Method for generating recombinant AAV particles

Abstract

A method for generating recombinant AAV particles is reported herein, which includes the steps of growing mammalian cells using perfusion until at least a first predetermined cell density is obtained; diluting an aliquot of the grown cells by adding fresh culture medium to obtain a production cell solution having a second predetermined cell density; culturing the production cell solution for 1 to 36 hours; directly transfecting one or more nucleic acids encoding recombinant AAV particles into the cells in the cultured production cell solution; and culturing the transfected production cell solution for 24 to 144 hours to thereby generate recombinant AAV particles.

Description

Technical Field

[0001] The present invention belongs to the field of gene therapy. More precisely, the present specification reports a method for generating recombinant AAV particles, in which cells are cultured using perfusion prior to transfection, transiently produce recombinant AAV particles, and the production is carried out without using perfusion.

Background Art

[0002] Background Gene therapy, in a broad sense, refers to the therapeutic administration of genetic material to modify gene expression in living cells and thereby change their biological properties. After decades of research, gene therapy has advanced into the market and is expected to become increasingly important. Generally, gene therapy can be divided into either in vivo or ex vivo approaches.

[0003] Today, most in vivo therapies rely on DNA delivery by recombinant adeno-associated virus (rAAV) vectors. AAV is a small naturally occurring non-pathogenic parvovirus, which is composed of a non-enveloped icosahedral capsid. It contains a linear single-stranded DNA genome of approximately 4.7 kb. The genome of wild-type AAV vectors carries two genes, rep and cap, adjacent to the inverted terminal repeats (ITRs). The ITRs are cis-required for viral replication and packaging. The rep gene encodes four different proteins, and its expression is driven by two alternative promoters, P5 and P19. Furthermore, different forms are generated by alternative splicing. Rep proteins have multiple functions such as, for example, DNA binding, endonuclease and helicase activities. They play roles in gene regulation, site-specific integration, excision, replication and packaging. The cap gene encodes three capsid proteins and one assembly activation protein. Differential expression of these proteins is achieved by alternative splicing and alternative start codon usage and is driven by a single promoter, P40, located in the coding region of the rep gene.

[0004] In engineered therapeutic rAAV vectors, the viral genes remain adjacent to the viral ITRs but are replaced by a transgene expression cassette encoding the gene of interest under the control of a selected promoter. Unlike wild-type viruses, engineered rAAV vectors do not undergo site-specific integration into the host genome and instead remain mainly episomal in the nuclei of transduced cells.

[0005] AAV is not itself replication-competent and requires the function of helper genes. These are naturally provided, for example, by co-infecting helper viruses such as adenovirus or herpes simplex virus. For example, five adenoviral genes, namely, E1A, E1B, E2A, E4 and VA, are known to be essential for AAV replication. In contrast to other helper genes that encode proteins, VA is a small RNA gene.

[0006] For the production of rAAV vectors, DNA carrying a transgene adjacent to the ITR is introduced into a packaging host cell line that also contains the rep gene, the cap gene, and the necessary helper genes. There are many ways to introduce these three groups of DNA elements into cells and ways to combine them on different DNA plasmids (see, for example, Robert, M.A., et al. Biotechnol. J. 12 (2017) 1600193 (Non-Patent Document 1)).

[0007] Two common production methods are widely used. In the triple transfection method, an adenoviral helper plasmid carrying the necessary adenoviral helper genes is transiently co-transfected into a plasmid containing rep / cap and a plasmid containing the rAAV transgene. This process can be carried out using CHO or HEK cells. Alternatively, the rep / cap gene and the viral helper genes can be combined on one large plasmid (double transfection method). The second method involves the infection of insect cells (Sf9) with two baculoviruses, one carrying the rAAV genome and the other carrying rep and cap. In this system, the helper function is provided by the baculovirus plasmid itself. Similarly, herpes simplex virus is used in combination with HEK293 or BHK cells. More recently, Mietzsch et al. (Hum. Gene Ther. 25 (2014) 212 - 222 (Non-Patent Document 2); Hum. Gene Ther. Methods 28 (2017) 15 - 22 (Non-Patent Document 3)) engineered Sf9 cells in which rep and cap were stably integrated into the genome. In these cells, a single baculovirus with the rAAV transgene is sufficient to produce the rAAV vector. Clark et al. (Hum. Gene Ther. 6 (1995) 1329 - 1341) (Non-Patent Document 4) generated a HeLa cell line in which the rep / cap gene and the rAAV transgene were integrated into its genome. By transfecting the cells with wild-type adenovirus, the production of the rAAV vector is induced, generating a mixed stock of the rAAV vector and adenovirus.

[0008] Perfusion culture has been reported to be applicable to the production of classical recombinant proteins (see Woodgate, J.N., in “Biopharmaceutical Processing: Development, Design and Implementation of Manufacturing Processes” (2018), pages 755 - 768 (Non - Patent Document 5)). In the case of mammalian cell lines in such classical recombinant protein production, cell culture takes, on average, 10 - 14 days for stable recombinant protein products, where the production bioreactor is at the end of a long train of 1000L, 100L, 10L, and 1L scale seed bioreactors for generating a sufficient starting number of cells to inoculate the production bioreactor. Cells are continuously diluted with fresh cell culture medium before reaching the mid - exponential growth phase. This ensures that cells can be maintained in exponential growth as they are not exposed to nutrient limitation (e.g., when growth - related amino acids are limiting) or a toxic environment (e.g., when metabolic by - products such as lactate and ammonia accumulate at high levels).

[0009] Yang, W.C., et al. (Biotechnol. Prog. 30 (2014) 616 - 625) (Non - Patent Document 6) demonstrated a proof - of - concept of alternating tangential flow (ATF) perfusion seed culture, which is later used in a standard fed - batch process, by using a high - cell - density seed culture (400×10 5 cells / mL) to inoculate the production bioreactor at a much higher concentration (standard 100×10 5 cells / mL). They completed this study using two CHO cell lines that express monoclonal antibodies, both achieving a typical 5 g / L productivity titer in 12 days instead of 17 days, and thereby increasing the manufacturing capacity by 30% while maintaining (and in one case improving) the product quality profile.

[0010] U.S. Patent No. 6,566,118 (Patent Document 1) reports a method and composition for generating a substantially purified preparation of recombinant adeno-associated virus (AAV) at high titer that can be used as a vector for gene delivery.

[0011] International Publication No. 2020 / 154607 (Patent Document 2) reports a method for generating adeno-associated virus (AAV) that includes culturing an AAV-producing cell line in a seed culture and subsequently performing an AAV production culture.

Prior Art Documents

Patent Documents

[0012]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0013]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

[0014] Disclosed herein is a method for generating recombinant AAV particles using mammalian cells, wherein the mammalian cells are i) grown in perfusion - based culture prior to producing AAV particles, ii) diluted / spread to a lower cell density after growth, iii) cultured without using perfusion after transfection with the nucleic acids required for the production of recombinant AAV particles. The method is reported.

[0015] The present invention is based, at least in part, on the finding that it is advantageous to grow mammalian cells for the production of recombinant AAV particles in perfusion culture for at least a certain period of time before, and more specifically before, transfection with the nucleic acids required for the production of recombinant AAV particles, and then, but before transfection with the nucleic acids required for the production of recombinant AAV particles, to split / dilute the cells in fresh medium.

[0016] By using mammalian cells grown according to the present invention for the production of recombinant AAV particles, the viable cell density in the production culture was found to be higher compared to a production culture using cells grown with fed-batch only starting from the same inoculum cell density. The cells further proliferate during a short period after transfection, in other words, the cell density increases after transfection. Without being bound by this theory, this is presumably the result of a better metabolic state of the cells resulting from the process using N-1 perfusion. As a result, the cells can better resist the stress caused by transfection, resulting in a higher yield of recombinant AAV particles.

[0017] In one aspect, a method for generating recombinant AAV particles comprises the following steps: (a) growing mammalian cells using perfusion until at least a first predetermined cell density is obtained / achieved; (b) diluting an aliquot / fraction of the cells obtained in step (a) by adding unused / fresh culture medium to obtain a production cell solution having a second predetermined cell density; (c) culturing the production cell solution for 1 to 36 hours; (d) directly transfecting the cells with one or more nucleic acids encoding recombinant AAV particles in the cultured production cell solution obtained in step (c); and (e) culturing the transfected production cell solution obtained in step (d) for 24 to 96 hours to thereby generate recombinant AAV particles.

[0018] The present invention encompasses the following independent aspects and dependent embodiments. 1. A method for generating recombinant AAV particles, comprising: a) growing mammalian cells using perfusion until at least a first predetermined cell density is obtained / achieved; b) diluting an aliquot / fraction of the cells obtained in step (a) by adding unused / fresh culture medium to obtain a production cell solution having a second predetermined cell density; c) culturing the production cell solution for 1 to 36 hours; d) In the cultured production cell solution obtained in step (c), directly transfecting the cells with one or more nucleic acids encoding recombinant AAV particles; e) Culturing the transfected production cell solution obtained in step (d) for 20 to 240 hours to thereby produce recombinant AAV particles; A method comprising the steps. 2. The method according to embodiment 1, wherein the first predetermined cell density is at least 80×10 5 cells / mL. 3. The method according to embodiment 1 and embodiment 2, wherein the first predetermined cell density is at least 100×10 5 cells / mL. 4. The method according to embodiment 1 or any one of embodiments 2 to 3, wherein the mammalian cell is a CHO cell, a HEK cell, or a human amniotic cell. 5. The method according to embodiment 1 or any one of embodiments 2 to 4, wherein the mammalian cell is a CHO-K1 cell or a HEK293 cell. 6. The one or more nucleic acids are i) in the 5' to 3' direction, alpha) a first ITR sequence; beta) a promoter; gamma) a nucleic acid sequence encoding a therapeutic molecule; delta) a polyadenylation signal sequence; epsilon) a transgene comprising a second ITR sequence; ii) a rep open reading frame; iii) a cap open reading frame; iv) adenovirus E1A, E1B, E2A, E4orf6, and VA RNA open reading frames The method according to embodiment 1 or any one of embodiments 2 to 5. 7. The method according to embodiment 1 or any one of embodiments 2 to 6, wherein in step (b), an aliquot of fresh culture medium having a volume 4 to 6 times that of the culture medium is added. 8. The method according to embodiment 1 or any one of embodiments 2 to 7, wherein in step (b), an aliquot of fresh culture medium having a volume approximately 5 times that of the culture medium is added. 9. The second predetermined cell density is 10×10 5 cells / mL to 30×10 5 cells / mL, the method according to any one of Embodiment 1 or Embodiments 2 to 8. 10. The second predetermined cell density is about 20×10 5 cells / mL, the method according to any one of Embodiment 1 or Embodiments 2 to 9. 11. The culture in step (c) lasts for 16 to 30 hours, the method according to any one of Embodiment 1 or Embodiments 2 to 10. 12. The culture lasts for 20 to 28 hours, the method according to any two of Embodiment 1 or Embodiments 2 to 11. 13. The culture lasts for about 24 hours, the method according to any one of Embodiment 1 or Embodiments 2 to 12. 14. The culture in step (e) is a batch culture, the method according to any one of Embodiment 1 or Embodiments 2 to 13. 15. The culture in step (e) lasts for about 24 to 144 hours, the method according to any one of Embodiment 1 or Embodiments 2 to 14. 16. The culture in step (e) lasts for about 48 to 120 hours, the method according to any one of Embodiment 1 or Embodiments 2 to 15. 17. The culture lasts for about 60 to 96 hours, the method according to any one of Embodiment 1 or Embodiments 2 to 16. 18. The culture lasts for about 72 hours, the method according to any one of Embodiment 1 or Embodiments 2 to 17. 19. Step (e) does not involve feeding, the method according to any one of Embodiment 1 or Embodiments 2 to 18. 20. f) A step of recovering cells from the culture solution of step (e), and g) A step of lysing the cells obtained in step (f), and h) A step of separating AAV particles from the lysed cells obtained in step (g), and i) Optionally, a step of purifying the AAV particles and further comprising the method according to any one of Embodiment 1 or Embodiments 2 to 19. 21. The cell density after step (c) is 35 to 40×10 5The method according to any one of embodiment 1 or embodiments 2 to 20, which is in cells / ml. 22. After step (c) and before step (d), the following step (cd): cd) Adding a further fresh medium of about 20% of the initial culture volume The method according to any one of embodiment 1 or embodiments 2 to 21, wherein the step is performed. 23. The method according to any one of embodiment 1 or embodiments 2 to 22, wherein the step of transfecting is by adding one or more nucleic acids as a mixture of a PEI-complexed nucleic acid and free PEI. 24. The method according to any one of embodiment 1 or embodiments 2 to 23, wherein the step of transfecting is a step in the presence of valproic acid at a final concentration of 5 mM. 25. Use of mammalian cells for the production of recombinant AAV particles, wherein the mammalian cells are grown using perfusion prior to transfection with all AAV particles encoding the nucleic acid. 26. Use of mammalian cells obtained by the method according to any one of embodiment 1 or embodiments 2 to 24 for the production of recombinant AAV particles.

[0019] In addition to the various embodiments shown and claimed, the disclosed subject matter also contemplates other embodiments having other combinations of the features disclosed and claimed herein. Accordingly, the specific features presented herein can be combined with each of the other features in other manners within the scope of the disclosed subject matter, and thus the disclosed subject matter includes any suitable combination of the features disclosed herein. The foregoing description of specific embodiments of the disclosed subject matter has been presented for purposes of illustration and description. The description is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed.

Brief Description of the Drawings

[0020]

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Mode for Carrying Out the Invention

[0021] Detailed Description of Specific Embodiments of the Invention Definitions Useful methods and techniques for carrying out the present invention are described, for example, in Ausubel, F.M. (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997), Glover, N.D., and Hames, B.D., ed., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press, Freshney, R.I. (ed.), Animal Cell Culture - a practical approach, IRL Press Limited (1986), Watson, J.D., et al., Recombinant DNA, Second Edition, CHSL Press (1992), Winnacker, E.L., From Genes to Clones, N.Y., VCH Publishers (1987), Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998), Freshney, R.I., Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., N.Y. (1987).

[0022] By using recombinant DNA technology, it becomes possible to produce derivatives of nucleic acids. Such derivatives can be modified at individual or several nucleotide positions, for example, by substitution, modification, exchange, deletion, or insertion. Modification or derivatization can be carried out, for example, by site-directed mutagenesis. Such modifications can be easily implemented by those skilled in the art (see, for example, Sambrook, J., et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA, Hames, B.D., and Higgins, S.G., Nucleic acid hybridization - a practical approach (1985) IRL Press, Oxford, England).

[0023] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes plural such cells and their equivalents known to those skilled in the art. Similarly, the terms "a" (or "an"), "one or more", and "at least one" may be used interchangeably in this specification. It should also be noted that the terms "comprising", "including", and "having" may be used interchangeably.

[0024] Deoxyribonucleic acid contains a coding strand and a non-coding strand. The terms "5'" and "3'" as used in this specification refer to positions on the coding strand.

[0025] The term "3'-adjacent sequence" means a sequence located at the 3'-end (downstream, below) of a nucleotide sequence.

[0026] The term "5'-flanking sequence" means a sequence located at the 5'-end (downstream, below) of a nucleotide sequence.

[0027] As used in this specification and the appended claims, it should be noted that the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells and their equivalents known to those skilled in the art. Similarly, the terms "a" (or "an"), "one or more", and "at least one" may be used interchangeably herein. It should also be noted that the terms "comprising", "including", and "having" may be used interchangeably.

[0028] The term "AAV helper function" refers to AAV gene products that function in trans for the replication and packaging of productive AAV and AAV-derived coding sequences (proteins) that can be expressed to provide AAV particles. Thus, the AAV helper function includes AAV open reading frames (ORFs) such as rep and cap, and others such as AAP for specific AAV serotypes. The rep gene expression product has been shown to have many functions including, among other things, recognition, binding, and nicking of the AAV origin of DNA replication; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. The cap gene expression product (capsid) provides the necessary packaging function. The AAV helper function is used to complement the trans AAV functions missing from the AAV vector genome.

[0029] The term "about" means a range of ±20% of the numerical value that follows. In certain embodiments, the term "about" means a range of ±10% of the numerical value that follows. In certain embodiments, the term "about" means a range of ±5% of the numerical value that follows.

[0030] The term "batch culture" refers to a culture in which, at the start of the culture process, all components for cell culture (including cells and all culture nutrients) are supplied to the culture bioreactor.

[0031] The terms "comprise(s)", "include(s)", "having", "has", "can", "contain(s)" and their variations are intended, as used herein, to be open-ended transitional phrases, terms or words that do not preclude the possibility of additional acts or structures. The term "comprising" includes the term "consisting of". The present disclosure also contemplates other embodiments that "comprise", "consist of" and "consist essentially of" the embodiments or elements presented herein, whether or not explicitly recited.

[0032] The term "culturing" as used herein refers to the process of maintaining cells in a culture medium under conditions in which the cells are transfected to produce AAV particles.

[0033] The terms "empty capsid" and "empty particle" refer to AAV particles that have an AAV protein shell but lack all or part of the nucleic acid that encodes a protein or that is transcribed into the transcription product of interest adjacent to the AAV ITR, i.e., the vector. Thus, an empty capsid does not function to transfer to a host cell the nucleic acid that encodes a protein or that is transcribed into the transcription product of interest.

[0034] The term "endogenous" refers to that which occurs naturally within a cell and is naturally produced by the cell. Similarly, an endogenous locus / cell endogenous locus is a locus that occurs naturally within a cell.

[0035] As used herein, the term "exogenous" indicates that a nucleotide sequence is not derived from a particular cell and is introduced into the cell by a DNA delivery method, such as a transfection method, an electroporation method, or transformation by a viral vector. Thus, an exogenous nucleotide sequence is an artificial sequence, and this artificial construct can be, for example, from a combination of subsequences of different origins (e.g., a combination of a recombinase recognition sequence having an SV40 promoter and a coding sequence of a green fluorescent protein is an artificial nucleic acid), or from a partial deletion of a sequence (e.g., a sequence encoding only the extracellular domain of a membrane-bound receptor or cDNA) or a mutation of a nucleobase. The term "endogenous" means a nucleotide sequence derived from a cell. An "exogenous" nucleotide sequence can have an "endogenous" counterpart that has the same base composition but whose sequence is becoming an "exogenous" sequence, for example, by introduction into a cell via recombinant DNA technology.

[0036] As used herein, the term "fed-batch cell culture" refers to a culture in which cells and medium are initially supplied to a culture bioreactor and additional culture nutrients are supplied continuously or incrementally to the culture during the culture process, regardless of the periodic harvesting of cells and / or product before the end of the culture.

[0037] An "isolated" composition is a composition that has been separated from one or more components of its natural environment. In some embodiments, the composition is purified by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing electrophoresis (IEF), capillary electrophoresis, CE-SDS) or chromatography (e.g., size exclusion chromatography, or ion exchange or reverse phase HPLC) until it is determined to be of a purity of 95% or greater than 99%. For a review of methods for assessing antibody purity, see, for example, Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0038] "Isolated" nucleic acid refers to a nucleic acid molecule separated from one or more components (plural) of its natural environment. Isolated nucleic acids typically include nucleic acid molecules contained within cells that normally contain nucleic acid molecules, but the nucleic acid molecules are present extrachromosomally or at a chromosomal location different from their natural chromosomal location.

[0039] "Isolated" polypeptide or antibody means a polypeptide molecule or antibody molecule separated from one or more components (plural) of its natural environment.

[0040] The term "mammalian cell containing an exogenous nucleotide sequence" encompasses cells into which one or more exogenous nucleic acids (plural) have been introduced, including the progeny of such cells. These can serve as starting points for further genetic modification. Thus, the term "mammalian cell containing an exogenous nucleotide sequence" includes cells containing an exogenous nucleotide sequence integrated at a single site within a locus of the genome of the mammalian cell, and this exogenous nucleotide sequence includes at least one first and at least one second recombination recognition site (which are different) adjacent to at least one first selectable marker. In certain embodiments, a mammalian cell containing an exogenous nucleotide sequence is a cell containing an exogenous nucleotide sequence integrated at a single site within a locus of the genome of the cell, and this exogenous nucleotide sequence includes a first recombination recognition sequence and a second recombination recognition sequence adjacent to at least one first selectable marker, and a third recombination recognition sequence located between the first recombination recognition sequence and the second recombination recognition sequence, and the recombination recognition sequences are all different.

[0041] Both "mammalian cell containing an exogenous nucleotide sequence" and "recombinant cell" are "transfected cells". This term includes primary transfected cells and their progeny, regardless of the number of passages. Progeny may, for example, contain mutations and not have exactly the same nucleic acid content as the parental cell. Mutant progeny having the same function or biological activity as the initially transfected cell are included.

[0042] The term "nucleic acid encoding AAV packaging protein" generally refers to one or more nucleic acid molecules containing nucleotide sequences that provide AAV functions deleted from an AAV vector and are used to produce transduction-competent recombinant AAV particles. Nucleic acids encoding AAV packaging proteins are commonly used to provide expression of the AAV rep and / or cap genes to complement the defective AAV functions required for AAV replication. However, the nucleic acid construct lacks AAV ITRs and cannot replicate or be packaged. Nucleic acids encoding AAV packaging proteins can be in the form of plasmids, phages, transposons, cosmids, viruses, or particles. Many nucleic acid constructs are described, such as the commonly used plasmids pAAV / Ad and pIM29+45 that encode both the rep gene expression product and the cap gene expression product. See, for example, Samulski et al. (1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945. Several plasmids encoding the rep and / or cap gene expression products are described (e.g., U.S. Patent No. 5,139,941 and U.S. Patent No. 6,376,237). Any one of these nucleic acids encoding AAV packaging proteins can contain a DNA element or nucleic acid according to the present invention.

