A method for the simultaneous production of multiple recombinant adeno-associated viruses

Abstract

The application discloses a method for simultaneously producing multiple recombinant adeno-associated viruses, and belongs to the field of gene therapy. The method comprises the following steps: stably integrating multiple target genes into a host cell genome or stably integrating multiple serotype capsid protein Cap genes into the host cell genome; screening a single clone cell which stably expresses the target genes or the serotype capsid protein Cap genes; co-transfecting a transfer plasmid containing AAV inverted terminal repeat sequences, a Rep gene plasmid and an adenovirus auxiliary gene plasmid into the single clone cell which stably expresses the target genes or the serotype capsid protein Cap genes; and harvesting and purifying recombinant AAV viruses to obtain an AAV virus mixture with a specific ratio of target genes or a hybrid AAV virus with a specific ratio of capsid proteins.

Description

Technical Field

[0001] This application relates to the field of gene therapy, and more specifically, to a method for simultaneously producing multiple recombinant adeno-associated viruses. Background Technology

[0002] In the field of gene therapy, adeno-associated virus (AAV) has become a favored vector for delivering genetic material into cells to treat a variety of diseases. AAV is considered one of the preferred vectors for gene therapy due to its low immunogenicity, high transduction efficiency in non-dividing cells, long-term expression characteristics, and high safety. AAV can infect multiple cell and tissue types, and its viral system has made significant progress in the past decade, being used to treat both rare and non-rare diseases.

[0003] Currently, common systems for producing adeno-associated virus (rAAV) and recombinant adeno-associated virus (Recombinant AAV) rely on transient transfection, which introduces all the genes necessary for AAV production, including the target gene, Rep / Cap gene, and helper genes, into the production cells.

[0004] Current AAV production typically employs a three-plasmid transient transfection method: a transfer vector containing the target gene GOI; a pHelper with adenovirus helper function; and pAAV-Rep2CapXCapX, which supplies the capsid and replicase functions, i.e., capsid functions for different AAV serotypes. If multiple AAVs or heterozygous AAVs need to be produced simultaneously, multiple GOI or Cap plasmids must be transfected concurrently. Due to batch-to-batch variability and capsid heterogeneity, the quality standards of the viral products are uncontrollable. Summary of the Invention

[0005] The summary section of this application is intended to provide a brief overview of the concepts, which will be described in detail in the detailed description section below. This summary section is not intended to identify key or essential features of the claimed technical solutions, nor is it intended to limit the scope of the claimed technical solutions.

[0006] To address the technical problems mentioned in the background section, some embodiments of this application provide a method for simultaneously producing multiple recombinant adeno-associated viruses, including:

[0007] Stable integration of multiple target genes into the host cell genome or stable integration of capsid protein Cap genes of multiple serotypes into the host cell genome;

[0008] Screening for monoclonal cells that stably express the target gene or stably express the serotype capsid protein Cap gene;

[0009] co-transfecting a transfer plasmid containing AAV inverted terminal repeat sequences, a Rep gene plasmid, and an adenovirus helper gene plasmid into a monoclonal cell stably expressing a target gene or stably expressing a capsid protein Cap gene of a serum type;

[0010] Harvest and purify the recombinant AAV virus to obtain an AAV virus mixture with a specific ratio of target genes or a hybrid AAV virus with a specific ratio of capsid proteins.

[0011] Further,

[0012] The method for stably integrating multiple target genes into the host cell genome includes:

[0013] Prepare a helper plasmid for recognizing and cutting the transposon plasmid sequence;

[0014] Prepare a transposon plasmid containing two inverted terminal repeat sequences and a transposed region between them; the transposed region carries a target gene;

[0015] Co-transfect the helper plasmid and the transposon plasmid into the host genome, and the transposase will recognize the ITR element in the transposon of the transposon plasmid, and insert the transposed region and the ITR element into the host genome.

[0016] Further,

[0017] The helper plasmid is SuperPiggyBac Transposase;

[0018] The transposon plasmid is constructed into the PiggyBac Dualpromoter PB513B-1 backbone vector by gene synthesis or PCR amplification of the sequences in PiggyBac R-ITR and L-ITR to obtain the expression EGFP transfer plasmid and the expression mcherry transfer plasmid;

[0019] The gene sequence of the PiggyBac-EGFP expression transfer plasmid is SEQ ID NO. 1;

[0020] The PiggyBac-mcherry expression transfer plasmid is SEQ ID NO. 2.

[0021] Further,

[0022] The method for inserting the transposed region and the ITR element into the host genome by the transposase recognizing the ITR element in the transposon of the transposon plasmid includes:

[0023] The transposase recognizes and cuts the 5'TR and 3'TR sequences, cuts the target gene of the transposon region from the transfer plasmid expressing EGFP and the transfer plasmid expressing mcherry, and inserts the target gene into the TTAA characteristic insertion site of the host genome by the transposase.

[0024] Further,

[0025] The method of co-transfecting the Cap / Rep gene plasmid and the adenovirus helper gene plasmid into the single clone cells stably expressing the target gene containing the AAV inverted terminal repeat sequence comprises:

[0026] The pAAV-R2C9 and pAdDelta6 are respectively transfected into the screened single clone cells stably expressing the target gene at a molar ratio of 2.2:1.

[0027] Further,

[0028] The method of harvesting and purifying the recombinant AAV virus to obtain the AAV virus mixture with a specific ratio of target genes comprises:

[0029] The cell suspension is obtained 48 hours after transfection, 0.25% trypsin-EDTA is added to the cell suspension, and the cell suspension is repeatedly frozen and thawed three times to lyse the cells and release the recombinant AAV virus particles;

[0030] Centrifugation at 12,000 rpm for 5 minutes to remove cell debris, collect the supernatant, and obtain the AAV virus mixture with a specific ratio of target genes.

[0031] Further,

[0032] The sequences in the R-ITR and L-ITR of the PiggyBac transposon region are genetically synthesized or PCR amplified, and are constructed into the PiggyBac Dualpromoter PB513B-1 backbone vector by homologous recombination to obtain the transfer plasmid expressing EGFP and the transfer plasmid expressing mcherry;

[0033] Substituted for

[0034] The sequences in the R-ITR and L-ITR of the PiggyBac transposon region are genetically synthesized or PCR amplified, and are constructed into the PiggyBac Dualpromoter PB513B-1 backbone vector by homologous recombination to obtain the transfer plasmid expressing cap9 and the transfer plasmid expressing cap9-EGFP;

[0035] The gene sequence of PiggyBac-cap9 is SEQ ID NO. 3;

[0036] The gene sequence of PiggyBac-cap9-EGFP is SEQ ID NO. 4.

[0037] Further,

[0038] The method for stably integrating the capsid protein Cap gene of multiple serotypes into the host cell genome comprises:

[0039] The host cell is inoculated into a shake flask;

[0040] When the density of the host cell reaches 2.5E+06 / ml, the helper plasmid pAdDelta6, the Rep gene plasmid, and the transfer plasmid containing the AAV inverted terminal repeat sequence expressing EGFP are co-transfected into the monoclonal cell capable of stably expressing Cap containing two serotypes of capsid proteins;

[0041] The gene sequence of the transfer plasmid expressing pAAV-EGFP is SEQ ID NO. 5.

[0042] Further,

[0043] The method for screening the monoclonal cell stably expressing the target gene comprises:

[0044] The monoclonal cell is obtained by antibiotic screening and limited dilution, and the ratio between different integrated genes is determined by quantitative PCR, and the screened monoclonal cell can stably express a specific ratio of target genes.

[0045] Further,

[0046] The method for screening the monoclonal cell stably expressing the capsid protein Cap gene of multiple serotypes comprises:

[0047] The monoclonal cell line capable of expressing a specific ratio of capsid proteins is screened by antibiotic screening or flow cytometry sorting FACS and other technologies.

