Vector system, transformed host cell, pharmaceutical composition and its uses.

Description

[0001] Split from patent application BR 11 2021 007221 7 filed on October 15, 2019. TECHNICAL FIELD

[0002] The present invention relates to constructs, vectors, related host cells and pharmaceutical compositions that enable effective gene therapy, in particular, for diseases due to mutations in genes with a coding sequence (CDS) greater than 5 kb. BACKGROUND OF THE INVENTION

[0003] Gene therapy with adeno-associated viral vectors (AAVs) is safe and effective in humans. AAV-based gene therapy products have been approved in recent years in the US and Europe for hereditary and metabolic diseases, while clinical trials for AAV-based gene therapy approaches for diseases in different therapeutic areas ranging from ophthalmology to hematology and musculoskeletal and metabolic disorders are constantly increasing.

[0004] However, the limited carrying capacity of AAV vectors prevents the development of AAV-based therapies for diseases due to mutations in genes with a coding sequence (CDS) larger than 5 kb (here also referred to as large genes).

[0005] Genetic diseases due to mutations in large genes (listed in Table 1 below) include, among others, Duchenne muscular dystrophy due to mutations in the DMD gene, cystic fibrosis due to mutations in the CFTR gene, Petition 870260074145, dated 07 / 24 / 2026, p. 13 / 259 2 / 192 hemophilia A due to mutations in the F8 gene, dysferlinopathies due to mutations in the DYSF gene, polycystic kidney disease due to a mutation in the PKD gene, Wilson's disease due to a mutation in the ATP7B gene, Huntington's disease due to a mutation in the HTT gene, Niemann-Pick type C due to a mutation in the NPC1 gene. Table 1: Genetic diseases due to mutations in large genes Disease Gene CDS Accession Number Duchenne Muscular Dystrophy DMD 11 Kb NM_000109 Cystic Fibrosis CFTR 4.4 Kb NM_000492 Hemophilia A F8 7 Kb NM_000132 Dysferlinopathies DYSF 6.2 Kb NM_001130455 Polycystic Kidney Disease PKD1 12.9 Kb NM_000296 Wilson's Disease ATP7B 4.4 KB NM_000053 Huntington's Disease HTT 9.4 KB NM_002111 Niemann-Pick Type C NPC1 3.8 KB NM_000271

[0006] In addition, several hereditary retinal degenerations (IRDs) are due to mutations in large genes, as listed in Table 2 below. IRDs affect about 1 in 3,000 people in Europe and the United States (58).

[0007] Among the most frequent and serious IRDs are retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), and Stargardt disease (STGD), which are most often inherited as monogenic conditions, with an overall global prevalence of 1 / 2,000 (1), and are a leading cause of blindness worldwide. Most mutations that cause IRDs occur in genes expressed in photoreceptors. Petition 870260074145, dated 07 / 24 / 2026, p. 14 / 259 3 / 192 neuronal (PR), rods and / or cones in the retina (2).

[0008] Gene therapy holds great promise for the treatment of IRDs. The first adeno-associated viral vector (AAV)-based gene therapy product for a hereditary form of blindness was approved in December 2017 (3). In addition, several other AAV-based products are currently in clinical development for gene therapy of rare and common forms of blindness (4). Although it is now well established that AAV represents, to date, the most efficient gene therapy vehicle for the retina (4,5), its limited payload capacity has hindered its use for conditions requiring the delivery of DNA sequences exceeding 5 kb in size (6) that include not only the transgene but also the cis regulatory elements that are necessary for its expression.

[0009] Examples of disease genes that exceed 5 kb in size are summarized in Table 2 below. Disease Gene CDS Accession Number Expression Stargardt disease and associated ABCA4 diseases ABCA4 6.8 Kb NM_000350 Rod & cone PRs Usher 1B MYO7A 6.7 Kb NM_000260 RPE and PRs Leber congenital amaurosis CEP290 7.5 Kb NM_025114 Mainly PRs (pan retinal) Usher 1D, CDH23 10.1 Kb NM_001171930 PRs Petition 870260074145, dated 07 / 24 / 2026, p. 15 / 259 4 / 192 Non-syndromic, autosomal recessive deafness (DFNB12) Retinitis pigmentosa EYS 9.4 Kb NM_001142800 PR ECM Usher 2A USH2a 15.6 Kb NM_007123 Rod&cone PRs Usher 2C ADGRV1 18.0 Kb NM_032119 Primarily PRs Alstrom syndrome ALMS1 12.5 Kb NM_015120 Rod&cone PRs

[0010] Stargardt disease (STGD; MIM # 248200) is the most common form of inherited macular degeneration caused by mutations in the ABCA4 gene (CDS: 6822 bp), which encodes the all-trans retinal transporter located in the outer PR segment (7); Usher syndrome type IB (USH1B; MIM # 276900) is the most severe form of RP and deafness caused by mutations in the MY07A gene (CDS: 6648 bp) (8) which encodes the unconventional MY07A, an actin-based motor expressed in both PR and RPE in the retina (9-11).

[0011] Conical rod dystrophy type 3, fundus flavimaculatus, age-related macular degeneration type 2, severe early-onset retinal dystrophy, and retinitis pigmentosa type 19 are also associated with ABCA4 mutations (referred to herein as ABCA4-associated diseases).

[0012] The inventors and others have shown that this limitation can be overcome by using double (up to 9 kb) (6, 12, 13) or triple (up to 14 kb) (14) AAV vectors, each Petition 870260074145, dated 07 / 24 / 2026, page 16 / 259 5 / 192 containing fragments of the coding sequence (CDS) of the large transgene expression cassette. Double and triple AAV vectors exploit the concatemerization and recombination of AAV genomes to reconstitute full-length genomes in cells co-infected with multiple AAV vectors. However, the efficiency of transgene expression achieved with double or triple AAV vectors in photoreceptors, which are the main therapeutic targets for most hereditary retinal diseases, is lower than that achieved with single AAV vectors (6, 14, 15). This may be due to the various limiting steps required for efficient transduction, including proper DNA concatemer formation, heterogeneous mRNA stability, and splicing efficiency across vector junctions.

[0013] The present inventors showed in document WO 2014 / 170480 and Colella et al (15) dual AAV vectors that reconstitute a large gene by splicing (transsplicing), homologous recombination (overlap) or a combination of the two (hybrid), finding that dual transsplicing and hybrid vectors are particularly efficient for the treatment of hereditary retinal degenerations. In addition, Maddalena et al. (14) demonstrated a triple AAV vector approach for genes up to 14 kb. However, the efficiency of transgene expression achieved with dual or triple AAV vectors is lower than that achieved with single AAV vectors (6, 13, 14). This may be due to the various limiting steps required for efficient transduction, including: proper formation of the DNA concatemer, stability of the heterogeneous mRNA and Petition 870260074145, dated 07 / 24 / 2026, page 17 / 259 6 / 192 splicing efficiency through vector junctions. Furthermore, the AAV triple vector strategy produces gene expression levels below the threshold required for a therapeutic approach.

[0014] Therefore, there is still a need for constructs and vectors that can be explored to reconstruct the expression of large genes for effective gene therapy.

[0015] The inventors have now discovered that delivering multiple AAV vectors, each encoding one of the fragments of either reporter or large therapeutic proteins flanked by short split inteins, results in protein trans-splicing and full-length protein reconstitution both in vitro and in vivo.

[0016] Inteins are genetic elements transcribed and translated within a host protein from which they self-excise in a manner similar to a protein intron, leaving no amino acid modifications in the final protein product, in the absence of energy supply, exogenous host-specific proteases, or cofactors (16, 17, 27, 28). Intein activity is context-dependent, with certain peptide sequences surrounding its linking junction (called N- and C-exteins) being necessary for efficient trans-splicing, the most important of which is an amino acid containing a thiol or hydroxyl group (i.e., Cys, Ser, or Thr) as the first residue in the C-extein (18). Split-inteins are a subset of inteins that are expressed as two separate polypeptides. Petition 870260074145, dated 07 / 24 / 2026, page 18 / 259 7 / 192 at the ends of two host proteins and catalyze their trans-splicing resulting in the generation of a single larger polypeptide (19). Inteins, including split inteins, are widely used in biotechnological applications including protein purification and labeling steps (19, 20), as well as the reconstitution of the widely used CRISPR / Cas9 genome editing nuclease (21, 22).

[0017] Several attempts have been made to explore intein-based protein splicing to reconstitute the expression of therapeutic genes, including the Factor gene. VIII, in which heavy and light chain genes fused with Synechocystis sp (Ssp) DnaB intein of Factor VIII have been shown to lead to the reconstitution of Factor VIII in cell culture and in animal models (23, 24). Similarly, a highly functional form of the dystrophin gene was expressed in vitro and in vivo, in which the 6.3 kb Becker dystrophin gene was split into two AAV vectors and each half was fused to split inteins obtained from Synechocystis sp. PCC 6803 (Ssp) DnaB intein or Rhodothermus marinus (Rma) DnaB intein (25). Furthermore, the split intein-mediated protein trans-splicing strategy (namely N. punctiforme DnaE split DnaE) has been reported to reconstitute the large pore-forming subunit of L-type calcium channels from two separate fragments in cardiac cells, (26). US patent 6,544,786 further reports the use of split inteins to deliver a dystrophin minigene.

[0018] The present inventors took advantage of the Petition 870260074145, dated 07 / 24 / 2026, page 19 / 259 8 / 192 intrinsic capacity of split inteins to mediate protein trans-splicing to reconstitute large full-length proteins after their fragmentation into two or three polypeptides flanked by split inteins, whose coding sequences fit into unique AAV vectors. The present invention, therefore, implements large protein cellular reconstitution by providing a target cell with two or more fragments of said large protein fused to split inteins to promote intein-mediated trans-splicing and reconstitute the functional protein. SUMMARY OF THE INVENTION

[0019] The present invention provides gene therapy with AAV vectors for diseases due to gene mutations, in particular, genes with coding regions exceeding 5 kb.

[0020] Based on findings that split-intein-mediated protein transsplicing is used by single-celled organisms to reconstitute proteins, the inventors constructed several AAV vectors, each encoding one of the fragments of either large reporter or therapeutic proteins flanked by short split inteins, resulting in protein trans-splicing and full-length protein reconstitution in vitro and in vivo.

[0021] Advantageously, the reconstitution of disease proteins mediated by AAV-based protein trans-splicing achieved by the present invention provided the expression of greater amounts of target proteins than AAV-based methods for large proteins known in Petition 870260074145, dated 07 / 24 / 2026, page 20 / 259 9 / 192 technique. This is likely due to overcoming several limiting steps required for efficient transduction of double vector-based systems, including: proper DNA concatemer formation, heterogeneous mRNA stability, and splicing efficiency between vector junctions.

[0022] The present invention provides a vector system for expressing a coding sequence in a cell, said coding sequence consisting of a first part (CDS1), a second part (CDS2) and, optionally, a third part (CDS3), said vector system comprising: a) a first vector comprising: - the aforementioned first portion of the aforementioned encoding sequence (CDS1), - a first nucleotide sequence of intein that codes for an N-intein, with said sequence located at the 3' end of CDS1; and b) a second vector comprising: - the aforementioned second portion of the aforementioned encoding sequence (CDS2), - a second nucleotide sequence of intein that codes for a C-intein, said sequence being located at the 5' end of CDS2; whereby when the first vector and the second vector are inserted into a cell, the protein product of the coding sequence is produced by protein splicing; or the aforementioned vector system comprising: a') a first vector comprising: Petition 870260074145, dated 07 / 24 / 2026, page 21 / 259 10 / 192 - the aforementioned first portion of the aforementioned encoding sequence (CDS1),

[0023] - a first intein nucleotide sequence encoding a first N-intein, said sequence being located at the 3' end of CDS1; and b') a second vector comprising: - the aforementioned second portion of the aforementioned encoding sequence (CDS2), - a second nucleotide sequence of intein that codes for a first C-intein, with said sequence located at the 5' end of CDS2; - a third nucleotide sequence of intein that codes for a second N-intein, with said sequence located at the 3' end of CDS2; and c') a third vector comprising: — the aforementioned third part of the said encoding sequence (CDS3) - a fourth intein nucleotide sequence that codes for a second C-intein, said sequence being located at the 5' end of CDS3, wherein the first intein nucleotide sequence is different from the third intein nucleotide sequence and the second intein sequence is different from the fourth intein nucleotide sequence, wherein when the first vector, the second vector, the third vector are inserted into a cell, the protein product of the coding sequence is produced by protein trans-splicing.

[0024] Preferably, in the vector system, the first integer, the second integer, the third integer and the Petition 870260074145, dated 07 / 24 / 2026, page 22 / 259 11 / 192 fourth protein codes for a split protein, preferably, said split protein has a maximum length of 150 amino acids, more preferably, said split protein is a DnaE or DnaB protein.

[0025] According to the present invention, an intein is a segment of a protein that is capable of excising itself and joining the remaining portions (the exteins) with a peptide bond in a process known as protein splicing. The segments are called inteins for the inner protein sequence and exteinos for the outer protein sequence, with the upstream exteins called N-exteins and the downstream exteins called C-exteins, the upstream intein called N-intein and the downstream intein called C-intein.Therefore, in the context of the present invention, an N-intein is an intein fragment located at the N-terminus of (and fused with) the first polypeptide, and a C-intein is an intein fragment located at the C-terminus of (and fused with) the second polypeptide, wherein after the expression of the two polypeptides, the two intein fragments undergo protein trans-splicing and are joined to form a complete intein, and the two polypeptides are joined, wherein when the two polypeptides form a full-length protein, said full-length protein is reconstituted.

[0026] According to the present invention, the first intein sequence is an N-intein sequence and the second intein sequence is a C-intein sequence, wherein said N-intein and said C-intein are preferably derived from the same intein or gene split. Petition 870260074145, dated 07 / 24 / 2026, page 23 / 259 12 / 192 intein. Alternatively, said N-intein and said C-intein derive from two different intein genes that are capable of undergoing the trans-splicing reaction naturally or are modified for this purpose. Consequently, the same gene may be from the same organism or from different organisms. For example, the widely used split inteins derive from the DnaE gene of different organisms. According to the present invention, when the coding sequence of the protein of interest is split into two portions, the N-intein coding sequence is fused in frame with the coding sequence for the N-terminal portion of the protein of interest; the C-intein coding sequence is fused in frame with the coding sequence for the C-terminal portion of the sequence of interest. After expression of the two precursor fusion proteins, the inteins undergo autocatalytic excision and form a linked extein, for example, the reconstituted protein of interest.

[0027] According to the present invention, the coding sequence of the protein of interest can be divided into three portions. Consequently, the first intein sequence is an N-intein sequence and the second intein sequence is a C-intein sequence, wherein the first intein coding sequence is fused into the C-terminus structure to the coding sequence for the N-portion of the protein of interest, and the second intein coding sequence is fused into the N-terminus structure to the coding sequence for the intermediate portion of the protein of interest. Consequently, said N-intein and said C-intein are preferably Petition 870260074145, dated 07 / 24 / 2026, page 24 / 259 13 / 192 derived from the same intein gene or split intein gene. Alternatively, said N-intein and said kintein derive from two different intein genes that are capable of undergoing the trans-splicing reaction naturally or are modified for this purpose. Consequently, the same gene may be from the same organism or from different organisms. Within the present configuration, the third intein is an N-intein coding sequence fused in-frame to the coding sequence for the C-terminus of the mid-portion of the protein of interest, and the fourth intein is a C-intein coding sequence fused in-frame to the sequence coding for the N-terminus of the C-portion of the protein of interest. Consequently, said third and fourth inteins are preferably derived from the same intein or split intein gene.Alternatively, said N-intein and said Kintein derive from two different intein genes that are capable of undergoing the trans-splicing reaction naturally or are modified for this purpose. Consequently, the same gene may be from the same organism or from different organisms. Within the scope of the present invention, said first and second inteins and said third and fourth inteins derive from different intein genes, and the first intein selectively ligates to the second intein, while the third intein selectively binds to the fourth intein.

[0028] In the present invention, when the first vector, the second vector and, optionally, the third vector are inserted into a cell, at least two fusion proteins or three fusion proteins are formed and upon entering Petition 870260074145, dated 07 / 24 / 2026, p. 25 / 259 14 / 192 contact with the aforementioned two or three fusion proteins, the protein product of the coding sequence is produced. The contact step is performed under conditions that allow the N-intein to bind to the C-intein.

[0029] In the present invention, when the first vector, the second vector, and the third vector are inserted into a cell, three independent polypeptides are produced and the full-length protein is produced via transsplicing. Fundamental to the development of the three AAV intein vectors has been the use of different inteins, i.e., DnaE and DnaB, which do not exhibit cross-reaction, thus avoiding improper trans-splicing between the polypeptides produced by the first and third vectors.

[0030] According to preferred embodiments of the present invention, a vector system for expressing the coding sequence of a gene of interest in a cell comprises two vectors, each vector comprising a portion of said coding sequence flanked by an intein sequence, wherein the 5' end of said coding sequence is flanked at the 3' end by an N-intein sequence, and the 3' end of the coding sequence of the gene of interest is flanked by a C-intein sequence, such that when both vectors are expressed in a cell, two fusion proteins are produced and the full-length protein of interest is generated as a result of a spontaneous trans-splicing reaction.

[0031] According to another embodiment Petition 870260074145, dated 07 / 24 / 2026, page 26 / 259 15 / 192 preferred version of the invention, the vector system for expressing the coding sequence of a gene of interest in a cell comprises three vectors, each vector comprising a portion of said coding sequence flanked by an intein sequence, wherein the coding sequence is divided into three portions such that the 5' end of said coding sequence is flanked at the 3' end by a first N-intein sequence; the middle portion of said coding sequence is flanked at the 5' end by a first C-intein, and at the 3' end with a second N-intein;The 3' portion of the aforementioned coding sequence is flanked at the 5' end by a second C-intein, so that when all three vectors are expressed in a cell, three fusion proteins are produced and the full-length protein of interest is generated as a result of a spontaneous trans-splicing reaction, in which the first N-intein reacts with the first C-intein and the second N-intein reacts with the second C-intein.

[0032] The split inteins of the invention may be encoded by a gene that is then modified to encode two separate fragments of inteins, for example, split inteins; alternatively, naturally occurring split inteins are encoded by two separate genes; for example, in cyanobacteria, DnaE, ​​the catalytic α subunit of DNA polymerase III, is encoded by two separate genes, dnaE-n and dnaE-c. Preferred inteins within the present invention are inteins derived from protein inteins (e.g., mini inteins) or inteins Petition 870260074145, dated 07 / 24 / 2026, page 27 / 259 16 / 192 divided into proteins that form integers through the trans-splicing reaction, which are 150 aa in length or less.

[0033] The split inteins of the invention may be 100% identical, 98%, 80%, 75%, 70%, 65%, 60%, 55%, 50% identical to naturally occurring inteins or SEQ ID No. 1 to 14 (homologous), wherein said inteins retain the ability to undergo trans-splicing reactions. Within the scope of the present invention are fragments or variants of naturally occurring or modified inteins that retain trans-splicing activity.

[0034] Conveniently, the split inteins of the invention can be derived from the same gene isolated from different organisms. Preferred intein genes are Dna B and Dna E.

[0035] In a preferred embodiment, the intein of the invention is a split intein derived from the DnaE gene (e.g., alpha subunit of DNA polymerase III) of cyanobacteria including Nostoc punctiforme (Npu), Synechocystis sp. PCC6803 (Ssp), Fischerella sp. PCC 9605, Scytonema tolypothrichoides, Cyanobacteria bacterium SW_9_4 7_5, Nodularia spumigena, Nostoc flagelliforme, Crocosphaera watsonii WH 8502, Chroococcidiopsis cubana CCALA 043, Trichodesmium erythraeum; preferably, the intein of the invention is derived from the DnaE gene isolated from Nostoc punctiforme or Synechocystis sp. PCC6803.

[0036] In another preferred embodiment, the intein of the invention is a split intein derived from the DnaB gene of cyanobacteria including R. marinus (Rma), Synechocystis sp. PC6803 (Ssp), Porphyra purpurea Petition 870260074145, dated 07 / 24 / 2026, p. 28 / 259 17 / 192 chloroplast (Ppu) which are described for example in (59).

[0037] Preferably, - the first nucleotide sequence of the intein codes for an intein selected from the group consisting of: SEQ. ID No 1, 3, 5, 7, 9, 11, 13 or a variant thereof or a fragment thereof or a homolog thereof; - the second nucleotide sequence of the intein codes for an intein selected from the group consisting of: SEQ ID No. 2, 4, 6, 8, 10, 12, 14 or a variant thereof or a fragment thereof or a homolog thereof; - the third nucleotide sequence of the intein codes for an intein selected from the group consisting of: SEQ ID No: 1, 3, 5, 7, 9, 11, 13 or a variant thereof or a fragment thereof or a homolog thereof; - the fourth nucleotide sequence of the intein codes for an intein selected from the group consisting of: SEQ ID No. 2, 4, 6, 8, 10, 12, 14 or a variant thereof or a fragment thereof or a homolog thereof.

[0038] Preferably, wherein when the first or third inteina is SEQ ID: 1, the second or fourth is SEQ ID: 2; or when the first or third inteina is SEQ ID: 3, the second or fourth inteina is SEQ ID: 4; or when the first or third inteina is SEQ ID: 5, the second or fourth is SEQ ID: 6; or when the first or third inteina is SEQ ID: 7, the second or fourth is SEQ ID: 8; or when the first or third inteina is SEQ ID: 9, the second or fourth is SEQ ID: 10; or when the first or third inteina is SEQ ID: 11, the second or fourth is SEQ ID: 12.

[0039] Preferably, when the first integer is Petition 870260074145, dated 07 / 24 / 2026, page 29 / 259 18 / 192 If the first integer has SEQ ID: 1 and the second is SEQ ID: 2, the third integer is not SEQ ID: 1 and the fourth integer is not SEQ ID: 2; preferably, if the first integer has SEQ ID: 3 and the second is SEQ ID: 4, the third integer is not SEQ ID: 3 and the fourth is not SEQ ID: 4; preferably, if the first integer has SEQ ID: 5 and the second is SEQ ID: 6, the third integer is not SEQ ID: 5 and the fourth is not SEQ ID: 6; preferably, if the first integer has SEQ ID: 7 and the second is SEQ ID: 8, the third integer is not SEQ ID: 7 and the fourth is not SEQ ID: 8; Preferably, when the first internal part is SEQ ID: 9 and the second internal part is SEQ ID: 10, the third internal part is not SEQ ID: 9 and the fourth internal part is not SEQ ID: 10; preferably, when the first internal part is SEQ ID: 11 and the second internal part is SEQ ID: 12, the third internal part is not SEQ ID: 11 and the fourth internal part is not SEQ ID: 12.

[0040] In a particular embodiment, the first inteina is SEQ ID: 1, the second inteina is SEQ ID: 2, the third inteina is SEQ: ID 3, the fourth inteina is SEQ ID: 4; or, the first inteina is SEQ ID: 5, the second inteina is SEQ ID: 6, the third inteina is SEQ ID: 3 and the fourth inteina is SEQ ID: 4.

[0041] In a preferred embodiment, the first vector, the second vector and the third vector further comprise a promoter sequence operationally linked to the 5' end portion of said first portion of the encoding sequence (CDS1) or said second portion of the encoding sequence (CDS2) or said third portion of the encoding sequence (CDS3). Petition 870260074145, dated 07 / 24 / 2026, p. 30 / 259 19 / 192

[0042] Preferred promoters are ubiquitous, artificial or tissue-specific promoters, including fragments and variants thereof that retain transcriptional promoter activity. Particularly preferred promoters are photoreceptor-specific promoters, including photoreceptor-specific human G protein-coupled receptor kinase 1 (GRK1), interphotoreceptor retinoid-binding protein (IRBP) promoter, rhodopsin (RHO) promoter, vitelliform macular dystrophy 2 (VMD2) promoter, rhodopsin kinase promoter (RK); Other particularly preferred promoters are muscle-specific promoters, including MCK, MYODI; liver-specific promoters, including thyroxine-binding globulin (TBG), liver-specific hybrid promoter (HLP) (67); neuron-specific promoters, including hSYNl, CaMKIla; kidney-specific promoters, including Ksp-caderinl6, NKCC2.Ubiquitous promoters according to the present invention are, for example, the promoters of ubiquitous cytomegalovirus (CMV) (32) and short CMV (33). The most preferred promoters within the scope of the present invention are GRK1, TBG, CaMKIla, Ksp-caderinl6.

[0043] In a still preferred embodiment, the first vector, the second vector and the third vector further comprise a 5'-end repeating nucleotide sequence (5'-TR) and a 3'-end repeating nucleotide sequence (3'-TR), preferably, the 5'-TR is a 5'-end inverted repeating nucleotide sequence (5'-ITR) and the 3'-TR is a 3'-end inverted repeating nucleotide sequence (3'-TR). Petition 870260074145, dated 07 / 24 / 2026, page 31 / 259 20 / 192 ITR).

[0044] In a still preferred embodiment, the first vector, the second vector and the third vector further comprise a polyadenylation signal nucleotide sequence.

[0045] In a still preferred embodiment, the coding sequence is split into a first part, a second part, and optionally a third part, at a position consisting of a nucleophilic amino acid that does not fall within a structural domain or a functional domain of the encoded protein product, wherein the nucleophilic amino acid is selected from serine, threonine, or cysteine.

[0046] Preferably, at least one of the first vector, the second vector and the third vector also comprise at least one enhancer or regulatory nucleotide sequence, operationally linked to the coding sequence.

[0047] The preferred enhancer or regulatory nucleotide sequence is the chimeric IgG intron of Oglobin, the post-transcriptional regulatory element of the marmot hepatitis virus. Optionally, at least one of the first vector, the second vector, and the third vector further comprises at least one degradation signal to decrease the stability of the reconstituted intein protein.

[0048] Preferably, the said degradation signal is a CL1 degron or a PB29 degron. More preferably, the said degradation signal is ecDHFR or a fragment thereof, preferably the ecDHFR degradation signal is a DHFR variant that functions as Petition 870260074145, dated 07 / 24 / 2026, page 32 / 259 21 / 192 internal degron as described in this document. More preferably, the fragment retains the ecDHFR degradation property, preferably the property of a variant DHFR that functions as an internal degron, preferably the fragment is miniecDHFR wherein the miniecDHFR is a variant that functions as an internal degron.

[0049] Preferably, the coding sequence encodes a protein capable of correcting a pathological state or disorder, preferably the disorder is retinal degeneration, a metabolic disorder, a blood disorder, a neurodegenerative disorder, hearing loss, channelopathy, lung disease, myopathy, heart disease, muscular dystrophy.

[0050] Preferably, the coding sequence encodes a protein capable of correcting a pathological state or disorder, preferably the disorder is retinal degeneration, preferably the retinal degeneration is inherited, preferably the pathology or disease is selected from the group consisting of: retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), Stargardt disease (STGD), Usher disease (USH), Alstrom syndrome, congenital stationary night blindness (CSNB), macular dystrophy, occult macular dystrophy, a disease caused by a mutation in the ABCA4 gene. More preferably, the coding sequence is the coding sequence of a gene selected from the group consisting of: ABCA4, MY07A, CEP290, CDH23, EYS, PCDH15, CACNA1, SNRNP200, RP1, PRPF8, RP1L1, ALMS1, USH2A, GPR98, HMCN1 or a fragment thereof or an ortholog thereof or a minigene thereof with a sequence Petition 870260074145, dated 07 / 24 / 2026, page 33 / 259 22 / 192 coding fragment longer than 5 kb, i.e., a minimal gene fragment that includes one or more exons and the regulatory elements necessary for the gene to express itself in the same way as a wild-type gene fragment.

[0051] However, preferably, the coding sequence encodes a protein capable of correcting muscular dystrophy, such as Duchenne muscular dystrophy, cystic fibrosis, hemophilia A, Wilson's disease, phenylketonuria, dysferlinopathies, Rett syndrome, polycystic kidney disease, Niemann-Pick type C disease, Huntington's disease.

[0052] More preferably, the coding sequence is the coding sequence of a gene selected from the group consisting of: ABCA4, MY07A, CEP290, CDH23, EYS, PCDH15, CACNA1, SNRNP200, RP1, PRPF8, RP1L1, ALMS1, USH2A, GPR98, HMCN1 or a fragment thereof or an ortholog thereof or a minigene thereof with a coding sequence exceeding 5 kb in length, i.e., a minimal gene fragment that includes one or more of the control regions required for the gene to express itself in the same manner as a wild-type gene fragment.

[0053] Preferably, the coding sequence is the coding sequence of a gene selected from the group consisting of: DMD, CFTR, F8, ATP7B, PAH, DYSF, MECP2, PKD, NPC1, HTT or a fragment thereof or an ortholog or a minigene thereof with a coding sequence greater than 5 kb in length, i.e., a minimal gene fragment that includes one or more of the regulatory elements necessary for the gene to be expressed. Petition 870260074145, dated 07 / 24 / 2026, p. 34 / 259 23 / 192 in the same way as a wild-type gene fragment.

[0054] In a particularly preferred embodiment of the invention, the coding sequence encodes the ABCA4 gene. Preferably, said coding sequence is split at a nucleotide corresponding to aa Cysll50, Serll68, Ser 1090 of said ABCA4 protein, and a split intein is inserted at the split point.

[0055] In another preferred embodiment, the coding sequence encodes the CEP290 gene. Preferably, said coding sequence is split into a nucleotide corresponding to aa Cysl076; Serl275. More preferably, said coding sequence is split into a nucleotide sequence corresponding to aa Cys 929 and 1474; Ser 453 and Cys 1474 of said CEP290 protein, and two split inteins are inserted at the split points.

[0056] EGFP - SEQ ID No. 15

[0057] The first amino acid of c-exteine ​​is highlighted within the Split Cys.71 sequence (bold). MVSKGEELFTG WPILVELDGDVNGHK FSVSGEGEGDATYG KLTLKFJ CTTGK LPVP W PTLVTTLTYGVqCfSRYPDH MKOH D FF KSAM PEGYVQERTIF FKDOGN YKTRAE VKFEGDTLVN RIE LKGIDFKEDG NILG HKLEYNYNSHHVYIM ADKQKN Gl KVNF Kl RH NIE DGSVQUkDHYQQNTPlSDGPVLLPDNHYLSTQSALS KD PN AND KRDH M VLLEFVTAAGÍTLGM DE LYKDYKDH DGDYKD HtUDVKDDDDK*

[0058] ABCA4 - SEQ ID NO: 16

[0059] The first amino acid of c-exteine ​​is highlighted in the sequence. Split setl Cys.1150 (bold) Split set2 Ser.1168 (underlined) Split set3 Ser.1090 (italic) Petition 870260074145, dated 07 / 24 / 2026, p. 35 / 259 24 / 192 MGFVRQIQLLLWKNWTLRKRQKIRFVVELVWPLSLFLVLJWLRNANPLYSHHECHFPNKAMPSAGMLP WLQGIFCNVNNPCFQSPTPGESPGIVSNYNNSILARVYRDFQELLMNAPESaHLGRIWTELHILSQFMDT LRTHPERIAGRGIRIRDILKDEETLTLFLIKNIGLSDSVVYLLINSQVRPEQFAHGVPDLALKDIACSEALLERFII FSQR RG A KTVR YALCSLSQGTLQWi E DTLY AN VD F FK LFR VLPTLLDS RS QGIN LRS WGGILS DM SP RIQE fihrpsmqdllwvtrplmqnggpetftklmgilsdllcgypegggsrvlsfnwyednnykaflgidstr KDPIVSYDRRTTSFCNALIQSLESNPLTKIAWRAAKPLLMGKILYTPDSPAARRILKNANSTFEELEHVRKLV kaweevgpqiwyffdnstqmnmjrdtlgnptvkdflnrqlgeegitaeailnflykgpresqaddman FDWRDIFNITDRTLRLVNQYLECLVLDKFESYNDETQLTQRALSLLEENMFWAGVVFPDMYPWTSSLPP HVKYKIRMDEDWEKTNKIKDRYWDSGPRADPVEDFRYIWGGFAYLQDMVEQGITRSQVQAEAPVGIYL QQMPYPCFVDDSFMIILNRCFPIFMVLAWIYSV5MTVKSIVLEKELRLKETLKNQGVSNAVIWCTWFLDS FSIMSMSIFLLTIFIMHGRILHYSDPFILFLFLLAFSTATIMLCFLLSTFFSKASLAAACSGVIYFTLYLPHILCFA WQD RM TAELK K AVSLLSP VAFGFGTEY LVRF E EQG LG LQWSNIG NS PT EG D EFSFLLSMQM M LLD AAV YGLLAWYLDQVFPGDYGTPLPWYFLLQESYWLGGEGCSTREERALEKTEPLTEETEDPEHPEGIHDSFFER EH PG WV Ρ GV CV KNLVKIFEP CG R PAVD RLNITFYE N QITAF LGH N GAG KTTTL51LTG LLP PTSGTVLVGG RDIETSLDAVRQSLGMCPQHNILFHHLTVAEHMLFYAQLKGKSQEEAQLEMEAMLEDTGLHHKRNEEA QD LSG GM QR KLSVAIAF VGDAKV VILD E PTSG VD PYSRR SIW DLLLKY R SG RTHM5TH Η M DE AD LLG DR I AIIAQGRLYCSGTPLFLKNCFGTGLYLTLVRKMKNIQSQRKGSEGTCSCSSKGFSTTCPAHVDDLTPEQVLD GDVNELMDVVLHHVPEAKLVECIGQELIFLLPNKNFKHRAYASLFRELEETLADLGLSSFGISDTPLEEIFLKV TEDSDSGPLFAGGAQQKRENVNPRHPCLGPREKAGQTPQDSNVCSPGAPAAHPEGQPPPEPECPGPÍ1L NTGTQLV LQH V QALL V KRFQHTI RS H KDF LAQI VLP ATFVFLA LM LSI VIP P FG E YP ALTLH P WIYG QQ YTF FSM DE PG SE QFT VLA D VLLN KPG FG N RCLK EG WLP E YPCG N STP W KT P 5 VS P NITQLFQKQK WTQ VNPS PSCR CST RE KLT M LP EC P EG AGG LP PPQRTQRSTEILQD LTDRNISD FL VKTYP AL IRSSLKS KF W VN EQR YGGISIGGKLPVVPITGEALVGFL5DLGRIMNV5GGPITREA5KEIPDFLKHLETEDNIKVWFNNKGWHAL VSF LN V AH N Al LR ASLP KDRSP EE YG IT VISQP LN LTK E QLSE IT V LKSVD AW AICV1FSM SFVP AS FVL Y LI QERVNKSKHLQF] SG VSPTTY W VT NFLW DIM N YSVSAG LV VG E Fl G FQKKA YT5 P ENLP ALV ALLLLYG WA VI PM Μ YP AS F LF D VPST AYVA LSC AN LAG IN SS AITFILE LFlE'N N RT'LLR FNAV LR K LLIV F PH FC LG RG LID LALSQAVTDVYARFGEEHSANPFHWDLIGKNLFAMVVEGVVYFLLTLLLVQRHFFLSQWIAEPTKEPIVDE DDD V AEER QR11TGG N KTDI LR LH ELT KI Y PGTSSP A VD RLC VG VRPG ECFG LLG VNG AG KTTTF KM LTGD TTVTSG DATVAGK51LTNISEVHQN MGYCPQFDAf DELLTGREHLYLYARLRGVPAEEIEKVAN WSEKSLGL TVYADCLAGTYSGGNKRKLSTAIALIGCPPLLVLLDEPTGMDPQARRMLWNVIVSIIREGRAWLTSHSME ECEALCTRLAIMVKGAFRCMGTIQHLKSKFGDGYIVTMKIKSPKDDLLPDLNPVEQFFQGNFPGSVQRER HYNMLQ.FQVSSSSLARIFQLLLSHKDSLLIEEYSVTQTTLDQVFVNFAKQQTESHDLPLHPRAAGASRQAQ DDYKDHDGDYKDHDfDYKDDD* Petition 870260074145, on 07 / 24 / 2026, page. 36 / 259 25 / 192

[0060] CEP290 SEQ ID No. 17

[0061] The primary amino acid in the chest is placed in the sequence. Split setl Cys.1076 (negrito) Split set 2-3 Ser.1275 (sublinhado) Split Set4 Cys.929 and Cys.1474 (itálico) Split Set5 Ser.453 and Cvs.1474 (double underline) MPPNINWKEIMKVDPDDLPRQEELADNLLISLSKVEVNELKSEKQENVIHLFRITQSLMKMKACiEVELAL EEVEKAGEE QAKFENQLKTKVM KLEN ELEMAQQSAGGRDTRFLRNEICQLEKQLEQKDRELEDM EKELE KEKKVNEQLALRNEEAENENSKLRRENKRLKKKNEQLCQDIIDYQKQIDSQKETLLSRRGEDSDYRSQLSK KNYELIQYLDEfQTLTEANEKIEVQNQEMRKNLEESVQEMEKMTDEYNRMKAIVHQTDNVIDQLKKEND HYQLQVQELTDLLKSKNEEDDPIMVAVNAKVEEWKULSSKDDEIIEYQQMLHNLREKLKNAQLDADKSN VMALQQGIQERD5QIKMLTEQVEQYTKEMEKNTCIIEDLKNELQRNKGASTL5QQTHMKIQ5TLDILKEK TKEAERTAELAEADAREKDKELVEALKRLKDYESGVYGLEDAVVEIKNCKNQIKIRDREIEILTKE1NKLELKIS DFLDENEALRERVGLEPKTMIDLTEFRNSKHLKQQQ.YRAENQ1LLKEIESLEEERLDLKKKSRQMAQERGKR SATSGLTTEDLNLTENISQGDRISERKLDLLSLKNMSEAQSKNEFLSRELiEKERDLERSRTVIAKFQNKLKEL VEENKQLEEGMKEILQAIKEMQKDPDVKGGETSLIIPSLERLVNAIESKNAEGIFDASLHLKAQVDQLTGR NEELRQELRESRKEAÍNYSQQLAKANLK1DHLEKET5LLRQSEGSNWFKGIDLPDGIAPS5ASHNSQNEYLI HLLQELENKEKKLKNLEDSLEDYNRKFAVIRHQQSLLYKEYLSEKETWKTESKTIKEEKRKLEDQVQQDAIK VKEYNNLLNALQMDSDEMKKILAENSRKITVLQVNEKSLIRQYTTLVELERQLRKENEKQKNELLSMEAEV CEKIGCLQRFKEMAIFK1AALQKWDNSVSLSELELANKQ.YNELTAKYRDILQKDNMLVQRTSNLEHLECE NISLKEQVESINKELErTKEKLHTIEQAWEQETKLGNESSMDKAKKSITNSDiVS1SKKITMLEMKELNERQ.R AEHCQKMYEHLRTSLKQMEERNFELETKFAELTKINLDAQKVEQMLRDELADSVSKAVSDADRQRILELE KNEMELKVEVSKLREISDiARRQVEfLNAQQQSRDKEVESLRMQLLDYQAQSDEKSLIAKLHQHNVSLQLS EATALGKLESITSKLQKMEAYNLRLEQKLDEKEQALYYARIEGRNRAKHLRQTIQSLRRQFSGALPLAQQE KFSKTMIQLQNDKLKIMQEMKNSQQEHRNMENKTLEMELKLKGLEELISTLKDTKGAQKVINWHMKIEE LRLQELKLNRELVKDKEEIKYLNNIISEYERTISSLEEEIVQQNKFHEERQMAWDQREVDLERQLDIFDRQQ NEÍLNAAQKFEEATGSIPDP5LPLPNQLEIALRKIKENIRIILETRATÇKSLEEKLKEKESALRLAEQNÍLSRDKVI NELRLRLPATAEREKLIAELGRKEMEPKSHHTLKIAHQTIANMClARLNQKEEVLKKYQRLLEKAREEaREIV KKHEEDLHILHHRLELCWDSSLNKFKQTAWOLMKQSPTPVPTNKHFIRLAEMEQWAEQODSLSSLLVKL ΚΚν5α0ΕΕΡαΡΕΙΤΕΕΚνΚΕΡΕΝΙΚΕαΕαΕΝΗΕ&ΕνΚΚνΚΑΕνΕ0ΕΚΥίΕ0α$αΚΕ5ααΚ$ΕΕ0ΑαΚΕΑΝ SRAPTTTMRNLVERLKSQLALKEKQCIKALSRALLELRAEMTAAAEERIISATSQKEAHLNVCtQlVDRHTRE LKTQVEDLNENLLKLKEALKT5KNREN5LTDNLNDLNNELQKKQKAYNKILREKEEIDQENDELKRQIKRLT 5GLQGKPLTDNKQSLIEELQRKVKKLENQLEGKVEEVDLKPMKEKNAKEEURWEEGKKWQAKIEGIRNK. Petition 870260074145, of 24 / 07 / 2026, p. 37 / 259 26 / 192 LKEKEG E VRLTKQl NTL KDLFA K ADK EK LTLQRKLKTTGMTV DQ VLGIR ALESE KE LEE LKKR N LD LE N DIL YMRAH QA LP R DS WE D LH LQN RY LQE K LH A LE K QFSK DTYSK PSISK DDESGI QELQRSKEN LKSSEN LKSS IE L KF QLEQAN KDLPR LK N QV RD LKE M CE F LCK EK AEV QR KLG H VRGSG R SG KTI PELE KTIG LM KK VV EK VQRENEQLKKASGILTSEKMANIEQENEKLKAELEKKHLKAHLGHQLSMHYESKKGTE KELEKLKAHKLKLKGTE I LN ΕΚ M TVQLE ETGK R LQF AE Ξ RG PQLEG AD S KS WKS1WTRMYETKLK ELET DIA CK N QSITDLK QLVK E ATE REQKVN KY N EDLEQQIKI LK H VP E GA ETEQG LK EKL EKL QL KL A H E AN KDQSG AESTI PD ADQLKEKIKD LETQLK M SDL E KQH LKE EI KK LK KELE N FDPS F FE EIE D LKY NYK EEVKKNILLEEKVKKLSEQLGVELTSPVAASEEFEDEEESPVNFPIYDYKDGDYKDHIDDKDDDKDKDK*

[0062] F8 SEQ ID NO. 18

[0063] The first amino acid of c-extein is highlighted in the sequence. Split set Cys.1312 (underlined) set Split set2 Ser.984 (bold) M Ql ELSTCFF LC LLR FCFS ATR RYYLG AV E L5 W DYM QS D LG EL P VDA RFPPRVPKSFPF NTS WY KKTLF AND FTDH LFNIA KP RPPWMG LLG PTIQA E VYDTV VITLK N MASH P VS LHAVG VEG DDS QTS A QY AS WY ARE KEDDK VF PGGS HTYV WQVLKENG PM ASD P LC LTYSY LS H VDLV K DLN SG LIG ALLVC REGS LAK E KTQTL H KF ILLF A VF D EG KS W HS ETK NS LM QD RDA AS ARA W PK Μ Η Η GN VN VG LW CHR VSG VSL VSL V VIG MGTTP E VHSIF LE G HT FLV RNHR QA5 LEIS PITF LTAQTLLM DLGQFLLFCH ÍSSH QH DG Μ EAY VK VD SCP EE PQLRM KN N EEA E DYD □ DLTDSÉ MO WRF DDDN SPSFI Ql RSVA K H A K H E W H E W OR PL V LAP DD RSY KSQYLNNG P QR | G RK YK KV R FM A YTD ETF KTRE AIQH E SG I LG PLLYG E VG DTL LI IFK N QAS RPYNIYP HGITD VRP LYS RR LPKG VKH LK DF Pl LPG EIFKYK WTVTV E DG PT KS D PRCLTRYY MESS V RDF V LIG LIG LIGK VDQRG N Ql M 5 D KR NV IL FSVFD EN RS WY LTE NIQR F LP NP AG VQLEDP EF QAS NI Μ H SINGYV FDSLQLS VCLH EV AY WYl LStG AQTDF LS VF FSGYT FK Η K Η K Μ Μ Μ TL Μ FL DSMGE TL FS FS PFSPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQK QFN ATTI PEN Dl E KT DP WF AH RTP MP KIQN VS 5SDLLM LLR QS PTP HG LSLSD LQEA KY ESNTF SPG DLP QNS AI EMT AI FRP HRP H HSG D Μ V FTP E SG LQLR LN E KLGTTA ATE LK K LD FK VSSTS NN LI STi PS DN LAA GTD NTSS LG P PSM PV HY DSQLDTTLFG K KS SP LTESGG PLSLS EEN N D5 KLLESG LM N 5QESS W RLSSK PAG KRLESK GRN SSG KSGTE LLTK DNA LF KVSISLLKT N KTSN N SATN R KTHI DG PSLL! E NSP Ξ VWQNI AND SDTE FKK VTP LI HDRM LM DKN ATA LR LN Η M 5 N KTTSSKN M EM VQQCKEG P IP PD AQN P DM SF F KM LF LPES AR WIQRTH GK N SLNSGQG PS P KQLV LPES GK GK VQN WQN G KG E FTKDVG LK E Μ V FP SSR N LF LTN LD N LH EN NTH NQE KK IQEEI ΕΚ K ETLIQE N VV LPQJ HTVTGTK NF Μ KN LFLLST RQN VE GS YDG AY APV LQD F RSLN DSKTN HRTK KH KHENK FGEEI LEG LG N QTK Ql VE KYACHKR ISP NTSQQN F VTQRS KRA LK QF RLP LE ETELE CRIIV D DT5TQW 5 KN Μ KH LTPST LTQID YN EKE KG AITQ5 P L5 DC LTR S HSi P QAN RSP LP IA CSI K R VQL VQLTRSSH LP AA5Y R KK DSG VQESSH FLQG AK KN N LSLAILTL EM TG DQR E VG SL GTS ATN S VTY K KVENTVLP KP DL P KTSG K VELLP KVHIYQK D LF PTETSNG S PG H LD LVE GSLLQG GTE KPF A KV R PG LR VATESSA KT PS KL LD PLA WD N HYGTQi PKEE WKSQEK S PE KTA FKK KDT! LSLN AC ESN H Al AA IN EG QN KP EEE VT WA KQGRTE R LCSQN PP VLK RH QR E IT RTTLQSDQE EIDYDDTIS VEM K KE DFDIY DE DENQSP RS FQK KT RHY FIA AVE RL WDYG M SSSPQ AFQ VK RFQ Η KLRWSG VFQE FT DGS FT QP LYRG E LN EH LG LLG PYIR AE WE DN t M VT FRNQAS RP VSFYSSU SYE E DQR QG AE PRK NF VK PN ETKTY F Petition 870260074145, of 24 / 07 / 2026, p. 38 / 259 27 / 192 WK VQH Η Μ APTK DE FD C KA WAV FSDV D LE KD VH SGLIG P LL VC HTN TLN PA HG RQVTVQE F ALFFTi FDE TKS WY FTE N Μ ERN CRA PCNIQM E 0 PTF KENYR FH Al N GYM D LPGRVM WSMGRILL SN E NI H SIH FSGH VFTVRKK E EYK M ALY N LYPG V FETVE M LPSKAGIW R VECLl GE H LH AG M STLFLVYS N KCQT P LG M ASG Hi R DF QI TASGQYG QW AP K LARL HY SGSIN A WST KEP FS WIK 1GDL Μ Μ LIKH AR QK F SSLYI SQF 11MYSLDG KK W QTYRG N 5TGTLM V FFG N VDSSGIKHN1FNPPIIARYIR LH PTHYSI RSTl+lM ELM GC D LN SCS Μ P LG Μ E SK AIS D AQ.IT ASSY FAT M WRPSN WRPSN LQ LQ A LQ A L. QVN NPKEW LQV DF QKT Μ K VTGVTTQG VKSLLTSMY VKEFLISSSQDG H QWTLFFQN GK VKV FQG N QD S FTP WNSLD PP LLTR Y LRIH PQS WVHQIALRM E VLG CE AQDLY *

[0064] ecDHFR SEQ ID no. 19 MiSLiAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESiGRPLPGRKNI ILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFLPKAQXLYLTHIDAEVE GDTHFPDDYEPDWESVFSEFHDADAQNSSYCFEILER*

[0065] mini ecDHFR SEQ ID no. 20 MISILIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWÊSÈGRPLPGRKNI ILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFLP*

[0066] In a preferred embodiment, the vector system of the invention comprises: a) a first vector comprising the 5'-3 direction - a reversed terminal repeat sequence 5' (5'ITR); - a promotional sequence; - a portion of the 5' end of an encoding sequence (CDS1), said portion of the 5' end being operationally linked and under the control of said promoter; - a first nucleotide sequence called an intein that codes for an N-intein; and - a reversed terminal repeat sequence 3' (3'- ITR); and b) a second vector comprising the 5'-3 direction - a reversed terminal repeat sequence 5' (5'- Petition 870260074145, dated 07 / 24 / 2026, page 39 / 259 28 / 192 ITR); - a promotional sequence; - a second nucleotide sequence called an Intein that codes for a C-Intein; - a 3' end portion of the encoding sequence (CDS2); and - a reversed terminal repeat sequence 3' (3'- ITR); or includes: a') a first vector comprising the direction 5'-3': - a reversed terminal repeat sequence 5' (5'- ITR); - a promotional sequence; - a portion of the 5' end of an encoding sequence (CDS1'), said portion of the 5' end being operationally linked and under the control of said promoter; - a first nucleotide sequence called an Intein that codes for a first N-Intein; and - a reversed terminal repeat sequence 3' (3'- ITR); and b') a second vector comprising the 5'-3' direction: - a reversed terminal repeat sequence 5' (5'- ITR); - a promotional sequence; - a second nucleotide sequence called an Intein that codes for a first C-Intein; - the second part of the encoding sequence (CDS2'); and Petition 870260074145, dated 07 / 24 / 2026, page 40 / 259 29 / 192 - a third nucleotide sequence called an Intein that codes for a second N-Intein; - a reversed terminal repeat sequence 3' (3'- ITR); and c') a third vector comprising the 5'-3' direction: - a reversed terminal repeat sequence 5' (5'- ITR); - a promotional sequence; - a fourth nucleotide sequence called an intein that codes for a second C-intein; - the third portion of the encoding sequence (CDS3'); and - a reversed terminal repeat sequence 3'(3'- ITR).