[0043] The term "nucleic acid encoding helper protein(s)" generally refers to one or more nucleic acid molecule(s) containing nucleotide sequences that encode protein(s) and / or RNA molecule(s) that provide adenovirus helper function(s). A plasmid having a nucleic acid(s) encoding helper protein(s) can be transfected into a suitable cell, and as a result, the plasmid can assist in the production of AAV particles in the cell. Any one of these nucleic acids encoding helper protein(s) can contain a DNA element or nucleic acid according to the present invention. Infectious virus particles existing in nature, such as adenovirus, herpesvirus, or vaccinia virus particles, are specifically excluded from this term.

[0044] As used herein, the term "operably linked" refers to the juxtaposition of two or more components such that they are in a relationship that enables them to function as intended. For example, when a promoter and / or enhancer functions to regulate the transcription of a coding sequence / open reading frame / gene, the promoter and / or enhancer is operably linked to the coding sequence / open reading frame / gene. In certain embodiments, "operably linked" DNA sequences are contiguous. In certain embodiments, for example, when it is necessary to join the coding regions of two proteins such as a secretion leader and a polypeptide, these sequences are contiguous and are in the same reading frame. In certain embodiments, an operably linked promoter is located upstream of the coding sequence / open reading frame / gene and can be adjacent to the coding sequence. In certain embodiments, for example, with respect to an enhancer sequence that regulates the expression of a coding sequence / open reading frame / gene, the two components may not be adjacent but can be operably linked. When an enhancer increases the transcription of a coding sequence / open reading frame / gene, the enhancer is operably linked to the coding sequence / open reading frame / gene. An operably linked enhancer can be located upstream, within, or downstream of the coding sequence / open reading frame / gene, and can also be located at a considerable distance from the promoter of the coding sequence / open reading frame / gene.

[0045] The term "packaging protein" refers to non-AAV-derived viral and / or cellular functions upon which AAV depends for its replication. Thus, this term encompasses the proteins and RNAs necessary for AAV replication, including those involved in AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of the Cap expression products, and activation of AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type I), and vaccinia virus.

[0046] As used herein, "AAV packaging protein" refers to AAV-derived sequences that function in trans for the replication of productive AAV. Thus, AAV packaging proteins are encoded by the major AAV open reading frames (ORFs), rep and cap. The rep proteins have been shown to have numerous functions including, inter alia, recognition, binding, and nicking of the AAV origin of DNA replication; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. The cap (capsid) proteins provide the necessary packaging function. AAV packaging proteins are used herein to complement the trans AAV functions that are missing from an AAV vector.

[0047] As used herein, "perfusion" or "perfusion culture", sometimes referred to as a continuous culture, refers to a culture in which cells are retained in the culture, for example, by filtration, encapsulation, anchoring to microcarriers, etc., and the medium is introduced continuously, stepwise, or intermittently (or any combination thereof) and removed from the culture bioreactor.

[0048] The terms "propagate" and "pre-culture" are used interchangeably herein and refer to the process of increasing the number of cells in a cell culture, starting from the inoculation of fresh culture medium containing an aliquot of cells and maintaining the culture conditions for the cells to grow exponentially and divide until a desired cell density, i.e., a first predetermined cell density, is achieved. Generally, cells can be propagated using different methods such as batch, fed-batch or perfusion culture. The term "propagate" includes the division of cells during propagation, i.e., removing an aliquot of the culture medium containing the cells and replacing it with a defined aliquot of fresh culture medium / adding a defined aliquot of fresh culture medium, which results in a decrease in the number of cells (cell density).

[0049] As used herein, the term "proteinaceous compound" means a heteromultimeric molecule comprising at least one polypeptide produced in a functional form in mammalian cells. Exemplary proteinaceous compounds are adeno-associated virus particles (AAV particles) comprising a capsid formed from capsid polypeptides and a single-stranded DNA molecule which is a non-polypeptide component.

[0050] As used herein, the term "recombinant cell" means a cell after final genetic modification, e.g., a cell that expresses a polypeptide of interest or produces rAAV particles of interest and can be used for the production of the polypeptide of interest or rAAV particles of interest at any scale. For example, a "mammalian cell containing an exogenous nucleotide sequence" that has been subjected to recombinase-mediated cassette exchange (RMCE) and thereby had the coding sequence of a polypeptide of interest introduced into the genome of the host cell is a "recombinant cell". This cell can still perform further RMCE reactions, but this is not the purpose.

[0051] A "recombinant AAV vector" is obtained from the wild-type genome of a virus (e.g., AAV) by using molecular biological methods to remove the wild-type genome from the virus and replace it with a non-natural nucleic acid, such as a nucleic acid transcribed into a transcript or a nucleic acid encoding a protein. Typically, for AAV, one or both of the inverted terminal repeat (ITR) sequences of the wild-type AAV genome are retained in the recombinant AAV vector. A "recombinant" AAV vector is distinguished from the wild-type viral AAV genome because all or part of the viral genome has been replaced with a non-natural (i.e., heterologous) sequence with respect to the viral genomic nucleic acid. Thus, the incorporation of the non-natural sequence defines the viral vector (e.g., AAV) as a "recombinant" vector, which, in the case of AAV, can be referred to as an "rAAV vector".

[0052] A recombinant vector (e.g., AAV) is packaged for subsequent infection (transduction) of cells ex vivo, in vitro, or in vivo and can be referred to herein as a "particle". When the recombinant vector sequence is encapsulated or packaged into an AAV particle, the particle can also be referred to as an "rAAV". Such particles contain proteins that encapsulate or package the vector genome. Specific examples include viral envelope proteins and, in the case of AAV, capsid proteins such as AAV VP1, VP2, and VP3.

[0053] As used herein, the term "selectable marker" refers to a gene that enables a cell carrying a certain gene to be specifically selected or specifically eliminated in the presence of a corresponding selective agent. For example, without limitation, a selectable marker can enable a host cell transformed with a selectable marker gene to be positively selected in the presence of each selective agent (selective culture conditions), and untransformed host cells cannot grow or survive under selective culture conditions. A selectable marker can be positive, negative, or bifunctional. A positive selectable marker can enable the selection of cells carrying the marker, and a negative selectable marker can enable the selective elimination of cells carrying the marker. A selectable marker can confer resistance to a drug or can complement a metabolic or catabolic defect of the host cell. In prokaryotic cells, in particular, genes conferring resistance to ampicillin, tetracycline, kanamycin, or chloramphenicol can be used. Useful resistance genes as selectable markers in eukaryotic cells include, but are not limited to, aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin and G418 (APH)), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), genes for histidinol dehydrogenase (histidinol D), and genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid. Further marker genes are described in WO 92 / 08796 and WO 94 / 28143.

[0054] Beyond facilitating selection in the presence of the corresponding selection agent, selectable markers alternatively may be molecules not normally present in cells, such as green fluorescent protein (GFP), enhanced GFP (eGFP), synthetic GFP, yellow fluorescent protein (YFP), enhanced YFP (eYFP), cyan fluorescent protein (CFP), mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire. For example, cells expressing such molecules can be distinguished from cells that do not carry this gene, based respectively on the detection or absence of fluorescence emitted by the encoded polypeptide.

[0055] As used herein, the term "serotype" is a distinction based on serologically distinct AAV capsids. Serological specificity is determined based on the lack of cross-reactivity between antibodies to one AAV as compared to other AAVs. Such differences in cross-reactivity are usually due to differences in the capsid protein sequence / epitope (e.g., differences in the VP1, VP2, and / or VP3 sequences of the AAV serotype). AAV variants, including capsid variants, may have at least one nucleotide or amino acid residue different compared to a reference AAV or other AAV serotype, even though they may not be serologically distinguishable from the reference AAV or other AAV serotypes.

[0056] Under the conventional definition, a serotype means that the virus of interest is tested against sera specific to all existing and characterized serotypes for neutralizing activity, and no antibodies that neutralize the virus of interest have been found. As more naturally occurring virus isolates are discovered and / or capsid variants are generated, there may or may not be serological differences from any of the currently existing serotypes. Thus, if a new virus (e.g., AAV) has no serological differences, this new virus (e.g., AAV) is a subgroup or variant of the corresponding serotype. In many cases, serological tests for neutralizing activity have not yet been performed on mutant viruses with capsid sequence modifications to determine whether they are other serotypes according to the conventional definition of serotypes. Thus, for convenience and to avoid repetition, the term "serotype" broadly refers to both serologically distinct viruses (e.g., AAV) and serologically indistinguishable viruses (e.g., AAV) that can be within a subgroup or variant of a given serotype.

[0057] The terms "transduction" and "transfect" refer to the introduction of molecules such as nucleic acids (viral vectors, plasmids) into cells. When exogenous nucleic acid is introduced inside the cell membrane, the cell has been "transduced" or "transfected". Thus, a "transduced cell" is a cell into which a "nucleic acid" or "polynucleotide" has been introduced, or its progeny into which exogenous nucleic acid has been introduced. In certain embodiments, a "transduced" cell (e.g., in a mammal, e.g., a cell or tissue or organ cell) has a genetic change after integration of an exogenous molecule, e.g., a nucleic acid (e.g., a transgene). The "transduced" cell(s) can be propagated to transcribe the introduced nucleic acid and / or express the protein.

[0058] In cells that have been "transfected" or "transformed", the nucleic acid (viral vector, plasmid) may or may not be integrated into the genomic nucleic acid. When the introduced nucleic acid is integrated into the nucleic acid (genomic DNA) of the recipient cell or organism, it is stably maintained within the cell or organism and can be passed on to or inherited by the progeny cells or organisms of the recipient cell or organism. Finally, the introduced nucleic acid may exist extrachromosomally or only transiently in the recipient cell or host organism. Several techniques are known, see, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Using such techniques, one or more exogenous DNA moieties can be introduced into a suitable host cell.

[0059] The term "transgene" is used herein to conveniently refer to a nucleic acid that has been intentionally introduced into a cell or organism. Transgenes include any nucleic acid, for example, a gene that is transcribed into a transcript or encodes a polypeptide or protein.

[0060] "Vector" refers to a portion of a recombinant plasmid sequence that is ultimately packaged or encapsulated, either directly or in the form of single-stranded or RNA, to form virus (e.g., AAV) particles. When using a recombinant plasmid to construct or generate recombinant virus particles, the virus particles do not contain the "plasmid" portion that does not correspond to the vector sequence of the recombinant plasmid. This non-vector portion of the recombinant plasmid is called the "plasmid backbone", which is important for the cloning and amplification of the plasmid, a process necessary for growth and recombinant virus production, but is not itself packaged or encapsulated in virus (e.g., AAV) particles. Thus, "vector" refers to the nucleic acid packaged or encapsulated by virus particles (e.g., AAV).

[0061] Generation of Recombinant Cell Lines Generally, for the efficient and large-scale production of a proteinaceous compound of interest, e.g., rAAV particles or a therapeutic polypeptide, cells that express and, if possible, secrete the proteinaceous compound are required. Such cells are referred to as "recombinant cells" or "recombinant production cells".

[0062] In the first step, an appropriate host cell is transfected with the nucleic acid sequence necessary to encode the proteinaceous compound of interest. Transfection of additional helper polypeptides may be required.

[0063] The expression of a coding sequence, i.e., an open reading frame, requires additional regulatory elements such as a promoter and a polyadenylation signal (sequence). Therefore, the open reading frame is operably linked to such additional regulatory elements for transcription. This can be achieved by incorporating it into a so-called expression cassette. The minimal control elements required for an expression cassette to function in mammalian cells are a promoter functional in the mammalian cells, located upstream, i.e., on the 5'-side, of the open reading frame, and a polyadenylation signal (sequence) functional in the mammalian cells, located downstream, i.e., on the 3'-side, of the open reading frame. Further, a terminator sequence may be present on the 3'-side of the polyadenylation signal (sequence). For expression, the promoter, open reading frame / coding region, and polyadenylation signal sequence must be arranged in an operably linked form.

[0064] Similarly, a nucleic acid transcribed into a non-protein-coding RNA is called an "RNA gene". For the expression of an RNA gene as well, additional regulatory elements such as a promoter and a transcription termination signal or a polyadenylation signal (sequence) are required. The nature and localization of such elements depend on the RNA polymerase intended to drive the expression of the RNA gene. Therefore, an RNA gene is usually also incorporated into an expression cassette.

[0065] If the proteinaceous compound of interest is composed of different (monomeric) polypeptides and single-stranded DNA molecules and requires other accessory factors for production and encapsidation, a number of expression cassettes with different open reading frames / coding sequences are required. In this case, at least one expression cassette is required for each of the transgene, the different polypeptides forming the capsid of the AAV vector, and VA RNA for the necessary helper functions. Thus, individual expression cassettes are required for each of the helper E1A, E1B, E2A, E4orf6, VA RNA, rep, and cap genes.

[0066] As outlined in the previous paragraph, the more complex the proteinaceous compound of interest or the greater the number of additional helper polypeptides and / or RNAs required, the greater the number of different expression cassettes required, respectively. In essence, along with the number of expression cassettes, the size of the nucleic acid incorporated into the genome of the host cell also increases. However, there is a practical upper limit to the size of transferable nucleic acids, which is in the range of about 15 kbps (kilobase pairs). Beyond this limit, handling and processing efficiency are significantly reduced. This problem can be addressed by using two or more separate nucleic acids. This allows different expression cassettes to be assigned to different nucleic acids, with each nucleic acid containing only a portion of the expression cassette.

[0067] Recombinant cells Generally, for the efficient and large-scale production of a proteinaceous compound of interest, such as rAAV particles or a therapeutic polypeptide, cells that express and, if possible, secrete the proteinaceous compound are required. Such cells are referred to as "recombinant cells" or "recombinant production cells".

[0068] For the generation of "recombinant production cells", appropriate mammalian cells are transfected with the necessary nucleic acid sequences encoding the proteinaceous compound of interest. Transfection of additional helper polypeptides may be required.

[0069] For the generation of stable recombinant production cells, a second step follows, in which a single cell that stably expresses the proteinaceous compound of interest is selected. This can be done, for example, based on the co-expression of a selection marker co-transfected with the nucleic acid sequence encoding the proteinaceous compound of interest, or it can be the expression of the proteinaceous compound itself.

[0070] For the expression of a coding sequence, i.e., an open reading frame, additional regulatory elements such as a promoter and a polyadenylation signal (sequence) are required. Thus, the open reading frame is operably linked to the said additional regulatory elements for transcription. This can be achieved by incorporating it into a so-called expression cassette. The minimal control elements required for an expression cassette to function in mammalian cells are a promoter functional in the mammalian cells, located upstream, i.e., on the 5'-side, of the open reading frame, and a polyadenylation signal (sequence) functional in the mammalian cells, located downstream, i.e., on the 3'-side, of the open reading frame. Furthermore, a terminator sequence may be present on the 3'-side of the polyadenylation signal (sequence). For expression, the promoter, open reading frame / coding region, and polyadenylation signal sequence must be arranged in an operably linked form.

[0071] Similarly, a nucleic acid transcribed into non-protein-coding RNA is called an "RNA gene". For the expression of an RNA gene, additional regulatory elements such as a promoter and a transcription termination signal or a polyadenylation signal (sequence) are also required. The nature and localization of such elements depend on the RNA polymerase intended to drive the expression of the RNA gene. Thus, an RNA gene is usually also incorporated into an expression cassette.

[0072] If the proteinaceous compound of interest is composed of different (monomeric) capsid polypeptides and single-stranded DNA molecules and further requires other adenoviral helper functions for production and encapsulation, a number of expression cassettes with different open reading frames / coding sequences contained therein are required. In this case, at least one expression cassette is required for each of the transgene, the different polypeptides forming the capsid of the AAV vector, and VA RNA for the required helper functions. Thus, individual expression cassettes for each of the helper E1A, E1B, E2A, E4orf6, VA RNA, rep, and cap genes are required.

[0073] As outlined in the previous paragraph, the more complex the proteinaceous compound of interest or the greater the number of additional helper polypeptides and / or RNAs required, the greater the number of different expression cassettes required, respectively. In essence, along with the number of expression cassettes, the total size of the nucleic acid is also. However, there is a practical upper limit to the size of transferable nucleic acids, which is in the range of about 15 kbps (kilobase pairs). Beyond this limit, handling and processing efficiency significantly decreases. This problem can be addressed by using two or more separate plasmids. Thereby, different expression cassettes are assigned to different plasmids, and each plasmid contains only a part of the expression cassette.

[0074] For the development of stable cell lines, random integration (RI) of nucleic acid(s) carrying the expression cassette for the proteinaceous compound of interest can be used. Generally, by using RI, the nucleic acid or a fragment thereof is randomly integrated into the genome of the host cell.

[0075] Alternatively, for RI, targeted integration (TI) into the CLD can be used. In TI CLD, one or more nucleic acids containing different expression cassettes are introduced into a predetermined locus within the genome of the host cell.

[0076] At TI, either homologous recombination or recombinase-mediated cassette exchange reaction (RMCE) can be used to integrate nucleic acid (a) containing each expression cassette into a specific locus in the genome of a TI host cell.

[0077] In certain embodiments of all aspects and embodiments, each expression cassette contains, in the 5' to 3' direction, a promoter, an open reading frame / coding sequence or RNA gene, and a polyadenylation signal sequence, and / or a terminator sequence. In certain embodiments, the open reading frame encodes a polypeptide and the expression cassette contains a polyadenylation signal sequence with or without an additional terminator sequence. In certain embodiments, the expression cassette contains an RNA gene, the promoter is a type 2 Pol III promoter, and there is a polyadenylation signal sequence or polyU terminator. See, for example, Song et al. Biochemical and Biophysical Research Communications 323 (2004) 573-578. In certain embodiments, the expression cassette contains an RNA gene, the promoter is a type 2 Pol III promoter and a polyU terminator sequence.

[0078] In certain embodiments of all aspects and embodiments, the open reading frame encodes a polypeptide, the promoter is a human CMV promoter with or without intron A, the polyadenylation signal sequence is a bGH (bovine growth hormone) polyA signal sequence, and the terminator is hGT (human gastrin terminator).

[0079] In certain embodiments of all aspects and embodiments, the promoter is a human CMV promoter having intron A, the polyadenylation signal sequence is a bGH polyadenylation signal sequence, the terminator is hGT, and except for the expression cassette of the RNA gene and the expression cassette of the selectable marker, for the selectable marker, the promoter is an SV40 promoter, the polyadenylation signal sequence is an SV40 polyadenylation signal sequence, and the terminator is absent; for the RNA gene, the promoter is a wild-type type 2 polymerase III promoter and the terminator is a polymerase II or III terminator.

[0080] It should be noted that in certain embodiments, the present invention does not encompass a permanent human cell line containing nucleic acid sequences for adenoviral gene functions E1A and E1B simultaneously with nucleic acid sequences for SV40 large T antigen or Epstein - Barr virus (EBV) nuclear antigen 1 (EBNA - 1).

[0081] Adeno - associated virus (AAV) For a general review of the helper functions of AAV and adenovirus or herpesvirus, see Berns and Bohensky, Advances in Virus Research, Academic Press., 32(1987)243-306. The AAV genome is described in Srivastava et al., J. Virol., 45(1983)555-564. U.S. Patent No. 4,797,368 describes design considerations for constructing recombinant AAV vectors (see also International Publication No. 93 / 24641). Further references describing AAV vectors are West et al., Virol. 160(1987)38-47, Kotin, Hum. Gene Ther. 5(1994)793-801, and Muzyczka J.Clin.Invest. 94(1994)1351. For the construction of recombinant AAV vectors, see U.S. Patent No. 5,173,414; Lebkowski et al., Mol. Cell. Biol. 8(1988)3988-3996, Tratschin et al., Mol. Cell. Biol. 5(1985)3251-3260, Tratschin et al., Mol. Cell. Biol., 4(1994)2072-2081, Hermonat and Muzyczka Proc. Natl. Acad. Sci. USA 81(1984)6466-6470, Samulski et al. J. Virol. 63(1989)3822-3828.

[0082] Adeno-associated virus (AAV) is a replication-defective parvovirus. It can replicate only in cells in which specific viral functions are provided by a co-infecting helper virus such as adenovirus, herpesvirus, and in some cases poxviruses such as vaccinia. Nevertheless, AAV can replicate in substantially any cell line of human, simian, or rodent origin if appropriate helper virus functions are present.

[0083] In the absence of helper virus genes, AAV establishes a latent period in its host cells. Its genome integrates into a specific site on chromosome 19 [(Chr)19(q13.4)], called the adeno-associated virus integration site 1 (AAVS1). For certain serotypes such as AAV-2, other integration sites have been found, such as on chromosome 5 [(Chr)5(p13.3)] called AAVS2 and on chromosome 3 [(Chr)3(p24.3)] called AAVS3.

[0084] AAV is classified into different serotypes. These are assigned based on parameters such as hemagglutination, tumorigenicity, and DNA sequence homology. To date, more than 10 different serotypes and more than 100 sequences corresponding to different clades of AAV have been identified.

