Production of recombinant adeno-associated virus (RAAV) in insect cells.

Abstract

Materials for efficient production of recombinant baculovirus seed stocks containing a nucleic acid sequence that encodes a gene of interest and methods of producing recombinant baculovirus seed stocks containing a nucleic acid sequence that encodes a gene of interest are provided. Also provided are rapid methods of producing rAAV comprising a nucleic acid having a nucleic acid sequence that encodes a gene of interest; the method allows production of rAAV at a high titer.

Description

1 / 76 “PRODUCTION OF RECOMBINANT ADENOASSOCIATED VIRUS (RAAV) IN INSECT CELLS” CROSS-REFERENCE TO RELATED REQUESTS

[001] This application claims priority under 35 U.S.C. § 119(e) for Provisional Application No. U.S. 63 / 489,656, filed March 10, 2023, and Provisional Application No. U.S. 63 / 465,669, filed May 11, 2023, each of which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION

[002] The present invention relates to the field of recombinant AAV production, particularly large numbers of rAAV particles. BACKGROUND OF THE INVENTION

[003] AAV is a linear, single-stranded DNA virus of 4.7 kb. rAAV is associated with excellent clinical safety, as wild-type AAV is non-pathogenic and has no etiological association with any known disease. Recent clinical and non-clinical success with recombinant adeno-associated virus (rAAV) vector as an in vivo gene delivery vehicle has increased interest in producing high-purity and high-efficacy viral vectors for various natural and engineered serotypes.

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

[005] The open reading frames of rep encode four rep proteins (Rep78, Rep68, Rep52, and Rep40) that are synthesized from mRNAs 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 for packaging AAV DNA into capsids. VP1, VP2, and VP3 are Petition 870250080612, dated 08 / 09 / 2025, page 7 / 111 2 / 76 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 an alternative splicing mechanism for VP1 and a less efficient start codon (CTG) for VP2 to reduce its protein levels, but maintains a highly efficient start codon for VP3. The N-terminal sequence present in VP1 contains a phospholipase A2 domain that is necessary for AAV infectivity. Furthermore, the VP2 / VP3 mRNA encodes an activator assembly protein (AAP) with a weak CTG start codon, but in a different reading frame. AAP facilitates nuclear import of the major capsid protein VP3 and promotes capsid assembly and maturation, but AAP is not present in the mature capsid.

[006] AAV vectors used for therapeutic nucleic acid delivery typically have approximately 96% of the parental genome deleted, so that only terminal repeats (ITRs) containing recognition signals for DNA replication and packaging remain. This reduces immunological or toxic side effects due to viral gene expression. In addition, the delivery of specific AAV proteins to producing cells allows for the integration of the AAV vector comprising AAV ITRs into a specific region of the cellular genome, if desired. See, for example, U.S. Patents 6342390 and 6821511. Host cells comprising an integrated AAV genome exhibit little or no alteration in cell growth or morphology (see, for example, U.S. Patent No. 4797368).

[007] Inverted terminal repeats (ITRs) flank the unique coding sequences for non-structural replication (Rep) and the structural capsid proteins (Cap) (also known as virion proteins (VPs)). ITRs are self-complementary and organized in such a way that an energetically stable, intramolecular, T-shaped hairpin duplex can be formed. The hairpin structures function as an origin for replication of Petition 870250080612, dated 08 / 09 / 2025, page 8 / 111 3 / 76 Viral DNA. The ITR is a site that plays a cis role in replication and serves as a recognition site for trans-acting replication proteins. An exception to the symmetry of the ITR sequence occurs in the “D” region of the ITR. Single-strand DNA cleavage occurs at the junction between the A and D regions; this is the region where de novo DNA synthesis begins. The Rep genes encode the Rep, Rep78, Rep68, Rep52, and Rep40 proteins. The Cap genes encode the VP, VP1, VP2, and VP3 proteins. These proteins form the capsid. An AAV that replicates in a mammalian cell typically has two ITR sequences. AAVs require the expression of helper genes for efficient replication.

[008] Several AAV serotypes and more than 100 AAV variants have been isolated from adenovirus stocks or tissues of human or non-human primates. Generally, AAV serotypes have genomic sequences of significant homology at the nucleic acid sequence and amino acid sequence levels, such that different serotypes have an identical set of genetic functions, produce virions that are physically and functionally equivalent, and replicate and assemble by nearly identical mechanisms.

[009] The Rep and ITR sequences of AAV are particularly conserved in many AAV serotypes. Furthermore, the Rep and ITR sequences of many AAV serotypes are known to efficiently complement (i.e., functionally replace) corresponding sequences from other serotypes during the production of AAV particles in mammalian cells. Generally, Cap proteins, which determine the cellular tropicity of the AAV particle, and sequences related to Cap protein coding, are significantly less conserved than Rep genes in different AAV serotypes. Given the ability of Rep and ITR sequences to cross-complement corresponding sequences from other serotypes, the AAV vector may comprise a mixture of serotypes and therefore be a “chimeric” or “pseudotyped” AAV vector. A vector Petition 870250080612, dated 08 / 09 / 2025, page 9 / 111 4 / 76 of chimeric AAV comprises AAV capsid proteins derived from two or more (e.g., 2, 3, 4, etc.) different AAV serotypes. In contrast, a pseudotyped AAV vector comprises one or more ITRs of one AAV serotype packaged in a capsid of another AAV serotype. Chimeric and pseudotype vectors are described in more detail, for example, in US 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.

[010] The most commonly used method for producing recombinant adeno-associated viruses (rAAVs) in research laboratories is by transient triple transfection of 293 cells with cis and trans AAV plasmids and an adenovirus helper plasmid. However, most HEK293 fabrication 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 entirely prohibitive. For example, a clinical study may require more than 1015 rAAV particles. To produce this number of rAAV particles in a mammalian cell line, such as human 293 cells, would require the transfection and culture of approximately 1011 cells, equivalent to 5,000 175 ml vials of cells.There is also concern that a vector intended for clinical use, produced in mammalian cell culture, could be contaminated with undesirable and potentially pathogenic material from a mammalian cell.

[011] Patent No. US 6,723,551 B2 by Kotin et al. and Urabe et al. 2006 J. Virol. 80:1874-1885 discloses methods for producing rAAV vectors in insect cells. The AAV sequences employed for AAV production in insect cells can be derived from the genome of any AAV serotype. Generally, AAV serotypes have genomic sequences of significant sequence identity. Petition 870250080612, dated 08 / 09 / 2025, page 10 / 111 5 / 76 at the amino acid and nucleic acid levels, provide a virtually identical set of genetic functions, produce virions that are physically and functionally similar, and replicate and assemble by virtually identical mechanisms. Kotin and Urabe describe baculovirus vectors constructed with nucleic acids encoding Rep78 / 68 and Rep52 / 40 in head-to-tail palindromic arrays under the control of an independent promoter. These vectors include inverted terminal repeats (ITRs), coding sequences of genes of interest, and AAV capsid genes. Although initial results suggested that high-titer rAAV could be obtained with these methods, later work indicated that rAAV vectors were less infectious than those produced in mammalian cells (Merten et al 2005 Gene Therapy 12:S51-S61 and Kohlbrenner et al 2005 Mol. Ther. 12:1217-1225).

[012] US Patent No. 6,723,551 suggests that insect cells perform Rep mRNA splicing differently from the process in mammalian cells. US Patent No. 6,723,551 avoids splicing or removing an intron before translation.

[013] In AAVs produced by mammalian cells, the best yield of “complete” virions (i.e., viral particles incorporating an AAV genome) that are fully functional and can reach the nucleus is obtained when all three VP proteins are expressed in a stoichiometry close to 1:1:10 (VP1:VP2:VP3). The splicing event required to produce a 1:1:10 stoichiometry of 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 modify the VP1 expression level. Attempts to use large homologous repeats of Rep78 and Rep52 indicated that the homologous repeats were unstable. Attempts to separate the coding sequences of Rep78 and Rep52 required two baculoviruses and an additional baculovirus to provide the Petition 870250080612, dated 08 / 09 / 2025, page 11 / 111 6 / 76 VP1 protein. Cao et al 2000 J. Virol 74:11456-11463 described a method for producing high-titer, wild-type recombinant AAV vectors using helper plasmids. However, Cao's methods used human cells and did not include a promoter that could direct transcription in insect cells.

[014] U.S. Patent No. 8,945,918 by Chen provides methods for expressing genes in insect cells and producing infectious rAAV vectors. The infectious rAAV vectors produced by Chen's methods exhibit loss of the gene of interest; by the second passage, only about 50% of the viral vectors contain the gene of interest. The viral particles continue to lose the gene of interest with each passage. By the third passage, only about 25% of the viral particles contain the gene of interest. Less than 10% of the viral particles contain the gene of interest after the fourth passage. The methods described in U.S. Patent No. 8,945,918 require multiple passages to obtain sufficient seed stock to produce a large number of rAAV particles. Large volumes of seed stock material are needed for rAAV production using Chen's methods. SUMMARY OF THE INVENTION

[015] Methods and systems for producing high-titer rAAV are provided in this document. The rAAV produced by these methods and systems can be used in pharmaceutical compositions, gene therapy compositions, for research purposes and for other uses.

[016] Systems are provided for the production of recombinant bacmids with reduced residual DNA in the production of rAAV. The systems for the production of recombinant bacmids with reduced residual DNA comprise (i) a donor plasmid comprising a first antibiotic resistance gene, a counterselectable marker and a transfer cassette, wherein the transfer cassette comprises a left transposon arm, a gene of interest Petition 870250080612, dated 08 / 09 / 2025, page 12 / 111 7 / 76 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.

[017] In aspects of the system, each of the first, second, and third antibiotic resistance genes confers resistance to a different antibiotic. In some aspects, the first antibiotic resistance gene, the second antibiotic resistance gene, and the third antibiotic resistance gene are selected from the group comprising 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 aspects, the first antibiotic resistance gene, the second antibiotic resistance gene, and the third antibiotic resistance gene are selected from the group comprising ampicillin resistance genes, tetracycline resistance genes, and kanamycin resistance genes.In one aspect, 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 a counterselectable marker which is rpsl.

[018] In several aspects of the system, the counterselectable marker is an antibiotic-sensitive gene. In some aspects, the antibiotic-sensitive gene is selected from the group comprising rpsl, URA3, and thymidine kinase. In certain aspects, the antibiotic-sensitive gene is rpsl.

[019] In some respects, the donor plasmid does not comprise an active gentamicin resistance gene. In some respects, the transfer cassette is optimized to reduce GC-rich regions in the transfer cassette. In several respects, the donor plasmid does not comprise an active gentamicin resistance gene. Petition 870250080612, dated 08 / 09 / 2025, page 13 / 111 8 / 76 gentamicin nor a GC-rich region in the transfer cassette.

[020] The left transposon arm is Tn7R and the right transposon arm is Tn7L in some aspects of the system.

[021] In some aspects of the system, the reporter cassette comprises a lacZα gene. In some aspects, the transposon insertion site is selected from the group comprising a Tn7 insertion site, a modified Tn7 insertion site, and a mini-att-Tn7 site. In certain aspects, the transposon insertion site is a mini-att-Tn7 site.

[022] In some aspects of the system, the gene of interest encodes AAV RepCap proteins or a therapeutic gene product. In some aspects, the gene of interest encoding AAV RepCap proteins comprises the 7m8 cassette.

[023] In several respects, the system is selected from the group comprising a modified Bac-to-Bac system, a modified baculovirus infectious cell (BIC) system and a modified Bac-plus system.

[024] Methods for producing recombinant bacmids with reduced residual DN are provided.Methods for producing recombinant bacmids comprising reduced residual DNA comprise the steps of (a) introducing a system for producing recombinant bacmids comprising a gene of interest into bacterial cells, wherein the system comprises (i) a donor plasmid comprising a first antibiotic resistance gene, a counterselectable marker and a transfer cassette, and wherein the transfer 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 and a transposon insertion site localized with a reporter cassette; (b) growing the transformed bacteria in the presence of three antibiotics to which the first, the second and the... Petition 870250080612, dated 08 / 09 / 2025, page 14 / 111 9 / 76 third antibiotic resistance genes confer resistance; (c) selection of at least one bacterial colony comprising a recombinant bacmid; (d) incubating bacteria from at least one bacterial colony comprising a recombinant bacmid selected in step (c) with a compound that allows counterselection against the donor plasmid; and (e) collecting recombinant bacmids with reduced residual DNA from a bacterial colony after counterselection.

[025] In some aspects of the methods, a bacterial colony comprising a recombinant bacmid is identified by a change in reporter activity. In several aspects, reporter activity is selected from the group comprising fluorescence, antibiotic sensitivity, and bacterial colony color. In some aspects of the methods, the transformed bacteria are cultured in the presence of a beta-galactosidase substrate. In certain aspects, the beta-galactosidase substrate is selected from the group comprising xgal and BluoGal. In several aspects, the presence of a recombinant bacmid is indicated by a white colony formed in the presence of the beta-galactosidase substrate.

[026] In several aspects of the methods, each of the first, second, and third antibiotic resistance genes confers resistance to a different antibiotic. In some aspects, the first antibiotic resistance gene, the second antibiotic resistance gene, and the third antibiotic resistance gene are selected from the group comprising 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 aspects, the first antibiotic resistance gene, the second antibiotic resistance gene, and the third antibiotic resistance gene are selected from the group comprising ampicillin resistance genes, tetracycline resistance genes, and Petition 870250080612, dated 08 / 09 / 2025, page 15 / 111 10 / 76 kanamycin resistance genes. In one aspect, 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 a counterselectable marker which is rpsl. In some aspects, the compound that allows counterselection against the donor plasmid is an antibiotic. In certain aspects, the compound that allows counterselection against the donor plasmid is streptomycin.

[027] The application embodiments provide efficient methods for producing a recombinant baculovirus seed stock comprising a gene of interest, wherein at least 50% of the recombinant baculovirus (rBV) in the recombinant baculovirus seed stock comprise a gene of interest.Efficient methods comprise the steps of (a) introducing a system for producing recombinant bacmids comprising a gene of interest into bacterial cells, wherein the system comprises (i) a donor plasmid comprising a first antibiotic resistance gene, a counterselectable marker and a transfer cassette, and wherein the transfer cassette comprises a left transposon arm, a 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 a transposon insertion site located within a reporter cassette.The methods comprise the steps of (b) cultivating the transformed bacteria in the presence of three antibiotics to which the first, second, and third antibiotic resistance genes confer resistance; (c) selecting at least one bacterial colony comprising a recombinant bacmid; (d) incubating a bacterium from at least one bacterial colony comprising a recombinant bacmid selected in step (c) with a compound that allows counterselection against the donor plasmid; (e) collecting recombinant bacmids that... Petition 870250080612, dated 08 / 09 / 2025, page 16 / 111 11 / 76 comprises a gene of interest from a bacterial colony after counterselection; (f) transform insect cells with recombinant bacmids comprising a gene of interest collected in step (e); (g) incubate the transformed insect cells and collect rBV comprising the gene of interest from the insect cells; (h) perform a first passage, wherein a passage comprises the steps of infecting insect cells with rBV comprising the gene of interest and incubating the infected insect cells; (i) harvest rBV comprising the gene of interest from at least two insect cell plates and identify a plate comprising rBV comprising a gene of interest and wherein the rBV exhibits a low loss rate of the gene of interest;and perform a second passage (p1 rBV), wherein the second passage comprises infecting insect cells with rBV from the plate identified in step (i), incubating the infected insect cells and harvesting recombinant baculovirus seed stock comprising rBV containing the gene of interest from insect cells.

[028] In some aspects of the method, after the first passage, at least 80% of the rBV comprises the gene of interest. In several aspects of the method, after the second passage, at least 70% of the rBV comprises the gene of interest. In certain aspects of the methods, the method produces a stock of recombinant baculovirus seeds after two passages through insect cells, wherein the stock of baculovirus seeds is at least 2 x 1010 vector genomes / ml (vg / ml).

[029] In several aspects of the methods, a bacterial colony comprising a recombinant bacmid is identified by a change in reporter activity. In some aspects, reporter activity is selected from the group comprising fluorescence, antibiotic sensitivity, and color of the bacterial colony. In some aspects of the methods, the transformed bacteria are cultured in the presence of a beta-galactosidase substrate. In certain aspects, Petition 870250080612, dated 08 / 09 / 2025, page 17 / 111 12 / 76 The beta-galactosidase substrate is selected from the group comprising xgal and BluoGal. In several respects, the presence of a recombinant bacmid is indicated by a white colony formed in the presence of the beta-galactosidase substrate.

[030] In several aspects of the methods, each of the first, second, and third antibiotic resistance genes confers resistance to a different antibiotic. In some aspects, the first antibiotic resistance gene, the second antibiotic resistance gene, and the third antibiotic resistance gene are selected from the group comprising 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 aspects, the first antibiotic resistance gene, the second antibiotic resistance gene, and the third antibiotic resistance gene are selected from the group comprising ampicillin resistance genes, tetracycline resistance genes, and kanamycin resistance genes.In one aspect, 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 a counterselectable marker which is rpsl. In some aspects, the compound that allows counterselection against the donor plasmid is an antibiotic. In certain aspects, the compound that allows counterselection against the donor plasmid is streptomycin.

[031] In some aspects of the methods, the incubation step of the transformed insect cells takes place at approximately 28°C and without CO2 for approximately four days. In several aspects of the methods, the incubation step of the transformed insect cells takes place in a shaker incubator. In certain aspects, the insect cells are cultured in 30 ml of medium. Petition 870250080612, dated 08 / 09 / 2025, page 18 / 111 13 / 76

[032] In certain aspects of the methods, insect cells are selected from the group comprising Sf-RVN cells, Sf9 cells and Hi5 cells. In one aspect, insect cells are selected from the group comprising Sf-RVN cells and Sf9 cells.

[033] In several respects, insect cells are transformed with between about 1 μg and 20 μg of bacmids. In certain respects, insect cells are transformed with about 6-9 μg of bacmids.

[034] In some respects, the gene of interest encodes Rep / Cap. In some respects, the gene of interest that encodes Rep / Cap comprises cassette 7m8. In certain respects, the gene of interest comprises cassette 7m8.

