trans-splicing molecules

Abstract

The present invention is characterized by nucleic acid trans-splicing molecules (e.g., pre-mRNA trans-splicing molecules (RTMs)) capable of correcting one or more mutations in the ABCA4 or CEP290 genes. Such molecules can be used to treat conditions associated with ABCA4 mutations, such as Stargardt disease (e.g., Stargardt disease 1), and conditions associated with CEP290 mutations, such as Leber congenital amaurosis 10 (LCA 10). The invention also provides methods for using nucleic acid trans-splicing molecules to correct mutations in ABCA4 and CEP290, and for treating conditions associated with mutations in ABCA4 and CEP290, such as Stargardt disease and LCA 10.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application Serial Nos. 62 / 658,658 and 62 / 658,667, both filed on April 17, 2018, the entire contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list, which has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on March 17, 2019, and is named 51219-016WO2_Sequence_Listing_04.16.19_ST25, with a size of 608,489 bytes. Technical Field

[0005] Typically, this invention is characterized by ABCA4 and CEP290 trans-splicing molecules. Background Technology

[0006] Stargardt disease is a progressive eye disorder characterized by loss of central and color vision. It can develop rapidly or over several years. Peripheral vision is usually intact. Various mutations along the length of the ABCA4 gene can contribute to Stargardt disease. Treatments currently under development for Stargardt disease include lentiviral delivery of ABCA4, chemically modified variants of vitamin A, and retinal pigment epithelial cell therapy.

[0007] Leber congenital amourosis 10 (LCA 10) is a condition characterized by severe visual impairment beginning in infancy. Vision loss is associated with photoreceptor cell death due to ciliary defects. The most common known mutation associated with LCA 10 is a point mutation in which adenine at nucleotide 1655 of intron 26 of the CEP290 gene is replaced by guanine, resulting in a splicing defect where a recessive stop codon is spliced ​​between exons 26 and 27. This autosomal recessive mutation leads to the production of nonfunctional centrosome proteins, resulting in the blindness characteristic of LCA10.

[0008] Adeno-associated viral (AAV) vector-mediated gene therapy has demonstrated safety in humans and is a promising approach for treating a variety of genetic defects. However, AAV vectors may have limitations determined by viral biology, such as packaging size restrictions, which may hinder the delivery of large nucleic acid molecules, such as those necessary to replace the ABCA4 or CEP290 genes. Therefore, there is a need in this field for compositions and methods for correcting mutations in ABCA4 and CEP290. Summary of the Invention

[0009] This invention relates to nucleic acid trans-splicing molecules and methods for correcting mutations in the ABCA4 or CEP290 genes. The compositions and methods provided herein can be used to treat or prevent diseases associated with ABCA4 mutations, such as Stargardt disease (e.g., Stargardt disease 1) or CEP290 mutations, such as LCA (e.g., LCA10).

[0010] ABCA4

[0011] In a first aspect, the present invention is characterized by 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 a target ABCA4 intron selected from the group consisting of introns 19, 22, 23, or 24, operably linked in a 3' to 5' or 5' to 3' direction; (b) a splicing domain configured to mediate trans-splicing; and (c) a coding domain comprising a functional ABCA4 exon; wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain to an endogenous ABCA4 exon adjacent to the target ABCA4 intron, thereby replacing the endogenous ABCA4 exon with the functional ABCA4 exon and correcting mutations in ABCA4.

[0012] In some embodiments, the binding domain binds to a target ABCA4 intron at the 3' end of the mutation, wherein 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, 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 intron 19 at a binding site comprising any one or more of nucleotides 990 to 2174 of SEQ ID NO: 25 (e.g., any one or more of nucleotides 1670 to 2174 of SEQ ID NO: 25, e.g., any one or more of nucleotides 1810 to 2000 of SEQ ID NO: 25, e.g., any one or more of nucleotides 1870 to 2000 of SEQ ID NO: 25, e.g., any one or more of nucleotides 1920 to 2000 of SEQ ID NO: 25).

[0013] In some embodiments, the target ABCA4 intron is intron 23, the mutation being in any one of ABCA4 exons 1-23 or introns 1-23, and / or the coding domain comprising ABCA4 exons 1-23. In some embodiments, the binding domain is configured to bind intron 23 at a binding site comprising any one or more of nucleotides 80 to 570 or nucleotides 720 to 1081 of SEQ ID NO: 29.

[0014] In some embodiments, the binding domain is configured to bind ABCA4 intron 23 at a binding site comprising any one or more of nucleotides 261 to 410 of SEQ ID NO: 29 (e.g., 1 to 200, 6 to 150, 12 to 100, or 20 to 80 nucleotides within or around nucleotides 261 to 410 of SEQ ID NO: 29, e.g., 1 to 6, 6 to 12, 12 to 18, 18 to 24, 24 to 50, 50 to 100, 100 to 150, or 150 to 200 nucleotides within or around nucleotides 261 to 410 of SEQ ID NO: 29, e.g., SEQ ID NO: 29). (The binding site comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 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 within or around nucleotides 261 to 410 of SEQ ID NO: 29.) For example, in a particular embodiment, the binding site comprises six or more of the nucleotides 261 to 410 of SEQ ID NO: 29. In some embodiments, the binding domain comprises six or more consecutive nucleic acid residues complementary (e.g., antisense) to six or more nucleotides of the binding site.In some embodiments, the binding domain comprises a set of consecutive nucleic acid residues complementary to a set of complementary nucleotides corresponding to the ABCA4 binding sites of one or more nucleotides having SEQ ID NO: 29, wherein the length of the set of consecutive nucleic acid residues of the binding domain is from 6 to 500 residues (e.g., from 8 to 400, from 12 to 300, from 16 to 200, from 24 to 280, or from 50 to 150 residues, e.g., from 100 to 200, from 6 to 10, from 10 to 20, from 20 to 30, from 30 to 40, from 40 to 50, from 50 to 80, from 80 to 100). From 100 to 120, from 120 to 150, from 150 to 200, or from 200 to 300 residues, for example, lengths of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55. 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or more residues).

[0015] In some embodiments, the binding domain is configured to bind ABCA4 intron 23 at a binding site comprising any one or more of nucleotides 801 to 950 of SEQ ID NO: 29 (e.g., 1 to 200, 6 to 150, 12 to 100, or 20 to 80 nucleotides within or around nucleotides 801 to 950 of SEQ ID NO: 29, e.g., 1 to 6, 6 to 12, 12 to 18, 18 to 24, 24 to 50, 50 to 100, 100 to 150, or 150 to 200 nucleotides within or around nucleotides 801 to 950 of SEQ ID NO: 29, e.g., SEQ ID NO: 29). (The binding site comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 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 within or around nucleotides 801 to 950 of SEQ ID NO: 29.) For example, in a particular embodiment, the binding site comprises six or more of the nucleotides 801 to 950 of SEQ ID NO: 29. In some embodiments, the binding domain comprises six or more consecutive nucleic acid residues complementary (e.g., antisense) to six or more nucleotides of the binding site.In some embodiments, the binding domain comprises a set of consecutive nucleic acid residues complementary to a set of complementary nucleotides corresponding to the ABCA4 binding sites of one or more nucleotides having SEQ ID NO: 29, wherein the length of the set of consecutive nucleic acid residues of the binding domain is from 6 to 500 residues (e.g., from 8 to 400, from 12 to 300, from 16 to 200, from 24 to 280, or from 50 to 150 residues, e.g., from 100 to 200, from 6 to 10, from 10 to 20, from 20 to 30, from 30 to 40, from 40 to 50, from 50 to 80, from 80 to 100). From 100 to 120, from 120 to 150, from 150 to 200, or from 200 to 300 residues, for example, lengths of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55. 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or more residues).

[0016] In some embodiments, the binding domain is configured to bind ABCA4 intron 23 at a binding site comprising any one or more of nucleotides 841 to 990 of SEQ ID NO: 29 (e.g., 1 to 200, 6 to 150, 12 to 100, or 20 to 80 nucleotides within or around nucleotides 841 to 990 of SEQ ID NO: 29, e.g., 1 to 6, 6 to 12, 12 to 18, 18 to 24, 24 to 50, 50 to 100, 100 to 150, or 150 to 200 nucleotides within or around nucleotides 841 to 990 of SEQ ID NO: 29, e.g., SEQ ID NO: 29). (The binding site comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 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 within or around nucleotides 841 to 990 of SEQ ID NO: 29.) For example, in a particular embodiment, the binding site comprises six or more of the nucleotides 841 to 990 of SEQ ID NO: 29. In some embodiments, the binding domain comprises six or more consecutive nucleic acid residues complementary (e.g., antisense) to six or more nucleotides of the binding site.In some embodiments, the binding domain comprises a set of consecutive nucleic acid residues complementary to a set of complementary nucleotides corresponding to the ABCA4 binding sites of one or more nucleotides having SEQ ID NO: 29, wherein the length of the set of consecutive nucleic acid residues of the binding domain is from 6 to 500 residues (e.g., from 8 to 400, from 12 to 300, from 16 to 200, from 24 to 280, or from 50 to 150 residues, e.g., from 100 to 200, from 6 to 10, from 10 to 20, from 20 to 30, from 30 to 40, from 40 to 50, from 50 to 80, from 80 to 100). From 100 to 120, from 120 to 150, from 150 to 200, or from 200 to 300 residues, for example, lengths of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55. 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or more residues).

