AAV Production Methods

Abstract

Methods for producing recombinant adeno-associated virus (sAAV) particles include an expansion phase that includes expanding the number of cells in at least one culture vessel containing a culture medium; introducing into cells a first polynucleotide sequence that includes a transgene flanked by AAV inverted repeats, and optionally a second polynucleotide sequence that includes AAV rep and cap genes, and / or a third polynucleotide sequence that includes one or more helper genes; culturing the cells into which the one or more polynucleotide sequences have been introduced; and culturing the cells by inducing calcium (Ca) ions (e.g., Ca 2+ a production phase comprising adding an rAAV salt to the production phase culture medium, and isolating the rAAV particles.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 211,877, filed June 17, 2021, which is incorporated by reference in its entirety.

[0002] The present disclosure provides methods for producing adeno-associated virus (AAV) vectors by adding certain salts, particularly calcium salts, such as calcium chloride, to recombinant AAV production media. [Background technology]

[0003] Adeno-associated virus (AAV) is a replication-deficient parvovirus. AAV particles contain a capsid with three capsid proteins - VP1, VP2, and VP3 - that contains a single-stranded DNA genome of about 4.8 kb in length, which may be either positive or negative strand. Particles containing either strand are infectious and replicate by converting the single strand that infects the parent cell into a double-stranded form, which then amplifies, from which the single strand of the progeny cell is displaced and packaged into the capsid.

[0004] AAV depends on co-infection with other viruses, primarily adenovirus, to replicate. Its single-stranded genome contains three genes, rep (replication), cap (capsid), and aap (assembly), which generate at least nine gene products through the use of three promoters, alternative translation initiation sites, and differential splicing. These coding sequences are flanked by inverted terminal repeats (ITRs) that are necessary for genome replication and packaging. The rep genes encode four proteins (Rep78, Rep68, Rep52, and Rep40) that are involved in viral genome replication and packaging, whereas cap expression generates viral capsid proteins (VP1, VP2, and VP3) that form the outer capsid shell that protects the viral genome and are actively involved in cell binding and internalization. The aap gene encodes an assembly-activating protein (AAP) in an alternative reading frame that overlaps with the cap gene. This AAP protein is thought to provide a scaffolding function for capsid assembly.

[0005] AAV particles have characteristics that make them attractive as vectors for therapeutic applications, including gene therapy and genetic vaccines. AAV infects a wide range of cell types, including many mammalian cells, allowing the possibility of targeting many different tissues in vivo. AAV infects slowly dividing and non-dividing cells. For therapeutic applications, recombinant AAV (rAAV) is used, in which the genome contains a heterologous transgene and typically retains the ITRs but lacks the viral rep, cap, and aap genes. In the absence of Rep proteins, the transgene flanked by the ITRs can form a transcriptionally active extranuclear chromosomal element or episome that can persist essentially for the life of the transduced cell.

[0006] Cell culture systems for producing rAAV vectors include transient transfection of human cell lines and infection of mammalian or insect cell lines. rAAV vector production methods generally involve a germline cell type that provides the biosynthetic machinery for vector production in combination with a helper vector (e.g., helper plasmid, helper virus) as a source of additional gene products required for rAAV replication and packaging.

[0007] A key goal of rAAV vector production methods is to achieve stable, high vector productivity while minimizing the generation of product-associated impurities, including residual AAV-encapsidated DNA impurities and empty capsids. Measured as vector genomes (VG) produced per cell, rAAV vector productivity can reach 10 per cell. 3 Less than ~2×10 5 In addition to being cost-effective, a key advantage of high productivity is that purification may be more efficient when the starting material has a higher ratio of rAAV vector product to total harvested biomass.

[0008] Product-associated impurities are similar to the rAAV vector itself and cannot be easily separated from the rAAV vector during the purification process. Current rAAV vector production systems produce high levels of AAV empty capsids. For AAV2, 50-95% of all AAV particles produced during cell culture are empty capsids. However, to limit potential harmful immune responses to AAV capsids, it is prudent to minimize the generation of empty capsids during cell culture and / or substantially remove them during vector purification. Thus, there is a need for methods to increase rAAV productivity and / or reduce product-associated impurities such as empty capsids. Summary of the Invention

[0009] The present disclosure provides methods for making AAV vectors by adding certain salts, particularly calcium salts, such as calcium chloride, to recombinant adeno-associated virus (rAAV) production media. Described herein are methods for producing recombinant adeno-associated virus (rAAV) particles, comprising: (i) an expansion phase, which comprises increasing the number of cells in at least one culture vessel containing a culture medium; (ii) introducing into the cell a first polynucleotide sequence comprising a transgene flanked by AAV inverted terminal repeats (ITRs), and optionally a second polynucleotide sequence comprising AAV rep and cap genes, and / or a third polynucleotide sequence comprising one or more helper genes; (iii) culturing the cells from step (ii) and adding calcium (Ca) ions (i.e., Ca) to the culture medium from about 0 to about 24 hours after introduction of the first polynucleotide sequence such that the total concentration of Ca ions in the culture medium is greater than 0.3 mM and less than 10 mM; 2+ a production phase, in which the rAAV particles are produced, comprising adding (iv) Isolating rAAV particles.

[0010] In an embodiment of the method, the addition of Ca ions to the culture medium comprises the addition of a Ca salt, such as CaCl2. The calcium ions are added to the culture medium to a concentration of Ca ions between about 0.5 mM and about 9 mM, between about 1 mM and about 9 mM, between about 1 mM and about 8 mM, between about 1 mM and about 7 mM, between about 2 mM and about 9 mM, between about 2 mM and about 8 mM, between about 2 mM and about 7 mM, between about 2 mM and about 6 mM, between about 2 mM and about 5 mM, or between about 2 mM and about 4 mM. In embodiments, calcium ions are added to a concentration of about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, or about 9.5 mM. In embodiments, calcium ions are added to the production culture medium to a concentration of about 0.5 mM to about 6 mM; about 2 mM to about 6 mM, about 2 mM to about 4 mM, or about 1 mM to about 3 mM.

[0011] In some embodiments, Ca ions (Ca salts, e.g., CaCl2) are added after introduction of at least one of the first, second, and third polynucleotide sequences or at the start of the production phase (i.e., about 0 hours after introduction). In embodiments, Ca ions are added any time from about 0 hours to about 24 hours (e.g., 0, about 1, about 2, about 5, about 8, about 12, about 18, about 23, about 24, about 24.5 hours) after introduction of the first polynucleotide sequence or after the start of the production phase. In embodiments, Ca ions are added any time from about 0 hours to about 18 hours after introduction of the first polynucleotide sequence or after the start of the production phase. In embodiments, Ca ions are added any time from about 6 hours to about 20 hours, about 6 hours to about 18 hours, or about 6 hours to about 12 hours after introduction of the first polynucleotide sequence or after the start of the production phase. In some embodiments, Ca ions are added about 12 hours after introduction of at least one of the first, second, and third polynucleotide sequences (or the initiation of the production phase) such that the total concentration of Ca in the culture medium is about 2 mM.

[0012] In an embodiment of the method, the expansion phase culture medium and / or the production phase culture medium are serum-free. In an embodiment, the expansion phase and / or the production phase comprises adding one or more of glutamine, glutamine precursors, or amino acid dipeptides containing glutamine to the expansion phase culture medium and / or the production phase culture medium. The one or more of glutamine, glutamine precursors, or amino acid dipeptides containing glutamine may be, for example, one or more of L-alanyl-L-glutamine, L-glutamine, glutamate, glycyl-L-glutamine, glutamine protein hydrolysate, L-glutamic acid, and glutamine dipeptide. A solution comprising at least one of glutamine, a glutamine precursor, or an amino acid dipeptide comprising glutamine may be added to the production phase culture medium, for example, about 6 hours, about 12 hours, about 24 hours, about 48 hours, and / or about 72 hours after introduction of at least one of the first, second, and third polynucleotide sequences, and about 6 hours, about 12 hours, about 24 hours, about 48 hours, and / or about 72 hours after initiation of the production phase.

