Method for simultaneously producing multiple recombinant adeno-associated viruses

Abstract

The invention discloses a method for simultaneously producing multiple recombinant adeno-associated viruses, and belongs to the field of gene therapy. Comprising the following steps: stably integrating multiple target genes into a host cell genome or stably integrating capsid protein Cap genes of multiple serotypes into the host cell genome; screening monoclonal cells of capsid protein Cap genes which stably express target genes or stably express serotypes; the method comprises the following steps: co-transfecting a transfer plasmid containing an AAV reverse terminal repetitive sequence, a Rep gene plasmid and an adenovirus auxiliary gene plasmid into a monoclonal cell of a capsid protein Cap gene for stably expressing a target gene or stably expressing a serotype; and harvesting and purifying the recombinant AAV virus to obtain an AAV virus mixture with a specific proportion of target genes or a hybrid AAV virus with a specific proportion of capsid proteins.

Description

Technical Field

[0001] This application relates to the field of gene therapy. Specifically, it relates to a method for simultaneously producing multiple recombinant adeno-associated viruses. Background Art

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

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

[0004] Existing AAV production generally uses the three-plasmid transient transfection method: a transfer vector containing the gene of interest (GOI); pHelper with adenovirus helper functions; and pAAV-Rep2CapXCapX, which supplies the capsid and replicase functions, i.e., the capsid functions of different AAV serotypes. If multiple AAVs or hybrid AAVs need to be produced simultaneously in one batch, multiple GOI or Cap plasmids need to be transfected simultaneously. Due to batch-to-batch differences and capsid heterogeneity, the quality standards of virus products are uncontrollable. Summary of the Invention

[0005] This section of the application is used to briefly introduce concepts, which will be described in detail in the following Detailed Description section. This section of the application is not intended to identify the key features or essential features of the claimed technical solution, nor is it intended to limit the scope of the claimed technical solution.

[0006] To solve the technical problems mentioned in the above Background Art section, some embodiments of this application provide a method for simultaneously producing multiple recombinant adeno-associated viruses, including: Stably integrating multiple genes of interest into the host cell genome or stably integrating capsid protein Cap genes of multiple serotypes into the host cell genome; Screening monoclonal cells stably expressing the gene of interest or stably expressing the capsid protein Cap gene of the serotype; A transfer plasmid containing AAV inverted terminal repeats, a Rep gene plasmid, and an adenovirus helper gene plasmid are co-transfected into monoclonal cells stably expressing a target gene or a capsid protein Cap gene of a stable expression serotype; The recombinant AAV virus is harvested and purified to obtain a mixture of AAV viruses with a specific ratio of the target gene or a hybrid AAV virus with a specific ratio of capsid proteins.

[0007] Furthermore, The method for stably integrating multiple target genes into the host cell genome includes: Prepare a helper plasmid for identifying and cleaving the transposon plasmid sequence; Prepare a transposon plasmid containing two inverted terminal repeats and the transposable region therebetween; the transposable region carries the target gene; The helper plasmid and the transposon plasmid are co-transfected into the host genome, and the transposase will recognize the ITR element in the transposon of the transposon plasmid and insert the transposable region and the ITR element into the host genome.

[0008] Furthermore, The helper plasmid is SuperPiggyBacTransposase; The transposon plasmid is obtained by gene synthesis or PCR amplification of the sequences in PiggyBac R-ITR and L-ITR and constructing them into the PiggyBacDualpromoterPB513B-1 backbone vector by homologous recombination to obtain the transfer plasmid expressing EGFP and the transfer plasmid expressing mcherry; The gene sequence of the transfer plasmid expressing PiggyBac-EGFP is SEQ ID NO.1; The transfer plasmid expressing PiggyBac-mcherry is SEQ ID NO.2.

[0009] Furthermore, The method by which the transposase recognizes the ITR element in the transposon of the transposon plasmid and inserts the transposable region and the ITR element into the host genome includes: The transposase recognizes and cleaves the 5'TR and 3'TR sequences, excises the target gene in the transposable region from the transfer plasmid expressing EGFP and the transfer plasmid expressing mcherry, and the transposase inserts the target gene into the TTAA characteristic insertion site of the host genome.

[0010] Furthermore, The method for co-transfecting the Cap / Rep gene plasmid and the adenovirus helper gene plasmid into monoclonal cells stably expressing the target gene containing AAV inverted terminal repeats includes: Transfect the monoclonal cells stably expressing the target gene screened with pAAV-R2C9 and pAdDelta6 at a molar ratio of 2.2:1 respectively.

[0011] Furthermore, The method for harvesting and purifying recombinant AAV virus to obtain a mixture of AAV viruses with a specific proportion of the target gene includes: After 48 hours of transfection, a cell suspension is obtained. Add 0.25% Trypsin-EDTA to the cell suspension, and repeatedly freeze-thaw the cell suspension three times to lyse the cells and release recombinant AAV virus particles; Centrifuge at 12,000 rpm for 5 minutes to remove cell debris, collect the supernatant to obtain a mixture of AAV viruses with a specific proportion of the target gene.

[0012] Furthermore, Synthesize or PCR amplify the sequences in the R-ITR and L-ITR of PiggyBac in the transposon region, and construct them into the PiggyBacDualpromoterPB513B-1 backbone vector by homologous recombination method to obtain the transfer plasmid expressing EGFP and the transfer plasmid expressing mcherry; Replace with Synthesize or PCR amplify the sequences in the PiggyBac R-ITR and L-ITR in the transposon region, and construct them into the PiggyBacDualpromoterPB513B-1 backbone vector by homologous recombination method to obtain the transfer plasmid expressing cap9 and the transfer plasmid expressing cap9-EGFP; The gene sequence of PiggyBac-cap9 is SEQ ID NO.3; The gene sequence of PiggyBac-cap9-EGFP is SEQ ID NO.4.

[0013] Furthermore, The method for stably integrating the capsid protein Cap genes of multiple serotypes into the host cell genome includes: Inoculate the host cells into a shake flask; When the host cell density reaches 2.5E+06 / ml, co-transfect the helper plasmid pAdDelta6, the Rep gene plasmid, and the transfer plasmid containing the AAV inverted terminal repeat sequence expressing EGFP into the monoclonal cells containing two serotype capsid proteins and stably expressing Cap; The gene sequence of the transfer plasmid expressing pAAV-EGFP is SEQ ID NO.5.

[0014] Furthermore, Methods for screening monoclonal cells stably expressing a target gene include: Monoclonal cells are obtained by antibiotic screening and limiting dilution methods, and the ratio between different integrated genes is determined by quantitative PCR. The screened monoclonal cells can stably express a specific ratio of the target gene.

[0015] Furthermore, Methods for screening monoclonal cells stably expressing the capsid protein Cap gene of a serotype include: By techniques such as antibiotic screening or fluorescence-activated cell sorting (FACS), monoclonal cell lines capable of expressing a specific ratio of capsid protein are screened out.