[0017] 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 intron 24 at a binding site comprising one or more of nucleotides 600 to 1250 or 1490 to 2660 of SEQ ID NO: 30. In other embodiments, the binding site comprises one or more of nucleotides 1,000 to 1,200 of SEQ ID NO: 30.

[0018] In some embodiments, the binding domain binds to a target ABCA4 intron at the 5' end of the mutation, wherein the mutation is in any of ABCA4 exons 23-50 or introns 22-49. For example, in some embodiments, the target ABCA4 intron is intron 23, the mutation is in any 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 intron 23 at a binding site comprising any one or more of nucleotides 80 to 1081 of SEQ ID NO: 29. In some embodiments, the binding site comprises any one or more of nucleotides 230 to 1081 of SEQ ID NO: 29, such as 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.

[0019] In some embodiments, the target ABCA4 intron is intron 24, mutated 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 intron 24 at a binding site comprising one or more of nucleotides 1 to 250, nucleotides 300 to 2100, or nucleotides 2200 to 2692 of SEQ ID NO: 30. In some embodiments, the binding site comprises one or more of nucleotides 360 to 610 of SEQ ID NO: 30. In other embodiments, the binding site comprises one or more of nucleotides 750 to 1110 of SEQ ID NO: 30.

[0020] In another aspect, the present invention features a nucleic acid trans-splicing molecule comprising: (a) a binding domain configured to bind ABCA4 intron 22 at a binding site comprising any one or more of nucleotides 60 to 570, 600 to 800, or 900 to 1350 of SEQ ID NO: 28, operably linked in a 5' to 3' direction; (b) a splicing domain configured to mediate trans-splicing; and (c) a coding domain comprising functional ABCA4 exons 23-50; wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain to endogenous ABCA4 exons 22, thereby replacing endogenous ABCA4 exons 23-50 with functional ABCA4 exons 23-50. In some embodiments, the binding site comprises any one or more of nucleotides 70 to 250 of SEQ ID NO: 28.

[0021] In another aspect, the present invention is characterized by a nucleic acid trans-splicing molecule comprising: (a) a binding domain configured to bind ABCA4 intron 22 at a binding site comprising any one or more of nucleotides 1 to 510 or 880 to 1350 of SEQ ID NO: 28; (b) a splicing domain configured to mediate trans-splicing; 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 exons 23, thereby replacing endogenous ABCA4 exons 1-22 with functional ABCA4 exons 1-22.

[0022] In some embodiments, the binding domain is configured to bind ABCA4 intron 22 at a binding site comprising any one or more of nucleotides 1041 to 1190 of SEQ ID NO: 28 (e.g., 1 to 200, 6 to 150, 12 to 100, or 20 to 80 nucleotides within or around nucleotides 1041 to 1190 of SEQ ID NO: 28, e.g., 1 to 6, 6 to 12, 12 to 18, 18 to 24, 24 to 50, 50 to 100, 100 to 150, or 150 to 200 nucleotides within or around nucleotides 1041 to 1190 of SEQ ID NO: 28, e.g., SEQ ID NO: 28). (The binding site comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 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 within or around nucleotides 1041 to 1190 of SEQ ID NO: 28.) In a particular embodiment, the binding site comprises six or more of the nucleotides 1041 to 1190 of SEQ ID NO: 28. In some embodiments, the binding domain comprises six or more consecutive nucleic acid residues complementary (e.g., antisense) to six or more nucleotides of the binding site.In some embodiments, the binding domain comprises a set of consecutive nucleic acid residues complementary to a set of complementary nucleotides corresponding to the ABCA4 binding sites of one or more nucleotides having SEQ ID NO: 28, wherein the length of the set of consecutive nucleic acid residues of the binding domain is from 6 to 500 residues (e.g., from 8 to 400, from 12 to 300, from 16 to 200, from 24 to 280, or from 50 to 150 residues, e.g., from 100 to 200, from 6 to 10, from 10 to 20, from 20 to 30, from 30 to 40, from 40 to 50, from 50 to 80, from 80 to 1...). 00, from 100 to 120, from 120 to 150, from 150 to 200, or from 200 to 300 residues, for example, lengths of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or more residues).

[0023] In some embodiments, the binding domain is configured to bind any one or more of nucleotides 1171 to 1320 of SEQ ID NO: 28 (e.g., 1 to 200, 6 to 150, 12 to 100, or 20 to 80 nucleotides within or around the binding sites of nucleotides 1171 to 1320 of SEQ ID NO: 28, e.g., 1 to 6, 6 to 12, 12 to 18, 18 to 24, 24 to 50, 50 to 100, 100 to 150, or 150 to 200 nucleotides within or around the binding sites of nucleotides 1171 to 1320 of SEQ ID NO: 28, e.g., SEQ ID NO: 28). (The binding site comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 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 within or around nucleotides 1171 to 1320 of SEQ ID NO: 28.) In a particular embodiment, the binding site comprises six or more of the nucleotides 1171 to 1320 of SEQ ID NO: 28. In some embodiments, the binding domain comprises six or more consecutive nucleic acid residues complementary (e.g., antisense) to six or more nucleotides of the binding site.In some embodiments, the binding domain comprises a set of consecutive nucleic acid residues complementary to a set of complementary nucleotides corresponding to the ABCA4 binding sites of one or more nucleotides of SEQ ID NO: 28, wherein the length of the consecutive set of nucleic acid residues of the binding domain is from 6 to 500 residues (e.g., from 8 to 400, from 12 to 300, from 16 to 200, from 24 to 280, or from 50 to 150 residues, e.g., from 100 to 200, from 6 to 10, from 10 to 20, from 20 to 30, from 30 to 40, from 40 to 50, from 50 to 80, from 80 to 1...). 00, from 100 to 120, from 120 to 150, from 150 to 200, or from 200 to 300 residues, for example, lengths of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or more residues).

[0024] In some embodiments, the binding domain is configured to bind any one or more of nucleotides 1201 to 1350 of SEQ ID NO: 28 (e.g., 1 to 200, 6 to 150, 12 to 100, or 20 to 80 nucleotides within or around the binding sites of nucleotides 1201 to 1350 of SEQ ID NO: 28, e.g., 1 to 6, 6 to 12, 12 to 18, 18 to 24, 24 to 50, 50 to 100, 100 to 150, or 150 to 200 nucleotides within or around the binding sites of nucleotides 1201 to 1350 of SEQ ID NO: 28, e.g., SEQ ID NO: 28). The binding site comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 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 within or around nucleotides 1201 to 1350 of SEQ ID NO: 28. In certain embodiments, the binding site comprises six or more of the nucleotides 1201 to 1350 of SEQ ID NO: 28. In some embodiments, the binding domain comprises six or more consecutive nucleic acid residues complementary (e.g., antisense) to six or more nucleotides of the binding site.In some embodiments, the binding domain comprises a set of consecutive nucleic acid residues complementary to a set of complementary nucleotides corresponding to the ABCA4 binding sites of one or more nucleotides having SEQ ID NO: 28, wherein the length of the consecutive set of nucleic acid residues of the binding domain is from 6 to 500 residues (e.g., from 8 to 400, from 12 to 300, from 16 to 200, from 24 to 280, or from 50 to 150 residues, e.g., from 100 to 200, from 6 to 10, from 10 to 20, from 20 to 30, from 30 to 40, from 40 to 50, from 50 to 80, from 80 to 1...). 00, from 100 to 120, from 120 to 150, from 150 to 200, or from 200 to 300 residues, for example, lengths of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or more residues).

[0025] In any of the foregoing embodiments, the length of the binding domain can be 20-1,000 nucleotides (e.g., 25-900 nucleotides, 30-800 nucleotides, 40-700 nucleotides, 50-600 nucleotides, 75-500 nucleotides, 100-400 nucleotides, 125-200 nucleotides, or about 150 nucleotides, e.g., 20-30 nuclei). nucleotides, with lengths of 30-40 nucleotides, 40-50 nucleotides, 50-75 nucleotides, 75-100 nucleotides, 125-150 nucleotides, 150-175 nucleotides, 175-200 nucleotides, 200-250 nucleotides, 250-500 nucleotides, 500-750 nucleotides, or 750-1000 nucleotides.

