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

Abstract

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

Description

[Technical Field]

[0001] Background technology Parvovirus gene therapy vectors, such as vectors based on adeno-associated virus (AAV), are an attractive method for delivering therapeutic transgenes to patients who need them.For example, AAV vectors have been used to successfully induce therapeutic protein expression in various loss-of-function disorders.However, despite these promising results, there is still a need for improved processes for producing AAV vectors.In particular, there is an increasing need for compositions and methods that can produce AAV particles with higher efficiency and higher titers. Summary of the Invention

[0002] This disclosure features compositions and methods useful for producing adeno-associated virus (AAV) particles. The compositions and methods described herein can be used to produce AAV particles at high titers and with improved efficiency compared to currently available methods.

[0003] 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 one or more nucleic acids encoding one or more AAV components into the host cells, thereby producing transfected host cells; (c) incubating the transfected host cells in cell culture medium for a time sufficient for the cells to produce AAV particles, wherein during the incubation, a portion of the cell culture medium is replaced with fresh medium; and (d) after step (c), lysing the host cells, thereby releasing the AAV particles.

[0004] 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 that include one or more nucleic acids encoding one or more AAV components; (b) incubating the transfected host cells in cell culture medium for a time sufficient for the cells to produce AAV particles, wherein a portion of the cell culture medium is replaced with fresh medium during the incubation; and (c) after step (b), lysing the host cells, thereby releasing the AAV particles.

[0005] 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 to allow the host cells to grow; (b) introducing one or more nucleic acids encoding one or more AAV components into the host cells or their progeny, thereby producing transfected host cells; (c) incubating the transfected host cells in cell culture medium for a time sufficient to allow the cells to produce AAV particles, wherein a portion of the cell culture medium is replaced with fresh medium during the incubation; and (d) after step (c), lysing the host cells, thereby releasing the AAV particles.

[0006] In some embodiments of the first and / or third aspects above, upon introduction of one or more nucleic acids into the host cell or its progeny, the host cell or its progeny may produce at least 5×10 5 cells / ml~400×10 5 cells / ml (e.g., approximately 5 x 10 5 cells / ml, 6×10 5 cells / ml, 7×10 5 cells / ml, 8×10 5 cells / ml, 9×10 5 cells / ml, 10×10 5 cells / ml, 20×10 5 cells / ml, 30×10 5 cells / ml, 40×10 5 cells / ml, 50×105 cells / ml, 60×10 5 cells / ml, 70×10 5 cells / ml, 80×10 5 cells / ml, 90×10 5 cells / ml, 100×10 5 cells / ml, 110×10 5 cells / ml, 120×10 5 cells / ml, 130×10 5 cells / ml, 140×10 5 cells / ml, 150×10 5 cells / ml, 160×10 5 cells / ml, 170×10 5 cells / ml, 180×10 5 cells / ml, 190×10 5 cells / ml, 200×10 5 cells / ml, 210×10 5 cells / ml, 220×10 5 cells / ml, 230×10 5 cells / ml, 240×10 5 cells / ml, 250×10 5 cells / ml, 260×10 5 cells / ml, 270×10 5 cells / ml, 280×10 5 cells / ml, 290×10 5 cells / ml, 300×10 5 cells / ml, 310×10 5 cells / ml, 320×10 5 cells / ml, 330×10 5 cells / ml, 340×10 5 cells / ml, 350×10 5 cells / ml, 360×10 5 cells / ml, 370×10 5 cells / ml, 380×10 5 cells / ml, 390×10 5 cells / ml, or 400 x 10 5 In some embodiments, upon introduction of the one or more nucleic acids into the host cell or its progeny, the host cell or its progeny has a density of: (a) 10 x 10 5 cells / ml~400×10 5cells / ml, 20×10 5 cells / ml~400×10 5 cells / ml, 30×10 5 cells / ml~400×10 5 cells / ml, 40×10 5 cells / ml~400×10 5 cells / ml, or 50 x 10 5 cells / ml~400×10 5 cells / ml; or (b) 100 × 10 5 cells / ml~200×10 5 cells / ml, 110×10 5 cells / ml~190×10 5 cells / ml, 120×10 5 cells / ml~180×10 5 cells / ml, 100×10 5 cells / ml~180×10 5 cells / ml, or 100 x 10 5 cells / ml~160×10 5 In some embodiments, upon introduction of the one or more nucleic acids into the host cell or its progeny, the host cell or its progeny has a density of 100×10 5 cells / ml~160×10 5 cells / ml (e.g., approximately 100 x 10 5 cells / ml, 110×10 5 cells / ml, 120×10 5 cells / ml, 130×10 5 cells / ml, 140×10 5 cells / ml, 150×10 5 cells / ml, or 160 x 10 5 The cells have a density of 1000 cells / ml.

[0007] In some embodiments of the second aspect above, the population of transfected host cells is 5×10 5 cells / ml~400×10 5 cells / ml (e.g., approximately 5 x 10 5 cells / ml, 6×10 5 cells / ml, 7×10 5 cells / ml, 8×10 5 cells / ml, 9×105 cells / ml, 10×10 5 cells / ml, 20×10 5 cells / ml, 30×10 5 cells / ml, 40×10 5 cells / ml, 50×10 5 cells / ml, 60×10 5 cells / ml, 70×10 5 cells / ml, 80×10 5 cells / ml, 90×10 5 cells / ml, 100×10 5 cells / ml, 110×10 5 cells / ml, 120×10 5 cells / ml, 130×10 5 cells / ml, 140×10 5 cells / ml, 150×10 5 cells / ml, 160×10 5 cells / ml, 170×10 5 cells / ml, 180×10 5 cells / ml, 190×10 5 cells / ml, 200×10 5 cells / ml, 210×10 5 cells / ml, 220×10 5 cells / ml, 230×10 5 cells / ml, 240×10 5 cells / ml, 250×10 5 cells / ml, 260×10 5 cells / ml, 270×10 5 cells / ml, 280×10 5 cells / ml, 290×10 5 cells / ml, 300×10 5 cells / ml, 310×10 5 cells / ml, 320×10 5 cells / ml, 330×10 5 cells / ml, 340×10 5 cells / ml, 350×10 5 cells / ml, 360×10 5 cells / ml, 370×10 5 cells / ml, 380×10 5 cells / ml, 390×10 5 cells / ml、または400×105 In some embodiments, the population of transfected host cells is provided at a density of: (a) 10 x 10 5 cells / ml~400×10 5 cells / ml, 20×10 5 cells / ml~400×10 5 cells / ml, 30×10 5 cells / ml~400×10 5 cells / ml, 40×10 5 cells / ml~400×10 5 cells / ml, or 50 x 10 5 cells / ml~400×10 5 cells / ml; or (b) 100 × 10 5 cells / ml~200×10 5 cells / ml, 110×10 5 cells / ml~190×10 5 cells / ml, 120×10 5 cells / ml~180×10 5 cells / ml, 100×10 5 cells / ml~180×10 5 cells / ml, or 100 x 10 5 cells / ml~160×10 5 In some embodiments, the population of transfected host cells is provided at a density of 100 x 10 cells / ml. 5 cells / ml~160×10 5 cells / ml (e.g., approximately 100 x 10 5 cells / ml, 110×10 5 cells / ml, 120×10 5 cells / ml, 130×10 5 cells / ml, 140×10 5 cells / ml, 150×10 5 cells / ml, or 160 x 10 5 The cells are provided at a density of 1000 cells / ml.

[0008] In some embodiments of any of the foregoing aspects or embodiments of the present disclosure, the host cell is a mammalian cell. In some embodiments, the mammalian cell is a human embryonic kidney (HEK) cell or a derivative thereof. In some embodiments, the HEK cell is a HEK293 cell. In some embodiments, the host cell is an insect cell. In some embodiments, the insect cell is an Sf9 cell.

[0009] In some embodiments, the one or more nucleic acids are introduced into a host cell by contacting the cell 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 embodiments, the cationic polymer is FectoVIR®, polyethyleneimine (PEI), or PEI-pro®. In some embodiments, the cationic lipid is AAV-MAX.

