Recombinant adeno-associated virus (rAAV) production in insect cells

Abstract

Materials for the efficient production of recombinant baculovirus seed stocks containing nucleic acid sequences encoding a target gene, and methods for producing recombinant baculovirus seed stocks containing nucleic acid sequences encoding a target gene are provided. A rapid method for producing rAAV containing nucleic acid having a nucleic acid sequence encoding a target gene is also provided; this method enables the production of rAAV at high titers. Methods and systems for producing high-titer rAAV are provided herein. rAAV produced by these methods and systems may be used in pharmaceutical compositions, gene therapy compositions, for research purposes and other uses.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 489,656 filed March 10, 2023 and U.S. Provisional Patent Application No. 63 / 465,669 filed May 11, 2023; these are each incorporated herein by reference in their entirety.

[0002] Field of Invention This invention relates to recombinant AAV, particularly to the production of high numbers of rAAV particles. [Background technology]

[0003] Background of the Invention AAV is a 4.7kb linear single-stranded DNA virus. Wild-type AAV is nonpathogenic and has no etiological association with any known disease, thus rAAV is associated with superior clinical safety. Recent clinical and nonclinical successes with recombinant adeno-associated virus (rAAV) vectors as in vivo gene delivery vehicles have increased interest in producing highly pure and effective viral vectors for various natural and engineered serotypes.

[0004] The wild-type AAV genome encodes three open reading frames—rep, cap, and AAP—that are flanked by terminal inverted repeats (ITRs) and aid in replication, packaging, and rescue. AAV enters host cells via specific receptors on the cell surface.

[0005] The rep open reading frame encodes four rep proteins (Rep78, Rep68, Rep52, and Rep40) synthesized from mRNA transcribed from the p5 and p19 promoters. Rep78 and Rep68 are required for adeno-associated virus (AAV) DNA replication. Rep52 and Rep40 contain helicase activity and are required to package AAV DNA within the capsid. VP1, VP2, and VP3 are synthesized from mRNA transcribed from the p40 promoter. To maintain a 1:1:10 ratio of VP1:VP2:VP3 for viral particle assembly, AAV uses alternative splicing mechanisms for VP1, a less efficient start codon (ACG) for VP2, and a highly efficient start codon for VP3 to reduce their protein levels. The N-terminal sequence present in VP1 contains the phospholipase A2 domain required for AAV infectivity. Furthermore, VP2 / VP3 mRNA encodes an assembly activation protein (AAP) from a weak CTG start codon, albeit in a different reading frame. AAP facilitates nuclear transport of the major VP3 capsid protein and promotes capsid assembly and maturation, although AAP is not present in mature capsids.

[0006] AAV vectors used for the administration of therapeutic nucleic acids typically have approximately 96% of the parent genome deleted so that only terminal repeats (ITRs) containing recognition signals for DNA replication and packaging remain. This reduces immunological or toxic side effects caused by the expression of viral genes. Furthermore, by delivering specific AAV proteins to productive cells, it becomes possible to incorporate the AAV vector, including AAV ITRs, into specific regions of the cell genome, as desired. See, for example, U.S. Patent Nos. 6,342,390 and 6,821,511. Host cells containing the incorporated AAV genome show little to no changes in cell growth or morphology (see, for example, U.S. Patent No. 4,797,368).

[0007] Terminal inverted repeats (ITRs) are adjacent to the unique coding sequences of unstructured replication (Rep) and structural capsid (Cap) proteins (also known as virion proteins (VP)). ITRs are self-complementary and organized to form an energetically stable intramolecular double-stranded T-shaped hairpin. This hairpin structure functions as an origin for viral DNA replication. ITRs are the cis-functioning sites in replication and function as recognition sites for trans-functioning replication proteins. One exception to the symmetry of ITR sequences occurs in the "D" region of the ITR. Nick formation of single-stranded DNA occurs at the junction between the A and D regions; this is the region where new DNA synthesis begins. Rep genes encode Rep proteins Rep78, Rep68, Rep52, and Rep40. Cap genes encode VP proteins VP1, VP2, and VP3. These proteins form capsids. AAV replicating in mammalian cells typically has two ITR sequences. AAV requires the expression of helper genes for efficient replication.

[0008] Several AAV serotypes and over 100 AAV variants have been isolated from adenovirus stocks or from human or non-human primate tissues. Generally, AAV serotypes have remarkably homologous genomic sequences at the nucleic acid and amino acid sequence levels, so that different serotypes have the same set of genetic functions, produce physically and functionally equivalent virions, and replicate and assemble by nearly identical mechanisms.

[0009] AAV Rep and ITR sequences are particularly conserved across many AAV serotypes. Furthermore, it is known that Rep and ITR sequences of many AAV serotypes efficiently cross-complement (i.e., functionally substitute) with corresponding sequences from other serotypes during AAV particle production in mammalian cells. Generally, the Cap protein, which determines the tropicity of AAV particles and associated Cap protein-coding sequences, is significantly less conserved across different AAV serotypes than the Rep gene. Given the ability of Rep and ITR sequences to cross-complement with corresponding sequences from other serotypes, AAV vectors may contain a mixture of serotypes and thus be "chimeric" or "pseudotyped" AAV vectors. Chimeric AAV vectors contain AAV capsid proteins derived from two or more (e.g., 2, 3, 4, etc.) different AAV serotypes. In contrast, pseudotyped AAV vectors contain one or more ITRs of one AAV serotype, packaged within a capsid of another AAV serotype. Chimeric and pseudotypes are further described, for example, in U.S. Patent No. 6,723,551; Flotte (2006) Mol Ther 13(1):1-2; Gao et al (2004) J. Virol 6381-6388; Gao et al (2002) Proc Natl Acad Sci USA 99:11854-11859; De et al (2006) Mol Ther 13:67-76 and Gao et al (2006) Mol Ther 13:77-87.

[0010] The most commonly used method for recombinant adeno-associated virus (rAAV) production in the laboratory is transient triple transfection of HEK293 cells with AAV cis and transplasmids as well as adenovirus helper plasmids. However, most HEK293 production platforms currently in use produce low titers and are not scalable. The difficulties associated with scaling up rAAV production using mammalian cell production systems can be significant, if not completely insurmountable. For example, clinical studies are 10 15 More than 10 rAAV particles may be required. To produce this number of rAAV particles in a mammalian cell system, for example, human 293 cells, approximately 10 rAAV particles would be needed, which is equivalent to 5,000 175 ml flasks of cells. 11 Transfection and culture of individual cells are required. There are concerns that vectors intended for clinical use, produced in mammalian cell cultures, may be contaminated with undesirable, potentially pathogenic, material derived from mammalian cells.

[0011] U.S. Patent No. 6,723,551B2 to Kotin et al. and Urabe et al. 2006 J. Virol. 80:1874-1885 disclose methods for producing rAAV vectors in insect cells. The AAV sequences used for AAV production in insect cells can be derived from the genome of any AAV serotype. Generally, AAV serotypes have genomic sequences with remarkable sequence identity at the amino acid and nucleic acid levels, provide a virtually identical set of gene functions, produce physically and functionally similar virions, and replicate and assemble by substantially identical mechanisms. Kotin and Urabe describe baculovirus vectors constructed using nucleic acids encoding Rep78 / 68 and Rep52 / 40 in a palindromic head-to-tail arrangement under the control of independent promoters. These vectors contain terminal inverted repeats (ITRs), the gene coding sequence of interest, and the AAV capsid gene. Initial results suggested that high-titer rAAV could be obtained by these methods, but later studies showed that these rAAV vectors were less infectious than their counterparts produced in mammalian cells (Merten et al 2005 Gene Therapy 12:S51-S61 and Kohlbrenner et al 2005 Mol. Ther. 12:1217-1225).

[0012] U.S. Patent No. 6,723,551 suggests that insect cells splice Rep mRNA differently from the process in mammalian cells. U.S. Patent No. 6,723,551 avoids splicing out or removing introns before translation. In mammalian cells, the best yield of fully functional, nuclear-targeting "complete" virions (i.e., viral particles incorporating the AAV genome) is obtained when all three VP proteins are expressed in a near 1:1:10 (VP1:VP2:VP3) stoichiometric ratio. The splicing event required to produce 1:1:10 stoichiometric VP1:VP2:VP3 is not adequately reproduced in insect cells when the original AAV Cap coding sequence is used. Modifications of the VP1 sequence have been used to alter VP1 expression levels. Attempts using large homologous repeats of Rep78 and Rep52 have shown that homologous repeats are unstable. Attempts to isolate the coding sequences of Rep78 and Rep52 required two baculoviruses and one additional baculovirus to supply the VP1 protein. Cao et al. (2000 J. Virol 74:11456-11463) described a method for producing a high-titer wild-type-free recombinant AAV vector using a helper plasmid. However, Cao's method used human cells and did not include a promoter that could direct transcription in insect cells. U.S. Patent No. 8,945,918 to Chen provides a method for expressing a gene in insect cells and producing an infectious rAAV vector. Infectious rAAV vectors produced by Chen's method exhibit loss of the target gene; after the second passage, only about 50% of the viral vector contains the target gene. Viral particles continue to lose the target gene with each passage. After the third passage, only about 25% of the viral particles contain the target gene. Less than 10% of the viral particles contain the target gene after the fourth passage. The method described in U.S. Patent No. 8,945,918 requires multiple passages to obtain a sufficient seed stock to produce a large number of rAAV particles. A high volume of seed stock material is required for rAAV production using Chen's method. [Prior art documents] [Patent Documents]

[0013] [License 1] U.S. Patent No. 6342390 [License 2] U.S. Patent No. 6821511 [License 3] U.S. Patent No. 4797368 [License 4] U.S. Patent No. 6723551 [Patent Document 5] U.S. Patent No. 8945918 [Non-licensed literature]

[0014] [Non-licensed Document 1] Flotte (2006) Mol Ther 13(1):1-2 [Non-licensed Document 2] Gao et al (2004) J. Virol 6381-6388 [Non-licensed Document 3] Gao et al (2002) Proc Natl Acad Sci USA 99:11854-11859 [Non-licensed Document 4] De et al (2006) Mol Ther 13:67-76 [Non-licensed Document 5] Gao et al (2006) Mol Ther 13:77-87 [Non-licensed Document 6] Urabe et al 2006 J. Virol.80:1874-1885 [Non-licensed Document 7] Merten et al 2005 Gene Therapy 12:S51-S61 [Non-licensed Document 8] Kohlbrenner et al 2005 Mol. Ther. 12:1217-1225 [Non-licensed Document 9] Cao et al 2000 J. Virol 74:11456-11463 [Overview of the Initiative] [Means for solving the problem]

[0015] Summary of the Invention Methods and systems for producing high-titer rAAV are provided herein. rAAV produced by these methods and systems may be used in pharmaceutical compositions and gene therapy compositions for research purposes and other uses.

[0016] A system is provided for the production of recombinant bacmids having reduced residual DNA in rAAV production. The system for the production of recombinant bacmids having reduced residual DNA comprises: (i) a donor plasmid comprising a first antibiotic resistance gene, an anti-selection marker and an import cassette, wherein the import cassette comprises a left transposon arm, a gene of interest and a right transposon arm; (ii) a helper plasmid comprising a second antibiotic resistance gene; and (iii) a bacmid comprising a third antibiotic resistance gene and a transposon insertion site located within a reporter cassette.

[0017] In embodiments of this system, each of the first, second, and third antibiotic resistance genes confers resistance to a different antibiotic. In some embodiments, the first, second, and third antibiotic resistance genes are selected from a group including kanamycin resistance genes, ampicillin resistance genes, tetracycline resistance genes, penicillin resistance genes, streptomycin resistance genes, erythromycin resistance genes, and penicillin / streptomycin resistance genes. In certain embodiments, the first, second, and third antibiotic resistance genes are selected from a group including ampicillin resistance genes, tetracycline resistance genes, and kanamycin resistance genes. In some embodiments, bacmid contains a kanamycin resistance gene, the helper plasmid contains a tetracycline resistance gene, and the donor plasmid contains an ampicillin resistance gene and an anti-selection marker which is rpsl.

[0018] In various embodiments of this system, the counter-selection marker is an antibiotic susceptibility gene. In some embodiments, the antibiotic susceptibility gene is selected from a group including rpsl, URA3, and thymidine kinase. In a particular embodiment, the antibiotic susceptibility gene is rpsl.

[0019] In some embodiments, the donor plasmid does not contain an active gentamicin resistance gene. In some embodiments, the transfer cassette is optimized to reduce GC-rich regions within the transfer cassette. In various embodiments, the donor plasmid does not contain an active gentamicin resistance gene or GC-rich regions in the transfer cassette.

[0020] In some embodiments of this system, the left transposon arm is Tn7R and the right transposon arm is Tn7L.

[0021] In this embodiment of the system, the reporter cassette contains the lacZα gene. In some embodiments, the transposon insertion site is selected from a group including Tn7 insertion sites, modified Tn7 insertion sites, and mini-att-Tn7 sites. In a particular embodiment, the transposon insertion site is a mini-att-Tn7 site.

[0022] In this embodiment of the system, the gene of interest encodes an AAV RepCap protein or a therapeutic gene product. In some embodiments, the gene of interest encoding the AAV RepCap protein comprises a 7m8 cassette.

[0023] In various embodiments, the system is selected from a group that includes a modified Bac-to-Bac system, a modified baculovirus-infected cell (BIC) system, and a modified Bac-plus system.

[0024] A method is provided for producing recombinant bacmid having reduced residual DNA. The method for producing recombinant bacmid containing reduced residual DNA comprises (a) introducing a system for producing recombinant bacmid containing a gene of interest into a bacterial cell, the system comprising (i) a donor plasmid comprising a first antibiotic resistance gene, an anti-selection marker and an import cassette, wherein the import cassette comprises a left transposon arm, a gene of interest and a right transposon arm, (ii) a helper plasmid comprising a second antibiotic resistance gene, and (iii) a reporter cassette containing a third antibiotic resistance gene and a transposon insertion site. The method comprises the steps of: (b) growing the transformed bacteria in the presence of three antibiotics, the first, second, and third antibiotic resistance genes conferring resistance to the bacteria; (c) selecting at least one bacterial colony containing recombinant bacmid; (d) incubating the bacteria from at least one bacterial colony containing recombinant bacmid selected in step (c) with a compound that enables counterselection against the donor plasmid; and (e) collecting recombinant bacmid having reduced residual DNA from the bacterial colony after counterselection.

[0025] In aspects of these methods, bacterial colonies containing recombinant bacmid are identified by a change in reporter activity. In various aspects, reporter activity is selected from a group including fluorescence, antibiotic susceptibility, and bacterial colony color. In some aspects of these methods, transformed bacteria are grown in the presence of a beta-galactosidase substrate. In certain aspects, the beta-galactosidase substrate is selected from a group including x-gal and BluoGal. In various aspects, the presence of recombinant bacmid is indicated by white colonies formed in the presence of a beta-galactosidase substrate.

[0026] In various embodiments of these methods, each of the first, second, and third antibiotic resistance genes confers resistance to a different antibiotic. In some embodiments, the first, second, and third antibiotic resistance genes are selected from a group including kanamycin resistance genes, ampicillin resistance genes, tetracycline resistance genes, penicillin resistance genes, streptomycin resistance genes, erythromycin resistance genes, and penicillin / streptomycin resistance genes. In certain embodiments, the first, second, and third antibiotic resistance genes are selected from a group including ampicillin resistance genes, tetracycline resistance genes, and kanamycin resistance genes. In some embodiments, bacmid contains a kanamycin resistance gene, a helper plasmid contains a tetracycline resistance gene, and a donor plasmid contains an ampicillin resistance gene and a counter-selection marker which is rpsl. In some embodiments, the compound enabling counter-selection against the donor plasmid is an antibiotic. In certain embodiments, streptomycin is a compound that enables counterselection against a donor plasmid.

[0027] Embodiments of this application provide an efficient method for producing a recombinant baculovirus seed stock containing a gene of interest, wherein at least 50% of the recombinant baculoviruses (rBV) in the recombinant baculovirus seed stock contain the gene of interest. The efficient method includes the step of (a) introducing a system for producing a recombinant bacmid containing the gene of interest into a bacterial cell, wherein the system comprises (i) a donor plasmid comprising a first antibiotic resistance gene, an antiselection marker and an import cassette, the donor plasmid comprising a left transposon arm, the gene of interest and a right transposon arm; a helper plasmid comprising a second antibiotic resistance gene; and a bacmid comprising a third antibiotic resistance and transposon insertion site located within a reporter cassette.The method involves (b) growing transformed bacteria in the presence of three antibiotics, the first, second, and third antibiotic resistance genes conferring resistance; (c) selecting at least one bacterial colony containing recombinant bacmid; (d) incubating bacteria from at least one bacterial colony containing recombinant bacmid selected in step (c) with a compound that enables counterselection against a donor plasmid; (e) collecting recombinant bacmid containing the target gene from the bacterial colony after counterselection; and (f) creating insect cells containing the target gene collected in step (e). The process includes the steps of: (g) transforming with recombinant bacmid; (h) incubating the transformed insect cells and recovering rBV containing the gene of interest from the insect cells; (i) recovering rBV containing the gene of interest from at least two plaques of insect cells and identifying the plaques containing rBV containing the gene of interest, wherein the rBV shows a low percentage loss of the gene of interest; and the process of performing a second passage (p1 rBV), wherein the second passage includes the steps of: (i) infecting insect cells with rBV from the plaques identified in step (i), incubating the infected insect cells, and recovering a recombinant baculovirus seed stock containing rBV containing the gene of interest from the insect cells.

[0028] In some embodiments of this method, after the first passage, at least 80% of the rBV contains the gene of the target. In various embodiments of this method, after the second passage, at least 70% of the rBV contains the gene of the target. In certain embodiments of these methods, the method produces recombinant baculovirus seed stock after two passages via insect cells, and the baculovirus seed stock contains at least 2 × 10⁶ cells. 10 The vector genome is in ml (vg / ml) format.

[0029] In various embodiments of these methods, bacterial colonies containing recombinant bacmid are identified by a change in reporter activity. In some embodiments, reporter activity is selected from a group including fluorescence, antibiotic sensitivity, and bacterial colony color. In some embodiments of these methods, transformed bacteria are grown in the presence of a beta-galactosidase substrate. In certain embodiments, the beta-galactosidase substrate is selected from a group including x-gal and BluoGal. In various embodiments, the presence of recombinant bacmid is indicated by white colonies formed in the presence of a beta-galactosidase substrate.