[0013] In embodiments of the method, the expansion phase and / or production phase includes adding an aggregation-inhibiting supplement to the expansion phase culture medium and / or production phase culture medium. In embodiments, the aggregation-inhibiting supplement includes dextran sulfate, heparin, and / or other sulfated glycosaminoglycans that inhibit cell aggregation. In embodiments, the aggregation-inhibiting supplement includes sodium heparin, which may be added to the medium to a concentration of about 25 μg / ml to about 250 μg / ml, e.g., about 25 μg / ml, about 50 μg / ml, about 100 μg / ml, about 150 μg / ml, and / or about 200 μg / ml. In embodiments, the aggregation-inhibiting supplement is not added to the production phase or is added only at the end of the production phase, immediately prior to the isolation step, e.g., within about 10 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour of the step of isolating the produced rAAV particles.

[0014] In an embodiment, the production phase includes adding sorbitol to the production phase medium. In an embodiment, sorbitol is added to the production phase medium at one or more time points during the production phase, for example, about 6 hours, about 12 hours, about 20 hours, about 24 hours, about 48 hours after introduction of at least one of the first, second, and third polynucleotide sequences (i.e., about 6 hours, about 12 hours, about 20 hours, about 24 hours, about 48 hours after the start of the production phase). In an embodiment, sorbitol is added to the production medium to a concentration of about 50 mM to about 200 mM, or about 80 mM to about 120 mM. In an embodiment, sorbitol is added to the production medium to a concentration of about 100 mM.

[0015] In embodiments, the production phase is at least about 48 hours (e.g., 47.5, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 96.5, or 97 hours). In preferred embodiments, the production phase is about 72 hours to about 100 hours, about 90 hours to about 100 hours, about 92 hours to about 98 hours, or about 94 hours to about 98 hours. In one embodiment, the production phase is about 96 hours.

[0016] In embodiments of the method, at least one of the first, second, and third polynucleotide sequences is introduced into the cell by transfection of one or more vectors comprising the first, second, and third polynucleotide sequences, by infection with one or more viral vectors comprising one or more of the first, second, and third polynucleotide sequences, by a combination of transfection of one or more vectors and infection with one or more viruses, or by electroporation of the first, second, and third polynucleotide sequences. In embodiments, at least one of the first, second, and third polynucleotide sequences is introduced into the cell by transfection.

[0017] In an embodiment of the method, the transgene encodes a therapeutic or reporter protein. A non-exclusive list of examples of transgenes includes RPGR, RPE65, GAD65, GAD67, and CNGB3. In some embodiments, the two AAV ITRs are AAV2 ITRs. In the method, the AAVcap gene may be an AAV serotype or AAV variant, such as, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrhlO, AAV-APHP.5, AAV-PHP.B, AAV-PHP.eB, AAV2-retro, AAV9-retro, and hybrids thereof. In an embodiment, the one or more helper genes may include all or part of one or more adenovirus genes, herpes simplex virus type 1 genes, or baculovirus genes.

[0018] In embodiments of the method, the cells are mammalian cells, e.g., HEK293 cells. The cells (e.g., HEK293 cells) may be adapted for culture in suspension. The at least one culture vessel may be, for example, one or more of a shaker flask, a spinner flask, a cell bag, or a bioreactor. In embodiments, the culture medium for the expansion phase may have a pH of about 7.1 to about 7.5 (e.g., about 7.2 to about 7.4), and the step of culturing the cells may include CO2 sparging. In some embodiments, the pH of the culture medium is allowed to drift between about 6.9 to about 7.3 prior to the step of introducing at least one of the first, second, and third polynucleotide sequences. In embodiments, the pH of the culture medium is changed to about 6.9 and the CO2 sparging is stopped prior to introducing one or more polynucleotide constructs into the cells.

[0019] In embodiments, isolating the rAAV particles includes a step of lysing the cells after the production phase. In embodiments, the rAAV particles may be isolated about 48 hours or more (e.g., 47.5, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 96.5, 97 hours) after introduction of the first, second, and / or third polynucleotide sequences (step (ii) above). For example, the rAAV particles may be isolated about 90 to about 100 hours, about 92 hours to about 98 hours, or about 94 to about 98 hours after introduction of the first, second, and / or third polynucleotide sequences. In embodiments, the rAAV particles are isolated (e.g., lysing the cells) about 96 hours after introduction of the first, second, and / or third polynucleotide sequences (e.g., about 96 hours after step (ii) above). In embodiments, the isolated rAAV particles are present in a lysate and the lysate is clarified using at least one filter to provide a clarified lysate. In such embodiments, the method may further comprise subjecting the clarified lysate to one or more purification steps. Any suitable purification step(s) may be used, e.g., one or more of ultracentrifugation, affinity chromatography, and / or ion exchange chromatography. In embodiments, the method provides at least about 40,000 to about 200,000 rAAV particles per cell and / or about 8×10 purified rAAV particles from one liter of transfected culture medium. 12 ~Approx. 1×10 14 In embodiments, the method provides an rAAV particle to empty AAV particle ratio of at least about 30%, e.g., about 30%-40%, at least 65%, such as about 65%-90%. In embodiments, the method further includes determining at least one of capsid concentration, viral genome titer, and rAAV particle to empty AAV particle ratio from the lysate or a portion of the clarified lysate. In embodiments of this method, the isolated rAAV particles are separated from empty AAV particles.

[0020] The invention also provides rAAV particle populations produced by the methods, and pharmaceutical compositions comprising the rAAV particle populations. [Brief description of the drawings]

[0021] [Figure 1A] Figure 1 shows the results of Ca2+ supplementation in a flask model for producing rAAV5 vectors containing the RPGR transgene. HEK293 cells were transfected with the RPGR transgene plasmid, helper plasmid, and Rep / Cap plasmid, and 0.5mM or 2mM CaCl2 was added either 6 hours or 20 hours after transfection. Cells were lysed either 72 hours or 96 hours after transfection. The total:empty (F:E) ratios of AAV5-RPGR, vector genome (VG), and capsid in the lysates at 72 hours and 96 hours after transfection are provided. [Figure 1B] Figure 1 shows the results of Ca2+ supplementation in a flask model for producing rAAV2 vectors containing a GAD67 transgene. HEK293 cells were transfected with GAD67 transgene plasmid, helper plasmid, and Rep / Cap plasmid. 20 or 24 hours after transfection, 100 mM sorbitol, 50 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, or 2 mM CaCl2 / MgCl2 were added to the culture medium. Cells were lysed either 72 or 96 hours after transfection. Vector genomes (VG) of AAV2-GAD67 in lysates at 72 and 96 hours after transfection are provided. [Diagram 2] (A and B) are graphs showing rAAV2 production in 250 mL bioreactors after Ca2+ supplementation, addition of aggregation inhibitor (ACA), and / or addition of concentrated essential nutrient feed during the production phase. (A) shows VG titer as VG / mL in lysates from 96 hour (post-transfection) harvests. (B) shows total to empty (F:E) ratio (percentage) in lysates from 96 hour (post-transfection) harvests. [Diagram 3](A and B) are graphs showing the effect on rAAV2 production from the addition of different salts 12 hours post-transfection. (A) shows VG titers as VG / mL, and (B) shows F:E ratios (percentages). [Figure 4] (A and B) are graphs showing the effect on rAAV2 production from addition of CaCl2 to 2 mM from -1 to 24 hours post-transfection. The control was AAV2-no salt. (A) shows VG titer as VG / mL and (B) shows F:E ratio (percentage). [Diagram 5] (A and B) are graphs showing the effect on rAAV2 production from the addition of CaCl2 to concentrations from 0 mM (control) to 10 mM 12 hours post-transfection. (A) shows VG titers as VG / mL and (B) shows F:E ratios (percentages). [Figure 6] (A and B) are graphs showing the effect of adding CaCl2 to 2 mM on rAAV2, rAAV5, or rAAV8 production 12 hours post-transfection. (A) shows VG titers as VG / mL, and (B) shows F:E ratios (percentages). Detailed Description of the Invention

[0022] The present disclosure provides a method for producing recombinant adeno-associated virus (rAAV). The method for producing rAAV particles includes the following steps: (i) an expansion phase, which comprises increasing the number of cells in at least one culture vessel containing a culture medium; (ii) introducing into the cell a first polynucleotide sequence comprising a transgene flanked by AAV inverted terminal repeats (ITRs), and optionally a second polynucleotide sequence comprising AAV rep and cap genes, and / or a third polynucleotide sequence comprising one or more helper genes; (iii) a production phase comprising culturing the cells from step (ii) and adding calcium (Ca) ions to the culture medium from about 0 to about 24 hours after introduction of the first polynucleotide sequence such that the total concentration of Ca ions in the culture medium is greater than 0.3 mM and less than 10 mM; and (iv) isolating the rAAV particles.

