Stable cell line for production of viral vectors

Abstract

Method of generating a cell for producing a viral vector, the method comprising the following steps: providing a cell; targeted insertion into a first genomic safe harbour site of the cell of a nucleic acid molecule comprising a nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell; and targeted insertion into a second genomic safe harbour site of the cell of a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector proteins; wherein the first and second genomic safe harbour sites are different. A cell with a modified genome for producing a viral vector comprising: a nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell inserted into a first genomic safe harbour site of the cell; and a nucleotide sequence encoding one or more viral vector proteins inserted into a second genomic safe harbour site of the cell; wherein the first and second genomic safe harbour sites are different. Uses of the produced viral vectors and the cell, for example as a medicament or in the production thereof.

Description

[0001] Stable cell line for production of viral vectors

[0002] Field of the invention

[0003] The present invention relates to a method of generating a stable cell line for the production of viral vectors, in particular lentiviral vectors. The method comprises targeted insertion of several nucleotide sequences expressing viral components into multiple different genomic safe harbour sites. The sequences are controllably inserted and expression of their products remains stable over a high number cell passages. The method may be used for any cell type, but has a particular application for animal or human cells. Cells transformed by the method of the invention may be used for production of viral vectors with a high functional titer. Such stable cell producer cell lines and produced viral vectors may be used in the preparation of a medicament, for example in the field of gene therapy.

[0004] Background

[0005] Gene therapy requires the delivery of a gene of interest to a target cell. Particularly advantageous means of delivery are viral vectors. Examples of viral vectors in use for clinical trials for gene therapy are adeno-associated viral, adenoviral, and lentiviral vectors.

[0006] Lentiviral vectors (LV) are integrating vectors that permit long-term transgene expression. LV systems derived from the HIV-1 virus have evolved to address safety and efficacy concerns regarding transactivation of protooncogenes and the unintended generation of replication-competent provirus. At present, third-generation HIV-1 -based self-inactivating (SIN) LV systems have been successfully used in various clinical trials to treat rare genetic disorders as well as in cancer therapy using chimeric antigen receptor T (CAR-T) cell therapy products.

[0007] Production of third-generation SIN-LVs typically requires the expression of four different expression cassettes in a producer cell: one containing the transgene of interest flanked by modified long-terminal repeats (LTRs), one containing the gag and pol genes, encoding viral structural proteins, one containing the rev gene, which is required for replication, and one containing the envelope gene, encoding glycoprotein(s) essential for viral entry into target cells, which typically is the glycoprotein of the vesicular stomatitis virus (VSV-G) .

[0008] Large-scale good manufacturing practice (GMP) of LVs for clinical application is challenging and involves various production, purification, and quality assessment steps. The production of LVs typically involves transient transfection of plasmids comprising the aforementioned expression cassettes. However, scalability of such a production method is restricted and limits reproducibility across patients in large clinical trials. High cost of production associated with GMP of plasmids, potential plasmid contamination in harvested virus, and optimization of transfection conditions are additional hurdles for clinical application.

[0009] Alternatively, LVs can be produced via a stable producer cell line that comprises functional lentiviral helper and packaging genes. Producer cell lines are typically more advantageous compared to transient transfection methods in terms of cost, reproducibility, and scalability. However, the generation of a stable cell line is also challenging and time-intensive. For example, expression of VSV- G, the most preferred envelope protein, and gag-pol results in cytotoxicity to the producer cell line. Furthermore, the cell line may become unstable over time and / or produce low LV titers.

[0010] The generation of producer cell lines via conventional transfection methods has proven to be challenging. Viral vectors such as murine leukemia virus and y-retrovirus have been used to introduce LV components into host cell genomes. However, employing viral vectors for cell line generation raises safety concerns regarding the potential generation of replication-competent LV due to similarities among LTRs and packaging sequences. Additionally, the random integration of viral elements into the genome poses risks of activating or silencing neighboring genes, or increasing the potential for oncogenic events due to insertional mutagenesis. Furthermore, the introduced viral elements themselves may be silenced as well, for example via epigenetic mechanisms such as DNA methylation or histone modifications.

[0011] As such, producer cell line instability and low LV titers (including low functional to physical titer ratios) continue to be a problem in the field. Hence, there remains a need for improved cell lines for the production of viral vectors.

[0012] Brief description of the invention

[0013] The present invention discloses a method of generating a cell for producing a viral vector via targeted insertion of different nucleic acid molecules encoding different viral vector components into respective different genomic safe harbour (GSH) sites of the cell. A first GSH site is used to introduce a nucleic acid sequence encoding at least a protein capable of exporting a viral vector transcript from a nucleus of the cell. A second GSH site is used to introduce a nucleic acid sequence encoding at least one or more viral vector proteins. Further nucleic acid sequence(s) may be inserted into further GSH sites, wherein the further GSH sites are different from the first and second GSH site.

[0014] A GSH site is, at a high level, a specific location within the genome where new genetic material can be inserted without disrupting the normal functions of the host cell. These sites allow for stable and safe expression of a sequence of interest.

[0015] As such, GSH sites offer a more reliable and stable platform for gene expression. Combined with targeted genome editing systems that enable the targeting and cleavage of a specific nucleotide sequence and subsequent insertion of a nucleic acid molecule carrying a sequence of interest, for example via the CRISPR / Cas (clustered regularly interspaced short palindromic repeats / CRISPR- associated protein) system, controlled and targeted gene insertion at GSH sites can be achieved.

[0016] The targeted integration approach of the invention ensures both the long-term stability of the producer cell line and controlled, non-random integration of essential viral production components. In contrast to known producer cell lines that comprise heterogeneous populations, clonal selection of edited cells according to the method of the invention results in a well-defined homogeneous population of producer cells.

[0017] The inventors have used the method of the invention to generate a stable packaging cell line for lentivirus production, demonstrating a functional virus titer of 105to 106TU / ml in small-scale production. In addition, the ratio of functional to physical virus titerwas much greater using the method of the invention compared to traditional complete transient transfection methods, thus resulting in a greatly improved production efficiency and effective clinical dosage.

[0018] The object of present invention is therefore to provide a method of generating a cell for producing a viral vector, the method comprising the following steps: a. providing a cell; b. targeted insertion into a first genomic safe harbour site of the cell of a nucleic acid molecule comprising a nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell; and c. targeted insertion into a second genomic safe harbour site of the cell of a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector proteins; wherein the first and second genomic safe harbour sites are different.

[0019] Suitably, the method may further comprise a step d. of: d. targeted insertion into a third genomic safe harbour site of the cell of a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins; wherein the first, second, and third genomic safe harbour sites are different.

[0020] Suitably, the method may further comprise a step d. of: d. introducing into the cell a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins.

[0021] Suitably, the method may further comprise a step e. of: e. i. targeted insertion into a fourth genomic safe harbour site of the cell of a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest; wherein the first, second, third, and fourth genomic safe harbour sites are different; or ii. introducing into the cell a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest.

[0022] Suitably, the first genomic safe harbour site of the cell may be selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene, preferably the AAVS1 locus, the CCR5 gene, and the CLYBL gene, more preferably the CCR5 gene.

[0023] Suitably, the second genomic safe harbour site of the cell may be selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene, preferably the AAVS1 locus, the CCR5 gene, and the CLYBL gene, more preferably the AAVS1 locus.

[0024] Suitably, the third genomic safe harbour site of the cell may be selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene, preferably the AAVS1 locus, the CCR5 gene, and the CLYBL gene, more preferably the CLYBL gene.

[0025] Suitably, the fourth genomic safe harbour site of the cell may be selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene. Suitably, the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell may comprise the gene rev or a functional equivalent thereof, preferably rev derived from human immunodeficiency virus 1 .

[0026] Suitably, the nucleotide sequence encoding one or more viral vector proteins may comprise a reverse transcriptase, an integrase, a protease, and / or a polyprotein comprising one or more core structural proteins, preferably wherein the nucleotide sequence encoding one or more viral vector proteins may comprise the gene gag and / or pol, or functional equivalents thereof, preferably gag and / or pol derived from human immunodeficiency virus 1 .

[0027] Suitably, the nucleotide sequence encoding one or more viral vector envelope proteins may comprise a nucleotide sequence encoding a vesicular stomatitis virus G glycoprotein envelope protein or a variant thereof.

[0028] Suitably, the first genomic safe harbour may be the CCR5 gene and the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell may comprise the gene rev; the second genomic safe harbour may be the AAVS1 locus and the nucleotide sequence encoding one or more viral vector proteins may comprise the genes gag and pol; the third genomic safe harbour may be the CLYBL gene and the nucleotide sequence encoding one or more viral vector envelope proteins may comprise a nucleotide sequence encoding a vesicular stomatitis virus G glycoprotein envelope protein; and the method may further comprise introducing into the cell a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest.

[0029] Suitably, the nucleotide sequence encoding one or more viral vector proteins may further comprise a HIV-1 -derived Rev response element (RRE) or a functional equivalent thereof.

[0030] Suitably, the viral vector genome may be a self-inactivating viral vector genome, preferably wherein the self-inactivating viral vector genome may further comprise a nucleotide sequence encoding: a HIV- 1-derived central polypurine tract (cPPT), a HIV-1-derived Rev response element (RRE), and / or a Woodchuck Hepatitis Virus (WHV)-derived WHV Posttranscriptional Regulatory Element (WPRE).

[0031] Suitably, the nucleotide sequence of interest may encode a transgene of interest or a non-coding RNA of interest.

[0032] Suitably, the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell may be operably linked to a promoter; the nucleotide sequence encoding one or more viral vector proteins may be operably linked to a promoter; the nucleotide sequence encoding one or more viral vector envelope proteins may be operably linked to a promoter; and / or the nucleotide sequence of interest may be operably linked to a promoter; preferably wherein the promoter may be an inducible promoter, a repressible promoter, or a constitutively active promoter such as an cytomegalovirus (CMV), Rous Sarcoma Virus (RSV) promoter, or a CAG synthetic promoter. Suitably, the method may comprise performing steps b and c, steps b, c, and d, or steps b, c, d, and e sequentially.

[0033] Suitably, the method may further comprise: determining targeted insertion into the first genomic safe harbour site of the cell of the nucleic acid molecule before performing step c, and optionally selecting for a cell comprising the nucleic acid molecule targeted inserted into the first genomic safe harbour site of the cell; and determining targeted insertion into the second genomic safe harbour site of the cell of the nucleic acid molecule before performing step d, and optionally selecting for a cell comprising the nucleic acid molecule targeted inserted into the second genomic safe harbour site of the cell; and optionally, determining targeted insertion into the third genomic safe harbour site of the cell of the nucleic acid molecule before performing step e, and optionally selecting for a cell comprising the nucleic acid molecule targeted inserted into the third genomic safe harbour site of the cell; and optionally, determining targeted insertion into the fourth genomic safe harbour site of the cell of the nucleic acid molecule after performing step e, and optionally selecting for a cell comprising the nucleic acid molecule targeted inserted into the fourth genomic safe harbour site of the cell.

[0034] Suitably, targeted insertion into a first, second, third, or fourth genomic safe harbour site of the cell may comprise using a targeted or site-directed nuclease, preferably a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) protein, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), a recombinase, a homing nuclease or meganuclease, a recombinase and a bridge RNA, or a modified version of any one of the aforementioned.

[0035] Suitably, introducing into the cell may comprise introducing the nucleic acid molecule and the nucleotide sequence into the cell transiently and / or not into the genome of the cell, preferably by transiently transfecting the cell or by transducing the cell using non-integrating viral vectors.

[0036] Suitably, the cell may be an animal cell, preferably a mammalian cell, more preferably a human cell, even more preferably a human embryonic kidney (HEK) 293 cell or a derivative thereof, for example a HEK 293T cell, a HEK 293G cell, or a HEK 293SF cell, most preferably a HEK 293T cell.

[0037] Suitably, the viral vector may be a retroviral vector, preferably a lentiviral vector.

[0038] Suitably, the method may be performed in vitro or ex vivo and / or the method may not comprise a process for modifying the germ line genetic identity of a human being.

[0039] Suitably, the targeted insertion of a nucleic acid molecule into the first, second, third, and / or fourth genomic safe harbour site of the cell may result in a stable expression of: the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell; and the nucleotide sequence encoding one or more viral vector proteins; and optionally, the nucleotide sequence encoding one or more viral vector envelope proteins; and optionally, the nucleotide sequence of interest; wherein the stable expression of the nucleotide sequence is defined as less than 50% difference in normalized expression of the transcript of the nucleotide sequence from passage to passage over at least 5 passages, preferably at least 10 passages, wherein the expression of the transcript of the nucleotide sequence is normalized to the expression of a housekeeping gene, for example GAPDH, as determined by quantitative PCR (qPCR).

[0040] It is a further object to provide a cell for producing a viral vector obtainable by the method of the invention.

[0041] It is yet a further object to provide a cell with a modified genome for producing a viral vector comprising: a nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell inserted into a first genomic safe harbour site of the cell; and a nucleotide sequence encoding one or more viral vector proteins inserted into a second genomic safe harbour site of the cell; wherein the first and second genomic safe harbour sites are different.

[0042] Suitably, the cell may further comprise: a nucleotide sequence encoding one or more viral vector envelope proteins inserted into a third genomic safe harbour site of the cell; wherein the first, second, and third genomic safe harbour sites are different; or a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins.

[0043] Suitably, the cell may further comprise: a viral vector genome comprising a nucleotide sequence of interest inserted into a fourth genomic safe harbour site of the cell; wherein the first, second, third, and fourth genomic safe harbour sites are different; or a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest.

[0044] Suitably, expression in the cell of: the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell; the nucleotide sequence encoding one or more viral vector proteins; and the nucleotide sequence encoding one or more viral vector envelope proteins; may be stable, wherein the stable expression of the nucleotide sequence is defined as less than 50% difference in normalized expression of the transcript of the nucleotide sequence from passage to passage over at least 5 passages, preferably at least 10 passages, wherein the expression of the transcript of the nucleotide sequence is normalized to the expression of a housekeeping gene, for example GAPDH, as determined by quantitative PCR (qPCR).

[0045] Suitably, the first genomic safe harbour site of the cell may be selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene, preferably the AAVS1 locus, the CCR5 gene, and the CLYBL gene, more preferably the CCR5 gene.

[0046] Suitably, the second genomic safe harbour site of the cell may be selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene, preferably the AAVS1 locus, the CCR5 gene, and the CLYBL gene, more preferably the AAVS1 locus.

[0047] Suitably, the third genomic safe harbour site of the cell may be selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene, preferably the AAVS1 locus, the CCR5 gene, and the CLYBL gene, more preferably the CLYBL gene.

[0048] Suitably, the fourth genomic safe harbour site of the cell may be selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene.

[0049] Suitably, the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell may comprise the gene rev or a functional equivalent thereof, preferably rev derived from human immunodeficiency virus 1 .

[0050] Suitably, the nucleotide sequence encoding one or more viral vector proteins may comprise a reverse transcriptase, an integrase, a protease, and / or a polyprotein comprising one or more core structural proteins, preferably wherein the nucleotide sequence encoding one or more viral vector proteins may comprise the gene gag and / or pol, or functional equivalents thereof, preferably gag and / or pol derived from human immunodeficiency virus 1 .

