Methods and kits for improved fermentative production of recombinant viruses

Abstract

The present invention relates to methods and kits for improved fermentative production of recombinant adeno-associated viruses (AAV), adenoviruses, lentiviruses or retroviruses such as gamma retroviruses in HEK293 cells. In addition, the use of the virus to increase the fermentative production in HEK293 cells is also described.

Description

Technical Field

[0001] This invention relates to an improved fermentation production method and kit for recombinant adeno-associated virus (rAAV), adenovirus, lentivirus, or retrovirus such as gamma retrovirus in HEK293 cells. Furthermore, its use in increasing the fermentation production of said virus in HEK293 cells is described. Background Art

[0002] Since the first clinical trial for cystic fibrosis approximately 18 years ago, the field of gene therapy using recombinant adeno-associated virus (rAAV) has matured. rAAV possesses the gene therapy properties of interest. Although rAAV induces a transient increase in cytokine levels when injected into mice, it has been found to induce minimal immunogenicity and show almost no inflammation several hours later. rAAV is suitable for many medical applications because it can transduce non-dividing cells in a variety of tissues or organs. Treatment of lipoprotein lipase deficiency (alipogene tiparvovec or...) with rAAV is also being explored. Clinical evidence following gene therapy for congenital hepatic amaurosis also indicates that rAAV is well-tolerated and can drive transgene expression in humans for several years.

[0003] rAAV production is carried out using mammalian cell production systems. The cells can support the replication of viral vectors (e.g., recombinant adenovirus, Adv).

[0004] Transient transfection methods using two or three plasmids have also been developed for rAAV production. These methods can be used for rapid testing of rAAV constructs and can be scaled up to 1000L. A key advantage of transient transfection is the absence of residual helper viral contaminants. Several stable cell lines (production / packaging cells) have also been developed to facilitate rAAV production.

[0005] The HEK293 cell line has been used for rAAV production. They can be readily adapted for suspension culture in serum-free medium. Furthermore, many viral vectors have been approved for Phase I / II / III clinical trials. Initially, HEK293 cells grew adherently, thus limiting the scalability of rAAV production. This limitation was overcome by adapting the HEK293 cell line to suspension growth.

[0006] While efficient large-scale production of rAAV can be achieved in specially genetically engineered HEK293 cells (such as HEK293T cells with inserted SV40 large T antigen) or through proprietary high-yield HEK293 cells, there is a need for HEK293 cell-based rAAV production systems that do not require genetic modification of HEK293 cells or the use of proprietary high-yield cells. Summary of the Invention

[0007] In a first aspect, the present invention provides a method for fermentation production of recombinant adeno-associated virus (rAAV), adenovirus, lentivirus, or retrovirus, comprising the following steps:

[0008] Step 1: Culture rAAV, lentivirus, adenovirus, or retrovirus in HEK293 cells for an incubation period in a culture medium containing a certain amount of interferon (IFN) inhibitor; and

[0009] Step 2: Recover rAAV, lentivirus, adenovirus, or retrovirus from cell culture.

[0010] In a second aspect, the present invention provides a method for fermentation production of adeno-associated virus (AAV), lentivirus, adenovirus, or retrovirus, comprising the following steps:

[0011] Step 1: Perform transcriptomic analysis on HEK293 cells transfected with rAAV, lentivirus, adenovirus, or retrovirus, and ensure that the transcription of at least one of the following genes is enriched compared to the negative control:

[0012] (i) TNFA signal transduction occurs via NFKB;

[0013] (ii) Interferon-γ response;

[0014] (iii) Interferon α response;

[0015] (iv) TGF-β signal transduction; and / or

[0016] (v)IL6_JAK_STAT3_Signal Transduction

[0017] Preferably, if

[0018] (1) Interferon-γ response; and / or

[0019] (2) Interferon α response

[0020] Transcription enrichment occurs; therefore, the HEK293 cells are selected.

[0021] Step 2: Incubate rAAV, lentivirus, adenovirus, or retrovirus in selected HEK293 cells for an incubation period in a medium containing a certain amount of interferon (IFN) inhibitor; and

[0022] Step 3: Recover rAAV, lentivirus, adenovirus, or retrovirus from the cell culture.

[0023] In a third aspect, the present invention provides the use of IFN inhibitors transfected or infected with rAAV, lentivirus, adenovirus or retrovirus.

[0024] In a fourth aspect, the present 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 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 the pharmaceutical composition.

[0025] In a fifth aspect, the present invention provides a kit for cell culture, comprising:

[0026] (a) HEK293 cells; and

[0027] (b) An IFN inhibitor, preferably selected from the group consisting of ruxolitinib, tofacitinib, baricitinib, delgocitinib, emapalumab, and fontolizumab, and any structural analogues thereof having IFN inhibitory activity. More preferably, the inhibitor is ruxolitinib or any structural analogue of ruxolitinib that has inhibitory activity against JAK family kinases in the IFN signaling pathway. Particularly preferred, the structural analogue has at least 50%, more preferably 80%, even more preferably at least 90%, and most preferably at least 95% inhibitory activity against the Jak1 component compared to ruxolitinib.

[0028] Through transcriptomic analysis, the inventors were surprised to discover that various available HEK293 cells were characterized by unique patterns of gene activation during triple transfection for rAAV production. For example, the inventors found that interferon-responsive genes were activated in some HEK293 cells that exhibited relatively low rAAV production yields in fermentation production.

[0029] According to Strasser et al., Int. J. Mol. Sci 22 (2021), 11499, this was unexpected. In a later paper, the effects of rAAV5 production on the proteome of HEK293 cells were investigated. The authors found that the EIF2 signaling pathway, oxidative phosphorylation, and spliceosome cycling were activated. Another paper, Barnes et al., Mol. Ther. Nucl. Acids 26 (2021), 94, used genome-wide activation screening to identify gRNAs that could potentially increase rAAV production in HEK293T cells. They found that SKA2 or ITPRIP could be used to increase rAAV production in HEK293T cells.

[0030] Therefore, the inventors' discovery is surprising given that this detailed study did not provide any relevant instructions. The inventors further utilized this unexpected finding by administering IFN inhibitors, particularly IFN signaling inhibitors that lead to increased rAAV production in HEK293 cells. By adding IFN inhibitors, rAAV viral yield increased by more than 100% after several days, even up to 200%, which is undoubtedly significant for the desired large-scale rAAV production in HEK293 cells. Attached Figure Description

[0031] Figure 1: rAAV production yields in HEK293 cells (CL1) identified by vector genomic droplet digital PCR (ddPCR) in supernatant and cell culture, respectively, for untreated (ctrl) cells, cells treated with inhibitor solvent (DMSO), and cells treated with different inhibitor concentrations. Figure 1A and 1B The figure shows that the rAAV production yield in HEK293 cells (CL1), determined by capsid particles in the supernatant and cell culture, was [data missing]. Figure 1C and 1D Shown in.

[0032] Figure 2: rAAV yields of three different cell lines (CL1, CL2, and CL3) were compared in a single set of experiments with or without (ctrl). The rAAV yield of CL1 ctrl was used as a baseline (100%) for comparing yields among CL1, CL2, and CL3, see Figures 2(A), 2(B), and 2(C). The potential effects of the corresponding solvents (DMSO, water for injection (wfi), PBS) without inhibitors were also tested. The results determined by AAV8 ELISA in the supernatant are shown in Figure 2. Figure 2(A) involves the CL1 cell line; Figure 2(B) involves the CL2 cell line; and Figure 2(C) involves the CL3 cell line.

[0033] Figure 3: To test the effect of IFN inhibitors on rAAV yield in large-scale bioreactor fermentation of HEK293 cells, a 10L bioreactor was used. Results were... Figure 3A (Determined by AAV8 ELISA of capsid particles) and Figure 3B (As shown in (determined by ddPCR)).

[0034] Figure 4: To test the effect of IFN inhibitors on the rAAV yield in adherent HEK293 cells, cells were cultured in a T75 flask system. Results were... Figure 4A (As determined by ddPCR) and B (as determined by capsid particle AAV8 ELISA) are shown.

[0035] Figure 5 To test the effect of IFN inhibitors on HEK293 suspension cells (CL1) used for lentiviral production, a shake-flask system was used. Results were obtained in... Figure 5 As shown in the image (determined via p24 ELISA). Detailed Implementation

[0036] In a first aspect, the present invention provides a method for fermentation production of recombinant adeno-associated virus (rAAV), adenovirus, lentivirus, or retrovirus, comprising the following steps:

[0037] Step 1: Culture rAAV, adenovirus, lentivirus, or retrovirus in HEK293 cells for an incubation period in a medium containing a certain amount of interferon (IFN) inhibitor; and

[0038] Step 2: Recover rAAV, adenovirus, lentivirus, or retrovirus from cell culture.

