Cell lines overexpressing vesicular stomatitis virus g

Abstract

The present disclosure generally relates to the manufacturing of gene therapy products, and specifically to methods of producing an enveloped virus from a stable producer cell clone or a stable producer cell pool overexpressing vesicular stomatitis virus G (VSV-G) in suspension cell culture.

Description

[0001] CELL LINES OVEREXPRESSING VESICULAR STOMATITIS VIRUS G

[0002] RELATED APPLICATION DATA

[0003] The present application claims priority from United States Patent Application No. 63 / 636,201 filed 19 April 2024 entitled “Cell lines overexpressing vesicular stomatitis virus G”. The entire contents of this application are hereby incorporated by reference.

[0004] SEQUENCE LISTING

[0005] The present application is filed together with a Sequence Listing in electronic form. The entire contents of the Sequence Listing is hereby incorporated by reference.

[0006] FIELD

[0007] The present disclosure generally relates to the manufacturing of gene therapy products, and specifically to methods of producing an enveloped virus from a stable producer cell clone or a stable producer cell pool overexpressing vesicular stomatitis virus G (VSV-G) in suspension cell culture.

[0008] BACKGROUND

[0009] Retroviruses, e.g., lentiviruses are one of the most studied viral vectors for gene therapy. Retroviruses in general are RNA-based viruses which integrate their genetic information into the target cell chromosomes permanently. The advantages of retroviruses include long-term transgene expression in target cells, a low immunogenic potential, and the ability to transduce into dividing and non-dividing cells.

[0010] Lentiviruses are genetically engineered and usually based on human immunodeficiency virus 1 (HIV-1). To increase safety, modem vectors contain only those HIV genes which are necessary for infection and gene delivery, but the genes necessary for replication and virulence factors have been removed. Often, the envelope protein of HIV-1 is exchanged with that of another vims to allow infection of a wide range of target cells, e.g., VSV-G protein from Vesicular stomatitis Indiana virus (VSV). However, VSV-G is toxic to cell cultures and is generally controlled by an inducible expression system, e.g., a Tet-off expression system.

[0011] To produce lentiviruses, cells are transfected with at least 3-4 plasmids. These include the transfer plasmid with the gene of interest and several packaging plasmids and essential viral proteins responsible for gene integration or self-assembly. These plasmids can be transiently transfected into the cells, or a producer cell line is created with stable integration of one or more of the plasmids with inducible promoters, in which lentivirus production can be induced.

[0012] Once the virus production has been induced, the release of the virus occurs by budding after successful assembly within the cells. The lentivirus is harvested from the producer cells and subsequently purified and concentrated in the downstream process. The resultant lentivirus can be used to modify patient cells, such as hemopoietic stem cells, for clinical benefit.

[0013] Clinical-grade lentiviral vectors are most often produced by transient transfection of adherent cell lines. These production methods are cost intensive, require large amounts of GMP-grade plasmids and hamper process scalability and reproducibility.

[0014] Thus, there is a need in the art for an efficient process for producing lentiviruses in a cell culture system, e.g., for gene therapy.

[0015] SUMMARY

[0016] In work leading up to the present invention, the inventors sought to produce a method for producing enveloped viruses, e.g., for gene therapy, at commercial scale and suitable for regulatory requirements.

[0017] When adapting adherent cell lines producing VSV-G-pseudotyped virus to suspension cell culture, the inventors found that the tetracycline -induced cultures exhibited high physical titres but low infectious titres, compared to adherent production processes using the same cell clones.

[0018] In developing the cell lines of the disclosure, the inventors recognised that VSV- G is toxic to cell cultures and the cells did not demonstrate reduced cell growth and viability which would have been expected with high levels of VSV-G. One theory of the inventors was that during adaptation from adherent to suspension culture, cells with a lower level of VSV-G expression may have been selected. Another theory was that there was epigenetic silencing of VSV-G.

[0019] To address this problem, the inventors identified that they could increase the level of VSV-G expressed by the enveloped virus by stably re-transfecting the suspension adapted cell lines with a VSV-G encoding plasmid. The inventors found that cell lines re-transfected with the VSV-G plasmid produce virus with significantly higher VSV-G levels and surprisingly the re-transfected VSV-G cell lines also had higher infectious titres and higher ratios of infectious titre to physical titre, indicating better quality virus. In addition, viruses derived from these re-transfected cell lines have significantly increased transduction efficiencies of CD34+ cells and vector copy numbers / cell compared to virus produced with the parental cell lines. Accordingly, the inventors have developed a scalable serum-free enveloped virus production platform using stable packaging cell lines for efficient transduction e.g., of human CD34+cells. The generated producer clonal cell lines with over-expressed VSV- G, which is known to be cytotoxic, were surprisingly able to sustain production of high infectious enveloped virus titers for an extended period of time without a significant decline in viability.

[0020] Thus, the findings by the inventors provide the basis for packaging cell lines, stable producer pools, stable producer cell clones, and producer cell lines thereof, capable of producing an enveloped virus in a suspension cell culture.

[0021] It will be apparent to the skilled person from the disclosure herein that the cell lines of the present disclosure are stable producer cell lines, i.e., cells having stably incorporated therein the genetic material required to produce an enveloped virus (i.e., a lentivirus). Such cells are distinguished from cells having the genetic elements transiently incorporated therein.

[0022] The present disclosure provides a stable producer cell line capable of producing an enveloped virus in a suspension cell culture, wherein the stable producer cell line is derived from a packaging cell line transfected with vesicular stomatitis virus G protein (VSV-G), and wherein the stable producer cell line is stably re-transfected with a VSV- G encoding plasmid.

[0023] In one example, the cell line is capable of producing the enveloped virus with an infectious titre of at least 5.0 x 106TU / mL of culture medium at 6 days following induction of enveloped virus production.

[0024] The present disclosure also provides a stable producer cell line stably expressing VSV-G, wherein the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with an infectious titre of at least 5.0 x 106TU / mL of culture medium at 6 days following induction of enveloped virus production.

[0025] In one example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer yield of at least 5 x 106transducing units (TU) / mL. For example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer yield of between about 5 x 106TU / mL and 1 x 1010TU / mL. In one example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer yield of about 5 x 106TU / mL, or about 7 x 106TU / mL, or about 10 x 106TU / mL. In a further example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer yield of about 1 x 107TU / mL, or about 5 x 107TU / mL, or about 10 x 108TU / mL, or about 5 x 108TU / mL, or about 10 x 108TU / mL, or about 5 x 109TU / mL, or about 1 x IO10TU / mL.

[0026] In one example, the stable producer cell line is capable of producing an enveloped virus with an infectious titer of at least 5.5 x 106TU / mL of culture medium at day 6 of culture. In one example, the stable producer cell line is capable of producing an enveloped virus with an infectious titer of at least 6 x 106TU / mL of culture medium at 6 days following induction of enveloped virus production. For example, the stable producer cell line is capable of producing an enveloped virus with an infectious titer of at least 6.5 x 106TU / mL of culture medium day 6 of culture. In another example, the stable producer cell line is capable of producing an enveloped virus with an infectious titer of at least 7 x 106TU / mL of culture medium at day 6 of culture. In a further example, the stable producer cell line is capable of producing an enveloped virus with an infectious titer of at least 8 x 106TU / mL of culture medium at day 6 of culture. In one example, the stable producer cell line is capable of producing an enveloped virus with an infectious titer of at least 9 x 106TU / mL of culture medium at day 6 of culture. In another example, the stable producer cell line is capable of producing an enveloped virus with an infectious titer of at least 1 x 107TU / mL of culture medium at day 6 of culture.

[0027] In one example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 5 x 106transducing units (TU) / m of culture medium at day 14 of culture. For example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 7 x 106TU / mL, or about 10 x 106TU / mL of culture medium at day 14 of culture. In another example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 1 x 107TU / mL of culture medium at day 14 of culture. For example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of about 1.1 x 107TU / mL, or about 1.2 x 107TU / mL, or about 1.3 x 107TU / mL, or about 1.4 x 107TU / mL, or about 1.5 x 107TU / mL of culture medium at day 14 of culture. In one example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 1.5 x 107TU / mL of culture medium at day 14 of culture. For example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of about 1.6 x 107TU / mL, or about 1.7 x 107TU / mL, or about 1.8 x 107TU / mL, or about 1.9 x 107TU / mL, or about 2.0 x 107TU / mL of culture medium at day 14 of culture. In one example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 2 x 107TU / mL of culture medium at day 14 of culture. For example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of about 2.1 x 107TU / mL, or about 2.2 x 107TU / mL, or about 2.3 x 107TU / mL, or about 2.4 x 107TU / mL, or about 2.5 x 107TU / mL, or about 2.6 x 107TU / mL, or about 2.7 x 107TU / mL, or about 2.8 x 107TU / mL, or about 2.9 x 107TU / mL, or about 3.0 x 107TU / mL of culture medium at day 14 of culture.

[0028] In one example, the method results in a viral infectious titer of at least 5 x 106transducing units (TU) / mL of culture medium at day 20 of culture. For example, stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 5 x 106TU / mL, or about 7 x 106TU / mL, or about 10 x 106TU / mL of culture medium at day 20 of culture. In another example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 1 x 107TU / mL of culture medium at day 20 of culture. For example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of about 1.1 x 107TU / mL, or about 1.2 x 107TU / mL, or about 1.3 x 107TU / mL, or about 1.4 x 107TU / mL, or about

[0029] 1.5 x 107TU / mL of culture medium at day 20 of culture. In one example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 1.5 x 107TU / mL of culture medium at day 20 of culture. For example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of about 1.6 x 107TU / mL, or about 1.7 x 107TU / mL, or about 1.8 x 107TU / mL, or about 1.9 x 107TU / mL, or about 2.0 x 107TU / mL of culture medium at day 20 of culture. In one example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 2 x 107TU / mL of culture medium at day 20 of culture. For example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with viral infectious titer of about 2.1 x 107TU / mL, or about 2.2 x 107TU / mL, or about 2.3 x 107TU / mL, or about 2.4 x 107TU / mL, or about

[0030] 2.5 x 107TU / mL, or about 2.6 x 107TU / mL, or about 2.7 x 107TU / mL, or about 2.8 x 107TU / mL, or about 2.9 x 107TU / mL of culture medium at day 20 of culture.

[0031] In one example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 5 x 106transducing units (TU) / mL of culture medium at day 25 of culture. For example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at about 5 x 106TU / mL, or about 7 x 106TU / mL, or about 10 x 106TU / mL of culture medium at day 25 of culture. In another example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 1 x 107TU / mL of culture medium at day 25 of culture. For example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of about 1.1 x 107TU / mL, or about 1.2 x 107TU / mL, or about 1.3 x 107TU / mL, or about 1.4 x 107TU / mL, or about

[0032] 1.5 x 107TU / mL of culture medium at day 25 of culture. In one example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 1.5 x 107TU / mL of culture medium at day 25 of culture. For example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of about 1.6 x 107TU / mL, or about 1.7 x 107TU / mL, or about 1.8 x 107TU / mL, or about 1.9 x 107TU / mL, or about 2.0 x 107TU / mL of culture medium at day 25 of culture. In one example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 2 x 107TU / mL of culture medium at day 25 of culture. For example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of about 2.1 x 107TU / mL, or about 2.2 x 107TU / mL, or about 2.3 x 107TU / mL, or about 2.4 x 107TU / mL, or about

[0033] 2.5 x 107TU / mL, or about 2.6 x 107TU / mL, or about 2.7 x 107TU / mL, or about 2.8 x 107TU / mL, or about 2.9 x 107TU / mL of culture medium at day 25 of culture.

[0034] In one example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 5 x 106transducing units (TU) / m of culture medium at day 30 of culture. For example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of about 5 x 106TU / mL, or about 7 x 106TU / mL, or about 10 x 106TU / mL of culture medium at day 30 of culture. In another example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 1 x 107TU / mL of culture medium at day 30 of culture. For example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of about 1.1 x 107TU / mL, or about 1.2 x 107TU / mL, or about 1.3 x 107TU / mL, or about 1.4 x 107TU / mL, or about 1.5 x 107TU / mL of culture medium at day 30 of culture. In one example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 1.5 x 107TU / mL of culture medium at day 30 of culture. For example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of about 1.6 x 107TU / mL, or about 1.7 x 107TU / mL, or about 1.8 x 107TU / mL, or about 1.9 x 107TU / mL, or about 2.0 x 107TU / mL of culture medium at day 30 of culture. In one example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 2 x 107TU / mL of culture medium at day 30 of culture. For example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of about 2.1 x 107TU / mL, or about

[0035] 2.2 x 107TU / mL, or about 2.3 x 107TU / mL, or about 2.4 x 107TU / mL, or about 2.5 x 107TU / mL, or about 2.6 x 107TU / mL, or about 2.7 x 107TU / mL, or about 2.8 x 107TU / mL, or about 2.9 x 107TU / mL of culture medium at day 30 of culture.

[0036] In one example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 5 x 106transducing units (TU) / m of culture medium at day 35 of culture. For example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of about 5 x 106TU / mL, or about 7 x 106TU / mL, or about 10 x 106TU / mL of culture medium at day 35 of culture. In another example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 1 x 107TU / mL of culture medium at day 35 of culture. For example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of about 1.1 x 107TU / mL, or about

[0037] 1.2 x 107TU / mL, or about 1.3 x 107TU / mL, or about 1.4 x 107TU / mL, or about 1.5 x 107TU / mL of culture medium at day 35 of culture. In one example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 1.5 x 107TU / mL of culture medium at day 35 of culture. For example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of about 1.6 x 107TU / mL, or about 1.7 x 107TU / mL, or about 1.8 x 107TU / mL, or about 1.9 x 107TU / mL, or about 2.0 x 107TU / mL of culture medium at day 35 of culture. In one example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of at least 2 x 107TU / mL of culture medium at day 35 of culture. For example, the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with a viral infectious titer of about 2.1 x 107TU / mL, or about

[0038] 2.2 x 107TU / mL, or about 2.3 x 107TU / mL, or about 2.4 x 107TU / mL, or about 2.5 x 107TU / mL, or about 2.6 x 107TU / mL, or about 2.7 x 107TU / mL, or about 2.8 x 107TU / mL, or about 2.9 x 107TU / mL of culture medium at day 35 of culture.

[0039] In one example, the stable producer cell line is capable of producing an enveloped virus with an average infectious titre of at least 3.0 x 107on day 2 through day 9 following induction of enveloped virus production. In one example, the stable producer cell line is capable of producing an enveloped virus with an average infectious titre of at least 3.1 x 107on day 2 through day 9 following induction of enveloped virus production. In one example, the stable producer cell line is capable of producing an enveloped virus with an average infectious titre of at least 3.2 x 107on day 2 through day 9 following induction of enveloped virus production. In one example, the stable producer cell line is capable of producing an enveloped virus with an average infectious titre of at least 3.3 x 107on day 2 through day 9 following induction of enveloped virus production. In one example, the stable producer cell line is capable of producing an enveloped virus with an average infectious titre of at least 3.4 x 107on day 2 through day 9 following induction of enveloped virus production. In one example, the stable producer cell line is capable of producing an enveloped virus with an average infectious titre of at least 3.5 x 107on day 2 through day 9 following induction of enveloped virus production. In one example, the stable producer cell line is capable of producing an enveloped virus with an average infectious titre of at least 3.6 x 107on day 2 through day 9 following induction of enveloped virus production. In one example, the stable producer cell line is capable of producing an enveloped virus with an average infectious titre of at least 3.7 x 107on day 2 through day 9 following induction of enveloped virus production. In one example, the stable producer cell line is capable of producing an enveloped virus with an average infectious titre of at least 3.8 x 107on day 2 through day 9 following induction of enveloped virus production. In one example, the stable producer cell line is capable of producing an enveloped virus with an average infectious titre of at least 3.9 x 107on day 2 through day 9 following induction of enveloped virus production.

[0040] In one example, the stable producer cell line is capable of producing an enveloped virus with an average infectious titre of at least 4.0 x 107on day 2 through day 9 following induction of enveloped virus production. In one example, the stable producer cell line is capable of producing an enveloped virus with an average infectious titre of at least 4.1 x 107on day 2 through day 9 following induction of enveloped virus production. In one example, the stable producer cell line is capable of producing an enveloped virus with an average infectious titre of at least 4.2 x 107on day 2 through day 9 following induction of enveloped virus production. In one example, the stable producer cell line is capable of producing an enveloped virus with an average infectious titre of at least 4.3 x 107on day 2 through day 9 following induction of enveloped virus production. In one example, the stable producer cell line is capable of producing an enveloped virus with an average infectious titre of at least 4.4 x 107on day 2 through day 9 following induction of enveloped virus production. In one example, the stable producer cell line is capable of producing an enveloped virus with an average infectious titre of at least 4.5 x 107on day 2 through day 9 following induction of enveloped virus production. In one example, the stable producer cell line is capable of producing an enveloped virus with an average infectious titre of at least 4.6 x 107on day 2 through day 9 following induction of enveloped virus production. In one example, the stable producer cell line is capable of producing an enveloped virus with an average infectious titre of at least 4.7 x 107on day 2 through day 9 following induction of enveloped virus production. In one example, the stable producer cell line is capable of producing an enveloped virus with an average infectious titre of at least 4.8 x 107on day 2 through day 9 following induction of enveloped virus production. In one example, the stable producer cell line is capable of producing an enveloped virus with an average infectious titre of at least 4.9 x 107on day 2 through day 9 following induction of enveloped virus production.

