Methods and kits for improved fermentative production of recombinant viruses

Abstract

The present invention relates to methods and kits for the improved fermentative production of recombinant adeno-associated virus (AAV), adenovirus, lentivirus or retroviruses such as gamma-retrovirus in HEK293 cells. Further described is the use for increasing the fermentative production of said virus in HEK293 cells.

Description

Technical Field

[0001] The present invention relates to methods and kits for the improved fermentative production of recombinant adeno-associated virus (rAAV), adenovirus, lentivirus, or retroviruses such as gamma-retrovirus in HEK293 cells. Further described is the use for increasing the fermentative production of said virus in HEK293 cells.

Background Art

[0002] The field of gene therapy using recombinant adeno-associated virus (rAAV) has reached maturity after about 18 years since the first clinical trial for cystic fibrosis. rAAV has interesting properties for gene therapy. When injected into mice, rAAV can induce a transient increase in cytokine levels, but it has been found that the induced immunogenicity is minimal and there is little inflammation after a few hours. Since rAAV can transduce non-dividing cells in various tissues or organs, it is suitable for many medical applications. Clinical evidence after gene therapy treatment with rAAV for lipoprotein lipase deficiency (alipogene tiparvovec or Glybera®) and Leber congenital amaurosis also shows that rAAV has good tolerance and can induce transgene expression in humans for several years.

[0003] For the production of rAAV, mammalian cell production systems are used. These cells can assist in the replication of viral vectors (e.g., recombinant adenovirus, AdV).

[0004] To produce rAAV, transient transfection methods using two or three plasmids have also been developed. These methods can be used not only to rapidly test rAAV constructs but also to scale up to 1000 L. The advantage of transient transfection is that there are no residual helper virus contaminants. To facilitate the production of rAAV, several stable cell lines (producer / packaging cells) have also been created.

[0005] The HEK293 cell line is used for rAAV production. The HEK293 cell line can be easily adapted to suspension culture in serum-free medium. Furthermore, a number of viral vectors have already been approved for Phase I / II / III clinical trials. Originally, HEK293 cells grow in an adherent state, so there are limitations in scalability for rAAV production. This limitation has been overcome by adapting the HEK293 cell line to grow in a floating state.

[0006] Efficient large-scale production of rAAV can be achieved with specially genetically engineered HEK293 such as HEK293T cells into which the SV40 large T antigen has been inserted or unique high-producer HEK293 cells, but there is a need for an rAAV production system based on HEK293 cells that does not require genetic modification of HEK293 cells and does not require the use of unique high-producer cells.

Summary of the Invention

Means for Solving the Problems

[0007] In a first aspect, the present invention is a method for the fermentative production of recombinant adeno-associated virus (rAAV), adenovirus, lentivirus or retrovirus, comprising the following steps: Step 1: Culturing rAAV, lentivirus, adenovirus or retrovirus in HEK293 cells over an incubation period in a culture medium containing an amount of interferon (IFN) inhibitor; Step 2: Recovering rAAV, lentivirus, adenovirus or retrovirus from cell cultures and providing a method comprising the same.

[0008] In a second aspect, the present invention provides a method for the fermentative production of adeno-associated virus (AAV), lentivirus, adenovirus or retrovirus, the method comprising the following steps: Step 1: Performing transcriptome analysis of HEK293 cells transfected with rAAV, lentivirus, adenovirus or retrovirus and comparing with a negative control to determine if the transcription of at least one of the following genes: (i) TNFA_signaling_via_NFKB; (ii) Interferon_Gamma_Response; (iii) Interferon_Alpha_Response; (iv) TGF_beta_Signaling; and / or (v) IL6_JAK_STAT3_Signaling is enriched, preferably, if the transcription of the following: (1) interferon_gamma response; and / or (2) interferon_alpha_respone is enriched, selecting HEK293 cells; Step 2: Culturing rAAV, lentivirus, adenovirus or retrovirus in the selected HEK293 cells in a culture medium containing an amount of interferon (IFN) inhibitor for an incubation period; Step 3: Recovering rAAV, lentivirus, adenovirus or retrovirus from cell cultures and providing a method comprising the same.

[0009] In a third aspect, the present invention provides the use of an IFN inhibitor transfected or infected with rAAV, lentivirus, adenovirus or retrovirus.

[0010] In a fourth aspect, the present invention provides a method for preparing a pharmaceutical composition, comprising: (i) performing the method according to the present invention, wherein rAAV, adenovirus, lentivirus or retrovirus contains a therapeutic gene; and (ii) adding one or more pharmaceutically acceptable excipients to the prepared rAAV particles, adenovirus particles, lentivirus particles or retrovirus particles to obtain a pharmaceutical composition.

[0011] In a fifth aspect, the present invention provides a kit for use in cell culture, (a) HEK293 cells, and (b) an IFN inhibitor, preferably an IFN inhibitor selected from the group consisting of ruxolitinib, tofacitinib, baricitinib, delgocitinib, emapalumab and fontolizumab, and any structural analog thereof having IFN inhibitory activity. More preferably, the inhibitor is ruxolitinib and any structural analog thereof having inhibitory activity against JAK family kinases in the IFN signaling pathway. Particularly preferably, the structural analog has inhibitory activity against the Jak1 component of at least 50%, more preferably 80%, even more preferably at least 90%, and most preferably at least 95% compared to ruxolitinib.

[0012] By performing transcriptome analysis, the inventors surprisingly discovered that various available HEK293 cells can be characterized by the unique pattern of genes activated during triple transfection for rAAV production. For example, the inventors discovered that interferon response genes are activated in some HEK293 cells with relatively low rAAV production yields in fermentation production.

[0013] This was unexpected in light of Strasser et al., Int. J. Mol. Sci 22 (2021), 11499. In the latter paper, the impact of rAAV5 production on the proteome of HEK293 cells was investigated. The authors discovered that the EIF2 signaling pathway, oxidative phosphorylation, and spliceosome cycle were activated. In a further paper by Barnes et al., Mol. Ther. Nucleic Acids 26 (2021), 94, genome-wide activation screening was used to identify gRNAs that might be useful for increasing rAAV production in HEK293T cells. It was found that SKA2 or ITPRIP might be useful for increasing rAAV production in HEK293T cells.

[0014] Therefore, the inventors' discovery is surprising in view of this detailed research that provided no relevant guidance. The inventors further explored this unexpected discovery by administering IFN inhibitors, particularly IFN signaling inhibitors, and found that rAAV production in these HEK293 cells increased. By adding IFN inhibitors, rAAV virus production increased to more than 100% and even up to 200% after several days. This is extremely important for the desired large-scale rAAV production in HEK293 cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0015]

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[0016] In a first aspect, the present invention is a method for the fermentative production of recombinant adeno-associated virus (rAAV), adenovirus, lentivirus or retrovirus, comprising the following steps: Step 1: culturing rAAV, adenovirus, lentivirus or retrovirus in HEK293 cells over an incubation period in a culture medium containing an amount of an interferon (IFN) inhibitor; and Step 2: recovering rAAV, adenovirus, lentivirus or retrovirus from the cell culture. A method is provided.

[0017] "AAV" as used herein includes AAV serotypes, their chimeras or hybrids. The AAV serotype can be selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, and their chimeric or hybrid variants. Preferably, the AAV serotype is selected from AAV5, AAV6, AAV8, AAV9, AAV-DJ, more preferably AAV8. The chimeric AAV variant contains two or more AAV serotypes. The hybrid AAV variant contains a mutant AAV serotype containing modifications.

[0018] AAV is a small virus that infects humans and some other primate species. AAV belongs to the genus Dependoparvovirus and the family Parvoviridae. AAV is a small (20 - 25 nm), non-enveloped virus lacking replication, and has a linear single-stranded DNA (ssDNA) genome of approximately 4.8 kilobases (kb). rAAV is thought to be maintained mainly as an episome.

[0019] For safety reasons, rAAV is usually prepared from two, three or even several individual expression units. Thus, many vector systems are produced by transient co-transfection of multiple plasmids. Stable transfection is also possible.

[0020] In the case of AAV, the so-called triple transfection method is well known. For this purpose, (i) a plasmid carrying the therapeutic gene of interest, (ii) a plasmid carrying AAV genes such as rep and cap, and (iii) a plasmid carrying adenovirus 5 genes such as E4, E2a and VA are transfected into host cells.

[0021] Alternatively, recombinant AAV can also be obtained by the double transfection method. In this method, (i) a plasmid having a therapeutic gene of interest, and (ii) a plasmid having adenovirus 5 genes such as E4, E2a and VA and further containing the AAV rep and cap genes are transfected into a host cell.

[0022] As used herein, "adenovirus" includes an adenovirus packaging system such as adenovirus serotype 5 (Adv5) using an adenovirus shuttle vector that transports a target gene sequence to an E1 expression-producing cell line. E1 and E3, which are viral early transcription units, are deleted in the most commonly used recombinant adenovirus packaging system. This system can be a two-vector system consisting of an adenovirus expression plasmid containing different promoters and tags and an adenovirus genomic backbone with a deletion in the E1 region on the plasmid.

[0023] As used herein, "lentivirus" includes HIV and its variants, including lentiviruses having a pseudotyped VSV-G envelope protein. Preferably, the lentivirus is derived from HIV-1.

