Systems and methods for RAAV production using a biphasic culture

Abstract

Embodiments herein include systems and methods for rAAV production using a biphasic culture. Various embodiments culture a cell line in a first cell culture media, then inoculate second cell culture media with an aliquot from the first cell culture media. One or more triggering agents can be added to the second cell culture media to induce production of rAAVs. In various instances, the cell line is a stable cell line, where one or more polynucleotides that encode components of rAAVs are integrated into the genome of the cell. Also provided are systems for rAAV production, including a bioreactor, first and second bioreactors in fluid communication, a sensor to measure one or more components within a cell culture, and a control system to initiate a transfer between the bioreactors. Some embodiments include a media reservoir to allow feeding the culture with additional media and / or a perfusion system to allow for removal of cellular waste components.

Description

SYSTEMS AND METHODS FOR RAAV PRODUCTION USING A BIPHASIC CULTURECross-Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 606,526, filed December 5, 2023, U.S. Provisional Application No. 63 / 612,821, filed December 20, 2023, and U.S. Provisional Application No. 63 / 626,439, filed January 29, 2024, each entitled “SYSTEMS AND METHODS FOR RAAV PRODUCTION USING A BIPHASIC CULTURE,” the contents of which are incorporated by reference in their entireties.Incorporation by Reference of Sequence Listing

[0002] The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 324632001040SeqList.xml, created December 4, 2024, which is 73,902 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.BACKGROUND

[0003] Adeno-associated virus (AAV) belongs to the Parvoviridae family and Dependovirus genus, of which some members require co-infection with a helper virus such as adenovirus to promote replication. AAV establishes a latent infection in the absence of a helper virus. AAV virions are composed of a 25 nm icosahedral capsid encompassing a 4.7 kb single-stranded DNA genome with two open reading frames: rep and cap. The non-structural rep gene encodes four AAV Rep proteins that are regulatory proteins essential for viral replication, whereas cap encodes three structural AAV Capsid proteins (Virion proteins 1-3 “VP 1-3”) that assemble into a 60-mer capsid shell. This viral capsid mediates the ability of AAV vectors to overcome many of the biological barriers of viral transduction, including cell surface receptor binding, endocytosis, intracellular trafficking, and unpackaging in the nucleus.

[0004] There is a need in the art for better methods and systems of recombinant AAV (rAAV) production, including cell culture, which produces rAAV effectively and efficiently.SUMMARY

[0005] Embodiments herein include systems and methods for rAAV production using a biphasic culture. Various embodiments culture a cell line in a first cell culture media, then inoculatesecond cell culture media with an aliquot from the first cell culture media. One or more triggering agents (e.g., doxycycline and tamoxifen) can be added to the second cell culture media to induce production of rAAVs. In various instances, the cell line is a stable cell line, where one or more polynucleotides that encode components of rAAVs are integrated into the genome of the cell. Also provided are systems for rAAV production, including first and second bioreactors in fluid communication, a sensor to measure one or more components within a cell culture, and a control system to initiate a transfer between the bioreactors. Some embodiments include a media reservoir to allow feeding the culture with additional media and / or a perfusion system to allow for removal of cellular waste components.

[0006] In some aspects, provided herein is a method for producing recombinant adeno- associated virus (rAAV), the method comprising: culturing a stable cell line, capable of inducibly producing rAAV, in a first cell culture media capable of supporting cell division; inoculating a second cell culture media, capable of supporting rAAV production, with an aliquot from the first cell culture media comprising the stable cell line; adding one or more triggering agents to the second cell culture media to induce production of rAAV from the stable cell line; and culturing the stable cell line in the second cell culture media. In some of any of the provided embodiments, culturing the stable cell line in the first cell culture media results in a peak cell density greater than 5 million cells / mL. In some of any of the provided embodiments, culturing the stable cell line in the second cell culture media results in a peak titer level greater than 5.0 x IO9vg / mL.

[0007] In some of any of the provided embodiments, the stable cell line comprises a sequence comprising an inducible promoter operably linked to a sequence encoding a recombinase. In some of any of the provided embodiments, the inducible promoter is a tetracycline inducible promoter. In some of any of the provided embodiments, the one or more triggering agents comprises a first triggering agent capable of activating the inducible promoter. In some of any of the provided embodiments, the first triggering agent is doxycycline. In some of any of the provided embodiments, the recombinase is an inducible recombinase. In some of any of the provided embodiments, the inducible recombinase is a recombinase fused to an estrogen receptor. In some of any of the provided embodiments, the one or more triggering agents comprises a second triggering agent capable of activating the inducible recombinase. In some of any of the provided embodiments, the second triggering agent is an estrogen agonist or selective estrogen receptor modulator. In some of any of the provided embodiments, the second triggering agent is tamoxifen.

[0008] In some of any of the provided embodiments, the adding step occurs prior to the inoculating step or simultaneously with the inoculating step. In some of any of the provided embodiments, the adding step occurs after the inoculating step. In some of any of the provided embodiments, after the inoculating step, the second cell culture media comprises between approximately 5 million cells per mL and approximately 10 million cells per mL, or comprises approximately 5 million cells per mL. In some of any of the provided embodiments, after the inoculating step, the second cell culture media comprises approximately 10 million cells per mL. In some of any of the provided embodiments, culturing the stable cell line in the first and / or second cell culture media comprises periodically adding additional media to the culture. In some of any of the provided embodiments, culturing the stable cell line in the first and / or second cell culture media comprises removing a waste metabolite from the culture. In some of any of the provided embodiments, culturing the stable cell line in the first and / or second cell culture media occurs in a bioreactor with perfusion. In some of any of the provided embodiments, perfusion is used to exchange the first cell culture media with the second cell culture media.

[0009] In some of any of the provided embodiments, culturing in the first cell culture media occurs until the stable cell line reaches a viable cell density of (VCD) of between approximately 1 million cells per mL and approximately 50 million cells per mL. In some of any of the provided embodiments, culturing in the first cell culture media occurs until the stable cell line reaches a viable cell density of (VCD) of between approximately 5 million cells per mL and approximately 10 million cells per mL. In some of any of the provided embodiments, culturing in the first cell culture media occurs until the stable cell line reaches a viable cell density of (VCD) of approximately 5 million cells per mL. In some of any of the provided embodiments, culturing in first cell culture media occurs until the stable cell line reaches a viable cell density of (VCD) of approximately 10 million cells per mL. In some of any of the provided embodiments, culturing in first cell culture media occurs until the stable cell line reaches a viable cell density of (VCD) of between approximately 10 million cells per mL and approximately 50 million cells per mL. In some of any of the provided embodiments, culturing in first cell culture media occurs until the stable cell line reaches a viable cell density of (VCD) of approximately 15 million cells per mL, 30 million cells per mL, or 50 million cells per mL.

[0010] In some of any of the provided embodiments, culturing in the first cell culture media occurs in a vessel of from approximately 100 mL to approximately 10 L. In some of any of theprovided embodiments, the first cell culture media has a volume of approximately 5 mL to approximately 9500 mL. In some of any of the provided embodiments, culturing in the second culture media occurs in a vessel of from approximately 1 L to approximately 2000 L. In some of any of the provided embodiments, the second culture media has a volume of approximately 500 mL to approximately 1800 L. In some of any of the provided embodiments, the inoculating step, the second cell culture media comprises less than or equal to 20% of the first cell culture media. In some of any of the provided embodiments, the triggering agents comprise doxycycline and tamoxifen.

[0011] In some of any of the provided embodiments, the stable cell line comprises a plurality of cells, each cell comprising: a first polynucleotide comprising a sequence encoding AAV Rep and AAV Cap proteins; a second polynucleotide comprising a sequence encoding one or more AAV helper proteins and-the sequence comprising the inducible promoter operably linked to the sequence encoding recombinase; and a third polynucleotide comprising a sequence encoding a payload flanked by AAV inverted terminal repeats (ITRs); wherein expression of the AAV Rep, AAV Cap and the one or more AAV helper proteins is induced by the one or more triggering agents. In some of any of the provided embodiments, the inducible promoter comprises tetracyclineresponse promoter elements (TREs). In some of any of the provided embodiments, transcription of the recombinase is induced in the presence of the first triggering agent. In some of any of the provided embodiments, the inducible recombinase translocates to the cell nucleus in the presence of the second triggering agent.

[0012] In some aspects, provided herein is a system for biphasic cell culture, the system comprising: a first bioreactor and a second bioreactor each configured to culture cells and in fluid communication with each other; a transfer apparatus configured to move a portion of a cell culture from the first bioreactor to the second bioreactor; a sensor located in the first bioreactor configured to measure one or more components within a cell culture in the first bioreactor; and a control system in electronic communication with the sensor and configured to initiate a transfer between the first bioreactor and the second bioreactor upon a reading from the sensor indicating a threshold condition; wherein the biphasic cell culture comprises a growth phase that occurs in the first bioreactor and a rAAV production phase that occurs in the in the second bioreactor.

[0013] In some of any of the provided embodiments, the sensor measures viable cell density (VCD) within the first bioreactor. In some of any of the provided embodiments, VCD is measuredby one or both of capacitance and optical cell density. In some of any of the provided embodiments, the threshold condition is a VCD greater than or equal to a target VCD that maximizes rAAV production. In some of any of the provided embodiments, the target VCD is between approximately 5 million cells / mL and approximately 10 million cells per mL. In some of any of the provided embodiments, the target VCD is 10 million cells / mL. In some of any of the provided embodiments, the threshold condition is a VCD of between approximately 5 million cells per mL and approximately 50 million cells per mL, or approximately 5, 10, 15, 20, 30, 40, or 50 million cells per mL. In some of any of the provided embodiments, the sensor measures one or more of pH, temperature, ion concentration, ion content, oxygen concentration, carbon dioxide concentration, and glucose concentration.

[0014] In some of any of the provided embodiments, the system further comprises a media reservoir in fluid communication with the first bioreactor, wherein the media reservoir is configured to periodically deliver fresh media to the first bioreactor. In some of any of the provided embodiments, the system further comprises a perfusion system in fluid communication with the first bioreactor and configured to remove cellular waste products from the first bioreactor. In some of any of the provided embodiments, the perfusion system comprises a cell retention device to recirculate cells into the first bioreactor. In some of any of the provided embodiments, the second bioreactor is larger than the first bioreactor. In some of any of the provided embodiments, the first bioreactor has a size of approximately 100 mL to approximately 10 L. In some of any of the provided embodiments, the second bioreactor has a size of approximately 1 L to approximately 2000 L.

[0015] In some of any of the provided embodiments, the system further comprises a second sensor located in the second bioreactor configured to measure one or more components within a cell culture in the second bioreactor. In some of any of the provided embodiments, the control system is in electronic communication with the second sensor and configured to initiate an addition of a triggering agent to the second bioreactor to induce rAAV production upon a reading from the second sensor indicating a threshold condition. In some of any of the provided embodiments, the second sensor measures viable cell density (VCD) within the second bioreactor. In some of any of the provided embodiments, the threshold condition is a target VCD that maximizes rAAV production. In some of any of the provided embodiments, the threshold condition is a target VCD of between approximately 5 million cells per mL and 10 million cells per mL. In some of any ofthe provided embodiments, the threshold condition is a target VCD of approximately 10 million cells per mL.

[0016] In some of any of the provided embodiments, the system further comprises a third bioreactor in fluid communication with the first bioreactor and configured to culture cells, and wherein the control system is configured to initiate a transfer between the first bioreactor and the third bioreactor upon a reading from the sensor located in the first bioreactor indicating a threshold condition. In some of any of the provided embodiments, the third bioreactor is larger than the first bioreactor. In some of any of the provided embodiments, the first bioreactor has a size of approximately 100 mL to approximately 10 L. In some of any of the provided embodiments, the third bioreactor has a size of approximately 1 L to approximately 2000 L.

[0017] In some of any of the provided embodiments, the system further comprises a third sensor located in the third bioreactor configured to measure one or more components within a cell culture in the third bioreactor. In some of any of the provided embodiments, the control system is in electronic communication with the third sensor and configured to initiate an addition of a triggering agent to the third bioreactor to induce rAAV production upon a reading from the third sensor indicating a threshold condition. In some of any of the provided embodiments, the third sensor measures viable cell density (VCD) within the third bioreactor. In some of any of the provided embodiments, the threshold condition is greater than or equal to a target VCD that maximizes rAAV production. In some of any of the provided embodiments, the target VCD is between approximately 5 million cells / mL and approximately 10 million cells / mL. In some of any of the provided embodiments, the target VCD is 10 million cells / mL. In some of any of the provided embodiments, the threshold condition is a viable cell density of (VCD) of between approximately 5 million cells per mL and 50 million cells per mL, or approximately 5, 10, 15, 20, 30, 40, or 50 million cells per mL.

[0018] In some of any of the provided embodiments, the fluid communication between the first and second bioreactors comprises a pump. In some of any of the provided embodiments, the pump is selected from one or more of: a centrifugal pump, a positive displacement pump, a peristaltic pump, a diaphragm pump, a reciprocating pump, a gear pump, an axial piston pump, a jet pump, a screw pump, and a vane pump. In some of any of the provided embodiments, the first bioreactor and the second bioreactor are a single bioreactor or wherein the first bioreactor, the second bioreactor, and the third bioreactor are a single bioreactor.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGS. 1A-1C illustrate exemplary polynucleotides for rAAV production that can be used in various embodiments. El a or EFla in the schematics refers a EFla promoter.

[0020] FIG. 2A illustrates an exemplary schematic of a biphasic culture system in accordance with various embodiments.

[0021] FIGS. 2B-2C illustrate a flow chart (FIG. 2B) and a diagram (FIG. 2C) of rAAV production phases.

[0022] FIG. 3 illustrates exemplary data showing effect of viable cell density (VCD) on production of rAAVs.

[0023] FIGS. 4A-4B illustrate exemplary data indicating different nutritional needs of cells during cell growth and rAAV production phases.

[0024] FIG. 5A illustrates viable cell density (VCD) reached from different culturing strategies, including batch, fed-batch, and perfusion.

[0025] FIG. 5B illustrates a table showing increased VCD maximus for stable cells using a control (batch) growth or using fed-batch strategies in 250 mL flasks or 10L bioreactors the produced titer levels from all three samples.