[0048] The present application stably integrates multiple target genes or different serotypes of capsid protein Cap into the host cell genome, and combines with the monoclonal screening technology, to screen the cell line stably expressing a specific ratio of target genes or capsid proteins, which has the following advantages:

[0049] It can effectively solve the challenge of gene packaging size limitation, meet the demand of multiple gene combination therapy or complex gene therapy, and realize the stable production of hybrid rAAV.

[0050] Overcome the limitations of traditional multi-plasmid transfection or cross packaging method, provide a high-efficiency, repeatable production rAAV production platform for gene therapy and biomedical research, significantly improve the stability, scalability and cost-effectiveness of production, while ensuring the consistency of product quality, with wide application prospect.

[0051] Gene integration and screening are carried out simultaneously: significantly saving time and cost, and each cell of the integrated monoclonal cells expresses the target gene or capsid protein, which can achieve rAAV production comparable to traditional three-plasmid transfection.

[0052] Stable production of multiple fixed ratio AAV viruses: the target gene in the screened monoclonal cells has a specific ratio, ensuring consistent ratio in different batches of production, ensuring the consistency and controllability of product function and quality, and avoiding the batch heterogeneity problem caused by unstable plasmid ratio in traditional methods.

[0053] Preparation of specific ratio hybrid AAV virus: precise regulation of the integration ratio of multiple AAV capsids to stably and flexibly produce hybrid rAAV with different chimeric ratios, and the virus particles have high homogeneity, which is easy for product characterization analysis, and has scalability and production reproducibility.

[0054] Simplify the operation process and production process: no need to rely on multi-plasmid mixing or cross packaging, one-time production of multiple AAVs, eliminating multiple virus purification and mixing steps, significantly reducing the complexity of production process, and improving production efficiency.

[0055] Reduce costs: reduce the use of GOI plasmid, reduce the number of plasmid production batches, reduce production costs, and reduce the residual plasmid backbone DNA in rAAV products and infected tissues

[0056] Improve scalability and robustness: the use of stable cell lines makes the production process more controllable, improves production reproducibility, and is suitable for large-scale production.

[0057] Ensure product quality consistency: ensure the functionality and quality consistency of rAAV complexes, meet the high standards of gene therapy. BRIEF DESCRIPTION OF DRAWINGS

[0058] The accompanying drawings, which form a part of this application, are intended to provide further understanding of the application and are incorporated herein in their entirety. The schematic embodiment drawings of the present application and their descriptions are used to explain the present application, and do not constitute undue limitation on the present application.

[0059] In the drawings:

[0060] Figure 1shows the framework of the transfer plasmid, containing the gene of interest and the two ITRs shown and the elements in between expressing EGFP;

[0061] Figure 2 shows the framework of the transfer plasmid, containing the gene of interest and the two ITRs shown and the elements in between expressing mcherry;

[0062] Figure 3 shows the fluorescence images of the monoclonal cells, showing the double positive fluorescence of EGFP and mCherry in the screened monoclonal cells;

[0063] Figure 4 shows the fluorescence data of the monoclonal cells detected by flow cytometry, showing the first example of the proportion of EGFP and mCherry fluorescence in the monoclonal cells;

[0064] Figure 5 shows the fluorescence data of the monoclonal cells detected by flow cytometry, showing the second example of the proportion of EGFP and mCherry fluorescence in the monoclonal cells;

[0065] Figure 6 shows the fluorescence data of the monoclonal cells detected by flow cytometry, showing the third example of the proportion of EGFP and mCherry fluorescence in the monoclonal cells;

[0066] Figure 7 shows the fluorescence data of the monoclonal cells detected by flow cytometry, showing the fourth example of the proportion of EGFP and mCherry fluorescence in the monoclonal cells;

[0067] Figure 8 shows the two transfer plasmid maps, containing the gene of interest and the two ITRs shown and the elements in between expressing cap9 and cap9-EGFP, respectively;

[0068] Figure 9 is the green fluorescence image of the cells 12 days after transfection and pressure at a ratio of 1:0;

[0069] Figure 10 is the green fluorescence image of the cells 12 days after transfection and pressure at a ratio of 1:1;

[0070] Figure 11 is the green fluorescence image of the cells 12 days after transfection and pressure at a ratio of 1:1;

[0071] Figure 12 is the green fluorescence image of the cells 12 days after transfection and pressure at a ratio of 1:50;

[0072] Figure 13 is the green fluorescence image of the cells 12 days after transfection and pressure at a ratio of 0:1;

[0073] Figure 14 First example of fluorescence data from flow cytometry 12 days post transfection and pressure

[0074] Figure 15 Second example of fluorescence data from flow cytometry 12 days post transfection and pressure

[0075] Figure 16 Third example of fluorescence data from flow cytometry 12 days post transfection and pressure

[0076] Figure 17 Fourth example of fluorescence data from flow cytometry 12 days post transfection and pressure

[0077] Figure 18 Fifth example of fluorescence data from flow cytometry 12 days post transfection and pressure

[0078] Figure 19 First example of 1:1 mixed transduction of hybrid and homozygous viruses representing 1:1 capsid packaging

[0079] Figure 20 Second example of 1:1 mixed transduction of hybrid and homozygous viruses representing 1:1 capsid packaging

[0080] Figure 21 Transduction of ARPE with viruses representing different ratios of capsid packaging DETAILED DESCRIPTION

[0081] Embodiments of the present disclosure will be described in more detail with reference to the drawings. Although certain embodiments of the present disclosure are shown in the drawings, it is understood that the present disclosure can be embodied through various forms and should not be interpreted as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that the present disclosure can be more thoroughly and completely understood. It should be understood that the drawings of the present disclosure are only for illustrative purposes and are not intended to limit the scope of protection of the present disclosure.

[0082] In addition, it should be further noted that only parts related to the present application are shown in the drawings for ease of description. The embodiments in the present disclosure and the features in the embodiments can be combined with each other without conflict.

[0083] The present disclosure will be described in detail with reference to the drawings and in conjunction with embodiments.

[0084] For the purposes of the present specification, the following definitions and explanations of terms are applicable to the particular use of the invention. Unless defined otherwise, technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art. Explanations of relevant terms can be found in standard textbooks and literature known to those of skill in the art. The definitions provided herein are intended to apply uniformly throughout the specification and claims unless an otherwise expressly different definition is expressly provided herein. In addition, all references cited herein are incorporated by reference to the extent that they support the technical content of the present invention.

[0085] The term "vector" as used herein refers to any molecule, such as a nucleic acid, plasmid or virus, which is capable of transporting a heterologous nucleic acid contained therein into a host cell either in a self-replicating fashion or as an integral part of the host cell genome.

[0086] "Transfection" as used herein refers to the transfer of a polynucleotide, such as a nucleic acid molecule, plasmid, etc., from outside a cell to inside a cell, so that the polynucleotide has a function in the cell. Methods of transfection are well known to those of skill in the art, such as calcium phosphate or liposome-mediated transfection.

[0087] As described herein, adeno-associated virus (AAV) is a non-enveloped, naturally replication-defective DNA virus belonging to the Parvoviridae family, and is one of the simplest single-stranded DNA viruses ever discovered, which requires a helper virus, usually adenovirus, to replicate. Its single-stranded DNA genome is about 4.7 kb in length, including genes encoding replication protein Rep, responsible for replication, integration and packaging of AAV genome, and capsid Cap, determining the serotype and tissue tropism of AAV, as well as two 145 bp terminal inverted repeats ITRs flanking the genes. ITR is the minimum self-sequence necessary for AAV replication and packaging, and is also a cis-acting element necessary for AAV replication, packaging and integration.