[0024] Lentiviruses are a genus of retroviruses that cause chronic and lethal diseases characterized by long latency periods in humans and other mammalian species. The human immunodeficiency virus (HIV) that causes AIDS is also included in this genus. Lentiviruses are distributed worldwide and are known to infect primates, cows, goats, horses, cats, and sheep, as well as several other mammals. To enhance the safety of lentiviruses, the components required for virus production are split into multiple plasmids (three in the second-generation system and four in the third-generation system). The components of both systems are as follows:

[0025] A lentiviral transfer plasmid encoding the insert of interest. The transgene sequence is flanked by long terminal repeat (LTR) sequences that facilitate the integration of the transfer plasmid sequence into the host genome. Typically, upon viral transduction, the sequence between the LTRs and the LTRs are integrated into the host genome. Many lentiviral transfer plasmids are based on the HIV-1 virus. For safety reasons, the transfer plasmid is replication-incompetent and can also contain additional deletions in the 3' LTR to "self-inactivate" (SIN) the virus after integration.

[0026] Packaging plasmid(s). The packaging plasmid may contain the gag, pol, rev, and tat genes in one plasmid or dispersed among two plasmids.

[0027] Envelope plasmid. Preferably, the envelope plasmid encodes VSV-G.

[0028] Lentivirus is one of the most efficient methods of gene delivery because it can integrate a substantial amount of viral nucleic acid DNA into the DNA of host cells and can efficiently infect non-dividing cells. The virus can integrate into the host's germline genome and become endogenous, thereby being passed on to the host's offspring thereafter.

[0029] "Retrovirus" as used herein includes gammaretrovirus. Preferably, the retrovirus is murine leukemia virus. In a more preferred embodiment, the retrovirus is derived from HIV-1.

[0030] "HEK293 cells" as used herein includes human fetal kidney cells first isolated and cultured by Alex van der Eb. These were transfected with sheared adenovirus 5 (Ad5) DNA by Frank Graham (Graham FL, Smiley J, Russell WC, Nairn R (July 1977). The Journal of General Virology. 36(1):59-74). When the adenovirus gene is integrated into the HEK293 cell genome, the cells have become capable of efficiently producing large amounts of recombinant protein from plasmid vectors. This term also includes any derivative strains of HEK293 cells, such as HEK293-F, HEK293-H, HEK293-T and HEK293FT, as well as HEK293-E, also known as HEK293-EBNA1. HEK293 cell lines and their derivative strains are available from cell culture banks such as ATCC or ECACC and private suppliers.

[0031] Preferably, when used in the method of the present invention, HEK293 cells produce increased yields of rAAV, adenovirus, lentivirus or retrovirus in the culture medium in the presence of an IFN inhibitor. "Increased yield" can be defined as the yield of rAAV, lentivirus or retrovirus produced in the HEK293 cell line in the presence of an IFN inhibitor, which is higher compared to the yield produced in the HEK293 cell line under the same culture conditions and for the same incubation period in the absence of the IFN inhibitor. Preferably, "increased yield" means that the yield is at least 0.2-fold higher, at least 0.3-fold higher, at least 0.5-fold higher, at least 0.7-fold higher, at least 1.0-fold higher, at least 1.5-fold higher, at least 2.0-fold higher, at least 2.5-fold higher, at least 3.0-fold higher, at least 4-fold higher, or at least 5-fold higher compared to the yield produced in the HEK293 cell line in the absence of the IFN inhibitor. Preferably, the yield is determined over an incubation period of 96 hours in the presence of 5 μM ruxolitinib as the IFN inhibitor, as outlined in the examples.

[0032] In certain embodiments, "increased yield" is at least about 1×10 9 vector genome-containing particles (vg / mL) per milliliter of cell culture from HEK293 cells in the presence of an inhibitor, not only after concentration of the product, but also in all upstream and downstream process steps, e.g., at least about 5×10 9 、1×10 10 、5×10 10 、1×10 11 、1×10 12 、1×10 13 or 1×10 14 or more vector genome-containing particles, and can be defined by the rAAV yield. Preferably, the yield is determined as described in the Examples. The yield can also be measured as capsid particles per liter or milliliter of cell culture. In certain embodiments, at least about 1×10 9 vector genome-containing particles (vg / mL) per milliliter of cell culture from HEK293 cells in the presence of an inhibitor, e.g., at least about 5×10 9 、1×10 10 、5×10 10 、1×10 11 、1×10 12 、1×10 13 or 1×10 14 or more capsid particles of AAV yield are obtained.

[0033] In preferred embodiments, the HEK293 cells of the invention are at least one of the following cell pathways at the indicated time points: (1)interferon_gamma response (0 - 48 hours); (2)interferon_alpha_respone (0 - 48 hours) characterized by activation of.

[0034] Activation of the cellular pathway is achieved by triple transfection of the HEK293 cells to be transfected with the plasmids necessary for rAAV8 replication. Further details are outlined in the Examples section.

[0035] In another preferred embodiment, the HEK293 cells of the present invention are at least one of the following cellular pathways: (i) TNFA_signaling_via_NFKB; (ii) Interferon_Gamma_Response; (iii) Interferon_Alpha_Response; (iv) TGF_beta_Signaling; (v) IL6_JAK_STAT3_Signaling characterized by activation of.

[0036] Preferably, the HEK293 cells show activation of at least two, at least three, at least four, more preferably at least five of the cellular pathways listed above.

[0037] More preferably, at least two of the activated cellular pathways are selected from the group consisting of TNFA_signaling_via_NFKB, interferon_gamma response, interferon_alpha_response, TGF_beta_Signaling, IL6_JAK_STAT3_Signaling.

[0038] More preferably, at least two of the activated genes are selected from the group consisting of interferon_gamma response, interferon_alpha_response and IL6_JAK_STAT3_Signaling.

[0039] Most preferably, the HEK293 cells used in the method of the present invention are CRL-1573.

[0040] In a preferred embodiment, the HEK293 cells are an adherent cell line. In an even more preferred embodiment, the HEK293 cells are a suspension cell line or are adapted to grow in a suspension state. Depending on whether the HEK293 cells are an adherent cell line or a suspension cell line, different culture methods are available to those skilled in the art. The culture can be carried out in a Petri dish, a shaker flask or a roller bottle. Culture in a shaker flask or a roller bottle is preferred.

[0041] "Culture medium" as used herein includes any medium suitable for culturing mammalian cells. The medium can be a complex medium, a semi-defined medium, or a defined medium. Preferably, the culture medium is a medium with chemically defined components. More preferably, a commercially available medium such as a mammalian cell culture medium known to those skilled in the art is used.

[0042] The culture conditions can vary according to the specific requirements of the cells in terms of parameters such as pH, salt concentration, temperature, etc. The process can be carried out in a batch mode or a fed-batch mode as a continuous process well known to those skilled in the art. Preferably, the process is carried out in a batch mode or a fed-batch mode.

[0043] As used herein, "IFN inhibitor" includes inhibitors of the interferon immune response pathway. Interferons are a group of signaling proteins made by host cells in response to the presence of some viruses. In a typical scenario, virus-infected cells release interferons to enhance the antiviral defenses of neighboring cells. Type I interferons can be broadly classified into three groups: IFN-I, type II (IFN-II), and type III (IFN-III), and are further classified based on differences in the gene loci of IFN transcription genes and cognate receptors. IFN-I is the largest and best-characterized group, with seven classes: IFNα, IFNβ, IFNδ, IFNε, IFNκ, IFNω, and IFNτ, while IFN-II includes IFNγ. IFN-I and IFN-II signal through IFNαR1 / R2 (IFNAR) and IFNγR1 / R2 (IFNGR), respectively. The last class, IFN-III, is classified as an "IFN-like cytokine" and consists of interleukin (IL)-28A (IFNλ2), IL28B (IFNλ3), and IL29 (IFNλ1), and signals through the IL-28RI / IL10R2 receptor chain.

[0044] When microbial products are recognized by various cell surface and intracellular pattern recognition receptors, including Toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I), genes encoding type I interferons (IFNs), which are mediated by several different signaling pathways, are induced. When type I IFN binds to its receptor (IFNAR), multiple downstream signaling pathways are induced, which can lead to diverse biological effects. The canonical signal transducer and activator of transcription 1 (STAT1)-STAT2-interferon regulatory factor 9 (IRF9) signaling complex (also known as the interferon-stimulated gene factor 3 (ISGF3) complex) binds to interferon-stimulated response elements (ISREs) in gene promoters, resulting in the induction of numerous interferon-stimulated genes (ISGs). Type I IFN can also signal through STAT1 homodimers, which is more commonly associated with the signaling pathway through IFNγ. Other STAT heterodimers and homodimers, including STAT3, STAT4, and STAT5, can also be activated downstream. Other signaling pathways that are independent of Janus kinase (JAK) and / or STAT activity, including the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways, can also be activated, thereby having diverse effects on cells.