[0026] In some embodiments, the coding domain is a cDNA sequence. In some embodiments, the coding domain comprises a natural sequence. In other embodiments, the coding domain comprises a codon-optimized sequence. In some embodiments, the trans-splicing molecule comprises an artificial intron containing a spacer sequence.

[0027] In some embodiments of any of the foregoing methods, the length of the nucleic acid trans-splicing molecule is 3,000 to 4,000 nucleotides (e.g., 3,100-3,900 nucleotides, 3,200-3,800 nucleotides, 3,300-3,700 nucleotides, 3,400-3,600 nucleotides), or about 3,500 nucleotides (e.g., 3,000-3,100 nucleotides). nucleotides, with lengths of 3,100-3,200 nucleotides, 3,200-3,300 nucleotides, 3,300-3,400 nucleotides, 3,400-3,500 nucleotides, 3,500-3,600 nucleotides, 3,600-3,700 nucleotides, 3,800-3,900 nucleotides, or 3,900-4,000 nucleotides.

[0028] In some implementations, mutations in the ABCA4 gene are associated with Stargardt disease. In some implementations, mutations in the ABCA4 gene associated with Stargardt disease are expressed in photoreceptor cells.

[0029] On the other hand, this document provides a nucleic acid trans-splicing molecule comprising: (a) a binding domain configured to bind ABCA4 intron 23 at a binding site comprising six or more nucleotides 261 to 410 of SEQ ID NO: 29, wherein the binding domain comprises six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) an artificial intron comprising the 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 to endogenous ABCA4 exons 24, thereby replacing endogenous ABCA4 exons 1-23 with functional ABCA4 exons 1-23.

[0030] In another aspect, the present invention provides a nucleic acid trans-splicing molecule comprising: (a) a binding domain configured to bind ABCA4 intron 23 at a binding site comprising six or more nucleotides 801 to 950 of SEQ ID NO: 29, wherein the binding domain comprises six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) an artificial intron comprising the 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 to endogenous ABCA4 exons 24, thereby replacing endogenous ABCA4 exons 1-23 with functional ABCA4 exons 1-23.

[0031] On the other hand, this document provides a nucleic acid trans-splicing molecule comprising: (a) a binding domain configured to bind ABCA4 intron 23 at a binding site comprising six or more nucleotides 841 to 990 of SEQ ID NO: 29, wherein the binding domain comprises six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) an artificial intron comprising the 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 to endogenous ABCA4 exons 24, thereby replacing endogenous ABCA4 exons 1-23 with functional ABCA4 exons 1-23.

[0032] In another aspect, the present invention provides a nucleic acid trans-splicing molecule comprising: (a) a binding domain configured to bind ABCA4 intron 22 at a binding site comprising six or more nucleotides 1041 to 1190 of SEQ ID NO: 28, wherein the binding domain comprises six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) an artificial intron comprising the 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 exons 23, thereby replacing endogenous ABCA4 exons 1-22 with functional ABCA4 exons 1-22.

[0033] In another aspect, the present invention is characterized by a nucleic acid trans-splicing molecule comprising: (a) a binding domain configured to bind ABCA4 intron 22 at a binding site comprising six or more nucleotides 1171 to 1320 of SEQ ID NO: 28, wherein the binding domain comprises six or more consecutive nucleic acid residues complementary to the six or more nucleotides at the binding site; (b) an artificial intron comprising the 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 exons 23, thereby replacing endogenous ABCA4 exons 1-22 with functional ABCA4 exons 1-22.

[0034] On the other hand, this document provides a nucleic acid trans-splicing molecule comprising: (a) a binding domain configured to bind ABCA4 intron 22 at a binding site comprising six or more nucleotides 1201 to 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 at the binding site; (b) an artificial intron comprising the 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 exons 23, thereby replacing endogenous ABCA4 exons 1-22 with functional ABCA4 exons 1-22.

[0035] In another aspect, the present invention is characterized by a proviral plasmid comprising a nucleic acid trans-splicing molecule of any of the foregoing embodiments.

[0036] In another aspect, the present invention is characterized by an adeno-associated virus (AAV) comprising the nucleic acid molecules of any of the foregoing embodiments. In some embodiments, the AAV preferentially targets photoreceptor cells. In some embodiments, the AAV comprises the AAV5 capsid protein, the AAV8 capsid protein, the AAV8(b) capsid protein, or the AAV9 capsid protein.

[0037] In another aspect, the present invention is characterized by a pharmaceutical composition comprising a nucleic acid trans-splicing molecule, proviral plasmid, or AAV as described in any of the foregoing aspects.

[0038] On the other hand, this document provides pharmaceutical compositions having any 5' nucleic acid trans-splicing molecule of any of the foregoing embodiments and any 3' nucleic acid trans-splicing molecule of any of the foregoing embodiments.

[0039] In another aspect, the present invention is characterized by a method for correcting mutations in the ABCA4 gene in the target cells of a subject by administering a pharmaceutical composition of any of the foregoing aspects to the subject.

[0040] On the other hand, this document provides a method for correcting mutations in one or more ABCA4 exons 1-24 in a subject with such a need by administering to the subject a pharmaceutical composition having any of the nucleic acid trans-splicing molecules of the foregoing embodiments. In a particular embodiment, the mutated ABCA4 exon to be corrected by the ABCA4 trans-splicing molecule of the present invention is exon 2. Alternatively or additionally, the mutated ABCA4 exon to be corrected by the ABCA4 trans-splicing molecule of the present invention is exon 3. Alternatively or additionally, the mutated ABCA4 exon to be corrected by the ABCA4 trans-splicing molecule of the present invention is exon 4.

[0041] In another aspect, the present invention includes a method for correcting mutations in one or more ABCA4 exons 23-50 in a subject in need by administering a pharmaceutical composition comprising any of the nucleic acid trans-splicing molecules of the foregoing embodiments to the subject.

[0042] On the other hand, the present invention is characterized by a method for correcting a mutation in any ABCA4 exons 1-24 and a second mutation in any exons 23-50 in a subject with such need, the method comprising administering to the subject a pharmaceutical composition having a 5' nucleic acid trans-splicing molecule of any of the foregoing embodiments and a 3' nucleic acid trans-splicing molecule of any of the foregoing embodiments.

[0043] In yet another embodiment, the invention is characterized by a method of treating a subject with a condition associated with a mutation in ABCA4, the method comprising administering any of the aforementioned pharmaceutical compositions to the subject. In some embodiments, a subject with a condition associated with a mutation in any one or more ABCA4 exons 1-24 or introns 1-24 is treated by administering a pharmaceutical composition comprising a nucleic acid trans-splicing molecule from any of the aforementioned embodiments. In some embodiments, a subject with a condition associated with a mutation in any one or more ABCA4 exons 23-50 or introns 22-49 is treated by administering a pharmaceutical composition comprising a nucleic acid trans-splicing molecule from any of the aforementioned embodiments.

[0044] In another aspect, the present invention is characterized by a method of treating a subject with a condition associated with a first mutation in any of the exons 1-24 of ABCA4 and a second mutation in any of the exons 23-50 by administering a pharmaceutical composition to the subject, the pharmaceutical composition having a 5' nucleic acid trans-splicing molecule of any of the foregoing embodiments and a 3' nucleic acid trans-splicing molecule of any of the foregoing embodiments.

[0045] In any of the foregoing methods, the subject may have Stargardt disease. In some embodiments, the composition is administered via subretinal injection, intravitreal injection, or intravenous injection.

[0046] 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., an increase of 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-50%, or 50-100% in ABCA4 protein expression after administration, e.g., relative to the same subject's ABCA4 protein expression before administration, or relative to a reference sample, reference subject, or reference subject group).

[0047] CEP290

[0048] In another aspect, the present invention is characterized by a CEP290 trans-splicing molecule. For example, the present invention provides a nucleic acid trans-splicing molecule comprising, operably linked in a 3' to 5' direction: (a) a binding domain configured to bind 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 functional CEP290 exons 2-26 and correcting pathogenic point mutations. In some embodiments, the pathogenic point mutation is an A to G mutation at nucleotide 1,655 of SEQ ID NO: 85.