[0023] The invention also provides rAAV particle populations produced by the methods, and pharmaceutical compositions comprising the rAAV particle populations.

[0024] In embodiments, the methods provide increased production titers and / or higher total:empty capsid ratios (F:E). More specifically, the disclosure provides methods of increasing the production titer and / or F:E ratio of rAAV by transfected cells, for example, by adding Ca ions (e.g., a calcium salt such as CaCl2) to the production medium. In embodiments, the production titer is increased by about 1.5-fold, about 1.8-fold, about 2-fold, about 3-fold, about 5-fold, about 10-fold, or more compared to the production titer when Ca ions are not added to the production medium. In embodiments, the F:E ratio is increased by about 1.3-fold, about 1.5-fold, about 2-fold, about 5-fold, about 8-fold, about 10-fold, about 12-fold, or more compared to the F:E ratio when Ca ions are not added to the production medium.

[0025] The method is scalable to production scales, e.g., about 5 to about 10 liters, about 10 to about 20 liters, about 20 to about 50 liters, about 50 to about 100 liters, about 100 to about 200 liters, or more, and is applicable to rAAVs, including a variety of AAV serotypes / capsid variants.

[0026] The rAAV vector produced by the method disclosed herein is useful for expressing transgenes in target cells. These rAAV vectors can be used for gene therapy because they can introduce polynucleotides containing transgenes that can be maintained and expressed in target cells into target cells, and rAAV vectors can deliver heterologous polynucleotide sequences (e.g., polynucleotide sequences encoding therapeutic or reporter proteins, and regulators for protein expression) to target cells of human patients. A non-exclusive list of examples of transgenes includes RPGR, RPE65, GAD65, GAD67, and CNGB3. In some embodiments, the two AAV ITRs are AAV2 ITRs. In the method, the AAVcap gene may be an AAV serotype or AAV variant, such as, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrhlO, AAV-PHP.5, AAV-PHP.B, AAV-PHP.eB, AAV2-retro, AAV9-retro, and hybrids thereof. In embodiments, the one or more helper genes may include all or a portion of one or more adenovirus genes, herpes simplex virus type 1 genes, or baculovirus genes.

[0027] The term "vector" refers to a vehicle for introducing polynucleotide into target cells.Vector may be a non-viral vector such as a viral vector (e.g., rAAV vector, HSV vector) or a plasmid, or a DNA associated with a compound such as liposome, gelatin, or polyamine.An expression vector is a vector that contains a polynucleotide sequence that codes for a gene product (e.g., protein or RNA) with a regulatory element for expression in a host cell or target cell.

[0028] "rAAV", "rAAV vector", "rAAV particle", or "rAAV virion" refers to a recombinant AAV vector genome that is packaged into (i.e., encapsidated by) capsid proteins for subsequent infection of a target cell, ex vivo, in vitro, or in vivo. These terms exclude empty AAV capsids and AAV capsids that lack a complete recombinant AAV genome, including a transgene to be expressed in a target cell. Thus, in addition to the capsid, an rAAV vector includes an rAAV genome. "rAAV genome" or "rAAV vector genome" refers to a polynucleotide sequence that includes a transgene of interest that is ultimately packaged or encapsidated to form an rAAV particle. Typically, for rAAV, most of the AAV genome (including, for example, the rep, cap, and aap genes) has been deleted, and one or both ITR sequences remain as part of the rAAV genome along with the transgene. As used herein, "transgene" refers to a polynucleotide sequence that encodes a gene product (e.g., a therapeutic or reporter protein) and regulatory elements for the expression of the gene product in a target cell.

[0029] "Empty capsid" and "empty particle" refer to an AAV particle that has an AAV capsid shell but lacks all or part of the recombinant AAV genome, including the transgene sequence and one or two ITRs. Such empty capsids are incapable of transferring a transgene into one or more target cells. In an embodiment, the isolated rAAV particles are separated from the empty AAV particles.

[0030] The rAAV genomes (e.g., including ITRs) may be based on the same strain or serotype (or subgroup or variant), or they may be different from each other. As a non-limiting example, a rAAV plasmid, or vector genome or particle (capsid) based on one serotype genome may be identical to one or more capsid proteins that package the vector genome. Furthermore, the rAAV genome may be derived from an AAV genome that is different from one or more capsid proteins that package the rAAV vector genome (e.g., including one or more ITRs derived from the AAV2 genome).

[0031] The rAAV vectors that may be produced by the methods disclosed herein include any rAAV vector comprising a capsid and genome from any AAV strain or serotype. As non-limiting examples, the rAAV vector capsid and / or genome may be based on AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-PHP-5, AAV-PHP-B, AAV-PHP-eB, AAV2-retro, AAV9-retro, AAVrh74, AAVrh, AAVrh.10 (i.e., AAV comprising AAVrh.10 ITRs and AAVrh.10 capsid proteins), and the like. In embodiments, the rAAV vector comprises a genome and capsid proteins from the same AAV strain or serotype. For example, the rAAV vector can be an rAAV2 vector (i.e., an rAAV comprising AAV2 ITRs and AAV2 capsid proteins).

[0032] In embodiments, the AAV vector is a pseudotyped rAAV vector that includes ITRs from one AAV serotype and capsid proteins from a different AAV serotype. In some embodiments, the pseudotyped rAAV is rAAV2 / 5 (i.e., rAAV that includes AAV2 ITRs and AAV5 capsid proteins), rAAV2 / 8 (i.e., rAAV that includes AAV2 ITRs and AAV8 capsid proteins), rAAV2 / 9 (i.e., AAV that includes AAV2 ITRs and AAV9 capsid proteins), rAAV2 / 10 (i.e., rAAV that includes AAV2 ITRs and AAV10 capsid proteins). In embodiments, the rAAV vector includes capsid proteins that are mutant AAV capsids, such as the AAV2 mutant rAAV2-retro (SEQ ID NO: 44 from WO 2017 / 218842, incorporated herein by reference).

[0033] cell line

[0034] The rAAV vector production methods described herein generally require certain elements, including, for example, (i) a permissive host cell for rAAV production (the producer cell), (ii) helper virus functions, which may be provided by an appropriate construct including, for example, genes providing adenovirus helper functions, (iii) a trans-packaging rep / cap construct, and (iv) a suitable production medium.

[0035] A producer cell is any cell that is a permissive host cell for producing rAAV when the rAAV genome construct, helper function construct, and constructs that provide AAV functions (e.g., rep and cap) are present. The term may also include the progeny of the original transfected cell. Thus, a producer cell is also the progeny of a host cell that has been transfected with an exogenous DNA sequence or that has integrated that DNA sequence into the host cell genome. It is understood that the progeny of a single parent cell need not necessarily be completely identical in morphology or genomic or total DNA complement to the original parent due to natural, accidental, or deliberate mutations.

[0036] In embodiments, the cells used to produce rAAV particles are mammalian cells, including HEK293 cells, BHK cells, and HeLa cells. Exemplary producer / host cells include human embryonic kidney (HEK) cells, such as HEK293. In preferred embodiments, the producer cells are adapted for growth in suspension, including suspension-adapted HEK293 cells. In further preferred embodiments, the producer cells are adapted for growth in serum-free media. In embodiments, the producer cells are grown in at least one culture vessel, which may be one or more of, for example, a shaker flask, a spinner flask, a cell bag, or a bioreactor.