[0067] Preferably, said first, second and third vectors are independently a viral vector, preferably an adenoviral vector or viral adeno-associated vector (AAV), preferably said first, second and third viral adeno-associated vectors (AAVs) are selected from the same or different AAV serotypes, preferably the serotype is selected from serotype 2, serotype 8, serotype 5, serotype 7 or serotype 9, serotype 7m8, serotype shlO; serotype 2 (quad YF). The present invention also provides a host cell transformed with the vector system as defined above.

[0068] Preferably, the vector system or host cell is for medical use, preferably for use in gene therapy, preferably for use in the treatment and / or prevention of a pathology or disease characterized by Petition 870260074145, dated 07 / 24 / 2026, page 41 / 259 30 / 192 retinal degeneration, a metabolic disorder, a blood disorder, a neurodegenerative disorder, hearing loss, channelopathy, lung disease, myopathy, heart disease, muscular dystrophy.

[0069] Preferably, retinal degeneration is inherited, preferably the pathology or disease is selected from the group consisting of: retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), Stargardt disease (STGD), Usher disease (USH), Alstrom syndrome, stationary congenital night blindness (CSNB), macular dystrophy, occult macular dystrophy, a disease caused by a mutation in the ABCA4 gene.

[0070] Preferably, the vector system or host cell is for use in the prevention and / or treatment of Duchenne muscular dystrophy, cystic fibrosis, hemophilia A, Wilson's disease, phenylketonuria, dysferlinopathies, Rett syndrome, polycystic kidney disease, Niemann-Pick type C, Huntington's disease.

[0071] The present invention also provides a pharmaceutical composition comprising the vector system or host cell of the invention and a pharmaceutically acceptable vehicle. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Fig. 1 - AAV inteina reconstitutes EGFP in vitro and in the retina of mice and pigs at levels that are higher than double AAV and even higher than those achieved with a single AAV. (A) Schematic representation of AAV interenin-mediated protein trans-splicing. ITR: repeats Petition 870260074145, dated 07 / 24 / 2026, page 42 / 259 31 / 192 inverted terminals of AAV2; CDS: encoding sequence; ·: Tag 3xflag; PolyA: polyadenylation signal. (B) Western blot (WB) analysis of HEK293 lysates transfected with full-length CMV-EGFP intein or AAV plasmids. pEGFP: full-length EGFP plasmid; pAAV l + ll: AAV-EGFP intein plasmids l + ll; pAAV I: single AAV-EGFP I intein plasmid; pAAV II: single AAV-EGFP II intein plasmid; Neg: untransfected cells. Arrows indicate both the full-length EGFP protein (EGFP), the N- and C-terminal halves of the EGFP protein (B and A, respectively), and the reconstituted intein excised from the full-length EGFP protein (C). The WBs are representative of n = 3 independent experiments. (C) WB analysis of HEK293 lysates infected with single, integer, or double AAV2 / 2-CMV-EGFP vectors. The WB are representative of n = 5 independent experiments. (D) Retinal cryosections of C57BL / 6J mice injected subretinally with AAV2 / 8-CMVEGFP intein vectors. Scale bar: 50 hours. RPE: retinal pigment epithelium; OS: outer segments; ONL: outer nuclear layer. (EF) Retinal cryosections of C57BL / 6J mice (E) or Large White pigs (F) injected subretinally with single, intein, or double AAV2 / 8-GRK1-EGFP vectors. Scale bar: 50 pm (E); 200 pm (F). OS: outer segment; ONL: outer nuclear layer. (G) Fluorescence analysis of retinal organoids infected with AAV2 / 2-GRKl-EGFP-intein vectors at 293 days of culture. Scale bar: 100 pm. Petition 870260074145, dated 07 / 24 / 2026, page 43 / 259 32 / 192

[0073] Fig. 2 - Optimization of the AAV protein allows for the appropriate reconstitution of the large ABCA4 and CEP290 proteins. (AB) Western blot (WB) analysis of HEK293 lysates transfected with different sets of AAVshCMV-ABCA4 or -CEP290 intein plasmids (set 1 and set 5, respectively). A schematic representation of the various sets used is illustrated in Fig. 16. The WBs are representative of n = 3 independent experiments. (CD) Representative images of immunofluorescence analysis of HeLa cells transfected with AAV-shCMV-ABCA4 (C) or AAV-shCMV-CEP290 (D) intein plasmids. pABCA4 (C) or pCEP290 (D): plasmid including full-length expression cassette; pAAV intein: AAV-intein plasmids (Set 1 in C or Set 5 in D); l + ll + lll: AAV l + ll + lll intein plasmids; l + ll: AAV l + ll intein plasmids; l + lll: AAV l + lll intein plasmids; ll + lll: AAV ll + lll intein plasmids; I: single AAV I intein plasmid; II: AAV intein plasmid II: single; III: single AAV III plasmid; Neg: untransfected cells. Cells were stained for 3xFLAG and VAP-B (endoplasmic reticulum marker) and TGN46 (trans-Golgi network marker) in C, or acetylated tubulin (microtubule marker) in D. White arrows point to the cells shown at higher magnification in Fig. 18.

[0074] Fig. 3 - The AAV interin reconstitutes the large ABCA4 and CEP290 proteins more efficiently than the double AAV vectors. Western blot (WB) analysis of cell lysates Petition 870260074145, dated 07 / 24 / 2026, page 44 / 259 33 / 192 HEK293 infected with AAV2 / 2-shCMV-ABCA4 (A) or CEP290 (B) double or intein vectors. AAV integer vectors: AAV-ABCA4 (set 1, A) or -CEP290 (set 5, B) integer vectors; l + ll + lll: AAV l + ll + l11 integer vectors; l + ll: integer vectors AAV l + ll; l + lll: integer vectors AAV l + lll; ll + lll: integer vectors AAV ll + lll; I: single vector interior AAV I; II: single vector interior AAV II; III: single vector interior AAV III; Double AAV: double AAV vectors; Neg: AAV-EGFP vectors. (A) The arrows indicate the full-length ABCA4 protein and A: protein product derived from AAV I; B: protein product derived from AAV II. * protein product with a potentially different post-translational modification. (B) The arrows indicate the full-length CEP290 protein and A: protein product derived from AAV II + III; B: protein product derived from AAV I + II; C: protein product derived from AAV II; D: protein product derived from AAV III; E: protein product derived from AAV I. The WB are representative of n = 3 independent experiments.

[0075] Fig. 4 - AAV protein reconstitutes large proteins in mouse, pig, and human photoreceptors at therapeutic levels. (AC) Western blot (WB) analysis of retinal lysates from wild-type mice (A, B) or large white pigs (C) injected with AAV2 / 8-GRK1-ABCA4 (A, C) or CEP290 vectors (B) (set 1 and set 5, respectively). AAV intein: AAV intein vectors; AAV double: AAV double vectors; Neg: AAV-EGFP or PBS vectors. (D) WB analysis of 3D retinal organoid lysates Petition 870260074145, dated 07 / 24 / 2026, page 45 / 259 34 / 192 derived from human iPSCs infected with AAV2 / 2-GRK1-ABCA4 intein vectors. AAV intein: AAV-ABCA4 intein vectors; Neg: uninfected organoids; - / -: organoids derived from STGD1 patients. (A, C, D) The arrows indicate the full-length ABCA4 protein (ABCA4) and A: protein product derived from AAV I; B: protein product derived from AAV II. * protein product with a potentially different post-translational modification. (B) The arrows indicate the full-length CEP290 protein (CEP290); A: protein product derived from AAV II + III and D: protein product derived from AAV III.

[0076] Fig. 5 - Subretinal administration of AAV intein improves the retinal phenotype of mouse models of hereditary retinal degenerations. (A) Quantification of the average area occupied by lipofuscin in the RPE of AbcaΛ mice treated with AAV intein. Each point represents the average value measured for each eye. The average lipofuscin area for each group is indicated in the graph. + / + or + / - ·. Abca4 injected control <+ / +> or <+ / > eyes (PBS); - / -: AbcaΛ eyes injected with negative control (AAV I ABCA4 or AAV II ABCA4 or PBS); - / - AAV intein: AbcaΛ eyes injected with AAV intein vectors (set 1). * ANOVA p-value <0.05; *** ANOVA p-value <0.001. (B) Representative images of retinal sections from uninjected wild-type mice and rdl6 mice injected subretinally with AAV2 / 8-GRK1-CEP290 intein vectors (AAV intein, set 5) or injected with controls Petition 870260074145, dated 07 / 24 / 2026, page 46 / 259 35 / 192 negatives (Neg; i.e., AAV I + II or AAV II + III or PBS). Scale bar: 25 miti. The ONL thickness measured in each image is indicated by the vertical black line. RPE: retinal pigment epithelium; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. (C) Representative images of wild-type uninjected and rdl6 subretinally injected mouse eyes with AAV2 / 8-GRK1-CEP290 intein vectors (AAV intein, set 5) or injected with negative controls (Neg; i.e. AAV l + ll or AAV ll + lll or PBS). White circles define the pupils.

[0077] Fig. 6 - Schematic representation of the trans-splicing-mediated reconstitution of a large protein The coding sequence (CDS) of a large gene is split into two halves (5' and 3'), flanked by inverted terminal repeats (ITRs), which are packaged separately into two AAV capsids. After co-transduction in the same cell, different mechanisms are exploited to reconstitute full-length protein expression by joining the two halves at the protein level. The 5' vector includes the 5' CDS, the 5' intein (n-intein), and the degron, while the 3' vector includes the 3' CDS and the 3' intein (c-intein); both vectors include the promoter and polyA. Pairing of the two polypeptide halves is mediated through intein self-recognition; subsequent intein self-excision from the host protein results in full-length protein reconstitution. The degron, now incorporated into the excised intein, is rapidly... Petition 870260074145, dated 07 / 24 / 2026, page 47 / 259 36 / 192 ubiquitinated and degraded by the proteasome.

[0078] Fig. 7 - In vitro EGFP expression of AAV internal vectors with and without degradation signal. Western blot (WB) analysis of lysates from HEK293 cells transfected with AAV intein plasmids containing ecDHFR (+) or not (-). Arrows indicate full-length EGFP protein (EGFP), excised intein containing degron (DnaE + ecDHFR) or not (DnaE). [007 9] Fig. 8 - In vitro ABCA4 expression of AAV internal vectors with and without degradation signal. Western blot (WB) analysis of lysates from HEK293 cells transfected with AAV intein plasmids containing ecDHFR (+) or not (-). Arrows indicate full-length ABCA4 protein (ABCA4), excised intein containing degron (DnaE + ecDHFR) or not (DnaE).

[0080] Fig. 9 - The expression of DnaE-ecDHFR Intein is TMP dependent. Western blot (WB) analysis of lysates from HEK293 cells transfected with AAV_ABCA4 intein plasmids containing ecDHFR (pAAV intein + ecDHFR) or not (pAAV intein) and treated with an increased dose of trimethoprim (from 1 to 50 0mI). Arrows indicate excised intein containing degron (DnaE + ecDHFR) or not (DnaE).

[0081] Fig. 10 - In vitro EGFP expression of AAV integer vectors with and without degradation signal. Western blot (WB) analysis of lysates from HEK293 cells transfected with AAV intein plasmids containing mini ecDHFR (+) or not (-). Arrows indicate the full-length EGFP protein (EGFP), the excised intein containing the Petition 870260074145, dated 07 / 24 / 2026, page 48 / 259 37 / 192 degron (DnaE + mini ecDHFR) or not (DnaE).

[0082] Fig. 11. In vitro ABCA4 expression of AAV integer vectors with and without degradation signal. Western blot (WB) analysis of lysates from HEK293 cells transfected with AAV intein plasmids containing mini ecDHFR (+) or not (-). Arrows indicate full-length ABCA4 protein (ABCA4), excised intein containing degron (DnaE + mini ecDHFR) or not (DnaE).

[0083] Fig. 12 - EGFP fluorescence in cells HEK293 transfected with AAV I + II, but not with individual AAV I or AAV II intein plasmids. Fluorescence analysis of HEK293 cells transfected with full-length or intein CMV-EGFP plasmids. pEGFP: plasmid including full-length EGFP expression cassette; pAAV l + ll: AAV l + ll intein plasmids; pAAV I: single intein plasmid of AAV I; pAAV II: single intein plasmid of AAV II; Neg: untransfected cells. Scale bar: 100 µm.

[0084] Fig. 13 - Protein content as a percentage of total protein length varies between species. Western blot (WB) analysis of lysates from HEK293 cells (A), C57BL / 6J mice (B), and large white pig retinas (C) infected with AAV-CMV-EGFP vectors (A) or AAV-GRK1-EGFPin vectors (BC). AAV intein: infected cells (A) or eyes injected (B, C) with AAV intein vectors; Neg: uninfected cells (A) or eyes injected with PBS (B, C). Arrows indicate full-length EGFP protein (EGFP) and excised intein (DnaE). Petition 870260074145, dated 07 / 24 / 2026, page 49 / 259 38 / 192

[0085] Fig. 14 - Characterization of 3D retinal organoids derived from human iPSCs. (A) Light microscopy analysis of retinal organoids after 183 days of culture. (B) Immunofluorescence analysis with antibodies targeting mature photoreceptor markers. Scale bar: 100 pm. (C) Fluorescence analysis of retinal organoids infected with AAV2 / 2-CMV-EGFP and AAV2 / 2-IRBPDsRed vectors. Scale bar: 100 mih. (D) Outer segment-like structures were observed projecting from the surface of retinal organoids at 230 days of culture. The inset shows the presence of outer segment-like (OS) structures with radial architecture. NR: neural retina; RPE: retinal pigment epithelium. (E) Scanning electron microscopy analysis reveals the presence of inner segments (IS), connecting cilia (CC), and outer segment-like structures (OS). Scale bar: 16h. (F) Electron microscopy analysis reveals the presence of the outer limiting membrane (*), centriole (C), basal bodies (BB), connecting cilia (CC) and outlines of outer segments (OS). The inset shows the presence of disorganized membranous discs in the OS. Scale bar: 500 nm. D: Days of Culture.

[0086] Fig. 15 - Low protein content relative to total protein length in human 3D retinal organoids. Petition 870260074145, dated 07 / 24 / 2026, page 50 / 259 39 / 192 Western blot (WB) analysis of 3D retinal organoid lysates derived from human iPSCs infected with AAV2 / 2-GRK1-EGFP intein vectors. AAV intein: AAV intein vectors; Neg: uninfected organoids. Arrows indicate full-length EGFP protein (EGFP) and excised intein (DnaE).

[0087] Fig. 16 - Schematic representation of the various AAV-ABCA4 and -CEP290 integer sets. (A) AAV-ABCA4-intein constructs. (Set 1-2, as exemplified by the construct) n-DnaE: DnaE n-intein from Npu; c-DnaE: Npu DnaE c-intein; (Set 3) n-mDnaE: Npu-mutated DnaE n-intein (mNpu); c-mDnaE: mNpu DnaE cystein. (B) AAV-CEP290-intein constructs. (Set 1) nDNAE: Npu DnaE n-intein; c-DnaE: DnaE c-intein from Floor; shPolyA: short synthetic polyA; (Set 2) n-DNAE: DnaE nintein from mNpu; c-DNAE: mNpu DnaE c-intein; (Set 3) n-mDNAE: mNpu DnaE n-intein; c-mDNAE: mNpu DNAE cysteine; (Set 4) n-DnaE: Npu DnaE n-intein; c-DnaE: DnaE c-intein of Npu between AAV I and AAV II; n-DnaB: N-intein of DnaB from Rhodothermus marinus (Rma); c-DnaB: Rma DnaE c-intein between AAV II and AAV III; wpre: Marmot hepatitis virus post-transcriptional regulatory element. (Set 5) n-mDNAE: mNpu DnaE n-intein; c-mDNAE: mNpu DnaE c-intein between AAV l and AAV II; n-DnaB: n-intein of DnaB from Rhodothermus marinus (Rma); c-DnaB: Rma DnaE c-intein between AAV II and AAV III; wpre: Marmot hepatitis virus post-transcriptional regulatory element. (AB) ITR: terminal repeats Petition 870260074145, dated 07 / 24 / 2026, page 51 / 259 40 / 192 inverted AAV2; Tag 3xflag; Promoter: short CMV for in vitro experiments and human G protein-coupled receptor (GRK1) promoter for in vivo experiments; PolyA: simian virus 40 polyadenylation signal (for ABCA4, A) and bovine growth hormone polyadenylation signal (for CEP290, B). The amino acids at the split points of each set are represented in the figure. Predicted protein molecular weights are described below each AAV vector.

[0088] Fig. 17 - Combination of heterologous N- and C-proteins does not result in the reconstitution of detectable EGFP protein in vitro. Fluorescence analysis of HEK293 cells transfected with full-length or intein AAV-CMV-EGFP plasmids. N + C-DnaE: AAV l + ll fused to DnaE inteins; N + C-DnaB: AAV l + ll fused to DnaB inteins; N + C-mDnaE: AAV l + ll fused to separate mDnaE inteins; N-DnaE + C-DnaB: AAV I fused to DnaE n-intein and AAV II fused to DnaB c-intein; N-DnaB + C-DnaE: AAV I fused to DnaB n-intein and AAV II fused to DnaE c-intein; N-mDnaE + C-DnaB: AAV I fused to mDnaE n-intein and AAV II fused to DnaB c-intein; N-DnaB + C-mDnaE: AAV I fused to the n-intein of DnaB and AAV II fused to the c-intein of mDnaE; pEGFP: plasmid including the full-length EGFP expression cassette; Neg: untransfected cells. Scale bar: 100 pm.

[0089] Fig. 18 - CEP290 aligns along the microtubules. Enlargement of individual cells in the 2D Figure. Analysis Petition 870260074145, dated 07 / 24 / 2026, p. 52 / 259 41 / 192 immunofluorescence of HeLa cells transfected with a plasmid including the full-length CEP290 expression cassette (pCEP290) or with CEP290 intein plasmids (set 5, pAAV l + ll + lll). Cells were stained for 3xFLAG and acetylated tubulin (microtubule marker). Scale bar: 50 hours. Western blot (WB) analysis of lysates from HEK293 cells transfected with whole plasmids or AAV intein encoding for CMV-ABCA4 short (set 1, A) or -CEP290 (set 5, B). (A) pABCA4: full-length ABCA4 expression cassette; Set 1: ABCA4 (Cys.ll50)-intein plasmids. (B) pCEP290: full-length CEP290 expression cassette; Set 5: CEP290 (Ser.453 and Cys.1474) - intein plasmids. Negative: AAV EGFP plasmids. The WB are representative of n = 3 independent experiments.

[0090] Fig. 19 - Transfection of AAV intein plasmids reconstitutes ABCA4 and CEP290 proteins in lower quantities than transfection of single plasmids with full-length expression cassettes. Western blot (WB) analysis of lysates from HEK293 cells transfected with whole plasmids or AAV intein encoding for CMV-short-ABCA4 (A) or -CEP290 (B). (A) pABCA4: full-length ABCA4 expression cassette; Set 1: ABCA4 (Cys.1150)-intein plasmids. (B) pCEP290: full-length CEP290 expression cassette; Set 5: CEP290 (Ser.453 and Cys.1474)-intein plasmids. Neg: AAV EGFP plasmids. The WB are representative Petition 870260074145, dated 07 / 24 / 2026, page 53 / 259 42 / 192 of n = 3 independent experiments.

[0091] Fig. 20 Subretinal delivery of AAV intraepithelial vectors results in ABCA4 expression in the mouse retina. Western blot (WB) analysis of retinal lysates from wild-type mice injected with double AAV2 / 8GRK1-ABCA4 vectors or intein (set 1). AAV intein: AAV intein vectors; AAV double: double AAV vectors; Neg: AAV-EGFP vectors.

[0092] Fig. 21 - AAV intein reconstitutes approximately 10% of endogenous Abca4.

[0093] Western blot (WB) analysis of retinal lysates from Abca4 <+ / ~> or Abca4 <~ / ~> mice Injected with AAV2 / 8-GRK1-ABCA4 retinal vectors (set 1). mAbca4: Abca4 <+ / ~> retina; AAV retinal: retina injected with AAV retinal; Neg: retina not injected. The Abca4 + / - loaded retinal lysates in Gel #2 and #3 are the same. The percentage of ABCA4 AAV retinal expression relative to endogenous expression is shown below each track.

[0094] Fig. 22 - AAV protein reconstitutes the protein Full-length ABCA4 cells in human retinal organoids.

[0095] Western blot analysis of 3D retinal organoid lysates derived from human iPSCs infected with AAV2 / 2-GRK1-ABCA4 intein vectors (set 1). AAV intein: AAV intein vectors; Neg: uninfected organoids. - / -: organoids derived from STGD1 patients; + / +: organoids derived from healthy donors. Petition 870260074145, dated 07 / 24 / 2026, page 54 / 259 43 / 192

[0096] Fig. 23 Subretinal administration of AAV vectors results in reduced lipofuscin accumulation in Abca4 - / - mice.

[0097] Representative transmission electron microscopy images showing lipofuscin granules in the RPE of wild-type and Abca4 ~ / ~ mice injected with negative control (Neg) or AAV intein vectors (set 1). White arrows indicate lipofuscin granules; M: mitochondria.

[0098] Fig. 24 - Subretinal delivery of AAV internal vectors in mice does not modify ONL thickness.

[0099] Spectral domain optical coherence tomogram analysis of C57BL / 6J mouse eyes injected subretinally with AAV intein vectors, unrelated AAV vectors (AAV neg), or PBS. Black bars represent eyes 6 months after injection with AAV-ABCA4 intein vectors (set 1) and their corresponding controls; white bars represent eyes 4.5 months after injection with AAV-CEP290 intein vectors (set 5) and their corresponding controls. Data are represented as mean ± sec. Mean values ​​are indicated above the corresponding bar.

[0100] Fig. 25. - AAV integer vectors can deliver full-length wild-type F8 A) Schematic representation of a single deleted B-domain variant 3 Factor VIII (F8-V3) AAV and integer vectors AAV F8. The coding sequence of the F8 gene is divided into Petition 870260074145, dated 07 / 24 / 2026, page 55 / 259 44 / 192 two halves (5' and 3' of F8), flanked by inverted terminal repeats (ITRs), which are packaged separately in two AAV capsids. The 5' vector includes the 5' F8 and 5'(n-DnaE) interteins, while the 3' vector includes the 3' F8 and 3'(c-DnaE) interteins; both vectors include the HLP promoter and synthetic polyA. V3, variant 3; SS, signal sequence. B) The F8 intein is properly packaged in AAV capsids with defined vector genomes, unlike the one-size-fits-all F8-V3 AAV. Southern blot analysis of the genome integrity of the vectors with a probe specific for the HLP promoter showed truncated products in the large-sized AAV F8-V3 vectors that were not present in the AAV F8.Neg, negative control. C) The AAV F8 intein vectors show slight correction of the bleeding phenotype in knockout mice for hemophilia A 8 weeks after injection. Analysis of aPTT in blood plasma samples from hemophilia A knockout mice 8 weeks after injection with AAV F8 intein (both split points) shows a slight phenotypic correction compared to the control group injected with PBS. aPTT, activated partial thromboplastin time. DETAILED DESCRIPTION OF THE INVENTION Gene Therapy

[0101] During the last decade, gene therapy has been applied to the treatment of diseases in hundreds of clinical trials. Several tools have been developed to deliver genes to human cells; among them, viruses Petition 870260074145, dated 07 / 24 / 2026, page 56 / 259 Genetically modified 45 / 192 viruses, including adeno-associated viruses, are currently among the most popular tools for gene delivery.

[0102] Most systems contain vectors that are capable of accommodating genes of interest and helper cells that can provide the viral structural proteins and enzymes to enable the generation of infectious viral particles containing vectors.

[0103] Adeno-associated viruses are a family of viruses that differ in nucleotide and amino acid sequence, genome structure, pathogenicity, and host range. This diversity offers opportunities to use viruses with different biological characteristics to develop different therapeutic applications.

[0104] As with any delivery tool, the efficiency, ability to target a particular tissue or cell type, expression of the gene of interest, and safety of adeno-associated virus-based systems are important for the successful application of gene therapy. Significant efforts have been devoted to these research areas in recent years. Several modifications have been made to adeno-associated virus-based vectors and helper cells to alter gene expression, target delivery, improve viral titers, and increase safety.

[0105] The present invention represents an improvement in this design process, insofar as it acts to efficiently deliver genes of interest with a size that exceeds the payload limit for a single vector based on Petition 870260074145, dated 07 / 24 / 2026, page 57 / 259 46 / 192 adeno-associated virus.

[0106] Viruses are logical tools for gene delivery. They replicate inside cells and have therefore developed mechanisms to enter cells and use the cellular machinery to express their genes. The concept of virus-based gene delivery is to manipulate the virus so that it can express the gene of interest. Depending on the specific application and type of virus, most viral vectors contain mutations that hinder their ability to replicate freely as wild-type viruses in the host.

[0107] Viruses from several different families have been modified to generate viral vectors for gene delivery. These viruses include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, picornaviruses, and alphaviruses.

[0108] The present invention preferably employs adeno-associated viruses. Therefore, virus-based vectors for gene delivery include, without limitation, adenoviral vectors, adeno-associated viral vectors (AAVs), pseudotyped AAV vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, baculoviral vectors.

[0109] An ideal adeno-associated virus-based vector for gene delivery must be efficient, cell-specific, regulated, and safe. Delivery efficiency is important because it can determine the effectiveness of the therapy. Current efforts aim to achieve cell-type-specific infection and gene expression with adeno-associated viral vectors. Furthermore, vectors Petition 870260074145, dated 07 / 24 / 2026, page 58 / 259 47 / 192 adeno-associated viral genes are being developed to regulate the expression of the gene of interest, since therapy may require regulated or long-lasting expression. Safety is an important issue for viral gene delivery because most viruses are pathogenic or have pathogenic potential.

[0110] Adeno-associated virus (AAV) is a small virus that infects humans and some other primate species. Currently, AAV is not known to cause disease, and consequently, the virus causes a very mild immune response. Gene therapy vectors using AAV can infect dividing and quiescent cells and persist in an extrachromosomal state without integrating into the host cell genome. These characteristics make AAV a very attractive candidate for creating viral vectors for gene therapy and for creating isogenic models of human diseases.

[0111] Wild-type AAV has attracted considerable interest from gene therapy researchers due to a number of characteristics. The main one is the apparent lack of pathogenicity of the virus. It can also infect non-dividing cells and has the ability to stably integrate into the host cell genome at a specific location (designated AAVS1) on human chromosome 19. This characteristic makes it somewhat more predictable than retroviruses, which present the threat of random insertion and mutagenesis, sometimes followed by the development of cancer. The AAV genome integrates more frequently at the aforementioned site, while random incorporations at Petition 870260074145, dated 07 / 24 / 2026, page 59 / 259 48 / 192 genome sequencing occurs with negligible frequency. The development of AAVs as gene therapy vectors, however, eliminated this integrative capacity by removing the rep and cap from the vector DNA. The desired gene, along with a promoter to drive gene transcription, is inserted between inverted terminal repeats (ITRs) that aid in the formation of the concatemer in the nucleus after the single-stranded vector DNA is converted by host cell DNA polymerase complexes into double-stranded DNA. AAV-based gene therapy vectors form episomal concatemers in the host cell nucleus. In non-dividing cells, these concatemers remain intact throughout the life of the host cell. Upon cell division, the AAV DNA is lost through cell division, since episomal DNA is not replicated along with host cell DNA.Random integration of AAV DNA into the host genome is detectable, but occurs at a very low frequency. AAVs also exhibit very low immunogenicity, apparently restricted to the generation of neutralizing antibodies, although they do not induce any clearly defined cytotoxic response. This characteristic, along with the ability to infect quiescent cells, demonstrates their dominance over adenoviruses as vectors for human gene therapy. AAV genome, transcriptome and proteome

[0112] The AAV genome is constructed of single-stranded deoxyribonucleic acid (ssDNA), with positive or negative detection, which is approximately 4.7 kilobases in length. The genome comprises terminal repeats Petition 870260074145, dated 07 / 24 / 2026, page 60 / 259 49 / 192 inverted receptors (ITRs) at both ends of the DNA strand and two open reading frames (ORFs): rep and cap. The former is composed of four overlapping genes that encode Rep proteins necessary for the AAV life cycle, and the latter contains overlapping nucleotide sequences of capsid proteins: VP1, VP2, and VP3, which interact with each other to form a capsid of icosahedral symmetry. ITR Sequences

[0113] Inverted Terminal Repeat (ITR) sequences comprise 145 bases each. They were named for their symmetry, which has proven necessary for the efficient multiplication of the AAV genome. Another property of these sequences is their ability to form a hairpin, which contributes to so-called self-priming, allowing for primase-independent synthesis of the second DNA strand. ITRs have also been shown to be necessary for the integration of AAV DNA into the host cell genome (19 chromosomes in humans) and its rescue, as well as for the efficient encapsulation of AAV DNA combined with the generation of fully assembled deoxyribonuclease-resistant AAV particles.

[0114] With regard to gene therapy, ITRs appear to be the only sequences required in cis alongside the therapeutic gene: structural (cap) and packaging (rep) genes can be delivered in trans. With this assumption, many methods have been established for the efficient production of recombinant AAV vectors (rAAV) containing a reporter or therapeutic gene. However, it has also been published that ITRs are not the only elements required in cis for the Petition 870260074145, dated 07 / 24 / 2026, page 61 / 259 50 / 192 effective replication and encapsidation. Some research groups have identified a sequence designated as cis-acting Rep-dependent element (CARE) within the coding sequence of the rep gene. CARE has been shown to enhance replication and encapsidation when present in cis. AAV serotypes

[0115] To date, dozens of different AAV variants (serotypes) have been identified and classified (60). All known serotypes can infect cells of various types of diverse tissues. Tissue specificity is determined by the capsid serotype, and pseudotyping AAV vectors to alter their tropism range will likely be important for their use in therapy. Pseudotyped AAV vectors are those that contain the genome of one AAV serotype in the capsid of a second AAV serotype; for example, an AAV2 / 8 vector contains the AAV8 capsid and the AAV2 genome (61). These vectors are also known as chimeric vectors. SEROTYPE 2

[0116] Serotype 2 (AAV2) has been the most extensively examined to date. AAV2 exhibits natural tropism for skeletal muscle, neurons, vascular smooth muscle cells, and hepatocytes. Three cellular receptors have been described for AAV2: heparan sulfate proteoglycan (HSPG), integrin qnb5, and fibroblast growth factor receptor 1 (FGFR-1). The first functions as a primary receptor, while the latter two have coreceptor activity and allow AAV to enter the cell via receptor-mediated endocytosis. The results of this study were Petition 870260074145, dated 07 / 24 / 2026, page 62 / 259 51 / 192 contested by Qju, Handa, et al. HSPG functions as the primary receptor, although its abundance in the extracellular matrix may eliminate AAV particles and impair infection efficiency.

[0117] Studies have shown that serotype 2 of the virus (AAV-2) apparently kills cancer cells without harming healthy ones. Our results suggest that adeno-associated virus type 2, which infects most of the population but has no known harmful effects, kills several types of cancer cells but has no effect on healthy cells, said Craig Meyers, professor of immunology and microbiology at Penn State College of Medicine in Pennsylvania. This could lead to a new anticancer agent. OTHER SEROTYPES

[0118] Although AAV2 is the most popular serotype in several AAV-based studies, other serotypes have been shown to be more effective as gene delivery vectors. For example, AAV6 appears much better at infecting airway epithelial cells, AAV7 exhibits a very high transduction rate of murine skeletal muscle cells (similar to AAV1 and AAV5), AAV8 excels at transducing hepatocytes and photoreceptors, and AAV1 and 5 have proven very efficient at delivering genes to vascular endothelial cells. In the brain, most AAV serotypes show neuronal tropism, while AAV5 also translates astrocytes. AAV6, a hybrid of AAV1 and AAV2, also shows lower immunogenicity than AAV2.

[0119] Serotypes may differ with respect to the receptors to which they are bound. For example, the Petition 870260074145, dated 07 / 24 / 2026, page 63 / 259 52 / 192 transduction of AAV4 and AAV5 can be inhibited by soluble sialic acids (differently for each of these serotypes), and AAV5 has been shown to enter cells via the platelet-derived growth factor receptor. Novel AAV variants, such as quad tyrosine mutants or AAV 2 / 7m8, have been shown to transduce the outer retina from the vitreous in small animal models (62, 63). Another AAV mutant, termed ShHIO, is an AAV6 variant with enhanced glial tropism after intravitreal administration (64). Another AAV mutant with particularly advantageous tropism for the retina is AAV2 (quad YF) (65).

[0120] The gene delivery vehicles of the present invention can be administered to a patient. Such administration can be in vivo or ex vivo. A skilled worker would be able to determine the appropriate dosage rates. The term administered includes delivery by viral or non-viral techniques. Viral delivery mechanisms include, but are not limited to, adenoviral vectors, adeno-associated viral vectors (AAVs), herpes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors, etc., as described above.

[0121] Nonviral delivery systems include DNA transfection, such as electroporation, lipid-mediated transfection, compacted DNA-mediated transfection; liposomes, immunoliposomes, lipofectin, cationic amphiphilic facies (CFAs) and combinations thereof. Delivery of one or more therapeutic genes by a vector system according to the present invention may be used alone or in combination with other vectors. Petition 870260074145, dated 07 / 24 / 2026, page 64 / 259 53 / 192 combination with other treatments or components of the treatment. Pharmaceutical Compositions

[0122] The present invention also provides a pharmaceutical composition for the treatment of an individual by gene therapy, wherein the composition comprises a therapeutically effective amount of the vector / construct or host cell of the present invention comprising one or more administrable therapeutic and / or diagnostic transgene(s) or a viral particle produced by or obtained therefrom. The pharmaceutical composition may be for human or animal use. Typically, a physician will determine the actual dosage that will be most appropriate for an individual subject and will vary with the age, weight and response of the particular individual.

[0123] The composition may optionally comprise a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant. The choice of pharmaceutical carrier, excipient, or diluent may be selected in relation to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions may comprise, as—or in addition to—the carrier, excipient, or diluent, any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilizing agent(s), and other carrier agents that may assist or enhance viral entry at the target site (such as, for example, a lipid delivery system). When appropriate, pharmaceutical compositions may be administered by either or Petition 870260074145, dated 07 / 24 / 2026, page 65 / 259 54 / 192 more than: inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or powder for sprinkling, by the use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules, either alone or in a mixture with excipients, or in the form of elixirs, solutions or suspensions containing flavoring or coloring agents; preferably, they may be injected parenterally, for example, intracavernosally, intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example, salts or monosaccharides sufficient to make the solution isotonic with blood. For oral or sublingual administration, the compositions can be administered in the form of tablets or lozenges, which can be formulated in a conventional manner.A preferred formulation is one in which the vector system is administered topically into the conjunctival sac or subconjunctivally, preferably administered 1 to 10 times daily, preferably for 1 to 6 months, preferably for 1 to 30 days.

[0124] The preferred administration method is anterior chamber administration, intravitreal injection, subretinal injection, parabulbar and / or retrobulbar injection, intrastromal corneal injection.

[0125] Preferably, the pharmaceutical composition of the invention is for topical ocular use, and is therefore an ophthalmic composition. Petition 870260074145, dated 07 / 24 / 2026, page 66 / 259 55 / 192

[0126] The vector system, according to the present invention, can be administered by any convenient route, however the preferred route of administration is topically on the ocular surface and especially topically on the cornea. An even more preferred route is instillation into the conjunctival sac.

[0127] It is a specific objective of the present invention, the use of the vector system for the production of an ophthalmic composition to be administered topically to the eye for medical use.

[0128] More generally, a preferred embodiment of the present invention is a composition formulated for topical application to a local, superficial or restricted area of ​​the eye and / or eye adnexa comprising the vector system optionally in conjunction with one or more pharmaceutically acceptable additives (such as diluents or vehicles).

[0129] As used in this descriptive report, the terms vehicle, diluent, carrier and additive are interchangeable.

[0130] The ophthalmic compositions of the invention may be in the form of a solution, emulsion or suspension (eye drops), ointment, gel, aerosol, mist or liniment together comprising a pharmaceutically acceptable, eye-tolerated and compatible ophthalmic vehicle of the active ingredient.

[0131] Also within the scope of the present invention are particular routes for delayed-release ophthalmic administration, e.g., such as erodible ocular inserts or polymeric membrane reservoir systems. Petition 870260074145, dated 07 / 24 / 2026, page 67 / 259 56 / 192 to be located in the conjunctival sac or in contact lenses. The ophthalmic compositions of the invention can be administered topically, for example, the composition is dispensed and directly contacts the eye and / or the eye's adnexa.

[0132] The pharmaceutical composition containing at least one vector system of the present invention can be prepared by any conventional technique, e.g., as described in Remington: The Science and Practice of Pharmacy 1995, edited by E.W. Martin, Mack Publishing Company, 19th edition, Easton, Pa.

[0133] In one embodiment, the composition is formulated as a liquid, wherein the carrier system may be in solution or in suspension. The composition may be formulated in any liquid form suitable for topical application, such as eye drops, artificial tears, eye washes or contact lens adsorbents comprising a liquid carrier, such as a cellulose ether (e.g., methylcellulose).

[0134] Preferably, the liquid is an aqueous liquid. In addition, it is preferred that the liquid be sterile. Sterility can be conferred by any conventional method, for example, filtration, irradiation or heating, or by conducting the manufacturing process under aseptic conditions.

[0135] The liquid may comprise one or more lipophilic vehicles.

[0136] In one embodiment of the present invention, the composition is formulated as an ointment. Preferably, a Petition 870260074145, dated 07 / 24 / 2026, page 68 / 259 57 / 192 carrier in the ointment may be a petroleum jelly carrier.

[0137] Pharmaceutically acceptable vehicles can generally be any pharmaceutically acceptable vehicle used, which should be selected according to the specific formulation, intended route of administration, etc. In addition, a pharmaceutically acceptable vehicle can be any accepted additive from the FDA's list of inactive ingredients, which, for example, is available at the following internet address: http: / / www.fda.gov / cder / drug / iig / default.htm.

[0138] At least one pharmaceutically acceptable diluent or vehicle may be a buffer. For some purposes, it is often desirable that the composition comprise a buffer capable of buffering a solution to a pH in the range of 5 to 9, for example, pH 5 to 6, pH 6 to 8, or pH 7 to 7.5. However, in other embodiments of the invention, the pharmaceutical composition may comprise no buffer or only micromolar amounts of buffer. The buffer may, for example, be selected from the group consisting of TRIS buffer, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, borate, carbonate, glycinate, histidine, glycine, succinate, and triethanolamine. Therefore, the buffer may be K2HPO4, Na2HPO4, or sodium citrate.

[0139] In a preferred embodiment, the buffer is a TRIS buffer. The TRIS buffer is known by several other names, for example tromethamine, including tromethamine USP, THAM, Trizma, Trisamine, Tris amino and trometamol. The designation TRIS covers all the above-mentioned designations. Petition 870260074145, dated 07 / 24 / 2026, page 69 / 259 58 / 192

[0140] The buffer may, in addition, for example, be selected from buffers compliant with USP for parenteral use, in particular when the pharmaceutical formulation is for parenteral use. For example, the buffer may be selected from the group consisting of monobasic acids, such as acetic, benzoic, gluconic, glyceric and lactic, dibasic acids, such as aconitic, adipic, ascorbic, carbonic, glutamic, malic, succinic and tartaric, polybasic acids, such as citric and phosphoric, and bases such as ammonia, diethanolamine, glycine, triethanolamine and TRIS.

[0141] The compositions may contain preservatives such as thimerosal, chlorobutanol, benzalkonium chloride or chlorhexidine, buffering agents such as phosphates, borates, carbonates and citrates and thickening agents such as high molecular weight carboxyvinyl polymers such as those sold under the name Carbopol which is a trademark of BF Goodrich Chemical Company, hydroxymethylcellulose and polyvinyl alcohol, all in accordance with the state of the art.

[0142] In some embodiments of the invention, the pharmaceutically acceptable additives comprise a stabilizer. The stabilizer may be, for example, a detergent, an amino acid, a fatty acid, a polymer, a polyhydric alcohol, a metal ion, a reducing agent, a chelating agent or an antioxidant; however, any other suitable stabilizer may also be used with the present invention. For example, the stabilizer may be selected from the group consisting of poloxamers, Tween-20, Tween-40, Tween-60, Tween-80, Brij, ions Petition 870260074145, dated 07 / 24 / 2026, p. 70 / 259 59 / 192 metallic, amino acids, polyethylene glycol, Triton and ascorbic acid. In addition, the stabilizer can be selected from the group consisting of amino acids such as glycine, alanine, arginine, leucine, glutamic acid and aspartic acid, surfactants such as polysorbate 20, polysorbate 80 and poloxamer 407, fatty acids such as phosphatidylcholine ethanolamine and acetyltryptophanate, polymers such as polyethylene glycol and polyvinylpyrrolidone, polyhydric alcohols such as sorbitol, mannitol, glycerin, sucrose, glucose, propylene glycol, ethylene glycol, lactose and trehalose, antioxidants such as ascorbic acid, cysteine ​​HCl, thioglycerol and thioglycerol, glutathione, reducing agents such as various thiols, chelating agents such as EDTA salts, glutamic acid and aspartic acid.

[0143] Pharmaceutically acceptable additives may comprise one or more selected from the group consisting of isotonic salts, hypertonic salts, hypotonic salts, buffers and stabilizers.

[0144] In preferred embodiments, other pharmaceutically graded excipients, such as preservatives, are present. In one embodiment, said preservative is a paraben, such as, but not limited to, methyl parahydroxybenzoate or propyl parahydroxybenzoate.

[0145] In some embodiments of the invention, pharmaceutically acceptable additives comprise mucolytic agents (e.g., N-acetylcysteine), hyaluronic acid, cyclodextrin, petroleum.