[0085] The type and symmetry of the capsid protein determine the tissue tropism of each AAV. For example, AAV-2, AAV-4, and AAV-5 are specific for the retina, AAV-2, AAV-5, AAV-8, AAV-9, and AAVrh-10 are specific for the brain, AAV-1, AAV-2, AAV-6, AAV-8, and AAV-9 are specific for heart tissue, AAV-1, AAV-2, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, and AAV-10 are specific for the liver, and AAV-1, AAV-2, AAV-5, and AAV-9 are specific for the lung.

[0086] Pseudotyping refers to the process involving cross-packaging of the AAV genome between various serotypes, i.e., the genome is packaged with capsid proteins of different origins.

[0087] The wild-type AAV genome has a size of approximately 4.7 kb. The AAV genome further contains two overlapping genes called rep and cap, which include multiple open reading frames (see, for example, Srivastava et al., J. Viral., 45 (1983) 555-564, Hermonat et al., J. Viral. 51 (1984) 329-339, Tratschin et al., J. Virol., 51 (1984) 611-619). The Rep proteins encoded by the open reading frames provide four proteins of different sizes called Rep78, Rep68, Rep52, and Rep40. These are involved in AAV replication, rescue, and integration. The open reading frame encoding the Cap proteins provides four proteins called VP1, VP2, VP3, and AAP. VP1, VP2, and VP3 are part of the proteinaceous capsid of the AAV particle. The combined rep and cap open reading frames are flanked at their 5' and 3' ends by so-called inverted terminal repeats (ITRs). For replication, AAV requires, in addition to the Rep and Cap proteins, the products of the adenovirus genes E1A, E1B, E4orf6, E2A, and VA or the corresponding factors of another helper virus.

[0088] For example, in the case of AAV of serotype 2 (AAV-2), the ITRs each have a length of 145 nucleotides and flank a coding sequence region of approximately 4470 nucleotides. Of the 145 nucleotides of the ITR, 125 nucleotides have a palindromic sequence and can form a T-shaped hairpin structure. This structure has the function of a primer during virus replication. The remaining 20 unpaired nucleotides are shown as the D sequence.

[0089] The AAV genome has three transcription promoters, P5, P19, and P40, for the expression of the rep and cap genes (Laughlin et al., Proc. Natl. Acad. Sci. USA 76 (1979) 5567-5571).

[0090] The ITR array must be present cis to the coding region. The ITR provides a functional origin of replication (ori), signals necessary for integration into the genome of the target cell, and efficient excision and rescue from the host cell chromosome or recombinant plasmid. The ITR further contains origin-like elements such as the Rep protein binding site (RBS) and the terminal resolution site (TRS). It has been found that the ITR itself can function as a transcriptional promoter in the AAV vector (Flotte et al., J. Biol. Chem. 268 (1993) 3781 - 3790, Flotte et al., Proc. Natl. Acad. Sci. USA 93 (1993) 10163 - 10167).

[0091] Trans - splicing of the rep gene product and the cap gene product is required for replication of the viral single - stranded DNA genome and capsid formation, respectively.

[0092] The rep locus contains two internal promoters called P5 and P19. It contains open reading frames for four proteins. Promoter P5 is operably linked to a nucleic acid sequence that provides a non - splicing type 4.2 kb mRNA encoding the Rep protein Rep78 (a chromatin nickase for arresting the cell cycle) and a splicing type 3.9 kb mRNA encoding the Rep protein Rep68 (a site - specific endonuclease). Promoter P19 is operably linked to a nucleic acid sequence that provides a non - splicing type mRNA encoding the Rep protein Rep52 and a splicing type 3.3 kb mRNA encoding the Rep protein Rep40 (a DNA helicase for accumulation and packaging).

[0093] The two larger Rep proteins, Rep78 and Rep68, are essential for AAV double-stranded DNA replication, while the smaller Rep proteins, Rep52 and Rep40, appear to be essential for progeny single-stranded DNA accumulation (Chejanovsky & Carter, Virology 173 (1989) 120-128).

[0094] The larger Rep proteins, Rep68 and Rep78, can specifically bind to the hairpin conformation of the AAV ITR. They exhibit certain enzymatic activities required for resolving replication at the AAV termini. Expression of Rep78 or Rep68 may be sufficient for infectious particle formation (Holscher, C., et al. J. Virol. 68 (1994) 7169-7177 and 69 (1995) 6880-6885).

[0095] All Rep proteins, mainly Rep78 and Rep68, are thought to exhibit regulatory activities such as induction and repression of AAV genes and inhibitory effects on cell proliferation (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894, Labow et al., Mol. Cell. Biol., 7 (1987) 1320-1325; Khleif et al., Virology, 181 (1991) 738-741).

[0096] Overexpression of recombinant Rep78 results in a phenotype associated with a decrease in cell proliferation due to induction of DNA damage. This causes the host cell to arrest in the S phase, thereby promoting latent infection by the virus (Berthet, C., et al., Proc. Natl. Acad. Sci. USA 102 (2005) 13634-13639).

[0097] Tratschin et al. reported that the P5 promoter is negatively auto-regulated by Rep78 or Rep68 (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894). Due to the toxic effects of Rep protein expression, very low expression has been reported for certain cell lines after stable integration of AAV (see, for example, Mendelson et al., Virol. 166 (1988) 154-165).

[0098] The cap locus contains one promoter, designated P40. Promoter P40 is operably linked to a nucleic acid sequence that provides a 2.6 kb mRNA encoding the Cap proteins VP1 (87 kDa, unspliced mRNA transcript), VP2 (72 kDa from a spliced mRNA transcript), and VP3 (61 kDa from an alternative start codon) by alternative splicing and alternative start codon usage. VP1-VP3 constitute the components of the viral capsid. The capsid has the function of binding to cell surface receptors and enabling intracellular transport of the virus. VP3 accounts for approximately 90% of the total viral particle protein. Nevertheless, all three proteins are essential for efficient capsid production.

[0099] Inactivation of all three capsid proteins VP1-VP3 has been reported to prevent accumulation of single-stranded progeny AAV DNA. Mutations at the VP1 amino terminus ("lipid negative" or "Inf negative") still allow assembly of single-stranded DNA into viral particles, thereby greatly reducing the infectivity titer.

[0100] The AAP open reading frame encodes an assembly activation protein (AAP). This has a size of approximately 22 kDa and transports the native VP proteins to the nucleolar region for capsid assembly. This open reading frame is located upstream of the VP3 protein coding sequence.

[0101] Each individual AAV particle contains only one single-stranded DNA molecule. This can be either the "plus" or "minus" strand. An AAV viral particle containing a DNA molecule is infectious. Inside an infected cell, the parental infecting single strand is converted to double-stranded and then amplified. The amplification results in a large pool of double-stranded DNA molecules from which single strands are displaced and packaged into capsids.

[0102] Adeno-associated virus (AAV) vectors can transduce both dividing and quiescent cells. Transgenes introduced into target cells using AAV vectors are thought to be expressed for a long time. One drawback of using AAV vectors is the size limitation of the transgenes that can be introduced into cells.

[0103] Viral vectors such as parvovirus particles including AAV serotypes and their variants provide a means of delivering nucleic acids to cells ex vivo, in vitro, and in vivo that encode proteins such that the cells express the encoded proteins. AAV is a virus useful as a gene therapy vector because it can penetrate cells and introduce nucleic acid / genetic material so that the nucleic acid / genetic material can be stably maintained within the cells. Furthermore, these viruses can introduce nucleic acid / genetic material into specific sites, for example. Since AAV is not associated with pathogenic diseases in humans, AAV vectors can deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and agents) to human patients without causing substantial AAV etiology or disease.

[0104] Viral vectors that can be used include, but are not limited to, adeno-associated virus (AAV) particles of multiple serotypes (such as AAV-1 to AAV-12, etc.) and hybrid / chimeric AAV particles.

[0105] AAV particles can be advantageously used as vehicles for effective gene delivery. Such particles have several desirable features for such uses, including tropism for both dividing and non-dividing cells. Even in the early clinical experience with these vectors, persistent toxicity was not shown and the immune response was minimal or undetectable. AAV is known to infect a wide variety of cell types in vivo and in vitro by receptor-mediated endocytosis or transcytosis. These vector systems have been tested in humans targeting retinal epithelium, liver, skeletal muscle, airway, brain, joints, and hematopoietic stem cells.

[0106] Recombinant AAV particles typically do not contain viral genes associated with the etiology. Such vectors typically have one or more of the wild-type AAV genes that are wholly or partially deleted, such as, for example, the rep and / or cap genes, but retain at least one functional flanking ITR sequence as required for rescue, replication, and packaging of the recombinant vector into AAV particles. For example, only the essential parts of the vector, such as the ITR element and the LTR element respectively, are included. Thus, the AAV vector genome will contain the sequences necessary in cis for replication and packaging (e.g., functional ITR sequences).

[0107] Recombinant AAV vectors, and methods and uses thereof, include any viral strain or serotype. By way of non-limiting example, recombinant AAV vectors can be based on any AAV genome, e.g., AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, 2i8, AAV rh74 or AAV 7m8. Such vectors can be based on the same strain or serotype (or subgroup or variant), or can be different from one another. By way of non-limiting example, a recombinant AAV vector based on one serotype genome can be identical to one or more of the capsid proteins that package the vector. Further, a recombinant AAV vector genome can be based on an AAV (e.g., AAV2) serotype genome that is different from one or more of the AAV capsid proteins that package the vector. For example, the AAV vector genome can be based on AAV2, but at least one of the three capsid proteins can be, e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, AAV 7m8 or a variant thereof. AAV variants include variants and chimeras of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74 and AAV 7m8 capsids.

[0108] In certain embodiments of all aspects and embodiments, adeno-associated virus (AAV) vectors include, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, and AAV 7m8, and variants thereof (e.g., capsid variants, e.g., amino acid insertions, additions, substitutions and deletions), as described in, e.g., International Publication No. WO 2013 / 158879, International Publication No. WO 2015 / 013313 and U.S. Patent Application Publication No. 2013 / 0059732 (disclosing LK01, LK02, LK03, etc.).

[0109] AAV and AAV variants (e.g., capsid variants) serotypes (e.g., VP1, VP2, and / or VP3 sequences) may or may not be distinguished from other AAV serotypes including, for example, AAV1 to AAV12 (e.g., different from the VP1, VP2, and / or VP3 sequences of any of the AAV1 to AAV12 serotypes).

[0110] In certain embodiments of all aspects and embodiments, the AAV particles related to the reference serotype have a polynucleotide, polypeptide, or a partial sequence thereof that contains or consists of a sequence that is at least 80% or more identical (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) to one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, or AAV 7m8 (e.g., ITR sequence, or VP1 sequence, VP2 sequence, and / or VP3 sequence, etc.).

[0111] The compositions, methods, and uses of the present invention include AAV sequences (polypeptides and nucleotides), and have less than 100% sequence identity to reference AAV serotypes such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, or AAV 7m8, but are different from and not identical to known AAV genes or proteins such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, or AAV 7m8, and include subsequences of these AAV sequences. In certain embodiments of all aspects and embodiments, the AAV polypeptide or a subsequence thereof comprises or consists of a sequence that is at least 75% or more identical to any reference AAV sequence or a subsequence thereof, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, or AAV 7m8 (e.g., VP1, VP2, and / or VP3 capsid or ITR), such as 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, up to 100% identical. In certain embodiments, the AAV variant has 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions.

[0112] Recombinant AAV particles comprising AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, or AAV 7m8, as well as variant, related, hybrid, and chimeric sequences, can be constructed using recombinant techniques known to those of skill in the art to contain one or more nucleic acid sequences (transgenes) adjacent to one or more functional AAV ITR sequences.

[0113] Recombinant particles (e.g., rAAV particles) can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are useful, inter alia, for in vivo or ex vivo administration and delivery to a subject. In certain embodiments, the pharmaceutical composition contains a pharmaceutically acceptable carrier or excipient. Such excipients include any pharmaceutical that can be administered without inducing an immune response harmful to the individual receiving the composition itself and without undue toxicity.

[0114] Protocols for the production of adenoviral vectors are described in U.S. Patent No. 5,998,205, U.S. Patent No. 6,228,646, U.S. Patent No. 6,093,699, U.S. Patent No. 6,100,242, International Publication No. 94 / 17810, and International Publication No. 94 / 23744, which are hereby incorporated by reference in their entirety.

[0115] Recombinant AAV particles (rAAV particles) Various methods known in the art for generating rAAV particles. For example, transfection using an AAV plasmid and an AAV helper sequence in conjunction with co-infection with one AAV helper virus (e.g., an adenovirus, herpesvirus, or vaccinia virus), or transfection with a recombinant AAV plasmid, an AAV helper plasmid, and a helper function plasmid. Non-limiting methods for generating rAAV particles are described, for example, in U.S. Patent No. 6,001,650, U.S. Patent No. 6,004,797, International Publication No. 2017 / 096039, and International Publication No. 2018 / 226887. After the production of recombinant rAAV particles (i.e., the generation of particles in a cell culture system), the rAAV particles can be obtained and purified from host cells and cell culture supernatants.

[0116] The production of recombinant AAV particles requires the expression of Rep protein and Cap protein, helper proteins E1A, E1B, E2A and E4orf6, and adenovirus VA RNA in a single mammalian cell. The helper proteins E1A, E1B, E2A and E4orf6 can be expressed using any promoter shown by Matsushita et al. (Gene Ther. 5 (1998) 938-945), particularly the CMV IE promoter. Therefore, any promoter can be used.

[0117] Generally, to produce recombinant AAV particles, different complementary plasmids are co-transfected into host cells. One of the plasmids contains a transgene flanked by two cis-acting AAV ITRs. The defective AAV elements required for replication of the progeny recombinant genome and subsequent packaging, namely the open reading frames of the Rep protein and Cap protein, are contained in trans on a second plasmid. Overexpression of the Rep protein results in an inhibitory effect on cell growth (Li, J., et al., J. Virol. 71 (1997) 5236-5243). Furthermore, a third plasmid containing the genes of the helper virus, namely E1, E4orf6, E2A and VA derived from adenovirus, is required for AAV replication.

[0118] To reduce the number of plasmids required, the Rep, Cap and adenovirus helper genes may be combined on a single plasmid.

[0119] Alternatively, the host cell may already stably express the E1 gene product. Such cells are HEK293 cells. The human embryonic kidney clone designated 293 was generated in 1977 by the incorporation of adenovirus DNA into human embryonic kidney cells (HEK cells) (Graham, F. L., et al., J. Gen. Virol. 36 (1977) 59-74). The HEK293 cell line contains base pairs 1 to 4344 of the adenovirus serotype 5 genome. This includes the E1A and E1B genes as well as the adenovirus packaging signal (Louis, N., et al., Virology 233 (1997) 423-429).

[0120] When using HEK293 cells, the missing E2A, E4orf6, and VA genes can be introduced by co-infection with adenovirus or by co-transfection with E2A, E4orf6, and VA expression plasmids (see, for example, Samulski, R. J., et al., J. Virol. 63 (1989) 3822-3828, Allen, J. M., et al., J. Virol. 71 (1997) 6816-6822, Tamayose, K., et al., Hum. Gene Ther. 7 (1996) 507-513, Flotte, T. R., et al., Gene Ther. 2 (1995) 29-37, Conway, J. E., et al., J. Virol. 71 (1997) 8780-8789, Chiorini, J. A., et al., Hum. Gene Ther. 6 (1995) 1531-1541, Ferrari, F. K., et al., J. Virol. 70 (1996) 3227-3234, Salvetti, A., et al., Hum. Gene Ther. 9 (1998) 695-706, Xiao, X., et al., J. Virol. 72 (1998) 2224-2232, Grimm, D., et al., Hum. Gene Ther. 9 (1998) 2745-2760, Zhang, X., et al., Hum. Gene Ther. 10 (1999) 2527-2537). Alternatively, adenovirus / AAV or herpes simplex virus / AAV hybrid vectors can be used (see, for example, Conway, J. E., et al., J. Virol. 71 (1997) 8780-8789, Johnston, K. M., et al., Hum. Gene Ther. 8 (1997) 359-370, Thrasher, A. J., et al., Gene Ther. 2 (1995) 481-485, Fisher, J. K., et al., Hum. Gene Ther. 7 (1996) 2079-2087, Johnston, K. M., et al., Hum. Gene Ther. 8 (1997) 359-370).

[0121] Thus, cell lines in which the rep gene is integrated and expressed tend to grow slowly or express the Rep protein at very low levels.

[0122] To restrict transgene activity to specific tissues, i.e., to restrict the site of integration, the transgene can be operably linked to an inducible or tissue-specific promoter (see, e.g., Yang, Y., et al. Hum. Gene. Ther. 6 (1995) 1203-1213).

[0123] The main difficulty in the production of rAAV particles is the inefficient packaging of the rAAV vector, which results in low titers. Packaging is preferred when wild-type AAV genomes are present, difficult to generate sufficient complementing functions as provided by the wild-type rep and cap genes due to the inhibitory effects associated with the rep gene products, and difficult for several reasons such as the limited efficiency of co-transfection of plasmid constructs.

[0124] All of this is based on the biological properties of the Rep protein. In particular, the inhibitory (cell growth-suppressing and cytotoxic) properties of the Rep protein, as well as the ability to reverse the immortalized phenotype of cultured cells, are problematic. Furthermore, the Rep protein down-regulates its own expression when the widely used AAV P5 promoter is used (see, e.g., Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894).

[0125] In certain embodiments of all aspects and embodiments, the rAAV particles are derived from an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74 and 7m8.

[0126] In certain embodiments of all aspects and embodiments, the rAAV particles comprise a capsid sequence having at least 70% sequence identity with the capsid sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74, or 7m8.

[0127] In certain embodiments of all aspects and embodiments, the rAAV particles comprise an ITR sequence having at least 70% sequence identity with the ITR sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

[0128] E1A, E1B, E2 and E4 The coding sequences of E1A and E1B (open reading frames) can be derived from human adenoviruses, such as, in particular, human adenovirus serotype 2 or serotype 5. Exemplary sequences of human Ad5 (adenovirus serotype 5) can be found in GenBank entry X02996, AC_000008, and exemplary sequences of human Ad2 can be found in GenBank entry AC_000007. Nucleotides 505-3522 contain the nucleic acid sequences encoding E1A and E1B of human adenovirus serotype 5. Plasmid pSTK146 reported in European Patent No. 1230354, and plasmids pGS119 and pGS122 reported in International Publication No. 2007 / 056994 can also be used as sources of the E1A and E1B open reading frames.

[0129] E1A is the first viral helper gene expressed after adenoviral DNA enters the cell nucleus. The E1A gene encodes 12S and 13S proteins based on the same E1A mRNA by alternative splicing. The expression of the 12S and 13S proteins leads to the activation of other viral functions E1B, E2, E3 and E4. Furthermore, the expression of the 12S and 13S proteins pushes the cell into the S phase of the cell cycle. When only E1A-derived proteins are expressed, the cells die (apoptosis).

[0130] E1B is the second viral helper gene that is expressed. It is activated by E1A-derived proteins 12S and 13S. The mRNA derived from the E1B gene can be spliced in two different ways, resulting in the first 55 kDa transcript and the second 19 kDa transcript. The E1B 55 kDa protein is involved in the regulation of the cell cycle, the prevention of the transport of cellular mRNA in the late stage of infection, and the prevention of E1A-induced apoptosis. The E1B 19 kDa protein is involved in the prevention of E1A-induced apoptosis in cells.

[0131] The E2 gene encodes different proteins. The E2A transcript encodes a single-stranded binding protein (SSBP) that is essential for AAV replication.

[0132] Also, the E4 gene encodes several proteins. The 34 kDa protein (E4orf6) derived from the E4 gene, together with the E1B 55 kDa protein, prevents the accumulation of cellular mRNA in the cytoplasm but also promotes the transport of viral RNA from the nucleus to the cytoplasm.

[0133] Adenovirus VA RNA gene Viral-associated RNA (VA RNA) is a non-coding RNA of adenovirus (Ad) that regulates translation. The adenovirus genome contains two independent copies: VAI (VA RNAI) and VAII (VA RNAII). Both are transcribed by RNA polymerase III from the type 2 polymerase III promoter (see, for example, Machitani, M., et al., J. Contr. Rel. 154 (2011) 285-289). For recombinant production, the adenovirus VA RNA gene can be driven by any promoter.

[0134] The structure, function, and evolution of adenovirus-related RNAs using a systems biology approach were investigated by Ma, Y. and Mathews, M. B. (J. Virol. 70 (1996) 5083-5099). They provided alignments and consensus VA RNA sequences based on 47 known human adenovirus serotypes. This disclosure is incorporated herein by reference in its entirety.

[0135] VA RNAs, VAI and VAII, consist of 157-160 nucleotides (nt).

[0136] Depending on the serotype, adenoviruses contain one or two VA RNA genes. VA RNAI is thought to play a dominant proviral role, while VA RNAII can partially compensate for the absence of VA RNAI (Vachon, V. K. and Conn, G. L., Virus Res. 212 (2016) 39-52).

[0137] VA RNA is not essential, but plays an important role in efficient virus growth by overcoming the cellular antiviral machinery. That is, VA RNA is not essential for virus growth, but VA RNA-deleted adenoviruses may not be able to grow during the early stages of vector production where only a few copies of the viral genome per cell are present, perhaps because viral genes other than VA RNA that block the cellular antiviral machinery are not sufficiently expressed (see Maekawa, A., et al. Nature Sci. Rep. 3 (2013) 1136).

