Use of PARP-specific inhibitor in improving yield or quality of viral packaging in cell

Abstract

Provided is a method for improving the yield and / or quality of viral packaging in a cell, wherein the PARP gene expression, activity or function in the cell is specifically downregulated or inhibited. The present invention further relates to a use of a PARP-specific inhibitor in improving the yield and / or quality of viral packaging in a cell, wherein the PARP-specific inhibitor can downregulate or inhibit the PARP gene expression, activity or function.

Description

Use of PARP-specific inhibitors in improving viral packaging yield or quality in cells

[0001] Related applications

[0002] This disclosure claims priority to international application PCT / CN2024 / 079139, filed on February 28, 2024, the entire contents of which are expressly incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the field of medicine and biology, and in particular to a method for improving the yield and / or quality of virus packaging in cells, wherein the expression, activity or function of the PARP gene in the cells is specifically downregulated or inhibited. Background Art

[0004] With the rapid growth of the global gene therapy market, adeno-associated virus (AAV) has become one of the most important viral 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, and development of cell feeds. Studies on the molecular mechanisms of AAV replication, infection and other processes have found that many cytokines in host cells play a key role in the replication and packaging of AAV. The method of improving rAAV packaging by regulating key signal transduction pathways in host cells has a certain theoretical basis and feasibility.

[0005] 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 shortcomings are also very obvious. First, their effect on increasing virus production is limited; second, traditional feeds are complex in composition, many components have redundant functions, and are expensive, requiring multiple additions during the cell culture process; finally, while traditional feeds increase virus production, they do not improve virus quality and may even reduce virus quality.

[0006] Currently, there are few reports in the industry on methods for improving the yield and quality of rAAV products by regulating the signaling pathways of host cells. The present disclosure aims to develop small molecule compounds that inhibit the PARP pathway as new enhancers to significantly improve the yield and quality of rAAV production, which is of great significance for improving the yield and quality of rAAV and reducing the cost of rAAV production.

[0007] PARP protein can sense DNA single-strand break (SSB) damage in cells and accumulate at the damage site. Its main functions are divided into two aspects: on the one hand, it accumulates at the damage site to prevent DNA replication, prevent DNA replication errors, and maintain genome stability; on the other hand, PARP protein can catalyze the ADP-ribosylation modification of protein substrates to convert NAD into + The ADP-ribose (ADPR) group produced by cleavage is covalently linked to the target protein. According to the form of the modification group, it can be divided into two categories: mono ADP-ribosylation (MARylation) and poly ADP-ribosylation (PARylation). ADP-ribosylation modification can change the activity, stability and ligand binding ability of proteins, thereby regulating various signal transduction pathways in cells. In addition to playing a role in the DNA damage repair signaling pathway, PARP protein is also involved in many other signaling pathways, such as apoptosis, cell cycle regulation, oxidative stress response, energy metabolism, innate immune signaling pathways, etc.

[0008] The PARP family has numerous members, all expressed in a variety of tissue cells. Its functions involve DNA repair, transcriptional regulation, signal transduction, and metabolic control. Chemical drugs that target PARP inhibition have been approved for use in prostate cancer, breast cancer, and ovarian cancer. Summary of the Invention

[0009] The PARP-specific inhibitors disclosed herein target the viral genome synthesis pathway. Compared to existing viral packaging enhancers, the small molecule compounds disclosed herein are more precise and specific, not only having a better effect on improving viral yield, but also significantly improving viral quality, and having simple ingredients and being easy to prepare. In general, the PARP inhibitors disclosed herein can not only significantly increase the yield of viruses such as AAV (or rAAV), but also improve viral quality, and are suitable for different serotypes and different cell lines. Compared to traditional cell culture supplements and enhancers, the small molecule compounds disclosed herein have significant advantages in improving viral packaging, are easy to use, and can be easily combined with existing AAV production processes.

[0010] In one aspect of the present disclosure, a method for improving the yield and / or quality of viral packaging in a cell is provided, wherein the expression, activity or function of the PARP gene in the cell is specifically downregulated or inhibited.

[0011] In one embodiment, PARP gene expression, activity or function is specifically downregulated or inhibited in the cell by using a PARP specific inhibitor.

[0012] Another aspect of the present disclosure provides use of a PARP-specific inhibitor in improving viral packaging yield and / or quality in cells, wherein the PARP-specific inhibitor can downregulate or inhibit PARP gene expression, activity or function.

[0013] In some embodiments, the virus is selected from adeno-associated virus or recombinant adeno-associated virus (rAAV).

[0014] In some embodiments, the PARP-specific inhibitor is an antibody, a small molecule compound, an RNAi molecule, a sgRNA, or an antisense nucleic acid.

[0015] In some embodiments, the PARP-specific inhibitor is a small molecule compound.

[0016] In some embodiments, the PARP specific inhibitor is selected from the group consisting of Olaparib, Niraparib, Fluzoparib, Veliparib, Rucaparib, Pamiparib, Talazoparib, Saruparib, PARP7-IN-4, PARP7-IN-15, PARP7-IN-16, PARP7-IN-17, UPF 1069, PJ34, Pamiparib, RBN-2397, Dehydrocorydaline, RBN012759, 3-Aminobenzamide, AG14361, PJ34 hydrochloride, Talazoparib tosylate, AZD-2461, Rucaparib monocamsylate, EB-47dihydrochloride, Rucaparib The present invention relates to a group consisting of phosphate, Iniparib, Venadaparib, DPQ, Senaparib, BGP-15 and structural analogues thereof.

[0017] In a preferred embodiment, the PARP specific inhibitor is one or more selected from Olaparib, Veliparib, Rucaparib, AZD-2461, UPF 1069 and structural analogs thereof.

[0018] In a preferred embodiment, the PARP-specific inhibitor is Olaparib, Veliparib or a structural analogue thereof.

[0019] In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody.

[0020] In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0021] In some embodiments, the RNAi molecule is siRNA, saRNA, shRNA, dsRNA, or miRNA.

[0022] In some embodiments, the RNAi molecule is a polynucleotide 15-40 bases long.

[0023] In some embodiments, the RNAi molecule is modified to enhance its stability.

[0024] In some embodiments, the final concentration of the small molecule compound is greater than 0.001 μM, for example, 0.001 μM-1000 μM, 0.008 μM-750 μM, 0.008 μM-500 μM, 0.008 μM-100 μM, 0.008 μM-50 μM, 0.008 μM-30 μM, 0.008 μM-20 μM, 0.008 μM-10 μM, 0.008 μM-5 μM, 0.01 μM-2 50μM, 0.01μM-200μM, 0.05μM-100μM, 0.1μM-100μM, 0.1μM-50μM, 0.1μM-30μM, 0.1μM-20μM, 0.1μM -10μM, 0.1μM-5μM, 0.15μM-50μM, 0.2μM-100μM, 0.2μM-50μM, 0.2μM-30μM, 0.2μM-20μM, 0.2μM-10μ M, 0.2μM-5μM, 0.25μM-30μM, 0.4μM-100μM, 0.4μM-50μM, 0.4μM-30μM, 0.4μM-25μM, 0.4μM-20μM, 0 .4μM-10μM, 0.4μM-5μM, 0.5-10μM, 0.8μM-20μM, 1μM-100μM, 1μM-50μM, 1μM-30μM, 1μM-20μM, 1μM-1 0 μM, 1 μM-5 μM, 1.2 μM-10 μM, 1.8 μM-7.5 μM, 2.5 μM-5 μM, 3.5 μM-4.5 μM, 5 μM-100 μM, 5 μM-50 μM, 5 μM-30 μM, 5 μM-20 μM, 5 μM-10 μM, 10 μM-100 μM, 10 μM-50 μM, 10 μM-30 μM, 10 μM-20 μM, 20 μM-50 μM or 30 μM-50 μM. The final concentration of the aforementioned small molecule compound is 0.008 μM-50 μM or 0.04 μM-50 μM.

[0025] According to certain embodiments of the present disclosure, the final concentration of the small molecule compound is 0.008 μM, 0.04 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 3 μM, 4 μM, 5 μM, 7.5 μM, 10 μM, 20 μM, 25 μM, 35 μM, 50 μM, 100 μM, 200 μM, 250 μM, 500 μM, 750 μM, or 1000 μM.

