Method for producing recombinant AAV particles

Abstract

Herein is reported a method for producing recombinant AAV particles comprising the steps of propagating mammalian cells using perfusion until at least a first predetermined cell density is obtained; diluting the aliquot of the propagated cells by adding a fresh 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 the cells in a cultured production cell solution with one or more nucleic acids encoding the recombinant AAV particles; and culturing the transfected production cell solution for 24 to 144 hours, thereby producing the recombinant AAV particle.

Description

[0001] The present invention belongs to the field of gene therapy. More precisely, a method for producing recombinant AAV particles is reported herein, wherein cells have been cultured using perfusion prior to transfection and transient production of recombinant AAV particles, which is performed without perfusion. Background Art

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

[0003] Today, most in vivo therapies rely on DNA delivery using recombinant adeno-associated virus (rAAV) vectors. AAV is a small, naturally occurring, non-pathogenic parvovirus that consists of a non-enveloped icosahedral capsid. It contains a linear single-stranded DNA genome of approximately 4.7 kb. The genome of the wild-type AAV vector carries two genes, rep and cap, which are flanked by inverted terminal repeats (ITRs). The ITRs are in cis and are required for viral replication and packaging. The rep gene encodes four different proteins, whose expression is driven by two alternative promoters, P5 and P19. In addition, different forms are generated by alternative splicing. Rep proteins have multiple functions, such as, for example, DNA binding, endonuclease and helicase activity. They play a role in gene regulation, site-specific integration, excision, replication and packaging. The cap gene encodes three capsid proteins and an assembly activation protein. The differential expression of these proteins is achieved by alternative splicing and selective 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 are replaced with a transgene expression cassette that still flanks the viral ITRs but encodes the gene of interest under the control of a selected promoter. Unlike wild-type viruses, engineered rAAV vectors do not integrate site-specifically into the host genome, but instead remain primarily episomal in the nucleus of transduced cells.

[0005] AAV itself does not have the ability to replicate, but requires the function of auxiliary genes. These are provided in nature by co-infected helper viruses, such as adenovirus or herpes simplex virus. For example, five adenovirus genes (i.e., E1A, E1B, E2A, E4, and VA) are known to be essential for AAV replication. Compared with other auxiliary genes that encode proteins, VA is a small RNA gene.

[0006] To produce rAAV vectors, DNA carrying a transgenic flanked by ITRs is introduced into a packaging host cell line that also contains rep and cap genes and required accessory genes. There are many ways to introduce these three sets of DNA elements into cells and many ways to combine them on different DNA plasmids (see, e.g., Robert, MA et al., Biotechnol. J. 12 (2017) 1600193).

[0007] Two general production methods have been widely used. In the triple transfection method, a plasmid containing rep / cap and a plasmid containing rAAV transgenes are transiently co-transfected with an adenoviral helper plasmid carrying the desired adenoviral helper gene. This method can be performed using CHO or HEK cells. Alternatively, rep / cap and viral helper genes can be combined on a larger plasmid (double transfection method). The second method covers infection of insect cells (Sf9) with two baculoviruses, one carrying the rAAV genome and the other carrying rep and cap. In this system, auxiliary functions are provided by the baculovirus plasmid itself. Similarly, herpes simplex virus is used in combination with HEK293 cells or BHK cells. Recently, Mietzsch et al. (Hum. Gene Ther. 25 (2014) 212-222; Hum. Gene Ther. Methods 28 (2017) 15-22) stably integrated rep and cap into the genome of engineered Sf9 cells. For these cells, a single baculovirus carrying the rAAV transgene is sufficient to produce rAAV vectors. Clark et al. (Hum. Gene Ther. 6 (1995) 1329-1341) generated a HeLa cell line with the rep / cap gene and the rAAV transgene integrated into its genome. The production of rAAV vectors was induced by transfecting cells with wild-type adenovirus, and a mixed stock of rAAV vectors and adenovirus was produced.

[0008] It is reported that perfusion culture is suitable for typical recombinant protein production (see Woodgate, JN, "Biopharmaceutical Processing: Development, Design and Implementation of Manufacturing Processes" (2018), pp. 755-768). For mammalian cell lines in such typical recombinant protein production, cell culture requires an average of 10 to 14 days to obtain a stable recombinant protein product, wherein the production bioreactor is the end of a long series of seed bioreactors with a scale of 1000L, 100L, 10L and 1L, and these seed bioreactors produce a sufficient number of starting cells, which are used to inoculate the production bioreactor. Before reaching mid-term exponential growth, cells are continuously diluted with new cell culture medium. This ensures that cells can maintain exponential growth because they are not exposed to nutrient-restricted environments (e.g., wherein growth-related amino acids are restricted) or toxic environments (e.g., wherein metabolic byproducts such as lactate and ammonia accumulate to high levels).

[0009] Yang, WC et al. (Biotechnol. Prog. 30 (2014) 616-625) demonstrated proof of concept for alternating tangential flow (ATF) perfusion seed culture for subsequent standard fed-batch process by using high cell density seed culture (400*10^5 cells / mL) to inoculate production bioreactors at much higher concentrations (standard 100*10^5 cells / mL). They completed the study with two CHO cell lines expressing monoclonal antibodies and showed that both reached their typical 5g / L product titers in 12 days instead of 17 days, thus increasing production capacity by 30% while maintaining (and improving for one cell line) product quality profile.

[0010] US 6,566,118 reports methods and compositions for producing high titer, substantially purified recombinant adeno-associated virus (AAV) preparations that can be used as gene delivery vectors.

[0011] WO 2020 / 154607 reports methods for producing adeno-associated viruses (AAV), which methods include culturing an AAV production cell line in a seed culture and then performing an AAV production culture. Summary of the invention

[0012] This article reports a method for producing recombinant AAV particles using mammalian cells, wherein the mammalian cells

[0013] i) have been propagated in perfusion culture prior to AAV particle production,

[0014] ii) have been diluted / divided to a lower cell density after propagation, and

[0015] iii) Cultivation without perfusion after transfection with nucleic acid required for production of recombinant AAV particles.

[0016] The present invention is based, at least in part, on the discovery that it is advantageous to propagate mammalian cells intended for the production of recombinant AAV particles in perfusion culture for at least a period of time prior to actual recombinant AAV particle production, even prior to transfection with nucleic acids required for the production of recombinant AAV particles, and thereafter to split the cells / dilute with fresh culture medium but prior to transfection with nucleic acids required for the production of recombinant AAV particles.

[0017] It has been found that by using mammalian cells propagated according to the present invention to produce recombinant AAV particles, the viable cell density in the production culture is higher than the production culture in which cells propagated only via fed-batch are used when starting from the same inoculation cell density. The cells even further propagate within a short period after transfection, i.e., the cell density increases after transfection. Without being bound by this theory, it is believed that this is a result of the cells having better metabolic conditions derived from the method using N-1 perfusion. Therefore, the cells can better resist the stress generated by transfection, resulting in a higher yield of recombinant AAV particles.

[0018] In one aspect, a method for producing recombinant AAV particles comprises the following steps: a) propagating mammalian cells using perfusion until at least a first predetermined cell density is obtained / reached; b) diluting an aliquot / portion of the cells obtained in step a) by adding 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) transfecting cells directly in the cultured production cell solution obtained in step c) with one or more nucleic acids encoding the recombinant AAV particles; e) culturing the transfected production cell solution obtained in step d) for 24 to 96 hours, thereby producing recombinant AAV particles.

[0019] The present invention encompasses the following independent aspects and dependent embodiments:

[0020] 1. A method for producing recombinant AAV particles, the method comprising the following steps:

[0021] a) Use perfusion to propagate mammalian cells until get / reaching at least a first predetermined cell density;

[0022] b) diluting an aliquot / portion 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;

[0023] c) culturing the production cell solution for 1 to 36 hours;

[0024] d) transfecting cells directly in the cultured producer cell solution obtained in step c) with one or more nucleic acids encoding the recombinant AAV particles;

[0025] e) culturing the transfected producer cell solution obtained in step d) for 20 to 240 hours, thereby producing recombinant AAV particles.

[0026] 2. The method according to aspect 1, wherein the first predetermined cell density is at least 80*10^5 cells / mL.

[0027] 3. The method according to aspect 1 and embodiment 2, wherein the first predetermined cell density is at least 100*10^5 cells / mL.

[0028] 4. The method according to any one of aspect 1 or embodiments 2 to 3, wherein the mammalian cell is a CHO cell or a HEK cell or a human amniotic fluid cell.

[0029] 5. The method according to any one of Aspect 1 or Embodiments 2 to 4, wherein the mammalian cell is a CHO-K1 or HEK293 cell.

[0030] 6. The method according to any one of aspect 1 or embodiments 2 to 5, wherein the one or more nucleic acids comprise

[0031] i) a transgene comprising, in the 5' to 3' direction:

[0032] α) the first ITR sequence;

[0033] β) Promoter;

[0034] γ) a nucleic acid sequence encoding a therapeutic molecule;

[0035] δ) polyadenylation signal sequence;

[0036] ε) the second ITR sequence;

[0037] ii) rep open reading frame;

[0038] iii) cap open reading frame; and

[0039] iv) Adenovirus E1A, E1B, E2A, E4orf6 and VA RNA open reading frames.

[0040] 7. The method according to any one of aspect 1 or embodiments 2 to 6, wherein 4 to 6 times the volume of the aliquot is added with fresh culture medium in step b).

[0041] 8. The method according to aspect 1 or any one of embodiments 2 to 7, wherein about 5 times the volume of the aliquot is added with fresh culture medium in step b).

[0042] 9. The method according to any one of aspect 1 or embodiments 2 to 8, wherein the second predetermined cell density is between 10*10^5 cells / mL and 30*10^5 cells / mL.

[0043] 10. The method according to any one of Aspect 1 or Embodiments 2 to 9, wherein the second predetermined cell density is about 20*10^5 cells / mL.

[0044] 11. The method according to any one of Aspect 1 or Embodiments 2 to 10, wherein the culturing in step c) lasts for 16 to 30 hours.

[0045] 12. The method according to any one of Aspect 1 or Embodiments 2 to 11, wherein the culturing lasts for 20 to 28 hours.

[0046] 13. The method according to any one of Aspect 1 or Embodiments 2 to 12, wherein the culturing lasts for about 24 hours.

[0047] 14. The method according to aspect 1 or any one of embodiments 2 to 13, wherein the culturing in step e) is a batch culture.

[0048] 15. The method according to any one of aspect 1 or embodiments 2 to 14, wherein the culturing in step e) lasts for about 24 to 144 hours.

[0049] 16. The method according to any one of Aspect 1 or Embodiments 2 to 15, wherein the culturing in step e) lasts for about 48 to 120 hours.

[0050] 17. The method according to any one of Aspect 1 or Embodiments 2 to 16, wherein the culturing lasts for about 60 to 96 hours.

[0051] 18. The method according to any one of Aspect 1 or Embodiments 2 to 17, wherein the culturing lasts for about 72 hours.

[0052] 19. The method according to any one of aspect 1 or embodiments 2 to 18, wherein step e) is free of feed.

[0053] 20. The method according to any one of aspect 1 or embodiments 2 to 19, wherein the method further comprises the following steps:

[0054] f) harvesting cells from the cultured solution of step e);

[0055] g) lysing the cells obtained in step f);

[0056] H) isolating AAV particles from the lysed cells obtained in step g);

[0057] i) Optionally purifying the AAV particles.

[0058] 21. The method according to any one of aspect 1 or embodiments 2 to 20, wherein the cell density after step c) is 35 to 40*10^5 cells / ml.

[0059] 22. The method according to any one of aspect 1 or embodiments 2 to 21, wherein after step c) and before step d) the following steps cd) are performed:

[0060] cd) Add additional fresh medium of approximately 20% of the initial culture volume.

[0061] 23. The method according to any one of aspect 1 or embodiments 2 to 22, wherein the transfection is performed by adding the one or more nucleic acids as a mixture of PEI complex nucleic acid and free PEI.

[0062] 24. The method according to any one of aspect 1 or embodiments 2 to 23, wherein the transfection is performed in the presence of a final concentration of 5 mM valproic acid.

[0063] 25. Use of a mammalian cell for producing recombinant AAV particles, wherein the mammalian cell has been propagated using perfusion prior to transfection with all of the AAV particle encoding nucleic acid.

[0064] 26. Use of a mammalian cell obtained by the method according to aspect 1 or any one of embodiments 2 to 24 for producing recombinant AAV particles.

[0065] In addition to the various embodiments depicted and claimed, the disclosed subject matter also relates to other embodiments having other combinations of features disclosed and claimed herein. Thus, the specific features presented herein may be combined with each other in other ways within the scope of the disclosed subject matter, such that the disclosed subject matter includes any suitable combination of features disclosed herein. For purposes of illustration and description, the foregoing description of specific embodiments of the disclosed subject matter has been presented. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 Viable cell density: fed-batch, N-2 (blue); fed-batch, N-1 (green); production phase, N (red) culture.

[0067] Figure 2 Viable cell density: N-2, fed-batch, 10 L scale, N-2 (blue); uncontrolled perfusion, 25 L scale, N-1 (green); production phase, 100 L scale, N (red) culture.

[0068] Figure 3 Viable cell density of N-1 controlled perfusion culture at 1 L scale.

[0069] Figure 4 Viable cell density of VCD controlled perfusion at 100 l scale: green = N-1, controlled perfusion; red = N, production phase culture.

[0070] Figure 5 Surviving cell density during dielectric constant-controlled perfusion; N-1, dielectric constant-controlled perfusion culture.

[0071] Figure 6 Production of AAV particles containing subtype 2 variant 7m8 containing a transgene encoding a therapeutic Fab. Viable cell density during biomass sensor controlled perfusion, N-1 (green); production phase, N (red) culture.

[0072] Figure 7 Viable cell density in VCD controlled perfusion and biosensor controlled perfusion cultures: red (VCD) vs. purple (biosensor); blue (VCD) vs. green (biomass sensor).

[0073] Figure 8 Viable densities obtained in different production scale cultures inoculated with cells obtained with perfusion at the N-1 stage (i.e. according to the invention) and without perfusion at the N-1 stage; the red curve corresponds to the production scale culture with N-1 culture without perfusion and the green curves (marked "1", "2", "3", "4") correspond to the production scale culture with N-1 culture with perfusion according to the invention.

[0074] Fig. 9 The viability of the cells in the production scale culture N depends on the culture of the cells used to inoculate the N stage at the N-1 stage. The red curve corresponds to the production culture inoculated with cells obtained from the batch N-1 culture. The green curves (denoted as "1", "2", "3" and "4") correspond to the production scale culture inoculated with cells obtained from the N-1 perfusion culture according to the present invention.

[0075] Fig.10Capsid titers in production cultures in a bioreactor system >10 L in N after N-1 with and without perfusion. Red curves ("5", "6") correspond to production cultures after batch N-1. Green curves ("1", "2", "3" and "4") correspond to production cultures after N-1 with perfusion. For the red curves, batch culture N-1 was performed in the same bioreactor (thus, N scale culture day zero corresponds to day 4 in the figure) DETAILED DESCRIPTION

[0076] definition

[0077] Methods and techniques that can be used to practice the present invention are described in, for example, Ausubel, FM (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, ND, and Hames, BD, eds., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, RI (ed.), Animal Cell Culture—a practical approach, IRL Press Limited (1986); Watson, JD et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, EL, From Genes to Clones; NY, VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, Second Edition, Alan R. Liss, Inc., NY (1987).

[0078] Derivatives of nucleic acids can be generated using recombinant DNA technology. Such derivatives can be modified, for example, by substitution, change, exchange, deletion or insertion at a single or several nucleotide positions. Modification or derivatization can be performed, for example, by means of site-directed mutagenesis. Such modifications can be easily performed 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, BD, and Higgins, SG, Nucleic acid hybridization-apractical approach (1985) IRL Press, Oxford, England).

[0079] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. Likewise, the terms "a", "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.

[0080] Deoxyribonucleic acid comprises a coding strand and a non-coding strand. The terms "5'" and "3'" when used herein refer to positions on the coding strand.

[0081] The term "3' flanking sequence" refers to a sequence located at the 3' end (downstream; below) of a nucleotide sequence.

[0082] The term "5' flanking sequence" refers to a sequence located at the 5' end (upstream, above) of a nucleotide sequence.

[0083] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. Likewise, the terms "a", "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.

[0084] The term "AAV helper function" refers to AAV-derived coding sequences (proteins) that can be expressed to provide AAV gene products and AAV particles, which in turn act in trans to carry out productive AAV replication and packaging. Thus, AAV helper functions include AAV open reading frames (ORFs), including rep and cap, and others, such as AAP for certain AAV serotypes. The rep gene expression product has been shown to have many functions, including: recognition, binding and cleavage 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. AAV helper functions are used to supplement trans-AAV functions that are missing in the AAV vector genome.

[0085] The term "about" means a range of + / -20% of the value that follows. In certain embodiments, the term "about" means a range of + / -10% of the value that follows. In certain embodiments, the term "about" means a range of + / -5% of the value that follows.

[0086] The term "batch culture" refers to a culture in which all components for cell culture (including cells and all culture nutrients) are supplied to a culture bioreactor at the beginning of the culture process.

[0087] As used herein, the terms "comprises," "including," "having," "having," "may," "containing," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. The term "comprising" also encompasses the term "including..." The present disclosure also contemplates other embodiments "comprising," "consisting of," and "consisting essentially of" the embodiments or elements set forth herein, whether or not explicitly set forth.

[0088] The term "culturing" herein refers to the step of maintaining cells in a culture medium under conditions in which the cells are transfected and produce AAV particles.

[0089] The terms "empty capsid" and "empty particle" refer to an AAV particle that has an AAV protein capsid but lacks in whole or in part a nucleic acid encoding a protein or a nucleic acid transcribed into a target transcript flanked by the AAV ITRs (i.e., the vector). Thus, an empty capsid cannot function to transfer a nucleic acid encoding a protein or transcribed into a target transcript into a host cell.

[0090] The term "endogenous" means that something occurs naturally within a cell; is naturally produced by the cell; similarly, an "endogenous locus / cell endogenous locus" is a locus that occurs naturally in a cell.

[0091] As used herein, the term "exogenous" refers to a nucleotide sequence that is not derived from a specific cell, but is introduced into the cell by a DNA delivery method (e.g., by transfection, electroporation, or transduction by a viral vector). Therefore, an exogenous nucleotide sequence is an artificial sequence, wherein the artificiality can be derived from, for example, a combination of subsequences from different sources (e.g., a combination of a recombinase recognition sequence with an SV40 promoter and a coding sequence for a green fluorescent protein is an artificial nucleic acid) or from a sequence (e.g., a sequence encoding only the extracellular domain of a membrane-bound receptor or a cDNA) of a portion of the deletion, or a nuclear base mutation. The term "endogenous" refers to a nucleotide sequence derived from a cell. An "exogenous" nucleotide sequence can have an "endogenous" counterpart with the same base composition, but wherein the sequence becomes an "exogenous" sequence, for example, by being introduced into a cell via recombinant DNA technology.

[0092] As used herein, the term "fed-batch cell culture" refers to a culture in which cells and culture medium are first supplied to a culture bioreactor, and additional culture nutrients are fed to the culture continuously or in discontinuous increments during the culture, with or without periodic cell and / or product harvesting prior to terminating the culture.

[0093] An "isolated" composition is one that has been separated from one or more components of its natural environment. In some embodiments, the composition is purified to a purity greater than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis, CE-SDS) or chromatography (e.g., size exclusion chromatography or ion exchange or reversed phase HPLC). For a review of methods for assessing, for example, antibody purity, see Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0094] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from one or more components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0095] An "isolated" polypeptide or antibody is one that has been separated from one or more components of its natural environment.