[0045] The IFN inhibitor for use in the present invention is intended to be used to target the IFN induction and / or IFN signaling cascade. Activation of multiple signaling pathways by the engagement of IFN with its receptor is essential for the occurrence of biological functions mediated by IFN. As used herein, the term "IFN induction cascade" encompasses any viral induction pathway leading to the activation of the transcription and expression of IFN-a / β. Molecules of the IFN induction cascade that can be targeted by the inhibitor may include, but are not limited to, TLR3, MDA5, RIG-I, Cardif, TBK1 / IKKe, IKKa / , IRF3, NFKB or ATF-2 / C-JUN. As used herein, the term "IFN signaling cascade" encompasses any IFN-activated signaling pathway that is activated to bring about antiviral changes in cells. Targeted signaling pathways may include, but are not limited to, the JAK-STAT pathway.

[0046] The inhibitor for use in the present invention can target one or more components of the IFN induction and / or IFN signaling cascade shown in Table 1. The inhibitors used in the present invention can be used alone or in combination.

[0047]

Table 1

[0048] In a preferred embodiment of the present invention, the inhibitor for use in the present invention targets the JAK / STAT signaling pathway. The inhibitor can target any component of the JAK / STAT pathway. Target components of the JAK / STAT pathway may include, but are not limited to, Tyk2, Jak1, Jak2, STAT-1, STAT-2 and IRF-9. In a more preferred embodiment, the inhibitor targets Jak1. In an even more preferred embodiment of the present invention, the inhibitor for use in the present invention targets the TBK-1 / IKKe / IKK2 induction pathway.

[0049] The inhibitor for use in the present invention can be any molecule that reduces the activity of IFN induction and / or the IFN signaling cascade. Non-limiting examples of inhibitors that can be used in accordance with the present invention include small molecule inhibitors, siRNA, miRNA, lipocalin, plastic antibodies, antibodies or antibody fragments.

[0050] The IFN inhibitor is preferably not selected from niacin and / or niacinamide.

[0051] Preferably, the IFN inhibitor is not selected from (i) methyl nicotinate, (ii) myo-inositol, and (iii) choline.

[0052] In a more preferred embodiment, the IFN inhibitor is not selected from (iv) a combination of methyl nicotinate and myo-inositol, (v) a combination of methyl nicotinate and choline, and (vi) a combination of myo-inositol and choline.

[0053] In a more preferred embodiment, the IFN inhibitor is not a combination of (vii) methyl nicotinate, myo-inositol, and choline.

[0054] When the inhibitor is designed to target Jak1, an antibody, plastic antibody or antibody fragment against Jak1 is envisioned, whereby, upon use, the antibody, plastic antibody or antibody fragment binds to Jak1 and, for example, phosphorylation of the STAT transcription factor is inhibited.

[0055] Alternatively, when the inhibitor is miRNA or siRNA or shRNA, a miRNA or siRNA or shRNA having a sequence complementary to a part of the Jak1 mRNA sequence is envisioned, whereby the miRNA or siRNA or shRNA inhibitor binds to Jak1 mRNA, reduces the translation of Jak1, and the intracellular abundance is decreased.

[0056] Preferably, the inhibitor is a small molecule. As used herein, the term "small molecule" refers to an organic or inorganic compound having a low molecular weight (less than approximately 800 Da).

[0057] The small molecule inhibitor for use in the present invention can function by competitive inhibition, non-competitive inhibition, mixed inhibition or uncompetitive inhibition. In one embodiment, the inhibitor is a competitive inhibitor.

[0058] Preferably, the IFN inhibitor is selected from the group consisting of ruxolitinib (CAS: 941678-49-5), tofacitinib, baricitinib, delgocitinib, emapalumab and fontolizumab, and preferably, the inhibitor is ruxolitinib.

[0059] Ruxolitinib is an inhibitor of the JAK family kinase component of the IFN signaling pathway. Ruxolitinib is also known as INC 424, INCB 018424, and (3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propanenitrile.

[0060] Tofacitinib is an inhibitor of the Jak1 component of the IFN signaling pathway.

[0061] Baricitinib is an inhibitor of Jak and blocks subtypes Jak1 and Jak2.

[0062] Delgocitinib is an inhibitor of Jak.

[0063] Emapalumab is a human monoclonal antibody against interferon-γ (IFN-γ).

[0064] Fontolizumab is a humanized antibody against interferon-γ (IFN-γ).

[0065] In a preferred embodiment, the final concentration of the IFN inhibitor in the medium is in the range of about 0.0001 to 50 mM, preferably about 1 to about 100 μM, more preferably about 0.5 to about 25 μM. Particularly preferably, the concentration of the IFN inhibitor is 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, or 25 μM, with 5 μM and 10 μM being more preferred. In particular, 5 μM or 10 μM of ruxolitinib is preferred.

[0066] The interferon inhibitor can be added either (i) to the culture medium containing HEK293 cells before the rAAV production plasmid is added in the transfection step, or (ii) during the transfection step to the culture medium containing HEK293 cells and the rAAV production plasmid. Process variant (i) is preferred.

[0067] A preferred process for rAAV production according to the present invention comprises the following steps: (a) culturing HEK293 cells in a culture medium containing an amount of IFN inhibitor for a first incubation period; (b1) transfecting HEK293 cells with three plasmids required for rAAV production, preferably (i) an adenovirus helper plasmid, (ii) a packaging rep-cap plasmid, and (iii) a recombinant rAAV plasmid containing the transgene, or (b2) transfecting HEK293 cells with two plasmids required for rAAV production, preferably (i) a plasmid containing the adenovirus helper gene and the rep-cap gene, and (ii) a recombinant rAAV plasmid containing the transgene; (c) culturing the transfected HEK293 cells for a second incubation period; (d) recovering the rAAV particles. and

[0068] An even more preferred process for rAAV production according to the present invention comprises the following steps: (a) Culturing HEK293 cells in a culture medium over a first incubation period, and (b1) Transfecting HEK293 cells with (i) an adenovirus helper plasmid, (ii) a packaging rep-cap plasmid, and (iii) a recombinant rAAV plasmid containing a transgene in a culture medium containing an amount of an IFN inhibitor and a transfection reagent, or (b2) Transfecting HEK293 cells with two plasmids necessary for rAAV production, preferably (i) a plasmid containing an adenovirus helper gene and a rep-cap gene, and (ii) a recombinant rAAV plasmid containing a transgene in a culture medium containing an amount of an IFN inhibitor and a transfection reagent, and (c) Culturing the transfected HEK293 cells in the presence of an IFN inhibitor over a second incubation period, and (d) Recovering rAAV particles comprising.

[0069] A more preferred process for rAAV production according to the present invention comprises the following steps: (a) Culturing HEK293 cells in a culture medium over a first incubation period, and (b1) Triple-transfecting HEK293 cells with (i) an adenovirus helper plasmid, (ii) a packaging rep-cap plasmid, and (iii) a recombinant rAAV plasmid containing a transgene in a culture medium containing a transfection reagent, or (b2) Transfecting HEK293 cells with two plasmids necessary for rAAV production, preferably (i) a plasmid containing an adenovirus helper gene and a rep-cap gene, and (ii) a recombinant rAAV plasmid containing a transgene in a culture medium containing a transfection reagent, and (c) Culturing the transfected HEK293 cells in a culture medium containing an IFN inhibitor over a second incubation period, (d) Recovering rAAV particles and comprising

[0070] Step (a) Before step (b) is carried out, HEK293 cells can be cultured with an IFN inhibitor for a period of about 1 hour to about 48 hours, preferably 1 to 12 hours, preferably 1 to 6 hours, more preferably 1, 2 or 3 hours in step (a).

[0071] Step (b) (i) The adenovirus helper plasmid can contain one or more of the adenovirus genes selected from the group consisting of E1A, E1B, E2A, E4ORF6 and VA. More preferably, the adenovirus helper plasmid contains the adenovirus genes E2A, E4ORF6 and VA.

[0072] (ii) The packaging rep-cap plasmid contains the rep and cap genes of rAAV. Alternatively, the required rep and cap genes are provided by HEK293 cells stably transfected with the rep and cap genes.

[0073] Preferably, the rep-cap plasmid contains the rep2 gene and the cap8 gene.

[0074] (iii) The rAAV plasmid can contain any desired heterologous gene(s). The gene of interest includes a nucleic acid encoding a polypeptide or RNA, including a reporter, therapeutic (e.g., for medical or veterinary use), immunogenic (e.g., for vaccines), or diagnostic polypeptide or RNA. As a further alternative, the heterologous nucleic acid can encode any polypeptide or RNA that is desired to be produced in cells in vitro, ex vivo, or in vivo.

[0075] In one embodiment of the present invention, the rAAV vector is self-complementary. Self-complementary vectors can advantageously overcome the rate-limiting step of second-strand DNA synthesis and result in earlier onset and more robust gene expression. Preferably, the rAAV vector containing the gene of interest is self-complementary. In one embodiment, the vector contains single-stranded DNA.

[0076] The rAAV vector may further comprise regulatory sequences operably linked to the gene of interest so as to enable one or more of transcription, translation, and expression in cells infected with the virus containing the vector.

[0077] Regulatory sequences can include appropriate transcription start sequences, transcription termination sequences, promoter sequences and enhancer sequences; RNA processing signals such as splicing and polyadenylation (polyA) signal sequences; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance secretion of the encoded product, if desired. The polyA signal sequence may be synthetic or may be derived from a number of suitable species including, for example, SV-40, human and bovine. Regulatory sequences can also include introns. Regulatory sequences can also include promoters. The promoter can be any promoter suitable for expressing the gene of interest in the target cell. The promoter can be inducible or constitutive.