[0026] In some specific embodiments, the PARP-specific inhibitor is Olaparib. In some preferred embodiments, the final concentration of Olaparib is 0.001 μM-1000 μM, preferably 0.008 μM-50 μM, 0.008 μM-30 μM, 0.01 μM-30 μM, 0.04 μM-30 μM, 0.008 μM-10 μM or 0.04 μM-10 μM, more preferably 0.04 μM-5 μM.

[0027] In some specific embodiments, the PARP-specific inhibitor is Veliparib. In some preferred embodiments, the final concentration of Veliparib is 0.001 μM-1000 μM, preferably 1 μM-100 μM, 5 μM-70 μM or 10 μM-50 μM, 10 μM-20 μM, more preferably 20 μM-50 μM.

[0028] In some specific embodiments, the PARP-specific inhibitor is AZD-2461. In some preferred embodiments, the final concentration of AZD-2461 is 0.001 μM-1000 μM, preferably 0.005 μM-100 μM, 0.01 μM-50 μM or 0.1 μM-30 μM, more preferably 0.2 μM-10 μM.

[0029] In some specific embodiments, the PARP-specific inhibitor is Rucaparib. In some preferred embodiments, the final concentration of Rucaparib is 0.001 μM-1000 μM, preferably 0.005 μM-50 μM, 0.1 μM-20 μM or 0.5 μM-10 μM, more preferably 1 μM-5 μM.

[0030] In some specific embodiments, the PARP-specific inhibitor is UPF 1069. In some preferred embodiments, the final concentration of UPF 1069 is 0.001 μM-1000 μM, preferably 1 μM-100 μM, 1 μM-50 μM or 1 μM-30 μM, more preferably 1 μM-10 μM or 10 μM-30 μM.

[0031] In some embodiments, the PARP-specific inhibitor is added to the cells 24, 18, 12, 6, 4, 2, 1, or 0.5 hours before transfection or 0.5, 1, 2, 4, 6, 12, 18, 24, 28, or 48 hours after transfection.

[0032] In one embodiment, the one or more PARP-specific inhibitors are added to the cells within 24 hours before transfection to 48 hours after transfection, within 18 hours before transfection to 24 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 0.5 hour before transfection to 0.5 hour after transfection, within 2 hours before transfection to 4 hours after transfection, within 4 hours before transfection to 4 hours after transfection, or within 0.5 hour before transfection to 24 hours after transfection. Preferably, the one or more PARP-specific inhibitors are added to the cells within 0.5 hour before transfection to 24 hours after transfection.

[0033] In some embodiments, 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.

[0034] 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.

[0035] 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 shell rate of virus packaging to increase the proportion of virus packaging, or any combination thereof.

[0036] 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.

[0037] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG1 shows the effect of different concentrations of Olaparib on rAAV9 production in HEK293 suspension cells.

[0039] FIG2 shows the effect of different concentrations of Olaparib on the quality of rAAV9 in HEK293 suspension cells.

[0040] FIG3 shows the effect of the timing of Olaparib addition on rAAV9 production in HEK293 suspension cells.

[0041] FIG4 shows the effect of Olaparib on rAAV9 production and quality in different cell lines.

[0042] FIG5 shows the effect of Olaparib on the yield of rAAV of different serotypes in HEK293 suspension cells.

[0043] FIG6 shows the effects of other specific PARP inhibitors on rAAV production in HEK293 suspension cells.

[0044] FIG. 7 shows the effects of other specific PARP inhibitors on rAAV quality in HEK293 suspension cells.

[0045] FIG8 shows the effect of PARP knockdown on rAAV9 virus yield and quality in suspension HEK293 cells.

[0046] FIG9 shows a comparison of the effects of PARP knockdown.

[0047] FIG10 shows a comparison of PARP protein expression levels.

[0048] FIG11 shows the effect of PARP knockdown on rAAV virus yield and quality in adherent HEK293T cells.

[0049] FIG12 shows the effect of Olaparib on rAAV9 yield and quality in HEK293T adherent cells.

[0050] FIG13 shows the effects of different PARP inhibitors on rAAV9 production in HEK293T adherent cells.

[0051] FIG14 shows the effects of Olaparib and Veliparib on rAAV2 production in HeLa adherent cells. DETAILED DESCRIPTION

[0052] 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.

[0053] 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.

[0054] 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.

[0055] definition

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] As used herein, the term "composition" refers to a combination or mixture of two or more different ingredients, components or substances.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] In one aspect of the present disclosure, there is provided use of a PARP-specific inhibitor for improving viral packaging yield and / or quality in cells, wherein the PARP-specific inhibitor can downregulate or inhibit PARP gene expression, activity or function.

[0065] Another aspect of the present disclosure provides a use of a cell for improving virus packaging yield and / or quality in the cell, wherein PARP gene expression, activity or function is specifically downregulated or inhibited in the cell.

[0066] In another aspect of the present disclosure, a method for improving the yield and / or quality of virus packaging in cells is provided, wherein the expression, activity or function of the PARP gene in the cells is specifically downregulated or inhibited.

[0067] According to certain embodiments of the present disclosure, PARP gene expression, activity or function in the cells is specifically downregulated or inhibited by using a PARP-specific inhibitor.

[0068] According to certain embodiments of the present disclosure, the virus is selected from adeno-associated virus or recombinant adeno-associated virus (rAAV).

[0069] Another aspect of the present disclosure provides use of a PARP-specific inhibitor in improving the yield and / or quality of adeno-associated virus (AAV) in cells, wherein the PARP-specific inhibitor can downregulate or inhibit PARP gene expression, activity or function.

[0070] According to certain embodiments of the present disclosure, the aforementioned PARP-specific inhibitor is an antibody, a small molecule compound, an RNAi molecule, an sgRNA, or an antisense nucleic acid.

[0071] According to certain embodiments of the present disclosure, the aforementioned PARP-specific inhibitor is a small molecule compound.

[0072] According to certain embodiments of the present disclosure, the PARP specific inhibitor is selected from the group consisting of Olaparib, Niraparib, Fluzoparib, Veliparib, Rucaparib, Pamiparib, Talazoparib, Saruparib, PARP7-IN-4, PARP7-IN-15, PARP7-IN-16, PARP7-IN-17, UPF 1069, PJ34, Pamiparib, RBN-2397, Dehydrocorydaline, RBN012759, 3-Aminobenzamide, AG14361, PJ34 hydrochloride, Talazoparib tosylate, AZD-2461, Rucaparib monocamsylate, EB-47dihydrochloride, Rucaparib The group consisting of phosphate, Iniparib, Venadaparib, DPQ, Senaparib, BGP-15, and structural analogs thereof.

[0073] In a preferred embodiment, the PARP specific inhibitor is one or more selected from Olaparib, Veliparib, Rucaparib, AZD-2461, UPF 1069 and structural analogs thereof.

[0074] In a preferred embodiment, the aforementioned PARP-specific inhibitor is Olaparib, Veliparib, or a structural analogue thereof.

[0075] According to certain embodiments of the present disclosure, the antibody is a monoclonal antibody or a polyclonal antibody.

[0076] According to certain embodiments of the present disclosure, the antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0077] According to certain embodiments of the present disclosure, the RNAi molecule is siRNA, saRNA, shRNA, dsRNA, or miRNA.

[0078] According to certain embodiments of the present disclosure, the RNAi molecule is a polynucleotide of 15-40 bases in length.

[0079] According to certain embodiments of the present disclosure, the RNAi molecule is modified to enhance its stability.