[0041] In one example, the cell line is derived from a producer pool that is capable of producing the enveloped virus with a VSV-G content at least 2-fold greater, or at least 3-fold greater, or at least 4-fold greater, or at least 5-fold greater, or at least 6-fold greater than the VSV-G content of an enveloped virus produced from a stable producer cell line derived from a producer pool that that has not been stably re-transfected with a VSV-G encoding plasmid. For example, the stable producer cell line is derived from a producer pool that is capable of producing an enveloped virus in a suspension cell culture with a normalized VSV-G content of at least 2-fold greater than a stable producer cell line that is derived from a producer pool that has not been stably re-transfected with a VSV-G encoding plasmid. In another example, the stable producer cell line is derived from a producer pool that is capable of producing an enveloped virus in a suspension cell culture with a normalized VSV-G content of at least 2.5-fold greater than a stable producer cell line that is derived from a producer pool that has not been stably re-transfected with a VSV-G encoding plasmid. In a further example, the stable producer cell line is derived from a producer pool that is capable of producing an enveloped virus in a suspension cell culture with a normalized VSV-G content of at least 3-fold greater than a stable producer cell line that is derived from a producer pool that has not been stably re-transfected with a VSV-G encoding plasmid. In one example, the stable producer cell line is derived from a producer pool that is capable of producing an enveloped virus in a suspension cell culture with a normalized VSV-G content of at least 3.5-fold greater than a stable producer cell line that is derived from a producer pool that has not been stably retransfected with a VSV-G encoding plasmid. In another example, the stable producer cell line is derived from a producer pool that is capable of producing an enveloped virus in a suspension cell culture with a normalized VSV-G content of at least 4-fold greater than a stable producer cell line that is derived from a producer pool that has not been stably re-transfected with a VSV-G encoding plasmid. In a further example, the stable producer cell line is derived from a producer pool that is capable of producing an enveloped virus in a suspension cell culture with a normalized VSV-G content of at least 4.5-fold greater than a stable producer cell line that is derived from a producer pool that has not been stably re-transfected with a VSV-G encoding plasmid. In one example, the stable producer cell line is derived from a producer pool that is capable of producing an enveloped virus in a suspension cell culture with a normalized VSV-G content of at least 5-fold greater than a stable producer cell line that is derived from a producer pool that has not been stably re-transfected with a VSV-G encoding plasmid.

[0042] In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.0 x 10’4TU / RNA copies, or at least 1.5 x 10’4TU / RNA copies, or at least 2 x 10’4TU / RNA copies, or at least 2.5 x 10’4TU / RNA copies, or at least 3.0 x 10’4TU / RNA copies, or at least 3.3 x 10’4TU / RNA copies, or at least 3.5 x 10’4TU / RNA copies, or at least 3.7 x 10’4TU / RNA copies. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of between about 1.5 x 10’4TU / RNA copies and 4.0 x 10’4TU / RNA copies. For example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.0 x 10’4TU / RNA copies. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.5 x 10’4TU / RNA copies. In another example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 2 x 10’4TU / RNA copies. In a further example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 2.5 x 10’4TU / RNA copies. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3 x 10’4TU / RNA copies. In another example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.3 x 10’4TU / RNA copies. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.5 x 10’4TU / RNA copies. In a further example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.7 x 10’4TU / RNA copies. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.0 x 10’4TU / RNA copies, or at least 1.5 x 10’4TU / RNA copies, or at least 2 x 10’4TU / RNA copies, or at least 2.5 x 10’4TU / RNA copies, or at least 3.0 x 10’4TU / RNA copies, or at least 3.3 x 10’4TU / RNA copies at day 6 of culture, or at least 3.5 x 10’4TU / RNA copies at day 6 of culture, or at least 3.7 x 10’4TU / RNA copies at day 6 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of between about 1.5 x 10’4TU / RNA copies and 4.0 x 10’4TU / RNA copies at day 6 of culture. For example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.0 x 10’4TU / RNA copies at day 6 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.5 x 10’4TU / RNA copies at day 6 of culture. In another example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 2 x 10’4TU / RNA copies at day 6 of culture. In a further example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 2.5 x 10’4TU / RNA copies at day 6 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3 x 10'4TU / RNA copies at day 6 of culture. In another example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.3 x 10’4TU / RNA copies at day 6 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.5 x 10’4TU / RNA copies at day 6 of culture. In another example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.7 x 10’4TU / RNA copies at day 6 of culture.

[0043] In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.0 x 10’4TU / RNA copies, or at least 1.5 x 10’4TU / RNA copies, or at least 2 x 10’4TU / RNA copies, or at least 2.5 x 10’4TU / RNA copies, or at least 3.0 x 10’4TU / RNA copies, or at least 3.3 x 10’4TU / RNA copies at day 14 of culture, or at least 3.5 x 10’4TU / RNA copies at day 14 of culture, or at least 3.7 x 10’4TU / RNA copies at day 14 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of between about 1.5 x 10’4TU / RNA copies and 4.0 x 10’4TU / RNA copies at day 14 of culture. For example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.0 x 10’4TU / RNA copies at day 14 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.5 x 10’4TU / RNA copies at day 14 of culture. In another example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 2 x 10’4TU / RNA copies at day 14 of culture. In a further example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 2.5 x 10’4TU / RNA copies at day 14 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3 x 10’4TU / RNA copies at day 14 of culture. In another example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.3 x 10’4TU / RNA copies at day 14 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.5 x IO-4TU / RNA copies at day 14 of culture. In another example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.7 x 10’4TU / RNA copies at day 14 of culture.

[0044] In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.0 x 10’4TU / RNA copies, or at least 1.5 x 10’4TU / RNA copies, or at least 2 x 10’4TU / RNA copies, or at least 2.5 x 10’4TU / RNA copies, or at least 3.0 x 10’4TU / RNA copies, or at least 3.3 x 10’4TU / RNA copies at day 20 of culture, or at least 3.5 x 10’4TU / RNA copies at day 20 of culture, or at least 3.7 x 10’4TU / RNA copies at day 20 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of between about 1.5 x 10’4TU / RNA copies and 4.0 x 10’4TU / RNA copies at day 20 of culture. For example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.0 x 10’4TU / RNA copies at day 20 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.5 x 10’4TU / RNA copies at day 20 of culture. In another example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 2 x 10’4TU / RNA copies at day 20 of culture. In a further example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 2.5 x 10’4TU / RNA copies at day 20 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3 x 10’4TU / RNA copies at day 20 of culture. In another example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.3 x 10’4TU / RNA copies at day 20 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.5 x IO-4TU / RNA copies at day 20 of culture. In another example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.7 x 10’4TU / RNA copies at day 20 of culture.

[0045] In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.0 x 10’4TU / RNA copies, or at least 1.5 x 10’4TU / RNA copies, or at least 2 x 10’4TU / RNA copies, or at least 2.5 x 10’4TU / RNA copies, or at least 3.0 x 10’4TU / RNA copies, or at least 3.3 x 10’4TU / RNA copies at day 25 of culture, or at least 3.5 x 10’4TU / RNA copies at day 25 of culture, or at least 3.7 x 10’4TU / RNA copies at day 25 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of between about 1.5 x 10’4TU / RNA copies and 4.0 x 10’4TU / RNA copies at day 25 of culture. For example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.0 x 10’4TU / RNA copies at day 25 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.5 x 10’4TU / RNA copies at day 25 of culture. In another example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 2 x 10’4TU / RNA copies at day 25 of culture. In a further example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 2.5 x 10’4TU / RNA copies at day 25 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3 x 10’4TU / RNA copies at day 25 of culture. In another example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.3 x 10’4TU / RNA copies at day 25 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.5 x IO-4TU / RNA copies at day 25 of culture. In another example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.7 x 10’4TU / RNA copies at day 25 of culture.

[0046] In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.0 x 10’4TU / RNA copies, or at least 1.5 x 10’4TU / RNA copies, or at least 2 x 10’4TU / RNA copies, or at least 2.5 x 10’4TU / RNA copies, or at least 3.0 x 10’4TU / RNA copies, or at least 3.3 x 10’4TU / RNA copies at day 30 of culture, or at least 3.5 x 10’4TU / RNA copies at day 30 of culture, or at least 3.7 x 10’4TU / RNA copies at day 30 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of between about 1.5 x 10’4TU / RNA copies and 4.0 x 10’4TU / RNA copies at day 30 of culture. For example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.0 x 10’4TU / RNA copies at day 30 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.5 x 10’4TU / RNA copies at day 30 of culture. In another example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 2 x 10’4TU / RNA copies at day 30 of culture. In a further example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 2.5 x 10’4TU / RNA copies at day 30 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3 x 10’4TU / RNA copies at day 30 of culture. In another example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.3 x 10’4TU / RNA copies at day 30 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.5 x IO-4TU / RNA copies at day 30 of culture. In another example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.7 x 10’4TU / RNA copies at day 30 of culture.

[0047] In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.0 x 10’4TU / RNA copies, or at least 1.5 x 10’4TU / RNA copies, or at least 2 x 10’4TU / RNA copies, or at least 2.5 x 10’4TU / RNA copies, or at least 3.0 x 10’4TU / RNA copies, or at least 3.3 x 10’4TU / RNA copies at day 35 of culture, or at least 3.5 x 10’4TU / RNA copies at day 35 of culture, or at least 3.7 x 10’4TU / RNA copies at day 35 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of between about 1.5 x 10’4TU / RNA copies and 3.5 x 10’4TU / RNA copies at day 35 of culture. For example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.0 x 10’4TU / RNA copies at day 35 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.5 x 10’4TU / RNA copies at day 35 of culture. In another example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 2 x 10’4TU / RNA copies at day 35 of culture. In a further example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 2.5 x 10’4TU / RNA copies at day 35 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3 x 10’4TU / RNA copies at day 35 of culture. In another example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.3 x 10’4TU / RNA copies at day 35 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.5 x IO-4TU / RNA copies at day 35 of culture. In another example, the stable producer cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 3.7 x 10’4TU / RNA copies at day 35 of culture. In one example, the stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1011copies / mL, or at least 5.0 x 1011copies / mL, or at least 1.0 x 1012copies / mL, or at least 5.0 x 1012copies / mL, or at least 1.0 x 1013copies / mL cell culture medium. For example, the stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1011copies / mL. In another example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 5.0 x 1011copies / mL. In a further example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1012copies / mL. In one example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 5.0 x 1012copies / mL. In another example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1013copies / mL.

[0048] In one example, the stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1011copies / mL, or at least 5.0 x 1011copies / mL, or at least 1.0 x 1012copies / mL, or at least 5.0 x 1012copies / mL, or at least 1.0 x 1013copies / mL cell culture medium at day 6 of culture. For example, the stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1011copies / mL at day 6 of culture. In another example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 5.0 x 1011copies / mL at day 6 of culture. In a further example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1012copies / mL at day 6 of culture. In one example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 5.0 x 1012copies / mL at day 6 of culture. In another example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1013copies / mL at day 6 of culture.

[0049] In one example, the stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1011copies / mL, or at least 5.0 x 1011copies / mL, or at least 1.0 x 1012copies / mL, or at least 5.0 x 1012copies / mL, or at least 1.0 x 1013copies / mL cell culture medium at day 14 of culture. For example, the stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1011copies / mL at day 14 of culture. In another example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 5.0 x 1011copies / mL at day 14 of culture. In a further example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1012copies / mL at day 14 of culture. In one example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 5.0 x 1012copies / mL at day 14 of culture. In another example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1013copies / mL at day 14 of culture.

[0050] In one example, the stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1011copies / mL, or at least 5.0 x 1011copies / mL, or at least 1.0 x 1012copies / mL, or at least 5.0 x 1012copies / mL, or at least 1.0 x 1013copies / mL cell culture medium at day 20 of culture. For example, the stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1011copies / mL at day 20 of culture. In another example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 5.0 x 1011copies / mL at day 20 of culture. In a further example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1012copies / mL at day 20 of culture. In one example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 5.0 x 1012copies / mL at day 20 of culture. In another example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1013copies / mL at day 20 of culture.

[0051] In one example, the stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1011copies / mL, or at least 5.0 x 1011copies / mL, or at least 1.0 x 1012copies / mL, or at least 5.0 x 1012copies / mL, or at least 1.0 x 1013copies / mL cell culture medium at day 25 of culture. For example, the stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1011copies / mL at day 25 of culture. In another example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 5.0 x 1011copies / mL at day 25 of culture. In a further example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1012copies / mL at day 25 of culture. In one example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 5.0 x 1012copies / mL at day 25 of culture. In another example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1013copies / mL at day 25 of culture.

[0052] In one example, the stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1011copies / mL, or at least 5.0 x 1011copies / mL, or at least 1.0 x 1012copies / mL, or at least 5.0 x 1012copies / mL, or at least 1.0 x 1013copies / mL cell culture medium at day 30 of culture. For example, the stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1011copies / mL at day 30 of culture. In another example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 5.0 x 1011copies / mL at day 30 of culture. In a further example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1012copies / mL at day 30 of culture. In one example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 5.0 x 1012copies / mL at day 30 of culture. In another example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1013copies / mL at day 30 of culture.

[0053] In one example, the stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1011copies / mL, or at least 5.0 x 1011copies / mL, or at least 1.0 x 1012copies / mL, or at least 5.0 x 1012copies / mL, or at least 1.0 x 1013copies / mL cell culture medium at day 35 of culture. For example, the stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1011copies / mL at day 35 of culture. In another example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 5.0 x 1011copies / mL at day 35 of culture. In a further example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1012copies / mL at day 35 of culture. In one example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 5.0 x 1012copies / mL at day 35 of culture. In another example, stable producer cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1013copies / mL at day 35 of culture.

[0054] In one example, the stable producer cell line has a viable cell density of at least about 1 x 105cells / mL of culture medium at day 15 of culture. For example, the stable producer cell line has a viable cell density of at least about 1 x 106cells / mL of culture medium at day 15 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1.5 x 106cells / mL, or about 2 x 106cells / mL, or about 5 x 106cells / mL, or about 7 x 106cells / mL, or about 10 x 106cells / mL of culture medium at day 15 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1 x 107cells / mL of culture medium at day 15 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1.1 x 107cells / mL, or about 1.2 x 107cells / mL, or about 1.3 x 107cells / mL, or about 1.4 x 107cells / mL, or about 1.5 x 107cells / mL of culture medium at day 15 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1.5 x 107cells / mL of culture medium at day 15 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1.5 x 107cells / mL, or about 1.6 x 107cells / mL, or about 1.7 x 107cells / mL, or about 1.8 x 107cells / mL, or about 1.9 x 107cells / mL, or about 2.0 x 107cells / mL of culture medium at day 15 of culture. In one example, the stable producer cell line has a viable cell density of at least about 2.0 x 107cells / mL of culture medium at day 15 of culture.

[0055] In one example, the stable producer cell line has a viable cell density of at least about 1 x 105cells / mL of culture medium at day 20 of culture. For example, the stable producer cell line has a viable cell density of at least about 1 x 106cells / mL of culture medium at day 20 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1.5 x 106cells / mL, or about 2 x 106cells / mL, or about 5 x 106cells / mL, or about 7 x 106cells / mL, or about 10 x 106cells / mL of culture medium at day 20 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1 x 107cells / mL of culture medium at day 20 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1.1 x 107cells / mL, or about 1.2 x 107cells / mL, or about 1.3 x 107cells / mL, or about 1.4 x 107cells / mL, or about 1.5 x 107cells / mL of culture medium at day 20 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1.5 x 107cells / mL of culture medium at day 20 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1.5 x 107cells / mL, or about 1.6 x 107cells / mL, or about 1.7 x 107cells / mL, or about 1.8 x 107cells / mL, or about 1.9 x 107cells / mL, or about 2.0 x 107cells / mL of culture medium at day 20 of culture. In one example, the stable producer cell line has a viable cell density of at least about 2.0 x 107cells / mL of culture medium at day 20 of culture.

[0056] In one example, the stable producer cell line has a viable cell density of at least about 1 x 105cells / mL of culture medium at day 25 of culture. For example, the stable producer cell line has a viable cell density of at least about 1 x 106cells / mL of culture medium at day 25 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1.5 x 106cells / mL, or about 2 x 106cells / mL, or about 5 x 106cells / mL, or about 7 x 106cells / mL, or about 10 x 106cells / mL of culture medium at day 25 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1 x 107cells / mL of culture medium at day 25 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1.1 x 107cells / mL, or about 1.2 x 107cells / mL, or about 1.3 x 107cells / mL, or about 1.4 x 107cells / mL, or about 1.5 x 107cells / mL of culture medium at day 25 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1.5 x 107cells / mL of culture medium at day 25 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1.5 x 107cells / mL, or about 1.6 x 107cells / mL, or about 1.7 x 107cells / mL, or about 1.8 x 107cells / mL, or about 1.9 x 107cells / mL, or about 2.0 x 107cells / mL of culture medium at day 25 of culture. In one example, the stable producer cell line has a viable cell density of at least about 2.0 x 107cells / mL of culture medium at day 25 of culture.

[0057] In one example, the stable producer cell line has a viable cell density of at least about 1 x 105cells / mL of culture medium at day 30 of culture. For example, the stable producer cell line has a viable cell density of at least about 1 x 106cells / mL of culture medium at day 30 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1.5 x 106cells / mL, or about 2 x 106cells / mL, or about 5 x 106cells / mL, or about 7 x 106cells / mL, or about 10 x 106cells / mL of culture medium at day 30 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1 x 107cells / mL of culture medium at day 30 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1.1 x 107cells / mL, or about 1.2 x 107cells / mL, or about 1.3 x 107cells / mL, or about 1.4 x 107cells / mL, or about 1.5 x 107cells / mL of culture medium at day 30 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1.5 x 107cells / mL of culture medium at day 30 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1.5 x 107cells / mL, or about 1.6 x 107cells / mL, or about 1.7 x 107cells / mL, or about 1.8 x 107cells / mL, or about 1.9 x 107cells / mL, or about 2.0 x 107cells / mL of culture medium at day 30 of culture. In one example, the stable producer cell line has a viable cell density of at least about 2.0 x 107cells / mL of culture medium at day 30 of culture.

[0058] In one example, the stable producer cell line has a viable cell density of at least about 1 x 105cells / mL of culture medium at day 35 of culture. For example, the stable producer cell line has a viable cell density of at least about 1 x 106cells / mL of culture medium at day 35 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1.5 x 106cells / mL, or about 2 x 106cells / mL, or about 5 x 106cells / mL, or about 7 x 106cells / mL, or about 10 x 106cells / mL of culture medium at day 35 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1 x 107cells / mL of culture medium at day 35 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1.1 x 107cells / mL, or about 1.2 x 107cells / mL, or about 1.3 x 107cells / mL, or about 1.4 x 107cells / mL, or about 1.5 x 107cells / mL of culture medium at day 35 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1.5 x 107cells / mL of culture medium at day 35 of culture. In one example, the stable producer cell line has a viable cell density of at least about 1.5 x 107cells / mL, or about 1.6 x 107cells / mL, or about 1.7 x 107cells / mL, or about 1.8 x 107cells / mL, or about 1.9 x 107cells / mL, or about 2.0 x 107cells / mL of culture medium at day 35 of culture. In one example, the stable producer cell line has a viable cell density of at least about 2.0 x 107cells / mL of culture medium at day 35 of culture.