[0096] The term "mammalian cell comprising an exogenous nucleotide sequence" encompasses cells into which one or more exogenous nucleic acids have been introduced, including the offspring of such cells. These can serve as the starting point for further genetic modification. Therefore, the term "mammalian cell comprising an exogenous nucleotide sequence" encompasses cells comprising an exogenous nucleotide sequence integrated at a single site in the locus of the mammalian cell genome, wherein the exogenous nucleotide sequence at least comprises a first recombination recognition site and a second recombination recognition site (these recombination recognition sites are different) flanking at least one first selection marker. In certain embodiments, the mammalian cell comprising an exogenous nucleotide sequence is a cell comprising an exogenous nucleotide sequence integrated at a single site in the locus of the cell genome, wherein the exogenous nucleotide sequence comprises a first recombination recognition sequence and a second recombination recognition sequence flanking at least one first selection marker, and a third recombination recognition sequence between the first recombination recognition sequence and the second recombination recognition sequence, and all recombination recognition sequences are different.

[0097] "Mammalian cells comprising exogenous nucleotide sequences" and "recombinant cells" are both "transfected cells". The term includes the primary transfected cell and progeny derived therefrom, without regard to the number of passages. Progeny may, for example, not be completely identical to the nucleic acid content of the parent cell, but may contain mutations. Mutant progeny having the same function or biological activity as in the original transfected cell are encompassed.

[0098] "Nucleic acid encoding AAV packaging proteins" generally refers to one or more nucleic acid molecules, which include nucleotide sequences that provide AAV functions deleted from AAV vectors, and the nucleic acid molecules are used to produce transduction-active recombinant AAV particles. Nucleic acids encoding AAV packaging proteins are generally used to provide expression of AAV rep and / or cap genes to supplement the missing AAV functions required for AAV replication; however, the nucleic acid construct lacks AAV ITR and can neither replicate nor package itself. Nucleic acids encoding AAV packaging proteins can be in the form of plasmids, phages, transposons, cosmids, viruses or particles. Many nucleic acid constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45, which encode both rep gene expression products and cap gene expression products. See, for example, Samulski et al. (1989) J. Virol. 63: 3822-3828; and McCarty et al. (1991) J. Virol. 65: 2936-2945. Many plasmids encoding the expression products of the rep and / or cap genes have been described (eg, US 5,139,941 and US 6,376,237). Any of these nucleic acids encoding AAV packaging proteins may comprise a DNA element or nucleic acid according to the invention.

[0099] The term "nucleic acid encoding an auxiliary protein" generally refers to one or more nucleic acid molecules that include nucleotide sequences encoding proteins and / or RNA molecules that provide adenoviral auxiliary functions. Plasmids with nucleic acids encoding auxiliary proteins can be transfected into suitable cells, where the plasmid is then able to support the production of AAV particles in the cells. Any of these nucleic acids encoding auxiliary proteins may comprise a DNA element or nucleic acid according to the present invention. The term explicitly excludes infectious viral particles as they occur in nature, such as adenovirus, herpes virus or vaccinia virus particles.

[0100] As used herein, the term "operably connected" refers to the juxtaposition of two or more components, wherein the relationship of these components allows them to play a role in an expected manner. For example, if a promoter and / or enhancer plays a role in regulating the transcription of a coding sequence / open reading frame / gene, the promoter and / or enhancer are operably connected to the coding sequence / open reading frame / gene. In certain embodiments, the DNA sequence of "operably connected" is adjacent. In certain embodiments, for example, when two protein coding regions (such as a secretory leader and a polypeptide) must be joined, these sequences are adjacent and in the same reading frame. In certain embodiments, the promoter that is operably connected is located upstream of the coding sequence / open reading frame / gene and can be adjacent to the coding sequence / open reading frame / gene. In certain embodiments, for example, about the enhancer sequence regulating the expression of the coding sequence / open reading frame / gene, these two components can be operably connected, but not adjacent. If an enhancer increases the transcription of the coding sequence / open reading frame / gene, the enhancer is operably connected to the coding sequence / open reading frame / gene. An operably linked enhancer may be located upstream, within, or downstream of a coding sequence / open reading frame / gene and may be located at a considerable distance from the promoter of the coding sequence / open reading frame / gene.

[0101] The term "packaging protein" refers to non-AAV derived viral and / or cellular functions on which AAV replication depends. Thus, the term embodies proteins and RNAs required for AAV replication, including those involved in AAV gene transcriptional activation, stage-specific AAV mRNA splicing, AAV DNA replication, Cap expression product synthesis, and AAV capsid assembly. Virus-based helper functions can be derived from any of the known helper viruses, such as adenovirus, herpes virus (except herpes simplex virus type I) and vaccinia virus.

[0102] As used herein, "AAV packaging proteins" refer to AAV-derived sequences that function in trans for productive AAV replication. Thus, AAV packaging proteins are encoded by the major AAV open reading frames (ORFs), rep and cap. The rep protein has been shown to have many functions, including: recognition, binding and cleavage of the AAV origin of DNA replication; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. The cap (capsid) protein provides the necessary packaging function. AAV packaging proteins are used herein to supplement the trans-AAV functions missing in AAV vectors.

[0103] As used herein, "perfusion" or "perfusion culture", sometimes also referred to as continuous culture, refers to a culture in which cells are confined in culture by, for example, filtration, encapsulation, anchoring to microcarriers, etc., and culture medium is introduced and removed from the culture bioreactor continuously, stepwise, or intermittently (or any combination of these means).

[0104] The terms "propagation" and "pre-culture" are used interchangeably herein and refer to the steps of increasing the number of cells in a cell culture, starting with inoculating fresh culture medium with an aliquot of cells and maintaining culture conditions for cell growth (in a preferred embodiment, exponential growth) and division until a desired cell density (i.e., a first predetermined cell density) is reached. In general, cells can be propagated using different methods such as batch culture, fed-batch culture, or perfusion culture. The term "propagation" includes shunting cells during propagation, i.e., reducing the number of cells (cell density) by removing an aliquot of culture medium containing cells and replacing it with / adding a defined aliquot of fresh culture medium.

[0105] As used herein, the term "proteinaceous compound" refers to a heteromultimeric molecule comprising at least one polypeptide that has been produced in a functional form in a mammalian cell. An exemplary proteinaceous compound is an adeno-associated virus particle (AAV particle), which comprises a capsid formed by a capsid polypeptide and a single-stranded DNA molecule, which is a non-polypeptide component.

[0106] As used herein, the term "recombinant cell" refers to a cell that has been genetically modified, such as, for example, a cell that expresses a polypeptide of interest or produces rAAV particles of interest and can be used to produce the polypeptide of interest or rAAV particles of interest on any scale. For example, a "mammalian cell comprising an exogenous nucleotide sequence" that has undergone recombinase-mediated cassette exchange (RMCE) and has thus introduced the coding sequence of the polypeptide of interest into the host cell genome is a "recombinant cell". Although the cell is still capable of further RMCE reactions, it is not desirable to do so.

[0107] "Recombinant AAV vectors" are derived from the wild-type genome of a virus (such as AAV) by using molecular methods to remove the wild-type genome from the virus (e.g., AAV) and replace the wild-type genome with a non-native nucleic acid (such as a nucleic acid transcribed into a transcript or encoding a protein). For AAV, one or two inverted terminal repeat (ITR) sequences of the wild-type AAV genome are usually retained in the recombinant AAV vector. "Recombinant" AAV vectors are distinguished from the wild-type viral AAV genome because all or part of the viral genome has been replaced with a non-native (i.e., heterologous) sequence relative to the viral genomic nucleic acid. Therefore, the incorporation of non-native sequences defines a viral vector (e.g., AAV) as a "recombinant" vector, which in the case of AAV can be referred to as a "rAAV vector."

[0108] Recombinant vector (e.g., AAV) sequences can be packaged - referred to herein as "particles" - for subsequent cell infection (transduction) ex vivo, in vitro or in vivo. When recombinant vector sequences are encapsulated or packaged into AAV particles, the particles may also be referred to as "rAAV". Such particles include 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.

[0109] As used herein, the term "selection marker" refers to a gene that allows specific selection or exclusion of cells carrying the gene in the presence of a corresponding selective agent. For example, but not limited to, a selection marker can allow positive selection of host cells transformed with the selection marker gene in the presence of a corresponding selective agent (selective culture conditions); untransformed host cells will not be able to grow or survive under the selective culture conditions. Selection markers can be positive, negative or bifunctional. Positive selection markers can allow selection of cells carrying the marker, while negative selection markers can allow selective elimination of cells carrying the marker. Selection markers can confer resistance to drugs, or compensate for metabolic or catabolism defects in host cells. In prokaryotic cells, genes that confer resistance to ampicillin, tetracycline, kanamycin or chloramphenicol, as well as other genes, can be used. Resistance genes that can be used as selectable markers in eukaryotic cells include, but are not limited to, genes for aminoglycoside phosphotransferases (APHs) (e.g., hygromycin phosphotransferase (HYG), neomycin and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), 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.

[0110] In addition to facilitating selection in the presence of a corresponding selective agent, a selectable marker may alternatively be a molecule that is 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. Cells expressing such a molecule can be distinguished from cells not containing the gene, for example, by detecting fluorescence emitted by the encoded polypeptide or the absence of such fluorescence, respectively.

[0111] As used herein, the term "serotype" is a distinction based on serologically different AAV capsids. Serological distinctiveness is determined based on the lack of cross-reactivity between antibodies to one AAV compared to antibodies to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in VP1, VP2, and / or VP3 sequences of AAV serotypes). Although AAV variants, including capsid variants, may be serologically indistinguishable from reference AAV or other AAV serotypes, they differ in at least one nucleotide or amino acid residue compared to the reference AAV serotype or other AAV serotypes.

[0112] Under the traditional definition, serotype refers to the target virus that has been tested for serum, which has specificity for neutralizing activity to all existing and characterized serotypes, and no antibodies to neutralize the target virus are found. With the discovery of more naturally occurring virus isolates and / or the generation of capsid mutants, there may or may not be serological differences with any of the currently existing serotypes. Therefore, in the case where a new virus (such as AAV) does not have serological differences, the new virus (such as AAV) will be 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 modified capsid sequences to determine whether they have another serotype according to the traditional definition of serotype. Therefore, for convenience and to avoid duplication, the term "serotype" refers broadly to serologically different viruses (such as AAV) and serologically no different viruses (such as AAV), which may be within a subgroup or variant of a given serotype.

[0113] The terms "transduction" and "transfection" refer to the introduction of molecules such as nucleic acids (viral vectors, plasmids) into cells. When exogenous nucleic acids have been introduced into the cell membrane, the cell has been "transduced" or "transfected". Therefore, a "transduced cell" is a cell into which a "nucleic acid" or "polynucleotide" has been introduced, or its progeny into which an exogenous nucleic acid has been introduced. In specific embodiments, a "transduced" cell (e.g., in a mammal, such as a cell or a tissue or organ cell) has genetic changes after the incorporation of an exogenous molecule such as a nucleic acid (e.g., a transgene). A "transduced" cell can reproduce and transcribe the introduced nucleic acid and / or express a protein.

[0114] In "transduced" or "transfected" cells, the nucleic acid (viral vector, plasmid) may or may not be integrated into the genomic nucleic acid. If the introduced nucleic acid is integrated into the nucleic acid (genomic DNA) of the recipient cell or organism, it can be stably maintained in the cell or organism and further transmitted to or inherited by the daughter cells or organisms of the recipient cell or organism. Finally, the introduced nucleic acid can be present extrachromosomally in the recipient cell or host organism, or only transiently. Many techniques are known, for example, see, 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. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.

[0115] The term "transgene" is used herein for convenience to refer to a nucleic acid that is intended to be or has been introduced into a cell or organism. A transgene includes any nucleic acid, such as a gene, that is transcribed into a transcript or encodes a polypeptide or protein.

[0116] "Vector" refers to a portion of a recombinant plasmid sequence that is ultimately packaged or encapsulated directly or in the form of a single strand or RNA to form a virus (e.g., AAV) particle. In the case where a recombinant plasmid is used to construct or manufacture a recombinant virus particle, the virus particle does not include a "plasmid" portion that does not correspond to the vector sequence of the recombinant plasmid. This non-vector portion of the recombinant plasmid is called a "plasmid backbone," which is important for cloning and amplification of the plasmid, a process required for propagation and recombinant virus production, but it itself is not packaged or encapsulated into a virus (e.g., AAV) particle. Therefore, "vector" refers to a nucleic acid packaged or encapsulated by a virus particle (e.g., AAV).

[0117] Recombinant cell line generation

[0118] In general, for efficient and large-scale production of a target protein compound, such as, for example, rAAV particles or therapeutic polypeptides, cells that express and, if possible, also secrete the protein compound are necessary. Such cells are referred to as "recombinant cells" or "recombinant production cells".

[0119] In the first step, the desired nucleic acid sequence encoding the target protein compound is transfected into a suitable host cell. It may be necessary to transfect additional auxiliary polypeptides.

[0120] For expressing coding sequence, i.e. open reading frame, additional regulatory elements are needed, such as promoter and polyadenylation signal (sequence). Therefore, open reading frame is operably connected to the additional regulatory elements for transcription. This can be achieved by being integrated into the so-called expression cassette. The minimum regulatory elements required for the expression cassette to work in mammalian cells are the promoter working in the mammalian cell, the promoter is located at the upstream of the open reading frame, i.e. 5', and the polyadenylation signal (sequence) working in the mammalian cell, the polyadenylation signal is located at the downstream of the open reading frame, i.e. 3'. In addition, the terminator sequence may be present in the 3' of the polyadenylation signal (sequence). For expression, promoter, open reading frame / coding region and polyadenylation signal sequence must be arranged in the form that is operably connected.

[0121] Similarly, nucleic acids that are transcribed into RNA encoding non-proteins are referred to as "RNA genes". Also for the expression of RNA genes, additional regulatory elements are required, such as promoters and transcription termination signals or polyadenylation signals (sequences). The nature and positioning of such elements depend on the RNA polymerase intended to drive RNA gene expression. Therefore, RNA genes are also typically integrated into expression cassettes.

[0122] If the target protein compound is an AAV particle, which is composed of different (monomeric) polypeptides and single-stranded DNA molecules, and additionally requires other auxiliary factors for production and packaging, multiple expression cassettes are required, which differ in the required open reading frame / coding sequence. In this case, at least an expression cassette is required for each of the transgene, the different polypeptides forming the capsid of the AAV vector, the required auxiliary functions and the VA RNA. Thus, a separate expression cassette is required for each of the auxiliary E1A, E1B, E2A, E4orf6, VA RNA, rep and cap genes.

[0123] As outlined in the preceding paragraph, respectively, the more complex or additionally required auxiliary polypeptide and / or RNA quantity of the target protein compound is, the higher the quantity of the required different expression cassettes is. Intrinsically, as the number of expression cassettes increases, the size of the nucleic acid integrated into the host cell genome also increases. However, there is a practical upper limit to the transferable nucleic acid size, which ranges from about 15kbps (kilobase pairs). Exceeding this limitation, processing and processing efficiency will significantly decline. This problem can be solved by using two or more independent nucleic acids. Therefore, different expression cassettes are assigned to different nucleic acids, and each nucleic acid only comprises some expression cassettes thus.

[0124] Recombinant cells

[0125] In general, for efficient and large-scale production of a target protein compound, such as, for example, rAAV particles or therapeutic polypeptides, cells that express and, if possible, also secrete the protein compound are necessary. Such cells are referred to as "recombinant cells" or "recombinant production cells".

[0126] To generate a "recombinant producer cell", suitable mammalian cells are transfected with the desired nucleic acid sequence encoding the protein compound of interest. Additional auxiliary polypeptides may need to be transfected.

[0127] In order to generate stable recombinant production cells, a second step is then performed in which single cells that stably express the protein compound of interest are selected. This can be done, for example, based on the co-expression of a selection marker that has been co-transfected with the nucleic acid sequence encoding the protein compound of interest, or on the expression of the protein compound itself.

[0128] For expressing coding sequence, i.e. open reading frame, additional regulatory elements are needed, such as promoter and polyadenylation signal (sequence). Therefore, open reading frame is operably connected to the additional regulatory elements for transcription. This can be achieved by being integrated into the so-called expression cassette. The minimum regulatory elements required for the expression cassette to work in mammalian cells are the promoter working in the mammalian cell, the promoter is located at the upstream of the open reading frame, i.e. 5', and the polyadenylation signal (sequence) working in the mammalian cell, the polyadenylation signal is located at the downstream of the open reading frame, i.e. 3'. In addition, the terminator sequence may be present in the 3' of the polyadenylation signal (sequence). For expression, promoter, open reading frame / coding region and polyadenylation signal sequence must be arranged in the form that is operably connected.

[0129] Similarly, nucleic acids that are transcribed into RNA encoding non-proteins are referred to as "RNA genes". Also for the expression of RNA genes, additional regulatory elements are required, such as promoters and transcription termination signals or polyadenylation signals (sequences). The nature and positioning of such elements depend on the RNA polymerase intended to drive RNA gene expression. Therefore, RNA genes are also typically integrated into expression cassettes.

[0130] If the target protein compound is an AAV particle, which is composed of different (monomeric) capsid polypeptides and single-stranded DNA molecules, and additionally requires other adenoviral helper functions for production and packaging, multiple expression cassettes are required, which differ in the required open reading frame / coding sequence. In this case, at least an expression cassette is required for each of the transgene, the different polypeptides forming the capsid of the AAV vector, the required helper functions and the VA RNA. Thus, a separate expression cassette is required for each of the helper E1A, E1B, E2A, E4orf6, VA RNA, rep and cap genes.

[0131] As outlined in the previous paragraph, respectively, the more complex or additionally required auxiliary polypeptide and / or RNA quantity of the target protein compound is, the higher the number of required different expression cassettes is. The total size of nucleic acid is also inherently related to the number of expression cassettes. However, there is a practical upper limit to the transferable nucleic acid size, which ranges from about 15kbps (kilobase pairs). Exceeding this limit, processing and processing efficiency will significantly decrease. This problem can be solved by using two or more independent plasmids. Thus, different expression cassettes are assigned to different plasmids, and each plasmid only comprises some of the expression cassettes thus.

[0132] For stable cell line development, random integration (RI) of nucleic acids carrying expression cassettes for the protein compound of interest may be used. Typically, by using RI, the nucleic acid or fragments thereof will be randomly integrated into the genome of the host cell.

[0133] Alternatively to RI, targeted integration (TI) can be used for CLD. In TI CLD, one or more nucleic acids comprising different expression cassettes are introduced into the genome of the host cell at a predetermined locus.

[0134] In TI, homologous recombination or recombinase-mediated cassette exchange reaction (RMCE) can be used to integrate a nucleic acid (a) containing a corresponding expression cassette into a specific locus in the genome of the TI host cell.

[0135] In certain embodiments of all aspects and embodiments, each of the expression cassettes comprises a promoter, an open reading frame / coding sequence or an RNA gene and a polyadenylation signal sequence, and / or a terminator sequence along the 5' to 3' direction. In certain embodiments, the open reading frame encodes a polypeptide and the expression cassette comprises a polyadenylation signal sequence with or without an additional terminator sequence. In certain embodiments, the expression cassette comprises an RNA gene, the promoter is a type 2 Pol III promoter and there is a polyadenylation signal sequence or a polyU terminator. See, e.g., Song et al. Biochemical and Biophysical Research Communications 323 (2004) 573–578. In certain embodiments, the expression cassette comprises an RNA gene, the promoter is a type 2 Pol III promoter and a polyU terminator sequence.

[0136] In certain embodiments of all aspects and embodiments, the open reading frame encodes a polypeptide, the promoter is human CMV promoter with or without intron A, the polyadenylation signal sequence is the bGH (bovine growth hormone) polyA signal sequence, and the terminator is hGT (human gastrin terminator).

[0137] In certain embodiments of all aspects and embodiments, the promoter is a human CMV promoter with intron A, the polyadenylation signal sequence is a bGH polyadenylation signal sequence and the terminator is hGT, except for the expression cassette for the RNA gene and the expression cassette for the selection marker, wherein for the selection marker, the promoter is the SV40 promoter and the polyadenylation signal sequence is the SV40 polyadenylation signal sequence and the terminator is not present, and wherein 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.

