, and Rep <NUM>) from the rep gene. Rep proteins are responsible for genomic replication. The Cap gene is expressed from the p40 promoter, and encodes three capsid proteins (VP1, VP2, and VP3) which are splice variants of the cap gene. These proteins form the capsid of the AAV particle.

Because the cis-acting signals for replication, encapsidation, and integration are contained within the ITRs, some or all of the <NUM> kb internal genome may be replaced with foreign DNA, for example, an expression cassette for an exogenous protein of interest. In this case the rep and cap proteins are provided in trans on, for example, a plasmid. In order to produce an AAV vector, a host cell line permissive of AAV replication must express the rep and cap genes, the ITR-flanked expression cassette, and helper functions provided by a helper virus, for example AV genes E1a, E1b55K, E2a, E4orf6, and VA (<NPL>). Production of AAV vector can also result in the production of helper virus particles, which must be removed or inactivated prior to use of the AAV vector. Numerous cell types are suitable for producing AAV vectors, including HEK293 cells, COS cells, HeLa cells, BHK cells, Vero cells, and A549 cells, as well as insect cells, including Sf9, Sf-<NUM>, Tn-<NUM>, and BTI-Tn-5B1-<NUM> (High-Five) cells (See e.g. <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, and <CIT>). AAV vectors are typically produced in these cell types by one plasmid containing the ITR-flanked expression cassette, and one or more additional plasmids providing the additional AAV and helper virus genes.

AAV of any serotype may be used in the present invention. Similarly, it is contemplated that any AV type may be used, and a person of skill in the art will be able to identify AAV and AV types suitable for the production of their desired recombinant AAV vector (rAAV). AAV and AV particles may be purified, for example by affinity chromatography, iodixonal gradient, or CsCl gradient.

The genome of wild-type AAV is single-stranded DNA and is approximately <NUM> kb. AAV vectors may have single-stranded genomes that are <NUM> kb in size, or are larger or smaller than <NUM> kb, including oversized genomes that are as large as <NUM> kb, or as small as <NUM> kb. Further, vector genomes may be substantially self-complementary, so that within the virus the genome is substantially double stranded. AAV vectors containing genomes of all types are suitable for use in the method of the instant invention.

As discussed above, AAV requires co-infection with a helper virus in order to enter the infectious phase of its life cycle. Helper viruses include Adenovirus (AV), including recombinant AV, and herpes simplex virus (HSV), including recombinant HSV. Systems also exist for producing AAV in insect cells using baculovirus, including recombinant baculovirus. It has also been proposed that papilloma viruses may provide a helper function for AAV (see, e.g., <NPL>)), and suitable papilloma viruses may be used in the methods of the instant invention. Helper viruses include any virus capable of creating an allowing AAV replication. AV is a nonenveloped nuclear DNA virus with a double-stranded DNA genome of approximately <NUM> kb. AV is capable of rescuing latent AAV in a cell, by providing E1a, E1b55K, E2a, E4orf6, and VA genes, allowing AAV replication and encapsidation. HSV is a family of viruses that have a relatively large double-stranded linear DNA genome encapsidated in an icosahedral capsid, which is wrapped in a lipid bilayer envelope. HSV are infectious and highly transmissible. The following HSV-<NUM> replication proteins were identified as necessary for AAV replication: the helicase/primase complex (UL5, UL8, and UL52) and the DNA binding protein ICP8 encoded by the UL29 gene, with other proteins enhancing the helper function.

The present invention comprises the production of a recombinant adeno-associated virus vector (rAAV) from a host cell, using any suitable method known in the art. As used herein, the term "host cell" refers to any cell or cells capable of producing a rAAV. The host cell can be a mammalian cell, for example, a HeLa cell, COS cell, HEK293 cell, A549 cell, BHK cell, or Vero cell. The host cell can be an insect cell, for example, a Sf9 cell, Sf-<NUM> cell, Tn-<NUM> cell, or BTI- Tn-5B <NUM>-<NUM> (High-Five) cell. Unless otherwise indicated, the terms "cell" or "cell line" are understood to include modified or engineered variants of the indicated cell or cell line.

As discussed above, to allow for production of rAAV, the host cell must be provided with AAV inverted terminal repeats (ITRs) (which may, for example, flank a heterologous nucleotide sequence of interest), AAV rep and cap gene functions, as well as additional helper functions. These may be provided to the host cell using a variety of appropriate plasmids or vectors. Additional helper functions can be provided by, for example, an adenovirus (AV) infection, by a plasmid that carries all of the required AV helper function genes, or by other viruses such as HSV or baculovirus. Any genes, gene functions, or other genetic material necessary for rAAV production by the host cell may transiently exist within the host cell, or be stably inserted into the host cell genome. In some embodiments, the host cell is a producer cell comprising AAV rep and cap gene functions and a rAAV vector genome. In some embodiments, the host cell is a packaging cell comprising AAV rep and cap gene functions, which at the time of production is provided a rAAV vector genome by a separate recombinant virus. rAAV production methods suitable for use with the methods of the current invention include those disclosed in <NPL>), <NPL>), <NPL>), <NPL>), and <NPL>).