[0016] In this application, by stably integrating multiple target genes or capsid proteins Cap of different serotypes into the host cell genome and combining monoclonal screening techniques, cell lines stably expressing a specific ratio of target genes or capsid proteins are screened out, achieving the following advantages: It can effectively address the challenges brought by gene packaging size limitations, meet the therapeutic needs of multi-gene combination therapy or complex genes, and simultaneously achieve stable production of hybrid rAAV.

[0017] It overcomes the limitations of traditional multi-plasmid transfection or cross-packaging methods, provides an efficient and reproducible rAAV production platform for gene therapy and biomedical research, significantly improves the stability, scalability and cost-effectiveness of production, and at the same time ensures the consistency of product quality, with broad application prospects.

[0018] Gene integration and screening are carried out simultaneously: significantly saving time and cost. Each cell of the integrated monoclonal cells expresses the target gene or capsid protein, and the rAAV yield equivalent to that of traditional triple-plasmid transfection can be achieved.

[0019] Stable production of multiple AAV viruses with fixed ratios: The target genes in the screened monoclonal cells have specific ratios, ensuring consistent ratios in different batch productions, ensuring the consistency and controllability of product functions and quality, and avoiding batch-to-batch heterogeneity problems caused by unstable plasmid ratios in traditional methods.

[0020] Preparation of hybrid AAV viruses with specific ratios: Precisely regulating the integration ratios of multiple AAV capsids, thus stably and flexibly producing hybrid rAAV with different chimeric ratios. Moreover, the virus particles have high homogeneity, are easy to perform product characterization analysis, and have scalability and production reproducibility.

[0021] Simplifying the operation process and production technology: There is no need to rely on multi-plasmid mixing or cross-packaging. Multiple AAVs can be produced at one time, eliminating multiple virus purification and mixing steps, significantly reducing the complexity of the production process, and improving production efficiency.

[0022] Cost reduction: Reducing the usage amount of the plasmid of the gene of interest (GOI), reducing the number of plasmid production batches, lowering the production cost, and reducing the residual plasmid backbone DNA in the rAAV product and the infected tissue. Improving 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.

[0023] Ensuring product quality consistency: Ensuring the functionality and quality consistency of the rAAV complex to meet the high standards of gene therapy requirements. Brief Description of the Drawings

[0024] The drawings forming a part of this application are used to provide a further understanding of this application, making other features, objectives, and advantages of this application more obvious. The schematic embodiments and descriptions of the drawings of this application are used to explain this application and do not constitute an improper limitation of this application.

[0025] In the drawings: Figure 1 Shows the framework of the transfer plasmid, containing the gene of interest and the two ITRs shown and the elements therebetween expressing EGFP; Figure 2 Shows the framework of the transfer plasmid, containing the gene of interest and the two ITRs shown and the elements therebetween expressing mcherry; Figure 3 Shows the fluorescence photograph of monoclonal cells, showing the double-positive fluorescence of EGFP and mCherry in the selected monoclonal cells; Figure 4 Shows the fluorescence data of monoclonal cells detected by flow cytometry, showing the first example of the fluorescence proportion of EGFP and mCherry in monoclonal cells; Figure 5 Shows the fluorescence data of monoclonal cells detected by flow cytometry, showing the second example of the fluorescence proportion of EGFP and mCherry in monoclonal cells; Figure 6 Shows the fluorescence data of monoclonal cells detected by flow cytometry, showing the third example of the fluorescence proportion of EGFP and mCherry in monoclonal cells; Figure 7 Shows the fluorescence data of monoclonal cells detected by flow cytometry, showing the fourth example of the fluorescence proportion of EGFP and mCherry in monoclonal cells; Figure 8 Shows two transfer plasmid maps, containing the gene of interest and the two ITRs shown and the elements therebetween expressing cap9 and cap9-EGFP respectively; Figure 9It is a photograph of green fluorescence of cells 12 days after transfection and pressure application at a ratio of 1:0; Figure 10 It is a photograph of green fluorescence of cells 12 days after transfection and pressure application at a ratio of 1:1; Figure 11 It is a photograph of green fluorescence of cells 12 days after transfection and pressure application at a ratio of 1:1; Figure 12 It is a photograph of green fluorescence of cells 12 days after transfection and pressure application at a ratio of 1:50; Figure 13 It is a photograph of green fluorescence of cells 12 days after transfection and pressure application at a ratio of 0:1; Figure 14 The first example of fluorescence data detected by flow cytometry 12 days after transfection and pressure application; Figure 15 The second example of fluorescence data detected by flow cytometry 12 days after transfection and pressure application; Figure 16 The third example of fluorescence data detected by flow cytometry 12 days after transfection and pressure application; Figure 17 The fourth example of fluorescence data detected by flow cytometry 12 days after transfection and pressure application; Figure 18 The fifth example of fluorescence data detected by flow cytometry 12 days after transfection and pressure application; Figure 19 The first example representing the mixed transduction of capsid 1:1 packaged hybrid virus and homozygous virus at a ratio of 1:1; Figure 20 The second example representing the mixed transduction of capsid 1:1 packaged hybrid virus and homozygous virus at a ratio of 1:1; Figure 21 Represents the transduction of ARPE by viruses packaged with capsids in different ratios. Detailed implementation manners

[0026] Embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although some embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided to more thoroughly and completely understand the present disclosure. It should be understood that the drawings and embodiments of the present disclosure are only for exemplary purposes and are not used to limit the protection scope of the present disclosure.

[0027] In addition, it should be noted that for the sake of convenience of description, only parts related to the relevant invention are shown in the drawings. Without conflict, the embodiments in the present disclosure and the features in the embodiments can be combined with each other.

[0028] The present disclosure will be described in detail below with reference to the drawings and in combination with embodiments.

[0029] To clarify the content of this specification, the following definitions and explanations of terms apply to the specific use of the present invention. Unless otherwise defined, the technical and scientific terms used in the present invention are consistent with the ordinary understanding of those of ordinary skill in the art. The explanations of related terms can be referred to standard textbooks and literature known to those skilled in the art. The definitions provided herein are intended to clarify the specific meanings of these terms within the scope of the present invention. In addition, all references cited in the present invention are incorporated herein by reference to support the technical content of the present invention.

[0030] The term "vector" as used in the present invention refers to any molecule that can transport the heterologous nucleic acid contained therein into a host cell and exist in a free form or integrate into the host cell genome, such as nucleic acid, plasmid or virus, etc.

[0031] "Transfection" as used in the present invention refers to the transfer of polynucleotides, such as nucleic acid molecules, plasmids, etc., from outside the cell into the cell, such that the polynucleotide has a function within the cell. Transfection methods are well known to those skilled in the art, such as calcium phosphate or liposome-mediated transfection.