[0026] FIGs. 6A-6B illustrate exemplary data showing vg / mL (FIG. 6A) and vp / mL (FIG. 6B) for stable cells induced in various experimental production media.

[0027] FIG. 6C illustrates exemplary data showing improved volumetric titer produced from stable cells in an experimental media allowing for higher VCD for induction.

[0028] FIGS. 7A-7C illustrate exemplary data showing consistent patterns in VCD, viability, and titer (vg / mL) during production in both 250 mL flasks and 10L bioreactors.

[0029] FIG. 8 provides a table of exemplary data showing titer levels, total viral genome (vg) produced, and percent yield of a biphasic culture strategy for rAAV production using a 10 L bioreactor.

[0030] FIG. 9 illustrates exemplary data showing capacitance as an effective in situ method for measuring cell density as compared to optical measurements.

[0031] FIGS. 10A -10B provide exemplary schematics of polynucleotide constructs of a v 1.0 system for inducibly producing rAAV. A full description of a v 1.0 system is provided in US Pat. No. 12,054,738 and PCT Pub. No. WO 2022 / 026927; the disclosures of which are incorporated by reference in their entireties for all purposes. FIG. 10A depicts the system in an off state, in whichrAAV is not produced in the absence of the first and second triggering agents. FIG. 10B depicts the post-triggered state of the Rep / Cap construct (Construct 1), helper construct (Construct 2), and payload construct (Construct 3) shown in FIG. 10A following the addition of the first triggering agent, and the second triggering agent.

[0032] FIGS. 11 -11B provide exemplary schematics of polynucleotide constructs of a v 1.2 system for inducibly producing rAAV. A full description of a v 1.2 system is provided PCT Pub. No. WO 2024 / 112813; the disclosure of which is incorporated by reference in its entirety for all purposes. FIG. 11A depicts the system in an off state, in which rAAV is not produced in the absence of the first and second triggering agents. FIG. 11B depicts the post-triggered state of Constructs 1-4 shown in FIG. 11A following the addition of the first triggering agent and the second triggering agent.

[0033] FIGS. 12A-12B provide exemplary schematics of polynucleotide constructs of the v 1.3 system for inducibly producing rAAV. FIG. 12A depicts the system in an off state, in which rAAV is not produced in the absence of the first and second triggering agents. FIG. 12B depicts the posttriggered state of Constructs 1 -3 shown in FIG. 12A following the addition of the first triggering agent and the second triggering agent.

[0034] FIGS. 13A-13B provide exemplary schematics of polynucleotide constructs of the v 1.4 system for inducibly producing rAAV. FIG. 13A depicts the system in an off state, in which rAAV is not produced in the absence of the first and second triggering agents. FIG. 13B depicts the posttriggered state of Constructs 1 -3 shown in FIG. 13A following the addition of the first triggering agent and the second triggering agent.

[0035] FIG. 14A provides exemplary data for cell viability percent and viable cell density (VCD) for pools of vl.3 and vl.4 cells as measured for days post-induction by the first triggering agent and the second triggering agent. FIG. 14B provides exemplary data for titer level (Vg / mL) for pools vl.3 and vl.4 cells as measured for days post-induction by the first triggering agent and the second triggering agent.

[0036] FIG. 15A illustrates an exemplary biphasic single bioreactor production system with media exchange for volumes less than 10 L. FIG. 15B illustrates an exemplary biphasic multibioreactor production system with dilution for volumes greater than 10 L. FIG. 15C illustrates an exemplary biphasic multi-bioreactor production system with media exchange and dilution for volumes greater than 10 L. FIG. 15D illustrates an exemplary biphasic semi-continuousproduction system where cells are grown continuously in the growth phase (N-l) in one bioreactor and periodically transferred to other bioreactors for the production phase (N). FIG. 15E illustrates an exemplary biphasic multi-bioreactor production system with a perfused production phase (N).DETAILED DESCRIPTION

[0037] Systems and methods for culturing cells for rAAV production are provided. In certain aspects, these systems and methods may be used to produce higher amounts of rAAV due to increased production of both recombinant cells and the rAAV. Many embodiments utilize a biphasic culturing strategy, where a first culture media is used during cell growth and a second culture media is used for induction during rAAV production.

[0038] Before the systems and methods of the present disclosure are described in greater detail, it is to be understood that the systems and methods are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the systems and methods will be limited only by the appended claims.

[0039] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the systems and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the systems and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the systems and methods.

[0040] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the systems and methods belong. Although any systems and methods similar or equivalent to those described hereincan also be used in the practice or testing of the systems and methods, representative illustrative systems and methods are now described.

[0042] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present systems and methods are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

[0043] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0044] It is appreciated that certain features of the systems and methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the systems and methods, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present systems and methods and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0045] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recitedmethod can be carried out in the order of events recited or in any other order that is logically possible.Definitions

[0046] The term "about", particularly in reference to a given quantity, is meant to encompass deviations of up to plus or minus five percent.

[0047] AAV" is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. The components of the AAV DNA genome consists of two open reading frames, Rep and Cap, flanked by two 145 base inverted terminal repeats (ITRs). Rep gene encodes multiple distinct proteins including Rep78, Rep68, Rep52, and Rep40. These proteins are also referred to herein as Rep proteins or Rep and may encompass one or more of Rep78, Rep68, Rep52, and Rep40 and functional variants thereof and homologs thereof. Rep78 and Rep68 and functional variants thereof and homologs thereof are referred to herein as large Rep. Rep52, and Rep40 and functional variants thereof and homologs thereof are referred to herein as small Rep. Rep proteins from an AAV of a particular serotype may also be referred to as Repl, Rep2, etc. where the Rep protein is derived from an AAV1 or an AAV2 serotype, respectively. Cap gene encodes capsid proteins VP1, VP2, and VP3 required for production of rAAV capsids. These proteins are also referred to herein as Cap proteins or Cap and may encompass one or more of VP1, VP2, and VP3 and functional variants thereof and homologs thereof. Cap proteins from an AAV of a particular serotype may also be referred to as Capl, Cap2, Cap4, etc. where the Rep protein is derived from an AA1, an AAV2, or an AAV5 serotype, respectively. In addition to Rep and Cap, AAV requires a helper plasmid containing genes from a helper virus such as adenovirus, including Ela, Elb, E4, E2a, and VA genes for AAV replication.

[0048] Recombinant virus" is meant to describe a virus that has been genetically altered, e.g., by the addition or insertion of a heterologous nucleic acid construct into the virus.

[0049] The abbreviation "rAAV" refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or "rAAV vector"). The term “AAV” includes any AAV serotype as well as AAV vectors based on the combination of different serotypes (also referred to as "hybrid AAV vectors" or "pseudotype AAV vectors"). AAV serotype may be AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6(AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), AAV type 10 (AAV- 10), AAV type 11 (AAV- 11), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, nonprimate AAV, ovine AAV, AAV-7m8, AAV-6.2, AAV-Dj, AAV-DJ / 8, AAV2-retro, AAV2-QuadYF and AAV2.7m8, AAV-PHP.B, AAV-PHPB2, AAV-PHPB3, AAV-PHPA, AAV-PHPeB, AAV- PHPeS, evolved capsids that are less immunogenic to mice and humans, and variants thereof and combinations thereof. “Primate AAV” refers to AAV isolated from a primate, “non-primate AAV” refers to AAV isolated from a non-primate mammal, “bovine AAV” refers to AAV isolated from a bovine mammal (e.g., a cow), etc. An "rAAV vector" comprises a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a polynucleotide sequence of interest for introducing into a target cell. In general, the heterologous polynucleotide is flanked by at least one, and usually by two AAV inverted terminal repeat sequences (ITRs). The heterologous polynucleotide can also be referred to as a polynucleotide payload. The term rAAV vector encompasses both rAAV virions and rAAV vector plasmids.

[0050] An "AAV virus" or "AAV viral particle" or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein (typically by all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide rAAV vector. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "rAAV vector particle" or simply an "rAAV vector". Thus, production of a rAAV particle necessarily includes production of a rAAV vector, as such a vector contained within an rAAV particle.

[0051] A “bioreactor” refers to a controlled environment system used for the cultivation of cells, microorganisms, or biological entities. Bioreactors can provide an environment where conditions such as temperature, pH, nutrient supply, and oxygenation can be maintained to support the growth and productivity of the cells or microorganisms being cultured. Such devices can include one or more of a vessel (or chamber), an agitation system, a temperature control, aeration or oxygenation system, ports (e.g., to add an inoculant, a sensor or probe, and / or remove a sample or aliquot), and / or any other component that can assist with cultivation.

[0052] AAV "rep" and "cap" genes refer to polynucleotide sequences encoding replication and capsid proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

[0053] By "AAV Rep coding region" or “sequence encoding one or more Rep proteins” or “Rep encoding sequence” and grammatical equivalents thereof is meant the art-recognized region of the AAV genome which encodes the replication proteins of the virus which are required to replicate the viral genome and / or a payload flanked by ITRs. The rep coding region, as used herein, may be derived from any viral serotype, such as those described above. The region need not include all of the wild-type genes but may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the rep genes provide for expression Rep proteins. Rep coding sequences are further described below.

[0054] By "AAV cap coding region" or “sequence encoding one or more cap proteins,” or “Cap encoding sequence” and grammatical equivalents thereof it is meant the art-recognized region of the AAV genome which encodes the coat proteins of the virus which are required for the capsid that viral genome or a payload is packaged into by the Rep proteins. For a further description of the cap coding region, see, e.g., Muzyczka, N. (1992) Current Topics in Microbiol, and Immunol. 158, 97-129; Kotin, R. M. (1994) Human Gene Therapy 5, 793-801. The AAV cap coding region, as used herein, may be derived from any AAV serotype, as described above. The region need not include all of the wild-type cap genes but may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the genes provide for sufficient packaging functions. Cap coding sequences are further described below.

[0055] By "adeno-associated virus inverted terminal repeats" or "AAV ITRs" is meant the art- recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the viral genome. The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin, R. M. (1994) Human Gene Therapy 5, 793-801; Berns, K. I. "Parvoviridae and their Replication" in Fundamental Virology, 2d ed., (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 ITRs sequence. As used herein, an "AAV ITR" need not have a wild-type nucleotide sequence, but may be altered, e.g., by the insertion, deletion or substitution of nucleotides. The AAV ITR may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-7, etc. Furthermore, 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate. The ITRs may be single stranded (ssITRs) or self-complementary (scITRs).

[0056] A "helper virus" for AAV refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by a mammalian cell. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.

[0057] "Helper virus function(s)" refers to function(s) encoded in a helper virus genome which allow AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, "helper virus function" may be provided in a number of ways, including by providing helper virus or providing, for example, polynucleotide sequences encoding the requisite function(s) to a producer cell in trans.

[0058] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0059] As used herein, the term “polynucleotide construct” refers to a DNA segment of any size that includes one or more sequences encoding an RNA or protein and at least one promoter for driving expression from the one or more sequences. A polynucleotide construct may be a circular DNA or a linear DNA. A polynucleotide construct may be single stranded or double stranded. As used herein, the term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which may transfer gene sequences into and between cells. Thus, the term includes cloning and expression vehicles, as wellas viral vectors. The use of the term "vector" throughout this specification encompasses plasmid or viral vectors, which permit the desired components to be transferred to the host cell via transfection or infection. For example, an adeno-associated viral (AAV) vector is a plasmid comprising a recombinant AAV genome. In some embodiments, useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter. A vector may be linear or circular, single stranded or double stranded, DNA or RNA. In certain aspects, the vector may be circular, double stranded DNA.

[0060] As used herein, the term “vector system” refers to two or more vectors that are used together, e.g., by simultaneous or sequential introduction into a cell, to provide at least two different components into the cell. The two different components may then work together in the cell.

[0061] For purposes herein, reference to a polynucleotide sequence (e.g., a DNA sequence or an RNA sequence) also encompasses the reverse complement of the polynucleotide sequence. For example, a sequence of AAAAAGGGGG also encompasses a sequence of CCCCCTTTTT.

[0062] A "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated.

[0063] The term "host cell" denotes, for example, microorganisms, yeast cells, insect cells, and mammalian cells, that may be, or have been, used as recipients of an AAV vector system as described herein, or other transfer DNA. The term includes the progeny of the original cell which has been transfected. Thus, a "host cell" as used herein generally refers to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to natural, accidental, or deliberate mutation.

[0064] As used herein, the term "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes may occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.

[0065] The term "cell culture," refers to cells grown adherent or in suspension, bioreactors, roller bottles, hyperstacks, microspheres, macrospheres, flasks and the like, as well as thecomponents of the supernatant or suspension itself, including but not limited to rAAV particles, cells, cell debris, cellular contaminants, colloidal particles, biomolecules, host cell proteins, nucleic acids, and lipids, and flocculants. Large scale approaches, such as bioreactors, including suspension cultures and adherent cells growing attached to microcarriers or macrocarriers in stirred bioreactors, are also encompassed by the term "cell culture." Cell culture procedures for both large and small-scale production of proteins are encompassed by the present disclosure.

[0066] The terms “tetracycline” is used generically herein to refer to all antibiotics that are structurally and functionally related to tetracycline, including tetracycline, doxycycline, demeclocycline, minocycline, sarecycline, oxytetracycline, omadacycline, or eravacycline.

[0067] Recombinant," as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature. A recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.

[0068] A cell is said to be "stably" altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during extended culture of the cell in vitro. Generally, such a cell is "heritably" altered (genetically modified) in that a genetic alteration is introduced which is also inheritable by progeny of the altered cell. For example, a gene integrated into the nuclear genome of the cell and is available to perform its function during extended culture of the cell in vitro. A gene integrated into the nuclear genome of the cell is inheritable by progeny of the cell.

[0069] As used herein, the term “cell culture media” refers to a nutrient solution used for growing mammalian cells in vitro that typically provides at least one component from one or more of the following categories: 1) an energy source, usually in the form of a carbohydrate such as, for example, glucose; 2) one or more of all essential amino acids, and usually the basic set of twenty amino acids plus cysteine; 3) vitamins and / or other organic compounds required at low concentrations; 4) free fatty acids; and 5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range. The nutrient solution may optionally be supplemented with additional components to optimize growth and / or transfection of cells.