[0080] According to certain embodiments of the present disclosure, the final concentration of the small molecule compound is greater than 0.001 μM, for example, 0.001 μM-1000 μM, 0.008 μM-750 μM, 0.008 μM-500 μM, 0.008 μM-100 μM, 0.008 μM-50 μM, 0.008 μM-30 μM, 0.008 μM-20 μM, 0.008 μM-10 μM, 0.008 μM-5 μM, 0.01 μM M-250μM, 0.01μM-200μM, 0.05μM-100μM, 0.1μM-100μM, 0.1μM-50μM, 0.1μM-30μM, 0.1μM-20μM, 0.1 μM-10μM, 0.1μM-5μM, 0.15μM-50μM, 0.2μM-100μM, 0.2μM-50μM, 0.2μM-30μM, 0.2μM-20μM, 0.2μM-1 0μM, 0.2μM-5μM, 0.25μM-30μM, 0.4μM-100μM, 0.4μM-50μM, 0.4μM-30μM, 0.4μM-25μM, 0.4μM-20μM, 0.4μM-10μM, 0.4μM-5μM, 0.5-10μM, 0.8μM-20μM, 1μM-100μM, 1μM-50μM, 1μM-30μM, 1μM-20μM, 1μM- 10 μM, 1 μM-5 μM, 1.2 μM-10 μM, 1.8 μM-7.5 μM, 2.5 μM-5 μM, 3.5 μM-4.5 μM, 5 μM-100 μM, 5 μM-50 μM, 5 μM-30 μM, 5 μM-20 μM, 5 μM-10 μM, 10 μM-100 μM, 10 μM-50 μM, 10 μM-30 μM, 10 μM-20 μM, 20 μM-50 μM or 30 μM-50 μM. The final concentration of the aforementioned small molecule compound is 0.008 μM-50 μM or 0.04 μM-50 μM.

[0081] According to certain embodiments of the present disclosure, the final concentration of the small molecule compound is 0.008 μM, 0.04 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 3 μM, 4 μM, 5 μM, 7.5 μM, 10 μM, 20 μM, 25 μM, 35 μM, 50 μM, 100 μM, 200 μM, 250 μM, 500 μM, 750 μM, or 1000 μM.

[0082] According to certain specific embodiments of the present disclosure, the PARP-specific inhibitor is Olaparib. In some preferred embodiments, the final concentration of Olaparib is 0.001 μM-1000 μM, preferably 0.008 μM-50 μM, 0.01 μM-30 μM or 0.04 μM-10 μM, more preferably 0.04 μM-5 μM.

[0083] According to certain specific embodiments of the present disclosure, the PARP-specific inhibitor is Veliparib. In some preferred embodiments, the final concentration of Veliparib is 0.001 μM-1000 μM, preferably 1 μM-100 μM, 5 μM-70 μM or 10 μM-50 μM, more preferably 20 μM-50 μM.

[0084] According to certain specific embodiments of the present disclosure, the PARP-specific inhibitor is AZD-2461. In some preferred embodiments, the final concentration of AZD-2461 is 0.001 μM-1000 μM, preferably 0.005 μM-100 μM, 0.01 μM-50 μM or 0.1 μM-30 μM, more preferably 0.2 μM-10 μM.

[0085] According to certain specific embodiments of the present disclosure, the PARP-specific inhibitor is Rucaparib. In some preferred embodiments, the final concentration of Rucaparib is 0.001 μM-1000 μM, preferably 0.005 μM-50 μM, 0.1 μM-20 μM or 0.5 μM-10 μM, more preferably 1 μM-5 μM.

[0086] According to certain specific embodiments of the present disclosure, the PARP-specific inhibitor is UPF 1069. In some preferred embodiments, the final concentration of UPF 1069 is 0.001 μM-1000 μM, preferably 1 μM-100 μM, 1 μM-50 μM, or 1 μM-30 μM, more preferably 1 μM-10 μM or 10 μM-30 μM. According to certain embodiments of the present disclosure, the PARP-specific inhibitor is added to the cells 24, 18, 12, 6, 4, 2, 1, or 0.5 hours before transfection or 0.5, 1, 2, 4, 6, 12, 18, 24, 28, or 48 hours after transfection.

[0087] In one embodiment, the one or more PARP-specific inhibitors are added to the cells within 24 hours before transfection to 48 hours after transfection, within 18 hours before transfection to 24 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 0.5 hour before transfection to 0.5 hour after transfection, within 2 hours before transfection to 4 hours after transfection, within 4 hours before transfection to 4 hours after transfection, or within 0.5 hour before transfection to 24 hours after transfection. Preferably, the one or more PARP-specific inhibitors are added to the cells within 0.5 hour before transfection to 24 hours after transfection.

[0088] According to certain embodiments of the present disclosure, 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.

[0089] According to certain 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] Another aspect of the present disclosure provides a method for producing a virus, comprising the step of culturing cells for producing the virus in the presence of a PARP-specific inhibitor.

[0094] According to certain embodiments of the present disclosure, the virus is selected from adeno-associated virus or recombinant adeno-associated virus (rAAV).

[0095] According to certain embodiments of the present disclosure, the PARP-specific inhibitor is an antibody, a small molecule compound, an RNAi molecule, an sgRNA, or an antisense nucleic acid.

[0096] According to certain embodiments of the present disclosure, the PARP-specific inhibitor is a small molecule compound.

[0097] According to certain embodiments of the present disclosure, the PARP specific inhibitor is selected from the group consisting of Olaparib, Niraparib, Fluzoparib, Veliparib, Rucaparib, Pamiparib, Talazoparib, Saruparib, PARP7-IN-4, PARP7-IN-15, PARP7-IN-16, PARP7-IN-17, UPF 1069, PJ34, Pamiparib, RBN-2397, Dehydrocorydaline, RBN012759, 3-Aminobenzamide, AG14361, PJ34 hydrochloride, Talazoparib tosylate, AZD-2461, Rucaparib monocamsylate, EB-47dihydrochloride, Rucaparib The group consisting of phosphate, Iniparib, Venadaparib, DPQ, Senaparib, BGP-15, and structural analogs thereof.

[0098] In a preferred embodiment, the PARP specific inhibitor is one or more selected from Olaparib, Veliparib, Rucaparib, AZD-2461, UPF 1069 and structural analogs thereof.

[0099] In a preferred embodiment, the PARP-specific inhibitor is Olaparib, Veliparib or a structural analogue thereof.

[0100] According to certain embodiments of the present disclosure, the antibody is a monoclonal antibody or a polyclonal antibody.

[0101] According to certain embodiments of the present disclosure, the antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0102] According to certain embodiments of the present disclosure, the RNAi molecule is siRNA, saRNA, shRNA, dsRNA, or miRNA.

[0103] According to certain embodiments of the present disclosure, the RNAi molecule is a polynucleotide of 15-40 bases in length.

[0104] According to certain embodiments of the present disclosure, the RNAi molecule is modified to enhance its stability.

[0105] According to certain embodiments of the present disclosure, the final concentration of the small molecule compound is greater than 0.001 μM, for example, 0.001 μM-1000 μM, 0.008 μM-750 μM, 0.008 μM-500 μM, 0.008 μM-100 μM, 0.008 μM-50 μM, 0.008 μM-30 μM, 0.008 μM-20 μM, 0.008 μM-10 μM, 0.008 μM-5 μM ,0.01μM-250μM, 0.01μM-200μM, 0.05μM-100μM, 0.1μM-100μM, 0.1μM-50μM, 0.1μM-30μM, 0.1μM -20μM, 0.1μM-10μM, 0.1μM-5μM, 0.15μM-50μM, 0.2μM-30μM, 0.2μM-20μM, 0.2μM-10μM, 0.2μM-5μ M, 0.25μM-30μM, 0.4μM-100μM, 0.4μM-50μM, 0.4μM-30μM, 0.4μM-25μM, 0.4μM-20μM, 0.4μM-10μ M, 0.4μM-5μM, 0.5-10μM, 0.8μM-20μM, 1μM-100μM, 1μM-50μM, 1μM-30μM, 1μM-20μM, 1μM-10μM, 1 μM-5μM, 1.2μM-10μM, 1.8μM-7.5μM, 2.5μM-5μM, 3.5μM-4.5μM, 5μM-100μM, 5μM-50μM, 5μM-30μM , 5μM-20μM, 5μM-10μM, 10μM-100μM, 10μM-50μM, 10μM-30μM, 10μM-20μM, 20μM-50μM or 30μM-50μM. The final concentration of the aforementioned small molecule compound is 0.008 μM-50 μM or 0.04 μM-50 μM.

[0106] According to certain embodiments of the present disclosure, the final concentration of the small molecule compound is 0.008 μM, 0.04 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 3 μM, 4 μM, 5 μM, 7.5 μM, 10 μM, 20 μM, 25 μM, 35 μM, 50 μM, 100 μM, 200 μM, 250 μM, 500 μM, 750 μM, or 1000 μM.

