Methods for enhancing recombinant adeno-associated virus yield

Abstract

The present invention provides a method for the production of a recombinant adeno-associated viral vector (rAAV), comprising contacting a host cell with a solution comprising at least one compound of formula (I), (IA), (IB), (II), (III), or (IV), or a salt thereof, or vitamin B, or any combination thereof. Also provided is a method for increasing production of rAAV by a host cell, comprising contacting the host cell with a solution comprising at least one compound of formula (I), (IA), (IB), (II), (III), or (IV), or a salt thereof, or vitamin B, or any combination thereof.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 990,099, filed on 16 March 2020, the entire contents of which are incorporated herein by reference in their entirety for all purposes. Field of Invention The present invention relates, as a whole, to a method for enhancing recombinant adeno-associated virus vector (rAAV) yield, and more specifically, to the use of compounds for enhancing rAAV yield. [Background technology]

[0002] background Adeno-associated viruses (AAVs) are non-pathogenic, replication-defective parvoviruses. Recombinant AAV vectors (rAAVs) possess numerous unique properties that make them attractive as vectors for gene therapy. In particular, rAAV vectors can deliver therapeutic genes to both dividing and non-dividing cells, and these genes can persist for extended periods without being integrated into the genome of the targeted cells. Given the widespread therapeutic applications of rAAVs, there is a continuing need for improved rAAV vector production methods, including methods for achieving high-titer rAAV vector yields. Previous attempts to improve the production of various viral vectors have included the use of cell culture additives, such as metals, trace supplements, and salts (see, for example, Williams, J. Gen. Virol., 9(3): 251-5 (1970), Weinbauer et al., Limnology and Oceanography, 54(3): 774-784 (2009), Yang et al., Hepatology, 48(5): 1396-403 (2008), and U.S. Publication No. 20150353899). [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] U.S. Patent Application Publication No. 2015 / 0353899 [Non-patent literature]

[0004] [Non-Patent Document 1] Weinbauer et al., Limnology and Oceanography (2009) 54(3):774~784 [Non-Patent Document 2] Yang et al., Hepatology (2008) 48(5):1396~403 [Overview of the Initiative] [Means for solving the problem]

[0005] Summary of the Invention This invention is in part based on the discovery that host cells used to produce recombinant adeno-associated virus vectors (rAAV) produce increased amounts of rAAV when they are brought into contact with a solution containing the compounds described herein. Therefore, in one embodiment, a method for producing recombinant adeno-associated virus (rAAV) is provided, wherein the host cell is given formula (I) [ka] The step includes contacting with a solution containing at least one compound or salt thereof, in which, [ka] However, it represents a single bond or a double bond. n is an integer selected from 1 to 5. Each R 1 However, independently, for each occurrence, -OH, C 1~6 Alkyl, C 1~6 alkyl-OH, C 1~6 Alkyl-OC 1~6 Alkyl, -OC 1~6 Alkyl, -C(O)N(Ra ) 2, and -C(O)OR b selected from the group consisting of R a is, independently for each occurrence, hydrogen or C 1~6 alkyl, R b is, independently for each occurrence, hydrogen or C 1~6 alkyl, X is CR c R d or N, R c and R d are, independently for each occurrence, hydrogen, C 1~6 alkyl, -OH, and -O-C 1~6 alkyl selected from the group consisting of, R c when is hydrogen, R d is C 1~6 alkyl, -OH, and -O-C 1~6 alkyl selected from, R c when is C 1~6 alkyl, -OH, and -O-C 1~6 alkyl selected from, R d is hydrogen, the at least one compound is

Chemical formula

[0006] In another aspect, a method for increasing rAAV titer yield, comprising the step of contacting a host cell with a solution containing a compound of formula (I) or a salt thereof, is provided herein.

[0007] In certain embodiments, the solution is of formula (I-A)

Chemical formula

[0008] In some embodiments, the solution is [ka] and includes at least one compound selected from the group consisting of any combination thereof.

[0009] In a particular embodiment, the solution is of formula (IB) [ka] It comprises at least one compound or salt thereof, in which, X is CR c R d And, R 5a , R 5b , R 5c , R 5d , and R 5e These are independent of each other, and for each occurrence, -OH and -OC. 1~6 Alkyl, C 1~6 Alkyl, C 1~6 alkyl-OH, and C 1~6 Alkyl-OC 1~6 It is alkyl, R c and R d Independently, for each appearance, hydrogen and C 1~6 Alkyl, -OH, and -OC 1~6 Selected from the group consisting of alkyl groups, R c However, if it is hydrogen, R dC 1~6 Alkyl, -OH, and -OC 1~6 Selected from alkyl, R c However, C 1~6 Alkyl, -OH, and -OC 1~6 If selected from alkyl, R d It is hydrogen.

[0010] In some embodiments, the solution is formula (III) [ka] It contains the compound.

[0011] In another embodiment, a method for producing rAAV, wherein a host cell is given formula (II) [ka] The step includes contacting with a solution containing at least one compound or salt thereof, in which, R 6 and R 7 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 Selected from the group consisting of alkyl groups, R 8 and R 9 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 Selected from the group consisting of alkyl groups, Y is either C or N, R 10 and R 11 However, independently, for each appearance, hydrogen, -OH, and C 1~6 Selected from the group consisting of alkyl groups, A is hydrogen, C 1~6 Alkyl and -C(O)N(R f Selected from the group consisting of )2, R f However, independently, for each appearance, hydrogen, C 1~6 Alkyl, C 1~6 alkyl-C(O)OH, and C 1~6Selected from the group consisting of alkyl-OH, The above at least one compound, [ka] But it's not that salt either. A method is provided herein.

[0012] In another embodiment, a method for increasing rAAV titer yield is provided herein, comprising the step of contacting host cells with a solution containing a compound of formula (II) or a salt thereof.

[0013] In a particular embodiment, at least one compound of formula (II) is of formula (IV) [ka] It is a compound of [the compound].

[0014] In another embodiment, a method for producing rAAV or increasing rAAV titer yield, wherein host cells are infused with vitamin B2, vitamin B7, vitamin B9, and vitamin B 12 A method is provided herein that includes the step of contacting a solution containing a vitamin B selected from the above.

[0015] In some embodiments, the methods described herein may further include the step of collecting and purifying rAAV.

[0016] Compounds described herein in solution (e.g., compounds of formula (I), (IA), (IB), (II), (III), or (IV)), or vitamin B (e.g., vitamin B2, vitamin B7, vitamin B9, or vitamin B 12The concentration of )) is intended to be higher than or equal to 10 mM, higher than or equal to 12 mM, higher than or equal to 15 mM, higher than or equal to 1 mM, higher than or equal to 0.5 mM, higher than 0.5 mM, between 0.5 mM and 15 mM, or between 1 mM and 10 mM. In one embodiment, the solution contains at least one compound (e.g., a compound of formula (I), (IA), (IB), (II), (III), or (IV)), or vitamin B (e.g., vitamin B2, vitamin B7, vitamin B9, or vitamin B 12 The concentration of )) is sufficient to produce at least 1.2 times greater amounts of secreted rAAV compared to that produced by host cells not in contact with a solution containing at least one compound. In another embodiment, the solution contains at least one compound (e.g., a compound of formula (I), (IA), (IB), (II), (III), or (IV)), or vitamin B (e.g., vitamin B2, vitamin B7, vitamin B9, or vitamin B 12 The concentration of )) is sufficient to produce at least 1.2 times greater total rAAV than that produced by host cells that have not been in contact with a solution containing at least one compound. In one embodiment, host cells are in contact with a solution containing at least one compound for at least two days.

[0017] The host cell may be a mammalian cell, e.g., HeLa, HEK293, COS, A549, BHK, or Vero cell. The host cell may also be an insect cell, e.g., Sf9, Sf-21, Tn-368, or BTI-Tn-5B1-4 (High-Five) cell. In one embodiment, the host cell is a HeLa cell. In another embodiment, the host cell is a HEK293 cell. The host cell may contain a recombinant nucleic acid construct containing heterologous nucleotide sequences with AAV terminal inversions adjacent to each other. In one embodiment, the host cell may contain rep and cap genes (e.g., AAV rep and cap genes). In one embodiment, the host cell may contain helper virus genes supplied, for example, via a helper plasmid or adenovirus infection. In one embodiment, the host cell includes i) heterologous nucleotide sequences adjacent to an AAV terminal inversion sequence, ii) rep and cap genes, and iii) helper virus genes.

[0018] In one embodiment, the host cell is given at least one compound (e.g., a compound of formula (I), (IA), (IB), (II), (III), or (IV)), or vitamin B (e.g., vitamin B2, vitamin B7, vitamin B9, or vitamin B 12 In another embodiment, the host cell produces at least 1.2 times greater amounts of secreted rAAV compared to that produced by host cells not in contact with a solution containing )). 12 It produces at least 1.2 times greater amounts of total rAAV compared to that produced by host cells that are not in contact with the solution containing )).

[0019] In some embodiments, the present invention relates to a method for producing recombinant adeno-associated virus (rAAV), wherein a host cell is... [ka] and contacting with a solution comprising at least one compound selected from the group consisting of these and any combination thereof, A method is provided wherein the host cell comprises i) a heterologous nucleotide sequence adjacent to the AAV terminal inverted repeat sequence, ii) the AAV rep and cap genes, and iii) optionally a helper virus gene.

[0020] In some embodiments, the present invention is a method for increasing the rAAV titer yield, comprising contacting a host cell with

Chemical formula

[0021] In some embodiments, the present invention provides a composition comprising a host cell and

Chemical formula

[0022] In some embodiments, the present invention is a method for producing recombinant adeno-associated virus (rAAV), comprising contacting a host cell with

Chemical formula

[0023] In some embodiments, the present invention is a method for increasing rAAV titer yield, wherein host cells [ka] The step includes contacting with a solution containing, The present invention provides a method in which a host cell comprises i) a heterologous nucleotide sequence adjacent to an AAV terminal inversion sequence, ii) AAV rep and cap genes, and iii) a helper virus gene, if necessary.

[0024] In some embodiments, the present invention involves a host cell and [ka] A composition comprising, The present invention provides a composition comprising, in the host cell, i) a heterologous nucleotide sequence adjacent to an AAV terminal inversion sequence, ii) AAV rep and cap genes, and iii) a helper virus gene as an option.

[0025] In some embodiments, the present invention relates to a method for producing recombinant adeno-associated virus (rAAV), wherein a host cell is... [ka] The step includes contacting with a solution containing, The present invention provides a method in which a host cell comprises i) a heterologous nucleotide sequence adjacent to an AAV terminal inversion sequence, ii) AAV rep and cap genes, and iii) a helper virus gene, if necessary.

[0026] In some embodiments, the present invention is a method for increasing rAAV titer yield, wherein host cells [ka] The step includes contacting with a solution containing, The present invention provides a method in which a host cell comprises i) a heterologous nucleotide sequence adjacent to an AAV terminal inversion sequence, ii) AAV rep and cap genes, and iii) a helper virus gene, if necessary.

[0027] In some embodiments, the present invention involves a host cell and [ka] A composition comprising, The present invention provides a composition comprising, in the host cell, i) a heterologous nucleotide sequence adjacent to an AAV terminal inversion sequence, ii) AAV rep and cap genes, and iii) a helper virus gene as an option.