[0138] Maekawa, A. et al. (Nature Sci. Rep. 3 (2013) 1136) reported the efficient production of adenovirus vectors lacking virus-related RNA genes that disrupt the cellular RNAi machinery, where HEK293 cells constitutively and highly expressing flippase recombinase were infected to obtain VA RNA-deleted adenoviruses by FLP recombinase-mediated excision of the VA RNA locus.

[0139] Human adenovirus 2 VA RNAI corresponds to nucleotides 10586 to 10810 of the GenBank entry AC_000007 sequence. Human adenovirus 5 VA RNAI corresponds to nucleotides 10579 to 10820 of the GenBank entry AC_000008 sequence.

[0140] Method for generating rAAV particles Carter et al. showed that the entire rep and cap open reading frames within the wild-type AAV genome can be deleted and replaced with a transgene (Carter, B. J., in “Handbook of Parvoviruses”, ed. by P. Tijssen, CRC Press, pp. 155 - 168 (1990)). Furthermore, it has been reported that the ITRs must be maintained to retain the functions of replication, rescue, packaging, and integration of the transgene into the genome of the target cell.

[0141] When cells containing each viral helper gene are transduced with an AAV vector, or vice versa, or when cells containing the integrated AAV provirus are transduced with an appropriate helper virus, the AAV provirus is activated and enters the lytic infection cycle again (Clark, K. R., et al., Hum. Gene Ther. 6 (1995) 1329 - 1341, Samulski, R. J., Curr. Opin. Genet. Dev. 3 (1993) 74 - 80).

[0142] The production cells contain the rep and cap gene sequences, as well as a transgene cassette adjacent to the ITR sequences on one or more plasmids retained via drug selection. The production of rAAV particles in these cell lines generally occurs after their infection by the necessary helper functions. Thus, to provide the helper viral proteins and initiate the production of rAAV particles, the cells are infected with either replication-competent AdV (usually wild-type Ad5) or a plasmid containing the respective helper genes. Packaging cell lines are different from producer cell lines as they contain only the rep and cap genes.

[0143] Aspect of the present invention is a method of transducing a cell with a nucleic acid (e.g., a plasmid) containing all the elements necessary for the production of recombinant AAV particles, wherein the cells prior to transfection are grown (at least for some time) using perfusion. Thus, since the plasmid encodes viral packaging proteins and / or helper proteins, the cells can produce recombinant viral particles containing a nucleic acid encoding the protein of interest or containing sequences transcribed into the transcript of interest.

[0144] The present invention provides a viral (e.g., AAV) particle production platform that includes features that distinguish it from current "industry standard" viral (e.g., AAV) particle production processes by using the method according to the present invention.

[0145] More generally, cells transfected or transduced with DNA for the recombinant production of AAV particles can be referred to as "recombinant cells". Such cells can be, for example, yeast cells, insect cells or mammalian cells, and are used as recipients of nucleic acids (plasmids) encoding packaging proteins such as AAV packaging proteins, nucleic acids (plasmids) encoding helper proteins, and nucleic acids (plasmids) encoding proteins or transcribed into the transcription product of interest, i.e., the transgene placed between two AAV ITRs. This term includes the progeny of the original transfected or transduced cells. It is understood that the progeny of a single parental cell may not necessarily be completely identical in form or genomic or total nucleic acid complement to the original parent due to natural, accidental or intentional mutations.

[0146] A number of suitable cell growth media are commercially available to maintain cell viability or to provide cell growth and / or proliferation. Examples of such media include serum-free eukaryotic growth media such as media for maintaining viability or for providing growth of mammalian (e.g., human) cells. Non-limiting examples include Ham's F12 or F12K medium (Sigma-Aldrich), FreeStyle (FS) F17 medium (Thermo-Fisher Scientific), MEM, DMEM, RPMI-1640 (Thermo-Fisher Scientific), and mixtures thereof. Such media can be supplemented with vitamins and / or trace minerals and / or salts and / or amino acids, such as essential amino acids for mammalian (e.g., human) cells.

[0147] The helper protein plasmid can be in the form of a plasmid, phage, transposon or cosmid. In particular, it has been demonstrated that the perfect complement of the adenovirus gene is not necessary for helper function. For example, adenovirus mutants that are unable to perform DNA replication and late gene synthesis have been shown to permit AAV replication. Ito et al., J. Gen. Virol. 9(1970)243; Ishibashi et al, Virology 45(1971)317.

[0148] Mutants within the E2B and E3 regions have been shown to support AAV replication, indicating that the E2B and E3 regions are probably not involved in providing helper functions. Carter et al., Virology 126(1983)505. However, adenoviruses that are defective in the E1 region or deleted for the E4 region are unable to assist AAV replication. Thus, in the case of adenovirus helper proteins, the E1A and E4 regions are likely required for AAV replication, either directly or indirectly (see, for example, Laughlin et al., J. Virol. 41(1982)868; Janik et al., Proc. Natl. Acad. Sci. USA 78(1981)1925; Carter et al., Virology 126(1983)505). Other characteristic adenovirus mutants include the following: the above-mentioned E1B (Laughlin et al. (1982), the above-mentioned Janik et al. (1981), Ostrove et al., Virology 104(1980)502), E2A (Handa et al., J. Gen. Virol. 29(1975)239, Strauss et al., J. Virol. 17(1976)140, Myers et al., J. Virol. 35(1980)665, Jay et al., Proc. Natl. Acad. Sci. USA 78(1981)2927, Myers et al., J. Biol. Chem. 256(1981)567), E2B (Carter, Adeno-Associated Virus Helper Functions, in I CRC Handbook of Parvoviruses (P. Tijssen ed., 1990)), E3 (the above-mentioned Carter et al. (1983)), and E4 (the above-mentioned Carter et al. (1983), Carter(1995)).

[0149] Studies of helper proteins provided by adenoviruses having mutations in E1B have reported that the E1B 55 kDa protein is required for the production of AAV particles, but the E1B 19 kDa protein is not. Further, WO 97 / 17458 and Matshushita et al. (Gene Therapy 5 (1998) 938-945) described helper function plasmids encoding various adenovirus genes. Examples of helper plasmids include the adenovirus VA RNA coding region, the adenovirus E4orf6 coding region, the adenovirus E2A 72 kDa coding region, the adenovirus E1A coding region, and the adenovirus E1B region lacking the intact E1B 55 kDa coding region (see, for example, WO 01 / 83797).

[0150] Accordingly, provided herein is a method for generating a recombinant AAV vector comprising a nucleic acid encoding a protein or a nucleic acid transcribed into a transcript of interest, or an AAV particle comprising the recombinant AAV vector, using the method of the invention for mammalian cell growth prior to transfection and viral particle production.

[0151] For this purpose, three plasmids are co-transfected into mammalian cells grown using perfusion according to the invention. The transgene plasmid (pTransgen) encodes an expression cassette cloned between AAV ITRs, while the rep and cap genes are provided in trans by co-transfecting a second packaging plasmid (pRep / Cap) to ensure AAV replication and packaging. A third plasmid, also called the helper plasmid (pHelper), contains minimal helper virus factors, generally the adenovirus E2A, EV, and VA genes, but lacks AAV ITRs.

[0152] One aspect of the invention is a method for producing a recombinant AAV vector comprising a nucleic acid encoding a protein or a nucleic acid transcribed into a transcript of interest, or an AAV particle comprising the recombinant AAV vector, comprising (i) Preparing one or more plasmids containing a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein; (ii) Preparing a plasmid containing a nucleic acid encoding a protein of interest or a nucleic acid transcribed into a transcription product of interest; (iii) Contacting the prepared plasmid with one or more mammalian cells or insect cells obtained by the method of the present invention; (iv) Further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture, or introducing the nucleic acid into the cells by physical means such as an electric current; (v) Culturing the transfected cells; (vi) Recovering the cultured cells and / or the culture medium from the cultured cells to produce a recovery of the cells and / or the culture medium; (vii) Lysing the cells and optionally isolating recombinant AAV vectors or AAV particles from the cell and / or medium recovery lysate; Thereby, producing a recombinant AAV vector or AAV particle containing a nucleic acid encoding a protein of interest or a nucleic acid transcribed into a transcription product of interest is a method comprising.

[0153] One aspect of the present invention is a method for producing a recombinant AAV vector containing a nucleic acid encoding a protein or a nucleic acid transcribed into a transcription product of interest, or an AAV particle containing the recombinant AAV vector, comprising: (i) Preparing one or more plasmids containing a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein; (ii) Preparing a plasmid containing a nucleic acid encoding a protein of interest or a nucleic acid transcribed into a transcription product of interest; (iii) (a) Prepare one or more mammalian cells obtained by the method according to the present invention and contact them with the prepared plasmid in (i) to produce stably transfected cells, further add a transfection reagent, and optionally incubate the plasmid / transfection reagent / cell mixture or provide physical means such as an electric current to introduce the nucleic acid into the cells. Select the first stably transfected cells, contact the selected first stably transfected cells with the prepared plasmid in (ii), further add a transfection reagent, and optionally incubate the plasmid / transfection reagent / cell mixture or provide physical means such as an electric current to introduce the nucleic acid into the cells. Or, (b) Prepare transiently transfected cells by contacting one or more mammalian cells or insect cells obtained by the method of the present invention with the prepared plasmids in (i) and (ii), further add a transfection reagent, and optionally incubate the plasmid / transfection reagent / cell mixture or provide physical means such as an electric current to introduce the nucleic acid into the cells. Either of the steps of performing either, (iv) Culturing the transfected cells in (iii); (v) Recovering the cultured cells and / or the culture medium from the cultured cells to produce a recovery of the cells and / or the culture medium; (vi) Lysing the cells and optionally isolating recombinant AAV vectors or AAV particles from the cell and / or medium recovery lysate, Thereby, generating recombinant AAV vectors or AAV particles containing nucleic acids encoding the protein of interest or nucleic acids transcribed into the transcription product of interest A method comprising.

[0154] One aspect of the present invention is a method for generating a recombinant AAV vector containing a nucleic acid encoding a protein or a nucleic acid transcribed into a transcription product of interest, or an AAV particle containing the recombinant AAV vector, comprising: (i) Preparing mammalian cells or insect cells comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein; (ii) Preparing a plasmid comprising a nucleic acid encoding a protein of interest or a nucleic acid transcribed into a transcription product of interest; (iii) (a) By contacting one or more mammalian cells or insect cells obtained by the method according to the present invention with the plasmid prepared in (i), producing stably transfected cells, further adding a transfection reagent, and optionally incubating the plasmid / transfection reagent / cell mixture or providing physical means such as an electric current to introduce the nucleic acid into the cells, selecting the first stably transfected cells, contacting the selected first stably transfected cells with the plasmid prepared in (ii), further adding a transfection reagent, and optionally incubating the plasmid / transfection reagent / cell mixture or providing physical means such as an electric current to introduce the nucleic acid into the cells, or (b) By contacting one or more mammalian cells or insect cells obtained by the method of the present invention with the plasmids prepared in (i) and (ii) to produce transiently transfected cells, further adding a transfection reagent, and optionally incubating the plasmid / transfection reagent / cell mixture or providing physical means such as an electric current to introduce the nucleic acid into the cells, either of the steps; (iv) Culturing the transfected cells of (iii); (v) Recovering the cultured cells and / or the culture medium from the cultured cells to produce a recovery of the cells and / or the culture medium; (vi) Lysing the cells and optionally isolating and / or purifying recombinant AAV vectors or AAV particles from the cell and / or medium recovery; Thereby, producing recombinant AAV vectors or AAV particles comprising a nucleic acid encoding a protein of interest or a nucleic acid transcribed into a transcription product of interest; A method comprising...

[0155] The introduction of nucleic acids (plasmids) into cells can be carried out by a plurality of methods.

[0156] A variety of methods for DNA transfer into mammalian cells have been reported in the art. All of these are useful in the method according to the present invention. In certain embodiments of all aspects and embodiments, electroporation, nucleofection or microinjection for nucleic acid transfer / transfection is used. In certain embodiments of all aspects and embodiments, inorganic substances (e.g., calcium phosphate / DNA coprecipitation, etc.), cationic polymers (e.g., polyethyleneimine, DEAE-dextran, etc.) or cationic lipids (lipofection) are used for nucleic acid transfer / transfection. Calcium phosphate and polyethyleneimine are the most commonly used reagents for transfection for large-scale nucleic acid transfer (see, for example, Baldi et al., Biotechnol. Lett. 29 (2007) 677-684), and polyethyleneimine is preferred.

[0157] Improvements in the efficiency and reproducibility of transfection conditions using PEI as a transfection reagent for growth in serum-free suspension culture enable easy scale-up of AAV production using shake flasks, waves or stirred tank bioreactors.

[0158] In certain embodiments of all aspects and embodiments, the nucleic acid (plasmid) is provided as a composition in combination with polyethyleneimine (PEI), optionally in combination with cells. In certain embodiments, the composition comprises the following plurality of components: (a) one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein, (b) a plasmid comprising a nucleic acid encoding a protein or transcribed into a transcript of interest, (c) a plasmid / PEI mixture having a polyethyleneimine (PEI) solution. In certain embodiments, the plasmids are in a molar ratio range of about 1:0.01 to about 1:100, or in a molar ratio range of about 100:1 to about 1:0.01, and the mixture of components (a), (b) and (c) is optionally incubated for a period of about 10 seconds to about 4 hours.

[0159] In certain embodiments of all aspects and embodiments, the composition further comprises cells. In certain embodiments, the cells are in contact with the plasmid / PEI mixture of components (a), (b) and / or (c).

[0160] In certain embodiments of all aspects and embodiments, the composition further comprises free PEI, optionally in combination with cells. In certain embodiments, the cells are in contact with free PEI.

[0161] In certain embodiments of all aspects and embodiments, the cells are in contact with the mixture of components (a), (b) and / or (c) for at least about 4 hours, or about 4 hours to about 140 hours, or about 4 hours to about 96 hours. In a preferred embodiment, the cells are in contact with the mixture of components (a), (b) and / or (c), and optionally free PEI, for at least about 4 hours.

[0162] The composition may comprise additional plasmids and / or cells. Such plasmids and cells may be in contact with free PEI. In certain embodiments, the plasmids and / or cells are in contact with free PEI for at least about 4 hours, or about 4 hours to about 140 hours, or about 4 hours to about 96 hours.

[0163] The present invention also provides a method for generating transfected cells. The method includes the steps of providing one or more plasmids, preparing a solution containing polyethyleneimine (PEI), and mixing the plasmid(s) with the PEI solution to produce a plasmid / PEI mixture. In certain embodiments, such a mixture is incubated for a period ranging from about 10 seconds to about 4 hours. In such a method, the cells are then contacted with the plasmid / PEI mixture to produce a plasmid / PEI cell culture, and then free PEI is added to the plasmid / PEI cell culture produced to produce a free PEI / plasmid / PEI cell culture, and then the resulting free PEI / plasmid / PEI cell culture is incubated for at least about 4 hours, thereby generating transfected cells. In certain embodiments, the plasmid includes one or more or all of the rep open reading frame, the cap open reading frame, the E1A, E1B, E2, and E4orf6 open reading frames, and the nucleic acids encoding a protein or transcribed into a transcript of interest.

[0164] A method for generating transfected cells that produce recombinant AAV vectors or AAV particles, comprising providing one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein, providing a plasmid comprising a nucleic acid encoding a protein or transcribed into a transcription product of interest, providing a solution comprising polyethyleneimine (PEI), mixing the aforementioned plasmid with the PEI solution to produce a plasmid / PEI mixture, wherein the plasmid is in a molar ratio range of about 1:0.01 to about 1:100 or in a molar ratio range of about 100:1 to about 1:0.01, mixing the aforementioned plasmid with the PEI solution to produce a plasmid / PEI mixture (and optionally incubating the plasmid / PEI mixture for a period ranging from about 10 seconds to about 4 hours), contacting mammalian cells with the plasmid / PEI mixture to produce a plasmid / PEI cell culture, adding free PEI to the resulting plasmid / PEI cell culture to produce a free PEI / plasmid / PEI cell culture, and incubating the free PEI / plasmid / PEI cell culture for at least about 4 hours, thereby generating transfected cells that produce recombinant AAV vectors or particles comprising a nucleic acid encoding a protein or transcribed into a transcription product of interest, and thereby obtaining mammalian cells by the method according to the present invention, is further provided.

[0165] Furthermore, a method for generating a recombinant AAV vector or AAV particle comprising a nucleic acid encoding a protein or a nucleic acid transcribed into a transcription product of interest, the method comprising preparing one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein, preparing a plasmid comprising a nucleic acid encoding a protein of interest or a nucleic acid transcribed into a transcription product of interest, preparing a solution comprising polyethyleneimine (PEI), mixing the aforementioned plasmid with the PEI solution to produce a plasmid / PEI mixture, wherein the plasmid is in a molar ratio range of about 1:0.01 to about 1:100 or in a molar ratio range of about 100:1 to about 1:0.01, mixing the aforementioned plasmid with the PEI solution to produce a plasmid / PEI mixture (and optionally incubating the plasmid / PEI mixture for a period in the range of about 10 seconds to about 4 hours), contacting mammalian cells with the plasmid / PEI mixture produced as described to produce a plasmid / PEI cell culture, adding free PEI to the plasmid / PEI cell culture produced as described to produce a free PEI / plasmid / PEI cell culture, incubating the produced plasmid / PEI cell culture or free PEI / plasmid / PEI cell culture for at least about 4 hours to produce transfected cells, recovering the produced transfected cells and / or the culture medium from the produced transfected cells to produce a cell and / or culture medium recovery, lysing the cells and optionally isolating a recombinant AAV vector or particle from the cell and / or medium recovery lysate, obtaining mammalian cells using the method according to the invention, and thereby generating a recombinant AAV vector particle comprising a nucleic acid encoding a protein or a nucleic acid transcribed into a transcription product of interest, is provided.

[0166] Methods for generating recombinant AAV vectors or AAV particles using the method according to the present invention can include one or more additional steps or features. Exemplary steps or features include, but are not limited to, the step of recovering the cultured mammalian cells produced and / or recovering the culture medium from the cultured cells produced to generate a cell and / or culture medium recovery. Further exemplary steps or features include, but are not limited to, lysing the collected cells and optionally isolating the recombinant AAV vector or AAV particles from the cell and / or culture medium recovery lysate, thereby obtaining mammalian cells prior to transfection using the method according to the present invention, thereby generating a recombinant AAV vector or AAV particle comprising a nucleic acid encoding a protein or transcribed into a transcript of interest.

[0167] In certain embodiments of all aspects and embodiments, PEI is added to the plasmid and / or cells at various times. In certain embodiments, free PEI is added to the cells before, at the same time as, or after contacting the plasmid / PEI mixture with the cells.

[0168] In certain embodiments of all aspects and embodiments, the cells are at a particular density and / or cell growth phase and / or viability when contacted with the plasmid / PEI mixture and / or free PEI. In a preferred embodiment, the cells are at a density in the range of about 1×10 5 cells / mL to about 1×10 8 cells / mL. In certain embodiments, the viability of the cells when contacted with the plasmid / PEI mixture or free PEI is about 60% or more than 60%, or the cells are in the logarithmic growth phase when contacted with the plasmid / PEI mixture, or the viability of the cells when contacted with the plasmid / PEI mixture or free PEI is about 90% or more than 90%, or the cells are in the logarithmic growth phase when contacted with the plasmid / PEI mixture or free PEI.

[0169] In addition to PEI, valproic acid (VPA) can be used to improve transfection efficiency. VPA is a branched short-chain fatty acid that inhibits histone deacetylase activity. For this reaction, it is commonly added to mammalian cell cultures as an enhancer of recombinant protein production.

[0170] In certain embodiments of all aspects and embodiments, the encoded AAV packaging proteins include AAV rep and / or AAV cap. In certain embodiments of all aspects and embodiments, such AAV packaging proteins include AAV rep and / or AAV cap proteins of any AAV serotype.

[0171] In certain embodiments of all aspects and embodiments, the encoded helper proteins include adenovirus E1A and E1B, adenovirus E2 and / or E4, VA RNA, and / or non-AAV helper proteins.

[0172] In certain embodiments of all aspects and embodiments, the nucleic acid (plasmid) is used in a specific amount or ratio. In certain embodiments, the total amount of a plasmid containing a nucleic acid encoding a protein or transcribed into a transcript of interest and one or more plasmids containing a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein ranges from about 0.1 μg to about 15 μg per mL of cells. In certain embodiments, the molar ratio of a plasmid containing a nucleic acid encoding a protein or transcribed into a transcript of interest to one or more plasmids containing a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein is within the range of about 1:5 to about 1:1, or within the range of about 1:1 to about 5:1.

[0173] In certain embodiments of all aspects and embodiments, the first plasmid contains a nucleic acid encoding an AAV packaging protein, and the second plasmid contains a nucleic acid encoding a helper protein.

[0174] In certain embodiments of all aspects and embodiments, the molar ratio of a plasmid containing a nucleic acid encoding a protein or a nucleic acid transcribed into a transcription product of interest, a first plasmid containing a nucleic acid encoding an AAV packaging protein, and a second plasmid containing a nucleic acid encoding a helper protein is in the range of about 1-5:1:1, or 1:1-5:1, or 1:1:1-5 in co-transfection.

[0175] In certain embodiments of all aspects and embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a mammalian cell. In a preferred embodiment, the cell is a HEK293 cell or a CHO cell.