[0107] According to certain specific embodiments of the present disclosure, the PARP-specific inhibitor is Olaparib. In some preferred embodiments, the final concentration of Olaparib is 0.001 μM-1000 μM, preferably 0.008 μM-50 μM, 0.01 μM-30 μM or 0.04 μM-10 μM, more preferably 0.04 μM-5 μM.

[0108] According to certain specific embodiments of the present disclosure, the PARP-specific inhibitor is Veliparib. In some preferred embodiments, the final concentration of Veliparib is 0.001 μM-1000 μM, preferably 1 μM-100 μM, 5 μM-70 μM or 10 μM-50 μM, more preferably 20 μM-50 μM.

[0109] According to certain specific embodiments of the present disclosure, the PARP-specific inhibitor is AZD-2461. In some preferred embodiments, the final concentration of AZD-2461 is 0.001 μM-1000 μM, preferably 0.005 μM-100 μM, 0.01 μM-50 μM or 0.1 μM-30 μM, more preferably 0.2 μM-10 μM.

[0110] According to certain specific embodiments of the present disclosure, the PARP-specific inhibitor is Rucaparib. In some preferred embodiments, the final concentration of Rucaparib is 0.001 μM-1000 μM, preferably 0.005 μM-50 μM, 0.1 μM-20 μM or 0.5 μM-10 μM, more preferably 1 μM-5 μM.

[0111] According to certain specific embodiments of the present disclosure, the PARP-specific inhibitor is UPF 1069. In some preferred embodiments, the final concentration of UPF 1069 is 0.001 μM-1000 μM, preferably 1 μM-100 μM, 1 μM-50 μM, or 1 μM-30 μM, more preferably 1 μM-10 μM or 10 μM-30 μM. According to certain embodiments of the present disclosure, the PARP-specific inhibitor is added to the cells within 0.5 hours before transfection to 24 hours after transfection.

[0112] According to certain embodiments of the present disclosure, in some embodiments, 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] The genes required for packaging AAV viruses can 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 an auxiliary gene. Optionally, the nucleic acid template can 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.

[0119] According to the present disclosure, the virus is obtained by introducing genes including but not limited to genes required for packaging related AAV viruses into production cells through plasmid transfection or viral infection, or a combination of the two methods.

[0120] According to the present disclosure, the virus is prepared by a plasmid system for producing AAV virus. A "plasmid system" includes a plasmid of AAV production-related genes and can be used to produce AAV without the need for additional plasmids. The present disclosure can be carried out using various plasmid systems for AAV production, which can express the gene of interest as needed. The plasmid system can provide AAV rep sequences, cap sequences, auxiliary gene coding sequences and target gene coding sequences by any method known in the art. As needed, the AAV rep sequence, cap sequence (encoding VP1, VP2 and VP3), auxiliary gene coding sequences and target gene coding sequences can be expressed on a single or multiple plasmids, thereby forming a three-plasmid system, a two-plasmid system or a single-plasmid system.

[0121] 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).

[0122] In the present disclosure, the cells used to prepare the AAV plasmid system can be any cells that allow the AAV virus to replicate. In some embodiments, the cells are mammalian cells. In some embodiments, the cells can be trans-complementing packaging cell lines that provide functions missing from the replication-defective helper virus, such as 293 cells or other Ela trans-complementing cells. In some embodiments, the cells are HEK293 cells.

[0123] 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.

[0124] 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.

[0125] 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. Preferably, the vector described herein is selected from an adenoviral vector, an adeno-associated viral vector, or a recombinant adeno-associated viral vector.

[0126] As used herein, the term "adeno-associated virus" or "AAV" 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 exhibit remarkably similar replication characteristics mediated by homologous rep genes; and all carry three related capsid proteins. Non-limiting exemplary serotypes for use in the methods disclosed herein include naturally occurring serotypes known in the art and variants thereof, such as AAV2, AAV8, AAV9, or variant serotypes such as AAV-DJ and AAV PHP.B, among others. AAV particles comprise, consist essentially of, or consist of the three major viral proteins VP1, VP2, and VP3.

[0127] As used herein, the term "PARP-specific inhibitor" refers to any substance that can specifically reduce, decrease, or eliminate the transcription or translation of the PARP gene, and / or the activity of the PARP protein. In some embodiments, the PARP-specific inhibitor can reduce the activity of PARP by at least 5%, 10%, 20%, 40%, 50%, 80%, 90%, 95%, or more. As used herein, "activity," when used in conjunction with increase or decrease, refers to a detected functional activity, which can be manifested as a change in content, or a change in functional activity while the content remains unchanged.

[0128] As used herein, the term "PARP" refers to a family of enzymes that are widely present in eukaryotic cells and are primarily involved in key biological processes such as DNA damage repair, genome stability, and cellular stress response. PARP enzymes that can repair DNA are discoveries of modern genetics. PARP is a cleavage substrate for caspase, a core member of apoptosis. As used herein, PARP can be, for example, PARP1, PARP2, PARP3, PARP4, and / or PARP5. When "PARP activity" is used, it refers to the activity of the enzyme, such as the activity of a DNA repair enzyme.

[0129] In some embodiments, the PARP specific inhibitor inhibits PARP at least 1-fold, 2-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 10,000-fold more potently than other targets. For example, in some embodiments, the PARP specific inhibitor has an IC 50 Greater than or equal to 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 80 μM, 100 μM, 150 μM, 200 μM or 500 μM.

[0130] In some embodiments, the specific inhibitor inhibits PARP at least 1-fold, 2-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 10,000-fold more potently than it inhibits other target genes.

[0131] In some embodiments, the PARP-specific inhibitor has an IC of 50 Less than or equal to 100 μM, 80 μM, 50 μM, 30 μM, 20 μM, 10 μM, 5 μM, 3 μM, 2 μM, 1 μM, 0.5 μM, 0.2 μM, 0.1 μM, 0.05 μM, 0.02 μM, 0.01 μM, 0.005 μM, 0.002 μM or 0.001 μM. In some embodiments, the PARP-specific inhibitor is an antibody, a small molecule compound, an RNAi molecule, an sgRNA or an antisense nucleic acid.

[0132] In some embodiments, the PARP-specific inhibitor is an antibody.

[0133] The term "antibody" as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody or antibody that can bind to a specific antigen. The term "antibody" herein is intended to broadly encompass conventional four-chain antibodies as well as less conventional antibodies that do not have four chains (e.g., antibodies that naturally lack light chains).

[0134] A typical intact antibody is a heterotetramer composed of two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, each consisting of a variable region (VH) and the first, second, and third constant regions (CH1, CH2, and CH3, respectively). Mammalian light chains are classified as λ or κ, each consisting of a variable region (VL) and a constant region. Conventional antibodies are Y-shaped. The neck of the Y is composed of the second and third constant regions of two heavy chains, which are linked by disulfide bonds. Each arm of the Y contains the variable region and first constant region of one heavy chain, which are bound to the variable region and constant region of one light chain. The variable regions of the light and heavy chains determine antigen binding. The variable region of each chain contains three hypervariable regions, called complementarity-determining regions (CDRs). The light chain CDRs consist of LCDR1, LCDR2, and LCDR3, and the heavy chain CDRs consist of HCDR1, HCDR2, and HCDR3. The three CDRs are separated by flanking continuous segments called framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but have various effector functions.

[0135] In some embodiments of the invention, the antibody is a full-length antibody or an antigen-binding fragment.

[0136] As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed by an antibody portion containing one or more CDRs but without the complete antibody structure. Examples of antigen-binding fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, single-chain antibody molecules (scFv), scFv dimers, camelized single domain antibodies, and nanobodies. Antigen-binding fragments can bind to the same antigen as the parent antibody.

[0137] The "Fab" fragment of an antibody refers to the portion of the antibody composed of a light chain (including the variable and constant regions) and the variable region and first constant region of a heavy chain bound together by disulfide bonds.

[0138] The "Fab'" fragment refers to the Fab fragment including part of the hinge region.

[0139] "F(ab')2" fragment refers to a dimer of Fab'.