[0088] The PiggyBac transposon described herein is a self-contained eukaryotic transposon derived from Trichoplusia ni, and its gene transposition principle is based on the "cut-and-paste" mechanism mediated by transposase. The full-length PiggyBac transposon is 2476 bp, with 13 bp terminal inverted repeats ITR at both ends, and a 2.1 kb open reading frame ORF in the middle, which can encode a transposase with autonomous transposition function. In application, the PiggyBac system consists of two plasmids: the helper plasmid is responsible for expressing the transposase, and the transposon plasmid carries the optimized ITR sequences on both sides and the target gene inserted therein. The transposase specifically recognizes and binds to the ITR sequence, accurately cutting the target gene from the transposon plasmid, and then inserting it into the TTAA tetranucleotide site in the host genome.

[0089] The "gene integration" described in the present application can be achieved by other transposon strategies, lentivirus, homologous recombination and other gene insertion strategies in addition to PiggyBac transposon. Lentivirus is a subclass of retroviridae, and its gene integration mechanism depends on the process of reverse transcription of viral RNA genome into DNA and insertion into host genome by integrase. When infecting host cells, lentivirus releases its positive single-stranded RNA ssRNA genome into the cytoplasm, and then reverse transcriptase converts it into double-stranded DNA proviral DNA, and integrase mediates the permanent integration of the DNA into the chromosomal DNA of the host cell. Gene homologous recombination is a process of exchanging genetic material by breaking, pairing and rejoining of two DNA molecules with the same or highly similar sequences. Its core mechanism involves the Holliday model, that is, after the homologous region forms a joint molecule, nucleases and ligases catalyze the cleavage and reconnection of the chains, and complete the cross exchange of double-stranded DNA. Other gene insertion strategies include Prime editing and CRISPR-mediated transposon CAST, etc.

[0090] The "recombination" described in the present application refers to a heterologous nucleic acid sequence that does not naturally exist in the vector, host cell containing it, which can be a nucleic acid of interest for expressing a protein of interest.

[0091] The recombinant AAV vector rAAV described in the present application refers to a viral vector constructed by replacing the Rep and Cap genes in the AAV genome with an exogenous gene of interest GOI. rAAV usually includes an AAV capsid and a recombinant viral genome and a nucleic acid of interest packaged in the capsid. The Rep and Cap genes are provided in trans, that is, expressed by other plasmids, while the ITR sequences on both sides are retained to support the replication and packaging of the virus. When it does not have rep and cap genes in its viral genome, the AAV vector does not integrate into the genome of the transduced cell, but exists in the form of episome in the nucleus of the transduced cell, and can stably express the exogenous gene for a long time. In the natural system, the infection of AAV requires the involvement of helper viruses such as adenovirus or herpes virus, which provide genes required for replication, such as E1A, E1B, E2A, E4, VARNA, etc. In the production of rAAV vectors, helper viruses can not be used, but the necessary genes contained in the helper virus are provided by plasmids to support the replication and packaging of AAV. The recombinant virus that can be used to express the gene of interest of the present application can be a viral vector derived from various types of viruses, including but not limited to adeno-associated virus AAV vector, adenovirus vector, lentivirus vector, retrovirus vector, herpes simplex virus HSV vector, etc.

[0092] The term "nucleic acid of interest" according to the present invention refers to a nucleic acid to be packaged by the recombinant AAV viral particle, which encodes any prophylactic or therapeutic protein or a marker detection protein such as GFP, mCherry, Cap proteins, etc. The nucleic acid of interest is contained in an expression cassette and is packaged within the AAV capsid. In some embodiments, the expression cassette comprises at least one ITR sequence to ensure that the vector genome can be assembled by the capsid. The expression cassette can be single-stranded DNA, double-stranded DNA or single-stranded RNA or double-stranded RNA. In some embodiments, the expression cassette can comprise one or more regulatory sequences to direct the expression of the nucleic acid of interest coding sequence in a cell. The regulatory sequences can be selected from the group consisting of a transcription initiation sequence, a termination sequence, a promoter and / or an enhancer sequence operably linked to the coding sequence; efficient RNA processing signals such as splicing and polyadenylation polyA regions, for example the human growth hormone polyadenylation region; inverted repeat sequences, for example L-ITR or R-ITR; a selectable marker or reporter gene, for example a resistance gene including but not limited to ampicillin, hygromycin, neomycin, methotrexate, kanamycin, gentamycin, Zeocin, or tetracycline; a post-transcriptional regulatory sequence, for example the post-transcriptional regulatory sequence of the WPRE woodchuck hepatitis virus; a sequence that stabilizes cytoplasmic mRNA; a nucleic acid restriction site; a homologous recombination sequence; a sequence that enhances translation efficiency such as a Kozak sequence; a sequence that enhances protein stability; and a sequence that enhances secretion of the encoded product. In some preferred embodiments, the regulatory sequences are selected from one or more of the following: an inverted repeat sequence, an enhancer, a promoter, a polyadenylation region, a selectable marker or reporter gene. Examples of promoters suitable for use in the present invention include, but are not limited to, promoters from bacteria, yeast, plants, viruses, and mammals including simians and humans. The promoter can be constitutive or can be inducible.

[0093] According to the present invention, the genes encoding the components required for the production of AAV are selected from the group consisting of genes encoding AAV Cap proteins VP1, VP2 and VP3; genes encoding AAV Rep proteins Rep78, Rep68, Rep52 and Rep40; genes encoding adenovirus helper functions E4orf6, E2A and VA-RNA; and a gene of interest GOI flanked by AAV ITRs. In a particular embodiment, the genes encoding the components required for the production of AAV consist of two different genes of interest GOI flanked by AAV ITRs, which are not particularly limited and include any gene that needs to be delivered to a target cell, such as GFP green fluorescent protein and mcherry red fluorescent protein in some cases.

[0094] The term "capsid protein" refers to a protein that is part of the viral capsid. For adeno-associated virus, the capsid proteins are generally referred to as VP1, VP2, and VP3. For AAV, these three capsid proteins are generated via alternative mRNA splicing and / or different translation initiation codons. The amino acid sequences of AAV capsid proteins are generally conserved within the Parvoviridae family, particularly in functionally critical regions. Relevant sequence information can be found in Rutledge et al. 1998 J. Virol. 72:309-19.

[0095] AAV serotype refers to different variants of AAV, which differ in the amino acid sequence of their capsid proteins, resulting in different tropism of the different serotypes for specific tissues or cell types. The AAVs in the present invention are not limited to a specific AAV serotype. Thus, the AAV can be selected from the group consisting of AAV serotype 1 AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, AAVDJ8, AAVrh10, a hybrid of different of said serotypes, and said serotypes with mutations that alter the tropism of the AAV serotype. Preferably, the AAV is selected from the group consisting of AAV2, AAV8, and AAV9. According to the present invention, some or all of the genes encoding components required for the production of the AAV are stably integrated into the host cell genome. Preferably, at least 2 or all of said genes are stably integrated into the host cell genome. In some embodiments, the ITR in the AAV vector can be from a different AAV serotype, for example, AAV2 ITR can be used with a capsid that is not AAV2, for example, AAV5, AAV8, or AAV9 capsid.

[0096] The term "tropism" refers to the targeting of a recombinant viral vector to a specific cell, tissue, and / or organ. The term "chimeric AAV capsid" refers to an AAV capsid protein comprising amino acid sequences from two or more different AAV serotypes and is capable of forming an AAV capsid, examples of which include but are not limited to the capsid of AAVDJ. The term "modified AAV capsid" refers to an AAV capsid protein comprising one or more insertions, substitutions, or deletions of amino acids, or an AAV capsid formed by the insertion of a polypeptide derived from other binding domains, examples of which include but are not limited to AAV2-7m8. The "cross-packing" described in the present invention is a process of generating a hybrid rAAV with mixed properties by combining capsid proteins of different serotypes with a gene of interest. Cross-packing can alter the cellular targeting properties of AAV, enhancing its efficiency and specificity of infection of specific cells or tissues.

