Compositions and methods for improved production of adeno-associated viral particles

Abstract

The present disclosure provides compositions and methods for producing adeno-associated virus (AAV) particles. The compositions and methods described herein may be used to produce AAV particles with improved efficiency and / or titer relative to currently available methods.

Description

[0001] COMPOSITIONS AND METHODS FOR IMPROVED PRODUCTION OF ADENO-ASSOCIATED VIRAL PARTICLES

[0002] Background of the Invention

[0003] Parvoviral gene therapy vectors, such as those based on adeno-associated virus (AAV), are an attractive modality for delivering therapeutic transgenes to patients in need of the same. For example, AAV vectors have been used to successfully effectuate therapeutic protein expression in various loss-of-function disorders. However, despite these promising results, there remains a need for improved processes for manufacturing AAV vectors. Particularly, there is a growing need for compositions and methods capable of producing AAV particles with greater efficiency and at higher titers.

[0004] Summary of the Invention

[0005] The present disclosure features compositions and methods useful for the production of adeno-associated virus (AAV) particles. Using the compositions and methods described herein, one can produce AAV particles at high titer and with increased efficiency relative to currently available methods.

[0006] In a first aspect, the disclosure features a method of producing a population of AAV particles by: (a) providing a population of host cells; (b) introducing into the host cells one or more nucleic acids that encode one or more AAV components, thereby producing transfected host cells; (c) incubating the transfected host cells in a cell culture medium for a time sufficient for the cells to produce AAV particles, wherein during the incubating, a portion of the cell culture medium is exchanged with new medium; and (d) after step (c), lysing the host cells, thereby releasing the AAV particles.

[0007] In a second aspect, the disclosure features a method of producing a population of AAV particles by: (a) providing a population of transfected host cells comprising one or more nucleic acids that encode one or more AAV components; (b) incubating the transfected host cells in a cell culture medium for a time sufficient for the cells to produce AAV particles, wherein during the incubating, a portion of the cell culture medium is exchanged with new medium; and (c) after step (b), lysing the host cells, thereby releasing the AAV particles.

[0008] In a third aspect, the disclosure features a method of producing a population of AAV particles by: (a) culturing a population of host cells for a time sufficient for the host cells to proliferate; (b) introducing into the host cells, or progeny thereof, one or more nucleic acids that encode one or more AAV components, thereby producing transfected host cells; (c) incubating the transfected host cells in a cell culture medium for a time sufficient for the cells to produce AAV particles, wherein during the incubating, a portion of the cell culture medium is exchanged with new medium; and (d) after step (c), lysing the host cells, thereby releasing the AAV particles.

[0009] In some embodiments of the first and / or third aspects above, upon introducing the one or more nucleic acids into the host cells or progeny thereof, the host cells or progeny thereof have a density of from 5 x 105cells / ml to 400 x 105cells / ml (e.g., a density of about 5 x 105cells / ml, 6 x 105 cells / ml , 7 x 105cells / ml, 8 x 105cells / ml, 9 x 105cells / ml, 10 x 105cells / ml, 20 x 105cells / ml, 30 x 105cells / ml, 40 x 105cells / ml, 50 x 105cells / ml, 60 x 105cells / ml, 70 x 105cells / ml, 80 x 105cells / ml, 90 x 105cells / ml, 100 x 105cells / ml, 1 10 x 105cells / ml, 120 x 105cells / ml, 130 x 105cells / ml, 140 x 105cells / ml, 150 x 105cells / ml, 160 x 105cells / ml, 170 x 105cells / ml, 180 x 105cells / ml, 190 x 105cells / ml, 200 x 105cells / ml, 210 x 105cells / ml, 220 x 105cells / ml, 230 x 105cells / ml, 240 x 105cells / ml, 250 x 105cells / ml, 260 x 105cells / ml, 270 x 105cells / ml, 280 x 105cells / ml, 290 x 105cells / ml, 300 x 105cells / ml, 310 x 105cells / ml, 320 x 105cells / ml, 330 x 105cells / ml, 340 x 105cells / ml, 350 x 105cells / ml, 360 x 105cells / ml, 370 x 105cells / ml, 380 x 105cells / ml, 390 x 105cells / ml, or 400 x 105cells / ml). In some embodiments, upon introducing the one or more nucleic acids into the host cells or progeny thereof, the host cells or progeny thereof have a density of: (a) from 10 x 105cells / ml to 400 x 105cells / ml, from 20 x 105cells / ml to 400 x 105cells / ml, from 30 x 105cells / ml to 400 x 105cells / ml, from 40 x 105cells / ml to 400 x 105cells / ml, or from 50 x 105cells / ml to 400 x 105cells / ml; or (b) from 100 x 105cells / ml to 200 x 105cells / ml, from 1 10 x 105cells / ml to 190 x 105cells / ml, from 120 x 105cells / ml to 180 x 105cells / ml, from 100 x 105cells / ml to 180 x 105cells / ml, or from 100 x 105cells / ml to 160 x 105cells / ml. In some embodiments, upon introducing the one or more nucleic acids into the host cells or progeny thereof, the host cells or progeny thereof have a density of from 100 x 105cells / ml to 160 x 105cells / ml (e.g., a density of about 100 x 105cells / ml, 1 10 x 105cells / ml, 120 x 105cells / ml, 130 x 105cells / ml, 140 x 105cells / ml, 150 x 105cells / ml, or 160 x 105cells / ml).

[0010] In some embodiments of the second aspect above, the population of transfected host cells is provided at a density of from 5 x 105cells / ml to 400 x 105cells / ml (e.g., a density of about 5 x 105cells / ml, 6 x 105cells / ml, 7 x 105cells / ml, 8 x 105cells / ml, 9 x 105cells / ml, 10 x 105cells / ml, 20 x 105cells / ml, 30 x 105cells / ml, 40 x 105cells / ml, 50 x 105cells / ml, 60 x 105cells / ml, 70 x 105cells / ml, 80 x 105cells / ml, 90 x 105cells / ml, 100 x 105cells / ml, 1 10 x 105cells / ml, 120 x 105cells / ml, 130 x 105cells / ml, 140 x 105cells / ml, 150 x 105cells / ml, 160 x 105cells / ml, 170 x 105cells / ml, 180 x 105cells / ml, 190 x 105cells / ml, 200 x 105cells / ml, 210 x 105cells / ml, 220 x 105cells / ml, 230 x 105cells / ml, 240 x 105cells / ml, 250 x 105cells / ml, 260 x 105cells / ml, 270 x 105cells / ml, 280 x 105cells / ml, 290 x 105cells / ml, 300 x 105cells / ml, 310 x 105cells / ml, 320 x 105cells / ml, 330 x 105cells / ml, 340 x 105cells / ml, 350 x 105cells / ml, 360 x 105cells / ml, 370 x 105cells / ml, 380 x 105cells / ml, 390 x 105cells / ml, or 400 x 105cells / ml). In some embodiments, the population of transfected host cells is provided at a density of: (a) from 10 x 105cells / ml to 400 x 105cells / ml, from 20 x 105cells / ml to 400 x 105cells / ml, from 30 x 105cells / ml to 400 x 105cells / ml, from 40 x 105cells / ml to 400 x 105cells / ml, or from 50 x 105cells / ml to 400 x 105cells / ml; or (b) from 100 x 105cells / ml to 200 x 105cells / ml, from 1 10 x 105cells / ml to 190 x 105cells / ml, from 120 x 105cells / ml to 180 x 105cells / ml, from 100 x 105cells / ml to 180 x 105cells / ml, or from 100 x 105cells / ml to 160 x 105cells / ml. In some embodiments, the population of transfected host cells is provided at a density of from 100 x 105cells / ml to 160 x 105cells / ml (e.g., a density of about 100 x 105cells / ml, 1 10 x 105cells / ml, 120 x 105cells / ml, 130 x 105cells / ml, 140 x 105cells / ml, 150 x 105cells / ml, or 160 x 105cells / ml).

[0011] In some embodiments of any of the foregoing aspects or embodiments of the disclosure, the host cells are mammalian cells. In some embodiments, the mammalian cells are human embryonic kidney (HEK) cells or derivatives thereof. In some embodiments, the HEK cells are HEK293 cells. In some embodiments, the host cells are insect cells. In some embodiment, the insect cells are Sf9 cells.

[0012] In some embodiments, the one or more nucleic acids are introduced into the host cells by contacting the cells with (i) the one or more nucleic acids and (ii) a transfection reagent. In some embodiments, the transfection reagent is a cationic polymer or a cationic lipid. In some embodiments, the transfection reagent is FectoVIR®. In some embodiments, the transfection reagent is AAV-MAX. In some embodiments, the transfection reagent is polyethyleneimine (PEI) or PEI-pro®. In some embodiment, a cationic polymer is FectoVIR®, polyethyleneimine (PEI), or PEI-pro®. In some embodiment, cationic lipid is AAV-MAX.

