Host cell lines and methods for identifying and using such host cell lines

Abstract

A host cell line and a producing cell line derived from the HeLaS3 parent cell line, as well as a method for producing the host cell line and the producing cell line, are disclosed.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to European Patent Application No. 24315118.0 filed April 4, 2024, and U.S. Provisional Patent Application No. 63 / 462,215 filed April 26, 2023, the disclosures of which are incorporated herein by reference.

[0002] This disclosure relates to a method and cell line for producing adeno-associated virus (AAV) particles. Host cells derived from the HeLa cell line are also disclosed. [Background technology]

[0003] Mammalian cell lines such as CHO cells, HEK293, and human cervical cancer (HeLa) cell lines are commonly used in the biotechnology industry to produce biological molecules such as recombinant AAV (rAAV) vectors, and sometimes proteins. HeLa cells adapted for growth in suspension in serum-free medium (e.g., HeLaS3) are desirable for developing large-scale cell culture processes for producing rAAV, for example, for use in human gene therapy.

[0004] To reduce the risk of contamination, there remains a need to develop host cells that exhibit suitable attributes for the efficient production of rAAV, including host cells developed without the use of animal-derived products (e.g., serum). [Overview of the Initiative] [Means for solving the problem]

[0005] This disclosure provides, at least in part, compositions and methods for using HeLa cells.

[0006] In one embodiment, the disclosure provides a host cell line derived from the HeLaS3 parent cell line. The host cell line may have a difference of approximately 0.5% to approximately 25% in cell doubling time (in hours) compared to the parent cell line. The host cell line may have a difference of approximately 0.5% to approximately 25% in transfection efficiency compared to the parent cell line. The host cell line may have a difference of approximately 0.5% to approximately 25% in peak viable cell density compared to the parent cell line. The host cell line may have a difference of approximately 0.5% to approximately 10% in the percentage of cell viability after one cell freeze-thaw cycle compared to the parent cell line. The host cell line can be transfected with one or more nucleic acid molecules encoding xenotransgenes adjacent to AAV ITR, AAV rep, and AAV cap, and may have a difference of approximately 1 to 20 times in adeno-associated virus (AAV) vector production titer (vg / mL) compared to the parent cell line.

[0007] In one embodiment, the host cell line can be selected by a method comprising: (a) growing one or more populations of HeLaS3 parental cell lines in serum-free medium; (b) selecting and isolating one or more single-cell clones from step (a); (c) growing each of the one or more single-cell clones from step (b) in serum-free medium; (d) selecting one or more single-cell clones from each of the one or more single-cell clones from step (c) and analyzing one or more of them for at least one of the following characteristics: (i) cell doubling time, (ii) transfection efficiency, (iii) peak viable cell density; and (iv) any combination thereof; and (e) growing the one or more single-cell clones selected from step (d) in serum-free medium; thereby obtaining the host cell line. One or more single-cell clones can be further analyzed for growth characteristics after seeding at low cell density, rAAV production titer, or a combination thereof.

[0008] In one embodiment of this method, step (d) further includes an assessment of recovery from cell freeze-thaw cycles, the degree of cell aggregation, or an analysis of the metabolic profile. The metabolic profile may include measuring glucose or glutamine depletion or lactate secretion or any combination thereof in serum-free medium for 1 to 7 days. In one embodiment of this method, one or more single-cell clones from step (d) may differ from the parent cell line by about 0.5% to about 25% in cell doubling time (in hours); may differ from the parent cell line by about 0.5% to about 25% in peak viable cell density; and / or may differ from the parent cell line by about 0.5% to about 25% in the percentage of cell viability after one cell freeze-thaw cycle. In one embodiment of this method, one or more single-cell clones from step (d) may have a difference of approximately 1 to 20 times in AAV vector production titer (vg / mL) compared to the parent cell line when transfected with one or more nucleic acid molecules encoding xenotransgenes adjacent to AAV ITR, AAV rep, and AAV cap, and infected with a helper virus. In one embodiment of this method, one or more single-cell clones from step (c) may have a difference of approximately 0.5% to approximately 25% in transfection efficiency compared to the parent cell line. In one embodiment of this method, step (d) further includes selection based on principal component analysis. In one embodiment of this method, the host cell line is grown in suspension.

[0009] In one embodiment, recombinant AAV (rAAV) particles are produced by a method comprising: (a) transfecting a host cell as described herein under conditions that produce rAAV particles, transfecting the host cell line with one or more nucleic acid molecules encoding a xenotransgene adjacent to inverted terminal repeats (ITRs), AAVreps, AAV caps, and optionally selectable markers; and growing one or more populations from the transfected parent cell line; (b) providing or infecting cells with an AAV helper virus or a derivative thereof; and (c) recovering the rAAV particles. rAAV particle host cell lines can be transfected with nucleic acid molecules encoding the AAV cap, derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, goat AAV, AAV1 / AAV2 chimera, bovine AAV, or mouse AAV capsid rAAV2 / HBoV1, or their variants. The AAV helper virus may be Ad5, such as human Ad5. Host cell lines can be infected with helper viruses. The host cell line can be infected with helper viruses, including adenovirus, herpes simplex virus, vaccinia virus, or cytomegalovirus. The transfected host cell line can be recovered to obtain AAV particles. One or more nucleic acid molecules can be stably transfected into the host cell line.

[0010] In one embodiment, the method provides a candidate cell line for the production of recombinant AAV (rAAV) particles. The method may include (a) generating a producing cell line by stably transfecting a host cell line in serum-free medium with one or more nucleic acids encoding (i) xenotransgenes adjacent to two AAV inverted terminal repeat sequences, (ii) an AAV rep gene and an AAVcap gene; (b) infecting the producing cell line with an AAV helper virus to produce rAAV particles; and (c) selecting the producing cell line as a candidate for rAAV particle production if the producing cell line produces a titer of at least approximately 1E9 vg / mL of rAAV particles. The method may further include, after step (d), growing the producing cell line from step (c) to a cell density of 3.5E5 or more cells; and (d) selecting the producing cell line as a candidate for rAAV particle production if the producing cell line produces a titer of at least approximately 1E9 vg / mL of rAAV particles. This method may further include the step of growing cells to a cell density of 3.5E5 or more and selecting a producing cell line as a candidate for rAAV particle production if the producing cell line generates a titer of at least approximately 1E10 vg / mL of rAAV particles. This method may further include determining the rAAV titer by quantitative polymerase chain reaction (qPCR). This method may further determine cell viability or cell viability density by freeze-thaw cycles, shear stress, or a combination thereof. A method for determining cell viability may include selecting cells with a cell viability of 70% or more compared to the HeLaS3 parent cell line. Cells with a cell viability of 90% or more compared to the HeLaS3 parent cell line can be selected. Cells with a cell doubling time of 32 hours or less compared to the HeLaS3 parent cell line can be selected. Cells with a cell doubling time of 32 hours or less compared to the HeLaS3 parent cell line can be selected. Compared to the HeLaS3 parent cell line, cells with a viability of 3×10 6Cells with a peak live cell density of cells / mL or higher can be selected. Cells with more than 30% transfected cells compared to the HeLaS3 parent cell line can be selected. Cells with reduced macroscopic cell aggregation compared to the HeLaS3 parent cell line can be selected. The producing cell line may be a mammalian host cell. The producing cell line may be a HeLa3 cell line. The serum-free medium used to generate the cells may not contain any animal-derived components. The serum-free medium may include a glutamine-supplemented medium. The serum-free medium may include a glutamine-supplemented medium of approximately 6 mM.

[0011] In one embodiment, (a) EX-CELL HeLa growth medium supplemented with 6 mM L-glutamine, 50% DMEM / F-12 (supplemented with 6 mM L-glutamine), 20% culture supernatant and 1x InstiGRO A method for producing a host cell line, comprising the steps of: (a) growing one or more populations of HeLaS3 parent cell lines at a density of 0.5 cells / well in a medium containing a CHO supplement (all components of the medium and culture supernatant are serum-free and free of animal-derived components); (b) selecting and isolating one or more single-cell clones from step (a); (c) growing each of the one or more single-cell clones from step (b) in serum-free medium; (d) selecting from each of the one or more single-cell clones from step (c) for at least one of the following characteristics: (i) cell viability, (ii) cell doubling time, (ii) transfection efficiency, (iv) peak viable cell density; (v) aggregation; (vi) any combination thereof; and (e) isolating and growing the single cells selected from step (d) in serum-free medium to produce a host cell line. The method may further include selection for cell proliferation after seeding at low cell density; rAAV production potency; and combinations thereof.

[0012] In one embodiment, a method is provided for producing a producing cell line, comprising the steps of (a) transfecting (or infecting the host cell with a helper virus) a host cell line selected by any of the host cell lines described herein or by any of the methods described herein with one or more nucleic acids encoding (i) a xenotransgene adjacent to two AAV inverted terminal repeat sequences, (ii) an AAV rep gene and an AAV cap gene, (iii) an AAV helper gene; and (b) selecting a producing cell line in which the producing cell line produces a titer of at least about 1E9 to about 1E11 vg / ml of rAAV particles.

[0013] One embodiment provides a method for producing recombinant adeno-associated virus (rAAV), comprising the steps of (a) stably transfecting a host cell line produced by any of the host cell lines described herein or any of the methods described herein with (i) a heterogene, (ii) an AAV rep gene and an AAV cap gene, and (iii) one or more nucleic acids encoding an AAV inverted terminal repeat (ITR); (b) infecting the host cell with the helper virus; and (c) isolating rAAV particles having a titer of at least about 1E9 vg / mL. The titer of the rAAV particles may be about 1E9 vg / mL to about 1E11 vg / mL.