[0032] As described herein, adeno-associated virus (AAV) is an enveloped, naturally replication-defective DNA virus belonging to the Parvoviridae family. It is one of the simplest single-stranded DNA viruses discovered so far and requires a helper virus, usually adenovirus, to participate in replication. Its single-stranded DNA genome is about 4.7 kb in length and includes genes encoding replication proteins Rep, which are responsible for the replication, integration, and packaging of the AAV genome, and viral capsid Cap, which determines the serotype and tissue tropism of AAV, as well as two 145-bp inverted terminal repeats (ITRs) located on both sides of the genes. The ITR is the minimum self-sequence required for AAV replication and packaging and is also a cis-acting element required for AAV replication, packaging, and integration.

[0033] The PiggyBac transposon described herein is an autonomous eukaryotic transposon derived from the cabbage looper moth. Its gene transposition principle is based on the "cut-and-paste" mechanism mediated by transposase. The full length of this transposon is 2476 bp, with 13-bp inverted terminal repeats (ITRs) at both ends, and contains an open reading frame (ORF) of 2.1 kb in the middle, which can encode transposase with autonomous transposition function. In application, the PiggyBac system consists of two plasmids: the helper plasmid is responsible for expressing transposase, and the transposon plasmid carries the optimized ITR sequences on both sides and the target gene inserted therein. Transposase precisely cuts the target gene from the transposon plasmid by specifically recognizing and binding to the ITR sequence, and then inserts it into the TTAA tetranucleotide site in the host genome.

[0034] The "gene integration" described in the present invention can be achieved not only through the PiggyBac transposon, but also through other transposon strategies, lentivirus, homologous recombination, and other gene insertion strategies. Lentivirus belongs to a subclass of the Retroviridae family, and its gene integration mechanism depends on the process of reverse transcription of the viral RNA genome into DNA and insertion into the host genome by integrase. When infecting host cells, lentivirus releases its positive-sense single-stranded RNA (ssRNA) genome into the cytoplasm, and then reverse transcriptase converts it into double-stranded DNA (proviral DNA), which is permanently integrated into the chromosomal DNA of the host cell by integrase. Gene homologous recombination is a process in which two DNA molecules with the same or highly similar sequences exchange genetic material through strand breakage, pairing, and re-ligation. Its core mechanism involves the Holliday model, that is, after the homologous regions form joint molecules, endonucleases and ligases catalyze the cleavage and re-ligation of the strands to complete the crossover of double-stranded DNA. Other gene insertion strategies include Prime editing and CRISPR-mediated transposon CAST, etc.

[0035] "Recombination" as described in the present invention refers to heterologous nucleic acid sequences that do not naturally exist in the vectors and host cells containing them, and the heterologous sequences can be nucleic acids of interest for expressing target proteins.

[0036] The recombinant AAV vector (rAAV) described in the present invention refers to a viral vector constructed by replacing the Rep and Cap genes in the AAV genome with exogenous genes of interest (GOI). rAAV generally includes an AAV capsid and a recombinant viral genome and target nucleic acid 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 viral replication and packaging. When there are no rep and cap genes in its viral genome, the AAV vector does not integrate into the genome of the transduced cells, but exists in the nucleus of the transduced cells in the form of an episome, and can stably express exogenous genes for a long time. In the natural system, the infection of AAV requires the participation of helper viruses such as adenovirus or herpesvirus, and the helper viruses provide genes required for replication, such as E1A, E1B, E2A, E4, VA RNA, etc. In the production of rAAV vectors, helper viruses can be not used, but the essential genes contained in the helper viruses can be provided by plasmids to support the replication and packaging of AAV. The recombinant viruses that can be used to express the genes of interest in the present invention can be viral vectors derived from various types of viruses, including but not limited to adeno-associated virus (AAV) vectors, adenovirus vectors, lentivirus vectors, retrovirus vectors, herpes simplex virus (HSV) vectors, etc.

[0037] As used herein, the term "target nucleic acid" refers to a nucleic acid to be packaged into recombinant AAV viral particles, which encodes a nucleic acid for any prophylactic or therapeutic protein or a protein for marker detection such as GFP, mCherry, Cap protein, etc. The target nucleic acid is contained in an expression cassette and is packaged within the AAV capsid. In some embodiments, the expression cassette contains at least one ITR sequence to ensure that the vector genome can be assembled into the capsid. The expression cassette can be single-stranded DNA, double-stranded DNA, single-stranded RNA, or double-stranded RNA. In some embodiments, the expression cassette may contain one or more regulatory sequences to direct the expression of the target nucleic acid coding sequence in cells. The regulatory sequences can be selected from transcriptional initiation sequences, termination sequences, promoters, and / or enhancer sequences operably linked to the coding sequence; effective RNA processing signals such as splicing and polyadenylation polyA regions, such as the human growth hormone polyadenylation region; inverted repeat sequences such as L-ITR or R-ITR; selectable markers or reporter genes, such as resistance genes including but not limited to ampicillin, hygromycin, neomycin, methotrexate, kanamycin, gentamicin, Zeocin, or tetracycline; post-transcriptional regulatory sequences, such as the post-transcriptional regulatory sequence of woodchuck hepatitis virus WPRE; sequences that stabilize cytoplasmic mRNA; nucleic acid restriction sites; homologous recombination sequences; sequences that enhance translation efficiency such as Kozak sequences; sequences that enhance protein stability; and sequences that enhance the secretion of the encoded product. In some preferred embodiments, the regulatory sequences are selected from one or more of the following: inverted repeat sequences, enhancers, promoters, polyadenylation regions, selectable markers or reporter genes. 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.

[0038] According to the present invention, the genes encoding the components required for the production of AAV are selected from the genes encoding AAV Cap proteins VP1, VP2, and VP3; the genes encoding AAV Rep proteins Rep78, Rep68, Rep52, and Rep40; the genes encoding adenovirus helper functions E4orf6, E2A, and VA-RNA; and the gene of interest GOI flanked by AAV ITRs. In a specific embodiment, the genes encoding the components required for the production of AAV consist of two different genes of interest GOI flanked by AAV ITRs, and these genes of interest are not particularly limited and include any gene that needs to be delivered to target cells, such as GFP green fluorescent protein and mcherry red fluorescent protein in some cases.

[0039] The term "capsid protein" refers to a protein that is part of a 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 start 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.

[0040] AAV serotype refers to different variants of AAV, which have differences in the amino acid sequences of their capsid proteins, resulting in different serotypes showing different tropisms for specific tissues or cell types. The AAV in the present invention is not limited to a specific AAV serotype. Thus, AAV can be selected from AAV serotype 1 (AAV1), AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, AAVDJ8, AAVrh10, hybrids of different said serotypes, and said serotypes with mutations that alter AAV serotype tropism. Preferably, AAV is selected from AAV2, AAV8, and AAV9. According to the present invention, some or all of the genes encoding the components required for AAV production 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 and capsid in the AAV vector can be from different AAV serotypes. For example, AAV2 ITR can be selected to be used with a capsid other than AAV2, such as the capsid of AAV5, AAV8, or AAV9.