[0049] In some embodiments, the binding site comprises any one or more of nucleotides 4,980 to 5,838 of SEQ ID NO: 85. In some embodiments, the binding site comprises any one or more of nucleotides 5,348 to 5,838 of SEQ ID NO: 85. In some embodiments, the binding site comprises any one or more of nucleotides 5,348 to 5,700 of SEQ ID NO: 85. In some embodiments, the binding site comprises any one or more of nucleotides 5,400 to 5,600 of SEQ ID NO: 85. In some embodiments, the binding site comprises any one or more of nucleotides 5,460 to 5,560 of SEQ ID NO: 85. In some embodiments, the binding site comprises nucleotide 5,500 of SEQ ID NO: 85.

[0050] In another aspect, the present invention features a nucleic acid trans-splicing molecule comprising, operably linked in the 3' to 5' direction: (a) a binding domain configured to bind CEP290 to any one of target introns 27, 28, 29, or 30; (b) a splicing domain configured to mediate trans-splicing; and (c) a coding domain comprising a functional CEP290 exon at the 5' end of the target intron; wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain to endogenous CEP290, thereby replacing the endogenous CEP290 exon at the 5' end of the target intron with a functional CEP290 exon and correcting a pathogenic point mutation. In some embodiments, the pathogenic point mutation is an A-G mutation at nucleotide 1,655 of SEQ ID NO: 85.

[0051] 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 intron 27 at a binding site comprising any one or more of nucleotides 120 to 680, 710 to 2,200, or 2,670 to 2,910 of SEQ ID NO: 86. In some embodiments, the binding site comprises any one or more of nucleotides 790 to 2,100 of SEQ ID NO: 86, for example, any one or more of nucleotides 1,020 to 1,630 of SEQ ID NO: 86. In other embodiments, the binding site comprises any one or more of nucleotides 1,670 to 2,000 of SEQ ID NO: 86.

[0052] 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 intron 28 at a binding site comprising any one or more of nucleotides 1 to 390, nucleotides 410 to 560, or nucleotides 730 to 937 of SEQ ID NO: 87. In some embodiments, the binding site comprises any one or more of nucleotides 1 to 200 of SEQ ID NO: 87. In other embodiments, the binding site comprises any one or more of nucleotides 720 to 900 of SEQ ID NO: 87.

[0053] 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 functional CEP290 exons 2-29. In some embodiments, the binding domain is configured to bind intron 28 at a binding site comprising 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.

[0054] 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 intron 29 at a binding site comprising any one or more of nucleotides 880 to 1,240 of SEQ ID NO: 89, such as any one or more of nucleotides 950 to 1,240 of SEQ ID NO: 89, such as any one or more of nucleotides 1,060 to 1,240 of SEQ ID NO: 89.

[0055] In any of the foregoing embodiments, the binding domain is 20-1,000 nucleotides in length (e.g., 25-900 nucleotides, 30-800 nucleotides, 40-700 nucleotides, 50-600 nucleotides, 75-500 nucleotides, 100-400 nucleotides, 125-200 nucleotides, or about 150 nucleotides, e.g., 20-30 nucleotides). (Length of 30-40 nucleotides, length of 40-50 nucleotides, length of 50-75 nucleotides, length of 75-100 nucleotides, length of 125-150 nucleotides, length of 150-175 nucleotides, length of 175-200 nucleotides, length of 200-250 nucleotides, length of 250-500 nucleotides, length of 500-750 nucleotides, or length of 750-1,000 nucleotides).

[0056] In some implementations, the coding domain is a cDNA sequence. In some implementations, the coding domain is a naturally occurring sequence. In other implementations, the coding domain is a codon-optimized sequence.

[0057] In some implementations, the artificial intron comprises an artificial intron and a spacer sequence.

[0058] In any of the foregoing embodiments, the length of the nucleic acid trans-splicing molecule can be 3,000 to 4,000 nucleotides.

[0059] In any of the foregoing embodiments, the mutated CEP290 exon may be associated with LCA 10. In some embodiments, the LCA 10-associated mutated CEP290 exon is expressed in photoreceptor cells.

[0060] In another aspect of the invention, a proviral plasmid is provided herein comprising a nucleic acid trans-splicing molecule of any of the foregoing aspects.

[0061] In another aspect, the present invention provides an adeno-associated virus (AAV) comprising the nucleic acid molecule of any of the foregoing aspects. In some embodiments, the AAV preferentially targets photoreceptor cells. In some embodiments, the AAV comprises the AAV5 capsid protein, the AAV8 capsid protein, the AAV8(b) capsid protein, or the AAV9 capsid protein.

[0062] In another aspect, the present invention is characterized by a pharmaceutical composition comprising a nucleic acid trans-splicing molecule, proviral plasmid, or AAV as described in any of the foregoing aspects.

[0063] On the other hand, this article is characterized by a method for correcting pathogenic point mutations in CEP290 intron 26 in target cells of a subject, the method comprising administering to the subject any of the aforementioned nucleic acid trans-splicing molecules, the proviral plasmid, the AAV, or the pharmaceutical composition. In some embodiments, the subject has LCA 10.

[0064] In another aspect, the present invention provides a method for treating a subject with LCA 10 caused by a pathogenic point mutation in intron 26 of CEP290, the method comprising administering to the subject any of the aforementioned nucleic acid trans-splicing molecules, the proviral plasmid, the AAV, or the pharmaceutical composition.

[0065] In any of the foregoing methods, the pathogenic point mutation can be an A to G mutation at nucleotide 1655 of intron 26 of CEP290 (SEQ ID NO: 85). In some embodiments, the nucleic acid trans-splicing molecule, the proviral plasmid, the AAV, or the pharmaceutical composition is administered via subretinal injection, intravitreal injection, or intravenous injection.

[0066] In another aspect, the present invention provides a kit comprising any one or more of the aforementioned nucleic acid trans-splicing molecules, proviral plasmids, AAVs, or pharmaceutical compositions, wherein the kit further comprises instructions for using one or more nucleic acid trans-splicing molecules, proviral plasmids, AAVs, or pharmaceutical compositions to correct mutations in the subject's CEP290 gene (e.g., disease-related mutations, such as LCA 10). Attached Figure Description

[0067] Figure 1This is a schematic diagram of several exemplary nucleic acid trans-splicing molecules used to correct mutations in ABCA4 exons using functional ABCA4 exons. Dark shaded boxes represent native ABCA4 exons. Dashed lines connecting the dark shaded boxes represent native introns. Light shaded boxes with dark borders represent functional ABCA4 exons in nucleic acid trans-splicing molecules. The splicing domains, represented by curves, attach to one end of each functional ABCA4 exon and guide to the introns of the ABCA4 pre-mRNA.

[0068] Figure 2 This is a graph showing the trans-splicing efficiency (relative fold change) conferred by the 150-mer binding domain of the 150-mer of ABCA4 intron 19 (SEQ ID NO: 25) using a 5' trans-splicing molecule across ten nucleotide intervals. 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).

[0069] Figure 3 This is a graph showing the trans-splicing efficiency (relative fold change) conferred by the 150-mer binding domain across intron 22 (SEQ ID NO: 28) of ABCA4 using 5' trans-splicing molecules within a ten-nucleotide interval. 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).

[0070] Figure 4 This is a graph showing the trans-splicing efficiency (relative fold change) conferred by the 150-mer binding domain across intron 22 (SEQ ID NO: 28) of ABCA4 using 3' trans-splicing molecules within a ten-nucleotide interval. 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).

[0071] Figure 5 This is a graph showing the trans-splicing efficiency (relative fold change) conferred by the 150-mer binding domain across intron 23 (SEQ ID NO: 29) of ABCA4 using 5' trans-splicing molecules within a ten-nucleotide interval. 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).

[0072] Figure 6 This is a graph showing the trans-splicing efficiency (relative fold change) conferred by the 150-mer binding domain across intron 23 (SEQ ID NO: 29) of ABCA4 using 3' trans-splicing molecules within a ten-nucleotide interval. 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).

[0073] Figure 7 This is a graph showing the trans-splicing efficiency (relative fold change) conferred by the 150-mer binding domain across intron 24 (SEQ ID NO: 30) of ABCA4 using 5' trans-splicing molecules within a ten-nucleotide interval. 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).

[0074] Figure 8 This is a graph showing the trans-splicing efficiency (relative fold change) conferred by the 150-mer binding domain across intron 24 (SEQ ID NO: 30) of ABCA4 using 3' trans-splicing molecules within a ten-nucleotide interval. 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).

[0075] Figure 9 This is a schematic diagram showing the TALEN protein, which consists of DNA-binding domains linked to transcription activation domains. 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 the putative transcription start site are shown. The sequences targeted by the three different DNA-binding domains of TALEN are also shown. 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.