[0070] As used herein, the term “configured to” has the same meaning as “capable of’.Cell Line

[0071] Many embodiments of the present disclosure include a method for culturing a cell line and inducing production of rAAV from the cultured cell line. In some embodiments, the cell line is a stable cell line. Within this disclosure, a “stable cell line” refers to cell line, wherein polynucleotides required for rAAV production are integrated into the genome of the cells. In some embodiments, the cell line is an inducible cell line or the stable cell line is an inducible stable cell line, wherein the components for rAAV production are inducible in the presence of one or more triggering agents (e.g., doxycycline and tamoxifen), as further described herein. In some embodiments, the polynucleotides required for rAAV production comprise a sequence comprising an inducible promoter, wherein the inducible promoter is activated in the presence of a first triggering agent. For example, in some embodiments, the inducible promoter is a tetracycline inducible promoter and the first triggering agent is doxycycline. In some embodiments, the polynucleotides required for rAAV production comprise a sequence encoding an inducible recombinase, wherein the inducible recombinase is activated in the presence of a second triggering agent. For example, in some embodiments, the inducible recombinase is a CRE recombinase fused to an estrogen receptor and the second triggering agent is tamoxifen. In some embodiments, the polynucleotides required for rAAV production comprise an inducible promoter and an inducible recombinase, wherein the components for rAAV production are induced in the presence of the first triggering agent and the second triggering agent. In some embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen.

[0072] FIGS. 1A-1C illustrate exemplary schematics of polynucleotide constructs that supply components for inducible rAAV production.The polynucleotide constructs comprise a first polynucleotide encoding AAV Rep and AAV Cap genes operably linked to their native promoters and a first half of a split blasticidin resistance gene operably linked to a constitutive El a promoter, a second polynucleotide encoding a tetracycline inducible promoter operably linked to a Cre recombinase upstream of AAV helper genes, and a third polynucleotide encoding an ITR-flanked gene of interest (GOI) and a second half of a blasticidin resistance gene operably linked to a constitutive El a promoter. Specifically, FIG. 1 illustrates the polynucleotide constructs in an uninduced or “off’ state, while FIG. IB illustrates the polynucleotide constructs in an induced or “on” state following addition of one or more triggering agents and subsequent recombination by the recombinase. Additionally, FIG. 1C illustrates an alternative configuration for apolynucleotide encoding the Rep and Cap proteins where the Cap protein is operably linked to a tetracycline inducible promoter.

[0073] In some embodiments, a first polynucleotide comprises a sequence encoding for Rep and Cap proteins and spacer or excisable sequence elements. This first polynucleotide is referred to as a Rep / Cap construct. In some embodiments, the Rep / Cap construct includes a Rep coding sequence comprising a first part of the Rep coding sequence and a second part of the Rep coding sequence which are separated by an excisable element. The excisable element prevents expression of all large Rep proteins and small Rep proteins. In some embodiments, the excisable element is positioned in the small Rep coding sequence since the small Rep coding sequence is common with the large Rep coding sequence such that expression of both small and large Rep proteins can be controlled. The excisable element includes a sequence comprising a stop signaling sequence (e.g., a stop codon and a polyA signal sequence) which prevents translation of the full-length Rep proteins, resulting in expression of truncated Rep proteins that are non-functional and lack toxicity associated with the full-length Rep proteins. The excisable element includes a first recombination site and a second recombination site flanking the sequence comprising the stop signaling sequence (e.g., a stop codon). In certain embodiments, the stop signaling sequence is present downstream of an exon. The first recombination site and the second recombination site are oriented in the same direction and recombination between the first and second recombination sites by an inducible recombinase results in excision of the sequence comprising the stop signaling sequence (e.g., a stop codon) allowing expression of full-length large Rep proteins and full-length small Rep proteins.

[0074] In certain aspects, the excisable element is flanked by a split intron to prevent read- through from the stop signaling sequence. For example, the polynucleotide construct comprises from 5 ’ to 3 ’ : one or more native AAV Rep promoters operably linked to a first part of an AAV Rep coding sequence, a 5’ splice site (SS), a first part of an intron, a first recombination site, a first 3’ SS, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3’ SS, and a second part of the AAV Rep coding sequence, wherein the first recombination site, the first 3’ splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form an excisable element, wherein the first recombination site and the second recombination site are oriented in the same direction, and wherein the one or more promoters are not operably linked to the second part of the AAV Repcoding sequence. The first and second recombination sites are recombined by an inducible recombinase resulting in excision of the excisable element which results in a polynucleotide in which the first part of the AAV Rep coding sequence and the first part of the intron are joined to the second part of the intron and the second part of the AAV Rep coding sequence to form a complete AAV Rep coding sequence comprising the intron. Upon transcription, the intron is spliced out by the endogenous cellular machinery to generate a mature mRNA which is translated to produce AAV Rep proteins.

[0075] In some embodiments, the polynucleotide comprising the AAV Rep coding sequence further includes an AAV Cap coding sequence. In certain embodiments, the AAV Cap coding sequence is positioned downstream of the AAV Rep coding sequence and is operably linked to a native p40 promoter present in the AAV Rep coding sequence. In some embodiments, expression of the AAV Cap proteins from the native p40 promoter requires the expression of full length Rep proteins. In certain embodiments, the native p40 promoter present in the AAV Rep coding sequence is mutated. In some embodiments, the AAV Cap coding sequence is operably linked to an inducible promoter. In certain cases, the AAV Cap coding sequence that is operably linked to an inducible promoter is separated from the AAV Rep coding sequence by an intervening sequence to provide spatial separation between the inducible promoter and the Rep coding sequence and the native Rep promoters and the AAV Cap coding sequence. In some embodiments, the AAV Cap coding sequence is positioned in a head-to-head orientation relative to the AAV Rep sequence. In some embodiments, the AAV Cap coding sequence and the Rep coding sequence are divergently transcribed.

[0076] In certain aspects, the AAV capsid proteins coding sequence is separated from the Rep coding sequence by an intervening sequence and the polynucleotide further comprises an inducible or constitutive promoter operably linked to the Cap coding sequence. In certain aspects, the intervening sequence comprises a transcriptional blocking element (TBE). In certain aspects, a separate polynucleotide comprises an inducible promoter operably linked to the AAV capsid proteins coding sequence.

[0077] In some embodiments, a second polynucleotide comprises a sequence encoding one or more adenoviral helper proteins, a recombinase, and an inducible promoter. This second polynucleotide is referred to as a helper construct. In some embodiments, the helper constructs provide inducible production of the helper proteins E2A and E4 and the recombinase. In someembodiments, the recombinase is a Cre recombinase. In some embodiments, the recombinase is an inducible recombinase, such as an inducible Cre recombinase. In certain embodiments, a second polynucleotide comprises an inducible promoter operably linked to a sequence encoding an inducible recombinase; a self-excising element comprising a third recombination site and a fourth recombination site flanking the sequence encoding the inducible recombinase, wherein the third recombination site and the fourth recombination site are oriented in the same direction. The selfexcising element separates the inducible promoter from a sequence encoding the one or more AAV helper proteins such that the inducible promoter is not operably linked to the sequence encoding the one or more AAV helper proteins and the one or more AAV helper proteins are not expressed. In certain embodiments, the second polynucleotide comprises a constitutive promoter operably linked to a sequence encoding an activator, where the activator is unable to activate the inducible promoter in absence of a first triggering agent. The second polynucleotide also comprises a constitutive promoter operably linked to a sequence encoding a selectable marker.

[0078] In certain aspects, the inducible promoter operably linked to the sequence encoding the inducible recombinase and the inducible promoter operably linked to the AAV capsid proteins coding sequence are the same promoters. In certain embodiments, the inducible promoter is a tetracycline inducible promoter. In some embodiments, the inducible promoter comprises a plurality of tetracycline (Tet) operator elements capable of binding to a Tet responsive activator protein in the presence of a tetracycline. In some embodiments, the plurality of tetracycline (Tet) operator elements form a Tetracycline Responsive element (TRE). In certain aspects, the inducible promoter comprises a tetracycline-responsive promoter element (TRE).. In certain aspects, the inducible promoter is the TRE3G promoter. In certain aspects, the activator that binds to the TRE in the presence of the first triggering agent is Tet-on 3G. In certain aspects, the first triggering agent is capable of activating the inducible promoter. In certain aspects, the first triggering agent is doxycycline. In certain aspects, the inducible recombinase is a recombinase fused to an estrogen receptor (ER2). In certain aspects, the inducible recombinase translocates to the nucleus of a cell comprising the second polynucleotide in the presence of a second triggering agent. In certain aspects, the second triggering agent is capable of activating the inducible recombinase. In certain aspects, the second triggering agent is an estrogen agonist or selective estrogen receptor modulator.. In certain aspects, the second triggering agent is tamoxifen. In certain aspects, the first triggering agent is doxycycline and the second triggering agent is tamoxifen.

[0079] In the presence of the first triggering agent, e.g., doxycycline, the tetracycline inducible promoter is activated resulting in the transcription of the inducible recombinase. In the presence of the second triggering agent, e.g., tamoxifen, the inducible recombinase translocates to the nucleus of the cell. The expression and nuclear localization of the recombinase results in recombination of the first polynucleotide to remove the self-excising element such that the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins, thereby triggering the expression of the one or more AAV helper proteins. The inducible recombinase also recombines the second polynucleotide to remove the excisable element positioned in the small Rep coding sequence, thereby allowing the expression of full length Rep proteins and transcription of Cap proteins.

[0080] In some embodiments, a third polynucleotide comprises a sequence encoding a payload (e.g., gene of interest) flanked by AAV inverted terminal repeats (ITRs). This third polynucleotide is referred to as a payload construct. The sequence coding a payload when delivered into a cell using a rAAV may provide DNA to the cell or may be transcribed into RNA (e.g., siRNA, guide RNA) in the cell and / or may be transcribed and subsequently translated into a protein in the cell.

[0081] In certain embodiments, the first polynucleotide comprises a sequence encoding a first portion of a first selectable marker operably linked to a constitutive promoter and the third polynucleotide comprises a sequence encoding a second portion of the first selectable marker operably linked to a constitutive promoter, wherein the first and second portions associate to form functional first selectable marker, e.g., a blasticidin resistance gene. In certain embodiments, the selectable marker encoded by the second polynucleotide is a second selectable marker which is different from the first selectable marker.Method for Producing rAAV

[0082] As described above, the present disclosure provides methods for culturing a cell line for rAAV production. In many instances, the cell line is cultured in a first cell culture media configured to support growth of the cell line. In some embodiments, the cell line is a stable cell line. Following expansion of viable cells in the first cell culture media, the cells are introduced into a second cell culture media configured to support induction of rAAV production and rAAV production. This method of using two different cell culture media, one for cell growth and the other for induction of rAAV production and rAAV production, is referred to herein as biphasic culture.

[0083] As described above, a cell line (e.g., a stable cell line) includes cells having integrated into its nuclear genome, polynucleotides that inducibly produce rAAV (e.g., as illustrated in FIGS. 1A-1C).

[0084] In many embodiments, the first cell culture media is configured to support cell division or is capable of supporting cell division. The first cell culture media is referred to as growth media. Cell division can increase the total number of cells through mitosis. In some instances, the first culture media is optimized or tailored to a metabolic profile of the cell line (e.g., HEK 293). In many instances, the first culture media includes a compound to apply selective pressure to the cell line. The compound can be the antibiotic or toxin to which the cells have resistance. In some embodiments, culturing of the cell line in the first cell culture media results in a peak cell density greater than 5 million cells / mL. In some embodiments, culturing of the cell line in the first cell culture media results in a peak cell density greater than 10 million cells / mL. In some embodiments, culturing of the cell line in the first cell culture media results in a peak cell density of 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / mL.

[0085] In many embodiments, the second cell culture media is configured to support AAV production or is capable of supporting AAV production. The second cell culture media is referred to as production media. In some instances, the second culture media is optimized or tailored to a metabolic profile of the cell line (e.g., HEK 293). In many embodiments, one or more triggering agents is added to the second cell culture media to induce rAAV production. In some embodiments, the culturing of the cell line in the second culture media results in a peak cell titer greater than 5xl09viral genomes per milliliter. (vg / mL). In some embodiments, the culturing of the cell line in the second culture media results in a peak cell titer greater than 1 x I011or no less than 5 x 1011, 1 x IO12, 5 x IO12, 1 x IO13or 1 x IO14vg / mL prior to purification. In some embodiments, the culturing of the cell line in the second culture media results in a peak cell titer of 2 x 1011, 3 x 1011, 4 x 1011, 5 x 1011, 6 x 1011, 7 x 1011, 8 x 1011, or 1 x IO12vg / mL prior to purification. In some embodiments, the culturing of the cell line in the second culture media results in a peak cell titer of 3 x I011vg / mL prior to purification. In some embodiments, the culturing of the cell line in the second culture media results in a peak cell titer of 8 x I011vg / mL prior to purification.

[0086] In some embodiments, cells cultured in the first cell culture media have or have about 50%, or have less than or less than about 50%, of the amount of viral titer (vg / ml) as compared to cells cultured in the second cell culture media. In some embodiments, cells cultured in the secondcell culture media have or have about 50%, or have less than or less than about 50%, of the amount of peak cell density (cells / mL) as compared to cells cultured in the first cell culture media.

[0087] In some embodiments, the first cell culture media (e.g., growth media) is used during the growth phase. In some embodiments, the first cell culture media allows for the cell culture to reach a higher peak cell density relative to cells cultured in the second cell culture media. In some embodiments, the first cell culture media allows for the cell culture to reach a peak cell density of between or between about 5 million cells / mL and 50 million cells / mL. In some embodiments, the first cell culture media allows for the cell culture to reach a peak cell density of 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / mL.

[0088] In some embodiments, the first cell culture media is a commercially available media. Exemplary commercially available media include, for example, Ham's F10 (SIGMA), Minimal Essential Medium (MEM, SIGMA), RPMI-1640 (SIGMA), Dulbecco's Modified Eagle's Medium (DMEM, SIGMA), DMEM / F12 (Life Technologies), and BalanCD® HEK293 Medium (Fujifilm Irvine Scientific). Any of these or other suitable media may be supplemented as necessary with hormones and / or other growth factors (such as but not limited toinsulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES or bicarbonate), nucleosides (such as adenosine and thymidine), antibiotics (such as puromycin, neomycin, hygromycin, blasticidin, or Gentamycin™), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) lipids (such as linoleic or other fatty acids) and their suitable carriers, and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.