[0176] The culture can be carried out using conditions of about 37 °C, 95% humidity and 8% by volume CO2, which are commonly used for culturing eukaryotic cells. The culture can be carried out in serum-containing medium or serum-free medium, adherent culture or suspension culture. Suspension culture can be carried out in any fermentation vessel such as, for example, a stirred tank reactor, a wave reactor, a rocking bioreactor, a shaker vessel or a spinner vessel, or a so-called roller bottle. Transfection can be carried out in high-throughput format and screening, respectively, for example, in 96 or 384 well format.

[0177] The method according to the present invention includes AAV particles of any serotype or variants thereof. In certain embodiments of all aspects and embodiments, the recombinant AAV particles include any of AAV serotypes 1-12, AAV VP1, VP2 and / or VP3 capsid proteins, or modified or variant AAV VP1, VP2 and / or VP3 capsid proteins, or wild-type AAV VP1, VP2 and / or VP3 capsid proteins. In certain embodiments of all aspects and embodiments, the AAV particles include an AAV serotype or an AAV pseudotype, and the AAV pseudotype includes an AAV capsid serotype different from the ITR serotype.

[0178] The methods according to the invention that provide or contain AAV vectors or particles can also contain other elements. Examples of such elements include, but are not limited to, introns, expression control elements, one or more adeno-associated virus (AAV) inverted terminal repeat sequences (ITRs) and / or filler / stuffer polynucleotide sequences. Such elements can be present in or adjacent to a nucleic acid encoding a protein or transcribed into a transcript of interest, or an expression control element can be operably linked to a nucleic acid encoding a protein or transcribed into a transcript of interest, or an AAV ITR(s) can be adjacent to the 5' or 3' end of a nucleic acid encoding a protein or transcribed into a transcript of interest, or a filler polynucleotide sequence can be adjacent to the 5' or 3' end of a nucleic acid encoding a protein or transcribed into a transcript of interest.

[0179] Expression control elements include constitutive or regulatable control elements such as tissue-specific expression control elements or promoters.

[0180] The ITR can be of any of the AAV2 or AAV6 or AAV8 or AAV9 serotypes, or a combination thereof. The AAV particles can contain any VP1, VP2 and / or VP3 capsid protein having at least 75% sequence identity to any of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV-2i8, AAV rh74 or AAV 7m8 VP1, VP2 and / or VP3 capsid proteins, or can contain a modified or variant VP1, VP2 and / or VP3 capsid protein selected from any of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV-2i8, AAV rh74 and AAV 7m8 AAV serotypes.

[0181] After the production of the recombinant virus (e.g., AAV) particles described herein, if desired, the virus (e.g., rAAV) particles can be purified and / or isolated from host cells using various conventional methods. Such methods include column chromatography, CsCl gradient, iodixanol gradient, and the like.

[0182] For example, multiple column purification steps such as purification by an anion exchange column, an affinity column, and / or a cation exchange column can be used. (See, for example, WO 02 / 12455 and US 2003 / 0207439). Alternatively or additionally, an iodixanol or CsCl gradient step can be used (see, for example, US 2012 / 0135515 and US 2013 / 0072548). Further, when using infectious virus to express packaging and / or helper proteins, various methods can be used to inactivate residual virus. For example, adenovirus can be inactivated by heating to a temperature of approximately 60°C for, for example, 20 minutes or more. Since AAV is heat stable while helper adenovirus is heat labile, this treatment effectively inactivates the helper virus.

[0183] The purpose of the rAAV vector production and purification system is to implement a method for minimizing / controlling the generation of product-related impurities such as wild-type / pseudo-wild-type AAV species (wtAAV) and protein, nucleic acid, and vector-related impurities including AAV encapsidated residual DNA impurities.

[0184] Considering that rAAV particles are a very small fraction of the biomass, rAAV particles need to be purified to a level of purity that can be used as a clinical human gene therapy product (see, for example, Smith P.H., et al., Mo. Therapy 7 (2003) 8348, Chadeuf G., et al, Mo. Therapy 12 (2005) 744, report from the CHMP gene therapy expert group meeting, European Medicines Agency EMEA / CHMP 2005, 183989 / 2004).

[0185] As a first step, typically, the cultured cells that produce rAAV particles are recovered, optionally in combination with the recovered cell culture supernatant (medium) in which the cells (suspension or adherent) that produce rAAV particles were cultured. The recovered cells and optionally the cell culture supernatant can be used as is, lysed, or concentrated. Further, if infection is used to express the helper function, residual helper virus can be inactivated. For example, adenovirus can be inactivated by heating to a temperature of approximately 60 °C for, for example, 20 minutes or more, which inactivates only the helper virus since AAV is heat stable while the helper adenovirus is heat labile.

[0186] The cells and / or the supernatant of the recovery are lysed by disrupting the cells, for example, by chemical or physical means such as detergents, microfluidization, and / or homogenization to release the rAAV particles. During or after cell lysis, a nuclease, for example, benzonase, is added to degrade contaminating DNA. Typically, the resulting lysate is clarified to remove cell debris, for example, by filtration or centrifugation, giving a clarified cell lysate. In certain examples, the lysate is filtered through a filter with a pore diameter in microns (for example, a filter with a pore diameter of 0.1 - 10.0 μm, for example, a filter with a pore diameter of 0.45 μm and / or 0.2 μm) to produce a clarified lysate.

[0187] The lysate (optionally clarified) contains AAV particles (including rAAV vectors and empty capsids) as well as product / process-related impurities, such as soluble cell components from host cells that can include, inter alia, cellular proteins, lipids and / or nucleic acids, and cell culture medium components. The optionally clarified lysate is then subjected to a purification process to purify the AAV particles (including rAAV vectors) from the impurities using chromatography. The clarified lysate can be diluted or concentrated with an appropriate buffer prior to the first chromatography step.

[0188] After cell lysis, any clarification, and any dilution or concentration, multiple subsequent consecutive chromatography steps can be used to purify the rAAV particles.

[0189] For example, the removal of empty capsids from full capsids during downstream processing is based on their different isoelectric points (pI) in anion exchange chromatography. The average pI calculated across all serotypes is 5.9 for full capsids and 6.3 for empty capsids (Venkatakrishnan, B. et al., J. Virol. 87 (2013) 4974 - 4984).

[0190] The first chromatography step can be cation exchange chromatography or anion exchange chromatography. If the first chromatography step is cation exchange chromatography, the second chromatography step can be anion exchange chromatography or size exclusion chromatography (SEC). Thus, in certain embodiments of all aspects and embodiments, rAAV particle purification is by cation exchange chromatography, followed by purification by anion exchange chromatography.

[0191] Alternatively, if the first chromatography step is cation exchange chromatography, the second chromatography step can be size exclusion chromatography (SEC). Thus, in certain embodiments of all aspects and embodiments, rAAV particle purification is by cation exchange chromatography, followed by purification by size exclusion chromatography (SEC).

[0192] Further alternatively, the first chromatography step may be affinity chromatography. If the first chromatography step is affinity chromatography, the second chromatography step can be anion exchange chromatography. Thus, in certain embodiments of all aspects and embodiments, rAAV particle purification is by affinity chromatography, followed by purification by anion exchange chromatography.

[0193] Optionally, a third chromatography can be added to the aforementioned chromatography steps. Typically, any third chromatography step follows cation exchange, anion exchange, size exclusion or affinity chromatography.

[0194] Thus, in certain embodiments of all aspects and embodiments, rAAV particle purification is by cation exchange chromatography, followed by purification by anion exchange chromatography, followed by purification by size exclusion chromatography (SEC).

[0195] Furthermore, in certain embodiments of all aspects and embodiments, further rAAV particle purification is by cation exchange chromatography, followed by purification by size exclusion chromatography (SEC), followed by purification by anion exchange chromatography.

[0196] In further embodiments of all aspects and embodiments, rAAV particle purification is by affinity chromatography, followed by purification by anion exchange chromatography, followed by purification by size exclusion chromatography (SEC).

[0197] In further embodiments of all aspects and embodiments, rAAV particle purification is by affinity chromatography, followed by purification by size exclusion chromatography (SEC), followed by purification by anion exchange chromatography.

[0198] Cation exchange chromatography functions to separate AAV particles from cell components and other components present in the clarified lysate and / or column eluate from affinity chromatography or size exclusion chromatography. Examples of strong cation exchange resins that can bind rAAV particles over a wide pH range include, but are not limited to, any sulfonic acid-based resin indicated by the presence of a sulfonate functional group, including aryl and alkyl substituted sulfonates such as sulfopropyl or sulfoethyl resins. Representative matrices include, but are not limited to, POROS HS, POROS HS 50, POROS XS, POROS SP, and POROS S (strong cation exchangers available from Thermo Fisher Scientific, Inc., Waltham, MA, USA). Further examples include Capto S, Capto S ImpAct, Capto S ImpRes (strong cation exchangers available from GE Healthcare, Marlborough, MA, USA), and the commercially available DOWEX®, AMBERLITE®, and AMBERLYST® resin families available from Aldrich Chemical Company (Milliwaukee, WI, USA). Examples of weak cation exchange resins include, but are not limited to, any carboxylic acid-based resin. Exemplary cation exchange resins include carboxymethyl (CM), phospho (based on a phosphate functional group), sulfomethyl (S), and sulfopropyl (SP) resins.

[0199] Anion exchange chromatography functions to separate AAV particles from proteins, cell components, and other components present in the clarified lysate and / or column eluate from affinity chromatography or cation exchange chromatography or size exclusion chromatography. The amount of empty capsids in the eluate can also be reduced and thereby controlled using anion exchange chromatography. For example, an anion exchange column bound with rAAV particles can be washed with a solution containing a moderate concentration (e.g., about 100 - 125 mM, e.g., 110 - 115 mM) of NaCl, and a portion of the empty capsids can be eluted in the flow-through without substantially eluting the rAAV particles. Subsequently, the rAAV particles bound to the anion exchange column can be eluted using a solution containing a higher concentration (e.g., about 130 - 300 mM NaCl) of NaCl, thereby generating a column eluate having a reduced or depleted amount of empty capsids and a proportionally increased amount of rAAV particles containing the rAAV vector.

[0200] Exemplary anion exchange resins include, but are not limited to, those based on polyamine resins and other resins. Examples of strong anion exchange resins include, but are not limited to, those generally based on quaternized nitrogen atoms, including quaternary ammonium salt resins such as trialkylbenzylammonium resins. Suitable exchange chromatography materials include, but are not limited to, MACRO PREP Q (a strong anion exchanger available from BioRad, Hercules, California, USA), UNOSPHERE Q (a strong anion exchanger available from BioRad, Hercules, California, USA), POROS 50HQ (a strong anion exchanger available from Applied Biosystems, Foster City, California, USA), POROS XQ (a strong anion exchanger available from Applied Biosystems, Foster City, California, USA), POROS SOD (a weak anion exchanger available from Applied Biosystems, Foster City, California, USA), POROS 50PI (a weak anion exchanger available from Applied Biosystems, Foster City, California, USA), Capto Q, Capto XQ, Capto Q ImpRes, and SOURCE 30Q (powerful anion exchangers available from GE healthcare, Marlborough, Massachusetts, USA), DEAE Sepharose (a weak anion exchanger available from Amersham Biosciences, Piscataway, New Jersey, USA), Q Sepharose (a powerful anion exchanger available from Amersham Biosciences, Piscataway, New Jersey, USA). Further exemplary anion exchange resins include aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE), and quaternary aminoethyl (QAE).

[0201] The manufacturing process for purifying recombinant AAV particles intended as a product for treating human diseases should achieve the following objectives: 1) consistent particle purity, potency, and safety, 2) scalability of the manufacturing process, 3) acceptable manufacturing costs.

[0202] An exemplary process for purifying recombinant AAV particles is reported in International Publication No. WO 2019 / 006390.

[0203] The methods for purifying and generating recombinant adeno-associated virus particles (rAAV particles) outlined below are scalable to large scales. For example, up to suspension cultures with volumes of 5, 10, 10 - 20, 20 - 50, 50 - 100, 100 - 200 liters or more. The methods for purifying and generating recombinant adeno-associated virus particles are applicable to a wide variety of AAV serotypes / capsid variants.

[0204] In certain embodiments of all aspects and embodiments, the purification of rAAV particles a) recovering a cell culture supernatant containing mammalian cells and / or rAAV particles to generate a recovery product; b) optionally, concentrating the recovery product generated in step (a) to generate a concentrated recovery product; c) lysing the recovery product generated in step (a) or the concentrated recovery product generated in step (b) to generate a lysate; d) treating the lysate generated in step (c) to reduce contaminating nucleic acids in the lysate, thereby generating a nucleic acid-reduced lysate; e) optionally, filtering the nucleic acid-reduced lysate generated in step (d) to generate a clarified lysate, and optionally diluting the clarified lysate to generate a diluted clarified lysate; f) subjecting the nucleic acid-reduced lysate of step (d), the clarified lysate of step (e), or the diluted clarified lysate generated in step (e) to cation exchange column chromatography to generate a column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities or other product / process-related impurities, and optionally diluting the column eluate to generate a diluted column eluate. g) Subjecting the column eluate or diluted column eluate generated in step (f) to anion exchange chromatography to generate a second column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities or product / process-related impurities, and optionally concentrating the second column eluate to generate a concentrated second column eluate; h) Subjecting the second column eluate or concentrated second column eluate generated in step (g) to size exclusion column chromatography (SEC) to generate a third column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities or product / process-related impurities, and optionally concentrating the third column eluate to generate a concentrated third column eluate; and i) Filtering the third column eluate or concentrated third column eluate generated in step (h) to thereby generate purified rAAV particles, whereby the mammalian cells before transfection / production are obtained by the method according to the present invention.

[0205] In certain embodiments, steps (a)-(f) are maintained and combined with the following steps: g) Subjecting the column eluate or concentrated column eluate generated in step (f) to size exclusion column chromatography (SEC) to generate a second column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities or other product / process-related impurities, and optionally diluting the second column eluate to generate a concentrated second column eluate; h) Subjecting the second column eluate or diluted second column eluate generated in step (g) to anion exchange chromatography to generate a third column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities of product / process-related impurities, and optionally diluting the third column eluate to generate a diluted third column eluate; i) filtering the third column eluate produced in step (h) or the concentrated third column eluate, thereby producing purified rAAV particles, is combined.

[0206] In certain embodiments, steps (a)-(g) are maintained and combined with the following steps: h) filtering the second column eluate produced in step (g) or the concentrated second column eluate, thereby producing purified rAAV particles.

[0207] In an embodiment, steps (a)-(e) are maintained and combined with the following steps: f) subjecting the nucleic acid-reduced lysate of step (d), or the clarified lysate or diluted clarified lysate produced in step (e), to AAV affinity chromatography to produce a column eluate containing rAAV particles, thereby separating rAAV particles from protein impurities or other product / process-related impurities, and optionally concentrating the column eluate to produce a concentrated column eluate; and g) subjecting the column eluate or concentrated column eluate produced in step (f) to size exclusion column chromatography (SEC) to produce a second column eluate containing rAAV particles, thereby separating rAAV particles from protein impurities or other product / process-related impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; and h) optionally, subjecting the second column eluate or diluted second column eluate produced in step (g) to anion exchange chromatography to produce a third column eluate containing rAAV particles, thereby separating rAAV particles from protein impurities or other product / process-related impurities, and optionally diluting the third column eluate to produce a diluted third column eluate; and i) Filtering the second column eluate or diluted second column eluate produced in step (g), or filtering the third column eluate or concentrated third column eluate produced in step (h), thereby producing purified rAAV particles.

[0208] In certain embodiments of all aspects and embodiments, the concentration in step (b) and / or step (f) and / or step (g) and / or step (h) is by ultrafiltration / diafiltration, such as tangential flow filtration (TFF).

[0209] In certain embodiments of all aspects and embodiments, the concentration in step (b) reduces the volume of the recovered cells and cell culture supernatant to about 1 / 2 to 1 / 20.

[0210] In certain embodiments of all aspects and embodiments, the concentration in step (f) and / or step (g) and / or step (h) reduces the volume of the column eluate to about 1 / 5 to 1 / 20.

[0211] In certain embodiments of all aspects and embodiments, the lysis of the recoverate produced in step (a) or the concentrated recoverate produced in step (b) is by physical or chemical means. Non-limiting examples of physical means include microfluidization and homogenization. Non-limiting examples of chemical means include detergents. Detergents include non-ionic detergents and ionic detergents. A non-limiting example of a non-ionic detergent is Triton X-100. A non-limiting example of the detergent concentration is about 0.1 to 1.0% (v / v) or (w / v) (including both ends).

[0212] In certain embodiments of all aspects and embodiments, step (d) includes treating with a nuclease, thereby reducing contaminating nucleic acids. Non-limiting examples of nucleases include benzonase.

[0213] In certain embodiments of all aspects and embodiments, the filtration of the clarified lysate or diluted clarified lysate in step (e) is by a filter. Non-limiting examples of the filter are those having a pore size of about 0.1 micron to 10.0 microns (including both ends).

[0214] In certain embodiments of all aspects and embodiments, the dilution of the clarified lysate in step (e) is by a buffered phosphate, acetic acid, or Tris aqueous solution. Non-limiting examples of the solution pH are about pH 4.0 to pH 7.4 (including both ends). Non-limiting examples of the pH of the Tris solution are greater than pH 7.5, for example, about pH 8.0 to pH 9.0 (including both ends).

[0215] In certain embodiments of all aspects and embodiments, the dilution of the column eluate in step (f) or the second column eluate in step (g) is by a buffered phosphate, acetic acid, or Tris aqueous solution. Non-limiting examples of the solution pH are about pH 4.0 to pH 7.4 (including both ends). Non-limiting examples of the pH of the Tris solution are greater than pH 7.5, for example, about pH 8.0 to pH 9.0 (including both ends).

[0216] In certain embodiments of all aspects and embodiments, the rAAV particles obtained from step (i) are formulated with a surfactant to produce an rAAV particle formulation.

[0217] In certain embodiments of all aspects and embodiments, the anion exchange column chromatography in steps (f), (g), and / or (h) includes polyethylene glycol (PEG)-regulated column chromatography.

[0218] In certain embodiments of all aspects and embodiments, the anion exchange column chromatography in steps (g) and / or (h) is washed with a PEG solution before elution of the rAAV particles from the column.

[0219] In certain embodiments of all aspects and embodiments, PEG has an average molecular weight in the range of about 1,000 g / mol to 80,000 g / mol (including both ends).

[0220] In certain embodiments of all aspects and embodiments, the PEG is at a concentration of about 4% to about 10% (w / v) (including both ends).

[0221] In certain embodiments of all aspects and embodiments, the anion exchange column in step (g) and / or (h) is washed with an aqueous surfactant solution prior to elution of the rAAV particles from the column.

[0222] In certain embodiments of all aspects and embodiments, the cation exchange column in step (f) is washed with a surfactant solution prior to elution of the rAAV particles from the column.

[0223] In certain embodiments of all aspects and embodiments, the PEG solution and / or the surfactant solution comprises an aqueous Tris-HCl / NaCl buffer, an aqueous phosphate / NaCl buffer, or an aqueous acetate / NaCl buffer.

[0224] In certain embodiments of all aspects and embodiments, the NaCl concentration in the buffer or solution is in the range of about 20 to 300 mM NaCl (including both ends) or about 50 to 250 mM NaCl (including both ends).

[0225] In certain embodiments of all aspects and embodiments, the surfactant comprises a cationic or anionic surfactant.

[0226] In certain embodiments of all aspects and embodiments, the surfactant comprises a 12-carbon chain surfactant.

[0227] In certain embodiments of all aspects and embodiments, the surfactant comprises dodecyltrimethylammonium chloride (DTAC) or sarcosyl.

[0228] In certain embodiments of all aspects and embodiments, the rAAV particles are eluted from the anion exchange column in step (f), (g) and / or (h) with an aqueous Tris-HCl / NaCl buffer.

[0229] In certain embodiments of all aspects and embodiments, the Tris-HCl / NaCl buffer contains 100 to 400 mM NaCl (including both ends), optionally at a pH in the range of about pH 7.5 to about pH 9.0 (including both ends).

[0230] In certain embodiments of all aspects and embodiments, the anion exchange column of step (f), (g) and / or (h) is washed with an aqueous Tris-HCl / NaCl buffer.

[0231] In certain embodiments of all aspects and embodiments, the NaCl concentration in the aqueous Tris-HCl / NaCl buffer is in the range of about 75 to 125 mM (including both ends).

[0232] In certain embodiments of all aspects and embodiments, the aqueous Tris-HCl / NaCl buffer has a pH of about pH 7.5 to about pH 9.0 (including both ends).

[0233] In certain embodiments of all aspects and embodiments, the anion exchange column of step (f), (g) and / or (h) is washed one or more times to reduce the amount of empty capsids in the second or third column eluate.

[0234] In certain embodiments of all aspects and embodiments, anion exchange column washing removes empty capsids from the column before and / or instead of rAAV particle elution, thereby reducing the amount of empty capsids in the second or third column eluate.

[0235] In certain embodiments of all aspects and embodiments, anion exchange column washing removes at least about 50% of the total empty capsids from the column before and / or instead of rAAV particle elution, thereby reducing the amount of empty capsids in the second or third column eluate by about 50%.

[0236] In certain embodiments of all aspects and embodiments, the NaCl concentration in the aqueous Tris-HCl / NaCl buffer is in the range of about 110 to 120 mM (including both ends).

[0237] In certain embodiments of all aspects and embodiments, the ratio and / or amount of eluted rAAV particles and empty capsids is controlled by the wash buffer.