[0039] The term "AAV" as used herein includes AAV serotypes, their chimeras, or heterozygotes. AAV serotypes may be selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, and their chimeric or heterozygous variants. Preferably, the AAV serotype is selected from AAV5, AAV6, AAV8, AAV9, and AAV-DJ, and more preferably AAV8. Chimeric AAV variants include more than one AAV serotype. Heterozygous AAV variants include modified mutant AAV serotypes.

[0040] AAVs are small viruses that infect humans and some other primate species. They belong to the genus *Dependoparvovirus*, which in turn belongs to the family *Parvoviridae*. They are small (20-25 nm) replication-defective, non-enveloped viruses with a linear single-stranded DNA (ssDNA) genome of approximately 4.8 kilobases (kb). rAAVs are thought to remain predominantly free-form.

[0041] For safety reasons, rAAV is typically prepared from 2, 3, or even several individual expressible units. Therefore, many vector systems are fabricated via transient co-transfection of multiple plasmids. Stable transfection is also possible.

[0042] For AAV, the so-called triple transfection method is well known. For this purpose, host cells are transfected with (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.

[0043] Alternatively, recombinant AAV can be obtained via a double transfection method. In this method, host cells are transfected with (i) a plasmid carrying the therapeutic gene of interest and (ii) a plasmid carrying adenovirus 5 genes such as E4, E2a, and VA, and also containing the rep and cap genes of AAV.

[0044] The term "adenovirus" in this article includes adenovirus packaging systems, such as adenovirus serotype 5 (Adv5), which use adenovirus shuttle vectors to deliver target gene sequences into production cell lines expressing E1. The early viral transcription units E1 and E3 are defective in the most commonly used recombinant adenovirus packaging systems. This system can be a dual-vector system consisting of an adenovirus expression plasmid with different promoters and tags, and an adenovirus genomic backbone with the E1 region deleted from the plasmid.

[0045] The term "lentivirus" as used herein includes HIV and its variants, and includes lentiviruses possessing the pseudotyped VSV-G envelope protein. Preferably, the lentivirus is derived from HIV-1.

[0046] Lentivirals are a genus of retroviruses that cause chronic and fatal diseases characterized by long latency periods in humans and other mammalian species. This genus includes human immunodeficiency virus (HIV), which causes AIDS. Lentivirals are distributed worldwide and are known to reside in apes, cattle, goats, horses, cats, sheep, and several other mammals. To increase the safety of lentiviruses, the components required for viral production are split into multiple plasmids (three for the second-generation system and four for the third-generation system). The components of both systems are as follows:

[0047] Lentiviral transfer plasmids encoding the insert fragment of interest The flanking structures of the transgenic sequence are long terminal repeats (LTRs), which facilitate the integration of the transfer plasmid sequence into the host genome. Typically, the sequences between LTRs (including the LTRs) are integrated into the host genome during viral transduction. Many lentiviral transfer plasmids are based on HIV-1. For safety reasons, transfer plasmids are non-replicable and may contain additional deletions in the 3' LTR, causing the virus to "self-inactivate" (SIN) after integration.

[0048] Packaging plasmid. Packaging plasmids may contain the gag, pol, rev, and tat genes on one plasmid or be distributed on two plasmids.

[0049] Encapsulated plasmids. Preferably, the envelope plasmid encodes VSV-G.

[0050] Lentivirals can integrate significant amounts of viral nucleic acid DNA into the host cell's DNA and can effectively infect non-dividing cells, making them one of the most efficient gene delivery methods. They can become endogenous, thereby integrating their genome into the host's germline genome, allowing the virus to be subsequently inherited by the host's offspring.

[0051] The term "retrovirus" as used herein includes gamma retroviruses. Preferably, the retrovirus is murine leukemia virus. In another preferred embodiment, the retrovirus is derived from HIV-1.

[0052] The term “HEK293 cells” in this article refers to human embryonic kidney cells originally isolated and cultured by Alex van der Eb. Frank Graham transfected these cells with spliced ​​adenovirus 5 (Ad5) DNA (Graham FL, Smiley J, Russell WC, Nairn R (July 1977). The Journal of General Virology. 36(1): 59–74). Incorporation of adenovirus genes into the HEK293 cell genome resulted in the cells becoming efficient in producing large quantities of recombinant proteins from plasmid vectors. The term also includes any derivatives 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 derivatives are available from cell culture collections such as ATCC or ECACC and from commercial providers.

[0053] Preferably, when used in the method of the present invention, HEK293 cells produce an increased yield of rAAV, adenovirus, lentivirus, or retrovirus in the presence of an IFN inhibitor in the culture medium. "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 that is higher than the yield produced in the HEK293 cell line in the absence of an IFN inhibitor under the same culture conditions and after the same incubation period. Preferably, "increased yield" means a yield that is at least 0.2 times, at least 0.3 times, at least 0.5 times, at least 0.7 times, at least 1.0 times, at least 1.5 times, at least 2.0 times, at least 2.5 times, at least 3.0 times, at least 4 times, or at least 5 times that produced in the HEK293 cell line in the absence of an IFN inhibitor. Preferably, as outlined in the examples, the yield is determined after an incubation period of 96 hours in the presence of 5 μM ruxolitinib (as an IFN inhibitor).

[0054] In some implementations, the "increased yield" can be defined by the rAAV yield from HEK293 cells in the presence of inhibitors, being at least about 1 x 10⁻⁶ per milliliter of cell culture. 9 Each vector-containing genome particle (vg / mL), for example, at least approximately 5 x 10^6 per milliliter of cell culture at all upstream and downstream processing steps and after product concentration. 9 , 1x10 10 5 x 10 10 1x10 11 1x10 12 1x10 13 Or 1x 10 14 One or more vector-containing genome particles. Preferably, the yield is determined as described in the examples. The yield can also be measured as capsid particles per L or mL of cell culture. In some embodiments, at least about 1 x 10⁻⁶ particles are obtained per mL of cell culture. 9 AAV yield from HEK293 cells containing vector genome particles (vg / mL) in the presence of inhibitors, for example, at least approximately 5 x 10⁻⁶ per mL of cell culture. 9 1x10 10 , 5x10 10 1x10 11 1x10 12 1x10 13 Or 1x 10 14 One or more capsid particles.

[0055] In a preferred embodiment, the HEK293 cells of the present invention are characterized by activation of at least one of the following cellular pathways at a specified time point:

[0056] (1) Interferon-γ response (0-48h);

[0057] (2) Interferon α response (0-48h);

[0058] Cellular pathway activation was achieved through triple transfection of the plasmid required for rAAV8 replication in transfected HEK293 cells. Further details are outlined in the Examples section.

[0059] In another preferred embodiment, the HEK293 cells of the present invention are characterized by activation via at least one of the following cellular pathways:

[0060] (i) TNFA signal transduction occurs via NFKB;

[0061] (ii) Interferon-γ response;

[0062] (iii) Interferon α response;

[0063] (iv) TGF_β_ signal transduction;

[0064] (v)IL6_JAK_STAT3_Signal Transduction;

[0065] Preferably, HEK293 cells exhibit activation of at least two, at least three, at least four, and more preferably at least five of the above-mentioned cellular pathways.

[0066] More preferably, at least two activated cellular pathways are selected from the group consisting of: TNFA signaling via NFKB, interferon-γ response, interferon-α response, TGF-β signaling, and IL6-JAK-STAT3 signaling.

[0067] Particularly preferred, at least two activating genes are selected from the group consisting of: interferon-γ response, interferon-α response, and IL6-JAK-STAT3 signal transduction.

[0068] Most preferably, the HEK293 cells to be used in the method of the present invention are CRL-1573.

[0069] In a preferred embodiment, HEK293 cells are an adherent cell line. In an even more preferred embodiment, HEK293 cells are a suspension cell line, or adapted for suspension growth. Depending on whether HEK293 cells are adherent or suspension cell lines, those skilled in the art can employ different culture methods. Culture can be carried out in culture dishes, shake flasks, or roller flasks. Culture in shake flasks or roller flasks is preferred.

[0070] The term "culture medium" as used herein includes any culture medium suitable for culturing mammalian cells. The culture medium can be a complex medium, a semi-defined medium, or a defined medium. Preferably, the culture medium is a chemically defined medium. More preferably, commercially available culture media, such as mammalian cell culture media known to those skilled in the art, are used.

[0071] Culture conditions can be varied according to the specific requirements of cells in parameters such as pH, salt concentration, and temperature. This method can be carried out as a continuous process, in batch mode, or in fed-batch mode, as is well known to those skilled in the art. Preferably, the method is carried out in batch mode or fed-batch mode.