[0030] In various embodiments of these methods, each of the first, second, and third antibiotic resistance genes confers resistance to a different antibiotic. In some embodiments, the first, second, and third antibiotic resistance genes are selected from a group including kanamycin resistance genes, ampicillin resistance genes, tetracycline resistance genes, penicillin resistance genes, streptomycin resistance genes, erythromycin resistance genes, and penicillin / streptomycin resistance genes. In certain embodiments, the first, second, and third antibiotic resistance genes are selected from a group including ampicillin resistance genes, tetracycline resistance genes, and kanamycin resistance genes. In some embodiments, bacmid contains a kanamycin resistance gene, a helper plasmid contains a tetracycline resistance gene, and a donor plasmid contains an ampicillin resistance gene and a counter-selection marker which is rpsl. In some embodiments, the compound enabling counter-selection against the donor plasmid is an antibiotic. In certain embodiments, streptomycin is a compound that enables counterselection against a donor plasmid.

[0031] In some embodiments of these methods, the step of incubating the transformed insect cells is carried out at approximately 28°C without CO2 for about 4 days. In various embodiments of these methods, the step of incubating the transformed insect cells is carried out in a shaker incubator. In a particular embodiment, the insect cells are grown in 30 ml of culture medium.

[0032] In certain embodiments of these methods, insect cells are selected from a group including Sf-RVN cells, Sf9 cells, and Hi5 cells. In some embodiments, insect cells are selected from a group including Sf-RVN cells and Sf9 cells.

[0033] In various embodiments, insect cells are transformed with bacmid between approximately 1 μg and approximately 20 μg. In a particular embodiment, insect cells are transformed with approximately 6–9 μg of bacmid.

[0034] In some embodiments, the gene of interest encodes Rep / Cap. In some embodiments, the gene of interest encoding Rep / Cap includes a 7m8 cassette. In certain embodiments, the gene of interest includes a 7m8 cassette.

[0035] In certain embodiments, the step of recovering rBV containing the gene of interest includes the step of recovering rBV from at least 10 plaques of insect cells, and the step of identifying plaques containing stable rBV with the gene of interest.

[0036] In some embodiments, the method further includes the step of performing a third passage, the third passage (p2 rBV) comprising the steps of infecting insect cells with rBV from p1 rBV identified in step (j), incubating the infected insect cells, and recovering a recombinant baculovirus seed stock from the insect cells containing the rBV containing the gene of interest or Rep / Cap.

[0037] A method is provided for producing a recombinant baculovirus seed stock containing a stable gene of interest, wherein at least 50% of the recombinant baculoviruses (rBV) in the recombinant baculovirus seed stock contain the stable gene of interest. The method comprises (a) introducing a system for the production of a recombinant bacmid containing the gene of interest into a bacterial cell, wherein the system comprises (i) a donor plasmid comprising a first antibiotic resistance gene, an anti-selection marker and an import cassette, the import cassette comprising a left transposon arm, the gene of interest and a right transposon arm; (ii) a helper plasmid comprising a second antibiotic resistance gene; and (iii) a bacmid comprising a third antibiotic resistance gene and a transposon insertion site located within a reporter cassette.The method involves (b) growing transformed bacteria in the presence of three antibiotics, the first, second, and third antibiotic resistance genes conferring resistance; (c) selecting at least one bacterial colony containing recombinant bacmid; (d) incubating bacteria from at least one bacterial colony containing recombinant bacmid selected in step (c) with a compound that enables counterselection against a donor plasmid; (e) collecting recombinant bacmid containing the target gene from the bacterial colony after counterselection; (f) transforming insect cells with recombinant bacmid containing the target gene collected in step (e); and (g) incubating the transformed insect cells and collecting the target gene from the insect cells. (h) a step of recovering rBV containing the gene of interest; (i) a step of recovering rBV containing the gene of interest from one or more insect cell plaques and identifying plaques containing stable rBV containing the gene of interest; and (j) a step of recovering rBV containing the gene of interest from the insect cells.

[0038] In one embodiment, a recombinant baculovirus seed stock containing a stable gene of interest is provided.

[0039] In one embodiment, a recombinant baculovirus seed stock containing a stable gene of interest produced by the method of this application is provided. In this embodiment of the recombinant baculovirus seed stock, the stable gene of interest comprises a 7m8 cassette. In this embodiment of the recombinant baculovirus seed stock, the stable gene of interest encodes Rep / Cap.

[0040] A composition is provided comprising a recombinant baculovirus seed stock containing a stable target gene.

[0041] A method for producing rAAV containing a target gene is provided. The method for producing rAAV containing a target gene comprises: (a) producing a first recombinant baculovirus seed stock by the above method, wherein the first recombinant baculovirus seed stock contains an rBV containing a first target gene; (b) producing a second recombinant baculovirus seed stock by the above method, wherein the second baculovirus seed stock contains an rBV encoding a Rep / Cap protein; (c) co-infecting insect cells with the first recombinant baculovirus seed stock and the second recombinant baculovirus seed stock; (d) adding a supply to the insect cell medium after co-infection; (e) incubating the infected insect cells; and (f) recovering rAAV containing the target gene from the insect cells. In embodiments of this method, the step of co-infecting insect cells includes co-infecting insect cells with a first recombinant baculovirus seed stock and a second recombinant baculovirus seed stock at a low multiple of infection (MOI). In various embodiments, the low MOI is less than 0.01. In various embodiments, the low MOI is less than 0.009. In certain embodiments, the low MOI is in the range of about 0.0005 to about 0.009. In certain embodiments, the low MOI is in the range of about 0.001. In various embodiments, the MOI of the first recombinant baculovirus seed stock is different from the MOI of the second recombinant baculovirus seed stock. In other embodiments, the MOI of the first recombinant baculovirus seed stock and the MOI of the second recombinant baculovirus seed stock are the same.

[0042] In this embodiment of the method, the recovered rAAV is at least 1 11 It has a titer of vg / ml. In some embodiments, the recovered rAAV is at least 5 11It has a titer of vg / ml. In a particular embodiment, the recovered rAAV is at least 1 12 It has a potency of vg / ml.

[0043] In various embodiments, the supply is added between approximately 1 hour and 8 hours after co-infection. In some embodiments, the supply is added approximately 4 hours after co-infection.

[0044] In some embodiments, the insect culture medium is ESF-AF.

[0045] In various embodiments, insect cells are selected from a group including Sf-RVN cells, Sf9 cells, and Hi5 cells. In a particular embodiment, the insect cells are Sf-RVN cells.

[0046] In some embodiments of these methods, rAAV containing the target gene is recovered at a high titer less than 25 days after transformation of insect cells with recombinant bacmid. In some embodiments, rAAV containing the target gene is recovered at a high titer approximately 23 days after transformation of insect cells with recombinant bacmid. In a specific embodiment, rAAV containing the target gene is recovered at a high titer 5 days after co-infection. In other embodiments, p2 rBV is recovered at a high titer 18 days after transformation of insect cells with recombinant bacmid.

[0047] In various embodiments of these methods, the volume of either the first recombinant baculovirus seed stock or the second recombinant baculovirus seed stock used to co-infect insect cells, or the individual volumes of the first recombinant baculovirus seed stock and the second recombinant baculovirus seed stock, is less than 1 liter. In other embodiments, the volume of either the first recombinant baculovirus seed stock or the second recombinant baculovirus seed stock used to co-infect insect cells, or the individual volumes of the first recombinant baculovirus seed stock and the second recombinant baculovirus seed stock, is less than 500 ml. In some embodiments, the volume of either the first recombinant baculovirus seed stock or the second recombinant baculovirus seed stock used to co-infect insect cells, or the individual volumes of the first recombinant baculovirus seed stock and the second recombinant baculovirus seed stock, is less than 100 ml. The volume of either the first recombinant baculovirus seed stock or the second recombinant baculovirus seed stock used to co-infect insect cells, or the individual volumes of the first recombinant baculovirus seed stock and the second recombinant baculovirus seed stock, is less than 30 ml.

[0048] A method for rapidly producing rAAV containing a gene of interest, comprising: (a) producing a first recombinant baculovirus seed stock by the method described herein, wherein the first recombinant baculovirus seed stock contains rBV containing the gene of interest; (b) producing a second recombinant baculovirus seed stock by the method described herein, wherein the second recombinant baculovirus seed stock contains rBV encoding the Rep / Cap protein; transducing insect cells with the first recombinant baculovirus seed stock at a low infection multiplicity and the second recombinant baculovirus seed stock at a low MOI; (d) adding a supply to the insect cell medium after co-infection; (e) incubating the infected insect cells for 5 days; and (f) recovering rAAV containing the gene of interest from the insect cells less than 27 days after the insect cells have been transformed with recombinant bacmid.

[0049] In various embodiments of these methods, the primary target gene encodes a therapeutic gene product for use in treating an eye disease or disorder. In certain embodiments, the eye disease or disorder is selected from a group including glaucoma, retinitis pigmentosa, macular degeneration, retinoschiosis, Leber congenital amaurosis, diabetic retinopathy, achromotopsia, and color vision deficiency. In some embodiments, macular degeneration is selected from a group including dry macular degeneration, wet macular degeneration, and age-related macular degeneration. In some embodiments of these methods, the therapeutic gene product is selected from a group including anti-angiogenic polypeptides, vascular endothelial growth factor (VEGF) binding proteins, opsin proteins, anti-C3 antibodies, anti-C5 antibodies, complement factor I (CFI), and anti-dry AMD gene products. In certain embodiments of these methods, the therapeutic gene product is selected from a group including aflibercept, sFLT1, CFI, ranibizumab, and bevacizumab.

[0050] In one embodiment, a method for rapidly producing rAAV containing a gene of interest is provided. The method for rapidly producing rAAV containing a gene of interest comprises: (a) producing a first recombinant baculovirus seed stock, wherein the first baculovirus seed stock contains an rBV containing a gene of interest; (b) producing a second recombinant baculovirus seed stock, wherein the second recombinant baculovirus seed stock contains an rBV encoding a Rep / Cap protein; (c) transducing insect cells with the first recombinant baculovirus seed stock at a low MOI and the second recombinant baculovirus seed stock at a low MOI; (d) adding a supply to the insect cell medium about 4 hours after co-infection; (e) incubating the infected insect cells for 5 days; and (f) recovering rAAV containing a gene of interest from the insect cells 5 days after co-infection.

[0051] In one embodiment, a method is provided for producing rAAV containing a gene of interest. The method includes (a) producing a first recombinant baculovirus seed stock, wherein the first recombinant baculovirus seed stock comprises rBV containing a first gene of interest; (b) providing a second recombinant baculovirus seed stock comprising rBV containing a stable gene of interest encoding a Rep / Cap protein; (c) co-infecting insect cells with the first recombinant baculovirus seed stock at a low MOI and the second recombinant baculovirus seed stock at a low MOI; (d) after co-infection, adding a supply to an insect cell medium; incubating the infected insect cells; and recovering rAAV containing the gene of interest from the insect cells. In various embodiments, the second recombinant baculovirus seed stock comprises rBV containing a 7m8 cassette.

[0052] In one embodiment, a composition is provided comprising a recombinant bacmid having reduced residual DNA produced by the method described herein.

[0053] A method for producing rAAV containing a gene of interest, comprising the steps of: (a) providing a first recombinant baculovirus seed stock containing a stable rBV containing a first gene of interest; (b) providing a second recombinant baculovirus seed stock containing a stable rBV containing a Rep / Cap protein; (c) co-infecting insect cells with the first recombinant baculovirus seed stock and the second recombinant baculovirus seed stock at a low MOI; (d) adding the supply to an insect cell medium after co-infection; (e) incubating the infected insect cells; and (f) recovering rAAV containing the gene of interest from the insect cells.

[0054] A method for rapidly producing rAAV containing a target gene is provided. The method for rapidly producing rAAV containing a target gene comprises: (a) producing a first recombinant baculovirus seed stock containing rBV containing a first target gene; (b) providing a second recombinant baculovirus seed stock containing rBV containing a stable target gene encoding a Rep / Cap protein; (c) co-infecting insect cells with the first recombinant baculovirus seed stock at a low MOI and the second recombinant baculovirus seed stock at a low MOI; (d) adding the supply to the insect cell medium after co-infection; (e) incubating the infected insect cells; and (f) recovering rAAV containing the target gene from the insect cells approximately 5 days after co-infection. The step of producing the first or second recombinant baculovirus seed stock may utilize the method described herein.

[0055] In one embodiment, a composition is provided comprising recombinant bacmid having reduced residual DNA produced by the method of the present application. In one embodiment, the composition comprising recombinant bacmid having reduced residual DNA is for use in the production of a pharmaceutical composition.

[0056] In one embodiment, insect cells capable of producing a stable rAAV containing the target gene are provided.

[0057] In various systems, recombinant bacmids with reduced residual DNA are intended for use in rAAV production. In embodiments of these systems, the helper plasmid is an AAV helper plasmid.

[0058] A modified Bac-to-Bac system for the production of recombinant bacmids having reduced residual DNA in rAAV production is provided herein. The modified Bac-to-Bac system comprises a donor plasmid containing a first antibiotic resistance gene, an anti-selection marker, and an import cassette. The donor plasmid does not contain an active gentamicin resistance gene or a GC-rich region in the import cassette. The import cassette contains a left transposon arm, the gene of interest, and a right transposon arm. The modified Bac-to-Bac system comprises an AAV helper plasmid containing a second antibiotic resistance gene. The modified Bac-to-Bac system comprises a bacmid containing a third antibiotic resistance gene and a transposon insertion site located within a reporter cassette. In some embodiments of these systems, the first, second, and third antibiotic resistance genes are selected from the group including ampicillin resistance genes, tetracycline resistance genes, and kanamycin resistance genes. In embodiments of these systems, bacmid contains a kanamycin resistance gene, the helper plasmid contains a tetracycline resistance gene, and the donor plasmid contains an ampicillin resistance gene and a counter-selection marker which is rpsl. In some embodiments, the counter-selection marker is rspl. Embodiments of these systems include cases where the left transposon arm is Tn7R and the right transposon arm is Tn7L. In various embodiments of these systems, the reporter cassette contains the lacZα gene. In certain embodiments of these systems, the transposon insertion site is the mini-att-Tn7 site. In various embodiments of this system, the gene of interest encodes the AAV RepCap protein or a therapeutic gene product.

[0059] In some embodiments, the present application provides an efficient method for producing a recombinant baculovirus seed stock containing a target gene, wherein at least 50% of the recombinant baculoviruses (rBV) in the recombinant baculovirus seed stock contain the target gene. In embodiments of these methods, after two passages, at least 70% of the rBV contain the target gene. In some embodiments, after one passage, at least 80% of the rBV contain the target gene. In certain embodiments, the method produces a recombinant baculovirus seed stock after two passages via insect cells, wherein the recombinant baculovirus seed stock contains at least 2 × 10⁶ cells. 10 The vector genome is in ml (vg / ml) format.

[0060] Some aspects of an efficient method for producing recombinant baculovirus seed stock containing a gene of interest include the step of introducing a modified Bac-to-Bac system for the production of recombinant rBV containing a gene of interest into bacterial cells, the modified Bac-to-Bac system comprising: (i) a donor plasmid comprising a first antibiotic resistance gene, an anti-selection marker and an import cassette, wherein the donor plasmid does not contain an active gentamicin resistance gene or a GC-rich region in the import cassette, and the import cassette comprises a left transposon arm, a gene of interest and a right transposon arm; (ii) a helper plasmid comprising a second antibiotic resistance gene; and (iii) a bacmid plasmid comprising a third antibiotic resistance gene.An efficient method involves: (b) growing transformed bacteria in the presence of three antibiotics, IPTG and BluoGal, whose resistance genes confer resistance to the first, second, and third antibiotics; (c) selecting at least one bacterial colony (white colony) containing recombinant bacmid; (d) incubating bacteria from at least one bacterial colony containing recombinant bacteria selected in step (c) with a compound that enables counterselection against the donor plasmid; (e) collecting bacmid from bacterial cells containing recombinant bacmid; (f) transforming insect cells with the recombinant bacmid collected in step (e); and (g) sterilizing the transformed insect cells in a shaker incubator in culture medium for 28 minutes. The method further comprises the steps of: (h) incubating at °C for 4 days to collect P0 recombinant baculovirus (rBv, BEV); (i) performing a first passaging, the passaging comprising the steps of infecting insect cells with P0 recombinant baculovirus (rBv) containing the gene of interest or Rep / Cap, and incubating the infected insect cells; and (j) performing a second passaging, the second passaging comprising the steps of infecting insect cells with rBV from the plaque purification step (step i), incubating the infected insect cells, and recovering a recombinant baculovirus seed stock containing p1 rBV containing the gene of interest from the insect cells. In some embodiments, the presence of recombinant bacmid in bacterial cells is indicated by white colonies. In certain embodiments of these methods, the donor plasmid contains an ampicillin resistance gene and rpsl, the helper plasmid contains a tetracycline resistance gene, and the bacmid plasmid contains a kanamycin resistance gene. In various embodiments of these methods, the compound enabling counterselection against the donor plasmid is streptomycin. In some embodiments, insect cells are selected from a group including Sf-RVN cells and Sf9 cells.In certain embodiments of these methods, insect cells are transformed with approximately 6–9 μg of bacmid. In embodiments of these methods, the transformed insect cells are grown in 30 ml of culture medium. In some embodiments, the method further includes a step of performing a third passage, the third passage comprising the steps of infecting insect cells with BEV from the P1 rBV step (step j), incubating the infected insect cells, and recovering a baculovirus seed stock containing the BEV with the gene of interest from the insect cells (P2 rBV). For example, in some cases in epidemic diseases, P2 rBV may be replastically formed to regenerate the starting virus bank in P1 rBV. In some embodiments of the third passage, the MOI is 0.1.