[0140] The "Fv" fragment of an antibody consists of the variable region of one light chain and the variable region of one heavy chain.

[0141] "Single-chain antibody molecule" or "scFv" refers to an engineered antibody in which the light chain variable region is directly linked to the heavy chain variable region or connected through a peptide chain. For detailed description, see, for example, Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988).

[0142] "scFv dimer" refers to an aggregate formed by two scFvs.

[0143] Camelized single-domain antibodies (also known as heavy-chain antibodies or HCAbs) are antibodies that contain two heavy-chain variable regions but no light chains. Heavy-chain antibodies were originally derived from camelids (camels, dromedaries, and llamas). Despite lacking light chains, camelized antibodies retain full antigen-binding function.

[0144] "Nanobodies" are composed of a heavy chain variable region and two constant regions CH2 and CH3 from a heavy chain antibody.

[0145] In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody.

[0146] In some embodiments, the antibody is a murine antibody, a rabbit antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0147] As used herein, the term "fully human" when applied to an antibody or antigen-binding fragment means that the amino acid sequence of the antibody or antigen-binding fragment corresponds to the amino acid sequence of an antibody produced by a human or human immune cell, or derived from a non-human source, such as a transgenic non-human animal utilizing a human antibody library, or other sequences encoding a human antibody.

[0148] As used herein, the term "humanized" when used for antibodies or antigen-binding fragments refers to antibodies or antigen-binding fragments that include CDRs derived from non-human animals, FR regions derived from humans, and constant regions (when applicable) derived from humans. Because humanized antibodies or antigen-binding fragments have lower immunogenicity, they can be used as therapeutic agents for humans in certain embodiments. In certain embodiments, the non-human animal is a mammal (e.g., mouse, rat, rabbit, goat, sheep, guinea pig, or hamster). In certain embodiments, the humanized antibody or antigen-binding fragment, except that the CDR sequences are non-human, is substantially entirely composed of human sequences.

[0149] As used herein, the term "chimeric" when applied to an antibody or antigen-binding fragment refers to an antibody or antigen-binding fragment that has a portion of a heavy chain and / or light chain derived from one species, while the remainder of the heavy chain and / or light chain is derived from a different species. In some embodiments, a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human animal (e.g., a mouse or rabbit).

[0150] In some embodiments, the antibodies described herein are monospecific antibodies, bispecific antibodies, or multispecific antibodies.

[0151] In some embodiments, the antibodies described herein can be further labeled.

[0152] In some embodiments, the PARP-specific inhibitor is an RNAi molecule.

[0153] As used herein, the term "RNAi molecule" refers to RNA or its analogs that have sufficient sequence complementarity with a target RNA to direct RNA interference. In some embodiments, DNA that can be used to generate RNA is also included. RNA interference (RNAi) refers to a sequence-specific selective process by which a target molecule (e.g., a target gene, protein, or RNA) is downregulated.

[0154] In some embodiments, the RNAi molecule is capable of reducing the expression of PARP, eg, knocking down the PARP gene.

[0155] In some embodiments, the RNAi molecule is 15-100 bases in length, preferably 15-40 bases in length.

[0156] In some embodiments, the RNAi molecule is modified to enhance its stability.

[0157] In some embodiments, the RNAi molecule is a small interfering RNA (siRNA), a guide RNA (sgRNA), a short hairpin RNA (shRNA), or a micro RNA (miRNA).

[0158] The term "small interfering RNA (siRNA)" as used herein refers to an RNA molecule, preferably a double-stranded molecule, having a length of about 10 to 50 nucleotides, preferably a length of about 15 to 25 nucleotides, more preferably a length of about 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides, the strand optionally having an overhanging end comprising, for example, 1, 2 or 3 overhanging nucleotides (or nucleotide analogs), which is capable of directing or mediating the degradation of the RNA.

[0159] The term "short hairpin RNA (shRNA)" as used herein refers to an RNA molecule having a stem-loop structure, which includes a first region and a second region of complementary sequence, the degree of complementarity and the orientation of the regions are sufficient to allow base pairs to occur between the regions, the first region and the second region are connected by the loop region, and the loop is generated by the lack of base pairing between nucleotides (or nucleotide analogs) in the loop region.

[0160] The term "microRNA (microRNA or miRNA)" as used herein is a short, naturally occurring, non-coding single-stranded RNA molecule of about 16-26 nucleotides (nt) in length (e.g., about 16-29nt, 19-22nt, 20-25nt, or 21-23nt), which is typically involved in regulating gene expression in vivo. In eukaryotic cells, miRNA genes are transcribed into "primary products" (pri-miRNA) by DNA transcriptase II. Pri-miRNAs are quickly processed into miRNA "precursors" (pre-miRNAs) by a ribonuclease III (Drosha). Pre-miRNAs are transported from the nucleus to the cytoplasm and then recognized and sheared into mature miRNAs by another ribonuclease III (Dicer). Mature miRNA molecules are partially complementary to one or more mRNAs and regulate protein expression. The sequences of known miRNAs can be obtained from public databases, such as the miRBase database (www.mirbase.org), which provides information including miRNA sequence information, functional annotations, and predicted gene targets. In the present invention, miRNA also includes RNA molecules expressed in cells by artificially synthesized plasmids and having structures and functions similar to natural miRNAs, which can target corresponding mRNAs like natural miRNAs and hinder their translation into proteins.

[0161] In some embodiments, the PARP-specific inhibitor is an antisense nucleic acid.

[0162] As used herein, the term "antisense nucleic acid" includes nucleotides that are completely complementary to a target sequence, as well as those that have one or more nucleotide mismatches, so long as the antisense nucleic acid can specifically hybridize to the target sequence. For example, antisense nucleic acids herein include polynucleotides that have at least 70% or greater, preferably 80% or greater, more preferably 90% or greater, and even more preferably 95% or greater homology over a length of at least 15 consecutive nucleotides. Due to the formation of the hybrid, transcription of the target gene and / or translation of the target mRNA is reduced or blocked.

[0163] In some embodiments, the PARP-specific inhibitor is a small molecule compound.

[0164] As used herein, the term "small molecule compound" refers to an organic compound having a molecular weight of less than 3000, 2500, 2000, 1500, 1000 or 500 Daltons, which may be natural or chemically synthesized.

[0165] Example

[0166] Reagents, models, instruments and experimental methods

[0167] I. Reagents and Models Used

[0168] 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.

[0169] All reagents and compounds synthesized can be purchased through general commercial channels in China (excluding Hong Kong, Macao and Taiwan). Please see Table 1 below for specific information.

[0170] Table 1. Reagent information

[0171] II. Instruments used

[0172] Table 2. Instrument information

[0173] Table 3. Primer and probe sequences

[0174] III. Experimental Methods

[0175] 3.1 Cell culture

[0176] Remove the suspended HEK293 cell line cryovial from the liquid nitrogen tank and quickly transfer it to a 37°C water bath. Gently shake for 2-3 minutes until the frozen cell suspension thaws. Transfer the cell suspension in the cryovial to a 125mL cell culture flask in a biosafety cabinet, mix with 30mL of culture medium (preheated to 37°C), and then culture in a CO2 shaking incubator (ZCZY-CS9) at 37°C, 8% (v / v) CO2, 135 rpm, and 80% humidity. Subculture every three days at a density of 0.3 to 0.6E+06 cells / mL. After five consecutive subcultures, the cells can be used for virus packaging experiments.

[0177] 3.2 Drug preparation

[0178] To a reagent bottle containing 5 mg of Olaparib (MCE, HY-10162) powder, add 1.15 mL of dimethyl sulfoxide (DMSO) until completely dissolved to obtain a 10 mM Olaparib stock solution. Then dilute to 0.008 mM, 0.04 mM, 0.2 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, and 5 mM working solutions for later use.