[0238] In certain embodiments of all aspects and embodiments, the rAAV particles are eluted from the cation exchange column of step (f) in an aqueous phosphate / NaCl buffer or an aqueous acetate / NaCl buffer. The non-limiting NaCl concentration in the buffer ranges from about 125 to 500 mM NaCl (including both ends). Non-limiting examples of the buffer pH are from about pH 5.5 to about pH 7.5 (including both ends).

[0239] In certain embodiments of all aspects and embodiments, the anion exchange columns of steps (f), (g) and / or (h) contain a quaternary ammonium functional group such as quaternized polyethyleneimine.

[0240] In certain embodiments of all aspects and embodiments, the size exclusion column (SEC) of steps (g) and / or (h) has a separation / fractionation range (molecular weight) from about 10,000 g / mol to about 600,000 g / mol (including both ends).

[0241] In certain embodiments of all aspects and embodiments, the cation exchange column of step (f) contains a functional group such as sulfonic acid or sulfopropyl.

[0242] In certain embodiments of all aspects and embodiments, the AAV affinity column contains a protein or ligand that binds to the AAV capsid protein. Non-limiting examples of the protein include antibodies that bind to the AAV capsid protein. More specific non-limiting examples include single-chain camelid antibodies (Camelid) that bind to the AAV capsid protein.

[0243] In certain embodiments of all aspects and embodiments, the method excludes the step of cesium chloride gradient ultracentrifugation.

[0244] In certain embodiments of all aspects and embodiments, the method recovers approximately 50-90% of all rAAV particles from the recovered material generated in step (a) or the concentrated recovered material generated in step (b).

[0245] In certain embodiments of all aspects and embodiments, the method generates rAAV particles having a higher purity than rAAV particles generated or purified by single AAV affinity column purification.

[0246] In certain embodiments of all aspects and embodiments, steps (c) and (d) are performed substantially simultaneously.

[0247] In certain embodiments of all aspects and embodiments, the NaCl concentration is adjusted to be in the range of about 100-400 mM NaCl (including both ends), or in the range of about 140-300 mM NaCl (including both ends), after step (c) but before step (f).

[0248] In certain embodiments of all aspects and embodiments, the cells are suspension-growing cells or adherent-growing cells.

[0249] In certain embodiments of all aspects and embodiments, the cells are mammalian cells. Non-limiting examples include HEK cells such as HEK-293 cells, and CHO cells such as CHO-K1 cells.

[0250] Methods for determining the infectivity titer of rAAV particles containing a transgene are known in the art (see, for example, Zhen et al., Hum. Gene Ther. 15 (2004) 709). Methods for assaying empty capsids and rAAV particles packaged with a transgene are known (see, for example, Grimm et al., Gene Therapy 6 (1999) 1322-1330; Sommer et al., Malec. Ther. 7 (2003) 122-128).

[0251] To determine the presence or amount of disassembled / denatured capsids, purified rAAV particles are subjected to SDS-polyacrylamide gel electrophoresis consisting of any gel capable of separating the three capsid proteins, e.g., a gradient gel, and then the gel is run until the samples are separated and the gel can be blotted onto a nylon or nitrocellulose membrane. Next, an anti-AAV capsid antibody is used as the primary antibody that binds to the denatured capsid protein (see, e.g., Wobus et al., J. Viral. 74 (2000) 9281-9293). The secondary antibody that binds to the primary antibody includes means for detecting the primary antibody. The binding between the primary and secondary antibodies is detected semi-quantitatively to determine the amount of capsid. Another method is analytical HPLC using an SEC column or an analytical ultracentrifuge.

[0252] Perfusion Continuous exchange of the medium in a perfusion bioreactor provides both continuous supply of nutrients by feeding and continuous removal of waste via the permeate. Thus, it is possible to overcome the accumulation of waste that adversely affects cell growth and viability observed in fed-batch processes. Furthermore, continuous medium renewal provides a more constant cell environment, which is beneficial for cell growth, health, metabolism, and product quality (Chotteau, V., “Perfusion Processes”, in Animal Cell Culture, M. Al-Rubeai, (Ed.) Cham: Springer International Publishing, 2015, pp. 407-443, Karst, D. J., et al., Curr. Opin. Biotechnol. 53 (2018) 76-84, Walther, J., et al., Biotechnol. J., 14 (2019) e1700733).

[0253] In the context of viral vector production, there has been an increasing amount of research focusing on perfusion techniques to further increase the yield of products, improve the full empty (F / E) ratio, and reduce the cost of viral vector production. The perfusion mode has already been demonstrated to be an advantageous method for the production of adenovirus, retrovirus, lentivirus, and AAV vectors in HEK293 cells (Ghani, K., et al., Biotechnol. Bioeng. 95 (2006) 653-660, Henry, O., et al., J. Process Cont. 17 (2007) 241-251, Henry, O., et al., Biotechnol. Bioeng. 86 (2004) 765-774, Ansorge, S., et al., J. Gene Med. 11 (2009) 868-876, Merten, O.W., J. Gene Med. 6 (2004) S105-S124, Benskey, M.J., et al., Hum. Gene Ther. Meth. 27 (2016) 32-45, Coronel, J., et al., Gen. Engin. Biotechnol. News 41 (2021) S23-S23).

[0254] Regarding perfusion in viral vector production, in contrast to the classical biotechnological production of recombinant proteins, the most important goal is no longer to achieve the highest possible cell density and maintain a long production period to increase volumetric productivity. Instead, for efficient transfection, the cells are about 2×10 6It is necessary to maintain at cells / mL. At higher cell densities, transfection is more difficult due to the widely described cell density effect (CDE) (Petiot, E., et al., Vaccine 33 (2015) 5974 - 5981, Le Ru, A., et al., Vaccine 28 (2010) 3661 - 3671, Bernal, V., et al., Biotechnol. Bioeng. 104 (2009) 162 - 180). When performed at higher cell densities, transfection efficiency and productivity decrease (Lavado - Garcia, J., et al., Front. Bioeng. Biotechnol. 8 (2020) Article 617).

[0255] Use continuous perfusion to continuously update the medium while maintaining a high percentage of cells in the exponential growth phase for efficient transfection (Henry, O., et al., Biotechnol. Bioeng. 86 (2004) 765 - 774, Cortin, V., et al., Biotechnol. Prog. 20 (2004) 858 - 863). Thus, viral vectors are produced up to a specific limited process period, and the viral vectors released into the medium can be rapidly recovered.

[0256] The perfusion rate represents the rate of medium regeneration in the perfusion process and is generally expressed as the volume of additional medium per working volume per day of the bioreactor (vvd). In the literature, various methods for perfusion rate control have been described, including methods based on cell density, the concentration of main substrates or by - products.

[0257] When the medium regeneration in the perfusion process is normalized with respect to the cell density, it is expressed as the specific cell medium supply rate. As explained by Dowd et al., perfusion control based on CSPR directly correlates the perfusion rate with the cell density (Cytotechnol. 42 (2003) 35 - 45). The lower the CSPR, the more cells can be maintained with a certain amount of medium. Therefore, by keeping the CSPR constant during the process, a consistent microenvironment for the cells in culture can be ensured.

[0258] To enhance the economic viability of the process, it is necessary to minimize the volumetric perfusion rate. However, it must be considered that if the CSPR falls below the lower limit, production may be impaired or become impossible due to poor cell growth, decreased viability, product degradation, or reduced specific productivity. Therefore, it is necessary to adjust the optimal CSPR close to the minimum CSPR during process development (Konstantinov, K., et al., Adv. Biochem. Eng. Biotechnol. 101 (2006) 75 - 98, Bielser, J.M., et al., Biotechnol. Prog. 36 (2020) e3026).

[0259] Inoculation train Conventional inoculation trains usually start by thawing a small amount of frozen cells, e.g., 1 mL of cryopreserved vials from the working cell bank. Subsequently, the cell culture grows into a larger culture system. For this purpose, the cells are cultured while increasing the volume in shake flasks, spinner flasks, and stirred - tank bioreactors. The production bioreactor is often called the N - th stage, while the bioreactors before the production stage are called N - 1, N - 2, N - 3 in sequence.

[0260] The use of high - density cell banking and disposable systems improves the operational outcome by reducing the duration and the potential for contamination of the inoculation train. This enables the simplification and reduction of the inoculation train, resulting in a reduction in process complexity, a shortening of the operation time, and an increase in yield.

[0261] Dielectric spectroscopy Dielectric spectroscopy, also called capacitance measurement or high-frequency impedance, is a method mainly used for on-line monitoring of viable cell density or viable cell volume. The measurement principle of this tool is based on the properties of living cells with intact non-conductive plasma membranes. This enables them to accumulate charges, so that living cells in an aqueous ionic suspension behave like small capacitors when introduced into an alternating electric field that periodically alternates in the radio frequency range (0.1 - 10 MHz).

[0262] The movement of intracellular and extracellular ions is restricted by the plasma membrane, which acts as an insulating physical barrier. This results in polarization, which is the separation of charges across the membrane in the polar direction of the cell. The magnitude of this polarization is measured by the capacitance ΔC in picofarads (pF).

[0263] Therefore, by measuring the capacitance of the suspension, its viable cell density can be estimated (J.P. Carvell and J.E. Dowd, Cytotechnol. 50 (2006) 35 - 48, Justice, C., et al., Biotechnol. Adv. 29 (2011) 391 - 401, Cole, H.E., et al., Processes 3 (2015) 384 - 405).

[0264] In most cells, this sigmoid curve is concentrated between 0.5 MHz and 3 MHz around the critical frequency fc. This is the frequency at which the fall of the capacitance is half completed (Cannizzaro, C., et al., Biotechnol. Bioeng. 84 (2003) 597 - 610). Usually, since the capacitance does not depend on the cell size, dielectric spectroscopy is performed at a frequency close to this frequency (Justice, C. cited above).

[0265] The capacitance increases as the volume fraction of the cells increases due to the presence of more polarization membranes, thereby resulting in a higher measured capacitance. Generally, the capacitance depends not only on the VCD but also on the cell type, cell size, and physiological state of the cells.

[0266] For VCD prediction, a mathematical model is required to correlate the capacitance signal with the offline biomass data. The cell constant K is multiplied by the capacitance ΔC, and the cell constant K is given in 1 / cm to obtain the absolute permittivity ε (pF / cm). TIFF2025519228000001.tif4128

[0267] The values of the biocapacitance for the control of perfusion processes in which perfusion feeding is automatically adjusted based on the viable cell concentration from an online capacitance probe have been demonstrated in several studies (Carvell and Dowd cited above, Cannizzaro, C., et al. cited above). Furthermore, dielectric spectroscopy has emerged as a relevant tool for monitoring the viral vector production process since it provides important information regarding the physiological state of the cells (Pais, D.A.M., et al., Processes 8 (2020) 1456, Escandell, J.M., et al., Curr. Opin. Biotechnol. 74 (2022) 271 - 277).

[0268] Specific embodiments of the method according to the present invention The scale - up of the production process of recombinant AAV particles to 100 L, 500 L, or even 1,000 L presents significant challenges for both cell growth and plasmid transfection.

[0269] Achieving a high capsid titer is the goal in the production of recombinant AAV particles. Thus, generating large cell aggregates results in the production of empty capsids and is not suitable for achieving this goal.

[0270] Therefore, the focus in the perfusion process for the production of viral vectors lies not in achieving high cell density, but in alleviating nutrient limitation by ensuring a constant medium replacement.

[0271] A variety of cell retention devices are available for cell retention in the process for manufacturing viral vectors. The most commonly used ones are alternating tangential flow filtration (ATF), tangential flow filtration (TFF), acoustic filters, and membrane-based internal retention systems.

[0272] The ATF technology uses a hollow fiber module connected to the bioreactor via a single port. Using a diaphragm pump, the cell suspension is pumped through the hollow fiber module, where the cells are retained and a clear supernatant containing the product of interest is recovered. Then, to remove membrane deposits, the flow is reversed and the remaining cell suspension is flowed back into the bioreactor. The diaphragm pump generates rapid low-shear flow, ensuring that the cells quickly return to the bioreactor. Recently, Coronel et al. showed that perfusion by an ATF system can be applied to increase the production of rAAV particles (Gen. Engin. Biotechnol. News 41(2021)S23 - S23).

[0273] However, in a specific embodiment of the method according to the present invention, perfusion is not performed by ATF.

[0274] More specifically, the TFF technology uses a hollow fiber system equivalent to ATF, but uses a low-shear circulation pump and two bioreactor ports instead of a diaphragm pump. One is for withdrawing liquid from the bioreactor, and the other is for returning liquid from the external cell retention device to the bioreactor. Various TFF systems are commercially available for this purpose (Xcellerex APS (trademark) by Cytiva, KML100 (trademark) by Repligen, or Cellicon (trademark) by Merck).

[0275] Suspension-based cell culture has been an important step towards achieving scalability from adherent culture. However, the cell density per volume is generally low compared to adherent cells. Therefore, to approach the transfection efficiency in adherent cultures, it is necessary to achieve a high cell density per volume. Furthermore, to ensure high productivity, consistent good cell growth and viability are required throughout the inoculation train and the production process. As the amount of product increases, this becomes more difficult, for example, with respect to sufficient gas sparging, efficient mixing, and nutrient supply. Therefore, the cells need to adapt to the changing environment. Furthermore, any change in the culture parameters can activate the internal control mechanisms of the cells, trigger responses, and lead to a decrease in cell and product yields or an increase in the formation of by-products.

[0276] In addition, regarding the transfection process, often a decrease in transfection efficiency occurs during scale-up.

[0277] To improve the production of recombinant AAV particles, it is necessary to improve the inoculation train to prevent substrate depletion and high accumulation of waste products.

[0278] This was achieved by ensuring the optimal physiological state of the cells before transfection, which enabled high transfection efficiency. This was achieved by reducing the proportion of spent medium in the production bioreactor from about 40% to about 20%. As a result, the concentration of inhibitory waste products present was lower, while at the same time the concentration of nutrients increased. This enabled the improvement of the cell growth rate at the start of the N process from about 0.3 / day to about 1.0 / day. Furthermore, the inoculation train could be simplified by omitting one bioreactor, which reduced the complexity of the process. Additionally, the required perfusion medium could be reduced by about 1 / 3, for example, by applying online viable cell density (VCD) measurement by dielectric spectroscopy and specific cell perfusion rate (CSPR).

[0279] Furthermore, by using the method according to the present invention, it was possible to more accurately control the cell density during transfection in the main fermentation tank, i.e., the production bioreactor. Thereby, it was possible to reduce or prevent the so-called cell density effect, i.e., the excess of cell density during transfection that results in a lower production yield in AAV production.

[0280] The present invention is at least partially based on the finding that it is advantageous for the host cells to be growing prior to transfection for the production of AAV particles, and thus it is advantageous to produce AAV particles using perfusion. More specifically, it has been found that for the recombinant production of AAV particles, it is advantageous to grow mammalian cells in a preculture using perfusion. An advantage of the present invention is that mammalian cells grown using perfusion prior to transfection maintain growth with a higher mitotic activity and a higher growth rate, and exhibit a higher survival rate after transfection, i.e., during the production culture. Thereby, a higher titer can be achieved at the same inoculation cell density as compared to a culture using mammalian cells cultured prior to transfection using only a batch method or a fed-batch method.

[0281] The present invention is at least partially based on the finding that it is advantageous to grow mammalian cells by perfusion starting from a low cell density in a cell culture and to dilute the mammalian cells grown prior to transfection with the transgene-encoding nucleic acid.

[0282] It has been found that without using perfusion, the nutrients are excessively reduced and the waste products accumulate to such an amount during the preculture that the resulting cells are severely affected and are no longer suitable for subsequent AAV particle production due to the impairment of cell growth in the culture.

[0283] In the following examples, parameters such as cell lines, AAV serotypes, media, transfection reagents, etc., as well as process parameters such as temperature, pH, and pO2 were kept constant or very similar, enabling a direct comparison of the results obtained with different processes. Therefore, the observed improvements are directly due to the differences in the culture conditions used for cell growth before transfection, i.e., the feeding.

[0284] In the comparative example, the production of recombinant AAV particles was carried out on a 100 L scale.

[0285] The main fermenter was set to be inoculated at a cell density of 20×10 5 cells / mL and an initial volume of 60 L, and it was necessary to reach the target of 1.2×10 11 cells after fermentation of N - 1. For the 25 L working volume of N - 1, in N - 1, a final density of at least 48×10 5 cells / mL was required. Since cells are known to double approximately daily, to provide a safety margin, the inoculation of N - 1 was carried out at 8×10 5 cells / mL, and a final density of approximately 60×10 5 cells / mL was reached. For the N - 2 fermentation, 5×10 5 cells / mL was inoculated according to the same considerations, and after 3 days, it reached approximately 40×10 5 cells / mL. The pH in the inoculation train was selected to support high cell growth while suppressing glucose consumption and the accumulation of toxic waste products. In the main fermenter, the pH was slightly increased to increase the production of AAV and their release into the medium.

[0286] However, in the N - 1 25L Rocker, a major problem occurred because cell growth and viability decreased after the N - 2 stage. Furthermore, the cells had already been too damaged and did not recover after being transferred to the main fermenter. This means that transfection could not be carried out because very high viability is required for efficient transfection.

[0287] At the N-1 stage, poorer cell growth was observed compared to the N-2 stage. At N-2, the cells grew from approximately 4×10 5 to almost 40×10 5 cells / mL during the 3-day growth period. However, at the subsequent N-1 stage, the cells started at 7.8×10 5 cells / mL and reached only 28.6×10 5 cells / mL within the same time frame.

[0288] The high growth rate of 0.79 / day at N-2 began to decline after the first day at N-1, decreased sharply, and reached only 0.47 / day after 3 days. At the same time, the viability, which had been consistently maintained at a high level at N-2, gradually decreased from 97.5% to 92.4% at N-1. After transferring the cell suspension to the main fermenter, the cells did not recover. Instead, the viability continued to decline from 94% to 86.9% before the planned transfection. Therefore, transfection could not be performed. The viability continued to decline to 70% after 4 days, and the growth rate also decreased similarly, reaching 0.14 / day by the end of the process.

[0289] There was no limitation at the end of the N-2 fermenter. Furthermore, the addition of fresh medium accompanied by the transfer to the N-1 fermenter increased the substrate concentration and restored it to its initial values. However, glucose limitation occurred at N-1 at the end of the process at a concentration of 0.4 g / L. Without being bound by this theory, another reason for poor cell growth could be the high production and accumulation of waste products. During N-2, a large amount of lactate and ammonium had already accumulated. From this, the fact that the starting concentrations at N-1 were already higher than those at N-2 is derived. Thus, at the end of the N-2 stage, lactate reached approximately 5 g / L and ammonium reached 52 mg / L.

[0290] Due to the low VCD at the end of N-1, it was necessary to transfer the entire fermentation volume to the main fermenter, reducing the starting volume to 50 L instead of 60 L to reach the desired inoculation density of 20×10 5 cells / mL.

[0291] This can be seen in Figure 1. Blue = N-2, fed batch; Green = N-1, fed batch; Red = production phase. The cells do not grow sufficiently in culture and invalidate transfection for AAV production.

[0292] It has been found here that when perfusion is used for the growth of mammalian cells prior to AAV particle production by the method according to the invention, an improvement is brought about regardless of whether the perfusion is uncontrolled or controlled perfusion.

[0293] In a first example according to the invention, a constant perfusion rate was set using N-1 stage perfusion. The perfusion rate increased once a day from 0.21 vvd (day 1) through 0.42 vvd (day 2) to 0.63 vvd (day 3). Thus, in this example, a total of 29.1 L of medium or 1.16 bioreactor volumes were supplied to the cell culture during perfusion.

[0294] However, since the pO2 value fluctuated greatly and decreased to almost 0% immediately after inoculation, it was necessary to adjust the aeration control. To improve the oxygen input to the fermenter, the rocking speed was increased after 3.5 hours and again after 25.5 hours. The O2 controller used here was a proportional-integral-derivative (PID) controller. Its basic operating principle is to read the sensor, calculate the difference between the desired setpoint (SP) and the measured process value (PV), and then apply a correction by setting the desired actuator output. This output is calculated by computing the proportional, integral, and derivative responses and summing those three components. To reduce the overshoot of the controller, the integral term was turned off after 6.5 hours. Thereafter, only the proportional and derivative gains were used. The aeration rate increased from 500 mL / min to 1000 mL / min after 26.5 hours.

[0295] The cells were from an initial VCD of 10.9×10 5 cells / mL to 92.3×10 5Higher cell growth was shown to the VCD of cells / mL. Therefore, compared to the first run of the comparison without perfusion, a cell density 3.2 times higher was reached at an inoculation density only 1.4 times higher. The viability was maintained at a high level throughout all runs and was at least 96.9% one day after inoculation, in contrast to the sharp decline in viability when perfusion was not used. The growth rate was very high at 1.05 / day at the start but decreased to 0.76 / day after 3 days during the process. Thus, the growth rate was 1.3 times higher at the start and 1.6 times higher at the end than the growth rate in the comparison N-1 fermentation without perfusion. Therefore, not only did the absolute growth rate increase, but the decrease in the growth rate in the process using perfusion also decreased.

[0296] The cells resumed growth after a 1.5-hour lag phase after inoculation in this fermentation.

