WO2020219897A1 - Procédés de production de virus adéno-associés recombinants - Google Patents

Procédés de production de virus adéno-associés recombinants Download PDF

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WO2020219897A1
WO2020219897A1 PCT/US2020/029836 US2020029836W WO2020219897A1 WO 2020219897 A1 WO2020219897 A1 WO 2020219897A1 US 2020029836 W US2020029836 W US 2020029836W WO 2020219897 A1 WO2020219897 A1 WO 2020219897A1
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rhsv
production
cell
cells
raav
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PCT/US2020/029836
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Mary Elizabeth GRIBBLE
Terrence Michael DOBROWSKY
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Biogen Ma Inc.
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Publication of WO2020219897A1 publication Critical patent/WO2020219897A1/fr

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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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16641—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16643—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16641—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16644—Chimeric viral vector comprising heterologous viral elements for production of another viral vector
    • 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
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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

  • rAAVs recombinant adeno-associated viruses
  • a heterologous nucleic acid of interest e.g., a gene encoding a therapeutic protein, an antisense nucleic acid molecule, a ribozyme, a miKNA, an siRNA, a nucleic acid encoding a CRISPR/Cas system, or the like
  • a heterologous nucleic acid of interest e.g., a gene encoding a therapeutic protein, an antisense nucleic acid molecule, a ribozyme, a miKNA, an siRNA, a nucleic acid encoding a CRISPR/Cas system, or the like
  • these recombinant AAVs are engineered by deleting, in whole or in part, the internal portion of the AAV genome and inserting the heterologous nucleic acid of interest or“payload” between the inverted terminal repeats (ITRs).
  • ITRs inverted terminal repeats
  • the ITRs remain functional in such vectors allowing replication and packaging of the AAV particle containing the nucleic acid payload enclosed within the AAV capsid.
  • the nucleic acid payload is also operably linked to regulatory sequences (e.g., promoter, enhancer, etc.) capable of driving expression of the payload in the patient’s target cells.
  • the upstream production phase can involve transfection of vectors (e.g., plasmids) expressing AAV replication, structural components and the heterologous nucleic acid of interest.
  • vectors e.g., plasmids
  • rAAV production cells can be infected with a virus, e.g., one or more recombinant herpes simplex viruses (rHSVs), that delivers AAV replication and structural components and the heterologous nucleic acid of interest.
  • rHSVs herpes simplex viruses
  • rAAV production cells cells that produce rAAV (“rAAV production cells”), are typically infected by a first herpes simplex virus (rHSV) that expresses AAV essential proteins (e.g., Rep and Cap) and a second rHSV encoding a proviral AAV construct that includes the heterologous nucleic acid of interest.
  • rHSV herpes simplex virus
  • AAV essential proteins e.g., Rep and Cap
  • the present disclosure is based in part on the insight that the specific productivity of these rAAV production cells can be significantly improved by including an amount of dextran sulfate during production of the underlying rHSVs.
  • the present disclosure also recognizes that the specific productivity of the production cells used to produce the underlying rHSVs (“rHSV production cells”) can be significantly improved by including an amount of dextran sulfate during production of the rHSVs.
  • the present disclosure provides a method of producing rAAV comprising the steps of: contacting a first rHSV production cell with a first rHSV that encodes essential AAV proteins in an infection medium that comprises dextran sulfate;
  • a second rHSV production cell with a second rHSV that encodes a proviral AAV construct in an infection medium that comprises dextran sulfate; culturing the first and second rHSV production cells to produce rHSV; isolating rHSV that encode essential AAV proteins produced by the first rHSV production cells and rHSV that encode a proviral AAV construct produced by the second rHSV production cells; co-infecting an rAAV production cell with the isolated rHSV that encode essential AAV proteins and the isolated rHSV that encode a proviral AAV construct; and culturing the rAAV production cell to produce rAAV that include the proviral AAV construct.