[0097] The present invention relates to a method for producing recombinant AAV viral particles rAAV and a culture system related thereto: the rAAV production culture comprises the following key components:

[0098] 1. Suitable host cells, such as cell lines derived from humans, e.g. HEK-293T cells, or cell lines derived from insects, e.g. SF-9, are used in a baculovirus production system;

[0099] 2. Suitable helper functions are provided by wild-type or mutant adenoviruses, e.g. temperature-sensitive adenoviruses, herpes viruses, baculoviruses or the plasmid pHelper construct providing helper functions;

[0100] 3. AAV rep and cap gene pRC for supporting replication of AAV and assembly of capsid proteins;

[0101] 4. Transfer plasmids containing the recombinant AAV genome comprising the gene of interest flanked by at least one fully functional ITR and, preferably, under the control of an operably linked promoter; and suitable culture systems to support efficient production of rAAV.

[0102] 5. Furthermore, the present application allows for the integration of the rep and cap genes of AAV into the genome of the packaging cell in advance, so that only the nucleic acid molecule containing the recombinant AAV genome and the nucleic acid molecule of the helper virus genes need to be transfected at the time of production, further simplifying the production process and increasing stability.

[0103] The stable AAV production cell lines used in the present application can be derived from a variety of cell lines known in the art. For example, packaging cell lines that can be used include, but are not limited to, mammalian, including human cells, insect cells, plant cells, microorganisms or yeast, such as the HEK293 series of cells, e.g. HEK293A, HEK293T or HEK293FT, HeLa cells, A549 cells, Vero cells, hARPE-19 cells and cell lines derived from insects, such as SF-9. As known to the skilled person, the appropriate packaging cell line can be selected depending on the type of recombinant viral vector. The culture medium used for the production of recombinant AAV can be commercially available or custom-made and can be supplemented with cell culture components known in the art, such as glucose, vitamins, amino acids or growth factors, to increase the titer of viral particles. The culture conditions can be optimized depending on the specific host cell used.

[0104] The term "viral genome, vg" as used herein refers to a polynucleotide sequence that can be packaged into a viral capsid, which at least comprises an expression cassette of a nucleic acid of interest such as a gene of interest and its regulatory elements and cis nucleic acid elements required for virus production such as ITR sequences of AAV, and can further contain other viral nucleic acid elements such as enhancers, promoters, polyadenylation signals. Depending on the different types of recombinant viral vectors, the recombinant viral vector genome can comprise single-stranded DNA, double-stranded DNA, or single-stranded RNA or double-stranded RNA. In some embodiments, the titer of the recombinant viral vector genome is measured in vg / mL, referring to the number of copies of the recombinant viral genome contained per milliliter of solution, which is used to quantify the concentration of the viral preparation.

[0105] The term "transduction" or "infection" and the like refer to the process of introducing nucleic acid into a target cell by a viral vector and releasing its genetic material including the nucleic acid of interest. The term "transduction efficiency" refers to the proportion of cells expressing the nucleotide of interest after incubation with a set number of viral vectors containing the nucleotide of interest, usually expressed as a percentage. Transduction efficiency is an important indicator for evaluating the delivery effect of viral vectors. Common methods for determining transduction efficiency include, but are not limited to, fluorescence-activated cell sorting (FACS): by transducing cells with viral vectors carrying a fluorescent reporter gene such as GFP, the proportion of fluorescent positive cells is detected by flow cytometry, thereby evaluating the transduction efficiency. Polymerase chain reaction (PCR): by detecting the expression level of the nucleotide of interest in the target cells, the transduction efficiency is indirectly evaluated. Other methods: including immunofluorescence staining, Western blotting or enzyme activity detection, etc., for quantitative or qualitative analysis of the expression of the gene of interest. These methods are well known to those skilled in the art, and suitable techniques can be selected according to the specific experimental requirements.

[0106] In the "wild type" wtAAV form of AAV, the AAV genome size is about 4.7 kb, and the structure includes two inverted terminal repeat sequences ITRs, and two major open reading frames: rep responsible for viral replication and cap encoding capsid proteins Merten, O.-W. Microorganisms 2024, 12, 384. The AAV genome is packaged into a viral capsid, and after infecting cells, the molecular mechanisms of the cells convert the single-stranded DNA into a double-stranded form, which is then transcribed and translated into polypeptides. However, the load of AAV is limited, and usually only about 4.1-4.7 kb of exogenous gene fragments can be accommodated, which is mainly due to the physical size of the AAV capsid and the genome packaging mechanism.

[0107] Currently, the number of gene therapy trials and products based on AAV vectors is rapidly increasing, but the main challenge in translating this technology into clinical applications is how to efficiently deliver a large amount of high-quality AAV viral vectors. Existing AAV production mainly relies on two systems: a HEK293 cell-based transient transfection system and

[0108] Sf9 / baculovirus system. These systems can usually only deliver a single AAV, and the limited load of AAV poses a challenge to their application in gene editing and gene therapy, especially when the target gene is large, such as certain large genes or genes requiring additional regulatory elements. To overcome this limitation, more and more studies use dual or multi-vector systems to achieve the delivery of large genes Hum Gene Ther. 2023, 3413-14: 616-628; Nat Commun. 2024, 151: 6141. In addition, many diseases are caused by abnormalities in multiple genes or signaling pathways, so treatment needs to be targeted at multiple targets simultaneously Hum Mol Genet. 2013, 17; 2224: 4929-4937.

[0109] The serotypes of AAV are an important resource in the field of gene therapy, and there are currently more than 200 registered AAV serotypes, among which there are 13 different serotypes of AAV in primates, i.e. AAV1-AAV13, of which AAV2 is the earliest to be cloned and the most widely studied serotype. The AAV capsid plays a key role in determining its tissue specificity, and different serotypes such as AAV1, AAV2, AAV5, etc. exhibit different tropism to different tissues or cell types due to differences in their capsid proteins, and this specificity is determined by the interaction of AAV capsid with specific receptors on the surface of host cells.

[0110] As research continues, researchers have discovered that different serotypes of AAV can hybridize, generating hybrid or chimeric AAVs that combine the characteristics of both hybrids, significantly expanding the potential applications of AAV in gene therapy. Hybrid or chimeric AAVs refer to viruses obtained by fusing or genetically modifying the capsid proteins of different viruses, and the composition of their capsids has a significant impact on the gene transduction characteristics of the vector. This strategy can compensate for the deficiencies of a single serotype, realize the hybrid advantages between AAV serotypes, and generate unique AAV vectors with serotype specificity. Hybrid or chimeric AAVs can change tissue tropism, more accurately target specific tissues or cell types, and other advantages such as improving receptor binding efficiency, optimizing tissue tropism, and improving new purification strategies Mol Ther, 2005, 116:856-65. Recombination between serotypes has been proven to occur in nature: for example, AAV6 is a natural variant of AAV1 and AAV2 recombination, with only 71 bp exchanged J. Virol, 1999, 73:3994-4003. Researchers use cross-packaging to generate hybrid AAVs, improving targeting and treatment effectiveness in treating muscle and nervous system diseases. For example: AAV2.1 vector is composed of capsid proteins from AAV1 and AAV2. After injecting it into the frontal cortex of macaques, the vector simultaneously exhibits excellent exogenous gene expression ability and neuron specificity Nat Commun. 2023, 8, 141:4762.