[0013] In some embodiments, the one or more nucleic acids are introduced into the host cells by way of a technique selected from the group consisting of electroporation, nucleofection, squeeze-poration, sonoporation, optical transfection, magnetofection, and impalefection.

[0014] In some embodiments, during the incubating, the host cells are cultured until reaching a density of from 5 x 105cells / ml to 400 x 105cells / ml (e.g., a density of about 5 x 105cells / ml, 6 x 105cells / ml, 7 x 105cells / ml, 8 x 105cells / ml, 9 x 105cells / ml, 10 x 105cells / ml, 20 x 105cells / ml, 30 x 105cells / ml, 40 x 105cells / ml, 50 x 105cells / ml, 60 x 105cells / ml, 70 x 105cells / ml, 80 x 105cells / ml, 90 x 105cells / ml, 100 x 105cells / ml, 1 10 x 105cells / ml, 120 x 105cells / ml, 130 x 105cells / ml, 140 x 105cells / ml, 150 x 105cells / ml, 160 x 105cells / ml, 170 x 105cells / ml, 180 x 105cells / ml, 190 x 105cells / ml, 200 x 105cells / ml, 210 x 105cells / ml, 220 x 105cells / ml, 230 x 105cells / ml, 240 x 105cells / ml, 250 x 105cells / ml, 260 x 105cells / ml, 270 x 105cells / ml, 280 x 105cells / ml, 290 x 105cells / ml, 300 x 105cells / ml, 310 x 105cells / ml, 320 x 105cells / ml, 330 x 105cells / ml, 340 x 105cells / ml, 350 x 105cells / ml, 360 x 105cells / ml, 370 x 105cells / ml, 380 x 105cells / ml, 390 x 105cells / ml, or 400 x 105cells / ml). In some embodiments, during the incubating, the host cells are cultured until reaching a density of: (a) from 10 x 105cells / ml to 400 x 105cells / ml, from 20 x 105cells / ml to 400 x 105cells / ml, from 30 x 105cells / ml to 400 x 105cells / ml, from 40 x 105cells / ml to 400 x 105cells / ml, or from 50 x 105cells / ml to 400 x 105cells / ml; or (b) from 100 x 105cells / ml to 200 x 105cells / ml, from 1 10 x 105cells / ml to 190 x 105cells / ml, from 120 x 105cells / ml to 180 x 105cells / ml, from 100 x 105cells / ml to 180 x 105cells / ml, or from 100 x 105cells / ml to 160 x 105cells / ml. In some embodiments, during the incubating, the host cells are cultured until reaching a density of from 100 x 105cells / ml to 160 x 105cells / ml (e.g., a density of about 100 x 105cells / ml, 1 10 x 105cells / ml, 120 x 105cells / ml, 130 x 105cells / ml, 140 x 105cells / ml, 150 x 105cells / ml, or 160 x 105cells / ml).

[0015] In some embodiments, the method further includes: (i) exchanging cell culture medium with new medium during step (a); (ii) temporarily ceasing to exchange cell culture medium with new medium during step (b); and (Hi) resuming the exchanging of cell culture medium with new medium during step (c). In some embodiments, the exchanging comprises replacing a portion of the cell culture medium with the new medium in one or discrete steps. In some embodiments, each of the discrete steps comprises separating the host cells from surrounding cell culture media by way of centrifugation, removing the surrounding cell culture media as a supernatant resulting from the centrifugation, and supplementing the host cells with new cell culture media. In some embodiments, the exchanging comprises continuously replacing a portion of the cell culture medium with the new medium.

[0016] In some embodiments, the exchanging is performed by way of alternating tangential flow filtration (ATF). In some embodiments, the exchanging comprises replacing at least 25% of the cell culture medium with new medium (e.g., replacing at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the cell culture medium with new medium).

[0017] In some embodiments, the transfected cells are incubated in a volume of cell culture medium of from 1 L to 1000 L (e.g., a volume of cell culture medium of about 1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, 10 L, 20 L, 30 L, 40 L, 50 L, 60 L, 70 L, 80 L, 90 L, 100 L, 200 L, 300 L, 400 L, 500 L, 600 L, 700 L, 800 L, 900 L, or 1000 L). In some embodiments, the transfected cells are incubated in a volume of cell culture medium of from 1 L to 10 L, 1 L to 2 L, 1 .6 L to 2 L, 8 L to 10 L, 40 L to 50 L, or 160 L to 200 L. In some embodiments, the transfected cells are incubated in a volume of cell culture medium of from 1 mL to 1 L, 10 mL to 30 mL, or 20 mL to 40 mL, 50 mL to 100 mL, 100 mL to 180 mL, or 200 mL to 400 mL.

[0018] In some embodiments, the host cells are lysed by contacting the cells with a surfactant. In some embodiments, the host cells are lysed in the presence of one or more reagents such as TritonX- 100, PS-20, PS-80, sodium deoxycholate, poloxamer, Tris-HCI, sodium chloride, and / or citric acid.

[0019] In some embodiments, the exchanging is performed from 1 hour to 24 hours following transfection of the host cells (e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours following transfection of the host cells). In some embodiments, the exchanging is performed from 1 hour to 2 hours following transfection of the host cells.

[0020] In some embodiments, the exchanging is initiated from 1 hour to 24 hours following transfection of the host cells (e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours following transfection of the host cells). In some embodiments, the exchanging is initiated from 1 hour to 24 hours following transfection of the host cells (e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours following transfection of the host cells) and continued until 48 hours after the transfection. In some embodiments, the exchanging is initiated from 1 hour to 2 hours following transfection of the host cells. In some embodiments, the exchanging is initiated from 1 hour to 2 hours following transfection of the host cells and continued until 48 hours after the transfection.

[0021] In some embodiments, the one or more nucleic acids that encode one or more AAV components are introduced into the host cells by providing to the cells (i) a first nucleic acid encoding one or more AAV capsid (Cap) and AAV replication (Rep) proteins and, separately, (ii) a second nucleic acid comprising a transgene of interest and inverted terminal repeats (ITRs). In some embodiments, the host cells are further provided with (Hi) a third nucleic acid comprising one or more adenoviral helper genes.

[0022] In some embodiments, the one or more AV Cap proteins are from an AAV serotype selected from AAV serotype 1 , serotype 2, serotype 3, serotype 4, serotype 5, serotype 6, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11 , serotype 12, and serotype 13.

[0023] In some embodiments, the one or more AAV Rep proteins include AAV Rep40, AAV Rep52, AAV Rep68, and / or AAV Rep78.

[0024] In some embodiments, the method further comprises purifying the released AAV particles following the lysing. In some embodiments, the AAV particles are purified by way of a process comprising one or more of affinity chromatography, ion exchange chromatography (e.g., anion exchange chromatography), and filtration.

[0025] In a further aspect, the disclosure features a population of AAV particles produced by the method of any one of the foregoing aspects or embodiments of the disclosure.

[0026] In another aspect, the disclosure features a cell culture mixture resulting from steps (a) to (c) of the first aspect of the disclosure (or any one or more embodiments thereof).

[0027] In another aspect, the disclosure features a cell culture mixture resulting from steps (a) and (b) of the second aspect of the disclosure (or any one or more embodiments thereof).

[0028] In another aspect, the disclosure features a cell culture mixture resulting from steps (a) to (c) of the third aspect of the disclosure (or any one or more embodiments thereof).

[0029] Brief Description of the Drawings

[0030] FIG. 1 is a graph showing the quantities of adeno-associated virus (AAV) particles produced from the experiments described in Examples 1 -1 , 1 -2, and 1 -3, below. The vertical axis shows vector genome (vg) titer (calculated with ITR primers) in cell lysates.

[0031] FIG. 2 is a graph showing the quantities of AAV particles produced from the experiments described in Examples 2-1 , 2-2, and 2-3, below. The vertical axis shows vector genome (vg) titer (calculated with gene of interest (GOI) specific primers) in cell lysates.

[0032] FIG. 3 is a graph showing the quantities of AAV particles produced from the experiments described in Example 3, below. The vertical axis shows vector genome (vg) titer (calculated with ITR primers) in cell lysates.

[0033] FIG. 4 is a graph showing the quantities of AAV particles produced from the various cell density conditions described in Example 4, below. The vertical axis shows vector genome (vg) titer (calculated with ITR primers) in cell lysates.

[0034] FIG. 5 is a graph showing the quantities of AAV particles produced from the experiments described in Examples 1 -1 , 5-1 , and 5-3, below. The vertical axis shows vector genome (vg) titer (calculated with GOI specific primers) in cell lysates.