[035] In particular aspects, the rBV collection step comprising the gene of interest involves collecting rBV from at least ten insect cell plates and identifying one plate comprising rBV having a stable gene of interest.

[036] In some respects, the methods further comprise performing a third passage, wherein the third passage (rBV p2) comprises infecting insect cells with rBV from the p1 rBV identified in step (j), incubating the infected insect cells and collecting a stock of recombinant baculovirus seeds comprising rBVs comprising the gene of interest or Rep / Cap from insect cells.

[037] Methods are provided for producing a recombinant baculovirus seed stock comprising a stable gene of interest, wherein at least 50% of the recombinant baculovirus (rBV) in the recombinant baculovirus seed stock comprise a stable gene of interest. The methods comprise the steps of (a) introducing a system for producing recombinant bacmids comprising a gene of interest into bacterial cells, wherein the system comprises (i) a donor plasmid comprising a first Petition 870250080612, dated 09 / 08 / 2025, p. 19 / 111 14 / 76 antibiotic resistance gene, a counterselectable marker and a transfer cassette, wherein the transfer 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. The methods comprise the steps of (b) culturing the transformed bacteria in the presence of three antibiotics to which the first, second and third antibiotic resistance genes confer resistance; (c) selecting at least one bacterial colony comprising a recombinant bacmid; (d) incubating bacteria from at least one bacterial colony comprising a recombinant bacmid selected in step (c) with a compound that allows counterselection against the donor plasmid;(e) collect recombinant bacmids comprising a gene of interest from a bacterial colony after counterselection; (f) transform insect cells with recombinant bacmids comprising a gene of interest collected in step (e); (g) incubate the transformed insect cells and collect rBV comprising the gene of interest from the insect cells; (h) perform a first passage, wherein a passage comprises the steps of infecting insect cells with rBV comprising the gene of interest and incubating the infected insect cells; (i) collect rBV comprising the gene of interest from one or more insect cell plates and identify a plate comprising rBVs comprising a stable gene of interest;and (j) perform a second passage, wherein the second passage comprises infecting insect cells with rBV comprising a stable gene of interest, incubating the infected insect cells and harvesting a seed stock of recombinant baculovirus comprising rBV comprising the stable gene of interest from the insect cells.

[038] In one embodiment, a stock of seeds is provided Petition 870250080612, dated 08 / 09 / 2025, page 20 / 111 15 / 76 recombinant baculovirus comprising a stable gene of interest.

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

[040] Compositions are provided comprising a recombinant baculovirus seed stock comprising a stable gene of interest.

[041] Methods for producing rAAV comprising a gene of interest are provided. The methods for producing rAAV comprising a gene of interest comprise the steps of (a) producing a first recombinant baculovirus seed stock by a method above, wherein the first recombinant baculovirus seed stock comprises rBV comprising a first gene of interest; (b) producing a second recombinant baculovirus seed stock by a method above, wherein the second baculovirus seed stock comprises rBV encoding Rep / Cap proteins; (c) co-infecting insect cells with the first recombinant baculovirus seed stock and with the second recombinant baculovirus seed stock; (d) adding food to the insect cell culture medium after co-infection; (e) incubating the infected insect cells; and (f) collecting rAAV comprising the gene of interest from the insect cells.In aspects of the method, the insect cell co-infection step comprises co-infecting insect cells with a first stock of recombinant baculovirus seeds with a low multiplicity of infection (MOI) and with a second stock of recombinant baculovirus seeds with a low multiplicity of infection. In several aspects, the low MOI is below 0.01. In several aspects, the low MOI is below 0.009. In certain aspects... Petition 870250080612, dated 08 / 09 / 2025, page 21 / 111 16 / 76 aspects, the low MOI is in the range of about 0.0005 to about 0.009. In certain aspects, the low MOI is in the range of about 0.001. In several aspects, the MOI of the first recombinant baculovirus seed stock differs from the MOI of the second recombinant baculovirus seed stock. In other aspects, the MOI of the first recombinant baculovirus seed stock and the MOI of the second recombinant baculovirus seed stock is the same.

[042] In some aspects of the method, the rAAV collected has a titer of at least 111vg / ml. In some aspects, the rAAV collected has a titer of at least 511vg / ml. In certain aspects, the rAAV collected has a titer of at least 112vg / ml.

[043] In several respects, feeding is added between about 1 hour and 8 hours after coinfection. In some respects, feeding is added about 4 hours after coinfection.

[044] In some respects, the insect culture medium is ESF-AF.

[045] In several respects, insect cells are selected from the group comprising Sf-RVN cells, Sf9 cells, and Hi5 cells. In certain respects, insect cells are Sf-RVN cells.

[046] In some aspects of the methods, rAAV comprising a gene of interest is harvested at a high titer less than 25 days after transformation of insect cells with recombinant bacmids. In some aspects, rAAV comprising a gene of interest is harvested at a high titer approximately 23 days after transformation of insect cells with recombinant bacmids. In certain aspects, rAAV comprising a gene of interest is harvested at a high titer five days after co-infection. In other aspects, rBV p2 is harvested at a high titer 18 days after transformation of insect cells with recombinant bacmids.

[047] In several aspects of the methods, the volume of the first stock of recombinant baculovirus seeds, of the second stock of seeds Petition 870250080612, dated 08 / 09 / 2025, page 22 / 111 17 / 76 recombinant baculovirus or the volumes of each of the first recombinant baculovirus seed stock and the second recombinant baculovirus seed stock used to co-infect insect cells is less than 1 liter. In other respects, the volume of the first recombinant baculovirus seed stock, the second recombinant baculovirus seed stock, or the volumes of each of the first recombinant baculovirus seed stock and the second recombinant baculovirus seed stock used to co-infect insect cells is less than 500 ml. In some respects, the volume of the first recombinant baculovirus seed stock, the second recombinant baculovirus seed stock, or the volumes of each of the first recombinant baculovirus seed stock and the second recombinant baculovirus seed stock used to co-infect insect cells is less than 100 ml.The volume of the first recombinant baculovirus seed stock, the second recombinant baculovirus seed stock, or the combined volumes of each of the first and second recombinant baculovirus seed stocks used to co-infect insect cells is less than 30 ml.

[048] Methods for rapid production of rAAV comprising a gene of interest comprising the steps of (a) producing a first recombinant baculovirus seed stock by a method described above in this document, wherein the first recombinant baculovirus seed stock comprises rBV comprising a first gene of interest; (b) producing a second recombinant baculovirus seed stock by a method described above in this document, wherein the second recombinant baculovirus seed stock comprises rBV encoding Rep / Cap proteins; transducing insect cells with the first recombinant baculovirus seed stock at a low multiplicity of infection and with the second seed stock Petition 870250080612, dated 08 / 09 / 2025, page 23 / 111 18 / 76 recombinant baculovirus seeds at a low MOI; (d) add food to insect cell culture medium after co-infection; (e) incubate infected insect cells for five days; and (f) collect rAAV comprising the first gene of interest from insect cells less than 27 days after transformation of insect cells with recombinant bacmids.

[049] In several aspects of the methods, the first gene of interest encodes a therapeutic gene product for use in the treatment of eye diseases or disorders. In certain aspects, the eye disease or disorder is selected from the group comprising glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis, Leber congenital amaurosis, diabetic retinopathy, acromotopsia, and color blindness. In some aspects, macular degeneration is selected from the group comprising dry macular degeneration, wet macular degeneration, and age-related macular degeneration. In some aspects of the methods, the therapeutic gene product is selected from the group comprising an anti-angiogenic polypeptide, a vascular endothelial growth factor (VEGF) binding protein, an opsin protein, an anti-C3 antibody, an anti-C5 antibody, complement factor I (CFI), and a dry anti-AMD gene product.In certain aspects of the methods, the therapeutic gene product is selected from the group that includes aflibercept, sFLT1, CFI, ranibizumab, and bevacizumab.

[050] In one embodiment, methods for the rapid production of rAAV comprising a gene of interest are provided. The methods for the rapid production of rAAV comprising a gene of interest comprise the steps of (a) producing a first recombinant baculovirus seed stock wherein the first baculovirus seed stock comprises rBV comprising a first gene of interest; (b) producing a second recombinant baculovirus seed stock wherein the second recombinant baculovirus seed stock comprises rBV encoding Rep / Cap proteins; (c) transducing insect cells with Petition 870250080612, dated 08 / 09 / 2025, page 24 / 111 19 / 76 the first recombinant baculovirus seed stock at a low MOI and with the second recombinant baculovirus seed stock at a low MOI; (d) add food to the insect cell culture medium approximately four hours after coinfection; (e) incubate the infected insect cells for five days; and (f) collect rAAV comprising the first gene of interest from the insect cells five days after coinfection.

[051] In one embodiment, methods are provided for producing rAAV comprising a gene of interest. The methods comprise the steps of (a) producing a first recombinant baculovirus seed stock, wherein the first recombinant baculovirus seed stock comprises rBV comprising a first gene of interest; (b) providing a second recombinant baculovirus seed stock comprising rBV comprising a stable gene of interest encoding Rep / Cap proteins; (c) co-infecting insect cells with the first recombinant baculovirus seed stock at a low MOI and with the second recombinant baculovirus seed stock at a low MOI; (d) adding food to the insect cell culture medium after co-infection; incubating the infected insect cells; and collecting rAAV comprising the gene of interest from the insect cells.In several respects, the second recombinant baculovirus seed stock comprises rBV which includes cassette 7m8.

[052] In one embodiment, a composition is provided comprising recombinant bacmids with reduced residual DNA produced by a method described above.

[053] Methods for producing rAAV comprising a gene of interest comprising the steps of (a) providing a first stock of recombinant baculovirus seeds comprising rBV comprising a first stable gene of interest; (b) providing a second stock of baculovirus seeds Petition 870250080612, dated 08 / 09 / 2025, page 25 / 111 (a) 20 / 76 recombinant comprising rBV comprising a stable gene of interest encoding Rep / Cap proteins; (b) co-infect insect cells with the first recombinant baculovirus seed stock at a low MOI and with the second recombinant baculovirus seed stock at a low MOI; (c) add food to the insect cell culture medium after co-infection; (d) incubate the infected insect cells; and (f) collect rAAV comprising the gene of interest from the insect cells.

[054] Methods are provided for the rapid production of rAAV comprising a gene of interest. The methods for the rapid production of rAAV comprising a gene of interest comprise the steps of (a) producing a first stock of recombinant baculovirus seed comprising rBV comprising a first gene of interest; (b) providing a second stock of recombinant baculovirus seed comprising rBV comprising a stable gene of interest encoding Rep / Cap proteins; (c) co-infecting insect cells with the first stock of recombinant baculovirus seed at a low MOI and with the second stock of recombinant baculovirus seed at a low MOI; (d) adding food to the insect cell culture medium after co-infection; (e) incubating the infected insect cells; and (f) collecting rAAV comprising the gene of interest from the insect cells approximately five days after co-infection.The production steps for a first or second batch of recombinant baculovirus seeds can utilize a method described above.

[055] In one embodiment, compositions are provided comprising recombinant bacmides with reduced residual DNA produced by a method of application. In one aspect, a composition comprising recombinant bacmides with reduced residual DNA is for use in the production of a pharmaceutical composition.

[056] In one embodiment, an insect cell capable of producing Petition 870250080612, dated 08 / 09 / 2025, page 26 / 111 21 / 76 rAAV comprising a stable gene of interest.

[057] In several systems, recombinant bacmids with reduced residual DNA are used in the production of rAAV. In some aspects of the systems, the helper plasmid is an AAV helper plasmid.

[058] Modified Bac-to-Bac systems for producing recombinant bacmids with reduced residual DNA in the production of rAAV are provided in this document. The modified Bac-to-Bac systems comprise a donor plasmid containing a first antibiotic resistance gene, a counterselectable marker, and a transfer cassette. The donor plasmid does not contain an active gentamicin resistance gene and a GC-rich region in the transfer cassette. The transfer cassette comprises a left transposon arm, a gene of interest, and a right transposon arm. The modified Bac-to-Bac systems comprise an AAV helper plasmid containing a second antibiotic resistance gene. The modified Bac-to-Bac systems comprise a bacmid containing a third antibiotic resistance gene and a transposon insertion site located within a reporter cassette.In some aspects of the systems, the first antibiotic resistance gene, the second antibiotic resistance gene, and the third antibiotic resistance gene are selected from the group comprising an ampicillin resistance gene, a tetracycline resistance gene, and a kanamycin resistance gene. In aspects of the systems, 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 a counterselectable marker which is rpsl. In some aspects, the counterselectable marker is rspl. Aspects of the systems include where the left transposon arm is Tn7R and the right transposon arm is Tn7L. In several aspects of the systems, the reporter cassette comprises a lacZα gene. In certain... Petition 870250080612, dated 08 / 09 / 2025, page 27 / 111 22 / 76 aspects of the systems, the transposon insertion site is a mini-att-Tn7 site. In several aspects of the system, the gene of interest encodes AAV RepCap proteins or a therapeutic gene product.

[059] In one embodiment, the application provides efficient methods for producing a recombinant baculovirus seed stock comprising a gene of interest, wherein at least 50% of the recombinant baculovirus (rBV) in the recombinant baculovirus seed stock comprises a gene of interest. In some aspects of the methods, after two passages, at least 70% of the rBV comprises the gene of interest. In some aspects, after one passage, at least 80% of the rBV comprises the gene of interest. In certain aspects, the method produces a recombinant baculovirus seed stock after two passages through insect cells, wherein the recombinant baculovirus seed stock is at least 2 x 1010 vector genomes / ml (vg / ml).

[060] Some aspects of efficient methods for producing a recombinant baculovirus seed stock comprising a gene of interest comprise the step of introducing a modified Bac-to-Bac system for producing recombinant rBV comprising a gene of interest into bacterial cells, wherein the modified Bac-to-Bac system comprises (i) a donor plasmid comprising a first antibiotic resistance gene, a counter-selective marker and a transfer cassette, wherein the donor plasmid does not comprise either an active gentamicin resistance gene or a GC-rich region in the transfer cassette and wherein the transfer 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.Efficient methods further comprise the steps of (b) cultivating the transformed bacteria in the presence of three antibiotics to which the. Petition 870250080612, dated 08 / 09 / 2025, page 28 / 111 23 / 76 first, second and third antibiotic resistance genes confer resistance, IPTG and BluoGal; (c) select at least one bacterial colony comprising a recombinant bacmid (blank colony); (d) incubate bacteria from at least one bacterial colony comprising a recombinant bacterium selected in step (c) with a compound that allows counterselectivity against the donor plasmid; (e) collect bacmids from a bacterial cell comprising a recombinant bacmid; (f) transform insect cells with the recombinant bacmids collected in step (e); (g) incubate the transformed insect cells in medium at 28°C for four days in a shaker incubator and collect recombinant baculovirus P0 (rBv,BEVs); (h) perform a first passage, in which one passage comprises the steps of infecting insect cells with recombinant baculovirus P0 (rBv) comprising the gene of interest or Rep / Cap and incubating the infected insect cells; (i) harvest RBV comprising the gene of interest from at least ten insect cell plates and selecting the most stable plate; and (j) perform a second passage, in which the second passage comprises infecting insect cells with rBV from the plate purification step (step i), incubating the infected insect cells, and harvesting a seed stock of recombinant baculovirus comprising p1 rBV comprising the gene of interest from insect cells. In some aspects, the presence of a recombinant bacmid in a bacterial cell is indicated by a white colony. In certain aspects of the methods, 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. In several aspects of the methods, the compound that allows counterselection against the donor plasmid is streptomycin. In some aspects, insect cells are selected from the group comprising Sf-RVN cells and Sf9 cells. In certain aspects of the methods, insect cells are transformed with approximately 6-9 μg of bacmids. Petition 870250080612, dated 08 / 09 / 2025, page 29 / 111 24 / 76 In some aspects of the methods, transformed insect cells are cultured in 30 ml of medium. In some aspects, the methods further comprise performing a third passage, wherein the third passage comprises infecting insect cells with BEVs from the rBV P1 step (step j), incubating the infected insect cells, and collecting a baculovirus seed bank comprising BEVs containing the gene of interest from the insect cells (rBV P2). In some cases, for example in the prevalent disease, rBV P2 may be replanted to regenerate the initial viral bank in rBV P1. In some aspects of the third passage, the MOI is 0.1.

[061] In one embodiment, the application provides methods for producing rAAV comprising a gene of interest comprising the steps of (a) producing a first recombinant baculovirus seed stock by a method described herein, wherein the first recombinant baculovirus seed stock comprises rBV comprising a first gene of interest, (b) producing a second recombinant baculovirus seed stock by the method of claim 13, wherein the second recombinant baculovirus seed stock comprises rBV encoding Rep / Cap proteins; (c) co-infecting insect cells with the first recombinant baculovirus seed stock at a low multiplicity of infection (MOI) and with the second recombinant baculovirus seed stock at a low MOI;(d) add food to the insect cell culture medium after co-infection with the first recombinant baculovirus seed stock and the second recombinant baculovirus seed stock; (e) incubate the infected insect cells and harvest rAAV comprising the gene of interest from the insect cells. In some aspects of the methods, the low MOI is in the range between about 0.01 and 0.001. In some aspects of the methods, the low MOI is in the range of about 0.001. In several aspects of the methods, the harvested rAAV has a titer of at least 112vg / ml. In certain aspects; Petition 870250080612, dated 08 / 09 / 2025, page 30 / 111 25 / 76 The feed is added approximately four hours after coinfection. In some aspects of the methods, the insect culture medium is ESF-AF. In certain aspects, insect cells are selected from the group comprising SfRVN cells and Sf9 cells. In some aspects of the methods, rAAV comprising a gene of interest is harvested at a high titer five days after coinfection. In aspects of the methods, rBV p2 are collected at a high titer 18 days after transformation of insect cells with bacmids collected from a bacterial colony comprising a recombinant bacmid. In several aspects of the methods, the volume of the first recombinant baculovirus seed stock, the second recombinant baculovirus seed stock, or both the first recombinant baculovirus seed stock and the second recombinant baculovirus seed stock used to coinfect the cells is less than 30 ml.