[0037] Producer cell lines that may be used in the rAAV production methods disclosed herein include mammalian or insect cell lines. The term "cell line" refers to a cell population capable of continuous or long-term growth and division in vitro under appropriate culture conditions. A cell line may be, but is not necessarily, a clonal population derived from a single progenitor cell. Spontaneous or induced changes in karyotype may occur in cell lines during storage or transfer of such clonal populations, as well as during long-term passage in tissue culture. Thus, progeny cells derived from a cell line may not be exactly the same as the progenitor cells or cultures.

[0038] To effect rAAV production, producer cell lines may require one or more of the following to be present in the producer cell: rAAV genome production construct, helper function construct, and / or AAVrep / cap construct. These may be introduced as three constructs (e.g., three plasmids), or the producer cell may already have one or more constructs that stably integrate some or all of these functions into the producer cell genome. As used herein, the term "stable" or "stably integrated" with respect to a cell means that a nucleic acid sequence, such as a selectable marker and / or a heterologous nucleic acid sequence or a plasmid or vector (or a portion thereof), has been inserted into a chromosome (e.g., by homologous recombination, non-homologous end joining, transfection, etc.) or has been maintained extrachromosomally in a recipient cell or host organism and has remained intrachromosomally or has been maintained extrachromosomally for a period of time.

[0039] Expansion of producer cell lines

[0040] In embodiments of the methods described herein, an expansion phase or step is used to increase the number of producer cells prior to the step of introducing the rAAV genome production construct (including the rAAV genome) and / or other constructs providing helper virus and AAV functions. The expansion phase or step may be performed in one or more cell culture vessels. For example, the expansion phase or step may be performed in a series of cell culture vessels of increasing volume. The cell culture medium used to expand the producer cell line may be any medium suitable for the growth (i.e., increase in number) of producer cells. In preferred embodiments, the expansion phase culture medium is animal component-free, e.g., does not contain serum or other components derived from animals. Chemically defined, animal component-free media are commercially available.

[0041] In embodiments, an anti-aggregation supplement, sometimes referred to herein as an anti-aggregation agent (ACA), is added to the expansion medium to reduce cell aggregation. ACA is commercially available, for example, from Irvine Scientific. The anti-aggregation supplement may be added to the expansion phase culture medium at one or more time points. In embodiments, the anti-aggregation supplement includes dextran sulfate, heparin, and / or other sulfated glycosaminoglycans that inhibit cell aggregation. In embodiments, the anti-aggregation supplement includes sodium heparin, which may be added to the medium to a concentration of about 25 μg / ml to about 250 μg / ml, for example, about 25 μg / ml, about 50 μg / ml, about 100 μg / ml, about 150 μg / ml, and / or about 200 μg / ml.

[0042] In embodiments, the expansion phase culture medium includes and / or is supplemented to include one or more of glutamine, glutamine precursors, or amino acid dipeptides containing glutamine at a concentration of about 2 mM to about 6 mM (e.g., about 2 mM, about 3 mM, about 4 mM, about 5 mM, or about 6 mM). The one or more of glutamine, glutamine precursors, or amino acid dipeptides containing glutamine may be, for example, one or more of L-alanyl-L-glutamine, L-glutamine, glutamate, glycyl-L-glutamine, glutamine protein hydrolysate, L-glutamic acid, and glutamine dipeptide. A commercially available example of a glutamine supplement provided as the dipeptide L-alanyl-L-glutamine is GlutaMAX (ThermoFisher).

[0043] In embodiments, the expansion phase culture medium includes and / or is supplemented to include a non-ionic polyol surfactant, such as poloxamer 188 (a copolymer of polyethylene and polypropylene ether glycol). In embodiments, the non-ionic polyol surfactant is present in the expansion phase culture medium at about 0.05% to about 0.2% (w:v) (e.g., about 0.05%, about 0.1%, about 0.1%, or about 0.2%). In embodiments, the expansion phase culture medium includes about 4 mM L-alanyl-L-glutamine dipeptide, and 0.1% (w:v) poloxamer 188.

[0044] In embodiments, the pH of the expansion phase culture medium is maintained at a pH of about 7.1 to about 7.5 (e.g., about 7.1, about 7.2, about 7.3, about 7.4 or about 7.5). In embodiments, the pH is maintained at about 7.2 to about 7.4 by CO2 sparging. In embodiments, prior to introducing one or more polynucleotide constructs into the cells, the pH of the culture medium is changed to about 6.9 and CO2 sparging is stopped.

[0045] Introduction of one or more polynucleotide constructs

[0046] Production of rAAV vectors typically requires a producer cell line that provides the basic biosynthetic machinery, as well as (i) a construct that provides the rAAV genome (the transgene of interest and associated regulatory elements flanking the AAV ITRs), and (ii) one or more constructs that contain additional genes that provide gene products necessary to direct rAAV vector production. These additional genes include genes from AAV (e.g., AAVrep and cap) and helper viruses (e.g., adenovirus E1a, E1b, E2a, E4, and VA) necessary to support replication and packaging of the vector genome.

[0047] In embodiments of the method, at least one of the first, second, and third polynucleotide sequences are introduced into the cell by transfection of one or more vectors comprising the first, second, and third polynucleotide sequences, by infection with one or more viruses comprising one or more of the first, second, and third polynucleotide sequences, by a combination of transfection of one or more vectors and infection with one or more viruses, or by electroporation of the first, second, and third polynucleotide sequences.

[0048] "Helper virus genes" or "genes from helper viruses" refer to non-AAV-derived viral genes on which AAV gene products are dependent for replication. The term includes proteins and / or RNAs required for AAV replication, including those involved in activation of AAV gene transcription, stage-specific AAV mRNA splicing, and AAV DNA replication. Helper virus genes may be derived from any of the known AAV helper viruses, such as adenovirus, herpesvirus, and vaccinia virus. Thus, "helper virus functions" refers to those functions provided by helper virus genes (e.g., adenovirus E1a, E1b, E2a, E4, and VA) required for AAV production. These helper virus functions may be provided on one or more vectors introduced into the producer cell, stably expressed by the producer cell, or a combination of both.

[0049] As used herein, "AAV functions" or "AAV accessory functions" refer to coding sequences from AAV that can be expressed in producer cells to provide AAV gene products that function in trans for productive AAV replication and packaging. Thus, AAV functions include AAV open reading frames (ORFs), including rep and cap, and others, such as aap of certain AAV serotypes. Such AAV functions are provided by one or more polynucleotide constructs, which may be plasmid vectors, non-plasmid vectors, or polynucleotide constructs integrated into the chromosome of the producer cell that provide AAV helper functions. Plasmids that provide AAV functions that may be used in the methods disclosed herein are commercially available.

[0050] In an embodiment of the method, one or more helper virus genes are constitutively expressed by the producer cell (e.g., HEK293 cell), while other helper virus genes are introduced into the producer cell, for example, by transfection of one or more polynucleotide constructs encoding the remaining helper virus genes required for AAV production. Genes from AAV (e.g., rep and cap) may be included in the same polynucleotide construct containing one or more helper virus genes or may be included on separate polynucleotide constructs, and rAAV particles are generated after a polynucleotide construct containing a rAAV genome (e.g., a rAAV genome production vector) is introduced into a producer cell line. In an embodiment, rAAV particles are produced after transient transfection of producer cells with (i) a rAAV genome production vector, and (ii) one or more vectors providing helper virus genes (e.g., E4, E2a, and VA) and AAV genes (e.g., rep and cap). In an embodiment, these vectors are plasmids.

[0051] In embodiments of the methods disclosed herein, following the expansion phase, a first polynucleotide construct comprising a transgene flanked by ITRs and a second polynucleotide construct comprising helper virus genes and AAV rep and cap genes are introduced into the expanded producer cells. When the first and second polynucleotide constructs are plasmids, the system may be referred to as a two-plasmid system.

