Trans-splicing molecules

Abstract

To provide nucleic acid trans-splicing molecules capable of correcting mutations in the ABCA4 gene or the CEP290 gene.SOLUTION: A nucleic acid trans-splicing molecule comprises, operatively linked in either a 3'-to-5' direction or a 5'-to-3' direction: (a) a binding domain configured to bind a target ABCA4 intron selected from the group consisting of introns 19, 23, or 24; (b) a splicing domain configured to mediate trans-splicing; and (c) a coding domain comprising a functional ABCA4 exon. Compositions including the nucleic acid trans-splicing molecule can be useful in treatment or prevention of diseases associated with mutations in ABCA4, such as Stargardt Disease (e.g., Stargardt Disease 1) or mutations in CEP290, such as LCA (e.g., LCA10).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application Nos. 62 / 658,658 and 62 / 658,667, both filed April 17, 2018. The contents of both of these provisional patent applications are incorporated herein by reference in their entirety.

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

[0003] FIELD OF THE INVENTION In general, the 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 intact. Various mutations along the entire length of the ABCA4 gene can cause Stargardt disease. Treatments currently under development for Stargardt disease include lentiviral delivery of ABCA4, chemically modified variants of vitamin A, and treatment of retinal pigment epithelial cells.

[0005] Leber congenital amourosis type 10 (LCA 10) is a condition characterized by severe visual impairment beginning in early childhood. This visual loss is associated with photoreceptor death due to a ciliary body defect. The most common known mutation associated with LCA 10 is a point mutation in intron 26 of the CEP290 gene, where an adenine is replaced by a guanine at nucleotide 1655, resulting in a splicing defect that results in the splicing of a cryptic stop codon between exons 26 and 27. This autosomal recessive mutation leads to the production of a nonfunctional centrosomal protein, resulting in the blindness characteristic 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 various genetic defects.However, AAV vectors may have limitations determined by virus biology, such as the packaging size constraints that can hinder the delivery of large nucleic acid molecules, such as those required to replace ABCA4 gene or CEP290 gene.Therefore, there is a need in the field for compositions and methods for correcting mutations in ABCA4 and CEP290. Summary of the Invention

[0007] overview The present invention relates to nucleic acid trans-splicing molecules and methods of using them to correct mutations in the ABCA4 gene or the CEP290 gene. The compositions and methods provided herein may be useful for 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 a 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, and 24; (b) a splicing domain configured to mediate trans-splicing; and (c) a coding domain containing a functional ABCA4 exon, operably linked in either the 3' to 5' direction or the 5' to 3' direction, 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 in ABCA4.

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

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

[0011] In some embodiments, the binding domain comprises a binding site comprising any one or more of nucleotides 261-410 of SEQ ID NO:29 (e.g., a binding site of 1-200, 6-150, 12-100, or 20-80 nucleotides within or encompassing nucleotides 261-410 of SEQ ID NO:29, e.g., a binding site of 1-6, 6-12, 12-18, 18-24, 24-50, 50-100, 100-150, or 150-20 ... The ABCA4 intron 23 is configured to bind to ABCA4 intron 23 at a binding site of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, 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 comprises six or more of nucleotides 261-410 of SEQ ID NO:29. In some embodiments, the binding domain comprises six or more contiguous nucleic acid residues that are complementary (e.g., antisense) to the six or more nucleotides of the binding site.In some embodiments, a binding domain comprises a set of contiguous nucleic acid residues that are complementary to a corresponding complementary set of nucleotides of a binding site of ABCA4 having one or more of nucleotides 261-410 of SEQ ID NO:29, wherein the set of contiguous nucleic acid residues of the binding domain is 6-500 residues in length (e.g., 8-400, 12-300, 16-200, 24-280, or 50-150 residues in length, e.g., 100-200, 6-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-110, 100-120, 120-140, 140-160, 160-200, 180-220, 180-240, 180-280, 190-210, 210-220, 220-240, 230-240, 250-260, 260-280, 270-290, 280-300, 300-310, 310-320, 320-330, 330-340, 340-350, 350-400, 360-370, 370-410, 380-420, 390-430, 440-450, 450-460, 460-470, 480-490, 490-500, 500-510, 510-5 a length of 0 to 50, 50 to 80, 80 to 100, 100 to 120, 120 to 150, 150 to 200, or 200 to 300 residues, 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, 161, 162, 163, 164, 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).

[0012] In some embodiments, the binding domain comprises a binding site that includes any one or more of nucleotides 801-950 of SEQ ID NO:29 (e.g., a binding site of 1-200, 6-150, 12-100, or 20-80 nucleotides within or encompassing nucleotides 801-950 of SEQ ID NO:29, e.g., a binding site of 1-6, 6-12, 12-18, 18-24, 24-50, 50-100, 100-150, or 150-20 ... The ABCA4 intron 23 is configured to bind to ABCA4 intron 23 at a binding site of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, 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 comprises six or more of nucleotides 801-950 of SEQ ID NO:29. In some embodiments, the binding domain comprises six or more contiguous nucleic acid residues that are complementary (e.g., antisense) to the six or more nucleotides of the binding site.In some embodiments, a binding domain comprises a set of contiguous nucleic acid residues that are complementary to a corresponding complementary set of nucleotides of a binding site of ABCA4 having one or more of nucleotides 801-950 of SEQ ID NO:29, wherein the set of contiguous nucleic acid residues of the binding domain is 6-500 residues in length (e.g., 8-400, 12-300, 16-200, 24-280, or 50-150 residues in length, e.g., 100-200, 6-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-120, 120-140, 140-160, 160-200, 180-220, 180-240, 180-280, 190-290, 200-300, 220-340, 240-360, 260-380, 280-390, 300-400, 300-420, 320-440, 320-460, 320-480, 320-540, 320-560, 320-640, 320-760, 320-800, 320-840, 320-900, 320-940, 320-960, 320-180, 320-190, 320-240, 320-250, 320-3 a length of 0 to 50, 50 to 80, 80 to 100, 100 to 120, 120 to 150, 150 to 200, or 200 to 300 residues, 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, 161, 162, 163, 164, 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).

[0013] In some embodiments, the binding domain comprises a binding site that includes any one or more of nucleotides 841-990 of SEQ ID NO:29 (e.g., a binding site of 1-200, 6-150, 12-100, or 20-80 nucleotides within or encompassing nucleotides 841-990 of SEQ ID NO:29, e.g., a binding site of 1-6, 6-12, 12-18, 18-24, 24-50, 50-100, 100-150, or 150-20 ... The ABCA4 intron 23 is configured to bind to ABCA4 intron 23 at a binding site of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, 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 comprises six or more of nucleotides 841-990 of SEQ ID NO:29. In some embodiments, the binding domain comprises six or more contiguous nucleic acid residues that are complementary (e.g., antisense) to the six or more nucleotides of the binding site.In some embodiments, a binding domain comprises a set of contiguous nucleic acid residues that are complementary to a corresponding complementary set of nucleotides of a binding site of ABCA4 having one or more of nucleotides 841-990 of SEQ ID NO:29, wherein the set of contiguous nucleic acid residues of the binding domain is 6-500 residues in length (e.g., 8-400, 12-300, 16-200, 24-280, or 50-150 residues in length, e.g., 100-200, 6-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-120, 120-140, 140-160, 160-200, 180-220, 180-240, 180-280, 190-210, 210-220, 220-240, 230-240, 250-260, 260-280, 270-300, 280-300, 300-320, 320-340, 340-360, 360-380, 380-400, 390-410, 410-420, 420-430, 430-440, 440-450, 450-460, 460-470, 470-480, 480-500, 490-510, 510-520, 520-5 a length of 0 to 50, 50 to 80, 80 to 100, 100 to 120, 120 to 150, 150 to 200, or 200 to 300 residues, 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, 161, 162, 163, 164, 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).

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

[0015] In some embodiments, the binding domain binds to a target ABCA4 intron 5' to the mutation, and the mutation is present in any one of ABCA4 exons 23-50 or introns 22-49. For example, in some embodiments, the target ABCA4 intron is intron 23, the mutation is present in any one of ABCA4 exons 24-50 or introns 23-49, and the coding domain comprises ABCA4 exons 24-50. In some embodiments, the binding domain is configured to bind to intron 23 at a binding site that comprises any one or more of nucleotides 80-1,081 of SEQ ID NO:29. In some embodiments, the binding site comprises any one or more of nucleotides 230-1,081 of SEQ ID NO:29, e.g., any one or more of nucleotides 250-400 of SEQ ID NO:29 or any one or more of nucleotides 690-850 of SEQ ID NO:29.

[0016] In some embodiments, the target ABCA4 intron is intron 24, the mutation is in any one of ABCA4 exons 25-50 or introns 24-49, and the coding domain comprises ABCA4 exons 25-50. In some embodiments, the binding domain is configured to bind to intron 24 at a binding site comprising any one or more of nucleotides 1-250, nucleotides 300-2,100, or nucleotides 2,200-2,692 of SEQ ID NO:30. In some embodiments, the binding site comprises any one or more of nucleotides 360-610 of SEQ ID NO:30. In other embodiments, the binding site comprises any one or more of nucleotides 750-1,110 of SEQ ID NO:30.

[0017] In another aspect, the invention features a nucleic acid trans-splicing molecule including, operably linked in a 5' to 3' direction, (a) a binding domain configured to bind to ABCA4 intron 22 at a binding site including any one or more of nucleotides 60-570, 600-800, or 900-1,350 of SEQ ID NO:28; (b) a splicing domain configured to mediate trans-splicing; and (c) a coding domain including 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 the functional ABCA4 exons 23-50. In some embodiments, the binding site includes any one or more of nucleotides 70-250 of SEQ ID NO:28.

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

[0019] In some embodiments, the binding domain comprises a binding site that includes any one or more of nucleotides 1041-1190 of SEQ ID NO:28 (e.g., a binding site of 1-200, 6-150, 12-100, or 20-80 nucleotides within or encompassing nucleotides 1041-1190 of SEQ ID NO:28, e.g., a binding site of 1-6, 6-12, 12-18, 18-24, 24-50, 50-100, 100-150, or 150-20 ... The ABCA4 intron 22 is configured to bind to ABCA4 intron 22 at a binding site of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, 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 comprises six or more of nucleotides 1041-1190 of SEQ ID NO:28. In some embodiments, the binding domain comprises six or more contiguous nucleic acid residues that are complementary (e.g., antisense) to the six or more nucleotides of the binding site.In some embodiments, a binding domain comprises a set of contiguous nucleic acid residues that are complementary to a corresponding complementary set of nucleotides of a binding site of ABCA4 having one or more of nucleotides 1041-1190 of SEQ ID NO:28, wherein the set of contiguous nucleic acid residues of the binding domain is 6-500 residues in length (e.g., 8-400, 12-300, 16-200, 24-280, or 50-150 residues in length, e.g., 100-200, 6-10, 10-20, 20-30, 30-40, 40-50, 50-80, 80-100, 100-120, 120-150, 150-200, or 200-300 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, 110, 111, 112, 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, 115 140, 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).

[0020] In some embodiments, the binding domain is selected from any one or more of nucleotides 1171-1320 of SEQ ID NO:28 (e.g., a binding site of 1 to 200, 6 to 150, 12 to 100, or 20 to 80 nucleotides within or encompassing nucleotides 1171-1320 of SEQ ID NO:28, e.g., a binding site of 1 to 6, 6 to 12, 12 to 18, 18 to 24, 24 to 50, 50 to 100, 100 to 150, or 150 to 20 ... The binding domain is configured to bind to a binding site of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, 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 1171-1320 of SEQ ID NO:28. In certain embodiments, the binding site comprises six or more of nucleotides 1171-1320 of SEQ ID NO:28. In some embodiments, the binding domain comprises six or more contiguous nucleic acid residues that are complementary (e.g., antisense) to the six or more nucleotides of the binding site.In some embodiments, a binding domain comprises a set of contiguous nucleic acid residues that are complementary to a corresponding complementary set of nucleotides of a binding site of ABCA4 having one or more of nucleotides 1171-1320 of SEQ ID NO:28, wherein the set of contiguous nucleic acid residues of the binding domain is 6-500 residues in length (e.g., 8-400, 12-300, 16-200, 24-280, or 50-150 residues in length, e.g., 100-200, 6-10, 10-20, 20-30, 30-40, 40-50, 50-80, 80-100, 100-120, 120-150, 150-200, or 200-300 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, 110, 111, 112, 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, 115 140, 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 located within any one or more of nucleotides 1201-1350 of SEQ ID NO:28 (e.g., a binding site of 1-200, 6-150, 12-100, or 20-80 nucleotides within or encompassing nucleotides 1201-1350 of SEQ ID NO:28, e.g., a binding site of 1-6, 6-12, 12-18, 18-24, 24-50, 50-100, 100-150, or 150-20 ... The binding domain is configured to bind to a binding site of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, 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 1201-1350 of SEQ ID NO:28. In certain embodiments, the binding site comprises six or more of nucleotides 1201-1350 of SEQ ID NO:28. In some embodiments, the binding domain comprises six or more contiguous nucleic acid residues that are complementary (e.g., antisense) to the six or more nucleotides of the binding site.In some embodiments, a binding domain comprises a set of contiguous nucleic acid residues that are complementary to a corresponding complementary set of nucleotides of a binding site of ABCA4 having one or more of nucleotides 1201-1350 of SEQ ID NO:28, wherein the set of contiguous nucleic acid residues of the binding domain is 6-500 residues in length (e.g., 8-400, 12-300, 16-200, 24-280, or 50-150 residues in length, e.g., 100-200, 6-10, 10-20, 20-30, 30-40, 40-50, 50-80, 80-100, 100-120, 120-150, 150-200, or 200-300 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, 110, 111, 112, 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, 115 140, 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).

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

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

[0024] In some embodiments of any of the foregoing methods, the nucleic acid trans-splicing molecule is 3,000 to 4,000 nucleotides in length (e.g., 3,100 to 3,900 nucleotides in length, 3,200 to 3,800 nucleotides in length, 3,300 to 3,700 nucleotides in length, 3,400 to 3,600 nucleotides in length, or about 3,500 nucleotides in length, e.g., 3,000 to 3,100 nucleotides in length, 3,100 to 3,200 nucleotides in length, 3,200 to 3,300 nucleotides in length, 3,300 to 3,400 nucleotides in length, 3,400 to 3,500 nucleotides in length, 3,500 to 3,600 nucleotides in length, 3,600 to 3,700 nucleotides in length, 3,800 to 3,900 nucleotides in length, or 3,900 to 4,000 nucleotides in length).

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

[0026] In another aspect, provided herein is a nucleic acid trans-splicing molecule comprising, operably linked in a 3' to 5' direction: (a) a binding domain configured to bind to ABCA4 intron 23 at a binding site comprising six or more nucleotides 261-410 of SEQ ID NO:29, wherein the binding domain comprises six or more consecutive nucleic acid residues that are complementary to the six or more nucleotides of 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 and endogenous ABCA4 exon 24, thereby replacing endogenous ABCA4 exons 1-23 with the 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 comprising six or more nucleotides 801-950 of SEQ ID NO:29, the binding domain comprising six or more consecutive nucleic acid residues complementary to the six or more nucleotides of the binding site; (b) an artificial intron comprising a splicing domain; and (c) a coding domain comprising functional ABCA4 exons 1-23, operably linked in a 3' to 5' direction; 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 the functional ABCA4 exons 1-23.

[0028] In another aspect, provided herein is a nucleic acid trans-splicing molecule comprising, operably linked in a 3' to 5' direction: (a) a binding domain configured to bind to ABCA4 intron 23 at a binding site comprising six or more nucleotides 841-990 of SEQ ID NO:29, wherein the binding domain comprises six or more consecutive nucleic acid residues that are complementary to the six or more nucleotides of 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 and endogenous ABCA4 exon 24, thereby replacing endogenous ABCA4 exons 1-23 with the 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 six or more nucleotides 1041 to 1190 of SEQ ID NO:28, the binding domain comprising six or more consecutive nucleic acid residues complementary to the six or more nucleotides of the binding site; (b) an artificial intron comprising a splicing domain; and (c) a coding domain comprising functional ABCA4 exons 1 to 22, operably linked in a 3' to 5' direction; wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain to endogenous ABCA4 exon 23, thereby replacing endogenous ABCA4 exons 1 to 22 with the functional ABCA4 exons 1 to 22.