[062] In one embodiment, methods are provided for the rapid production of rAAV comprising a gene of interest.The methods comprise the steps of (a) producing a first recombinant baculovirus seed stock by a method described herein, wherein the first recombinant baculovirus seed stock comprises rBV containing the first gene of interest, (b) producing a second recombinant baculovirus seed stock by a method described herein, wherein the second recombinant baculovirus seed stock comprises rBV encoding Rep / Cap proteins, (c) transducing insect cells with the first recombinant baculovirus seed stock at a low multiplicity of infection and with the second recombinant baculovirus seed stock at a low multiplicity of infection, (d) adding food to the insect cell culture medium approximately four hours after co-infection with the first recombinant baculovirus seed stock and the second recombinant baculovirus seed stock, (e). Petition 870250080612, dated 09 / 08 / 2025, p. 31 / 111 26 / 76 Incubate the infected insect cells for five days and harvest rAAV comprising a gene of interest from insect cells five days after co-infection. INCORPORATION BY REFERENCE

[063] The following passages describe different aspects of the invention in more detail. Each aspect, embodiment or feature of the invention can be combined with any other aspect, embodiment or feature of the invention, unless clearly indicated otherwise. All publications, patents and patent applications mentioned in this descriptive report are incorporated herein by reference in their entirety to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[064] Figure 1 provides an overview of the modifications to the donor plasmid and the process for obtaining recombinant bacmids without the donor plasmid (pFastBac) in a modified bac-to-bac process. The media contains the indicated additives. The modified bac-to-bac process is an example of a system for producing recombinant bacmids with reduced residual DNA.

[065] Figure 2 provides images of E. coli colonies grown under various conditions. The modified Bac-to-Bac system allows the selection of colonies without the donor plasmid (2) and with reduced residual DNA in the bacmids collected from bacteria after incubation of the bacteria with a compound that allows counterselectivity against the donor plasmid. The donor plasmid (pFastBac) is not significantly visible in lane 2 of the gel, indicating a significant reduction of the donor plasmid.

[066] Figure 3 provides a schematic summarizing the previous process for obtaining rAAV from bacmids compared to the current (new) process. The new methods eliminate necessary steps. Recombinant bacmids Petition 870250080612, dated 08 / 09 / 2025, page 32 / 111 27 / 76 produced by the systems of this application show reduced residual DNA.

[067] Figure 4 provides images of infected cell plaques obtained during the plaque purification step for two different rBVs. The two different rBVs comprise different expression cassettes (CFI 1.0 and 7m8) comprising either CFI 1.0 as the gene of interest or comprising 7m8 Rep / Cap as the gene of interest. The plaque purification process described in this document is similar to that described in Example 4. The images shown depict plaques with a control and 10⁻¹, 10⁻², 10⁻⁴, 10⁻⁶, and 10⁻⁸ dilutions.

[068] Figure 5 provides a schematic overview of the rAAV production process using a modified Bac-to-Bac system in contrast to the standard production process. Note the shorter time between bacmid and rAAV collection from Sf-RVN cells using the modified system compared to traditional methods. Sf-RVN cells are rhabdovirus-free.

[069] Figure 6 provides graphs summarizing the stability of rBaculovirus (BEV) during different passages and its effect on rAAV titer. The rBV comprising 7m8 was obtained from two different plates (plate 1 and plate 2). The ratio of the gene of interest (Rep / Cap 7m8 cassette) to GP64 is indicated for rBV from each plate at P0, Passage 1 (P1), and Passage 2 (P2). The rBV from plate 2 has a gene of interest to structure ratio equal to or greater than 0.5 after Passage 2. The rBV from plate 2 has a stable gene of interest (7m8). The rBV from each plate and each passage was co-infected with rBV containing a stable gene of interest (GFP-Fluc) in insect cells to obtain rAAV. The rAAV titer obtained from co-infection with rBV comprising a stable gene of interest (GFPFluc) and rBV comprising 7m8 at stages P0, P1, and P2 of each plate is indicated in vg / ml.The AAV titers obtained with rBV from stages P0, P1, and P2 were compared for each plate, and the ratio of titers is shown. The rAAV titers obtained from rBV comprising a stable gene of interest after P1 and P2. Petition 870250080612, dated 09 / 08 / 2025, p. 33 / 111 28 / 76 (plate 2) are significantly higher than the rAAV titles of rBV P1 and P2 of plate 1.

[070] Figure 7 provides graphs summarizing the rAAV titer results obtained in several experiments. The graph on the left summarizes the rAAV titers obtained from insect cells cultured in the indicated media (Sf-900™ SFM, Sf-900™ II SFM, EX-CELL™ TiterHigh, and ESF AF) in Sf-RVN cells with CFI / 7m8. The central graph summarizes the rAAV titers obtained from Sf-RVN cells cultured in ESF AF medium or Sf-900™ II SFM medium with and without feeding after coinfection. The rAAV titer obtained from ESF AF medium was substantially higher than with Sf-900™ II SFM with or without feeding. The addition of feeding to ESF-AF medium increased the obtained titer. The right panel provides a graph summarizing the results obtained from coinfection at an MOI of 0.001 or 3 in ESF AF medium or Sf-900™ II SFM medium. Surprisingly, the results obtained with an MOI of 0.001 were significantly higher than the titer obtained when an MOI of 3 was used in ESF AF medium.

[071] Figure 8 presents a summary of an assessment of the donor plasmid (pFB) level in three different recombinant bacmid preparations. The donor plasmid level was assessed by finger droplet PCR (ddPCR) and the results are summarized in Figure 8A. Bacmid prep. 1 contains recombinant bacmids after counterselection of the donor plasmid (Bacmid-GOI-without donor plasmid). The ratio between the gene of interest and DNApol was 1.19. The ratio between the donor plasmid and rBacmid was 0 (measured in times). Bacmid prep. 2 contains recombinant bacmids obtained using a donor plasmid comprising the ampicillin resistance gene (instead of a kanamycin resistance gene) without counterselection for the donor plasmid (Bacmid-GOI-with donor plasmid (Amp)). The ratio between the gene of interest and DNA polymerase was 5.45. The ratio between the donor plasmid and rBacmid was 4.6 times. The preparation of Bacmid 3 Petition 870250080612, dated 08 / 09 / 2025, page 34 / 111 29 / 76 contains recombinant bacmids obtained using a donor plasmid comprising a kanamycin resistance gene (Kan), wherein the recombinant bacmid also comprises a kan resistance gene. The ratio between the gene of interest and DNApol was 309.18. The ratio between the donor plasmid and rBacmid was 261 times. Figures 8B and 8C provide a graphical representation of the number of copies of rBacmid versus the donor plasmid comprising the number of copies of the gene of interest (pFB) of the three Bacmid preparations (Figure 8B). The results of Bacmid Prep. 3 (donor plasmid and bacmid plasmid with the same antibiotic resistance gene) are excluded from Figure 8C. DETAILED DESCRIPTION OF THE INVENTION

[072] As gene therapies are developed for use in treating common or prevalent diseases or disorders, the difficulties associated with the large-scale production of recombinant adeno-associated viruses (rAAVs) comprising a gene of interest have become apparent. There is a need in the art for efficient methods to produce large volumes of rAAVs for clinical use. This application provides modified systems and methods for efficiently producing large quantities of rAAVs. In addition, baculovirus expression vectors (rBVs) used to produce rAAVs exhibit a significant loss rate of the gene of interest from the BEV. This application provides rBVs comprising a stable gene of interest, methods for obtaining rBVs comprising a stable gene of interest, and compositions and methods for obtaining recombinant bacmids and rBVs comprising a stable gene of interest.Compositions comprising recombinant bacmides and rBVs comprising a stable gene of interest can also be used as a master source or master viral bank (MVB). A master source or master viral bank can facilitate large-scale manufacturing, testing, and clinical-grade regulatory approvals. Many approved gene therapies target rare or uncommon diseases or disorders. A. Petition 870250080612, dated 09 / 08 / 2025, p. 35 / 111 30 / 76 The quantity of delivery vector needed for a rare or uncommon disease or disorder is limited. As gene therapies are developed for prevalent diseases or disorders, it will be necessary to produce much larger quantities of gene therapy delivery vectors, such as rAAV. Prior to this work, there were limited methods for producing large quantities of rAAV. Previous methods were subject to challenges, including, but not limited to, high risk of contamination, substantial declines in the quality of rAAV produced over time, low production efficiency, and long production times.

[073] Except where otherwise defined, all technical and scientific terms used in this document have the meaning commonly understood by a person skilled in the art to which this invention pertains.

[074] Numerical ranges include the numbers that define the range. The term about is used in this document 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” a range refers to that range minus 10% of its lowest value and plus 10% of its highest value. The reference to “about” a value or parameter in this document includes (and describes) modalities that are directed to that value or parameter itself.

[075] As a convention and as used throughout the application, a scientific exponential notation format may be used, replacing part of the number with En, where E (exponent) multiplies the preceding number by 10 raised to the nth power. For example, a scientific format with 2 decimal places displays 12345678901 as 1.23E 10, which is 1.23 times 10 raised to the tenth power and can alternatively be written as 1.23χ1010. Similarly, 1.23E-10 can alternatively be written as 1.23x10'1°.

[076] Unless otherwise indicated, nucleic acids are written from left to right in the 5' to 3' orientation; amino acid sequences Petition 870250080612, dated 09 / 08 / 2025, p. 36 / 111 31 / 76 are written from left to right in amino to carboxyl orientation, respectively.

[077] The headings provided in this document do not constitute limitations of the various aspects or embodiments of the invention, which can be understood by reference to the specification as a whole. Consequently, the terms defined immediately below are more fully defined by reference to the descriptive report as a whole.

[078] The terminology used in this document is for the sole purpose of describing examples and is not intended to be limiting. As used in this document, the singular forms “a”, “an”, “the” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with” or variants thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term “comprising”. The term “comprising”, as used in this document, is synonymous with “that includes” or “that contains” and is inclusive or open-ended.

[079] By “essentially consisting of”, we mean a limitation of the scope of, for example, composition, method, kit, etc., described to the specified materials that do not materially affect the basic and novel feature(s) of, for example, composition, method, kit, etc. For example, an expression cassette “essentially consisting of” a coding sequence encoding a polynucleotide operationally linked to a promoter and a polyadenylation sequence may include additional sequences, for example, linkage sequences, provided they do not materially affect the transcription or translation of the coding sequence. As another example, a variant or mutant polypeptide “essentially consisting of” a recited sequence has the amino acid sequence of the recited sequence plus or minus about 10 amino acid residues within the boundaries Petition 870250080612, dated 09 / 08 / 2025, p. 37 / 111 32 / 76 of the sequence based on the full-length naive polypeptide from which it was derived, for example, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 residue less than the recited limiting amino acid residue or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues more than the recited limiting amino acid residue.

[080] Any reference to “or” in this document is intended to include “and / or”, unless otherwise indicated.

[081] The donor plasmids comprise a first antibiotic resistance gene, a counterselectable marker, and a transfer cassette, wherein the transfer cassette comprises a left transposon arm, a gene of interest, and a right transposon arm. By “counterselectorable marker” is meant a selectable marker that eliminates or inhibits the growth of the host organism after selection. Counterselective markers are known in the art and include, but are not limited to, antibiotic sensitivity genes. Antibiotic sensitivity genes include, but are not limited to, rpsl, a thymidine kinase gene, and URA3. The donor plasmids for use in the systems and methods of the present application are suitable for use in bacterial cells.

[082] The AAV “rep” and “cap” genes refer to polynucleotide sequences that encode replication and encapsulation proteins of the adeno-associated virus. AAV rep and cap are referred to herein as AAV “packaging genes.” In some embodiments, the AAV rep gene may be of any AAV serotype or may be a modified AAV rep gene. In some respects, the AAV rep gene is of the same serotype as the ITRs of the rAAV vector genome. In some respects, the AAV rep gene is of a different serotype from the ITRs of the rAAV vector genome or from the Cap serotype. In some respects, AAV Rep is a chimeric Rep. In some respects, AAV Cap is a chimeric Cap.

[083] The “rep” and “cap” genes of AAV refer to polynucleotide sequences that encode proteins for virus replication and encapsulation. Petition 870250080612, dated 09 / 08 / 2025, p. 38 / 111 33 / 76 adeno-associated cells and variants thereof. The Rep genes encode the Rep proteins Rep78, Rep68, Rep52, and Rep40. Rep79 and Rep68 proteins are multifunctional DNA-binding proteins that perform helicase and nickase functions during productive replication to allow resolution of AAV termini (see, for example, Im et al. 1990 Cell 61:447-57). These proteins also regulate the transcription of endogenous AAV promoters and promoters within helper viruses (see, for example, Pereira et al. (1997) J. Virol 71:1079-1088). Other Rep proteins modify the function of Rep78 and Rep68. “Rep” encompasses variant Rep, chimeric Rep, and modified Rep. The Cap genes encode the capsid proteins VP1, VP2, and VP3. Cap encompasses variant Cap, chimeric Cap, pseudotyped Cap, and modified Cap. A person skilled in the art would know which Rep and Cap forms are preferential for joint use and would select appropriately to form Rep / Cap.The term Rep / Cap refers to a nucleotide sequence that encodes functional forms of Rep and Cap. In some embodiments, the polynucleotide comprising the polynucleotide encoding Cap additionally comprises a polynucleotide encoding Rep.

[084] AAV Rep and ITR sequences efficiently complement other AAV Rep and ITR sequences in insect cells. Generally, Cap proteins, which determine the cellular tropicity of the rAAV particle, and sequences related to Cap protein coding, are significantly less conserved than Rep proteins and genes among different AAV serotypes. Given the ability of Rep and ITR sequences to cross-complement corresponding sequences from other serotypes, pseudotyped AAV particles comprising the capsid proteins of one serotype (e.g., AAV6) and the Rep and / or ITR sequences of another AAV serotype (e.g., AAV2) can be easily generated. As used herein, “pseudotyped” refers to the source of the Cap protein in an adeno-associated virus. See Halbert et al. 2000 J. Virol. 74:1524-1532 and Halbert et al. Petition 870250080612, dated 08 / 09 / 2025, page 39 / 111 34 / 76 2001 J. Virol 75:6615-6624. rAAV2 / 6 and rAAV2 / 8 (i.e., pseudotyped AAV comprising the ITRs and Rep sequences of AAV2 and VP sequences derived from AAV6 and AAV8, respectively, in insect cells. The production of a pseudotyped AAV vector comprising the Cap genes of a specific AAV serotype indicates that non-pseudotyped vectors of that serotype can be successfully produced in this system.

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

[086] In some aspects of the methods, an AAV ITR may be an AAV1, aAV2, or AAV6 ITR; a nucleic acid comprising the Rep ORFs may comprise an AAV1, AAV2, or AAV6 Rep gene, and a nucleic acid comprising the Cap ORFs may comprise an AAV1, AAV2, or AAV6 Cap gene. Modified AAV sequences may also be used to produce rAAV in insect cells. A nucleotide sequence with at least about 70%, at least about 80%, at least about 90%, at least about 95% sequence identity with an AAV1, AAV2, AAV3, and / or AAV4 sequence may be used in place of wild-type AAV Rep, AAV-ITR, or AAV-Cap sequences, provided that the rAAV particles are produced in infected cells.

[087] In some modalities, one or more ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, rhesus monkey AAV or ITRs Petition 870250080612, dated 09 / 08 / 2025, p. 40 / 111 35 / 76 of ovine AAV or variants thereof. In some modalities, one or more ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, rhesus monkey AAV, or ovine AAV ITRs. In some forms, one or more ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, rhesus monkey AAV, or ovine AAV ITR comprising one or more nucleotide insertions, deletions, and / or substitutions.

[088] Any vector known in the art may be employed with the present teachings, provided it is compatible with insect cells. The presence of a vector in the insect cell need not be permanent. Vectors may be introduced by any known method, for example, by chemical treatment of cells, electroporation, or infection. A “vector,” as used in this document, refers to a macromolecule or association of macromolecules comprising or associating with a polynucleotide and which can be used to mediate the delivery of the polynucleotide to a cell. Illustrative vectors include, but are not limited to, plasmids, viral vectors (i.e., adeno-associated viruses), liposomes and other gene delivery vehicles, and bacmids.

[089] An “AAV vector” or “rAAV vector” as used herein refers to an adeno-associated virus (AAV) vector or a recombinant AAV (rAAV) vector comprising a non-AAV-origin polynucleotide sequence (e.g., a heterologous polynucleotide for AAV, such as a nucleic acid sequence encoding a therapeutic transgene, e.g., human complement factor inhibitor (CFI)) for transduction into a target cell or target tissue. In general, the heterologous polynucleotide is usually flanked by two inverted terminal repeat (ITR) sequences of AAV. The term rAAV vector encompasses rAAV vector particles and rAAV vector plasmids. A vector of Petition 870250080612, dated 08 / 09 / 2025, page 41 / 111 36 / 76 rAAV can be single-strip (ssAAV) or self-complementing (scAAV).

[090] An “AAV virus” or “AAV viral particle” or “rAAV vector particle” or “rAAV particle” refers to a viral particle comprising at least one AAV capsid protein and an rAAV polynucleotide vector. In some cases, at least one AAV capsid protein is from a wild-type AAV or is a variant AAV capsid protein. By “variant AAV capsid protein” is meant that the AAV capsid protein comprises at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) relative to a corresponding parental AAV capsid protein. The variant capsid protein may confer greater infectivity to a retinal cell compared to the infectivity of a retinal cell by an AAV virion comprising an amino acid sequence present in a naturally occurring AAV capsid protein.Variant AAV capsid proteins may include, but are not limited to, an AAV capsid protein with an insertion, a 7m8 amino acid sequence insertion, an R100 insertion, a 7m8-like insertion, an LSV1 sequence substitution, and any other engineered capsid protein generated by other strategies (e.g., DNA scrambling, directed evolution, peptide insertion, ancestral reconstruction, among others). The LSV1 substitution sequence and the 7m8 insertion sequence are known in the art (see, for example, U.S. Patent No. 9,193,956; U.S. Patent No. 9,233,133; Pub. No. U.S. US2021 / 0040501; and PCT / US2020 / 029895). Variants of AAVs of particular interest may include, but are not limited to, those disclosed in Patent No. US 9,193,956, WO2017197355, WO2018022905, WO2019104279 and / or US20210371879A1. In some embodiments, the AAV variant comprises or consists of the variant capsid protein 7m8 (which may be referred to as AAV2).7m8 and 7m8AAV2). In some embodiments, AAV comprises or consists of a capsid protein AAV2.5T. Petition 870250080612, dated 09 / 08 / 2025, p. 42 / 111 37 / 76 as provided in U.S. Patent No. 9,233,131. In some embodiments, AAV comprises the capsid protein AAVShH10 or AAV6 (Pub. of Patent Application No. U.S. 20120164106 and Klimczak et al. PLOS One 4(10):e7467 (October 14, 2009)). In some embodiments, AAV comprises or consists of an AAV2.5T_LSV1 variant disclosed in U.S. Patent Application No. WO2020219933.