[0028] In some embodiments, the present invention relates to a method for producing recombinant adeno-associated virus (rAAV), wherein host cells are infused with vitamin B2, vitamin B7, vitamin B9, and vitamin B 12 The present invention provides a method comprising the step of contacting a host cell with a solution containing, and at least one compound selected from the group consisting of any combination thereof, wherein the host cell contains i) a heterologous nucleotide sequence adjacent to an AAV terminal inversion sequence, ii) AAV rep and cap genes, and iii) optionally a helper virus gene.

[0029] In some embodiments, the present invention is a method for increasing rAAV titer yield, wherein host cells are supplied with vitamin B2, vitamin B7, vitamin B9, and vitamin B 12 The present invention provides a method comprising the step of contacting a host cell with a solution containing, and at least one compound selected from the group consisting of any combination thereof, wherein the host cell contains i) a heterologous nucleotide sequence adjacent to an AAV terminal inversion sequence, ii) AAV rep and cap genes, and iii) optionally a helper virus gene.

[0030] In some embodiments of the provided method, the solution [Chemical Formula] does not contain either it or its salts.

[0031] In some embodiments of the provided method, the solution [Chemical Formula] does not contain either it or its salts.

[0032] In some embodiments, the provided composition [Chemical Formula] does not contain either it or its salts.

[0033] In some embodiments, the provided composition [Chemical Formula] does not contain either it or its salts.

[0034] In some embodiments, the present invention provides a composition comprising a host cell and at least one compound selected from the group consisting of vitamin B2, vitamin B7, vitamin B9, vitamin B 12 , and any combination thereof, wherein the host cell comprises: i) a heterologous nucleotide sequence adjacent to an AAV terminal inverted repeat sequence, ii) AAV rep and cap genes, and iii) optionally, a helper virus gene.

[0035] In other aspects, the present invention provides rAAV produced by any of the contemplated methods, a composition comprising rAAV produced by any of the contemplated methods, or a host cell and at least one compound described herein (e.g., a compound of formula (I), (I-A), (I-B), (II), (III), or (IV), or a vitamin B (e.g., vitamin B2, vitamin B7, vitamin B9, vitamin B 12The present invention provides a composition comprising, or any combination thereof, or any combination thereof.

[0036] These and other aspects and characteristics of the present invention are described in the following detailed description and claims.

[0037] The aforementioned and other objects, characteristics, and advantages of the present invention will become apparent from the following description of preferred embodiments, as illustrated in the accompanying drawings. Similar reference elements identify common characteristics in the corresponding drawings. [Brief explanation of the drawing]

[0038] [Figure 1] Figure 1 is a schematic bar graph of normalized volume-recombinant AAV (rAAV) titers for various clade E and F rAAVs produced from HeLa-producing cells at fluctuating niacinamide concentrations (0.1 mM to 40 mM). Titers, measured in genome copy number per mL (GC / mL), are normalized to rAAV production in the absence of niacinamide.

[0039] [Figure 2] Figure 2 shows the cubic polynomial fit of data points for normalized volume rAAV titer (GC / mL) of clade E and F rAAV produced from different HeLa-producing cell clones at various niacinamide concentrations.

[0040] [Figure 3] Figure 3 is a bar graph showing the effect of container scaling of niacinamide on rAAV production from HeLa-producing cell lines capable of producing clade E rAAV (AAVrh10-FIX), which encodes a factor IX transgene. Two common container scales were used: a 3 mL deep well (24-DW) and a 100 mL shaking flask (flask). Niacinamide was added at the indicated concentrations (1 mM to 50 mM), and the rAAV yield (GC / mL) was measured.

[0041] [Figure 4A] Figure 4A is a bar graph showing the effect of niacinamide on rAAV production from HeLa-producing cell line clones capable of producing recombinant clade E AAV (AAVrh10-FIX) encoding factor IX transgene. Niacinamide was added at the indicated concentrations (5 mM to 40 mM), and the rAAV yield (GC / mL) was measured.

[0042] [Figure 4B] Figure 4B is a bar graph showing the effect of niacinamide on rAAV production from a first HeLa-producing cell line clone capable of producing recombinant clade E AAV (AAV8-G6Pase) encoding a glucose-6-phosphatase (G6Pase) transgene. Niacinamide was added at the indicated concentrations (5 mM to 40 mM), and the rAAV yield (GC / mL) was measured.

[0043] [Figure 4C] Figure 4C is a bar graph showing the effect of niacinamide on rAAV production from a second HeLa-producing cell line clone capable of producing recombinant clade E AAV (AAV8-G6Pase) encoding a glucose-6-phosphatase (G6Pase) transgene. Niacinamide was added at the indicated concentrations (1 mM to 40 mM), and the rAAV yield (GC / mL) was measured.

[0044] [Figure 4D] Figure 4D is a bar graph showing the effect of niacinamide on rAAV production from a third HeLa-producing cell line clone capable of producing recombinant clade E AAV (AAVhu37-FVIII) encoding factor VIII transgene. Niacinamide was added at the indicated concentrations (1 mM to 40 mM), and the rAAV yield (GC / mL) was measured.

[0045] [Figure 5]Figure 5 is a bar graph showing the effect of niacinamide on rAAV production from two HeLa-producing cell line clones capable of producing recombinant clade F AAV (AAV9-ATP7B) encoding a truncated ATP7B transgene. The clones were cultured in a 2 L bioreactor. Niacinamide was added at the indicated concentration (3 mM), and the rAAV yield (GC / mL) was measured.

[0046] [Figure 6] Figure 6 is a bar graph showing the effect of niacinamide on rAAV production from HEK293 cells cultured in a 30 mL flask. HEK293 cells were transfected with a plasmid that enables the production of recombinant clade E AAV (rAAV8-OTC) encoding ornithine transcarbamylase. Niacinamide was added at the indicated concentrations (0.03 mM to 30 mM), and the rAAV yield (GC / mL) was measured. [Modes for carrying out the invention]

[0047] Detailed explanation This invention is partly based on the discovery that host cells used to produce recombinant adeno-associated virus vectors (rAAV) produce increased amounts of rAAV when a compound, such as the compound of formula (I), the compound of formula (IA), the compound of formula (IB), the compound of formula (II), the compound of formula (III), the compound of formula (IV), or a salt of any one of these, or vitamin B, is added to the host cell culture. I. Compounds used in this method

[0048] In one embodiment, the present invention relates to a method for producing recombinant adeno-associated virus (rAAV), wherein a host cell is subjected to formula (I) [ka] The step includes contacting with a solution containing at least one compound or salt thereof, in which, [ka] represents a single bond or a double bond, and n is an integer selected from 1 to 5, and each R 1 is independently, for each occurrence, -OH, C 1~6 alkyl, C 1~6 alkyl-OH, C 1~6 alkyl-O-C 1~6 alkyl, -O-C 1~6 alkyl, -C(O)N(R a )2, and -C(O)OR b is selected from the group consisting of, R a is independently, for each occurrence, hydrogen or C 1~6 alkyl, and R b is independently, for each occurrence, hydrogen or C 1~6 alkyl, and X is CR c R d or N, and R c and R d are independently, for each occurrence, hydrogen, C 1~6 alkyl, -OH, and -O-C 1~6 alkyl, and when R c is hydrogen, R d is selected from C 1~6 alkyl, -OH, and -O-C 1~6 alkyl, and when R c is C 1~6 alkyl, -OH, and -O-C 1~6 alkyl, R d is hydrogen, and the at least one compound is

Chemical formula

[0049] In another embodiment, the present invention provides a method for increasing rAAV titer yield, comprising the step of contacting host cells with a solution containing a compound of formula (I) or a salt thereof.

[0050] In certain embodiments, the compound of formula (I) described herein is a compound in which X is N. In some embodiments, the compound of formula (I) described herein is a compound in which X is N and R 1 However, independently, for each occurrence, -OH, C 1~6 Alkyl, C 1~6 alkyl-OH, C 1~6 Alkyl-OC 1~6 Alkyl, -OC 1~6 Alkyl, -C(O)N(R a )2, and -C(O)OR b A compound selected from. In certain embodiments, the compound of formula (I) described herein is such that X is N and R 1 However, independently, for each occurrence, C 1~6 alkyl-OH, -C(O)N(R a )2, and -C(O)OR b A compound selected from. In certain embodiments, the compound of formula (I) described herein is such that X is N and R 1 However, C 1~6 The compound is an alkyl-OH (e.g., -CH2OH). In certain embodiments, the compound of formula (I) described herein is such that X is N and R 1 However, -C(O)N(R a )2, R a However, it is a compound where X is hydrogen (e.g., -C(O)NH2). In certain embodiments, the compound of formula (I) described herein is where X is N and R 1 However, -C(O)OR b And R b However, independently, each occurrence is a compound that is either hydrogen or methyl (e.g., C(O)OH and -C(O)OCH3).

[0051] In certain embodiments, the compound of formula (I) described herein is such that X is CR c R d And R c However, it is hydrogen, and R d However, C 1~6 Alkyl, -OH, and -OC 1~6 Selected from alkyl groups, each R 1 However, independently, for each occurrence, -OH, C 1~6 Alkyl, C 1~6 alkyl-OH, C 1~6 Alkyl-OC 1~6 Alkyl, -OC 1~6 Alkyl, -C(O)N(R a )2, and -C(O)OR b A compound selected from the following. In some embodiments, the compound of formula (I) described herein is a compound of formula (I) in which X is CR c R d And R c However, C 1~6 Alkyl, -OH, and -OC 1~6 Selected from alkyl, R d However, it is -OH, and R 1 However, independently, for each occurrence, -OH, C 1~6 Alkyl, C 1~6 alkyl-OH, C 1~6 Alkyl-OC 1~6 Alkyl, -OC 1~6 Alkyl, -C(O)N(R a )2, and -C(O)OR b A compound selected from the following. In certain embodiments, the compound of formula (I) described herein is such that X is CR c R d And R c However, it is hydrogen, and R d However, it is -OH, and R 1 However, independently, for each occurrence, -OH, C 1~6 Alkyl, C 1~6 alkyl-OH, C 1~6 Alkyl-OC 1~6 Alkyl, -OC 1~6 Alkyl, -C(O)N(R a)2, and -C(O)OR b A compound selected from the following. In certain embodiments, the compound of formula (I) described herein is such that X is CR c R d And R c However, it is hydrogen, and R d However, it is -OH, and R 1 However, it is a compound with an -OH group.

[0052] In certain embodiments, the compound of formula (I) described herein is such that X is N, n is 1, and R 1 However, independently, for each occurrence, C 1~6 alkyl-OH, -C(O)N(R a )2, and -C(O)OR b Selected from (for example, R 1 However, independently, for each occurrence, the compound is selected from -CH2OH, -C(O)NH2, -C(O)OH, and -C(O)OCH3). In certain embodiments, the compound of formula (I) described herein is such that X is CR c R d And R c However, it is hydrogen, and R d However, it is -OH, n is 5, and R 1 However, it is a compound with an -OH group.

[0053] In certain embodiments, the present invention provides a method for producing recombinant adeno-associated virus (rAAV), comprising the step of contacting host cells with a solution containing at least one compound of formula (IA) or a salt thereof. In certain embodiments, the present invention provides a method for increasing rAAV titer yield, comprising contacting host cells with a solution containing formula (IA) [ka] The step includes contacting with a solution containing the compound or a salt thereof, in which, R 2 and R 3 However, independently, for each occurrence, is it hydrogen or -OH? or R2 and R 3 However, together they can form an oxo, R 4 However, hydrogen, -OH, -OC 1~6 Alkyl, and -N(R e Selected from the group consisting of )2, R e However, independently, for each appearance, hydrogen or C 1~6 It is alkyl. Provide a method.