[0010] In some embodiments, one or more nucleic acids are introduced into the host cell using a technique selected from the group consisting of electroporation, nucleofection, squeezeporation, sonoporation, optical transfection, magnetofection, and impalefection.

[0011] In some embodiments, during incubation, the host cells are cultured at a density of 5×10 5 cells / ml~400×10 5 cells / ml (e.g., approximately 5 x 10 5 cells / ml, 6×10 5 cells / ml, 7×10 5 cells / ml, 8×10 5cells / ml, 9×10 5 cells / ml, 10×10 5 cells / ml, 20×10 5 cells / ml, 30×10 5 cells / ml, 40×10 5 cells / ml, 50×10 5 cells / ml, 60×10 5 cells / ml, 70×10 5 cells / ml, 80×10 5 cells / ml, 90×10 5 cells / ml, 100×10 5 cells / ml, 110×10 5 cells / ml, 120×10 5 cells / ml, 130×10 5 cells / ml, 140×10 5 cells / ml, 150×10 5 cells / ml, 160×10 5 cells / ml, 170×10 5 cells / ml, 180×10 5 cells / ml, 190×10 5 cells / ml, 200×10 5 cells / ml, 210×10 5 cells / ml, 220×10 5 cells / ml, 230×10 5 cells / ml, 240×10 5 cells / ml, 250×10 5 cells / ml, 260×10 5 cells / ml, 270×10 5 cells / ml, 280×10 5 cells / ml, 290×10 5 cells / ml, 300×10 5 cells / ml, 310×10 5 cells / ml, 320×10 5 cells / ml, 330×10 5 cells / ml, 340×10 5 cells / ml, 350×10 5 cells / ml, 360×10 5 cells / ml, 370×10 5 cells / ml, 380×10 5 cells / ml, 390×10 5cells / ml, or 400 x 10 5 In some embodiments, during incubation, the host cells are cultured to reach a density of: (a) 10 x 10 5 cells / ml~400×10 5 cells / ml, 20×10 5 cells / ml~400×10 5 cells / ml, 30×10 5 cells / ml~400×10 5 cells / ml, 40×10 5 cells / ml~400×10 5 cells / ml, or 50 × 10 5 cells / ml~400×10 5 cells / ml; or (b) 100 × 10 5 cells / ml~200×10 5 cells / ml, 110×10 5 cells / ml~190×10 5 cells / ml, 120×10 5 cells / ml~180×10 5 cells / ml, 100×10 5 cells / ml~180×10 5 cells / ml, or 100 x 10 5 cells / ml~160×10 5 In some embodiments, the host cells are cultured until they reach a density of 100×10 cells / ml during incubation. 5 cells / ml~160×10 5 cells / ml (e.g., approximately 100 x 10 5 cells / ml, 110×10 5 cells / ml, 120×10 5 cells / ml, 130×10 5 cells / ml, 140×10 5 cells / ml, 150×10 5 cells / ml, or 160 x 10 5 The cells are cultured until they reach a density of 1000 cells / ml.

[0012] In some embodiments, the method further comprises (i) replacing the cell culture medium with fresh medium during step (a), (ii) temporarily pausing to replace the cell culture medium with fresh medium during step (b), and (iii) resuming replacing the cell culture medium with fresh medium during step (c). In some embodiments, the replacement comprises replacing a portion of the cell culture medium with fresh medium in one or separate steps. In some embodiments, each of the separate steps comprises separating the host cells from the surrounding cell culture medium by centrifugation, removing the surrounding cell culture medium as a supernatant resulting from the centrifugation, and replenishing the host cells with fresh cell culture medium.

[0013] In some embodiments, this exchange involves continuously replacing a portion of the cell culture medium with fresh medium.

[0014] In some embodiments, the exchange is performed by alternating tangential flow filtration (ATF). In some embodiments, the exchange 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).

[0015] In some embodiments, the transfected cells are incubated in a volume of 1 L to 1000 L of cell culture medium (e.g., 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 of cell culture medium). In some embodiments, the transfected cells are incubated in a volume of 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 to 200 L of cell culture medium. In some embodiments, the transfected cells are incubated in a volume of cell culture medium between 1 mL and 1 L, between 10 mL and 30 mL, or between 20 mL and 40 mL, between 50 mL and 100 mL, between 100 mL and 180 mL, or between 200 mL and 400 mL.

[0016] In some embodiments, the host cells are lysed by contacting the cells with a detergent, hi some embodiments, the host cells are lysed in the presence of one or more reagents such as Triton X-100, PS-20, PS-80, sodium deoxycholate, poloxamer, Tris-HCl, sodium chloride, and / or citric acid.

[0017] In some embodiments, the exchange occurs between 1 hour and 24 hours after 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 after transfection of the host cells). In some embodiments, the exchange occurs between 1 hour and 2 hours after transfection of the host cells.

[0018] In some embodiments, the exchange begins 1 hour to 24 hours after 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 after transfection of the host cells). In some embodiments, the exchange occurs between 1 hour and 24 hours after 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 after transfection of the host cells) and is continued for up to 48 hours post-transfection. In some embodiments, the exchange begins between 1 hour and 2 hours after transfection of the host cells. In some embodiments, the exchange begins between 1 hour and 2 hours after transfection of the host cells and is continued for up to 48 hours post-transfection.

[0019] In some embodiments, one or more nucleic acids encoding one or more AAV components are introduced into a host cell by providing the cell with (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 cell is further provided with (iii) a third nucleic acid comprising one or more adenoviral helper genes.

[0020] In some embodiments, the one or more AV Cap proteins are derived 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.

[0021] In some embodiments, the one or more AAV Rep proteins include AAV Rep40, AAV Rep52, AAV Rep68, and / or AAV Rep78.

[0022] In some embodiments, the method further comprises purifying the released AAV particles after lysis, hi some embodiments, the AAV particles are purified by a process comprising one or more of affinity chromatography, ion exchange chromatography (e.g., anion exchange chromatography), and filtration.

[0023] In a further aspect, the disclosure features a population of AAV particles produced by the method of any one of the aforementioned aspects or embodiments of the disclosure.

[0024] In another aspect, the disclosure features a cell culture mixture obtained from steps (a)-(c) of the first aspect of the disclosure (or any one or more embodiments thereof).

[0025] In another aspect, the disclosure features a cell culture mixture obtained from steps (a)-(b) of the second aspect of the disclosure (or any one or more embodiments thereof).

[0026] In another aspect, the disclosure features a cell culture mixture obtained from steps (a)-(c) of the third aspect of the disclosure (or any one or more embodiments thereof). [Brief explanation of the drawings]

[0027] [Figure 1] 1 is a graph showing the amount 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 the vector genome (vg) titer in cell lysates (calculated using ITR primers). [Figure 2]1 is a graph showing the amount 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) titers in cell lysates (calculated using gene of interest (GOI)-specific primers). [Figure 3] 1 is a graph showing the amount of AAV particles produced from the experiment described in Example 3 below. The vertical axis shows the vector genome (vg) titer in cell lysates (calculated using ITR primers). [Figure 4] 1 is a graph showing the amount of AAV particles produced from various cell density conditions described below in Example 4. The vertical axis shows the vector genome (vg) titer in the cell lysate (calculated using ITR primers). [Figure 5] 1 is a graph showing the amount of AAV particles produced from the experiments described in Examples 1-1, 5-1, and 5-3 below. The vertical axis shows the vector genome (vg) titer in cell lysates (calculated using GOI-specific primers). DETAILED DESCRIPTION OF THE INVENTION

[0028] definition As used herein, the term "about" refers to a value that is within 10% of the stated value. For example, "100 liters (L)" as used in the context of volumes described herein includes amounts within 10% above and below 100 L. Furthermore, when used in the context of a list of numerical quantities, the term "about" preceding the list of numerical quantities should be understood to apply to each individual amount recited in the list.