[0052] In embodiments of the methods disclosed herein, following the expansion phase, a first polynucleotide construct comprising a transgene flanked by ITRs, a second polynucleotide construct comprising helper virus genes, and a third polynucleotide construct comprising AAVrep and cap genes are introduced into the expanded producer cells. When the first, second, and third polynucleotide constructs are plasmids, the system may be referred to as a three-plasmid system.

[0053] When one or more recombinant plasmids are used to produce an rAAV vector, "rAAV genome-producing plasmid" refers to a plasmid that contains a transgene (operably linked to regulatory sequences) and one or more ITRs intended for packaging into an rAAV, as well as non-rAAV genome components (plasmid backbone) that are important for plasmid cloning and amplification, but are not packaged or encapsidated into the rAAV vector.

[0054] The terms "introduction" and "transfect" refer to the introduction of a polynucleotide into a host cell or target cell. In embodiments, the host cell is a producer cell, e.g., a HEK293 cell. In embodiments, the rAAV genome production plasmid is introduced into the producer cell by transient transfection methods together with one or more plasmids providing helper virus and AAV functions. Transient transfection of the producer cell to introduce a first polynucleotide construct including a transgene and ITR(s) (e.g., a rAAV genome production vector), and optionally a second and / or third polynucleotide construct providing AAV functions (rep and cap genes) and helper virus functions, may be accomplished by standard transfection methods, including, for example, calcium phosphate co-precipitation, cationic lipid-based transfection, and cationic polymer-based transfection. Cationic lipid-based transfection includes, for example, lipofectamine (a 3:1 mixture of DOSPA (2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propandinium trifluoroacetate) and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine)). Cationic polymer-based transfection includes, for example, linear and / or branched polyethyleneimine (PEI), poly-L-lysine, poly-L-arginine, and polyamidoamine dendrimers. In an embodiment, transient transfection of producer cells is performed using a PEI-based transfection reagent. The PEI may be a linear or branched polymer. In an embodiment, the PEI is a linear PEI of 20-25 kD. For example, in an embodiment, the PEI is jetPEI or PEIpro (available from Polyplus). In addition, transient transfection of producer cells may be performed using a transfection reagent that contains both a cationic lipid and a cationic polymer.

[0055] rAAV may alternatively be produced in insect cells (e.g., sf9 cells) using baculovirus vectors, or in HSV-infected baby hamster kidney (BHK) cells (e.g., BHK21). In either method, rAAV production is induced in host cells, insect cells, or mammalian cells, respectively, by co-infection with two or more recombinant viruses carrying the rAAV genome and one or more AAVrep and cap, and helper virus functions required for rAAV replication and packaging.

[0056] Production of rAAV particles

[0057] The methods disclosed herein include a production phase (also referred to as the production step) following the step of introducing the rAAV genomic vector and / or a vector providing helper virus function and / or AAV function into the producer cells. In the methods described herein, rAAV particles are produced by culturing the cells for at least about 48 hours (e.g., 47.5, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 96.5, or 97 hours) following the introduction of the rAAV genomic vector. In embodiments, the transfected producer cells are cultured (i.e., maintaining the production phase) for about 72 to about 100 hours, about 90 to about 100 hours, about 92 to about 98 hours, or about 94 to about 98 hours. In embodiments, the production phase is maintained for about 96 hours.

[0058] The production phase medium may be any cell culture medium suitable for the production of rAAV in producer cells. In embodiments, the production medium is free of animal products, such as serum. "Free" in this context means that the medium has undetectable levels of animal products, such as serum. In embodiments, the pH of the production medium is reduced compared to the pH of the expansion phase medium. In embodiments, the production medium is maintained at a pH of about 6.8 to about 7.4 (e.g., about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, or about 7.14). In embodiments, the pH is maintained at about 6.9 to about 7.3.

[0059] In an embodiment of the method, the production phase includes the addition of calcium ions to the production phase cell culture medium. In the prior art, calcium ion levels are typically maintained at low levels (usually about 0.1 mM) in medium formulations used in suspension cell culture processes to reduce or prevent cell aggregation. Proteins involved in cell adhesion, such as E-cadherin, are calcium-dependent. The extracellular domain of E-cadherin forms a calcium-dependent homophilic trans-dimer, leading to specific interactions with adjacent cells, while the cytoplasmic domain is linked to the actin cytoskeleton. Thus, it is generally considered desirable in the art to maintain low levels of calcium.

[0060] The addition of calcium ions to the production medium, also referred to herein as calcium supplementation, provides calcium ions (Ca 2+ ) in the form of a calcium salt, e.g., CaCl2. Calcium ions may be added one or more times during the production phase after introduction of the rAAV genome vector (e.g., after transfection). For example, calcium ions may be added at one or more times between about 0 hours and about 48 hours from the start of the production phase (i.e., after transfection), e.g., about 1 hour, about 6 hours, about 10 hours, about 12 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, and / or about 48 hours. In embodiments, calcium ions are added to the production medium at one or more times between about 0 hours and about 24 hours, about 0 hours to about 20 hours, about 0 hours to about 18 hours, or about 6 hours to about 12 hours after the start of the production phase (i.e., after transfection). In embodiments, calcium ions are added to the production phase medium about 6 hours after transfection.

[0061] Calcium ions may be added to the production medium so that the total concentration of calcium ions in the culture medium is greater than 0.3 mM and less than 10 mM. In an embodiment, calcium ions are added to a total concentration of between about 1 mM and about 9 mM, between about 1 mM and about 8 mM, between about 1 mM and about 7 mM, between about 2 mM and about 9 mM, between about 2 mM and about 8 mM, between about 2 mM and about 7 mM, between about 2 mM and about 6 mM, between about 2 mM and about 5 mM, or between about 2 mM and about 4 mM. In embodiments, calcium ions (e.g., CaCl2) are added to a concentration of about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, or about 9.5 mM.

[0062] In an embodiment, the production phase includes adding one or more of glutamine, glutamine precursors, or amino acid dipeptides containing glutamine to the production phase medium. The one or more of glutamine, glutamine precursors, or amino acid dipeptides containing glutamine may be, for example, one or more of L-alanyl-L-glutamine, L-glutamine, glutamate, glycyl-L-glutamine, glutamine protein hydrolysate, L-glutamic acid, and glutamine dipeptide. A solution containing at least one of glutamine, glutamine precursors, or amino acid dipeptides containing glutamine may be added to the production phase medium, for example, at one or more of about 6 hours, about 12 hours, about 24 hours, about 48 hours, or about 72 hours after transfection.

[0063] In embodiments, the production phase includes adding sorbitol to the production phase medium. Sorbitol may be added to the production phase medium at one or more time points during the production phase, for example, about 6 hours, about 12 hours, about 20 hours, about 24 hours, and / or about 48 hours after transfection. In embodiments, sorbitol is added to the production medium to a concentration of about 50 mM to about 200 mM, or about 80 mM to about 120 mM. In embodiments, sorbitol is added to the production medium to a concentration of about 100 mM.

[0064] In embodiments, the production phase includes adding an aggregation-inhibiting supplement to the production phase medium. The aggregation-inhibiting supplement may be added to the production phase culture medium at one or more time points (e.g., at one or more of about 6 hours, about 10 hours, about 12 hours, about 20 hours, about 24 hours, about 48 hours, or about 72 hours after transfection). In embodiments, the aggregation-inhibiting supplement includes dextran sulfate, heparin, and / or other sulfated glycosaminoglycans that inhibit cell aggregation. In embodiments, the aggregation-inhibiting supplement includes heparin sodium, which may be added to the medium to a concentration of about 25 μg / ml to about 250 μg / ml, e.g., about 25 μg / ml, about 50 μg / ml, about 100 μg / ml, about 150 μg / ml, and / or about 200 μg / ml.

[0065] In embodiments, the aggregation inhibitor supplement is not added to the production phase medium or is added to the production phase medium only shortly before the end of the production phase, for example within about 24 hours, within about 12 hours, within about 6 hours, within about 3 hours, within about 2 hours, or within about 1 hour of the end of the production phase.

