The present invention provides a method of releasing viral vectors from cells producing those viral vectors by contacting the cells with a photosensitising agent which is then irradiated to disrupt the plasma membrane of the cells to release the viral vectors which may be collected and/or purified. The product of such methods as well as kits and apparatuses for performing the methods are also provided.
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A method of releasing a viral vector from a cell in which said viral vector has been produced, comprising: a) contacting a cell in which said viral vector is present with a photosensitising agent, b) irradiating said cell with light of a wavelength effective to activate said photosensitising agent, wherein said irradiation is conducted at a dose of light and for a time sufficient to disrupt the plasma membrane of said cell, thereby releasing said viral vector, and c) optionally collecting and/or purifying said released viral vector.
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The method of claim 1 , wherein said cell is a mammalian cell, preferably a human cell.
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The method of claim 1 , wherein said cell is selected from a HEK293 cell, a Vero cell, a sf9 cell and a PER.C6 cell.
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The method of any one of claims 1 to 3, wherein the viral vector is a virus that lacks an envelope, preferably an adenovirus or an adeno-associated virus.
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The method of any one of claims 1 to 4, wherein the photosensitising agent is an amphiphilic or hydrophobic photosensitising agent, preferably TPCS2 a or TPPS 2a .
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The method of any one of claims 1 to 5, wherein the light has a wavelength of 400-700 nm (visible) or 400-475 nm (blue light).
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The method of any one of claims 1 to 6, wherein said contacting step a) is performed for 0.5-120 minutes, preferably 2-30 minutes.
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The method of any one of claims 1 to 7, wherein the irradiation in step b) is performed for 0.5-120 minutes.
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The method of any one of claims 1 to 8, wherein a lysing agent is added to said cell in step a), b) and/or c).
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The method of any one of claims 1 to 9, wherein the cell is in an aqueous medium during steps a) and b).
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The method of any one of claims 1 to 10, wherein the plasma membrane is disrupted by the generation of pores in said membrane.
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The method of any one of claims 1 to 11, wherein at least 30% of the genomic DNA in said cell prior to illumination remains in said cell after release of said viral vector.
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The method of any one of claims 1 to 12, wherein said collection is by removal of cell debris.
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The method of claim 13, wherein the cell is in an aqueous medium during steps a) and b) and collection is performed by separation of the aqueous medium from cell debris which is not suspended in said medium, preferably by centrifugation.
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The method of any one of claims 1 to 14, wherein said viral vector is subject to purification, preferably using at least one of the following methods selected from centrifugation, sonication, freeze-thawing, enzyme digestion and liquid chromatography, preferably to a purity of at least 50% (w/w, dry weight).
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The method of any one of claims 1 to 15, wherein prior to said contacting step a) a step in which said viral vector is produced in said cell is performed.
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The method of claim 16, wherein said viral vector is produced by culturing said cell to allow said cell to produce said viral vector and optionally the culture supernatant is removed before step a).
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The method of claim 17, wherein said cell produces said viral vector after infection of said cell with one or more of said viral vectors and/or transfection with one or more polynucleotides which allow the production of said viral vector in said cell.
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The method of any one of claims 16 to 18, wherein prior to said step in which said viral vector is produced a step is performed in which said cell is infected with one or more of said viral vectors or transfected with one or more polynucleotides and/or viral vectors which allow the production of said viral vector in said cell.
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A cell harvesting kit or apparatus for releasing a viral vector from a cell comprising: a) a photosensitising agent; and b) a light source to irradiate said cell.
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The kit or apparatus of claim 20, wherein said kit or apparatus additionally comprises a container in which said cell may be contained.
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The kit or apparatus of claim 21 , wherein said container is suitable for cell culture or purification of said cell.
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The kit or apparatus of claim 21 or 22, wherein said container is a bag or a tank and/or said light source is attached to said container.
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The kit of any one of claims 20 to 23, wherein said kit or apparatus additionally comprises a means to agitate said cells.
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A preparation of viral vectors obtainable by a method as defined in any one of claims 1 to 19.
