Modified REP-CAP plasmids and their uses.

Description

1 / 94 “MODIFIED REP-CAP PLASMIDES AND THEIR USES”

[001] This application claims priority of Application Serial No. US63 / 545,351, filed October 23, 2023, and Serial No. US63 / 456,632, filed April 3, 2023. These applications are incorporated herein by reference. Field of Inventions

[002] The present inventions relate to modified rep-Cap plasmids and uses of modified rep-Cap plasmids, such as to produce recombinant adeno-associated viruses (rAAVs). The rAAV may comprise a transgene of interest, which includes any gene that you wish to express and may fit within an AAV capsid. Background

[003] Research and clinical applications of recombinant adeno-associated virus have increased significantly in recent years, along with regulatory approvals of rAAV gene therapy products. For gene therapy to be effective, the rAAV vector must be present in sufficient concentration to exert a therapeutic effect. The standard approach used to produce rAAV vectors leads to a concentration that is too low for patient treatment. Therefore, to achieve an effective dose in a reasonable volume, rAAV vectors must be further concentrated. Concentrating vectors can also result in the concentration of contaminants or impurities and is therefore not without disadvantages. Consequently, there is a need for new approaches to increase the production of AAV vectors. Reference to Electronic Sequence Listing

[004] The request contains a Sequence List, which was sent electronically in .XML format and is incorporated herein by reference. The .XML copy, created on March 27, 2024, is named 11635.xml and is 68,473 bytes in size. A Petition 870250105146, dated 11 / 17 / 2025, page 7 / 125 2 / 94 The sequence listing contained in this .XML file is part of the descriptive report and is incorporated herein by reference. Summary of Inventions

[005] The present inventions provide modified adeno-associated virus (AAV) rep-cap plasmids and methods of using them to produce AAV vectors with an AAV capsid (e.g., an AAV1 capsid). The modified rep-cap plasmids may contain a tetracycline-inducible expression system (e.g., a Tet-Off or Tet-On system) positioned between a polynucleotide encoding an AAV rep protein and a polynucleotide encoding an AAV capsid protein (e.g., an AAV1 capsid protein). For example, the rep-cap plasmids may have an arrangement according to Figures 1, 3, 5, 6, or 7 and may include one or more sequences set forth herein.

[006] When a modified rep-cap plasmid is introduced into a cell along with a helper plasmid and a transgenic plasmid, the production of rAAV (e.g., rAAV1) is increased compared to the production of rAAV (e.g., rAAV1) with the use of a traditional rep-cap plasmid (e.g., a rep-cap plasmid lacking a tetracycline-inducible expression system). The tetracycline-inducible expression system can be switched off or inactivated (e.g., by exposing a plasmid containing a Tet-Off system to an inducer such as doxycycline, or by keeping a plasmid containing a Tet-On system in the absence of an inducer) to increase the percentage of complete rAAV vectors produced (i.e., rAAV vectors containing a transgene of interest to be expressed).Consequently, the compositions and methods described in this document can be used to produce rAAV vectors, such as rAAV1 vectors, for use in gene therapy (for example, for a gene therapy approach that would utilize AAV1 vectors, such as...). Petition 870250105146, dated 11 / 17 / 2025, page 8 / 125 3 / 94 a gene therapy approach targeting the central nervous system, heart, skeletal muscle, retinal pigment epithelium, or inner ear).

[007] The inventions provide adeno-associated virus (AAV) rep-cap plasmid, including, in a 5' to 3' orientation: (a) a polynucleotide encoding an AAV rep protein; (b) a tetracycline-induced expression system; (c) an AAV promoter, such as p41 or TRE-tight; and (d) a polynucleotide encoding an AAV1 capsid protein.

[008] The AAV rep protein may be an AAV2 rep protein.

[009] The polynucleotide that codes for an AAV rep protein can code for Rep78 and Rep52. Rep78 can have the sequence SEQ ID NO: 1. Rep52 can have the sequence SEQ ID NO: 2.

[0010] The polynucleotide that codes for the AAV rep protein may have the sequence SEQ ID NO: 3.

[0011] The tetracycline-inducible expression system may be a tetracycline (Tef-off) system. The Tet-Off system may include, in a 5' to 3' orientation: (a) a first polynucleotide encoding a tetracycline transactivator (tTA), wherein the tTA may include a tetracycline repressor-derived DNA-binding domain and a transcriptional activation domain; and (b) a second polynucleotide containing 1 to 12 tet operator sequences (tetO). The first polynucleotide can encode a polypeptide with an amino acid sequence of SEQ ID NO: 5. The first polynucleotide can have at least 80% sequence identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) with SEQ ID NO: 6 (e.g., it can have at least 80% sequence identity with SEQ ID NO: 6 and can encode a polypeptide with an amino acid sequence of SEQ ID NO: 5).The first polynucleotide may have a... Petition 870250105146, dated 11 / 17 / 2025, page 9 / 125 4 / 94 sequence with SEQ ID NO: 6. Each tetO sequence can have the sequence with SEQ ID NO: 4. Each tetO sequence can be separated from an adjacent tetO sequence by 0-20 nucleotides (e.g., by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides). The second polynucleotide can comprise eight tetO sequences. The second polynucleotide must have at least 80% sequence identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) with SEQ ID NO: 7. The second polynucleotide must have at least 80% sequence identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) with the sequence SEQ ID NO: 7 in addition to the eight tetO sequences, each of which may have the sequence SEQ ID NO: 4.The second polynucleotide may have the sequence SEQ ID NO: 7. The Tet-Off system may also comprise a polyA signal sequence located between the first polynucleotide and the second polynucleotide.

[0012] The tetracycline-inducible expression system may be a Tet-On system. The Tet-On system may include, in a 5' to 3' orientation: (a) a first polynucleotide encoding a tetracycline reverse transactivator (rtTA), wherein the rtTA may include a tetracycline repressor-derived DNA-binding domain (e.g., a tetracycline reverse repressor) and a transcriptional activation domain; and (b) a second polynucleotide containing 1 to 12 tet operator sequences (tetO). The first polynucleotide can encode a polypeptide with an amino acid sequence of SEQ ID NO: 20. The first polynucleotide can have at least 80% sequence identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) with SEQ ID NO: 21 (e.g., it can have at least Petition 870250105146, dated 11 / 17 / 2025, p. 10 / 125 5 / 94 80% sequence identity with SEQ ID NO: 21 and can encode a polypeptide with an amino acid sequence of SEQ ID NO: 20). The first polynucleotide can have the sequence of SEQ ID NO: 21. Each tetO sequence can have the sequence of SEQ ID NO: 4. Each tetO sequence can be separated from an adjacent tetO sequence by 0-20 nucleotides (e.g., by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides). The second polynucleotide can comprise eight tetO sequences. The second polynucleotide must have at least 80% sequence identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) with SEQ ID NO: 7.The second polynucleotide may have at least 80% sequence identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) with the sequence of SEQ ID NO: 7 in addition to the eight tetO sequences, each of which may have the sequence of SEQ ID NO: 4. The second polynucleotide may have the sequence of SEQ ID NO: 7. The Tet-On system may additionally comprise a polyA signal sequence located between the first polynucleotide and the second polynucleotide.

[0013] Promoter p41 may have sequence SEQ ID NO: 8.

[0014] The polynucleotide encoding an AAV1 capsid protein may encode a polypeptide with an amino acid sequence of SEQ ID NO: 9. The polynucleotide encoding an AAV capsid protein may have the sequence of SEQ ID NO: 10. The polynucleotide encoding an AAV capsid protein may have the sequence of SEQ ID NO: 11.

[0015] The rep-cap plasmid may include a polynucleotide sequence with SEQ ID NO: 12. The rep-cap plasmid may include a polynucleotide sequence with SEQ ID NO: 13. The plasmid of Petition 870250105146, dated 11 / 17 / 2025, page 11 / 125 6 / 94 rep-cap may include a polynucleotide sequence with SEQ ID NO: 22.

[0016] The present inventions also provide methods for manufacturing recombinant adeno-associated viruses by introducing into a mammalian cell: (a) a rep-cap plasmid of any of the prior inventions, such as using the p41 or TRE-tight promoters; (b) a helper plasmid containing one or more helper genes selected from E4, E2a, and VA; and (c) a transgenic plasmid including a transgene of interest flanked by terminal inverse repeats. The introduction step can be performed under conditions that allow the formation of the recombinant adeno-associated virus. The methods may additionally include the step of collecting the recombinant adeno-associated virus. The transgenic plasmid may additionally include a promoter operationally linked to the transgene of interest. The mammalian cell may be an HEK-293 cell. The rep-cap plasmid may include a polynucleotide sequence of SEQ ID NO: 12.The rep-cap plasmid may include a polynucleotide sequence with SEQ ID NO: 13. The rep-cap plasmid may include a polynucleotide sequence with SEQ ID NO: 22. The transgene of interest may encode a protein that is endogenously expressed in the human inner ear. The transgene of interest may encode, for example, solute carrier family 26, member 4 (Pendrin), Otoferlin (OTOF), Stereocilin (STRC), BHLH Atonal Transcription Factor 1 (ATOH1), gap junction protein beta 2 (GJB2), or SRY-Box 2 (Sox2), for example. Other transgenes that may be expressed include, for example, GJB6; WFS1; COCH; EYA4; MYO7A; POU4F3; ACTG1; MYO6; REST; NLRP3; COL11A1; TJP2; TBC1D24; SLC26A4; ELMOD3; EPSN; WHRN; IGF1; IGF1R; MYO15A; TMIE; TMC1; TMC2; TMPRSS3; OTOF; CDH23; GIPC3; USH1C; OTOG; TECTA; OTOA; PDCH15; CLDN14; WHRN; ESRRB; MYO3A; HGF, ILDR1, ADCY1; CIB2; MARVELD2;. Petition 870250105146, dated 11 / 17 / 2025, page 12 / 125 7 / 94 SOX2; SLC26A5; COL4A3; COL4A4; COL4A5; CLPP; PJVK; LRTOMT / COMT2; LOXHD1; TPRM; SYNE4; KCNJ10; PTPRQ; OTOGL; LHFPL5; A1PR2; CABP2; MET; GRXCR2; EPS8; CLIC5; EPS8L2; WBP2; ROR1; CLDN9; USH1G; PKHD1L1; DIAPH3; NDP; USH2A; CLRN1; SANS; HARS1; TRIOBP and other genes. A transgene of interest includes any gene that one wishes to express and that can fit into an AAV capsid. More than one transgene of interest is possible if the totality of the genes is of a combined size that can fit into an AAV capsid.

[0017] Viral yield may be at least 1 x 1011vg / ml (e.g., 1 x 1011vg / ml, 2 x 1011vg / ml, 3 x 1011vg / ml, 4 x 1011vg / ml, 5 x 1011vg / ml, or higher, such as 1 x 1011vg / ml to 5 x 1011vg / ml, 1 x 1011vg / ml to 1 x 1012vg / ml, 1 x 1011vg / ml to 5 x 1012vg / ml, or 1 x 1011vg / ml to 1 x 1013vg / ml) as measured using ddPCR of clarified lysate.

[0018] The inventions also provide methods for manufacturing recombinant adeno-associated viruses by introducing into a mammalian cell: (a) a rep-cap plasmid including, in a 5' to 3' orientation: (i) a polynucleotide encoding an AAV rep protein; ii) a tetracycline-inducible expression system; (iii) an AAV promoter (such as p41 or TRE-tight); and (iv) a polynucleotide encoding an AAV capsid protein; (b) a helper plasmid containing one or more helper genes selected from E4, E2a, and VA; and (c) a transgenic plasmid including a transgene of interest flanked by terminal inverse repeats. The introduction step can be performed under conditions that allow the formation of the recombinant adeno-associated virus. The methods may additionally include the step of collecting the recombinant adeno-associated virus.The polynucleotide encoding an AAV capsid protein can be a polynucleotide encoding an AAV1, AAV2, AAV2quad (YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, rh10 capsid protein. Petition 870250105146, dated 11 / 17 / 2025, page 13 / 125 8 / 94 rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S. The tetracycline-inducible expression system may be a Tet-Off system or a Tet-On system, as described herein. The transgenic plasmid of the inventions may additionally include a promoter operationally linked to the transgene of interest. The mammalian cell may be a HEK-293 cell. The transgene of interest may encode a protein that is endogenously expressed in the human inner ear. The transgene of interest may encode for the solute carrier family 26, member 4 (Pendrin), Otoferlin (OTOF), Stereocilin (STRC), BHLH Atonal Transcription Factor 1 (ATOH1), gap junction protein beta 2 (GJB2), or SRY-Box 2 (Sox2), for example.Other transgenes that may be expressed include, for example, GJB6; WFS1; COCH; EYA4; MYO7A; POU4F3; ACTG1; MYO6; REST; NLRP3; COL11A1; TJP2; TBC1D24; SLC26A4; ELMOD3; EPSN; WHRN; IGF1; IGF1R; MYO15A; TMIE; TMC1; TMC2; TMPRSS3; OTOF; CDH23; GIPC3; USH1C; OTOG; TECTA; OTOA; PDCH15; CLDN14; WHRN; ESRRB; MYO3A; HGF; ILDR1; ADCY1; CIB2; MARVELD2; SOX2; SLC26A5; COL4A3; COL4A4; COL4A5; CLPP; PJVK; LRTOMT / COMT2; LOXHD1; TPRM; SYNE4; KCNJ10; PTPRQ; OTOGL; LHFPL5; A1PR2; CABP2; MET; GRXCR2; EPS8; CLIC5; EPS8L2; WBP2; ROR1; CLDN9; USH1G; PKHD1L1; DIAPH3; NDP; USH2A; CLRN1; SANS; HARS1; TRIOBP and other genes. A transgene of interest includes any gene that one wishes to express and that can fit into an AAV capsid. More than one transgene of interest is possible if the totality of the genes is of a combined size that can fit into an AAV capsid.

[0019] The viral yield may be at least 1 x 1011vg / ml (e.g., 1 x 1011vg / ml, 2 x 1011vg / ml, 3 x 1011vg / ml, 4 x 1011vg / ml, 5 x 1011vg / ml), or higher, such as 1 x 1011vg / ml to 5 x 1011vg / ml, 1 x 1011vg / ml to 1 x 1012vg / ml, 1 x 1011vg / ml to 5 x 1012vg / ml, or 1 x 1011vg / ml to 1 x Petition 870250105146, dated 11 / 17 / 2025, page 14 / 125 9 / 94 1013vg / ml) as measured using ddPCR of clarified lysate.

[0020] In any of the foregoing inventions, the tetracycline-inducible expression system may be a Tet-Off system and the methods may include manufacturing recombinant adeno-associated virus, exposing the mammalian cell to a Tet-Off system inducer (e.g., manufacturing in the presence of doxycycline). These methods lead to a higher percentage of complete AAV vectors (i.e., AAV vectors containing the transgene or nucleic acid to be expressed) than manufacturing recombinant adeno-associated virus using the same modified rep-cap plasmid in the absence of the Tet-Off system inducer (e.g., exposing the Tet-Off system to an inducer that can increase the percentage of complete AAV vectors by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more percentage points, such as from 4% complete to 6%, 8%, 10%, 12%, 14% complete or more).

[0021] In any of the foregoing inventions, the tetracycline-inducible expression system may be a Tet-On system and the methods may include fabrication of recombinant adeno-associated virus while maintaining the mammalian cell in the absence of a Tet-On system inducer (e.g., fabrication without exposure to doxycycline). These methods lead to a higher percentage of complete AAV vectors (i.e., AAV vectors containing the transgene or nucleic acid to be expressed) than manufacturing recombinant adeno-associated virus using the same modified rep-cap plasmid in the presence of the Tet-On system inducer (e.g., manufacturing AAV vectors without exposing the Tet-On system to an inducer that can increase the percentage of complete AAV vectors by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more percentage points, such as from 10% complete to 12%, 14%, 16%, 18%, 20%, 22% complete or more).

[0022] Inventions also provide methods for increasing the Petition 870250105146, dated 11 / 17 / 2025, p. 15 / 125 10 / 94 yield of a recombinant adeno-associated virus capped by AAV1 by at least 2.5 times (e.g., 2.5 times, 2.7 times, 3.0 times, 3.1 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 20 times or more) over a recombinant adeno-associated virus capped by AAV1 produced with pAAV-RC1, including the step of replacing a rep-cap plasmid of any of the prior inventions for pAAV-RC1 in the manufacture of the adeno-associated virus. The yield of the recombinant adeno-associated virus capped by AAV1 can be increased by at least 7 times. The yield of recombinant adeno-associated virus capped by AAV1 can be increased by at least 8 times. The yield of recombinant adeno-associated virus capped by AAV1 can be increased by at least 9 times.The yield of recombinant adeno-associated virus capped by AAV1 can be increased by at least 10 times. The rep-cap plasmid can include a polynucleotide sequence with SEQ ID NO: 12. The rep-cap plasmid can include a polynucleotide sequence with SEQ ID NO: 13. The rep-cap plasmid can include a polynucleotide sequence with SEQ ID NO: 22. The tetracycline-inducible expression system can be a Tet-Off system, and the methods can be performed in the presence of a Tet-Off system inducer. The tetracycline-inducible expression system can be a Tet-On system, and the methods can be performed in the absence of a Tet-On system inducer. The methods also increase the percentage of complete vectors produced.

[0023] The inventions further provide methods for increasing the yield and percentage of complete AAV vectors (i.e., AAV vectors containing the transgene or nucleotide sequence of interest to be expressed) recombinant adeno-associated virus capped by AAV1 compared to an adeno-associated virus Petition 870250105146, dated 11 / 17 / 2025, p. 16 / 125 11 / 94 recombinant AAV1-capped virus produced with pAAV-RC1, including the step of replacing a rep-cap plasmid from any of the prior inventions, which may include a Tet-On system for pAAVRC1 in the fabrication of the adeno-associated virus. The formation of the recombinant AAV1-capped adeno-associated virus in such methods can be achieved without the addition of an inducer (e.g., without the addition of doxycycline, meaning that the Tet-On system is not induced). The formation of the recombinant AAV1-capped adeno-associated virus in such methods can also be achieved with an added inducer (e.g., tetracycline or doxycycline).

[0024] The inventions further provide cells comprising rep-cap plasmids according to the inventions and cell cultures comprising the cells according to the inventions. Brief Description of the Drawings

[0025] FIGURE 1 depicts a schematic map of the modified rep-cap plasmid P929.

[0026] FIGURE 2 depicts a schematic plasmid map of the standard rep-cap P374 plasmid.

[0027] FIGURE 3 depicts a schematic map of the modified rep-cap plasmid P2056.

[0028] FIGURE 4 is a graph showing the percentage of complete vectors produced using the P929 modified rep-cap plasmid in the presence and absence of doxycycline as measured by SEC-MALS.

[0029] FIGURE 5 depicts a schematic plasmid map of the rep-cap P2057 plasmid.

[0030] FIGURE 6 depicts a schematic map of the rep-cap P2058 plasmid.

[0031] FIGURE 7 depicts a schematic plasmid map of Petition 870250105146, dated 11 / 17 / 2025, page 17 / 125 12 / 94 rep-cap P2059 plasmid.

[0032] FIGURES 8A AND 8B depict data from the production of AAV1 containing a GJB2 transgene using an RC1 p5AS or P929 plasmid. FIGURE 8A depicts the genomic titer and FIGURE 8B shows the capsid titer.

[0033] FIGURES 9A to 9C depict the production of AAV1 containing an enhanced green fluorescent protein (eGFP) transgene using plasmids P374, P929, P2057, P2058, or P2059. A FIGURE 9A compares the capsid titer for each of the five plasmids. FIGURE 9B compares the genomic titer using digital droplet PCR (ddPCR) for each of the five plasmids. FIGURE 9C compares the genomic titer estimated based on the percentage of complete capsids for each of the five plasmids.

[0034] FIGURES 10A to 10B depict the production of AAV1 containing an eGFP transgene using plasmids P374, P929, P2057, P2058, or P2059 and use mass photometry to assess capsid fullness achieved by each of the five plasmids. FIGURE 10A shows the percentage of complete capsids. FIGURE 10B shows the percentage of partially filled capsids. Detailed Description of the Inventions Definitions

[0035] Unless defined otherwise, all technical and scientific terms used in this document have the same meaning commonly understood by someone with ordinary skill in the art to which this invention pertains.

[0036] As used in this document, the terms AAV and adeno-associated virus refer to a Dependoparvovirus within the Parvoviridae virus genus. AAV may refer to an AAV derived from a naturally occurring wild-type virus, an AAV derived from Petition 870250105146, dated 11 / 17 / 2025, page 18 / 125 13 / 94 an rAAV genome packaged in a capsid derived from capsid proteins encoded by a naturally occurring cap gene and / or an rAAV genome packaged in a capsid derived from capsid proteins encoded by a non-natural capsid cap gene.