[0041] The term "tropism" refers to the targeting of a recombinant viral vector to specific cells, tissues, and / or organs. The term "chimeric AAV capsid" refers to an AAV capsid protein that includes amino acid sequences from two or more different AAV serotypes and is capable of forming an AAV capsid. Examples include but are not limited to the capsid of AAVDJ. The term "modified AAV capsid" refers to an AAV capsid protein that includes an insertion, substitution, or deletion of one or more amino acids, or an AAV capsid formed by inserting a polypeptide derived from another binding domain. Examples include but are not limited to AAV2-7m8. The "cross-packaging" described in the present invention is a method of generating a hybrid rAAV with mixed characteristics by combining capsid proteins of different serotypes with a target gene. Cross-packaging can change the cell targeting characteristics of AAV and enhance its infection efficiency and specificity for specific cells or tissues.

[0042] The present invention relates to a method for preparing recombinant AAV virus particles (rAAV) and its related culture system: the rAAV production culture includes the following key components: 1. Suitable host cells, such as cell lines derived from humans like HEK-293T cells, or cell lines derived from insects are used in the baculovirus production system; 2. Suitable helper functions, which are provided by wild-type or mutant adenoviruses such as temperature-sensitive adenoviruses, herpesviruses, baculoviruses, or plasmid pHelper constructs that provide helper functions; 3. AAVrep and cap gene pRC, which are used to support the replication of AAV and the assembly of capsid proteins; 4. A transfer plasmid containing a recombinant AAV genome, comprising a gene of interest flanked by at least one intact functional ITR, and preferably under the drive of an operably linked promoter; and a suitable culture system to support the efficient production of rAAV.

[0043] 5. Additionally, the present invention also allows for the prior integration of the AAV rep and cap genes into the packaging cell genome, so that during production, only the nucleic acid molecule containing the recombinant AAV genome and the nucleic acid molecule of the helper virus gene need to be transfected, further simplifying the production process and improving stability.

[0044] The stable AAV production cell line used in the present invention 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 yeast, such as cells of the HEK293 series like 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 those skilled in the art, a suitable packaging cell line can be selected according to the different types of recombinant viral vectors. The 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 increase the titer of virus particles. The culture conditions can be optimized according to the specific host cell used.

[0045] The "viral genome (ViralGenome, vg)" as described in the present invention refers to a polynucleotide sequence that can be packaged into a viral capsid, which at least contains an expression cassette of the 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 the ITR sequence of AAV, and may also contain other viral nucleic acid elements such as enhancers, promoters, polyadenylation signals. According to the different types of recombinant viral vectors, the recombinant viral vector genome can contain single-stranded DNA, double-stranded DNA, single-stranded RNA, or double-stranded RNA. In some embodiments, the titer of the recombinant viral vector genome is measured in vg / mL, which refers to the number of copies of the recombinant viral genome contained in each milliliter of solution and is used to quantify the concentration of the viral preparation.

[0046] The terms "transduction" or "infection", etc. refer to the process of introducing nucleic acids into target cells through viral vectors and releasing their genetic materials, including the nucleic acids of interest. The term "transduction efficiency" refers to the proportion of cells expressing the nucleic acids of interest, usually expressed as a percentage, after incubation with a set number of viral vectors containing the nucleic acids of interest. 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 fluorescent reporter genes such as GFP, and using flow cytometry to detect the proportion of fluorescent-positive cells, thereby evaluating transduction efficiency. Polymerase chain reaction (PCR): By detecting the expression level of the nucleic acids of interest in target cells, indirectly evaluating transduction efficiency. 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 applicable techniques can be selected according to specific experimental requirements.

[0047] Among them, in the "wild-type" (wtAAV) form of AAV, the AAV genome size is approximately 4.7 kb, and its structure includes two inverted terminal repeats (ITRs), and two major open reading frames: rep is responsible for viral replication and cap encodes the capsid protein [Merten, O.-W. Microorganisms 2024, 12, 384]. The AAV genome is packaged into the viral capsid. After infecting cells, the molecular mechanism of the cells converts single-stranded DNA into double-stranded form, and then transcribes and translates it into polypeptides. However, the loading capacity of AAV is limited, usually only able to accommodate exogenous gene fragments of about 4.1 - 4.7 kb, and this limitation mainly stems from the physical size of the AAV capsid and the genome packaging mechanism.

[0048] Currently, the number of gene therapy trials and products based on AAV vectors is increasing rapidly, but the main challenge in translating this technology into clinical applications lies in how to efficiently deliver a large number of high-quality AAV viral vectors. Existing AAV production mainly relies on two systems: the transient transfection system based on HEK293 cells and Sf9 / Baculovirus systems. These systems usually can only deliver a single AAV, and the AAV load limit poses challenges to their applications in gene editing and gene therapy, especially when the target gene is large, such as certain large genes or genes that require additional regulatory elements. To break through this limitation, more and more studies have adopted dual-vector or multi-vector systems to achieve the delivery of large genes (Hum Gene Ther. 2023, 34(13 - 14): 616 - 628; Nat Commun. 2024, 15(1): 6141). In addition, many diseases are caused by abnormalities in multiple genes or signaling pathways, so simultaneous treatment of multiple targets is required (Hum Mol Genet. 2013, 17(22): 4929 - 4937).

[0049] The serotypes of AAV are important resources in the gene therapy field. Currently, more than 200 AAV serotypes have been registered. Among them, there are 13 different AAV serotypes in primates, namely AAV1 - AAV13, and AAV2 is the earliest cloned and most widely studied serotype. The AAV capsid plays a key role in determining its tissue specificity. Different serotypes, such as AAV1, AAV2, AAV5, etc., show different tropisms for different tissues or cell types due to the differences in their capsid proteins. This specificity is determined by the interaction between the AAV capsid and specific receptors on the surface of host cells.

[0050] With the continuous deepening of research, researchers have found that different serotypes of AAV can hybridize, and the generated hybrid or chimeric AAVs have the characteristics of both hybrid partners, significantly expanding the application potential of AAV in gene therapy. Hybrid or chimeric AAV viruses refer to viruses obtained by fusing or genetically modifying the capsid proteins of different viruses, and their capsid composition has an important impact on the gene transduction characteristics of the vector. This strategy can make up for the defects of a single serotype, achieve the heterotic advantage between AAV serotypes, and generate unique AAV vectors with serotype specificity. Hybrid or chimeric AAVs can change tissue tropism and more precisely target specific tissues or cell types. Other advantages include improved receptor binding efficiency, optimized tissue tropism, and improved new purification strategies, etc. (Mol Ther, 2005, 116:856 - 65). Recombination between serotypes has been proven to occur in nature: for example, AAV6 is a natural variant resulting from the recombination of AAV1 and AAV2, with only a 71bp exchange (J. Virol, 1999, 73:3994 - 4003). Researchers have used cross-packaging to generate hybrid AAVs, improving the targeting and therapeutic effects of AAV in the treatment of muscle and nervous system diseases. For example, the AAV2.1 vector is composed of the capsid proteins of AAV1 and AAV2. After injection into the frontal cortex of macaques, this vector simultaneously exhibits excellent exogenous gene expression ability and neuron specificity (Nat Commun. 2023, 8, 141:4762).