[0030] In another aspect, the invention features a nucleic acid trans-splicing molecule including, operably linked in a 3' to 5' direction: (a) a binding domain configured to bind to ABCA4 intron 22 at a binding site comprising six or more 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 of 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 to endogenous ABCA4 exon 23, thereby replacing endogenous ABCA4 exons 1-22 with the functional ABCA4 exons 1-22.

[0031] In yet another aspect, provided herein is a nucleic acid trans-splicing molecule comprising, operably linked in a 3' to 5' direction: (a) a binding domain configured to bind to ABCA4 intron 22 at a binding site comprising six or more nucleotides 1201-1350 of SEQ ID NO:28, wherein the binding domain comprises six or more consecutive nucleic acid residues complementary to the six or more nucleotides of 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 to endogenous ABCA4 exon 23, thereby replacing endogenous ABCA4 exons 1-22 with the functional ABCA4 exons 1-22.

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

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

[0034] In another aspect, the invention features a pharmaceutical composition including the nucleic acid trans-splicing molecule, proviral plasmid, or AAV of any of the previous aspects.

[0035] In another aspect, provided herein is a pharmaceutical composition comprising any of the 5' nucleic acid trans-splicing molecules of any of the above embodiments and any of the 3' nucleic acid trans-splicing molecules of any of the above embodiments.

[0036] In yet another aspect, the invention features a method of correcting a mutation in an ABCA4 gene in a target cell of a subject by administering to the subject a pharmaceutical composition of any of the previous aspects.

[0037] In another aspect, provided herein is a method for correcting a mutation in any one or more of ABCA4 exons 1-24 in a subject in need thereof by administering to the subject a pharmaceutical composition having a nucleic acid trans-splicing molecule of any of the above embodiments. In a specific 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 a mutation in any one or more of ABCA4 exons 23-50 in a subject in need thereof by administering to the subject a pharmaceutical composition comprising the nucleic acid trans-splicing molecule of any of the above embodiments.

[0039] In another aspect, the invention features a method for correcting a mutation in any one of ABCA4 exons 1-24 and a second mutation in any one of exons 23-50 in a subject in need thereof, comprising administering to the subject 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.

[0040] In another aspect, the invention features a method of treating a subject having a disorder associated with a mutation in ABCA4, comprising administering to the subject any of the pharmaceutical compositions described above. In some aspects, 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 aspects described above. In some aspects, 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 aspects described above.

[0041] In another aspect, the invention features a method of treating a subject having a disorder associated with a first mutation in any one of ABCA4 exons 1-24 and a second mutation in any one of exons 23-50 by administering to the subject 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.

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

[0043] In some embodiments of any of the foregoing methods, the subject exhibits at least a 1% increase in ABCA4 protein expression after administration (e.g., a 1-5% increase, a 5-10%, a 10-15% increase, a 15-20% increase, a 20-25% increase, a 25-50% increase, or a 50-100% increase in ABCA4 protein expression after administration, e.g., compared to ABCA4 protein expression in the same subject before administration, or compared to a reference sample, reference subject, or reference subject group).

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

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

[0046] In another aspect, the invention features a nucleic acid trans-splicing molecule comprising, operably linked in a 3' to 5' direction, (a) a binding domain configured to bind to CEP290 in any one of target introns 27, 28, 29, or 30; (b) a splicing domain configured to mediate trans-splicing; and (c) a coding domain including a functional CEP290 exon 5' to the target intron, wherein the nucleic acid trans-splicing molecule is configured to trans-splice between the coding domain and an endogenous CEP290, thereby replacing the endogenous CEP290 exon 5' to the target intron with the functional CEP290 exon and 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 comprises 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 comprising any one or more of nucleotides 120-680, nucleotides 710-2,200, or nucleotides 2,670-2,910 of SEQ ID NO:86. In some embodiments, the binding site comprises any one or more of nucleotides 790-2,100 of SEQ ID NO:86, such as any one or more of nucleotides 1,020-1,630 of SEQ ID NO:86. In other embodiments, the binding site comprises any one or more of nucleotides 1,670-2,000 of SEQ ID NO:86.

[0048] In some embodiments, the target intron is intron 28, the coding domain comprises 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 comprising any one or more of nucleotides 1-390, nucleotides 410-560, or nucleotides 730-937 of SEQ ID NO:87. In some embodiments, the binding site comprises any one or more of nucleotides 1-200 of SEQ ID NO:87. In other embodiments, the binding site comprises any one or more of nucleotides 720-900 of SEQ ID NO:87.

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

[0050] In some embodiments, the target intron is intron 30, the coding domain comprises 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 comprising any one or more of nucleotides 880-1,240 of SEQ ID NO:89, e.g., any one or more of nucleotides 950-1,240 of SEQ ID NO:89, e.g., any one or more of 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 in length (e.g., 25 to 900 nucleotides in length, 30 to 800 nucleotides in length, 40 to 700 nucleotides in length, 50 to 600 nucleotides in length, 75 to 500 nucleotides in length, 100 to 400 nucleotides in length, 125 to 200 nucleotides in length, or about 150 nucleotides in length, e.g., 20 to 30 nucleotides in length, 30 to 40 nucleotides in length, 40 to 50 nucleotides in length, 50 to 75 nucleotides in length, 75 to 100 nucleotides in length, 125 to 150 nucleotides in length, 150 to 175 nucleotides in length, 175 to 200 nucleotides in length, 200 to 250 nucleotides in length, 250 to 500 nucleotides in length, 500 to 750 nucleotides in length, or 750 to 1,000 nucleotides in length).

[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 sequence.

[0054] In any of the above embodiments, the nucleic acid trans-splicing molecule can be 3,000 to 4,000 nucleotides in length.

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

[0056] In another aspect of the present invention, there is provided herein a proviral plasmid comprising a nucleic acid trans-splicing molecule of any of the preceding aspects.

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

[0058] In another aspect, the invention features a pharmaceutical composition including the nucleic acid trans-splicing molecule, proviral plasmid, or AAV of any of the previous aspects.

[0059] In another aspect, featured herein is a method of correcting a pathogenic point mutation in CEP290 intron 26 in a target cell of a subject, comprising administering to the subject a nucleic acid trans-splicing molecule, proviral plasmid, AAV, or pharmaceutical composition of any of the previous aspects. In some embodiments, the subject has LCA 10.

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

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

[0062] In another aspect, the present invention provides a kit comprising any one or more of the aforementioned nucleic acid trans-splicing molecules, proviral plasmids, AAV, or pharmaceutical compositions, further comprising instructions for using the one or more nucleic acid trans-splicing molecules, proviral plasmids, AAV, or pharmaceutical compositions to correct a mutation in a CEP290 gene in a subject (e.g., a mutation associated with a disorder, e.g., LCA 10). [The present invention 1001] operably linked in either a 3' to 5' or 5' to 3' orientation, (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 trans-splicing; and (c) Coding domain containing functional ABCA4 exons 1. A nucleic acid trans-splicing molecule comprising: 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 the mutation in ABCA4. The nucleic acid trans-splicing molecule. [The present invention 1002] 1001. A nucleic acid trans-splicing molecule of the present invention, wherein the binding domain binds to a target ABCA4 intron 3' to a mutation, and the mutation is present in any one of ABCA4 exons 1-24 or introns 1-24. [The present invention 1003] 1002. The nucleic acid trans-splicing molecule of the present invention, wherein the target ABCA4 intron is intron 19, the mutation is present in any one of ABCA4 exons 1-19 or introns 1-19, and the coding domain comprises ABCA4 exons 1-19. [The present invention 1004] 1003. A nucleic acid trans-splicing molecule of the present invention, wherein the binding domain is configured to bind to intron 19 at a binding site comprising any one or more of nucleotides 990 to 2,174 of SEQ ID NO:25. [The present invention 1005] 1004. The nucleic acid trans-splicing molecule of the present invention, wherein the binding site comprises any one or more of nucleotides 1,670 to 2,174 of SEQ ID NO:25. [The present invention 1006] 1005. The nucleic acid trans-splicing molecule of the present invention, wherein the binding site comprises any one or more of nucleotides 1,810 to 2,000 of SEQ ID NO:25. [The present invention 1007] 1006. The nucleic acid trans-splicing molecule of the present invention, wherein the binding site comprises any one or more of nucleotides 1,870 to 2,000 of SEQ ID NO:25. [The present invention 1008] 1007. The nucleic acid trans-splicing molecule of the present invention, wherein the binding site comprises any one or more of nucleotides 1,920 to 2,000 of SEQ ID NO:25. [The present invention 1009] 1002. The nucleic acid trans-splicing molecule of the present invention, wherein the target ABCA4 intron is intron 23 and the mutation is present in any one or more of ABCA4 exons 1-23 or introns 1-23. [The present invention 1010] 1009. The nucleic acid trans-splicing molecule of the present invention, wherein the coding domain comprises functional ABCA4 exons 1-23. [The present invention 1011] 1010. A nucleic acid trans-splicing molecule of the present invention, wherein the binding domain is configured to bind to intron 23 at a binding site comprising any one or more of nucleotides 80 to 570 or nucleotides 720 to 1,081 of SEQ ID NO:29. [The present invention 1012] The binding domain is (a) any one or more of nucleotides 261 to 410 of SEQ ID NO:29; (b) any one or more of nucleotides 801-950 of SEQ ID NO:29; or (c) any one or more of nucleotides 841 to 990 of SEQ ID NO: 29 and configured to bind to ABCA4 intron 23 at a binding site comprising: A nucleic acid trans-splicing molecule of the present invention. [The present invention 1013] The binding site is (a) six or more of nucleotides 261 to 410 of SEQ ID NO:29; (b) six or more nucleotides between nucleotides 801 and 950 of SEQ ID NO:29; or (c) six or more nucleotides from nucleotides 841 to 990 of SEQ ID NO:29 Including, A nucleic acid trans-splicing molecule of the present invention. [The present invention 1014] 1013. The nucleic acid trans-splicing molecule of the invention, wherein the binding domain comprises six or more consecutive nucleic acid residues that are complementary to said six or more nucleotides of the binding site. [The present invention 1015] 1002. The nucleic acid trans-splicing molecule of the present invention, wherein the target ABCA4 intron is intron 24, the mutation is present in any one of ABCA4 exons 1-24 or introns 1-24, and the coding domain comprises ABCA4 exons 1-24. [The present invention 1016] 1011. A nucleic acid trans-splicing molecule of the present invention, wherein the binding domain is configured to bind to intron 24 at a binding site comprising any one or more of nucleotides 600 to 1,250 or nucleotides 1,490 to 2,660 of SEQ ID NO:30. [The present invention 1017] 1012. A nucleic acid trans-splicing molecule of the present invention, wherein the binding site comprises any one or more of nucleotides 1,000 to 1,200 of SEQ ID NO:30. [The present invention 1018] 1001. A nucleic acid trans-splicing molecule of the present invention, wherein the binding domain binds to a target ABCA4 intron 5' to a mutation, and the mutation is present in any one of ABCA4 exons 23-50 or introns 22-49. [The present invention 1019] 1014. The nucleic acid trans-splicing molecule of the present invention, wherein the target ABCA4 intron is intron 23, the mutation is present in either one of ABCA4 exons 24-50 or intron 23-49, and the coding domain comprises ABCA4 exons 24-50. [The present invention 1020] 1015. A nucleic acid trans-splicing molecule of the present invention, wherein the binding domain is configured to bind to intron 23 at a binding site comprising any one or more of nucleotides 80 to 1,081 of SEQ ID NO:29. [The present invention 1021] 1016. The nucleic acid trans-splicing molecule of the present invention, wherein the binding site comprises any one or more of nucleotides 230 to 1,081 of SEQ ID NO:29. [The present invention 1022] 1017. The nucleic acid trans-splicing molecule of the present invention, wherein the binding site comprises any one or more of nucleotides 250 to 400 of SEQ ID NO:29. [The present invention 1023] 1017. The nucleic acid trans-splicing molecule of the present invention, wherein the binding site comprises any one or more of nucleotides 690 to 850 of SEQ ID NO:29. [The present invention 1024] 1014. The nucleic acid trans-splicing molecule of the present invention, wherein the target ABCA4 intron is intron 24, the mutation is present in either one of ABCA4 exons 25-50 or introns 24-49, and the coding domain comprises ABCA4 exons 25-50. [The present invention 1025] 1020. The nucleic acid trans-splicing molecule of the present invention, wherein the binding domain is configured to bind to intron 24 at a binding site comprising any one or more of nucleotides 1 to 250, nucleotides 300 to 2,100, or nucleotides 2,200 to 2,692 of SEQ ID NO:30. [The present invention 1026] 1021. A nucleic acid trans-splicing molecule of the present invention, wherein the binding site comprises any one or more of nucleotides 360 to 610 of SEQ ID NO:30. [The present invention 1027] 1021. A nucleic acid trans-splicing molecule of the present invention, wherein the binding site comprises any one or more of nucleotides 750 to 1,110 of SEQ ID NO:30. [The present invention 1028] operably linked in the 5' to 3' direction, (a) a binding domain configured to bind to ABCA4 intron 22 at a binding site comprising any one or more of nucleotides 60-570, 600-800, or 900-1,350 of SEQ ID NO:28; (b) a splicing domain configured to mediate trans-splicing; and (c) Coding domain containing functional ABCA4 exons 23–50 1. A nucleic acid trans-splicing molecule comprising: 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 the functional ABCA4 exons 23-50; The nucleic acid trans-splicing molecule. [The present invention 1029] 1024. The nucleic acid trans-splicing molecule of the present invention, wherein the binding site comprises any one or more of nucleotides 70 to 250 of SEQ ID NO:28. [The present invention 1030] operably linked in the 3' to 5' direction, (a) 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; (b) a splicing domain configured to mediate trans-splicing; and (c) Coding domain containing functional ABCA4 exons 1–22 1. A nucleic acid trans-splicing molecule comprising: 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 the functional ABCA4 exons 1-22. The nucleic acid trans-splicing molecule. [The present invention 1031] The binding domain is (a) any one or more of nucleotides 1041 to 1190 of SEQ ID NO:28; (b) any one or more of nucleotides 1171 to 1320 of SEQ ID NO:28; (c) any one or more of nucleotides 1201 to 1350 of SEQ ID NO: 28 and configured to bind to ABCA4 intron 22 at a binding site comprising: A nucleic acid trans-splicing molecule of the present invention 1030. [The present invention 1032] The binding site is (a) six or more of nucleotides 1041 to 1190 of SEQ ID NO:28; (b) six or more of nucleotides 1171 to 1320 of SEQ ID NO:28; (c) six or more of nucleotides 1201 to 1350 of SEQ ID NO: 28 Including, The nucleic acid trans-splicing molecule of the present invention 1031. [The present invention 1033] 1032. The nucleic acid trans-splicing molecule of the present invention, wherein the binding domain comprises six or more consecutive nucleic acid residues that are complementary to said six or more nucleotides of the binding site. [The present invention 1034] 1034. The nucleic acid trans-splicing molecule of any of claims 1001 to 1033, wherein the binding domain is 100 to 200 nucleotides in length. [This invention 1035] The nucleic acid trans-splicing molecule of any one of claims 1001 to 1034, wherein the coding domain is a cDNA sequence. [The present invention 1036] 1035. The nucleic acid trans-splicing molecule of any one of claims 1001 to 1034, wherein the coding domain comprises a naturally occurring sequence. [This invention 1037] 1035. The nucleic acid trans-splicing molecule of any one of claims 1001 to 1034, wherein the coding domain comprises a codon-optimized sequence. [The present invention 1038] 8. The nucleic acid trans-splicing molecule of any one of claims 1001 to 1037, wherein the artificial intron comprises a spacer sequence. [This invention 1039] 1038. The nucleic acid trans-splicing molecule of any one of claims 1001 to 1038, which is 3,000 to 4,000 nucleotides in length. [The present invention 1040] The nucleic acid trans-splicing molecule of any of claims 1001 to 1039, wherein a mutation in the ABCA4 gene is associated with Stargardt disease. [This invention 1041] 1040. The nucleic acid trans-splicing molecule of the present invention, wherein a mutation in the ABCA4 gene associated with Stargardt disease is expressed in photoreceptor cells. [The present invention 1042] operably linked in the 3' to 5' direction, (a) a binding domain configured to bind to ABCA4 intron 23 at a binding site comprising six or more of nucleotides 261-410 of SEQ ID NO:29, wherein the binding domain comprises six or more consecutive nucleic acid residues that are complementary to the six or more nucleotides of the binding site; (b) an artificial intron containing a splicing domain; and (c) Coding domain containing functional ABCA4 exons 1–23 1. A nucleic acid trans-splicing molecule comprising: 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 the functional ABCA4 exons 1-23. The nucleic acid trans-splicing molecule. [This invention 1043] operably linked in the 3' to 5' direction, (a) a binding domain configured to bind to ABCA4 intron 23 at a binding site comprising six or more of nucleotides 801-950 of SEQ ID NO:29, wherein the binding domain comprises six or more consecutive nucleic acid residues that are complementary to the six or more nucleotides of the binding site; (b) an artificial intron containing a splicing domain; and (c) Coding domain containing functional ABCA4 exons 1–23 1. A nucleic acid trans-splicing molecule comprising: 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 the functional ABCA4 exons 1-23. The nucleic acid trans-splicing molecule. [This invention 1044] operably linked in the 3' to 5' direction, (a) a binding domain configured to bind to ABCA4 intron 23 at a binding site comprising six or more of nucleotides 841-990 of SEQ ID NO:29, wherein the binding domain comprises six or more consecutive nucleic acid residues that are complementary to the six or more nucleotides of the binding site; (b) an artificial intron containing a splicing domain; and (c) Coding domain containing functional ABCA4 exons 1–23 1. A nucleic acid trans-splicing molecule comprising: 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 the functional ABCA4 exons 1-23. The nucleic acid trans-splicing molecule. [This invention 1045] operably linked in the 3' to 5' direction, (a) a binding domain configured to bind to ABCA4 intron 22 at a binding site comprising six or more of nucleotides 1041-1190 of SEQ ID NO:28, wherein the binding domain comprises six or more consecutive nucleic acid residues that are complementary to the six or more nucleotides of the binding site; (b) an artificial intron containing a splicing domain; and (c) Coding domain containing functional ABCA4 exons 1–22 1. A nucleic acid trans-splicing molecule comprising: 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 the functional ABCA4 exons 1-22. The nucleic acid trans-splicing molecule. [The present invention 1046] operably linked in the 3' to 5' direction, (a) a binding domain configured to bind to ABCA4 intron 22 at a binding site comprising six or more of nucleotides 1171-1320 of SEQ ID NO:28, wherein the binding domain comprises six or more consecutive nucleic acid residues that are complementary to the six or more nucleotides of the binding site; (b) an artificial intron containing a splicing domain; and (c) Coding domain containing functional ABCA4 exons 1–22 1. A nucleic acid trans-splicing molecule comprising: 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 the functional ABCA4 exons 1-22. The nucleic acid trans-splicing molecule. [This invention 1047] operably linked in the 3' to 5' direction, (a) a binding domain configured to bind to ABCA4 intron 22 at a binding site comprising six or more of nucleotides 1201-1350 of SEQ ID NO:28, wherein the binding domain comprises six or more consecutive nucleic acid residues that are complementary to the six or more nucleotides of the binding site; (b) an artificial intron containing a splicing domain; and (c) Coding domain containing functional ABCA4 exons 1–22 1. A nucleic acid trans-splicing molecule comprising: 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 the functional ABCA4 exons 1-22. The nucleic acid trans-splicing molecule. [This invention 1048] A proviral plasmid comprising any one of the nucleic acid trans-splicing molecules of the present inventions 1001 to 1047. [This invention 1049] An adeno-associated virus (AAV) comprising any one of the nucleic acid molecules of the present inventions 1001 to 1048. [The present invention 1050] The AAV of the present invention 1049 preferentially targets photoreceptor cells. [This invention 1051] The AAV of the present invention 1049 or 1050, comprising an AAV5 capsid protein, an AAV8 capsid protein, an AAV8(b) capsid protein, or an AAV9 capsid protein. [This invention 1052] A pharmaceutical composition comprising the nucleic acid trans-splicing molecule of any one of claims 1001 to 1047, the proviral plasmid of claim 1048, or the AAV of any one of claims 1049 to 1051. [This invention 1053] A pharmaceutical composition comprising a 5' nucleic acid trans-splicing molecule and a 3' nucleic acid trans-splicing molecule, wherein the 5' nucleic acid trans-splicing molecule is any one of the nucleic acid trans-splicing molecules 1002 to 1013 or 1030 to 1047 of the present invention, and the 3' nucleic acid trans-splicing molecule is any one of the nucleic acid trans-splicing molecules 1014 to 1025 of the present invention. [This invention 1054] A method for correcting a mutation in the ABCA4 gene in a target cell of a subject, comprising the step of administering to the subject the pharmaceutical composition of invention 1052 or 1053. [This invention 1055] A method for correcting a mutation in any one or more of ABCA4 exons 1 to 24 in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a nucleic acid trans-splicing molecule of any one of 1002 to 1013 or 1030 to 1047. [This invention 1056] A method for correcting a mutation in any one or more of ABCA4 exons 23 to 50 in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising any one of the nucleic acid trans-splicing molecules of the present invention 1014 to 1025. [This 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 in need thereof, comprising administering to the subject a pharmaceutical composition of the present invention. [This invention 1058] A method for treating a subject having a disorder associated with a mutation in ABCA4, comprising administering to the subject a pharmaceutical composition of invention 1052 or 1053. [This invention 1059] A method for treating a subject having a disorder associated with a mutation in any one or more of ABCA4 exons 1 to 24 or introns 1 to 24, comprising administering to the subject a pharmaceutical composition comprising a nucleic acid trans-splicing molecule of any one of 1002 to 1013 or 1030 to 1047. [The present invention 1060] A method for treating a subject having a disorder associated with a mutation in any one or more of ABCA4 exons 23-50 or introns 22-49, comprising administering to the subject a pharmaceutical composition comprising any one of the nucleic acid trans-splicing molecules of the present invention 1014 to 1025. [This 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 administering to the subject a pharmaceutical composition of the present invention 1053. [This invention 1062] 1062. The method of any one of claims 1054 to 1061, wherein the subject has Stargardt disease. [This invention 1063] 1063. The method of any of claims 1054 to 1062, wherein said composition is administered by subretinal injection, intravitreal injection, or intravenous injection. [This invention 1064] An adeno-associated virus (AAV) comprising an assembled capsid in which a genome vector comprising the AAV 5'ITR, any one of the nucleic acid molecules of the present invention 1001 to 1047 under the functional control of a regulatory sequence, and the AAV 3'ITR is packaged. [This invention 1065] 1064. The method of any of claims 1054 to 1063, wherein the subject exhibits at least a 10% increase in ABCA4 protein expression after administration. [The present invention 1066] operably linked in the 3' to 5' direction, (a) a binding domain configured to bind to CEP290 intron 26 at a binding site comprising any one or more of nucleotides 4,800 to 5,838 of SEQ ID NO:32; (b) a splicing domain configured to mediate trans-splicing; and (c) Coding domain containing functional CEP290 exons 2–26 1. A nucleic acid trans-splicing molecule comprising: 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. [This invention 1067] operably linked in the 3' to 5' direction, (a) a binding domain configured to bind to CEP290 in any one of target introns 27, 28, 29, or 30; (b) a splicing domain configured to mediate trans-splicing; and (c) a coding domain containing a functional CEP290 exon 5' to the target intron; 1. A nucleic acid trans-splicing molecule comprising: the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain and the endogenous CEP290, thereby replacing the endogenous CEP290 exon 5' to the target intron with the functional CEP290 exon and correcting the pathogenic point mutation. The nucleic acid trans-splicing molecule. [The present invention 1068] A proviral plasmid comprising a nucleic acid trans-splicing molecule of the present invention 1066 or 1067. [This invention 1069] An AAV comprising any one of the nucleic acid molecules of the present inventions 1066 to 1068. [The present invention 1070] A pharmaceutical composition comprising the nucleic acid trans-splicing molecule of the present invention 1066 or 1067, the proviral plasmid of the present invention 1068 or the AAV of the present invention 1069. [This invention 1071] A method for correcting a pathogenic point mutation in CEP290 intron 26 in a target cell of a subject, comprising 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. [This invention 1072] A method for treating a subject having LCA 10 caused by a pathogenic point mutation in CEP290 intron 26, comprising 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 drawings]