[0037] As used in this document, the term rAAV refers to a recombinant AAV. Recombinant AAV refers to an AAV genome in which some or all of the rep and cap genes have been replaced by heterologous sequences.

[0038] As used in this document, the term AAV capsid protein-coding polynucleotide refers to the nucleic acid sequences that encode the capsid proteins that form or contribute to the formation of the capsid, or protein shell, of the virus. In the case of AAV, the AAV capsid protein-coding polynucleotide encodes the capsid proteins VP1, VP2, and VP3.

[0039] As used in this document, administration refers to providing or giving an individual a therapeutic agent (e.g., an rAAV1 vector produced using a modified rep-cap plasmid described in this document) by any effective route. Exemplary routes of administration are described below.

[0040] As used in this document, the term cell type refers to a group of cells that share a phenotype that is statistically separable based on gene expression data. For example, cells of a common cell type may share similar structural and / or functional characteristics, such as gene activation patterns and similar antigen presentation profiles. Cells of a common cell type may include those that are isolated from a common tissue (e.g., epithelial tissue, neural tissue, connective tissue, or muscle tissue) and / or those that are isolated from an organ, tissue system, blood vessel, or other common structure and / or region in an organism. Petition 870250105146, dated 11 / 17 / 2025, page 19 / 125 14 / 94

[0041] As used in this document, the terms conservative mutation, conservative substitution, and conservative amino acid substitution refer to a substitution of one or more amino acids by one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric bulk. These properties are summarized for each of the twenty naturally occurring amino acids in Table 1 below. Table 1. Representative physicochemical properties of natural amino acids Amino Acid | 3-Letter Code | 1-Letter Code | Side Chain Polarity | Electrostatic Character at Physiological pH (7.4) | Steric Volume |+ | Alanine | Ala | A | nonpolar | neutral | small | Arginine | Arg | R | polar | cationic | large | Asparagine | Asn | N | polar | neutral | intermediate | Aspartic Acid | Asp | D | polar | anionic | intermediate | Cysteine ​​| Cys | C | nonpolar | neutral | intermediate | Glutamic Acid | Glu | E | polar | anionic | intermediate | Glutamine | Gln | Q | polar | neutral | intermediate | Glycine | Gly | G | nonpolar | neutral | small | Histidine | His | H | polar | Both neutral and large forms Petition 870250105146, dated 11 / 17 / 2025, page 20 / 125 15 / 94 Cationic compounds in equilibrium at pH 7.4: Isoleucine (Ile), I (nonpolar, neutral, large); Leucine (Leu), L (nonpolar, neutral, large); Lysine (Lys), K (polar, cationic, large); Methionine (Met), M (nonpolar, neutral, large); Phenylalanine (Phe), F (nonpolar, neutral, large); Proline (Pro), P (nonpolar, neutral, intermediate); Serine (Ser), S (polar, neutral, small); Threonine (Thr), T (polar, neutral, intermediate); Tryptophan (Trp), W (nonpolar, neutral, bulky); Tyrosine (Tyr), Y (polar, neutral, large); Valine (Val), V (nonpolar, neutral, intermediate). ^Based on volume in A3: 50-100 is small, 100-150 is intermediate, 150-200 is large, and >200 is bulky.

[0042] From this table, it should be recognized that the conservative amino acid families include (i) G, A, V, L and I; (ii) D and E; (iii) C, S and T; (iv) H, K and R; (v) N and Q; and (vi) F, Y and W. A conservative mutation or substitution is therefore one that replaces an amino acid with a member of the same amino acid family (for example, a substitution of Ser for Thr or Lys for Arg). Petition 870250105146, dated 11 / 17 / 2025, page 21 / 125 16 / 94

[0043] As used in this document, the term endogenous refers to a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is found naturally in a specific organism (e.g., a human) or in a specific location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell, for example, a human cochlear support cell).

[0044] As used in this document, the term expression refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation and / or 3' end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein. The term expression product refers to a protein or RNA molecule produced by any of these events.

[0045] As used in this document, the term exogenous describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not found naturally in a specific organism (e.g., a human) or in a specific location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell, for example, a human cochlear support cell). Exogenous materials include those that are supplied from a source external to an organism or to cultured matter extracted from it.

[0046] As used in this document, the term heterologous refers to a combination of non-naturally occurring elements. For example, a heterologous transgene refers to a transgene that is not naturally expressed by the promoter to which it is operationally attached.

[0047] As used in this document, the term target host cell refers to a cell that has been transformed, or has the Petition 870250105146, dated 11 / 17 / 2025, page 22 / 125 17 / 94 capacity to be transformed, with a nucleic acid sequence and therefore expresses a gene of interest and, preferably, will be from a cell lineage. The term includes the progeny of the precursor cell, regardless of whether the progeny is identical in morphology or genetic composition to the original precursor cell, as long as the gene of interest is present.

[0048] Target host cells that are suitable for use with the inventions can be readily selected by persons skilled in the art. In some embodiments, the cell line is a eukaryotic cell line, such as a yeast cell line, insect cell line (e.g., Sf9 and Sf21 cells), or mammalian cell lines. Preferred mammalian cells include primate cells (including human cells), canine cells, and rodent cells. The cells can be primary cells or immortalized cells. Suitable cells can be selected from Vero cells, COS cells, HEK 293 cells, HeLa cells, CHO cells, BHK cells, MDCK cells, amniotic (human) cells, embryonic cells, cell lines transfected with viral genes, e.g., AD5 E1, including but not limited to an immortalized human retinal cell transfected with an adenovirus gene, e.g., a PER.C6 or an NSO cell.In some embodiments, the cell is a Chinese hamster ovary (CHO) cell line. Some examples of CHO cells include, but are not limited to, CHO-ori, CHO-K1, CHO-s, CHO-DHB11, CHO-DXB11, CHO-K1SV, and mutants and variants thereof. In other embodiments, the cell is a HEK293 cell. Some examples of HEK293 cells include, but are not limited to, HEK293, HEK293A, HEK293E, HEK293F, HEK293FT, HEK293FTM, HEK293H, HEK293MSR, HEK293S, HEK293SG, HEK293SGGD, HEK293T, and mutants and variants thereof.

[0049] As used in this document, the term of Petition 870250105146, dated 11 / 17 / 2025, page 23 / 125 18 / 94 natural occurrence refers to materials that are found in nature or a form of material that is found in nature.

[0050] As used in this document, the term operationally linked refers to a first molecule linked to a second molecule, wherein the molecules are arranged in such a way that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent. For example, a promoter is operationally linked to a transcriptional polynucleotide molecule if the promoter modulates the transcription of the transcriptional polynucleotide molecule of interest in a cell. Additionally, two parts of a transcriptional regulatory element are operationally linked to each other if they are joined in such a way that the transcriptional activation functionality of one part is not adversely affected by the presence of the other part.Two regulatory elements of transcription may be operationally linked to each other via a linker polynucleotide (e.g., a non-coding polynucleotide intermediate) or may be operationally linked to each other without the presence of intermediate nucleotides.

[0051] As used in this document, the term pAAV-RC1 refers to a vector containing a polynucleotide encoding an AAV2 rep protein and a polynucleotide encoding an AAV1 capsid protein that does not include a tetracycline-inducible expression system. This vector is produced by Cell Biolabs, Inc. (cellbiolabs.com / sites / default / files / VPK-421-aav-rc1-vector.pdf).

[0052] As used in this document, the term plasmid refers to an extrachromosomal circular double-stranded DNA molecule to which additional DNA segments can be attached. A plasmid is a type of vector, a nucleic acid molecule capable of carrying another nucleic acid to which it has been attached. Certain Petition 870250105146, dated 11 / 17 / 2025, page 24 / 125 19 / 94 Plasmids have the capacity for autonomous replication in a host cell into which they are introduced (e.g., bacterial plasmids of bacterial origin and episomal mammalian plasmids). Other vectors (e.g., non-episomic mammalian vectors) can be integrated into the genome of a host cell after introduction into the host cell and are therefore replicated along with the host genome. Certain plasmids have the ability to direct the expression of genes to which they are operationally linked.

[0053] As used in this document, the term polynucleotide refers to a polymer of nucleosides. Typically, a polynucleotide is composed of nucleosides that are found naturally in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) linked by phosphodiester bonds. The term encompasses molecules comprising nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified structures, etc., whether or not found in natural nucleic acids, and such molecules may be preferred for certain applications. When such application refers to a polynucleotide, it is understood that DNA, RNA, and in each case, the single-stranded and double-stranded forms (and complements of each single-stranded molecule) are provided.Polynucleotide sequence, as used herein, may refer to the polynucleotide material itself and / or to the sequence information (i.e., the succession of letters used as base abbreviations) that biochemically characterizes a specific nucleic acid. A polynucleotide sequence presented herein is presented in the 5' to 3' direction, unless otherwise indicated.

[0054] As used in this document, the term promoter refers to a recognition site on DNA that is bound by RNA. Petition 870250105146, dated 11 / 17 / 2025, page 25 / 125 20 / 94 polymerase. The polymerase drives transgene transcription. AAV promoters include, but are not limited to, the p41 and TRE-tight preferential promoters. Other AAV promoters that may be used include the p40 promoter.

[0055] The percent identity (%) of the sequence with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after sequence alignment and introduction of gaps, if necessary, to achieve maximum percent sequence identity. Alignment for purposes of determining the percent identity of nucleic acid or amino acid sequences can be achieved in several ways that are within the capabilities of a person skilled in the art, for example, with the use of publicly available computer software such as BLAST, BLAST-2, or Megalign software.Experts in the technique can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment across the entire length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the BLAST sequence comparison computer program. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which may alternatively be formulated as a given nucleic acid or amino acid sequence, A, having a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:

[0056] 100 multiplied by (the fraction X / Y) Petition 870250105146, dated 11 / 17 / 2025, p. 26 / 125 21 / 94

[0057] where X is the number of nucleotides or amino acids classified as identical matches by a sequence alignment program (e.g., BLAST) in that program's alignment of A and B, and where Y is the total number of nucleic acids in B. It will be recognized that when the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent identity of sequence A to B will not be equal to the percent identity of sequence B to A.

[0058] As used in this document, the term pharmaceutical composition refers to a mixture containing a therapeutic agent, optionally in combination with one or more pharmacologically acceptable excipients, diluents and / or carriers, to be administered to an individual, such as a mammal, for example, a human being, in order to prevent, treat or control a particular disease or condition affecting or potentially affecting the individual.

[0059] As used in this document, the term pharmaceutically acceptable refers to compounds, materials, compositions and / or pharmaceutical forms that are suitable for contact with the tissues of an individual, such as a mammal (e.g., a human being), without excessive toxicity, irritation, allergic response and other problematic complications proportionate to a reasonable benefit / risk ratio.

[0060] As used in this document, the term polynucleotide encoding an AAV rep protein refers to the nucleic acid sequences that encode the non-structural proteins (i.e., rep78, rep68, rep52, and rep40) required for virus replication and production.

[0061] As used in this document, the term rep-cap plasmid refers to a plasmid that provides the functions of viral rep. Petition 870250105146, dated 11 / 17 / 2025, page 27 / 125 22 / 94 and capsid gene. This plasmid may be useful for producing AAVs from rAAV genomes lacking functional representation and / or capsid gene sequences.

[0062] As used in this document, the term individual refers to any organism to which a composition according to the invention may be administered, for example, for experimental, diagnostic, prophylactic and / or therapeutic purposes. Typical individuals include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates and humans). An individual may seek and require treatment, require treatment, be receiving treatment, be receiving treatment in the future, and be a human being and an animal that is under the care of a trained professional for a particular disease and condition. Preferably, the individual is a human being.

[0063] As used in this document, the terms transcription regulatory element and regulatory sequence refer to a polynucleotide that controls, at least in part, the transcription of a gene of interest. Transcription regulatory elements may include promoters, enhancers, and other polynucleotides (e.g., polyadenylation signals) that control or help control gene transcription. Examples of transcription regulatory elements are described, for example, in Lorence, Recombinant Gene Expression: Reviews and Protocols (Humana Press, New York, NY, 2012).

[0064] As used in this document, the term transfection refers to any of a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, for example, electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, nucleofection, compression, sonoporation, optical transfection, magnetofection, impalement and the like. Petition 870250105146, dated 11 / 17 / 2025, page 28 / 125 23 / 94

[0065] As used in this document, the terms transduction and transduce refer to a method of introducing a vector construct or part thereof into a cell. Where the vector construct is contained within a viral vector, such as an AAV vector, transduction refers to viral infection of the cell and subsequent transfer and integration of the vector construct or part thereof into the cell's genome.

[0066] As used in this document, the term transgene refers to a recombinant nucleic acid (e.g., DNA) that encodes a gene product, such as a peptide, protein, or RNA (e.g., a protein-coding mRNA or an inhibitory RNA, such as a miRNA, shRNA, or the like). In addition to the gene product coding region, the transgene may include or be operationally linked to one or more elements to facilitate or enhance expression, such as promoter(s), enhancer(s), destabilizing domain(s), response element(s), reporter element(s), insulating element(s), polyadenylation signal(s), and / or other functional elements. According to the present inventions, any suitable promoter(s), enhancer(s), destabilizing domain(s), response element(s), reporter element(s), insulating element(s), polyadenylation signal(s), and / or other functional elements may be used.Examples of transgenes are identified in this document.

[0067] As used in this document, treatment and treating, in reference to a disease or condition, refer to an approach to obtain beneficial or desired results, for example, clinical results. Beneficial or desired results may include, but are not limited to, relief or improvement of one or more symptoms or conditions; reduction in the extent of the disease or condition; stabilization (i.e., not worsening) of a disease, disorder, or condition; preventing the spread of diseases or conditions; delaying or slowing the Petition 870250105146, dated 11 / 17 / 2025, page 29 / 125 24 / 94 disease or condition progression; disease or condition improvement or palliation; and detectable or undetectable remission (partial or total). Improving or palliating a disease or condition means that the extent and / or undesirable clinical manifestations of the disease, disorder, or condition are diminished and / or the time course of progression is slowed or prolonged, compared to the extent or time course in the absence of treatment. Treatment can also mean prolonging survival compared to the expected survival if not receiving treatment. Those who need treatment include those who already have the condition or disorder, as well as those likely to have the condition or disorder or those in whom the condition or disorder should be prevented.

[0068] As used in this document, the term vector refers to a nucleic acid vector, for example, a DNA vector, such as a plasmid, cosmid or artificial chromosome, an RNA vector, a virus or any other suitable replicon (e.g., viral vector). A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a prokaryotic or eukaryotic cell. Examples of such expression vectors are described, for example, in Gellissen, Recombinant Protein Production: Novel Microbial and Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, MA, 2006). The expression vectors suitable for use with the compositions and methods described in this document contain a polynucleotide sequence as well as, for example, additional sequence elements used for protein expression.Certain vectors that can be used for the expression of a viral capsid protein, as described in this document, include vectors containing regulatory sequences, such as promoter and enhancer regions, that direct gene transcription. Other vectors useful for the expression of a transgene contain sequences of... Petition 870250105146, dated 11 / 17 / 2025, page 30 / 125 25 / 94 polynucleotides that increase the translation rate of the transgene or improve the stability or nuclear export of the mRNA resulting from genetic transcription. These sequence elements include, for example, untranslated 5' and 3' regions and a polyadenylation signal site to direct efficient transcription of the gene carried in the expression vector. Suitable expression vectors for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selecting cells containing that vector. Examples of a suitable marker include genes encoding antibiotic resistance, such as ampicillin, chloramphenicol, kanamycin, or nourseotricin.

[0069] As used in this document, the term viral capsid protein refers to a capsid protein that makes up a protein shell. This protein shell is generally composed of one or more viral capsid proteins and, when assembled, has the ability to be loaded with one or more polynucleotide molecules. A viral capsid protein described in this document may, for example, be a viral protein (VP) 1, VP2, and VP3. Additionally, a viral capsid protein described in this document may refer to a viral capsid protein of an AAV1.

[0070] As used in this document, the term VP1 refers to a capsid protein that is a component of an AAV capsid, such as AAV1. The AAV1 protein VP1 has the sequence SEQ ID NO: 9. As used in this document, a VP1 may possess a surface binding site that interacts with one or more molecules on the surface of a cell to initiate the process of cell entry (e.g., endocytic entry and receptor-mediated fusion). As used in this document, a VP1 may self-assemble into a structure consisting of VP1, VP2, and / or VP3 molecules. VP1 may exhibit self-linking and self-assembly properties around the exterior. Petition 870250105146, dated 11 / 17 / 2025, page 31 / 125 26 / 94 of a respective capsid containing VP1.

[0071] As used herein, the term VP2 refers to a capsid protein that is a component of an AAV capsid, such as AAV1. The AAV1 VP2 protein has the sequence SEQ ID NO: 14. As used herein, a VP2 can facilitate capsid entry into a host cell, for example, by mediating associations and exit from the endoplasmic reticulum of a host cell and by facilitating the entry of a nucleic acid molecule into the nucleus of a host cell. As used herein, a VP2 can self-assemble into a structure consisting of VP1, VP2, and / or VP3 molecules. The VP2 can self-assemble within a respective capsid containing VP2.

[0072] As used in this document, the term VP3 refers to a capsid protein that is a component of an AAV capsid, such as AAV1. The AAV1 VP3 protein has the sequence SEQ ID NO: 15. As used in this document, a VP3 can facilitate capsid entry into a host cell, for example, by mediating associations and exit from the endoplasmic reticulum of a host cell and by facilitating the entry of a nucleic acid molecule into the nucleus of a host cell. As used in this document, a VP3 can self-assemble into a structure consisting of VP1, VP2, and / or VP3 molecules. The VP3 can self-assemble within a respective capsid containing VP3.

[0073] As used in this document, the term wild type refers to a genotype with the highest frequency for a given gene in a given organism. Description

[0074] This document describes compositions and methods for producing AAV vectors (e.g., AAV1 vectors). The present inventions provide modified rep-cap plasmids that Petition 870250105146, dated 11 / 17 / 2025, p. 32 / 125 27 / 94 contain a tetracycline-inducible expression system (e.g., a Tet-Off or Tet-On system) positioned between a polynucleotide encoding an AAV rep protein and a polynucleotide encoding an AAV capsid protein (e.g., an AAV1 capsid protein). The polynucleotide encoding an AAV capsid protein (e.g., AAV1 capsid protein) can encode the AAV capsid proteins VP1, VP2, and VP3. Furthermore, the inventions provide methods for producing AAV vectors (e.g., AAV1 vectors) using the modified rep-cap plasmid (e.g., by transducing a cell with the modified rep-cap plasmid, a helper plasmid, and a transgenic plasmid).The methods may include deactivating the tetracycline-inducible expression system (e.g., producing AAV vectors using a modified rep-cap plasmid containing a Tet-Off system in the presence of a Tet-Off system inducer, or producing AAV vectors using a modified rep-cap plasmid containing a Tet-On system in the absence of a Tet-On system inducer). The compositions and methods described herein can be used to increase AAV production (e.g., AAV1 production) compared to approaches using unmodified rep-cap plasmids (e.g., rep-cap plasmids containing the same polynucleotides encoding rep and capsid proteins, but without the tetracycline-inducible expression system).Furthermore, the compositions and methods described in this document can be used to increase the percentage of complete AAV vectors produced (e.g., AAV vectors containing a transgene or polynucleotide to be expressed) compared to approaches using the same modified rep-cap plasmids in which the tetracycline-inducible expression system is activated. The AAV vectors (e.g., AAV1 vectors) produced using these methods... Petition 870250105146, dated 11 / 17 / 2025, page 33 / 125 The 28 / 94 specimens described in this document may contain a transgene of interest and may be used in gene therapy applications. Modified Rep-Cap Plasmids

[0075] The production of AAV vectors requires three plasmids: 1) a rep-cap plasmid, which contains the structural and packaging genes of AAV; 2) a helper plasmid, which contains genes encoding proteins necessary for viral replication; and 3) a transgenic plasmid (also called a transfer plasmid), which contains a transgene to be expressed by the AAV vector. The rep-cap plasmid contains two genes, the replication gene (rep) and the capsid gene (cap). The rep gene encodes four proteins involved in the replication and packaging of the viral genome: Rep78, Rep68, Rep52, and Rep40. The cap gene encodes three AAV viral capsid proteins, VP1, VP2, and VP3, which together form the outer capsid shell that protects the viral genome. Alternative splicing and different translational initiation sites generate the three VP proteins.The cap gene also encodes the Activator Assembly Protein (AAP) and the Membrane-Associated Accessory Protein (MAAP) from an alternative open reading frame. AAP is believed to provide a scaffolding function for capsid assembly.