[0072] The term "IFN inhibitors" in this article refers to inhibitors of the interferon immune response pathway. Interferons are a group of signaling proteins produced by host cells in response to the presence of certain viruses. Typically, virus-infected cells release interferons, leading nearby cells to enhance their antiviral defenses. Type I interferons can be broadly characterized into three groups: IFN-I, type II (IFN-II), and type III (IFN-III), with subclassifications based on differences in IFN transcription gene loci and their homologous receptors. IFN-I is the largest and most well-characterized group, comprising seven subclasses: IFNα, IFNβ, IFNδ, IFN∈, IFNκ, IFNω, and IFNτ, while IFN-II includes IFNγ. IFN-I and IFN-II signal via IFNαR1 / R2 (IFNAR) and IFNγR1 / R2 (IFNGR), respectively. The last class of IFN-III (also classified as "IFN-like cytokines") consists of interleukin (IL)-28A (IFNλ2), IL28B (IFNλ3), and IL29 (IFNλ1), and signals are transmitted through the IL-28RI / IL10R2 receptor chain.

[0073] Recognition of microbial products by a series of cell surface and intracellular pattern recognition receptors, including Toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I), can lead to the induction of genes encoding type I interferon (IFN), mediated by several different signaling pathways. When type I IFN binds to its receptor (IFNAR), it can induce a variety of downstream signaling pathways, resulting in a wide range of biological effects. The canonical signal transducer and activator of the transcription 1 (STAT1)-STAT2-IFN-regulatory factor 9 (IRF9) signaling complex (also known as the IFN-stimulatory gene factor 3 (ISGF3) complex) binds to the IFN-stimulatory response element (ISRE) in the gene promoter, leading to the induction of numerous IFN-stimulatory genes (ISGs). Type I IFN can also signal via the STAT1 homodimer, which is more commonly associated with IFNγ-mediated signaling pathways. Other STAT heterodimers and homodimers can also be activated downstream, including STAT3, STAT4, and STAT5. Other signaling pathways independent of Janus kinase (JAK) and / or STAT activity can also be activated, including the mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K) pathways, leading to different effects on cells.

[0074] The IFN inhibitors used in this invention are intended to target IFN-induced and / or signaling cascades. Activation of multiple signaling pathways via IFN binding to its receptor is crucial for the generation of IFN-mediated biological functions. As used herein, the phrase "IFN-induced cascade" encompasses any viral-induced pathway leading to activation of IFN-α / β transcription and expression. Molecules in the IFN-induced cascade that can be targeted by the inhibitor may include, but are not limited to, TLR3, MDA5, RIG-I, Cardif, TBK1 / IKKE, 25IKKa / , IRF3, NFKB, or ATF-2 / C-JUN. As used herein, the phrase "IFN signaling cascade" encompasses any IFN-activated signaling pathway whose activation leads to antiviral changes in cells. Targeted signaling pathways may include, but are not limited to, the JAK-STAT pathway.

[0075] The inhibitors used in this invention can target one or more components of the IFN-induced and / or signaling cascades shown in Table 1. The inhibitors used in this invention can be used alone or in combination.

[0076] Table 1: Possible targets for interferon pathway inhibition

[0077]

[0078]

[0079] In a preferred embodiment of the invention, the inhibitor of the present invention targets the JAK / STAT signaling pathway. The inhibitor can target any component of the JAK / STAT pathway. Targeting components of the JAK / STAT pathway may include, but are not limited to, Tyk2, Jak1, Jak2, STAT-1, STAT-2, and IRF-9. In another preferred embodiment, the inhibitor targets Jak1. In yet another preferred embodiment of the invention, the inhibitor of the present invention targets the TBK-1 / IKKE / IKK2 induction pathway.

[0080] The inhibitors used in this invention can be any molecule that reduces the activity of IFN-induced and / or signaling cascades. Non-limiting examples of inhibitors that can be used according to this invention include small molecule inhibitors, siRNAs, miRNAs, lipid transport proteins, plastic antibodies, antibodies, or antibody fragments.

[0081] IFN inhibitors are preferably not selected from nicotinic acid and / or nicotinamide.

[0082] Preferably, the IFN inhibitor is not selected from (i) nicotinic acid methyl ester, (ii) inositol and (iii) choline.

[0083] In another preferred embodiment, the IFN inhibitor is not selected from (iv) a combination of methyl nicotinic acid and inositol, (v) a combination of methyl nicotinic acid and choline, or (vi) a combination of inositol and choline.

[0084] In another preferred embodiment, the IFN inhibitor is not (vii) a combination of nicotinic acid methyl ester, inositol, and choline.

[0085] If the inhibitor is designed to target Jak1, it is conceivable to generate antibodies, plastic antibodies, or antibody fragments against Jak1 such that, in use, the antibodies, plastic antibodies, or antibody fragments bind to Jak1 and prevent it from phosphorylating, for example, STAT transcription factors.

[0086] Alternatively, if the inhibitor is a miRNA, siRNA, or shRNA, it can be envisioned that the miRNA, siRNA, or shRNA has a sequence complementary to a portion of the Jak1 mRNA sequence, such that the miRNA, siRNA, or shRNA inhibitor will bind to Jak1 mRNA, thereby reducing the translation and abundance of Jak1 in the cell.

[0087] Preferably, the inhibitor is a small molecule. As used herein, the phrase "small molecule" refers to a low molecular weight (less than about 800 Da) organic or inorganic compound.

[0088] The small molecule inhibitors used in this invention can function through competitive, non-competitive, mixed, or non-competitive inhibition. In one embodiment, the inhibitor is a competitive inhibitor.

[0089] Preferably, the IFN inhibitor is selected from the group consisting of ruxolitinib (CAS: 941678-49-5), tofacitinib, baricitinib, degotinib, imamalub and fenestrazumab, and preferably, the inhibitor is ruxolitinib.

[0090] Ruxotinib is an inhibitor of the JAK family kinase component in the IFN signaling pathway. Ruxotinib 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]propionitrile.

[0091] Tofacitinib is an inhibitor of the Jak1 component in the IFN signaling pathway.

[0092] Baricitinib is an inhibitor of Jak, blocking subtypes Jak1 and Jak2.

[0093] Degatinib is an inhibitor of Jak.

[0094] Imalumab is a human monoclonal antibody targeting interferon-γ (IFN-γ).

[0095] Fontuzumab is a humanized antibody against interferon-γ (IFN-γ).

[0096] In a preferred embodiment, the final concentration of the IFN inhibitor in the culture medium is in the range of about 0.0001 to 50 mM, preferably about 1 to about 100 μM, and more preferably about 0.5 to about 25 μM. Particularly preferred are IFN inhibitor concentrations of 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, or 25 μM, with 5 μM and 10 μM being more preferred. Specifically, 5 μM or 10 μM ruxotinib is preferred.

[0097] The interferon inhibitor can be added to the culture medium containing HEK293 cells (i) before adding the plasmid for rAAV production in the transfection step, or (ii) during the transfection step, to the culture medium containing HEK293 cells and the plasmid for rAAV production. Method variant (i) is preferred.

[0098] The preferred method for rAAV production according to the present invention includes the following steps:

[0099] (a) HEK293 cells were cultured in a medium containing a certain amount of IFN inhibitor for the first incubation period;

[0100] (b1) Transfect HEK293 cells with the 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

[0101] (b2) Transfect HEK293 cells with the two plasmids required 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;

[0102] (c) The transfected HEK293 cells were cultured for a second incubation period; and

[0103] (d) Recover rAAV particles.

[0104] Another preferred method for rAAV production according to the present invention includes the following steps:

[0105] (a) HEK293 cells were cultured in a culture medium for the first incubation period;

[0106] (b1) HEK293 cells were transfected in a culture medium containing a certain amount of IFN inhibitor and transfection reagent using (i) adenovirus helper plasmid, (ii) packaged rep-cap plasmid and (iii) recombinant rAAV plasmid containing transgene; or

[0107] (b2) HEK293 cells were transfected with two plasmids required for rAAV production in a culture medium containing a certain amount of IFN inhibitor and transfection reagent, preferably (i) a plasmid containing an adenovirus helper gene and a rep-cap gene and (ii) a recombinant rAAV plasmid containing a transgene.

[0108] (c) Transfected HEK293 cells were cultured for a second incubation period in the presence of an IFN inhibitor; and

[0109] (d) Recover rAAV particles.

[0110] Another preferred method for rAAV production according to the present invention includes the following steps:

[0111] (a) HEK293 cells were cultured in a culture medium for the first incubation period;

[0112] (b1) HEK293 cells were triple-transfected in a medium containing transfection reagents with (i) an adenovirus helper plasmid, (ii) a packaged rep-cap plasmid, and (iii) a recombinant rAAV plasmid containing transgenes; or

[0113] (b2) Transfect HEK293 cells with the two plasmids required for rAAV production in a medium containing transfection reagent, preferably (i) a plasmid containing an adenovirus helper gene and a rep-cap gene and (ii) a recombinant rAAV plasmid containing a transgene;

[0114] (c) Transfected HEK293 cells were cultured in a medium containing an IFN inhibitor for a second incubation period; and

[0115] (d) Recover rAAV particles.