[0051] Although hybrid AAVs show great potential in gene therapy, their production still faces the following challenges: Traditional methods mainly prepare hybrid AAVs through multi-plasmid cross-packaging transfection, which has problems such as AAV heterogeneity, functional inconsistency, and high difficulty in commercial preparation.

[0052] In the field of gene therapy, the cross-packaging and multi-gene packaging strategies of AAV are becoming key technologies for a new generation of gene delivery tools. These strategies significantly improve the precision, effectiveness, and applicable scope of gene therapy by optimizing the capsid proteins and gene loading capacity of AAV.

[0053] Cross-packaging can generate hybrid rAAVs with mixed characteristics by combining the capsid proteins of different serotypes with the target gene. Hybrid rAAVs have new phenotypes and tropisms. Researchers can select the best vector transduction according to the treatment needs and deliver it specifically to the target tissues or cells of patients. This strategy not only improves the therapeutic effect but also reduces the impact on non-target cells, thereby reducing the side effects of the treatment.

[0054] Multigene packaging overcomes the limitation of the single AAV payload by integrating multiple genes or regulatory elements into the AAV vector, providing greater flexibility for gene therapy. Targeting multiple disease treatment targets simultaneously allows more gene information to be carried and transmitted, thus opening up new possibilities for treating a greater variety of diseases.

[0055] For these strategies, the development of gene therapy products requires a cell line capable of stably producing such AAVs for commercialization. In the future, we can expect these strategies to play an even more important role in treating various genetic diseases, cancers, and other chronic diseases, bringing more benefits to human health.

[0056] Refer to Figures 1 - 21 , Example 1

[0057] A method for simultaneously producing multiple recombinant adeno-associated viruses, specifically a method for producing, in a single batch, AAV viruses expressing two fixed ratios through monoclonal cells screened by the PiggyBac stable transfection system. This method includes the following steps: S1. Stably integrate multiple target genes into the host cell genome or stably integrate the capsid protein Cap genes of multiple serotypes into the host cell genome; S2. Screen monoclonal cells stably expressing the target gene or stably expressing the capsid protein Cap gene of the serotype; S3. Co-transfect the Cap / Rep gene plasmid and the adenovirus helper gene plasmid into the monoclonal cells containing the AAV inverted terminal repeat sequence and stably expressing the target gene or the capsid protein Cap gene of the serotype; S4. Harvest and purify the recombinant AAV viruses to obtain a mixture of AAV viruses with specific ratios of target genes or hybrid AAV viruses with specific ratios of capsid proteins.

[0058] Specifically, The PiggyBac system contains two vectors. One vector is called the helper plasmid, which is responsible for encoding the transposase for recognizing and cleaving the transposon sequence. The other vector is called the transposon plasmid, which contains two inverted terminal repeat sequences ITRs and the transposable region between them, carrying the target gene GOI to be transposed into the host genome.

[0059] Achieve the integration of foreign genes through the PiggyBac transposon gene expression system; the PiggyBac system has the advantages of a large vector capacity, high integration efficiency, and strong stability, and can achieve the co-expression of multiple genes The method for stably integrating multiple target genes into the host cell genome includes: S11. Prepare an auxiliary plasmid for identifying and cutting the transposon plasmid sequence; the auxiliary plasmid is used to encode a transposase; S12. Prepare a transposon plasmid containing two inverted terminal repeats and the transposable region therebetween; the transposable region carries a target gene; S13. Co-transfect the auxiliary plasmid and the transposon plasmid into a host cell, and the transposase will recognize the ITR element in the transposon of the transposon plasmid and insert the transposable region and the ITR element into the host genome.

[0060] The method by which the transposase recognizes the ITR element in the transposon of the transposon plasmid and inserts the transposable region and the ITR element into the host genome includes: S131. Construct a vector, perform gene synthesis or PCR amplification on the sequences in PiggyBac-R-ITR and PiggyBac-L-ITR, and construct them into the PiggyBacDualpromoterPB513B-1 backbone vector by homologous recombination to obtain the transfer plasmid expressing EGFP and the transfer plasmid expressing mcherry; The overall sequence containing EGFP-Puromycin inserted in PiggyBac-L-ITR and PiggyBac-R-ITR is SEQ ID NO.6; the overall sequence containing mCherry-Neomycin inserted in PiggyBac-ITR is SEQ ID NO.7. Among them, the single R-ITR sequence is SEQ ID NO.10, and the single R-L-ITR sequence is SEQ ID NO.11.

[0061] 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 the two AAV2-ITRs shown and the elements therebetween. The sequences of the two AAV2-ITRs are SEQ ID NO.8 and SEQ ID NO.9 respectively.

[0062] The full-length gene sequence of the transfer plasmid expressing mcherry is SEQ ID NO.2. Refer to Figure 2 . The transfer plasmid expressing mcherry contains the two AAV2-ITRs shown and the elements therebetween.

[0063] The PiggyBac transposase recognizes and cleaves the PiggyBac-L-ITR and PiggyBac-R-ITR sequences, excises the target gene in the transposable region from the transfer plasmid, and inserts the target genes EGFP-puromycin and mcherry-neomycin into the characteristic TTAA insertion sites of the host genome, ultimately achieving gene integration. The target genes EGFP-puromycin and mcherry-neomycin are located between PiggyBac-R-ITR and PiggyBac-L-ITR.

[0064] In addition, the AAV2ITR 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.

[0065] S2. The method for screening monoclonal cells stably expressing the target gene or the capsid protein Cap gene of the serotype includes: S21. Screening a stable AAV-producing cell line: Obtaining monoclonal cells by antibiotic screening and limiting dilution methods. The antibiotics can be Puromycin and Neomycin; and determining the integration ratio of the target gene among various products obtained after integrating multiple target genes into the host cell by quantitative PCR to ensure that the screened monoclonal cells can stably express a specific ratio of the target gene. The stable AAV-producing cell line used in the present invention 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, such as cells of the HEK293 series like HEK293A, HEK293T or HEK293FT, HeLa cells, A549 cells, Vero cells, hARPE-19 cells, and insect-derived cell lines such as SF-9. As known to those skilled in the art, an appropriate packaging cell line can be selected according to the different types of recombinant virus vectors. The 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 increase the titer of virus particles. The culture conditions can be optimized according to the specific host cells used.

