Trans-splicing molecules

Abstract

This invention provides a nucleic acid trans-splicing molecule that can correct mutations in the ABCA4 gene or the CEP290 gene. [Solution] A nucleic acid trans-splicing molecule comprising (a) a binding domain configured to bind to a target ABCA4 intron selected from the group consisting of introns 19, 23, or 24, functionally linked in either the 3' to 5' direction or the 5' to 3' direction; (b) a splicing domain configured to mediate trans-splicing; and (c) a coding domain containing a functional ABCA4 exon. A composition comprising the nucleic acid trans-splicing molecule may be useful in treating or preventing diseases associated with mutations within ABCA4, such as Stargardt disease (e.g., Stargardt disease type 1), or diseases associated with mutations in CEP290, such as LCA (e.g., LCA10).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Applications No. 62 / 658,658 and No. 62 / 658,667, both filed on April 17, 2018. The contents of both of these Provisional Patent Applications are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing, which has been submitted electronically in ASCII format and is incorporated in its entirety by reference herein. The ASCII copy, created on 17 March 2019, is named 51219-016WO2_Sequence_Listing_04.16.19_ST25 and is 608,489 bytes in size.

[0003] Field of Invention Generally, the present invention features ABCA4 trans-splicing molecules and CEP290 trans-splicing molecules. [Background technology]

[0004] background Stargardt disease is a progressive eye disease characterized by a rapid or long-term decline in central vision and color vision. Peripheral vision generally remains unaffected. Various mutations along the full length of the ABCA4 gene can cause Stargardt disease. Treatments currently under development for Stargardt disease include lentiviral delivery of ABCA4, chemically modified vitamin A variants, and treatment of retinal pigment epithelial cells.

[0005] Leber congenital amourosis type 10 (LCA 10) is a condition characterized by severe visual impairment that begins in infancy. This vision loss is associated with the death of photoreceptors due to a defect in the ciliary body. The most common known mutation associated with LCA 10 is a point mutation in the CEP290 gene in which adenine at nucleotide 1,655 of intron 26 is replaced with guanine, resulting in a splice defect where a hidden stop codon is spliced ​​between exons 26 and 27. This autosomal recessive mutation leads to the production of a non-functional centrosome protein, causing the characteristic blindness of LCA 10.

[0006] Adeno-associated virus (AAV) vector-mediated gene therapy has a proven safety profile in humans and is a promising approach for treating a variety of genetic defects. However, AAV vectors may have packaging size constraints that can hinder the delivery of large nucleic acid molecules, such as those required to replace the ABCA4 or CEP290 genes, as determined by viral biology. Therefore, there is a need in this field for compositions and methods to correct mutations within ABCA4 and CEP290. [Overview of the project]

[0007] overview The present invention relates to nucleic acid trans-splicing molecules and methods for using them to correct mutations in the ABCA4 gene or the CEP290 gene. The compositions and methods provided herein may be useful in treating or preventing diseases associated with mutations in ABCA4, such as Stargardt disease (e.g., Stargardt disease type 1), or diseases associated with mutations in CEP290, such as LCA (e.g., LCA10).

[0008] ABCA4 In the first aspect, the present invention features an ABCA4 trans-splicing molecule. For example, the present invention provides a nucleic acid trans-splicing molecule comprising (a) a binding domain configured to bind to a target ABCA4 intron selected from the group consisting of introns 19, 22, 23, or 24, functionally linked in either the 3'-to-5' direction or the 5'-to-3' direction; (b) a splicing domain configured to mediate trans-splicing; and (c) a coding domain containing a functional ABCA4 exon, wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and an endogenous ABCA4 exon adjacent to the target ABCA4 intron, thereby replacing the endogenous ABCA4 exon with the functional ABCA4 exon and correcting a mutation within ABCA4.

[0009] In some embodiments, the binding domain binds to a target ABCA4 intron at the 3' end of the mutation, and the mutation is located in either ABCA4 exons 1-24 or introns 1-24. In some embodiments, the target ABCA4 intron is intron 19, the mutation is located in either ABCA4 exons 1-19 or introns 1-19, and the coding domain includes ABCA4 exons 1-19. In some embodiments, the binding domain is configured to bind to the intron 19 at a binding site that includes one or more nucleotides from SEQ ID NO:25 990 to 2,174 (for example, one or more nucleotides from SEQ ID NO:25 1,670 to 2,174, for example, one or more nucleotides from SEQ ID NO:25 1,810 to 2,000, for example, one or more nucleotides from SEQ ID NO:25 1,870 to 2,000, for example, one or more nucleotides from SEQ ID NO:25 1,920 to 2,000).

[0010] In some embodiments, the target ABCA4 intron is intron 23, the mutation is located in either ABCA4 exons 1-23 or introns 1-23, and / or the coding domain contains ABCA4 exons 1-23. In some embodiments, the binding domain is configured to bind to intron 23 at a binding site containing one or more nucleotides from either nucleotides 80-570 or nucleotides 720-1,081 of SEQ ID NO:29.

[0011] In some embodiments, the binding domain is a binding site containing one or more nucleotides from SEQ ID NO:29, specifically nucleotides 261-410 (for example, a binding site of 1-200, 6-150, 12-100, or 20-80 nucleotides within the range of nucleotides 261-410 of SEQ ID NO:29, or encompassing nucleotides 261-410 of SEQ ID NO:29, for example, a binding site of 1-6, 6-12, 12-18, 18-24, 24-50, 50-100, 100-150, or 150-200 nucleotides within the range of nucleotides 261-410 of SEQ ID NO:29, or encompassing nucleotides 261-410 of SEQ ID The organism is configured to bind to the ABCA4 intron 23 at a binding site of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least fifteen, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 150, or at least 200 nucleotides encompassing nucleotides 261-410 of SEQ ID NO:29. For example, in certain embodiments, the binding site contains six or more nucleotides from nucleotides 261-410 of SEQ ID NO:29. In some embodiments, the binding domain contains six or more consecutive nucleic acid residues that are complementary (e.g., antisense) to the six or more nucleotides of the binding site.In some embodiments, the binding domain comprises a set of sequential nucleic acid residues complementary to the corresponding complementary nucleotide set of the binding site of ABCA4 having one or more nucleotides from nucleotides 261 to 410 of SEQ ID NO:29, wherein the set of sequential nucleic acid residues of the binding domain has a length of 6 to 500 residues (e.g., 8 to 400, 12 to 300, 16 to 200, 24 to 280, or 50 to 150 residues, e.g., 100 to 200, 6 to 10, 10 to 20, 20 to 30, 30 to 40, 4 Lengths of 0-50, 50-80, 80-100, 100-120, 120-150, 150-200, or 200-300 residues, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 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, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 11 This is the length of 5, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 160 or more residues.

[0012] In some embodiments, the binding domain is a binding site containing one or more nucleotides from SEQ ID NO:29, specifically nucleotides 801-950 (for example, a binding site of 1-200, 6-150, 12-100, or 20-80 nucleotides within the range of nucleotides 801-950 of SEQ ID NO:29, or encompassing nucleotides 801-950 of SEQ ID NO:29, for example, a binding site of 1-6, 6-12, 12-18, 18-24, 24-50, 50-100, 100-150, or 150-200 nucleotides within the range of nucleotides 801-950 of SEQ ID NO:29, or encompassing nucleotides 801-950 of SEQ ID NO:29, for example, a binding site containing one or more nucleotides from the range of nucleotides 801-950 of SEQ ID NO:29, or SEQ ID The nucleotides are configured to bind to the ABCA4 intron 23 at binding sites of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least fifteen, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 150, or at least 200 nucleotides encompassing nucleotides 801-950 of SEQ ID NO:29. For example, in certain embodiments, the binding site contains six or more nucleotides from nucleotides 801-950 of SEQ ID NO:29. In some embodiments, the binding domain contains six or more consecutive nucleic acid residues that are complementary (e.g., antisense) to the six or more nucleotides of the binding site.In some embodiments, the binding domain comprises a set of sequential nucleic acid residues complementary to the corresponding complementary nucleotide set of the binding site of ABCA4 having one or more nucleotides from nucleotides 801 to 950 of SEQ ID NO:29, wherein the set of sequential nucleic acid residues of the binding domain has a length of 6 to 500 residues (e.g., 8 to 400, 12 to 300, 16 to 200, 24 to 280, or 50 to 150 residues, e.g., 100 to 200, 6 to 10, 10 to 20, 20 to 30, 30 to 40, 4 Lengths of 0-50, 50-80, 80-100, 100-120, 120-150, 150-200, or 200-300 residues, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 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, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 11 This is the length of 5, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 160 or more residues.

[0013] In some embodiments, the binding domain is a binding site containing one or more nucleotides from SEQ ID NO:29, specifically nucleotides 841-990 (for example, a binding site of 1-200, 6-150, 12-100, or 20-80 nucleotides within the range of nucleotides 841-990 of SEQ ID NO:29, or encompassing nucleotides 841-990 of SEQ ID NO:29, for example, a binding site of 1-6, 6-12, 12-18, 18-24, 24-50, 50-100, 100-150, or 150-200 nucleotides within the range of nucleotides 841-990 of SEQ ID NO:29, or SEQ ID The nucleotides are configured to bind to the ABCA4 intron 23 at binding sites of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least fifteen, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 150, or at least 200 nucleotides encompassing nucleotides 841-990 of SEQ ID NO:29. For example, in certain embodiments, the binding site contains six or more nucleotides from nucleotides 841-990 of SEQ ID NO:29. In some embodiments, the binding domain contains six or more consecutive nucleic acid residues that are complementary (e.g., antisense) to the six or more nucleotides of the binding site.In some embodiments, the binding domain comprises a set of sequential nucleic acid residues complementary to the corresponding complementary nucleotide set of the ABCA4 binding site having one or more nucleotides from nucleotides 841-990 of SEQ ID NO:29, wherein the set of sequential nucleic acid residues of the binding domain has a length of 6-500 residues (e.g., 8-400, 12-300, 16-200, 24-280, or 50-150 residues, e.g., 100-200, 6-10, 10-20, 20-30, 30-40, 4 Lengths of 0-50, 50-80, 80-100, 100-120, 120-150, 150-200, or 200-300 residues, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 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, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 11 This is the length of 5, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 160 or more residues.

[0014] In other embodiments, the target ABCA4 intron is intron 24, the mutation is located in either ABCA4 exons 1-24 or introns 1-24, and the coding domain contains ABCA4 exons 1-24. In some embodiments, the binding domain is configured to bind to intron 24 at a binding site containing one or more nucleotides from SEQ ID NO:30, either nucleotides 600-1,250 or nucleotides 1,490-2,660. In other embodiments, the binding site contains one or more nucleotides from SEQ ID NO:30, either nucleotides 1,000-1,200.

[0015] In some embodiments, the binding domain binds to a target ABCA4 intron at the 5' end of the mutation, and the mutation is located in either ABCA4 exons 23-50 or introns 22-49. For example, in some embodiments, the target ABCA4 intron is intron 23, the mutation is located in either ABCA4 exons 24-50 or introns 23-49, and the coding domain includes ABCA4 exons 24-50. In some embodiments, the binding domain is configured to bind to intron 23 at a binding site containing one or more nucleotides from SEQ ID NO:29 80-1081. In some embodiments, the binding site contains one or more nucleotides from SEQ ID NO:29 230-1081, for example, one or more nucleotides from SEQ ID NO:29 250-400 or one or more nucleotides from SEQ ID NO:29 690-850.

[0016] In some embodiments, the target ABCA4 intron is intron 24, the mutation is located in either ABCA4 exon 25-50 or intron 24-49, and the coding domain includes ABCA4 exon 25-50. In some embodiments, the binding domain is configured to bind to intron 24 at a binding site containing one or more nucleotides from SEQ ID NO:30, including nucleotides 1-250, 300-2,100, or 2,200-2,692. In some embodiments, the binding site contains one or more nucleotides from SEQ ID NO:30, including nucleotides 360-610. In other embodiments, the binding site contains one or more nucleotides from SEQ ID NO:30, including nucleotides 750-1,110.

[0017] In another aspect, the present invention features a nucleic acid trans-splicing molecule comprising (a) a binding domain configured to bind to ABCA4 intron 22 at a binding site containing one or more nucleotides 60-570, 600-800, or 900-1,350 of SEQ ID NO:28, functionally linked in the 5' to 3' direction; (b) a splicing domain configured to mediate trans-splicing; and (c) a coding domain containing functional ABCA4 exons 23-50, wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous ABCA4 exon 22, thereby replacing endogenous ABCA4 exons 23-50 with functional ABCA4 exons 23-50. In some embodiments, the binding site contains one or more nucleotides 70-250 of SEQ ID NO:28.

[0018] In another aspect, the present invention provides a nucleic acid trans-splicing molecule comprising a binding domain configured to bind to ABCA4 intron 22 at a binding site comprising any one or more of nucleotides 1 to 510 or 880 to 1,350 of SEQ ID NO:28, functionally linked in the 3' to 5' direction; (b) a splicing domain configured to mediate trans-splicing; and (c) a coding domain comprising functional ABCA4 exons 1 to 22, wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous ABCA4 exon 23, whereby endogenous ABCA4 exons 1 to 22 are replaced with the functional ABCA4 exons 1 to 22.

[0019] In some embodiments, the binding domain is a binding site containing one or more nucleotides from SEQ ID NO:28, specifically nucleotides 1041-1190 (for example, a binding site of 1-200, 6-150, 12-100, or 20-80 nucleotides within the range of nucleotides 1041-1190 of SEQ ID NO:28, or encompassing nucleotides 1041-1190 of SEQ ID NO:28, for example, a binding site of 1-6, 6-12, 12-18, 18-24, 24-50, 50-100, 100-150, or 150-200 nucleotides within the range of nucleotides 1041-1190 of SEQ ID NO:28, or SEQ ID The nucleotides are configured to bind to the ABCA4 intron 22 at binding sites of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least fifteen, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 150, or at least 200 nucleotides encompassing nucleotides 1041-1190 of SEQ ID NO:28. In certain embodiments, the binding site contains six or more nucleotides from nucleotides 1041-1190 of SEQ ID NO:28. In some embodiments, the binding domain contains six or more consecutive nucleic acid residues that are complementary (e.g., antisense) to the six or more nucleotides of the binding site.In some embodiments, the binding domain comprises a set of sequential nucleic acid residues complementary to the corresponding complementary nucleotide set of the binding site of ABCA4 having one or more nucleotides from nucleotides 1041 to 1190 of SEQ ID NO:28, wherein the set of sequential nucleic acid residues of the binding domain has a length of 6 to 500 residues (e.g., 8 to 400, 12 to 300, 16 to 200, 24 to 280, or 50 to 150 residues, e.g., 100 to 200, 6 to 10, 10 to 20, 20 to 30, 30 to 40, Lengths of 40-50, 50-80, 80-100, 100-120, 120-150, 150-200, or 200-300 residues, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 5 3, 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, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 11 This is the length of 5, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 160 or more residues.

[0020] In some embodiments, the binding domain is one or more of the nucleotides 1171-1320 of SEQ ID NO:28 (for example, binding sites of 1-200, 6-150, 12-100, or 20-80 nucleotides within or encompassing the range of nucleotides 1171-1320 of SEQ ID NO:28, for example, binding sites of 1-6, 6-12, 12-18, 18-24, 24-50, 50-100, 100-150, or 150-200 nucleotides within or encompassing the range of nucleotides 1171-1320 of SEQ ID NO:28, or SEQ ID The device is configured to bind to a binding site of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least fifteen, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 150, or at least 200 nucleotides comprising nucleotides 1171-1320 of SEQ ID NO:28. In certain embodiments, the binding site contains six or more nucleotides from nucleotides 1171-1320 of SEQ ID NO:28. In some embodiments, the binding domain contains six or more consecutive nucleic acid residues that are complementary (e.g., antisense) to the six or more nucleotides of the binding site.In some embodiments, the binding domain comprises a set of contiguous nucleic acid residues that is complementary to a corresponding set of complementary nucleotides of a binding site of ABCA4 having one or more of nucleotides 1171 to 1320 of SEQ ID NO:28, wherein the set of contiguous nucleic acid residues of the binding domain is of a length of 6 to 500 residues (e.g., 8 to 400, 12 to 300, 16 to 200, 24 to 280 or 50 to 150 residues in length, e.g., 100 to 200, 6 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 80, 80 to 100, 100 to 120, 120 to 150, 150 to 200 or 200 to 300 residues in length, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 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, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or more residues in length).

[0021] In some embodiments, the binding domain is one or more of the nucleotides 1201-1350 of SEQ ID NO:28 (for example, binding sites of 1-200, 6-150, 12-100, or 20-80 nucleotides within or encompassing the range of nucleotides 1201-1350 of SEQ ID NO:28, for example, binding sites of 1-6, 6-12, 12-18, 18-24, 24-50, 50-100, 100-150, or 150-200 nucleotides within or encompassing the range of nucleotides 1201-1350 of SEQ ID NO:28, or SEQ ID The device is configured to bind to a binding site of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least fifteen, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 150, or at least 200 nucleotides comprising nucleotides 1201-1350 of SEQ ID NO:28. In certain embodiments, the binding site contains six or more nucleotides from nucleotides 1201-1350 of SEQ ID NO:28. In some embodiments, the binding domain contains six or more consecutive nucleic acid residues that are complementary (e.g., antisense) to the six or more nucleotides of the binding site.In some embodiments, the binding domain comprises a set of sequential nucleic acid residues complementary to the corresponding complementary nucleotide set of the binding site of ABCA4 having one or more nucleotides from nucleotides 1201 to 1350 of SEQ ID NO:28, wherein the set of sequential nucleic acid residues of the binding domain has a length of 6 to 500 residues (e.g., 8 to 400, 12 to 300, 16 to 200, 24 to 280, or 50 to 150 residues, e.g., 100 to 200, 6 to 10, 10 to 20, 20 to 30, 30 to 40, Lengths of 40-50, 50-80, 80-100, 100-120, 120-150, 150-200, or 200-300 residues, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 5 3, 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, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 11 This is the length of 5, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 160 or more residues.

[0022] In any of the above embodiments, the binding domain may be 20 to 1,000 nucleotides long (for example, 25 to 900 nucleotides long, 30 to 800 nucleotides long, 40 to 700 nucleotides long, 50 to 600 nucleotides long, 75 to 500 nucleotides long, 100 to 400 nucleotides long, 125 to 200 nucleotides long, or about 150 nucleotides long, for example, 20 to 30 nucleotides long, 30 to 40 nucleotides long, 40 to 50 nucleotides long, 50 to 75 nucleotides long, 75 to 100 nucleotides long, 125 to 150 nucleotides long, 150 to 175 nucleotides long, 175 to 200 nucleotides long, 200 to 250 nucleotides long, 250 to 500 nucleotides long, 500 to 750 nucleotides long, or 750 to 1,000 nucleotides long).

[0023] In some embodiments, the coding domain is a cDNA sequence. In some embodiments, the coding domain contains a naturally occurring sequence. In other embodiments, the coding domain contains a codon-optimized sequence. In some embodiments, the trans-splicing molecule contains an artificial intron that includes a spacer sequence.

[0024] In some aspects of the above-described method, the nucleic acid trans-splicing molecule is 3,000 to 4,000 nucleotides long (e.g., 3,100 to 3,900 nucleotides long, 3,200 to 3,800 nucleotides long, 3,300 to 3,700 nucleotides long, 3,400 to 3,600 nucleotides long, or about 3,500 nucleotides long, e.g., 3,000 to 3,100 nucleotides long, 3,100 to 3,200 nucleotides long, 3,200 to 3,300 nucleotides long, 3,300 to 3,400 nucleotides long, 3,400 to 3,500 nucleotides long, 3,500 to 3,600 nucleotides long, 3,600 to 3,700 nucleotides long, 3,800 to 3,900 nucleotides long, or 3,900 to 4,000 nucleotides long).

[0025] In some embodiments, mutations in the ABCA4 gene are associated with Stargardt disease. In some embodiments, mutations in the ABCA4 gene associated with Stargardt disease are expressed in photoreceptor cells.

[0026] In another aspect, the Specified Information provides a nucleic acid trans-splicing molecule comprising: (a) a binding domain configured to bind to ABCA4 intron 23 at a binding site comprising six or more nucleotides from SEQ ID NO: 29 nucleotides 261-410, functionally linked in the 3' to 5' direction, and comprising six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) an artificial intron comprising a splicing domain; and (c) a coding domain comprising functional ABCA4 exons 1-23, wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain with endogenous ABCA4 exon 24, thereby replacing endogenous ABCA4 exons 1-23 with functional ABCA4 exons 1-23.

[0027] In another aspect, the present invention provides a nucleic acid trans-splicing molecule comprising: (a) a binding domain configured to bind to ABCA4 intron 23 at a binding site containing 6 or more nucleotides from nucleotides 801-950 of SEQ ID NO:29, functionally linked in the 3' to 5' direction, and containing 6 or more consecutive nucleic acid residues complementary to the 6 or more nucleotides at the binding site; (b) an artificial intron containing a splicing domain; and (c) a coding domain containing functional ABCA4 exons 1-23, wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous ABCA4 exon 24, thereby replacing endogenous ABCA4 exons 1-23 with functional ABCA4 exons 1-23.

[0028] In another aspect, the Specified Information provides a nucleic acid trans-splicing molecule comprising: (a) a binding domain configured to bind to ABCA4 intron 23 at a binding site comprising six or more nucleotides from SEQ ID NO: 29 nucleotides 841-990, functionally linked in the 3' to 5' direction, and comprising six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) an artificial intron comprising a splicing domain; and (c) a coding domain comprising functional ABCA4 exons 1-23, wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain with endogenous ABCA4 exon 24, thereby replacing endogenous ABCA4 exons 1-23 with functional ABCA4 exons 1-23.

[0029] In another aspect, the present invention provides a nucleic acid trans-splicing molecule comprising: (a) a binding domain configured to bind to ABCA4 intron 22 at a binding site comprising 6 or more nucleotides from nucleotides 1041 to 1190 of SEQ ID NO: 28, functionally linked in the 3' to 5' direction, and comprising 6 or more consecutive nucleic acid residues complementary to the 6 or more nucleotides at the binding site; (b) an artificial intron comprising a splicing domain; and (c) a coding domain comprising functional ABCA4 exons 1 to 22, wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous ABCA4 exon 23, thereby replacing endogenous ABCA4 exons 1 to 22 with functional ABCA4 exons 1 to 22.

[0030] In another aspect, the present invention features a nucleic acid trans-splicing molecule comprising: (a) a binding domain configured to bind to ABCA4 intron 22 at a binding site comprising six or more nucleotides from SEQ ID NO: 28 nucleotides 1171-1320, functionally linked in the 3' to 5' direction, and comprising six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) an artificial intron comprising a splicing domain; and (c) a coding domain comprising functional ABCA4 exons 1-22, wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain with endogenous ABCA4 exon 23, thereby replacing endogenous ABCA4 exons 1-22 with functional ABCA4 exons 1-22.

[0031] In another aspect, this specification provides a nucleic acid trans-splicing molecule comprising: (a) a binding domain configured to bind to ABCA4 intron 22 at a binding site comprising six or more nucleotides from SEQ ID NO: 28 nucleotides 1201-1350, functionally linked in the 3' to 5' direction, and comprising six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) an artificial intron comprising a splicing domain; and (c) a coding domain comprising functional ABCA4 exons 1-22, wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain with endogenous ABCA4 exon 23, thereby replacing endogenous ABCA4 exons 1-22 with functional ABCA4 exons 1-22.

[0032] In another aspect, the present invention features a proviral plasmid comprising a nucleic acid trans-splicing molecule in any of the above embodiments.

[0033] In another aspect, the present invention features an adeno-associated virus (AAV) comprising any of the nucleic acid molecules described above. In some embodiments, the AAV preferentially targets photoreceptor cells. In some embodiments, the AAV comprises AAV5 capsid protein, AAV8 capsid protein, AAV8(b) capsid protein, or AAV9 capsid protein.

[0034] In another aspect, the present invention features a pharmaceutical composition comprising a nucleic acid trans-splicing molecule, a proviral plasmid, or an AAV in any of the aforementioned aspects.

[0035] In another aspect, this specification provides a pharmaceutical composition having any 5' nucleic acid trans-splicing molecule of any embodiment described above and any 3' nucleic acid trans-splicing molecule of any embodiment described above.

[0036] In another aspect, the present invention features a method for correcting mutations in the ABCA4 gene within target cells by administering the aforementioned pharmaceutical composition to the target.

[0037] In another aspect, this specification provides a method for correcting a mutation in one or more of the ABCA4 exons 1 to 24 in a subject requiring such correction by administering a pharmaceutical composition having the nucleic acid trans-splicing molecule of any of the above embodiments to the subject. In a particular embodiment, the mutant ABCA4 exon corrected by the ABCA4 trans-splicing molecule of the present invention is exon 2. Additionally or alternatively, the mutant ABCA4 exon corrected by the ABCA4 trans-splicing molecule of the present invention is exon 3. Additionally or alternatively, the mutant ABCA4 exon corrected by the ABCA4 trans-splicing molecule of the present invention is exon 4.

[0038] In another aspect, the present invention includes a method for correcting mutations in one or more of the ABCA4 exons 23-50 in a subject requiring such correction by administering a pharmaceutical composition comprising a nucleic acid trans-splicing molecule of any of the above embodiments to the subject.

[0039] In another aspect, the present invention features a method for correcting a mutation in any one of ABCA4 exons 1 to 24 and a second mutation in any one of exons 23 to 50 in a subject requiring such correction, comprising the step of administering a pharmaceutical composition having a 5' nucleic acid trans-splicing molecule of any of the above embodiments and a 3' nucleic acid trans-splicing molecule of any of the above embodiments to a subject.

[0040] In another embodiment, the present invention features a method for treating a subject having a disorder associated with a mutation in ABCA4, comprising the step of administering the subject any of the above-mentioned pharmaceutical compositions. In some embodiments, a subject having a disorder associated with a mutation in any one or more of ABCA4 exons 1-24 or introns 1-24 is treated by administering a pharmaceutical composition comprising the nucleic acid trans-splicing molecule of any of the above embodiments. In some embodiments, a subject having a disorder associated with a mutation in any one or more of ABCA4 exons 23-50 or introns 22-49 is treated by administering a pharmaceutical composition comprising the nucleic acid trans-splicing molecule of any of the above embodiments.

[0041] In another aspect, the present invention features a method for treating a subject having a disorder associated with a first mutation in any one of ABCA4 exons 1 to 24 and a second mutation in any one of exons 23 to 50 by administering a pharmaceutical composition having a 5' nucleic acid trans-splicing molecule of any of the above embodiments and a 3' nucleic acid trans-splicing molecule of any of the above embodiments to the subject.