[091] The AAV2.7m8 capsid was engineered from wild-type AAV2 and exhibits highly efficient retinal transduction after intravitreal injection (IVT). The AAV2.7m8 capsid has a 7m8 insertion in the IV loop, reduces interactions between the virus capsid and the HSPG receptor, and allows diffusion across the internal limiting membrane (ILM) into the retina. AAV2.7m8 can transduce retinal cells, including photoreceptors, Müller glial cells, retinal ganglion cells, bipolar cells, and RPE cells.

[092] If the particle comprises a heterologous polynucleotide (e.g., a polynucleotide different from a wild-type AAV genome, such as a transgene to be delivered to a target cell or target tissue), this is called an “rAAV particle”, “rAAV vector particle” or “rAAV vector”. Thus, the production of rAAV particles necessarily includes the production of an rAAV vector, as such a vector is contained within an rAAV particle. In general, the heterologous polynucleotide is flanked by inverted terminal repeat sequences (ITRs) of the AAV. A heterologous polynucleotide may comprise a polynucleotide cassette. A polynucleotide cassette of the present application may be packaged into an AAV variant particle to promote the delivery of the cassette to a cell type of interest, such as, but not limited to, a retinal cell, in a target tissue.

[093] The term “packaging”, as used in this document, may refer to a series of intracellular events that may result in assembly. Petition 870250080612, dated 08 / 09 / 2025, page 43 / 111 38 / 76 and encapsulation of an rAAV particle.

[094] By “passage” is meant a process comprising the steps of infecting insect cells with rBV comprising a gene of interest and incubating the infected insect cells.

[095] The terms “gene of interest,” “GOI,” and “transgene” are used interchangeably in this document. A gene of interest comprises an open reading frame encoding a gene product of interest. In many respects, a nucleic acid may comprise two or more nucleic acid sequences, each comprising a gene of interest encoding a gene product. A “gene product” is a molecule resulting from the expression of a specific gene. Gene products include, but are not limited to, a polypeptide, an aptamer, an interfering RNA, an mRNA, and the like. In specific embodiments, a “gene product” is a polypeptide, peptide, protein, or interfering RNA, including short interfering RNA (siRNA), miRNA, or small hairpin RNA (shRNA). In some embodiments, the gene of interest may be a reporter gene.Reporter genes are known in the art and include, but are not limited to, chloramphenicol acetyltransferase, a β-galactosidase, a β-glucuronidase, a renilla luciferase, a firefly luciferase, a green fluorescent protein (GFP), a red fluorescent protein (RFP), and an alkaline phosphatase, such as secreted alkaline phosphatase. 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, Rep78, Rep68, Rep58, Rep40 proteins, Cap, VP1, VP2, VP3 proteins, and fragments and variants thereof, including, but are not limited to, Rep / Cap. In some embodiments, the gene of interest may encode a reporter gene product, such as, but are not limited to, GFP and RFP. In some modalities, the gene of interest may encode a therapeutic gene product, such as a therapeutic protein. Therapeutic gene products. Petition 870250080612, dated 09 / 08 / 2025, p. 44 / 111 39 / 76 are known in the art and include, but are not limited to, a polypeptide hormone, cytokine or growth factor (e.g., insulin or erythropoietin), an interferon, a blood clotting factor, a vaccine, an anti-angiogenic polypeptide, a vascular endothelial growth factor (VEGF) binding protein, an anti-VEGF agent, an anti-VEGF protein, an opsin protein, an anti-C3 antibody, an anti-C5 antibody, a hormone receptor (such as, but not limited to, mineral corticosteroid, glucocorticoid and thyroid hormone receptors), intramembrane proteins (such as, but not limited to, TM-1 and TM-7), intracellular receptors (such as, but not limited to, orphan, retinoid, vitamin D3 and vitamin A receptors), signaling molecules (such as, but not limited to, kinases, transcription factors and signal transducers and activators of transcription receptors of the cytokine superfamily (e.g., erythropoietin, growth hormone,Interferons, interleukins, and colony-stimulating factors; G protein-coupled receptors, such as, but not limited to, hormones, calcitonin, epinephrine, gastrin, paracrine or autocrine mediators such as somatostatin or prostaglandins; neurotransmitter receptors (norepinephrine, dopamine, serotonin, or acetylcholine); tyrosine kinase receptor ligands, such as insulin-like growth factor and nerve growth factor; and a product of the seca anti-DMAE gene. Anti-VEGF agents are known in the art and include, but are not limited to, bevacizumab, brolucizumab, ranibizumab, faricimab, abicipar pegol, conbercept, OPT-302, KSI-301, sunitinib maleate injection (GB-102), PAN90806 (PanOptica), and / or aflibercept.

[096] The term “anti-VEGF agent” includes any therapeutic agent, including proteins, polypeptides, peptides, fusion protein, multimeric proteins, gene products, 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, Petition 870250080612, dated 08 / 09 / 2025, page 45 / 111 40 / 76 to block and / or inhibit the activity or function of an endogenous VEGF and / or an endogenous VEGF receptor (VEGFR), or the VEGF-VEGFR interaction or pathway in vivo. An anti-VEGF agent may be any of the known therapeutic agents that can reduce the growth or formation of new blood vessels and / or edema or swelling when administered to a cell, tissue, or subject in vivo, for example, ranibizumab, brolucizumab, or bevacizumab. In some embodiments, an anti-VEGF agent may be natural, non-natural, or synthetic. In some embodiments, an anti-VEGF agent may be derived from a natural molecule that has been subsequently modified or mutated to confer anti-VEGF activity. In some embodiments, an anti-VEGF agent is a fusion or chimeric protein.In such proteins, functional domains or polypeptides are artificially fused to a fraction or polypeptide to form a fusion or chimeric protein that can sequester VEGF in vivo or function as a VEGFR decoy. In some embodiments, an anti-VEGF agent is a fusion or chimeric protein that blocks the interaction of endogenous VEGFR with its ligands.

[097] As used in this document, “VEGF” may refer to any isoform of VEGF, unless otherwise required, 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 thereof. Unless otherwise required, “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, functional fragment or variant thereof. As used in this document, “VEGF receptor” or “VEGFR” or “VEGFR” may be used to refer to any of the VEGF receptors, including, but not limited to, VEGFR-1 (or Flt-1), VEGFR-2 (or Flk-1 / KDR), and VEGFR-3 (or Flt4). VEGFR may be a soluble or membrane-bound form, or a functional fragment or truncation of a receptor. Petition 870250080612, dated 09 / 08 / 2025, p. 46 / 111 41 / 76

[098] Therapeutic gene products for use in the treatment of an eye disease or disorder may include, but are not limited to, an anti-angiogenic polypeptide, a VEGF-binding protein, an opsin protein, an anti-C3 antibody, an anti-C5 antibody, an anti-dry AMD gene product, aflibercept, sFLT-1, CFI, ranibizumab, and bevacizumab. An anti-dry AMD gene product may include, but are not limited to, inhibitors of C3, C5, HtrA1, C1qm, and natural complement pathway inhibitors such as CFI, CFH, and CD59.

[099] As used in this document, a “therapeutic gene” refers to a gene that, when expressed, produces a therapeutic gene product that confers a beneficial effect to the cell or tissue where it is present or to a mammal in which the gene is expressed. Examples of beneficial effects include the improvement of a sign or symptom of a condition or disease, the prevention or inhibition of a condition or disease, or the conferral of a desired characteristic. Therapeutic genes include, but are not limited to, genes that correct a genetic deficiency in a cell or mammal and genes that express a therapeutic gene product.

[0100] “Helper function(s)” refers to the function(s) encoded in the genome of a helper virus that enables AAV replication and packaging (in conjunction with other requirements for replication and packaging). As described herein, the “helper function” can be provided in a variety of ways, including, but not limited to, providing a helper virus or providing, for example, a helper plasmid comprising polynucleotide sequences encoding the functions required for a trans-producing cell. The required functions include, but are not limited to, the functions provided by the AdenoVA, E4, and E2A genes. For example, a plasmid or other expression vector comprising nucleotide sequences encoding one or more adenoviral helper proteins can be transfected or cotransfected into a cell. Petition 870250080612, dated 08 / 09 / 2025, page 47 / 111 42 / 76 producer.

[0101] By “plasmid” is meant a small extrachromosomal DNA molecule, usually circular, that can replicate independently in a cell. A plasmid may comprise one or more expression cassettes, open reading frames, polynucleotide cassettes, or expression vectors. A helper plasmid of the present system provides an auxiliary function and comprises an antibiotic resistance gene. In some cases, multiple copies of a helper plasmid are present in a bacterial cell comprising a donor plasmid and a bacmid. A larger number of copies of the helper plasmid may be beneficial in methods of obtaining a recombinant bacmid or BEV comprising 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. A person skilled in the art is able to select suitable antibiotic resistance genes for use in the methods claimed.

[0103] As used in this document, “VEGF” may refer to any isoform of VEGF unless otherwise required, 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 thereof. Unless otherwise required, “VEGF” may refer to any member of the VEGF family, including members: VEGF-A, placental growth factor (PGF), VEGF-B, Petition 870250080612, dated 09 / 08 / 2025, p. 48 / 111 43 / 76 VEGF-C and VEGF-D, or any combination, functional fragment, or variant thereof. As used herein, “VEGF receptor” or “VEGFR” or “VEGFR” may be used to refer to any of the VEGF receptors, including, but not limited to, VEGFR-1 (or Flt-1), VEGFR-2 (or Flk-1 / KDR), and VEGFR-3 (or Flt4). VEGFR may be a soluble or membrane-bound form, or a functional fragment or truncation of a receptor. The modified Bac-to-Bac systems of this application make use of a first antibiotic resistance gene, a second antibiotic resistance gene, and a third antibiotic resistance gene.A person skilled in the art may select any three separate antibiotic resistance genes for use in the modified Bac-to-Bac system, provided that each of the three selected antibiotic resistance genes confers resistance to a different antibiotic and the antibiotic resistance gene from the donor plasmid does not interfere with the counterselective marker used in the system. In several embodiments, the antibiotic resistance gene from the donor plasmid is designated as a first antibiotic resistance gene, the antibiotic resistance gene from the helper plasmid is designated as a second antibiotic resistance gene, and the antibiotic resistance gene from the bacmid is designated as a third antibiotic resistance gene.In one embodiment, the first antibiotic resistance gene, the second antibiotic resistance gene, and the third antibiotic resistance gene are selected from the group comprising ampicillin resistance genes, tetracycline resistance genes, and kanamycin resistance genes. In some aspects of the 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 comprises a gene of interest encoding Rep / Cap operationally linked to Petition 870250080612, dated 08 / 09 / 2025, page 49 / 111 44 / 76 a promoter sequence that directs the expression of the polynucleotide in a cell. In some embodiments, the baculovirus expression vector comprises a gene of interest encoding a therapeutic gene product operationally linked to a promoter sequence that directs the expression of the polynucleotide in a cell. In certain embodiments, the cell is a production cell. By “production cell” is meant a host cell used to produce rAAV virions. Exemplary host cells include insect cells including, but not limited to, Sf-9 cells, Sf-21 cells, Sf-RVN cells, Drosophila cell lines, Hi-5 cells, and cell lines derived from Aedes albopictus. The production cell may be used to produce virions comprising Rep / Cap and a gene of interest. In certain embodiments, the cell is a subject cell. By "subject cell" is meant the cell of a subject who has received rAAV gene therapy.It is recognized that different promoters can be used to target expression in a production cell rather than a target cell. For example, a gene of interest encoding Rep / Cap can be operationally linked to an insect-operable promoter. Insect-operable promoters include, but are not limited to, the polyhedron promoter (polh). A gene of interest encoding a therapeutic gene product can be operationally linked to a promoter sequence that is operable in a target cell. The gene of interest can be operationally linked to one or more regulatory regions that impact expression in a given cell.

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

[0106] The terms “rBVs”, “baculovirus expression vectors” and “BEVs” are used interchangeably in this document. A baculovirus expression vector is a eukaryotic DNA viral vector for cloning and / or expression in Petition 870250080612, dated 08 / 09 / 2025, page 50 / 111 45 / 76 cells of lepidopteran insects or cultured insects. Most baculovirus expression vectors are derived from the nuclear polyhedrosis virus Autographa californica (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 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 can be used in the methods and systems of the application. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in 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 the CAG promoter. 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 (Patents under US Nos. 5464758 and 5814618), 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 tamoxifen-inducible recombinase system Cre-ERT (Indra et al (1999) Nuc Acid Res 27:4324-4327, Patent No. US 7112715, Kramer & Fussenegger 2005 Methods Mol Biol 308:123144) and the LacSwitch™ system (Stratagene San Diego CA). Insect-operable promoters 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, 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, attR1 / attR2. Petition 870250080612, dated 08 / 09 / 2025, page 51 / 111 46 / 76

[0109] “Multiplicity of infection” or “MOI” refers to the number of virions or viral particles per cell during an infection. A “low MOI” is an MOI below about 2, below about 1, below about 0.1, below about 0.01, below about 0.009, below about 0.008, below about 0.007, below about 0.006, below about 0.005, below about 0.004, below about 0.003, below about 0.002, about 0.001, about 0.0005, or about 0.0001.A low MOI may be in the range of about 0.0005 to about 0.09, about 0.0008 to about 0.05, about 0.001 to about 0.04, about 0.001 to about 0.03, about 0.001 to about 0.02, about 0.001 to about 0.01, about 0.001 to about 0.009, about 0.001 to about 0.008, about 0.001 to about 0.007, about 0.001 to about 0.006, about 0.001 to about 0.005, about 0.001 to about 0.004, about 0.001 to about 0.003 and about 0.001 to approximately 0.002. The MOIs suitable for use with rBV and insect cells may differ substantially from the MOIs suitable for use with rAAVs and subjects or subject cells. A standard or regular MOI can be in the range of approximately 2.3, 3, 4, 5, 6, 7, 8, 9, 1 x 101, 1 x 102, 1 x 103, 1 x 104, 1 x 105, 1 x 106, 1 x 107, 1 x 108, 1 x 109, 1 x 1010, 1 x 1011, 1 x 1012, 1 x 1013, 1 x 1014, 1 x 1015, 1 x 1016, 1 x 1017 and 1 x 1018.

[0110] The terms “harvested” and “collected” may be used interchangeably. Methods of collecting virions, viral 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 of collecting viral particles known in the art may be used in the methods and systems of the application. The quantity of viral particles collected from insect cells in the various modalities of the method can be in the range of approximately 1 x 10¹⁰vg, 5 x 10¹⁰vg, 6 x 10¹⁰vg, 7 x 10¹⁰vg, 8 x 10¹⁰vg, 9 x 10¹⁰vg, 1 x 10¹¹vg, 2 x 10¹¹vg, 3 x 10¹¹vg, 4 x 10¹¹vg, 5 x 10¹¹vg, 6 x 10¹¹vg, 7 x 10¹¹vg, 8 x 10¹¹vg, 9 x 10¹¹vg, 1 x 10¹²vg, 2 x 10¹²vg, 3 x 10¹²vg, 4 x 10¹²vg, 5 x 10¹²vg, 6 x 1012vg, 7 x 1012vg, Petition 870250080612, dated 08 / 09 / 2025, page 52 / 111 47 / 76 x 1012vg, 9 x 1012vg, 1 x 1013vg, 2 x 1013vg, 3 x 1013vg, 4 x 1013vg, 5 x 1013vg, 6 x 1013vg, 7 x 1013vg, 8 x 1013vg, 9 x 1013vg, 1 x 1014vg, 2 x 1014vg, 3 x 1014vg, 4 x 1014vg, 5 x 1014vg, 6 x 1014vg, 7 x 1014vg, 8 x 1014vg, 9 x 1014vg, 1 x 1015vg, 2 x 1015vg, 3 x 1015vg, 4 x 1015vg, 5 x 1015vg, 6 x 1015vg, 7 x 1015vg, 8 x 1015vg, 9 x 1015vg, 1 x 1016vg, 2 x 1016vg, 3 x 1016vg, 4 x 1016vg, 5 x 1016vg, 6 x 1016vg, 7 x 1016vg, 8 x 1016vg and approximately 9 x 1016vg. It is recognized that the rBV collection methods used in the methods may be the same as or different from the rAAV collection methods used in the methods.

[0111] The amount of rBV harvested from insect cells in the various modalities of the method may be in the range of approximately 1 x 1010vg, 5 x 1010vg, 6 x 1010vg, 7 x 1010vg, 8 x 1010vg, 9 x 1010vg, 1 x 1011vg, 2 x 1011vg, 3 x 1011vg, 4 x 1011vg, 5 x 1011vg, 6 x 1011vg, 7 x 1011vg, 8 x 1011vg, 9 x 1011vg, 1 x 1012vg, 2 x 1012, 3 x 1012, 4 x 1012vg, 5 x 1012vg, 6 x 1012vg, 7 x 1012vg, 8 x 1012vg, 9 x 1012vg, 1 x 1013vg, 2 x 1013vg, 3 x 1013vg, 4 x 1013vg, 5 x 1013vg.A stock of recombinant baculovirus seeds produced by the methods presented may be in the range of approximately 1 x 10⁸vg / ml, 5 x 10⁸vg / ml, 1 x 10⁹vg / ml, 2 x 10⁹vg / ml, 3 x 10⁹vg / ml, 4 x 10⁹vg / ml, 5 x 10⁹vg / ml, 6 x 10⁹vg / ml, 7 x 10⁹vg / ml, 8 x 10⁹vg / ml, 9 x 10⁹vg / ml, 1 x 10¹⁰vg / ml, 2 x 10¹⁰vg / ml, 3 x 10¹⁰vg / ml, 4 x 10¹⁰vg / ml, 5 x 10¹⁰vg / ml, 6 x 10¹⁰vg / ml, 7 x 10¹⁰vg / ml, 8 x1010vg / ml, 9 x 1010vg / ml, 1 x 1011vg / ml, 2 x 1011vg / ml, 3 x 1011vg / ml, 4 x 1011vg / ml, 5 x 1111vg / ml, 6 x 1011vg / ml, 7 x 1011vg / ml, 8 x 1011vg / ml, 9 x 1011vg / ml or about 1 x 1012vg / ml.