[0066] Gene integration: S211. Cell culture: Resuscitate 293FT cells, inoculate 1.5E+05 cells per well in a 24-well plate, and culture with DMEM / 10% fetal bovine serum complete medium at 37°C and 5% carbon dioxide for a period of time, preferably overnight. When the cell confluence reaches 70%-80%, perform transfection; S212, Transfection: Use the lipo3000 transfection reagent from Thermo to co-transfect 293FT cells with PiggyBac-EGFP-Puromycin, PiggyBac-mCherry-Neomyocin, and the transposase PB200PA-1 at a total amount of 500 ng in a ratio of 1:1:1. After 48 hours of transfection, passage the cells and simultaneously add puromycin and G-418 for screening. After one week of screening, obtain monoclonal cells that co-express EGFP and mcherry. Continuously dilute the cell suspension and seed it into a 96-well plate at a theoretical value of 1 cell per well for further culture. Observe the monoclonal growing cells under a microscope, mark them, and passage them for expansion culture.

[0067] Screening and identification of stable cell lines: S213, Monoclonal screening: Four monoclonal cells that are double positive for EGFP and mCherry are found under a microscope. Refer to Figure 2 . Expand their culture. After digestion with Typsin, add DMEM containing 10% FBS to terminate the digestion, and detect the proportion of the two fluorescences of the cells by flow cytometry.

[0068] S214, Result analysis: Figure 4 The results show that these cells are double positive for EGFP and mCherry, further confirming the success of gene integration.

[0069] Analysis of gene integration copy number S215, Detection: After expanding the culture of monoclonal cells, extract the cell genome according to the instructions of the Dneasy Blood & Tissue Kit #69506 from Qiagen. Use the primers / probes targeting the gene of interest (GOI), as shown in Table 1. Analyze the integration copy number and ratio of the two genes using digital PCR (dPCR). Briefly introduce the process of cell genome extraction and dPCR sample pretreatment below: Cell genomic extraction: Centrifuge 1E6 cells at 300 g, discard the supernatant, resuspend in 200 μl PBS, add 20 μl Proteinase K and vortex thoroughly, then add 4 μl of 100 mg / ml Rnase A solution and let stand for 5 min. Add 200 μl Buffer AL to the cell suspension, vortex thoroughly, and incubate in a water bath at 56 °C for 10 min. After incubation, add 200 μl of absolute ethanol and mix by oscillation. Transfer the supernatant to the genomic extraction column of the Dneasy Blood & Tissue Kit, centrifuge at 12,000 rpm for 1 min, and discard the filtrate. Add 500 μl AW1 to the filter column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate. Add 500 μl AW2 to the filter column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate. Transfer the filter column to a clean centrifuge tube, add 50 μl of preheated sterile water to the center of the membrane, centrifuge at 12,000 rpm for 1 min, collect the centrifuged DNA and measure its concentration.

[0070]

[0071] Sample processing: Prepare the digestion system - Take 1 μl of the extracted genome, add 1 μl of EcoRI - HF NEB, R3101S, 5 μl of rCutsmart buffer, and 43 μl of H2O, mix well and place in a PCR instrument for digestion at 37 °C for 30 min. After completion, dilute 2 - fold with H2O for dPCR reaction.

[0072] The dPCR reaction contains the following components: 3 μl of 4×Probe PCR Master Mix QIAGEN, 1.6 μl of nuclease - free H2O, 0.96 μl each of 10 μM primer / probe, and 5 μl of the diluted sample. According to the program: pre - denaturation at 95 °C for 2 min; denaturation at 95 °C for 15 s, annealing and extension at 60 °C for 30 s, 40 cycles to complete the detection.

[0073] S216. Results: As shown in Table 2, there are multiple monoclonal cells with different ratios of two genes, such as 1:1, 1:2, etc. Fixed values are detected by quantitative PCR, and thus the integration ratio of the two target genes can be confirmed.

[0074]

[0075] S3. The method of co - transfecting the Cap / Rep gene plasmid and the adenovirus helper gene plasmid into monoclonal cells stably expressing the target gene containing the AAV inverted terminal repeat sequence includes: S31. Transfect the selected monoclonal cells stably expressing the target gene with pAAV - R2C9 and pAdDelta6 at a molar ratio of 2.2:1 respectively.

[0076] S311. Plasmid construction: Construction of the pAAV-R2C9 plasmid, i.e., the pR2C9 packaging plasmid, which expresses the Rep gene of AAV2 and the Cap gene of AAV9. The pAAV-R2C9 plasmid was constructed by removing the AAV8 Cap gene from Addgene plasmid #112864 pAAV.R2C8 and inserting the AAV9 Cap gene. The AAV9 genome sequence refers to GeneBank: AY530579.1.

[0077] The helper plasmid pAdDelta6, i.e., the pHelper plasmid, was constructed by synthesizing DNA according to the sequence of Addgene plasmid #112867.

[0078] S312. Transfection: Transfect pAAV-R2C9 and pAdDelta6 into the monoclonal cells stably expressing the target gene (integrated with the target genes EGFP and mCherry) at a molar ratio of 2.2:1 with a total mass of 26 μg.

[0079] S4. Method for harvesting and purifying recombinant AAV virus to obtain a mixture of AAV viruses with a specific proportion of the target gene includes: S41. After 48 hours of transfection, a cell suspension is obtained. There are recombinant vectors in the cell suspension. Add 0.25% Trypsin-EDTA to the cell suspension, and freeze-thaw the cell suspension three times to lyse the cells and release the recombinant AAV virus particles. S42. Centrifuge at 12,000 rpm for 5 minutes to remove cell debris, collect the supernatant, and obtain a mixture of AAV viruses with a specific proportion of the target gene.

[0080] Obtaining a mixture of AAV viruses with a specific proportion of the target gene, i.e., a hybrid AAV virus.

[0081] The method for simultaneously producing multiple recombinant adeno-associated viruses also includes: S5. Determine the viral genome titer of the crude cell lysate of the recombinant AAV vector by digital PCR (dPCR).

[0082] S51. Treat the AAV virus mixture with DNaseI at 37°C for 30 minutes to remove the residual plasmid DNA during cell transfection and the free DNA of the cell's own genome. Add EDTA with a final concentration of 12 mM, pH 8.0, to inactivate DNaseI. Then add a 0.1% SDS solution and heat at 95°C for 30 minutes to release the viral genome. Use the primers / probes for the target gene in Table 3 to determine the titer of AAV in vg / mL by dPCR. Run dPCR on QIAcuity QIAGEN. After outputting the data, the titer in VG / ml = output result * dilution factor * 1000 * 2.4.

[0083] S52. As shown in the results of Table 4, in the AAVs produced by the four clones, the ratio of EGFP to mcherry was basically consistent with the gene integration situation, verifying that the production of the recombinant AAV virus met the expectations.