[0042] In any of the above methods, the subject may have Stargardt disease. In some embodiments, the composition is administered by subretinal injection, intravitreous injection, or intravenous injection.

[0043] In some aspects of the above-described method, the subject exhibits an increase of at least 1% in ABCA4 protein expression after administration (for example, an increase of 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-50%, or 50-100% in ABCA4 protein expression after administration compared to, for example, ABCA4 protein expression in the same subject before administration, or compared to a reference sample, reference subject, or reference group).

[0044] CEP290 In another aspect, the present invention features a CEP290 trans-splicing molecule. For example, the present invention provides a nucleic acid trans-splicing molecule comprising (a) a binding domain configured to bind to a CEP290 intron 26 at a binding site containing one or more nucleotides from SEQ ID NO: 85, any one of 4,800 to 5,838, functionally linked in the 3' to 5' direction; (b) a splicing domain configured to mediate trans-splicing; and (c) a coding domain containing functional CEP290 exons 2 to 26, wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain with endogenous CEP290 exon 27, thereby replacing endogenous CEP290 exons 2 to 26 with functional CEP290 exons 2 to 26, thereby correcting a pathogenic point mutation. In some embodiments, the pathogenic point mutation is a mutation from A to G at nucleotide 1,655 of SEQ ID NO:85.

[0045] In some embodiments, the binding site contains one or more nucleotides from SEQ ID NO: 85, specifically nucleotides 4,980 to 5,838. In some embodiments, the binding site contains one or more nucleotides from SEQ ID NO: 85, specifically nucleotides 5,348 to 5,838. In some embodiments, the binding site contains one or more nucleotides from SEQ ID NO: 85, specifically nucleotides 5,348 to 5,700. In some embodiments, the binding site contains one or more nucleotides from SEQ ID NO: 85, specifically nucleotides 5,400 to 5,600. In some embodiments, the binding site contains one or more nucleotides from SEQ ID NO: 85, specifically nucleotides 5,460 to 5,560. In some embodiments, the binding site contains nucleotide 5,500.

[0046] In another aspect, the present invention features a nucleic acid trans-splicing molecule comprising (a) a binding domain configured to bind to CEP290 in any one of target introns 27, 28, 29, or 30, functionally linked in the 3' to 5' direction; (b) a splicing domain configured to mediate trans-splicing; and (c) a coding domain containing a functional CEP290 exon on the 5' side of the target intron, wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain with endogenous CEP290, thereby replacing the endogenous CEP290 exon on the 5' side of the target intron with the functional CEP290 exon, thereby correcting a pathogenic point mutation. In some embodiments, the pathogenic point mutation is an A-to-G mutation at nucleotide 1,655 of SEQ ID NO:85.

[0047] In some embodiments, the target intron is intron 27, the coding domain contains functional CEP290 exons 2-27, and the nucleic acid trans-splicing molecule is configured to replace endogenous CEP290 exons 2-27 with functional CEP290 exons 2-27. In some embodiments, the binding domain is configured to bind to intron 27 at a binding site containing one or more nucleotides from SEQ ID NO: 86, including nucleotides 120-680, 710-2,200, or 2,670-2,910. In some embodiments, the binding site contains one or more nucleotides from SEQ ID NO: 86, including nucleotides 790-2,100, for example, one or more nucleotides from SEQ ID NO: 86, including nucleotides 1,020-1,630. In other embodiments, the binding site contains one or more nucleotides from SEQ ID NO: 86, including nucleotides 1,670-2,000.

[0048] In some embodiments, the target intron is intron 28, the coding domain contains functional CEP290 exons 2-28, and the nucleic acid trans-splicing molecule is configured to replace endogenous CEP290 exons 2-28 with functional CEP290 exons 2-28. In some embodiments, the binding domain is configured to bind to intron 28 at a binding site containing one or more nucleotides from SEQ ID NO: 87, including nucleotides 1-390, nucleotides 410-560, or nucleotides 730-937. In some embodiments, the binding site contains one or more nucleotides from SEQ ID NO: 87, including nucleotides 1-200. In other embodiments, the binding site contains one or more nucleotides from SEQ ID NO: 87, including nucleotides 720-900.

[0049] In some embodiments, the target intron is intron 29, the coding domain contains functional CEP290 exons 2-29, and the nucleic acid trans-splicing molecule is configured to replace endogenous CEP290 exons 2-29 with functional CEP290 exons 2-29. In some embodiments, the binding domain is configured to bind to intron 29 at a binding site containing one or more nucleotides from SEQ ID NO:88, including nucleotides 1-600, nucleotides 720-940, or nucleotides 1,370-1,790.

[0050] In some embodiments, the target intron is intron 30, the coding domain contains functional CEP290 exons 2-30, and the nucleic acid trans-splicing molecule is configured to replace endogenous CEP290 exons 2-30 with functional CEP290 exons 2-30. In some embodiments, the binding domain is configured to bind to intron 29 at a binding site containing one or more nucleotides 880-1,240 of SEQ ID NO:89, for example, one or more nucleotides 950-1,240 of SEQ ID NO:89, for example, one or more nucleotides 1,060-1,240 of SEQ ID NO:89.

[0051] In any of the above embodiments, the binding domain is 20 to 1,000 nucleotides long (for example, 25 to 900 nucleotides long, 30 to 800 nucleotides long, 40 to 700 nucleotides long, 50 to 600 nucleotides long, 75 to 500 nucleotides long, 100 to 400 nucleotides long, 125 to 200 nucleotides long, or about 150 nucleotides long, for example, 20 to 30 nucleotides long, 30 to 40 nucleotides long, 40 to 50 nucleotides long, 50 to 75 nucleotides long, 75 to 100 nucleotides long, 125 to 150 nucleotides long, 150 to 175 nucleotides long, 175 to 200 nucleotides long, 200 to 250 nucleotides long, 250 to 500 nucleotides long, 500 to 750 nucleotides long, or 750 to 1,000 nucleotides long).

[0052] In some embodiments, the coding domain is a cDNA sequence. In some embodiments, the coding domain is a naturally occurring sequence. In other embodiments, the coding domain is a codon-optimized sequence.

[0053] In some embodiments, the artificial intron is composed of an artificial intron and a spacer array.

[0054] In any of the above embodiments, the nucleic acid trans-splicing molecule may have a length of 3,000 to 4,000 nucleotides.

[0055] In any of the above embodiments, the mutant CEP290 exon may be associated with LCA 10. In some embodiments, the mutant CEP290 exon associated with LCA 10 is expressed in photoreceptor cells.

[0056] In another aspect of the present invention, this specification provides a proviral plasmid comprising a nucleic acid transsplicing molecule in any of the aforementioned aspects.

[0057] In another aspect, the present invention provides adeno-associated viruses (AAVs) comprising nucleic acid molecules in any of the aforementioned aspects. In some embodiments, the AAV preferentially targets photoreceptor cells. In some embodiments, the AAV comprises AAV5 capsid protein, AAV8 capsid protein, AAV8(b) capsid protein, or AAV9 capsid protein.

[0058] In another aspect, the present invention features a pharmaceutical composition comprising a nucleic acid trans-splicing molecule, a proviral plasmid, or an AAV in any of the aforementioned aspects.

[0059] In another aspect, the foregoing describes a method for correcting a pathogenic point mutation in the CEP290 intron 26 within a target cell, comprising the step of administering the subject any of the above-mentioned nucleic acid trans-splicing molecules, proviral plasmids, AAVs, or pharmaceutical compositions to the subject. In some embodiments, the subject has LCA 10.

[0060] In another aspect, the present invention provides a method for treating a subject having LCA 10 caused by a pathogenic point mutation in CEP290 intron 26, comprising the step of administering a nucleic acid trans-splicing molecule, proviral plasmid, AAV, or pharmaceutical composition of any of the aforementioned aspects to the subject.

[0061] In any of the above methods, the pathogenic point mutation may be a mutation from A to G at nucleotide 1,655 of CEP290 intron 26 (SEQ ID NO: 85). In some embodiments, a nucleic acid trans-splicing molecule, proviral plasmid, AAV, or pharmaceutical composition is administered by subretinal injection, intravitreous injection, or intravenous injection.

[0062] In another aspect, the present invention provides a kit comprising one or more of the aforementioned nucleic acid trans-splicing molecules, proviral plasmids, AAVs, or pharmaceutical compositions, further comprising instructions for use of the one or more nucleic acid trans-splicing molecules, proviral plasmids, AAVs, or pharmaceutical compositions for correcting mutations in the target CEP290 gene (e.g., defects, e.g., mutations associated with LCA 10). [Invention 1001] Functionally connected in either the 3' to 5' direction or the 5' to 3' direction, (a) A binding domain configured to bind to a target ABCA4 intron selected from the group consisting of introns 19, 23, or 24; (b) a splicing domain configured to mediate transsplicing; and (c) Code domain containing functional ABCA4 exons A nucleic acid transsplicing molecule containing, The nucleic acid transsplicing molecule is configured to transsplice the coding domain and the endogenous ABCA4 exon adjacent to the target ABCA4 intron, thereby replacing the endogenous ABCA4 exon with the functional ABCA4 exon and correcting the mutation in ABCA4. The nucleic acid trans-splicing molecule. [Invention 1002] A nucleic acid transsplicing molecule according to the present invention 1001, wherein the binding domain binds to the 3' target ABCA4 intron of the mutation, and the mutation is present in any one of ABCA4 exons 1-24 or introns 1-24. [Invention 1003] A nucleic acid transsplicing molecule according to the present invention 1002, wherein the target ABCA4 intron is intron 19, the mutation is located in either ABCA4 exons 1-19 or introns 1-19, and the coding domain contains ABCA4 exons 1-19. [Invention 1004] A nucleic acid transsplicing molecule according to the present invention 1003, wherein the binding domain is configured to bind to intron 19 at a binding site containing one or more nucleotides from nucleotide 990 to 2,174 of SEQ ID NO:25. [Invention 1005] A nucleic acid transsplicing molecule according to Invention 1004, wherein the binding site contains one or more nucleotides from SEQ ID NO:25, specifically nucleotides 1,670 to 2,174. [Invention 1006] A nucleic acid transsplicing molecule according to the present invention 1005, wherein the binding site contains one or more nucleotides from SEQ ID NO:25, specifically from nucleotides 1,810 to 2,000. [Invention 1007] A nucleic acid transsplicing molecule according to the present invention 1006, wherein the binding site contains one or more nucleotides from among nucleotides 1,870 to 2,000 of SEQ ID NO:25. [Invention 1008] A nucleic acid transsplicing molecule according to Invention 1007, wherein the binding site contains one or more nucleotides from SEQ ID NO:25, specifically from nucleotide 1,920 to 2,000. [Invention 1009] The nucleic acid transsplicing molecule of the present invention 1002, wherein the target ABCA4 intron is intron 23, and the mutation is present in one or more of the ABCA4 exons 1-23 or introns 1-23. [Invention 1010] A nucleic acid transsplicing molecule according to the present invention 1009, wherein the coding domain contains functional ABCA4 exons 1-23. [Invention 1011] A nucleic acid transsplicing molecule according to the present invention 1010, wherein the binding domain is configured to bind to intron 23 at a binding site containing one or more nucleotides from nucleotides 80-570 or nucleotides 720-1,081 of SEQ ID NO:29. [Invention 1012] The binding domain is (a) One or more nucleotides from nucleotides 261 to 410 of SEQ ID NO:29; (b) One or more nucleotides from nucleotides 801 to 950 of SEQ ID NO:29; or (c) One or more nucleotides from nucleotides 841-990 of SEQ ID NO:29 It is configured to bind to the ABCA4 intron 23 at the binding site including the binding site. Nucleic acid trans-splicing molecule according to the present invention 1011. [Invention 1013] The binding site is (a) Six or more nucleotides from nucleotides 261-410 of SEQ ID NO:29; (b) Six or more nucleotides from nucleotides 801-950 of SEQ ID NO:29; or (c) Six or more nucleotides from nucleotides 841-990 of SEQ ID NO:29 including, Nucleic acid trans-splicing molecule according to the present invention 1012. [Invention 1014] A nucleic acid trans-splicing molecule according to the present invention 1013, wherein the binding domain comprises six or more consecutive nucleic acid residues complementary to the six or more nucleotides of the binding site. [Invention 1015] A nucleic acid transsplicing molecule according to the present invention 1002, wherein the target ABCA4 intron is intron 24, the mutation is located in either ABCA4 exons 1-24 or introns 1-24, and the coding domain contains ABCA4 exons 1-24. [Invention 1016] A nucleic acid transsplicing molecule according to the present invention 1011, wherein the binding domain is configured to bind to intron 24 at a binding site containing one or more nucleotides from SEQ ID NO:30, either nucleotides 600-1,250 or nucleotides 1,490-2,660. [Invention 1017] A nucleic acid transsplicing molecule according to the present invention 1012, wherein the binding site contains one or more nucleotides from among 1,000 to 1,200 of SEQ ID NO:30. [Invention 1018] A nucleic acid transsplicing molecule according to the present invention 1001, wherein the binding domain binds to the 5' target ABCA4 intron of the mutation, and the mutation is located in either ABCA4 exons 23-50 or introns 22-49. [Invention 1019] The nucleic acid transsplicing molecule of the present invention 1014, wherein the target ABCA4 intron is intron 23, the mutation is located in either ABCA4 exons 24-50 or introns 23-49, and the coding domain comprises ABCA4 exons 24-50. [Invention 1020] A nucleic acid transsplicing molecule according to the present invention 1015, wherein the binding domain is configured to bind to intron 23 at a binding site containing one or more nucleotides from nucleotide 80 to 1,081 of SEQ ID NO:29. [Invention 1021] A nucleic acid transsplicing molecule according to the present invention 1016, wherein the binding site contains one or more nucleotides from nucleotides 230 to 1,081 of SEQ ID NO:29. [Invention 1022] A nucleic acid transsplicing molecule according to the present invention 1017, wherein the binding site contains one or more nucleotides from among nucleotides 250 to 400 of SEQ ID NO:29. [Invention 1023] A nucleic acid transsplicing molecule according to the present invention 1017, wherein the binding site contains one or more nucleotides from nucleotide 690 to 850 of SEQ ID NO:29. [Invention 1024] The nucleic acid transsplicing molecule of the present invention 1014, wherein the target ABCA4 intron is intron 24, the mutation is located in either ABCA4 exons 25-50 or introns 24-49, and the coding domain comprises ABCA4 exons 25-50. [Invention 1025] A nucleic acid transsplicing molecule according to the present invention 1020, wherein the binding domain is configured to bind to intron 24 at a binding site containing one or more nucleotides from among nucleotides 1-250, nucleotides 300-2,100, or nucleotides 2,200-2,692 of SEQ ID NO:30. [Invention 1026] A nucleic acid transsplicing molecule according to the present invention 1021, wherein the binding site contains one or more nucleotides from nucleotide 360 ​​to 610 of SEQ ID NO:30. [Invention 1027] A nucleic acid transsplicing molecule according to Invention 1021, wherein the binding site contains one or more nucleotides from nucleotides 750 to 1,110 of SEQ ID NO:30. [Invention 1028] Functionally connected in the direction from 5' to 3', (a) A binding domain configured to bind to ABCA4 intron 22 at a binding site containing one or more nucleotides from among nucleotides 60-570, 600-800, or 900-1,350 of SEQ ID NO:28; (b) a splicing domain configured to mediate transsplicing; and (c) Code domain including functional ABCA4 exons 23-50 A nucleic acid transsplicing molecule containing, The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous ABCA4 exon 22, thereby replacing endogenous ABCA4 exons 23-50 with functional ABCA4 exons 23-50. The nucleic acid trans-splicing molecule. [Invention 1029] A nucleic acid transsplicing molecule according to the present invention 1024, wherein the binding site contains one or more nucleotides from nucleotides 70 to 250 of SEQ ID NO:28. [Invention 1030] Functionally connected in the direction from 3' to 5', (a) A binding domain configured to bind to ABCA4 intron 22 at a binding site containing one or more nucleotides from SEQ ID NO:28, either nucleotides 1-510 or 880-1,350; (b) a splicing domain configured to mediate transsplicing; and (c) Code domain including functional ABCA4 exons 1-22 A nucleic acid transsplicing molecule containing, The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous ABCA4 exon 23, thereby replacing endogenous ABCA4 exons 1-22 with functional ABCA4 exons 1-22. The nucleic acid trans-splicing molecule. [Invention 1031] The binding domain is (a) One or more nucleotides from nucleotides 1041 to 1190 of SEQ ID NO:28; (b) One or more nucleotides from nucleotides 1171-1320 of SEQ ID NO:28; (c) One or more nucleotides from nucleotides 1201 to 1350 of SEQ ID NO:28 It is configured to bind to the ABCA4 intron 22 at the binding site including the binding site. Nucleic acid trans-splicing molecule according to the present invention 1030. [Invention 1032] The binding site is (a) Six or more nucleotides from nucleotides 1041-1190 of SEQ ID NO:28; (b) Six or more nucleotides from nucleotides 1171-1320 of SEQ ID NO:28; (c) Six or more nucleotides from nucleotides 1201-1350 of SEQ ID NO:28 including, Nucleic acid trans-splicing molecule according to the present invention 1031. [Invention 1033] A nucleic acid trans-splicing molecule according to the present invention 1032, wherein the binding domain comprises six or more consecutive nucleic acid residues complementary to the six or more nucleotides of the binding site. [Invention 1034] A nucleic acid transsplicing molecule according to any of the invention 1001 to 1033, wherein the binding domain is 100 to 200 nucleotides long. [Invention 1035] A nucleic acid transsplicing molecule according to any of the present invention 1001 to 1034, wherein the coding domain is a cDNA sequence. [Invention 1036] A nucleic acid transsplicing molecule according to any of the present invention 1001 to 1034, wherein the coding domain contains a naturally occurring sequence. [Invention 1037] A nucleic acid transsplicing molecule according to any of the present invention 1001 to 1034, wherein the coding domain contains a codon-optimized sequence. [Invention 1038] A nucleic acid transsplicing molecule according to any of invention 1001 to 1037, wherein the artificial intron contains a spacer sequence. [Invention 1039] A nucleic acid transsplicing molecule of any of the present invention 1001 to 1038, having a length of 3,000 to 4,000 nucleotides. [Invention 1040] A nucleic acid transsplicing molecule according to any of the invention items 1001 to 1039, wherein a mutation in the ABCA4 gene is associated with Stargardt disease. [Invention 1041] A nucleic acid trans-splicing molecule of the present invention 1040, wherein a mutation in the ABCA4 gene associated with Stargardt disease is expressed in photoreceptor cells. [Invention 1042] Functionally connected in the direction from 3' to 5', (a) A binding domain configured to bind to intron 23 of ABCA4 at a binding site containing six or more nucleotides from nucleotides 261 to 410 of SEQ ID NO:29, wherein the binding domain contains six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) Artificial introns containing splicing domains; and (c) Code domain including functional ABCA4 exons 1-23 A nucleic acid transsplicing molecule containing, The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous ABCA4 exon 24, thereby replacing endogenous ABCA4 exons 1-23 with functional ABCA4 exons 1-23. The nucleic acid trans-splicing molecule. [Invention 1043] Functionally connected in the direction from 3' to 5', (a) A binding domain configured to bind to intron 23 of ABCA4 at a binding site containing six or more nucleotides from nucleotides 801-950 of SEQ ID NO:29, wherein the binding domain contains six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) Artificial introns containing splicing domains; and (c) Code domain including functional ABCA4 exons 1-23 A nucleic acid transsplicing molecule containing, The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous ABCA4 exon 24, thereby replacing endogenous ABCA4 exons 1-23 with functional ABCA4 exons 1-23. The nucleic acid trans-splicing molecule. [Invention 1044] Functionally connected in the direction from 3' to 5', (a) A binding domain configured to bind to intron 23 of ABCA4 at a binding site containing six or more nucleotides from nucleotides 841-990 of SEQ ID NO:29, wherein the binding domain contains six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) Artificial introns containing splicing domains; and (c) Code domain including functional ABCA4 exons 1-23 A nucleic acid transsplicing molecule containing, The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous ABCA4 exon 24, thereby replacing endogenous ABCA4 exons 1-23 with functional ABCA4 exons 1-23. The nucleic acid trans-splicing molecule. [Invention 1045] Functionally connected in the direction from 3' to 5', (a) A binding domain configured to bind to intron 22 of ABCA4 at a binding site containing six or more nucleotides from nucleotides 1041 to 1190 of SEQ ID NO:28, wherein the binding domain contains six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) Artificial introns containing splicing domains; and (c) Code domain including functional ABCA4 exons 1-22 A nucleic acid transsplicing molecule containing, The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous ABCA4 exon 23, thereby replacing endogenous ABCA4 exons 1-22 with functional ABCA4 exons 1-22. The nucleic acid trans-splicing molecule. [Invention 1046] Functionally connected in the direction from 3' to 5', (a) A binding domain configured to bind to intron 22 of ABCA4 at a binding site containing six or more nucleotides from nucleotides 1171-1320 of SEQ ID NO:28, wherein the binding domain contains six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) Artificial introns containing splicing domains; and (c) Code domain including functional ABCA4 exons 1-22 A nucleic acid transsplicing molecule containing, The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous ABCA4 exon 23, thereby replacing endogenous ABCA4 exons 1-22 with functional ABCA4 exons 1-22. The nucleic acid trans-splicing molecule. [Invention 1047] Functionally connected in the direction from 3' to 5', (a) A binding domain configured to bind to intron 22 of ABCA4 at a binding site containing six or more nucleotides from nucleotides 1201 to 1350 of SEQ ID NO:28, the binding domain comprising six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) Artificial introns containing splicing domains; and (c) Code domain including functional ABCA4 exons 1-22 A nucleic acid transsplicing molecule containing, The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous ABCA4 exon 23, thereby replacing endogenous ABCA4 exons 1-22 with functional ABCA4 exons 1-22. The nucleic acid trans-splicing molecule. [Invention 1048] A proviral plasmid comprising any nucleic acid transsplicing molecule according to invention 1001 to 1047. [Invention 1049] Adeno-associated virus (AAV) comprising any nucleic acid molecule of invention 1001 to 1048. [Invention 1050] AAV according to the present invention 1049, which preferentially targets photoreceptor cells. [Invention 1051] AAV according to Invention 1049 or 1050, comprising AAV5 capsid protein, AAV8 capsid protein, AAV8(b) capsid protein, or AAV9 capsid protein. [Invention 1052] A pharmaceutical composition comprising any nucleic acid transsplicing molecule of Invention 1001 to 1047, a proviral plasmid of Invention 1048, or any AAV of Invention 1049 to 1051. [Invention 1053] A pharmaceutical composition comprising a 5' nucleic acid transsplicing molecule and a 3' nucleic acid transsplicing molecule, wherein the 5' nucleic acid transsplicing molecule is a nucleic acid transsplicing molecule according to any of Invention 1002 to 1013 or 1030 to 1047, and the 3' nucleic acid transsplicing molecule is a nucleic acid transsplicing molecule according to any of Invention 1014 to 1025. [Invention 1054] A method for correcting a mutation in the ABCA4 gene within a target cell, comprising the step of administering a pharmaceutical composition of the present invention 1052 or 1053 to the target. [Invention 1055] A method for correcting a mutation in one or more of the ABCA4 exons 1 to 24 in a subject requiring such correction, comprising the step of administering a pharmaceutical composition containing any nucleic acid trans-splicing molecule of the present invention 1002 to 1013 or 1030 to 1047 to the subject. [Invention 1056] A method for correcting a mutation in one or more of the ABCA4 exons 23-50 in a subject requiring such correction, comprising the step of administering a pharmaceutical composition containing any nucleic acid trans-splicing molecule of the present invention 1014-1025 to the subject. [Invention 1057] A method for correcting a mutation in any one of ABCA4 exons 1 to 24 and a second mutation in any one of exons 23 to 50 in a subject requiring such correction, comprising the step of administering the pharmaceutical composition of the present invention 1053 to the subject. [Invention 1058] A method for treating a subject having a disorder associated with a mutation in ABCA4, comprising the step of administering a pharmaceutical composition of the present invention 1052 or 1053 to the subject. [Invention 1059] A method for treating a subject having a disorder associated with a mutation in one or more of the ABCA4 exons 1-24 or introns 1-24, comprising the step of administering a pharmaceutical composition containing any nucleic acid trans-splicing molecule of the present invention 1002-1013 or 1030-1047 to the subject. [Invention 1060] A method for treating a subject having a disorder associated with a mutation in one or more of the ABCA4 exons 23-50 or introns 22-49, comprising the step of administering a pharmaceutical composition containing any nucleic acid trans-splicing molecule of the present invention 1014-1025 to the subject. [Invention 1061] A method for treating a subject having a disorder associated with a first mutation in any one of ABCA4 exons 1 to 24 and a second mutation in any one of exons 23 to 50, comprising the step of administering the pharmaceutical composition of the present invention 1053 to the subject. [Invention 1062] A method according to any of the present invention 1054 to 1061, wherein the subject has Stargardt disease. [Invention 1063] A method according to any one of items 1054 to 1062 of the present invention, wherein the composition is administered by subretinal injection, intravitreous injection, or intravenous injection. [Invention 1064] Adeno-associated virus (AAV) comprising an assembled capsid in which a genome vector containing AAV 5'ITR, any nucleic acid molecule of the present invention 1001-1047 under functional control of a regulatory sequence, and AAV 3'ITR is packaged internally. [Invention 1065] A method according to any one of the present invention 1054 to 1063, wherein the subject shows an increase of at least 10% in ABCA4 protein expression after administration. [Invention 1066] Functionally connected in the direction from 3' to 5', (a) A binding domain configured to bind to CEP290 intron 26 at a binding site containing one or more nucleotides from among nucleotides 4,800 to 5,838 of SEQ ID NO:32; (b) a splicing domain configured to mediate transsplicing; and (c) Code domain including functional CEP290 exons 2-26 A nucleic acid transsplicing molecule containing, The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous CEP290 exon 27, thereby replacing endogenous CEP290 exons 2-26 with the functional CEP290 exons 2-26, and correcting the pathogenic point mutation. The nucleic acid trans-splicing molecule. [Invention 1067] Functionally connected in the direction from 3' to 5', (a) A binding domain configured to bind to CEP290 in any one of the target introns 27, 28, 29, or 30; (b) a splicing domain configured to mediate transsplicing; and (c) Code domain including the functional CEP290 exon on the 5' side of the target intron A nucleic acid transsplicing molecule containing, The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous CEP290, thereby replacing the 5' endogenous CEP290 exon of the target intron with the functional CEP290 exon, and correcting the pathogenic point mutation. The nucleic acid trans-splicing molecule. [Invention 1068] A proviral plasmid comprising the nucleic acid trans-splicing molecule of the present invention 1066 or 1067. [Invention 1069] AAV comprising any nucleic acid molecule according to invention 1066 to 1068. [Invention 1070] A pharmaceutical composition comprising a nucleic acid transsplicing molecule of Invention 1066 or 1067, a proviral plasmid of Invention 1068, or an AAV of Invention 1069. [Invention 1071] A method for correcting a pathogenic point mutation in CEP290 intron 26 within a target cell, comprising the step of administering to the target a nucleic acid trans-splicing molecule of the present invention 1066 or 1067, a proviral plasmid of the present invention 1068, an AAV of the present invention 1069, or a pharmaceutical composition of the present invention 1070. [Invention 1072] A method for treating a subject having LCA 10 caused by a pathogenicity point mutation in CEP290 intron 26, comprising the step of administering to the subject a nucleic acid trans-splicing molecule of the present invention 1066 or 1067, a proviral plasmid of the present invention 1068, an AAV of the present invention 1069, or a pharmaceutical composition of the present invention 1070. [Brief explanation of the drawing]