[0089] In some embodiments, the first cell culture media is supplemented with fetal bovine serum. In some embodiments, the first media is supplemented with fetal bovine serum at between or between about 20% and 10% of the total volume of the first cell culture media. In some embodiments, the first media is supplemented with fetal bovine serum at about 20%, 15%, or 10% of the total volume of the first cell culture media. In some embodiments, the first cell culture media is supplemented with between or between about 2 mM and 6 mM L-glutamine. In some embodiments, the first cell culture media is supplemented with 2 mM, 3 mM, 4 mM, 5 mM, or 6 mM L-glutamine.

[0090] In some embodiments, the first cell culture media comprises one or more of the following amino acids: glycine, L-alanine, L-arginine hydrochloride, L-asparagine, L-aspartic acid, L-cystine 2HCL, L-glutamic acid, L-histidine hydrochloride, L-isoleucine, L-leucine, L- lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L- tryptophan, L-tyrosine disodium salt dihydrate, and L-valine. In some embodiments, the first cell culture media comprises one or more of the following vitamins: ascorbic acid phosphate, choline chloride, D-Calcium pantothenate, folic acid, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, and i-inositol. In some embodiments, the first cell culture media comprises one or more of the following inorganic salts: calcium chloride (CaCh) (anhyd.), ferric nitrate (Fe(NO3)3), magnesium sulfate (MgSO4) (anhyd.), potassium chloride (KC1), sodium bicarbonate (NaHCO3), sodium chloride (NaCl), and sodium phosphate dibasic (Na2HPO4-H2O). In some embodiments, the first cell culture media comprises one or more of the following proteins: AlbuMAX® II, Human Transferrin (Holo), and Insulin Recombinant (Full Chain). In some embodiments, the first cell culture media comprises one or more of the following trace elements: ammonium metavanadate, cupric sulfate, manganous chloride, and sodium selenite. In some embodiments, the first cell culture media comprises one or more of the following components: D- glucose (dextrose), ethanolamine, glutathione (reduced), phenol red, and sodium pyruvate.

[0091]

[0092] In some embodiments, the first cell culture media comprises about 6 g / L glucose. In some embodiments, the first cell culture media lacks L-glutamine. In some embodiments, the first cell culture media is supplemented with GlutaMAX™ supplement (ThermoFisher Scientific). In some embodiments, the first cell culture media comprises BalanCD® HEK293 medium (Fujifilm Irvine Scientific). In some embodiments, the first cell culture media is BalanCD® HEK293 medium. In some embodiments, the first cell culture media is BalanCD® HEK293 medium supplemented with GlutaMAX™ supplement. GlutaMAX™ supplement includes L-alanyl-L- glutamine, which is an alternative to L-glutamine having increased stability. Accordingly, in some embodiments, the first cell culture media is supplemented with L-alanyl-L-glutamine. In some embodiments, the first cell culture media is BalanCD® HEK293 medium supplemented with L- alanyl-L-glutamine.In some embodiments, the second cell culture media (e.g., production media) is used during the AAV production phase. In some embodiments, the second cell culture media allows for the cell culture to reach a higher viral titer relative to cells cultured in the first cellculture media. In some embodiments, the second cell culture media allows for the cell culture to reach a peak cell titer of between or between about 2 x 1011and 1 x 1012vg / mL prior to purification. In some embodiments, the second cell culture media allows for the cell culture to reach a peak cell titer of 2 x 1011, 3 x 1011, 4 x 1011, 5 x 1011, 6 x 1011, 7 x 1011, 8 x 1011, or 1 x 1012vg / mL prior to purification.

[0093] In some embodiments, the second cell culture media is a commercially available media. Exemplary commercially available media include, for example, Ham's F10 (SIGMA), Minimal Essential Medium (MEM, SIGMA), RPML1640 (SIGMA), Dulbecco's Modified Eagle's Medium (DMEM, SIGMA), and DMEM / F12 (Life Technologies). Any of these or other suitable media may be supplemented as necessary with hormones and / or other growth factors (such as but not limited toinsulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES or bicarbonate), nucleosides (such as adenosine and thymidine), antibiotics (such as puromycin, neomycin, hygromycin, blasticidin, or Gentamycin™), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) lipids (such as linoleic or other fatty acids) and their suitable carriers, and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.

[0094] In some embodiments, the second cell culture media is supplemented with fetal bovine serum. In some embodiments, the second media is supplemented with fetal bovine serum at between or between about 20% and 10% of the total volume of the second cell culture media. In some embodiments, the second cell culture media is supplemented with fetal bovine serum at about 20%, 15%, or 10% of the total volume of the second cell culture media. In some embodiments, the second cell culture media is supplemented with between or between about 2 mM and 6 mM L- glutamine. In some embodiments, the second cell culture media is supplemented with 2 mM, 3 mM, 4 mM, 5 mM, or 6 mM L-glutamine.

[0095] In some embodiments, the second cell culture media comprises one or more of the following amino acids: glycine, L-alanine, L-arginine hydrochloride, L-asparagine, L-aspartic acid, L-cystine 2HCL, L-glutamic acid, L-histidine hydrochloride, L-isoleucine, L-leucine, L- lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L- tryptophan, L-tyrosine disodium salt dihydrate, and L-valine. In some embodiments, the second cell culture media comprises one or more of the following vitamins: ascorbic acid phosphate,choline chloride, D-Calcium pantothenate, folic acid, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, and i-inositol. In some embodiments, the second cell culture media comprises one or more of the following inorganic salts: calcium chloride (CaCh) (anhyd.), ferric nitrate (Fe(NO3)3"9H2O), magnesium sulfate (MgSO4) (anhyd.), potassium chloride (KC1), sodium bicarbonate (NaHC03), sodium chloride (NaCl), and sodium phosphate dibasic (Na2HPO4-H2O). In some embodiments, the second cell culture media comprises one or more of the following proteins: AlbuMAX® II, Human Transferrin (Holo), and Insulin Recombinant Full Chain). In some embodiments, the second cell culture media comprises one or more of the following trace elements: ammonium metavanadate, cupric sulfate, manganous chloride, and sodium selenite. In some embodiments, the second cell culture media comprises one or more of the following components: D-glucose (dextrose), ethanolamine, glutathione (reduced), phenol red, and sodium pyruvate. In many embodiments, the second cell culture media differs from the of first cell culture media in the level of amino acids, vitamins, lipids, trace metals and / or poly amines.

[0096] In some embodiments, the second cell culture media comprises M21 medium from Fujifilm. In some embodiments, the second cell culture media is M21 medium from Fujifilm.

[0097] In various embodiments, the culture is a suspension culture (e.g., where cells grow freely in the culture medium without attaching to a surface), while in some instances the culture is an adherent cell culture (e.g., cells attach to a substrate or surface). The vessel for culturing can be of any applicable mechanism to encourage cell growth, including a petri dish, a deep well plate, a culture flask, an Erlenmeyer flask, and / or any other applicable vessel for a culture.

[0098] A culture of various embodiments can include one or more of a “batch” culture, a “fed- batch” culture, and / or a “perfusion” culture. A batch culture refers to traditional culture methods, where a culture media is inoculated and growth proceeds under the cells are harvested; fed-batch refers to a strategy that periodically adds additional culture media to the vessel to provide additional nutrients or other compounds that can aid in cellular growth or division. A perfusion strategy further allows for removal of waste products or other metabolites that may hamper cellular growth and / or division. Perfusion strategies can circulate fresh media into a culture vessel while removing older media. In some instances, perfusion may include filtering or collecting cells from the older media and returning the cells to the vessel. In some instances, fed-batch and / or perfusion may alter the components of the culture media to maintain growth conditions, such as salt contentand / or concentration, sugar (e.g., glucose) content and / or concentration, pH, oxygen concentration, carbon dioxide concentration, temperature, and / or any other component.

[0099] The cell culture vessel and / or volume may be of any applicable size for experimental, production, and / or any other reason. For example, small scale production my occur in volumes ranging from approximately 5 mL to approximately 5 L, while commercial production can occur in volumes of approximately 1 L up to 5000 L or greater. Thus, various instances culture the cell line in volumes such as 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 50 mL, 75 mL, 100 mL, 150 mL, 200 mL, 250 mL, 500 mL, 750 mL, 1 L, 1.25 L, 1.5 L, 1.75 L, 2 L, 2.5 L, 3 L, 3.5 L, 4 L, 4.5 L, 5 L, 7.5 L, 10 L, 15 L, 20 L, 25 L, 50 L, 100 L, 150 L, 200 L, 250 L, 500 L, 750 L, 1000 L, 1500 L, 2000 L, 2500 L, 3000 L, 3500 L, 4000 L, 4500 L, 5000 L, or greater.

[0100] Culturing the cell line can proceed for a defined period of time and / or until a desired endpoint, such as a quantifiable metric. In certain instances, the quantifiable metric is viable cell density (VCD). VCD can be measured by various mechanisms, including optical (e.g., absorbance or optical density), electrochemical (e.g., impedance, capacitance, etc.), cell counting (including flow cytometry), and / or any other mechanism that can measure VCD. In some instances, the target VCD is from approximately 1 million cells per mL to approximately 50 million cells per mL, including approximately 1 million cells per mL, 5 million cells per mL, 10 million cells per mL, 15 million cells per mL, 20 million cells per mL, 25 million cells per mL, 30 million cells per mL, 40 million cells per mL and / or 50 million cells per mL, or any value in between any of the foregoing. In some instances, the target VCD is a target VCD of from approximately 5 million cells per mL to approximately 15 million cells per mL, or from approximately 5 million cells per mL to approximately 10 million cells per mL. In some embodiments, the target VCD is approximately 10 million cells per mL.

[0101] Additional embodiments inoculate second cell culture media with one or more cells from the first culture media. In some embodiments, after the inoculating step, the second cell culture media comprises between approximately 5 million cells per mL and approximately 10 million cells per mL. In some embodiments, after the inoculating step, the second cell culture media comprises approximately 5 million cells per mL. In some embodiments, after the inoculating step, the second cell culture media comprises approximately 10 million cells per mL. In certain instances, cells are harvested from the first cell culture media and transferred to the second cell culture media. In certain instances, the inoculation is an aliquot from the first cellculture media. The aliquot can be of any volume up to 100% of the volume of the first culture media. In certain instances, the volume of the aliquot is selected so the final volume of the second culture media has a VCD for increased rAAV production. This dilution via an aliquot can be calculated using the equation:represents the VCD of the first cell culture media; represents the volume of the aliquot; Cf represents the desired concentration of the second cell culture media; and Vf represents the volume of the second cell culture media.

[0102] In some embodiments, an aliquot of the first cell culture media is combined with the second cell culture media such that the volume of the first cell culture media that is added to the second culture media presents less than or less than about 30%, 25%, 20%, 15%, or 10% of the total volume of the second cell culture media following addition of the aliquot of the first cell culture media. In some embodiments, the volume of the first cell culture media that is added to the second culture media presents less than or less than about 20% of the total volume of the second cell culture media following addition of the aliquot of the first cell culture media. In some embodiments, the volume of the second cell culture media that the first cell culture media is added to is at least 3, 4, 5, 6, 7, 8, or 9-fold greater than the volume of the first cell culture media. In some embodiments, the volume of the second cell culture media that the first cell culture media is added to is at least 4-fold greater than the volume of the first cell culture media. In some embodiments, after the inoculating step, the second cell culture media comprises less than or equal to 20% of the first cell culture media.

[0103] The second cell culture media can be in any cell culture vessel or volume, such as described above (e.g., bioreactor, flask, petri dish, etc.). In some instances, the second cell culture media may be at a larger volume than the first cell culture media — for example, the first cell culture media may be a 3 L culture, while the second cell culture media may be a 5 L culture. In certain embodiments, multiple vessels with a second cell culture media are inoculated — for example, a 3 L first cell culture media could be used to inoculate multiple (e.g., 2 or more) 5 L second cell culture media. Additionally, the second cell culture media may be subject to one or more of the culturing strategies described above — e.g., batch, fed-batch, and / or perfusion.

[0104] In many embodiments, a triggering agent to induce production of rAAVs can be added to the second cell culture media. The addition of the triggering agent can occur prior to inoculatingthe second cell culture media, such that rAAV production begins immediately, while other embodiments add the triggering agent after inoculating the second cell culture media. A postinoculation addition of a triggering agent can allow cells to equilibrate and / or acclimatize to the second cell culture media.

[0105] The particular triggering agent (or agents) are specific to an inducible promoter, such as described previously. In the absence of a triggering agent, the inducible promoter is not active. For example, the inducible promoter of the second polynucleotide in FIGS. 1A-1B and the polynucleotide of FIG. 1C is a Tet-inducible promoter and the triggering agent is a tetracycline, such as doxycycline (“Dox”). In some embodiments, the particular triggering agent (or agents) are specific to an inducible recombinase, such as described previously. In the absence of a triggering agent, the inducible recombinase is not active. In some embodiments, an inducible recombinase can be a CRE recombinase fused to an estrogen receptor that requires binding of an estrogen agonist or a selective modulator, such as tamoxifen, for translocation from the cytoplasm to the nucleus. For example, the triggering agent for a CRE recombinase fused to an estrogen receptor is tamoxifen. In some embodiments, triggering agents can include one or more of tetracycline, doxycycline, or tamoxifen, including any combination thereof, such as tetracycline and tamoxifen or doxycycline and tamoxifen. In some embodiments, the triggering agents are added sequentially. In some embodiments, the triggering agents comprise doxycycline, tamoxifen, or doxycycline followed by tamoxifen. In some particular embodiments, the triggering agents are doxycycline and tamoxifen

[0106] In some embodiments, doxycycline is added in a concentration of between or between about 20 ng / ul and 300 ng / ul, 20 ng / ul and 250 ng / ul, 20 ng / ul and 200 ng / ul, 75 ng / ul and 300 ng / ul, 75 ng / ul and 250 ng / ul, 75 ng / ul and 200 ng / ul, 100 ng / ul and 300 ng / ul, 100 ng / ul and 250 ng / ul, or 100 ng / ul and 200 ng / ul. In some embodiments, doxycycline is added in concentrations of 20ng / ul, 50 ng / ul, 100 ng / ul, 125 ng / ul, 150 ng / ul, 175 ng / ul, 200 ng / ul, 225 ng / ul, 250 ng / ul, 275 ng / ul, or 300 ng / ul, or a value between any of the foregoing. In some embodiments, doxycycline is added in a concentration of or of about 20ng / ul, 50 ng / uL, 60ng / ul, 70ng / ul, 80 ng / ul, 90 ng / ul, 100 ng / ul, 110 ng / ul, 120 ng / ul, 130 ng / ul, 140 ng / ul, 150 ng / ul, 160 ng / ul, 170 ng / ul, 180 ng / ul, 190 ng / ul, or 200 ng / ul, or a value between any of the foregoing. In some embodiments, doxycycline is added in a concentration of or of about 140 ng / ul, 145 ng / ul, 150 ng / ul, 151 ng / ul, 152 ng / ul, 153 ng / ul, 154 ng / ul, 155 ng / ul, 156 ng / ul, 157 ng / ul, 158 ng / ul, 159ng / ul, 160 ng / ul, 161 ng / ul, 162 ng / ul, 163 ng / ul, 164 ng / ul, or 165 ng / ul, or a value between any of the foregoing. In some embodiments, doxycycline is added in concentration of 160 ng / ul.