[0051] Suitably, the nucleotide sequence encoding one or more viral vector envelope proteins may comprise a nucleotide sequence encoding a vesicular stomatitis virus G glycoprotein envelope protein or a variant thereof.

[0052] Suitably, the first genomic safe harbour may be the CCR5 gene and the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell may comprise the gene rev; the second genomic safe harbour may be the AAVS1 locus and the nucleotide sequence encoding one or more viral vector proteins may comprise the genes gag and pol; the third genomic safe harbour may be the CLYBL gene and the nucleotide sequence encoding one or more viral vector envelope proteins may comprise a nucleotide sequence encoding a vesicular stomatitis virus G glycoprotein envelope protein; and the method may further comprise introducing into the cell a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest.

[0053] Suitably, the nucleotide sequence encoding one or more viral vector proteins may further comprise a HIV-1 -derived Rev response element (RRE) or a functional equivalent thereof.

[0054] Suitably, the viral vector genome may be a self-inactivating viral vector genome, preferably wherein the self-inactivating viral vector genome may further comprise a nucleotide sequence encoding: a HIV- 1-derived central polypurine tract (cPPT), a HIV-1-derived Rev response element (RRE), and / or a Woodchuck Hepatitis Virus (WHV)-derived WHV Posttranscriptional Regulatory Element (WPRE).

[0055] Suitably, the nucleotide sequence of interest may encode a transgene of interest or a non-coding RNA of interest.

[0056] Suitably, the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell may be operably linked to a promoter; the nucleotide sequence encoding one or more viral vector proteins may be operably linked to a promoter; the nucleotide sequence encoding one or more viral vector envelope proteins may be operably linked to a promoter; and / or the nucleotide sequence of interest may be operably linked to a promoter; preferably wherein the promoter may be an inducible promoter, a repressible promoter, or a constitutively active promoter such as an cytomegalovirus (CMV), Rous Sarcoma Virus (RSV) promoter, or a CAG synthetic promoter.

[0057] Suitably, the cell may be an animal cell, preferably a mammalian cell, more preferably a human cell, even more preferably a human embryonic kidney (HEK) 293 cell or a derivative thereof, for example a HEK 293T cell, a HEK 293G cell, or a HEK 293SF cell, most preferably a HEK 293T cell.

[0058] Suitably, the viral vector may be a retroviral vector, preferably a lentiviral vector.

[0059] It is a further object to provide a method of producing a viral vector comprising culturing the cell according to the invention under conditions suitable forthe production of the viral vector, and optionally obtaining the viral vector from the cell.

[0060] Suitably, producing the viral vector may result in a functional titer of at least 105TU / ml, and / or producing the viral vector may result in the functional titer (TU / ml) being at least 10% of the physical titer (VP / ml), preferably at least 20%, at least 50%, or at least 90%.

[0061] It is yet a further object to provide a cell transduced by the viral vector produced by the cell according to the invention or produced by the method of producing a viral vector comprising culturing the cell according to the invention under conditions suitable for the production of the viral vector.

[0062] It is yet another object to provide a pharmaceutical composition comprising the viral vector produced by the cell according to the invention or by the method of producing a viral vector comprising culturing the cell according to the invention under conditions suitable for the production of the viral vector; and a pharmaceutically acceptable carrier, diluent, and / or excipient.

[0063] It is a further object to provide a viral vector produced by the cell according to the invention or by the method of producing a viral vector comprising culturing the cell according to the invention under conditions suitable for the production of the viral vector, or a pharmaceutical composition according to the invention, for use as a medicament.

[0064] It is yet a further object to provide a method of treatment comprising administering the viral vector produced by the cell according to the invention or by the method of producing a viral vector comprising culturing the cell according to the invention under conditions suitable for the production of the viral vector, or a pharmaceutical composition according to the invention to a subject in need of the same.

[0065] It is yet another object to provide a use of the cell according to the invention for producing a viral vector or in the production of a medicament.

[0066] It is yet another object to provide a use of a viral vector produced by the cell according to the invention or by the method of producing a viral vector comprising culturing the cell according to the invention under conditions suitable for the production of the viral vector, in the production of a medicament. Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0067] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0068] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0069] The patent, scientific, and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published, and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail. Various aspects of the invention are described in further detail below.

[0070] Description of the figures

[0071] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0072] Figure 1 shows experimental design of cell line generation. A) Schematic design of expression cassettes. B) Overview of cell line generation. C) Experimental design for cell line generation.

[0073] Figure 2 shows generation of 293T-Rev cell line. A) Schematic representation of targeted insertion in CCR5 site. B) Representation of primer design for integration screening and PCR gel. The leftmost arrows above and below the schematic representation represent a first primer pair which was used in PCR reactions followed by gel analysis. The rightmost arrows above and below the schematic representation represent a second primer pair which was used in PCR reactions followed by gel analysis. M= Marker. C) FACS plot shows expression of EGFP. D) Plots show physical and functional titer of LVs produced by 293T and 293-Rev cell line (n=2-3, independent virus batches).

[0074] Figure 3 shows generation of 293T-Rev-gp cell line. A) Schematic representation of the AAVS1 gene targeting components for stable gagpol expression. B) Schematic representation of primers for integration screening and PCR gel. The leftmost arrows above and below the schematic representation represent a first primer pair which was used in PCR reactions followed by gel analysis as can be observed in the lanes to the left of the first marker lane M. The middle arrows above and below the schematic representation represent a second primer pair which was used in PCR reactions followed by gel analysis as can be observed in the lanes between the two marker lanes M. The rightmost arrows above and below the schematic representation represent a third primer pair which was used in PCR reactions followed by gel analysis as can be observed in the lanes to the right of the second marker lane M. Although faint, bands forCI are present after using the first and second primer pairs. M= Marker, C1=Clone 1 , C2=Clone 2. C) FACS contour plot showing mCherry and EGFP coexpression in the 293T-Rev-gp cell line. D) Graphs show physical and functional LV titers produced in parental 293T cells and in the 293T-Rev-gp cell lines (n=3, independent viral vector batches).

[0075] Figure 4 shows establishment of Osiris cell line. A) Schematic representation of integration of VSV.G into CLYBL site. B) Representation of primers targeting genomic sites and insert. PCR gel shows corresponding bands on selected clone (C1). The leftmost arrows above and below the schematic representation represent a first primer pair which was used in PCR reactions followed by gel analysis. The rightmost arrows above and below the schematic representation represent a second primer pair which was used in PCR reactions followed by gel analysis. C) FACS plot demonstrates mTurquoise expressing in Osiris cell line. The Osiris cell line is indicated by the contour plot furthest to the right, while the 293T control is indicated by the contour plot furthest to the left. It can be observed that the Osiris cell line has a much greater mTurquoise expression compared to control. D) Bar plots show physical and functional titers produced by Osiris and 293T cell lines (n=3, independent virus productions). E) Dot Plot shows relative expression of Rev, Gagpol (T26S) and VSV.G to GAPDH (a housekeeping gene) in Osiris cell line over different passages number during continuous culture. It can be observed that the Osiris cell line, followed by transfection of a transfer plasmid comprising EGFP, produces lentiviral vectors with an average functional to physical titer ratio of about 0.5 : 1 , while comparative production by traditional transfection of a 293T cell line by four plasmids for the production of lentiviral vectors produces lentiviral vectors with a much lower average functional to physical titer ratio of about 0.00001 : 1 .

[0076] Figure 5 shows Proof of concept of seamless integration of mCherry into CCR5 safe harbor site. A) schematic design of targeting mCherry in CCR5. B) representation of primer design. PCR gel represent integration of selected of clones out of 31 clones. Bar plot shows percentage of mCherry+ clones with correct integration.

[0077] Further details of the figures are disclosed in the Examples section below.

[0078] Detailed description

[0079] The inventors have developed a method that is useful for the large-scale production of viral vectors, specifically lentiviral vectors, for clinical use. In particular, the inventors have developed a method of generating a cell for producing a viral vector comprising targeted insertion into a first genomic safe harbour site of a nucleic acid molecule comprising a first nucleotide sequence encoding a first component for viral vector production and targeted insertion into a second genomic safe harbour site of a nucleic acid molecule comprising a second nucleotide sequence encoding a second component for viral vector production.

[0080] Further nucleic acid molecule(s) comprising further nucleic acid sequence(s) may be inserted into further genomic safe harbour sites, wherein the further genomic safe harbour sites are different from the first and second genomic safe harbour site. Alternatively, or additionally, further nucleic acid molecule(s) comprising further nucleotide sequence(s) may be introduced into the cell via other means than targeted insertion into a genomic safe harbour site. Using the method of the invention, the inventors have generated, as an example, a stable packaging cell line capable of producing a functional virus titer of 105to 106TU / ml in small-scale production (see the section Examples, in particular Figure 4D). In addition, the ratio of functional to physical virus titer was unexpectedly much greater compared to a traditional complete (four-plasmid) transient transfection method, which resulted in a lentiviral product of higher purity. As can be observed in Figure 4D, the complete (four-plasmid) transient transfection method as commonly used in the art results in an average functional to physical viral vector titer of about 0.00001 : 1 (i.e. 100000 fold lower), while the stable packaging cell line according to the invention resulted in an average functional to physical viral vector titer of about 0.5 : 1.

[0081] Functional titer using methods of the prior art are typically reported as being 100-1000 fold lower than physical titer. A low functional to physical titer can impact the reproducibility of viral production, which results in high inter-batch variability. Moreover, a high physical to functional titer ratio results in a low effective dose in clinical use as a high proportion of non-functional viral particles are included during administration to the patient.

[0082] In contrast, the present invention can provide for a functional titer which is nearly identical to the physical titer, which enables clinical administration of much lower doses of viral particles.

[0083] The stable packaging or producer cell line according to the invention offers the significant advantage of continuous viral vector production, contributing to a simpler and more efficient manufacturing process for GMP-grade viral vectors for clinical use.

[0084] Thus, the present invention relates to a method of generating a cell for producing a viral vector, the method comprising the following steps: a. providing a cell; b. targeted insertion into a first genomic safe harbour site of the cell of a nucleic acid molecule comprising a nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell; and c. targeted insertion into a second genomic safe harbour site of the cell of a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector proteins; wherein the first and second genomic safe harbour sites are different.

[0085] The terms "cell for producing a viral vector" and "viral vector production system" as used herein are to be understood as respectively a cell or a system comprising the necessary components for production of viral vectors. Accordingly, they comprise nucleic acid sequences encoding the components necessary to generate viral vectors. The term "cell for producing a viral vector" is to be understood as a cell that is capable of producing viral vectors. The cell may be a producer cell or a packaging cell. The term "packaging cell" refers to a cell which contains the elements necessary for production of infectious viral vectors but which do not comprise a viral vector genome comprising a nucleic acid sequence of interest. A packaging cell typically comprises nucleotide sequences encoding one or more viral vector (structural) proteins, for example gag and pol, and one or more viral vector envelope proteins, for example env. Genomic safe harbour (GSH) sites have been defined previously as "intragenic or extragenic regions of the human genome that are able to accommodate the predictable expression of newly integrated DNA without adverse effects on the host cell or organism. A useful safe harbour must permit sufficient transcription of the inserted nucleotide sequence to yield desired levels of the protein (via further translation) or non-coding RNA. A GSH also must not predispose cells to malignant transformation nor alter cellular functions" (Sadelain et al., 2012, Nature Reviews Cancer, 12(1), 51-8, doi:10.1038 / nrc3179).

[0086] A GSH site is a locus within the genome wherein a gene or other genetic material may be inserted without any deleterious effects on the cell or on the inserted genetic material. Most beneficial is a GSH site in which expression of the inserted gene sequence is not perturbed by any read-through expression from neighbouring genes and expression of the inducible cassette minimizes interference with the endogenous transcription programme. More formal criteria have been proposed that assist in the determination of whether a particular locus is a GSH site in future (Papapetrou et al., 2011 , Nature Biotechnology, 29(1), 73-78, doi: 10.1038 / nbt.1717). These criteria include a site that is (i) at least 50 kb from the 5' end of any gene, (ii) at least 300 kb from any cancer-related gene, (iii) at least 300 kb from any microRNA (miRNA), (iv) located outside a transcription unit and (v) located outside ultraconserved regions (UCR). It may not be necessary to satisfy all of these proposed criteria, since GSH sites already identified do not fulfil all of the criteria, including the following GSH sites: the adeno- associated virus site 1 (AAVS1), a naturally occurring site of integration of AAV virus on chromosome 19; (ii) the chemokine (C-C motif) receptor 5 (CCR5) gene, a chemokine receptor gene known as an HIV-1 coreceptor; and (iii) the human ortholog of the mouse Rosa26 locus (Papapetrou et al., 2016, Mol Ther, 24(4), 678-684, doi: 10.1038 / mt.2016.38). It is thought that a suitable GSH will satisfy at least 2, 3, 4, or all of the aforementioned five criteria.

[0087] Further GSH sites may be identified by identifying sites where viruses naturally integrate without disrupting natural gene expression.

[0088] Any suitable GSH site may be used in the method of the invention, on the basis that the site allows insertion of genetic material without deleterious effects to the cell and permits transcription of the inserted genetic material. Those skilled in the art may use this simplified criteria to identify a suitable GSH site, and / or the more formal criteria set out above. Various genomic and epigenetic tools can be used by those skilled in the art to identify and validate suitable GSH sites (for example as disclosed in Papapetrou et al., 2016, Mol Ther, 24(4), 678-684, doi: 10.1038 / mt.2016.38, Aznauryan et al., 2022, Cell Reports Methods, 2(1), 100154, doi: https: / / doi.Org / 10.1016 / j.crmeth.2021.100154), or Quezada-Ramirez et al., 2024, bioRxiv, doi: https: / / doi.org / 10.1101 / 2023.09.08.556857).

[0089] It is noted that for the generation of a cell for (e.g. ex vivo or in vitro) producing a viral vector according to the method of the present invention, less strict criteria may be applicable.

[0090] Suitably, the targeted insertion into into a first, second, third, or fourth GSH site according to the disclosure may occur on one chromosome or on both chromosomes of a diploid organism. Targeted insertion may suitably be achieved via genome editing systems or methods that enable specific insertion of the nucleic acid molecule into the target GSH site. Genome editing systems or methods typically rely on sequence-specific cleavage by endonucleases or recombinases. Such genome editing systems or methods therefore enable the targeting and cleaving of a specific custom nucleotide sequence. The nucleic acid molecule may then be introduced into the genome (e.g. at a GSH site) using any suitable mechanism, such as homologous-directed repair or homologous recombination. Sequence-specific targeting, and cleaving and inserting of a nucleic acid molecule comprising a nucleic acid sequence of interest (targeted genome editing) may be achieved by one skilled in the art using any suitable means. The field of targeted genome editing and related systems and methods is expansive and rapidly developing (see for example Khalil, J Genet Eng Biotechnol. 2020, 18, 68, doi: 10.1186 / s43141-020-00078-y. Van der Oost and Patinios, Trends in Biotechnology, 2023, 41 (3), 396-409, https: / / doi.Org / 10.1016 / j.tibtech.2022.12.022. Hillary and Cesar, Mol Biotechnol. 2023, 65(3), 311-325, doi: 10.1007 / s12033-022-00567-0). Hence, any system or method of targeted genome editing may be suitably used by one skilled in the art for targeted insertion into a GSH site according to the present invention. These systems typically comprise introducing specific nucleases, specific nucleic acid sequence targeting components, and the nucleic acid molecule to be inserted into a cell, e.g. via one or more rounds of transfection of plasmid(s) expressing the respective components into the cell, but any other suitable means may be employed. Preferably, such systems include CRISPR / Cas system, ZFN system, TALEN system, programmable RNA bridge recombination system (Durrant et al, 2024, Nature, 630, 984-993, doi: https: / / doi.org / 10.1038 / s41586-024-07552-4), and modified versions of any one of the aforementioned. Controlled and targeted insertion of nucleic acid molecules at GSH sites can thusly be achieved. Components of systems of targeted genome editing are commercially available and can optionally be further adapted by one skilled in the art for targeted insertion into a GSH site according to the present invention. For example, for the CRISPR / Cas system, vectors expressing Cas9 nickase and variants thereof such as Cas9D10Aand / or gRNAs, related donor plasmids, all optionally comprising custom designed sequences, and other associated components can be obtained commercially via various providers.