[0179] To a reagent bottle containing 5 mg of Veliparib (MCE, HY-10129) powder, 0.41 mL of DMSO was added until completely dissolved to obtain a 50 mM Veliparib stock solution for use; to a reagent bottle containing 5 mg of AZD-2461 (MCE, HY-13536) powder, 1.26 mL of DMSO was added until completely dissolved to obtain a 10 mM AZD-2461 stock solution for use; to a reagent bottle containing 5 mg of Rucaparib (MCE, HY-10617A) powder, 1.55 mL of DMSO was added until completely dissolved to obtain a 10 mM Rucaparib stock solution for use; to a reagent bottle containing 5 mg of UPF 1069 (MCE, HY-14478) powder, 1.79 mL of DMSO was added until completely dissolved to obtain a 10 mM UPF 1069 stock solution for use;

[0180] 3.3 Cell Preparation

[0181] 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, and cultured in a CO2 shaking incubator (ZCZY-CS9) under the conditions of 37°C, 8% (v / v) CO2, 135 rpm, and 80% humidity.

[0182] 3.4 Cell transfection

[0183] After culturing the cells for 24 hours, recount the cells and adjust the cell density to approximately 3.3E+06 cells / mL with fresh culture medium. Ensure that the cell viability is greater than 95%. Inoculate 27 mL of the cell suspension into a 125 mL cell culture flask. Add Helper, RepCap, and the GOI plasmid (Table 1) and PEI to the culture medium and mix thoroughly to create a transfection complex. Let the mixture stand for 10-20 minutes, then transfer the entire suspension to a cell culture flask. After transfection, continue culturing the cells in a CO2 shaking incubator.

[0184] 3.5 Addition of small molecule compounds

[0185] Dilute the stock solutions of different compounds to the corresponding working solutions according to the drug concentration. Add the Olaparib (or other small molecule) working solution to the cell culture flask at a ratio of 1:1000 (drug volume: culture system) within 4 hours after transfection and mix thoroughly.

[0186] 3.6 Cell Harvest

[0187] 72 hours after transfection, 10 mL of cell suspension (mixed before sampling) was transferred to a 15 mL centrifuge tube, and 20% (v / v) Tween 20 was added to a final concentration of 0.2-1% (v / v). The tube was mixed by inversion and lysed at room temperature, and allowed to stand for 10-20 minutes. 1 M MgCl2 was then added to a final concentration of 1-2 mM, and the tube was mixed by inversion and allowed to stand for 5-10 minutes. SuperNuclease (Sino Biological) was added to a final concentration of 10-50 U / mL and mixed. The tube was then incubated in a CO2 shaking incubator at 37°C for 2-3 hours. 5 M NaCl was then added to a final concentration of 200-400 mM and mixed. After incubation at room temperature for 10 minutes, the tube was centrifuged at 4000 rpm for 10-20 minutes. The supernatant was transferred to a centrifuge tube and stored at 4°C or -20°C.

[0188] 3.7 ddPCR detection

[0189] After DNase I treatment, the sample supernatant was 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 generate droplets. The droplets were then transferred to a 96-well PCR reaction plate, sealed, and 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.

[0190] Table 4. PCR amplification program in ddPCR

[0191] 3.8 ELISA

[0192] 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 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. The plate was incubated at room temperature in the dark for 15 minutes. 50 μL of stop solution was added to each well. The absorbance (OD) at a wavelength of 450 nm was measured using a microplate reader, and the viral capsid titer in the sample was calculated.

[0193] Example 1: Effects of different concentrations of Olaparib on rAAV9 yield and quality in HEK293 suspension cells

[0194] Cell culture was performed according to Experimental Method 3.1, drug preparation was performed according to Experimental Method 3.2, cell preparation was performed according to Experimental Method 3.3, and cells were transfected with the rAAV9 packaging plasmid according to Experimental Method 3.4. Within 4 hours after cell transfection, olaparib working solution was added to each culture flask at a ratio of 1:1000 to achieve final concentrations of 0 μM, 0.008 μM, 0.04 μM, 0.2 μM, 1 μM, 5 μM, and 10 μM (Experiment 1), and 0 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 3 μM, and 5 μM (Experiment 2).

[0195] 72 hours after transfection, cells were harvested and lysed according to Experimental Method 3.6. The supernatant was collected and assayed by ddPCR and ELISA according to Experimental Methods 3.7 and 3.8, respectively, to quantify the rAAV9 genomic DNA and capsid protein in the supernatant.

[0196] The culture flask with DMSO added was the blank control group, and the culture flask with Olaparib added was the experimental group. The results showed that the addition of different concentrations of Olaparib significantly increased the titer of rAAV9, among which 0.5μM Olaparib had the most significant improvement effect (Figure 1). At the same time, Olaparib significantly reduced the empty shell rate E:F (Empty:Full, also known as the empty-full shell ratio, empty shell rate, etc., the lower the E:F value, the less empty capsid protein content in the virus product, and conversely, the higher the E:F value, the more empty capsid protein content in the virus) of rAAV9, among which 0.5μM Olaparib had the most significant effect (Figure 2). It is proved that the addition of Olaparib can effectively increase the yield of rAAV9 in the HEK293 suspension cell production system and reduce the empty shell rate, thereby improving the quality of rAAV9.

[0197] Referring to the experimental methods of this example, when preparing rAAV9 using a two-plasmid or single-plasmid system, adding different concentrations of olaparib can achieve the same results as the three-plasmid viral packaging system. Olaparib can increase rAAV9 titer and reduce empty shell fraction, improving rAAV9 virus quality and yield.

[0198] Example 2: Effect of Olaparib Addition Time on rAAV9 Yield in HEK293 Suspension Cells

[0199] Perform cell culture according to Protocol 3.1, drug preparation according to Protocol 3.2, and cell preparation according to Protocol 3.3. Transfect cells with the rAAV9 packaging plasmid according to Protocol 3.4. Add olaparib working solution at a 1:1000 dilution to each culture flask at 0.5 h before, 0 h after, 2 h after, 4 h after, and 24 h after transfection to a final concentration of 0.5 μM.

[0200] 72 hours after transfection, complete cell lysis according to experimental method 3.6, remove the supernatant and perform ddPCR detection according to experimental method 3.7 to quantify the rAAV9 genomic DNA in the supernatant.

[0201] The results showed that the addition of Olaparib from 0.5 hours before transfection to 24 hours after transfection significantly increased the titer of rAAV9, with no significant difference in the effect (Figure 3). This indicates that the addition of Olaparib from 0.5 hours before transfection to 24 hours after transfection significantly increased the yield of rAAV9.

[0202] Referring to the experimental method of this example, when preparing rAAV9 virus using a two-plasmid or single-plasmid system, adding olaparib at different times can also achieve the same effect as the three-plasmid viral packaging system. Olaparib can significantly increase the titer of rAAV9 and increase the yield of rAAV9.

[0203] Example 3: Effect of Olaparib on rAAV9 Yield and Quality in Different Cell Lines

[0204] Recovery VPC 1.0, Wayne LVPro TM HEK293 and VPC2.0 cells were cultured to the appropriate passages. The three consecutively cultured cells were counted and diluted to a density of 1.0 to 2.0E+06 cells / mL by adding fresh culture medium (preheated at 37°C) according to the count results. The volume of each cell was 100 mL, and the viable cell ratio was ensured to be greater than 95%.

[0205] Cells were transfected with the rAAV9 packaging plasmid according to Experimental Method 3.4. Within 4 hours after transfection, DMSO and 0.5 mM Olaparib working solution were added to the culture flask at a 1:1000 ratio to reach final concentrations of 0 μM and 0.5 μM, respectively. (A control group and a 0.5 μM Olaparib group were set up for each cell line.)

[0206] 72 hours after transfection, cells were harvested and lysed according to Experimental Method 3.6. The supernatant was collected and assayed by ddPCR and ELISA according to Experimental Methods 3.7 and 3.8, respectively, to quantify the rAAV9 genomic DNA and capsid protein in the supernatant.

[0207] The results showed that compared with the control group, VPC1.0, VPC2.0, WayneLVPro TM Adding 0.5 μM Olaparib to HEK293 cells increased rAAV9 titers by 243%, 198%, and 196%, respectively, while significantly decreasing the empty shell fraction (Figure 4). This suggests that Olaparib's effect on improving rAAV9 yield and quality is applicable to a variety of cell lines.

[0208] Referring to the experimental methods described in this example, olaparib can achieve the same results as a three-plasmid viral packaging system when using a two-plasmid or single-plasmid system to prepare rAAV9 in different cell lines. Olaparib effectively increases rAAV9 titer and reduces empty shell fraction in various cell lines, improving rAAV9 quality and yield.