[0146] Examples of compounds that can be Petition 870260074145, dated 07 / 24 / 2026, page 71 / 259 60 / 192 incorporated into the pharmaceutical composition of the invention to facilitate and expedite the transdermal delivery of topical compositions to ocular or adnexal tissues include, but are not limited to, alcohol (ethanol, propanol and nonanol), fatty alcohol (lauryl alcohol), fatty acid (valeric acid, caproic acid and capric acid), fatty acid ester (isopropyl myristate and isopropyl n-hexanoate), alkyl ester (ethyl acetate and butyl acetate), polyol (propylene glycol, propanedione and hexanotriol), sulfoxide (dimethyl sulfoxide and decyl methyl sulfoxide), amide (urea, dimethylacetamide and pyrrolidone derivatives), surfactant (sodium lauryl sulfate, cetyltrimethylammonium bromide, poloxamers, spans, tweens, bile salts and lecithin), terpene (d-limonene, alpha-8), cineole and menthone) and alkanone (N-heptane and N-nonane).Furthermore, topically administered compositions may comprise surface adhesion modulating agents, including, but not limited to, a cadherin antagonist, a selectin antagonist, and an integrin antagonist. Additionally, the ophthalmic solution may contain a thickener, such as hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, methylcellulose, polyvinylpyrrolidone, or similar substances, to improve drug retention in the conjunctival sac.

[0147] In one embodiment, the vector system for use according to the invention can be combined with ophthalmologically acceptable preservatives, surfactants, viscosity enhancers, penetration enhancers, buffers, sodium chloride and water to form suspensions or Petition 870260074145, dated 07 / 24 / 2026, page 72 / 259 61 / 192 sterile, aqueous, ophthalmic solutions. The ophthalmic solution may also include an ophthalmologically acceptable surfactant to aid in the dissolution of the vector system. Ophthalmic solution formulations may be prepared by dissolving the vector system in a physiologically acceptable isotonic aqueous buffer.

[0148] In order to prepare sterile ophthalmic ointment formulations, the Vector system can be combined with a preservative in a suitable vehicle, such as mineral oil, liquid lanolin or white petrolatum. Sterile ophthalmic gel formulations can be prepared by suspending the Vector system in a hydrophilic base prepared from the combination of, for example, carbopol-940 or similar, according to the published formulations for analogous ophthalmic preparations; preservatives and tonicity agents may be incorporated.

[0149] Preferably, the formulation of the present invention is a non-irritating aqueous ophthalmic composition for topical application to the eye, comprising: a therapeutically effective amount of a vector system for topical treatment; a xanthine derivative being present in an amount between the amount of water-soluble derivative of said composition and 0.05% by weight / volume of said composition which is effective in reducing discomfort associated with the vector system after topical application of said composition, the xanthine derivative being selected from the group consisting of theophylline, caffeine, theobromine and mixtures thereof; an ophthalmic preservative; and a buffer, to provide an isotonic, aqueous, non-irritating ophthalmic composition. Petition 870260074145, dated 07 / 24 / 2026, page 73 / 259 62 / 192 annoying. Drug delivery devices

[0150] In one embodiment, the invention comprises a drug delivery device consisting of at least one delivery system and a pharmaceutically compatible polymer. For example, the composition is incorporated into or coated within said polymer. The composition is chemically bonded or physically trapped by the polymer. The polymer is hydrophobic or hydrophilic. The polymer device comprises various physical arrangements. Exemplary physical forms of the polymer device include, but are not limited to, a film, a frame, a chamber, a sphere, a microsphere, a stent, or other structure. The polymer device has internal and external surfaces. The device has one or more internal chambers. These chambers contain one or more compositions. The device contains polymers of one or more chemically differentiated monomers. The subunits or monomers of the device polymerize in vitro or in vivo.

[0151] In a preferred embodiment, the invention comprises a device comprising a polymer and a bioactive composition incorporated in or on said polymer, wherein said composition includes a delivery system and wherein said device is implanted or injected into an ocular surface tissue, an adnexal tissue in contact with an ocular surface tissue, a fluid-filled ocular or adnexal cavity, or an ocular or adnexal cavity.

[0152] Natural polyanionic mucoadhesive polymers Petition 870260074145, dated 07 / 24 / 2026, page 74 / 259 Examples of 63 / 192 or semi-synthetic materials from which the device can be formed include, but are not limited to, polygalacturonic acid, hyaluronic acid, carboxymethylamylose, carboxymethylchitin, chondroitin sulfate, heparin sulfate, and mesoglycan. In one embodiment, the device comprises a biocompatible polymer matrix that may optionally be biodegradable in whole or in part. A hydrogel is an example of a suitable polymer matrix material. Examples of materials that can form hydrogels include polylactic acid, polyglycolic acid, PLGA polymers, alginates and alginate derivatives, gelatin, collagen, agarose, natural and synthetic polysaccharides, polyamino acids such as polypeptides, particularly poly(lysine), polyesters such as polyhydroxybutyrate and poly-epsilon.- caprolactone, polyanhydrides; polyphosphazines, polyvinyl alcohols), poly(alkylene oxides) particularly polyethylene oxides), poly(allylamines) (PAM), poly(acrylates), modified styrene polymers such as poly(4-aminomethylstyrene), pluronic polyols, polyoxamers, poly(uronic acids), poly(vinylpyrrolidone) and copolymers of the foregoing, including graft copolymers. In another embodiment, the structures may be fabricated from a variety of synthetic polymers and naturally occurring polymers, such as, but not limited to, collagen, fibrin, hyaluronic acid, agarose and laminin-rich gels.

[0153] A preferred material for hydrogel is alginate or modified alginate material. The molecules of Petition 870260074145, dated 07 / 24 / 2026, page 75 / 259 64 / 192 Alginate polysaccharides are composed of monomers of β-D-mannuronic acid (M units) and L-guluronic acid (G units) that vary in proportion and sequential distribution along the polymer chain. Alginate polysaccharides are polyelectrolyte systems that have a strong affinity for divalent cations (e.g., Ca+2, Mg+2, Ba+2) and form stable hydrogels when exposed to these molecules.

[0154] The device is administered topically, subconjunctivally or into the episcleral space, subcutaneously or intraductally. Specifically, the device is placed on or just below the surface of an ocular tissue. Alternatively, the device is placed within a lacrimal duct or gland. The composition incorporated in or on the polymer is released or diffuses from the device.

[0155] In one embodiment, the composition is incorporated or coated onto a contact lens or drug delivery device, from which one or more molecules diffuse away from the lens or device or are released in a temporally controlled manner. In this embodiment, the contact lens composition remains on the ocular surface, e.g., if the lens is required for vision correction, or the contact lens dissolves over time, simultaneously releasing the composition into adjacent tissues. Similarly, the drug delivery device is optionally biodegradable or permanent in various embodiments.

[0156] For example, the composition is incorporated into or coated onto said lenses. The composition is chemically bonded or physically held in place by the lens polymer. Petition 870260074145, dated 07 / 24 / 2026, page 76 / 259 65 / 192 contact. Alternatively, a color additive is chemically bound or physically trapped by the polymer composition that is released at the same rate as the therapeutic drug composition, so that changes in the intensity of the color additive indicate changes in the amount or dose of therapeutic drug composition remaining bound or trapped in the polymer. Alternatively, or in addition, an ultraviolet (UV) absorber is chemically bound or physically trapped within the polymer of the contact lens. The contact lens is hydrophobic or hydrophilic.

[0157] Exemplary materials used to fabricate a hydrophobic lens with means for dispensing the compositions of the invention include, but are not limited to, amefocon A, amsilfocon A, aquilafocon A, arfocon A, cabufocon B, carbosfoconfocon A, cabufoconfocon A, carbofoconfocon A, cabufoconfocon A. B, dimefocon A, enflufocon A, enflofocon B, erifocon A, flurofocon A, flusilfocon A, flusilfocon B, flusilfocon C, flusilfocon D, flusilfocon E, hexafocon A, hofocon A, hybufocon A, itabisfluorofocon B, itafluorofocon B, itafluocon a kol A A, colfocon B, colfocon C, colfocon D, lotifocon A, lotifocon B, lotifocon C, melafocon A, migafocon A, nefocon A, nefocon B, nefocon C, onsifocon A, oprifocon A, oxyfluflocon A, paflufocon B, F paflufocon C, paflufocon C Pacificocon A, Pacificocon B, Pacificocon C, Pacificocon D, Pacificocon E, Pemufocon A, Porofocon A, Porofocon B, Roflufocon A, Roflufocon B, Roflufocon C, Roflufocon D, Roflufocon E, Rosilfocon A, Satafoconuf A, , silafocon A, , silafoconsterafocon A, sulfocon A, sulfocon B, telafocon A, tisilfocon A, tolofocon A, trifocon, Petition 870260074145, dated 07 / 24 / 2026, page 77 / 259 66 / 192 A, unifocon A, vinafocon A, e wilofocon A. Materiais exemplificativos usados para fabricar uma lente hidrofílica com meios para admistrar as composições da invenção inclui, mas não está limitada a, abafilcon A, acofilcon A, acofilcon B, acquafilcon A, alofilcon A, alphafilcon A, amfilcon A, astifilcon A, atlafilcon A, balafilcon A, bisfilcon A, bufilcon A, comfilcon A, crofilcon A, ciclofilcon A, darfilcon A, deltafilcon A, deltafilcon B, dimefilcon A, droxfilcon A, elastofilcon A, epsilfilcon A, esterifilcon A, etafilcon A, focofilcon A, galyfilcon A, genfilcon A, govafilcon A, hefilcon A, hefilcon B, hefilcon C, hilafilcon A, hilafilcon B, hioxifilcon A, hioxifilcon B, hioxifilcon A, hioxifilcon B, hioxifilcon hidrofilcon A, lenefilcon A, licrifilcon A, licrifilcon B, lidofilcon A, lidofilcon B, lotrafilcon A, lotrafilcon B, mafilcon A, mesafilcon A, metafilcon B, mipafilcon A, nelfilcon A, netrafilcon A, ocufilcon A, ocufilcon A, ocufilcon, ocufilcon D, ocufilcon E, ofilcon A,omafilcon A, oxyfilcon A, pentafilcon A, profilecon A, pevafilcon A, phemfilcon A, polymacon, senofilcon A, silafilcon A, siloxifilcon A, surfilcon A, tefilcon A, tefilcon A, vifilcon A, vifilcon B and xilofilcon A.,

[0158] Within the scope of the invention are compositions formulated as a gel or gel-like substance, cream, or viscous emulsions. It is preferred that said compositions comprise at least one gelling component, polymer, or other suitable agent for increasing the viscosity of the composition. Any gelling component known to a person skilled in the art, which Petition 870260074145, dated 07 / 24 / 2026, page 78 / 259 67 / 192 has no harmful effect on the area to be treated and is applicable in the formulation of pharmaceutical compositions and compounds for topical administration to the skin, eyes or mucous membranes, it may be used. For example, the gelling component can be selected from the group of: acrylic acids, carbomer, carboxypolymethylene, such materials sold by BF Goodrich under the trade name Carbopol (e.g., Carbopol 940), polyethylene-polypropylene glycols, such materials sold by BASF under the trade name Poloxamer (e.g., Poloxamer 188), a cellulose derivative, e.g., hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxyethylene cellulose, methyl cellulose, carboxymethyl cellulose, alginic acid-propylene glycol ester, polyvinylpyrrolidone, veegum (aluminum magnesium silicate), Pemulen, Simulgel (such as Simulgel 600, Simulgel EG and Simulgel NS), Capigel, Colafax, Plasdones and the like and mixtures thereof.

[0159] A gel or gel-like substance according to the present invention comprises, for example, less than 10% w / w water, for example, less than 20% w / w water, for example, at least 20% w / w water, such as at least 30% w / w water, for example, at least 40% w / w water, such as at least 50% w / w water, for example, at least 75% w / w water, such as at least 90% w / w water, for example, at least 95% w / w water. Preferably, said water is deionized water.

[0160] The gel-like substances of the invention include a hydrogel, a colloidal gel formed as a dispersion in water or another aqueous medium. Thus, a hydrogel Petition 870260074145, dated 07 / 24 / 2026, p. 79 / 259 68 / 192 is formed after the formation of a colloid in which a dispersed phase (the colloid) has combined with a continuous phase (i.e., water) to produce a viscous, jelly-like product; for example, coagulated silicic acid. A hydrogel is a three-dimensional network of hydrophilic polymer chains that are cross-linked by chemical or physical bonds. Due to the hydrophilic nature of the polymer chains, hydrogels absorb water and swell. The swelling process is the same as the dissolution of non-cross-linked hydrophilic polymers. By definition, water constitutes at least 10% of the total weight (or volume) of a hydrogel.

[0161] Examples of hydrogels include synthetic polymers such as polyhydroxyethyl methacrylate and chemically or physically crosslinked polyvinyl alcohol, polyacrylamide, poly(N-vinylpyrrolidone), polyethylene oxide, and hydrolyzed polyacrylonitrile. Examples of hydrogels that are organic polymers include hydrogels based on covalently or ionically crosslinked polysaccharides, such as polyvalent metal salts of alginate, pectin, carboxymethylcellulose, heparin, hyaluronate, and hydrogels of chitin, chitosan, pullulan, gellan, and xanthan gum. The particular hydrogels used in our experiment were a cellulose compound (i.e., hydroxypropylmethylcellulose [HPMC]) and a high molecular weight hyaluronic acid (HA). Hyaluronic acid is a polysaccharide produced by various tissues of the body. US patent 5,166,331 discusses the purification of different fractions of hyaluronic acid for use as a substitute for intraocular fluids and as a Petition 870260074145, dated 07 / 24 / 2026, page 80 / 259 69 / 192 topical ophthalmic drug carrier. Other US patent applications discussing ocular uses of hyaluronic acid include serial numbers US 11 / 859,627; US 11 / 952,927; US 10 / 966,764; US 11 / 741,366; and US 11 / 039,192. Macromolecular formulations for intraocular use are known. See, for example, US patent applications serial numbers US 11 / 370,301; US ​​11 / 364,687; US 60 / 721,600; US 11 / 116,698 and US 60 / 567,423; US 11 / 695,527. The use of multiple active agents is known as high-viscosity hyaluronic acid. See, for example, the serial numbers of patent applications US 10 / 966,764; US 11 / 091,977; US 11 / 354,415; US 60 / 519,237; US 60 / 530,062 and US 11 / 695,527.

[0162] Sustained-release formulations, as described in document WO 2010 / 048086, are within the scope of the invention.

[0163] The technician versed in the subject is well aware of the standard methods for incorporating a polynucleotide or vector into a host cell, for example, transfection, lipofection, electroporation, microinjection, viral infection, heat shock, transformation after chemical permeabilization of the membrane or cell fusion.

[0164] As used herein, the term host cell or genetically modified host cell refers to host cells that have been transduced, transformed or transfected with the construct or vector described above.

[0165] As representative examples of appropriate host cells, one can cite bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium, cells Petition 870260074145, dated 07 / 24 / 2026, page 81 / 259 70 / 192 fungal cells, such as yeast, insect cells, such as Sf9, animal cells, such as CHO or COS, plant cells, etc. The selection of a suitable host is considered to be within the scope of those skilled in the art based on the teachings of this descriptive report. Preferably, said host cell is an animal cell and, more preferably, a human cell. The invention further provides a host cell comprising any of the recombinant expression vectors described herein. The host cell may be a cultured cell or a primary cell, i.e., isolated directly from an organism, for example, a human. The host cell may be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for example, DH5a, E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells and the like.In the case of ex vivo gene therapy, a host cell can be a cell isolated from a patient, for example, hematopoietic stem cells, which after the introduction of the transgene is reintroduced into the patient in need. Viral delivery systems based on AAVs

[0166] The construction of an AAV vector can be carried out following procedures and using techniques known to a skilled technician. The theory and practice for the construction of adeno-associated viral vectors and their use in therapy are illustrated in several scientific and patent publications (the following bibliography is here). Petition 870260074145, dated 07 / 24 / 2026, page 82 / 259 71 / 192 incorporated by reference: Flotte TR. Adeno-associated virus-based gene therapy for hereditary diseases. Pediatr Res. 2005 Dec; 58(6): 1143-7; Gonçalves MA. Adeno-associated virus: from defective virus to effective vector, Virol J. 2005 May 6; 2: 43; Surace EM, Auricchio A. Adenoassociated viral vectors for retinal gene transfer. Prog Retin Eye Res. 2003 Nov; 22(6): 705-19; Mandel RJ, Manfredsson FP, Foust KD, Rising A, Reimsnider S, Nash K, Burger C. Recombinant adeno-associated viral vectors as therapeutic agents to treat neurological disorders. Mol Ther. 2006 Mar; 13(3): 463-83).

[0167] Suitable forms of administration of a pharmaceutical composition containing AAV vectors include, but are not limited to, injectable solutions or suspensions, eye lotions, and ophthalmic ointment. In a preferred embodiment, the AAV vector is administered by intrathecal injection. In a particularly preferred embodiment, the AAV vector is administered by subretinal injection, into the anterior chamber or retrobulbar space, and intravitreal. Preferably, viral vectors are delivered via a subretinal approach (as described in Bennicelli J, et al Mol Ther. 2008, January 22; Reversal of Blindness in Animal Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated Gene Transfer).

[0168] Virus doses for use in therapy will be determined on a case-by-case basis, depending on the route of administration, disease severity, patient's general condition, and other clinical parameters. In general, appropriate dosages will range from 108 to 1013 vg (vector genomes) / eye. Petition 870260074145, dated 07 / 24 / 2026, page 83 / 259 72 / 192 Proteins

[0169] An intein is a segment of a protein that is capable of being excised and joined to the remaining portions (the exteins) with a peptide bond in a process known as protein splicing. The segments are called intein for the inner protein sequence and extein for the outer protein sequence, with upstream exteins called N-exteins and downstream exteins called C-exteins. The products of the protein splicing process are two stable proteins: the mature protein and the intein.

[0170] Inteins can also exist as two fragments encoded by two genes transcribed and translated separately, here called split inteins.

[0171] Proteins of the present invention include, without limitation, split proteins listed in the New England Biolabs Protein database, disclosed in (66).

[0172] Split inteins can be produced from inteins by first removing the endonuclease homing domain sequence to produce a mini intein. This mini intein can then be split at one or more designed sites via protein sequence alignments with inteins of known crystal structures to generate split inteins, tested for trans-splicing activity according to the protocols included in this disclosure.

[0173] Split proteins can be further improved in desirable characteristics, including Petition 870260074145, dated 07 / 24 / 2026, page 84 / 259 73 / 192 activity, efficiency, generality and stability through site-directed mutagenesis or modifications of intein sequences based on rational design and / or through directed evolution using methods such as functional selection, phage display and ribosome display.

[0174] An example of split inteins are the inteins derived from DnaE, ​​which is the catalytic α subunit of DNA polymerase III in cyanobacteria, encoded by two separate genes, dnaE-n and dnaE-c. The intein encoded by the dnaE-n gene is referred to here as the N-intein. The intein encoded by the dnaE-c gene is referred to here as the C-intein. Generally, the N-part of a split intein is referred to as the N-intein and the C-part of a split intein is referred to as the C-intein. Split inteins self-associate and catalyze the trans-splicing activity of the protein (here trans splicing).

[0175] Other examples of split inteins of the present invention comprise Nostoc punctiforme (Npu) DnaE inteins (27, 28)), indicated in Table 3 below as SEQ ID 1 encoded by the nucleotide sequence NpuDnaE-n and SEQ ID 2 encoded by the nucleotide sequence Npu-DnaE-c; the Rhodothermus marinus DnaB (Rma) (29) intein indicated in the table below as SEQ ID 4 encoded by the nucleotide sequence Rma-DnaB-n and SEQ ID 5 encoded by the nucleotide sequence Rma-DnaB-c; N- and C-inteins mutated wherein the Nl intein is from Npu DnaE (SEQ ID 5) and the Cl intein is from Synechocystis strain PCC6803 (Ssp (SEQ ID 6), respectively (30); the Synechocystis strain PCC6803 Nintein and Cintein are Petition 870260074145, dated 07 / 24 / 2026, page 85 / 259 74 / 192 included as SEQ ID 13 and 14, respectively, in the Table below. Other intein systems may also be used. For example, a synthetic fast intein based on dnaE intein, the Cfa-N and Cfa-C intein pair, has been described (e.g., (31) and in WO 2017 / 132580, incorporated herein by reference). Additional internals have been described in U.S. Patent No. 8,394,604, including Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3, Ter ThyX intein, and Cne Prp8 intein. Other internals within the present invention are the internals disclosed in WO2018071868, wherein the first pair of internals is listed in the table below and named as SEQ ID 9 (Nintein) and SEQ ID 10 (Kintein); A second pair of internals is listed, for example, SEQ ID 11 and SEQ ID 12.

[0176] Alternatively, the intein system may be a ligand-dependent intein that exhibits no or minimal protein splicing activity in the absence of ligand (e.g., small molecules such as 4-hydroxytamoxifen, peptides, proteins, polynucleotides, amino acids, and nucleotides). Ligand-dependent inteins include, for example, those described in US document 2014 / 0065711 A1, which is incorporated herein by reference. Table 3. Examples of split inteins of the present invention. Intein SEQ ID Nu. Sequence AípU- DruE-n 1 CLSYETEILTVEYGLLP-PGKIVEKRIECTVYSVDNNONÍYTO.PVAQWHDRGEQtVFEYCL EDGSLIRATKDHKFMTVDQQJVILPIDCLFCRELDIMRVDNLPH JVpLf- DildE< 2 1K1AT RK YtG KQNVV DIGV ER DH N FAI K NGF IAS N 3 C LAG DTLITLA DOR RVPIR ELV5QQN FSV WA LTJ P-QTYR LE R AR VSR Af CTGIKPVY R LT Petition 870260074145, dated 07 / 24 / 2026, p. 86 / 259 75 / 192 DnaB-n TRLGRS1R AT AN HR FLTPQGWK RVD ELQPG DYLA LPRR1 PT ASTPTL Rma- DnaB-c 4 AAA CPELRQLAQSDVYWDPIVSIE PDGV EEVFDLTVPGPH N FVAN Dl IAH W mNpu DnaE-n 5 El CVLT YG YG! LPE G K1 AND K RIECT WSVDN NG NIYTQPVAQWH D RG EQE VF EVC LE DG 5LIRATKDH KF MTV DGQMM P1 DE 1 FER ELDLM RVD N LP N mNpu- DnaE't 5 VKVI GR RSLGVQR1F LPQAMGÁLLY 1GÁLLY Cfa-n 7 CLSYDTEILTVEYGFLP IGKIVE E RI E CTVYTVD KNGF VYTQP1AQW HN RG EQE VFEYCL EDG 511RATK DH KFMTT DGQMLP1 DE 1 FE RGL DL KQV DGLP Cfa-c 8 VDHRK VGTDIGVQ15N KYGDIGVQ LLK NG LVAS N N-intein SEQ 351 W0_2D15 _071868_ 351 9 CLS YETEILTVEY6 LLP IGKIVEKFt 1ECTVYSVD N NGN 1YTQPVAQW H DRG EQEVF EYCL EDGSLIRATDHKfGFERTVKfFERTVKF ELDLMRVDNLPN C-Intein SEQ 353 LU IKFATRKYLGKQNVYDIGVERDHNFALKNGFIASN N- Intein SEQ 354 11 CLSYDTEILTVEYGFLP IGKIVEERIECTWTV D KNGFVYTQPIAQWHNRGEQEDGEVEDK FEYCL 511 KFMTTDGQMLP1 DE 1F ERG LD L KQV DG LP Q Intein SEQ 357 12 KRTADGSEFESPKKKRKVKH5RKSLGTQNVYDIGVEKDHNFLLKNGLVA5N 5sp DnaEn 13 CLS FGTE1LTVEYG PLLCPIG K1V5EEINCSVY5V DP EGR V YTQA1AQWH DR GEQEV LEYE L EDG 5 V1RATSDH RF LTTD VQLLA1E E ÍF ARQW L LTLE N1KQTEE ALDN HR LPf PLLDAGT IK Petition 870260074145, of 24 / 07 / 2026, p. 87 / 259 76 / 192 PCC6SD3 Ssp DnaEc PCC6SD3 14 VKVIG RR5LG VQP1F DIG LPQD HNF LLA NG Al A ANC

[0177] As described in this descriptive report, within the scope of the present invention are inteins originating from the same gene of different organisms, retaining trans-splicing activity. As a non-limiting example, the split DNA-E intein can be derived from split inteins of the DnaE gene (e.g., alpha subunit of DNA polymerase III) from cyanobacteria including Nostoc punctiforme (Npu), Synechocystis sp. PCC6803 (Ssp), Fischerella sp. PCC 9605, Scytonema tolypothrichoidesf, Cyanobacteria bacterium SW_9_47_5, Nodularia spumigena, Nostoc flagelliformi, Crocosphaera watsonii WH 8502, Chroococcidiopsis cubana CCALA 043, Trichodesmium erythraeum. As another example, the DNA-B ssplit intein can be derived from the DnaB gene of cyanobacteria including R. marinus (Rma), Synechocystis sp. PC6803 (Ssp), Porphyra purpurea chloroplast (Ppu) which are described for example in (59).

[0178] Therefore, the split inteins of the invention can be 100% identical, 98%, 80%, 75%, 70%, 65%, or 50% identical to naturally occurring inteins, wherein said inteins retain the ability to undergo trans-splicing reactions. Within the scope of the present invention are Petition 870260074145, dated 07 / 24 / 2026, p. 88 / 259 77 / 192 fragments of naturally occurring or modified inteins that retain trans-splicing activity.

[0179] See, for example, the alignment between Npu (Nostoc punti forme) DnaE and Synechocytis sp.

[0180] PCC6803 N-intein: Score Positive Identities Gaps ί4&&ιΐψ73) 63 / 100(68¾) 83 / 10D(83%) 0 / 100(0%) CLSYETE lETVEYGLLEIGKTTEKR I ECTVYSVDtnTGtTIYTQEVAQWH DRGEQEVFEYC1EDG2 LI RA^KDJiKFHTVDGQML P CLS+ TEILTVEYG LPIGKIV + I C+VYSVD G +YTQ 1-AQWHDRGEQEV EY EDGSt-IRAT DH+F+T □ Q*-L CLSrcTEILTVEYGPLPIGKIVSEEIHCSVYSVDPEGRVY^ÜAIAQWHDRGEÜEVLEYELEDGÍiVIRATSDHRPLTTDYQLLA 1DEIPERELDLMRVDUL SEQ ID Nd. 21 I+EIF JU-LDL+ +4H SEQ ID No. 22 ΤΕΕΤΡΑΙΐΰΙΰΙ-ΕΤΙΕΙΙΊ SEQ ID No. 23

[0181] And the alignment between Npu (Point-shaped Nostoc) DNA and Synechocytis sp.

[0182] PCC6803 C- Internal: Positive Identity Score 46.6 bits(109) 19 / 36(53%) 27 / 36(75%) Gaps 0 / 36(0%) MIKIATRKYLGKQNVYDIG^TRPHNFALKNGFIASN SEQ ID No. 24 M+K+ R+· LG Q ++DIG+ *DHHF L MG IA+H SEQ ID NO. 25 MVKVÍGRRSLGVQRIFDIGLPQDHNFLLANGAIAAN SEQIDNo. 26

[0183] Therefore, within the scope of the present invention are also variants and fragments of split inteins of the inteins of the invention that retain trans-splicing activity.

[0184] Interestingly, it has been reported that internal Petition 870260074145, dated 07 / 24 / 2026, p. 89 / 259 78 / 192 retained functional features that ensure their splicing activity. In particular, four intein motifs were identified (see below for their consensus sequence): AH blocks (Pietrokovski 1994 and Perler 1997) and N2 and N4 blocks (Pietrokovski 1998). The A, N2, B, N4, F, and G blocks are involved in protein splicing. The O, D, E, and H blocks are in the endonuclease domain, which is absent from split inteins. Thus, split inteins retain conserved motifs that are essential for trans-splicing activity. (Intein Database, published in [Perler, FB (2002). InBase, the Intein Database. Nucleic Acids Res. 30, 383-384.]) Hast protein N-termil splicing region Homing endonuclease or linker domain C-teimma ... Host protein splicing region Motifs: N-erteln DOD Endo nue lease Domain C-exteln Conserved C TxxH Residues: L§J to <1 P Key to Conserved Residues: Boxed amno acids nudcop Miles in stand aid spli&rg reaction. Upper case = conserved ammo acids In standard into us. Lowercase = smew sods in polymorphic rte ins that may splice by moaned mechantsms.

[0185] Although no residue is invariant, Ser and Cys in Block A, His in Block B, His, Asn and Ser / Cys / Thr in Block G are the most conserved residues in splice motifs.

[0186] Alignment of the N-inteins of the present invention: CLUSTAL W alignment of all listed N-inteins: Petition 870260074145, dated 07 / 24 / 2026, p. 90 / 259 79 / 192 5EQ1 C LS E TE ILTVE YG LL PIG KI AND CRI EC TV YS VD WGNIYTQP VAQ WHDRGEQEV FE YC L SEQ? CLSYETEILTVEYGL LPIGKIVECRIECTVYSVDNNGNTYTQPVAQWHDRGEQEVF EYCL SEQ5 C LSYDTEILTVE YG1L PIG KIVEKRI EC TV YSVDNNGNIYTQP VAQWHDfiG EQEV F EYC L SEQ7 CLSYDTMLTVEYGFLPIGKIVÉEftlECWYTVDKNGFVVTQPIAlQldHNRGEQÉVFÉYCL SEQll CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQI*™ SEQ13 CL 5 FGTEIL YGGE VGPS VIGS INCY SW INCY EGRVYTQAIAQWHDRGEQEV L EY EL SEQ3 C LAG DT LIT LADGR RVPIR E LVSQQNF 5VWA L NPQTYR L ERARVS RA FC TGIK PVYR LIT ** * · + - ψψ -+ * - - -- . I.· * d + .«.* SEQ1 E0G5 LIRATKDHKFMT VDGQMLPIDEIFERE LDLMRVDNLPN- - - --------------- SEQ? EDGSLIFWKDHKFmVDGQMLPIDEI^^ ..................SEQ5 EDGS LIRATKDHKFMTVDGQMMPIDEIFERE LDLMRVDNLPN------------------ SEQ7 EDGSIIRATKDHK FMTTDGQMLPTDEIFERGLDLKQVDGLP----------------- - - SEQ11 EDG S11 RAT KDHK FMTTDGQM L PID EI FE RG LD L KQVDG LP------------------- 5EQ1B EDGSVI RA T SDHRF LT TDYQL L AIE EI F AAQ LDL LT LE NIK QT EEAL DNHR LP FP L LD AG SEQ? RLGRSIRATANHRFLTPQGNKRWELQPGDYÍALPRRIPTASTPTL.............. ψ 4 * ** .ΐ.*. . 4*. 5EQ1 - - - SEQ? -- - SÊQS — SEQ7 — 5EQ11 — SEQ13 TIK SEQS —

[0187] Alinhamento de sequência múltipla CLUSTAL 2.1 Petition 870260074145, on 07 / 24 / 2026, page. 91 / 259 80 / 192 todas as N-inteinas listadas: SEQ2... MKRTAMSEFESPKKKRKVKnSRKSLGTQWYDIGVEKDHNFLLKNGLVASRh SEQ6 ---...........- - - - VK VIG R RS LG VQRIF DIG L PQYHN FL LANGAIAANΞ E Ql 4 -----------------MVK VIG RR Ξ LG VQR IF DIG L PQDHN FL LANGAIAANC S EQ4 -AAAC PEL RQ LAQSDVYWDPIVSIE PDGVÊE VFD LTVPG P HN F VAN-DllAHN- # - ΐ ΐΐΐ r ψ ψ

[0188] In summary, the activity of interna is context-dependent, with certain peptide sequences around its linking junction (called N- and C-exsteines) being required for efficient trans-splicing to occur, the most important of which is an amino acid containing a nucleophilic element thiol or hydroxyl group (i.e., Cys, Ser, or Thr) as the first residue in the C-exsteine.

[0189] The present inventors used intein-mediated protein transplication in order to reconstitute large proteins in vivo. The split intes encoded by the inteina gene sequences are produced as precursor polypeptides, which through their structural complementarity can reassemble and catalyze a protein transsplicing reaction.

[0190] In the context of protein trans-splicing, the N-intein gene is fused into the structure with the sequence encoding the N-terminal portion of the protein of interest; the C-intein gene is fused into the structure with the sequence encoding the C-terminal portion of the sequence of interest. After Petition 870260074145, dated 07 / 24 / 2026, page 92 / 259 81 / 192 the expression of the two precursor fusion proteins, the inteins undergo autocatalytic excision and form an extein linked, for example, to the reconstituted protein of interest.

[0191] Therefore, the reconstitution of a protein of interest requires the division of said protein into two or three fragments, whose coding sequences are cloned separately into the AAV vector, fused to an N- or C-Intein and under the control of a promoter. The cleavage points for each protein are selected taking into account the need for amino acids at the junction point (e.g., the presence of an amino acid containing a nucleophilic thiol or hydroxyl group (i.e., Cys, Ser, or Thr) as the first residue in the C-Intein), as well as the preservation of the integrity of critical protein domains in order to favor proper protein folding and the stability of each precursor polypeptide of the Intein polypeptide and the resulting reconstituted protein.

[0192] In particular, the present inventors selected junction points within two proteins of interest: the ABCA4 protein is split into the amino acids Cys1150, Ser1168, Ser1090 and a split intein is inserted at the junction point. The CEP290 protein is split into amino acids Cys1076, Ser1275, Cys929 and 1474; Ser453 and Cys1474. Signs of degradation

[0193] Regulated protein degradation protects cells from misfolded, aggregated, or otherwise abnormal proteins, and also controls the levels of proteins that have evolved for a short lifespan in vivo and is largely mediated by the ubiquitin (Ub) proteasome system (UPS) and Petition 870260074145, dated 07 / 24 / 2026, page 93 / 259 82 / 192 via autophagy-lysosome pathways, with molecular chaperones being a part of both systems. Degradation signals are protein characteristics that make them targets of protein degradation pathways, resulting in a decrease in their half-life. In particular, N-degrons and C-degrons are degradation signals whose main determinants are, respectively, the N-terminal and C-terminal residues of cellular proteins. N-degrons and C-degrons include, to varying extents, adjacent sequence motifs and also internal lysine residues that function as polyubiquitylation sites.

[0194] Within the meaning of the present invention, internal degrons are defined as degradation signals located within a protein sequence neither at the N-terminus nor the C-terminus and whose functionally essential elements do not include N-terminus residues or C-terminus residues and mediate protein degradation.

[0195] Degron pathways comprise sets of proteolytic systems whose unifying characteristic is the ability to recognize proteins containing N- or C- or internal degrons, thus causing the degradation of these proteins by the 26S proteasome or autophagy.

[0196] E. coli dihydrofolate reductase (ecDHFR) is a 159-residue enzyme that catalyzes the reduction of dihydrofolate to tetrahydrofolate, an essential cofactor for several steps in primary prokaryotic metabolism. Numerous DHFR inhibitors have been developed as drugs, and one such inhibitor, trimethoprim (TMP), inhibits ecDHFR much more potently than mammalian DHFR. This Petition 870260074145, dated 07 / 24 / 2026, page 94 / 259 83 / 192 large therapeutic window makes TMP biologically silent in mammalian cells. The specificity of the ecDHFR-TMP interaction, along with the commercial availability and attractive pharmacological properties of TMP, makes this protein-ligand pair ideal for development as a degradation system. (69) Thus, the presence of the DHFR amino acid sequence, preferably the ecDHFR amino acid sequence, within a protein, functions as a target signal for the proteasome system, resulting in protein degradation. In the presence of TMP, the protein in question is stabilized.

[0197] Conveniently, degron signals derived from ecDHFR carrying dot points developed by Iwamoto et al. include three amino acid mutations, R12Y, Y100I and G67S (69) which confers functional activity (e.g., fusion protein degradation) only when placed at the N-terminus or within an internal position.

[0198] Further improvements to the ecDHFR-derived degron have been made by the present inventors who have identified the shortest active peptide. Conveniently, a shorter sequence allows for the fitting of longer coding sequences within the same AAV vector.

[0199] In the present invention, the degron derived from ecDHFR was fused to the N-terminal of the Intein where it is inactive. After protein transplication, the degron is located within the reconstituted Intein and mediates its degradation. The ecDHFRs of the present invention are WT ecDHFR, mutant DHFR, full-length ecDHFR, and shorter ecDHFR.

[0200] DHFR can be 105 to 159 aa in length, Petition 870260074145, dated 07 / 24 / 2026, page 95 / 259 84 / 192 in which shortening occurs at the C-terminal end ecDHFR derived from wild-type E. coli Nucleotide sequence (623 nt) SEQ ID NO: 27 AtcagectgatcgccgccctggccgtKRactacgtgatcggcatggaKaacgccatgxctggaacttecccgccgacctggcctgg ttcaagaKgaacaccctgaacaagcccgtgatcatggKcagficacacCtgERagagcatcKgCaEEcCcCtgcwEEçaREaaga scat ca tcctgagca gccaficccagcaccgacgaca gggtfia cctggg tRaagagcatfigacRa ggccatcgccgcctgcRgcga cfitgcccgagatcat&Etflatcfigcggcggcagggtfiatcgaficagttcctgcccaagficccafiaagctgtacctgacccacatcg acgccga gEtgga ggRCgaca cccacttccccga ctacflagcccgaceacti^aflagcgtEttc^^ cflcccagaacagccacaEctactgcÍtcgaRatcctgfiagafigafigtga amino acid sequence 159 aa- WT SEQ !□ Na. 28 Ml 15 LI A AL AVDR VIG MEN AM PWNL PA D LAW F KR NTL DKP VI MG RHTW ESI GRP LPG RK N11LSSQP G TD D R VT W VKS VD EA IAACG DV PE IM VIG GG R VY E QF LPKA QK LYLT HID AEV EG DTH F PDY EPDDWES VFS EF HDADAQNSHSYCFEiLERR ecDHFR E.Coli derived. Internal deeron mutant (159 aa) Positions of mutações and negrito - SEQ ID NO: 29 ΜI SLi A AUX VDY VIG MEN AM PWN LP AD LA WF KRNTLN KP VIM GR HT WESIG R PLPG RK N11LSSQP STD DR VT W VKSV D EA I AACG DV PEi M VIG GG RV1E QF LPK AQKLY LTHID AE V EG DTH FP DV E PD DWE SVFS EF HD AD AQN SH SY CFEILERR ecDHFR E.Coti derived, wild type, minimum active fragment Nucleotide sequence: (SEQ ID NO: 30 atcagcctaatcgccg<cctggcgtáaachtacgtgattgg.catggagajacKÇcatgccctggjiachetgccgc cgacctggechtgg ttcaaeaggaacaccctgaacaagcccRtRatcatfiftgeagficacacctj^afiaficatcfiflcaeflcccctficecaicaefiaafia acatcatcctfiagcagccagcccaflcaccfiacgacagegtgacctfiggtfiaagaficgtggacaaficctcflcctgcgcgg cfitgcccfiagatcatfiEtfiatcggcgfiCfiEcagEgtfiatcgagcafittcctgcctfia Amino acid sequence SEQ ID NO: 31 ΜISLIAALAVD R VIGM EN AM PWN LP AD LAW F KR N TLDKP VI MG RHT WES IG RP LPG RKN11LSSQPGTD D R VT WV KS VDEA! A ACG D VP Ei Μ VI GG GRVYE QFLP Petition 870260074145, dated 07 / 24 / 2026, p. 96 / 259 85 / 192 ecDHFR derived from E. coli, internal pe degron mutant, minimum active fragment (104 aa) Mutation positions in bold - SEQ ID NO: 32 ΜISLI AA LA VDY VIG MEN AM PWN LP AD LA WF KRN TLN KPV! MGRH TW ESIG RP LP GRK N11LSSQPSTD DR Ví WV KS VD ÊAIA ACG D VP EIM V!G GG R VIÉQF LP Sequences

[0201] The coding sequences of the invention can be operatively linked to a promoter sequence optionally followed by an intron sequence, capable of regulating its expression in a mammary cell, preferably a mammalian retinal cell, particularly a photoreceptor cell, or a liver cell, a muscle cell, a cardiac cell, a neuronal cell, a renal cell, an endothelial cell.Illustrative promoters include, without limitation, ubiquitous, artificial or tissue-specific promoters, including fragments and variants thereof that retain transcriptional promoter activity, such as photoreceptor-specific promoters, including photoreceptor-specific human G protein-coupled receptor kinase 1 (GRK1), interphotoreceptor retinoid-binding protein promoter (I RBP), rhodopsin promoter (RHO), vitelliform macular dystrophy promoter 2 (VM D2), rhodopsin kinase promoter (RK); muscle-specific promoters, including MCK, MYODI; liver-specific promoters, including thyroxine-binding globulin (TBG), liver-specific hybrid promoter (HLP) (67); neuron-specific promoters, including hSYNl, CaM Klla; kidney-specific promoters, including Kspcaderinl6, N KCC2. Ubiquitous promoters according to the present invention are, for example, ubiquitous promoters. Petition 870260074145, dated 07 / 24 / 2026, p. 97 / 259 86 / 192 cytomegalovirus (CMV) (32) and short CMV (33).

[0202] Optionally, the promoter sequence includes an enhancer sequence, such as the chimeric IgG globin 0 intron.

[0203] For the purposes of this invention, an EGFP (YP_009062989), ABCA4 and CEP290 coding sequence, which are preferably selected respectively from the sequences included herein, or sequences that encode the same amino acid sequence due to genetic code degeneracy, is functionally linked to a promoter sequence capable of regulating its expression in a mammalian retinal cell, particularly in photoreceptor cells.

[0204] Illustrative polyadenylation signals include, without limitation, the bovine growth hormone (bGHpA) polyadenylation signal, the human beta globin polyadenylation signal or a short synthetic version (68), the SV40 polyadenylation signal or other artificial or naturally occurring polyadenylation signal.

[0205] The present invention provides the use of a nucleotide sequence of a degradation signal in order to decrease the stability of the reconstituted protein. Conveniently, one or more sequences can be repeated in order to retain the maximum effect.

[0206] Suitable degradation signals according to the present invention include: (i) the short degron CL1, a C-terminal destabilizing peptide that shares structural similarities with misfolded proteins and is therefore recognized by the ubiquitination system, (ii) Petition 870260074145, dated 07 / 24 / 2026, page 98 / 259 87 / 192 ubiquitin, whose fusion at the N-terminal of a donor protein mediates both direct protein degradation and degradation via the N-end rule pathway, (iii) the N-terminal degron PB29 which is a 9 amino acid-long peptide that, similarly to degron CL1, is predicted to fold into structures that are recognized by enzymes of the ubiquitination pathway, variant ecDHFR and fragments thereof as described herein and in (69), particularly ecDHFR-derived degron signals carrying point mutations that include three amino acid mutations, R12Y, Y100I and G67S conferring functional activity (e.g., fusion protein degradation) only when placed at the N-terminal or within an internal position.

[0207] Exemplary signs of degradation are described in document WO 2016 / 13932, which is incorporated herein by reference.

[0208] As those skilled in the art can readily appreciate, there may be a number of variant sequences of a protein found in nature, in addition to those variants that can be artificially created by the laboratory specialist. The polynucleotides and polypeptides of the present invention encompass those specifically exemplified herein, as well as any natural variants thereof, as well as any variants that may be artificially created, provided that such variants retain the desired functional activity. Furthermore, within the scope of the present invention are polypeptides that have the same amino acid sequences as a polypeptide exemplified herein, except for Petition 870260074145, dated 07 / 24 / 2026, page 99 / 259 88 / 192 amino acid substitutions, additions, or deletions in the polypeptide sequence, provided that these variant polypeptides retain substantially the same relevant functional activities as the polypeptides specifically exemplified herein. For example, conservative amino acid substitutions within a polypeptide that do not affect the polypeptide's function would fall within the scope of the present invention. Thus, the polypeptides disclosed herein should be understood as including variants and fragments, as discussed above, of the sequences specifically exemplified. The present invention further includes nucleotide sequences encoding the polypeptides disclosed herein. These nucleotide sequences can be easily constructed by those skilled in the art having knowledge of the protein and amino acid sequences presented herein.As would be appreciated by a person skilled in the art, the degeneracy of the genetic code allows the specialist to construct a variety of nucleotide sequences that encode a particular polypeptide or protein. The choice of a particular nucleotide sequence may depend, for example, on the use of codons from a particular expression system or host cell. Polypeptides with amino acid substitutions different from those specifically exemplified in the polypeptides in question are also contemplated within the scope of the present invention. For example, non-natural amino acids may be substituted for the amino acids of a polypeptide of the invention, provided that the polypeptide possesses amino acids. Petition 870260074145, dated 07 / 24 / 2026, pp. 100 / 259 89 / 192 substituted amino acids retain substantially the same activity as the polypeptide in which the amino acids have not been substituted. Examples of unnatural amino acids include, but are not limited to, ornithine, citrulline, hydroxyproline, homoserine, phenylglycine, taurine, iodotyrosine, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, γ-aminobutyric acid, ε-aminohexanoic acid, 6-aminohexanoic acid, 2-aminoisobutylic acid, 3-aminopropionic acid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids such as β-methyl amino acids, C-methyl amino acids, N-methyl amino acids, and amino acid analogs in general. Unnatural amino acids also include amino acids with derivatized side groups. Furthermore, any of the amino acids in the protein can be of the D (dextrorotatory) or L (levorotatory) form.Amino acids can generally be classified into the following classes: nonpolar, uncharged polar, basic, and acidic. Conservative substitutions, in which a polypeptide with an amino acid from one class is replaced by another amino acid from the same class, fall within the scope of the present invention, provided that the polypeptide with the substitution still retains substantially the same biological activity as a polypeptide that does not have the substitution. Table 4 provides a list of examples of amino acids belonging to each class. Petition 870260074145, dated 07 / 24 / 2026, p. 101 / 259 90 / 192 Table 4. List of examples of amino acids belonging to each class. Amino acid classes Examples of amino acids Nonpolar Ala, Val, Leu, Ile, Pro, Met, Phe, Trp Polar Uncharged Gly, Ser, Thr, Cys, Tyr, Asn Acidic Gln Asp, Glu Basic Lys, Arg, His

[0209] Also within the scope of the present invention are polynucleotides that have the same nucleotide sequences as a polynucleotide exemplified herein, except for substitutions, additions, or deletions of nucleotides in the polynucleotide sequence, provided that such variant polynucleotides retain substantially the same relevant functional activity as the polynucleotides specifically exemplified herein (for example, they encode a protein having the same amino acid sequence or the same functional activity as encoded by the exemplified polynucleotide). Thus, the polynucleotides disclosed herein should be understood as including variants and fragments, as discussed above, of the sequences specifically exemplified.

