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WO2011097447A2 - Production de virus recombinant - Google Patents

Production de virus recombinant Download PDF

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Publication number
WO2011097447A2
WO2011097447A2 PCT/US2011/023699 US2011023699W WO2011097447A2 WO 2011097447 A2 WO2011097447 A2 WO 2011097447A2 US 2011023699 W US2011023699 W US 2011023699W WO 2011097447 A2 WO2011097447 A2 WO 2011097447A2
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Prior art keywords
virus
transfection
host cell
culture
cells
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WO2011097447A3 (fr
Inventor
Steven Ye
Christine V. Sapan
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Neurologix Inc
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Neurologix Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14151—Methods of production or purification of viral material

Definitions

  • the present invention is directed to a transfected host cell culture system for producing higher yields of virus.
  • the present invention relates generally to methods for producing recombinant virus, and more particularly, to such methods that can be employed on a large scale and in some embodiments in a serum-free manner.
  • Adeno-associated virus are emerging as the vectors of choice for long- term, stable in vivo gene transfer. These vectors are attractive because, while the wildtype vector can integrate into a site specific location on human chromosome 19, the recombinant vector does not integrate and is capable of stably transducing both dividing and quiescent cells without known pathogenesis caused by the AAV viruses. (Coura and Nardi, Virol. J., 16;4:99, 2007.)
  • AAV vectors are produced by cotransfecting human cell lines with plasmid DNA that encodes the viral components required for packaging. Transient transfection of these cell lines is often accomplished using the conventional transfection techniques known by one of ordinary skill in the art. For example, calcium phosphate coprecipitation is a commonly practiced technique.
  • packaging cell lines that facilitate the production of virus by reducing the need for multi- plasmid transfections. Although the use of packaging cell lines has streamlined the packaging procedure, the resulting viral titers have not been significantly higher than those obtained using transient cotransfection methods.
  • the advantages of these new cell lines are often offset by the need to develop new lines for each generation of improved AAV vector. (Coleman et al., Physiol Genomics, 12:221-228, 2003.)
  • rAAV recombinant AAV vector
  • Serum-free media allows users to standardize their cell culture conditions by avoiding the use of undefined and highly variable serum products derived from humans or animals, e.g. human serum or Fetal Bovine Serum (FBS).
  • FBS Fetal Bovine Serum
  • the high variability in the biological properties of different serum batches also makes it necessary to pre-screen many batches in order to obtain a single one which is well suited for a given application. For example, even a brief exposure of PBMC (peripheral blood mononuclear cells) to a mitogenic serum batch during washing or freezing of these cells can result in a high background in cytokine assays, and toxic/inhibitory serum batches can jeopardize the assay results.
  • PBMC peripheral blood mononuclear cells
  • the present invention is generally directed to methods of producing and purifying virus from a host cell.
  • the method can be directed to large scale production of the high titer virus.
  • the method can be directed to production of adeno-associated virus.
  • the method can be directed to production and purification of the virus.
  • the method can be directed to continuous production of the virus.
  • the present invention also discloses methods for rapid production of high titer virus in substantially serum-free culture conditions.
  • Standard procedures require long periods of cell growth or culturing that can prolong the transfection process and slow the production of virus.
  • little or no culturing step is needed prior to transfection.
  • automated steps can also be utilized for efficiency and reproducibility of the virus production process.
  • An apparatus based on a combination of an incubation and transfection chamber, can allow for automatic transfer of transfection reagents to the cells.
  • the present invention relates to the production and purification of high titer virus.
  • At least 5xl0 8 viral particles/mL of culture can be obtained through a combination of multiple purifications methods, such as density gradient centrifugation followed by either affinity heparin, affinity sepharose, hydroxyapatite, or anion/cation exchange chromatography.
  • the purification methods can further facilitate a high- throughput purification of the viruses.
  • additives e.g. insulin, EDTA and/or citrate
  • use of serum free media and/or maintaining the host cells in suspension culture can result in substantial increases in cost savings, efficiency and viral titer.
  • the methods can be scaled-up to larger culture volumes, as well as for continuous production methods to increase viral yield.
  • the method for large scale production of the virus can comprise the steps of growing a host cell in a suspension culture, introducing a vector into the host cell, culturing the host cell to produce a virus and harvesting the virus.
  • the present invention finds particular utility in that the cultures can be substantially serum free. This means that the culture media is sufficiently free of serum products derived from humans or animals. In addition to the cost savings and convenience of serum free conditions, similar viral titers can be consistently achieved from one batch to another.
  • the culture, transfection and recovery media can be different or the same media.
  • the media can contain less than about 20% serum.
  • the culture, transfection and/or the recovery media can contain less than about 10%, 8%, 5%, 4%, 3%, 2%, 1% or 0.5% serum.
  • the culture, transfection and/or the recovery media can contain less than about 1% serum. In one embodiment, the culture, transfection and/or the recovery media can contain less than about 2% serum. In yet another embodiment, the culture, transfection and/or the recovery media can contain less than about 0.5% serum.
  • the culture media can be growth media, transfection media, post-transfection media and/or recovery media of the host cell.
  • the media can provide adequate support for the cells with subsequent growth, proliferation, transfection and viral production.
  • Factors including nutrients, growth factors, inducers of differentiation or dedifferentiation, products of secretion, immunomodulators, inhibitors of inflammation, regression factors, biologically active compounds, and drugs, can be incorporated into the media or provided in conjunction with the media.
  • Growth factors, proteins and other additives e.g., epidermal growth factor (EGF), heparin-binding epidermal-like growth factor (HBGF), fibroblast growth factor (FGF), cytokines, genes, yeast extract, glycerol, insulin, pluronic, dipase, H-albumin, collagenase, EDTA, bovine serum albumin (BSA) and citrate, and the like
  • EGF epidermal growth factor
  • HBGF heparin-binding epidermal-like growth factor
  • FGF fibroblast growth factor
  • cytokines genes
  • yeast extract glycerol
  • insulin pluronic, dipase, H-albumin, collagenase, EDTA, bovine serum albumin (BSA) and citrate
  • BSA bovine serum albumin
  • citrate bovine serum albumin
  • Addition of insulin can be in a range of about 20 ⁇ g/mL to about 150 ⁇ g/mL to increase transfection efficiency. Addition of insulin can also be in a range of about 32 ⁇ g/mL to about 96 ⁇ g/mL.
  • Addition of insulin can be greater than about 5 ⁇ g/mL, 10 ⁇ g/mL, 15 ⁇ g/mL, 20 ⁇ g/mL, 25 ⁇ g/mL, 30 ⁇ g/mL, 35 ⁇ g/mL, 40 ⁇ g/mL, 45 ⁇ g/mL, 50 ⁇ g/mL, 55 ⁇ g/mL, 60 ⁇ g/mL, 65 ⁇ g/mL, 70 ⁇ g/mL, 75 ⁇ g/mL, 80 ⁇ g/mL, 85 ⁇ g/mL, 90 ⁇ g/mL, 95 ⁇ g/mL, 100 ⁇ g/mL, 125 ⁇ g/mL, 150 ⁇ g/mL, 175 g/mL, 200 ⁇ g/mL, 250 ⁇ g/mL and 300 ⁇ g/mL.
  • Addition of EDTA can be greater than about 0.05 mM, 0.1 mM,
  • EDTA can also be added in a range of about 0.1 mM to about 5 mM to increase transfection efficiency.
  • Addition of citrate can be greater than about 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.75 mM, 1 mM, 2 niM, 3 mM, 4 mM and 5 mM.
  • Citrate can also be added in a range of about 0.05 mM to about 1 mM to increase transfection efficiency.
  • Additives can also be provided in an amount sufficient to promote the growth of the host cells, efficient transfection, and recovery from transfection and/or viral production.
  • at least one additive is added to the media.
  • multiple additives can be added to the media.
  • Other useful additives can include antibacterial agents such as antibiotics.
  • the media comprises albumin.
  • the media comprises bovine serum albumin.
  • the media comprises human albumin.
  • the media comprises insulin.
  • suspension culture can increase culture volume, ease of virus production and lead to substantial increases in viral titer.
  • the suspension culture can be maintained in standard culture dishes, bottles, flasks, spinner flasks, wave-bags or other suspension cultures known by those skilled in the art. Some nonlimiting examples can be plates, dishes, wave-bag cultures, spinner flasks or any other container that allows for suspension culture. Some embodiments include suspension cultures with volumes greater than 500mL.
  • One aspect of the invention is directed to large-scale production of virus.
  • Large- scale production can also refer to cultures which allow for the generation of large amounts of the desired product as compared to standard protocols.
  • Large-scale cell culture can range from about 500 mL to about 20,000 L. It is to be understood that large amounts of recombinant virus can mean higher numbers of virus as compared to those produced using standard procedures.
  • the present invention makes use of large volume culture containers (e.g., containers such as those described herein) to produce at least 10 12 , 10 11 , 10 10 or 10 9 viral particles per batch.
  • large-scale production refers to production of more recombinant virus as compared to amounts or titers produced using standard culture conditions.
  • the present invention allows for large-scale production of recombinant virus, for example, from suspension cultures, from cells grown in culture containers that are larger than the standard cell culture containers.
  • large-volume culture containers suitable for use in the present invention include roller bottles (from about 500 ml to about 2000 ml), wave bag culture (from about 100 ml to about 1,000 L), spinner flask (from about 100 ml to about 36 L) or any other container that allows for production of significantly more product than the standard culture containers.
  • the nucleic acid can be transferred to host cells for production of the virus.
  • the vector of the invention can be transferred to the host cell to produce an adeno-associated virus or a virus other than an adeno-associated virus, or a portion thereof, which allows for expression of a nucleic acid molecule.
  • the method produces an adeno-associated virus.
  • the method is directed to the production of recombinant adeno-associated virus by growing the host cell in at least 500 mL of suspension culture, introducing a vector into the host cell, culturing the host cell to produce new AAV and harvesting the new AAV.
  • the method discloses transfecting the host cell with a nucleic acid.
  • transfection techniques are generally known in the art. Methods of transfection can be, but are not limited to, calcium phosphate co-precipitation, direct micro-injection into cultured cells, liposome mediated gene transfer, lipid-mediated transduction and nucleic acid delivery using high-velocity microprojectiles.
  • the present invention can also encompass a media change prior to transfection.
  • the media change can be a gradual media exchange or can include resuspending the host cells in the transfection media.
  • the transfection media can be the same media used for growth and propagation of the host cells, or the transfection media can be a different media.
  • the transfection media can also be substantially serum-free, or containing optimal levels of serum and/or containing additives to promote optimal transfection efficiencies.
  • the transfection media can comprise less than about 1% serum.
  • the transfection media can comprise less than about 0.5% serum.
  • media exchange may not be desirable and the transfection of the host cells can be performed in the growth media used to sustain the host cell population.
  • infusion of new media can occur to create optimal transfection conditions for the host cells.
  • the host cells can be seeded prior to transfection at a concentration optimal for transfection.
  • the present invention finds particular utility by transfecting the host cells with little, if any, delay after seeding the cells at an optimal concentration. By transfecting the cells substantially immediately after seeding, production of virus can be faster, amounting to substantial time savings, while achieving equivalent or increased viral titers.