[0078] In one embodiment of the present invention, the rAAV vector comprises an ITR or a functional fragment thereof. Preferably, the vector comprises a 5' AAV ITR and a 3' AAV ITR. The ITR can be of any suitable rAAV serotype, including any of the AAV serotypes described herein. The ITR can also be easily isolated using techniques known in the art or obtained from public or commercial sources (e.g., American Type Culture Collection, Manassas, VA). Alternatively, the ITR sequence may be obtained by synthesis or other suitable means with reference to the published sequences. Preferably, the vector comprises a 5' AAV2 ITR and a 3' AAV2 ITR.

[0079] Preferably, the recombinant rAAV plasmid comprises a 5' AAV ITR, a gene of interest, and a 3' AAV ITR.

[0080] The 5' AAV ITR and the 3' AAV ITR can be derived from the same serotype or different serotypes. More preferably, the 5' AAV ITR and the 3' AAV ITR are derived from the AAV8 serotype.

[0081] The transfection step can be carried out using polyethyleneimine or FectoVIR®-AAV as a transfection reagent. Preferably, polyethyleneimine is used as the transfection reagent.

[0082] In one embodiment, the transfection reagent is added and the IFN inhibitor remains in the medium. The transfection step can be carried out using the transfection reagent and the IFN inhibitor. Further, the transfection step can be carried out using the transfection reagent and the IFN inhibitor in a transfection split medium.

[0083] Step (c) The second incubation period is at least 24 hours, preferably at least 48 hours, more preferably at least 72 hours, still more preferably at least 96 hours, and most preferably at least 120 hours.

[0084] Overall, the incubation period in the presence of the IFN inhibitor is at least 24 hours, preferably at least 48 hours, more preferably at least 72 hours, even more preferably at least 96 hours, and most preferably at least 120 hours, regardless of the time point at which the inhibitor is added to the medium.

[0085] The rAAV template and the rAAV rep and cap sequences are provided under conditions such that a viral vector containing the rAAV template packaged within the rAAV capsid is produced intracellularly. The method may further include the step of recovering the viral vector from the culture. In one embodiment, the viral vector can be recovered by lysing the cells, for example, after removing the cells from the culture medium, for example, by pelleting the cells. In another embodiment, the viral vector can be recovered from the medium in which the cells are cultured, for example, to isolate the vector secreted from the cells. A portion or all of the medium can be removed from the culture at regular intervals (e.g., every 12, 18, 24, or 36 hours, or at a longer time compatible with cell viability and vector production), starting, for example, 48 hours after transfection and one or more times during the culture step for recovering rAAV. After removing the medium, fresh medium, which may or may not contain additional nutrient supplements, can be added to the culture. In one embodiment, the cells can be cultured in a perfusion system in which the medium constantly flows over the cells and is recovered for isolation of the secreted rAAV.

[0086] Step (d) As long as rAAV is secreted from HEK293 cells, step (d) includes recovering the supernatant of the culture medium and purifying rAAV particles from the supernatant. As long as rAAV is not secreted from HEK293 cells, step (d) includes disrupting HEK293 cells to obtain rAAV particles and then purifying the rAAV particles.

[0087] Purification may include, but is not limited to, any of the following methods: ion exchange, hydrophobic chromatography, affinity chromatography, filtration, precipitation, density gradient centrifugation, and heparin sulfate matrix.

[0088] In representative embodiments, the methods of the invention are fully scalable and can be performed in any desired volume of culture medium, e.g., from 10 ml (e.g., in a shaker flask) to 10 L, 50 L, 100 L, or more (e.g., in a bioreactor such as a wave bioreactor system and a stirred tank). The methods are suitable for the production of all serotypes and chimeras of AAV, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, and any chimera thereof.

[0089] The yield of rAAV from HEK293 cells can be determined, for example, as the number of vector genomes per liter of cell culture in a droplet digital PCR assay (e.g., available from Bio-Rad). Alternatively, the yield can be determined, for example, by evaluating the amount of capsid particles per liter or milliliter of cell culture in an ELISA assay. Details of the assays are outlined in the Examples section.

[0090] In certain embodiments, the method provides at least about 1×10 9 purified vector genome-containing particles (vg / mL) per milliliter of cell culture, e.g., at least about 5×10 9 , 1×10 10 , 5×1010 、 1×10 11 、 1×10 12 、 1×10 13 or 1×10 14 or more vector genome-containing particles are provided. Yield can also be measured as capsid particles per liter or milliliter of cell culture. In certain embodiments, the method provides at least about 1×10 9 purified vector genome-containing particles (vg / mL) per milliliter of cell culture, e.g., at least about 5×10 9 、 1×10 10 、 5×10 10 、 1×10 11 、 1×10 12 、 1×10 13 or 1×10 14 or more capsid particles.

[0091] Yield can be measured in the supernatant or cell suspension. The cell suspension includes the virus content in the supernatant and the virus content in the disrupted cells. Preferably, yield is determined in the cell suspension after at least one freeze-thaw cycle. The absolute titer depends, for example, on the analytical method used, the AAV serotype, the therapeutic gene of interest inserted, and the transfection method.

[0092] In a further aspect, the invention provides the use of an IFN inhibitor to increase the yield of rAAV, adenovirus, lentivirus or retrovirus in a culture of HEK293 cells. The IFN inhibitor and HEK293 cells are as defined above. Preferably, the IFN inhibitor is selected from the group consisting of ruxolitinib, tofacitinib, baricitinib, delgocitinib, emapalumab and fontolizumab and any structural analog thereof having IFN inhibitory activity, and preferably, the IFN inhibitor is ruxolitinib and any structural analog thereof having inhibitory activity against the Jak1 component of the IFN signaling pathway.

[0093] In a further aspect, the invention provides a method for preparing a pharmaceutical composition, comprising: (i) performing the method according to the invention, wherein the rAAV, adenovirus, lentivirus, or retrovirus comprises a therapeutic gene; and (ii) adding one or more pharmaceutically acceptable excipients to the prepared rAAV particles, adenovirus particles, lentivirus particles or retrovirus particles, thereby obtaining a pharmaceutical composition.

[0094] Any pharmaceutically acceptable excipient can be used within the context of the present invention, and such pharmaceutically acceptable excipients are well known in the art. The choice of pharmaceutically acceptable excipient is determined in part by the particular site to which the composition is administered and the particular method used to administer the composition. The pharmaceutical composition can optionally be sterile or can be sterile except for one or more recombinant adeno-associated virus vectors.

[0095] Formulations suitable for pharmaceutical compositions can include aqueous and non-aqueous solutions, antioxidants, buffers, and isotonic sterile solutions that may contain bacteriostatic agents, as well as aqueous and non-aqueous sterile suspensions that may include suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives. The formulations can be provided in sealed containers of unit dose or multiple dose, such as ampoules and vials, and can be stored in a freeze-dried state that requires only the addition of a sterile liquid carrier, such as water, immediately prior to use. Immediate solutions and suspensions can be prepared from sterile powders, granules, and tablets. Preferably, the carrier is buffered saline. More preferably, the pharmaceutical composition for use in the present method is formulated to protect the adeno-associated virus vector from damage prior to administration. For example, the pharmaceutical composition can be formulated to reduce the loss of adeno-associated virus vectors in devices such as glassware, syringes, or needles used to prepare, store, or administer the expression vector. The pharmaceutical composition can be formulated to reduce the photosensitivity and / or temperature photosensitivity of the adeno-associated virus vector. For this purpose, the pharmaceutical composition preferably includes a pharmaceutically acceptable liquid carrier, such as those described above, and a stabilizer selected from the group consisting of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, mannitol, and combinations thereof. The use of such compositions can extend the shelf life of the vector, facilitate administration, and enhance the efficiency of the method. The pharmaceutical composition can also be formulated to increase the transduction efficiency of the recombinant adeno-associated virus vector. In addition, those skilled in the art will understand that the pharmaceutical composition can include other therapeutic or biologically active agents. For example, factors that control inflammation, such as ibuprofen or steroids, can be part of the pharmaceutical composition to reduce swelling and inflammation associated with in vivo administration of the adeno-associated virus vector. Antibiotics, i.e., bactericidal and fungicidal agents, may be present to treat existing infections and / or reduce the risk of future infections, such as those associated with gene transfer procedures.

[0096] In a further aspect, the present invention is a kit for use in cell culture, comprising (a) HEK293 cells and (b) an IFN inhibitor selected from the group consisting of ruxolitinib, tofacitinib, baricitinib, delgocitinib, emapalumab, and fontolizumab and any structural analog thereof having IFN inhibitory activity, preferably, the IFN inhibitor is ruxolitinib, and any structural analog thereof has at least 50%, more preferably 80%, even more preferably at least 90%, most preferably at least 95% inhibitory activity against JAK family kinases, a kit is provided.

[0097] The kit may include additional components. Instructions for the user may also be included.