It is contemplated that any cell culture medium appropriate for propagation of the host cell may be used in the instant invention. Exemplary suitable cell culture media include minimum essential medium (MEM) such as Eagle's culture medium, Dulbecco's modified Eagle's medium (DMEM), minimum essential medium alpha (MEM-alpha), mesenchymal cell basal medium (MSCBM), Ham's F-<NUM> medium and Ham's F-<NUM> medium, DMEM/F12 medium, William's medium E, RPMI-<NUM> medium, MCDB medium, medium <NUM>, Fisher's medium, Iscove's modified Dulbecco's medium (IMDM), Leibovitz's L-<NUM> medium, and McCoy's modified medium.

Osmolality is a measure of the number of dissolved particles per kilogram of solvent. The osmolality of a solution can be measure by freezing point depression or vapor pressure depression. A tonicifying agent is any agent capable of increasing the osmolality of a solution without substantially adversely affecting other important attributes of the solution. Appropriate tonicifying agents will thus vary depending on the nature and purpose of the solution. In the case of cell culture media, an appropriate tonicifying agent is one that is non-toxic, and will not substantially alter the pH, buffering capacity, and nutrient density of the media. Ionic tonicifying agents are those that dissociate into ions, for example, salts such as NaCl, KCI, NaNO<NUM>, NaHCO<NUM>, Na<NUM>SO<NUM>, Na<NUM>HPO<NUM>, NaH<NUM>PO<NUM>, NaNO<NUM>, KNO<NUM>, K<NUM>SO<NUM>, K<NUM>HPO<NUM>, KH<NUM>PO<NUM>, and KNO<NUM>. Non-ionic tonicifying agents are those that do not dissociate into ions, for example, sugars such as sucrose, fructose, glucose, galactose, mannose, maltose, and trehalose.

In some embodiments of the current invention, host cells are cultured in suspension culture. During suspension culture, metabolically active cells will acidify the cell culture medium in which they are grown, and the pH of the culture medium must be periodically adjusted via the addition of sterile Na<NUM>CO<NUM> or NaOH to return the pH to optimal levels. It will be apparent the addition of Na<NUM>CO<NUM> or NaOH, as well as the metabolic activity of the cells in culture will increase the osmolality of the culture media. For this reason, the increased osmolality of the instant invention is the lowest osmolality to which the cells are exposed, and the osmolality increases over the duration of culture.

In some embodiments of the current invention, the rAAV particles are harvested and/or purified from the host cell that has been incubated in cell culture media containing one or more tonicifying agents and having an osmolality of <NUM> mOsm/kg or higher at the start of the incubation period. rAAV particles may be obtained from host cells by lysing the cells. Lysis of host cells can be accomplished by methods that chemically or enzymatically treat the cells in order to release infections viral particles. These methods include the use of nucleases such as benzonase or DNAse, proteases such as trypsin, or detergents or surfactants. Physical disruption, such as homogenization or grinding, or the application of pressure via a microfluidizer pressure cell, or freeze-thaw cycles may also be used. Alternatively, supernatant may be collected from host cells without the need for cell lysis. As used herein, "total rAAV" refers to the total rAAV produced by a host cell, and "secreted rAAV" or "extracellular rAAV" refers to rAAV that can be can be harvested from a host cell without a cell lysis step.

After harvesting rAAV particles, it may be necessary to purify the sample containing rAAV to remove, for example, the cellular debris resulting from cell lysis. Methods of minimal purification of AAV particles are known in the art. Two exemplary purification methods are Cesium chloride (CsCl)- and iodixanol-based density gradient purification. Both methods are described in <NPL>). Minimal purification can also be accomplished using affinity chromatography using, for example AVB Sepharose affinity resin (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Methods of AAV purification using AVB Sepharose affinity resin are described in, for example, <NPL>). Following purification, rAAV particles may be filtered and stored at ≤-<NUM>.

