Use of tonicifying agents to enhance recombinant adeno-associated virus yield
AssigneeDimension Therapeutics, Inc.
InventorsYing Jing, Jan Panteli
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Abstract
The invention provides methods for the production of recombinant adeno-associated virus vectors (rAAV), comprising culturing producer cells in media with increased osmolality. Also provided are methods for decreasing the production of helper virus by a rAAV producer cell, comprising culturing the producer cell in media with increased osmolality.
Claims100
A method for producing a recombinant adeno-associated virus vector (rAAV) using a helper virus, the method comprising incubating a host cell capable of producing rAAV in the presence of helper virus for an incubation period in a cell culture medium containing helper virus and one or more tonicifying agents and having an osmolality of 360 mOsm/kg or higher at the start of the incubation period.
The method of any preceding claim, wherein the host cell comprises a genome, said genome comprising one or more AAV genes stably integrated therein.
The method of any preceding claim, wherein the cell culture medium has an osmolality of 375 mOsm/kg or higher at the start of the incubation period.
The method of any preceding claim, wherein the cell culture medium has an osmolality of 400 mOsm/kg or higher at the start of the incubation period.
The method of any preceding claim, wherein the cell culture medium has an osmolality at the start of the incubation period sufficient to produce a 20% reduction in total helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.
The method of any preceding claim, wherein the cell culture medium has an osmolality at the start of the incubation period sufficient to produce a 30% reduction in total helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.
The method of any preceding claim, wherein the cell culture medium has an osmolality at the start of the incubation period sufficient to produce a 40% reduction in total helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.
The method of any preceding claim, wherein the cell culture medium has an osmolality at the start of the incubation period sufficient to produce a 50% reduction in total helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.
The method of any preceding claim, wherein the cell culture medium has an osmolality at the start of the incubation period sufficient to produce at least a 50% increase in total rAAV production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.
The method of any preceding claim, wherein the cell culture medium has an osmolality at the start of the incubation period sufficient to produce at least a 100% increase in total rAAV production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.
The method of any preceding claim, wherein the cell culture medium has an osmolality at the start of the incubation period sufficient to produce at least a 150% increase in total rAAV production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.
The method of any preceding claim, wherein the cell culture medium has an osmolality at the start of the incubation period sufficient to produce at least a 200% increase in total rAAV production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.
The method of any preceding claim, wherein at least one tonicifying agent is an ionic tonicifying agent.
The method of any preceding claim, wherein at least one tonicifying agent is selected from the group comprising:
- NaCl, KCl, NaNO3, NaHCO3, Na2SO4, Na2HPO4, NaH2PO4, NaNO3, KNO3, K2SO4, K2HPO4, KH2PO4, and KNO3.
The method of any preceding claim, wherein the cell culture medium is a serum-free cell culture medium.
The method of any preceding claim, wherein the cell culture medium is a protein-free cell culture medium.
The method of any preceding claim, wherein the cell culture medium is selected from the group consisting of MEM, DMEM, RPMI, Ham's F-12 medium, Leibovitz's L-15 medium, and mixtures thereof, said medium being supplemented with one or more tonicifying agents.
The method of any preceding claim, wherein the cell culture medium consists essentially of DMEM supplemented with one or more tonicifying agents.
The method of any preceding claim, wherein the host cell is a mammalian cell.
The method of any preceding claim, wherein the host cell is selected from the group consisting of HeLa, HEK293, COS, A549, BHK, and Vero cells.
The method of any preceding claim, wherein the helper virus is selected from the group consisting of adenovirus, herpes virus, baculovirus, and recombinant forms of any of the foregoing viruses.
The method of any preceding claim, wherein the host cell comprises a heterologous nucleotide sequence flanked by AAV inverted terminal repeats.
The method of any preceding claim, wherein the host cell comprises rep and cap genes.
The method of any preceding claim, wherein the host cell comprises helper virus genes.
The method of any preceding claim, wherein the host cell comprises a heterologous nucleotide sequence flanked by AAV inverted terminal repeats, rep and cap genes, and helper virus genes.
The method of any preceding claim, wherein the host cell is incubated in the cell culture medium for a period of at least 2 days.
The method of any preceding claim, further comprising the steps of harvesting and purifying the rAAV.
A cell culture system comprising:
- a) a host cell capable of producing rAAV;
- b) a helper virus;
- and
- c) a cell culture medium with an osmolality of 360 mOsm/kg or higher when measured immediately after the host cell is introduced into the cell culture medium.
Dependencies are inferred from the claim text.