[0091] Suitably, targeted insertion into a first, second, third, or fourth genomic safe harbour site of the cell may comprise using a targeted or site-directed nuclease. Suitably, the targeted or site-directed nuclease may be a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR- associated (Cas) protein, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), a recombinase, a homing nuclease or meganuclease, or a recombinase and a bridge RNA, or a modified version of any one of the aforementioned.

[0092] Suitably, targeted insertion into a first, second, third, or fourth genomic safe harbour site of the cell may comprise using a targeted genome editing system. Suitably, the targeted genome editing system may comprise CRISPR / Cas, ZFN, TALEN, programmable RNA bridge recombination, or a modified version of any one of the aforementioned.

[0093] Further nucleic acid sequence(s) may be inserted into further GSH sites, wherein the further GSH sites are different from the first and second GSH site. This may advantageously allow additional viral vector (production) components (e.g. genes) to be included in the producer cell via targeted insertion in GSH sites. Hence, the targeted integration approach of the invention is expanded to additional components ensuring the long-term stability of the producer cell line and controlled, non-random integration of multiple viral production components.

[0094] Suitably, the method may further comprise a step d. of: d. targeted insertion into a third genomic safe harbour site of the cell of a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins; wherein the first, second, and third genomic safe harbour sites are different.

[0095] Suitably, the method may further comprise a step e. of: e. targeted insertion into a fourth genomic safe harbour site of the cell of a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest; wherein the first, second, third, and fourth genomic safe harbour sites are different.

[0096] Suitably, the method may further comprise a step d. of: d. targeted insertion into a third genomic safe harbour site of the cell of a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest; wherein the first, second, and third genomic safe harbour sites are different.

[0097] Suitably, the method may further comprise a step e. of: e. targeted insertion into a fourth genomic safe harbour site of the cell of a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins; wherein the first, second, third, and fourth genomic safe harbour sites are different.

[0098] Suitably, the method may further comprise a step d. of: d. targeted insertion into a third genomic safe harbour site of the cell of a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins; wherein the first, second, and third genomic safe harbour sites are different; and a step e. of: e. targeted insertion into a fourth genomic safe harbour site of the cell of a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest; wherein the first, second, third, and fourth genomic safe harbour sites are different.

[0099] Several GSH sites have been identified and utilized for targeted transgenesis in the human genome. Identification of further GSH sites in the field is ongoing. The skilled person can select suitable GSH sites for use in the present invention.

[0100] Suitably, the first, second, third, and fourth genomic safe harbour site of the cell are selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene.

[0101] Suitably, the first, second, and third genomic safe harbour site of the cell are selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the Olonne- 18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene.

[0102] Suitably, the first, second, and third genomic safe harbour site of the cell are selected from any one of: the AAVS1 locus, the CCR5 gene, and the CLYBL gene. Suitably, the first genomic safe harbour site of the cell is selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene. Suitably, the first genomic safe harbour site of the cell is selected from any one of: the AAVS1 locus, the CCR5 gene, and the CLYBL gene. Suitably, the first genomic safe harbour site of the cell is the CCR5 gene.

[0103] Suitably, the second genomic safe harbour site of the cell is selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene. Suitably, the second genomic safe harbour site of the cell is selected from any one of: the AAVS1 locus, the CCR5 gene, and the CLYBL gene. Suitably, the second genomic safe harbour site of the cell is the AAVS1 locus.

[0104] Suitably, the third genomic safe harbour site of the cell is selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene. Suitably, the third genomic safe harbour site of the cell is selected from any one of: the AAVS1 locus, the CCR5 gene, and the CLYBL gene. Suitably, the third genomic safe harbour site of the cell is the CLYBL gene.

[0105] Suitably, the fourth genomic safe harbour site of the cell is selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene. Suitably, the fourth genomic safe harbour site of the cell is selected from any one of: the AAVS1 locus, the CCR5 gene, and the CLYBL gene. Suitably, the fourth genomic safe harbour site of the cell is the CLYBL gene.

[0106] Suitably, the first genomic safe harbour is the CCR5 gene, the second genomic safe harbour is the AAVS1 locus, and the third genomic safe harbour is the CLYBL gene.

[0107] Alternatively or additionally, further nucleic acid molecule(s) comprising further nucleic acid sequence(s) may be introduced into the cell via a different means than targeted insertion into a genomic safe harbour site.

[0108] Suitably, the method may further comprise a step d. of: d. introducing into the cell a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins.

[0109] Suitably, the method may further comprise a step e. of: e. introducing into the cell a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest.

[0110] Herein it is understood that introducing into the cell a nucleic acid molecule comprising a nucleotide sequence does not comprise targeted insertion of the nucleic acid molecule into a genomic safe harbour site. For example, the nucleic acid molecule may be episomally maintained in the cell. Methods for introducing nucleic acid molecules into cells are well known in the art, examples of which include transfection and transduction.

[0111] “Transfection” and “transduction” refer generally to techniques for introducing foreign (exogenous) nucleic acid sequences into a host cell, and therefore encompass methods such as electroporation, microinjection, gene gun delivery, lipofection, superfection, etc. and transduction with retroviral, lentiviral or adeno-associated vectors, etc. The specific method used typically depends on both the type of vector and the cell. Appropriate methods for introducing nucleic acid sequences and vectors into host cells such as human cells are well known in the art; see for example Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y; Ausubel et al (1987) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY; Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110; Luchansky et al (1988) Mol. Microbiol. 2, 637- 646. Further conventional methods that are suitable for preparing expression vectors and introducing them into appropriate host cells are described in detail in WO2016 / 071758 for example.

[0112] In contrast to stably transfected genes, transiently transfected genes are only expressed for a limited period of time and are not integrated into the host cell genome. Similarly, transiently transduced genes (e.g. by transducing the cell using non-integrating viral vectors) are only expressed for a limited period of time and are not integrated into the host cell genome. In other words, transient expression is not stable over a prolonged period of time. Transiently transfected or transduced nucleic acid molecules may be lost by environmental factors and cell division. Suitably, the nucleotide molecule which provides for the transient expression may not integrate into the genome of the cell and may not be episomally maintained in the cell.

[0113] Suitably, introducing into the cell may comprise introducing the nucleic acid molecule and the nucleotide sequence into the cell transiently and / or not into the genome of the cell, preferably by transiently transfecting the cell or by transducing the cell using non-integrating viral vectors.

[0114] Suitably, introducing into the cell may comprise introducing the nucleic acid molecule and the nucleotide sequence into the cell transiently and / or not into the genome of the cell. Suitably, introducing into the cell may comprise introducing the nucleic acid molecule and the nucleotide sequence into the cell transiently. Suitably, introducing into the cell may comprise introducing the nucleic acid molecule and the nucleotide sequence not into the genome of the cell.

[0115] Suitably, introducing into the cell may comprise transiently transfecting the cell. Suitably, introducing into the cell may comprise transducing the cell using non-integrating viral vectors.

[0116] Introducing into the cell a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins advantageously allows a selection of different envelope proteins for the produced viral vectors by the generated cell of the invention. Pseudotyping the viral vectors as such may for example allow the production of viral vectors with a specific envelope protein directed to enter a specific target cell with a corresponding host receptor for the specific envelope protein. Hence, the advantages of a stable cell line and flexibility of selection of viral vector envelope proteins can be combined, resulting in flexible manufacturing of different viral vectors.

[0117] Similarly, introducing into the cell a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest allows a selection of different viral vector genomes and / or nucleotide sequences of interest. Hence, the advantages of a stable cell line and flexibility of selection of viral vector genomes and / or nucleotide sequences of interest can be combined, resulting in flexible manufacturing of different viral vectors.

[0118] Similarly, introducing into the cell a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins and a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest may allow the selection and production of different pseudotyped viral vectors targeted to different target cells for delivery of different nucleotide sequences of interest.

[0119] Suitably, one of the nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins and the nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest may be targeted inserted into a genomic safe harbour site and the other of the nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins and the nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest may be introduced into the cell.

[0120] Suitably, both of the nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins and the nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest may be targeted inserted into a genomic safe harbour site.

[0121] Suitably, both of the nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins and the nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest may be introduced into the cell.

[0122] Suitably, the method may further comprise a step d. of: d. i. targeted insertion into a third genomic safe harbour site of the cell of a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest; wherein the first, second, and third genomic safe harbour sites are different; or ii. introducing into the cell a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest.

[0123] Suitably, the method may further comprise a step d. of: d. i. targeted insertion into a third genomic safe harbour site of the cell of a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins; wherein the first, second, and third genomic safe harbour sites are different; or ii. introducing into the cell a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest.

[0124] Suitably, the method may further comprise a step e. of: e. i. targeted insertion into a fourth genomic safe harbour site of the cell of a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest; wherein the first, second, third, and fourth genomic safe harbour sites are different; or ii. introducing into the cell a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest.

[0125] Suitably, the method may further comprise a step e. of: e. i. targeted insertion into a fourth genomic safe harbour site of the cell of a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins; wherein the first, second, third, and fourth genomic safe harbour sites are different; or ii. introducing into the cell a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest. Suitably, the method may further comprise a step d. of: d. i. targeted insertion into a third genomic safe harbour site of the cell of a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins; wherein the first, second, and third genomic safe harbour sites are different; or ii. introducing into the cell a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest; and a step e. of: e. i. targeted insertion into a fourth genomic safe harbour site of the cell of a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest; wherein the first, second, third, and fourth genomic safe harbour sites are different; or ii. introducing into the cell a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest.

[0126] Suitably, the method may further comprise a step d. of: d. i. targeted insertion into a third genomic safe harbour site of the cell of a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest; wherein the first, second, and third genomic safe harbour sites are different; or ii. introducing into the cell a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest; and a step e. of: e. i. targeted insertion into a fourth genomic safe harbour site of the cell of a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins; wherein the first, second, third, and fourth genomic safe harbour sites are different; or ii. introducing into the cell a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest.

[0127] Suitably, the first, second, and third genomic safe harbour site of the cell may be selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the Olonne- 18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene; and the method according to the invention may further comprise introducing into the cell: a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest, or a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins.

[0128] Suitably, the first genomic safe harbour may be the CCR5 gene, the second genomic safe harbour may be the AAVS1 locus, and the third genomic safe harbour may be the CLYBL gene; and the method according to the invention may further comprise introducing into the cell: a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest, or a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins.

[0129] A viral vector may be defined as a modified virus designed to deliver a nucleic acid molecule comprising a nucleic acid sequence of interest, e.g. a transgene or non-coding RNA, into a cell.

[0130] Suitably, the viral vector according to the invention may be a retroviral vector. Suitably, the viral vector may be derived from a retrovirus. Suitably, the viral vector may be a lentiviral vector. Suitably, the viral vector may be derived from a lentivirus. Suitably, the retroviral vector may be derived from or may be derivable from any suitable retrovirus. Many different retroviruses are known. Examples include, but are not limited to: murine leukemia virus (MLV), human T-cell leukemia virus (HTLV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV).

[0131] Lentivirus is a genus of the family of retroviruses. Many different lentiviruses are known. Lentiviruses may be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to: the human immunodeficiency virus (HIV) and the simian immunodeficiency virus (SIV). Examples of non-primate lentiviruses include, but are not limited to: the prototype "slow virus" visna / maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV), feline immunodeficiency virus (FIV), and bovine immunodeficiency virus (BIV). Suitably, the lentiviral vector may be derived from a primate lentivirus or a non-primate lentivirus. Suitably, the lentiviral vector may be derived from HIV-1 , HIV-2, SIV, FIV, BIV, EIAV, CAEV, or VMV. Suitably, the lentiviral vector may be derived from FIV, BIV, EIAV, CAEV, or VMV. Suitably, the lentiviral vector may be derived from HIV-1 , HIV-2, or SIV. Suitably, the lentiviral vector may be derived from HIV-1 . Suitably, the lentiviral vector may be derived from EIAV.

[0132] Retroviruses share a similar virion morphology, which comprises a nucleocapsid core containing the RNA genome, a capsid surrounding the nucleocapsid core containing the enzymes protease, reverse transcriptase, and integrase, and the viral envelope composed of lipids and viral glycoproteins. All retroviruses comprise RNA encoding the genes gag, pol, and env, which express polypeptides for assembly of viral particles. Other regulatory and accessory genes can be present that may for example coordinate and regulate viral gene expression.

[0133] On a basic level, the retrovirus and lentivirus genomes typically share common features such as a 5' long terminal repeat (LTR) and a 3' LTR, comprising in between a packaging signal for packaging the viral genome into the viral vector, a primer binding site, integration sites for enabling integration of the viral genome into a target cell genome and gag, pol, and env genes encoding the packaging components. The genes gag and pol may be present as a single gag / pol gene, which encodes a Gag / Pol fusion polyprotein. The LTRs are responsible for proviral integration, and transcription. LTRs also serve as enhancer-promoter sequences and can control the expression of the viral genes.

[0134] Lentiviruses have additional features, such as the rev gene and rev-response element (RRE) sequences in HIV, which enable the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell. HIV-1 RRE is a 240 base-pair sequence located in the second intron of the HIV-1 genome. An HIV-1-derived RRE sequence may promote high levels of env mRNA in the cytoplasm. The RRE may also facilitate multimerization of Rev proteins, which promotes Rev binding and function. The Rev protein may bind unspliced gag and pol transcripts and incompletely spliced env, vif, vpr, and vpu transcripts at the RRE, facilitating export to the cytoplasm. Viral vector systems based on retroviruses and lentiviruses have been used in various clinical studies to date. Essential components of the original virus genomes on which these systems are based have been identified and are typically incorporated in viral vector production systems. The viral genome components are typically further altered in order to improve safety of the viral vectors produced by the productions systems, for example by making the viral vectors replication-defective. In addition, further optimizations have been engineered into the viral vector systems and production systems enhancing their efficacy. Munis, 2020 (Viruses, 2020, 12(10), 1106, doi: https: / / doi.org / 10.3390 / v12101106) is an example of the current state of the art of lentiviral vectors for clinical use, e.g. in gene therapy. A person skilled in the art can suitably identify the components that need to be introduced into a cell for the cell to become an effective viral vector producer cell that produces clinically safe viral vectors. As an example of such possible components: in HIV-1-based lentiviral vectors, the gene rev encodes a protein capable of exporting a (HIV-1 based) viral vector transcript from a nucleus of the cell. In another example, viral vector proteins such as structural and enzymatic components are typically encoded by respectively the genes gag (group-specific antigen) and pot, and optionally pro (protease). In a further example, viral vector envelope proteins are encoded by the gene env. In another example, a viral vector genome comprising a nucleotide sequence of interest (e.g. a transgene or non-coding RNA) typically comprises the nucleotide sequence of interest flanked by two modified viral long-terminal repeats (LTRs).