[0063] [Figure 1] Figure 1 is a schematic diagram of several exemplary nucleic acid trans-splicing molecules for correcting mutant ABCA4 exons with functional ABCA4 exons. Darkly shaded boxes represent native ABCA4 exons. Dotted lines connecting darkly shaded boxes represent native introns. Lightly shaded boxes with dark outlines represent functional ABCA4 exons within the nucleic acid trans-splicing molecule. A curved splicing domain is attached to one end of each functional ABCA4 exon, resulting in an intron in the ABCA4 pre-mRNA. [Figure 2]Figure 2 is a graph showing the trans-splicing efficiency (relative fold change) provided by a 150-base binding domain spanning ABCA4 intron 19 (SEQ ID NO:25) at 10-nucleotide intervals using a 5' trans-splicing molecule. The x-axis label indicates the number 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 fold change) mediated by a 150-base binding domain spanning ABCA4 intron 22 (SEQ ID NO:28) at 10-nucleotide intervals using a 5' trans-splicing molecule. The x-axis label indicates the number 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 fold change) mediated by a 150-base binding domain spanning ABCA4 intron 22 (SEQ ID NO:28) at 10-nucleotide intervals using a 3' trans-splicing molecule. The x-axis labels indicate the number of each binding site, starting from the 5' end of the intron (i.e., the first nucleotide of the intron sequence). [Figure 5] 5 is a graph showing the trans-splicing efficiency (relative fold change) mediated by a 150-base binding domain spanning ABCA4 intron 23 (SEQ ID NO:29) at 10-nucleotide intervals using a 5' trans-splicing molecule. The x-axis labels indicate the number 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 fold change) mediated by a 150-base binding domain spanning ABCA4 intron 23 (SEQ ID NO:29) at 10-nucleotide intervals using a 3' trans-splicing molecule. The x-axis labels indicate the number of each binding site, starting from the 5' end of the intron (i.e., the first nucleotide of the intron sequence). [Figure 7] 7 is a graph showing the trans-splicing efficiency (relative fold change) mediated by a 150-base binding domain spanning ABCA4 intron 24 (SEQ ID NO:30) at 10-nucleotide intervals using a 5' trans-splicing molecule. The x-axis label indicates the number of each binding site, starting from the 5' end of the intron (i.e., the first nucleotide of the intron sequence). [Figure 8] 8 is a graph showing the trans-splicing efficiency (relative fold change) mediated by a 150-base binding domain spanning ABCA4 intron 24 (SEQ ID NO:30) at 10-nucleotide intervals using a 3' trans-splicing molecule. The x-axis labels indicate the number 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 transcription activation domain. The VP64 transcription activation domain is shown. The right panel shows a portion of the 5'-untranslated region (5'-UTR) of ABCA4. The TATA box and putative transcription start site are also shown. Also shown are the sequences targeted by the three different DNA-binding domains of the TALENs. As shown, 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 showing that 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 clones were harvested and analyzed by Western blot. The positive control (+) represents cells transfected with a plasmid expressing ABCA4 cDNA. Cell lysates were prepared 48 hours post-transfection, and membrane fractions were probed for ABCA4 expression using antibody ab72955 (Abcam). Clones ZT-22 and ZT-48 showed ABCA4 protein expression. [Figure 11] FIG. 11 is a schematic diagram showing a CAG promoter cell line. [Figure 12A] Figures 12A and 12B show a site-specific guide designed to insert a CAG promoter and puromycin selectable marker (Figure 12A) using homology arms (Figure 12B). [Figure 12B] See legend to Figure 12A. [Figure 13] FIG. 13 is a schematic diagram showing a CAG promoter cell line. [Figure 14] Figures 14A and 14B are a graph and gel, respectively, showing expression results for several clonal lines selected for further analysis. Figure 14A shows RNA expression for the cell lines, and Figure 14B shows protein expression. Membrane preparations of the indicated cell lines were probed for 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] FIG. 15 is a schematic diagram showing CRISPR guide RNAs for targeting exons 3 and 4. [Figure 16] FIG. 16 is a graph showing RNA expression and a gel showing protein profiles of single clone cells derived after treatment with CRISPR / Cas9 as shown in FIG. 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 disrupted. [Figure 17B] See legend to Figure 17A. [Figure 18] Figure 18 is a pair of tables showing that mutational analysis of Figures 17A and 17B confirmed that exons 3 and 4 were targeted and disrupted in the alleles in the 17+06 and 17+21 cell lines. [Figure 19] Figures 19A and 19B are schematic diagrams of trans-splicing molecules targeting ABCA4 pre-mRNA. Figure 19A shows a general 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 targeting 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) showing the results of the trans-splicing reactions. Figures 20A and 20B show the protein and RNA levels, respectively, of the intron 22 trans-splicing reaction, and Figures 20C and 20D show the protein and RNA levels, respectively, of the intron 23 trans-splicing reaction. [Figure 20B] See legend to Figure 20A. [Figure 20C] See legend to Figure 20A. [Figure 20D] See legend to Figure 20A. [Figure 21]Figure 21 is a schematic diagram of several exemplary nucleic acid trans-splicing molecules for correcting a mutation in CEP290 intron 26 with a functional 5' portion of the CEP290 gene. Darkly shaded boxes represent native CEP290 exons. The dotted lines connecting the darkly shaded boxes represent native introns. Lightly shaded boxes with dark outlines represent functional CEP290 exons within the nucleic acid trans-splicing molecules. A curved splicing domain is attached to one end of each functional CEP290 exon sequence, resulting in an intron in the CEP290 pre-mRNA. [Figure 22] Figure 22 is a graph showing the trans-splicing efficiency (relative fold change) provided by a 150-base binding domain spanning CEP290 intron 26 (SEQ ID NO:85) at 10-nucleotide intervals. 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 23] Figure 23 is a graph showing the trans-splicing efficiency (relative fold change) provided by a 150-base-long binding domain spanning 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 fold change) provided by a 150-base-long binding domain spanning 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 fold change) provided by a 150-base-long binding domain spanning 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 fold change) provided by a 150-base-long binding domain spanning 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). DETAILED DESCRIPTION OF THE INVENTION

[0064] Detailed Description 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 use pre-mRNA trans-splicing as gene therapy (e.g., ex vivo gene therapy and in vivo gene therapy) for the treatment of diseases caused by mutations in ABCA4, such as Stargardt disease (e.g., Stargardt disease type 1).

[0065] Alternatively, 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 CEP290 gene, such as LCA 10. Such methods use pre-mRNA trans-splicing as gene therapy (e.g., ex vivo gene therapy and in vivo gene therapy) for the treatment of diseases caused by mutations in CEP290, such as LCA 10.

[0066] The trans-splicing molecules and methods of use exemplified herein offer several advantages over conventional therapies. First, the use of AAV-mediated trans-splicing molecule delivery provides efficient and specific delivery of gene therapy agents to photoreceptors while overcoming problems associated with AAV packaging limitations. Second, such compositions and methods allow for correction of genetic defects at their source. Furthermore, the compositions and methods provided herein are useful for treating any type of mutation within ABCA4 (or other large cDNA / transgene cassettes). Correction of the photoreceptor defect provides collateral rescue for retinal pigment epithelial cells. Furthermore, the methods and compositions of the present invention are generally immunologically harmless. The use of subretinal delivery and other features ensure that the effect is specific to target cells, such as photoreceptors, thereby reducing toxicity due to 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) have the requirement that cells undergo mitosis for homology-directed repair to occur, which is disadvantageous in post-mitotic tissues such as the retina.