[0076] The present inventions are based, in part, on the discovery of modifications to the rep-cap plasmid that can result in increased production of AAVs, such as AAV1 (for example, when the modified rep-cap plasmid is introduced into a cell with a helper plasmid and a transgenic plasmid). These modifications may include the introduction of a tetracycline-inducible expression system between a polynucleotide encoding an AAV rep protein and a polynucleotide encoding an AAV1 capsid protein. Producing larger quantities of AAV vector, such as AAV1, is desirable because it facilitates manufacturing and can reduce the amount of concentration needed to achieve an effective dose for applications. Petition 870250105146, dated 11 / 17 / 2025, page 34 / 125 29 / 94 of gene therapy in a reasonable volume. The current inventors also made the surprising discovery that a higher percentage of complete vectors could be produced using the modified rep-cap plasmids, manufacturing AAV vectors under conditions that would cause the tetracycline-inducible expression system to be deactivated (e.g., producing AAV vectors using a modified rep-cap plasmid containing a Tet-Off system in the presence of a Tet-Off system inducer, or producing AAV vectors using a modified rep-cap plasmid containing a Tet-On system in the absence of a Tet-On system inducer). Producing a higher percentage of complete vectors is desirable because it means that more of the resulting product can be used for therapy.The compositions and methods described herein can therefore be used to manufacture the AAV1 vector for use in gene therapy (e.g., a therapy involving the expression of a transgene in a cell that can be transduced by AAV1 vectors).

[0077] The modified rep-cap plasmids described herein may include a polynucleotide encoding an AAV rep protein and a polynucleotide encoding an AAV cap protein, such as an AAV1 cap protein. The plasmids may contain any of the polynucleotide sequences disclosed or identified herein, as well as other existing polynucleotides, including regulatory sequences, that are useful for making the rep-cap plasmids described herein.

[0078] The polynucleotide encoding an AAV rep protein may be a polynucleotide encoding an AAV2 rep protein, for example. The polynucleotide encoding an AAV rep protein may encode Rep78 and Rep52. The Rep78 protein encoded by the polynucleotide encoding an AAV rep protein (e.g., an AAV2 rep protein) may have the amino acid sequence of SEQ ID Petition 870250105146, dated 11 / 17 / 2025, page 35 / 125 30 / 94 NO: 1 provided below. MPGFYEIVIKVPSDLDEHLPGISDFVNWVAEKEWELPPDSDMDLNLIE QAPLTVAEKLQRDFLTEWRRVSKAPEALFFVQFEKGESYFHMHVLVET TGVKSMVLGRFLSQIREKLIQRIYRGIEPTLPNWFAVTKTRNGAGGGNK VVDECYIPNYLLPKTQPELQWAWTNMEQYLSACLNLTERKRLVAQHLT HVSQTQEQNKENQNPNSDAPVIRSKTSARYMELVGWLVDKGITSEKQ WIQEDQASYISFNAASNSRSQIKALDNAGKIMSLTKTAPDYLVGQQQPV EDISSNRIYKILELNGYDPQYAASVFLGWATKKFGKRNTIWLFGPATTGK TNIAEAIAAHTVPFYGCVNWTNENFPFNDCVDKMVIWWEEGKMTAKVVE SAKAILGGSKVRVDQKCKSSAQIDPTPVIVTSNTNMCAVIDGNSTTFEH QQPLQDRMFKFELTRRLDHDFGKVTKQEVKDFFRWAKDHVVEVEHEF YVKKGGAKKRPAPSDADISEPCRVRESVAQPSTSDAEASINYADRYQN KCSRHVGMNLMLFPCRQCERLNQNSNICFTHGVKDCLECFPVSESQP VSVVKKAYQKLCYIHIMGKVPDACTACDLVNVDLDDCVSEQ (SEQ ID NO: 1)

[0079] The Rep52 protein encoded by the polynucleotide encoding an AAV rep protein (e.g., an AAV2 rep protein) may have the amino acid sequence of SEQ ID NO: 2 provided below. MELVGWLVDKGITSEKQWIQEDQASYISFNAASNSRSQIKAALDNAGKI MSLTKTAPDYLVGQQPVEDISSNRIYKILELNGYDPQYAASVFLGWATK KFGKRNTIWLFGPATTGKTNIAEAIAHTVPFYGCVNWTNENFPFNDCVD KMVIWWEEGKMTAKVVESAKAILGGSKVRVDQKCKSSAQIDPTPVIVTS NTNMCAVIDGNSTTFEHQQPLQDRMFKFELTRRLDHDFGKVTKQEVKD FFRWAKDHVVEVEHEFYVKKGGAKKRPAPSDADISEPKRVRESVAQPS TSDAEASINYADRYQNKCSRHVGMNLMLFPCRQCERLNQNSNICFTHG VKDCLECFPVSESQPVSVVKKAYQKLCYIHHIMGKVPDACTACDLVNVD LDDCVSEQ (SEQ ID NO: 2)

[0080] The polynucleotide encoding an AAV rep protein included in the rep-cap plasmids described herein may be a polynucleotide that may have the sequence SEQ ID NO: 3 Petition 870250105146, dated 11 / 17 / 2025, page 36 / 125 31 / 94 provided below. ATGCCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTG ACGAGCATCTGCCCGGCATTTCTGACAGCTTTGTGAACTGGGTGGC CGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAAT CTGATTGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGC GACTTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGGAGGCT CTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCACATGCA CGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGTTTTGGGACGT TTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGG GATCGAGCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGA AATGGCGCCGGAGGCGGGAACAAGGTGGTGGATGAGTGCTACATC CCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGGCGT GGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAG CGTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACG CAGGAGCAGAACAAAGAGAATCAGAATCCCAATTCTGATGCGCCGG TGATCAGATCAAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTG GCTCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGA GGACCAGGCCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGG TCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGCC TGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGG AGGACATTTCCAGCAATCGGATTTATAAAATTTTGGAACTAAACGGG TACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAAC TACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCC TTCTACGGGTGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGA CTGTGTCGACAAGATGGTGATCTGGTGGGAGGAGGGGAAGATGAC CGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAA GGTGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCC GACTCCCGTGATCGTCACCTCCAACACCAACATGTGCGCCGTGATT GACGGGAACTCAACGACCTTCGAACACCAGCAGCCGTTGCAAGACC Petição 870250105146, de 17 / 11 / 2025, pág. 37 / 125 32 / 94 GGATGTTCAAATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGG AAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGG ATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGG AGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCC AAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCG GAAGCTTCGATCAACTACGCGGACAGGTACCAAAACAAATGTTCTCG TCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAG AGACTGAATCAGAATTCAAATATCTGCTTCACTCACGGTGTCAAAGA CTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCG TCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCACATCATGGGA AAGGTGCCAGACGCTTGCACTGCTTGCGACCTGGTCAATGTGGACT TGGATGACTGTGTTTCTGAACAA (SEQ ID NO: 3)

[0081] The modified rep-cap plasmids described in this document may also include a tetracycline-inducible expression system. The tetracycline-inducible expression system may be positioned 3' from the polynucleotide encoding an AAV rep protein within the modified rep-cap plasmid.

[0082] The tetracycline-inducible expression system can be a tetracycline-off (Tet-Off) system. The Tet-Off system can include a tetracycline-controlled transactivator (tTA) and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more) tetracycline operator (tetO) sequences. The tTA can contain a tetracycline repressor-derived DNA-binding domain (tetR) and a transcriptional activation domain. The tTA can be a fusion of a tetR and the C-terminal domain of VP16 (virion protein 16), an essential transcriptional activation domain of HSV (herpes simplex virus). The Tet-Off system can contain 7 tetO sequences. The Tet-Off system can contain 8 tetO sequences. One or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more) tetO sequences can be positioned directly next to each other (e.g., joined without any intervening sequence between them). Petition 870250105146, dated 11 / 17 / 2025, p. 38 / 125 33 / 94 tetO sequences, for example, the 3' end of a first tetO sequence is positioned directly before the 5' end of a second tetO sequence) or can be joined by a nucleic acid linker (for example, a nucleic acid linker can be positioned between each tetO sequence included in the Tet-Off system or between at least two of the tetO sequences in the Tet-Off system). According to the present inventions, where one or more tetO sequences can be joined by a linker, each linker can contain 1-20 or more nucleotides (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides) and does not disrupt the function of the tetO sequences. Each tetO sequence in the Tet-Off system can be separated from the next tetO sequence by a ligand containing 20 or more nucleotides. Each tetO sequence in the Tet-Off system can be separated from the next tetO sequence by a ligand containing 18 nucleotides.Within the Tet-Off system, the polynucleotide sequence encoding tTA can be positioned 5' from one or more tetO sequences. The tetracycline-inducible expression system may include a polyadenylation signal sequence (polyA) positioned 3' from the polynucleotide sequence encoding tTA and 5' from the polynucleotide sequence encoding tetO sequences (i.e., the polyA signal sequence can be positioned between the tTA sequence and the tetO sequences in the modified rep-cap plasmid).

[0083] A tTA may include a protein with at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) with SEQ ID NO: 5 and that retains function as a tTA (i.e., excludes proteins with a sequence that functions as an rtTA). A tTA may include a protein with an amino acid sequence that contains one or more amino acid substitutions conservative with respect to SEQ ID NO: 5 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, Petition 870250105146, dated 11 / 17 / 2025, p. 39 / 125 34 / 94 13, 14, 15, 16, 17, 18, 19, 20 or more conservative amino acid substitutions), provided that the encoded tTA analog retains the ability to bind to tetO sequences and promote expression in the absence of an effector. The inventions provide that no more than 10% of the amino acids in the tTA protein may be substituted by conservative amino acid substitutions. The polynucleotide sequence encoding a tTA protein may be any polynucleotide sequence that, by redundancy of the genetic code, encodes SEQ ID NO: 5. tTA may have the amino acid sequence SEQ ID NO: 5 provided below. MSRLDKSKVINGALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKR ALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGA KVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVL EEQEHQVAKEERETPTTDSMPPLLRQAIELFDRQGAEPAFLFGLELIICG LEKQLKCESGGPADALDDFDLDMLPADALDDFDLDMLPADALDDFDLD MLPG (SEQ ID NO: 5)

[0084] The tTA protein with SEQ ID NO: 5 can be encoded by a polynucleotide with at least 80% sequence identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) with the sequence of SEQ ID NO: 6. The tTA protein with SEQ ID NO: 5 can be encoded by the polynucleotide sequence of SEQ ID NO: 6 provided below. ATGAGTCGGCTGGATAAATCTAAAGTCATAAACGGCGCTCTGGAATT ACTCAATGAAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCT CAAAAGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGA ACAAGCGGGCCCTGCTCGATGCCCTGGCCATCGAGATGCTGGACA GGCATCATACCCACTTCTGCCCCCTGGAAGGCGAGTCATGGCAAGA CTTTCTGCGGAACAACGCCAAGTCATTCCGCTGTGCTCTCCTCTCAC ATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGAA Petition 870250105146, dated 11 / 17 / 2025, p. 40 / 125 35 / 94 ACAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAA GGCTTCTCCCTGGAGAACGCACTGTACGCTCTGTCCGCCGTGGGCC ACTTTACACTGGGCTGCGTATTGGAGGAACAGGAGCATCAAGTAGC AAAAGAGGAAAGAGAGACACCTACCACCGATTCTATGCCCCCACTTC TGAGACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCCGAACCTGC CTTCCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGC TAAAGTGCGAAAGCGGCGGGCCGGCCGACGCCCTTGACGATTTTGA CTTAGACATGCTCCCAGCCGATGCCCTTGACGACTTTGACCTTGATA TGCTGCCTGCTGACGCTCTTGACGATTTTGACCTTGACATGCTCCCC GGGTAA (SEQ ID NO: 6)

[0085] The tetracycline-inducible expression system may be a tetracycline system (Tet-On). The Tet-On system may include a reverse tetracycline-controlled transactivator (rtTA) and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more) tetracycline operator (tetO) sequences. The rtTA may contain a variant of a tetracycline repressor-derived DNA-binding domain (tetR) that can bind to tetO only in the presence of an effector (called the reverse tetR). The inventions also establish that the rtTA may be a fusion of the reverse tetR and the C-terminal domain of VP16 (virion protein 16), an essential transcriptional activation domain of HSV (herpes simplex virus). The Tet-On system can contain 7 tetO sequences. The TetOn system can contain 8 tetO sequences.One or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more) tetO sequences can be positioned directly next to each other (e.g., joined without any intermediate sequence between the tetO sequences, for example, the 3' end of a first tetO sequence is positioned directly before the 5' end of a second tetO sequence) or they can be joined by a nucleic acid linker (e.g., a nucleic acid linker can be positioned between each tetO sequence included in the Tet-On system or between at least two of the tetO sequences). Petition 870250105146, dated 11 / 17 / 2025, p. 41 / 125 36 / 94 in the Tet-On system). According to the current inventions, where one or more tetO sequences can be joined by a linker, each linker can contain 1-20 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides) and does not disrupt the function of the tetO sequences. Each tetO sequence in the Tet-On system can be separated from the next tetO sequence by a linker containing 20 nucleotides. Each tetO sequence in the Tet-On system can be separated from the next tetO sequence by a linker containing 18 nucleotides. Within the Tet-On system, the polynucleotide sequence encoding rtTA can be positioned 5' from one or more tetO sequences.The tetracycline-inducible expression system may include a polyadenylation signal sequence (polyA) positioned 3' from the polynucleotide sequence encoding rtTA and 5' from the polynucleotide sequence encoding tetO sequences (i.e., the polyA signal sequence may be positioned between the rtTA sequence and the tetO sequences in the modified rep-cap plasmid). The rtTA may include a protein with at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) with SEQ ID NO: 20, and which may retain function as an rtTA (i.e., excludes proteins with a sequence that functions as a tTA).The rtTA may include a protein with an amino acid sequence that may contain one or more amino acid conservative substitutions relative to SEQ ID NO: 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid conservative substitutions), provided that the encoded rtTA analog can retain the ability to bind to tetO sequences and promote expression in the presence of an effector. The inventions provide that no more than 10% of the amino acids in the rtTA protein may be substituted by amino acid conservative substitutions. The polynucleotide sequence encoding an rtTA protein may be any. Petition 870250105146, dated 11 / 17 / 2025, p. 42 / 125 37 / 94 polynucleotide sequence which, due to redundancy in the genetic code, may encode SEQ ID NO: 20. rtTA may have the amino acid sequence of SEQ ID NO: 20 provided below. MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKR ALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGA KVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVL EEQEHQVAKEERETPTTDSMPPLLKQAIELFDRQGAEPAFLFGLELIICG LEKQLKCESGGPTDALDDFDLDMLPADALDDFDLDMLPADALDDFDLD MLPG (SEQ ID NO: 20)

[0086] The rtTA protein with SEQ ID NO: 20 can be encoded by a polynucleotide with at least 80% sequence identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) with the sequence with SEQ ID NO: 21. The rtTA protein with SEQ ID NO: 20 can be encoded by the polynucleotide sequence with SEQ ID NO: 21 provided below. ATGAGTCGGCTGGATAAATCTAAAGTCATAAACTCTGCTCTGGAATT ACTCAATGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCT CAAAAGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGA ACAAGCGGGCCCTGCTCGATGCCCTGCCAATCGAGATGCTGGACAG GCATCATACCCACTCCTGCCCCCTGGAAGGCGAGTCATGGCAAGAC TTTCTGCGGAACAACGCCAAGTCATACCGCTGTGCTCTCCTCTCACA TCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGAAA CAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAG GCTTCTCCCTGGAGAACGCACTGTACGCTCTGTCCGCCGTGGGCCA CTTTACACTGGGCTGCGTATTGGAGGAACAGGAGCATCAAGTAGCA AAAGAGGAAAGAGAGACACCTACCACCGATTCTATGCCCCCACTTCT GAAACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCCGAACCTGCC TTCCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCT AAAGTGCGAAAGCGGCGGGCCGACCGACGCCCTTGACGATTTTGAC Petição 870250105146, de 17 / 11 / 2025, pág. 43 / 125 38 / 94 TTAGACATGCTCCCAGCCGATGCCCTTGACGACTTTGACCTTGATAT GCTGCCTGCTGACGCTCTTGACGATTTTGACCTTGACATGCTCCCCG GGTAA (SEQ ID NO: 21)

[0087] Each of the tetO sequences included in the tetracycline-inducible expression system (e.g., Tet-Off or Tet-On system) in the modified rep-cap plasmids described in this document may have the sequence: TCCCTATCAGTGATAGAGA (SEQ ID NO: 4). The tetracycline-inducible expression system (e.g., Tet-Off or Tet-On system) may include eight tetO sequences, and the polynucleotide containing the eight tetO sequences may have at least 80% sequence identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) with the sequence SEQ ID NO NO: 7.The polynucleotide containing the 8 tetO sequences may have at least 80% sequence identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) with the SEQ ID sequence NO:7 in addition to the eight tetO sequences, each of which may have the SEQ ID sequence NO:4. The portion of the tetracycline-inducible expression system containing the eight tetO sequences may have the SEQ ID sequence NO:7 provided below. TCCCTATCAGTGATAGAGAAGTCGACACGTCTCGAGCTCCCTATCAG TGATAGAGAAGGTACGTCTAGAACGTCTCCCTATCAGTGATAGAGAA GTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGAAGGTACGTCT AGAACGTCTCCCTATCAGTGATAGAGAAGTCGACACGTCTCGAGCT CCCTATCAGTGATAGAGAAGGTACGTCTAGAACGTCTCCCTATCAGT GATAGAGAAGTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGA (SEQ ID NO: 7)

[0088] The modified rep-cap plasmids described in this document may include an AAV promoter positioned between the Petition 870250105146, dated 11 / 17 / 2025, p. 44 / 125 39 / 94 tetracycline-inducible expression system and the polynucleotide encoding an AAV1 capsid protein. The AAV promoter may be an AAV p41 promoter. The p41 promoter may have the sequence SEQ ID NO: 8 provided below. TGTTCAAATTTGAACTGACTAAGCGGCTCCCGCCAGATTTTGGCAAG ATTACTAAGCAGGAAGTCAAGGACTTTTTTGCTTGGGCAAAGGTCAA TCAGGTGCCGGTGACTCACGAGTTTAAAGTTCCCAGGGAATTGGCG GGAACTAAAGGGGCGGAGAAATCTCTAAAACGCCCACTGGGTGACG TCACCAATACTAGCTATAAAAGTCTGGAGAAGCGGGCCAGGCTCTC ATTTGTTCCCGAGACGCCTCGCAGTTCAGACGTGACTGTTGATCCC GCTCCTCTGCGACCGCT (SEQ ID NO: 8)

[0089] The modified rep-cap plasmids described in this document can be used to produce AAV vectors containing an AAV1 capsid.