[0076] Figure 10 This is a gel showing the transfection of 293T cells with a TALEN construct designed to induce endogenous ABCA4 expression. (The gel contains cells from...) Figure 9 All three TALENS were stably introduced into 293 cells, and single-cell clones were selected and analyzed by Western blotting. A positive control (+) indicated cells transfected with the ABCA4 cDNA plasmid. Cell lysates were prepared 48 hours post-transfection, and ABCA4 expression in the membrane fraction was examined using antibody ab72955 (Abcam). Clones ZT-22 and ZT-48 showed ABCA4 protein expression.

[0077] Figure 11 This is a schematic diagram showing the CAG promoter cell line.

[0078] Figure 12A and 12B The site-specific guide (shown) Figure 12A It was designed to use homologous arms ( Figure 12B Insert the CAG promoter and puromycin selection marker.

[0079] Figure 13This is a schematic diagram showing the CAG promoter cell line.

[0080] Figure 14A and 14B These are graphs and gel plots, which show the expression results from several clone lines selected for further analysis. Figure 14A RNA expression was shown. Figure 14B Protein expression in the cell lines is shown. ABCA4 protein was detected in the membrane formulation of the specified cell lines using a rabbit polyclonal antibody against ABCA4 (Abcam, ab72955). Exposure time was 23 seconds. Cell 293 was a parental cell that did not express ABCA4. The highest band represents nonspecific background present in all cells.

[0081] Figure 15 This is a schematic diagram showing the CRISPR guide RNA used to target exons 3 and 4.

[0082] Figure 16 This is a graph showing RNA expression and a gel, which displays the protein profiles of single-cell clones derived after CRISPR / Cas9 treatment, such as... Figure 15 As shown.

[0083] Figure 17A and 17B This is a schematic diagram showing PCR used for mutation analysis on cDNA. Figure 17A ) and PCR for genotyping on cDNA ( Figure 17B This confirmed that exons 3 and 4 were targeted and interrupted.

[0084] Figure 18 It is a set of tables that display data from... Figure 17A and 17B Mutation analysis confirmed that exons 3 and 4 were targeted and interrupted in the alleles of the 17+06 and 17+21 cell lines.

[0085] Figure 19A and 19B This is a schematic diagram of a trans-splicing molecule that targets ABCA4 pre-mRNA. Figure 19A It shows a universal trans-splicing molecule that includes codon-optimized exons (or exon sets), binding domains that hybridize with target RNA, and artificial intron adapters. Figure 19B Various trans-splicing molecules targeting specific regions within introns 22 and 23 of ABCA4 are shown.

[0086] Figure 20A-20D It is a gel ( Figure 20A and 20C ) and diagram ( Figure 20B and 20DThis shows the results from the trans-splicing reaction. Figure 20A and 20B The protein and RNA levels of the intron 22 trans-splicing reaction are shown separately. Figure 20C and 20D The protein and RNA levels of the intron 23 trans-splicing reaction are shown separately.

[0087] Figure 21 This is a schematic diagram of several exemplary nucleic acid trans-splicing molecules that correct mutations in CEP290 intron 26 using the functional 5' portion of the CEP290 gene. Dark shaded boxes represent native CEP290 exons. Dashed lines connecting the dark shaded boxes represent native introns. Light shaded boxes with dark borders represent functional CEP290 exons in nucleic acid trans-splicing molecules. The splicing domains, represented by curves, attach to one end of each functional CEP290 exon sequence and guide to the intron of the CEP290 pre-mRNA.

[0088] Figure 22 This is a graph showing the trans-splicing efficiency (relative fold change) conferred by the 150-mer binding domain across CEP290 intron 26 (SEQ ID NO: 85) in ten nucleotide spacers. The X-axis labels indicate the "base number" (i.e., the first nucleotide of the intron sequence) of each binding site starting from the 5' end of the intron.

[0089] Figure 23 This is a graph showing the trans-splicing efficiency (relative fold change) conferred by the 150-mer binding domain across intron 27 (SEQ ID NO: 86) of CEP290 in ten nucleotide spacers. Each of the three rows represents an independent experiment. The X-axis labels indicate the "base number" (i.e., the first nucleotide of the intron sequence) of each binding site starting from the 5' end of the intron.

[0090] Figure 24 This is a graph showing the trans-splicing efficiency (relative fold change) conferred by the 150-mer binding domain across intron 28 (SEQ ID NO: 87) of CEP290 in ten nucleotide spacers. Each of the three rows represents an independent experiment. The X-axis labels indicate the "base number" (i.e., the first nucleotide of the intron sequence) of each binding site starting from the 5' end of the intron.

[0091] Figure 25This is a graph showing the trans-splicing efficiency (relative fold change) conferred by the 150-mer binding domain across intron 29 (SEQ ID NO: 88) of CEP290 in ten nucleotide spacers. Each of the three rows represents an independent experiment. The X-axis labels indicate the "base number" (i.e., the first nucleotide of the intron sequence) of each binding site starting from the 5' end of the intron.

[0092] Figure 26 This is a graph showing the trans-splicing efficiency (relative fold change) conferred by the 150-mer binding domain across CEP290 intron 30 (SEQ ID NO: 89) in ten nucleotide spacers. Each of the three rows represents an independent experiment. The X-axis labels indicate the "base number" (i.e., the first nucleotide of the intron sequence) of the number of each binding site starting from the 5' end of the intron. Detailed Implementation

[0093] The compositions and methods described herein relate to trans-splicing molecules (e.g., pre-mRNA splicing molecules delivered by adeno-associated virus (AAV)) for treating diseases or conditions caused by mutations in the ABCA4 gene. The methods and compositions described herein employ pre-mRNA trans-splicing as a gene therapy (e.g., in vitro and in vivo gene therapy) for treating diseases caused by ABCA4 mutations, such as Stargardt disease (e.g., Stargardt disease 1).

[0094] Alternatively, the compositions and methods described herein relate to trans-splicing molecules (e.g., pre-mRNA trans-splicing molecules delivered by adeno-associated virus (AAV)) for treating diseases or conditions caused by mutations in the CEP290 gene (e.g., LCA 10). These methods employ pre-mRNA trans-splicing as a gene therapy (e.g., ex vivo and in vivo gene therapy) for treating diseases caused by CEP290 mutations, such as LCA 10.

[0095] The trans-splicing molecules and their methods of use illustrated in this article offer several advantages over conventional therapies. First, the use of trans-splicing molecules via AAV provides efficient and specific delivery of gene therapy to photoreceptors, while overcoming difficulties associated with AAV packaging limitations. Second, these compositions and methods allow for correction of genetic defects at the source. Furthermore, the compositions and methods presented herein can be used to treat any type of mutation in ABCA4 (or other large cDNA / genetic cassettes). Correction of photoreceptor defects provides secondary rescue for retinal pigment epithelial cells. Moreover, this method and composition are generally immunologically benign. The use of subretinal delivery and other features makes the effect specific to target cells (e.g., photoreceptors), thereby reducing toxicity caused by off-target splicing. Furthermore, unlike nucleases, trans-splicing does not require genomic alteration. Finally, RNA repair does not require cell division, while DNA repair methods (e.g., CRISPR-Cas9 or zinc fingers) require cells to undergo mitosis for homologous targeted repair, which is disadvantageous in post-mitotic tissues (like the retina).

[0096] I. Definition

[0097] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains, and reference is made to the published texts, which provide general guidance to those skilled in the art regarding the many terms used in this application. In the event of any conflict between the definitions set forth herein and those in the referenced publications, the definitions provided herein shall prevail.

[0098] A “nucleic acid trans-splicing molecule” or “trans-splicing molecule” has three main elements: (a) a binding domain that imparts specificity by tethering the trans-splicing molecule to its target gene (e.g., pre-mRNA); (b) a splicing domain (e.g., a splicing domain having a 3' or 5' splicing site); and (c) a coding sequence configured to be trans-spliced ​​onto the target gene, which may replace one or more exons in the target gene (e.g., one or more mutated exons). A “pre-mRNA trans-splicing molecule” or “RTM” refers to a nucleic acid trans-splicing molecule that targets pre-mRNA. In some embodiments, the trans-splicing molecule, such as an RTM, may include cDNA, for example as part of a functional exon (e.g., a functional ABCA4 or CEP290 exon, e.g., a codon-optimized exon), for replacing or correcting mutated ABCA4 or CEP290 exons.

[0099] "Trans-splicing" refers to the connection of a nucleic acid molecule containing one or more exons (e.g., exogenous exons, such as exons that are part of the coding domain of a trans-splicing molecule) to a single RNA molecule (e.g., a pre-mRNA molecule, such as an endogenous pre-mRNA molecule) by replacing the second part of the RNA molecule through a spliceosome-mediated mechanism.