[0111] Despite the great potential of hybrid AAVs in gene therapy, their production still faces the following challenges: traditional methods mainly use multi-plasmid cross-packaging transfection to prepare hybrid AAVs, which has problems such as AAV heterogeneity, inconsistent function, and difficulty in commercial production.

[0112] In the field of gene therapy, cross-packaging and multi-gene packaging strategies of AAV are becoming key technologies for the next generation of gene delivery tools. These strategies optimize the capsid proteins and gene loading capacity of AAV, significantly improving the precision, effectiveness, and scope of gene therapy.

[0113] Cross-packaging can generate hybrid rAAV with mixed characteristics by combining capsid proteins of different serotypes with target genes. Hybrid rAAV has new phenotypes and tropisms, and researchers can select the best vector transduction according to treatment needs and direct it to the target tissues or cells of patients. This strategy not only improves treatment effectiveness but also reduces the impact on non-target cells, thereby reducing the side effects of treatment.

[0114] Multi-gene packaging overcomes the single AAV load limit by integrating multiple genes or regulatory elements into AAV vectors, providing greater flexibility for gene therapy. At the same time, targeting multiple disease targets allows more genetic information to be carried and delivered, opening up new possibilities for treating more types of diseases.

[0115] For these strategies, the development of gene therapy products requires a cell line that can be used to stably produce such AAVs for commercialization. In the future, we can expect these strategies to play an increasingly important role in the treatment of various genetic diseases, cancers, and other chronic diseases, bringing more well-being to human health.

[0116] Reference Figures 1-21 ,

[0117] Example 1

[0118] A method for simultaneously producing multiple recombinant adeno-associated viruses, specifically a single clone cell selected by PiggyBac stable transfection system, and a method for producing AAV viruses expressing two fixed ratios of genes at one time. The method comprises the following steps:

[0119] S1, stably integrating multiple target genes into the host cell genome or stably integrating multiple serotype capsid proteins Cap genes into the host cell genome;

[0120] S2, screening single clone cells stably expressing target genes or stably expressing serotype capsid proteins Cap genes;

[0121] S3, co-transfecting Cap / Rep gene plasmids and adenovirus helper gene plasmids into single clone cells containing AAV inverted terminal repeat sequences stably expressing target genes or stably expressing serotype capsid proteins Cap genes;

[0122] S4, harvesting and purifying recombinant AAV viruses to obtain AAV virus mixtures with specific ratios of target genes or hybrid AAV viruses with specific ratios of capsid proteins.

[0123] Specifically,

[0124] The PiggyBac system contains two vectors, one of which is called a helper plasmid, which is responsible for encoding a transposase for recognizing and cutting a transposon sequence; the other vector is called a transposon plasmid, which contains two inverted terminal repeat sequences ITRs and a transposition region between them, carrying the target genes GOI that need to be transposed into the host genome.

[0125] The exogenous gene is integrated by a PiggyBac transposon gene expression system; the PiggyBac system has the advantages of large vector capacity, high integration efficiency and strong stability, and can realize the co-expression of multiple genes

[0126] S1, a method for stably integrating a plurality of target genes into the genome of a host cell comprises:

[0127] S11, an auxiliary plasmid for recognizing and cutting the transposon plasmid sequence is prepared; the auxiliary plasmid is used to encode a transposase;

[0128] S12, a transposon plasmid containing two inverted terminal repeat sequences and a transposed region between them is prepared; the transposed region carries a target gene;

[0129] S13, the auxiliary plasmid and the transposon plasmid are co-transfected into the host cell, and the transposase will recognize the ITR element in the transposon of the transposon plasmid, and insert the transposed region and the ITR element into the host genome.

[0130] The method for the transposase to recognize the ITR element in the transposon of the transposon plasmid and insert the transposed region and the ITR element into the host genome comprises:

[0131] S131, construct a vector, gene synthesize or PCR amplify the sequences in PiggyBac-R-ITR and PiggyBac-L-ITR, and construct into the PiggyBac Dualpromoter PB513B-1 skeleton vector by homologous recombination to obtain the transfer plasmid expressing EGFP and the transfer plasmid expressing mcherry;

[0132] The whole sequence containing EGFP-puromycin inserted in PiggyBac-L-ITR and PiggyBac-R-ITR is SEQ ID NO. 6; the whole sequence containing mCherry-neomycin inserted in PiggyBac-ITR is SEQ ID NO. 7. Wherein, the sequence of R-ITR alone is SEQ ID NO. 10, and the sequence of R-L-ITR alone is SEQ ID NO. 11.

[0133] The full-length gene sequence of the transfer plasmid expressing EGFP is SEQ ID NO. 1; refer to Figure 1 The transfer plasmid expressing EGFP contains two AAV2-ITRs and the elements therebetween as shown. The sequences of the two AAV2-ITRs are SEQ ID NO. 8 and SEQ ID NO. 9, respectively.

[0134] The full-length gene sequence of the mcherry-expressing transfer plasmid is SEQ ID NO. 2. Referring to Figure 2 The mcherry-expressing transfer plasmid comprises the two AAV2-ITRs and the elements therebetween as shown.

[0135] The PiggyBac transposase recognizes and cuts the PiggyBac-L-ITR and PiggyBac-R-ITR sequences, excises the target gene from the transfer plasmid, and the transposase inserts the target genes EGFP-puromycin and mcherry-neomycin into the TTAA signature insertion site of the host genome, finally achieving the integration of the genes. The target genes EGFP-puromycin and mcherry-neomycin are located between the PiggyBac-R-ITR and PiggyBac-L-ITR.

[0136] In addition, the AAV2 ITR sequences flanking the reporter gene EGFP or the reporter gene mCherry can be used to package AAV viruses. The reporter gene EGFP sequence is SEQ ID NO. 12. The reporter gene mCherry sequence is SEQ ID NO. 13.

[0137] S2, a method for screening a monoclonal cell stably expressing a target gene or a monoclonal cell stably expressing a capsid protein Cap gene of a serum type, comprises:

[0138] S21, screening a stable AAV production cell line: obtain a monoclonal cell by an antibiotic screening method and a limiting dilution method, the antibiotic can be puromycin or neomycin; and determine the proportion of the target gene integration among various products obtained after the integration of the target gene into the host cell by a quantitative PCR method, to ensure that the screened monoclonal cell can stably express a specific proportion of the target gene. The stable AAV production cell line used in the present application can be derived from various types of cell lines known in the art. For example, the packaging cell lines that can be used include, but are not limited to, mammalian cells including human cells, insect cells, plant cells, microorganisms or yeasts, for example, cells of the HEK293 series such as HEK293A, HEK293T or HEK293FT, HeLa cells, A549 cells, Vero cells, hARPE-19 cells, and cell lines derived from insects such as SF-9. As known by those skilled in the art, a suitable packaging cell line can be selected according to the different types of recombinant viral vectors. The culture medium for producing recombinant AAV can be commercially available or customized, and can be supplemented with cell culture components known in the art such as glucose, vitamins, amino acids or growth factors to improve the titer of viral particles. The culture conditions can be optimized according to the specific host cells used.

[0139] Gene integration:

[0140] S211. Cell Culture: Resuscitate 293FT cells and seed 1.5E+05 cells per well in a 24-well plate. Culture in DMEM / 10% fetal bovine serum complete medium at 37°C and 5% CO2 for a period of time, preferably overnight. Transfect when the cell confluence reaches 70%-80%.

[0141] S212. Transfection: PiggyBac-EGFP-Puromycin and PiggyBac-mCherry-Neomyocin, along with transposase PB200PA-1 at a ratio of 1:1:1 (500 ng total) were co-transfected into 293FT cells using the Lipo3000 Thermo transfection reagent. After 48 hours of transfection, the cells were passaged, and puromycin and G-418 were added for selection. One week after selection, monoclonal cells expressing both EGFP and mCherry were obtained. The cell suspension was serially diluted, and one cell per well was seeded into 96-well plates for further culture. The monoclonal cells were observed under a microscope, labeled, and passaged for expansion.

