Method for producing adeno-associated virus vector using cell physiological index control and plasmid sequential injection

Abstract

The present invention provides a method for producing an adeno-associated virus (AAV) vector, wherein the method can improve production efficiency. In one embodiment, the method comprises: (a) a step for controlling the G1 cell ratio of a produced cell to 40% to 70%; and (b) a step for introducing a Rep / Cap plasmid, a Transgene plasmid, and a Helper plasmid into the produced cell, wherein in step (b), the Transgene plasmid may be introduced prior to the other plasmids or together with one of the other plasmids.

Description

Method for producing adeno-associated virus vectors using control of cellular physiological markers and sequential plasmid delivery

[0001] The present invention relates to the field of viral vector production technology, more specifically to process optimization technology for the mass production of adeno-associated virus (AAV) vectors for gene therapy.

[0002] Unless otherwise indicated in this specification, the contents described in this identification item are not prior art for the claims of this application, and are not recognized as prior art simply because they are described in this identification item.

[0003] The Importance of Gene Therapy and Adeno-Associated Viruses (AAV) With the development of modern pharmaceutical biotechnology, gene therapy is establishing itself as a new paradigm for the treatment of rare genetic diseases, neurodegenerative diseases, and cancer. Among various gene vectors, the Adeno-Associated Virus (AAV) is currently the most preferred viral vector due to its advantages, such as being non-pathogenic, capable of infecting both dividing and non-dividing cells, having a low risk of inducing mutations upon insertion into the host genome, and being able to express genes for a long period.

[0004] Existing AAV Production Technology and Its Limitations General production of recombinant AAV (rAAV) is achieved through a triple transfection method in which a Rep / Cap plasmid, a helper plasmid, and a transgene plasmid containing a therapeutic gene are simultaneously introduced into HEK293 (Human Embryonic Kidney 293)-derived cell lines. However, while this transient transfection method is useful on a laboratory scale, it has the following critical limitations when applied to large-scale manufacturing required in clinical and commercial stages.

[0005] First, there is a limitation in production yield. Although continuous attempts have been made to increase cell density within the bioreactor, specific productivity per cell tends not to increase beyond a certain level or even decreases. Second, there is non-uniformity in quality and low packaging efficiency. A frequent problem is the high proportion of 'empty capsids'—where genes are not packaged—among the produced AAV particles. This reduces therapeutic efficacy and can induce unnecessary immune responses in patients, thereby increasing the burden on the purification process. Third, there is a lack of reproducibility in the production process. Even when using the same medium and plasmid ratio, significant variations in productivity occur between batches, suggesting that process parameters do not accurately reflect the internal physiological state of the cells.

[0006] Existing process optimization efforts to address the 'black box' issues of cell physiological state and virus production have primarily focused on controlling the 'extracellular environment,' such as media formulation, pH, dissolved oxygen (DO), and temperature. However, AAV production inherently depends entirely on the host cell's replication, energy metabolism, and protein synthesis mechanisms. Consequently, despite the fact that the intracellular physiological state is a key factor determining AAV production efficiency, existing technologies merely regard the cell as a simple 'virus production factory' and have failed to specifically elucidate the impact of internal operating conditions—such as the cell cycle or metabolic status—on virus packaging. In other words, the intracellular state remains a 'black box.'

[0007] In an attempt to address the "double-edged sword" nature of the cell cycle and energy metabolism, as well as the difficulties of optimization, some prior studies have attempted to inhibit cell growth or arrest cells at specific cycles; however, this has resulted in side effects, such as reduced cell vitality and decreased overall protein expression. Furthermore, since viral replication is a process that consumes massive amounts of energy, the prevailing conventional wisdom was that higher intracellular ATP concentrations or mitochondrial activity (MMPs) are advantageous for production. However, excessive mitochondrial activity can induce cellular stress due to the accumulation of reactive oxygen species (ROS), which can actually hinder virus assembly or induce apoptosis. Conversely, if energy is too insufficient, viral genome replication and capsid formation are inhibited. In other words, cell cycle (e.g., G1 Phase) and energy metabolism indicators (MMPs, ATP) are not simply "better the higher"; rather, it is highly likely that a specific "sweet spot" optimized for viral production exists. However, to date, no integrated analysis has been conducted on the specific 'optimal mid-range' of G1 ratio, MMP, and ATP for maximizing AAV productivity, as well as the 'bell-shaped' correlation between them and productivity.

[0008] Furthermore, traditional triple transfusion involves the simultaneous introduction of three plasmids. However, considering the life cycle of AAV, the timing of capsid protein expression, viral genome replication, and helper function activation must be precisely coordinated in terms of timing. If all genetic material is released into the nucleus at the same time, resource competition arises over the intracellular transcription / translation machinery. This leads to imbalances, such as the capsid forming before the viral genome is replicated, or conversely, the genome being excessively replicated without a capsid, ultimately causing a decrease in packaging efficiency (Full / Empty Ratio).

[0009] Necessity of the present invention: Therefore, in order to overcome the limitations of the AAV production process, it is necessary to develop a new process that goes beyond a one-dimensional approach of simply culturing cells at high density or increasing the amount of plasmid.

[0010] Prior Art: PCT Application Publication WO2020 / 154607 A1 (Published July 30, 2020)

[0011] The problem that the present invention aims to solve is to provide a method for improving AAV production efficiency by maintaining a combination of cellular physiological indicators, namely G1 cell cycle ratio, mitochondrial membrane potential (MMP), and intracellular ATP concentration (IAL), within a specific range.

[0012] In addition, the present invention provides a method for improving AAV production efficiency by controlling the conditions of the plasmid insertion sequence and time interval.