[0135] In a typical viral vector of the present disclosure, at least part of one or more genomic (e.g. protein coding) regions essential for replication may be removed from the viral vector genome or the produced viral vector. Suitably, in a retroviral or lentiviral vector of the present disclosure, gag, pol and / or env may be absent, mutated, or not functional. This may result in a replication-defective produced viral vector. Suitably, the viral vector may comprise a viral vector genome comprising a nucleic acid sequence of interest, wherein the viral vector is capable of transducing a target dividing or non-dividing host cell and / or integrating the viral vector genome into a genome of a target cell. Suitably, the retroviral vector may be a vector comprising at least one component derivable from a retrovirus. Suitably, the lentiviral vector may be a vector comprising at least one component derivable from a lentivirus. Suitably, the component may be involved in biological mechanisms by which the viral vector infects cells, expresses genes, or is replicated.

[0136] The produced viral vector according to the disclosure is a viral particle capable of infecting a target cell. Infection of the target cell may suitably include reverse transcription and integration into the target cell genome. Suitably, the viral vector comprises at least one non-viral nucleic acid sequence of interest which is to be delivered by the viral vector to the target cell. The viral vector should be incapable of independent replication to produce infectious viral particles in the target cell. Typically, the viral vector may lack a functional gag, pol, and / or env gene, and / or other genes essential for replication.

[0137] A typical retroviral vector production system involves the separation of the viral vector genome from viral packaging functions. As for example illustrated in Munis, 2020 (Viruses, 2020, 12(10), 1106, doi: https: / / doi.org / 10.3390 / v12101106), these components are typically provided to viral vector production cells on separate DNA expression cassettes (alternatively known as plasmids, expression plasmids, DNA constructs or expression constructs). The packaging functions may include the gag, pot, and env genes. Providing these functions in trans to the viral vector genome may facilitate the production of replication-defective virus.

[0138] Production systems for gammaretroviral vectors are typically three-component systems including viral vector genome, gag / pol, and env expression constructs. Production systems for HIV-1 - based lentiviral vectors additionally typically include the gene rev to be provided in trans and for the viral vector genome, and optionally other viral vector production components, to include the rev- responsive element (RRE).

[0139] In the art, production of retroviral vectors typically involves transiently transfecting production cells with DNA expression cassettes expressing the aforementioned components or using stable producer cell lines wherein the components are stably integrated in the genome of the production cells. Both approaches can also be combined by using a stable producer cell (into which e.g. gag / pol, env, and optionally rev expressing constructs are stably integrated) and then transiently transfecting a DNA expression cassette comprising a viral vector genome comprising a nucleic acid sequence of interest (e.g. a transgene or non-coding RNA).

[0140] Suitably, the nucleic acid molecule comprising a nucleotide sequence of interest according to the disclosure may comprise a viral vector genome comprising the nucleotide sequence of interest. The term "viral vector genome" is herein understood to mean a nucleic acid sequence comprising one or more elements derived from one or more viral genomes. Such a viral vector genome is typically substantially modified from the virus where it is derived from to improve safety and enhance efficacy. An example of a (modified) viral vector genome is the transfer vector element of the self-inactivating (SIN) third-generation HIV-1 -based lentiviral vector system which comprises two modified LTRs (a 5' promoter and SIN 3' U3 element) flanking a nucleotide sequence of interest (e.g. a transgene or noncoding RNA) and further comprises enhancing elements of other viruses such as the Woodchuck hepatitis virus post-transcriptional regulatory element. In a further example, SIN retroviral vector systems have been developed by deleting the transcriptional enhancers or the enhancers and promoter in the U3 region of the 3' LTR. In general, transcriptional inactivation of the LTR in a typical SIN provirus should prevent mobilization by replication-competent virus.

[0141] Suitably, the viral vector produced according to the method of the present invention may be used in a self-inactivating (SIN) configuration in which viral enhancer and / or promoter sequences in the viral vector genome have been deleted, mutated, or modified.

[0142] Suitably, the viral vector genome comprising a nucleotide sequence of interest comprised in the viral vector produced according to the method of the invention may have a minimal viral genome. Suitably, the nucleotide molecule comprising a viral vector genome comprising a nucleotide sequence of interest may have a minimal viral genome. Suitably, the viral vector genome may be a modified viral vector genome. Suitably, the minimal viral genome may be a modified viral genome. Suitably, the viral vector genome may be a self-inactivating viral vector genome. Herein, the term "minimal viral genome" is understood to mean that the viral vector genome has been modified to remove non-essential elements whilst at least retaining the essential elements that facilitate the integration of the nucleotide sequence of interest into the genome of a target cell. Suitably, the viral vector having a minimal viral genome may comprise the nucleotide sequence of interest, and optionally a promoter and / or an internal ribosome entry site (IRES), flanked by two long terminal repeats. Suitably, a minimal HIV-1-derived viral vector genome does not comprise gag, pol, rev, env, vif, vpr, vpu, tat, and nef. An example of a nucleotide molecule comprising a minimal HIV-1- derived viral vector genome is the transfer vector of the SIN third-generation HIV-1-based lentiviral vector system.

[0143] Suitably, the nucleic acid molecule comprising a nucleotide sequence of interest may comprise a long-terminal repeat (LTR), a HIV-1-derived central polypurine tract (cPPT), a HIV-1-derived rev response element (RRE), a viral packaging signal, and / or a Woodchuck Hepatitis Virus (WHV)-derived WHV Posttranscriptional Regulatory Element (WPRE). Suitably, the viral packaging signal may be a viral packaging signal psi (qj). Suitably, the LTR and / or the viral packaging signal may be HIV-1- derived.

[0144] Herein, the term "packaging signal", which is referred to interchangeably as "packaging sequence" or "psi", is used in reference to the non-coding, c / s-acting sequence required for encapsidation of retroviral RNA strands during viral particle formation.

[0145] However, it is noted that the nucleic acid molecule (e.g. introduced in a cell on a plasmid or stably integrated into a GSH site according to the invention) used to produce the viral vector genome comprising a nucleotide sequence of interest within a production cell preferably comprises transcriptional regulatory control sequences operably linked to the viral vector genome to direct transcription of the genome in the production cell. Suitably, these regulatory sequences may be the natural sequences associated with the transcribed retroviral sequence, i.e. the 5' U3 region, or they may be a heterologous promoter such as another viral promoter, for example the CMV promoter. Some lentiviral vector genomes may require additional sequences for efficient virus production. Suitably, the viral vector genome may comprise an RRE sequence or a variant thereof.

[0146] Alternative sequences which perform the same function as the rev / RRE system are also known. For example, a functional analogue of the rev / RRE system is found in the Mason Pfizer monkey virus. This is known as the constitutive transport element (CTE) and comprises an RRE-type sequence in the genome which is believed to interact with a factor in the infected cell. The cellular factor can be thought of as a rev analogue. Thus, CTE may suitably be used as an alternative to the rev / RRE system. Any other functional equivalents which are known or become available may also be used accordingly. For example, it is also known that the Rex protein of HTLV-I can functionally replace the Rev protein of HIV-1 .

[0147] The majority of lentiviral systems comprise the rev / RRE system and is preferably included in the present invention. Thus, suitably, the nucleic acid molecule comprising a nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell may comprise rev and / or RRE, or a functional equivalent thereof, preferably rev or a functional equivalent thereof. Suitably, the nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector proteins may comprise rev and / or RRE, or a functional equivalent thereof, preferably RRE or a functional equivalent thereof. Suitably, the nucleotide molecule comprising a viral vector genome comprising a nucleotide sequence of interest may comprise rev and / or RRE, or a functional equivalent thereof, preferably RRE or a functional equivalent thereof.

[0148] Suitably, the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell may be a functional equivalent thereof.

[0149] Suitably, the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell may be a protein that increases or promotes export of viral vector transcripts from a nucleus of the cell.

[0150] Suitably, the viral vector transcript may be messenger RNA (mRNA). Suitably, the mRNA may be unspliced and / or incompletely spliced mRNA. Suitably, the mRNA may be derived from one or more of the genes gag, pol, and / or env.

[0151] Suitably, the viral vector transcript may be a retroviral vector transcript. Suitably, the viral vector transcript may be a lentiviral vector transcript. Suitably, the viral vector transcript may comprise one or more elements derived from human immunodeficiency virus 1 .

[0152] Suitably, the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell may comprise the gene rev or a variant thereof. Suitably, the gene rev may be derived from human immunodeficiency virus 1 .

[0153] The nucleic acid molecule comprising a nucleotide sequence of interest, typically a transgene of interest, may be incorporated in the viral vector during production by various means based on the desired clinical application. For example, for integration of the nucleotide sequence of interest of a retroviral vector, a nucleic acid molecule flanking the nucleotide sequence of interest by LTRs may be used. In another example, the nucleic acid molecule may comprise or be derived from a viral vector genome that is naturally incorporated into the produced viral vectors because of the viral origin of the different viral vector components.

[0154] The nucleotide sequence of interest may include any genetic material capable of transcription. Suitably, the nucleotide sequence of interest may encode a transgene of interest or a non-coding RNA of interest. Suitably, the transgene may encode a protein.

[0155] A non-coding RNA may be any suitable RNA. Suitably, the non-coding RNA may be a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicersubstrate RNA (dsRNA), a small hairpin RNA (shRNA), an antisense RNA (asRNA), an aptamer, an anti-miRNA, or a long non-coding RNA (IncRNA). Suitably, the non-coding RNA may reduce or block expression of a target gene.

[0156] Suitably, the one or more viral vector proteins according to the disclosure may comprise one or more structural viral proteins. Suitably, the one or more structural viral proteins may comprise a viral capsid protein. Suitably, the one or more structural viral proteins may comprise a polyprotein comprising one or more core structural proteins. Suitably, the one or more viral vector proteins may comprise one or more viral vector proteins encoded by the gene gag or a functional equivalent thereof. Suitably, the gene gag may be derived from human immunodeficiency virus 1 . Suitably, the one or more viral vector proteins may comprise one or more enzymes. Suitably, the one or more enzymes may comprise one or more proteins that enable synthesis of viral DNA and integration of the synthesized viral DNA into host DNA after transduction of a target cell by the viral vector. Suitably, the one or more enzymes may comprise a reverse transcriptase, an integrase, and / or a protease. Suitably, the one or more viral vector proteins may comprise a reverse transcriptase, an integrase, and / or a protease. Suitably, the one or more viral vector proteins may comprise one or more viral vector proteins encoded by the gene pol and / or pro or functional equivalents thereof. Suitably, the gene pol may be derived from human immunodeficiency virus 1. Suitably, the gene pro may be derived from human immunodeficiency virus 1 .

[0157] Suitably, the one or more viral vector proteins may comprise one or more viral vector proteins encoded by the genes gag / pol, gag, pol, and / or pro, or functional equivalents thereof.

[0158] Suitably, the one or more viral vector proteins may comprise a HIV-1-derived protease having an amino acid substitution on position 26 of threonine to serine. Suitably, the HIV-1 -derived protease may be encoded by the gene gag / pol. Cytotoxicity associated with protease activity of HIV-1 -derived gag / pol is known to be reduced by using a T26S mutant of gag / pol (Konvalinka et al., J Virol. Nov 1995;69(11 ):7180-6. doi: 10.1128 / jvi.69.11.7180-7186.1995). This T26S mutant has reduced protease activity without compromising efficacy of the produced viral vectors.

[0159] Suitably, the nucleotide sequence encoding one or more viral vector proteins may further comprise a HIV-1 -derived rev response element (RRE) or a functional equivalent thereof.

[0160] Suitably, the viral vector may be pseudotyped. Suitably, the one or more viral vector envelope proteins according to the disclosure may be modified, e.g. mutated. Modifications may suitably be made or selected by the skilled person to for example introduce specific targeting or to reduce toxicity of the viral vector. Suitably, the nucleotide sequence encoding one or more viral vector envelope proteins may comprise the gene env or a functional equivalent thereof. Suitably, the gene env may be derived from vesicular stomatitis virus. Suitably, the nucleotide sequence encoding one or more viral vector envelope proteins may comprise a nucleotide sequence encoding a vesicular stomatitis virus G glycoprotein envelope protein or a variant thereof. Suitably, the nucleotide sequence encoding one or more viral vector envelope proteins may comprise a nucleotide sequence encoding a Ross River virus envelope protein or a variant thereof. Suitably, the nucleotide sequence encoding one or more viral vector envelope proteins may comprise a nucleotide sequence encoding a baculovirus GP64 envelope protein or a variant thereof. Suitably, the one or more viral vector envelope proteins may comprise a glycoprotein. Suitably, the one or more viral vector envelope proteins may be a protein capable of binding a target cell membrane receptor expressed by a target cell. Suitably, the one or more viral vector envelope proteins may be a protein capable of facilitating entry of the viral vector into a target cell.

[0161] Suitably, the first genomic safe harbour may be the CCR5 gene and the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell may comprise the gene rev, the second genomic safe harbour may be the AAVS1 locus and the nucleotide sequence encoding one or more viral vector proteins may comprise the genes gag and pol, the third genomic safe harbour may be the CLYBL gene and the nucleotide sequence encoding one or more viral vector envelope proteins may comprise a nucleotide sequence encoding a vesicular stomatitis virus G glycoprotein envelope protein; and the method may further comprise introducing into the cell a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest.

[0162] Any of the nucleotide sequences as disclosed herein may be operably linked to a promoter in order to suitably control or enhance expression of the protein encoded by the nucleotide sequence.

[0163] Suitably, the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell may be operably linked to a promoter. Suitably, the nucleotide sequence encoding one or more viral vector proteins may be operably linked to a promoter. Suitably, the nucleotide sequence encoding one or more viral vector envelope proteins may be operably linked to a promoter. Suitably, the nucleotide sequence of interest may be operably linked to a promoter.

[0164] Suitably, the promoter may be an inducible promoter. Suitably, the promoter may be a repressible promoter. Suitably, the promoter may be a constitutively active promoter. Suitably, the constitutively active promoter may be a cytomegalovirus (CMV), Rous Sarcoma Virus (RSV) promoter, or a CAG synthetic promoter.