[0210] The present invention also contemplates those polynucleotide molecules having sequences that are sufficiently homologous to the polynucleotide sequences of the invention so as to permit hybridization with that sequence under strict standard conditions and standard methods (Maniatis, T. et al, 1982). The Petition 870260074145, dated 07 / 24 / 2026, p. 102 / 259 The 91 / 192 polynucleotides described in this document may also be defined in terms of more particular identity and / or similarity ranges with those exemplified in this document. Sequence identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and may be greater than 95%. The identity and / or similarity of a sequence may be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% or more compared to a sequence exemplified here. Unless otherwise specified, as used herein, the percentile sequence identity and / or similarity of two sequences can be determined using the Karlin and Altschul (1990) algorithm, modified as in Karlin and Altschul (1993).This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990). BLAST searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain sequences with the desired sequence identity percentage. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997). When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) can be used. See the NCBI / N1H website.

[0211] Plasmids of the invention Petition 870260074145, dated 07 / 24 / 2026, page 103 / 259 92 / 192 Plasmid Sets $lie ITS- FTlt AAV teratvpe 2 / 2 Z / B ESFP pAAVÍ.l <W5' EGFPiniein PnaB ΪΕ59 PÂAV2.1-CMV-3' EGFP Inteln DnaB (1AAV2.1 CMV-5' EGFP intein mPnSE 2704 3557 PAAV2.1-CMV-3' EÜFP lnie«n mDnaE 2È56 - Ϊ557- H pM«.l CMV-5' EGFP im*in X PMV2.1-CMV-3' ESFP Inlein 2656 X X pAAVJ.l-CMW EGFP inrcin.^PHFR 3031 PAAV2.1-CMV-5' ESFP in1e.n_minl kDHFH 2869 pAAVZ-l-GRKl·^ FGFP irilKírt 1090 X 1 1 1 1 ρΜν2.1-ΰΒΚ1·Τ EGFP iirtein 21®t X PAAV2.1-TBG-5' EGFP iniein X pWP.Vl-lBÜU1 EGFP Intern 2764 X A0CA4 piac-CMVitfr S' ABCA4 Intein Set 1 487S X pzac<MV260· 3'ABCA4 Iniein 4602 X pAWJ |.CMV3W· 5' A&CA4 |n<trin_ecDHFH Set 1 5086 pAAV2 1-CMV2W 5' ABCA4 inwin.mioi rÜHFR 5« 1 4924 pzac-GRKl- 5' A6CA1 Intein Set 1 49C8 X pzaC-GRIÍl' 3ΆΜΑ4 intein 4634 X pAAVJ 1-GRKt- 5' A&CA4 ifttejn_eeDHFIl Sei 1 5059 X pAAV2.Hjfi.KL- &' AÈCA4 lmein_mlni CDHFR Set 1 49ΉΙ X pi3C-£MV26fr i1 ARCA4 Inteln Set 2 4979 pxac-CMV2fiO- 3'ARCA4 im^n 45 4a pzac-CMV2tO- i' Inteln AHCM Set 46 395 pzac-CW26O- 3'ABCA4 Intern 4782 & o ' «J Φ . U « I pAAVimCMVIGO 5'CEPIBO rnteirt Set 1 4281. Petition 870260074145, of 24 / 07 / 2026, p. 104 / 259 93 / 192 Plasmid Sets Size AAV serotype ITR* itr 2 / 2 2 / 8 (tip} pAAV2.1-CMV260-3* CEP 290 intein 5070 pMV2,l-CMV260-^ CEP29O intein 5et2 5051 pMVZl’CMWeO-y CEP29O intein 46d6 PAAV2.KMV260-5' CEP29O intern Set 3 SD51 PMV2.1-CMV2Ê0-T CEP290 Intein 4646 pAAV2.1-CMV260-5' CEP29O intein 4631 pAAV2.1-CMV260-CEP290 body intein Seta 3602 pMW- 1-CMV260-3' CEP29O intein 4536 pAAV2.1-CMV260-5' CEP290 Intein 3074 X PAAV2.1-CMV260-CEP290 body intein Sets 4906 X PAAV2.1-CMV260-3' CEP29O intein 4536 X PAAV2.1-GRK1-S' CÉP290 intein 3118 X pAAVO. 1-GRK1-CEP29Ü body intern Sets 4945 X pAAWA-GRKl-T CEP290 intein 4630 X ρΑΑν2Λ_ΗίΡ_5ρ FS intein Set 1 4919 X pAAV2.1_HLP_3' F8 intein 3962 X . —___-. ........___________ ....____ - . pAAV2.1_HLP_5r FS intein Set2 3935 X pAAV2.1_HLP_ 31 FS Intein 4946 X Petição 870260074145, de 24 / 07 / 2026, pág. 105 / 259 94 / 192 EGFP p915^pAAVZ.lTBG5' EGFP intein (SEQ ID No. 33) 5' ITR: dashed underline (seq A at 5' beginning of the sequence) TBG promoter: bold (seq B) 5' EGFP; underline (seq C) JNJ.ntej.n Npu DnaE.i doublejinderline (seq D) 3*flag: Italic (seq E) lA / PKE: italic underline (seq F) Bgh Poly A: bold underline (seq G) 3' ITR: dashed underline (seq H at 3' end of the sequence) ctgcacECtcgctcactcactRafiECCECCCfij^caaagcccj^gcgtcfiggCEacctttEEtcacccgECCtcaEtaaecgagcft aHCfiCECafiaHaggfiafitfificcaactccatcactdEfiEEttccttgtaEttaatsattaacccatfiCtacttatctacEtaHcca tgctctagga a gatcgga a ttcgccctta agctagcaggttaatttttaaaaagcagtcaaaagtccaagtggccctt tattctctttgtttgctctggttaataatctcBggagcacaaacattccagatccaggttaatttttaaaagcagtcaaaagtcca agtggcccttggfagcamarrtctctgtttgctrtggttaataauttaggcacaaacattccagatccggcgcgccagggct ggaagctacctttgacatcatttcctcgaatgoatgtataatttctacagaacctattagaaaggatcacccagcctctgrttttt gtacaa cttcccttaaa aactgcca attccactgctgtttggccc atgcccctattctgcctgctgaagacactcttgccagcatggacttaaacccctccagctctgacaatcctetttctctttgttttac atga agggtctggcagccaa agca atca ctcaaagttca aaccttatcattttttgttttgttctcttegccttggttttgtacatca gctttgaaaataccaUccagggttaatgctggggti:aatttataactaagagtgctrtagttttgcaatacaggacatgctataaa a stggaa agatgttgcittctgagagactgcaga agttggtcgtga ggcactgggcdggiaa gtatcae ggttaca a gacaggttt aaggagaccaata ga aactgggc ttgtcgagacagagaaga ctcttgcgtttctga taggcacctattggtctta ctgacatccactt Petition 870260074145, of 24 / 07 / 2026, p. 106 / 259 95 / 192 tgccttctctctccacaggtgtccaggcggccgccatggtgagcaafigficgaggagçt^ttcaccfieggtEgtgccca.tcctagtcaa gc tEfiacgRcgacgtaaacggccacaagtlcagcg tgtcchgficgaggfiCfigRgcg tgacc tgac tgac ccctgaag ttcatctflcaccaccEgcaafictgcccgtgccctgEcccaccctcgtgaccacectfiacctacggcgtgcagtgcctgaactacgag acc^agatcctgacqjtggagtaeggcctectgcccatcggcaagatcgtggagaagcjigatcgyLcac aa&HcatEacceteaacgECcagatactgetxatcaa c&aaa tcttcaaaceãEa UatcGogotco tgcca tcgadoca aggatgocgatgiicaogtgaaagcnggitccggtcgacctgggttocgggamgttiaagQgttggctggtgttcttagct atattqctccttttacqctgtq tqaqtqcactqctttaatqccttta to tea tacta ttacttcecattccattcacctta tgtaggtccctsqttgctQtccttctgtggQgagttgtcgKcgttgtcggcaachgtggggtggtgcachtgtQtQgchtggcgc aacccQcactaattaaQQcattQccaccacctatcaactcctttccaaQactttcactttcccccccctQtaQtaQtaQact eatcacctiãtqccttqcccqctactqacaaactcqqctqttqcnctaqcaattecqtqattattcaaqtiaaQctqac atcctttccataactQctc(icctQtQttaccacc.taaattctacQCQaQacatccttciaactacatcccttcQaccutcatccaQCi aaccttccttcceacQQcctactaccaacKtacaacctcttccacatcttcaaE.atctscictcgact&s.ccttctagiiía.cicagiea tcj|jttgttt^cccctccccgKtgccttccttgac«tggaajqjt^ccact«xactgtcctttcctaataaaatftaggaaattt;catc gcattgtctgagtaggtatcattctattctgagmtEEgEtgggEcggaacagcaaEggggatGgacacGcacGcac gtta agggega a ttcccgattaggatcttcctagagcatggcta cgtaga taagtagca rggcgggttaa tcatta actacaaggaa cccctagtga tggagttggcca ctccctctctgçgçgctcgc tcgctcac tgaaaccgggcaa cca aaggt cgcccga çgccqgg&chtttgcccfigficg&cçtcagtgagcga gcgagcgcgcag p917_pAAV2.1-TBG-3' EGFP intein. 5' ITR (seq A) TBG promoter (seq B) C-intein Npu DnaE (seq 1} SEQ3D No. 34 atgatcaagatcgccacccggaagtacctgggca agcaga a cgtgtacgaca tcggcgtggagcgggaccaca a rttcgccrtga agaa eggettea tcgccagwat Petition 870260074145, of 24 / 07 / 2026, p. 107 / 259 96 / 192 3' ÉGFP (seq L) SEQ ID no. 3S tgcttcagccgcta ecccgaccacatgaa gcacgacttettcaa gtccgcea tgcccga aggctacgtccaggagcgcaccatc ttctt ca aggacgacggca a data agaccegcgccgaggtgaagttcgagggcga ca ccctcaggggagtga gcatcgacttcaaggaeggca a ca tcctggggcacaagctgEagtaca a eta ca a cageca ca a cgtctatatcatggccgat aagcagaacacggcatcaggtgaacttcagatccgccacacatcgaggacggcagcgtgcagctcgccgaccacaccac agaacacccatcgggacggccgtgctgccgacaaccacctgagcccagtccgcctgegcaaag^ccccaac gagaagcgcgatcacacatgtctgctgtcgtgaccgccgccggtcactcggcatggacgagcctgtacaag Exflag (seq E) WPRE(seq F) Bgh PolyA (seq G) 3′ 1TR (seq Hj p914_pAAV2.1^MV-5′ EGFP Intein 5′ 1TR (seq A) CMV promoter (seq M} SEQ ID No, 36 tagt tatta a tag taatcaattacggggtca ttagttca ta gccca tatatggagttccgcgtta ca taaettaeggt aaa tggcccgc ctggctgaccgccca acgacc cccgccca ttgaegteaata algacgtatg a ccctagta ccctag caccag teccag atgggt ggagta ttt aeggtaa a ctgccca cttggcagtacatca agtgta tcatatgccaagta cgcccccta ttgaegteaat gacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctactcttggcagtacatctacgtagtcatcgc aatgggcgtgga tagcggtttgactcacgggga t ttccaagtctccacccc attg a egtea atggga gtttgttttggca cca aa atca a cgggactttcca a aatgcgta a ca a ctccgccccattgacgcaaatgggcg gtaggcgtgtacggtgggaggtcctcttactactaggtcaggttagt 5' EGFP (seq C) N-|nteln Npu DnaE (seq D) Petition 870260074145, of 24 / 07 / 2026, p. 108 / 259 97 / 192 3xflag (seq E) WERE (seq F} Bgh PolyA (seq G) 3' ITR (seq H) of p916pAAV2.KMV-3' EGFP Intern 5' ITR (seq A) CMV promoter (seq M) C-intein Npu DnaE (seq I) 3' EGFP (seq L) 3xflag (seq E) WPRE (seq F) Sgh PolyA (seq G) 3' ]TR (seq H) p932_pAAV2.1-GRKl-5' EGFP intein 5' STR (seq A) GRK1 promoter (seq N) SEQ ID no. 37 eta gtgggccccagaagcctggtggl tgtttgtccttctcaggggaaaaa gtgaggqggccccttggagga aggggccgggca ga at gatctaatcggattccaagcagctcaggggattgtctttttctageaccttcttgctaetcctaagcgtcctecgtgaecccggggga Petition 870260074145, of 24 / 07 / 2026, p. 109 / 259 98 / 192 tttagcctggtgctgtgtcagccccgggctc ceàggggcttcccagtggtcúccaggaaccctcEa ca gggoca gggçgtctctcg tccagcaagggcaggga cgggcca caggcaagggcgc 5* EGFP (seq C) N-intein Npu DnaE (seq D) 3xflag (seq E) WPREfseq F) 6gh PolyA {seq G) 3' ITR (seq H) of p933 pAAV2,l-GRKl-3' EGFP intein S' ITR {seq A) GRK1 promoter (seq N) C-Intein Downstream DNAE (sequence I) 3′ EGFP (seq L) 3xflag ($eq E) WPRE(seqF) 6gtl PolyA {seq G) 3′ ITR (seq H) p36 pAAV2.1-CMV-5fEGFP intein_ecDHFR Petition 870260074145, dated 24 / 07 / 2026, p. 110 / 259 99 / 192 5′ ITR (seq A) CMV promoter (seq M) S’ EGFP(seq C) N-intein Npu DnaE (sequence D) Bxflag (seq E) ecDHFR (seq 0} SEQ. ID No. 38 atcagcctgatcgccgccctggccgtggacgtgatcgtcgcgtggcgcgcctgcctgcctgccgcgcgcgttgg ttcaga gga a ca ccctgaacaagcccgtgatcatcatgggcaggcaggcacctgggagcatcggcaggCccctgcccggcaggaga a catcatcctgag^gccaECceaEcaccgacgacaEEgtEacctEEEtga a gagcgtggacgacgagccatcgccgaccga cggtgcgggg tcggcggcggcagggtgatcga gcagttcctgcccaaggccca ga agctgtacctgaccca catcg a cgccgaggtggagggcg aca cccacttccccga eta ega gcccgacgac tgggagcgtgttc a gcgagttcca egaccacgacgacga ccgc great grandmother! WPRE (seq F) Bgl·PolyA (seq G) ITR {seq H) p37 pAAV2.1-CMV-S' EGEP intein_mini ecDHFR S' ITR (seq A) CMV promoter (seq M) 5' EGFP(seqC} N-intein Npu DnaE (seq D) Bxflag (seq E) mini ecDHFR (seq P) SEQ !D No. 39 Petition 870260074145, on 07 / 24 / 2026, page. 111 / 259 100 / 192 a teagcc (gatcgccgccctggccgtggactacgtga tcggcatggagaacgccatgccctgga acclgcc cgccgacctggcctgg ttcaagagga acaccctgaaca agcccgtga tcatgEgcaggca ca cctgggagagcatcEgcaggcccctgcc cggcagga a ga a ca tea tcctgagcagccagcccagca ccga ega cagggtga cctgggtga a gagcgtggacgaggccatcgccgcctgcggega cgtgcccgagatcatggtgatcggcggcggcagggtgatcga gca gttcctgccctga WPRE (seq F) Bgh PoFyA (seq G) 3' ITR (seq H) p902_pAAV2.1-CMV-5' EGFP inteJn DnaB 5' ITR (seq A) CMV promoter (seq M) 5' EGFP (seq C) N tein RmaDnae (seq Q) SEQ ID No. 40 tgc ctggccggcga ca ccctga tea ccctggccgacggcaggagggtgcccatca gggagctggtgagccagcaga acttcagcgt gtgggccctga a cccccaga ccta ca ggctgga gagggccagggt gagcaggggccttctgcaccggcatca agcccgtgtacagg ctgac ca ccaggctgggcaggagca tea gggccaccgcca a ccacaggttcctgaccccccagggctggaagagggtgga ega gc tgcagcccggcgacta cctggccctgcccaggagga tcccca ccgccagcacccccaccctg N-lnteln Npu DnaE (seq D) Ixfiag {seq e) WPRE (seq F) Bgh PolyA (seq GJ 3' ITR (seq H) Petition 870260074145, on 07 / 24 / 2026, page. 112 / 259 101 / 192 p9C3_pAAV2.1 <MV 3' EGFP intein DnaB 5' JTR (seq A) CMV promoter ($eq M) C-intein Rma DnaB {seq R) SEQ. ID No. 41 a tggccgccgcctgccccgagctgaggcagctggcctaga gcga cgtgta ctgggacccca tcgtgagcatcgagcccgacggcgt ggagga ggtgttcgacctgaccgtgcccggcccccacaacttcgtggcca acgacatcatcgcccaca ac 3' EGFP (seq L) 3xfag (seq E) WPRE(seqF) Bgh PolyA (seq G) 3' JTR (seq H)pl256_pAAV2.1-CMV-SrEGFP intein mDnaE 5' ITR (seq A) CMV promoter (seq M) 5' EGFP (seq C) N-intein mDnaE (seq S) SEQ ID No. 42 tgcc tgagcra cga ca ccga gatcctgacogtgga gtacggcatcctgcccatcggca ag a tcgtggagaaga ggatcgagtgcac cgtgtacagcgtggacaacaacggca a catcta ca ccca gcccgtggcccagtggcacga caggggcga gca ggaggtgttcgag ta ctgcctggagga cggcagcctgatca gggccacca aggaccacaagttca tgaccg tgga cggccagatgatgcccatcgacg a gatcttcgagagggagctggacctgatga gggtgga ca acctgccca ac BxfJag (seq E) WPRE(seqF) Petition 870260074145, on 07 / 24 / 2026, page. 113 / 259 102 / 192 Bgh PolyA ($eq G) 3' ITR (seq H) P1257 pAAV2.1-CMV-3' EGFP intein mDn&E S' ITR (seq A) CMV promoter (seq M) C-intein mDnaE (seq T) SEQ ID no. 43 atggtgaaggtgatcggcaggaggagcctgggcgtgcagaggatctcgacatcggcctgccccagtaccacaacttcctgctggcc aa cggcgccatcgccgcca ac 3' EGFP (seq L) 3xflag (seq E) WPRE ($eq F) Bgh PolyA (seq G) 3' ITR (seq H) CEP29Q plOCS pAAV2.1 <MV260-5' CEP290 intein (set 1) 5' ITR (seq A) CMV26O (seq U) SEQ ID no. 44 ctagcgttgacattgattattgactagtacggtaeatggcccgcctggctgactcacgEggatttecaagtctccaccccattgac gtcaatgggagtttgttttggca ccaa aa tea aeggga ctttccaa aa tgcgta a ca a ctcqgcccca ageggat egggt Petition 870260074145, of 24 / 07 / 2026, p. 114 / 259 103 / 192 aggcgtgtacggtgggaggtctataataagcagctggtttagtgaactagaacecactgcttactggctlctcgagattccacca tggtgC 5JCEP290: SEQ ID No. 45 a tgccacctaatata aa ctggaaa gaaataatga aagttgacc ca gatga cctgccccgtca agaa ga a ctggcagata attta tl ga ttccttatcca aggtaagtaaatgagcta aaa agtga aa agcagaa aa tgtgatacacctttca agga ga attack aaggagtcas gtca gctggctttggaaga agta ga aaa agctggagagaga a ca agcaaatttga aa atca a ttaaaa eta a agtaatgaactgga aa atga a ctggagatggdxagcagtctgcaggtggacgagat actcggttttacgta a tgaatttgccaacuga aaa aaggaagattaggaca acaagattagaca a aagagaag aaa gttaatga gca at tggctcttcgaaatgaggaggcagaaa a tgaaaacagca aa traga a gagaga a ca aa eg teta aa ga aaa agaatga aca a ctttgtcaggata ttattga ctacca gaacaaa tagattcacaga a gacaagacactttt agacagtgactaccgatcacagttgtctaaa aa aaac tatgagcttatccaata tetga tga aattca ga ctttaaca ga aget aa tgagaaaa ttgaagttcagaatea aga aatga ga aaaaatttagaaga gtctgta eaggaaatgg aga a gatgaetgatcattagaat tgatag atag ta atgta atagatcagttaaaaaa agaaaaegatea ttatcaacttcaa gtg ca ggagetta caga tctcclga aa tea aaatga ag aaga tgatccaatta tggtagctgtc aatgea aa agtagaa gatgga a getta âtttgtctctaa gatgatga a atta ttgAgta tcagcaattagatta attagtta cataa tgatgctga la a aagta a tgtta tggeteta ca gcaggta tacagga a cgagacagtcaa ttaagatg ctcaccg aacaagtag aacaatatacaaagaatgaaagaatacttgtattattgagatttgaaaatgagetccaaagaaaaagtca tgctca tcaagaaccte gtcaacgttagacatttta aaaga ga aa acta aa gaggctgagagaacagctgaact ggctgaggctgatgetagggaaa a ggataagagtta gttgaggetetgaagagg tta aa agattatgaatcgggagta ta tggtt tagagstgctgtcgttgaat3â gaágaatagattagaatata aagata caaigga a at caa ta aact tga at tgaaga tcag tga tttcc tga tgaaaatgaggcactta gagagcgtgtgggccttgaaccaaa gata a tga tgattaactgaatttaga ata gca aa cacttaaa gacagcagcagta ca gagetgaaaa «a ga aga tgactgattgacttga ettgatetga aaaaaaaaa do tcaatggctc aga aa gaggaa aaagaagtgea acttcaggatt aa cca ctgaggacctga acctaac tgaaa a ca tt tctcaggagata ga a taagtga aagaa aat tgga tttattgagcctcaaa a tatgagtg aagcaca a tea a agaattca atttettca agagaacta attgatta aa gaagaaggaagagaa gatagccaaatttcagaata aa ttaaaaga attagttgaaga a ataagcaa ettgaaga a ggtatga aagaaa tattgea a gca a tta aggaa atgcagaagatcctgatgtt aaggaggaga aa catctctaatt a tcctagccttga a agactagttaatgctat tgtca gcaatcaagagaga tgcatttg aaagcccaa gttgatcagcttaccggaa ga a atga a ga an aa gac aggagctcagggaa teteggaaa gaggetata aattattca ca gcagttggcaaagcta a tttaaaga taga ccatcttg aaaa agaaa eta gtcttta cgacaakagagaattgattgatta t aa tggtagcaecatcta g Petition 870260074145, dated 24 / 07 / 2026, p. 115 / 259 104 / 192 tgccagtattatta a ttCtca ga atgaatàtttâ at aCatttgtta caggaactagaaa a ta aa gaaaaaaagtts aa ga atttága agattctcttgaagattaoaacagaaaatttgcttaattcgcatcaaaagtttgttgttaagtataaggaaa acctggaaaacagaatctaaa a caata aa agaggaaaaga ga aaa cttgagga tea a gtcca a ca a ga tgctataaaagta aaa ga atataastttgctcaatgctcttcagatgga ttegga tga a atgaaaaaaa ta ettgeaga aa atagtagga aagcttat tcaagtat acagaaa ggcaatataca a ixttaggaattggagcgacaaettagaaaaga aaa tgagaagca aaagaatgaattgttgtcaatggaggctgaagtttgtgaaaaattgggtttgcaaagatttaaggaaalggccatttcdagatt gcagctctccaaaaagttEtagataatagtgtttctttgtctgaactagaactggctaataaacagtacaatgaactgactgctaagt a cagggacatct tgcaaaaa ga taa tatgcttgtt ca a aga a ca agtaact tgga a cacctgga gtgtgaa a ata ctcc atta atta aggata agat agat agat a actggagattaccaagga aaaa ettea ca eta ttgaacaagectggga a ca gga aacta aattaggtaatgaatctagcatggataaggcaagaaatcaataaccacagtgacattgttccatttcaaaaaaataactatgc tggaaatga aggaat taaatga aa ggcagcggggctga a cat N-intein DnaE (seq D) 3xflag (seq E) shPolyA (seq V) SEQ ID No. 46 a attcaa taa a agatcttttcattaga tctgtgtgttggttttgtgtgcggcc 3'ITR (seq H) pl093 pAAV2,l-CMV26O-3' CEP29O intein (set 1) 5′ ITR (seq A) CMV260 (seq U) 3′ CEP290: SEQ ID No. 47 tgtcaaaaaatgta tgaacacttacggacttcgtta a agca a atggagga a cgt a attttga a ttggaaccaaatttgctgagctta cca aaa tea a tt igga tgea caga a ggtggaacaga tgt gatagagatga a ttagcagtag ta tcaggtca gtgtg eggattetagaattagaga aga atgaaatgga a ctaaaagttga a gtgtcaaaactgagaga ga tttetgata ttgc taga agacaagttgaa a tttgaa tgcacaacaatcttagggaca agg aagtagagt ccctca ga atgea actgctagactatc Petition 870260074145, dated 24 / 07 / 2026, p. 116 / 259 105 / 192 aggcacagtctgatga a aagtcgctcattgcca agttgcaccacacataatEtctcttca actgagtgaggtactgcttgEtaa gttggagtca attacatctaa etgea ga agatggaggcctacaacttgcgacagatt agagagagagaga tctattatgctcgtttgga gggaagaa a caga gca aaa ca tctgcgccaaacaattcagctcta cgacg acagttta gtggagcttt acccttgca ca a cag&aaagttctcca aaa ca atgattcaactacaa as tg acaaettaaga a ta a tgactagaa tggaga a ca aaa ca tiggaga tggaatta aaa ttaaagggcctggagagttaataagcactt a aaggata ccaaggagccca aa aggteat ca a ctggcatatga aa atagaagaacttcgttca agaacttaa a eta aa tegg ga attagteaa gg caatagata tt agaataaga caatca gca gtcttgaa ga aga a a attgtgea a caga a ca agtttcatga agaaaga caaatggcctgggatcaagaga agttga cctggaacgccaactagacattt tgaccgtcagca aaa tga a atactaa tgeggecta ca aa agtttga ag caengtagettag accaccacccatca tegeta aggaaatta aggagaa do a atatteta gaacacgggcaacttgcaa at ca ctagaa gagaasetaaa a gagaaaga a tctgctttaaggttagcagascaaa ta ta etgteaagaga ca aa gtaatcaatgaa etga ggc negattgcctgcca etgea ga a agagaaa a getea tagc tgageta ggca ga aaagaga tggaaccaaccattagtegca a aaccaaccaaccattagtectca ca tgcaagcaaggtta aatcaaaaaga a ga agtatta a agaagtatcaacgtcttctaga ad aagccagagagcaagaga aa ttgtgaaga aaca tgagga agaccttca uttctt«t«»ga n agaactacaggct gatagttca eta a ataaattcaaaacatgaccattaccagtggcatta. ccaacaagca ttttattcg tctggctga gatggaacaga cagtagcagaacaaga tgactctttttccteactctggt ca aa eta a agaaagtacaca agattt ggagaga ca a agagaatcactga a it gaaagta aaa gaantga aa atcaaattaca gettcat cataagaagaaagaaaa agtagaggatnaaa gtatcttctgga ceagtca case agg sgtcacagtgtttaaaatctgaa c ttcaggctcaaaa ga a gca aattcaagagctcca a caacta ca a tg agaaateta gt agaaeggeta a agage a attageettg a aggagag aa ccag ca a cagat ctccgggcagaatga ca gca gctgctgaagaacgtattantctgcaacnctcaaaagaggcccatcaatgtcaaaatcgttgatcgacatactcatagagctaagacacaagttgaagagatn aa atgaaa a tctttaaattga a agaagcactta aaa ctagtaagtaaa ca ga gaaaactcactactgata a ttcaatgaaa aagaatga atage etta etta ta aa ataetta ga gag aa aga ggaa al tga tea a gagaa tga t gaac tga aa aggcaaatta aa agactaaccagtgga ttacagggcaa cccctga ca gataataacaaagtcta attgaa ga a ctccaa ag gaaagtta a aaaactagaga a cca a ggtagaggaag aaccagaag aacctagaaa gatgetaaa ga agaatta a ttaggtggga agaagg t aaa aagtggca age ca aaa taga a gga attegaa a ca a gtt aaaga gaaagagggg gaagtetta ctttaacaagcagttgaatacttg a aggatcttttgccaagccga ta a agagaaacttacagaacttacttt ggttt tgggaatacgagctttggagtca gaaaaagaattggaagaatta a aaagaga a atettgaettaga a aatga ta tattgta tatgaggcccacc aagctctcctcga ga ttctgttgtagaagattta catttacaaa tagatacctccagaaaaaaca aattaga aettea ggatacatattcta a getettea at ttcagga atagagtea gatgatcattgtc richness a caggagettc ga a ggaaaacttga a gttgtca tetgaaaatattga a etgaaatttea gettgaa Petition 870260074145, 07 / 24 / 2026, p. 117 / 2 106 / 192 ca agca a ataaaga ttigc ca agatta a agaatcaagtcagagatttga a gga a atgtgtgaatttctraagaaaga aaa agcag a agttcagcgga aa cttggccatgttagagggtctgEtageagtgEaaagacaa tcccaga a ctggaaaatta atta tccag aaaaaagtagttgaaaaagtccagagagaaaatgaacagttgaaaaaagcatcaggaatattgactagtgaaaaaatggctaat attgagcagga aa atgaaaa a ttgaaggctga a ttagaa aaa ctta a agctcatcttgggcatcagttga gcatgca ctatgaatcc adgacf;3daggCAcag33aaa3ttattgctgaaaarg3aaggL:ttLgUaag3acttaaaa?33gaaactgatgctgcagag3a3 tt acggata gca a agaataattragagata ttaaatgaga a cagacagttca a cta gagagac ctgaccattagattcat gcttgaaggtgctgae agtaa gagctgaastccattgtggtta ca agaatgtatga aacca agttaa a agaattgga a actgatattgcca aaaaa aatcaaagcatta ctga ccttaaacagcttgta aa agaagcaacaga ga gagaca aaa agttaaga caa acc taca atga ttcttaaacatgttcrtgaaggtgctgagacagagceaggcc ttaaacgggagcttca agttctta ga ttagctaatca tcagctggataagagaaagcagaatta a tcca tc agatagaa get a aca aggaccaaagtgagctgaagca ccatecctga aagaata tg aaggataacca tgtcagatctaga aa ageagea tttga aggagga aa tAAagaagctga a aa aagaactgga aaatttgatccttcattttg aag aa attgaa gatetta agta ta a ttacagga a ga agtga a ga agaatattctctcttagaagaga agtaaaaaa ctttcaga aca àitÊggagttgdattaactagccctgttgctgcttctgaagagttÊdagatgaaga3gaaagtcctgttaatttccccattac; C-intein DnaE (sequence i) Sxflag (seq E) shPolyA (seq V) 3' ITR (seq H) P1065 PAAV2. 1-CMV260-5 'CEP290 Intein (set 2) 5' ITR (seq A) CMV260(seq U) 5' CEP290; SEQ ID No. 48 atgctacctaatataaattggaaagedataetgaaagttgacccdgdtgacctgccccgtcaagaagaactggcagdtaatttatt gatttccttatccaaggtgga a gta a atgageta aaa agtgaaaagca aga a aa tgtgatacaccttttcagaattactcagtcact Petition 870260074145, dated 07 / 24 / 2026, p. 118 / 259 107 / 192 aatgaatgaagctcaaga agtggagctggctttggagaagta gaaaaa gc tggagaaga acaagca aa atttgaaaa tea atttaaaactaaEtaatgaactggaaatgaactgactgEctcegcagtctgcagEtcggaggttgaatgaat atgaaatttgccaacttgaaaaaattagaaaaagatagagagaatlggagacatggaaaaggagttggagagagagaga aagt tatgagcaattggctcttcgaa a tgaggaggca gaa a tgaa aa cagcaaa ttaga agaga gacaaa egtetaa tegaga acaaactat atgaat a aaaaa tagattcagaaa gaacactttca agagaggg gagacagtgatgatccgatcacagttgtctaaaaaaactatgagcttatccaatctcttgatgaattcagactttaacagagct aatgaga a attga agttca gatca a gaaa tgaga aaaca gagatgaattt a tgaat ataatagaatgaagctattgtgcatcagacagataatgtaatagatcagttaaaaaaagaaacgatcattatcaacttcaagtg caggagc tta ca gatctcctgaatca aa aaatgaaga a gatgatcc a atta tggtagctgtc aatgetta t tga aga tga tgata agatatt agatta gtatca gcaaatgttaca ta acctaagggaga a ac tta gatgctcagct tga tgctga t aaaa gtaatgttatggctctacagcagggtata caggaa cgagacagtcaaatt aagatgctcaccga a ca agta g aaca a tatacaaaagaaa tggaa a agaa ta cttgta tta ttgaa ga tttgaaaa a tgagctcca a agaaa ca a aggtgettea a cc ctttcaacacattcaa^ aagtcagtatcaa^ aagtcagtatcaa^ gagaaa a rtaaagaggetgagaEa a cagctga act ggctgaggctgatgt tagggaaaggataa agagtt agttgaggctctgaa gaggttaaa gatta tgaatcggga gtata tggtt taga agatg etgtegt tga aa taaaga attgtaaaa ac gat a gaga attaga a aettga attga agatca gtga tttccttgatga aa atga ggcacttagagagcgtgtgggccttgaa cca a aga ca atgat tgatttaa etgaatttaga aa tagca aa cacttaa a cagcagcagtacagagctga aaaccaga ttcttgaaa a egatattgaatgaagaaa attegt caaatggctcaaga agaggaa aa agaagtgcaacttcaggatt aaccactgagga cctgaaccta a etgaaa acatttca agga gata ga ata a gtga a agaaa ttgga tgagcctca a aa ata tgagtgaagca a tcaaaga a tcaaaga a tga atgata aagtaga aggata aggata ggacagt gatagccaaa tttcaga ataaa ttaaaaga attagt tga agaa aa taagca a ettga agaaggtatgaaaga aa tattgeaagea atta ag£aatgeagaaaga tcctgatgttaa a ggaggagaa a catctccta geettga a agacta gtta atgc tata gaa tcaattgaagg agga a tgcat a tgcat agcccaa gttga tcagcttaccggaagaaatgaaga a 11 aagacaEgagctcagggaatctcggaa agaggeta taa a ttca cagcagttggca aa ageta ttata agata gaccatctg aaaagaaactagctcttttacgacaatcagaaggatcgattaagccagccatgattgattgattt tgcca gtatcattaattctcagaatgaatatt taata ca t ttgttacaggaacta gaaa a taaa gaaaaa a agtta a aga a ttta ga ags ttettgaagattawaw ga aaatttgctgtaattcgtcatca a aaagtttgttgta ta a gaatacctaaggaagaatcatagaatgaaaa agaga a aacttgagga tea agtccaaca agatgetata aa agta a aa gatataataatttgctcaatgcttcagattcggattgaatgaaaaaaaacttgcagaaatagtdggaaaattacttt tge agtga agagaat aatagaatcacc aatgagaa gca aagaatga a ttgttgtcaa tgga ggctgaagtttgtga aa aaattgggtgittgcaaaga ttaa ggaaa tggccattttcaa gatt Petition 870260074145, of 24 / 07 / 2026, p. 119 / 259 108 / 192 gcagctctci: aa aagt tgtagata a tagtgtttctttgtctgaactagaactggciaata a acagta ca atga aciga ctgctaagt acagggacatcttgcaaaa agata a tatgcttgttca a agaa ca agtaacttgga acacctggagtgagtgaa cagtact aacc aacc agtggagtctsatsaaaagaa ct ggagattacca a ggaaa a arttca ca ctattga acaagcetggga a caggaa acta aattaggta atgaatctagcatggata aggcaa agaaa teaata a cca acagtgacattgtttccatttcaaaa aa aataa ctatgc tgga aatga agga a atta iga aaa ggcagcgggctgaacattgtcaaa aa tgta tga a ca cttacggacttcgttaaagca aatg gaggaacgta a ttttgaattggaa a ccaaatttgc tga get tacca a aatcaa tttgga tgcacagaaggtgga a cagatgttaa ga gatgaattagctgatagtgtÊagcaaggcaEtaagtgatgctgatóggcaâtggattctgaattagaagaatgaaatggaact aaaagttga agtgtcaaactgagagagatttga tattgccagaaga ca agttga aattrtga a tgeacaaca a ca ateta gggaccgactactactactactactac teaggca cagtctgatga a aagtcgeteattgcca agttgcacca a cataatgtctctcttca aetgagtga ggetaetget cttggtaagttggagtcaa ttacatcta aaetgea gaaga tgga ggccta caacttgcgcttagagcaga a aettga tgaa a agaa ca ggeteteta ttatgc tcgtttggaggga agaaa cag agcaaa acatctg cgccaaaca a ttcagt ctct aega cgacagtt t DnaE assays (seq D) Sxflag (seq E) Bgh PolyA (seq G) 3′ITR (seq H) P1O&7 pAAVZ. 1-CMV26O*3′ CEP290 intein (set 2) 5′ITR (seq A) CMV2S0 (seqUi 3' CEP290;SEQID No. 49 ^etggagctttacccttggcacacacaggaaagttctccaaaacaatgattcaactaeaaatgacaacttaagatagaatgcaaga atgaaaaattctc aacaagaacata gaata tgga gacaa aa cattggaga tggaa tta aaa tt aaagggctgga g taccaccaa aggaatta ataa ggta a tcaactggcata tgaaa a tagaga ga acttcgtcttcaagaactta a actaaa teggga a ttagteaagga taa gaga aa taaa a tatttgaata acataatttctga a tatga a cgta ca a tcagcagtcl agaga aattgtgtcagatcagatcaag aga gaagttgacctggaacgcca actaga cattttgsccgtcagca aaatg ssatactaaa tgcggcaca aa agtttga agaageta cagga tea atccctgaecctag ttgccccttccaaa tea aettga gategetetagga aaattaaggagaacattcgaata aacagcag ga atteta Petition 870260074145, dated 24 / 07 / 2026, p. 120 / 259 109 / 192 at ca ct agaa gagaaactaa aa gagaaagaa tctgcttta aggttagcaga a ca aa ata tactgt ca agaga ca a agtaatca at ga a ctgaggctcg attgctgc ca ctgcagaaa gag aaa agctcatagctga a caccagaa tggac ga atggac ga aa ttgctcatcaa a cca ttgc aa acatgca a gc a aggtta aatcaaaaa ga agaagt a tta a ga agtatca a cgtcttetãgaaaâgccaagagagca a agagaaa ttgtga agaaacatgaggaaga ccttcatattcttcatca cagattag aactacaggctgatagttcactaataaattcaacaaacggcttgggatttaatgaaacagtctcccactccagttcctaccaca agca ttcgtctggctgagatggacaga ca gtagcagaa ca a gatga ctet cttcctcactt aggcaactaa a ga ca ca agatt at gagatt a tea ttaaaagtaaaagaatttga aaa tatcaaatta cage ttcaaga aa ac ca tgaatgaagtga aa aaagta aa agegga agtaga gga Etta a agta tcttctggaccagtca ca aaaggagt l aaa atetga a cttcaggctcaaa a agaagccatagaa aaggeaa aa ae ate atagct gage ca a tt ageettga agga ga a aca acaga aa gcacttagtcgggcactttagaactccgggcagaaatgacagcagctgctga ag aacEtatttttctctgcaacttctcaaaagagEcccatctcaatgtcaacaatcgttcgacatactagagctaagacaca a cttagagattta a atgaa aa tctataetta aagataa ga atatga ctca eta aetgataa ttt ga atgaettaaataatgaetgeaaaga aaaaaaagectat aaaaataettagagagasa ga ggaattgatca ga ga gaa tgatgaactgaaaggcaattaagaactaaccagtggattacaaggcaaaccctgacagaata gaatgaaaag aaggaaaaccaaaccaattgaa ttagagggaaa ggtgga ggaagt agacctaaaacctatgaa aga aaa ga atgeta aa gagaa tttatta ggtggga agaagtaaaagtggcaa gcca aa atagaa ggaattega a aca agtta aa aga gaagaggggga a gtcttta ettt agcaaagccattt g ttgagact taccagaa gaaaetta ctttg ctaaaaacaaetggcatgaetgttgateaggttttgggaata cgagrtttggagtcaga aa sags a ttggaaga a1t aaaa aaga ga a atettgaettagaaa atgata tattgta ta tgaggccca cca ca agcttcctcga gatcatcatta a gatcatta aagct aacttcatgctttaga aa acagtttt caaagga tacatattcta a gccttcaattt tag gatagagtcagatcattgtca gagaga a caggagettcagaagga aa aettga a gttgtca tetgaaaa tattgaaetgaaa ttcagcttgaacaagcaaata a agatttgccaagatta a agaatcaagttaga aagatttgaat gaaaaagca gaagttcagggaaacttggccatgtta gaggtetggtagaagtgaagacaaa tccca gaaetgga aaaacc a tlggttraatga aa aaagta gttgaaaa gtccagagagaaatga acagttga aaaaagcatca gg atatlgaata tgragactaaaggatgaaa ttgaaggetgaatta gaaaaaetta aagctcatcttgggcatca gttga gcatg ca ctgaatccaa ga cca a aggcacaga aaa a atta ttgctgaa a atgaaggettegtaa a gaaettaa aa agaaaetgatg etgeagagaaa ttagaatt1gaatgaattagcaatata a ctagaaga gactgta a gag a ttgcagtttgcaga a agcagaggtcca cagcttga aggtgetga cagtaagagctgga a atccattgtggtta ca agaatgta tga aa cca ag ttaa a agaattgga aactga ta ((gccaa aaaa aatcaaagca tt gacagac acagac attaac) aaaaagtt aaaaataca tgaga ccttgaacacagatt aagattcttaaacatgttctgaaggtgjctgaga c agagcaaggccttaaacgggagcttcaagttcttaga ttagetaa tea tc agetgga ta aaga ga a agcagaanaa tccatca ga Petition 870260074145, dated 24 / 07 / 2026, p. 121 / 259 110 / 192 taga a geta a ca aggaccaaagtggagctgaaagca ccatacctga tgctgatcaactaaagga a aaaataaa a gattagaga cacagctca a atgtca ga tctagaaagcagca ttgaagga ggaataa a ga a getgaaaaaga a ttgagatgattaa a ttgatgattaa a ctgatgattaa agtataattacaaggaa ga a gtgaaga a ga a tattetetta gagagaa ggtaaaaaa actttcaga a ca a ttggga gttgaatta a eta gccctgttgctgcttga ag agttga agatgaaga agaaagtcctgtt a atttcc ccatttac C-intein DnaE (seq I) Jxflag (seq E) Bgh PolyA (seq G) 3′ ITR (seq H) pl087 pAAV2.1-CMV26O-5′ CÉP29Ü intein (set 3) 5′ ITR (seq A) CMV26O (seq U) 5′ CEP29O: SEQ ID No. 50 atgccacctaatata aa etggaaaga aa ta a tgaaagtgacc caga tga cctgccgtcaagaa ga a ctggcagata a tttatt gatttcctta tccaaggtgga agtaaatga geta aaaagtga aa agcaagaaatgtgatacaccttca ga attactcagegtea a gactcatca gctggctttgga agaagtaga aa aagetggaga agaac aagcaaatttgaaatca atta aaaa eta a agtaatga aa ctggaaatga actggagatggctcagcagtctgca ggtggacgagata ctcggtttta cgta atga aa ttgccaacttga taggagasaaaaggaaaaa aaaggagttggagaa aggaga aagtta a tgagcaatttggcttq;aaatga ggaggcaga aaa tgaaaacagcaaatta a gaagaga ga aa aaegtetaaaga áaaaga a tga ata attttgtCa ggatattattga CtaccagaaaCâatagattcacagâaag3aa CaCttttatCaagàa ga ggg gaaga ca gtgaetac cgatcac agttgtcta aa aaaaac ta tga gcttatccaatatcttgatgaaa ttca ga ctttaacagaa get aatga ga aa attgaagttcagaatcaagaaa tgagaaaaa a tttagaagagtctgtacagga a atggaga a gatgaetgatga at ataata ga a tgaaageta ttgtgcatcaga ca ga taatgta a tag atcagtta aaa aaagaa aa ega tea ttatcaacttcaagtg caggagctta ca gatctcctgaatcaaaa aa tgaagaga tgatcca attatggtagctgtcaa tgea aa attagaga atgga agctaattttgtcttcta aa ga tgatga aa tt attgagt atcagcaaatgtt a ca taacctaagga ga a aettaaga atgctcagct tgatgctgataaaagtaatgttatgEctctacagcagggtatacaggaacgagacagtcaaatttaagatgctcaccgaacaagtag aacaatata caaa aga aatggaaaa ga atacttgta ttattga aga tttgaa aa atgagctccaaagaaacaaaggtgcttcaacc ctttctcaa cage ctcata tgaaaattca gtea a cgttagacatttta aaagagaaa ctaa a gaggctgagagacagctga a ct; Petition 870260074145, dated 07 / 24 / 2026, pp. 122 / 259 111 / 192 ggctgaggctgatgctaEggaaaaggataaagagttagttgaggctagagaaggttaaaagattatgaatcggEagtatatggtt tagagaatgc tgtcgttgaaata aagaattgtaaaaaccaaattaaaaata a gagatcgagagattgaaatatta acaaagga aat ca at aaacttga a ttgaa