[0013] To solve the above problems, the present invention provides a method for producing an adeno-associated virus (AAV) vector, comprising: (a) controlling the ratio of G1 cells of the production cells to 40% to 70%; and (b) introducing a Rep / Cap plasmid, a Transgene plasmid, and a Helper plasmid into the production cells; wherein, in step (b), the Transgene plasmid is introduced before other plasmids or together with any one of the other plasmids.

[0014] At this time, the G1 cell ratio of step (a) above may be 42% to 61%.

[0015] Additionally, the above step (a) further includes a step of controlling the mitochondrial membrane potential (MMP) of the production cell, and the MMP may include a relative MMP index of 0.85 to 1.25 when the production cell under normal culture conditions is used as a control.

[0016] Additionally, the above step (a) further comprises a step of controlling the intracellular ATP concentration of the production cell, wherein the ATP concentration is 20 to 40 pmol / 10 6 It can include cells.

[0017] In addition, (a-1) a step of controlling the G1 cell ratio of the production cells to 42% to 61%; (a-2) a step of controlling the mitochondrial membrane potential (MMP) of the production cells, wherein the relative MMP index relative to normal culture conditions is 0.85 to 1.25; and (a-3) an intracellular ATP concentration of the production cells of 20 to 40 pmol / 10 6 It may include a step of controlling with cells.

[0018] Additionally, the above step (b) may sequentially include: (b-1) a step of introducing a Transgene plasmid; (b-2) a step of introducing a Rep / Cap plasmid after step (b-1); and (b-3) a step of introducing a Helper plasmid after step (b-2).

[0019] Additionally, the above step (b) may include (b-1) a step of introducing a Transgene plasmid; and (b-2) a step of introducing a Rep / Cap plasmid and a Helper plasmid after the above step (b-1).

[0020] Meanwhile, the time interval between the above steps (b-1) and (b-2) may be 1 hour to 6 hours.

[0021] At this time, the above time interval may be 2 to 5 hours.

[0022] At this time, the above time interval may be 3 hours.

[0023] The present invention also relates to a method for producing an adeno-associated virus (AAV) vector, comprising: (a) a step of controlling physiological indicators of the production cells, wherein (a-1) the G1 cell ratio is controlled to 42% to 61%, (a-2) the relative mitochondrial membrane potential (MMP) index relative to normal culture conditions is controlled to 0.85 to 1.25, and (a-3) the intracellular ATP concentration is controlled to 20 to 40 pmol / 10 6 A method for producing an AAV vector is provided, comprising: a step of controlling with cells; and (b) a step of sequentially introducing plasmids, wherein (b-1) introducing a Transgene plasmid, (b-2) introducing a Rep / Cap plasmid 2 to 5 hours after step (b-1), and (b-3) introducing a Helper plasmid 2 to 5 hours after step (b-2).

[0024] According to the present invention, AAV productivity can be improved by controlling physiological indicators (G1 ratio, MMP, ATP) of the producing cells.

[0025] In addition, according to the present invention, the order of plasmid insertion and the time interval thereof can be controlled according to the cell's preparation state to improve AAV productivity.

[0026] Figure 1 is a graph showing the production of AAV2 vector according to the G1 cell ratio.

[0027] Figure 2 is a graph showing the production of AAV2 vectors according to the MMP index.

[0028] Figure 3 is a graph showing the production of AAV2 vector according to intracellular ATP concentration.

[0029] Figure 4 is a graph comparing the production of AAV2 vectors according to the order of plasmid input.

[0030] Figure 5 is a graph showing the production of AAV2 vector according to the plasmid input time interval.

[0031] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but can be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0032]

[0033] In this invention, AAV refers to an adeno-associated virus, a concept that encompasses recombinant adeno-associated viruses expressing foreign genes; however, unless otherwise specifically stated in this document, it refers to a wild-type virus. The adeno-associated virus is also referred to as an adeno-related virus or an adeno-associated virus.

[0034] In the present invention, the G1 ratio of the production cell line refers to the ratio of cells remaining in the G1 phase (Gap 1 Phase) of the cell cycle in a population of host cells (e.g., HEK293 lineage cells) that produce an AAV vector. Here, G1 may refer to G1 when the cell cycle is divided into G1 (Gap 1, the stage where the cell grows and prepares for the next stage, DNA replication), S (Synthesis, the stage where DNA is replicated), G2 (Gap 2, the stage where division is prepared), and M (Mitosis, the stage of cell division).

[0035]

[0036] After various trials and errors to develop a technique to maintain the physiological indicators (G1 ratio, MMP, ATP) of the production cell at an optimal range most favorable for AAV production, and a technique to control the timing and sequence of plasmid insertion according to the cell's readiness, the inventors of the present invention confirmed that the G1 cell cycle ratio, mitochondrial membrane potential, and intracellular ATP concentration of the production cell line have a bell-shaped correlation with AAV productivity, thereby deriving the optimal range for each indicator. Furthermore, by optimizing the sequence of plasmid insertion, they were able to significantly improve the production yield and quality of the AAV vector.

[0037]

[0038] Specifically, the applicant of the present invention confirmed that the production efficiency of an adeno-associated virus vector can be improved by controlling at least one of the physiological indicator conditions of the production cell and the plasmid insertion conditions.

[0039] Regarding the physiological indicator conditions of the production cells, the applicant of the present invention has confirmed the following.

[0040] First, when the G1 ratio of the production cell line was controlled, the productivity curve exhibited a clear bell shape as shown in Fig. 1, and the highest yield (2.8-3.4 × 10⁻⁶) was achieved under conditions with an intermediate to G1 ratio (G2-G3). 14 vg / L) was confirmed. Through this, it was confirmed that both excessive proliferation (S / G2M) and excessive cessation (G1 / G0) conditions reduce AAV2 productivity.