[0107] In some embodiments, tamoxifen is added in a concentration of between or between about 1 uM and 20 uM, 1 uM and 15 uM, 1 uM and 10 uM, 1 uM and 8 uM, 1 uM and 5 uM, 2 uM and 20 uM, 2 uM and 15 uM, 2 uM and 10 uM, 2 uM and 8 uM, 2 uM and 5 uM, 3 uM and 20 uM, 3 uM and 15 uM, 3 uM and 10 uM, 3 uM and 8 uM, 3 uM and 5 uM. In some embodiments, tamoxifen is added in a concentration of or of about 1 uM, 2 uM, 3 uM, 4uM, 5 uM, 6 uM, 7 uM, 8 uM, 9 uM, 10 uM, 11 uM, 12 uM, 13 uM, 14 uM, 15 uM, 16 uM, 17 uM, 18 uM, 19 uM, or 20 uM, or a value between any of the foregoing. In some embodiments, tamoxifen is added in a concentration of or of about 3 uM, 3.5 uM, 3.75 uM, 4 uM, 4.25 uM, 4.5 uM, 4.75 uM, 5 uM, 5.5 uM, 6 uM, 6.5 uM, 7 uM, or 7.5 uM, or a value between any of the foregoing. In some embodiments, tamoxifen is added in a concentration of or of about 4 uM. In some embodiments, doxycycline is added in concentration of or of about 160 ng / ul and tamoxifen is added in concentration of or of about 4 uM.

[0108] After production of rAAVs, additional embodiments can comprise harvest and / or collection of rAAVs from the culture. Harvesting and collecting methods are generally known in the art. Harvesting can include various methods to induce cellular lysis, such as enzymatic lysis, thermal shock, adding a hypotonic solution, and / or any other applicable means to lyse a cell. Collecting can involve collecting the rAAV particles from the solution or culture. Such methods can include affinity purification, size selection, centrifugation (e.g., gradient centrifugation), and / or any other appropriate method for AAV capture.Systems for rAAV Production with Biphasic Culture

[0109] Further embodiments are directed to systems that assist with biphasic culturing. FIG. 2A illustrates an exemplary biphasic culturing system 200. Many embodiments include a first bioreactor 202 and a second bioreactor 204 in fluid communication. In the illustrated example, the first bioreactor 202 is intended for cell growth and / or division, while the second bioreactor 204 is intended for rAAV production; however, both bioreactors may be configured for cell cultivation, including one or more components described herein. As described above, the bioreactors 202, 204 can be of any volume. In certain embodiments, bioreactors 202, 204 are the same size; in other embodiments, the first bioreactor 202 is smaller than the second bioreactor 204; in additional embodiments, second bioreactor 204 is smaller than first bioreactor 202. Some embodimentsinclude multiple bioreactors intended for rAAV production (e.g., second bioreactor), such cells from the first bioreactor 202 may transferred to each rAAV production bioreactor. While Figure 2 illustrates a first bioreactor 202 and a second bioreactor 204, in some instances, the first bioreactor 202 and second bioreactor 204 may be a single bioreactor used for both cell growth and / or division and rAAV production.

[0110] As described herein, in some embodiments, the first bioreactor 202 and the second bioreactor 204 are in fluid communication. Such fluid communication can be provided via a conduit 206, such as a tube, pipe, and / or other mechanism to allow fluid transfer between the first bioreactor 202 and the second bioreactor 204. Such transfer can be unidirectional or bidirectional between the first bioreactor 202 and the second bioreactor 204. Additional embodiments can include a transfer mechanism 208. Such transfer mechanisms can allow for active transfer (e.g., a pump) or can allow for passive transfer (e.g., a valve to allow gravity transfer. Transfer mechanism 208 can be integrated with conduit 206 and / or replace conduit 206. For example, a transfer mechanism 208 may be the only conduit between bioreactors, a transfer mechanism may be connected to conduit 206 on one or both sides of the transfer mechanism 208. When using a pump, a pump can include one or more of: a centrifugal pump, a positive displacement pump, a peristaltic pump, a diaphragm pump, a reciprocating pump, a gear pump, an axial piston pump, a jet pump, a screw pump, a vane pump, and / or any other applicable pumping mechanism.

[0111] Additional embodiments include a sensor 210 to measure one or more components within the first bioreactor. In some instances, the sensor 210 measures one or more of: VCD, pH, capacitance, inductance, temperature, ion concentration, ion content, oxygen concentration, carbon dioxide concentration, glucose concentration, and / or any other component. It should be noted that some embodiments measure multiple components with a single sensor, while some embodiments use multiple sensors to measure any component of interest. In some preferred embodiments the sensor 210 measures VCD.

[0112] Further instances include a controller 212 (or microcontroller) in electronic communication with one or more of the sensor 210 and transfer mechanism 208. Such controllers may include a processor or logic switch that can perform an action in response to a metric from the sensor 210. In certain embodiments, the action is performed in response to a threshold condition or target amount. For example, once the first bioreactor 202 reaches a target VCD (as measured by sensor 210), controller 212 can activate the transfer mechanism 208 to transfer analiquot from the first bioreactor 202 to the second bioreactor 204. In other instances, 212 may provide an alert or alarm in response to a target condition. Controller 212 can further include an input device, output device, memory, log, display, and / or any other system to allow for monitoring, programming, troubleshooting, and / or otherwise providing commands to controller 212 and / or system 200.

[0113] Many embodiments can be automated using a controller 212. Such automation can follow a flow chart 250, such as illustrated in FIG. 2B. The flow chart 250 is divided into three stages representing cell growth (or N-l) phase 252, a dilution / induction phase 254, and a production (or N) phase 256. FIG. 2C graphically illustrates these phases as a function of VCD versus time. During the growth phase 252, VCD increases until reaching the desired, or target, amount, in growth media. Sensor 210 can be used to determine the VCD amount, such as through periodic measurements or constant monitoring. Once the target VCD is reached, the controller 212 can turn on a production media pump, where the production media is a media that improves rAAV production.

[0114] During the dilution / induction phase 254, sensor 210 can monitor VCD until a target dilution VCD is reached. This target dilution VCD can be a VCD that increases rAAV production or titer. Once the target dilution VCD is reached, the controller 212 can turn off a production media pump and turn on induction reagent pump(s), where the induction reagents can include triggering agents to induce rAAV production from cells (e.g., stable cells). In some embodiments, the triggering agents can be selected from one or both of tamoxifen and doxycycline. Once the induction reagents are dispensed, the induction reagent pump(s) can be turned off.

[0115] With the addition of induction reagents, production phase 256 begins, and the process can continue across desirable set points for rAAV production, such as a particular VCD, a change in trend of the VCD, flattening of VCD and / or any other characteristic.

[0116] This type of system allows for removing manual timing of the dilution step and to hit a target viable cell density for the induction step.

[0117] System 200 also illustrates components that can be used with one or both of fed-batch and perfusion type culture strategies. In particular, some embodiments can include a media reservoir 214 in fluid communication with first bioreactor 202. Additionally, a perfusion system 216 to assist in the removal of waste products can also be in fluid communication with first bioreactor 202. A perfusion system can allow for cyclical flow, such that a portion of media can bemoved into the perfusion system, where a waste portion is sent to waste, while non-waste (e.g., cells) are returned or recirculated to the bioreactor. Such perfusion systems can include a filter media, cell retention device, pump, and / or any other component to assist its performance. In certain instances, filter media acts as a cell retention device. For example, filter media can include one or more size exclusion media, such that small moieties (e.g., waste metabolites may pass through the filter (e.g., to waste) while cells are directed toward the bioreactor. Alternatively or additionally, filter media can include affinity media that shows specificity to certain components to either capture or enrich for these components. Additionally, in certain embodiments using a perfusion system 216, reservoir 214 may be in fluid connection with the perfusion system instead of (or in addition to) the bioreactor. A controller (e.g., controller 212) can control or activate perfusion system 216. While not illustrated, second bioreactor 204 may include a reservoir 214 and / or perfusion system 216. In embodiments using a single bioreactor, once the threshold condition is reached (e.g., target VCD), a perfusion system 216 or other cell capture system can be used to collect cells and add a second cell culture media to the bioreactor. SPECTS OF THE INVENTION

[0118] The below items disclose various aspects of the invention. Each of the aspects described below can be combined with other aspects and embodiments disclosed elsewhere herein, including the claims, where the combinations are clearly compatible.

[0119] Certain aspects include:Aspect 1. A method for producing recombinant adeno-associated virus (rAAV), the method comprising: culturing a cell line, configured to inducibly produce rAAV, in a first cell culture media; inoculating a second cell culture media with an aliquot from the first cell culture media comprising the cell line; adding one or more triggering agents to the second cell culture media to induce production of rAAV from the cell line; and culturing the cell line in the second cell culture media.Aspect 2. The method of Aspect 1 , wherein the adding step occurs prior to the inoculating step or simultaneously with the inoculating step.Aspect 3. The method of Aspect 1, wherein the adding step occurs after the inoculating step.Aspect 4. The method of Aspect 1 or 3, wherein after the inoculating step, the second cell culture media comprises approximately 5 million cells per mL.Aspect 5. The method of any one of Aspects 1-4, wherein culturing the cell line comprises periodically adding additional media to the culture.Aspect 6. The method of any one of Aspects 1-5, wherein culturing the cell line comprises removing a waste metabolite from the culture.Aspect 7. The method of any one of Aspects 1-6, wherein culturing in first cell culture media occurs until the cell line reaches a viable cell density of (VCD) of from 1 million cells per mLto 30 million cells per mL.Aspect 8. The method of any one of Aspects 1-7, wherein culturing in first cell culture media occurs until the cell line reaches a viable cell density of (VCD) of approximately 5 million cells per mL.Aspect 9. The method of any one of Aspects 1-7, wherein culturing in first cell culture media occurs until the cell line reaches a viable cell density of (VCD) of approximately 15 million cells per mL.Aspect 10. The method of any one of Aspects 1-9, wherein the culturing step occurs in a vessel of from approximately 100 mL to approximately 10 L.Aspect 11. The method of any one of Aspects 1-10, wherein the culturing has a volume of approximately 5 mL to approximately 9500 mL.Aspect 12. The method of any one of Aspects 1-11, wherein the first cell culture media is configured to support cell division.Aspect 13. The method of any one of Aspects 1-12, wherein the second culture media is in a vessel of from approximately 1 L to approximately 2000 L.Aspect 14. The method of any one of Aspects 1-13, wherein the second culture media has a volume of approximately 500 mL to approximately 1800 L.Aspect 15. The method of any one of Aspects 1-14, wherein the second cell culture media is configured to support r AAV production.Aspect 16. The method of any one of Aspects 1-15, wherein the triggering agents comprise doxycycline and tamoxifen.Aspect 17. The method of anyone of Aspects 1-16, wherein the cell line comprises a plurality of cells, each cell comprising: a first polynucleotide comprising a sequence encoding AAV Rep and Cap proteins; a second polynucleotide comprising a sequence encoding one or more AAV helper proteins; and a third polynucleotide comprising a sequence encoding a payload flanked by AAV inverted terminal repeats (ITRs).Aspect 18. A system for biphasic cell culture, the system comprising: a first bioreactor and a second bioreactor each configured to culture cells and in fluid communication with each other; a transfer apparatus configured to move a portion of a cell culture between the first bioreactor and the second bioreactor; a sensor located in the first bioreactor configured to measure one or more components within a cell culture in the first bioreactor; and a control system in electronic communication with the sensor and configured to initiate a transfer between the first bioreactor and the second bioreactor upon a reading from the sensor indicating a threshold condition.Aspect 19. The system of Aspect 18, wherein the sensor measures viable cell density (VCD) within the first bioreactor.Aspect 20. The system of Aspect 19, wherein viable cell density of (VCD) is measured by one or both of capacitance and optical cell density.Aspect 21. The system of Aspect 19 or 20, wherein the threshold condition is a viable cell density of (VCD) for rAAV production.Aspect 22. The system of any one of Aspects 19-21, wherein the threshold condition is a viable cell density of (VCD) of approximately 15 million cells per mL.Aspect 23. The system of any one of Aspects 18-22, wherein the sensor measures one or more of pH, temperature, ion concentration, ion content, oxygen concentration, carbon dioxide concentration, and glucose concentration.Aspect 24. The system of any one of Aspects 18-23, further comprising a media reservoir in fluid communication with the first bioreactor, wherein the media reservoir is configured to periodically deliver fresh media to the first bioreactor.Aspect 25. The system of any one of Aspects 18-24, further comprising a perfusion system in fluid communication with the first bioreactor and configured to remove cellular waste products from the first bioreactor.Aspect 26. The system of Aspect 25, wherein the perfusion system comprises a cell retention device to recirculate cells into the first bioreactor.Aspect 27. The system of any one of Aspects 18-26, wherein the second bioreactor is larger than the first bioreactor.Aspect 28. The system of any one of Aspects 18-27, wherein the first bioreactor has a size of approximately 100 mL to approximately 10 L.Aspect 29. The system of any one of Aspects 18-28, wherein the second bioreactor has a size of approximately 1 L to approximately 2000 L.Aspect 30. The system of any one of Aspect 18-29, further comprising a second sensor located in the second bioreactor configured to measure one or more components within a cell culture in the second bioreactor.Aspect 31. The system of Aspect 30, wherein the control system is in electronic communication with the second sensor and configured to initiate an addition of a triggering agent to the second bioreactor upon a reading from the second sensor indicating a threshold condition.Aspect 32. The system of Aspect 30 or 31 , wherein the second sensor measures viable cell density of (VCD) within the second bioreactor.Aspect 33. The system of Aspect 31 or 32, wherein the threshold condition is a viable cell density of (VCD) for rAAV production.Aspect 34. The system of any one of Aspects 31-33, wherein the threshold condition is a viable cell density of (VCD) of approximately 5 million cells per mL.Aspect 35. The system of any one of Aspects 18-34, further comprising a third bioreactor in fluid communication with the first bioreactor and configured to culture cells, and wherein the control system is configured to initiate a transfer between the first bioreactor and the third bioreactor upon a reading from the sensor indicating a threshold condition.Aspect 36. The system of Aspect 35, wherein the third bioreactor is larger than the first bioreactor.Aspect 37. The system of Aspect 35 or 36, wherein the first bioreactor has a size of approximately 100 mL to approximately 10 L.Aspect 38. The system of any one of Aspects 35-37, wherein the third bioreactor has a size of approximately 1 L to approximately 2000 L.Aspect 39. The system of any one of Aspect 18-38, further comprising a third sensor located in the third bioreactor configured to measure one or more components within a cell culture in the third bioreactor.Aspect 40. The system of Aspect 39, wherein the control system is in electronic communication with the third sensor and configured to initiate an addition of a triggering agent to the third bioreactor upon a reading from the third sensor indicating a threshold condition.Aspect 41. The system of Aspect 39 or 40, wherein the third sensor measures viable cell density of (VCD) within the third bioreactor.Aspect 42. The system of Aspect 40 or 41, wherein the threshold condition is a viable cell density of (VCD) for rAAV production.Aspect 43. The system of any one of Aspects 40-42, wherein the threshold condition is a viable cell density of (VCD) of approximately 15 million cells per mL.Aspect 44. The system of any one of Aspects 18-34, wherein the fluid communication between the first and second bioreactors comprises a pump.Aspect 45. The system of Aspect 44, wherein the pump is selected from one or more of: a centrifugal pump, a positive displacement pump, a peristaltic pump, a diaphragm pump, areciprocating pump, a gear pump, an axial piston pump, a jet pump, a screw pump, and a vane pump.Aspect 46. The system of any one of Aspects 18-26 and 35, wherein the first bioreactor and the second bioreactor are a single bioreactor or wherein the first bioreactor, the second bioreactor, and the third bioreactor are a single bioreactor.

