WO2025180499A1 - Method for improving virus packaging yield or quality in cells - Google Patents

Abstract

Provided are a compound of formula (I), and a pharmaceutically acceptable salt and stereochemically isomer form thereof, a compound of formula (II) and a pharmaceutically acceptable salt and stereochemically isomer form thereof, or a use of a composition comprising any one of the compounds in improving the virus packaging yield and/or quality in cells.

Description

A method for improving the yield or quality of virus packaging in cells

Related applications

This disclosure claims priority to international application PCT/CN2024/079140, filed on February 28, 2024, the entire contents of which are expressly incorporated herein by reference.

Technical Field

The present disclosure relates to the field of medicine and biology, and in particular to use of a specific compound or a composition comprising the compound in improving the yield and/or quality of virus packaging in cells.

Background Art

With the rapid growth of the global gene therapy market, adeno-associated virus (AAV) has become one of the most important gene vectors in the field of gene therapy due to its advantages such as long-term expression, low toxicity, low immunogenicity, and high tissue specificity. However, the high R&D and production costs limit the application of recombinant adeno-associated virus (rAAV) in the field of cell and gene therapy. Currently, rAAV is mainly produced by transient transfection of suspended HEK293 cells with three plasmids. The current methods for optimizing the yield and quality of recombinant adeno-associated virus (rAAV) mainly focus on traditional cell culture, adjustment of transfection process, optimization of cell lines, cell feeding, etc. However, with the study of the molecular mechanisms of AAV replication, infection and other processes, it has been found that many factors in the host cell play a key role in the replication and packaging process of AAV. The method of improving rAAV packaging by regulating key signal transduction pathways in host cells has a certain theoretical basis and feasibility.

Traditional cell culture feeds and enhancers mainly regulate cell status and metabolic processes by changing the concentration of nutrients and key components in the cell culture medium. Although they can increase virus production, their disadvantages are also very obvious. First, the composition of traditional feeds is complex, many components have redundant functions, and they are expensive and need to be added multiple times during the cell culture process. Second, traditional feeds have limited effects on increasing virus production, cannot improve virus quality, and may even reduce virus quality.

The present disclosure aims to develop the application of small molecule compounds as a new type of scalable enhancer that significantly improves the yield and quality of rAAV production, which is of great significance for improving the yield and/or quality of AAV and/or rAAV and reducing the cost of rAAV production.

Summary of the Invention

The small molecule compounds disclosed in the present invention are more accurate and specific, more efficient, more effective, have a single component, and are easy to prepare. Compared with existing feed supplements or Enhancers, the present invention has a more obvious improvement effect on the production of AAV or rAAV. It can not only significantly increase the yield of viruses such as AAV (or rAAV), but also improve the efficiency of virus packaging, reduce the empty shell rate of the product, and improve the quality of the virus product. At the same time, the small molecule compound is used in small amounts, is safe to use and easy to operate, and there is no need to modify the existing production process (such as cell culture and transfection steps). Finally, the small molecule compound has a broad-spectrum effect on improving the efficiency of virus packaging and is suitable for a variety of serotypes and different cell lines.

According to an embodiment of the present disclosure, there is provided a compound defined by formula (I), a pharmaceutically acceptable salt thereof, or a stereochemical isomeric form thereof, or a composition comprising any of the above for use in improving the yield and/or quality of viral packaging in cells:

wherein R ₁ is each independently selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, and cyclopropyl;

R ₂ is selected from the group consisting of H, F, Cl, Br, I, NH ₂ , NO ₂ , OH, CN, methyl, ethyl, propyl, isopropyl, cyclopropyl, difluoromethyl, trifluoromethyl, substituted or unsubstituted phenyl, pyrrolyl, and pyridyl;

L ₁ is selected from the group consisting of methylene, ethylene, propylene, butylene, cyclopentylene, cyclohexylene, and phenylene.

L ₂ is selected from the group consisting of absence, carbonyl, -C(O)O-, -C(O)NR ₁ -, and -NR ₁ -;

R ₃ is selected from the group consisting of H, NH ₂ , methyl, ethyl, propyl, isopropyl, acetamido, propionamido, butyramido, isobutyramido, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, substituted or unsubstituted phenyl, pyridyl, pyrrolyl, furanyl, thienyl, thiazolyl, imidazolyl, pyrazolyl, pyrimidinyl, pyridazinyl, pyranyl, piperazinyl, and piperidinyl;

L ₃ is selected from the group consisting of methylene, ethylene, propylene, -NR ₁ -, -NR ₁ C(O)-, and -NR ₁ C(O)O-;

_R4 is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, tetrahydropyrrolyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, and morpholinyl.

In another embodiment, each R ₁ is independently H or methyl.

In another embodiment, R ₂ is selected from the group consisting of Cl, Br, I, CN, methyl, difluoromethyl, trifluoromethyl, cyclopropyl, and phenyl.

In another embodiment, L ₁ is selected from the group consisting of methylene, ethylene, propylene, cyclopentylene, and cyclohexylene.

In another embodiment, L ₂ is selected from the group consisting of absent, carbonyl, and -C(O)NR ₁ -.

In another embodiment, R ₃ is selected from the group consisting of acetamido, isopropylamido, tert-butylamido, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, pyridyl, pyrrolyl, thienyl, thiazolyl, imidazolyl, pyrazolyl, and pyrimidinyl.

In another embodiment, L ₃ is -NR ₁ C(O)- or methylene.

In another embodiment, R ₄ is selected from the group consisting of cyclopentyl, cyclohexyl, tetrahydropyrrolyl, piperidinyl, and morpholinyl.

In one embodiment, the compound of formula (I) has the structure of formula (Ia):

wherein R ₁ , R ₂ , R ₃ , R ₄ , L ₁ and L ₂ are as defined above.

In one embodiment, the compound of formula (I) has the structure of formula (Ib):

wherein R ₂ , R ₃ , R ₄ , L ₁ and L ₂ are as defined above.

In one embodiment, the compound of formula (I) has the structure of formula (Ic):

wherein R ₁ , R ₂ , R ₃ , R ₄ and L ₃ are as defined above.

In one embodiment, the compound of formula (I) has the structure of formula (Id):

wherein R ₂ , R ₃ , R ₄ and L ₃ are as defined above.

In one embodiment, the compound of formula (I) has the structure of formula (Ie):

wherein R ₁ , R ₂ , R ₃ and R ₄ are as defined above.

In one embodiment, the compound of formula (I) has the structure of formula (If):

wherein R ₂ , R ₃ and R ₄ are as defined above.

In one embodiment, the compound of formula (I) has a structure selected from:

wherein R ₂ is selected from Br, I or cyclopropyl;

L ₁ is selected from ethylene or propylene;

_L2 is -C(O)NH- or absent;

_R3 is selected from the group consisting of tert-butylamido, cyclobutyl, thienyl, and imidazolyl;

L ₃ is -NR ₁ C(O)- or methylene;

R ₄ is tetrahydropyrrolyl or morpholinyl.

Another aspect of the present disclosure provides a compound having the following structure, a pharmaceutically acceptable salt thereof, or a stereochemically isomeric form thereof

According to another aspect of the present disclosure, there is provided a compound defined by formula (II), a pharmaceutically acceptable salt thereof, or a stereochemically isomeric form thereof:

wherein R ₁ is each independently selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, and cyclopropyl;

R ₂ is selected from the group consisting of H, F, Cl, Br, NH ₂ , NO ₂ , OH, CN, methyl, ethyl, propyl, isopropyl, cyclopropyl, difluoromethyl, trifluoromethyl, substituted or unsubstituted phenyl, pyrrolyl, and pyridyl;

R ₃ is -C(O)R ₆ or -C(O)OR ₆ ;

R ₆ is selected from the group consisting of NH ₂ , methyl, ethyl, propyl, isopropyl, cyclopropyl, and phenyl;

R ₄ and R ₅ are each independently selected from the group consisting of F, Cl, Br, NH ₂ , NO ₂ , OH, CN, methyl, ethyl, difluoromethyl, and trifluoromethyl;

m and n are each independently 0 or 1.

In one embodiment, each R ₁ is independently H or methyl.

In one embodiment, _R2 is selected from the group consisting of CN, methyl, difluoromethyl, trifluoromethyl, and phenyl.

In one embodiment, R ₃ is -C(O)R ₆ .

In one embodiment, _R6 is selected from the group consisting of _NH2 , methyl, ethyl, propyl, isopropyl, cyclopropyl, and phenyl.

In one embodiment, m and n are 0.

Another aspect of the present disclosure provides a compound having the following structure, a pharmaceutically acceptable salt thereof, or a stereochemical isomeric form thereof for use in improving the yield and/or quality of virus packaging in cells,

In one embodiment, the virus is an adeno-associated virus or a recombinant adeno-associated virus (rAAV).

In one embodiment, the cells include but are not limited to HEK293 or Hela cells cultured adherently or in suspension, or cell lines obtained by gene editing, transformation, domestication, and/or screening of the above cells, such as HEK293T, HEK293F, or HEK293FT cells.

In one embodiment, the final concentration of the one or more compounds is greater than 0.05 μM, such as 0.05-60 μM, 0.15-40 μM, 0.2-30 μM, 0.3-35 μM, 0.4-25 μM, 0.5-10 μM, 0.6-8 μM, 1-5 μM, 0.1-5 μM, 0.2-2 μM or 0.2-2.5 μM, such as 0.1-5 μM, or such as 0.2-2.5 μM. The final concentration of the one or more compounds is preferably 0.2-30 μM or 1-30 μM.

In some embodiments, the concentration of one or more of the aforementioned compounds is 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 2.5 μM, 5 μM, 7.5 μM, 10 μM, 15 μM, 20 μM, 30 μM, or 50 μM.