[0067] I. Definition Unless otherwise defined, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and as commonly understood by reference to publications which provide those skilled in the art with general guidance on many of the terms used in this application. In the event of a conflict between definitions set forth herein and those in the referenced publications, the definitions provided herein shall control.

[0068] A "nucleic acid trans-splicing molecule" or "trans-splicing molecule" has three major elements: (a) a binding domain that confers specificity by tethering the trans-splicing molecule to its target gene (e.g., pre-mRNA); (b) a splicing domain (e.g., a splicing domain with a 3' splice site or a 5' splice site); and (c) a coding sequence configured to allow trans-splicing to occur in the target gene, where one or more exons (e.g., one or more mutant exons) within the target gene can be replaced. A "pre-mRNA trans-splicing molecule" or "RTM" refers to a nucleic acid trans-splicing molecule that targets a pre-mRNA. In some embodiments, a trans-splicing molecule, e.g., an RTM, can include a cDNA as part of a functional exon (e.g., a functional ABCA4 exon or CEP290 exon, e.g., a codon-optimized exon) for replacing or correcting, e.g., a mutant ABCA4 exon or CEP290 exon).

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

[0070] "Binding" between a binding domain and a target intron, as used herein, refers to hydrogen bonding between the binding domain and the target intron sufficient to bring the trans-splicing molecule into association with the target gene (e.g., pre-mRNA) and thereby mediate trans-splicing. In some embodiments, the hydrogen bonding between the binding domain and the target intron is between nucleotide bases that are complementary to each other and in antisense orientation (e.g., hybridized to each other).

[0071] As used herein, an "artificial intron" refers to a nucleic acid sequence that links (directly or indirectly) a binding domain to a coding domain. The artificial intron contains a splicing domain and may further contain one or more spacer sequences and / or other regulatory elements.

[0072] As used herein, a "splice domain" refers to a nucleic acid sequence having a motif that is recognized by the spliceosome and mediates trans-splicing. A splice domain includes a splice site (e.g., a single splice site, i.e., a unique splice site), which may be a 3' splice site or a 5' splice site. A splice domain may also include other regulatory elements. For example, in some embodiments, a splice domain includes a splicing enhancer sequence (e.g., an exonic splicing enhancer sequence (ESE) or an intronic splicing enhancer sequence (ISE)). In some embodiments, a splice domain includes a branch point (e.g., a highly conserved branch point) or a branch site sequence and / or a polypyrimidine tract (PPT). In some embodiments, the splice domain of a 5' trans-splicing molecule does not include a branch point or PPT, but includes a 5' splice acceptor or a 3' splice donor.

[0073] As used herein, a "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 that is produced in lower or higher amounts than normal). 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 a functional variant thereof. In some embodiments, a mutation refers to a nucleic acid sequence that encodes a protein having an amino acid sequence that differs from the corresponding wild-type protein or a functional variant thereof. A "mutant exon" (e.g., a mutant ABCA4 exon) refers to an exon containing a mutation or an exon sequence that reflects a mutation in a different region, e.g., a cryptic exon resulting from a mutation in 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 a functional human ABCA4 gene is shown in SEQ ID NO:6. An exemplary genomic DNA sequence of a functional (wild-type) human ABCA4 gene is shown in NCBI Reference Sequence:NG_009073. The amino acid sequence of an exemplary ABCA4 protein is shown in Protein Accession No. P78363.

[0075] The exons and introns of ABCA4 can be identified herein as shown in Table 1 below and mapped onto the ABCA4 pre-mRNA molecule of SEQ ID NO:6. Each exon and intron of ABCA4 is identified herein according to the reference number in the first (left) column. The size (in base pairs; bp) of each exon and intron is shown in columns two and three. Column four indicates the length of the cDNA molecule corresponding to the exon 5' of the corresponding intron number. Column five indicates the length of the cDNA molecule corresponding to the mRNA 3' of the corresponding intron number.

[0076] Table 1. Summary of exons and introns of ABCA4 TIFF2024099770000002.tif221138

[0077] As used herein, a "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 set forth in Table 2 below. It is understood that the term "target ABCA4 intron" also encompasses variants of the ABCA4 introns provided herein, such as 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 the adjacent exons as shown in Table 1).

[0078] Table 2: ABCA4 intron sequence TIFF2024099770000003.tif21764

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

[0080] The exons and introns of CEP290 can be identified herein as shown in Table 3 below and mapped onto the CEP290 pre-mRNA molecule of SEQ ID NO:112. Each exon and intron of CEP290 is identified herein according to the reference number in the first (left) column. The size (in base pairs; bp) of each exon and intron is shown in columns two and three. Column four indicates the length of the cDNA molecule corresponding to the exon 5' of the corresponding intron number. Column five indicates the length of the cDNA molecule corresponding to the mRNA 3' of the corresponding intron number.

[0081] Table 3. Summary of exons and introns of CEP290 TIFF2024099770000004.tif238123

[0082] As used herein, a "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 set forth in Table 4 below. It is understood that the term "target CEP290 intron" also encompasses variants of the CEP290 introns provided herein, such as 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 the adjacent exons as shown in Table 3).

[0083] Table 4: CEP290 intron sequence TIFF2024099770000005.tif23364

[0084] As used herein, the term "subject" includes any mammal, e.g., a human, in need of such a treatment or prevention method. Other mammals in need of such treatment or prevention include dogs, cats or other domestic animals, horses, livestock, and 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., an 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 subject can be of any age for which treatment or prophylactic therapy may be beneficial. For example, in some embodiments, the subject is 0-5 years old, 5-10 years old, 10-20 years old, 20-30 years old, 30-50 years old, 50-70 years old, or over 70 years old. In other embodiments, the subject is 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 subject has viable retinal cells.

[0085] As used herein, the term "mutation-associated disorder" or "mutation associated with disorder" refers to the correlation between the disorder and the mutation. In some embodiments, the mutation-associated disorder is known to be caused entirely or in part, directly or indirectly, or is suspected to be caused entirely or in part, directly or indirectly, by the mutation. For example, a subject with the mutation may be at risk of developing the disorder, and this risk may further depend on other factors, such as other (e.g., unrelated) mutations (e.g., in the same gene or in different genes) or environmental factors.

[0086] As used herein, the term "treat" or grammatical derivatives thereof is defined as reducing the progression of a disease, reducing the severity of disease symptoms, slowing the progression of disease symptoms, relieving disease symptoms, or delaying the onset of a disease.

[0087] As used herein, the term "prevention" of a disorder or its grammatical derivatives is defined as reducing the risk of developing a disease, for example, as a preventative 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 according to any suitable method known in the art or described herein.In some embodiments, a subject at risk of developing a disorder has one or more ABCA4 mutations or CEP290 mutations associated with the disorder.In addition, 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 a disorder in a subject can be achieved by administering a trans-splicing molecule (e.g., in an AAV vector or AAV particle) directly to the subject, or by administering host cells harboring the trans-splicing molecule to the subject.

[0089] The term "administering" or grammatical variations thereof, as used in the methods described herein, refers to the delivery of a composition or ex vivo treated cells to a subject in need thereof, e.g., having a mutation or defect in the targeted gene of interest. For example, in one embodiment where ocular cells are targeted, the method involves delivering the composition to photoreceptor cells or other ocular cells via subretinal injection. In another embodiment, intravitreal injection into ocular cells or injection into ocular cells via the palpebral vein can be used. In another embodiment, the composition is administered intravenously. Still other methods of administration can be selected by one of skill in the art in light of the present disclosure.

[0090] Codon optimization refers to modifying a nucleic acid sequence to alter individual nucleic acids without resulting in any changes to the encoded amino acids. Sequences modified in this manner are referred to herein as "codon-optimized." This process can be performed on any of the sequences described herein to promote expression or stability. Codon optimization can be performed, for example, in a manner such as that described in U.S. Patent Nos. 7,561,972, 7,561,973, and 7,888,112, each of which is incorporated herein by reference in its entirety. The sequence surrounding the translation start site can 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 by reference in its entirety.

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

[0092] The term "pharmaceutically acceptable" means safe for administration to mammals, such as humans. In some embodiments, a pharmaceutically acceptable composition is approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, more particularly humans.

[0093] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic molecule (e.g., a trans-splicing molecule of the present invention or a vector or cell containing the trans-splicing molecule) is administered. Examples of suitable pharmaceutical carriers are found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA., 2002. nd It is described in the 2005 edition.

[0094] The words "a" and "an" mean "one or more of." For example, "a gene" should be understood to refer to one or more such genes. Thus, the terms "a" and "an," "one or more of a (or an)," and "at least one of a (or an)" are used interchangeably herein.

[0095] As used herein, the term "about" refers to a value within a ±10% variation from the reference value, unless otherwise specified.

[0096] II. Trans-splicing molecules Provided herein are 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 in the ABCA4 gene (e.g., an ABCA4 gene having a mutant ABCA4 exon). In some embodiments, the nucleic acid trans-splicing molecule is a pre-RNA trans-splicing molecule (RTM). The design of the trans-splicing molecule allows for the replacement of defective or mutated portions of pre-mRNA exon(s) with a nucleic acid sequence, e.g., 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 some other modification, e.g., codon optimization.

[0098] In one embodiment, the trans-splicing molecule is configured to correct one or more mutations located in the 3' portion of the ABCA4 gene. In one embodiment, the trans-splicing molecule is configured to correct one or more mutations located in the 5' portion of the ABCA4 gene. The trans-splicing molecules provided herein function to repair a defective gene in a target cell of a subject by replacing the defective pre-mRNA gene sequence to remove the defective portion of the target pre-mRNA, resulting in a functional ABCA4 gene that can transcribe a functional gene product in the cell.

[0099] The present invention provides trans-splicing molecules 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, splice site, and binding domain are operably linked in a 5' to 3' direction, such that the trans-splicing molecule is configured to replace the 5' end of the endogenous gene with a coding domain containing a functional ABCA4 exon that replaces the mutant ABCA4 exon. Conversely, in a 3' trans-splicing molecule, the coding domain, splice site, and binding domain are operably linked in a 3' to 5' direction, such that the trans-splicing molecule is configured to replace the 3' end of the endogenous gene with a coding domain containing a functional ABCA4 exon that replaces the mutant ABCA4 exon. In some embodiments, the splicing domain is present within an artificial intron that links the binding domain to the coding domain. The artificial intron may comprise further components, such as a spacer.

[0100] In some embodiments, the trans-splicing molecule or its coding domain is up to 4,700 nucleotide bases in length (e.g., 200-300 nucleotide bases in length, 300-400 nucleotide bases in length, 400-500 nucleotide bases in length, 500-600 nucleotide bases in length, 600-700 nucleotide bases in length, 700-800 nucleotide bases in length, 800-900 nucleotide bases in length, 900-1000 nucleotide bases in length, 1000-1200 nucleotide bases in length, 1200-1400 nucleotide bases in length, 1400-1600 nucleotide bases in length, 1600-1800 nucleotide bases in length, 1800-2000 nucleotide bases in length, 1900-2100 nucleotide bases in length, 2000-2200 nucleotide bases in length, 2100-2400 nucleotide bases in length, 2200-2500 nucleotide bases in length, 2300-2600 nucleotide bases in length, 2400-2600 nucleotide bases in length, 2500-2700 nucleotide bases in length, 2600-2800 nucleotide bases in length, 2700-2800 nucleotide bases in length, 2800-2900 nucleotide bases in length, 2900-3000 nucleotide bases in length, 3000-3100 nucleotide bases in length, 3100-3200 nucleotide bases in length, 3200-3300 nucleotide bases in length, 3300-3400 nucleotide bases in length, 3400-3500 nucleotide bases nucleotide bases in length, 1,000 to 1,000 nucleotide bases in length, 1,000 to 1,500 nucleotide bases in length, 1,500 to 2,000 nucleotide bases in length, 2,000 to 2,500 nucleotide bases in length, 2,500 to 3,000 nucleotide bases in length, or 3,000 to 4,000 nucleotide bases in length, e.g., 3,100 to 3,800 nucleotide bases in length, 3,200 to 3,700 nucleotide bases in length, or 3,300 to 4,000 nucleotide bases in length. 3,500 nucleotide bases in length, e.g., 3,000 to 3,100 nucleotide bases in length, 3,100 to 3,200 nucleotide bases in length, 3,200 to 3,300 nucleotide bases in length, 3,300 to 3,400 nucleotide bases in length, 3,400 to 3,500 nucleotide bases in length, 3,500 to 3,600 nucleotide bases in length, 3,600 to 3,700 nucleotide bases in length, 3,700 to 3,800 nucleotide bases in length The nucleic acid sequence may be about 2,918 nucleotide bases in length, about 3,800 to 3,900 nucleotide bases in length, or about 3,900 to 4,000 nucleotide bases in length, e.g., about 2,918 nucleotide bases in length, about 3,328 nucleotide bases in length, about 3,522 nucleotide bases in length, about 3,607 nucleotide bases in length, about 3,632 nucleotide bases in length, about 3,494 nucleotide bases in length, or about 3,300 nucleotide bases in length).

[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 into an AAV vector may not cover all mutations in the ABCA4 gene that may be associated with a disorder, and therefore, mutation correction may not be possible across the entire length of the ABCA4 gene. Therefore, the trans-splicing molecule of the present invention may be adapted as part of the method described below 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 a functional (wild-type) human ABCA4 gene is set forth in NCBI Reference Sequence: NG_009073. The amino acid sequence of an exemplary retina-specific ATP-binding cassette transporter protein expressed by ABCA4 is set forth in Protein Accession No. P78363.

[0103] In addition to these published sequences, any subsequent modifications or naturally occurring conservative non-disease-causing variant sequences present in human or other mammalian populations are also included. Additional conservative nucleotide substitutions or codon optimizations are also included. Sequences obtained by database accession numbers may also be used to search for homologous sequences in the same or other mammalian organisms.

[0104] It is anticipated that the ABCA4 nucleic acid sequences and resulting truncated proteins or amino acid fragments may tolerate certain minor nucleic acid level modifications, e.g., modifications to silent nucleotide bases, e.g., to include preference codons. In other embodiments, nucleic acid base modifications (e.g., codon optimization) that alter amino acids to improve expression of the resulting peptide / protein are anticipated. Similar modifications of fragments also include allelic variations caused by the natural degeneracy of the genetic code.

[0105] Also included are analogs or modified forms of the encoded protein fragments provided herein as modifications of the ABCA4 gene. Typically, such analogs differ from the specifically identified protein by only one to four codon changes. Conservative substitutions are those that occur within a family of amino acids that are related in side chains and chemical properties.

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

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

[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 with a single trans-splicing molecule having a coding region that includes a functional ABCA4 exon. Mutations within specific ABCA4 exons are also described in International Patent Application Publication No. WO 2017 / 087900, which is incorporated herein by reference.

[0109] ABCA4 coding domain In some embodiments, the coding domain of a 5' trans-splicing molecule comprises all ABCA4 exons (e.g., functional ABCA4 exons) present 5' to the targeted ABCA4 intron. For example, in embodiments where a 5' trans-splicing molecule targets ABCA4 intron 19, the coding domain comprises functional ABCA4 exons 1-19. In such embodiments featuring a 5' trans-splicing molecule having a coding domain comprising functional ABCA4 exons 1-19, the coding domain is approximately 2918 bp in length. In embodiments where a 5' trans-splicing molecule targets ABCA4 intron 22, the coding domain comprises functional ABCA4 exons 1-22. In such embodiments featuring a 5' trans-splicing molecule having a coding domain comprising functional ABCA4 exons 1-22, the coding domain is approximately 3,328 bp in length. In embodiments where a 5' trans-splicing molecule targets ABCA4 intron 23, the coding domain comprises functional ABCA4 exons 1-23. In such embodiments featuring 5' trans-splicing molecules having a coding domain comprising functional ABCA4 exons 1-23, the coding domain is approximately 3,522 bp in length. In embodiments where the 5' trans-splicing molecule targets ABCA4 intron 24, the coding domain comprises functional ABCA4 exons 1-24. In such embodiments featuring 5' trans-splicing molecules having a coding domain comprising functional ABCA4 exons 1-24, the coding domain is approximately 3,607 bp in length. Illustrative examples of the foregoing embodiments of 5' trans-splicing molecules targeting ABCA4 are shown in the lower left portion of Figure 1.