[0063] [Figure 1] Figure 1 is a schematic diagram of several exemplary nucleic acid trans-splicing molecules for modifying mutant ABCA4 exons using functional ABCA4 exons. The darkly shaded boxes represent native ABCA4 exons. The dotted lines connecting the darkly shaded boxes represent native introns. The lightly shaded boxes with dark outlines represent functional ABCA4 exons within the nucleic acid trans-splicing molecule. The splicing domains shown by curves bind to one end of each functional ABCA4 exon, becoming introns of the ABCA4 premRNA. [Figure 2]Figure 2 is a graph showing the trans-splicing efficiency (relative change ratio) brought about by a 150-nucleotide-long binding domain spanning the entire ABCA4 intron 19 (SEQ ID NO: 25) at 10-nucleotide intervals using a 5' trans-splicing molecule. The X-axis labels indicate the numbers of each binding site, starting from the 5' end of the intron (i.e., the first nucleotide of the intron sequence). [Figure 3] Figure 3 is a graph showing the trans-splicing efficiency (relative change rate) brought about by a 150-base-length binding domain spanning the entire ABCA4 intron 22 (SEQ ID NO: 28) at 10-nucleotide intervals using a 5' trans-splicing molecule. The X-axis labels indicate the numbers of each binding site, starting from the 5' end of the intron (i.e., the first nucleotide of the intron sequence). [Figure 4] Figure 4 is a graph showing the trans-splicing efficiency (relative change rate) brought about by a 150-nucleotide-long binding domain spanning the entire ABCA4 intron 22 (SEQ ID NO: 28) at 10-nucleotide intervals using a 3' trans-splicing molecule. The X-axis labels indicate the numbers of each binding site, starting from the 5' end of the intron (i.e., the first nucleotide of the intron sequence). [Figure 5] Figure 5 is a graph showing the trans-splicing efficiency (relative change rate) brought about by a 150-nucleotide-long binding domain spanning the entire ABCA4 intron 23 (SEQ ID NO: 29) at 10-nucleotide intervals using a 5' trans-splicing molecule. The X-axis labels indicate the numbers of each binding site, starting from the 5' end of the intron (i.e., the first nucleotide of the intron sequence). [Figure 6]Figure 6 is a graph showing the trans-splicing efficiency (relative change ratio) brought about by a 150-nucleotide-long binding domain spanning the entire ABCA4 intron 23 (SEQ ID NO: 29) at 10-nucleotide intervals using a 3' trans-splicing molecule. The X-axis labels indicate the numbers of each binding site, starting from the 5' end of the intron (i.e., the first nucleotide of the intron sequence). [Figure 7] Figure 7 is a graph showing the trans-splicing efficiency (relative change rate) brought about by a 150-nucleotide-long binding domain spanning the entire ABCA4 intron 24 (SEQ ID NO: 30) at 10-nucleotide intervals using a 5' trans-splicing molecule. The X-axis labels indicate the numbers of each binding site, starting from the 5' end of the intron (i.e., the first nucleotide of the intron sequence). [Figure 8] Figure 8 is a graph showing the trans-splicing efficiency (relative change rate) brought about by a 150-base-length binding domain spanning the entire ABCA4 intron 24 (SEQ ID NO: 30) at 10-nucleotide intervals using a 3' trans-splicing molecule. The X-axis labels indicate the numbers of each binding site, starting from the 5' end of the intron (i.e., the first nucleotide of the intron sequence). [Figure 9] Figure 9 is a schematic diagram showing a TALEN protein consisting of a DNA-binding domain linked to a transcriptional activation domain. The VP64 transcriptional activation domain is shown. The right panel shows the 5' untranslated region (5'-UTR) of ABCA4. The TATA box and putative transcription start site are also shown. The sequences targeted by the three different DNA-binding domains of TALEN are also shown. As shown in the figure, TALEN 1 binds to the first underlined sequence, TALEN 2 binds to the second underlined sequence, and TALEN 3 binds to the third underlined sequence. [Figure 10]Figure 10 shows a gel in which 293T cells were transfected with TALEN constructs designed to induce endogenous ABCA4 expression. All three TALENs in Figure 9 were stably introduced into 293 cells, and single-clonal cells were collected and analyzed by Western blotting. Positive controls (+) indicate cells transfected with plasmids expressing ABCA4 cDNA. Cell lysates were prepared 48 hours after transfection, and the membrane fraction was examined for ABCA4 expression using the antibody ab72955 (Abcam). Clones ZT-22 and ZT-48 showed ABCA4 protein expression. [Figure 11] Figure 11 is a schematic diagram showing a CAG promoter cell line. [Figure 12A] Figures 12A and 12B show a site-specific guide (Figure 12A) designed to insert a CAG promoter and a puromycin-selectable marker using a homologous arm (Figure 12B). [Figure 12B] See the explanation in Figure 12A. [Figure 13] Figure 13 is a schematic diagram showing a CAG promoter cell line. [Figure 14] Figures 14A and 14B are graphs and gels, respectively, showing expression results in several clonal cell lines selected for further analysis. Figure 14A shows RNA expression in the cell lines, and Figure 14B shows protein expression. Membrane preparations of the displayed cell lines were probe-searched for the ABCA4 protein using a rabbit polyclonal antibody against ABCA4 (Abcam, ab72955). The exposure time was 23 seconds. 293 cells are parental cells that do not express ABCA4. The top band is nonspecific background present in all cells. [Figure 15] Figure 15 is a schematic diagram showing CRISPR guide RNAs for targeting exons 3 and 4. [Figure 16] Figure 16 is a gel showing a graph of RNA expression and protein profiles of single-clonal cells induced after treatment with CRISPR / Cas9 as shown in Figure 15. [Figure 17A] Figures 17A and 17B are schematic diagrams showing PCR for mutation analysis in cDNA (Figure 17A) and PCR for genotyping in cDNA (Figure 17B), confirming that exons 3 and 4 were targeted and fragmented. [Figure 17B] See the explanation in Figure 17A. [Figure 18] Figure 18 is a set of tables showing that mutation analysis of Figures 17A and 17B confirmed that exons 3 and 4 were targeted and fragmented in the alleles within the 17+06 and 17+21 cell lines. [Figure 19] Figures 19A and 19B are schematic diagrams of trans-splicing molecules targeting ABCA4 premRNA. Figure 19A shows a typical trans-splicing molecule containing a codon-optimized exon (or set of exons), a binding domain that hybridizes to the target RNA, and an artificial intron linker. Figure 19B shows various trans-splicing molecules that target specific regions within intron 22 and intron 23 of ABCA4. [Figure 20A] Figures 20A–20D show gels (Figures 20A and 20C) and graphs (Figures 20B and 20D) illustrating the results of the trans-splicing reaction. Figures 20A and 20B show the protein and RNA levels of the intron 22 trans-splicing reaction, respectively, while Figures 20C and 20D show the protein and RNA levels of the intron 23 trans-splicing reaction, respectively. [Figure 20B] See the explanation in Figure 20A. [Figure 20C] See the explanation in Figure 20A. [Figure 20D] See the explanation in Figure 20A. [Figure 21]Figure 21 is a schematic diagram of several exemplary nucleic acid trans-splicing molecules for correcting mutations within CEP290 intron 26 using the functional 5' portion of the CEP290 gene. The darkly shaded boxes represent native CEP290 exons. The dotted lines connecting the darkly shaded boxes represent native introns. The lightly shaded boxes with dark outlines represent functional CEP290 exons within the nucleic acid trans-splicing molecules. The splicing domains shown by curves are bound to one end of each functional CEP290 exon sequence, becoming introns of the CEP290 premRNA. [Figure 22] Figure 22 is a graph showing the trans-splicing efficiency (relative change rate) brought about by a 150-nucleotide-long binding domain that spans the entire CEP290 intron 26 (SEQ ID NO: 85) at 10-nucleotide intervals. The X-axis labels indicate the "motif number," which is the number of each binding site starting from the 5' end of the intron (i.e., the first nucleotide of the intron sequence). [Figure 23] Figure 23 is a graph showing the trans-splicing efficiency (relative change rate) brought about by a 150-nucleotide-long binding domain spanning the entire CEP290 intron 27 (SEQ ID NO: 86) at 10-nucleotide intervals. Each of the three lines represents an independent experiment. The X-axis label indicates the "motif number," which is the number of each binding site starting from the 5' end of the intron (i.e., the first nucleotide of the intron sequence). [Figure 24] Figure 24 is a graph showing the trans-splicing efficiency (relative change rate) brought about by a 150-nucleotide-long binding domain spanning the entire CEP290 intron 28 (SEQ ID NO: 87) at 10-nucleotide intervals. Each of the three lines represents an independent experiment. The X-axis label indicates the "motif number," which is the number of each binding site starting from the 5' end of the intron (i.e., the first nucleotide of the intron sequence). [Figure 25]Figure 25 is a graph showing the trans-splicing efficiency (relative change rate) brought about by a 150-nucleotide-long binding domain spanning the entire CEP290 intron 29 (SEQ ID NO: 88) at 10-nucleotide intervals. Each of the three lines represents an independent experiment. The X-axis label indicates the "motif number," which is the number of each binding site starting from the 5' end of the intron (i.e., the first nucleotide of the intron sequence). [Figure 26] Figure 26 is a graph showing the trans-splicing efficiency (relative change rate) brought about by a 150-nucleotide-long binding domain spanning the entire CEP290 intron 30 (SEQ ID NO: 89) at 10-nucleotide intervals. Each of the three lines represents an independent experiment. The X-axis label indicates the "motif number," which is the number of each binding site starting from the 5' end of the intron (i.e., the first nucleotide of the intron sequence). [Modes for carrying out the invention]

[0064] Detailed explanation The compositions and methods described herein involve trans-splicing molecules (e.g., pre-mRNA trans-splicing molecules delivered by adeno-associated virus (AAV)) for treating diseases or disorders caused by mutations in the ABCA4 gene. The methods and compositions described herein utilize pre-mRNA trans-splicing as gene therapy (e.g., ex vivo gene therapy and in vivo gene therapy) for treating diseases caused by mutations in ABCA4, such as Stargardt disease (e.g., Stargardt disease type 1).

[0065] Alternatively, the compositions and methods described herein may also involve trans-splicing molecules (e.g., pre-mRNA trans-splicing molecules delivered by adeno-associated virus (AAV)) for treating diseases or disorders caused by mutations in the CEP290 gene, such as LCA 10. Such methods utilize pre-mRNA trans-splicing as gene therapy (e.g., ex vivo gene therapy and in vivo gene therapy) for treating diseases caused by mutations in CEP290, such as LCA 10.

[0066] The trans-splicing molecules and methods of use exemplified herein offer several advantages compared to conventional treatments. Firstly, the use of trans-splicing molecule delivery via AAV provides efficient and specific delivery of gene therapies to photoreceptors, while addressing the problems associated with AAV packaging limitations. Secondly, such compositions and methods enable modification at the source of gene defects. Furthermore, the compositions and methods provided herein are useful for treating any type of mutation within ABCA4 (or other large cDNA / transgene cassettes). Modification of photoreceptor defects provides secondary relief to retinal pigment epithelial cells. Moreover, the methods and compositions of the present invention are generally immunologically harmless. The use of subretinal delivery and other features makes the effect specific to target cells, e.g., photoreceptors, thus reducing the toxicity of off-target splicing. Furthermore, unlike nucleases, trans-splicing does not require genome modification. Finally, while RNA repair does not require cell division, DNA repair methodologies (e.g., CRISPR-Cas9 or zinc fingers) require mitosis for homologous recombination repair to occur, which is unsuitable for post-mitotic tissues such as the retina.

[0067] I. Definition Unless otherwise defined, scientific and technical terms used herein have the same meanings as commonly understood by those skilled in the art in the field to which this invention pertains, and as commonly understood by referring to publications that provide many of the general terms used herein. In the event of any conflict between the definitions given herein and those in the reference publications, the definitions provided herein shall prevail.

[0068] A “nucleic acid trans-splicing molecule” or “trans-splicing molecule” has three main elements: (a) a binding domain that confers specificity to the trans-splicing molecule by tethering it to its target gene (e.g., premRNA); (b) a splicing domain (e.g., a splicing domain having a 3' splice site or a 5' splice site); and (c) a coding sequence configured such that trans-splicing occurs in the target gene, in which one or more exons (e.g., one or more mutant exons) within the target gene can be replaced. A “premRNA trans-splicing molecule” or “RTM” refers to a nucleic acid trans-splicing molecule that targets premRNA. In some embodiments, the trans-splicing molecule, e.g., RTM, may include cDNA as part of a functional exon (e.g., a functional ABCA4 exon or CEP290 exon, e.g., a codon-optimized exon) for replacement or modification of (e.g., a mutant ABCA4 exon or CEP290).

[0069] "Trans-splicing" is intended to link a nucleic acid molecule containing one or more exons (e.g., exogenous exons, e.g., exons that are part of the coding domain of a trans-splicing molecule) to a separate RNA molecule (e.g., a pre-mRNA molecule, e.g., an endogenous pre-mRNA molecule) by a spliceosome-mediated mechanism, where a second portion of the RNA molecule replaces a first portion of the RNA molecule.

[0070] As used herein, "binding" between the binding domain and the target intron refers to a hydrogen bond between the binding domain and the target intron that is sufficient to mediate transsplicing by enabling the transsplicing molecule to associate with the target gene (e.g., premRNA). In some embodiments, the hydrogen bond between the binding domain and the target intron is between nucleotide bases that are complementary and antisense in orientation (e.g., hybridize with each other).

[0071] As used herein, “artificial intron” refers to a nucleic acid sequence that ligates a binding domain to a coding domain (directly or indirectly). The artificial intron includes a splicing domain and may further include one or more spacer sequences and / or other regulatory elements.

[0072] As used herein, "splicing domain" refers to a nucleic acid sequence having a motif recognized by a spliceosome and mediating trans-splicing. The splicing domain includes a splice site (e.g., a single splice site, i.e., a sole splice site), which may be a 3' splice site or a 5' splice site. The splicing domain may also include other regulatory elements. For example, in some embodiments, the splicing domain includes a splicing-promoting sequence (e.g., an intra-exon splicing-promoting sequence (ESE) or an intra-intron splicing-promoting sequence (ISE)). In some embodiments, the splicing domain includes a branching point (e.g., a strongly conserved branching point) or a branching site sequence and / or a polypyrimidine tract (PPT). In some embodiments, the splicing domain of a 5' trans-splicing molecule does not include a branching point or PPT but includes a 5' splice acceptor or a 3' splice donor.

[0073] As used herein, “mutation” refers to any abnormal nucleic acid sequence that results in a defective protein product (e.g., a non-functional protein product, a protein product with reduced function, a protein product with abnormal function, and / or a protein product produced in less or more than normal amounts). Examples of mutations include base pair mutations (e.g., single nucleotide polymorphisms), missense mutations, frameshift mutations, deletions, insertions, and splice mutations. In some embodiments, a mutation refers to a nucleic acid sequence in which one or more portions of the sequence differ from the corresponding wild-type nucleic acid sequence or its functional variant. In some embodiments, a mutation refers to a nucleic acid sequence that codes for a protein having a different amino acid sequence from the corresponding wild-type protein or its functional variant. A “mutant exon” (e.g., a mutant ABCA4 exon) refers to an exon sequence that reflects a mutation within an exon or a different region, such as a hidden exon resulting from a mutation within an intron.

[0074] As used herein, the term "ABCA4" refers to a polynucleotide (e.g., RNA (e.g., pre-mRNA or mRNA) or DNA) encoding a retina-specific ATP-binding cassette transporter. An exemplary pre-mRNA sequence of the functional human ABCA4 gene is shown in SEQ ID NO:6. An exemplary genomic DNA sequence of the functional (wild-type) human ABCA4 gene is shown in NCBI Reference Sequence:NG_009073. The amino acid sequence of the exemplary ABCA4 protein is shown in Protein Accession No.P78363.

[0075] The exons and introns of ABCA4 are identified herein as shown in Table 1 below and can be mapped onto the ABCA4 premRNA molecule of SEQ ID NO:6. Each exon and intron of ABCA4 is identified herein according to the reference number in the first (leftmost) column. The size (base pairs; bp) of each exon and intron is shown in the second and third columns. The fourth column shows the length of the cDNA molecule corresponding to the 5' end exon of the corresponding intron number. The fifth column shows the length of the cDNA molecule corresponding to the 3' end mRNA of the corresponding intron number.

[0076] (Table 1) Overview of exons and introns of ABCA4 TIFF2026095408000002.tif221138

[0077] As used herein, “target ABCA4 intron” refers to one of the 49 ABCA4 introns identified in Table 1 above. The nucleic acid sequence identification of each ABCA4 intron sequence is shown in Table 2 below. The term “target ABCA4 intron” encompasses variants of the ABCA4 introns provided herein, for example, intron sequences having 90-100% homology to the sequences provided herein (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the sequences provided herein), where the location of the variant intron on the ABCA4 gene corresponds to that provided herein (e.g., with respect to adjacent exons as shown in Table 1).

[0078] (Table 2) ABCA4 Intron Sequence TIFF2026095408000003.tif21764

[0079] As used herein, the term "CEP290" refers to the polynucleotide (e.g., RNA (e.g., pre-mRNA or mRNA) or DNA) encoding centrosome protein 290. An exemplary pre-mRNA sequence of the functional human CEP290 gene is shown in SEQ ID NO:113. An exemplary genomic DNA sequence of the functional (wild-type) human CEP290 gene is shown in NCBI Reference Sequence:NG_008417. The amino acid sequence of an exemplary human centrosome protein 290 protein is shown in Protein Accession No.O15078.

[0080] The exons and introns of CEP290 are identified herein as shown in Table 3 below and can be mapped onto the CEP290 premRNA molecule with SEQ ID NO: 112. Each exon and intron of CEP290 is identified herein according to the reference number in the first (leftmost) column. The size (base pairs; bp) of each exon and intron is shown in the second and third columns. The fourth column shows the length of the cDNA molecule corresponding to the 5' end exon of the corresponding intron number. The fifth column shows the length of the cDNA molecule corresponding to the 3' end mRNA of the corresponding intron number.

[0081] (Table 3) Overview of exons and introns of CEP290 TIFF2026095408000004.tif238123

[0082] As used herein, “target CEP290 intron” refers to one of the 53 CEP290 introns identified in Table 3 above. The nucleic acid sequence identification of each CEP290 intron sequence is shown in Table 4 below. The term “target CEP290 intron” encompasses variants of the CEP290 introns provided herein, for example, intron sequences having 90-100% homology to the sequences provided herein (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the sequences provided herein), where the location of the variant intron on the CEP290 gene corresponds to that provided herein (e.g., with respect to adjacent exons as shown in Table 3).

[0083] (Table 4) CEP290 intron sequence TIFF2026095408000005.tif23364

[0084] As used herein, the term “subject” includes any mammal requiring such treatment or preventive measures, such as humans. Other mammals requiring such treatment or preventive measures include dogs, cats or other domesticated animals, horses, livestock, laboratory animals, such as non-human primates. The subject may be male or female. In one embodiment, the subject has a disease or disorder caused by a mutation in the ABCA4 gene (e.g., Stargardt disease, e.g., Stargardt disease type 1) or a disease or disorder caused by a mutation in the CEP290 gene (e.g., autosomal recessive disorder, e.g., LCA 10). In another embodiment, the subject is at risk of developing a disease or disorder caused by a mutation in the ABCA4 gene or a disease or disorder caused by a mutation in the CEP290 gene. In another embodiment, the subject exhibits clinical signs of a disease or disorder caused by a mutation in the ABCA4 gene (e.g., Stargardt disease) or a disease or disorder caused by a mutation in the CEP290 gene (e.g., LCA 10). The subjects may be of any age at which treatment or prophylactic care may be beneficial. For example, in some embodiments, the subjects are 0–5 years, 5–10 years, 10–20 years, 20–30 years, 30–50 years, 50–70 years, or over 70 years. In other embodiments, the subjects are 12 months or older, 18 months or older, 2 years or older, 3 years or older, 4 years or older, 5 years or older, 6 years or older, 7 years or older, 8 years or older, 9 years or older, or 10 years or older. In other embodiments, the subjects have viable retinal cells.

[0085] As used herein, the terms “disorder associated with mutation” or “disorder associated with mutation” indicate a correlation between the disorder and the mutation. In some embodiments, a disorder associated with a mutation is known, or suspected, to be caused entirely, partially, directly, or indirectly, by the mutation. For example, a person having the mutation may be at risk of developing the disorder, and that risk may further depend on other factors, such as other (e.g., unrelated) mutations (e.g., within the same gene or a different gene) or environmental factors.

[0086] As used herein, the term “treatment” or its grammatical derivatives are defined as reducing the progression of a disease, reducing the severity of disease symptoms, delaying the progression of disease symptoms, eliminating disease symptoms, or delaying the onset of a disease.

[0087] As used herein, the term “prevention” of a disorder, or its grammatical derivatives thereof, is defined as reducing the risk of developing a disease, for example, as a preventive treatment for a subject at risk of developing a disorder associated with a mutation. A subject can be characterized as being at “risk” of developing a disorder by identifying the mutation associated with the disorder in accordance with any appropriate method known in the art or described herein. In some embodiments, a subject at risk of developing a disorder has one or more ABCA4 or CEP290 mutations associated with the disorder. Additionally or alternatively, a subject can be characterized as being at “risk” of developing a disorder if they have a family history of the disorder.

[0088] Treatment or prevention of the target disorder may be carried out by directly administering the trans-splicing molecule (e.g., within an AAV vector or AAV particle) to the target. Alternatively, the trans-splicing molecule may be administered to host cells containing it.

[0089] The term “administer” or its grammatical derivatives, as used in the methods herein, indicates the delivery of the composition or ex vivo-treated cells to a subject that requires it, for example, a subject having a mutation or defect in a target gene. For example, in one embodiment where ocular cells are targeted, the method involves delivering the composition to photoreceptor cells or other ocular cells by subretinal injection. In another embodiment, intravitreal injection into ocular cells or injection into ocular cells via the eyelid vein may be used. In yet another embodiment, the composition is administered intravenously. Further methods of administration may be selected by those skilled in the art in view of this disclosure.

[0090] Codon optimization refers to the modification of a nucleic acid sequence to alter individual nucleic acids without altering the encoded amino acids. Sequences thus modified are referred to herein as “codon-optimized.” This process may be performed on any sequence described herein to enhance expression or stability. Codon optimization may be performed in the manner described, for example, in U.S. Patents 7,561,972, 7,561,973, and 7,888,112, each incorporated herein in whole by reference. Sequences surrounding the translation initiation site may be converted to a consensus Kozak sequence according to known methods. See, for example, Kozak et al, Nucleic Acids Res. 15(20):8125-8148, incorporated herein in whole by reference.

[0091] The term "homologous" refers to the degree of sequence identity between two nucleic acid sequences. Homologous sequences are determined by comparing two sequences aligned under optimal conditions across the entire comparison sequence. The comparison sequence in this specification may have additions or deletions (e.g., gaps) within the two sequences in an optimally aligned state. Such sequence homology can be calculated, for example, by performing alignment using the ClustalW algorithm (Nucleic Acid Res., 1994, 22(22):4673 4680). Alternatively, commonly available sequence analysis software, such as Vector NTI, GENETYX, BLAST, or analysis tools provided by public databases may also be used.

[0092] The term "pharmaceutically acceptable" means that it is safe for administration to mammals, such as humans. In some embodiments, a pharmacopoeia is approved by a federal or state regulatory agency, or is listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, and more specifically, in humans.

[0093] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle administered together with the therapeutic molecule (e.g., the trans-splicing molecule of the present invention or a vector or cell containing the trans-splicing molecule). Examples of suitable pharmaceutical carriers are found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA., 2 nd This is described in edition 2005.

[0094] The terms “a” and “an” mean “one or more of.” For example, “a gene” should be understood to mean one or more such genes. Therefore, the terms “a” and “an,” “one or more of a (or an),” and “at least one of a (or an)” are used interchangeably in this specification.

[0095] In this specification, the term "approximately" refers to a value within ±10% of the baseline value, unless otherwise specified.

[0096] II. Trans-splicing molecules This specification provides for ABCA4 trans-splicing molecules and CEP290 trans-splicing molecules.

[0097] ABCA4 trans-splicing molecule The present invention features nucleic acid trans-splicing molecules useful for treating diseases and disorders associated with mutations in the ABCA4 gene by replacing one or more exons within the ABCA4 gene (e.g., an ABCA4 gene having a mutant ABCA4 exon). In some embodiments, the nucleic acid trans-splicing molecule is a preRNA trans-splicing molecule (RTM). The design of the trans-splicing molecule allows for the replacement of a defective or mutant portion of a premRNA exon(s) with a nucleic acid sequence, for example, an exon(s) having a functional (e.g., normal) sequence without the mutation. The functional sequence may be a wild-type, naturally occurring sequence, or a modified sequence with several other modifications, such as codon optimization.

[0098] In one embodiment, the trans-splicing molecule is configured to modify one or more mutations located in the 3' portion of the ABCA4 gene. In another embodiment, the trans-splicing molecule is configured to modify one or more mutations located in the 5' portion of the ABCA4 gene. The trans-splicing molecules provided herein perform the function of repairing defective genes in target cells by replacing the defective premRNA gene sequence, thereby removing the defective portion of the target premRNA and resulting in a functional ABCA4 gene that can transcribe a functional gene product within the cell.