[0029] As used herein, the terms "adeno-associated virus component," "AAV component," and the like refer to elements of a recombinantly expressed AAV particle. That is, AAV components include AAV capsid (Cap) and AAV replication (Rep) proteins, as well as a gene of interest flanked by one or more AAV inverted terminal repeats (ITRs). AAV components may also include adenovirus helper genes that facilitate the formation of packaged AAV particles. Exemplary AAV Cap proteins, Rep proteins, ITRs, and adenovirus helper genes include those known in the art and described herein.

[0030] As used herein, the terms "administering," "administration," and the like refer to providing a therapeutic agent directly to a patient by any effective route. Exemplary routes of administration are described herein and include systemic routes such as intravenous injection.

[0031] As used herein, the terms "cell culture medium," "cell culture medium," and the like refer to a mixture (e.g., an aqueous mixture) containing one or more components that promote the growth, proliferation, and / or survival of cells (e.g., host cells described herein).

[0032] As used herein, the term "exchange" when used with respect to cell culture medium refers to the process of replacing at least a portion of existing cell culture medium with new medium. New medium may be fresh medium that has not previously been exposed to cells (e.g., host cells as described herein), or it may be medium that has been exposed to a population of cells (e.g., host cells) but that retains components that promote cell growth, proliferation, and / or survival. Cell culture medium exchange may be continuous or may occur in one or more discrete steps.

[0033] Cell culture medium exchange may be performed, for example, by 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 tangentially (e.g., at high velocity) relative to the plane of a membrane. In such filtration, a pressure differential may be applied along the length of the membrane, which in turn causes the fluid and filterable solutes to flow through the filter. This filtration is suitably performed as a batch process and as a continuous flow process. For example, a solution may be repeatedly passed over the membrane while the fluid passing through the filter is continuously discharged to another unit, or the solution may be passed once through the membrane and the fluid passing through the filter is continuously processed downstream.

[0034] In some embodiments, cell culture medium exchange can be performed by alternating tangential flow filtration (ATF). ATF is a filtration technology similar to TFF. In TFF, the liquid in contact with the membrane flows in only one direction, whereas in ATF, the direction of liquid flow alternates at regular intervals. In some ATF embodiments, a diaphragm pump alternates between positive pressure and vacuum to move the retentate back and forth through the membrane, while removing and collecting the permeate. The retentate may, for example, return to the bioreactor during each cycle and mix with the next batch of fluid to be filtered. Because the retentate is continuously pushed back into the bioreactor, it is desirable to use ATF to continuously exchange the medium in the cell culture mixture.

[0035] As used herein, the term "pharmaceutical composition" refers to a mixture containing therapeutic compounds that is administered to a subject, such as a mammal, e.g., a human, to prevent, treat, or control a particular disease or condition from which the subject is suffering or may suffer.

[0036] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human), without undue toxicity, irritation, allergic response, and other significant complications, commensurate with a reasonable benefit / risk ratio.

[0037] As used herein, the term "promoter" refers to a recognition site on DNA to which RNA polymerase binds. The polymerase promotes transcription of the transgene. Exemplary promoters suitable for use in the compositions and methods described herein are described, for example, in Sandelin et al., Nature Reviews Genetics 8:424 (2007), the disclosure of which pertains to nucleic acid regulatory elements and is incorporated herein by reference. Additionally, the term "promoter" can refer to a synthetic promoter, which is a regulatory DNA sequence that does not naturally occur in a biological system. Synthetic promoters contain portions of naturally occurring promoters combined with non-naturally occurring polynucleotide sequences and can be optimized for recombinant DNA expression using a variety of transgenes, vectors, and target cell types.

[0038] As used herein, a therapeutic agent is considered to be "provided" to a host cell when the agent is administered directly to the host cell, or when a substance is administered to the host cell that is processed or metabolized by the cell such that the therapeutic agent is obtained endogenously. For example, a host cell can be provided with a nucleic acid molecule encoding a therapeutic protein by administering the nucleic acid molecule directly to the cell, or by administering a substance (e.g., a viral vector) that is processed intracellularly to produce the desired nucleic acid molecule.

[0039] As used herein, the terms "patient" and "subject" refer to an organism being treated for a particular disease or condition described herein. Examples of subjects and patients include mammals, such as humans, undergoing treatment for a disease or condition described herein.

[0040] As used herein, the terms "transfect," "transfection," and the like refer to the process by 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) to express that gene in the desired cell (e.g., to produce AAV in the host cell). Exemplary transfection techniques include the use of non-viral plasmids to introduce heterologous genes into the desired cell. The section below, entitled "Methods for Transfecting a Target Cell to Express AAV Components," provides additional non-limiting examples of transfection techniques that can be used in conjunction with the compositions and methods of the present disclosure.

[0041] As used herein, the term "transgene" refers to a recombinant nucleic acid (e.g., DNA or cDNA) that encodes a gene product (e.g., a gene product described herein). The gene product may be RNA, a peptide, or a 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 promote or enhance expression, such as a promoter, enhancer(s), destabilization domain(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s), and / or other functional elements. Embodiments of the present disclosure may utilize any known suitable promoter(s), enhancer(s), destabilization domain(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s), and / or other functional elements.

[0042] As used herein, the terms "treat" and "treatment" refer to therapeutic treatment, where the objective is to prevent or delay (alleviate) an undesired physiological change or disorder. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, a stable (i.e., not worsening) disease state, delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or complete).

[0043] As used herein, the term "vector" refers to a nucleic acid, e.g., DNA or RNA, that can function as a vehicle to deliver a gene of interest to a cell (e.g., a mammalian cell, such as a human cell), for purposes such as 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 replicons (e.g., viral vectors). Various vectors have been developed for delivering polynucleotides encoding exogenous proteins to prokaryotic or eukaryotic cells. Examples of such expression vectors are disclosed, for example, in WO 1994 / 11026, the disclosure of which is incorporated herein by reference. The expression vectors described herein contain not only polynucleotide sequences but also additional sequence elements used, for example, for protein expression and / or integration of these polynucleotide sequences into the genome of a mammalian cell. Particular vectors that can be used to express the transgenes described herein include plasmids containing regulatory sequences, such as promoter and enhancer regions, that drive gene transcription.

[0044] Detailed Description The present disclosure provides compositions and methods that can be used to produce adeno-associated virus (AAV) particles. The compositions and methods described herein can be used to produce AAV particles at increased titers and / or with improved efficiency compared to currently available methods. In some embodiments of the present disclosure, AAV particles are produced by: (a) providing a population of host cells; (b) introducing one or more nucleic acids encoding one or more AAV components into the host cells, thereby producing transfected host cells; (c) incubating the transfected host cells in cell culture medium for a time sufficient for the cells to produce AAV particles, wherein a portion of the cell culture medium is replaced with fresh medium during incubation; and (d) after step (c), lysing the host cells, thereby releasing the AAV particles.

[0045] In some embodiments, the disclosure features a method of producing a population of AAV particles by: (a) providing a population of transfected host cells that include one or more nucleic acids encoding one or more AAV components; (b) incubating the transfected host cells in cell culture medium for a time sufficient for the cells to produce AAV particles, wherein a portion of the cell culture medium is replaced with fresh medium during the incubation; and (c) after step (b), lysing the host cells, thereby releasing the AAV particles.

[0046] 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 one or more nucleic acids encoding one or more AAV components into the host cells or their progeny, thereby producing transfected host cells; (c) incubating the transfected host cells in cell culture medium for a time sufficient for the cells to produce AAV particles, wherein a portion of the cell culture medium is replaced with fresh medium during the incubation; and (d) after step (c), lysing the host cells, thereby releasing the AAV particles.

[0047] The compositions and methods of the present disclosure are based, in part, on the discovery that changing the cell culture medium after transfecting the host cells to express the corresponding AAV component(s) surprisingly increases the production of AAV particles from a population of host cells. As shown in the Examples below, it has now been discovered that performing a cell culture medium change after transfecting such host cells to express one or more AAV components results in unexpected improvements in AAV yield and efficiency.