[0138] It must be noted that, in certain embodiments, the present invention does not encompass permanent human cell lines comprising nucleic acid sequences of adenoviral gene functions E1A and E1B and concomitantly nucleic acid sequences of SV40 large T antigen or Estein-Barr virus (EBV) nuclear antigen 1 (EBNA-1).

[0139] Adeno-associated virus (AAV)

[0140] For a general review of AAV and adenovirus or herpes helper functions, see Berns and Bohensky, Advances in Virus Research, Academic Press., 32 (1987) 243-306. The genome of AAV is described in Srivastava et al., J. Virol., 45 (1983) 555-564. Design considerations for constructing recombinant AAV vectors are described in US 4,797,368 (see also WO 93 / 24641). Other 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. The construction of recombinant AAV vectors is described in US5,173,414; 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.

[0141] Adeno-associated virus (AAV) is a replication-defective parvovirus. It can only replicate in cells where certain viral functions are provided by a co-infecting helper virus, such as adenovirus, herpes virus, and in some cases poxvirus, such as vaccinia. Nevertheless, AAV can replicate in almost any cell line of human, simian or rodent origin, provided that appropriate helper virus functions are present.

[0142] If helper viral genes are not present, AAV establishes latency in its host cells. Its genome integrates into a specific site in chromosome 19 [(Chr)19(q13.4)], called adeno-associated virus integration site 1 (AAVS1). For specific serotypes, such as AAV-2, other integration sites have been found, such as, for example, on chromosome 5 [(Chr)5(p13.3)], called AAVS2, and on chromosome 3 [(Chr)3(p24.3)], called AAVS3.

[0143] AAV is divided 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 a hundred sequences corresponding to different branches of AAV have been identified.

[0144] The capsid protein type and symmetry determine the tissue tropism of the corresponding 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 cardiac 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.

[0145] Pseudotyping refers to a process that involves cross-packaging of the AAV genome between different serotypes, i.e., the genome is packaged with capsid proteins of different origin.

[0146] The wild-type AAV genome has a size of about 4.7 kb. The AAV genome further comprises two overlapping genes, called rep and cap, which contain 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 open reading frame encoding the Rep protein provides four proteins of different sizes, which are called Rep78, Rep68, Rep52 and Rep40. These are related to the replication, rescue and integration of AAV. The open reading frame encoding the Cap protein provides four proteins, called VP1, VP2, VP3 and AAP. VP1, VP2 and VP3 are part of the protein capsid of the AAV particle. The combined rep and cap open reading frames are flanked by so-called inverted terminal repeats (ITRs) at their 5' and 3' ends. For replication, AAV requires, in addition to the Rep and Cap proteins, the products of the adenoviral genes E1A, E1B, E4orf6, E2A and VA or the corresponding factors of another helper virus.

[0147] For example, in the case of AAV of serotype 2 (AAV-2), each ITR has a length of 145 nucleotides and is flanked by a coding sequence region of about 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 functions as a primer during viral replication. The remaining 20 non-paired nucleotides are represented as D sequences.

[0148] The AAV genome contains three transcription promoters P5, P19 and P40 (Laughlin et al., Proc. Natl. Acad. Sci. USA 76 (1979) 5567-5571) for the expression of rep and cap genes.

[0149] ITR sequence must be present in the coding region in cis. ITR provides a functional origin of replication (ori), is integrated into the target cell genome required signal, and effectively excised and rescued from the host cell chromosome or recombinant plasmid. ITR further comprises an origin of replication-like element, such as Rep protein binding site (RBS) and terminal resolution site (TRS). It has been found that ITR itself can have the function of a transcription promoter in an AAV vector (Flotte et al., J.Biol.Chem.268 (1993) 3781-3790; Flotte et al., Proc.Natl.Acad.Sci.USA 93 (1993) 10163-10167).

[0150] The trans organization of the rep and cap gene products is required for the replication and encapsidation of the viral single-stranded DNA genome, respectively.

[0151] The rep locus comprises two internal promoters, called P5 and P19. It comprises open reading frames for four kinds of proteins. Promoter P5 is operably connected to a nucleotide sequence providing a non-spliced ​​4.2kb mRNA encoding Rep protein Rep78 (chromatin nickase that blocks the cell cycle) and a spliced ​​3.9kb mRNA encoding Rep protein Rep68 (site-specific endonuclease). Promoter P19 is operably connected to a nucleotide sequence providing a non-spliced ​​mRNA encoding Rep protein Rep52 and a spliced ​​3.3kb mRNA encoding Rep protein Rep40 (DNA helicase for accumulation and packaging).

[0152] The two larger Rep proteins, Rep78 and Rep68, are essential for AAV duplex DNA replication, whereas the smaller Rep proteins, Rep52 and Rep40, appear to be essential for progeny single-stranded DNA accumulation (Chejanovsky & Carter, Virology 173 (1989) 120-128).

[0153] The larger Rep proteins Rep68 and Rep78 can specifically bind to the hairpin conformation of AAV ITR. They exhibit defined enzymatic activity, which is required for resolving replication at the AAV termini. Expression of Rep78 or Rep68 may be sufficient for the formation of infectious particles (Holscher, C. et al. J. Virol. 68 (1994) 7169-7177 and 69 (1995) 6880-6885).

[0154] All Rep proteins, primarily Rep78 and Rep68, are believed to exhibit regulatory activities such as induction and repression of AAV genes and inhibition of cell growth (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).

[0155] Recombinant overexpression of Rep78 produces a phenotype of reduced cell growth caused by induction of DNA damage. As a result, host cells are arrested in the S phase, thereby promoting latent infection of the virus (Berthet, C. et al., Proc. Natl. Acad. Sci. USA 102 (2005) 13634-13639).

[0156] Tratschin et al. reported that the P5 promoter is negatively autoregulated by Rep78 or Rep68 (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894). Due to the toxic effects of Rep protein expression, it has been reported that after stable integration of AAV, expression for some cell lines is only very low (see, e.g., Mendelson et al., Virol. 166 (1988) 154-165).

[0157] The cap locus contains a promoter, called P40. The promoter P40 is operably linked to a nucleic acid sequence that provides 2.6 kb mRNA, which encodes the Cap proteins VP1 (87 kDa, non-spliced ​​mRNA transcripts), VP2 (72 kDa, from spliced ​​mRNA transcripts) and VP3 (61 kDa, from alternative start codons) by alternative splicing and using alternative start codons. VP1 to VP3 constitute the components of the viral capsid. The capsid has the function of binding to cell surface receptors and allowing intracellular transport of the virus. VP3 accounts for about 90% of the total protein of the viral particle. However, all three proteins are essential for effective capsid production.

[0158] Inactivation of all three capsid proteins VP1 to VP3 reportedly prevents the accumulation of single-stranded progeny AAV DNA. Mutations at the amino terminus of VP1 (“Lip negative” or “Inf negative”) still allow single-stranded DNA to assemble into viral particles, thereby greatly reducing infectious titers.

[0159] The AAP open reading frame encodes the assembly activation protein (AAP). It has a size of about 22 kDa and transports the native VP protein to the nucleolar region for capsid assembly. This open reading frame is located upstream of the VP3 protein coding sequence.

[0160] A single AAV particle contains only one single-stranded DNA molecule. This can be either the "plus" strand or the "minus" strand. AAV virus particles containing DNA molecules are infectious. Within infected cells, the parental infectious single strand is converted to a double strand and subsequently amplified. Amplification produces a large number of double-stranded DNA molecules, which displace the single strand and are packaged into capsids.

[0161] Adeno-associated virus (AAV) vectors can transduce both dividing and quiescent cells. It can be assumed that transgenes introduced into target cells using AAV vectors will be expressed long term. One disadvantage of using AAV vectors is that the size of the transgene that can be introduced into cells is limited.

[0162] Viral vectors such as parvovirus particles, including AAV serotypes and variants thereof, provide a way to deliver nucleic acids in vitro, in vitro and in vivo to cells encoding proteins, so that the cells express the encoded proteins. AAV is a virus that can be used as a gene therapy vector because they can penetrate cells and introduce nucleic acids / genetic material so that nucleic acids / genetic material can be stably maintained in cells. In addition, for example, these viruses can introduce nucleic acids / genetic material into specific sites. Since AAV is not related to human pathogenic diseases, AAV vectors are able to deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and agents) to human patients without causing substantial AAV pathogenesis or disease.

[0163] Viral vectors that can be used include, but are not limited to, adeno-associated virus (AAV) particles of various serotypes (eg, AAV-1 to AAV-12, etc.) and hybrid / chimeric AAV particles.

[0164] Advantageously, AAV particles can be used as vehicles for efficient gene delivery. Such particles have many characteristics required for such applications, including tropism for dividing and non-dividing cells. Early clinical experience with these vectors also showed no persistent toxicity, and the immune response was small or undetectable. AAV is known to infect a variety of cell types in vivo and in vitro through receptor-mediated endocytosis or through transcytosis. These vector systems have been tested in humans targeting retinal epithelium, liver, skeletal muscle, airways, brain, joints, and hematopoietic stem cells.

[0165] Recombinant AAV particles generally do not include viral genes associated with pathogenesis. Such vectors generally have one or more of the wild-type AAV genes that are completely or partially deleted, such as rep and / or cap genes, but retain at least one functional flanking ITR sequence, which is necessary for rescuing, replicating, and packaging the recombinant vector into AAV particles. For example, only the essential parts of the vector are included, such as ITR and LTR elements, respectively. Therefore, the AAV vector genome will include cis sequences (e.g., functional ITR sequences) required for replication and packaging.

[0166] Recombinant AAV vectors and methods and uses thereof include any viral strain or serotype. As a non-limiting example, the recombinant AAV vector can be based on any AAV genome, such as, for example, 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 different from each other. As a non-limiting example, a recombinant AAV vector based on a serotype genome can be the same as one or more of the capsid proteins that package the vector. In addition, the 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, and at least one of the three capsid proteins can be 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 AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74 and AAV 7m8 capsids.

[0167] In certain embodiments of all aspects and embodiments, adeno-associated virus (AAV) vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74 and AAV7m8 and variants thereof (e.g., capsid variants, such as amino acid insertions, additions, substitutions and deletions), for example, as shown in WO 2013 / 158879, WO2015 / 013313 and US2013 / 0059732 (disclosing LK01, LK02, LK03, etc.).

[0168] AAV and AAV variant (e.g., capsid variant) serotypes (e.g., VP1, VP2 and / or VP3 sequences) may or may not be different from other AAV serotypes, including, for example, AAV1-AAV12 (e.g., different from the VP1, VP2 and / or VP3 sequences of any of the AAV1 to AAV12 serotypes).

[0169] In certain embodiments of all aspects and embodiments, the AAV particles associated with a reference serotype have a polynucleotide, polypeptide, or subsequence thereof that comprises or consists of a sequence that is at least 80% or more (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, or AAV 7m8 (e.g., such as ITRs, or VP1, VP2, and / or VP3 sequences).

[0170] The compositions, methods and uses of the present invention include AAV sequences (polypeptides and nucleotides) and subsequences thereof that exhibit less than 100% sequence identity to a reference AAV serotype (such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74 or AAV7m8), but are different 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 genes or proteins, etc.). 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 (e.g., 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%, etc., up to 100% 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 ITRs). In certain embodiments, the AAV variant has 1, 2, 3, 4, 5, 5 to 10, 10 to 15, 15 to 20, or more amino acid substitutions.

[0171] Recombinant AAV particles, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74 or AAV7m8, as well as variants, related, hybrid and chimeric sequences, can be constructed using recombinant techniques known to those of skill in the art to include one or more nucleic acid sequences (transgenes) flanked by one or more functional AAV ITR sequences.

[0172] Recombinant particles (e.g., rAAV particles) can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are particularly useful for in vivo or ex vivo administration and delivery to a subject. In certain embodiments, the pharmaceutical composition contains a pharmaceutical carrier or excipient. Such excipients include any medicament that does not itself induce an immune response that is harmful to the individual receiving the composition and can be administered without excessive toxicity.

[0173] Protocols for generating adenoviral vectors have been described in US 5,998,205; US 6,228,646; US 6,093,699; US 6,100,242; WO 94 / 17810 and WO 94 / 23744, the entire contents of which are incorporated herein by reference.

[0174] Recombinant AAV particles (rAAV particles)

[0175] Different methods for generating rAAV particles are known in the art. For example, transfection using AAV plasmids and AAV auxiliary sequences and co-infection with an AAV helper virus (e.g., adenovirus, herpes virus or vaccinia virus), or transfection using recombinant AAV plasmids, AAV helper plasmids and auxiliary function plasmids. Non-limiting methods for generating rAAV particles are described in, for example, US 6,001,650, US6,004,797, WO 2017 / 096039 and WO 2018 / 226887. After recombinant rAAV particle production (i.e., particle generation in a cell culture system), rAAV particles can be obtained from host cells and cell culture supernatants and purified.

[0176] For the production of recombinant AAV particles, it is necessary to express the Rep and Cap proteins, the accessory proteins E1A, E1B, E2A and E4orf6, and the adenovirus VA RNA in a single mammalian cell. The accessory proteins E1A, E1B, E2A and E4orf6 can be expressed using any promoter, as shown by Matsushita et al. (Gene Ther. 5 (1998) 938-945), in particular the CMV IE promoter. Therefore, any promoter can be used.

[0177] In general, in order to produce recombinant AAV particles, different complementary plasmids are co-transfected into host cells. One of these plasmids contains a transgene sandwiched between two cis-acting AAV ITRs. The deleted AAV elements (i.e., open reading frames for Rep and Cap proteins) required for offspring recombinant genome replication and subsequent packaging are contained in the second plasmid in trans. Overexpression of Rep protein causes an inhibitory effect on cell growth (Li, J. et al., J. Virol. 71 (1997) 5236-5243). In addition, AAV replication requires a third plasmid containing genes of helper viruses (i.e., E1, E4orf6, E2A and VA from adenovirus).

[0178] To reduce the number of plasmids required, Rep, Cap, and adenoviral helper genes can be combined on a single plasmid.

[0179] Alternatively, the host cell may have stably expressed the E1 gene product. Such cells are HEK293 cells. The human embryonic kidney clone, denoted 293, was generated as early as 1977 by integrating adenovirus DNA into human embryonic kidney cells (HEK cells) (Graham, FL 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 covers E1A and E1B genes and adenovirus packaging signals (Louis, N. et al., Virology 233 (1997) 423-429).

[0180] When HEK293 cells are used, the deleted E2A, E4orf6 and VA genes can be introduced by co-infection with adenovirus or by co-transfection with plasmids expressing E2A, E4orf6 and VA (see, e.g., Samulski, RJ et al., J. Virol. 63 (1989) 3822-3828; Allen, JM et al., J. Virol. 71 (1997) 6816-6822; Tamayose, K. et al., Hum. Gene Ther. 7 (1996) 507-513; Flotte, TR et al., Gene Ther. 2 (1995) 29-37; Conway, JE et al., J. Virol. 71 (1997) 8780-8789; Chiorini, JA et al., Hum. Gene Ther. 6 (1995) 1531-1541; Ferrari, FK 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 may be used (see, e.g., Conway, JE et al., J. Virol. 71 (1997) 8780-8789; Johnston, KM et al., Hum. Gene Ther. 8 (1997) 359-370; Thrasher, AJ et al., Gene Ther. 2 (1995) 481-485; Fisher, JK et al., Hum. Gene Ther. 7 (1996) 2079-2087; Johnston, KM et al., Hum. Gene Ther. 8 (1997) 359-370).

[0181] Therefore, cell lines in which the rep gene is integrated and expressed tend to grow slowly or express the Rep protein at very low levels.

[0182] To restrict transgene activity to specific tissues, ie, to limit integration sites, the transgene can be operably linked to an inducible or tissue-specific promoter (see, eg, Yang, Y. et al. Hum. Gene. Ther. 6 (1995) 1203-1213).

[0183] One difficulty in rAAV particle production is the inefficiency of rAAV vector packaging, resulting in low titers. Packaging has been difficult for a number of reasons, including:

[0184] Preferred encapsidation of the wild-type AAV genome when present;

[0185] Due to the repression associated with the rep gene product, it is difficult to generate adequate complementary functions, such as those provided by wild-type rep and cap genes;

[0186] Co-transfection of plasmid constructs has limited efficiency.

[0187] All of this is based on the biological properties of the Rep protein. In particular, the inhibitory (cytostatic and cytotoxic) properties of the Rep protein and its ability to reverse the immortalized phenotype of cultured cells are problematic. In addition, when the widely used AAV P5 promoter is adopted, the Rep proteins downregulate their own expression (see, for example, Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894).

[0188] 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.

[0189] In certain embodiments of all aspects and embodiments, the rAAV particle comprises a capsid sequence having 70% or greater sequence identity to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74, or 7m8 capsid sequence.

[0190] In certain embodiments of all aspects and embodiments, the rAAV particle comprises an ITR sequence having 70% or greater sequence identity to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 ITR sequence.

[0191] E1A, E1B, E2 and E4

[0192] The coding sequences (open reading frames) of E1A and E1B may be derived from human adenoviruses, such as, for example, in particular human adenovirus serotype 2 or serotype 5. Exemplary sequences of human Ad5 (adenovirus serotype 5) are found in GenBank entries X02996, AC_000008, and the sequence of an exemplary human Ad2 is found in GenBank entry AC_000007. Nucleotides 505 to 3522 comprise the nucleic acid sequence encoding E1A and E1B of human adenovirus serotype 5. Plasmid pSTK146 reported in EP 1 230 354 and plasmids pGS119 and pGS122 reported in WO2007 / 056994 may also be used as sources of E1A and E1B open reading frames.

[0193] E1A is the first viral helper gene expressed after adenoviral DNA enters the cell nucleus. The E1A gene encodes 12S and 13S proteins, which are based on the same E1A mRNA by alternative splicing. The expression of 12S and 13S proteins causes the activation of other viral functions E1B, E2, E3 and E4. In addition, the expression of 12S and 13S proteins forces the cell to enter the S phase of the cell cycle. If only E1A-derived proteins are expressed, the cell will die (apoptosis).

[0194] E1B is the second viral helper gene 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 a first 55kDa transcript and a second 19kDa transcript. The E1B 55kDa protein is involved in regulating the cell cycle, preventing mRNA trafficking in cells late in infection, and preventing E1A-induced apoptosis. The E1B 19kDa protein is involved in preventing E1A-induced apoptosis.

[0195] The E2 genes encode different proteins. The E2A transcript encodes the single-stranded binding protein (SSBP), which is essential for AAV replication.

[0196] The E4 gene also encodes several proteins. The 34kDa protein (E4orf6) derived from the E4 gene, together with the E1B55kDa protein, prevents the accumulation of cellular mRNA in the cytoplasm, but also promotes the transport of viral RNA from the nucleus to the cytoplasm.

[0197] Adenovirus VA RNA gene

[0198] Virus-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 from a type 2 polymerase III promoter by RNA polymerase III (see, e.g., 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.

[0199] Ma, Y. and Mathews, MB (J. Virol. 70 (1996) 5083-5099) used a phylogenetic approach to study the structure, function and evolution of adenovirus-associated RNA. They provided an alignment based on 47 known human adenovirus serotypes and a consensus VA RNA sequence. The disclosure is hereby incorporated by reference into this application in its entirety.

[0200] VA RNA, VAI and VAII consist of 157 to 160 nucleotides (nt).

[0201] Depending on the serotype, adenovirus contains one or two VA RNA genes. VA RNAI is thought to play a major proviral role, while VA RNAII can partially compensate for the absence of VA RNAI (Vachon, VK and Conn, GL, Virus Res. 212 (2016) 39-52).

[0202] VA RNA is not essential, but plays an important role in efficient viral growth by overcoming cellular antiviral mechanisms. That is, although VA RNA is not essential for viral growth, adenoviruses lacking VA RNA fail to grow during the initial steps of vector generation, where only a few copies of the viral genome are present per cell, probably because viral genes other than VARNA that block cellular antiviral mechanisms may not be adequately expressed (see Maekawa, A. et al. Nature Sci. Rep. 3 (2013) 1136).