[0014] The aforementioned and other features and advantages of this disclosure will be better understood from the following detailed description of exemplary embodiments in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0015] [Figure 1]This flowchart outlines the isolation and initial selection of HeLaS3-derived host cells, illustrating the workflow for isolating and selecting host cell lines with desired characteristics. First, the HeLaS3 parent strain was subcloned into 96-well plates to achieve a seeding density of less than one cell per well. For wells containing clone-derived cell proliferation, the cells were expanded into 24-well plates, then 6-well plates, and finally shaking flasks. The culture media used were free of animal-derived components (i.e., no animal-derived components or products). Each subclone was cryopreserved in multiple cryovials. The subclones were thawed and screened for freeze-thaw tolerance (including low aggregation) and cell proliferation characteristics, yielding 54 lead candidate subclones for further evaluation. [Figure 2] This schematic diagram illustrates some of the preferred cellular characteristics exhibited by the improved host cell lines exemplified by this disclosure. These factors include improvements in growth characteristics such as population doubling time (PDT) and batch culture peak live cell density (VCD), improved transfection efficiency with larger plasmids of approximately 4, 5, and 10 kilobases or more, and the production of preferred viral vector titers. [Figure 3] This outlines the selection of improved host cell lines from 54 candidate cell populations. First, 54 candidate cell populations were isolated from HeLa parent cells, as shown in Figure 1. These populations were then narrowed down to 23 candidate cell populations based on a screening procedure that evaluated parameters such as freeze-thaw tolerance, population doubling time, transfection efficiency, degree of cell aggregation, and peak VCD. The 23 candidate cell populations were then grown in serum-free medium and subjected to additional screening, which included culturing the 23 candidate cell populations in an automated bioreactor system (labeled "Process Suitability Assessment (Ambr®)" + shear stress test), which allowed for a further narrowing down to 5 candidate cell populations. [Figure 4A-B]This shows the candidate cell populations evaluated by the freeze-thaw tolerance (FTT) assessment. Figure 4A is a schematic diagram of the freeze-thaw tolerance (FTT) assay. Thirty frozen candidate cell populations were thawed on day 0. The candidate cell populations were grown in liquid culture and passaged twice (passages 1 and 2 are labeled "P1 FTT1" and "P2 FTT2," respectively). Two replicas from FTT1 and FTT2 were tested for cell viability. Figure 4B is a graph measuring the percentage of cell viability from the FFT1 and FFT2 samples. The parental HeLa cell line was used as a control (indicated by the dashed box "HeLa parental line"). [Figure 5A-B] This shows the candidate cell populations evaluated by population doubling time (PDT) assessment. Figure 5A is a schematic diagram of the population doubling time (PDT) assay. Using the same experimental setup as the FTT assay, the rate at which the candidate population doubled was measured in hours. Figure 5B is a graph measuring the rate at which the candidate cell population doubled over two passages. The parental HeLa cell line was used as a control (indicated by the dashed box "HeLa parental line"). [Figure 6A] The candidate cell populations evaluated in the nucleofection transfection efficiency assessment are shown. Figure 6A is a schematic diagram of the nucleofection transfection (NTx) efficiency assay. The candidate populations were transiently transfected with plasmids containing the fluorescent reporter GFP. [Figure 6B] The candidate cell populations evaluated by nucleofection transfection efficiency assessment are shown. Figure 6B is a graph measuring the percentage of GFP-positive cells calculated 24 hours after transfection. The parental HeLa cell line was used as a control (indicated by the dashed box "HeLa parental line"). [Figure 7A-B] This shows the candidate cell populations evaluated by peak viable cell density (VCD) assessment. Figure 7A is a schematic diagram of the peak viable cell density (VCD) assay in which candidate populations were thawed and grown over two passages (P1-replication 1 and P2-replication 2). Figure 7B is a graph calculating the normalized magnification change in viable cell density (relative to the HeLa parent strain) for 32 candidate populations. The parent HeLa cell line was used as a control (indicated by the dashed box "HeLa parent strain"). [Figure 8A-B] Principal component analysis (PCA) plots obtained for the candidate cell populations after the first relative clone property screening. Figure 8A is an exemplary PCA biplot of all 32 candidate populations (represented by individual points). Figure 8B is a graph and exemplary score contribution of the candidate cell populations for FTT, PDT, and peak VCD. The GFP transfection efficiency was evaluated at 24 hours and 48 hours. PCA is a multivariate tool that combines all the original variables and identifies correlations (structured variations) that exclude noise (unstructured variations). [Figure 9] The timeline when adding media and cell candidate populations to small-scale bioreactors (such as Ambr® 15) for cell growth kinetics and metabolic profiling analysis is shown. [Figure 10A] The cell growth evaluation of 21 candidate populations cultured for 7 days under small-scale bioreactor (such as Ambr® 15 bioreactor) conditions is reported. Figure 10A is a line graph measuring the viable cell density (E6 cells / mL) of each candidate cell population. [Figure 10B] The cell growth evaluation of 21 candidate populations cultured for 7 days under small-scale bioreactor (such as Ambr® 15 bioreactor) conditions is reported. Figure 10B is a line graph measuring the percentage viability of each candidate cell population over 7 days. [Figure 11A] The cell metabolic profiling of 21 candidate populations cultured for 7 days under small-scale bioreactor conditions is reported. Figure 11A is a line graph measuring the glucose consumption rate of each candidate cell population. [Figure 11B] The cell metabolic profiling of 21 candidate populations cultured for 7 days under small-scale bioreactor conditions is reported. Figure 11B is a line graph measuring the lactate production rate of each candidate cell population over 7 days. [Figure 12]Cell growth evaluations measured in terms of peak VCD and cell viability from the top 7 out of 21 candidate cell populations cultured over 7 days under small-scale bioreactor conditions, and metabolic profiling measured in terms of glutamine depletion, glucose consumption, and lactate production are reported. [Figure 13A-B] Measure the amount of AAV titer (vg / mL) released into the cell culture medium from each of the top 7 candidate cell populations 72 hours after a media change, which is the 5th day after transfection with the viral plasmid. Figure 13A is a schematic diagram of an assay where the candidate population is transfected with the viral plasmid on day 1, the culture medium is exchanged on day 2 (24 hours after transfection), and the cell culture medium is recovered for viral titer analysis on day 5 (72-hour harvest). Figure 13B is a graph of the amount of AAV titer (vg / mL) released into the cell culture medium from each of the top 7 candidate cell populations on day 5 along with the viral plasmid. [Figure 14] Shows one aspect of the process for generating an AAV-producing cell line starting from the HeLaS3 host cell line. First, the HeLaS3 host cells are transfected with a heterologous transgene, AAV rep, AAV cap, and a selectable marker using a plasmid (see the "Plasmid components" and "Transfection" displays in the figure). The transfected cells are cultured in the presence of a selection agent, and the stably transfected cell population is grown, cryopreserved, and screened for productivity. The lead population (based on several metrics including growth and productivity) is seeded into a well plate to isolate single cell clones, which are then grown, cryopreserved, and screened for the desired characteristics. [Figure 15] Shows the workflow of an exemplary clone characterization process. [Figure 16] Graphically shows the transfection efficiency represented as the percentage of green fluorescent protein (GFP) cells (black solid bars) and cell viability (white bars) of subclones and HeLaS3 parental cells. [Figure 17]This document describes a process for evaluating cell proliferation performance in the cell culture bioreactor system, Ambr® 15. [Figure 18] This document describes a process for evaluating cell proliferation performance and viability in the Ambr® 15 cell culture bioreactor system under shear stress testing. [Figure 19] The graphs show the viable cell density and cell viability profiles of the top 11 clones evaluated for shear stress resistance. VCD (solid black line) and cell viability (dashed line) profiles of HeLaS3 parental cells are included as controls. To increase the shear stress of the cell cultures, the shear rate and % DO (black circles) were increased. Clones MQ, E, S, and P showed high resistance to shear stress and, combined with other characteristics, were classified as the top five clones. [Figure 20A-B] The graph shows the top 5 clones selected for nucleovector efficiency or viability and CAR level. [Figure 21] The graphs show the viable cell density (VCD) and cell viability (Viab) from Ambr® 15 runs performed on clone M and the parental HeLa strain. Shear stress was induced by increasing the stirring speed and dissolved oxygen (DO) settings. Shear stress runs are shown as dark black lines. The average of three Ambr® 15 runs for VCD and viability are shown for the parental HeLa strain used as a control. [Figure 22] The graphs show the viable cell density (VCD) and cell viability (Viab) from Ambr® 15 runs performed on clone R and the parental HeLa strain. Shear stress was induced by increasing the stirring speed and dissolved oxygen (DO) settings. Shear stress runs are shown as dark black lines. The average of three Ambr® 15 runs for VCD and viability are shown for the parental HeLa strain used as a control. [Figure 23]The graphs show the viable cell density (VCD) and cell viability (Viab) from Ambr® 15 runs performed on clone Q and the parental HeLa strain. Shear stress was induced by increasing the stirring speed and dissolved oxygen (DO) settings. Shear stress runs are shown as dark black lines. The average of three Ambr® 15 runs for VCD and viability are shown for the parental HeLa strain used as a control. [Figure 24] The graphs show the viable cell density (VCD) and cell viability (Viab) from Ambr® 15 runs performed on clone C and the parental HeLa strain. Shear stress was induced by increasing the stirring speed and dissolved oxygen (DO) settings. Shear stress runs are shown as dark black lines. The average of three Ambr® 15 runs for VCD and viability are shown for the parental HeLa strain used as a control. [Figure 25] The graphs show the viable cell density (VCD) and cell viability (Viab) from Ambr® 15 runs performed on clone E and the parental HeLa strain. Shear stress was induced by increasing the stirring speed and dissolved oxygen (DO) settings. Shear stress runs are shown as dark black lines. The average of three Ambr® 15 runs for VCD and viability are shown for the parental HeLa strain used as a control. [Figure 26] The graph shows the plating efficiency of selected clones under serum-free conditions. HeLaS3 parental cells and clones M, B, Q, E, and C were seeded at a low cell density (3 cells / well) using a cell dispenser. Their ability to grow into cell colonies was evaluated 14 days after deposition. The graph shows the percentage of colony growth, calculated by dividing the number of counted colonies by the number of seeded cells and multiplying by 100. [Modes for carrying out the invention]

[0016] Host cell lines and producing cell lines derived from the HeLaS3 parental cell line are disclosed. The host cell lines are derived from the HeLaS3 parental cell line and have different properties from the parental cell line. In some embodiments, the host cell lines have improved properties compared to the HeLaS3 parental cell line, such as doubling time, improved transfection efficiency, peak viable cell density, cell viability after one cell-free thawing cycle, AAV vector production titer (vg / mL), reduced aggregation, improved shear stress, or a combination thereof. In some embodiments, the host cell is a cell derived from a single cell.

[0017] general technology Certain techniques and procedures described or referenced herein are based on Molecular Cloning: A Laboratory Manual (Sambrook et al., 4 th ed.,Cold Spring Harbor Laboratory Press,ColdSpring Harbor,NY,2012);Current Protocols in Molecular Biology(FMAusubel et al.eds.,2003);the series Methods in Enzymology(Academic Press,Inc.);PCR 2:A Practical Approach(MJMacPherson,BDHames and GRTaylor eds.,1995);Antibodies,A Laboratory Manual(Harlow and Lane,eds.,1988);Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications(RIFreshney,6 thed.,J.Wiley and Sons,2010);Oligonucleotide Synthesis(M.J.Gait,ed.,1984);Methods in Molecular Biology,Humana Press;Cell Biology:A Laboratory Notebook(J.E.Cellis,ed.,Academic Press,1998);Introduction to Cell and Tissue Culture(J.P.Mather and P.E.Roberts,Plenum Press,1998);Cell and Tissue Culture:Laboratory Procedures(A.Doyle,J.B.Griffiths,and D.G.Newell,eds.,J.Wiley and Sons,1993-8);Handbook of Experimental Immunology(D.M.Weir and C.C.Blackwell,eds.,1996);Gene Transfer Vectors for Mammalian Cells(J.M.Miller and M.P.Calos,eds.,1987);PCR:The Polymerase Chain Reaction,(Mullis et al.,eds.,1994);Current Protocols in Immunology(J.E.Coligan et al.,eds.,1991);Short Protocols in Molecular Biology(Ausubel et al.,eds.,J.Wiley and Sons,2002); Immunobiology(CAJaneway et al.,2004);Antibodies(P.Finch,1997);Antibodies:A Practical Approach(D.Catty.,ed.,IRL Press,1988-1989);Monoclonal Antibodies:A Practical Approach(P.Shepherd and C.Dean,eds.,Oxford University Press,2000);Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JDCapra, eds., Harwood Academic Publishers, 1995); and DeVita Jr, VT, Lawrence, T., & Rosenberg, SA (2012). Cancer: principles&practice of oncology: annual advances in oncology. Lippincott Described in Williams & Wilkins.

[0018] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure pertains.

[0019] As used herein, "vector" refers to a recombinant plasmid or virus containing a nucleic acid molecule that is delivered to a host cell in vitro or in vivo.

[0020] The terms “polynucleotide” or “nucleic acid molecule,” as used herein, refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Therefore, the terms include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically, or biochemically modified, unnatural, or derivatized nucleotide bases. The backbone of a polynucleotide may contain sugars and phosphate groups (typically found in RNA or DNA), or modified or substituted sugars or phosphate groups.