[0112] The amount of AAV viral particles collected from insect cells using various methods can range from approximately 1 x 1011vg, 2 x 1011vg, 3 x 1011vg, 4 x 1011vg, 5 x 1011vg, 6 x 1011vg, 7 x 1011vg, 8 x 1011vg, 9 x 1011vg, 1 x 1012vg, 2 x 1012, 3 x 1012, 4 x 1012vg, 5 x 1012vg, 6 x 1012vg, 7 x 1012vg, 8 x 1012vg, 9 x 1012vg, 1 x 1013vg, 2 x 1013vg, 3 x 1013vg, 4 x 1013vg, 5 x 1013vg, 6 x 1013vg, 7 x 1013vg, 8 x 1013vg, 9 x 1013vg, 1 x 1014vg, 2 x 1014vg, 3 x 1014vg, 4 x 1014vg, 5x Petition 870250080612, dated 08 / 09 / 2025, page 53 / 111 48 / 76 1014vg, 6 x 1014vg, 7 x 1014vg, 8 x 1014vg, 9 x 1014vg, 1 x 1015vg, 2 x 1015vg, 3 x 1015vg, 4 x 1015vg, 5 x 1015vg, 6 x 1015vg, 7 x 1015vg, 8 x 1015vg, 9 x 1015vg, 1 x 1016vg, 2 x 1016vg, 3 x 1016vg, 4 x 1016vg, 5 x 1016vg, 6 x 1016vg, 7 x 1016vg, 8 x 1016vg and approximately 9 x 1016vg. The titer of the harvested rAAV can be at least approximately 111vg / ml, 211vg / ml, 311vg / ml, 411vg / ml, 511vg / ml, 611vg / ml, 711vg / ml, 811vg / ml, 911vg / ml, at least 112vg / ml, 212vg / ml, 312vg / ml, 412vg / ml, 512vg / ml, 612vg / ml, 712vg / ml, 812vg / ml, 912vg / ml, at least 113vg / ml, at least 114vg / ml, or at least approximately 115vg / ml.

[0113] A “polynucleotide cassette” is understood to be a polynucleotide sequence comprising two or more functional polynucleotide sequences, for example, regulatory elements, translation initiation sequences, coding sequences, termination sequences, etc., typically in operable linkage to at least one other functional polynucleotide sequence in the polynucleotide cassette. Generally, a polynucleotide cassette in question is composed of DNA.

[0114] The polynucleotide cassettes of the present disclosure typically comprise a promoter region. A “promoter,” as used herein, encompasses a DNA sequence that directs RNA polymerase binding and thus promotes RNA synthesis. Promoters and the expression of corresponding proteins or polypeptides may be ubiquitous, i.e., strongly active in a wide range of cells, tissues, and species, or specific to cell type, tissue, or species. Promoters may be “constitutive,” i.e., continuously active, or “inducible,” i.e., the promoter may be activated or deactivated by the presence or absence of biotic or abiotic factors. In certain embodiments, the promoter region promotes the expression of the coding sequence in mammalian cells. Suitable examples include actin promoters, chicken β-actin (CBA), cytomegalovirus (CMV), CMV / β-immediate enhancer. Petition 870250080612, dated 08 / 09 / 2025, page 54 / 111 49 / 76 actin (CAG), elongation factor 1 alpha (EF1a), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). A promoter may show tissue- or cell-specific expression.

[0115] As used in this document, the term “operationally linked” refers to a juxtaposition of genetic elements, for example, promoter, enhancer, termination signal sequence, polyadenylation sequence, Kozak sequence, etc., where the elements are in a relationship that allows them to operate in the expected manner. For example, a promoter is operationally linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intermediate residues between the promoter and the coding sequence or between any two elements, provided the functional relationship is maintained.

[0116] Any concentration of viral particles suitable for effectively transducing mammalian cells can be prepared for contact with mammalian cells in vitro or in vivo. For example, viral particles can be formulated at a concentration of 10⁸ vector genomes per ml (vg / ml) or more, for example, 5 x 10⁸vg / ml; 10⁹vg / ml, for example, 5 x 10⁹vg / ml; 10¹⁰vg / ml, for example, 5 x 10¹⁰vg / ml; 10¹¹vg / ml, for example, 5 x 10¹¹vg / ml; 10¹²vg / ml, for example, 5 x 10¹²vg / ml; 10¹³vg / ml, for example, 5 x 10¹³vg / ml; 10¹³vg / ml, for example, 1.5 x 10¹³vg / ml; 1014vg / ml, for example 1 x 1014vg / ml and 5 x 1014vg / ml or more, but normally not more than 1 x 1015vg / ml. Similarly, any total number of viral particles suitable to provide appropriate cell transduction to confer the desired effect or treat the disease may be administered to the mammal.

[0117] The viral vector in question may be formulated into a pharmaceutical composition comprising any suitable unit dose of the vector that can be administered to a subject to produce an alteration in the subject or to treat a disease in the subject. In some embodiments, a unit dose comprises, Petition 870250080612, dated 08 / 09 / 2025, page 55 / 111 50 / 76 without limitation, 1 x 108vg or more, for example, at least approximately 1 x 109vg, 1 x 1010vg, 1 x 1011vg, 1 x 1012vg, 1 x 1013vg, 1 x 1014vg or 1 x 1015vg. In some modalities, a unit dose is approximately 1 x 10⁹ to approximately 4 x 10¹² vg, approximately 1 x 10¹⁰ to approximately 4 x 10¹¹ vg, approximately 2 x 10¹⁰ to approximately 3 x 10¹¹ vg, approximately 2 x 10¹⁰ to approximately 2 x 10¹¹ vg, approximately 2.5 x 10¹⁰ to approximately 2 x 10¹¹ vg, approximately 2 x 10¹⁰ to approximately 1 x 10¹¹ vg, approximately 5 x 10⁹ to approximately 8 x 10¹¹ vg, approximately 1 x 10¹⁰ to approximately 2 x 10¹¹ vg, approximately 5 x 10¹⁰ to approximately 2 x 10¹¹ vg, or approximately 8 x 1010 for approximately 1 x 1011vg.

[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 that exhibit a reduced rate of loss of the gene of interest are provided in this document. The methods and compositions allow the production of a recombinant baculovirus seed stock comprising a gene of interest. Methods for producing a recombinant baculovirus seed stock comprising a gene of interest, wherein at least approximately 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 65%, at least about 80%, at least about 55%, at least about 70%, at least about 85%, at least about 60%, at least about 75%, at least about 90%, at least about 95% of the rBV in the recombinant baculovirus seed stock comprising the gene of interest are provided in this document. In some cases, a recombinant baculovirus seed stock containing rBVs containing a stable gene of interest can be replanted and regenerated. In some cases, the recombinant baculovirus seed stock containing rBVs containing Petition 870250080612, dated 08 / 09 / 2025, page 56 / 111 51 / 76 A stable gene of interest can be stored. Storage may involve the transfer of recombinant baculovirus seed stock. Thus, a recombinant baculovirus seed stock containing rBV with a stable gene of interest can be used as a master viral bank, an initial viral bank, a regenerative bank, or an original supply.

[0119] The rBV of the present application may exhibit a low loss rate of the gene of interest. A low gene loss rate of interest is defined as a gene loss of interest of less than 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%, 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 less of approximately 80% of the rBV per passage.

[0120] By “stable gene of interest” is meant that the gene of interest is maintained within a vector at a high retention rate over one or more cycles through cells. The vector may be a recombinant bacmid and the cells may be bacterial cells or insect cells. The vector may be a recombinant BEV and the cells may be insect cells. By cycle through a cell is meant the introduction of the vector into a cell and the collection of vectors from one or more cells. A cycle through a cell may involve additional steps, including, but not limited to, a purification step or a concentration step. It is recognized that the retention rate of a gene of interest in a bacmid may be the same as or different from the retention rate of the same gene of interest constructed in a BEV.It is recognized that the retention rate of a gene construct of interest in rBV may be higher than the retention rate of the same gene construct of interest in a recombinant bacmid. It is recognized that the retention rate of a gene construct of interest in rBV may be lower than the retention rate of the same gene construct of interest in a recombinant bacmid. Petition 870250080612, dated 09 / 08 / 2025, p. 57 / 111 52 / 76 The retention rate is the ratio between the gene of interest and the vector in a vector population.

[0121] The retention rate of a gene of interest in a vector can vary from 0 to 1. The 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 a retention rate above approximately 0.5, above approximately 0.55, above approximately from 0.6, above about 0.65, above about 0.7, above about 0.75, above about 0.8, above about 0.85, above about 0.9, above about 0.95 and about 1 after pass 1 (or after cycle 1);a retention rate 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 approximately 1 after pass 2 (or after cycle 2); and a retention rate 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 approximately 1 after pass 3 (or after cycle 3).

[0122] A stable gene of interest may exhibit a gene-to-vector ratio equal to or greater than about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95 and about 1 after Passage 1; equal to or greater than about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95 and about 1 after Passage 1. Petição 870250080612, de 08 / 09 / 2025, pág. 58 / 111 53 / 76 Passage 2; or equal to or greater than 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 after three or more passages. It is recognized that the presence of a donor plasmid containing the gene of interest may artificially increase the ratio between the gene of interest and the vector.

[0123] The vector level can be assessed by evaluating any suitable portion of the vector, including, but not limited to, a promoter region and a major structure region, with particularly preferential portions of the vector being those regions unique to the bacmid plasmid.

[0124] Compositions and methods for producing a vector comprising a stable gene of interest are provided in this document. While not linked to a single mechanism, a stable gene of interest may result from a process involving concatemer formation, increased transposase levels, altered integration, or a combination of any of these elements. Maintaining selection for the helper plasmid up to and including during the counterselection process for the donor plasmid may be beneficial for producing vectors comprising a stable gene of interest. Increased transposase production may be beneficial for the formation of vectors comprising a stable gene of interest.

[0125] In some cases, the unit dose of a pharmaceutical composition can be measured using the multiplicity of infection (MOI). By MOI is meant the ratio or multiple of vector or viral genomes to the cells to which the nucleic acid can be administered. In some cases, the MOI may be 1 x 10⁴ to 1 x 10⁸, 1 x 10⁵ to 1 x 10⁷, or 1 x 10⁶. In some cases, recombinant revelation viruses are infected with subject cells at least approximately 1 x 10¹, 1 x 10², 1 x 10³, 1 x 10⁴, 1 x 10⁵, 1 x 10⁶, 1 x 10⁷, 1 x 10⁸, 1 x 10⁹, 1 x 10¹⁰, 1 x 10¹¹, 1 x 10¹², Petition 870250080612, dated 08 / 09 / 2025, page 59 / 111 54 / 76 x 1013, 1 x 1014, 1 x 1015, 1 x 1016, 1 x 1017 and 1 x 1018I. In some respects, the quantity of the pharmaceutical composition comprises approximately 1 x 108 for approximately 1 x 1015 recombinant viruses, approximately 1 x 108 for approximately 1014 recombinant viruses, approximately 1 x 1010 for approximately 1 x 1013 recombinant viruses or approximately 1 x 1010 for approximately 3 x 1012 recombinant viruses. It is recognized that the MOI for administration of rAAV to subject cells may differ substantially from the MOI suitable for production or preparation of rAAV.

[0126] The present invention includes pharmaceutical compositions comprising a polynucleotide cassette or gene delivery vector produced by a method described herein and a pharmaceutically acceptable carrier, diluent, or excipient. For example, one embodiment is a pharmaceutical composition comprising a polynucleotide and a pharmaceutically acceptable excipient. In one specific embodiment, the recombinant virus is a recombinant adeno-associated virus (AAV). The polynucleotide cassettes or gene delivery vector in question may be combined with pharmaceutically acceptable carriers, diluents, and reagents, useful in preparing a formulation that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for use in primates. These excipients may be solid, liquid, semi-solid, or, in the case of an aerosol composition, gaseous.Examples of such carriers or diluents may include, but are not limited to, water, saline solution, Ringer's solutions, dextrose solution, and 5% human serum albumin. Additional active compounds may also be incorporated into the formulations. Solutions or suspensions used for the formulations may include a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial compounds, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating compounds, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as... Petition 870250080612, dated 08 / 09 / 2025, pages 60 / 111 55 / 76 acetates, citrates, or phosphates; detergents, such as Tween 20, to prevent clumping, and compounds for adjusting tonicity, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. In specific embodiments, the pharmaceutical compositions are sterile.

[0127] Pharmaceutical compositions suitable for use with instant compositions and methods also include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In some cases, the composition is fluid enough to be easily administered with a syringe. In certain embodiments, the compositions are stable under manufacturing and storage conditions. Liquid pharmaceutical compositions generally include a liquid vehicle, such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline solution, magnesium chloride, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may be included. In some cases, a surfactant such as 0.001% pluronic acid may be used.

[0128] For delayed release, the drug may be included in a pharmaceutical composition formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal delivery systems according to methods known in the art.

[0129] The term “combination” or the terms “in combination,” “used in combination with,” and “combined preparation,” as used herein, may refer to the combined administration of two or more agents simultaneously, sequentially, or separately. The term “simultaneously,” as used herein, means that the agents are administered concurrently or at the same time. The term “sequential,” as used herein, means that the agents are administered one after the other. The term “separate,” as used herein, means that the agents Petition 870250080612, dated 08 / 09 / 2025, page 61 / 111 56 / 76 are administered independently of each other, but within a time interval that allows the agents to show a combined, preferably synergistic, effect.

[0130] Methods for determining expression levels are known in the art. Any method for determining the expression level can be used in the application methods. Methods for determining the expression level include, but are not limited to, immunoassay methods and activity assays.

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

[0132] Immunoassay methods for measuring the presence and quantity of a protein in a biological or cellular sample are 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 are generally based on the reaction between a target protein and an antibody or antibody fragment that binds specifically to the target protein. The immunoassay can be performed in liquid or solid phase. Suitable immunoassays include, but are not limited to, sandwich and competitive assays, Western blotting, ELISAs, radioimmunoassays, fluoroimmunoassays, and the like.A biological sample can be a cell culture medium or supernatant (a sample taken from the culture without lysing the cells), cell lysate, whole cells, blood, serum, plasma, aqueous humor, vitreous humor, or other body fluid or tissue. It is recognized that a biological sample from a subject can be enriched by separating whole cells from the sample, particularly when the polypeptide of interest can be secreted from a cell. The separation can be done by any technique. Petition 870250080612, dated 08 / 09 / 2025, page 62 / 111 57 / 76 convenient separation known in the art, including, but not limited to, fluorescence-activated cell sorting (FACS), magnetic separation, affinity chromatography, panning with an affinity reagent, centrifugation and ultracentrifugation.

[0133] As used in this document, 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 between two or more polypeptide sequences when aligned using an amino acid sequence alignment program. Similarly, the terms “identical” and “percent identity”, when used in this document in the context of two or more nucleotide or amino acid sequences, refer to two sequences that are equal or have a specified percentage of amino acid or nucleotide residues when compared and aligned for maximum match, for example, as measured using a sequence comparison algorithm, for example, the Smith-Waterman algorithm, etc., or by visual inspection.The percentage of identity between amino acid sequences can be determined using, for example, the Needleman and Wunsch algorithm (1970, J. Mol. Biol. 48:444453) which was incorporated into the GAP program in the GCG software package, using a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. As another example, the percentage of identity between two nucleotide sequences can be determined using the GAP program in the GCG software package, using an NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless specified) (Otherwise) is a Blossom 62 scoring matrix with a gap penalty of 12, one. Petition 870250080612, dated 08 / 09 / 2025, page 63 / 111 58 / 76 gap length penalty of 4 and a frame shift gap penalty of 5. The percentage of 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) which was incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Additional search and alignment tools known in the art include, but are not limited to, the NBLAST and XBLAST programs (version 2.0) by Altschul et al (1990) J. Mol. Biol. 215:403-410 and the Gapped BLAST program.

[0134] The term “subject”, “patient” or “individual” refers to a mammal, including, but not limited to, primates such as humans and non-human primates, for example, African green monkeys and rhesus monkeys, mammalian sport animals, mammalian farm animals, mammalian pets and rodents. In some modalities, the subject is a human being.

[0135] The terms “treat”, “treating”, “treatment”, “improve” or “improving” and other grammatical equivalents, as used in this document, refer to relieving, abateing or improving a disease or disorder, or symptoms of a disease or disorder, preventing further symptoms of the disease or disorder, improving or preventing the underlying causes of the symptoms, inhibiting a disease or disorder, for example, halting the development of a disease or disorder, alleviating a disease or disorder, causing the regression of a disease or disorder, or halting the symptoms of a disease or disorder, and are intended to include prophylaxis and prevention. The terms also include obtaining a therapeutic benefit and / or a prophylactic benefit. The term “therapeutic benefit” refers to the eradication or improvement of a disease or disorder that is being treated.Furthermore, a therapeutic benefit is achieved by eradicating or improving one or more physiological symptoms associated with a disease or disorder, such that an improvement is observed in the subject, despite... Petition 870250080612, dated 08 / 09 / 2025, page 64 / 111 59 / 76 In some modalities, the subject is still suffering from a disease or disorder. For prophylactic benefit, pharmaceutical compositions are administered to a subject at risk of developing a disease or disorder, or to a subject reporting one or more physiological symptoms of a disease or disorder, even if a diagnosis of the disease or disorder has not been made.