[0099] The present invention provides a trans-splicing molecule having a binding domain configured to bind to a target ABCA4 intron, a splicing domain configured to mediate trans-splicing, and a coding domain having one or more functional ABCA4 exons. In a 5' trans-splicing molecule, the coding domain, splicing site, and binding domain are functionally linked in the 5' to 3' direction so that the trans-splicing molecule is configured such that the 5' end of an endogenous gene is replaced by a coding domain containing a functional ABCA4 exon that replaces a mutant ABCA4 exon. Conversely, in a 3' trans-splicing molecule, the coding domain, splicing site, and binding domain are functionally linked in the 3' to 5' direction so that the trans-splicing molecule is configured such that the 3' end of an endogenous gene is replaced by a coding domain containing a functional ABCA4 exon that replaces a mutant ABCA4 exon. In some embodiments, the splicing domain is located within an artificial intron that links the binding domain to the coding domain. The artificial intron may include further components, such as spacers.

[0100] In some embodiments, the transsplicing molecule or its coding domain has a length of up to 4,700 nucleotide bases (e.g., a length of 200-300 nucleotide bases, a length of 300-400 nucleotide bases, a length of 400-500 nucleotide bases, a length of 500-600 nucleotide bases, a length of 600-700 nucleotide bases, a length of 700-800 nucleotide bases, a length of 800-900 nucleotide bases, 9 Lengths of 00-1,000 nucleotide bases, 1,000-1,500 nucleotide bases, 1,500-2,000 nucleotide bases, 2,000-2,500 nucleotide bases, 2,500-3,000 nucleotide bases, or 3,000-4,000 nucleotide bases, for example, 3,100-3,800 nucleotide bases, 3,200-3,700 nucleotide bases, or 3,300- Length of 3,500 nucleotide bases, for example, length of 3,000-3,100 nucleotide bases, length of 3,100-3,200 nucleotide bases, length of 3,200-3,300 nucleotide bases, length of 3,300-3,400 nucleotide bases, length of 3,400-3,500 nucleotide bases, length of 3,500-3,600 nucleotide bases, length of 3,600-3,700 nucleotide bases, length of 3,700-3,800 nucleotides The length of the rheotide base is the length of 3,800 to 3,900 nucleotide bases, or the length of 3,900 to 4,000 nucleotide bases (for example, the length of approximately 2,918 nucleotide bases, approximately 3,328 nucleotide bases, approximately 3,522 nucleotide bases, approximately 3,607 nucleotide bases, approximately 3,632 nucleotide bases, approximately 3,494 nucleotide bases, or approximately 3,300 nucleotide bases).

[0101] Due to the large size of the ABCA4 gene and the size constraints of AAV delivery, a single trans-splicing molecule configured for packaging within an AAV vector may not cover the entire range of all mutations in the ABCA4 gene that may be associated with the disorder, thereby preventing mutation correction across the entire length of the ABCA4 gene. Therefore, the trans-splicing molecule of the present invention may be adapted as part of a method described later for correcting multiple mutations across the entire length of the ABCA4 gene.

[0102] The ABCA4 gene targeted by the trans-splicing molecules described herein contains one or more mutations associated with (e.g., causing or correlated with) a disease, such as Stargardt disease (e.g., Stargardt disease type 1). An exemplary DNA sequence of the functional (wild-type) human ABCA4 gene is shown in NCBI Reference Sequence:NG_009073. The amino acid sequence of an exemplary retina-specific ATP-binding cassette transporter protein expressed by ABCA4 is shown in Protein Accession No.P78363.

[0103] In addition to these publicly available sequences, the database also includes any subsequently acquired modifications or naturally occurring, conserved, non-disease-causing variant sequences present in human or other mammalian populations. This also includes those that result in further conserved nucleotide substitutions or codon optimizations. Furthermore, sequences obtained by accession numbers in the database may be used to search for homologous sequences in the same or a different mammalian organism.

[0104] It is expected that the ABCA4 nucleic acid sequence and the resulting cleaved protein or amino acid fragments may tolerate certain minor nucleic acid-level modifications, such as silent modifications to nucleotide bases, including preference codons. In other embodiments, nucleic acid base modifications that alter amino acids to improve the expression of the resulting peptide / protein (e.g., codon optimization) are expected. Similar modifications to the fragments also include allelic mutations caused by the innate degeneracy of the genetic code.

[0105] This also includes analogs or modified forms of coding protein fragments provided herein as modified versions of the ABCA4 gene. Typically, such analogs differ from the specifically identified protein by only one to four codon changes. Conservative substitutions occur within amino acid families related to side chains and chemical properties.

[0106] The nucleic acid sequence of the functional ABCA4 gene may be derived from any mammal that expresses the functional retina-specific ATP-binding cassette transporter in its native state, or a homolog thereof. In other embodiments, certain modifications are made to the ABCA4 gene sequence to promote its expression in target cells. Such modifications include codon optimization.

[0107] In some embodiments, the disorder associated with the mutation within ABCA4 is an autosomal recessive disorder, such as Stargardt disease. In some specific cases involving subjects with autosomal recessive disorders, the subject has the mutation in both alleles of ABCA4. Compositions containing trans-splicing molecules can correct the mutation in both alleles regardless of the location of the mutation within the ABCA4 gene. For example, for a subject having a mutant ABCA4 exon 1 in the first allele and a mutant ABCA4 exon 30 in the second allele, this specification provides a composition having a 5' trans-splicing molecule that replaces mutant ABCA4 exon 1 and a 3' trans-splicing molecule that replaces mutant ABCA4 exon 30. In such embodiments, these two trans-splicing molecules may be co-delivered as part of the same AAV vector or delivered in separate AAV vectors (e.g., if both trans-splicing molecules exceed the AAV packaging limit).

[0108] Alternatively, in embodiments in which two or more mutations that can be replaced by the same trans-splicing molecule are located in a portion of the ABCA4 gene, one or more exons containing the mutations can be replaced by a single trans-splicing molecule having a coding region containing a functional ABCA4 exon. Specific mutations within the ABCA4 exon are also described in International Patent Application Publication No. 2017 / 087900, which is incorporated herein by reference.

[0109] ABCA4 Code Domain In some embodiments, the coding domain of the 5' trans-splicing molecule includes all ABCA4 exons (e.g., functional ABCA4 exons) located on the 5' side of the target ABCA4 intron. For example, in an embodiment where the 5' trans-splicing molecule targets ABCA4 intron 19, the coding domain includes functional ABCA4 exons 1-19. In such embodiments, the coding domain is approximately 2918 bp long. In an embodiment where the 5' trans-splicing molecule targets ABCA4 intron 22, the coding domain includes functional ABCA4 exons 1-22. In such embodiments, the coding domain is approximately 3328 bp long. In an embodiment where the 5' trans-splicing molecule targets ABCA4 intron 23, the coding domain includes functional ABCA4 exons 1-23. In an embodiment characterized by a 5' trans-splicing molecule having a coding domain containing functional ABCA4 exons 1-23, the coding domain is approximately 3,522 bp in length. In an embodiment in which the 5' trans-splicing molecule targets ABCA4 intron 24, the coding domain contains functional ABCA4 exons 1-24. In an embodiment characterized by a 5' trans-splicing molecule having a coding domain containing functional ABCA4 exons 1-24, the coding domain is approximately 3,607 bp in length. An example of the aforementioned embodiment of a 5' trans-splicing molecule targeting ABCA4 is shown in the lower left portion of Figure 1.

[0110] In some embodiments, the coding domain of the 3' trans-splicing molecule contains one or more of the ABCA4 exons 20-50. For example, in an embodiment in which the 3' trans-splicing molecule targets ABCA4 intron 22, the coding domain contains functional ABCA4 exons 23-50. In such embodiments featuring a 3' trans-splicing molecule having a coding domain containing functional ABCA4 exons 23-50, the coding domain is approximately 3,632 bp long. In an embodiment in which the 3' trans-splicing molecule targets ABCA4 intron 23, the coding domain contains functional ABCA4 exons 24-50. In such embodiments featuring a 3' trans-splicing molecule having a coding domain containing functional ABCA4 exons 24-50, the coding domain is approximately 3,494 bp long. In an embodiment in which the 3' trans-splicing molecule targets ABCA4 intron 24, the coding domain contains functional ABCA4 exons 25-50. In this embodiment, which features a 3' trans-splicing molecule having a coding domain containing functional ABCA4 exons 25-50, the coding domain is approximately 3,300 bp in length. An example of the above embodiment of a 3' trans-splicing molecule targeting ABCA4 is shown in the upper right portion of Figure 1.

[0111] In some embodiments, the code domain includes a functional ABCA4 exon of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28.

[0112] In some cases, both mutations are located in the 5' portion of the target gene, and a 5' trans-splicing molecule is selected such that both mutations are corrected. In one embodiment, the binding domain binds to intron 19 and the coding domain contains functional ABCA4 exons 1-19. In another embodiment, the binding domain binds to intron 22 and the coding domain contains functional ABCA4 exons 1-22. In another embodiment, the binding domain binds to intron 23 and the coding domain contains functional ABCA4 exons 1-23. In another embodiment, the binding domain binds to intron 24 and the coding domain contains functional ABCA4 exons 1-24. Alternatively, in cases where both mutations are located in the 3' portion of the target gene, a 3' trans-splicing molecule is selected such that both mutations are corrected. In one embodiment, the binding domain binds to intron 22 and the coding domain contains functional ABCA4 exons 23-50. In another embodiment, the binding domain binds to intron 23 and the coding domain contains functional ABCA4 exons 24-50. In one embodiment, the binding domain is bound to intron 24, and the coding domain includes functional ABCA4 exons 25-50.

[0113] As an example, the premRNA ABCA4 3' trans-splicing molecule functions as follows: A chimeric mRNA is created by a trans-splicing reaction mediated by a spliceosome, combining the 5' splice site of an endogenous target premRNA with the 3' splice site of the trans-splicing molecule. The trans-splicing molecule binds to the target ABCA4 intron of the endogenous target premRNA through specific base pairing, replacing the entire 3' sequence upstream of the targeted intron of the endogenous ABCA4 gene with the coding domain of the trans-splicing molecule, which contains a functional ABCA4 exon sequence.

[0114] The 3' trans-splicing molecule comprises a binding domain that binds to the mutated or defective 5' side target ABCA4 intron, an artificial intron including an optional spacer and a 3' splice site, and a coding domain encoding all exons of the ocular target gene on the 3' side of the binding domain to the target. The 5' trans-splicing molecule comprises a binding domain that binds to the mutated or defective 3' side target ABCA4 intron, a 5' splice site, an optional spacer, and a coding domain encoding all exons of the ocular target gene on the 5' side of the binding domain to the target.

[0115] In some embodiments, the coding domain includes a complementary DNA (cDNA) sequence. For example, one or more functional ABCA4 exons within the coding domain may be cDNA sequences. In some embodiments, the entire coding domain is a cDNA sequence. Additionally or alternatively, all or part of the coding domain, or one or more functional ABCA4 exons, may be naturally occurring sequences (e.g., sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with endogenous ABCA4 exons).

[0116] In some embodiments, all or part of the coding domain or one or more functional ABCA4 exons are codon-optimized sequences in which the nucleic acid sequence is modified to promote expression or stability without, for example, altering the encoded amino acids. Codon optimization can be performed in the form of, for example, those described in U.S. Patents 7,561,972, 7,561,973 and 7,888,112, each of which is incorporated herein by reference in whole. For delivery by recombinant AAV as described herein, in one embodiment, the coding domain is a length of up to 4,000 nucleotide bases (e.g., a length of 3,000 to 4,000 nucleotide bases, a length of 3,100 to 3,800 nucleotide bases, a length of 3,200 to 3,700 nucleotide bases, or a length of 3,300 to 3,500 nucleotide bases, e.g., a length of 3,000 to 3,100 nucleotide bases, a length of 3,100 to 3,200 nucleotides) The nucleic acid sequence may have a length of one base, a length of 3,200-3,300 nucleotide bases, a length of 3,300-3,400 nucleotide bases, a length of 3,400-3,500 nucleotide bases, a length of 3,500-3,600 nucleotide bases, a length of 3,600-3,700 nucleotide bases, a length of 3,700-3,800 nucleotide bases, a length of 3,800-3,900 nucleotide bases, or a length of 3,900-4,000 nucleotide bases.

[0117] ABCA4 binding domain The trans-splicing molecule of the present invention features a binding domain configured to bind to a target ABCA4 intron. In one embodiment, the binding domain is a nucleic acid sequence complementary to the sequence of the target ABCA4 premRNA (e.g., the target ABCA4 intron), thereby promoting trans-splicing between the trans-splicing molecule and the target ABCA4 premRNA while suppressing endogenous target cis-splicing, for example, creating a chimeric molecule having a portion of endogenous ABCA4 mRNA and a coding domain having one or more functional ABCA4 exons. In some embodiments, the binding domain is antisense-oriented with respect to the sequence of the target ABCA4 intron.

[0118] 5' trans-splicing molecules typically bind to the 3' target ABCA4 intron of the mutation, while 3' trans-splicing molecules typically bind to the 5' target ABCA4 intron of the mutation. In one embodiment, the binding domain contains a portion of a sequence complementary to the target ABCA4 intron. In one embodiment of this specification, the binding domain is a nucleic acid sequence complementary to the intron closest to (i.e., adjacent to) the exon sequence to be modified.

[0119] In another embodiment, the binding domain is configured to target an intron sequence adjacent to the 3' splice signal sequence or 5' splice signal sequence of the target intron. In yet another embodiment, the binding domain sequence may bind to a portion of an adjacent exon in addition to the target intron.

[0120] Therefore, in some cases, the binding domain specifically binds to the endogenous mutant target premRNA, anchoring the coding domain of the trans-splicing molecule to the premRNA, allowing trans-splicing to occur at a precise location within the target ABCA4 gene. Subsequently, processing by spliceosomes in the nucleus may mediate successful trans-splicing of the modified exon with the disease-causing mutant exon.

[0121] In some specific embodiments, the trans-splicing molecule features a binding domain containing sequences that bind to more than one site on the target pre-mRNA. The binding domain may contain any number of nucleotides necessary to stably bind to the target pre-mRNA and enable trans-splicing with the coding domain. In one embodiment, the binding domain is selected using mFOLD structural analysis of the resulting loop (Zuker, Nucleic Acids Res. 2003, 31(13):3406-3415).

[0122] A suitable target-binding domain may be 10 to 500 nucleotides long. In some embodiments, the binding domain is 20 to 400 nucleotides long. In some embodiments, the binding domain is 50 to 300 nucleotides long. In some embodiments, the binding domain is 100 to 200 nucleotides long. In some embodiments, the binding domain may be 10 to 20 nucleotides long (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides long), 20 to 30 nucleotides long (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides long), or 30 to 40 nucleotides long (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides long). Length of nucleotides), length of 40-50 nucleotides (e.g., 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides), length of 50-60 nucleotides (e.g., 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides), length of 60-70 nucleotides (e.g., 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 nucleotides) Length), length of 70-80 nucleotides (e.g., length of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 nucleotides), length of 80-90 nucleotides (e.g., length of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90 nucleotides), length of 90-100 nucleotides (e.g., length of 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides) , lengths of 100-110 nucleotides (e.g., lengths of 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110 nucleotides), lengths of 110-120 nucleotides (e.g., lengths of 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 or 120 nucleotides), lengths of 120-130 nucleotides (e.g., lengths of 120, 121, 122, 123, 124, 125,Lengths of 126, 127, 128, 129 or 130 nucleotides), lengths of 130-140 nucleotides (for example, lengths of 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 or 140 nucleotides), lengths of 140-150 nucleotides (for example, lengths of 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 nucleotides), lengths of 150-160 nucleotides (for example (For example, lengths of 150, 151, 152, 153, 154, 155, 156, 157, 158, 159 or 160 nucleotides), lengths of 160-170 nucleotides (for example, lengths of 160, 161, 162, 163, 164, 165, 166, 167, 168, 169 or 170 nucleotides), lengths of 170-180 nucleotides (for example, lengths of 170, 171, 172, 173, 174, 175, 176, 177, 178, 179 or 180 nucleotides) Length of nucleotides), length of 180-190 nucleotides (e.g., length of 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 or 190 nucleotides), length of 190-200 nucleotides (e.g., length of 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 or 200 nucleotides), length of 200-210 nucleotides, length of 210-220 nucleotides, length of 220-230 nucleotides The length of the target binding domain is 230–240 nucleotides, 240–250 nucleotides, 250–260 nucleotides, 260–270 nucleotides, 270–280 nucleotides, 280–290 nucleotides, 290–300 nucleotides, 300–350 nucleotides, 350–400 nucleotides, 400–450 nucleotides, or 450–500 nucleotides. In some embodiments, the binding domain is approximately 150 nucleotides long. In other embodiments, the target binding domain may contain nucleic acid sequences up to 750 nucleotides long. In other embodiments,The target-binding domain may contain a nucleic acid sequence with a length of up to 1000 nucleotides. In another embodiment, the target-binding domain may contain a nucleic acid sequence with a length of up to 2000 nucleotides or more.

[0123] In some embodiments, the specificity of the trans-splicing molecule can be increased by increasing the length of the target-binding domain. Other lengths may be used depending on the lengths of the other components of the trans-splicing molecule.

[0124] The binding domain may be 80% to 100% complementary to the target intron so that it can stably hybridize with the target intron. For example, in some embodiments, the binding domain may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the target intron. The degree of complementarity is selected by those skilled in the art based on the need to maintain the nucleic acid construct, which includes the trans-splicing molecule and the sequences required for expression and inclusion in rAAV, within a nucleotide base limit of 3,000 or up to 4,000. This selection of sequences and hybridization strength depends on the complementarity and length of the nucleic acid.

[0125] Any of the aforementioned binding domains may bind to binding sites within intron 19 (SEQ ID NO: 25), intron 22 (SEQ ID NO: 28), intron 23 (SEQ ID NO: 29), or intron 24 (SEQ ID NO: 30).

[0126] In some specific examples of the present invention, the trans-splicing molecule is a 5' trans-splicing molecule characterized by a binding domain that binds to intron 19 of ABCA4 (SEQ ID NO: 25) and includes a coding domain having functional ABCA4 exons 1-19. In some embodiments, the binding site includes one or more nucleotides from SEQ ID NO: 25 (e.g., one or more nucleotides from SEQ ID NO: 25 1,670-2,174, one or more nucleotides from SEQ ID NO: 25 1,810-2,000, one or more nucleotides from SEQ ID NO: 25 1,870-2,000, or one or more nucleotides from SEQ ID NO: 25 1,920-2,000).

[0127] In some embodiments, the trans-splicing molecule is a 5' trans-splicing molecule characterized by a binding domain that binds to intron 22 of ABCA4 (SEQ ID NO: 28) and includes a coding domain having functional ABCA4 exons 1-22. In some embodiments, the binding site includes one or more nucleotides from SEQ ID NO: 28, specifically nucleotides 60-570, 600-800, or 900-1,350 (for example, one or more nucleotides from SEQ ID NO: 28, specifically nucleotides 70-250).

[0128] Alternatively, the trans-splicing molecule may be a 3' trans-splicing molecule and may feature a binding domain that binds to intron 22 of ABCA4 (SEQ ID NO: 28). This trans-splicing molecule may contain a coding domain having functional ABCA4 exons 23-50. In some embodiments, the binding site contains one or more nucleotides from SEQ ID NO: 28, either nucleotides 1-510 or 880-1,350.

[0129] In other embodiments, the trans-splicing molecule is a 5' trans-splicing molecule characterized by a binding domain that binds to intron 23 of ABCA4 (SEQ ID NO: 29) and includes a coding domain having functional ABCA4 exons 1-23. In some embodiments, the binding site includes one or more nucleotides from SEQ ID NO: 29, either nucleotides 80-570 or 720-1,081.

[0130] Alternatively, the trans-splicing molecule may be a 3' trans-splicing molecule, characterized by a binding domain that binds to intron 23 of ABCA4 (SEQ ID NO: 29) and a coding domain having functional ABCA4 exons 24-50. In some embodiments, the binding site includes one or more nucleotides from SEQ ID NO: 29 (e.g., one or more nucleotides from SEQ ID NO: 29 230-1081, one or more nucleotides from SEQ ID NO: 29 250-400, or one or more nucleotides from SEQ ID NO: 29 690-850).

[0131] In some embodiments, the trans-splicing molecule is a 5' trans-splicing molecule characterized by a binding domain that binds to intron 24 of ABCA4 (SEQ ID NO: 30) and includes a coding domain having functional ABCA4 exons 1-24. In some embodiments, the binding site includes one or more nucleotides from SEQ ID NO: 30, either 600-1,250 or 1,490-2,660 (e.g., one or more nucleotides from SEQ ID NO: 30, either 1,000-1,200).

[0132] In other embodiments, the trans-splicing molecule is a 3' trans-splicing molecule characterized by a binding domain that binds to intron 24 of ABCA4 (SEQ ID NO: 30) and includes a coding domain having functional ABCA4 exons 25-50. In some embodiments, the binding site includes one or more nucleotides from SEQ ID NO: 30, including nucleotides 1-250, 300-2,100, or 2,200-2,692 (for example, one or more nucleotides from SEQ ID NO: 30, including nucleotides 360-610, or one or more nucleotides from SEQ ID NO: 30, including nucleotides 750-1,110).

[0133] CEP290 trans-splicing molecule The present invention features nucleic acid trans-splicing molecules useful for treating diseases and disorders associated with mutations in the CEP290 gene by replacing one or more exons within the CEP290 gene (e.g., a CEP290 gene with a mutation in intron 26). In some embodiments, the nucleic acid trans-splicing molecule is a preRNA trans-splicing molecule (RTM). The design of the trans-splicing molecule allows for the replacement of a defective or mutated portion of premRNA with a nucleic acid sequence, for example, one or more exons having a functional (e.g., normal) sequence without the mutation. The functional sequence may be a wild-type, naturally occurring sequence, or a modified sequence with several other modifications, such as codon optimization.

[0134] In one embodiment, the trans-splicing molecule is configured to modify one or more mutations located in the 5' portion of the CEP290 gene. The trans-splicing molecule provided herein functions to repair a defective gene in a target cell by replacing the defective premRNA gene sequence, thereby resulting in a functional CEP290 gene capable of transcribing a functional gene product within the cell.

[0135] The present invention provides a trans-splicing molecule having a binding domain configured to bind to a target CEP290 intron, a splicing domain configured to mediate trans-splicing, and a coding domain having one or more functional CEP290 exons. In a 5' trans-splicing molecule, the coding domain, splice site, and binding domain are functionally linked in the 5'-to-3' direction such that the trans-splicing molecule is configured such that the 5' end of an endogenous gene is replaced by a coding domain containing a functional CEP290 exon for modifying a mutant CEP290 premRNA. In some embodiments, the splicing domain is located within an artificial intron that links the binding domain to the coding domain. The artificial intron may include further components, such as spacers.

[0136] In some embodiments, the trans-splicing molecule has a length of up to 4,700 nucleotide bases (for example, a length of 3,000-4,000 nucleotide bases, a length of 3,100-3,800 nucleotide bases, a length of 3,200-3,700 nucleotide bases, or a length of 3,300-3,500 nucleotide bases, for example, a length of 3,000-3,100 nucleotide bases, a length of 3,100-3,200 nucleotide bases, a length of 3,200-3,300 nucleotide bases, a length of 3,300-3,400 nucleotide bases, 3, The lengths are approximately 400-3,500 nucleotide bases, 3,500-3,600 nucleotide bases, 3,600-3,700 nucleotide bases, 3,700-3,800 nucleotide bases, 3,800-3,900 nucleotide bases, or 3,900-4,000 nucleotide bases (for example, approximately 2,991 nucleotide bases, approximately 3,103 nucleotide bases, approximately 3,309 nucleotide bases, approximately 3,461 nucleotide bases, or approximately 3,573 nucleotide bases).

[0137] The CEP290 gene targeted by the trans-splicing molecules described herein contains one or more mutations associated with (e.g., causing or correlated with) a disease, such as Leber congenital amaurosis (e.g., LCA 10). An exemplary DNA sequence of the functional (wild-type) human CEP290 gene is shown in NCBI Reference Sequence:NG_008417. An exemplary amino acid sequence of centrosome protein 290 is shown in Protein Accession No.O15078.

[0138] In addition to these publicly available sequences, the database also includes any subsequently acquired modifications or naturally occurring, conserved, non-disease-causing variant sequences present in human or other mammalian populations. This also includes those that result in further conserved nucleotide substitutions or codon optimizations. Furthermore, sequences obtained by accession numbers in the database may be used to search for homologous sequences in the same or a different mammalian organism.

[0139] It is expected that the CEP290 nucleic acid sequence and the resulting cleaved protein or amino acid fragments may tolerate certain minor nucleic acid-level modifications, such as silent nucleotide base modifications, including preferred codons. In other embodiments, nucleic acid base modifications that alter amino acids to improve the expression of the resulting peptide / protein (e.g., codon optimization) are expected. Similar modifications to the fragments also include allelic mutations caused by the innate degeneracy of the genetic code.

[0140] This also includes analogs or modified forms of coding protein fragments provided herein as modified versions of the CEP290 gene. Typically, such analogs differ from the specifically identified protein by only one to four codon changes. Conservative substitutions occur within amino acid families related to side chains and chemical properties.

[0141] The nucleic acid sequence of the functional CEP290 gene may be derived from any mammal that expresses functional centrosome protein 290 in its native state, or a homolog thereof. In other embodiments, certain modifications are made to the CEP290 gene sequence to promote its expression in target cells. Such modifications include codon optimization.

[0142] For information on CEP290 mutations, please refer to the CCHMC Molecular Genetics Laboratory Mutation Database, LOVD v.2.0. Specific mutations within the CEP290 exon are also described in International Patent Application Publication No. 2017 / 087900, which is incorporated herein by reference. Table 3 above shows information regarding the size and location of each exon and intron of CEP290.

[0143] In some embodiments, disorders associated with mutations within CEP290 are autosomal recessive disorders, such as LCA 10.

[0144] Code Domain In some embodiments, the coding domain of the 5' trans-splicing molecule includes all CEP290 exons (e.g., functional CEP290 exons) located on the 5' side of the target CEP290 intron. For example, in an embodiment in which the 5' trans-splicing molecule targets CEP290 intron 26, the coding domain includes functional CEP290 exons 2-26. In such embodiments, the coding domain is approximately 2,991 bp long, and the 5' trans-splicing molecule targets CEP290 intron 27, the coding domain includes functional CEP290 exons 2-27. In such embodiments, the coding domain is approximately 3,103 bp long, and the 5' trans-splicing molecule has a coding domain including functional CEP290 exons 2-27. In embodiments in which the 5' trans-splicing molecule targets CEP290 intron 28, the coding domain contains functional CEP290 exons 2-28. In such embodiments, the coding domain is approximately 3,309 bp long, and the 5' trans-splicing molecule targets CEP290 intron 29, and the coding domain contains functional CEP290 exons 2-29. In such embodiments, the coding domain is approximately 3,461 bp long, and the 5' trans-splicing molecule targets CEP290 intron 30, and the coding domain contains functional CEP290 exons 2-30. In this embodiment, which features a 5' trans-splicing molecule having a coding domain containing functional CEP290 exons 2-30, the coding domain is approximately 3,573 bp in length. An example of the above embodiment of a 5' trans-splicing molecule targeting CEP290 is shown in Figure 21.