Quantification of rAAV particles is complicated by the fact that AAV infection does not result in cytopathic effect in vitro, and therefore plaque assays cannot be used to determine infectious titers. rAAV particles can be quantified using a number of methods, however, including quantitative polymerase chain reaction (qPCR) (<NPL>)) or dot-blot hybridization (<NPL>)), or by optical density of highly purified vector preparations (<NPL>)). DNase-resistant particles (DRP) can be quantified by real-time quantitative polymerase chain reaction (qPCR) (DRP-qPCR) in a thermocycler (for example, an iCycler iQ <NUM>-well block format thermocycler (Bio-Rad, Hercules, CA)). In this technique, samples containing rAAV particles are incubated in the presence of DNase I (<NUM> U/ml; Promega, Madison, WI) at <NUM> for <NUM>, followed by proteinase K (Invitrogen, Carlsbad, CA) digestion (<NUM> U/ml) at <NUM> for <NUM>, and then denatured at <NUM> for <NUM>. The primer-probe set used should be specific to a non-native portion of the rAAV vector genome, for example, the poly(A) sequence of the protein of interest. The PCR product can be amplified using any appropriate set of cycling parameters, based on the length and composition of the primers, probe, and amplified sequence. Alternative protocols are disclosed in, for example, <NPL>).

The infectivity of rAAV particles can be determined using a TCID50 (tissue culture infectious dose at <NUM>%) assay, as described for example in <NPL>). In this assay, rAAV vector particles are serially diluted and used to co-infect a Rep/Cap-expressing cell line along with AV particles in <NUM>-well plates. <NUM> hours post-infection, total cellular DNA from infected and control wells is extracted. rAAV vector replication is then measured using qPCR with transgene-specific probe and primers. TCID50 infectivity per milliliter (TCID50/ml) is calculated with the Kärber equation, using the ratios of wells positive for AAV at <NUM>-fold serial dilutions.

Throughout the description, where apparatus, devices, and systems are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are apparatus, devices, and systems of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

Practice of the invention will be more fully understood from the foregoing examples, which are presented herein for illustrative purposes only, and should not be construed as limiting the invention in any way.

A HeLa producer cell line derived from the HeLa S3 parental cell line was used to produce rAAV in this experiment. In this system, a single plasmid containing three components: the vector sequence, the AAV rep and cap genes and a selectable marker gene was transfected into HeLa S3 cells, and a stable integrant was selected. The cells were cultured in protein free, chemically defined cell culture medium with a <NUM>% fraction of culture medium carried over through inoculation. Culture media were supplemented with NaCl to a final osmolality either <NUM>, <NUM>, or <NUM> mOsmo/Kg in <NUM> shake flasks with a starting volume of <NUM> and an initial cell density of <NUM> × <NUM><NUM> cells/mL. Cells were maintained at <NUM> and <NUM>% CO<NUM> for <NUM> days with <NUM>% fraction of feed for all conditions. The cultures were sampled daily to monitor cell growth and metabolites and the pH was adjusted equally across flasks as needed using <NUM> Na<NUM>CO<NUM>. At the end of <NUM> days in culture, total and extracellular yield of rAAV and Ad5 was determined.

Extracellular yield was determined by removing samples of cell culture media containing suspended cells from each shake flask at the end of <NUM> days, filtering with <NUM> PES (polyethersulfone) syringe filter and quantifying the rAAV and Ad5 genomes in each sample using quantitative real-time PCR.

Total yield was determined by removing samples of cell culture media containing suspended cells from each shake flask at the end of <NUM> days, lysing cells with detergent, filtering with <NUM> PES (polyethersulfone) syringe filter, and quantifying the rAAV and Ad5 genomes in each sample using quantitative real-time PCR.

Results of this experiment are presented in <FIG>. As depicted in <FIG>, increasing the osmolality of culture media by adding an ionic tonicifying agent, NaCl, resulted in an increase in the production of extracellular rAAV, with no significant change in the production of extracellular Ad5. As depicted in <FIG>, increasing the osmolality of culture media by adding an ionic tonicifying agent, NaCl, resulted in an increase in the production of total rAAV, together with a decrease in production of total Ad5.

Cells were cultured as described in Example <NUM>, with the exception that media were supplemented with the non-ionic tonicifying agent sucrose to a final osmolality of <NUM>, <NUM>, <NUM>, <NUM> or <NUM> mOsmo/Kg and no additional NaCl was added.

Results from this experiment are presented in <FIG>. As depicted in <FIG>, increasing the osmolality of culture media by adding a non-ionic tonicifying agent, sucrose, resulted in an increase in extracellular rAAV production and a decrease in extracellular Ad5 production. As depicted in <FIG>, increasing the osmolality of culture media by adding a non-ionic tonicifying agent, sucrose, resulted in no change in total rAAV production and a decrease in total Ad5 production.

Claim 1:
A cell culture system for use in increasing total rAAV production compared to production from a cell culture system comprising a cell culture medium having an osmolality of <NUM> mOsm/kg, the cell culture system comprising:
a) a HeLa host cell capable of producing rAAV;
b) a helper virus; and
c) a cell culture medium comprising a non-ionic tonicifying agent, and having an osmolality of <NUM> mOsm/kg or higher when measured immediately after the HeLa host cell is introduced into the cell culture medium.