[0116] Step (a)

[0117] Before proceeding to step (b), HEK293 cells may be cultured with an IFN inhibitor for approximately 1 hour to approximately 48 hours, preferably 1 to 12 hours, more preferably 1 to 6 hours, and even more preferably 1, 2 or 3 hours.

[0118] Step (b)

[0119] (i) The adenovirus helper plasmid may contain one or more adenovirus genes selected from the group consisting of E1A, E1B, E2A, E4ORF6 and VA. More preferably, the adenovirus helper plasmid contains adenovirus genes E2A, E4ORF6 and VA.

[0120] (ii) The packaged rep-cap plasmid contains the rep and cap genes of rAAV. Alternatively, the necessary rep and cap genes are provided by HEK293 cells stably transfected with said rep and cap genes.

[0121] Preferably, the rep-cap plasmid contains the rep2 gene and the cap8 gene.

[0122] (iii) rAAV plasmids may contain any heterologous gene of interest. Genes of interest include nucleic acids or RNA encoding polypeptides, including reporter genes, therapeutic (e.g., for medical or veterinary use), immunogenic (e.g., for vaccines), or diagnostic polypeptides or RNAs. Alternatively, the heterologous nucleic acid may encode any polypeptide or RNA that is desired to be produced in vitro, ex vivo, or in cells in vivo.

[0123] In one embodiment of the invention, the rAAV vector is self-complementary. Self-complementary vectors can advantageously overcome the rate-limiting step of second-strand DNA synthesis and confer earlier and stronger gene expression. Preferably, the rAAV vector containing the gene of interest is self-complementary. In one embodiment, the vector contains single-stranded DNA.

[0124] The rAAV vector may also contain regulatory sequences that are operablely linked to the gene of interest in a manner that allows transcription, translation, and expression in cells infected with a virus containing the vector.

[0125] Regulatory sequences may include appropriate transcription initiation, termination, promoter, and enhancer sequences; RNA processing signals, such as splicing and polyadenylation (polyA) signal sequences; sequences stabilizing cytoplasmic mRNA; sequences enhancing translation efficiency (i.e., Kozak concordant sequences); sequences enhancing protein stability; and sequences enhancing the secretion of the encoded product when desired. PolyA signal sequences may be synthetic or derived from many suitable species, including, for example, SV-40, humans, and cattle. Regulatory sequences may also include introns. Regulatory sequences may also include promoters. Promoters may be any promoter suitable for expressing the gene of interest in target cells. Promoters may be inducible or constitutive.

[0126] In one embodiment of the invention, the rAAV vector comprises an ITR or a functional fragment thereof. Preferably, the vector comprises 5'AAV ITR and 3'AAV ITR. The ITR can be any suitable rAAV serotype, including any AAV serotype described herein. The ITR can be readily isolated using techniques known in the art and can be isolated or obtained from public or commercial sources (e.g., the American Type Culture Collection, Manassas, VA). Alternatively, the ITR sequence can be obtained by synthesis or other suitable means with reference to published sequences. Preferably, the vector comprises 5'AAV2 ITR and 3'AAV2 ITR.

[0127] Preferably, the recombinant rAAV plasmid contains a 5'AAV ITR, the gene of interest, and a 3'AAV ITR.

[0128] The 5'AAV ITR and 3'AAV ITR can be from the same serotype or from different serotypes. More preferably, the 5'AAV ITR and 3'AAV ITR are from the AAV8 serotype.

[0129] The transfection step can use polyethyleneimine or It is used as a transfection reagent. Preferably, polyethyleneimine is used as the transfection reagent.

[0130] In one implementation, a transfection reagent is added, and the IFN inhibitor remains in the culture medium. The transfection step can be performed using both the transfection reagent and the IFN inhibitor. Alternatively, the transfection step can be performed using transfection split media containing both the transfection reagent and the IFN inhibitor.

[0131] Step (c)

[0132] The second incubation period 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.

[0133] In general, the incubation period in the presence of IFN inhibitors, independent of when they are added to the culture medium, 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.

[0134] The rAAV template, along with the rAAV rep and cap sequences, are provided under conditions that allow a viral vector containing the rAAV template packaged within an rAAV capsid to be produced in cells. The method may also include a step of collecting the viral vector from the culture. In one embodiment, the viral vector may be collected by lysing cells, for example, after removing cells from the culture medium, such as by cell pelleting. In another embodiment, the viral vector may be collected from the culture medium of the cultured cells, for example, to isolate the vector secreted from the cells. Some or all of the culture medium may be removed from the culture once or more, for example, periodically during the culture step to collect rAAV (such as every 12, 18, 24, or 36 hours, or a longer extended time compatible with cell viability and vector production), for example, starting approximately 48 hours after transfection. After removing the culture medium, fresh culture medium, with or without additional nutrient supplements, may be added to the culture. In one embodiment, cells may be cultured in a perfusion system such that the culture medium flows constantly through the cells and collected for the isolation of secreted rAAV.

[0135] Step (d)

[0136] When rAAV is secreted from HEK293 cells, step (d) includes collecting the supernatant of the culture medium and purifying the rAAV particles from the supernatant. When rAAV is not secreted from HEK293 cells, step (d) includes cytotoxicity of HEK293 cells to obtain rAAV particles and subsequently purifying the rAAV particles.

[0137] 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.

[0138] In representative embodiments, the method of the present invention is fully scalable, and therefore can be carried out in any desired volume of culture medium, for example, from 10 ml (e.g., in a shake flask) to 10 L, 50 L, 100 L or more (e.g., in a bioreactor system such as a wave bioreactor system and a stirred tank). The method is applicable to the production of all serotypes and chimeras of AAV, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, and any chimeras thereof.

[0139] The yield of rAAV from HEK293 cells can be determined, for example, in a droplet digital PCR assay (available from, for example, Bio-Rad) as a vector genome / L cell culture. Alternatively, the yield can be determined by assessing the amount of capsid particles per L or mL of cell culture in, for example, an ELISA assay. Details of the assays are outlined in the Examples section.

[0140] In some embodiments, the method provides at least about 1 x 10⁻⁶ cells per milliliter of cell culture. 9 One purified vector-containing genome particle (vg / mL), for example, at least approximately 5 x 10^6 particles per milliliter of cell culture. 9 1x10 10 5 x 10 10 1x10 11 1x10 12 1x10 13 Or 1x 10 14 One or more vector-containing genome particles. Yield can also be measured as capsid particles per L or mL of cell culture. In some embodiments, the method provides at least about 1 x 10⁻⁶ particles per mL of cell culture. 9 One purified vector-containing genome particle (vg / mL), for example, at least approximately 5 x 10^6 particles per milliliter of cell culture. 9 1x10 10 5 x 10 10 1x10 11 1x10 12 1x10 13 Or 1x 10 14 One or more capsid particles.

[0141] The yield can be determined in the supernatant or cell suspension. The cell suspension contains the virus content from the supernatant as well as the virus content in the destroyed cells. Preferably, the yield in the cell suspension is determined after at least one freeze-thaw cycle. The absolute titer depends on, for example, the analytical method used, the AAV serotype, the inserted therapeutic gene of interest, and the transfection method.

[0142] In another aspect, the present invention provides the use of an IFN inhibitor for increasing the yield of rAAV, adenovirus, lentivirus, or retrovirus in HEK293 cell cultures. The IFN inhibitor and HEK293 cells are as defined above. Preferably, the IFN inhibitor is selected from the group consisting of ruxotinib, tofacitinib, baricitinib, degotinib, imarumumab, and fustatinumab, and any structural analogues thereof having IFN inhibitory activity. Preferably, the IFN inhibitor is ruxotinib and any structural analogue thereof having inhibitory activity against the Jak1 component of the IFN signaling pathway.

[0143] In another aspect, the present invention provides a method for preparing a pharmaceutical composition, comprising (i) the method according to the invention, wherein the 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.

[0144] In the context of this invention, any pharmaceutically acceptable excipient may be used, and such pharmaceutically acceptable excipients are well known in the art. The selection of a pharmaceutically acceptable excipient will be determined in part by the specific site at which the composition is to be administered and the specific method of administering the composition. The pharmaceutical composition may optionally be sterile or sterile except for one or more recombinant adeno-associated virus vectors.