[0021] The terms “polypeptide” and “protein” are used interchangeably to refer to polymers of amino acid residues and are not limited to minimum length. Such polymers of amino acid residues may include natural or unnatural amino acid residues and include, but are not limited to, amino acid peptides, oligopeptides, dimers, trimers, and polymers. Both full-length proteins and their fragments are included in the definition. The term also includes post-translational modifications of polypeptides, such as glycosylation, sialylation, acetylation, and phosphorylation. Furthermore, for the purposes of this disclosure, “polypeptide” refers to proteins that include modifications such as deletions, additions, and substitutions (generally inherently conserved) of the natural sequence, as long as the protein maintains the desired activity. These modifications may be intentional, such as through site-directed mutagenesis, or accidental, such as mutations in the host producing the protein or errors in PCR amplification.

[0022] A "recombinant viral vector" refers to a polynucleotide viral vector containing one or more heterogeneous nucleic acid molecules (i.e., nucleic acid molecules not naturally associated with the viral vector). In the case of a recombinant AAV viral vector, the heterogeneous nucleic acid molecules may be adjacent to two ITRs.

[0023] A "recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector containing one or more heterogeneous nucleic acid molecules (i.e., nucleic acid molecules that do not naturally associate with the wild-type AAV vector) adjacent to two AAV inverted terminal repeat sequences (ITRs). Such rAAV vectors, when present in a host cell that is infected with a suitable helper virus (or expresses suitable helper function) and expresses the AAVrep and cap gene products (i.e., AAVRep and Cap proteins), can be replicated and packaged into infectious viral particles. When an rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection), the rAAV vector may be referred to as a "provector" that can be "rescued" by replication and capsid formation in the presence of AAV packaging function and suitable helper function. rAAV vectors can be any of several forms, including, but not limited to, plasmids that are complexed with lipids, encapsulated in liposomes, and capsid-formed within viral particles, particularly AAV particles, or linear artificial chromosomes. The rAAV vector can be packaged into an AAV virus capsid to generate "recombinant adeno-associated virus particles (rAAV particles)."

[0024] A "host cell line" refers to a population of cells capable of continuous or long-term proliferation and division in vitro. A host cell line may have spontaneous or induced changes in its karyotype that may occur during storage or transport. Therefore, a host cell line does not necessarily have to be identical to the ancestral cell or culture, and thus host cell lines may contain variants.

[0025] A "producing cell line" refers to cells derived from a host cell line. Producing cells can be generated by incorporating (e.g., stably incorporating) one or more AAV genes (e.g., rep and cap) along with the target ITR flanking transgene into a host cell.

[0026] "Heterogeneous" means that it originates from an entity with a different genotype from the rest of the entity being compared to, or into which it is introduced, or incorporated. For example, a polynucleotide introduced into a different cell type by genetic engineering is a heterogeneous polynucleotide (and can encode a heterogeneous polypeptide when expressed). Similarly, a cellular sequence (e.g., a gene or a portion thereof) incorporated into a viral vector is a heterogeneous nucleotide sequence relative to the vector.

[0027] The term "transgene" refers to a polynucleotide introduced into a cell, which is transcribed into RNA and can be translated and / or expressed under appropriate conditions by choice. In some embodiments, this confers a desired characteristic to the cell into which it is introduced, or otherwise results in a desired therapeutic or diagnostic outcome. In other embodiments, the transgene may be transcribed into an RNA interference-mediating molecule such as miRNA, siRNA, or shRNA.

[0028] An "AAV inverted terminal repeat (ITR)" sequence refers to a relatively short sequence (e.g., a sequence of about 145 nucleotides) found at the ends of a viral genome, oriented in opposite directions. The outermost 125 nucleotides of an ITR can exist in one of two different orientations, resulting in heterogeneity between different AAV genomes and between the ends of a single AAV genome. The outermost 125 nucleotides also contain several short self-complementary regions (A, A', B, B', C, and C' regions), which enable intrachain base pairing within this portion of the ITR.

[0029] "AAV helper function" refers to a function that enables host cells to replicate and package AAV. AAV helper function may be provided in any of several forms, including, but not limited to, a helper virus or helper virus gene that facilitates AAV replication and packaging. Other AAV helper functions are known in the art, such as genotoxic agents.

[0030] A "helper virus" for AAV refers to a virus that enables host cells to replicate and package AAV (which is an abnormal parvovirus). Several such helper viruses have been identified, including adenoviruses, herpesviruses, poxviruses such as vaccinia virus, and baculoviruses. Adenoviruses encompass several different subgroups, but adenovirus type 5 (Ad5) of subgroup C is the most commonly used. Numerous adenoviruses originating from humans, non-human mammals, and birds are known and available from depositary organizations such as ATCC. Examples of herpes family viruses available from depositary organizations such as ATCC include, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and pseudorabies virus (PRV). Examples of adenovirus helper functions for AAV replication include E1A, E1B, E2A, VA, and E4 or E6 functions. Baculoviruses available from depositary institutions include Autographa californica nuclear polyhedron virus.

[0031] host cell In some embodiments, the host cell line or producing cell line is derived from the HeLaS3 parental cell line. In some embodiments, the HeLaS3 cell line is a subclone of the HeLa cell line. In some embodiments, the HeLaS3 parental cell line is a clonal derivative of the parental HeLa cell line (ATCC CL-2). In some embodiments, the host cell is derived from the HeLaS3 parental cell line (e.g., from a single cell or a single precursor). In some embodiments, host cells or producing cells derived from the HeLaS3 parental cell line can be manipulated to develop novel cellular phenotypes that may be useful for the large-scale production of recombinant AAV virus vectors.

[0032] In some embodiments, host cells or producing cells are in a suspension, without serum, at high cell density, on a large scale (e.g., more than 10, 15, 20, or 50 liters of culture medium, or at least 5, 10, 15, or 20 m³). 2Suitable for culturing in culture media exceeding [a certain value].

[0033] In some embodiments, suspension cultures have scale-up advantages because such cells have advantages such as ease of production, lower cost, less space requirement as they do not require the use of proteolytic enzymes, and the ability to be cultured in a bioreactor with complete environmental control. Furthermore, suspension cell cultures can be characterized by nutrient homogeneity and a homogeneous cell population with good inter-experimental reproducibility. In some embodiments, host cells or producing cells are grown in serum-free medium. In some embodiments, host cells or producing cells are grown in a medium that does not contain animal-derived components or products. In some embodiments, host cells or producing cells are grown in a medium that does not contain mammalian or avian-derived components or products. In some embodiments, the medium may contain animal-derived products (e.g., cod liver oil) that are biosafe and do not pose a risk of contamination with infectious spongiform encephalopathy (TSE) or bovine spongiform encephalopathy (BSE). In some embodiments, the host cell or producing cell medium is biosafe and free of animal-derived components, except for cod liver oil that is TSE / BSE-free. In some embodiments, host cells or producing cells are grown in Ex-CELL® HeLa serum-free medium (Sigma-Aldrich, St. Louis, USA). In some embodiments, the medium (e.g., Ex-CELL® HeLa serum-free medium) is supplemented with glutamine at, for example, 4, 5, 6, or 7 mM.

[0034] In some embodiments, the method for selecting the host cell line is described in the Examples section. In some embodiments, the producing cells are selected by the following method: (a) Growing one or more populations of HeLaS3 parental cell lines in serum-free medium; (b) Select and isolate one or more single-cell clones from step (a); (c) Growing each of the one or more single-cell clones from step (b) in serum-free medium; (d) Select one or more single-cell clones from each of the single-cell clones from step (c) and give them the following characteristics: (i) cell doubling time; (ii) Transfection efficiency, and (iii) Peak live cell density To analyze at least one of the following; and (e) Grow one or more single-cell clones selected from step (d) in serum-free medium; thereby obtaining a host cell line; (f) Transfect the host cell line selected by steps (a) to (e) with one or more nucleic acids encoding (i) a xenotransgene adjacent to two AAV inverted terminal repeat sequences, (ii) an AAV rep gene and an AAVcap gene; and (g) Select a cell line that produces rAAV particles and generates their titer.

[0035] In some embodiments, a host cell line can be transfected with a xenotransgene flanking two AAV inverted terminal repeats, an AAV rep, an AAV cap, and, optionally, one or more nucleic acid molecules encoding a selectable marker encoding an adenovirus helper virus gene and / or a nucleic acid sequence to obtain a producing cell line. The AAV titer per cell can be quantified.

[0036] In some cases, (a) A step of growing one or more populations of parental cell lines in serum-free medium; (b) A step of selecting and isolating one or more single cell clones from step (a), (c) Growing each of the one or more single-cell clones from step (b) in serum-free medium; (d) From each of the one or more single-cell clones from step (c), the following characteristics: (i) cell viability; (ii) cell doubling time; (iii) Transfection efficiency, (iv) Peak live cell density; and (v) agglomeration The step of selecting at least one of the following; and (e) A step in which a single cell selected from step (d) is isolated in serum-free medium and grown to generate a host cell line. A host cell line containing the above is obtained.

[0037] In some embodiments, the host cell line is obtained by repeating steps (d) to (e) until the desired result is obtained, selecting a single cell to be further isolated and grown in a serum-free medium, thereby generating the host cell line.

[0038] In some embodiments, the host cell line may be selected alternatively or additionally for at least one of the following characteristics: (i) cell viability; (ii) cell doubling time; (ii) transfection or nucleofection efficiency; (iv) peak viable cell density; (v) aggregation; (vi) metabolic profiling (e.g., changes in lactate and glucose consumption); (vii) cell proliferation after seeding at low cell density (e.g., 0.5, 1, 2, 3, 4, or 5 cells / well); (viii) rAAV production potency; (ix) quality of the rAAV product (in some cases, assessed by the percentage of complete capsid); or (x) any combination thereof. In some embodiments, method steps (d) to (e) may be repeated multiple times to obtain the desired characteristics. In a particular selection round, the same or different characteristics, or combinations of characteristics, may be selected. For example, in the first selection round, cells may be selected for cell viability. In the second selection round, cells may be selected for different characteristics such as cell viability and cell doubling time. In a third selection round, cells may be selected for yet another characteristic, such as cell viability, doubling time, and aggregation. In some embodiments, characterization of parameters for selection was carried out in parallel, and clones were selected based on an overall evaluation of the parameters (e.g., cell viability, doubling time, transfection or nucleofection efficiency, peak viable cell density, aggregation, metabolic profiling, cell proliferation after seeding at low cell density, or rAAV production titer).

[0039] In some embodiments, the host cell line is (a) Growing one or more populations of HeLaS3 parental cell lines at a density of 0.5 cells / well in a medium containing EX-CELL HeLa growth medium supplemented with 6 mM L-glutamine, 50% DMEM / F-12 (supplemented with 6 mM L-glutamine), 20% culture supernatant, and 1x InstiGRO CHO supplement (the medium may be serum-free and free of animal-derived components); (b) A step of selecting and isolating one or more single-cell clones from step (a); (c) Growing each of the one or more single-cell clones from step (b) in serum-free medium; (d) From each of the one or more single-cell clones from step (c), the following characteristics: (i) cell viability; (ii) cell doubling time; (iii) Transfection efficiency, (iv) Peak live cell density; and (v) agglomeration The step of selecting at least one of the following; and (e) A step in which a single cell selected from step (d) is isolated in serum-free medium and grown to produce a host cell line. It is produced by a method that includes [a specific process].

[0040] In some embodiments, host cells are cultured in a culture supernatant. Once the culture medium has been incubated with the cells, it is called “used” or “culture supernatant.” The culture supernatant contains many of the original components of the medium, as well as various cellular metabolites and secreted proteins, which may include, for example, growth factors, inflammatory mediators, and other extracellular proteins. In one embodiment, the culture supernatant can be obtained by (a) culturing host cells derived from a parental cell line at an initial density to produce a second cell culture medium containing higher densities of cells and factors secreted by the cultured cells; and (b) separating the cells from the second culture medium to produce the culture supernatant.