[0061] In one embodiment, the present application provides a method for producing rAAV containing a gene of interest, comprising: (a) producing a first recombinant baculovirus seed stock by a method described herein, wherein the first recombinant baculovirus seed stock contains an rBV containing a first gene of interest; (b) producing a second recombinant baculovirus seed stock by the method described in claim 13, wherein the second recombinant baculovirus seed stock contains an rBV encoding a Rep / Cap protein; (c) co-infecting insect cells with the first recombinant baculovirus seed stock at a low MOI and the second recombinant baculovirus seed stock at a low MOI; (d) adding a supply to an insect cell medium having the first recombinant baculovirus seed stock and the second recombinant baculovirus seed stock after co-infection; and (e) incubating the infected insect cells and recovering rAAV containing the gene of interest from the insect cells. In embodiments of these methods, the low MOI is in the range between about 0.01 and about 0.001. In embodiments of these methods, the low MOI is in the range of about 0.001. In various embodiments of these methods, the recovered rAAV is at least 1 12It has a titer of vg / ml. In certain embodiments, the supply is added approximately 4 hours after co-infection. In some embodiments of these methods, the insect medium is ESF-AF. In certain embodiments, the insect cells are selected from a group including Sf-RVN cells and Sf9 cells. In embodiments of these methods, rAAV containing the gene of interest is recovered at a high titer 5 days after co-infection. In embodiments of these methods, p2 rBV is recovered at a high titer 18 days after transforming insect cells with bacmid collected from bacterial colonies containing recombinant bacmid. In various embodiments of these methods, the volume of either the first recombinant baculovirus seed stock, the second recombinant baculovirus seed stock, or both the first and second recombinant baculovirus seed stocks used to co-infect the cells is less than 30 ml.

[0062] In one embodiment, a method is provided for rapidly producing rAAV containing a gene of interest. The method includes: (a) producing a first recombinant baculovirus seed stock by the method described herein, wherein the first recombinant baculovirus seed stock contains an rBV containing a first gene of interest; (b) producing a second recombinant baculovirus seed stock by the method described herein, wherein the second recombinant baculovirus seed stock contains an rBV encoding a Rep / Cap protein; (c) transducing insect cells with the first recombinant baculovirus seed stock and the second recombinant baculovirus seed stock at a low infection multiplicity; (d) adding a supply to an insect cell medium about 4 hours after co-infection with the first recombinant baculovirus seed stock and the second recombinant baculovirus seed stock; and (e) incubating the infected insect cells for 5 days and recovering rAAV containing the gene of interest from the insect cells 5 days after co-infection.

[0063] Embedding by reference The following section describes different aspects of the present invention in more detail. Each aspect, embodiment, or feature of the present invention may be combined with any other aspect, embodiment, or feature of the present invention unless the reverse is expressly indicated. All publications, patents, and patent applications referenced herein are incorporated herein by reference in whole to the same extent that each individual publication, patent, or patent application is specifically and individually indicated as being incorporated herein by reference. [Brief explanation of the drawing]

[0064] [Figure 1] Figure 1 provides an overview of the modified bac-to-bac process, including modifications to the donor plasmid and the process for obtaining recombinant bacmid without the donor plasmid (pFastBac). The culture medium contains the additives shown. The modified bac-to-bac process is an example of a system for producing recombinant bacmid with reduced residual DNA.

[0065] [Figure 2] Figure 2 provides images of E. coli colonies grown under various conditions. The modified Bac-to-Bac system allows for the selection of colonies with reduced residual DNA in the bacmid collected from the bacteria after incubation with a compound that enables counterselection against the donor plasmid (2). The donor plasmid (pFastBac) is significantly less visible in lane 2 of the gel, indicating a significant reduction in the donor plasmid.

[0066] [Figure 3] Figure 3 provides a schematic diagram summarizing the previous process for obtaining rAAV from bacmid, compared to the (novel) process of the present invention. The novel method reduces the number of steps required. Recombinant bacmid produced by the system of this application has reduced residual DNA.

[0067] [Figure 4]Figure 4 provides images of plates of infected cells obtained during the plaque purification step for two different rBVs. The two different rBVs contain different expression cassettes (CFI 1.0 and 7m8) containing either CFI 1.0 as the gene of interest or 7m8 Rep / Cap as the gene of interest. The plaque purification process described herein is similar to that described in Example 4. The images shown show plates using control as well as 10-1, 10-2, 10-4, 10-6, and 10-8 dilutions.

[0068] [Figure 5] Figure 5 provides a schematic overview of the process of producing rAAV using a modified Bac-to-Bac system, in contrast to a standard production process. Note the shorter timeline from bacmid to rAAV recovery from Sf-RVN cells using the modified system compared to conventional methods. Sf-RVN cells are rhabdovirus-free.

[0069] [Figure 6]Figure 6 provides a chart summarizing the stability of rBaculovirus (BEV) between different passages and its effect on rAAV titer. rBV containing 7m8 was obtained from two different plaques (plaque 1 and plaque 2). The ratio of the gene of interest (7m8 Rep / Cap cassette) to GP64 is shown for rBV from each plaque in passage 0, passage 1 (P1), and passage 2 (P2). rBV from plaque 2 has a ratio of the gene of interest to the skeleton equal to or greater than 0.5 after passage 2. rBV from plaque 2 contains a stable gene of interest (7m8). rBV from each plaque and each passage was co-infected with rBV containing a stable gene of interest (GFP-Fluc) in insect cells to obtain rAAV. The rAAV titers obtained from co-infection with rBV containing the stable target gene (GFP-Fluc) and rBV containing 7m8 at P0, P1, and P2 stages from each plaque are shown in vg / ml. The AAV titers obtained using rBV from P0, P1, and P2 stages are compared for each plaque, and the titer ratios are shown. The rAAV titer obtained from rBV containing the stable target gene after P1 and P2 (plaque 2) is significantly higher than the rAAV titer from P1 and P2 rBV from plaque 1.

[0070] [Figure 7]Figure 7 provides graphs summarizing the rAAV titer results obtained in various experiments. The graph on the left summarizes the rAAV titers obtained from insect cells (Sf-RVN cells) grown in the indicated media (Sf-900™ SFM, Sf-900™ II SFM, EX-CELL™ TiterHigh, and ESF AF) having CFI / 7m8. The graph in the center summarizes the rAAV titers obtained from Sf-RVN cells grown in either ESF AF medium or Sf-900™ II SFM medium, with and without supplementation after co-infection. The rAAV titers obtained from ESF AF medium were substantially higher than those obtained using Sf-900™ II SFM, with or without supplementation. The addition of supplementation to ESF-AF medium increased the obtained titers. The panel on the right provides a graph summarizing the results obtained from co-infection at either an MOI of 0.001 or 3 in ESF AF medium or Sf-900(trademark) II SFM medium. Surprisingly, the results obtained at an MOI of 0.001 were significantly higher titers than those obtained when using an MOI of 3 in ESF AF medium.

[0071] [Figure 8]Figure 8 summarizes the evaluation of donor plasmid (pFB) levels in three different recombinant bacmid preparations. Donor plasmid levels were evaluated by droplet digital (ddPCR), and the results are summarized in Figure 8A. Bacmid preparation 1 contains recombinant bacmid after counter-selection for the donor plasmid (bacmid-GOI-donor plasmid not included). The ratio of the gene of interest to DNApol was 1.19. The ratio of the donor plasmid to rBacmid was 0 (measured at 1 / 2). Bacmid preparation 2 contains recombinant bacmid obtained using a donor plasmid containing an ampicillin resistance gene (not a kanamycin resistance gene) without counter-selection for the donor plasmid (bacmid-GOI-donor plasmid (Amp) included). The ratio of the gene of interest to DNApol was 5.45. The ratio of the donor plasmid to rBacmid was 4.6-fold. Bacmid preparation 3 contains recombinant bacmid obtained using a donor plasmid containing the kanamycin resistance gene (Kan), and this recombinant bacmid also contains the Kan resistance gene. The ratio of the target gene to DNApol was 309.18. The ratio of the donor plasmid to rBacmid was 261-fold. Figures 8B and 8C provide a graphical representation of the rBacmid copy number relative to the donor plasmid (pFB) copy number containing the target gene from three bacmid preparations (Figure 8B). Results from bacmid preparation 3 (donor plasmid and bacmid plasmid with the same antibiotic resistance gene) are excluded from Figure 8C. [Modes for carrying out the invention]

[0072] Detailed description of the invention As gene therapy is developed for use in treating common or epidemic diseases or disorders, the challenges associated with the large-scale production of recombinant adeno-associated viruses (rAAVs) containing the gene of interest have become apparent. An efficient method for producing large volumes of rAAV for clinical use is needed in the field. This application provides a modified system and method for the efficient production of large quantities of rAAV. Furthermore, the baculovirus expression vector (rBV) used to produce rAAV exhibits a significant loss of the gene of interest from the BEV. This application provides a stable rBV containing the gene of interest, a method for obtaining a stable rBV containing the gene of interest, and compositions and methods for obtaining a stable recombinant bacmid and rBV containing the gene of interest. The compositions containing a stable recombinant bacmid and rBV containing the gene of interest may also be used as a master source or master virus bank (MVB). A master source or master virus bank can facilitate large-scale clinical-grade production, testing, and regulatory approval. Many approved gene therapies target rare or unusual diseases or disorders. The amount of delivery vector required for these rare or unusual diseases or disorders is limited. When gene therapies are developed for prevalent diseases or disorders, it is necessary to produce considerably large quantities of gene therapy delivery vectors, such as rAAV. Prior to this study, limited methods existed for producing large quantities of rAAV. Previous methods had challenges including, but not limited to, a high risk of contamination, substantial degradation in the quality of produced rAAV over time, low production efficiency, and long production times.

[0073] Unless otherwise defined, all technical and scientific terms used herein have meanings that are generally understood by those skilled in the art to which this invention pertains.

[0074] A numerical range includes the numbers defining that range. The term “about” is used herein to mean plus or minus ten percent (10%) of a value. For example, “about 100” refers to any number between 90 and 110. The term “about” with respect to a range refers to that range which is from minus 10% of the lowest value to plus 10% of the highest value thereof. References herein to “about” a value or parameter include (and describe) embodiments that are directed to that value or parameter itself.

[0075] As a matter of convention and as used throughout this application, a scientific notation of exponential expression in which a portion of a number is replaced with E+n may be used, where E (the exponent) multiplies the preceding number by ten to the nth power. For example, a scientific notation with two digits after the decimal point shows 12345678901 as 1.23E+10, which is 1.23 multiplied by ten to the tenth power, 1.23×10 10 and may alternatively be written as. Similarly, 1.23E-10 may be alternatively written as 1.23×10 -10 and may alternatively be written as.

[0076] Unless otherwise indicated, each nucleic acid is written in the 5’ to 3’ orientation, left to right; an amino acid sequence is written in the amino to carboxy orientation, left to right.

[0077] The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be understood by reference to the specification as a whole. Thus, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0078] The technical terms used herein are for illustrative purposes only and are not intended to be limiting. Where used herein, the singular forms “a,” “an,” and “the” are intended to include the plural unless the context explicitly indicates otherwise. Furthermore, where the terms “including,” “includes,” “having,” “has,” “with,” or their variants are used in any of the detailed description and / or claims, such terms are intended to be inclusive, in a manner similar to that of the term “comprising.” Where used herein, the term “comprising” is synonymous with “including” or “containing,” and is inclusive or open-ended.

[0079] "Essentially derived from" is intended to limit the scope of a described composition, method, kit, etc., to specific materials that do not substantially affect the basic and novel features(s) of the composition, method, kit, etc. For example, an expression cassette "essentially derived from" a coding sequence encoding a polynucleotide operably linked to a promoter and a polyadenylation sequence may include further sequences, e.g., linker sequences, as long as they do not substantially affect the transcription or translation of the coding sequence. As another example, a variant or mutant polypeptide "essentially derived from" an enumerated sequence has an amino acid sequence of approximately 10 amino acid residues plus or minus the enumerated sequence at the boundary of the sequence based on the full-length naive polypeptide from which it is derived, e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue less than the amino acid residues that indicate the enumerated boundary, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues more than the amino acid residues that indicate the enumerated boundary.

[0080] Any reference to “or” in this specification is intended to include “and / or” unless otherwise stated.

[0081] The donor plasmid comprises a first antibiotic resistance gene, a counter-selection marker, and an import cassette, the import cassette comprising a left transposon arm, the gene of interest, and a right transposon arm. The “counter-selection marker” is intended to be a selectable marker that eliminates or inhibits the growth of the host organism upon selection. Counter-selection markers are known in the art and include, but are not limited to, antibiotic susceptibility genes. Antibiotic susceptibility genes include, but are not limited to, rpsl, thymidine kinase genes, and URA3. The donor plasmid for use in the system and method of this application is suitable for use in bacterial cells.

[0082] The AAV "rep" and "cap" genes refer to polynucleotide sequences encoding the replication and encapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes." In some embodiments, the AAV rep gene may be derived from any AAV serotype or may be a modified AAV rep gene. In some embodiments, the AAV rep gene is of the same serotype as the ITR of the rAAV vector genome. In some embodiments, the AAV rep gene is of a different serotype than the ITR of the rAAV vector genome or the Cap serotype. In some embodiments, the AAV Rep is a chimeric Rep. In some embodiments, the AAV Cap is a chimeric Cap.

[0083] The AAV "rep" and "cap" genes refer to polynucleotide sequences encoding the replication and capsid inclusion proteins of adeno-associated viruses and their variants. The Rep genes encode the Rep proteins Rep78, Rep68, Rep52, and Rep40. The Rep79 and Rep68 proteins are multifunctional DNA-binding proteins that perform helicase and nickase functions during productive replication, enabling the resolution of the AAV terminus (see, e.g., Im et al 1990 Cell 61:447-57). These proteins also regulate transcription from the endogenous AAV promoter and promoters within helper viruses (see, e.g., Periera et al (1997) J. Virol 71:1079-1088). Other Rep proteins modify the functions of Rep78 and Rep68. "Rep" encompasses variant Rep, chimeric Rep, and modified Rep. The Cap gene encodes the capsid proteins VP1, VP2, and VP3. Cap encompasses variant Cap, chimeric Cap, pseudotyped Cap, and modified Cap. Those skilled in the art will know which Rep and Cap forms are preferable to use together and will appropriately select them to form Rep / Cap. The term Rep / Cap refers to nucleotide sequences encoding functional forms of Rep and Cap. In some embodiments, a polynucleotide containing a polynucleotide encoding Cap further contains a polynucleotide encoding Rep.

[0084] AAV Rep and ITR sequences efficiently cross-complement other AAV Rep and ITR sequences in insect cells. Generally, the Cap protein, which determines the cytotropy of rAAV particles and associated Cap protein-coding sequences, is significantly less conserved among different AAV serotypes than the Rep protein and gene. Given the ability of Rep and ITR sequences to cross-complement corresponding sequences of other serotypes, pseudotyped AAV particles containing the capsid protein of one serotype (e.g., AAV6) and the Rep and / or ITR sequences of another AAV serotype (e.g., AAV2) can be readily generated. As used herein, “pseudotyped” refers to the source of the Cap protein in adeno-associated viruses. See Halbert et al 2000 J. Virol. 74:1524–1532 and Halbert et al 2001 J. Virol 75:6615–6624. rAAV2 / 6 and rAAV2 / 8 are pseudotyped AAVs in insect cells that contain the ITR and Rep sequences of AAV2 and VP sequences derived from AAV6 and AAV8, respectively. The production of pseudotyped AAV vectors containing the Cap gene of a particular AAV serotype demonstrates that non-pseudotyped vectors of that serotype can be successfully produced in the system.

[0085] Sequences derived from more than one AAV serotype may be combined for AAV production in insect cells. For example, a nucleic acid containing at least one AAV ITR nucleotide sequence may be derived from one serotype, while other nucleic acids may contain open reading frames or coding sequences derived from one or more other serotypes. Any nucleic acid from AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 may provide the Rep gene, Cap gene, and / or AAV ITR in the method of the present invention.

[0086] In some embodiments of these methods, the AAV ITR may be AAV1, aAV2, or AAV6 ITR; the nucleic acid containing the Rep ORF may contain the AAV1, AAV2, or AAV6 Rep gene; and the nucleic acid containing the Cap ORF may contain the AAV1, AAV2, or AAV6 Cap gene. Modified AAV sequences may also be used to produce rAAV in insect cells. Nucleotide sequences having at least about 70%, at least about 80%, at least about 90%, or at least about 95% sequence identity to the AAV1, AAV2, AAV3, and / or AAV4 sequences may be used instead of wild-type AAV Rep, AAV-ITR, or AAV-Cap sequences, provided that rAAV particles are produced in infected cells.

[0087] In some embodiments, one or more ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, bird AAV, cattle AAV, dog AAV, horse AAV, primate AAV, non-primate AAV, rhesus macaque AAV, or sheep AAV ITR, or variants thereof. In some embodiments, one or more ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, bird AAV, cattle AAV, dog AAV, horse AAV, primate AAV, non-primate AAV, rhesus macaque AAV, or sheep AAV ITRs, comprising one or more insertions, deletions, and / or substitutions of nucleotides.

[0088] Any vector known in the art may be used in conjunction with the teachings herein, provided that it is compatible with insect cells. The presence of the vector in insect cells does not need to be permanent. The vector may be introduced by any known method, for example, by chemical treatment of cells, electroporation, or infection. As used herein, “vector” means a macromolecule or macromolecule association containing or relating to a polynucleotide and which may be used to mediate the delivery of a polynucleotide to a cell. Scenario vectors include, but are not limited to, plasmids, viral vectors (i.e., adeno-associated viruses), liposomes and other gene delivery vehicles, and bacmids.

[0089] When used herein, “AAV vector” or “rAAV vector” refers to an adeno-associated virus (AAV) vector, or a recombinant AAV (rAAV) vector containing a polynucleotide sequence that is not of AAV origin (e.g., a polynucleotide heterologous to AAV, e.g., a nucleic acid sequence encoding a therapeutic transgene for transduction into or into a target cell or tissue, e.g., a human complement factor inhibitor (CFI)). Generally, heterologous polynucleotides typically have two AAV terminal inverted repeat sequences (ITRs) adjacent to each other. The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. rAAV vectors can be either single-stranded (ssAAV) or self-complementary (scAAV).