[0048] The sections that follow provide descriptions of exemplary host cells, transfection techniques, and AAV components that can be used in conjunction with the compositions and methods of the present disclosure.

[0049] Exemplary AAV Vectors of the Disclosure Recombinant AAV vectors useful in the present invention are recombinant nucleic acid constructs that can contain (1) a transgene to be expressed (e.g., a polynucleotide encoding a therapeutic protein or therapeutic RNA molecule) and (2) viral nucleic acid that facilitates the integration and expression of the heterologous gene. The viral nucleic acid can include those AAV sequences required in cis for DNA replication and packaging into virions (e.g., functional ITRs). rAAV vectors of the present disclosure can also contain marker or reporter genes. Useful rAAV vectors can have one or more AAV wild-type genes deleted, in whole or in part, but retain functional flanking ITR sequences. AAV ITRs can be of any serotype (e.g., from serotype 2) suitable for a particular application. Methods for using rAAV vectors are described, for example, in Tal et al., J. Biomed. Sci. 7:279-291 (2000), and Monahan and Samulski, Gene Delivery 7:24-30 (2000), the disclosures of each of which are incorporated herein by reference with respect to AAV vectors for gene delivery.

[0050] The nucleic acids described herein can be incorporated into AAV virions to facilitate the introduction of the nucleic acid into cells. The AAV capsid protein constitutes the outer, non-nucleic acid portion of the virion and is encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2, and VP3, required for virion assembly. Construction of AAV virions is described, for example, in U.S. Pat. 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 relate to AAV vectors for gene delivery and are incorporated herein by reference.

[0051] AAV virions useful in connection with the compositions and methods described herein include those derived from various AAV serotypes, including AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. The 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 relate to AAV vectors for gene delivery and are incorporated herein by reference).

[0052] AAV virions with mutations in the virion capsid can be used to infect certain cell types more efficiently than non-mutated capsid virions. Other AAV virions that can be used in the methods of the present invention include capsid hybrids.

[0053] Host cells for producing AAV particles Various types of host cells can be used to produce AAV particles using the compositions and methods of the present disclosure. In some embodiments, the host cell is a mammalian cell. In some embodiments, the mammalian cell is a human embryonic kidney (HEK) cell or a derivative thereof. In some embodiments, the HEK cell is a HEK293 cell. In some embodiments, the host cell is an insect cell. In some embodiments, the insect cell is an Sf9 cell.

[0054] Methods for transfecting target cells to express AAV components Various methods can be used to transfect target cells to express AAV components (and thus produce AAV particles). Transfection techniques that can be used to express one or more desired AAV components in host cells include methods in which nucleic acids encoding the AAV component(s) are introduced into host cells (e.g., human cells, particularly mammalian cells such as HEK cells 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, included in the Gibco™ AAV-MAX Transfection Kit (see, e.g., Thermo Fisher Scientific catalog number 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.

[0055] Additional techniques that can be used to transfect the host cells of the present disclosure to express one or more AAV components include electroporation, which can be used to permeabilize mammalian cells (e.g., human target cells) by applying an electrostatic potential to the target cells.Mammalian cells, such as human cells, exposed to an external electric field in this way are then susceptible to the uptake of exogenous nucleic acid.Electroporation of mammalian cells is described in detail, for example, in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference.

[0056] A similar technique, Nucleofection™, utilizes an applied electric field to stimulate the uptake of exogenous polynucleotides into the nuclei of eukaryotic cells. Nucleofection™ and protocols useful for carrying out this technique are described in detail, for example, in Distler et al., Experimental Dermatology 14:315 (2005), and U.S. Patent Application Publication No. 2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0057] Another useful technique for transfecting host cells is squeezeporation. This technique induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membrane pores that form in response to applied stress. This technique is advantageous in that it does not require a vector to deliver nucleic acid to cells, such as human target cells. Squeezeporation is described in detail, for example, in Sharei et al., J. Vis. Exp. 81: e50980 (2013), the disclosure of which is incorporated herein by reference.

[0058] Lipofection is another technique useful for transfecting host cells. This method involves loading nucleic acids into liposomes, which often present cationic functional groups, such as quaternary amines or protonated amines, on the exterior of the liposomes. This, due to the anionic nature of the cell membrane, promotes electrostatic interactions between the liposomes and cells, ultimately leading to the uptake of 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 U.S. Patent No. 7,442,386, the disclosure of which is incorporated herein by reference. A similar technique that utilizes ionic interactions with the cell membrane to induce the uptake of exogenous nucleic acids is to contact cells with a cationic polymer-nucleic acid complex. Exemplary cationic molecules that associate with polynucleotides to impart a positive charge favorable for interaction with cell membranes are activated dendrimers (described, e.g., in Dennig, Top Curr Chem. 228:227 (2003), the disclosure of which is incorporated herein by reference), polyethyleneimine, and DEAE-dextran, the use of which as transfection agents is described in detail, e.g., 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.

[0059] Another useful tool for inducing the uptake of exogenous nucleic acids by host cells is laser transfection, also known as phototransfection, which is a technique that involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. The biological activity of this technique is similar to, and in some cases, superior to, electroporation.

[0060] Impalefection is another technique that can be used to deliver genetic material into 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 genes intended for intracellular delivery is attached to the surface of the nanostructures. A tip with an array of these needles is then pressed against cells or tissues. Cells impaled by the 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.

[0061] Magnetofection™ may also be used to deliver nucleic acids into host cells. The principle of Magnetofection™ is to associate nucleic acids with cationic magnetic nanoparticles. The magnetic nanoparticles are made entirely of biodegradable iron oxide and are coated with specific cationic specific molecules that vary depending on the application. Their association with gene vectors (DNA or RNA vectors) is achieved by salt-induced colloidal aggregation and electrostatic interactions. The magnetic particles are then concentrated on target cells under the influence of an external magnetic field generated by a magnet. This technique is described in detail in Scherer et al., Gene Ther. 9:102 (2002), the disclosure of which is incorporated herein by reference.

[0062] Magnetic beads are another tool that can be used to transfect host cells in a gentle and efficient manner, as this method utilizes an applied magnetic field to induce the uptake of nucleic acids. This technique is described in detail, for example, in US2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0063] Another useful tool for inducing the uptake of exogenous nucleic acid by host cells is sonoporation, which is a technique that involves using sound (usually ultrasonic frequencies) to change the permeability of cell plasma membrane, thereby permeabilizing the cell and allowing polynucleotides to penetrate the cell membrane.This technique is described in detail, for example, in Rhodes et al., Methods Cell Biol.82:309 (2007), the disclosure of which is incorporated herein by reference.

[0064] Microvesicles represent another potential vehicle that can be used to transfect desired host cells according to the methods described herein. For example, microvesicles induced by the co-overexpression of glycoprotein VSV-G and a genome-modifying protein, such as a nuclease, can be used to efficiently deliver proteins that subsequently catalyze the site-specific cleavage of endogenous polynucleotide sequences to cells, preparing the genome of the cell for the covalent integration of a target polynucleotide, such as a gene or regulatory sequence. The use of such vesicles, also known as gesicles, for the genetic modification of eukaryotic cells is described in detail, for example, 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.

[0065] Exemplary Cell Culture Medium Exchange Techniques In a preferred embodiment of the present disclosure, host cells are subjected to cell culture medium exchange before being transfected to express desired AAV component(s).During transfection, cell culture medium exchange is temporarily stopped.Then, after transfection is completed, cell culture medium exchange is resumed.

[0066] In some embodiments, the replacement involves replacing a portion of the cell culture medium with new medium in one or separate steps, each of which includes separating the host cells from the surrounding cell culture medium by centrifugation, removing the surrounding cell culture medium as a supernatant resulting from the centrifugation, and replenishing the host cells with the new cell culture medium.

[0067] In some embodiments, this exchange involves continuously replacing a portion of the cell culture medium with fresh medium. An exemplary method for continuously exchanging cell culture medium includes tangential flow filtration (TFF). In some embodiments, the continuous cell culture medium exchange is performed by alternating tangential flow filtration (ATF).