  • the step of isolating comprises collecting a culture supernatant and clarifying the supernatant by depth filtration or centrifugation. In some embodiments, the step of isolating further comprises concentrating the clarified supernatant by tangential flow filtration (TFF). In some embodiments, the dextran sulfate is present at a concentration in the range of 0.0005 g/L to 0.03 g/L.
  • the present disclosure provides a method of producing rAAV comprising: co-infecting an rAAV production cell with a first rHSV that encodes essential AAV proteins and a second rHSV that encodes a proviral AAV construct; and culturing the rAAV production cell to produce rAAV that include the proviral AAV construct, wherein the rHSVs were produced by infection of rHSV production cells with rHSVs in the presence of dextran sulfate.
  • Figure 1 depicts a representative process of generating rAAVs from rHSVs generated in the presence of dextran sulfate (DS).
  • DS dextran sulfate
  • Figure 2 shows titers of rHSV generated by infection of adherent V27 producer cells by rHSV in the presence of varying concentrations of dextran sulfate, and the associated lot-to-lot variability.
  • Figure 3 shows the specific productivity of cells producing rAAVs generated from rHSVs produced in the presence or absence of dextran sulfate.
  • cell density refers to that number of cells present in a given volume of medium or the number of cells present in a given surface area.
  • culture and“cell culture” refer to a cell population (e.g., eukaryotic cell population) that is suspended in or covered by a medium under conditions suitable to survival and/or growth of the cell population. As will be clear to those of ordinary skill in the art, these terms can also refer to the combination comprising the cell population and the medium.
  • an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single sample) under otherwise comparable conditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent.
  • an appropriate reference measurement may be or comprise a measurement in a comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.
  • the terms“medium”,“cell culture medium”,“culture medium”, and“growth medium” refer to a solution containing nutrients which nourish growing cells (e.g., eukaryotic cells). Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cell for minimal growth and/or survival.
  • the solution can also contain components that enhance growth and/or survival above the minimal rate, including hormones and growth factors.
  • the solution is formulated to a pH and salt concentration optimal for cell survival and proliferation.
  • the medium can also be a“defined medium” or“chemically defined medium”, i.e., a serum-free medium that contains no proteins, hydrolysates or components of unknown composition.
  • defined media are free of animal-derived components and all components have a known chemical structure.
  • a defined medium can comprise recombinant polypeptides or proteins, for example, but not limited to, hormones, cytokines, interleukins and other signaling molecules.
  • seeding refers to the process of providing a cell culture to a bioreactor or another vessel.
  • the cells have been propagated previously in another bioreactor or vessel.
  • the cells have been frozen and thawed immediately prior to providing them to the bioreactor or vessel.
  • the term refers to any number of cells, including a single cell.
  • “specific productivity” or“cell specific productivity” refers to the amount of product (e.g., rAAVs) that can be produced per viable production cell at the time of infection.
  • Specific productivity for example, can be expressed as viral genome copies (vg) per viable production cell (vc) in culture at the time of infection. .
  • TFF tangential flow filtration
  • Titer refers to the quantity of virus in a given volume. Titer, for example, can be expressed as plaque forming units (pfu) per given volume or viral genome copies (vg) per given volume.
  • pfu plaque forming units
  • vg viral genome copies
  • the present disclosure describes improved methods for the production of recombinant AAVs (rAAVs).
  • Production of rAAV requires delivery of a proviral AAV construct to an rAAV production cell wherein the proviral AAV construct encodes for a payload and is intended to be packaged in the rAAV.
  • the payload is a
  • heterologous protein with a therapeutic purpose e.g., an enzyme or antibody.
  • the payload is a heterologous nucleic acid with a therapeutic purpose, e.g., an miRNA, siRNA, shRNA, mRNA, snRNA, CRISPR/Cas guide RNA, etc.
  • a therapeutic purpose e.g., an miRNA, siRNA, shRNA, mRNA, snRNA, CRISPR/Cas guide RNA, etc.
  • the payload can be selected from any heterologous protein or nucleic acid of interest.