[0059] In one example, the stable producer cell line has a viability of at least 70% at day 15 of culture. For example, the stable producer cell line has a viability of about 70%, or about 75% or about 80% at day 15 of culture. In one example, the stable producer cell line has a viability of at least 75% at day 15 of culture. In another example, the stable producer cell line has a viability of at least 80% at day 15 of culture. For example, the stable producer cell line has a viability of about 80% or about 85% or about 90% at day 15 of culture. In one example, the stable producer cell line has a viability of about 80% at day 15 of culture. For example, a viability of about 81%, or about 82%, or about 83%, or about 84%. In another example, the stable producer cell line has a viability of about 85% at day 15 of culture. For example, a viability of about 86%, or about 87%, or about 88%, or about 89% at day 15 of culture. In a further example, the cell line has a viability of about 90% at day 15 of culture. In one example, the cell line has a viability of at least 90% at day 15 of culture. For example, the cell line has a viability of about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95% at day 15 of culture. In one example, the cell line has a viability of at least 95% at day 15 of culture. For example, the cell line has a viability of about 96%, or about 97%, or about 98%, or about 99% at day 15 of culture.

[0060] In one example, the cell line has a viability of at least 70% at day 20 of culture. For example, the cell line has a viability of about 70%, or about 75% or about 80% at day 20 of culture. In one example, the cell line has a viability of at least 75% at day 20 of culture. In another example, the cell line has a viability of at least 80% at day 20 of culture. For example, the cell line has a viability of about 80% or about 85% or about 90% at day 20 of culture. In one example, the cell line has a viability of about 80% at day 20 of culture. For example, a viability of about 81%, or about 82%, or about 83%, or about 84%. In another example, the cell line has a viability of about 85% at day 20 of culture. For example, a viability of about 86%, or about 87%, or about 88%, or about 89% at day 20 of culture. In a further example, the cell line has a viability of about 90% at day 20 of culture. In one example, the cell line has a viability of at least 90% at day 20 of culture. For example, the cell line has a viability of about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95% at day 20 of culture. In one example, the cell line has a viability of at least 95% at day 20 of culture. For example, the cell line has a viability of about 96%, or about 97%, or about 98%, or about 99% at day 20 of culture.

[0061] In one example, the cell line has a viability of at least 70% at day 25 of culture. For example, the cell line has a viability of about 70%, or about 75% or about 80% at day 25 of culture. In one example, the cell line has a viability of at least 75% at day 25 of culture. In another example, the cell line has a viability of at least 80% at day 25 of culture. For example, the cell line has a viability of about 80% or about 85% or about 90% at day 25 of culture. In one example, the cell line has a viability of about 80% at day 25 of culture. For example, a viability of about 81%, or about 82%, or about 83%, or about 84%. In another example, the cell line has a viability of about 85% at day 25 of culture. For example, a viability of about 86%, or about 87%, or about 88%, or about 89% at day 25 of culture. In a further example, the cell line has a viability of about 90% at day 25 of culture. In one example, the cell line has a viability of at least 90% at day 25 of culture. For example, the cell line has a viability of about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95% at day 25 of culture. In one example, the cell line has a viability of at least 95% at day 25 of culture. For example, the cell line has a viability of about 96%, or about 97%, or about 98%, or about 99% at day 25 of culture.

[0062] In one example, the cell line has a viability of at least 70% at day 30 of culture. For example, the cell line has a viability of about 70%, or about 75% or about 80% at day 30 of culture. In one example, the cell line has a viability of at least 75% at day 30 of culture. In another example, the cell line has a viability of at least 80% at day 30 of culture. For example, the cell line has a viability of about 80% or about 85% or about 90% at day 30 of culture. In one example, the cell line has a viability of about 80% at day 30 of culture. For example, a viability of about 81%, or about 82%, or about 83%, or about 84%. In another example, the cell line has a viability of about 85% at day 30 of culture. For example, a viability of about 86%, or about 87%, or about 88%, or about 89% at day 30 of culture. In a further example, the cell line has a viability of about 90% at day 30 of culture. In one example, the cell line has a viability of at least 90% at day 30 of culture. For example, the cell line has a viability of about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95% at day 30 of culture. In one example, the cell line has a viability of at least 95% at day 30 of culture. For example, the cell line has a viability of about 96%, or about 97%, or about 98%, or about 99% at day 30 of culture.

[0063] In one example, the cell line has a viability of at least 70% at day 35 of culture. For example, the cell line has a viability of about 70%, or about 75% or about 80% at day 35 of culture. In one example, the cell line has a viability of at least 75% at day 35 of culture. In another example, the cell line has a viability of at least 80% at day 35 of culture. For example, the cell line has a viability of about 80% or about 85% or about 90% at day 35 of culture. In one example, the cell line has a viability of about 80% at day 35 of culture. For example, a viability of about 81%, or about 82%, or about 83%, or about 84%. In another example, the cell line has a viability of about 85% at day 35 of culture. For example, a viability of about 86%, or about 87%, or about 88%, or about 89% at day 35 of culture. In a further example, the cell line has a viability of about 90% at day 35 of culture. In one example, the cell line has a viability of at least 90% at day 35 of culture. For example, the cell line has a viability of about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95% at day 35 of culture. In one example, the cell line has a viability of at least 95% at day 35 of culture. For example, the cell line has a viability of about 96%, or about 97%, or about 98%, or about 99% at day 35 of culture.

[0064] In one example, the virus infectious titer yield is increased by at least 2% or 3% or 4% or 5% or 10% or 15% or 20% or 30% or 40% or 50% compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid. For example, the virus infectious titer yield is increased by at least 2% compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid. In one example, the virus infectious titer yield is increased by at least 3% compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid. In another example, the virus infectious titer yield is increased by at least 4% compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid. In a further example, the virus infectious titer yield is increased by at least 5% compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid. In one example, the virus infectious titer yield is increased by at least 10% compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid. In one example, the virus infectious titer yield is increased by at least 15% compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid. In one example, the virus infectious titer yield is increased by at least 20% compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid. In one example, the virus infectious titer yield is increased by at least 30% compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid. In one example, the virus infectious titer yield is increased by at least 40% compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid. In one example, the virus infectious titer yield is increased by at least 50% compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid.

[0065] The present disclosure also provides a method of producing a stable producer cell line stably expressing vesicular stomatitis virus G protein (VSV-G), wherein the cell line is capable of producing an enveloped virus in a suspension cell culture.

[0066] The present disclosure provides a method of producing a stable producer cell line stably expressing vesicular stomatitis virus G protein (VSV-G), wherein the cell line is capable of producing an enveloped virus in a suspension cell culture, the method comprising stably integrating a VSV-G encoding plasmid into a stable producer cell line, wherein the stable producer cell line is derived from a packaging cell line transfected with VSV-G.

[0067] The present disclosure provides a method of producing a stable producer cell line stably expressing vesicular stomatitis virus G protein (VSV-G), wherein the cell line is capable of producing an enveloped virus in a suspension cell culture, the method comprising stably integrating a plasmid comprising a gene of interest and a VSV-G encoding plasmid into a packaging cell line, wherein the packaging cell line is transfected with VSV-G.

[0068] The present disclosure provides a method of producing a VSV-G re-transfected packaging cell line, the method comprising stably integrating a VSV-G encoding plasmid into a packaging cell line, wherein the packaging cell line is transfected with VSV-G.

[0069] The present disclosure provides a method of producing a stable producer cell line stably expressing VSV-G, wherein the cell line is capable of producing an enveloped virus in a suspension cell culture, the method comprising stably integrating a plasmid comprising a gene of interest into a VSV-G re-transfected packaging cell line.

[0070] The present disclosure provides a packaging cell line transfected with vesicular stomatitis virus G protein (VSV-G), that has been stably re-transfected with a VSV-G encoding plasmid. The present disclosure further provides a method of producing an enveloped virus in a suspension cell culture, the method comprising culturing a stable producer suspension cell line, wherein the stable producer suspension cell line is transfected with a VSV-G encoding plasmid and wherein the stable producer cell line is derived from a packaging cell line transfected with VSV-G.

[0071] In one example, the suspension cell culture is operated in a batch, fed batch, continuous, semi-continuous, or perfusion mode. In one example, the cell culture is operated in batch mode. In another example, the cell culture is operated in fed batch mode. In a further example, the cell culture is operated in semi-continuous mode. In another example, the cell culture is operated in perfusion mode. In one example, the cell culture is operated in batch and perfusion mode. For example, the cell culture is initially operated in batch mode and subsequently operated in perfusion mode.

[0072] In one example, the suspension cell culture has a volume of greater than about 1 L, about 2 L, about 5 L, about 10 L, about 50 L, about 100 L, about 500 L, about 1000 L, about 5,000 L, about 10,000 L, or about 15,000 L. For example, the suspension cell culture has a volume of between about 1 L and 1000 L. In one example, the suspension cell culture has a volume of about 1 L. In another example, the suspension cell culture has a volume of about 5 L. In a further example, the suspension cell culture has a volume of about 10 L. In one example, the suspension cell culture has a volume of about 50 L. In another example, the suspension cell culture has a volume of about 100 L. In a further example, the suspension cell culture has a volume of about 500 L. In one example, the suspension cell culture has a volume of about 1000 L. In another example, the suspension cell culture has a volume of about 5000 L. In another example, the suspension cell culture has a volume of about 10,000 L. In another example, the suspension cell culture has a volume of about 15,000 L.

[0073] In one example, the suspension cell culture is operated with a dissolved carbon dioxide (CO2) level of between 4% and 8%. For example, the suspension cell culture is operated with about 4% CO2. In another example, the suspension cell culture is operated with about 5% CO2. In a further example, the suspension cell culture is operated with about 6% CO2. In a further example, the suspension cell culture is operated with about 7% CO2. In a further example, the suspension cell culture is operated with about 8% CO2.

[0074] In one example, the suspension cell culture is operated at a pH of between 6.0 and 8.0. In one example, the suspension cell culture is at a pH of between about 6.5 and 7.5. For example, the pH is between about 6.90 and about 7.3. In one example, the pH is about 7.1. In one example, the pH is about 6.5. In one example, the pH is about 6.6. In one example, the pH is about 6.7. In one example, the pH is about 6.8. In one example, the pH is about 6.9. In one example, the pH is about 7.0. In one example, the pH is about 7.1. In one example, the pH is about 7.2. In one example, the pH is about 7.3. In one example, the pH is about 7.4. In one example, the pH is about 7.5.

[0075] In one example, the suspension cell culture is operated at a temperature of between about 35 °C and 39 °C. For example, the suspension cell culture is at a temperature of about 35 °C, or about 35.5 °C, or about 36 °C, or about 36.5 °C, or about 37 °C, or about 37.5 °C, or about 38 °C, or about 38.5 °C, or about 39 °C. In one example, the suspension cell culture is at a temperature of between about 36.5 °C and about 37.5 °C. For example, the suspension cell culture is at a temperature of about 37.0 °C. In one example, the suspension cell culture is at a temperature of between about 38 °C and about 39 °C. For example, the suspension cell culture is at a temperature of about 38.5 °C.

[0076] In one example, the suspension cell culture is at a pH of between 6.0 and 8.0 and / or at a temperature of between 35-39 °C. In one example, the suspension cell culture is at a pH of between 6.0 and 8.0 and / or at a temperature of between 37-38.5 °C. For example, the suspension cell culture is at a pH of between about 6.8 and about 7.1 and / or at a temperature of between about 37 °C and about 38.5 °C. In one example, the suspension cell culture is at a pH of between about 6.8 and about 7.1 and at a temperature of between about 37 °C and about 38.5 °C. In one example, the suspension cell culture is at a pH of about 6.9 to about 7.0 and at a temperature of about 37.0 °C.

[0077] It will be apparent to the skilled person from the disclosure herein that cell lines of the disclosure express a tetracycline-suppressible gene expression system or “Tet- OFF” system. The skilled person will understand that this refers to cell line that stably expresses tetracycline-controlled transactivator (tTA) such that the presence of tetracycline or a derivative thereof (e.g., doxycycline) silences transcription from tetracycline responsive element promoters.

[0078] The skilled person will understand that initially cells are cultured in the presence of tetracycline or a derivative thereof to suppress virus production but allow cell growth or expansion of the cell line. In one example, the suspension cell line is initially cultured in a cell culture medium comprising a sufficient amount of tetracycline or a derivative thereof to suppress production of the enveloped virus and allow expansion of the suspension cell line. In one example, the amount of tetracycline or a derivative thereof in the cell culture medium required to suppress virus production is at least 0.01 ng / mL.

[0079] It will be apparent to the skilled person that the tetracycline or a derivative thereof may be added to the cell culture medium as a single bolus feed, as multiple feeds, or continuously over the duration of the culture. In one example, the method comprises reducing the concentration of tetracycline or a derivative thereof in the cell culture medium such that production of the enveloped virus is induced. It will be apparent to the skilled person that withdrawal (or a reduction in the concentration) of tetracycline or a derivative thereof results in expression of the enveloped virus. For example, the concentration of tetracycline or derivative thereof in the cell culture medium is reduced to a concentration of 0.1 ng / mL or less, 0.5 ng / mL or less, or 1.0 ng / mL or less.

[0080] In one example, the method further comprises selecting a cell line capable of producing the enveloped virus at a high infectious titer and viability for at least 15 days. For example, the method further comprises selecting a cell line capable of producing the enveloped virus at an infectious titer of at least 5 x 106TU / mL of culture medium and / or a viability of at least 75% for at least 15 days and / or a viable cell density of at least 1 x 107cells / mL of culture medium.

[0081] In one example, the method further comprises purifying the enveloped virus from the suspension cell culture. For example, the enveloped virus is harvested from the suspension cell culture fluid.

[0082] In one example, the enveloped virus is harvested from the suspension cell culture at least 7 days following induction of enveloped virus production. For example, the enveloped virus is harvested from the suspension cell culture at least 14 days following induction of enveloped virus production.

[0083] In one example, the enveloped virus is harvested from the suspension cell culture daily following induction of enveloped virus production. For example, the enveloped virus is harvested daily beginning on day 1 following induction of enveloped virus production. In another example, the enveloped virus is harvested daily beginning on day 2 following induction of enveloped virus production. In other examples, the enveloped virus is harvested daily beginning on day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, or day 20, following induction of enveloped virus production.

[0084] In another example, cell culture media containing virus particles is removed and discarded (i.e., not harvested for downstream processing) following induction of enveloped virus production, for 1 or more days prior to the first day of harvesting. For example, cell culture media containing virus particles is removed and discarded on day

[0085] 1 following induction of enveloped virus production, and harvesting commences on day

[0086] 2 after induction of enveloped virus production. In another example, cell culture media containing virus particles is removed and discarded on days 1 and 2 following induction of enveloped virus production, and harvesting commences on day 3 after induction of enveloped virus production. In other examples, cell culture media containing virus particles is removed and discarded on days 1 to 3, days 1 to 4, days 1 to 5, days 1 to 6, days 1 to 7, days 1 to 8, days 1 to 9, days 1 to 10, days 1 to 11, days 1 to 12, days 1 to 13, days 1 to 14, days 1 to 15, days 1 to 16, days 1 to 17, days 1 to 18, or days 1 to 19, following induction of enveloped virus production, and harvesting commences on day 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, respectively, after induction of enveloped virus production.

[0087] In one example, harvesting commences on the first day following induction in which infectious titre is predicted to be greater than about 5.0 x 105TU / mL. In other examples, harvesting commences on the first day following induction in which infectious titre is predicted to be greater than about 6.0 x 105TU / mL, greater than about 7.0 x 105TU / mL, greater than about 8.0 x 105TU / mL, greater than about 9.0 x 105TU / mL, greater than about 1.0 x 106TU / mL, greater than about 2.0 x 106TU / mL, greater than about 3.0 x 106TU / mL, greater than about 4.0 x 106TU / mL, greater than about 5.0 x 106TU / mL, greater than about 6.0 x 106TU / mL, greater than about 7.0 x 106TU / mL, greater than about 8.0 x 106TU / mL, greater than about 9.0 x 106TU / mL, greater than about 1.0 x 107TU / mL, greater than about 2.0 x 107TU / mL, greater than about 3.0 x 107TU / mL, greater than about 4.0 x 107TU / mL, or greater than about 5.0 x 107TU / mL. In examples, cell culture media containing virus particles is removed and discarded on days prior to commencement of harvesting. After the first day on which infectious titre is predicted to be greater than about 5.0 x 105TU / mL, greater than about 6.0 x 105TU / mL, greater than about 7.0 x 105TU / mL, greater than about 8.0 x 105TU / mL, greater than about 9.0 x 105TU / mL, greater than about 1.0 x 106TU / mL, greater than about 2.0 x 106TU / mL, greater than about 3.0 x 106TU / mL, greater than about 4.0 x 106TU / mL, greater than about 5.0 x 106TU / mL, greater than about 6.0 x 106TU / mL, greater than about 7.0 x 106TU / mL, greater than about 8.0 x 106TU / mL, greater than about 9.0 x 106TU / mL, greater than about 1.0 x 107TU / mL, greater than about 2.0 x 107TU / mL, greater than about 3.0 x 107TU / mL, greater than about 4.0 x 107TU / mL, or greater than about 5.0 x 107TU / mL, the enveloped virus is harvested on each subsequent day for a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 harvest days.

[0088] In one example, harvesting commences on the first day following induction in which bulk VCN / cell is predicted to be greater than about 0.1. In other examples, harvesting commences on the first day following induction in which bulk VCN / cell is predicted to be greater than about 0.2, greater than about 0.3, greater than about 0.4, greater than about 0.5, greater than about 0.6, greater than about 0.7, greater than about 0.8, greater than about 0.9, greater than about 1.0, greater than about 1.1, greater than about 1.2, greater than about 1.3, greater than about 1.4, or greater than about 1.5. In examples, cell culture media containing virus particles is removed and discarded on days prior to commencement of harvesting. After the first day on which bulk VCN / cell is predicted to be greater than about 0.1, greater than about 0.2, greater than about 0.3, greater than about 0.4, greater than about 0.5, greater than about 0.6, greater than about 0.7, greater than about 0.8, greater than about 0.9, greater than about 1.0, greater than about 1.1, greater than about 1.2, greater than about 1.3, greater than about 1.4, or greater than about 1.5, the enveloped virus is harvested on each subsequent day for a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 harvest days. Methods of purifying the enveloped virus from the suspension cell culture will be apparent to the skilled person and / or are described herein. In one example, purifying the enveloped virus comprises one or more steps selected from the group consisting of clarification filtration, anion exchange chromatography, concentration and diafiltration.