  • the seeding can occur less than about 24 hours prior to transfection.
  • the seeding can also occur in a range between about 0 hours to about 24 hours prior to transfection.
  • the seeding can also occur no greater than 16 hours prior to transfection.
  • the transfection can be performed about 12 hours prior to transfection.
  • the transfection can also be performed about 10 hours, 8 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes or substantially immediately after seeding the host cells at a concentration optimal for transfection.
  • the transfection and seeding of the cells can be performed simultaneously.
  • One embodiment of the invention can encompass a recovery step after the transfection.
  • the recovery step can include, but is not limited to, washing the transfection reagents from the transfected host cells and/or culturing the transfected host cells in recovery media.
  • the transfected host cells can be washed prior to or after culturing in recovery media to remove transfection reagents.
  • the recovery media can be the same media used for growth and propagation of the host cells, same as the transfection media or it can be a different media.
  • the recovery media can also be substantially serum-free, or containing optimal levels of serum and/or containing additives to promote optimal transfection efficiencies.
  • the recovery media can comprise less than about 1% serum. In another embodiment, the recovery media can comprise less than about 0.5% serum.
  • the transfected host cells can be allowed a recovery period by culturing for at most about 12 hours post transfection.
  • the transfected host cells can be cultured for at most about 2 hours post transfection.
  • the transfected host cells can also be cultured for between about 2 hours to about 72 hours post transfection.
  • the transfected host cells can be cultured for between about 2 hours to about 48 hours post transfection.
  • the transfected host cells can be cultured for between about 2 hours to about 24 hours post transfection.
  • the transfected host cells can also be cultured for between about 2 hours to about 12 hours post transfection and prior to harvesting the virus.
  • the method is directed to large scale production and purification of the virus by growing the host cell in at least a 500 niL suspension culture.
  • the purification procedures will result in the extensive removal of cellular DNA, other cellular components, and adventitious agents.
  • the host cells can be harvested from the culture media and lysed.
  • Nonlimiting examples of lysis methods can be freeze-thaw, solid shear, hypertonic and/or hypotonic lysis, liquid shear, sonication, high-pressure extrusion, surfactant lysis, and combinations of the above, and the like.
  • the host cells can be lysed by repeated freeze-thaws with or without surfactants added.
  • the virus is purified from the lysed host cell.
  • Methods of purification can fractionate the virus from the lysate using, for example, fractionation by ultracentrifugation using density gradients, either alone or in combination with column chromatography to isolate the virus.
  • density gradients can be iodixanol gradient.
  • Another embodiment further comprises column chromatography.
  • the column chromatography is affinity chromatography or combination of affinity, ion exchange and hydrophobic interaction chromatography.
  • the method provides for continuous production of the virus by growing the host cells in a suspension culture, introducing a vector into at least one of the host cells, culturing the host cell to produce a virus and harvesting a portion of the suspension culture to purify the virus from the host cells.
  • the host cells are grown in suspension.
  • the culture can be continuously maintained by methods such as fed-batch processing or perfusion processing or continuous reactors such as stirred tank reactors or air-life reactors.
  • the method can also comprise continuous ultracentrifugation processes for harvesting cells from the continuous cultures.
  • another embodiment can comprise continuous membrane filtration to harvest cells from the culture.
  • the harvested host cells from the continuous culture can be dispersed in a transfection chamber or reactor.
  • the transfection chamber or reactor can be very similar to the continuous or fed-batch culture, however with culture conditions optimized for transfection.
  • the transfection mixture i.e. DNA
  • the transfection mixture can also be fed-batch into the reactor, continuously added to the transfection reactor, multiple bonus additions, or added by other means known by those skilled in the art.
  • the transfected host cells can be harvested by continuous centrifugation, continuous membrane filtration or other means known by those skilled in the art.
  • the harvested transfected cells can be harvested into lysis buffer for purification of the virus.
  • the harvested transfected cells can be released into a reactor for further processing.
  • the reactor can be an incubation chamber for release of the recombinant virus from the transfected host cells.
  • the reactor can also be a chamber to produce additional virus.
  • the reactor can be an induction chamber that induces production of the virus in the host cells.
  • the reactor can be an infection chamber to infect additional host cells.
  • the transfected host cells can be harvested for lysis and virus purification. Density gradient centrifugation and chromatography techniques, such as ion-exchange, hydrophobic interaction or affinity chromatography can be utilized for continuous purification of the virus. Continuous centrifugation can be used to harvest large-scale cell culture broth ranging from 500 mL to 20,000 L, resulting in a cell-free supernatant.
  • FIG. 1 is a schematic illustration of an apparatus for automatic preparation of DNA mixtures for transfection of cells according to the invention
  • Figure 2 depicts T75 GFP transfection rating of the experiments designed to test inhibition of transfection by various potential growth media, including CD293 and AEM;
  • Figure 3 depicts varying DNA amounts and cell density and effect on GFP transfection ratings
  • Figure 4 shows a silver stained gel of two-column purified AAV-GAD65 samples.
  • Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., Current Edition); Transcription and Translation (B. Hames & S. Higgins, eds., Current Edition); CRC Handbook of Parvoviruses, vol. I & II (P. Tijssen, ed.); Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.); Freshney Culture of Animal Cells, A Manual of Basic Technique (Wiley-
  • vector any genetic element, such as a nucleic acid, plasmid, phage, transposon, cosmid, chromosome, virus, recombinant virus, etc.
  • vector includes cloning and expression vehicles, as well as viral vectors and viral particles.
  • recombinant virus is meant a virus that has been genetically altered, e.g., by the addition or insertion of a heterologous nucleic acid construct into the particle.
  • AAV is meant a complete virus particle, such as a wild-type (wt) AAV virus particle (comprising a linear, single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat).
  • wt wild-type
  • AAV virus particle comprising a linear, single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat.
  • single-stranded AAV nucleic acid molecules of either complementary sense, e.g., "sense” or “antisense” strands can be packaged into any one AAV virus and both strands are equally infectious.
  • a "recombinant AAV,” or “rAAV” is defined herein as an infectious, replication- defective virus composed of an AAV protein shell, encapsidating a heterologous nucleotide sequence of interest which is flanked on both sides by AAV ITRs.
  • An rAAV is produced in a suitable host cell which has had an AAV vector, AAV helper functions and accessory functions introduced therein. In this manner, the host cell is rendered capable of encoding AAV polypeptides that are required for packaging the AAV vector
  • an “AAV vector” is meant a vector derived from an adeno-associated virus serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, etc.
  • AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, in some instances the rep and/or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virus.
  • an AAV vector is defined herein to include at least those sequences required in cis for replication and packaging (e.g., functional ITRs) of the virus.
  • AAV helper functions refer to AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication.
  • AAV helper functions include both of the major AAV open reading frames (ORFs), rep and cap.
  • the Rep expression products have been shown to possess many functions, including, among others: recognition, binding and nicking of the AAV origin of DNA replication; DNA helicase activity; and modulation of transcription from AAV (or other heterologous) promoters.
  • the Cap expression products supply necessary packaging functions.
  • AAV helper functions are used herein to complement AAV functions in trans that are missing from AAV vectors.
  • AAV helper construct refers generally to a nucleic acid molecule that includes nucleotide sequences providing AAV functions deleted from an AAV vector which is to be used to produce a transducing vector for delivery of a nucleotide sequence of interest.
  • AAV helper constructs are commonly used to provide transient expression of AAV rep and/or cap genes to complement missing AAV functions that are necessary for lytic AAV replication; however, helper constructs lack AAV ITRs and can neither replicate nor package themselves.
  • AAV helper constructs can be in the form of a plasmid, phage, transposon, cosmid or virus.
  • a number of AAV helper constructs have been described, such as the commonly used plasmids pAAV/Ad and pIM29+45 which encode both Rep and Cap expression products. See, e.g., Samulski et al. (1989) J. Virol.
  • Accessory functions refers to non-AAV derived viral and/or cellular functions upon which AAV is dependent for its replication.
  • captures proteins and RNAs that are required in AAV replication including those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products and AAV capsid assembly.
  • Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1) and vaccinia virus.
  • helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1) and vaccinia virus.
  • adenovirus-derived accessory functions have been widely studied, and a number of adenovirus genes involved in accessory functions have been identified and partially characterized. See, e.g., Carter, B. J. (1990) "Adeno-Associated Virus Helper Functions," in CRC Handbook of Par
  • accessory function vector refers generally to a nucleic acid molecule that includes nucleotide sequences providing accessory functions.
  • An accessory function vector can be transfected into a suitable host cell, wherein the vector is then capable of supporting AAV virus production in the host cell.
  • infectious viral particles as they exist in nature, such as adenovirus, herpesvirus or vaccinia virus particles.
  • accessory function vectors can be in the form of a plasmid, phage, transposon or cosmid.
  • transfection is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected” when exogenous DNA has been introduced inside the cell membrane.
  • transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene
  • Such techniques can be used to introduce one or more exogenous DNA moieties, such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells.
  • host cell denotes, for example, microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of an AAV helper construct, an AAV vector plasmid, an accessory function vector, or other transfer DNA.
  • the term includes the progeny of the original cell which has been transfected.
  • a "host cell” as used herein generally refers to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
  • cell line refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.
  • heterologous as it relates to nucleic acid sequences such as coding sequences and control sequences, denotes sequences that are not normally joined together, and/or are not normally associated with a particular cell.
  • heterologous region of a nucleic acid construct or a vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature.
  • a heterologous region of a nucleic acid construct could include a coding sequence flanked by sequences not found in association with the coding sequence in nature.
  • Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene).
  • a cell transformed with a construct which is not normally present in the cell would be considered heterologous for purposes of this invention. Allelic variation or naturally occurring mutational events do not give rise to heterologous DNA, as used herein.
  • a "coding sequence” or a sequence which "encodes” a particular protein is a nucleic acid sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • the boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.
  • a transcription termination sequence will usually be located 3' to the coding sequence.
  • nucleic acid sequence refers to a DNA or RNA sequence.
  • the term captures sequences that include any of the known base analogues of DNA and RNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1 -methyl guanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7- methylguanine, 5-methylaminomethyl
  • oligonucleotide is defined as a molecule comprised of two or more deoxyribonucleotides, in some cases more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.
  • primer refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a
  • the primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent.
  • the exact length of the primer will depend upon many factors, including temperature, source of primer and use the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • control sequences refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.
  • promoter is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence.
  • Transcription promoters can include "inducible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “repressible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and
  • operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
  • the control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • isolated when referring to a nucleotide sequence, is meant that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type.
  • an "isolated nucleic acid molecule which encodes a particular polypeptide" refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties which do not deleteriously affect the basic characteristics of the composition.
  • “Homology” refers to the percent identity between two polynucleotide or two polypeptide moieties. The correspondence between the sequences from one moiety to another can be determined by techniques known in the art. For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs such as ALIGN, Dayhoff, M. O. (1978) in Atlas of Protein Sequence and Structure 5:Suppl. 3, National biomedical Research Foundation, Washington, D.C. Default parameters are used for alignment. One alignment program is BLAST, used with default parameters.
  • homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
  • Two DNA, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 70%, 80%, or 85%, in some instances at least about 90%, and can be at least about 95%, 96%, 97%, 98% or 99% sequence identity over a defined length of the molecules, as determined using the methods above.
  • substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence. DNA sequences that are substantially homologous can be identified in a Southern
  • hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid
  • a “functional homologue,” or a “functional equivalent” of a given polypeptide includes molecules derived from the native polypeptide sequence, as well as recombinantly produced or chemically synthesized polypeptides which function in a manner similar to the reference molecule to achieve a desired result.
  • a functional homologue of AAV Rep68 or Rep78 encompasses derivatives and analogues of those polypeptides— including any single or multiple amino acid additions, substitutions and/or deletions occurring internally or at the amino or carboxy termini thereof— so long as its original activity remains.
  • a “functional homologue,” or a “functional equivalent” of a given adenoviral nucleotide region includes similar regions derived from a heterologous adenovirus serotype, nucleotide regions derived from another virus or from a cellular source, as well as recombinantly produced or chemically synthesized polynucleotides which function in a manner similar to the reference nucleotide region to achieve a desired result.
  • a functional homologue of an adenoviral VA RNA gene region or an adenoviral E2a gene region encompasses derivatives and analogues of such gene regions—including any single or multiple nucleotide base additions, substitutions and/or deletions occurring within the regions, so long as the homologue retains the ability to provide its inherent accessory function to support AAV virus production at levels detectable above background.
  • suspension or “suspension culture” is meant a cell culture maintained in a liquid. Although not required, suspension cultures are frequently maintained in suspension by stirring or shaking or other means of agitation.
  • adhered to a substrate refers to cells that are maintained adhered to a substrate.
  • HEK293 HEK293 cells
  • HEK cells are used interchangeably and refer to a cell line derived from human embryonic kidney cells.
  • Large-scale production refers to culture processes which allow for the generation of large amounts of the desired product as compared to standard protocols. It is to be understood that large amounts of rAAV virus can mean higher numbers of virus as compared to those produced using standard procedures.
  • the present invention makes use of large volume culture containers (e.g., containers such as those described herein) to produce at least 10 12 , 10 1 1 , 10 10 or 10 9 AAV particles per batch.
  • large-scale production refers to production of more rAAV virus as compared to amounts or titers produced using standard culture conditions.
  • the present invention allows for large-scale production of rAAV virus, for example, from suspension cultures, from cells grown in culture containers that are larger than the standard cell culture containers.
  • large-volume culture containers suitable for use in the present invention include roller bottles (from about 500 ml to about 2000 ml), wave bag culture (from about 100 ml to about 1,000 L), spinner flask (from about 100 ml to about 36 L) or any other container that allows for production of significantly more product than the standard culture containers.
  • wave and wave-bag refer to a cell culture system that contains medium and cells in contact with a chamber or sealed bad that is placed on a rocking platform.
  • the rocking motion of the platform induces waves in the cell culture fluid.
  • the waves provide mixing and oxygen transfer, resulting in an optimized environment for cell growth.
  • the cultures can also be substantially “serum-free” or cultured in “serum-free media” or “SFM.”
  • SFM serum-free media
  • a number of SFM formulations are commercially available, such as those designed to support the culture of endothelial cells, keratinocytes,
  • monocytes/macrophages lymphocytes, hematopoietic stem cells, fibroblasts, chondrocytes or hepatocytes which are available from Life Technologies, Inc.
  • SFM serum-derived keratinocytes
  • the cultures can be selected to be "substantially" serum free. This means that the culture media is sufficiently free of serum products derived from humans or animals. Alternatively, the culture media can contain less than about 5% serum. The culture media can contain less than about 4%, 3%, 2%, 1% or 0.5% serum. In some instances, the culture media can contain less than about 2% serum. The culture media can be growth media, transfection media, post-transfection media and/or recovery media of the host cell. Virus
  • Adeno-associated virus are emerging as the vectors of choice for long-term, stable in vivo gene transfer. These vectors are attractive because they are capable of stably transducing both dividing and quiescent cells and have no known pathogenesis.
  • AAV is maintained as a latent provirus until superinfection with a helper virus (e.g., adenovirus) induces the lytic phase of the AAV life cycle. Subsequently, AAV gene expression is activated and leads to rescue of the provirus from the chromosome, replication of the viral genome, and production of progeny virus.
  • helper virus e.g., adenovirus
  • AAV is unique among eukaryotic DNA viruses in its ability to integrate at a specific site within the human chromosome (19ql 3.3-qter) and for this reason, and because it is nonpathogenic, has become increasingly attractive as a vector for gene delivery. Due to these features, wild- type AAV has attracted considerable interest from gene therapy researchers.
  • the AAV genome integrates most frequently into the site mentioned, while random incorporations into the genome take place with a negligible frequency.
  • ITR inverted terminal repeats
  • AAV -based gene therapy vectors form episomal concatamers in the host cell nucleus. In non-dividing cells, these concatamers remain intact for the life of the host cell. In dividing cells, AAV DNA is lost through cell division, since the episomal DNA is not replicated along with the host cell DNA.
  • the AAV genome is built of single-stranded deoxyribonucleic acid, which is about 4.7 kilobase long.
  • the genome comprises inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames, rep and cap.
  • ITRs inverted terminal repeats
  • the former is composed of four overlapping genes encoding Rep proteins required for the AAV life cycle, and the latter contains overlapping nucleotide sequences of capsid proteins: VP1 , VP2 and VP3, which interact together to form a capsid of an icosahedral symmetry.
  • the nucleotide sequences of AAV ITR regions are known.
  • the AAV Inverted Terminal Repeat (ITR) sequences comprise 145 bases each. They were named so because of their symmetry, which was shown to be required for efficient multiplication of the AAV genome. ITRs were also shown to be required for both integration of the AAV DNA into the host cell genome (19 th chromosome in humans) and rescue from it, as well as for efficient encapsidation of the AAV DNA combined with generation of a fully-assembled DNase resistant AAV particles.
  • ITR Inverted Terminal Repeat
  • AAV-2 The ITR sequences for AAV-2 are described, for example by Kotin et al. (1994) Human Gene Therapy 5:793-801 ; Berns "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (B. N. Fields and D. M. Knipe, eds.)
  • AAV ITR's can be modified using standard molecular biology techniques. Accordingly, AAV ITRs used in the vectors of the invention need not have a wild-type nucleotide sequence, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides.
  • AAV ITRs may be derived from any of several AAV serotypes, including but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, AAV-8 and the like.
  • 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV expression vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as the ITR's function as intended, i.e., to allow for excision and replication of the bounded nucleotide sequence of interest when AAV rep gene products are present in the cell.
  • regulatory sequences can often be provided from commonly used promoters derived from viruses such as, polyoma,
  • Adenovirus 2, cytomegalovirus and Simian Virus 40 Use of viral regulatory elements to direct expression of the protein can allow for high level constitutive expression of the protein in a variety of host cells.
  • Ubiquitously expressing promoters which can also be used include, for example, the early cytomegalovirus promoter Boshart et al. (1985) Cell 41 :521-530, herpesvirus thymidine kinase (HSV-TK) promoter (McKnight et al.
  • ⁇ -actin promoters e.g., the human ⁇ -actin promoter as described by Ng et al. (1985) Mol. Cell Biol. 5: 2720-2732
  • CSF-1 colony stimulating factor-1
  • the regulatory sequences of the AAV vector can direct expression of the gene preferentially in a particular cell type, i.e., tissue-specific regulatory elements can be used.
  • tissue-specific promoters which can be used include, central nervous system (CNS) specific promoters such as, neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477) and glial specific promoters (Morii et al. (1991) Biochem. Biophys Res. Commun. 175: 185-191).
  • the AAV vector harboring the nucleotide sequence encoding a protein of interest, e.g., GAD, and a post-transcriptional regulatory sequence (PRE) flanked by
  • AAV ITRs can be constructed by directly inserting the nucleotide sequence encoding the protein of interest and the PRE into an AAV genome which has had the major AAV open reading frames ("ORFs") excised therefrom. Other portions of the AAV genome can also be deleted, as long as a sufficient portion of the ITRs remain to allow for replication and packaging functions.
  • ORFs major AAV open reading frames
  • AAV ITRs can be excised from the viral genome or from an AAV nucleic acid containing the same and fused 5' and 3' of a selected nucleic acid construct that is present in another nucleic acid using standard ligation techniques, such as those described in Sambrook et al , Supra.
  • standard ligation techniques such as those described in Sambrook et al , Supra.
  • ATCC American Type Culture Collection
  • Host cells can also be capable of providing AAV helper functions in order to replicate and encapsidate the expression cassette flanked by the AAV ITRs to produce recombinant AAV particles.
  • AAV helper functions are generally AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication.
  • AAV helper functions are used herein to complement necessary AAV functions that are missing from the AAV vectors.
  • AAV helper functions include one, or both of the major AAV open reading frames (ORFs), namely the rep and cap coding regions, or functional homologues thereof.
  • ORFs major AAV open reading frames
  • the AAV rep coding region of the AAV genome encodes the replication proteins Rep 78, Rep 68, Rep 52 and Rep 40. These Rep expression products have been shown to possess many functions, including recognition, binding and nicking of the AAV origin of DNA replication, DNA helicase activity and modulation of transcription from AAV (or other exogenous) promoters. The Rep expression products are collectively required for replicating the AAV genome.
  • the AAV cap coding region of the AAV genome encodes the capsid proteins VP1, VP2, and VP3, or functional homologues thereof.
  • AAV helper functions can be introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of the AAV vector comprising the expression cassette, AAV helper constructs are thus used to provide at least transient expression of AAV rep and/or cap genes to complement missing AAV functions that are necessary for productive AAV infection.
  • AAV helper constructs lack AAV ITRs and can neither replicate nor package themselves. These constructs can be in the form of a plasmid, phage, transposon, cosmid or virus.
  • a number of AAV helper constructs have been described, such as the commonly used plasmids pAAV/Ad and pIM29+45 which encode both Rep and Cap expression products. (See, e.g., Samulski et al. (1989) J. Virol. 63:3822-3828; and
  • a vector of the invention can be a virus other than the adeno- associated virus, or portion thereof, which allows for expression of a nucleic acid molecule introduced into the viral nucleic acid.
  • a virus other than the adeno- associated virus, or portion thereof which allows for expression of a nucleic acid molecule introduced into the viral nucleic acid.
  • replication defective retroviruses, adenoviruses, herpes simplex virus, and lentivirus can be used. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals.
  • retroviruses examples include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art.
  • suitable packaging virus lines include psi-Crip, psi-Cre, psi-2 and psi-Am.
  • the genome of adenovirus can be manipulated such that it encodes and expresses the protein of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See e.g.,
  • Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus are well known to those skilled in the art.
  • mammalian cell culture in vitro comprise, in addition to basic nutritional substances, a complex series of growth factors (Werner, R. G. et al., Mammalian Cell Cultures Part I: Characterization, morphology and metabolism, in: Arzneim.-Forsch./Drug Res. 43: 1134-1 139 (1993)).