[0110] In some embodiments, the coding domain of a 3' trans-splicing molecule comprises any one or more of ABCA4 exons 20-50. For example, in embodiments where a 3' trans-splicing molecule targets ABCA4 intron 22, the coding domain comprises functional ABCA4 exons 23-50. In such embodiments featuring a 3' trans-splicing molecule having a coding domain comprising functional ABCA4 exons 23-50, the coding domain is approximately 3,632 bp in length. In embodiments where a 3' trans-splicing molecule targets ABCA4 intron 23, the coding domain comprises functional ABCA4 exons 24-50. In such embodiments featuring a 3' trans-splicing molecule having a coding domain comprising functional ABCA4 exons 24-50, the coding domain is approximately 3,494 bp in length. In embodiments where a 3' trans-splicing molecule targets ABCA4 intron 24, the coding domain comprises functional ABCA4 exons 25-50. In one such embodiment featuring 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 aforementioned embodiment of a 3' trans-splicing molecule targeting ABCA4 is shown in the upper right portion of Figure 1.

[0111] In some embodiments, the coding domain comprises 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 functional ABCA4 exons.

[0112] In some instances, when both mutations are present in the 5' portion of the target gene, a 5' trans-splicing molecule is selected that corrects both mutations. In one embodiment, the binding domain binds to intron 19 and the coding domain comprises functional ABCA4 exons 1-19. In one embodiment, the binding domain binds to intron 22 and the coding domain comprises functional ABCA4 exons 1-22. In one embodiment, the binding domain binds to intron 23 and the coding domain comprises functional ABCA4 exons 1-23. In one embodiment, the binding domain binds to intron 24 and the coding domain comprises functional ABCA4 exons 1-24. Alternatively, when both mutations are present in the 3' portion of the target gene, a 3' trans-splicing molecule is selected that corrects both mutations. In one embodiment, the binding domain binds to intron 22 and the coding domain comprises functional ABCA4 exons 23-50. In one embodiment, the binding domain binds to intron 23 and the coding domain comprises functional ABCA4 exons 24-50. In one embodiment, the binding domain binds to intron 24 and the coding domain comprises functional ABCA4 exons 25-50.

[0113] As an example, the pre-mRNA ABCA4 3' trans-splicing molecule functions as follows: a chimeric mRNA consisting of the 5' splice site of the endogenous target pre-mRNA and the 3' splice site of the trans-splicing molecule is generated by a spliceosome-mediated trans-splicing reaction. The trans-splicing molecule binds to the target ABCA4 intron of the endogenous target pre-mRNA by specific base pairing and replaces the entire 3' sequence upstream of the targeted intron of the endogenous ABCA4 gene with the coding domain of the trans-splicing molecule containing functional ABCA4 exon sequence.

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

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

[0116] In some embodiments, all or a portion of the coding domain or one or more functional ABCA4 exons thereof are codon-optimized, e.g., in which the nucleic acid sequence is modified to promote expression or stability without resulting in an alteration of the encoded amino acid. Codon optimization can be performed in a manner such as that described in U.S. Pat. Nos. 7,561,972, 7,561,973, and 7,888,112, each of which is incorporated herein by reference in its entirety. For delivery by recombinant AAV as described herein, in one embodiment, the coding domain is up to 4,000 nucleotide bases in length (e.g., 3,000-4,000 nucleotide bases in length, 3,100-3,800 nucleotide bases in length, 3,200-3,700 nucleotide bases in length, or 3,300-3,500 nucleotide bases in length, e.g., 3,000-3,100 nucleotide bases in length, 3,100-3,200 nucleotide bases in length). The nucleic acid sequence may be 3,200 to 3,300 nucleotide bases in length, 3,300 to 3,400 nucleotide bases in length, 3,400 to 3,500 nucleotide bases in length, 3,500 to 3,600 nucleotide bases in length, 3,600 to 3,700 nucleotide bases in length, 3,700 to 3,800 nucleotide bases in length, 3,800 to 3,900 nucleotide bases in length, or 3,900 to 4,000 nucleotide bases in length).

[0117] ABCA4 binding domain The trans-splicing molecules of the present invention feature 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 a target ABCA4 pre-mRNA (e.g., a target ABCA4 intron), promoting trans-splicing between the trans-splicing molecule and the target ABCA4 pre-mRNA while suppressing endogenous target cis-splicing, e.g., creating a chimeric molecule having a portion of an endogenous ABCA4 mRNA and a coding domain having one or more functional ABCA4 exons. In some embodiments, the binding domain is in an antisense orientation relative to the sequence of the target ABCA4 intron.

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

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

[0120] Thus, in some instances, the binding domain specifically binds to the endogenous mutant target pre-mRNA and anchors the coding domain of the trans-splicing molecule to the pre-mRNA, allowing trans-splicing to occur at a precise location within the target ABCA4 gene. The nuclear spliceosomal processing machinery can then mediate successful trans-splicing of the correct exon to the disease-causing mutant exon.

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

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

[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 can be used depending on the lengths of the other components of the trans-splicing molecule.

[0124] The binding domain can be 80% to 100% complementary to the target intron so as to stably hybridize with the target intron. For example, in some embodiments, the binding domain is 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 will be selected by those skilled in the art based on the need to maintain the nucleic acid construct containing the trans-splicing molecule and sequences required for expression and inclusion within a rAAV within a 3,000 or up to 4,000 nucleotide base limit. The selection of the sequence and hybridization strength will depend on the complementarity and length of the nucleic acid.

[0125] Any of the aforementioned binding domains may bind to a binding site 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 certain examples of the present invention, the trans-splicing molecule is a 5' trans-splicing molecule and features a binding domain that binds to intron 19 of ABCA4 (SEQ ID NO:25), and contains a coding domain with functional ABCA4 exons 1 to 19. In some embodiments, the binding site comprises any one or more of nucleotides 990-2,174 of SEQ ID NO:25 (e.g., any one or more of nucleotides 1,670-2,174 of SEQ ID NO:25, any one or more of nucleotides 1,810-2,000 of SEQ ID NO:25, any one or more of nucleotides 1,870-2,000 of SEQ ID NO:25, or any one or more of nucleotides 1,920-2,000 of SEQ ID NO:25).

[0127] In some embodiments, the trans-splicing molecule is a 5' trans-splicing molecule and features a binding domain that binds to intron 22 of ABCA4 (SEQ ID NO:28) and contains a coding domain with functional ABCA4 exons 1-22. In some embodiments, the binding site comprises any one or more of nucleotides 60-570, nucleotides 600-800, or nucleotides 900-1,350 of SEQ ID NO:28 (e.g., any one or more of nucleotides 70-250 of SEQ ID NO:28).

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

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

[0130] Alternatively, the trans-splicing molecule can be a 3' trans-splicing molecule and can feature a binding domain that binds to intron 23 of ABCA4 (SEQ ID NO:29) and a coding domain having functional ABCA4 exons 24 to 50. In some embodiments, the binding site comprises any one or more of nucleotides 80 to 1,081 of SEQ ID NO:29 (e.g., any one or more of nucleotides 230 to 1,081 of SEQ ID NO:29, any one or more of nucleotides 250 to 400 of SEQ ID NO:29, or any one or more of nucleotides 690 to 850 of SEQ ID NO:29).

[0131] In some embodiments, the trans-splicing molecule is a 5' trans-splicing molecule and features 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 any one or more of nucleotides 600-1,250 or 1,490-2,660 of SEQ ID NO:30 (e.g., any one or more of nucleotides 1,000-1,200 of SEQ ID NO:30).

[0132] In other embodiments, the trans-splicing molecule is a 3' trans-splicing molecule and features a binding domain that binds to intron 24 of ABCA4 (SEQ ID NO:30) and contains a coding domain with functional ABCA4 exons 25 to 50. In some embodiments, the binding site comprises any one or more of nucleotides 1-250, nucleotides 300-2,100, or nucleotides 2,200-2,692 of SEQ ID NO:30 (e.g., any one or more of nucleotides 360-610 of SEQ ID NO:30 or any one or more of nucleotides 750-1,110 of SEQ ID NO:30).

[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 (e.g., a CEP290 gene having a mutation in intron 26) by replacing one or more exons in the CEP290 gene. In some embodiments, the nucleic acid trans-splicing molecule is a pre-RNA trans-splicing molecule (RTM). The trans-splicing molecule is designed to allow for the replacement of a defective or mutated portion of a pre-mRNA with a nucleic acid sequence, e.g., 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 corrected sequence with some other modification, e.g., codon optimization.

[0134] In one embodiment, the trans-splicing molecule is configured to correct one or more mutations located in the 5' portion of the CEP290 gene. The trans-splicing molecule provided herein serves to repair a defective gene in a target cell of a subject by replacing the defective pre-mRNA gene sequence and resulting in a functional CEP290 gene capable of transcribing a functional gene product in the cell.

[0135] The present invention provides trans-splicing molecules 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 5' trans-splicing molecules, the coding domain, splice site, and binding domain are operably linked in a 5' to 3' direction such that the trans-splicing molecule is configured to replace the 5' end of the endogenous gene with a coding domain containing a functional CEP290 exon for correcting a mutant CEP290 pre-mRNA. In some embodiments, the splicing domain is present within an artificial intron that links the binding domain to the coding domain. The artificial intron may include additional components, such as a spacer.

[0136] In some embodiments, the trans-splicing molecule is up to 4,700 nucleotide bases in length (e.g., 3,000-4,000 nucleotide bases in length, 3,100-3,800 nucleotide bases in length, 3,200-3,700 nucleotide bases in length, or 3,300-3,500 nucleotide bases in length, e.g., 3,000-3,100 nucleotide bases in length, 3,100-3,200 nucleotide bases in length, 3,200-3,300 nucleotide bases in length, 3,300-3,400 nucleotide bases in length, 3, The nucleic acid sequence may be 400 to 3,500 nucleotide bases in length, 3,500 to 3,600 nucleotide bases in length, 3,600 to 3,700 nucleotide bases in length, 3,700 to 3,800 nucleotide bases in length, 3,800 to 3,900 nucleotide bases in length, or 3,900 to 4,000 nucleotide bases in length, e.g., about 2,991 nucleotide bases in length, about 3,103 nucleotide bases in length, about 3,309 nucleotide bases in length, about 3,461 nucleotide bases in length, or about 3,573 nucleotide bases in length).

[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 a functional (wild-type) human CEP290 gene is set forth in NCBI Reference Sequence: NG_008417. The amino acid sequence of an exemplary centrosomal protein 290 is set forth in Protein Accession No. O15078.

[0138] In addition to these published sequences, any subsequent modifications or naturally occurring conservative non-disease-causing variant sequences present in human or other mammalian populations are also included. Additional conservative nucleotide substitutions or codon optimizations are also included. Sequences obtained by database accession numbers may also be used to search for homologous sequences in the same or other mammalian organisms.

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

[0140] Also included are analogs or modified forms of the encoded protein fragments provided herein as modifications of the CEP290 gene. Typically, such analogs differ from the specifically identified protein by only one to four codon changes. Conservative substitutions are those that occur within a family of amino acids that are related in side chains and chemical properties.

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

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

[0143] In some embodiments, the disorder associated with a mutation in CEP290 is an autosomal recessive disease, eg, LCA 10.

[0144] Code Domain In some embodiments, the coding domain of a 5' trans-splicing molecule comprises all CEP290 exons (e.g., functional CEP290 exons) present 5' to the targeted CEP290 intron. For example, in embodiments where a 5' trans-splicing molecule targets CEP290 intron 26, the coding domain comprises functional CEP290 exons 2-26. In such embodiments featuring a 5' trans-splicing molecule having a coding domain comprising functional CEP290 exons 2-26, the coding domain is approximately 2,991 bp in length. In embodiments where a 5' trans-splicing molecule targets CEP290 intron 27, the coding domain comprises functional CEP290 exons 2-27. In such embodiments featuring a 5' trans-splicing molecule having a coding domain comprising functional CEP290 exons 2-27, the coding domain is approximately 3,103 bp in length. In embodiments where the 5' trans-splicing molecule targets CEP290 intron 28, the coding domain comprises functional CEP290 exons 2-28. In such embodiments featuring 5' trans-splicing molecules with a coding domain comprising functional CEP290 exons 2-28, the coding domain is approximately 3,309 bp in length. In embodiments where the 5' trans-splicing molecule targets CEP290 intron 29, the coding domain comprises functional CEP290 exons 2-29. In such embodiments featuring 5' trans-splicing molecules with a coding domain comprising functional CEP290 exons 2-29, the coding domain is approximately 3,461 bp in length. In embodiments where the 5' trans-splicing molecule targets CEP290 intron 30, the coding domain comprises functional CEP290 exons 2-30. In one such embodiment featuring 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 aforementioned embodiment of a 5' trans-splicing molecule targeting CEP290 is shown in Figure 21.

[0145] In some embodiments, the coding domain comprises 25, 26, 27, 28, or 29 functional CEP290 exons.

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

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

[0148] Binding domain The trans-splicing molecules of the present invention feature a binding domain configured to bind to a target CEP290 intron. In one embodiment, the binding domain is a nucleic acid sequence complementary to a sequence of a target CEP290 pre-mRNA (e.g., a target CEP290 intron), promoting trans-splicing between the trans-splicing molecule and the target CEP290 pre-mRNA while suppressing endogenous target cis-splicing, e.g., creating a chimeric molecule having a portion of an endogenous CEP290 mRNA and a coding domain having one or more functional CEP290 exons. In some embodiments, the binding domain is in an antisense orientation relative to the sequence of the target CEP290 intron.

[0149] The 5' trans-splicing molecule generally binds to the target CEP290 intron 3' to the mutation. In one embodiment, the binding domain comprises a portion of the sequence complementary to the target CEP290 intron.

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

[0151] Thus, in some instances, the binding domain specifically binds to the endogenous mutant target pre-mRNA and anchors the coding domain of the trans-splicing molecule to the pre-mRNA, allowing trans-splicing to occur at a precise location within the target CEP290 gene. The nuclear spliceosomal processing machinery can then mediate successful trans-splicing of the correct exon to the disease-causing mutant exon.

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

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

[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 can be used depending on the lengths of the other components of the trans-splicing molecule.

[0155] The binding domain can be 80% to 100% complementary to the target intron so as to stably hybridize with the target intron. For example, in some embodiments, the binding domain is 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 will be selected by those skilled in the art based on the need to maintain the nucleic acid construct containing the trans-splicing molecule and sequences required for expression and inclusion within a rAAV within a 3,000 or up to 4,000 nucleotide base limit. The selection of the sequence and hybridization strength will depend on the complementarity and length of the nucleic acid.

[0156] Any of the aforementioned binding domains may bind to a binding site within intron 26 (SEQ ID NO:85; e.g., at the site of a mutation, e.g., a substitution, at nucleotide 1,655 of intron 26 or 3' to a mutation, e.g., a substitution, at nucleotide 1,655 of intron 26), 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 certain 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 contains a coding domain having functional CEP290 exons 2-26. In some embodiments, the binding site comprises any one or more of nucleotides 4,980-5,383 of SEQ ID NO:85. In one embodiment, the binding site comprises any one or more of nucleotides 5,348-5,838 of SEQ ID NO:85 (e.g., any one or more of nucleotides 5,348-5,700 of SEQ ID NO:85, e.g., any one or more of nucleotides 5,400-5,600 of SEQ ID NO:85, e.g., any one or more of nucleotides 5,460-5,560 of SEQ ID NO:85, e.g., at least nucleotide 5,500 of SEQ ID NO:85).

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

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

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

[0161] In other embodiments, the trans-splicing molecule features a binding domain that binds to intron 30 of CEP290 (SEQ ID NO:89) and contains a coding domain with functional CEP290 exons 2 to 30. In some embodiments, the binding site comprises any one or more of nucleotides 880 to 1,240 of SEQ ID NO:89, e.g., any one or more of nucleotides 950 to 1,240 of SEQ ID NO:89, e.g., any one or more of nucleotides 1,060 to 1,240 of SEQ ID NO:89.