[0041] In some embodiments, host cells are produced by selecting host cells that have at least one of the following characteristics compared to the parent cell line: (i) Improvement of cell viability; (ii) Improvement of peak live cell density; (iii) Improvement in group doubling time (in units of time); (iv) Improvement of transfection or nucleofection efficiency; (v) Increased expression levels of one or more genes related to the immune response; (vii) Increased cell proliferation after seeding at low cell density; and (vii) Increase in AAV vector titer

[0042] In some embodiments, the transfection efficiency of host cells can be determined by transiently transfecting the cells with the required components. In some embodiments, the transfection efficiency can be determined by transfection in: a single plasmid containing a vector genome, which is an expression cassette having a promoter, a target transgene flanked by an ITR, and nucleic acid molecules encoding Rep and Cap proteins specific to the desired serotype. In some embodiments, the target transgene used to assess transfection efficiency is a reporter protein such as green fluorescent protein or mCherry.

[0043] In some embodiments, the production of viral particles in cells is determined by transiently transfecting host cells with a plasmid containing a vector genome, which is an expression cassette having (i) a promoter and the desired transgene adjacent to the ITR, (ii) nucleic acid molecules encoding Rep and Cap proteins specific to the desired serotype, and (iii) a plasmid (Ad helper) having the minimum adenovirus genes necessary to support AAV replication (e.g., E2, E4, and VARNA). In some embodiments, the genes encoding AAV and Ad helper functions can be cloned into a single plasmid. In some embodiments, host cells are transfected with a second plasmid, which is a pAd helper plasmid containing an AAV vector sequence and encoding AAV genes (Rep and Cap) necessary to support AAV genome replication and packaging, and a plasmid containing a gene from a helper virus genome (e.g., wild-type adenovirus 5 (wtAd5) genome) necessary to support AAV gene expression, replication, and packaging. In some embodiments, wild-type adenovirus 5 (wtAd5) is used as the helper. In some embodiments, Rep and Cap are transcribed from endogenous AAV promoters p5, P19, and P40 or modified versions thereof (e.g., modified p5 promoter).

[0044] In some embodiments, viral particles are produced from host cells by stably incorporating nucleic acids for target transgenes adjacent to the AAV ITR and AAV Rep and Cap proteins into the host cell, thereby generating a producing cell line. The producing cell line is then infected with a helper virus, such as wild-type Ad5 adenovirus, to produce rAAV particles. The titer of rAAV particles produced from host cells with stable incorporation of the elements necessary for rAAV production indicates the robustness of the host cell as a candidate for generating a producing cell line.

[0045] In some embodiments, cell viability or live cell density is determined after freeze-thaw cycles, shear stress, or a combination thereof. In some embodiments, shear stress involves agitating cells in culture medium in a cell culture bioreactor system (e.g., Ambr® 15) at an agitation speed of approximately 1500 rpm to approximately 2000 rpm. In some embodiments, shear stress can be determined by measuring cell viability and cell proliferation at increasing agitation speeds of 1500, 1650, 1800, or 2000 rpm. In some embodiments, shear stress can be determined by measuring cell viability and cell proliferation at increasing agitation speeds of 1500, 1650, 1800, or 2000 rpm and 80% dissolved oxygen. In some embodiments, Ambr® is a benchtop microbioreactor system, which is a multi-parallel bioreactor that enables testing multiple cell culture conditions in parallel and evaluating cell viability and live cell density of candidate cell populations under those conditions. Cell viability was measured using Vi-CELL.

[0046] Host cell lines or producing cell lines derived from the HeLaS3 parent cell line may have several different characteristics compared to the parent cell line. For example, in some embodiments, host cells or producing cells have a difference of approximately 0.5% to approximately 25% in cell doubling time compared to the parent cell line (e.g., HeLaS3) (e.g., an increase or decrease of approximately 0.5, 1.0, 5.0, 10.0, 15.0, 20.0, or 25.0%). In some embodiments, host cells have a difference of approximately 0.5% to approximately 25% in cell doubling time (in hours) compared to the parent cell line (e.g., an increase or decrease of approximately 0.5, 1.0, 5.0, 10.0, 15.0, 20.0, or 25.0%).

[0047] In some embodiments, host cells derived from the HeLaS3 parental cell line may have a cell doubling time (in hours) of less than 32 hours, less than 30 hours, less than 28 hours, less than 26 hours, or less than 24 hours compared to the parental cell line. In some embodiments, host cells derived from the HeLaS3 parental cell line may have a cell doubling time (in hours) of 30±4 hours, 28±4 hours, 26±4 hours, or less, compared to the parental cell line.

[0048] In some embodiments, host cells exhibit a difference of approximately 0.5% to approximately 25% in transfection efficiency compared to the parent cell line (e.g., an increase or decrease of approximately 0.5%, 1.0%, 5.0%, 10.0%, 15.0%, 20.0%, or 25.0% or more).

[0049] In some embodiments, transfection is performed by nucleofection. In some embodiments, host cells have a difference of approximately 0.5% to approximately 25% in nucleofection efficiency compared to the parent cell line (e.g., an increase or decrease of approximately 0.5, 1.0, 5.0, 10.0, 15.0, 20.0, or 25.0%). In some embodiments, transfection (e.g., nucleofection) is determined, for example, by the presence of an intracellular fluorescent protein (e.g., green fluorescent protein), flow cytometry, or other preferred method. In some embodiments, transfection is performed on cells that have been passaged 2 to 10 times after thawing. In some embodiments, host cells have a transfection efficiency of 12% or more. In some embodiments, host cells have a transfection efficiency of more than 30%. In some embodiments, host cells have a transfection efficiency of 30-90%, 40-90%, or 60-90%. In some embodiments, host cells have transfection efficiencies of 30% or more, 30–90% or more, 40–90% or more, or 60–90% or more. In some embodiments, transfection efficiency is determined using a reporter transgene such as green fluorescent protein or mChery.

[0050] In some embodiments, host cells selected after freeze-thaw, shear stress, or a combination thereof have a cell viability of 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more. In some embodiments, cells selected after freeze-thaw, shear stress, or a combination thereof have a cell viability ranging from 70% to 99%. In some embodiments, cells selected after freeze-thaw, shear stress, or a combination thereof have a cell viability ranging from 70% to 99% after a first selection step, a second selection step, a third selection step, or any additional step thereafter. In some embodiments, cell viability is determined by a standard viability assay, such as trypan blue exclusion or other preferred methods.

[0051] In some embodiments, host cells have a difference of approximately 0.5% to approximately 25% in peak viable cell density compared to the parent cell line (e.g., an increase or decrease of approximately 0.5%, 1.0%, 5.0%, 10.0%, 15.0%, 20.0%, or 25.0% or more).

[0052] In some embodiments, host cells exhibit a difference of approximately 0.5% to approximately 25% in cell survival percentage after a single cell freeze-thaw cycle compared to the parent cell line (e.g., an increase or decrease of approximately 0.5%, 1.0%, 5.0%, 10.0%, 15.0%, 20.0%, or 25.0% or more).

[0053] In some embodiments, host cells exhibit a difference of approximately 0.5% to approximately 25% in cell survival percentage after one round of shear stress compared to the parent cell line (e.g., an increase or decrease of approximately 0.5%, 1.0%, 5.0%, 10.0%, 15.0%, 20.0%, or 25.0% or more).

[0054] In some embodiments, the host cells have a peak viable cell density of 3E6 cells / mL or higher. In some embodiments, the host has a peak viable cell density of 3E6-6E6, 3E6-5E6, or 5E6-6E6. In some embodiments, the host cells have a transfection efficiency of 12%, 15%, 20%, 30%, 40%, 50%, 60%, or more. In some embodiments, the host cells have a transfection efficiency of more than 30%. In some embodiments, the host cells have a transfection efficiency of 30% or more of cells containing a transgene such as green fluorescent protein.

[0055] In some embodiments, the host cells reduce macroscopic cell aggregation or aggregation, as determined by culture on day 3 or 4 after seeding.

[0056] In some embodiments, the production cells generated using host cells isolated from a parent cell line using the method herein have a difference of about 1 to 20 times (e.g., an increase or decrease) in AAV vector production titer (vg / mL) compared to the parent cell line. In one embodiment, AAV vector production may be 1E8, 1E9, or 1E10 vg / mL or higher. In some embodiments, the method includes producing an AAV titer of about 1E11 AAV vector genome / cell (vg / cell). In some embodiments, the method includes the production of about 1E9 to about 1E10 AAV (vg / mL), about 2E9 to about 6E10 vg / mL, or about 5E9 to about 6E10 vg / mL, or about 1E10 to about 6E10 vg / mL. In some embodiments, vg / mL and vg / cell can be determined by standard methods, such as, but not limited to, droplet digital PCR (ddPCR) or quantitative polymerase chain reaction (qPCR) of whole cell lysates or purified vector particles.

[0057] In some embodiments, host cells may be further selected by measuring AAV vector production titer and metabolic profiling. In some embodiments, the rate of glucose consumption or lactate production of the host cells, or both, is evaluated. In some embodiments, the rate of change in glucose consumption or lactate production of the host cells, or both, is evaluated in comparison to the parent cell line.

[0058] In some embodiments, the host cell line is grown in a selective medium.

[0059] In some embodiments, one or more nucleic acid molecules can be stably transfected into a host cell line.

[0060] In some embodiments, the selection may further include evaluating cell freeze-thaw cycles, assessing the degree of cell aggregation, or a combination thereof.

[0061] In some embodiments, the host cells are selected according to preferred characteristics described in the section and drawings of the examples.

[0062] In some embodiments, a cell line for producing rAAV particles can be prepared by a method comprising: (a) Transfect a host cell line selected according to the desired characteristics with (i) a heterologous gene, (ii) an AAV rep gene and an AAV cap gene, or (iii) one or more nucleic acids encoding at least one AAV inverted terminal repeat (ITR). The host cell line may be infected with the helper virus. (b) Select a producing cell line that produces an rAAV particle titer of at least approximately 1E9 to approximately 1E11 vg / ml. In some embodiments, after the transfection and selection process is complete, the cells are grown to a higher density (e.g., 3.5E5 cells) and subjected to further primary screening, by which rAAV titer production is measured. In some embodiments, the steps of growing the selected cells and further selecting cells for rAAV production titer can be repeated several times until the desired producing cell line is generated. In some embodiments, the rAAV titer is determined by quantitative polymerase chain reaction (qPCR).

[0063] In some embodiments, the method for generating a producible cell line includes: (a) Transfect a host cell line selected by the method described herein with (i) a heterologous gene, (ii) an AAV rep gene and an AAV cap gene, or (iii) one or more nucleic acids encoding two AAV inverted terminal repeats (ITRs). The host cell may be infected with the helper virus. (b) Select a producing cell line that produces a titer of at least approximately 1E9 to approximately 1E11 vg / ml of rAAV particles.

[0064] In some embodiments, a method for generating candidate cell lines for rAAV particle production includes the following: (a) Stably transfecting the host cell line described herein with (i) a heterologous transgene adjacent to two AAV inverted terminal repeat sequences, (ii) one or more nucleic acids encoding the AAV rep gene and the AAVcap gene in serum-free medium to produce a producible cell line, and (b) Infect a producing cell line with AAV helper virus to produce rAAV particles; if the producing cell line generates a titer of rAAV particles of at least approximately 1E9vg / mL, select the producing cell line as a candidate for rAAV particle production.