  • these are added to the culture medium by supplying it with animal sera or protein-fractions from animal sources.
  • animal sera or protein-fractions from animal sources.
  • these chemically non-defined mixtures exhibit variable lot to lot composition.
  • Such mixtures also represent a potential source of contaminants, including viruses and mycoplasmas.
  • the high price of the supplements and difficulties in downstream processing are additional considerations.
  • Cell culture media provide the nutrients necessary to maintain and grow cells in a controlled, artificial and in vitro environment. Characteristics and compositions of the cell culture media vary depending on the particular cellular requirements. Important parameters include osmolarity, pH, and nutrient formulations.
  • cell culture media formulations are supplemented with a range of additives, including undefined components such as fetal bovine serum (FBS) (10-20% v/v) or extracts from animal embryos, organs or glands (0.5-10% v/v). While FBS is the most commonly applied supplement in animal cell culture media, other serum sources are also routinely used, including newborn calf, horse and human. Organs or glands that have been used to prepare extracts for the supplementation of culture media include submaxillary gland (Cohen, S., J. Biol. Chem. 237: 1555-1565 (1961)), pituitary (Peehl, D. M., and Ham, R.
  • FBS fetal bovine serum
  • these supplements provide carriers or chelators for labile or water-insoluble nutrients; bind and neutralize toxic moieties; provide hormones and growth factors, protease inhibitors and essential, often unidentified or undefined low molecular weight nutrients; and protect cells from physical stress and damage.
  • sera are commonly used as relatively low-cost supplements to provide an optimal culture medium for the cultivation of animal cells.
  • the cultures can also be substantially “serum-free” or cultured in "serum-free media” or "SFM.”
  • SFM serum-free media
  • a number of SFM formulations are commercially available, such as those designed to support the culture of endothelial cells, keratinocytes, monocytes/macrophages, lymphocytes, hematopoietic stem cells, fibroblasts, chondrocytes, hepatocytes or retinoblasts which are available from Life
  • SFM can be media devoid of serum and some protein fractions (e.g., serum albumin).
  • serum-free media can include, 293 SFM II, CD 293, Freestyle 293 (all Invitrogen), Pro 293s CDM (BioWhittaker), Hektor S (Cell Culture Technologies) and EX-CELL VPRO (JRH Biosciences), as well as any other serum-free formulation capable of supporting the culture of the host cells.
  • the cultures can be selected to be “substantially" serum free. This means that the culture media is sufficiently free of serum products derived from humans or animals. Alternatively, the culture media can contain less than about 10% serum.
  • the culture media can contain less than about 10%, 5%, 4%, 3%, 2%, 1% or 0.5% serum. In some cases, the culture media can contain less than about 2% serum.
  • the culture media can be growth media, transfection media, post-transfection media and/or recovery media of the host cell. Host Cells
  • any cell which supports efficient replication of the virus can be employed in the invention, including mutant cells which express reduced or decreased levels of one or more receptors for the virus.
  • Suitable host cells for producing recombinant AAV particles can include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of a exogenous nucleic acid molecule.
  • a "host cell” as used herein generally refers to a cell which has been transfected with an exogenous nucleic acid molecule.
  • the host cell includes any eukaryotic cell or cell line so long as the cell or cell line is not incompatible with the protein to be expressed, the selection system chosen or the fermentation system employed.
  • Non-limiting examples include CHO DHFR- cells (Urlaub and Chasin (1980) Proc. Natl. Acad. Sci.
  • the cells are WHO certified, or certifiable, continuous cell lines.
  • the requirements for certifying such cell lines include characterization with respect to at least one of genealogy, growth characteristics, immunological markers, virus susceptibility tumorigenicity and storage conditions, as well as by testing in animals, eggs, and cell culture. Such characterization is used to confirm that the cells are free from detectable adventitious agents. Sometimes, karyology or analysis of the chromosomes may also be required.
  • Data that can be used for the characterization of a cell to be used in the present invention includes (a) information on its origin, derivation, and passage history; (b) information on its growth and morphological characteristics; (c) results of tests of adventitious agents; (d) distinguishing features, such as biochemical, immunological, and cytogenetic patterns which allow the cells to be clearly recognized among other cell lines; and (e) results of tests for tumorigenicity.
  • the passage level, or population doubling, of the host cell used is as low as possible.
  • the host cell for use in the present invention can comprise any mammalian cell line which supports replication of the virus, for example any host cell line known in the art which will support infection and replication of an adeno-associated virus.
  • Any cell line may be used to generate recombinant virus, but not limited to, cell lines including 293 cells, PER.C6® cells, 91 1 cells from a human embryonic retinal cell line (Fallaux et al. 1996, Human Gene Therapy 7: 215-222); El -transformed amniocytes (Schiedner et al. 2000, Human Gene Therapy 1 1 :2105-21 16); an El -transformed A549 cell line for a human lung carcinoma (Imler et al. 1996, Gene Therapy 3:75-84) and GH329: HeLa
  • cells from the stable human cell line, 293 are used in the practice of the present invention.
  • the human cell line 293 which is a human embryonic kidney cell line that has been transformed with adenovirus type-5 DNA fragments (Graham et al. (1977) J. Gen. Virol. 36:59), and expresses the adenoviral Ela and Elb genes (Aiello et al. (1979) Virology 94:460).
  • the 293 cell line is readily transfected, and provides a particularly convenient platform in which to produce rAAV virus.
  • Such a cell lines can be transformed to support replication and packaging of a respective recombinant virus.
  • Additional cell lines which may be utilized in the present invention are again cell lines which have been adapted to act as host cells for a particular virus.
  • the cell line be a continuous cell line and the source of the cultured cells originating from a non-neoplastic tissue.
  • the source can also be mammalian, most likely from a primate origin, and especially of human origin.
  • cell lines can be
  • 293 cells epidermal cells from human kidney
  • PER C6® human embryonic retinoblasts
  • Other cell types include but are not limited to HeLa cells, A549 cells, KB cells, CKT1 cells, NIH/sT3 cells, Vero cells, Chinese Hamster Ovary (CHO) cells, or any eukaryotic cells which support the virus life cycle.
  • the present invention also relates to the production of high titer virus through optimized culture conditions.
  • High titer virus refers to at least 5x10 8 viral particles/mL of culture.
  • the viral titer can be at least l lO 9 viral particles/mL, 5xl0 9 viral particles/mL, lxlO 10 viral particles/mL, 5xl0 10 viral particles/mL, lxlO 11 viral particles/mL, 5xl O n viral particles/mL, lxlO 12 viral particles/mL, 5xl0 12 viral particles/mL, and at least lxlO 13 viral particles/mL of culture.
  • suspension cultures in similar-sized vessels, however, can only grow two-dimensionally on the vessel surface.
  • suspension cultures typically result in higher cell yields, and correspondingly higher yields of biologicals (e.g., viruses, recombinant polypeptides, etc.) compared to monolayer cultures.
  • biologicals e.g., viruses, recombinant polypeptides, etc.
  • suspension cultures are often easier to feed and scale-up, via simple addition of fresh culture media (dilution subculturing) to the culture vessel rather than trypsinization and centrifugation as is often required with monolayer adherent cultures.
  • Suspension cultures can also be maintained in standard culture dishes, bottles, flasks, spinner flasks, wave bags or other suspension cultures depending on size of culture and desired output. Other methods for suspension cultures may be known by those skilled in the art. Some nonlimiting examples of suspension culture containers can be dishes, plates, wave bag cultures, spinner flasks or any other container that allows for suspension culture. Non-limiting examples of large-volume culture containers suitable for use in the present invention include roller bottles (from about 500 ml to about 2000 ml), wave bag culture (from about 100 ml to about 1,000 L), spinner flask (from about 100 ml to about 36 L) or any other container that allows for production of significantly more product than the standard culture containers.
  • the culture be a suspension culture which is maintained in a suitable medium which supports cell growth, virus infection and virus production.
  • the culture medium can be subjected to various growth conditions which are suitable for virus production, including but not limited to batch, fed-batch or continuous perfusion operations to introduce fresh medium into the culture medium.
  • the culture medium can be any suitable medium for maintaining, the cells and permitting the propagation of the respective virus.
  • media suitable for use in the practice of the present invention, and the principles to generate modified or new suitable media are widely known in the art. For a review, see Chapter 8 (serum-based media) and Chapter 9 (serum-free media) from Culture of Animal Cells:
  • either serum-based or serum free media can be manipulated to enhance growth of the respective cell line in culture, with a potential for inclusion of any of the following: a selection of secreted cellular proteins, diffusible nutrients, amino acids, organic and inorganic salts, vitamins, trace metals, sugars, and lipids as well as perhaps other compounds such as growth promoting substances (e.g., cytokines).
  • growth promoting substances e.g., cytokines
  • the present invention also discloses methods for rapid production of high titer virus.
  • Standard procedures require long periods of cell growth or culturing that can prolong the transfection process and slow the production of virus. Unlike standard procedures, it has been discovered that little or no culturing step is needed prior to transfection. Moreover, automated steps can also be utilized for efficiency and reproducibility of the virus production process.
  • an AAV vector can be introduced into a suitable host cell using known techniques, such as by transfection. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al.
  • transfection methods include calcium phosphate co-precipitation (Graham et al. (1973) Virol. 52:456-467), direct micro-injection into cultured cells (Capecchi (1980) Cell 22:479-488), electroporation (Shigekawa et al. (1988) BioTechniques 6:742-751), liposome mediated gene transfer (Mannino et al. (1988) BioTechniques 6:682-690), lipid-mediated transduction (Feigner et al. (1987) Proc. Natl. Acad. Sci. USA
  • Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo.
  • the following characteristics should be present: (1) encapsulation of the genetic material at high efficiency while not compromising the biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al. (1988) Biotechniques, 6:682).
  • lipids liposomes production examples include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Additional examples of lipids include, but are not limited to, polylysine, protamine, sulfate and 3b -[N- ( ⁇ ', ⁇ ' dimethylaminoethane) carbamoyl] cholesterol.
  • the AAV vector is introduced into the host cell by transfection.
  • the present invention can encompass a media change prior to the transfection.
  • the media change can be a gradual media exchange or can include resuspending the host cells in the transfection media.
  • the transfection media can be the same media used for growth and propagation of the host cells, or the transfection media can be different media.
  • the transfection media can also be substantially serum-free, or containing optimal levels of serum and/or containing additives to promote optimal transfection efficiencies.
  • rapid production of high titer virus can be desired.
  • media exchange may not be desirable and the transfection of the host cells can be performed in the growth media used to sustain the host cell population. Removing the step of media exchange can reduce the time required for production of the virus.
  • infusion of new media growth media, transfection media etc. can occur to create optimal transfection conditions for the host cells.
  • Infusion of new media can dilute the host cell population to an optimal cell
  • the host cells can be cultured for less than about 12 hours post transfection. Moreover, rapid production of high titer virus can also be achieved by transfecting the host cells on the same day as seeding the host cells in transfection media. Transfection of the host cells can occur less than about 6 hours from seeding the host cells at an optimal concentration for transfection. The host cells can also be seeded at optimal concentrations between about 1 hour to about 6 hours prior to transfection. The host cells can also be seeded at optimal concentrations no greater than 1 hour prior to transfection. In another embodiment, transfection can also occur less than about 12 hours, 8 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes or substantially immediately after seeding the host cells at an optimal concentration for transfection. In one embodiment, transfection of the host cells and seeding of the host cells can be simultaneous.