In some specific embodiments, the compound is BX795; in some preferred embodiments, the concentration of BX795 is 0.05-60 μM, preferably 0.2-10 μM, 0.2-2.5 μM or 0.2-2 μM, more preferably 1-2 μM.

In some specific embodiments, the compound is BX912; in some preferred embodiments, the concentration of BX912 is 0.05-60 μM, preferably 1-10 μM, and more preferably 2-5 μM.

In some specific embodiments, the compound is BX517; in some preferred embodiments, the concentration of BX517 is 0.05-60 μM, preferably 1-10 μM, and more preferably 2-5 μM.

In some specific embodiments, the compound is BX320; in some preferred embodiments, the concentration of BX320 is 0.05-60 μM, preferably 0.1-40 μM, more preferably 10-30 μM.

In some specific embodiments, the compound is MRT67307; in some preferred embodiments, the concentration of MRT67307 is 0.05-60 μM, preferably 1-10 μM, and more preferably 1-5 μM.

In one embodiment, one or more of the aforementioned compounds are added to the cells 24, 18, 12, 6, 4, 2, or 1 hour before transfection, or 1, 2, 4, 6, 12, 18, 24, 36, or 48 hours after transfection.

In one embodiment, the one or more aforementioned compounds are added to the cells within 24 hours before transfection to 48 hours after transfection, within 18 hours before transfection to 36 hours after transfection, within 12 hours before transfection to 24 hours after transfection, within 6 hours before transfection to 18 hours after transfection, within 4 hours before transfection to 12 hours after transfection, within 2 hours before transfection to 6 hours after transfection, within 1 hour before transfection to 4 hours after transfection, within 1 hour before transfection to 2 hours after transfection, within 1 hour before transfection to 1 hour after transfection, within 2 hours before transfection to 4 hours after transfection, or within 4 hours before transfection to 4 hours after transfection, for example, one or more aforementioned compounds are added to the cells within 4 hours before transfection to 4 hours after transfection.

In one embodiment, the virus is prepared by transfecting relevant plasmids into cells for packaging. The relevant plasmid system can be a one-plasmid, two-plasmid, three-plasmid or multi-plasmid adeno-associated virus packaging system composed of one or more plasmids containing AAV Rep protein coding region sequences, AAV capsid protein VP1, VP2, VP3 coding region sequences, target gene coding sequences, auxiliary gene coding sequences and ITR sequences.

In one embodiment, as a non-limiting example, improving the yield and/or quality of virus packaging in cells includes one or more of the following: increasing virus yield, reducing the empty envelope rate of virus packaging, or any combination thereof.

As a non-limiting example, the serotype of rAAV herein is selected from the group consisting of wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.39, AAVHSC15, AAVHSC17, and novel AAV serotypes engineered based on AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.39, AAVHSC15, and AAVHSC17.

Another aspect of the present disclosure provides a method for improving the yield and/or quality of virus packaging in cells, comprising culturing cells for producing viruses in the presence of a compound of formula (I), a pharmaceutically acceptable salt thereof, or a stereochemically isomeric form thereof.

wherein R ₁ is each independently selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, and cyclopropyl;

R ₂ is selected from the group consisting of H, F, Cl, Br, I, NH ₂ , NO ₂ , OH, CN, methyl, ethyl, propyl, isopropyl, cyclopropyl, difluoromethyl, trifluoromethyl, substituted or unsubstituted phenyl, pyrrolyl, and pyridyl;

L ₁ is selected from the group consisting of methylene, ethylene, propylene, butylene, cyclopentylene, cyclohexylene, and phenylene.

L ₂ is selected from the group consisting of absence, carbonyl, -C(O)O-, -C(O)NR ₁ -, and -NR ₁ -;

R ₃ is selected from the group consisting of H, NH ₂ , methyl, ethyl, propyl, isopropyl, acetamido, propionamido, butyramido, isobutyramido, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, substituted or unsubstituted phenyl, pyridyl, pyrrolyl, furanyl, thienyl, thiazolyl, imidazolyl, pyrazolyl, pyrimidinyl, pyridazinyl, pyranyl, piperazinyl, and piperidinyl;

L ₃ is selected from the group consisting of methylene, ethylene, propylene, -NR ₁ -, -NR ₁ C(O)-, and -NR ₁ C(O)O-;

_R4 is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, tetrahydropyrrolyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, and morpholinyl.

In another embodiment, each R ₁ is independently H or methyl.

In another embodiment, R ₂ is selected from the group consisting of Cl, Br, I, CN, methyl, difluoromethyl, trifluoromethyl, cyclopropyl, and phenyl.

In another embodiment, L ₁ is selected from the group consisting of methylene, ethylene, propylene, cyclopentylene, and cyclohexylene.

In another embodiment, L ₂ is selected from the group consisting of absent, and -C(O)NR ₁ -.

In another embodiment, R ₃ is selected from the group consisting of acetamido, isopropylamido, tert-butylamido, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, pyridyl, pyrrolyl, thienyl, thiazolyl, imidazolyl, pyrazolyl, and pyrimidinyl.

In another embodiment, L ₃ is -NR ₁ C(O)- or methylene.

In another embodiment, R ₄ is selected from the group consisting of cyclopentyl, cyclohexyl, tetrahydropyrrolyl, piperidinyl, and morpholinyl.

In one embodiment, the compound of formula (I) has the structure of formula (Ia):

wherein R ₁ , R ₂ , R ₃ , R ₄ , L ₁ and L ₂ are as defined above.

In one embodiment, the compound of formula (I) has the structure of formula (Ib):

wherein R ₂ , R ₃ , R ₄ , L ₁ and L ₂ are as defined above.

In one embodiment, the compound of formula (I) has the structure of formula (Ic):

wherein R ₁ , R ₂ , R ₃ , R ₄ and L ₃ are as defined above.

In one embodiment, the compound of formula (I) has the structure of formula (Id):

wherein R ₂ , R ₃ , R ₄ and L ₃ are as defined above.

In one embodiment, the compound of formula (I) has the structure of the following formula (Ie):

wherein R ₁ , R ₂ , R ₃ and R ₄ are as defined above.

In one embodiment, the compound of formula (I) has the structure of formula (If):

wherein R ₂ , R ₃ and R ₄ are as defined above.

In one embodiment, the compound of formula (I) has a structure selected from:

wherein R ₂ is selected from Br, I or cyclopropyl;

L ₁ is selected from ethylene or propylene;

_L2 is -C(O)NH- or absent;

_R3 is selected from the group consisting of tert-butylamido, cyclobutyl, thienyl, and imidazolyl;

L ₃ is -NR ₁ C(O)- or methylene;

R ₄ is tetrahydropyrrolyl or morpholinyl.

Another aspect of the present disclosure provides a method for improving the yield and/or quality of virus packaging in cells, comprising culturing cells for virus production in the presence of a compound of the following structure, a pharmaceutically acceptable salt thereof, or a stereochemical isomeric form thereof,

Another aspect of the present disclosure provides a method for improving the yield and/or quality of virus packaging in cells, comprising the step of culturing cells for virus production in the presence of a compound of formula (II), a pharmaceutically acceptable salt thereof, or a stereochemical isomeric form thereof.

wherein R ₁ is each independently selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, and cyclopropyl;

R ₂ is selected from the group consisting of H, F, Cl, Br, NH ₂ , NO ₂ , OH, CN, methyl, ethyl, propyl, isopropyl, cyclopropyl, difluoromethyl, trifluoromethyl, substituted or unsubstituted phenyl, pyrrolyl, and pyridyl;

R ₃ is -C(O)R ₆ or -C(O)OR ₆ ;

R ₆ is selected from the group consisting of NH ₂ , methyl, ethyl, propyl, isopropyl, cyclopropyl, and phenyl;

R ₄ and R ₅ are each independently selected from the group consisting of F, Cl, Br, NH ₂ , NO ₂ , OH, CN, methyl, ethyl, difluoromethyl, and trifluoromethyl;

m and n are each independently 0 or 1.

In one embodiment, each R ₁ is independently H or methyl.

In one embodiment, _R2 is selected from the group consisting of CN, methyl, difluoromethyl, trifluoromethyl, and phenyl.

In one embodiment, R ₃ is -C(O)R ₆ .

In one embodiment, _R6 is selected from the group consisting of _NH2 , methyl, ethyl, propyl, isopropyl, cyclopropyl, and phenyl.

In one embodiment, m and n are 0.

Another aspect of the present disclosure provides a method for improving the yield and/or quality of virus packaging in cells, comprising culturing cells for virus production in the presence of a compound of the following structure, a pharmaceutically acceptable salt thereof, or a stereochemical isomeric form thereof,

In an embodiment of the present disclosure, the virus selects adeno-associated virus or recombinant adeno-associated virus (rAAV). In one embodiment, the final concentration of the aforementioned one or more of the above compounds is greater than 0.05 μM, such as 0.05-60 μM, 0.15-40 μM, 0.2-30 μM, 0.3-35 μM, 0.4-25 μM, 0.5-10 μM, 0.6-8 μM, 1-5 μM, 0.1-5 μM, 0.2-2 μM or 0.2-2.5 μM, such as 0.1-5 μM, or for example 0.2-2.5 μM. The final concentration of the aforementioned one or more of the above compounds is preferably 0.2-30 μM or 1-30 μM. In some embodiments, the concentration of one or more of the aforementioned compounds is 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 2.5 μM, 5 μM, 7.5 μM, 10 μM, 15 μM, 20 μM, 30 μM, or 50 μM.