[0035] Definitions

[0036] As used herein, the term “about” refers to a value that is within 10% above or below the value being described. For example, “100 liters (L)” as used in the context of volume described herein includes quantities that are within 10% above or below 100 L. Additionally, when used in the context of a list of numerical quantities, it is to be understood that the term “about,” when preceding a list of numerical quantities, applies to each individual quantity recited in the list.

[0037] As used herein, the terms “adeno-associated virus components,” “AAV components,” and the like refer to elements of a recombinantly expressed AAV particle. Namely, AAV components include AAV capsid (Cap) and AAV replication (Rep) proteins, as well as a gene of interest that is flanked by one or more AAV inverted terminal repeats (ITRs). AAV components may also include adenoviral helper genes that promote the formation of packaged AAV particles. Exemplary AAV Cap proteins, Rep proteins, ITRs, and adenoviral helper genes include those known in the art and described herein.

[0038] As used herein, the terms “administering,” “administration,” and the like refer to directly giving a patient a therapeutic agent by any effective route. Exemplary routes of administration are described herein and include systemic administration routes, such as intravenous injection.

[0039] As used herein, the terms “cell culture media,” “cell culture medium,” and the like refer to a mixture (e.g., an aqueous mixture) containing one or more components that promote growth, proliferation, and / or survival of a cell (e.g., a host cell described herein).

[0040] As used herein, the term “exchange,” when used in the context of a cell culture medium, refers to a process of replacing at least a portion of an existing cell culture medium with new media. The new media may be fresh media that has not been previously exposed to cells (e.g., host cells described herein) or may be media that has been exposed to a population of cells (e.g., host cells) but has retained components that promote cell growth, proliferation, and / or survival. Exchange of cell culture media may be continuous or may occur in one or more discrete steps.

[0041] Exchange of cell culture media may be carried out by way of, for example, tangential flow filtration (TFF). As used herein, the term "tangential flow filtration" refers to a process in which a fluid mixture containing components is recirculated (e.g., at high velocities) tangential to the plane of a membrane. In such filtrations, a pressure differential may be applied along the length of the membrane. This may, in turn, cause the fluid and filterable solutes to flow through the filter. This filtration is suitably conducted as a batch process as well as a continuous-flow process. For example, the solution may be passed repeatedly over the membrane while that fluid which passes through the filter is continually drawn off into a separate unit or the solution is passed once over the membrane and the fluid passing through the filter is continually processed downstream.

[0042] Exchange of cell culture media may, in some embodiments, be carried out by way of alternating tangential flow filtration (ATF). ATF is a similar filtration technique to TFF. In TFF, the liquid in contact with the membrane flows only in one direction, whereas in ATF, the direction of the liquid flow alternates at regular intervals. In some embodiments of ATF, permeate is removed to a collection while a diaphragm pump alternates between positive pressure and vacuum to move retentate back and forth across a membrane. The retentate may return, for example, to a bioreactor between each cycle, mixing with the next fluid batch to be filtered. ATF is desirably used for continuously exchanging media within a cell culture mixture, as retentate is continually pushed back into the bioreactor. As used herein, the term “pharmaceutical composition” refers to a mixture containing a therapeutic compound to be administered to a subject, such as a mammal, e.g., a human, in order to prevent, treat, or control a particular disease or condition affecting or that may affect the subject.

[0043] As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms, which are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human) without excessive toxicity, irritation, allergic response, and other problem complications commensurate with a reasonable benefit / risk ratio.

[0044] As used herein, the term “promoter” refers to a recognition site on DNA that is bound by an RNA polymerase. The polymerase drives transcription of the transgene. Exemplary promoters suitable for use with the compositions and methods described herein are described, for example, in Sandelin et al., Nature Reviews Genetics 8:424 (2007), the disclosure of which is incorporated herein by reference as it pertains to nucleic acid regulatory elements. Additionally, the term “promoter” may refer to a synthetic promoter, which are regulatory DNA sequences that do not occur naturally in biological systems. Synthetic promoters contain parts of naturally occurring promoters combined with polynucleotide sequences that do not occur in nature and can be optimized to express recombinant DNA using a variety of transgenes, vectors, and target cell types.

[0045] As used herein, a therapeutic agent is considered to be “provided” to a host cell if the agent is directly administered to the host cell or if the host cell is administered a substance that is processed or metabolized by the cell so as to yield the therapeutic agent endogenously. For example, a host cell may be provided a nucleic acid molecule encoding a therapeutic protein by direct administration of the nucleic acid molecule to the cell or by administration of a substance (e.g., viral vector) that is processed in the cell so as to yield the desired nucleic acid molecule.

[0046] As used herein, the terms “patient” and “subject” refer to an organism that receives treatment for a particular disease or condition as described herein. Examples of subjects and patients include mammals, such as humans, receiving treatment for a disease or condition described herein.

[0047] As used herein, the terms “transfect,” “transfection,” and the like refer to a process in which a heterologous gene (e.g., a gene encoding an AAV component described herein) is introduced into a desired cell (e.g., a host cell described herein) so as to express the gene in the desired cell (e.g., produce AAV in the host cell). Exemplary transfection techniques include the use of non-viral plasmids to introduce a heterologous gene into the desired cell. The section, below, entitled “Methods for Transfecting a Target Cell to Express AAV Components” provides additional, nonlimiting examples of transfection techniques that may be used in conjunction with the compositions and methods of the disclosure.

[0048] As used herein, the term "transgene" refers to a recombinant nucleic acid (e.g., DNA or cDNA) encoding a gene product (e.g., a gene product described herein). The gene product may be an RNA, peptide, or protein. In addition to the coding region for the gene product, the transgene may include or be operably linked to one or more elements to facilitate or enhance expression, such as a promoter, enhancer(s), destabilizing domain(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s), and / or other functional elements. Embodiments of the disclosure may utilize any known suitable promoter, enhancer(s), destabilizing domain(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s), and / or other functional elements.

[0049] As used herein, the terms “treat” and “treatment” refer to therapeutic treatment, in which the object is to prevent or slow down (lessen) an undesired physiological change or disorder. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. , not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.

[0050] As used herein, the term “vector” refers to a nucleic acid, e.g., DNA or RNA, that may function as a vehicle for the delivery of a gene of interest into a cell (e.g., a mammalian cell, such as a human cell), such as for purposes of replication and / or expression. Exemplary vectors useful in conjunction with the compositions and methods described herein are plasmids, DNA vectors, RNA vectors, virions, or other suitable replicon (e.g., viral vector). A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a prokaryotic or eukaryotic cell. Examples of such expression vectors are disclosed in, e.g., WO 1994 / 11026, the disclosure of which is incorporated herein by reference. Expression vectors described herein contain a polynucleotide sequence as well as, e.g., additional sequence elements used for the expression of proteins and / or the integration of these polynucleotide sequences into the genome of a mammalian cell. Certain vectors that can be used for the expression of transgenes described herein include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription.

[0051] Detailed Description

[0052] The present disclosure provides compositions and methods that can be used for manufacturing adeno-associated virus (AAV) particles. Using the compositions and methods described herein, one can produce AAV particles with heightened titer and / or increased efficiency relative to currently utilized methods. In some embodiments of the disclosure, AAV particles are produced by: (a) providing a population of host cells; (b) introducing into the host cells one or more nucleic acids that encode one or more AAV components, thereby producing transfected host cells; (c) incubating the transfected host cells in a cell culture medium for a time sufficient for the cells to produce AAV particles, wherein during the incubating, a portion of the cell culture medium is exchanged with new medium; and (d) after step (c), lysing the host cells, thereby releasing the AAV particles.

[0053] In some embodiments, the disclosure features a method of producing a population of AAV particles by: (a) providing a population of transfected host cells comprising one or more nucleic acids that encode one or more AAV components; (b) incubating the transfected host cells in a cell culture medium for a time sufficient for the cells to produce AAV particles, wherein during the incubating, a portion of the cell culture medium is exchanged with new medium; and (c) after step (b), lysing the host cells, thereby releasing the AAV particles.

[0054] In some embodiments, the disclosure features a method of producing a population of AAV particles by: (a) culturing a population of host cells for a time sufficient for the host cells to proliferate; (b) introducing into the host cells, or progeny thereof, one or more nucleic acids that encode one or more AAV components, thereby producing transfected host cells; (c) incubating the transfected host cells in a cell culture medium for a time sufficient for the cells to produce AAV particles, wherein during the incubating, a portion of the cell culture medium is exchanged with new medium; and (d) after step (c), lysing the host cells, thereby releasing the AAV particles.