[0120] Certain aspects also include:Aspect 1. A method for producing recombinant adeno-associated virus (rAAV), the method comprising: culturing a stable cell line, capable of inducibly producing rAAV, in a first cell culture media capable of supporting cell division; inoculating a second cell culture media, capable of supporting rAAV production, with an aliquot from the first cell culture media comprising the stable cell line; adding one or more triggering agents to the second cell culture media to induce production of rAAV from the stable cell line; and culturing the stable cell line in the second cell culture media.Aspect 2. The method of aspect 1 , wherein culturing the stable cell line in the first cell culture media results in a peak cell density greater than 5 million cells / mL.Aspect 3. The method of aspect 1 or aspect 2, wherein culturing the stable cell line in the second cell culture media results in a peak titer level greater than 5.0 x 109vg / mL.Aspect 4. The method of any of aspects 1-3, wherein the stable cell line comprises a sequence comprising an inducible promoter operably linked to a sequence encoding a recombinase.Aspect 5. The method of aspect 4, wherein the inducible promoter is a tetracycline inducible promoter.Aspect 6. The method of aspect 4 or aspect 5, wherein the one or more triggering agents comprises a first triggering agent capable of activating the inducible promoter.Aspect 7. The method of aspect 6, wherein the first triggering agent is doxycycline.Aspect 8. The method of any of aspects 4-7, wherein the recombinase is an inducible recombinase.Aspect 9. The method of aspect 8, wherein the inducible recombinase is a recombinase fused to an estrogen receptor.Aspect 10. The method of aspect 8 or aspect 9, wherein the one or more triggering agents comprises a second triggering agent capable of activating the inducible recombinase.Aspect 11. The method of aspect 10, wherein the second triggering agent is an estrogen agonist or selective estrogen receptor modulator.Aspect 12. The method of aspect 10 or aspect 11, wherein the second triggering agent is tamoxifen.Aspect 13. The method of any one of aspects 1-12, wherein the adding step occurs prior to the inoculating step or simultaneously with the inoculating step.Aspect 14. The method of any one of aspects 1-12, wherein the adding step occurs after the inoculating step.Aspect 15. The method of any one of aspects 1-12 or 14, wherein after the inoculating step, the second cell culture media comprises between approximately 5 million cells per mL and approximately 10 million cells per mL, or comprises approximately 5 million cells per mL.Aspect 16. The method of any one of aspects 1-12 or 14, wherein after the inoculating step, the second cell culture media comprises approximately 10 million cells per mL.Aspect 17. The method of any one of aspects 1-16, wherein culturing the stable cell line in the first and / or second cell culture media comprises periodically adding additional media to the culture.Aspect 18. The method of any one of aspects 1-17, wherein culturing the stable cell line in the first and / or second cell culture media comprises removing a waste metabolite from the culture.Aspect 19. The method of any one of aspects 1-18, wherein culturing the stable cell line in the first and / or second cell culture media occurs in a bioreactor with perfusion.Aspect 20. The method of aspect 19, wherein perfusion is used to exchange the first cell culture media with the second cell culture media.Aspect 21. The method of any one of aspects 1-20, wherein culturing in the first cell culture media occurs until the stable cell line reaches a viable cell density of (VCD) of between approximately 1 million cells per mL and approximately 50 million cells per mL.Aspect 22. The method of any one of aspects 1-21, wherein culturing in the first cell culture media occurs until the stable cell line reaches a viable cell density of (VCD) of between approximately 5 million cells per mL and approximately 10 million cells per mL.Aspect 23. The method of any one of aspects 1-21, wherein culturing in the first cell culture media occurs until the stable cell line reaches a viable cell density of (VCD) of approximately 5 million cells per mL.Aspect 24. The method of any one of aspects 1-21, wherein culturing in first cell culture media occurs until the stable cell line reaches a viable cell density of (VCD) of approximately 10 million cells per mL.Aspect 25. The method of any one of aspects 1-21, wherein culturing in first cell culture media occurs until the stable cell line reaches a viable cell density of (VCD) of between approximately 10 million cells per mL and approximately 50 million cells per mL.Aspect 26. The method of any one of aspects 1-21, wherein culturing in first cell culture media occurs until the stable cell line reaches a viable cell density of (VCD) of approximately 15 million cells per mL, 30 million cells per mL, or 50 million cells per mL.Aspect 27. The method of any one of aspects 1-26, wherein culturing in the first cell culture media occurs in a vessel of from approximately 100 mL to approximately 10 L.Aspect 28. The method of any one of aspects 1-27, wherein the first cell culture media has a volume of approximately 5 mL to approximately 9500 mL.Aspect 29. The method of any one of aspects 1-28, wherein culturing in the second culture media occurs in a vessel of from approximately 1 L to approximately 2000 L.Aspect 30. The method of any one of aspects 1-29, wherein the second culture media has a volume of approximately 500 mL to approximately 1800 L.Aspect 31. The method of any one of aspects 1 -30, wherein after the inoculating step, the second cell culture media comprises less than or equal to 20% of the first cell culture media.Aspect 32. The method of any one of aspects 1-31, wherein the triggering agents comprise doxycycline and tamoxifen.Aspect 33. The method of any one of aspects 4-32, wherein the stable cell line comprises a plurality of cells, each cell comprising: a first polynucleotide comprising a sequence encoding AAV Rep and AAV Cap proteins; a second polynucleotide comprising a sequence encoding one or more AAV helper proteins and the sequence comprising the inducible promoter operably linked to the sequence encoding recombinase; and a third polynucleotide comprising a sequence encoding a payload flanked by AAV inverted terminal repeats (ITRs) wherein expression of the AAV Rep, AAV Cap and the one or more AAV helper proteins is induced by the one or more triggering agents.Aspect 34. The method of any one of aspects 4-33, wherein the inducible promoter comprises tetracycline-response promoter elements (TREs).Aspect 35. The method of any one of aspects 4-34, wherein the transcription of the recombinase is induced in the presence of the first triggering agent.Aspect 36. The method of any one of aspects 8-35, wherein the inducible recombinase translocates to the cell nucleus in the presence of the second triggering agent.Aspect 37. A system for biphasic cell culture, the system comprising: a first bioreactor and a second bioreactor each configured to culture cells and in fluid communication with each other; a transfer apparatus configured to move a portion of a cell culture from the first bioreactor to the second bioreactor; a sensor located in the first bioreactor configured to measure one or more components within a cell culture in the first bioreactor; and a control system in electronic communication with the sensor and configured to initiate a transfer between the first bioreactor and the second bioreactor upon a reading from the sensor indicating a threshold condition;wherein the biphasic cell culture comprises a growth phase that occurs in the first bioreactor and a rAAV production phase that occurs in the in the second bioreactor.Aspect 38. The system of aspect 37, wherein the sensor measures viable cell density (VCD) within the first bioreactor.Aspect 39. The system of aspect 38, wherein VCD is measured by one or both of capacitance and optical cell density.Aspect 40. The system of aspect 38 or 39, wherein the threshold condition is a VCD greater than or equal to a target VCD that maximizes rAAV production.Aspect 41. The system of aspect 40, wherein the target VCD is approximately between 5 million cells / mL and approximately 10 million cells / mL.Aspect 42. The system of aspect 40, wherein the target VCD is approximately 10 million cells / mL.Aspect 43. The system of any one of aspects 38-40, wherein the threshold condition is a VCD of between approximately 5 million cells per mL and approximately 50 million cells per mL, or approximately 5, 10, 15, 20, 30, 40, or 50 million cells per mL.Aspect 44. The system of any one of aspects 37-43, wherein the sensor measures one or more of pH, temperature, ion concentration, ion content, oxygen concentration, carbon dioxide concentration, and glucose concentration.Aspect 45. The system of any one of aspects 37-44, further comprising a media reservoir in fluid communication with the first bioreactor, wherein the media reservoir is configured to periodically deliver fresh media to the first bioreactor.Aspect 46. The system of any one of aspects 37-45, further comprising a perfusion system in fluid communication with the first bioreactor and configured to remove cellular waste products from the first bioreactor.Aspect 47. The system of aspect 46, wherein the perfusion system comprises a cell retention device to recirculate cells into the first bioreactor.Aspect 48. The system of any one of aspects 37-47, wherein the second bioreactor is larger than the first bioreactor.Aspect 49. The system of any one of aspects 37-48, wherein the first bioreactor has a size of approximately 100 mL to approximately 10 L.Aspect 50. The system of any one of aspects 37-49, wherein the second bioreactor has a size of approximately 1 L to approximately 2000 L.Aspect 51. The system of any one of aspect 37-50, further comprising a second sensor located in the second bioreactor configured to measure one or more components within a cell culture in the second bioreactor.Aspect 52. The system of aspect 51 , wherein the control system is in electronic communication with the second sensor and configured to initiate an addition of a triggering agent to the second bioreactor to induce rAAV production upon a reading from the second sensor indicating a threshold condition.Aspect 53. The system of aspect 51 or aspect 52, wherein the second sensor measures viable cell density (VCD) within the second bioreactor.Aspect 54. The system of aspect 52 or aspect 53, wherein the threshold condition is a target VCD that maximizes rAAV production.Aspect 55. The system of any one of aspects 52-54, wherein the threshold condition is a target VCD of between approximately 5 million cells per mL and approximately 10 million cells per mL.Aspect 56. The system of any one of aspects 52-54, wherein the threshold condition is a target VCD of approximately 10 million cells per mL.Aspect 57. The system of any one of aspects 37-56, further comprising a third bioreactor in fluid communication with the first bioreactor and configured to culture cells, and wherein the control system is configured to initiate a transfer between the first bioreactor and the third bioreactor upon a reading from the sensor located in the first bioreactor indicating a threshold condition.Aspect 58. The system of aspect 57, wherein the third bioreactor is larger than the first bioreactor.Aspect 59. The system of aspect 57 or aspect 58, wherein the first bioreactor has a size of approximately 100 mL to approximately 10 L.Aspect 60. The system of any one of aspects 57-59, wherein the third bioreactor has a size of approximately 1 L to approximately 2000 L.Aspect 61. The system of any one of aspects 37-60, further comprising a third sensor located in the third bioreactor configured to measure one or more components within a cell culture in the third bioreactor.Aspect 62. The system of aspect 61 , wherein the control system is in electronic communication with the third sensor and configured to initiate an addition of a triggering agent to the third bioreactor to induce rAAV production upon a reading from the third sensor indicating a threshold condition.Aspect 63. The system of aspect 61 or aspect 62, wherein the third sensor measures viable cell density (VCD) within the third bioreactor.Aspect 64. The system of aspect 62 or aspect 63, wherein the threshold condition is greater than or equal to a target VCD that maximizes rAAV production.Aspect 65. The system of aspect 64, wherein the target VCD is between approximately 5 million cells / mL and approximately 10 million cells / mL.Aspect 66. The system of aspect 64, wherein the target VCD is approximately 10 million cells / mL.Aspect 67. The system of any one of aspects 62-64, wherein the threshold condition is a viable cell density (VCD) of between approximately 5 million cells per mL and approximately 50 million cells per mL, or approximately 5, 10, 15, 20, 30, 40, or 50 million cells per mL.Aspect 68. The system of any one of aspects 37-56, wherein the fluid communication between the first and second bioreactors comprises a pump.Aspect 69. The system of aspect 68, wherein the pump is selected from one or more of: a centrifugal pump, a positive displacement pump, a peristaltic pump, a diaphragm pump, a reciprocating pump, a gear pump, an axial piston pump, a jet pump, a screw pump, and a vane pump.Aspect 70. The system of any one of aspects 37-47 and 57, wherein the first bioreactor and the second bioreactor are a single bioreactor or wherein the first bioreactor, the second bioreactor, and the third bioreactor are a single bioreactor.EXAMPLES