[0065] Virus particles: Certain embodiments provide a method for producing recombinant adeno-associated virus (rAAV) particles containing an rAAV genome. The rAAV may contain a xenotransgene flanked by two AAV inverted terminal repeats (ITRs) packaged in a capsid. A nucleic acid molecule can be encapsulated in the AAV particle. The AAV particle may also contain a capsid protein. In some embodiments, the nucleic acid molecule includes a regulatory sequence comprising a transcription start sequence and a termination sequence, which are components in which the coding sequence of interest (e.g., a xenotransgene) is operatively linked in the transcription direction, thereby forming an expression cassette. The expression cassette may have two functional AAV ITR sequences flanked at its 5' and 3' ends. “Functional AAV ITR sequences” means that the ITR sequences function as intended for the rescue, replication, and packaging of the AAV particle. See Davidson et al., PNAS, 2000, 97(7)3428-32; Passini et al., J. Virol, 2003, 77(12):7034-40; and Pechan et al., Gene Ther., 2009, 16:10-16 (all of which are incorporated herein by reference in their entirety). To carry out several embodiments, the recombinant vector contains at least the AAV sequence essential for capsid formation and all the physical structures for infection by rAAV. The AAV ITR for use in the vector does not need to have a wild-type nucleotide sequence (as described, e.g., Kotin, Hum. Gene Ther., 1994, 5:793-801), and can be modified by nucleotide insertion, deletion, or substitution, or the AAV ITR may be derived from any of several AAV serotypes. Currently, more than 40 AAV serotypes are known, and new serotypes and variants of existing serotypes continue to be identified. See Gao et al., PNAS, 2002, 99(18):11854-6; Gao et al., PNAS, 2003, 100(10):6081-6; and Bossis et al., J. Virol., 2003, 77(12):6799-810. The use of any AAV serotype is considered to be within the scope of this application.In some embodiments, the rAAV vector is a vector derived from an AAV serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV. For example, in some embodiments, the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10. In some embodiments, the nucleic acid molecules in AAV ITRs include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV serotype ITRs. In certain embodiments, the nucleic acid molecules in AAV include AAV2 ITRs.In a further embodiment, the rAAV particles include AAVl capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid (e.g., wild-type AAV6 capsid, or variant AAV6 capsid such as ShHIO as described in U.S. Patent Application Publication No. 2012 / 0164106), AAV7 capsid, AAV8 capsid, AAVrh8 capsid, and AAVrh8 R capsid, AAV9 capsid (e.g., wild-type AAV9 capsid, or modified AAV9 capsid as described in U.S. Patent Application Publication No. 2013 / 0323226), AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, tyrosine capsid variant, heparin-binding capsid variant, AAV2R471A capsid, AAVAAV2 / 2-7m8 capsid, AAV This may include DJ capsids (e.g., AAV-DJ / 8 capsid, AAV-DJ / 9 capsid, or any other capsid described in U.S. Patent Application Publication No. 2012 / 0066783), AAV2 N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2 / HBoVl capsid, or AAV capsids described in U.S. Patent No. 8,283,151 or International Publication No. 2003 / 042397. In some embodiments, the mutant capsid protein retains the ability to form AAV capsids. In some embodiments, the rAAV particles contain the AAV5 tyrosine mutant capsid (Zhong L. et al., (2008) Proc Natl Acad Sci USA 105(22):7827-7832). In further embodiments, the rAAV particles contain the capsid protein of an AAV serotype derived from clades A-F (Gao, et al., J. Virol. 2004, 78(12):6381). In some embodiments, the rAAV particles contain the AAVl capsid protein or a variant thereof. In other embodiments, the rAAV particles contain the AAV2 capsid protein or a variant thereof.In some embodiments, the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10. In some embodiments, the rAAV particle contains an AAV serotype 1 (AAVl) capsid. In some embodiments, the rAAV particle contains an AAV serotype 2 (AAV2) capsid. In some embodiments, the rAAV particle contains an AAVrh8R capsid or a variant thereof.

[0066] Xenotransferred genes In some embodiments, the viral particle is a recombinant AAV particle containing a heterologous nucleic acid molecule (e.g., a xenotransgene) adjacent to two AAV inverted terminal repeats (ITRs). The heterologous nucleic acid molecule can be encapsulated within the AAV particle. In some embodiments, the rAAV genome of this disclosure contains one or more AAV inverted terminal repeats (ITRs) and a xenotransgene. For example, in some embodiments, the rAAV genome of this disclosure contains two AAV inverted terminal repeats (ITRs). In certain embodiments, the rAAV genome of this disclosure contains two AAV inverted terminal repeats (ITRs) and a xenotransgene. In some embodiments, the vector genome is approximately 4.7 kb to approximately 10 kb. In some embodiments, the vector genome is greater than approximately 5 kb. In some embodiments, the vector genome is approximately 5 kb to approximately 7 kb, approximately 4.7 kb to approximately 9.4 kb, or approximately 4.7 kb to approximately 6.7 kb, or any value in between. In some embodiments, the vector genome is approximately 5.0kb, 5.1kb, 5.2kb, 5.3kb, 5.4kb, 5.5kb, 5.6kb, 5.7kb, 5.8kb, 5.9kb, 6.0kb, 6.1kb, 6.2kb, 6.3kb, 6.4kb, 6.5kb, 6.6kb, 6.7kb, 6.8kb, 6.9kb, 7.0kb, 7.1kb, 7.2kb, It is greater than or any of the following values: 7.3kb, 7.4kb, 7.5kb, 7.6kb, 7.7kb, 7.8kb, 7.9kb, 8.0kb, 8.1kb, 8.2kb, 8.3kb, 8.4kb, 8.5kb, 8.6kb, 8.7kb, 8.8kb, 8.9kb, 9.0kb, 9.2kb, 9.3kb, 9.4kb or any value between them.

[0067] In some embodiments, a xenotransgene encodes a therapeutic transgene product. In some embodiments, the therapeutic transgene product is a therapeutic polypeptide. Therapeutic polypeptides can, for example, supply polypeptide and / or enzyme activity that is absent or present at reduced levels in a cell or organism. Alternatively, therapeutic polypeptides can supply polypeptide and / or enzyme activity that indirectly counteract imbalances in a cell or organism. For example, a therapeutic polypeptide for a disorder associated with the accumulation of metabolites caused by a deficiency of a metabolic enzyme or activity can supply the deficient metabolic enzyme or activity, or supply an alternative metabolic enzyme or activity that results in a reduction of the metabolite. Therapeutic polypeptides can, for example, be used to reduce the activity of a polypeptide (e.g., one that is overexpressed, activated by a gain-of-function mutation, or whose activity is otherwise misregulated) by acting as a dominant-negative polypeptide.

[0068] In some embodiments, the therapeutic transgene product is a therapeutic nucleic acid molecule. In some embodiments, the therapeutic nucleic acid molecule may be, but is not limited to, siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozyme, or DNAzyme. Thus, the therapeutic nucleic acid molecule may encode an RNA molecule that, when transcribed from the nucleic acid molecule of the vector, can treat a disorder by interfering with the translation or transcription of abnormal or excess proteins associated with the disorder. For example, a xenotransgene may encode an RNA molecule that treats a disorder by highly specific removal or reduction of mRNA encoding abnormal and / or excess proteins. Examples of therapeutic RNA sequences that can treat a disorder by highly specific removal or reduction of mRNA encoding abnormal and / or excess proteins include RNAi, low-inhibitory RNA (siRNA), microRNA (miRNA), and / or ribozymes (such as hammerhead and hairpin ribozymes).

[0069] One embodiment provides a method for producing recombinant adeno-associated virus (rAAV) particles containing a recombinant AAV genome using a producing cell line. Several embodiments provide a method for generating rAAV. Generating rAAV may include (a) transfecting a selected host cell (e.g., a host cell produced by the method described herein) with nucleic acid molecules encoding AAV rep and cap genes and nucleic acid molecules encoding transgenes adjacent to two ITRs. This results in a producing cell line. rAAV can then be produced by infecting the producing cell line with an AAV helper function (e.g., Ad5 helper virus or other suitable helper virus) to generate rAAV. In some embodiments, the rAAV genome is about 4.7 kb to about 10 kb. In other embodiments, the AAV genome is about 4.7 kb to 5.1 kb.

[0070] In some embodiments, the nucleic acid molecule encoding the AAV cap is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, goat AAV, AAV1 / AAV2 chimera, bovine AAV, or mouse AAV capsid rAAV2 / HBoV1, or variants thereof. In some embodiments, the AAV helper is an AAV helper virus or vector. In some embodiments, the host cell is infected with the helper virus. In some embodiments, the helper virus is an adenovirus, herpes simplex virus, vaccinia virus, or cytomegalovirus.

[0071] In some embodiments, the helper is adenovirus type 5 (Ad5) of subgroup C. In some embodiments, the Ad5 helper uses CAR receptors for uptake into cells.

[0072] Adenovirus helper (Ad) is a small, non-enveloped virus. The Ad genome encodes approximately 39 genes, which are classified as either early or late depending on whether they are expressed before or after DNA replication. Helper function for AAV expression is provided by the early Ad transcription unit, which encodes proteins ElA, ElB, E2A, and E4, as well as the transcription unit VA RNA. The major late proteins are organized into transcription units L1-L5.

[0073] In certain embodiments, the helper nucleic acid molecule or virus comprises an Ad nucleic acid molecule. The helper nucleic acid molecule comprises genes E1A, E1B, E2, E3, and E4, and may encode proteins IX and IVa2, regions L1-L5, and virus-associated (VA) RNAI and VA RNAII, or fragments thereof. The Ad helper nucleic acid molecule may be derived from any hAd type, such as Ad5 or Ad2. In certain embodiments, the Ad nucleic acid molecule comprises the Ad5 gene. In certain aspects, the helper nucleic acid molecule or virus may encode one or more of Ad E4, E2A, VA RNA, or fragments thereof. Adenovirus helper genes may be present in the vector, in the helper adenovirus, or may be incorporated into the cell genome.

[0074] In some embodiments, the producing cell line contains stably maintained nucleic acid molecules encoding the AAV rep and cap genes. In some embodiments, the AAV replication and / or capsid genes are stably maintained in the producing cell line. In some embodiments, an AAV vector genome containing one or more, e.g., two AAV ITRs and heterologous nucleic acid molecules (e.g., xenotransgenes) is stably maintained in the producing cell line. In some embodiments, the AAV replication and / or capsid genes, as well as an AAV vector genome containing one or more, e.g., two AAV ITRs and heterologous nucleic acid molecules (e.g., xenotransgenes), are stably maintained in the producing cell line. In some embodiments, one or more of the AAV replication genes, capsid genes, or AAV vector genomes containing one or more, e.g., two AAV ITRs, are stably incorporated into the genome of the producing cell line. The stably maintained nucleic acid molecules are maintained in the producing cell line for multiple passages (e.g., 5, 10, 15, 25, or more passages).

[0075] In some embodiments, an AAV vector genome containing one or more AAV ITRs, for example two, and heterologous nucleic acid molecules (e.g., xenogenes) is transiently transfected in host cells. In some embodiments, an AAV vector genome containing one or more AAV ITRs, for example two, and heterologous nucleic acid molecules (e.g., xenogenes) is stably transfected in host cell lines.

[0076] In some embodiments, recombinant AAV (rAAV) particles are produced by a method comprising: a) Culturing a host cell line under conditions that produce rAAV particles, wherein the AAV-producing cell line i) Nucleic acid molecules encoding AAV rep and cap, which are incorporated into the genome of AAV-producing cell lines. ii) A nucleic acid molecule encoding a heterologous transgene adjacent to an inverted terminal repeat (ITR), and iii) Nucleic acid molecules encoding selectable markers Including; b) To provide AAV helper virus or derivatives thereof; and c) To recover rAAV particles.