  • the proviral AAV construct comprises a sequence encoding the payload (e.g., a cDNA expression cassette) flanked by AAV inverted terminal repeats (ITRs).
  • the ITRs are the cis acting viral DNA sequences required to direct replication and packaging of the proviral AAV construct.
  • the proviral AAV construct will also typically include other regulatory sequences, e.g., promoters, introns, enhancers, etc. to regulate expression of the payload in the cells or tissue of interest.
  • the essential AAV proteins include Rep and Cap proteins.
  • the Rep proteins (Rep78, 68, 52 and 40) are involved in viral DNA replication, resolution of replicative intermediates and generation of single-strand genomes. It will be appreciated that not all four Rep proteins need to be delivered to an rAAV production cell in order to produce rAAVs.
  • the Cap proteins (VP1, VP2 and VPS) are the structural proteins that make up the viral capsid.
  • rAAV production is achieved by delivery of the proviral AAV construct encoding a payload and the delivery of proteins essential for AAV production to an rAAV production cell.
  • the delivery of the proviral AAV construct encoding a payload and the delivery of the AAV essential proteins is achieved by co-infection of an rAAV production cell with a first rHS V encoding the proviral AAV construct and a second rHS V encoding the AAV essential proteins.
  • the AAV essential proteins may be delivered using a second rHSV that encodes the Rep proteins and a third rHSV that encodes the Cap proteins.
  • the rAAV production cell already encodes the essential AAV proteins and is infected with an rHSV that encodes the proviral AAV construct. In some embodiments, the rAAV production cell already encodes the proviral AAV construct and is infected with an rHSV that encodes the essential AAV proteins. In some embodiments the rHSVs have been modified so that they do not express an HSV protein necessary for replication in host cells. In some embodiments the rHSVs have been modified so that they do not express the ICP27 protein (infected cell culture polypeptide 27 protein) which is essential for infection of most cells.
  • ICP27 protein infected cell culture polypeptide 27 protein
  • Figure 1 provides a schematic for a representative process of producing rAAVs by co-infection of an rAAV production cell with two rHSVs.
  • Stock rHSVs encoding for the essential AAV proteins (Rep and Cap) or encoding a gene of interest (GOI) or other payload are initially used to infect separate cultures of rHSV production cells.
  • the rHSV production cells can support replication of rHSVs that do not express an HSV protein necessary for replication.
  • a gene that expresses a protein necessary for replication is deleted from the rHSV.
  • the gene that expresses a protein necessary for replication that is deleted for the rHSV is ICP27.
  • the rHSV production cells provide the HSV protein necessary for replication in trans. In some
  • the rHSV production cells can support replication of rHSV that do not express the ICP27 protein. In some embodiments, the rHSV production cells provide ICP27 in trans. In some embodiments, the rHSV production cell is a Vero cell. In some embodiments, the rHSV production cell is the Vero cell line V27.
  • the rHSV production cell is infected with stock rHSV at a multiplicity of infection (MOI) in the range of 0.05 to 3 pfu/vc. In some embodiments, the rHSV production cell is infected with stock rHSV at a MOI in the range of 0.1 to 2 pfu/vc. In some embodiments, the rHSV production cell is infected with stock rHSV at a MOI of 0.15 pfu/vc.
  • MOI multiplicity of infection
  • the rHSV production cell is seeded at a density in the range of 1.2e4 to 1.6e4 vc/cm 2 .
  • the infection of the rHSV production cells with rHSVs is done in the presence of dextran sulfate.
  • Dextran sulfate is a sulfated polysaccharide and is commercially available form a variety of chemical manufacturers including Sigma-Aldrich of St. Louis, MO and Cayman Chemicals of Ann Arbor, MI. It is typically used as a sodium salt.
  • dextran sulfate is added to culture medium before the rHSV production cells are contacted with stock rHSVs.