[0054] In certain embodiments, the compound of formula (IA) described herein is R 2 and R 3 However, together they form an oxo compound. In a particular embodiment, the compound of formula (IA) is R 4 However, -OH, -OC 1~6 Alkyl, and -N(R e )2 (e.g., -NH2, -OH, and -OCH3) is a compound selected from these. In certain embodiments, the compound of formula (IA) described herein is R 4 However, the compound is selected from -NH2, -OH, and -OCH3. In certain embodiments, the compound of formula (IA) described herein is R 2 and R 3 However, together they form an oxo, R 4 However, -OH, -OC 1~6 Alkyl, and -N(R e )2 (e.g., -NH2, -OH, and -OCH3) is a compound selected from these. In some embodiments, the compound of formula (IA) described herein is R 2 and R 3 However, together they form an oxo, R 4 However, the compound is selected from -NH2, -OH, and -OCH3. In certain embodiments, the compound of formula (IA) described herein is R 2 and R 3 However, together they form an oxo, R 4However, it is a compound that is -NH2. In certain embodiments, the compound of formula (IA) described herein is R 2 and R 3 However, together they form an oxo, R 4 However, it is a compound that is -OH. In certain embodiments, the compound of formula (IA) described herein is R 2 and R 3 However, together they form an oxo, R 4 However, it is a compound that is -OCH3.

[0055] In certain embodiments, the compound of formula (IA) described herein is R 2 and R 3 However, all of them are hydrogen compounds. In a particular embodiment, the compound of formula (IA) is R 4 However, hydrogen, -OH, -OC 1~6 Alkyl, and -N(R e )2 (e.g., -NH2, -OH, and -OCH3) is a compound selected from these. In certain embodiments, the compound of formula (IA) described herein is R 4 However, the compound is selected from hydrogen, -NH2, -OH, and -OCH3. In certain embodiments, the compound of formula (IA) described herein is R 2 and R 3 However, all of them are hydrogen, R 4 However, the compound is selected from the group consisting of hydrogen, -NH2, -OH, and -OCH3. In certain embodiments, the compound of formula (IA) described herein is R 2 and R 3 However, all of them are hydrogen, R 4 However, it is a compound that is hydrogen. In certain embodiments, the compound of formula (IA) described herein is R 2 and R 3 However, all of them are hydrogen, R 4 However, it is a compound that is -NH2. In certain embodiments, the compound of formula (IA) described herein is R 2 and R 3However, all of them are hydrogen, R 4 However, it is a compound that is -OH. In certain embodiments, the compound of formula (IA) described herein is R 2 and R 3 However, all of them are hydrogen, R 4 However, it is a compound that is -OCH3.

[0056] In certain embodiments, the compound of formula (IA) described herein is R 2 and R 3 However, independently, for each occurrence, it is either hydrogen or -OH, or R 2 and R 3 However, together they can form an oxo, R 4 However, it is a compound that is -OH. In certain embodiments, the compound of formula (IA) described herein is R 2 and R 3 However, independently, for each occurrence, it is either hydrogen or -OH, or R 2 and R 3 However, together they can form an oxo, R 4 However, it is a compound that is -OCH3. In certain embodiments, the compound of formula (IA) described herein is R 2 and R 3 However, independently, for each occurrence, it is either hydrogen or -OH, or R 2 and R 3 However, together they can form an oxo, R 4 However, it is a compound with the -NH2 group.

[0057] In a particular embodiment, the present invention provides a method for producing rAAV, wherein a host cell is... [ka] The present invention provides a method comprising the step of contacting a solution containing at least one compound selected from the group consisting of any combination thereof.

[0058] In a particular embodiment, a method for producing rAAV is provided, comprising the step of contacting a host cell with a solution containing at least one compound selected from the group consisting of niacinamide, niacin, methyl nicotinate, nicotinyl alcohol, and any combination thereof.

[0059] In a particular embodiment, the present invention is a method for increasing rAAV titer yield, wherein host cells [ka] The present invention provides a method comprising the step of contacting a solution containing at least one compound selected from the group consisting of any combination thereof.

[0060] In a particular embodiment, a method is provided for increasing rAAV titer yield, comprising the step of contacting host cells with a solution containing at least one compound selected from the group consisting of niacinamide, niacin, methyl nicotinate, nicotinyl alcohol, and any combination thereof.

[0061] In certain embodiments, the present invention provides a method for producing rAAV, comprising the step of contacting host cells with a solution containing at least one compound of formula (IB) or a salt thereof. In some embodiments, the present invention provides a method for increasing rAAV titer yield, comprising contacting host cells with a solution containing formula (IB) [ka] The step includes contacting with a solution containing at least one compound or salt thereof, in which, X, CR c R d And, R 5a , R 5b , R 5c , R 5d , and R 5e However, independently, for each occurrence, -OH, -OC 1~6 Alkyl, C1~6 Alkyl, C 1~6 alkyl-OH, and C 1~6 Alkyl-OC 1~6 It is alkyl, R c and R d However, independently, for each appearance, hydrogen, C 1~6 Alkyl, -OH, and -OC 1~6 Selected from alkyl, R c However, if it is hydrogen, R d C 1~6 Alkyl, -OH, and -OC 1~6 Selected from alkyl, R c However, C 1~6 Alkyl, -OH, and -OC 1~6 If selected from alkyl, R d It is hydrogen. Provide a method.

[0062] In certain embodiments, the compound of formula (IB) described herein is R c However, it is hydrogen, and R d However, C 1~6 Alkyl, -OH, and -OC 1~6 A compound selected from alkyl groups. In certain embodiments, the compound of formula (IB) described herein is R c However, C 1~6 Alkyl, -OH, and -OC 1~6 Selected from alkyl, R d However, it is a compound that is hydrogen. In some embodiments, the compound of formula (IB) described herein is R c However, it is hydrogen, and R d However, it is a compound that is -OH. In certain embodiments, the compound of formula (IB) described herein is R c However, it is hydrogen, and R d However, -OC 1~6 It is an alkyl compound. In certain embodiments, the compound of formula (IB) described herein is R c However, it is hydrogen, and R d However, C 1~6It is an alkyl compound.

[0063] In certain embodiments, the compound of formula (IB) described herein is R 5a , R 5b , R 5c , R 5d , and R 5e However, it is a compound that is -OH. In certain embodiments, the compound of formula (IB) described herein is R c However, it is hydrogen, and R d However, it is a compound that is -OH. In certain embodiments, the compound of formula (IB) described herein is R 5a , R 5b , R 5c , R 5d , and R 5e However, it is -OH, and R c and R d However, independently, for each appearance, hydrogen, C 1~6 Alkyl, -OH, and -OC 1~6 Selected from alkyl, R c However, if it is hydrogen, R d C 1~6 Alkyl, -OH, and -OC 1~6 Selected from alkyl, R c However, C 1~6 Alkyl, -OH, and -OC 1~6 If selected from alkyl, R d is a compound, which is hydrogen. In certain embodiments, the compound of formula (IB) described herein is R 5a , R 5b , R 5c , R 5d , and R 5e However, it is -OH, and R c However, it is hydrogen, and R d However, C 1~6 Alkyl, -OH, and -OC 1~6 A compound selected from alkyl groups. In certain embodiments, the compound of formula (IB) described herein is R 5a , R 5b , R 5c , R 5d, and R 5e However, it is -OH, and R c However, it is hydrogen, and R d However, it is a compound selected from -OH.

[0064] In a particular embodiment, the present invention provides a method for producing rAAV, wherein a host cell is given formula (III) [ka] The present invention provides a method comprising the step of contacting a compound or a salt thereof with a solution containing the compound or a salt thereof.

[0065] In a particular embodiment, the present invention provides a method for producing rAAV, comprising the step of contacting host cells with a solution containing myo-inositol or a salt thereof.

[0066] In a particular embodiment, the present invention provides a method for increasing rAAV titer yield, wherein host cells are given formula (III) [ka] The present invention provides a method comprising the step of contacting a compound or a salt thereof with a solution containing the compound or a salt thereof.

[0067] In a particular embodiment, the present invention provides a method for increasing rAAV titer yield, comprising the step of contacting host cells with a solution containing myo-inositol or a salt thereof.

[0068] In another embodiment, the present invention provides a method for producing rAAV, comprising the step of contacting host cells with a solution containing a compound of formula (II) or a salt thereof. In another embodiment, the present invention provides a method for increasing rAAV titer yield, comprising contacting host cells with a solution containing a compound of formula (II) [ka] The step includes contacting with a solution containing at least one compound or salt thereof, in which, R6 and R 7 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 Selected from the group consisting of alkyl groups, R 8 and R 9 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 Selected from the group consisting of alkyl groups, Y is either C or N, R 10 and R 11 However, independently, for each appearance, hydrogen, -OH, and C 1~6 Selected from the group consisting of alkyl groups, A is hydrogen, C 1~6 Alkyl and -C(O)N(R f Selected from the group consisting of )2, R f However, independently, for each appearance, hydrogen, C 1~6 Alkyl, C 1~6 alkyl-C(O)OH, and C 1~6 Selected from the group consisting of alkyl-OH, The above at least one compound, [ka] But it's not that salt either. Provide a method.

[0069] In certain embodiments, the compound of formula (II) described herein is R 6 and R 7 However, it is hydrogen, a compound. In certain embodiments, the compound of formula (II) described herein is R 8 and R 9 However, it is hydrogen, a compound. In a particular embodiment, the compound of formula (II) is R 6 and R 7 However, it is hydrogen, and R 8 and R 9 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6A compound selected from alkyl groups. In certain embodiments, the compound of formula (II) described herein is R 8 and R 9 However, it is hydrogen, and R 6 and R 7 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 A compound selected from alkyl groups. In certain embodiments, the compound of formula (II) described herein is R 6 , R 7 , R 8 , and R 9 However, it is hydrogen, a compound.

[0070] In certain embodiments, the compound of formula (II) described herein is a compound in which Y is C. In certain embodiments, the compound of formula (II) described herein is a compound in which Y is C and R 6 and R 7 However, it is hydrogen, and R 8 and R 9 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 It is a compound selected from alkyl. In certain embodiments, the compound of formula (II) described herein is such that Y is C and R 8 and R 9 However, it is hydrogen, and R 6 and R 7 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 It is a compound selected from alkyl. In certain embodiments, the compound of formula (II) described herein is such that Y is C and R 6 , R 7 , R 8 , and R 9 However, it is hydrogen, a compound.

[0071] In certain embodiments, the compound of formula (II) described herein is a compound in which Y is N. In certain embodiments, the compound of formula (II) described herein is a compound in which Y is N and R 6and R 7 However, it is hydrogen, and R 8 and R 9 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 It is a compound selected from alkyl groups. In certain embodiments, the compound of formula (II) described herein is such that Y is N and R 8 and R 9 However, it is hydrogen, and R 6 and R 7 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 It is a compound selected from alkyl groups. In certain embodiments, the compound of formula (II) described herein is such that Y is N and R 6 , R 7 , R 8 , and R 9 However, it is hydrogen, a compound.