[0142] Screening and identification of stable cell lines:

[0143] S213, Monoclonal Screening: Microscopic observation revealed four monoclonal cells that were double-positive for both EGFP and mCherry. (Refer to...) Figure 2 The cells were cultured on a larger scale, digested with Typsin, and then the digestion was terminated by adding DMEM containing 10% FBS. The proportion of the two fluorescent substances in the cells was detected by flow cytometry.

[0144] S214. Results Analysis: Figure 4 The results showed that these cells were double positive for EGFP and mCherry, further confirming the success of gene integration.

[0145] Gene integration copy number analysis

[0146] S215. Detection: After expanding the culture of single-clonal cells, the genomic DNA was extracted according to the instructions of Dneasy Blood & Tissue Kit #69506 (Qiagen). Primers / probes targeting the GOI gene were used (see Table 1). Digital PCR (dPCR) was used to analyze the integration copy number and ratio of the two genes. The following is a brief introduction to the cell genomic DNA extraction and dPCR sample pretreatment process:

[0147] Cell genomic extraction: 1E6 cells 300g centrifugation, discard supernatant, add 200ul PBS resuspension, add 20ul Proteinase K vortex mixing, add 4ul 100mg / ml Rnase A solution static 5min. Add 200ul Buffer AL to the cell resuspension, vortex mixing, 56℃ water bath 10min. Incubation end, add 200ul anhydrous ethanol, shake mixing. The supernatant is added to the Dneasy Blood & Tissue Kit genomic extraction column, 12000rpm centrifugation for 1min, discard the filtrate. Add 500uL AW1 to the filter column, 12000rpm centrifugation for 1min, discard the filtrate. Add 500uL AW2 to the filter column, 12000rpm centrifugation for 1min, discard the filtrate. The filter column is transferred to a clean centrifuge tube, 50ul of preheated sterile water is added to the center of the membrane, 12000rpm centrifugation for 1min, collect the centrifuged DNA and conduct concentration determination.

[0148]

[0149] Sample processing: Configure the enzyme cutting system - take 1ul extracted genome, add 1ul EcoRI-HFNEB, R3101S, 5ul rCutsmartbuffer, 43ul H20, mix and put into PCR instrument, 37℃ enzyme cutting 30min. After the end, dilute 2 times with H20 for dPCR reaction.

[0150] The dPCR reaction contains the following components: 4x Probe PCR Master Mix QIAGEN 3ul, 1.6ul nuclease-free H2O, 10uM primer / probe each 0.96ul, 5ul diluted sample. According to the procedure: pre-denaturation 95℃, 2min; denaturation 95℃ 15s, annealing extension 60℃, 30s, 40 cycles to complete detection.

[0151] S216, Results: As shown in Table 2, there are various ratios of two genes in monoclonal cells, such as 1:1, 1:2, etc. The fixed value is detected by quantitative PCR, so the ratio of the integration of the two target genes can be confirmed.

[0152]

[0153] S3, The method for co-transfecting Cap / Rep gene plasmid and adenovirus auxiliary gene plasmid into monoclonal cells stably expressing target genes containing AAV inverted terminal repeat sequences includes:

[0154] S31, respectively, pAAV-R2C9, pAdDelta6 are transfected into monoclonal cells stably expressing target genes screened at a molar ratio of 2.2:1.

[0155] S311, Plasmid construction: Construct pAAV-R2C9 plasmid, i.e., pR2C9 packaging plasmid: express Rep gene of AAV2 and Cap gene of AAV9. The pAAV-R2C9 plasmid is constructed by removing the AAV8 Cap gene from Addgene plasmid #112864 pAAV.R2C8 and inserting the AAV9 Cap gene. The AAV9 genome sequence is referenced to GeneBank: AY530579.1.

[0156] Helper plasmid pAdDelta6, i.e., pHelper plasmid: DNA is synthesized by referring to the sequence of Addgene plasmid #112867.

[0157] S312, Transfection: Transfect the selected single clone cells stably expressing the target gene with pAAV-R2C9 and pAdDelta6 at a total mass of 26 ug and a molar ratio of 2.2:1, so that the target genes EGFP and mCherry are integrated.

[0158] S4, Harvest and purify the recombinant AAV virus to obtain an AAV virus mixture with a specific ratio of target genes. The method comprises:

[0159] S41, After 48 hours of transfection, a cell suspension is obtained, and the recombinant vector is present in the cell suspension. 0.25% Trypsin-EDTA is added to the cell suspension, and the cell suspension is repeatedly frozen and thawed three times to lyse the cells and release the recombinant AAV virus particles.

[0160] S42, Centrifuge at 12,000 rpm for 5 minutes to remove cell debris, collect the supernatant, and obtain an AAV virus mixture with a specific ratio of target genes.

[0161] An AAV virus mixture with a specific ratio of target genes, i.e., a hybrid AAV virus, is obtained.

[0162] The method for simultaneously producing multiple recombinant adeno-associated viruses further comprises:

[0163] S5, Determine the viral genome titer of the recombinant AAV vector crude cell lysate by digital PCR dPCR.

[0164] S51, the AAV virus mixture is treated with DNase I at 37°C for 30 minutes to remove residual plasmid DNA and free DNA of the cell genome during cell transfection, and 12 mM EDTA, pH 8.0 is added to inactivate DNase I. Add a final concentration of 0.1% SDS solution at 95°C for 30 minutes to release the viral genome. The titer of AAV vg / mL is determined by dPCR using the primers / probes in Table 3 for the target gene. Run dPCR on QIAcuity QIAGEN, and after outputting the data, the titer VG / ml = output result * dilution factor * 1000 * 2.4.

[0165] S52, according to the results shown in Table 4: the ratio of EGFP to mcherry in the AAV produced by the four clones is basically consistent with the gene integration situation, verifying that the production of recombinant AAV virus meets the expectations.

[0166]

[0167]

[0168] Summary: This implementation case integrates two different target genes EGFP and mCherry into host cells, selects single clone cells with a specific ratio, and successfully produces recombinant AAV virus from single clone cells stably integrated with target genes. The experimental results show that the ratio of EGFP to mCherry in the AAV virus is consistent with the gene integration situation, verifying the reliability and stability of the method. This method realizes the packaging of two target genes into AAV virus at a fixed ratio, providing an efficient and stable production platform for multi-gene combination therapy and complex gene research.

[0169] Example 2

[0170] Example 2 adjusts the transfection amount of the two target genes to obtain different ratios of stable transfection cells.

[0171] The steps of Example 2 are basically the same as those described in Material and Method 1 of Example 1;

[0172] The method for obtaining different ratios of stable transfection cells by adjusting the transfection amount of the two target genes includes the following steps:

[0173] S1, stably integrate multiple target genes into the host cell genome;

[0174] S2, screen single clone cells stably expressing target genes;

[0175] S3, co-transfect Rep gene plasmid and adenovirus helper gene plasmid into single clone cells stably expressing target genes containing AAV inverted terminal repeats;

[0176] S4, harvesting and purifying the recombinant AAV virus to obtain an AAV virus mixture with a specific ratio of target genes.