[0165] A “promoter” is a nucleotide sequence which initiates and regulates transcription of a polynucleotide. In order to drive a high level of expression, it may be beneficial to use a high efficiency promoter. An “inducible promoter” is a nucleotide sequence where expression of a genetic sequence operably linked to the promoter is controlled by an analyte, co-factor, regulatory protein, etc. It is intended that the term “promoter” or “control element” includes full-length promoter regions and functional (e.g., controls transcription or translation) segments of these regions. “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a nucleotide sequence is capable of effecting the expression of that sequence when the proper enzymes are present. The promoter need not be contiguous with the sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the nucleotide sequence and the promoter sequence can still be considered “operably linked” to the nucleotide sequence. Thus, the term “operably linked” is intended to encompass any spacing or orientation of the promoter element and the nucleotide sequence which allows for initiation of transcription of the nucleotide sequence upon recognition of the promoter element by a transcription complex.

[0166] Suitably, any of the nucleic acid sequences used in the present invention (including the nucleotide sequence of interest and / or components of the viral vector production system) may be codon- optimised.

[0167] Many viruses, including lentiviruses, use a large number of rare codons. Changing these to correspond to commonly used mammalian codons, increased expression may be achieved. Codon usage tables are known in the art for mammalian cells, as well as for a variety of other organisms. Codon optimisation may result in increased viral titer and / or improved safety.

[0168] Steps b, c, d, and e of the method of the invention may appropriately be performed in any order. Preferably, one or both of a step of: according to the disclosure, introducing into the cell a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins, and, according to the disclosure, introducing into the cell a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest may be performed after the steps of targeted insertion into the first, second, and optionally third genomic safe harbour site of the cell. In other words, the establishment of a stable cell line via targeted insertion into GSH sites may be preferably performed before introducing into the cell of further nucleic acid molecules other than targeted insertion into a GSH site.

[0169] Suitably, steps b and c, steps b, c, and d, or steps b, c, d, and e of the method of the invention may be performed sequentially. Suitably, each of steps b and c, steps b, c, and d, or steps b, c, d, and e comprises targeted insertion into a genomic safe harbour site of the cell of a nucleic acid molecule. Each instance of targeted insertion into a GSH site may be followed by a controlled selection process to assess proper targeted insertion of the nucleic acid molecule into the GSH site and optionally proper expression of the nucleic acid sequence encoding the protein or nucleic acid sequence of interest.

[0170] Additional nucleic acid sequences may be suitably targeted inserted into a respective genomic safe harbour site of the cell together with the nucleic acid sequence encoding the respective protein or the nucleic acid sequence of interest. Such additional nucleic acid sequences may comprise a marker enabling detection or selection of successful targeted insertion of the nucleic acid sequence encoding the respective protein or the nucleic acid sequence of interest. Suitably, the marker may be a detectable protein, for example a fluorescent protein such as green fluorescent protein (GFP) or variants thereof. Suitably, the marker may be a resistance gene. Resistance genes are well-known in the art; they enable the cell to metabolize otherwise toxic selection compounds such as antibiotics. Such resistance genes thereby may allow for selection of successfully targeted inserted nucleic acid sequences.

[0171] Suitably, the method according to the invention may further comprise: a. determining targeted insertion into the first genomic safe harbour site of the cell of the nucleic acid molecule before performing step c, and optionally selecting for a cell comprising the nucleic acid molecule targeted inserted into the first genomic safe harbour site of the cell; and b. determining targeted insertion into the second genomic safe harbour site of the cell of the nucleic acid molecule before performing step d, and optionally selecting for a cell comprising the nucleic acid molecule targeted inserted into the second genomic safe harbour site of the cell; and c. optionally, determining targeted insertion into the third genomic safe harbour site of the cell of the nucleic acid molecule before performing step e, and optionally selecting for a cell comprising the nucleic acid molecule targeted inserted into the third genomic safe harbour site of the cell; and d. optionally, determining targeted insertion into the fourth genomic safe harbour site of the cell of the nucleic acid molecule after performing step e, and optionally selecting for a cell comprising the nucleic acid molecule targeted inserted into the fourth genomic safe harbour site of the cell.

[0172] The cell for producing a viral vector according to the invention (e.g. a producer cell or a packaging cell) may suitably be any type of cell. Suitable cells for producing viral vectors are cells which are capable of producing viral vectors when cultured under appropriate conditions.

[0173] Suitably, the cell may be an animal cell. Suitably, the cel may be an insect cell. Suitably, the cell may be an SF9 cell.

[0174] Suitably, the cell may be a mammalian cell. Suitably, the cell may be a human cell. Suitably, the cell may be a human embryonic kidney (HEK) 293 cell or a derivative thereof. Suitably, the cell may be a HEK 293T cell, a HEK 293G cell, or a HEK 293SF cell. Suitably, the cell may be a HEK 293T cell. Suitably, the cell may be a CAP cell or a derivative thereof. Suitably, the cell may be a CAP- T cell. Suitably, the cell may be a CHO cell. Suitably, the cell may be a HEK293 cell, a HEK293T cell, a HEK 293G cell, a HEK 293SF cell, a CAP cell, a CAP-T cell, or a CHO cell.

[0175] Suitably, the cell according to the invention may be a modified cell.

[0176] Suitably, the method according to the invention may be performed in vitro or ex vivo. Suitably, the method may not comprise a process for modifying the germ line genetic identity of a human being.

[0177] The cell for producing a viral vector according to the invention may also be termed a host cell. Once a nucleic acid molecule, vector, or viral vector has been introduced into the cell, including targeted insertion of a nucleic acid molecule into a GSH site, it may be referred to as a “modified cell” herein. Once the nucleic acid molecule, vector, or viral vector is introduced into the host cell as above, the resultant modified cell should be capable of expressing the encoded polypeptide or non-coding RNA (and e.g. correctly localising the encoded polypeptide such as an encoded binding protein for its intended function e.g. transporting the encoded binding protein to the cell surface).

[0178] The term “modified cell” as used herein refers to a genetically altered (e.g. transformed, transduced, or transfected) cell. The modified cell includes at least one exogenous nucleic acid sequence (i.e. a nucleic acid sequence that is not naturally found in the host cell). The term refers to the particular subject cell and also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. The particular subject cell and the progeny or potential progeny of such a cell may also be referred to as a cell line.

[0179] In one example, a modified cell comprises (part of) a nucleic acid molecule or a viral vector provided herein. In another example, a modified cell comprises a nucleic acid molecule or part thereof comprising a nucleic acid sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell inserted into a first genomic safe harbour site of the cell; and a nucleic acid molecule or part thereof comprising a nucleotide sequence encoding one or more viral vector proteins inserted into a second genomic safe harbour site of the cell; wherein the first and second genomic safe harbour sites are different, as provided herein.

[0180] The term "modified genome" as used herein refers to a genetically altered genome, e.g. of a cell. The modified genome includes at least one exogenous nucleic acid sequence.

[0181] The inventors have developed a method for enabling the stable introduction of multiple viral vector production components into the genome of a cell.

[0182] Suitably, the targeted insertion of a nucleic acid molecule into the first, second, third, and / or fourth genomic safe harbour site of the cell may result in a stable expression of: the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell; and the nucleotide sequence encoding one or more viral vector proteins; and optionally, the nucleotide sequence encoding one or more viral vector envelope proteins; and optionally, the nucleotide sequence of interest; wherein the stable expression of the nucleotide sequence is defined as less than 50% difference in normalized expression of the transcript of the nucleotide sequence from passage to passage over at least 5 passages, preferably at least 10 passages, wherein the expression of the transcript of the nucleotide sequence is normalized to the expression of a housekeeping gene, for example GAPDH, as determined by quantitative PCR (qPCR). Suitably, the difference may be less than 45%, 40%, 35%, 30%, or 25% difference in normalized expression of the transcript of the nucleotide sequence from passage to passage over at least 5 passages, preferably at least 10 passages.

[0183] Suitably, the cell for producing a viral vector may be a stable producer cell. Suitably, the cell for producing a viral vector may be a stable producer cell line.

[0184] Another aspect of the disclosure relates to the nucleic acid molecules according to the disclosure for use in generating the cell for producing a viral vector of the invention.

[0185] The present invention further relates to a cell for producing a viral vector obtainable by the method of the invention.

[0186] The present invention also relates to a cell with a modified genome for producing a viral vector comprising: a nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell inserted into a first genomic safe harbour site of the cell; and a nucleotide sequence encoding one or more viral vector proteins inserted into a second genomic safe harbour site of the cell; wherein the first and second genomic safe harbour sites are different. Suitably, the cell according to the invention may be a modified cell.

[0187] As set out supra, further nucleotide sequence(s) may have been suitably inserted into further

[0188] GSH sites of the cell. Alternatively or additionally, further nucleic acid molecule(s) comprising nucleic acid sequence(s) may be comprised in the cell via a different means than targeted insertion into a genomic safe harbour site. This may have been suitably achieved by introducing the nucleic acid molecule and the nucleotide sequence into the cell transiently and / or not into the genome of the cell as set out supra.

[0189] Suitably, the cell may further comprise: a nucleotide sequence encoding one or more viral vector envelope proteins inserted into a third genomic safe harbour site of the cell; wherein the first, second, and third genomic safe harbour sites are different; or a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins.

[0190] Suitably, the cell may further comprise: a viral vector genome comprising a nucleotide sequence of interest inserted into a third genomic safe harbour site of the cell; wherein the first, second, and third genomic safe harbour sites are different; or a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest.

[0191] Suitably, the cell may further comprise: a nucleotide sequence encoding one or more viral vector envelope proteins inserted into a fourth genomic safe harbour site of the cell; wherein the first, second, third, and fourth genomic safe harbour sites are different; or a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins.

[0192] Suitably, the cell may further comprise: a viral vector genome comprising a nucleotide sequence of interest inserted into a fourth genomic safe harbour site of the cell; wherein the first, second, third, and fourth genomic safe harbour sites are different; or a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest.

[0193] Suitably, the cell may further comprise: a nucleotide sequence encoding one or more viral vector envelope proteins inserted into a third genomic safe harbour site of the cell; wherein the first, second, and third genomic safe harbour sites are different; and a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest.

[0194] Suitably, the cell may further comprise: a viral vector genome comprising a nucleotide sequence of interest inserted into a third genomic safe harbour site of the cell; wherein the first, second, and third genomic safe harbour sites are different; and a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins.

[0195] Suitably, expression in the cell of: the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell; the nucleotide sequence encoding one or more viral vector proteins; and the nucleotide sequence encoding one or more viral vector envelope proteins; may be stable, wherein the stable expression of the nucleotide sequence is defined as less than 50% difference in normalized expression of the transcript of the nucleotide sequence from passage to passage over at least 5 passages, preferably at least 10 passages, wherein the expression of the transcript of the nucleotide sequence is normalized to the expression of a housekeeping gene, for example GAPDH, as determined by quantitative PCR (qPCR).

[0196] Suitably, the difference may be less than 45%, 40%, 35%, 30%, or 25% difference in normalized expression of the transcript of the nucleotide sequence from passage to passage over at least 5 passages, preferably at least 10 passages. Suitably, the cell for producing a viral vector may be a stable producer cell. Suitably, the cell for producing a viral vector may be a stable producer cell line.

[0197] Suitably, the first, second, third, and fourth genomic safe harbour site of the cell are selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene.

[0198] Suitably, the first, second, and third genomic safe harbour site of the cell are selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the Olonne- 18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene.

[0199] Suitably, the first, second, and third genomic safe harbour site of the cell are selected from any one of: the AAVS1 locus, the CCR5 gene, and the CLYBL gene.

[0200] Suitably, the first genomic safe harbour site of the cell is selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene. Suitably, the first genomic safe harbour site of the cell is selected from any one of: the AAVS1 locus, the CCR5 gene, and the CLYBL gene. Suitably, the first genomic safe harbour site of the cell is the CCR5 gene.

[0201] Suitably, the second genomic safe harbour site of the cell is selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene. Suitably, the second genomic safe harbour site of the cell is selected from any one of: the AAVS1 locus, the CCR5 gene, and the CLYBL gene. Suitably, the second genomic safe harbour site of the cell is the AAVS1 locus.

[0202] Suitably, the third genomic safe harbour site of the cell is selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene. Suitably, the third genomic safe harbour site of the cell is selected from any one of: the AAVS1 locus, the CCR5 gene, and the CLYBL gene. Suitably, the third genomic safe harbour site of the cell is the CLYBL gene.

[0203] Suitably, the fourth genomic safe harbour site of the cell is selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene. Suitably, the fourth genomic safe harbour site of the cell is selected from any one of: the AAVS1 locus, the CCR5 gene, and the CLYBL gene. Suitably, the fourth genomic safe harbour site of the cell is the CLYBL gene.

[0204] Suitably, the first genomic safe harbour is the CCR5 gene, the second genomic safe harbour is the AAVS1 locus, and the third genomic safe harbour is the CLYBL gene.

[0205] Suitably, the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell may be a functional equivalent thereof.

[0206] Suitably, the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell may be a protein that increases export of viral vector transcripts from a nucleus of the cell. Suitably, the viral vector may be a retroviral vector. Suitably, the viral vector may be a lentiviral vector.

[0207] Suitably, the viral vector transcript may be messenger RNA (mRNA). Suitably, the mRNA may be unspliced and / or incompletely spliced mRNA. Suitably, the mRNA may be derived from one or more of the genes gag, pol, and / or env.

[0208] Suitably, the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell may comprise the gene rev or a variant thereof. Suitably, the gene rev may be derived from human immunodeficiency virus 1 .

[0209] Suitably, the one or more viral vector proteins may comprise one or more structural viral proteins. Suitably, the one or more structural viral proteins may comprise a viral capsid protein. Suitably, the one or more structural viral proteins may comprise a polyprotein comprising one or more core structural proteins. Suitably, the one or more viral vector proteins may comprise one or more viral vector proteins encoded by the gene gag or a functional equivalent thereof. Suitably, the gene gag may be derived from human immunodeficiency virus 1 .

[0210] Suitably, the one or more viral vector proteins may comprise one or more enzymes. Suitably, the one or more enzymes may comprise one or more proteins that enable synthesis of viral DNA and integration of the synthesized viral DNA into host DNA after transduction of a target cell by the viral vector. Suitably, the one or more enzymes may comprise a reverse transcriptase, an integrase, and / or a protease. Suitably, the one or more viral vector proteins may comprise a reverse transcriptase, an integrase, and / or a protease. Suitably, the one or more viral vector proteins may comprise one or more viral vector proteins encoded by the gene pol and / or pro or functional equivalents thereof. Suitably, the gene pol may be derived from human immunodeficiency virus 1. Suitably, the gene pro may be derived from human immunodeficiency virus 1 .

[0211] Suitably, the one or more viral vector proteins may comprise a HIV-1-derived protease having an amino acid substitution on position 26 of threonine to serine.

[0212] Suitably, the nucleotide sequence encoding one or more viral vector proteins may further comprise a HIV-1 -derived rev response element (RRE) or a functional equivalent thereof.