[0041] Second, regarding mitochondrial membrane potential (MMP) control, as shown in Fig. 2, productivity decreased due to insufficient energy supply when the membrane potential was excessively low (0.3); conversely, productivity decreased again in the region where the membrane potential became excessively high (1.5-1.8) due to metabolic stress and the potential for increased ROS. Through this, at the optimal range of mitochondrial membrane potential (MMP) (0.6-1.2), 3.1-4.4×10 14 It was confirmed that the highest yield of vg / L was observed.

[0042] Third, when controlling intracellular ATP concentration, a similar bell-shaped curve was observed as shown in Fig. 3, and at intermediate ATP levels (25-35 pmol / 10⁻¹⁰ 6It was confirmed that cells) improve AAV2 productivity. It was found that intracellular ATP concentrations too low (12-18 pmol) resulted in reduced protein synthesis and vector replication ability, while intracellular ATP concentrations too high (45-60 pmol) exhibited mitochondrial overload and stress-linked inhibition.

[0043] Thus, the present invention confirmed the existence of an "optimal mid-range" that encompasses all three key physiological indicators of the production cell line: G1 ratio, mitochondrial membrane potential (MMP), and intracellular ATP concentration, and confirmed that productivity decreased in both low and high states deviating from the "optimal mid-range."

[0044] Regarding the plasmid input conditions, the applicant of the present invention has confirmed the following.

[0045] First, regarding AAV2 production, as shown in Figure 4, AAV2 productivity is improved when the transgenic expression plasmid, REP / CAP plasmid, and Helper plasmid are introduced sequentially compared to when they are introduced simultaneously, and among these, AAV2 productivity is further improved when the transgenic expression plasmid is introduced first.

[0046] Second, it was confirmed that AAV2 productivity is improved when the time interval is controlled to 2 to 6 hours, particularly 3 hours, even when the plasmid is sequentially injected, as shown in Fig. 5.

[0047]

[0048] The present invention will be described in detail below by way of examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples. Furthermore, the following examples provide examples for implementing the features of the present invention, and areas not described can be easily reproduced by a person skilled in the art by applying techniques well known in the art.

[0049]

[0050] Preparation Example 1: Cell Culture

[0051] HEK293-based suspension culture cells were cultured in DMEM-based culture medium under stirring conditions.

[0052] Preparation Example 1-1: Cell line and medium

[0053] HEK293-F cells (Thermo Fisher Scientific, Cat. R79007) or equivalent suspension-adapted HEK293 cells were used.

[0054] The culture medium used was FreeStyle™ 293 Expression Medium (Gibco, Cat. 12338018) or GeneScript Probiotics' serum-free medium for AAV production, supplemented with the following composition:

[0055] - L-Glutamine: 2-4 mM (final concentration)

[0056] - Pluronic F-68: 0.1% (w / v)

[0057] Preparation Example 1-2: Culture Conditions

[0058] - Culture vessel: 1-5L single-use bioreactor (Stirred-tank single-use bioreactor)

[0059] - Stirring speed: 80-120 rpm

[0060] - Incubation temperature: 37 ± 0.5℃

[0061] - CO₂ concentration: 5 ± 0.5%

[0062] - Humidity: 85% or higher

[0063] - pH: 7.0-7.4 (Automatic control)

[0064] - Dissolved Oxygen (DO): 40-50% air saturation

[0065] Preparation Example 1-3: Cell Maintenance and Subculture

[0066] The cell is 0.3-0.5 × 10⁶ 6 Subcultured at a density of cells / mL, with a viable cell density of 2.0–3.0 × 10⁶ 6 When cells / mL was reached, subculture was performed at a ratio of 1:3 to 1:5.

[0067] Viability was measured using trypan blue staining and maintained over 95%.

[0068] The criteria applied to the statistical analysis used in the present embodiment are as follows:

[0069] -. Display values ​​as mean ± standard deviation (SD).

[0070] Comparisons between groups were performed using Tukey's post-hoc test following one-way ANOVA, and a p-value < 0.05 was considered statistically significant.

[0071] Statistical analysis was performed using GraphPad Prism 9.0 software.

[0072] Below, based on the standards applied to quality control used in this embodiment, the following was verified for each experimental batch:

[0073] - Cell viability: Over 95% at the time of transfection

[0074] - Plasmid quality: Confirmed supercoiled form by agarose gel electrophoresis

[0075] - Mycoplasma contamination test: Confirmed negative via PCR-based test

[0076]

[0077] Example 1: Control of physiological indicators of producing cells

[0078] Example 1-1: Control of G1 ratio in production cell lines

[0079] To quantitatively evaluate the effect of G1 cell ratio on AAV2 productivity, six culture populations (G1-G6) with different G1%s were established and AAV2 production was compared (Table 1).

[0080] Group average G1 cell ratio (±5%) G133 % G242 % G351 % G461 % G571 % G680 %

[0081] Each group was configured to reflect different cellular physiological states, and independent cultures of N=3 were performed under each condition.

[0082] As a result of the analysis, AAV2 productivity showed a bell-shaped non-linear pattern as G1% increased (Table 2, Fig. 1).

[0083] GroupReplicateTiter_vg_per_LG115.25E+13G124.93E+13G135.32E+13G213.23E+14G222.73E+14G232.73E+14G313.82E+14G323.55E +14G333.15E+14G411.69E+14G421.53E+14G431.53E+14G517.17E+13G525.66E+13G535.79E+13G611.98E+13G621.89E+13G632.17E+13

[0084] In particular, the highest yields were observed in the G2 and G3 groups at approximately 2.8×10^14 vg / L and 3.3×10^14 vg / L, respectively, which were significantly higher than the remaining conditions.

[0085] This range suggests that an intermediate G1 ratio, in which cells neither lean toward an excessively proliferative state (increased S / G2M ratio) nor fall into an excessively quiescent state (G0 / G1 stagnation), provides the most suitable cellular physiological environment for AAV2 production.