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

[0163] The splicing domain may include a splice site, a branch point, and / or a PPT tract that mediates trans-splicing. In some embodiments, the splicing domain has a single splice site, which means that the splice site has no corresponding splice site, and therefore trans-splicing is possible, but cis-splicing is not possible. In some embodiments, the splicing domain of a 3' trans-splicing molecule includes a highly conserved branch point or branch 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 include a branch point or PPT, but includes a 5' splice acceptor / or a 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 the essential consensus motif recognized by the spliceosome. The use of the branch point and PPT follows the consensus sequence required for the execution of the two phosphorylation transfer reactions involved in trans-splicing. In one embodiment, the branch point consensus sequence in mammals is YNYURAC (Y = pyrimidine; N = any nucleotide). A polypyrimidine tract is located between the branch point and the splice acceptor site and is important for the utilization of different branch points and the recognition of the 3' splice site. Consensus sequences for 5' splice donor sites and 3' splice regions used in RNA splicing are well known in the art. Modified consensus sequences that maintain the ability to function as 5' donor splice sites and 3' splice regions can 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, the endogenous splice junction corresponding to the exon proximal to the splice junction can be used to maintain splicing regulatory signal sequences, if any.

[0165] In one aspect, a suitable 5' splice site with a spacer is TIFF2024099770000006.tif4128. In one embodiment, the preferred 5' splice site is AGGT.

[0166] In one embodiment, the branch site of a suitable 3' trans-splicing molecule is 5'-TACTAAC-3'. In one embodiment, a suitable 3' splice site is TIFF2024099770000007.tif4155. In one embodiment, the PPT of a suitable 3' trans-splicing molecule is TIFF2024099770000008.tif4128.

[0167] Further components and modifications In some embodiments of any trans-splicing molecule of the present invention (e.g., any ABCA4 or CEP290 trans-splicing molecule described herein), the splicing domain is included as part of an artificial intron, which may include one or more additional components. For example, a spacer region may be included within the artificial intron to separate 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 translation of any unspliced ​​trans-spliced ​​molecule and / or (ii) a sequence that promotes trans-splicing to the target pre-mRNA. The spacer may be 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 suitable 5' trans-splicing molecule is: TIFF2024099770000009.tif4128. In one aspect, a suitable 3' spacer is The file is TIFF2024099770000010.tif4128.

[0168] Further optional components of the trans-splicing molecule (e.g., as part of an artificial intron) include mini-introns and intra-intronic or intra-exonic enhancers (e.g., intra-intronic splice enhancers, e.g., downstream intra-intronic splice enhancers) or silencers that may regulate trans-splicing.

[0169] In another embodiment, the trans-splicing molecule further comprises at least one safety sequence for suppressing non-specific trans-splicing, incorporated within the spacer (e.g., as part of an artificial intron), within the binding domain, or elsewhere within the trans-splicing molecule. This is a region of the trans-splicing molecule that covers the 3' and / or 5' splice site elements of the trans-splicing molecule with relatively weak complementarity, thereby suppressing non-specific trans-splicing. The trans-splicing molecule is designed in such a way that upon hybridization of the binding / targeting moiety(s) of the trans-splicing molecule, the 3' or 5' splice site is uncovered and becomes fully active. Such safety sequences include the complementary strand of a cis sequence (or may be a separate second nucleic acid strand) that may bind to one or both sides of the branch point of the trans-splicing molecule, the pyrimidine tract, the 3' splice site and / or the 5' splice site (splice element), or may bind to a portion of the splice element itself. The binding of the safety sequence can be disrupted by the target binding region of the trans-splicing molecule binding to the target pre-mRNA, thus exposing and activating the splice element (making it available for trans-splicing with the target pre-mRNA). In another embodiment, the trans-splicing molecule has a 3'UTR sequence or a ribozyme sequence added to the 3' or 5' end.

[0170] In one embodiment, splicing-promoting sequences, such as sequences referred to as intra-exon splicing-promoting sequences, can also be included in the structure of the artificial intron. Additional structural elements, such as polyadenylation signal sequences for modifying RNA expression / stability, or 5' splice sequences for promoting splicing, additional binding regions, safety self-complementary regions, additional splice sites, or protecting groups for adjusting the stability of the molecule and preventing degradation, can also be added to the artificial intron. A stop codon can also be included in the trans-splicing molecule structure (e.g., as part of the artificial intron) to suppress the translation of unspliced ​​trans-splicing molecules. Additional elements, such as a 3' hairpin structure, circularized RNA, nucleotide base modifications, or synthetic analogs, can be incorporated into the trans-splicing molecule to promote or facilitate nuclear localization and spliceosome incorporation and intracellular stability.

[0171] In some embodiments, the binding of the trans-splicing molecule to the target pre-mRNA is mediated by complementarity (i.e., based on the base-pairing properties of nucleic acids), triple helix formation, or protein-nucleic acid interactions (as described in the documents cited herein). In one embodiment, the nucleic acid trans-splicing molecule includes DNA, RNA, or a DNA / RNA hybrid molecule, where the DNA or RNA is either single-stranded or double-stranded. Also encompassed herein is an RNA or DNA that can hybridize to one of the aforementioned RNAs or DNAs, preferably under stringent conditions, for example, in 2.5x SSC buffer at 60°C and several washes in low buffer concentrations, for example, 0.5x SSC buffer at 37°C. Such nucleic acids may encode proteins that exhibit lipid phosphate phosphatase activity and / or plasma membrane association. When a trans-splicing molecule is synthesized in vitro, the base moiety, sugar moiety, or phosphate backbone of the trans-splicing molecule can be modified to improve, for example, the molecule's stability, hybridization with target mRNA, intracellular transport, or stability against intracellular enzymatic cleavage. For example, modifying a trans-splicing molecule to reduce its overall charge can facilitate its cellular uptake. Modifications can also be made to reduce its susceptibility to nucleases or chemical degradation. The nucleic acid molecule can be synthesized in such a way that it is conjugated to another molecule, such as a peptide, a hybridization-triggered cross-linking agent, a transport agent, or a hybridization-triggered cleavage agent.

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

[0173] III. Recombinant AAV molecules Any suitable nucleic acid vector can be used to design and assemble the components of trans-splicing molecule and recombinant adeno-associated virus (AAV) in the context of the compositions and methods of the present invention.In one embodiment, the vector is a recombinant AAV carrying a trans-splicing molecule and driven by a promoter that expresses the trans-splicing molecule in selected cells of a subject.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.Medicine, 2001,7(1):33-40; and Walther W. and Stein U., Drugs 2000,60(2):249-71.

[0174] In certain embodiments described herein, a trans-splicing molecule carrying the binding domain and coding domain 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 of AAV capsids exist, allowing the identification and use of AAVs with properties specifically suited to ocular cells. AAV viruses can be engineered using conventional molecular biology techniques to optimize such particles for cell-specific delivery of trans-splicing molecule nucleic acid sequences, minimize immunogenicity, fine-tune stability and particle lifespan, efficient degradation, precise delivery to the nucleus, and the like.

[0175] Expression of the trans-splicing molecules described herein can be achieved in selected cells by delivery of recombinant engineered or engineered AAVs containing sequences encoding the desired trans-splicing molecules. The use of AAVs is a common mode of exogenous DNA delivery because they are relatively non-toxic, provide efficient gene transfer, and can be easily optimized for specific purposes. Among the well-characterized AAV serotypes isolated from humans or non-human primates, human serotype 2 has been widely used for 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 ITRs and other selected AAV components described herein can be readily selected from any AAV serotype, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or other known and unknown AAV serotypes. In one embodiment, the ITRs are derived from AAV2. Such ITRs or other AAV components can be readily isolated from AAV serotypes using techniques available to those skilled in the art. Such AAV can be isolated or obtained from academic, commercial, or public sources (e.g., American Type Culture Collection, Manassas, VA). Alternatively, AAV sequences can be obtained by synthetic or other suitable means, e.g., by reference to published sequences, such as those available in the literature or databases, e.g., GenBank, PubMed, etc.

[0176] Desirable AAV fragments for assembly into vectors include cap proteins, including vp1, vp2, vp3 and hypervariable regions, rep proteins, including 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 viral sequences or non-AAV viral sequences. As used herein, artificial AAV serotypes include, but are not limited to, AAVs having capsid proteins that do not occur in nature. Such artificial capsids can be generated by any suitable technique using selected AAV sequences (e.g., fragments of the vp1 capsid protein) in combination with heterologous sequences that may be derived from a selected different AAV serotype, non-contiguous portions of the same AAV serotype, heterologous sequences that may be derived from non-AAV viral sources, or heterologous sequences that may be derived from non-viral sources. Artificial AAV serotypes can be, but are not limited to, pseudotyped AAV, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids. Pseudotyped vectors in which the capsid of one AAV is used with ITRs from an AAV with a different capsid protein are useful in the present invention. In one embodiment, the AAV is AAV2 / 5 (i.e., an AAV with AAV2 ITRs and an AAV5 capsid). In another embodiment, the AAV is AAV2 / 8 (i.e., an AAV with AAV2 ITRs and an AAV8 capsid). In one embodiment, the AAV comprises an AAV8 capsid. Such an AAV8 capsid comprises the amino acid sequence found in NCBI Reference Sequence: YP_077180.1 (SEQ ID NO:56). In another embodiment, the AAV8 capsid comprises the capsid encoded by nt 2121 to 4337 of GenBank accession: AF513852.1 (SEQ ID NO:57).

[0177] In one embodiment, the AAV comprises a capsid sequence derived from AAV8. In some embodiments, the AAV derived from AAV8 is AAV8(b), described in U.S. Patent No. 9,567,376, incorporated herein by reference in its entirety. AAV(b) (SEQ ID NO:58) comprises 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 by SEQ ID NO:59.

[0178] In one embodiment, vectors useful in the compositions and methods described herein minimally comprise a sequence encoding a selected AAV serotype capsid, e.g., an AAV2 capsid, or a fragment thereof. In another embodiment, useful vectors minimally comprise a sequence encoding a selected AAV serotype rep protein, e.g., an AAV2 rep protein, or a fragment thereof. Optionally, such vectors can contain both AAV cap and rep proteins. In vectors comprising both AAV rep and cap, the AAV rep and AAV cap sequences can both be of one serotype origin, e.g., AAV2 origin.

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

[0180] Suitable recombinant AAV (rAAV) is produced by culturing host cells containing a nucleic acid sequence encoding an AAV serotype capsid protein, as defined herein, or a fragment thereof; a functional rep gene; a minigene composed, for example, of AAV ITRs and trans-splicing molecule nucleic acid sequences; and sufficient helper function sites to allow packaging of the minigene into the AAV capsid protein. The components required for culturing the host cell so that the AAV minigene is packaged into the AAV capsid are supplied in trans to the host cell. Alternatively, any one or more of the required components (e.g., minigene, rep sequence, cap sequence, and / or helper function sites) may be supplied by stable host cells that have been engineered using methods known to those skilled in the art to contain one or more of the required components.

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

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

[0183] Other conventional regulatory sequences for inclusion within minigenes or rAAVs are disclosed in literature such as WO 2014 / 124282 and others cited herein and incorporated by reference. One of skill in the art may select from among these and other expression control sequences without departing from the scope described herein.

[0184] The AAV minigene can include the trans-splicing molecule and its regulatory sequences, as well as the 5' and 3' AAV ITRs, as described herein. In one embodiment, the ITRs of AAV serotype 2 are used. In another embodiment, the ITRs of AAV serotypes 5 or 8 are used. However, ITRs from other suitable serotypes may be selected. In some embodiments, the minigene is packaged into a capsid protein and delivered to a selected host cell.

[0185] The minigene, rep sequence, cap sequence, and helper function units required to generate rAAV may be delivered to packaging host cells in the form of any genetic element that transfers the sequences carried by the genetic element. The selected genetic element may 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 and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for producing rAAV virions are well known, and the selection of an appropriate method is not a limitation on the present invention. See, for example, K. Fisher et al., J. Virol., 1993 70:520-532 and U.S. Patent No. 5,478,745, each of which is incorporated herein by reference.

[0186] In another embodiment, a minigene of a trans-splicing molecule is prepared in 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 contains a modular recombinant AAV genome, including a cytomegalovirus (CMV) ITR sequence flanked by unique restriction sites that allow for the immediate removal or replacement of the wild-type 5'AAV2 ITR, contained in a functionally related state; a promoter containing a cytomegalovirus sequence of 49 nucleic acids upstream of a CMV-chicken beta actin sequence, or a photoreceptor-specific promoter / enhancer, the promoter flanked by unique restriction sites that allow for the immediate removal or replacement of the entire promoter sequence, and the upstream sequence flanked by unique restriction sites that allow for the immediate removal or replacement of only the CMV sequence or enhancer sequence upstream of the promoter sequence. The trans-splicing molecule described herein can be inserted into the multicloning polylinker site, whereby the trans-splicing molecule is operably linked to the promoter and under the regulatory control of the promoter. Also part of this plasmid are a bovine growth hormone-derived polyadenylation sequence flanked by unique restriction sites that allow for the immediate removal or replacement of the polyA sequence, and a 3' ITR sequence flanked by unique restriction sites that allow for the immediate removal or replacement of the wild-type 3' AAV2 ITR. The plasmid backbone contains elements necessary for replication in bacterial cells, such as a kanamycin resistance gene, and the backbone itself is flanked by transcription terminator / insulator sequences.

[0187] In one embodiment, a proviral plasmid comprises a modular recombinant AAV genome comprising: (a) operably associated (i) wild-type 5' AAV2 ITR sequences flanked by unique restriction sites that allow for the immediate removal or replacement of said ITRs; (ii) a promoter comprising 49 nucleic acids of CMV sequence upstream of (A) the CMV-chicken beta actin sequence; (b) a photoreceptor-specific promoter / enhancer; or (c) a neuron-specific promoter / enhancer. The promoter is flanked by upstream sequences flanked by unique restriction sites that allow for the immediate removal or replacement of the entire promoter sequence, and unique restriction sites that allow for the immediate removal or replacement of only the CMV sequence or enhancer sequence upstream of the promoter sequence. Also included in this proviral plasmid are a multicloning polylinker sequence that allows for the insertion of a trans-splicing molecule sequence, such as any of those described herein, operably linked to a promoter under the regulatory control of the promoter; a bovine growth hormone-derived polyadenylation sequence flanked by unique restriction sites that allow for the immediate removal or replacement of the polyA sequence; and a 3' AAV2 ITR sequence flanked by unique restriction sites that allow for the immediate removal or replacement of the wild-type 3' AAV2 ITR. The proviral plasmid also contains elements necessary for replication in bacterial cells and a plasmid backbone that further contains a kanamycin resistance gene, with the plasmid backbone flanked by transcription terminator / insulator sequences. The proviral plasmids described herein may also contain a 5.1 kb non-coding stuffer sequence from lambda phage within the plasmid backbone to increase the backbone length and suppress reverse packaging of non-functional AAV genomes.

[0188] In some embodiments, the proviral plasmid contains multiple copies of the trans-splicing molecule. For example, the present invention features a trans-splicing molecule that is less than half the packaging limit of AAV and thus can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times in a single proviral plasmid.

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

[0190] This proviral plasmid is then used in a conventional packaging methodology currently in use to produce a recombinant virus that expresses the trans-splicing molecule transgene carried by the proviral plasmid. Suitable producer cell lines can be easily 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, which may exist transiently. Alternatively, a minigene or gene expression cassette with flanking ITRs is stably integrated into the genome of the host cell, either chromosomally or episomally. Suitable transfection techniques are known and can be easily used to deliver recombinant AAV genomes into host cells. Typically, the proviral plasmid is cultured in a host cell that expresses cap and / or rep proteins. Within the host cell, a minigene consisting of a trans-spliced ​​molecule with flanking AAV ITRs is rescued and packaged into capsid or envelope proteins to form infectious viral particles. Thus, recombinant AAV infectious particles are generated by culturing packaging cells carrying a proviral plasmid in the presence of sufficient viral sequences to allow packaging of the gene expression cassette viral genome into the infectious AAV envelope or capsid.