  • dextran sulfate is added to culture medium when the rHSV production cells are contacted with stock rHSVs. In some embodiments dextran sulfate is added to culture medium after the rHSV production cells are contacted with stock rHSVs. In some embodiments, the presence of dextran sulfate increases the titer of rHSV produced by the rHSV production cell.
  • the dextran sulfate is present at a concentration range of 0.0005 g/L to 0.03 g/L. In some embodiments the dextran sulfate is present at a concentration range of 0.01 g/L to 0.03 g/L. In some embodiments the dextran sulfate is present at a concentration range of 0.01 g/L to 0.02 g/L. In some embodiments the dextran sulfate is present at a concentration range of 0.005 g/L to 0.025 g/L. In some embodiments the dextran sulfate is present at a concentration of 0.01 g/L. In some embodiments the dextran sulfate is present at a concentration of 0.02 g/L. In some embodiments the dextran sulfate is present at a concentration of 0.015 g/L.
  • the dextran sulfate has a weight-average molecular weight (Mw) in the range of 4 to 40 kDa. In some embodiments the dextran sulfate has a weight- average molecular weight (Mw) in the range of 5 to 12 kDa. In some embodiments the dextran sulfate has a weight-average molecular weight (Mw) in the range of 7 to 12 kDa. In some embodiments the dextran sulfate has a weight-average molecular weight (Mw) in the range of 5 to 10 kDa. In some embodiments the dextran sulfate has a weight-average molecular weight (Mw) of 4, 5, 6, 7, 8, 9, 10, 11, or 12 kDa.
  • the culture supernatant comprising rHSVs is collected and the rHSVs are purified.
  • the culture supernatant is clarified by depth filtration and concentrated by tangential flow filtration.
  • the rHSVs encoding for the essential AAV proteins (rHSV-Rep/Cap) and rHSVs encoding the gene of interest (GOI) or other payload (rHSV-GOI) are then used to co-infect the rAAV production cells.
  • the rHSVs encoding the GOI are used to infect rAAV production cells at a MOI in the range of 0.5 to 4 pfu/viable cell (vc).
  • the rHSVs encoding the GOI are used to infect rAAV production cells at a MOI in the range of 0.5 to 3 or 1 to 4 pfu/vc.
  • the rHSVs encoding the GOI are used to infect rAAV production cells at a MOI of about 0.5, 1, 2, 3, 4 pfu/vc. In some embodiments, the rHSVs encoding for the essential AAV proteins are used to infect rAAV production cells at a MOI in the range of 1.5 to 8 pfu/vc. In some embodiments, the rHSVs encoding for the essential AAV proteins are used to infect rAAV production cells at a MOI in the range of 1.5 to 6 pfu/vc (e.g., 3 to 8, 2 to 5, 1 to 2, 1.5 to 3, or 2 to 4 pfu/vc). In some embodiments, the rHSVs encoding for the essential AAV proteins are used to infect rAAV production cells at a MOI of about 1.5, 2, 3, 4, or 5. In some embodiments, the rAAV
  • the rAAV production cell is an adherent cell. In some embodiments, the rAAV production cell is a suspension-adapted cell. In some embodiments, the rAAV production cell is a BHK cell, e.g., a suspension-adapted BHK cell as shown in Figure 1. In some embodiments, the rAAV
  • HEK293 cell e.g., a suspension-adapted HEK293 cell.
  • the rAAVs are recovered.
  • the rAAVs may be recovered by lysing the rAAV production cells and recovering the rAAVs from the lysate, e.g., after centrifugation.
  • the rAAVs may be recovered from the culture supernatant.
  • the rAAVs are isolated using one or more of affinity chromatography, ion- exchange chromatography (e.g., cation exchange chromatography), filtration (e.g., UF/DF filtration), e.g., as shown in Figure 1.
  • the present disclosure recognizes that the specific productivity of an rHSV production cell at a given cell density is increased when dextran sulfate is present. In some embodiments, the present disclosure recognizes that the specific productivity of an rAAV production cell at a given cell density is increased when the underlying rHSVs are produced in the presence of dextran sulfate. In some embodiments, the present disclosure recognizes that the specific productivity of an rAAV production cell per unit of underlying rHSV is increased when the underlying rHSVs are produced in the presence of dextran sulfate.