[0077] The compositions and methods are described more specifically below, and the examples described herein are intended to be illustrative only, as numerous modifications and variations will be apparent to those skilled in the art. The terms used herein generally have the ordinary meanings in the art within the context of the compositions and methods described herein and in the specific context in which each term is used. Some terms are defined more specifically herein to provide additional guidance to practitioners regarding the description of the compositions and methods.

[0078] As used herein, the term "and / or" includes any combination of one or more of the enumerated items relating to it. As used herein and throughout the claims, the meanings of "a," "an," and "the" include both plural and singular references unless the context explicitly indicates otherwise. As used in relation to a number, the term "about" means that the value can fluctuate by 5% above or below it. For example, a value of about 100 means between 95 and 105 (or any value between 95 and 105).

[0079] All patents, patent applications, and other scientific or technical documents referenced in any part of this Spec. are incorporated herein by reference in their entirety. The embodiments described herein as exemplary may be well practiced in the absence of any elements or elements, limitations or restrictions, whether or not they are specifically disclosed herein. Accordingly, for example, in each case herein, any of the terms “including,” “essentially consisting of,” and “consisting of” may be replaced with any of the other two terms, while retaining their ordinary meanings. The terms and expressions used are not limiting and are used as descriptive terms, and in the use of such terms and expressions there is no intention to exclude any equivalents of any or any part thereof of the features shown and described, but it should be recognized that various modifications are possible within the claims. Accordingly, although the methods and compositions are specifically disclosed by embodiments and optional features, it should be understood that modifications and variations of the concepts disclosed herein are practicable by those skilled in the art, and such modifications and variations are considered to be within the scope of compositions and methods as defined herein and in the appended claims.

[0080] Any single term, single element, single phrase, group of terms, group of phrases, or group of elements described herein may be specifically excluded from the claims.

[0081] Where a range is described herein, for example, a temperature range, a time range, a composition or concentration range, all intermediate and partial ranges, and all individual values ​​within a given range are intended to be included in this disclosure. Any partial range or individual value within a range or partial range described herein may be excluded from the embodiments described herein. Any element or step described herein may be excluded from the claimed composition or method.

[0082] In addition, if the features or aspects of a composition and method are described in terms of the Markush group or other group of options, a person skilled in the art will recognize that the composition and method are also described in terms of the individual components or subgroups of components of the Markush group or other groups.

[0083] The following are provided for illustrative purposes only and are not intended to limit the scope of the embodiments described in the broad terminology above. [Examples]

[0084] Example 1: Generation of host cells derived from the HeLaS3 parent cell line To induce improved host cell lines, the methodology shown in Figure 1 was established. First, the HeLaS3 parent strain was subcloned into 96-well plates to achieve a seeding density of less than one cell per well. For wells with clone-derived cell proliferation, the cells were expanded into 24-well plates, then 6-well plates, and then shaking flasks. Each subclone was cryopreserved in multiple cryovials. The subclones were thawed and screened for freeze-thaw tolerance (including low aggregation) and cell proliferation characteristics, yielding 54 lead candidate subclones for further evaluation. The schematic diagram in Figure 2 summarizes some of the desirable cellular attributes present in the improved host cell lines.

[0085] Using a screening method, 54 different candidate cell populations were generated by subcloning of the HeLaS3 parental cell line. These populations were then narrowed down to 23 candidate cell populations based on parameters such as freeze-thaw tolerance, population doubling time, transfection efficiency, degree of cell aggregation, and peak VCD. These 23 candidate cell populations were then evaluated in a secondary screening by culturing them in an automated bioreactor system (shear stress test), which further narrowed the field to 5 candidate cell populations. Finally, the remaining 5 candidate cell populations were stably transfected with components necessary for rAAV production as described herein, and then infected with helper viruses, and their ability to produce high viral titers was evaluated.

[0086] conclusion From the remaining five candidate cell populations, the remaining top candidate cell populations were cultured and frozen individually. These top candidate cell populations were selected because their host cell lines were more suitable for large-scale rAAV production and for cGMP process development than the HeLa parent cell line. Phenotypic profiling of each host cell line can be established by the screening method described herein and can be supplemented by genome sequencing analysis.

[0087] Example 2 - Initial screening to identify improved host cell lines Introduction As described in Example 1, an improved host cell line was identified using a screening approach. This example focuses on the results obtained from an initial screening process performed on 32 of the candidate cell populations. Relative production screening included evaluation of (i) freeze-thaw tolerance, (ii) cell doubling time, (iii) transfection efficiency, and (iv) peak viable cell density. All cell culture media used for these evaluations were serum-free.

[0088] result Freeze-thaw tolerance (FTT) was evaluated by thawing 32 frozen candidate cell populations, growing them in liquid culture, and assessing their viability after two passages. A schematic of the FTT experiment setup is shown in Figure 4A. As shown in Figure 4B, the majority of the candidate cell populations showed favorable freeze-thaw tolerance with cell viability exceeding 90%.

[0089] Population doubling time (PDT) was evaluated by monitoring the rate at which the candidate cell population doubled over approximately 30 hours. A schematic of the PDT experiment setup is shown in Figure 5A. As shown in Figure 5B, the PDT rate was similar to or slightly lower than that of the parental HeLa cell line.

[0090] Transfection efficiency was evaluated by transiently transfecting candidate cell populations with plasmids containing a fluorescent reporter (GFP). A schematic of the transfection efficiency experiment setup is shown in Figure 6A. As shown in Figure 6B, the rate of transfection efficiency, expressed as the percentage of GFP-positive cells, varied, indicating that approximately half of the candidate populations had a reduced number of GFP-positive cells compared to the parental HeLa S2 cell population.

[0091] Peak viable cell density (VCD) was measured over time for the candidate cell populations. A schematic of the VCD experiment setup is shown in Figure 7A. As shown in Figure 7B, the viable cell densities of the candidate populations differed significantly not only from those of the parental HeLa cell line but also from those of the parental HeLa cell line. Table 1 shows the subclone IDs of the variants shown in Figures 4B, 5B, 6B, and 7B.

[0092] [Table 1]

[0093] Principal component analysis (PCA) was performed to determine which of the above evaluations showed the most variation. Exemplary PCA biplots of the results for all 32 candidate populations (represented by individual points) are shown in Figure 8A. As shown in Figure 8B, the score contribution of transfection efficiency and the peak VCD evaluation had the most sample variation.

[0094] Overall, the results from the initial cell performance assessment indicate that several evaluation parameters are necessary during initial screening, as each candidate cell population exhibited a unique sequence of phenotypic characteristics, particularly in relation to their peak VCD and transfection efficiency.

[0095] Example 3 - Screening to identify improved host cell lines Introduction In Example 2, initial cell performance evaluations narrowed the original 54 candidate cell populations down to 23. This example describes further evaluations for narrowing down the candidate cell populations. These evaluations were performed using serum-free media in strict compliance with GMP guidelines. For general information, see the article “Serum-free media: ask the experts” (published in 2022, available at regmednet.com / serum-free-media-ask-the-experts / ). In the second stage, the candidate cell populations underwent a series of assays aimed at mimicking large-scale cell culture conditions and specifically evaluating the host cell line’s ability to establish subsequent AAV-producing cell lines. Schulze et al. (2021), J. Biotechnol., vol.335:65-75.

[0096] result To evaluate the cell growth conditions and metabolic profiling of 23 candidate cell populations, they were cultured in a benchtop small-scale microbioreactor system as shown in Figure 9A. Over 7 days, 21 of the candidate cell populations were evaluated for their growth conditions (see Figure 10A) and metabolic profiling (see Figures 11A-B). As shown in Figures 10A and 10B, the majority of the candidate cell populations showed peaks in viable cell density and high cell viability, respectively. However, certain candidate cell populations, such as subclone B, did not show a VCD peak (see Figure 10A), and subclone H showed a dramatic decrease in viability by day 5 of culture (see Figure 10B). As shown in Figure 11, the glucose depletion (Figure 11A) and lactate production (Figure 11B) curves showed variability in the metabolic profiles of each candidate cell population compared to each other and to the HeLaS3 parental cell line.

[0097] Based on the results of dynamic and metabolic profiling evaluations, the top seven candidate cell populations were analyzed for their specific ability to produce AAV titers. AAV titers released into cell culture medium were collected 72 hours after viral plasmid transfection and measured for each candidate, as shown in Figure 13A. As shown in Figure 13B, the mean viral titers of each candidate cell population varied, with the cell populations labeled as subclones B and C having the highest viral titers compared to the HeLaS3 parental control.

[0098] In summary, the results suggest that screening in Ambr(registered trademark)15 may lead to a further reduction in the candidate cell population. However, since each candidate cell population has its own unique profile and can exhibit variability, it is important to consider several parameters simultaneously.

[0099] Example 4: Generation of host cells derived from the HeLaS3 parent cell line HeLaS3 parental cell lines were thawed and subcultured (in an orbital shaker platform set to 37°C, 5% CO2, 125 rpm, and orbital diameter 25 mm), and grown in a culture medium free of animal-derived components (EX-CELL® HeLa growth medium supplemented with 6 mM L-glutamine). After thawing, cells were measured at 0.2 × 10⁶. 6 The cells were subcultured twice at a density of cells / mL. In the third subculture, the cells were used to prepare (i) culture supernatant (CM); and (ii) a culture for limiting dilution cloning (LDC), called N-1 culture. CM is a medium used to promote cell proliferation.

[0100] Limiting dilution cloning (LDC) was used to generate single-cell subclones. For the LDC process, an N-1 culture was first prepared, in which 0.2 × 10⁶ HeLaS3 cells were added. 6 Cells were seeded at a density of cells / mL into 125 mL shaking flasks. Cell culture was maintained for 3 days at 37°C and 5% CO2 on an orbital shaker platform set to an orbital diameter of 25 mm and 125 RPM.

[0101] In the LDC process, cells with a viability exceeding 90% and population doubling of less than 26 hours were seeded at a density of 0.5 cells / well in 96-well plates with EX-CELL® HeLa (supplemented with 6 mM L-glutamine), 50% DMEM / F-12 (supplemented with 6 mM L-glutamine), 20% CM, and 1× InsiGRO CHO additives. This medium was serum-free and free of animal-derived cells, except for the EX-CELL® HeLa medium component (cod liver oil), which does not pose a biosafety or TSE / BSE risk.

[0102] Throughout the entire process, cells were imaged on days 0, 4, 7, and 12 to assess clonality (determining whether the cell line originated from a single cell).

[0103] If the colonies were sufficiently large and the cells appeared healthy, the cells were ready to proliferate approximately 21 days after seeding. The cells were sequentially expanded into 24-well plates and then 6-well plates, and finally transferred to 125 mL shaking flasks. For newly generated subclones, expansion into 12 mL shaking flasks was considered passage 0.

[0104] Subclones were expanded over 2-3 passages in suspension culture, and then selected clones were banked in EX-CELL® HeLa medium containing 6 mM L-glutamine and 10% DMSO. Subsequently, four frozen vials of each subclone were frozen with 10 million cells per vial. These cryopreserved and banked clones were referred to as premaster cell bank (MCB) generation.

[0105] To prepare MCBs, subclones that had been generated and cryopreserved as pre-MCBs were thawed. One vial of each subclone was amplified. The thawed clones were expanded over three passages in EX-CELL® HeLa growth medium supplemented with 6 mM L-glutamine. After the third passage, the cells were banked in EX-CELL® HeLa growth medium supplemented with 6 mM L-glutamine and 10% DMSO. 10 million cells per cryovial were frozen in a total of 14 cryovials per clone.

[0106] Example 5 - Clone selection process under serum-free conditions Based on Example 4, 61 clones were identified for further selection and characterization to ensure consistency and improve the host cell line. The 61 clones were characterized based on the process shown in Figure 15.

[0107] In short, the process was divided into a series of screening or characterization steps.