[0145] Suitable formulations of the pharmaceutical composition include aqueous and non-aqueous solutions; isotonic sterile solutions that may contain antioxidants, buffers, and antibacterial agents; and aqueous and non-aqueous sterile suspensions that may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The formulations may be available in single-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored under lyophilized (freeze-dried) conditions, requiring only the addition of a sterile liquid carrier, such as water, just before use. Temporary solutions and suspensions may be prepared from sterile powders, granules, and tablets. Preferably, the carrier is a buffered saline solution. More preferably, the pharmaceutical composition used in the method of the present invention is formulated to protect the adeno-associated virus vector from damage before administration. For example, the pharmaceutical composition may be formulated to reduce loss of the adeno-associated virus vector in devices used for preparing, storing, or administering the expression vector, such as glassware, syringes, or needles. The pharmaceutical composition may be formulated to reduce the photosensitivity and / or temperature sensitivity of the adeno-associated virus vector. Therefore, the pharmaceutical composition preferably comprises 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 a composition can extend the shelf life of the carrier, facilitate administration, and improve the efficiency of the method of the present invention. The pharmaceutical composition can also be formulated to improve the transduction efficiency of the recombinant adeno-associated virus vector. Furthermore, those skilled in the art will recognize that the pharmaceutical composition may contain other therapeutic agents or bioactive agents. For example, anti-inflammatory factors such as ibuprofen or steroids may 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., antimicrobial and antifungal agents, may be present to treat existing infections and / or reduce the risk of future infections such as those associated with gene transfer procedures.

[0146] In another aspect, the present invention provides a kit for cell culture, comprising:

[0147] (a) HEK293 cells; and

[0148] (b) An IFN inhibitor selected from the group consisting of ruxolitinib, tofacitinib, baricitinib, degotinib, imarumab and fenestrazumab and any structural analogues thereof having IFN inhibitory activity, preferably, the IFN inhibitor is ruxolitinib and any structural analogue thereof having at least 50%, more preferably 80%, even more preferably at least 90%, and most preferably at least 95% inhibitory activity against JAK family kinases compared to ruxolitinib.

[0149] The kit may include other components. It may also include instructions for the user.

[0150] The project of this invention

[0151] 1. A method for fermenting to produce adeno-associated virus (AAV), adenovirus, lentivirus, or retrovirus, comprising the following steps:

[0152] Step 1: The rAAV, the lentivirus, the adenovirus, or the retrovirus were cultured in HEK293 cells for an incubation period in a culture medium containing a certain amount of interferon (IFN) inhibitor; and

[0153] Step 2: Recover the rAAV, the adenovirus, the lentivirus, or the retrovirus from the cell culture.

[0154] 2. The method of Project 1, wherein the yield of the rAAV, adenovirus, lentivirus, or retrovirus after an incubation period of at least 96 hours is increased by at least 10%, preferably at least 50%, more preferably at least 100%, and even more preferably at least 200%, compared to the yield of the rAAV, adenovirus, lentivirus, or retrovirus in the absence of the IFN inhibitor.

[0155] 3. The method as described in Project 1 or 2, wherein, in the presence of the IFN inhibitor from the culture medium, the yield of rAAV, adenovirus, lentivirus, or retrovirus produced by the HEK293 cells is at least about 1 x 10⁻⁶ per milliliter of cell culture. 9 One purified vector-containing genome particle (vg / mL), preferably at least about 5 x 10^6 per milliliter of cell culture. 9 One, more preferably at least 1 x 10 10 One, more preferably at least 5 x 10 10 And the optimal choice is 1x10. 11 1x10 12 1x10 131x10 14 Or 5 x 10 14 One or more genome particles containing vectors.

[0156] 4. The method of any one of items 1 to 3, wherein the HEK293 cells are characterized upon rAAV transfection by activation of at least one of the following genes:

[0157] (1) Interferon-γ response; and / or

[0158] (2) Interferon α response.

[0159] 5. The method as described in Project 4, wherein the HEK293 cells are characterized by activation of at least interferon-γ and interferon-α responses upon rAAV transfection.

[0160] 6. The method of any one of items 1 to 3, wherein the HEK293 cell line is characterized upon rAAV transfection by activation of at least one of the following genes:

[0161] (i) TNFA signal transduction occurs via NFKB;

[0162] (ii) Interferon-γ response;

[0163] (iii) Interferon α response;

[0164] (iv) TGF-β signal transduction; and / or

[0165] (v)IL6_JAK_STAT3_Signal Transmission.

[0166] 7. The method of Project 6, wherein at least two activating genes are selected from the group consisting of: TNFA signaling via NFKB, interferon γ response, interferon α response, TGF β signaling and IL6 JAK STAT3 signaling, more preferably the at least two activating genes are selected from the group consisting of: interferon γ response, interferon α response and IL6 JAK STAT3 signaling.

[0167] 8. The method as described in item 6 or 7, wherein the HEK293 cells exhibit activation of at least two, at least three, at least four, more preferably each of five genes.

[0168] 9. The method according to any one of items 1 to 8, wherein the HEK293 cells are administered at a dose of at least 1 x 10⁻⁶ ppm of the IFN inhibitor from the culture medium. 10 One, more preferably at least 5 x 10 10One, and the optimal choice is at least 1x10 11 1x10 12 1x10 13 1x10 14 Or 5 x 10 14 The AAV yield of each capsid particle is used for characterization.

[0169] 10. The method according to any one of items 1 to 9, wherein the HEK293 cells are adherent cell lines.

[0170] 11. The method according to any one of items 1 to 9, wherein the HEK293 cells are a suspension cell line or adapted for suspension growth.

[0171] 12. The method according to any one of items 1 to 11, wherein the HEK293 cells are selected from the group consisting of CRL-1573 and NRC (HEK293SF-3F6).

[0172] 13. The method according to any one of items 1 to 12, wherein step 1 is performed in a petri dish, shake flask or roller flask.

[0173] 14. The method according to any one of items 1 to 12, wherein step 1 is carried out in a bioreactor, preferably the bioreactor having a volume of at least 10 L.

[0174] 15. The method according to any one of items 1 to 14, 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, chimeric and heterozygous variants thereof, preferably the AAV serotype selected from AAV5, AAV6, AAV8 and AAV9, more preferably AAV8.

[0175] 16. The method according to any one of items 1 to 14, wherein the lentivirus is derived from human immunodeficiency virus (HIV), preferably the lentivirus is derived from HIV-1.

[0176] 17. The method of claim 16, wherein the lentiviral vector is obtained by transfecting the HEK293 cells with at least one packaging plasmid, the packaging plasmid preferably being combined with an envelope plasmid derived from vesicular stomatitis virus (VSV), more preferably the envelope plasmid being derived from VSV-G.

[0177] 18. The method according to any one of items 1 to 14, wherein the retrovirus is murine leukemia virus (MuLV).

[0178] 19. The method according to any one of items 1 to 18, wherein the IFN inhibitor is a molecule that reduces the activity of IFN-induced and / or signal transduction cascades.

[0179] 20. The method according to Item 19, wherein the IFN inhibitor targets the JAK / STAT signaling pathway.

[0180] 21. The method according to item 20, wherein the IFN inhibitor targets Jak1.

[0181] 22. The method according to any one of items 19 to 21, wherein the IFN inhibitor is selected from the group consisting of ruxolitinib, tofacitinib, baricitinib, degotinib, imarumumab and fenestrazumab and any structural analogues thereof having IFN inhibitory activity, preferably, the IFN inhibitor is ruxolitinib and any structural analogue thereof having at least 50% inhibitory activity against JAK family kinases in the IFN signaling pathway compared to ruxolitinib.

[0182] 23. The method according to any one of items 1 to 22, wherein the final concentration of the IFN inhibitor in the culture 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, 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.

[0183] 24. The method according to any one of items 1 to 23, wherein the culture medium is a culture medium with a defined chemical composition.

[0184] 25. The method according to any one of items 1 to 24 further includes the following steps:

[0185] (a) HEK 293 cells were cultured in a medium containing a certain amount of IFN inhibitor for the first incubation period;

[0186] (b1) Transfect the HEK293 cells with the 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

[0187] (b2) Transfect the HEK293 cells with the two plasmids required for rAAV production, preferably (i) a plasmid containing an adenovirus helper gene and a rep-cap gene and (ii) a recombinant rAAV plasmid containing the transgene;

[0188] (c) The transfected HEK293 cells were cultured for a second incubation period; and

[0189] (d) Recover the AAV particles.

[0190] 26. The method of item 25, wherein, prior to step (b), the HEK293 cells are cultured with the IFN inhibitor in step (a) for a period of about 1 hour to about 12 hours, preferably 1 to 6 hours, more preferably 1 to 3 hours.

[0191] 27. The method of Item 25 or 26, wherein (i) the adenovirus helper plasmid comprises one or more adenovirus genes selected from the group consisting of E1A, E1B, E2A, E4ORF6 and VA; and / or (ii) the packaging rep-cap plasmid comprises the Rep2 gene and the Cap8 gene; and / or (iii) the AAV plasmid comprises a 5'AAV ITR and a 3'AAV ITR from the same AAV serotype, preferably from AAV2.

[0192] 28. The method of any one of items 25 to 27, wherein the transfection step uses polyethyleneimine or FectoVIR. TM -AAV was used as the transfection reagent.