[0090] The AAV1 VP1 capsid protein may have the amino acid sequence SEQ ID NO: 9. MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLV LPGYKYLGPFNGLDKGEPVNAADAAAALEHDKAYDQQLKAGDNPYLRYN HADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGK KRPVEQSPQEPDSSSGIGKTGQQPAKRLNFGQTGDSESVPDPQPLG EPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWL GDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYF DFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVT TIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLT LNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHS QSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPK NWLPGPCYRQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTA MASHKDDEDKFFPMSGVMIFGKESAGASTALDNVMITDEEEIKATNPV ATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYLQGPIW AKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFAS Petition 870250105146, of 17 / 11 / 2025, p. 45 / 125 40 / 94 FITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDN NGLYTEPRPIGTRYLTRPL (SEQ ID NO: 9)

[0091] The capsid protein of AAV1 VP2 may have the amino acid sequence of SEQ ID NO: 14. TAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPD PQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHC DSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTN DGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQY GYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSS YAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMS VQPKNWLPGPCYRQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINP GTAMASHKDDEDKFFPMSGVMIFGKESAGASTALDNVMITDEEEIKAT NPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYLQ GPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPAEPPAEFSAT KFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFVDNPGLQL IDPRPLY (14) IDPRPLY:

[0092] The capsid protein of AAV1 VP3 may have the amino acid sequence of SEQ ID NO: 15. MASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWA LPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRD WQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFS DSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQVGRSSFY CLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYL YYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRV SKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPM SGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQS SSTDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSP LMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEI EWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTR Petition 870250105146, dated 11 / 17 / 2025, page 46 / 125 41 / 94 YLTRPL (SEQ ID NO: 15)

[0093] The polynucleotide encoding an AAV1 capsid protein may have the sequence SEQ ID NO: 10 provided below. ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTC TGAGGGCATTCGCGAGTGGTGGGACTTGAAACCTGGAGCCCCGAA GCCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGT GCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAG GGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGA CAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTG CGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAG ATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAA GAAGCGGGTTCTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAG ACGGCTCCTGGAAAGAAACGTCCGGTAGAGCAGTCGCCACAAGAGC CAGACTCCTCCTCGGGCATCGGCAAGACAGGCCAGCAGCCCGCTA AAAAGAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTCCCC GATCCACAACCTCTCGGAGAACCTCCAGCAACCCCCGCTGCTGTGG GACCTACTACAATGGCTTCAGGCGGTGGCGCACCAATGGCAGACAA TAACGAAGGCGCCGACGGAGTGGGTAATGCCTCAGGAAATTGGCAT TGCGATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCC GCACCTGGGCCTTGCCCACCTACAATAACCACCTCTACAAGCAAATC TCCAGTGCTTCAACGGGGGCCAGCAACGACAACCACTACTTCGGCT ACAGCACCCCCTGGGGGTATTTTGATTTCAACAGATTCCACTGCCAC TTTTCACCACGTGACTGGCAGCGACTCATCAACAACAATTGGGGATT CCGGCCCAAGAGACTCAACTTCAAACTCTTCAACATCCAAGTCAAGGAGGTCACGACGAATGATGGCGTCACAACCATCGCTAATAACCTTACC AGCACGGTTCAAGTCTTCTCGGACTCGGAGTACCAGCTTCCGTACG TCCTCGGCTCTGCGCACCAGGGCTGCCTCCCTCCGTTCCCGGCGG ACGTGTTCATGATTCCGCAATACGGCTACCTGACGCTCAACAATGGC AGCCAAGCCGTGGGACGTTCATCCTTTTACTGCCTGGAATATTTCCC TTCTCAGATGCTGAGAACGGGCAACAACTTTACCTTCAGCTACACCT Petition 870250105146, dated 11 / 17 / 2025, page 47 / 125 42 / 94 TTGAGGAAGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGCCT GGACCGGCTGATGAATCCTCTCATCGACCAATACCTGTATTACCTGA ACAGAACTCAAAATCAGTCCGGAAGTGCCCAAAACAAGGACTTGCT GTTTAGCCGTGGGTCTCCAGCTGGCATGTCTGTTCAGCCCAAAAACT GGCTACCTGGACCCTGTTATCGGCAGCAGCGCGTTTCTAAAACAAA AACAGACAACAACAACAGCAATTTTACCTGGACTGGTGCTTCAAAAT ATAACCTCAATGGGCGTGAATCCATCATCAACCCTGGCACTGCTATG GCCTCACACAAAGACGACGAAGACAAGTTCTTTCCCATGAGCGGTG TCATGATTTTTGGAAAAGAGAGCGCCGGAGCTTCAAACACTGCATTG GACAATGTCATGATTACAGACGAAGAGGAAATTAAAGCCACTAACCC TGTGGCCACCGAAAGATTTGGGACCGTGGCAGTCAATTTCCAGAGC AGCAGCACAGACCCTGCGACCGGAGATGTGCATGCTATGGGAGCAT TACCTGGCATGGTGTGGCAAGATAGAGACGTGTACCTGCAGGGTCC CATTTGGGCCAAAATTCCTCACACAGATGGACACTTTCACCCGTCTC CTCTTATGGGCGGCTTTGGACTCAAGAACCCGCCTCCTCAGATCCT CATCAAAAACACGCCTGTTCCTGCGAATCCTCCGGCGGAGTTTTCAG CTACAAAGTTTGCTTCATTCATCACCCAATACTCCACAGGACAAGTG AGTGTGGAAATTGAATGGGAGCTGCAGAAAGAAAACAGCAAGCGCT GGAATCCCGAAGTGCAGTACACATCCAATTATGCAAAATCTGCCAAC GTTGATTTTACTGTGGACAACAATGGACTTTATACTGAGCCTCGCCC CATTGGCACCCGTTACCTTACCCGTCCCCTGTAA (SEQ ID NO:10)

[0094] The polynucleotide encoding an AAV1 capsid protein may have the sequence of SEQ ID NO: 11 provided below. ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTCTC TGAGGGCATTCGCGAGTGGTGGGACTTGAAACCTGGAGCCCCGAA GCCCAAAGCCAACCAGCAAAAGCAGGACGGCCGGGGTCTGGT GCTTCCTGGCTACAAGTACCGGACCTCCCCAGAGAGGAGGA GGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGA CAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTT CGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAG Petition 870250105146, of 17 / 11 / 2025, p. 48 / 125 43 / 94 ATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAA GAAGCGGGTTCTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAG ACGGCTCCTGGAAAGAAACGTCCGGTAGAGCAGTCGCCACAAGAGC CAGACTCCTCCTCGGGCATCGGCAAGACAGGCCAGCAGCCCGCTA AAAAGAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTCCCC GACCCACAACCTCTCGGAGAACCTCCAGCAACCCCCGCTGCTGTGG GACCTACTACAATGGCTTCAGGCGGTGGCGCACCAATGGCAGACAA TAACGAAGGCGCCGACGGAGTGGGTAATGCCTCAGGAAATTGGCAT TGCGATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCC GCACCTGGGCCTTGCCCACCTACAATAACCACCTCTACAAGCAAATC TCCAGTGCTTCAACGGGGGCCAGCAACGACAACCACTACTTCGGCT ACAGCACCCCCTGGGGGTATTTTGATTTCAACAGATTCCACTGCCAC TTTTCACCACGTGACTGGCAGCGACTCATCAACAACAATTGGGGATT CCGGCCCAAGAGACTCAACTTCAAACTCTTCAACATCCAAGTCAAGG AGGTCACGACGAATGATGGCGTCACAACCATCGCTAATAACCTTACC AGCACGGTTCAAGTCTTCTCGGACTCGGAGTACCAGCTTCCGTACG TCCTCGGCTCTGCGCACCAGGGCTGCCTCCCTCCGTTCCCGGCGG ACGTGTTCATGATTCCGCAATACGGCTACCTGACGCTCAACAATGGC AGCCAAGCCGTGGGACGTTCATCCTTTTACTGCCTGGAATATTTCCC TTCTCAGATGCTGAGAACGGGCAACAACTTTACCTTCAGCTACACCT TTGAGGAAGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGCCTGGACCGGCTGATGAATCCTCTCATCGACCAATACCTGTATTACCTGA ACAGAACTCAAAATCAGTCCGGAAGTGCCCAAAACAAGGACTTGCT GTTTAGCCGTGGGTCTCCAGCTGGCATGTCTGTTCAGCCCAAAAACT GGCTACCTGGACCCTGTTATCGGCAGCAGCGCGTTTCTAAAACAAA AACAGACAACAACAACAGCAATTTTACCTGGACTGGTGCTTCAAAAT ATAACCTCAATGGGCGTGAATCCATCATCAACCCTGGCACTGCTATG GCCTCACACAAAGACGACGAAGACAAGTTCTTTCCCATGAGCGGTG TCATGATTTTTGGAAAAGAGAGCGCCGGAGCTTCAAACACTGCATTG GACAATGTCATGATTACAGACGAAGAGGAAATTAAAGCCACTAACCC Petição 870250105146, de 17 / 11 / 2025, pág. 49 / 125 44 / 94 TGTGGCCACCGAAAGATTTGGGACCGTGGCAGTCAATTTCCAGAGC AGCAGCACAGACCCTGCGACCGGAGATGTGCATGCTATGGGAGCAT TACCTGGCATGGTGTGGCAAGATAGAGACGTGTACCTGCAGGGTCC CATTTGGGCCAAAATTCCTCACACAGATGGACACTTTCACCCGTCTC CTCTTATGGGCGGCTTTGGACTCAAGAACCCGCCTCCTCAGATCCT CATCAAAAACACGCCTGTTCCTGCGAATCCTCCGGCGGAGTTTTCAG CTACAAAGTTTGCTTCATTCATCACCCAATACTCCACAGGACAAGTG AGTGTGGAAATTGAATGGGAGCTGCAGAAAGAAAACAGCAAGCGCT GGAATCCCGAAGTGCAGTACACATCCAATTATGCAAAATCTGCCAAC GTTGATTTTACTGTGGACAACAATGGACTTTATACTGAGCCTCGCCC CATTGGCACCCGTTACCTTACCCGTCCCCTG (SEQ ID NO: 11)

[0095] O plasmídeo de rep-cap modificado pode conter, em ordem de 5' to 3', a polynucleotide encoding an AAV rep protein (e.g., an AAV2 rep protein, such as the polynucleotide at SEQ ID NO: 3), a tetracycline-inducible expression system (e.g., a Tet-Off system containing a tTA encoded by SEQ ID NO: 6 or a Tet-On system containing an rtTA encoded by SEQ ID NO: 21 and a series of tetO sequences from SEQ ID NO: 4, such as the series of tetO sequences contained in SEQ ID NO: 7), an AAV promoter (e.g., an AAV p41 promoter from SEQ ID NO: 8), and a polynucleotide encoding an AAV1 capsid protein (e.g., an AAV1 VP1, VP2, and VP3 protein, such as a polynucleotide at SEQ ID NO: 10 or SEQ ID NO: 11). The modified rep-cap plasmid may include the sequence SEQ ID NO: 12 provided below. AAACCTGCAGGACTAGAGGTCCTGTATTAGAGGTCACGTGAGTGTTT TGCGACATTTTGCGACACCATGTGGTCACGCTGGGTATTTAAGCCC GAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGC GCAGCCGCCAAGCCGAATTCTGCAGATATCCATCACACTGGCGGCC GCTCGACTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTT CCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAG Petição 870250105146, de 17 / 11 / 2025, pág. 50 / 125 45 / 94 CTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATA CGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGA GCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCG GGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGG GGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCAC TGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGC TCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAAC GCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACC GTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCC TGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAAC CCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCC TCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTC CGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGC TGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCT GTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGG TAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCAC TGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGG CGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACT AGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTT CGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCT GGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTA TCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTA AATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTAGAAAA ACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTAT CAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACT CACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGC GATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTC AAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAAT Petição 870250105146, de 17 / 11 / 2025, pág. 51 / 125 46 / 94 CCGGTGAGAATGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCA ACAGGCCACCATTAGGCTCGTCATCAAAATCACTCGCATCAACCAA ACCGTTATTCATTCGTGATTGCGCCTGAGCGACGAAATACGCGAT CGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCG CAGGAACACTGCCAGCGCATCAACAACATATTTTCACCTGAATCCAGTGGTG AGTAACCATGCATCATCACAGGAGTACGGATAAAATGCTTGATGGTCGG AAGAGGCATAAATTCCGTCAGCCAGTTAGTCTGACCATCTCTG TAACATATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCT GGCGCATCGGGCTTCCCATACAAGCGATATAGATTGTCGCACCTGATT GCCCGACATATTATCGCGAGCCCATTATACCCATATAAATCAGCATCC ATGTTGGAATTTAATCGCGGGCCTCGACGTTTCCCGTTGAATATGGCT CATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGT CTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATA GGGGTTCCGCGCACATTTCCCGAAAAGTGCCACCTAAATTGTAAG CGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCA TTTTTTAACCAATAGGCCGAAATCGGCAAATCCCTATAAATCAAAA GAATAGACCGAGATAGGGTTGAGTTGTTCCAGTTTGGAACAAGA GTCCACTATTAAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAAC CGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAA AGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGT GGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGG CGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCG CCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAG GCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCT ATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGT TGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGG CCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTAC CGGGCCCCCCCTCGATCGAGGTCGACGGTATCGGGGGAGCTCGCA Petição 870250105146, de 17 / 11 / 2025, pág. 52 / 125 47 / 94 GGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGCCGCCATGC CGGGGTTTACGAGATTGTGATTAAGGTCCCCAAGCGACCTTGACGA GCATCTGCCCGGCATTTCTGACAGCTTTTGTGAACTGGGTGGCCGAG AAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGAT TTGACGGAATGGGCGCCGTGTGAGTAAGGCCCCGGAGAGCTCTTTTC TTTGTGCAATTTGAGAAGGGAGAGAGAGAGACTACTTCCACATGCACGTGCT CGTGGAAAACCACCGGGGTGAAATCCATGGTTTTGGGACGTTTCCT AGTCAGATTCGCGGAAAAACTGATTCAGAGAATTTACCGCGGGATCGA GCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGC GCCGGAGGCGGGAACAAGGTGGTGGATGAGTGCTACATCCCCAATT ACTTGCTCCCCAAAAACCAGCCTGAGCTCCATGTGGGCGGTGGACTAA TATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAAC GGTTGGTGGCGCAGCATCTGACGCACGTGTCCAGACGCAGGAGC AGAACAAAGGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGA TCAAAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTGGTCGTGG ACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGACCAGG CCTCATACATCTCCTTCAATGCGGCTCCAACTCGCGGTCCCAAATC AAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGACTAAAA CCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTT CCAGCAATCGGATTTATAAAATTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTTCG GCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAA GACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGG TGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGA CAAGATGGTGATCTGGTGGGAGGAGGGGAAGATGACCGCCAAGGT CGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGCGCGT GGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTG ATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACGGGAACT CAACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAA Petição 870250105146, de 17 / 11 / 2025, pág. 53 / 125 48 / 94 ATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCA AGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGT TGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAA AGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGC GCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGAT CAACTACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGC ATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGACTGAATCA GAATTCAAATATCTGCTTCACTCACGGTGTCAAAGACTGTTTAGAGT GCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCG TATCAGAAACTGTGCTACATTCATCACATCATGGGAAAGGTGCCAGA CGCTTGCACTGCTTGCGACCTGGTCAATGTGGACTTGGATGACTGT GTTTCTGAACAATAAATGACTTAAACCAGGTATGAGTCGGCTGGATA AATCTAAAGTCATAAACGGCGCTCTGGAATTACTCAATGAAGTCGGT ATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTTG AGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCT CGATGCCCTGGCCATCGAGATGCTGGACAGGCATCATACCCACTTC TGCCCCCTGGAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACG CCAAGTCATTCCGCTGTGCTCTCCTCTCACATCGCGACGGGGCTAA AGTGCATCTCGGCACCCGCCCAACAGAGAAACAGTACGAAACCCTG GAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTGGAGA ACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAGGAACAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAG ACACCTACCACCGATTCTATGCCCCCACTTCTGAGACAAGCAATTGA GCTGTTCGACCGGCAGGGAGCCGAACCTGCCTTCCTTTTCGGCCTG GAACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGCGAAAGCG GCGGGCCGGCCGACGCCCTTGACGATTTTGACTTAGACATGCTCCC AGCCGATGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGAC GCTCTTGACGATTTTGACCTTGACATGCTCCCCGGGTAAATGCATGA ATTCGATCTAGAGGGCCCTATTCTATAGTGTCACCTAAATGCTAGAG CTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTT Petition 870250105146, dated 11 / 17 / 2025, page 54 / 125 49 / 94 GTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTC CCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGA GTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCA AGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGG TGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGAA TCAAGCTATCAAGTGCCACCTGACGTCTCCCTATCAGTGATAGAGAA GTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGAAGGTACGTCT AGAACGTCTCCCTATCAGTGATAGAGAAGTCGACACGTCTCGAGCT CCCTATCAGTGATAGAGAAGGTACGTCTAGAACGTCTCCCTATCAGT GATAGAGAAGTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGAA GGTACGTCTAGAACGTCTCCCTATCAGTGATAGAGAAGTCGACACGT CTCGAGCTCCCTATCAGTGATAGAGAAGGTACCCCCTATATAAGCAG AGAGATCTGTTCAAATTTGAACTGACTAAGCGGCTCCCGCCAGATTT TGGCAAGATTACTAAGCAGGAAGTCAAGGACTTTTTTGCTTGGGCAA AGGTCAATCAGGTGCCGGTGACTCACGAGTTTAAAGTTCCCAGGGA ATTGGCGGGAACTAAAGGGGCGGAGAAATCTCTAAAACGCCCACTG GGTGACGTCACCAATACTAGCTATAAAAGTCTGGAGAAGCGGGCCA GGCTCTCATTTGTTCCCGAGACGCCTCGCAGTTCAGACGTGACTGTT GATCCCGCTCCTCTGCGACCGCTAGCTTCGATCAACTACGCGGACA GGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTG TTTCCCTGCAGACAATGCGAGAGACTGAATCAGAATTCAAATATCTGCTTCACTCACGGTGTCAAAGACTGTTTAGAGTGCTTTCCCGTGTCAG AATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGC TACATTCATCACATCATGGGAAAGGTGCCAGACGCTTGCACTGCTTG CGACCTGGTCAATGTGGACTTGGATGACTGTGTTTCTGAACAATAAA TGACTTAAACCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCT CGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACTTGAAA CCTGGAGCCCCGAAGCCCAAAGCCAACCAGCAAAAGCAGGACGAC GGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCA ACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCG Petição 870250105146, de 17 / 11 / 2025, pág. 55 / 125 50 / 94 GCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGT GACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGG AGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGC AGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTCTGGTT GAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAACGTCCGGTAGAG CAGTCGCCACAAGAGCCAGACTCCTCCTCGGGCATCGGCAAGACAG GCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAGACTGGCGA CTCAGAGTCAGTCCCCGATCCACAACCTCTCGGAGAACCTCCAGCA ACCCCCGCTGCTGTGGGACCTACTACAATGGCTTCAGGCGGTGGCG CACCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAATGC CTCAGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTC ATCACCACCAGCACCCGCACCTGGGCCTTGCCCACCTACAATAACC ACCTCTACAAGCAAATCTCCAGTGCTTCAACGGGGGCCAGCAACGA CAACCACTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGATTTCA ACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAGCGACTCATC AACAACAATTGGGGATTCCGGCCCAAGAGACTCAACTTCAAACTCTT CAACATCCAAGTCAAGGAGGTCACGACGAATGATGGCGTCACAACC ATCGCTAATAACCTTACCAGCACGGTTCAAGTCTTCTCGGACTCGGA GTACCAGCTTCCGTACGTCCTCGGCTCTGCGCACCAGGGCTGCCTC CCTCCGTTCCCGGCGGACGTGTTCATGATTCCGCAATACGGCTACC TGACGCTCAACAATGGCAGCCAAGCCGTGGGACGTTCATCCTTTTACTGCCTGGAATATTTCCCTTCTCAGATGCTGAGAACGGGCAACAACT TTACCTTCAGCTACACCTTTGAGGAAGTGCCTTTCCACAGCAGCTAC GCGCACAGCCAGAGCCTGGACCGGCTGATGAATCCTCTCATCGACC AATACCTGTATTACCTGAACAGAACTCAAAATCAGTCCGGAAGTGCC CAAAACAAGGACTTGCTGTTTAGCCGTGGGTCTCCAGCTGGCATGT CTGTTCAGCCCAAAAACTGGCTACCTGGACCCTGTTATCGGCAGCA GCGCGTTTCTAAAACAAAAACAGACAACAACAACAGCAATTTTACCT GGACTGGTGCTTCAAAATATAACCTCAATGGGCGTGAATCCATCATC AACCCTGGCACTGCTATGGCCTCACACAAAGACGACGAAGACAAGT Petição 870250105146, de 17 / 11 / 2025, pág. 56 / 125 51 / 94 TCTTTCCCATGAGCGGTGTCATGATTTTTGGAAAAGAGAGCGCCGGA GCTTCAAACACTGCATTGGACAATGTCATGATTACAGACGAAGAGGA AATTAAAGCCACTAACCCTGTGGCCACCGAAAGATTTGGGACCGTG GCAGTCAATTTCCAGCAGCAGCAGACCATGGACCATGGACCATGGCCA TGCATGCTATGGGAGCATTACCTGGCATGGTGTGGCAAGATAGAGA CGTGTACCTGCAGGGTCCCATTTGGGCCAAAATTCCTCACACACAGAT GGACACTTTTCACCCGTCTCCTCTTATGGGCGGCTTTGGACTCAAGAA CCCGCCTCCTCAGATCCTCATCAAAAACCTGACTGAGGCTCCATCC CTCCGGCGGAGTTTTCAGCTACAAGTTTGCTTCATTCATCACCCAA TACTCCACAGGACAAGTGAGTGTGGAAATTGAATGGGAGCTGCAGA AAGAAAACAGCAAGCGCTGGAATCCCGAAGTGCAGTACACATCCAA TTATGCAAAATCTGCCAACGTTGATTTTACTGGACTGGACTGGACT TTATACTGAGCCTCGCCCCATTGGCACCCGTTACCTTACCCGTCCCC TGTAATAATTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGA ACTTTGGTCTCTGCGAAGGGCGAATTCGTTT (SEQ ID NO: 12)