[0297] Cell growth in N-2 and N-1 was very good, and the final VCD was 44×10 5 cells / mL in N-2. In N-1, a higher VCD of 92×10 5 cells / mL was reached by perfusion. Furthermore, the viability was always maintained at a high level exceeding 97%, and the final growth rate was 0.64 / day in N-2 and 0.77 / day in N-1, confirming good cell growth in the inoculum strain. This growth continued at a growth rate of 0.80 / day at the start of the main fermenter, which means that the transfer of the cell suspension from N-1 to the N bioreactor was successful.

[0298] As expected, the growth rate decreased after transfection of the cell culture on day 1, and the final value just before harvesting on day 4 was 0.12 / day. It should also be noted that the cell density of 26.77×10 5 cells / mL was inoculated instead of 20×10 5 cells / mL in the main fermentation. Therefore, the cell density of 52×10 5 cells / mL before medium addition was higher than the desired 36×10 5 cells / mL.

[0299] The results of uncontrolled perfusion at different culture volumes (scales) are shown in Figure 2. Blue = N-2, fed-batch at 10 L scale; green = N-1, uncontrolled perfusion at 25 L scale; red = production phase at 100 L scale. The perfusion rate was changed daily from 0.21 vvd to 0.42 vvd and then to 0.63 vvd. This led to consistent growth of the growing cells and high viability. Successful production of AAV particles of serotype 8 was achieved.

[0300] However, despite nutrient replenishment by perfusion, the glucose and glutamine concentrations decreased as in the process without using perfusion. As a result, substrate limitation occurred at the end of fermentation. Without being bound by this theory, this could be explained by the fact that perfusion achieved a significantly higher cell density, and accordingly, more cells consumed a larger absolute amount of substrate. This means that the applied perfusion rate was not sufficient to supply enough nutrients to keep their concentrations constant in rapidly growing cell cultures. Therefore, the perfusion rate was adjusted so that substrate depletion decreased or rather did not occur. Therefore, viable cell density control (VCD control) perfusion was used.

[0301] Therefore, the perfusion rate was calculated as outlined in the Examples section. Using the average specific growth rate from the first example according to the invention at 0.764 / day, the VCD was planned. The goal of the N-1 fermentation was to reach a final VCD of about 60×10 5 cells / mL in a 25 L working volume to provide sufficient cells for inoculation of the main fermenter. As a result, the working volume of the N-1 fermentation could be reduced while still achieving the required number of cells at a higher cell density. If the working volume was reduced to 10 L, a final cell density of at least 120×10 5 cells / mL was required. In light of the growth rate of 0.764 brought about by the first perfusion run, an inoculation density of 15×10 5 cells / mL would be required to reach a final cell density of 150×10 5 cells / mL. A scale-down model with a 1 L working volume was used.

[0302] Specific cell substrate consumption was determined according to the predicted VCD. For glucose (7.50×10 -10 g / cell / day) and glutamine (1.20×10 -10 g / cell / day), the specific cell consumption rates were calculated as the average of two previous processes using HEK293 cells in a 10 L rocker without perfusion. These were then used to determine the perfusion rate necessary to provide sufficient substrate for this specific cell consumption. Since the perfusion rate to keep the glutamine concentration constant was slower than that of glucose, the perfusion rate of glucose was used. The perfusion rate was increased once a day. To avoid nutrient limitation due to temporary interruption of medium supply or process variations, the daily rate was calculated using the predicted VCD at the end of each day.

[0303] The planned VCD was only slightly higher than the actually measured VCD reached at the end of the process. A growth rate of 0.673 / day was determined, which was lower than the predicted one and was 0.9-fold. As a result, a final cell density of 128×10 5 cells / mL was obtained after 2.9 days. Using the substrate concentration and VCD measured daily, the perfusion rate was manually readjusted during the process. The resulting perfusion rate was in very good agreement with the perfusion rate based on the process conditions. Generally, 2.36 L of medium was required for perfusion, which corresponded to a bioreactor volume of 2.36.

[0304] In the last process section, the VCD increased more during the run at a controlled perfusion rate, reaching a final value of 128×10 5 cells / mL instead of 92.3×10 5 cells / mL at the first perfusion run. This trend was also reflected in the growth rate. In the first perfusion process, the growth rate decreased from 0.90 / day to 0.77 / day during the process, especially between 2.2 and 2.9 days. In the process with controlled perfusion rate, the growth rate was generally low, with an average of 0.67 / day, but was maintained relatively constant and finally increased.

[0305] The viability also showed a slight decrease at the end of the first perfusion process, but was kept constant or slightly increased at the end of the perfusion process with controlled perfusion rate.

[0306] The controlled perfusion process using a regulated perfusion rate prevented the limitation of glucose and glutamine at the end of the culture. However, the substrate concentration could not be kept constant, and the concentrations of lactate and glutamate were the same as or slightly higher than those in the case of the first perfusion process.

[0307] The results of viable cell density control (VCD control) perfusion at 1 L scale are shown in Figure 3 (controlled perfusion). The perfusion rate was calculated using the specific consumption rate of cells based on cell density measurement and cell growth calculation. No substrate limitation was observed. To achieve a target cell density of 80 - 100×10 5 Cell division was performed after 3 days to achieve a target cell density of cells / mL.

[0308] The prediction of VCD using the growth rate from the previous perfusion process further improved the method according to the present invention. The VCD prediction provided a basis necessary for determining the subsequent perfusion rate. When using controlled perfusion, the substrate concentration could not be kept constant, but substrate limitation was prevented. This enabled consistent good cell growth and viability until the process ended at a constant growth rate. Compared with the first perfusion process, the controlled perfusion process used twice the amount of perfusion medium.

[0309] The results of VCD control perfusion at 100 L scale are shown in Figure 4. Green = N - 1, controlled perfusion; Red = production stage. The production of AAV particles of subtype 2 mutant 7m8 was successful.

[0310] The fermentation tank volume of the N - 1 bioreactor was increased to 15 L to achieve a cell density high enough to inoculate the main fermentation tank. However, after 3 days, to reach a target density of 100×10 5 cells / mL, approximately 13×10 5An inoculation VCD of cells / mL was required, and the volume of the necessary pre-culture was relatively large. Therefore, the N-1 process was carried out for a longer period, 6 days in this example. However, this requires a very low inoculation VCD of 1.6×10 5 cells / mL. Such a low inoculation VCD is difficult to set accurately because cell aggregates can cause measurement inaccuracies. Even a slight deviation from this can exponentially increase the medium consumption with the VCD, potentially resulting in a final VCD that is too low or an exorbitant increase in medium consumption. The solution to this problem is to inoculate at a higher and more easily adjustable VCD and split the cell culture during the process to reach the desired final VCD as accurately as possible. As a result, in the following perfusion process, inoculation was carried out at 5×10 5 cells / mL and split to 13×10 5 cells / mL on the 3rd day, reaching a final VCD of approximately 100×10 5 cells / mL after 6 days.

[0311] The cell culture was split from 55.4×10 5 to 10.5×10 5 cells / mL on the 3rd day and a final VCD of 97.4×10 5 cells / ml was obtained. The average growth rate in the first growth phase after splitting was determined to be 0.77 / day, and the average growth rate in the second growth phase was 0.78 / day. This correlated well with the predicted average growth rate of 0.72 / day in the previous two perfusion processes. The survival rate immediately after inoculation was 96%.

[0312] A total of 3.3 bioreactor volumes of perfusion medium was added throughout this process. No substrate limitation occurred during the first growth phase, and the substrate concentration was maintained even higher than in the previous perfusion process. However, the inoculation VCD was lower at 5.8×10 5 cells / mL compared to 17.4×10 5 cells / mL in the first 1L perfusion process.

[0313] This new setting of the N-1 fermenter did not shorten the overall duration of the inoculation train, but due to the low inoculation density of N-1, it enabled the removal of the N-2 fermenter.

[0314] The process was repeated using a capacitance probe to generate a dielectric constant signal to control the perfusion rate including the split 3 days after growth. Thereby, a method according to the invention with automated perfusion control was obtained. A linear correlation between the VCD measured offline and the dielectric constant measured online was demonstrated. The found correlation between viable cell density and dielectric constant enabled more economical control of the perfusion process, resulting in a reduction of about 30% of the perfusion culture medium (from 3.3 reactor volumes to 2.2 reactor volumes). The cell density curve of the dielectric constant controlled perfusion process is shown in Figure 5 (N-1, dielectric constant controlled perfusion, 10 L scale). The applied cell-specific perfusion rate was 107 pL / cell / day in this exemplary process. In other examples, a CSPR of 101 pL / cell / day was applied.

[0315] The process was repeated using an online biomass sensor to control the perfusion rate including the split 3 days after growth. Successful production of AAV particles of subtype 2 mutant 7m8 containing a transgene encoding a therapeutic Fab was achieved. The cell density curve of the biomass sensor controlled perfusion process is shown in Figure 6. Green = N-1, biomass sensor controlled perfusion; Red = production stage.

[0316] Figure 7 shows the two viable cell densities of two pairs of VCD controlled perfusion vs. biosensor controlled perfusion: Red (VCD) vs. Purple (biosensor); Blue (VCD) vs. Green (biomass sensor)).

[0317] Figure 8 shows the viable densities obtained in cultures of different production scales inoculated with cells obtained using perfusion at the N-1 stage, i.e., the cells according to the present invention, and cells obtained without using perfusion at the N-1 stage. The red curve corresponds to the production-scale culture inoculated with cells from the N-1 culture without perfusion, and the green curves (labeled "1", "2", "3", "4") correspond to the production-scale cultures inoculated with cells from the N-1 culture using perfusion according to the present invention. It can be seen that the viable cell density obtained in the culture inoculated with cells grown by perfusion at the N-1 stage is higher, and as a result, a higher AAV particle yield is obtained (transfection 24 hours after the start of the culture).

[0318] Figure 9 shows the cell viability in production-scale culture N according to the N-1 stage culture of the cells used for inoculation at the N stage. The red curve corresponds to the production culture inoculated with cells obtained from the batch N-1 culture. The green curves (labeled "1", "2", "3", "4") correspond to the production-scale cultures inoculated with cells obtained from the N-1 culture using perfusion according to the present invention. It can be seen that the cells obtained from N-1 perfusion grow better and have a better response to transfection 24 hours after the start, i.e., maintain a higher viability. These cells still continue to grow and produce rAAV particles.

[0319] Figure 10 shows the capsid titers of the production cultures in a bioreactor system of more than 10 L at N after N-1 with and without perfusion. The red curves ("5", "6") correspond to the production cultures after batch N-1. The green curves ("1", "2", "3", "4") correspond to the production-scale cultures after N-1 with perfusion. For the red curves, batch culture N-1 was carried out in the same bioreactor (thus, day 0 of the N-scale culture corresponds to day 4 in the figure). In an equivalent bioreactor system, the effect of the increase in capsid titer using cells cultured by perfusion at the N-1 stage for inoculation is significant.

[0320] Thus, the method according to the present invention achieves higher final cell density as well as capsid titer. When cells are grown using perfusion, the range of variation also becomes smaller. Without being bound by this theory, cells grown using perfusion are thought to have higher resistance to stress during transfection, recover faster, maintain a higher viable cell density, and are still growing slowly but steadily.

[0321] All references mentioned in this specification are incorporated herein by reference.

[0322] The following examples, sequences, and drawings are provided to assist in understanding the present invention, the true scope of which is set forth in the appended claims. It is understood that changes can be made to the described procedures without departing from the spirit of the present invention.

Examples

[0323] General Techniques 1) Recombinant DNA Technology DNA was manipulated using standard methods as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y, (1989). Molecular biology reagents were used according to the manufacturer's instructions.

[0324] 2) DNA and Protein Sequence Analysis and Sequence Data Management The EMBOSS (European Molecular Biology Open Software Suite) software package, Invitrogen's Vector NTI, and Geneious Prime were used for sequence creation, mapping, analysis, annotation, and illustration.

[0325] 3) Gene and Oligonucleotide Synthesis The desired gene segment is prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragment is cloned into an E. coli plasmid for propagation / amplification. The DNA sequence of the subcloned gene fragment is confirmed by DNA sequencing. Alternatively, short synthetic DNA fragments are constructed by annealing chemically synthesized oligonucleotides or via PCR. Each oligonucleotide is prepared by metabion GmbH (Planegg-Martinsried, Germany).

[0326] 4) Reagents Unless otherwise specified, all commercially available chemicals, antibodies, and kits are used as provided according to the manufacturer's protocol.

[0327] 5) Cloning Overview Cloning at the R site depends on the DNA sequence adjacent to the gene of interest (GOI), which is the same as the sequence in the following fragment. Similarly, fragment assembly is possible by overlapping equal sequences and subsequent sealing of nicks in the assembled DNA by DNA ligase. Therefore, cloning of a single gene, especially a preliminary plasmid containing an appropriate R site, is required. After successful cloning of these preliminary plasmids, the gene of interest flanked by the R sites is excised via restriction digestion with an enzyme that cleaves immediately adjacent to the R sites. The final step is to assemble all DNA fragments in one step. More specifically, 5' exonuclease removes the 5' end (R site) of the overlapping region. Subsequently, annealing of the R sites can be performed, and DNA polymerase extends the 3' end to fill the sequence gaps. Finally, DNA ligase seals the nicks between nucleotides. When an assembly master mix containing different enzymes such as exonuclease, DNA polymerase, and ligase is added and then the reaction mix is incubated at 50 °C, the single fragments are assembled into one plasmid. Subsequently, competent E. coli cells are transformed with the plasmid.

[0328] For some plasmids, cloning methods via restriction enzymes were used. By selecting appropriate restriction enzymes, the gene of the desired target can be excised and then inserted into another plasmid by ligation. Therefore, by using enzymes that cut at the multiple cloning site (MCS) and selecting in a smart way, ligation of the fragments within the correct array can be carried out. If the plasmid and the fragment have been previously cut with the same restriction enzyme, the sticky ends of the fragment and the plasmid will perfectly match and can then be ligated with DNA ligase. After ligation, competent E. coli cells are transformed with the newly created plasmid.

[0329] Cloning via Restriction Digestion For digestion of the plasmid with restriction enzymes, pipette the components shown in the following table together on ice.

[0330] (Table) Restriction Digestion Reaction Mixture TIFF2025519228000002.tif38170

[0331] If using more enzymes in a single digestion, use 1 μL of each enzyme and add more or less PCR-grade water to adjust the volume. All enzymes are qualified for use with New England Biolabs' CutSmart buffer (100% activity) and are selected on the premise that they have the same incubation temperature (all 37°C).

[0332] Use a thermomixer or thermal cycler for incubation to enable incubation of the sample at a constant temperature (37°C). During incubation, the sample is not stirred. Set the incubation time to 60 minutes. Then, directly mix the sample with loading dye and load it onto an agarose electrophoresis gel or store it at 4°C / on ice for further use.

[0333] Prepare a 1% agarose gel for gel electrophoresis. To do this, weigh 1.5 g of multipurpose agarose into a 125 - triangular flask and fill it with 150 mL of TAE buffer. Heat the mixture in a microwave until the agarose is completely dissolved. Add 0.5 μg / mL of ethidium bromide to the agarose solution. Then, pour the gel into the mold. After the agarose solidifies, place the mold into the electrophoresis chamber and fill the chamber with TAE buffer. Then, load the samples. (From left) Load an appropriate DNA molecular weight marker into the first pocket, and then load the samples subsequently. Run the gel at less than 130 V for about 60 minutes. After electrophoresis, remove the gel from the chamber and analyze it with a UV imager.

[0334] Cut the target band and transfer it to a 1.5 - mL Eppendorf tube. For gel purification, use Qiagen's QIAquick Gel Extraction Kit according to the manufacturer's instructions. Store the DNA fragment at - 20 °C for further use.

[0335] Fragments for ligation are pipetted together at a molar ratio of plasmid to insert fragment of 1:2, 1:3, or 1:5, depending on the lengths of the insert fragment and the plasmid fragment and their mutual correlation. When the fragment to be inserted into the plasmid is short, use a ratio of 1:5. If the insert fragment is longer, the amount of insert fragment used in relation to the plasmid will be less. Use 50 ng of plasmid amount for each ligation, and calculate the specific insert fragment amount with NEBioCalculator. Use NEB's T4 DNA Ligation Kit for ligation. An example of the ligation mixture is shown in the following table.

[0336] (Table) Ligation reaction mixture TIFF2025519228000003.tif63170

[0337] Starting from the mixing of DNA and water, the addition of buffer, and finally the addition of enzyme, all components are pipetted together on ice. The reaction mixture is gently mixed by pipetting up and down, briefly microcentrifuged, and then incubated at room temperature for 10 minutes. After incubation, T4 ligase is heat inactivated at 65°C for 10 minutes. The sample is cooled on ice. In the final step, 10 beta-competent Escherichia coli cells are transformed with 2 μL of the ligation plasmid (see below).

[0338] Transformation of 10-beta-competent Escherichia coli cells For transformation, 10 beta-competent Escherichia coli cells are thawed on ice. Then, 2 μl of plasmid DNA is pipetted directly into the cell suspension. The tube is flicked and placed on ice for 30 minutes. Then, the cells are placed in a thermal block at 42°C and given a heat shock for exactly 30 seconds. Immediately afterwards, the cells are cooled on ice for 2 minutes. 950 μL of NEB10-beta growth medium is added to the cell suspension. The cells are incubated at 37°C for 1 hour with shaking. Next, 50 - 100 μL is pipetted onto a pre-warmed (37°C) LB-Amp agar plate and spread with a disposable spatula. The plate is incubated at 37°C overnight. Only bacteria that have successfully incorporated the plasmid and have the resistance gene to ampicillin can grow on these plates. The next day, single colonies are picked and cultured in LB-Amp medium for subsequent plasmid preparation.

[0339] Bacterial culture The culture of Escherichia coli is carried out in LB medium, which is the abbreviation of Luria Bertani, and 1 mL / L of 100 mg / mL ampicillin is added to make the ampicillin concentration 0.1 mg / mL. For different plasmid preparation amounts, a single bacterial colony is inoculated with the following amounts.

[0340] (Table) Escherichia coli culture amounts TIFF2025519228000004.tif42170

[0341] For Mini-Prep, fill a 96-well 2 mL deep well plate with 1.5 mL of LB-Amp medium per well. Pick colonies and push the inoculating loop into the medium. Once all colonies have been picked, close the plate with an adhesive air-permeable membrane. Incubate the plate in a 37 °C incubator at a shaking speed of 200 rpm for 23 hours.

[0342] For Mini-Prep, fill a 15 mL tube (with vented cap) with 3.6 mL of LB-Amp medium and evenly inoculate with bacterial colonies. Leave the inoculating loop in the tube during incubation without removing it. Incubate the tube at 37 °C and 200 rpm for 23 hours as with the 96-well plate.

[0343] For Maxi-Prep, fill a 1 L autoclaved glass Erlenmeyer flask with 200 mL of LB-Amp medium and inoculate with 1 mL of a midday bacterial culture that has been growing for approximately 5 hours. Close the Erlenmeyer flask with a paper stopper and incubate at 37 °C and 200 rpm for 16 hours.

[0344] Plasmid Preparation For Mini-Prep, transfer 50 μL of the bacterial suspension to a 1 mL deep well plate. Then, centrifuge the bacterial cells in the plate at 3000 rpm and 4 °C for 5 minutes. Remove the supernatant and place the plate containing the bacterial pellet in the EpMotion. After approximately 90 minutes, the run is complete and the eluted plasmid DNA can be removed from the EpMotion for further use.

[0345] For Mini-Prep, remove the 15 mL tube from the incubator and divide the 3.6 mL of bacterial culture into two 2 mL Eppendorf tubes. Centrifuge the tubes in a benchtop microcentrifuge at 6,800×g at room temperature for 3 minutes. Then, perform Mini-Prep using the Qiagen QIAprep Spin Mini-prep kit according to the manufacturer's instructions. Measure the plasmid DNA concentration with a Nanodrop.

[0346] Perform Maxi-Prep using the Macherey-Nagel NucleoBond® Xtra Maxi EF kit according to the manufacturer's instructions. Measure the DNA concentration with a Nanodrop.

[0347] Ethanol precipitation Mix the volume of the DNA solution with 2.5 volumes of 100% ethanol. Incubate the mixture at -20 °C for 10 minutes. Then centrifuge the DNA at 14,000 rpm at 4 °C for 30 minutes. Carefully remove the supernatant and wash the pellet with 70% ethanol. Again, centrifuge the tube at 14,000 rpm at 4 °C for 5 minutes. Carefully remove the supernatant by pipetting and dry the pellet. Once the ethanol has evaporated, add an appropriate amount of endotoxin-free water. Redissolve the DNA in water overnight at 4 °C. Take a small aliquot and measure the DNA concentration with a Nanodrop device.

[0348] Composition of the expression cassette For the expression of an open reading frame, a transcription unit containing at least the following functional elements is used: - A promoter, - A nucleic acid containing each open reading frame, optionally including a signal sequence, - A polyadenylation signal sequence A double-stranded DNA element containing the above in the above order.

[0349] In addition to the expression unit / cassette containing the desired gene to be expressed, a basic / standard mammalian expression plasmid contains - An origin of replication from plasmid pUC18 that enables the replication of this plasmid in E. coli, and - A beta-lactamase gene that confers ampicillin resistance in E. coli and includes.

[0350] 6) Cell culture techniques Standard cell culture techniques are used as described in Current Protocols in Cell Biology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J. and Yamada, K.M. (eds.), John Wiley & Sons, Inc.