[0193] 29. The method of any one of items 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.

[0194] 30. The method of any one of items 25 to 29, wherein, in the case of the rAAV being secreted from the HEK293 cells, step (d) comprises collecting the supernatant of the culture medium and purifying the rAAV particles from the supernatant.

[0195] 31. The method of any one of items 25 to 30, wherein, in the absence of rAAV being secreted from the HEK293 cells, step (d) comprises the step of destroying the HEK293 cells to obtain the rAAV particles and subsequently purifying the rAAV particles.

[0196] 32. A method for fermenting to produce adeno-associated virus (AAV), adenovirus, lentivirus, or retrovirus, comprising the following steps:

[0197] Step 1: Perform gene set enrichment analysis on HEK293 cells transfected with rAAV, adenovirus, lentivirus, or retrovirus, and ensure that the transcription of at least one of the following genes is enriched compared to the negative control:

[0198] (i) TNFA signal transduction occurs via NFKB;

[0199] (ii) Interferon-γ response;

[0200] (iii) Interferon α response;

[0201] (iv) TGF-β signal transduction; and / or

[0202] (v)IL6_JAK_STAT3_Signal Transduction

[0203] Preferably, if

[0204] (1) Interferon-γ response; and / or

[0205] (2) Interferon α response

[0206] Transcription enrichment occurs; therefore, the HEK293 cells are selected.

[0207] Step 2: The rAAV, the adenovirus, the lentivirus, or the retrovirus are cultured in selected HEK293 cells for an incubation period in a culture medium containing a certain amount of interferon (IFN) inhibitor; and

[0208] Step 3: Recover the rAAV, the adenovirus, the lentivirus, or the retrovirus from the cell culture.

[0209] 33. Use of IFN inhibitors for increasing the yield of said rAAV, said adenovirus, said lentivirus or said retrovirus in cultures of HEK293 cells transfected or infected with rAAV, said adenovirus, said lentivirus or said retrovirus.

[0210] 34. The use as described in item 33, wherein the HEK293 cells are characterized by activation via at least one of the following cellular pathways upon transfection or infection with the rAAV, the adenovirus, the lentivirus, or the retrovirus:

[0211] (1) Interferon-γ response; and / or

[0212] (2) Interferon α response.

[0213] 35. According to the use described in item 34, the IFN inhibitor is selected from the group consisting of ruxolitinib, tofacitinib, baricitinib, degotinib, imarumab and fenestrazumab and any structural analogues thereof having IFN inhibitory activity, preferably, the IFN inhibitor is ruxolitinib and any structural analogue thereof having at least 50% inhibitory activity against JAK family kinases in the IFN signaling pathway compared to ruxolitinib.

[0214] 36. A method for preparing a pharmaceutical composition, comprising performing (i) the method as described in any one of items 1 to 32, wherein the rAAV, the adenovirus, the lentivirus, or the retrovirus contains a therapeutic gene; and (ii) adding another pharmaceutically acceptable excipient to the prepared rAAV particles, adenovirus particles, lentivirus particles, or retrovirus particles to obtain the pharmaceutical composition.

[0215] 37. A kit for cell culture, comprising:

[0216] (a) HEK293 cells; and

[0217] (b) An IFN inhibitor selected from the group consisting of ruxolitinib, tofacitinib, baricitinib, degotinib, imarumab and fenestrazumab and any structural analogues thereof having IFN inhibitory activity, preferably, the IFN inhibitor being ruxolitinib and any structural analogue thereof having at least 50% inhibitory activity against the Jak1 component of the IFN signaling pathway.

[0218] Example

[0219] Example 1:

[0220] Materials and methods

[0221] Cell culture, fermentation

[0222] For this study, serum-free culture media with a defined chemical composition and suitable for suspension growth were used. TM Three derivatives of HEK293 cells cultured in F17 expression medium (Thermo Fisher, NY, USA) were used to produce the rAAV8 vector. All cell lines were exclusively HEK293 and treated in the same manner. All HEK293 cell lines used originated from the Frank Grahams experiment of 1977, which enabled the cells to produce rAAV. Cell line CRL-1573, also known as CL1, was purchased from the American Type Culture Collection (ATCC) and adapted for serum-free suspension culture. Cell line NRCHEK293SF-3F6, also known as CL2, was commercially available from the National Research Council of Canada. Cell line HEK293... Also known as CL3, it is available commercially from AskBio.

[0223] Each cell line is stored as a separate cell bank, which is 1.0 x 10 6Cells / mL were dispensed into cryopreservation vials from Thermo Fisher's FreeStyle. TM The F17 expression medium contains 7.5% DMSO to protect cells from damage. To thaw, the frozen storage vials are placed from -120°C to -80°C overnight and then thawed in a water bath to rapidly transfer the cells to preheated Freestyle thawed medium. TM 125 mL of F17 expression medium In a disposable rotating flask. This allows the cells to further develop... Propagation was carried out in disposable spin bottles for two weeks, with subculturing every two or three days to allow for a fresh culture medium supply. The propagation process was conducted in a Thermo Fisher Heracell 150 incubator with a humid atmosphere, at 37°C and a 5% carbon dioxide supply.

[0224] Adherent HEK293 cell lines (Agilent Technologies catalog number 240073, batch number 0006218516) were cultured in T75 flasks with a total volume of 23 mL in DMEM medium containing 2% FCS. 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 solid white powder in DMSO according to the manufacturer's instructions before use and stored at -20°C. Ruxolitinib was added to the cell culture at a defined concentration during the final passaging process before transfection. Each condition was tested in duplicate. On the day of transfection, confluence was achieved in the range of 60% to 80%.

[0225] transfection

[0226] To perform transfection, HEK293 cells were transferred to a 250 mL Sartorius SU bioreactor (Sartorius, 250 modular vessels, Mammalian; 10 Leppendorf glass bioreactors (Eppendorf) 320) or 125mL In disposable shake flasks. Adjust all systems to 37°C and 5% CO2. The 250 mL bioreactor was continuously stirred at 492 rpm, and the 10 L bioreactor was continuously stirred at 120 rpm, with pH controlled using 0.5 M sodium hydroxide solution. The shake flasks were placed in an Eppendorf New Brunswick S41i incubator with stirring at 150 rpm and a humid atmosphere.

[0227] Transient transfection of HEK293 cells to generate rAAV8 capsids was performed using a three-plasmid system and with polyethyleneimine (PEI) according to the vendor's transfection protocol (Merck KGaA, Darmstadt, Germany). An adenovirus 5 helper gene was delivered using one plasmid. The Rep2Cap8 gene, which identifies the rAAV serotype, was delivered using a second plasmid, and a third plasmid encoding a potential therapeutic target gene, a human FIX sequence. The ratio of helper plasmid, RepCap plasmid, and transgenic plasmid was 1:2:1.5. The plasmid to PEI ratio was 1:2.5. The transfection mixture volume was equivalent to 10% of the final working volume and was prepared by Freestyle. TM The F17 expression medium, plasmid, and PEI were used. Cells were passaged and adjusted to 3.0 and 5.0 x 10⁶ cells / mL before transfection. 6 The target range is between cells / mL.

[0228] Transient transfection of HEK293 for lentivirus production was performed using the pALDI-Lenti system (Aldevron). This is a four-plasmid system comprising pALD-Lenti, pALD-VSV-G, pALD-GagPol, and pALD-Rev, suitable for producing HIV-1-derived viral vectors. The transfection mixture consisted of the four-plasmid system, DMEM medium, and PEI as a transfection reagent. The ratio of plasmids used was 1:1:1:1, and the plasmid DNA to PEI ratio was 1:3.

[0229] Gene set enrichment analysis

[0230] Gene set enrichment analysis was performed based on the Hallmark gene set available from UC San Diego and Broad Institute (https: / / www.gsea-msigdb.org / gsea / msigdb / genesets.jsp?collection=H); see also Liberzon et al., Cell Syst.23 (2015), 417-425.

[0231] Next-generation sequencing

[0232] To preliminarily analyze the differences in mRNA expression patterns among the three HEK293 cell lines used, next-generation sequencing was performed on four biological replicates of each cell line at five different time points under both transfection and simulated transfection conditions. Simulated transfection was performed without plasmids, with only polyethyleneimine (PEI) and Thermo Fisher Freestyle added. Culture medium. The selected time points were 0h (before transfection), 4h, 24h, 48h, and 72h post-transfection. For each time point, the same sampling procedure was performed, including 5.0x10⁻⁶ samples. 6 Cells / mL were collected and washed twice with PBS. Cell pellet was analyzed using Illumina sequencing. The output metadata contained mRNA reads from the three HEK293 cell lines at all five time points and was used to explore differences in transcriptomics.

[0233] result

[0234] The HEK293 cell line used for fermentation production produced different yields.