[0072] In certain embodiments, the compound of formula (II) described herein is R 10 and R 11 However, C 1~6 It is an alkyl compound. In certain embodiments, the compound of formula (II) described herein is R 10 and R 11 However, it is a compound with CH3.

[0073] In certain embodiments, the compound of formula (II) described herein is such that Y is C and R 10 and R 11 However, C 1~6 It is alkyl, R 6 and R 7 However, it is hydrogen, and R 8 and R 9 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 It is a compound selected from alkyl. In certain embodiments, the compound of formula (II) described herein is such that Y is C and R 10 and R 11 However, C 1~6It is alkyl, R 8 and R 9 However, it is hydrogen, and R 6 and R 7 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 It is a compound selected from alkyl. In certain embodiments, the compound of formula (II) described herein is such that Y is C and R 10 and R 11 However, C 1~6 It is alkyl, R 6 , R 7 , R 8 , and R 9 However, it is hydrogen, a compound.

[0074] In certain embodiments, the compound of formula (II) described herein is such that Y is N and R 10 and R 11 However, C 1~6 It is alkyl, R 6 and R 7 However, it is hydrogen, and R 8 and R 9 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 It is a compound selected from alkyl groups. In certain embodiments, the compound of formula (II) described herein is such that Y is N and R 10 and R 11 However, C 1~6 It is alkyl, R 8 and R 9 However, it is hydrogen, and R 6 and R 7 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 It is a compound selected from alkyl groups. In certain embodiments, the compound of formula (II) described herein is such that Y is N and R 10 and R 11 However, C 1~6 It is alkyl, R 6 , R 7 , R 8 , and R 9 However, it is hydrogen, a compound.

[0075] In certain embodiments, the compound of formula (II) described herein is such that Y is C and R 10 and R 11 However, it is CH3, and R 6 and R 7 However, it is hydrogen, and R 8 and R 9 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 It is a compound selected from alkyl. In certain embodiments, the compound of formula (II) described herein is such that Y is C and R 10 and R 11 However, it is CH3, and R 8 and R 9 However, it is hydrogen, and R 6 and R 7 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 It is a compound selected from alkyl. In certain embodiments, the compound of formula (II) described herein is such that Y is C and R 10 and R 11 However, it is CH3, and R 6 , R 7 , R 8 , and R 9 However, it is hydrogen, a compound.

[0076] In certain embodiments, the compound of formula (II) described herein is such that Y is N and R 10 and R 11 However, it is CH3, and R 6 and R 7 However, it is hydrogen, and R 8 and R 9 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 It is a compound selected from alkyl groups. In certain embodiments, the compound of formula (II) described herein is such that Y is N and R 10 and R 11 However, it is CH3, and R 8 and R 9However, it is hydrogen, and R 6 and R 7 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 It is a compound selected from alkyl groups. In certain embodiments, the compound of formula (II) described herein is such that Y is N and R 10 and R 11 However, it is CH3, and R 6 , R 7 , R 8 , and R 9 However, it is hydrogen, a compound.

[0077] In certain embodiments, the compound of formula (II) described herein is A, C 1~6 It is an alkyl compound. In certain embodiments, the compound of formula (II) described herein is a compound in which A is CH3.

[0078] In certain embodiments, the compound of formula (II) described herein is such that Y is C and A is C 1~6 It is alkyl, R 10 and R 11 However, it is CH3, and R 6 and R 7 However, it is hydrogen, and R 8 and R 9 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 It is a compound selected from alkyl groups. In certain embodiments, the compound of formula (II) described herein is such that Y is C and A is C 1~6 It is alkyl, R 10 and R 11 However, it is CH3, and R 8 and R 9 However, it is hydrogen, and R 6 and R 7 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 It is a compound selected from alkyl groups. In certain embodiments, the compound of formula (II) described herein is such that Y is C and A is C 1~6It is alkyl, R 10 and R 11 However, it is CH3, and R 6 , R 7 , R 8 , and R 9 However, it is hydrogen, a compound.

[0079] In a particular embodiment, the compound of formula (II) described herein is such that Y is N and A is C 1~6 It is alkyl, R 10 and R 11 However, it is CH3, and R 6 and R 7 However, it is hydrogen, and R 8 and R 9 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 It is a compound selected from alkyl groups. In certain embodiments, the compound of formula (II) described herein is such that Y is N and A is C 1~6 It is alkyl, R 10 and R 11 However, it is CH3, and R 8 and R 9 However, it is hydrogen, and R 6 and R 7 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 It is a compound selected from alkyl groups. In certain embodiments, the compound of formula (II) described herein is such that Y is N and A is C 1~6 It is alkyl, R 10 and R 11 However, it is CH3, and R 6 , R 7 , R 8 , and R 9 However, it is hydrogen, a compound.

[0080] In certain embodiments, the compound of formula (II) described herein is R 6 , R 7 , R 8 , and R 9 However, Y is hydrogen, and N is R. 10 and R11 However, it is a compound in which A is CH3.

[0081] In a particular embodiment, the present invention provides a method for producing rAAV, wherein a host cell is given formula (IV) [ka] The present invention provides a method comprising the step of contacting a compound or a salt thereof with a solution containing the compound or a salt thereof.

[0082] In a particular embodiment, the present invention provides a method for producing rAAV, comprising the step of contacting a host cell with a solution containing choline or a salt thereof.

[0083] In a particular embodiment, the present invention provides a method for increasing rAAV titer yield, wherein host cells are given formula (IV) [ka] The present invention provides a method comprising the step of contacting a compound or a salt thereof with a solution containing the compound or a salt thereof.

[0084] In a particular embodiment, the present invention provides a method for increasing rAAV titer yield, comprising the step of contacting host cells with a solution containing choline or a salt thereof.

[0085] In another embodiment, the present invention relates to a method for producing rAAV, wherein a host cell is subjected to vitamin B2, vitamin B7, vitamin B9, and vitamin B 12 The present invention provides a method comprising the step of contacting the solution with a solution containing at least one vitamin B selected from, or any combination thereof.

[0086] In another embodiment, the present invention relates to a method for increasing rAAV titer yield, wherein host cells are subjected to vitamin B2, vitamin B7, vitamin B9, and vitamin B 12The present invention provides a method comprising the step of contacting the solution with a solution containing at least one vitamin B selected from, or any combination thereof.

[0087] II.Culture conditions In certain embodiments, the present invention relates to a method for producing rAAV or increasing rAAV titer yield, wherein host cells are subjected to at least one compound described herein (e.g., a compound of formula (I), (IA), (IB), (II), (III), or (IV)), or vitamin B (e.g., vitamin B2, vitamin B7, vitamin B9, vitamin B 12 The present invention provides a method comprising the step of contacting a solution containing (or any combination thereof), wherein the final concentration of at least one compound in the solution is greater than 0.5 mM. In other embodiments, the final concentration of at least one compound in the solution is greater than or equal to 0.5 mM.

[0088] In certain embodiments, the present invention relates to a method for producing rAAV or increasing rAAV titer yield, wherein host cells are subjected to at least one compound described herein (e.g., a compound of formula (I), (IA), (IB), (II), (III), or (IV)), or vitamin B (e.g., vitamin B2, vitamin B7, vitamin B9, vitamin B 12The step includes contacting a solution containing (1) or (2) a compound (1), (2) a compound (2), or any combination thereof (3), and (2) a compound (2), wherein the final concentration of at least one compound in the solution is between 0.5 mM and 15 mM (for example, between 0.5 mM and 14 mM, between 0.5 mM and 13 mM, between 0.5 mM and 12 mM, between 0.5 mM and 11 mM, between 0.5 mM and 10 mM, between 0.5 mM and 9 mM, between 0.5 mM and 8 mM, between 0.5 mM and 7 mM, between 0.5 mM and 6 mM, between 0.5 mM and 5 mM, 0.5 mM The present invention provides methods for concentrations between M and 4 mM, between 0.5 mM and 3 mM, between 0.5 mM and 2 mM, between 0.5 mM and 1 mM, between 1 mM and 15 mM, between 2 mM and 15 mM, between 3 mM and 15 mM, between 4 mM and 15 mM, between 5 mM and 15 mM, between 6 mM and 15 mM, between 7 mM and 15 mM, between 8 mM and 15 mM, between 9 mM and 15 mM, between 10 mM and 15 mM, between 11 mM and 15 mM, between 12 mM and 15 mM, between 13 mM and 15 mM, and between 14 mM and 15 mM. In some exemplary embodiments, the final concentration of at least one compound in the solution is between 1 mM and 10 mM. In some embodiments, the final concentration of at least one compound in the solution is selected from 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM, 5.5mM, 6mM, 6.5mM, 7mM, 7.5mM, 8mM, 8.5mM, 9mM, 9.5mM, 10mM, 11mM, 12mM, 13mM, 14mM, or 15mM, as well as all decimal and fractional values ​​between 0.5mM and 15mM.

[0089] In certain embodiments, the present invention relates to a method for producing rAAV or increasing rAAV titer yield, wherein host cells are subjected to at least one compound described herein (e.g., a compound of formula (I), (IA), (IB), (II), (III), or (IV)), or vitamin B (e.g., vitamin B2, vitamin B7, vitamin B9, vitamin B 12The present invention provides a method comprising the step of contacting a host cell with a solution containing (1) or (2) a solution containing (1) a solution containing (1) a solution containing (1) a solution containing (1) a solution containing (2

[0090] In certain embodiments, the present invention relates to a method for producing rAAV or increasing rAAV titer yield, wherein host cells are subjected to at least one compound described herein (e.g., a compound of formula (I), (IA), (IB), (II), (III), or (IV)), or vitamin B (e.g., vitamin B2, vitamin B7, vitamin B9, vitamin B 12 The present invention provides a method comprising the step of contacting a host cell with a solution containing (1) or (2) a solution containing (1) a solution containing (1) a solution containing (1) a solution containing (2) a solution containing (1) a solution containing (2) a solution containing (1) a solution containing (2) a solution containing (3) a solution containing (1) a solution containing (2) a solution containing (3) a solution containing (1), a solution containing (2) a solution containing (1

[0091] In certain embodiments, the present invention relates to a method for producing rAAV or increasing rAAV titer yield, wherein host cells are subjected to at least one compound described herein (e.g., a compound of formula (I), (IA), (IB), (II), (III), or (IV)), or vitamin B (e.g., vitamin B2, vitamin B7, vitamin B9, vitamin B 12The present invention provides a method comprising the step of contacting a host cell with a solution containing (1, 2, or any combination thereof), wherein the final concentration of at least one compound in the solution is sufficient to produce a secreted amount of rAAV that is 1.2 to 2.5 times greater than that produced by a host cell that has not been in contact with a solution containing at least one compound.

[0092] In certain embodiments, the present invention relates to a method for producing rAAV or increasing rAAV titer yield, wherein host cells are subjected to at least one compound described herein (e.g., a compound of formula (I), (IA), (IB), (II), (III), or (IV)), or vitamin B (e.g., vitamin B2, vitamin B7, vitamin B9, vitamin B 12 The present invention provides a method comprising the step of contacting a host cell with a solution containing (or any combination thereof), wherein the final concentration of at least one compound in the solution is sufficient to produce a total amount of rAAV that is 1.2 to 2.5 times greater than that produced by a host cell that has not been in contact with a solution containing at least one compound.