[0055] The compositions and methods of the disclosure are based, in part, on the discovery that production of AAV particles from a population of host cells is surprisingly increased when cell culture media is exchanged after the host cells are transfected to express the corresponding AAV component(s). As is shown in the Examples, below, it has presently been discovered that performing a cell culture medium exchange after such host cells are transfected to express one or more AAV components effectuates an unexpected improvement in AAV yield and efficiency.

[0056] The sections that follow provide a description of exemplary host cells, transfection techniques, and AAV components that may be used in conjunction with the compositions and methods of the disclosure.

[0057] Exemplary AAV Vectors of the Disclosure

[0058] Recombinant AAV vectors useful in the invention are recombinant nucleic acid constructs that may include (1 ) a transgene to be expressed (e.g., a polynucleotide encoding a therapeutic protein or a therapeutic RNA molecule) and (2) viral nucleic acids that facilitate integration and expression of the heterologous genes. The viral nucleic acids may include those sequences of AAV that are required in cis for replication and packaging (e.g., functional ITRs) of the DNA into a virion. AAV vectors of the disclosure may also contain marker or reporter genes. Useful AAV vectors may have one or more of the AAV wild type genes deleted in whole or in part but retain functional flanking ITR sequences. The AAV ITRs may be of any serotype (e.g., derived from serotype 2) suitable for a particular application. Methods for using rAAV vectors are described, for example, in Tai et al., J. Biomed. Sci. 7:279-291 (2000), and Monahan and Samulski, Gene Delivery 724-30 (2000), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0059] The nucleic acids described herein can be incorporated into an AAV virion in order to facilitate introduction of the nucleic acid into a cell. The capsid proteins of AAV compose the exterior, non- nucleic acid portion of the virion and are encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1 , VP2, and VP3, which are required for virion assembly. The construction of AAV virions has been described, for example, in US Patent Nos. 5,173,414; 5,139,941 ; 5,863,541 ; 5,869,305; 6,057,152; and 6,376,237; as well as in Rabinowitz et al., J. Virol. 76:791 -801 (2002) and Bowles et al., J. Virol. 77:423-432 (2003), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0060] AAV virions useful in conjunction with the compositions and methods described herein include those derived from a variety of AAV serotypes including AAV 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, and 13. Construction and use of AAV vectors and AAV proteins of different serotypes are described, for example, in Chao et al., Mol. Ther. 2:619-623 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428-3432 (2000); Xiao et al., J. Virol. 72:2224-2232 (1998); Halbert et al., J. Virol. 74:1524-1532 (2000); Halbert et al., J. Virol. 75:6615-6624 (2001 ); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081 (2001 ), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0061] AAV virions that have mutations within the virion capsid may be used to infect particular cell types more effectively than non-mutated capsid virions. Other AAV virions that can be used in methods of the invention include capsid hybrids.

[0062] Host Cells for Producing AAV Particles

[0063] Various types of host cells may be used to produce an AAV particle using the compositions and methods of the disclosure. In some embodiments, the host cells are mammalian cells. In some embodiments, the mammalian cells are human embryonic kidney (HEK) cells or derivatives thereof. In some embodiments, the HEK cells are HEK293 cells. In some embodiments, the host cells are insect cells. In some embodiments, the insect cells are Sf9 cells.

[0064] Methods for Transfecting a Target Cell to Express AAV Components

[0065] A variety of methods can be used to transfect a target cell to express AAV components (and, thus, produce an AAV particle). The transfection techniques that may be used to express one or more desired AAV components in a host cell include methods in which a nucleic acid encoding the AAV component(s) is introduced into the host cells (e.g., a mammalian cell, such as a human cell, and particularly, a HEK cell described herein) in the presence of a transfection reagent. In some embodiments, the transfection reagent is a cationic polymer or a cationic lipid. In some embodiments, the transfection reagent is FectoVIR® or PEI, or PEIpro® (see, e.g., Polyplus® Transfection Reagents for Protein & Viral Vector Production (2020, Catalog), the disclosure of which is incorporated herein by reference). In some embodiments, the transfection reagent is FectoVIR® In some embodiments, the transfection reagent is AAV-MAX which is included in Gibco™ AAV-MAX Transfection kit (see, e.g., Thermo Fisher scientific catalog No. A50515, the disclosure of which is incorporated herein by reference). In some embodiments, the cationic polymer is FectoVIR®, polyethyleneimine (PEI), or PEI-pro®. In some embodiments, the cationic lipid is AAV-MAX.

[0066] Additional techniques that can be used to transfect a host cell of the disclosure so as to express one or more AAV components include electroporation, which can be used to permeabilize mammalian cells (e.g., human target cells) by the application of an electrostatic potential to the cell of interest. Mammalian cells, such as human cells, subjected to an external electric field in this manner are subsequently predisposed to the uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail, e.g., in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference.

[0067] A similar technique, Nucleofection™, utilizes an applied electric field in order to stimulate the uptake of exogenous polynucleotides into the nucleus of a eukaryotic cell. Nucleofection™ and protocols useful for performing this technique are described in detail, e.g., in Distler et al., Experimental Dermatology 14:315 (2005), as well as in US 2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0068] An additional technique useful for the transfection of host cells is the squeeze-poration methodology. This technique induces the rapid mechanical deformation of cells in order to stimulate the uptake of exogenous DNA through membranous pores that form in response to the applied stress. This technology is advantageous in that a vector is not required for delivery of nucleic acids into a cell, such as a human target cell. Squeeze-poration is described in detail, e.g., in Sharei et al., J. Vis. Exp. 81 :e50980 (2013), the disclosure of which is incorporated herein by reference.

[0069] Lipofection represents another technique useful for transfection of host cells. This method involves the loading of nucleic acids into a liposome, which often presents cationic functional groups, such as quaternary or protonated amines, towards the liposome exterior. This promotes electrostatic interactions between the liposome and a cell due to the anionic nature of the cell membrane, which ultimately leads to uptake of the exogenous nucleic acids, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in US 7,442,386, the disclosure of which is incorporated herein by reference. Similar techniques that exploit ionic interactions with the cell membrane to provoke the uptake of foreign nucleic acids are contacting a cell with a cationic polymer-nucleic acid complex. Exemplary cationic molecules that associate with polynucleotides so as to impart a positive charge favorable for interaction with the cell membrane are activated dendrimers (described, e.g., in Dennig, Top Curr Chem. 228:227 (2003), the disclosure of which is incorporated herein by reference) polyethylenimine, and DEAE-dextran, the use of which as a transfection agent is described in detail, for example, in Gulick et al., Curr Protoc Mol Biol. 40:1 :9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference.

[0070] Another useful tool for inducing the uptake of exogenous nucleic acids by host cells is laserfection, also called optical transfection, a technique that involves exposing a cell to electromagnetic radiation of a particular wavelength in order to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. The bioactivity of this technique is similar to, and in some cases found superior to, electroporation.

[0071] Impalefection is another technique that can be used to deliver genetic material to host cells. This technique relies on the use of nanomaterials, such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. DNA containing the gene, intended for intracellular delivery, is attached to the nanostructure surface. A chip with arrays of these needles is then pressed against cells or tissue. Cells that are impaled by nanostructures can express the delivered gene(s). An example of this technique is described in Shalek et al., PNAS 107:25 1870 (2010), the disclosure of which is incorporated herein by reference.

[0072] Magnetofection™ can also be used to deliver nucleic acids to host cells. The principle of Magnetofection™ is to associate nucleic acids with cationic magnetic nanoparticles. The magnetic nanoparticles are made of iron oxide, which is fully biodegradable, and coated with specific cationic proprietary molecules varying upon the applications. Their association with the gene vectors (DNA or RNA vectors) is achieved by salt-induced colloidal aggregation and electrostatic interaction. The magnetic particles are then concentrated on the target cells by the influence of an external magnetic field generated by magnets. This technique is described in detail in Scherer et al., Gene Ther. 9:102 (2002), the disclosure of which is incorporated herein by reference.

[0073] Magnetic beads are another tool that can be used to transfect host cells in a mild and efficient manner, as this methodology utilizes an applied magnetic field in order to direct the uptake of nucleic acids. This technology is described in detail, for example, in US2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0074] Another useful tool for inducing the uptake of exogenous nucleic acids by host cells is sonoporation, a technique that involves the use of sound (typically ultrasonic frequencies) for modifying the permeability of the cell plasma membrane in order to permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, e.g., in Rhodes et al., Methods Cell Biol. 82:309 (2007), the disclosure of which is incorporated herein by reference.