[0177] Example 2 differs from Example 1 in that:

[0178] S431, construct vectors, and construct sequences cap9 and cap9-EGFP in PiggyBac-R-ITR and PiggyBac-L-ITR into PiggyBacDualpromoter backbone vectors by gene synthesis to obtain a transfer plasmid expressing cap9 and a transfer plasmid expressing cap9-EGFP. The full-length gene sequence of the transfer plasmid expressing cap9 is SEQ ID NO. 3; the gene sequence of the transfer plasmid expressing cap9-EGFP is SEQ ID NO. 4. The sequence cap9 in PiggyBac-ITR is SEQ ID NO. 14, and the sequence cap9-EGFP in PiggyBac-L-ITR is SEQ ID NO. 15.

[0179] The AAV2 ITR sequences flanking the reporter gene cap9 and the reporter gene cap9-EGFP can be used to package AAV viruses. The sequence of the reporter gene cap9 is SEQ ID NO. 16, and the sequence of the reporter gene cap9-EGFP is SEQ ID NO. 17.

[0180] Example 2 differs from Example 1 in that:

[0181] S2, the method for screening single clone cells stably expressing target genes comprises:

[0182] S21, screening a stable AAV production cell line: obtain single clones by antibiotic screening method and limiting dilution method, the antibiotic can be Puromycin, Neomycin; and determine the ratio of target gene integration among various products obtained after the integration of the target gene into the host cell by quantitative PCR method to ensure that the screened single clone cells can stably express a specific ratio of target genes.

[0183] 6-well plates were inoculated with 1E+06 293T-pro cells, and DMEM / 10% FBS complete medium was used for culture at 37°C, 5% carbon dioxide overnight. Two transfer plasmids Cap9 and Cap9-EGFP and transposase were co-transfected into cells using lipo3000 transfection reagent according to the amount in Table 5, and the cells were passaged 48 h after transfection, and puromycin was added for screening. The cells were subcultured every 2 to 3 days, and stable target gene integrated cells were obtained after 3 weeks of screening. The fluorescence expression of the cells was detected using a flow cytometer.

[0184] Example 2 differs from Example 1 in that:

[0185] Gene integration copy number analysis

[0186] The genomic DNA of cells stably integrated with the target genes was extracted, and the integration copy numbers of the two genes Cap9 and Cap9-EGFP were analyzed by dPCR using the primer / dual-labeled probe combination for the GOI, as shown in Table 6. The specific integration copy numbers of the two genes were calculated using PTBP2 as the internal reference.

[0187] As shown in Table 7, after transfection, the cells had various different copy numbers of Cap9 and Cap9-EGFP. As the ratio of the cap transfer plasmid increased, the integration copy number of cap-EGFP decreased in a gradient; when the two transfer plasmids were mixed at a 1:1 ratio for transfection, the ratio of integrated Cap9 and Cap9-EGFP in the cells was close to 1:1. By adjusting the transfection amount of the two transfer plasmids, as shown in Table 5, stably transfected cells with different ratios were successfully obtained.

[0188]

[0189]

[0190]

[0191] Summary: In this example, by adjusting the transfection amount of the two target genes Cap9 and Cap9-EGFP, the integration copy numbers of the two genes Cap9 and Cap9-EGFP were successfully precisely regulated to meet the experimental requirements and expectations. In addition, it was found in the actual operation process that by adjusting the ratio of transposase to transfer plasmid, the integration efficiency can be further optimized. For example, increasing the amount of transposase and reducing the amount of transfer plasmid can reduce the total copy number after integration. This method provides a flexible and efficient technical means for precise regulation of the integration ratio of multiple genes.

[0192] Example 3

[0193] The hybrid virus produced by cross packaging in Example 3 has the characteristics of the capsids of both hybrid partners.

[0194] Example 3 differs from Example 1 in that:

[0195] The method for stably integrating multiple serotype capsid protein Cap genes into the genome of a host cell comprises:

[0196] 1. Packaging and purification of recombinant AAV virus

[0197] The operation adopts a transient transfection method to produce a hybrid capsid, inoculates HEK293 into a shake flask, and when the cell density reaches 2.5E+06 / ml, the capsid plasmid pAAV8, the helper plasmid pAdDelta6, and the transfer plasmid containing the AAV inverted terminal repeat sequence expressing EGFP are subjected to four-plasmid transfection.

[0198] The gene sequence of the target plasmid expressing AAV-EGFP is SEQ ID NO. 5. The pAAV2 plasmid is constructed by removing the AAV8 Cap gene from Addgene plasmid #112864 pAAV.R2C8 and inserting the AAV2 Cap gene. The AAV2 genome sequence is referenced to GeneBank: AF043303, and the pAAV2-7M8 is inserted with the polypeptide LALGETTRPA at the 588 position of cap8, and the sequence is derived from the patent NCT04645212. AAV2-7m8 has been used in clinical experiments and has stronger retinal targeting ability than AAV2.

[0199] Example 3 differs from Example 1 in that:

[0200] For each 30ml shake flask packaging system, the transfection mixture preparation is carried out according to the following system: 1. Add 45ug plasmid to OPM-293CD05 culture medium in the ratio of pAdDelta6: pAAV.RC: target gene = 2.2: 1: 1, wherein pAAV.RC contains two plasmid mixtures with the following combinations, respectively, pAAV2-7m8: pAAV8 = 1:0, 15:1, 5:1, 1:1 and 0:1; 2. Add 90μL of FectoVIR-AAV to the plasmid dilution, vortex for 3 seconds, and stand at room temperature for 30 min. 72h after transfection, add lysis solution, shake at 37℃ for 1h, centrifuge at 4000rpm for 10min to collect the supernatant, filter the supernatant with a 0.45μm filter, remove impurities and empty shells by iodixanol density gradient centrifugation, and obtain pure rAAV. The mixed serotype virus prepared above is detected for physical titer using the BGHpolyA primer and probe in Table 6.

[0201] Example 3 differs from Example 1 in that:

[0202] 2. Transduction activity of hybrid serotype capsid in in vitro cell lines

[0203] Inoculate 1E+05 APRE19 cells per well in 24-well plates with DMEM-F12 / 10% FBS complete medium, and incubate at 37℃, 5% CO2 overnight. According to the different MOI of viruses: 5E5, 1E5, 2E4, 4E3, 8E2, 1.6E2, 3.2E1, calculate the required virus volume vg per well, and dilute the AAV with complete medium. Take out the overnight cultured cells, aspirate the complete medium from the well plate, add 300 μL of medium containing diluted virus, and incubate in a 37℃, 5% CO2 cell incubator for 3 hours, then add 1 mL of fresh complete medium and continue to culture for 48 hours.

[0204] Collect the cells and use flow cytometry to detect the proportion of cell fluorescence. Derive the data and analyze the flow cytometry results using FlowJo. Figures 19-21 The transduction activity of hybrid viruses and mixed viruses in ARPE19 cells at different MOIs is shown, and the comparison with parent viruses AAV2.7m8 and AAV8 is shown. Figures 19-20 As can be seen from the figure, the hybrid virus mixed at a ratio of 1:1 has different transduction efficiencies from the homozygous virus mixed at a ratio of 1:1. Compared with the wild-type serotypes AAV2-7m8 and AAV8, the transduction efficiency of the hybrid virus is between the two, verifying the advantages of the hybrid virus with the characteristics of both capsids. According to the present application, by integrating two capsids into cells, a hybrid AAV can be stably produced by screening single clones.

[0205] The present embodiment successfully generates a hybrid AAV virus with the characteristics of AAV2-7m8 and AAV8 capsids through cross-packaging technology. The experimental results show that the hybrid virus has the advantages of both capsids. This method provides an efficient and stable technical platform for the production and application of hybrid AAV viruses, which is suitable for gene therapy and tissue-specific delivery research.

[0206] The present application realizes the stable production of multiple AAVs or hybrid AAVs by constructing a stable cell line that simultaneously integrates different target genes or capsid protein genes. Compared with the prior art, the present application has the following advantages:

[0207] 1. Simplified operation process and reduced production cost: By using gene integration technology, the dependence on high-cost plasmid DNA is avoided, the production process is simplified, and the production cost is significantly reduced.