gatcagtgatttccttga tga&a a tgaggcacttagagagcgtgtgggccttga a cca a agacaatgat tgattta actgaatttagaaata gca a acacttaaaa ca gcagcagca ca gagctgaaa a cca ga ttctttg aaaga ga ttgaa a gtctagaggaagaa ega cttgatctga aaaaaaaa a ttcgtcaaatggcteaaga a sgaggaaaaaga^gtgcaacttcaggatt aa cca ctgagga cctga acctaactgaa aa ca tttctcaa ggagataga at aagtgaaagaa aa ttgga tttattgagcctcaaaa a tatgagtgaagca ca a tea aagaatga at ttctttca agagaa ctaattga a aaagaaagagattta gaaaggagt agga ca gt gatagccaaatttcagaata □ attaaaa gaattagtgaagaaaaataa gca a ettgaaga aggta tgaaagaaatatgea agCa a ttaagga aatgeaga a agatcctga tgttaa a ggagga gaaacatctcta a it a tccctagcct tga a agactagttaatgc ta ta gaatcaaaga a tgcagaagga atctttgatgegagtctgcatttgaaagcccaa gttgatca gcttaccgga a gaa atga a gaatt a agac aggagctcaggga a teteggaa ag aggeta ta aattattcaca gcagttggcaaaagcta a tttaa a gataga ccatcttg aaa aagaa a eta gtcttttacgacaatcaga aggatega a tgttgtttta a aggaattgaetta cctga iggga t agcaccactcatca tatgatca g tgt tttgtt acagga a ctagaaaata a agaaaa aa agttaa a ga atttaga a gattetettgaagatta ca acagaaaatt tgctgtaattcgtcatcaaca aagttg ttgttg taagaa t acctaagtga a aagga ga cetggaaaacaga atetaaaatgaca gaagga atagagga agtccaacaagatgcttaaaagt aaaa gatatãataatttgctcaatgcttcagatgãttcggatgdaatgaaãadaatacttgcagaaatagtaggaaattdcttt tgcaagtga a tgaaa aa teaettata aggca ata ta caac a aggatca aggagacagacaat agaatgaa ttgttgtcaatggaggctga agtttgtgaaaaattgggtgtttgca a agatttaaggaa a tggccattttca agatt gcagctctccaaaagttg ta ga ta ta gtgttctt tgtctga actagaactggctaata a gtgaetgeatca cagggacatcttgcaaaagaatatatgcttgttca a agaacaagta aettgga a ca cctggagtgtgaaa catctccttaaaa ga a caagtggagtcta ta a aaaga aetggaga ttaccaagga aaa acttcacacta ttgaaca agcctgggaa cagga aacta a attsggt Mtga at etageMgga taaggca a agaaatcaataacca acagtga cattgtttcca ttcaaaaa aa at aactaggac a tggagga tggaaa ca ttgt ca aaaa atgta tgaacactta eggaettegtta a agcaatg gaggaaegt aatttga attggaaccaaatttgctgagctta cca aaatcaatttggatgcacagaaggtggaacaga tgtta aga gatgaattagetga tagtgagggcagagcagca aggagatte gtaagct ( gagaatgaaatgga act aaagttgaagtgtcaaactgagagagatttctgaattgccagacacaagttgaatttttgaatgcacaacaatctctaggg acaggaagtagtccctCagsatgcactgctagactatcaggcagtctgatgaaagtcgctagcattCgccaagt tgtctctcttcactgagtgaggcta ctgctcttggtaagttgagtca a ttacatctaa a etgeagaga tggaggcctaca a cttgcgcttagagcaga aa ettgatga aaagaaca ggetetetatta tgctcgttggagggaaga aa cagagcaagcacacacacaca cat N-intel rtiDnaE (seq 5) Petition 870260074145, dated 24 / 07 / 2026, p. 123 / 259 112 / 192 3xflag (seq E) Bgh PolvA($eq G) 3'ITR (seq H) plQSB pAAV2.1-CMV2G0-3' CEP29O intein (set 3) 5' ITR (seq A) CMV26O (seq U) 3rCEP290r5EQIDNo.51 agtggagctttacccttggca caacagga a aagttctcca a aaca atgattcaacta ca a a atgaca aa ctta agata atgca a ga aattctca a caagaa ca ta gaatatggagaaca a aacattggag atgga agga a gact a tta agg ataccaaaggagcccaaaaggt aa tcaactggca ta tga aaatagaagaa cttcgtcttca aga acttaa act aaa tcgggaattagtcagga taaagagaa a ta aaa tatttgaataaca taa ttt ctgaatatgaacgt a caatcagca gt caatt aacat tgagag aga gaaagaca aa tggcctgggatca aa gagaagttgacctgga a cgcca ga cattttgaccgtca gCaaa atga aa ta ctaaatgcggcacaa a gtttga aga agCtacaggatcaatccctgacccta g ttgccccttcca a atcaacttgaga tgagcttaga atttacattagaga a aacacgggcaacttgca a atcactagagagagaaactaagagagaaagaatctctgctttaaggttagcagacaaaatactgtcaagacaagtaatcaat ga actga ggctcgattgc ctgccactgca gaagagaaaaccaaat cactcatagctgagctaggcag accaaaaacaat gactgaat attgctcatca aacca tt gcaaca tgca attgcatcacaaaaaga aga a gtattaaagaagtatca acgtcttctagaa aaa gccagagaggagca aagagaaa tgtga agaa a catgaggaagaccttcatattcttca tcac aga tt ag aacta caggctagttca ctaataa a ttca aaaa cggcttca agattatcattaccatta gcaccact a ca g ttcgtctggctgagatggaa ca gagagtagca ga acag a tga ctCtctttcctcacttggtcaaacta a aga aa gt atcacaagdtttggagacaagagaaatcactgaattaaagtaagaattgaaataatcaattacagctcagaaaac agac agatga aaggaaga tga taaa gta t cttctggacca gtcac aaaa gga gtcacagtgttt aaa a tctga acttcaggctcaa a aga a gcaattca aga gctcca accacta catgagaatcta gta gaacggcta a aggact ca attagccttgaggag aaa caacattagtta aa gcagcagtta aa gcagtcaaa tga cagcagctgctgaag aa cgtatta tttctgca acttctcaaaa agaggcccatctcaatgttc a acaaatcgttgatcga catactagaga geta aagaca ca agttg aga ttta a atga aa atctttaaa a ttga aa ga ageaetta a aa ctagttaa aag a ctacacta aa a cagacagaa a cagagaetta atatgaetgeaaaaaaaaaagccta taaaaataetta gagaga aagaggaa a ttga tea a gaga tgatg aa etga a aaggcaa atta aaa gacta accagtggattacagggcaaacccctgaca ga ta taaaca aa gtctaattgaa Petition 870260074145, dated 24 / 07 / 2026, p. 124 / 259 113 / 192 gaa ctccaaagga a agttaaaa aa eta ga ga a tea a tlagaggga aa gg tggagga agtaga cctatga a aga aaa gatgeta a agaagaatta attaggtgga agaaggta aaa agtggcaagccaaataga aggacaatttaga agaa gtctttactttaacaaagta gttgaataetttga aggatctttt tgea aa geega t aa agagaa a ett a ct ttg cagagga aa ctaaaaacaetggcatgttgttcaggttttgggaata ega gett tggagtea ga aaaagaattgga agaa tt aaaccaattagae a gattaga tagac a gaga a ccaa gctcttcctcga gattctgttgt agagattta cattta ta aaa tagatacctccaagaaaacttcatgctttagaaaaacagttca aa ggatacatattctaa getttea mcag gaa tagaglcagatga tea tig tcagagaga a ea gaatt aaggatt cgattaca tegattaga etgaaa ttcagcttgaacaagcaaataaa ga tttgcca ag attaagaatca agtcagagatttga aggaaatgtgtgaatttctta aga aa gag aaa gcagaagttcagcgga aa ett ggccatgttagagggtctgtaga agtgga aa ga ea a tcggattgaatta aatggaatta a etggaat aaagteeagagaga aaa tga acagttga aaaaa gcatcaggaatattga ct agtgaaa aaatggcta atattga gca gg aaaatgaaaa a ttga aggetga a ttagaaaaaetta aa getea tctgggcatcagttgagcatg eactatga a tcea a ga ecaaa ggca ea ga aaaaa ttattggactgaaa agaaettaaaaaga aa etga tg ctgcagagaaattacggatagcaaagaataatttagagattaatgagagagagatgacagttcactagagactggtaagag attgcagtttgcagaa a gca gaggtccacacagcttgaggtgctg acagta aagattcagctgattag acca agaattgga aa ctgatattgccaaaaaaaateaaa gca 11 a ttgaccttaaacagc tgta aa aga agcaaca ga ga gagacaaaagtta a ea ta ea a tgagacct tgacacagatta agattetta aa catgttcctgaggt agcaagctagggaggac acu gttuttagattageta atcatcagctgata aagagaaagcagaatta a teCa tea ga taga ageta a ta a ggaccaaagtggagttgaagcaccat acctgatgctga tcactaaa gg a aaaataaagateta gaga cacagctcaaatgtca gatctagaaaggaaggagagagaa aataagaaggagattt ctggaaaattttgatcc ttcatttttgaga a attgaagatetta agtatatta ea a ggaaga agtgaaga agatattetettaga agagaaggta aaaaa actttcaga a ea a ttgggagttga attaactagccc tgttgctg cttctgaagagtttgaagatga agaagaaag tcctgtta a tttcc ccatttac Cintein mDnaE (seq T} 3xflag (seq E) Bgl· Poly A (seq G) 3' ITR(seq H) pll82 pAAV2. 1-CMV260-5' CEP29Ú intein (conjunto 4) 5'1 TR {seq A) CMV26Q (seq U) Petition 870260074145, dated 07 / 24 / 2026, pp. 125 / 259 114 / 192 S' CEP290: SEQ ID No. 52 atgcca cctaa ta ta aactggaaaga aa ta a tga aa gttgacccaga tga cctgccccgtca a ga a a ctgca ga taa tttatt gattccttatcea a ggtggaagtaaatgagcta a saagtga aaagcagactca gaaatgtgata ca accttagactcatcagagagata gctggctttgga agaagtagaaaagctggagaàga acaagCaaaatttgaaastca at taaaact aa agtaatgaactggaaatgaactggagatggctcageagtc tgcaggtgga cgaga ta ctcggttt t tacgta atgaatttgcaacaact aggagta tgaaaact tggaaaaggagtt ggag aaagag aagag ggca ga aa atga aaa ca gcaatta a ga a ga ga a ca aa cgtctaa ga aaa agaatg a ac aa g cttgtcaggatattgattattgactaccagaaca gatta gaaa tegagattca agacagtgactaccga tea cagttgtcta aa aaaa a etatgagetta tccaatatctcttgaaattca gaettta a cagaagct aa tgagaaaattgaagttcagã a tcaagaatgaga aa aaattta ga a gagtetgt aca gg aa atggagaattgat tag atgacd etgá summer a ta gatcagttaaa aaa agaaa a ega tcatta tea a cttcaagtg caggagct tacaga tctcctga aa tcaaaaaatgaagaaga tgatccaattatggt agetgtea a tgea aa agt a ga aga a tgga ag ctaat 11 tgtcttctaaagatga aa tta gtatcaa gttaca gttag taccaat a ct ta a ga atgctcagct tgatgctgataaaagtaatgttatgctctacagcagggtatacaggaacgagacagtcaaattaagatgctcaccgaacaagtag aa ca a ta caaaaga aa tgga aaa ga ata ettgtau attgaagatttga aaa atga gctccaagaagtacactacactat aaa cagactcatatgaaaattca gtcaacgttagacattttaaa a ga ga aaa etaaagaggetga gaga a ca getga a ct EgetEaggetEatgetaggga aa agga ta aagagttaEttgaggctctgaagaggtta aa agattatEaatcgggagtetatggtt tagaagatgetgtegt tga aataata aa aata aa aa aa ca tegaga ga ttga aa ta ttaaca a aggaaat ca ataaa ettga a ttgaagatcag tgatttccttga tgaaaatga ggca ettagagagegtg tgggccttgaacca aa gaca a tga t tgatttaactgaatttagaatagcaaacacttaaaacagcagcagtacagact gtctagaggaagaacgacttgatctgaaaaaaaaa attegtea aatggetea aga a agaggaa aa agaagtgcaacttcaggatt aa cca etgagga cctga acc taac tga aa ac a ttetea a ggagatag a ataagtgaaagaaattgga tttattgagctca aaa at atgagtgaaggaatcacaatcaaga atgaattttcagattagagagagaactaattgaat gatageca aa tttcaga ata aa ttaaa agaattagttgagasaata a gcaacttgaagaaggt atgaaagaa atattgeaagea at taggaagga ggaga aa catctctaattatccct ageettga a agact agttaa tgetata gatcaaagaatgcagaaggaatctttgatgcgagtctgcatttgaaagcccaagttgatcagcttaccggaagaaaEgaagaatt a agac a ggagetea ggga atetegga aa gaggeta taa a ttcacagca gttggca aa aget a atttaaagatagacc atc ttg aaaaagaaaetagtcttttacgacaatcagaaggatcgaatgttgttttaaggaattgacttagatgatgatggatcaccatctag tgea gt atcatta attctcaga a tgaatatttaataeatttgtta cagga a ctagaa aaaagaaaaaa gttaaaaaatte agaattga atttaga tttgetgtaattcgtcatcaaea aa gtttgttgtata aaga a ta cctaagtga aaa gga g acetgga aaa ca gateta aa acataaaagagga aa agaga aaaettgagga teaa gt snowflake Petition 870260074145, 07 / 24 / 2026, p. 126 / 2 115 / 192 ga atat aa ta atttgctcaatgctcttcagatggattcggatga aa tgaaaaaaat a cttgcagaaa ata gtagga a aattactgttt tgca agt ga a tga a aaatcactttataaggca a ta ta caacctta gtagaattggagcgaca a cttagaaaag aaaa tga ga a gca aa aga a tga attgttgtcaatggaggc from a gt t N-intein DnaE (seq D) 3xflag (seq E) WPRE (seq F} Bgh PolvA(seq G) 3'ITR (seq H) pll83 pAAV2.1-CMV2ÊÜ-CEP290 body intein (set 4) . 5' ITR (seq A} CMV26O (seq U) C-intein DnaE (seq I) CEP290 body: SEClID No. 53 tgtgaaaaa a 1t gggtgtttgca aagatttaaggaaatggccattca aga tgca gctcca aaaagt tgtag a ta (agtgttc tugtctgaa ct a ga actggctaata aacagtaca a tga a cttgactcaggact aagat t agtagta t agtag haaagaacaagtaacttggacacctggagtgtgaaaacatctccttaaaagaacaagtggaEtcttaataagaactggaga ttactaaggaaaa cttcac acta tga a caagcctgggaacagga aa Ctaaaattaggtaatga atctaggata aggca aa gaccat ttcaaat aatcag a aaatactatgctggaaa tgaaggaa tta a atga aa ggcagcggg ctga a cattgtcaa aaaa tgtatgacactta cggact tcgt taa agcaaatggagg aa cgta a tttga a ttgga aacca a atttg actgagctta ccaa aa tcagagattgacttggacattagt t agtgtgagcaggcagta agtgatgctgat aggcaacgga ttcta ga a ttaga gagaatga aa tgga a ctaaaagttgaagtgtca a aact gagagaga t e tgatdttgccagagacaagttga aa tttgaa tgcaca a caact a tcta gggacaagggat gattactaga gt tcaggcacagtctgatg aaagtcgctcattgcca a gttgcacca acataatgtctctcttcaactga gtgaggctactgc tcttggtaagttggagtca atta catcta aa ctgcaga a ga tgga ggcctaca a cttgcgctt agagcaga aacttga tga a aaaga aca ggctctcta 11 a tgctcgtttggagggaagaaa ca gagcaaaa catctgcgccaaaca a ttca gtctctacga cgacagt ttag tggagcttla cccttggca ca acagga aaa gttctcca aaa caatga ttca a ctaca aaa tgacaaactta agataatgc aaga aa Petition 870260074145, dated 07 / 24 / 2026, pp. 127 / 259 116 / 192 tga a a attctcaacaagaacatagaatatggaga a ca aaa ca ttggagatggaattaaatta a agggcctgga agagttaat aagcact ttaaaggataecaaggagcccaaagtaatcaac tggc a tatgaaa a ta ga aga a cttcgat a gagt a cttacca cttca ggata aagagaa taaaatatttga atacata antctgaatatga a cgta caatca gcagtcttg aga a gaaattgtgca aca ga a ca agttca tga agaagac aa atggcctggga tcaaaga ga a gttgacctggaacgcca a ct agaatcaatttga tgcggcacaaagtttga aga agctaca gg atca atccctga ccctagttt gcccttccaaatcaacttga t egetetagga aaat ta aggacacattcgaa taattetaga aacacgggcaact N-intein Rma DNAB (seq Q) 3xflag (seq E) WPRE (seq F) Bgh PolyA (seqG) 3'ITR (seq H) pllBl pAAV2.1 <MV26O 3’ CEP29O intein (set 4 / set5) 5′ ITR (seq A) CMV260 (seq U) C-intein Rma DNAB (seq R) 3rCEP290: SEQ ID No.54 tgcaatcactgagagaaactaaaagaga aa gaa tetgetttaaggttagcagacaaaatactgtcaagacaagtaa tcaatgaactgaggcttcga tgectgecactgcagaagagaaagetcatagctga getaggea ga aa aga gatggaacc aaa atctcaccacacattgaaattgctcaatcaaccattgcaaacatgcaagcaagttaaateaaaagaagcagagagagcaagt atca acgtcttctgaaaaagccagagagcaaga gaattgtgaagaa a ca tgaggagacct tcatattcat tacataga aacatt aaggata ttaga cggcttggga tttaatga aa cagtctccca ctccagttcctacca aca agca tttattcgtctggctga ga tgga acagtagcag aaca agatgactctctct ca ctcttggtca aactaagaa agtatcacacagatttggaga gata a agaga ttca etgaaactataaagtaa aaccatga agatgaagtga aaa 3 a gta aa agegga agt agaggattta aagtacttctctctggaccagtcaca aa aggagtcagt gttta aaa tetga a cttca ggetcaaaga a gcaaattca a ga gctccaacaactacaatgagaatctagta gagaacca aa eggattagaet a cagaaagcacttagtcgggca cttttaga a ctccgggca ga aa tgaca gca getgetg. Petition 870260074145, dated 07 / 24 / 2026, pp. 128 / 259 117 / 192 a a a cgta tt atttetgca acttctcaaaa a gaggcccatctca atgttcaaca a atcgttgatcgacata eta gaga geta a a ca caagttgaa ga ttta a atga aa atctttaa a attgaagaagcacttaaaactagtegaaaactaactacagaca atgacttaaataatgaactgcaaagaaaca aaaa gccta taa ta aaa ta etta gagaga agaggaa a ttgatcaag a gaa tga tgaaetgaaaaggea aa ttaa agacta accagtgga ttacaggca aa cccctgaeagat aa taaaca a agteggtega aaaaaa tccaagga acga acga aa ggtggaggaagta gacetaaaaccta tgaaag aaaa ga atgeta a a ga a aa ttagtggga a ga a ggt aaaa agtggcaagcca aaa taga a gga a ttcgaa aca a gtta aa agaga a agagggaagtcttta ectttaacaaagcattagaggattgattgattacttact etttgeaga gga aa Cta aa àa CaaCtggcatg3ctgttgatcaggttttgggaatacgagctttggagteagaa aaa ga attgga a gattaa aaa agag aaa tettgaettagaaatgata tattgtatatgaggcc ca ccaagctcttectcga gattctatttgtaga t aaca tga t tagatacctccaagaaaaacttcatgc tttagaaaaacag ttttcaaaggata cata ttctaagccttcaatt tea gga a tagagt ca ga tga t ca ttgtcagagagaacaggagct tc agaaggaaaacttgaagttgtca tetga aaa ta ttgaact gaa atttcagcttgaaca a gca aa ta aa ga tttgcca a ga atta gca a gaa a gaa a tttgaaggaaatgtgtga a tttettaa gaa aga aaaa gcaga agtt ca gegga aa cttggccatgtta gagggtctggtagaagtgga a aga ca a tcccagaactggaaaaa aa ccattggttta atgaaaaaa gtagttgaaaaaagtccagagagaaaatgaac agttgagagacatgaatta c gaaaaaatggctaatattgagcaggaaaatgaaaattgadggctgaattagaaadcttaaagctcatcttgggcatcagttgag ca tgcactatgaatccaagacca aa ggca cagaaaa a attattgetga aaa tga a aggettegtaaagaaettaa aaa agaaactgactga tgaga tga attagaa attagaa tttagagatatta aa tga ga a ga tgaeagttc aa ctaga a gaga ctggta a gag attgeag tttgcagaaagcagaggtccacagcttg a aggtgctgacagtaagagctgga aatccattgtggtt aca a ga a tg ta tgaa a cca a gtta aaa ga a tttagaactaactaaccaaa tcaaagcattactgaccttaaacagcttgtaaaagaagc a ca gaga ga ga a caaaaagtta acaatacaatgaaga ccttga caac a gattaagattcttaacatgttcctgaggtgctg agacagagcagccttaaaqgggagcttcaagttcttagttagctaatcagctggataagagaaagcaatcatcatcatcatca a ga taga a geta a caaggaccaa agtcaggagat cactaga cactagag aaa ga teta gagacagctcaaatgtcagatctgaaaagcagcatttgaggaaaattttttttgaagaa ttgaagatetta agtatattaca agga a ga agtgaaga agaatattetettagagaga aggtagg aattagaaaaa gttgaattaactagccctgttgctgcttga agag tttga a gatga a ga aga aa gtcctgtt a atttccccatttac 3xflag (seq E) WPRE (seq F) Bgh Poly A (seq G) Petition 870260074145, dated 24 / 07 / 2026, p. 129 / 259 118 / 192 3'rTR (seq HI pll79 pAAV2.1-CMV260-5' CEP290 intein (set 5) 5′ ITR (seq A) CMV260 (seq U) 5′ CEP290: SEQ ID No. 55 atgc ca cctaatata aa ctgga aa ga ataa tgaaagttga cccagatgacc tgcccgtca a ga a ga a ctggcaga taatttatt gatttccttatccaggtggaagtaatgagctaaaagtgaagaagcaagaaatgtgtacacctttcagagaact aactcagat agtggagctggctttgga a ga agt aga aaagc tggagaga a caagcaaaatuga aaa tea attaaaactaaagtaatgaactgga aatga a ctggagatggctcagcagtctgcaggtgga egaga ta ctcggtttttacgta atgaatttgccaagaattagagaaaaaaa ttggaggacatggaaaagga gttggagaaagagaga a agttaatga gcaatggct ett egaa a tgaggaggcagaaa atga aaa ca gcaatta a ga a gagagaaca aa egteta a aga agaatgacaacttgtcaggata tta ttgact a cea ga catt e aaggata ga aaca agacagtgactaccgatca ca gttgtctaaaa aaactatgagcttatccaatatcttga tga a attca ga ettta acagaagct a atgagaaaattga agttca ga atca agaaatgagaaaaattta ga aga gtctgt a cagga a atgga ga a tgattga etgatgaat ta tgt a atagatcagtta aa aa aaga aaa egatea tta tea a ettea a gtg caggagcttaeagatctcetga aa tea aa aaatga a gaaga tgatccaa ttatggta getgt ca a tgea aaa gtagaagaatgga agctaattttgtcttcta aa ga tgatgaaatttattga gtatca gca aa tgttagaca gcactactta gcacttag aacttag tgatgctgataaaagta atgttatggctctacagc agggtatacaggaacgaga cagtca aatta a ga tgetea ccgea caagta g aaca atata ca aaa ga a atggaaaagaatacttgtattattgaagatttga aaaa tgagctcca aa gaa acaaaggtgcttcame a cc ga tteca a tca tca tcame tea a cgttagaca tttuaaagaga aaa ctaaagaggctgagagaacagetgaact ggctgaggctga tgctagggaa a aggataaagagtta gttgaggetetgaaga ggtta aa agattatgaa N-intein mDnaE (seq S) 3xf!ag (seq E) WPRE (seq F) Bgh PolyA (seq G) 3TTR (seq H) Petition 870260074145, of 24 / 07 / 2026, p. 130 / 259 119 / 192 pl 180 pAAV2.1-CMV250-CEP290 body inteln (set 5) 5' ITR (seq A) CMV260(seq U} C-intein mDnaE (seq T} CEP29O body: SEQ ID Na. 56 tcgggagtatatggtttaga agatgctgtcgttga a ata agaattgtaa a accaaattaaa taagagatcgagaga ttga a at atta acaaa ggaaa tea ata a aettga attga agatcagtgattccttgatgaaa tgaggcattcatcattagaggaggag tgattgatttaactga a tttagaa a tagcaa a ca etta aa acagca gc agta cagagctgaaa a cca gattcttt ga agagattgaaagtetaga ggaaga aega ettgattgaa aa aaaaa a ttegtea aatggctcaagaaaga ggaa aaetgaat actcacc ag tgcaca ttgaa aa tattt ttcaggaga tagaata agtgaaaga Mattggattt attgagcctca aaaatatgagtga agcawatca aga atgaatttcttcaagagaacta attga aaagaaagagatttagsa a ggagtaggecdgtgatgaccaaa ttcaga ata attaacaa agattagaa aa gaattgaa ttag tattgea agcaa ttaggaaatgeagaa agatcctgatgttaa aggaggaga aa catctctaatta tccrtagccttgaa agact agt taa tgetata gatea a aga a tgcaga agga atctttgcgagtctgca tttga a caccactagttga tcagcttaccgga ag aaggactgag agagact tga gaggetataaa ttattcacagca gttggca aagetaattt aaag atagacca tettga aa aaga a ac tagtet ttta cgacaa tcagaa ggatcgaatgt tgtt t tta a agga attgact tacctgatggga ta gcacca tetag tgccag tatea ttaattc tcacca a tga a I atttaataca tttgttacaggaagata agatta agaaaast agattetettga agatt scaacagaaaatttgctgtaa ttcgtcatcaacaa agttgttgtataaa gatacct a agtgaa agga gacctggaaaa cagaatcta aaaaaa gagga aaaga gaaa a eftga ggatca agtcca a ca agatgtaa tattagta tatagactaaactaa gatggattcggatga atga aaaa a ataettgeagaa atagtag ga aaatta ctgttt tgca agtga a tga a aaatca ettata aggca a tatacaa ccttagtagaa ttgga gcgaca a etta gaaaga aaatgagaagcaaagaatgaattgttgtcaatggaggetgaagtttgtgaaaaaattgggtgtttgcaagatttaaggaatgg ccattt teaaga t tgeagctctcca aa aagttgtagata a tagtgttcttcttgtgaa ctagaa etgaa tgeta aggata acag taact tettgea aaaa gataa ta tgcttgttca a aga a caagta acttggacacctggagtgtga aaa catctcttaa a agaaca agtggagtctataa ataagaaetggaga ttaccaaggaa^aa rttcacactattga a caagcctggg aa caggaa actaaattaggtaatga atctagcatggataa ggcaaâgaaatca ataaccaa cagtga cattgtttccatttcaaaa aa a ataactatgctggaaa tgaaggaattaaatgaaaggeagcgggctga acattgtcaaa aa atgt atgaae eggaatta ggattaa ttttgaa ttgga a ac eaaatttgctgagcttaccaaaa tcaa tttgga tgcacaga aggtgga aca ga tgttaaga gatga a tta getga tag tgtgagcaaggca gtaa gtgatgetga taggca aegga ttetagaattagaga aga Petition 870260074145, of 24 / 07 / 2026, p. 131 / 259 120 / 192 a tgaaatgga actaaaagttgaa gtgteaaaactgagaga ta ttgcca ga agacaagltgaaatttgaatgcaca a caa ca a tctaggga ca aggaagt agagtccctcaga a tgeaactgctagattatcagca cagtctgaaag tgctctcttcaactgagtga ggctactgctcttggta a gttggagtcaatta ca tctaaactgca ga ag a tggaggceta ca a cttgcgcttagcaga aa cttgatga aaaaga a ea ggctctctatt a tgctcgtttgga gggaaga aa cag a at gca ac a ca a a ca ttcagtctctacgacgacagtttagtggagcttta cccttggcacaacagga a aagttctcca aaa ca a tgattca a ct a ca a aa tga ca aa cttaaga ta a tgcaaga aatgaaaa at tctcaacaaga aca tagaaata tgga ga ac aaaa cattEgagatEga atta attagaggga taggatta accta aggata ccaa aggagcccaaaa ggtaa tea a ctggcatatga aaataga ag aacttcgtcttc a agaa etta a actaaa tegggaattagteagga ta a aga a ga aataaa a ta tttgdataácataattt Ctgaata tgaacgta Ca a tCagCagtCttgaaga attagaga attagaCa attagaa gtttcàtgíagaa a gaca a atggcctgggatca aa gagaagttgacctgga a cgcca a ctagacatttttgaccgtca gca aaatga a at actaaatgc ggcacaaagtttgaaga a getacagga icatccetgacctagtt tgccccttccaaatcaa ettgagategtt ct aagga aa a11 aa ggagaacattcgaataattctagaacacgggca act N-mtein RmaDnaB (seq Q) Sxflag (seq E) WPRE (seq F) Bgh PalyA (seq G) 3ΊΤΒ (seq H) pil52 pAAV2.1-GRKl-5′ CEP290 intein (set 5) 5′ ITR (seq A) GRK1 promoter (seq N) 5′ CEP290: SEQ ID No. 57 a tgeea ccta a tata aa ctggaaagaaataatga aagttga tceaga tgacctgtcccgtca agaga a etgge agata attta ga ttcttatccaggtgga agtaaatgageta aaa agtgaaa agcagaaa a tgctga agcttca ga agactcaga tgactcaga tggctttggaaga ag ta ga aaa agetggagaaga a ca agcaaaa tttgaaa tea a ttaaaaacta a agtaatgaactgga aa atga a ctggagatggttcagcagtetgeaggtggacgaga t acteggtttttacgta a tgaatttgcca aetttaga aaggataga aaga caa aca aggagttggaga a agagaa ga aa gttaa tgagcaattggcttcgaaa tgagga ggcagaaaa tgaaaacagca aa ttaaga agagaga a ca a aegtetaa a ga Petition 870260074145, dated 24 / 07 / 2026, p. 132 / 259 121 / 192 aa aagaatgaaeactttgtcaggatattattga ctaccaga a ca aa ta ga t tcacaga a agaaacact t ttatcaagaa ga ggg ga agacagtga eta ccgatca cagttgcta aaaa aaactatgagcttatccaatatcttga tga aattcaa cagaattta getgaatttat gttcagaatcaagaaatgaga aaaa atttagaaga gtctgtacaggaaatgga ga agatgaetga tga at ataatagaatga a agctattgtgcatcaga cagata a tgtsatagatcagtta aaaaaa gas aacgatcattatcaactteaagtg caggagcttacagatetcctgaateaaagata agatagata getatta agattag aatgcaaaagta ga a ga atgga agctaattttgtcttctaa a gatgattgaaatta rtgagtatca gcaaatgtta ca ta aecta agggagaaaettaaga a tgetea gc t tgatgctga ta aaa gtaatgtta tggctctacagcagggtatacagga a ega ga cagtcag acagat acagat acct acagat acagat attac aaagaaatgga aaa ga ataet tgtatta t tgaagatttga aaaa tgagctcea a aga a aca a aggtgcttcaacc ctttctcaacagactcatatgaaaattcagtcaacgttagacattttaaagagaaactaaagaggctgagagaacagctgaact ggctgagggac aagagttagtigaggctttgaagaggtta a aagattatga a Ν-intein mDnaE (S-seq) 3xflag (seq E) WPRE(seq F) Bgh PolyA (seqG) 3′ ITR (seq H) pll53 pAAV2.1-GRKl-CEP290 body Intel n (set 5) 5′ ITR (seq A) GRK1 promoter (seq N) C-intein mDNAE (T-seq) CEP29O body: SEQ ID NO. 58 ttgggagtatatggtttagaagaattgctgtcgttgaataagaattgtaaaaaccaaataagagatcgagattgaat attackaca a tea a ta aa ettga at tgaagatca gtga t ttcdlgatgaa a caggcactta tgaggagttgact tgaggt tgaggt tgaggt a tttaga aa ta gca aacactta aaa ca gcagcagta ca ga gctgaaaacca gattctttt gaaagagàttga ^agtetagagga agaa ega ettgat etgaaaaaaaaaaaattegteaa tggetea ettgat aga a agaggactaaaga ag tgcagaacctt eegatata teggatta ggagataga ata agtga aagaaaattgga tttgagcctcaaaaatga gtgaagcacaatca a agaatgaatttctttca agagaactattga a aa aga a agagatttaga aa Petition 870260074145, dated 24 / 07 / 2026, p. 133 / 259 122 / 192 ggagtaggàcagtga tàgCca a atttcagaataaatta aaa gaa tt agttgàga aa ata agca atttgaagaggta tga a aga a ata ttgcaagc a atttagga aa tgcaga aagatcctgttaa a gaggagaaacatctctatta a teattag aacctagcc atgcagaagga atctttgatgcgagtctgca tttgaaagccca agttgatcagctta ccggaag aa atgagaatta a ga caggagctcagggaatacggaaaga ggct a ta a attattcagcagttggca aaa geta a ttataag atagaccatctgaaatta aagatagatagattat egattactacta gt agg aattgactta cctgatggga ta gcacca tet agtgccagt a tea ttaattctcagaatga a ta tttatacaittgt t acaggaactagaa aa ta aa ga aaaaa agtt aa agaatttaga a ga ttctctgaagatta acacagaaatttgctgta atcaattatac aatttca agtgaaaaggaga cctgga aa aca ga atctaaa acataa aagagga aaa ga ga a a acttgagga tcaagtcca a caa ga tgc ta taaaagtaaa a ga a ta ataatttgctcaatgcttcaga tggattcggatga aa tga aaaaatacttgcaga ga atctaa ag aaagatta ta gt attack the ggcccttagtagaattggagcgac aa cttagaaaga aa atgagaagcaaagaatga aa ttgttgtcaatggaggctgaagtttgtgaaaaaatígggtgttgca aa ga ttta aggaaatgg cea ttttca ag at tgcagctctcca aaaagttgta ga taa tagtgtttcmgtctgaac ta ga a ctggcta a uaacagtaca atgaa ctga ctgct a agtacaggggacatcttgca aa aaga (aa tatgcttgtteaa aga a ca a gtaact iggaacacctggag tgtgaaaa ca tctectta aa aga a ca agtggagtcta ta aa taaagaactggaga tta ccaagga aaaa cttcaca eta ttgaaca age ctggg aa ca gga aacta aattaggta a tga atctagcatggata a ggcaaa ga a atea ata a cea a cagtga cattgtttccatttcaaaa aaa at aactatgctggaaatga aggaattaaatgaa aggcagcgggctgaacattgtcaaaa aa tgtatgaacacttacggacttc gttaaagcaa atggagga a cgt a attttga attggaaacca aa ittgctga gctta cea aaa tea a tttggatgcacagaaggtgga a ca ga tgttaaga tga attagctgatagtgtgagcaaggcagta a gtgatgctga taggca a cggattct agaattaga ga a ga a tga aa tggaactaaaagttga agtgtcaaaactgaga ga gattctgatattgcca ga aga ca a gttgaaattttgaatgcacaa ca a ca a teta ggga ca aggaagtagagtccctcagaatgca a etgeta ga eta tea ggea cagtctga tgaaaagtcgctcattgc ca a gttgcacca a ca ta a tgtctcttca actgagtgaggcta ctgctcttgta agttgga gtea attacatctaaagcagagaga atggaggcctacaacttgcgcttagagcagaacattgatgaaaaagaacaggctctcttattgctcgtttggagggaagaaacag agca a aaca tctgcgccaacaatca gteteta ega ega cagttt ag Lgga gcttt a cccttgca ca a ca gatacaa aacata caat at tgacaaacttaagataatgea a ga aa tgaaa aattctcacagaacatagaatatggagaac aaaa cattggagatgga atta aaattaaagggcetgga agagttaatagca cttta aa ggatacca a aggagccc aaaaggtaa tea a etggaa t atgaaataga a ettaacgta a ettaacgacgt aggaaagaaga aa ta aaa tattgaatacataatttctgaatatgaegtacaatcagcagtcttgaagaaattgtgcaaagaacaagtttcatgagaa a ga ca aa tggctggcaagaga agttgacctggacgaacca a cta gatatttga cegtca agatc aag eggataaaca t cagga tea a tccctga ccctagttgccccttccaatcaacttgagatcgctcta aggaaatt aa ggagaaca ttcgaataattcta gaaa ca cgggca act N-labeled RmaDnaB (seq Q) Petition 870260074145, of 24 / 07 / 2026, p. 134 / 259 123 / 192 3xfiag (&eq E) WPRE (seq F} Bgh PolyA (seq G) 31TR (seq H) pll56 pAAV2.1-GRKl-3' CEP290 intein (set 5) 5' JTR (seq A) GRK1 promoter (seq N) C-intein Rma DnaB (seq R) 3′ CEP290: SEQ ID No. 59 tgcaa atcactaga agagaaactaaaa ga ga a aga atctgctttaaggttagcagaaca aaat atactgtcaagagae^aagta a tea a tgaactgaggcttcgattgcctgcactgcaga aa ga ga aa age tea tagetgagctaggcaga aaccaa gacactcatcaccacca aa ccattgca aa ca tgea a gcaa gcaa ggttaaatcaaaagaagaagtaaagaagt atca a cgtuttctcaaaaagccagagaga gcaaga ga a attgtga a gaa a catgagg aagaccttcatattcttcatcacaga ttagaactaeaggetga ta gcaattcaatgea ata aatgaaacagtctcccactceagtl ccta tea a ca a gca tilt a ttcgtctggctgaga iggaacagacagta gcagaacaaga tgactcf ttcctca ctctgte aa act aaa gaa agtatcac a agatttgga gagaca a agagaaattcaattga aacactgaaattagaaa agcttcaaga aaa cca tga agatgaagtga aaaaa gt aaa ageggaagta ga ggatttaaagta tcttctggacca gtcaca aa aggagtcacagt gtttaaaatctga acttcaggctcaa aa agaa gca aa ttca a gagctcca a ca a eta ca at aggagaaggetaagta ttagccttgagggaaacagaa ageaettagtcgggc a ctttagaa ctccgggcagaaatga ca gcagctgctg a agaaegtattatttettetgea a cttctcaaaaga ggcccatca a tgttca a ca a atcgttcgacatactagaga cata aa ga ca ca a gttga agatttaaat agataa gaaat tgaa taaaaaacagaga aa a ctcactaactga t aatttgaatgacttaaataatgaaclgcaaaaaaaaagcctataataaacttagagagaagaattgatcaag a gatgatgea etga aa aggga a attaaagactaccagtggatta caggca aa cccct aacagata at aa tgagaactecaaggaagttaaaaactagagaaccaattagagggaaggtgaggaagtagacctaaaacctagaaag aaa agaatgetaagaga a tta a ttaggtgggaaggta aaagtgcaagcca aa atagactgaa tt ega aa ca agttaa a cagattac aacagact tttagagg ggatctttttgccaaagegataagaga aa etta ctttgcag aggaaactaaaaeaaclggcatga ctgttgatcaggttttgggaata cgagctttggagtcagaaaaga a ttggaaa Petition 870260074145, dated 24 / 07 / 2026, p. 135 / 259 124 / 192 EaattaaaaaaEaEaaatcttgacttagaaetgatatattgtatatga^cccaagcttcctcgagattctgttgtaEaag atta ca tttacaaata ga tacctca agaaaaacttca tgctt ta ga aaa acagtttca tt aagga tacattca ta tctagta atagagtcagatcagatcattgtcagagaga ata ggagcttca gaagga aaa cttgaagttgtcatctgaa a atattga a Ct gaa a tt cagcttga a ca agca aaaaga tt tgccaagatta a aga atca a gtc agagatt tgaagga aa tgtgtgaatttctt aaa gaaagaaaaagcagaagttcagcggaa.acttggccatgttagaggtctggtagaagtggaagacaatcccagaacrggaaa aaccat Eggtttaatga a aaagta gttgaaa a gtccag agaga aaa tgaacagttgaaaagca ttcagag taggatag aagata gaatattga aaa attga a ggctgaa tta gaaaaaetta aa gctcatatggcatcagttgag ca tgcacta tga a tccaagaccaaaggca caga aaaaatta ttgctgaaa a tgaaggettegtaaaga a ct ta aaaaga aa ct gatgetgeagagaaatta a tta agatagataga agatagataga cagttcaactaga a gagactgta agaga tgcagtttgcagaaagcagaggtccacagcttgaggtgctgacagtaagagctggaatccattgtggttacagaatg tatga aa ccaagttaaa a ga a attgga aa ctgauttgcca aaa aaaatcaa gca ttactgaccttaaacagcttgtaa aga aacagagagagacaaaa a gtlaacaataca tgagaccttga a ca a caga tta ga tlctta aa ca tgt teetga a ggtacatteagg aggegactg gttettagatt agetaatea tea getggata a agagaaagca ga attaatccat cag ata ga age taacaagga ccaa a gtgga getga aagcaccata cctga tgctgatcaactaa aggaaaa a ta aa agateta gaga ca ca getea aaa tgteaga tetagaaaagcaggagaa aataaa gaatgaa agaactggaaaattt gatccttca tttttga aga aattgaagatetta agt ata ca aggaga agtgaga aga ata ttettag^agaga aggta aaaaactttcaEaacattgggagttgaattaactaaaaacttgctgctctgaagagttgatgatta agatta gatta t 3xflag (seq E) WPRE (seq F) Bgh PolyA (seq G) 3'ITR(seq H) pzac-GRKl- 5' AB€A4 intern (sell) SEQ ID No. SO 5′ ITR (seq A) GRK1: bold 5' ABCA4: underline N-intein Npu DnaE: double underlining Petition 870260074145, dated 07 / 24 / 2026, pp. 136 / 259 125 / 192 3«flag; italic 5V40; bold underline 3' ITR (seq H) ctgçgcgctcgctegctcactgaegecBcccgggeaaagcccgggcgtcgggceacetttggtcBcccggcctcagtgagcgagCB agcgcgcagagagggagt tgctctaggaagatcttcaata ttggccattagceatattattcattggtutatagca ta aatcaatattggcuttggcca ttgcata eg ttgta tetata tea ta a ta tgtacattta ta t tggctcatgtcca ata tgaccgccatg ttggca t tgat ta ttgactagtgggcccca gaagtctggtggttgtttgtccttctcagggaaaagtgaggcggccccttggaaggggcggcgaatgatctaatcgga ttccaagcgctcaggggattgtctttctagcaecttcttgccactcctaagcgtcctcgtgaccccgggggatttagcctggt gctgtgtcagccccgggctcccaggcttcccagtggtccccaggaaccctcgacagggccaggcgtctctcgtccagcaag phircafi^acj^c^^aaacaagfificgcggc^ccatj^gcttcEtEaaa^aatacaEctttactctfia^a^a^ cagaaaagEcaaaafiattcgctttEtggtEEaactcgtgtEgcctttatctttattctaEtcttEatctEgttaageaatKccaaccc gctctdcagcc ate ​​ataaatecca tttcocca a ca aRgcflatgccctcagcagga a tgctgccfltgRfiatcRfictt gcagcat gcatc tccctgtt t tcaaaflccccaccccaagaflaatctcctflga a ttg tgtea aaeta taacaactcca tet tagcaagRgtat ategaea tttcaagaa ctcctcatga atRcaccaRagagccaflcaccttgfiCcgtatttgRa cagagctacacatcttgtcccaatt catflflacaccctccRRactcacccflR agaga at Rgdattc tt aaggga at aagggata tgaaactaga tggaa a-^ÇtdÍVÇ-tÇdttaaacatçgEcctEtçteactcartEEetct^ fflagtcccggacctggcflctflaaggacatcgcctgcagcgaflflCcctcctfiflagcgcttcatcatcttcaRccaRagacgcflgggc aaagacflgtECKctatflccctgtEctccctcttxxaRflacaccctacafltEEataEaaEacactctatatEccaacfltgEacttcttc aagchtchttchcgtÊtÊchttccchacaçt£chtafiaçagç£gttchtcaaÊgtatçaat€jÊaÊ3tçttHÊÊgaÊEaatattatchtÊatatÊÍÇ accaaflaattcaafiagtttatçcatcgEccgagtatgcaggacttEctgtEgRtgaccagEccccttcatEcagaatefltggtccafia flacctttacaaaEctgatflRRcatcctgtrtflaGGtcctfitgtggcccgaRRgaggtEEctctcflfcttcgtcgtaccgaRRgaggtEctctcflfcttcgtcaut act act act cat ctataafiEcctttctflgflga ttgactccaca aRgaagga tcctatct a ttcttatga caflaagaacaacatcctt ttgtaa^carttigatccajgagccjgggjgtcaaatcctttaacca^aat^ggttggagggcjggcaaagcctt^gctg^ggg^aactcctcgcacgc aggataetga agaatgccaacctcaacttttgaagaactflgaa ca cgt taggaagt t flgtcaaagçctgEgaaBa.agt.agggcc çca£a.tctggta.ct.tçttttggc.aaçagç3cacagatga.acatgatcagagat.a.