[0093] In a particular embodiment, host cells are in contact with a solution containing at least one compound for at least two days.

[0094] In a particular embodiment, the method further includes the step of collecting and purifying rAAV.

[0095] In certain embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is selected from HeLa, HEK293, COS, A549, BHK, and Vero cells.

[0096] In one embodiment, the host cell is a HeLa cell. It will be understood and readily apparent to those skilled in the art that the meaning of HeLa cell includes any clonal derivative, such as HeLa S3 cells, which are subclones of a HeLa cell line that can grow in serum-free medium and suspension culture.

[0097] In another embodiment, the host cell is a HEK293 cell. It will be understood and readily apparent to those skilled in the art that the meaning of HEK293 cells includes any clonal derivative, such as HEK293-F cells, HEK293-T cells, or HEK-EXPI293 cells.

[0098] In certain embodiments, the host cells are insect cells. In some embodiments, the host cells are selected from the group consisting of Sf9, Sf-21, Tn-368, and BTI-Tn-5B1-4 (High-Five) cells.

[0099] In some embodiments, the host cell contains a heterogeneous nucleotide sequence flanked by an AAV terminal inversion sequence. In some embodiments, the host cell contains rep and cap genes (e.g., AAV rep and cap genes). In some embodiments, the host cell contains a helper virus gene. In certain embodiments, the host cell contains i) a heterogeneous nucleotide sequence flanked by an AAV terminal inversion sequence, ii) rep and cap genes (e.g., AAV rep and cap genes), and iii) a helper virus gene.

[0100] In certain embodiments, the present invention relates to a method for producing rAAV or increasing rAAV titer yield, wherein host cells are subjected to at least one compound described herein (e.g., a compound of formula (I), (IA), (IB), (II), (III), or (IV)), or vitamin B (e.g., vitamin B2, vitamin B7, vitamin B9, and vitamin B 12 The present invention provides a method comprising the step of contacting a host cell with a solution containing (or any combination thereof), or any combination thereof, such that the host cell produces at least 1.2 times greater amounts of secreted rAAV compared to that produced by a host cell that has not been in contact with a solution containing at least one compound.

[0101] In certain embodiments, the present invention relates to a method for producing rAAV or increasing rAAV titer yield, wherein host cells are subjected to at least one compound described herein (e.g., a compound of formula (I), (IA), (IB), (II), (III), or (IV)), or vitamin B (e.g., vitamin B2, vitamin B7, vitamin B9, and vitamin B 12 The present invention provides a method comprising the step of contacting a host cell with a solution containing (or any combination thereof), or any combination thereof, such that the host cell produces at least 1.2 times greater total rAAV than that produced by a host cell that has not been in contact with a solution containing at least one compound.

[0102] In certain embodiments, the present invention relates to a method for producing rAAV or increasing rAAV titer yield, wherein host cells are subjected to at least one compound described herein (e.g., a compound of formula (I), (IA), (IB), (II), (III), or (IV)), or vitamin B (e.g., vitamin B2, vitamin B7, vitamin B9, and vitamin B 12 The present invention provides a method comprising the step of contacting a host cell with a solution containing (or any combination thereof), causing the host cell to produce 1.2 to 2.5 times greater amounts of secreted rAAV compared to that produced by a host cell that has not been in contact with a solution containing at least one compound.

[0103] In certain embodiments, the present invention relates to a method for producing rAAV or increasing rAAV titer yield, wherein host cells are subjected to at least one compound described herein (e.g., a compound of formula (I), (IA), (IB), (II), (III), or (IV)), or vitamin B (e.g., vitamin B2, vitamin B7, vitamin B9, and vitamin B 12The present invention provides a method comprising the step of contacting a host cell with a solution containing (at least one of these, or any combination thereof), wherein the host cell produces a total amount of rAAV that is 1.2 to 2.5 times greater than that produced by a host cell that has not been in contact with a solution containing at least one of these compounds.

[0104] In certain embodiments, the solution includes a cell culture medium. In some embodiments, the solution includes a production medium.

[0105] In certain embodiments, the method includes culturing the host cells in suspension culture. In certain embodiments, the method includes culturing the host cells in adherent culture. In certain embodiments, the method includes culturing the host cells in a 1 L bioreactor. In certain embodiments, the method includes culturing the host cells in a 2 L bioreactor. In certain embodiments, the method includes culturing the host cells in a 3 L bioreactor. In certain embodiments, the method includes culturing the host cells in a 250 L bioreactor. In certain embodiments, the method includes culturing the host cells in a 500 L bioreactor. In certain embodiments, the method includes culturing the host cells in a 2,000 L bioreactor.

[0106] III. Composition In one embodiment, the present invention provides a composition comprising a host cell and a compound of formula (I) or a salt thereof.

[0107] In another embodiment, the present invention provides a composition comprising a host cell and a compound of formula (IA) or a salt thereof.

[0108] In some embodiments, the present invention involves a host cell and [ka] The present invention provides a composition comprising at least one compound selected from the group consisting of any combination thereof.

[0109] In some embodiments, the present invention provides a composition comprising a host cell and at least one compound selected from the group consisting of niacinamide, niacin, methyl nicotinate, nicotinyl alcohol, and any combination thereof.

[0110] In one embodiment, the present invention provides a composition comprising a host cell and at least one compound of formula (IB) or a salt thereof.

[0111] In certain embodiments, the present invention provides a composition comprising a host cell and a compound of formula (III) or a salt thereof. [ka]

[0112] In certain embodiments, the present invention provides a composition comprising a host cell and myo-inositol or a salt thereof.

[0113] In another embodiment, the present invention provides a composition comprising a host cell and a compound of formula (II) or a salt thereof.

[0114] In certain embodiments, the present invention provides a composition comprising a host cell and a compound of formula (IV) or a salt thereof. [ka]

[0115] In certain embodiments, the present invention provides a composition comprising a host cell and a choline compound or a salt thereof.

[0116] In one embodiment, the present invention relates to a host cell and vitamin B2, vitamin B7, vitamin B9, vitamin B 12The present invention provides a composition comprising, or at least one vitamin B selected from any combination thereof. IV.Chemical definition

[0117] The definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are given in the CAS version of Handbook of Chemistry and Physics, 75 th Identified according to the periodic table of elements on the inside cover of the ed., specific functional groups are generally defined as described therein. Furthermore, general principles of organic chemistry, as well as specific functional parts and reactivity, are referred to in Thomas Sorrell, *Organic Chemistry*, University Science Books, Sausalito, 1999; Smith and March, *March's Advanced Organic Chemistry*, 5 th Edition, John Wiley & Sons, Inc., New York, 2001, Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989, and Carruthers, Some Modern Methods of Organic Synthesis, 3 rd This information is found in Edition, Cambridge University Press, Cambridge, 1987.

[0118] The articles “a” and “an” may be used herein to refer to one or more than one (i.e., at least one) grammatical object of the article. For example, “one analog” means one analog or more than one analog.

[0119] When a range of values ​​is listed, it is intended to include each value and subrange within that range. For example, "C 1~6 "Alkyl" refers to C1, C2, C3, C4, C5, C6, C1~6 , C 1~5 , C 1~4 , C 1~3 , C 1~2 , C 2~6 , C 2~5 , C 2~4 , C 2~3 , C 3~6 , C 3~5 , C 3~4 , C 4~6 , C 4~5 , and C 5~6 It is intended to include alkyl groups.

[0120] The following terms are intended to have the meanings presented together below and are useful for understanding the description and intended scope of this invention.

[0121] "Alkyl" refers to a radical of a linear or branched saturated hydrocarbon group having 1 to 20 carbon atoms ("C 1~20 ("alkyl"). In some embodiments, the alkyl group has 1 to 12 carbon atoms ("C"). 1~12 ("alkyl"). In some embodiments, the alkyl group has 1 to 10 carbon atoms ("C"). 1~10 ("alkyl"). In some embodiments, the alkyl group has 1 to 9 carbon atoms ("C"). 1~9 (alkyl). In some embodiments, the alkyl group has 1 to 8 carbon atoms ("C"). 1~8 (alkyl). In some embodiments, the alkyl group has 1 to 7 carbon atoms ("C"). 1~7 ("alkyl"). In some embodiments, the alkyl group has 1 to 6 carbon atoms ("C"). 1~6 ("alkyl"). In some embodiments, the alkyl group has 1 to 5 carbon atoms ("C"). 1~5 ("alkyl"). In some embodiments, the alkyl group has 1 to 4 carbon atoms ("C"). 1~4 ("alkyl"). In some embodiments, the alkyl group has 1 to 3 carbon atoms ("C"). 1~3 (alkyl). In some embodiments, the alkyl group has 1 to 2 carbon atoms ("C"). 1~2In some embodiments, the alkyl group has one carbon atom ("C1 alkyl"). In some embodiments, the alkyl group has two to six carbon atoms ("C1 alkyl"). 2~6 Alkyl). C 1~6 Examples of alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Further examples of alkyl groups include n-heptyl (C7) and n-octyl (C8). Unless otherwise indicated, each alkyl group can be independently substituted as needed, i.e., unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent ("substituted alkyl"). In certain embodiments, the alkyl group is unsubstituted C 1~10 It is an alkyl group (e.g., -CH3). In certain embodiments, the alkyl group is a substituted C 1~10 It is an alkyl group. Common abbreviations for alkyl groups include Me(-CH3), Et(-CH2CH3), iPr(-CH(CH3)2), nPr(-CH2CH2CH3), n-Bu(-CH2CH2CH2CH3), or i-Bu(-CH2CH(CH3)2).

[0122] The "oxo group" refers to -C(=O)-.

[0123] "Halo" or "halogen" refers to fluoro(F), chloro(Cl), bromo(Br), and iodine(I). In certain embodiments, the halogen group is either fluoro or chloro.

[0124] A "counterion" or "anionic counterion" is a negatively charged group associated with a cationic quaternary amino group to maintain electronic neutrality. Examples of counterions include halide ions (e.g., F-, Cl-, Br-, I-), NO3-, ClO4-, OH-, H2PO4-, HSO4-, sulfonate ions (e.g., methanesulfonate ion, trifluoromethanesulfonate ion, p-toluenesulfonate ion, benzenesulfonate ion, 10-camphorsulfonate ion, naphthalene-2-sulfonate ion, naphthalene-1-sulfonic acid-5-sulfonate ion, ethane-1-sulfonic acid-2-sulfonate ion, etc.), and carboxylate ions (e.g., acetate ion, ethaneate ion, propanoate ion, benzoate ion, glycerate ion, lactate ion, tartrate ion, glycolate ion, etc.).

[0125] "Pharmacologically acceptable salt" refers to a salt of the compound of the present invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. Specifically, such salts may be nontoxic and may be inorganic or organic acid addition salts and base addition salts. Specifically, such salts may be (1) formed using an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., or organic acids, such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid Examples of salts include acid addition salts formed using acids, such as 4-methylbicyclo[2.2.2]-octo-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and muconic acid; and (2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion, such as an alkali metal ion, an alkaline earth metal ion, or an aluminum ion, or when it coordinates with an organic base, such as ethanolamine, diethanolamine, triethanolamine, or N-methylglucamine. Examples of salts include sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium, as well as, if the compound contains a basic functional group, salts of non-toxic organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, and oxalate. The term "pharmaceutically acceptable cation" refers to an acceptable cationic counterion of an acidic functional group. Such cations are exemplified by sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium cations.For example, see Berge, et al., J. Pharm. Sci. (1977) 66(1): 1-79.