[0089] In one example, a method of the disclosure additionally comprises performing sterile filtration. For example, the sterile filtration is performed prior to concentrating and diafiltering the eluted virus. In an alternative example, the sterile filtration is performed after concentrating and diafiltering the eluted virus.

[0090] In one example, the method additionally comprising formulating the purified enveloped virus into a pharmaceutical formulation or into a solution suitable for infecting a cell.

[0091] The present disclosure also provides a purified enveloped virus produced by the method described herein.

[0092] An exemplary enveloped virus is a retrovirus. For example, the retrovirus is a lentivirus. For example, the lentivirus is HIV or a derivative thereof.

[0093] In one example, the cell is a GPR, GPRG, GPRT, GPRGT or GPRTG cell or a derivative thereof. For example, the cell is a GPRG or GPRTG cell.

[0094] The present disclosure further provides a method of transducing a cell with an enveloped virus in a cell culture, the method comprising contacting the cells with the enveloped virus, wherein the enveloped virus is produced from the stable producer cell line of the disclosure or is produced according to the method of the disclosure.

[0095] The present disclosure also provides a method of increasing integrated vector copy number (VCN) of a cell transduced with an enveloped virus in a cell culture, the method comprising contacting the cells with the enveloped virus, wherein the enveloped virus is produced from the stable producer cell line of the disclosure or is produced according to the method of the disclosure. In one example, the method results in a transduction efficiency of at least 10% 14 days post transduction. For example, the method results in a transduction efficiency of at least 11%, or at least 12%, or at least 13%, or at least 14%, or at least 15%, or at least 16%, or at least 17%, or at least 18% 14 days post transduction. In one example, the method results in a transduction efficiency of at least 11% 14 days post transduction. In another example, the method results in a transduction efficiency of at least 12% 14 days post transduction. In a further example, the method results in a transduction efficiency of at least 13% 14 days post transduction. In one example, the method results in a transduction efficiency of at least 14% 14 days post transduction. In another example, the method results in a transduction efficiency of at least 15% 14 days post transduction. In a further example, the method results in a transduction efficiency of at least 16% 14 days post transduction. In one example, the method results in a transduction efficiency of at least 17% 14 days post transduction. In another example, the method results in a transduction efficiency of at least 18% 14 days post transduction.

[0096] In one example, the method results in an increase in transduction efficiency by at least 2% 14 days post transduction compared to a cell line that has not been stably retransfected with a VSV-G encoding plasmid. For example, the method results in an increase in transduction efficiency by at least 3% 14 days post transduction compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid. In one example, the method results in an increase in transduction efficiency by at least 4% 14 days post transduction compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid. In one example, the method results in an increase in transduction efficiency by at least 5% 14 days post transduction compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid. For example, the method results in an increase in transduction efficiency by at least 6% 14 days post transduction compared to a cell line that has not been stably re-transfected with a VSV- G encoding plasmid. In one example, the method results in an increase in transduction efficiency by at least 7% 14 days post transduction compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid. In another example, the method results in an increase in transduction efficiency by at least 8% 14 days post transduction compared to a cell line that has not been stably re-transfected with a VSV- G encoding plasmid. In a further example, the method results in an increase in transduction efficiency by at least 9% 14 days post transduction compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid. In one example, the method results in an increase in transduction efficiency by at least 10% 14 days post transduction compared to a cell line that has not been stably re-transfected with a VSV- G encoding plasmid.

[0097] In one example, the cell culture has a multiplicity of infection (MOI) of at least 0.1. For example, the cell culture has a MOI of at least 0.3. In another example, the cell culture has a MOI of at least 1.0.

[0098] In one example, the method results in a vector copy number (VCN) of at least 0.1 copies / cell 14 days post transduction of the cell 14 days post transduction of the cell at MOI of 1. For example, the method results in a VCN of about 0.2 copies / cell 14 days post transduction of the cell at MOI of 1. In another example, the method results in a VCN of about 0.25 copies / cell 14 days post transduction of the cell at MOI of 1. In a further example, the method results in a VCN of about 0.3 copies / cell 14 days post transduction of the cell at MOI of 1. In one example, the method results in a VCN of about 0.35 copies / cell 14 days post transduction of the cell at MOI of 1.

[0099] In one example, the method results in an increase in VCN of at least 0.05 copies / cell 14 days post transduction of the cell 14 days post transduction of the cell at MOI of 1 compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid. For example, the method results in an increase in VCN of at least 0.1 copies / cell 14 days post transduction of the cell 14 days post transduction of the cell at MOI of 1 compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid. In another example, the method results in an increase in VCN of at least 0.15 copies / cell 14 days post transduction of the cell 14 days post transduction of the cell at MOI of 1 compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid. In a further example, the method results in an increase in VCN of at least 0.2 copies / cell 14 days post transduction of the cell 14 days post transduction of the cell at MOI of 1 compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid. In one example, the method results in an increase in VCN of at least 0.25 copies / cell 14 days post transduction of the cell 14 days post transduction of the cell at MOI of 1 compared to a cell line that has not been stably re-transfected with a VSV-G encoding plasmid.

[0100] In one example, the cell is a hematopoietic stem cell (HSC) or a hematopoietic progenitor cell (HPC). For example, the HSC or HPC is a CD34 positive (CD34+) cell. In one example, the cell is a CD34+HSC. In another example, the cell is a CD34+HPC.

[0101] The present disclosure also provides stable producer cell lines re-transfected with VSV-G that are stable for at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, or at least 70 days. The present disclosure also provides methods of producing an enveloped virus in a suspension cell culture, the method comprising culturing a VSV-G re-transfected stable producer cell line for at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, or at least 70 days.

[0102] BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 is a series of graphical representations showing stable GPRGs and GPRTGs producer pools. Average integrated copy number per cell of the transfer vector and ble resistance vector in the (A) GPRGs and (B) GPRTGs producer pools. Infectious titers produced by established stable (C) GPRGs and (D) GPRTGs producer pools. The error bars are representing the assay standard deviation, dpi = days post induction.

[0104] Figure 2 is a series of graphical representations showing lentivirus production using stable GPRGs and GPRTGs producer pools and transduction of human CD34+cells. (A) VCD (solid lines) and viability (dotted lines) of each stable producer pool during production in semi -perfusion mode. (B) Normalized LV VSV-G content in the harvest collected at 3 and 7 dpi determined using a VSV-G ELISA. (C) Proportion of GFP+transduced CD34+cells at MOIs of 0.1, 0.3 and 1.0 determined using a flow cytometer 14 days post transduction. Statistical analysis was performed using a two-way ANOVA. p values: parental GPRTGs vs. GPRTGs.p VSV-G = 0.057; parental GPRGs vs. GPRGs.p VSV-G <0.001. (D) Bulk VCN of transduced CD34+cells at MOIs of 0.1, 0.3 and 1.0 determined 14 days post transduction by ddPCR. Values are shown as mean and the error bars are representing the standard deviation. Statistical analysis was performed using a two-way ANOVA. p values: parental GPRTGs vs. GPRTGs_pVSV- G = 0.042; parental GPRGs vs. GPRGs_pVSV-G <0.001. dpi = days post induction; MOI = multiplicity of infection; VCN = vector copy number

[0105] Figure 3 is a series of graphical representations showing screening and evaluation of monoclonal stable producer clones isolated from stable GPRGs, GPRTGs, GPRGs_pVSV-G and GPRTGs_pVSV-G producer pools. (A) Infectious titers produced by isolated stable producer clones in the supernatant collected at 6 days post induction (dpi). (B) Cell specific productivities based on viable cell densities and infectious titers determined at 6 dpi. Integrated copy numbers of the transfer and ble resistance vector in (C) stable GPRTGs and GPRTGs_pVSV-G producer pools and (D) GPRGs and GPRGs_pVSV-G producer pools. Functional stability of the selected top clones for (E) parental GPRTG (F) GPRG (G) GPRTGs_pVSV-G and (H) GPRGs_pVSV-G after continuous passaging. Figure 4 is a series of graphical representations showing assessment of four stable producer clones in 5 L perfusion stirred-tank bioreactor set-up and purification of 20 L virus containing harvest. (A) Viable cell densities for LV production in 5 L perfusion bioreactors. (B) Cell viabilities for LV production in 5 L perfusion bioreactors. (C) Infectious titers in the cell-free harvest collected in 24-hour intervals at 4°C. (D) Virus containing harvests collected at 15-18 dpi from the 5 L bioreactor inoculated with clone 102 were pooled, filtered, purified using a chromatography capture step, and further concentrated by a subsequent tangential-flow filtration step. The bars are representing infectious titers and total infectious units at different stages of the process. Error bars are showing the standard deviation of the infectious titer assay.

[0106] Figures 5 is a series of graphical representations showing transduction of human CD34+cells at MOI of 0.3, 1.0 and 3 using LV derived from stable producer clones with harvest collected at 7 and 14 dpi. (A) C45, parental GPRTGs, (B) C56, parental GPRGs (C) C102, GPRTGs_pVSV-G and (D) C155, GPRGs_pVSV-G. Values are shown as mean and the error bars are representing the standard deviation, dpi = days post induction; MOI = multiplicity of infection; VCN = vector copy number.

[0107] DETAILED DESCRIPTION

[0108] General

[0109] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise. For example, reference to “a” includes a single as well as two or more; reference to “an” includes a single as well as two or more; reference to “the” includes a single as well as two or more and so forth.

[0110] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0111] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0112] Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise. Stated another way, any specific example of the present disclosure may be combined with any other specific example of the disclosure (except where mutually exclusive).

[0113] Any example of the present disclosure disclosing a specific feature or group of features or method or method steps will be taken to provide explicit support for disclaiming the specific feature or group of features or method or method steps.

[0114] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, molecular biology, microbiology, virology).

[0115] Unless otherwise indicated, the conventional techniques of molecular biology, microbiology, virology, recombinant protein, cell culture, and immunological techniques utilised in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0116] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0117] The term “about”, unless stated to the contrary, refers to + / - 20%, more for example + / - 10%, of the designated value. For the avoidance of doubt, the term “about” followed by a designated value is to be interpreted as also encompassing the exact designated value itself (for example, “about 10” also encompasses 10 exactly).

[0118] As used herein the term “from” in the shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source (i.e., includes recombinantly obtained). Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0119] All publications cited herein are hereby incorporated by reference in their entirety. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0120] Any discussion of documents, acts, materials, devices, articles or the like that has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

[0121] Selected Definitions

[0122] As used herein, the term “enveloped virus” refers to DNA and RNA viruses that have a viral envelope. Envelopes are typically derived from host cell membranes (e.g., phospholipids and proteins), but may include viral glycoproteins on the surface of the envelope. Enveloped viruses also comprise a “capsid”, which is a protein layer between the envelope and viral genome. In one example, the enveloped virus is a retrovirus. For example, the enveloped virus is a lentivirus, e.g., human immunodeficiency virus.

[0123] As used herein, the term “cell culture fluid” or “cell culture medium” will be understood to encompass the fluid or medium in which cells are grown for the purpose of producing an enveloped virus. The fluid or medium does not comprise the cells (e.g., the cells may have been removed, e.g., by centrifugation and / or removal of supernatant).

[0124] As used herein, the term “cell culture” or “suspension cell culture” will be understood to refer to the collective of the cell culture fluid or medium and the cultured cells.

[0125] The term “suspension” in reference to the cell lines will be understood to refer to single cells or small aggregates of cells that are free-floating in the cell culture medium. For example, such cells function and multiply in an agitated growth medium, thus forming a suspension. The term “purify” or “purifying” or “purification” shall be taken to mean the removal, whether completely or partially, of at least one impurity present in the cell culture fluid, which thereby improves the level of purity of enveloped virus in solution.

[0126] Production of Stable Producer Cell Lines

[0127] The present disclosure provides packaging cell lines, stable producer pools, stable producer cell clones, and stable producer cell lines thereof, capable of producing an enveloped virus.

[0128] Stable producer cells of the present disclosure can be derived from packaging cell lines, including any of the cell lines disclosed herein, transfected with a transfer plasmid with the gene of interest and several packaging (or helper) plasmids encoding VSV-G and essential viral proteins responsible for gene integration or assembly (Miller (2001) Curr. Protoc. Hum. Genet. Chapter 12: Unit 12.5.; Rodrigues et al. 2011, supra).

[0129] In one example, stable producer cell line cells are generated by synthesizing a vector by cloning one or more genes into a recombinant plasmid; forming a concatemeric array from an expression cassette excised from the synthesized vector, and an expression cassette obtained from an antibiotic resistance cassette plasmid; transfecting packaging cell line cells with the formed concatemeric array; and selecting and isolating the stable producer cell line cells.

[0130] It will be apparent to the skilled person from the disclosure herein, that the stable producer cells of the present disclosure are derived from a packaging cell line transfected with VSV-G, and then subsequently re-transfected with a VSV-G encoding plasmid. Methods of generating a VSV-G plasmid for re-transfection of the packaging cell line will be apparent to the skilled person and / or described herein. For example, the VSV-G plasmid is generated by amplification of a VSV-G sequence under control of a promotor (e.g., a 7tetO promoter). In one example, the VSV-G sequence is from the same provirus that was used to generate the parental packaging cell line.

[0131] As used herein, reference to a ‘stable’ cell clone or cell line, refers to a cell or population of cells that have been genetically modified to express a specific gene or genetic construct in a stable and heritable manner. This stable expression is due to the integration of the foreign DNA into the cell's chromosomes, which allows for consistent production of the desired protein or function.

[0132] In one example, the stable producer cell clone is cultured to produce a stable producer cell line. Methods of culturing the stable producer cell clone to generate a stable producer cell line are described herein and / or are apparent to the skilled person. The packaging cell may be selected from any cell allowing production of an enveloped virus. According to one example, the cell is selected from a human cell (HEK293, HEK293T, HEK293FT, HEK293OX, Te671, HT1080, CEM), a musteli cell (NIH-3T3), a mustelidae cell (Mpf), a canid cell (D17), and derivatives thereof. According to one example, the cell is selected from CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY I, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211 A cells, and derivatives thereof.

[0133] In some embodiments, the packaging cell lines are GPRG or GPRTG cell lines (Throm et al. (2009) Blood 113(21):5104-5110; and Bonner et al. (2015) Molecular Therapy, Vol. 23, Suppl. 1, S35). For example, the cell is selected from the GPR, GPRG, GPRT, GPRGT, and GPRTG cell lines. In another example, the cell is selected from a cell line derived from any of the above cell lines.

[0134] In one example, the cell line is a suspension cell line selected from GPR, GPRG, GPRT, GPRGT, and GPRTG cell lines. For example, the suspension cell line is a cell line derived from any of the above cell lines. In one example, the suspension cell line is a cell line derived from a GPRG cell line. In another example, the suspension cell line is a cell line derived from a GPRGT cell line. In a further example, the suspension cell line is a cell line derived from a GPRTG cell line.

[0135] Methods of adapting an adherent cell line to grow in suspension will be apparent to the skilled person and / or described herein.

[0136] The present disclosure provides a stable producer cell line capable of producing an enveloped virus with an infectious titer of at least 5.0 x 106TU / mL of culture medium at 6 days following induction of enveloped virus production. For example, the stable producer cell line is capable of producing an enveloped virus with an infectious titer of at least 5.5 x 106TU / mL of culture medium at day 6 of culture. In one example, the stable producer cell line is capable of producing an enveloped virus with an infectious titer of at least 6 x 106TU / mL of culture medium at 6 days following induction of enveloped virus production. For example, the stable producer cell line is capable of producing an enveloped virus with an infectious titer of at least 6.5 x 106TU / mL of culture medium at 6 days following induction of enveloped virus production. In another example, the stable producer cell line is capable of producing an enveloped virus with an infectious titer of at least 7 x 106TU / mL of culture medium at 6 days following induction of enveloped virus production. In a further example, the stable producer cell line is capable of producing an enveloped virus with an infectious titer of at least 8 x 106TU / mL of culture medium at 6 days following induction of enveloped virus production. In one example, the stable producer cell line is capable of producing an enveloped virus with an infectious titer of at least 9 x 106TU / mL of culture medium at 6 days following induction of enveloped virus production. In another example, the stable producer cell line is capable of producing an enveloped virus with an infectious titer of at least 1 x 107TU / mL of culture medium at 6 days following induction of enveloped virus production.

[0137] Methods of measuring infectious titer will be apparent to the skilled person and / or described herein. In one example, infectious titer is determined by transduction with a GFP LV followed by flow cytometry. For example, the method comprises incubating virus containing supernatants with HEK293T cells seeded on plates, followed by trypsinization and washing, and using flow cytometry to determine the percentage of GFP positive cells and calculating the infectious titer in transducing units (TU) / mL of media.

[0138] The present disclosure provides a stable producer cell line capable of producing an enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.0 x IO-4TU / RNA copies. For example, at least 1.5 x IO-4TU / RNA copies, or at least 2 x 10’4TU / RNA copies, or at least 2.5 x 10’4TU / RNA copies, or at least 3.0 x 10’4TU / RNA copies, or at least 3.3 x 10’4TU / RNA copies. In one example, the cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of between about 1.5 x 10’4TU / RNA copies and 3.5 x IO’5TU / RNA copies.

[0139] Methods of determining the ratio of infectious particles to viral vector genome RNA copies will be apparent to the skilled person and / or described herein.

[0140] The present disclosure further provides a stable producer cell line capable of producing an enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1011copies / mL cell culture medium. For example, at least 5.0 x 1011copies / mL, or at least 1.0 x 1012copies / mL, or at least 5.0 x 1012copies / mL, or at least 1.0 x 1013copies / mL cell culture medium.

[0141] Method of determining viral vector genome RNA concentration will be apparent to the skilled person and / or described herein.

[0142] In one example, the stable cell line is capable of producing an enveloped virus with a viability of at least 75% at day 15 of culture. For example, a viability of at least 75% at day 20 of culture, or a viability of at least 75% at day 25 of culture, or a viability of at least 75% at day 30 of culture, or a viability of at least 75% at day 35 of culture.