In some embodiments, the compound is BX795; in some preferred embodiments, the concentration of BX795 is 0.05-60 μM, preferably 0.2-10 μM, 0.2-2.5 μM, or 0.2-2 μM, more preferably 1-2 μM. In some embodiments, the compound is BX912; in some preferred embodiments, the concentration of BX912 is 0.05-60 μM, preferably 1-10 μM, more preferably 2-5 μM. In some embodiments, the compound is BX517; in some preferred embodiments, the concentration of BX517 is 0.05-60 μM, preferably 1-10 μM, more preferably 2-5 μM. In some embodiments, the compound is BX320; in some preferred embodiments, the concentration of BX320 is 0.05-60 μM, preferably 0.1-40 μM, more preferably 10-30 μM. In some specific embodiments, the compound is MRT67307; in some preferred embodiments, the concentration of MRT67307 is 0.05-60 μM, preferably 1-10 μM, and more preferably 1-5 μM.

In one embodiment, one or more of the aforementioned compounds are added to the cells 24, 18, 12, 6, 4, 2, or 1 hour before transfection, or 1, 2, 4, 6, 12, 18, 24, 36, or 48 hours after transfection.

In one embodiment, the one or more aforementioned compounds are added to the cells within 24 hours before transfection to 48 hours after transfection, within 18 hours before transfection to 36 hours after transfection, within 12 hours before transfection to 24 hours after transfection, within 6 hours before transfection to 18 hours after transfection, within 4 hours before transfection to 12 hours after transfection, within 2 hours before transfection to 6 hours after transfection, within 1 hour before transfection to 4 hours after transfection, within 1 hour before transfection to 2 hours after transfection, within 1 hour before transfection to 1 hour after transfection, within 2 hours before transfection to 4 hours after transfection, or within 4 hours before transfection to 4 hours after transfection, for example, one or more aforementioned compounds are added to the cells within 4 hours before transfection to 4 hours after transfection.

In one embodiment, the aforementioned cells can be, for example, including but not limited to adherent or suspension cultured HEK293, Hela cells, or cell lines obtained by gene editing, transformation, domestication, and/or screening of the above cells, such as HEK293T, HEK293F, or HEK293FT.

In one embodiment, the virus is prepared by transfecting relevant plasmids into cells for packaging. The relevant plasmid system can be a one-plasmid, two-plasmid, three-plasmid or multi-plasmid adeno-associated virus packaging system composed of one or more plasmids containing AAV Rep protein coding region sequences, AAV capsid protein VP1, VP2, VP3 coding region sequences, target gene coding sequences, auxiliary gene coding sequences and ITR sequences.

In one embodiment, as a non-limiting example, improving the yield and/or quality of virus packaging in cells includes one or more of the following: increasing virus yield, reducing the empty envelope rate of virus packaging, or any combination thereof.

As a non-limiting example, the serotype of AAV herein is selected from the group consisting of wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.39, AAVHSC15, AAVHSC17, and novel AAV serotypes engineered based on AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.39, AAVHSC15, and AAVHSC17.

As a non-limiting example, the serotype of rAAV herein is selected from the group consisting of wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.39, AAVHSC15, AAVHSC17, and novel AAV serotypes engineered based on AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.39, AAVHSC15, and AAVHSC17.

A "derivative" or "variant" of a virus refers to a virus obtained by selecting a virus under different growth conditions, a virus that has been subjected to various selective pressures, a virus that has been genetically modified using recombinant techniques known in the art, or a virus that has been engineered to be replication-defective and/or to express an introduced gene, or any combination thereof. Examples of such viruses are known in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the effects of different concentrations of the compounds of the present application on the yield and quality of rAAV9 in HEK293 suspension cells.

FIG2 shows the effect of the addition time of the compounds of the present application on the rAAV9 yield in HEK293 suspension cells.

FIG3 shows the effects of the compounds of the present application on the yield and quality of rAAV9 in different cell lines.

FIG4 shows the effects of the compounds of the present application on the yields of rAAV of different serotypes in HEK293 suspension cells.

FIG5 shows the effects of the compounds of the present application on the yield and quality of rAAV9 in HEK293 suspension cells.

FIG6 shows the effects of the compounds of the present application on rAAV9 production in HEK293T adherent cells.

FIG7 shows the effects of the compounds of the present application on rAAV2 production in Hela cells.

DETAILED DESCRIPTION

Before further describing the present disclosure, the following sections collect certain terms used in the specification, examples, and appended claims. The definitions listed herein should be read and understood by those skilled in the art in light of the remainder of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs.

The present disclosure is not limited to the specific systems, devices, and methods described, as they may vary. The terminology used in this description is for the purpose of describing a particular version or implementation only and is not intended to limit the scope. These aspects of the present disclosure may be embodied in many different forms; rather, these implementations are provided so that this disclosure will be thorough and complete and will fully convey its scope to those skilled in the art.

The present disclosure is not limited to the specific embodiments described in this disclosure which are intended to serve as illustrations of various aspects. As will be apparent to those skilled in the art, many modifications and variations can be made without departing from its spirit and scope. Based on the foregoing description, in addition to those listed herein, functionally equivalent methods and devices within the scope of this disclosure will be apparent to those skilled in the art. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is limited only by the terms of the appended claims and the full scope of equivalents to which these claims are entitled. It should be understood that the present disclosure is not limited to specific methods, reagents, compounds, compositions or biological systems, which can of course vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not restrictive.

definition

With respect to the use of substantially any plural and/or singular terms herein, those skilled in the art can transform from plural to singular and/or from singular to plural depending on the context and/or application. For clarity, various different singular/plural permutations may be explicitly set forth herein.

Unless otherwise indicated, when any type of range is disclosed or claimed, it is intended to disclose or claim individually every possible value that the range may reasonably encompass, including any subranges encompassed therein. For example, a radical number of 1 to 6 indicates an integer within the range, where 1-6 is understood to include 1, 2, 3, 4, 5, 6, and also includes subranges of 1-5, 1-4, and 1-3.

The words “include”, “contain” or “comprises” and the like used in this disclosure mean that the elements preceding the word include the elements listed after the word and their equivalents, but do not exclude unrecited elements.

The term "about" as used herein refers to a change in the numerical quantity that may occur, for example, by actual measurement or processing procedures, by negligent errors in these procedures, by the preparation of compositions or reagents, sources or purity differences, etc. Typically, the term "about" as used herein means a stated value or range of values that is greater than or less than 1/10 of the stated value, for example, ±10%. The term "about" also refers to a change that is considered to be equivalent by those skilled in the art, as long as such change does not encompass known values practiced in the prior art. Each value or range of values preceded by the term "about" is also intended to encompass the embodiment of the absolute value or range of values being described. Regardless of whether or not modified by the term "about", the quantitative values described in this disclosure include equivalents of the values described, such as variations in the numerical amount of such values that may occur, but are considered to be equivalent by those skilled in the art. Where the context of this disclosure indicates otherwise or is inconsistent with such an explanation, the above explanation may be modified, which is obvious to those skilled in the art. For example, in a list of values such as "about 49, about 50, about 55," "about 50" means a range that extends to less than half of the interval between the preceding and following values, e.g., greater than 49.5 to less than 52.5. Additionally, the phrases "less than about" a value or "greater than about" a value should be understood according to the definition of the term "about" provided herein.

As used herein, the term "composition" refers to a combination or mixture of two or more different ingredients, components or substances.

The term "pharmaceutically acceptable" as used herein means that the compound or composition is chemically and/or toxicologically compatible with the other ingredients constituting the formulation and/or with humans or mammals for the prevention or treatment of a disease or condition.

In addition, where features or aspects of the present disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. The description of the present disclosure should be interpreted in accordance with the laws and principles of chemical bonding. In some cases, a hydrogen atom may be removed in order to accommodate a substituent at a given position.

The present disclosure includes all possible salts of the disclosed compounds, either as a single salt or as any mixture of such salts in any ratio.

The compounds of the present disclosure may contain one or more asymmetric centers, depending on the position and properties of the various substituents desired. Asymmetric carbon atoms can exist in the (R) or (S) configuration, resulting in racemic mixtures in the case of one asymmetric center and diastereomeric mixtures in the case of multiple asymmetric centers. In some cases, asymmetry may also exist due to hindered rotation about a particular bond, such as where the central bond connects two substituted aromatic rings of a particular compound.

The compounds of the present disclosure may also be those that produce more desirable biological activities. Isolation, purification or partial purification of isomers and stereoisomers, or racemic mixtures or diastereomeric mixtures of the compounds of the present disclosure are included within the scope of the present disclosure. Purification and separation of such substances can be achieved by standard techniques known in the art.

By retaining the right to exclude or exclude any individual member of any such group (including any subrange or subrange combination of such groups), this can be claimed based on scope or any similar means, or you can choose to claim less than the complete measure of the disclosure for any reason. In addition, by retaining the right to exclude or exclude any individual substituent, structure or group thereof, or any member of the required group, you can claim less than the complete measure of the disclosure for any reason. Various patents, patent applications and publications are cited throughout this disclosure. The disclosures of these patents, patent applications and publications are incorporated into the disclosure as a whole by reference to more fully describe the state of the art known to those skilled in the art as of the date of this disclosure. In the event of any inconsistency between the cited patents, patent applications and publications and the disclosure, the disclosure will prevail.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Nothing in this disclosure should be construed as an obligation that the embodiments described herein do not have an antedate status by virtue of prior invention.

Another aspect of the present disclosure provides use of any one of the aforementioned compounds, a pharmaceutically acceptable salt thereof, or a stereochemical isomer thereof in improving the yield and/or quality of cellular viruses.