[0084]

[0085]

[0086] Summary: In this example, by integrating two different target genes, EGFP and mCherry, into host cells, monoclonal cells with specific ratios were screened, and recombinant AAV viruses were successfully produced from the monoclonal cells stably integrating the target genes. The experimental results showed that the ratio of EGFP to mCherry in the AAV virus was consistent with the gene integration situation, verifying the reliability and stability of this method. This method achieved the packaging of two target genes into AAV viruses in a fixed ratio, providing an efficient and stable production platform for multi-gene combination therapy and the study of complex genes.

[0087] Example 2

[0088] In Example 2, stable transfected cells with different ratios were obtained by adjusting the transfection dosages of the two target genes.

[0089] The steps of Example 2 were basically the same as those described in Materials and Methods 1 of Example 1; The method for obtaining stable transfected cells with different ratios by adjusting the transfection dosages of the two target genes included the following steps: S1. Stably integrate multiple target genes into the host cell genome; S2. Screen monoclonal cells stably expressing the target genes; S3. Co-transfect the Rep gene plasmid and the adenovirus helper gene plasmid into the monoclonal cells stably expressing the target genes containing the AAV inverted terminal repeats; S4. Harvest and purify the recombinant AAV virus to obtain a mixture of AAV viruses with specific ratios of target genes.

[0090] The difference between Example 2 and Example 1 was that: S431. Construct vectors. Synthesize the sequences cap9 and cap9-EGFP in PiggyBac-R-ITR and PiggyBac--L-ITR into the PiggyBac Dualpromoter backbone vector 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.

[0091] The AAV2 ITR sequences on both sides of 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.

[0092] The difference between Example 2 and Example 1 also lies in: S2. The method for screening monoclonal cells stably expressing the target gene includes: S21. Screen a stable AAV-producing cell line: Obtain monoclonal cells through antibiotic screening and limited dilution methods. The antibiotics can be Puromycin and Neomycin; and use quantitative PCR to determine the proportion of the target gene integration among multiple products obtained after the integration of multiple target genes into the host cell, to ensure that the screened monoclonal cells can stably express a specific proportion of the target gene.

[0093] Inoculate 1E+06 293T-pro cells into each well of a 6-well plate, use DMEM / 10% FBS complete medium, and culture overnight at 37°C and 5% carbon dioxide. According to the dosage in Table 5, use lipo3000 transfection reagent to co-transfect the cells with two transfer plasmids Cap9 and Cap9-EGFP and the transposase. After 48 hours of transfection, passage the cells and add puromycin for screening at the same time. Pressurize and passage the cells every 2 to 3 days. After 3 weeks of screening, obtain cells stably integrated with the target gene, and use a flow cytometer to detect the fluorescence expression of the cells.

[0094] The difference between Example 2 and Example 1 also lies in: Analysis of gene integration copy number Extract the genomic DNA of the cells stably integrated with the target gene. The primer / double-labeled probe combination targeting the GOI is shown in Table 6. Analyze the integrated copy numbers of the two genes Cap9 and Cap9-EGFP by digital PCR (dPCR), and calculate the specific integrated copy numbers of the two genes using PTBP2 as an internal reference.

[0095] As shown in Table 7, after transfection, there are various copy numbers of Cap9 and Cap9-EGFP in the cells. As the proportion of the cap transfer plasmid increases, the integrated copy number of cap-EGFP shows a gradient decrease; when the two transfer plasmids are transfected in a 1:1 ratio, the ratio of integrated Cap9 and Cap9-EGFP in the cells is close to 1:1. By adjusting the transfection dosages of the two transfer plasmids as shown in Table 5, stable transfected cells with different ratios were successfully obtained.

[0096]

[0097]

[0098]

[0099] Summary: In this example, by adjusting the transfection dosages of the two target genes Cap9 and Cap9-EGFP, precise regulation of the integrated copy numbers of the two genes Cap9 and Cap9-EGFP was successfully achieved to meet the experimental requirements and was in line with expectations. In addition, it was found during the actual operation process that the integration efficiency could be further optimized by adjusting the ratio of the transposase to the transfer plasmid. For example, increasing the transposase dosage and reducing the transfer plasmid dosage could reduce the total copy number after integration (data not shown). This method provides a flexible and efficient technical means for the precise regulation of the integration ratio of multiple genes.

[0100] Example 3

[0101] The hybrid virus generated by cross-packaging in Example 3 has the characteristics of the capsids of both hybrid partners.

[0102] The difference between Example 3 and Example 1 is as follows: The method for stably integrating the Cap genes of capsid proteins of multiple serotypes into the host cell genome includes: 1. Packaging and purification of recombinant AAV viruses This operation uses the transient transfection method to generate hybrid capsids. HEK293 cells are inoculated into a shake flask. When the cell density reaches 2.5E+06 / ml, the capsid plasmids pAAV8, pAAV2-7m8, the helper plasmid pAdDelta6, and the transfer plasmid containing the AAV inverted terminal repeat sequence expressing EGFP are transfected with four plasmids.

[0103] The gene sequence of the target plasmid expressing AAV-EGFP is SEQ ID NO.5. The pAAV2 plasmid was constructed by removing the AAV8Cap gene from Addgene plasmid #112864 pAAV.R2C8 and inserting the AAV2Cap gene. The AAV2 genome sequence refers to GeneBank: AF043303. pAAV2-7M8 inserts the polypeptide LALGETTRPA at position 588 of cap8. The sequence is derived from patent NCT04645212. AAV2-7m8 has been used in clinical trials and has a stronger retinal targeting ability than AAV2.

[0104] Example 3 is also different from Example 1 in that: For each 30 ml shake flask packaging system, the transfection mixture was prepared according to the following system: 1 Add 45 μg of plasmid to the OPM-293CD05 medium at a ratio of pAdDelta6: pAAV.RC: target gene = 2.2: 1: 1. Among them, 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 90 μL of FectoVIR-AAV to the plasmid diluent, vortex for 3 seconds, and let stand at room temperature for 30 min. 72 h after transfection, add lysis buffer and lyse on a shaker at 37 °C for 1 h. Centrifuge at 4000 rpm for 10 min to collect the supernatant. Filter the supernatant through a 0.45 μm filter, and use iodixanol density gradient centrifugation to remove impurities and empty capsids to obtain pure rAAV. Use the BGH polyA primer and probe in Table 6 to detect the physical titer of the mixed serotype virus prepared above.

[0105] Example 3 is also different from Example 1 in that: 2. Transduction activity of heterozygous serotype capsids in vitro cell lines Seed 1E+05 APRE19 cells in each well of a 24-well plate and culture overnight at 37 °C in 5% carbon dioxide using DMEM-F12 / 10% FBS complete medium. Calculate the amount of virus vg required for each well according to different multiplicity of infection MOI of the virus: 5E5, 1E5, 2E4, 4E3, 8E2, 1.6E2, 3.2E1. Dilute AAV using complete medium. Remove the cells cultured overnight, aspirate the complete medium from the well plate, add 300 μL of medium containing the diluted virus, place it in a cell culture incubator at 37 °C and 5% carbon dioxide for 3 hours, and then add 1 mL of fresh complete medium and continue to culture for 48 hr.