[0143] Methods of determining cell viability will be apparent to the skilled person and / or are described herein. For example, the cell viability is determined using a hemocytometer after trypan blue staining. For example, cell viability is determined by trypan blue exclusion and determining the percentage of living cells to total cells in the sample.

[0144] In one example, the stable cell line is capable of producing an enveloped virus with a viable cell density of at least 1.0 x 107cells / mL of culture medium at day 15 of culture. For example, a viable cell density of at least 1.5 x 107cells / mL of culture medium at day 15 of culture. In one example, the stable cell line is capable of producing an enveloped virus with a viable cell density of at least 1.5 x 107cells / mL of culture medium at day 20 of culture. For example, a viable cell density of at least 1.5 x 107cells / mL of culture medium at day 25 of culture, or a viable cell density of at least 1.5 x 107cells / mL of culture medium at day 30 of culture, viable cell density of at least 1.5 x 107cells / mL of culture medium at day 35 of culture.

[0145] Methods of determining viable cell density will be apparent to the skilled person and / or are described herein. In one example, viable cell density is determined using a hemocytometer after trypan blue staining. For example, viable cell density is determined by trypan blue exclusion and determining the total number of living cells per mL of sample.

[0146] In one example, the packaging cell line, stable producer pool, stable producer cell clone and / or stable producer cell line comprises a feature of any cell line described herein.

[0147] In one example, the packaging cell line, stable producer pool, stable producer cell clone and / or the stable producer cell line expresses a tetracycline-suppressible gene expression system in a cell culture medium.

[0148] In one example, the packaging cell line, stable producer pool, stable producer cell clone and / or the stable producer cell line is adapted to grow in suspension cell culture.

[0149] In one example, the packaging cell line, stable producer pool, stable producer cell clone and / or stable producer cell line is selected from or derived from a GPR, a GPRG, a GPRT, a GPRGT or a GPRTG cell line.

[0150] The present disclosure also provides methods for producing a stable producer cell line as described herein capable of producing an enveloped virus in a suspension cell culture.

[0151] The present disclosure provides a method of producing an enveloped virus in a suspension cell culture, the method comprising culturing a stable producer suspension cell line, wherein the stable producer suspension cell line is transfected with a VSV-G encoding plasmid and wherein the stable producer cell line is derived from a packaging cell line transfected with VSV-G. In one example, the packaging cell line transfected with VSV-G is re-transfected with a VSV-G encoding plasmid using concatemeric array transfection. For example, the concatemeric arrays are prepared as previously described, e.g., Throm et al., (2009) Blood, 113(21), 5104-5110. In one example, the VSV-G plasmid is generated by amplifying a VSV-G sequence under control of a promotor (e.g., a 7tetO-promoter). In one example, the VSV-G sequence is the same sequence used to generate the packaging cell lines. For example, the packaging cell line is re-transfected with the same VSV-G encoding plasmid. In one example, the VSV-G construct is cloned into a vector backbone. For example, the vector comprises one or more antibiotic resistance genes (e.g., blasticidin or hygromycin) driven by a promotor (e.g., SV40). In one example, antibiotics are added to cells transfected with the VSV-G plasmid and cells carrying an antibiotic resistance gene selected.

[0152] In one example, the disclosure provides a packaging cell line that is re-transfected. In one example, the disclosure provides a stable producer cell line that is re-transfected. In one example, the disclosure provides a method of generating a producer cell line by co-transfecting a packaging cell line with the VSV-G plasmid and a plasmid carrying a gene of interest. In one example, the disclosure provides a method of re-transfecting a packaging cell line with VSV-G to generate a re-transfected packaging cell line. In one example, the disclosure provides a method of generating a stable producer cell line by transfecting a re-transfected packaging cell line with a gene of interest.

[0153] Production of Enveloped Viruses

[0154] Methods for the production of enveloped viruses will be apparent to the skilled artisan and / or described, for example, in Ansorge et al., (2010) Biochem. Eng. J. 48: 362- 377; Schweizer and Merten (2010) Curr. Gene Ther. 10: 474-486; and Rodrigues et al., (2011) Viral Gene Therapy. Xu, InTech. Chapter 2: 15-40.

[0155] Enveloped viruses

[0156] In one example, the virus is a retrovirus, for example, a lentivirus. Exemplary retroviruses are from alpha retroviruses (such avian leukosis virus (ALV)), from beta retroviruses (such as mouse mammary tumor virus (MMTV)), from gamma retroviruses (such as murine leukemia virus (MLV)), from delta retroviruses (such as human T- lymphotropic virus (HTLV)), from epsilon retroviruses (such as Walleye dermal sarcoma virus (WDSV)), from spumavirus (such as human foamy virus (HFV) or simian foamy virus (SFV)), from primate lentiviruses such as the different types of human immunodeficiency viruses (HIV), the different types of simian immunodeficiency viruses (SIV), or from non-primate mammal lentiviruses such as the equine infectious anemia virus (EIAV), from the feline immunodeficiency virus (FIV), the caprine arthritis-encephalitis virus (CAEV), or the ovine visna-maedi virus (VMV).

[0157] Trans gene expression

[0158] In some examples, the enveloped virus comprises a transgene introduced into its genome. The transgene will depend on the specific use for which the enveloped viral vector is intended. Exemplary transgenes include a transgene coding for a therapeutic RNA (e.g. encoding an antisense complementary RNA of a target RNA or DNA sequence), a transgene encoding for a protein that is deficient or absent in a subject affected with a pathology, or a transgene used for vaccination with DNA, i.e. a transgene coding for a protein, the expression of which will induce vaccination of the recipient body against said protein. In some examples, the transgene encodes a protein or nucleic acid useful for treating a hemoglobinopathy, e.g., sickle cell disease or a thalassemia. In some examples, the transgene encodes a protein or nucleic acid useful for treating a primary immunodeficiency. In some examples, the transgene encodes a protein or nucleic acid useful for treating Wiskott-Aldrich Syndrome. In some examples, the transgene encodes a protein or nucleic acid useful for treating X linked agammaglobulinemia. In some examples, the transgene encodes a protein or nucleic acid useful for treating Deficiency of Interleukin- 1 Receptor Antagonist (DIRA). In some examples, the transgene encodes a protein or nucleic acid useful for treating Adenosine deaminase 2 deficiency (DADA2).

[0159] In some examples, an enveloped virus is produced by introducing the four following elements into a host cell: an expression cassette comprising a lentiviral gene gagpol, an expression cassette comprising a lentiviral gene rev, a transgene, all positioned between a lentiviral LTR-5’ and a lentiviral LTR-3’, and an expression cassette encoding envelope glycoprotein(s).

[0160] Cell culture medium

[0161] The cells are cultivated in a medium suitable for cultivation of mammal cells and for producing an enveloped virus. As described herein, the cells are cultivated in a suspension environment, e.g., suspended in the medium. The medium may moreover be supplemented with additives known in the field such as antibiotics, serum (notably fetal calf serum, etc.) added in suitable concentrations. The medium may be supplemented with GlutaMax™, Pluronic™ F-68 (ThermoFisher), LONG® R3 IGF-I (Sigma- Aldrich), Cell Boost™ 5, and / or an antidumping agent. The medium used may notably comprise serum or be serum-free. Culture media for mammal cells are known and include, for example, DMEM (Dulbecco’s Modified Eagle’s medium) medium, RPMI1640 or a mixture of various culture media, including for example DMEM / F12, or a serum-free medium like optiMEM®, optiPRO®, optiPRO-SFM®, CD293® (ThermoFisher), TransFx™ (Cytiva), BalanCD® (Irvine), Freestyle Fl 7® (Fife Technologies), Ex-Cell® 293 (Sigma- Aldrich), or OptiMEM™ medium (ThermoFisher).

[0162] According to some examples, the culture medium used has a neutral pH (e.g. comprised between 7 and 7.4, notably 7, 7.1, 7.2, 7.3 or 7.4) conventionally used in the state of the art for cultivating cells and producing viruses. In one example, the suspension cell culture is at a pH of between 6.0 and 8.0. For example, the pH of the culture medium is 7.1 ± 0.15. In other examples, the production process used comprises the cultivation of producing cells in a moderately acid medium. The expression “moderately acid condition” designates the pH of an aqueous solution comprised between 5 and 6.8, for example between 5.5 and 6.5, such as between 5.8 and 6.2. The selected pH will also depend on the buffering power of the culture medium used, which one skilled in the art may easily determine taking into account his / her general knowledge. One skilled in the art is able to modify the pH of a solution.

[0163] Suspension cell culture

[0164] The present disclosure provides a method of producing an enveloped virus in a suspension cell culture.

[0165] Methods of the disclosure are applicable to producing enveloped viruses from both small- and large-scale productions. The methods are particularly useful for their ability to be scaled up for manufacturing pharmaceutical products at commercial scale.

[0166] It will be apparent to the skilled person that production of an enveloped virus includes a cell expansion phase and a viral production phase.

[0167] In one example, the cell culture is operated in a batch, fed batch, continuous, semi- continuous, or perfusion mode.

[0168] In one example, the cell expansion phase and / or viral production phase are operated in a batch, fed batch, continuous, semi-continuous, or perfusion mode.

[0169] In one example, the cell expansion phase is carried out in batch, fed batch, continuous, semi-continuous, or perfusion mode. In one example, the cell expansion phase is carried out in batch mode. In another example, the cell expansion phase is carried out in fed-batch mode. In a further example, the cell expansion phase is carried out in continuous mode. In one example, the cell expansion phase is carried out in perfusion mode. In another example, the cell expansion phase is carried out in batch and perfusion mode. For example, the cell expansion phase is initially carried out in batch mode and subsequently carried out in perfusion mode.

[0170] In one example, the viral production phase is carried out in batch, fed batch, continuous, semi-continuous, or perfusion mode. In one example, the viral production phase is carried out in batch mode. In another example, the viral production phase is carried out in fed-batch mode. In a further example, the viral production phase is carried out in continuous mode. In one example, the viral production phase is carried out in perfusion mode. In another example, the viral production phase is carried out in batch and perfusion mode. For example, the viral production phase is initially carried out in batch mode and subsequently carried out in perfusion mode.

[0171] In one example, the cell expansion and the viral production phases are carried out in batch mode. In another example, the cell expansion and the viral production phases are carried out in perfusion mode. In a further example, the cell expansion phase is carried out in batch mode and the viral production phase is carried out in perfusion mode.

[0172] It will be apparent to the skilled person that reference to a batch, fed-batch, continuous and / or perfusion mode for a particular phase of cell culture (i.e., cell expansion and / or viral production) does not mean that the entire culture phase is carried out in that mode. For example, it only means that a period of the cell culture phase (e.g., at least 1 day) is carried out in that mode. It will also be understood that the mode does not necessary commence on day 0 of the culture phase. For example, the culture may commence on day 0 and perfusion mode only commence on day 2 of the cell culture phase.

[0173] In one example, the suspension cell culture is operated in batch mode. It will be apparent to the skilled person that “batch mode” refers to a process where cells are initially cultured in a medium and this medium is neither removed, replaced, nor supplemented, i.e., the cells are not “fed” with new medium, during or before the end of cultivation.

[0174] In one example, the suspension cell culture is operated in fed-batch mode. It will be apparent to the skilled person that “fed-batch mode” refers to a process where one or more nutrients are fed to the bioreactor during the cultivation period. In one example, the cell expansion phase and / or the virus production phase are operated in fed-batch mode. In one example, the cell expansion phase is operated in fed-batch mode.

[0175] In one example, the suspension cell culture is operated in perfusion mode. It will apparent to the skilled person that “perfusion mode” involves the constant feeding of fresh media and removal of spent media while retaining high numbers of viable cells (i.e., continuous media exchange). In one example, the cell expansion phase and / or the virus production phase are operated in perfusion mode. In one example, the virus production phase is operated in perfusion mode.

[0176] In one example, the cells are cultured in fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, or stirred tank bioreactors. In one example, cells are cultured in a stirred tank bioreactor. In examples, the cells are cultured in a Biostat® or Univessel® bioreactor (Sartorius).

[0177] In one example, the volume of the cell culture can be for example, about 0.01 L to about 0.1 L, or about 0.1 L to about 1 L, or about 1 L to about 5 L. In another example, the volume of the cell culture can be about 5 L to about 10 L, about 10 L to about 50 L, about 50 L to about 100 L, about 100 L to about 200 L, about 200 L to about 500 L, about 500 L to about 1000 L, about 1000 L to about 2000 L, or about 2000 L to about 5000 L. In one example, the volume of the cell culture is between about 35 and 150 L. In one example, the volume of the cell culture is about 35-150 L. In one example, the volume of the cell culture is about 50-70 L.

[0178] In one example, the suspension cell culture is operated at a temperature that permits cell growth and viral production. For example, the cell culture has a temperature conventionally used in the state of the art for cultivating cells and producing viruses. In one example, the suspension cell culture is at a temperature of between 35-39°C. For example, at a temperature of 37 ± 0.5°C or at a temperature of 38 ± 0.5°C.

[0179] In one example, the suspension cell culture is operated at large-scale. For example, the suspension cell culture is operated at commercial-scale.

[0180] In one example, the suspension cell culture is operated for a period of at least 10 days. For example, the suspension cell cultures is operated for a period of between about 10 and 50 days. In one example, the suspension cell culture is operated for a period of between 10 and 35 days, for example, about 10 days or about 12 days, or about 15 days, or about 18 days, or about 20 days, or about 22 days, or about 25 days, or about 28 days, or about 30 days or about 32 days or about 35 days. In one example, the suspension cell culture is operated for at least 15 days. For example, the suspension cell culture is operated for about 20 days. In another example, the suspension cell culture is operated for at least 20 days. In a further example, the suspension cell culture is operated for at least 25 days. For example, the suspension cell culture is operated for about 28 days. In one example, the suspension cell culture is operated for at least 30 days. In one example, the suspension cell culture is operated for at least 32 days. For example, the suspension cell culture is operated for a period of 35 days. In one example, the suspension cell culture is operated for at least 35 days. Purifying Enveloped Viruses

[0181] In one example, the enveloped virus is purified from the cell culture comprising one or more steps selected from the group consisting of clarification filtration, anion exchange chromatography, concentration and diafiltration.

[0182] The downstream process for purifying and concentrating viral vector from a cell culture includes a harvest filtration step (also known as “clarification filtration” or “harvest clarification filtration” or “bioburden reduction”) to remove cellular debris and components from the harvest, a purification step, e.g., anion exchange chromatography, to reduce overall volume and to separate viral vector from host cell DNA, proteins, and media components, and an ultrafiltration / diafiltration step to concentrate the viral vector into a final formulation buffer. In some examples, the downstream step further includes a sterile filtration step for removal of microorganisms from the final product.

[0183] As used herein, “harvesting” refers to removal of the cell culture media containing virus particles from the producer cells for downstream processing, and “harvest” refers to the cell culture media containing virus particles that has been removed for the purpose of downstream processing. A harvesting process may include collecting one or more harvests. “Harvest filtration” refers to either a harvest that has been fdtered or cell culture media containing virus particles that has been filtered to remove the producer cells for downstream processing.

[0184] In one example, a harvested cell culture fluid is filtered following production of the enveloped virus.

[0185] As used herein, the term “fdtered cell culture fluid” will be understood to encompass the cell culture fluid after it has been subjected to harvest filtration.

[0186] Following harvest filtration, the enveloped virus is purified using anion exchange.

[0187] In one example, the anion exchange is performed in bind-elute mode. In this regard, the enveloped virus binds to the anion exchanger while contaminants flow through. The virus is subsequently eluted from the anion exchanger. Performing anion exchange in this manner reduces the volume of liquid in which the virus is suspended and removes contaminants such as host cell DNA, host cell proteins, and medium components like fetal bovine serum.

[0188] Suitable anion exchangers will be apparent to the skilled artisan. Exemplary anion exchangers are a column comprising a resin or a membrane or another suitable substrate.

[0189] In one example, the anion exchanger is a weak anion exchanger, e.g., comprising an ion exchange group selected from a diethylaminoethyl (DEAE) or aminoethyl group. In another example, the anion exchanger is a strong anion exchanger, e.g., comprising an ion exchange group selected from a quaternary ammonium (Q), diethyl- 2-hydroxypropylaminoethyl (QAE), triethylaminoethyl (TEAE), or trimethyl aminoethyl group. Exemplary anion exchangers include MUSTANG® E, MUSTANG® Q, SARTOBIND® Q, CHROMASORB®, POSSIDYNE®, CAPTO® Q, QSFF, POROS® Q, FRACTOGEL® Q, NATRIX® Q.

[0190] In one example, the anion exchanger comprises a Q ion exchange group.

[0191] In one example, the anion exchanger is a membrane anion exchanger comprising a Q ion exchange group. For example, the anion exchanger is MUSTANG® Q.

[0192] In one example, an enveloped virus eluted from anion exchange column is further purified on the basis of its size. In one example, the buffer in which virus was eluted from the anion exchange column, is exchanged more or less at the same time. In the process of the disclosure, tangential flow filtration is preferred. This method permits impurity removal and buffer exchange at almost the same time.

[0193] Tangential flow ultrafiltration / diafiltration is a method which may be used to remove residual protein and nucleic acids as well as for exchanging working buffer into a final formulation buffer. Ultrafiltration using tangential flow is preferred and different devices can be used (e.g. Proflux and LABSCALE (ultrafiltration system) TFF System, both Millipore or the KR2i system from Repligen). The particular ultrafiltration membrane selected will be of a filter pore size sufficient small to retain enveloped virus but large enough to allow penetration of impurities. Depending on the manufacturer and membrane type, nominal molecular weight cut-offs between 100 and 1000 kDa may be appropriate (e.g. UFP-750-E-5A, GE Healthcare; BIO MAX (ultrafiltration device) NMWC 1000, Millipore). In one example, the molecular weight cut-off is 500kDa. The membrane composition may be, but it is not limited to, regenerate cellulose, (modified) polyethersulfone, polysulfone. Membranes can be of flat sheet or hollow fibre type. The main parameters that must be optimized are flux rate and trans-membrane pressure. In combination with nominal molecular weight cut-off these two parameters will enable efficient purification and buffer exchange and high virus yield.