[0086] On the other hand, productivity in the G1 group was significantly lowered to about 5×10^13 vg / L, which is consistent with the tendency for AAV packaging efficiency and protein expression balance to decline when cells are in a relatively high-proliferative state.

[0087] In addition, in the G4-G6 groups, the yield gradually decreased with increasing G1%, and G6 showed a minimum value of approximately 2.1×10^13 vg / L.

[0088] Therefore, in subsequent experiments, the G3 group, which showed the maximum value, was used.

[0089] Example 1-2: Mitochondrial Membrane Potential (MMP) Control

[0090] To evaluate the effect of changes in mitochondrial membrane potential (MMP) on AAV2 productivity based on the G3 group of Example 1, six culture conditions (G1-G6) corresponding to different relative MMP indices (0.3, 0.6, 0.9, 1.2, 1.5, 1.8) were established. Independent cultures with N=3 were performed under each condition, and the AAV2 vector production was quantified in vg / L at the end of production (Table 3).

[0091] Group average MMP (ΔΨm)G1 0.3G2 0.6G3 0.9G4 1.2G5 1.5G6 1.8

[0092] As a result of the analysis, the AAV2 yield showed a bell-shaped non-linear change depending on the MMP index (Fig. 2, Table 4).

[0093] GroupMMP_indexReplicateTiter_vg_per_LG10.313.03E+13G10.323.74E+13G10.33 3.5E+13G20.612.72E+14G20.623.01E+14G20.633.73E+14G30.912.88E+14G30.923.6 4E+14G30.934.28E+14G41.213.93E+14G41.224.01E+14G41.234.26E+14G51.511.72E +14G51.521.4E+14G51.531.43E+14G61.815.74E+13G61.827.32E+13G61.837.31E+13

[0094] In particular, the highest productivity was observed under G2-G4 conditions (exponent 0.6-1.2) with intermediate levels of MMP, with average yields of approximately 3.2×10^14 vg / L, 3.8×10^14 vg / L, and 4.3×10^14 vg / L, respectively, showing high yields in the range of 3.1-4.4×10^14 vg / L for all three conditions. This suggests that an intermediate mitochondrial membrane potential, which is neither excessively low nor excessively high, provides the most favorable energy state for AAV2 packaging and capsid assembly.

[0095] In contrast, G1 (exponent 0.3) with low MMP showed a minimum yield of about 3.4×10^13 vg / L, and G5-G6 (exponent 1.5-1.8) with high MMP also showed reduced productivity, remaining at levels of about 1.5×10^14 and 6.0×10^13 vg / L, respectively.

[0096] Examples 1-3: Control of Intracellular ATP Concentration

[0097] To evaluate the effect of intracellular ATP levels on AAV2 productivity, intracellular ATP concentration (unit: pmol / 10 6G1-G6 groups were established by dividing the cells into six stages. Each group had 12, 18, 25, 35, 45, and 60 pmol / 10 6 The conditions were designed to be representative of the cells, and the G1 cell ratio was maintained at approximately 50% in all cultures. After performing N=3 independent cultures under each condition, AAV2 production was quantified in vg / L (Table 5).

[0098] Group Name Average IAL (pmol / 10 6 cell)G112G218G325G435G545G660

[0099] As a result of the analysis, AAV2 yield showed a bell-shaped non-linear change depending on the ATP level (Fig. 3, Table 6).

[0100] In particular, G3 with intermediate levels of ATP (25 pmol / 10 6 cells) and G4 (35 pmol / 10 6 Maximum yields were observed at approximately 3.2×10^14 vg / L and 3.3×10^14 vg / L, respectively, in cells, which corresponded to the highest productivity among all conditions. This range falls within the previously defined optimal metabolic zone (20-35 pmol / 10⁻¹⁰ cells). 6 It corresponds to cells) and can be interpreted as the state in which the energy supply required for AAV2 replication and capsid assembly and the cellular stress levels are in the best balance.

[0101] In contrast, the low ATP state G1-G2 (12 and 18 pmol / 10 6 In cells), productivity decreased significantly, showing yields of approximately 2.4×10^13 vg / L at G1 and less than 1×10^14 at G2. This suggests that when energy supply is insufficient, vector genome replication and structural protein synthesis are limited, leading to a decrease in overall productivity. Additionally, G5-G6 cells (45 and 60 pmol / 10⁻¹⁰), which exhibit high ATP levels... 6In cells as well, the yield decreased again, remaining at levels of approximately 1.7×10^14 and 5.0×10^13 vg / L, respectively.

[0102] GroupATP_pmol_per_1e6cellsReplicateTiter_vg_per_LG11212.24E+13G11222.28E+13G11232.55E+13G21818.45E+13G21829.08E+13G21839.3E+13G32513.3E+14G3 2523.34E+14G32533.09E+14G43513.2E+14G43522.63E+14G43532.93E+14G54511.5 1E+14G54521.74E+14G54531.64E+14G66015.97E+13G66025.96E+13G66035.46E+13

[0103]

[0104] Experimental Example 1: Method for controlling physiological indicators of production cells

[0105] Experimental Example 1-1: Method for controlling the G1 ratio of production cell lines

[0106] The G1 ratio adjustment was a combination of the following methods.

[0107] (1) Control of inoculated cell density:

[0108] - G1 (35% Target): 0.8-1.0 × 10 6 Inoculated at cells / mL, harvested during the mid-explosive growth phase (24-30 hours).

[0109] - G2 (45% target): 0.5-0.7 × 10 6 Inoculated at cells / mL, harvested in the late exponential growth phase (36-42 hours).

[0110] - G3 (55% Target): 0.3-0.5 × 10 6 Inoculate at cells / mL and harvest early in the stationary phase (48-54 hours).

[0111] - G4 (65% Target): 0.3-0.5 × 10 6 Inoculate at cells / mL, harvest during the mid-stationary phase (60-72 hours).