[0136] The terms “treat,” “treating,” “treatment,” “improve,” or “improving,” and other grammatical equivalents as used herein may refer to alleviating, abateing, or improving dry age-related macular degeneration (dry AMD) disease or disorder, or symptoms of dry AMD disease or disorder, preventing further symptoms of dry AMD disease or disorder, improving or preventing the underlying causes of symptoms, inhibiting dry AMD disease or disorder, for example, halting the development of dry AMD disease or disorder, alleviating dry AMD disease or disorder, causing regression of dry AMD disease or disorder, or halting the symptoms of dry AMD disease or disorder, and may include prophylaxis and prevention of wet AMD. The terms also include obtaining a therapeutic benefit and / or a prophylactic benefit.The term “therapeutic benefit” of dry AMD refers to the eradication or improvement of the dry AMD disease or disorder being treated. Furthermore, a therapeutic benefit is achieved by eradicating or improving one or more physiological symptoms associated with the dry AMD disease or disorder, such that an improvement is observed in the subject even though, in some modalities, the subject still suffers from the dry AMD disease or disorder. For prophylactic benefit, pharmaceutical compositions are administered to a subject at risk of developing dry AMD disease or disorder, or to a subject reporting one or more physiological symptoms of dry AMD disease or disorder, even if a diagnosis of the disease or disorder has not been made.

[0137] The signs and symptoms of dry AMD include, but are not limited to, Petition 870250080612, dated 09 / 08 / 2025, p. 65 / 111 60 / 76 proliferation of endothelial cells and atrophy of the retinal pigment epithelium (RPE).

[0138] The terms “administer”, “administering”, “administration” and the like, as used in this document, may refer to methods that are used to enable the delivery of therapeutic or pharmaceutical compounds to the desired site of biological action. These methods include intravitreal or subretinal injection into the eye.

[0139] The terms “effective amount,” “therapeutically effective amount,” or “pharmaceutically effective amount,” as used herein, may refer to a sufficient quantity of at least one pharmaceutical composition or compound being administered that will alleviate to some extent one or more signs or symptoms of the eye disease, eye disorder, or eye condition being treated. An “effective amount,” “therapeutically effective amount,” or “pharmaceutically effective amount” of a pharmaceutical composition may be administered to a subject in need thereof as a unit dose (as described in more detail elsewhere in this document). The subject may be a human or non-human mammal.

[0140] The term “pharmaceutically acceptable”, as used herein, may refer to a material, such as a carrier or diluent, that does not abrogate the biological activity or properties of a compound disclosed herein and that is relatively non-toxic (i.e., when the material is administered to a subject, it does not cause undesirable biological effects nor does it interact deleteriously with any of the components of the composition in which it is contained).

[0141] The term “pharmaceutical composition”, or simply “composition” as used herein, may refer to a biologically active compound, optionally mixed with at least one pharmaceutically acceptable chemical component, such as, but not limited to, carriers, Petition 870250080612, dated 08 / 09 / 2025, page 66 / 111 61 / 76 Stabilizers, diluents, dispersing agents, suspending agents, thickening agents, excipients and similar products.

[0142] Complement Factor I (also known as Factor I, CFI, and C3b / C4b inactivator) is a protein that, in humans, is encoded by the CFI gene. CFI is a serine protease that circulates in a zymogen-like state, typically at a concentration of about 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, and analogously, C4b into C4c and C4d. Thus, CFI activity negatively regulates the complement cascade in all complement pathways (alternative, classical, and lectin). CFI requires the presence of one or more cofactor proteins to perform its functions; 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. Once C3b is cleaved into iC3b, iC3b does not perpetuate complement cascade amplification or activation via the alternative pathway.The iC3b promotes a pro-inflammatory action by activating complement receptor 3 (CR3) in certain cell types. CFI is able to process iC3b into C3dg in the presence of the cofactor CR1. C3dg cannot bind to CR3. The binding of C3b to CR3 is involved in complement activation, leading to inflammation; the breakdown of iC3b into C3dg reduces complement-induced inflammation (Lachmann (2009) Adv. Immunol. 104:115-149). CFI is able to process iC3b into an inactive degradation product.

[0143] Eye diseases and disorders are known in the art. Eye diseases and disorders include, but are not limited to, achromatopsia, glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis, Leber congenital amaurosis, diabetic retinopathy, color blindness, diabetic macular edema, choroidal neovascularization, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branching retinal vein occlusion, diabetic macular edema, Petition 870250080612, dated 09 / 08 / 2025, p. 67 / 111 62 / 76 Diabetic retinal ischemia, ischemic retinopathy, 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 acuity. Conditions affecting the macula reduce central vision but leave peripheral vision intact. In severe cases, the disease can lead to central blindness. AMD is a notable 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 macular degeneration. Dry AMD is more common than wet AMD, but dry AMD can progress to wet AMD. Dry AMD is CHARACTERIZED by thinning of the macular tissues 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, overlying photoreceptor cells, and often also underlying cells in the choroidal capillary layer. Confluent areas of RPE cell death accompanied by atrophy of overlying photoreceptors are called geographic atrophy (GA). As dry AMD progresses and GA increases, central vision slowly worsens and the ability to see fine details is gradually lost. Dry AMD tends to progress more slowly than wet AMD.

[0146] In some modalities, ocular neovascular disease is recurrent and / or persistent wAMD. In some modalities, ocular neovascular disease Petition 870250080612, dated 09 / 08 / 2025, p. 68 / 111 63 / 76 is active subfoveal CNV secondary to AMD. In some modalities, active subfoveal CNV secondary to AMD occupies > 50% of the total lesion size. In some modalities, active subfoveal CNV secondary to AMD occupies > 50% of the total lesion size with 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 modalities, active subfoveal CNV secondary to AMD occupies > 50% of the total lesion size with 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 lesion size does not exceed 12 macular photocoagulation study disk areas.In some embodiments, one eye and / or the contralateral eye of the individual exhibited better corrected visual acuity (BCVA) based on an ETDRS letter assessment of 78-25 (e.g., less than any of approximately 78, approximately 75, approximately 70, approximately 65, approximately 60, approximately 55, approximately 50, approximately 45, approximately 40, approximately 35, approximately 30, or approximately 25) prior to administration of the unit dose of rAAV particles of the present disclosure. In some embodiments, one eye and / or the contralateral eye of the individual exhibited better corrected visual acuity (BCVA) based on an ETDRS letter assessment of more than 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 the unit dose of rAAV particles of the present disclosure.

[0147] In some modalities, the individual had polypoidal choroidal vasculopathy (PCV) in one eye and / or the contralateral eye prior to administration of the unit dose of rAAV particles.

[0148] Acromotopsia is a rare autosomal recessive disease that results Petition 870250080612, dated 08 / 09 / 2025, page 69 / 111 64 / 76 retinal degeneration affecting all three types of cone photoreceptor cells, resulting in reduced visual acuity, photophobia, hemeralopia, and severe loss of color discrimination. Mutations in the CNGB3 genes are responsible for more than 90% of patients and result in complete achromatopsia, meaning they have significant impairment in color discrimination and central visual acuity.

[0149] In some embodiments, a unit dose of rAAV particles is administered in combination with steroid treatment. In some embodiments, the steroid treatment is corticosteroid treatment. In some embodiments, the steroid treatment is systemic steroid treatment. In some embodiments, the steroid treatment is oral steroid treatment. In some embodiments, the steroid treatment is prednisone treatment. In some embodiments, the steroid treatment is ophthalmic steroid treatment. In some embodiments, the ophthalmic steroid treatment is topical steroid treatment (e.g., eye drops), periocular steroid treatment (e.g., subtenon, subconjunctival), intravitreal steroid treatment, or superchoroid steroid treatment.In some modalities, ophthalmic steroid treatment is a glucocorticoid, including but not limited to an anti-inflammatory glucocorticoid. In some modalities, topical steroid treatment is a glucocorticoid, including but not limited to an anti-inflammatory glucocorticoid. In some modalities, topical steroid treatment is treatment with difluprednate, treatment with medrysone, treatment with loteprednol, treatment with prednisolone, treatment with fluocinolone, treatment with triamcinolone, treatment with rimexolone, treatment with dexamethasone, treatment with fluorometholone, treatment with fluocinolone, treatment with rimexolone, or treatment with prednisone. Anti-inflammatory glucocorticoids. Petition 870250080612, dated 08 / 09 / 2025, pp. 70 / 111 65 / 76 may include, but are not limited to, difluprednate, dexamethasone, prednisolone, triamcinolone, fluorometholone, rimexolone, fluocinolone, loteprednol, and bioequivalents thereof. In some embodiments, topical steroid treatment is difluprednate treatment. “Dexamethasone” means dexamethasone, dexamethasone biosimilars, dexamethasone bioequivalents, and pharmaceutical compositions comprising dexamethasone, a dexamethasone biosimilar, or a dexamethasone bioequivalent. Pharmaceutical compositions comprising 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 comprising difluprednate, a difluprednate biosimilar, or a difluprednate bioequivalent.Pharmaceutical compositions comprising difluprednate include, but are not limited to, Durezol™ and difluprednate emulsions. "Triamcinolone" means triamcinolone, triamcinolone biosimilars, triamcinolone bioequivalents, and pharmaceutical compositions comprising triamcinolone, a triamcinolone biosimilar, or a triamcinolone bioequivalent. Pharmaceutical compositions comprising triamcinolone include, but are not limited to, Triesence™, Xipere™, and Trivaris™. In some embodiments, steroid treatment is administered before, during, and / or after administration of the unit dose of rAAV particles. In some embodiments, steroid treatment is administered before administration of the unit dose of rAAV particles. In some embodiments, steroid treatment is administered during administration of the unit dose of rAAV particles.In some modalities, steroid treatment is administered after the administration of the unit dose of rAAV particles. In some modalities, steroid treatment is administered before and during the dose administration. Petition 870250080612, dated 09 / 08 / 2025, pp. 71 / 111 66 / 76 unit dose of rAAV particles. In some embodiments, steroid treatment is administered before and after administration of the unit dose of rAAV particles. In some embodiments, steroid treatment is administered during and after administration of the unit dose of rAAV particles. In some embodiments, steroid treatment is administered before, during, and after administration of the unit dose of rAAV particles.

[0150] In some embodiments, steroid treatment is ophthalmic steroid treatment (e.g., difluprednate). In some embodiments, ophthalmic steroid treatment (e.g., difluprednate) is daily steroid treatment for up to about 4 weeks, about 6 weeks, or about 8 weeks from the administration of the unit dose of rAAV particles. In some embodiments, ophthalmic steroid treatment comprises about four administrations of ophthalmic steroids around week 1, about three administrations of ophthalmic steroids around week 2, about two administrations of ophthalmic steroids around week 3, and about one administration of ophthalmic steroids around week 4; time begins with and after the administration of the unit dose of rAAV particles. In some formulations, the ophthalmic steroid is approximately 0.005% to approximately 0.5% difluprednate.In some embodiments, the ophthalmic steroid is any of 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% difluprednate. In some embodiments, the ophthalmic steroid is 0.05% difluprednate. In some embodiments, a dose of 0.05% difluprednate is one drop of ophthalmic solution. In some embodiments, a drop is approximately 50 μl (e.g., approximately 25 μl to approximately 50 μl, approximately 50 μl to approximately 100 μl). In some embodiments, a dose of difluprednate comprises approximately 1 μg to approximately... Petition 870250080612, dated 09 / 08 / 2025, p. 72 / 111 67 / 76 of 5 μg, or about 2 μg to about 3 μg, or about 2.5 μg of difluprednate. In some embodiments, a dose of difluprednate comprises about 2.5 μg of difluprednate.

[0151] In some embodiments, steroid treatment is ophthalmic steroid treatment (e.g., difluprednate). In some embodiments, ophthalmic steroid treatment (e.g., difluprednate) is daily topical steroid treatment for up to about 4 weeks, about 6 weeks, or about 8 weeks from the administration of the unit dose of rAAV particles. In some embodiments, topical steroid treatment comprises about four topical steroid administrations around week 1, about three topical steroid administrations around week 2, about two topical steroid administrations around week 3, and about one topical steroid administration around week 4; time begins with and after the administration of the unit dose of rAAV particles.In some embodiments, topical steroid treatment comprises approximately four topical steroid administrations (i.e., QID) per day for approximately 3 weeks following administration of the unit dose of rAAV particles, followed by approximately three topical steroid administrations per day (i.e., TID) for approximately 1 week, followed by approximately two topical steroid administrations per day (i.e., BID) for approximately 1 week, and followed by approximately one topical steroid administration per day (i.e., QD) for approximately 1 week. In some embodiments, the topical steroid comprises difluprednate 0.05% at a dose of approximately 1 pg to approximately 3 μg. In some embodiments, the topical steroid comprises difluprednate 0.05% at a dose of approximately 2.5 μg. In some formulations, the topical steroid is approximately 0.005% to approximately 0.5% difluprednate.In some formulations, the topical steroid is anywhere from approximately 0.005%, approximately 0.006%, approximately 0.007%, approximately 0.008%, approximately 0.009%, approximately. Petition 870250080612, dated 09 / 08 / 2025, p. 73 / 111 68 / 76 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% of difluprednate. In some embodiments, the topical steroid is difluprednate 0.05%. In some embodiments, a dose of difluprednate 0.05% is one drop of ophthalmic solution. In some embodiments, a drop is approximately 50 μl (e.g., approximately 25 μl to approximately 50 μl, approximately 50 μl to approximately 100 μl). In some embodiments, a dose of difluprednate comprises 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, a dose of difluprednate comprises approximately 2.5 μg of difluprednate.

[0152] The methods, systems, and kits described herein may employ, unless otherwise indicated, conventional techniques and descriptions of molecular biology (including recombinant techniques), cell biology, biochemistry, immunochemistry, and virology techniques that are within the ability of those practicing the art. Such conventional techniques include methods for cloning and propagating recombinant viruses, formulating a pharmaceutical composition, and biochemical and immunochemical purification. Specific illustrations of suitable techniques may be obtained by reference to the examples described herein. However, equivalent conventional procedures may also be used. Such conventional techniques and descriptions may 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 Molecular Cloning Protocols: 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] It is understood that the reference to the examples below is for purposes only. Petition 870250080612, dated 09 / 08 / 2025, pp. 74 / 111 69 / 76 are illustrative and do not limit the scope of the claims. Any of the various alternative compositions and methods are applicable and suitable for use in the practice of the claimed methods and compositions. It is also understood that an evaluation of the expression constructions and methods of application can be performed using standard procedures in the art. EXAMPLES Example 1. Cell Culture

[0154] Sf9 rhabdovirus-negative insect cells (Sf-RVN®) (GlycoBac) were replaced with Sf9 insect cells to increase the rAAV production titer. Sf-RVN cells were maintained at a constant rotation of 125 rpm at 28 °C in polycarbonate Erlenmeyer flasks (Corning) using serum-free Sf-900II™ SFM medium (Gibco™) or ESF AF medium (Expression Systems) on a Multitron shaker (Infors HT). Cells were checked in the logarithmic phase (3.0-4.0E⁶ cells / ml) with viability greater than 90% using Vi-Cell BLU (Beckman Coulter). Example 2. Construction of recombinant baculovirus.

[0155] Recombinant baculoviruses were constructed using the Bac-to-Bac baculovirus expression system (Invitrogen). This method relies on site-specific transposition of donor plasmid sequences (pFastBac™) into a baculovirus bridge vector (bacmid) propagated in E. coli DH10Bac™ cells. pFastBac™ vectors containing the gene(s) of interest or rep-cap, ampicillin resistance gene and polyhedrin (polh) of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and P10 promoters 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 a gentamicin resistance gene and an SV40 polyadenylation signal to form a mini Tn7. In our modified system, the gentamicin resistance gene and the GC-rich regions near the Tn7 sites were removed. Petition 870250080612, dated 08 / 09 / 2025, page 75 / 111 70 / 76 to avoid unnecessary transfer of antibiotic resistance genes to patients and also to prevent inflammation from occurring.

[0156] The pFastBac™ vector was transformed into the E. coli DH10Bac™ strain, which contains a baculovirus bridge vector (bacmid) with a mini-attTn7 target site and a helper plasmid, using the heat shock method. 1 μL of the 1 mg / ml pFastBac™ vector was added to 20 μL of thawed DH10Bac™ cells (Invitrogen) on ice. The cells were incubated on ice for 30 minutes and then transferred to a water bath (PolyScience) at 40 °C for 25 seconds. The cells were then returned to ice for 2 minutes, followed by the addition of 1000 μL of SOC medium (Thermo Scientific). DH10Bac™ cells containing the plasmid were cultured for 6 hours at 37 °C at 220 rpm in an Innova43 shaker (New Brunswick). Transposition of the desired sequence occurs between the mini-Tn7 element in the pFastBac™ vector and the mini-attTn7 target site in the bacmid to generate a recombinant bacmid (rbacmid) in the presence of transposable proteins provided by the helper plasmid.After 6 hours, the cells were spread on an agar plate (Teknova) and incubated at 37 °C for 48 hours. The agar plate contained 50 μg / ml of kanamycin and 10 μg / ml of tetracycline, as bacmid and pHelper confer resistance to kanamycin and tetracycline, respectively. The agar plate also contained 200 μg / ml of isopropyl e-D-1-thiogalactopyranoside (IPTG) and 20 μg / ml of Bluegal, which is a chromogenic substrate. This aids in screening bacmid-containing clones using blue vs. white colonies. After the formation of recombinant bacmid, lacZ exclusion in the bacmid is complete, resulting in the absence of β-galactosidase production in the reporter cassette. Otherwise, the β-galactosidase produced by lacZ would hydrolyze Blue-gal to form 5-bromo-4-chloroindoxyl, which produces an insoluble blue pigment called 5,5'-dibromo-4,4'-dichloroindigo. Colonies formed by non-recombinant cells, therefore, appear blue, while recombinant cells appear white. Petition 870250080612, dated 09 / 08 / 2025, p. 76 / 111 71 / 76

[0157] A single white colony was seeded again onto a new agar plate with the same ingredients and allowed to grow for 48 hours to confirm the color. In the original Bac-to-Bac system, a single colony from the second agar plate is used to purify the bacmid. However, we modified the system to remove pFastBac™ from the colonies. To do this, a streptomycin-sensitive gene (rpsl), which functions as a counter-selective marker, was added to pFastBac™. After confirmation of the white colonies on the second agar plate, individual colonies were propagated onto the third agar plate, which contained 50 μg / ml kanamycin, 10 μg / ml tetracycline, and 100 μg / ml streptomycin. Each of the colonies on the 3rd plate was cultured onto the 4th agar plate with 100 μg / ml of carbenicillin. One of the colonies that failed to grow on the carbenicillin plate was selected for the next step. This colony was spread onto the 5th plate with the 3 antibiotics.Next, a single colony from the last plate was cultured overnight in 170 ml of 2XYT broth with animal-free soybean culture medium (Teknova) with kanamycin and tetracycline at 37°C at 220 rpm on an Innova 43 shaker. The bacmid from the culture was purified using the PureLink™ HiPure Plasmid Filter Maxiprep kit (Invitrogen) following the manufacturer's protocol. An overview of a process with representative steps is shown in Figure 1. The presence of rbacmid and the absence of donor plasmid were confirmed using 0.5% agarose gel and ddPCR. See Figure 2. Example 3. P0 passage of recombinant baculovirus (rBV)

[0158] rBacmid DNA purified from the bacterial colony was used to transfect Sf-RVN cells to generate recombinant baculovirus. rBacmid GFP / rBacmid Fluc was engineered as the gene of interest, whose reporter gene GFP / Fluc was flanked by ITRs. rBacmid 7m8 was engineered to include modified Rep / Cap AAV2 genes and targeted by polh and P10 promoters.