[0209] Example 4: Effect of Olaparib on the yield of rAAV of different serotypes in HEK293 suspension cells

[0210] Perform cell culture according to Protocol 3.1, drug preparation according to Protocol 3.2, and cell preparation according to Protocol 3.3. Transfect cells with rAAV2, rAAV5, rAAV6, rAAV8, rAAV9, or rAAV9 variant packaging plasmids according to Protocol 3.4. Within 4 hours of transfection, add DMSO and a 0.5 mM working solution of olaparib to the culture medium at a 1:1000 ratio to final concentrations of 0 μM and 0.5 μM, respectively.

[0211] 72 hours after transfection, cells were lysed according to Experimental Method 3.6, and the supernatant was collected and subjected to ddPCR detection according to Experimental Method 3.7 to quantify rAAV genomic DNA.

[0212] In HEK293 suspension cells, rAAV yields varied across serotypes, but the addition of 0.5 μM Olaparib significantly increased the titers of rAAV2, rAAV5, rAAV6, rAAV8, rAAV9, and rAAV9 variants compared to the control group (Figure 5). This suggests that Olaparib can increase the yields of rAAV2, rAAV5, rAAV6, rAAV8, rAAV9, and rAAV9 variants, and that the rAAV yield-enhancing effect of Olaparib is applicable to multiple serotypes and their variants.

[0213] Referring to the experimental methods of this example, when using a two-plasmid or single-plasmid system to prepare rAAV2, rAAV5, rAAV6, rAAV8, rAAV9, or rAAV9 variant viruses, the use of olaparib can also achieve the same results as a three-plasmid viral packaging system. Olaparib can effectively increase the yield of different rAAV serotypes and their variants.

[0214] Example 5: Effects of specific PARP inhibitors on rAAV9 yield and quality in HEK293 suspension cells

[0215] As non-limiting embodiments, the experiments in this example were carried out using Veliparib, AZD-2461, Rucaparib, UPF 1069 and Olaparib, respectively.

[0216] Cell culture was performed according to Experimental Method 3.1, drug preparation was performed according to Experimental Method 3.2, cell preparation was performed according to Experimental Method 3.3, and cells were transfected with the rAAV9 packaging plasmid according to Experimental Method 3.4. Within 4 hours after transfection, DMSO, 20mM veliparib, 0.2mM AZD-2461, 1mM Rucaparib, 10mM UPF 1069, and 0.5mM Olaparib working solutions were added to each culture flask at a 1:1000 ratio, resulting in final concentrations of 0μM, 20μM veliparib, 0.2μM AZD-2461, 1μM Rucaparib, 10μM UPF 1069, and 0.5μM Olaparib, respectively.

[0217] 72 hours after transfection, cells were lysed according to the experimental method 3.6, and the supernatant was collected for ddPCR and ELISA detection to quantify the rAAV9 genomic DNA and capsid protein in the supernatant.

[0218] The culture flasks with DMSO added served as the blank control group, and the culture flasks with final concentrations of 20 μM Veliparib, 0.2 μM AZD-2461, 1 μM Rucaparib, 10 μM UPF 1069, and 0.5 μM Olaparib served as the experimental group. The results showed that the addition of different specific PARP inhibitors significantly increased the titer of rAAV9, with 20 μM Veliparib, 0.2 μM AZD-2461, 1 μM Rucaparib, 10 μM UPF 1069, and 0.5 μM Olaparib increasing rAAV9 production by 288%, 227%, 250%, 148%, and 265%, respectively (Figure 6), while reducing the virus empty shell rate (Figure 7). This demonstrates that specific PARP inhibitors can significantly improve the yield and quality of rAAV9.

[0219] Referring to the experimental methods of this example, when using a two-plasmid or single-plasmid system to produce rAAV, the use of specific PARP inhibitors can also achieve the same effect as the three-plasmid viral packaging system. Specific PARP inhibitors can effectively improve the yield and quality of rAAV in HEK293 suspension cells.

[0220] Example 6: Effect of siRNA Knockdown of PARP on rAAV9 Yield and Quality in HEK293 Suspension Cells

[0221] Three siRNAs specific for PARP mRNA were designed and synthesized (SEQ ID NOs: 4-6).

[0222] HEK293 (VPC1.0) cells were cultured according to Experimental Method 3.1. After reaching a packaging-ready passage, cells were seeded at a density of 0.4–0.5E+06 cells / mL on the day of passage. The first siRNA transfection was performed 24 hours after seeding. siRNA#2 (SEQ ID NO: 5) and Lipofectamine 3000 were added to the culture medium and mixed thoroughly to obtain a transfection complex. The complex was allowed to stand for 10–20 minutes before being transferred to a cell culture flask. Following transfection, the cells were placed in a CO2 shaking incubator and continued to be cultured. A second siRNA transfection was performed 24 hours later, following the same procedures as the first. Cell counts were performed 72 hours after seeding, and the cell density was adjusted to 2.0–2.5E+06 cells / mL based on the counts. Flasks transfected with control siRNA (siCON (SEQ ID NO: 7)) served as the control group, while flasks transfected with siRNA targeting the PARP gene (siRNA#2 (SEQ ID NO: 5)) served as the experimental group.

[0223] Cells were transfected with rAAV9 plasmid according to experimental method 3.4. 72 hours after transfection, cells were lysed according to experimental method 3.6. The supernatant was collected and tested by ddPCR and ELISA according to experimental methods 3.7 and 3.8, respectively, to quantify the rAAV9 genomic DNA and capsid protein in the supernatant.

[0224] The results are shown in Figure 8. The specific knockdown of PARP using siRNA increased the rAAV9 virus yield in suspended HEK293 cells by about 1.3 times; the specific knockdown of PARP using siRNA significantly reduced the empty shell rate of rAAV9 virus in suspended HEK293 cells.

[0225] Referring to the experimental method of this embodiment, when using a two-plasmid or single-plasmid system to produce rAAV, the same effect as the three-plasmid virus packaging system can be obtained.

[0226] Example 7: Effect of siRNA knockdown of PARP on rAAV9 virus yield and quality in adherent HEK293T cells

[0227] Three siRNAs specific to PARP mRNA were designed and synthesized (SEQ ID NO: 4-6).

[0228] After recovery, HEK293T adherent cells were cultured continuously at 37° C. and 5% (v / v) CO 2 for virus packaging after the fifth passage.

[0229] HEK293T adherent cells were plated at 1E+5 cells / mL in continuous culture. After the cells reached an appropriate density, the first siRNA transfection was performed. siRNA#1, siRNA#2, and siRNA#3 (corresponding to SEQ ID NOs: 4-6, respectively) and Lipofectamine 3000 were added to the culture medium and mixed thoroughly to obtain a transfection complex. The complex was allowed to stand for 10-20 minutes and then transferred to the cell suspension. After transfection, the cells were placed in a CO2 incubator and continued to be cultured. 24 hours later, the second siRNA transfection was performed using the same procedure as the first. After 48 hours, the cell suspension was replaced, and 2 mL of the cell suspension was collected and centrifuged at 1400 rpm for 5 minutes. The supernatant was discarded and the cells were lysed. The expression levels of PARP and GAPDH proteins in the cells were analyzed by Western Blot to verify the knockdown effect of the siRNA. 72 hours later, the cells were transfected with rAAV9 plasmid according to Experimental Method 3.4. After 144 hours, cells were lysed according to Experimental Method 3.6. The supernatant was collected and subjected to ddPCR and ELISA assays according to Experimental Methods 3.7 and 3.8, respectively, to quantify the rAAV9 genomic DNA and capsid protein in the supernatant. The wells transfected with control siRNA (siCON (SEQ ID NO: 7)) served as the control group, and the wells transfected with siRNA targeting the PARP gene (siPARP) served as the experimental group. The comparison of PARP knockdown effects is shown in Figure 9, and the comparison of PARP protein expression levels is shown in Figure 10.