[0126] These and other exemplary substituents are described in more detail in the detailed description and claims. The present invention is not intended to be limited by the above-mentioned list of exemplary substituents.

[0127] V. adeno-associated virus Adeno-associated viruses (AAVs) are small, non-enveloped, dodecahedral viruses belonging to the genus Dependparvovirus and the family Parvoviridae. AAVs have a single-stranded linear DNA genome of approximately 4.7 kb. AAVs include numerous serologically distinguishable types, including over 100 serotypes derived from non-human primates, as well as serotypes AAV-1 to AAV-12 (see, e.g., Srivastava, J. Cell Biochem., 105(1): 17-24 (2008), and Gao et al., J. Virol., 78(12), 6381-6388 (2004)). Any AAV type can be used in the methods of the present invention. AAVs can infect both dividing and dormant cells of several tissue types, and different AAV serotypes exhibit different histotropies. AAVs replicate non-autonomously and have a life cycle with an incubation period and an infectious period. During the incubation period, after a cell is infected with AAV, the AAV is site-specifically integrated into the host genome as a provirus. The infection phase does not occur unless a helper virus (e.g., adenovirus (AV) or herpes simplex virus) that enables AAV replication also infects the cell.

[0128] The wild-type AAV genome contains two 145-nucleotide terminal inversion sequences (ITRs) that contain signal sequences directing AAV replication, genomic capsid inclusion, and integration. In addition to the ITRs, three AAV promoters, p5, p19, and p40, drive the expression of two open reading frames encoding rep and cap genes. The two rep promoters, combined with differential splicing of a single AAV intron, result in the production of four rep proteins (Rep78, Rep68, Rep52, and Rep40) from the rep gene. These rep proteins are responsible for genomic replication. The cap gene is expressed from the p40 promoter and encodes three capsid proteins (VP1, VP2, and VP3), which are splice variants of the Cap gene. These proteins form the capsid of the AAV particle.

[0129] Because cis-acting signals for replication, capsid inclusion, and integration are contained within the ITR, some or all of the 4.3kb internal genome can be replaced with foreign DNA, such as an expression cassette for the foreign protein of interest. In this case, the rep and cap proteins (e.g., AAV rep and cap proteins) are provided trans, for example, on a plasmid. To produce an AAV vector, a host cell line that allows AAV replication should typically express the rep and cap genes, the ITR flanking expression cassette, and helper functions provided by helper viruses, such as adenovirus (AV) genes E1a, E1b55K, E2a, E4orf6, and VA (Weitzman et al., Adeno-associated virus biology. Adeno-Associated Virus: Methods and Protocols, pp. 1-23, 2011). AAV vector production may also result in the production of helper virus particles that must be removed or inactivated before use of the AAV vector in some embodiments. Numerous cell types, including HEK293 cells, COS cells, HeLa cells, BHK cells, Vero cells, and insect cells, are suitable for producing AAV vectors (see, for example, U.S. Patents 6,156,303, 5,387,484, 5,741,683, 5,691,176, 5,688,676, 8,163,543, U.S. Publication 20020081721, PCT Publications WO00 / 47757, WO00 / 24916, and WO96 / 17947). AAV vectors are typically produced in these cell types by a single plasmid containing an ITR-facing expression cassette, as well as one or more additional plasmids providing additional AAV and helper virus genes. In certain embodiments, the AAV vector is produced in these cell types by a plasmid containing an ITR-adjacent expression cassette and the AAV viral gene, as well as one or more additional plasmids providing helper viral genes.

[0130] Any serotype of AAV can be used in this invention. Similarly, any AV type can be used, and those skilled in the art can identify the AAV and AV type suitable for the production of their desired recombinant AAV vector (rAAV). AAV and AV particles can be purified, for example, by affinity chromatography, iodixanol gradient, or CsCl gradient.

[0131] The wild-type AAV genome is single-stranded DNA and measures 4.7 kb. AAV vectors may have single-stranded genomes larger or smaller than 4.7 kb, including an oversized genome of approximately 5.2 kb or a small genome of about 3.0 kb. Furthermore, vector genomes may be substantially self-complementary, such that the genome within the virus is substantially double-stranded. AAV vectors containing all types of genomes are suitable for use in the methods of the present invention.

[0132] As discussed above, AAV requires co-infection with a helper virus to enter the infectious phase of its life cycle. Examples of helper viruses include adenoviruses (AV) and herpes simplex viruses (HSV), and systems exist for producing AAV in insect cells using baculoviruses. It has also been proposed that papillomaviruses can provide helper function for AAV (see, for example, Hermonat et al., Molecular Therapy 9, S289-S290 (2004)). Any virus capable of generating and enabling AAV replication can be considered a helper virus. AV is a non-enveloped nuclear DNA virus with a double-stranded DNA genome of approximately 36 kb. AV provides the E1a, E1b55K, E2a, E4orf6, and VA genes, which can rescue latent AAV proviruses in cells by enabling AAV replication and capsid inclusion. HSVs are a family of viruses in which a relatively large double-stranded linear DNA genome is capsid-encapsulated in an icosahedral capsid enclosed in a lipid bilayer envelope. HSVs are infectious and highly contagious. The following HSV-1 replication proteins: helicase / primase complexes (UL5, UL8, and UL52) and the DNA-binding protein ICP8 encoded by the UL29 gene were identified as typically required for HSV-1 helper function for AAV replication, while other proteins enhanced helper function.

[0133] VI. Production of rAAV The present invention comprises the production of recombinant adeno-associated virus vectors (rAAV) from host cells using any preferred method known in the art. As used herein, the term “host cell” means any one or more cells capable of producing rAAV. In some embodiments, the host cell is a mammalian cell, e.g., HeLa cells, COS cells, HEK293 cells, A549 cells, BHK cells, or Vero cells. In other embodiments, the host cell is an insect cell, e.g., Sf9 cells, Sf-21 cells, Tn-368 cells, or BTI-Tn-5B1-4 (High-Five) cells. Unless otherwise indicated, the terms “cell” or “cell line” are understood to include modified or manipulated variants of the indicated cell or cell line.

[0134] As described above, to enable rAAV production, the host cell should typically include the terminal inversion sequence (ITR) of AAV (which may be, for example, adjacent to the heterologous nucleotide sequence of interest), AAV rep and cap gene functions, and additional helper functions. These can be provided to the host cell using any number of plasmids or vectors. Additional helper functions can be provided, for example, by adenovirus (AV) infection, by a plasmid containing all the necessary AV helper function genes, or by a virus such as HSV or baculovirus. Any genes, gene functions, or other genetic material required for rAAV production by the host cell can, for example, be transiently present in the host cell or stably inserted into the host cell genome. In some embodiments, the host cell is a producing cell containing AAV rep and cap gene functions and an rAAV vector genome. In some embodiments, the host cell is a packaging cell containing AAV rep and cap gene functions, which, at the time of production, are provided by a separate recombinant virus for the rAAV vector genome. Suitable rAAV production methods for use in conjunction with the method of the present invention include those disclosed in Clark et al., Human Gene Therapy, Vol. 6: pp. 1329-1341 (1995), Martin et al., Human Gene Therapy Methods, Vol. 24: pp. 253-269 (2013), Thorne et al., Human Gene Therapy, Vol. 20: pp. 707-714 (2009), Fraser Wright, Human Gene Therapy, Vol. 20: pp. 698-706 (2009), and Virag et al., Human Gene Therapy, Vol. 20: pp. 807-817 (2009).

[0135] VII. Purification of rAAV particles In some embodiments of the present invention, rAAV particles are used by host cells to obtain compounds described herein (e.g., compounds of formula (I), (IA), (IB), (II), (III), or (IV)), or vitamin B (e.g., vitamin B2, vitamin B7, vitamin B9, and vitamin B 12rAAV particles are collected and / or purified from host cells after contact with a solution containing rAAV or a salt thereof. rAAV particles can be obtained from host cells by lysing them. Lysis of host cells can be achieved by chemically or enzymatically treating the cells to release infectious viral particles. These methods include the use of nucleases such as benzonase or DNAse, proteases such as trypsin, or detergents or surfactants. Physical disruption such as homogenization or grinding, pressurization by a microfluidizer pressure cell, or freeze-thaw cycles may also be used. In certain embodiments, lysates derived from manipulated rAAV packaging and / or producing cells can be used to collect rAAV particles. Alternatively, the supernatant may be collected from host cells without requiring cell lysis. As used herein, “total rAAV” refers to the total rAAV produced by host cells, and “secreted rAAV” refers to rAAV that can be collected from host cells without requiring cell lysis.

[0136] After recovering the rAAV particles, it may be desirable to purify the sample containing the rAAV particles, for example, to remove cell debris resulting from cell lysis. Methods for minimal purification of AAV particles are known in the art. Two exemplary purification methods are density gradient purification based on cesium chloride (CsCl) and iodixanol. Both methods are described in Strobel et al., Human Gene Therapy Methods, Vol. 26 (No. 4): pp. 147-157 (2015). Minimal purification can also be achieved, for example, using affinity chromatography with AVB Sepharose affinity resin (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). A method for AAV purification using AVB Sepharose affinity resin is described, for example, in Wang et al., Mol Ther Methods Clin Dev., Vol. 2: pp. 15040 (2015). After purification, the rAAV particles can be filtered and stored at -60°C or below. VIII. Quantification of rAAV particles

[0137] The quantification of rAAV particles is complicated by the fact that AAV infection does not result in in vitro cytopathic effects, and therefore plaque assays cannot be used to determine infectivity titers. However, rAAV particles can be quantified using several methods, including quantitative polymerase chain reaction (qPCR) (Clark et al., Hum. Gene Ther., Vol. 10, pp. 1031-1039 (1999)), dot blot hybridization (Samulski et al., J. Virol., Vol. 63, pp. 3822-3828 (1989)), and by the optical density of highly purified vector preparations (Sommer et al., Mol. Ther., Vol. 7, pp. 122-128 (2003)). DNase-resistant particles (DRPs) can be quantified by real-time quantitative gene expression reduction polymerase chain reaction (qPCR) (DRP-qPCR) in a thermocycler (e.g., iCycler iQ 96-well block format thermocycler (Bio-Rad, Hercules, CA)). Samples containing rAAV particles are incubated at 37°C for 60 minutes in the presence of DNase I (100 U / ml; Promega, Madison, WI), followed by digestion with proteinase K (Invitrogen, Carlsbad, CA) (10 U / mL) at 50°C for 60 minutes, and then denatured at 95°C for 30 minutes. The primer-probe set used should be specific to the non-native portion of the rAAV vector genome, e.g., the poly(A) sequence of the protein of interest. PCR products can be amplified using any suitable set of cycling parameters based on the length and composition of the primers, probes, and amplified sequence. An alternative protocol is disclosed, for example, in Lock et al., Human Gene Therapy Methods, Vol. 25 (No. 2): pp. 115-125 (2014).

[0138] Throughout this specification, where apparatus, devices, and systems are described as having, including, or comprising certain components, or where processes and methods are described as having, including, or comprising certain steps, it is intended that, in addition, there exist apparatus, devices, and systems of the present invention that are essentially composed of or comprise the listed components, and processes and methods of the present invention that are essentially composed of or comprise the listed processes.