[0108] In the first screening step, Tier 1, the clones were tested for the following: Step 1.1 Characterization of the shaking flask culture system a. Freeze-thaw resistance, and b. Population doubling time (PDT) in a shaking flask culture system. Step 1.2 Characterization of the shaking flask culture system a. Freeze-thaw resistance b. PDT and maximum viable cell density (maximum VCD) c. Transfection efficiency and cell viability after nucleofection using triple-play plasmids (to produce rAAV2) Step 2: Characterization of the top 23 selected clones in a high-throughput cell culture bioreactor system known as Ambr(registered trademark)15. a. PDT and maximum VCD b.Agglutination Step 3: Characterization of the top 11 selected clones (Ambr(registered trademark)15) a. Shear stress resistance

[0109] In the second screening (Tier 2), the clones were characterized in the following respects: 1. Coxsackie adenovirus receptor (CAR) expression levels (11 clones) 2. Immune-related gene expression (7 clones)

[0110] Step 1.1 Freeze-thaw tolerance (FTT) was assessed by thawing 61 frozen candidate cell populations, growing them in liquid culture, and evaluating their viability. Photodynamic therapy (PDT) was assessed by monitoring the time rate at which the candidate cell population doubled. PDT was monitored after three passages, and the mean PDT after passages 2 and 3 was used to identify the relevant clones. Cell viability after thawing was measured by Vi-CELL.

[0111] Clones that underwent more than 32 hours of PDT, or showed cell viability after thawing of less than 70% (i.e., approximately 20% lower than the cell viability before freezing), or combinations thereof, were excluded.

[0112] Based on freeze-thaw cycles, PDT, and the characteristics shown in Table 2, 54 clones were selected, and the process proceeded to the next characterization step 1.2.

[0113] [Table 2]

[0114] Step 1.2 Characterization in a shaking flask Fifty-four frozen candidate cell populations were thawed and grown in liquid culture (EX-CELL® HeLa growth medium supplemented with 6 mM L-glutamine). Freeze-thaw tolerance (FTT) was assessed by evaluating their viability. Photodynamic therapy (PDT) was assessed by monitoring the time it took for the candidate cell population's cell concentration to double. Cells were subcultured every three days for three passages, and both VCD and cell viability were measured.

[0115] PDT was monitored after three passages, and the mean PDT after passages 2 and 3 was used to identify the relevant clones. Cell viability after thawing was measured by Vi-CELL. Cells were subcultured every 3 days for 3 passages, and both VCD and cell viability were measured.

[0116] Aggregation was another characteristic to be evaluated. Cell aggregates were visible from the first day of culture and were generally observed to disappear over several days. Therefore, 0.2 × 10⁻⁶ 6 After seeding at a cell / mL concentration, agglutination was observed visually on days 3 and 4 of culture.

[0117] Agglutination assessments of the cultures over these two consecutive days were performed by two operators, and the clones for agglutination were evaluated using the mean of all measurements (n=4). Qualitative scores (1-5) were assigned daily by each operator, where, A relative value of 1 corresponded to many aggregates compared to the parent HeLaS3. A relative value of 5 corresponds to the absence of aggregates compared to the parent HeLaS3. HeLaS3 parental cells were used as a control, and the results were classified on a relative scale of 4. The selected clones that were tested were classified on a relative scale of 2 to 5.

[0118] Cell proliferation curve and maximum VCD Growth curves were measured twice during the third passaging after cell thawing. The cells were 0.2 × 10⁶. 6 Cells were seeded at a concentration of cells / mL, and VCD and cell viability were assessed daily using Vi-CELL. PDT and maximum VCD during the exponential growth phase were calculated. Parental HeLaS3 cells were used as controls in all experiments.

[0119] Transfection efficiency Cells in the exponential growth phase were transfected with pTPK (thiamine pyrophosphokinase 1) herpes simplex virus (HSV), TK (thymidine kinase), ITR (inverted terminal repeat), CBA (chicken beta-actin promoter), and enhanced green fluorescent protein (EGFP) plasmids. 48 hours after transfection, transfection levels were measured by GFP expression using flow cytometry (BD FACSCELESTA® SORP flow cytometer) and cell viability using Vi-CELL. The experiment was repeated twice (Figure 16). In Figure 16, transfection efficiency is shown as the percentage of GFP (solid black bars) and cell viability (white bars) of 54 clones compared to HeLaS3 parent cells.

[0120] From the 54 clones (cones), 23 clones are selected based on the characteristics shown in Table 3 below, and the process proceeds to the next characterization step (Step 2).

[0121] [Table 3]

[0122] Some clones were selected to proceed to the next characterization step even though they showed less than 90% cell viability at P1 after thawing, but higher cell viability at P2.

[0123] Step 2 - Cell culture in the Ambr® 15 system Cell growth performance In Step 1.2, 23 clones with the highest performance based on the characteristics selected from Table 2 were thawed and expanded over 2 passages. HeLaS3 parental cells were used as a control. After 2 passages, the clones were seeded in Ambr® 15 under the following conditions: 1. Seeding = 0.2×10 6 cells / mL 2. pH control at 7.2±0.05 3. Constant stirring speed of 1500 rpm, bottom stirring 4. Dissolved oxygen (DO) = 50% 5. Antifoam: Foam Away diluted 1:10 and added daily

[0124] The experimental design for Step 2 is shown in Figure 17.

[0125] Clones were able to show excellent performance in some characteristics, but as shown in Table 4, a minimum performance level was required for all characteristics.

[0126]

Table 4

[0127] Some clones showed cell viability slightly below 90% at P1 after thawing, but were still selected if they could show higher cell viability at P2. Of the 23 clones, 11 clones were selected to proceed to the next characterization step (Step 3) and were tested for shear stress resistance in the Ambr® 15 system.

[0128] Step 3 - Shear stress Eleven clones with the best performance identified in Step 2 were thawed and expanded over two passages. HeLaS3 parental cells were used as a control. Shear stress resistance tests are shown in Figure 18.

[0129] After two passages, the clones were seeded into Ambr(registered trademark)15 containers and cell culture was performed according to the following initiation conditions: 1.Seeding=0.2×10 6 cells / mL 2.7.2±0.05 pH control 3. Steady-state stirring speed 1500 rpm, bottom stirring 4. Dissolved oxygen (DO) = 50% 5. Antifoaming agent: Add Foam Away diluted 1:10 daily.

[0130] On the second day, the stirring speed was increased to 1650 rpm, and then to 1800 and 2000 rpm on the third and fourth days, respectively (Table 5). On the sixth day, since some clones were still unaffected, the DO setting was changed to increase aeration and bubble formation, resulting in an increased shear rate. The calculation of the Kolmogorov vortex size is shown in Table 5.

[0131] [Table 5]

[0132] Sampling for cell number and viability determination was performed approximately 8 hours and 24 hours after changing the setpoint (agitation or DO). Therefore, the 24-hour sampling was performed just before increasing the shear stress again. Sampling was performed daily until day 2 and after day 6. The selected clones (M, Q, E, S, and P, Figure 19) were considered the top five clones.

[0133] Example 6 - Characterization of selected clones Five clones were selected for further characterization following the process described in Example 5. These clones were tested in shaking flasks for their PDT and peak VCD. The five clones were further evaluated for aggregation by testing their PDT and peak VCD in a high-throughput automated cell culture bioreactor system, Ambr® 15.

[0134] Table 6 below shows a comparison of five clones designated as clones M, R, Q, C, and E with some of their characteristics compared to the HeLa parent cell line.

[0135] [Table 6]

[0136] Other characteristics of the clones tested were their (i) transfection efficiency and viability, (nucleo-vector efficiency and viability), and (iii) the expression ratio of several immune response gene transcript levels to those of the HeLaS3 parental strain. The housekeeping gene HRPT1 was used as a control.

[0137] Figure 20 shows the characteristics of the top five clones M, R, Q, C, and E related to nucleofector efficiency and viability (Figure 20A). Growth curves for various clones are also shown: clone M (Figure 21); clone R (Figure 22); clone QM (Figure 23); clone C (Figure 24); and clone M (Figure 25).

[0138] Table 7 shows the productivity and robustness of the selected clones, as determined by their AAV titer data. Clones were more robust when the recommended AAV titer was lower.

[0139] [Table 7]

[0140] Figure 26 also shows the productivity and robustness of the selected clones at the limiting dilution cloning (LDC) stage, as determined by plate efficiency growth assessed two weeks after seeding. The lower the recommended seeding value, the more robust the clones were.

[0141] Example 7: Generation of a producing cell line Thawing and subculturing: The five clones identified in Example 6 were each stored in 1 mL of 1E7 cells in cryovials in a liquid nitrogen tank (LN2). The vials were transferred from the LN2 tank to the laboratory via filled cryopods (150°C and -180°C). The vials were thawed in a water bath set to 37°C and kept in the water bath until approximately 90% thawed, at which point ethanol was sprayed and the vials were transferred to a work hood. The contents of the vials were transferred to either a 15 mL or 50 mL conical flask, to which 9.2 mL of fresh EX-CELL® growth medium (EX-CELL® medium and L-glutamine) was added (final volume after medium addition: 10.2 mL). Cells were counted using VICELL® to determine cell density and viability (200 μL was required for counting). Next, the remaining 10 mL was spun down and resuspended in a 125 mL shake flask (SF) in a volume ranging from 10 to 40 mL at a target density of 3E5 cells / mL. The SF was maintained in a Kuhner shaking incubator at 110 RPM, 80% humidity, 5% CO2, and 37°C. Passing was performed every 3–4 days, and after completely resuspending the culture, cell density and viability counts were performed using VICEL®. Cells were subcultured at a seeding density of 2E5 cells / mL for any passage after initial thawing.

[0142] Transfection: Nucleofection (Ntx) is an electroporation-based transfection technique using the Lonza 4D Nucleofector. Nucleofection was performed 3 days after two passages following thawing. In the case of transfection, the population doubling time (PDT) was less than 26 hours, and the cell viability (CV%) was greater than 98%. Cells were transfected with specified amounts of various reagents (with DNA / buffer additives and a given cell density). Each reaction required 4E6 cells and 6 μg of plasmid DNA (diluted with LONZA nucleofection reagent) as recommended by the manufacturer. Transfection was performed according to established transfection protocols with settings appropriate for each cell type. After Ntx, cells were placed in either a static incubator (for 6-well plates) or a shaking incubator (for shaking flasks). The transfection efficiency of Ntx was evaluated 24 and 48 hours after transfection using a cytometer (CELIGO®) and the number of SF cells was calculated using VICELL®.

[0143] Plating and selection of transfected cells: 72 hours after Ntx, cell cultures were counted using VICELL®. Cells were seeded in selective medium (EX-CELL® medium, L-glutamine, and puromycin) at a rate of 2.5K cells / well in 150 μL into 96-well plates. These were placed in a static incubator at 37°C, 80% humidity, and 5% CO2 and left there until scale-up began. Every 7 days, 30 μL of fresh selective medium was added to each well. Plates were scanned weekly to determine the growth pattern, which allowed for the next steps in scale-up.

[0144] Scaling up: The process began 14–18 days after selection, with wells being scaled up from 96-well plates to 24-well plates containing 1 mL of fresh selection medium. Scans were evaluated on the day of or one day before scale-up, and wells with sufficient colony size (3D growth) were scaled up using an automated liquid handling system. Scale-up was typically performed over three rounds, or until the target number of wells to be scaled up was achieved (1000–1200 wells in total).