  • Recombinant virus can be produced by transfecting cells with multiple constructs, i.e. the gag, pol and env protein coding sequences required for replication of the virus can be contained in one or more plasmids.
  • the gag, pol and env protein coding sequences may all be contained in one plasmid or each may be contained in a separate plasmid.
  • the virus is purified from total cell lysates. Because of the low efficiency of DNA transfection, it has been difficult in the past to generate high titer virus. Cell lines have been used in the literature to stably express at least one component of the viral genome. However, long term expression of viral components can lead to reduction of expression.
  • the host cells are transiently transfected with the viral components.
  • the viral components are integrated within the host cell genome and can be induced to express the viral components.
  • rapid production of high titer virus is desired.
  • the present invention finds particular utility by transfecting the host cells with little, if any, delay after seeding the cells at an optimal concentration. By transfecting the cells substantially immediately after seeding, production of virus can be faster, amounting to substantial time savings, while achieving equivalent or increased viral titers. Maintaining the host cells in suspension culture during transfection can also influence the speed with which virus is produced. Suspension culture transfection can reduce the time required for viral production by eliminating the need for host cells to become adherent after seeding at an optimal concentration when static transfection protocols are used.
  • the present invention can also encompass a recovery step after the transfection.
  • the recovery step can include, but is not limited to, washing the transfection reagents from the transfected host cells and/or culturing the transfected host cells in recovery media.
  • the transfected host cells can be washed prior to or after culturing in recovery media to remove transfection reagents.
  • the recovery media can be the same media used for growth and propagation of the host cells, same as the transfection media or it can be a different media.
  • the recovery media can also be substantially serum-free, or containing optimal levels of serum and/or containing additives to promote optimal transfection efficiencies.
  • the transfected host cells can be allowed a recovery period by culturing for less than about 2 hours post transfection. In some embodiments, the transfected host cells can be cultured for less than about 12 hours post transfection. The transfected host cells can also be cultured for about 12 hours to about 24 hours post transfection.
  • the transfected host cells can be cultured for about 24 hours to about 48 hours post transfection.
  • the transfected host cells can be cultured for greater than about 24 hours or for less than about 72 hours after transfection and prior to harvesting the virus.
  • transfection of the host cells can be performed by an automated method.
  • a transfection cocktail comprising all the transfection reagents and vector DNA, can be added to the culture of host cells through a pumping mechanism.
  • the transfection cocktail can be added to the culture at a rate of about 5 mL/min, 6 mL/min, 7 mL/min, 8 mL/min, 9 mL/min, 10 mL/min, 11 mL/min, 12 mL/min, 13 mL/min, 14 mL/min, 15 mL/min, 16 mL/min, 17 mL/min, 18 mL/min, 19 mL/min, 20 mL/min, 21 mL/min, 22 mL/min, 23 mL/min, 24 mL/min, 25 mL/min, 26 mL/min, 27 mL/min, 28 mL/min, 29 mL/min, 30 mL/min, 35 mL/min, 40 mL/min, 45 mL/min, 50 mL/min, 55 mL/min and 60 mL/min.
  • the transfection cocktail can be added to the culture at a rate of about 10 mL/min to about 50 mL/min. In another embodiment, the transfection cocktail can be added to the culture at a rate of about 20 mL/min to about 40 mL/min.
  • An apparatus based on a combination of an incubation and transfection chamber, can allow for automatic transfer of transfection reagents to the cells.
  • Figure 1 provides a schematic view of the apparatus 100 for automatic transfer of transfection reagents to cells.
  • the apparatus 100 can comprise a platform 1 10 to hold the formulation vessel 120.
  • the platform 1 10 can be a rotating or shaking platform. In one embodiment the platform is an orbital shaking platform.
  • the formulation vessel 120 can hold the transfection reagents and/or cells to be transfected.
  • Tubing 130 can be used to transfer the transfection reagents into the formulation vessel 120.
  • the tubing 130 can be sterilized by methods known to those skilled in the art, such as autoclaving.
  • the tubing 130 can be attached to a pump 140 on one end and the other end to deliver the transfection reagents to the formulation vessel 120.
  • the pump 140 can be a peristaltic or roller pump to control the amount of transfection reagents delivered to the formulation vessel 120 via the tubing 130.
  • the tubing can further be connected to a reagent vessel 150.
  • the reagent vessel 150 can hold the transfection reagents that are to be transferred to the formulation vessel 120 via the tubing 130.
  • the transfection reagents can be placed in the reagent vessel and then pumped via tubing and a peristaltic pump into the formulation vessel that is shaking on a orbital shaker platform.
  • a general transfection method can be utilized.
  • DNA plasmids can be added to the formulation vessel.
  • the remaining transfection reagents can be premixed in the reagent vessel 150.
  • the transfection reagents can be transferred to the formulation vessel via the tubing and pump at a set flow rate to mix the transfection reagents with the DNA plasmids.
  • a calcium chloride transfection can be used for transfection.
  • DNA plasmids and calcium chloride can be added to the formulation vessel for premixing.
  • the remaining transfections reagents can be added to the reagent vessel.
  • the transfection reagents can be pumped into the formulation vessel at a set flow rate.
  • the transfection mix in the formulation vessel can then be transferred to a transfection chamber holding the cells for transfection.
  • the transfection mix can be pumped via the tubing and pump or manually transferred.
  • the transfection chamber can be a separate vessel to hold the transfection mix and cells.
  • the media can provide adequate support for the cells with subsequent growth, proliferation, transfection and viral production.
  • Factors including nutrients, growth factors, inducers of differentiation or dedifferentiation, products of secretion, immunomodulators, inhibitors of inflammation, regression factors, biologically active compounds, and drugs, can be incorporated into the media or provided in conjunction with the media.
  • EGF epidermal growth factor
  • HBGF heparin-binding epidermal-like growth factor
  • fibroblast growth factor e.g., fibroblast growth factor
  • FGF cytokines
  • genes yeast extract, glycerol, insulin, pluronic, dipase, Human- albumin, collagenase, EDTA, bovine serum albumin (BSA) and citrate, and the like
  • BSA bovine serum albumin
  • Such additives can be provided in an amount sufficient to promote the growth of the host cells, efficient transfection and recovery from transfection and/or viral production.
  • at least one additive is added to the media.
  • multiple additives can be added to the media. It has been demonstrated that addition of additives, such as insulin, EDTA and citrate, can increase transfection efficiency, thereby increasing viral titer.
  • Addition of insulin can be in a range of about 20 ug/mL to about 150 ug/mL to increase transfection efficiency. Addition of insulin can also be in a range of about 32 ug/mL to about 96 ug/mL.
  • Addition of insulin can be greater than about 5 ug/mL, 10 ug/mL, 15 ug/mL, 20 ug/mL, 25 ug/mL, 30 ug/mL, 35 ug/mL, 40 ug/mL, 45 ug/mL, 50 ug/mL, 55 ug/mL, 60 ug/mL, 65 ug/mL, 70 ug/mL, 75 ug/mL, 80 ug/mL, 85 ug/mL, 90 ug/mL, 95 ug/mL, 100 ug/mL, 125 ug/mL, 150 ug/mL, 175 ug/mL, 200 ug/mL, 250 ug/mL and 300 ug/mL.
  • Addition of EDTA can be greater than about 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM and 5 mM. EDTA can also be added in a range of about 0.1 mM to about 5 mM to increase transfection efficiency.
  • Addition of citrate can be greater than about 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.75 mM, 1 mM, 2 mM, 3 mM, 4 mM and 5 mM. Citrate can also be added in a range of about 0.05 mM to about 1 mM to increase transfection efficiency.
  • the additive can also be added in amounts of at least 20 mM, 15 mM, lOmM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, 0.5 mM, 0.25 mM, 0.2 mM, 0.1 mM, 0.05 mM, 0.025 mM and 0.01 mM.
  • the additive can range from at least about 5 percent by weight to about 20 percent by weight.
  • the additive can range from about 5 percent to about 50 percent, in some embodiments from about 25 percent to about 75 percent by weight.
  • the additive can be about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2,5%, 2%, 1,5%, 1%, 0.75%, 0.5%, 0.25%, 0.2%, 0.1%, 0.075%, 0.05%, 0.025%, 0.02%, 0.01% by weight.
  • Other useful additives can include antibacterial agents such as antibiotics.
  • the additives can be added to the growth media, the transfection media and/or the recovery media for post-transfection.
  • the additives can be the same for each media or different depending on the effect desired, such as growth of host cells, efficient transfection, recovery from transfection and/or viral production.
  • the additives can also be added alone to the media, with other additives and/or in varying concentrations depending on the desired outcome. Viral Purification
  • the present invention also relates to the purification of the high titer virus.
  • High titer viral yields can be at least 5xl0 8 viral particles/mL of culture.
  • the viral titer can be at least lxlO 9 viral particles/mL, 5xl0 9 viral particles/mL, lxlO 10 viral particles/mL, 5xl0 10 viral particles/mL, lxlO 1 1 viral particles/mL, 5xlO n viral particles/mL, lxl O 12 viral particles/mL, 5xl0 12 viral particles/mL, and at least lxlO 13 viral particles/mL of culture.
  • the virus produced in the host cell can be highly purified prior to vaccine or gene therapy formulation. Generally, the purification procedures will result in the extensive removal of cellular DNA, other cellular components, and adventitious agents.
  • the present application has demonstrated that the purification of virus utilizing at least one of the following methods of purification can yield high titer virus with little host cell impurities.
  • cell lysis refers to the disruption of cell membrane of a cell and the subsequent release of all or part of the content of the cell.
  • purifying the virus refers to the act of collecting the produced virus from a host cell that has propagated the virus.
  • the virus can be collected by separating the host cellular debris from the virus and harvesting the portion which comprises the virus.
  • the virus can be further separated from the soluble substances, e.g., by centrifugation.
  • Host cells can be harvested from the culture media and/or transfection chamber and lysed. Host cell lysis can be performed by methods known by those skilled in the art that will not damage or otherwise affect the virus. Such methods have, for instance, been discussed in WO 98/22588, p. 28-35. Useful methods in this respect are, for example, freeze-thaw, solid shear, hypertonic and/or hypotonic lysis, liquid shear, sonication, high-pressure extrusion, surfactant lysis, combinations of the above, and the like. In one embodiment, the host cells can be lysed by repeated freeze-thaws. Use of freeze-thaw for lysis has the advantage that it is an easy method and that it inexpensive.
  • the cells can be lysed using at least one detergent.
  • a detergent for lysis has the advantage that it is also an easy method and can be easily scaled to larger quantities.
  • the cells can be lysed by shear using hollow fiber ultrafiltration, such as described in WO 03/084479.
  • Enzymes can also be added to the host cells prior to lysis to remove host cell debris.
  • Such enzymes can include nucleases. The nuclease can be added just seconds prior to (or virtually concomitant with) the lysis step, and in some embodiments, the nuclease can be added to the culture at least one minute before the lysis step.