[0106] Collect the cells and use flow cytometry to detect the proportion of cell fluorescence. Export the data and analyze the flow cytometry results using FlowJo. Figures 19 - 21Shows the transduction activity of hybrid viruses and mixed viruses in ARPE19 cells at different MOIs and the comparison with the parental viruses AAV2.7m8 and AAV8. Figures 19 - 20 As can be seen from Figures 19 - 20 , the transduction efficiency of the hybrid virus generated by mixing and packaging in a 1:1 ratio is different from that of the homozygous virus with a 1:1 capsid mixture. Compared with the wild-type serotype AAV2-7m8 and AAV8, the transduction efficiency of the hybrid virus is between the two, verifying the advantage of its combination of the characteristics of both capsids. According to the present invention, by integrating two capsids into cells and screening monoclonal clones, hybrid AAV can be stably produced.

[0107] In this example, a hybrid AAV virus with the characteristics of both AAV2-7m8 and AAV8 capsids was successfully generated 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.

[0108] The present invention realizes the stable production of various 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 invention has the following significant advantages: 1 Simplify the operation process and save production costs: By gene integration technology, the dependence on high-cost plasmid DNA is avoided, the production process is simplified, and the production costs are significantly reduced.

[0109] 2 Can stably produce multiple AAV viruses simultaneously, and the ratio of different AAVs is determined, ensuring the consistency and controllability of product functions and quality; 3 Greatly reduce 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.

[0110] 4 Produce hybrid AAV viruses: Can stably produce AAV viruses with different capsid chimeric ratios, and the virus particles have high homogeneity, meeting the needs of precise gene therapy; 5 More suitable for industrial production. The use of a stable cell line makes the production process more controllable, can meet the high demands of large-scale production of gene therapy drugs, and provides reliable technical support for industrial production.

[0111] In summary, the present invention overcomes the limitations of the existing transient transfection method by constructing a stable cell line, and provides an efficient, stable and economical AAV production platform. This method not only simplifies the operation process and reduces the production costs, but also can stably produce multiple AAVs or hybrid AAVs, significantly improving the quality and consistency of products, and providing strong technical support for gene therapy and biomedical research.

[0112] The above description is only some preferred embodiments of the present disclosure and an explanation of the applied technical principles. Those skilled in the art should understand that the scope of the invention involved in the embodiments of the present disclosure is not limited to the technical solutions formed by the specific combination of the above technical features, but 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 technical solutions formed by mutually replacing the above features with the technical features disclosed in the embodiments of the present disclosure but not limited to having similar functions.

Claims

1. A method for simultaneously producing multiple recombinant adeno-associated viruses, characterized in that, Comprising: Stably integrating multiple target genes into the host cell genome or stably integrating capsid protein Cap genes of multiple serotypes into the host cell genome; Screening monoclonal cells stably expressing the target gene or stably expressing the capsid protein Cap gene of the serotype; Co-transfecting a transfer plasmid containing AAV inverted terminal repeats, a Rep gene plasmid, and an adenovirus helper gene plasmid into the monoclonal cells stably expressing the target gene or stably expressing the capsid protein Cap gene of the serotype; Harvesting and purifying the recombinant AAV virus to obtain a mixture of AAV viruses with a specific proportion of target genes or a hybrid AAV virus with a specific proportion of capsid proteins.

2. The method for simultaneously producing multiple recombinant adeno-associated viruses according to claim 1, wherein: The method for stably integrating multiple target genes into the host cell genome includes: Preparing an auxiliary plasmid for recognizing and cleaving the transposon plasmid sequence; Preparing a transposon plasmid containing two inverted terminal repeats and the transposable region therebetween; the transposable region carries the target gene; Co-transfecting the auxiliary plasmid and the transposon plasmid into the host cell, and the transposase encoded by the auxiliary plasmid will recognize the ITR element in the transposon of the transposon plasmid and insert the transposable region and the ITR element into the host genome.

3. The method for simultaneously producing multiple recombinant adeno-associated viruses according to claim 2, wherein: The auxiliary plasmid is used to encode the transposase; The transposon plasmid contains two inverted terminal repeats and the transposable region therebetween; Performing gene synthesis or PCR amplification on the sequences in PiggyBac-R-ITR and PiggyBac-L-ITR on the transposable region, and constructing them into the PiggyBacDualpromoter backbone vector by homologous recombination method 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 transposable region and the ITR element into the host genome includes: The transposase recognizes and cleaves the 5'TR and 3'TR sequences, excises the target gene in the transposable region from the transfer plasmid expressing EGFP and the transfer plasmid expressing mcherry, and the transposase randomly inserts the target gene 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 monoclonal cells stably expressing the target gene containing the AAV inverted terminal repeats includes: Transfect pAAV-RC and pAdDelta6 into the screened monoclonal cells stably expressing the target gene respectively.

6. The method for simultaneously producing multiple recombinant adeno-associated viruses according to claim 5, characterized in that: The method for harvesting and purifying recombinant AAV viruses to obtain a mixture of AAV viruses with a specific proportion of target genes includes: After transfection to obtain a cell suspension, add Trypsin-EDTA to the cell suspension, lyse the cell suspension to release recombinant AAV virus particles; After centrifugation, remove cell debris, collect the supernatant to obtain a mixture of AAV viruses with a specific proportion of target genes.

7. The method for simultaneously producing multiple recombinant adeno-associated viruses according to claim 3, characterized in that: Synthesize or PCR amplify the sequences in PiggyBac-R-ITR and PiggyBac-L-ITR of the transposon region, and construct them into the PiggyBacDualpromoter backbone vector by homologous recombination to obtain the transfer plasmid expressing EGFP and the transfer plasmid expressing mcherry; Replace with Synthesize or PCR amplify the sequences in PiggyBac-R-ITR and PiggyBac-L-ITR of the transposon region, and construct them into the PiggyBacDualpromoter backbone 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 1, characterized in that: The method for stably integrating the capsid protein Cap genes of multiple serotypes into the host cell genome includes: Inoculate the host cells into a shake flask; co-transfect the helper plasmid pAdDelta6, the Rep gene plasmid, and the transfer plasmid containing the AAV inverted terminal repeat sequence expressing EGFP into the monoclonal cells containing two serotype 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 1, characterized in that: The method for screening monoclonal cells stably expressing the target gene includes: Determine the ratio between different integrated genes by quantitative PCR, and the screened monoclonal cells can stably express a specific proportion of the target gene.

10. The method for simultaneously producing multiple recombinant adeno-associated viruses according to claim 1, characterized in that: The method for screening monoclonal cells stably expressing the capsid protein Cap gene of the serotype includes: Screen out the monoclonal cell line that can express a specific proportion of the capsid protein.

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