[0145] Primary screening: Seven days after scale-up, counting was performed on 24-well plates using CELIGO®. Wells with a cell density of 3.5E5 cells or greater were further processed for primary screening. 100,000 cells were transferred from the 24-well plates to 96-well V-bottom plates via an automated liquid handling system. After the transfer was complete, these plates were spun down at 300 g for 4 minutes. After spinning down the medium, the medium was aspirated and 200 μL of fresh infection medium containing EX-CELL® was added to the wells. The entire contents of the plates were further transferred to new 96-well plates and subsequently incubated at 37°C, 80% humidity, and 10% CO2 for 72 hours. After 72 hours, the wells were treated with TWEEN® (polysorbate) and benzonase for 1 hour and further incubated under static conditions of 37°C, 80% humidity, and 5% CO2. After incubation, 100 μL of the treated sample was transferred to a new 96-well plate and further processed for viral particle or vector production titer using qPCR (as reported in Marin 2013). Table 8 below shows the titers of the selected clones in the primary screening, with viral production titers close to 1E10.

[0146] Scaling up results and secondary screening: Once qPCR data became available, top-producing cells were selected from each screening round and scaled up from 24 wp to T25 culture flasks. As part of the scale-up process, cells were transferred from T25 flasks to SF. Cultures showing PDT of ≤40 hours and ≥90% CV% were banked and processed in three consecutive passage rounds (5E6 cells in 10 mL of infection medium, under shaking conditions) for secondary screening. Samples in shaking flasks were analyzed for virus production by qPCR. Data for secondary screening were reviewed, and overall, the performance of each candidate was evaluated across one primary screening and three secondary screenings.

[0147] Table 8 shows the results of secondary screening, where titer improved from primary screening to secondary screening. For example, for clone M, the highest titer in primary screening was approximately 1E10, and the highest average titer observed during secondary screening was 3E10. Similarly, for clone C, the highest titer in primary screening was approximately 4E10, and the highest average titer observed during secondary screening was 1.1E11.

[0148] [Table 8]

Claims

1. Host cells derived from the HeLaS3 parent cell line.

2. The host cell according to claim 1, wherein the host cell has a difference of about 0.5% to about 25% in cell doubling time (in hours) compared to the parent cell line.

3. The host cell according to claim 1 or 2, wherein the host cell has a transfection efficiency of about 0.5% to about 25% compared to the parent cell line.

4. The host cell according to any one of claims 1 to 3, wherein the host cell has a difference of about 0.5% to about 25% in peak viable cell density compared to the parent cell line.

5. The host cell according to any one of claims 1 to 4, wherein the host cell has a difference of about 0.5% to about 10% in the percentage of cell viability after one cell freeze-thaw cycle compared to the parent cell line.

6. The host cell according to any one of claims 1 to 5, wherein the host cell is transfected with one or more nucleic acid molecules encoding heterologous transgenes adjacent to AAV ITR, AAV rep, and AAV cap, and has an adeno-associated virus (AAV) vector production titer (vg / mL) that is about 1 to 20 times lower than that of the parent cell line.

7. A method for selecting a host cell line according to any one of claims 1 to 6, (a) Growing one or more populations of the HeLaS3 parent cell line in serum-free medium; (b) Select and isolate one or more single-cell clones from step (a); (c) Growing each of the one or more single-cell clones from step (b) in serum-free medium; (d) Select from each of the one or more single-cell clones from step (c) and give the one or more single-cell clones the following characteristics: (i) cell doubling time; (ii) Transfection efficiency, (iii) Peak live cell density; (iv) Cell proliferation after seeding at low cell density; and (iv) Any combination of them To analyze at least one of the following; and (e) Growing one or more single-cell clones selected from step (d) in serum-free medium; This will allow you to obtain the aforementioned host cell line; Methods that include...

8. The method according to claim 7, wherein step (d) further comprises evaluating the recovery from the cell freeze-thaw cycle.

9. The method according to any one of claims 7 to 8, wherein step (d) further comprises evaluating the degree of cell aggregation.

10. The method according to any one of claims 7 to 9, wherein step (d) further includes selection based on principal component analysis.

11. The method according to any one of claims 7 to 10, further comprising step (d) evaluating the metabolic profile.

12. The method according to claim 11, wherein evaluating the metabolic profile includes measuring glucose or glutamine depletion or lactate secretion or any combination thereof in serum-free medium for 1 to 7 days.

13. The method according to any one of claims 7 to 12, wherein one or more single-cell clones from step (d) have a difference of about 0.5% to about 25% in cell doubling time (hours) compared to the parent cell line.

14. The method according to any one of claims 7 to 13, wherein one or more single-cell clones from step (d) have a difference of about 0.5% to about 25% in peak viable cell density compared to the parent cell line.

15. The method according to any one of claims 7 to 14, wherein one or more single-cell clones from step (d) have a difference of about 0.5% to about 25% in the percentage of cell viability after one cell freeze-thaw cycle compared to the parent cell line.

16. The method according to any one of claims 7 to 15, wherein one or more single-cell clones from step (c) have a transfection efficiency of about 0.5% to about 25% compared to the parent cell line.

17. The method according to any one of claims 7 to 16, wherein when one or more single-cell clones from step (d) are transfected with one or more nucleic acid molecules encoding heterologous transgenes adjacent to AAV ITR, AAV rep, and AAV cap, and infected with a helper virus, they have a difference of about 1 to 20 times in AAV vector production titer (vg / mL) compared to the parent cell line.

18. The method according to any one of claims 7 to 17, wherein the host cell line can be grown in a suspension.

19. Recombinant AAV (rAAV) particles produced by the following method: a) Transfecting a host cell according to any one of claims 1 to 6 or a host cell line selected using the method according to any one of claims 7 to 18, under conditions that generate the rAAV particles, wherein the host cell line is transfected with one or more nucleic acid molecules encoding a heterologous transgene adjacent to an inverted terminal repeat (ITR), AAVrep, AAVcap, and optionally selectable markers; and growing one or more populations from the transfected parent cell line; b) Infecting one or more of the aforementioned populations with the AAV helper virus or its derivatives; and c) Recover the rAAV particles.

20. The rAAV particle according to claim 19, wherein the nucleic acid molecule encoding AAV cap is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, goat AAV, AAV1 / AAV2 chimera, bovine AAV, or mouse AAV capsid rAAV2 / HBoV1, or a variant thereof.

21. The rAAV particle according to any one of claims 19 to 20, wherein the AAV helper virus is Ad5.

22. The rAAV particle according to any one of claims 19 to 21, wherein the host cell line is infected with a helper virus.

23. The rAAV particle according to claim 22, wherein the helper virus is an adenovirus, herpes simplex virus, vaccinia virus, or cytomegalovirus.

24. The rAAV particle according to claim 23, wherein the adenovirus is human adenovirus 5.

25. The rAAV particles according to any one of claims 19 to 24, wherein the transfected host cell line is recovered and the AAV particles are recovered.

26. The rAAV particle according to any one of claims 19 to 25, wherein one or more nucleic acid molecules are stably transfected into the host cell line.

27. A method for generating candidate cell lines for the production of recombinant AAV (rAAV) particles, (a) Stably transfecting a host cell line according to any one of claims 1 to 6 in serum-free medium, or a host cell line selected by the method according to any one of claims 7 to 18 in serum-free medium with (i) one or more nucleic acids encoding a heterologous transgene adjacent to two AAV inverted terminal repeat sequences, (ii) an AAV rep gene and an AAVcap gene, to produce a producible cell line, and (b) Infect the producing cell line with AAV helper virus to produce rAAV particles; if the producing cell line generates a titer of rAAV particles of at least about 1 E9 vg / mL, select the producing cell line as a candidate for rAAV particle production. Methods that include...

28. The method according to claim 27, further comprising step (d) growing the producing cell line of step (b) to a cell density of 3.5E5 or more; and step (c) selecting the producing cell line as a candidate for rAAV particle production if the producing cell line produces a titer of at least about 1E9 vg / mL of rAAV particles.

29. The method according to claim 28, further comprising step (e) of growing the cells of step (d) to a cell density of 3.5E5 or more, and selecting the producing cell line as a candidate for rAAV particle production if the producing cell line produces a titer of at least about 1E10 vg / mL of rAAV particles.

30. The method according to any one of claims 27 to 29, wherein the rAAV titer is determined by quantitative polymerase chain reaction (qPCR).

31. The method according to claim 7, wherein the cell viability rate or cell viability density is determined by freeze-thaw cycles, shear stress, or a combination thereof.

32. The method according to claim 7, comprising selecting cells having a cell viability rate of 70% or more compared to the HeLaS3 parent cell line.

33. The method according to claim 7, comprising selecting cells having a cell viability rate of 90% or more compared to the HeLaS3 parent cell line.

34. The method according to claim 7, comprising selecting cells having a cell doubling time of 32 hours or less in viability compared to the HeLaS3 parent cell line.

35. The aforementioned peak viable cell density was 3 × 10⁻⁶ compared to the HeLaS3 parent cell line. ６ The method according to claim 7, comprising selecting cells having a peak live cell density of cells / mL or more.

36. The method according to claim 7, wherein the transfection efficiency is such that the cells have 30% or more transfected cells compared to the HeLaS3 parent cell line.

37. The method according to claim 7, wherein the aggregation is reduced in cells with reduced macroscopic cell aggregation compared to the HeLaS3 parent cell line.

38. The method according to claim 7, wherein the host cell line is a mammalian host cell.

39. The method according to claim 7, wherein the host cell is a HeLa3 cell line.

40. The method according to claim 7 or claim 27, wherein the serum-free culture medium does not contain any animal-derived components.

41. The method according to claim 7 or claim 27, wherein the serum-free medium includes a medium supplemented with glutamine.

42. The method according to claim 41, wherein the culture medium is supplemented with approximately 6 mM glutamine.

43. A method for producing a host cell line, (a) Growing one or more populations of HeLaS3 parental cell lines at a density of 0.5 cells / well in a medium comprising EX-CELL HeLa growth medium supplemented with 6 mM L-glutamine, 50% DMEM / F-12 (supplemented with 6 mM L-glutamine), 20% culture supernatant, and 1x InstiGRO CHO supplement (all components of the medium and culture supernatant are serum-free and free of animal-derived components); (b) A step of selecting and isolating one or more single-cell clones from step (a); (c) A step of growing each of the one or more single-cell clones from step (b) in serum-free medium; (d) From each of the one or more single-cell clones from step (c), the following characteristics: (i) Cell viability; (ii) cell doubling time; (iii) Transfection efficiency, (iv) Peak live cell density; (v) agglomeration; (vi) Cell proliferation after seeding at low cell density; and (vii) Any combination of them A step of selecting at least one of the following; and (e) A method comprising the step of isolating a single cell selected from step (d) in serum-free medium and growing it to produce the host cell line.

44. A method for generating a producible cell line, (a) transfecting a host cell line according to any one of claims 1 to 6, or a host cell line selected by the method according to any one of claims 7 or 43, with one or more nucleic acids encoding (i) a xenotransgene adjacent to two AAV inverted terminal repeat sequences, (ii) an AAV rep gene and an AAV cap gene, (iii) an AAV helper gene; and (b) step of selecting a producing cell line which produces a titer of rAAV particles of at least about 1E9 to about 1E11 vg / mL Methods that include...

45. A method for producing recombinant adeno-associated virus (rAAV), (a) transfecting a host cell line according to any one of claims 1 to 6, or a host cell line produced by the method of claim 7 or 43, with (i) a heterogene, (ii) an AAV rep gene and an AAV cap gene, and (iii) one or more nucleic acids encoding an AAV inverted terminal repeat (ITR); (b) the step of infecting the host cells with a helper virus; and (b) A step of isolating the rAAV particles, wherein the titer of the rAAV particles produced is at least about 1E9vg / mL. Methods that include...

46. The method according to claim 45, wherein the titer of the rAAV particles is about 1E9 vg / mL to about 1E11 vg / mL.

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