Another aspect of the present disclosure provides the use of a compound of formula (I), a pharmaceutically acceptable salt thereof, a stereochemical isomeric form thereof, or a composition comprising any of the above in improving the yield and/or quality of virus packaging in cells,

wherein R ₁ is each independently selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, and cyclopropyl;

R ₂ is selected from the group consisting of H, F, Cl, Br, I, NH ₂ , NO ₂ , OH, CN, methyl, ethyl, propyl, isopropyl, cyclopropyl, difluoromethyl, trifluoromethyl, substituted or unsubstituted phenyl, pyrrolyl, and pyridyl;

_L1 is selected from the group consisting of methylene, ethylene, propylene, butylene, cyclopentylene, cyclohexylene, and phenylene;

L ₂ is selected from the group consisting of absence, carbonyl, -C(O)O-, -C(O)NR ₁ -, and -NR ₁ -;

R ₃ is selected from the group consisting of H, NH ₂ , methyl, ethyl, propyl, isopropyl, acetamido, propionamido, butyramido, isobutyramido, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, substituted or unsubstituted phenyl, pyridyl, pyrrolyl, furanyl, thienyl, thiazolyl, imidazolyl, pyrazolyl, pyrimidinyl, pyridazinyl, pyranyl, piperazinyl, and piperidinyl;

L ₃ is selected from the group consisting of methylene, ethylene, propylene, -NR ₁ -, -NR ₁ C(O)-, and -NR ₁ C(O)O-;

_R4 is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, tetrahydropyrrolyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, and morpholinyl.

According to an embodiment of the present disclosure, each R ₁ is independently H or methyl.

According to an embodiment of the present disclosure, R ₂ is selected from the group consisting of Cl, Br, I, CN, methyl, difluoromethyl, trifluoromethyl, cyclopropyl, and phenyl.

According to an embodiment of the present disclosure, L ₁ is selected from the group consisting of methylene, ethylene, propylene, cyclopentylene, and cyclohexylene.

According to an embodiment of the present disclosure, L ₂ is selected from the group consisting of absent, and -C(O)NR ₁ -.

According to an embodiment of the present disclosure, R ₃ is selected from the group consisting of acetamido, isopropylamido, tert-butylamido, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, pyridyl, pyrrolyl, thienyl, thiazolyl, imidazolyl, pyrazolyl, and pyrimidinyl.

According to an embodiment of the present disclosure, L ₃ is -NR ₁ C(O)- or methylene.

According to an embodiment of the present disclosure, R ₄ is selected from the group consisting of cyclopentyl, cyclohexyl, tetrahydropyrrolyl, piperidinyl, and morpholinyl.

According to an embodiment of the present disclosure, the compound of formula (I) has the structure of the following formula (Ia):

wherein R ₁ , R ₂ , R ₃ , R ₄ , L ₁ and L ₂ are as defined above.

According to an embodiment of the present disclosure, the compound of formula (I) has the structure of the following formula (Ib):

wherein R ₂ , R ₃ , R ₄ , L ₁ and L ₂ are as defined above.

According to an embodiment of the present disclosure, the compound of formula (I) has the structure of the following formula (Ic):

wherein R ₁ , R ₂ , R ₃ , R ₄ and L ₃ are as defined above.

According to an embodiment of the present disclosure, the compound of formula (I) has the structure of the following formula (Id):

wherein R ₂ , R ₃ , R ₄ and L ₃ are as defined above.

According to an embodiment of the present disclosure, the compound of formula (I) has the structure of the following formula (Ie):

wherein R ₁ , R ₂ , R ₃ and R ₄ are as defined above.

According to an embodiment of the present disclosure, the compound of formula (I) has the structure of the following formula (If):

wherein R ₂ , R ₃ and R ₄ are as defined above.

According to an embodiment of the present disclosure, the compound of formula (I) has a structure selected from the group consisting of:

wherein R ₂ is selected from Br, I or cyclopropyl;

L ₁ is selected from ethylene or propylene;

_L2 is -C(O)NH- or absent;

_R3 is selected from the group consisting of tert-butylamido, cyclobutyl, thienyl, and imidazolyl;

L ₃ is -NR ₁ C(O)- or methylene;

R ₄ is tetrahydropyrrolyl or morpholinyl.

Another aspect of the present disclosure provides the use of a compound having the following structure, a pharmaceutically acceptable salt thereof, a stereochemical isomeric form thereof, or a composition comprising any of the above in improving the yield and/or quality of virus packaging in cells,

Another aspect of the present disclosure provides the use of a compound of formula (II), a pharmaceutically acceptable salt thereof, a stereochemical isomeric form thereof, or a composition comprising any of the above in improving the yield and/or quality of virus packaging in cells,

wherein R ₁ is each independently selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, and cyclopropyl;

R ₂ is selected from the group consisting of H, F, Cl, Br, NH ₂ , NO ₂ , OH, CN, methyl, ethyl, propyl, isopropyl, cyclopropyl, difluoromethyl, trifluoromethyl, substituted or unsubstituted phenyl, pyrrolyl, and pyridyl;

R ₃ is -C(O)R ₆ or -C(O)OR ₆ ;

R ₆ is selected from the group consisting of NH ₂ , methyl, ethyl, propyl, isopropyl, cyclopropyl, and phenyl;

R ₄ and R ₅ are each independently selected from the group consisting of F, Cl, Br, NH ₂ , NO ₂ , OH, CN, methyl, ethyl, difluoromethyl, and trifluoromethyl;

m and n are each independently 0 or 1.

According to an embodiment of the present disclosure, each R ₁ is independently H or methyl.

According to an embodiment of the present disclosure, R ₂ is selected from the group consisting of CN, methyl, difluoromethyl, trifluoromethyl, and phenyl.

According to an embodiment of the present disclosure, R ₃ is -C(O)R ₆ .

According to an embodiment of the present disclosure, R ₆ is selected from the group consisting of NH ₂ , methyl, ethyl, propyl, isopropyl, cyclopropyl, and phenyl.

According to an embodiment of the present disclosure, m and n are 0.

Another aspect of the present disclosure provides a compound having the following structure, a pharmaceutically acceptable salt thereof, or a stereochemical isomeric form thereof, or a composition comprising any of the above for use in improving the yield and/or quality of viral packaging in cells,

According to an embodiment of the present disclosure, the aforementioned virus is an adeno-associated virus or a recombinant adeno-associated virus (rAAV).

According to an embodiment of the present disclosure, the aforementioned AAV serotype is selected from the group consisting of wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.39, AAVHSC15, AAVHSC17, and novel AAV serotypes modified based on AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.39, AAVHSC15, and AAVHSC17.

According to an embodiment of the present disclosure, the final concentration of the one or more aforementioned compounds is greater than 0.05 μM, such as 0.05-60 μM, 0.15-40 μM, 0.2-30 μM, 0.3-35 μM, 0.4-25 μM, 0.5-10 μM, 0.6-8 μM, 1-5 μM, 0.1-5 μM, 0.2-2 μM or 0.2-2.5 μM, such as 0.1-5 μM, or such as 0.2-2.5 μM. The final concentration of the one or more aforementioned compounds is preferably 0.2-30 μM or 1-30 μM.

In some embodiments, the concentration of one or more of the aforementioned compounds is 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 2.5 μM, 5 μM, 7.5 μM, 10 μM, 15 μM, 20 μM, 30 μM, or 50 μM.

In some embodiments, the compound is BX795; in some preferred embodiments, the concentration of BX795 is 0.05-60 μM, preferably 0.2-10 μM, 0.2-2.5 μM, or 0.2-2 μM, more preferably 1-2 μM. In some embodiments, the compound is BX912; in some preferred embodiments, the concentration of BX912 is 0.05-60 μM, preferably 1-10 μM, more preferably 2-5 μM. In some embodiments, the compound is BX517; in some preferred embodiments, the concentration of BX517 is 0.05-60 μM, preferably 1-10 μM, more preferably 2-5 μM. In some embodiments, the compound is BX320; in some preferred embodiments, the concentration of BX320 is 0.05-60 μM, preferably 0.1-40 μM, more preferably 10-30 μM. In some specific embodiments, the compound is MRT67307; in some preferred embodiments, the concentration of MRT67307 is 0.05-60 μM, preferably 1-10 μM, and more preferably 1-5 μM.

In one embodiment, one or more of the aforementioned compounds are added to the cells 24, 18, 12, 6, 4, 2, or 1 hour before transfection, or 1, 2, 4, 6, 12, 18, 24, 36, or 48 hours after transfection.

In one embodiment, the one or more aforementioned compounds are added to the cells within 24 hours before transfection to 48 hours after transfection, within 18 hours before transfection to 36 hours after transfection, within 12 hours before transfection to 24 hours after transfection, within 6 hours before transfection to 18 hours after transfection, within 4 hours before transfection to 12 hours after transfection, within 2 hours before transfection to 6 hours after transfection, within 1 hour before transfection to 4 hours after transfection, within 1 hour before transfection to 2 hours after transfection, within 1 hour before transfection to 1 hour after transfection, within 2 hours before transfection to 4 hours after transfection, or within 4 hours before transfection to 4 hours after transfection, for example, one or more aforementioned compounds are added to the cells within 4 hours before transfection to 4 hours after transfection.

According to the embodiments of the present disclosure, the aforementioned cells include but are not limited to HEK293 or Hela cells cultured adherently or in suspension, or cell lines obtained by gene editing, transformation, domestication, and/or screening of the above cells, such as HEK293T, HEK293F, or HEK293FT.

According to an embodiment of the present disclosure, as a non-limiting example, improving the yield and/or quality of adeno-associated viruses in cells includes one or more of the following: increasing virus yield, reducing virus emptying rate, or any combination thereof.