[0145] In some embodiments, the code domain includes a functional CEP290 exon of 25, 26, 27, 28, or 29.

[0146] In some embodiments, the coding domain includes a complementary DNA (cDNA) sequence. For example, one or more functional CEP290 exons within the coding domain may be cDNA sequences. In some embodiments, the entire coding domain is a cDNA sequence. Additionally or alternatively, all or part of the coding domain, or one or more functional CEP290 exons, may be naturally occurring sequences (e.g., sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with endogenous CEP290 exons).

[0147] In some embodiments, the codon-optimized sequence is one in which all or part of the coding domain or one or more functional CEP290 exons are modified in a nucleic acid sequence to promote expression or stability without, for example, altering the encoded amino acids. Codon optimization can be performed in the manner described, for example, U.S. Patents 7,561,972, 7,561,973, and 7,888,112, each of which is incorporated herein by reference in whole. For delivery by recombinant AAV as described herein, in one embodiment, the coding domain may have a length of up to 4,000 nucleotide bases (for example, a length of 3,000 to 4,000 nucleotide bases, a length of 3,100 to 3,800 nucleotide bases, a length of 3,200 to 3,700 nucleotide bases, or a length of 3,300 to 3,500 nucleotide bases, for example, a length of 3,000 to 3,100 nucleotide bases, a length of 3,100 to 3,200 nucleotide bases, a length of 3,200 to 3,300 nucleotide bases, a length of 3,300 to 3,400 nucleotide bases, a length of 3,400 to 3,500 nucleotide bases, or a length of 3,500 to 3,600 nucleotide bases). The nucleic acid sequence may be of a length of 3,600 to 3,700 nucleotide bases, 3,700 to 3,800 nucleotide bases, 3,800 to 3,900 nucleotide bases, or 3,900 to 4,000 nucleotide bases (for example, a length of approximately 3,108 nucleotide bases, approximately 3,285 nucleotide bases, approximately 3,375 nucleotide bases, approximately 3,503 nucleotide bases, approximately 3,630 nucleotide bases, approximately 3,540 nucleotide bases, approximately 3,363 nucleotide bases, approximately 3,273 nucleotide bases, approximately 3,145 nucleotide bases, or approximately 3,018 nucleotide bases).

[0148] Joint domain The trans-splicing molecule of the present invention features a binding domain configured to bind to a target CEP290 intron. In one embodiment, the binding domain is a nucleic acid sequence complementary to the sequence of the target CEP290 premRNA (e.g., the target CEP290 intron), thereby promoting trans-splicing between the trans-splicing molecule and the target CEP290 premRNA while suppressing endogenous target cis-splicing, creating, for example, a chimeric molecule having a portion of endogenous CEP290 mRNA and a coding domain having one or more functional CEP290 exons. In some embodiments, the binding domain is antisense-oriented with respect to the sequence of the target CEP290 intron.

[0149] 5' trans-splicing molecules typically bind to the 3' side of the target CEP290 intron of the mutation. In one embodiment, the binding domain contains a portion of the sequence complementary to the target CEP290 intron.

[0150] In another embodiment, the binding domain is configured to target an intron sequence adjacent to the 3' splice signal sequence or 5' splice signal sequence of the target intron. In yet another embodiment, the binding domain sequence may bind to a portion of an adjacent exon in addition to the target intron.

[0151] Therefore, in some cases, the binding domain specifically binds to the endogenous mutant target premRNA, anchoring the coding domain of the trans-splicing molecule to the premRNA, allowing trans-splicing to occur at a precise location within the target CEP290 gene. Subsequently, processing by spliceosomes in the nucleus may mediate successful trans-splicing of the modified exon with the disease-causing mutant exon.

[0152] In some specific embodiments, the trans-splicing molecule features a binding domain containing sequences that bind to more than one site on the target pre-mRNA. The binding domain may contain any number of nucleotides necessary to stably bind to the target pre-mRNA and enable trans-splicing with the coding domain. In one embodiment, the binding domain is selected using mFOLD structural analysis of the resulting loop (Zuker, Nucleic Acids Res. 2003, 31(13):3406-3415).

[0153] A suitable target-binding domain may be 10 to 500 nucleotides long. In some embodiments, the binding domain is 20 to 400 nucleotides long. In some embodiments, the binding domain is 50 to 300 nucleotides long. In some embodiments, the binding domain is 100 to 200 nucleotides long. In some embodiments, the binding domain may be 10 to 20 nucleotides long (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides long), 20 to 30 nucleotides long (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides long), or 30 to 40 nucleotides long (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides long). Length of nucleotides), length of 40-50 nucleotides (e.g., 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides), length of 50-60 nucleotides (e.g., 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides), length of 60-70 nucleotides (e.g., 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 nucleotides) Length), length of 70-80 nucleotides (e.g., length of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 nucleotides), length of 80-90 nucleotides (e.g., length of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90 nucleotides), length of 90-100 nucleotides (e.g., length of 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides) , lengths of 100-110 nucleotides (e.g., lengths of 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110 nucleotides), lengths of 110-120 nucleotides (e.g., lengths of 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 or 120 nucleotides), lengths of 120-130 nucleotides (e.g., lengths of 120, 121, 122, 123, 124, 125,Lengths of 126, 127, 128, 129 or 130 nucleotides), lengths of 130-140 nucleotides (for example, lengths of 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 or 140 nucleotides), lengths of 140-150 nucleotides (for example, lengths of 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 nucleotides), lengths of 150-160 nucleotides (for example (For example, lengths of 150, 151, 152, 153, 154, 155, 156, 157, 158, 159 or 160 nucleotides), lengths of 160-170 nucleotides (for example, lengths of 160, 161, 162, 163, 164, 165, 166, 167, 168, 169 or 170 nucleotides), lengths of 170-180 nucleotides (for example, lengths of 170, 171, 172, 173, 174, 175, 176, 177, 178, 179 or 180 nucleotides) Length of nucleotides), length of 180-190 nucleotides (e.g., length of 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 or 190 nucleotides), length of 190-200 nucleotides (e.g., length of 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 or 200 nucleotides), length of 200-210 nucleotides, length of 210-220 nucleotides, length of 220-230 nucleotides The length of the target binding domain is 230–240 nucleotides, 240–250 nucleotides, 250–260 nucleotides, 260–270 nucleotides, 270–280 nucleotides, 280–290 nucleotides, 290–300 nucleotides, 300–350 nucleotides, 350–400 nucleotides, 400–450 nucleotides, or 450–500 nucleotides. In some embodiments, the binding domain is approximately 150 nucleotides long. In other embodiments, the target binding domain may contain nucleic acid sequences up to 750 nucleotides long. In other embodiments,The target-binding domain may contain a nucleic acid sequence with a length of up to 1000 nucleotides. In another embodiment, the target-binding domain may contain a nucleic acid sequence with a length of up to 2000 nucleotides or more.

[0154] In some embodiments, the specificity of the trans-splicing molecule can be increased by increasing the length of the target-binding domain. Other lengths may be used depending on the lengths of the other components of the trans-splicing molecule.

[0155] The binding domain may be 80% to 100% complementary to the target intron so that it can stably hybridize with the target intron. For example, in some embodiments, the binding domain may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the target intron. The degree of complementarity is selected by those skilled in the art based on the need to maintain the nucleic acid construct, which includes the trans-splicing molecule and the sequences required for expression and inclusion in rAAV, within a nucleotide base limit of 3,000 or up to 4,000. This selection of sequences and hybridization strength depends on the complementarity and length of the nucleic acid.

[0156] The aforementioned arbitrary binding domains may bind to binding sites within intron 26 (SEQ ID NO: 85; for example, a mutation at nucleotide 1,655 of intron 26, e.g., a substitution, or the 3' side of a mutation at nucleotide 1,655 of intron 26, e.g., a substitution), intron 27 (SEQ ID NO: 86), intron 28 (SEQ ID NO: 87), intron 29 (SEQ ID NO: 88), or intron 30 (SEQ ID NO: 89).

[0157] In some specific examples of the present invention, the trans-splicing molecule features a binding domain that binds to intron 26 of CEP290 (SEQ ID NO: 85) and includes a coding domain having functional CEP290 exons 2-26. In some embodiments, the binding site includes one or more nucleotides from SEQ ID NO: 85,980-5,383. In one embodiment, the binding site includes one or more nucleotides from SEQ ID NO: 85,348-5,838 (e.g., one or more nucleotides from SEQ ID NO: 85,348-5,700, one or more nucleotides from SEQ ID NO: 85,400-5,600, one or more nucleotides from SEQ ID NO: 85,460-5,560, at least nucleotide 5,500).

[0158] In other embodiments, the trans-splicing molecule features a binding domain that binds to intron 27 of CEP290 (SEQ ID NO: 86) and includes a coding domain having functional CEP290 exons 2-27. In some embodiments, the binding site includes one or more nucleotides from SEQ ID NO: 86, specifically nucleotides 120-680, 710-2,200, or 2,670-2,910. In some embodiments, the binding site includes one or more nucleotides from SEQ ID NO: 86, specifically nucleotides 790-2,100, for example, one or more nucleotides from SEQ ID NO: 86, specifically nucleotides 1,020-1,630. In other embodiments, the binding site includes one or more nucleotides from SEQ ID NO: 86, specifically nucleotides 1,670-2,000.

[0159] In some embodiments, the trans-splicing molecule features a binding domain that binds to intron 28 of CEP290 (SEQ ID NO: 87) and includes a coding domain having functional CEP290 exons 2-28. In some embodiments, the binding site includes one or more nucleotides from SEQ ID NO: 87, specifically nucleotides 1-390, 410-560, or 730-937. In some embodiments, the binding site includes one or more nucleotides from SEQ ID NO: 87, specifically nucleotides 1-200. In other embodiments, the binding site includes one or more nucleotides from SEQ ID NO: 87, specifically nucleotides 720-900.

[0160] In some embodiments, the trans-splicing molecule features a binding domain that binds to intron 29 of CEP290 (SEQ ID NO: 88) and includes a coding domain having functional CEP290 exons 2-29. In some embodiments, the binding site includes one or more nucleotides from SEQ ID NO: 88, specifically nucleotides 1-600, nucleotides 720-940, or nucleotides 1,370-1,790.

[0161] In other embodiments, the trans-splicing molecule features a binding domain that binds to intron 30 of CEP290 (SEQ ID NO: 89) and includes a coding domain having functional CEP290 exons 2-30. In some embodiments, the binding site includes one or more nucleotides from SEQ ID NO: 89 (880-1240), for example, one or more nucleotides from SEQ ID NO: 89 (950-1240), for example, one or more nucleotides from SEQ ID NO: 89 (1060-1240).

[0162] Splicing Domain The following splicing domains may be used in any trans-splicing molecule of the present invention (for example, any ABCA4 trans-splicing molecule or CEP290 trans-splicing molecule described herein).

[0163] The splicing domain may contain a splice site, a branching point, and / or a PPT tract that mediates trans-splicing. In some embodiments, the splicing domain has a single splice site, which indicates that the splice site is trans-splicable but not cis-splicable because there is no corresponding splice site. In some embodiments, the splicing domain of a 3' trans-splicing molecule contains a highly conserved branching point or branching site sequence, a polypyrimidine tract (PPT), and a 3' splice acceptor (AG or YAG) site and / or a 5' splice donor site. The splicing domain of a 5' trans-splicing molecule does not contain a branching point or PPT, but it does contain a 5' splice acceptor / or 3' splice donor splice site.

[0164] The splicing domain can be selected by those skilled in the art according to known methods and principles. The splicing domain provides an essential consensus motif recognized by the spliceosome. The use of the branching point and PPT follows the consensus sequence required for the execution of two phosphate transfer reactions involved in trans-splicing. In one embodiment, the branching point consensus sequence in mammals is YNYURAC (Y = pyrimidine; N = any nucleotide). The polypyrimidine tract is located between the branching point and the splice acceptor site and is important for the use of different branching points and recognition of the 3' splice site. Consensus sequences for the 5' splice donor site and 3' splice region used in RNA splicing are well known in the art. Modified consensus sequences that retain the ability to function as the 5' donor splice site and 3' splice region may also be used. Briefly, in one embodiment, the consensus sequence for the 5' splice site is the nucleic acid sequence AG / GURAGU (where / indicates the splice site). In another embodiment, if a splicing regulatory signal sequence is present, an endogenous splice site corresponding to a proximal exon may be used to maintain it at the splice site.

[0165] In one embodiment, a preferred 5' splice portion having a spacer is, This is TIFF2026095408000006.tif4128. In one embodiment, a preferred 5' splice site is AGGT.

[0166] In one embodiment, a preferred branching site for the 3'-trans-splicing molecule is 5'-TACTAAC-3'. In one embodiment, a preferred 3'-splice site is TIFF2026095408000007.tif4155. In one embodiment, a preferred 3' trans-splicing molecule PPT is The filename is TIFF2026095408000008.tif4128.

[0167] Further components and modifications In some embodiments of any trans-splicing molecule of the present invention (e.g., any ABCA4 trans-splicing molecule or CEP290 trans-splicing molecule described herein), the splicing domain is included as part of an artificial intron which may contain one or more further components. For example, the artificial intron may include a spacer region for separating the splicing domain from the target-binding domain within the trans-splicing molecule. The spacer region may be designed to include features such as (i) a stop codon that would function to block the translation of any unspliced ​​trans-splicing molecule and / or (ii) a sequence that promotes trans-splicing to the target pre-mRNA. The spacer may consist of 3 to 25 or more nucleotides, depending on the length of the other components of the trans-splicing molecule and the limitations of rAAV. In one embodiment, the spacer of a preferred 5' trans-splicing molecule is: This is TIFF2026095408000009.tif4128. In one embodiment, a preferred 3' spacer is The filename is TIFF2026095408000010.tif4128.

[0168] Other optional components of the trans-splicing molecule (e.g., as part of an artificial intron) include mini-introns and intra-intron or intra-exon enhancers (e.g., intra-intron splice enhancers, e.g., downstream intra-intron splice enhancers) or silencers that would modulate trans-splicing.

[0169] In another embodiment, the trans-splicing molecule further includes at least one safety sequence for suppressing nonspecific trans-splicing, which is incorporated within the spacer, within the binding domain, or somewhere within the trans-splicing molecule (e.g., as part of an artificial intron). This is a region of the trans-splicing molecule that suppresses nonspecific trans-splicing by covering elements of the 3' splice site and / or 5' splice site of the trans-splicing molecule with relatively weak complementarity. The trans-splicing molecule is designed such that when hybridization occurs of the binding / targeting site(s) of the trans-splicing molecule, the 3' splice site or 5' splice site is uncovered and becomes fully active. Such safety sequences include a complementary cis sequence (or possibly a separate second nucleic acid strand) that may bind to one or both sides of the branching point of the trans-splicing molecule, the pyrimidine tract, the 3' splice site and / or the 5' splice site (splicing element), or to a portion of the splicing element itself. Binding of the safety sequence can be disrupted by the target binding region of the trans-splicing molecule binding to the target pre-mRNA, thereby exposing and activating the splicing element (making trans-splicing with the target pre-mRNA available). In another embodiment, the trans-splicing molecule has a 3'UTR sequence or a ribozyme sequence appended to its 3' or 5' end.

[0170] In one embodiment, splicing-promoting sequences, such as those referred to as intra-exon splicing-promoting sequences, may also be included within the structure of the artificial intron. Further structural elements, such as polyadenylation signal sequences for modifying RNA expression / stability, or 5' splice sequences for promoting splicing, further binding regions, safety self-complementary regions, further splice sites, or protecting groups for regulating molecular stability and suppressing degradation, may be added to the artificial intron. Additionally, stop codons may be included within the trans-splicing molecular structure (e.g., as part of the artificial intron) to suppress translation of unspliced ​​trans-splicing molecules. Further elements, such as 3' hairpin structures, cyclic RNA, nucleotide base modifications, or synthetic analogs, may be incorporated into the trans-splicing molecule to promote or enhance nuclear localization and spliceosome integration, as well as intracellular stability.

[0171] In some embodiments, the binding of a trans-splicing molecule to a target premRNA is mediated by complementarity (i.e., based on the base-pairing properties of the nucleic acid), triple-helical formation, or protein-nucleic acid interactions (as described in the literature cited herein). In one embodiment, the nucleic acid trans-splicing molecule may be DNA, RNA, or a DNA / RNA hybrid molecule, where the DNA or RNA is either single-stranded or double-stranded. Also, as used herein, one of the aforementioned RNAs or DNAs may be RNA or DNA that can be hybridized, preferably under stringent conditions, for example, in 2.5 × SSC buffer at 60°C and with several washes at a low buffer concentration, for example, in 0.5 × SSC buffer at 37°C. Such nucleic acids may encode proteins that exhibit lipid phosphate phosphatase activity and / or binding to the plasma membrane. When trans-splicing molecules are synthesized in vitro, such trans-splicing molecules may be modified, for example, by modifying the base portion, glycosylation portion, or phosphate backbone to improve their stability, hybridization with target mRNA, intracellular transport, or resistance to enzymatic cleavage within the cell. For example, modification of a trans-splicing molecule to reduce its total charge may promote its intracellular uptake. Modifications may also be made to reduce its sensitivity to nucleases or chemical degradation. The nucleic acid molecule may be synthesized in a manner in which it is conjugated with another molecule, such as a peptide, a hybridization-induced crosslinking agent, a transporter, or a hybridization-induced cleavage agent.

[0172] Various other known modifications to the nucleic acid molecule can be introduced as means of increasing its intracellular stability and half-life (see also above for oligonucleotides). Possible modifications are known in the art. Modifications that can be performed on the structure of synthetic trans-splicing molecules include main chain modifications.

[0173] III. Recombinant AAV molecules Any suitable nucleic acid vector can be used in connection with the compositions and methods of the present invention to design and assemble trans-splicing molecules and recombinant adeno-associated virus (AAV) components. In one embodiment, the vector is a recombinant AAV that carries a trans-splicing molecule and is driven by a promoter that expresses the trans-splicing molecule in a selected cell of interest. Methods for assembling recombinant vectors are known in the art. See, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, MA et al., Nat. Medic, 2001, 7(l):33-40; and Walther W. and Stein U., Drugs 2000, 60(2):249-71.

[0174] In some specific embodiments described herein, a trans-splicing molecule carrying the binding and coding domains of the ABCA4 gene is delivered to selected cells, such as photoreceptor cells, that require treatment with an AAV vector. More than 30 naturally occurring AAV serotypes are available. Many natural variants exist in AAV capsids, and it is possible to identify and use AAVs with properties specifically suited to ophthalmic cells. AAV viruses can be modified using conventional molecular biology techniques, and such particles can be optimized for cell-specific delivery of trans-splicing molecule nucleic acid sequences, minimization of immunogenicity, fine-tuning of stability and particle lifetime, efficient degradation, and precise delivery to the nucleus.

[0175] The expression of trans-splicing molecules described herein may be achieved by delivery in selected cells using recombinant modified AAV or artificial AAV containing a sequence encoding the desired trans-splicing molecule. The use of AAV is a common form of exogenous DNA delivery because it is relatively non-toxic, results in efficient gene transfer, and can be easily optimized for specific purposes. Among the AAV serotypes isolated from humans or non-human primates and well-characterized, human serotype 2 is widely used in efficient gene transfer experiments in various target tissues and animal models. Other AAV serotypes include, but are not limited to, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. Unless otherwise specified, AAV ITR and other selected AAV components described herein can be readily selected from any AAV serotype, including, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or other known and unknown AAV serotypes. In one embodiment, the ITR is derived from AAV2. Such ITRs or other AAV components can be readily isolated from AAV serotypes using methods available to those skilled in the art. Such AAVs may be isolated, obtained from academic organizations, commercial or public sources (e.g., the American Type Culture Collection, Manassas, VA), or otherwise. Alternatively, AAV sequences can be obtained by synthetic means or other suitable means by referring to publicly available sequences, such as those available in the literature or databases, e.g., GenBank, PubMed, etc.

[0176] Desired AAV fragments for assembly within a vector include cap proteins containing vp1, vp2, vp3 and the hypervariable region, rep proteins containing rep 78, rep 68, rep 52 and rep 40, and sequences encoding these proteins. Such fragments can be readily used in a variety of vector systems and host cells. Such fragments can be used alone, in combination with sequences or fragments of other AAV serotypes, or in combination with elements derived from other AAV virus sequences or non-AAV virus sequences. As used herein, artificial AAV serotypes include, non-limitingly, AAVs having capsid proteins that do not exist naturally. Such artificial capsids can be created by any suitable method using a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) in combination with a selected different AAV serotype, a heterologous sequence that may be obtained from a discontinuous portion of the same AAV serotype, a heterologous sequence that may be obtained from a non-AAV virus source, or a heterologous sequence that may be obtained from a non-viral source. Artificial AAV serotypes may be, without limitation, pseudotyped AAVs, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids. Pseudotyped vectors in which the capsid of a certain AAV is used together with an ITR derived from an AAV having a different capsid protein are useful in this invention. In one embodiment, the AAV is AAV2 / 5 (i.e., an AAV having an AAV2 ITR and an AAV5 capsid). In another embodiment, the AAV is AAV2 / 8 (i.e., an AAV having an AAV2 ITR and an AAV8 capsid). In one embodiment, the AAV contains an AAV8 capsid. Such an AAV8 capsid contains the amino acid sequence found in NCBI Reference Sequence: YP_077180.1 (SEQ ID NO: 56). In another embodiment, the AAV8 capsid contains capsids coded as nt 2121~4337 in GenBank accession:AF513852.1(SEQ ID NO:57).

[0177] In one embodiment, the AAV includes a capsid sequence derived from AAV8. In several embodiments, the AAV derived from AAV8 is AAV8(b) as described in U.S. Patent No. 9,567,376, which is incorporated herein by reference in its entirety. AAV(b) (SEQ ID NO: 58) contains the amino acid sequence Pro-Glu-Arg-Thr-Ala-Met-Ser-Leu-Pro at amino acid positions 587-595 compared to wild-type AAV8. In another embodiment, the AAV8(b) capsid is encoded as SEQ ID NO: 59.

[0178] In one embodiment, a vector useful for the compositions and methods described herein comprises at least a sequence or fragment encoding a selected AAV serotype capsid, e.g., AAV2 capsid. In another embodiment, a useful vector comprises at least a sequence or fragment encoding a selected AAV serotype rep protein, e.g., AAV2 rep protein. Optionally, such a vector may contain both the AAV cap protein and the rep protein. In a vector containing both AAV rep and cap, both the AAV rep and AAV cap sequences may originate from a single serotype, e.g., AAV2.

[0179] Alternatively, a vector may be used in which the rep sequence is derived from a different AAV serotype than the one from which the cap sequence was obtained. In one embodiment, the rep sequence and the cap sequence are expressed in separate sources (e.g., separate vectors, or host cells and vector). In another embodiment, such a rep sequence is fused in frame with a cap sequence from a different AAV serotype to form a chimeric AAV vector as described in U.S. Patent No. 7,282,199, which is incorporated herein by reference.

[0180] A suitable recombinant AAV (rAAV) is produced by culturing a host cell containing a nucleic acid sequence or fragment thereof encoding an AAV serotype capsid protein as defined herein; a functional rep gene; a minigene composed of, for example, an AAV ITR and a trans-splicing molecular nucleic acid sequence; and a helper functional region sufficient to enable the packaging of the minigene into the AAV capsid protein. The components required for the AAV minigene to be cultured in the host cell so as to be packaged into the AAV capsid are supplied trans to the host cell. Alternatively, one or more of the required components (e.g., minigene, rep sequence, cap sequence, and / or helper functional region) may be supplied by a stable host cell that has been modified using methods known to those skilled in the art to contain one or more of the required components.

[0181] In one aspect, the AAV contains a promoter (or a functional fragment of a promoter). The selection of the promoter to be used for rAAV can be made from several broad constitutive or inducible promoters that can drive the expression of the selected transgene in the desired target cells. See, for example, the list of promoters identified in International Patent Application Publication No. WO 2014 / 012482, which is incorporated herein by reference. In one aspect, the promoter is cell-specific. The term "cell-specific" means that the specific promoter selected for the recombinant vector can direct the expression of the selected transgene in a particular cell type. In one aspect, the promoter is specific for the expression of the transgene in photoreceptor cells. In another aspect, the promoter is specific for expression in rods and / or cones. In another aspect, the promoter is specific for the expression of the transgene in retinal pigment epithelial (RPE) cells. In another aspect, the promoter is specific for the expression of the transgene in ganglion cells. In another aspect, the promoter is specific for the expression of the transgene in Müller cells. In another aspect, the promoter is specific for the expression of the transgene in bipolar cells. In another aspect, the promoter is specific for the expression of the transgene in horizontal cells. In another aspect, the promoter is specific for the expression of the transgene in amacrine cells. In another aspect, the transgene is expressed in any of the above cells.

[0182] In another aspect, the promoter is the native promoter in which the target gene is expressed. Useful promoters include, but are not limited to, the rod opsin promoter, the red / green opsin promoter, the blue opsin promoter, the cGMP-phosphodiesterase promoter, the mouse opsin promoter, the rhodopsin promoter, the alpha subunit of the cone transducin, the beta phosphodiesterase (PDE) promoter, the retinitis pigmentosa promoter, the NXNL2 / NXNL1 promoter, the RPE65 promoter, the retinal degeneration slow (RDS) / peripherin-2 (Rds / perph2) promoter, and the VMD2 promoter.

[0183] Mini - genes or other conventional regulatory sequences included within rAAV are also disclosed in documents such as WO 2014 / 124282 and others incorporated herein by reference. Those skilled in the art can make selections from these and other expression control sequences without departing from the scope described herein.

[0184] The AAV mini - gene can contain the trans - splicing molecules and their regulatory sequences described herein, as well as the 5' AAV ITR and 3' AAV ITR. In one embodiment, the ITR of AAV serotype 2 is used. In another embodiment, the ITR of AAV serotype 5 or 8 is used. However, ITRs from other suitable serotypes may be selected. In some embodiments, the mini - gene is packaged within the capsid protein and delivered to the selected host cell.