€cctEg gaga cccaatagttttaa agaggtttagatagacagaggatta agatta ctgctgaasccatcctaaacttccta caa gg flccctcEflflaa8gccaEE€tflacfla£.atggccaacttcgactggaflflgacatttaacatcactgatcgcaccctccgccttgca atcaatacctggagtEchttggtcctgEataaRtttEaaagct3caatEatflaaachtcaEctcaciççaacgtEccctctctctact£Ea flgaa aa ca tgttctgggccggagtflgta ttccctgaca tgta tccctgga ccagctctcgtacca ag cctataccacgacga Petition 870260074145, of 24 / 07 / 2026, p. 137 / 259 126 / 192 atagacataga cetggtRRaga a aaccaata aaatta a agaca ggtattgREa ttctggtccca gagctaa tcccgtREaagattl ccggtacatctggggcgggtugcctatugcaagacatggttgaacagggEatcacaaggagccaggtgcaggcggt a tggag^ tgga tccta cctccagcagatgccctacccctgcttcfitgga cga ttctttca tgatca tcctgaa ccgctgtttcccta tcttcatggt RctfiRcatRfiatctactctfitctccatfiactfitfiaaRaRcatCfitrttfifiaRaaRgaRttfiCRactRacctRaaaafiafia ggEtgtctccaatgcagtEatttRgtgtacctEgttcctggacagctctctcatcatEtcgatgagcatctctcctcgacgatattcatc atgcaTggaafiaatcctacattacagcgaccccattchatcctcttcttcttgttgctttcccctcctctcctc ÊçtçagçaççttçttčUçaaggoçaÊtçtÊgçaÊçaEÉÇÍÉÍêÊÍÊÍÊÍÊêiçíaítiÇêítitçtaçtÊççaçacatççtEtÊítt CBtetgficaHtaccficatjgccagacgagfiira^^ SiiMlgaajgagcaaggkciggg&lgMgtggagcaaGatcgg^^ gtçcatgca^a tgatgctccttgatgciEtt^ tgEcttactcgcttggta ccttgatcaggtEtnccaggagacta tggaa ccçc achtccttgR ta cttct tcta caaRagtcRtattggcttggcagtEaaaggtgtt ca accagaga agaaa gagcmgga aaaga c cgagcccctaa ca gagga aa ca cRactccttctttgaaCRtgaficatccaggfitgg gttcctggggtatgcEteaaKaafctggtaaaEattttteagccctgtggccggccaficffi agaa cca ga tcaccgcattcctgggcca ca a tgeagctfiggaaaccaccaccttfitccatcctaacfigatctgttgcca cca acct ctgctcg cattggg aggagga accagcctggatgcagtccggcagagcc ttggca tgtgtccacagcaca ac atcctEttccaccacctcacREtEgctEaRcacatfictEttctatRcccagctRaaHRdaagtcccaRRagHaERcccaRcteEag ata£aaEccatEttEEaEEacacaKRCctccaccacaa£C££aatEaafiaE£ctcaagacctatcaEgtEEcatgcagaEaaafi ctgtcgfittgcca ttgcctttg tgggagatgcca aggtggtga ttctggacgaacccacctfiggtflfiacccctcctcgatcggatctc agtatcgct caggcaga a cca tcatcatgtcactca ccaca tggacgaggccga cctccttgggga ccRcattaccatcdttaccCciREgdaREctctdctRctcagEcaccGcactcttGGtfiaafiaactECCtEaEctacgaeaGcgaEat acaacatctacacccagccgt££cccag.tE£cacgaccagEgcgagcaggaeEtattcEagtactgcct£EaggacEgcagcct gatcgggccaccaaggaccacaagttcalgaccgtggacgEccagatgcccatcgacga&atctggacgcgcgcte tEatBCBegtEBacaacctecccaaactacaaaaacccrtacJCOqtaa ttptaoagotcatgacütcgactüCapggo tgac qatÍfacaoqtíjaEcgECCECttcfiagcaEacatgataagatacattgatRagtttgRacaaaccacaactajÍHatECBgtaaaa aaaatgctttatttgtaaaatttgtgatEctattgacctttattgtaa cacaf a ca attgc attcattttatgtttcaggttca g^gggagatgtgggaggttttttaa agcaagtaaaacctcta caaatgtggtaa s atcga ta agg atcttcctag agcatggcta cgtagsta agtagca tggcgggtta a tcatta a ctacaaggaacccctagtggagttggccactc cctctctgcgcgctcgctcactgaggccgggcgaccaaaggtcgccgacgccgggccgcggcgcgtgagcg agcgagcgcgcag pzac GRKl- 3' ABCA4 intein ($etl) SEQ ID NO. 61 Petition 870260074145, of 24 / 07 / 2026, p. 138 / 259 127 / 192 ITR does* A) GRClbold 3' ABC A4: underline C-intein Npu DnaE: double underline 3xflag: italic SV4Ü: bold underline 3'1 TR (seqH) ctflcgcgctcgctefictcacWficq,cccRfificaaagcc^^ aECECSiSfiagaggggagtEgccaactccatcactaggEEttottfitagttaatEattaacMgccatECtacttatctacgtiEcca tgctcta ggaat a^atgcatTccatctta accatc ttcattggt tatatagca ta a atcaatattgact a ttggccattaca ta CEttEtaKtatatcataatatRtacatttatattEfictcatcatgt£caatat£3CCECçatgttfi£cattgaftaHgacl:agtggg«:cca fiaagcctWflEttRtttRtccttcΐcaflfiggaaaaflgcfigccccttEgaEeaaRRg£ccaggcagaatgatctaal·cgRa ttccaagcagctcaggag&ttgtctttttctagcaccttcctcctaagcgtcctcgigaccccccgcggtggtggtggt RctRtRtcaffccccRfíEctcccasRRRCttcccagtRRUcccaRRaa ccctcga caRggccamcgt ctctcgtccaRcaag ggcagggacgggcaggcaaggg£g£ggccgcatcaagatcgi€acccggaagtacctgggcaagcagaacgtgtacga catCflfiCfiteFafícRREaccdcaacttcfíccteaaaaacERCttcatcRCCãficaattectttgficacãfflÍCttRtdCttaaccttfi gtgcgca aga tgaa aaa ca tccaga gcca aa gga aaggca Rtgagggga cctgcagctcgctcgctcgctcttcttc RtccdRCCCacatcgatRacctaactccafidacaa£tcctfigatfíR£RatgtãaatfidfiCtfiatRRatRÍaattctCGaccatgttccaaaggca a afictaatRgagtRcattggtcaaaaactta t cttccttcttcc a aataagaa cttca agca cagaRcatatRccagcctt ttcaRafiaEct£RaERafiac.Rc.teRctaacctteRtctcaKcaEttttKRaatttctKacactc. cacaaa Rga t tctgattca Rgacct ctg tt tfiCfigEtaacfíctcafícafia a aagagaaa a cgtca a cccccgacacccctacttRRR tcccagaga Ra aggctgga caga ca ccccagga ctccaa tgtc tgctccccaggfificgcccRgctgca cccagacgc tccagcc ccccagaRccafíaÉtRcecaRfícccRcafictca a ca cRRRgacacaftctgRtcctcca gca tRtecagficgctfict / atcaaga g attccaacacaccatccRcafíccaca aggacttcctfiflcgca gatcgtgctcccggctt cctttgctctctgtg tgcttc tgtctc cc ttttRRCga a ta ccccRCttteacccttca cccctRfiatatatRfificaficaRtacaccttcttcaRcatgRa tsaacc aggcagtgagcagttcacggtacttgcagacgtcctctgaataagccaggctttggcaaccgctgcctgaaggaagggtggctc EgagtaOcCttgtggCactcaacaccctggaagaCtCcttrtgtEtCcCcaaacatcacccagcttCcagaagcagaaalEgac acaggtca a cccttcaccatectgeagRtgcagcaccafiafia agctcacca tflctficcagaRtgcccaa rRRccr taectcacca Petition 870260074145, dated 07 / 24 / 2026, pp. 139 / 259 128 / 192 tcccccccccagafíaa ca cagcficagcacggaaattcta ca aga cctgacgaacafigaaca tctccgacttcttggtaaaa a cg tatcctgctctta taa gaagca gctta aagaacaaa tttegRtca a tea acaga gta tEga cttactc ccag icglccc catcacgaaagaaficacttgttfifigtt tttaagcga ccttfigccgga tcatgaatgtea gcaaaagccctatcacta Eafíaggcctctaaaga a ata cctga tttcctta aacatct agaaactga aga ca acatta agEtetg^tttaa aggctaa aggc atgccctfiRteaficttti3caatetegccracaacgccatcttaffigg«agçctg^ tcacc^tcattagccaacccctgaacctgaccaag^agcagctctcagagattacagtgcteaccacttcagtefiatfícttegttfic cateteCfíteattttctccateccttCfittctc afifiagCfiRgteaaca a atccaa gcacctcca gttta tcagteaafiteagccccaccaccta cteERta a ccaacttcctctgggaca tcatgaatta ttccgtea gtgctggactgatge tefigcatcttcatcttefltttcafíd agaa afíCCta eacttccatctcttctcttefltttcafíd agaa afíCCta eacttccatcctacctacctactaccta ta>a tefi geggtea ttccca tgatgtacccagca tccttcctatttga tgtccccagcacagcctatetfigcttta tet tetecta atctgHcatcggcatcaacaflcaatectattaccttEatctteaaattattteafiaataaccfiaacfictectcaggttcaacEccfitecteaaEaagct gctcatteÍcttcccccacttctecctEgaccflgaficcteattaaccttecactfiaficcaRgctEtaacafiatRtctatecccgfitttefi cteaccctectefit<tcaacaccacttcttcctctcccaateEatteccãaficccactaa££aficccattetteateaaaateatfiatet ggetgaaga aaga caaaga a tta ttactggtggaa ataa a actga ca tetta agRctacatgaacta accaa ga tttatccaggca cctccafiCccagcaat^acafiactetetRtcggagttcflccctfifiagaatgcttt^cctccteggafitgaat^tgccggcaaaa ca accacatt caagatgctca ctagfiga caccacafitgacctcaflaEfiatgccaccgtaficagficaafiaata t ttt aacca a t a ttt ctgaagtccatcaaaatatgggctactgtcctcagntgatgcaatcgatgagctgctcacaggacgagaacatetttacctttatgcc cfificttcfiaagtetaccagcaflaagaaatcfiaaaafifittecaaactafiaatattaaRagçctefígcctedctetctacficcaacte ccte^ctg^cacgtacagteKAgfica acaagcgga a actctcca cagcca tcgcactca ttggctgccca ccactggtgctectgga tfiaficçcaçc acaggga tgga cccccaggca cgccgcatgc tetgga ac^tca tçgteagça tcatcaga&aafigaagaEç tgtg gtcctcacatcccacagca tggaaga ateteaRgcactfitetaccccagctggcca tea tggta aflagcgcctttcfiatgtatgaac aj^jtgaficaicjLcaagtcca aattteeaga tagctatatcgtcaca a tea agatea a atccccEaagEacaacçtjgcttçççç.tg,açç tgaa ccctet Egagcagcagcagca gtetgcagagggagficacta caacatgctccafittccaggtct cctcctcctcctgacgagga tcttccagctcctcetctccc acaa ggacagcctgctca tcgaggagtactcagtca cacaga ccac actgEaccaggtgtttaataaatttgctaaaca£cagactg.aaaatcatgacctccctctgcaccctcgaactfict£gaficcaatcfla caagcccaggacg ac tacaaagacca tgaeggtgattataaagatea tgacatcgactacaagga tgacgatgacaagtga gcctactacactactac atacattgaitgaKtttiBzaca33.ccacaactagaatacaRtBaaa a a aatgctttatt tgtgaaattte^atEctattflctttatttgtaaceatlataaEctacaataaaagtta acaa ca acaattgcattcattlta tgttt caggtt caggaggatt tgtgggaggttcagcagcttaa agtcaggtaa aaategata agatcttcctaga gca t ggeta cgtagata agta gcatggcgggtta atca tt aacta ca aggaacccctagtga tggagttggcca ctccctctctgcgcgct Petition 870260074145, of 24 / 07 / 2026, p. 140 / 259 129 / 192 cgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgg^ctttgcccgggcggcctcagtgagcgagcgagcgcgca Ê pzac-CMV260 5' ABCA4 intein (set 1) SEQ ID No. 62 5' ITR (seq Al CMV26Ü: bold 5' A0CA4: underline N-intein Npu DnaE: double underline 3xflag: italic SV4O: bold underline 3' ITR (seq H) ctecgcgctcgctcgctcactgaggcgcccgggcaaagcccgggcgtcgggcgaccttggtcgccggcctcgtgagcgagcg agcgcgcaga gagggagtggccaactccatcactaggggttctgtcgg atgacttacttacgctc tgctctaggaagatettca at attggccattagccatatts ttcattggttat stages ta a at ca atattggctsttggccattgcata cgttgta tetatatcata a ta igta ca ttt atattggetea tg tccaata tgaccgccatgttggcattga tta t tgactagcgttgacat tgattattgactagtacggtasatggcccgcctggctgatgactcacggggattccaagtrtccaccccattgacgtcaatgggag tttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaaactccgccccattgacgcaaatgggcggtaggcgtc getgEEaEEetttatataagcagagctgEttaBtgaactagagaacccactgcttattggcttctcgagattccaccatggcggcc gccataggcttcgtga gaca gatacagctttgctctgga aga a ctggaccctgcgga aaaggcaa a aga ttcgcttigtggtggaa ctcBtfitRAccmatctttatttctaEtcttgatctEtett&iigEaataccaacccActctacaRCcattaaateccatttccccaeca aggega tgccctcaficafiEaa tgctficcgtgEctccafiggaatcttctRC a atgtga a ca atccctgtttca a agcccca agcctat tcactat tcactat caggat a ctcca t ct tggcaagggtatatcfiagatttca a gaactcctcatgaatgca cc afiagagccaficaccttggccgtatttggacagagctacacatcttgtcccaattcatfigacaccctccggactcacccggagaat tgea ggaagagga a ttcgaataagggatatcttgaaaga tgaagactga cacatta etacta ttac acatcggcctgt etga ctcaatggtctaccttcteatcaBctctcaagtcqgtccagagcagttcgctcatfigagtcccfiRacctggcgctgaafigacatcgcc tgcagcgaggccctcctEEagcRcttcatcatcttcagccaRaRacEcggfiCasafiacggtgcgctatgGcctgtgctccctctccc agggcaccccta cagtggataga agacachctgta tgcca aegtgga cttcttcaagcttcctcgtcgcgccccccc CCEttctcaaRgtatCaatCtgaEatcttgggEÍggaatattatCtaa tatgtCaCcaaga a ttcaagagtttatCCatCggccgagta Petition 870260074145, of 24 / 07 / 2026, p. 141 / 259 130 / 192 cctgtgtggcta ccccga gggaggtggc tctcgggtgctc tcc ttcaactggtatgaagacaat aactata a ggc ctttctggggatt gactccaca aggaaggatcct atetattettataacagd aga a ca acatcctttgtaataca ttgatccagagaatcctaEagt cgtt taacaatc ggagggcggca aagcct ttgctgatgggaaaaatcctgta ca ctcctga t tcacctgcagcacga agga Tactgaaga a tgcca actcaacttttga agaactEEa aca CEttagga a EttEEtca a agcctEEEa a gaaetaEEECccca gate tggtacttct acagcagac agatcaga cagatcaga taccctgggga accca acagtaa aa gactttttga a taggca flettggtea aaaaEEtattactgctgaaEccatcctaaacttcctdta caaeggccctcgEga a agccaggctgacga ca tggcca a ct tcgactggagggacatattt aacatcactga tegea cccttccata tglcata cctggagtgcttggtcctggata ag tttg a aagctacaatgatgaaactcagctca cccaacgtgccctctcta ctEgaaga a aacatettctg^cj#^ gtggta ttccctga ca tgtatccctgga cca geteteta cca ccccacgtga agtata a gatccgaat ca tagaggt agga agga at gagaagga ttaaageca ggtattgggattctggtccca gagetga tcccgtggaaga ttccEEtacatctggfificggEtttEcctatctEcaREa ca tggttgaacaggggatca ca aggagccaggtgcaggcggaggctccagttggaatctacctccagcaga tgee ct acccctgctt cgtgEacgatl·ctttcatEatcatcΐtgaaccEctEtttccctatcttcatEgtEctgEcatEgatt;tactctRtctccatEact agcactEggacttgac etgaagga gaccttgaaaa a tea gggtgtctcca a tgcagtga tuagtgtacctggtt cc tfigacaECttctcca t catEtcgataagcatcttotcctaacgatattcatcatgCd tggaaga atccta catt acagceacccattc a tcctcttc ctgttctttg tecca acca etge tgctgtgctttctgctcagca ccttcttctc ca aggccagtctggcagc agcctgtagl·gRtgtcatctaΐttcaocctctácctECcacacatcctgtgctt^:gcctgEcaggaccRCatgaccEctgaRίtgaaEa aggctgtgagctctctctcttc ttggcactgagtacctggttcgctttga agagcaaggcctggggctgcagtgg agca acatcgggaacagtcccacggaaggggacgaattca gene ctgctgtccatgcagatgatgctccttgatgctgctgtctatg gcttactcgcttgglaccttgatcaggttcgctatcttacttacttac agagtegta ttggct tggcggtgaaggatgitcaaccagagaagaaagagccctfigaaaafiaccaaficccctaacagaggaaacgRaggatccagagc a cccaga aggaata ca cgactcctlctttga acgtgagcatccagggtgggttcctggggta tgcgtga aga atctggta aagatttt tgaaccctatEgccEgccagctgtEEaccgtctgaacatcaccttctacEagaaccafiatcaccgcattcctgggccacaatggagc tgggaaaccaccaccttgtccatcctgacgggtctgttgccaccaaccttgggactgtgctcgttgggggaagggacattgaaac cagcctfiBdtecafitcCEgcagaBcctteficatBtgtcca ca eca ca a ca tcctgttccaccacctcacggtgfictgagcacatgct gttçtatgcccagchtga.aaEga.aagjçç.caggaggaggccccagchtEeaflatgEaagccatgttggaggacacaggcchtçcaçcaç aaRcgEa&tRagaggctcaggacctdtcagEgcatEcaEaRaaEctEcttcggcgc ggtEa ttctggacga a cccacctctEEEEetega ccctta tceagacectcaa tctEggatctgctcctgaagta tegetea ggcaga accatcatgtccactcaccacatggacfiaggccgacctccttgggfiaccgcattgccatcattgcccafiggaafifictctactctct cajMcaccccactcttcrtgaagaactgcctea£cht <EagaccgagatcrtEaq«tgg3Eta!CEgc^^ atcgtEfiaeaagcfiEatc&a&tEcaccatgtacagcatEgacaacaacEgcaacatctacacccaecccetEgcccafitgacacE Petition 870260074145, of 24 / 07 / 2026, p. 142 / 259 131 / 192 g£g£ggwg£gt££j£iX£ah^aj^Lgjll£ih^g££^ gacca tgacgg (gaita taaagat ca tgacatcgactacaagg a tgacga tgacaagt^agcggccgcttcgagçagacatg ataagatacBttgaJgagtttggaçaaaaçcaaachtagaatgcagtgaaaaaatgchttatttgtgaaatttgtgattgctattgçt ttatttgtaaccajttataagctgcaataaacaagttaacaacaattEcattcattttatgttcagEttcaggggagatgtjtt aggcacta agcacta aacct gtggtaaaatcgataagEa tcttcctaga gcatggcta cgtagata agtagcat ggcgggtta atca tt aa etacaa gga a ccctagtga tggagttggccactccctctcgcgctcgctcgctca ctgaggccgggc a aggtcgccccgcggcgcggcgc agtgagcga gcga gcgcgca g pzacCMV260- 3' ABCA4 intein (setl) SEQ ID No. 63. 5FITR (seq A) CMV260: bold 3' ABCA4: underline C-intein Npu DnaE: double underline 3xflag: Italic SV40: bold underline 3' ITR (seq H) ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacclttggcgcccggcctcaglgagcgagcg agcgcgcag agagggagtggcca a ctccatcactaggggtt gccttgtta acc acctag tc cttatctacgtagcca tgctctaggaa ga tcttca ata ttggcca ttagccata ttattcattggttatategcata aa tea atattgget attggecattgeata cgttgtatctata tea ta atatgtacatttatattggctcatgtcca atatga ccgccatgttggca ttga ttattg^ctag agcttgact ttgcatta atta cggtaaatggcccgcctggctgatgactcacggggatttcca agtctccaccccattgacgtca atgggag tttgttttggcacca a aatca a cgggactttcca aaatgcgta acaactccgccccatt ga cgcaaatgggcggtaggcgtc EgtEgga>cta tataagcaggEBEctgtacccactgactc actggcttcgagattcca cca tggeggee KC£jeiUatcaa£aicfi.cca£Ccfi£^£t^ctEE&ca^c^ acgtgtacfiaatasffiteEaa^ acttcgixct EdaaaacgECttcatcflcca gca a ttECtltgRcacaggcttEtacttaa ccttggtECgca a Bataa aaaacatecaga Eccaaag gaaaggcagtgaggggacctgcagctgctcgtctaagggtttctccacgtgtccageccacgtcgatgacctaactccagaaca aatcctEgatEKfifiatataatgaRctRatagatatatagttctccaccatattccaaaaaaaaagctflatRRaatficattgatcaafja. Petition 870260074145, of 24 / 07 / 2026, p. 143 / 259 132 / 192 acttatcttccttcttccaaataagaacttcaagcacaRagcatatgccagccttttcagagaRCtEgaEgaRdcgctgEctgacctt REtctcagcagttttEEa a tttctgacactcCcc3⁄4EaaBaRattΐttcta^a3⁄4TcactaEgga Eggatca CCtctRtttRcg gEtErcgctcaRcagaaaagaRaa aacgtca a cccccgacacccctRCttREgtcc ca gaga ga a ggctgRa casaca cccag ga ctccaatRtctEctccccaEEEEcgccEEctgctcacccaEaaEECCaEcctxcaga Eccca gcacGca gEcct cacgfiEEBca caEctEgtcctccagca tgtgcaEgcEctgctggtcaagagattccaaca ccatccgcagccaca agga ct tcctgECECEcaEdtcatactcccEgctacctttRtattttt EEctctaatgctttctattEttatccctctttggcBataHcct cccttcacccctggatatatgggcagcagtaca ccttcttcagca tgga tga a ccaggcagtgagcagttca cggta cttgcagacgt çchtçchtgaataagçça^EchttggçaaçxEcjgçchtgaaggaagggtggjttçcagcagcagccac gactccttctgtgtcccca a acatca cccagctgttccaga agcagaaatgga cacaggtca a cccttcaccatcctgcaggtgcag caccagEEaE3agctcaccatgctEccagagtficccc.RaggEtEcc.gfiEgEcctcccgc.€cccccagagagacagcagcagcagcta cagaccgc cgga ca gga a catctccgacttcttggtaaaaacfitatcctgctcttataagaagcagc tta a agagc aaa ttctgggtca a tga a cagaggtatgEagga a ttcçattggaggaaagctçcçagtcgjtçccçatc a cgggggaagca cttgtt gggttttta agcgaccttggcc ctagagaggcctcta aa gaaa ta cc tgattcctta aacatctagaaactgaagacaacattaagfitgtgRttaataaaaggctggcatgccctggtcafictttctcaatgtggcccaca acgccatcttacggEccagcctgcctaaEEacaggagcccgaagccctatgccattagc aaggagcagctctcagagattacagtgctgaccacttcagtggatgctgtggttgccatctgegtgatttctccatgtccttcgtccca gcca gctttgtcctttatttga tccaggagçgggtgaaca aa tccaagcacctcc agtttatcagtggagtgagcccca cc acctactcc gggctctt taacct tcagcat tgagtatt tgctggflctggtggtEggcatcttc a tcgggtttcaga aga a age eta cacttctcca gaa aa ccttcctgcccttgtggc actgctcctgtatggatggatgggcggtcattccca tgatgtacccagc a teett cctgtttga tgtccccagc a cagccctg tgtc tgtc tgtc a tcggcatcaac agca gtgeta tta ccttcatct tg ga attatttgagaataaccgga cgctgctcaggttca acgccgtgctgaggaagctgctcattgtcttccccca cttctgcctgggccg gggcctcattgaecttgcactga gccaggctgtgac agatgtctatgcccggttigfitga ggagca ctctgcaaa tccgttccactgg ga cctga ttggga aga a cctgttgccatgggtggtggt cttcctcctgaccctgctggtcc agegccacttcttcct etcecaa tggattgccgagcccacta a ggagecca ttgttgatga aga tgatga tfitgRctga aga aa gacaa agaa ttattacigg tggaaa taa aa etga catcttaaaactaca tga actaacca a gat ta t tccaggcacctccagcccagcagtEgacaggctatgtgt cgfiagttcgccctgfiagagtgctttggcctcctggga gtga a tggtgccggcaaaaca accaca ttca aga tgctcactggggacae ca caEtaacctcagEEEatgecaccEtâEcattEC&agagtattcaattcaattcaatt tgggctactatcct ca gtt tga tgea a tega tgagetgetea ca Efia cgaaaacatct t taccttta tgcccggcttg aggtgtacca gcagaagaaatcg aaaaggttgcaaactggagttaaEaRCCtãggcctgactEtctac cgEaactctccacaEccatcgcactcattagctficccaccactEgtEctEctggatgagcccaccacagggatEEacccccaggca cgccgcatgctgtggaa egtea tegtga gcatcatcagaga agggagggctgtgRtcctcacatcccacagcatRgaaga atgtga Petition 870260074145, dated 07 / 24 / 2026, pp. 144 / 259 133 / 192 ggcactatfitacccggctfigcca tea Wtãappgcaccttcgãtata tgggca ccattcagcatctcaagtccaaattiaaaeat ggctatatcgtcacaatgaagatcaaatccccga a gga ega cctgcttcctgacctaaacmgtfigagctfigagfigfigfigfig ncccaggcagtgtacagafifigaEaggcactacaaiatficticagttccafigtctcctcctccctEgqgaaflatcttccagctcc tcctctcccaca aEEacagcctgctca tcgaggagtactcagtcaca caca ccacactBgaccaggtatttfita aatttgctaca gcagactfiaa.agtcajtEacctccccttEcacc.ctcgagctgctggagccagt.çfiacaagcççaggaçqocfqcooaqoccatqgt; ggtga ttataeaga tcetgaco tcqactüCGtjqaotüacootqüCfJowtaogcggccgcttcgagcagaeatgataagatac attgatgaetttagacaaaccacaactaaaatgcagtfiaaaaaaatgctttatttataaaatttfitfiatattattRctttatttgtaa ecattataagctgcaataaacaagtta a ca acaacaaattgcattcautta tgtttcaggt tcagggggaga tg tggga ggllt tt ta a agca agta aaa cctctacaaatgtggtaaa atcgataaggatcttcctagagcatggcta cgta ga ta agtagcatggcgggtta atcattaactacaaggaacccctagtgatggagttggcc3CtccCtctctgcgCgCtCgCtcgçtcactgaggccgggcgaCCaaag gtCECCcgaCECccgggctttgcccggEcggcctcagtgagcgagegagCECECaE p38 pAAV2.1-CMV260- 5' ABCA4 intein_ecDHFR (setl). 5'ITR (seq A) CMV260{seqU) 5' ABCA4 (from set 1) N protein Npu DnaE (seq D) 3xfhg (seq E) ecDHFR (seq O) WPRE (seq F) SV40 PolyA (seqW) 3′ ITR (seq H) p39 PAAV2.1-CMV260- 5TABCA4 intefn_mini ecDHFR (set 1); 5' ITR (seq A) CMV260(seq U) 5' ABCA4 (from set 1) Petition 870260074145, 07 / 24 / 2026, p. 145 / 2 134 / 1 N-intein Npu DnaE (seq D) 3xf lag (seq E} mini ecDHFR (seq P) WPRE (seq F) 5V40 PotyA (sec W) 3' ITR (seq H) p40 pAAV2.1-GRKl- 5' ABCA4 intein„ecDHFR (setl) . 5' ITR (seq A) GRK1 (seq N) 5' ABCA4 (from set 1} N-intein Npu DnaE (seq D) 3xflag (seq E) ecDHFR (seq 0) WPRE (seq F) SV40 PoiyA(seqW) 3' ITR (seq H) p41 pAAVZ.l-GRKl- 5' ABCA4 intermediate ecDHFR (setl) SEQ ID NO-64 5' ITR (see Al· GR KEY: bold 5' ABCA4: underline N-intestinal Npu DnaE: double underline 3xflag: italic Petition 870260074145, 07 / 24 / 2026, p. 146 / 2 135 / 1 Mini ecDHFR: thick underline SV40: ball underline 3' ÍTR (seq H) etgcgcgctcgctcgctcactgaggcgc cggc a a aagcccgggcgtcgggcgaccttggcgcccggcctca gtg agcgagcg agcgcgcagagagEgagtggccaa ctcca tea ctaggggttcctgctagcctagtgggcixcagaaEcctggtgEttgtttgtcctt ccaggggaaaagtgaggeggttccttggaggaaggggccgcgaatgatctaBtcggBttccaagcagctcaggggattgtgt cttttcttagcaccttcttgccactcctaagcgtcctccgtgaccccggctgggatttagcctgctgtgtcgccccgggctccca gggcttcccagtggtccccaggaatcrtcgacagggccagggcUttcgtccagcaaEggcagggacggc gEecaECCECcatEEECttcgtEaEacaEatacagcttttgctctEgaaEaactEgaccctRCEEaeaaEEcaaaaEattcEctttg tggtggaactcgtgtggcct ttatcttta 1I tc tggtet tgatctggtta aggaatgcca a cccgct tgccat tccatgat ccccaacaaggegatgccctcagcagga a tgçtgc cgtgEçtççaggggatčttctgçaa tgtgaacaatcçctEttttcaaaaçcçç aceccaggagaatctcctggaa ttgtgtcaaactataacaactcca tet tggcaagggtatatcga aa tttcttca a tcctc atgcaccagagaficcagcaccttfiaccgtatttERBcagagctacacatcttgrcccaattcatggacaccctccggactcacccgg agaeaa ttgcaggaagaggaattcgaa ta agEEatatettgaaaga taagaaacactEaçaçtatttctcatta a aaa ca tegge ctgtctga ctcagtggtctaccttga tea a ctctca agte cgtccagagcagttcgctca tggagtcccgga cctggcactgaagga ratcgcctecagcgaggccctcctKagcgcttcatcatcattcagccagacccgcgcgcgc ga agacaetetgtgtatgecaa cgtggacttcttca agctcttccgtg tgcttcccacactcc tagacagccattctca afigtatcaatctgagatcttgEggaggaa ta ttatctgatatgtçacca aaaattcaagagtttatccatcg g«KagtatBcaj&acttgctgtMgteacecctcatRccctGga^tgatfig^ cctcctgtfitggctaccccfiagggagfitggctctcggEtgctctcc ttcaactggta tgaaga ca a ta a eta taaggeettte tgfiggattgacucacaagfia aggatcctatctattcttatgac agaaga a ca a ca t cctt ttgtaatgca ttgatccagagcctgga g tccaaa a tettage tttagea geficagcaaagcctttfictfiataEgaaaa atcctgtacactcctga ttcacctgcagca egaaggataetgaagaa tgccaactcaa cttttgaagaactggaacacgttagga agttggtca a agcctggga aga agta gggc cccaga tctggta cttcttgacacga tca tca tca gaca tca gaca cctggggaacccaacagta aa aga et tttga ataEgcagcttggtEaa ga aggtattactg ctgaaEccatcctaaacttccctacaaEEgccctcgggaaaEccaEEctga cgac atggccaacttcgactggagggacatatt taaca tcactgatcgca ccctccgcc ttgtca a tea at acctggagtgct tggtcctgga taaEttgaaaEctacaatgatfiaaactcaEctcacccaacgtgccctctchtçtactEgagaaaaacatgttctggficcggagtggt a ttccc tgacatgtatccctggaccagctctctaccaccccacgtgaagtata agatccgaatggacatagaegtggtgaagaaac caataagattaaagacaggtattgggattctggtcccagagctgatcccgtggaafiatttccggtacatctfigEgGgggtttgcctat ctficaggacatgEttgaacaggRgatcacaaggagccagRtgcaggcggaRRCtccaRttgRaatctacctccagcaRatgccta Petition 870260074145, of 24 / 07 / 2026, p. 147 / 259 136 / 192 cccctfiÇttÇÊtSÊaçÊittchttchatÊatchatcchtEaacccgctÊtttícctatcttcatggtÊÇtggchatggatchtacctÊtccchatga cctaattccttEEacaCatCtCtCtCtRCAt tcttcctoctRa csa ta ttcatcatgcatRRaaRaatccta cattacaRCR acccattcatctctcttcctEttcttEttaactttctcCBctfiCCaccatcatgctgtECtttctectcaECaccHeitctccaagBccaEtc tgEcagcagcctRtagtggtgtcatcttatttcaccctctacctEccacaca.tcctEtgcttcecctgfica^gaccEcatEaccgctgag chtgaagaagggctgaEcttactetctccggtKRC3tttagatttj«cactre^ tgcagtggagcaacatcggEaacajgtcccacgga.a£EEEacgaa.tt.cagchtcctRchtetg.catgcagatga.tgctcd:.tgatEchtg EtattEecttgEcgEaagEEettcaaccaEagaaeaaaRaEocctREaaaaEaccRagcccctaaaaaaaaaacEEaEEa tccaaagcacccaaaaaaaaaaa cga ctccttctttEaacRtaaRCa tccattRRgtaRgta teCRtagta aggga aggga eactteaaa EatttttRaficcctEECCEgccBgctEtEEa ccgtctaaacatca ccttctacga ga a ccaaatca ccacattcctesEcca caa tgga RctggRa a aaocaccaccttateca tcctaacgggtctattgccaccaaccfctRERa çtEtRCtcgcgcRRRa egga attEaaacçaRcçtfiEatgcaEtccEEcaEagccttgEcatgtfitccacaRcacaacatcctRttccaccacctcacgEtEEctgag ca catgctgttct atgcccagctgaa agga a afitcccaggageaggcccagctEgaga tgga agccatgttcc ggacacaggcagct caaECfiEaatgaagaEEctcagRa cctatcaRgtEECatgcaRaRaa aEctgtcEEttaccattEcctttgtggRa Eatg ccaagBtggtgattctggacgaacccaccictgEfiEtggacccttactcEagacgctcaatctgggatctgctcctgaagtatcgctacca Escacatca tggcatc ccacateea^ga ggcggacctccttEEEEa ccacattacca tcattecceaagg aaggcht ctBctgcfcaggcacccca ctçttççtga aga a ctgcctgagctacgagaccgagatcctgaccEtgfiagta cggc^tgctgcccatc ggcacgaccc gaggcac Ega^gtgtcEagta^tgcc tEgageacMcagcctEatccegeccaccaaggacca ca a gttcat gaccgtggacggccagatgctgcccatcga cgaga tcttcgagcgggagctggac.ctgatgcgggtggaca a cctgccca acqgcr gctjüüaüccütaocqraarrütaüaaatcüíqüCutcqacrücauqquraocqatgüt:oücatcaacctaat€£€CECcct£ RccetEEactacEtEatcgEcatEEaeaacEccatgcctEEaacctEcccECRacctEECacctEECaCaEact caaecccetRatca teaeca eeca cctEEEaea eca tcEEcagacccctecccEEca EEa a eaacatcatcctga ecaeccaa cccaaca ccgacea ca gaetaa cctgEEtaa a ga Ecgteea cEaeccatcficcgcct acRRcccea cEcccea teetsaga teetc tcEEECEEcaEcagEgtEatcaa gc a gttectEcc tgattcgaKcaaacatgataaaata cattaatgagtttggacaaa ccacaa etaaaatgeagt gaaa aaaatgctttattgtEaaatttgtRatgctattgctttattgt aacca ttata aaetgea gt ataa taacaaacaattgcattcattttatEtttcaggttcagjraEgagatgtgggaggttttaaagcaaataaaacctctacaaatg tggtaaaatchgataaggatçcaattgaggaaccecta gtga tgga gttggcca ctccctctgcgcgctcgctcgdca etga ggc cgggcgaccaaaggtcgcccgacgcccgggcutgcecgggcggcctcagtgagcgagcgacgcgcag piac-CMV260- 5* ABCA4 intein (setZ} SEQ ID No. 65 Petition 870260074145, dated 07 / 24 / 2026, pp. 148 / 259 137 / 192 5' ITR (seq A) CMV260: bold 5' ABCA4: underline N-intein Npu DnaE: double underscore Bxflag; italic SV40: bold underline 3' ITR (seq H) ctgcgcgctcgctcgctcactgaggcgccgggcaaagcccgggcgtcgggcgaccttggcgccggcgtcgtgagcgagcg agcgcgcagaeagggggccactccatcaetaggggttcttgtagttaccctctactactactcg tgc tetaggaaga t cttca a ta ttggcca it agcc a tattattca t tggtta ta tagcata aa tea a tattggeta ttggccattgcata cgttgtatctatatcataatatgtacatttatattggacatgtccaatatgaccgccatgltggcattfattattgactaEcgttgacat tgattattgactagtacggtaaatgg«cgcrtggctgatgactcacg^ggatttccaagtctccacrccattg3cgtcaatgggag tttgttttggcaccaaaatcaacgggactttccaaa a tgtcgta acaactccgcccca ttgacgcaaatgggcggtaggcgtgta c ggtgggaggtctatataagcagagctggtttagtgaactagagaacccactgcttactggctctcgagattccaccatggcg^cc gcca tgggchtcgteagaçagatacagçttttg^^ aga a ctggaccctgcgga aa aggcaaaaga ttçgchtttgtggtgggttctcttcttcttcttctt tetaettaa ggaa tgccaacccgctctacagccatca tga a tgcca 11 tcccca a ca agficgatgccctcagcaggaatfict£ccgtEE£tccagg£atctttctgcaat£tj;aacaatccctgttncaaaaccccaccccag fiafiaatctcctgeaattatEtcaaactataaactccatcttggcaagfi|atatatcgaEattttcaaaactCEtcatEaatfícacc afiagagccagcaccttfigccgtatttggacagaflctacacatcttgtcccaattfiaactcccc tgcagaa a gaaaaattcaa ata a agga ta ttgaaaaa tgaagaaacactga ca eta ttdcattaaaaa catcggcctgtctaa ctcagtggtctaccttctfiatcaactctcaafltccgtccagagcafittcfictcatgaaEtcccggacctfigcccatcftcftcftc agaagacactctgta tgcca a cgtgga ettettea age tetteeg tgtgcttcccacactcctaga ca g tgcagga cttgclgtgEEtgaccaggcccctca Lgcagaatggtggtccagaga ccttta ca a agetga tgggca tcetgtctga cct cctEtetfigcta cccctcctgagattcgtcgtcg EE ctEfitatga a Eacaataactata a EEcetttetgEEgatt ga ctcca ca aggaagga tecta tetattetta tgaca gaaga a caa ca tccttttg taatgea ttgatccagaacctggagtcaaatc cttaaccaaaatcgcttggagggcgEcaaaEcctttgctgatgRgaaaaatcctgtacactcctEattcacetgcagcacgaagga; Petition 870260074145, of 24 / 07 / 2026, p. 149 / 259 138 / 192 tactga aga a igccaactcaacttttgaaga a ctggaacacgttagga agttggt ca aagcc tggga agaagtagggccccagatc tggtacttctttgacaecegcacacagatEaacaXgatcagagataccctEfigfiaacccaacagtaaagacltttjEatagagatggt aggtattactgctgaagccatcctaaacttcctctac a agggccc tcggga a agccaggctga ega ca tggccaa titcgSctggagfigscatatttaacatcSctEatsEcaccCtGcgcCttEtcaatCaatatCtgEagtgcttgEtcttEgata aat catageta aat tcagata caccca a cgtgcccLct ct ct aetggagga a aacatgttctgggccgga gtggta ttccctga catgtatLortggaors^ctcl / gT^acccca / ^tEiiagtakmg^^ ttaa aga cagg tattgggattctgEtcccagagctga tcccgtggaaga »tcccgggtaggtgggc caiggttgaacaggggatcacaaRgagccaggtgcaggcfigaggctccagttggaatctai:ctccaRcagatgccctacccctgctt cgtggacgattctcatcatcatcatcctgaaccfictgtttccctatctcatggtectggcatctactctgtclccatgactgtgaattctactctgtclccatgactgttgaattctacttag aga ggagttgcgactgaaggagaccttgaaaa atcagggtgtctcca a tgcagtga tttggtgtacctsgttcc tgga cagcttc tc ca tea tgtegatgagea tcttcctcctgacga ta ttcatcatgca tggaaga a tecta cattacagcgacccattc atcctcttcctgttcttgttgctttctccactgccaccatcatEctgtgctttctgctcagcaccttcttccaaggccagtctggcaac agcctgtagtggtcatcttatttcaccctctacctgccacacatcctfitgcttcgcctc aggetgtEagetta ctgtctccggtggcatttgga mggca ctaa gtacctREttcgctttaa a RagcaaRECCtfigRgctgcaEtgE afiç^aç.atçgggaaçagtcççacfigaagfigRaçga atuafiçttçchtfic.tgtç..caTggcagatjtjgtjgtcgctatc. gctta ctcgcttggta cttaa tcaggtgtt tccaggaga eta tggaaccccacttccttggtactttcttcta ca agagtegct tggct tggcEEtgaaEEErgttcaaccaEagaagaaagagccctEBaaaagaccgaEccctaacaEagggaacgEagEatgacgacgactaaccgactac acgtgagcatccagggtgfigttcctRgEEtatgcatga agaatctggt aaaga ttt tEaEccctEtEECCEECcaEctEtEEaccEtctEaacatcaccttctaceaEdaccaEatcaccEcattcctEgEccacaatEEagc tgggaaaaccacca ccttcatctctc ccaacctctgggactgtgct cgttgggggaagggac attgaa ac cagcctg£ÍtECagtccgEcaEaficcttEEcatEtccacagcacaacatcctgttccaccacctcacgEtgECtgagcacatEctgttctatgcccagctgaaaggaaagtcccaggaggcccgctggagatgfiaagccatgttggaggacacaggcctccaccac aagcgKaatgaagagECtcagg&cctatcafiEtggcatECaEagaaagctEtcggttECCattgcctttEtfiEgagatEccaggt t cctctggggtggaccct tactcgagacgctca at ctgggatctgcicctgaagta tcgctcaggcaga accatcatcatEtccactcacçacatggacxaggçcgacctjctggggaccgcat^gccatcattecccaggEaafiEctctactEct caggcaccccactt gagctt agggact gcagta cttacgett ettggtgege aagatga aaaa ca tccagtgcctgag rta <aagac.cga£atafia«giggaetaraGc.tg!tec£c.alira<a^^ gcatggacaacaacEacaacatctacacccagcccgtEgcccaEtEEcacgaccggEECEagcaEEaEgtgttcgagtactgcct agcgEEagctg^iacctgatECEEEtEEacaacctgcccaacaoctgcoaaaocct? tgacggtgai taiaaagaicazgaca tc goctacaoggatguega tgocong tqügcRgecEcttcgagcagacatflataaBatacattRatgaEtttEEacaaaccacaa Petition 870260074145, of 24 / 07 / 2026, p. 150 / 259 139 / 192 taacaacaattgcatttatgttttcaggucagggggagatgtgggaggttttltaaagcaagtaaaacctctacaaatgt ggta aa atcgata a ggatcttccta ga gca tggcta cgtagataagt agca tggcgggttaatcattaacctaca acctaca ggaggt cagaggt ctccctctctgçcgctcgctcgctcactga ggccgggcga ccaa a ggcgcccgacgcccgggctttgcccg ggcggcctcagtgagcgagqjagcEcgcag piac^MV26C- 3' ABCA4 Intern (set2) SEQ ID No. 66 5ΓITR (seq A) CMV260: bold 3' ABC A4; underline C-intein Npu DnaE; double underline 3xflag: italic SV40; bold underline 3' ITR (seq H) ctgcgcgctcgctcfiacaajjagiycgcccTOgcaaagc^^^ agcgcficagagagafiagtflficca a ctccatca ctaggggttccttgtagtta a tgatta a cccgccatgctacttacta cgtagcca tEctctaHEaaaBtcttcaatattgECcattaEccatattattcattfigttatatagcataaatcjiatattEEctattfiEccattacatd cgttgt atctatatca tatatgta ca tttata ttggctcatgtcca a ta tgaccgccatgttggcattga ttattgactagcgttgacat teattattgactagtacgRtaaatEgcccKcctggctRatBacRacggfiRatttccaaKtctccaccccattRacEtcaatRRBaE tttgttttggcaccaaaatcaaqowactttccaaaatgtcgtaaactccgccccattEacacaaatEggcggtagtagEcgtgtac RRtEEgaRRtctataaRcaEaEctEEtttaRtRaactagaEaacccactecttactggcttcsagattccaccatggCgEcc: Eccatga tea agatcgccacccggaaEta cctgggcaagcaRa.acEtgta.cEa ca tçgEçgtagagçgfigaccacaacttcEççct gM£a^gE£ttç^ç£ççai£aala£^^^^^^^^ gtccagcccacgtcgatgacctaactccagaacaagtcctggatggggatgtaaatgagctgatggatEtagttctccatcccagcaegcagcAggtcAggt aget age aga acttatcttccttcttcca aataaaaa ettea agcacagagcata tgccagcctt ttcegaRaRctggaflggecgcttKctaaccttgEtctcaa^^ cacgga eeattetaa ttcajggacctctEtttgcggKtKEcectcagcaEa aa gagacct ca&ccct ca&gcct tcccagaga ga aggetgga çagacacccca EEactccaatgtctEctccccaggfiEcgccfifictgctca ceca gagaficcagcctc. Petition 870260074145, of 24 / 07 / 2026, p. 151 / 259 140 / 192 ccccagasccagagtgcccaRgcctacagctca aca cerres cacaRCtERtcctccagcatEtgcaggcgctgctgRtca a gag attççaaçaçaççatçgayjíxaçaaÊÊaçttççíÊÊÇÊçaÊaiQíiÊítwíÊÊÉtaçüttÊü^^ tgUatccctccttttggcgaataccc^ctttgacccttcacccctggatatatgggcagcagcctc age a gt tea eggt a cttgcagacgtcctcctgaa taagccaggct ttggcaaccgctgcctga aggaagggtggcttcc ggagtacccctgtggca a ctcaacaccctgga a ga ctccttctgtgtccc ca