[0100] As used herein, “binding” between the binding domain and the target intron refers to a hydrogen bond binding between the binding domain and the target intron that is strong enough to mediate trans-splicing by associating the trans-splicing molecule with the target gene (e.g., pre-mRNA). In some embodiments, the hydrogen bond between the binding domain and the target intron is between nucleotide bases that are complementary to each other and in an antisense orientation (e.g., hybridizing with each other).

[0101] As used herein, an "artificial intron" refers to a nucleic acid sequence that directly or indirectly links a binding domain to a coding domain. Artificial introns include splicing domains and may further include one or more spacer sequences and / or other regulatory elements.

[0102] As used herein, a "splicing domain" refers to a nucleic acid sequence having a motif recognized by the spliceosome and mediating trans-splicing. A splicing domain includes a splicing site (e.g., a single splicing site, i.e., one and only one splicing site), which may be a 3' splicing site or a 5' splicing site. A splicing domain may include other regulatory elements. For example, in some embodiments, a splicing domain includes a splicing enhancer (e.g., an exon splicing enhancer (ESE) or an intron splicing enhancer (ISE)). In some embodiments, a splicing domain includes a branch point (e.g., a strongly conserved branch point) or a branch point sequence and / or a polypyrimidine bundle (PPT). In some embodiments, the splicing domain of a 5' trans-splicing molecule does not contain a branch point or PPT, but instead contains a 5' splice acceptor or a 3' splice donor.

[0103] As used herein, “mutation” refers to any abnormal nucleic acid sequence that causes a defective protein product (e.g., a nonfunctional protein product, a protein product with reduced function, a protein product with abnormal function, and / or a protein product produced in quantities lower or higher than normal). Mutations include base pair mutations (e.g., single nucleotide polymorphisms), missense mutations, frameshift mutations, deletions, insertions, and splicing mutations. In some embodiments, a mutation refers to a nucleic acid sequence in which one or more portions of a nucleic acid 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 encoding a protein having an amino acid sequence different from the corresponding wild-type protein or a functional variant thereof. A “mutated exon” (e.g., a mutated ABCA4 exon) refers to an exon containing a mutation or a sequence of exons reflecting a mutation in a different region, such as a recessive exon resulting from a mutation in an intron.

[0104] As used herein, the term "ABCA4" refers to a polynucleotide (e.g., RNA (e.g., pre-mRNA or mRNA) or DNA) encoding a retinal-specific ATP-binding cassette transporter. SEQ ID NO: 6 provides an exemplary pre-mRNA sequence of the functional human ABCA4 gene. An exemplary genomic DNA sequence of the functional (wild-type) human ABCA4 gene is provided by NCBI reference sequence: NG_009073. The amino acid sequence of the exemplary ABCA4 protein is provided by protein accession number P78363.

[0105] The exons and introns of ABCA4 identified in this paper are shown in Table 1 below, which can be mapped to the ABCA4 pre-mRNA molecule of SEQ ID NO: 6. Each exon and intron of ABCA4 is identified in this paper according to the reference number in the first column (left). The size (base pairs; bp) of each exon and intron is indicated in the second and third columns. The fourth column indicates the length of the cDNA molecule of the corresponding exon located at the 5' end of the corresponding intron number. The fifth column indicates the length of the cDNA molecule of the corresponding mRNA located at the 3' end of the corresponding intron number.

[0106] Table 1. Summary of ABCA4 exons and introns

[0107]

[0108]

[0109]

[0110] As used herein, “target ABCA4 intron” refers to one of the 49 ABCA4 introns identified in Table 1 above. Nucleic acid sequence identifiers for each ABCA4 intron sequence are provided in Table 2 below. It should be understood that the term “target ABCA4 intron” 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), wherein the position of the variant intron on the ABCA4 gene corresponds to the position provided herein (e.g., relative to its adjacent exons listed in Table 1).

[0111] Table 2. Intron sequences of ABCA4

[0112]

[0113]

[0114]

[0115] 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. SEQ ID NO: 113 provides an exemplary pre-mRNA sequence of the functional human CEP290 gene. An exemplary genomic DNA sequence of the functional (wild-type) human CEP290 gene is provided by NCBI reference sequence: NG_008417. The amino acid sequence of an exemplary human centrosomal protein 290 protein is provided by protein accession number O15078.

[0116] The CEP290 exons and introns identified in this paper are shown in Table 3 below and can be mapped to the CEP290 pre-mRNA molecule of SEQ ID NO: 112. Each exon and intron of CEP290 is identified in this paper according to the reference number in the first column (left). The size (base pairs; bp) of each exon and intron is indicated in the second and third columns. The fourth column indicates the length of the cDNA molecule of the corresponding exon, which is located at the 5' end of the corresponding intron number. The fifth column indicates the length of the cDNA molecule of the corresponding mRNA, which is located at the 3' end of the corresponding intron number.

[0117] Table 3. Summary of CEP290 exons and introns

[0118]

[0119]

[0120] As used herein, “target CEP290 intron” refers to one of the 53 CEP290 introns identified in Table 3 above. Nucleic acid sequence identifiers for each CEP290 intron sequence are provided in Table 4 below. It should be understood that the term “target CEP290 intron” encompasses variants of the CEP290 introns provided herein, such as intron sequences with 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), wherein the position of the variant intron on the CEP290 gene corresponds to the position provided herein (e.g., relative to its adjacent exon listed in Table 3).

[0121] Table 4. CEP290 Intron Sequences

[0122]

[0123]

[0124]

[0125] As used herein, the term "subject" includes any mammal, including humans, that requires these treatments or preventative measures. Other mammals requiring such treatment or preventative measures include dogs, cats or other domesticated animals, horses, livestock, laboratory animals, including non-human primates, etc. Subjects may be male or female. In one embodiment, the subject suffers from a disease or condition caused by a mutation in the ABCA4 gene (e.g., Stargardt disease, such as Stargardt disease 1) or the CEP290 gene (e.g., an autosomal recessive genetic disorder, such as LCA 10). In another embodiment, the subject is at risk of developing a disease or condition caused by a mutation in the ABCA4 or CEP290 gene. In yet another embodiment, the subject has shown clinical signs of a disease or condition caused by a mutation in the ABCA4 gene (e.g., Stargardt disease) or the CEP290 gene (e.g., LCA 10). Subjects may be of any age from which treatment or preventative therapy may be beneficial. For example, in some embodiments, the subject is aged 0-5 years, 5-10 years, 10-20 years, 20-30 years, 30-50 years, 50-70 years, or 70 years and older. In another embodiment, 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 another embodiment, the subject has viable retinal cells.

[0126] As used herein, the terms "mutation-related condition" or "condition-related mutation" refer to the correlation between the condition and the mutation. In some embodiments, a known or suspected mutation-related condition is caused wholly or partially, or directly or indirectly, by the mutation. For example, a subject with the mutation may be at risk of developing the condition, and that risk may additionally depend on other factors, such as other (e.g., independent) mutations (e.g., the same or different genes) or environmental factors.

[0127] As used in this article, the term “treatment” or its grammatical derivatives are defined as reducing the progression of disease, lessening the severity of disease symptoms, slowing the progression of disease symptoms, eliminating disease symptoms, or delaying the onset of disease.

[0128] As used herein, the term “prevention” of a disease, or its grammatical derivatives, is defined as reducing the risk of disease onset, for example, prophylactic treatment for a subject at risk of developing a mutation-related disease. A subject can be characterized as “at risk” of having the disease by identifying a mutation associated with the disease, according to any suitable method known in the art or described herein. In some embodiments, a subject at risk of having the disease has one or more ABCA4 or CEP290 mutations associated with the disease. Alternatively or alternatively, a subject can be characterized as “at risk” if they have a family history of the disease.

[0129] The condition of a subject can be treated or prevented by administering trans-splicing molecules (e.g., within an AAV carrier or AAV particle) directly to the subject. Alternatively, host cells containing trans-splicing molecules can be administered to the subject.

[0130] As used in the methods described herein, the term "application" or its grammatical derivatives refer to the delivery of a composition or ex vivo treated cells to a subject in need, such as a subject with a mutation or defect in a target gene. For example, in one embodiment targeting ocular cells, the method involves delivering the composition to photoreceptor cells or other ocular cells via subretinal injection. In another embodiment, intravitreal injection or injection via a palpebral vein may be used to deliver the composition to ocular cells. In yet another embodiment, the composition is administered intravenously. In view of this disclosure, those skilled in the art may choose other methods of application.

[0131] Codon optimization refers to modifying a nucleic acid sequence to alter a single nucleic acid without causing any change in the encoded amino acid. Sequences modified in this manner are referred to herein as "codon-optimized". This process can be performed on any sequence described in this specification to enhance expression or stability. Codon optimization can be performed, for example, as 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. Sequences around translation start sites can be converted into shared Kozak sequences according to known methods. See, for example, Kozak et al., Nucleic Acids Res. 15(20):8125-8148, which is incorporated herein by reference in its entirety.