  • the cell culture with the added nuclease can then be incubated above process temperature, e.g., around 40 °C, or at the culturing temperature (e.g., between about 35 °C to about 37 °C), or at room temperature (around 20 °C) or lower (e.g., around 0 °C), wherein, in general, longer incubation times can be required at lower temperatures to achieve the same result.
  • the incubation can, for instance, be performed at about 37 ° C for about 10 minutes, after which the cells are lysed.
  • the nuclease can, and in some instances will, still actively degrade nucleic acid after the lysis step, and in certain embodiments according to the invention, the incubation of the cells with endonuclease after lysis is prolonged for about 50 minutes (resulting in a total time of the nuclease treatment of about one hour, although this time may effectively be still longer, because it is likely that the nuclease will still be functional until it is removed in subsequent purification steps). This is considerably shorter than the overnight incubation disclosed in WO 98/22588. Longer incubation, such as, up to two hours, up to 8 hours or overnight or even longer incubation is also possible according to the methods of the invention, but is not required to obtain acceptable results.
  • the virus After host cell lysis, the virus can be purified from the cellular debris.
  • Highly concentrated solutions of sugar e.g., sucrose or sorbitol
  • salt e.g. CsCl 2 or NaBr
  • a sugar can be used to create a density gradient.
  • Combinations of sugars i.e., two or more sugars, either in the same or different layers, can also be used to generate density gradients, provided that the corresponding sugar solutions that constitute the layers have specified densities.
  • the volume of the gradient forming preparation is between about 5% to about 100% of the effective volume of the ultracentrifugation rotor or device applying centrifugal forces to the sample, in some instances the volume of the gradient preparation is above about 5%, 10% 15%, 20%, 25%, 30%, 32%, 35%, 40%, 45% or 50% or less than 100%, 90%, 80%; 75%, 70%, 65%, 60%, 55%, 50%, 48%, 45%, 40%, 35%, 30% or 25%, of the volume of the ultracentrifugation rotor or device. In other embodiments, the volume of gradient preparation is between about 1% to about 75% of the volume of the ultracentrifugation rotor or device.
  • Iodixanol is an iodinated density gradient media originally produced as an X-ray contrast compound for injection into humans. Unlike the hyper-osmotic inorganic salt
  • iodixanol solutions can be made iso-osmotic at all densities. This property makes iodixanol an ideal media for analysis and downstream purification steps.
  • iodixanol has the capacity to significantly reduce free capsid proteins and empty capsids from genome-containing (full) capsids.
  • iodixanol is one of the methods in the invention, other suitable density gradient media can be substituted.
  • a method for increasing the concentration of recombinant virus is also provided that generally involves centrifuging a sample containing recombinant virus through an iodixanol gradient, collecting the virus from the iodixanol gradient at least a first fraction comprising the recombinant virus, contacting the recombinant virus with a matrix comprising an affinity matrix, under conditions effective to permit binding of the virus to the matrix, removing any non-bound species from the matrix, and eluting the virus from the matrix.
  • virus can be purified by column chromatography. Any chromatography method that allows purification of virus can be used.
  • ion exchange chromatography can be used. Ion exchange chromatography is a method that relies on charge interactions between the protein of interest and the ion exchange matrix, which is generally composed of resins, such as agarose, dextran, and cross-linked cellulose and agarose, that are covalently bound to a charged group. Charged groups are classified according to type (cationic and anionic) and strength (strong or weak).
  • Ion exchange chromatography is directly upgradable from a small-scale to a large-scale level.
  • Anionic exchange chromatography is a type of ionic exchange chromatography in which a negatively charged resin will bind proteins with a net positive charge.
  • One embodiment of the present invention for purifying virus can also comprise subjecting a culture of recombinant viral particles to high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • Standard methods for performing HPLC are well known to those of ordinary skill in the art.
  • Ion exchange columns that can be utilized in the methods of the present invention include both cationic and anionic ion exchange columns. Weak cationic and strong cationic ion exchange columns can also be used in the methods of this invention.
  • Strong cationic exchange columns generally can have a surface coated with a polyhydroxylated polymer and functionalized with sulfopropyl, a dextran matrix functionalized with a sulfopropyl group, or a surface coated with a polyhydroxylated polymer functionalized with sulfoethyl.
  • Examples of strong cationic ion exchange columns using each of these materials include, respectively, a PO OS HS®, a SP-Sephadex® column and a POROS S® column.
  • Weak cationic exchange columns can have a dextran matrix functionalized by carboxymethyl or an acrylic matrix functionalized by a carboxylic group.
  • Examples of weak cationic exchange columns using each of these materials include, respectively, CM-Sephadex® and Bio-Rex 70®.
  • Weak anionic and strong anionic ion exchange columns can also be used in the methods of the present invention.
  • Weak anionic ion exchange columns can have a surface coated with polyethyleneimine that is capable of surface ionization up to a pH of about 9, a styrene-divinylbenzene copolymer containing sulfonic acid groups or a dextran matrix functionalized by diethylaminoethyl.
  • Examples of weak anionic exchange columns using each of these materials include, respectively, a POROS PI® column, a Dowex 50® column and a DEAE-Sephadex®.
  • Strong anionic exchange columns can have a surface coated with quaternized polyethyleneimine with a surface ionization over a pH range of about 1 to about 14.
  • An example of a strong anionic ion exchange column is a POROS HQ® column.
  • the resins for the columns listed above can be obtained from Amersham/Pharmacia (Piscataway, N.J.), PerSeptive Biosystems (Foster City, Calif.), TosoHaas (Montgomeryville, Pa.) and other suppliers.
  • the ion exchange column can be equilibrated at a pH ranging from about 6.0 to about 9.0 and at a salt concentration that can range from about 50 mM to 200 mM. Therefore, the column can be equilibrated at a pH of about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0 or any other pH in between these pH values and at a salt concentration of about 50 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM or any other concentration in between the salt concentration values listed above.
  • the salts that can be utilized to equilibrate the column include NaCl, KC1 or any other salt that can be adjusted to match the ionic strength of KG. Bound AAV can be eluted from the ion exchange column with an appropriate salt or pH change.
  • Affinity chromatography is also a technique that can provide for ligand specific purification of the virus. As such, the technique exploits the structural and functional characteristics of the viral capsid by binding based on these specific characteristics under certain conditions.
  • affinity column matrices can be contemplated for use with the disclosed invention.
  • antibodies directed against coat proteins of virus can be used to generate affinity column media that can be used to purify the virus.
  • Another example of an affinity column utilizes one or more cell receptors of the virus. These receptors can often be produced and used to generate affinity column media.
  • a number of molecules are thought to serve as AAV receptors. For example,
  • AlphaV-beta5 integrin (Summerford, C, et al., Nat Med January 5: 1 78-82 (1999)), human fibroblast growth factor receptor 1 (Qing K, et al., (Nat Med January 5:1 71-7 (1 99)), and a membrane-associated heparin sulfate proteoglycan (Summerford & Samulski, J Virol. February 72:2 1438-45 (1998)), have all been reported as AAV receptors. Any of these receptors or other receptor molecules can be used to generate affinity chromatography media.
  • One embodiment of the disclosed invention can contemplate the use of heparin as the adsorbent group.
  • Affinity chromatography media containing heparin is commercially available from a variety of sources. For example, PerSeptive Biosystems, Inc. (Framingham, Mass.) markets a heparin-based medium (POROS 20HE®).
  • AVB Sepharose as the adsorbent group, for example AVB Sepharose from GE Life Sciences.
  • POROS 20HE® or AVB Sepharose are used as the affinity chromatography medium, the rAAV containing solution is applied to the affinity medium and subsequently eluted with an appropriate salt concentration or pH change.
  • Hydroxyapatite chromatography is another example of a suitable
  • Hydroxyapatite is a crystalline form of calcium phosphate.
  • the mechanism of hydroxyapatite chromatography involves nonspecific interactions between negatively charged protein carboxyl groups and positively charged calcium ions on the resin, and positively charged protein amino groups and negatively charged.
  • phosphate ions on the resin examples include Bio-Gel HT and CHT ceramic resins by Bio-Rad (Hercules, Calif.);
  • hydroxyl apatite high resolution and hydroxylapatite fast flow by Calbiochem (San Diego, Calif.); HA Ultrogel by Ciphergen (Fremont, Calif.); and hydroxyapatite by Sigma-Aldrich (St. Louis, Mo.).
  • An example of a hydroxyapatite resin is ceramic hydroxyapatite by Bio-Rad, Hercules, Calif., as it has a porous form of hydroxyapatite with an improved calcium :phosphate ratio, which overcomes low binding capacity due to excess phoshpate.
  • the density gradient, affinty, ion exchange and/or chromatography column can be a 0.5 ml column, a 1.5 ml column, a 10 ml column, a 20 ml column, a 30 ml column, a 50 ml column, a 100 ml column, a 200 ml column, a 300 ml column, a 400 ml column, a 500 ml column, a 600 ml column, a 700 ml column, an 800 ml column, a 900 ml column, a 1000 ml (1L) column, a 2000 ml (2L) a 10L column, a 20L column, a 30L column, a 40L column, a 50L column, a 60L column, a 70L column, an 80L column a 90L column, a 100L column or a column with a capacity greater than 100L as well as any other column with a capacity between the volumes listed above.
  • a combination of density gradient centriiugation followed by either affinity heparin, affinity sepharose, hydroxyapatite, or anion/cation exchange chromatography can be used to facilitate the high-throughput of several viruses for purification of the viruses from host cell debris.
  • a combination of multiple chromatography purifications can be used to for purification of the viruses from host cell debris. Examples of such combinations can be affinity chromatography, like heparin columns chromatography, and anion/cation exchange chromatography to further purify the viruses.
  • the present invention also relates to a continuous process of producing virus. It has been discovered that a high viral production can be continuous by culturing a population of cells, transfecting the cells with a vector for the virus, removing a portion of the cells from the culture that have been transfected, then adding fresh cells can be repeatedly performed to produce high viral titers.
  • Culturing can be done, for instance, in dishes, roller bottles or in bioreactors, using batch, fed-batch, continuous systems, hollow fiber, and the like.
  • it can be advantageous to have cells capable of growing in suspension, and it can also be advantageous to have cells capable of being cultured in the absence of animal- or human-derived serum or animal- or human-derived serum components.
  • Suitable conditions for culturing cells are known (see, e.g., Tissue Culture, Academic Press, Kruse and Paterson, editors (1973), and R. I. Freshney, Culture of animal cells: A manual of basic technique, fourth edition (Wiley-Liss Inc., 2000, ISBN 0-471-34889-9).
  • Two cell culture processes are primarily used for large-scale culturing: the fed- batch process and the perfusion process.
  • the primary goals of these methods are to add nutrients, e.g., glucose, as they are being consumed, and the removal of metabolic waste products, e.g., lactic acid and ammonia, as they are being produced.
  • cells receive inoculation medium containing glucose at the initiation of the culture and at one or more points after initiation, but before termination, of the culture.
  • one fed-batch method is an invariant, constant-rate feeding of glucose (Ljunggren and Haggstrom (1994) Biotechnol. Bioeng. 44:808-18; Haggstrom et al. (1996) Annals N.Y. Acad. Sci. 782:40-52).