[0066] Isolation of rAAV

[0067] Embodiments of the methods described herein include a step of isolating the rAAV particles at the end of the production phase. In embodiments, the rAAV particles may be isolated about 48 hours or more (e.g., 47.5, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 96.5, 97 hours) after introduction of the first, second, and / or third polynucleotide sequences (after step (ii) above, in other words, after the start of the production phase). For example, the rAAV particles may be isolated about 90 to about 100 hours, about 92 hours to about 98 hours, or about 94 to about 98 hours after introduction of the first, second, and third polynucleotide sequences. In embodiments, the rAAV particles are isolated (e.g., cells are lysed) about 96 hours (e.g., about 96 hours after step (ii) above) after introduction of the first, second, and / or third polynucleotide sequences. Isolation of rAAV may involve multiple steps including, for example, lysing the producer cells, clarifying the lysate, and subsequent purification steps.

[0068] rAAV particles may be retained within producer cells after production, and methods for releasing intracellular rAAV vectors include physical and chemical disruption, e.g., the use of detergents, microfluidization, and / or homogenization. During and / or simultaneously after cell lysis, a nuclease such as benzonase may be added to degrade contaminating DNA.

[0069] Typically, the resulting lysate is clarified to remove cellular debris and provide a clarified cell lysate. Filtration techniques are useful for separating rAAV vectors from relatively large particles such as cellular debris (microfiltration) and also for separating rAAV vectors from relatively small molecules such as soluble protein impurities (ultrafiltration). For example, the disrupted rAAV vector-containing cells may be passed through one or more micron pore size filters (e.g., filters with pore sizes between 0.1 and 10.0 μm, e.g., 0.45 μm and / or 0.2 μm pore size filters). A two-stage 0.45 / 0.2 μm cartridge filter may be used to separate the rAAV vectors recovered in the filter permeate from the cellular debris retained by the filter. In embodiments, the method further includes determining at least one of the capsid concentration, viral genome titer, and ratio of rAAV particles to empty AAV particles from the lysate or a portion of the clarified lysate.

[0070] The rAAV vector in the clarified lysate may be further isolated (purified) by using one or more additional purification methods, including ultracentrifugation, affinity chromatography, ion exchange chromatography, and tangential flow filtration (TFF).

[0071] In embodiments, the isolated rAAV particles are separated from empty AAV particles, and the method provides a ratio of rAAV particles to empty AAV particles of at least about 30%, e.g., about 30%-40%, at least 65%, about 65%-90%, etc. The level of nuclease-resistant AAV encapsulated DNA impurities may be assessed by qPCR using primers and probes designed for relevant sequences in the helper plasmid or against high copy number genomic sequences. Nuclease-sensitive "naked" residual DNA impurities can be distinguished from nuclease-insensitive encapsulated residual DNA impurities by their susceptibility to nuclease treatment prior to qPCR. Total AAV capsids may be measured using a capsid-specific ELISA assay and the amount of empty capsids determined by comparison of capsid particle titers to VG titers. Spectrophotometry may be used for samples where non-AAV capsid impurities have been substantially removed. EXAMPLES

[0072] Example 1. Evaluation of calcium chloride supplementation in AAV production in a shaker flask model

[0073] Calcium chloride addition at different production time points and concentrations, as well as production length, was evaluated in suspension adapted HEK293 cells cultured in E125 shaker flasks. At the end of the flask procedure, cultures were harvested and chemically lysed. Performance between conditions was evaluated based on physical capsid and viral genome titers.

[0074] Seed Train and Expansion:

[0075] HEK293 cells were expanded to sufficient numbers for testing in Erlenmeyer flasks (E1000) and cultured in a CO2 shaking incubator (New Brunswick S41i). Seed train flasks were cultured with 2 x 10 cells (per mL in a seed volume of 200 ml). 5The cells were seeded at a seeding density of 100 viable cells (VC). The seed medium was a chemically defined medium supplemented with 4 mM GlutaMAX and 0.1% w:v Poloxamer 188. 48 hours after seeding, 180 mL of chemically defined medium was added (9:10, feed:seed medium volume ratio).

[0076] To control cell aggregation, an aggregation inhibitor (ACA, Fujifilm Irvine Scientific) was added at 1 mL / L during the first passage and periodically thereafter.

[0077] HEK293 cells were cultured at 37°C and 8% CO2 with 1 inch orbital agitation at 150 rpm. Cells were cultured in chemically defined medium supplemented with 4 mM GlutaMAX and 0.1% w:v Poloxamer 188.

[0078] Transfection conditions for AAV5-RPGR:

[0079] HEK293 cells were cultured in E1000 non-baffled flasks (Corning) at approximately 1.8–2.4 × 10 6 At a cell density of 10 VC / mL, 96 h after inoculation, plasmid:helper (pFD6k, 15.4 kb); Rep / Cap (pRC2~5k, 8.1 kb); transgene (pRPGR-k, 7.7 kb) was transfected at a plasmid copy number ratio of 1:1:2, 10 at the time of transfection. 6 A total of 0.95 μg of DNA was transfected per viable cell.

[0080] The transfection mixture was prepared at a PEI:DNA ratio of 1.3:1 (v:w) with a DNA concentration of 30 mg / L. The medium for the transfection mixture was chemically defined culture medium (without supplements). Helper, Rep / Cap, and transgene constructs were added to the medium. PEI was added to the medium and mixed, then the mixture was added to the DNA mix, followed by rotation and incubation for 15 minutes. After the 15 minute incubation, the transfection mix was added to the flask, which was returned to the shaking incubator.

[0081] Transfection conditions for rAAV2-GAD67:

[0082] HEK293 cells were cultured in E1000 non-baffled flasks (Corning) at approximately 1.8–2.4 × 10 6 At a cell density of 10 VC / mL, 96 h after inoculation, plasmid:helper (pFD6k, 15.4 kb); Rep / Cap (pNLRep2-2~2k, 10.3 kb); transgene (pGAD67k, 7.1 kb) was transfected at a plasmid copy number ratio of 2:1.5:1, 10 at the time of transfection. 6 A total amount of DNA of 1.0 μg per viable cell was transfected.

[0083] The transfection mixture was prepared at a FectoVIR:DNA ratio of 1.5:1 (v:w) with a DNA concentration of 80 mg / L. The medium for the transfection mixture was chemically defined culture medium (no supplements). Helper, Rep / Cap, and transgene constructs were added to the medium. FectoVIR was added to the medium and mixed, then the mixture was added to the DNA mix, followed by rotation and incubation for 15 minutes. After the 15 minute incubation, the transfection mix was added to the flask, which was returned to the shaking incubator.

[0084] AAV production conditions:

[0085] For HEK293 cells transfected with the AAV5-RPGR plasmid, cells were dropped into E125 unbaffled flasks (Corning) 1 hour after transfection and incubation continued at 37°C, 8% CO2 with 1 inch orbital rotation at 150 RPM. Transfected HEK293 cells were incubated at 37°C, 8% CO2 with 1 inch orbital rotation at 180 RPM, and 1M CaCl2 stock solution was added to a concentration of 0.5 mM or 2.0 mM either 6 or 24 hours after transfection. For control flasks, no stock solution was added.

[0086] For HEK293 cells transfected with the AAV5-GAD67 plasmid, cells were plated 1 hour post-transfection into E125 non-baffled flasks (Corning) and continued to incubate at 37°C, 8% CO2 with 1 inch orbital rotation at 150 RPM. Transfected HEK293 cells were incubated at 37°C, 8% CO2 with a 1 inch orbital rotation at 150 RPM and one of the following was added: 4M D-sorbitol to a 100 mM sorbitol concentration 20 hours after transfection; 4M NaCl stock solution to a 50 mM NaCl concentration 20 hours after transfection; 1M CaCl2 stock solution to a 2 mM CaCl2 concentration 24 hours after transfection; 1M MgCl2 stock solution to a 2 mM MgCl2 concentration 24 hours after transfection; 1M CaCl2 and 1M MgCl2 stock solutions to 2 mM CaCl2 and 2 mM MgCl2 concentrations 24 hours after transfection. To the control flasks, no stock solutions were added.