[0194] As an additional step sterile filtration may be performed to eliminate bioburden. Therefore diluted eluate or final retentate from the ultrafiltration step may be filtered through a filter, for example a 0.22 pm filter. The filter may be constructed from various materials, which may include but are not limited to polypropylene, hydrophilic PVDF, cellulose, hydrophilic regenerated cellulose, cellulose esters, wetting agent-free cellulose acetate, cellulose acetate, nylon, hydrophilic nylon membrane, polyethersulfone, hydrophilic polyethersulfone, hydrophilic asymmetric PES, or any other material which is consistent with low unspecific influenza virus binding. The filter may have a single membrane layer or more than one layer or may incorporate a prefilter of the same or different material, for example a 0.45 pm prefilter. The sterile filtrated virus can be held frozen for subsequent manipulation.

[0195] Embodiments of the Disclosure

[0196] The invention is further disclosed in the following numbered paragraphs:

[0197] 1. A stable producer cell line capable of producing an enveloped virus in a suspension cell culture, wherein the stable producer cell line is derived from a packaging cell line transfected with vesicular stomatitis virus G protein (VSV-G), and wherein the stable producer cell line is stably re-transfected with a VSV-G encoding plasmid.

[0198] 2. The stable producer cell line of paragraph 1, wherein the cell line is capable of producing the enveloped virus with an infectious titre of at least 5.0 x 106TU / mL of culture medium at 6 days following induction of enveloped virus production.

[0199] 3. The stable producer cell line of paragraphs 1 or 2, wherein the cell line is capable of producing the enveloped virus with an average infectious titre of at least 3.0 x 107on day 2 through day 9 following induction of enveloped virus production.

[0200] 4. The stable producer cell line of any one of paragraphs 1 to 3, wherein the cell line is capable of producing the enveloped virus with an average infectious titre of at least 4.0 x 107on day 2 through day 9 following induction of enveloped virus production.

[0201] 5. A stable producer cell line stably expressing VSV-G, wherein the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with an infectious titre of at least 5.0 x 106TU / mL of culture medium at 6 days following induction of enveloped virus production.

[0202] 6. The stable producer cell line of any one of paragraphs 1 to 5, wherein the cell line is capable of producing the enveloped virus with an infectious titre of at least 5.5 x 106TU / mL, or at least 6.0 x 106TU / mL, or at least 7.0 x 106TU / mL, or at least 8.0 x 106TU / mL, or at least 9.0 x 106TU / mL, or at least 1.0 x 107TU / mL of culture medium at 6 days following induction of enveloped virus production. 7. The stable producer cell line of any one of paragraphs 1 to 6, wherein the cell line is capable of producing the enveloped virus with an infectious titre of at least 1.0 x 107TU / mL, or at least 1.5 x 107TU / mL, or at least 2.0 x 107TU / mL, or at least 2.5 x 107TU / mL, or at least 3.0 x 107TU / mL of culture medium at 14 days following induction of enveloped virus production.

[0203] 8. The stable producer cell line of any one of paragraphs 1 to 7, wherein the cell line is derived from a producer pool capable of producing the enveloped virus with a VSV-G content at least 2-fold greater, or at least 3-fold greater, or at least 4-fold greater, or at least 5-fold greater, or at least 6-fold greater than the VSV-G content of an enveloped virus produced from a stable producer cell line derived from a producer pool that has not been stably re-transfected with a VSV-G encoding plasmid.

[0204] 9. The stable producer cell line of any one of paragraphs 1 to 8, wherein the cell line is derived from a producer pool capable of producing the enveloped virus with a VSV-G content of at least 5-fold greater than the VSV-G content of an enveloped virus produced from a stable producer cell line derived from a producer pool that has not been stably retransfected with a VSV-G encoding plasmid.

[0205] 10. The stable producer cell line of any one of paragraphs 1 to 9, wherein the cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.0 x 10’4TU / RNA copies, or at least 1.5 x 10’4TU / RNA copies, or at least 2 x 10’4TU / RNA copies, or at least 2.5 x 10’4TU / RNA copies, or at least 3.0 x 10’4TU / RNA copies, or at least 3.3 x 10’4TU / RNA copies.

[0206] 11. The stable producer cell line of any one of paragraphs 1 to 10, wherein the cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of between about 1.5 x 10’4TU / RNA copies and 4.0 x IO’4TU / RNA copies.

[0207] 12. The stable producer cell line of any one of paragraphs 1 to 11, wherein the cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1011copies / mL, or at least 5.0 x 1011copies / mL, or at least 1.0 x 1012copies / mL, or at least 5.0 x 1012copies / mL, or at least 1.0 x 1013copies / mL cell culture medium. 13. The stable producer cell line of any of paragraphs 1 to 12, wherein the cell line is stable for at least 70 days.

[0208] 14. A method of producing a stable producer cell line stably expressing VSV-G, wherein the cell line is capable of producing an enveloped virus in a suspension cell culture, the method comprising stably integrating a plasmid comprising a gene of interest and a VSV-G encoding plasmid into a packaging cell line, wherein the packaging cell line is transfected with VSV-G.

[0209] 15. A method of producing a stable producer cell line stably expressing vesicular stomatitis virus G protein (VSV-G), wherein the cell line is capable of producing an enveloped virus in a suspension cell culture, the method comprising stably integrating a VSV-G encoding plasmid into a stable producer cell line, wherein the stable producer cell line is derived from a packaging cell line transfected with VSV-G.

[0210] 16. A method of producing an enveloped virus in a suspension cell culture, the method comprising culturing a stable producer suspension cell line, wherein the stable producer suspension cell line is transfected with a VSV-G encoding plasmid and wherein the stable producer cell line is derived from a packaging cell line transfected with VSV- G.

[0211] 17. The method of paragraphs 15 or 16, wherein the enveloped virus is harvested from the suspension cell culture fluid.

[0212] 18. The method of paragraph 17, wherein the enveloped virus is harvested from the suspension cell culture at least 7 days following induction of enveloped virus production.

[0213] 19. The method of paragraphs 17 or 18, wherein the enveloped virus is harvested from the suspension cell culture at least 14 days following induction of enveloped virus production.

[0214] 20. The method of any one of paragraphs 15 to 19, wherein the suspension cell culture is at a pH of between 6.0 and 8.0 and / or at a temperature of between 35-39 °C. 21. The method of any one of paragraphs 15 to 20, wherein the suspension cell culture has a volume of greater than about 1 L, about 2 L, about 5 L, about 10 L, about 50 L, about 100 L, about 500 L, about 1,000 L.

[0215] 22. The method of any one of paragraphs 17 to 21, wherein the method further comprises purifying the enveloped virus from the harvested suspension cell culture fluid.

[0216] 23. The method of paragraph 22, wherein purifying the enveloped virus comprises one or more steps selected from the group consisting of clarification filtration, anion exchange chromatography, concentration and diafiltration.

[0217] 24. The method of paragraph 22 or 23, additionally comprising formulating the purified enveloped virus into a pharmaceutical formulation or into a solution suitable for infecting a cell.

[0218] 25. The stable producer cell line of any one of paragraphs 1 to 14, or the method of any one of paragraphs 15 to 24, wherein the cell is a GPRG, GPRGT or GPRTG cell or a derivative thereof.

[0219] 26. The stable producer cell line or the method of paragraph 25, wherein the cell is a GPRG or GPRTG cell.

[0220] 27. The stable producer cell line of any one of paragraphs 1 to 14 or 25 to 26, or the method of any one of paragraphs 15 to 26, wherein the enveloped virus is a retrovirus.

[0221] 28. The stable producer cell line or the method of paragraph 27, wherein the retrovirus is a lentivirus.

[0222] 29. A purified enveloped virus produced by the method according to any one of paragraphs 15 to 28.

[0223] 30. A method of transducing a cell with an enveloped virus in a cell culture, the method comprising contacting the cells with the enveloped virus, wherein the enveloped virus is produced from the stable producer cell line of any one of paragraphs 1 to 14 or 25 to 28 or is produced according to the method of any one of paragraphs 15 to 28. 31. A method of increasing integrated vector copy number (VCN) of a cell transduced with an enveloped virus in a cell culture, the method comprising contacting the cells with the enveloped virus, wherein the enveloped virus is produced from the stable producer cell line of any one of paragraphs 1 to 14 or 25 to 28 or is produced according to the method of any one of paragraphs 15 to 28.

[0224] 32. The method of paragraph 30 or 31, wherein the method results in a transduction efficiency of at least 10% 14 days post transduction of the cell.

[0225] 33. The method of any one of paragraphs 30 to 32, wherein the method results in a transduction efficiency of at least 15% 14 days post transduction of the cell.

[0226] 34. The method of any one of paragraphs 30 to 33, wherein the cell culture has a multiplicity of infection (MOI) of at least 0.1.

[0227] 35. The method of any one of paragraphs 30 to 34, wherein the cell culture has a MOI of at least 0.3.

[0228] 36. The method of any one of paragraphs 30 to 35, wherein the cell culture has a MOI of at least 1.0.

[0229] 37. The method of any one of paragraphs 30 to 36, wherein the method results in a vector copy number (VCN) of at least 0.1 copies / cell 14 days post transduction of the cell at MOI of 1.

[0230] 38. The method of any one of paragraphs 30 to 37, wherein the method results in a VCN of about 0.2 copies / cell 14 days post transduction of the cell at MOI of 1.

[0231] 39. The method of any one of paragraphs 30 to 38, wherein the cell is a hematopoietic stem cell (HSC) or a hematopoietic progenitor cell (HPC).

[0232] 40. The method of paragraph 39, wherein the HSC or HPC is a CD34 positive (CD34+) cell.

[0233] 41. The method of any of the preceding paragraphs, wherein the cell line is stable for at least 70 days. 42. A method of producing a VSV-G re-transfected packaging cell line, the method comprising stably integrating a VSV-G encoding plasmid into a packaging cell line, wherein the packaging cell line is transfected with VSV-G.

[0234] 43. A method of producing a stable producer cell line stably expressing VSV-G, wherein the cell line is capable of producing an enveloped virus in a suspension cell culture, the method comprising stably integrating a plasmid comprising a gene of interest into the VSV-G re-transfected packaging cell line of paragraph 42.

[0235] 44. A packaging cell line transfected with vesicular stomatitis virus G protein (VSV- G), that has been stably re-transfected with a VSV-G encoding plasmid.

[0236] SEQUENCES OF THE DISCLOSURE

[0237] The present disclosure is described further in the following non-limiting examples.

[0238] EXAMPLES

[0239] Example 1: Materials and Methods

[0240] Cell culture

[0241] GPRGs or GPRTGs LV packaging cells adapted to growth in suspension were generated as previously described (Klimpel et al., 2023). In brief, for generation of the GPRG cell line, the rTA encoding for the tetracycline-controlled transactivator, gag-pol and rev encoding for the viral packaging elements, and VSV-G encoding for the envelope were integrated subsequently into the cell line using SIN MLVs. The GPRTG cell line has the tat gene additionally integrated and was further optimized regarding expression levels of the introduced elements (Bonner et al., 2015). The transcription of rev, VSV-G, and tat in case of the GPRTG cell line, is under control of a 7 / c / O-promotor and thus suppressed by doxycycline supplementation.

[0242] For general cell maintenance GPRGs or GPRTGs packaging cell lines were cultivated in TransFx-H medium supplemented with 6 mM GlutaMAX (ThermoFisher) and 0.1% poloxamer 188 (Merck), hereafter called TransFx-H basal medium. A final concentration of 2.5 ng / mL doxycycline (MP Biomedicals) was added to suppress gene expression of Tet-controlled genetic elements, unless otherwise indicated. Cells were cultivated in shaken environment using shake flasks (Corning) at a maximum relative working volume of maximum 32% at 120 rpm in a 5% CO2 atmosphere (25 mm orbit, ZEFA SOK-3190). Cells were regularly passaged at a VCD of 4 x 105cells / mL for a 3- day split and at 3 x 105cells / mL for a 4-day split. Static cell cultivation during single cell sorting and antibiotic selection for stable transfections was performed in static phase in an 8% CO2 atmosphere (Thermo HERAcell 250i) using CD293 medium supplemented with 6 mM GlutaMAX (Thermo Fisher), 0.1% poloxamer 188 (Merck) and 100 pL / mL LONG®R3 IGF-I (Sigma- Aldrich), hereafter called CD293 basal medium. Cell counts were performed using Nucleocounter NC-200 (Chemometec) or ViCellBLU (Beckmann Coulter).

[0243] Concatemeric array preparation and VSV-G plasmid generation

[0244] For the generation of stable producer cell lines, concatemeric arrays were prepared for the generation of stable producer cell lines as previously described by Throm et al. (2009). A WAS-T2A-GFP construct encoding for the WASp and GFP separated by a T2A self-cleaving site was cloned into a tetracycline -repressible pTL20 vector cassette like previously described (Klimpel et al., 2023). The pTL20c vector and a pPGK-ble resistance vector were amplified on separate plasmids, linearized using Sfil and PflMI (New England Biolabs) and purified. The pTL20c- and pPGK-ble fragments are mixed at a molar ratio of 25: 1 and directionally ligated using T4 DNA ligase (New England Biolabs). The mixture was incubated overnight and the generated WAS-T2A- GFP_ble concatemers purified using the Genomic-tip DNA extraction kit (Qiagen).

[0245] VSV-G plasmids were generated by amplification of the VSV-G sequence controlled by 7 / c / O-pro motor from a provirus that was initially used to generate the packaging cell lines (Lee & Bartlett, 2018; Throm et al., 2009). The VSV-G construct was cloned into a vector backbone carrying a blasticidin or hygromycin resistance gene driven by a SV40 promotor, resulting in a pVSV-G_BSD or pVSV-G_Hyg vector.

[0246] Generation of stable producer cell lines and stable VSV-G transfections

[0247] To generate stable producer pools a total of 2.21 x 106GPRGs or GPRTGs packaging cells were seeded into 60 mm dishes using DMEM medium supplemented with 5% FBS (Thermo Fisher) and 2.5 ng / mL doxycycline. 24 h later the medium was changed to OptiMEM medium (Thermo Fisher) supplemented with 1 ng / mL doxycycline, and cells were transfected with 11 pg WAS-T2A-GFP_ble concatemer using Lipofectamine 3000 (Thermo Fisher). 18 h post transfection the medium was changed to CD293 basal medium supplemented with 2% FBS and 2.5 ng / mL doxycycline in static phase. Zeocin® was added at a concentration of 50 pg / mL starting from 72 h post transfection. When the cell number started to increase, the Zeocin® concentration was reduced to 20 pg / mL and further cultivation was performed under serum-free conditions in shake flask.

[0248] For the stable integration of the generated VSV-G plasmids, WAS-T2A-GFP producer pools were transfected like previously described for the concatemeric array transfection. 72 h post transfection, a final concentration of 4 pg / mL blasticidin (Merck) was added to cells that were transfected with the pVSV-G_BSD, or 20 pg / mL hygromycin (Merck) was added to cells that were transfected with the pVSV-G_Hyg. When the cell number started to increase, antibiotic concentrations were reduced to 2 pg / mL for blasticidin and 10 pg / mL for hygromycin and further cultivation was performed under serum-free conditions in shake flask.

[0249] To investigate the generated stable WAS-T2A-GFP_pVSV-G producer pools with respect to virus production, cells were seeded at a VCD of 1.5 x 106in doxycycline- free basal TransFx-H medium supplemented with 5% of the chemically defined feed Cell Boost 5 (Cytiva), hereafter called TransFx-H+CB5. Starting from 2 dpi, a daily medium exchange was performed by centrifuging the cell suspension for 10 min at 100 g and resuspending the pellet in fresh TransFx-H+CB5 medium. Six consecutive medium exchanges were performed and virus containing supernatants were frozen at -80°C for infectious titer determination.

[0250] Virus production and purification from stable producer pools

[0251] Four virus preparations were produced using a GPRTGs-WAS-T2A-GFP_pVSV- G and a GPRGs-WAS-T2A-GFP_pVSV-G pool as well as the corresponding parental producer pools. Cells were seeded at a VCD of 1.5 x 106using doxycycline-free TransFx-H+CB5 medium in 1 L shake flasks at a working volume of 250 mL. A daily medium exchange was performed starting from 2 dpi by centrifuging the cell suspension for 5 min at 200 g and resuspending the pellet in fresh TransFx-H+CB5 medium. Eight consecutive harvests were collected and the virus containing supernatant was frozen at - 80°C. For vector purification all production sublots produced by each pool were thawed in a water bath at 37°C. 1 pL / L benzonase (Merck) resulting in a target concentration of 0.3 U / mL was added to the pooled vector harvest and filtered using a Sartopore® 2 0.8 / 0.45 pm filter (Sartorius). 1.8 L of the filtered vector material was loaded on a Mustang® Q XT capsule (Pall) with a 0.86 mL bed volume using an AKTA Pure system (Cytiva). For the purification process, a 10 mM HEPES buffer at pH 7.5 was used with different indicated NaCl concentrations. The Mustang® Q XT capsule was pre- conditioned according to manufactures instructions and equilibrated using HEPES buffer containing 150 mM NaCl. The filtered harvest was loaded at a flow rate of 8.084 mL / min with an in-line spike of a 5M NaCl solution at a flow rate of 0.516 mL / min to reduce the residence time of the LV at high osmolarities. A wash was performed using 8.6 mL of HEPES buffer containing 750 mM NaCl. The virus was then eluted using 9.46 mL of HEPES buffer containing 1.5 mM NaCl. The first 0.86 mL of the eluate were discarded, and the following 8.6 mL directly eluted into 77.4 mL chilled HEPES buffer without NaCl. Further vector concentration and diafiltration was performed using the KrosFlo® KR2i TFF system (Repligen) with a 28 cm2500 kDa cut-off PS -membrane (Repligen) at a shear rate of 1136 s’1. First, the eluate was concentrated 10-fold to 20-fold, followed by a diafiltration using the sevenfold volume of X-VIVO medium (Lonza). A final concentration step to a final volume of approximately 4 mL was performed and the vector material frozen at -80°C.