[0096] The modified rep-cap plasmid may include the sequence given SEQ ID NO: 13 provided below. AAACCTGCAGGACTAGAGGTCCTGTATTAGAGGTCACGTGAGTGTTT TGCGACATTTTGCGACACCATGTGGTCACGCTGGGTATTTAAGCCC GAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGC GCAGCCGCCAAGCCGAATTCTGCAGATATCCATCACACTGGCGGCC GCTCGACTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTT CCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAG CTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATA CGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGA GCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCG GGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGG GGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCAC TGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGC TCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAAC Petição 870250105146, de 17 / 11 / 2025, pág. 57 / 125 52 / 94 GCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACC GTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCC TGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAAC CCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCC TCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTC CGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGC TGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCT GTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGG TAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCAC TGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGG CGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACT AGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTT CGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCT GGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAA AAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTA TCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTA AATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTAGAAAA ACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTAT CAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACT CACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTC AAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAAT CCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCA ACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAA ACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGAT CGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCG CAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGAT ATTCTTCTAATACCTGGAATGCTGTTTTTCCGGGGATCGCAGTGGTG AGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGG Petição 870250105146, de 17 / 11 / 2025, pág. 58 / 125 53 / 94 AAGAGGCATAAATTCCGTCAGCCAGTTAGTCTGACCATCTCATCTG TAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCT GGCGCATCGGGCTTCCCATACAAGCGATAGATTGTCGCACCTGATT GCCCGACATTATCCGGAGCCCATTTATACCCATATAAATCAGCATCC ATGTTGGAATTTAATCGCGGCCCTCGAGTTTCCCGTTGAATATGGCT CATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGGTTGT CTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATA GGGGTTCCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAG CGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTTAAAATCAGCTCA TTTTTTAACCAATAGGCCGCAAAATCCCCTATAAATCAAAA GAATAGACCGAGATAGGGTTGAGTTGTTTCCAGTTTTGGAACAAGA GTCCACTATTAAAGACGTGGACTCCAACGTCAAAGGGCGAAAAAC CGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCA AGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAA AGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGT GGCGAGAAAGGAAGGGAAAAGCGAAAGGAGGCGGCTAGGG CGCTGGCAAGTGTAGCGGTCACGCTGCGTAACCACCACCCG CCGCGCTTAATGCGCGCCTACAGGGCGCGTCCCATTCGCCATTCAG GCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCT ATTAGCGCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGT TGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTAC CGGGCCCCCCCTCGATCGAGGTCGACGGTATCGGGGGAGCTCGCA GGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGCCGCCATGC CGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGA GCATCTGCCCGGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAG AAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGAT TGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTT TCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGGAGGCTCTTTTC TTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCACATGCACGTGCT Petição 870250105146, de 17 / 11 / 2025, pág. 59 / 125 54 / 94 CGTGGAAACCACCGGGGTGAAATCCATGGTTTTGGGACGTTTCCTG AGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGA GCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGC GCCGGAGGCGGGAACAAGGTGGTGGATGAGTGCTACATCCCCAATT ACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGGCGTGGACTAA TATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAAC GGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGC AGAACAAAGAGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGA TCAAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTGGCTCGTGG ACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGG CCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATC AAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGACTAAAA CCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTT CCAGCAATCGGATTTATAAAATTTTGGAACTAAACGGGTACGATCCC CAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTTCG GCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAA GACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGG TGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGA CAAGATGGTGATCTGGTGGGAGGAGGGGAAGATGACCGCCAAGGT CGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGCGCGT GGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACGGGAACT CAACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAA ATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCA AGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGT TGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAA AGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGC GCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGAT CAACTACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGC ATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGACTGAATCA Petição 870250105146, de 17 / 11 / 2025, pág. 60 / 125 55 / 94 GAATTCAAATATCTGCTTCACTCACGGTGTCAAAGACTGTTTAGAGT GCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCG TATCAGAAACTGTGCTACATTCATCACATCATGGGAAAGGTGCCAGA CGCTTGCACTGCTTGCGACCTGGTCAATGTGGACTTGGATGACTGT GTTTCTGAACAATAAATGACTTAAACCAGGTATGAGTCGGCTGGATA AATCTAAAGTCATAAACGGCGCTCTGGAATTACTCAATGAAGTCGGT ATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTTG AGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCT CGATGCCCTGGCCATCGAGATGCTGGACAGGCATCATACCCACTTC TGCCCCCTGGAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACG CCAAGTCATTCCGCTGTGCTCTCCTCTCACATCGCGACGGGGCTAA AGTGCATCTCGGCACCCGCCCAACAGAGAAACAGTACGAAACCCTG GAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTGGAGA ACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTG CGTATTGGAGGAACAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAG ACACCTACCACCGATTCTATGCCCCCACTTCTGAGACAAGCAATTGA GCTGTTCGACCGGCAGGGAGCCGAACCTGCCTTCCTTTTCGGCCTG GAACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGCGAAAGCG GCGGGCCGGCCGACGCCCTTGACGATTTTGACTTAGACATGCTCCC AGCCGATGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGAC GCTCTTGACGATTTTGACCTTGACATGCTCCCCGGGTAAATGCATGAATTCGATCTAGAGGGCCCTATTCTATAGTGTCACCTAAATGCTAGAG CTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTT GTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTC CCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGA GTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCA AGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGG TGGGCCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGAA TCAAGCTATCAAGTGCCACCTGACGTCTCCCTATCAGTGATAGAGAA GTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGAAGGTACGTCT Petition 870250105146, dated 11 / 17 / 2025, page 61 / 125 56 / 94 AGAACGTCTCCCTATCAGTGATAGAGAAGTCGACACGTCTCGAGCT CCCTATCAGTGATAGAGAAGGTACGTCTAGAACGTCTCCCTATCAGT GATAGAGAAGTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGAA GGTACGTCTAGAACGTCTCCCTATCAGTGATAGAGAAGTCGACACGT CTCGAGCTCCCTATCAGTGATAGAGAAGGTACCCCCTATATAAGCAG AGAGATCTGTTCAAATTTGAACTGACTAAGCGGCTCCCGCCAGATTT TGGCAAGATTACTAAGCAGGAAGTCAAGGACTTTTTTGCTTGGGCAA AGGTCAATCAGGTGCCGGTGACTCACGAGTTTAAAGTTCCCAGGGA ATTGGCGGGAACTAAAGGGGCGGAGAAATCTCTAAAACGCCCACTG GGTGACGTCACCAATACTAGCTATAAAAGTCTGGAGAAGCGGGCCA GGCTCTCATTTGTTCCCGAGACGCCTCGCAGTTCAGACGTGACTGTT GATCCCGCTCCTCTGCGACCGCTAGCTTCGATCAACTACGCGGACA GGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTG TTTCCCTGCAGACAATGCGAGAGACTGAATCAGAATTCAAATATCTG CTTCACTCACGGTGTCAAAGACTGTTTAGAGTGCTTTCCCGTGTCAG AATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGC TACATTCATCACATCATGGGAAAGGTGCCAGACGCTTGCACTGCTTG CGACCTGGTCAATGTGGACTTGGATGACTGTGTTTCTGAACAATAAA TGACTTAAACCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCT CGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACTTGAAA CCTGGAGCCCCGAAGCCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTCA ACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCG GCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGT AGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGC AGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTCTGGTT GAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAACGTCCGGTAGAG CAGTCGCCACAAGAGCCAGACTCCTCCTCGGCCAGCAAGGCAG GCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAGACTGGCGA Petition 870250105146, of 17 / 11 / 2025, p. 62 / 125 57 / 94 CTCAGAGTCAGTCCCCGACCCACAACCTCTCGGAGAACCTCCAGCA ACCCCCGCTGCTGTGGGACCTACTACAATGGCTTCAGGCGGTGGCG CACCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAATGC CTCAGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTC ATCACCACCAGCACCCGCACCTGGGCCTTGCCCACCTACAATAACC ACCTCTACAAGCAAATCTCCAGTGCTTCAACGGGGGCCAGCAACGA CAACCACTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGATTTCA ACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAGCGACTCATC AACAACAATTGGGGATTCCGGCCCAAGAGACTCAACTTCAAACTCTT CAACATCCAAGTCAAGGAGGTCACGACGAATGATGGCGTCACAACC ATCGCTAATAACCTTACCAGCACGGTTCAAGTCTTCTCGGACTCGGA GTACCAGCTTCCGTACGTCCTCGGCTCTGCGCACCAGGGCTGCCTC CCTCCGTTCCCGGCGGACGTGTTCATGATTCCGCAATACGGCTACC TGACGCTCAACAATGGCAGCCAAGCCGTGGGACGTTCATCCTTTTA CTGCCTGGAATATTTCCCTTCTCAGATGCTGAGAACGGGCAACAACT TTACCTTCAGCTACACCTTTGAGGAAGTGCCTTTCCACAGCAGCTAC GCGCACAGCCAGAGCCTGGACCGGCTGATGAATCCTCTCATCGACC AATACCTGTATTACCTGAACAGAACTCAAAATCAGTCCGGAAGTGCC CAAAACAAGGACTTGCTGTTTAGCCGTGGGTCTCCAGCTGGCATGT CTGTTCAGCCCAAAAACTGGCTACCTGGACCCTGTTATCGGCAGCA GCGCGTTTCTAAAACAAAAACAGACAACAACAACAGCAATTTTACCTGGACTGGTGCTTCAAAATATAACCTCAATGGGCGTGAATCCATCATC AACCCTGGCACTGCTATGGCCTCACAAAGACGACGAAGACAAGT TCTTTCCCATGAGCGGTGTCATGATTTGGAAAAGAGAGCGCCGGA GCTTCAAACACTGCATTGGACAATGTCATGAGAGGAGAGAGGAAGGAAG AATTAAAGCCACTAACCCTGTGGCCACCGAAAGATTTGGGACCGTG GCAGTCAATTTCCAGAGCAGCAGCACAGACCCTGCGACCGGAGATG TGCATGCTATGGGAGCATTACCTGGCATGGTGTGGCAAGATAGAGA CGTGTACCTGCAGGGTCCCATTTGGCCAAAATCCATCCATTCATTT GGACACTTTCACCCGTCTCCTCTTATGGGCGGCTTTGGACTCAAGAA Petition 870250105146, of 17 / 11 / 2025, p. 63 / 125 58 / 94 CCCGCCTCCTCAGATCCTCATCAAACACGCCTGTTCCTGCGAATC CTCCGGCGGAGTTTTCAGCTACAAGTTTGCTTCATTCATCACCCAA TACTCCACAGGACAAGTGAGTGTGGAAATTGAATGGGAGCTGCAGA AAGAAAACAGCAAGCGCTGGAATCCCGAAGTCCAGTCAGTCAATCAA TTATGCAAAATCTGCCAACGTTGATTTTACTGTGGACAACAATGGACT TTATACTGAGCCTCGCCCCATTGGCACCCGTTACCTTACCCGTCCCC TGTAATTGCTTGTCAATAAACCGTTTAATTCGTTTCAGTTGAACT TTGGTCTCTGCGAAGGGCGAATTCGTTT (SEQ 13) ID:

[0097] The modified rep-cap plasmid can include the sequence given SEQ ID NO: 22 fornecida abaixo. AAACCTGCAGGACTAGAGGTCCTGTATTAGAGGTCACGTGAGTGTTT TGCGACATTTTGCGACACCATGTGGTCACGCTGGGTATTTAAGCCC GAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGC GCAGCCGCCAAGCCGAATTCTGCAGATATCCATCACACTGGCGGCC GCTCGACTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTT CCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAG CTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATA CGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGA GCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCG GGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGG GGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCAC TGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGC TCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAAC GCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACC GTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCC TGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAAC CCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCC TCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTC CGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGC TGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCT Petition 870250105146, dated 11 / 17 / 2025, p. 64 / 125 59 / 94 GTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGG TAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCAC TGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGG CGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACT AGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTT CGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCT GGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAA AAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTA TCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTA AATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTAGAAAA ACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTAT CAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACT CACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGC GATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTC AAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAAT CCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCA ACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAA ACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGAT CGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGAT ATTCTTCTAATACCTGGAATGCTGTTTTTCCGGGGATCGCAGTGGTG AGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGG AAGAGGCATAAATTCCGTCAGCCAGTTTAGTCCATCCATCCATGCTG TAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAAACAACTCT GGCGCATCGGGCTTCCCATACAAGCGATAGATTGTCGCACCTGATT GCCCGACATTATCGCGAGCCCATTTATACCCCATATAAATCAGCATCC ATGTTGGAATTTAATCGCGGCCTCGACGTTTCCCGTTGAATGGGCTGATT CATACTCTTCCTTTTTCAATATTTGAAGCATTTATCAGGGTTATTGT CTCATGAGCGGATACATATTTGAATGTTTGAAAAAATAAACAAATA Petition 870250105146, of 17 / 11 / 2025, p. 65 / 125 60 / 94 GGGGTTCCGCGCACATTCCCCGAAAAGTGCCACCTAAATTGTAAG CGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCA TTTTTTAACCAATAGGCCGAAATCGGCAAATCCCTATAAATCAAAA GAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGA GTCCACTATTAAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAAC CGTCTATCAGGGGCGATGGCCCACTACGTGAACCATCACCTAATCA AGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAA AGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGT GGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGCGGCTAGGG CGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACCCG CCGCGCTTAATGCGCGCCTACAGGGCGCGTCCCATTCGCCATTCAG GCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCT ATTACGCCAGCTGGCGAAAGGGGATGTGCTGCAAGGCGATTAAGT TGGGTAACGCCAGGGTTTTCCCAGTCACGACGGTTGTAAAACGACGG CCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTAC CGGGCCCCCCTCGATCGAGGTCGACGGTTATCGGGGGAGCTCGCA GGGTCTCCATTTTGAAGCGGGAGTTTGAACGCGCGCAGCCCGCCATGC CGGGGTTTACGAGATTGTGATTAAGGTCCCCAGCCGACCCTTGACGA GCATCTGCCCGGCATTTCTGACAGCCTTTGTGAACTGGGTGGCCGAG AAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGAT TGAGCAGCACCCCTGACCGTGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGGAGGCTCTTTTC TTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCACATGCACGTGCT CGTGGAAACCACCGGGGTGAAATCCATGGTTTTGGGACGTTTCCTG AGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGA GCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGC GCCGGAGGCGGGAACAAGGTGGTGGATGAGTGCTACATCCCCAATT ACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGGCGTGGACTAA TATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAAC GGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGC Petição 870250105146, de 17 / 11 / 2025, pág. 66 / 125 61 / 94 AGAACAAAGAGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGA TCAAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTGGCTCGTGG ACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGG CCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATC AAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGACTAAAA CCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTT CCAGCAATCGGATTTATAAAATTTTGGAACTAAACGGGTACGATCCC CAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTTCG GCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAA GACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGG TGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGA CAAGATGGTGATCTGGTGGGAGGAGGGGAAGATGACCGCCAAGGT CGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGCGCGT GGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTG ATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACGGGAACT CAACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAA ATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCA AGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGT TGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAA AGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGC GCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGC ATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGACTGAATCA GAATTCAAAATATCTGCTTCACTCACGGTGTCAAAGACTGTTTAGAGT GCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAAGGCG TATCAGAAACTGTGCTACATTCATCACATCATGGGAAAGGTGCCAGA CGCTTGCACTGCTTGCGACCTGGTCAATGTGGACTTGGATGACTGT GTTTCTGAACAATAAATGACTTAAACCAGGTATGAGTCGGCTGGATA AATCTAAAGTCATAAACTCTGCTCTGGAATTACTCAATGGAGTCGGT ATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTTG Petição 870250105146, the 17 / 11 / 2025, pág. 67 / 125 62 / 94 AGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCT CGATGCCCTGCCAATCGAGATGCTGGACAGGCATCATACCCACTCC TGCCCCCTGGAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACG CCAAGTCATACCGCTGTGCTCTCCTCTCACATCGCGACGGGGCTAA AGTGCATCTCGGCACCCGCCCAACAGAGAAACAGTACGAAACCCTG GAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTGGAGA ACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTG CGTATTGGAGGAACAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAG ACACCTACCACCGATTCTATGCCCCCACTTCTGAAACAAGCAATTGA GCTGTTCGACCGGCAGGGAGCCGAACCTGCCTTCCTTTTCGGCCTG GAACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGCGAAAGCG GCGGGCCGACCGACGCCCTTGACGATTTTGACTTAGACATGCTCCC AGCCGATGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGAC GCTCTTGACGATTTTGACCTTGACATGCTCCCCGGGTAAATGCATGA ATTCGATCTAGAGGGCCCTATTCTATAGTGTCACCTAAATGCTAGAG CTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTT GTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTC CCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGA GTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCA AGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGG TGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGAATCAAGCTATCAAGTGCCACCTGACGTCTCCCTATCAGTGATAGAGAA GTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGAAGGTACGTCT AGAACGTCTCCCTATCAGTGATAGAGAAGTCGACACGTCTCGAGCT CCCTATCAGTGATAGAGAAGGTACGTCTAGAACGTCTCCCTATCAGT GATAGAGAAGTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGAA GGTACGTCTAGAACGTCTCCCTATCAGTGATAGAGAAGTCGACACGT CTCGAGCTCCCTATCAGTGATAGAGAAGGTACCCCCTATATAAGCAG AGAGATCTGTTCAAATTTGAACTGACTAAGCGGCTCCCGCCAGATTT TGGCAAGATTACTAAGCAGGAAGTCAAGGACTTTTTTGCTTGGGCAA Petição 870250105146, de 17 / 11 / 2025, pág. 68 / 125 63 / 94 AGGTCAATCAGGTGCCGGTGACTCACGAGTTTAAAGTTCCCAGGGA ATTGGCGGGAACTAAAGGGGCGGAGAAATCTCTAAAACGCCCACTG GGTGACGTCACCAATACTAGCTATAAAAGTCTGGAGAAGCGGGCCA GGCTCTCATTTGTTCCCGAGACGCCTCGCAGTTCAGACGTGACTGTT GATCCCGCTCCTCTGCGACCGCTAGCTTCGATCAACTACGCGGACA GGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTG TTTCCCTGCAGACAATGCGAGAGACTGAATCAGAATTCAAATATCTG CTTCACTCACGGTGTCAAAGACTGTTTAGAGTGCTTTCCCGTGTCAG AATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGC TACATTCATCACATCATGGGAAAGGTGCCAGACGCTTGCACTGCTTG CGACCTGGTCAATGTGGACTTGGATGACTGTGTTTCTGAACAATAAA TGACTTAAACCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCT CGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACTTGAAA CCTGGAGCCCCGAAGCCCAAAGCCAACCAGCAAAAGCAGGACGAC GGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCA ACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCG GCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGT GACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGG AGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGC AGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTCTGGTT GAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAACGTCCGGTAGAGCAGTCGCCACAAGAGCCAGACTCCTCCTCGGGCATCGGCAAGACAG GCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAGACTGGCGA CTCAGAGTCAGTCCCCGATCCACAACCTCTCGGAGAACCTCCAGCA ACCCCCGCTGCTGTGGGACCTACTACAATGGCTTCAGGCGGTGGCG CACCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAATGC CTCAGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTC ATCACCACCAGCACCCGCACCTGGGCCTTGCCCACCTACAATAACC ACCTCTACAAGCAAATCTCCAGTGCTTCAACGGGGGCCAGCAACGA CAACCACTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGATTTCA Petition 870250105146, dated 11 / 17 / 2025, page 69 / 125 64 / 94 ACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAGCGACTCATC AACAACAATTGG GGATTCCGGCCCAAGACTCAACTTCAAACTCTT CAACATCCAAGTCAAGGAGGTCACGACGAATGGCGTCACAACC ATCGCTAATAACCTTACCAGCACGGTTCAAGTCGACTCGTTCGGA GTACCAGCTTCCGTACGTCCTCGGCTCTGCGCACCAGGGCTGCCTC CCTCCGTTCCCGGCGGACGTGTTCATGATTCCGCAATACGGCTACC TGACGCTCAACAATGGCAGCCAAGCCGTGGGACGTTCATCCTTTTA CTGCCTGGAATATTTCCCTCTTCAGATGCTGAACGAACGAACGACTGGGCT TTACCTTCAGCTACACCTTTGAGGAAGTGCCTTTCCACAGCAGCTAC GCGCACAGCCAGAGCCTGGACCGGCTGATGAATCCTCTCATCGACC AATACCTGTATTACCTGAACAGAACTCAAAATCAGTCCGGAAGTGCC CAAAACAAGGACTTGCTGTTTAGCCGTGGGGTCTGCTGGCTGCTGAT CTGTTCAGCCCAAAAACTGGCTACCTGGACCCTGTTATCGGCAGCA GCGCGTTTCTAAAACAAAAACAGACAACAACAAGCAATTTTACCT GGACTGGTGCTTCAAAATAATAACCTCAATGGGCGTGAATCCATCATC AACCCTGGCACTGCTATGGCCTCACACAAAGAAGCAAGGAAGCAAGCATC TCTTTCCCATGAGCGGTGTCATGATTTTTGGAAAAGAGAGCGCCGGA GCTTCAAACACTGCATTGGACAATGTCATGATTACAGACGAAGAGGA AATTAAAGCCACTAACCCTGTGGCCACCGAAAGATTTGGGACCGTG GCAGTCAATTTCCAGCAGCAGCAGACCATGGACCATGGACCATGGCCA TGCATGCTATGGGAGCATTACCTGGCATGGTGTGGCAAGATAGAGACGTGTACCTGCAGGGTCCCATTTGGGCCAAAATTCCTCACACACAGAT GGACACTTTTCACCCGTCTCCTCTTATGGGCGGCTTTGGACTCAAGAA CCCGCCTCCTCAGATCCTCATCAAACGCCTGTTCCTGCGAATC CTCCGGCGGAGTTTTCAGCTCAAAGTTTACTCCCATTCATCA TACTCCACAGGACAAGTGAGTGTGGAAATTGAATGGGAGCTGCAGA AAGAAAACAGCAAGCGCTGGAATCCCGAAGTGCAGTACACATCCAA TTATGCAAAATCTGCCAACGTTGATTTTACTGTGGACAACAATGGACT TTATACTGAGCCTCGCCCCATTT TGTAATATTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGA Petition 870250105146, of 17 / 11 / 2025, p. 70 / 125 65 / 94 ACTTTGGTCTCTGCGAAGGGCGAATTCGTTT (SEQ ID NO: 22). Methods for Releasing Exogenous Polynucleotides to Target Host Cells

[0098] The modified rep-cap plasmids described herein can be introduced into a cell along with an auxiliary plasmid and a transgenic plasmid to produce an rAAV vector (e.g., rAAV1 vector) using methods known in the art. Techniques that can be used to introduce a polynucleotide, such as DNA and RNA (e.g., encoding a rep protein, tetracycline-inducible expression system, and AAV1 capsid protein described herein), into a target host cell (e.g., a mammalian cell) are well known in the art. For example, electroporation can be used to permeabilize mammalian cells by applying an electrostatic potential to the cell of interest. Mammalian cells, like human cells, subjected to an external electric field in this way are subsequently predisposed to the uptake of exogenous nucleic acids (e.g., nucleic acids with the capacity for expression in cells).Electroporation of mammalian cells is described in detail, for example, in Chu et al., Nucleic Acids Research 15:1311 (1987). A similar technique... NUCLEOFECTION™ uses an applied electric field to stimulate the uptake of exogenous polynucleotides into the nucleus of a eukaryotic cell. NUCLEOFECTION™ and protocols useful for performing this technique are described in detail, for example, in Distler et al., Experimental Dermatology 14:315 (2005), as well as in document no. US2010 / 0317114.