[0121] The following examples are offered by way of illustration and not by way of limitation.Example 1: Identifying VCD for rAAV Production

[0122] A stable cell line for inducible production of rAAV was cultured to various VCDs (measured as million cells per mL) prior to induction for rAAV production. After induction, the titer level was measured for each sample, where titer was measured as number of viral genomes (vg) per mL (i.e., vg / mL). FIG. 3 illustrates the data and a trendline illustrating a cell density effect, where rAAV production increases from approximately 1 million cells per mL to approximately 5 million cells per mL, then the rAAV production decreases and levels off at approximately 15 million cells per mL. This data indicates that a VCD of approximately 5 million cells per mL provides for the best rAAV production.Example 2: Cellular Growth and rAAV Production have Different Nutritional Needs

[0123] A stable cell line for inducible production of rAAV was grown in two different culture media (Media I and Media II), where the peak cell density was measured. Additionally, the stable cell line was further induced for rAAV production at 5 million cells per mL in each media. FIG. 4A illustrates how the cells cultured in Media I reached a peak cell density of approximately 5 million cells per mL, while the cells cultured in Media II reached a peak of approximately 15 million cells per mL. FIG. 4B illustrates how cells induced in Media I produced a titer level (vg / mL) of approximately 1.0 x IO10vg / mL, while cells induced in Media II produced a titer level of less than 5.0 x IO9vg / mL. FIGs. 4A-4B indicate that the nutritional needs of growth and production phases differ, and overall production can be altered with a biphasic strategy of using a first media for growth and a second media for production.Example 3: Culturing Strategies

[0124] A stable cell line for inducible production of rAAV was cultured in a growth media according to different feeding culturing strategies, including batch, fed-batch, and mock perfusion. As described above, batch is the culturing of a cell in media. Fed-batch involves adding additional volumes of media to the culture to supplement the nutritional needs. Finally, mock perfusion involves harvesting cells via centrifugation and resuspension in media to simulate a perfusion system to remove waste products and supplement the culture with fresh media. FIG. 5A illustrates that fed-batch and mock perfusion produced and maintained a higher VCD than the batch method.

[0125] To assess scalability, a 250 mL shaker flask and a 10L bioreactor were used to culture cells via a fed-batch strategy. These fed-batch scales were compared to a control strategy (batchmedia, 250 mL shaker flask). Cells were induced at a cell density of 5 million cells / mL with triggering agents, doxycycline and tamoxifen, to produce rAAV. The resulting titer (vg / mL) was measured from each culture. FIG. 5B provides a table of results from culturing and production, which show that the fed-batch strategies increase VCD maximum and retain high titer levels.Example 4: Selecting Optimal Media for Production

[0126] Media for production (N phase) was optimized by comparing twenty-one different media recipes to a standard “control” media. FIGs. 6A-6B illustrate bar graphs showing titer levels after induction in the various media. Stable cells in media M21 produced increased vg / mL (FIG. 6A) and vp / mL (FIG. 6B) over the control. This media resulted in cell specific productivity increase by 30% and improved packaging efficiency by 1.7 fold.

[0127] Using an improved media can further increase volumetric productivity. Cells were induced with triggering agents, doxycycline and tamoxifen, at various VCDs (from 2 million cells / mL up to 25 million cells / mL) in either a control media or M21 media. The resultant titers were measured. FIG. 6C illustrates a graph showing titer levels (vg / mL) produced in control or M21 media as a function of VCD at induction. Stable cells in the M21 media produced a maximum titer of approximately 8 x 1011vg / mL when inducing cells at 10 million cells / mL, while stable cells in the control media produced a maximum titer of approximately 3 x 1011vg / mL when inducing cells at 5 million cells / mL. This improvement allows for increased volumetric productivity when using a media that improves production.Example 5: Production Consistency at Different Volumes

[0128] To assess scalability, a 250 mL shaker flask versus a 10L bioreactor were used to culture cells. Cell lines were cultured in a 250 mL shaker flask or a 10L bioreactor. When the cell cultures reached a density of 5 million cells per mL, they were induced with doxycycline and tamoxifen as triggering agents. Viable Cell Density (VCD) and viability were measured periodically and titer was measured at harvest, using methods described herein. FIG. 7A illustrates a plot of the VCD versus production day, while FIG. 7B illustrates a plot of cell viability versus production day. FIG. 7C provides a table of titer measured from the 250mL shaker flask and 10L bioreactor, where both production scales show similar titer levels. FIGs. 7A-7C show that the small scale (250 mL shaker flask) and 10L bioreactor possess similar profiles for VCD, viability, and titer, indicating scalability.Example 6: Biphasic Culture, Harvest, and Capture

[0129] A stable cell line for inducible production of rAAV was cultured in a growth media (i.e., a media configured to support cell division) in a shaker flask using a fed-batch strategy. The cells included a payload construct that included a self-complementary progranulin gene (scPGRN). When the cells reached a VCD of approximately 15 million cells / mL, a 10 L bioreactor with production media (e.g., a media configured to support rAAV production) was inoculated with the cells at approximately 5 million cells / mL. rAAV production was induced by adding doxycycline and tamoxifen. The cells underwent harvest and capture steps to collect rAAV particles.

[0130] FIG. 8 provides a table summarizing the volume, titer, and percent step yield. As illustrated, the production step a volume of 7.9 L and produced a titer of 5.9 x 1014vg / L, which equates to approximately 4.7 x 1015vg in total. The harvest step occurred at a volume of 9.5 L and produced a titer of 3.6 x 1014vg / mL, equating to 3.4 x 1015vg in total and a 74% step yield. Capture occurred using a 3 L aliquot volume from the solution after harvest, in which the harvest was split in half to give 2.44 x 1015vg that was used for subsequent calculations. The capture produced a titer of 5.2 x 1014vg / mL and 1.6 x 1015vg in total for a 64% step yield.Example 7: Measurement of Cell Density Using Capacitance

[0131] Traditionally, cell density is measured optically. However, optical measurements utilize cuvettes or other optical sensors that take cells offline or remove cells from a culture. An in situ probe or methodology can allow for more efficient measurements, by using constant and / or real time measurement. FIG. 9 illustrates a plot of capacitance (pF / cm) versus optical measurement (cells / mL). This figure shows that capacitance has a linear relationship with optical measurement with an R2of 0.94, indicating a strong correlation, such that capacitance can be used as a constant measurement method for VCD.Example 8: Automated versus Manually Produced Titers

[0132] Automated production of rAAV was compared to an average of manually produced rAAV. For the automated production, a pool from a stable cell line was cultured in a continuous stir tank reactor (CSTR) bioreactor, using a capacitance probe to monitor cell growth. A computing system running SCADA software was used to control peristaltic pumps for media and induction reagents. The CSTR was inoculated with the cells from a shaker flask at half the maximum working volume, creating the N-l expansion reactor. The VCD was monitored using thecapacitance probe. Once the target viable cell density was reached, the SCADA triggered the peristaltic pumps to dilute the cells with production media and activated pumps with induction reagents.

[0133] After production, the titer levels and percent of capsids containing viral genomes were measured by droplet digital PCR and AEX HPLC, respectively. These results were compared to a manual production method, and the results are displayed in the following table. As can be seen in this table, the titer and % full for both automated and manual production are similar and within one standard deviation (std dev = 5.7 x 1013vg / mL).Example 9: Cell lines for inducible production of rAAV

[0134] Stable mammalian cell lines capable of inducible expression of rAAV encapsidating a payload were constructed by integrating three nucleic acid constructs into the nuclear genome of a cell line that expresses adenovirus E1A and E1B (e.g., HEK 293 cells). The first construct (the Rep / Cap construct) encoded AAV Rep and Cap proteins. The second construct (the helper construct) encoded one or more AAV helper proteins and an inducible recombinase operably linked to an inducible promoter. The third construct (the payload construct) encoded a payload flanked by AAV inverted terminal repeats (ITRs). The first and second constructs included excisable elements that prevented the expression of the AAV Rep, Cap and helper proteins in the absence of triggering agents that activate the inducible promoter and inducible recombinase.

[0135] To induce rAAV production, the transcription and nuclear localization of the recombinase were induced by the addition of a first triggering agent and second triggering agent. In this example, the expression of the recombinase was driven by a tetracycline inducible promoter and the localization of the recombinase was regulated by fusion of the recombinase to an estrogen receptor (ER2). In the presence of a first triggering agent (e.g, doxycycline), the tetracycline inducible promoter was activated resulting in transcription of the recombinase. In the presence of the second triggering agent (e.g. tamoxifen), the recombinase translocated to the nucleus of a cell comprising the constructs. The expression and nuclear localization of the recombinase resulted inthe excision of the excisable elements in constructs 1 and 2 and expression of the Rep, Cap, and helper proteins.

[0136] FIG. 10A provides a schematic of constructs of the vl.O system for inducibly producing rAAV. Constructs of the vl.O system were used to produce stably transfected cells referred to as vl.O cells. In the vl.O system, the Rep / Cap construct was designed to permit expression of AAV Rep and Cap proteins from their endogenous promoters after induction. Construct 1 shown in FIG. 10A is pre-induction of the integrated nucleic acid Rep / Cap construct. An intervening spacer interrupts the Rep coding sequence. The intervening spacer comprises a first spacer segment, a second spacer segment which is excisable (BFP flanked by Lox sites), and a third spacer segment. The transcript contains a single intron flanked by 5’ and 3’ splice sites. An exemplary polynucleotide sequence for the vl.O Rep / Cap construct is set forth in SEQ ID NO: 1. An exemplary polynucleotide sequence for the helper construct is set forth in SEQ ID NO: 2. An exemplary polynucleotide sequence for the payload construct, where the gene of interest (GOI) is a sequence encoding progranulin, is set forth in SEQ ID NO: 3. FIG. 10B depicts the posttriggered state of the Rep / Cap construct (Construct 1), helper construct (Construct 2), and payload construct (Construct 3) shown in FIG. 10A following the addition of the first triggering agent, doxycycline, and the second triggering agent, tamoxifen.

[0137] FIG. 11A provides a schematic of constructs of the vl.2 system for inducibly producing rAAV. Constructs of the vl.2 system were used to produce stably transfected cells referred to as vl.2 cells. In addition to the Rep / Cap (Construct 1), helper (Construct 3) and payload (Construct 4) constructs of the vl.O system, the vl.2 system includes an additional construct (Construct 2) encoding Cap proteins under control of a Tet-inducible promoter. An exemplary polynucleotide sequence for the Tet-inducible Cap construct is set forth in SEQ ID NO: 4. FIG. 11B depicts the post-triggered state of Constructs 1-4 shown in FIG. 11A following the addition of the first triggering agent, doxycycline, and the second triggering agent, tamoxifen.

[0138] FIG. 12A provides a schematic of constructs of the vl.3 system for inducibly producing rAAV. Constructs of the vl.3 system were used to produce stably transfected cells referred to as vl.3 cells. In the vl.3 system, the Rep / Cap construct (Construct 1) is modified to: a) include a transcriptional blocking element (TBE) between the divergently oriented promoters operably linked to the Rep and Cap coding sequences, b) have the Cap coding sequence operably linked to an inducible promoter and include a SV40 poly A signal sequence, and c) have the Repcoding sequence include a bGH polyA signal sequence followed by a downstream enhancer sequence. An exemplary polynucleotide sequence for the vl.3 Rep / Cap construct is set forth in SEQ ID NO: 5. FIG. 12B depicts the post-triggered state of Constructs 1-3 shown in FIG. 12A following the addition of the first triggering agent, doxycycline, and the second triggering agent, tamoxifen.

[0139] FIG. 13 provides a schematic of constructs of the vl.4 system for inducibly producing rAAV. Constructs of the vl.4 system were used to produce stably transfected cells referred to as vl.4 cells. In the vl.4 system, the native p5 promoter for the large Rep coding sequence is replaced with a first heterologous promoter (pl), the TATA box of the native pl9 promoter for the small Rep coding sequence is mutated, and a second heterologous promoter (p2) is operably linked to the small Rep coding sequence. In this example, pl is a ubiquitin C (UBC) promoter and p2 is a CMV enhancer / chicken beta actin (CAG) promoter. An exemplary polynucleotide sequence for the vl.4 Rep / Cap construct is set forth in SEQ ID NO: 6. FIG. 13B depicts the post-triggered state of Constructs 1 -3 shown in FIG. 13A following the addition of the first triggering agent, doxycycline, and the second triggering agent, tamoxifen.Example 10: Cell growth and AAV titer production kinetics for vl.3 and vl.4 cells

[0140] To determine the time for harvesting cells to collect rAAV particles, a pool of vl.3 cells and a pool of vl.4 cells were induced with doxycycline and tamoxifen and assessed for cell viability percent, viable cell density (VCD) and titer level at multiple time points post-induction. FIG. 14A provides cell viability percent and viable cell density (VCD) for vl.3 and vl.4 cell pools over the course of seven days post-induction. For both the vl.3 and vl.4 cell pools, cell viability percent and VCD decreased over time post-induction. The largest decrease occurred between days 2 and 4 post-induction. FIG. 14B shows the titer level (Vg / mL) for vl.3 and vl.4 cell pools over the course of seven days post-induction. The vl.4 cell pool reached a peak of 1.28 x IO11vg / mL on day 3 and seemed to plateau there with a slight decrease the rest of the production days to ~1.10 x I011vg / mL. Even a 2-day production of the vl.4 cell pool yielded values in the 1011decade. The vl.3 cell pool reached 1.01 x I011vg / mL by day 3 and continued to increase, reaching 1.88 x I011vg / mL on day 7. These results indicate that harvesting cells at day 3 post- induction yields high rAAV titers.Example 11: Biphasic Bioreactor Production Systems

[0141] Single or multi-reactor production systems can be used for biphasic culture of a stable cell line for inducible production of rAAV. The transition between the growth and production phase can occur through exchange of the media in the same bioreactor (media exchange), transfer of the cells to another larger volume bioreactor with the production media (dilution), or a combination of media exchange and dilution. The growth and production phases can include a perfusion system.