[0098] The terms of the present invention 1. A method for the fermentative production of adeno-associated virus (AAV), adenovirus, lentivirus or retrovirus, comprising the following steps: Step 1: Culturing the rAAV, the lentivirus, the adenovirus or the retrovirus in HEK293 cells over an incubation period in a culture medium containing an amount of interferon (IFN) inhibitor; and Step 2: Recovering the rAAV, the adenovirus, the lentivirus or the retrovirus from the cell culture. The method as described in the above clause.

[0099] 2. The method according to clause 1, wherein the yield of the rAAV, the adenovirus, the lentivirus or the retrovirus over an incubation period of at least 96 hours is increased by at least 10%, preferably at least 50%, more preferably at least 100%, even more preferably at least 200% compared to the yield of the rAAV, the adenovirus, the lentivirus or the retrovirus in the absence of the IFN inhibitor.

[0100] 3. The HEK293 cells produce at least about 1×10 9 per milliliter of cell culture, preferably at least about 5×10 9 per milliliter of cell culture, more preferably at least 1×10 10 purified vector genome-containing particles (vg / mL), more preferably at least 5×10 10 per milliliter of cell culture, and most preferably at least 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 or 5×10 14 or more vector genome-containing particles of rAAV, adenovirus, lentivirus or retrovirus, according to the method of item 1 or 2.

[0101] 4. The HEK293 cells, during rAAV transfection, have at least one of the following genes: (1) interferon_gamma response; and / or (2) interferon_alpha_response characterized by activation, according to any one of items 1 to 3.

[0102] 5. The HEK293 cells, during rAAV transfection, are characterized by activation of at least interferon_gamma response and interferon_alpha_response, according to the method of item 4.

[0103] 6. The HEK293 cell line, during rAAV transfection, has at least one of the following genes: (i) TNFA_signaling_via_NFKB; (ii) Interferon_Gamma_Response; (iii) Interferon_Alpha_Response; (iv) TGF_beta_Signaling; and / or (v) IL6_JAK_STAT3_Signaling The method according to any one of clauses 1 to 3, characterized by the activation of

[0104] 7. The method according to clause 6, wherein the at least two activated genes are selected from the group consisting of TNFA_signaling_via_NFKB, interferon_gamma response, interferon_alpha_response, TGF_beta_Signaling, and IL6_JAK_STAT3_Signaling, and more preferably, the at least two activated genes are selected from the group consisting of interferon_gamma response, interferon_alpha_response, and IL6_JAK_STAT3_Signaling.

[0105] 8. The method according to clause 6 or 7, wherein the HEK293 cells show at least two, at least three, or at least four activations, and more preferably, all of the five genes are activated.

[0106] 9. The method according to any one of clauses 1 to 8, wherein the HEK293 cells, in the presence of the IFN inhibitor, produce at least 1×10 10 per milliliter of the culture medium, more preferably at least 5×10 10 per milliliter of the culture medium, and most preferably at least 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 or 5×10 14 AAV yields of capsid particles per milliliter of the culture medium.

[0107] 10. The method according to any one of clauses 1 to 9, wherein the HEK293 cells are an adherent cell line.

[0108] 11. The method according to any one of clauses 1 to 9, wherein the HEK293 cells are a suspension cell line or are adapted to grow in a suspended state.

[0109] 12. The method according to any one of clauses 1 to 11, wherein the HEK293 cells are selected from the group consisting of CRL-1573 and NRC (HEK293SF-3F6).

[0110] 13. The method according to any one of clauses 1 to 12, wherein step 1 is carried out in a Petri dish, a shaker flask or a roller bottle.

[0111] 14. The method according to any one of clauses 1 to 12, wherein step 1 is carried out in a bioreactor, preferably, the bioreactor has a volume of at least 10 L.

[0112] 15. The rAAV is selected from any AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13, its chimeras and hybrid variants, preferably, the AAV serotype is selected from AAV5, AAV6, AAV8, and AAV9, more preferably selected from AAV8. The method according to any one of clauses 1 to 14.

[0113] 16. The lentivirus is derived from the human immunodeficiency virus (HIV), preferably, the lentivirus is derived from HIV-1. The method according to any one of clauses 1 to 14.

[0114] 17. The lentiviral vector is obtained by transfecting the HEK293 cells by combining at least one packaging plasmid, preferably, an envelope plasmid derived from the vesicular stomatitis virus (VSV). More preferably, the envelope plasmid is derived from VSV-G. The method according to clause 16.

[0115] 18. The method according to any one of clauses 1 to 14, wherein the retrovirus is murine leukemia virus (MuLV).

[0116] 19. The method according to any one of clauses 1 to 18, wherein the IFN inhibitor is a molecule that reduces the activity of IFN induction and / or the IFN signaling cascade.

[0117] 20. The method according to clause 19, wherein the IFN inhibitor targets the JAK / STAT signaling pathway.

[0118] 21. The method according to clause 20, wherein the IFN inhibitor targets Jak1.

[0119] 22. The IFN inhibitor is selected from the group consisting of ruxolitinib, tofacitinib, baricitinib, delgocitinib, emapalumab, and fontolizumab, and any of its structural analogs having IFN inhibitory activity. Preferably, the IFN inhibitor is ruxolitinib and any of its structural analogs having at least 50% inhibitory activity against the JAK family kinases of the IFN signaling pathway compared to ruxolitinib. The method according to any one of clauses 19 to 21.

[0120] 23. The final concentration of the IFN inhibitor in the medium ranges from about 0.0001 to 50 mM, preferably from about 1 to about 100 μM, more preferably from about 0.5 to about 25 μM. Even more preferably, the final concentration is about 1 μM, about 5 μM, about 10 μM, about 15 μM, about 20 μM, or about 25 μM. The method according to any one of clauses 1 to 22.

[0121] 24. The method according to any one of clauses 1 to 23, wherein the culture medium is a medium with chemically defined components.

[0122] 25. The following steps: (a) Culturing HEK293 cells in a culture medium containing an amount of IFN inhibitor for a first incubation period. (b1) Transfecting the HEK293 cells with the three plasmids necessary for rAAV production, preferably (i) an adenovirus helper plasmid, (ii) a packaging rep-cap plasmid, and (iii) a recombinant rAAV plasmid containing the transgene, or (b2) Transfecting the HEK293 cells with the two plasmids necessary for rAAV production, preferably (i) a plasmid containing the adenovirus helper gene and the rep-cap gene, and (ii) a recombinant rAAV plasmid containing the transgene, and (c) Culturing the transfected HEK293 cells over a second incubation period, and (d) Recovering the AAV particles The method according to any one of clauses 1 to 24, comprising:

[0123] 26. The method according to clause 25, wherein the HEK293 cells are cultured with the IFN inhibitor for a period of about 1 hour to about 12 hours, preferably 1 to 6 hours, more preferably 1 to 3 hours, in step (a) before step (b) is carried out.

[0124] 27. (i) The adenovirus helper plasmid contains one or more of the adenovirus genes selected from the group consisting of E1A, E1B, E2A, E4ORF6, and VA, and / or (ii) the packaging rep-cap plasmid contains the Rep2 gene and the Cap8 gene, and / or (iii) the AAV plasmid contains the 5' AAV ITR and the 3' AAV ITR derived from the same AAV serotype, preferably AAV2. The method according to clause 25 or 26.

[0125] 28. The method according to any one of clauses 25 to 27, wherein the transfection step is carried out using polyethyleneimine or FectoVIR™-AAV as the transfection reagent.

[0126] 29. The method according to any one of clauses 25 to 28, wherein the second incubation period is at least 24 hours, preferably at least 48 hours, more preferably at least 72 hours, and most preferably at least 96 hours.

[0127] 30. The method according to any one of clauses 25 to 29, wherein as long as the rAAV is secreted from the HEK293 cells, step (d) includes collecting the supernatant of the culture medium and purifying rAAV particles from the supernatant.

[0128] 31. The method according to any one of clauses 25 to 30, wherein as long as the rAAV is not secreted from the HEK293 cells, step (d) includes destroying the HEK293 cells to obtain rAAV particles, and then purifying the rAAV particles.

[0129] 32. A method for the fermentative production of adeno-associated virus (AAV), adenovirus, lentivirus or retrovirus, comprising the following steps: Step 1: Perform gene set enrichment analysis on HEK293 cells transfected with rAAV, adenovirus, lentivirus or retrovirus, and compare with a negative control, and at least one of the following genes: (i) TNFA_signaling_via_NFKB; (ii) Interferon_Gamma_Response; (iii) Interferon_Alpha_Response; (iv) TGF_beta_Signaling; and / or (v) IL6_JAK_STAT3_Signaling is enriched in transcription, preferably, the following: (1) interferon_gamma response; and / or (2) interferon_alpha_respone When the transcription of Step 2: culturing the rAAV, the adenovirus, the lentivirus or the retrovirus in the selected HEK293 cells in a culture medium containing an amount of interferon (IFN) inhibitor over an incubation period; Step 3: recovering the rAAV, the adenovirus, the lentivirus or the retrovirus from the cell culture The method comprising.

[0130] 33. Use of an IFN inhibitor to increase the yield of rAAV, adenovirus, lentivirus or retrovirus in the culture of HEK293 cells transfected or infected with the rAAV, the adenovirus, the lentivirus or the retrovirus.