[0203] Maekawa, A. et al. (Nature Sci. Rep. 3 (2013) 1136) reported the efficient production of adenoviral vectors lacking genes for virus-associated RNAs that disrupt the cellular RNAi machinery, in which HEK293 cells that constitutively and highly express flippase recombinase were infected to obtain VA RNA-deficient adenoviruses through FLP recombinase-mediated excision of the VA RNA locus.

[0204] Human adenovirus 2VA RNAI corresponds to nucleotides 10586-10810 of the sequence of GenBank entry AC_000007. Human adenovirus 5VA RNAI corresponds to nucleotides 10579-10820 of the sequence of GenBank entry AC_000008.

[0205] Methods for producing rAAV particles

[0206] Carter et al. have shown that the entire rep and cap open reading frames in the wild-type AAV genome can be deleted and replaced with a transgene (Carter, BJ, "Handbook of Parvoviruses", P. Tijssen, ed., CRC Press, pp. 155-168 (1990)). Further, it has been reported that the ITR must be maintained to retain the functions of transgene replication, rescue, packaging, and integration into the target cell genome.

[0207] When cells containing the corresponding viral helper genes are transduced with AAV vectors, or vice versa, when cells containing integrated AAV provirus are transduced with appropriate helper virus, the AAV provirus is activated and enters the lytic infection cycle again (Clark, KR et al., Hum. Gene Ther. 6 (1995) 1329-1341; Samulski, RJ, Curr. Opin. Genet. Dev. 3 (1993) 74-80).

[0208] The production cells contain rep and cap gene sequences, and a transgenic cassette flanked by ITR sequences on one or more plasmids retained by drug selection. The production of rAAV particles in these cell lines usually occurs after they are infected with the required helper functions. Therefore, the cells are infected with AdV (generally wild-type Ad5) or plasmids containing corresponding helper genes to provide helper viral proteins and initiate rAAV particle production. Packaging cell lines are different from production cell lines because they only contain rep and cap genes.

[0209] Aspects of the invention are methods of transducing cells with a nucleic acid (e.g., a plasmid) comprising all of the required elements for producing recombinant AAV particles, wherein the cells prior to transfection have been propagated (at least for a period of time) using perfusion. Thus, when the plasmid encodes viral packaging proteins and / or accessory proteins, the cells can produce recombinant viral particles that include a nucleic acid encoding a protein of interest or a nucleic acid comprising a sequence that is transcribed into a transcript of interest.

[0210] The present invention provides a viral (eg, AAV) particle production platform that includes features that distinguish it from current 'industry standard' viral (eg, AAV) particle production processes through the use of methods according to the present invention.

[0211] More generally, cells transfected or transduced with DNA for 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 have been used as receptors for nucleic acids (plasmids) encoding packaging proteins (such as AAV packaging proteins), nucleic acids (plasmids) encoding auxiliary proteins, and nucleic acids (plasmids) encoding proteins or transcribed into target transcripts (i.e., transgenes placed between two AAV ITRs). The term includes the descendants of the original cells that have been transduced or transfected. It should be understood that the descendants of a single parental cell may not necessarily be exactly the same as the original parent in terms of morphology or genome or total nucleic acid complement due to natural, accidental or deliberate mutations.

[0212] Many cell growth mediums suitable for maintaining cell viability or providing cell growth and / or proliferation are commercially available. The example of such culture medium includes serum-free eukaryotic growth medium, such as for maintaining mammalian (e.g., human) cell viability or providing mammalian (e.g., human) cell growth medium. Non-limiting examples include Ham's F12 or F12K culture medium (Sigma-Aldrich), FreeStyle (FS) F17 culture medium (Thermo-Fisher Scientific), MEM, DMEM, RPMI-1640 (Thermo-FisherScientific) and mixtures thereof. Such culture medium 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.

[0213] The helper protein plasmid can be in the form of a plasmid, phage, transposon or cosmid. In particular, it has been shown that helper function does not require a complete complement of adenoviral genes. For example, adenoviral mutants that are unable to carry out DNA replication and late gene synthesis have been shown to allow AAV replication. Ito et al., J. Gen. Virol. 9 (1970) 243; Ishibashi et al., Virology 45 (1971) 317.

[0214] Mutants within the E2B and E3 regions have been shown to support AAV replication, indicating that the E2B and E3 regions may not be involved in providing auxiliary functions. Carter et al., Virology 126 (1983) 505. However, adenoviruses with defects in the E1 region or deletions in the E4 region cannot support AAV replication. Therefore, for adenovirus auxiliary proteins, the E1A and E4 regions may be directly or indirectly required for AAV replication (see, e.g., 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 characterized adenovirus mutants include: E1B (Laughlin et al. (1982), supra; Janik et al. (1981), supra; 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, ICRCH and book of Parvoviruses (P. Tijssen, ed., 1990)); E3 (Carter et al. (1983), supra); and E4 (Carter et al. (1983), supra; Carter (1995)).

[0215] Studies on helper proteins provided by adenoviruses with E1B mutations have reported that the E1B 55 kDa protein is essential for AAV particle production, whereas the E1B 19 kDa is not. In addition, WO 97 / 17458 and Matshushita et al. (Gene Therapy 5 (1998) 938-945) describe helper plasmids encoding various adenovirus genes. Examples of helper plasmids include adenovirus VA RNA coding regions, adenovirus E4orf6 coding regions, adenovirus E2A 72 kDa coding regions, adenovirus E1A coding regions, and adenovirus E1B regions lacking the complete E1B 55 kDa coding region (see, e.g., WO 01 / 83797).

[0216] Thus, provided herein is a method for producing a recombinant AAV vector or an AAV particle comprising the recombinant AAV vector comprising a nucleic acid encoding a protein or a nucleic acid transcribed into a target transcript using mammalian cell propagation prior to transfection and viral particle production according to the method of the present invention.

[0217] For this purpose, three plasmids were co-transfected into mammalian cells propagated by perfusion according to the invention. The transgenic plasmid (pTransgen) encodes the expression cassette cloned between the AAV ITRs, while the rep and cap genes are provided in trans by co-transfection of the second packaging plasmid (pRep / Cap) to ensure AAV replication and packaging. The third plasmid, also called the helper plasmid (pHelper), contains minimal helper viral factors (usually adenoviral E2A, EV and VA genes), but lacks the AAV ITRs.

[0218] One aspect of the present invention is a method for producing a recombinant AAV vector or an AAV particle comprising the recombinant AAV vector, wherein the recombinant AAV vector comprises a nucleic acid encoding a protein or transcribed into a target transcript, the method comprising the following steps:

[0219] (i) providing one or more plasmids comprising nucleic acids encoding AAV packaging proteins and / or nucleic acids encoding accessory proteins;

[0220] (ii) providing a plasmid comprising a nucleic acid encoding a target protein or transcribed into a target transcript;

[0221] (iii) contacting one or more mammalian or insect cells obtained by the method according to the present invention with a provided plasmid;

[0222] (iv) further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture; or providing a physical means, such as an electric current, to introduce the nucleic acid into the cells;

[0223] (v) culturing the transfected cells;

[0224] (vi) harvesting the cultured cells and / or medium from the cultured cells to produce a cell and / or medium harvest; and

[0225] (vii) lysing the cells and optionally isolating the recombinant AAV vector or AAV particles from the cells and / or culture medium harvest lysate;

[0226] Recombinant AAV vectors or AAV particles are thereby produced that contain a nucleic acid encoding a protein of interest or transcribed into a transcript of interest.

[0227] One aspect of the present invention is a method for producing a recombinant AAV vector or an AAV particle comprising the recombinant AAV vector, wherein the recombinant AAV vector comprises a nucleic acid encoding a protein or transcribed into a target transcript, the method comprising the following steps:

[0228] (i) providing one or more plasmids comprising nucleic acids encoding AAV packaging proteins and / or nucleic acids encoding accessory proteins;

[0229] (ii) providing a plasmid comprising a nucleic acid encoding a target protein or transcribed into a target transcript;

[0230] (iii) or

[0231] (a) generating stably transfected cells by contacting one or more mammalian cells obtained by the method according to the present invention with a plasmid provided in (i); further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture or providing a physical means such as an electric current to introduce nucleic acid into the cells; selecting a first stably transfected cell; contacting the selected first stably transfected cell with a plasmid provided in (ii); and further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture; or providing a physical means such as an electric current to introduce nucleic acid into the cells; or

[0232] (b) generating transiently transfected cells by contacting one or more mammalian or insect cells obtained by the method according to the present invention with the plasmids provided by (i) and (ii); and further adding a transfection agent and optionally incubating the plasmid / transfection agent / cell mixture; or providing physical means such as an electric current to introduce the nucleic acid into the cells;

[0233] (iv) culturing the transfected cells of (iii);

[0234] (v) harvesting the cultured cells and / or medium from the cultured cells to produce a cell and / or medium harvest; and

[0235] (vi) lysing the cells and optionally isolating the recombinant AAV vector or AAV particles from the cells and / or culture medium harvest lysate;

[0236] Recombinant AAV vectors or AAV particles are thereby produced that contain a nucleic acid encoding a protein of interest or transcribed into a transcript of interest.

[0237] One aspect of the present invention is a method for producing a recombinant AAV vector or an AAV particle comprising the recombinant AAV vector, wherein the recombinant AAV vector comprises a nucleic acid encoding a protein or transcribed into a target transcript, the method comprising the following steps:

[0238] (i) providing a mammalian or insect cell comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding an accessory protein;

[0239] (ii) providing a plasmid comprising a nucleic acid encoding a target protein or transcribed into a target transcript;

[0240] (iii) or

[0241] (a) generating stably transfected cells by contacting one or more mammalian or insect cells obtained by the method according to the present invention with a plasmid provided in (i); further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture or providing a physical means such as an electric current to introduce nucleic acid into the cells; selecting a first stably transfected cell; contacting the selected first stably transfected cell with a plasmid provided in (ii); and further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture; or providing a physical means such as an electric current to introduce nucleic acid into the cells; or

[0242] (b) generating transiently transfected cells by contacting one or more mammalian or insect cells obtained by the method according to the present invention with the plasmids provided by (i) and (ii); and further adding a transfection agent and optionally incubating the plasmid / transfection agent / cell mixture; or providing physical means such as an electric current to introduce the nucleic acid into the cells;

[0243] (iv) culturing the transfected cells of (iii);

[0244] (v) harvesting the cultured cells and / or medium from the cultured cells to produce a cell and / or medium harvest; and

[0245] (vi) lysing the cells and optionally isolating and / or purifying the recombinant AAV vector or AAV particles from the cells and / or culture medium harvest;

[0246] Recombinant AAV vectors or AAV particles are thereby produced that contain a nucleic acid encoding a protein of interest or transcribed into a transcript of interest.

[0247] Introduction of nucleic acids (plasmids) into cells can be done in a variety of ways.

[0248] The various methods of transferring DNA to mammalian cells have been reported in the art. These can be used according to the method of the present invention. In certain embodiments of all aspects and embodiments, electroporation, nuclear transfection or microinjection for nucleic acid transfer / transfection is used. In certain embodiments of all aspects and embodiments, inorganic substances (such as calcium phosphate / DNA coprecipitation), cationic polymers (such as polyethyleneimine, DEAE-dextran) or cationic lipids (lipofection) for nucleic acid transfer / transfection are used. Calcium phosphate and polyethyleneimine are the most commonly used reagents in the transfection for larger-scale nucleic acid transfer (see, for example, Baldi et al., Biotechnol.Lett.29 (2007) 677-684), wherein polyethyleneimine is preferred.

[0249] Growing in serum-free suspension culture and using PEI as a transfection reagent improves the efficiency and reproducibility of transfection conditions, allowing real-time scale-up of AAV production using shake flasks, wave or stirred tank bioreactors.

[0250] In certain embodiments of all aspects and embodiments, nucleic acid (plasmid) is provided in the form of a composition in combination with polyethyleneimine (PEI), optionally in combination with cells. In certain embodiments, the composition includes a plasmid / PEI mixture having multiple components: (a) one or more plasmids comprising nucleic acids encoding AAV packaging proteins and / or nucleic acids encoding auxiliary proteins; (b) a plasmid comprising a nucleic acid encoding a protein or transcribed into a target transcript; and (c) a polyethyleneimine (PEI) solution. In certain embodiments, the molar ratio of the plasmid ranges from about 1:0.01 to about 1:100, or a molar ratio ranges from 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.

[0251] In certain embodiments of all aspects and embodiments, the composition further comprises a cell. In certain embodiments, the cell is contacted with the plasmid / PEI mixture of components (a), (b) and / or (c).

[0252] In certain embodiments of all aspects and embodiments, the composition optionally combined with the cells further comprises free PEI. In certain embodiments, the cells are contacted with free PEI.

[0253] In certain embodiments of all aspects and embodiments, the cell has been contacted with a mixture of components (a), (b) and / or (c) for at least about 4 hours, or from about 4 hours to about 140 hours, or from about 4 hours to about 96 hours. In a preferred embodiment, the cell has been contacted with a mixture of components (a), (b) and / or (c) and optionally free PEI for at least about 4 hours.

[0254] Said composition can further comprise plasmid or / and cell.Such plasmid and cell can contact with free PEI.In certain embodiments, plasmid and / or cell have contacted with free PEI at least about 4 hours, or about 4 hours to about 140 hours, or about 4 hours to about 96 hours.

[0255] The present invention also provides a method for producing transfected cells. The method includes providing one or more plasmids; providing a solution comprising polyethyleneimine (PEI); and mixing the plasmid with the PEI solution to produce a plasmid / PEI mixture. In certain embodiments, such a mixture is incubated for a period of about 10 seconds to about 4 hours. In such methods, the cells are then contacted with the plasmid / PEI mixture to produce a plasmid / PEI cell culture; free PEI is then added to the produced plasmid / PEI cell culture to produce free PEI / plasmid / PEI cell culture; the produced free PEI / plasmid / PEI cell culture is then incubated for at least about 4 hours, thereby producing transfected cells. In certain embodiments, the plasmid comprises one or more or all of a rep open reading frame, a cap open reading frame, an E1A, E1B, E2 and an E4orf6 open reading frame, and a nucleic acid encoding a protein or transcribed into a target transcript.

[0256] Further provided is a method for producing transfected cells that produce recombinant AAV vectors or AAV particles, the method comprising providing one or more plasmids comprising nucleic acids encoding AAV packaging proteins and / or nucleic acids encoding auxiliary proteins; providing a plasmid comprising a nucleic acid encoding a protein or transcribed into a target transcript; providing a solution comprising polyethyleneimine (PEI); mixing the above plasmids with the PEI solution, wherein the molar ratio of the plasmids ranges from about 1:0.01 to about 1:100, or the molar ratio ranges from about 100:1 to about 1:0.01, 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 produced 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 producing transfected cells that produce recombinant AAV vectors or particles comprising nucleic acids encoding proteins or transcribed into transcripts of interest, wherein the mammalian cells have been obtained using a method according to the present invention.

[0257] Also provided is a method for producing a recombinant AAV vector or AAV particle comprising a nucleic acid encoding a protein or transcribed into a target transcript, the method comprising providing one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding an auxiliary protein; providing a plasmid comprising a nucleic acid encoding a target protein or transcribed into a target transcript; providing a solution comprising polyethyleneimine (PEI); mixing the above plasmids with the PEI solution, wherein the molar ratio of the plasmids ranges from about 1:0.01 to about 1:100, or the molar ratio ranges from about 100:1 to about 1:0.01, to produce a plasmid / PEI mixture (and optionally incubating the plasmid / PEI mixture for a period of time ranging from about 10 seconds to about 4 hours); incubating mammalian cells with the plasmid / PEI mixture produced as described above; PEI mixture to produce a plasmid / PEI cell culture; adding free PEI to the plasmid / PEI cell culture produced as described above to produce a free PEI / plasmid / PEI cell culture; incubating the produced plasmid / PEI cell culture or the free PEI / plasmid / PEI cell culture for at least about 4 hours to produce transfected cells; harvesting the produced transfected cells and / or the culture medium from the produced transfected cells to produce a cell and / or culture medium harvest; lysing the cells and optionally isolating the recombinant AAV vector or particle from the cell and / or culture medium harvest lysate, whereby mammalian cells have been obtained using the method according to the present invention; and thereby producing a recombinant AAV vector or particle comprising a nucleic acid encoding a protein or transcribed into a target transcript.

[0258] Methods for producing recombinant AAV vectors or AAV particles using methods according to the invention may include one or more additional steps or features. Exemplary steps or features include, but are not limited to, steps of harvesting the cultured cells produced and / or harvesting the culture medium from the cultured mammalian cells produced to produce a cell and / or culture medium harvest. Additional exemplary steps or features include, but are not limited to, lysing the harvested cells and optionally isolating the recombinant AAV vector or AAV particle from the cell and / or culture medium harvest lysate; whereby the mammalian cells have been obtained using methods according to the invention prior to transfection; and whereby a recombinant AAV vector or AAV particle comprising a nucleic acid encoding a protein or transcribed into a target transcript is produced.

[0259] In certain embodiments of all aspects and embodiments, PEI is added to plasmid and / or cells at different time points. In certain embodiments, free PEI is added to cells before, simultaneously with or after contacting the plasmid / PEI mixture with cells.

[0260] In some embodiments of all aspects and embodiments, when contacting with plasmid / PEI mixture and / or when contacting with free PEI, cell is in specific density and / or cell growth stage and / or vigor.In a preferred embodiment, when contacting with plasmid / PEI mixture and / or when contacting with free PEI, the density of cell is in the range of about 1*10^5 individual cells / mL to about 1*10^8 individual cells / mL.In certain embodiments, when contacting with plasmid / PEI mixture or with free PEI, cell viability is about 60% or greater than 60%, or wherein when contacting with plasmid / PEI mixture, cell is in logarithmic growth stage, or when contacting with plasmid / PEI mixture or with free PEI, cell viability is about 90% or greater than 90%, or wherein when contacting with plasmid / PEI mixture or with free PEI, cell is in logarithmic growth stage.

[0261] In addition to PEI, valproic acid (VPA) can be used to improve transfection efficiency. VPA is a branched short-chain fatty acid and inhibits histone deacetylase activity. For this reason, it is often added to mammalian cell cultures as an enhancer for recombinant protein production.

[0262] In certain embodiments of all aspects and embodiments, the encoded AAV packaging proteins include AAVrep and / or AAVcap. In certain embodiments of all aspects and embodiments, such AAV packaging proteins include AAVrep and / or AAVcap proteins of any AAV serotype.

[0263] In certain embodiments of all aspects and embodiments, the encoded accessory proteins include adenoviral E1A and E1B, adenoviral E2 and / or E4, VA RNA and / or non-AAV accessory proteins.

[0264] In certain embodiments of all aspects and embodiments, nucleic acids (plasmids) are used in specific amounts or ratios. In certain embodiments, the total amount of a plasmid comprising a nucleic acid encoding a protein or transcribed into a target transcript and one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding an auxiliary protein is in the range of about 0.1 μg to about 15 μg per mL of cells. In certain embodiments, the molar ratio of a plasmid comprising a nucleic acid encoding a protein or transcribed into a target transcript to one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding an auxiliary protein is in the range of about 1:5 to about 1:1, or in the range of about 1:1 to about 5:1.

[0265] In certain embodiments of all aspects and embodiments, the first plasmid comprises a nucleic acid encoding an AAV packaging protein and the second plasmid comprises a nucleic acid encoding an accessory protein.

[0266] In certain embodiments of all aspects and embodiments, the molar ratio of the plasmid comprising a nucleic acid encoding a protein or transcribed into a target transcript to the first plasmid comprising a nucleic acid encoding an AAV packaging protein to the second plasmid comprising a nucleic acid encoding an auxiliary protein in the co-transfection is in the range of about 1-5:1:1 or 1:1-5:1 or 1:1:1-5.

[0267] 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.