[0132] The term "homologous" refers to the degree of identity between two nucleic acid sequences. Homology of homologous sequences is determined by comparing two sequences aligned under optimal conditions with the sequence to be compared. The sequences to be compared in this paper may have additions or deletions (e.g., gaps, etc.) in the optimal alignment of the two sequences. Such sequence homology can be calculated by creating alignments using, for example, the ClustalW algorithm (Nucleic Acid Res., 1994, 22(22): 4673 4680). Commonly used sequence analysis software, such as Vector NTI, GENETYX, BLAST, or analysis tools provided by public databases, can also be used.

[0133] The term "pharmaceuticalally acceptable" means that it is safe for use in mammals such as humans. In some implementations, a pharmaceutically acceptable composition is approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, especially humans.

[0134] The term "carrier" refers to a diluent, adjuvant, excipient, or medium that is administered together with a therapeutic molecule (e.g., the trans-splicing molecule of the present invention or a trans-splicing molecule comprising a carrier or cell). Examples of suitable drug carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 2nd edition, 2005.

[0135] The term "a" means "one or more". For example, "a gene" should be understood to represent one or more such genes. Thus, the terms "a", "one or more of one or more", and "at least one of one (or one or more)" are used interchangeably in this document.

[0136] As used herein, unless otherwise stated, the term “about” means a value that is within ±10% of a reference value.

[0137] II. Trans-splicing molecules

[0138] This article provides information on the ABCA4 trans-splicing molecule and the CEP290 trans-splicing molecule.

[0139] ABCA4 trans-splicing molecule

[0140] The present invention is characterized by a nucleic acid trans-splicing molecule that, by substituting one or more exons in the ABCA4 gene (e.g., the ABCA4 gene with mutated ABCA4 exons), can be used to treat diseases and conditions associated with ABCA4 gene mutations. 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 the substitution of defective or mutated portions of one or more pre-mRNA exons with a nucleic acid sequence (e.g., an exon with a functional (e.g., normal) sequence without mutations). This functional sequence can be wild-type, a naturally occurring sequence, or a corrected sequence with some other modifications, such as codon-optimized correction sequences.

[0141] In one embodiment, the trans-splicing molecule is configured to correct one or more mutations located at the 3' portion of the ABCA4 gene. In one embodiment, the trans-splicing molecule is configured to correct one or more mutations located at the 5' portion of the ABCA4 gene. The trans-splicing molecule provided herein functions to repair a defective gene in a subject's target cells by replacing the defective pre-mRNA gene sequence and removing the defective portion of the target pre-mRNA, thereby generating a functional ABCA4 gene capable of transcribing a functional gene product in the cell.

[0142] This invention provides a trans-splicing molecule having a binding domain configured to bind a target ABCA4 intron, a splicing domain configured to mediate trans-splicing, and a coding domain having one or more functional ABCA4 exons. In the 5' trans-splicing molecule, the coding domain, splice site, and binding domain are operably connected in the 5' to 3' direction, such that the trans-splicing molecule is configured to replace the 5' end of an endogenous gene with a coding domain comprising a functional ABCA4 exon in place of a mutated ABCA4 exon. Conversely, in the 3' trans-splicing molecule, the coding domain, splice site, and binding domain are operably connected in the 3' to 5' direction, such that the trans-splicing molecule is configured to replace the 3' end of an endogenous gene with a coding domain comprising a functional ABCA4 exon in place of a mutated ABCA4 exon. In some embodiments, the splicing domain is located within an artificial intron that links the binding domain to the coding domain. Artificial introns can include other elements, such as spacers.

[0143] In some embodiments, the trans-splicing molecule or its coding domain is up to 4,700 nucleotides long (e.g., 200 to 300 nucleotides, 300 to 400 nucleotides, 400 to 500 nucleotides, 500 to 600 nucleotides, 600 to 700 nucleotides, 700 to 800 nucleotides, 800 to 900 nucleotides, 900 to 1...). 1,000 nucleotide bases, 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, for example, 3,100 to 3,800 nucleotide bases in length, 3,200 to 3,700 nucleotide bases in length, or 3,300 to 3500 nucleotide bases, for example, 3000 to 3100 nucleotide bases in length, 3100 to 3200 nucleotide bases in length, 3200 to 3300 nucleotide bases in length, 3300 to 3400 nucleotide bases in length, 3400 to 3500 nucleotide bases in length, 3500 to 3600 nucleotide bases in length, 3600 to 3700 nucleotide bases in length, and 3700 to 3,800 nucleotide bases, with a length of 3,800 to 3,900 nucleotide bases, or with a length of 3,900 to 4,000 nucleotide bases, for example, with a length of approximately 2,918 nucleotide bases, approximately 3,328 nucleotide bases, approximately 3,522 nucleotide bases, approximately 3,607 nucleotide bases, approximately 3,632 nucleotide bases, approximately 3,494 nucleotide bases, or approximately 3,300 nucleotide bases).

[0144] Due to the large size of the ABCA4 gene and the size limitations of AAV delivery, a single trans-splicing molecule configured for packaging in an AAV vector may not cover all mutations in the ABCA4 gene that may be disease-related, and therefore may not be able to correct mutations along the entire length of the ABCA4 gene. Therefore, the trans-splicing molecule of the present invention is adapted as part of the method described below to correct multiple mutations spanning the entire length of the ABCA4 gene.

[0145] The ABCA4 gene targeted by the trans-splicing molecules described herein contains one or more mutations associated with (e.g., causing or related to) diseases such as Stargardt disease (e.g., Stargardt disease 1). An exemplary DNA sequence of the functional (wild-type) human ABCA4 gene is provided by NCBI reference sequence: NG_009073. The amino acid sequence of an exemplary protein expressed by ABCA4, a retinal-specific ATP-binding cassette transporter, is provided by protein accession number P78363.

[0146] In addition to these publicly available sequences, this includes all subsequently obtained corrected or naturally occurring conserved and nonpathogenic sequences in humans or other mammalian populations. It also includes other conserved nucleotide substitutions or substitutions that cause codon optimization. Sequences provided by the database accession number can also be used to search for homologous sequences in the same or another mammalian organism.

[0147] It is anticipated that the ABCA4 nucleic acid sequence and the resulting protein truncated or amino acid fragments can tolerate certain minor modifications at the nucleic acid level, including, for example, modifications to silent nucleotide bases, such as codon preference. In other embodiments, nucleic acid base modifications that alter amino acids are anticipated, for example, to improve the expression of the resulting peptide / protein (e.g., codon optimization). Allelic variations due to the natural degeneracy of the genetic code are also included as possible fragment modifications.

[0148] Modifications to the ABCA4 gene also include analogs or modified forms of the protein fragments encoded as described in this article. Typically, these analogs differ from the specifically labeled protein by only one to four codon changes. Conserved substitutions are those that occur within amino acid families and are related to the side chains and chemical properties of those amino acids.

[0149] The nucleic acid sequence of the functional ABCA4 gene can be derived from any mammal that naturally expresses a functional retinal-specific ATP-binding cassette transporter or its homologs. In other embodiments, the ABCA4 gene sequence is modified to enhance expression in target cells. Such modifications include codon optimization.

[0150] In some embodiments, the condition associated with mutations in ABCA4 is an autosomal recessive genetic disorder, such as Stargardt disease. In certain cases involving subjects with an autosomal recessive genetic disorder, the subject has ABCA4 mutations in both alleles. Regardless of the location of the mutation in the ABCA4 gene, compositions containing trans-splicing molecules can correct for mutations in both alleles. For example, for a subject with a mutated ABCA4 exon 1 in the first allele and a mutated ABCA4 exon 30 in the second allele, this document provides compositions having a 5' trans-splicing molecule to replace the mutated ABCA4 exon 1 and a 3' trans-splicing molecule to replace the mutated ABCA4 exon 30. In such embodiments, the two trans-splicing molecules can be delivered co-delivered as part of the same AAV vector or delivered in separate AAV vectors (e.g., where the two trans-splicing molecules exceed the packaging limits of AAV).

[0151] Alternatively, in embodiments where two or more mutations are located on a portion of the ABCA4 gene, and that portion of the ABCA4 gene can be replaced by the same trans-splicing molecule, a single trans-splicing molecule having a coding region containing a functional ABCA4 exon can replace one or more exons containing mutations. Mutations in specific ABCA4 exons are also listed in International Patent Publication No. WO 2017 / 087900, which is incorporated herein by reference.