[0112] - G5 (70% Target): 0.2-0.3 × 10 6 Inoculated at cells / mL, harvested in the late stationary phase (84-96 hours).

[0113] - G6 (80% target): Use nutrient-depleted medium, maintain stationary phase (96-120 hours)

[0114] (2) Use of cell cycle inhibitors:

[0115] For G4-G6 groups, one of the following can be added to induce G1 arrest:

[0116] - Lovastatin: 5-20 μM (final concentration)

[0117] - Rapamycin: 20-100 nM (final concentration)

[0118] - Serum starvation: 24-48 hours

[0119] (3) Adjustment of medium composition:

[0120] - Glucose concentration: 1-4 g / L (G1 increases as the concentration decreases)

[0121] - Glutamine concentration: 1-6 mM (lower values ​​increase G1)

[0122] (4) Check G1 ratio:

[0123] Cells were collected at each culture time point and analyzed as follows:

[0124] - Cell fixation: 70% ethanol, 4℃, at least 2 hours

[0125] - DNA staining: Propidium Iodide (PI) 50 μg / mL + RNase A 100 μg / mL, treated at room temperature for 15 minutes

[0126] - Flow cytometry analysis: BD FACSCanto™ II

[0127] * Excitation wavelength: 488 nm

[0128] * Detection wavelength: 585 / 42 nm (PE channel)

[0129] * Cell count: Minimum 10,000 events

[0130] - Data Analysis: FlowJo™ v10 or higher, using the Dean-Jett-Fox model

[0131] - G1 Ratio Allowable Range: Target Value ± 3%

[0132] Independent cultures of N=3 were performed under each of the above conditions.

[0133] Experimental Example 1-2: Method for Controlling Mitochondrial Membrane Potential (MMP)

[0134] To set culture conditions such that the MMP indices of the six groups (G1–G6) in Example 1-2 ranged from 0.3, 0.6, 0.9, 1.2, 1.5, and 1.8, respectively, based on the G3 group (G1 ratio 52%) of Example 1-1 (Table 3), MMP was adjusted by the following method:

[0135] (1) Induction of MMP reduction (G1-G2, target index 0.3-0.6):

[0136] - FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone): 0.5-2.0 ㎛, treatment 30-60 minutes

[0137] - Oligomycin A: 1-5 μg / mL, 1-2 hours after treatment

[0138] - Or hypoxic conditions: maintain DO 10-20%

[0139] (2) Maintain MMP at an intermediate level (G3-G4, target index 0.9-1.2):

[0140] - Maintain normal culture conditions

[0141] - DO: 40-50%, pH 7.2-7.4

[0142] (3) Induction of increased MMP (G5-G6, target index 1.5-1.8):

[0143] - Pyruvate addition: 10-20 mM (final concentration)

[0144] - High concentration glucose: 6-8 g / L

[0145] - or ATP synthase activator treatment

[0146] (4) MMP Measurement and Quantification:

[0147] - Staining: MitoTracker® Red CMXRos (Invitrogen, Cat. M7512)

[0148] * Final concentration: 100-200 nM

[0149] Add directly to culture medium, incubate at 37℃ for 30 minutes.

[0150] - Washing: PBS twice, 300×g, 5 minutes

[0151] - Flow cytometry analysis: BD FACSCanto™ II

[0152] * Excitation wavelength: 561 nm or 488 nm

[0153] * Detection wavelength: 585 / 42 nm

[0154] * Cell count: Minimum 10,000 events

[0155] - MMP Index Calculation:

[0156] MMP Index = (Average Fluorescence Intensity - Background) / Average Fluorescence Intensity of Control Group

[0157] - Tolerance: Target value ± 0.15

[0158] Three independent cultures (N=3) were performed for each of the above conditions.

[0159] Experimental Example 1-3: Method for Controlling Intracellular ATP Concentration

[0160] Based on the G3 group (G1 ratio 52%) of Example 1-1, the ATP concentrations of the six groups (G1–G6) in Example 1-3 were 12, 18, 25, 35, 45, and 60 pmol / 10, respectively. 6 To set the culture conditions to form cells (Table 5), intracellular ATP concentration was controlled by the following methods:

[0161] (1) Induction of ATP reduction (G1-G2, target 12-18 pmol / 10 6 cells):

[0162] - 2-Deoxy-D-glucose (2-DG): 2-10 mM, treatment 2-4 hours

[0163] - Decrease in glucose concentration: 0.5-1.0 g / L

[0164] - Antimycin A: 0.1-1 μM

[0165] (2) Maintain moderate ATP levels (G3-G4, target 25-35 pmol / 10 6 cells):

[0166] - Normal glucose concentration: 2-4 g / L

[0167] - Normal culture conditions

[0168] (3) Induction of ATP increase (G5-G6, target 45-60 pmol / 10 6 cells):

[0169] - High concentration glucose: 6-8 g / L

[0170] - Pyruvate addition: 10-20 mM

[0171] - Galactose added: 5-10 mM (OXPHOS enrichment)

[0172] (4) ATP measurement:

[0173] - Reagent: CellTiter-Glo® 3D Cell Viability Assay (Promega, Cat. G9681)

[0174] - Cell preparation:

[0175] * Cell collection from culture medium: 300×g, 5 min

[0176] 1 PBS wash

[0177] * Cell counting: Automatic cell counter or hemocytometer

[0178] - Measurement Procedure:

[0179] 100 μL cell suspension + 100 μL CellTiter-Glo reagent

[0180] * Room temperature, 10 min incubation (orbital shaker, 300 rpm)

[0181] * Luminescence measurement: GloMax® Navigator (Promega) or equivalent equipment

[0182] * Integration time: 0.5-1 second

[0183] - ATP concentration calculation:

[0184] * ATP Standard Curve Construction: 0-10 μM ATP (Sigma, Cat. A2383)

[0185] * Normalize to cell number → pmol / 10 6 cells

[0186] - Tolerance: Target value ± 5 pmol / 10 6 cells

[0187] Three independent cultures (N=3) were performed for each of the above conditions.