[0268] The usual conditions for culturing eukaryotic cells at about 37° C., 95% humidity and 8% CO2 by volume can be used for culturing. Cultivation can be carried out in serum-containing or serum-free medium, adherent culture or suspension culture. Suspension culture can be carried out in any fermentation vessel, such as, for example, in a stirred tank reactor, a wave reactor, a rocking bioreactor, a shaking container or a rotating container or a so-called roller bottle. Transfection can be carried out in a high-throughput format and screening, for example, in a 96 or 384-well format, respectively.

[0269] The methods according to the invention include AAV particles of any serotype or variants thereof. In certain embodiments of all aspects and embodiments, the recombinant AAV particles comprise any one of the following: AAV serotypes 1 to 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 comprise an AAV serotype or an AAV pseudotype, wherein the AAV pseudotype comprises an AAV capsid serotype different from the ITR serotype.

[0270] The method according to the invention that provides or includes AAV vectors or particles may also include 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 repeats (ITRs) and / or filler / filler polynucleotide sequences. Such elements may be located within or flanked by a nucleic acid encoding a protein or transcribed into a target transcript, or an expression control element may be operably linked to a nucleic acid encoding a protein or transcribed into a target transcript, or an AAVITR may flank the 5' or 3' end of a nucleic acid encoding a protein or transcribed into a target transcript, or a filler polynucleotide sequence may flank the 5' or 3' end of a nucleic acid encoding a protein or transcribed into a target transcript.

[0271] Expression control elements include constitutive or regulatable control elements, such as tissue-specific expression control elements or promoters.

[0272] The ITR may be any of AAV2 or AAV6 or AAV8 or AAV9 serotypes, or a combination thereof. The AAV particles may include any VP1, VP2 and / or VP3 capsid proteins having 75% or greater sequence identity with any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV-2i8, AAV rh74 or AAV 7m8 VP1, VP2 and / or VP3 capsid proteins, or a modified or variant VP1, VP2 and / or VP3 capsid protein selected from any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV-2i8, AAV rh74 and AAV 7m8 AAV serotypes.

[0273] After producing recombinant virus (e.g., AAV) particles as described herein, if desired, a variety of conventional methods can be used to purify and / or separate virus (e.g., rAAV) particles from host cells. Such methods include column chromatography, CsCl gradients, iodixanol gradients, etc.

[0274] For example, multiple column purification steps can be used, such as purification by anion exchange column, affinity column and / or cation exchange column. (See, for example, WO 02 / 12455 and US2003 / 0207439). Alternatively or in addition, iodixanol or CsCl gradient steps can be used (see, for example, US2012 / 0135515; and US2013 / 0072548). Further, if infectious viruses are used to express packaging proteins and / or auxiliary proteins, various methods can be used to inactivate residual viruses. For example, adenovirus can be inactivated by heating to a temperature of about 60° C. for, for example, 20 minutes or longer. This treatment effectively inactivates the helper virus because AAV is heat stable, while the helper adenovirus is heat unstable.

[0275] One goal of rAAV vector production and purification systems is to implement strategies to minimize / control the generation of production-related impurities, such as proteins, nucleic acids, and vector-related impurities, including wild-type / pseudo-wild-type AAV species (wtAAV) and residual DNA impurities encapsulated by AAV.

[0276] Considering that rAAV particles account for only a small part of the biomass, rAAV particles need to be purified to a purity level that can be used as a clinical human gene therapy product (see, for example, Smith PH et al., Mo. Therapy 7 (2003) 8348; Chadeuf G. et al., Mo. Therapy 12 (2005) 744; report from CHMP gene therapy expert group meeting, European Medicines Agency EMEA / CHMP2005, 183989 / 2004).

[0277] As an initial step, cultured cells producing rAAV particles are typically harvested and optionally combined with harvested cell culture supernatant (culture medium) in which cells (suspension cells or adherent cells) producing rAAV particles have been cultured. The harvested cells and optional cell culture supernatant can be used as is, optionally lysed or concentrated. Further, if infection is used to express auxiliary functions, residual helper virus can be inactivated. For example, adenovirus can be inactivated by heating to a temperature of about 60°C for, for example, 20 minutes or more, which only inactivates the helper virus because AAV is heat stable, while helper adenovirus is heat unstable.

[0278] The supernatant of the cells and / or harvest is lysed by destroying the cells (e.g., by chemical or physical means, such as detergents, microfluidization and / or homogenization) to release rAAV particles. During cell lysis or subsequently after cell lysis, a nuclease (such as, for example, benzonase) is added to degrade contaminated DNA. Typically, the resulting lysate is clarified to remove cell debris, for example, by filtering or centrifugation to obtain a clarified cell lysate. In a specific example, the lysate is filtered with a micron diameter pore size filter (such as a 0.1 μm to 10.0 μm pore size filter, for example, a 0.45 μm and / or pore size 0.2 μm filter) to produce a clarified lysate.

[0279] The lysate (optionally clarified) contains AAV particles (including rAAV vectors as well as empty capsids) and production / process-related impurities, such as soluble cellular components from host cells, which may include cell proteins, lipids and / or nucleic acids, and cell culture medium components, among others. The optionally clarified lysate is then subjected to a purification step to purify the AAV particles (including rAAV vectors) from the impurities using chromatography. Prior to the first chromatography step, the clarified lysate may be diluted or concentrated with an appropriate buffer.

[0280] Following cell lysis, optional clarification, and optional dilution or concentration, multiple subsequent and sequential chromatography steps may be used to purify the rAAV particles.

[0281] For example, during downstream processing, the removal of empty capsids from intact capsids is based on their different isoelectric points (pi) in anion exchange chromatography. The average pI calculated for all serotypes is 5.9 for intact capsids and 6.3 for empty capsids (Venkatakrishnan, B. et al., J. Virol. 87 (2013) 4974-4984).

[0282] 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). Therefore, in certain embodiments of all aspects and embodiments, the rAAV particle purification is a purification via cation exchange chromatography followed by a purification via anion exchange chromatography.

[0283] 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, the rAAV particle purification is purification via cation exchange chromatography followed by purification via size exclusion chromatography (SEC).

[0284] Still alternatively, the first chromatography step can 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, the rAAV particle purification is purification via affinity chromatography followed by purification via anion exchange chromatography.

[0285] Optionally, a third chromatography may be added to the preceding chromatography steps. Typically, the optional third chromatography step follows cation exchange, anion exchange, size exclusion or affinity chromatography.

[0286] Thus, in certain embodiments of all aspects and embodiments, rAAV particle purification is purification via cation exchange chromatography, followed by purification via anion exchange chromatography, followed by purification via size exclusion chromatography (SEC).

[0287] Additionally, in certain embodiments of all aspects and embodiments, further purification of the rAAV particles is purification via cation exchange chromatography, followed by purification via size exclusion chromatography (SEC), followed by purification via anion exchange chromatography.

[0288] In yet further embodiments of all aspects and embodiments, the rAAV particle purification is purification via affinity chromatography followed by purification via anion exchange chromatography followed by purification via size exclusion chromatography (SEC).

[0289] In yet further embodiments of all aspects and embodiments, the rAAV particle purification is purification via affinity chromatography followed by purification via size exclusion chromatography (SEC) followed by purification via anion exchange chromatography.

[0290] The role of cation exchange chromatography is to separate AAV particles from cells 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 capable of binding 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 ThermoFisher Scientific, Inc., Waltham, MA, USA). Additional examples include Capto S, Capto SImp Act, Capto S ImpRes (strong cation exchangers available from GE Healthcare, Marlborough, MA, USA), and commercial preparations of resins available from Aldrich Chemical Company (Milliwaukee, WI, USA). and Family. Weak cation exchange resins include, but are not limited to, any carboxylic acid-based resins. Exemplary cation exchange resins include carboxymethyl (CM) resins, phosphate resins (based on phosphate functional groups), methanesulfonate (S) resins, and sulfopropyl (SP) resins.

[0291] The effect of anion exchange chromatography is to separate AAV particles from proteins, cells and other components present in the lysate and / or column eluate of the clarification from affinity or cation exchange or size exclusion chromatography. Anion exchange chromatography can also be used to reduce and thus control the amount of empty capsids in the eluate. For example, the anion exchange column with the rAAV particles combined therewith can be washed with a solution of NaCl comprising a moderate concentration (e.g., about 100mM to 125mM, such as 110mM to 115mM), and a portion of empty capsids can be eluted when flowing through without eluting rAAV particles in large quantities. Subsequently, the rAAV particles bonded to the anion exchange column can be eluted using a solution comprising a higher concentration of NaCl (e.g., about 130mM to 300mM NaCl), thereby producing the column eluate of the rAAV particles comprising rAAV vectors with empty capsids reduced or depleted and proportionally increased amounts.

[0292] 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 those generally based on quaternized nitrogen atoms, including but not limited to quaternary ammonium salt resins such as trialkylbenzylammonium resins. Suitable exchange chromatography materials include, but are not limited to, MACROPREP Q (strong anion exchanger available from BioRad, Hercules, CA, USA); UNOSPHERE Q (strong anion exchanger available from BioRad, Hercules, CA, USA); POROS 50HQ (strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS XQ (strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS SOD (weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS 50PI (weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); Capto Q, Capto XQ, Capto Q ImpRes, and SOURCE30Q (strong anion exchangers available from GE Healthcare, Marlborough, MA, USA); DEAE SEPHAROSE (a weak anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA); Q SEPHAROSE (a strong anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA). Additional exemplary anion exchange resins include aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE), and quaternary aminoethyl (QAE).

[0293] Manufacturing processes for purifying recombinant AAV particles intended as products for treating human disease should achieve the following goals: 1) consistent particle purity, potency, and safety; 2) manufacturing process scalability; and 3) acceptable manufacturing costs.

[0294] An exemplary process for purification of recombinant AAV particles is reported in WO 2019 / 006390.

[0295] The recombinant adeno-associated virus particle (rAAV particle) purification and production methods outlined below are scalable to large scale. For example, to a suspension culture volume of 5, 10, 10-20, 20-50, 50-100, 100-200 or more liters. The recombinant adeno-associated virus particle purification and production methods are applicable to a variety of AAV serotypes / capsid variants.

[0296] In certain embodiments of all aspects and embodiments, purification of rAAV particles comprises the following steps:

[0297] a) harvesting mammalian cells and / or cell culture supernatant containing rAAV particles to produce a harvest;

[0298] b) optionally concentrating the harvest produced in step (a) to produce a concentrated harvest;

[0299] c) lysing the harvest produced in step (a) or the concentrated harvest produced in step (b) to produce a lysate;

[0300] d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate;

[0301] e) optionally filtering the nucleic acid-reduced lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate;

[0302] f) subjecting the nucleic acid-reduced lysate of step (d), the clarified lysate of step (e), or the diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprising rAAV particles, thereby separating the rAAV particles from protein impurities or other production / process related impurities, and optionally diluting the column eluate to produce a diluted column eluate;

[0303] g) subjecting the column eluate produced in step (f) or the diluted column eluate to anion exchange chromatography to produce a second column eluate comprising rAAV particles, thereby separating the rAAV particles from protein impurities or production / process related impurities, and optionally concentrating the second column eluate to produce a concentrated second column eluate;

[0304] h) subjecting the second column eluate or the concentrated second column eluate produced in step (g) to size exclusion column chromatography (SEC) to produce a third column eluate comprising rAAV particles, thereby separating the rAAV particles from protein impurities or production / process-related impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate; and

[0305] i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV particles;

[0306] Wherein the mammalian cells prior to transfection / production have been obtained using the method according to the invention.

[0307] In certain embodiments, steps (a) to (f) are retained and combined with the following steps:

[0308] g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography (SEC) to produce a second column eluate comprising rAAV particles, thereby separating the rAAV particles from protein impurities or other production / process related impurities, and optionally diluting the second column eluate to produce a concentrated second column eluate;

[0309] h) subjecting the second column eluate or the diluted second column eluate produced in step (g) to anion exchange chromatography to produce a third column eluate comprising rAAV particles, thereby separating the rAAV particles from protein impurity production / process related impurities, and optionally diluting the third column eluate to produce a diluted third column eluate; and

[0310] i) filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV particles.

[0311] In certain embodiments, steps (a) to (g) are retained and combined with the following steps:

[0312] h) filtering the second column eluate or the concentrated second column eluate produced in step (g), thereby producing purified rAAV particles.

[0313] In an embodiment, steps (a) to (e) are retained and combined with the following steps:

[0314] f) subjecting the nucleic acid-reduced lysate of step (d) or the clarified lysate or the diluted clarified lysate produced in step (e) to AAV affinity column chromatography to produce a column eluate comprising rAAV particles, thereby separating the rAAV particles from protein impurities or other production / process related impurities, and optionally concentrating the column eluate to produce a concentrated column eluate;

[0315] g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography (SEC) to produce a second column eluate comprising rAAV particles, thereby separating the rAAV particles from protein impurities or other production / process related impurities, and optionally diluting the second column eluate to produce a diluted second column eluate;

[0316] h) optionally subjecting the second column eluate or the diluted second column eluate produced in step (g) to anion exchange chromatography to produce a third column eluate comprising rAAV particles, thereby separating the rAAV particles from protein impurities or other production / process related impurities, and optionally diluting the third column eluate to produce a diluted third column eluate; and

[0317] i) filtering the second column eluate or the diluted second column eluate produced in step (g), or filtering the third column eluate or the concentrated third column eluate produced in step (h), thereby producing purified rAAV particles.

[0318] In certain embodiments of all aspects and embodiments, the concentration of step (b) and / or step (f) and / or step (g) and / or step (h) is performed via ultrafiltration / diafiltration, such as by tangential flow filtration (TFF).

[0319] In certain embodiments of all aspects and embodiments, the concentration of step (b) reduces the volume of harvested cells and cell culture supernatant by about 2 to 20 fold.

[0320] In certain embodiments of all aspects and embodiments, the concentrating of step (f) and / or step (g) and / or step (h) reduces the volume of the column eluate by about 5 to 20 fold.

[0321] In certain embodiments of all aspects and embodiments, the lysis of the harvest produced in step (a) or the concentrated harvest produced in step (b) is carried out 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 nonionic and ionic detergents. Non-limiting examples of non-ionic detergents include TritonX-100. Non-limiting examples of detergent concentrations are between about 0.1% (v / v) or (w / v) and 1.0% (v / v) or (w / v), including end values.

[0322] In certain embodiments of all aspects and embodiments, step (d) comprises treating with a nuclease to thereby reduce contaminating nucleic acids. Non-limiting examples of nucleases include benzonase.

[0323] In certain embodiments of all aspects and embodiments, the filtration of the clarified lysate or diluted clarified lysate of step (e) is performed via a filter. Non-limiting examples of filters are those having a pore size between about 0.1 microns and 10.0 microns (including end values).

[0324] In certain embodiments of all aspects and embodiments, the dilution of the clarified lysate of step (e) is performed with an aqueous buffered phosphate, acetate or Tris solution. Non-limiting examples of solution pH are between about pH 4.0 and pH 7.4, including end values. Non-limiting examples of Tris solution pH are greater than pH 7.5, such as between about pH 8.0 and pH 9.0, including end values.

[0325] In certain embodiments of all aspects and embodiments, the dilution of the column eluate of step (f) or the second column eluate of step (g) is carried out with an aqueous buffered phosphate, acetate or Tris solution. Non-limiting examples of solution pH are between about pH 4.0 and pH 7.4, including end values. Non-limiting examples of Tris solution pH are greater than pH 7.5, such as between about pH 8.0 and pH 9.0, including end values.

[0326] In certain embodiments of all aspects and embodiments, the rAAV particles produced in step (i) are formulated with a surfactant to produce a rAAV particle formulation.

[0327] In certain embodiments of all aspects and embodiments, the anion exchange column chromatography of steps (f), (g) and / or (h) comprises polyethylene glycol (PEG)-modulated column chromatography.

[0328] In certain embodiments of all aspects and embodiments, the anion exchange column chromatography of steps (g) and / or (h) is washed with a PEG solution prior to eluting the rAAV particles from the column.

[0329] In certain embodiments of all aspects and embodiments, the average molecular weight of PEG is in the range of about 1,000 g / mol to 80,000 g / mol, inclusive.

[0330] In certain embodiments of all aspects and embodiments, the concentration of PEG is about 4% (w / v) to about 10% (w / v), inclusive.

[0331] In certain embodiments of all aspects and embodiments, the anion exchange column of steps (g) and / or (h) is washed with an aqueous surfactant solution prior to eluting the rAAV particles from the column.

[0332] In certain embodiments of all aspects and embodiments, the cation exchange column of step (f) is washed with a surfactant solution prior to eluting the rAAV particles from the column.

[0333] 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.

[0334] In certain embodiments of all aspects and embodiments, the NaCl concentration in the buffer or solution ranges between about 20 mM to 300 mM NaCl, inclusive, or between about 50 mM to 250 mM NaCl, inclusive.

[0335] In certain embodiments of all aspects and embodiments, the surfactant comprises a cationic or anionic surfactant.

[0336] In certain embodiments of all aspects and embodiments, the surfactant comprises a twelve carbon chain surfactant.

[0337] In certain embodiments of all aspects and embodiments, the surfactant comprises dodecyltrimethylammonium chloride (DTAC) or Sarkosyl.

[0338] In certain embodiments of all aspects and embodiments, the rAAV particles are eluted from the anion exchange column of steps (f), (g) and / or (h) with aqueous Tris-HCl / NaCl buffer.

[0339] In certain embodiments of all aspects and embodiments, the Tris-HCl / NaCl buffer comprises 100 mM to 400 mM NaCl (inclusive), optionally at a pH in the range of about pH 7.5 to about pH 9.0 (inclusive).

[0340] In certain embodiments of all aspects and embodiments, the anion exchange column of steps (f), (g) and / or (h) is washed with aqueous Tris-HCl / NaCl buffer.

[0341] 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 mM to 125 mM, inclusive.

[0342] 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, inclusive.

[0343] In certain embodiments of all aspects and embodiments, the anion exchange column of steps (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.

[0344] In certain embodiments of all aspects and embodiments, washing of the anion exchange column removes empty capsids from the column prior to and / or in place of elution of the rAAV particles, thereby reducing the amount of empty capsids in the second or third column eluate.

[0345] In certain embodiments of all aspects and embodiments, washing the anion exchange column removes at least about 50% of the total empty capsids from the column prior to and / or in place of elution of the rAAV particles, thereby reducing the amount of empty capsids in the second or third column eluate by about 50%.

[0346] 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 mM to 120 mM, inclusive.

[0347] In certain embodiments of all aspects and embodiments, the ratio and / or amount of eluted rAAV particles and empty capsids is controlled by a wash buffer.

[0348] 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. Non-limiting NaCl concentrations in the buffer range from about 125 mM to 500 mM NaCl, inclusive. Non-limiting examples of buffer pH range from about pH 5.5 to about pH 7.5, inclusive.

[0349] In certain embodiments of all aspects and embodiments, the anion exchange column of steps (f), (g) and / or (h) comprises quaternary ammonium functional groups, such as quaternized polyethyleneimine.

[0350] In certain embodiments of all aspects and embodiments, the size exclusion column (SEC) of step (g) and / or (h) has a separation / fractionation range (molecular weight) of about 10,000 g / mol to about 600,000 g / mol (inclusive).

[0351] In certain embodiments of all aspects and embodiments, the cation exchange column of step (f) comprises a sulfonic acid or a functional group, such as a sulfopropyl group.

[0352] In certain embodiments of all aspects and embodiments, the AAV affinity column comprises a protein or ligand that binds to an AAV capsid protein. Non-limiting examples of proteins include antibodies that bind to an AAV capsid protein. More specific non-limiting examples include single-chain llama antibodies (Camelidae) that bind to an AAV capsid protein.

[0353] In certain embodiments of all aspects and embodiments, the method does not comprise the step of cesium chloride gradient ultracentrifugation.

[0354] In certain embodiments of all aspects and embodiments, the method recovers about 50% to 90% of the total rAAV particles from the harvest produced in step (a) or the concentrated harvest produced in step (b).

[0355] In certain embodiments of all aspects and embodiments, the method produces rAAV particles having a higher purity than rAAV particles produced or purified by a single AAV affinity column purification.