Another aspect of the present disclosure provides a method for producing a virus, comprising the step of culturing cells for virus production in the presence of a compound of formula (I), a pharmaceutically acceptable salt thereof, or a stereochemically isomeric form thereof.

wherein R ₁ is each independently selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, and cyclopropyl;

R ₂ is selected from the group consisting of H, F, Cl, Br, I, NH ₂ , NO ₂ , OH, CN, methyl, ethyl, propyl, isopropyl, cyclopropyl, difluoromethyl, trifluoromethyl, substituted or unsubstituted phenyl, pyrrolyl, and pyridyl;

_L1 is selected from the group consisting of methylene, ethylene, propylene, butylene, cyclopentylene, cyclohexylene, and phenylene;

L ₂ is selected from the group consisting of absence, carbonyl, -C(O)O-, -C(O)NR ₁ -, and -NR ₁ -;

R ₃ is selected from the group consisting of H, NH ₂ , methyl, ethyl, propyl, isopropyl, acetamido, propionamido, butyramido, isobutyramido, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, substituted or unsubstituted phenyl, pyridyl, pyrrolyl, furanyl, thienyl, thiazolyl, imidazolyl, pyrazolyl, pyrimidinyl, pyridazinyl, pyranyl, piperazinyl, and piperidinyl;

L ₃ is selected from the group consisting of methylene, ethylene, propylene, -NR ₁ -, -NR ₁ C(O)-, and -NR ₁ C(O)O-;

_R4 is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, tetrahydropyrrolyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, and morpholinyl.

According to an embodiment of the present disclosure, each R ₁ is independently H or methyl.

According to an embodiment of the present disclosure, R ₂ is selected from the group consisting of Cl, Br, I, CN, methyl, difluoromethyl, trifluoromethyl, cyclopropyl, and phenyl.

According to an embodiment of the present disclosure, L ₁ is selected from the group consisting of methylene, ethylene, propylene, cyclopentylene, and cyclohexylene.

According to an embodiment of the present disclosure, L ₂ is selected from the group consisting of absent, and -C(O)NR 1 -.

According to an embodiment of the present disclosure, R ₃ is selected from the group consisting of acetamido, isopropylamido, tert-butylamido, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, pyridyl, pyrrolyl, thienyl, thiazolyl, imidazolyl, pyrazolyl, and pyrimidinyl.

According to an embodiment of the present disclosure, L ₃ is -NR ₁ C(O)- or methylene.

According to an embodiment of the present disclosure, R ₄ is selected from the group consisting of cyclopentyl, cyclohexyl, tetrahydropyrrolyl, piperidinyl, and morpholinyl.

In one embodiment, the compound of formula (I) has the structure of formula (Ia):

wherein R ₁ , R ₂ , R ₃ , R ₄ , L ₁ and L ₂ are as defined above.

In one embodiment, the compound of formula (I) has the structure of formula (Ib):

wherein R ₂ , R ₃ , R ₄ , L ₁ and L ₂ are as defined above.

In one embodiment, the compound of formula (I) has the structure of formula (Ic):

wherein R ₁ , R ₂ , R ₃ , R ₄ and L ₃ are as defined above.

In one embodiment, the compound of formula (I) has the structure of formula (Id):

wherein R ₂ , R ₃ , R ₄ and L ₃ are as defined above.

In one embodiment, the compound of formula (I) has the structure of formula (Ie):

wherein R ₁ , R ₂ , R ₃ and R ₄ are as defined above.

In one embodiment, the compound of formula (I) has the structure of formula (If):

wherein R ₂ , R ₃ and R ₄ are as defined above.

In one embodiment, the compound of formula (I) has a structure selected from:

wherein R ₂ is selected from Br, I or cyclopropyl;

L ₁ is selected from ethylene or propylene;

_L2 is -C(O)NH- or absent;

_R3 is selected from the group consisting of tert-butylamido, cyclobutyl, thienyl, and imidazolyl;

L ₃ is -NR ₁ C(O)- or methylene;

R ₄ is tetrahydropyrrolyl or morpholinyl.

Another aspect of the present disclosure provides a method for producing a virus, comprising culturing cells for producing the virus in the presence of a compound of the following structure, a pharmaceutically acceptable salt thereof, or a stereochemical isomeric form thereof,

Another aspect of the present disclosure provides a method for producing a virus, comprising culturing cells for producing the virus in the presence of a compound of formula (II), a pharmaceutically acceptable salt thereof, or a stereochemically isomeric form thereof.

wherein R ₁ is each independently selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, and cyclopropyl;

R ₂ is selected from the group consisting of H, F, Cl, Br, NH ₂ , NO ₂ , OH, CN, methyl, ethyl, propyl, isopropyl, cyclopropyl, difluoromethyl, trifluoromethyl, substituted or unsubstituted phenyl, pyrrolyl, and pyridyl;

R ₃ is -C(O)R ₆ or -C(O)OR ₆ ;

R ₆ is selected from the group consisting of NH ₂ , methyl, ethyl, propyl, isopropyl, cyclopropyl, and phenyl;

R ₄ and R ₅ are each independently selected from the group consisting of F, Cl, Br, NH ₂ , NO ₂ , OH, CN, methyl, ethyl, difluoromethyl, and trifluoromethyl;

m and n are each independently 0 or 1.

According to an embodiment of the present disclosure, each R ₁ is independently H or methyl.

According to an embodiment of the present disclosure, R ₂ is selected from the group consisting of CN, methyl, difluoromethyl, trifluoromethyl, and phenyl.

According to an embodiment of the present disclosure, R ₃ is -C(O)R ₆ .

According to an embodiment of the present disclosure, R ₆ is selected from the group consisting of NH ₂ , methyl, ethyl, propyl, isopropyl, cyclopropyl, and phenyl.

According to an embodiment of the present disclosure, m and n are 0.

Another aspect of the present disclosure provides a method for producing a virus, comprising culturing a cell for producing the virus in the presence of a compound of the following structure, a pharmaceutically acceptable salt thereof, or a stereochemical isomeric form thereof,

According to an embodiment of the present disclosure, the aforementioned is an adeno-associated virus or a recombinant adeno-associated virus (rAAV).

In one embodiment, the final concentration of the one or more aforementioned compounds is greater than 0.05 μM, for example, 0.05-60 μM, 0.15-40 μM, 0.2-30 μM, 0.3-35 μM, 0.4-25 μM, 0.5-10 μM, 0.6-8 μM, 1-5 μM, 0.1-5 μM, 0.2-2 μM or 0.2-2.5 μM, for example, 0.1-5 μM, or for example, 0.2-2.5 μM. The final concentration of the one or more aforementioned compounds is preferably 0.2-30 μM or 1-30 μM. In some embodiments, the concentration of the one or more aforementioned compounds is 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 2.5 μM, 5 μM, 7.5 μM, 10 μM, 15 μM, 20 μM, 30 μM, or 50 μM.

In some specific embodiments, the compound is BX795; in some preferred embodiments, the concentration of BX795 is 0.05-60 μM, preferably 0.2-10 μM, 0.2-2.5 μM or 0.2-2 μM, more preferably 1-2 μM.

In some embodiments, the compound is BX912; in some preferred embodiments, the concentration of BX912 is 0.05-60 μM, preferably 1-10 μM, and more preferably 2-5 μM. In some embodiments, the compound is BX517; in some preferred embodiments, the concentration of BX517 is 0.05-60 μM, preferably 1-10 μM, and more preferably 2-5 μM. In some embodiments, the compound is BX320; in some preferred embodiments, the concentration of BX320 is 0.05-60 μM, preferably 0.1-40 μM, and more preferably 10-30 μM. In some embodiments, the compound is MRT67307; in some preferred embodiments, the concentration of MRT67307 is 0.05-60 μM, preferably 1-10 μM, and more preferably 1-5 μM.

In one embodiment, one or more of the aforementioned compounds are added to the cells 24, 18, 12, 6, 4, 2, or 1 hour before transfection, or 1, 2, 4, 6, 12, 18, 24, 36, or 48 hours after transfection.

In one embodiment, the one or more aforementioned compounds are added to the cells within 24 hours before transfection to 48 hours after transfection, within 18 hours before transfection to 36 hours after transfection, within 12 hours before transfection to 24 hours after transfection, within 6 hours before transfection to 18 hours after transfection, within 4 hours before transfection to 12 hours after transfection, within 2 hours before transfection to 6 hours after transfection, within 1 hour before transfection to 4 hours after transfection, within 1 hour before transfection to 2 hours after transfection, within 1 hour before transfection to 1 hour after transfection, within 2 hours before transfection to 4 hours after transfection, or within 4 hours before transfection to 4 hours after transfection, for example, one or more aforementioned compounds are added to the cells within 4 hours before transfection to 4 hours after transfection.

According to the embodiments of the present disclosure, the aforementioned cells include but are not limited to HEK293 or Hela cells cultured in adherent or suspension culture, or cell lines obtained by gene editing, transformation, domestication, and/or screening of the above cells, such as HEK293T, HEK293F, or HEK293FT.

According to the embodiments of the present disclosure, the virus is prepared by transfecting relevant plasmids into cells for packaging. The relevant plasmid system can be a one-plasmid, two-plasmid, three-plasmid or multi-plasmid adeno-associated virus packaging system composed of one or more plasmids containing AAV Rep protein coding region sequences, AAV capsid protein VP1, VP2, VP3 coding region sequences, target gene coding sequences, auxiliary gene coding sequences and ITR sequences.

According to an embodiment of the present disclosure, as a non-limiting example, improving the yield and/or quality of adeno-associated virus in cells includes one or more of the following: increasing virus yield, reducing virus packaging empty shell rate, or any combination thereof.

According to an embodiment of the present disclosure, as a non-limiting example, the serotype of AAV herein is selected from the group consisting of wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.39, AAVHSC15, AAVHSC17, and novel AAV serotypes modified based on AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.39, AAVHSC15, and AAVHSC17.

According to an embodiment of the present disclosure, as a non-limiting example, the serotype of rAAV herein is selected from the group consisting of wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.39, AAVHSC15, AAVHSC17, and novel AAV serotypes modified based on AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.39, AAVHSC15, and AAVHSC17.