[0188] Experimental Examples 1-4: AAV2 Production and Vector Quantification Method

[0189] Cultured cells were collected in bulk at the time of harvest, and vector quantification was calculated in vg / L units using vector genome-based analysis. DNA quantification was performed using the Bio-Rad QX200 Droplet Digital PCR System, and the final vg concentration was calculated via a standard curve or absolute quantification analysis. The specific methods are as follows.

[0190] (1) AAV harvesting and purification:

[0191] - Harvest time: 48-72 hours after transfection

[0192] - Harvesting method:

[0193] * Collect the cells + the entire culture medium

[0194] * Freeze-thaw cycle: Freeze at -80°C / Thaw at 37°C, repeat 3 times (cell lysis and AAV release)

[0195] * Or detergent treatment: Triton X-100 0.1%, 37℃, 30 minutes

[0196] - Refining:

[0197] * Benzonase treatment: 50 U / mL, 37℃, 30 min (removal of residual DNA)

[0198] * Centrifugation: 3,000×g, 15 min, 4℃ (removal of cell debris)

[0199] * Recovery of supernatant (crude lysate)

[0200] (2) AAV vector genome quantification (ddPCR):

[0201] - DNA extraction:

[0202] * Crude lysate 10 μL + DNase I treatment (external DNA removal)

[0203] * Heat at 95℃ for 10 minutes (DNase inactivation and capsid destruction)

[0204] * Proteinase K treatment: 50 μg / mL, 55℃, 30 minutes

[0205] * 95℃, 10 min (Proteinase K inactivation)

[0206] - ddPCR reaction composition (20 μL reaction solution):

[0207] * QX200™ ddPCR EvaGreen Supermix (Bio-Rad): 10 μL

[0208] * Forward primer (10 μM): 1 μL

[0209] * Reverse primer (10 μM): 1 μL

[0210] * Template DNA: 1-2 μL (1:100-1:10,000 dilution)

[0211] * Nuclease-free water: up to 20 μL

[0212] - Primer Design:

[0213] * Forward: 5'-GGAACCCCTAGTGATGGAGTT-3'

[0214] * Reverse: 5'-CGGCCTCAGTGAGCGA-3'

[0215] * Amplicon size: 80-150 bp

[0216] - ddPCR Protocol:

[0217] * Droplet generation: QX200 Droplet Generator (Bio-Rad)

[0218] * PCR conditions:

[0219] - 95℃, 5 min (initial denaturation)

[0220] - [95℃ 30 sec → 60℃ 1 min] × 40 cycles

[0221] - 4℃, 5 min (signal stabilization)

[0222] - 90℃, 5 min (droplet stabilization)

[0223] * Droplet analysis: QX200 Droplet Reader (Bio-Rad)

[0224] * Data Analysis: QuantaSoft™ Software

[0225] - vg / L Calculation: vg / L = (copies / μL PCR reaction solution) × (dilution factor) × (extraction volume) × (1000 / harvest volume mL)

[0226] - Standard deviation: Mean ± SD of biological triplicates (N=3)

[0227] (3) Quantitative criteria:

[0228] - Number of positive droplets: At least 3

[0229] - Number of negative droplets: At least 10,000

[0230] - Dilution conditions: copies / μL in the range of 10–1000

[0231]

[0232] Example 2: Condition control of plasmid insertion sequence and time interval

[0233] Example 2-1: Control of Plasmid Injection Sequence

[0234] To evaluate the effect of the order of administration of Rep / Cap, Helper, and Transgene plasmids on productivity in AAV2 production, a total of 13 infection conditions (G1-G13) were designed for the G3 group of Example 1-1.

[0235] G1 was established as a condition in which the three plasmids were administered at the same time and was used as a reference group to represent the average yield. In the remaining groups, Rep / Cap, Helper, and Transgene were administered in different sequences at 3-hour intervals in either 2 or 3 stages, and after performing N=3 independent cultures under each condition, the AAV2 yield (vg / L) was compared (Table 7).

[0236] Group NameInfection OrderStep 1Step 2Step 3G1Rep / Cap, Helper, Transgene--G2Rep / CapHelper, Transgene-G3HelperRep / Cap, Transgene-G4TransgeneRep / Cap, Helper-G5Helper, TransgeneRep / Cap-G6Rep / Cap, TransgeneHelper-G7Rep / Cap, HelperTransgene-G8TransgeneRep / CapHelperG9TransgeneHelperRep / CapG10Rep / CapTransgeneHelperG11Rep / CapHelperTransgeneG12HelperTransgeneRep / CapG13HelperRep / CapTransgene

[0237] As a result of the analysis, the results confirming the productivity of AAV2 according to the order of plasmid administration are shown in Figure 4 and Table 8.

[0238] GroupReplicateTiter_vg_per_LG119.91E+13G121.07E+14G138.56E+13G214.67E+13G224.95E+13G236.07E+13G311.08E+14G329.3E+13G 331.02E+14G413.06E+14G422.69E+14G432.75E+14G512.5E+14G522. 78E+14G532.84E+14G618.62E+13G621.16E+14G631.05E+14G713.93E +13G723.87E+13G734.64E+13G813.63E+14G823.63E+14G833.97E+14G913.1E+14G923.34E+14G933.13E+14G1011.11E+14G1021.23E+14G1 031.09E+14G1115.46E+13G1125.24E+13G1136.03E+13G1218.13E+13 G1229.67E+13G1239.25E+13G1319.5E+13G1329.16E+13G1339.73E+13

[0239] Under G8 conditions (Stage 1: Transgene, Stage 2: Rep / Cap, Stage 3: Helper), the highest yield among all conditions was observed at approximately 3.7×10^14 vg / L.