[0087] Melting conditions:

[0088] Cells were harvested 96 hours after transfection. 50X lysis buffer (0.1% Triton™ X-100, 2 mM MgCl2, 1 mM Tris, 125 U / 10 7Benzonase (mixed to achieve a final concentration of 100 µg / ml) was added to the cell culture at 2% v / v and incubated at 37°C and 150 RPM for 2 hours.

[0089] Measurement of viral capsid and viral genome titers:

[0090] Viral capsid (VC) titers were performed by ELISA using the AAV Titration ELISA Kit from Progen® or the GYROLAB® AAVX Titration Kit from Gyros Protein Technologies according to the manufacturer's guidelines.

[0091] Viral genome (VG) concentrations and titers were determined by qPCR. Samples were subjected to DNAse I treatment followed by proteinase K treatment. Four serial dilutions were assessed by mixing an appropriate amount of sample with a qPCR master mix containing nucleotides, forward primers, reverse primers, and Taqman probes targeting specific regions of each transgene. Plasmid DNA or DNA oligonucleotides containing the target regions were used to generate a seven-point standard curve, which was used to assess the number of viral genomes per sample.

[0092] result:

[0093] After addition of 0.5 mM or 2 mM CaCl2 at 6 or 20 hours post-transfection, viral genome (VG) titers increased 3.5-fold for rAAV5 compared to control cultures without supplementation. See Figure 1A. 2+ Supplementation increased viral genome (VG) titers for rAAV2 by approximately 4-5 fold compared to control cultures without supplementation; see Figure 1B. In addition, the total-to-empty capsid (F:E) ratio increased from 10-15% in control cultures to 30-40% for rAAV5; see Figure 1A. 2+When supplementation was combined with an extended production time to 96 hours post-transfection, both the viral genome titer and the F:E ratio were significantly increased. See Figures 1A and 1B. Supplementation with NaCl or MgCl2 did not increase VG titer compared to controls, but sorbitol supplementation appears to provide some improvement in VG titer of rAAV2 when harvested 96 hours post-transfection. See Figure 1B.

[0094] Example 2: Evaluation of calcium chloride addition during AAV production in bioreactors (250 ml scale)

[0095] In this study, a 250 mL scale stirred tank bioreactor will be used to evaluate CaCl2 addition, CaCl2 addition time, ACA addition, GlutaMAX addition, and specific feed addition during the AAV production phase.

[0096] Seed Train: Suspension-adapted HEK293 cells were expanded to sufficient numbers for testing in Erlenmeyer flasks (E125, E250, E500, and E1000) and cultured in a CO2 shaking incubator (New Brunswick S41i). Seed train flasks were seeded at densities of 1.5 or 3.0 × 10 5 Cells were seeded at either 100 ng / mL VC / mL and expanded for 96 and 72 h culture times, respectively.

[0097] To control cell aggregation, an aggregation inhibitor (ACA, Irvine Scientific) was added at 1 mL / L during the first passage and periodically thereafter.

[0098] HEK293 cells were cultured at 37°C and 8% CO2 with 1 inch orbital agitation at 150 rpm. Cells were cultured in chemically defined medium supplemented with 4 mM GlutaMAX and 0.1% w:v Poloxamer 188.

[0099] Bioreactor Cell Expansion

[0100] Seed medium for expansion in 250 mL stirred tank bioreactors was supplemented with chemically defined medium supplemented with 4 mM GlutaMAX and 0.1% w:v Poloxamer 188. HEK293 cells were seeded at 2.0 × 10 in a volume of 45% of the total working volume (113 mL). 5 The cells were seeded at a target seed density of VC / mL.

[0101] 48 h after inoculation, cells were fed with chemically defined medium (unsupplemented medium) at a feeding volume of 40% of the total working volume (100 mL).

[0102] The temperature setpoint was 37°C, the pH setpoint was 7.30±0.05, and the DO setpoint was 50%. The bioreactor was sparged with air and pure oxygen to control the DO concentration, and CO2 (acid) and sodium hydroxide (base) were added to control the pH. Agitation was at 7.5 W / m at 45% of the total working volume. 3 and 17.5 W / m at 85% of the total working volume. 3 The value was changed between 0.01 and 0.1.

[0103] Transfection

[0104] HEK293 cells were cultured at approximately 1.6–2.2 × 10 6 At a cell density of 10 VC / mL, 96 h after inoculation, plasmid:helper (pFD6k, 15.4 kb); Rep / Cap (pNLRep2-2-k, 10.3 kb); transgene (pGAD67k, 7.1 kb) was transfected at a plasmid copy number ratio of 2:1.5:1, 10 at the time of transfection. 6 A total amount of DNA of 1.0 μg per viable cell was transfected.

[0105] The transfection mixture was prepared with a FectoVIR:DNA ratio of 1.5:1 (v:w) and a DNA concentration of 80 mg / L in the transfection mixture. The medium for the transfection mixture was chemically defined culture medium (without supplements). Helper, Rep / Cap, and transgene constructs were added to the medium. FectoVIR was added to the culture medium and mixed, and the mixture was then added to the DNA mix, followed by rotation and incubation for 15 minutes. After the 15 minute incubation, the transfection mix was added to the bioreactor.

[0106] rAAV production

[0107] rAAV production parameters: temperature set at 37°C; DO set point at 50%; pH set point at 6.9, no CO2 addition; and agitation set point at 17.5 W / m 3 , and the total working volume was 85%. The production conditions tested are listed in Table 1.

[0108] For conditions where feed was added, BalanCD HEK293 feed was added at 3% v / v per addition (6.4 mL per addition) four times during the production phase (at the first calcium addition, 24 hours, 48 ​​hours, and 72 hours after transfection). For studies where CaCl2 was added, a 1 M stock solution of CaCl2 was added to a concentration of 2 mM CaCl2 either 6 hours, 12 hours, or 20 hours after transfection. For studies where ACA was added, 2 mL / L ACA was added 20 hours after transfection. For studies where GlutaMAX was added during the production phase, GlutaMAX was added four times (6 hours, 24 hours, 48 ​​hours, and 72 hours after transfection) to achieve a concentration of 1 mM GlutaMAX per addition (1.1 mL per addition) in the bioreactor. When CaCl2, ACA, BalanCD HEK293 feed, or GlutaMAX were added simultaneously, the individual solutions were combined. [Table 1]

[0109] Cell lysis

[0110] Cells were harvested 96 hours after transfection. 50X lysis buffer (formulated to achieve a final concentration of 0.1% Triton™ X-100, 2 mM MgCl2, and 1 mM Tris) was added with 125 U / 10 benzonase. 7 It was added to the bioreactor at 2% v / v along with the whole cells and incubated for 2 hours at 37° C. and 17.5 W / m 3 at 85% of the total working volume.

[0111] Determination of viral capsid and viral genome titers

[0112] Determination of viral capsid and viral genome titers was performed as described above.

[0113] result:

[0114] In a bioreactor scale-down model, calcium supplementation during the rAAV production phase increased VG titers 7-8 fold compared to controls and increased F:E ratios from 5-10% to 65-90% for rAAV2. See Figures 2A and 2B. 2+ In addition to ACA supplementation, the addition of ACA and glucose-based feed during the production phase reduces the Ca content of the fermentation medium without ACA or glucose-based feed. 2+ Compared to (ACA) supplementation, the F:E ratio was reduced. However, the F:E ratio was still about 4-fold higher than the control. See FIG. 2B. Glutamine supplementation appears to improve VG titers during the production phase compared to a test with the same supplement (with CaCl2) but no glutamine. See FIG. 2A.