[0068] In some embodiments, the exchange is stopped while the host cells are transfected to express one or more AAV components. In the context of the ATF procedure, the exchange can be "stopped" by stopping continuous medium exchange during the transfection process (i.e., when the host cells are contacted with the genetic material encoding one or more AAV components). Alternatively, the exchange can be reduced to a lower exchange rate during the transfection process so that the transfection efficiency of the host cells is not affected.

[0069] In some embodiments, if the host cells are subjected to a medium change after the transfection step, the medium change can be initiated any time after completion of transfection (i.e., after introducing one or more nucleic acid molecules encoding one or more AAV components into the host cells). For example, the medium change can be initiated 1 to 24 hours after transfection of the host cells (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after transfection of the host cells). In some embodiments, the exchange occurs between 1 hour and 24 hours after 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 after transfection of the host cells) and is continued for up to 48 hours post-transfection. In some embodiments, the exchange begins between 1 hour and 2 hours after transfection of the host cells. In some embodiments, the exchange begins between 1 hour and 2 hours after transfection of the host cells and is continued for up to 48 hours post-transfection.

[0070] Exemplary cell densities In a preferred embodiment of the present disclosure, after transfecting the host cells to express one or more desired AAV components, the host cells are cultured until a target density is reached. For example, after transfecting the host cells to express the desired AAV component(s), the host cells are cultured to a density of 5×10 5 cells / ml~400×10 5 cells / ml (e.g., approximately 5 x 10 5 cells / ml, 6×10 5 cells / ml, 7×10 5 cells / ml, 8×10 5 cells / ml, 9×10 5 cells / ml, 10×105 cells / ml, 20×10 5 cells / ml, 30×10 5 cells / ml, 40×10 5 cells / ml, 50×10 5 cells / ml, 60×10 5 cells / ml, 70×10 5 cells / ml, 80×10 5 cells / ml, 90×10 5 cells / ml, 100×10 5 cells / ml, 110×10 5 cells / ml, 120×10 5 cells / ml, 130×10 5 cells / ml, 140×10 5 cells / ml, 150×10 5 cells / ml, 160×10 5 cells / ml, 170×10 5 cells / ml, 180×10 5 cells / ml, 190×10 5 cells / ml, 200×10 5 cells / ml, 210×10 5 cells / ml, 220×10 5 cells / ml, 230×10 5 cells / ml, 240×10 5 cells / ml, 250×10 5 cells / ml, 260×10 5 cells / ml, 270×10 5 cells / ml, 280×10 5 cells / ml, 290×10 5 cells / ml, 300×10 5 cells / ml, 310×10 5 cells / ml, 320×10 5 cells / ml, 330×10 5 cells / ml, 340×10 5 cells / ml, 350×10 5 cells / ml, 360×10 5 cells / ml, 370×10 5 cells / ml, 380×10 5 cells / ml, 390×10 5 cells / ml、または400×10 5The cells are cultured until they reach a density of 1000 cells / ml.

[0071] In some embodiments, after transfection of the host cells to express one or more desired AAV components, the host cells are grown at the following densities: (a) 10 x 10 5 cells / ml~400×10 5 cells / ml, 20×10 5 cells / ml~400×10 5 cells / ml, 30×10 5 cells / ml~400×10 5 cells / ml, 40×10 5 cells / ml~400×10 5 cells / ml, or 50 × 10 5 cells / ml~400×10 5 cells / ml; or (b) 100 × 10 5 cells / ml~200×10 5 cells / ml, 110×10 5 cells / ml~190×10 5 cells / ml, 120×10 5 cells / ml~180×10 5 cells / ml, 100×10 5 cells / ml~180×10 5 cells / ml, or 100 x 10 5 cells / ml~160×10 5 In some embodiments, after transfection of the host cells to express one or more desired AAV components, the host cells are cultured until they reach a density of 100×10 cells / ml. 5 cells / ml~160×10 5 cells / ml (e.g., approximately 100 x 10 5 cells / ml, 110×10 5 cells / ml, 120×10 5 cells / ml, 130×10 5 cells / ml, 140×10 5 cells / ml, 150×10 5 cells / ml, or 160 x 10 5 The cells are cultured until they reach a density of 1000 cells / ml.

[0072] Pharmaceutical Composition AAV particles produced using the compositions and methods described herein can include a transgene, such as a transgene encoding a therapeutic protein or a therapeutic RNA molecule. AAV particles produced using the compositions and methods described herein can be incorporated into a vehicle for administration to a patient, such as a human patient suffering from a disease or disorder. Pharmaceutical compositions of the present disclosure can be formulated into a desired form, such as a lyophilized or aqueous formulation, using, for example, physiologically acceptable carriers, excipients, or stabilizers (see, for example, Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) (incorporated herein by reference)).

[0073] The AAV vectors described herein can include one or more transcriptional regulatory elements operably linked to a desired therapeutic transgene. Such vectors can be administered to a patient (e.g., a human patient) by a variety of administration routes. The administration route can vary, for example, depending on the onset and severity of the disease, and can include, for example, intravenous administration. [Example]

[0074] The following examples are presented to provide one of ordinary skill in the art with an understanding of how the compositions and methods described herein can 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.

[0075] Example 1-1: Production of rAAV8 vectors by general methods Viral Production Cells 2.0 (VPC2.0, Thermo Fisher Scientific, catalog number A49784) in Viral Production Medium (VPM, Thermo Fisher Scientific, catalog number A4817901) containing 4% GlutaMAX™ Supplement (GultaMAX, Thermo Fisher Scientific, catalog number 35050061) were cultured at a density of 38 × 10 5 The cells were diluted to a density of 1000 cells / ml. 20 mL of the cell suspension was placed in a 125 mL shake flask. Gibco AAV-MAX Enhancer, a component of the AAV-MAX Transfection Kit (Thermo Fisher Scientific, catalog number A50515), was added to the cell suspension. The transgene plasmid construct and the helper plasmids RC_AAV and helper were added to Viral-Plex™ Complexation Buffer (Viral-Plex, Thermo Fisher Scientific, catalog number 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. After 10 minutes, the complex was added to the flask. Three days after transfection, Triton X-100 was added to the medium at a concentration of 0.2% and allowed to stand at room temperature for 1 hour to lyse the cells. The resulting solution was centrifuged at 10,000 g for 5 minutes at room temperature. The supernatant was collected as the rAAV sample.

[0076] Example 1-2: Production of rAAV8 vectors using the ATF method Viral producer cells 2.0 (VPC2.0, Thermo Fisher Scientific, Cat. No. A49784) in Viral Production Medium (VPM, Thermo Fisher Scientific, Cat. No. A4817901) containing 4% GlutaMAX™ Supplement (GultaMAX, Thermo Fisher Scientific, Cat. No. 35050061) were cultured at a density of 38 × 10 5 The cells were diluted to a density of 90 x 10 cells / ml. The cell suspension was centrifuged at 400 g for 4 minutes at room temperature. A portion of the supernatant was then removed to obtain a high cell density, and the cells were resuspended. 5 A cell suspension of 1000 cells / ml was prepared. 20 mL of the cell suspension was placed in a 125 mL shake flask. The transgene plasmid construct and the helper plasmids RC_AAV and helper were added to Viral-Plex™ complexing buffer (Viral-Plex, Thermo Fisher Scientific, catalog number A4983901). The remaining two reagents from the AAV-MAX transfection kit were added to the complexing buffer in the order described in the kit protocol. After 10 minutes, the complex was added to the flask. Three days after transfection, Triton X-100 was added to the medium at a concentration of 0.2% and allowed to stand at room temperature for 1 hour to lyse the cells. The resulting solution was centrifuged at 10,000 g for 5 minutes at room temperature. The supernatant was collected as the rAAV sample.