[0090] "AAV virus," "AAV virus particle," "rAAV vector particle," or "rAAV particle" refers to viral particles and polynucleotide rAAV vectors containing at least one AAV capsid protein. In some cases, at least one AAV capsid protein is either derived from wild-type AAV or is a variant AAV capsid protein. A "variant AAV capsid protein" is intended to be an AAV capsid protein that contains at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) compared to the corresponding parent AAV capsid protein. Variant capsid proteins may confer increased infectivity to retinal cells compared to the infectivity of retinal cells by AAV virions containing amino acid sequences present in naturally occurring AAV capsid proteins. Variant AAV capsid proteins may include, but are not limited to, AAV capsid proteins having insertions, 7m8 amino sequence insertions, R100 insertions, 7m8-like insertions, LSV1 sequence substitutions, and any other manipulated capsid proteins produced by other strategies (e.g., DNA shuffling, directional evolution, peptide insertion, ancestral reconstruction). LSV1 substitution sequences and 7m8 insertion sequences are publicly known in the art (see, e.g., U.S. Patent No. 9,193,956; U.S. Patent No. 9,233,133; U.S. Patent Application Publication No. 2021 / 0040501; and PCT / US2020 / 029895). Particularly targeted variant AAVs may include, but are not limited to, those disclosed in U.S. Patent No. 9,193,956, WO2017197355, WO2018022905, WO2019104279 and / or U.S. Patent Application Publication No. 20210371879A1. In some embodiments, the variant AAV comprises or consists of a 7m8 variant capsid protein (which may be referred to as AAV2.7m8 and 7m8AAV2). In some embodiments, the AAV comprises or consists of an AAV2.5T capsid protein, for example, provided in U.S. Patent No. 9,233,131.In some embodiments, AAV comprises the AAVShH10 or AAV6 capsid protein (U.S. Patent Application Publication No. 20120164106 and Klimczak et al. PLOS One 4(10):e7467 (Oct. 14, 2009)). In some embodiments, AAV comprises or consists of the AAV2.5T_LSV1 variant disclosed in U.S. Patent Application Publication No. WO2020219933.

[0091] The AAV2.7m8 capsid, engineered from wild-type AAV2, exhibits highly efficient retinal transduction after intravitreous (IVT) injection. The AAV2.7m8 capsid has 7m8 insertion in loop IV, reducing interaction between the viral capsid and HSPG receptors, allowing diffusion into the retina across the internal limiting membrane (ILM). AAV2.7m8 can transduce retinal cells, including photoreceptor cells, Müller glial cells, retinal ganglion cells, bipolar cells, and RPE cells.

[0092] When particles contain heterologous polynucleotides (e.g., polynucleotides other than the wild-type AAV genome, e.g., transgenes delivered to target cells or target tissues), they are called “rAAV particles,” “rAAV vector particles,” or “rAAV vectors.” Therefore, the production of rAAV particles necessarily involves the production of rAAV vectors, and consequently, vectors contained within the rAAV particles. Generally, heterologous polynucleotides are flanked by AAV terminal inverted repeat sequences (ITRs). Heterologous polynucleotides may contain polynucleotide cassettes. The polynucleotide cassettes of this application may be packaged within variant AAV particles to facilitate delivery of the cassette to a target cell type in a target tissue, such as retinal cells, but not limited to retinal cells.

[0093] The term "packaging," as used herein, may refer to a series of intracellular events that can result in the assembly and capsid encapsulation of rAAV particles.

[0094] "Subculturing" refers to a process that includes the steps of infecting insect cells with rBV containing the target gene, and incubating the infected insect cells.

[0095] The terms “target gene,” “GOI,” and “transgene” are used interchangeably herein. The target gene includes an open reading frame encoding the target gene product. In various embodiments, the nucleic acid may contain two or more nucleic acid sequences, each containing the target gene encoding the gene product. The “gene product” is a molecule resulting from the expression of a particular gene. Gene products include, but are not limited to, polypeptides, aptamers, interfering RNA, mRNA, etc. In certain embodiments, the “gene product” is a polypeptide, peptide, protein, or interfering RNA, including small interfering RNA (siRNA), miRNA, or small hairpin RNA (shRNA). In some embodiments, the target gene may be a reporter gene. Reporter genes are known in the art and include, but are not limited to, chloramphenicol acetyltransferase, β-galactosidase, β-glucoronidase, sea urchin luciferase, firefly luciferase, green fluorescent protein (GFP), red fluorescent protein (RFP), and alkaline phosphatases, such as secreted alkaline phosphatases. In some embodiments, the gene of interest may encode one or more AAV proteins or polypeptides. AAV proteins or polypeptides may include, but are not limited to, Rep proteins Rep78, Rep68, Rep58, Rep40, Cap proteins VP1, VP2, VP3, and Rep / Cap fragments and variants thereof. In some embodiments, the gene of interest may encode reporter gene products, such as GFP and RFP. In some embodiments, the gene of interest may encode therapeutic gene products, such as therapeutic proteins.Therapeutic gene products are known in the field and include polypeptide hormones, cytokines or growth factors (e.g., insulin or erythropoietin), interferons, blood coagulation factors, vaccines, anti-angiogenic polypeptides, vascular endothelial growth factor (VEGF) binding proteins, anti-VEGF agents, anti-VEGF proteins, opsin proteins, anti-C3 antibodies, anti-C5 antibodies, hormone receptors (e.g., mineralocorticosteroids, glucocorticoids and thyroid hormone receptors, but not limited to these), intramembrane proteins (e.g., TM-1 and TM-7, but not limited to these), intracellular receptors (e.g., orphan, retinoid, vitamin D3 and vitamin A receptors, but not limited to these), signaling molecules (e.g., This includes, but is not limited to, kinases, transcription factors, and signaling molecules, as well as transcription activator receptors of the cytokine superfamily (e.g., erythropoietin, growth hormone, interferon, interleukin, and colony-stimulating factor); G protein-coupled receptors, such as hormones, calcitonin, epinephrine, gastrin, paracrine or autocrine mediators, such as somatostatin or prostaglandins, but is not limited to these; neurotransmitter receptors (norepinephrine, dopamine, serotonin, or acetylcholine); ligands for tyrosine kinase receptors, such as insulin growth factor and nerve growth factor; and anti-dry AMD gene products. Anti-VEGF agents are known in the art and include, but are not limited to, bevacizumab, brolucizumab, ranibizumab, falisimab, abisipalpegol, conbercept, OPT-302, KSI-301, sunitinib maleate for injection (GB-102), PAN-90806 (PanOptica), and / or aflibercept.

[0096] The term “anti-VEGF agent” includes any therapeutic agent, such as a protein, polypeptide, peptide, fusion protein, multimeric protein, gene product, antibody, human monoclonal antibody, antibody fragment, aptamer, small molecule, kinase inhibitor, receptor or receptor fragment, or nucleic acid molecule, that can reduce, interfere with, disrupt, block, and / or inhibit the activity or function of endogenous VEGF and / or endogenous VEGF receptor (VEGFR), or the VEGF-VEGFR interaction or pathway in vivo. An anti-VEGF agent may be any known therapeutic agent, such as ranibizumab, brolucizumab, or bevacizumab, that, when delivered in vivo in cells, tissues, or subjects, can reduce the growth or formation of new blood vessels and / or edema or swelling. In some embodiments, the anti-VEGF agent may be naturally occurring, not naturally occurring, or synthetic. In some embodiments, the anti-VEGF agent may be derived from a naturally occurring molecule that has been subsequently modified or mutated to confer anti-VEGF activity. In some embodiments, the anti-VEGF agent may be a fusion or chimeric protein. In such proteins, a functional domain or polypeptide is artificially fused to a portion or polypeptide to create a fusion or chimeric protein that can sequester VEGF in vivo or function as a VEGFR decoy. In some embodiments, the anti-VEGF agent is a fusion or chimeric protein that blocks endogenous VEGFR from interacting with its ligand.

[0097] As used herein, “VEGF” may refer to any isoform of VEGF, including but not limited to VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, or any combination thereof, or any functional fragment or variant, unless otherwise specified. Unless otherwise specified, “VEGF” may refer to any member of the VEGF family, including members: VEGF-A, placental growth factor (PGF), VEGF-B, VEGF-C, and VEGF-D, or any combination thereof, functional fragment or variant. As used herein, “VEGF receptor” or “VEGFR” or “VEGF-R” may be used to refer to any one of the receptors of VEGF, including but not limited to VEGFR-1 (or Flt-1), VEGFR-2 (or Flk-1 / KDR), and VEGFR-3 (or Flt-4). VEGFR may be in membrane-bound or soluble form, or a functional fragment or shortened form of the receptor.

[0098] Therapeutic gene products for use in treating eye diseases or disorders may include, but are not limited to, anti-angiogenic polypeptides, VEGF-binding proteins, opsin proteins, anti-C3 antibodies, anti-C5 antibodies, anti-dry AMD gene products, aflibercept, sFLT-1, CFI, ranibizumab, and bevacizumab. Anti-dry AMD gene products may include, but are not limited to, inhibitors of C3, C5, HtrA1, C1qm, and natural inhibitors of the complement pathway, such as CFI, CFH, and CD59.

[0099] As used herein, “therapeutic gene” means a gene that, when expressed, produces a therapeutic gene product that, if present, confers a beneficial effect to a cell or tissue, or to the mammal in which the gene is expressed. Examples of beneficial effects include amelioration of signs or symptoms of a condition or disease, prevention or inhibition of a condition or disease, or conferring a desired characteristic. Therapeutic genes include, but are not limited to, genes that correct gene defects in cells or mammals, and genes that express therapeutic gene products.

[0100] "Helper function(s)" refers to a function(s) encoded in the helper viral genome that enables the replication and packaging of AAV (in conjunction with other requirements for replication and packaging). Where used herein, "Helper function(s)" can be provided in several ways, including, but not limited to, providing a helper virus or providing, transfected into producing cells, a helper plasmid containing a polynucleotide sequence encoding the desired function(s). Desired functions include, but are not limited to, those provided by the Adeno VA, E4, and E2A genes. For example, a plasmid or other expression vector containing a nucleotide sequence encoding one or more adenovirus helper proteins may be transfected or co-transfected into producing cells.

[0101] A "plasmid" refers to a small, often circular, extrachromosomal DNA molecule that can replicate independently in a cell. A plasmid may comprise one or more expression cassettes, open reading frames, polynucleotide cassettes, or expression vectors. The helper plasmid in the system of the present invention provides helper function and contains antibiotic resistance genes. In some examples, multiple copies of the helper plasmid are present in bacterial cells containing the donor plasmid and bacmid. An increased helper plasmid copy number may be beneficial in methods for obtaining recombinant bacmid or BEV containing a stable gene of interest.

[0102] Antibiotic resistance genes are known in the art and include, but are not limited to, ampicillin resistance genes, chloramphenicol resistance genes, erythromycin resistance genes, gentamicin resistance genes, kanamycin resistance genes, penicillin resistance genes, streptomycin resistance genes, penicillin / streptomycin resistance genes, rifampicin resistance genes, and tetracycline resistance genes. See, for example, Martinez et al 2008 Science 321:365-367 and Pal et al 2014 Nuc. Acids Res. 42:D737-43. Those skilled in the art can select an appropriate antibiotic resistance gene for use in the claimed method.

[0103] As used herein, “VEGF” may refer to any isoform of VEGF, including but not limited to VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, or any combination thereof, or any functional fragment or variant, unless otherwise specified. Unless otherwise specified, “VEGF” may refer to any member of the VEGF family, including members: VEGF-A, placental growth factor (PGF), VEGF-B, VEGF-C, and VEGF-D, or any combination thereof, functional fragment or variant. As used herein, “VEGF receptor” or “VEGFR” or “VEGF-R” may be used to refer to any one of the receptors of VEGF, including but not limited to VEGFR-1 (or Flt-1), VEGFR-2 (or Flk-1 / KDR), and VEGFR-3 (or Flt-4). VEGFR may be in membrane-bound or soluble form, or a functional fragment or shortened form of the receptor. The modified Bac-to-Bac system of this application utilizes a first antibiotic resistance gene, a second antibiotic resistance gene, and a third antibiotic resistance gene. Each of the three selected antibiotic resistance genes confers resistance to a different antibiotic, and as long as the antibiotic resistance gene in the donor plasmid does not interfere with the counter-selection markers utilized in the system, a person skilled in the art can select any three separate antibiotic resistance genes for use in the modified Bac-to-Bac system. In various embodiments, the antibiotic resistance gene in the donor plasmid is referred to as the first antibiotic resistance gene, the antibiotic resistance gene in the helper plasmid is referred to as the second antibiotic resistance gene, and the antibiotic resistance gene in the bacmid is referred to as the third antibiotic resistance gene. In some embodiments, the first, second, and third antibiotic resistance genes are selected from the group including ampicillin resistance genes, tetracycline resistance genes, and kanamycin resistance genes.In some aspects of this method, the first antibiotic resistance gene is an ampicillin resistance gene, the second antibiotic resistance gene is a tetracycline resistance gene, and the third antibiotic resistance gene is a kanamycin resistance gene.

[0104] In some embodiments, the baculovirus expression vector includes a target gene encoding Rep / Cap operably ligated to a promoter sequence that drives the expression of polynucleotides in cells. In some embodiments, the baculovirus expression vector includes a target gene encoding a therapeutic gene product operably ligated to a promoter sequence that drives the expression of polynucleotides in cells. In certain embodiments, the cells are producing cells. “Producing cells” are intended to be host cells used to produce rAAV virions. Exemplary host cells include, but are not limited to, Sf-9 cells, Sf-21 cells, Sf-RVN, Drosophila cell lines, Hi-5 cells, and cell lines derived from Aedes albopictus, as well as insect cells. Producing cells may be used to produce virions containing Rep / Cap and the target gene. In certain embodiments, the cells are reference cells. “Reference cells” are intended to be reference cells that have received rAAV gene therapy. Different promoters may be used to drive expression in producing cells rather than in reference cells. For example, the target gene encoding Rep / Cap can be operably ligated to a promoter that is activatable in insects. Insect-activatable promoters include, but are not limited to, polyhedron promoters (polh). The target gene encoding a therapeutic gene product can be operably ligated to a promoter sequence that is activatable in the target cell. The target gene can be operably ligated to one or more regulatory regions that influence its expression in the target cell.

[0105] Bacmid is a baculovirus plasmid that can grow in both bacterial and insect cells; in some cases, bacmid can be considered a shuttle vector.

[0106] The terms “rBV,” “baculovirus expression vector,” and “BEV” are used interchangeably herein. A baculovirus expression vector is a eukaryotic DNA viral vector for cloning and / or expression in cultured lepidopteran insect cells or insects. Most baculovirus expression vectors are derived from Autographa californica nuclear polyhedron disease virus (AcNPV). Methods for producing and using rBV are known in the art. See, for example, Chambers et al 2018 Curr Protoc Prot Sci 91:5.4.1-5.4.6 and Felberbaum 2015 Biotechnol. 10:702-714.

[0107] A "promoter" is a region of DNA that initiates the transcription of a specific gene. Promoters from a wide variety of sources are well known in the art, and any promoter known in the art may be used in the methods and systems of this application. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in either the 3' or 5' direction). Promoters include, but are not limited to, constitutively active promoters, inducible promoters, and cell type-specific promoters. Constitutively active promoters include, but are not limited to, human beta-actin, chicken beta-actin, cytomegalovirus (CMV), SV40, and CAG promoters. Cell type-specific promoters include, but are not limited to, the CD19 gene promoter, CaMKII1, and UAS. Inducible promoters include, but are not limited to, the Tet system (U.S. Patent Nos. 5,464,758 and 5,814,618), the ecdysone-inducible system (No et al, (1996) Proc. Natl Acad Sci 93:3346-3351), the Cre-Lox system, the T-Rex® system (Invitrogen, Carlsbad CA), the Cre-ERT tamoxifen-inducible recombinase system (Indra et al (1999) Nuc Acid Res 27:4324-4327, U.S. Patent No. 7,112,715, Kramer & Fussenegger 2005 Methods Mol Biol 308:123-144), and the LacSwitch® system (Stratagene San Diego CA). Insect promoters that can be activated include, but are not limited to, polh, IE-1, IE-0, IE-2, 39K, gp64, p6.9, VLf-1, p10, p10-p6.9, pCap / polh, promoter, ORF46 promoter, polh-L21, hsp70, Mtn promoter, pTre-CMV, pB2, pB2-p10, GAPDH promoter, OPie2, and hr5-ie1-p10 promoter.

[0108] Transposon insertion sites are known in the art and include, but are not limited to, Tn7, mini-att-Tn7, loxP, and attR1 / attR2.

[0109] "Multiplicity of Infection" or "MOI" refers to the number of virions or viral particles per cell during infection. "Low MOI" is an MOI below approximately 2, below approximately 1, below approximately 0.1, below approximately 0.01, below approximately 0.009, below approximately 0.008, below approximately 0.007, below approximately 0.006, below approximately 0.005, below approximately 0.004, below approximately 0.003, below approximately 0.002, below approximately 0.001, below approximately 0.0005, or below approximately 0.0001. Low MOIs may be within the ranges of approximately 0.0005–0.09, 0.0008–0.05, 0.001–0.04, 0.001–0.03, 0.001–0.02, 0.001–0.01, 0.001–0.009, 0.001–0.008, 0.001–0.007, 0.001–0.006, 0.001–0.005, 0.001–0.004, 0.001–0.003, and 0.001–0.002. The appropriate MOI for use with rBV and insect cells may differ substantially from the appropriate MOI for use with rAAV and target or target cells. Standard or typical MOI are approximately 2.3, 3, 4, 5, 6, 7, 8, 9, and 1 × 10⁻⁶. 1 , 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 , 1 x 10 15 , 1 x 10 16 , 1 x 10 17 and 1 × 10 18 It may be within the range.

[0110] The terms “recovered” and “collected” may be used interchangeably. Methods for recovering virions, virus particles, and rAAV are known in the art and may include, but are not limited to, filtration, flow filtration, depth filtration, membrane filtration, centrifugation, and combinations thereof. Any method for recovering virus particles known in the art may be used in the methods and systems of this application. In various embodiments of the method, the amount of virus particles recovered from insect cells is about 1 × 10⁻⁶. 10 vg, 5×10 10 vg, 6×10 10 vg, 7×10 10 vg, 8×10 10 vg, 9×10 10 vg, 1×10 11 vg, 2×10 11 vg, 3×10 11 vg, 4×10 11 vg, 5×10 11 vg, 6×10 11 vg, 7×10 11 vg, 8×10 11 vg, 9×10 11 vg, 1×10 12 vg, 2×10 12 , 3 x 10 12 , 4×10 12 vg, 5×10 12 vg, 6×10 12 vg, 7×10 12 vg, 8×10 12 vg, 9×10 12 vg, 1×10 13 vg, 2×10 13 vg, 3×10 13 vg, 4×10 13 vg, 5×10 13 vg, 6×10 13 vg, 7×10 13 vg, 8×10 13 vg, 9×10 13 vg, 1×10 14 vg, 2×10 14 vg, 3×10 14 vg, 4×10 14vg, 5×10 14 vg, 6×10 14 vg, 7×10 14 vg, 8×10 14 vg, 9×10 14 vg, 1×10 15 vg, 2×10 15 vg, 3×10 15 vg, 4×10 15 vg, 5×10 15 vg, 6×10 15 vg, 7×10 15 vg, 8×10 15 vg, 9×10 15 vg, 1×10 16 vg, 2×10 16 vg, 3×10 16 vg, 4×10 16 vg, 5×10 16 vg, 6×10 16 vg, 7×10 16 vg, 8×10 16 vg and approximately 9×10 16 It may be within the range of vg. It is recognized that the method for recovering rBV used in these methods may be the same as or different from the method for recovering rAAV used in these methods.