[0139] The implementation of the present invention is presented herein for illustrative purposes only and should not be construed as limiting the invention in any way, and will be better understood from the examples described herein. [Examples]

[0140] (Example 1: Effects of different concentrations of niacinamide on rAAV production in HeLa cells) In this example, the effects of various concentrations of niacinamide, ranging from 0.1 mM to 40 mM, on rAAV production were tested in various HeLa-producing cell lines. The experiment involved using 3 mL of working volume cell culture medium and 0.2 × 10⁶ niacinamide. 6 The analysis was performed in a 24-deep-well plate culture system at a seeding density of 100 cells and an Ad5 infection multiplicity (MOI) of 200 vp / cell. Figure 1 shows a schematic graph of normalized volume rAAV titers for various clade E and F rAAVs produced from HeLa-producing cells at varying niacinamide concentrations (0.1 mM–40 mM). Increases in rAAV titers were observed between 0.1 mM and 10 mM, with the most significant increases observed between 1 mM and 10 mM. The capsid serotypes included in this analysis were AAV8, AAV9, rh10, and hu37.

[0141] Figure 2 shows a graph of the normalized volume-based rAAV titer (GC / mL) for varying niacinamide concentrations, as well as the predicted maximum value when using a cubic polynomial fit of the data points. A significant increase in rAAV titer was observed between 1 mM and 10 mM, with a predicted maximum value at 5 mM.

[0142] (Example 2: Effect of container scaling on rAAV production) In this example, the effect of scales using two common containers containing niacinamide (a 3 mL deep well ("24-DW") and a 100 mL shaking flask ("Flask")) on rAAV production was tested. The analysis was performed using a HeLa-producing cell line capable of producing clade E rAAV (AAVrh10-FIX), which encodes a factor IX transgene. Figure 3 shows a bar graph illustrating rAAV titer (GC / mL) at varying niacinamide concentrations at different scales. A two-way ANOVA considering factors of niacinamide, scale, and their interactions showed a statistically significant effect only from niacinamide (p<0.0001). Factors of scale and interaction were not significant (p=0.3915 and p=0.1353, respectively). Dunnett's test was performed, which showed significance at all concentrations when scale was removed as a factor.

[0143] (Example 3: Effect of niacinamide on rAAV production in clade E HeLa clones) Four different HeLa clones were tested using the conditions described in Example 1. Figures 4A–D show the rAAV titer (GC / mL) per varying niacinamide concentration for clone 1 (AAVrh10-FIX) follow-up supernatant, clone 2 (AAV8-G6Pase) supernatant, clone 3 (AAV8-G6Pase) broad range without supplement, and clone 4 (AAVhu37-FVIII) broad range suspension (i.e., Triton-dissolved samples), respectively. An increase in titer was observed in all four clones upon addition of niacinamide between 1 mM and 5 mM.

[0144] (Example 4: Effect of niacinamide on rAAV production in clade F HeLa clones) In this example, the effect of 3 mM niacinamide on two HeLa-producing cell line clones expressing recombinant clade F AAV (AAV9-ATP7B), which encodes a truncated ATP7B transgene, was tested in a 2 L bioreactor. As shown in Figure 5, adding 3 mM niacinamide to the bioreactor resulted in a significant increase in titer in both clones.

[0145] (Example 5: Effects of different concentrations of niacinamide on rAAV production in HEK293 cells) In this example, the effects of various niacinamide concentrations ranging from 0.03 mM to 30 mM on rAAV production were tested in HEK293 cells. The experiment involved 1.3 × 10⁶ cells. 6 ~2.0×10 6 Transfection was performed in a 30 mL flask using a transfection density of 10 cells / mL. HEK293 cells used in this experiment were transfected with a plasmid that enables the production of rAAV8 (rAAV8-OTC), which encodes ornithine transcarbamylase. As shown in Figure 6, the addition of 0.3 mM and 3 mM niacinamide resulted in increased titer in HEK293 cells. Repeated trials on a 2 L bioreactor scale also showed increased titer in HEK293 cells with the addition of niacinamide (data not shown).

[0146] (Example 6: Effect of additional compounds on rAAV production in HEK293 cells) In this example, the effects of compounds other than niacinamide on rAAV production in HEK293 cells were tested. The compounds analyzed in this example included niacin, methyl nicotinate, myo-inositol, pantothenic acid, pyridoxine, choline, thiamine, glucosamine, caffeine, n-acetylglucosamine, and thymidine.

[0147] The experiment was performed in 24-deep-well plates using the same cells as described in Example 5, under similar transfection and culture conditions. The concentrations of each compound tested were 1 mM, 3 mM, and 9 mM. Among the compounds tested, niacin resulted in a 20% increase in titer at 1 mM, methyl nicotinate in a 42% increase at 3 mM, myo-inositol in an approximately 20% increase at 3 mM and 9 mM, while choline in a 65% increase at 3 mM. None of the other compounds tested provided a significant benefit to rAAV titer at any of the three concentrations tested.

[0148] This example demonstrates that while alternative compounds, including niacin, methyl nicotinate, myo-inositol, and choline, can boost rAAV titer, none offered the same level of benefit provided by niacinamide, which appeared to be substantially and remarkably superior to the other 11 compounds tested.

[0149] Numbered Embodiments The embodiments disclosed herein include embodiments P1 to P68 provided in the numbered embodiments of this disclosure.

[0150] Embodiment P1: A method for producing recombinant adeno-associated virus (rAAV), wherein a host cell is subjected to formula (I) [ka] The step includes contacting with a solution containing the compound or a salt thereof, in which, [ka] However, it represents a single bond or a double bond. n is an integer selected from 1 to 5. R 1 However, -OH, C 1~6 Alkyl, C 1~6alkyl-OH, C 1~6 Alkyl-OC 1~6 Alkyl, -OC 1~6 Alkyl, -C(O)N(R a )2, and -C(O)OR b Selected from the group consisting of, R a However, independently, for each appearance, hydrogen or C 1~6 It is alkyl, R b However, independently, for each appearance, hydrogen or C 1~6 It is alkyl, X, CR c R d or N, R c and R d However, independently, for each appearance, hydrogen, C 1~6 Alkyl, -OH, and -OC 1~6 Selected from the group consisting of alkyl groups, R c However, if it is hydrogen, R d C 1~6 Alkyl, -OH, and -OC 1~6 Selected from alkyl, R c However, C 1~6 Alkyl, -OH, and -OC 1~6 If selected from alkyl, R d It is hydrogen, The aforementioned compound, [ka] But it's not that salt either. method.

[0151] Embodiment P2: A method for increasing rAAV titer yield, wherein host cells are subjected to formula (I) [ka] The step includes contacting with a solution containing the compound or a salt thereof, in which, [ka] However, it represents a single bond or a double bond. n is an integer selected from 1 to 5. R 1 However, -OH, C 1~6 Alkyl, C 1~6 alkyl-OH, C 1~6 Alkyl-OC 1~6 Alkyl, -OC 1~6 Alkyl, -C(O)N(R a )2, and -C(O)OR b Selected from the group consisting of, R a However, independently, for each appearance, hydrogen or C 1~6 It is alkyl, R b However, independently, for each appearance, hydrogen or C 1~6 It is alkyl, X, CR c R d or N, R c and R d However, independently, for each appearance, hydrogen, C 1~6 Alkyl, -OH, and -OC 1~6 Selected from the group consisting of alkyl groups, R c However, if it is hydrogen, R d C 1~6 Alkyl, -OH, and -OC 1~6 Selected from alkyl, R c However, C 1~6 Alkyl, -OH, and -OC 1~6 If selected from alkyl, R d It is hydrogen, The aforementioned compound, [ka] But it's not that salt either. method.

[0152] Embodiment P3: The method according to Embodiment P1 or P2, wherein X is N.

[0153] Embodiment P4: The method according to any one of Embodiments P1 to P3, wherein n is 1.

[0154] Embodiment P5:R 1 However, C 1~6 alkyl-OH, -C(O)N(R a )2, and -C(O)OR b A method according to any one of embodiments P1 to P4, selected from the group consisting of the following.

[0155] Embodiment P6:R 1 The method according to any one of Embodiments P1 to P5, wherein the method is selected from the group consisting of CH2OH, -C(O)NH2, -C(O)OH, and -C(O)OCH3.

[0156] Embodiment P7:X is CR c R d The method according to embodiment P1 or P2.

[0157] Embodiment P8:R c However, it is hydrogen, and R d The method according to any one of embodiments P1, P2, and P7, wherein the compound is -OH.

[0158] Embodiment P9: The method according to any one of Embodiments P1, P2 and P7 to P8, wherein n is 5.

[0159] Embodiment P10:R 1 The method according to any one of embodiments P1, P2 and P7 to P9, wherein the compound is -OH.

[0160] Embodiment P11: The compound is of formula (IA) [ka] It is a compound or salt thereof, in which, R 2 and R 3 However, independently, for each occurrence, is it hydrogen or -OH? or R 2 and R3 However, together they can form an oxo, R 4 However, hydrogen, -OH, -OC 1~6 Alkyl, and -N(R e Selected from the group consisting of )2, R e However, independently, for each appearance, hydrogen or C 1~6 It is alkyl. The method according to embodiment P1 or P2.

[0161] Embodiment P12:R 2 and R 3 The method according to embodiment P11, wherein the two components combine to form an oxo.

[0162] Embodiment P13:R 4 The method according to embodiment P11 or P12, wherein the group is selected from -NH2, -OH, and -OCH3.

[0163] Embodiment P14:R 2 and R 3 However, in all cases, the method according to Embodiment P11 is hydrogen.

[0164] Embodiment P15:R 4 The method according to embodiment P11 or P14, wherein the compound is -OH.

[0165] Embodiment P16: The compound is, [ka] Selected from the group consisting of, The method according to any one of embodiments P1, P2, and P11.

[0166] Embodiment P17: The compound is of formula (IB) [ka] It is a compound or salt thereof, in which, X, CR c Rd And, R 5a , R 5b , R 5c , R 5d , and R 5e However, independently, for each occurrence, -OH, -OC 1~6 Alkyl, C 1~6 Alkyl, C 1~6 alkyl-OH, and C 1~6 Alkyl-OC 1~6 It is alkyl, R c and R d However, as defined in Embodiment P1, The method according to embodiment P1 or P2.

[0167] Embodiment P18:R c However, it is hydrogen, and R d However, the method according to embodiment P17, wherein it is -OH.

[0168] Embodiment P19:R 5a , R 5b , R 5c , R 5d , and R 5e The method according to embodiment P17 or P18, wherein the compound is -OH.

[0169] Embodiment P20: The compound is, [ka] The method according to any one of embodiments P17 to P19.

[0170] Embodiment P21: A method for producing rAAV, wherein a host cell is subjected to formula (II) [ka] The step includes contacting with a solution containing the compound or a salt thereof, in which, R 6 and R 7 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C1~6 Selected from the group consisting of alkyl groups, R 8 and R 9 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 Selected from the group consisting of alkyl groups, Y is either C or N, R 10 and R 11 However, independently, for each appearance, hydrogen, -OH, and C 1~6 Selected from the group consisting of alkyl groups, A is hydrogen, C 1~6 Alkyl and -C(O)N(R f Selected from the group consisting of )2, R f However, independently, for each appearance, hydrogen, C 1~6 Alkyl, C 1~6 alkyl-C(O)OH, and C 1~6 Selected from the group consisting of alkyl-OH, The aforementioned compound, [ka] No, method.