[0185] The mini - gene, rep sequence, cap sequence, and helper function required to produce rAAV may be delivered to the packaging host cell in the form of any gene element that transfers the sequences carried by itself. The selected gene element can be delivered by any suitable method, such as those described herein. The methods used to construct any of the embodiments described herein are known to those skilled in the art of nucleic acid manipulation, including genetic modification operations, recombinant modification operations, and synthetic techniques. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, the method for producing rAAV virions is well - known, and the selection of a suitable method is not a limitation to the present invention. See, for example, K. Fisher et al., J. Virol., 1993 70:520 - 532 and U.S. Patent 5,478,745, each incorporated herein by reference.

[0186] In another embodiment, a minigene of the trans-splicing molecule is prepared within a proviral plasmid, such as that disclosed in International Patent Application Publication No. 2012 / 158757, which is incorporated herein by reference. Such a proviral plasmid comprises a modular recombinant AAV genome including the ITR sequence franked to a unique restriction site enabling immediate removal or replacement of the wild-type 5'AAV2 ITR, included in a functionally related state; a promoter containing cytomegalovirus (CMV)-chicken beta-actin sequence of 49 nucleic acid cytomegalovirus sequences; or a photoreceptor-specific promoter / enhancer, the promoter franked to a unique restriction site enabling immediate removal or replacement of the entire promoter sequence, and the upstream sequence franked to a unique restriction site enabling immediate removal or replacement of only the CMV sequence or enhancer sequence upstream of the promoter sequence. The trans-splicing molecules described herein may be inserted into a multicloning polylinker site, where the trans-splicing molecule is functionally linked to the promoter under its regulatory control. The plasmid also includes a bovine growth hormone-derived polyadenylated sequence flanked at a unique restriction site that allows for immediate removal or replacement of the aforementioned polyA sequence, and the 3'ITR sequence flanked at a unique restriction site that allows for immediate removal or replacement of the wild-type 3'AAV2 ITR. The plasmid backbone contains elements necessary for replication in bacterial cells, such as the kanamycin resistance gene, and the backbone itself is flanked at a transcriptional terminator / insulator sequence.

[0187] In one embodiment, the proviral plasmid comprises a modular recombinant AAV genome comprising (a) a functionally related ITR sequence (i) flanked to a unique restriction site enabling immediate removal or replacement of the wild-type 5'AAV2 ITR; (ii) a promoter comprising (A) a CMV sequence of 49 nucleic acids upstream of the CMV-chicken beta-actin sequence; (b) a photoreceptor-specific promoter / enhancer; or (c) a neuron-specific promoter / enhancer. The promoter is flanked to an upstream sequence flanked to a unique restriction site enabling immediate removal or replacement of the entire promoter sequence, and to a unique restriction site enabling immediate removal or replacement of only the CMV sequence or enhancer sequence upstream of the promoter sequence. Furthermore, the proviral plasmid also includes a sequence of trans-splicing molecules, for example, a multi-cloning polylinker sequence that allows insertion of any of those described herein, wherein the trans-splicing molecule is functionally linked to a promoter under the regulatory control of the promoter; a bovine growth hormone-derived polyadenylated sequence flanked to a unique restriction site that allows immediate removal or replacement of the poly-A sequence; and the 3'ITR sequence flanked to a unique restriction site that allows immediate removal or replacement of the wild-type 3'AAV2 ITR. The proviral plasmid also includes a plasmid backbone containing elements necessary for replication in bacterial cells and further containing a kanamycin resistance gene, the plasmid backbone flanked to a transcriptional terminator / insulator sequence. The proviral plasmid described herein may also include within its plasmid backbone a 5.1 kb non-coding stuffer sequence of lambda phage for increasing the backbone length and for suppressing the reverse packaging of the non-functional AAV genome.

[0188] In some embodiments, the proviral plasmid contains multiple copies of the transsplicing molecule. For example, the present invention features a transsplicing molecule that is less than half the packaging limit of AAV and can therefore be repeated once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times, fifteen times, sixteen times, seventeen times, eighteen times, nineteen times, twenty times, or more in a single proviral plasmid.

[0189] In further contexts, the promoter of a proviral plasmid is modified to reduce its size in order to allow the insertion of a large trans-splicing molecule sequence into the rAAV. In one embodiment, as described in International Patent Application Publication No. 2017 / 087900, which is incorporated in its entirety herein by reference, a CMV / CBA hybrid promoter, typically containing about 1,000 base pairs of non-coding exons and introns in total, is replaced with a 130-base-pair chimeric intron.

[0190] Such proviral plasmids are then used in conventional packaging methodologies currently in use to produce recombinant viruses that express the transgene of the trans-splicing molecule supported on the proviral plasmid. Suitable producing cell lines are readily selected by those skilled in the art. For example, suitable host cells can be selected from any biological organism, such as prokaryotic (e.g., bacterial) cells and eukaryotic cells, such as insect cells, yeast cells, and mammalian cells. Briefly, the proviral plasmid is transfected into the selected packaging cell, in which case it may be transient. Alternatively, a minigene or gene expression cassette with flanking ITRs is stably incorporated into the genome of the host cell, either on the chromosome or as an episome. Suitable transfection techniques are known and can be readily used to deliver recombinant AAV genomes to host cells. Typically, the proviral plasmid is cultured in host cells expressing the cap protein and / or rep protein. Within host cells, minigenes consisting of trans-splicing molecules with flanking AAV ITRs are rescued and packaged within capsid or envelope proteins to form infectious viral particles. Therefore, recombinant AAV infectious particles are produced by culturing packaging cells carrying a proviral plasmid in the presence of sufficient viral sequences to enable the packaging of gene-expressing cassette viral genomes into the envelope or capsid of infectious AAV.

[0191] IV. Pharmaceutical compositions and kits This specification provides a pharmaceutical composition comprising a nucleic acid trans-splicing molecule, a proviral plasmid, or rAAV comprising a nucleic acid trans-splicing molecule described herein. In some embodiments, the pharmaceutical composition comprises any 5' trans-splicing molecule described herein. In other embodiments, the pharmaceutical composition comprises any 3' trans-splicing molecule described herein. In some embodiments, the pharmaceutical composition comprises a 5' trans-splicing molecule and a 3' trans-splicing molecule, for example, in which the 5' trans-splicing molecule and the 3' trans-splicing molecule collectively comprise functional ABCA4 exons 1-50 and bind to the same target ABCA4 intron.

[0192] The pharmaceutical compositions described herein may be evaluated for impurities by conventional methods and then formulated into pharmaceutical compositions intended for a suitable route of administration. Further compositions containing trans-splicing molecules, such as compositions as naked DNA or proteins, may similarly be formulated with a suitable carrier. Such formulations involve the use of a pharmaceutically and / or physiologically acceptable vehicle or carrier specifically directed for administration to target cells. In one embodiment, suitable carriers for administration to target cells include buffered saline, isotonic sodium chloride solution, or other buffers for maintaining pH at an appropriate physiological level, such as HEPES, as well as optionally other pharmacokinetic agents, pharmaceutical agents, stabilizers, buffers, carriers, aids, diluents, etc.

[0193] In some embodiments, the carrier is an injection solution. Exemplary physiologically acceptable carriers include sterile pyrogen-free water and sterile pyrogen-free phosphate-buffered saline. Various such known carriers are described in U.S. Patent No. 7,629,322, incorporated herein by reference. In one embodiment, the carrier is an isotonic sodium chloride solution. In another embodiment, the carrier is a buffer salt solution. In one embodiment, the carrier contains tween. If the virus is to be stored for an extended period, it may be frozen in the presence of glycerol or Tween20.

[0194] In other embodiments, the composition comprising the trans-splicing molecule described herein comprises a surfactant. Useful surfactants, such as Pluronic F68 (also known as Poloxamer 188, LUTROL® F68), may be included to inhibit the adhesion of AAV to inert surfaces and thus ensure delivery of the desired dose. As an example, an example of an exemplary composition designed for the treatment of an eye disease described herein comprises a recombinant adeno-associated vector on which a nucleic acid sequence encoding a 3' trans-splicing molecule as described herein is supported under the control of a regulatory sequence that expresses the trans-splicing molecule in ophthalmic cells of a mammalian subject, and a pharmaceutically acceptable carrier. The carrier is an isotonic sodium chloride solution and comprises the surfactant Pluronic F68. In one embodiment, the trans-splicing molecule is any of those described herein.

[0195] In yet another exemplary embodiment, the composition comprises a recombinant AAV2 / 5 pseudotyped adeno-associated virus carrying a 3' trans-splicing molecule or a 5' trans-splicing molecule for the replacement of the ABCA4 gene, the nucleic acid sequence being under the control of a promoter that directs the expression of the trans-splicing molecule in the photoreceptor cell, and the composition is formulated with a carrier suitable for subretinal injection and further components. In yet another embodiment, the kit may include a composition or components for the preparation or assembly of the composition, such as a carrier, rAAV particles, a surfactant and / or components for producing rAAV, and appropriate laboratory hardware for preparing the composition.

[0196] In some examples, the composition comprises a recombinant AAV2 / 5 pseudotyped adeno-associated virus carrying a 5' trans-splicing molecule for the replacement of the CEP290 gene, the nucleic acid sequence being under the control of a promoter that directs the expression of the trans-splicing molecule in the photoreceptor cell, and the composition is formulated with a carrier suitable for subretinal injection and further components. In yet another embodiment, the kit may include a composition or components for the preparation or assembly of the composition, such as a carrier, rAAV particles, a surfactant and / or components for producing rAAV, as well as appropriate laboratory hardware for preparing the composition.

[0197] Furthermore, this specification provides a kit comprising a first pharmaceutical composition comprising a 5' trans-splicing molecule and a second pharmaceutical composition comprising a 3' trans-splicing molecule, wherein, for example, the 5' trans-splicing molecule and the 3' trans-splicing molecule collectively comprise functional ABCA4 exons 1-50 and bind to the same target ABCA4 intron (for example, the trans-splicing molecule is packaged within any AAV vector described herein). In some embodiments, the kit includes instructions for use for mixing the two pharmaceutical compositions before administration.

[0198] Furthermore, this specification provides a kit comprising a first pharmaceutical composition containing a 5' trans-splicing molecule that binds to a target CEP290 intron.

[0199] V. Method The above compositions involving ABCA4 trans-splicing are useful in methods for treating diseases or disorders caused by mutations in the ABCA4 gene, such as Stargardt disease (e.g., Stargardt disease type 1), for example, in methods for delaying or alleviating the symptoms associated with the disease as described herein. Such methods involve contacting a target ABCA4 gene (e.g., ABCA4 premRNA) with a trans-splicing molecule as described herein (e.g., one or more 3' trans-splicing molecules, 5' trans-splicing molecules, or both 3' and 5' trans-splicing molecules) to modify ABCA4 expression in target cells, under conditions in which splicing occurs between the coding domain of the trans-splicing molecule and the target ABCA4 gene, and one or more defective or mutated portions of the targeted gene are replaced with functional (i.e., healthy), normal, wild-type, or modified mRNA of the targeted gene. Thus, the methods and compositions are used to treat eye diseases / conditions associated with specific mutations and / or gene expression.

[0200] In one embodiment, the contact involves direct administration to the affected subject. In another embodiment, the contact may be performed ex vivo to cultured cells and treated ophthalmoplasmic cells to be re-implanted into the subject. In one embodiment, the method involves administering rAAV carrying a 3' trans-splicing molecule. In another embodiment, the method involves administering rAAV carrying a 5' trans-splicing molecule. In yet another embodiment, the method involves administering a mixture of rAAV carrying a 3' trans-splicing molecule and rAAV carrying a 5' trans-splicing molecule. Such a method involves administering to a subject requiring it a composition of any of the substances described herein at an effective concentration.

[0201] In some embodiments, the method includes selecting one or more trans-splicing molecules to treat a subject having a disorder associated with a mutation within ABCA4, such as Stargardt disease (e.g., Stargardt disease type 1). Such selection may be based on the subject's genotype. In some embodiments, the disorder associated with ABCA4 may be an autosomal recessive disorder. In some examples, the subject may be homozygous or compound heterozygous with respect to the mutation within ABCA4. Methods for screening and identifying specific mutations within ABCA4 are known in the art.

[0202] In other examples, the compositions described above, with CEP290 trans-splicing, are useful in methods for treating diseases or disorders caused by mutations in the CEP290 gene, such as Leber congenital amaurosis (e.g., LCA 10), or for delaying or alleviating the symptoms associated with such diseases as described herein. Such methods involve contacting a target CEP290 gene (e.g., CEP290 premRNA) with a trans-splicing molecule (e.g., a 5' trans-splicing molecule) as described herein, under conditions that splicing occurs between the coding domain of the trans-splicing molecule and the target CEP290 gene, and that a portion of the targeted gene having one or more defects or mutations is replaced with functional (i.e., healthy), normal, wild-type, or modified mRNA of the targeted gene. Thus, the methods and compositions are used to treat ocular diseases / conditions associated with specific mutations and / or gene expression. The present invention involves modifying a pathogenic point mutation within intron 26 of CEP290 (e.g., at nucleotide 1,655 of intron 26) by administering a 5' trans-splicing molecule (e.g., any 5' trans-splicing molecule described herein) or a pharmaceutically acceptable composition thereof. Thus, the present invention provides a method for treating a subject having a disease or disorder associated with a mutation within CEP290 (e.g., a disease or disorder associated with a mutation within intron 26 of CEP290, e.g., at nucleotide 1,655 of intron 26) by administering a trans-splicing molecule described herein. Any of the aforementioned trans-splicing molecules may be included in a pharmaceutically acceptable composition (e.g., a single pharmaceutically acceptable composition as part of a kit, for example, containing molecules that are either pre-prepared or mixed before administration).

[0203] In one aspect, the contact involves direct administration to the affected subject. In another aspect, the contact can be performed ex vivo on cultured cells and treated ocular system cells to be re-transplanted into the subject. In one aspect, the method involves administering an rAAV carrying a 5' trans-splicing molecule. Such a method includes administering to a subject in need thereof a composition of any of those described herein at an effective concentration.

[0204] In some aspects, the method includes selecting one or more trans-splicing molecules for treating a subject having a disorder associated with a mutation in CEP290, such as LCA 10. Such selection can be made based on the genotype of the subject. In some aspects, the disorder associated with CEP290 can be an autosomal recessive disorder. In some examples, the subject is homozygous or compound heterozygous with respect to a mutation in CEP290. Methods for screening and identifying specific mutations in CEP290 are known in the art.

[0205] A single trans-splicing molecule for correcting a single mutation The method of the present invention includes selecting a single trans-splicing molecule based on the location of a single mutation (e.g., a mutation in one allele of the subject) in ABCA4. In some examples in the context of an autosomal recessive mutation, correction of only one of the two mutations may be sufficient to restore the activity of the functional protein. For example, at this time, the second allele has a mutation in the opposing portion of the ABCA4 gene outside the range of a single AAV delivery trans-splicing molecule configured such that the first mutation is corrected.

[0206] Accordingly, in some embodiments, the method of the present invention involves selecting a single trans-splicing molecule to correct a single mutation in the 5' portion of a target gene, regardless of the location of the mutation, for example, within the other allele. In one embodiment, the mutant exon is exon 1, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exon 1. In another embodiment, exon 1 or exon 2 is mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1 and 2. In another embodiment, one of exons 1, 2, and 3 is mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-3. In another embodiment, one of exons 1, 2, 3, and 4 is mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-4. In one embodiment, one of exons 1, 2, 3, 4, and 5 is mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1 to 5. In another embodiment, one of exons 1, 2, 3, 4, 5, and 6 is mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1 to 6. In another embodiment, one of exons 1, 2, 3, 4, 5, 6, or 7 is mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1 to 7. In another embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, or 8 is mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1 to 8. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, or 9 is a variant, the target intron is intron 19, 22, 23, or 24, and the coding domain contains functional ABCA4 exons 1-9.In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 is mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-10. In another embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 is mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-11. In yet another embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 is mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-12. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 is mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-13. In another embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 is mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-13. In yet another embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 is mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-14. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 is mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-15. In another embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 is mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-16.In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 is mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-17. In another embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 is mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-18. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 is mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-19. In another embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-20. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-21. In another embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-22. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 is a variant, the target intron is intron 23 or 24, and the coding domain contains functional ABCA4 exons 1-23.In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 is a variant, the target intron is intron 24, and the coding domain contains functional ABCA4 exons 1-24.

[0207] Alternatively, in cases where the mutation is located in the 3' portion of the target gene, the 3' trans-splicing molecule is selected so that the mutation is corrected regardless of the location of the mutation in the other allele, for example. In one embodiment, one of exons 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, and the coding domain contains functional ABCA4 exons 23-50. In one embodiment, one of exons 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22 or 23, and the coding domain includes functional ABCA4 exons 24-50. In another embodiment, one of exons 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 25-50. In one embodiment, one of exons 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 26-50. In another embodiment, one of exons 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 27-50.In one embodiment, one of exons 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 28-50. In another embodiment, one of exons 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 29-50. In one embodiment, one of exons 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 30-50. In another embodiment, one of exons 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 31-50. In one embodiment, one of exons 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 32-50. In another embodiment, one of exons 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 33-50. In one embodiment, one of exons 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is a variant, the target intron is intron 22, 23, or 24, and the coding domain contains functional ABCA4 exons 34-50.In one embodiment, one of exons 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 35-50. In another embodiment, one of exons 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 36-50. In one embodiment, one of exons 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 37-50. In another embodiment, one of exons 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 38-50. In one embodiment, one of exons 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 39-50. In another embodiment, one of exons 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 40-50. In yet another embodiment, one of exons 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 41-50. In one embodiment, one of exons 42, 43, 44, 45, 46, 47, 48, 49, or 50 is a variant, the target intron is intron 22, 23, or 24, and the coding domain contains functional ABCA4 exons 42-50.In one embodiment, one of exons 43, 44, 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 43-50. In another embodiment, one of exons 44, 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 44-50. In yet another embodiment, one of exons 45, 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 45-50. In one embodiment, one of exons 46, 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 46-50. In another embodiment, one of exons 47, 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 47-50. In another embodiment, one of exons 48, 49, or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 48-50. In another embodiment, one of exons 49 or 50 is mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exon 49 or 50. In one embodiment, exon 50 is a variant, the target intron is intron 22, 23, or 24, and the coding domain contains the functional ABCA4 exon 50.

[0208] Single trans-splicing molecules for correcting multiple mutations The method of the present invention includes selecting a single trans-splicing molecule based on the location of the mutations within ABCA4 within each allele of interest, such that if two mutations are present in either the 5' portion or the 3' portion of the gene, the single trans-splicing molecule that can be packaged within an AAV vector covers the range of both mutations and thereby corrects both mutations.

[0209] For example, in cases where both mutations are located in the 5' portion of the target gene, a 5' trans-splicing molecule is selected such that both mutations are corrected. In one embodiment, the mutant exon is exon 1 (i.e., both mutations are located in exon 1), the target intron is intron 19, 22, 23, or 24, and the coding domain contains functional ABCA4 exon 1. In another embodiment, exon 1 and / or exon 2 are mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain contains functional ABCA4 exons 1 and 2. In another embodiment, one or two of exons 1, 2, and 3 are mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain contains functional ABCA4 exons 1-3. In one embodiment, one or two of exons 1, 2, 3, and 4 are mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-4. In another embodiment, one or two of exons 1, 2, 3, 4, and 5 are mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-5. In another embodiment, one or two of exons 1, 2, 3, 4, 5, and 6 are mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-6. In another embodiment, one or two of exons 1, 2, 3, 4, 5, 6, or 7 are mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-7. In one embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, or 8 are mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-8. In another embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, or 9 are mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-9.In one embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 are mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-10. In another embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 are mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-11. In yet another embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 are mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-12. In one embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 are mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-13. In another embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 are mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-13. In yet another embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 are mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-14. In one embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 are mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-15. In another embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 are mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-16.In one embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 are mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-17. In another embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 are mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-18. In one embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 are mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-19. In another embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-20. In one embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-21. In another embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-22. In one embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 are mutant, the target intron is intron 23 or 24, and the coding domain contains functional ABCA4 exons 1-23.In one embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 are mutant, the target intron is intron 24, and the coding domain contains functional ABCA4 exons 1-24.

[0210] Alternatively, in cases where both mutations are located in the 3' portion of the target gene, a 3' trans-splicing molecule is selected such that both mutations are corrected. In one embodiment, one or two of exons 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, and the coding domain contains functional ABCA4 exons 23-50. In one embodiment, one or two of exons 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22 or 23, and the coding domain contains functional ABCA4 exons 24-50. In one embodiment, one or two of exons 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain contains functional ABCA4 exons 25-50. In one embodiment, one or two of exons 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 26-50. In another embodiment, one or two of exons 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 27-50.In one embodiment, one or two of exons 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 28-50. In another embodiment, one or two of exons 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 29-50. In one embodiment, one or two of exons 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 30-50. In another embodiment, one or two of exons 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 31-50. In one embodiment, one or two of exons 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 32-50. In another embodiment, one or two of exons 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 33-50.In one embodiment, one or two of exons 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 34-50. In another embodiment, one or two of exons 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 35-50. In one embodiment, one or two of exons 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 36-50. In another embodiment, one or two of exons 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 37-50. In one embodiment, one or two of exons 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 38-50. In another embodiment, one or two of exons 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 39-50. In one embodiment, one or two of exons 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain contains functional ABCA4 exons 40-50.In one embodiment, one or two of exons 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 41-50. In another embodiment, one or two of exons 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 42-50. In yet another embodiment, one or two of exons 43, 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 43-50. In one embodiment, one or two of exons 44, 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 44-50. In one embodiment, one or two of exons 45, 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 45-50. In one embodiment, one or two of exons 46, 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 46-50. In one embodiment, one or two of exons 47, 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 47-50. In another embodiment, one or two of exons 48, 49, or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 48-50. In yet another embodiment, one or two of exons 49 or 50 are mutant, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exon 49 or 50.In one embodiment, exon 50 is a variant, the target intron is intron 22, 23, or 24, and the coding domain contains the functional ABCA4 exon 50.

[0211] Two types of trans-splicing molecules for correcting multiple mutations Furthermore, this specification provides a method for correcting multiple mutations within the ABCA4 gene using two types of trans-splicing molecules: a 5' trans-splicing molecule and a 3' trans-splicing molecule. In some embodiments, the binding of both trans-splicing molecules replaces the entire ABCA4 gene, for example, in which case the 5' trans-splicing molecule and the 3' trans-splicing molecule bind to the same target ABCA4 intron and replace the upstream and downstream exons of the target intron, respectively.

[0212] For example, in some embodiments of the present invention, the 5' trans-splicing molecule and the 3' trans-splicing molecule each bind to the target ABCA4 intron 22; the 5' trans-splicing molecule replaces endogenous exons 1-22 with functional exons 1-22; and the 3' trans-splicing molecule replaces endogenous exons 23-50 with functional exons 23-50. In other embodiments, the 5' trans-splicing molecule and the 3' trans-splicing molecule each bind to the target ABCA4 intron 23; the 5' trans-splicing molecule replaces endogenous exons 1-23 with functional exons 1-23; and the 3' trans-splicing molecule replaces endogenous exons 24-50 with functional exons 24-50. In other embodiments, the 5'-trans-splicing molecule and the 3'-trans-splicing molecule each bind to the target ABCA4 intron 24; the 5'-trans-splicing molecule replaces endogenous exons 1-24 with functional exons 1-24; and the 3'-trans-splicing molecule replaces endogenous exons 25-50 with functional exons 25-50. Any of the aforementioned combinations of the 5'-trans-splicing molecule and the 3'-trans-splicing molecule may be included in a pharmaceutical composition (e.g., a single pharmaceutical composition as part of a kit, which includes both molecules either pre-prepared or mixed before administration).

[0213] Medication, monitoring, and combination therapy The effective concentration of recombinant adeno-associated virus carrying the trans-splicing molecule described herein is approximately 10 8 ~10 13 The range is genome vector / milliliter (vg / mL). rAAV infection units are measured as described in McLaughlin et al., J. Virol. 1988, 62:1963. In one embodiment, the concentration is 10 9 ~10 13 The range is vg / mL. In another embodiment, the effective concentration is approximately 1.5 × 10⁻⁶. 11 The concentration is vg / mL. In one embodiment, the effective concentration is approximately 1.5 × 10⁻⁶. 10 The concentration is vg / mL. In another embodiment, the effective concentration is approximately 2.8 × 10⁻⁶.11 vg / mL. In another embodiment, the effective concentration is about 5 x 10 11 vg / mL. In yet another embodiment, the effective concentration is about 1.5 x 10 12 vg / mL. In another embodiment, the effective concentration is about 1.5 x 10 13 vg / mL.

[0214] To reduce undesirable effects, such as toxicity, and other problems associated with ocular administration, such as the risk of abnormal retinogenesis and retinal detachment, it is desirable to use the virus at the lowest effective dosage (total genome copies delivered). The effective dosage of recombinant adeno-associated virus carrying a trans-splicing molecule as described herein is about 10 8 ~10 13 genome vectors (vg) / dose (i.e., / injection). In one embodiment, the dosage is 10 9 ~10 13 vg. In another embodiment, the effective dosage is about 1.5 x 10 11 vg. In another embodiment, the effective dosage is about 5 x 10 11 vg. In one embodiment, the effective dosage is about 1.5 x 10 10 vg. In another embodiment, the effective dosage is about 2.8 x 10 11 vg. In yet another embodiment, the effective dosage is about 1.5 x 10 12 vg. In another embodiment, the effective concentration is about 1.5 x 10 13 vg. Still other dosages within these ranges or in other units may be selected by the attending physician, taking into account the physical condition of the subject being treated, such as the age of the subject; the composition being administered, and the specific disorder; the cells targeted and, if the disorder is progressive, the degree of progression.

[0215] The composition may be delivered in a volume of about 50 μL to about 1 mL, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method, and the volume includes any value within this range. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 70 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 250 μL. In another embodiment, the volume is about 300 μL. In another embodiment, the volume is about 350 μL. In another embodiment, the volume is about 400 μL. In another embodiment, the volume is about 450 μL. In another embodiment, the volume is about 500 μL. In another embodiment, the volume is about 600 μL. In another embodiment, the volume is approximately 750 μL. In yet another embodiment, the volume is approximately 850 μL. In yet another embodiment, the volume is approximately 1,000 μL.

[0216] In one embodiment, the volume and concentration of the rAAV composition are selected so that only a specific anatomical region containing target cells is affected. In another embodiment, the volume and / or concentration of the rAAV composition is high to reach a broader portion of the eye. Similarly, the dosage is adjusted for administration to other organs.