[0159] Sf-RVN cells were checked in the logarithmic phase (3.0-4.0E6 cells / ml) with viability greater than 90% using Vi-Cell BLU. The cells were Petition 870250080612, dated 08 / 09 / 2025, page 77 / 111 72 / 76 cells were centrifuged at 230 G for 10 minutes and resuspended in fresh SF-900 II SFM at a density of 2E6 / ml. 9 μg of rbacmid were added to 1 ml of 1X PBS (pH 7.4, Calcium-free, Magnesium-free) (Gibco™), and 45 μg of Cellfectin™ II reagent (Gibco™) were added to another 1 ml of 1X PBS. The mixtures were incubated for 5 minutes, and then the rbacmid was added to the Cellfectin and incubated for 30 minutes at room temperature. The Cellfectin reagent needs to be at room temperature and well mixed before use. After 30 minutes, the DNA and oil mixture was added dropwise to 30 ml of the cells. On the 4th day after transfection, the cells were checked with Vi-Cell BLU and then centrifuged for 10 minutes at 230 G using a Sorvall Legend XFR centrifuge (Thermo Scientific). The supernatant was collected in amber canonical centrifuge tubes (Avantor) as P0 stock of rBVs. 5% sucrose was added to the rBVs for long-term storage at -80 °C.The rBV titer was verified by ddPCR. Example 4. Plate Assay Purification

[0160] After the Sf-RVN cells were checked in the logarithmic phase (3,04,0E6 cells / ml) with viability greater than 90%, they were resuspended in Sf900II SFM at a density of 0.5E6 / ml. 2 ml of the diluted cells were added to each well of a 6-well plate and incubated at room temperature for 30 minutes. The 4% agarose gel (Gibco) was transferred from 4 °C to a water bath at 70 °C. An empty 100 ml vial and Sf-900TM 1.3X (Gibco) were placed in a water bath at 40 °C. Then, serial dilution of rBVs P0 to E10 vg / ml was performed using SF900II SFM from 10-1 to 10-7. A person versed in the technique recognizes that the 1E# notation format is equivalent to the 1 x 10# notation format.

[0161] After the cells were fixed in the 6-well plate, they were checked with an inverted microscope (Zeiss Invertroskop) to verify if there was 50-70% cell confluence. Then, 1000 μl of Sf 900II SFM were added to the first well as CTRL and 1000 μl of 10⁻³ to 10⁻⁷ dilutions were Petition 870250080612, dated 08 / 09 / 2025, page 78 / 111 73 / 76 added to the corresponding wells. The 6-well plate was incubated at 28 °C for 1 hour. After the agarose melted, 30 ml of SF-900 medium (1.3X) and 10 ml of 4% agarose gel were added to the empty vial and gently mixed to avoid bubble formation. This caused the 1% agarose overlay to be returned to a water bath at 40 °C until use.

[0162] At the end of 1 hour of incubation, the virus was aspirated and replaced with 2 ml of 1% agarose coating. The agarose coating was allowed to solidify in BSC for 20 minutes. Then, 1 ml of SF900 II SFM was added over the agarose layer and the plate was incubated at 28°C for 7 to 10 days. On days 7 to 10, depending on plate formation and growth, the medium was removed from the agarose layer and individual plates were collected using a 2 ml pipette, inserting the tip into the agarose layer with gradual suction so that the agarose plugs and some liquid were drawn into the tip. The agarose plugs were transferred to sterile tubes containing 1 ml of SF900II SFM, pipetting up and down several times to rinse the agarose and liquid in the sterile tubes. The tubes were kept at 4°C overnight to produce rBVs P0 or BEVs. Example 5. P1 passage of rBVs

[0163] One day before infection, Sf-RVN cells were checked to be in the logarithmic phase (3.0-4.0E6 cells / ml) with viability greater 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 checked again using Vi-Cell BLU and 500 μl of P0' rBVs were added to each flask. On the 4th day after infection, the cells were harvested as described in Example 3. ddPCR was performed on P1 rBVs to determine the most stable clones. Example 6. rBVs of Passage P2

[0164] Passage P2 was made as described in Example 5 using the Petition 870250080612, dated 08 / 09 / 2025, pp. 79 / 111 74 / 76 more stable clones of rBVs P1 for Gene of Interest (GOI) and Rep-Cap. However, to improve protocol consistency, instead of the 1:1000 dilution of rBVs P1 used previously, MOI 0.1 was used to infect Sf-RVN cells. On the third day post-infection, rBVs P2 were collected and ddPCR was performed to determine the viral titer in vg. Example 7. Environment Optimization and MOI

[0165] To optimize rAAV generation, SF900II SFM, SF900III SFM (Gibco), ESF-AF, and EX-Cell CD (SAFC) media were used to find the highest rAAV titer. The ESF-AF medium resulted in the highest rAAV titer in the E12 vg / ml range (E8 IFU / ml). Therefore, the ESF-AF medium was chosen for rAAV production.

[0166] Insect cells were co-infected with 7m8 rBV and CFI rBV at an MOI of 3 or 0.001 in two types of media (SF900II SFM or ESF-AF). rAAV were collected and rAAV titers were determined. The rAAV titer obtained from co-infection at an MOI of 0.001 was significantly higher than that obtained from co-infection at an MOI of 3. The results of one of these experiments are presented in Figure 7. Example 8. Production of rAAV

[0167] One day before infection, Sf-RVN cells in ESF-AF medium were checked in the logarithmic phase (3.0-4.0E6 cells / ml) with viability greater than 90% and were prepared at a density of 1.2E6 / ml in 30 ml cultures for each rAAV production. The following day, P2 rBVs carrying the GOI and P2 rBVs carrying the Rep / Cap were added to the cells with MOI 0.001. Previous methods used MOI 5. After the cells were incubated in shakers at 28°C for 4 hours, 225 μl of Advantage™ 66765 40X immediate insect feed (SAFC) and 4.5 μl of fatty acid (SAFC) were added to each culture. On the 5th day after infection, the cells were checked using Vi-Cell BLU. For harvesting, use 10x AAV-MAX lysis buffer (Gibco) to achieve a final concentration of 1x in culture, or 1M MgCl2 for a... Petition 870250080612, dated 08 / 09 / 2025, pages 80 / 111 75 / 76 final concentration of 2-4 mM in the culture and 90 U / ml of Benzonase were added to each 30 ml of culture. The cultures were then incubated on a shaker at 37°C for 2 hours. After cell lysion, they were pelleted at 4000 rpm for 30 minutes in a Sorvall Legend XFR centrifuge. The supernatant was filtered using a 0.2 μm PES membrane (Thermo Scientific) and stored at -80°C for later purification. The titer of rAAV viruses was determined using ddPCR.

[0168] In some cases, no insect food was added to the cultures. rAAV titers obtained from methods with and without added food were determined. The addition of food increased the rAAV titers obtained from insect cells cultured in ESF AF medium and Sf-900II™ SFM medium. The results of one of these experiments are shown in Figure 7. Example 9: Evaluation of rBV stability during different passages and the effect of the stability of the gene of interest on rAAV titer.

[0169] After the generation of P0 rBV, the plate purification assay was performed and 10 different plates for GOI (GFP-Fluc) and 7m8 were selected. The gene of interest / vector structure relationship was evaluated in 10 different plates. The results of one of these experiments are presented in Figure 6.

[0170] For rBV-GOI (GFP-Fluc), 78% of rBV retains the gene of interest at passage 0 (P0), 75% of rBV retains the gene of interest after passage 1 (P1), and 50% of rBV retains the gene of interest after passage 2 (P2).

[0171] For rBV-7m8, two different plates were selected for further evaluation. In plate 1 of 7m8, 71% of rBVs retain the rep / cap gene of interest at P0, 31% of rBVs retain the rep / cap gene of interest at P1, and 20% of rBVs retain the rep / cap gene of interest at P2.

[0172] In plate 2 of 7m8, 71% of rBVs retain the rep / cap gene of interest at P0, 81% of rBVs retain the rep / cap gene of interest at P1, and 54% of rBVs retain the rep / cap gene of interest at P2. Petition 870250080612, dated 09 / 08 / 2025, pp. 81 / 111 76 / 76

[0173] rAAVs were produced with rBVs P0, P1 and P2 (BEVs) from two different 7m8 plates (plates 1 and 2) with the same GOI plate (rBV-GOI: GFP-Fluc). The rAAV titer with plate 1-7m8 drops 20 times, but with plate 2-7m8, the rAAV titer drops only about 25%. Example 9. Evaluation of the pHelper effect on the formation of stable genes of interest.

[0174] Donor plasmids containing a gene of interest, bacmids, and pHelper plasmids are transformed into bacterial cells. Bacterial cells are transformed with multiple amounts of pHelper plasmid. Bacterial cells are cultured under selection for all three plasmids and under conditions that allow the identification of recombinant bacmids. In some experiments, bacteria are transformed with a standard amount of pHelper and cultured in media with varying amounts of an antibiotic to which pHelper provides resistance. Bacteria from bacterial colonies comprising recombinant bacmids are cultured under conditions that counterselect the donor plasmid. Bacmids are harvested from bacterial colonies comprising recombinant bacmids. Recombinant bacmids are evaluated by next-generation sequencing (NGS) of long read sequences. Recombinant bacmids comprising concatemers are identified.The number of copies of the gene of interest is evaluated.

[0175] Insect cells are infected with recombinant bacmids. P0 rBVs are obtained. NGS is used to assess the copy number of the gene of interest. Population heterogeneity is evaluated. P0 rBVs are infected in insect cells and P1 rBVs are harvested. NGS is used to assess the copy number of the gene of interest. Population heterogeneity is evaluated in the P1 rBV population. Petition 870250080612, dated 08 / 09 / 2025, page 82 / 111

Claims

1 / 20 CLAIMS 1. A system for producing recombinant bacmids with reduced residual DNA, wherein said system is CHARACTERIZED in that it comprises: i. a donor plasmid comprising a first antibiotic resistance gene, a counterselectable marker and a transfer cassette, wherein the transfer 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; iii. a bacmid comprising a third antibiotic resistance gene and a transposon insertion site located within a reporter cassette.

2. System according to claim 1, CHARACTERIZED in that each of the first, second and third antibiotic resistance genes confers resistance to a different antibiotic.

3. System according to claim 1, CHARACTERIZED in that the first antibiotic resistance gene, the second antibiotic resistance gene and the third antibiotic resistance gene are selected from the group comprising kanamycin resistance genes, ampicillin resistance genes, tetracycline resistance genes, penicillin resistance genes, streptomycin resistance genes, erythromycin resistance genes and penicillin / streptomycin resistance genes.

4. System according to claim 1, CHARACTERIZED in that the first antibiotic resistance gene, the second antibiotic resistance gene, and the third antibiotic resistance gene are selected from the group comprising the ampicillin resistance gene, the tetracycline resistance gene, and the kanamycin resistance gene. Petition 870250080612, dated 08 / 09 / 2025, p. 83 / 111 2 / 20 5. System, according to claim 1, CHARACTERIZED in that 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 a counterselectable marker that is rpsl.

6. System according to claim 1, CHARACTERIZED in that the counterselectable marker is an antibiotic-sensitive gene.

7. System according to claim 6, CHARACTERIZED in that the antibiotic-sensitive gene is selected from the group comprising rpsl, URA3 and thymidine kinase.

8. System according to claim 6, CHARACTERIZED in that the antibiotic-sensitive gene is rpsl.

9. System according to claim 1, CHARACTERIZED in that the donor plasmid does not comprise an active gentamicin resistance gene.

10. System according to claim 1, CHARACTERIZED in that the transfer cassette is optimized to reduce GC-rich regions in the transfer cassette.

11. System according to claim 1, CHARACTERIZED in that the donor plasmid does not comprise an active gentamicin resistance gene nor a GC-rich region in the transfer cassette.

12. System according to claim 1, CHARACTERIZED in that the left transposon arm is Tn7R and the right transposon arm is Tn7L.

13. System according to claim 1, CHARACTERIZED in that the reporter cassette comprises a lacZa gene.

14. System, according to claim 1, CHARACTERIZED in that the transposon insertion site is selected from the group comprising a Tn7 insertion site, a modified Tn7 insertion site and a mini-att-Tn7 site.

15. System according to claim 1, CHARACTERIZED in that the transposon insertion site is a mini-att-Tn7 site.

16. System according to claim 1, CHARACTERIZED in that the gene of interest encodes AAV RepCap proteins or a therapeutic gene product.

17. System according to claim 16, CHARACTERIZED in that the gene of interest encoding AAV RepCap proteins comprises the 7m8 cassette.

18. System according to claim 1, CHARACTERIZED in that the system is selected from the group comprising a modified Bacto-Bac system, a modified baculovirus infectious cell (BIC) system and a modified Bac-plus system.

19. Method for producing recombinant bacmids with reduced residual DNA, wherein said method is CHARACTERIZED in that it comprises the steps of: (a) introducing a system for producing recombinant bacmids comprising a gene of interest into bacterial cells, wherein the system comprises: i. a donor plasmid comprising a first antibiotic resistance gene, a counterselectable marker and a transfer cassette, wherein the transfer 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; iii. a bacmid plasmid comprising a third antibiotic resistance gene. Petition 870250080612, dated 08 / 09 / 2025, p.85 / 111 4 / 20 an antibiotic and a transposon insertion site located within a reporter cassette; (b) cultivate the transformed bacteria in the presence of three antibiotics to which the first, second, and third antibiotic resistance genes confer resistance; (c) select at least one bacterial colony comprising a recombinant bacmid; (d) incubate bacteria from at least one bacterial colony comprising a recombinant bacmid selected in step (c) with a compound that allows counterselection against the donor plasmid; and (e) collect recombinant bacmids with reduced residual DNA from a bacterial colony after counterselection.

20. Method according to claim 19, CHARACTERIZED in that a bacterial colony comprising a recombinant bacmid is identified by a change in reporter activity.

21. Method according to claim 19, CHARACTERIZED in that the reporter activity is selected from the group comprising fluorescence, antibiotic sensitivity and color of the bacterial colony.

22. Method according to claim 19, CHARACTERIZED in that the transformed bacteria are grown in the presence of a beta-galactosidase substrate.

23. Method according to claim 19, CHARACTERIZED in that the beta-galactosidase substrate is selected from the group comprising x-gal and BluoGal.

24. Method according to claim 23, CHARACTERIZED in that the presence of a recombinant bacmid is indicated by a white colony formed in the presence of the beta-galactosidase substrate. Petition 870250080612, dated 08 / 09 / 2025, page 86 / 111 5 / 20 25. Method according to claim 19, CHARACTERIZED in that each of the first, second and third antibiotic resistance genes confers resistance to a different antibiotic.

26. Method according to claim 19, CHARACTERIZED in that the first antibiotic resistance gene, the second antibiotic resistance gene and the third antibiotic resistance gene are selected from the group comprising kanamycin resistance genes, ampicillin resistance genes, tetracycline resistance genes, penicillin resistance genes, streptomycin resistance genes, erythromycin resistance genes and penicillin / streptomycin resistance genes.

27. Method, according to claim 19, CHARACTERIZED in that the donor plasmid comprises an ampicillin and rpsl resistance gene, the helper plasmid comprises a tetracycline resistance gene, and the bacmid plasmid comprises a kanamycin resistance gene.

28. Method according to claim 19, CHARACTERIZED in that the compound that enables counterselection against the donor plasmid is an antibiotic.

29. Method according to claim 19, CHARACTERIZED in that the compound that allows counterselection against the donor plasmid is streptomycin.

30. An efficient method for producing a recombinant baculovirus seed stock comprising a gene of interest, wherein at least 50% of the recombinant baculovirus (rBV) in the recombinant baculovirus seed stock comprises a gene of interest, said method being CHARACTERIZED in that it comprises the steps of: (a) introducing a system for producing recombinant bacmids comprising a gene of interest into bacterial cells, wherein the system Petition 870250080612, dated 08 / 09 / 2025, page 87 / 111 6 / 20 comprises: i. a donor plasmid comprising a first antibiotic resistance gene, a counterselectable marker and a transfer cassette, wherein the transfer 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;iii. a bacmid comprising a third antibiotic resistance gene and a transposon insertion site located within a reporter cassette; (b) cultivate the transformed bacteria in the presence of three antibiotics to which the first, second, and third antibiotic resistance genes confer resistance; (c) select at least one bacterial colony comprising a recombinant bacmid; (d) incubate bacteria from at least one bacterial colony comprising a recombinant bacmid selected in step (c) with a compound that allows counterselection against the donor plasmid; (e) collect recombinant bacmids comprising a gene of interest from a bacterial colony after counterselection; (f) transform insect cells with the recombinant bacmids comprising a gene of interest collected in step (e); (g) incubate the transformed insect cells and harvest rBV comprising the gene of interest from the insect cells;(h) perform a first passage, in which a passage comprises the steps of infecting insect cells with rBV comprising the gene of interest and incubating the infected insect cells; (i) harvest rBV comprising the gene of interest from at least two insect cell plates and identify a plate comprising rBV comprising a gene of interest and in which the rBV exhibits a low loss rate of the gene of interest; and (j) perform a second passage (rBV P1), in which the second passage comprises infecting insect cells with rBV from the plate identified in step (i), incubating the infected insect cells, and harvesting a seed stock of recombinant baculovirus comprising rBV comprising the gene of interest from the insect cells.