A "derivative" or "variant" of a virus refers to a virus obtained by selecting a virus under different growth conditions, a virus that has been subjected to various selective pressures, a virus that has been genetically modified using recombinant techniques known in the art, or a virus that has been engineered to be replication-defective and/or to express an introduced gene, or any combination thereof. Examples of such viruses are known in the art.

The genes required for packaging AAV virus generally include: (a) a nucleic acid template comprising at least one AAV ITR sequence, (b) an AAV sequence encoding genes required for viral replication and packaging (e.g., an AAV rep sequence and an AAV cap sequence encoding an AAV capsid), and (c) a coding sequence comprising auxiliary genes. Optionally, the nucleic acid template may further comprise at least one heterologous nucleic acid sequence. In some embodiments, the nucleic acid template comprises two AAV ITR sequences located at the 5' and 3' ends of the heterologous nucleic acid sequence, respectively.

According to the present disclosure, the virus is obtained by, but not limited to, introducing genes required for packaging the relevant AAV virus into production cells through plasmid transfection or viral infection, or a combination of the two methods.

According to the present disclosure, the virus is prepared by transfecting relevant plasmids into cells for packaging. The relevant plasmid system can be a one-plasmid, two-plasmid, three-plasmid or multi-plasmid adeno-associated virus packaging system composed of one or more plasmids containing AAV Rep protein coding region sequences, AAV capsid protein VP1, VP2, VP3 coding region sequences, target gene coding sequences, auxiliary gene coding sequences and ITR sequences.

In order to produce viruses using the plasmid system for producing AAV viruses, the virus can be produced by transfecting cells with the plasmid system or introducing the plasmid system into cells by other means. The compounds of the present disclosure can be added to the cell culture medium at a desired concentration before or after the cells containing the AAV plasmid system are prepared (i.e., transfection or introduction is completed).

In the present disclosure, cells used in virus packaging production include but are not limited to adherent or suspension cultured HEK293, Hela cells, or cell lines obtained by gene editing, transformation, domestication, and/or screening of the above cells, such as HEK293T, HEK293F, or HEK293FT.

When producing AAV virus, different serotypes of AAV can be used (for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.39, AAVHSC15, AAVHSC17, and new AAV serotypes modified based on AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.39, AAVHSC15, or AAVHSC17).

In some embodiments, complete or partial domains, functional regions, epitopes, etc. from one AAV serotype or another parvovirus can be substituted in any combination for corresponding wild-type domains, functional regions, epitopes, etc. of a different AAV serotype to generate chimeric capsid proteins.

Furthermore, the AAV capsid or genomic elements may contain other modifications, including insertions, deletions, and/or substitutions. In some embodiments, the amino acid sequence of the capsid protein may comprise substitutions in one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues, insertions in one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues, and/or deletions relative to the wild type.

The term "vector" as used herein refers to a nucleic acid comprising, consisting essentially of, or consisting of a complete replicon such that the vector can be replicated when placed within a cell by, for example, a transfection, infection, or transformation process. As will be appreciated in the art, once within the cell, the vector can replicate as an extrachromosomal (episomal) element, or can be integrated into the host cell chromosome. The vector can include nucleic acids derived from retroviruses, adenoviruses, herpesviruses, baculoviruses, modified baculoviruses, papillomaviruses, AAV viral vectors, lentiviral vectors, adenoviral vectors, alphaviral vectors, and the like, such as vectors described herein selected from adenoviral vectors, adeno-associated viral vectors, or recombinant adeno-associated viral vectors.

The term "adeno-associated virus" or "AAV" as used herein refers to members of the class of viruses associated with that name and belonging to the genus Dependoviridae of the family Parvoviridae. Adeno-associated virus is a single-stranded DNA virus that grows exclusively in cells where certain functions are provided by a co-infecting helper virus. All AAV serotypes remarkably exhibit very similar replication characteristics mediated by homologous rep genes; and all possess three related capsid proteins. At least 13 sequentially numbered naturally occurring AAV serotypes are known in the art. Non-limiting exemplary serotypes for use in the methods disclosed herein include any of these 13 serotypes, such as AAV2, AAV8, AAV9, or variant serotypes such as AAV-DJ and AAV PHP.B. The AAV virus comprises, consists essentially of, or consists of three major viral proteins, VP1, VP2, and VP3.

Example

Reagents, instruments and experimental methods

I. Reagents and consumables used

The starting materials of the embodiments are commercially available and/or can be prepared by a variety of methods known to those skilled in the art of organic synthesis. Those skilled in the art of organic synthesis will appropriately select reaction conditions (including solvent, reaction atmosphere, reaction temperature, duration of experiment and aftertreatment) in the following synthetic methods. Those skilled in the art of organic synthesis will appreciate that the functional groups present in each part of the molecule should be compatible with the proposed reagents and reactions.

All reagents and compounds synthesized can be purchased through general commercial channels. For detailed information, please see Table 1 below.

Table 1. Reagent information

II. Instruments used

Table 2. Instrument information

Table 3. Primer and probe sequences

III. Experimental Methods

3.1 Cell culture

Remove the cryovial of suspended HEK293 cells from the liquid nitrogen tank and quickly transfer to a 37°C water bath. Gently shake for 2-3 minutes until the frozen cell suspension thaws. Transfer the cell suspension from the cryovial to a 125mL cell culture flask in a biosafety cabinet and mix thoroughly with 30mL of culture medium (preheated to 37°C). Culture the cells in a _CO2 shaking incubator (ZCZY-CS9) at 37°C, 8% (v/v) _CO2 , 135 rpm, and 80% humidity. Subculture the cells every three days at a density of 0.3 to 0.6E+06 cells/mL. Cells can be used for viral packaging experiments after five consecutive subcultures.

3.2 Drug preparation

To a reagent bottle containing 10 mg of BX795 (MCE, HY-10514) powder, add 1.69 mL of dimethyl sulfoxide (DMSO) until completely dissolved to obtain a 10 mM BX795 stock solution. Dilute to 0.2 mM, 0.5 mM, 1 mM, and 2.5 mM working solutions for later use.

To a reagent bottle containing 1 mg of BX320 (MCE, HY-10515) powder, add 0.61 mL of DMSO until completely dissolved to obtain a 3 mM BX320 working solution for use; to a reagent bottle containing 1 mg of BX517 (MCE, HY-13842) powder, add 0.708 mL of DMSO until completely dissolved to obtain a 5 mM BX517 working solution for use; to a reagent bottle containing 1 mg of BX912 (MCE, HY-11005) powder, add 0.424 mL of DMSO until completely dissolved to obtain a 5 mM BX912 working solution for use; to a reagent bottle containing 5 mg of MRT67307 (MCE, HY-13018) powder, add 1.08 mL of DMSO until completely dissolved to obtain a 10 mM MRT67307 working solution for use.

3.3 Cell Preparation

1 mL of continuously cultured suspended HEK293 cells was taken and counted using a Vi-Cell cell counter (Vi-cell XR). Fresh culture medium (preheated at 37°C) was added according to the counting results to dilute the cell density to 1.0-2.0E+06 cells/mL. The cells were cultured in a _CO2 shaking incubator (ZCZY-CS9) at 37°C, 8% (v/v) _CO2 , 135 rpm, and 80% humidity.

3.4 Cell transfection

After 24 hours of culture, recount the cells and adjust the cell density to approximately 3E+06 cells/mL with fresh culture medium, ensuring a viable cell ratio greater than 95%. Inoculate 27 mL of the cell suspension into a 125 mL cell culture flask. Mix the Helper, RepCap, and GOI plasmids with the transfection reagent PEI in a fixed ratio in culture medium to create a transfection complex. After 10-20 minutes, transfer the complex to a cell culture flask. After transfection, continue culturing the cells in a _CO2 shaking incubator.

3.5 Addition of small molecule compounds

Depending on the drug concentration, within 4 hours after transfection, add BX795 or other compound working solution to the cell culture flask at a ratio of 1:1000 (drug volume: culture system) and mix well.

3.6 Cell Harvest

72 hours after transfection, 10 mL of cell suspension (mixed before sampling) was transferred to a 15 mL centrifuge tube, 20% (v/v) Tween 20 was added to a final concentration of 0.2-1% (v/v), the tube was inverted to mix, and lysed at room temperature for 10-20 minutes; 1 M MgCl2 was then added to a final concentration of _1-2 mM, the tube was inverted to mix, and the tube was allowed to stand for 5-10 minutes; SuperNuclease (Sino Biological) was added to a final concentration of 10-50 U/mL and mixed, and the tube was incubated in a 37°C _CO2 shaking incubator for 2-3 hours. 5 M NaCl was then added to a final concentration of 100-400 mM and mixed. After incubation at room temperature for 10-20 minutes, the tube was centrifuged at 4000 rpm for 10 minutes, and the supernatant was transferred to a centrifuge tube and stored at 4°C or -20°C.

3.7 ddPCR detection

The sample supernatant was treated with DNase I and mixed with primers, probes, and ddPCR premix. The mixed ddPCR reaction system was added to the droplet generator chip and the QX200 AutoDG droplet generator was run to obtain droplets. The droplets were then transferred to a 96-well PCR reaction plate and sealed. PCR cycles were performed using a T100 PCR instrument (Table 4). Finally, the PCR reaction plate was transferred to a QX200 droplet reader to detect and calculate the genomic titer of the sample to be tested.