[0131] 34. When the HEK293 cells are transfected or infected with the rAAV, the adenovirus, the lentivirus or the retrovirus, at least one of the following cell pathways: (1) interferon_gamma response; and / or (2) interferon_alpha_response The use according to clause 33, characterized by the activation of.

[0132] 35. The IFN inhibitor is selected from the group consisting of ruxolitinib, tofacitinib, baricitinib, delgocitinib, emapalumab and fontolizumab, and any structural analog thereof having IFN inhibitory activity, preferably, the IFN inhibitor is ruxolitinib and any structural analog thereof having inhibitory activity against at least 50% of the JAK family kinases of the IFN signaling pathway compared to ruxolitinib. The use according to clause 34.

[0133] 36. A method for preparing a pharmaceutical composition, comprising: (i) performing the method according to any one of clauses 1 to 32, wherein the rAAV, the adenovirus, the lentivirus or the retrovirus contains a therapeutic gene, and performing the method; and (ii) adding one or more pharmaceutically acceptable excipients to the prepared rAAV particles, adenovirus particles, lentivirus particles or retrovirus particles, thereby obtaining a pharmaceutical composition.

[0134] 37. A kit for use in cell culture, comprising: (a) HEK293 cells, and (b) an IFN inhibitor selected from the group consisting of ruxolitinib, tofacitinib, baricitinib, delgocitinib, emapalumab, fontolizumab and any structural analog thereof having IFN inhibitory activity, preferably, the IFN inhibitor is ruxolitinib and any structural analog thereof having inhibitory activity against the Jak1 component of at least 50% of the IFN signaling pathway.

Examples

[0135] Example 1: Materials and Methods Cell Culture, Fermentation In this study, three derivative strains of HEK293 cells that were fully adapted to suspension culture were used and cultured in a chemically defined serum-free medium (FreeStyle™ F17 Expression Medium, Thermo Fisher, NY, USA) to produce rAAV8 vectors. All cell lines were defined as HEK293 and were treated similarly. All HEK293 cell lines used originated from the 1977 experiment by Frank Grahams, which enabled the cells to produce rAAV. Cell line CRL-1573 (also called CL1) was purchased from the American Type Culture Collection (ATCC) and adapted to serum-free suspension culture. Cell line NRC HEK293SF-3F6 (also called CL2) is commercially available from the National Research Council, Canada. Cell line HEK293 Pro10® (also called CL3) is commercially available from AskBio.

[0136] All cell lines were stored as individual cell banks aliquoted at 1.0 × 10 6 cells / mL in Thermo Fisher's FreeStyle™ F17 Expression Medium containing 7.5% DMSO to protect the cells from damage. To thaw, the frozen stock vial was placed at -120 °C to -80 °C overnight and thawed in a water bath, and the cells were quickly transferred into a pre-warmed 125 mL Corning® single-use spinner flask containing Freestyle™ F17 Expression Medium. The cells were further grown in a Corning® disposable spinner flask for two weeks, passaged regularly every two or three days, and supplied with fresh medium. The growth process was carried out in a Thermo Fisher Heracell 150 incubator at 37 °C and 5% carbon dioxide supply in a humidified atmosphere.

[0137] Subsequently, the HEK293 cell line (Agilent Technologies catalog number 240073, lot number 0006218516) was cultured in a T75 flask with a total volume of 23 mL in DMEM medium containing 2% FCS. The cell culture was carried out in an incubator at +37°C and 5% CO2. A 1 mM ruxolitinib stock solution was prepared by dissolving the white solid powder in DMSO according to the manufacturer's instructions before use and stored at -20°C. At the last passage before transfection, ruxolitinib was added to the cell culture at the specified concentration. All conditions were tested in duplicate. On the day of transfection, a confluence range of 60 - 80% was achieved.

[0138] Transfection For transfection, HEK293 cells were transferred into a 250 mL Sartorius SU bioreactor (Sartorius, Ambr® 250 modular vessel, mammalian), a 10 L Eppendorf glass bioreactor (Eppendorf BioFlo® 320) or a 125 mL Corning® disposable shake flask. All systems were adjusted to 37°C and 5% carbon dioxide. The 250 mL bioreactor was constantly stirred at 492 rpm, the 10 L bioreactor was constantly stirred at 120 rpm, and the pH was adjusted using a 0.5 M sodium hydroxide solution. The shake flask was placed in an Eppendorf New Brunswick S41i incubator, stirred at 150 rpm, and humidified.

[0139] Transient transfection of HEK293 cells for rAAV8 capsid production was performed with a three-plasmid system using polyethyleneimine (PEI) (Merck KGaA, Darmstadt, Germany) according to the supplier's transfection protocol. The adenovirus 5 helper gene was delivered with one plasmid. The second plasmid was used to deliver the Rep2Cap8 gene that determines the rAAV serotype, and the third plasmid encoded the human FIX sequence, a potential therapeutic target gene. The ratio of helper, RepCap, and transgene plasmids was 1:2:1.5. The ratio of plasmid to PEI was 1:2.5. The volume of the transfection mix corresponded to 10% of the final working volume and was composed of Freestyle™ F17 expression medium, plasmid, and PEI. Prior to transfection, the cells were passaged and adjusted to a target range of 3.0 - 5.0×10 6 cells / mL.

[0140] Transient transfection of HEK293 for lentivirus production was performed with the pALDI-lenti system (Aldevron). This is a four-plasmid system containing pALD-lenti, pALD-VSV-G, pALD-GagPol, and pALD-Rev, which is suitable for the production of HIV-1-derived viral vectors. The transfection mix was composed of the four-plasmid system, DMEM medium, and the transfection reagent PEI. The ratio of plasmids used was 1:1:1:1, and the ratio of plasmid DNA to PEI was 1:3.

[0141] Gene Set Enrichment Analysis Gene set enrichment analysis was performed based on the Hallmark gene sets available from UC San Diego and the Broad Institute (https: / / www.gsea - msigdb.org / gsea / msigdb / genesets.jsp?collection = H). See also Liberzon et al., Cell Syst. 23 (2015), 417 - 425.

[0142] Next - generation sequencing To first analyze the differences in mRNA expression patterns of the three HEK293 cell lines used, next - generation sequencing of four biological replicates of all cell lines under transfection conditions and mock - transfection conditions was performed at five different time points. Mock - transfection was performed without plasmid, adding only polyethyleneimine (PEI) and Thermo Fisher Freestyle F17 (registered trademark) medium. The selected time points were 0 hours (before transfection), 4 hours, 24 hours, 48 hours, and 72 hours after transfection. At each time point, the same sampling procedure was performed, including washing 5.0×10 6 cells / mL twice with PBS. The cell pellets were analyzed by the Illumina sequencing method. The output metadata contained mRNA reads of the three HEK293 cell lines at all five time points and was used to investigate transcriptome differences.

[0143] Results The HEK293 cell lines used for fermentation production showed different yields.

[0144] Results of gene set enrichment analysis of HEK293 cell lines transfected with rAAV showed differences in activated cell pathways.

[0145] Upon triple - transfection with the rAAV - producing plasmid, the CL1 cell line showed activation of the following genes: (1)interferon_gamma response (0 - 48 hours); and (2)interferon_alpha_respone (0 - 48 hours).

[0146] Activation of these genes was not observed in the CL3 cell line.

[0147] Therefore, the genes activated by rAAV are significantly different between the CL3 cell line and the CL1 cell line.

[0148] In further experiments, the activation of cell pathways in the CL1 cell line by triple transfection for rAAV production was compared with mock transfection. The CL1 cell line was found to show activation of the following cell pathways by triple transfection for rAAV production: (i)TNFA_signaling_via_NFKB; (ii)Interferon_Gamma_Response; (iii)Interferon_Alpha_Response; (iv)TGF_beta_Signaling;

[0149] Therefore, this experiment confirmed that specific genes were specifically activated in the CL1 cell line transfected with the plasmids required for rAAV production. Since this activation could not be detected by mock transfection, it is rAAV - specific.

[0150] Example 2: Treatment with inhibitors From the fact that the CL1 cell line showed activation of interferon - alpha and gamma response genes in contrast to the CL3 cell line, it was considered possible that the lower rAAV yield of the CL1 cell line compared to the CL3 cell line was due in part to the activation of these genes.

[0151] For treatment with the inhibitor, the solubilized inhibitor and cells were incubated for 2 to a maximum of 3 hours and then transfected. As the inhibitor, ruxolitinib (Stemcell Technologies), a Janus-associated kinase (JAK) family inhibitor, was used and prepared according to the product's instruction manual. After adding the inhibitor and the transfection mix to the cell culture, the cells were further cultured in batch mode for 48 to a maximum of 96 hours.

[0152] AAV8 ELISA A commercially available enzyme-linked immunosorbent assay (ELISA; Progen AAV8 Titration ELISA Kit, catalog number PRAAV8) uses a monoclonal antibody (ADK8) specific for conformational epitopes on the assembled AAV8 capsid. This antibody immobilized on the plate captures the rAAV-8 particles of the test sample. The captured particles are then detected by the binding of biotinylated anti-AAV8 ADK8. This is because the target epitope is repeatedly expressed on the assembled AAV8 capsid. Then, streptavidin peroxidase and a peroxidase substrate are used to measure the bound anti-AAV8, thereby measuring the concentration of the AAV8 capsid. The color reaction was photometrically measured at 450 nm. The kit contains an rAAV2 / 8 particle preparation with the rAAV8 particle concentration noted as a calibration standard.