[0213] Suitably, the nucleotide sequence encoding one or more viral vector envelope proteins may comprise the gene env or a functional equivalent thereof. Suitably, the gene env may be derived from vesicular stomatitis virus. Suitably, the nucleotide sequence encoding one or more viral vector envelope proteins may comprise a nucleotide sequence encoding a vesicular stomatitis virus G glycoprotein envelope protein or a variant thereof. Suitably, the nucleotide sequence encoding one or more viral vector envelope proteins may comprise a nucleotide sequence encoding a Ross River virus envelope protein or a variant thereof. Suitably, the nucleotide sequence encoding one or more viral vector envelope proteins may comprise a nucleotide sequence encoding a baculovirus GP64 envelope protein or a variant thereof. Suitably, the one or more viral vector envelope proteins may comprise a glycoprotein. Suitably, the one or more viral vector envelope proteins may be a protein capable of binding a target cell membrane receptor expressed by a target cell. Suitably, the one or more viral vector envelope proteins may be a protein capable of facilitating entry of the viral vector into a target cell. The nucleic acid molecule comprising a nucleotide sequence of interest, typically a transgene of interest, may be incorporated in the viral vector during production by various means based on the desired clinical application. For example, for integration of the nucleotide sequence of interest of a retroviral vector, a nucleic acid molecule flanking the nucleotide sequence of interest by LTRs may be used. In another example, the nucleic acid molecule may comprise or be derived from a viral vector genome that is naturally incorporated into the produced viral vectors because of the viral origin of the different viral vector components.

[0214] Suitably, the nucleic acid molecule comprising a nucleotide sequence of interest may comprise a viral vector genome comprising the nucleotide sequence of interest. Suitably, the nucleotide molecule comprising a viral vector genome comprising a nucleotide sequence of interest may have a minimal viral genome. Suitably, the viral vector genome may be a modified viral vector genome. Suitably, the minimal viral genome may be a modified viral genome. Suitably, the viral vector genome may be a self-inactivating viral vector genome.

[0215] Suitably, the nucleic acid molecule comprising a nucleotide sequence of interest may comprise a long-terminal repeat (LTR), a HIV-1-derived central polypurine tract (cPPT), a HIV-1-derived rev response element (RRE), a viral packaging signal, and / or a Woodchuck Hepatitis Virus (WHV)-derived WHV Posttranscriptional Regulatory Element (WPRE). Suitably, the viral packaging signal may be a viral packaging signal psi (qj). Suitably, the LTR and / or the viral packaging signal may be HIV-1 - derived.

[0216] Suitably, the viral vector genome may comprise an RRE sequence or a variant thereof.

[0217] Suitably, the first genomic safe harbour may be the CCR5 gene and the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell may comprise the gene rev, the second genomic safe harbour may be the AAVS1 locus and the nucleotide sequence encoding one or more viral vector proteins may comprise the genes gag and pol, the third genomic safe harbour may be the CLYBL gene and the nucleotide sequence encoding one or more viral vector envelope proteins may comprise a nucleotide sequence encoding a vesicular stomatitis virus G glycoprotein envelope protein; and the cell may further comprise a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest.

[0218] The nucleotide sequence of interest may include any genetic material capable of transcription. Suitably, the nucleotide sequence of interest may encode a transgene of interest or a non-coding RNA of interest. Suitably, the transgene may encode a protein.

[0219] A non-coding RNA may be any suitable RNA. Suitably, the non-coding RNA may be a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicersubstrate RNA (dsRNA), a small hairpin RNA (shRNA), an antisense RNA (asRNA), an aptamer, an anti-miRNA, or a long non-coding RNA (IncRNA). Suitably, the non-coding RNA may reduce or block expression of a target gene. Suitably, the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell may be operably linked to a promoter. Suitably, the nucleotide sequence encoding one or more viral vector proteins may be operably linked to a promoter. Suitably, the nucleotide sequence encoding one or more viral vector envelope proteins may be operably linked to a promoter. Suitably, the nucleotide sequence of interest may be operably linked to a promoter.

[0220] Suitably, the promoter may be an inducible promoter. Suitably, the promoter may be a repressible promoter. Suitably, the promoter may be a constitutively active promoter. Suitably, the constitutively active promoter may be a cytomegalovirus (CMV), Rous Sarcoma Virus (RSV) promoter, or a CAG synthetic promoter.

[0221] Suitably, any of the nucleic acid sequences used in the present invention (including the nucleotide sequence of interest and / or components of the viral vector production system) may be codon- optimised.

[0222] The cell for producing a viral vector according to the invention (e.g. a producer cell or a packaging cell) may suitably be any type of cell. Suitable cells for producing viral vectors are cells which are capable of producing viral vectors when cultured under appropriate conditions.

[0223] Suitably, the cell may be an animal cell. Suitably, the cel may be an insect cell. Suitably, the cell may be an SF9 cell.

[0224] Suitably, the cell may be a mammalian cell. Suitably, the cell may be a human cell. Suitably, the cell may be a human embryonic kidney (HEK) 293 cell or a derivative thereof. Suitably, the cell may be a HEK 293T cell, a HEK 293G cell, or a HEK 293SF cell. Suitably, the cell may be a HEK 293T cell. Suitably, the cell may be a CAP cell or a derivative thereof. Suitably, the cell may be a CAP- T cell. Suitably, the cell may be a CHO cell. Suitably, the cell may be a HEK293 cell, a HEK293T cell, a HEK 293G cell, a HEK 293SF cell, a CAP cell, a CAP-T cell, or a CHO cell.

[0225] The present invention also relates to the nucleic acid molecules suitable for insertion of the respective nucleotide sequences into the respective genomic safe harbour sites. Suitably, the nucleic acid molecule may be a vector.

[0226] The invention further relates to a method of producing a viral vector comprising culturing the cell according to the invention under conditions suitable forthe production of the viral vector, and optionally obtaining the viral vector from the cell.

[0227] Various methods for determining the titer of viral vectors are known in the art. The (functional) titer is typically expressed in transducing units / mL (TU / mL). The titer may for example be increased by increasing the number of infectious particles or by increasing the specific activity of a vector preparation. Physical titers measure the amount of viral particles in a sample and are usually based on the presence of a viral protein, such as p24, or viral nucleic acid. However, in unpurified lentiviral supernatant, significant concentrations of p24 protein may be present that are not assembled into viral particles. The physical titer is typically expressed in viral particles / mL (VP / mL). Functional titers, or infectious titers, measure how many of the viral particles produced can actually infect cells and are typically expressed in transducing units / mL (TU / mL). Functional titers may for example be determined by measuring expression in transduced cells of reporter genes that are to be included in the produced viral vector; e.g. fluorescent proteins via flow cytometry or other genes via qPCR.

[0228] The present invention surprisingly resulted in very high ratios of functional to physical titer.

[0229] Suitably, producing the viral vector may result in a functional titer of at least 105TU / ml. Suitably, producing the viral vector may result in a functional titer of at least 106or 107TU / ml. Suitably, producing the viral vector may result in the functional titer (TU / ml) being at least 1 % of the physical titer (VP / ml). Suitably, producing the viral vector may result in the functional titer (TU / ml) being at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the physical titer (VP / ml).

[0230] Suitably, producing the viral vector may result in a functional titer of at least 105TU / ml and producing the viral vector may result in the functional titer (TU / ml) being at least 10% of the physical titer (VP / ml). Suitably, producing the viral vector may result in a functional titer of at least 105TU / ml and producing the viral vector may result in the functional titer (TU / ml) being at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the physical titer (VP / ml).

[0231] Suitably, producing the viral vector may result in a functional titer of at least 106TU / ml and producing the viral vector may result in the functional titer (TU / ml) being at least 10% of the physical titer (VP / ml). Suitably, producing the viral vector may result in a functional titer of at least 106TU / ml and producing the viral vector may result in the functional titer (TU / ml) being at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the physical titer (VP / ml).

[0232] Suitably, producing the viral vector may result in a functional to physical titer ratio of from 0.01 to 1 : 1 . Suitably, producing the viral vector may result in a functional to physical titer ratio of from 0.01 to 0.99 : 1 , of from 0.1 to 1 : 1 , of from 0.1 to 0.99 : 1 , of from 0.1 to 0.95 : 1 , of from 0.1 to 0.9 : 1 , of from 0.1 to 0.8 : 1 , of from 0.1 to 0.7 : 1 , of from 0.2 to 1 : 1 , of from 0.2 to 0.99 : 1 , of from 0.2 to 0.95 : 1 , of from 0.2 to 0.9 : 1 , of from 0.2 to 0.8 : 1 , of from 0.2 to 0.7 : 1 , of from 0.5 to 1 : 1 , of from 0.5 to 0.99 : 1 , of from 0.5 to 0.95 : 1 , of from 0.5 to 0.9 : 1 , of from 0.5 to 0.8 : 1 , of from 0.5 to 0.7 : 1 , of from 0.7 to 1 : 1 , of from 0.7 to 0.99 : 1 , of from 0.7 to 0.95 : 1 , or of from 0.7 to 0.9 : 1.

[0233] The invention further relates to a cell transduced by the viral vector produced by the cell according to the invention or produced by the method of producing a viral vector comprising culturing the cell according to the invention under conditions suitable for the production of the viral vector. "A cell transduced by the viral vector" is to be understood as a cell, in particular a target cell, into which the nucleic acid sequence of interest (e.g. a transgene or non-coding RNA) comprised by the viral vector has been transferred. The invention also relates to a pharmaceutical composition comprising the viral vector produced by the cell according to the invention or by the method of producing a viral vector comprising culturing the cell according to the invention under conditions suitable for the production of the viral vector; and a pharmaceutically acceptable carrier, diluent, and / or excipient.

[0234] A produced viral vector as described herein may be provided as part of a pharmaceutical composition. Advantageously, such compositions may be administered to a human subject in need thereof (as described elsewhere herein).

[0235] Suitably, the pharmaceutical composition may comprise a pharmaceutically acceptable excipient, adjuvant, diluent and / or carrier.

[0236] Compositions may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune suppressing agents, and optionally other therapeutic agents or compounds.

[0237] As used herein, "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the produced viral vector without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0238] Excipients are natural or synthetic substances formulated alongside an active ingredient (e.g. produced viral vector as provided herein), included for the purpose of bulking-up the formulation or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. A suitable excipient is therefore easily identifiable by one of ordinary skill in the art. Byway of example, and without limitation, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, and ethanol.

[0239] Adjuvants are pharmacological and / or immunological agents that modify the effect of other agents in a formulation. Pharmaceutically acceptable adjuvants are well known in the art. A suitable adjuvant is therefore easily identifiable by one of ordinary skill in the art.

[0240] Diluents are diluting agents. Pharmaceutically acceptable diluents are well known in the art. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art.

[0241] Carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation. The term “carrier’’ denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. Pharmaceutically acceptable carriers are well known in the art. A suitable carrier is therefore easily identifiable by one of ordinary skill in the art.

[0242] The produced viral vectors described herein can be used in treatment strategies in which viral vectors are injected or otherwise administered to a patient to effectuate a medicinal effect. The invention further relates to a viral vector produced by the cell according to the invention or by the method of producing a viral vector comprising culturing the cell according to the invention under conditions suitable for the production of the viral vector, or a pharmaceutical composition according to the invention, for use as a medicament.

[0243] The invention further relates to a method of treatment comprising administering the viral vector produced by the cell according to the invention or by the method of producing a viral vector comprising culturing the cell according to the invention under conditions suitable for the production of the viral vector, or a pharmaceutical composition according to the invention to a subject in need of the same. Suitably, administering may comprise administering to the subject a therapeutically effective amount of the viral vectors or the pharmaceutical composition. Suitably, the subject may be a mammal. Suitably, the mammal may be a human.

[0244] The invention further relates to a use of the cell according to the invention for producing a viral vector or in the production of a medicament.

[0245] The invention further relates to a use of a viral vector produced by the cell according to the invention or by the method of producing a viral vector comprising culturing the cell according to the invention under conditions suitable for the production of the viral vector, in the production of a medicament.

[0246] A therapeutically effective amount herein refers to an amount sufficient to reduce the severity and / or duration of a defect, disorder, disease, deficiency, or a symptom thereof. Progression, development, or onset of the defect, disorder, disease, deficiency may thereby be reduced or prevented. The amount of viral vectors or the pharmaceutical composition according to the invention actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound(s) administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

[0247] As used herein, the terms “treat”, “treating” and "treatment" are taken to include an intervention performed with the intention of preventing the development or altering the pathology of a condition, disorder or symptom. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted condition, disorder or symptom.

[0248] As used herein the term “subject” refers to an individual, e.g., a human, having or at risk of having a specified condition, disorder or symptom. The subject may be a patient i.e. a subject in need of treatment in accordance with the invention. The subject may have received treatment for the condition, disorder or symptom. Alternatively, the subject has not been treated prior to treatment in accordance with the present invention.

[0249] The compositions described herein can be administered to the subject by any conventional route, including injection, implantation, or by gradual infusion over time. The administration may, for example, be by infusion or by intramuscular, intravascular, intracavity, intracerebral, intralesional, rectal, subcutaneous, intradermal, epidural, intrathecal, percutaneous administration. The compositions described herein may be in any form suitable for the above modes of administration. For example, compositions comprising viral vectors may in any form be suitable for infusion or implantation. As further examples, suitable forms for parenteral injection (including, subcutaneous, intramuscular, intravascular or infusion) include a sterile solution, suspension or emulsion. Alternatively, the route of administration may be by direct injection into the target area, or by regional delivery or by local delivery. The identification of suitable dosages of the compositions of the invention is well within the routine capabilities of a person of skill in the art.

[0250] The compositions described herein are for administration in an effective amount. An “effective amount” is an amount that alone, or together with further doses, produces the desired (therapeutic or non-therapeutic) response. The effective amount to be used will depend, for example, upon the therapeutic (or non-therapeutic) objectives, the route of administration, and the condition of the patient / subject. For example, the suitable dosage of the composition of the invention for a given patient / subject will be determined by the attending physician (or person administering the composition), taking into consideration various factors known to modify the action of the composition of the invention for example severity and type of haematological malignancy, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors. The dosages and schedules may be varied according to the particular condition, disorder or symptom and the overall condition of the patient / subject. Effective dosages may be determined by either in vitro or in vivo methods. The pharmaceutical compositions described herein are advantageously presented in unit dosage form.

[0251] General definitions

[0252] As used herein, the term “ex vivo” refers to “outside” the body. The term “in vitro” can be used to encompass “ex vivo” components, compositions and methods.

[0253] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention.

[0254] Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

[0255] As used herein, the term “substantially” refers to a great extent or degree. The exact allowable degree may depend on the specific context. For example, the term “substantially” in the context of a viral vector genome being a nucleic acid sequence substantially consisting of elements derived from one or more viral genomes as disclosed herein would mean that the viral vector genome may comprise at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, of elements derived from one or more viral genomes.

[0256] A "functional equivalent" in relation to genes, nucleic acid sequences, proteins, or polypeptides of the present disclosure as used herein may also comprise a variant of the instant genes, nucleic acid sequences, proteins, or polypeptides. The term "variant" as used herein, in relation to genes, nucleic acid sequences, proteins, or polypeptides of the present disclosure includes any substitution of, variation of, modification of, replacement of, deletion of and / or addition of one (or more) nucleic acid or amino acid residues from or to the sequence providing that the resultant gene, nucleic acid sequence, protein, or polypeptide retains at least one of its endogenous functions.

[0257] The term "expression construct" or "expression cassette" as used herein refers to a functional expression unit, capable of driving the expression of one or more incorporated nucleic acid sequences, e.g. a DNA sequence comprising one or more genes and sequences that control their expression. For example, the expression cassette may comprise a promoter and / or a transcriptional terminator. For example, the expression cassette may comprises two genes separated by an IRES that are both transcribed from a single promoter.