Table 4. PCR amplification program in ddPCR

3.8 ELISA test

ELISA assays were performed using the AAV Titration ELISA kit (PROGEN). 100 μL of standard and diluted test sample were added to the ELISA plate and incubated at 37°C for 1 hour. Each well was then washed three times with 200 μL of 1×ASSB. 100 μL of anti-AAV biotin conjugate was added and incubated at 37°C for 1 hour. Each well was then washed three times with 200 μL of 1×ASSB. 100 μL of streptavidin-enzyme conjugate was then added and incubated at 37°C for 10 minutes. Each well was washed three times with 200 μL of 1×ASSB. 100 μL of TMB solution was added and incubated at room temperature in the dark for 15 minutes. 50 μL of stop solution was added to each well. The absorbance (OD) at 450 nm was measured using a microplate reader to calculate the viral capsid titer.

Example 1: Effects of different concentrations of the compounds of the present application on the yield and quality of rAAV9 in HEK293 suspension cells

As a non-limiting embodiment, a compound BX795 having the structure of formula (I) was added to the HEK293 cell adeno-associated virus (rAAV) production system. The specific experimental method is as follows:

Perform cell culture according to Protocol 3.1, drug preparation according to Protocol 3.2, cell preparation according to Protocol 3.3, and transfection of cells with the rAAV9 packaging plasmid according to Protocol 3.4. Four hours after transfection, add DMSO, 0.2 mM, 0.5 mM, 1 mM, or 2.5 mM BX795 working solution to the culture flask at a 1:1000 ratio, to final concentrations of 0 M, 0.2 μM, 0.5 μM, 1 μM, and 2.5 μM, respectively.

72 hours after transfection, cells were harvested according to Experimental Method 3.6, lysed, and the supernatant was collected. ddPCR and ELISA were performed according to Experimental Methods 3.7 and 3.8, respectively, to quantify the rAAV9 genomic DNA and protein capsid in the supernatant.

The culture flasks with DMSO added served as the blank control group, and the culture flasks with BX795 added at final concentrations of 0.2μM, 0.5μM, 1μM, and 2.5μM served as the experimental group. The results showed that the rAAV9 titer was significantly increased after adding different concentrations of BX795, and the empty shell ratio E:F (Empty:Full, also known as the empty-full shell ratio or empty shell rate, the lower the E:F value, the less empty protein capsids in the virus product, and conversely, the higher the E:F value, the more empty protein capsids in the virus) decreased significantly. Compared with the control group, the addition of 1μM BX795 increased the rAAV9 titer by 165% and significantly reduced the empty shell rate, with the most significant effect on improving the yield and quality of the rAAV9 virus (Figure 1). This demonstrates that BX795 can effectively increase the yield of rAAV9 and reduce the empty shell rate, thereby improving the quality and yield of the rAAV9 virus.

Referring to the experimental method of this example, when preparing rAAV9 virus using a two-plasmid or single-plasmid system, adding different concentrations of BX795 can also achieve the same effect as the three-plasmid virus packaging system. The compounds of the present application can increase the titer of rAAV9 and reduce the empty shell rate, thereby improving the quality and yield of rAAV9 virus.

Example 2: Effect of the Addition Time of the Compound of the Present Application on the Yield of rAAV9 in HEK293 Suspension Cells

As a non-limiting embodiment, the experiments of this example were carried out using a compound BX795 having the structure of formula (I).

Perform cell culture according to Protocol 3.1, drug preparation according to Protocol 3.2, cell preparation according to Protocol 3.3, and transfection with the rAAV9 packaging plasmid according to Protocol 3.4. Add 1 mM BX795 working solution at a dilution of 1:1000 to each culture flask 4 hours before transfection, 2 hours before transfection, 0 hour after transfection, 1 hour after transfection, 2 hours after transfection, and 4 hours after transfection.

72 hours after transfection, cells were harvested according to Experimental Method 3.6, lysed, and the supernatant was collected. ddPCR was performed according to Experimental Method 3.7 to quantify the rAAV9 genomic DNA in the supernatant.

The experimental results showed that adding BX795 from 4 hours before to 4 hours after transfection significantly increased the titer of rAAV9 (Figure 2), and there was no significant difference in the effect. This indicates that adding BX795 from 4 hours before to 4 hours after transfection can significantly increase the yield of rAAV9.

Referring to the experimental method of this example, when using a two-plasmid or single-plasmid system to prepare rAAV9 virus, adding BX795 at different times can also achieve the same effect as the three-plasmid virus packaging system. The compound of the present application can significantly increase the titer of rAAV9 and increase the yield of rAAV9.

Example 3: Effects of the compounds of the present application on rAAV9 production and quality in different cell lines

As a non-limiting embodiment, the experiments of this example were carried out using a compound BX795 having the structure of formula (I).

VPC1.0, VPC2.0, and WayneLV Pro ^™ HEK293 cells were cultured and transfected according to the aforementioned experimental methods. Within 4 hours after transfection, DMSO and 1 mM BX795 working solution were added to each culture flask at a 1:1000 ratio to reach final concentrations of 0 μM and 1 μM, respectively. Each cell line had a blank control group and a 1 μM BX795 control group.

72 hours after transfection, cells were harvested according to Experimental Method 3.6, lysed, and the supernatant was collected. ddPCR and ELISA were performed according to Experimental Methods 3.7 and 3.8, respectively, to quantify the rAAV9 genomic DNA and protein capsid in the supernatant.

The experimental results showed that compared with the control group, the addition of 1 μM BX795 significantly increased the titer of rAAV9 in all three cell lines and significantly decreased the empty shell rate (Figure 3). This demonstrates that BX795 can improve the yield and quality of rAAV9 in multiple cell lines.

Referring to the experimental methods of this example, when using a two-plasmid or single-plasmid system to prepare rAAV9 virus in different cell lines, the use of BX795 can also achieve the same effect as the three-plasmid viral packaging system. The compounds of this application can effectively increase the titer of rAAV9 and reduce the empty shell rate in various cell lines, thereby improving the quality and yield of rAAV9 virus.

Example 4: Effects of the compounds of the present application on the yield of rAAV of different serotypes in HEK293 suspension cells

As a non-limiting embodiment, the experiments of this example were carried out using a compound BX795 having the structure of formula (I).

Culture cells according to Protocol 3.1, prepare drugs according to Protocol 3.2, prepare cells according to Protocol 3.3, and transfect cells with rAAV2, rAAV5, rAAV6, rAAV8, rAAV9, and rAAV9 variant packaging plasmids according to Protocol 3.4. Within 4 hours of transfection, add DMSO and 1 mM BX795 working solution at a ratio of 1:1000 to the cell culture flask to a final concentration of 0 μM and 1 μM, respectively.

72 hours after transfection, cells were harvested according to Experimental Method 3.6, lysed, and the supernatant was collected. ddPCR was performed according to Experimental Method 3.7 to quantify the rAAV genomic DNA in the supernatant.

In HEK293 suspension cells, rAAV yields varied across serotypes. However, addition of 1 μM BX795 significantly increased the titers of rAAV2, rAAV5, rAAV6, rAAV8, rAAV9, and rAAV9 variants compared to the control (Figure 4). This suggests that BX795 can increase the yields of rAAV2, rAAV5, rAAV6, rAAV8, rAAV9, and rAAV9 variants, and that the rAAV yield enhancement effect of BX795 is applicable to multiple serotypes and their variants.

Referring to the experimental method of this example, when using a two-plasmid or single-plasmid system to prepare rAAV2, rAAV5, rAAV6, rAAV8, rAAV9, and rAAV9 variant viruses, the use of BX795 can also achieve the same effect as the three-plasmid virus packaging system. The compound of the present application can effectively increase the yield of different serotypes of rAAV and their variants.

Example 5: Effects of different compounds of the present application on rAAV9 yield and quality in HEK293 suspension cells

As a non-limiting embodiment, the experiments of this example were carried out using compounds BX795, BX912, BX320 and MRT67307 having the structure of formula (I) and BX517 having the structure of formula (II).

Cell culture was performed according to Protocol 3.1, drug preparation was performed according to Protocol 3.2, cell preparation was performed according to Protocol 3.3, and cells were transfected with the rAAV9 packaging plasmid according to Protocol 3.4. Within 4 hours after transfection, DMSO, 1 mM BX795, 5 mM BX912, 5 mM BX517, 3 mM BX320, and 5 mM MRT67307 working solutions were added to each shake flask to achieve final concentrations of 0 μM, 1 μM BX795, 5 μM BX912, 5 μM BX517, 30 μM BX320, and 5 μM MRT67307, respectively.

72 hours after transfection, cells were harvested according to Experimental Method 3.6, lysed, and the supernatant was collected. ddPCR and ELISA were performed according to Experimental Methods 3.7 and 3.8, respectively, to quantify the rAAV9 genomic DNA and protein capsid in the supernatant.

The experimental results are shown in Figure 5. The culture flasks supplemented with DMSO served as the blank control group, while the culture flasks supplemented with 1μM BX795, 5μM BX912, 5μM BX517, 30μM BX320, and 5μM MRT67307 served as the experimental groups. The results showed that the addition of BX795 and its structural analogs significantly increased rAAV9 titers; 1μM BX795, 5μM BX912, 5μM BX517, 30μM BX320, and 5μM MRT67307 increased rAAV9 titers by 204%, 180%, 152%, 136%, and 144%, respectively, while also reducing the empty capsid fraction (Figure 5). This demonstrates that BX795 and its structural analogs can effectively improve rAAV9 yield and quality.

Referring to the experimental method of this example, when using a two-plasmid or single-plasmid system to produce rAAV, the use of the compounds of the present application can also achieve the same effect as the three-plasmid viral packaging system. The compounds of the present application can effectively improve the yield and quality of rAAV9 in HEK293 suspension cells.

Example 6: Effects of the compounds of the present application on the yield and quality of rAAV9 in HEK293T adherent cells

As a non-limiting embodiment, two compounds BX795 and BX912 having the structure of formula (I) were used to carry out the experiments of this example.