[0240] Similarly, under G4, G5, and G9 conditions, where the transgene was introduced in the initial stage and the Rep / Cap and Helper were provided sequentially in subsequent stages, high yields of 3.0×10^14, 2.8×10^14, and 3.1×10^14 vg / L were observed, respectively, which corresponded to a high yield range that was reduced by approximately 20-30% compared to G8.

[0241] On the other hand, under conditions where Rep / Cap and Helper were administered simultaneously (e.g., G2, G7, G11) or Transgene was administered relatively late, the overall yield was low.

[0242] The average yields of G2, G7, and G11 were 5.0×10^13, 4.0×10^13, and 6.0×10^13 vg / L, respectively, showing a significant decrease compared to the reference group G1 (1.0×10^14 vg / L).

[0243] The remaining groups (G3, G6, G10, G12, G13) exhibited productivity within a range similar to G1 (within ±10%), indicating that changes in the plasmid insertion order had a moderate effect on productivity.

[0244] Example 2-2: Control of Plasmid Injection Time Interval

[0245] Based on the G8 conditions of Example 2-1 (sequential addition of Transgene → Rep / Cap → Helper), a total of eight experimental groups (G1-G8) were constructed with intervals of 1 to 8 hours adjusted to 1-hour increments to evaluate the effect of the time interval between each step on AAV2 productivity. Each condition was repeated with N=3 in the same cell culture environment, and the yield was quantified in vg / L at the end of production (Table 9).

[0246] Group Name Plasmid Injection Time Interval Step 1->Step 2 Step 2->Step 3 G111G222G333G444G555G666G777G888

[0247] Analysis results showed that AAV2 productivity exhibited a bell-shaped non-linear change over time intervals (Fig. 5, Table 10).

[0248] GroupReplicateTiter_vg_per_LG119.57E+13G128.61E+13G131.03E+14G212.6E+14 G222.98E+14G232.68E+14G313.03E+14G322.77E+14G333.5E+14G412.71E+14G422.78 E+14G432.63E+14G512E+14G522.73E+14G532.62E+14G611.12E+14G621.17E+14G639. 63E+13G717.18E+13G725.81E+13G736.22E+13G815.37E+13G823.64E+13G835.84E+13

[0249] In particular, the highest yield was observed at approximately 3.3×10^14 vg / L under G3 (3-hour interval) conditions. In comparison, G2 (2-hour interval), G4 (4 hours), and G5 (5 hours) showed levels of 2.6-2.8×10^14 vg / L, respectively, corresponding to a high-yield region that was about 20-30% lower than the peak value. This suggests that there exists a specific optimal point for plasmid expression timing alignment that maximizes AAV2 packaging efficiency.

[0250] On the other hand, under conditions where the time interval was very short (G1, 1 hour) or excessively long (G7-G8, 7-8 hours), the yield decreased significantly and remained at the level of 5-6×10^13 vg / L. This implies that early interference or over-separation of expression timing can impede production efficiency.

[0251] G6 (6 hours), with an intermediate interval, maintained productivity within a range (±10%) similar to the reference yield (G1).

[0252] In summary, the temporal arrangement of expression between plasmids is a key upstream structural variable determining AAV2 productivity, and G3 conditions (3-hour intervals) were found to provide the most efficient expression window.

[0253]

[0254] Experimental Example 2: Method for Controlling Conditions of Plasmid Injection Sequence and Time Interval

[0255] Experimental Example 2-1: Method for Controlling Plasmid Injection Conditions

[0256] To evaluate the effect of the delivery intervals of individual plasmids (Rep / Cap, Transgene, Helper) on productivity during AAV2 production, a timing panel based on three-step sequential plasmid delivery was constructed. The basic configuration maintained the plasmid delivery sequence used under G8 conditions—Step 1: Transgene → Step 2: Rep / Cap → Step 3: Helper—but a total of eight experimental groups (G1–G8) were established by varying the time intervals between each step in 1-hour increments within the range of 1–8 hours. Meanwhile, all conditions were performed under identical culture environments, and the total amount of each plasmid set was maintained at the same level. In other words, this experiment compared the relative effect of plasmid expression interaction timing on AAV2 productivity by varying only the temporal arrangement between steps. The experimental conditions are described below.

[0257] (1) Plasmid preparation:

[0258] - Rep / Cap Plasmid: pAAV2 / 2 (contains AAV2 rep and cap genes)

[0259] * Size: Approx. 7.3 kb

[0260] * Backbone: pBR322 origin

[0261] * Purification: EndoFree Plasmid Maxi Kit (Qiagen)

[0262] * Purity: A260 / A280 = 1.8-2.0

[0263] * Concentration: 1-2 mg / mL

[0264] - Helper plasmid: pHelper (adenovirus helper gene)

[0265] * Size: Approx. 11.8 kb

[0266] * Genes: E2A, E4, VA RNA

[0267] * Refining and Purity: Same as above

[0268] - Transgene plasmid: pAAV-CMV-GFP (or target gene)

[0269] * Size: 4.5-5.0 kb (including ITR)

[0270] * ITR sequence: AAV2 145 bp inverted terminal repeats

[0271] * Expression Cassette: CMV promoter-transgene-polyA

[0272] * Refining and Purity: Same as above

[0273] (2) Cell preparation:

[0274] - Vaccination: 0.8-1.2 × 10 6 cells / mL (24 hours prior to transfection)

[0275] - Survival rate: 95% or higher

[0276] - G1 ratio: 50 ± 3% (G3 condition of Example 1-1)

[0277] (3) Transfection conditions:

[0278] - Reagent: PEI MAX (Polysciences, Cat. 24765-1)

[0279] or FectoPRO® (Polyplus-transfection)

[0280] - PEI:DNA ratio (w / w): 3:1 to 5:1

[0281] - Plasmid ratio (Rep / Cap : Helper : Transgene): 1:1:1 (w / w / w)