[0235] Gene set enrichment analysis of rAAV-transfected HEK293 cell lines revealed differences in cell activation pathways.

[0236] When triple transfected with plasmids for rAAV production, the CL1 cell line showed activation of the following genes:

[0237] (1) Interferon-γ response (0-48h); and

[0238] (2) Interferon α response (0-48h).

[0239] Activation of these genes was not observed in the CL3 cell line.

[0240] Therefore, the genes activated by rAAV are significantly different in the CL3 cell line and the CL1 cell line.

[0241] In another experiment, the activation of cellular pathways in the CL1 cell line during triple transfection for rAAV production was compared to that during simulated transfection. The following cellular pathways were found to be activated during triple transfection for rAAV production:

[0242] (i) TNFA signal transduction occurs via NFKB;

[0243] (ii) Interferon-γ response;

[0244] (iii) Interferon α response;

[0245] (iv) TGF_β_ signal transduction;

[0246] Therefore, this experiment confirmed the specific activation of a particular gene in the CL1 cell line during transfection with the plasmid required for rAAV production. This activation was rAAV-specific because it was undetectable in simulated transfection.

[0247] Example 2:

[0248] Inhibitor treatment

[0249] Because the CL1 cell line exhibits activation of interferon α and γ response genes, unlike the CL3 cell line, it is believed that the activation of these genes may lead to a lower rAAV yield in the CL1 cell line compared to the CL3 cell line.

[0250] For inhibitor treatment, cells were incubated with the dissolved inhibitor for 2 to 3 hours prior to transfection. Ruxotinib (Stemcell Technologies), a Janus-associated kinase (JAK) family inhibitor, was used as the inhibitor and prepared according to the product instructions. After adding the inhibitor and transfection mixture to the cell culture, cells were further cultured in batches for 48 to 96 hours.

[0251] AAV8 ELISA

[0252] Commercially available enzyme-linked immunosorbent assay (ELISA; Progen AAV8 Titration ELISA Kit, catalog number PRAAV8) uses a monoclonal antibody (ADK8) specific for a conformational epitope on the assembled AAV8 capsid. The plate-immobilized antibody captures rAAV-8 particles from the sample. The captured particles are then detected by binding to biotinylated anti-AAV8 ADK8, as the targeted epitope is repeatedly expressed on the assembled AAV8 capsid. The concentration of the AAV8 capsid is then measured using streptavidin peroxidase and peroxidase substrate. The color reaction is measured spectrophotometrically at 450 nM. The kit contains an rAAV2 / 8 particle formulation as a calibration standard for labeled rAAV8 particle concentration.

[0253] P24 ELISA

[0254] ELISA was used to quantify the amount of structural HIV1 p24 capsid protein in cell culture and supernatant samples. It was a quantitative sandwich ELISA method. HIV1 p24 ELISA kit ab218268 was used to titrate lentiviral expression. Cell Biolabs Quick Titer was used to distinguish between viral p24 produced by lentiviral cells and associated p24 produced by HEK293 cells. TM The kit includes LV-Origine / TR30021 / lot number 134641F and Cell Biolabs Reference Standard #310809. Viral pulldown technology ensures that only lentivirus-related p24 is detected.

[0255] Droplet digital PCR (ddPCR)

[0256] For vector genome quantification, a Bio-Rad-based droplet digital PCR method was used, employing either a fully automated QXOne System or an automated QX 200AutoDG system. This method provides absolute quantification of the vector genome without the use of a standard curve. Samples are dispensed into droplets, with each droplet serving as an independent compartment for the PCR reaction. Capsid denaturation occurs in the initial phase of PCR within a thermal cycler, making the DNA readily amplifiable. The vector genome titer is then determined using a droplet reader. Samples are treated with DNase I (NEB) to remove foreign DNA sequences. Prior to treatment, the samples are pre-diluted to increase the efficiency of DNase I activity. After droplet generation, PCR was performed using Bio-Rad ddPCR Supermix (dUTP-free) and FIX-specific primers and probes: 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), and probe 5'-6FAM-AGACCAAGC TGA CCT GAT-MGBNFQ-3' (SEQ ID NO:3). Vector genomic concentrations were calculated using appropriate Bio-Rad software.

[0257] result

[0258] Shaking bottle experiment

[0259] HEK293 cells (CL1) were treated with different concentrations of the IFN inhibitor ruxotinib. The final concentrations of the IFN inhibitor are shown in Table 1 below:

[0260] Shaker condition Ruxotinib concentration 1 CL1 control 0 2 CL1 control 0 3 DMSO control 0 4 DMSO control 0 5 Concentration 1 1 μM 6 Concentration 2 5μM 7 Concentration 3 10μM 8 Concentration 4 15μM 9 Concentration 5 20μM 10 Concentration 6 25μM

[0261] The rAAV production yields in supernatant and cell culture, as determined by vector genomic droplet digital PCR (ddPCR), were respectively at [values ​​missing]. Figure 1A and 1B As shown in the diagram. To determine the genome in the supernatant, the vector genome was exported to the culture medium. To determine the genome in cell cultures, samples of the cell cultures were subjected to freeze-thaw cycles.

[0262] Compared to the negative control without the inhibitor, a concentration of 10 μM ruxotinib resulted in an approximately 2.5-fold increase in vector genome (vg) in the supernatant and an approximately 2-fold increase when determined in cell suspension. Approximately 2 x 10^6 cells / g were observed in cell suspension at inhibitor concentrations of 5 μM and 10 μM ruxotinib. 13rAAV yield of vg / L cell cultures.

[0263] The rAAV production yields, determined by the capsid particles in the supernatant and cell culture, were respectively at [percentages missing]. Figure 1C and 1D As shown in the figure, similar results were observed compared to ddPCR determination.

[0264] As can be seen, the addition of IFN inhibitors significantly increased the rAAV yield from CL1 cells treated with these inhibitors compared to the yield without IFN inhibitors. The optimal concentrations of IFN inhibitors were 5 μM and 10 μM. The increase in rAAV yield in cell suspension was approximately 3-fold compared to the yield without inhibitors. A decrease in effect was observed at concentrations above 10 μM.

[0265] In a separate set of experiments, rAAV yields of three different cell lines (CL1, CL2, and CL3) were compared with and without inhibitors. The rAAV yield of CL1 ctrl was used as a baseline (100%) for comparing yields among the different cell lines used, see Figures 2(A), 2(B), and 2(C). To exclude any effect of solvents on inhibitors, experiments were also performed in the presence of the corresponding solvents (DMSO, water for injection (wfi), PBS) without inhibitors. The results determined by rAAV8 ELISA in the supernatant are shown in Figure 2. Figure 2(A) involves the CL1 cell line; Figure 2(B) involves the CL2 cell line; and Figure 3(C) involves the CL3 cell line.

[0266] It can be seen that for each different HEK293 cell line, adding an IFN inhibitor to the culture medium can increase the rAAV8 yield. The lower the rAAV yield in HEK293 cells without an IFN inhibitor, the higher the yield.

[0267] Therefore, this invention is generally applicable to HEK293 cells.

[0268] 10L bioreactor experiment

[0269] To test the effect of IFN inhibitors on rAAV yield in large-scale bioreactor fermentation of HEK293 cells, a 10L bioreactor was used. Results were... Figure 3A (Determined by AAV8 ELISA of capsid particles) and Figure 3B(As shown in the figure) (determined by ddPCR). ddPCR results confirmed the improvement achieved with interferon inhibitors (approximately 1.3-fold improvement determined in the supernatant). ELISA results for rAAV8 capsid particles showed an improvement of approximately 1.5-fold or even higher in the supernatant. Experiments determining rAAV8 capsid particles by ELISA showed an increase of approximately 1.5-fold or even higher in inhibitor-treated experiments compared to the negative control without inhibitors.

[0270] Results obtained from a 3L single-use bioreactor show performance highly comparable to that of a 10L bioreactor.

[0271] Experiment with HEK293 adhering to the wall in a T-flask

[0272] Adherent HEK293 cells were treated with different concentrations of the IFN inhibitor ruxotinib. The final concentrations of the IFN inhibitor are shown in Table 2 below:

[0273] Shaker condition Ruxotinib concentration 1 Wall-mounted comparison (ctrl) 0 2 DMSO control 0 5 Concentration 1 1 μM 6 Concentration 2 5μM 7 Concentration 3 10μM 8 Concentration 4 30μM

[0274] The rAAV production yield in the supernatant, determined by vector genomic droplet digital PCR (ddPCR), was [data missing]. Figure 4A As shown in the figure. To determine the concentration of capsid particles, an AAV8 ELISA was used, such as... Figure 4B As shown. The statistical mean of the control conditions is calculated as 100%.