[0252] Single cell sorting of stable producer cell lines

[0253] A single cell sorting was performed to isolate monoclonal cells from the generated producer pools. Cells were maintained for 10 days in basal CD293 medium supplemented with 5% CB5 and 2.5 ng / mL doxycycline, hereafter called CD293+CB5 medium. Two days prior to the sorting the VCD was adjusted to 6 x 105cells / mL. On the day of sorting the cells were dissociated by diluting the culture twofold in TypleLE (Thermo Fisher) and incubation for 15 min at 37°C. Cells were centrifuged, resuspended in non-supplemented CD293 medium and printed into flat bottom tissue-culture treated 96-well plates (Corning) using F.SIGHT™ single-cell dispenser (Cytena). The 96-well plates were pre-filled with CD293+CB5 medium supplemented with 5% InstiGRO (Solentim) and 7.5 ng / mL doxycycline. To verify monoclonality and observe the colony growth, images were taken on day 0, day 12, day 15, and day 20 post sorting using Cell Metric® imager (Solentim). A final concentration of 5 ng / mL doxycycline was added 7 days and 15 days post sorting. The best growing colonies were scaled up to 24- well plate format and maintained in CD293+CB5 medium. To rank the isolated clones with respect to virus production, a duplicate of each clone culture was scaled up to shaken 6-well format at 120 rpm and induced at a VCD of 1 x 106in TransFx-H+CB5 medium. Starting from 2 dpi the medium was daily exchanged. 6 dpi cell counts were determined and the virus containing supernatant was collected for infectious titer determination. Three stable producer clones derived from GPRTGs-WAS-T2A-GFP and a GPRGs-WAS-T2A-GFP pools stably transfected with the designed pVSV-G vector, as well as three clones from the respective parental producer pools were selected and banked as a research cell bank. Stability study of stable producer clones

[0254] A vial of each banked producer clone was thawed and cultivated in continuous culture for 71 days in TransFx-H+CB5 medium at a doxycycline concentration of 2.5 ng / mL. 15 days after thawing the culture was split, and a 125 mL shake flask of each clone was seeded at a VCD of 1.5 x 106cells / mL in doxycycline-free TransFx-H+CB5 medium. A daily medium exchange was performed starting from 3 dpi by centrifuging the cell suspension for 10 min at 100 g and resuspending the pellet in fresh TransFx- H+CB5 medium. Five consecutive harvests were collected and the virus containing supernatant was frozen at -80°C. The procedure was periodically repeated every 14 days for a total of five productions. For infectious titer determination all five collected harvests of each clone were thawed and pooled at the same ratio right before performing the assay.

[0255] LV production in 5 L perfusion bioreactor

[0256] Clone 45 (GPRTGs-WAS-T2A-GFP), clone 56 (GPRGs-WAS-T2A-GFP), clone 102 (GPRTGs-WAS-T2A-GFP_pVSV-G_BSD) and clone 155 (GPRTGs-WAS-T2A- GFP_pVSV-G_Hyg) were expanded for 21 days in shake flask using TransFx-H+CB5 medium supplemented with 2.5 ng / mL doxycycline. Four days before inoculation of the bioreactor the doxycycline concentration was reduced to 1 ng / mL. Virus production was performed at 4.5 L working volume in 5 L stirred-tank glass reactors (Biostat B-DCU, Sartorius) equipped with a double pitch blade impeller and a cell separation chamber based on acoustic wave separation with a chamber volume of 30 ml (SinePhase Instruments) to allow operation in perfusion mode. The reactor was operated at a stirring speed of 100 rpm, 37°C, 50% dissolved oxygen and pH of 6.95 with a dead band of + / - 0.15. Lactate and glucose concentrations in the supernatant were determined using EPOC Blood Analysis System (Siemens Healthcare). The bioreactors were preconditioned with doxycycline-free medium TransFx-H+CB5 medium supplemented with 100 pL / L Antidumping Agent (Thermo Fisher) and inoculated at a VCD of 1.5 x 106cells / mL on consecutive days to allow a purification of vector material from each producer clone at the same time point post induction. After a 48-h cultivation in batch mode, perfusion was initiated at an exchange rate of approximately one reactor volume per day with the following settings for the acoustic wave separation device: stop time: 10s, run time: 300s, power: 5W. The cell-free harvest was continuously collected at 4°C in 24 h intervals. Samples for infectious titer determination were directly taken from the harvest bag after mixing. A continuous cell bleed was applied to maintain a cell density of approximately 1.5-2.0 x 107cells / mL. The harvests collected at 7 and 14 dpi were purified and concentrated. The previously described LV purification was scaled up using a Mustang® Q XT capsule (Pall) with 2 x 5 mL bed volume. The TFF concentration step was performed like previously described for the concentration of producer pool derived vector material but scaled-up using a 390 cm2500 kDa cut-off PS-membrane (Repligen). The final vector material was sterile filtered using Mini Kleenpak™ EKV capsules (Pall) and frozen at -80°C.

[0257] The harvest collected at 15-18 dpi produced by clone 102 was used for a purification at larger scale using a 40 mL weak anion exchange prototype nanofiber membrane and an AKTA pilot 600S system (both Cytiva). 1 pL / L benzonase (Merck) resulting in a target concentration of 0.3 U / mL and a final concentration of 2 mM magnesium chloride (Merck) was added to the vector harvest and filtered using a Sartopore® 2 Midicap® 0.8 / 0.45 pm filter (Sartorius). The nanofiber membrane was previously equilibrated using 100 mL of 10 mM HEPES buffer at pH 7.5 at a flow rate of 320 mL / min. 20 L of filtered vector material were loaded at an initial flow rate of 320 mL / min, which was gradually reduced to 120 mL / min. LV elution was performed at a flow rate of 80 mL / min using 400 mL of 10 mM HEPES buffer with 650 mM NaCl at a pH of 8. The elution fraction was directly diluted into 1335 mL of 10 mM HEPES buffer at pH 7.5. Further vector concentration and diafiltration was performed using the KrosFlo® KR2i TFF system with a 1000 cm2500 kDa cut-off PS-membrane (both Repligen). First, the eluate was concentrated 20-fold, followed by a diafiltration using the sevenfold volume of X-VIVO medium (Lonza). A final concentration step to a final volume of approximately 90 mL was performed and the vector material frozen at -80°C.

[0258] Transduction of human CD34+cells

[0259] Cryopreserved G-CSF mobilized human peripheral blood CD34+cells (Hemacare) from a male donor were thawed and cultured at a VCD of 2 x 106cells / mL using HSC-Brew medium (Miltenyi Biotec) supplemented with 2% human serum albumin (CSL Behring) as well as 100 ng / mL recombinant human Flt3L, 100 ng / mL recombinant human SCF and 100 ng / mL recombinant human TPO (all Miltenyi Biotec), hereafter called complete HSC-Brew medium. After 16-24h, cells were centrifuged for 5 min at 300 g and seeded at a VCD of 1-2 x 106cells / mL in 24-well or 48-well plates (Coming) at a working volume of 0.25 mL / well using complete HSC-Brew medium. A final concentration of 0.5 mg / mL Poloxamer 407 (Spectrum Chemical) and 8 pg / mL protamine sulfate (Sigma Aldrich) was added for all performed transductions. For transductions using LV preparations derived from stable producer pools, 10 pM PGE2 (Stem Cell Technologies) was additionally added. Afterwards, cells were transduced at MOIs of 0.1, 0.3 and 1.0 for virus preparations derived from stable producer pools, or at MOIs of 0.3, 1.0 and 3.0 for virus preparations derived from stable producer clones in duplicates. For determination of bulk myeloid VCN, cells were washed and resuspended at 0.5 x 106cells / mL in StemSpan SFEM II supplemented with StemSpan CD34+expansion supplement (all Stem Cell Technologies) 24 h post transduction. Cells were passaged every 3-4 days in supplemented StemSpan SFEMII media and kept at a VCD of <lxl06cells / mL. VCNs were evaluated by ddPCR after genomic DNA extraction 14 days post transduction.

[0260] Extraction of genomic DNA

[0261] 14 days post-transduction, 1 x 106cells were collected for genomic DNA extraction. Genomic DNA was extracted using a Maxwell® RSC Cultured Cells DNA Kit and Maxwell® RSC Instruments (all Promega). DNA concentrations were measured using a BioTek Take 3 (Agilent) and adjusted to 10 ng / pl for VCN quantification using ddPCR.

[0262] Digital droplet PCRfor integrated vector copy number quantification

[0263] VCNs were assessed by ddPCR using QX200™ Droplet Digital PCR (Bio-Rad Laboratories) according to manufacturer’s instructions. In brief, 20 pL of the reaction mixture containing 50 ng template DNA, lx ddPCR Supermix for Probes (no dUTPs) (Bio-Rad), 50 units / pL of Haelll restriction enzyme (New England Biolabs), 900 nM of each primer, and 250 nM of each probe (Bio-Rad) was loaded into the sample wells in the QX100 Droplet Generator (Bio-Rad). A total of 40 pL of oil-water emulsion containing approximately 20,000 droplets was generated with the droplet generator and transferred into a separate well of a 96-well PCR plate. PCR was performed under the following thermocycling conditions: enzyme activation at 95 °C for 10 min, 40 cycles of denaturation at 94 °C for 30 sec and annealing / extension at 60 °C for 1 min, and final enzyme deactivation at 98°C for 10 min. After PCR amplification, positive and negative droplets were counted using QX200™ Droplet Reader and QuantaSoft software (BioRad).

[0264] The following primer-probe sets were used to amplify lentiviral psi (Y) and the human ribonuclease P protein subunit p30 (RPP30) as a control for normalization (Y: fwd 5’-TAGTGTGTGCCCGTCTGTTG-3’, rev 5’-CCTCTGGTTTCCCTTTCGCT-3’ and probe 5’-FAM-TCTCTAGCAGTGGCGCCCGA-3’; RPP30: fwd 5’- TGTAAGTGGTAGTGCATAGACTTTA-3’, rev 5’- GTCAAGAGTAGGAGGACATTTGA-3’ and probe 5’-HEX- AGGCAGACTGACACTAGAGTTCAC-3’). VCNs were determined by calculating the number of copies of Y per two copies of RPP30.

[0265] Digital droplet PCRfor determination of vector RNA concentration

[0266] For the determination of the WAS-T2A-GFP FV RNA concentration, all samples were spiked with a P-globin LV with a known vector concentration prior to the extraction process. Samples were then treated with DNAse (Qiagen) and incubated for 10 min at room temperature to digest host cell DNA. Vector RNA extraction was performed using QIAamp Viral RNA Mini Kit and Quicube connect (Qiagen) according to manufacturer’s instructions. The RT-PCR step was done using the High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific). Digital droplet PCR was performed like previously described for VCN determination. The calculated WAS-T2A-GFP LV copy number was corrected using the normalization factor calculated by the calculated P- globin LV copy number.

[0267] The following primer-probe sets were used to amplify the GFP gene and the P- globin gene (rGbG) as a control for normalization (GFP: fwd 5’- CTGCTGCCCGACAACCA-3’, rev 5’-TGTGATCGCGCTTCTCGTT-3’ and probe 5’- HEX-TACCTGAGC ACCCAGTCCGCCCT-3’; rGbG: fwd 5’

[0268] CCCCATACCATCAGTACAAATTGCT-3’, rev 5’-

[0269] TGTTAGAGGACACATGCTCACATACAT-3’ and probe 5’-FAM- CCTCCTTTGCAAGTGTATTTACGACGGT-3 ’ ).

[0270] Lentivirus-Associated HIV p24

[0271] Determination of lentivirus-associated HIV p24 core protein was performed using the commercially available QuickTiter™ Lentivirus Titer Kit (Cell Biolabs) according to manufacturer’s instructions.

[0272] VSV-G enzyme-linked immunosorbent assay (ELISA)

[0273] A two-step sandwich ELISA was developed for quantification of the VSV-G protein on LV. Therefore, 50 pL sample derived from the HIV p24 ELISA were used to determine the relative quantity of VSV-G content based on an optical density (OD) measurement. 100 pL of the primary anti-VSV-G antibody (clone 8G5F11; Absolute Antibody) previously diluted 1:2000 in carbonate buffer were added to each well and incubated for 2 h at 4 °C. After washing the plate three times with 100 pL Pierce™ protein-free blocking buffer (Thermo Fisher), 100 pl of the secondary anti-VSV-G- antibody (clone 1E9F9; Kerafast) diluted 1: 10000 in PBS-Tween20 solution (Sigma Aldrich) were added and incubated for 90 min at 22°C. The wells were washed three times with PBS-Tween20 solution, 100 pL of 1-Step™ Ultra TMB-ELISA solution (Thermo Fisher) were added and incubated for 3 min at 22°C. The reaction was stopped by adding 100 pL ELISA stop solution (Thermo Fisher). OD was determined at 450 / 650 nm using a plate reader (Tecan) and normalized by the previously determined p24 concentration.

[0274] Infectious titer determination

[0275] Functional lentiviral particles were quantified by transduction of adherent HEK293T / 17 cells (ATCC). The cells were cultivated in DMEM medium supplemented with 10% FBS and 1% Penicillin-Streptomycin (Thermo Fisher) at 5% CO2. For infectious titer determination, cells were seeded in 24-wells plates at a VCD of 1 x 105cells / well and a final working volume of 800 pL using DMEM medium supplemented with 10% FBS and 125 pg / mL polybrene (Merck), hereafter called DMEM transduction medium. Virus containing supernatants were thawed quickly and serially diluted in duplicates using DMEM transduction medium, starting from a 50-fold dilution. Fivefold serial dilutions were performed for harvest samples and ninefold serial dilutions for TFF samples. 200 pL of each dilution was added to seeded cells resulting in a final volume of 1 mL per well. Tracking controls were included by diluting a GFP LV preparation with a known virus concentration 25.000-fold using DMEM transduction medium. Negative controls were included by adding 200 pL DMEM medium with 10% FBS instead of virus containing supernatants. Cells were trypsinized 4 days post transduction, washed with PBS and resuspended in cold PBS supplemented with 0.05% glutaraldehyde solution (Sigma-Aldrich). Samples were incubated for 10 min at 4°C, centrifuged for 2 min at 800 g, and resuspended in 200 pL autoMACS® Running Buffer (Miltenyi Biotec). As we have previously reported that a similar proportion of WASp+and GFP+cells is obtained after transductions using the produced vector WAS-T2A-GFP vector (Klimpel et al., 2023), in the present work we only examined the GFP expression for determination of transduction efficiencies and infectious titers. The percentage of GFP+cells was determined using MACSQuant® Analyzer 16 Flow Cytometer (Miltenyi Biotec) and infectious titers were calculated in TU / mL using dilutions that led to a proportion of 5% to 30% GFP positive cells in the sample. Quantification of total DNA concentration

[0276] Total DNA was determined using the Qubit™ dsDNA Assay-Kit (Thermo Fisher). Samples and controls were diluted under light protection using the HS buffer and reagent dilutions supplied with the kit. Samples were incubated for a maximum of 15 min and read using a SPARK® microplate reader (Tecan) at 485 nm excitation and 530 nm emission.

[0277] Statistical analysis

[0278] Statistical analysis was performed using GraphPad Prism 9. The correlation between the integrated transfer vector copy number and infectious titer formation in producer cells was investigated by performing a Pearson correlation test. A two-way ANOVA was performed for the comparison of the transduction of CD34+cells using different LV preparations. A linear regression analysis was performed to assess functional stability of the producer clones. Comparisons that resulted in p values <0.05 were considered as statistically significant. The representation of the p values in the figures was performed as follows: >0.05 (ns), 0.01 to 0.05 (*), 0.001 to 0.01 (**), <0.001 ***)

[0279] Example 2: Suspension adaptation and stable producer pool generation

[0280] Suspension adaptation adherent growing cells were established as previously described (WO 2023 / 242783). Final growth rates of ~0.7 d-1at viabilities of >95% were reached 14 passages after serum removal for both the established GPRGs and GPRTGs packaging cell lines.

[0281] To establish stable producer pools, the suspension-adapted packaging cells were transfected with concatemeric arrays previously generated by in-vitro ligation of a bleomycin (ble) resistance vector and a WAS-T2A-GFP transfer vector at a ratio of 1:25, and stable producer pools were selected using 50 pg / mL Zeocin®. The average integrated copy number of the antibiotic resistance and transfer vector per cell was quantified by digital droplet polymerase chain reaction (ddPCR). The range for the average integrated copy number was 1-6 copies per cell for the ble resistance vector and 5-38 copies per cell for the WAS-T2A-GFP transfer vector (Figure 1A and IB). The generated stable producer pools were induced by doxycycline removal after centrifugation and seeded in doxycycline-free medium in shake flasks. A daily medium exchange was performed by centrifugation for six consecutive days starting from 2 days post induction (dpi), and supernatants were collected for infectious titer determination. Highest infectious titers were determined in the last two collected harvests for all producer pools, with peak titers of 2.37 x 107TU / mL for GPRTGs pool #4 and 3.01 x 107TU / mL for GPRGs pool #4, which were used for further development work (Figure 1C and D). No correlation was found between the integrated copy number of the transfer vector and the average infectious titer formation when a Pearson correlation test was performed ( / ? = 0.2115).

[0282] Example 3: Stable VSV-G transfections and LV production in shake flask

[0283] For the assessment of higher VSV-G expression levels during virus production, the already integrated VSV-G construct was cloned into two vector backbones with a hygromycin or blasticidin resistance cassette and transfected into the highest yielding WAS-T2A-GFP producer pool derived from each packaging cell line. Depending on the antibiotic resistance encoded on the vector, either hygromycin or blasticidin was added 72 h post transfection. After around 30 days of antibiotic selection, stable WAS-T2A- GFP_pVSV-G cell pools were obtained. Stable GPRTGs cell pools could only be generated using blasticidin, while stable GPRGs cell pools could be generated with blasticidin and hygromycin (data not shown). A hygromycin resistant GPRGs-WAS- T2A-GFP_p VSV-G, a blasticidin resistant GPRTGs-WAS-T2A-GFP_p VSV-G pool, and their corresponding parental producer pools were induced by doxycycline removal and seeded in shake flasks. A daily medium exchange was performed by centrifugation starting from 2 dpi and supernatants were collected for eight consecutive days. The range for the maximum viable cell density (VCD) reached during the production was 1.8-2.4 x 107cells / mL (Figure 2A). Viabilities at the end of the production were 86- 92%, while the profiles seem similar between the producer cell pools (Figure 2A). Harvests collected at 3 dpi and 7 dpi were analyzed for LV associated VSV-G content. The average VSV-G content for GPRTGs_pVSV-G-derived LVs was found to be sevenfold higher compared to LVs derived from the parental GPRTGs producer pool. Similarly, for GPRGs_pVSV-G-derived LVs, the VSV-G content was found to be fivefold higher than that of LVs derived from the parental GPRGs producer pool (Figure 2B). All harvests collected from each respective producer pool were combined, purified using an anion exchange chromatography step and concentrated by a subsequent tangential flow filtration (TFF) step.