[0111] In various embodiments of the method, the amount of rBV recovered from insect cells is about 1×10 10 vg, 5×10 10 vg, 6×10 10 vg, 7×10 10 vg, 8×10 10 vg, 9×10 10 vg, 1×10 11 vg, 2×10 11 vg, 3×10<000009​​​​​​​​​​​​​​​​​​​​​12 vg, 6×10 12 vg, 7×10 12 vg, 8×10 12 vg, 9×10 12 vg, 1×10 13 vg, 2×10 13 vg, 3×10 13 vg, 4×10 13 vg, 5×10 13 It may be within the range of vg. Recombinant baculovirus seed stock produced by the method of the present invention is approximately 1 × 10⁻⁶. 8 vg / ml, 5 x 10 8 vg / ml, 1 x 10⁻⁶ 9 vg / ml, 2 x 10 9 vg / ml, 3 x 10 9 vg / ml, 4 x 10 9 vg / ml, 5 x 10 9 vg / ml, 6 x 10 9 vg / ml, 7×10 9 vg / ml, 8 x 10 9 vg / ml, 9×10 9 vg / ml, 1 x 10⁻⁶ 10 vg / ml, 2 x 10 10 vg / ml, 3 x 10 10 vg / ml, 4 x 10 10 vg / ml, 5 x 10 10 vg / ml, 6 x 10 10 vg / ml, 7×10 10 vg / ml, 8 x 10 10 vg / ml, 9×10 10 vg / ml, 1 x 10⁻⁶ 11 vg / ml, 2 x 10 11 vg / ml, 3 x 10 11 vg / ml, 4 x 10 11 vg / ml, 5×11 11 vg / ml, 6 x 10 11 vg / ml, 7×10 11 vg / ml, 8 x 10 11 vg / ml, 9×10 11 vg / ml or approximately 1 x 10⁶ 12 It may be within the range of vg / ml.

[0112] The AAV vaccine was administered at 1 × 10 11 vg、2×10 11 vg、3×10 11 vg、4×10 11 vg、5×10 11 vg、6×10 11 vg、7×10 11 vg、8×10 11 vg、9×10 11 vg、1×10 12 vg、2×10 12 、3×10 12 、4×10 12 vg、5×10 12 vg、6×10 12 vg、7×10 12 vg、8×10 12 vg、9×10 12 vg、1×10 13 vg、2×10 13 vg、3×10 13 vg、4×10 13 vg、5×10 13 vg、6×10 13 vg、7×10 13 vg、8×10 13 vg、9×10 13 vg、1×10 14 vg、2×10 14 vg、3×10 14 vg、4×10 14 vg、5×10 14 vg、6×10 14 vg、7×10 14 vg、8×10 14 vg、9×10 14 vg、1×10 15 vg、2×10 15 vg、3×10 15 vg、4×10 15 vg、5×10 15 vg、6×10 15 vg、7×10 15 vg、8×10 15 vg、9×10 15 vg、1×10 16 vg、2×10 16 vg、3×10 16 vg、4×1016 vg, 5×10 16 vg, 6×10 16 vg, 7×10 16 vg, 8×10 16 VG and approximately 9 × 10 16 It may be within the range of vg. The titer of the recovered rAAV is at least about 1 11 vg / ml, 2 11 vg / ml, 3 11 vg / ml, 4 11 vg / ml, 5 11 vg / ml, 6 11 vg / ml, 7 11 vg / ml, 8 11 vg / ml, 9 11 vg / ml, at least 1 12 vg / ml, 2 12 vg / ml, 3 12 vg / ml, 4 12 vg / ml, 5 12 vg / ml, 6 12 vg / ml, 7 12 vg / ml, 8 12 vg / ml, 9 12 vg / ml, at least 1 13 vg / ml, at least 1 14 vg / ml or at least about 1 15 It could be vg / ml.

[0113] A "polynucleotide cassette" refers to a polynucleotide sequence containing two or more functional polynucleotide sequences, such as regulatory elements, translation initiation sequences, coding sequences, and termination sequences, typically operably ligated to at least one other functional polynucleotide sequence within the polynucleotide cassette. Generally, the polynucleotide cassette in question is composed of DNA.

[0114] The polynucleotide cassettes of this disclosure typically include a promoter region. “Promoter,” as used herein, encompasses a DNA sequence that directs the binding of RNA polymerase, thereby promoting RNA synthesis. Promoter and corresponding protein or polypeptide expression may be ubiquitous, meaning it is strongly active in a wide range of cells, tissues, and species, or it may be cell-type specific, tissue-specific, or species-specific. A promoter may be “constitutive,” meaning it is continuously active, or a promoter may be “inducible,” meaning it can be activated or deactivated by the presence or absence of biofactors or non-biological factors. In certain embodiments, the promoter region promotes the expression of coding sequences in mammalian cells. Suitable examples include actin, chicken β-actin (CBA), cytomegalovirus (CMV), CMV immediate enhancer / β-actin (CAG), elongation factor 1-alpha (EF1a), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoters. Promoters may exhibit tissue or cell-specific expression.

[0115] As used herein, the term “operatably linked” refers to the juxtaposition of gene elements, such as promoters, enhancers, termination signal sequences, polyadenylation sequences, and Kozak sequences, which are related in a way that allows them to function in the expected manner. For example, a promoter is operatably linked to a coding region if the promoter helps initiate transcription of the coding sequence. Intervening residues may exist between a promoter and a coding sequence, or between any two elements, as long as the functional relationship is maintained.

[0116] Viral particles at any concentration suitable for efficiently transducing mammalian cells can be prepared for in vitro or in vivo contact with mammalian cells. For example, the viral particles may be 10 8Vector genome / mL (vg / ml) or higher, e.g., 5 × 10⁻¹⁰ 8 vg / mL;10 9 vg / mL, for example, 5 × 10 9 vg / ml;10 10 vg / ml, for example, 5 × 10 10 vg / ml;10 11 vg / ml, for example, 5 × 10 11 vg / ml;10 12 vg / ml, for example, 5 × 10 12 vg / ml;10 13 vg / ml, for example, 5 × 10 13 vg / ml;10 13 vg / ml, for example, 1.5 × 10 13 vg / ml;10 14 vg / ml, for example, 1 × 10 14 vg / ml and 5×10 14 The concentration is vg / ml or higher, but typically 1 × 10⁻⁶ 15 It can be formulated at concentrations of vg / ml or less. Similarly, any total number of viral particles suitable to provide adequate transduction of cells to impart a desired effect or treat a disease can be administered to mammals.

[0117] The target viral vector can be formulated into a pharmaceutical composition containing any appropriate unit dose of the vector that can be administered to a target to induce a change in the target or to treat a disease in the target. In some embodiments, the unit dose is, without limitation, 1 × 10⁻⁶. 8 vg or more, for example, at least about 1 × 10⁻¹⁶ 9 vg, 1×10 10 vg, 1×10 11 vg, 1×10 12 vg, 1×10 13 vg, 1×10 14 VG or 1×10 15 Contains vg. In some embodiments, the unit dose is approximately 1 × 10⁻⁶. 9 ~Approx. 4×10 12 vg, approx. 1×10 10 ~Approx. 4×10 11 vg, approx. 2×10 10~Approx. 3×10 11 vg, approx. 2×10 10 ~about 2×10 11 vg, approx. 2.5×10 10 ~about 2×10 11 vg, approx. 2×10 10 ~Approx. 1×10 11 vg, approx. 5×10 9 ~Approx. 8×10 11 vg, approx. 1×10 10 ~about 2×10 11 vg, approx. 5×10 10 ~about 2×10 11 VG or approximately 8 x 10 10 ~Approx. 1×10 11 It is vg.

[0118] A previously unresolved problem in the field of rAAV production from baculovirus expression vectors is the loss of the gene of interest from rBV. Compositions and methods for obtaining baculovirus expression vectors exhibiting a reduced percentage loss of the gene of interest are provided herein. The methods and compositions enable the production of recombinant baculovirus seed stocks containing the gene of interest. Methods for producing recombinant baculovirus seed stocks containing the gene of interest are provided herein, wherein at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, and at least about 95% of the rBV in the recombinant baculovirus seed stock contains the gene of interest. In some cases, recombinant baculovirus seed stocks containing rBV with a stable gene of interest can be re-plaqued and regenerated. In some cases, recombinant baculovirus seed stocks containing rBV with a stable gene of interest can be stored. Storage may involve re-passaging the recombinant baculovirus seed stock. Thus, recombinant baculovirus seed stocks containing rBV with a stable gene of interest can be used as a master virus bank, a starting virus bank, a regeneration bank, or a source of originals.

[0119] The rBV of this application may exhibit a low percentage loss of the target gene. The low percentage loss of the target gene per passage is approximately 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 24%, 26% of the rBV. The intention is to achieve loss of the target gene from less than 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, or approximately less than 80%.

[0120] A “stable target gene” is intended to be maintained in the vector with a high retention rate through one or more cell-mediated cycles. The vector may be a recombinant bacmid, and the cells may be either bacterial or insect cells. The vector may be a recombinant BEV, and the cells may be insect cells. The cell-mediated cycle is intended to involve the introduction of the vector into cells and the recovery of the vector from the cell(s). The cell-mediated cycle may include, but is not limited to, further steps such as purification or enrichment. It is recognized that the retention rate of the target gene in the bacmid may be the same as or different from the retention rate of the same target gene construct in the BEV. It is recognized that the retention rate of the target gene construct in the rBV may be higher than that of the same target gene construct in the recombinant bacmid. It is recognized that the retention rate of the target gene construct in the rBV may be lower than that of the same target gene construct in the recombinant bacmid. Retention rate is the ratio of the target gene to the vector in a population of vectors.

[0121] The retention rate of the target gene in the vector can range from 0 to 1. rBV exhibits retention rates of 0, approximately 0.001, approximately 0.005, approximately 0.01, approximately 0.05, approximately 0.1, approximately 0.15, approximately 0.2, approximately 0.25, approximately 0.3, approximately 0.35, approximately 0.4, approximately 0.45, approximately 0.5, approximately 0.55, approximately 0.6, approximately 0.65, approximately 0.7, approximately 0.75, approximately 0.8, approximately 0.85, approximately 0.9, approximately 0.95, and approximately 1. High retention rates include, after passage 1 (or cycle 1), retention rates above approximately 0.5, above approximately 0.55, above approximately 0.6, above approximately 0.65, above approximately 0.7, above approximately 0.75, above approximately 0.8, above approximately 0.85, above approximately 0.9, above approximately 0.95, and approximately 1; after passage 2 (or cycle 2), retention rates above approximately 0.4, above approximately 0.45, above approximately 0.5, above approximately 0.55, above approximately 0.6, above approximately 0.65, above approximately 0.7, and approximately 0. Retention rates above 0.75, above approximately 0.8, above approximately 0.85, above approximately 0.9, above approximately 0.95, and above approximately 1; as well as retention rates after passage 3 (or after cycle 3) above approximately 0.35, above approximately 0.4, above approximately 0.45, above approximately 0.5, above approximately 0.55, above approximately 0.6, above approximately 0.65, above approximately 0.7, above approximately 0.75, above approximately 0.8, above approximately 0.85, above approximately 0.9, above approximately 0.95, and above approximately 1.

[0122] Stable target gene ratios to vector after passage 1 are approximately 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, and equal to or higher than 1; after passage 2, approximately 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, and 0. The ratio of the gene of interest to the vector may be 0.95, and equal to or greater than approximately 1; or, after three or more passages, the ratio of the gene of interest to the vector may be approximately 0.35, approximately 0.4, approximately 0.45, approximately 0.5, approximately 0.55, approximately 0.6, approximately 0.65, approximately 0.7, approximately 0.75, approximately 0.8, approximately 0.85, approximately 0.9, approximately 0.95, and equal to or greater than approximately 1. The presence of a donor plasmid containing the gene of interest is recognized as being able to artificially increase the ratio of the gene of interest to the vector.

[0123] Vector quality can be assessed by evaluating any suitable portion of the vector, including but not limited to the promoter region and the skeletal region, with preferred portions being those unique to the bacmid plasmid.

[0124] Compositions and methods for producing vectors containing a stable gene of interest are provided herein. While not bound by any mechanism, a stable gene of interest may arise from processes involving concatemerization, increased transposase levels, altered integration, or a combination of these elements. Maintaining selection for a helper plasmid up to such a process, including during the process of counterselection for a donor plasmid, may be beneficial for producing a vector containing a stable gene of interest. Increasing transposase production may be beneficial for the formation of a vector containing a stable gene of interest.

[0125] In some cases, the unit dose of a pharmaceutical composition may be measured using the multiplicity of infection (MOI). MOI refers to the ratio or multiple of the vector or viral genome to the cells to which the nucleic acid can be delivered. In some cases, the MOI is 1 × 10⁻⁶ 4 ~1 × 10 8 , 1 x 10 5 ~1 × 10 7 , or 1 × 10 6 It is possible that in some cases the recombinant viruses of this disclosure may be at least about 1 × 10 1 , 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 , 1 x 10 15 , 1 x 10 16 , 1 x 10 17 and 1 × 10 18 The target cells are infected by MOI. In some embodiments, the amount of the pharmaceutical composition is approximately 1 × 10⁻⁶. 8 ~Approx. 1×10 15 Recombinant virus, approximately 1 x 10 8 ~about 10 14 Recombinant virus, approximately 1 x 10 10 ~Approx. 1×10 13 Recombinant virus or approximately 1 x 10 10 ~Approx. 3×10 12 This includes recombinant viruses. It is recognized that the MOI for administering rAAV to target cells may differ substantially from the appropriate MOI for the production or preparation of rAAV.

[0126] The present invention comprises pharmaceutical compositions comprising a polynucleotide cassette or gene delivery vector produced by the method described herein and a pharmaceutically acceptable carrier, diluent, or additive. For example, one embodiment is a pharmaceutical composition comprising a polynucleotide and a pharmaceutically acceptable additive. In a specific embodiment, the recombinant virus is recombinant adeno-associated virus (AAV). The polynucleotide cassette or gene delivery vector of interest may be combined with pharmaceutically acceptable carriers, diluents, and reagents that are useful in preparing formulations containing additives that are generally safe, non-toxic, desirable, and acceptable for primate use. Such additives may be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous. Examples of such carriers or diluents may include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Supplementary active compounds may also be incorporated into the formulation. The solutions or suspensions used for the formulation may include sterile diluents, such as water for injection, saline solution, non-volatile oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial compounds, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelate compounds, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, or phosphates; detergents, such as Tween® 20 for preventing aggregation; and compounds for adjusting osmotic pressure, such as sodium chloride or dextrose. The pH may be adjusted using an acid or base, such as hydrochloride acid or sodium hydroxide. In certain embodiments, the pharmaceutical composition is sterile.

[0127] Suitable pharmaceutical compositions for use with the compositions and methods of the present invention further include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In some cases, the compositions are fluid to the extent that syringe ability exists. In certain embodiments, the compositions are stable under manufacturing and storage conditions. Liquid pharmaceutical compositions generally contain a liquid carrier, e.g., water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. They may also contain physiological saline solution, magnesium chloride, dextrose or other saccharide solution, or glycol, e.g., ethylene glycol, propylene glycol, or polyethylene glycol. In some cases, a surfactant, e.g., 0.001% pluronic acid, may be used.

[0128] For delayed release, the pharmaceutical may be included in a pharmaceutical composition formulated for slow release, for example, in microcapsules formed from biocompatible polymers, or in a liposome carrier system according to methods known in the art.

[0129] The terms “combination,” “combined,” “used in combination with,” and “combined preparation” may, as used herein, refer to the combined administration of two or more drugs simultaneously, sequentially, or separately. “Simultaneously” as used herein means that these drugs are administered concurrently or at the same time. “Sequentially” as used herein means that these drugs are administered one after the other. “Separately” as used herein means that these drugs are administered independently of each other but within a time interval that allows them to exhibit a combined, preferably synergistic, effect.

[0130] Methods for determining expression levels are known in the art. Any method for determining expression levels may be used in the method of this application. Methods for determining expression levels include, but are not limited to, immunoassays and activity assays.

[0131] Methods for determining concentration are known in the art. Any method for determining concentration may be used in the method of this application. Methods for determining concentration include, but are not limited to, immunoassays, activity assays, and serial dilutions.

[0132] Immunoassays for measuring the presence and quantity of proteins in biological or cellular samples are well known in the art. See, for example, Hage, DS (1999) "Immunoassays" Analytica Chemistry 71(12):294-304; The Immunoassay Handbook, 4th Edition: Theory and Applications of Ligand Binding, ELISA and Related Techniques by David Wild (Ed) Elsevier Science (2013). Immunoassays generally rely on a reaction between a target protein and an antibody or antibody fragment that specifically binds to that target protein. Immunoassays can be performed in liquid or solid phase. Appropriate immunoassays include, but are not limited to, sandwich and competitive assays, Western blotting, ELISA, radioimmunoassays, and fluorescence immunoassays. Biological samples may include cell culture media or supernatant (samples obtained from cultures without lysing cells), cell lysates, whole cells, blood, serum, plasma, aqueous humor, vitreous humor, or other body fluids or tissues. Biological samples derived from the subject are recognized as potentially enriched by the separation of whole cells from the sample, particularly if the polypeptide of interest may be secreted from the cells. Separation may be obtained by any simple separation technique known in the art, including but not limited to fluorescence-activated cell sorting (FACS), magnetic separation, affinity chromatography, "panning" with affinity reagents, centrifugation, and ultracentrifugation.