[0258] Examples

[0259] Aspects of the invention are demonstrated by the following non-limiting examples.

[0260] Introduction

[0261] Lentiviral vectors (LVs) are a prominent tool for research and clinical gene therapy applications, primarily owing to their ability to seamlessly integrate into genomes of both quiescent stem cells dividing and non-dividing cells, ensuring sustained and reliable expression of transferred genes1.

[0262] Self-inactivating LVs (SIN-LVs) have been developed to improve safety and efficacy concerns regarding transactivation of protooncogenes which observed in early gamma-retroviral (y-RV) clinical trials treating X-linked severe combined immunodeficiency (SCID-X1) patients23. SIN-LVs have been successfully used in various clinical trials to treat rare genetic disorders4'7as well as cancer therapy using chimeric antigen receptor T (CAR-T) cell therapy products8'10. With the approval of different gene and cell therapy products of Kymriah (2017), Yescarta (2017), Tecartus (2020), Zynteglo (2022) into market and increasing interest in the field of gene and cell therapy, large-scale production of clinical grade LV is an unmet need.

[0263] SIN-LVs, are replication-defective with modified long terminal repeats (LTRs) where strong viral enhancers and promoters are removed11. Therefore, their production requires the concurrent expression of four crucial viral components: 1) Gag / pol, encoding viral structural proteins; 2) Rev, a facilitator of nuclear exportation processes, 3) Envelope, encoding glycoproteins essential for viral entry into target cells; and 4) the transfer vector, which carries the gene of interest to encapsulate within viral particles. Transient transfection of these essential components is the predominant method employed to date for lentivirus vector production in a packaging cell line which saves time, allows expression of cytotoxic viral genes and provides high yields. However, transient production of LVs is challenging due to lacking scalability for large-scale production, the high cost of production associated with good manufacturing practice (GMP) of plasmids, potential plasmid contamination in harvested virus, and optimizing transfection conditions. As a result, LV production is primarily restricted to small- scale production using cell factories with titer yields ranging from 109to 1011TU / ml, which limits the reproducibility across patients in large clinical trials.

[0264] Stable producer cell lines can provide an affordable system compared to a transient system for LV production within an industrial context. Nonetheless, the generation of stable cell line is also challenging and time-intensive. Besides, the cytotoxicity of gagpol and glycoprotein from the vesicular stomatitis virus (VSV.G) envelope cell line result in instability over time and low viral titer have resulted in the setbacks for generation of LV producer cell lines12.

[0265] Various strategies have been employed to control cytotoxicity through inducible systems such as” Teton” or “Tet-off “system. Additionally, the incorporation of alternative envelopes or nontoxic variants of gagpol have been explored. However, VSV.G holds distinct advantage in clinical context because of their broad tropism and FDA approval for gene therapy purposes13 14-18.

[0266] Generating a LV packaging cell line through conventional transfection methods has proven to be challenging. Viral vectors such as murine leukemia virus (MLV) and y-RV have been used to introduce LV components into host cell genomes, to ensure high expression and high LV yield15. However, employing viral vectors in cell line generation raises safety concerns regarding the potential generation of replication-competent lentivirus (RCL) due to similarities among long terminal repeats (LTRs) and packaging sequences19. Additionally, the random integration of viral elements into the genome poses risks of activating or silencing neighboring genes, resulting in cell line instability. In contrast, genomic safe harbor (GSH) sites offer a more reliable and stable platform for gene expression. Importantly, the insertion of transgenes at genomic safe harbors does not adversely affect the integrity of the host cell genome. The advent of CRISPR technology provides a powerful means to achieve targeted gene insertion at these specific genomic loci.

[0267] This study successfully generated a packaging cell line for LV production using a CRISPR-Cas9 platform. Rev, Gag pol, and VSV.G were strategically integrated into three human genomic safe harbor (GSH) loci; CCR5, AAVS1 , and CLYBL, respectively. This targeted integration approach ensures both the stability of the cell line and controlled integration of essential components.

[0268] Results

[0269] Generation of Osiris packaging cell line via seamless gene integration.

[0270] The inventors have designed the Donor plasmids featuring homology arms, promoter and gene of interest tailored for each safe harbor site. In order to facilitate the identification of a population characterized by robust and sustained expression of LV packaging elements, a fluorochrome marker was linked to each viral component (Fig 1A). To control cytotoxicity associated with protease activity of Gagpol, the inventors used a mutated Gagpol (T26S) variant, which has reported to reduce protease activity without compromising virus functionality20 14. Furthermore, the expression of VSV.G was controlled under “Tet-off ” inducible system.

[0271] For cell line generation, viral components were introduced to each safe harbor site through sequential transfections of Donor, gRNA, and Cas9D10Aplasmids. The subsequent steps included single cell cloning facilitated by flow activated cell sorting and clone screening using PCR and flow cytometry (Fig 1 B-C). To ensure seamless homology directed integration of transgenes into safe harbor site, we incorporated cas9 nickase (Cas9D10A) that has previously described by Chen et al. Double nicking strategy using catalytic mutant Cas9D10Anickase has proven the advantage of minimal off-target effects21. As a proof of concept, the inventors knocked in mCherry into CCR5 site of HEK293T cells (Fig 5A). To confirm the specific integration in CCR5 site, a total of 31 randomly selected mCherry + clones were screened through PCR. The results indicated that 90% of screened clones had undergone accurate homology-directed gene targeting (Fig 5B).

[0272] Initially, the Rev element, essential for the nuclear export of vector genomes, was introduced into the CCR5 site (Fig 2A). The selected clones underwent screening to confirm accurate integration into the CCR5 safe harbor site on chromosome 3 (Fig 2B) and EGFP expression was assessed via flow cytometry (Fig 2C). Viral vector production utilizing the 293T-Rev cell line yielded a physical titer of 106VP / ml and a functional titer of 105TU / ml in small-scale production (Fig 2D).

[0273] Subsequently, Gagpol (T26S) was introduced into AAVS1 site located on chromosome 19 of 293T- Rev cell line (Fig 3A). Gagpol (T26S) was linked with mCherry to select and screen the cell population. We have screened clones for accurate integration of the transgene into AAVS1 site by PCR using primers targeting genomic AAVS1 and within transgene as depicted in (Fig 3B). mCherry expression in 293T-Rev-Gp cell line was confirmed with flow cytometry (Fig 3C). 293T-Rev-Gp cell lines was transfected with remaining viral component of VSV.G and transfer vector carrying EGFP for virus production in small scale, while 293T cells were transiently transfected with all four viral plasmids as a control. Small-scale virus production utilizing the 293T-Rev-Gp cell line resulted in a physical titer of 106VP / ml and a functional titer of 104TU / ml.

[0274] Finally, the 293T-Rev-Gp cell line was transfected with pDonor plasmid carrying VSV.G, pgRNA and pCas9D10A, to introduce VSV.G into CLYBL locus on chromosome 13 (Fig 4A). Single cell derived clones were screened for correct integration using primers targeting genomic sites and the insert (Fig 4B). Additionally, expression of mTurquoise in the packaging cell line (called Osiris) was confirmed via flow cytometry (Fig 4C). Lentiviral vectors produced in small scale after transfecting the final packaging cell line (Osiris) with transfer vector carrying EGFP, yielded a physical titer of 106VP / ml and a marginal drop in functional titer to 105TU / ml. Although higher physical and functional titers were achieved in transient quadruple-transfection in 293T cells, a substantial drop of five log in functional titer was observed (Fig 4D).

[0275] T o assess the stability of Osiris cell line over time, expression of integrated genes of Rev, GagPol and VSV.G in safe harbor loci, were measured over different passages up to three months of continuous culturing. qPCR results validated stable expression of Rev, Gagpol (T26S) and VSV.G (Fig 4E), proving the cell line stability.

[0276] Discussion

[0277] Working towards achieving scalable production of lentiviral vectors, the above experiments focus on generation of a stable packaging cell line for lentivirus production, demonstrating a virus titer of 105to 106TU / ml in small-scale production. To address the challenge of long-term cell line stability, a CRISPR-Cas9 platform was employed for the targeted integration of viral elements into safe harbor loci. Unlike conventional methods, this approach eliminates the need for antibiotic selection during cell thawing or reselection during culturing.

[0278] In orderto mitigate the cytotoxicity associated with gagpol, a point mutated variant (T26S) with reduced protease activity was utilized, as seen in the LentiPro26 cell line14. The production of high titer lentivirus relies on robust and stable expression of gagpol15. Thus the inventors have used strong promoter of CAG to ensure high expression of gagpol.

[0279] To control cytotoxicity of VSV.G, an inducible “Tet-off’ system was implemented, allowing for the induction of VSV.G expression upon the removal of tetracycline / doxycycline. While alternative nontoxic envelopes have been explored in previous cell line developments14 18, VSV.G was chosen not only for its ability to provide a high titer but also because it has received FDA approval for clinical purposes due to its broad tropism.

[0280] The stable packaging cell line offers the significant advantage of continuous lentiviral vector production, contributing to a more cost-effective manufacturing process. Although this study demonstrates stable LV production using Osiris cell line in small-scale settings as a proof of concept, further optimizations, including achieving high cell density, evaluating commercial culture mediums, and enhancing LV titer by disrupting the antiviral response in the stable cell line, are advantageous for scaling up production in larger settings.

[0281] Materials and methods

[0282] Cell culture. Human cervix carcinoma HeLa cells (American Type Culture Collection) and Human embryonic kidney (HEK) 293 T cells (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium supplemented with L-Glutamine (4,5 g / L) (DMEM; Capricorn) containing 10% fetal bovine serum (FBS; Sigma) and Penicilin-Streptomicine antibiotic (Gibco). These cells were kept at 37 °C in an humidified-air 5% CO2 atmosphere. During the selective pressure, if needed puromycin were added to the culture medium at appropriate concentrations (Invivogen, San Diego, USA). Cell concentration and viability were assessed by nuecleocounter 200 (Chemometec, Denmark).

[0283] Plasmids. The plasmid pMDLg / pRRE (Addgene #12251) codes for HIV-1 Gag-Pro-Pol under the control of a cytomegalovirus (CMV) promoter. The plasmid pRSV-REV (Addgene #12253) codes for HIV-1 Rev under the control of the rous sarcoma virus (RSV) U3 promoter. The plasmid pMD2.G (Addgene #12259) codes for envelope glycoprotein of the vesicular stomatitis virus (VSV.G) under the control of a CMV promoter. The plasmid pRRLSIN.cPPT.PGK-GFP.WPRE (Addgene #12252) harbors a SIN vector genome under the control of a LTR-RSV chimeric promoter and drives the expression of eGFP reporter from an internal promoter. All plasmids previously mentioned were kindly provided by Dr. Didier Trono through Addgene plasmid repository (Cambridge, MA)

[0284] Constructs of phPGK-Rev-eGFP, pCMV-Gagpol (T26S)-mCherry-Puro and pTet-off-VSVG-mTurq were designed in house using SnapGene and synthesized and cloned in pUC57 by BaseClear B.V. All Donor plasmids, gRNA plasmids and pCas9D10Awere kindly provided by Manuel Goncalves’s lab (Leiden University Medical Center, Netherlands), and include the plasmids pCas9 D10 (Addgene #199252), pgRNA CCR5 (Addgene #100294), and pgRNA CLYBL (Addgene #199238). Constructs of phPGK-REV-EGFP, pCMV-Gagpol (T26S)-mCherry-Puro and pTet-off-VSVG-mTurq were custom synthesized BaseClear (Netherlands). Each construct was cloned into designated targeting donor plasmids.

[0285] The GP(T26S) is the result of a point mutation on pMDLg / pRRE viral protease sequence. With this point mutation, the protease 26th amino acid is changed from a Threonine to a Serine as described in Konvalinka et al20. The plasmid was synthesized de novo linked to mcherry by base clear. The plasmid was cloned into pDonor.AAVSI .

[0286] Transfection for cell line generation. One day prior to transfection, Hek293T cells were seeded in 24-well plates (Greiner, one-Bio) at the density of 200000 cells per well. The transfections were performed by mixing of appropriate amount of plasmids with X-tremeGENE™ HP DNA transfection reagent (Sigma-Aldrich), at a mass ratio of 1 :3 (DNA:X-tremeGENE) in 50ul (appropriate amount) of serum free medium. After gentle mixing with pipet, the transfection mixtures were incubated for 15-20 min at room temperature (RT), after which the mixes were added in a dropwise manner to the cells. At 48h or 72h post transfection, the transfection efficiency were determined by measuring the relevant fluorochrome via flow cytometry. Subsequently, the cells were culture up to 2 weeks to remove episomal DNA, after which stable transfection level was measured by flowcytometry.

[0287] Flow Cytometry and flow activated sorting. The frequencies of cells expressing EGFP, mCherry and mTurquoise2 were determined by using a BD Fortessa flow cytometer (BD Biosciences) or Aurora (Cytek). Data was analyzed with FlowJo 10.9.0 software (Tree Star).Non-transfected cells were used to set background fluorescence levels. At least 10000 events, each representing a single viable cell, were measured per sample. Stable transfected cells were sorted using Aria (BD Biosciences) based of EGFP, mCherry and mTurquoise2 expression based on single cell in 96 well plates (Greiner-bio- One) as well as in bulk. To increase the efficiency of cell cloning the culture media were supplemented with 50uM s-thioglycerol (Sigma-Aldrich).

[0288] Clone Screening and PCR analyses. Two to three weeks after single sorting, the single cell-derived clones were selected and moved to 24 well-plates. Cells also were collected for genomic DNA analysis by PCR with the Phire Tissue Direct PCR Master Mix (ThermoFisher Scientific) according to the manufacturer’s recommendations. Cells were scaled up for cell banking.

[0289] Virus production and concentration. HEK 293T cells were seeded at density of 1-4 x 106cells in 6 well plate and 10cm2respectively. After 24 hours, cells were transfected using X-tremeGENE HP DNA transfection reagent (Sigma-Aldrich) at a mass ratio of 1 :3 (DNA:X-tremeGene), with the respective plasmids. The amount of each viral component per million of cells was: 2.5 pg of vector genome; 1 .5 pg of Gag-Pol 0.6 pg of Rev, 0.75 pg of envelope. Supernatant was harvested, clarified with 0.45 pm filter (Whatman) at 24h and 48h post transfection. Supernatant from different time points were pooled together. Virus concentration was performed using Vivaspin20 filter (Sartorius) according to the manufacturer’s instruction.

[0290] Physical virus titration. The physical LV titers were determined by qPCR measuring HIV, albumin multiplexing. RNA isolation was performed using QIAGEN miniRNA purification kit (QIAGEN) according to manufacturer’s instruction. cDNA libraries were created by Superscript Kit (Thermo Fisher Scientific) according to the manufacturer’s instruction performed on QuantStudio (Thermo Fisher Scientific). Physical titer (VP / ml) was determined by calculating viral particles based on HIV standard curve.