[0356] In certain embodiments of all aspects and embodiments, steps (c) and (d) are performed substantially simultaneously.

[0357] In certain embodiments of all aspects and embodiments, after step (c) but before step (f), the NaCl concentration is adjusted to within the range of about 100 mM to 400 mM NaCl (inclusive), or within the range of about 140 mM to 300 mM NaCl (inclusive).

[0358] In certain embodiments of all aspects and embodiments, the cells are cells growing in suspension or adherently.

[0359] In certain embodiments of all aspects or embodiments, the cell is a mammalian cell. Non-limiting examples include HEK cells, such as HEK-293 cells, and CHO cells, such as CHO-K1 cells.

[0360] Methods for determining the infectious titer of rAAV particles containing a transgene are known in the art (see, e.g., Zhen et al., Hum. Gene Ther. 15 (2004) 709). Methods for determining empty capsids and rAAV particles with packaged transgenes are known (see, e.g., Grimm et al., Gene Therapy 6 (1999) 1322-1330; Sommer et al., Malec. Ther. 7 (2003) 122-128).

[0361] In order to determine the presence or amount of degraded / denatured capsids, purified rAAV particles can be subjected to SDS-polyacrylamide gel electrophoresis, which consists of any gel (e.g., gradient gel) capable of separating the three capsid proteins, and then running the gel until the sample is separated and blotted onto a nylon or nitrocellulose membrane. Anti-AAV capsid antibodies are then used as primary antibodies that bind to denatured capsid proteins (see, e.g., Wobus et al., J. Viral. 74 (2000) 9281-9293). The secondary antibody that binds the primary antibody contains a tool for detecting the primary antibody. Semi-quantitative detection of the binding between the primary and secondary antibodies determines the amount of capsids. Another method is to use an analytical HPLC or analytical ultracentrifuge using a SEC column.

[0362] Perfusion

[0363] Due to the continuous exchange of culture medium in perfusion bioreactor, it is provided to continuously supply nutrients via feed and continuously remove waste via permeate. Therefore, the waste accumulation that negatively affects cell growth and viability observed in fed batch process can be overcome. In addition, continuous culture medium renewal provides a more stable cell environment, which is conducive to cell growth, health, metabolism and product quality (Chotteau, V., "Perfusion Processes", included in Animal Cell Culture, M. Al-Rubeai (Editor) Cham: Springer International Publishing, 2015, pp. 407-443; Karst, DJ et al., Curr. Opin. Biotechnol. 53 (2018) 76-84; Walther, J. et al., Biotechnol. J., 14 (2019) e1700733).

[0364] In the context of viral vector production, increasing research has focused on perfusion technology to further increase production yield, improve the full-empty (F / E) ratio, and reduce viral vector production costs. Perfusion mode has been validated as an advantageous strategy for producing 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, OW, J. Gene Med. 6 (2004) S105-S124; Benskey, MJ et al., Hum. Gene Ther. Meth. 27 (2016) 32-45; Coronel, J. et al., Gen. Engin. Biotechnol. News 41(2021)S23-S23).

[0365] For perfusion in viral vector production, compared with typical recombinant protein biotechnology production, the most important goal is no longer to achieve the highest possible cell density and maintain a long production phase to improve volume productivity. On the contrary, cells need to be maintained at about 2x 10^6 cells / mL to achieve efficient transfection. Due to the widely described cell density effect (CDE), the higher the cell density, the more difficult it is to transfect (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 transfection is performed at a higher cell density, transfection efficiency and productivity are both reduced (Lavado-García, J. et al., Front. Bioeng. Biotechnol. 8 (2020) Article 617).

[0366] Continuous perfusion is used to continuously update the culture medium while keeping most of the cells in the exponential growth phase to achieve efficient transfection (Henry, O. et al., Biotechnol. Bioeng. 86 (2004) 765-774; Cortin, V. et al., Biotechnol. Prog. 20 (2004) 858-863). Therefore, the production of viral vectors reaches a certain limited process span, and the viral vectors released into the culture medium can be quickly harvested.

[0367] The perfusion rate describes the rate of medium renewal during perfusion and is usually expressed as the volume of medium added per day per bioreactor working volume (vvd). Different perfusion rate control strategies have been described in the literature, including those based on cell density, major substrate or byproduct concentration.

[0368] When the culture medium renewal in the perfusion process is normalized to the cell density, it is expressed as the cell-specific perfusion rate. As described by Dowd et al., perfusion control based on CSPR directly links the perfusion rate to the cell density (Cytotechnol. 42 (2003) 35-45). A lower CSPR means that more cells can be maintained with a certain amount of culture medium. Therefore, by keeping the CSPR constant during the process, it is ensured that the cells have a consistent microenvironment in the culture.

[0369] To enhance the economic potential of the process, the volume perfusion rate should be minimized. However, it must be taken into account that if the CSPR is below the lower limit, poor cell growth, decreased viability, product degradation, or decreased specific productivity may affect production or make it impossible to produce. Therefore, the optimal CSPR must be adjusted to close to the minimum CSPR during process development (Konstantinov, K. et al., Adv. Biochem. Eng. Biotechnol. 101 (2006) 75-98; Bielser, JM et al., Biotechnol. Prog. 36 (2020) e3026).

[0370] Vaccination series

[0371] Traditional inoculation series usually starts with thawing a small amount of frozen cells (such as, for example, 1 mL of a cryopreserved vial from a working cell bank). Then, the cell culture is expanded into a larger culture system. For this reason, cells are cultured in shake flasks, rotary culture flasks, and stirred tank bioreactors with increased volumes. The production bioreactor is commonly referred to as the N stage, and the bioreactors before the production stage are named N-1, N-2, and N-3 in descending order.

[0372] The use of high-density cell banks and single-use systems improves operational success by reducing the duration of seeding series and potential contamination. This enables simplification and reduction of seeding series, thereby reducing process complexity, shortening operation time and increasing yield.

[0373] Dielectric spectroscopy

[0374] Dielectric spectroscopy, also known as capacitance measurement or radio frequency impedance, is a method mainly used for online monitoring of viable cell density or viable cell volume. The measurement principle of this tool is based on the characteristic of living cells with an intact non-conductive plasmid membrane. This enables them to store electrical charges, making living cells in aqueous ionic suspension behave like small capacitors when introduced to a periodic alternating electric field in the radio frequency range (0.1 to 10 MHz).

[0375] The movement of ions inside and outside the cell is restricted by the plasmid membrane, which acts as an insulating physical barrier. This results in polarization, which is the separation of charge across the membrane at the two poles of the cell. The magnitude of this polarization is measured by capacitance, ΔC, in picofarads (pF).

[0376] Therefore, by measuring the capacitance of a suspension, its viable cell density can be estimated (JP Carvell and JE Dowd, Cytotechnol. 50 (2006) 35-48; Justice, C. et al., Biotechnol. Adv. 29 (2011) 391-401; Cole, HE et al., Processes 3 (2015) 384-405).

[0377] For most cells, the S-shaped curve is centered between 0.5 MHz and 3 MHz with a critical frequency fc. The critical frequency is the frequency at which half of the capacitance drop is complete (Cannizzaro, C. et al., Biotechnol. Bioeng. 84 (2003) 597-610). Usually, dielectric spectroscopy is performed at a frequency close to this frequency because the capacitance here is independent of the cell size (Justice, C., supra).

[0378] The capacity increases with increasing cell volume fraction because there are more polarized membranes, which in turn provide higher measured capacitance. In general, capacitance depends not only on VCD, but also on cell type, cell size, and the physiological state of the cell.

[0379] For VCD prediction, a mathematical model is required to relate the capacitance signal to the offline biomass data: Multiply the capacitance ΔC by the cell constant K (given in 1 / cm) to obtain the absolute dielectric constant ε (given in pF / cm):

[0380] ε=ΔC*K (1)

[0381] Biocapacitance values ​​for controlling perfusion processes, where the perfusion feed is automatically adjusted based on the viable cell concentration from an online capacitance probe, have been demonstrated in several studies (Carvell and Dowd, supra; Cannizzaro, C. et al., supra). In addition, dielectric spectroscopy is becoming a relevant tool for monitoring viral vector production processes as it provides key information about the physiological state of cells (Pais, DAM et al., Processes 8 (2020) 1456; Escandell, JM et al., Curr. Opin. Biotechnol. 74 (2022) 271-277).

[0382] Embodiments of the method according to the invention

[0383] Scaling up the production process of recombinant AAV particles to 100 L, 500 L, or even 1,000 L presents significant challenges to both cell expansion and plasmid transfection.

[0384] Achieving high capsid titers is a goal of recombinant AAV particle production. Therefore, generating large cell aggregates that result in the production of empty capsids is not suitable for achieving this goal.

[0385] Therefore, the focus of perfusion processes for producing viral vectors is not on achieving high cell density but on alleviating nutrient limitation by ensuring constant replacement of the culture medium.

[0386] A variety of cell retention devices can be used for cell retention in viral vector manufacturing methods. The most commonly used are alternating tangential flow filtration (ATF), tangential flow filtration (TFF), acoustic filters, and membrane-based internal retention systems.

[0387] The ATF technology uses a hollow fiber module connected to a bioreactor via a single port. Using a diaphragm pump, the cell suspension is pumped through the hollow fiber module (where the cells are trapped), and a transparent supernatant containing the product of interest is harvested. Then, to reduce membrane fouling, the flow direction is reversed and the remaining cell suspension is flushed back into the bioreactor. The diaphragm pump produces a fast low shear flow to ensure that the cells return to the bioreactor quickly. Recently, Coronel et al. have shown that the ATF system can be used for perfusion to increase rAAV particle production (Gen.Engin.Biotechnol.News 41(2021)S23-S23).

[0388] However, in a particular embodiment of the method according to the invention, the perfusion is not done via ATF.

[0389] In more detail, TFF technology uses a hollow fiber system comparable to ATF, but uses a low shear circulation pump instead of a diaphragm pump and two bioreactor ports. One port extracts liquid from the bioreactor and the other port returns liquid from an external cell retention device to the bioreactor. Different TFF systems are commercially available (Cytiva's Xcellerex APS TM 、Repligen's KML100 TM or Merck's Cellicon TM ) for this application.

[0390] Suspension-based cell culture is an important step in adherent culture to provide scalability. However, compared with adherent cells, the cell density per unit volume is generally low. Therefore, it is necessary to reach a high cell density per unit volume to approach the transfection efficiency in adherent culture. In addition, good cell growth and viability are required throughout the entire inoculation series and production method to ensure high productivity. As production volume increases, this becomes more difficult, for example, with respect to sufficient gas input, efficient mixing and nutrient supply. Therefore, cells need to adapt to the ever-changing environment. Furthermore, every change in culture parameters triggers the internal control mechanism of the cell and activates the reaction, which may result in a reduction in cell and product yield or an increase in by-product formation.

[0391] Additionally, regarding the transfection process, a decrease in transfection efficiency often occurs during scale-up.

[0392] To improve the production of recombinant AAV particles, it is necessary to improve the inoculum series to prevent substrate depletion and excessive accumulation of waste products.

[0393] This is achieved by ensuring an optimal physiological state of the cells before transfection. This enables high transfection efficiencies. This has been achieved by reducing the proportion of spent culture medium in the production bioreactor from about 40% to about 20%. As a result, there is a lower concentration of inhibitory waste and at the same time the concentration of nutrients is increased. This allows the cell growth rate to be improved from about 0.3 / d to about 1.0 / d at the beginning of the N-process. In addition, the inoculation series can be simplified by omitting one bioreactor. This reduces process complexity. In addition, by using automatic perfusion based on online viable cell density (VCD) measurement (e.g. using dielectric spectroscopy) and applying cell-specific perfusion rate (CSPR), the required perfusion medium can be reduced by about one third.

[0394] In addition, by using the method according to the invention, the cell density at the time of transfection in the main fermenter (i.e. the production bioreactor) can be controlled more accurately. The so-called cell density effect (i.e. an overshoot of the cell density at the time of transfection), which leads to lower production yields in AAV production, can thereby be reduced or even prevented.

[0395] The present invention is based at least in part on the discovery that for the production of AAV particles, it is advantageous that the host cells have been propagated prior to transfection and that the AAV particles are thereby produced using perfusion. In more detail, it has been found to be advantageous to propagate mammalian cells in a preculture using perfusion for recombinant production of AAV particles. An advantage of the present invention is that mammalian cells propagated using perfusion prior to transfection maintain growth at a higher growth rate and higher mitotic activity and exhibit higher viability after transfection (i.e. during production culture). Thus, higher titers can be achieved compared to cultures in which mammalian cells are cultured using only a batch or fed-batch method prior to transfection, albeit with the same inoculation cell density.

[0396] The present invention is based, at least in part, on the discovery that for production of AAV particles in cell culture starting from low cell densities, it is advantageous to propagate mammalian cells by perfusion and dilute the propagated mammalian cells prior to transfection with a transgene encoding nucleic acid.

[0397] It has been found that without the use of perfusion, nutrients are excessively reduced and waste products accumulate during the pre-culture period in such an amount that the resulting cells are severely compromised and no longer suitable for subsequent AAV particle production due to the affected cell growth during the culture period.

[0398] In the following examples, parameters such as cell line, AAV serotype, culture medium, transfection reagent, as well as process parameters such as temperature, pH and pO2 were kept constant or highly similar in order to directly compare the results obtained with different processes. Therefore, the observed improvements were directly caused by the differences in culture conditions (i.e., the feed used to propagate the cells before transfection).

[0399] In the comparative example, the production of recombinant AAV particles was carried out at a 100 L scale.

[0400] The main fermenter was set to be inoculated with a cell density of 20*10^5 cells / mL and a starting volume of 60 L, with a target of 1.2*10^11 cells that had to be reached after the N-1 fermentation. Using a working volume of 25 L in N-1, the desired final density in N-1 was at least 48*10^5 cells / mL. Since it was known that the cells doubled almost every day and to increase the safety margin, the inoculation of N-1 was performed with 8*10^5 cells / mL to reach a final density of approximately 60*10^5 cells / mL. The N-2 fermentation was inoculated with 5*10^5 cells / mL based on the same considerations and reached approximately 40*10^5 cells / mL after three days. The pH values ​​in the inoculation series were selected to support high cell growth while limiting glucose consumption and accumulation of toxic wastes. In the main fermenter, the pH was slightly increased to increase AAV production and their release into the culture medium.

[0401] However, as cell growth and viability subsequently deteriorated in the N-2 stage, major problems occurred in the 25L shaker at N-1. Furthermore, the cells did not recover after being transferred to the main fermenter because they were too damaged. This meant that transfection might not be possible, as efficient transfection requires very high viability.

[0402] Poorer cell growth was observed in the N-1 stage than in the N-2 stage. In N-2, cells grew from 4*10^5 cells / mL to almost 40*10^5 cells / mL during the three-day growth phase. However, in the following N-1, cells started from 7.8*10^5 cells / mL and reached only 28.6*10^5 cells / mL in the same time span.

[0403] In N-2, the high growth rate of 0.79 / d began to decline after the first day of N-1 and dropped sharply, reaching only 0.47 / d after three days. At the same time, the viability, which had been maintained at a high level in N-2, gradually dropped from 97.5% to 92.4% in N-1. After the cell suspension was transferred to the main fermenter, the cells did not recover. On the contrary, before the planned transfection, the viability continued to drop from 94% to 86.9%. Therefore, transfection could not be performed. After four days, the viability continued to drop to 70%, and the growth rate also dropped, which dropped to 0.14 / d at the end of the process.

[0404] There is no limitation at the end of the N-2 fermentor. Furthermore, fresh medium is added when transferring to the N-1 fermentor to increase the substrate concentration back to its initial value. However, at the end of the process, glucose limitation occurs in N-1 with a concentration of 0.4g / L. Without being bound by this theory, another reason for poor cell growth may be the large-scale generation and accumulation of waste. During N-2, a large amount of lactate and ammonium have been accumulated. This results in the initial concentration of N-1 being higher than the initial concentration of N-2. Therefore, almost 5g / L lactate and 52mg / L ammonium are reached at the end of the N-2 stage.

[0405] Due to the lower VCD at the N-1 terminus, the entire fermentation volume had to be transferred to the main fermentor and the starting volume reduced to 50 L instead of 60 L to reach the required inoculum density of 20*10^5 cells / mL.

[0406] This can be obtained from Figure 1 Blue = N-2, fed-batch; Green = N-1, fed-batch; Red = production phase. Insufficient growth of cells in culture results in low transfection efficiency for AAV production.

[0407] It has now been found that the use of perfusion in the propagation of mammalian cells prior to the production of AAV particles according to the methods of the invention results in improvements, irrespective of whether the perfusion is uncontrolled perfusion or controlled perfusion.

[0408] In a first example according to the invention, perfusion was used in the N-1 phase, with a fixed perfusion rate. The perfusion rate was increased once a day, from 0.21 vvd (day 1) to 0.42 vvd (day 2) to 0.63 vvd (day 3). Thus, in this example, a total of 29.1 L or 1.16 times the bioreactor volume of culture medium was delivered to the cell culture during perfusion.

[0409] However, the ventilation control needed to be adjusted because the pO2 value fluctuated violently and dropped to almost 0% directly after inoculation. To improve the oxygen input into the fermenter, the shaking rate was increased after 3.5 hours and further increased after 25.5 hours. The O2 controller used here is a proportional-integral-derivative (PID) controller. Its basic working principle is: read the sensor, calculate the difference between the expected set point (SP) and the measured process value (PV), and then apply the correction by setting the desired actuator output. This output is calculated by calculating the proportional, integral and derivative response and adding these three components. To reduce controller overshoot, the integral term is turned off after 6.5 hours. Only proportional and derivative gains are used thereafter. After 26.5 hours, the ventilation rate is increased from 500mL / min to 1000mL / min.

[0410] Compared to the comparative N-1 process without perfusion, the cells showed higher cell growth, increasing from a starting VCD of 10.9*10^5 cells / mL to a VCD of up to 92.3*10^5 cells / mL. Thus, a 3.2-fold cell density was achieved using an inoculation density that was only increased to 1.4-fold compared to the comparative first run without perfusion. Viability remained high throughout the run, with a minimum of 96.9% one day after inoculation, in stark contrast to the sharp drop in viability when no perfusion was performed. The growth rate was very high at the beginning, at 1.05 / d, but decreased during the process, falling to 0.76 / d after three days. Thus, the growth rate was 1.3 and 1.6 times the growth rate in the comparative N-1 fermentation without perfusion at the beginning and end, respectively. Thus, not only the absolute growth rate increased, but also the drop in growth rate during perfusion was reduced.

[0411] After a lag period of 1.5 hours after inoculation in the main fermentation, cells resumed growth.

[0412] The cell growth in N-2 and N-1 was excellent, with a final VCD of 44*10^5 cells / mL in N-2. In N-1, a higher VCD of 92*10^5 cells / mL was achieved due to perfusion. In addition, the viability was always maintained at a high level above 97%, and the final growth rate of 0.64 / d in N-2 and 0.77 / d in N-1 confirmed the good cell proliferation in the inoculation series. This growth continued at the beginning of the main fermenter with a growth rate of 0.80 / d, which means that the cell suspension was successfully transferred from N-1 to the N bioreactor.

[0413] As expected, the growth rate decreased after transfection of the cell culture on day 1, and dropped directly to a final value of 0.12 / d before harvesting on day 4. It should also be noted that the main fermentation was inoculated with a cell density of 26.77*10^5 cells / mL instead of 20*10^5 cells / mL. Therefore, the cell density before adding medium was 52*10^5 cells / mL, which is higher than the expected 36*10^5 cells / mL.

[0414] The results of uncontrolled perfusion at different culture volumes (scales) are as follows Figure 2 As shown. 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 from 0.21 vvd to 0.42 vvd to 0.63 vvd every day. This resulted in sustained growth and high viability of the propagated cells. Production of AAV particles of serotype 8 was successfully completed.