[0171] Embodiment P22: A method for increasing rAAV titer yield, wherein host cells are subjected to formula (II) [ka] The step includes contacting with a solution containing the compound or a salt thereof, in which, R 6 and R 7 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 Selected from the group consisting of alkyl groups, R 8 and R 9 However, independently, for each appearance, hydrogen, -OH, -CN, halogen, and C 1~6 Selected from the group consisting of alkyl groups, Y is either C or N, R 10 and R 11 However, independently, for each appearance, hydrogen, -OH, and C 1~6 Selected from the group consisting of alkyl groups, A is hydrogen, C 1~6 Alkyl and -C(O)N(R f Selected from the group consisting of )2, R f However, independently, for each appearance, hydrogen, C 1~6 Alkyl, C 1~6 alkyl-C(O)OH, and C 1~6 Selected from the group consisting of alkyl-OH, The aforementioned compound, [ka] No, method.

[0172] Embodiment P23:R 6 and R 7 The method according to embodiment P21 or P22, wherein the hydrogen is hydrogen.

[0173] Embodiment P24:R 8 and R 9 The method according to any one of embodiments P21 to P23, wherein the hydrogen is hydrogen.

[0174] Embodiment P25: The method according to any one of Embodiments P21 to P24, wherein Y is N.

[0175] Embodiment P26:R 10 and R 11 The method according to any one of embodiments P21 to P25, wherein CH3 is present.

[0176] Embodiment P27: The method according to any one of Embodiments P21 to P26, wherein A is CH3.

[0177] Embodiment P28: The compound is [ka] The method according to any one of embodiments P21 to P27.

[0178] Embodiment P29: A method for producing rAAV or increasing rAAV titer yield, wherein host cells are infused with vitamin B2, vitamin B7, vitamin B9, and vitamin B 12 A method comprising the step of contacting a solution containing vitamin B selected from the group consisting of the following.

[0179] Embodiment P30: The method according to any one of Embodiments P1 to P29, wherein the final concentration of the compound in the solution is higher than 0.5 mM.

[0180] Embodiment P31: The method according to any one of Embodiments P1 to P29, wherein the final concentration of the compound in the solution is higher than or equal to 0.5 mM.

[0181] Embodiment P32: The method according to any one of Embodiments P1 to P29, wherein the final concentration of the compound in the solution is between 0.5 mM and 10 mM.

[0182] Embodiment P33: The method according to any one of Embodiments P1 to P29, wherein the final concentration of the compound in the solution is between 1 mM and 10 mM.

[0183] Embodiment P34: The method according to any one of Embodiments P1 to P33, wherein the final concentration of the compound in the solution is sufficient to produce at least 1.2 times greater amounts of secreted rAAV compared to that produced by host cells not in contact with the solution containing the compound.

[0184] Embodiment P35: The method according to any one of Embodiments P1 to P33, wherein the final concentration of the compound in the solution is sufficient to produce a total amount of rAAV that is at least 1.2 times greater than that produced by host cells that have not come into contact with the solution containing the compound.

[0185] Embodiment P36: The method according to any one of Embodiments P1 to P33, wherein the final concentration of the compound in the solution is sufficient to produce a secreted amount of rAAV that is 1.2 to 2.5 times greater than that produced by host cells that have not come into contact with the solution containing the compound.

[0186] Embodiment P37: The method according to any one of Embodiments P1 to P33, wherein the final concentration of the compound in the solution is sufficient to produce a total amount of rAAV that is 1.2 to 2.5 times greater than that produced by host cells that have not come into contact with the solution containing the compound.

[0187] Embodiment P38: The method according to any one of Embodiments P1 to P37, wherein the host cells are in contact with the solution containing the compound for at least two days.

[0188] Embodiment P39: The method according to any one of Embodiments P1 to P38, further comprising the step of collecting and purifying the rAAV.

[0189] Embodiment P40: The method according to any one of Embodiments P1 to P39, wherein the host cell is a mammalian cell.

[0190] Embodiment P41: The method according to Embodiment P40, wherein the host cells are selected from the group consisting of HeLa, HEK293, COS, A549, BHK, and Vero cells.

[0191] Embodiment P42: The method according to Embodiment P41, wherein the host cell is a HeLa cell.

[0192] Embodiment P43: The method according to Embodiment P41, wherein the host cell is a HEK293 cell.

[0193] Embodiment P44: The method according to any one of Embodiments P1 to P39, wherein the host cell is an insect cell.

[0194] Embodiment P45: The method according to Embodiment P44, wherein the host cells are selected from the group consisting of Sf9, Sf-21, Tn-368, and BTI-Tn-5B1-4 (High-Five) cells.

[0195] Embodiment P46: The method according to any one of Embodiments P1 to P45, wherein the host cell contains a heterogeneous nucleotide sequence adjacent to an AAV terminal inversion sequence.

[0196] Embodiment P47: The method according to any one of Embodiments P1 to P46, wherein the host cell comprises the rep and cap genes.

[0197] Embodiment P48: The method according to any one of Embodiments P1 to P47, wherein the host cell contains a helper virus gene.

[0198] Embodiment P49: The method according to any one of Embodiments P1 to P48, wherein the host cell comprises heterologous nucleotide sequences with adjacent AAV terminal inversion sequences, rep and cap genes, and a helper virus gene.

[0199] Embodiment P50: The method according to any one of Embodiments P1 to P49, wherein the host cell produces at least 1.2 times greater amounts of secreted rAAV than that produced by a host cell that has not come into contact with the solution containing the compound.

[0200] Embodiment P51: The method according to any one of Embodiments P1 to P49, wherein the host cells produce at least 1.2 times greater amounts of total rAAV than those produced by host cells that have not been in contact with the solution containing the compound.

[0201] Embodiment P52: The method according to any one of Embodiments P1 to P49, wherein the host cell produces 1.2 to 2.5 times larger amounts of secreted rAAV compared to that produced by a host cell that has not come into contact with the solution containing the compound.

[0202] Embodiment P53: The method according to any one of Embodiments P1 to P49, wherein the host cells produce a total amount of rAAV that is 1.2 to 2.5 times greater than that produced by host cells that have not been in contact with the solution containing the compound.

[0203] Embodiment P54: The method according to any one of Embodiments P1 to P53, wherein the solution comprises a cell culture medium.

[0204] Embodiment P55: The method according to any one of Embodiments P1 to P53, wherein the solution includes a production culture medium.

[0205] Embodiment P56: The method according to any one of Embodiments P1 to P55, further comprising the step of culturing the host cells in a suspension culture.

[0206] Embodiment P57: The method according to any one of Embodiments P1 to P55, further comprising the step of culturing the host cells in adherent culture.

[0207] Embodiment P58: The method according to any one of Embodiments P1 to P56, comprising the step of culturing the host cells in a 1 L bioreactor.

[0208] Embodiment P59: The method according to any one of Embodiments P1 to P56, comprising the step of culturing the host cells in a 2 L bioreactor.

[0209] Embodiment P60: The method according to any one of Embodiments P1 to 56, comprising the step of culturing the host cells in a 3 L bioreactor.

[0210] Embodiment P61: The method according to any one of Embodiments P1 to P56, comprising the step of culturing the host cells in a 250 L bioreactor.

[0211] Embodiment P62: The method according to any one of Embodiments P1 to P56, comprising the step of culturing the host cells in a 500 L bioreactor.

[0212] Embodiment P63: The method according to any one of Embodiments P1 to P56, further comprising the step of culturing the host cells in a 2,000 L bioreactor.

[0213] Embodiment P64: A composition comprising a host cell and a compound of formula (I) or a salt thereof.

[0214] Embodiment P65: A composition comprising a host cell and a compound of formula (IA) or a salt thereof.

[0215] Embodiment P66: A composition comprising a host cell and a compound of formula (IB) or a salt thereof.

[0216] Embodiment P67: A composition comprising a host cell and a compound of formula (II) or a salt thereof.

[0217] Embodiment P68: Host cells and vitamin B2, vitamin B7, vitamin B9, and vitamin B 12 A composition comprising vitamin B selected from the group consisting of the following. Built-in by reference

[0218] The entirety of each patent and scientific document disclosure referenced herein is incorporated by reference for all purposes. Equal parts

[0219] The present invention may be embodied in other specific forms without departing from its spirit or essential features. Therefore, the embodiments described herein should be considered illustrative in all respects rather than limiting the invention as described herein. The scope of the invention is therefore indicated more by the appended claims than by the foregoing description, and all variations occurring within the equivalent meaning and scope of the claims are intended to be included therein.

Claims

1. A method for producing recombinant adeno-associated virus (rAAV), wherein a host cell is subjected to (i) 【Chemistry 1】 and at least one compound or salt thereof selected from the group consisting of any combination thereof, (ii) formula 【Chemistry 2】 The step includes contacting with a production solution containing at least one compound or salt thereof, The final concentration of at least one compound or its salt in the production solution is between 1 mM and 10 mM. method.

2. A method for increasing rAAV titer yield, wherein host cells 【Transformation 3】 The step includes contacting the product with a production solution containing at least one compound or salt thereof selected from the group consisting of any combination thereof, The final concentration of at least one compound or its salt in the production solution is between 1 mM and 10 mM. method.

3. The at least one of the aforementioned compounds or a salt thereof 【Chemistry 4】 and If necessary, the final concentration of at least one compound or its salt in the production solution is between 3 mM and 5 mM. The method according to claim 1 or 2.

4. The method according to any one of claims 1 to 3, wherein the final concentration of the at least one compound or a salt thereof in the production solution is sufficient to produce at least 1.2 times greater amounts of secreted rAAV or total rAAV compared to that produced by host cells that have not come into contact with the production solution containing the at least one compound or a salt thereof.

5. The method according to any one of claims 1 to 4, wherein the host cells are in contact with the production solution containing at least one compound or a salt thereof for at least two days.

6. The method according to any one of claims 1 to 5, further comprising the step of collecting and purifying rAAV.

7. The method according to any one of claims 1 to 6, wherein the host cell is a mammalian cell or an insect cell.

8. i) The host cell is a mammalian cell selected from the group consisting of HeLa, HEK293, COS, A549, BHK, and Vero cells, or ii) The host cell is an insect cell selected from the group consisting of Sf9, Sf-21, Tn-368, and BTI-Tn-5B1-4 (High-Five) cells. The method according to claim 7.

9. The aforementioned host cells i) Heterogeneous nucleotide sequences adjacent to AAV terminal inversion sequences, ii) AAV rep and cap genes, iii) Helper virus gene, or iv) These combinations The method according to any one of claims 1 to 8, including the method described in any one of claims 1 to 8.

10. i) The production solution contains a cell culture medium or a production medium, ii) The method includes the step of culturing the host cells in suspension culture or adherent culture, or iii) The method comprises the step of culturing the host cells in a bioreactor of 1 L to 2,000 L, The method according to any one of claims 1 to 9.

11. host cells and, (i) 【Transformation 5】 and at least one compound or salt thereof selected from the group consisting of any combination thereof, (ii) formula 【Transformation 6】 It comprises at least one compound or salt thereof, The aforementioned host cells a) Heterogeneous nucleotide sequences adjacent to AAV terminal inversion sequences, b) AAV rep and cap genes, c) Helper gene, or d) These combinations A composition further comprising:

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