[0208] 2. Multiple AAV viruses can be stably produced at the same time, and the ratio of different AAVs is determined, ensuring the consistency and controllability of product function and quality;

[0209] 3. Greatly reduces the error between batches: By using a stable cell line, the differences between batches caused by transient transfection are avoided, and the reproducibility and stability of production are significantly improved.

[0210] 4 Production of hybrid AAV viruses: capable of stably producing AAV viruses with different chimeric capsid ratios, and the virus particles have high homogeneity, meeting the needs of precise gene therapy;

[0211] 5 More suitable for industrial production, the use of stable cell lines makes the production process more controllable, which can meet the high demand of large-scale production of gene therapy drugs, and provides reliable technical support for industrial production.

[0212] In summary, by constructing a stable cell line, the limitations of the existing transient transfection method are overcome, and a high-efficiency, stable and economical AAV production platform is provided. This method not only simplifies the operation process and reduces the production cost, but also can stably produce various AAV or hybrid AAV, significantly improving the quality and consistency of the product, and providing strong technical support for gene therapy and biomedical research.

[0213] The above description is only some of the preferred embodiments of the present disclosure and the explanation of the technical principles applied. Those skilled in the art should understand that the scope of the application involved in the embodiments of the present disclosure is not limited to the technical solutions formed by the specific combinations of the above technical features, and should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the above inventive concept. For example, the above features are replaced with each other to form a technical solution, which is not limited to the technical features disclosed in the embodiments of the present disclosure but has similar functions.

Claims

1. A method for the simultaneous production of multiple recombinant adeno-associated viruses, characterized in that, The method comprises the following steps: stably integrating multiple target genes into the genome of host cells by PiggyBac system; obtaining single clone cells by antibiotic screening and limited dilution method, and screening single clone cell lines stably expressing specific proportions of target genes by quantitative PCR; co-transfecting a transfer plasmid containing AAV inverted terminal repeat sequences, a Cap / Rep gene plasmid and an adenovirus helper gene plasmid into the single clone cell line stably expressing specific proportions of target genes; harvesting and purifying recombinant AAV viruses to obtain AAV virus mixture with specific proportions of target genes; The method for stably integrating multiple target genes into the genome of host cells comprises the following steps: preparing a helper plasmid for recognizing and cutting the transposon plasmid sequence; preparing a transposon plasmid containing two inverted terminal repeat sequences and a transposed region between the two inverted terminal repeat sequences; the transposed region carries target genes; co-transfecting the helper plasmid and the transposon plasmid into host cells, and the transposase encoded by the helper plasmid recognizes the ITR element in the transposon of the transposon plasmid, and inserts the transposed region and the ITR element into the host genome.

2. A method for the simultaneous production of multiple recombinant adeno-associated viruses, characterized in that, The method comprises the following steps: stably integrating multiple capsid protein Cap genes of multiple serotypes into the genome of host cells by PiggyBac system; obtaining single clone cells by antibiotic screening and limited dilution method, and screening single clone cell lines stably expressing specific proportions of capsid proteins by quantitative PCR; co-transfecting a transfer plasmid containing AAV inverted terminal repeat sequences, a Rep gene plasmid and an adenovirus helper gene plasmid into the single clone cell line stably expressing specific proportions of capsid protein Cap genes; harvesting and purifying recombinant AAV viruses to obtain hybrid AAV viruses with specific proportions of capsid proteins.

3. The method for simultaneously producing multiple recombinant adeno-associated viruses according to claim 2, wherein: the helper plasmid is used to encode the transposase; the transposon plasmid contains two inverted terminal repeat sequences and a transposed region between the two inverted terminal repeat sequences; the sequences in PiggyBac-R-ITR and PiggyBac-L-ITR in the transposed region are genetically synthesized or PCR amplified, and are constructed into a PiggyBac Dualpromoter backbone vector by homologous recombination to obtain a transfer plasmid expressing EGFP and a transfer plasmid expressing mcherry; the full-length gene sequence of the transfer plasmid expressing PiggyBac-EGFP is SEQ ID NO. 1; the full-length gene sequence of the transfer plasmid expressing PiggyBac-mcherry is SEQ ID NO.

2.

4. The method for simultaneously producing multiple recombinant adeno-associated viruses according to claim 3, wherein: the method for the transposase to recognize the ITR element in the transposon of the transposon plasmid and insert the transposed region and the ITR element into the host genome comprises: the transposase recognizes and cuts the 5'TR and 3'TR sequences, and cuts the target genes of the transposed region from the transfer plasmid expressing EGFP and the transfer plasmid expressing mcherry, and the transposase randomly inserts the target genes into the TTAA site of the host cell genome.

5. The method for simultaneously producing multiple recombinant adeno-associated viruses according to claim 4, wherein: The method for co-transfecting the Cap / Rep gene plasmid and the adenovirus helper gene plasmid into the single clone cells containing the AAV inverted terminal repeat sequence and stably expressing the target gene comprises: The pAAV-RC and pAdDelta6 are transfected into the single clone cells stably expressing the target gene, respectively.

6. The method for simultaneously producing multiple recombinant adeno-associated viruses according to claim 5, wherein: The method for harvesting and purifying the recombinant AAV virus to obtain the AAV virus mixture with specific proportions of the target genes comprises: The cell suspension is obtained by transfection, Trypsin-EDTA is added to the cell suspension, the cell suspension is lysed, and the recombinant AAV virus particles are released; After centrifugation, the cell debris is removed, the supernatant is collected, and the AAV virus mixture with specific proportions of the target genes is obtained.

7. The method for simultaneously producing multiple recombinant adeno-associated viruses according to claim 3, wherein: The sequences in the PiggyBac-R-ITR and PiggyBac-L-ITR of the transposition region are genetically synthesized or PCR amplified, and are constructed into the PiggyBacDualpromoter (PB513B-1) skeleton vector by homologous recombination to obtain the transfer plasmid expressing EGFP and the transfer plasmid expressing mcherry; is replaced by The sequences in the PiggyBac-R-ITR and PiggyBac-L-ITR of the transposition region are genetically synthesized or PCR amplified, and are constructed into the PiggyBacDualpromoter (PB513B-1) skeleton vector by homologous recombination to obtain the transfer plasmid expressing cap9 and the transfer plasmid expressing cap9-EGFP; The full-length gene sequence of PiggyBac-cap9 is SEQ ID NO. 3; The full-length gene sequence of PiggyBac-cap9-EGFP is SEQ ID NO.

4.

8. The method for simultaneously producing multiple recombinant adeno-associated viruses according to claim 2, wherein: The method for stably integrating multiple serotypes of capsid protein Cap genes into the host cell genome comprises: The host cells are inoculated into a flask; the helper plasmid pAdDelta6, the Rep gene plasmid, and the transfer plasmid containing the AAV inverted terminal repeat sequence expressing EGFP are co-transfected into the single clone cells containing two serotypes of capsid proteins and stably expressing Cap; The full-length sequence of the transfer plasmid expressing pAAV-EGFP is SEQ ID NO.

5.

9. The method for simultaneously producing multiple recombinant adeno-associated viruses according to claim 2, wherein: The method for screening the single clone cells stably expressing the target gene comprises: The ratio between different integrated genes is determined by quantitative PCR, and the screened single clone cells can stably express the target genes with specific proportions.

10. The method for simultaneously producing multiple recombinant adeno-associated viruses according to claim 2, wherein: The method for screening the monoclonal cells stably expressing the capsid protein Cap gene of the serotype comprises: The monoclonal cell line capable of expressing the specific proportion of the capsid protein is screened out.

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