[0077] Example 1-3: Production of rAAV8 vectors by HCD method Viral producer cells 2.0 (VPC2.0, Thermo Fisher Scientific, Cat. No. A49784) in Viral Production Medium (VPM, Thermo Fisher Scientific, Cat. No. A4817901) containing 4% GlutaMAX™ Supplement (GultaMAX, Thermo Fisher Scientific, Cat. No. 35050061) were cultured at a density of 38 × 10 5 The cells were diluted to a density of 99 x 10 cells / ml. The cell suspension was centrifuged at 400 g for 4 minutes at room temperature. A portion of the supernatant was then removed to obtain a high cell density, and the cells were resuspended. 5 A cell suspension of 20 cells / ml was prepared. 20 mL of the cell suspension was placed in a 125 mL shake flask.

[0078] The transgene plasmid construct and the helper plasmid RC_AAV were added to Viral-Plex™ complexing buffer (Viral-Plex, Thermo Fisher Scientific, catalog number A4983901). The remaining two reagents from the AAV-MAX transfection kit were added to the complexing buffer in the order described in the kit's protocol. After 10 minutes, the complex was added to the flask. One day after transfection, the cell suspension was centrifuged at 400g for 4 minutes at room temperature. The spent half was then removed, and an equal volume of fresh VPM containing 4% GultaMAX was added. The cells were resuspended, and the cell suspension was cultured in an incubator shaker. Two days after transfection, the cell suspension was centrifuged at 400g for 4 minutes at room temperature. The spent half was then removed, and an equal volume of fresh VPM containing 4% GultaMAX was added. The cells were resuspended, and the cell suspension was cultured in an incubator shaker. Three days after transfection, 0.2% Triton X-100 was added to the culture medium and allowed to stand at room temperature for 1 hour to lyse the cells. The resulting solution was centrifuged at 10,000 g for 5 minutes at room temperature. The supernatant was collected as the rAAV sample.

[0079] result rAAV samples produced using the methods described in Examples 1-1, 1-2, and 1-3 were quantified. After subsequent treatment with DNase I, AAV genomic DNA was extracted and the solution diluted with water. Real-time PCR was then performed using the AAVpro® Titration Kit for Real-Time PCR (Takara Bio Inc., Catalog No. 6233) and the ITR primers provided with the kit. A calibration curve was created using the standard DNA provided with the kit, and the vg concentration of the supernatant was calculated (Figure 1).

[0080] As shown in FIG. 1, it was confirmed that Example 1-3 exhibited improved productivity compared to that achieved in Examples 1-1 and 1-2.

[0081] Example 2-1: Production of rAAV8 vectors by general methods Virus-producing cells 2.0 (VPC2.0, Thermo Fisher Scientific, Cat. No. A49784) were cultured at 30 × 10 5 The cells were diluted to a density of 1000 cells / ml. 20 mL of the cell suspension was placed in a 125 mL shake flask. The transgene plasmid construct and helper plasmids RC_AAV and helper were added to PBS (PBS pH 7.4, Thermo Fisher Scientific, catalog number 10010023). FectoVIR (FectoVIR®-AAV 10 mL, Polyplus, catalog number 101000022) was added to the PBS. After 5 minutes, the complex was added to the flask. Two days after transfection, Triton X-100 was added to the culture medium at a concentration of 0.5% and left at room temperature for 1 hour to lyse the cells. The resulting solution was centrifuged at 10,000 g for 5 minutes at room temperature. The supernatant was collected as the rAAV sample.

[0082] Example 2-2: Production of rAAV8 vectors using the ATF method Virus-producing cells 2.0 (VPC2.0, Thermo Fisher Scientific, Cat. No. A49784) were cultured at 30 × 10 5 Dilute to a density of 90 x 10 cells / ml. Place 20 mL of cell suspension into a 125 mL shake flask. Centrifuge this cell suspension at 400 g for 4 minutes at room temperature. Then remove part of the supernatant to obtain a high cell density and resuspend the cells. 5 A cell suspension of 1000 cells / ml is prepared. 20 mL of the cell suspension is placed in a 125 mL shake flask. The transgene plasmid construct and the helper plasmid RC_AAV and helper are added to PBS (PBS pH 7.4, Thermo Fisher Scientific, catalog number 10010023). FectoVIR (FectoVIR®-AAV 10 mL, Polyplus, catalog number 101000022) is added to the PBS. After 5 minutes, the complex is added to the flask. Two days after transfection, Triton X-100 is added to the culture medium at a concentration of 0.5% and left at room temperature for 1 hour to lyse the cells. The resulting solution is centrifuged at 10,000 g for 5 minutes at room temperature. The supernatant is collected as the rAAV sample.

[0083] Example 2-3: Production of rAAV8 vectors by HCD method Virus-producing cells 2.0 (VPC2.0, Thermo Fisher Scientific, Cat. No. A49784) were cultured at 30 × 10 5 The cells were diluted to a density of 90 x 10 cells / ml. 20 mL of the cell suspension was placed in a 125 mL shake flask. The cell suspension was centrifuged at 400 g for 4 minutes at room temperature. A portion of the supernatant was then removed to obtain a high cell density, and the cells were resuspended. 5A cell suspension of 1000 cells / ml was prepared. 20 mL of the cell suspension was placed in a 125 mL shake flask. The transgene plasmid construct and the helper plasmid RC_AAV and helper were added to PBS (PBS pH 7.4, Thermo Fisher Scientific, catalog number 10010023). FectoVIR (FectoVIR®-AAV 10 mL, Polyplus, catalog number 101000022) was added to the PBS. After 5 minutes, the complexes were added to the flask. One day after transfection, the cell suspension was centrifuged at 400 g for 4 minutes at room temperature. Next, half of the spent suspension was removed, and the same volume of fresh VPM containing 4% GultaMAX was added. The cells were resuspended, and the cell suspension was cultured in an incubator shaker. Two days after transfection, Triton X-100 was added to the culture medium at a concentration of 0.5%, and the medium was left to stand at room temperature for 1 hour to lyse the cells. The resulting solution was centrifuged at 10,000 g for 5 minutes at room temperature, and the supernatant was collected as the rAAV sample.

[0084] result rAAV samples produced using the methods described in Examples 2-1 and 2-3 were quantified. After subsequent treatment with DNase I, the solution was diluted with water and then subjected to digital droplet PCR (QX ONEDroplet Digital PCR System, Bio-Rad Laboratories, Inc.) using ddPCR EvaGreen Supermix (Bio-Rad Laboratories, Inc., Cat. No. 186-4034), a FAM probe, and primers designed for the transgene construct. The vg concentration of the supernatant was calculated (Figure 2).

[0085] As shown in FIG. 2, it was confirmed that Example 2-3 exhibited improved productivity compared to that achieved in Example 2-1.

[0086] Example 3: Confirmation of medium exchange time Virus-producing cells 2.0 (VPC2.0, Thermo Fisher Scientific, catalog no. A49784) were cultured at 30 × 10 5 The cells were diluted to a density of 1000 cells / ml. 20 mL of the cell suspension was placed in a 125 mL shake flask. A total of 12 identical flasks were prepared. Gibco AAV-MAX Enhancer, a component of the AAV-MAX Transfection Kit (Thermo Fisher Scientific, catalog number A50515), was added to the cell suspension. The transgene plasmid construct and the helper plasmids RC_AAV and helper were added to Viral-Plex™ complexing buffer (Viral-Plex, Thermo Fisher Scientific, catalog number A4983901). The remaining two reagents of the AAV-MAX Transfection Kit were added to the complexing buffer in the order described in the kit protocol. After 10 minutes, the complex was added to the flask. The following procedure was performed for all flasks except flask 1. For conditions 2, 3, 4, 5, and 6, the cell suspension was centrifuged at 400 g for 4 minutes at room temperature 0, 1, 2, 4, and 6 hours after transfection, respectively. Then, half of the spent medium was removed, and fresh medium was added. The cells were resuspended, and the cell suspension was cultured in a shaker incubator. Conditions 7 to 12 were identical to conditions 1 to 6, respectively. Three days after transfection, Triton X-100 was added to the culture medium at a concentration of 0.2% and allowed to stand at room temperature for 1 hour to lyse the cells. The resulting solution was centrifuged at 10,000 g for 5 minutes at room temperature. The supernatant was collected as the rAAV sample.