[0351] Transient transfection in HEK293 cell line Cells producing recombinant AAV particles were generated by transient transfection using each plasmid with the HEK293 system (Invitrogen) according to the manufacturer's instructions. Briefly, HEK293 cells (Invitrogen) grown in suspension in serum-free FreeStyle (trademark) 293 expression medium (Invitrogen) in either a shake flask or a stirred fermenter were transfected with a mix of each plasmid and 293fectin (trademark) or fectin (Invitrogen). HEK293 cells were seeded at a density of 1×10 6 cells / mL in 600 mL in a 2 L shake flask (Corning) and incubated at 120 rpm and 8% CO2. At a later date, cells at a cell density of approximately 1.5×10 6 cells / mL were transfected with A) 20 mL of Opti-MEM (Invitrogen) containing a total of 600 μg of plasmid DNA (1 μg / mL) and B) approximately 42 mL of a mix of 20 mL of Opti-MEM + 1.2 mL of 293 fectin or fectin (2 μL / mL). A glucose solution was added during the course of fermentation according to glucose consumption.

[0352] 7) Monitoring and analysis methods Dielectric spectroscopy Dielectric spectroscopy of the BioStat® RM 20 / 50 Rocker was performed using the pre-installed disposable viable biomass sensor BioPAT® ViaMass. Before starting fermentation and before connecting the probe to the relevant port of the bag, signal tests were performed using low (0 pF / cm, 0 mS / cm) and high (100 pF / cm, 40 mS / cm) signal simulators.

[0353] For the HyClone® bioreactor, the Incyte system was used. The IncyteArc 220 probe was inserted into the probe assembly sheath, autoclaved, and installed in the bioreactor.

[0354] Before inoculation, the dielectric probe was zero-adjusted against the medium according to the manufacturer's instructions. For the IncyteArc 220, the ArcAir application was used for zero-adjustment. Both systems measure capacitance, which is calculated as the dielectric constant ε in pF / cm. The dielectric constant is linearly correlated with VCV or VCD and thus enables real-time estimation of viable cell density.

[0355] Cell culture analysis VCD, viability, mean cell diameter, and aggregation rate were measured using a Cedex® HiRes analyzer. This is a fully automated image-based cell analyzer that uses the trypan blue exclusion method in combination with a high-resolution image scanner.

[0356] To determine the above cell parameters, a predetermined sample volume of the cell suspension was aspirated through a syringe module and a manifold valve and mixed with the required amount of 0.2% trypan blue solution. Cells with non-intact cell membranes were stained blue by the diffusion of the dye into the cell interior. The intact cells could not take in the dye. The stained cell suspension was transferred to a flow chamber through a capillary tube and a manifold valve. After a certain sedimentation time, digital images were taken using a scanner. The scanned image of the flow chamber was divided into smaller images for evaluation and analyzed using analysis software on a relevant computer.

[0357] Depending on the estimated total cell density (TCD), prior to analysis, the samples of the process were diluted with PBS according to the following table to bring them within the working concentration range of the system (0.1 - 50 10 5 cells / mL).

[0358] (Table) Dilution ratios required for measurement according to the estimated total cell density using the Cedex® HiRes analyzer TIFF2025519228000005.tif39128

[0359] In addition to typical cell parameters, the Cedex® HiRes analyzer provides information on aggregates and morphological parameters. The cell line used is prone to aggregation, which can potentially reduce the transfection success rate, so information on cell aggregation is relevant to this study (Girard et al., P., Cytotechnol. 38 (2002) 15 - 21). The Cedex® HiRes software determines cells that are aggregated with at least one other cell and calculates the aggregation rate as the proportion of cells found in the aggregates relative to the total cell count.

[0360] The relative standard deviation of multiple measurement values using a Cedex (registered trademark) HiRes analyzer is given as 5%. However, due to additional dilution errors when exceeding the measurement range, a relative standard deviation of 6% can be assumed according to the internal specifications.

[0361] Separate from the directly measured cell parameters, the viable cell volume (VCV) was calculated because it was necessary for the correlation with the permittivity signal from the capacitance probe. In calculating the viable cell volume, a spherical cell shape was assumed. Using the VCD (cells / mL) and the average cell diameter (d) (μm) measured with a Cedex (registered trademark) HiRes analyzer, the VCV (mm3 / mL) was determined according to Equation (14). TIFF2025519228000006.tif9128

[0362] Example 1 - Reference Example Basic process for rAAV particle production without perfusion A frozen cell aliquot after thawing was inoculated into a 125 mL shaking flask to initiate rAAV particle production. Every 3 - 4 days, the cell culture was passaged and the volume was increased. After reaching a sufficient volume for inoculation of the N - 2 bioreactor, i.e., reaching the cell density, the preculture was transferred to a 10 L wave - mixed bioreactor with an inoculation density of about 5×10 5 cells / mL and cultured for 3 days. Subsequently, after reaching a cell density of about 40×10 5 cells / mL, the cell culture was transferred to a further 25 L wave - mixed bioreactor at an inoculation density of about 8×10 5 cells / mL. Only the base was added to both wave - mixed bioreactors. Then, using the cell suspension, the main fermenter was inoculated at a starting volume of 60 L and a target inoculation density of 20×10 5 cells / mL. After 24 hours, an additional 20% volume of fresh culture medium was added based on the actual culture volume. Then, the cells were transfected with their respective plasmids. After a total of 4 days, the manufacturing process was terminated and lysis was initiated by adding lysis buffer, nuclease, and an alkaline solution. Subsequently, the lysed cell solution was transferred to a recovery tank and filtered to recover rAAV particles.

[0363] Example 2 Biostat® RM Rocker operation Small-scale experiments, as well as N-2 and N-1 seed train fermentations, were carried out in a wave-mixed benchtop bioreactor and a Biostat® RM Rocker equipped with a Biostat® B control tower. These bioreactors utilize rocking motion mixing technology and are used with disposable Flexsafe® RM bags. A Biostat® Cultibag RM 50 was used for the 10 L scale N-2 fermentation. N-1 fermentations in 15 L or 25 L scales and 1 L small-scale perfusion experiments were carried out using a Biostat® RM 20 / 50 basic. Both have interchangeable bag holders compatible with bags with a total volume of 1 to 50 L. The rocking platform is equipped with an integrated local controller, an air and CO2 mixing module, and a load cell.

[0364] The waves generated within the RM bag ensure effective gas exchange through the gas-liquid interface via two mechanisms, namely surface aeration and air entrainment by breaking waves. For sufficient aeration, four different gas lines for compressed air, O2, N2, and CO2 were equipped with flow meters and four mass flow controllers were used. An integrated pressure sensor continuously measures the pressure within the bag and sets the aeration to be slightly overpressure. The Flexsafe® RM bag is equipped with an intake filter and an exhaust filter.

[0365] Two heating elements using electrical resistance heating are directly integrated into the bag holder and can be controlled separately. For small-scale experiments, only the left heating circuit was used, and a 1 L bag was placed on the left side of the bag holder. A Pt100 resistance thermometer inside the bag holder measures the temperature of the bag. Since heating the Biostat® RM causes slight condensation in the exhaust filter, the filter heater keeps the filter dry and prevents it from clogging. The hood functions as a safety cover with an opening in the front for handling the bag. This protects the attached bag from mechanical influences during operation and reduces heat loss.

[0366] The Flexsafe® RM bag is equipped with disposable photochemical pH and pO2 probes, and optionally a viable biomass probe (BioPAT® ViaMass). The pH control device adjusted the addition of sodium carbonate and the CO2 gas stream. Additionally, the bag has free ports with C-flex tubes and Luer connectors or MPC connectors for adding medium, inoculum material, and base. For sampling, a dip tube with a Luer receptum is used.

[0367] For perfusion experiments, a special bag with an integrated 1.2 μm perfusion membrane was used. The membrane was fixed to the bottom of the bag to form a compartment for removing cell-free medium. The surface of the membrane is washed by the waves generated by the rocking motion with each degree of rocking, ensuring low contamination and long culture times. The addition of medium and removal of permeate are performed using the peristaltic pump of the Biostat® B control tower. To enable weight control, the medium bag was placed on a scale.

[0368] Example 3 Operation of a 100 L disposable bioreactor HyClone® The main fermentation aimed at producing rAAV particles was carried out in a disposable stirred-tank bioreactor HyClone (registered trademark) with a total working volume of 100 L. This included a stainless-steel fermentation tank container, a disposable BioProcess (registered trademark) container (BPC), an operating station equipped with a DCU, a temperature control unit, and a medium station equipped with six scales and peristaltic pumps. The reactor was equipped with a double jacket for temperature control and a vertical viewing glass. Temperature control was performed by a temperature control unit. For agitation, a three-blade pitched impeller was used.

[0369] Aeration was carried out through an underwater aeration pipe equipped with a ring sparger and an open tube. The BPC was equipped with two intake filters at the bottom, two exhaust filters, and a vent filter heater at the top. In addition to the exhaust port at the top of the BPC, there were an inlet for headspace ventilation, a pressure sensor, and several free ports for adding antifoaming agent, base, and feed. The BPC included two side port levels at the lower one-third of the bag. The lower port level included one port for a temperature probe and four AseptiQuik (registered trademark) connector probe ports used for installing a pH probe, a pO2 probe, and a capacitance probe. For sampling, a built-in dip tube was used. Above these connectors, the BPC was characterized by a free port with a C-Flex tube used for adding fresh medium and transfection. For recovery, the BPC had a drain line at the bottom.

[0370] To enable weight control, bags containing base and different feeds were placed on the hanging scales of the medium station. The pH controller adjusted the addition of the base. Antifoaming agent was added as needed.

[0371] Example 4 Pre-culture and inoculation The pre-culture was carried out in a shaking flask containing the fermentation medium. While increasing the culture volume to the amount required for inoculation, the cells were cultured every 3 - 4 days until a cell density of about 40 - 60×10 5 cells / mL was reached.5 It was divided into cells / mL. During this time, the shaking flask was incubated at 37 °C, 85% humidity, and 5% CO2 while shaking.

[0372] For 100 L fermentation in a HyClone® bioreactor, the cells were further cultured in a Biostat® RM Rocker of N-2 and N-1 and then used to inoculate the main fermenter.

[0373] The inoculation procedures for fermentation in a Biostat® RM Rocker and a HyClone® bioreactor were equivalent. First, the volume of the inoculum was determined by the current and the target initial VCD. The preculture was pumped into the bioreactor using a low pump speed to prevent excessive shear stress.

[0374] For a large preculture volume required to inoculate a 100 L HyClone® bioreactor, the cells were gently and quickly transferred to the fermenter using the hydrostatic pressure difference.

[0375] Example 5 Transfection procedure 36×10 5 Triple transfection was performed 24 hours after inoculation at a target VCD of cells / mL.

[0376] Immediately before transfection, 20% (v / v) of the initial culture volume was added to the bioreactor as fresh medium. The transfection reagent (PEI MAX®) was prepared, mixed with the plasmid, and filled with the fermentation medium. After mixing, the solution was transferred to a 10 L FLEXBOY® bag for incubation. At the end of the incubation period, the contents of the bag containing the transfection complex were transferred to the bioreactor. Subsequently, the free PEI solution and then the VPA solution were pumped into the bioreactor to reach a final concentration of 5 mM VPA.

[0377] Example 6 Operation of the perfusion bioreactor The pH set value was defined, and the dead zone was set to 0.02 pH units. For pH control, a dual control loop was used that applied CO2 sparging or the addition of a 1 M sodium carbonate solution (hereinafter referred to as the base) to lower or raise the pH, respectively.

[0378] 1) Manual operation of the perfusion process - daily changes A fixed perfusion rate based on the amount of medium remaining from the previous process was used. The perfusion rate was gradually increased once a day.

[0379] 2) Manual operation of the perfusion process - based on specific cell mass A fixed perfusion rate adapted to the specific cell substrate consumption was used.

[0380] The specific cell substrate consumption rate was determined based on the previous culture and multiplied by the predicted VCD to calculate the amount of substrate required by the cells.

[0381] Prediction of VCD by specific growth rate VCD was predicted using the average specific growth rate over the entire process time calculated in the first perfusion culture above. The average specific growth rate was determined by plotting the natural logarithm of VCD against the process time t and inserting a linear fit whose absolute value of the slope was the specific growth rate μ according to the following equation (Zhang, X., et al., “1.21 - Modes of Culture / Animal Cells”, in Comprehensive Biotechnology (Second Edition), M. Moo - Young, (Ed.) Burlington: Academic Press, 2011, pp. 285 - 302). TIFF2025519228000007.tif4128

[0382] This simplifies because the specific growth rate can vary across the entire process. However, since perfusion processes typically allow for very stable cell growth, the average growth rate can be used to predict the VCD. Therefore, the course of the VCD in the next perfusion experiment could be predicted by using Equation (3). TIFF2025519228000008.tif4128

[0383] Calculation of the specific cell consumption rate At the same time, since no substrate was added or removed except for cell metabolites, the specific cell consumption rate was calculated from the previous N-2 fermentation, which simplified the calculation. Furthermore, since the weight of the fermenter remained constant, the substrate concentration could be used instead of their absolute weights to calculate the consumption rate. The specific cell consumption rate is defined as the amount of substrate consumed by a single cell per unit time and is usually measured in units of pg / cell / day. This calculation involves the integration of the viable cell density (IVCD), which is understood as the productivity of the cell culture (cells×day / mL) (see Zhang cited above). Therefore, the specific cell consumption rate q cons is obtained by taking the difference in substrate concentration between the end c t and the start c t-1 of the fermentation and dividing it by the difference in the associated IVCD according to Equation (4). TIFF2025519228000009.tif11128

[0384] IVCD is defined as follows. TIFF2025519228000010.tif12128

[0385] Next, the absolute consumption rate q cons,abs (g / L / day) was predicted by multiplying the obtained consumption rate by the predicted VCD. TIFF2025519228000011.tif6128

[0386] Determination of the perfusion rate Regarding this, the perfusion rate pvvd in vvd can be obtained by dividing the absolute consumption rate q cons,abs (g / L / day) by the concentration of the substrate in the medium (g / L). TIFF2025519228000012.tif13128

[0387] Furthermore, to take into account the fluctuations in substrate consumption, the maximum perfusion rate was used by calculating the rate for each day using the maximum predicted VCD. To enable comparison between different perfusion processes, the specific cell culture supply rate CSPR (pL / cell / day) was also calculated according to Bausch et al. (Biotechnol. J. 14 (2019) 1700721). Therefore, the perfusion rate pvvd in vvd was divided by the VCD. TIFF2025519228000013.tif12128

[0388] A scale-down model was established in a 1 L working volume Flexsafe® RM bag. This was a physically much smaller version under the same conditions. The perfusion rate was further adjusted by manually sampling daily based on trypan blue exclusion cell counting and measurements of substrate and metabolite concentrations as described below. The perfusion rate was adjusted when the VCD was higher than expected or the substrate concentration decreased too much. Furthermore, the calculations of the predicted VCD and substrate consumption were refined in each experiment by using the average values of the specific growth rate and specific cell consumption rate instead of individual values for further calculations.

[0389] Perfusion automation using a capacitance probe For the automation of the perfusion rate, a robust automatic perfusion rate control system based on the viable cell concentration from an online capacitance probe was established. This system operated in a fully closed loop so that it was not necessary to take samples to obtain process information. In the control algorithm, the CSPR was specified and the dielectric constant signal of the capacitance probe was converted into the perfusion rate through calculations and implementation by a variable speed control pump.

[0390] To achieve this, the dielectric constant output signal of the probe was converted to an online VCD. Accordingly, the correlation between the VCD measured offline and the dielectric constant signal was determined. The correlation coefficient c VCD,Perm To determine, the VCD was plotted against the dielectric constant ε, and a linear trend line was inserted according to Equation (9). TIFF2025519228000014.tif6128

[0391] Based on the current dielectric constant signal and the predetermined CSPR, to determine the pump output required to replenish the required amount of supply medium, the VCD was predicted, and the required perfusion rate pvvd was calculated using the above equation. The correlation between the perfusion rate pvvd and the required pump output pump out (%) was established using the data from previous perfusion experiments. The relevant correlation coefficient c pump,pvvd was determined by inserting a linear fit using the following equation. TIFF2025519228000015.tif6128

[0392] This correlation coefficient c pump,pvvd was used to determine the pump output from the required perfusion rate pvvd. To enable automatic control of the pump via the dielectric constant signal ε, the automation coefficient f auto was calculated using Equation (11) and input into the control software of the bioreactor. TIFF2025519228000016.tif10128

[0393] Thereby, the pump output could be set by multiplying the dielectric constant signal by the automation coefficient. In addition to the control loop for perfusion supply, another control loop for removing the permeate based on the weight of the bioreactor was used. This is a control loop that results in the outflow of the cell-free permeate to be recovered. Since the bioreactor is placed on a scale, the permeate pump is configured to start removing the medium as soon as the weight of the bioreactor becomes greater than the initial weight immediately after inoculation until the weight returns to the starting value again.

[0394] To evaluate the accuracy of VCD prediction, the mean absolute percentage error (MAPE) was determined. This is a means to evaluate the overall performance of the prediction model. As shown in Equation (12), MAPE (%) was determined by dividing the sum of the deviations of the predicted value xpred from the actual value xi normalized by the actual value by the number of data points n. TIFF2025519228000017.tif10128

[0395] Example 7 Evaluation of Cell Growth and Metabolism The specific growth rate μ represents the dynamic behavior of cells and provides information regarding their growth and, indirectly, the health of the cell culture. This is a good criterion for comparison of different processes (A.K. Srivastava and S. Gupta, “2.38 - Fed - Batch Fermentation - Design Strategies”, in Comprehensive Biotechnology (Second Edition), M. Moo - Young, (Ed.) Burlington: Academic Press, 2011, pp. 515 - 526). The course of the growth rate over the entire fermentation was determined by dividing the difference between the logarithmic VCD and the inoculation VCD (VCD0) by the corresponding time difference (Zhang, X., et al., “1.21 - Modes of Culture / Animal Cells”, in Comprehensive Biotechnology (Second Edition), M. Moo - Young, (Ed.) Burlington: Academic Press, 2011, pp. 285 - 302). TIFF2025519228000018.tif9128

[0396] Therefore, the final growth rate in the process also corresponds to the average growth rate over the entire process time. For fermentations involving splitting of the cell culture, VCD0 was set to VCD immediately after splitting.

[0397] The specific cell formation and consumption rates follow Equation (4) in the absence of feeding / perfusion and the following Equation (16) in the perfusion process: along with TIFF2025519228000019.tif9128, TIFF2025519228000020.tif14128 and TIFF2025519228000021.tif7128 can be used to determine, where the abbreviations are as follows. TIFF2025519228000022.tif47143

[0398] The metabolite production rate is positive, but the substrate consumption rate is negative because the substrate is removed from the medium and the amount in the medium is decreasing.

Claims

1. A method for producing recombinant AAV particles, a) A step of growing mammalian cells using perfusion until at least a first predetermined cell density is achieved, b) In order to obtain a production cell solution having a second predetermined cell density, the cell fraction obtained in step (a) is diluted by adding fresh culture medium, c) A step of culturing the production cell solution for 1 to 36 hours, d) A step of directly transfecting the cells in the cultured production cell solution obtained in step (c) with one or more nucleic acids encoding the recombinant AAV particles, e) The transfected production cell solution obtained in step (d) is cultured for 24 to 144 hours. This process generates recombinant AAV particles. The method, including the method described above.

2. The first predetermined cell density is at least 80 × 10 ５ The method according to claim 1, wherein the amount is cells / mL.

3. The method according to claim 1, wherein the mammalian cells are CHO-K1 or HEK293 cells.

4. The one or more nucleic acids mentioned above i) From 5' to 3', Alpha) The first ITR sequence, Beta promoter and, Gamma) Nucleic acid sequences encoding therapeutic molecules, Delta) Polyadenylation signal sequence and Epsilon) Second ITR sequence and Transgenes including, ii) Rep Open Reading Frame and, iii) Cap Open Reading Frame and, iv) Adenovirus E1A, E1B, E2A, E4 or f6, and VA RNA open reading frame The method according to any one of claims 1 to 3, including

5. The method according to any one of claims 1 to 3, wherein in step (b), an aliquot of fresh culture medium in an amount of 4 to 6 times the volume is added.

6. The second predetermined cell density is approximately 20 × 10 ５ The method according to any one of claims 1 to 3, wherein the amount is cells / mL.

7. The method according to any one of claims 1 to 3, wherein the culture in step (c) lasts for approximately 24 hours.

8. The method according to any one of claims 1 to 3, wherein the culture in step (e) lasts for approximately 72 hours.

9. The method according to any one of claims 1 to 3, wherein step (e) does not involve feeding.

10. f) A step of recovering cells from the culture solution of step (e), g) A step of lysing the cells obtained in step (f), h) A step of separating AAV particles from the lysed cells obtained in step (g), i) A step of optionally purifying the AAV particles and The method according to any one of claims 1 to 3, further comprising:

11. The cell density after step (c) is 35-40 × 10 ５ The method according to any one of claims 1 to 3, wherein the cell density is cells / ml.

12. After step (c) and before step (d), the following steps (cd): d) Adding an additional 20% of the culture volume in fresh culture medium. The method according to any one of claims 1 to 3, wherein the method is performed.

13. The method according to any one of claims 1 to 3, wherein the transfecting step involves adding one or more nucleic acids as a mixture of PEI-complexed nucleic acids and free PEI.

14. The method according to any one of claims 1 to 3, wherein the transfecting step is performed in the presence of valproic acid at a final concentration of 5 mM.

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