[0191] IV. Pharmaceutical Compositions and Kits Provided herein are pharmaceutical compositions comprising a nucleic acid trans-splicing molecule, a proviral plasmid, or an rAAV comprising the nucleic acid trans-splicing molecule described herein. In some embodiments, the pharmaceutical composition comprises any of the 5' trans-splicing molecules described herein. In other embodiments, the pharmaceutical composition comprises any of the 3' trans-splicing molecules described herein. In some embodiments, the pharmaceutical composition comprises a 5' trans-splicing molecule and a 3' trans-splicing molecule, for example, where 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 can be evaluated for contaminants by conventional methods and then formulated into pharmaceutical compositions intended for an appropriate administration route. Other compositions containing trans-splicing molecules, such as naked DNA or proteins, can also be similarly formulated with appropriate carriers. Such formulations involve the use of pharmaceutically and / or physiologically acceptable vehicles or carriers specifically designed for administration to target cells. In one embodiment, carriers suitable for administration to target cells include buffered saline, isotonic sodium chloride solution, or other buffers for maintaining pH at appropriate physiological levels, such as HEPES, as well as, optionally, other medicinal agents, pharmaceutical agents, stabilizers, buffers, carriers, adjuvants, diluents, and the like.

[0193] In some embodiments, the carrier is an injectable solution.Exemplary physiologically acceptable carriers include sterilized pyrogen-free water and sterilized pyrogen-free phosphate buffered saline.Such known carriers are listed in U.S. Patent No. 7,629,322, which is incorporated herein by reference.In one embodiment, the carrier is an isotonic sodium chloride solution.In another embodiment, the carrier is a buffered saline solution.In one embodiment, the carrier comprises Tween.When the virus is stored for a long period of time, it can be frozen in the presence of glycerol or Tween 20.

[0194] In other embodiments, compositions comprising the trans-splicing molecules described herein include a surfactant. Useful surfactants, such as Pluronic F68 (Poloxamer 188, also known as LUTROL® F68), can be included to prevent AAV from adhering to inert surfaces, thus ensuring delivery of the desired dose. For example, one illustrative composition designed for the treatment of an ocular disease described herein includes a recombinant adeno-associated vector carrying a nucleic acid sequence encoding a 3′ trans-splicing molecule as described herein under the control of regulatory sequences that cause the trans-splicing molecule to be expressed in ocular cells of a mammalian subject, and a pharmaceutically acceptable carrier. The carrier is an isotonic sodium chloride solution and includes the surfactant Pluronic F68. In one embodiment, the trans-splicing molecule is any of those described herein.

[0195] In yet another exemplary embodiment, a composition comprises a recombinant AAV2 / 5-pseudotyped adeno-associated virus carrying a 3' or 5' trans-splicing molecule for replacement of the ABCA4 gene, wherein the nucleic acid sequence is under the control of a promoter that directs expression of the trans-splicing molecule in the photoreceptor cells, and the composition is formulated with a carrier and additional components suitable for subretinal injection. In yet another embodiment, compositions or components for the production or assembly of the composition, such as carriers, rAAV particles, surfactants, and / or components for generating rAAV, as well as appropriate laboratory hardware for preparing the composition, can be incorporated into a kit.

[0196] In some examples, the composition comprises a recombinant AAV2 / 5 pseudotyped adeno-associated virus carrying a 5' trans-splicing molecule for replacement of the CEP290 gene, the nucleic acid sequence being under the control of a promoter that directs expression of the trans-splicing molecule in the photoreceptor cells, and the composition is then formulated with a carrier and additional components suitable for subretinal injection. In yet another embodiment, compositions or components for the production or assembly of the composition, such as carriers, rAAV particles, surfactants, and / or components for generating rAAV, as well as appropriate laboratory hardware for preparing the composition, can be incorporated into a kit.

[0197] Further provided herein are kits containing a first pharmaceutical composition comprising a 5' trans-splicing molecule and a second pharmaceutical composition comprising a 3' trans-splicing molecule, e.g., 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 (e.g., the trans-splicing molecules are packaged within any of the AAV vectors described herein). In some embodiments, the kits include instructions for mixing the two pharmaceutical compositions prior to administration.

[0198] Further provided herein is a kit comprising a first pharmaceutical composition comprising a 5' trans-splicing molecule that binds to a target CEP290 intron.

[0199] V. Method The above-described 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), e.g., methods for delaying or alleviating symptoms associated with the disease described herein. Such methods involve contacting a target ABCA4 gene (e.g., ABCA4 pre-mRNA) with a trans-splicing molecule (e.g., one or more 3' trans-splicing molecules, 5' trans-splicing molecules, or both 3' and 5' trans-splicing molecules as described herein) to correct ABCA4 expression in a target cell under conditions in which splicing of the coding domain of the trans-splicing molecule to the target ABCA4 gene occurs, replacing one or more defective or mutated portions of the targeted gene with functional (i.e., healthy), normal, wild-type, or corrected 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.

[0200] In one embodiment, the contacting involves direct administration to the affected subject. In another embodiment, the contacting can be performed ex vivo on cultured cells and treated ocular cells that are reimplanted into the subject. In one embodiment, the method involves administering an rAAV carrying a 3' trans-splicing molecule. In another embodiment, the method involves administering an rAAV carrying a 5' trans-splicing molecule. In yet another embodiment, the method involves administering a mixture of an rAAV carrying a 3' trans-splicing molecule and an rAAV carrying a 5' trans-splicing molecule. Such methods include administering to a subject in need thereof an effective concentration of any of the compositions described herein.

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

[0202] In another example, the above-described compositions involving 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), e.g., methods for delaying or alleviating symptoms associated with the disease described herein. Such methods involve contacting a target CEP290 gene (e.g., a CEP290 pre-mRNA) with a trans-splicing molecule (e.g., a 5' trans-splicing molecule) as described herein to correct CEP290 expression in a target cell under conditions in which splicing of the coding domain of the trans-splicing molecule to the target CEP290 gene occurs, and a portion of the targeted gene having one or more defects or mutations is replaced with functional (i.e., healthy), normal, wild-type, or corrected 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 methods of the present invention include correcting 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 of the 5' trans-splicing molecules described herein) or a pharmaceutical composition thereof. Thus, the present invention provides methods of treating a subject having a disease or disorder associated with a mutation in 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 can be included in a pharmaceutical composition (e.g., a single pharmaceutical composition, e.g., as part of a kit, containing both molecules, either pre-prepared or mixed prior to administration).

[0203] In one embodiment, the contacting involves direct administration to the affected subject. In another embodiment, the contacting can be performed ex vivo on cultured cells and treated ocular cells that are re-implanted into the subject. In one embodiment, the method involves administering rAAV carrying a 5' trans-splicing molecule. Such a method comprises administering to a subject in need thereof an effective concentration of any of the compositions described herein.

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

[0205] A single trans-splicing molecule to correct a single mutation The methods of the invention involve selecting a single trans-splicing molecule based on the location of a single mutation within ABCA4 (e.g., a mutation in one allele of interest). In some instances, in the context of an autosomal recessive mutation, correction of one of the two mutations may be sufficient to restore functional protein activity, e.g., where the second allele has a mutation in an opposing portion of the ABCA4 gene outside the range of the single AAV-delivered trans-splicing molecule configured to correct the first mutation.

[0206] Thus, in some embodiments, the methods of the present invention involve selecting a single trans-splicing molecule for correcting a single mutation in the 5' portion of a target gene, regardless of the location of the mutation, for example, in 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 comprises functional ABCA4 exon 1. In one embodiment, exon 1 or exon 2 is mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain comprises functional ABCA4 exons 1 and 2. In one embodiment, one of exons 1, 2, and 3 is mutant, the target intron is intron 19, 22, 23, or 24, and the coding domain comprises functional ABCA4 exons 1-3. In one 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 comprises functional ABCA4 exons 1-4. In one embodiment, one of exons 1, 2, 3, 4, and 5 is mutant, the targeted intron is intron 19, 22, 23, or 24, and the coding domain comprises functional ABCA4 exons 1-5. In one embodiment, one of exons 1, 2, 3, 4, 5, and 6 is mutant, the targeted intron is intron 19, 22, 23, or 24, and the coding domain comprises functional ABCA4 exons 1-6. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, or 7 is mutant, the targeted intron is intron 19, 22, 23, or 24, and the coding domain comprises functional ABCA4 exons 1-7. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, or 8 is mutant, the targeted intron is intron 19, 22, 23, or 24, and the coding domain comprises functional ABCA4 exons 1-8. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8 or 9 is mutated, the targeted intron is intron 19, 22, 23 or 24, and the coding domain comprises 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 targeted intron is intron 19, 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 1 to 10. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 is mutant, the targeted intron is intron 19, 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 1 to 11. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 is mutant, the targeted intron is intron 19, 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 1 to 12. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 is mutant, the targeted intron is intron 19, 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 1 to 13. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 is mutant, the targeted intron is intron 19, 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 1 to 13. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 is mutant, the targeted intron is intron 19, 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 1 to 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 targeted intron is intron 19, 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 1 to 15. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 is mutant, the targeted intron is intron 19, 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 1 to 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 targeted intron is intron 19, 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 1 to 17. In one 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 targeted intron is intron 19, 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 1 to 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 targeted intron is intron 19, 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 1 to 19. 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 or 20 is mutant, the targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 1 to 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 targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 1 to 21. 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 or 22 is mutant, the targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 1 to 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 mutated, the targeted intron is intron 23 or 24, and the coding domain comprises 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 mutated, the targeted intron is intron 24, and the coding domain comprises functional ABCA4 exons 1-24.

[0207] Alternatively, in instances where the mutation is present in the 3' portion of the target gene, a 3' trans-splicing molecule is selected to correct the mutation, e.g., regardless of the location of the mutation within the other allele. 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 mutated, the target intron is intron 22, and the coding domain comprises 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 targeted intron is intron 22 or 23, and the coding domain comprises functional ABCA4 exons 24 to 50. In one 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 targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 25 to 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 targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 26 to 50. In one 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 targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 27 to 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 targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 28 to 50. In one 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 targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 29 to 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 targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 30 to 50. In one 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 targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 31 to 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 targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 32 to 50. In one 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 targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 33 to 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 mutated, the targeted intron is intron 22, 23 or 24, and the coding domain comprises 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 targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 35 to 50. In one embodiment, one of exons 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 is mutant, the targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 36 to 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 targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 37 to 50. In one embodiment, one of exons 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 is mutant, the targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 38 to 50. In one embodiment, one of exons 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 is mutant, the targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 39 to 50. In one embodiment, one of exons 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 is mutant, the targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 40 to 50. In one embodiment, one of exons 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 is mutant, the targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 41 to 50. In one embodiment, one of exons 42, 43, 44, 45, 46, 47, 48, 49 or 50 is mutated, the targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 42-50.In one embodiment, one of exons 43, 44, 45, 46, 47, 48, 49 or 50 is mutant, the targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 43 to 50. In one embodiment, one of exons 44, 45, 46, 47, 48, 49 or 50 is mutant, the targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 44 to 50. In one embodiment, one of exons 45, 46, 47, 48, 49 or 50 is mutant, the targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 45 to 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 comprises functional ABCA4 exons 46-50. In one embodiment, one of exons 47, 48, 49 or 50 is mutant, the target intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 47-50. In one embodiment, one of exons 48, 49 or 50 is mutant, the target intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 48-50. In one embodiment, one of exons 49 or 50 is mutant, the target intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exons 49 or 50. In one embodiment, exon 50 is mutated, the targeted intron is intron 22, 23 or 24, and the coding domain comprises functional ABCA4 exon 50.

[0208] A single trans-splicing molecule for correcting multiple mutations The methods of the present invention involve selecting a single trans-splicing molecule based on the location of the mutations in ABCA4 within each allele of interest, such that if two mutations are present in either the 5' portion of the gene or the 3' portion of the gene, the single trans-splicing molecule that can be packaged into an AAV vector will span both mutations, thereby correcting both mutations.

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

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

[0211] Two trans-splicing molecules for correcting multiple mutations Further provided herein are methods for correcting multiple mutations in 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 this case, the 5' trans-splicing molecule and the 3' trans-splicing molecule bind to the same target ABCA4 intron and replace the upstream exon and downstream exon of the target intron, respectively.

[0212] For example, in some embodiments of the present invention, a 5' trans-splicing molecule and a 3' trans-splicing molecule each bind to 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, a 5' trans-splicing molecule and a 3' trans-splicing molecule each bind to 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 foregoing combinations of 5' trans-splicing molecules and 3' trans-splicing molecules can be included in a pharmaceutical composition (e.g., a single pharmaceutical composition, e.g., as part of a kit, containing both molecules, either pre-prepared or mixed prior to administration).

[0213] Dosing, monitoring and concomitant therapy The effective concentration of recombinant adeno-associated virus carrying a trans-splicing molecule as described herein is about 10 8 ~10 13 In one embodiment, the concentration is in the range of 10 genome vectors per milliliter (vg / mL). rAAV infectious units are measured as described by McLaughlin et al., J. Virol. 1988, 62:1963. In one embodiment, the concentration is in the range of 10 9 ~10 13 In another embodiment, the effective concentration is in the range of about 1.5 x 10 11 In one embodiment, the effective concentration is about 1.5 x 10 10 In another embodiment, the effective concentration is about 2.8 x 1011 In another embodiment, the effective concentration is about 5×10 11 In another embodiment, the effective concentration is about 1.5 x 10 12 In another embodiment, the effective concentration is about 1.5 x 10 13 vg / mL.

[0214] It is desirable to use the lowest effective dosage (total genome copy number delivered) of virus to reduce the risk of undesirable effects, such as toxicity, and other problems associated with ocular administration, such as retinal dysplasia and retinal detachment. An effective dosage of recombinant adeno-associated virus carrying a trans-splicing molecule as described herein is about 10 8 ~10 13 In one embodiment, the dosage ranges from 10 to 1000 genomic vectors (vg) / dose (i.e., / injection). 9 ~10 13 In another embodiment, the effective dosage is in the range of about 1.5×10 11 In another embodiment, the effective dosage is about 5×10 11 In one embodiment, the effective dosage is about 1.5×10 10 In another embodiment, the effective dosage is about 2.8×10 11 In another embodiment, the effective dosage is about 1.5×10 12 In another embodiment, the effective concentration is about 1.5 x 10 13 vg. Still other dosages within these ranges or other units can be selected by the attending physician, taking into consideration the physical condition of the subject being treated, e.g., the age of the subject; the composition being administered, and the particular disorder; the cells being targeted, and if the disorder is progressive, its progression.

[0215] The composition may be delivered in a volume of about 50 μL to about 1 mL, including any value within this range, 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. 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 about 750 μL. In another embodiment, the volume is about 850 μL. In another embodiment, the volume is about 1,000 μL.

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

[0217] In another aspect, the present invention provides a method for suppressing or stopping the decline in photoreceptor function or enhancing photoreceptor function in a subject. The composition can be administered before or after the onset of disease. For example, photoreceptor function can be evaluated using functional tests conventional in the art, such as ERG or visual field testing. As used herein, "decrease in photoreceptor function" refers to a decline in photoreceptor function compared to a normal eye without disease or the same eye at an earlier time point. As used herein, "enhancement of photoreceptor function" refers to improving photoreceptor function or increasing the number or proportion of functional photoreceptors compared to a diseased eye (having the same ocular disease), the same eye at an earlier time point, an untreated part of the same eye, or the opposite eye of the same subject.

[0218] In each of the described methods, treatment can be used to prevent further damage from occurring or to salvage tissue with mild or advanced disease. As used herein, the term "salvage" means to prevent the progression of disease, to prevent the spread of damage to uninjured cells, or to ameliorate damage to injured cells.

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

[0220] In another embodiment, any of the above methods is performed in combination with another treatment or secondary treatment. The treatment may be any currently known or yet unknown, a treatment that helps to suppress, stop, or alleviate such mutations or defects, or any effects associated with the mutations or defects. The secondary treatment may be administered before, simultaneously with, or after the administration of the trans-splicing molecule. In one embodiment, the secondary treatment involves a non-specific approach to maintain the health of retinal cells, such as the administration of neurotrophic factors, antioxidants, or anti-apoptotic agents. The non-specific approach is carried out by injecting 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 comprises carrying out functional test and imaging diagnostic test to determine the effectiveness of treatment.Such test includes electroretinogram (ERG) and in vivo retinal imaging diagnostic, as described in United States Patent No. 8,147,823; International Patent Application Publication No. 2014 / 011210 or International Patent Application Publication No. 2014 / 124282, which are incorporated herein by reference.In addition, visual field test, visual field test and microperimetry, motor function test, visual acuity and / or color vision test can also be carried out.