[0153] P24 ELISA ELISA quantifies the amount of the structural HIV-1 p24 capsid protein in cell cultures and supernatant samples. This is a quantitative sandwich ELISA assay. The HIV-1 p24 ELISA kit ab218268 was used for the titration of lentiviral expression. To distinguish between viral p24 produced by lentivirus and p24 produced by HEK293 cells, the Cell Biolabs Quick Titer™ Kit containing LV-Origine / TR30021 / lot number 134641F and Cell Biolabs Reference Standard #310809 was used. With the virus pull-down technique, only p24 associated with lentivirus is reliably detected.

[0154] Droplet digital PCR (ddPCR) For the quantification of the vector genome, the droplet digital PCR method by Bio-Rad was used, with the fully automated QX One System or the automated QX 200 AutoDG System. In this method, absolute quantification of the vector genome can be obtained without using a standard curve. The sample is divided into oil droplets, and each droplet becomes an independent compartment for the PCR reaction. Denaturation of the capsid occurs at the initial stage of PCR in the thermal cycler, enabling DNA amplification. Then, a droplet reader is used to determine the titer of the vector genome. The sample was treated with DNase I (NEB) to remove irrelevant DNA sequences. Before the treatment, the sample was pre-diluted to enhance the efficiency of DNase I activity. After droplet generation, PCR was performed using Bio-Rad ddPCR Supermix (without dUTP) and FIX-specific primers and probe: forward 5’-GGC ATC TAC ACC AAA GTC TCC AG -3’ (SEQ ID NO: 1), reverse 5’-CAG CGA GCT CTA GGC ATG CT -3’ (SEQ ID NO: 2), probe 5’-6FAM-AGA CCA AGC TGA CCT GAT-MGBNFQ -3’ (SEQ ID NO: 3). The vector genome concentration was calculated by appropriate Bio-Rad software.

[0155] Result Shaker flask experiment HEK293 cells (CL1) were treated with different concentrations of the IFN inhibitor ruxolitinib. The final concentrations of the IFN inhibitor are shown in Table 2 below:

[0156] [Table 2]

[0157] The yields of each rAAV production determined by droplet digital PCR (ddPCR) of the vector genome in the supernatant and cell culture are shown in Figures 1A and 1B, respectively. In the determination of the supernatant, the vector genome was transferred to the medium. In the determination of the cell culture, the samples of the cell culture were subjected to freeze-thaw cycles.

[0158] In the supernatant, at a concentration of 10 μM ruxolitinib, the vector genome (vg) increased by approximately 2.5-fold compared to the negative control in the absence of the inhibitor, and approximately 2-fold when determined in the cell suspension. In the cell suspension, at ruxolitinib inhibitor concentrations of 5 μM and 10 μM, approximately 2×10 13 vg / L of rAAV yield was observed.

[0159] The yields of rAAV production determined by capsid particles in the supernatant and cell culture are shown in Figures 1C and 1D, respectively. Similar results were observed compared to the determination by ddPCR.

[0160] It can be seen that the addition of the IFN inhibitor results in a significant increase in the rAAV yield of CL1 cells treated with these inhibitors compared to the yield in the absence of the IFN inhibitor. The optimal concentrations of the IFN inhibitor were 5 μM and 10 μM. The increase determined in the cell suspension was approximately 3-fold compared to the rAAV yield in the absence of the inhibitor. A decrease in the effect was observed at concentrations above 10 μM.

[0161] In another experimental set, the rAAV yields of three different cell lines (CL1, CL2, and CL3) were compared in the absence (control) or presence of the inhibitor. To compare the yields among the different cell lines used, the rAAV yield of the CL1 control was used as the reference (100%). See FIGS. 2(A), 2(B), and 2(C). To exclude any influence of the solvent on the inhibitor, experiments were also conducted in the presence of each solvent (DMSO, water for injection (wfi), PBS) without the inhibitor. The results determined by the rAAV8 ELISA assay in the supernatant are shown in FIG. 2. FIG. 2(A) relates to CL1 cells, FIG. 2(B) relates to the CL2 cell line, and FIG. 3(C) relates to the CL3 cell line.

[0162] For each of the different HEK293 cell lines, it can be seen that an increase in rAAV8 yield can be obtained by adding the IFN inhibitor to the culture medium. This effect is clearly higher the lower the rAAV production of HEK293 cells in the absence of the IFN inhibitor.

[0163] Therefore, the present invention is widely applicable to HEK293 cells.

[0164] 10 L bioreactor experiment To test the effect of the IFN inhibitor on the rAAV yield in the large-scale bioreactor fermentation of HEK293 cells, a 10 L bioreactor was used. The results are shown in FIGS. 3A (determined by capsid particle rAAV8 ELISA) and 3B (determined by ddPCR). The improvement by the interferon inhibitor was confirmed by the ddPCR results (an improvement of approximately 1.3-fold was determined in the supernatant). In the ELISA results of rAAV8 capsid particles, an even higher improvement of approximately 1.5-fold was shown in the supernatant. In the experiment for determining rAAV8 capsid particles by ELISA, a higher improvement of an increase of approximately 1.5-fold was shown in the experiment treated with the inhibitor compared to the negative control in the absence of the inhibitor.

[0165] The results obtained from the 3L single-use bioreactor showed highly comparable performance compared to the 10L bioreactor.

[0166] Adherent HEK293 experiments in T-flasks Adherent HEK293 cells were treated with different concentrations of the IFN inhibitor ruxolitinib. The final concentrations of the IFN inhibitor are shown in Table 3 below:

[0167] [Table 3]

[0168] The yields of rAAV production determined by droplet digital PCR (ddPCR) of the vector genome in the supernatant are shown in Figure 4A. To determine the capsid particle concentration, AAV8 ELISA was used as shown in Figure 4B. The statistical mean of the control conditions was calculated as 100%.

[0169] The ddPCR results of the supernatant samples showed a 48% increase in yield at an inhibitor concentration of 1 μM compared to the control conditions. Inhibitors at concentrations of 5 μM and 10 μM showed increases in vg yields of 38.5% and 31%, respectively.

[0170] The ELISA results for capsid particles in the supernatant samples of adherent HEK293 cells showed the highest increase in yield of 66.5% compared to the control conditions at an inhibitor concentration of 5 μM. A concentration of 10 μM showed a 62% increase in titer, and a concentration of 1 μM showed an improvement in 49% of the capsid particles in the supernatant.

[0171] All experiments were performed in duplicate and showed high comparability. Specific doses from 1 μM to a maximum of 5 μM resulted in increased yields in ddPCR vg and ELISA cp detection. A positive effect of the inhibitor can be observed in adherent HEK293 cells.

[0172] Suspension HEK293 (CL1) experiments using lentivirus The suspension HEK293 (CL1) experiment using 4 plasmid lentivirus transfection was carried out in a shake flask under the same conditions as shown in Table 2. The lentivirus cp titer was determined by the p24 sandwich ELISA shown in Figure 5. The statistical average of the control conditions was evaluated as 100%.

[0173] In the suspension HEK293 transfected with the lentivirus plasmid, a maximum cp titer increase of 81.5% was shown at an inhibitor concentration of 10 μM compared to the untreated control condition. An inhibitor concentration of 5 μM showed a 40% yield increase compared to the control.

[0174] A positive effect of IFN pathway inhibition was confirmed in HEK293 cells (CL1) transfected with the lentivirus plasmid.

Claims

1. A method for the fermentative production of adeno-associated virus (AAV), adenovirus, lentivirus or retrovirus, comprising the following steps: Step 1: Culturing the rAAV, the adenovirus, the lentivirus or the retrovirus in HEK293 cells over an incubation period in a culture medium containing an amount of interferon (IFN) inhibitor; Step 2: Recovering the rAAV, the adenovirus, the lentivirus or the retrovirus from the cell culture. The method as described above.

2. The method according to claim 1, wherein the IFN inhibitor is not selected from niacin and / or niacinamide.

3. The yield of the rAAV, the adenovirus, the lentivirus or the retrovirus over an incubation period of at least 96 hours is increased by at least 10%, preferably at least 50%, more preferably at least 100%, even more preferably at least 200% compared to the yield of the rAAV, the adenovirus, the lentivirus or the retrovirus in the absence of the IFN inhibitor. The method according to claim 1 or 2.

4. The HEK293 cells produce at least about 1×10 ９ per milliliter of cell culture, preferably at least about 5×10 ９ per milliliter of cell culture, more preferably at least 1×10 １０ purified vector genome-containing particles (vg / mL), more preferably at least 5×10 １０ per milliliter of cell culture, and most preferably at least 1×10 １１ , 1×10 １２ , 1×10 １３ , 1×10 １４ or 5×10 １４ or more vector genome-containing particles of rAAV, lentivirus or retrovirus, according to any one of claims 1 to 3.

5. The HEK293 cells are characterized by the activation of at least one of the following genes during rAAV transfection: (1) interferon_gamma response; and / or (2) interferon_alpha_response The method according to any one of claims 1 to 4.