  • the constant-rate feeding of glucose in a fed-batch process can help control lactic acid production by cultured cells to relatively low levels.
  • cells also receive inoculation base medium, and at the point when cells achieve a desired cell density, cell perfusion is initiated such that the spent medium is replaced by fresh medium.
  • the perfusion process allows the culture to achieve high cell density, and thus enables the production of a large quantity of product.
  • the entire culture medium can be exchanged. It is shown that high viable cell densities can be attained when this is applied to cells.
  • Exchanging culture medium can be performed by any means known to the person skilled in the art, including, but not limited to, collection of the cells by centrifugation, filtration, and the like, followed by re-suspension of the cells into fresh culture medium.
  • a perfusion system can be used, wherein culture medium is either continuously or intermittently exchanged using a cell separation device such as a Centritech centrifuge or passage through a hollow fiber cartridge, and the like.
  • fed-batch culture processes are known in the art and can be used in the methods of the present invention.
  • Nonlimiting examples of fed-batch processes to be used with the methods of the present invention include: invariant, constant-rate feeding of glucose in a fed-batch process (Ljunggren and Haggstrom (1994) Biotechnol. Bioeng. 44:808-18; Haggstrom et al. (1996) Annals N.Y. Acad. Sci. 782:40-52); a fed-batch process in which glucose delivery is dependent on glucose sampling (e.g., through flow- injection analysis, as by Male et al. (1997) Biotechnol. Bioeng. 55:497-504; Siegwart et al. (1999) Biotechnol.
  • CA 2309810 Al also: WO 99/283466 also describes the use of a fed-batch process for the recombinant production in CHO cells and HeLa cells of erythropoietin (EPO) with a high specific activity, characterized by a high proportion of N-acetyl- lactosamine units (lactosamine units) and/or tetra-antennary branches in the
  • US Patent Application to Hiller (2009/0042253) provides various methods related to improving protein production in cell cultures, e.g., animal cell cultures, wherein the cell culture is perfused for a period of time, either continuously or intermittently, and subsequently grown in a fed-batch culture.
  • the disclosure provides a method for production of a polypeptide comprising the steps of growing cells in a cell culture to a first critical level; perfusing the cell culture, wherein perfusing comprises replacing spent medium with fresh medium, whereby at least some portion of the cells are retained and at least one waste product is removed; growing cells in the cell culture to a second critical level; initiating a polypeptide production phase; and maintaining cells in a fed-batch culture during at least some portion of the polypeptide production phase.
  • Continuous ultracentrifugation processes can be useful for harvesting cells from continuous cultures.
  • Continuous flow centrifugation is a laboratory time-saver whereby large volumes of material can be centrifuged at high centrifugal forces without the tedium of filling and decanting a lot of centrifuge tubes, or frequently starting and stopping the rotor.
  • a batch of particle-containing liquid flows continuously into the rotor, which is running at the selected operating speed. Particles sediment out of the flowing stream that then emerges as a particle-depleted effluent. This process continues until the particle-containing capacity of the rotor is reached, or until the starting material
  • sample is completely processed.
  • the amount of starting material that can be handled in one run is governed by the concentration of the particles it contains as well as its volume.
  • the sediment, the particle-depleted effluent, or both can be collected, just as one collects the sediment and/or supernatant after differential centrifugation in conventional fixed angle or swinging bucket rotors.
  • continuous membrane filtration can be used to harvest cells from the culture.
  • Other means known by those skilled in the art can also be utilized to harvest the cells.
  • Removing a portion of the cells in culture can also facilitate continuous production of the viruses.
  • a portion of the culture containing host cells that have been transfected can be removed from the culture to harvest the virus produced by the harvested cells. Subsequent or in tandem with the removal of a portion of the culture, fresh cells can be added to the culture for transfection with the vector.
  • a continuous process of adding fresh cells, transfection of the cells and removing a portion of the cells from the culture can be repeatedly performed.
  • the harvested host cells can be dispersed in a transfection chamber or reactor.
  • the transfection chamber or reactor can be very similar to the continuous or fed-batch culture, however with culture conditions optimized for transfection.
  • the transfection mixture i.e. DNA
  • the transfection mixture can also be fed-batch into the reactor, continuously added to the transfection reactor, or added by other means known by those skilled in the art.
  • the transfected host cells can be harvested by means similar to described above, continuous centrifugation, continuous membrane filtration or other means known by those skilled in the art.
  • the harvested transfected cells can be harvested into lysis buffer for purification of the virus.
  • the harvested transfected cells can be released into a reactor for further culturing.
  • the reactor can be an incubation chamber for release of the recombinant virus from the transfected host cells.
  • the reactor can also be a propagation chamber to produce additional virus.
  • the reactor can be an induction chamber that induces production of the virus in the host cells.
  • the reactor can be an infection chamber to infect additional host cells.
  • the transfected host cells can be harvested for lysis and virus purification. Density gradient centrifugation and chromatography techniques, such as ion-exchange or affinity chromatography, have been utilized for large-scale purification of virus. Continuous centrifugation can be used to harvest large-scale cell culture broth ranging from 500 mL to 20,000 L, resulting in a cell-free supernatant. Other methods described above and those known by one of ordinary skill in the art can also be used to purify the virus.
  • Example 1 Use of media in sequential or combination
  • Transfection media were initially screened based on their ability to transfect T75 cultures (25 mis) as measured by visual inspection of GFP fluorescence after transfecting with a calcium phosphate transfection cocktail containing a plasmid with a GFP transgene. Confirmation of the ability of select , promising candidates to support transfection was done by repeating transfections on larger cultures, specifically T225 flasks, 500 ml spinners, 1L spinners, 1L wavebag , and 5L wavebags.
  • HEK 293 cells grown in suspension culture in CD293 medium were centrifuged at 150g for 4 minutes and the cell pellets were resuspended in suspension media (refer to Table 1 and Figure 2) at 5.0x10 5 /ml. Then 25 ml of cell suspension was added to a T75 Corning flask. Cells were maintained at 37 °C and 5 % CO2 for 24 hours before FBS and plasmid DNA addition. FBS was added to the media at 0% to 10% volume.
  • DNA mix was made of 1 1.79ug of pFdelta6, 7.9 lug of pNLrep, 5.45ug of GFP, 117.9ul of 2.5M CaCl 2 , 1.036ml of H 2 0 and 1.179ml of HeBS buffer.
  • the mixture was prepared by adding the reagents in order and adding HeBS buffer drop- wise.
  • the mixture was incubated at room temperature for about 10 minutes and precipitation formation observed under a microscope before addition of the mixture to the cells.
  • FBS was added to the cells from 0% ⁇ 10% ( Figure 2).
  • the DNA/Ca mixture was added to cells and the cells were incubated at 37 °C and 5 % C0 2 for another 72 hours. During the incubation, the cells were microscopically observed each day for fluorescence, rated based on the percent of cells having fluorescence and the intensity of the fluorescence.
  • CD293 and AEM Two candidate media were selected based on their ability to support cell growth: CD293 and AEM (Invitrogen), while two other types of media, Freestyle 293
  • Figure 2 illustrates the experiments designed to test inhibition of transfection by various potential growth media, including CD293 and AEM.
  • Table 1 Transfection and supplemental media for a sample experiment to determine media inhibition of transfection.
  • the cell pellets were re-suspended in a mixture of two different media (see Table 1) at 5.0xl0 5 /ml.
  • the mix ratios ranged from 80% to 90% of the main transfection media and supplemented by a single other medium to total 100%.
  • a calcium phosphate master mix was prepared by pipetting 1.156 ml of pFdelta6-kana (pFdelta6), 0.774 ml of pNLrep-kana(pNLrep), and pAM/EGFP-kana (pEFGP) into 101.545 ml of sterile water. To this, 1 1.554 ml of 250mM CaCI 2 was added and mixed.
  • Table 2 GFP fluorescence of transfected HEK293 cells with various transfection media/growth media combinations. T75 flasks were transfected by calcium phosphate with a mixture that included a GFP transgene-containing plasmid. Flasks were inspected by UV microscopy 2 days later and assigned a value of 0-5.
  • HEK 293 cells grown in suspension culture in CD293 medium were centrifuged at 150g for 4 minutes and the cell pellets were resuspended in suspension media (refer to Table 3) at 5.0xl0 5 /ml. Then 25 ml of cell suspension was added to a T- 75 Corning flask. The cells were maintained at 37 °C and 5 % C0 2 for 24 hours before FBS and piasmid DNA addition. FBS was added to the cells at 1% volume ratios. Then the additives (except human albumin, BSA, glycerol and yeast extract) were added to concentrations indicated in the Table 3. Human albumin, BSA, glycerol or yeast extract were added without combination with FBS.
  • CD293 medium Invitrogen
  • the DNA mix was made by adding 11.79ug of pFdelta6, 7.91ug of pNLrep, 5.45ug of GFP, 1 17.9ul of 2.5M CaCl 2 , 1.036ml of H 2 0 and 1.179ml of HeBS buffer.
  • the mixture was prepared by adding the reagents in order and adding HeBS buffer drop- wise to the mixture.
  • the mixture was then incubated at room temperature for about 10 minutes and the precipitation formation was observed under the microscope before the addition of the mixture to the cells.
  • the DNA/Ca mixture was added to cells and the cells were incubated at 37 °C and 5 % CO2 for another 24 to 72 hours. During the incubation, the cells were observed under the microscope each day for fluorescence, rating the fluorescence based on the percent of cells having fluorescence and the intensity.
  • HEK 293 cells grown in suspension culture in CD293 media were centrifuged at 150g for 4 minutes and the cell pellets were re-suspended in suspension media at
  • the mixture was made by adding the reagents in order and then adding the HeBS buffer drop-wise to the mixture.
  • the same mixture was also used for experiments of 78%> and 125% DNA with reduced (to 78%>) or increased (125%) amount of the total added to each T-75 flask.
  • the mixture was incubated at room temperature for about 10 minutes and the precipitation formation was observed under the microscope before adding the mixture to the cells.
  • the incubated DNA/Ca mixture was added to cells with 78%), 100% and 125%) total DNA amount individually.
  • the cells were incubated at 37 °C and 5 % CO2 for another 24 to 72 hours. During the incubation, the cells were observed under the microscope each day for fluorescence.
  • Example 2 Process of transfection by spinner, wave or static and preparing and purifying rAAV-GAD
  • Table 4 A summary of the side-by-side performance comparison of the old and two new processes. Data for all processes were calculated for 2 stack scale (or 300mL total transfection volume) on rAAV-GAD produced.
  • a frozen HEK293 vial at ⁇ - 70 °C was thawed at room temperature and grown in serum-free medium (CD293 or AEM) in t-flasks (T75 or T225, Corning), spinners (125, 500 or 1000 mL scale, Corning), and/or wave bags (1-5L) at 37 °C and 5-7 % C0 2 for multiple passages.