[0142] FIG. 15A illustrates an exemplary biphasic single bioreactor production system with media exchange for volumes less than 10 L. During the growth phase (N-l), the cells are cultured in growth media in a bioreactor with perfusion. Once the cells reach a target viable cell density (VCD), e.g., 10 million cells / mL, the growth media is switched to a production media. The production phase (N) is initiated once the triggering reagents (e.g., doxycycline and tamoxifen) are added to induce rAAV production.

[0143] FIG. 15B illustrates an exemplary biphasic multi-bioreactor production system with dilution for volumes greater than 10 L. During the growth phase (N-l), the cells are cultured in growth media in a bioreactor with perfusion. The cells are grown to a target viable cell density (VCD), e.g., 50 million cells / mL, and transferred to a larger volume bioreactor with production media. The target viable cell density for the production phase would be less than or equal to 20% of the production reactor by volume, e.g., 10 million cells / mL. The production phase (N) is initiated once the triggering reagents (e.g., doxycycline and tamoxifen) are added to the larger volume production reactor to induce rAAV production.

[0144] FIG. 15C illustrates an exemplary biphasic multi-bioreactor production system with media exchange and dilution for volumes greater than 10 L. During the growth phase (N-l), the cells are cultured in growth media in a bioreactor with perfusion. The cells are grown to a target viable cell density (VCD), e.g., 50 million cells / mL, and the growth media is exchanged for production media in the same small vessel. The cells are then transferred to a larger volume bioreactor with production media. The target viable cell density for the production phase would be less than or equal to 20% of the production reactor by volume, e.g., 10 million cells / mL. The production phase (N) is initiated once the triggering reagents (e.g., doxycycline and tamoxifen) are added to the larger volume production reactor induce to rAAV production.

[0145] FIG. 15D illustrates an exemplary biphasic semi-continuous production system where cells are grown continuously in the growth phase (N-l) in one bioreactor and periodicallytransferred to another bioreactor for the production phase (N). The cells are grown to a target viable cell density (VCD), e.g., 10 million cells / mL, and transferred to another bioreactor with production media. With continuous culture in the growth bioreactor, cells can be transferred to additional production bioreactors each time the cells reach the target VCD. This strategy reduces the time for the growth phase (N-l) and allows for smaller growth (N-l) and production (N) bioreactors. The production phase (N) is initiated once the triggering reagents (e.g., doxycycline and tamoxifen) are added to the production reactor(s) induce to rAAV production.

[0146] FIG. 15E illustrates an exemplary biphasic multi-bioreactor production system with a perfused production phase (N). Transfer to the production phase can be accomplished using either media exchange, dilution, or both. During the growth phase (N-l), the cells are cultured in growth media in a bioreactor with perfusion. In this example, the cells are grown to target viable cell density (VCD), e.g., 50 million cells / mL, and transferred to a larger volume bioreactor with production media. The target viable cell density for the production phase would be less than or equal to 20% of the production reactor by volume, e.g., 10 million cells / mL. The production phase (N) is initiated once the triggering reagents (e.g., doxycycline and tamoxifen) are added to the larger volume production bioreactor to induce rAAV production. Perfusion of the production (N) bioreactor, where fresh media is added and spent media is removed, may result in rAAV particles in the permeate (spent media) or the reactor so either may need to be collected to harvest the rAAV particles.

[0147] The preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless ofstructure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.SEQUENCES

Claims

CLAIMSWhat is claimed is:

1. A method for producing recombinant adeno-associated virus (rAAV), the method comprising: culturing a stable cell line, capable of inducibly producing rAAV, in a first cell culture media capable of supporting cell division; inoculating a second cell culture media, capable of supporting rAAV production, with an aliquot from the first cell culture media comprising the stable cell line; adding one or more triggering agents to the second cell culture media to induce production of rAAV from the stable cell line; and culturing the stable cell line in the second cell culture media.

2. The method of claim 1, wherein culturing the stable cell line in the first cell culture media results in a peak cell density greater than 5 million cells / mL.

3. The method of claim 1 or claim 2, wherein culturing the stable cell line in the second cell culture media results in a peak titer level greater than 5.0 x 109vg / mL.

4. The method of any of claims 1-3, wherein the stable cell line comprises a sequence comprising an inducible promoter operably linked to a sequence encoding a recombinase.

5. The method of claim 4, wherein the inducible promoter is a tetracycline inducible promoter.

6. The method of claim 4 or claim 5, wherein the one or more triggering agents comprises a first triggering agent capable of activating the inducible promoter.

7. The method of claim 6, wherein the first triggering agent is doxycycline.

8. The method of any of claims 4-7, wherein the recombinase is an inducible recombinase.. The method of claim 8, wherein the inducible recombinase is a recombinase fused to an estrogen receptor.

10. The method of claim 8 or claim 9, wherein the one or more triggering agents comprises a second triggering agent capable of activating the inducible recombinase.

11. The method of claim 10, wherein the second triggering agent is an estrogen agonist or selective estrogen receptor modulator.

12. The method of claim 10 or claim 11, wherein the second triggering agent is tamoxifen.

13. The method of any one of claims 1-12, wherein the adding step occurs prior to the inoculating step or simultaneously with the inoculating step.

14. The method of any one of claims 1-12, wherein the adding step occurs after the inoculating step.

15. The method of any one of claims 1-12 or 14, wherein after the inoculating step, the second cell culture media comprises between approximately 5 million cells per mL and approximately 10 million cells per mL, or comprises approximately 5 million cells per mL.

16. The method of any one of claims 1-12 or 14, wherein after the inoculating step, the second cell culture media comprises approximately 10 million cells per mL.

17. The method of any one of claims 1-16, wherein culturing the stable cell line in the first and / or second cell culture media comprises periodically adding additional media to the culture.

18. The method of any one of claims 1-17, wherein culturing the stable cell line in the first and / or second cell culture media comprises removing a waste metabolite from the culture.

19. The method of any one of claims 1-18, wherein culturing the stable cell line in the first and / or second cell culture media occurs in a bioreactor with perfusion.

20. The method of claim 19, wherein perfusion is used to exchange the first cell culture media with the second cell culture media.

21. The method of any one of claims 1-20, wherein culturing in the first cell culture media occurs until the stable cell line reaches a viable cell density of (VCD) of between approximately 1 million cells per mL and approximately 50 million cells per mL.

22. The method of any one of claims 1-21, wherein culturing in the first cell culture media occurs until the stable cell line reaches a viable cell density of (VCD) of between approximately 5 million cells per mL and approximately 10 million cells per mL.

23. The method of any one of claims 1-21, wherein culturing in the first cell culture media occurs until the stable cell line reaches a viable cell density of (VCD) of approximately 5 million cells per mL.

24. The method of any one of claims 1-21, wherein culturing in first cell culture media occurs until the stable cell line reaches a viable cell density of (VCD) of approximately 10 million cells per mL.

25. The method of any one of claims 1-21, wherein culturing in first cell culture media occurs until the stable cell line reaches a viable cell density of (VCD) of between approximately 10 million cells per mL and approximately 50 million cells per mL.

26. The method of any one of claims 1-21, wherein culturing in first cell culture media occurs until the stable cell line reaches a viable cell density of (VCD) of approximately 15 million cells per mL, 30 million cells per mL, or 50 million cells per mL.

27. The method of any one of claims 1-26, wherein culturing in the first cell culture media occurs in a vessel of from approximately 100 mL to approximately 10 L.

28. The method of any one of claims 1-27, wherein the first cell culture media has a volume of approximately 5 mL to approximately 9500 mL.

29. The method of any one of claims 1-28, wherein culturing in the second culture media occurs in a vessel of from approximately 1 L to approximately 2000 L.

30. The method of any one of claims 1-29, wherein the second culture media has a volume of approximately 500 mL to approximately 1800 L.

31. The method of any one of claims 1-30, wherein after the inoculating step, the second cell culture media comprises less than or equal to 20% of the first cell culture media.

32. The method of any one of claims 1-31, wherein the triggering agents comprise doxycycline and tamoxifen.

33. The method of any one of claims 4-32, wherein the stable cell line comprises a plurality of cells, each cell comprising: a first polynucleotide comprising a sequence encoding AAV Rep and AAV Cap proteins; a second polynucleotide comprising a sequence encoding one or more AAV helper proteins and the sequence comprising the inducible promoter operably linked to the sequence encoding recombinase; and a third polynucleotide comprising a sequence encoding a payload flanked by AAV inverted terminal repeats (ITRs); wherein expression of the AAV Rep, AAV Cap and the one or more AAV helper proteins is induced by the one or more triggering agents.

34. The method of any one of claims 4-33, wherein the inducible promoter comprises tetracycline-response promoter elements (TREs).

35. The method of any one of claims 4-34, wherein transcription of the recombinase is induced in the presence of the first triggering agent.

36. The method of any one of claims 8-35, wherein the inducible recombinase translocates to the cell nucleus in the presence of the second triggering agent.

37. A system for biphasic cell culture, the system comprising: a first bioreactor and a second bioreactor each configured to culture cells and in fluid communication with each other; a transfer apparatus configured to move a portion of a cell culture from the first bioreactor to the second bioreactor; a sensor located in the first bioreactor configured to measure one or more components within a cell culture in the first bioreactor; and a control system in electronic communication with the sensor and configured to initiate a transfer between the first bioreactor and the second bioreactor upon a reading from the sensor indicating a threshold condition; wherein the biphasic cell culture comprises a growth phase that occurs in the first bioreactor and a rAAV production phase that occurs in the in the second bioreactor.

38. The system of claim 37, wherein the sensor measures viable cell density (VCD) within the first bioreactor.

39. The system of claim 38, wherein VCD is measured by one or both of capacitance and optical cell density.

40. The system of claim 38 or 39, wherein the threshold condition is a VCD greater than or equal to a target VCD that maximizes rAAV production.

41. The system of claim 40, wherein the target VCD is between approximately 5 million cells / mL and approximately 10 million cells / mL.

42. The system of claim 40, wherein the target VCD is approximately 10 million cells / mL.

43. The system of any one of claims 38-40, wherein the threshold condition is a VCD of between approximately 5 million cells per mL and approximately 50 million cells per mL, or approximately 5, 10, 15, 20, 30, 40, or 50 million cells per mL.

44. The system of any one of claims 37-43, wherein the sensor measures one or more of pH, temperature, ion concentration, ion content, oxygen concentration, carbon dioxide concentration, and glucose concentration.

45. The system of any one of claims 37-44, further comprising a media reservoir in fluid communication with the first bioreactor, wherein the media reservoir is configured to periodically deliver fresh media to the first bioreactor.

46. The system of any one of claims 37-45, further comprising a perfusion system in fluid communication with the first bioreactor and configured to remove cellular waste products from the first bioreactor.

47. The system of claim 46, wherein the perfusion system comprises a cell retention device to recirculate cells into the first bioreactor.

48. The system of any one of claims 37-47, wherein the second bioreactor is larger than the first bioreactor.

49. The system of any one of claims 37-48, wherein the first bioreactor has a size of approximately 100 mL to approximately 10 L.

50. The system of any one of claims 37-49, wherein the second bioreactor has a size of approximately 1 L to approximately 2000 L.

51. The system of any one of claim 37-50, further comprising a second sensor located in the second bioreactor configured to measure one or more components within a cell culture in the second bioreactor.

52. The system of claim 51 , wherein the control system is in electronic communication with the second sensor and configured to initiate an addition of a triggering agent to the second bioreactor to induce rAAV production upon a reading from the second sensor indicating a threshold condition.

53. The system of claim 51 or claim 52, wherein the second sensor measures viable cell density (VCD) within the second bioreactor.

54. The system of claim 52 or claim 53, wherein the threshold condition is a target VCD that maximizes rAAV production.

55. The system of any one of claims 52-54, wherein the threshold condition is a target VCD of between approximately 5 million cells per mL and approximately 10 million cells per mL.

56. The system of any one of claims 52-54, wherein the threshold condition is a target VCD of approximately 10 million cells per mL.

57. The system of any one of claims 37-56, further comprising a third bioreactor in fluid communication with the first bioreactor and configured to culture cells, and wherein the control system is configured to initiate a transfer between the first bioreactor and the third bioreactor upon a reading from the sensor located in the first bioreactor indicating a threshold condition.

58. The system of claim 57, wherein the third bioreactor is larger than the first bioreactor.

59. The system of claim 57 or claim 58, wherein the first bioreactor has a size of approximately 100 mL to approximately 10 L.

60. The system of any one of claims 57-59, wherein the third bioreactor has a size of approximately 1 L to approximately 2000 L.

61. The system of any one of claims 37-60, further comprising a third sensor located in the third bioreactor configured to measure one or more components within a cell culture in the third bioreactor.

62. The system of claim 61, wherein the control system is in electronic communication with the third sensor and configured to initiate an addition of a triggering agent to the third bioreactor to induce rAAV production upon a reading from the third sensor indicating a threshold condition.

63. The system of claim 61 or claim 62, wherein the third sensor measures viable cell density (VCD) within the third bioreactor.

64. The system of claim 62 or claim 63, wherein the threshold condition is greater than or equal to a target VCD that maximizes rAAV production.

65. The system of claim 64, wherein the target VCD is between approximately 5 million cells / mL and approximately 10 million cells / mL.

66. The system of claim 64, wherein the target VCD is approximately 10 million cells / mL.

67. The system of any one of claims 62-64, wherein the threshold condition is a viable cell density (VCD) of between approximately 5 million cells per mL and approximately 50 million cells per mL, or approximately 5, 10, 15, 20, 30, 40, or 50 million cells per mL.

68. The system of any one of claims 37-56, wherein the fluid communication between the first and second bioreactors comprises a pump.

69. The system of claim 68, wherein the pump is selected from one or more of: a centrifugal pump, a positive displacement pump, a peristaltic pump, a diaphragm pump, a reciprocating pump, a gear pump, an axial piston pump, a jet pump, a screw pump, and a vane pump.

70. The system of any one of claims 37-47 and 57, wherein the first bioreactor and the second bioreactor are a single bioreactor or wherein the first bioreactor, the second bioreactor, and the third bioreactor are a single bioreactor.

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