[0133] As used herein, the terms “sequence identity,” “percent identity,” and “percent sequence identity” refer to the degree of identity between two or more polynucleotides when aligned using a nucleotide sequence alignment program; or the degree of identity between two or more polypeptide sequences when aligned using an amino acid sequence alignment program. Similarly, the terms “identical” and “percent identity,” as used herein in the context of two or more nucleotide or amino acid sequences, refer to two sequences that are identical, or two sequences that, when compared and aligned for the greatest match, have a specified percentage of amino acid residues or nucleotides, for example, using a sequence comparison algorithm, such as the Smith-Waterman algorithm, or measured by visual inspection. Percent identicality between amino acid sequences can be determined, for example, by using either a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, using the Needleman and Wunsch (1970, J. Mol. Biol. 48:444-453) algorithm incorporated into the GAP program in the GCG software package. As another example, percentage identicality between two nucleotide sequences can be determined using the NWSgapdna.CMP matrix, and gap weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6, using the GAP program in the GCG software package. A particularly preferred set of parameters (and one to use unless otherwise specified) is the Blossum 62 scoring matrix, using a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.Percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller (1989, Cabios, 4: 11-17), incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a 12-gap length penalty, and a 4-gap penalty. Further search and alignment tools known in the art include, but are not limited to, the NBLAST and XBLAST programs (version 2.0) of Altschul et al (1990) J. Mol. Biol. 215:403-410, as well as the Gapped BLAST program.

[0134] The terms “subject,” “patient,” or “individual” refer to mammals, including but not limited to primates such as humans and non-human primates such as African green monkeys and rhesus monkeys, mammalian sport animals, mammalian livestock, mammalian pets, and rodents. In some embodiments, the subject is human.

[0135] The terms “to treat,” “to treat,” “treatment,” “to improve,” or “to improve” and other grammatical equivalents, as used herein, mean to reduce, alleviate or improve a disease or disorder or its symptoms, prevent further symptoms of a disease or disorder, improve or prevent the underlying cause of symptoms, inhibit a disease or disorder, for example, to stop the onset of a disease or disorder, alleviate a disease or disorder, cause regression of a disease or disorder, or stop the symptoms of a disease or disorder, and are intended to include preventive measures and prevention. These terms further include achieving therapeutic benefits and / or preventive benefits. The term “therapeutic benefit” means the eradication or improvement of the disease or disorder being treated. Also, in some embodiments, therapeutic benefits are achieved by the eradication or improvement of one or more physiological symptoms associated with a disease or disorder such that improvement is observed in the subject, even though the subject still suffers from the disease or disorder. For preventive benefit, the pharmaceutical composition is administered to subjects at risk of developing the disease or disorder, or to subjects reporting one or more of the physiological symptoms of the disease or disorder, even if the disease or disorder has not been diagnosed.

[0136] The terms “to treat,” “to treat,” “treatment,” “to improve,” or “to improve” and other grammatical equivalents, as used herein, may mean reducing, alleviating or improving the symptoms of dry age-related macular degeneration (dry AMD) disease or disorder, preventing further symptoms of dry AMD disease or disorder, improving or preventing the underlying cause of symptoms, inhibiting dry AMD disease or disorder, for example, stopping the onset of dry AMD disease or disorder, alleviating dry AMD disease or disorder, causing regression of dry AMD disease or disorder, or stopping the symptoms of dry AMD disease or disorder, and may include preventive measures and prevention of wet AMD. These terms further include achieving therapeutic benefits and / or preventive benefits. The term “therapeutic benefits” for dry AMD means the eradication or improvement of the treated dry AMD disease or disorder. Furthermore, therapeutic benefits are achieved, in some embodiments, by the elimination or improvement of one or more physiological symptoms associated with dry AMD disease or disorder, even though the subject still suffers from the disease or disorder. For preventive benefits, the pharmaceutical composition is administered to subjects at risk of developing dry AMD disease or disorder, or subjects reporting one or more physiological symptoms of dry AMD disease or disorder, even if the disease or disorder has not been diagnosed.

[0137] Signs and symptoms of dry AMD include, but are not limited to, endothelial cell proliferation and retinal pigment epithelial (RPE) atrophy.

[0138] The terms “administer,” “give delivery,” and “dosage,” as used herein, may refer to methods used to enable the delivery of a therapeutic agent or pharmaceutical composition to a desired site of biological action. These methods include intravitreous or subretinal injection into the eye.

[0139] The terms “effective dose,” “therapeutic dose,” or “pharmaceutical dose,” as used herein, may refer to a sufficient amount of at least one pharmaceutical composition or compound administered to alleviate, to some extent, one or more signs or symptoms of an eye disease, eye disorder, or eye condition being treated. The “effective dose,” “therapeutic dose,” or “pharmaceutical dose” of a pharmaceutical composition may be administered as a unit dose to a subject requiring it (as further described elsewhere herein). The subject may be human or a non-human mammal.

[0140] When used herein, the term "pharmaceutically acceptable" may mean a relatively non-toxic material, such as a carrier or diluent, that does not negate the biological activity or properties of the compounds disclosed herein (i.e., does not cause undesirable biological effects when administered to an individual, nor does it interact in a harmful manner with any of the components of the composition in which it is contained).

[0141] The term “pharmaceutical composition” or simply “composition” may, as used herein, mean a biologically active compound that is optionally mixed with at least one pharmaceutically acceptable chemical component, not limited to, for example, a carrier, stabilizer, diluent, dispersant, suspending agent, thickener, or additive.

[0142] Complement factor I (also known as factor I, CFI, and C3b / C4b inactivator) is a protein encoded by the CFI gene in humans. CFI is a serine protease that typically circulates in an enzyme precursor-like state at a concentration of approximately 35 μg / mL (Roversi et al (2011) PNAS 108:12839-12844, Nilsson et al (2011) Mol Immunol 48:1611-1620). CFI inactivates C3b by cleaving it into iC3b, C3d, and C3d,g, and similarly inactivates C4b by cleaving C4b into C4c and C4d. Therefore, CFI activity downregulates the complement cascade in all complement pathways (secondary, classical, and lectin). CFI requires the presence of one or more cofactor proteins to perform its function; these cofactor proteins include, but are not limited to, C4BP, CFH, CR1 (also known as CR1 / CD35), and MCP (CD46); see Degn et al (2011) Am J Hum Genet 88:689-705. When C3b is cleaved to iC3b, iC3b does not perpetuate the amplification of the complement cascade or activation via the secondary pathway. iC3b promotes pro-inflammatory effects by activating complement receptor 3 (CR3) on certain cell types. CFI can process iC3b to C3dg in the presence of cofactor CR1. C3dg cannot bind to CR3. C3b binding to CR3 is involved in complement activation that leads to inflammation; the breakdown of iC3b to C3dg reduces complement-induced inflammation (Lachmann (2009) Adv. Immunol. 104:115-149). CFI can process iC3b into inactive degradation products.

[0143] Eye diseases and disorders are publicly known in the art. Eye diseases and disorders include, but are not limited to, monochromacy, glaucoma, retinitis pigmentosa, macular degeneration, retinal schizophrenia, Leber congenital amaurosis, diabetic retinopathy, color vision deficiency, diabetic macular edema, choroidal neovascularization, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branched retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, and diabetic retinal edema. Macular degeneration may include, but is not limited to, dry macular degeneration, wet macular degeneration, age-related macular degeneration, and acute macular degeneration.

[0144] Age-related macular degeneration (AMD) is a degenerative eye disease that affects the macula, a small, light-sensitive area in the center of the retina responsible for reading and high vision. The condition affecting the macula reduces central vision while leaving peripheral vision intact. In some cases, the disease can lead to central blindness. AMD is a significant cause of vision loss in the US population among people aged 65 and older, and the estimated prevalence of any AMD among people over 40 is approximately 6.5% (Klein et al., (2011) Arch Ophthalmol, 129(1):75-80).

[0145] There are two forms of age-related macular degeneration: dry (atrophic) and wet. Dry AMD is more common than wet AMD, but dry AMD can progress to wet AMD. Dry AMD is characterized by thinning of macular tissue as cells disappear; dry AMD can affect both eyes. Dry AMD is typically characterized by progressive apoptosis of cells in the retinal pigment epithelium (RPE) layer, covering photoreceptor cells, and also frequently, underlying cells in the choroidal capillary layer. Areas of dense RPE cell death accompanied by atrophy of covering photoreceptor cells are called geographic atrophy (GA). As dry AMD progresses and GA increases, central vision slowly deteriorates, and the ability to see fine details is gradually lost. Dry AMD tends to progress more slowly than wet AMD.

[0146] In some embodiments, ocular neovascularization is recurrent and / or persistent wAMD. In some embodiments, ocular neovascularization is active subfoveal CNV secondary to AMD. In some embodiments, active subfoveal CNV secondary to AMD accounts for ≥50% of the total lesion size. In some embodiments, active subfoveal CNV secondary to AMD accounts for ≥50% of the total lesion size, accompanied by evidence of leakage on fluorescein angiography (FA), fluid on spectral-domain optical coherence tomography (SD-OCT), and / or subretinal hemorrhage on color fundus photography. In some embodiments, active subfoveal CNV secondary to AMD accounted for ≥50% of the total lesion size, accompanied by evidence of leakage on fluorescein angiography (FA), fluid on spectral domain optical coherence tomography (SD-OCT), and / or subretinal hemorrhage on color fundus photography, and the total dimensions of the lesion did not exceed 12 macular photocoagulation study disc area. In some embodiments, one eye and / or the other eye of an individual exhibited a best corrected visual acuity (BCVA) based on ETDRS letter assessment of 78–25 (e.g., less than any of approximately 78, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, or 25) prior to administration of a unit dose of the rAAV particles of this disclosure. In some embodiments, one eye and / or the other eye of an individual exhibited a best corrected visual acuity (BCVA) based on the ETDRS letter rating that was higher than any of the following: about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100, prior to administration of a unit dose of the rAAV particles of the Disclosure.

[0147] In some embodiments, individuals had polypoid choroidal vasculopathy (PCV) in one eye and / or the other eye prior to administration of a unit dose of rAAV particles.

[0148] Monochromacy is a rare autosomal recessive disorder that causes retinal degeneration affecting all three types of cone photoreceptor cells, resulting in reduced visual acuity, photophobia, diurnal blindness, and severe loss of color discrimination. Mutations in the CNGB3 gene account for more than 90% of patients, resulting in complete monochromacy, which means they have significant impairments in color discrimination and central vision.

[0149] In some embodiments, a unit dose of rAAV particles is administered in combination with a steroid treatment. In some embodiments, the steroid treatment is a corticosteroid treatment. In some embodiments, the steroid treatment is a systemic steroid treatment. In some embodiments, the steroid treatment is an oral steroid treatment. In some embodiments, the steroid treatment is a prednisone treatment. In some embodiments, the steroid treatment is an ophthalmic steroid treatment. In some embodiments, the ophthalmic steroid treatment is a topical steroid treatment (e.g., eye drops), a periorbital steroid treatment (e.g., subtenon, subconjunctival), an intravitreal steroid treatment, or a superchoroidal steroid treatment. In some embodiments, the ophthalmic steroid treatment is a glucocorticoid, including but not limited to anti-inflammatory glucocorticoids. In some embodiments, the topical steroid treatment is a glucocorticoid, including but not limited to anti-inflammatory glucocorticoids. In some embodiments, topical steroid treatment is difluprednate treatment, medlisone treatment, loteprednol treatment, prednisolone treatment, fluocinolone treatment, triamcinolone treatment, rimexolone treatment, dexamethasone treatment, fluorometholone treatment, fluocinolone treatment, rimexolone treatment, or prednisone treatment. Anti-inflammatory glucocorticoids may include, but are not limited to, difluprednate, dexamethasone, prednisolone, triamcinolone, fluorometholone, rimexolone, fluocinolone, loteprednol, and their bioequivalents. In some embodiments, topical steroid treatment is difluprednate treatment. "Dexamethasone" refers to dexamethasone, dexamethasone biosimilars, dexamethasone bioequivalents, and pharmaceutical compositions comprising dexamethasone, dexamethasone biosimilars, or dexamethasone bioequivalents. Pharmaceutical compositions containing dexamethasone include, but are not limited to, Ozurdex®, Maxidex®, Decadron®, Dexamethasone Intensol®, Ocu-Dex®, Dexycu®, Dextenza®, and Zodex®.Ozurdex® is a pharmaceutical composition containing dexamethasone. "Difluprednate" refers to difluprednate, difluprednate biosimilars, difluprednate bioequivalents, and pharmaceutical compositions containing difluprednate, difluprednate biosimilars, or difluprednate bioequivalents. Pharmaceutical compositions containing difluprednate include, but are not limited to, Durezol® and difluprednate emulsion. "Triamcinolone" refers to triamcinolone, triamcinolone biosimilars, triamcinolone bioequivalents, and pharmaceutical compositions containing triamcinolone, triamcinolone biosimilars, or triamcinolone bioequivalents. Pharmaceutical compositions containing triamcinolone include, but are not limited to, Triesence®, Xipere®, and Trivaris®. In some embodiments, steroid treatment is administered before, during, and / or after the administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered before the administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered during the administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered after the administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered before and during the administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered before and after the administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered during and after the administration of a unit dose of rAAV particles. In some embodiments, steroid treatment is administered before, during, and after the administration of a unit dose of rAAV particles.

[0150] In some embodiments, the steroid treatment is an ophthalmic steroid treatment (e.g., difluprednate). In some embodiments, the ophthalmic steroid treatment (e.g., difluprednate) is a daily steroid treatment for up to about 4 weeks, about 6 weeks, or about 8 weeks, starting from the administration of a unit dose of rAAV particles. In some embodiments, the ophthalmic steroid treatment includes about 4 administrations of ophthalmic steroids in about week 1, about 3 administrations of ophthalmic steroids in about week 2, about 2 administrations of ophthalmic steroids in about week 3, and about 1 administration of ophthalmic steroids in about week 4; the timing begins with and is followed by the administration of a unit dose of rAAV particles. In some embodiments, the ophthalmic steroid is about 0.005% to about 0.5% difluprednate. In some embodiments, the ophthalmic steroid is difluprednate in any of the following concentrations: approximately 0.005%, approximately 0.006%, approximately 0.007%, approximately 0.008%, approximately 0.009%, approximately 0.01%, approximately 0.02%, approximately 0.03%, approximately 0.4%, approximately 0.05%, approximately 0.06%, approximately 0.07%, approximately 0.08%, approximately 0.09%, or approximately 0.1%. In some embodiments, the ophthalmic steroid is 0.05% difluprednate. In some embodiments, the dose of 0.05% difluprednate is one drop of ophthalmic solution. In some embodiments, one drop is approximately 50 μl (e.g., approximately 25 μl to approximately 50 μl, approximately 50 μl to approximately 100 μl). In some embodiments, the dose of difluprednate includes approximately 1 μg to approximately 5 μg, or approximately 2 μg to approximately 3 μg, or approximately 2.5 μg of difluprednate. In some embodiments, the dose of difluprednate includes approximately 2.5 μg of difluprednate.

[0151] In some embodiments, the steroid treatment is an ophthalmic steroid treatment (e.g., difluprednate). In some embodiments, the ophthalmic steroid treatment (e.g., difluprednate) is a daily topical steroid treatment for up to about 4 weeks, about 6 weeks, or about 8 weeks, starting from the administration of a unit dose of rAAV particles. In some embodiments, the topical steroid treatment includes about 4 administrations of topical steroids in about week 1, about 3 administrations of topical steroids in about week 2, about 2 administrations of topical steroids in about week 3, and about 1 administration of topical steroids in about week 4; the timing begins and is followed by the administration of a unit dose of rAAV particles. In some embodiments, topical steroid treatment includes approximately four daily doses of topical steroids for approximately three weeks following the administration of a unit dose of rAAV particles (i.e., QID), followed by approximately three daily doses of topical steroids for approximately one week (i.e., TID), followed by approximately two daily doses of topical steroids for approximately one week (i.e., BID), and then approximately one daily dose of topical steroids for approximately one week (i.e., QD). In some embodiments, the topical steroid contains 0.05% difluprednate in a dose of approximately 1 μg to approximately 3 μg. In some embodiments, the topical steroid contains 0.05% difluprednate in a dose of approximately 2.5 μg. In some embodiments, the topical steroid is approximately 0.005% to approximately 0.5% difluprednate. In some embodiments, the topical steroid is difluprednate in one of the following concentrations: approximately 0.005%, approximately 0.006%, approximately 0.007%, approximately 0.008%, approximately 0.009%, approximately 0.01%, approximately 0.02%, approximately 0.03%, approximately 0.04%, approximately 0.05%, approximately 0.06%, approximately 0.07%, approximately 0.08%, approximately 0.09%, or approximately 0.1%. In some embodiments, the topical steroid is 0.05% difluprednate. In some embodiments, the dose of 0.05% difluprednate is one drop of ophthalmic solution. In some embodiments, one drop is approximately 50 μl (e.g., approximately 25 μl to approximately 50 μl, approximately 50 μl to approximately 100 μl). In some embodiments, the dose of difluprednate includes approximately 1 μg to approximately 5 μg, or approximately 2 μg to approximately 3 μg, or approximately 2.5 μg of difluprednate.In some embodiments, the dose of difluprednate includes approximately 2.5 μg of difluprednate.

[0152] The methods, systems, and kits described herein may utilize conventional techniques and descriptions of molecular biology (including recombinant techniques), cell biology, biochemistry, immunochemistry, and virology techniques within the scope of the art of those skilled in the art, unless otherwise indicated. Such conventional techniques include methods for the cloning and propagation of recombinant viruses, the formulation of pharmaceutical compositions, and biochemical purification and immunochemical processes. Specific descriptions of suitable techniques may be made by reference to the examples herein. However, equivalent conventional procedures may also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals, such as Weiner et al., Eds, Genetic Variation: a Laboratory Manual (2007); Dieffenbach, Dveksler (Eds.), PCR Primer: a Laboratory Manual (2003); Sambrook and Russell, Condensed Protocols from Molecular Cloning: a Laboratory Manual (2006); Miller & Calos eds. (1987) "Gene Transfer Vectors for Mammalian Cells" and Current Protocols in Immunology (1999-2022) John Wiley & Sons, all of which are incorporated herein by reference in their entirety for all purposes.