aa catcaccca gctgttcca ga agcaga cagga caggat a cacca t tcctEGaggtgcagcaccaEggagaafictca ccatECtgccagaEtgccccgagggtECCggfiggcc tcccgcccccccagaaaaca cagçgcagcachggaaattçtaçaagaccchtgachgga ca tctcchgacttcttggta aa açg tatcctRCtcttataaEaaficaRCttaaaRafiCdaattctRRgtcaatRaacaEaggtatggagRaatttGcattRRaRfiaaaRCtc ccaRtCRtccçca tcacRggggaagca cttgttggRttttta agcgaccttRRCCgRatcatgaatRtgaccatRCcaatRRECTA a gagagga e cctgatttccttaaa ca tctagaaactgaagacaac a ttaaggtgtggtlta ata aca aa ggctggc a tgccctggtcagct ttctcaa tgtggcccaca acgccatcttacgggccagcctgcctaagga ca gga gccccgaggag tatgga a tcaccgtcattagcçaacccchtgaacctgaccaaggagcagctctcagagattacafitfictgaçcacttcagtggatfictgtggttgc catctficgtgattctcΐccatEtccttcgtcccagccaEctttEtcctttal·ttgaΐccaggagcgggΐEaacaaaΐccaagcacctCJca gtttatcagtfigagtgagcrcaccacchtctgggtaaçcaacttcctctfiggacatcattgattcgctEgctEgtEgt t^RcatcttcatCRREtttcaRaaRaaRcctacacttctccaRaaaaccttcctRCCÍttRtRRcactRctRtatRRatRR gcggtca ttcccatsatRta cccaRcatccttcctRtttgatgtocccaEcacaRCCtatgtRRctttatctgctgcttgctaactaacgcgcgc tgçtatta ccttcatct tggaattatttga ga ataa ccgga cgctgctcaggttcaacgccgtgctgaggaagct gctcattgtcttcccccacttgcctgggccggggcctcattfiaccttgcactgagccaggcttfiacagatatcttatgcccggttfig tgaggagcctc tgattggga aga a cctgtttgccatggtggtgga aggggtggtgta cttcctc etga ccctgctggtçcagcgcc acttcttcctctcccaa tggattgcc.gagccça ctaaggagcccattgttga tga a ga tgatgatgt ggctga aga a aga ca aa ctaactAtaAtaAtaac agRctacatgaacta accaaeatttatcca rrcb ca accacattca agatgetea ctggggaca ccacafitRacctcagfigRatgccaccgtagcaggca a ga gt at tttaacca a ta tt t etgaa gt ccatca aaa tatgRgctactetcctcafitttaatgcaatcgatgafictgctcacaaRacgaRaccatt tacctaca t taccat cggcttcgaggtgtacca gcaga a ga aa tega a aaggttgea aa ctgga gt attaaaagcctgagcctgactgtctacgccga etg cctggctggca cgtdcagtgggggca a caagcggaaactctcc a ca gccatcgcact ca ttggctgccca ccgct gccaggct gccagc tggacccccaggc acgccgca tgctgtgga a egtea tegtgagea tea tcagaga agggagggctfitg ^cctçacattxça caEcatEEaa ga a tEjfiaggcaçteÍEtacccggctggccatcatEgta aagEECgcctttcEatgtatgEEC a ccatRcatcact agtcaca tttEEagatEectatatcgtcaca ateaagat caa atccccEaagRaacctRcttcctgacc tgaaccctRtEgagcagttcttccaRgggaacttcccaggcagtRtacagaRggagaggcactacaacatRctccagttccaggtct cctcctcctcctcgctcctcacctc cagcctgctcatcgaggagtactcagtcacacaga ccac Petition 870260074145, of 24 / 07 / 2026, p. 152 / 259 141 / 192 actega ccaEEtgttteta a attttgcta aa carcagactEa a agtcatgacctccctctgca coctcgdEctEcteeaEC cagcga caas^g^s^tg^tacaaagaccatgacggtgattatgaagatca tgncatcgactaca ECEECcficttcaaEcagacatgataagatacattgatgHgtttggBcaaaccacaactagaBtgcBgtgaaaaaatgctttatt tgtgaaatttgtgatKctattgctttattgtaaccattataagctgcaataaacaagttaacaaacaattgca ttca tttatgttt tagEttcaggEggagatgt&BEaggttttttaaagcaagtaaaacctctacaaatgtEEtaaaatcgataaggatcttcctagca tggcta cgtag a tagtagca tggcgggtta a tea tta a eta ca aggaacccctagtgatgga gttggccactccc tctctgcgcgct ege tçgc tc act gaggccgggcgaccaaaggtcgcccga eg cccgggctttgcccgggcggcctcagtgagcgagcgagcgcgca g pzac-CMV260- 5rABCA4 intein (set3) SEQ ID No. 67 5rITR {seq A) CMV26O; bold 5' ABCA4: underline N-intein Npu DnaEi double underline 3xflag: italic 5V4O: bold underline 3* ITR {seq H) c Ecgcgctcgc tcgctcactga ggccgcccgggcaaa gcccgggcgtcgggcgacctt tggtcgcccggcctcagtga geg ageg agcgcgcagaga gggagtggcca a ctccatcactagggggttctt acctta accta aggta atgetta cgtagcca tgeteta gga ag atette aatattggcca ttagccat attattcattggttatatagca ta aatcaatattggcta ttggccattgcata cgttgta teta tatcataa ta tgtaca 11 tatattggetea tgtccaata tga ccgccatgttggca ttgatta ttgactagcgttgacattgattgactaggtactactactacta atggcccgcctggctga tgactcacggggatttcca agtctccaccccattgacgtcaatgggag tttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaaactccECCccattgacgtaaatgggcggtaggcgtc ggtgggaggtctatataagcagagctggttctcgagattccaccatggcggcc ETMtj%Ecttc|^aK?cafiatacaigcttttfictctKga3Kaact|^KCctfic^^ ctcatgtgftccttttatttatttagtgaactagatagaacccactgcttactttatttagtegategatega cccgctctacagccatcatga a teccatttcccca a ca agecEatficçtcagcaggaatfictfi.ccfitgEctc.caEfigEatcttctEcaatgtfia.aca.atccctfi,ttttcaaagçcccaccccag gaga a tctccteea attatEtca aa eta ta a ca a ctccatcttEgca aaegta ta tcgaeattttcaaea a ctcctca tea atgcacc. Petition 870260074145, of 24 / 07 / 2026, p. 153 / 259 142 / 192 agaRagccagcacchtfigçcgtatttgigacagagctacacatctrtcçcaattçat^açaccçtcca^ tgca eea ​​a gagga a ttcgaata aggga ta tcttgaa agatgaaga aacactga cactatttctc attaaa agt acact tcEECct ctcafitEEtctacc-ttctgatcaactctcaagtccEtccagagcagttcgctcatRgafitcc-cg£acctEECgctgaàggacatcgcc ijTca^cgaggcpctajgga^cgdjrahyitcttjq^jqy^^ agggca ccctacagtgga tagag cact actgtat cgt cttcttcaagctcttccgtgtgcttc ca cactcctaga cag ccgttctcaaggtatcaatctgaRatcttggEgagfiaattattatctfiatgtcaccaagaattcaagagtttatccatcggccgagta tgcafifiacttgctgtgggtgaccaggccctcatgcafiaatggtggtccagagacctttacaaafictgatgggcatcctgtctgacct ççtgtgtggcta ccccga gggaggtggctctcgggtgctctccttca a ctggtatgaa ga ca a taactataaggccttctgEEagat aggtacgat aggtactat c agaagaaca a catcc tttgtaatgcattgatccaga gcctggagtcaaatc cttΐaaccaaaatcgcttgEagggcEEcaaagcctttggatgggaaaaatcctgta€actccΐgaΐΐcacctgcagcacgaagga ta ctga aga a tgcca act acctttga a gaggaac a cgattac agttggtc aaa gcctggga aga a gtagggcccca ga tctggtacttctttgaca a cagçaca ca gatga acatgatcagagata ccctgggga a ccca a ca gtaaaagactttttga ataggca gcttggtg aagaaggtattac tgetga agccatccra aa cttcctcta ca agggccctcgggaa agccaggctgacgaca tggccatt cgactt caggaca ctgatcgcaccctccgccttgtca a tcaatacctggagtgcttggtcctgga ta agt ttg aa agcta caatgatgaaactca gctcaccca acgtgccctctctctactggaggaa aa ca tgttctgggccggagtggta ttccctga ca tgta tecctggaccagccct ctacta gta gta aa ΐ gatecgaatggacat agacgtggtggagaaa a ccaa taaga ttaaaga ca ggta ttggg a ttc tggtcecagagctgatcccgtgga aga ttt ccggta catctggggcgggtttgcctatc tgcagga ca tggt tg a aca ggggac tea caaggagccaggtgcaggccc tgcccta cccctgctt cfitggacga ttctttca tga tcatcctga accgctgtttccct atcttcatggtgctggcatgga teta ctctgtctcca tgactgtga ag agea tcgtcttggaga agga gttgcga çtgaaggagaçct tgaaaa at ça gggtgtc tggacagcttccatca tgcga tgagcatcttcctcctga cgata ttcatcatgcatgga aga a tcctacattacagcga ccca ttc atcctcttcctgt tet tgttggctttctcca ctgcc a ücatcatgctgtficΐttcΐgctcagcaccttcttcl·ccaaggccag^ctggcagc agcctgta gtggtgtca teta tttea ccccta cctgcca cacatcclgtgcttcgcctgcatcgcacgac agactEtgaaa ta ctgtctccgfitggcatttfigatttfigcactfiagtacctggttcgct ttg a agagea a figcctgagRCtgcagtgg agea acatcgggaacagtcccacgaaaggggacga a ttcagcttcctgctgtccatgcaga t ga tectccttaatgctctctctgttagttac ccttgatcaggtgtttccaggaga eta tgga a cccca cttccttggtactttcttctaca a ga gtcgta ttggct tggcgatgaag^tgttcaacca gaga a gaaagagccctRga aa agaccgagccctaacaga gga aa cgga gga tccagagc acccagaaggacatac tgaacgtgagcatccagggtgggttcctgggfitatgcgtgaagaatctggtaaagatttt tgagccctgtggccggcca gctetgga ccgtctgaaca tea ccttctacgaga a ccagatcaccgca ttcctgggeca caatggagc tgggaaaacca cea ccttgtccatcct cggcg tgccat agt cctctgggactgtgctcgttgggggaaggga cattgaaac cagcctggatgcagtccRgcagagccttggcatgtatcca ca gcaca aca tcctgttccaccacctca cggtggctgagcacatgct Petition 870260074145, of 24 / 07 / 2026, p. 154 / 259 143 / 192 RÊtâ4UctâÊÍ£ÊílíV«2LCíj££tí£íiâ£ÍâS££O££ÊâÊs^^ ggggl^Í^ll£^^^£ÇgXÊÍOg££Íggâ£ã01£g^0^1£Í^^Íg££Ç^Íig^ʣʣʣ ÊggÇ^^£gg^X^Í£^^â£Í^lÊSÍggÍ£ggÇigÇ^gâÍ^gg^^âaggi^£OÍt^Êã£<£ÍSã^ ü tgacggtgatta taaagatcatgaca tcgactacaaggatgaega twcg^t^fiCRfiCcgRacagaRacata gatacattEatgafftttggacaaccacaactagaatKcaatgaaaa&aatBctttattigigaaatttgts3tfictattgctttal· ttgtaaccattataagctgcaataaaaagtta a caacaaca a ttgca tt camt a tgt (tcaggttcagggggagatgtgggaggi ttttta agct agctaca aa cctaa atgtggtaaaatcgataaggatc ttccta ga gcatggcta cgtaga ta a gta gcatggcg ggt taa tcatt aa eta caaggaaccccta gtga tggagttggcca ctccctetctgcgcgctcgetea etga ggecgggcgacc aaa ggtcgccccccc gcgagcgagcgc ag P4ÔC-CMV260- 3' A0CA4 inteln (set3) SEQ ID No, 53 5JITR ^eu A) CMV26O: bold 3' A&CA4: underline C-intein Npu DnaE: double underline 3xfiag; italics SV4O; bold underline 3' ITR {seq H) ctgcgcgctcgctcgctcactgaggccccgggcaaagcccgggcgtcgggcga cctttggtcgcceggcctcegtga geg a gcgcgeagagaggggagtggccBactccatcactaggggttccttgtagtta a g tgaacta gtta gtta gtta gtagtta tgeteta gga a ga trttcaatattggccattagccatattattcattggttatatBgcataaatcaatattggctattggccattgcata cgttgta tetatatea taa ta tgta ca ttta tattggetea tgtccaatatgaccgcca tgttggcattgattattgactagcgttgacat tgattattgactag eggta Btggcccgcctggctgatgactcacggggatttccaagtctccaccccattgacgtcaatggga g tttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaaaactccg(xccattgacgcaaatgggcggtaggcgtc ggtgggaggtctatata agcagagctgacttagatagatt cccactgcttactggctctcgagattcca ccatggcggcc Petition 870260074145, of 24 / 07 / 2026, p. 155 / 259 144 / 192 ficcataataaa&tgatcfiacaft&aaaaacctggecet&caeaaaatcttcgacatc&eccteccccaataccacaacttcctfictg gccaa^ggÊgçcatggccgccaactctggggtgaccctctcgaegctcaatcctcctgctcgcgc aaaaccatcatCdtatccactcaccacatKEdCRaEECcgacctccttggEEacCfiÇattRccatcattRcccagEEaaEEctctac tgctcaggcacccca ct cttectgaaaaactactttfigca ca ggcttgtacttaaccttfigtgcRca agataaaaaaccagaaa aaaflfiaaagEcafitRaflggfiacctficaEclgctcfitctaaflfigtttctccaccacgtgtccaficccacgtcEatfiacctaactccag aacaafitccl·REatggκEatgtaaatEaRctRatRκatEtagttctccaccatgttccaEagRçaagatRcatRcatκT aagaacttatcttccttcttccaaataagaacttcaagcacagagcatatgccaficctttcagagagctfiaaggagacgctggctfi accttRgtchtcagcagttttEgaatttctRacactchccchtEE.aajgaBattttctigaaggtcacgEagEattctgattc. ttficgEEtEECRctcdECaEdaaagaaa aaa CRtcaaccctcaacccctgfigtccçagaga ga a EEctRfiacaga cace ccaggac tcca a tgtc taetcccca gaggcaccggctgctcacccafl agagccagcetcccca Eagccagaccca£çtcaaçaçg£EEaçaçdEçt:gEtcctçca£çat£teçafiaç£Ítaçt£EtÇ-aa£a£aÍt ç£aaçaçaçç^ aacttawcflcaaatcgtgct cccggcta cctttgtatttttgflctflatgct etacctta tccttgc tcttgc tttgacccttcacccc^gatatatgggcagcagtacaccHcttcagcatggat^aaccaggcagtgagcagttcacggtacttgca Raç£tçctçççt£aata agccaggcttiBgca ac.cgçtgc.ctgaagga afigRtggccttcc£fittccEggcagcagcac£a gacac ctccttctfitgtccccaaacatcacccagtgttecaga a gea ga aatfigacacaggtca a cccttcaccatcctgcagfl tRcagcaccagggga ga agetea cea tEctficcagagtgccccEafiggtEccgfiEfificctcccgccc ca ca ccagagagccaa flc3CfíRaaattctacaaRacctfiãcHfiacagflaacatctc¢gacttcttfiκίaa3aacRl·atcctfiCtcΐtataaκ3aRcaEcΐt^a agagcaaattctfigfitcaatRa acaga ggt atgaagga atttttcgalga aggctfccagtcec tcacfígggaaaaca cttgttgEEttttta agcEaccttEECCEBatcatga a tEtgaecEEBEECccta tea eta eaaaggcctctaaaga aa ta cctgatt tccttaaacatctgaaactga a gaca aca ttaaggtfitggtttaa ta a ca aacccat ggcat caggttca atcaggtc agggtc cgcca tetta cgggcca gcctgcctaagflacaagagccecgaagagta tggaateaccfltcanagccaacccctgaacc teacc a aEgaeca EctctcaEaeattacaEtga ccacttcaEtEEatEctEtEEttaccatagcEtEa ttitctccatetccttcg tcccagcca gett tgtcctttaat tRatc gt t ta tcafitfigagtgagccccaccac ctactgggtaaccaacttcctgggacatcatgaattattccgtgagtgetgggctggtgggcatcttcgggttca^aaga aaEcctacacttccagaaaaccttcctEcccttEtEEcactEcttEctEctEctatEcatEcatEcatEcatEcct tcctgtttga tgtccccagca ca gcctatfitgRcttta tcttgtflctaatetgttca teggea tcaacagcagtgctatta cettcat ettggaatta tttgaga a ta a ccgEaegctgc tcaflattcaacgccHtgctfiafiEaaactflctcattccctcttgctc ECCgRRECCtcattgaccttgcactgagccaHtctEtaacagatetctatECC^tttafitRaRgagcactctac&aatccfittcca ctRfifiacctgattflggaagaacctgtttflccatRfitggtflgaafigggtggtfltacttcctcctgcctcgcgcctcgctc diaaaactgacatctt&aggcta ca teaactaaccaagattta tccaggca cctccagcccagcagtgga ca ggctgt Petition 870260074145, of 24 / 07 / 2026, p. 156 / 259 145 / 192 fitetCSgagttCgÇttWlRaRtgttttERCCtCCtKRRPgte^^ acaçcacafitgacctcaa^atgccac^tagcafificaaEa£tattttaaíxaatamctfiaafitccatcaaaatatfiaactact RtictcaEtUEatgcaaTcfiaTaagctEctcacaEgacEaEaacatctttaatttatgcccgEcttcEagEtRtaccaficaaaaEa aatQaaaaRgttgctoactrçajftattaagaEçct^gçctEaçtgtctacKc^^ açaaÊçggaaaçtçtççaçaÊççat£ÊÊâçíwítÊÊSÊ£<ça ççÊÇíEÊtÊ£iÊÇtÊÊâlÊêÊÇÇ£ÊÇÊ»çâSÊÊatÊÊaççç€ç agRcacgccgcatgc.t£tEgaacEtcatc:gt£aEcatcatcaEagaa£B£a.gggct£tfí£tt:ctcacatcccacdEcatg£aafiaa t^EaRgcacfEtetacccEEçtggccatt^tgEtaaaEKgcgcctttcEatEtat^ gaflatagcta ta tcRtcaca a tgaaga tea aa tccccRaagga cga cctKcttcctgacctaa a ccctRtfiRagcaattcttcca gg figaacttcccaRgcaRtgtgcagafifigagaggcactacaacatfictccaEttccaRgtctcctcctcctccctgECfiaggatcttcca gqtmcctctcccacaaggac <igcctgctcatc^£K4&t4£lcagtcacaimgaccacactggaccag^tgtti&uaatiltfict a a a cagca gactgaaagtcatga cctccc tctgcaccctçgafictactaga gcca gtega ca agccca Egacgociqeooaqacc π tgaeggtgatta taaaga fcatoPcatcgortacoaqgotaocqgiaacomjtagECEgocEcttcEaEcaEacatEataaEatacattjgatgagtttWKaaaccacaactagaatecagtjaaaaa3atgctttatttEtEaaatttEtgatgctattectttat ttEtaaccattataaRCtgcaataaacaagttaacaacaacaa t tgcattcatttta tgtttcaggttcaggggga gatgtgggaggt tttttaaagca a gtaaaacctctaca a atgtggta a a atcgataagga tcttccta gagcatggctacgta ga t aagtagcatggcg ggttaatcattaactacaaggaacccctagtgatggagttEgccactccctctctgCECECtcgctcgctcactgaggccgggcgace aaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagçgagcgagcgcgcag pB36 (lRBP_DsRed) SEQ ID No. €9 S' ITR {sect Aj IRBP bald WPR£; Italic underline DsRed underline BghpA; bold underline 3' ITR (seq H) ctgcgcgctcgctcgctcactEaEgccgcccEggcaaaEccgggcgtcgggcgaccttggcgcccggcctcagtgagcgegcg agcgcgcagagaggga gtggccaactcc a tcact aggggttc aaataa tggcagtgattaatgttatgatatgcagacac^ cacagca agataagaatgea atgtaccttctgggtcaacca ccctggccact cctccccgatacccagggttgatgtgcttgaattagacaggattaaggcttactggagctggaagccttgccccaactcaggagtt tagccctccaccc ccggccaagggcgaattctgcagatatccatcaca ctggcatggatagcact Petition 870260074145, of 24 / 07 / 2026, p. 157 / 259 146 / 192 cgagggcaagcctacgagggcacecagaffigccaagctgtaggtgaccaagggcggfcccctgcccttcgggacatcctgt cccccajntccaatacjtectccaagfitgtaratfiaaficaccRccgacatcccfiactacaagaagctatcctcctcctccAgERCAgcAgERCAgc cttcEBjagacggcgEcgWRacc^gaccca^^ a cca cgtgaagncatcggcgtga acttcccctccgacggccccgtaatgcaga aga agactctgggctgggagccctcca ccgag cfiCCtgtaccccggacggcfitgctRaaragcccacaaggc^ caafitcaatctacatggccaagaagcccgtgaagctgcccggctactactac giggactccaagaggflcatcaccjcccacaacg aERactacaccHtREtfigaRcaKtacRagcECRCcgagficccRccaccacctgttccagtaaoatrooccttgatjntJCaoagt trqrgactat ttqactqa ttqtaqta tgiigctccttrfacqcforg tQggtacqctgctttaa tgcctttgtatca tgeta 11 g ttccccctactattQccacQgcQgaactcatcgccQcctgccttgcocQctgclQgaacqgqgctcqQctqttQgqcactqacaa tccataactat tcccatcatta taactQctcacctQtQttqccacGtaaattctacgcaatjaca teetteta ctacqtcccttcqQccctcaatccaQcgQaccttccttcccqqccctqctqccQqctctqcqQcctctcgcqcttcQficjctcftattgtgCrttctagttficcagccatctfittKtttRcccctccccgtgccttectteaccctfigaaBRtficCactCiCcactEtccttttccta ataaaatgaKgBaattgcatcgcattgtrtgagtaKKtgtcattctattctgKggKgtiaaatgaKgcagcagcagK gattEEgaaeacaatagcaracatECTEKEgaa^aacaxtafitfiatflfta gt tgecactccctctgcgcgct cgctcgctca ctgaggccgggcgaccaaaggtcgcccgacgcccgggctttgccgggcggcgtcagtgagcgagcgcgcgc22gcg pAAV2.1_HLP_5rF8 intein (set 1) 5' ITR (seq AJ HLP promoter (seq J) SEQ ID No, 70 tgt ttgetgettgea a tgtttgcccattttagggtgga ca ca ggacgcfgtggtttctgagcca gggggcga ctca ga tccca gccagt ggacttagcccctgtttgctcctcgataactgEg^tgaccttggttaatattcaccagcagcctcccccgttgcccctggatccact gctiaadtacggacgdggacaggEtcctgtctctcgcttcaggcaccactEacctEggacagtgaat FS signal sequence (seq K) SEQ ID no. 71 atgcaatagafictctccacctgcttctttctgtgccttngcgattctRctttaEt 5' F8: SEQ ID no. 72 gcca ccagàagata eta cctgggtgcagtggaactgtcatggga etatatgeasa gtgatet eggtga gctgcctgtgE^cficaag a tttcctcctagagtgccaaaatcttttcca ttca a ca cctcagtcgtgtacaaaaagactctgtttgtagaattca egga cattt a ca cacca a teggca ccctgga tgggctgcta ggtcct accatcca ggctg aggtttatgatacagtggtca ttacactt Petition 870260074145, of 24 / 07 / 2026, p. 158 / 259 147 / 192 aa gaacatggcttccca tcctgtcaEtcttcatgctgttggtgtatcctactggaaagcttctga gggagctga atatgatga tcaga c ca gtcaaagggaga aagaagatgata aa gtettccctggtggaagcca ta ca tetgtctggcaggtcctgaa ggagcca a tggcctctgacccactgtgccttacctac teat a ttte tcatgtggacctggta aaa ga ettgaattea ggcctcattggagcccta c tagtatgtaga gaa gggagtctggccaaggaaaagacac agaccttgc aca a attta tactactttttgctgta tttgatgaaggaa aa aaa aaca acact g ctccttgatgcaggataggga tgctgcatctgctcgggcctggccta aa atgea cacagtc aa tggttatgta aa ca ggt ctctgccaggtctga ttggatgccacaggaaatcagtctattggcatgtgat tggaatgggca ccactc ctgaagtgcactcaatattcctcgaaggtcacacatttcttgtgaggaaccatcgccaggcgtccttggaaatctcgccaataactttc etta etgetea aa cactcttgatgga ccttgga ca gilt ctactgttttgtca ta tctcttccca cca a ca tga tggca tggagcttatg ca a ca gette g gaggaaccccaactacgaatgaa aaatatga aga a gcggaagactatgatgatga tetta etga t tetga aatggatgtggtcaggtttgatga tga ca a ctctccttcctttatccaaattcgctcagttgccaaga ageatetta aaaettg ggtacattacattgctgaagaggactgggactatgctcccttagtcgcccccgatgacagaagttataaaagtcaatat ttgaaca a iggccetca gegga ttgg tagga a gta caaaaaa gtccgattta tggca taca ca ga tgaaacctt ta agactcga gctgatt agtca tcagca aggac ttgggaccttta ctttatggggaagttggaga ca ca ctgttga father tttaagaateaa gcaa gca ga ccata ta a catctaccclca egga a teaetga tgtccgtcctttgtattca aggagatta ccaaaaggtgta aa aca tttgaa ggatttccaattgccaggagaaatattcaataataaatggacagtgactgtagaagatgggccaactaaatcaE^tcctcggtg cctga cccgctattactctagttcgttaa ta tggagagagatetagettcagga ctcattggccctctctca tcctgttttctgtatttga tga ga a ccga a gctggt acct cacagagaatatacaacgctttctccecaatccagctggagtgcagcttgaggatccagagttccaagcctccaacatcatgcacag ca teaatggeta tgttttga ta gtttgcagttgtcagtttgtttgcatc tgacttcctttctgtcttcttctggatâtaccttca aaca caaaatggtctatga agacaca ctcaccctattcccattca gga aa etgtet tea tgtegatggaaaaccca ggtet atggattctggggtgccacaaetcagae ttcggaacagaggcatga ccgcct ta etga aggt ttctagttgtga ca a ga a ca ctggtga ttat taegagga cagttatga a ga tat ttcagca tacttgclgagta aaaa ca atgccattga a ccacacctact atcacactact gcacta ggcaa aa gcaa tta a tgcca ccacaattc cagaa a atgacatagagactgaoccttggtttgcacacagaacacctatgccta aaa ta ca aaa tgtctcctctagtgatttgttgatgc tcttgcgacagagtcctactccacatgggctatccttatctgatctccaagaagccaaatatgagaictttttctgatcatccatcacctg ga gca a tags cagt aa taacagcctgtctgaaa tgacacacttca ggeca ca gctccatcacagtgggga catggt a tttacccctg accatca accata aggta attac tgagaaactggggacaactgcagcaacagagttga a ga a aettga tttca a agttctagta ca teaaataatetga tttca a caattccatcaga caatttggca gcaggtactgataatacaagttcc ttaggacccccaagtatgcc agttcatt atgatagteaattaga ta cactatta acta gtcatctccccttactgagtctggtgga cctctga gettgagtga a gaaa ata atgattcaaagttgttaga atcaggtttaatgaa ta gccaagaaagtt ca tggggaaaaaatgtatcgtcaacagaga gtgetaggtta ttta aagggaaaagagctcatgga cctgctttgttgact aa agataatgccttattcaaagtta gcatctctttgtt a Petition 870260074145, of 24 / 07 / 2026, p. 159 / 259 148 / 192 a agacaaaaaacttCca ataattcagcaactaatàga a agattCaCa ttgatggCccatcatta t taa t tga ga a ta gtctatCa gtctggcaaaatata ttagaaagtga cactgagtttaaaaaagtgacaccmgattga ca a ga a cactat tga cacta cacta gctttgaggctaa a tea tatgte aa ataaaactaettcatcaaaaa aa ca tgga aatggtccaacagaaaaaa ga gggcccca t teca Ccâga tgea ca aa atccaga tàtgtcgttcttta agatgctattcttgccagaatcagcaaggtggataca a agactcatctgcaact aagact ggccccagtccaaa gcaattagutccttaggaccaga aa aa tctgtggaaggtcaga a ttt cttgtctgagaaaaaaagtggtagta gga a agggtga a tttacaaaggacgta ggactca aag agatggtttttccaagcagca gaaacctattKtta cttaaga cttggata catttagaata acaacaaaaa aaaa ttcaggaagaaata ga aa ag a aggaaaca tta a tccaaga ga a tgtagttttgc ctca gataca tacagtgaetggea eta agaatttcatgaa ga a ccttttct tac tgagca ctaggcaaaatgta ga aggttcatatga cggggcatatgctccagta ettcatta aggtaca aggtacat agattacat tagaaaagaaacacacagctcatttetcaaaa a aggggagga aga aaa cttggaa ggcttgggaaatcaaa ccaagcaaattgtagaaaatgea N-intel Npu DnaE (seq D) Bxflag (seq E} shPolyA (seq V) 3JITR (seq H} pl389 pAAV2.1_HLP_3' F8 inteir (set 1} 5′ ITR (seq A) HLP promoter (seq J) F8 signal sequence (seq K) C-intein Npti DnaE (sequence) 3' F8: SEQ ID No. 73 tgcacca sia ggatatetctcctaatacaagccagcagaatttgtcacgcaacgtagta agagagcttgaa a caa ttcagactccca ctaga agaacagaacttga aaaa ggatattgtggatga cacctcaaccca a gtggtccaaaaacatgaccgacc aacaccac ca gca tttga tgagaaggaga a agggceattac tcagtcccttatcagattgccttacgagtca ta gcatccctca agca a atagatctccacccattgcaaggta tea tcattccatctattagacctatatatctctga cca gggtcctat Petition 870260074145, dated 24 / 07 / 2026, p. 160 / 259 149 / 192 tccaagacaa ctcttctca tt tccagcagc atctta t agaaagaa agattetgggg tccaaga aa gca gtc a tΐ tc11 a ca aggagc caaaaa aa taaccttctttagccattctaacc.ttggag atga ctggtgatca acccggc aaattca gtcacatacaaga aa gt tgsga a ca ctg ttctcccgaaaccagacttgcccaa aa ca tctggeaaa gttgaattgct tcc aaaa gtt cacattta tcaga aggacctattcccta cggaaactagcaa tgggtctcctggccatgga tggcggagt cttcagggaacagagggagcgatta ag tgga a tgaagcaaa ca ga cctggaaaagttccctttctgagagt agcaacagaaa get etgeaaa ga ctccctcca agctattgEatcctrttgcttgggataaccactatggt actcagata cca aaa gaagagtggaaatccactc accagattac a aaagga tacca ttttgtccctga a cgcttgtga a agcaatcatgca at agca gcaata a atgagEgaca aaataagcccga a tagaagtcacctEEgca aagessEEtaBEsetga a aggctgtgctctcaaac ccaccagtcttga aa cgcca teaaeggga aattaactaactaactact gacagat gagatate ct atgatgataccata tcagttgaaatga agaagga agattttga catttatga tgagga tga a aatcagagcccccgcagctttca aa agaaa a ca egaca ctattt tat tgetgcagtgga gaggctctggga ttatgggatga gtagctcccca ca tgttcta a gaaaacagggctca gag tggca gt gtccctcagttca agaaagttgttttcca ggaattta etga tggctccttta ctcagcccttataccgtggagaactaaatg aacatttg c agaggt taggtactagca agaca gataatatcatcatggtaactttca gaaatcaggcctcgtcccta ttccttcta ttctagccttatttctta tgagga a gatcagaggca aggagcaga a cctaga a aaaactttgtcaagcctaatga a acca aa aetta cttttggaaagtgcaaca tcatatggca cccact accta accta ggat aggca tttctctga tgttgacctggaaa aaga tgtgcactcaggcctgattgga ccccttctggtctgccacacta a ca ca ctgaaccctgctcatggEa gaca agtga cagta c agga a tttgctetgtt tttca ccatctttgatga ga cca aa agctggta ettea etgaa tgaa aa aa aa ctgcagggctccctg caatatccag^tggaega tccca cttttaaagaga attatcgcttcca tgea atcaatggcta ca ta atggatacact acctggctta gta atggctcagga tea a aggattcgatggta tc tgctcagcatgggcagcaatga aacat tccattctattca tcaggca tca ctgtàcgaaa aaa agaggagtataiaa tggCactgtacaa tctctStCcaggtgtttttgaga cagtggSa atgttaccatC caaagctgga atttggcgggtgga atgccttattggcgagcatcta ca tgctggga tgagcacactttttctggtgtacagcaata ag tgtcagactcccctgggaatggcttctggacacBtta gagattttcaga 11a ca gettcaggaca a ta tggacagtgggcccca agettcat tcca aggatc aggatc tgcctgg agcaccaagga gccctttcttggatca aggtggatctgttggc accaatg attattcacggc atcaaga cccagggtgcccgtcagaagttctccagcctctacatetctcagttatcatca tgtatagctttgatggg aagaa gtggeagactta teg agga aa ttccattactctt tctggga ta aa ae a ca a ta ttttaaccctccaattattECtcgatacatccgtttgcacccaactcattatagcattcgcactcttcgcatggagttgatgggctg tga tttaaata gttgcagcatgcca ttggga a tggagagta a agcaata tea ga tgcacagctct ctta atcttaccat ttgccacctggtctccttcaa a agctcgacttcacc tccaagggaggagtaa tgcctgga gacctcaggtgaa taatccaaaa gagtggetgea a gtggacttccaga aga ca atgaa a gtcacaggagta actactcaggga gtaaaatctctgcttaccagc atgta tc tggccatcagtggactctct ttttcagaatggca a agtaaaggttttca gggaa Petition 870260074145, on 07 / 24 / 2026, page. 161 / 259 150 / 192 atca aga ctccttcaea cctgtggtgaactctet agacccaccgttac tgaetcgct a ccttcga a ttcacccccaga gttggg tgcac ca ga ttgecctgagga tggaggtttctggggc t gcga ggca caggacctetac Bxfiag (seq E| SÍiPolyA (seq V) 3' ITR (seq Hjpl207 pAAV2.1HLP5lF8 intein (set 2) 5' ITR (seq A) HLP promoter (seq J) F8 signal sequence (seq K) 5' F8 (set 2): SEQ ID no. 74 gcca ccagaagatactacctgggtgcagtgga a ctgtca tggga ctat atgc aa agtgatctcggtgagctgcc tgtggacgc aag atttcc tectaga gtgccaaaatcttttcc attcaaca cctcagtcgtgta ca a aa aga ctctgtttgt aga tcatttt tcattc a cc a a catcgctaagccaaggcca ccctggatgggtctgtaggtcctaccatcca ggctgaggtttatga tacagtggtca tta ca ctt aagaacatggctcccatcctgtcagtcttcatgctgttggtgtattctactggaaagcttgagggagctgaatgatgatcagac cagtea aaga aaga aaga tgataaagtcttccctggtggaagccataea ta tgtctggcaggtcctgaaagagaatggtccaa tggcctctgacceactgtgcct tacetactea ta tctttctca tgtgga cctggtaa a aga cttga a «ca ggcctcattggagccctae tagtatgtaga gacca gggagg aggac aggaca cacaca aa atttatactacuttgctgta tttga tgaaggga aa agttggc a ctca ga aa ca aa gaactccttgatgcagga taggga tgctgca tctgctcgggcctggcctaaaatgcacacagtc a atggttatgts aa ca ggtctctgccaggtctgattggatgcCaggac aatcagtctattggcatgtgattggaatgggcaccactc ctga agtgcactca atattcctcga a ggtcacacatttcttgtga gga accatcgccaggcgtccttggaaatctcgccaataaetttc ctta ctgctca aa cactcttgatggaccttggacagtttct3ctgttttgtta ta tCtcttCccacca a catga tggca tgga agdtatg tea a agtagacagctgt a ccagagga a cccca a eta ega at gagga aaa agagaa aaact atgatgatgatettaetga t tetgaaatggatgtggtca ggt ttgatgatgecaactctcctt ccttta t ccaaattcgctcagt tgccaaga ageatccta aaa ettg ggtaeatt aea ttgctgc tga agaggagga ctggga ctatgctcccttagtcctqgccccgatga agacaatc agta catgaat tggccctca gegga ttggtaggaagtacaa aaa agteega tttatggcatac a ca gatgaaaccttta a gactcgtga a gctattcagcatga atcagga atcttgggacctttactttatgggga agttggagacaca ctgttgattatttaagaatcaagcaa Petition 870260074145, of 24 / 07 / 2026, p. 162 / 259 151 / 192 gcagacca ta ta a ca tctaccctcacggaatca ctga tgtccgtcctt tgtattcaagga ga ttacca aaa ggtgtaaa a catttgaa ggattttccaattctgccagga ga a atattc aataataaatgga ca gtgac tgtaga a gatgggccacta tgagtcct tgagcct cccgctatta ctttcgttaata tggagagagatcta gctt caggactcattggtcctctcctca tctgcta ca a aga a tc tgtagatca aa ga gg saacca gataatgtcagacaaga ggaatg tcatcagttttctgta t ttgatga ga a ccga a gct caggct cagaccta ctatacactcact tccactt gctggagtgcagcttga gga tcca gagt tcca agcctccaacatcatgca cag catca atggctatgtttttgatagtttgcagttgtcagtttgtttgcatgaggtggcatactggta cattcta agca ttggagcacagac tgacttccttctgtctctctctctggatatacc atcacac ttctca ggaga aactgtcttcatgtcgatgga a aacccaggtcta tggattctggggtgc ca ca a ctca ga ctttcgga acagaggca tgaccgcctta ctgaaggtttcta gttgtgacaagaa cactggtgattatta cgagga ca gtta tga agatatttcagca tacttgca acctaga atgta attgaaccaagaagcttctcccagaattcaagaeacccta gca ctaggcaaaagca atttaatgccaccaca attcca gaaaatgacataga ga a gactga cccttggt ttgcacacagaacacctagcct aaaa tacaaaatgtctccctag tga ttgttgatgc tcttgcgacagtcctactca catgggctatccttatctga tctccaga a gccaaata tgagacttcagttga cgatga tg acagcctgtctga a atgacaca cttcaggcca ca gctccacacagtggggacatggtattt accctg agtcaggcctcca attagatta aa tgagaactgggga caactgca gcacagttgagasacttgattca a gttctagta catca aa taatctcgattgattcataca taggcaatccattca caagttcctta ggaccccaagtagcc agttcattatga tagtc aattagataccactctatttggca aaa agtcactccctta ctgagtctgtggacctgagcttga gtga agaaataatga tt ca a agttgtta gatcaggtttaatga a ta gccaga a aa attgtca N-intein Npu DnaE (sequence D) 3xflag (seq E) shPolyA (seq V} 3′ ITR (seq H) pl388 pAAV2.1mHLP_3′ F8 intein (set 2) 5′ ITR (seq A) HLP promoter (seq J) F8 signal sequence (seq K) C-intein Npu DnaE (sequence I) 3' FS: SEQ ID No. 75 Petition 870260074145, dated 07 / 24 / 2026, pp. 163 / 259 152 / 192 tcgtcaacagagagtggtaggttatttaaÊggaaaaE3gCtcâtggacctgCtttgttgactaBaÊâtaatgccttattcaaagtta gcatct ctttgtta a aga caa a caa aa cttccaataa ttcagcaa eta a ta gacatta agactcatta ttgacatta tagtceateagtetggcaaaata ta ttagaaagtga ca etga gtttaaaaaagtga ca cctttga tie atgacagaatgctta tg gaca aa aalgctacagctttgaggcta a atcatatgtcaaataaa a eta cttcatcaaaaacatgga a atggtcc aacacacagaccaccacacacaacaa atccaga tatgtcgttcttta a gatgetattettgeeaga atcagcaaggtggataca aagg actca tggaaaga a ctctctga a ctctgggca aggc cceagt cca aa gcaa tt aglatccttaggaccagaa a a a tctgtgg a aggteaga a tttcttctgagaaa cagagat aggta aggta caggaatta g aa gga cgtagg actea aagagatggtt tttcc a agcagcagaaacctatttcttactaacttgga taatttacatgaaaata ata ca ca a tcaaga aa a aa attcaggaa gaaatagaaaagaagga aa catt aatcca agagaatgta gttttgcctcagatacagtgagta a catgaatta gaatttac ctgagcaeta ggca aa atg tagaaggttca ta tga cggggcatatgctccagtacttcaa ga ftttaggtea tta a atgattcaaaataga a caa aga aa cacacagctc a tttctcaaaaaaggggaggaaga a a a a ettgga aggcttggga aa tc as acca agca aa ttgtagagaa atatgcatgcacca ca aggatatctccta atacaagcca gca gaattttgtagtt agacacagatt agacagett agacagett ttcagactcccactaga a ga aa ca ga a ettgaaaaaaggata a ttgtggatga ca cctcaacccagt gg tccaaaaaca tgaaacatttga ccccgageaccctca ca caga taga eta ca a tgagaaggaga aa ggggcca ttactcagtc tcccttatcagattgccttacgaggagtcatagcatccctcaagcBBatagatctccattacccattgcaaaggtatcatcatttccat et attagaectatata tetga ccagggtccta tteca a ga ca a ctcttctca tetteca gcagcatctta tagaaagaaagattctggg g tee a agaaageg tea aa aaggacata acctttctttageca ttctaaecttggaga tga ctggtgatcaaa g agaggttggctccctggggacaagtgccacaaatteagtcacatacaagaaagttgaga a ca ctgttc tcccga aa ccaga ettg ccca aaa ca tetggea a agttgaattgcttccaat cattcaaa at cagaca aggacctattecctacggaaattageaatgggtct c ctggccatctggatctcgtgga a ca ga gggagegattaa gtggaatgaagcaaacagaoctggaaaagttccctttctgagagtagca aca ga a age tetgea a agactccctccaagcta ttgg atcctettgeltggga ta accactatggta ctcagataceaaaagaagagtggaaatcccaagaagaagtcaccagaaaaacagctttaagaaaaaggataccattttgtccct gaacgcttgtgaaagcaatcatgcaatagcagcaataaatgagggacaaaataagcecgaataagaagtcacctgggcaaagca agg tagga etga a aggctgtgctctcaaaaecca cca gtcttgaaacgcca tea a cgggaaataactcgtaetactcttcagtcagctaga tcaggatagattga eccagtaga aa tgaagaaggaa ga tutga cattta tg atgagga tga aaa teaga gccccgcagctttca a aa ga aaa ca ega ca ctattttangctgcagtggagaggctctggga tt atgggatga gtagctccccac atgttctaaga aa ca gggctcagagtggcagtgtccctcagttcagttcagttc ctcag ecetta ta cegtggagaactaaatga acatttgggactcctggggccatatataaga gca gaagttgaaga ta a ta testggtaact ttcagaaatcaggcctctegtccct a ttcc ttctattctageettatttctta tgaggaaga tea gaggca a ggagcagaaectagaaa aaacctaatcagtca ccaaaacttactttggaaagtgcaacatcatatggca cccac taaagatgagtttgactgca a agcctgggcttstttcttga tgttgacetgga aa aagatgtgcactcaggectgattgga ccccttctggtctgcca cacteacacac Petition 870260074145, dated 07 / 24 / 2026, pp. 164 / 259 153 / 192 tgaaccctgcK atgggagacaagtgaca giacaggaamgctctgttttcacca tctttgatgagaccaa aagctggta cttcact ga aaata tgga aaga aactgcagggctc cctgca a ta tcca ga tggaaga tcccacttttaa a gaga at tatcgc tccag tcag tca tc tc tc tgga tac acta cctggcttagta a tggct caggatca a agga tcga tggtatctgctcagca tgggcagcaa t ga aaacatcca ttcta t tcatt tcagtgga ca tgtgttca ctgtacgaaa aa aagagga gtata aaatggcactgtacaa tcttt tatttc caggtc agctggaatttggcgggtgga atgcctt attggcga gca tc tacatgctgg ga tgagca cac 1111 tctgg tgt acagca a taagtg Lcagactcccctggga atggcttctggacaca itagagattttca ga tta cag cttcagga ca atatggacagtgggccccaaagctcatc tcaatgcctggagcacca agga gccct 11 tc ctggatca aggtggatctgttggca cca a tga ttattcacggca tcaagacccagggtgcccgtcagaa gttctccagcctctac atctctcagtttatca tcatgtatagtcttgatgggaaga agtggcagactta tcgac 111 ggcaatgtgga ttcatctgggataa a acacaa ta 11111a a ccctccaa Lta t tgctcgata ca tccgtttgcaccca ac tcattatagc attcgca gcactcttcgcatggagttgatgggctglga t lta atagttgcagcatgccattgggaa tggagagt aaagca ata tcag atgcacaga t tactgc ttca tcctactttaccaa tatgtttgcca cctgg Lctcc ttca a aagctcgactcgcgt aggac cctcaggtgaataatcca a aagagtggctgcaa gtggacttcca gaaga ca atgaa^gtcac^ggagta acta ctcagggagta aa atctctgcttaragc*tgtatgtga agg3 gttcctcatctccügCagtta ag^tggccatcagtggactctcttt tttcagt a tggcagt att tcaggatt tc aagactccttca cacctgtggtgaactctctagaccca ccgtta ctgactc geta ccttcga attca cccccagagttgggtgca ccagattgccctgagga tgga ggttctgggctgtgaggca cagga ettetac 3xfiag (seq E) shPofyA(seq B) 3μITR (seqH)