[0075] Microvesicles represent another potential vehicle that can be used to transfect a desired host cell according to the methods described herein. For example, microvesicles that have been induced by the co-overexpression of the glycoprotein VSV-G with, e.g., a genome-modifying protein, such as a nuclease, can be used to efficiently deliver proteins into a cell that subsequently catalyze the sitespecific cleavage of an endogenous polynucleotide sequence so as to prepare the genome of the cell for the covalent incorporation of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also referred to as Gesicles, for the genetic modification of eukaryotic cells is described in detail, e.g., in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract]. In: Methylation changes in early embryonic genes in cancer [abstract], in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122.

[0076] Exemplary Cell Culture Medium Exchange Techniques

[0077] In preferred embodiments of the disclosure, host cells are subject to cell culture medium exchange before being transfected to express the desired AAV component(s). During transfection, the cell culture medium exchange is temporarily ceased. Then, after transfection is completed, cell culture medium exchange is resumed.

[0078] In some embodiments, the exchanging comprises replacing a portion of the cell culture medium with the new medium in one or discrete steps. In some embodiments, each of the discrete steps comprises separating the host cells from the surrounding cell culture media by way of centrifugation, removing the surrounding cell culture media as a supernatant resulting from the centrifugation, and supplementing the host cells with new cell culture media.

[0079] In some embodiments, the exchanging comprises continuously replacing a portion of the cell culture medium with the new medium. Exemplary methods for continuously exchanging cell culture media include tangential flow filtration (TFF). In some embodiments, continuous cell culture medium exchange is carried out by way of alternating tangential flow filtration (ATF).

[0080] In some embodiments, the exchanging is ceased while host cells are transfected to express one or more AAV components. In the context of an ATF procedure, the exchange may be “ceased” by stopping the continuous medium exchange during the transfection process (i.e., when host cells are contacted with the genetic material encoding one or more AAV components). Alternatively, the exchange may be reduced to a lower exchange rate during the transfection process, such that the transfection efficiency of the host cells is not affected.

[0081] In some embodiments, when host cells are subjected to a medium exchange following the transfection step, the medium exchange may commence at any time following completion of transfection (i.e. , following the introduction of one or more nucleic acid molecule encoding one or more AAV components into the host cells). For example, the exchanging may be initiated from 1 hour to 24 hours following transfection of the host cells (e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours following transfection of the host cells). In some embodiments, the exchanging is initiated from 1 hour to 24 hours following transfection of the host cells (e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours following transfection of the host cells) and continued until 48 hours after the transfection. In some embodiments, the exchanging is initiated from 1 hour to 2 hours following transfection of the host cells. In some embodiments, the exchanging is initiated from 1 hour to 2 hours following transfection of the host cells and continued until 48 hours after the transfection.

[0082] Exemplary Cell Densities

[0083] In preferred embodiments of the disclosure, after transfecting the host cells so as to express one or more desired AAV components, the host cells are cultured until reaching a target density. For example, after transfecting the host cells to express the desired AAV component(s), the host cells may be cultured until reaching a density of from 5 x 105cells / ml to 400 x 105cells / ml (e.g., a density of about 5 x 105cells / ml, 6 x 105cells / ml, 7 x 105cells / ml, 8 x 105cells / ml, 9 x 105cells / ml, 10 x 105cells / ml, 20 x 105cells / ml, 30 x 105cells / ml, 40 x 105cells / ml, 50 x 105cells / ml, 60 x 105cells / ml, 70 x 105cells / ml, 80 x 105cells / ml, 90 x 105cells / ml, 100 x 105cells / ml, 1 10 x 105cells / ml, 120 x 105cells / ml, 130 x 105cells / ml, 140 x 105cells / ml, 150 x 105cells / ml, 160 x 105cells / ml, 170 x 105cells / ml, 180 x 105cells / ml, 190 x 105cells / ml, 200 x 105cells / ml, 210 x 105cells / ml, 220 x 105cells / ml, 230 x 105cells / ml, 240 x 105cells / ml, 250 x 105cells / ml, 260 x 105cells / ml, 270 x 105cells / ml, 280 x 105cells / ml, 290 x 105cells / ml, 300 x 105cells / ml, 310 x 105cells / ml, 320 x 105cells / ml, 330 x 105cells / ml, 340 x 105cells / ml, 350 x 105cells / ml, 360 x 105cells / ml, 370 x 105cells / ml, 380 x 105cells / ml, 390 x 105cells / ml, or 400 x 105cells / ml).

[0084] In some embodiments, after transfecting the host cells so as to express one or more desired AAV components, the host cells are cultured until reaching a density of: (a) from 10 x 105cells / ml to 400 x 105cells / ml, from 20 x 105cells / ml to 400 x 105cells / ml, from 30 x 105cells / ml to 400 x 105cells / ml, from 40 x 105cells / ml to 400 x 105cells / ml, or from 50 x 105cells / ml to 400 x 105cells / ml; or (b) from 100 x 105cells / ml to 200 x 105cells / ml, from 1 10 x 105cells / ml to 190 x 105cells / ml, from 120 x 105cells / ml to 180 x 105cells / ml, from 100 x 105cells / ml to 180 x 105cells / ml, or from 100 x 105cells / ml to 160 x 105cells / ml. In some embodiments, after transfecting the host cells so as to express one or more desired AAV components, the host cells are cultured until reaching a density of from 100 x 105cells / ml to 160 x 105cells / ml (e.g., a density of about 100 x 105cells / ml, 1 10 x 105cells / ml, 120 x 105cells / ml, 130 x 105cells / ml, 140 x 105cells / ml, 150 x 105cells / ml, or 160 x 105cells / ml).

[0085] Pharmaceutical Compositions

[0086] The AAV particles produced using the composition and methods described herein may contain a transgene, such as a transgene encoding a therapeutic protein or therapeutic RNA molecule. The AAV particles produced using the compositions and methods described herein may be incorporated into a vehicle for administration into a patient, such as a human patient suffering from a disease or disorder. Pharmaceutical compositions of the disclosure can be formulated using, for example, physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980); incorporated herein by reference), and in a desired form, e.g., in the form of lyophilized formulations or aqueous solutions.

[0087] AAV vectors described herein may contain one or more transcription regulatory elements operably linked to the desired therapeutic transgene. Such vectors may be administered to a patient (e.g., a human patient) by a variety of routes of administration. The route of administration may vary, for example, with the onset and severity of disease, and may include, e.g., intravenous administration.

[0088] Examples

[0089] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0090] Example 1-1: Production of rAAV8 Vector in Common method

[0091] Viral Production Cells 2.0 (VPC2.0, Thermo Fisher Scientific, catalog No. A49784) in Viral Production Medium (VPM, Thermo Fisher Scientific, catalog No. A4817901 ) containing 4% GlutaMAX™ Supplement (GultaMAX, Thermo Fisher Scientific, catalog No. 35050061 ) were diluted to a density of 38 x 105cells / mL using VPM containing 4% GultaMAX. 20mL of the cell suspension was put into the 125mL shaker flask. Gibco AAV-MAX Enhancer, one of the components of the AAV- MAX Transfection kit (Thermo Fisher Scientific, catalog No. A50515), was added into the cell suspension. The transgene plasmid construct and the helper plasmids RC_AAV and Helper were added into Viral-Plex™ Complexation Buffer (Viral-Plex, Thermo Fisher Scientific, catalog No. A4983901 ). The remaining two reagents of the AAV-MAX transfection kit were added to the complexation buffer in the order described in the kit protocol. 10 minutes later, the complex was added into the flask. 3 days after the transfection, Triton X-100 was added to the culture medium at a concentration of 0.2%, which was left to stand at room temperature for 1 hour to lyse the cells. The resulting solution was centrifuged at 10000g for 5 minutes at room temperature. The supernatant was collected as rAAV sample. Example 1-2: Production of rAA V8 Vector in ATF method

[0092] Viral Production Cells 2.0 (VPC2.0, Thermo Fisher Scientific, catalog No. A49784) in Viral Production Medium (VPM, Thermo Fisher Scientific, catalog No. A4817901 ) containing 4% GlutaMAX™ Supplement (GultaMAX, Thermo Fisher Scientific, catalog No. 35050061 ) were diluted to a density of 38 x 105cells / mL using VPM containing 4% GultaMAX. The cell suspension was centrifuged at 400g for 4 minutes at room temperature. Then a part of the supernatant was removed to obtain high cell density and cells were resuspended. Cell suspension at a density of 90 x 105cells / mL was prepared. 20mL of the cell suspension was put into the 125mL shaker flask. The transgene plasmid construct and the helper plasmids RC_AAV and Helper were added into Viral- Plex™ Complexation Buffer (Viral-Plex, Thermo Fisher Scientific, catalog No. A4983901 ). The remaining two reagents of the AAV-MAX transfection kit were added to the complexation buffer in the order described in the kit protocol. 10 minutes later, the complex was added into the flask. 3 days after the transfection, Triton X-100 was added to the culture medium at a concentration of 0.2%, which was left to stand at room temperature for 1 hour to lyse the cells. The resulting solution was centrifuged at 10000g for 5 minutes at room temperature. The supernatant was collected as rAAV sample.