[0153] References to the following examples are for illustrative purposes only and should not be understood as limiting the scope of the claims. Several alternative compositions and methods are applicable and suitable for use in carrying out the claimed methods and compositions. It should also be understood that evaluation of the expression constructs and methods of this application may be carried out using procedures standard in the art. [Examples]

[0154] (Example 1) cell culture

[0155] Sf9 rhabdovirus-negative (Sf-RVN®) insect cells (GlycoBac) were used as a substitute for Sf9 insect cells to increase the titer of rAAV production. Sf-RVN cells were maintained in a Multitron shaker (Infors HT) in a polycarbonate Erlenmeyer flask (Corning) at 28°C at a constant rotation of 125 rpm using serum-free Sf-900II® SFM (Gibco®) or ESF AF (Expression Systems) medium. The cells were verified to be in the logarithmic phase (3.0–4.0E6 cells / mL) with a viability of more than 90% using Vi-Cell BLU (Beckman Coulter).

[0156] (Example 2) Recombinant baculovirus construction

[0157] Recombinant baculoviruses were constructed using the Bac-to-Bac Baculovirus Expression System (Invitrogen). This method is based on site-specific transposition of a donor plasmid (pFastBac®) sequence into a baculovirus shuttle vector (bacmid) grown in DH10Bac® E.Coli cells. pFastBac® vectors containing the target gene(s) or rep-cap, ampicillin resistance gene, and Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedrin (polh) and P10 promoter for high-level expression in insect cells were ordered from Integrated DNA Technologies. The pFastBac® expression cassette is flanked by the left and right arms of Tn7 and also contains the gentamicin resistance gene and SV40 polyadenylation signal to form mini Tn7. In our modified system, the gentamicin resistance gene and GC-rich region near the Tn7 site are removed, preventing the unnecessary transfer of antibiotic resistance genes into the patient and also preventing the development of inflammation.

[0158] Using the heat shock method, pFastBac® vectors were transformed into DH10Bac® E. coli strains containing a baculovirus shuttle vector (Bacmid) with a mini-attTn7 target site and a helper plasmid. 1 μl of 1 mg / ml pFastBac® vector was added to 20 μl of DH10Bac® cells (Invitrogen) thawed on ice. The cells were incubated on ice for 30 minutes, then transferred to a water bath (PolyScience) at 40°C for 25 seconds. The cells were then returned to ice for 2 minutes, after which 1000 μl of SOC medium (Thermo Scientific) was added. DH10Bac® cells containing the plasmid were grown in an Innova43 shaker (New Brunswick) at 220 rpm at 37°C for 6 hours. Transposition of the desired sequence occurs between the mini-Tn7 element on the pFastBac™ vector and the mini-attTn7 target site on bacmid, generating recombinant bacmid (rbacmid) in the presence of a transposition protein supplied by a helper plasmid. After 6 hours, the cells were spread on an agar plate (Teknova) and incubated at 37°C for 48 hours. Since bacmid and pHelper confer resistance to kanamycin and tetracycline, respectively, the agar plate contains 50 μg / ml kanamycin and 10 μg / ml tetracycline. The agar plate also contains 200 μg / ml isopropyl β-D-1-thiogalactopyranoside (IPTG) and 20 μg / ml Blue-gal, a chromogenic substrate. This aids in screening bacmid-containing clones using blue vs. white colonies. During recombinant bacmid formation, lacZ deletion on the bacmid is completed, preventing the production of β-galactosidase from the reporter cassette. Otherwise, the β-galactosidase produced by lacZ hydrolyzes Blue-gal to form 5-bromo-4-chloro-indoxyl, which produces an insoluble blue pigment called 5,5'-dibromo-4,4'-dichloro-indigo. Therefore, colonies formed by non-recombinant cells appear blue, while recombinant cells appear white.

[0159] A single white colony was re-streaked onto a new agar plate with the same components and grown for 48 hours to confirm its color. In the original Bac-to-Bac system, a single colony from the second agar plate is used to purify bacmid. However, the inventors modified this system in such a way as to remove pFastBac™ from the colony. To do this, a streptomycin-sensitive gene (rpsl), which acts as an anti-selection marker, was added to pFastBac™. After confirming the white colony on the second agar plate, a single colony was grown on a third agar plate containing 50 μg / ml kanamycin, 10 μg / ml tetracycline, and 100 μg / ml streptomycin. Each of the colonies on the third plate was grown on a fourth agar plate containing 100 μg / ml carbenicillin. One of the colonies that could not grow on the carbenicillin plate was selected for the next step. The colony was spread on a fifth plate containing three antibiotics. Next, a single colony from the last plate was grown overnight at 37°C at 220 rpm in 170 mL of 2×YT Broth containing animal-free soytone medium (Teknova) with kanamycin and tetracycline in an Innova 43 shaker. Bacmid from the culture was purified using the PureLink® HiPure Plasmid Filter Maxiprep Kit (Invitrogen) according to the manufacturer's protocol. An overview of the process using representative steps is shown in Figure 1. The presence or absence of rbacmid and donor plasmid was confirmed using 0.5% agarose gel and ddPCR. See Figure 2.

[0160] (Example 3) P0 passage of recombinant baculovirus (rBV)

[0161] Sf-RVN cells were transfected to generate recombinant baculovirus using rBacmid DNA purified from bacterial colonies. rBacmid GFP / Fluc was designed as the target gene with a GFP / Fluc reporter gene adjacent to it via ITR. 7m8 rBacmid was designed to include modified AAV2 Rep / Cap genes driven by polh and P10 promoters.

[0162] Sf-RVN cells were verified to be in the logarithmic phase (3.0–4.0E6 cells / mL) with a viability of more than 90% using Vi-Cell BLU. Cells were centrifuged at 230G for 10 minutes and resuspended in fresh SF-900 II SFM at a density of 2E6 / mL. 9 μg rbacmid was added to 1 mL of 1× PBS (pH 7.4, Cal-free, Mag-free) (Gibco®), and 45 μL of Cellfectin® II Reagent (Gibco®) was added to another 1 mL of 1× PBS. The mixture was incubated for 5 minutes, then rbacmid was added to Cellfectin, and incubated at room temperature for 30 minutes. Cellfectin reagent must be at room temperature and thoroughly mixed before use. After 30 minutes, the DNA-oil mixture was added dropwise to 30 mL of cells. Four days after transfection, cells were checked with Vi-Cell BLU and then centrifuged at 230G for 10 minutes using a Sorvall Legend XFR centrifuge (Thermo Scientific). The supernatant was collected in amber canonical centrifuge tubes (Avantor) as the P0 stock of rBV. 5% sucrose was added to rBV for long-term storage at -80°C. The titer of rBV was checked using ddPCR.

[0163] (Example 4) Plaque assay purification

[0164] After verifying that Sf-RVN cells were in the logarithmic phase (3.0-4.0E6 cells / mL) with a viability rate higher than 90%, they were resuspended in Sf-900II SFM at a density of 0.5E6 / mL. 2 mL of diluted cells were added to each well of a 6-well plate and incubated at room temperature for 30 minutes. 4% agarose gel (Gibco) was transferred from 4°C to a 70°C water bath. A 100 mL empty bottle and Sf-900® 1.3× (Gibco) were placed in a 40°C water bath. Next, serial dilutions of P0 rBV at E10 vg / mL were performed. -1 from 10 -7 This was carried out using the SF900II SFM. A person skilled in the art will recognize that the 1E# notation is equivalent to the 1×10# notation.

[0165] After attaching the cells to a 6-well plate, they were checked for 50–70% cellular confluence using an inverted microscope (Zeiss Invertroskop). Next, 1000 μL of Sf 900II SFM was added to the first well as a control, and a 1000 μL dilution was added. -3 ~10 -7 The agarose was added to the corresponding wells. The 6-well plate was incubated at 28°C for 1 hour. After the agarose had thawed, 30 mL of SF-900 (1.3×) medium and 10 mL of 4% agarose gel were added to an empty bottle and gently mixed to avoid foam formation. This prepared a 1% agarose overlay, which was then returned to a 40°C water bath until use.

[0166] At the end of a 1-hour incubation, the virus was aspirated and replaced with a 2 mL 1% agarose overlay. The agarose overlay was allowed to solidify in BSC for 20 minutes. Next, 1 mL of SF900 II SFM was added on top of the agarose overlay, and the plate was incubated at 28°C for 7–10 days. On days 7–10, depending on plaque formation and growth, the medium was removed from the agarose overlay, and individual plaques were collected using a 2 mL pipette by inserting the tip into the agarose overlay with stepwise aspiration, such that the agarose plug and some liquid were aspirated into the tip. The agarose plug was transferred to a sterile tube containing 1 mL of SF900 II SFM, and the agarose and liquid were rinsed into the sterile tube by aspirating and discharging several times with a pipette. The tube was maintained overnight at 4°C to produce P0 rBV or BEV.

[0167] (Example 5) P1 passage of rBV

[0168] One day prior to infection, Sf-RVN cells were checked to be in the logarithmic phase (3.0-4.0E6 cells / mL) with a viability rate of more than 90% using Vi-Cell BLU. 30 mL of cells were cultured in 125 mL flasks at a density of 1.2E6 / mL using SF900II SFM for each P0 rBV. On the day of infection, the cells were re-examined using Vi-Cell BLU, and 500 μL of P0 rBV was added to each flask. Four days after infection, the cells were harvested as described in Example 3. ddPCR was performed on P1 rBV to determine the most stable clone.

[0169] (Example 6) P2 passage of rBV

[0170] P2 passage was carried out as described in Example 5 using the most stable clone of P1 rBV for the gene of interest (GOI) and Rep-Cap. However, to improve the consistency of the protocol, Sf-RVN cells were infected with an MOI of 0.1 instead of the previously used 1:1000 dilution of P1 rBV. Three days after infection, P2 rBV was harvested and ddPCR was performed to determine the vg titer of the virus.

[0171] (Example 7) Medium and MOI Optimization

[0172] To optimize rAAV production, the highest rAAV titers were found using SF900II SFM, SF900III SFM (Gibco), ESF-AF and EX-Cell CD (SAFC) media. ESF-AF medium yielded the highest rAAV titers within the range of E12 vg / ml (E8 IFU / ml). Therefore, ESF-AF medium was selected for rAAV production.

[0173] Insect cells were co-infected with 7m8 rBV and CFI rBV at either an MOI of 3 or 0.001 in two medium types (SF900II SFM or ESF-AF). rAAV was harvested and the rAAV titer was determined. The rAAV titers obtained from co-infection at an MOI of 0.001 were significantly higher than those obtained from co-infection at an MOI of 3. The results from one such experiment are shown in Figure 7.

[0174] (Example 8) rAAV Production

[0175] One day prior to infection, Sf-RVN cells in ESF-AF medium were verified to be in the logarithmic phase (3.0–4.0E6 cells / mL) with a viability rate of more than 90%, and these were prepared at a density of 1.2E6 / mL in 30mL culture for each rAAV production. The following day, P2 rBV containing GOI and P2 rBV containing Rep / Cap were added to the cells at an MOI of 0.001. The previous method used an MOI of 5. After incubating the cells in a shaker at 28°C for 4 hours, 225 μL of immediate Advantage® 66765 40× insect feed (SAFC) and 4.5 μL of fatty acids (SAFC) were added to each culture. Five days after infection, the cells were checked using Vi-Cell BLU. For recovery, 10×AAV-MAX lysis buffer (Gibco) was added to the culture to a final concentration of 1×, 1M MgCl2 was added to the culture to a final concentration of 2–4 mM, and 90 U / mL Benzonase was added to each 30 mL of culture. The culture was then incubated in a shaker at 37°C for 2 hours. After lysing the cells, they were pelleted in a Sorvall Legend XFR centrifuge at 4000 rpm for 30 minutes. The supernatant was filtered using a 0.2 μm PES membrane (Thermo Scientific) and stored at -80°C for further purification. The titer of rAAV virus was determined by ddPCR.

[0176] In some cases, insect feed was not added to the culture. rAAV titers were determined from both the feed-added and unadded methods. The addition of feed increased rAAV titers from insect cells grown in both ESF AF medium and Sf-900II™ SFM medium. Results from one such experiment are shown in Figure 7.

[0177] (Example 9) Evaluation of the effect of the stability of the target gene on rBV stability and rAAV titer between different passages.

[0178] After generating P0 rBV, a plaque purification assay was performed, and 10 different plaques were selected for GOI (GFP-Fluc) and 7m8. The target gene / vector skeleton ratio was evaluated in the 10 different plaques. The results from one such experiment are shown in Figure 6.

[0179] For rBV-GOI (GFP-Fluc), 78% of rBV cells retain the target gene in passage 0 (P0), 75% retain the target gene after passage 1 (P1), and 50% retain the target gene after passage 2 (P2).

[0180] For rBV-7m8, two different plaques were selected for further evaluation. In 7m8 plaque 1, 71% of rBV cells retained the target gene rep / cap at P0, 31% at P1, and 20% at P2.

[0181] In 7m8 plaque 2, 71% of rBVs retain the target gene rep / cap in P0, 81% retain the target gene rep / cap in P1, and 54% retain the target gene rep / cap in P2.

[0182] rAAV was produced using P0, P1, and P2 rBV (BEV) from two different 7m8 plaques (plaques 1 and 2), along with the same GOI plaque (rBV-GOI:GFP-Fluc). rAAV titers decreased to 1 / 20th in plaques 1-7m8, but only by approximately 25% in plaques 2-7m8.

[0183] (Example 9) Evaluation of the effect of pHelper on the formation of stable target genes.

[0184] Transform bacterial cells with a donor plasmid containing the target gene, bacmid, and pHelper plasmid. Transform bacterial cells with multiple amounts of pHelper plasmid. Grow bacterial cells under conditions that allow for selection for all three plasmids and enable identification of recombinant bacmid. In some experiments, bacteria are transformed with standard amounts of pHelper and grown in a medium with varying amounts of antibiotics to which pHelper provides resistance. Grow bacteria from bacterial colonies containing recombinant bacmid under conditions that allow for counterselection for the donor plasmid. Recover bacmid from bacterial colonies containing recombinant bacmid. Evaluate recombinant bacmid by next-generation sequencing (NGS) of long-read sequences. Identify recombinant bacmid containing concatemers. Assess the copy number of the target gene.

[0185] Insect cells are infected with recombinant bacmid. P0 rBV is obtained. The copy number of the target gene is evaluated using NGS. Heterogeneity of the population is evaluated. Insect cells are infected with P0 rBV and P1 rBV is recovered. The copy number of the target gene is evaluated using NGS. Heterogeneity of the population is evaluated in the P1 rBV population.

Claims

1. A system for producing recombinant bacmids having reduced residual DNA, i. A donor plasmid comprising a first antibiotic resistance gene, an anti-selection marker, and an import cassette, wherein the import cassette comprises a left transposon arm, the gene of interest, and a right transposon arm; ii. A helper plasmid containing a second antibiotic resistance gene; iii. Bacmid containing a third antibiotic resistance gene and transposon insertion site located within the reporter cassette A system that includes this.

2. The system according to claim 1, wherein each of the first, second, and third antibiotic resistance genes confers resistance to a different antibiotic.

3. The system according to claim 1, wherein the first antibiotic resistance gene, the second antibiotic resistance gene, and the third antibiotic resistance gene are selected from the group including kanamycin resistance gene, ampicillin resistance gene, tetracycline resistance gene, penicillin resistance gene, streptomycin resistance gene, erythromycin resistance gene, and penicillin / streptomycin resistance gene.

4. The system according to claim 1, wherein the first antibiotic resistance gene, the second antibiotic resistance gene, and the third antibiotic resistance gene are selected from the group including an ampicillin resistance gene, a tetracycline resistance gene, and a kanamycin resistance gene.

5. The system according to claim 1, wherein the bacmid comprises a kanamycin resistance gene, the helper plasmid comprises a tetracycline resistance gene, and the donor plasmid comprises an ampicillin resistance gene and an anti-selection marker which is rpsl.

6. The system according to claim 1, wherein the counter-selection marker is an antibiotic sensitivity gene.

7. The system according to claim 6, wherein the antibiotic sensitivity gene is selected from the group comprising rpsl, URA3, and thymidine kinase.

8. The system according to claim 6, wherein the antibiotic sensitivity gene is rpsl.

9. The system according to claim 1, wherein the donor plasmid does not contain an active gentamicin resistance gene.

10. The system according to claim 1, wherein the import cassette is optimized to reduce the GC-rich region within the import cassette.

11. The system according to claim 1, wherein the donor plasmid does not contain an active gentamicin resistance gene or a GC-rich region in the transfer cassette.

12. The system according to claim 1, wherein the left transposon arm is Tn7R and the right transposon arm is Tn7L.

13. The system according to claim 1, wherein the reporter cassette contains the lacZα gene.

14. The system according to claim 1, wherein the transposon insertion site is selected from the group including a Tn7 insertion site, a modified Tn7 insertion site, and a mini-att-Tn7 site.

15. The system according to claim 1, wherein the transposon insertion site is a mini-att-Tn7 site.

16. The system according to claim 1, wherein the target gene encodes an AAV RepCap protein or a therapeutic gene product.

17. The system according to claim 16, wherein the target gene encoding the AAV RepCap protein comprises a 7m8 cassette.

18. The system according to claim 1, selected from the group comprising a modified Bac-to-Bac system, a modified baculovirus-infected cell (BIC) system, and a modified Bac-plus system.

19. A method for producing recombinant bacmid having reduced residual DNA, (a) A step of introducing into a bacterial cell a system for producing recombinant bacmid containing the gene of the target, wherein the system i. A donor plasmid comprising a first antibiotic resistance gene, an anti-selection marker, and an import cassette, wherein the import cassette comprises a left transposon arm, the gene of interest, and a right transposon arm; ii. A helper plasmid containing a second antibiotic resistance gene; iii. Bacmid plasmid containing a third antibiotic resistance gene and transposon insertion site located within the reporter cassette. Steps including; (b) Growing the transformed bacteria in the presence of three antibiotics to which the first, second, and third antibiotic resistance genes confer resistance; (c) step of selecting at least one bacterial colony containing recombinant bacmid; (d) Incubating bacteria from at least one bacterial colony containing the recombinant bacmid selected in step (c) with a compound that enables counterselection against the donor plasmid; and (e) After competitor selection, the step of collecting recombinant bacmids with reduced residual DNA from bacterial colonies. Methods that include...