[0415] However, despite the supplementation of nutrients by perfusion, the concentrations of glucose and glutamine decreased as in the process without perfusion. Therefore, substrate limitation occurred at the end of the fermentation. Without being bound by this theory, this can be explained by the fact that significantly higher cell densities are achieved with perfusion, and more cells also consume higher absolute substrate amounts. This means that the applied perfusion rate is not enough to supply enough nutrients to keep the concentration constant in a fast-growing cell culture. Therefore, the perfusion rate is adjusted to reduce or even eliminate the depletion of substrate. Therefore, perfusion with viable cell density control (VCD control) is adopted.

[0416] Therefore, the perfusion rate is calculated as outlined in the Examples section. The VCD is planned using the average specific growth rate of 0.764 / d from the first example according to the present invention. The goal of the N-1 fermentation is to reach a final VCD of approximately 60*10^5 cells / mL at a 25L working volume to provide sufficient cells for the inoculation of the main fermentor. Therefore, the working volume of the N-1 fermentation can be reduced while still reaching the required number of cells at a higher cell density. If the working volume is reduced to 10L, a final cell density of at least 120*10^5 cells / mL is required. Based on the growth rate of 0.764 from the first perfusion run, an inoculation density of 15*10^5 cells / mL will be required to reach a final cell density of 150*10^5 cells / mL. A scaled-down model with a working volume of 1L is used.

[0417] Determine cell-specific substrate consumption based on predicted VCD. Cell-specific consumption rates for glucose (7.50*10^-10 g / cell / d) and glutamine (1.20*10^-10 g / cell / d) were calculated as the average of the first two processes using HEK293 cells in a 10L shaker without perfusion. These values ​​were then used to determine the perfusion rate required to provide sufficient substrate for the cell-specific consumption. Since the perfusion rate required to keep the glutamine concentration constant was lower than the perfusion rate required to keep the glucose concentration constant, the perfusion rate required to keep the glucose concentration constant was used. The perfusion rate was increased once a day. To avoid nutrient limitations caused by temporary medium supply interruptions or process variability, each daily rate was calculated using the predicted VCD at the end of the day.

[0418] The planned VCD was only slightly higher than the VCD actually measured at the end of the process. The growth rate determined was 0.673 / d, 0.9 times lower than the predicted value. This resulted in a final cell density of 128*10^5 cells / mL after 2.9 days. During the process, the perfusion rate was manually readjusted using the daily measured substrate concentration and VCD. The resulting perfusion rate then matched the perfusion rate based on the process conditions very well. Overall, 2.36L of culture medium was required for perfusion, corresponding to 2.36 times the bioreactor volume.

[0419] In the last process part, VCD increased more in the run with controlled perfusion rate, reaching a final value of 128*10^5 cells / mL, rather than 92.3*10^5 cells / mL in the first perfusion run. This trend is also reflected in the growth rate. In the first perfusion process, the growth rate decreased during the process, especially from 0.90 / d to 0.77 / d between 2.2 days and 2.9 days. In the process with controlled perfusion rate, the growth rate was generally lower, with an average of 0.67 / d, but remained relatively stable and even increased towards the end.

[0420] Viability also showed a slight decrease at the end of the first perfusion process, while remaining constant or increasing slightly towards the end of the perfusion process with the controlled perfusion rate.

[0421] The controlled perfusion process with adjusted perfusion rate prevented glucose and glutamine limitation at the end of the culture. However, the substrate concentrations could not be kept constant, and the lactate and glutamate concentrations were similar or slightly higher than in the first perfusion process.

[0422] The results of viable cell density-controlled (VCD-controlled) perfusion at 1L scale are shown in Figure 2. Figure 3 As shown (controlled perfusion). The perfusion rate was calculated using cell-specific consumption rates based on cell density measurements and calculations of cell growth. No substrate limitation was observed. To achieve a target cell density of 80 to 100*10^5 cells / mL, cell division was completed after 3 days.

[0423] The method according to the invention is further improved by using the growth rate prediction VCD from the previous perfusion process. The VCD prediction provides the necessary basis for the subsequent determination of the perfusion rate. With controlled perfusion, it is not possible to keep the substrate concentration constant, but substrate limitation can be avoided. This allows to obtain consistently good cell growth and viability, with a constant growth rate at the end of the process. The controlled perfusion process uses twice as much perfusion medium as the first perfusion process.

[0424] The results of VCD controlled perfusion at 100L scale are as follows Figure 4 Green = N-1, controlled perfusion; Red = production phase. Production of AAV particles of subtype 2 variant 7m8 was successfully completed.

[0425] The fermenter volume of the N-1 bioreactor is increased to 15 L to achieve a sufficiently high cell density for inoculation of the main fermenter. However, an inoculation VCD of approximately 13*10^5 cells / mL is required to reach the target density of 100*10^5 cells / mL after three days, resulting in the need for a relatively high volume of pre-culture. Therefore, the N-1 process is run for a longer period of time, in this example six days. However, this will require a very low inoculation VCD of 1.6*10^5 cells / mL. Such low inoculation VCDs are difficult to set accurately because cell aggregation can lead to inaccurate measurements. Even the resulting small deviations can result in a final VCD that is too low or a medium consumption that is too high, because medium consumption increases exponentially with VCD. The solution to this problem is to inoculate with a higher, easily adjustable VCD and to separate the cell culture in the middle of the process to achieve the desired final VCD as accurately as possible. Therefore, the next perfusion process was seeded with 5*10^5 cells / mL and split to 13*10^5 cells / mL on day 3, so that the final VCD reached approximately 100*10^5 cells / mL after six days.

[0426] After three days, the cell culture was split from 55.4*10^5 cells / mL to 10.5*10^5 cells / mL, resulting in a final VCD of 97.4*10^5 cells / ml. The average growth rate of the first growth phase after the split was determined to be 0.77 / d, and the average growth rate of the second growth phase was 0.78 / d. This is closely related to the average growth rate of 0.72 / d predicted based on the first two perfusion processes. The viability immediately after inoculation was 96%.

[0427] A total of 3.3 bioreactor volumes of perfusion medium were added throughout the process. No substrate limitation occurred during the first growth phase, and the substrate concentration was still higher than in the previous perfusion process. However, the inoculation VCD was lower, reaching 5.8*10^5 cells / mL, compared to 17.4*10^5 cells / mL in the first 1L perfusion process.

[0428] Although this new setup of the N-1 fermentor did not shorten the overall duration of the inoculation series, it allowed the elimination of the N-2 fermentor due to the lower inoculation density of N-1.

[0429] The process was repeated using the dielectric constant signal generated by the capacitance probe to control the perfusion rate (including splitting after propagation on day 3). This resulted in a process with automatic perfusion control according to the present invention. A linear correlation between the VCD measured off-line and the dielectric constant measured on-line was demonstrated. The correlation found between the viable cell density and the dielectric constant enabled a more economical control of the perfusion process, resulting in a reduction of about 30% in the perfusion medium (from 3.3 times the reactor volume to 2.2 times the reactor volume). The cell density curve for the dielectric constant controlled perfusion process is shown in Figure 1. Figure 5 As shown (N-1, dielectric constant controlled perfusion, 10L scale). For this exemplary process, the cell-specific perfusion rate applied was 107 pL / cell / day. In other examples, CSPR of 101 pL / cell / day has been applied.

[0430] This process was repeated using an online biomass sensor to control the perfusion rate (including splitting after propagation on day 3). Production of AAV particles containing the subtype 2 variant 7m8 encoding the transgene for the therapeutic Fab was successfully completed. The cell density curve for the biomass sensor controlled perfusion process is shown in Figure 2. Figure 6 Green = N-1, biomass sensor controlled perfusion; red = production phase.

[0431] Figure 7 Two viable cell densities for two pairs of VCD-controlled perfusion and biosensor-controlled perfusion are shown: red (VCD) and purple (biosensor); blue (VCD) and green (biomass sensor).

[0432] Figure 8 The viable densities obtained in different production-scale cultures inoculated with cells obtained by perfusion at the N-1 stage (i.e., according to the present invention) and without perfusion at the N-1 stage are shown. The red curve corresponds to a production-scale culture inoculated with cells from an N-1 culture without perfusion, and the green curves (marked "1", "2", "3", "4") correspond to production-scale cultures inoculated with cells from an N-1 culture perfused according to the present invention. It can be seen that the higher viable cell density obtained in the culture inoculated with cells propagated by perfusion at the N-1 stage leads to a higher AAV particle yield (transfected 24 hours after the start of the culture).

[0433] Fig. 9It is shown that the viability of the cells in the production scale culture N depends on the culture of the cells used to inoculate the N stage in the N-1 stage. The red curve corresponds to the production culture inoculated with cells obtained from the batch N-1 culture. The green curves (denoted as "1", "2", "3" and "4") correspond to the production scale culture inoculated with cells obtained from the N-1 perfusion culture according to the present invention. It can be seen that the cells obtained by the N-1 perfusion grow better and respond better to the transfection, i.e. maintain a higher viability, 24 hours after the start. These cells still grow further and continue to produce rAAV particles.

[0434] Fig.10 Capsid titer in production culture in a bioreactor system >10 L in N is shown after N-1 with and without perfusion. The red curves ("5", "6") correspond to production cultures after batch N-1. The green curves ("1", "2", "3" and "4") correspond to production-scale cultures after N-1 with perfusion. For the red curves, batch culture N-1 was performed in the same bioreactor (thus, N-scale culture day zero corresponds to day 4 in the figure). For comparable bioreactor systems, the effect of increasing capsid titer is significant when cells cultured with perfusion at the N-1 stage are used for inoculation.

[0435] Therefore, the method according to the present invention achieves higher final cell density and capsid titer. When using perfusion to propagate cells, the fluctuation range is also reduced. Without being bound by this theory, it is believed that cells propagated using perfusion have a higher tolerance to stress during transfection, recover faster, maintain a higher viable cell density and are still growing (although slowly).

[0436] ***

[0437] All references mentioned herein are hereby incorporated by reference.

[0438] ***

[0439] The following examples, sequences and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It should be understood that modifications may be made to the procedures set forth without departing from the spirit of the present invention.

[0440] Examples

[0441] Conventional techniques

[0442] 1) Recombinant DNA technology

[0443] Standard methods were used to manipulate DNA, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1989). Molecular biology reagents were used according to the manufacturers' instructions.

[0444] 2) DNA and protein sequence analysis and sequence data management

[0445] The EMBOSS (European Molecular Biology Open Software Suite) software package and Invitrogen's VectorNTI and Geneious Prime were used for sequence creation, mapping, analysis, annotation, and illustration.

[0446] 3) Gene and oligonucleotide synthesis

[0447] The desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into E. coli plasmids for propagation / amplification. The DNA sequences of the subcloned gene fragments were verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or via PCR. Individual oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany).

[0448] 4) Reagents

[0449] If not stated otherwise, all commercial chemicals, antibodies and kits were used as provided according to the manufacturers' protocols.

[0450] 5) Cloning

[0451] conventional

[0452] Cloning using the R site depends on a DNA sequence next to the target gene (GOI) that is equivalent to the sequence located in the following fragment. Similarly, the assembly of fragments is possible by overlapping of identical sequences and subsequent sealing of the nicks in the assembled DNA by DNA ligase. Therefore, it is necessary to clone individual genes, especially preliminary plasmids containing the correct R sites. After successful cloning of these preliminary plasmids, the target gene flanked by R sites is cut off by restriction digestion by enzyme cutting directly next to the R site. The last step is to assemble all DNA fragments in one step. In more detail, 5' exonuclease removes the 5' end of the overlapping region (R-site). Afterwards, annealing of the R site can be performed, and DNA polymerase extends the 3' end to fill the gap in the sequence. Finally, DNA ligase seals the gap between nucleotides. Adding an assembly master mix containing different enzymes (such as exonucleases, DNA polymerases and ligases), followed by incubation of the reaction mixture at 50°C, assembles the individual fragments into a plasmid. Afterwards, the plasmid is used to transform competent E. coli cells.

[0453] For some plasmids, a cloning strategy via a restriction enzyme is used. By selecting a suitable restriction enzyme, the desired target gene can be excised, and then inserted into different plasmids by connection. Therefore, it is preferred to use an enzyme that cuts at a multiple cloning site (MCS), and select in a clever manner, so that the connection of the fragment can be carried out in the correct array. If the plasmid and the fragment are cut with the same restriction enzyme before, the sticky ends of the fragment and the plasmid fit together perfectly, and can be connected by DNA ligase subsequently. After the connection, the competent E. coli cells are transformed with the newly generated plasmid.

[0454] Cloning by restriction digestion

[0455] For digestion of plasmids with restriction enzymes, pipette the components shown in the table below together onto ice.

[0456] Table: Restriction digestion reaction mixture

[0457]

[0458] If more enzymes were used in one digestion, 1 μL of each was used and the volume was adjusted by adding more or less PCR-grade water. All enzymes were chosen on the premise that they were qualified for use with CutSmart buffer from New England Biolabs (100% activity) and had the same incubation temperature (37°C for all).

[0459] The incubation was performed using a thermomixer or thermal cycler, allowing the samples to incubate at a constant temperature (37°C). During the incubation, the samples were not agitated. The incubation time was set to 60 min. The samples were then mixed directly with the loading dye and loaded onto an agarose electrophoresis gel or stored at 4°C / ice for further use.

[0460] A 1% agarose gel was prepared for gel electrophoresis. Therefore, 1.5 g of multipurpose agarose was weighed into a 125 conical shake flask and filled with 150 mL of TAE buffer. The mixture was heated in a microwave oven until the agarose was completely dissolved. 0.5 μg / mL of ethidium bromide was added to the agarose solution. The gel was then cast in a mold. After the agarose was shaped, the mold was placed in the electrophoresis chamber and the chamber was filled with TAE buffer. The sample was then loaded. In the first pocket (starting from the left), the appropriate DNA molecular weight marker was loaded, followed by the sample. The gel was run at <130V for approximately 60 minutes. After electrophoresis, the gel was removed from the chamber and analyzed in a UV imager.

[0461] The band was cut and transferred into a 1.5 mL Eppendorf tube. For purification of the gel, the QIAquick Gel Extraction Kit from Qiagen was used according to the manufacturer's instructions. The DNA fragments were stored at -20 °C for further use.

[0462] The fragments for ligation are pipetted together at a plasmid / insert molar ratio of 1:2, 1:3 or 1:5, depending on the length of the insert and plasmid fragments and their relationship to each other. If the fragment that should be inserted into the plasmid is short, a ratio of 1:5 is used. If the insert is long, a smaller amount of plasmid is used. A plasmid amount of 50 ng is used in each ligation, and the specific amount of insert is calculated using the NEBioCalculator. For ligation, a T4 DNA ligation kit from NEB is used. The table below describes an example of a ligation mixture.

[0463] Table: Ligation Reaction Mixture

[0464]

[0465]

[0466] Start by mixing the DNA and water, add the buffer and finally the enzyme, pipetting all components together onto ice. Gently mix the reaction by pipetting up and down, briefly microcentrifuge, and then incubate at room temperature for 10 minutes. After incubation, heat inactivate the T4 ligase at 65°C for 10 minutes. Cool the sample on ice. In the last step, transform 10-beta competent E. coli cells (see below) with 2 μL of the ligated plasmid.

[0467] Transformation of 10-β competent E. coli cells

[0468] For transformation, 10-β competent E. coli cells were thawed on ice. Afterwards, 2 μL of plasmid DNA was directly transferred to the cell suspension. The test tube was flicked and placed on ice for 30 minutes. Thereafter, the cells were placed in a 42°C heat block and heat-shocked for exactly 30 seconds. Immediately thereafter, the cells were cooled on ice for 2 minutes. 950 μL of NEB 10-β growth medium was added to the cell suspension. The cells were incubated at 37°C with shaking for one hour. Then, 50 μL to 100 μL were transferred to a preheated (37°C) LB-Amp agar plate and plated with a disposable spatula. The plates were incubated overnight at 37°C. Only bacteria that successfully incorporated a plasmid carrying a resistance gene for ampicillin could grow on these plates. Single colonies were picked the next day and cultured in LB-Amp medium for subsequent plasmid preparation.

[0469] Bacterial culture

[0470] The culture of E. coli was carried out in LB medium (abbreviation of Luria Bertani) into which 1 mL / L of 100 mg / mL ampicillin was added so that the ampicillin concentration was 0.1 mg / mL. For different plasmid preparation quantities, the following amounts were inoculated with a single bacterial colony.

[0471] Table: E. coli culture volume

[0472]

[0473]

[0474] For small-scale preparation, fill each well of a 96-well 2 mL deep-well plate with 1.5 mL of LB-Amp medium. Pick a colony and insert a toothpick into the medium. When all colonies are picked, seal the plate with a sticky air porous membrane. Incubate the plate in a 37°C incubator at a shaking speed of 200 rpm for 23 hours.

[0475] For small-scale preparation, 3.6 mL of LB-Amp medium was filled in a 15 mL tube (with vented cap) and inoculated with bacterial colonies. During incubation, the toothpick was not removed but left in the tube. As with the 96-well plate, the tube was incubated at 37°C, 200 rpm for 23 hours.

[0476] For large-scale preparation, 200 mL of LB-Amp medium was filled into an autoclaved 1L Erlenmeyer glass flask and inoculated with 1 mL of bacterial day culture approximately 5 hours later. The Erlenmeyer flask was closed with a paper stopper and incubated at 37°C, 200 rpm for 16 hours.

[0477] Plasmid preparation

[0478] For miniprep, 50 μL of bacterial suspension was transferred to a 1 mL deep well plate. Afterwards, the bacterial cells were centrifuged in the plate at 3000 rpm, 4°C for 5 min. The supernatant was removed and the plate with the bacterial pellet was placed in the EpMotion. After approximately 90 minutes, the run was completed and the eluted plasmid DNA could be removed from the EpMotion for further use.

[0479] For the miniprep, remove the 15 mL tube from the incubator and aliquot 3.6 mL of the bacterial culture into two 2 mL Eppendorf tubes. Centrifuge the tubes at 6,800 x g for 3 minutes at room temperature in a benchtop microcentrifuge. Afterwards, perform the miniprep using the Qiagen QIAprep Spin Miniprep Kit according to the manufacturer's instructions. Measure the concentration of the plasmid DNA using a Nanodrop.

[0480] Large quantities were prepared using the Macherey-Nagel method according to the manufacturer's instructions. DNA concentration was measured with Nanodrop.

[0481] Ethanol precipitation

[0482] Mix a volume of DNA solution with 2.5 volumes of 100% ethanol. Incubate the mixture at -20°C for 10 min. Then centrifuge the DNA at 14,000 rpm, 4°C for 30 min. Carefully remove the supernatant and wash the pellet with 70% ethanol. Centrifuge the tube again at 14,000 rpm, 4°C for 5 min. Carefully remove the supernatant by pipetting and dry the pellet. When the ethanol evaporates, add an appropriate amount of endotoxin-free water. Give the DNA time to redissolve in 4°C water overnight. Take a small aliquot and measure the DNA concentration using a Nanodrop device.

[0483] Expression cassette composition

[0484] For expression of open reading frames, transcription units are used that contain at least the following functional elements:

[0485] - promoter,

[0486] - if necessary, a nucleic acid comprising a corresponding open reading frame including a signal sequence,

[0487] - polyadenylation signal sequence.

[0488] In addition to the expression unit / cassette containing the desired gene to be expressed, the basic / standard mammalian expression plasmid also contains

[0489] - an origin of replication from the plasmid pUC18, which allows replication of this plasmid in E. coli, and

[0490] -β-lactamase gene, which confers ampicillin resistance in Escherichia coli.

[0491] 6) Cell culture technology

[0492] Standard cell culture techniques were used as described in Current Protocols in Cell Biology (2000), Bonifacino, JS, Dasso, M., Harford, JB, Lippincott-Schwartz, J. and Yamada, KM (Eds.), John Wiley & Sons, Inc.