[0093] Example 1-3: Production of rAA V8 Vector in HCD method

[0094] Viral Production Cells 2.0 (VPC2.0, Thermo Fisher Scientific, catalog No. A49784) in Viral Production Medium (VPM, Thermo Fisher Scientific, catalog No. A4817901 ) containing 4% GlutaMAX™ Supplement (GultaMAX, Thermo Fisher Scientific, catalog No. 35050061 ) were diluted to a density of 38 x 105cells / mL using VPM containing 4% GultaMAX. The cell suspension was centrifuged at 400g for 4 minutes at room temperature. Then a part of the supernatant was removed to obtain high cell density and cells were resuspended. Cell suspension at a density of 99 x 105cells / mL was prepared. 20mL of the cell suspension was put into the 125mL shaker flask.

[0095] The transgene plasmid construct and the helper plasmids RC_AAV and were added into Viral-Plex™ Complexation Buffer (Viral-Plex, Thermo Fisher Scientific, catalog No. A4983901 ). The remaining two reagents of the AAV-MAX transfection kit were added to the complexation buffer in the order described in the kit protocol. 10 minutes later, the complex was added into the flask. 1 day after the transfection, the cell suspension was centrifuged at 400g for 4 minutes at room temperature. Then half of spent was removed and the same amount of fresh VPM containing 4% GultaMAX was added. The cells were resuspended, and the cell suspension was cultured in the incubator shaker. 2 days after the transfection, the cell suspension was centrifuged at 400g for 4 minutes at room temperature. Then half of spent was removed and the same amount of fresh VPM containing 4% GultaMAX was added. The cells were resuspended, and the cell suspension was cultured in the incubator shaker. 3 days after the transfection, Triton X-100 was added to the culture medium at a concentration of 0.2%, which was left to stand at room temperature for 1 hour to lyse the cells. The resulting solution was centrifuged at 10000g for 5 minutes at room temperature. The supernatant was collected as rAAV sample. Results rAAV samples produced by using the method described in Examples 1 -1 , 1 -2, and 1 -3 were quantified. After the subsequent treatment with DNase I, AAV genomic DNA was extracted, the solution was diluted with water, and real-time PCR was then performed using an AAVpro (R) Titration Kit for Real Time PCR (Takara Bio Inc., catalog No. 6233) and ITR primers attached to the kit. A calibration curve was prepared by using standard DNA attached to the kit, and vg concentrations of the supernatant were calculated. (FIG. 1 )

[0096] As demonstrated in FIG. 1 , it was confirmed that enhanced productivity is achieved with Example 1 -3, as compared to what is achieved with Example 1 -1 and Example 1 -2.

[0097] Example 2-1 : Production of rAAV8 Vector in Common method

[0098] Viral Production Cells 2.0 (VPC2.0, Thermo Fisher Scientific, catalog No. A49784) were diluted to a density of 30 x 105cells / mL using medium. 20mL of the cell suspension was put into the 125mL shaker flask. The transgene plasmid construct and the helper plasmids RC_AAV and Helper were added into PBS (PBS pH 7.4, Thermo Fisher Scientific, catalog No. 10010023). FectoVIR (FectoVIR®-AAV 10 mL, Polyplus, catalog No. 101000022) was added to the PBS. 5 minutes later, the complex was added into the flask. 2 days after the transfection, Triton X-100 was added to the culture medium at a concentration of 0.5%, which was left to stand at room temperature for 1 hour to lyse the cells. The resulting solution was centrifuged at 10000g for 5 minutes at room temperature. The supernatant was collected as rAAV sample.

[0099] Example 2-2: Production of rAA V8 Vector in ATF method

[0100] Viral Production Cells 2.0 (VPC2.0, Thermo Fisher Scientific, catalog No. A49784) are diluted to a density of 30 x 105cells / mL using medium. 20mL of the cell suspension is put into the 125mL shaker flask. The cell suspension is centrifuged at 400g for 4 minutes at room temperature. Then a part of the supernatant is removed to obtain high cell density and cells were resuspended. Cell suspension at a density of 90 x 105cells / mL is prepared. 20mL of the cell suspension is put into the 125mL shaker flask. The transgene plasmid construct and the helper plasmids RC_AAV and Helper are added into PBS (PBS pH 7.4, Thermo Fisher Scientific, catalog No. 10010023). FectoVIR (FectoVIR®- AAV 10 mL, Polyplus, catalog No. 101000022) is added to the PBS. 5 minutes later, the complex is added into the flask. 2 days after the transfection, Triton X-100 is added to the culture medium at a concentration of 0.5%, which is left to stand at room temperature for 1 hour to lyse the cells. The resulting solution is centrifuged at 10000g for 5 minutes at room temperature. The supernatant is collected as rAAV sample.

[0101] Example 2-3: Production of rAA V8 Vector in HCD method

[0102] Viral Production Cells 2.0 (VPC2.0, Thermo Fisher Scientific, catalog No. A49784) were diluted to a density of 30 x 105cells / mL using medium. 20mL of the cell suspension was put into the 125mL shaker flask. The cell suspension was centrifuged at 400g for 4 minutes at room temperature. Then a part of the supernatant was removed to obtain high cell density and cells were resuspended. Cell suspension at a density of 90 x 105cells / mL was prepared. 20mL of the cell suspension was put into the 125mL shaker flask. The transgene plasmid construct and the helper plasmids RC_AAV and Helper were added into PBS (PBS pH 7.4, Thermo Fisher Scientific, catalog No. 10010023).

[0103] FectoVIR (FectoVIR®-AAV 10 mL, Polyplus, catalog No. 101000022) was added to the PBS. 5 minutes later, the complex was added into the flask. 1 day after the transfection, the cell suspension was centrifuged at 400g for 4 minutes at room temperature. Then half of spent was removed and the same amount of fresh VPM containing 4% GultaMAX was added. The cells were resuspended, and the cell suspension was cultured in the incubator shaker. 2 days after the transfection, Triton X-100 was added to the culture medium at a concentration of 0.5%, which was left to stand at room temperature for 1 hour to lyse the cells. The resulting solution was centrifuged at 10000g for 5 minutes at room temperature. The supernatant was collected as rAAV sample.

[0104] Results rAAV samples produced by using the method described in Examples 2-1 and 2-3 were quantified. After the subsequent treatment with DNase I, the solution was diluted with water, and digital droplet PCR (QX ONE Droplet Digita PCR system, Bio-Rad Laboratories, Inc.) was then performed using an ddPCR EvaGreen supermix (Bio-Rad Laboratories, Inc., catalog No. 186-4034), FAM probe, and primers designed for transgene construct. The vg concentrations of the supernatant were calculated. (FIG. 2)

[0105] As demonstrated in FIG. 2, it was confirmed that enhanced productivity is achieved with Example 2-3, as compared to what is achieved with Example 2-1 .

[0106] Example 3: Confirmation of medium exchange time

[0107] Viral Production Cells 2.0 (VPC2.0, Thermo Fisher Scientific, catalog No. A49784) in medium were diluted to a density of 34 x 105cells / mL. 20mL of the cell suspension was put into the 125mL shaker flask. A total of 12 identical flasks were prepared. Gibco AAV-MAX Enhancer, one of the components of the AAV-MAX Transfection kit (Thermo Fisher Scientific, catalog No. A50515), was added into the cell suspension. The transgene plasmid construct and the helper plasmids RC_AAV and Helper were added into Viral-Plex™ Complexation Buffer (Viral-Plex, Thermo Fisher Scientific, catalog No. A4983901 ). The remaining two reagents of the AAV-MAX transfection kit were added to the complexation buffer in the order described in the kit protocol. 10 minutes later, the complex was added into the flasks. The following operations were performed on all flasks except flasks 1 . For conditions 2, 3, 4, 5, and 6, respectively 0, 1 , 2, 4, 6 hour(s) after the transfection, the cell suspension was centrifuged at 400g for 4 minutes at room temperature. Then half of spent was removed and fresh medium was added. The cells were resuspended, and the cell suspension was cultured in the incubator shaker. Conditions 7-12 were exactly the same as conditions 1 -6, respectively. 3 days after the transfection, Triton X-100 was added to the culture medium at a concentration of 0.2%, which was left to stand at room temperature for 1 hour to lyse the cells. The resulting solution was centrifuged at 10000g for 5 minutes at room temperature. The supernatant was collected as rAAV sample. rAAV samples produced by using the method described in Example 3 were quantified. After the subsequent treatment with Dnase I, AAV genomic DNA was extracted, the solution was diluted with water, and real-time PCR was then performed using an AAVpro ® Titration Kit for Real Time PCR (Takara Bio Inc., catalog No. 6233) and ITR primers attached to the kit. A calibration curve was prepared by using standard DNA attached to the kit, and vg concentrations of the supernatant were calculated. (FIG. 3)

[0108] As demonstrated in FIG. 3, it was confirmed that productivities are constant in the conditions in which medium exchange was conducted 1 hour after transfection.