[0291] Functional virus titration. The functional LV titers were determined by flow cytometric analysis for EGFP expression of transduced Hela cells. Briefly, Hela cells were seeded at 30000 to 100000 cells in 12-well or 6 well plates respectively. After 24 hours, the concentrated virus was added to each well, as well as 1 ug / ml of Lentiboost (Sirion).The plates were centrifuged at 32 °C for 1 hour at 800 g (spininoculation). After centrifugation, the plates were incubated at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air. Cells were harvested and analyzed for EGFP fluorescence by flow cytometry (Aurora-Cytek) 5 to 8 days after transduction. The number of LV transducing units per volume (TU / mL) was determined by the equation:

[0292] Cell line stability test. Osiris cell line was cultured continuously in presence of doxycycline up to 3 months. An aliquot of cells were harvested at different time points for RNA isolation and cDNA creation as described before. To induce expression of VSV.G, doxycycline contained culture medium was replaced with normal culture medium 3-4 days before cell harvest. RNA isolation and cDNA creation were performed as previously described. qPCR performed to measure the expression of Rev, Gagpol (T26S) and VSV.G. GAPDH was used as a housekeeping gene to calculate relative expression. References

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[0301] 9. Davila ML, Riviere I, Wang X, et al. Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med. Feb 19 2014;6(224):224ra25. doi: 10.1126 / scitranslmed.3008226

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[0303] 11 . Zufferey R, Dull T, Mandel RJ, et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol. Dec 1998;72(12):9873-80. doi:10.1128 / jvi.72.12.9873-9880.1998

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[0306] 14. Tomas HA, Rodrigues AF, Carrondo MJT, Coroadinha AS. LentiPro26: novel stable cell lines for constitutive lentiviral vector production. Scientific Reports. 2018 / 03 / 27 2018;8(1 ):5271 . doi: 10.1038 / S41598-018-23593-y

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Claims

Claims1 . A method of generating a cell for producing a viral vector, the method comprising the following steps: a. providing a cell; b. targeted insertion into a first genomic safe harbour site of the cell of a nucleic acid molecule comprising a nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell; c. targeted insertion into a second genomic safe harbour site of the cell of a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector proteins; wherein the first and second genomic safe harbour sites are different.

2. The method according to claim 1 , the method further comprising a step d. of: d. targeted insertion into a third genomic safe harbour site of the cell of a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins; wherein the first, second, and third genomic safe harbour sites are different.

3. The method according to claim 1 , the method further comprising a step d. of: d. introducing into the cell a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins.

4. The method according to any one of claims 1 to 3, the method further comprising a step e. of: e. i. targeted insertion into a fourth genomic safe harbour site of the cell of a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest; wherein the first, second, third, and fourth genomic safe harbour sites are different; or ii. introducing into the cell a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest.

5. The method according to any one of claims 1 to 4, wherein: a. the first genomic safe harbour site of the cell is selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene, preferably the AAVS1 locus, the CCR5 gene, and the CLYBL gene, more preferably the CCR5 gene; and / or b. the second genomic safe harbour site of the cell is selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene, preferably the AAVS1 locus, the CCR5 gene, and the CLYBL gene, more preferably the AAVS1 locus; and / orc. the third genomic safe harbour site of the cell is selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene, preferably the AAVS1 locus, the CCR5 gene, and the CLYBL gene, more preferably the CLYBL gene; and / or d. the fourth genomic safe harbour site of the cell is selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene.

6. The method according to any one of claims 1 to 5, wherein: a. the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell comprises the gene rev or a functional equivalent thereof, preferably rev derived from human immunodeficiency virus 1 ; and / or b. the nucleotide sequence encoding one or more viral vector proteins comprises a reverse transcriptase, an integrase, a protease, and / or a polyprotein comprising one or more core structural proteins, preferably wherein the nucleotide sequence encoding one or more viral vector proteins comprises the gene gag and / or pol, or functional equivalents thereof, preferably gag and / or pol derived from human immunodeficiency virus 1 ; and / or c. the nucleotide sequence encoding one or more viral vector envelope proteins comprises a nucleotide sequence encoding a vesicular stomatitis virus G glycoprotein envelope protein or a variant thereof.

7. The method according to any one of claims 4 to 6, wherein: a. the first genomic safe harbour is the CCR5 gene and the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell comprises the gene rev; b. the second genomic safe harbour is the AAVS1 locus and the nucleotide sequence encoding one or more viral vector proteins comprises the genes gag and pol; c. the third genomic safe harbour is the CLYBL gene and the nucleotide sequence encoding one or more viral vector envelope proteins comprises a nucleotide sequence encoding a vesicular stomatitis virus G glycoprotein envelope protein; and d. the method further comprises introducing into the cell a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest.

8. The method according to any one of claims 1 to 7, wherein the nucleotide sequence encoding one or more viral vector proteins further comprises a HIV-1 -derived Rev response element (RRE) or a functional equivalent thereof.

9. The method according to any one of claims 4 to 8, wherein the viral vector genome is a selfinactivating viral vector genome, preferably wherein the self-inactivating viral vector genome further comprises a nucleotide sequence encoding: a HIV-1-derived central polypurine tract (cPPT), a HIV-1 -derived Rev response element (RRE), and / or a Woodchuck Hepatitis Virus (WHV)-derived WHV Posttranscriptional Regulatory Element (WPRE).

10. The method according to any one of claims 4 to 9, wherein the nucleotide sequence of interest encodes a transgene of interest or a non-coding RNA of interest.11 . The method according to any one of claims 1 to 10, wherein: a. the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell is operably linked to a promoter; b. the nucleotide sequence encoding one or more viral vector proteins is operably linked to a promoter; c. the nucleotide sequence encoding one or more viral vector envelope proteins is operably linked to a promoter; and / or d. the nucleotide sequence of interest is operably linked to a promoter; preferably wherein the promoter is an inducible promoter, a repressible promoter, or a constitutively active promoter such as an cytomegalovirus (CMV), Rous Sarcoma Virus (RSV) promoter, or a CAG synthetic promoter.

12. The method according to any one of claims 1 to 11 , wherein the one or more viral vector proteins comprises a HIV-1 -derived protease having an amino acid substitution on position 26 of threonine to serine.

13. The method according to any one of claims 1 to 12, the method comprising performing steps b and c, steps b, c, and d, or steps b, c, d, and e sequentially.

14. The method according to claim 13, the method further comprising: a. determining targeted insertion into the first genomic safe harbour site of the cell of the nucleic acid molecule before performing step c, and optionally selecting for a cell comprising the nucleic acid molecule targeted inserted into the first genomic safe harbour site of the cell; and b. determining targeted insertion into the second genomic safe harbour site of the cell of the nucleic acid molecule before performing step d, and optionally selecting for a cell comprising the nucleic acid molecule targeted inserted into the second genomic safe harbour site of the cell; and c. optionally, determining targeted insertion into the third genomic safe harbour site of the cell of the nucleic acid molecule before performing step e, and optionally selecting for acell comprising the nucleic acid molecule targeted inserted into the third genomic safe harbour site of the cell; and d. optionally, determining targeted insertion into the fourth genomic safe harbour site of the cell of the nucleic acid molecule after performing step e, and optionally selecting for a cell comprising the nucleic acid molecule targeted inserted into the fourth genomic safe harbour site of the cell.

15. The method according to any one of claims 1 to 14, wherein targeted insertion into a first, second, third, or fourth genomic safe harbour site of the cell comprises using a targeted or site-directed nuclease, preferably a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) protein, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), a recombinase, a homing nuclease or meganuclease, a recombinase and a bridge RNA, or a modified version of any one of the aforementioned.

16. The method according to any one of claims 3 to 15, wherein introducing into the cell comprises introducing the nucleic acid molecule and the nucleotide sequence into the cell transiently and / or not into the genome of the cell, preferably by transiently transfecting the cell or by transducing the cell using non-integrating viral vectors.

17. The method according to any one of claims 1 to 16, wherein the cell is an animal cell, preferably a mammalian cell, more preferably a human cell, even more preferably a human embryonic kidney (HEK) 293 cell or a derivative thereof, for example a HEK 293T cell, a HEK 293G cell, or a HEK 293SF cell, most preferably a HEK 293T cell.

18. The method according to any one of claims 1 to 17, wherein the viral vector is a retroviral vector, preferably a lentiviral vector.

19. The method according to any one of claims 1 to 18, wherein the method is performed in vitro or ex vivo and / or the method does not comprise a process for modifying the germ line genetic identity of a human being.

20. The method according to any one of claims 1 to 19, wherein the targeted insertion of a nucleic acid molecule into the first, second, third, and / or fourth genomic safe harbour site of the cell results in a stable expression of: a. the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell; and b. the nucleotide sequence encoding one or more viral vector proteins; and c. optionally, the nucleotide sequence encoding one or more viral vector envelope proteins; and d. optionally, the nucleotide sequence of interest;wherein the stable expression of the nucleotide sequence is defined as less than 50% difference in normalized expression of the transcript of the nucleotide sequence from passage to passage over at least 5 passages, preferably at least 10 passages, wherein the expression of the transcript of the nucleotide sequence is normalized to the expression of a housekeeping gene, for example GAPDH, as determined by quantitative PCR (qPCR).21 . A cell for producing a viral vector obtainable by the method of any one of claims 1 to 20.

22. A cell with a modified genome for producing a viral vector comprising: a. a nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell inserted into a first genomic safe harbour site of the cell; and b. a nucleotide sequence encoding one or more viral vector proteins inserted into a second genomic safe harbour site of the cell; wherein the first and second genomic safe harbour sites are different.

23. The cell according to claim 22, the cell further comprising: a. a nucleotide sequence encoding one or more viral vector envelope proteins inserted into a third genomic safe harbour site of the cell; wherein the first, second, and third genomic safe harbour sites are different; or b. a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector envelope proteins.

24. The cell according to claim 22 or 23, the cell further comprising: a. a viral vector genome comprising a nucleotide sequence of interest inserted into a fourth genomic safe harbour site of the cell; wherein the first, second, third, and fourth genomic safe harbour sites are different; or b. a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest.

25. The cell according to any one of claims 22 to 24, wherein expression of: a. the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell; b. the nucleotide sequence encoding one or more viral vector proteins; and c. the nucleotide sequence encoding one or more viral vector envelope proteins; are stable, wherein the stable expression of the nucleotide sequence is defined as less than 50% difference in normalized expression of the transcript of the nucleotide sequence from passage to passage over at least 5 passages, preferably at least 10 passages, wherein the expression of the transcript of the nucleotide sequence is normalized to the expression of a housekeeping gene, for example GAPDH, as determined by quantitative PCR (qPCR).

26. The cell according to any one of claims 22 to 25, wherein: a. the first genomic safe harbour site of the cell is selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene, preferably the AAVS1 locus, the CCR5 gene, and the CLYBL gene, more preferably the CCR5 gene; and / or b. the second genomic safe harbour site of the cell is selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene, preferably the AAVS1 locus, the CCR5 gene, and the CLYBL gene, more preferably the AAVS1 locus; and / or c. the third genomic safe harbour site of the cell is selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene, preferably the AAVS1 locus, the CCR5 gene, and the CLYBL gene, more preferably the CLYBL gene; and / or d. the fourth genomic safe harbour site of the cell is selected from any one of: the hROSA26 locus, the AAVS1 locus, the SHS231 locus, the Pansio-1 locus, the OI6nne-18 locus, the Keppel-19 locus, the CLYBL gene, the CCR5 gene, and the HPRT gene.

27. The cell according to any one of claims 22 to 26, wherein: a. the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell comprises the gene rev or a functional equivalent thereof, preferably rev derived from human immunodeficiency virus 1 ; and / or b. the nucleotide sequence encoding one or more viral vector proteins comprises a reverse transcriptase, an integrase, a protease, and / or a polyprotein comprising one or more core structural proteins, preferably wherein the nucleotide sequence encoding one or more viral vector proteins comprises the gene gag and / or pol, or functional equivalents thereof, preferably gag and / or pol derived from human immunodeficiency virus 1 ; and / or c. the nucleotide sequence encoding one or more viral vector envelope proteins comprises a nucleotide sequence encoding a vesicular stomatitis virus G glycoprotein envelope protein or a variant thereof.

28. The cell according to any one of claims 22 to 27, wherein: a. the first genomic safe harbour is the CCR5 gene and the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell comprises the gene rev; b. the second genomic safe harbour is the AAVS1 locus and the nucleotide sequence encoding one or more viral vector proteins comprises the genes gag and pol;c. the third genomic safe harbour is the CLYBL gene and the nucleotide sequence encoding one or more viral vector envelope proteins comprises a nucleotide sequence encoding a vesicular stomatitis virus G glycoprotein envelope protein; and d. the method further comprises introducing into the cell a nucleic acid molecule comprising a viral vector genome comprising a nucleotide sequence of interest.

29. The cell according to any one of claims 22 to 28, wherein the nucleotide sequence encoding one or more viral vector proteins further comprises a HIV-1 -derived Rev response element (RRE) or a functional equivalent thereof.

30. The cell according to any one of claims 22 to 29, wherein the viral vector genome is a selfinactivating viral vector genome, preferably wherein the self-inactivating viral vector genome further comprises a nucleotide sequence encoding: a HIV-1-derived central polypurine tract (cPPT), a HIV-1 -derived Rev response element (RRE), and / or a Woodchuck Hepatitis Virus (WHV)-derived WHV Posttranscriptional Regulatory Element (WPRE).31 . The cell according to any one of claims 22 to 30, wherein the nucleotide sequence of interest encodes a transgene of interest or a non-coding RNA of interest.

32. The cell according to any one of claims 22 to 31 , wherein: a. the nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell is operably linked to a promoter; b. the nucleotide sequence encoding one or more viral vector proteins is operably linked to a promoter; c. the nucleotide sequence encoding one or more viral vector envelope proteins is operably linked to a promoter; and / or d. the nucleotide sequence of interest is operably linked to a promoter; preferably wherein the promoter is an inducible promoter, a repressible promoter, or a constitutively active promoter such as an cytomegalovirus (CMV), Rous Sarcoma Virus (RSV) promoter, or a CAG synthetic promoter.

33. A method of producing a viral vector comprising culturing the cell according to any one of claims 21 to 32 under conditions suitable for the production of the viral vector, and optionally obtaining the viral vector from the cell.

34. The method according to claim 33, wherein producing the viral vector results in the functional titer (TU / ml) being at least 50% of the physical titer (VP / ml).

35. A cell transduced by the viral vector produced by the cell according to any one of claims 21 to 32 or produced by the method according to claim 33 or 34.

36. A pharmaceutical composition comprising the viral vector produced by the cell according to any one of claims 21 to 32 or by the method according to claim 33 or 34, or the cell according to claim 35; and a pharmaceutically acceptable carrier, diluent, and / or excipient.

37. A viral vector produced by the cell according to any one of claims 21 to 32 or by the method according to claim 33 or 34, a cell according to claim 35, or a pharmaceutical composition according to claim 36, for use as a medicament.

38. A method of treatment comprising administering the viral vector produced by the cell according to any one of claims 21 to 32 or by the method according to claim 33 or 34, a cell according to claim 35, or a pharmaceutical composition according to claim 36 to a subject in need of the same.

39. Use of the cell according to any one of claims 21 to 32 for producing a viral vector or in the production of a medicament.

40. Use of a viral vector produced by the cell according to any one of claims 21 to 32 or by the method according to claim 33 or 34 in the production of a medicament.

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