[0493] Transient transfection in HEK293 system

[0494] Cells producing recombinant AAV particles have been generated by transient transfection with the corresponding plasmids using the HEK293 system (Invitrogen) according to the manufacturer's instructions. Briefly, cells were grown in shake flasks or stirred fermenters in serum-free FreeStyle TM HEK293 cells (Invitrogen) grown in suspension in 293 expression medium (Invitrogen) were incubated with the corresponding plasmids and 293fectin TM For 2L shake flasks (Corning), HEK293 cells were transfected at 1*10 6 The cells were inoculated at a density of 10 cells / mL in 600 mL and incubated at 120 rpm and 8% CO2. The next day, the cells were cultured at a density of 1.5*10 6 Cells / mL were transfected with a cell density of about 42 mL of a mixture of the following: A) 20 mL Opti-MEM (Invitrogen) with 600 μg total plasmid DNA (1 μg / mL) and B) 20 mL Opti-MEM + 1.2 mL 293fectin or fectin (2 μL / mL). Glucose solution was added during the fermentation process according to glucose consumption.

[0495] 7) Monitoring and analysis methods

[0496] Dielectric spectroscopy

[0497] Dielectric spectroscopy in the RM 20 / 50 shaker is performed using a pre-installed disposable live biomass sensor. Before starting the fermentation, the signal was tested with low (0 pF / cm, 0 mS / cm) and high (100 pF / cm, 40 mS / cm) signal simulators, and then the probes were connected to the relevant ports of the bag.

[0498] for Bioreactor, using the Incyte system. The IncyteArc 220 probe was inserted into the probe assembly sheath, autoclaved, and installed into the bioreactor.

[0499] Prior to inoculation, the dielectric constant probe was zeroed to the culture medium according to the manufacturer's instructions. For the IncyteArc220, the ArcAir application was used for zeroing. Both systems measure capacitance, which is calculated as the dielectric constant ε in pF / cm. The dielectric constant is linearly related to VCV or VCD and can therefore estimate viable cell density in real time.

[0500] Cell culture analysis

[0501] use The HiRes Analyzer measures VCD, viability, mean cell diameter, and aggregation rate. It is a fully automated, image-based cell analyzer that combines trypan blue exclusion with a high-resolution image scanner.

[0502] To determine the above cell parameters, a predetermined volume of a cell suspension sample is drawn up via the syringe module and the manifold valve and mixed with the required amount of 0.2% trypan blue solution. Cells with non-intact cell membranes are stained blue by diffusion of the dye into the cell interior. In intact cells, the dye cannot enter. The stained cell suspension is transferred to the flow chamber via the capillary and the manifold valve. After a fixed sedimentation time, a digital image is taken using a scanner. The scanned image of the flow chamber is divided into smaller images for evaluation and analyzed using analysis software on the relevant computer.

[0503] Based on the estimated total cell density (TCD), process samples need to be diluted with PBS according to the table below prior to analysis to bring them within the working concentration range of the system (0.1 to 50 10^5 cells / mL).

[0504] Table: Based on the estimated total cell density, The required dilution is measured by the HiRes analyzer.

[0505] Total cell density [10^5 cells / mL] dilution 0–50 None (1:1) 51–100 1:2 101–200 1:5 201–500 1:10 >500 1:20

[0506] In addition to typical cell parameters, The HiRes analyzer also provides information on aggregates and morphological parameters. Information on cell aggregation is relevant for this work, since the cell lines used are prone to aggregation, which may reduce transfection success (Girard et al., P., Cytotechnol. 38 (2002) 15-21). HiRes software identified cells that aggregated with at least one other cell and calculated the aggregation ratio as the percentage of cells found in aggregates relative to the total cell count.

[0507] use The relative standard deviation of the multiple measurements made by the HiRes analyzer is 5%. However, due to the additional dilution error outside the measurement range, a relative standard deviation of 6% can be assumed according to internal specifications.

[0508] In addition to the directly measured cell parameters, the viable cell volume (VCV) is calculated because this parameter is needed to correlate with the dielectric constant signal from the capacitance probe. For the calculation of the viable cell volume, the cell shape is assumed to be spherical. Using The VCD in cells / mL and the mean cell diameter (d) in μm measured by the HiRes analyzer were used to determine the VCV in mm3 / mL according to equation (14):

[0509]

[0510] Example 1 - Reference Example

[0511] Basic process for rAAV particle production without perfusion

[0512] The production of rAAV particles begins with inoculating 125mL shake flasks with frozen cell aliquots after thawing. Every three to four days, the cell culture is passaged and the volume is increased. After reaching a sufficient volume (i.e., cell density) for inoculation of the N-2 bioreactor, the preculture is transferred to a 10L wave mixing bioreactor with an inoculation density of about 5*10^5 cells / mL, and the culture continues for three days. Then, after reaching a cell density of about 40*10^5 cells / mL, the cell culture is further transferred to a 25L wave mixing bioreactor with an inoculation density of about 8*10^5 cells / mL. In two wave mixing bioreactors, only alkali is added. Afterwards, the main fermenter is inoculated with a cell suspension, with a starting volume of 60L and a target inoculation density of 20*10^5 cells / mL. After 24 hours, 20% fresh culture medium based on the additional volume of the actual culture volume is added. The cells are then transfected with the corresponding plasmids. After a total of four days, the production process was terminated and lysis was initiated by adding lysis buffer, nuclease and alkaline solution. The lysed cell solution was then transferred to a harvest tank and filtered to harvest the rAAV particles.

[0513] Example 2

[0514] RM Shaker Operation

[0515] Small-scale experiments and series fermentation of N-2 and N-1 seeds RM shaker, which contains a wave mixing benchtop bioreactor and B control tower. These bioreactors utilize shaking mixing technology and are combined with disposable For 10L scale N-2 fermentation, use CultiBag RM 50. Use The RM 20 / 50basic performs N-1 fermentations at 15 L or 25 L scale and small-scale perfusion experiments at 1 L scale. Both have interchangeable bag holders to accommodate bags with total volumes from 1 to 50 L. The shaking platform includes an integrated local controller, air and CO2 mixing module and force gauge.

[0516] The waves generated in the RM bag ensure efficient gas exchange across the gas-liquid interface via two mechanisms, namely surface aeration and air entrainment by breaking waves. For adequate aeration, four different gas lines for compressed air, O2, N2 and CO2 are equipped with flow meters and four mass flow controllers are used. An integrated pressure sensor continuously measures the pressure inside the bag and sets the aeration to achieve a slight overpressure. RM bags are equipped with inlet and outlet filters.

[0517] Two heating elements using resistive heating are directly integrated into the bag holder and can be controlled individually. For small-scale experiments, only the left heating circuit is used and the 1L bag is placed on the left side of the bag holder. A Pt100 resistance thermometer in the bag holder measures the bag temperature. The heating of the RM produces a light condensation on the exhaust filter, so the filter heater keeps the filter dry and prevents it from clogging. The hood is used as a safety cover with an opening at the front for bag handling. It protects the connected bag from mechanical influences during operation and reduces heat losses.

[0518] RM bags are equipped with disposable photochemical pH and pO2 probes and, when required, a live biomass probe ( ViaMass). A pH controller regulates the addition of sodium carbonate and CO2 gas flow. In addition, the bag has a free port with C-Flex tubing and Luer or MPC connector for the addition of culture medium, inoculum and base. A dip tube with Luer septum is used for sampling.

[0519] For perfusion experiments, special bags with an integrated 1.2 μm perfusion membrane are used. The membrane is fixed to the bottom of the bag, forming a compartment for the removal of cell-free culture medium. The surface of the membrane is rinsed with each shaking of the waves thus generated, which ensures low contamination and long culture times. The peristaltic pump on the B control tower performs media addition and permeate removal. The media bag is placed on a scale for weight control.

[0520] Example 3

[0521] 100L disposable bioreactor operate

[0522] The main fermentation for the production of rAAV particles was carried out in a single-use bioreactor with a total working volume of 100 L. The stirred tank includes a stainless steel fermentation tank container, a disposable Vessel (BPC), operating station with DCU, temperature control unit and culture station with six scales and peristaltic pump. The reactor is equipped with a double jacket for temperature control and a longitudinal sight glass. Temperature control is performed via the temperature control unit. Agitation is performed using an inclined 3-blade impeller.

[0523] Aeration is done via a submerged aeration tube with an annular sparger and an open tube. The BPC is equipped with two inlet filters at the bottom and two exhaust filters and aeration filter heater at the top. In addition to the exhaust port at the top of the BPC, there are inlets for headspace aeration, a pressure sensor and several free ports for adding defoamer, base and feed. The BPC is closed with two side port levels in the lower third of the bag. The lower port level contains a port for a temperature probe and four Connector probe ports for pH, pO2 and capacitance probes. Sampling is done using the built-in dip tube. Above these connectors, the BPC has a free port with C-Flex tubing for fresh media addition and transfection. For harvesting, the BPC has a drain line at the bottom.

[0524] The bags with base and different feeds are placed on the hanging scale of the culture station to achieve weight control. The pH controller regulates the base addition. Defoamer is added as needed.

[0525] Example 4

[0526] Pre-cultivation and inoculation

[0527] Pre-culture is carried out in a shake flask with fermentation medium. The cells are split to 5*10^5 cells / mL every three to four days until they reach a cell density of approximately 40 to 60*10^5 cells / mL, and the culture volume is increased to the amount required for inoculation. During this period, the shake flask is incubated with shaking at 37°C, 85% humidity, and 5% CO2.

[0528] for 100 L fermentation in bioreactor, cells were placed in N-2 and N-1 The culture was further cultured in a RM shaker and then used to inoculate the main fermenter.

[0529] RM shaker and The fermentation inoculation procedures in the bioreactor are comparable. First, the inoculation volume is determined based on the current and target initial VCD. The preculture is pumped into the bioreactor using a low pump rate to prevent excessive shear stress.

[0530] For inoculation 100L The large preculture volumes required for the bioreactors use hydrostatic pressure differentials to gently and quickly transfer the cells to the fermenter.

[0531] Example 5

[0532] Transfection procedure

[0533] 24 hours after seeding, triple transfection was performed at 36*10^5 cells / mL of target VCD.

[0534] Before transfection, 20% (v / v) of the initial culture volume was added directly to the bioreactor as fresh medium. ), mixed with plasmid and filled with fermentation medium. After mixing, the solution was transferred to 10L At the end of the incubation period, the contents of the bag containing the transfection complex were transferred to the bioreactor. Next, the free PEI solution and the VPA solution were pumped into the bioreactor in sequence to reach a final concentration of 5 mMVPA.

[0535] Example 6

[0536] Perfusion Bioreactor Operation

[0537] The pH set point was defined and the dead band was set to 0.02 pH units. For pH control, a double-sided control loop was used, where CO2 sparging or addition of 1 M sodium carbonate solution (hereinafter referred to as base) was applied to lower or increase the pH, respectively.

[0538] 1) Manual operation of the perfusion process - daily change

[0539] Use a fixed perfusion rate based on the amount of medium remaining from the previous procedure. Gradually increase the perfusion rate once a day.

[0540] 2) Manual operation of the perfusion process - based on cell-specific rates

[0541] A fixed perfusion rate was used, which was adapted to the cell-specific substrate depletion.

[0542] Determine cell-specific substrate consumption rates based on previous cultures and multiply by the predicted VCD to calculate the amount of substrate required by the cells.

[0543] Predicting VCD using specific growth rate

[0544] VCD is predicted by the average specific growth rate of the whole process time calculated in the above-mentioned first perfusion culture. The average specific growth rate is determined by plotting the natural logarithm of VCD and the relationship between the treatment time t and inserting a linear fit, and the absolute value of the slope μ is the specific growth rate according to the following formula (Zhang, X. et al., "1.21-Modes of Culture / Animal Cells", included in: Comprehensive Biotechnology (Second Edition), M. Moo-Young (Editor) Burlington: Academic Press, 2011, pp. 285-302):

[0545] ln(VCD(t))=μ*t+b (2)

[0546] This leads to simplification, since the specific growth rate can fluctuate throughout the process. However, since the perfusion process generally allows very stable cell growth, the average growth rate can be used to predict the VCD. Therefore, the VCD process for the next perfusion experiment can be predicted by using equation (3):

[0547] VCD(t)=VCD 初始 *e μ*t (3)

[0548] Calculation of cell-specific depletion rates

[0549] At the same time, the cell-specific consumption rate was calculated based on the previous N-2 fermentation, since no substrate was added or removed except for cell metabolism, thus simplifying the calculation. In addition, the consumption rate can be calculated using the substrate concentration rather than its absolute weight, since the fermenter weight remains constant. The cell-specific consumption rate q 消耗It is defined as the amount of substrate consumed by a single cell per unit time, usually in pg / cell / d. The calculation involves the integral of the viable cell density (IVCD), which is interpreted as the production capacity of the cell culture, in units of cells*d / mL (see Zhang, supra). Therefore, the cell-specific consumption rate is calculated according to equation (4) by t The difference between the initial substrate concentration and t-1 and divide it by the difference in the associated IVCD to determine:

[0550]

[0551] IVCD is defined as

[0552]

[0553] Next, the absolute consumption rate q is predicted by multiplying the predicted VCD with the determined consumption rate 绝对消耗 , in units of g / L / d.

[0554] q 绝对消耗 =VCD*q 消耗 (6)

[0555] Determination of perfusion rate

[0556] Thus, the perfusion rate pvvd (in units of vvd) can be calculated using equation (7) by substituting the absolute consumption rate q 绝对消耗 (in g / L / d) divided by the concentration of substrate in the culture medium c 培养基 (in g / L) to obtain.

[0557]

[0558] In addition, the maximum perfusion rate was used by calculating each daily rate using the maximum predicted VCD to allow for fluctuations in substrate consumption. To be able to compare different perfusion processes, the cell-specific perfusion rate CSPR (in pL / cell / d) was also calculated according to Bausch et al. (Biotechnol. J. 14 (2019) 1700721). Therefore, the perfusion rate pvvd (in vvd) was divided by VCD:

[0559]

[0560] In a working volume of 1 L The reduced model is set up in the RM bag. Although under the same conditions, the model is a much smaller version physically. As described below, based on the determination of trypan blue exclusion cell counts and substrate and metabolite concentrations, the perfusion rate is further adjusted by manual daily sampling. If VCD is higher than expected or the substrate concentration drops too much, the perfusion rate is adjusted. In addition, by using the mean value of specific growth rate and cell-specific substrate consumption rate rather than individual values ​​for further calculation, the calculation of predicted VCD and substrate consumption is refined with each experiment.

[0561] Perfusion Automation Using Capacitance Probes

[0562] To automate the perfusion rate, a robust automatic perfusion rate control system based on the viable cell concentration from an online capacitance probe was established. The system operates in a completely closed loop, eliminating the need to collect samples to obtain process information. In the control algorithm, the CSPR is specified, and the dielectric constant signal of the capacitance probe is converted into the perfusion rate through calculation and implementation using a variable speed control pump.

[0563] To this end, the dielectric constant output signal of the probe is converted into an online VCD. Therefore, the correlation between the offline measured VCD and the dielectric constant signal is determined. To determine the correlation factor c VCD,介电常数 , plot the relationship between VCD and dielectric constant ε, and insert a linear trend line according to equation (9):

[0564] VCD=c VCD，介电常数 *ε (9)

[0565] To determine the pump output required to replenish the desired volume of feed medium based on the current dielectric constant signal and the previously determined CSPR, the VCD is predicted and the required perfusion rate pvvd is calculated using the above equation. 输出 The correlation between the values ​​(expressed in %) was determined using data from previous perfusion experiments. The correlation factor c 泵,pvvd Determined by interpolating the linear fit using the following equation:

[0566]

[0567] Using the correlation coefficient c 泵,pvvd The pump output is determined by the required perfusion rate pvvd. To achieve automatic control of the pump via the dielectric constant signal ε, the automation factor f is calculated using equation (11): 自动化 and enter it into the bioreactor control software:

[0568]

[0569] This allows the pump output to be set by multiplying the automation factor with the dielectric constant signal. In addition to the control loop for perfusion feeding, another control loop is used for removing permeate based on the bioreactor weight. This loop is the control loop that triggers the outflow of cell-free permeate for harvesting. Since the bioreactor is placed on a scale, the osmotic pump is configured to start removing medium once the bioreactor weight is greater than the initial weight after inoculation, until the weight is at the starting point again.

[0570] To assess the accuracy of the VCD predictions, the mean absolute percentage error (MAPE) was determined. This is a measure to assess the overall performance of the prediction model. As described in equation (12), the MAPE expressed in % is determined by the sum of the deviations of the predicted values ​​x predicted from the actual values ​​xi normalized to the actual values ​​(dividing it by the number of data points n).

[0571]

[0572] Example 7

[0573] Assessment of cell growth and metabolism

[0574] The specific growth rate μ represents the dynamic behavior of the cell and provides information about cell growth, and thus indirectly the health of the cell culture. It is a good basis for comparing different processes (AK Srivastava and S. Gupta, "2.38-Fed-Batch Fermentation-Design Strategies", included in: Comprehensive Biotechnology (Second Edition), M. Moo-Young (Editor) Burlington: Academic Press, 2011, pages 515-526). The growth rate of the entire fermentation process is 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", included in: Comprehensive Biotechnology (Second Edition), M. Moo-Young (Editor) Burlington: Academic Press, 2011, pages 285-302).

[0575]

[0576] Therefore, the final growth rate in the process also corresponds to the average growth rate over the entire process time.For fermentations with a cell culture split, VCD0 was set to VCD directly after the split.

[0577] Cell-specific formation and consumption rates were determined according to equation (4) in the absence of feeding / perfusion and the following equation (16) during perfusion:

[0578]

[0579] in

[0580]

[0581] and

[0582] m 输入 =c 培养基+进料 *p*(t n -t n-1 )+m 丸剂 (18)

[0583] The abbreviations are as follows:

[0584]

[0585] While the metabolite production rate is positive, the substrate consumption rate is negative because the substrate is removed from the culture medium and thus the amount in the culture medium decreases.

Claims

1. A method for producing recombinant AAV particles, the method comprising the following steps: a) propagating mammalian cells using perfusion until at least a first predetermined cell density is reached; b) diluting a portion of the cells obtained in step a) by adding 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) transfecting said cells directly in the cultured producer cell solution obtained in step c) with one or more nucleic acids encoding said recombinant AAV particles; e) culturing the transfected producer cell solution obtained in step d) for 24 to 144 hours; Recombinant AAV particles are thereby produced.

2. The method of claim 1, wherein the first predetermined cell density is at least 80*10^5 cells / mL.

3. The method according to any one of claims 1 to 2, wherein the mammalian cell is a CHO-K1 or HEK293 cell.

4. The method according to any one of claims 1 to 3, wherein the one or more nucleic acids comprise: i) a transgene comprising in the 5' to 3' direction α) the first ITR sequence; β) Promoter; γ) a nucleic acid sequence encoding a therapeutic molecule; δ) polyadenylation signal sequence; ε) the second ITR sequence; ii) rep open reading frame; iii) cap open reading frame; and iv) Adenovirus E1A, E1B, E2A, E4orf6 and VA RNA open reading frames.

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

6. The method according to any one of claims 1 to 5, wherein the second predetermined cell density is about 20*10^5 cells / mL.

7. The method according to any one of claims 1 to 6, wherein the culturing in step c) lasts for about 24 hours.

8. The method according to any one of claims 1 to 7, wherein the culturing in step e) lasts for about 72 hours.

9. The process according to any one of claims 1 to 8, wherein step e) is free of feed.

10. The method according to any one of claims 1 to 9, wherein the method further comprises the following steps: f) harvesting the cells from the cultured solution of step e); g) lysing the cells obtained in step f); h) isolating AAV particles from the lysed cells obtained in step g); i) optionally purifying the AAV particles.

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

12. The method according to any one of claims 1 to 11, wherein after step c) and before step d) the following steps cd) are carried out: cd) Add additional approximately 20% of the culture volume of fresh medium.

13. The method according to any one of claims 1 to 12, wherein the transfection is performed by adding the one or more nucleic acids as a mixture of PEI complex nucleic acid and free PEI.

14. The method according to any one of claims 1 to 13, wherein the transfection is performed in the presence of a final concentration of 5 mM valproic acid.

Images