[0109] Example 4: Confirmation of medium exchange time

[0110] Viral Production Cells 2.0 (VPC2.0, Thermo Fisher Scientific, catalog No. A49784) in medium were diluted to a density of 40 x 105cells / mL. The cell suspension was centrifuged at 400g for 4 minutes at room temperature. Then a part of the supernatant was removed to obtain high cell density and cells were resuspended. 13 cell suspensions in the flasks at a density of 94-185 x 105cells / mL were prepared. The actual cell densities and target cell densities (x 105cells / mL) are shown in Table 1 below.

[0111] Table 1. Cell densities of flasks 1-13

[0112] 20mL of the cell suspension was put into the 125mL shaker flask. A total of 13 identical flasks were prepared. Gibco AAV-MAX Enhancer, one of the components of the AAV-MAX Transfection kit (Thermo Fisher Scientific, catalog No. A50515), was added into the cell suspension. The transgene plasmid construct and the helper plasmids RC_AAV and Helper were added into Viral-Plex™ Complexation Buffer (Viral-Plex, Thermo Fisher Scientific, catalog No. A4983901 ). The remaining two reagents of the AAV-MAX transfection kit were added to the complexation buffer in the order described in the kit protocol. 10 minutes later, the complex was added into the flasks. The following operations were performed on all flasks except flask 1 . 1 day after the transfection, the cell suspension was centrifuged at 400g for 4 minutes at room temperature. Then half of spent was removed and the same amount of fresh medium was added. The cells were resuspended, and the cell suspension was cultured in the incubator shaker. 2 days after the transfection, Triton X-100 was added to the culture medium at a concentration of 0.2%, which was left to stand at room temperature for 1 hour to lyse the cells. The resulting solution was centrifuged at 10000g for 5 minutes at room temperature. The supernatant was collected as rAAV sample.

[0113] Results rAAV samples produced by using the method described in Example 4 were quantified. After the subsequent treatment with DNase I, AAV genomic DNA was extracted, the solution was diluted with water, and real-time PCR was performed using an AAVpro (R) Titration Kit for Real Time PCR (Takara Bio Inc., catalog No. 6233) and ITR primers attached to the kit. A calibration curve was prepared by using standard DNA attached to the kit, and vg concentrations of the supernatant were calculated. (FIG. 4)

[0114] As demonstrated in FIG. 4, it was confirmed that productivities are maximized at a density of 150.

[0115] Example 5-1: Production of rAA V8 Vector in Common method

[0116] 1600 mL of Viral Production Medium (VPM, Thermo Fisher Scientific, catalog No. A4817901 ) containing 4% GlutaMAX™ Supplement (GultaMAX, Thermo Fisher Scientific, catalog No. 35050061 ) was put into a 2L bioreactor and conditioned to 37 °C. The Viral Production Cells 2.0 (VPC2.0, Thermo Fisher Scientific, catalog No. A49784) at a density of 48 x 105cells / mL were seeded so that the cell density in the reactor was of 4 x 105cells / mL. The cell suspension was cultured at 37 °C for 3 days. 3 days thereafter, cell density reached 38 x 105cells / mL. Gibco AAV-MAX Enhancer, one of the components of the AAV-MAX Transfection kit (Thermo Fisher Scientific, catalog No. A50515), was added into the cell suspension. The transgene plasmid construct and the helper plasmids RC_AAV and Helper were added into Viral-Plex™ Complexation Buffer (Viral-Plex, Thermo Fisher Scientific, catalog No. A4983901 ). The remaining two reagents of the AAV-MAX transfection kit were added to the complexation buffer in the order described in the kit protocol. 10 minutes later, the complex was added into the bioreactor. 3 days after the transfection, Triton X-100 was added to the culture medium at a concentration of 0.2%, which was left to stand at room temperature for 1 hour to lyse the cells. The resulting solution was centrifuged at 10000g for 5 minutes at room temperature. The supernatant was collected as rAAV sample.

[0117] Example 5-2: Production of rAA V8 Vector in ATF method

[0118] 1600 mL of Viral Production Medium (VPM, Thermo Fisher Scientific, catalog No. A4817901 ) containing 4% GlutaMAX™ Supplement (GultaMAX, Thermo Fisher Scientific, catalog No. 35050061 ) is put into a 2L bioreactor and conditioned to 37 °C. The Viral Production Cells 2.0 (VPC2.0, Thermo Fisher Scientific, catalog No. A49784) at a density of 48 x 105cells / mL are seeded so that the cell density in the reactor was of 4 x 105cells / mL. The cell suspension is cultured at 37 °C for 3 days.

[0119] 3 days thereafter, cell density reached 38 x 105cells / mL. The Cell ATF 2 (ATF2, Repligen) system with 0.2 urn PES single use filter (Repligen, product number; suATF 2-S02PES) is used. The cell suspension was cultured with medium exchange rate 1 VVD.

[0120] 1 day thereafter, cell density reached 70 x 105cells / mL. ATF2 and medium exchange are stopped. Gibco AAV-MAX Enhancer, one of the components of the AAV-MAX Transfection kit (Thermo Fisher Scientific, catalog No. A50515), is added into the cell suspension. The transgene plasmid construct and the helper plasmids RC_AAV and Helper are added into Viral-Plex™ Complexation Buffer (Viral-Plex, Thermo Fisher Scientific, catalog No. A4983901 ). The remaining two reagents of the AAV-MAX transfection kit are added to the complexation buffer in the order described in the kit protocol. 10 minutes later, the complex was added into the bioreactor. 3 days after the transfection, 0.2% Triton X-100 is added into the culture solution, which is left to stand at room temperature for 1 hour to lyse the cells. The resulting solution is centrifuged at 10000g for 5 minutes at room temperature. The supernatant is collected as rAAV sample.

[0121] Example 5-3: Production of rAA V8 Vector in HCD method

[0122] 1600 mL of Viral Production Medium (VPM, Thermo Fisher Scientific, catalog No. A4817901 ) containing 4% GlutaMAX™ Supplement (GultaMAX, Thermo Fisher Scientific, catalog No. 35050061 ) was put into a 2L bioreactor and conditioned to 37 °C. The Viral Production Cells 2.0 (VPC2.0, Thermo Fisher Scientific, catalog No. A49784) at a density of 48 x 105cells / mL were seeded so that the cell density in the reactor was 4 x 105cells / mL. The cell suspension was cultured at 37 °C for 3 days.

[0123] 3 days thereafter, cell density reached 38 x 105cells / mL. The Cell ATF 2 (ATF2, Repligen) system with 0.2 urn PES single use filter (Repligen, product number; suATF 2-S02PES) was used. The cell suspension was cultured with medium exchange rate 1 VVD.

[0124] 2 days thereafter, cell density reached 130 x 105cells / mL. The medium exchange was stopped while the ATF2’s tangential flow continued. Gibco AAV-MAX Enhancer, one of the components of the AAV-MAX Transfection kit (Thermo Fisher Scientific, catalog No. A50515), was added into the cell suspension. The transgene plasmid construct and the helper plasmids RC_AAV and Helper were added into Viral-Plex™ Complexation Buffer (Viral-Plex, Thermo Fisher Scientific, catalog No. A4983901 ). The remaining two reagents of the AAV-MAX transfection kit were added to the complexation buffer in the order described in the kit protocol. 10 minutes later, the complex was added into the bioreactor. 2 hours later, the medium exchange was restarted.

[0125] 2 days after the transfection, Triton X-100 was added to the culture medium at a concentration of 0.2%, which was left to stand at room temperature for 1 hour to lyse the cells. The resulting solution was centrifuged at 10000g for 5 minutes at room temperature. The supernatant was collected as rAAV sample.

[0126] Results rAAV samples produced by using the method described in Examples 5-1 and 5-3 were quantified. After the subsequent treatment with DNase I, the solution was diluted with water, and digital droplet PCR (QX ONE Droplet Digita PCR system, Bio-Rad Laboratories, Inc.) was performed using an ddPCR EvaGreen supermix (Bio-Rad Laboratories, Inc., catalog No. 186-4034), FAM probe, and primers designed for transgene construct. The vg concentrations of the supernatant were calculated. (FIG. 5)

[0127] As demonstrated in FIG. 5, it was confirmed that enhanced productivity is achieved with Example 5-3, as compared to what is achieved with Example 5-1 and that it worked equally well at the reactor.