[0115] Example 3: Evaluation of the timing and concentration of salt addition during rAAV production

[0116] Production of rAAV2 and rAAV5 serotypes was evaluated by testing VG titers and F:E capsid ratios after addition of CaCl2 at different time points or concentrations, and by evaluating the effect of adding other salts. The timing of addition of CaCl2 (to a final concentration of 2 mM) was evaluated by adding CaCl2 1 hour before transfection (-1 hour), at the time of transfection (0 hour), or 6, 12, 18, and 24 hours after transfection. A range of CaCl2 concentrations was tested by adding CaCl2 to a final concentration of 0.3 mM, 2 mM, 4 mM, 6 mM, or 10 mM at 12 hours after transfection. Additionally, addition of CaCl2 to a final concentration of 2 mM was compared to addition of MgCl2 to a final concentration of 2 mM, or addition of NaCl to a final concentration of 4 mM (each added 12 hours after transfection).

[0117] Seed train and expansion, transfection, production, and lysis methods were essentially the same as those described above for rAAV2-GAD67 and rAAV5-RPGR, except that ACA was not added to any conditions post-transfection. The production phase was terminated and cells were lysed 96 hours post-transfection.

[0118] result: Addition of NaCl or MgCl2 to the production phase culture medium did not increase VG titer or F:E capsid ratio (Figures 3A and 3B). Calcium supplementation by adding CaCl2 to 2 mM 1 hour before transfection significantly reduced VG titer and F:E capsid ratio compared to control (no calcium supplementation). See Figures 4A and 4B. Calcium supplementation by adding CaCl2 to 2 mM 0-18 hours after transfection increased VG titer by 1.5-fold compared to control. See Figure 4A. An increase in F:E capsid ratio was observed when CaCl2 was added 0 hours after transfection. See Figure 4B. Improvement of VG titer was observed when CaCl2 was added to final concentrations of 2-6 mM (Figure 5A), and the highest F:E ratio occurred when CaCl2 was added to concentrations of 2 mM or 4 mM (Figure 5B).

[0119] Example 4: Production of AAV2, AAV5, and AAV8 in 250 ml bioreactors

[0120] Production of rAAV2-GAD67, rAAV5-RPE65, and rAAV8-CNGB3 was performed at 250 mL scale in stirred tank bioreactors to evaluate the effect of CaCl2 addition on VG titer and F:E ratio. Seed train, expansion, transfection, production, and lysis methods were performed as described in Example 3 above. Specifically, CaCl2 was added to the production cultures to 2 mM 12 hours post-transfection. No ACA was added to any condition post-transfection. The production phase was terminated and cells were lysed 96 hours post-transfection.

[0121] result:

[0122] After 12 hours of transfection, calcium supplementation by adding CaCl2 to 2 mM resulted in a 1.5-fold, 1.4-fold, and 1.5-fold improvement in VG titers for rAAV2, rAAV5, and rAAV8, respectively (Figure 6A). Furthermore, calcium supplementation increased the F:E ratio by 3-fold for rAAV5 (Figure 6B).

Claims

1. A method for producing recombinant adeno-associated virus (rAAV) particles, said method comprising: (i) an expansion phase comprising increasing the number of cells in at least one culture vessel containing a culture medium; (ii) introducing into said cells a first polynucleotide sequence comprising a transgene flanked by AAV inverted terminal repeat sequences (ITRs); (iii) culturing said cells from step (ii) and adding Ca ions to said culture medium at about 0 to about 24 hours after step (ii) such that the total concentration of calcium (Ca) ions in said culture medium is greater than 0.3 mM and less than 10 mM, a production phase; and (iv) isolating said rAAV particles, said method.

2. The method according to claim 1, wherein step (ii) further comprises introducing into said cells a second polynucleotide sequence comprising AAV rep and cap genes and / or a third polynucleotide sequence comprising one or more helper genes.

3. The method according to claim 1, wherein adding said Ca ions to said production phase culture medium comprises adding a calcium salt.

4. Said calcium salt comprises CaCl ２ The method according to claim 3.

5. The method according to any one of claims 1 to 4, wherein said Ca ions are added at a concentration of about 0.5 mM to about 6 mM.

6. The method according to claim 5, wherein said Ca ions are added at a concentration of about 2 mM to about 6 mM.

7. The method according to claim 5, wherein said Ca ions are added at a concentration of about 2 mM to about 4 mM.

8. The method according to claim 5, wherein said Ca ions are added at a concentration of about 1 mM to about 3 mM.

9. The method according to claim 5, wherein the Ca ions are added at a concentration of about 2 mM.

10. The method according to claim 1, wherein the Ca ions are added to the production phase culture medium at about 0 to about 24 hours after step (ii).

11. The method according to claim 10, wherein the Ca ions are added to the production phase culture medium at about 6 to about 18 hours after step (ii).

12. The method according to any one of claims 1 to 4, wherein the Ca ions are added at a concentration of about 2 mM to the Ca ion concentration in the culture medium about 12 hours after step (ii).

13. The method according to claim 1, wherein the production phase (step iii) is at least about 48 hours, about 72 to about 100 hours, about 90 to about 100 hours, about 92 to about 98 hours, about 94 to about 98 hours, or about 96 hours.

14. The method according to claim 1, wherein the production phase comprises adding one or more of (a) glutamine, a glutamine precursor, or an amino acid dipeptide containing glutamine, and / or (b) sorbitol to the production phase culture medium.

15. The method according to claim 14, wherein one or more of the glutamine, glutamine precursor, or amino acid dipeptide containing glutamine are selected from one or more of L-alanyl-L-glutamine, L-glutamine, glutamate, glycyl-L-glutamine, glutamine protein hydrolysate, L-glutamic acid, and glutamine dipeptide.

16. The method according to claim 14, wherein at least one of glutamine, a glutamine precursor, or an amino acid dipeptide containing glutamine is added to the culture medium at one or more of about 6 hours, about 12 hours, about 24 hours, about 48 hours, or about 72 hours after step (ii).

17. The method according to claim 1, wherein in step (i), an aggregation-inhibiting adjuvant is added to the culture medium.

18. The method according to claim 2, wherein at least one of the first, the second, and the third polynucleotide sequences is introduced into the cell by transfection of one or more vectors comprising the first, the second, and the third polynucleotide sequences, by infection with one or more viruses comprising one or more of the first, the second, and the third polynucleotide sequences, by a combination of transfection of the one or more vectors and infection with the one or more viruses, or by electroporation of the first, the second, and the third polynucleotide sequences.

19. The method according to claim 18, wherein at least one of the first, the second, and the third polynucleotide sequences is introduced into the cell by transfection.

20. The method according to claim 1, wherein the transgene encodes a therapeutic protein or a reporter protein.

21. The method according to claim 20, wherein the transgene is selected from the group consisting of RPE65, RPGR, GAD65, GAD67, and CNGB3.

22. The method according to claim 1, wherein the AAV ITR is AAV2 ITR.

23. The method according to claim 2, wherein the AAV cap gene is derived from an AAV serotype or an AAV variant selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, AAV-PHP.5, AAV-PHP.B, AAV-PHP.eB, AAV2-retro, AAV9-retro, and hybrids thereof.

24. The method according to claim 1, wherein the cell is a mammalian cell.

25. The method according to claim 24, wherein the mammalian cell is a HEK293 cell.

26. The method according to claim 24 or 25, wherein the cell is cultured in a suspension.

27. The method according to claim 26, wherein the at least one culture vessel is a shaker flask, a spinner flask, a cell bag, or a bioreactor.

28. In step (i), the culture medium has a pH of about 7.2 to about 7.4, and the culturing of the cells involves CO ２ sparging. The method according to claim 1.

29. Before step (ii), the pH of the culture medium is changed to about 6.9, and the CO ２ sparging is stopped. The method according to claim 28.

30. The method according to claim 1, wherein the step of isolating the rAAV particles comprises lysing the cells.

31. The method according to claim 30, wherein the step of isolating the rAAV particles further comprises clarifying the obtained lysate and subjecting the clarified lysate to a purification step.

32. The method according to claim 31, wherein the purification step is selected from the group consisting of ultracentrifugation, affinity chromatography, and ion exchange chromatography.

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