[0212] The present invention will now be illustrated by means of non-limiting examples. MATERIALS AND METHODS Generation of AAV Vector Plasmids

[0213] The plasmids used for the production of the AAV vector are derived from pAAV2.1 (36) or pZac (37) plasmids containing the ITRs of AAV serotype 2. The AAV intein plasmids were designed as detailed in Figure 1A and Figure S5. The EGFP protein was cleaved at amino acid (aa) C71. The ABCA4 protein was cleaved at the large cytoplasmic domain GDI (34, 35) at aa C1150 (Set 1), aa Petition 870260074145, dated 07 / 24 / 2026, pp. 165 / 259 154 / 192 S1168 (Set 2) and aa C1090 (Set 3). While aa C1150 (Set 1) and S1168 (Set 2) are within regions that are not associated with a known ABCA4 function, C1090 is included in the ABCA4 nucleotide-binding domain that extends from aa929 to aa1148. All CEP290 splitting points fall within the coiled-coil domains (36): when CEP290 was split into two polypeptides, this occurred at either aa C1076 (Set 1) or S1275 (Set 2-3), when it was split into three polypeptides, this was at aa C929 and C1474 (Set 4) or aa S453 and C1474 (Set 5).

[0214] The inteins included in the plasmids were the DnaE intein from Nostoc punctiforme (Npu) (27, 28), or an intein composed of mutated N- and C-inteins from DnaE of Npu and Synechocystis sp. strain PCC6803 (Ssp), respectively (30), or the DnaB intein from Rhodothermus marinus (Rma) (29). The plasmids used in the study were under the control of the promoters of ubiquitous cytomegalovirus (CMV) (38) and short CMV (39) or the promoters of the human photoreceptor-specific G protein-coupled receptor kinase 1 (GRK1) 40. Plasmids encoding EGFP and CEP290 include the bovine growth hormone polyadenylation signal (bGHpA), while plasmids encoding ABCA4 include the simian virus polyadenylation signal 40 (SV40). Production and characterization of the AAV vector

[0215] The AAV vectors were produced by TIGEM AAV Vector Core by triple transfection of HEK293 cells as previously described (14, 41). No differences were observed. Petition 870260074145, dated 07 / 24 / 2026, pp. 166 / 259 155 / 192 in vector yields between AAV vectors including or excluding intein sequences. Transfection and infection of AAV cells

[0216] HEK293 cells were maintained and transfected using the calcium phosphate method (1 pg of each plasmid / well in a 6-well plate format) as previously described (14). For the experiments described in Figure S9, a plasmid quantity encoding the full-length gene corresponding to the same number of molecules contained in 1 pg of AAV intein plasmids was used. The total amount of DNA transfected in each well was kept equal by adding an encoding plasmid when necessary. HeLa cells used for experiments in Figures 2C and 2D were transfected (1 or 0.5 pg of each plasmid / well in a 24-well plate format) using Lipofectamine LTX (Invitrogen). AAV infections were performed as previously described (14). iPSCs and retinal differentiation culture

[0217] Human induced pluripotent stem cells (iPSCs) were derived from fibroblasts that were cultured from skin biopsies using methods described in (42). The STGD1 cell lines carry the heterozygous variants of the ABCA4 compound c.4892T>C and c.4539+2001G>A, also described in (43), or the heterozygous variants of the compound c.[2919-?_3328+?del;4462T>C] and c.5196+1137G>A. c.[2919-?_3328+?del;4462T>C] is an allele consisting of two variations, c.2919-?3328+? Del constitutes a deletion of exons 20, 21, and 22, as well as unknown segments of introns 19 and 22. This deletion Petition 870260074145, dated 07 / 24 / 2026, pp. 167 / 259 156 / 192 was found in a c / s configuration with c.4462T>C. The iPSCs were maintained in matrigel (#354277, Corning®). Matrigel <®> hESC-Qualified Matrix; Corning, NY) coated with 6-well plates containing mTeSR™ medium (# 85850; stem cell technologies). The cells were passed at approximately 80% confluence using 0.5 mM EDTA (# AM9260G; Ambion) for 2–6 minutes. Retinal differentiation was based on a combination of previously described protocols (44, 45). Briefly, iPSCs were seeded in 96-well V-bottom plates (9,000 cells / well) containing RevitaCell supplement (# A-2644501; Gibco, ThermoFisher) and 1% matrigel to induce aggregate formation. The aggregates were then cultured to generate 3D retinal organoids, as reported in (46). Western blot and ELISA analysis

[0218] Samples (HEK293 cells, retinas, and retinal organoids) were lysed in RIPA buffer to extract EGFP, ABCA4, and CEP290 proteins. The lysis buffers were supplemented with protease inhibitors (complete protease inhibitor cocktail tablets; Roche, Basel, Switzerland) and 1 mM phenylmethylsulfonyl. After lysis, ABCA4 samples were denatured at 37°C for 15 minutes in Laemmli sample buffer IX supplemented with 2 M of urea. The EGFP and CEP290 samples were denatured at °C for 5 minutes in Laemmli sample buffer IX. The lysates were separated by electrophoresis on 12% SDS-polyacrylamide gel (for the EGFP sample) or 6% (for the ABCA4 and CEP290 samples). The antibodies used for immunoblotting are as follows: anti-3xflag (1:1000, A8592; Petition 870260074145, dated 07 / 24 / 2026, pp. 168 / 259 157 / 192 Sigma-Aldrich, Saint Louis, MO, USA) to detect EGFP, ABCA4, and CEP290 proteins; anti-ABCA4 (1:500, LSC87292; LifeSpan BioSciences, Inc., Seattle, USA) to detect ABCA4; anti-Filamin A (1:1000, #4762; Cell Signaling Technology, Danvers, MA, USA); anti-β-Actin (1:1000, NB600-501; Novus Biological LLC, Littleton, CO, USA) to detect Filamine A, β-Actin used as a loading control in in vitro experiments; anti-Dysferlin (1:500, Dysferlin, clone Haml / 7B6, MONX10795; Tebu-bio, Le Perrayen-Yveline, France) to detect Dysferlin used as a loading control in in vivo experiments. The quantification of EGFP, ABCA4, and CEP290 bands detected by Western blot was performed using ImageJ software (free download available at http: / / rsbweb.nih.gov / ijy).

[0219] For the experiments shown in Fig. 21, retinal lysates from both Abca4 - / - mice injected with AAV intein vectors and Abca4 + / control litter mice were lysed in 30 L of lysis buffer, as described above, and 25 or 50 L of lysate, respectively, were used for Western blotting using anti-ABCA4 antibodies (LS-C87292; epitope conservation: 100% for human ABCA4; 86% for murine Abca4). The amounts of ABCA4 in retinal lysates, measured by quantifying band intensity using ImageJ software, were then normalized to the volume of retinal lysate loaded onto the acrylamide gel. For the experiments in Fig. 9, HEK293 cells were treated daily with an increased dose of trimethoprim (T7883, Sigma-Aldrich), as reported in the figure. Petition 870260074145, dated 07 / 24 / 2026, pp. 169 / 259 158 / 192

[0220] ELISA was performed on mouse and pig retinal cells or lysates using the Max Discovery Green Fluorescent Protein ELISA kit (Bioo Scientific Corporation, Austin, TX, USA). Southern blot analysis of rAAV vector DNA.

[0221] DNA was extracted from 1.5 to 6 x 10 <10> Viral particles (measured as GC). To digest unpackaged genomes, the vector solution was incubated with 30 µg of DNase (Roche) in a total volume of 300 ml, containing 50 mM Tris, pH 7.5, and 1 mM MgCl2 for 2 hours at 37°C. The DNase was then inactivated with 50 mM EDTA, followed by incubation at 50°C for 1 hour with proteinase K and a 2.5% / V-laurylsarcosyl solution to lyse the capsids. DNA was extracted twice with phenol-chloroform and precipitated with 2 volumes of 100% ethanol and 10% sodium acetate (3 M) and 1 µg of glycogen (200 g). Alkaline agarose gel electrophoresis was performed as previously described (Sambrook, J., and Russel, DW 2001. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York, USA. 999 pp). The markers were produced by double digestion of pF8-V3 with Smal to produce a 5102 bp band. A specific probe for the HLP promoter was used. Activated partial thromboplastin time (aPTT)

[0222] Nine parts of blood were collected by retro-orbital swab in one part of 0.109 M buffered trisodium citrate (BD, Franklin Lakes, NJ, USA). Blood plasma was isolated by centrifuging the samples at 13,000 rpm for 15 minutes.

[0223] aPTT was measured in Coatron M4 (Teco, Biinde, Petition 870260074145, dated 07 / 24 / 2026, pp. 170 / 259 159 / 192 Germany) using the aPTT program following the manufacturer's manual. Immunoprecipitation analysis and liquid chromatography / mass spectrometry.

[0224] Cells were plated in 100 mm plates (cells / plate lx l07) and transfected in suspension with AAV-EGFP plasmids or ABCA4 intein using the calcium phosphate method (20 pg of each plasmid / plate). Cells were harvested 72 hours after transfection and both EGFP and ABCA4 proteins were immunoprecipitated using M2 anti-flag magnetic beads (M8823; Sigma-Aldrich), according to the manufacturer's instructions. Proteins were eluted from the beads by incubation for 15 minutes in sample buffer supplemented with 4 M urea at 37°C. The proteins were then loaded onto 12% (for EGFP) or 6% (for ABCA4) SDS-polyacrylamide gel electrophoresis. Twenty-six and thirty protein bands (from HEK293 cells transfected 2 and 3 times independently with AAV-EGFP and ABCA4 intein plasmids, respectively) cut after staining with Coomassie Blue were used for protein sequencing (Creative proteomics, Shirley, NY).In summary, 3 gel sheets were used for digestion by each of the following enzymes: Trypsin, Chymotrypsin, Glu-C, Arg-C, Asp-N, and LysN. Pepsin was also used to digest ABCA4. The resulting peptides were identified and quantified using nanoscale liquid chromatography coupled with tandem mass spectrometry analysis (nano LC-MS / MS). The mass spectrometry data obtained were analyzed using PEAKS STUDIO 8.5. The inventors achieved... Petition 870260074145, dated 07 / 24 / 2026, pp. 171 / 259 160 / 192 100% protein sequence coverage for EGFP and ABCA4 proteins. Animal models

[0225] The animals were housed at the TIGEM animal facility (Naples) and kept under a 12-hour light / dark cycle.

[0226] C57BL / 6J mice were acquired from Envigo (Italy). Abca4- / - albino mice were generated by successive crosses and backcrosses with BALB / c mice (homozygous for Rpe65 Leu450) and kept inbred. BXD24 / TyJ-Cep290rd16 / J mice (referred to as rdl6) were imported from Jackson Laboratory (stock JAX # 000031). The rdl6 mouse carries an 897 bp in-frame deletion spanning exons 35-39 (46). Mice were maintained by crossing homozygous females with homozygous males. Hemophilic B6;129S-F8tmlKaz / J mice (referred to as F8tml) were imported from The Jackson Laboratory (JAX stock # 004424). The F8tml mouse has a neomycin resistance cassette that replaces 293 bp of the sequence, including 7 bp at the 3' end of exon 16 and 286 bp at the 5' end of intron 16. The mouse colony was maintained by crossing homozygous females with hemizygous males.

[0227] The Large White female sows (Azienda Agricola Pasotti, Imola, Italy) used in this study were registered as purebred in the LWHerd Book of the National Association of Pig Breeders of Italy and were housed at the Centro di Biotecnologie AORN Antonio Cardarelli (Naples, Italy) and kept under a clear cycle / Petition 870260074145, dated 07 / 24 / 2026, pp. 172 / 259 161 / 192 dark for 12 hours. Subretinal injection of AAV vectors in mice and pigs

[0228] This study was conducted in accordance with the Declaration from the Association for Research in Vision and Ophthalmology regarding the Use of Animals in Ophthalmic and Vision Research and in accordance with the regulations of the Italian Ministry of Health for procedures on animals. All procedures on rats were approved by the Italian Ministry of Health; Department of Public Health, Animal Health, Nutrition and Food Safety on March 6, 2015.

[0229] Subretinal injections in mice and pigs were performed as previously described (e.g., in 14). Mouse eyes were injected with 1 pi or 0.5 pi (for rdl6 pups) of vector solution. AAV2 / 8 doses varied in different mouse experiments, as described in the Results section. Pig eyes were injected with 2 adjacent subretinal blebs of 100 ml of AAV2 / 8 vector solution. The AAV2 / 8 dose was 2 x 1011GC per vector / eye, therefore, co-injection of two AAV vectors resulted in a total dose of 4 x 10 GC / Eye. Histology, Light Microscopy and Fluorescence

[0230] To evaluate EGFP expression in histological sections, retinal organoids, C57BL / 6J mouse eyes, and Large White pig eyes were fixed and sectioned as previously described. EGFP-positive cryosections, mounted with Vectashield with DAPI (Vector Lab Inc., Peterborough, UK), were analyzed under an LSM-700 confocal microscope (Carl Zeiss, Oberkochen, Germany) using excitation Petition 870260074145, dated 07 / 24 / 2026, pp. 173 / 259 162 / 192 and appropriate detection settings acquired at 40x magnification. Due to the prevalence of red-green color blindness, to avoid the presence of red and green together, the colors in the original images were modified in Fig. 14.

[0231] To evaluate the thickness of the outer nuclear layer in rdl6 mice injected with AAV intein vectors. CEP290, the eyes were fixed in 4% paraformaldehyde (PFA) overnight, followed by dehydration in serial ethanols and then embedded in paraffin blocks. Serial sections of rdl6 mice (22h) were cut along the horizontal meridian, progressively distributed onto slides, and stained with hematoxylin and eosin (H&E). The sections were then analyzed under a microscope (Leica Microsystems GmbH; DM5000) and acquired at 20x magnification. For each eye, an image of the temporal side injected from a slice in the central region of the eye was used for analysis. Three ONL thickness measurements were made on each image by an operator masked to the genotype / treatment group using the freehand line tool of ImageJ software. Immunofluorescence analysis

[0232] HeLa cells transfected with ABCA4 intein plasmids or CEP290 AAV were fixed 24 hours after transfection in 4% PFA for 10 minutes. The cells were blocked in blocking buffer (0.05% saponin, 0.5% BSA, 50 mM NH4Cl, 0.02% NaI in PBS, pH 7.2) for 30 minutes and then incubated as follows: - for 1 hour with anti-FLAG M2 antibody (F1804, Sigma) Petition 870260074145, dated 07 / 24 / 2026, pp. 174 / 259 163 / 192 Aldrich) to detect ABCA4 proteins; with anti-VAP-B antibody [produced in the Antonella De Matteis laboratory ((47)], to stain the endoplasmic reticulum and with TGN46 (AHP-499, Serotech) to stain the Trans-Golgi network. After washing in PBS, the cells were incubated with secondary antibodies for 30 min: goat anti-mouse Alexa Fluor 568; goat anti-rabbit Alexa Fluor 488, donkey anti-sheep Alexa Fluor 633 directed against anti-FLAG, -VAP-B and -TGN46 antibodies, respectively. - overnight with anti-FLAG antibody (F7425, Sigma-Aldrich) to detect CEP290 proteins and with anti-acetylated tubulin antibody (T6793, Sigma-Aldrich) to stain microtubules. After washing in PBS, the cells were incubated with appropriate secondary antibodies for one hour: goat anti-rabbit Alexa Fluor 594 and Alexa Fluor 488 donkey anti-mouse, directed against the anti-FLAG and -Ac-Tubulin antibodies, respectively.

[0233] The nuclei were stained with DAPI. Due to the prevalence of red-green color blindness, to avoid the presence of red and green together, the colors of the original images were modified in Fig. 2 CD and Fig. 18.

[0234] The antibodies used for immunofluorescence of human retinal organoids are as follows: anti-human chone arrestin (CAR) (50, 51) (1:10000, 'Luminaire founders' hCAR; gift of Dr. Cheryl M. Craft, Doheny Eye Institute, Los Angeles, CA, USA); anti-Opsin, Red / Green (1:200, AB5405; Merck Millipore, Darmstadt, Germany); anti-Recoverin (1:500, AB5585; Merck Millipore); anti-CRX (A-9, 1:250, sc377138; Santa Cruz Biotechnology, Dallas, Petition 870260074145, dated 07 / 24 / 2026, pp. 175 / 259 164 / 192 Texas, USA); anti-rhodopsin (1D4, 1:200, ab5417, Abeam, Cambridge, MA, USA. Transmission electron microscopy and scanning electron microscopy analyses

[0235] For electron microscopy (EM) analyses, Abca4- / - mice 3 months after subretinal injection of AAV were dark-adapted overnight, and then the eyes were harvested. The eyes were fixed in 0.2% glutaraldehyde (GA)-2% PFA in 0.1 M PHEM buffer pH 6.9 for 18 hours and then rinsed in 0.1 M PHEM buffer. The eyes were then dissected under a light microscope to select the injected temporal area of ​​the eyepieces. This portion of the eyepieces was subsequently embedded in 12% gelatin infused with 2.3 M sucrose. Cryosections (60 nm) were frozen in liquid nitrogen and sectioned using a Leica Ultramicrotome EM FC7 (Leica Microsystems).To avoid bias in assigning data to the various experimental groups, measurements of the area occupied by lipofuscin granules in the retinal pigment epithelium were performed by an operator masked to the genotype / treatment group using iTEM software (Olympus SYS, Hamburg, Germany). The area of ​​each lipofuscin granule in each field was measured in at least 20 different images (areas of 25 pm2) using the 'Free hand polygon' tool of the iTEM software. For scanning electron microscopy (SEM) analysis, retinal organoids were fixed in GA, stained with Os04, dehydrated in ethanol, and dried using a critical point drying procedure. The dried specimens were then mounted on a SEM specimen outline and coated with a... Petition 870260074145, dated 07 / 24 / 2026, pp. 176 / 259 165 / 192 thin layer of gold. The three-dimensional organization of the sample surfaces was analyzed and images were acquired using a JEOL 6700F scanning electron microscope (JEOL Ltd., Tokyo, Japan).

[0236] For ultrastructure analysis, retinal organoids were fixed overnight with a mixture of 2% PFA and 1% GA in 0.2 M PHEM buffer, pH 7.3. After fixation, the specimens were subsequently fixed as previously described. They were then dehydrated, embedded in epoxy resin, and polymerized at 60°C for 72 hours. Thin serial sections of 60 nm were cut using a Leica EM UC7 microtome.

[0237] EM images were acquired using a FEI Tecnai-12 electron microscope equipped with a VELETTA CCD digital camera (FEI, Eindhoven, Netherlands). Electrophysiological recordings and spectral-domain optical coherence tomography

[0238] Functional and morphological analyses were performed as described above (14). Pupillary light response

[0239] Pupillary light responses of rdl6 mice were recorded in the dark using the TRC-50IX retinal camera connected to a NikonDlH digital camera with a charge-coupled device (Topcon Biomedical Systems, Oakland, NJ). Mice were exposed to 10 lux light stimuli for approximately 10 seconds, and one image per eye was acquired using IMAGEnet software (Topcon Biomedical Systems). For each eye, pupil diameter was normalized to eye diameter (from the temporal side to Petition 870260074145, dated 07 / 24 / 2026, pp. 177 / 259 166 / 192 (nasal). Statistical analysis

[0240] One-way ANOVA (parametric test) or Kruskal-Wallis rank-sum test (non-parametric test) were performed to determine whether there were statistically significant differences between two or more groups of an independent variable in a dependent variable. The P-values ​​are as follows: ELISA assay for EGFP protein quantification in vitro (Kruskal-Wallis p = 0.006036), in mouse retina (ANOVA p = 0.00585) and in pig retina (Kruskal-Wallis p = 0.009005); Figure 5A (p ANOVA = 0.00585); Figure 5B (p Kruskal-Wallis = 5.547E-5); Figure 5C (p ANOVA = 5.81E-10); ERG analyses (p ANOVA or p Kruskal-Wallis > 0.05 in all luminances analyzed for the amplitudes of the a and b waves); OCT analysis in Fig. 14 (p ANOVA = 0.52 for ABCA4 and p ANOVA = 0.965 for CEP290). The statistically significant differences between the groups determined with multiple pairwise comparisons between the group means are as follows: ELISA assay for quantification of EGFP protein in vitro (single AAV versus double AAV = 0.012; AAV intein versus double AAV = 0.012; single AAV versus AAV intein = 0.222), in mouse retina (single AAV versus double AAV = 0.0044; AAV intein versus double AAV = 0.3754; single AAV versus AAV intein = 0.0561) and in pig retina (single AAV versus double AAV = 0.012; Figure 5A: + / + versus - / - AAV inteina = 0.4530; + / + versus - / - = 0.0002; Figure 5B: wild type versus rdl6 AAV inteina = 0.00131; Figure 5C: type Petition 870260074145, dated 07 / 24 / 2026, pp. 178 / 259 167 / 192 wild type versus rdl6 AAV internal IE-07; wild type versus rdl6 negative <IE-06. EXAMPLES Example 1. AAV-EGFP protein reconstitutes the full-length protein in vitro.

[0241] The present inventors tested the efficiency of intein-mediated protein trans-splicing in the retina; two AAV vectors were generated, each encoding the N- or C-terminal half of the EGFP reporter protein fused to the N- and C-terminal halves of the Nostoc punctiform split-intein DnaE [Npu Fig. 1A], respectively. The EGFP protein was spliced ​​at amino acid (aa) C71. Each AAV vector included appropriate regulatory elements (i.e., the bovine growth hormone (bGHpA) polyadenylation promoter and signal) and a triple marker (3xflag) to allow detection of both halves as well as the reconstituted full-length EGFP protein (Fig. 1A).

[0242] AAV-EGFP DnaE intein plasmids were used to transfect human embryonic kidney cells 293 (HEK293) and evaluate the production of single N- and C-terminal halves, as well as full-length EGFP protein. EGFP fluorescence, comparable to that observed in cells transfected with a single AAV plasmid encoding full-length EGFP, was detected in cells co-transfected with AAV-EGFP intein plasmids, but not with the single AAV-EGFP N- and C-terminal intein plasmids, as shown in Fig. 12. The presence of transdivided EGFP protein of the expected size (~28 kDa) along with DnaE intein (~17 kDa) split from the mature protein was confirmed. Petition 870260074145, dated 07 / 24 / 2026, pp. 179 / 259 168 / 192 by Western blot (WB) analysis of HEK293 cell lysates only after co-transfection of both AAV-EGFP intein plasmids, as shown in Fig. IB. Furthermore, quantification of band intensity showed that the amounts of EGFP protein from the AAV intein plasmids were 76 ± 37% (n = 3 independent experiments) of those observed from a single AAV plasmid. To define the accuracy of protein reconstitution, EGFP was immunopurified from HEK293 cells transfected with the AAV-EGFP intein plasmids and liquid chromatography-mass spectrometry (LC-MS) analysis was performed to define its protein sequence. The 3539 peptides obtained from the proteolytic digestion of this sample, 7 of which included the split point (Table 5), covered the entire protein and confirmed that the amino acid sequence of EGFP reconstituted by AAV plasmid-containing proteins corresponds precisely to that of wild-type EGFP. Table 5. Peptides that include the EGFP cleavage point. 0 = Cysteine ​​71 Peptide sequence Length GVQÇFSR SEQ ID No. 76 7 LPVPWPTLmLTYGVQÇFSRY SEQ ID No. 77 22 PTLVTTLTYGVQÇFSR SEQ ID No. 78 16 Petition 870260074145, dated 07 / 24 / 2026, pp. 180 / 259 169 / 192 TYGVQ£FSR SEQ ID No. 79 9 YGVQ£FSR SEQID No. 80 8 VQÇFSR SEQID No. 81 6 Q£FSR SEQID No. 82 5 Example 2. AAV-EGFP internals are more efficient than dual AAV vectors in vitro.

[0243] To confirm the reconstitution of the EGFP protein from the AAV intein vectors, HEK293 cells were infected with AAV2 / 2-CMV-EGFP DnaE intein or with single and double AAV vectors that included the same expression cassette. Multiplicity of infection (moi), 5 x 104 genome copies (GC) / cell of each vector, meaning a similar dose among the 3 systems, assuming that the double vectors undergo complete recombination of DNA or protein. In order to precisely quantify the amounts of EGFP, cell lysates were harvested seventy-two hours after infection. EGFP expression was evaluated by WB and enzyme-linked immunosorbent assay (ELISA): EGFP expression obtained with AAV intein vectors was approximately half that achieved with a single AAV (single AAV = 0.735 ± 0.2 ng EGFP / pg total lysate, n = 5 independent experiments; AAV intein = 0.403 ± 0.04 ng EGFP / pg total lysate, n = 5). Petition 870260074145, dated 07 / 24 / 2026, pp. 181 / 259 170 / 192 independent experiments) and 10 times greater than that obtained with double AAV vectors, as shown in Fig. 1C (double AAV = 0.046 ± 0.01 ng EGFP / pg total lysate, n = 5 independent experiments). Furthermore, the intensity of full-length EGFP relative to that of excised intein was quantified by WB; its relative abundance was found to be 1:0.2 (n = 6 independent experiments, Fig. 13A). Example 3. Subretinal administration of AAV-EGFP integer vectors results in efficient reconstitution of full-length proteins in the mouse and pig retina.

[0244] To investigate whether trans-splicing mediated by AAV intein reconstitutes full-length protein expression in the retina, 4-week-old C57BL / 6J mice were injected subretinally with AAV2 / 8-CMV-EGFP Dna E intein vectors (dose of each vector / eye: 5.8 x 109GC). Eyes were harvested 1 month later and analyzed by microscopy. EGFP fluorescence was detected in all eyes in the retinal pigment epithelium and, most importantly, in the photoreceptors (Fig. ID). To compare the expression of the AAV intein transgene to that of single and double AAV in photoreceptors, AAV2 / 8 vectors encoding EGFP under the control of the photoreceptor-specific human G protein-coupled receptor kinase 1 (GRK1) promoter were subretinally injected into one-week-old 4-C57BL / 6J mice (dose of each vector / eye: 5c10 <l>9 GC). Eyes were collected 1 month after injection and analyzed by fluorescence microscopy, ELISA, or WB.

[0245] EGFP fluorescence was detected in the layer Petition 870260074145, dated 07 / 24 / 2026, pp. 182 / 259 171 / 192 of photoreceptor cells in eyes injected with all vector sets, as seen in Fig. IE. Precise quantification of EGFP protein amounts by ELISA confirmed that AAV intein reconstituted EGFP protein less efficiently than a single AAV and approximately 3 times more efficiently than double AAV (single AAV = 8.41 ± 2.48 ng EGFP / retina, n = 5 eyes; AAV intein = 3.72 ± 0.85 ng EGFP / retina, n = 7 eyes; double AAV = 1.38 ± 0.43 ng EGFP / retina, n = 7 eyes). The relative amounts of full-length EGFP to excised intein after quantification of WB band intensities were 1:3 (n = 14 eyes analyzed, Fig. 13B).

[0246] The inventors then evaluated the efficiency of AAV intein vectors in photoreceptor transduction in the porcine retina, which is an excellent preclinical model for evaluating viral vector transduction due to its size and architecture ((48). Thus, Large White pigs were injected subretinally with single and double AAV2 / 8-GRK1-EGFP vectors (dose of each vector / eye: 2 x 1011GC, delivered through two adjacent subretinal blebs). The eyes were harvested 1 month after injection and analyzed by either fluorescence microscopy, ELISA, or WB. Notably, the reconstitution of EGFP protein mediated by AAV intein in the photoreceptor cell layer was greater than that mediated by double AAV and indistinguishable from vectors. Single AAVs, as assessed by EGFP fluorescence (Fig. IF). Precise quantification of EGFP in retinal lysates confirmed that AAVs reconstitute the protein in amounts similar to those achieved with a single AAV. Petition 870260074145, dated 07 / 24 / 2026, pp. 183 / 259 172 / 192 AAV is approximately 3 times greater than those obtained with double AAV vectors (single AV AAV = 247.5 ± 45.1 ng EGFP / retina, n = 5 eyes; intein AAV = 227.0 ± 15.7 ng EGFP / retina, n = 5 eyes; double AAV = 82.3 ± 9.6 ng EGFP / retina, n = 5 eyes). The relative amount of full-length EGFP to excised intein after quantification of WB band intensities was 1:2 (n = 8 eyes, Fig. 13C). Example 4. Full-length EGFP is reconstituted by AAV-mediated protein trans-splicing in 3D human retinal organoids.

[0247] As an additional representative preclinical model of the human retina, the inventors generated 3D retinal organoids ((49, 50)) from human induced pluripotent stem cells (iPSCs). Six-month-old organoids (Fig. 14A) contained cells stained with mature photoreceptor markers, as shown in Fig. 14B; the organoids were successfully transduced by AAV2 vectors with a photoreceptor-specific promoter, namely AAV2 / 2 CMV EGFP and AAV2 / 2 IRBP DsRed vectors, as shown in Fig. 14C by fluorescence analysis. (Fig. 14D) and electron microscopies (Fig. 14E-F) show the presence of outer segment buttons of photoreceptors. Nine-month-old 3D human retinal organoids incubated for 30 days with AAV-GRK1-EGFP intein vectors (dose of each vector / organoid: 1 x 1012GC) show EGFP fluorescence (Fig. 1G). WB analysis of retinal organoid lysates (Fig.15) confirms the expression of full-length EGFP which was approximately 5 times more abundant than excised intein after band intensity quantification (n = 4. Petition 870260074145, dated 07 / 24 / 2026, pp. 184 / 259 173 / 192 organoids). Example 5. Trans-splicing mediated by the intein of large proteins (identification of ideal ABCA4 and CEP90 cleavage points is necessary for efficient AAV intein-mediated protein trans-splicing)

[0248] To test whether protein trans-splicing can be developed as a mechanism for reconstituting large therapeutic proteins, the inventors developed the AAV-ABCA4 and -CEP290 intein vectors. ABCA4 and CEP290 were split into two (AAV I, AAV II) or three (AAV I, AAV II, AAV III) fragments whose coding sequences were cloned separately into single AAV vectors, fused with the coding sequences of the split N- and C-terminal inteins as shown in Fig. 16. The AAV intein vectors included the ubiquitous short CMV [(shCMV), for all sets] or the GRK1 promoter (set 1 for ABCA4 and set 5 for CEP290).The splicing points for each protein were selected taking into account the amino acid residue requirements at the junction points for efficient protein trans-splicing 18, 51), as well as the preservation of the integrity of critical protein domains, which should favor proper folding and stability of each independent polypeptide and, therefore, of the final reconstituted protein. Additional split inteins were also considered. CEP290 sets in which the protein was split into 3 polypeptides (sets 4 and 5, Fig. 16B) were generated to allow the inclusion of the marmot hepatitis virus Post-transcriptional Regulatory Element [WPRE, (52)] to increase transgene expression. Para. Petition 870260074145, dated 07 / 24 / 2026, pp. 185 / 259 174 / 192 to avoid unwanted trans-splicing between AAV I and AAV III, which could reduce the amount of total length protein generated, sets 4 and 5 included two different split inteins at the two cleavage junctions, specifically DnaB intein from Rhodothermus marinus and any type or a mutated DnaE intein that the inventors show do not exhibit cross-reactivity (Fig. 17).

[0249] The inventors compared the ability of each set of AAV interen plasmids to reconstitute ABCA4 and CEP290 after transfection of HEK293 cells. The analysis of WB of cell lysates 72 hours after transfection showed that full-length ABCA4 and CEP290 proteins of the expected size (~250 kDa and ~290 kDa, respectively) were reconstituted from each set of AAV intein plasmids, although with variable efficiency (Fig. 2A-B). Sets 1 and 5 were considered the most efficient for the reconstitution of ABCA4 and CEP290 proteins, respectively, and therefore used in all subsequent experiments.

[0250] To determine the accuracy of protein reconstitution, the inventors immunopurified ABCA4 from HEK293 cells transfected with set 1 and performed LC-MS analysis to define its protein sequence. The 3108 peptides obtained from the proteolytic digestion of this sample, 22 of which included the split point (Table 6), covered the entire protein and confirmed that the amino acid sequence of ABCA4 reconstituted by AAV plasmids corresponds precisely to that of wild-type ABCA4. The amino acid sequence of reconstituted ABCA4 Petition 870260074145, dated 07 / 24 / 2026, pp. 186 / 259 The 175 / 192 sequence of AAV intein corresponds to that of wild-type ABCA4. Alignment was performed between the wild-type ABCA4 sequence and the peptides identified in the liquid chromatography-mass spectrometry analysis of ABCA4 reconstituted from AAV inteins. Table 6. Peptides that include the ABCA4 cleavage point. NB: C: Cysteine ​​1150 Peptide Sequence Length KNÇFGT SEQ ID No. 83 6 KNÇFGTGL(x3) SEQ ID No. 84 8 KNCFGTGLY (k2) SEQ ID No. 85 9 FLKNÇFGTGL SEQ ID No. 86 10 KNÇFGTGLYLT SEQ ID No. 87 11 KNÇFGTGLYLTL SEQ ID No. 88 12 LYCSGTPLFLKNÇ SEQ ID NO. 89 13 YCSGTPLFLKNÇF SEQ ID No. 90 13 Petition 870260074145, dated 07 / 24 / 2026, pp. 187 / 259 176 / 192 KNCFGTGLYLTLVR (x7) SEQ ID No. 91 14 KNÇFGTGLYLTLVRKM SEQ ID No. 92 16 IAíIAQGRLYCSGTPLFLKNÇFGTGLYLT SEQ ID No. 93 29 HQ RLYCSGTP LLFLKNÇFGTG LYLTLVR KM KN IQSQR SEQ ID NO. 94 36 G TP LFÍKNÇFGTGLY LTLVRKM K NI QSQRKGS EGTCSCS S SEQ ID No. 75 40

[0251] The inventors then evaluated the intracellular localization of the protein products of the different intein-containing plasmids, comparing them with the localization of the full-length protein. The full-length ABCA4 is known to localize in the endoplasmic reticulum (ER) when expressed in cultured cell lines (53, 54). The two ABCA4 polypeptides from set 1 were found to co-localize in the ER, while no colocalization was found in the Trans-Golgi network (Fig. 20). A similar localization was observed in cells co-transfected with both AAV intein plasmids, as well as in cells transfected with a plasmid encoding the full-length ABCA4 protein, thus confirming the predominant localization in the ER of ABCA4 exogenously expressed in cell lines).

[0252] Regarding CEP290, it has been reported that the full-length protein shows a mixed distribution pattern with a predominantly speckled pattern and a fibrillar pattern. Petition 870260074145, dated 07 / 24 / 2026, pp. 188 / 259 177 / 192 smaller (55). Dissection of the domains responsible for the subcellular targeting of CEP290 showed that the N-terminal domain (aa 1-362) directs the protein to vesicular structures thanks to its ability to interact with membranes, while a region near the C-terminal of CEP290, encompassing much of the myosin tail homology domain of the protein, mediates microtubule binding (aa 580-2479) and when expressed as a truncated form has a prominent fibrillar distribution coinciding with acetylated tubulin (Ac-Tub). According to Drivas et al.Immunofluorescence analysis in HeLa cells transfected with AAV I, II, or III intein plasmids individually or cotransfected with AAV I + II, AAV I + III, and AAV II + III showed that AAV I and AAV II products have a predominantly speckled pattern, while AAV III (encompassing the myosin tail homology domain of the protein) shows a fibrillar pattern and is the only one to completely co-localize to Ac-tub (Fig. 2D). Thus, AAV I + II products have a predominantly speckled pattern, while those of AAV I + III and AAV II + III have a combined fibrillar and speckled microtubule pattern. Cells cotransfected with the three AAV CEP290 intein plasmids showed a predominantly speckled signal partially aligned along the microtubules that is comparable to the signal observed in cells transfected with a plasmid encoding the full-length CEP290 protein (Fig. 2D and Fig. 18).

[0253] The present inventors then compared the amount of protein obtained with the best set of Petition 870260074145, dated 07 / 24 / 2026, pp. 189 / 259 178 / 192 AAV-ABCA4 and -CEP290 intein plasmids were compared with those obtained from a single AAV plasmid encoding the corresponding full-length protein. For this purpose, HEK293 cells were transfected with the same equimolar amounts of the single AAV intein plasmid, and 72 hours after transfection, cell lysates were analyzed by WB (Fig. 19). Quantification of band intensity showed that the expression of ABCA4 and CEP290 from AAV intein plasmids was 61 ± 4% (n = 3 independent experiments) and 58 ± 4% (n = 3 independent experiments) of that observed with the corresponding single AAV plasmids, respectively. Example 6. AAV intraocular vectors mediate the expression of large therapeutic proteins in vitro and in the retina.

[0254] The inventors compared the efficiency of large protein reconstitution mediated by AAV intein with that of dual AAV vectors both in vitro and in mouse and pig retinas. HEK293 cells were infected with dual AAV2 / 2 vectors or inteins encoding ABCA4 (set 1) or CEP290 (set 5) (moi: 5x 10 <l>4 GC / cell of each vector) and cell lysates were analyzed 72 hours later by WB. As shown in Figures 3A and 3B, the AAV-ABCA4 and -CEP290 intein vectors mediated the reconstitution of large proteins more efficiently than the double AAV vectors. As expected, in addition to full-length proteins, shorter polypeptides derived from either of the AAV intein vectors (in the case of ABCA4 and CEP290) or from trans-splicing occurring between AAV II and AAV III (in the case of CEP290) were observed (Fig. 3A and 3B). Petition 870260074145, dated 07 / 24 / 2026, pp. 190 / 259 179 / 192 3B).

[0255] In addition, 4-week-old wild-type mice were subretinally injected with AAVGRK1-ABCA4 or -CEP290 intein (sets 1 and 5, respectively) compared with double vectors (dose of each vector / eye ABCA4: 3.3 x 109GC, dose of each vector / eye CEP290: 1.1 x 109GC). Animals were sacrificed 47 weeks after injection and protein expression in retinal lysates was assessed by WB. Full-length proteins were detected in 10 / 11 (91%) of eyes injected with AAV-ABCA4 intein (Fig. 4A and 20) and in 5 / 10 (50%) of eyes injected with AAV-CEP290 intein (Fig. 4B). On the other hand, full-length protein expression was evident in 5 / 9 (56%) and in 0 / 5 eyes injected with the dual AAV vectors ABCA4 and CEP290, respectively.Similar to what was observed in vitro, polypeptides derived from single AAV integer vectors (in the case of ABCA4 and CEP290) and from transsplicing occurring between AAV II and AAV III (in the case of CEP290) were detected (Figs. 4A and 4B).

[0256] To investigate the efficiency of AAV-mediated protein reconstitution in relation to endogenous ABCA4, 1-4 month old Abca4- / - mice were injected subretinally with AAVGRK1-ABCA4 intein vectors (set 1) (dose of each ABCA4 vector / eye: 5.5 x 109GC). One month later, ABCA4 expression in retinal lysates from unaffected Abca4- / - mice injected with AAV intein was analyzed by WB using an antibody that recognizes human and murine ABCA4 (Fig. 21). AAV ABCA4 intein expression was 8.6 ± 1.3% of endogenous ABCA4. Petition 870260074145, dated 07 / 24 / 2026, pp. 191 / 259 180 / 192

[0257] To confirm the efficient reconstitution of large proteins in clinically relevant pig retinas, Large White pigs were injected subretinally with AAV2 / 8-GRK1-ABCA4 intein (set 1) or dual vectors (dose of each vector / eye: 2x 1011GC, delivered through two adjacent subretinal blebs) and protein expression after 1 month of injection was analyzed by WB. Notably, AAV intein was found to reconstitute full-length ABCA4 protein more efficiently than dual AAV vectors (Fig. 4C).

[0258] Lastly, human retinal organoids from iPSCs of healthy individuals or STGD1 patients at 121 days of culture [when photoreceptor maturation begins (20)] were infected with AAV2 / 2-GRK1-ABCA...

Claims

1. Vector system for expressing a coding sequence in a cell characterized in that the coding sequence is the coding sequence of a gene selected from the group consisting of: DMD, CFTR, F8, ATP7B, PAH, DYSF, MECP2, PKD, NPC1 and HTT, said coding sequence consisting of a first portion (CDS1), a second portion (CDS2) and, optionally, a third portion (CDS3), said vector system comprising: a) a first vector comprising: - said first portion of said coding sequence (CDS1), - a first intein nucleotide sequence encoding for an N-intein, said sequence being located at the 3' end of CDS1; (b) a second vector comprising: - the said second portion of the said coding sequence (CDS2), - a second intein nucleotide sequence encoding a C-intein, said sequence being located at the 5' end of CDS2;wherein, when the first vector and the second vector are inserted into a cell, the protein product of the coding sequence is produced by protein processing; or optionally, said vector system comprising: a') a first vector comprising: - said first portion of said coding sequence (CDS1), - a first intein nucleotide sequence encoding for a first N-intein, said sequence being located at the 3' end of CDS1; and b') a second vector comprising: - said second portion of said coding sequence (CDS2), - a second intein nucleotide sequence encoding for a first C-intein, said sequence being located at the 5' end of CDS2; - a third nucleotide sequence of intein that codes for a second N-intein, with said sequence located at the 3' end of CDS2;and c') a third vector comprising: - said third portion of said coding sequence (CDS3); - a fourth nucleotide sequence of the intein encoding a second C-intein, said sequence being located at the 5' end of CDS3, wherein the first nucleotide sequence of the intein is different from the third nucleotide sequence of the intein when present and the second nucleotide sequence of the intein is different from the fourth nucleotide sequence of the intein when present, wherein when the first vector, the second vector, and the third vector, when present, are inserted into a cell, the protein product of the coding sequence is produced by protein processing.

2. Vector system, according to claim 1, Petition 870260074145, dated 07 / 24 / 2026, pp. 206 / 259 3 / 8 characterized in that the first intein, the second intein, the third intein when present and the fourth intein when present encode for a split intein, preferably, said split intein has a maximum length of 150 amino acids, more preferably, said split intein is a DnaE or DnaB intein.

3. Vector system according to claim 1 or 2, characterized in that: - the first intein nucleotide sequence codes for an intein selected from the group consisting of: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or a variant thereof or a fragment thereof or a homolog thereof; - the second intein nucleotide sequence codes for an intein selected from the group consisting of: SEQ.ID NO: 2, 4, 6, 8, 10, 12, 14 or a variant thereof or a fragment thereof or a homolog thereof; - the third intein nucleotide sequence, when present, codes for an intein selected from the group consisting of: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or a variant thereof or a fragment thereof or a homolog thereof; - the fourth intein nucleotide sequence, when present, codes for an intein selected from the group consisting of: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or a variant thereof or a fragment thereof or a homolog thereof.

4. Vector system, according to any one of claims 1 to 3, characterized in that Petition 870260074145, dated 07 / 24 / 2026, p. 207 / 259 4 / 8 the first vector, the second vector and the third vector, when present, further comprise a promoter sequence operationally linked to the 5' end portion of said first portion of the encoding sequence (CDS1) or said second portion of the encoding sequence (CDS2) or said third portion of the encoding sequence (CDS3).

5. Vector system, according to any one of claims 1 to 4, characterized in that the first vector, the second vector and the third vector, when present, further comprise a 5'-terminal repeat nucleotide sequence (5'-TR) and a 3'-terminal repeat nucleotide sequence (3'-TR), preferably the 5'-TR is a 5'-terminal repeat nucleotide sequence (5'-ITR) inverted and the 3'-TR is a 3'-terminal repeat nucleotide sequence (3'-ITR) inverted.

6. Vector system, according to any one of claims 1 to 5, characterized in that the first vector, the second vector and the third vector, when present, further comprise a polyadenylation signal nucleotide sequence and / or in that at least one of the first vector or of the second vector or of the third vector, when present, further comprises a nucleotide sequence encoding a degradation signal.

7. Vector system according to claim 6, characterized in that the degradation signal is selected from the group consisting of CL1, PB29, SMN, CIITA, ODc, ecDHFR or a fragment thereof. Petition 870260074145, dated 24 / 07 / 2026, pp. 208 / 259 5 / 8 8. Vector system, according to any one of claims 1 to 7, characterized in that the coding sequence is split into a first portion, a second portion, and optionally a third portion, at a position consisting of a nucleophilic amino acid that does not fit into a structural domain or a functional domain of the encoded protein product, wherein the nucleophilic amino acid is selected from serine, threonine, or cysteine.

9. Vector system, according to any one of claims 1 to 8, characterized in that at least one of the first vector, the second vector and the third vector, when present, further comprises at least one enhancer or regulatory nucleotide sequence, operationally linked to the coding sequence.

10. Vector system, according to any one of claims 1 to 9, characterized in that it comprises: a) a first vector comprising in a 5'-3' direction: - a 5' inverted terminal repeat sequence (5'ITR); - a promoter sequence; - a 5' end portion of a coding sequence (CDS1), said 5' end portion being operationally linked to and under the control of said promoter; - a first nucleotide sequence intein encoding for an N-intein; and - a 3' inverted terminal repeat sequence (3'-ITR); and b) a second vector comprising in the 5'-3' direction: - a 5' inverted terminal repeat sequence (5'ITR); - a promoter sequence; - a second nucleotide sequence that codes for a C-intein; - a 3' end portion of the coding sequence (CDS2); and - a 3' inverted terminal repeat sequence (3'ITR);or optionally comprising: a') a first vector comprising in a 5'-3' direction: - a 5' inverted terminal repeat sequence (5'ITR); - a promoter sequence; - a 5' end portion of a coding sequence (CDS1'), said 5' end portion being operationally linked to and under the control of said promoter; - a first nucleotide sequence intein encoding for a first N-intein; and - a 3' inverted terminal repeat sequence (3'ITR); and b') a second vector comprising in the 5'-3' direction: - a 5' inverted terminal repeat sequence (5'ITR); - a promoter sequence; Petition 870260074145, dated 24 / 07 / 2026, p. 210 / 259 7 / 8 - a second nucleotide sequence intein encoding for a first C-intein; - a second portion of the coding sequence (CDS2'); and - a third nucleotide sequence (intein) that codes for a second N-intein;- a 3' terminal inverted repeat sequence (3'ITR); and c') a third vector comprising in the 5'-3' direction: - a 5' terminal inverted repeat sequence (5'ITR); - a promoter sequence; - a fourth nucleotide sequence of intein encoding a second C-intein; - the third portion of the coding sequence (CDS3'); and - a 3' terminal inverted repeat sequence (3'ITR).

11. Vector system, according to any one of claims 1 to 10, characterized in that said first, second and third vectors are independently a viral vector, preferably an adenoviral vector or adeno-associated viral vector (AAV), preferably said first, second and third adeno-associated viral vectors (AAVs) are selected from the same or different AAV serotypes, preferably the serotype is selected from the group consisting of serotype 2, serotype 8, serotype 5, serotype 7 or serotype 9, serotype 7m8, serotype shlO and serotype 2 (quad YF). Petition 870260074145, dated 24 / 07 / 2026, pp. 211 / 259 8 / 8 12. Microbial host cell characterized in that it is transformed with the vector system as defined in any one of claims 1 to 11.

13. Use of the vector system, as defined by any one of claims 1 to 11, or of the host cell, as defined in claim 12, characterized in that it is for the manufacture of a medicament.

14. Use of the vector system, as defined in any one of claims 1 to 11, or of the host cell, as defined in claim 12, characterized in that it is for the manufacture of a gene therapy drug, preferably in the treatment and / or prevention of a metabolic disorder, a hematological disorder, a neurodegenerative disorder, hearing loss, cannellopathy, lung disease, myopathy, or heart disease.

15. Use, in accordance with claim 14, characterized in that the medicine is for the prevention and / or treatment of Duchenne muscular dystrophy, cystic fibrosis, hemophilia A, Wilson's disease, phenylketonuria, dysferlinopathies, Rett syndrome, polycystic kidney disease, Niemann-Pique type C, or Huntington's disease.

16. Pharmaceutical composition characterized in that it comprises the vector system as defined in any one of claims 1 to 11 or the host cell as defined in claim 12 and a pharmaceutically acceptable vehicle.

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