20. The method according to claim 19, wherein bacterial colonies containing recombinant bacmid are identified by a change in reporter activity.

21. The method according to claim 19, wherein the reporter activity is selected from the group comprising fluorescence, antibiotic sensitivity, and bacterial colony color.

22. The method according to claim 19, wherein the transformed bacteria are grown in the presence of a beta-galactosidase substrate.

23. The method according to claim 19, wherein the beta-galactosidase substrate is selected from the group comprising x-gal and BluoGal.

24. The method according to claim 23, wherein the presence of recombinant bacmid is indicated by white colonies formed in the presence of the beta-galactosidase substrate.

25. The method according to claim 19, wherein each of the first, second, and third antibiotic resistance genes confers resistance to a different antibiotic.

26. The method according to claim 19, wherein the first antibiotic resistance gene, the second antibiotic resistance gene, and the third antibiotic resistance gene are selected from the group comprising kanamycin resistance gene, ampicillin resistance gene, tetracycline resistance gene, penicillin resistance gene, streptomycin resistance gene, erythromycin resistance gene, and penicillin / streptomycin resistance gene.

27. The method according to claim 19, wherein the donor plasmid comprises an ampicillin resistance gene and rpsl, the helper plasmid comprises a tetracycline resistance gene, and the bacmid plasmid comprises a kanamycin resistance gene.

28. The method according to claim 19, wherein the compound enabling counterselection against the donor plasmid is an antibiotic.

29. The method according to claim 19, wherein the compound that enables counterselection against the donor plasmid is streptomycin.

30. An efficient method for producing a recombinant baculovirus seed stock containing a target gene, wherein at least 50% of the recombinant baculovirus (rBV) in the recombinant baculovirus seed stock contains the target gene, and the method is (a) A step of introducing into a bacterial cell a system for producing recombinant bacmid containing the gene of the target, wherein the system i. A donor plasmid comprising a first antibiotic resistance gene, an anti-selection marker, and an import cassette, wherein the import cassette comprises a left transposon arm, the gene of interest, and a right transposon arm; ii. A helper plasmid containing a second antibiotic resistance gene; iii. Bacmid containing a third antibiotic resistance gene and transposon insertion site located within the reporter cassette Steps including; (b) Growing the transformed bacteria in the presence of three antibiotics to which the first, second, and third antibiotic resistance genes confer resistance; (c) step of selecting at least one bacterial colony containing recombinant bacmid; (d) Incubating bacteria from at least one bacterial colony containing the recombinant bacmid selected in step (c) with a compound that enables counterselection against the donor plasmid; (e) After competitor selection, a step of collecting recombinant bacmid containing the gene of interest from the bacterial colony; (f) A step of transforming insect cells with the recombinant bacmid containing the gene of interest collected in step (e); (g) Incubating the transformed insect cells and recovering rBV containing the gene of the objective from the insect cells; (h) A step of performing a first passaging, the passaging comprising the steps of infecting insect cells with rBV containing the gene of the objective, and incubating the infected insect cells; (i) recovering rBV containing the gene of the interest from at least two plaques of insect cells and identifying the plaques containing rBV containing the gene of the interest, wherein the rBV shows a low percentage loss of the gene of the interest; and (j) A step of performing a second passage (P1 rBV), the second passage comprising the steps of infecting insect cells with the rBV from the plaque identified in step (i), incubating the infected insect cells, and recovering a recombinant baculovirus seed stock containing the rBV containing the gene of the object from the insect cells. Methods that include...

31. The method according to claim 30, wherein, after the first passage, at least 80% of the rBV contains the target gene.

32. The method according to claim 30, wherein, after the second passage, at least 70% of the rBV contains the target gene.

33. The above method produces recombinant baculovirus seed stock after two passages via insect cells, wherein the recombinant baculovirus seed stock is at least 2 × 10 １０ The method according to claim 30, wherein the vector genome is in the form of a genomic molecule (vg / ml).

34. The method according to claim 30, wherein bacterial colonies containing recombinant bacmid are identified by a change in reporter activity.

35. The method according to claim 30, wherein the reporter activity is selected from the group comprising fluorescence, antibiotic sensitivity, and bacterial colony color.

36. The method according to claim 30, wherein the transformed plasmid is grown in the presence of a beta-galactosidase substrate.

37. The method according to claim 36, wherein the beta-galactosidase substrate is selected from the group comprising x-gal and BluoGal.

38. The method according to claim 30, wherein the presence of recombinant bacmid is indicated by white colonies formed in the presence of the beta-galactosidase substrate.

39. The method according to claim 30, wherein each of the first, second, and third antibiotic resistance genes confers resistance to a different antibiotic.

40. The method according to claim 30, wherein the first antibiotic resistance gene, the second antibiotic resistance gene, and the third antibiotic resistance gene are selected from the group comprising kanamycin resistance gene, ampicillin resistance gene, tetracycline resistance gene, penicillin resistance gene, streptomycin resistance gene, erythromycin resistance gene, and penicillin / streptomycin resistance gene.

41. The method according to claim 30, wherein the donor plasmid comprises an ampicillin resistance gene and rpsl, the helper plasmid comprises a tetracycline resistance gene, and the bacmid plasmid comprises a kanamycin resistance gene.

42. The method according to claim 30, wherein the compound enabling counterselection against the donor plasmid is an antibiotic.

43. The method according to claim 30, wherein the compound that enables counterselection against the donor plasmid is streptomycin.

44. The step of incubating the transformed insect cells is performed at approximately 28°C with CO2. ２ The method according to claim 30, which is carried out for approximately four days without any additional procedures.

45. The method according to claim 30, wherein the step of incubating the transformed insect cells is performed in a shaker incubator.

46. The method according to claim 30, wherein the insect cells are selected from the group comprising Sf-RVN cells, Sf9 cells, and Hi5 cells.

47. The method according to claim 46, wherein the insect cells are selected from the group including Sf-RVN cells and Sf9 cells.

48. The method according to claim 30, wherein the insect cells are transformed with bacmid in an amount between approximately 1 μg and approximately 20 μg.

49. The method according to claim 48, wherein insect cells are transformed with approximately 6 to 9 μg of bacmid.

50. The method according to claim 30, wherein the transformed insect cells are grown in 30 ml of culture medium.

51. The method according to claim 30, wherein the target gene encodes Rep / Cap.

52. The method according to claim 51, wherein the target gene comprises a 7m8 cassette.

53. The method according to claim 30, wherein the step of recovering rBV containing the target gene comprises the steps of recovering rBV from at least 10 plaques of insect cells and identifying plaques containing stable rBV having the target gene.

54. The method according to claim 30, further comprising the step of performing a third passage, wherein the third passage (P2 rBV) includes the steps of: infecting insect cells with rBV from P1 rBV identified in step (j); incubating the infected insect cells; and recovering a recombinant baculovirus seed stock from the insect cells containing the gene of the interest or rBV containing Rep / Cap.

55. A method for producing a recombinant baculovirus seed stock containing a stable target gene, wherein at least 50% of the recombinant baculovirus (rBV) in the recombinant baculovirus seed stock contains a stable target gene, and the method is (a) A step of introducing into a bacterial cell a system for producing recombinant bacmid containing the gene of the target, wherein the system i. A donor plasmid comprising a first antibiotic resistance gene, an anti-selection marker, and an import cassette, wherein the import cassette comprises a left transposon arm, the gene of interest, and a right transposon arm; ii. A helper plasmid containing a second antibiotic resistance gene; iii. Bacmid containing a third antibiotic resistance gene and transposon insertion site located within the reporter cassette Steps including; (b) Growing the transformed bacteria in the presence of three antibiotics to which the first, second, and third antibiotic resistance genes confer resistance; (c) step of selecting at least one bacterial colony containing recombinant bacmid; (d) Incubating bacteria from at least one bacterial colony containing the recombinant bacmid selected in step (c) with a compound that enables counterselection against the donor plasmid; (e) After competitor selection, a step of collecting recombinant bacmid containing the gene of interest from the bacterial colony; (f) A step of transforming insect cells with the recombinant bacmid containing the gene of interest collected in step (e); (g) Incubating the transformed insect cells and recovering rBV containing the gene of the objective from the insect cells; (h) A step of performing a first passaging, the passaging comprising the steps of infecting insect cells with rBV containing the gene of the objective, and incubating the infected insect cells; (i) recovering rBV containing the target gene from one or more insect cell plaques and identifying plaques containing stable rBV containing the target gene; and (j) A step of performing a second passage, the second passage comprising: infecting insect cells with rBV containing a stable target gene; incubating the infected insect cells; and recovering a recombinant baculovirus seed stock containing rBV containing the stable target gene from the insect cells. Methods that include...

56. Recombinant baculovirus seed stock containing stable target genes.

57. A recombinant baculovirus seed stock containing a stable gene of interest produced by the method described in claim 55.

58. The recombinant baculovirus seed stock according to claim 57, wherein the gene for the purpose contains a 7m8 cassette.

59. The recombinant baculovirus seed stock according to claim 57, wherein the target gene encodes Rep / Cap.

60. A composition comprising a recombinant baculovirus seed stock containing a stable target gene.

61. A composition comprising recombinant bacmid containing a stable target gene.

62. A method for producing rAAV containing the target gene, (a) A step of producing a first recombinant baculovirus seed stock by the method of claim 30, wherein the first recombinant baculovirus seed stock comprises an rBV containing a first target gene; (b) A step of producing a second recombinant baculovirus seed stock by the method of claim 30, wherein the second recombinant baculovirus seed stock comprises an rBV encoding the Rep / Cap protein; (c) The step of co-infecting insect cells with the first recombinant baculovirus seed stock and the second recombinant baculovirus seed stock; (d) After co-infection, add the supply to the insect cell culture medium; (e) the step of incubating the infected insect cells; and (f) A step of recovering rAAV containing the target gene from the insect cells. Methods that include...

63. The method according to claim 62, wherein the step of co-infecting insect cells comprises the step of co-infecting insect cells with a first recombinant baculovirus seed stock and a second recombinant baculovirus seed stock with a low MOI.

64. The method according to claim 63, wherein the low MOI is less than 0.

01.

65. The method according to claim 63, wherein the low MOI is less than 0.

009.

66. The method according to claim 63, wherein the low MOI is in the range of about 0.0005 to about 0.

009.

67. The method according to claim 63, wherein the low MOI is in the range of about 0.

001.

68. The method according to claim 63, wherein the MOI of the first recombinant baculovirus seed stock is different from the MOI of the second recombinant baculovirus seed stock.

69. The method according to claim 63, wherein the MOI of the first recombinant baculovirus seed stock and the MOI of the second recombinant baculovirus seed stock are the same.

70. The recovered rAAV is at least 1 １１ The method according to claim 62, having a titer of vg / ml.

71. The recovered rAAV is at least 5 １１ The method according to claim 70, having a titer of vg / ml.

72. The recovered rAAV is at least 1 １２ The method according to claim 70, having a titer of vg / ml.

73. The method according to claim 62, wherein the supply is added between approximately one hour and approximately eight hours after co-infection.

74. The method according to claim 73, wherein the supply is added approximately four hours after co-infection.

75. The method according to claim 62, wherein the insect culture medium is ESF-AF.

76. The method according to claim 62, wherein the insect cells are selected from the group comprising Sf-RVN cells, Sf9 and Hi5 cells.

77. The method according to claim 62, wherein the insect cells are Sf-RVN cells.

78. The method according to claim 62, wherein rAAV containing the target gene is recovered with a high titer less than 25 days after transforming insect cells with recombinant bacmid.

79. The method according to claim 78, wherein rAAV containing the target gene is recovered with a high titer approximately 23 days after transforming insect cells with recombinant bacmid.

80. The method according to claim 62, wherein rAAV containing the target gene is recovered with a high titer five days after co-infection.

81. The method according to claim 62, wherein p2 rBV is recovered with a high titer 18 days after transforming insect cells with recombinant bacmid.

82. The method according to claim 62, wherein the volume of either the first recombinant baculovirus seed stock or the second recombinant baculovirus seed stock used to co-infect the insect cells, or the volume of each of the first recombinant baculovirus seed stock and the second recombinant baculovirus seed stock, is less than 1 liter.

83. The method according to claim 62, wherein the volume of either the first recombinant baculovirus seed stock or the second recombinant baculovirus seed stock used to co-infect the insect cells, or the volume of each of the first recombinant baculovirus seed stock and the second recombinant baculovirus seed stock, is less than 500 ml.

84. The method according to claim 62, wherein the volume of either the first recombinant baculovirus seed stock or the second recombinant baculovirus seed stock used to co-infect the insect cells, or the volume of each of the first recombinant baculovirus seed stock and the second recombinant baculovirus seed stock, is less than 100 ml.

85. The method according to claim 62, wherein the volume of either the first recombinant baculovirus seed stock or the second recombinant baculovirus seed stock used to co-infect the insect cells, or the volume of each of the first recombinant baculovirus seed stock and the second recombinant baculovirus seed stock, is less than 30 ml.

86. A method for rapidly producing rAAV containing the target gene, (a) A step of producing a first recombinant baculovirus seed stock by the method of claim 30, wherein the first recombinant baculovirus seed stock comprises an rBV containing a first target gene; (b) A step of producing a second recombinant baculovirus seed stock by the method of claim 30, wherein the second recombinant baculovirus seed stock comprises an rBV encoding the Rep / Cap protein; (c) Transducing insect cells with the first recombinant baculovirus seed stock at a low multiple of infection (MOI) and the second recombinant baculovirus seed stock at a low multiple of infection; (d) After co-infection, add the supply to the insect cell culture medium; (e) the step of incubating the infected insect cells for five days; and (f) A step of recovering rAAV containing the first target gene from the insect cells less than 27 days after transforming the insect cells with recombinant bacmid. Methods that include...

87. The method according to claim 62, wherein the first target gene encodes a therapeutic gene product for use in treating an eye disease or disorder.

88. The method according to claim 87, wherein the eye disease or disorder is selected from the group including glaucoma, retinitis pigmentosa, macular degeneration, retinal schizophrenia, Leber congenital amaurosis, diabetic retinopathy, monochromacy, and color vision deficiency.

89. The method according to claim 88, wherein the macular degeneration is selected from the group including dry macular degeneration, wet macular degeneration, and age-related macular degeneration.

90. The method according to claim 87, wherein the therapeutic gene product is selected from the group comprising anti-angiogenic polypeptides, vascular endothelial growth factor (VEGF) binding proteins, opsin proteins, anti-C3 antibodies, anti-C5 antibodies, complement factor I (CFI), and anti-dry AMD gene products.

91. The method according to claim 87, wherein the therapeutic gene product is selected from the group comprising aflibercept, sFLT1, CFI, ranibizumab, and bevacizumab.

92. A method for rapidly producing rAAV containing the target gene, (g) A step of producing a first recombinant baculovirus seed stock by the method of claim 30, wherein the first recombinant baculovirus seed stock comprises an rBV containing a first target gene; (h) A step of producing a second recombinant baculovirus seed stock by the method of claim 30, wherein the second recombinant baculovirus seed stock comprises an rBV encoding the Rep / Cap protein; (i) Transducing insect cells with the first recombinant baculovirus seed stock at a low multiple of infection (MOI) and the second recombinant baculovirus seed stock at a low multiple of infection; (j) Approximately four hours after co-infection, add the supply to the insect cell culture medium; (k) the step of incubating the infected insect cells for 5 days; and (l) Five days after co-infection, the rAAV containing the first target gene is recovered from the insect cells. Methods that include...

93. A method for producing rAAV containing the target gene, (a) A step of producing a first recombinant baculovirus seed stock by the method of claim 30, wherein the first recombinant baculovirus seed stock comprises an rBV containing a first target gene; (b) Providing a second recombinant baculovirus seed stock, wherein the second recombinant baculovirus seed stock comprises an rBV containing a stable gene of interest encoding the Rep / Cap protein; (c) A step of co-infecting insect cells with the first recombinant baculovirus seed stock at a low MOI and the second recombinant baculovirus seed stock at a low MOI; (d) After co-infection, add the supply to the insect cell culture medium; (e) the step of incubating the infected insect cells; and (f) A step of recovering rAAV containing the target gene from the insect cells. Methods that include...

94. The method according to claim 89, wherein the second recombinant baculovirus seed stock comprises rBV including a 7m8 cassette.

95. A composition comprising recombinant bacmid having reduced residual DNA produced by the method described in claim 19.

96. A method for producing rAAV containing the target gene, (a) A step of providing a first recombinant baculovirus seed stock, wherein the first recombinant baculovirus seed stock comprises an rBV containing a stable first target gene; (b) Providing a second recombinant baculovirus seed stock, wherein the second recombinant baculovirus seed stock comprises an rBV containing a stable gene of interest encoding the Rep / Cap protein; (c) A step of co-infecting insect cells with the first recombinant baculovirus seed stock at a low MOI and the second recombinant baculovirus seed stock at a low MOI; (d) After co-infection, add the supply to the insect cell culture medium; (e) the step of incubating the infected insect cells; and (f) A step of recovering rAAV containing the target gene from the insect cells. Methods that include...

97. A method for rapidly producing rAAV containing the target gene, (a) A step of producing a first recombinant baculovirus seed stock by the method of claim 30, wherein the first recombinant baculovirus seed stock comprises an rBV containing a first target gene; (b) Providing a second recombinant baculovirus seed stock, wherein the second recombinant baculovirus seed stock comprises an rBV containing a stable gene of interest encoding the Rep / Cap protein; (c) A step of co-infecting insect cells with the first recombinant baculovirus seed stock at a low MOI and the second recombinant baculovirus seed stock at a low MOI; (d) After co-infection, add the supply to the insect cell culture medium; (e) the step of incubating the infected insect cells; and (f) Approximately five days after co-infection, the rAAV containing the target gene is recovered from the insect cells. Methods that include...

98. A composition comprising a recombinant bacmid having reduced residual DNA produced by the method described in claim 19, 30, 54, 61, 82, 88, 89, 92, or 93.

99. The composition according to claim 98 for use in the production of a pharmaceutical composition.

100. Insect cells capable of producing stable rAAV containing the target gene.

101. The system according to claim 1, wherein the recombinant bacmid having reduced residual DNA is for use in rAAV production.

102. The system according to claim 1, wherein the helper plasmid is an AAV helper plasmid.

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