Claims

CLAIMS1 . A method of producing a population of adeno-associated virus (AAV) particles, the method comprising:(a) providing a population of host cells;(b) introducing into the host cells one or more nucleic acids that encode one or more AAV components, thereby producing transfected host cells;(c) incubating the transfected host cells in a cell culture medium for a time sufficient for the cells to produce AAV particles, wherein during the incubating, a portion of the cell culture medium is exchanged with new medium; and(d) after step (c), lysing the host cells, thereby releasing the AAV particles.

2. A method of producing a population of AAV particles, the method comprising:(a) providing a population of transfected host cells comprising one or more nucleic acids that encode one or more AAV components;(b) incubating the transfected host cells in a cell culture medium for a time sufficient for the cells to produce AAV particles, wherein during the incubating, a portion of the cell culture medium is exchanged with new medium; and(c) after step (b), lysing the host cells, thereby releasing the AAV particles.

3. A method of producing a population of AAV particles, the method comprising:(a) culturing a population of host cells for a time sufficient for the host cells to proliferate;(b) introducing into the host cells, or progeny thereof, one or more nucleic acids that encode one or more AAV components, thereby producing transfected host cells;(c) incubating the transfected host cells in a cell culture medium for a time sufficient for the cells to produce AAV particles, wherein during the incubating, a portion of the cell culture medium is exchanged with new medium; and(d) after step (c), lysing the host cells, thereby releasing the AAV particles.

4. The method of claim 1 or 3, wherein upon introducing the one or more nucleic acids into the host cells or progeny thereof, the host cells or progeny thereof have a density of from 5 x 105cells / ml to 400 x 105cells / ml.

5. The method of claim 4, wherein upon introducing the one or more nucleic acids into the host cells or progeny thereof, the host cells or progeny thereof have a density of:(a) from 10 x 105cells / ml to 400 x 105cells / ml, from 20 x 105cells / ml to 400 x 105cells / ml, from 30 x 105cells / ml to 400 x 105cells / ml, from 40 x 105cells / ml to 400 x 105cells / ml, or from 50 x 105cells / ml to 400 x 105cells / ml; or(b) from 100 x 105cells / ml to 200 x 105cells / ml, from 1 10 x 105cells / ml to 190 x 105cells / ml, from 120 x 105cells / ml to 180 x 105cells / ml, from 100 x 105cells / ml to 180 x 105cells / ml, or from 100 x 105cells / ml to 160 x 105cells / ml.

6. The method of claim 4, wherein upon introducing the one or more nucleic acids into the host cells or progeny thereof, the host cells or progeny thereof have a density of from 100 x 105cells / ml to 160 x 105cells / ml.

7. The method of claim 2, wherein the population of transfected host cells is provided at a density of from 5 x 105cells / ml to 400 x 105cells / ml.

8. The method of claim 7, wherein the population of transfected host cells is provided at a density of:(a) from 10 x 105cells / ml to 400 x 105cells / ml, from 20 x 105cells / ml to 400 x 105cells / ml, from 30 x 105cells / ml to 400 x 105cells / ml, from 40 x 105cells / ml to 400 x 105cells / ml, or from 50 x 105cells / ml to 400 x 105cells / ml; or(b) from 100 x 105cells / ml to 200 x 105cells / ml, from 1 10 x 105cells / ml to 190 x 105cells / ml, from 120 x 105cells / ml to 180 x 105cells / ml, from 100 x 105cells / ml to 180 x 105cells / ml, or from 100 x 105cells / ml to 160 x 105cells / ml.

9. The method of claim 7, wherein the population of transfected host cells is provided at a density of from 100 x 105cells / ml to 160 x 105cells / ml.

10. The method of any one of claims 1 -9, wherein the host cells are mammalian cells.1 1 . The method of claim 10, wherein the mammalian cells are human embryonic kidney(HEK) cells or derivatives thereof.

12. The method of claim 1 1 , wherein the HEK cells are HEK293 cells.

13. The method of any one of claims 1 -12, wherein the one or more nucleic acids are introduced into the host cells by contacting the cells with (i) the one or more nucleic acids and (ii) a transfection reagent.

14. The method of claim 13, wherein the transfection reagent is a cationic polymer or a cationic lipid.

15. The method of any one of claims 1 -14, wherein during the incubating, the host cells are cultured until reaching a density of from 5 x 105cells / ml to 400 x 105cells / ml.

16. The method of claim 15, wherein during the incubating, the host cells are cultured until reaching a density of:(a) from 10 x 105cells / ml to 400 x 105cells / ml, from 20 x 105cells / ml to 400 x 105cells / ml, from 30 x 105cells / ml to 400 x 105cells / ml, from 40 x 105cells / ml to 400 x 105cells / ml, or from 50 x 105cells / ml to 400 x 105cells / ml; or(b) from 100 x 105cells / ml to 200 x 105cells / ml, from 1 10 x 105cells / ml to 190 x 105cells / ml, from 120 x 105cells / ml to 180 x 105cells / ml, from 100 x 105cells / ml to 180 x 105cells / ml, or from 100 x 105cells / ml to 160 x 105cells / ml.

17. The method of claim 15, wherein during the incubating, the host cells are cultured until reaching a density of from 100 x 105cells / ml to 160 x 105cells / ml.

18. The method of any one of claims 1 and 3-17, wherein the method comprises:(i) exchanging cell culture medium with new medium during step (a);(ii) temporarily ceasing to exchange cell culture medium with new medium during step (b); and(iii) resuming the exchanging of cell culture medium with new medium during step (c).

19. The method of any one of claims 1 -18, wherein the exchanging comprises replacing a portion of the cell culture medium with the new medium in one or discrete steps.

20. The method of claim 19, wherein each of the discrete steps comprises:(a) separating the host cells from surrounding cell culture media by way of centrifugation;(b) removing the surrounding cell culture media as a supernatant resulting from the centrifugation; and(c) supplementing the host cells with new cell culture media.21 . The method of any one of claims 1 -18, wherein the exchanging comprises continuously replacing a portion of the cell culture medium with the new medium.

22. The method of claim 21 , wherein the exchanging is performed by way of alternating tangential flow filtration (ATF).

23. The method of any one of claims 1 -22, wherein the exchanging comprises replacing at least 25% of the cell culture medium with new medium, optionally wherein the exchanging comprises replacing at least 30%, at least 40%, or at least 50% of the cell culture medium with new medium.

24. The method of claim 23, wherein the exchanging comprises replacing at least 60% of the cell culture medium with new medium, optionally wherein the exchanging comprises replacing at least 70%, at least 80%, or at least 90% of the cell culture medium with new medium.

25. The method of any one of claims 1 -24, wherein the transfected cells are incubated in a volume of cell culture medium of from 1 L to 1000 L.

26. The method of any one of claims 1 -24, wherein the transfected cells are incubated in a volume of cell culture medium of from 1 L to 10 L, 1 L to 2 L, 1 .6 L to 2 L, 8 L to 10 L, 40 L to 50 L, or 160 L to 200 L.

27. The method of any one of claims 1 -24, wherein the transfected cells are incubated in a volume of cell culture medium of from 1 mL to 1 L, 10 mL to 30 mL, or 20 mL to 40 mL, 50 mL to 100 mL, 100 mL to 180 mL, or 200 mL to 400 mL.

28. The method of any one of claims 1 -27, wherein the host cells are lysed by contacting the cells with a surfactant.

29. The method of any one of claims 1 -28, wherein the exchanging is performed from 1 hour to 24 hours following transfection of the host cells, optionally wherein the exchanging is performed about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours following transfection of the host cells.

30. The method of claim 29, wherein the exchanging is performed from 1 hour to 2 hours following transfection of the host cells.31 . The method of any one of claims 1 -30, wherein the one or more nucleic acids that encode one or more AAV components are introduced into the host cells by providing to the cells (i) a first nucleic acid encoding one or more AAV capsid (Cap) and AAV replication (Rep) proteins and, separately, (ii) a second nucleic acid comprising a transgene of interest and inverted terminal repeats (ITRs).

32. The method of claim 31 , wherein the host cells are further provided with (iii) a third nucleic acid comprising one or more adenoviral helper genes.

33. The method of any one of claims 1 -32, wherein the method further comprises purifying the released AAV particles following the lysing.

34. A population of AAV particles produced by the method of any one of claims 1 -33.

35. A cell culture mixture resulting from:(a) steps (a) to (c) of claim 1 ;(b) steps (a) and (b) of claim 2; or(c) steps (a) to (c) of claim 3.

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