31. Method according to claim 30, CHARACTERIZED in that after the first pass at least 80% of the rBV comprises the gene of interest.

32. Method according to claim 30, CHARACTERIZED in that after the second passes at least 70% of the rBV comprises the gene of interest.

33. Method according to claim 30, CHARACTERIZED in that said method produces a stock of recombinant baculovirus seeds after two passages through insect cells, wherein the stock of recombinant baculovirus seeds is at least 2 X 1010 vector genomes / ml (vg / ml).

34. Method according to claim 30, CHARACTERIZED in that a bacterial colony comprising a recombinant bacmid is identified by a change in reporter activity.

35. Method according to claim 30, CHARACTERIZED in that the reporter activity is selected from the group comprising fluorescence, antibiotic sensitivity and color of the bacterial colony.

36. Method according to claim 30, CHARACTERIZED in that the transformed plasmids are grown in the presence of a beta-galactosidase substrate. Petition 870250080612, dated 08 / 09 / 2025, page 89 / 111 8 / 20 37. Method according to claim 36, CHARACTERIZED in that the beta-galactosidase substrate is selected from the group comprising x-gal and BluoGal.

38. Method according to claim 30, CHARACTERIZED in that the presence of a recombinant bacmid is indicated by a white colony formed in the presence of the beta-galactosidase substrate.

39. Method according to claim 30, CHARACTERIZED in that each of the first, second and third antibiotic resistance genes confers resistance to a different antibiotic.

40. Method according to claim 30, CHARACTERIZED in that the first antibiotic resistance gene, the second antibiotic resistance gene and the third antibiotic resistance gene are selected from the group comprising kanamycin resistance genes, ampicillin resistance genes, tetracycline resistance genes, penicillin resistance genes, streptomycin resistance genes, erythromycin resistance genes and penicillin / streptomycin resistance genes.

41. Method according to claim 30, CHARACTERIZED in that the donor plasmid comprises an ampicillin and rpsl resistance gene, the helper plasmid comprises a tetracycline resistance gene, and the bacmid plasmid comprises a kanamycin resistance gene.

42. Method according to claim 30, CHARACTERIZED in that the compound that enables counterselection against the donor plasmid is an antibiotic.

43. Method according to claim 30, CHARACTERIZED in that the compound that allows counterselection against the donor plasmid is streptomycin.

44. Method, according to claim 30, CHARACTERIZED by the fact that the step of incubating the transformed insect cells occurs at approximately 28 °C and without CO2 for approximately four days.

45. Method according to claim 30, CHARACTERIZED in that the incubation step of the transformed insect cells takes place in a shaker incubator.

46. ​​Method according to claim 30, characterized in that the insect cells are selected from the group comprising Sf-RVN cells, Sf9 cells and Hi5 cells.

47. Method according to claim 46, CHARACTERIZED in that the insect cells are selected from the group comprising Sf-RVN cells and Sf9 cells.

48. Method according to claim 30, CHARACTERIZED in that insect cells are transformed with between about 1 μg and 20 μg of bacmids.

49. Method according to claim 48, CHARACTERIZED in that insect cells are transformed with about 6 to 9 μg of bacmids.

50. Method according to claim 30, characterized in that the transformed insect cells are grown in 30 ml of medium.

51. Method according to claim 30, CHARACTERIZED in that the gene of interest encodes Rep / Cap.

52. Method according to claim 51, CHARACTERIZED in that the gene of interest comprises cassette 7m8.

53. Method, according to claim 30, CHARACTERIZED in that the step of harvesting rBV comprising the gene of interest comprises harvesting rBV from at least ten insect cell plates and identifying one plate comprising rBV that has a stable gene of interest.

54. Method according to claim 30, CHARACTERIZED by the fact Petition 870250080612, dated 08 / 09 / 2025, page 91 / 111 10 / 20 that it further comprises performing a third passage, wherein the third passage (rBV P2) comprises infecting insect cells with rBVs of rBV P1 identified in step (j), incubating the infected insect cells and harvesting a stock of recombinant baculovirus seeds comprising rBVs comprising the gene of interest or Rep / Cap from the insect cells.

55. Method for producing a recombinant baculovirus seed stock comprising a stable gene of interest, wherein at least 50% of the recombinant baculovirus (rBV) in the recombinant baculovirus seed stock comprises a stable gene of interest, wherein the method is CHARACTERIZED in that it comprises the steps of: (a) introducing a system for producing recombinant bacmids comprising a gene of interest into bacterial cells, wherein the system comprises: i. a donor plasmid comprising a first antibiotic resistance gene, a counterselectable marker and a transfer cassette, wherein the transfer 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;iii. a bacmid comprising a third antibiotic resistance gene and a transposon insertion site located within a reporter cassette; (b) cultivate the transformed bacteria in the presence of three antibiotics to which the first, second, and third antibiotic resistance genes confer resistance; (c) select at least one bacterial colony comprising a recombinant bacmid; (d) incubate bacteria from at least one bacterial colony comprising a recombinant bacmid selected in step (c) with a compound that allows counterselection against the donor plasmid; (e) collect recombinant bacmids comprising a gene of interest from a bacterial colony after counterselection; (f) transform insect cells with the recombinant bacmids comprising a gene of interest collected in step (e);(g) incubate the transformed insect cells and harvest rBV comprising the gene of interest from the insect cells; (h) perform a first passage, wherein a passage comprises the steps of infecting insect cells with rBV comprising the gene of interest and incubating the infected insect cells; (i) harvest rBV comprising the gene of interest from one or more insect cell plates and identifying a plate comprising rBV comprising a stable gene of interest; and (j) perform a second passage, wherein the second passage comprises infecting insect cells with rBV comprising a stable gene of interest, incubating the infected insect cells, and harvesting a stock of recombinant baculovirus seeds comprising rBV comprising the stable gene of interest from the insect cells.

56. Recombinant baculovirus seed stock CHARACTERIZED by the fact that it comprises a stable gene of interest.

57. Recombinant baculovirus seed stock CHARACTERIZED in that it comprises a stable gene of interest produced by the method as defined in claim 55.

58. Recombinant baculovirus seed stock, according to claim 57, CHARACTERIZED in that the gene of interest comprises cassette 7m8. Petition 870250080612, dated 08 / 09 / 2025, p. 93 / 111 12 / 20 59. Recombinant baculovirus seed stock, according to claim 57, CHARACTERIZED in that the gene of interest encodes Rep / Cap.

60. Composition CHARACTERIZED by the fact that it comprises a stock of recombinant baculovirus seeds comprising a stable gene of interest.

61. Composition CHARACTERIZED by the fact that it comprises a recombinant bacmid comprising a stable gene of interest.

62. Method for producing rAAV comprising a gene of interest CHARACTERIZED in that it comprises the steps of: (a) producing a first stock of recombinant baculovirus seeds by the method as defined in claim 30, wherein the first stock of recombinant baculovirus seeds comprises rBV comprising a first gene of interest; (b) producing a second stock of recombinant baculovirus seeds by the method as defined in claim 30, wherein the second stock of recombinant baculovirus seeds comprises rBV encoding Rep / Cap proteins; (c) co-infecting insect cells with the first stock of recombinant baculovirus seeds and with the second stock of recombinant baculovirus seeds; (d) adding feed to the insect cell medium after co-infection; (e) incubating the infected insect cells; and (f) harvesting rAAV comprising the gene of interest from the insect cells.

63. Method, according to claim 62, CHARACTERIZED in that the insect cell coinfection step comprises coinfecting insect cells with a first stock of recombinant baculovirus seeds with low multiplicity of infection (MOI) and with a second stock of recombinant baculovirus seeds with low multiplicity of infection (MOI).

64. Method according to claim 63, CHARACTERIZED in that the low MOI is less than 0.

01.

65. Method according to claim 63, CHARACTERIZED in that the low MOI is less than 0.

009.

66. Method according to claim 63, CHARACTERIZED in that the low MOI is in the range of about 0.0005 to about 0.

009.

67. Method according to claim 63, CHARACTERIZED in that the low MOI is in the range of about 0.

001.

68. Method according to claim 63, CHARACTERIZED in that the MOI of the first stock of recombinant baculovirus seeds differs from the MOI of the second stock of recombinant baculovirus seeds.

69. Method according to claim 63, CHARACTERIZED in that the MOI of the first stock of recombinant baculovirus seeds and the MOI of the second stock of recombinant baculovirus seeds are equal.

70. Method according to claim 62, CHARACTERIZED in that the harvested rAAV has a titer of at least 111 vg / ml.

71. Method according to claim 70, CHARACTERIZED in that the harvested rAAV has a titer of at least 511 vg / ml.

72. Method according to claim 70, CHARACTERIZED in that the harvested rAAV has a titer of at least 112 vg / ml.

73. Method according to claim 62, CHARACTERIZED in that food is added between about 1 hour and 8 hours after coinfection.

74. Method according to claim 73, CHARACTERIZED in that the food is added approximately 4 hours after co-infection.

75. Method, according to claim 62, CHARACTERIZED by the fact that the insect medium is ESF-AF.

76. Method according to claim 62, characterized in that the insect cells are selected from the group comprising Sf-RVN cells, Sf9 cells and Hi5 cells.

77. Method according to claim 62, CHARACTERIZED in that the insect cells are Sf-RVN cells.

78. Method according to claim 62, CHARACTERIZED in that the rAAV comprising a gene of interest is harvested at a high titer less than 25 days after transformation of insect cells with recombinant bacmids.

79. Method according to claim 78, CHARACTERIZED in that the rAAV comprising a gene of interest is harvested at a high titer approximately 23 days after transformation of insect cells with recombinant bacmids.

80. Method according to claim 62, CHARACTERIZED in that the rAAV comprising a gene of interest is harvested at a high titer five days after coinfection.

81. Method according to claim 62, CHARACTERIZED in that rBV p2 are harvested at a high titer 18 days after transformation of insect cells with recombinant bacmids.

82. Method according to claim 62, CHARACTERIZED in that the volume of the first stock of recombinant baculovirus seeds, the second stock of recombinant baculovirus seeds, or the volumes of each of the first stock of recombinant baculovirus seeds and the second stock of recombinant baculovirus seeds used to co-infect insect cells is less than 1 liter.

83. Method according to claim 62, CHARACTERIZED by the fact that the volume of the first stock of recombinant baculovirus seeds, the second stock of recombinant baculovirus seeds, or the volumes of each of the first stock of recombinant baculovirus seeds and the second stock of recombinant baculovirus seeds used to co-infect insect cells is less than 500 ml.

84. Method according to claim 62, CHARACTERIZED in that the volume of the first stock of recombinant baculovirus seeds, the second stock of recombinant baculovirus seeds, or the volumes of each of the first stock of recombinant baculovirus seeds and the second stock of recombinant baculovirus seeds used to co-infect insect cells is less than 100 ml.

85. Method according to claim 62, CHARACTERIZED in that the volume of the first stock of recombinant baculovirus seeds, the second stock of recombinant baculovirus seeds, or the volumes of each of the first stock of recombinant baculovirus seeds and the second stock of recombinant baculovirus seeds used to co-infect insect cells is less than 30 ml.

86. Method for rapidly producing rAAV comprising a gene of interest CHARACTERIZED in that it comprises the steps of: (a) producing a first stock of recombinant baculovirus seeds by the method as defined in claim 30, wherein the first stock of recombinant baculovirus seeds comprises rBV comprising a first gene of interest; (b) producing a second stock of recombinant baculovirus seeds by the method as defined in claim 30, wherein the second stock of recombinant baculovirus seeds comprises rBV encoding Rep / Cap proteins; Petition 870250080612, dated 08 / 09 / 2025, p.97 / 111 16 / 20 (c) transduce insect cells with the first recombinant baculovirus seed stock at a low multiplicity of infection (MOI) and with the second recombinant baculovirus seed stock at a low multiplicity of infection; (d) add feed to the insect cell medium after co-infection; (e) incubate the infected insect cells for five days; and (f) harvest rAAV comprising the first gene of interest from insect cells less than 27 days after transformation of insect cells with recombinant bacmids.

87. Method according to claim 62, CHARACTERIZED in that the first gene of interest encodes a therapeutic gene product for use in the treatment of an eye disease or disorder.

88. Method, according to claim 87, CHARACTERIZED in that the eye disease or disorder is selected from the group comprising glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis, Leber congenital amaurosis, diabetic retinopathy, acromotopsia and color blindness.

89. Method according to claim 88, CHARACTERIZED in that macular degeneration is selected from the group comprising dry macular degeneration, wet macular degeneration and age-related macular degeneration.

90. Method according to claim 87, CHARACTERIZED in that the therapeutic gene product is selected from the group comprising an anti-angiogenic polypeptide, a vascular endothelial growth factor (VEGF) binding protein, an opsin protein, an anti-C3 antibody, an anti-C5 antibody, complement factor I (CFI) and an anti-AMD seca gene product.

91. Method, according to claim 87, CHARACTERIZED in that the therapeutic gene product is selected from the group comprising Petition 870250080612, dated 08 / 09 / 2025, page 98 / 111 17 / 20 aflibercept, sFLTI, CFI, ranibizumab and bevacizumab.

92. Method for rapidly producing rAAV comprising a gene of interest CHARACTERIZED in that it comprises the steps of: (g) producing a first recombinant baculovirus seed stock by the method as defined in claim 30, wherein the first recombinant baculovirus seed stock comprises rBV comprising a first gene of interest; (h) producing a second recombinant baculovirus seed stock by the method as defined in claim 30, wherein the second recombinant baculovirus seed stock comprises rBV encoding Rep / Cap proteins; (i) transducing insect cells with the first recombinant baculovirus seed stock at a low multiplicity of infection (MOI) and with the second recombinant baculovirus seed stock at a low multiplicity of infection; (j) adding feed to the insect cell culture medium approximately 4 hours after co-infection;(k) incubate the infected insect cells for five days; and (l) harvest rAAV comprising the first gene of interest from the insect cells five days after co-infection.

93. Method for producing rAAV comprising a gene of interest CHARACTERIZED in that it comprises the steps of: (a) producing a first stock of recombinant baculovirus seeds by the method as defined in claim 30, wherein the first stock of recombinant baculovirus seeds comprises rBV comprising a first gene of interest; (b) providing a second stock of recombinant baculovirus seeds, Petition 870250080612, dated 08 / 09 / 2025, p.99 / 111 18 / 20 wherein the second recombinant baculovirus seed stock comprises rBV comprising a stable gene of interest encoding Rep / Cap proteins; (c) co-infect insect cells with the first recombinant baculovirus seed stock at a low multiplicity of infection (MOI) and with the second recombinant baculovirus seed stock at a low multiplicity of infection; (d) add feed to the insect cell medium after co-infection; (e) incubate the infected insect cells; and (f) harvest rAAV comprising the gene of interest from the insect cells.

94. Method according to claim 89, CHARACTERIZED in that the second recombinant baculovirus seed stock comprises rBV comprising cassette 7m8.

95. Composition CHARACTERIZED in that it comprises recombinant bacmids with reduced residual DNA produced by the method as defined in claim 19.

96. Method for producing rAAV comprising a gene of interest CHARACTERIZED in that it comprises the steps of: (a) providing a first recombinant baculovirus seed stock wherein the first recombinant baculovirus seed stock comprises rBV comprising a first stable gene of interest; (b) providing a second recombinant baculovirus seed stock wherein the second recombinant baculovirus seed stock comprises rBV comprising a stable gene of interest encoding Rep / Cap proteins; (c) co-infecting insect cells with the first recombinant baculovirus seed stock at a low multiplicity of infection (MOI) and with the second recombinant baculovirus seed stock at a low multiplicity of infection; Petition 870250080612, dated 08 / 09 / 2025, p.100 / 111 19 / 20 (d) add food to insect cell medium after co-infection; (e) incubate infected insect cells; and (f) harvest rAAV comprising the gene of interest from insect cells.

97. Method for rapidly producing rAAV comprising a gene of interest CHARACTERIZED in that it comprises the steps of: (a) producing a first recombinant baculovirus seed stock by the method as defined in claim 30, wherein the first recombinant baculovirus seed stock comprises rBV comprising a first gene of interest; (b) providing a second recombinant baculovirus seed stock, wherein the second recombinant baculovirus seed stock comprises rBV comprising a stable gene of interest encoding Rep / Cap proteins; (c) co-infecting insect cells with the first recombinant baculovirus seed stock at a low multiplicity of infection (MOI) and with the second recombinant baculovirus seed stock at a low multiplicity of infection; (d) adding feed to the insect cell medium after co-infection; (e) incubating the infected insect cells;and (f) collect rAAV comprising the gene of interest from insect cells approximately five days after co-infection.; 98. Composition CHARACTERIZED in that it comprises recombinant bacmids with reduced residual DNA produced by the method as defined in claims 19, 30, 54, 61, 82, 88, 89, 92 or 93.

99. Composition according to claim 98, CHARACTERIZED in that it is for use in the production of a pharmaceutical composition.

100. Insect cell CHARACTERIZED by the fact that it is capable of producing a rAAV comprising a stable gene of interest. Petition 870250080612, dated 08 / 09 / 2025, pp. 101 / 111 20 / 20 101. System according to claim 1, CHARACTERIZED in that recombinant bacmids with reduced residual DNA are for use in the production of rAAV.

102. System according to claim 1, CHARACTERIZED in that the auxiliary plasmid is an AAV auxiliary plasmid. Petition 870250080612, dated 08 / 09 / 2025, pp. 102 / 111

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