[0087] rAAV samples produced using the method described in Example 3 were quantified. After subsequent treatment with DNase I, AAV genomic DNA was extracted and the solution diluted with water. Real-time PCR was then performed using the AAVpro® Titration Kit for Real-Time PCR (Takara Bio Inc., Catalog No. 6233) and the ITR primers provided with the kit. A calibration curve was generated using the standard DNA provided with the kit, and the vg concentration of the supernatant was calculated (Figure 3).

[0088] As shown in Figure 3, it was confirmed that the productivity was constant under the condition where the medium was changed 1 hour after transfection.

[0089] Example 4: Confirmation of medium exchange time Virus-producing cells 2.0 (VPC2.0, Thermo Fisher Scientific, Cat. No. A49784) in culture medium at 40 × 10 5 The cells were diluted to a density of 94–185 × 10 cells / ml. The cell suspension was centrifuged at 400 g for 4 minutes at room temperature. A portion of the supernatant was then removed to obtain a high cell density, and the cells were resuspended. 5 A cell suspension was prepared in 13 flasks at a density of 10 cells / mL. The actual cell density and the target cell density (×10 5 The cell counts (cells / ml) are shown in Table 1 below.

[0090] [Table 1]

[0091] 20 mL of cell suspension was placed in a 125 mL shake flask. A total of 13 identical flasks were prepared. Gibco AAV-MAX Enhancer, a component of the AAV-MAX Transfection Kit (Thermo Fisher Scientific, catalog number A50515), was added to the cell suspension. The transgene plasmid construct and the helper plasmids RC_AAV and helper were added to Viral-Plex™ complexing buffer (Viral-Plex, Thermo Fisher Scientific, catalog number A4983901). The remaining two reagents of the AAV-MAX Transfection Kit were added to the complexing buffer in the order described in the kit protocol. After 10 minutes, the complex was added to the flask. The following procedure was performed for all flasks except flask 1. One day after transfection, the cell suspension was centrifuged at 400 g for 4 minutes at room temperature. The spent half was then removed and an equal volume of fresh medium was added. The cells were resuspended and cultured in a shaker incubator. Two days after transfection, Triton X-100 was added to the culture medium at a concentration of 0.2% and allowed to stand at room temperature for 1 hour to lyse the cells. The resulting solution was centrifuged at 10,000 g for 5 minutes at room temperature. The supernatant was collected as the rAAV sample.

[0092] result rAAV samples produced using the method described in Example 4 were quantified. After subsequent treatment with DNase I, AAV genomic DNA was extracted, the solution was diluted with water, and real-time PCR was performed using the AAVpro® Titration Kit for Real-Time PCR (Takara Bio Inc., catalog number 6233) and the ITR primers provided with the kit. A calibration curve was generated using the standard DNA provided with the kit, and the vg concentration of the supernatant was calculated (Figure 4).

[0093] As shown in Figure 4, it was confirmed that productivity was maximized at a density of 150.

[0094] Example 5-1: Production of rAAV8 vectors by general methods 1600 mL of virus production medium (VPM, Thermo Fisher Scientific, catalog number A4817901) containing 4% GlutaMAX™ Supplement (GultaMAX, Thermo Fisher Scientific, catalog number 35050061) was placed in a 2 L bioreactor and adjusted to 37°C. 5 Virus producer cells 2.0 (VPC2.0, Thermo Fisher Scientific, Cat. No. A49784) at a density of 100 cells / ml were added to the reactor until the cell density in the reactor was 4 × 10 5 The cell suspension was cultured at 37°C for 3 days. After 3 days, the cell density was 38 x 10 5 The cell suspension reached a concentration of 1000 cells / ml. The Gibco AAV-MAX Enhancer, a component of the AAV-MAX Transfection Kit (Thermo Fisher Scientific, catalog number A50515), was added to the cell suspension. The transgene plasmid construct and the helper plasmids RC_AAV and helper were added to Viral-Plex™ Complexing Buffer (Viral-Plex, Thermo Fisher Scientific, catalog number A4983901). The remaining two reagents of the AAV-MAX Transfection Kit were added to the complexing buffer in the order described in the kit's protocol. After 10 minutes, the complex was added to the bioreactor. Three days after transfection, Triton X-100 was added to the culture medium at a concentration of 0.2% and allowed to stand at room temperature for 1 hour to lyse the cells. The resulting solution was centrifuged at 10,000 g for 5 minutes at room temperature. The supernatant was collected as the rAAV sample.

[0095] Example 5-2: Production of rAAV8 vectors using the ATF method 1600 mL of virus production medium (VPM, Thermo Fisher Scientific, Cat. No. A4817901) containing 4% GlutaMAX™ Supplement (GultaMAX, Thermo Fisher Scientific, Cat. No. 35050061) is placed in a 2 L bioreactor and adjusted to 37°C. 5 Virus producer cells 2.0 (VPC2.0, Thermo Fisher Scientific, Cat. No. A49784) at a density of 100 cells / ml were added to the reactor until the cell density in the reactor was 4 × 10 5 The cells are seeded at 1000 cells / ml and the cell suspension is cultured at 37°C for 3 days.

[0096] Three days later, the cell density was 38 × 10 5 The cell suspension was cultured using a Cell ATF 2 (ATF2, Repligen) system equipped with a 0.2 μm PES disposable filter (Repligen, product number: suATF 2-S02PES) at a medium exchange rate of 1 VVD.

[0097] One day later, the cell density was 70 × 10 5When the cell density reached 1000 cells / ml, ATF2 and medium changes were stopped. The Gibco AAV-MAX Enhancer, a component of the AAV-MAX Transfection Kit (Thermo Fisher Scientific, catalog number A50515), was added to the cell suspension. The transgene plasmid construct and the helper plasmids RC_AAV and helper were added to Viral-Plex™ complexing buffer (Viral-Plex, Thermo Fisher Scientific, catalog number A4983901). The remaining two reagents of the AAV-MAX Transfection Kit were added to the complexing buffer in the order described in the kit protocol. After 10 minutes, the complex was added to the bioreactor. Three days after transfection, 0.2% Triton X-100 was added to the culture solution, which was then left at room temperature for 1 hour to lyse the cells. The resulting solution was centrifuged at 10,000 g for 5 minutes at room temperature. The supernatant was collected as the rAAV sample.

[0098] Example 5-3: Production of rAAV8 vectors by HCD method 1600 mL of virus production medium (VPM, Thermo Fisher Scientific, catalog number A4817901) containing 4% GlutaMAX™ Supplement (GultaMAX, Thermo Fisher Scientific, catalog number 35050061) was placed in a 2 L bioreactor and adjusted to 37°C. 5 Virus producer cells 2.0 (VPC2.0, Thermo Fisher Scientific, Cat. No. A49784) at a density of 100 cells / ml were added to the reactor until the cell density in the reactor was 4 × 10 5 The cells were seeded at 1000 cells / ml and cultured at 37°C for 3 days.

[0099] Three days later, the cell density was 38 × 10 5The cell suspension was cultured at a medium exchange rate of 1 VVD using a Cell ATF 2 (ATF2, Repligen) system equipped with a 0.2 μm PES disposable filter (Repligen, product number: suATF 2-S02PES).

[0100] Two days later, the cell density was 130 × 10 5 The cell concentration reached 100 cells / ml. While the tangential flow of ATF2 was continued, medium exchange was stopped. Gibco AAV-MAX Enhancer, a component of the AAV-MAX Transfection Kit (Thermo Fisher Scientific, catalog number A50515), was added to the cell suspension. The transgene plasmid construct and the helper plasmids RC_AAV and helper were added to Viral-Plex™ complexing buffer (Viral-Plex, Thermo Fisher Scientific, catalog number A4983901). The remaining two reagents of the AAV-MAX Transfection Kit were added to the complexing buffer in the order described in the kit protocol. After 10 minutes, the complex was added to the bioreactor. After 2 hours, medium exchange was resumed.