[0217] In another embodiment, the present invention provides a method for suppressing or halting the decline in photoreceptor function, or for enhancing photoreceptor function, in a subject. The composition may be administered before or after the onset of disease. For example, photoreceptor function may be evaluated using conventional functional tests in the art, such as ERG or visual field testing. As used herein, “decline in photoreceptor function” means a decrease in photoreceptor function compared to a normal, non-disease eye or the same eye at an earlier point in time. As used herein, “enhance in photoreceptor function” means improving photoreceptor function compared to a diseased eye (having the same eye disease), the same eye at an earlier point in time, an untreated portion of the same eye, or the opposite eye of the same subject, or increasing the number or proportion of functional photoreceptors.

[0218] Each of the methods described herein may be used to prevent further damage or to salvage tissue with mild or advanced disease. As used herein, the term “salvage” means to prevent disease progression, to prevent the spread of damage to undamaged cells, or to improve damage to damaged cells.

[0219] Therefore, in one embodiment, the composition is administered before the onset of the disease. In another embodiment, the composition is administered before the onset of symptoms. In yet another embodiment, the composition is administered after the onset of symptoms. In yet another embodiment, the composition is administered when, for example, less than 90% of the target cells are functional or remaining compared to a reference tissue. In yet another embodiment, the composition is administered when, for example, more than 10% of the target cells are functional or remaining compared to a reference tissue. In yet another embodiment, the composition is administered when more than 20% of the target cells are functional or remaining. In yet another embodiment, the composition is administered when more than 30% of the target cells are functional or remaining.

[0220] In another embodiment, any of the above methods may be carried out in combination with another treatment or second-line treatment. The treatment may be any treatment that helps to suppress, suspend or alleviate any currently known or yet unknown mutation or defect or any effect associated with such mutation or defect. The second-line treatment may be carried out before, concurrently with, or after the administration of the trans-splicing molecule. In one embodiment, the second-line treatment may involve the administration of a nonspecific approach to maintain the health of retinal cells, such as neurotrophic factors, antioxidants, or anti-apoptotic agents. The nonspecific approach may be carried out by injection of proteins, recombinant DNA, recombinant viral vectors, stem cells, embryonic tissue, or genetically modified cells. The latter may also include encapsulated genetically modified cells.

[0221] In another embodiment, the method includes performing functional tests and imaging tests to determine the effectiveness of the treatment. Such tests include electroretinography (ERG) and in vivo retinal imaging as described in U.S. Patent No. 8,147,823; International Patent Application Publication No. 2014 / 011210 or 2014 / 124282, which are incorporated herein by reference. Visual field tests, visual field examinations and microperimetry, motor function tests, and visual acuity and / or color vision tests may also be performed.

[0222] In some specific embodiments, it is desirable to perform non-invasive retinal imaging and functional testing to identify the photoreceptor retention areas to be targeted for treatment. In such embodiments, clinical diagnostic tests are used to determine the precise location(s) for one or more subretinal injections. Such tests may include ERG, visual field testing, topographical mapping of retinal layers and measurement of their thickness by scanning confocal laser ophthalmography (cSLO) and optical coherence tomography (OCT), topographic mapping of cone density by adaptive optics (AO), and functional testing of the eye. In light of imaging and functional testing, in some embodiments, one or more injections are administered to the same eye to target different photoreceptor retention areas.

[0223] For use in this manner, the volume and viral titer of each injection are determined individually and may be the same as or different from other injections administered in the same or opposite eye. In another embodiment, a single, high-volume injection is administered to treat the entire eye. Dosage, administration, and regimen may be determined by a physician who has been instructed in this disclosure. [Examples]

[0224] The present invention is based, at least in part, on the applicant's findings that specific introns of ABCA4 and specific regions within those introns provide highly efficient binding sites for the binding domains of transsplicing molecules, thereby efficiently mediating transsplicing. The applicant created a series of mock transsplicing molecules having 150-nucleotide-length binding domains designed to hybridize to a series of corresponding 150-nucleotide-pair binding site sequences in (i) the ABCA4 introns of interest (introns 19 and 22-24) and (ii) the CEP290 introns of interest (introns 26-30). Each binding domain in the ABCA4 and CEP290 series was designed with 140 nucleotides overlapping, allowing for scanning of every 10 nucleotides of each intron between each sequential test binding domain. Transsplicing efficiency was quantified for each binding domain in ABCA4 introns 19 and 22-24 and CEP290 introns 26-30, respectively. Screening for ABCA4 is described in Example 1, and the results are shown in Figures 1-8. Screening for CEP290 is described in Example 2, and the results are shown in Figures 21-26.

[0225] Example 1.ABCA4 This embodiment describes, for example, the development of ABCA4 trans-splicing molecules by screening for effective binding sites within specific ABCA4 introns, the development of ABCA4 cell lines for testing trans-splicing molecules, and the testing of various ABCA4 trans-splicing molecules for the restoration of ABCA4 protein expression.

[0226] Screening of binding sites Screening of a series of binding domains configured to bind to ABCA4 intron 19 (SEQ ID NO: 25) via a continuous binding site revealed the 3' region of ABCA4 intron 19, specifically the nucleotide region 990–2,174 of intron 19, which is preferentially efficient during transsplicing of 5' transsplicing molecules (Figure 2). Binding sites within the nucleotide ranges of 1,670–2,174, 1,810–2,000, 1,870–2,000, or 1,920–2,000 were found to be particularly efficient in mediating 5' transsplicing in intron 19.

[0227] Preferred binding sites for 5' trans-splicing molecules within intron 22 were similarly identified (Figure 3). Binding sites within the range of nucleotides 1-150 or nucleotides 880-1,350 of intron 22 were particularly efficient compared to the rest of the intron.

[0228] Figure 4 shows the results of a similar screening for ABCA4 intron 22 (SEQ ID NO: 28) in 3' trans-splicing molecules. Binding sites with nucleotides 60-570, 600-800, or 900-1,350 were identified as preferentially suitable for trans-splicing of 3' trans-splicing molecules. In particular, binding domains designed to target binding sites in the range of nucleotides 70-250 were highly efficient during 3' trans-splicing.

[0229] Within intron 23, relatively efficient binding sites for 5' trans-splicing molecules were identified as those within the range of nucleotides 80-570 or 720-1,081 of SEQ ID NO:29 (Figure 5). For 3' trans-splicing molecules, particularly efficient binding sites were found within the range of nucleotides 80-1,081 of SEQ ID NO:29 (e.g., nucleotides 230-1,081, 250-400, or 690-850 of SEQ ID NO:29), as shown in Figure 6.

[0230] Similar screening in intron 24 of ABCA4 (SEQ ID NO: 30) revealed that binding sites within the range of nucleotides 600–1,250 or 1,490–2,660 were efficient during 5' trans-splicing (Figure 7). In particular, the binding site within the range of nucleotides 1,000–1,200 showed the highest 5' trans-splicing efficiency. Figure 8 shows the results of 3' trans-splicing efficiency screening, which revealed that binding sites within the range of nucleotides 1–250, 300–2,000, or 2,200–2,692 (especially binding sites within the range of nucleotides 750–1,110) were the most efficient.

[0231] ABCA4 cell line First, we created cell lines expressing ABCA4. The ABCA4 gene is only known to be expressed in photoreceptors in the retina of living organisms, and the full-length premRNA and protein of ABCA4 are generally undetectable in vitro in cultured cells. Therefore, to test trans-splicing strategies for ABCA4, we modified cells to express ABCA4 from its native genomic locus (1p22.1) on chromosome 1. Two strategies were employed. In the first case, a stable cell line was induced to express a site-specific (upstream of the ABCA4 transcription start site) DNA-binding TALEN fused with the VP64 viral transactivator. In the second case, a eukaryotic constitutive promoter was directly inserted into the genomic locus immediately upstream of the ABCA4 transcription start site (using CRISPR / Cas9). In both cases, the results were stable cell lines that robustly expressed the premRNA and protein of ABCA4.

[0232] TALEN cell lines We designed TALENs targeting a specific domain upstream of the ABCA4 transcription start site and fused them with the VP-64 transactivator sequence (Figure 9). 293 cells were transfected with this combination of three TALENs, inducing stable single-clonal cells. Two different clones were shown to signal the expression of the ABCA4 protein (Figure 10).

[0233] CAG Promoter Cell Line A general strategy for inducing CAG promoter cell lines is outlined in Figures 11–13. Site-specific guides for inserting the CAG promoter and puromycin-selectable marker (Figure 12A) were designed using homologous arms (Figure 12B). Puromycin-resistant cells were cloned and analyzed for desired insertions by PCR. Several clone lines were selected for further analysis. RNA and protein expression in two lines (B6 and C3) are shown in Figures 14A and 14B. As demonstrated by RNA and protein analysis, both lines clearly contained promoter insertions.

[0234] ABCA4 knockout cell lines Once stable ABCA4 expression was established in cultured cells, ABCA4 expression knockout cells were created for testing ABCA4 trans-splicing molecules designed to restore ABCA4 protein expression. Generally, guide RNA and Cas9 protein were co-transfected into B6 cells (which have the CAG promoter knocked into the ABCA4 locus and mediate ABCA4 expression). After 9 days, a second transfection was performed using guide RNA and Cas9 protein. The basic design targeting exons 3 and 4 is shown in Figure 15. Single cells were plated at limiting dilution and cultured once, and ABCA4 protein expression was evaluated by Western blotting.

[0235] Figure 16 shows the RNA and protein profiles induced after treatment with CRISPR / Cas9 as shown in Figure 15 for single-clonal cells. Various degrees of RNA and protein depletion were observed. Clones 17+06 and 17+21 were selected due to complete ABCA4 protein knockout. Mutation analysis (Figures 17A-17B and 18) confirmed that exons 3 and 4 were targeted and fragmented.

[0236] ABCA4 trans-splicing-mediated protein recovery Based on the high-throughput binding site screening described above, eight trans-splicing molecules were selected. The methods and results of these tests are described below.

[0237] method For the Western blot assay, 17+06 cells or 17+21 cells are used. 6 Cells were seeded in each well of a 12-well plate at a cell / well density. 1 μg of plasmid (RTM) was added to each individual well. xTransfection was performed using ). At 48 hours, cells were collected and membrane preparations for analysis were processed by standard Western blotting using the Mem-PER Plus Membrane Protein Extraction Kit (Thermo Fisher 89842) according to the manufacturer's protocol, with the addition of 1×HALT® Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher 78440) in all buffers. RNA was also processed for analysis as described below. The membrane lysates were denatured at room temperature for 30 minutes using 4×Laemmli Sample Buffer (Biorad 161-0737) containing 10% reducing agent TCEP 0.5M (Sigma 646547). The samples were electrophoresed on NuPage Precast 3-8% Tris-Acetate gel (Thermo Fisher), and proteins were transferred using iBlot 2 Mini PVDF Transfer Packs and run on iBlot 2 at 25V for 10 minutes. The primary antibody for ABCA4 was rabbit polyclonal Abcam ab72955 (at a dilution of 1:2500). The secondary antibody was anti-rabbit (at a dilution of 1:5000). The blots were exposed for various durations depending on the signal intensity.

[0238] For qPCR of RNA samples, RNA was collected using the RNeasy Plus Mini kit (Qiagen) as described above for qPCR analysis. cDNA was synthesized from 400 ng of RNA in a reaction volume of 20 μl using SuperScript IV VILO Master Mix (Thermo 11756500; diluted 1:4 with water). The native ABCA4 (Thermo's commercially available assay Hs00979594_m1) was in the range of exons 49-50. For the housekeeper gene as a control, the RNF20 assay was used (Thermo's commercially available assay Hs00219623_m1). The following primers and probes were used for qPCR of the chimeric ABCA4 codon-optimized exon 22-native exon 23. TIFF2026095408000011.tif35128

[0239] The primers and probes for qPCR of double-stranded assays with RNF20 were as follows: TIFF2026095408000012.tif38128

[0240] The PCT reaction was performed using QuantiFast 2×qPCR Mastermix.

[0241] result Trans-splicing molecules that bind to introns 19, 22, 23, and 24 were tested. No protein recovery was observed with trans-splicing molecules that bind to introns 19 and 24 (data not shown), but trans-splicing molecules that bind to introns 22 and 23 resulted in the recovery of ABCA4 protein and RNA expression, as discussed below (Figures 19A and 19B).

[0242] Figure 20A is a Western blot showing ABCA4 protein expression resulting from trans-splicing reactions in two different cell lines (17+06 and 17+21) of a mock GFP control or 5'A4In22 tethered to five different binding domains (no binding domain (NBD) control, numbers 92, 99, 105, 118, and 121 corresponding to the RTM# in Figure 3, where binding domain 92 binds to nucleotides 911-1060 of intron 22, binding domain 99 binds to nucleotides 981-1130 of intron 22, binding domain 105 binds to nucleotides 1041-1190 of intron 22, binding domain 118 binds to nucleotides 1171-1320 of intron 22, and binding domain 121 binds to nucleotides 1201-1350 of intron 22 (according to the 10-base shift intervals of 150 bases across the entire intron 22 as described above)). Four of these intron 22-binding constructs, 99, 105, 118, and 121, resulted in protein recovery, with 105, 118, and 121 showing particularly enhanced recovery, while 118 resulted in maximum protein expression in both cell lines. A similar pattern was observed in mRNA expression profiles, with the 118 construct yielding the highest levels of ABCA4 mRNA in both cell lines (Figure 20B). Units are relative to RNF20 housekeeping genes.

[0243] Figure 20C is a Western blot showing ABCA4 protein expression resulting from trans-splicing reactions in two different cell lines (17+06 and 17+21) with mock GFP control or 5'A4In23 tethered to three different binding domains (NBD control, numbers 27, 81, and 85 corresponding to RTM# in Figure 5, where binding domain 27 binds to nucleotides 261-410 of intron 23, binding domain 81 binds to nucleotides 801-950 of intron 23, and binding domain 85 binds to nucleotides 841-990 of intron 23 (according to the 10-base shift intervals of 150 bases length across the entire intron 23 mentioned above)). Trans-splicing was induced with all three intron 23 binding constructs, as indicated by the protein expression levels in both cell lines. Similar results were obtained in the mRNA expression profiles, where all three constructs resulted in robust ABCA4 mRNA expression in both cell lines (Figure 20D). The unit is the relative amount to the RNF20 housekeeping gene.

[0244] In summary, the protein and RNA expression data of ABCA4 obtained with intron-22 and intron-23 binding trans-splicing molecules correlated with the binding domain screening described above. In particular, intron-22 binding constructs 105, 118, and 121, and intron-23 binding constructs 27, 81, and 85 were predicted to bind with high efficiency (Figures 3 and 5). The recovery data for the ABCA4 protein indicate that the ABCA4 intron region, including the binding sites of these constructs, is suitable for binding to ABCA4 trans-splicing molecules to result in protein and RNA recovery. In this example, protein expression was recovered by 10–20%, and the recovery was comparable between the intron-22 and intron-23 binding trans-splicing molecules. Importantly, since ABCA4-related diseases (e.g., Stargardt disease) are recessive, asymptomatic carriers of the disease likely express less ABCA4 than usual, and while we do not wish to be constrained by theory, partial protein restoration as described herein may yield meaningful clinical benefits.

[0245] Example 2. CEP290 Screening of a series of binding domains configured to bind to CEP290 intron 26 (SEQ ID NO: 85) via a continuous binding site revealed the 3' region of CEP290 intron 26, specifically the region of nucleotides 4,980–5,838 of intron 26, which is preferentially suitable for transsplicing of 5' transsplicing molecules (Figure 22). Binding sites within the nucleotide ranges of 5,348–5,838, 5,348–5,700, 5,400–5,600, 5,460–5,560, or 5,500 were found to be particularly efficient in mediating transsplicing.

[0246] Figure 23 shows the results of a similar screening in CEP290 intron 27 (SEQ ID NO: 86). Binding sites with nucleotides 120-680, 710-2,200, and 2,670-2,910 were identified as preferentially suitable for transsplicing 5' transsplicing molecules. In particular, binding domains designed to target binding sites within the ranges of nucleotides 790-2,100, 1,020-1,630, or 1,670-2,000 were highly efficient during transsplicing.

[0247] In intron 27 (SEQ ID NO: 87), binding sites within the ranges of nucleotides 1-390 (e.g., nucleotides 1-200), nucleotides 410-560, or nucleotides 720-937 were identified as having relatively high trans-splicing efficiency (Figure 24).

[0248] Intron 28 (SEQ ID NO: 88) was similarly characterized and shown to have relatively efficient binding sites within nucleotides 1-600, 720-940, or 1,370-1,790 (Figure 25).

[0249] In intron 29 (SEQ ID NO: 89), the 3' portion of the intron was significantly more efficient than the rest of the intron in mediating 5' trans-splicing (Figure 26). In particular, the binding domain targeting binding sites within the range of nucleotides 95-1,240, for example, nucleotides 1,060-1,240, showed the highest trans-splicing efficiency.

[0250] Sequence information SEQUENCE LISTING <110> The Trustees of the University of Pennsylvania Ascidian Therapeutics, Inc. <120> Trans-splicing Molecules <150> US 62 / 658,667 <151> 2018-04-17 <150> US 62 / 658,658 <151> 2018-04-17 <160> 113 <170> PatentIn version 3.5 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 1 gtaagagagc tcgttgcgat attat 25 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 2 tactaactgg tacctcttct tttttttctg cag 33 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 3 tggtacctct tctttttttt ctg 23 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 4 agatctcgtt gcgatattat 20 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 5 gagaacatta ttatagcgtt gctcgag 27 <210> 6 <211> 135313 <212> RNA <213> Homo sapiens <400> 6 aguccccagu cuuugcuuag gccccuacgu acacaaacug 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cuugucugaa gauagaaug 50760 accuugaaug cauuuuaaaaaaaaa uuuuuuuuu uuuuuuua 50820 uuuuaccuua aguocuggga uacaagugca gaugogouag googouaca uaoghuaoug 50880 ugugccaugg ugguuugcug cccaucaucu agguuuuaag cccacaugc 50940 auuagcuauu uguccuaaug cucucccucg cuckoo cuckoo 51000 ggugugugau guuccccucc cuguguccau guguucucuu uguucagcuu ccacuuacau 51060 gugagaacou guguguua guuucugu cuguguuag uuggcugagg augauggcuu 51120 ccagcuucuu ccaugucccu gcaaaggaca ugaucucauu ccuuuuuaug gcugcauagu 51180 auucuaugu guauauugac cauauuuucc uuauccagcc uaucacugau gggcauuugg 51240 augguucca ugucuuugca auguuaaca uacaugugca ugaauuuuuua uaguagaaug 51300 auuauauuc cuuuuuuu auaacccagua augggauugc cuggucaaau uguuuucug 51360 51420 accaacagug uaaaagccuu ccuauuucuc aacagccuca ccagcaucua uuguuucuug 51480 aaauuuuau aaucaccauu ugacaugauga ugaugaugaua gauacccaau ugucagaugg 51540 guagauuaca aaaauuuucu cucauucugc agguugccug uucacgcuaa ugauaguuuc 51600 uuuugcug cagaagcucu uaagccuaau uagauccauu uuucaauuuu ggcuuuuguu 51660 ccaauugcuu uugguguuuu agucaugaag ucuuugccca ugcguauguc cugagugggua 51720 uugccuaggc uuucucuag uuuucaugau uuuagauuuu acauuuaagu cuuuaaucca 51780 gcuugaguua auuuuuguau aagguguaag gaagggaucc aguuuaaguu uucuacaauau 51840 ggcuagccag uuuucccaac accauuuauu aaauagga clothescccc auugcuugug 51960. snow snow snow snow snow snow snow snow 52020. uagucuguuc cauuggucua uagucuguu uacaaaacag auucuuaagc aucaacccag aucgacuggc ucagauuuc cagggagag gccugguuau cugcauguuu acagaccuau uagauuugug ggaccugcag uucccuugua caguaaguua cucaauuaac aucucccucc ucucauggug ccucuaccug cuaagcccuu auucccagcc aggcccacca ccauccaccc acugcuguua uaacauaagc aggaccugug cgagggggug uggacggagg agagaggcuc snow snow snow snow snow snow snow snow caauguuuuu gcaaaguaua uaaagaauac uccuugucua cuugacauuc auaaaugucu uguuuuccag aaggauuauu uuuuccaagc agcuuguucc uaaugcagcc ccaggcacca aacagauacu 52500. uaaaauauau uaauugcuua aaugguuaag aauucagucu cuggacccac acugccuggg uucaauucc uauuaucugu gcccaguuuc caagucuaua aaauagggau auuaauagca 52560 cuaccuau aggcuacgua ugagaauuaa augagcuau ucaugcaag cacugacaua 52620 uaguaagcac uaaaaaaaa uaagcuuuu aaaaaauac aagccaaaaa accugcuua 52680 ggagaggaaa ugauguuagua gccuccugua auaggccca gccuccaagc uggugcuccu 52740 cuaggaauca caacgcugca aucacaucc uccggggccg ccaggacuuc acgaggggccu 52800 cugagcagag ggguaugaug ggaggagaag cccagcagcu gugaugagu gguuucugau 52860 cuccugcccc uugggggggg agggagggaaaaaaaaaaaaaaaaaaggggaa 52920 uagcggggag gaaugugug aggaaac acaucacugu ggcuuguccu ggauuuuucu 52980 gcuucuguuc ucguguuuug ggaagucugg aggacuug aaaucauuc auguccccac 53040 cugaggaug gcuuaguagc aggaggcca ugaacucu uugcugaugg cucugaaagc 53100 aaggaugug cucacugggg cugcugagg cuggcuggg gguucuggc agagaguaca 53160 ggccccuccc aggaggcgg cuaccacc augcuggcau ucuguggac cuggucugc 53220 ugucucagac ccccuccaca auagggucug caaucucauu caccccauaa auacauucug 53280 ucuuuccucu gauccccucc cauuagcagg gggaaauaaa uggaagucag acggcccagu 53340 uagaaggcag gcaguggagu aggaaaauag augauggugg uuugggggagc cucacaucac 53400 ucauggggag acauucauuc ccaugggccu uccaaucacc cuuuucucca aaucuaagga 53460 cacaggacaa auggguccuc auacaggcaa auaucuuaaa cugguaugug uauucauuua 53520 uauguucuaau uuauauugu cuuuauucac auauuuuug cuucuggaga aaagcucaau 53580 uaagaaaaauu aauacauuau ucuucuuauu gcccuucagc uaaaacaagc auaacacacc 53640 cuccccuuug gauuuuuugu uuagcaaaag guuaggccug gcacagauga aauacuauuc 53700 agaguucaca guguauuuuc auuucauaau auuuugauu uucaggucuu gaauuuucaca 53760 ucaggaagcu gauauaggaa gcugaauuca gccagauuuu aauacgaaaa uaccucugau 53820 caaggcauaa aauuguacuu uaaccaguaa ccacuguauu ucucuaagcu gugaaaaaac 53880 53940 cuuauugucu acauggugau uaucuugcug augaauucuc aaaggccaga gauuuggacu 54000 auuuuucuc uguaaccuug cauguuccug gccacaugcc accaccaccc aaacagaaug 54060 uacgcaggga auguauuuuu caggauaacc uaagaaaaaa uaggauuaag aagauaaagc 54120 ugcugaucau guaauguacu uuagacucag auauauaaau auuugugaau uaucuguccu 54180 auuucuucu ucuauuaauu cauugacucu agaugugcau uggaaggcua gggagaaauc 54240 aggggaucgu gagaagagc acagaagucu gcaucacaca aacaauua uuucaagagc 54300 caugaacuag auccuaagca acuauaggc aaugacuca uuucauaccu cuagucucua 54360 agaaacauau aacuggccug agaaggaaa auguggcaa gggguagacc ggggucaugg 54420 guggaggucc aaauaguaau caauggagcu cauagggugg acugauauug aagcugcuau 54480 gagccagcca caugcugggc acuguacau gucaucucau gcauacucc caauuaccug 54540 ccuaguaagc auaauuguca uuuuauagaa uuaaaaacag acucaaagag guugacaguc 54600 uaaguaaca augggcua uggaauaua auccagagcu gccuggcucu 54660 gaugagaaag cucuuucugc ugucauaugc agcccacauu aauagggggc ucagaaagua 54720 uocucuggau aaaaaaaa augaauccaa ugaggaga cauaauuua uaaaugcag 54780 cauaauaggc acuauuauuga uggauuuuc cugcuugaaa guagcuagau uagaguagga 54840 aaccaaaaag augugaauuc aucagucau ucaugcauuu gcauggauug agcuaccuac 54900 auugaauaa augcuguaa uccugauuc cuoggaagcu cacauuggag agauaagcau 54960 cuauuaaau auugccauaa uagugguauc ucagaggacu agcagaacau auuucaaucu 55020 gagagaguag aaacagauug uacaaucca auucaaaaca ucauaaaucc ucuaagcacu 55080 aucuucuu ccuccaauu aucucugaaa uccuccuuc uuucccauuu auggccucca 55140 uuacagaag cguguacugu cucucuuagc uguuugccag gccgccaguc ucuugcugu 55200 cagcucucaa cugcuuccag caagaucuuu cuaaauccc aggcuugcca agacuuagcg 55260 cccacagcuc cacagugacu ccucauugcu guuaagguaa aggccuuccc agucuagccc 55320 uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu cuuuccaucc uucccccacu cgacugccag gucaacaccc acacccacgc uucaggacuc 55440 agguccuaug uuucgggccu ucuucugugc accauucccu ucccuguagc ccuugaucau 55500 gauuuuuua uacgccucccg caccuucaug gcccugaacc ccucaagggc cgaaacugcc 55560 uuacuuuucu uuuugacuuc ccaacuuacc uuaguggagc ugagucaca uagaauagac 55620 gcucauaaau gcuucucugg gcuguaaagg uugaauuuuc cagcuaagca aggaaaag 55680 acaauuuucag ccaggagaa gggcauaagc aaagugcaga gaugugaagc ucaagagaa 55740 uggauggcu gggcagaggu guggcugcag caucaggga gaagaugag ugccuggagu 55800 cagcaggcac ggcuugcaaa agcuucaccu auaggugaaa ggacaccauc ucuugcacca 55860 auaggcucug ugauuggagg caacuuugcu guuuuacugc cagaaaacug aggauuaa 55920 cccaaacugc aguucaagug gcauucacug guggcuga aauggguguu uguggccaga 55980 auguggucug auuggucagu gcccagcucu guugauuagc agauguuuug aauauaguag 56040 cauccaugug cccaaguugu ugggaugauu caacaagaaa cuuuaagagc ucaagugccc 56100 ugcaguuguc agccagguga uuccuuccu uuggacccag uuagacgcag gcauuaccuc 56160 guggcuuugc cccaguguga aucuuugucc uccaacuuga ucuuuuuauu uguuuucauua 56220 uuguauuuaa guuguuuuuuu uuagagacag acauuuuua acagcugugc auuuccuguc 56280 ccuuuguuuuu ccagucguca uguguuuuccu uacucucugu gggugaacgu uucagauguc 56340 uguuugcggu gcccagcgug caagauaaaa uuuauugcag ugccuuccgg cucuaacuca 56400 ccaauccaac caauucagau agcccaaggc uguuuuuaucc aguggauuuu uccauguagu 56460 gggaaauaaa ucuugaaugu uacuguuuag auuagccagg aaacucauuc ugggauguuu 56520 gcccacaucc auuggcauuu cucaaaaagga accccaggug ucuaccuuga caccagcagg 56580 gccacuugag cccuccgcug gcauucaucg cccgcuuuugu ucucagccug aguuuaggag 56640 uaacagaugu gagaggcgggg auuauacagc tornado aagcggggcag uggcucccuu 56700 acccucgaag acccucacucc uagcacgucc uggauguauu cgucaaaua uguccucuua 56760 ugccacguca gcacaggguu gcuccccacu uugaucauca aguuaaaaaaggaaaga 56820 uuuucuuucu ucucugccu cuacuggaca ucauuucca cuacagau auuuuaauugu 56880 aucuguacu gauuguuuu gauuacaga cagagagguc acaguuaag aaggcu 56940 gcugcuacug cagcugucc ucccaggag guguogau u uagcugugu aaaaaugac 57000 ugcauucucc agagguccug aacacagcug ccugcgcugg agagggcuca aaccucuucc 57060 gccaggguga acucugcuuc cuggugagug ccagcaaaac aaccaaaaa gagcuguagg 57120 acuugugugg acuucaaug gugguggucc ugccacuugg gcucagccac agcaguuagg 57180 aaacuaaagg gaggaggaa agcccuuucc uugcuuuuuu agcuuggcu agquaagggc 57240 auuacaaugg uucucuuuga gauucugagc uccggcuaua acauuugccc agaaucugcc 57300 ucugaggccu uaagacacug uguuuuuauu cagcaagau gcccuuugac uccuuuuccc 57360 acuaguggug cuagguuuga gcaccuuaca cuggccccuu acaauagcca guucuugucu 57420 accuacauuc uucccuaaca uucaugauug cauaguuacu cuuaguguag aagcagacag 57480 cuuuuacaca uagacuccau ggccguagcc ucauagaacc uacuauauuc uaacuugcaa 57540 gcuaaucaga ccaauauau caaaaucaaa aaccucugcu gagaguuuau ucauucaucu 57600 cugucuccca aacguacuua uguacauacg ugcacuauau uacaugucca uuagccaaga 57660 uuuugauuuc agggaucaaa gcaaguacca auagggaaug aggucacuug cugcauggca 57720 gguggcuucc ccaugagaau gcaaggccac cucaugacuc auacuucaga gggugacca 57780 ggaacuucug auucaugucc aaagcagcuu cuacaauugc ucuaccuuga ucuagggaag 57840 augugggag gaugacauuc gggauuagcu uuauaaggcc uuccugugg cagaguuguc 57900 ugacuuucac cuagugauca acaagcagcu agcaagcauc agugugugag gccccacgcc 57960 cucucagcuc cccuacugcc caccugggac augggcuuug gcaucugucc auagcauugu 58020 ucuaaccaaa ugagguguua uggaucagcu caggauggga uauguuccca gacauauuau 58080 uuaaagaaaa uagcucccuu ccuccccuga uaaacagcug ccauggcuaa aagguaaccu 58140 ggcuggggcu uaaaagucug uugacuuuca agauauuuug caaaaacagu cauaaaaaug 58200 guauuuauca gauccuaacu auuugugaga cgguuuggua uaccauaagug guuaaaaaaca 58260 caggcuuuu ccagaggagg uuuacuuugc uuagucgugu cuccuaagug aacuuggacc 58320 ucauaagguu guugugagaa ugaaaugggu gaauogagu aaaguccuug gaccaguuuu 58380 ggccguauag uaagccuuca gcaagcaucu gcuuuuuauuc cuacagggag gcaauuguaa 58440 gcccuucaca aacagcgucu aaugugaucc uuagaacaaa ccuaugagau agggcauauc 58500 ucaauuuugu agguagggaa acagaagcca cacaauuagg aaauggcaac agaucuguua 58560 gacucuuaaa cacuaugcua caccaauuug caaggcaagg aagacaaagc accuuugaaa 58620 augggucaga uguuuuaggg uaaaugaacg uuugagaauc uuuuaaguuu uuuuuccccc 58680 agagauuauc aagguaucau uguagggga ugcaucagga aacaugacua ugaaucagcu 58740 gccugauaaa ccagccagga uggagcccac gucaucacag cagucagcaa ugccacugaa 58800 aaacaucagc ugcuuauucc cguauagauu uccccuuaag acaugaaaag ggaguucaaa 58860 gagaaugggc cagauauuc ugagagucau auuacuaaaaa uauauuuauuu uuuacuagcu 58920 uuuuuguuuu aagagguaua cugucauuag cacuguagca aaaauucacg uuuuauuaau 58980 uucuccuagu uuaucaugug auucuagggu aggaugcaga guuauauuca aaauacacaa 59040 aucaacucaa cucaguaaac auauaucgag gcccuaucau gcaaaaugc uauucuagag 59100 accacggcga acaagccacg gccccagccu caaagaaugu acuaucuuug gaacugugcu 59160 ggccaauaca guaaccagca gccacgcagg gcauuuuaaa uuuaaauuaa uuaaaaguaa 59220 aaacacaaug ccucagaugc auuagccaca uuuuaaagugu ucaauagaua uuuguggcuc 59280 cugccugcca uauuggacag ggcagauaua gaacaauucc aucacugcag aaaguucuac 59340 ugaacaaugc ugcucuggag cagaacu ucuuguucag ggauguuaca cccccgcuug 59400 uggcuagagu guggcuuauc cucagagcaa ggauagggga accauggcac ucugcaggcu 59460 cagcacugaa gacacggaug caggcucugc uucugaccua gauugaccuu gggcaaggcc 59520 cuuugcuccu cugaucccaa uuucuucacc agccaaguaa gaacaucaga ccacaagccc 59580 ucuagggcuc uguccaaaug ccccaugacu gagugaacug guagaacauu cuaugugugu 59640 gucacaacau gaagagcaaa gacuuucauc uccccaaaua auuuuguuuu ucguuuuagg 59700 aauuaaauuu cagauucacu cuaauugcca auacuaaaau ucucuauaug caguucuaaa 59760 cuugacaaac caauaaaaaa agauuauuug acuacuuauc uuuguacaac auugaggucu 59820 cccuaaagca aauuuaaaug cauauuuuaa aaauguauuc uagcaguuca guucagaagc 59880 ccccuggccc aagcaucaca cugucaaucc uuuguccuca agcagcaugg uugggugggu 59940 uaaguacuga caaacacugg gugucaggcc cauggucagg gacugugcua acagucuaca 60000 uauuagaugc caccuacccc caccccucaac agacccaaac uauuuaucca auagcaaacc 60060 uugcauuauu ucuguccaga agaaacaaac auuuauugac aacuuuuggu gugugaccug 60120 uuuaaguccu acaucucauu uaaggacugg ucaauguuag gcuaggcaau gccuguuugu 60180 gagagaauca cugccuaaag aaaauucucc auuucccuua gcucuauggu gggugacuac 60240 acauacuggu auuucuuaaa gaaauaccaa uuccauuucc uuuuaacaua auuauuaaua 60300 ucucauuagc auggugucac ugaagccugg gcccaaagaa auaccaauuc cauaucauuu 60360 uaagaucauu auuaauaucu caucagcgug gugucacuua agccugggcc cuuuagaauu 60420 uuucauguac cuguguuccu cugcccauau cagcuggaac acuaauaguu uucuuccuuu 60480 uuaucuagaa gacugagaac auuacauggg accugccccc agggcaugga ggcugaggug 60540 ggacaguuua guucaggagg cccaagaagu guugggugug cagccccuug uucaaacaca 60600 gccucugaau cgccagaggc uuccggugca uacucugagg cgcagguggg acucgggagu 60660 gagagguuuc ggcgaaugaa uugggauugc cuacuucuuc ccagugcagu ggagcuuggu 60720 ucugugguca gguccuuacg cccugucugc cuuucucguu ucuuuauuuc ucggguagua 60780 guuguggaau caaugaccu gggguuugau accuacua ccacgccucu gggggaguca 60840 cucagacucg ugaaccuaa guuccggggc ugccaaguga ggauaguag uaauugcuga 60900 uccaccuacu ugacagaua guagugagggg cccugagcgc caggcugugg auccagccuu 60960 uccacgguu ccuggugugg cuckoo cuckoo cuckoo cuckoo cuckoo cuckoo cuckoo cuckoo ugggaggga 61020 guccagcuc guccaccuguc guccagggug accucaggcu guccaggcuck aaaaaaua...