[0099] An additional useful technique for transfecting target host cells is the compression methodology. This technique induces rapid mechanical deformation of the cells to stimulate the uptake of exogenous DNA through membrane pores that form in response. Petition 870250105146, dated 11 / 17 / 2025, page 71 / 125 66 / 94 to applied stress. This technology is advantageous because a vector is not required for the release of nucleic acids into a cell, such as a human target cell. Compression is described in detail, for example, in Sharei et al., Journal of Visualized Experiments 81: e50980 (2013).

[00100] Lipofection represents another useful technique for transfecting target host cells. This method involves loading nucleic acids into a liposome, which typically features cationic functional groups such as quaternary and protonated amines, towards the exterior of the liposome. This promotes electrostatic interactions between the liposome and a cell due to the anionic nature of the cell membrane, ultimately resulting in the uptake of exogenous nucleic acids, for example, by direct fusion of the liposome with the cell membrane and by endocytosis of the complex. Lipofection is described in detail, for example, in US patent 7,442,386. Similar techniques that exploit ionic interactions with the cell membrane to induce the uptake of foreign nucleic acids involve contacting a cell with a cationic polymer-nucleic acid complex.Exemplary cationic molecules that associate with polynucleotides in order to transmit a positive charge favorable to interaction with the cell membrane are activated dendrimers (described, for example, in Dennig, Topics in Current Chemistry 228:227 (2003)), polyethyleneimine, and DEAE-dextran, whose use as a transfection agent is described in detail, for example, in Gulick et al., Current Protocols in Molecular Biology 40:1:9.2:9.2.1 (1997).

[00101] Another useful tool for inducing the uptake of exogenous nucleic acids by target host cells is laserfection, also called optical transfection, a technique that involves exposing a cell to electromagnetic radiation of a specific wavelength in order to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. A Petition 870250105146, dated 11 / 17 / 2025, page 72 / 125 67 / 94 bioactivity of this technique is similar to, and in some cases considered superior to, electroporation.

[00102] Impaling is another technique that can be used to deliver genetic material to target host cells. This is based on the use of nanomaterials, such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. The DNA containing the gene, intended for intracellular release, is attached to the surface of the nanostructure. A chip with arrays of these needles is then pressed against cells and tissues. Cells that are impaled by nanostructures can express the released gene(s). An example of this technique is described in Shalek et al., PNAS 107:25, 1870 (2010).

[00103] MAGNETOFECTION™ can also be used to deliver nucleic acids to target host cells. The principle of MAGNETOFECTION™ is to associate nucleic acids with cationic magnetic nanoparticles. The magnetic nanoparticles are made of iron oxide, which is fully biodegradable, and coated with specific proprietary cationic molecules that vary according to the applications. Their association with genetic vectors (e.g., a plasmid or viral vector) is achieved by salt-induced colloidal aggregation and electrostatic interaction. The magnetic particles are then concentrated on the target host cells by the influence of an external magnetic field generated by magnets. This technique is described in detail in Scherer et al., Gene Therapy 9:102 (2002).Magnetic microspheres are another tool that can be used to transfect target cells gently and efficiently, as this methodology uses an applied magnetic field to direct the uptake of nucleic acids. This technology is described in detail, for example, in document no. US2010 / 0227406.

[00104] Another useful tool for inducing acid absorption Petition 870250105146, dated 11 / 17 / 2025, page 73 / 125 68 / 94 exogenous nucleic acids by target host cells is sonoporation, a technique that involves the use of sound (typically ultrasonic frequencies) to modify the permeability of the cell plasma membrane, permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, for example, in Rhodes et al., Methods in Cell Biology 82:309 (2007).

[00105] Microvesicles represent another potential vehicle that can be used to modify the genome of a target host cell according to the methods described in this document. For example, microvesicles that have been induced by co-overexpression of the glycoprotein VSV-G with, for example, a genome-modifying protein such as a nuclease, can be used to efficiently deliver proteins into a cell that subsequently catalyze site-specific cleavage of an endogenous polynucleotide sequence, so as to prepare the cell's genome for the covalent incorporation of a polynucleotide of interest, such as a gene and a regulatory sequence. The use of such vesicles, also known as Gesicles, for the genetic modification of eukaryotic cells is described in detail, for example, in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract].In: Methylation changes in early embryonic genes in cancer [abstract], in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; May 13, 2015, Abstract No. 122. AAV Vectors for Polynucleotide Release

[00106] The modified rep-cap plasmids described herein can be used to incorporate polynucleotides (e.g., polynucleotides contained in a transgenic plasmid) into rAAV vectors (e.g., rAAV1) and / or virions to facilitate their introduction into a cell. According to the present inventions, the rAAV vectors (e.g., rAAV1) produced using the Petition 870250105146, dated 11 / 17 / 2025, page 74 / 125 69 / 94 modified rep-cap plasmids described in this document are recombinant polynucleotide constructs that may include (1) a promoter, (2) a sequence to be expressed (e.g., a polynucleotide encoding a protein or an RNA molecule), and (3) viral sequences that facilitate the integration and expression of the sequence to be expressed. The viral sequences may include AAV sequences that are cis-required for replication and packaging (e.g., functional ITRs) of DNA into a virion.The sequence to be expressed can be a sequence encoding an RNA molecule or protein that is endogenously expressed in a cell that can be transduced with an AAV1 vector (e.g., an RNA molecule or protein that is endogenously expressed in a cell of the central nervous system, heart, skeletal muscle, retinal pigment epithelium, or inner ear) or a sequence encoding an RNA molecule or protein of interest suitable for expression in a cell that can be transduced with an AAV1 vector (e.g., an RNA molecule or protein intended to have a therapeutic effect in a cell of interest or to investigate the biology of the cell of interest). The sequence to be expressed can encode a protein that is endogenously expressed in the human ear.

[00107] The transgenic sequence to be expressed may encode any of the solute carriers of family 26, member 4 (Pendrin), Otoferlin (OTOF), Stereocilin (STRC), BHLH Atonal Transcription Factor 1 (ATOH1), gap junction protein beta 2 (GJB2), and SRYBox 2 (Sox2). The sequence to be expressed may encode Pendrin, for example. The sequence to be expressed may encode GJB2, for example. The sequence to be expressed may encode OTOF, for example. The sequence to be expressed may encode STRC, for example. The sequence to be expressed may encode BHLH Atonal Transcription Factor 1 (ATOH1), for example. The sequence to be expressed may encode SRY Petition 870250105146, dated 11 / 17 / 2025, page 75 / 125 70 / 94 Box 2 (Sox2), for example.

[00108] Other transgenes that may be expressed include, for example, GJB6; WFS1; COCH; EYA4; MYO7A; POU4F3; ACTG1; MYO6; REST; NLRP3; COL11A1; TJP2; TBC1D24; SLC26A4; ELMOD3; EPSN; WHRN; IGF1; IGF1R; MYO15A; TMIE; TMC1; TMC2; TMPRSS3; CDH23; GIPC3; STRC; USH1C; OTOG; TECTA; OTOA; PDCH15; CLDN14; WHRN; ESRRB; MYO3A; HGF, ILDR1, ADCY1; CIB2; MARVELD2; SOX2; SLC26A5; COL4A3; COL4A4; COL4A5; CLPP; PJVK; LRTOMT / COMT2; LOXHD1; TPRM; SYNE4; KCNJ10; PTPRQ; OTOGL; LHFPL5; A1PR2; CABP2; MET; GRXCR2; EPS8; CLIC5; EPS8L2; WBP2; ROR1; CLDN9; USH1G; PKHD1L1; DIAPH3; NDP; USH2A; CLRN1; SANS; HARS1; TRIOBP. A transgene of interest includes any gene that one wishes to express and that can fit into an AAV capsid. More than one transgene of interest is possible if the totality of the genes is of a combined size that can fit into an AAV capsid.

[00109] These rAAV vectors may also contain marker or reporter genes. Useful rAAV vectors may have one or more of the AAV WT genes deleted in whole or in part, but retain functional flanking ITR sequences. AAV ITRs may be of any serotype suitable for a specific application. ITRs may be AAV2 ITRs. Methods for using rAAV vectors are described, for example, in Pupo et al., Mol Ther. 7:3515-3541 (2022) and Wang et al., Nat Rev Drug Discov. 18:358-378 (2019).

[00110] A polynucleotide (for example, a polynucleotide contained in a transgenic plasmid that is cotransfected into a cell with a helper plasmid and a modified rep-cap plasmid described herein) can be incorporated into an rAAV virion to facilitate the introduction of the polynucleotide or vector into a cell. AAV capsid proteins comprise the non-acid outer portion Petition 870250105146, dated 11 / 17 / 2025, page 76 / 125 71 / 94 of the virion's nucleic acid and are encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2, and VP3, which are necessary for virion assembly. The construction of rAAV virions has been described, for example, in documents no. US 5,173,414; US 5,139,941; US ​​5,863,541; US ​​5,869,305; US 6,057,152; and US 6,376,237; as well as in Rabinowitz et al., J. Virol. 76:791 (2002) and Bowles et al., J. Virol. 77:423 (2003). Manufacturing

[00111] The production of rAAV vectors for gene therapy can be carried out in vitro using suitable producer cell lines. Producer cells can be any cell type that possesses the genes necessary to promote AAV genome replication, capsid assembly, and packaging. Exemplary producer cells may include human embryonic kidney cells 293 (HEK-293) or their derivatives, HeLa cells, human amniotic cells, CHO cells, BHK cells, insect cells, and others. One strategy to release all the elements necessary for rAAV production may utilize two plasmids (e.g., a modified rep-cap plasmid described herein and a transgenic plasmid) and a helper virus.This method relies on transfecting producer cells with plasmids containing genes encoding the necessary gene products, as well as infecting the cells with adenovirus to provide auxiliary functions. This system uses plasmids with two different genes. The first can be a proviral plasmid encoding the recombinant DNA to be packaged as rAAV (i.e., the transgenic plasmid). The second can be a plasmid containing polynucleotides encoding the rep and capsid proteins (e.g., a modified rep-cap plasmid described in this document). To introduce these various elements into the cells, the cells can be infected with adenovirus as well as transfected. Petition 870250105146, dated 11 / 17 / 2025, page 77 / 125 72 / 94 with both plasmids. Alternatively, the adenovirus infection step can be replaced by transfection with a helper plasmid containing the VA, E2A, and E4 genes. Another alternative approach may involve incorporating the helper genes (e.g., VA, E2A, and / or E4) into the rep-cap plasmid (e.g., into a modified repcap plasmid described herein), so that only two plasmids are needed to produce an rAAV vector.

[00112] Although adenovirus has been conventionally used as a helper virus for rAAV production, other DNA viruses, such as Herpes simplex virus type 1 (HSV-1), are known to also be usable. The minimum set of HSV-1 genes required for AAV2 replication and packaging has been identified and includes the first genes UL5, UL8, UL52, and UL29. These genes encode components of the HSV-1 core replication machinery, namely helicase, primase, primase accessory proteins, and single-stranded DNA binding protein. This rAAV helper property of HSV-1 was used in the design and construction of a recombinant herpes virus vector capable of delivering helper virus gene products necessary for rAAV production.

[00113] The production of an rAAV1 vector using a modified rep-cap plasmid described herein may result in an increased yield of rAAV1 virus compared to the production of rAAV1 virus using an unmodified rep-cap plasmid containing a polynucleotide encoding an AAV2 rep protein and a polynucleotide encoding an AAV1 capsid protein and without a tetracycline-inducible expression system (e.g., the pAAV-RC1 vector produced by Cell Biolabs, Inc.). For example, the production of an rAAV1 vector using a modified rep-cap plasmid described herein may result in an rAAV1 virus yield that can be increased by at least Petition 870250105146, dated 11 / 17 / 2025, page 78 / 125 73 / 94 2.5 times (e.g., 2.5 times, 2.7 times, 3.0 times, 3.1 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 20 times or more) compared to the production of an rAAV1 vector using an unmodified rep-AAV1 cap plasmid of AAV2 (e.g., pAAV-RC1). The yield can be increased by at least 7 times (e.g., 7 times, 8 times, 9 times, 10 times, 15 times, 20 times or more).The production of an rAAV1 virus using a modified rep-cap plasmid described in this document (e.g., by introducing the modified rep-cap plasmid into a cell with a transgenic plasmid and a helper plasmid) can result in a viral yield of at least 1 x 10¹¹vg / ml (e.g., 1 x 10¹¹vg / ml, 2 x 10¹¹vg / ml, 3 x 10¹¹vg / ml, 4 x 10¹¹vg / ml, 5 x 10¹¹vg / ml, or higher, such as 1 x 10¹¹vg / ml to 5 x 10¹¹vg / ml, 1 x 10¹¹vg / ml to 1 x 10¹²vg / ml, 1 x 10¹¹vg / ml to 5 x 10¹²vg / ml, or 1 x 10¹¹vg / ml to 1 x 1013vg / ml) as determined by ddPCR of clarified lysate or a viral yield of at least 1 x 1013vg / ml (e.g., 1 x 1013vg / ml, 2 x 1013vg / ml, 3 x 1013vg / ml, 4 x 1013vg / ml, 5 x 1013vg / ml, or higher, such as 1 x 1013vg / ml to 5 x 1013vg / ml, 1 x 1013vg / ml to 1 x 1014vg / ml, 1 x 1013vg / ml to 5 x 1014vg / ml, or 1 x 1013vg / ml to 1 x 1015vg / ml) as determined by ddPCR of purified vector.Increased rAAV1 production can be achieved when the tetracycline-inducible expression system is active (e.g., producing rAAV1 using a modified rep-cap plasmid containing a Tet-Off system in the absence of a Tet-Off system inducer, or producing rAAV1 using a modified rep-cap plasmid containing a Tet-On system in the presence of a Tet-On system inducer). Furthermore, increased rAAV1 production can be achieved when the tetracycline-inducible expression system is inactive (e.g., producing rAAV1 using a modified rep-cap plasmid that...). Petition 870250105146, dated 11 / 17 / 2025, page 79 / 125 74 / 94 contains a Tet-Off system in the presence of a Tet-Off system inducer or producing rAAVI with the use of a modified rep-cap plasmid containing a Tet-On system in the absence of a Tet-On system inducer). The tetracycline-inducible expression systems contained in the modified rep-cap plasmids described herein may have leaks, meaning that some rAAV1 production still occurs even when the tetracycline-inducible expression system is inactivated.

[00114] The production of an rAAV1 vector using a modified rep-cap plasmid described herein under conditions in which the tetracycline-inducible expression system is inactive (e.g., producing rAAV1 using a modified rep-cap plasmid containing a Tet-Off system in the presence of a Tet-Off system inducer or producing rAAV1 using a modified rep-cap plasmid containing a Tet-On system in the absence of a Tet-On system inducer) may result in a higher percentage of complete AAV1 vectors compared to the production of complete AAV1 vectors using the same rep-cap plasmid under conditions in which the tetracycline-inducible expression system is active (e.g.,Producing rAAV1 using a modified rep-cap plasmid containing a Tet-Off system in the absence of a Tet-Off system inducer, or producing rAAV1 using a modified rep-cap plasmid containing a Tet-On system in the presence of a Tet-On system inducer. Producing rAAV1 using a rep-cap plasmid described herein under inactivation conditions can increase the production of complete rAAV1 vectors by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more percentage points (e.g., from 4% complete to 6%, 8%, 10%, 12%, 14% complete or more, or from 10% complete to 12%, 14%, 16%, 18%, 20%, 22% complete or more).

[00115] The production of an rAAV1 virus using a plasmid of Petition 870250105146, dated 11 / 17 / 2025, page 80 / 125 The non-induced (i.e., inactive, for example, without the addition of doxycycline or any other inducer) modified rep-cap plasmid described herein (e.g., by introducing the modified rep-cap plasmid described herein into a cell with a transgenic plasmid and a helper plasmid) can result in a viral yield of at least 1 x 10¹¹vg / ml (e.g., 1 x 10¹¹vg / ml, 2 x 10¹¹vg / ml, 3 x 10¹¹vg / ml, 4 x 10¹¹vg / ml, 5 x 10¹¹vg / ml or higher, such as 1 x 10¹¹vg / ml to 5 x 10¹¹vg / ml, 1 x 10¹¹vg / ml to 1 x 10¹²vg / ml, 1 x 10¹¹vg / ml to 5 x 1012vg / ml or 1 x 1011vg / ml to 1 x 1013vg / ml) as determined by ddPCR of clarified lysate or a viral yield of at least 1 x 1013vg / ml (e.g., 1 x 1013vg / ml, 2 x 1013vg / ml, 3 x 1013vg / ml, 4 x 1013vg / ml, 5 x 1013vg / ml, or more, such as 1 x 1013vg / ml to 5 x 1013vg / ml, 1 x 1013vg / ml to 1 x 1014vg / ml, 1 x 1013vg / ml to 5 x 1014vg / ml,or 1 x 10¹³vg / ml to 1 x 10¹⁵vg / ml) as measured by ddPCR in clarified lysate and at least an estimated percentage of complete AAV1 vectors (AAV1 vectors that also contain the transgene) of at least 20% (e.g., 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, or more) as determined using SEC-MALS.

[00116] Although the rep-cap plasmids described in this document may include a polynucleotide encoding an AAV1 capsid protein and are for use in the fabrication of rAAV1 vectors, modified rep-cap plasmids may be used to produce rAAV vectors with a different capsid if the polynucleotide encoding an AAV1 capsid protein is replaced by a polynucleotide encoding a different AAV capsid protein. For example, the polynucleotide encoding an AAV1 capsid protein can be replaced by a polynucleotide encoding an AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S capsid protein. Petition 870250105146, dated 11 / 17 / 2025, page 81 / 125 76 / 94 any of the rep-cap plasmids described in this document. These rep-cap plasmids can be used to produce vectors of AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S using the methods described in this document (e.g., by introducing the rep-cap plasmid into a producer cell along with a transgenic plasmid and a helper plasmid). According to current inventions, vectors of AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S can be produced using these modified rep-cap plasmids, fabricating rAAV vectors under conditions that would cause the tetracycline-inducible expression system to become inactive (for example, producing AAV vectors using a modified rep-cap plasmid containing a Tet-Off system in the presence of a Tet-Off system inducer, or producing AAV vectors using a modified rep-cap plasmid containing a Tet-On system in the absence of a Tet-On system inducer). The fabrication of vectors of AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S with the use of modified rep-cap plasmids containing a polynucleotide encoding a capsid protein of AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S under inactivation conditions may lead to a higher percentage of complete vectors than fabricating rAAV vectors using the same rep-cap plasmids under activation conditions (e.g., conditions under which the tetracycline-inducible expression system would be). Petition 870250105146, dated 11 / 17 / 2025, page 82 / 125 77 / 94 linked, such as the production of AAV vectors using a modified rep-cap plasmid containing a Tet-Off system in the absence of a Tet-Off system inducer, or producing AAV vectors using a modified rep-cap plasmid containing a Tet-On system in the presence of a Tet-On system inducer).