[0101] Two days after transfection, 0.2% Triton X-100 was added to the culture medium and allowed to stand at room temperature for 1 hour to lyse the cells. The resulting solution was centrifuged at 10,000 g for 5 minutes at room temperature. The supernatant was collected as the rAAV sample.

[0102] result rAAV samples produced using the methods described in Examples 5-1 and 5-3 were quantified. After subsequent DNase I treatment, the solution was diluted with water and subjected to digital droplet PCR (QX ONEDroplet Digital PCR System, Bio-Rad Laboratories, Inc.) using ddPCR EvaGreen Supermix (Bio-Rad Laboratories, Inc., Cat. No. 186-4034), a FAM probe, and primers designed for the transgene construct. The vg concentration of the supernatant was calculated (Figure 5).

[0103] As shown in Figure 5, improved productivity was achieved in Example 5-3 compared to that achieved in Example 5-1, confirming that it performed equally well in the reactor.

Claims

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

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

3. 1. A method for producing a population of AAV particles, comprising: (a) culturing a population of host cells for a period of time sufficient to allow said host cells to grow; (b) introducing one or more nucleic acids encoding one or more AAV components into said host cell, or a progeny thereof, thereby producing a transfected host cell; (c) incubating the transfected host cells in cell culture medium for a time sufficient for the cells to produce AAV particles, wherein during said incubation, a portion of the cell culture medium is replaced with fresh medium; and (d) after step (c), lysing the host cells, thereby releasing the AAV particles.

4. Upon introduction of the one or more nucleic acids into the host cell or its progeny, the host cell or its progeny may be transformed into a ５ cells / ml~400×10 ５ The method of claim 1 or 3, wherein the cell culture medium has a density of 1000 cells / ml.

5. 5. The method of claim 4, wherein upon introduction of the one or more nucleic acids into the host cell or its progeny, the host cell or its progeny grows to a density of: (a) 10 x 10 ５ cells / ml~400×10 ５ cells / ml, 20 x 10 ５ cells / ml~400×10 ５ cells / ml, 30 x 10 ５ cells / ml~400×10 ５ cells / ml, 40 x 10 ５ cells / ml~400×10 ５ cells / ml, or 50 x 10 ５ cells / ml~400×10 ５ cells / ml, or (b) 100 x 10 ５ cells / ml~200×10 ５ cells / ml, 110 x 10 ５ cells / ml~190×10 ５ cells / ml, 120 x 10 ５ cells / ml~180×10 ５ cells / ml, 100 x 10 ５ cells / ml~180×10 ５ cells / ml, or 100 x 10 ５ cells / ml~160×10 ５ cells / ml.

6. Upon introduction of the one or more nucleic acids into the host cell or its progeny, the host cell or its progeny may be transformed into a ５ cells / ml~160×10 ５ The method of claim 4, wherein the cell culture medium has a density of 1000 cells / ml.

7. The population of transfected host cells is 5 x 10 ５ cells / ml~400×10 ５ The method of claim 2, wherein the cells are provided at a density of 1000 cells / ml.

8. 8. The method of claim 7, wherein the population of transfected host cells has a density of: (a) 10 x 10 ５ cells / ml~400×10 ５ cells / ml, 20 x 10 ５ cells / ml~400×10 ５ cells / ml, 30 x 10 ５ cells / ml~400×10 ５ cells / ml, 40 x 10 ５ cells / ml~400×10 ５ cells / ml, or 50 x 10 ５ cells / ml~400×10 ５ cells / ml; or (b) 100 x 10 ５ cells / ml~200×10 ５ cells / ml, 110 x 10 ５ cells / ml~190×10 ５ cells / ml, 120 x 10 ５ cells / ml~180×10 ５ cells / ml, 100 x 10 ５ cells / ml~180×10 ５ cells / ml, or 100 x 10 ５ cells / ml~160×10 ５ The method, wherein the cells are provided at a density of cells / ml.

9. The population of transfected host cells is 100 x 10 ５ cells / ml~160×10 ５ The method of claim 7, wherein the cells are provided at a density of 1000 cells / ml.

10. The method according to any one of claims 1 to 9, wherein the host cell is a mammalian cell.

11. 11. The method of claim 10, wherein the mammalian cells are human embryonic kidney (HEK) cells or derivatives thereof.

12. 12. The method of claim 11, wherein the HEK cells are HEK293 cells.

13. 13. The method of any one of claims 1 to 12, wherein the one or more nucleic acids are introduced into the host cell by contacting the cell 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. During the incubation, the host cells were added to 5×10 ５ cells / ml~400×10 ５ The method according to any one of claims 1 to 14, wherein the culture is cultured until a density of 1000 cells / ml is reached.

16. 16. The method of claim 15, wherein during the incubation, the host cells are cultured until they reach a density of: (a) 10 x 10 ５ cells / ml~400×10 ５ cells / ml, 20 x 10 ５ cells / ml~400×10 ５ cells / ml, 30 x 10 ５ cells / ml~400×10 ５ cells / ml, 40 x 10 ５ cells / ml~400×10 ５ cells / ml, or 50 x 10 ５ cells / ml~400×10 ５ cells / ml; or (b) 100 x 10 ５ cells / ml~200×10 ５ cells / ml, 110 x 10 ５ cells / ml~190×10 ５ cells / ml, 120 x 10 ５ cells / ml~180×10 ５ cells / ml, 100 x 10 ５ cells / ml~180×10 ５ cells / ml, or 100 x 10 ５ cells / ml~160×10 ５ cells / ml.

17. During the incubation, the host cells were added to 100×10 ５ cells / ml~160×10 ５ 16. The method of claim 15, wherein the cells are cultured until a density of 100 cells / ml is reached.

18. 18. The method of any one of claims 1 and 3 to 17, wherein the method comprises: (i) during step (a), replacing the cell culture medium with fresh medium; (ii) temporarily stopping the replacement of the cell culture medium with fresh medium during step (b); (iii) resuming replacing the cell culture medium with fresh medium during step (c).

19. 19. The method of any one of claims 1 to 18, wherein said exchanging comprises, in one or separate steps, replacing part of said cell culture medium with said fresh medium.

20. 20. The method of claim 19, wherein each of the individual steps comprises: (a) separating the host cells from the surrounding cell culture medium by centrifugation; (b) removing the surrounding cell culture medium as a supernatant resulting from the centrifugation; (c) replenishing the host cells with fresh cell culture medium.

21. 19. The method of any one of claims 1 to 18, wherein said exchanging comprises continuously exchanging a portion of said cell culture medium with said fresh medium.

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

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

24. 24. The method of claim 23, wherein said replacing comprises replacing at least 60% of the cell culture medium with new medium, and optionally, said replacing 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 to 24, wherein the transfected cells are incubated in a cell culture medium volume of 1 L to 1000 L.

26. 25. The method of any one of claims 1 to 24, wherein the transfected cells are incubated in a volume of cell culture medium between 1 L and 10 L, between 1 L and 2 L, between 1.6 L and 2 L, between 8 L and 10 L, between 40 L and 50 L, or between 160 L and 200 L.

27. 25. The method of any one of claims 1 to 24, wherein the transfected cells are incubated in a cell culture medium volume of 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. 28. The method of any one of claims 1 to 27, wherein the host cells are lysed by contacting the cells with a detergent.

29. 29. The method of any one of claims 1 to 24 hours after transfection of the host cells, optionally wherein the exchange occurs 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 after transfection of the host cells.

30. 30. The method of claim 29, wherein the exchange occurs 1 to 2 hours after transfection of the host cells.

31. 31. The method of any one of claims 1 to 30, wherein the one or more nucleic acids encoding one or more AAV components are introduced into a host cell by providing the cell with (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. 32. The method of claim 31 , wherein the host cell is further provided with (iii) a third nucleic acid comprising one or more adenoviral helper genes.

33. 33. The method of any one of claims 1 to 32, wherein the method further comprises purifying the released AAV particles after the lysis.

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

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

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