The effect of BX795 and BX912 on rAAV9 production in HEK293T adherent cells was tested with reference to the method in Example 3. HEK293T adherent cells were continuously cultured after recovery at 37°C and 5% (v/v) _CO2 until the fifth passage was used for virus packaging.

HEK293T adherent cells were plated in 6-well plates at a density of 6E+5 cells/well. After 24 hours, when the cells reached 80%-90% confluency, three plasmid transfections were performed. The Helper, RepCap, and GOI plasmids were mixed with the transfection reagent PEI at a fixed ratio in culture medium to create a transfection complex. After 10-20 minutes, the complex was transferred to the cell suspension. After transfection, the cells were placed in a _CO2 incubator and continued to culture. Within 4 hours of transfection, DMSO, 2mM BX795, and 2mM BX912 working solutions were added to the cell suspension at a 1:1000 ratio to achieve final concentrations of 0μM, 2μM, and 2μM, respectively. 72 hours after transfection, the cells were harvested and lysed according to Protocol 3.6 to obtain viral supernatant. rAAV9 genomic DNA in the supernatant was quantified by ddPCR according to Protocol 3.7.

The experimental results showed that after transfection of the adherent HEK293T cell line, the rAAV9 titer could be significantly increased by adding 2μM BX795 and 2μM BX912, as shown in Table 5 and Figure 6, indicating that BX795 can effectively improve the rAAV9 yield and quality in HEK293T cells.

Referring to the experimental method of this embodiment, when using a two-plasmid or single-plasmid system to prepare rAAV, the use of BX795, BX912 and their structural analogs can also achieve the same effect as the three-plasmid viral packaging system. Other compounds that meet the requirements of this application, such as BX517, BX320 and MRT67307, have been verified by experimental results to increase the yield and quality of rAAV in HEK293T adherent cells. In other words, the compounds of this application can effectively increase the yield and quality of rAAV in HEK293T adherent cells.

Table 5. Effects of BX795 and BX912 on rAAV9 in HEK293T cells

Example 7: Effects of the compounds of the present application on the yield and quality of rAAV2 in Hela adherent cells

As a non-limiting embodiment, the experiments of this example were carried out using compounds BX795 and BX912 having the structure of formula (I) and compound BX517 having the structure of formula (II).

The effect of BX795 and its structural analogs on rAAV2 production in Hela adherent cells was tested with reference to the method in Example 3. Hela adherent cells were cultured continuously after recovery at 37°C and 5% (v/v) CO ₂ until the fifth passage was used for virus packaging.

It should be pointed out that Hela cells lack key elements for virus packaging. On the basis of two-plasmid transfection, Ad5 infection is required to perform the function of helper virus (instead of Helper in the three-plasmid transfection system) to package AAV.

Adherent HeLa cells were plated in 6-well plates at a density of 6E+5 cells/well. After 24 hours, when the cells reached 80%-90% confluence, plasmid transfection was performed. RepCap and GOI plasmids were mixed with the transfection reagent Lipofectamine 3000 at a fixed ratio in culture medium to form a transfection complex. After 10-20 minutes, the complex was transferred to the cell suspension. Following transfection, the cells were incubated in a _CO2 incubator and continued to be cultured. Four to six hours after transfection, the cells were infected with Ad5 helper virus at an MOI of 50 PFU/cell. DMSO, 0.2mM BX795, 2mM BX517, and 3mM BX912 working solutions were then added to the cell suspension at a 1:1000 ratio, resulting in final concentrations of 0μM, 0.2μM, 2μM, and 3μM, respectively. 72 hours after transfection, cells were harvested and then lysed according to Experimental Method 3.6 to obtain viral supernatant. ddPCR was performed according to Experimental Method 3.7 to quantify rAAV2 genomic DNA in the supernatant.

Results indicate that 0.2 μM BX795, 2 μM BX517, and 3 μM BX912 significantly increased rAAV2 titers, as shown in Table 6 and Figure 7. Other compounds described herein, such as BX320 and MRT67307, have also been shown to improve rAAV production and quality in HeLa cells. This suggests that the compounds described herein also improve rAAV production and quality in HeLa cells.

Table 6. Effects of BX795, BX517, and BX912 on rAAV2 in Hela cell lines

Based on the results of the above non-limiting examples, it can be seen that the compounds of the present application can improve the yield and quality of rAAV in HEK293, Hela or other cell lines used for rAAV production.

Incorporated by Reference

Each patent and scientific document mentioned herein is incorporated by reference in its entirety for all purposes.

Equivalence

The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered in all cases as illustrative rather than limiting of the invention described herein. The scope of the present invention is therefore indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalence of the claims are intended to be embraced therein.

Claims (14)

Use of a compound of formula (I), a pharmaceutically acceptable salt thereof, a stereochemical isomeric form thereof, or a composition comprising any of the above in improving the yield and/or quality of virus packaging in cells,
wherein R ₁ is each independently selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, and cyclopropyl; R ₂ is selected from the group consisting of H, F, Cl, Br, I, NH ₂ , NO ₂ , OH, CN, methyl, ethyl, propyl, isopropyl, cyclopropyl, difluoromethyl, trifluoromethyl, substituted or unsubstituted phenyl, pyrrolyl, and pyridyl; _L1 is selected from the group consisting of methylene, ethylene, propylene, butylene, cyclopentylene, cyclohexylene, and phenylene; L ₂ is selected from the group consisting of absence, carbonyl, -C(O)O-, -C(O)NR ₁ -, and -NR ₁ -; R ₃ is selected from the group consisting of H, NH ₂ , methyl, ethyl, propyl, isopropyl, acetamido, propionamido, butanamido, isobutanamido, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, substituted or unsubstituted phenyl, pyridyl, pyrrolyl, furanyl, thienyl, thiazolyl, imidazolyl, pyrazolyl, pyrimidinyl, pyridazinyl, pyranyl, piperazinyl, and piperidinyl; L ₃ is selected from the group consisting of methylene, ethylene, propylene, -NR ₁ -, -NR ₁ C(O)-, and -NR ₁ C(O)O-; _R4 is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, tetrahydropyrrolyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, and morpholinyl. The use according to claim 1, wherein R ₁ is each independently H or methyl; _R2 is selected from the group consisting of Cl, Br, I, CN, methyl, difluoromethyl, trifluoromethyl, cyclopropyl, and phenyl; _L1 is selected from the group consisting of methylene, ethylene, propylene, cyclopentylene, and cyclohexylene; L ₂ is selected from the group consisting of absent, and -C(O)NR ₁ -; _R3 is selected from the group consisting of acetamido, isopropylamido, tert-butylamido, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, pyridyl, pyrrolyl, thienyl, thiazolyl, imidazolyl, pyrazolyl, and pyrimidinyl; L ₃ is -NR ₁ C(O)- or methylene; _R4 is selected from the group consisting of cyclopentyl, cyclohexyl, tetrahydropyrrolyl, piperidinyl, and morpholinyl. The use according to claim 1 or 2, wherein the compound has a structure selected from the following:
The use according to any one of claims 1 to 3, wherein the compound has a structure selected from the following:
The use according to claim 4, wherein _R2 is selected from Br, I or cyclopropyl; L ₁ is selected from ethylene or propylene; _L2 is -C(O)NH- or absent; _R3 is selected from the group consisting of tert-butylamido, cyclobutyl, thienyl, and imidazolyl; L ₃ is -NR ₁ C(O)- or methylene; R ₄ is tetrahydropyrrolyl or morpholinyl. The use according to any one of claims 1 to 4, wherein the compound has the following structure:
Use of a compound of formula (II), a pharmaceutically acceptable salt thereof, or a stereochemically isomeric form thereof in improving the yield and/or quality of virus packaging in cells,
wherein R ₁ is each independently selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, and cyclopropyl; R ₂ is selected from the group consisting of H, F, Cl, Br, NH ₂ , NO ₂ , OH, CN, methyl, ethyl, propyl, isopropyl, cyclopropyl, difluoromethyl, trifluoromethyl, substituted or unsubstituted phenyl, pyrrolyl, and pyridyl; R ₃ is -C(O)R ₆ or -C(O)OR ₆ ; R ₆ is selected from the group consisting of NH ₂ , methyl, ethyl, propyl, isopropyl, cyclopropyl, and phenyl; R ₄ and R ₅ are each independently selected from the group consisting of F, Cl, Br, NH ₂ , NO ₂ , OH, CN, methyl, ethyl, difluoromethyl, and trifluoromethyl; m and n are each independently 0 or 1. The use according to claim 7, wherein R ₁ is each independently H or methyl; _R2 is selected from the group consisting of CN, methyl, difluoromethyl, trifluoromethyl, and phenyl; R ₃ is -C(O)R ₆ ; R ₆ is selected from the group consisting of NH ₂ , methyl, ethyl, propyl, isopropyl, cyclopropyl, and phenyl; m and n are 0. The use according to claim 7 or 8, wherein the compound has the following structure:
The method according to any one of claims 1 to 9, wherein the virus is an adeno-associated virus or a recombinant adeno-associated virus. The use according to any one of claims 1 to 10, wherein the cells include adherent or suspension cultured HEK293, Hela cells, or cell lines obtained by gene editing, modification, domestication, and/or screening thereof, such as HEK293T, HEK293F, or HEK293FT cells. The use according to any one of claims 1 to 11, wherein the concentration of the compound is 0.05-60 μM, preferably 0.05-30 μM, more preferably 0.2-30 μM. The use according to any one of claims 1 to 12, wherein the compound is added to the cells 24, 18, 12, 6, 4, 2, or 1 hour before transfection, or 1, 2, 4, 6, 12, 18, 24, 36, or 48 hours after transfection. The use according to any one of claims 1 to 13, wherein increasing the yield and/or quality of virus packaging in cells comprises one or more of the following: increasing virus yield, reducing the empty shell rate of virus packaging, or any combination thereof.