[0222] In some specific embodiments, it is desirable to carry out non-invasive retinal imaging diagnosis and functional testing to identify the photoreceptor-retaining region that is targeted for treatment.In these embodiments, clinical diagnostic tests are used to determine the exact location(s) for one or more subretinal injections.Such tests may include ERG, visual field test, topographical mapping of retinal layers and the measurement of their thickness by scanning confocal laser ophthalmoscopy (cSLO) and optical coherence tomography (OCT), topographical mapping of cone density by adaptive optics (AO), eye function test, etc.In consideration of imaging diagnosis and functional testing, in some embodiments, one or more injections are carried out in the same eye to target different photoreceptor-retaining regions.

[0223] For use in such methods, the volume and viral titer of each injection is determined individually and may be the same or different from other injections given in the same or opposite eye. In another embodiment, a single large volume injection is given to treat the entire eye. Dosage, administration, and regimen can be determined by the attending physician after receiving the teachings of the present disclosure. [Example]

[0224] The present invention is based, at least in part, on the applicant's discovery that certain introns of ABCA4 and specific regions within those introns provide highly efficient binding sites for the binding domains of trans-splicing molecules, effectively mediating trans-splicing. Applicant created a series of mock trans-splicing molecules with 150-base-long binding domains designed to hybridize to corresponding sets of 150-base-pair binding site sequences in (i) the ABCA4 introns of interest (introns 19 and 22-24) and (ii) the CEP290 intron of interest (introns 26-30). Each binding domain in the ABCA4 and CEP290 series was designed to overlap by 140 nucleotides, allowing scanning of every 10 nucleotides of each intron between each successive test binding domain. Trans-splicing efficiency was quantified for each binding domain in each of ABCA4 introns 19 and 22-24 and CEP290 introns 26-30. Screening of ABCA4 is described in Example 1, and the results are shown in Figures 1 to 8. Screening of CEP290 is described in Example 2, and the results are shown in Figures 21 to 26.

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

[0226] Binding site screening Screening of a series of binding domains designed to bind to ABCA4 intron 19 (SEQ ID NO:25) at consecutive binding sites revealed a region in the 3' portion of ABCA4 intron 19 that is preferentially efficient in trans-splicing 5' trans-splicing molecules, namely, the region between nucleotides 990 and 2,174 of intron 19 (Figure 2). Binding sites within the nucleotide ranges 1,670 to 2,174, 1,810 to 2,000, 1,870 to 2,000, or 1,920 to 2,000 were found to be particularly efficient in mediating 5' trans-splicing in intron 19.

[0227] Preferred binding sites for 5' trans-splicing molecules within intron 22 were also identified (Figure 3). Binding sites within nucleotides 1 to 150 or nucleotides 880 to 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 of ABCA4 intron 22 (SEQ ID NO:28) for 3' trans-splicing molecules. Binding sites with nucleotides 60-570, nucleotides 600-800, or nucleotides 900-1,350 were identified as preferentially suitable for trans-splicing of 3' trans-splicing molecules. In particular, binding domains targeted to binding sites within nucleotides 70-250 were highly efficient in 3' trans-splicing.

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

[0230] A similar screening of intron 24 of ABCA4 (SEQ ID NO:30) revealed that binding sites within nucleotides 600 to 1,250 or 1,490 to 2,660 were efficient for 5' trans-splicing (Figure 7). In particular, binding sites within nucleotides 1,000 to 1,200 exhibited the highest 5' trans-splicing efficiency. Figure 8 shows the results of screening for 3' trans-splicing efficiency, revealing that binding sites within nucleotides 1 to 250, 300 to 2,000, or 2,200 to 2,692 (particularly those within nucleotides 750 to 1,110) were the most efficient.

[0231] ABCA4 cell line First, we generated cell lines expressing ABCA4. The ABCA4 gene is only known to be expressed in photoreceptors in the retina in vivo, and full-length ABCA4 pre-mRNA and protein are generally not detectable in vitro in cultured cells. Therefore, to test trans-splicing strategies for ABCA4, we engineered cells to express ABCA4 from its native genomic locus on chromosome 1 (1p22.1). Two strategies were pursued. In the first case, stable cell lines were induced to express a site-specific (upstream of the ABCA4 transcription start site) DNA-binding TALEN fused to the VP64 viral transactivator. In the second case, a eukaryotic constitutive promoter was directly inserted (using CRISPR / Cas9) into the genomic locus immediately upstream of the ABCA4 transcription start site. In both cases, the result was stable cell lines that robustly expressed ABCA4 pre-mRNA and protein.

[0232] TALEN cell lines We designed TALENs targeting specific domains upstream of the ABCA4 transcription start site and fused them to the VP-64 transactivator sequence (Figure 9). 293 cells were transfected with the three TALEN combinations to derive stable single clones. Two clones were shown to direct the expression of ABCA4 protein (Figure 10).

[0233] CAG promoter cell line The general strategy for deriving 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 homology arms (Figure 12B). Puromycin-resistant cells were cloned and analyzed for the desired insertion by PCR. Several clonal lines were selected for further analysis. RNA and protein expression in two lines (B6 and C3) are shown in Figures 14A and 14B. Both lines clearly contained the promoter insertion, as demonstrated by RNA and protein analysis.

[0234] ABCA4 knockout cell line Once stable ABCA4 expression was established in cultured cells, we generated an ABCA4 expression knockout for testing ABCA4 trans-splicing molecules designed to restore ABCA4 protein expression. Generally, guide RNA and Cas9 protein were co-transfected into B6 cells (in which the CAG promoter was knocked into the ABCA4 locus to mediate ABCA4 expression). Nine days later, a second transfection with guide RNA and Cas9 protein was performed. The basic design, targeting exons 3 and 4, is shown in Figure 15. Single cells were plated by limiting dilution, cultured once, and ABCA4 protein expression was assessed by Western blot.

[0235] Figure 16 shows the RNA and protein profiles induced after treatment with CRISPR / Cas9 of single clones as shown in Figure 15. Varying degrees of RNA and protein ablation were observed. Clones 17+06 and 17+21 were selected because they demonstrated complete ABCA4 protein knockout. Mutational analysis (Figures 17A-17B and 18) confirmed that exons 3 and 4 were targeted and disrupted.

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

[0237] method For Western blot assay, 17+06 or 17+21 cells were cultured at 10 6 Cells were seeded into each well of a 12-well plate at a density of 1000 cells / well. 1 μg of plasmid (RTM) was added to each well. xTransfection was performed using a 100-well platelet-free platelet-free (100-well platelet-free) PBS. At 48 hours, cells were harvested, and membrane preparations were processed for analysis 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 1x HALT™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher 78440) in all buffers. RNA was also processed for analysis as described below. Membrane lysates were denatured for 30 minutes at room temperature using 4x Laemmli Sample Buffer (Biorad 161-0737) containing 10% reducing agent TCEP 0.5M (Sigma 646547). Samples were run on NuPage Precast 3-8% Tris-Acetate gels (Thermo Fisher), and proteins were transferred using iBlot 2 Mini PVDF Transfer Packs and run at 25V for 10 minutes using the iBlot 2. 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). Blots were exposed for various times depending on the intensity of the signal.

[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 20 μl reaction volume using SuperScript IV VILO Master Mix (Thermo 11756500; diluted 1:4 with water). Native ABCA4 (Thermo commercial assay Hs00979594_m1) spans exons 49–50. For housekeeper gene control, an assay for RNF20 was used (Thermo commercial assay Hs00219623_m1). Primers and probes for qPCR of chimeric ABCA4 codon-optimized exon 22–native exon 23 were as follows: TIFF2024099770000011.tif35128

[0239] The primers and probes for qPCR of the double-stranded assay with RNF20 were as follows: TIFF2024099770000012.tif38128

[0240] PCR reactions were performed using QuantiFast 2x qPCR Mastermix.

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

[0242] Figure 20A is a Western blot showing ABCA4 protein expression resulting from the trans-splicing reaction in two different cell lines (17+06 and 17+21) with a mock GFP control or 5'A4In22 tethered to five different binding domains (no binding domain (NBD) control, 92, 99, 105, 118, and 121, corresponding to 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 across intron 22 of 150 bases 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, and 118 showing the highest protein expression in both cell lines. The mRNA expression profiles showed a similar pattern, with 118 construct producing the highest levels of ABCA4 mRNA in both cell lines (Figure 20B). Units are relative to the RNF20 housekeeping gene.

[0243] Figure 20C shows Western blots depicting 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, 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 (following the 150-nucleotide, 10-base shift interval across intron 23 described above). All three intron 23-binding constructs resulted in trans-splicing, as indicated by protein expression levels in both cell lines. Similar results were obtained for the mRNA expression profiles; all three constructs resulted in robust ABCA4 mRNA expression in both cell lines (Figure 20D). Units are relative amounts to the RNF20 housekeeping gene.

[0244] Taken together, the ABCA4 protein and RNA expression data obtained with intron 22- and intron 23-linked trans-splicing molecules correlate with the results of the binding domain screening described above. In particular, intron 22-linked constructs 105, 118, and 121 and intron 23-linked constructs 27, 81, and 85 were predicted to bind with high efficiency (Figures 3 and 5). The present ABCA4 protein recovery data indicate that the ABCA4 intron regions containing the binding sites of these constructs are 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 recovery was comparable between intron 22-linked and intron 23-linked trans-splicing molecules. Importantly, because ABCA4-related diseases (e.g., Stargardt disease) are recessive, asymptomatic carriers of the disease likely express less ABCA4 than normal, and without wishing to be bound by theory, partial restoration of the protein as shown herein likely provides a significant clinical benefit.

[0245] Example 2. CEP290 Screening of a series of binding domains designed to bind to CEP290 intron 26 (SEQ ID NO:85) with consecutive binding sites revealed a region in the 3' portion of CEP290 intron 26 that is preferentially suitable for trans-splicing of 5' trans-splicing molecules, namely, the region between nucleotides 4,980 and 5,838 of intron 26 (Figure 22). Binding sites within the nucleotide ranges 5,348 to 5,838, 5,348 to 5,700, 5,400 to 5,600, 5,460 to 5,560, or 5,500 were found to be particularly efficient in mediating trans-splicing.

[0246] Figure 23 shows the results of a similar screen on 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 trans-splicing of 5' trans-splicing molecules. In particular, binding domains targeted to binding sites within the ranges of nucleotides 790-2,100, nucleotides 1,020-1,630, or nucleotides 1,670-2,000 were highly efficient in trans-splicing.

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

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

[0249] In intron 29 (SEQ ID NO:89), the 3' portion of the intron was significantly more efficient at mediating 5' trans-splicing than the remainder of the intron (Figure 26). In particular, binding domains targeting binding sites within nucleotides 95-1,240, e.g., nucleotides 1,060-1,240, showed the greatest 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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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 augaaucca ugaggaga cauaauuua uaaaugcag 54780 cauaauaggc acuauuauuga uggauuuuc cugcuugaa 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 55140 auggccucca 55140 uuacagaag cguguacugu cucucuuagc uguuugccag gccgccaguc ucuugcugu 55200 cagcucucaa cugcuuccag caagaucuuu cuaaauccc aggcuugcca agacuuagcg 55260 cccacagcuc cacagugacu ccucauugcu guuaagguaa aggccuuccc agucuagccc 55320 uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu 55380 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 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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 ucuguggu...

Claims

1. operably linked in the 5' to 3' direction, (a) a binding domain configured to bind to ABCA4 intron 22 at a binding site within nucleotides 60-570, 600-800, or 900-1,350 of SEQ ID NO:28, comprising a sequence ranging in length from 50 to 300 nucleotides; (b) a splicing domain configured to mediate trans-splicing; and (c) Coding domain containing functional ABCA4 exons 23-50 A nucleic acid trans-splicing molecule comprising: 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 the functional ABCA4 exons 23-50; The nucleic acid trans-splicing molecule.

2. The nucleic acid trans-splicing molecule described in claim 1, wherein the binding site is within nucleotides 60 to 570 of SEQ ID NO:

28.

3. The nucleic acid trans-splicing molecule described in claim 2, wherein the binding site is within nucleotides 70 to 250 of SEQ ID NO:

28.

4. The nucleic acid trans-splicing molecule described in claim 1, wherein the binding site is within nucleotides 900 to 1,350 of SEQ ID NO:

28.

5. The binding site is (a) six or more of nucleotides 60 to 570 of SEQ ID NO:28; (b) six or more of nucleotides 600-800 of SEQ ID NO:28; or (c) six or more of nucleotides 900 to 1,350 of SEQ ID NO:28 Including, A nucleic acid trans-splicing molecule according to any one of claims 1 to 4.

6. The nucleic acid trans-splicing molecule of claim 5, wherein the binding site comprises six or more of nucleotides 331 to 570 of SEQ ID NO:

28.

7. A nucleic acid trans-splicing molecule as described in claim 5 or 6, wherein the binding domain comprises six or more consecutive nucleic acid residues that are complementary to the six or more nucleotides of the binding site.

8. The nucleic acid trans-splicing molecule of any one of claims 1 to 7, wherein the binding domain comprises a sequence in the length range of 50 to 75 nucleotides, in the length range of 75 to 100 nucleotides, in the length range of 125 to 150 nucleotides, in the length range of 150 to 175 nucleotides, in the length range of 175 to 200 nucleotides, in the length range of 200 to 250 nucleotides, in the length range of 100 to 200 nucleotides, or in the length of 150 nucleotides.

9. A nucleic acid trans-splicing molecule described in any one of claims 1 to 8, wherein the binding domain comprises at least 10, at least 12, at least 15, 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 consecutive nucleotides that are complementary to the binding site.

10. A nucleic acid trans-splicing molecule described in any one of claims 1 to 9, wherein the binding domain is 80% to 100% complementary to the binding site. (i) the binding domain is 100 to 200 nucleotides in length; (ii) the coding domain is a cDNA sequence; (iii) the coding domain comprises a naturally occurring sequence; (iv) the coding domain comprises a codon-optimized sequence; (v) the artificial intron comprises a spacer sequence; (vi) the nucleic acid trans-splicing molecule is 3,000 to 4,000 nucleotides in length; (vii) a mutation in the ABCA4 gene is associated with Stargardt disease, and / or (viii) the mutation is expressed in a photoreceptor cell; A nucleic acid trans-splicing molecule according to any one of claims 1 to 10.

12. A proviral plasmid comprising a nucleic acid trans-splicing molecule described in any one of claims 1 to 11.

13. An adeno-associated virus (AAV) comprising a nucleic acid trans-splicing molecule described in any one of claims 1 to 11.

14. The AAV of claim 13, which preferentially targets photoreceptor cells.

15. An AAV described in claim 13 or 14, comprising an AAV5 capsid protein, an AAV8 capsid protein, an AAV8(b) capsid protein or an AAV9 capsid protein.

16. An AAV described in any one of claims 13 to 15, comprising an assembled capsid in which a vector genome including an AAV 5'ITR, the nucleic acid trans-splicing molecule under the functional control of a regulatory sequence, and an AAV 3'ITR is packaged.

17. A pharmaceutical composition comprising a nucleic acid trans-splicing molecule described in any one of claims 1 to 11, a proviral plasmid described in claim 12, or an AAV described in any one of claims 13 to 16.

18. The pharmaceutical composition of claim 17 for correcting a mutation in the ABCA4 gene in a target cell of a subject.

19. The pharmaceutical composition of claim 17, for correcting a mutation in any one or more of ABCA4 exons 23-50 in a subject in need thereof.

20. The pharmaceutical composition of claim 18 or 19, wherein the subject has Stargardt disease.

21. The pharmaceutical composition of any one of claims 18 to 20, formulated for administration by subretinal injection, intravitreal injection, or intravenous injection.

22. A pharmaceutical composition described in any one of claims 17 to 21 for treating a subject having a disorder associated with a mutation in ABCA4.

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