[0282] - Total DNA amount: 1.0-1.5 μg / 10 6 cells

[0283] - Transfection Protocol:

[0284] Step 1) Dilute the plasmid in Opti-MEM (Gibco) or serum-free medium

[0285] Step 2) Dilute PEI with the same medium in a separate tube

[0286] Step 3) Add PEI solution to DNA solution, vortex immediately for 5 seconds

[0287] Step 4) Incubate at room temperature for 10-15 minutes (complex formation)

[0288] Step 5) Add to cell culture medium dropwise

[0289] Step 6) Gently mix

[0290] (4) Sequential input method (G8 condition):

[0291] - Step 1 (0 hours): Transgene plasmid only transfection

[0292] - Step 2 (after 3 hours): Rep / Cap Plasmid Transfection

[0293] - Step 3 (after 6 hours): Helper Plasmid Transfection

[0294] The above protocol was repeated at each transfection point. At this time, the time interval was calculated as 0 hours starting from the first transfection start time.

[0295] (5) Cultivation and Harvesting:

[0296] - Incubation after transfection: 48-72 hours

[0297] - Incubation temperature: 37℃ (or temperature shiftable to 32℃)

[0298] - Harvest: Collect cells + entire culture medium

[0299] Each of the above conditions was performed with independent culture N=3.

[0300] Experimental Example 2-2: AAV2 Production and Harvesting

[0301] In each experimental group, cells were harvested after being cultured for a certain period under identical upstream culture conditions. Vector productivity (AAV2 yield) was determined by collecting the entire culture medium and transferring it to the downstream quantitative analysis stage. All experimental groups were kept identical in terms of harvest time, culture duration, and culture environment to control for variables other than the timing of plasmid insertion, ensuring that they did not affect the results.

[0302] Experimental Example 2-3: Vector Genome Quantification Analysis

[0303] AAV2 vector yield was quantified based on the vector genome copy number, and absolute quantification was performed using the droplet digital PCR (ddPCR) platform. The vg / L value of each sample was calculated based on the absolute copy number derived from the manufacturer's algorithm. All analyses included three biological replicates (N=3) performed under identical conditions.

[0304]

[0305] Although preferred embodiments of the present invention have been described above with reference to the attached drawings, the embodiments described in this specification and the configurations illustrated in the drawings are merely the most preferred embodiments of the present invention and do not represent all technical concepts of the present invention. Therefore, it should be understood that various equivalents and modifications that can replace them may exist at the time of filing this application. Accordingly, the embodiments described above should be understood as illustrative in all respects and not restrictive, and the scope of the present invention is defined by the claims set forth below rather than by the detailed description. Furthermore, all modifications or variations derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included within the scope of the present invention.

Claims

1. A method for producing an adeno-associated virus (AAV) vector, (a) a step of controlling the G1 cell ratio of the production cells to 40% to 70%; and (b) a step of introducing the Rep / Cap plasmid, Transgene plasmid, and Helper plasmid into the production cells; Includes, A method for producing an AAV vector, characterized in that, in step (b) above, the Transgene plasmid is introduced before other plasmids or together with any one of the other plasmids.

2. In Claim 1, A method for producing an AAV vector in which the G1 cell ratio of step (a) above is 42% to 61%.

3. In Claim 1, The above step (a) further includes a step of controlling the mitochondrial membrane potential (MMP) of the production cell, and A method for producing an AAV vector, characterized in that the MMP has a relative MMP index of 0.85 to 1.25 when production cells under normal culture conditions are used as a control.

4. In Claim 1, The above step (a) further includes a step of controlling the intracellular ATP concentration of the production cell, and The above ATP concentration is 20 to 40 pmol / 10 6 A method for producing an AAV vector characterized by being cells.

5. In Claim 1, The above step (a) is, (a-1) A step of controlling the G1 cell ratio of production cells to 42% to 61%; (a-2) a step of controlling the mitochondrial membrane potential (MMP) of the production cell, wherein the relative MMP index relative to normal culture conditions is 0.85 to 1.25; and (a-3) Intracellular ATP concentration of producing cells 20 to 40 pmol / 10 6 Steps controlled by cells; A method for producing an AAV vector including 6. In Claim 1, The above step (b) is, (b-1) Step of administering a transgene plasmid; (b-2) A step of introducing a Rep / Cap plasmid after the above step (b-1); and (b-3) After step (b-2) above, a step of introducing a helper plasmid; A method for producing an AAV vector that sequentially includes 7. In Claim 1, The above step (b) is, (b-1) Step of administering a transgene plasmid; and (b-2) After step (b-1) above, a step of introducing the Rep / Cap plasmid and the Helper plasmid; A method for producing an AAV vector including 8. In claim 6 or 7, A method for producing an AAV vector, wherein the time interval between step (b-1) and step (b-2) is 1 hour to 6 hours.

9. In Claim 8, A method for producing an AAV vector, wherein the above time interval is 2 to 5 hours.

10. In Claim 9, A method for producing an AAV vector, wherein the above time interval is 3 hours.

11. A method for producing an adeno-associated virus (AAV) vector, (a) As a step of controlling physiological indicators of producing cells, (a-1) Control the G1 cell ratio to 42% to 61%, and (a-2) Control the relative mitochondrial membrane potential (MMP) index to 0.85 to 1.25 relative to normal culture conditions, and (a-3) Intracellular ATP concentration of 20 to 40 pmol / 10 6 Steps controlled by cells; and (b) A step of sequentially introducing plasmids, (b-1) Inject the transgene plasmid, and (b-2) 2 to 5 hours after step (b-1) above, Rep / Cap plasmid is added, and (b-3) A step of introducing a helper plasmid 2 to 5 hours after step (b-2) above; A method for producing an AAV vector including

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