[0275] ddPCR results from the supernatant samples showed a 48% increase in yield compared to control conditions at an inhibitor concentration of 1 μM. Inhibitor concentrations of 5 μM and 10 μM showed increases in vg yield of 38.5% and 31%, respectively.

[0276] At an inhibitor concentration of 5 μM, ELISA results for capsid particles in the supernatant samples of adherent HEK293 cells showed a maximum yield increase of 66.5% compared to control conditions. In the supernatant, a concentration of 10 μM showed a 62% titer increase, and a concentration of 1 μM showed a 49% improvement in capsid particles.

[0277] All experiments were performed in duplicate and showed high comparability. Specific doses from 1 to a maximum of 5 μM resulted in increased yields in ddPCR vg and ELISA cp assays. Positive effects of the inhibitor on adherent HEK293 cells were observed.

[0278] Experiments using lentivirus-based suspension HEK293(CL1)

[0279] Suspension HEK293(CL1) experiments using tetraplasmid lentivirus transfection were performed in shake flasks under the same conditions shown in Table 2. Lentiviral cp titers were determined by... Figure 5The p24 sandwich ELISA shown was used for determination. The statistical mean of the control condition was evaluated as 100%.

[0280] Compared to untreated control conditions, a 10 μM inhibitor concentration showed an 81.5% increase in the maximum cp titer in suspension HEK293 transfected with lentiviral plasmids. A 5 μM inhibitor concentration showed a 40% yield increase compared to the control.

[0281] This study confirmed the positive effects of IFN pathway inhibition on HEK293 cells (CL1) transfected with lentiviral plasmids.

Claims

1. A method for fermenting and producing 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 in a culture medium containing a certain amount of an interferon (IFN) inhibitor for an incubation period; and Step 2: Recovering the rAAV, the adenovirus, the lentivirus or the retrovirus from the cell culture.

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

3. The method of claim 1 or 2, 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.

4. The method of any one of claims 1 to 3, wherein the HEK293 cells produce rAAV, lentivirus or retrovirus at a yield of at least about 1 x 10 per milliliter of cell culture in the presence of the IFN inhibitor from the culture medium. 9 Purified vector genome-containing particles (vg / mL), preferably at least about 5 x 10 per mL of cell culture. 9 More preferably at least 1 x 10 10 More preferably at least 5 x 10 10 and most preferably 1x10 11 , 1x 10 12 , 1x 10 13 , 1x10 14 or 5x 10 14 More particles containing the vector genome.

5. The method of any one of claims 1 to 4, wherein the HEK293 cells are characterized by activation of at least one of the following genes upon rAAV transfection: (1) interferon-γ response; and / or (2) Interferon α response.

6. The method of claim 5, wherein the HEK293 cells are characterized by activation of at least an interferon-γ response and an interferon-α response upon rAAV transfection.

7. The method of any one of claims 1 to 6, wherein the HEK293 cell line is characterized by activation of at least one of the following genes upon rAAV transfection: (i) TNFA_signaling_via_NFKB; (ii) interferon_γ_response; (iii) interferon-α response; (iv) TGF-β signaling; and / or (v) IL6_JAK_STAT3_signaling.

8. The method of claim 7, wherein at least two activated genes are selected from the group consisting of: TNFA_signaling_via_NFKB, interferon_γ response, interferon_α_response, TGF_β_signaling and IL6_JAK_STAT3_signaling, more preferably, the at least two activated genes are selected from the group consisting of: interferon_γ response, interferon_α_response and IL6_JAK_STAT3_signaling.

9. The method of claim 7 or 8, wherein the HEK293 cells exhibit activation of at least two, at least three, at least four, preferably each of the five pathways.

10. The method according to any one of claims 1 to 9, wherein the HEK293 cells are cultured at least 1 x 10 10 More preferably at least 5 x 10 10 and most preferably at least 1x10 11 , 1x 10 12 , 1x 10 13 , 1x 10 14 or 5x 10 14 The yield of AAV containing 10 or more capsid particles can be characterized.

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 adapted to grow in suspension.

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 performed in a culture dish, a shake flask, a rocking bag or a roller bottle.

15. The method according to any one of claims 1 to 13, wherein step 1 is performed in a bioreactor, 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, chimeric and hybrid variants thereof, preferably the AAV serotype is selected from AAV5, AAV6, AAV8 and AAV9, 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), 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), 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 IFN induction and / or signaling cascade activity.

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

22. The method of claim 21, wherein the IFN inhibitor targets a JAK family kinase.

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, degotinib, imatinib and fontuzumab and any structural analogues thereof having IFN inhibitor activity, preferably, the IFN inhibitor is ruxolitinib and any structural analogues thereof having at least 50% inhibitory activity against JAK family kinases in 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 culture 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, 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 method according to any one of claims 1 to 25, further comprising the steps of: (a) culturing HEK 293 cells in a medium containing a certain amount of IFN inhibitor for a first incubation period; (b1) transfecting the 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 a transgene; or (b2) transfecting the HEK293 cells with two plasmids required for rAAV production, preferably with (i) a plasmid containing adenoviral helper genes and rep-cap gene and (ii) a recombinant rAAV plasmid containing a transgene; (c) culturing the transfected HEK293 cells for a second incubation period; and (d) recovering the AAV particles.

27. The method of claim 26, wherein the HEK293 cells are cultured with the IFN inhibitor in step (a) for a period of about 1 hour to about 12 hours, preferably 1 to 6 hours, more preferably 1 to 3 hours, prior to performing step (b).

28. The method of claim 26 or 27, wherein (i) the adenoviral helper plasmid comprises one or more adenoviral genes selected from the group consisting of E1A, E1B, E2A, E4ORF6 and VA; and / or (ii) the packaging rep-cap plasmid comprises the Rep2 gene and the Cap8 gene and / or (iii) the AAV plasmid comprises a 5'AAV ITR and a 3'AAV ITR from the same AAV serotype, preferably from AAV2.

29. The method of any one of claims 26 to 28, wherein the transfection step uses polyethyleneimine or FectoVIR TM -AAV was used as transfection reagent.

30. The method of 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, most preferably at least 96 hours.

31. The method of any one of claims 26 to 30, wherein when the rAAV is secreted from the HEK293 cells, step (d) comprises collecting the supernatant of the culture medium and purifying the rAAV particles from the supernatant.

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

33. A method for fermentative production of adeno-associated virus (AAV), lentivirus or retrovirus, comprising the following steps: Step 1: Perform transcriptomic analysis on HEK293 cells transfected with rAAV, adenovirus, lentivirus, or retrovirus and if transcripts of at least one of the following genes are enriched compared to negative controls: (i) TNFA_signaling_via_NFKB; (ii) interferon_γ_response; (iii) interferon-α response; (iv) TGF-β signaling; and / or (v) IL6_JAK_STAT3_signaling, Preferably, if (1) interferon-γ response; and / or (2) the transcription of interferon-α response is enriched; then the HEK293 cells are selected; Step 2: culturing the rAAV, the adenovirus, the lentivirus or the retrovirus in the selected HEK293 cells in a culture medium containing a certain amount of an interferon (IFN) inhibitor for an incubation period; and Step 3: Recovering the rAAV, the adenovirus, the lentivirus or the retrovirus from the cell culture.

34. Use of an IFN inhibitor for increasing the yield of rAAV, adenovirus, lentivirus or retrovirus in a culture of HEK293 cells transfected or infected with said rAAV, adenovirus, lentivirus or retrovirus.

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

36. The use of claim 34 or 35, wherein the HEK293 cells are characterized by activation of at least one of the following cellular pathways when transfected or infected with the rAAV, the adenovirus, the lentivirus or the retrovirus: (1) interferon-γ response; and / or (2) Interferon α response.

37. The use according to claim 36, wherein the IFN inhibitor is selected from the group consisting of: ruxolitinib, tofacitinib, baricitinib, degotinib, imatinib and fontuzumab and any structural analogues thereof having IFN inhibitor activity, preferably, the IFN inhibitor is ruxolitinib and any structural analogues thereof having at least 50% inhibitory activity against JAK family kinases in the IFN signaling pathway compared to ruxolitinib.

38. A method for preparing a pharmaceutical composition, comprising performing (i) the method of any one of claims 1 to 34, wherein the rAAV, the adenovirus, the lentivirus or the retrovirus comprises a therapeutic gene; and (ii) adding another pharmaceutically acceptable excipient to the prepared rAAV particles, adenovirus particles, lentivirus particles or retrovirus particles to obtain a pharmaceutical composition.

39. A kit for cell culture, comprising (a) HEK293 cells; and (b) an IFN inhibitor selected from the group consisting of ruxolitinib, tofacitinib, baricitinib, degotinib, imatinib and fontuzumab and any structural analogues thereof having IFN inhibitor activity, preferably, the IFN inhibitor is ruxolitinib and any structural analogues thereof having at least 50% inhibitory activity against JAK family kinases in the IFN signaling pathway.

Images