6. The method according to claim 5, wherein the HEK293 cells are characterized by the activation of at least interferon_gamma response and interferon_alpha_response during rAAV transfection.

7. The HEK293 cell line is characterized by the activation of at least one of the following genes during rAAV transfection: (i) TNF_A_signaling_via_NFKB; (ii) Interferon_Gamma_Response; (iii) Interferon_Alpha_Response; (iv) TGF_beta_Signaling; and / or (v) IL6_JAK_STAT3_Signaling The method according to any one of claims 1 to 6, characterized by the activation of

8. The method according to claim 7, wherein the at least two activated genes are selected from the group consisting of TNFα_signaling_via_NFκB, interferon_gamma response, interferon_alpha_response, TGF_beta_Signaling, and IL6_JAK_STAT3_Signaling, and more preferably, the at least two activated genes are selected from the group consisting of interferon_gamma response, interferon_alpha_response, and IL6_JAK_STAT3_Signaling.

9. The method according to claim 7 or 8, wherein the HEK293 cells show activation of at least two, at least three, or at least four pathways, and preferably all of the five pathways are activated.

10. The HEK293 cells produce at least 1×10 １０ particles, more preferably at least 5×10 １０ particles, and most preferably at least 1×10 １１ , 1×10 １２ , 1×10 １３ , 1×10 １４ or 5×10 １４ AAV yields of capsid particles or more, according to any one of claims 1 to 9 The method described in the section.

11. The method according to any one of claims 1 to 10, wherein the HEK293 cells are an adherent cell line.

12. The method according to any one of claims 1 to 10, wherein the HEK293 cells are a suspension cell line or are adapted to grow in a suspended state.

13. The method according to any one of claims 1 to 12, wherein the HEK293 cells are selected from the group consisting of CRL-1573 and NRC(HEK293SF-3F6).

14. The method according to any one of claims 1 to 13, wherein step 1 is carried out in a Petri dish, a shaker flask, a rocking bag, or a roller bottle.

15. The method according to any one of claims 1 to 13, wherein step 1 is carried out in a bioreactor, and preferably, the bioreactor has a volume of at least 10 L.

16. The method according to any one of claims 1 to 15, wherein the rAAV is selected from any AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13, its chimeric and hybrid mutants, and preferably, the AAV serotype is selected from AAV5, AAV6, AAV8, and AAV9, and more preferably AAV8.

17. The method according to any one of claims 1 to 15, wherein the lentivirus is derived from human immunodeficiency virus (HIV), and preferably, the lentivirus is derived from HIV-1.

18. The method according to claim 17, wherein the lentiviral vector is obtained by transfecting the HEK293 cells with at least one packaging plasmid, preferably in combination with an envelope plasmid derived from vesicular stomatitis virus (VSV), and more preferably, the envelope plasmid is derived from VSV-G.

19. The method according to any one of claims 1 to 15, wherein the retrovirus is murine leukemia virus (MuLV).

20. The method according to any one of claims 1 to 19, wherein the IFN inhibitor is a molecule that reduces the activity of IFN induction and / or the IFN signaling cascade.

21. The method according to claim 20, wherein the IFN inhibitor targets the JAK / STAT signaling pathway.

22. The method according to claim 21, wherein the IFN inhibitor targets JAK family kinases.

23. The method according to any one of claims 20 to 22, wherein the IFN inhibitor is selected from the group consisting of ruxolitinib, tofacitinib, baricitinib, delgocitinib, emapalumab, and fontolizumab, and any of its structural analogs having IFN inhibitory activity, and preferably, the IFN inhibitor is ruxolitinib and any of its structural analogs having at least 50% of the inhibitory activity against the JAK family kinases of the IFN signaling pathway compared to ruxolitinib.

24. The method according to any one of claims 1 to 23, wherein the final concentration of the IFN inhibitor in the medium ranges from about 0.0001 to 50 mM, preferably from about 1 to about 100 μM, more preferably from about 0.5 to about 25 μM, and even more preferably, the final concentration is about 1 μM, about 5 μM, about 10 μM, about 15 μM, about 20 μM, or about 25 μM.

25. The method according to any one of claims 1 to 24, wherein the culture medium is a chemically defined medium.

26. The following steps: (a) Culturing HEK293 cells in a culture medium containing an amount of IFN inhibitor for a first incubation period; (b1) Transfecting the HEK293 cells with three plasmids necessary for rAAV production, preferably (i) an adenovirus helper plasmid, (ii) a packaging rep-cap plasmid, and (iii) a recombinant rAAV plasmid containing the transgene, or (b2) Transfecting the HEK293 cells with two plasmids necessary for rAAV production, preferably (i) a plasmid containing the adenovirus helper gene and the rep-cap gene, and (ii) a recombinant rAAV plasmid containing the transgene, and (c) Culturing the transfected HEK293 cells over a second incubation period, and (d) Recovering the AAV particles The method according to any one of claims 1 to 25, comprising. (Claim 27) The method according to claim 26, wherein the HEK293 cells are cultured with the IFN inhibitor for a period of about 1 hour to about 12 hours, preferably 1 to 6 hours, more preferably 1 to 3 hours, in step (a) before step (b) is performed. (Claim 28) (i) The adenovirus helper plasmid contains one or more of the adenovirus genes selected from the group consisting of E1A, E1B, E2A, E4ORF6, and VA, and / or (ii) the packaging rep-cap plasmid contains the Rep2 gene and the Cap8 gene, and / or (iii) the AAV plasmid contains the 5' AAV ITR and the 3' AAV ITR derived from the same AAV serotype, preferably AAV2. The method according to claim 26 or 27. (Claim 29) The method according to any one of claims 26 to 28, wherein the transfection step is performed using polyethyleneimine or FectoVIR™-AAV as a transfection reagent. (Claim 30) The method according to any one of claims 26 to 29, wherein the second incubation period is at least 24 hours, preferably at least 48 hours, more preferably at least 72 hours, and most preferably at least 96 hours. (Claim 31) The method according to any one of claims 26 to 30, wherein as long as the rAAV is secreted from the HEK293 cells, step (d) comprises recovering the supernatant of the culture medium and purifying the rAAV particles from the supernatant.

32. The method according to any one of claims 26 to 31, wherein as long as the rAAV is not secreted from the HEK293 cells, step (d) comprises disrupting the HEK293 cells to obtain the rAAV particles, and then purifying the rAAV particles.

33. A method for the fermentative production of adeno-associated virus (AAV), lentivirus or retrovirus, comprising the following steps: Step 1: Performing transcriptome analysis of HEK293 cells transfected with rAAV, adenovirus, lentivirus or retrovirus, and comparing with a negative control, at least one of the following genes: (i) TNFα_signaling_via_NFκB; (ii) Interferon_Gamma_Response; (iii) Interferon_Alpha_Response; (iv) TGF_beta_Signaling; and / or (v) IL6_JAK_STAT3_Signaling is transcriptionally enriched, preferably the following: (1) interferon_gamma_response; and / or (2) interferon_alpha_response when the transcription of is transcriptionally enriched, selecting the HEK293 cells; Step 2: Culturing the rAAV, the adenovirus, the lentivirus or the retrovirus in the selected HEK293 cells in a culture medium containing an amount of interferon (IFN) inhibitor over an incubation period; Step 3: Recovering the rAAV, the adenovirus, the lentivirus or the retrovirus from the cell culture The method as described above.

34. Use of an IFN inhibitor to increase the yield of rAAV, adenovirus, lentivirus or retrovirus in the culture of HEK293 cells transfected or infected with the rAAV, the adenovirus, the lentivirus or the retrovirus.

35. The use according to claim 34, wherein the IFN inhibitor is not selected from niacin and / or niacinamide.

36. When the HEK293 cells are transfected or infected with the rAAV, the adenovirus, the lentivirus or the retrovirus, at least one of the following cell pathways: (1) interferon_gamma response; and / or (2) interferon_alpha_response The use according to claim 34 or 35, characterized by activation of.

37. The IFN inhibitor is selected from the group consisting of ruxolitinib, tofacitinib, baricitinib, delgocitinib, emapalumab and fontolizumab, and any structural analog thereof having IFN inhibitory activity, preferably, the IFN inhibitor is ruxolitinib and any structural analog thereof having inhibitory activity against JAK family kinases of at least 50% of the IFN signaling pathway compared to ruxolitinib. The use according to claim 36.

38. A method for preparing a pharmaceutical composition, comprising: (i) performing the method according to any one of claims 1 to 34, wherein the rAAV, the adenovirus, the lentivirus or the retrovirus comprises a therapeutic gene, said performing, and (ii) adding one or more pharmaceutically acceptable excipients to the prepared rAAV particles, adenovirus particles, lentivirus particles or retrovirus particles, thereby obtaining a pharmaceutical composition. The method described above.

39. A kit for use in cell culture, comprising: (a) HEK293 cells, and (b) an IFN inhibitor selected from the group consisting of ruxolitinib, tofacitinib, baricitinib, delgocitinib, emapalumab and fontolizumab and any structural analog thereof having IFN inhibitory activity, preferably, the IFN inhibitor is ruxolitinib and any structural analog thereof having inhibitory activity against JAK family kinases of at least 50% of the IFN signaling pathway. The kit described above.

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