  • the cultured cells were centrifuged and Medium- Exchanged (MEX) into a transfection medium of DMEM (Dulbecco's Modified Eagle Medium), Freestyle or Pro293 at 3x10 s to lxlO 6 viable cells/mL with a small amount of
  • DMEM Dens Modified Eagle Medium
  • DNA mixture was added, consisting of the three plasmids (pAM-GAD65 or pAM-GAD67, along with pNLrep and pFdelta-6 in CaCl 2 and HEPES buffer for calcium phosphate (CaP) transient transfection).
  • the cells with DNA mixture were maintained at 37 °C and 5 % CO2 .
  • the cells were centrifuged and Medium-Exchanged (MEX) into a serum-free or serum containing media of DMEM, Freestyle, AEM or Pro293.
  • MEX Medium-Exchanged
  • the cells were centrifuged/harvested and mixed with lysis buffer. For 200 to 300 mL of cells, the cells were resuspended with 15 ml of lysis buffer (20mM NaCl, 50mM Tris-Cl pH8.5, 1 raM MgCl) to give
  • Lysate containing AAV can be purified using iodixanol gradients. Typical gradients have been described in the literature and in general consist of 15%), 25%, 40%, and either a 54% or 60% iodixanol solution. Lysate was loaded first into an
  • the AAV containing fraction was collected in the 40% iodixanol layer, being careful to leave the cellular protein found in the 25% and 40% interphase.
  • Lysate containing AAV can be also be purified using AVB Sepharose from GE Life Sciences. This resin can be packed into columns and used to purify AAV containing lysates. ImL of resin can bind approximately lel2 vg. The lysate should be in a buffer at or near neutral pH, where of about pH 7.4 to about pH 8.5 is preferred. The AAV binds to the resin at neutral pH and elutes from the resin when exposed to lower pH conditions (pH 2 to 5).
  • Example 3 Variable times for transfection after seeding cells
  • Table 5 illustrates viral yield after transfecting HEK293 cells the same day as seeding the suspension cells.
  • Table 5 In process lysate titers from transfections of HEK293 cells seeded on the same day or one day prior to transfection. All flasks were 500 ml spinner flasks (except one was 1L wavebag as noted) with 250 ml of media and transfected with a calcium phosphate mixture using pGAD65 as the transgene.
  • a frozen HEK293 vial at ⁇ - 70 °C was thawed at room temperature and grown in serum-free medium (CD293 or AEM) in t-flasks (T75 or T225, Corning), spinners (125, 500 or 1000 mL scale, Corning), and/or wave bags (1-5L) at 37 °C and 5-7 % C0 2 for multiple passages. Most cultures were maintained in 10-Stacks (Corning) in lOOOmL. Aseptically, the cultured cells were centrifuged and Medium-Exchanged (MEX) into a transfection medium of DMEM (Dulbecco's Modified Eagle Medium), at
  • plasmids were thawed at ambient temperature for 5- 15 minutes for small volume transfections. After thawing, the three plasmids were added to the formulation container as listed in Table 8. (Note: plasmids were not maintained at ambient temperature.)
  • the DNA mixture was added, consisting of the three plasmids (pAM-GAD65 or pAM-GAD67, along with pNLrep and pFdelta-6 in CaCl 2 and HEPES buffer for CaP transient transfection).
  • the cells with DNA mixture were maintained at 37 °C and 5 % C0 2 .
  • Table 8 Manual transfection components and volumes
  • Reagents were pre-warmed (2.5M CaC12, 2X HEPES and Distilled Water) at 25 °C. For ease, 50 mL bottles of 2xHEPES were pooled by pouring them through either 250 mL or 1000 mL filtration units.
  • the autoclaved tubing was placed into the peristaltic pump with one end of the tubing in the 2X HEPES and the other in the formulation vessel. 309 mL of 2X HEPES was pumped into the formulation vessel at a set flow rate of 20 to 40 mL/min. It was ensured that no air bubbles were created during the 2X HEPES addition.
  • the cells were centrifuged and Medium- Exchanged (MEX) into a serum-free or serum containing media of DMEM, Freestyle, AEM or Pro293..
  • MEX Medium- Exchanged
  • the cells were centrifuged/harvested and mixed with lysis buffer. For 200 to 300 mL of cells, the cells were resuspended with 15 ml of lysis buffer (20mM NaCl, 50mM Tris-Cl pH8.5, 1 mM MgCl) to give ⁇ 5ml total and then the resuspended cells were frozen at - 20 °C.
  • lysis buffer 20mM NaCl, 50mM Tris-Cl pH8.5, 1 mM MgCl
  • the samples were frozen at -20°C and then thawed at 37°C. The freeze/thaw was repeated until a "flocculent mass" was formed. The lysate was then centrifuged at 5000g for 30 minutes. The iodixanol ultra-centrifugation purified rAAV-GAD was concentrated by Amicon Ultracel 100K concentrators.
  • Table 10 A summary of the side-by-side performance comparison of the manual and automatic DNA mixing. Data were calculated for 10 stack scale (CS 10) on rAAV-GAD produced.
  • NaH 2 P04 anhydrous and 58.44g of NaCl to a 1L volumetric flask and dilute with HPLC grade water and adjust the pH before filling to the volumetric mark.
  • Heparin column was equilibrated in 20 Column Volumes (CV) of Heparin equilibration buffer (IX PBS pH 7.4).
  • the column was cleaned with 10 CV of 0.1N NaOH. 1 1.
  • the column was stored in 20% EtOH by running 10 CV of 20% ethanol through the column then capping and storing.
  • the eluted fraction collected from the Heparin column was diluted to ⁇ 1 OOmM NaCl using 20mM Sodium Phosphate pH 7.4 from the rAAV-GAD containing elution buffer (IX PBS pH 7.4 + 350mM NaCl (500mM total NaCl)). This is approximately a 1 to 5 dilution with 20mM Sodium Phosphate pH 7.4 (50mL Heparin elution diluted to a final volume of 250mL).
  • the sample was eluted with 10 CV of cation elution buffer (20mM Sodium Phosphate, 370mM NaCl pH 7.4).
  • the column was cleaned with 5 CV of cation cleaning buffer (20mM Sodium Phosphate, 1M NaCl pH 7.4).
  • the cation exchange elution fraction was added to an Amicon Ultracel 100K concentrator (UFC910096) up to 15mL per concentrator.
  • the concentrator was centrifuged at 3000 x g in 10 to 15 minute intervals until 200 ⁇ to 500 ⁇ remained in the retentate cup.
  • wash 1 - sample formulation buffer (2X PBS, ImM MgC12) was added to the retentate cup and step 2 repeated.
  • wash 2 - sample formulation buffer (2X PBS, ImM MgC12) was added to the retentate cup and step 2 repeated.
  • wash 3 - sample formulation buffer (2X PBS, ImM MgC12) was added to the retentate cup and step 2 repeated.
  • wash 4 - sample formulation buffer (2X PBS, ImM MgC12) was added to the retentate cup and step 2 repeated.
  • Fractions were sterilized by using 0.2 ⁇ ultra centrifugal filters (Centrex 10 467 004) for small volumes or 0.2 ⁇ PVDF syringe filters with polypropylene housings (Millipore SLGV-013-SL) for larger volumes.
  • Table 12 Productivity summary of 5mL/4mL (Heparin/Poros - two runs from two different lysates and 185 mL/143 mL (-37 folds increase) scales.)
  • Presence of 3 vector proteins VPl, VP2, and VP3 between 50 and 90 kDa were visible on all runs.
  • the two-column purified AAV-GAD65 sample was cleaner (farthest right of Gel #2 in Figure 4) when compared with samples purified by the iodixanoi process and represented by individual purifications in four lanes of gel #1 in Figure 4.

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Abstract

La présente invention concerne le besoin d'améliorer les rendements en virus des transfections transitoires de plasmides lorsqu'ils sont mis en croissance dans des systèmes de cultures cellulaires. En particulier, il a été démontré que les systèmes de transfection et de culture cellulaire en suspension fournissent une production virale avec un titre élevé. Dans d'autres modes de réalisation, les inventeurs ont montré que la production de virus recombinants est améliorée par la croissance des cellules dans des conditions exemptes de sérum, et en particulier dans des cultures en suspension exemptes de sérum et puis transfectées de manière statique ou par liaison à des empilements de cellules, ou par le biais d'une transfection en suspension avec l'addition d'additifs sélectifs.
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CN103881984A (zh) * 2013-12-06 2014-06-25 深圳市赛百诺基因技术有限公司 无血清悬浮细胞生产重组腺病毒的方法及其药品制剂生产方法
US20140315294A1 (en) * 2011-11-24 2014-10-23 Genethon Scalable lentiviral vector production system compatible with industrial pharmaceutical applications
WO2017015102A1 (fr) * 2015-07-17 2017-01-26 The Trustees Of The University Of Pennsylvania Compositions et procédés pour obtenir des niveaux élevés de transduction dans des cellules hépatiques humaines
CN110168081A (zh) * 2016-11-04 2019-08-23 百深公司 腺相关病毒纯化方法
RU2709678C2 (ru) * 2013-03-15 2019-12-19 Дзе Чилдрен'З Хоспитал Оф Филадельфия Масштабный производственный способ получения рекомбинантных лентивирусных векторов в системе культивирования клеток в бессывороточной суспензии
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WO2022133051A1 (fr) * 2020-12-16 2022-06-23 Regenxbio Inc. Procédé de production d'une particule de virus adéno-associé recombinant
CN117089514A (zh) * 2023-10-13 2023-11-21 思鹏生物科技(苏州)有限公司 提升hek293细胞系aav生产效率的细胞筛选驯化方法
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WO2000022152A1 (fr) * 1998-10-13 2000-04-20 Avigen, Inc. Compositions et methodes de production de virus associe aux adenovirus recombine
US6593123B1 (en) * 2000-08-07 2003-07-15 Avigen, Inc. Large-scale recombinant adeno-associated virus (rAAV) production and purification

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RU2709678C2 (ru) * 2013-03-15 2019-12-19 Дзе Чилдрен'З Хоспитал Оф Филадельфия Масштабный производственный способ получения рекомбинантных лентивирусных векторов в системе культивирования клеток в бессывороточной суспензии
CN103881984A (zh) * 2013-12-06 2014-06-25 深圳市赛百诺基因技术有限公司 无血清悬浮细胞生产重组腺病毒的方法及其药品制剂生产方法
WO2017015102A1 (fr) * 2015-07-17 2017-01-26 The Trustees Of The University Of Pennsylvania Compositions et procédés pour obtenir des niveaux élevés de transduction dans des cellules hépatiques humaines
CN110168081A (zh) * 2016-11-04 2019-08-23 百深公司 腺相关病毒纯化方法
US12037599B2 (en) 2019-08-23 2024-07-16 Lonza Walkersville, Inc. Methods and constructs for production of lentiviral vector
US12391960B2 (en) 2019-08-23 2025-08-19 Lonza Walkersville, Inc. Methods and constructs for production of lentiviral vector
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WO2022133051A1 (fr) * 2020-12-16 2022-06-23 Regenxbio Inc. Procédé de production d'une particule de virus adéno-associé recombinant
CN113265425A (zh) * 2021-05-08 2021-08-17 苏州博腾生物制药有限公司 基于悬浮细胞的car-cd19慢病毒制备工艺
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