[00117] Recombinant AAV vectors (e.g., AAV1 virus) produced using a modified rep-cap plasmid described herein can be purified by any suitable purification method known in the art, such as chromatography, ultracentrifugation, centrifugation, flocculation, filtration, and / or ultrafiltration / diafiltration. For example, rAAV1 virus can be purified by chromatography. rAAV1 virus can be purified by ultracentrifugation. rAAV1 virus can be purified by centrifugation. rAAV1 virus can be purified by flocculation. rAAV1 virus can be purified by filtration. rAAV1 virus can also be purified by ultrafiltration / diafiltration. Kits

[00118] The modified rep-cap plasmids described herein may be provided in a kit. The kit may include a modified rep-cap plasmid and a helper plasmid, or a modified rep-cap plasmid, a helper plasmid, and a transgenic plasmid. The kit may also include a leaflet instructing a user of the kit, such as a scientist skilled in the art, to perform any of the methods described herein. For example, the kit may include an information leaflet instructing a user of the kit to fabricate an rAAV vector (e.g., an rAAV1 vector) using a modified rep-cap plasmid described herein. Examples

[00119] The following examples are presented in order to provide those of common skill in the technique with a description of how the Petition 870250105146, dated 11 / 17 / 2025, page 83 / 125 78 / 94 The compositions and methods described herein may be used, made and evaluated, and are intended to be purely illustrative of the inventions and are not intended to limit the scope of what the inventors consider to be their inventions. Example 1. Compare AAV1 Production Using Standard and Modified Rep-Cap Plasmids AAV Production

[00120] To enable AAV production, the AAV2 rep / AAV1 cap encoding plasmid, the pHelper plasmid, and the transfer plasmid in a 2:1:2 concentration ratio were combined with FectoVIR (Polypus) in a 1:1 FectoVIR:DNA (deoxyribonucleic acid) ratio and added to VPC (viral production cells, 293 origin) suspension cultures. The AAV2 rep / AAV1 cap encoding plasmid used for AAV production was modified into the rep-cap plasmid P929 (Figure 1) or standard rep-cap plasmid P374 (Figure 2). P929 was developed according to the inventions to provide AAV1 rep / cap on PHP Tet-Off backstrand. B. P929 contains 2 silent single-point mutations in the open CAP reading frame for VP1:2:3 (may not be silent in AAP ORF) compared to P374, considered to have the wild-type AAV1 CAP sequence.

[00121] The transfer plasmids used for AAV production were the P1381 transfer plasmid, which contained a CMV promoter operationally linked to an enhanced GFP-encoding polynucleotide; the P1900 transfer plasmid, which contained a pendrin promoter operationally linked to a pendrin-encoding polynucleotide; and the P707 transfer plasmid, which contained a CMV promoter linked to a GFP-encoding polynucleotide. The same pHelper plasmid (P376) was used with all combinations of rep-cap plasmid and transfer plasmid. The culture medium Petition 870250105146, dated 11 / 17 / 2025, page 84 / 125 79 / 94 cells were harvested after 72 hours of incubation at 37 °C, 8% CO2, and 125 RPM. The cell lysate was then treated with benzonase nuclease to degrade non-viral DNA and RNA (ribonucleic acid) and Triton X-100 (10% w / v Triton X-100) and lysed during incubation at 37 °C, 8% CO2, and 125 RPM. The cell lysates were then filtered through a 0.22 μm filter before purification.

[00122] AAV particles were enriched from cell culture lysate by gravity affinity chromatography using AAVX resin and subsequently buffer-exchanged using centrifugal filtration units. The AAV was formulated in 10 mM sodium phosphate, 180 mM NaCl, 5% (w / v) sucrose, and 0.001% (w / v) poloxamer 188, pH 7.3, and stored at -80 °C. The resulting AAV and its transfer plasmid sources are indicated in Table 2. Table 2. AAV particle content Sample Rep / Cap pHelper GOI (encoded protein) AAV1439 P929 P376 P1381 (GFP) AAV1441 P929 P376 P1900 (pendrin) AAV1440 P929 P376 P707 (GFP) AAV1442 P374 P376 P1381(GFP) AAV1444 P374 P376 P1900 (pendrin) AAV1443 P374 P376 P707 (GFP) Vector Genome Title by Droplet Digital Polymerase Chain Reaction

[00123] Digital droplet polymerase chain reaction (ddPCR) was used to quantify the total number of vector genomes within AAV. The ddPCR method used a primer and probe set specific to the gene of interest. The primer / probe set used for samples AAV1439, AAV1440, AAV1442, and AAV1443 was Petition 870250105146, dated 11 / 17 / 2025, page 85 / 125 80 / 94 targeting the Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element (WPRE), a sequence present in all four viruses. The primer and probe sequences included forward primer: TTGTGAAAGATTGACTGGTATTCT (SEQ ID NO: 16), reverse primer: AGGCATTAAAGCAGCGTA (SEQ ID NO: 17), and probe: TAACTATGTTGCTCCTTTTAC (SEQ ID NO: 18). For the two remaining samples (AAV1441 and AAV1444), the primers / probe included in the pre-designed TaqMan Gene Expression Assay Hs01070627_m1 from ThermoFisher were used. This proprietary set is specifically targeted to a sequence in the SLC26A4 gene – which encodes the pendrin protein – included in both viruses. The sequence is: AAGGGATGGCATATGCCCTACTAGCTGCAGTTCCTGTCGGATATGG TCTCTACTCTGCTTTTTTCCCTATCC (SEQ ID NO: 19).

[00124] To perform the ddPCR test, a DNase digestion (30 min at 37 °C) was performed to remove all non-encapsulated DNA and ensure that only endonuclease-resistant (encapsulated) vector genomes were quantified. This step used recombinant DNase I from Sigma-Aldrich (10 units / μL, catalog number 4716728001). Subsequently, a proteinase K treatment (ProK, > 600 mAU / ml, Qiagen catalog number 19133) was used to neutralize the DNase and digest the AAV capsid to release any encapsulated DNA, followed by a heat treatment to inactivate the enzymes and open the capsid. Incubation parameters were: 56°C for 30 minutes, 95°C for 5 minutes, and 4°C for 5 minutes to 1 hour. The sample was then diluted and the polymerase chain reaction (PCR) master mix, which contains Bio-Rad PCR Supermix (1863023) plus primer and probe sets, was added.

[00125] For the ddPCR assay, the PCR mixture (test sample, master mix with primers and probe) was partitioned into nanoliter-scale droplets using a water-in-oil emulsion system with the Petition 870250105146, dated 11 / 17 / 2025, page 86 / 125 81 / 94 using the Bio-Rad Automatic Droplet Generator. The test sample was randomly partitioned into droplets and amplified by PCR using a Bio-Rad C1000 Touch thermocycler. At the end of the PCR reaction, the fluorescence in each droplet was individually measured with the Bio-Rad QX200 droplet reader. The inner probe was labeled with a 5' fluorescent reporter dye and a non-fluorescent 3' blunt end. At each PCR cycle, the activity of the 5' Taq polymerase exonuclease hydrolyzed the inner probe, releasing fluorophore from the probe and the blunt end. Each droplet was assigned as negative (no measurable DNA) or positive (one or more copies of DNA) based on a fluorescence intensity threshold, which was determined from the non-template control.The absolute concentration of DNA in each sample was determined according to the Poisson distribution, based on the ratios of positive to negative droplets for different dilutions of the test sample. The dilution-corrected concentration of the gene of interest (GOI) was reported in units of vg / ml.

[00126] As shown in Table 3, AAV1 production using the modified rep-cap plasmid P929 resulted in a higher titer compared to AAV1 production using the standard rep-cap plasmid P374. For example, comparisons of ddPCR titers of clarified lysate (vg / ml) showed a 13.9-fold difference between the titer of the AAV1 vector genome produced with modified rep-cap plasmid P929 and transfer plasmid P1381 versus standard rep-cap plasmid P374 and transfer plasmid P1381, a 13.2-fold difference between the titer of the AAV1 vector genome produced with modified rep-cap plasmid P929 and transfer plasmid P1900 versus standard rep-cap plasmid P374 and transfer plasmid P1900, and a 9.1-fold difference between the titer of the AAV1 vector genome produced with modified rep-cap plasmid P929 and transfer plasmid P707 versus plasmid P1381. Petition 870250105146, dated 11 / 17 / 2025, page 87 / 125 82 / 94 standard rep-cap P374 and transfer plasmid P707. Comparisons of purified ddPCR titer (vg / ml) also showed an increase in AAV1 titer produced using the modified rep-cap plasmid P929 versus AAV1 produced using the standard rep-cap plasmid P374, with a difference of 16.5 times for AAV1 produced with transfer plasmid P1381, a difference of 15.2 times for AAV1 produced with transfer plasmid P1900, and a difference of 68.89 times for AAV1 produced with transfer plasmid P707. Vector Capsid Titer by Enzyme-Linked Immunosorbent Assay

[00127] A sandwich-type enzyme-linked immunosorbent assay (ELISA) was used to quantify the total capsid content in all samples.

[00128] The AAV1 capsid assay was performed as a two-step colorimetric ELISA using commercially available capture and detection reagents, calibration standards, and assay diluent. Two different versions of the same single-chain camelid antibody, which bind with high affinity to AAV capsids, were used. The capture antibody (ThermoFisher 7103522100, 1 mg / ml) was the biotin-conjugated anti-AAV antibody; the detection antibody (ThermoFisher 7303522100, 0.5 mg / ml) was the same anti-AAV antibody conjugated to horseradish peroxidase (HRP).

[00129] Each well of streptavidin-coated plates (ThermoFisher number 15124) was incubated for one hour at room temperature with 100 ng of biotin capture antibody conjugate. After washing, calibration standards and diluted test samples containing AAV1 capsids were added to the plate wells and incubated for one hour at room temperature. The standard curve consisted of 2.5 serial dilutions of empty AAV1 capsids (Vigene RS-AAV1-ET), in a range of 1.00 x 10¹¹ to 1.64 x 10⁸ capsids / ml. The plates were Petition 870250105146, dated 11 / 17 / 2025, page 88 / 125 83 / 94 washed samples and 100 μL of the HRP detection conjugate antibody, diluted 1:10,000, were added to the wells and incubated for one hour at room temperature. After washing, 3,3',5,5'-tetramethylbenzidine (TMB) substrate (ThermoFisher 34028) was added to the wells of the plate. After ten minutes, the reaction was stopped by adding 25% sulfuric acid (Sigma-Aldrich 84736-1L), resulting in a colorimetric reaction proportional to the concentration of AAV1 capsids in the calibration standards and diluted test samples. Absorbance was measured on a PerkinElmer EnVision multimode plate reader at 450 nm. Concentrations were determined by interpolation from a standard curve fit with a 4-parameter logistic model (4PL) using GraphPad Prism. Triplicate measurements were performed for each standard, control assay, and test sample. The dilution-corrected concentration of AAV1 capsids was reported in capsid units / ml.

[00130] As shown in Table 3, AAV1 production using the modified rep-cap plasmid P929 resulted in increased capsid titer (cp / ml) and total capsids compared to AAV1 production using the standard rep-cap plasmid P374.For example, comparisons of clarified lysate capsid titer (cp / ml) showed an 18.8-fold difference between AAV1 capsid titer produced with modified rep-cap plasmid P929 and transfer plasmid P1381 versus standard rep-cap plasmid P374 and transfer plasmid P1381, a 15.8-fold difference between AAV1 capsid titer produced with modified rep-cap plasmid P929 and transfer plasmid P1900 versus standard rep-cap plasmid P374 and transfer plasmid P1900, and a 27.2-fold difference between AAV1 capsid titer produced with modified rep-cap plasmid P929 and transfer plasmid P707 versus standard rep-cap plasmid P374 and transfer plasmid. Petition 870250105146, dated 11 / 17 / 2025, page 89 / 125 84 / 94 P707. Table 3. Vector Production Using Standard and Tet-Off Modified Rep-Cap Plasmids Sample Capsid Titer of Clarified Lysate (cp / ml) Total Capsids (per 120 ml of lysate) Estimated ddPCR Titer (based on 25% complete) Clarified ddPCR Lysate Actual (vg / ml) % Complete Estimated based on VG / CP ratio Estimated Purified ddPCR Titer (vg / ml) Purified ddPCR Titer (vg / ml) AAV1 439 1.45E+1 2 1.74E+1 4 3.63E+1 1 3.03E+1 1 21% 3.64E+1 3 4.20E+13 AAV1 441 1.22E+1 2 1.46E+1 4 3.05E+1 1 3.88E+1 1 32% 4.66E+1 3 3.36E+13 AAV1 440 7.30E+1 1 8.76E+1 3 1.83E+1 1 1.10E+1 1 15% 1.32E+1 3 2.26E+13 AAV1 442 7.68E+1 0 9.22E+1 2 1.92E+1 0 2.18E+1 0 28% 2.61E+1 2 2.54E+12 AAV1 444 7.69E+1 0 9.23E+1 2 1.92E+1 0 2.94E+1 0 38% 3.53E+1 2 2.20E+12 AAV1 443 2.68E+1 0 3.22E+1 2 6.70E+0 9 1.20E+1 0 45% 1.45E+1 2 3.28E+11 Example 2. Compare AAV1 Production Using Standard and Modified Rep-Cap Plasmids AAV Production

[00131] To enable AAV production, the AAV2 rep-encoding plasmid / AAV1 cap-encoding plasmid, the pHelper plasmid, and the transfer plasmid were combined in a 2:1:2 concentration ratio. Petition 870250105146, dated 11 / 17 / 2025, pp. 90 / 125 85 / 94 with FectoVIR (Polypus) in a 1:1 FectoVIR:DNA (deoxyribonucleic acid) ratio and added to VPC suspension cultures (viral production cells, 293 origin). The AAV2 / AAV1 rep-cap encoding plasmid used for AAV production was the modified rep-cap plasmid P2056 (Figure 3) or the standard rep-cap plasmid P374 (Figure 2). The transfer plasmid used for AAV production was the transfer plasmid P1381, which contained a CMV promoter operationally linked to a GFP-encoding polynucleotide. The same pHelper plasmid (P376) was used with each combination of rep-cap plasmid and transfer plasmid. The rep / cap plasmid P2056 contains an rtTA-inducible system that is induced by doxycycline. Three concentrations of doxycycline (1 μg / ml, 0.1 μg / ml and 0.01 pg / ml) were tested, added simultaneously after transfection with the P2056 / P1381 / P374 system, as well as an untreated control.The P376 / P1381 / P374 system, which does not contain a tetracycline-inducible repeat / cap, was also tested by adding 1 pg / ml of doxycycline simultaneously after transfection to determine the impact of doxycycline on transfection. Cell culture medium and cells were harvested after 72 hours of incubation at 37 °C, 8% CO2, and 125 RPM. The cell lysate was then treated with benzonase nuclease to degrade non-viral DNA and RNA (ribonucleic acid) and Triton X-100 (10% w / v Triton X-100) and lysed during incubation at 37 °C, 8% CO2, and 125 RPM. The cell lysates were then filtered through a 0.22 µm filter before purification.

[00132] AAV particles (AAV1556 for the P2056 / P1381 / P374 co-transfection; and AAV1442 for the P376 / P1381 / P374 co-transfection) were enriched from cell culture lysate by gravity affinity chromatography using AAVX resin and subsequently buffer-exchanged using centrifugal filtration units. The AAV was formulated in 10 mM sodium phosphate, NaCl Petition 870250105146, dated 11 / 17 / 2025, page 91 / 125 86 / 94 180 mM, 5% (w / v) sucrose and 0.001% (w / v) poloxamer, pH 7.3 and stored at -80°C. Vector Genome Title by Droplet Digital Polymerase Chain Reaction

[00133] Droplet digital polymerase chain reaction (ddPCR) was used to quantify the total number of vector genomes within the AAV and performed as described in Example 1 using the same forward and reverse primers (SEQ ID NOs: 16 and 17) and probe (SEQ ID NO: 18).

[00134] As shown in Table 5, AAV1 production using the modified rep-cap plasmid P2056 resulted in a higher titer compared to AAV1 production using the standard rep-cap plasmid P374, as determined by ddPCR. For example, comparisons of clarified lysate ddPCR titer (vg / ml) showed a 2.7-fold difference between the AAV1 vector genome titer produced with the modified rep-cap plasmid P2056 versus the standard rep-cap plasmid P374 in the absence of added doxycycline, and a 3.1-fold difference in the presence of 1.0 μg / ml of doxycycline. Comparisons of purified ddPCR titer (vg / ml) also showed an increase in AAV1 titer produced using the modified rep-cap plasmid P2056 versus AAV1 produced using the standard rep-cap plasmid P374, with a 5.0-fold difference for AAV1 produced in the absence of doxycycline and a 3.0-fold difference for AAV1 produced in the presence of 1 μg / ml of doxycycline.Interestingly, the AAV1 titer produced with the P2056-modified rep-cap plasmid in both the clarified lysate and the affinity purification afterward was minimally affected by the presence or absence of added doxycycline, as determined by ddPCR. This is believed to be due to the fact that the Tet-On system used in P2056 is leaky and allows capsid protein production even in the absence of doxycycline. Petition 870250105146, dated 11 / 17 / 2025, page 92 / 125 87 / 94 added. Vector Capsid and Genome Titer and Empty / Complete Analysis by Size Exclusion Chromatography - Multiangle Light Scattering

[00135] Multi-angle light scattering size exclusion chromatography (SEC-MALS) analysis was performed using an Agilent 1260 Infinity II HPLC instrument equipped with a multi-wavelength UV-Vis detector and a Wyatt Dawn MALS detector. Separation was performed using a 7.8 x 300 mm Agilent Bio SEC-5 column packed with 5 μm particles with 1000 Å pores and a mobile phase containing 300 mM NaCl, 20 mM sodium phosphate, pH 7.4, and 0.001% (w / v) Pluronic® F68, at a flow rate of 1.0 mL / min. The volume of each injection was 50 μL. The determination of capsid titers (cp / ml) and genome titers (vg / ml) was performed using the Viral Vector procedure, which is based on Conjugate Analysis, within the Astra software (version 8.1.2.1). The procedure is described in Wyatt Technology Application Note 1617 (Quantifying quality attributes of AAV gene therapy vectors by SEC-UV-MALS-dRI) and discussed in Werle et al., Mol Ther Methods Clin Dev.23:254-262, 2021, compared to other techniques used to determine empty / complete AAV ratios, involves the use of multi-angle light scattering signals along with two concentration sources, such as UV absorbance at 260 and 280 nm. These signals are used to calculate the total protein and DNA masses and the molar masses of the capsid and transgene (Mw), which in turn are converted into capsid and genome titers. Finally, the % of complete capsids is determined by the following formula: (capsid titer) / (genome titer) x 100. The processing parameters that were used for the SEC-MALS analysis are listed in Table 4. Table 4. Parameters used for SEC-MALS analysis Petition 870250105146, dated 11 / 17 / 2025, page 93 / 125 88 / 94 Parameter Value Protein Extraction Coefficient at 280 nm (ml mg -1 cm -1) 2.10 Protein Extraction Coefficient at 260 nm (ml mg -1 cm -1) 1.30 Expected Protein Molar Mass (MDa) 3.75 Nucleic Acid Extraction Coefficient at 280 nm (ml mg -1 cm -1) 14.6 Nucleic Acid Extraction Coefficient at 260 nm (ml mg -1 cm -1) 24.0 Expected Nucleic Acid Molar Mass (MDa) 0.94

[00136] The SEC-MALS analysis results are presented in Table 5. As measured by SEC-MALS, AAV1 production using the modified rep-cap plasmid P2056 resulted in a 4.7-fold increase in purified viral titer compared to AAV1 production using the standard rep-cap plasmid P374 in the absence of doxycycline and a 3.5-fold increase in the presence of 1.0 μg / ml doxycycline. The capsid titer using P2056 was also increased 4.1-fold in the absence of doxycycline and 6.7-fold in the presence of 1.0 pg / ml doxycycline, respectively, compared to P374. The SEC-MALS results also confirmed that doxycycline had a minimal effect on the purified viral titer using the modified rep-cap plasmid P2056. Surprisingly, the production of complete AAV1 vectors using P2056 was higher in the absence of added doxycycline (23.1%) than in its presence (10.1-18.5%). Table 5. Vector Production Using Standard and Tet-On Modified Rep-Cap Plasmids Sample Plasmid of Rep / C ap Doxycycline added to Lysate Clarified by ddPCR (vg / ml) Affinity purification by ddPCR Purified SECMALS Titer SEC- % Capsid Titer Complete based Petition 870250105146, dated 11 / 17 / 2025, pp. 94 / 125 89 / 94 (vg / ml) (vg / ml) MALS (cp / ml) in SEC- MALS AAV 1556 P2056 1.0 ug / ml 1.01E+11 1.10E+13 8.06E+1 2 7.96E+ 13 10.1 AAV 1556 P2056 0.1 ug / ml 8.14E+10 8.89E+12 6.42E+1 2 5.86E+ 13 11 AAV 1556 P2056 0.01 ug / ml 9.96E+10 1.25E+13 9.02E+1 2 4.88E+ 13 18.5 AAV 1556 P2056 0 1.05E+11 1.51E+13 1.09E+1 3 4.71E+ 13 23.1 AAV 1442 P374 1.0 ug / ml 3.30E+10 3.61E+12 2.29E+1 2 1.18E+ 13 19.4 AAV 1442 P374 0 3.95E+10 3.03E+12 2.33E+1 2 1.16E+ 13 20.1

[00137] In another experiment, the modified rep-cap plasmid P929 was used to produce AAV1 in the presence of 1.0 μg / ml doxycycline, 0.1 pg / ml doxycycline, or 0.01 μg / ml doxycycline, or in the absence of doxycycline. The percentage of complete AAV1 vectors was evaluated using SEC-MALS with a method similar to that described above. Surprisingly, the production of complete AAV1 vectors using P929 was higher in the presence of added doxycycline than in its absence (Figure 4). Example 3. Compare the Production of AAV1 Encoding the GJB2 Transgene Using Standard and Modified Rep-Cap Plasmid

[00138] The GJB2 transgene encodes the connexin 26 protein. Mutations in this gene are implicated in hearing loss.