[0230] Table 5. siRNA sequences

[0231] Table 6. Titers of rAAV9 in HEK293 adherent cells after PARP knockdown by different siRNAs

[0232] The experimental results are shown in Table 6 and Figure 11. The use of siRNA to specifically knock down PARP increased the rAAV9 virus yield in adherent HEK293T cells by 1.6 to 2.2 times; at the same time, the use of siRNA to specifically knock down PARP slightly reduced the rAAV9 virus empty shell rate in adherent HEK293T cells.

[0233] Referring to the experimental method of this embodiment, when using a two-plasmid or single-plasmid system to produce rAAV, the same effect as the three-plasmid virus packaging system can be obtained.

[0234] In addition, the inventors also studied whether knocking down PARP in Hela cells affects the yield and quality of their rAAV9 virus. The results showed a similar trend to that in HEK293 suspension cells and HEK293T adherent cells. That is, whether in HEK293, Hela or other cells used for rAAV production, using siRNA to specifically knock down PARP can increase the yield of rAAV virus.

[0235] Example 8: Effect of Olaparib on rAAV9 Yield and Quality in HEK293T Adherent Cells

[0236] After recovery, HEK293T adherent cells were cultured continuously at 37° C. and 5% (v / v) CO 2 for virus packaging after the fifth passage.

[0237] 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 and 0.5mM Olaparib working solution were added to the cell suspension at a 1:1000 ratio, respectively, to achieve final concentrations of 0μM and 0.5μM Olaparib. 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.

[0238] The experimental results are shown in Table 7 and Figure 12. Olaparib increased the rAAV9 virus yield in adherent HEK293T cells by approximately 2.8 times; at the same time, Olaparib significantly reduced the rAAV9 virus empty shell rate in adherent HEK293T cells.

[0239] Referring to the experimental method of this example, when using a two-plasmid or single-plasmid system to produce rAAV, the use of Olaparib can also achieve the same effect as the three-plasmid viral packaging system. Olaparib can effectively improve the yield and quality of rAAV in HEK293T cells.

[0240] Table 7. Titers of rAAV9 in HEK293T adherent cells after addition of Olaparib

[0241] Example 9: Effect of specific PARP inhibitors on rAAV9 production in HEK293T adherent cells

[0242] As non-limiting embodiments, the experiments in this example were carried out using Veliparib, AZD-2461, Rucaparib, UPF 1069 and Olaparib, respectively.

[0243] The effects of specific PARP inhibitors on rAAV9 production in HEK293T adherent cells were tested using the method described in Example 8. Within 4 hours after transfection, DMSO, 50 mM Veliparib, 0.2 mM AZD-2461, 1 mM Rucaparib, 10 mM UPF 1069, and 0.5 mM Olaparib working solutions were added to the cells at a 1:1000 dilution, achieving final concentrations of 0 μM, 50 μM Veliparib, 0.2 μM AZD-2461, 1 μM Rucaparib, 10 μM UPF 1069, and 0.5 μM Olaparib, respectively. All other procedures were the same as in Example 8.

[0244] The experimental results are shown in Figure 13. The results show that after transfection of adherent HEK293T cells, the addition of different PARP inhibitors can significantly increase the rAAV9 titer, with the yield increases of 351%, 182%, 226%, 142% and 250%, respectively, proving that specific PARP inhibitors also promote viral packaging in adherent HEK293T cells.

[0245] Referring to the experimental methods of this example, when using a two-plasmid or single-plasmid system to produce rAAV, the use of specific PARP inhibitors can also achieve the same effect as the three-plasmid viral packaging system. Specific PARP inhibitors can effectively improve the yield and quality of rAAV in HEK293T adherent cells.

[0246] Example 10: Effect of specific PARP inhibitors on rAAV2 production in Hela adherent cells

[0247] As a non-limiting embodiment, the experiments in this example were performed using Olaparib and Veliparib, respectively.

[0248] After recovery, HeLa adherent cells were cultured continuously at 37°C and 5% (v / v) CO2 for five generations before use for virus packaging.

[0249] Continuously cultured adherent HeLa cells were plated at 6E+5 cells / well in 6-well plates. After 24 hours, when the cells reached 80%-90% confluency, three plasmid transfections were 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. After transfection, the cells were placed in a CO2 incubator and continued to culture. 4-6 hours after transfection, the cells were infected with Ad5 helper virus at an MOI of 50 PFU / cell. DMSO and 0.2mM Olaparib and 10mM Veliparib working solutions were then added to the cell suspension at a ratio of 1:1000, respectively, to achieve final concentrations of 0μM, 0.2μM Olaparib, and 10μM Veliparib, respectively. 72 hours after transfection, cells were harvested and lysed according to Protocol 3.6 to obtain viral supernatant. ddPCR was performed according to Protocol 3.7 to quantify rAAV2 genomic DNA in the supernatant. It should be noted that HeLa cells lack key components for viral packaging. Therefore, in addition to the two-plasmid transfection system, Ad5 infection is required to function as a helper virus (replacing the helper in the three-plasmid transfection system) for AAV packaging.

[0250] The experimental results are shown in Table 8 and Figure 14, which indicate that 0.2 μM Olaparib and 10 μM Veliparib can increase the titer of rAAV2. This means that specific PARP inhibitors can also improve rAAV production in Hela cells.

[0251] Table 8. Titers of rAAV2 in Hela cells after addition of Olaparib and Veliparib

[0252] Based on the results of the above non-limiting examples, it can be seen that specific PARP inhibitors can improve rAAV yield and quality in HEK293, Hela or other cell lines used for rAAV production.

[0253] Incorporated by Reference

[0254] Each patent and scientific document mentioned herein is incorporated by reference in its entirety for all purposes.

[0255] Equivalence

[0256] 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

1. A method for improving the yield and / or quality of viral packaging in a cell, wherein the expression, activity or function of the PARP gene in the cell is specifically downregulated or inhibited.

2. The method of claim 1, wherein PARP gene expression, activity or function in the cell is specifically downregulated or inhibited by using a PARP specific inhibitor.

3. The method according to claim 2, wherein the PARP-specific inhibitor is an antibody, a small molecule compound, an RNAi molecule, a sgRNA or an antisense nucleic acid.

4. The method according to claim 1 or 2, wherein the PARP-specific inhibitor is a small molecule compound, preferably selected from the group consisting of Olaparib, Niraparib, Fluzoparib, Veliparib, Rucaparib, Talazoparib, Saruparib, PARP7-IN-4, PARP7-IN-15, PARP7-IN-16, PARP7-IN-17, UPF 1069, PJ34, Pamiparib, RBN-2397, Dehydrocorydaline, RBN012759, 3-Aminobenzamide, AG14361, PJ34hydrochloride, Talazoparib tosylate, AZD-2461, Rucaparib monocamsylate, EB-47dihydrochloride, Rucaparib phosphate, Iniparib, Venadaparib, DPQ, Senaparib, BGP-15 and structural analogs thereof.

5. The method according to any one of claims 1 to 4, wherein The PARP specific inhibitor is selected from one or more of Olaparib, Veliparib, Rucaparib, AZD-2461, UPF 1069 and structural analogs thereof, preferably Olaparib, Veliparib or structural analogs thereof. The method according to claim 3 , wherein the antibody is a monoclonal antibody or a polyclonal antibody. The method of claim 3 , wherein the antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.

8. The method of claim 3, wherein the RNAi molecule is siRNA, saRNA, shRNA, dsRNA, or miRNA.

9. The method of claim 3 or 8, wherein the RNAi molecule is a polynucleotide of 15-40 bases in length.

10. The method according to any one of claims 3 and 8-9, wherein the RNAi molecule is modified to enhance its stability.

11. The method of any one of claims 1-10, wherein the virus is an adeno-associated virus or a recombinant adeno-associated virus (rAAV).

12. The method according to any one of claims 1 to 11, wherein The cells include adherent or suspended Hela, HEK293, or cell lines obtained by gene editing, transformation, domestication, and / or screening thereof, such as HEK293T, HEK293F, or HEK293FT cells.

13. The method according to any one of claims 1 to 12, wherein Improving the yield and / or quality of virus packaging in cells includes one or more of the following: increasing virus yield, reducing the empty shell rate of virus packaging, or any combination thereof.

14. Use of a cell for improving virus packaging yield and / or quality in the cell, wherein PARP gene expression, activity or function is specifically downregulated or inhibited in the cell.

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