Claims

1. Functionally connected in either the 3' to 5' direction or the 5' to 3' direction, (a) A binding domain configured to bind to a target ABCA4 intron selected from the group consisting of introns 19, 23, or 24; (b) a splicing domain configured to mediate transsplicing; and (c) Code domain containing functional ABCA4 exons A nucleic acid transsplicing molecule containing, The nucleic acid transsplicing molecule is configured to transsplice the coding domain and the endogenous ABCA4 exon adjacent to the target ABCA4 intron, thereby replacing the endogenous ABCA4 exon with the functional ABCA4 exon and correcting the mutation in ABCA4. The nucleic acid trans-splicing molecule.

2. The nucleic acid transsplicing molecule according to claim 1, wherein the binding domain binds to the 3' target ABCA4 intron of the mutation, and the mutation is present in any one of ABCA4 exons 1 to 24 or introns 1 to 24.

3. The nucleic acid transsplicing molecule according to claim 2, wherein the target ABCA4 intron is intron 19, the mutation is located in either ABCA4 exons 1-19 or introns 1-19, and the coding domain comprises ABCA4 exons 1-19.

4. The nucleic acid transsplicing molecule according to claim 3, wherein the binding domain is configured to bind to intron 19 at a binding site containing one or more nucleotides from nucleotides 990 to 2,174 of SEQ ID NO:

25.

5. A nucleic acid transsplicing molecule according to claim 4, wherein the binding site contains one or more nucleotides from nucleotides 1,670 to 2,174 of SEQ ID NO:

25.

6. A nucleic acid transsplicing molecule according to claim 5, wherein the binding site contains one or more nucleotides from 1,810 to 2,000 of SEQ ID NO:

25.

7. A nucleic acid transsplicing molecule according to claim 6, wherein the binding site contains one or more nucleotides from among nucleotides 1,870 to 2,000 of SEQ ID NO:

25.

8. A nucleic acid transsplicing molecule according to claim 7, wherein the binding site contains one or more nucleotides from among nucleotides 1,920 to 2,000 of SEQ ID NO:

25.

9. The nucleic acid trans-splicing molecule according to claim 2, wherein the target ABCA4 intron is intron 23, and the mutation is present in one or more of the ABCA4 exons 1 to 23 or introns 1 to 23.

10. A nucleic acid transsplicing molecule according to claim 9, wherein the coding domain contains functional ABCA4 exons 1 to 23.

11. The nucleic acid transsplicing molecule according to claim 10, wherein the binding domain is configured to bind to intron 23 at a binding site containing one or more nucleotides from SEQ ID NO:29, either nucleotides 80-570 or nucleotides 720-1,081.

12. The binding domain is (a) One or more nucleotides from nucleotides 261 to 410 of SEQ ID NO:29; (b) One or more nucleotides from nucleotides 801 to 950 of SEQ ID NO:29; or (c) One or more nucleotides from nucleotides 841 to 990 of SEQ ID NO:29 It is configured to bind to the ABCA4 intron 23 at the binding site including the binding site. The nucleic acid trans-splicing molecule according to claim 11.

13. The binding site is (a) Six or more nucleotides from nucleotides 261–410 of SEQ ID NO:29; (b) Six or more nucleotides from nucleotides 801–950 of SEQ ID NO:29; or (c) Six or more nucleotides from nucleotides 841-990 of SEQ ID NO:29 including, The nucleic acid trans-splicing molecule according to claim 12.

14. The nucleic acid trans-splicing molecule according to claim 13, wherein the binding domain comprises six or more consecutive nucleic acid residues complementary to the six or more nucleotides of the binding site.

15. The nucleic acid transsplicing molecule according to claim 2, wherein the target ABCA4 intron is intron 24, the mutation is located in either ABCA4 exons 1-24 or introns 1-24, and the coding domain comprises ABCA4 exons 1-24.

16. The nucleic acid transsplicing molecule according to claim 11, wherein the binding domain is configured to bind to intron 24 at a binding site containing one or more nucleotides from SEQ ID NO:30, either nucleotides 600-1,250 or nucleotides 1,490-2,660.

17. A nucleic acid transsplicing molecule according to claim 12, wherein the binding site contains one or more nucleotides from among 1,000 to 1,200 of SEQ ID NO:

30.

18. The nucleic acid transsplicing molecule according to claim 1, wherein the binding domain binds to a target ABCA4 intron on the 5' side of the mutation, and the mutation is located in any one of ABCA4 exons 23-50 or introns 22-49.

19. The nucleic acid transsplicing molecule according to claim 14, wherein the target ABCA4 intron is intron 23, the mutation is located in either ABCA4 exons 24-50 or introns 23-49, and the coding domain comprises ABCA4 exons 24-50.

20. The nucleic acid transsplicing molecule according to claim 15, wherein the binding domain is configured to bind to intron 23 at a binding site containing one or more nucleotides from nucleotide 80 to 1,081 of SEQ ID NO:

29.

21. A nucleic acid transsplicing molecule according to claim 16, wherein the binding site contains one or more nucleotides from 230 to 1,081 of SEQ ID NO:

29.

22. A nucleic acid transsplicing molecule according to claim 17, wherein the binding site contains one or more nucleotides from 250 to 400 of SEQ ID NO:

29.

23. The nucleic acid transsplicing molecule according to claim 17, wherein the binding site contains one or more nucleotides from nucleotide 690 to 850 of SEQ ID NO:

29.

24. The nucleic acid transsplicing molecule according to claim 14, wherein the target ABCA4 intron is intron 24, the mutation is located in either ABCA4 exons 25-50 or introns 24-49, and the coding domain comprises ABCA4 exons 25-50.

25. The nucleic acid transsplicing molecule according to claim 20, wherein the binding domain is configured to bind to intron 24 at a binding site containing one or more nucleotides from among nucleotides 1-250, nucleotides 300-2,100, or nucleotides 2,200-2,692 of SEQ ID NO:

30.

26. A nucleic acid transsplicing molecule according to claim 21, wherein the binding site contains one or more nucleotides from 360 to 610 of SEQ ID NO:

30.

27. A nucleic acid transsplicing molecule according to claim 21, wherein the binding site contains one or more nucleotides from 750 to 1,110 of SEQ ID NO:

30.

28. Functionally connected in the direction from 5' to 3', (a) A binding domain configured to bind to ABCA4 intron 22 at a binding site containing one or more nucleotides from among nucleotides 60–570, 600–800, or 900–1,350 of SEQ ID NO:28; (b) a splicing domain configured to mediate transsplicing; and (c) Code domain including functional ABCA4 exons 23-50 A nucleic acid transsplicing molecule containing, The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous ABCA4 exon 22, thereby replacing endogenous ABCA4 exons 23-50 with functional ABCA4 exons 23-50. The nucleic acid trans-splicing molecule.

29. The nucleic acid transsplicing molecule according to claim 24, wherein the binding site contains one or more nucleotides from nucleotides 70 to 250 of SEQ ID NO:

28.

30. Functionally connected in the direction from 3' to 5', (a) A binding domain configured to bind to ABCA4 intron 22 at a binding site containing one or more nucleotides from SEQ ID NO:28, either nucleotides 1-510 or 880-1,350; (b) a splicing domain configured to mediate transsplicing; and (c) Code domain including functional ABCA4 exons 1-22 A nucleic acid transsplicing molecule containing, The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous ABCA4 exon 23, thereby replacing endogenous ABCA4 exons 1-22 with functional ABCA4 exons 1-22. The nucleic acid trans-splicing molecule.

31. The binding domain is (a) One or more nucleotides from nucleotides 1041 to 1190 of SEQ ID NO:28; (b) One or more nucleotides from nucleotides 1171 to 1320 of SEQ ID NO:28; (c) One or more nucleotides from nucleotides 1201 to 1350 of SEQ ID NO:28 It is configured to bind to the ABCA4 intron 22 at the binding site including the binding site. The nucleic acid trans-splicing molecule according to claim 30.

32. The binding site is (a) Six or more nucleotides from nucleotides 1041–1190 of SEQ ID NO:28; (b) Six or more nucleotides from nucleotides 1171–1320 of SEQ ID NO:28; (c) Six or more of nucleotides 1201-1350 of SEQ ID NO:28 including, The nucleic acid trans-splicing molecule according to claim 31.

33. The nucleic acid trans-splicing molecule according to claim 32, wherein the binding domain comprises six or more consecutive nucleic acid residues complementary to the six or more nucleotides of the binding site.

34. A nucleic acid transsplicing molecule according to any one of claims 1 to 33, wherein the binding domain is 100 to 200 nucleotides long.

35. A nucleic acid transsplicing molecule according to any one of claims 1 to 34, wherein the coding domain is a cDNA sequence.

36. A nucleic acid transsplicing molecule according to any one of claims 1 to 34, wherein the coding domain includes a naturally occurring sequence.

37. A nucleic acid transsplicing molecule according to any one of claims 1 to 34, wherein the coding domain includes a codon-optimized sequence.

38. A nucleic acid transsplicing molecule according to any one of claims 1 to 37, wherein the artificial intron includes a spacer sequence.

39. A nucleic acid transsplicing molecule according to any one of claims 1 to 38, having a length of 3,000 to 4,000 nucleotides.

40. A nucleic acid trans-splicing molecule according to any one of claims 1 to 39, wherein a mutation in the ABCA4 gene is associated with Stargardt disease.

41. A nucleic acid trans-splicing molecule according to claim 40, wherein a mutation in the ABCA4 gene associated with Stargardt disease is expressed in photoreceptor cells.

42. Functionally connected in the direction from 3' to 5', (a) A binding domain configured to bind to the ABCA4 intron 23 at a binding site containing six or more nucleotides from nucleotides 261 to 410 of SEQ ID NO:29, the binding domain comprising six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) Artificial introns containing splicing domains; and (c) Code domain including functional ABCA4 exons 1-23 A nucleic acid transsplicing molecule containing, The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous ABCA4 exon 24, thereby replacing endogenous ABCA4 exons 1-23 with functional ABCA4 exons 1-23. The nucleic acid trans-splicing molecule.

43. Functionally connected in the direction from 3' to 5', (a) A binding domain configured to bind to the ABCA4 intron 23 at a binding site containing six or more nucleotides from nucleotides 801 to 950 of SEQ ID NO:29, wherein the binding domain contains six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) Artificial introns containing splicing domains; and (c) Code domain including functional ABCA4 exons 1-23 A nucleic acid transsplicing molecule containing, The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous ABCA4 exon 24, thereby replacing endogenous ABCA4 exons 1-23 with functional ABCA4 exons 1-23. The nucleic acid trans-splicing molecule.

44. Functionally connected in the direction from 3' to 5', (a) A binding domain configured to bind to ABCA4 intron 23 at a binding site containing six or more nucleotides from nucleotides 841 to 990 of SEQ ID NO:29, wherein the binding domain contains six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) Artificial introns containing splicing domains; and (c) Code domain including functional ABCA4 exons 1-23 A nucleic acid transsplicing molecule containing, The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous ABCA4 exon 24, thereby replacing endogenous ABCA4 exons 1-23 with functional ABCA4 exons 1-23. The nucleic acid trans-splicing molecule.

45. Functionally connected in the direction from 3' to 5', (a) A binding domain configured to bind to intron 22 of ABCA4 at a binding site containing six or more nucleotides from nucleotides 1041 to 1190 of SEQ ID NO:28, wherein the binding domain contains six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) Artificial introns containing splicing domains; and (c) Code domain including functional ABCA4 exons 1-22 A nucleic acid transsplicing molecule containing, The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous ABCA4 exon 23, thereby replacing endogenous ABCA4 exons 1-22 with functional ABCA4 exons 1-22. The nucleic acid trans-splicing molecule.

46. Functionally connected in the direction from 3' to 5', (a) A binding domain configured to bind to ABCA4 intron 22 at a binding site containing six or more nucleotides from nucleotides 1171-1320 of SEQ ID NO:28, the binding domain comprising six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) Artificial introns containing splicing domains; and (c) Code domain including functional ABCA4 exons 1-22 A nucleic acid transsplicing molecule containing, The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous ABCA4 exon 23, thereby replacing endogenous ABCA4 exons 1-22 with functional ABCA4 exons 1-22. The nucleic acid trans-splicing molecule.

47. Functionally connected in the direction from 3' to 5', (a) A binding domain configured to bind to intron 22 of ABCA4 at a binding site containing six or more nucleotides from nucleotides 1201 to 1350 of SEQ ID NO:28, wherein the binding domain contains six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) Artificial introns containing splicing domains; and (c) Code domain including functional ABCA4 exons 1-22 A nucleic acid transsplicing molecule containing, The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous ABCA4 exon 23, thereby replacing endogenous ABCA4 exons 1-22 with functional ABCA4 exons 1-22. The nucleic acid trans-splicing molecule.

48. A proviral plasmid comprising a nucleic acid transsplicing molecule according to any one of claims 1 to 47.

49. Adeno-associated virus (AAV) comprising a nucleic acid molecule according to any one of claims 1 to 48.

50. The AAV according to claim 49, which preferentially targets photoreceptor cells.

51. The AAV according to claim 49 or 50, comprising AAV5 capsid protein, AAV8 capsid protein, AAV8(b) capsid protein, or AAV9 capsid protein.

52. A pharmaceutical composition comprising a nucleic acid transsplicing molecule according to any one of claims 1 to 47, a proviral plasmid according to claim 48, or an AAV according to any one of claims 49 to 51.

53. A pharmaceutical composition comprising a 5' nucleic acid transsplicing molecule and a 3' nucleic acid transsplicing molecule, wherein the 5' nucleic acid transsplicing molecule is a nucleic acid transsplicing molecule according to any one of claims 2 to 13 or 30 to 47, and the 3' nucleic acid transsplicing molecule is a nucleic acid transsplicing molecule according to any one of claims 14 to 25.

54. A method for correcting a mutation in the ABCA4 gene within a target cell, comprising the step of administering the pharmaceutical composition according to claim 52 or 53 to the target.

55. A method for correcting a mutation in one or more of the ABCA4 exons 1 to 24 in a subject requiring such correction, comprising the step of administering a pharmaceutical composition comprising a nucleic acid trans-splicing molecule according to any one of claims 2 to 13 or 30 to 47 to the subject.

56. A method for correcting a mutation in one or more of the ABCA4 exons 23 to 50 in a subject requiring such correction, comprising the step of administering a pharmaceutical composition comprising a nucleic acid trans-splicing molecule according to any one of claims 14 to 25 to the subject.

57. A method for correcting a mutation in any one of ABCA4 exons 1 to 24 and a second mutation in any one of exons 23 to 50 in a subject requiring such correction, comprising the step of administering the pharmaceutical composition according to claim 53 to the subject.

58. A method for treating a subject having a disorder associated with a mutation in ABCA4, comprising the step of administering the pharmaceutical composition according to claim 52 or 53 to the subject.

59. A method for treating a subject having a disorder associated with a mutation in one or more of the ABCA4 exons 1 to 24 or introns 1 to 24, comprising the step of administering a pharmaceutical composition comprising a nucleic acid trans-splicing molecule according to any one of claims 2 to 13 or 30 to 47 to the subject.

60. A method for treating a subject having a disorder associated with a mutation in one or more of the ABCA4 exons 23-50 or introns 22-49, comprising the step of administering a pharmaceutical composition comprising a nucleic acid trans-splicing molecule according to any one of claims 14 to 25.

61. A method for treating a subject having a disorder associated with a first mutation in any one of ABCA4 exons 1 to 24 and a second mutation in any one of exons 23 to 50, comprising the step of administering the pharmaceutical composition according to claim 53 to the subject.

62. The method according to any one of claims 54 to 61, wherein the subject has Stargardt disease.

63. The method according to any one of claims 54 to 62, wherein the composition is administered by subretinal injection, intravitreous injection, or intravenous injection.

64. Adeno-associated virus (AAV) comprising an assembled capsid in which a genome vector containing AAV 5'ITR, a nucleic acid molecule according to any one of claims 1 to 47 under functional control of a regulatory sequence, and AAV 3'ITR is packaged inside.

65. The method according to any one of claims 54 to 63, wherein the subject shows an increase of at least 10% in ABCA4 protein expression after administration.

66. Functionally connected in the direction from 3' to 5', (a) A binding domain configured to bind to CEP290 intron 26 at a binding site containing one or more nucleotides from among nucleotides 4,800 to 5,838 of SEQ ID NO:32; (b) a splicing domain configured to mediate transsplicing; and (c) Code domain including functional CEP290 exons 2-26 A nucleic acid transsplicing molecule containing, The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous CEP290 exon 27, thereby replacing endogenous CEP290 exons 2-26 with the functional CEP290 exons 2-26, and correcting the pathogenic point mutation. The nucleic acid trans-splicing molecule.

67. Functionally connected in the direction from 3' to 5', (a) A binding domain configured to bind to CEP290 in any one of the target introns 27, 28, 29, or 30; (b) a splicing domain configured to mediate transsplicing; and (c) Code domain containing the functional CEP290 exon at the 5' end of the target intron A nucleic acid transsplicing molecule containing, The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and endogenous CEP290, thereby replacing the 5' endogenous CEP290 exon of the target intron with the functional CEP290 exon, and correcting the pathogenic point mutation. The nucleic acid trans-splicing molecule.

68. A proviral plasmid comprising the nucleic acid trans-splicing molecule according to claim 66 or 67.

69. AAV comprising a nucleic acid molecule according to any one of claims 66 to 68.

70. A pharmaceutical composition comprising a nucleic acid transsplicing molecule according to claim 66 or 67, a proviral plasmid according to claim 68, or an AAV according to claim 69.

71. A method for correcting a pathogenic point mutation in a CEP290 intron 26 within a target cell, comprising the step of administering to the target a nucleic acid transsplicing molecule according to claim 66 or 67, a proviral plasmid according to claim 68, an AAV according to claim 69, or a pharmaceutical composition according to claim 70.

72. A method for treating a subject having LCA 10 caused by a pathogenic point mutation in CEP290 intron 26, comprising the step of administering to the subject a nucleic acid trans-splicing molecule according to claim 66 or 67, a proviral plasmid according to claim 68, an AAV according to claim 69, or a pharmaceutical composition according to claim 70.

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