[0284] Table 1 summarizes infectious titers, physical titers, and the calculated ratios for all four virus preparations at harvest stage (average of days 2-9 following induction) and after TFF concentration. A higher ratio of infectious particles to vector RNA copies was observed in harvests that were generated with producer cell pools that have additional integrated copies of the VSV-G construct (3.79 x 10’4TU / RNA copies for GPRTGs_pVSV-G and 3.36 x IO’4TU / RNA copies for GPRGs_pVSV-G derived LV) compared to their parental cell lines (2.33 x 10’4TU / RNA copies for GPRTGs and 1.75 x 1 O’4TU / RNA copies for GPRGs derived LV). A similar trend was seen for the ratio of infectious particles to RNA copies or to the amount of p24 after TFF concentration.

[0285] Table 1. Characteristics after Harvest and TFF concentration

[0286] Example 4: Transduction of CD34+cells using LV preparations derived from producer cell pools

[0287] The TFF concentrated LV preparations were used to transduce human CD34+cells at multiplicities of infection (MOIs) of 0.1, 0.3 and 1.0. The percentage of green fluorescent protein (GFP) positive cells and the integrated vector copy number (VCN) was determined 14 days post transduction (Figure 2C and 2D). For LV preparations derived from the parental producer pools, those produced by GPRTGs cells were showing a higher transduction efficiency of 17.4% and a VCN of 0.37 at an MOI of 1 compared to LVs derived from GPRGs cells, showing transduction efficiencies of 7.7% and a VCN of 0.14 (both p values <0.0001). However, when the GPRGs cell pool was stably transfected with the generated VSV-G plasmid and expressing LVs with higher VSV-G content, the transduction efficiency and VCN increased to 17.8% and 0.37 respectively, which is comparable those obtained by GPRTGs derived LVs. In contrast, when transduction data using LVs derived from the parental GPRTGs cell pool and the GPRTGs_pVSV-G cell pool with higher VSV-G content are compared, slightly lower transduction efficiencies (16.4% vs. 17.4%; p = 0.0566) and VCNs (0.34 vs. 0.37; p = 0.0425) were found. At the investigated MOIs, all virus preparations were transducing CD34+cells in a dose-dependent manner.

[0288] Example 5: Producer clone screening and stability study

[0289] Single cell sorting was performed as cell line monoclonality is considered as a requirement from the regulatory authorities. Single stable producer cells of each cell line were sorted, expanded and a certain proportion of each culture was split off and induced to screen for the best producing cell clones. The production was performed in semiperfusion mode in shaken 6-well format. Harvest collected at 6 dpi was used for determination of infectious titers and cell counts were performed to estimate cell specific productivities. Highest average infectious titers of 1.03 x 107TU / mL (n = 26) were found for GPRGs_pVSV-G cell clones compared to clones derived from parental GPRGs (5.04 x 106TU / mL; n = 19), parental GPRTGs (3.56 x 106TU / mL; n = 23) and GPRTGs_pVSV-G (5.00 x 106TU / mL; n = 6) cell lines (Figure 3A). However, average cell specific productivities were highest for GPRTGs_pVSV-G derived cell clones (1.75 TU / cell / day) compared to clones derived from parental GPRGs (0.24 TU / cell / day), GPRGs_VSV-G (0.71 TU / cell / day) and parental GPRTGs (0.88 TU / cell / day) cell lines (Figure 3B). In the small-scale model based on 6-well plates that was used for the LV production screening after the sorting process, we observed a slower cell growth for the GPRTGs clones compared to GPRGs clones.

[0290] Three clones from each type of cell line, resulting in a total of 12 clones, were selected based on cell specific productivities and cell growth. The average integrated copy number for the transfer vector was 6-9 copies per cell for GPRTGs-derived clones and 16-33 copies per cell for GPRGs-derived clones (Figure 3C and 3D). The integrated copy number for the ble resistance vector was 3-6 copies per cell for all investigated clones. All 12 clones were investigated for LV production in shake flask in semiperfusion mode and repetitively induced after continuous passaging to assess the functional stability (Figures 3E-H). Highest average infectious titers of 9.38 x 107TU / mL were obtained by clone 102 derived from a GPRTGs_pVSV-G producer pool (Figure 3G), which corresponds to a volumetric yield of 4.69 x 108TU / mL for a 7-day production. In contrast to the productions performed in 6-wells for the initial screening, a similar growth for all clones in shake flask (data not shown) was observed. A linear regression analysis was performed to assess functional stability after continuous passaging for 71 days and repetitive induction. Out of six clones derived from the GPRTGs packaging cell line (Figure 3E and 3G), only clone 45 was showing a significant change in productivity after more than 43 days of cultivation (p = 0.0148). Out of six clones derived from the GPRGs packaging cell line (Figure 3F and 3H), clone 52 (p = 0.0155), clone 56 (p = 0.0204) and clone 138 (p = 0.0081) were showing a significant change in productivity.

[0291] Example 6: LV production in 5L perfusion stirred-tank bioreactors

[0292] A producer clone manufacturability assessment was performed in parallel to the conducted stability study. Therefore, one producer clone derived from the parental GPRTGs (clones 45), parental GPRGs (clone 56), GPRTGs_pVSV-G (clone 102) and GPRGs_pVSV-G (clone 155) cell line was used for a LV production in 5L stirred-tank bioreactor in perfusion. The cells were inoculated in doxycycline-free medium without washing to mediate induction of LV expression by a previously described scalable dilution method (Klimpel et al., 2023). Perfusion was initiated at 2 dpi using a cell separation device based on acoustic wave separation. Clone 102 and clone 56 were able to maintain a target VCD of 1.5-2.0 x 107cells / mL up to 35 dpi and 36 dpi (Figure 4A) at viabilities higher than 88% and 75%, respectively (Figure 4B). Cell viabilities for clone 45 and clone 155 dropped below 70% at 16 dpi and 15 dpi respectively, and subsequently the VCD for clone 45 dropped below the target VCD. The cell-free harvest was continuously collected for a cycle of 24 h and sampled for infectious titer determination (Figure 4C). The productivity profile was found to be specific for each clone. Clone 56 was producing infectious titers of >1 x 107TU / mL for 29 consecutive days, reaching titers of >2 x 107TU / mL starting from 20 dpi with peak titers of up to 3.68 x 107TU / mL at 33 dpi. In comparison, clone 102 produced titers of > 1 x 107TU / mL for 16 consecutive days with the highest infectious titers of up to 2.55 x 107TU / mL at 15 dpi. Clone 45 produced titers of >1 x 107TU / mL for 12 consecutive days with the highest infectious titers of up to 2.81 x 107TU / mL at 10 dpi. Clone 155 was only able to produce infectious titers >1 x 107TU / mL at 7 dpi, with titers dropping below the detectable limit of the infectious titer assay after 11 dpi. Next-generation sequencing was performed to investigate the genomic integrity of the produced LVs at 5 dpi and a time point near the end of each process, depending on the production duration supported by each clone. Looking at the number of mismatches with a variant frequency >5%, no differences were found between early and late harvested LVs for clones 45, 56 and 102, and an increase from five to six mismatches was found for clone 155 (data not shown). The obtained data suggest that the length of the production process does not affect the integrity of the LV genome.

[0293] The vector harvest collected at 7 and 14 dpi were purified and concentrated for the transduction of CD34+cells. The obtained infectious titers, vector RNA copy concentrations, and residual total DNA concentration in the final TFF concentrate are summarized in Table 2. Infectious titers of 4.25 x 108-3.66 x 109TU / mL were determined in the final TFF retentates, except for preparations obtained by clone 155, showing a titer of 8.16 x 107TU / mL and 3.44 x 106TU / mL respectively. Residual total DNA concentrations in the TFF retentates were higher for harvest collected at 14 dpi compared to harvest collected at 7 dpi for all clones. Values of 3.2-72.4 ng / 107TU were determined for the clones 45, 56, and 102. For clone 155, a DNA concentration of 4928.3 ng / 107TU was determined in the TFF retentate of harvest collected at 14 dpi, while it should be noted that infectious titers in the supernatant were below the detection limit (Figure 4C), which may lead to increased DNA binding to the anion exchanger during the chromatography step.

[0294] Furthermore, 20 L of vector harvest collected at 15-18 dpi for clone 102 were purified and 44-fold concentrated to a vector concentration of 9.99 x 108TU / mL (Figure 4D). The overall yield from harvest to the final TFF retentate was 19%, and the residual determined total DNA concentration was 6.2 ng / 107TU.

[0295] Table 2, Characteristics of TFF retentate harvest a 7 dpi and 14 dpi

[0296] Example 7: Transduction of human CD34+cells using LV preparations derived from producer clones

[0297] The TFF concentrated vector preparations derived from harvest collected at 7 dpi (early vector material) and 14 dpi (late vector material) of each producer clone were used to transduce human CD34+cells at MOIs of 0.3, 1.0 and 3.0. The TFF concentrated late vector material from clone 155 was not tested due to the low vector concentration. Bulk VCNs of myeloid cultures were determined 14 days post transduction by ddPCR (Figures 5A-D). Furthermore, cell differentiation was characterized and colony specific VCNs were determined for cells transduced at an MOI of 1 (data not shown).

[0298] Overall, concentrated LV preparations derived from late vector material resulted in higher bulk VCNs compared to the respective early vector material. Comparing late vector material at an MOI of 3, the highest bulk VCN of 1.50 copies / cell was obtained by GPRTGs_VSV-G-derived LVs from clone 102 (Figure 5C), which is significantly higher compared to the bulk VCN of 0.89 copies / cell obtained by GPRTGs-derived LVs from clone 45 (p = 0.0055) (Figure 5A) and 0.18 copies / cell obtained by GPRGs-derived LVs from clone 56 (p <0.0001) (Figure 5B). Similar bulk VCNs were obtained by early and late vector material derived from clone 45 at an MOI of 3 (0.75 vs. 0.89; p = 0.9585). A larger, non-significant difference was found between the bulk VCNs obtained by early and late vector material derived from clone 56 (VCN of 0.18 vs. 0.59; p = 0.1191). In contrast, significantly higher bulk VCNs were obtained by late vector material compared to early vector material derived from clone 102 (VCN of 1.50 vs. 0.35; p < 0.0001).

Claims

CLAIMS1. A stable producer cell line capable of producing an enveloped virus in a suspension cell culture, wherein the stable producer cell line is derived from a packaging cell line transfected with vesicular stomatitis virus G protein (VSV-G), and wherein the stable producer cell line is stably re-transfected with a VSV-G encoding plasmid.

2. The stable producer cell line of claim 1, wherein the cell line is capable of producing the enveloped virus with an infectious titre of at least 5.0 x 106TU / mL of culture medium at 6 days following induction of enveloped virus production.

3. The stable producer cell line of claim 1, wherein the cell line is capable of producing the enveloped virus with an average infectious titre of at least 3.0 x 107on day 2 through day 9 following induction of enveloped virus production.

4. The stable producer cell line of claim 1, wherein the cell line is capable of producing the enveloped virus with an average infectious titre of at least 4.0 x 107on day 2 through day 9 following induction of enveloped virus production.

5. A stable producer cell line stably expressing VSV-G, wherein the stable producer cell line is capable of producing an enveloped virus in a suspension cell culture with an infectious titre of at least 5.0 x 106TU / mL of culture medium at 6 days following induction of enveloped virus production.

6. The stable producer cell line of claim 1, wherein the cell line is capable of producing the enveloped virus with an infectious titre of at least 5.5 x 106TU / mL, or at least 6.0 x 106TU / mL, or at least 7.0 x 106TU / mL, or at least 8.0 x 106TU / mL, or at least 9.0 x 106TU / mL, or at least 1.0 x 107TU / mL of culture medium at 6 days following induction of enveloped virus production.

7. The stable producer cell line of claim 1, wherein the cell line is capable of producing the enveloped virus with an infectious titre of at least 1.0 x 107TU / mL, or at least 1.5 x 107TU / mL, or at least 2.0 x 107TU / mL, or at least 2.5 x 107TU / mL, or at least 3.0 x 107TU / mL of culture medium at 14 days following induction of enveloped virus production.

8. The stable producer cell line of claim 1, wherein the cell line is derived from a producer pool capable of producing the enveloped virus with a VSV-G content at least 2-fold greater, or at least 3-fold greater, or at least 4-fold greater, or at least 5-fold greater, or at least 6-fold greater than the VSV-G content of an enveloped virus produced from a stable producer cell line derived from a producer pool that has not been stably retransfected with a VSV-G encoding plasmid.

9. The stable producer cell line of claim 1, wherein the cell line is derived from a producer pool capable of producing the enveloped virus with a VSV-G content of at least 5-fold greater than the VSV-G content of an enveloped virus produced from a stable producer cell line derived from a producer pool that has not been stably re-transfected with a VSV-G encoding plasmid.

10. The stable producer cell line of claim 1, wherein the cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of at least 1.0 x 10’4TU / RNA copies, or at least 1.5 x 10’4TU / RNA copies, or at least 2 x 10’4TU / RNA copies, or at least 2.5 x 10’4TU / RNA copies, or at least 3.0 x 10’4TU / RNA copies, or at least 3.3 x 10’4TU / RNA copies.

11. The stable producer cell line of claim 1, wherein the cell line is capable of producing the enveloped virus with a ratio of infectious particles to viral vector genome RNA copies of between about 1.5 x 10’4TU / RNA copies and 4.0 x 10’4TU / RNA copies.

12. The stable producer cell line of claim 1, wherein the cell line is capable of producing the enveloped virus with a viral vector genome RNA concentration of at least 1.0 x 1011copies / mL, or at least 5.0 x 1011copies / mL, or at least 1.0 x 1012copies / mL, or at least 5.0 x 1012copies / mL, or at least 1.0 x 1013copies / mL cell culture medium.

13. The stable producer cell line of claim 1, wherein the cell line is stable for at least 70 days.

14. A method of producing a stable producer cell line stably expressing VSV-G, wherein the cell line is capable of producing an enveloped virus in a suspension cell culture, the method comprising stably integrating a plasmid comprising a gene of interest and a VSV-G encoding plasmid into a packaging cell line, wherein the packaging cell line is transfected with VSV-G.

15. A method of producing a stable producer cell line stably expressing vesicular stomatitis virus G protein (VSV-G), wherein the cell line is capable of producing an enveloped virus in a suspension cell culture, the method comprising stably integrating a VSV-G encoding plasmid into a stable producer cell line, wherein the stable producer cell line is derived from a packaging cell line transfected with VSV-G.

16. A method of producing an enveloped virus in a suspension cell culture, the method comprising culturing a stable producer suspension cell line, wherein the stable producer suspension cell line is transfected with a VSV-G encoding plasmid and wherein the stable producer cell line is derived from a packaging cell line transfected with VSV- G.

17. The method of claim 15, wherein the enveloped virus is harvested from the suspension cell culture fluid.

18. The method of claim 17, wherein the enveloped virus is harvested from the suspension cell culture at least 7 days following induction of enveloped virus production.

19. The method of claim 17, wherein the enveloped virus is harvested from the suspension cell culture at least 14 days following induction of enveloped virus production.

20. The method of claim 15, wherein the suspension cell culture is at a pH of between 6.0 and 8.0 and / or at a temperature of between 35-39 °C.

21. The method of claim 15, wherein the suspension cell culture has a volume of greater than about 1 L, about 2 L, about 5 L, about 10 L, about 50 L, about 100 L, about 500 L, about 1,000 L.

22. The method of claim 17, wherein the method further comprises purifying the enveloped virus from the harvested suspension cell culture fluid.

23. The method of claim 22, wherein purifying the enveloped virus comprises one or more steps selected from the group consisting of clarification filtration, anion exchange chromatography, concentration and diafiltration.

24. The method of claim 22, additionally comprising formulating the purified enveloped virus into a pharmaceutical formulation or into a solution suitable for infecting a cell.

25. The stable producer cell line of claim 1, wherein the cell is a GPRG, GPRGT or GPRTG cell or a derivative thereof.

26. The stable producer cell line of claim 25, wherein the cell is a GPRG or GPRTG cell.

27. The stable producer cell line of claim 1, wherein the enveloped virus is a retrovirus.

28. The stable producer cell line of claim 27, wherein the retrovirus is a lentivirus.

29. A purified enveloped virus produced by the method according to claim 15.

30. A method of transducing a cell with an enveloped virus in a cell culture, the method comprising contacting the cells with the enveloped virus, wherein the enveloped virus is produced from the stable producer cell line of claim 1.

31. A method of increasing integrated vector copy number (VCN) of a cell transduced with an enveloped virus in a cell culture, the method comprising contacting the cells with the enveloped virus, wherein the enveloped virus is produced from the stable producer cell line of claim 1.

32. The method of claim 30, wherein the method results in a transduction efficiency of at least 10% 14 days post transduction of the cell.

33. The method of claim 30, wherein the method results in a transduction efficiency of at least 15% 14 days post transduction of the cell.

34. The method of claim 30, wherein the cell culture has a multiplicity of infection (MOI) of at least 0.1.

35. The method of claim 30, wherein the cell culture has a MOI of at least 0.3.

36. The method of claim 30, wherein the cell culture has a MOI of at least 1.0.

37. The method of claim 30, wherein the method results in a vector copy number(VCN) of at least 0.1 copies / cell 14 days post transduction of the cell at MOI of 1.

38. The method of claim 30, wherein the method results in a VCN of about 0.2 copies / cell 14 days post transduction of the cell at MOI of 1.

39. The method of claim 30, wherein the cell is a hematopoietic stem cell (HSC) or a hematopoietic progenitor cell (HPC).

40. The method of claim 39, wherein the HSC or HPC is a CD34 positive (CD34+) cell.

41. The method of claim 30, wherein the cell line is stable for at least 70 days.

42. A method of producing a VSV-G re-transfected packaging cell line, the method comprising stably integrating a VSV-G encoding plasmid into a packaging cell line, wherein the packaging cell line is transfected with VSV-G.

43. A method of producing a stable producer cell line stably expressing VSV-G, wherein the cell line is capable of producing an enveloped virus in a suspension cell culture, the method comprising stably integrating a plasmid comprising a gene of interest into the VSV-G re-transfected packaging cell line of claim 42.

44. A packaging cell line transfected with vesicular stomatitis virus G protein (VSV- G), that has been stably re-transfected with a VSV-G encoding plasmid.

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