[00139] A comparison was made between an AAV-pRC1 variant and P929 (see Figure 1) in the production of AAV1 encoding the transgene Petition 870250105146, dated 11 / 17 / 2025, pp. 95 / 125 90 / 94 GJB2. The AAV-pRC1 variant, designated RC1 p5AS, was used as a control. Production was carried out in the presence of 1.5 μg / ml of doxycycline or in the absence of doxycycline. The same pHelper (P376) plasmid was used.

[00140] Productions were carried out in 125 ml shaken flasks with a working volume of 30 ml. After 72 hours of incubation at 37 °C, 8% CO2 and 120 RPM, the cell culture was lysed using 1% Tween 20 and 100 U / ml of Denarase nuclease. Cell lysates were clarified by centrifugation before genomic quantification and capsid titer determination.

[00141] Figures 8A and 8B show data from the production of AAV1 containing a GJB2 transgene using an RC51 p5AS or P929. Figure 8A depicts the genomic titer and shows that P929 was superior to RC1 p5AS in the presence of doxycycline (more than 2 times superior) and in the absence of doxycycline (more than 3 times superior). Figure 8B depicts the capsid titer and shows that P929 was superior to RC1 p5AS in the presence of doxycycline (almost 3 times superior) and in the absence of doxycycline (more than 10 times superior). Table 6 below presents the tabulated data. Table 6. Tabulated data for p929 compared to RC1 p5AS Sample Rep / Cap Plasmid with added Doxycycline Clarified Lysate Capsid Titer (cp / ml) Genomic Titer of Clarified Lysate (vg / ml) CER5631 RC1 p5AS 0 3.63E+11 2.94E+11 CER5630 RC1 p5AS 1.5 ug / ml 2.45E+11 1.90E+11 CER5637 RC1 p5AS 1.5 ug / ml 1.14E+12 4.85E+11 CER5629 P929 0 5.65E+12 1.17E+12 CER5635 P929 0 7.18E+12 1.50E+12 Petition 870250105146, dated 11 / 17 / 2025, pp. 96 / 125 91 / 94 CER5628 P929 1.5 ug / ml 1.78E+12 5.80E+11 CER5634 P929 1.5 ug / ml 2.83E+12 8.15E+11 Example 4. AAV1 Expressing the Enhanced Green Fluorescent Protein Transgene

[00142] This example compares the plasmids P374 (Figure 2), P929 (Figure 1), P2057 (Figure 5), P2058 (Figure 6) and P2059 (Figure 7).

[00143] Plasmid 2057 is based on P929 with the promoter altered to a TRE-tight promoter. Plasmid 2058 has tTA (tetracycline transactivator) linked to the Rep gene by peptide 2A. Peptide 2A is an 18-22 amino acid peptide that mediates the autocleavage of polypeptides during translation in eukaryotic cells. Plasmid 2059 has tTA regulated by its own promoter and not regulated by Rep.

[00144] Each of the five plasmids was used to produce AAV1 vectors carrying an eGFP transgene. The vectors were produced in biological duplicates in the absence of doxycycline. A 2:1:2 plasmid ratio relative to the - cap encoding plasmid:helper plasmid:eGFP plasmid was used for productions. Figures 9A to 9C show the production titer of AAV1 containing an enhanced green fluorescent protein (eGFP) transgene using plasmids P374, P929, P2057, P2058, or P2059. The same pHelper plasmid (p376) was used. Productions were carried out in 125 ml shaken flasks using the method described in Example 3.

[00145] Figure 9A compares the capsid titer measured by ELISA in clarified lysate samples for each of the five plasmids. P2059 achieved the highest capsid titer (cp / ml), followed by P2058 and P929. P2057 achieved a slightly lower capsid titer compared to P374.

[00146] Figure 9B compares genomic titer with the use of PCR. Petition 870250105146, dated 11 / 17 / 2025, page 97 / 125 92 / 94 digital droplet assay (ddPCR) was performed on clarified lysate samples for each of the five plasmids. The results are similar to those in Figure 9A. All tetracycline-regulated constructs (P929, P2057, P2058, and P2059), which can be regulated by tetracycline and derivatives such as doxycycline, achieved higher genomic titers (vg / ml) than the control P374. P2059 performed best in terms of genomic titer, followed by P2058, P929, and P2057.

[00147] Figure 9C compares the estimated genomic titer based on the percentage of complete capsids for each of the five plasmids. The results are similar to those in Figure 9B.

[00148] Figures 10A to 10B show the production of AAV1 containing an eGFP transgene using plasmids P374, P929, P2057, P2058, or P2059 and use mass photometry to evaluate the capsid fullness achieved by each of the five plasmids. Before quantifying capsid fullness using mass photometry, the clarified lysate samples were purified.

[00149] Figure 10A establishes the percentage of complete capsids. P2057 showed the highest percentage of complete capsids at 64%, followed by P2058 at 35% and P929 at 34%. The percentage of complete vectors produced with P2059 was slightly lower than the control P374.

[00150] Figure 10B shows the percentage of partially filled capsids. P2057 showed the highest percentage. Vectors produced with all constructs showed minimal partially filled capsids. The tabulated data are below in Table 7. Table 7. Tabulated Data for a Comparison of P374, P929, P2057, P2058, and P2059 Plasmid Sample Titer % complete % partial Titer Petition 870250105146, dated 11 / 17 / 2025, pp. 98 / 125 93 / 94 Rep / Cap Clarified Lysate Capsid (cp / ml) based on Mass Photometry based on estimated ddPCR (based on % complete) CER5707 P374 5.30E+10 27.2 0.8 1.44E+10 CER5708 P374 2.40E+11 23.1 0.4 5.54E+10 CER5715 P929 1.30E+12 31.6 0.4 4.11E+11 CER5716 P929 4.10E+11 37 0.6 1.52E+11 CER5723 P2057 4.60E+10 63 1.2 2.90E+10 CER5724 P2057 1.40E+11 64.2 1.7 8.99E+10 CER5731 P2058 1.80E+12 36.3 0.9 6.53E+11 CER5732 P2058 2.90E+12 33.3 0.7 9.66E+11 CER5739 P2059 6.20E+12 20.3 0.8 1.26E+12 CER5740 P2059 7.10E+12 19.4 0.6 1.38E+12

[00151] The data in Example 7 are compelling. P2059 and 2058 performed best in terms of overall capsid and genomic titer. However, in terms of percentage of complete capsids, P2057 performed best. Thus, where complete capsid purification is readily achieved, P2059 and P2058 are a logical choice. However, where complete capsid purification is more difficult, P2057 may be the better choice, regardless of its inferior performance in terms of overall capsid and genomic titer. Other Inventions

[00152] Although the present inventions have been described in connection with specific examples thereof, it will be understood that they are subject to further modifications and this application is Petition 870250105146, dated 11 / 17 / 2025, page 99 / 125 94 / 94 is intended to cover any variations, uses or adaptations of the inventions generally following the principles of the inventions and including such deviations from the inventions that fall within known or customary practice in the art to which the inventions belong and can be applied to the essential features set forth herein and fall within the scope of the claims. Petition 870250105146, dated 11 / 17 / 2025, pages 100 / 125

Claims

1 / 9 CLAIMS 1. Adeno-associated virus (AAV) rep-cap plasmid, characterized in that it comprises, in a 5' to 3' orientation: (a) a polynucleotide encoding an AAV rep protein; (b) a tetracycline-off (Tet-Off) system or a tetracycline-on (Tet-On) system; (c) an AAV promoter; and (d) a polynucleotide encoding an AAV1 capsid protein.

2. Rep-cap plasmid according to claim 1, characterized in that the AAV promoter is a p41 AAV promoter.

3. Rep-cap plasmid, according to claim 2, characterized in that it comprises a tetracycline-off (Tet-off) system.

4. Rep-cap plasmid, according to claim 2, characterized in that it comprises a tetracycline-on (Tet-on) system.

5. Rep-cap plasmid, according to claim 2, characterized in that the AAV rep protein is an AAV2 rep protein.

6. Rep-cap plasmid, according to claim 2, characterized in that the polynucleotide encoding an AAV rep protein encodes Rep78 and Rep52.

7. Rep-cap plasmid according to claim 6, characterized in that Rep78 has the sequence SEQ ID NO:

1.

8. Rep-cap plasmid according to claim 6, characterized in that Rep52 has the sequence SEQ ID NO:

2.

9. Rep-cap plasmid according to claim 2, characterized in that the polynucleotide encoding the AAV rep protein has the sequence SEQ ID NO:

3. Petition 870250105146, dated 11 / 17 / 2025, pp. 101 / 125 2 / 9 10. Rep-cap plasmid according to claim 2, characterized in that the Tet-Off system comprises, in a 5' to 3' orientation: (a) a first polynucleotide encoding a tetracycline transactivator (tTA), wherein the tTA comprises a DNA-binding domain derived from the tetracycline repressor and a transcriptional activation domain; and (b) a second polynucleotide comprising 1 to 12 tet operator sequences (tetO).

11. Rep-cap plasmid, according to claim 10, characterized in that the first polynucleotide encodes a polypeptide with an amino acid sequence of SEQ ID NO:

5.

12. Rep-cap plasmid according to claim 11, characterized in that the first polynucleotide has at least 80% sequence identity with SEQ ID NO:

6.

13. Rep-cap plasmid, according to claim 12, characterized in that the first polynucleotide has the sequence SEQ ID NO:

6.

14. Rep-cap plasmid, according to claim 10, characterized in that each tetO sequence has the sequence SEQ ID NO:

4.

15. Rep-cap plasmid, according to claim 10, characterized in that each tetO sequence is separated from an adjacent tetO sequence by 0-20 nucleotides.

16. Rep-cap plasmid according to claim 10, characterized in that the second polynucleotide comprises eight tetO sequences.

17. Rep-cap plasmid according to claim 16, characterized in that the second polynucleotide has at least 80% sequence identity with SEQ ID NO:

7. Petition 870250105146, dated 11 / 17 / 2025, pp. 102 / 125 3 / 9 18. Rep-cap plasmid according to claim 17, characterized in that the second polynucleotide has the sequence SEQ ID NO:

7.

19. Rep-cap plasmid according to claim 2, characterized in that the Tet-On system comprises, in a 5' to 3' orientation: (a) a first polynucleotide encoding a reverse tetracycline transactivator (rtTA), wherein the rtTA comprises a DNA-binding domain derived from the tetracycline repressor and a transcriptional activation domain; and (b) a second polynucleotide comprising 1 to 12 tet operator sequences (tetO).

20. Rep-cap plasmid, according to claim 19, characterized in that the first polynucleotide encodes a polypeptide with an amino acid sequence of SEQ ID NO:

20.

21. Rep-cap plasmid according to claim 20, characterized in that the first polynucleotide has at least 80% sequence identity with SEQ ID NO:

21.

22. Rep-cap plasmid, according to claim 21, characterized in that the first polynucleotide has the sequence SEQ ID NO:

21.

23. Rep-cap plasmid according to claim 19, characterized in that each tetO sequence has the sequence SEQ ID NO:

4.

24. Rep-cap plasmid, according to claim 19, characterized in that each tetO sequence is separated from an adjacent tetO sequence by 0-20 nucleotides.

25. Rep-cap plasmid according to claim 19, characterized in that the second polynucleotide comprises eight tetO sequences. Petition 870250105146, dated 11 / 17 / 2025, pp. 103 / 125 4 / 9 26. Rep-cap plasmid according to claim 25, characterized in that the second polynucleotide has at least 80% sequence identity with SEQ ID NO:

7.

27. Rep-cap plasmid according to claim 26, characterized in that the second polynucleotide has the sequence SEQ ID NO:

7.

28. Rep-cap plasmid according to claim 2, characterized in that the p41 promoter has the sequence SEQ ID NO:

8.

29. Rep-cap plasmid, according to claim 2, characterized in that the polynucleotide encoding an AAV1 capsid protein encodes a polypeptide with an amino acid sequence of SEQ ID NO:

9.

30. Rep-cap plasmid, according to claim 29, characterized in that the polynucleotide encoding an AAV capsid protein has the sequence SEQ ID NO:

10.

31. Rep-cap plasmid, according to claim 29, characterized in that the polynucleotide encoding an AAV capsid protein has the sequence SEQ ID NO:

11.

32. Rep-cap plasmid, according to claim 2, characterized in that the Tet-Off system or Tet-On system further comprises a polyA signal sequence located between the first polynucleotide and the second polynucleotide.

33. Rep-cap plasmid, according to claim 2, characterized in that it comprises a polynucleotide sequence of SEQ ID NO:

12.

34. Rep-cap plasmid, according to claim 2, characterized in that it comprises a polynucleotide sequence of SEQ ID NO:

13.

35. Rep-cap plasmid, according to claim 2, Petition 870250105146, dated 11 / 17 / 2025, pp. 104 / 125 5 / 9, characterized in that it comprises a polynucleotide sequence of SEQ ID NO:

22.

36. Method for manufacturing a recombinant adeno-associated virus, characterized in that it comprises the steps of: (I) introducing into a mammalian cell: (a) a rep-cap plasmid, as defined in claim 1; (b) a helper plasmid comprising one or more helper genes selected from E4, E2a and VA; and (c) a transgene plasmid comprising a transgene of interest flanked by terminal inverse repeats, wherein the introduction step is under conditions that permit the formation of the recombinant adeno-associated virus; and (II) collecting the recombinant adeno-associated virus.

37. Method according to claim 36, characterized in that the AAV promoter is a p41 AAV promoter.

38. Method according to claim 37, characterized in that the mammalian cell is a HEK-293 cell.

39. Method according to claim 37, characterized in that the rep-cap plasmid comprises a polynucleotide sequence of SEQ ID NO:

12.

40. Method according to claim 37, characterized in that the rep-cap plasmid comprises a polynucleotide sequence of SEQ ID NO:

13.

41. Method according to claim 37, characterized in that the rep-cap plasmid comprises a polynucleotide sequence of SEQ ID NO:

22.

42. Method according to claim 37, characterized in that the transgene of interest encodes a protein that is normally expressed in the human ear.

43. Method, according to claim 42, characterized in Petition 870250105146, dated 11 / 17 / 2025, page 105 / 125 6 / 9 by the fact that the transgene of interest encodes the solute carrier family 26, member 4 (Pendrin), Otoferlin (Otof), Stereocilin (STRC), Atonal Transcription Factor of BHLH 1 (ATOH1), gap junction protein beta 2 (GJB2) or SRY-Box 2 (Sox2).

44. Method according to claim 42, characterized in that the transgene of interest encodes Pendrin, GJB2 or OTOF.

45. Method according to claim 37, characterized in that the viral yield is at least 1 x 1011 vg / ml, as measured using ddPCR of clarified lysate.

46. ​​Method according to claim 37, characterized in that: (a) the rep-cap plasmid comprises a Tet-On system; and (b) production of recombinant AAV1 virus is achieved without inducing the Tet-On system.

47. Method for increasing the yield of a recombinant AAV1-capped adeno-associated virus by at least 2.5 times compared to a recombinant AAV1-capped adeno-associated virus produced with pAAV-RC1, characterized in that it comprises the step of replacing a rep-cap plasmid, as defined in claim 2, with pAAV-RC1 in the fabrication of the adeno-associated virus.

48. Method according to claim 47, characterized in that the yield is increased by at least 7 times.

49. Method according to claim 47, characterized in that the rep-cap plasmid comprises a polynucleotide sequence of SEQ ID NO:

12.

50. Method according to claim 47, characterized in that the rep-cap plasmid comprises a polynucleotide sequence of SEQ ID NO:

13. Petition 870250105146, dated 11 / 17 / 2025, pp. 106 / 125 7 / 9 51. Method according to claim 47, characterized in that the rep-cap plasmid comprises a polynucleotide sequence of SEQ ID NO:

22.

52. Rep-cap plasmid according to claim 2, characterized in that the rep-cap plasmid has an arrangement according to Figure 1.

53. Rep-cap plasmid according to claim 2, characterized in that the rep-cap plasmid has an arrangement according to Figure 6.

54. Rep-cap plasmid according to claim 2, characterized in that the rep-cap plasmid has an arrangement according to Figure 7.

55. Cell, characterized in that it comprises a rep-cap plasmid, as defined in any one of claims 1 to 54.

56. Cell according to claim 55, characterized in that the cell further comprises a transgene of interest encoding a protein that is normally expressed in the human ear.

57. A cell according to claim 56, characterized in that the transgene of interest encodes the solute carrier family 26, member 4 (Pendrin), Otoferlin (Otof), Stereocilin (STRC), Atonal Transcription Factor bHLH 1 (ATOH1), gap junction protein beta 2 (GJB2) or SRY-Box 2 (Sox2).

58. Cell according to any one of claims 55 to 57, characterized in that the cell is a mammalian cell.

59. Cell according to claim 58, characterized in that the mammalian cell is a HEK-293 cell.

60. Cell culture, characterized in that it comprises a cell, as defined in any of the claims 55 to 59.

61. Adeno-associated virus (AAV) rep-cap plasmid, characterized in that it comprises, in a 5' to 3' orientation: (a) a polynucleotide encoding an AAV rep protein; (b) a tetracycline-off (Tet-Off) system or a tetracycline-on (Tet-On) system; (c) a TRE-tight promoter; and (d) a polynucleotide encoding an AAV1 capsid protein.

62. Rep-cap plasmid according to claim 51, characterized in that the rep-cap plasmid has an arrangement according to Figure 5.

63. Cell, characterized in that it comprises a rep-cap plasmid, as defined in claim 61 or 62.

64. Cell according to claim 63, characterized in that the cell further comprises a transgene of interest encoding a protein that is normally expressed in the human ear.

65. A cell according to claim 64, characterized in that the transgene of interest encodes the solute carrier family 26, member 4 (Pendrin), Otoferlin (Otof), Stereocilin (STRC), Atonal Transcription Factor bHLH 1 (ATOH1), gap junction protein beta 2 (GJB2) or SRY-Box 2 (Sox2).

66. A cell according to any one of claims 63 to 65, characterized in that the cell is a mammalian cell.

67. Cell according to claim 66, characterized in that the mammalian cell is a HEK-293 cell.

68. Cell culture, characterized in that it comprises a cell, as defined in any one of claims 63 to 67.

69. Method for manufacturing a recombinant adeno-associated virus, characterized in that it comprises the steps of: (I) introducing into a mammalian cell: (a) a rep-cap plasmid, as defined in any one of claims 61 to 62; (b) a helper plasmid comprising one or more helper genes selected from E4, E2a and VA; and (c) a transgene plasmid comprising a transgene of interest flanked by terminal inverse repeats, wherein the introduction step is under conditions that permit the formation of the recombinant adeno-associated virus; and (II) collecting the recombinant adeno-associated virus.

70. Method according to claim 69, characterized in that the mammalian cell is a HEK-293 cell.

71. Method according to claim 69 or 70, characterized in that the cell further comprises a transgene of interest encoding a protein that is normally expressed in the human ear.

72. Method according to claim 71, characterized in that the transgene of interest encodes the solute carrier family 26, member 4 (Pendrin), Otoferlin (Otof), Stereocilin (STRC), Atonal Transcription Factor of bHLH 1 (ATOH1), gap junction protein beta 2 (GJB2) or SRY-Box 2 (Sox2). Petition 870250105146, dated 11 / 17 / 2025, pp. 109 / 125

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