  • Example 1 Scheme for the Production of rAAV using rHSVs Produced in the Presence of Dextran Sulfate
  • Figure 1 provides a schematic for a representative process of producing rAAVs using rHSVs produced in the presence of dextran sulfate.
  • Stock rHSV encoding for the essential AAV proteins Rep and Cap or encoding a gene of interest (GOI) are used to infect separate cultures of rHSV production cells (e.g., Vero cells). The infection of the rHSV production cells is done in the presence of dextran sulfate.
  • the supernatants of the cultures comprising the separate rHSVs arecollected and the rHSVs encoding for the essential AAV proteins (rHSV-Rep/Cap) and rHSVs encoding the gene of interest (GOI) or other payload (rHSV-GOI) are then used to co-infect rAAV production cells. After the rAAV production cells have been infected and allowed to culture for a period of time, the rAAVs are recovered.
  • the present example describes an exemplary protocol for the production of rHSV in the presence of dextran sulfate.
  • V27 cells were thawed, plated and cultured in DMEM supplemented with 9.1% fetal bovine serum (FBS) for approximately 24 hours.
  • the V27 cells were trypsinized and seeded in a subsequent flasks at a density of 1.0e4 vc/cm 2 for a 3 day passage (72 ⁇ 3 hours) and 2.0e4 vc/cm 2 for a 2 day passage (48 ⁇ 3 hours).
  • V27 cells were passaged at least three times prior to infection.
  • the present example describes results of an rHSV production run using the protocol described in Example 1 with varying concentrations of dextran sulfate at the time of infection. Separate infections of V27 cells were performed with separate lots of dextran sulfate and concentrations of dextran sulfate ranging from 0.0005 g/L to 0.05 g/L. Each lot of dextran sulfate was tested multiple times under the same procedure with cells thawed at different times. The same host, MOI, infection procedure, harvest procedure, infection density and titer method was used for each lot.
  • Example 4 Production of rAAV using rHSV Produced in the Presence or Absence of Dextran Sulfate.
  • the present example describes the results of rAAV production using rHSV produced in the presence or absence of dextran sulfate.
  • BHK cells were seeded in an automated micro bioreactor (AMBR) at a density of 0.5 x 10 6 vc/ml.
  • the production has also been performed using 3L and 10L single use bioreactors.
  • GOI gene of interest
  • the rHSV expressing a GOI was used at an MOI in the range of 1 to 2 and the rHSV expressing essential AAV proteins Rep and Cap was used at an MOI in the range of 2-4. No significant difference in specific productivity was observed within these MOI ranges Approximately 24 hours after infection MgCl (final concentration of 0.002 M), benzonase (25 U/ml) and Triton (1% final concentration) were added to the culture. Two hours later NaCl at a final concentration of 0.5 M was added and the supernatant comprising rAAV was collected from the bioreactor and purified.
  • MgCl final concentration of 0.002 M
  • benzonase 25 U/ml
  • Triton 1% final concentration
  • rAAV produced by co-infection of BHK cells with rHSVs that were produced with dextran sulfate resulted in a significantly higher specific productivity.
  • dextran sulfate or other additive a significantly higher rAAV specific productivity was observed by use of rHSVs produced in the presence of dextran sulfate.

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Abstract

La présente invention concerne des procédés améliorés pour la production de virus adéno-associés recombinants (rAAV) générés lors de l'infection d'une cellule de production par un ou plusieurs virus herpès simplex recombinants (rHSV).
PCT/US2020/029836 2019-04-24 2020-04-24 Procédés de production de virus adéno-associés recombinants WO2020219897A1 (fr)

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WO2022159662A1 (fr) * 2021-01-21 2022-07-28 Regenxbio Inc. Production améliorée de polypeptides et de virus recombinés

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