[0152] ABCA4 encoding structure field

[0153] In some embodiments, the coding domain of the 5' trans-splicing molecule includes all ABCA4 exons (e.g., functional ABCA4 exons) at the 5' end of the target ABCA4 intron. For example, in an embodiment where the 5' trans-splicing molecule targets ABCA4 intron 19, the coding domain includes functional ABCA4 exons 1-19. In such embodiments of a 5' trans-splicing molecule having a coding domain including functional ABCA4 exons 1-19, the coding domain is approximately 2918 bp in length. In an embodiment where the 5' trans-splicing molecule targets ABCA4 intron 22, the coding domain includes functional ABCA4 exons 1-22. In such embodiments of a 5' trans-splicing molecule having a coding domain including functional ABCA4 exons 1-22, the coding domain is approximately 3,328 bp in length. In embodiments of 5' trans-splicing molecules targeting ABCA4 intron 23, the coding domain includes functional ABCA4 exons 1-23. In such embodiments of 5' trans-splicing molecules having coding domains including functional ABCA4 exons 1-23, the length of this coding domain is approximately 3,522 bp. In embodiments of 5' trans-splicing molecules targeting ABCA4 intron 24, the coding domain includes functional ABCA4 exons 1-24. In such embodiments of 5' trans-splicing molecules having coding domains including functional ABCA4 exons 1-24, the length of this coding domain is approximately 3,607 bp. The above embodiments of trans-splicing molecules targeting 5'ABCA4... Figure 1 The lower left portion is shown.

[0154] In some embodiments, the coding domain of the 3' trans-splicing molecule includes any one or more of ABCA4 exons 20-50. For example, in an embodiment where the 3' trans-splicing molecule targets ABCA4 intron 22, the coding domain includes functional ABCA4 exons 23-50. In such embodiments of a 3' trans-splicing molecule having a coding domain including functional ABCA4 exons 23-50, the coding domain is about 3,632 bp in length. In an embodiment where the 3' trans-splicing molecule targets ABCA4 intron 23, the coding domain includes functional ABCA4 exons 24-50. In such embodiments of a 3' trans-splicing molecule having a coding domain including functional ABCA4 exons 24-50, the coding domain is about 3,494 bp in length. In an embodiment where the 3' trans-splicing molecule targets ABCA4 intron 24, the coding domain includes functional ABCA4 exons 25-50. In such embodiments of 3' trans-splicing molecules having a coding domain including functional ABCA4 exons 25-50, the coding domain is approximately 3,300 bp in length. The above-described embodiments of trans-splicing molecules targeting 3'ABCA4... Figure 1 The upper right part is shown.

[0155] In some embodiments, the coding structure domain includes 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.

[0156] In some cases, both mutations occur in the 5' portion of the target gene, and a 5' trans-splicing molecule is selected to correct both mutations. In one embodiment, the binding domain binds to intron 19 and encodes a domain comprising functional ABCA4 exons 1-19. In one embodiment, the binding domain binds to intron 22 and encodes a domain comprising functional ABCA4 exons 1-22. In one embodiment, the binding domain binds to intron 23 and encodes a domain comprising functional ABCA4 exons 1-23. In one embodiment, the binding domain binds to intron 24 and encodes a domain comprising functional ABCA4 exons 1-24. Alternatively, if both mutations occur in the 3' portion of the target gene, a 3' trans-splicing molecule is selected to correct both mutations. In one embodiment, the binding domain binds to intron 22 and encodes a domain comprising functional ABCA4 exons 23-50. In one embodiment, the binding domain binds to intron 23, and the encoding domain includes functional ABCA4 exons 24-50. In another embodiment, the binding domain binds to intron 24, and the encoding domain includes functional ABCA4 exons 25-50.

[0157] As an example, the operation of the 3' pre-mRNA ABCA4 trans-splicing molecule is as follows: The chimeric mRNA is produced through a trans-splicing reaction mediated by the spliceosome between the 5' splice site of the endogenous target pre-mRNA and the 3' splice site of the trans-splicing molecule. The trans-splicing molecule binds to the target ABCA4 intron of the endogenous target pre-mRNA through specific base pairing and replaces the entire 3' sequence of the endogenous ABCA4 gene upstream of the target intron with the coding domain of the functional ABCA4 exon sequence of the trans-splicing molecule.

[0158] The 3' trans-splicing molecule includes a binding domain that binds to the target ABCA4 intron at the mutated or defective 5' end, an artificial intron containing an optional spacer and a 3' splice site, and a coding domain for all exons of a target gene encoding the eye, wherein the target gene is located at the 3' end where the binding domain binds to the target. The 5' trans-splicing molecule includes a binding domain that binds to the target ABCA4 intron at the mutated or defective 3' end, a 5' splice site, an optional spacer, and a coding domain for all exons of a target gene encoding the eye, wherein the target gene is located at the 5' end where the binding domain binds to the target.

[0159] In some embodiments, the coding domain comprises a complementary DNA (cDNA) sequence. For example, one or more functional ABCA4 exons within the coding domain may be a cDNA sequence. In some embodiments, the entire coding domain is a cDNA sequence. Alternatively or additionally, all or a portion of the coding domain, or one or more functional ABCA4 exons thereof, may be naturally occurring sequences (e.g., sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with endogenous ABCA4 exons).

[0160] In some embodiments, all or part of the coding domain or one or more functional ABCA4 exons thereof are codon-optimized sequences, wherein the nucleic acid sequence has been modified, for example, to enhance expression or stability without resulting in changes to the encoded amino acids. Codon optimization can be performed, for example, in a manner 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 via recombinant AAV, as described herein, in one embodiment, the coding domain can be a nucleic acid sequence up to 4,000 nucleotides in length (e.g., 3,000 to 4,000 nucleotides in length, 3,100 to 3,800 nucleotides in length, 3,200 to 3,700 nucleotides in length, or 3,300 to 3,500 nucleotides in length, such as 3,000 to 3,100 nucleotides in length, 3,100 to 3,500 nucleotides in length, etc.). 200 nucleotide bases, 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.

[0161] ABCA4 combined structural domain

[0162] The trans-splicing molecule of the present invention is characterized by a binding domain configured to bind to the target ABCA4 intron. In one embodiment, the binding domain is a nucleic acid sequence complementary to the sequence of the target ABCA4 pre-mRNA (e.g., the target ABCA4 intron) to inhibit endogenous target cis-splicing while enhancing trans-splicing between the trans-splicing molecule and the target ABCA4 pre-mRNA, for example, to produce a chimeric molecule having a portion of the endogenous ABCA4 mRNA and a coding domain having one or more functional ABCA4 exons. In some embodiments, the binding domain is in the antisense direction of the target ABCA4 intron sequence.

[0163] The 5' trans-splicing molecule typically binds to the target ABCA4 intron at the 3' end of the mutation, while the 3' trans-splicing molecule typically binds to the target ABCA4 intron at the 5' end of 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 of the nearest (i.e., adjacent) corrected exon sequence.

[0164] In another implementation, the binding domain targets an intron sequence that is very close to the 3' or 5' splicing signal of the target intron. In yet another implementation, the binding domain sequence can also bind the target intron, in addition to partially adjacent exons.

[0165] Therefore, in certain cases, binding domains specifically bind to the mutated endogenous target pre-mRNA to anchor the coding domain of the trans-splicing molecule to the pre-mRNA, thereby allowing trans-splicing to occur at the correct location in the target ABCA4 gene. The nuclear spliceosome processing machinery can then mediate successful trans-splicing of the corrective exon targeting the mutated exon causing the disease.

[0166] In some embodiments, the trans-splicing molecule is characterized by a binding domain comprising a sequence that binds to more than one site on the pre-target mRNA. The binding domain may contain any number of nucleotides necessary to stably bind to the pre-target mRNA to allow trans-splicing with the coding domain. In one embodiment, the binding domain is selected using mFOLD structural analysis for accessible loops (Zuker, Nucleic Acids Res. 2003, 31(13):3406-3415).

[0167] The appropriate length of the target-binding domain can be from 10 to 500 nucleotides. In some embodiments, the length of the binding domain is from 20 to 400 nucleotides. In some embodiments, the length of the binding domain is from 50 to 300 nucleotides. In some embodiments, the length of the binding domain is from 100 to 200 nucleotides. In some embodiments, the binding domain is 10-20 nucleotides long (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides), 20-30 nucleotides long (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides), 30-40 nucleotides long (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides), or 40-50 nucleotides long (e.g., 40, 41, 42, 43, 44). 45, 46, 47, 48, 49, 50 nucleotides), 50-60 nucleotides (e.g., 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides), 60-70 nucleotides (e.g., 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 nucleotides), 70-80 nucleotides (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides), 80-90 nucleotides (e.g., 80, 81, 82, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides), and 80-90 nucleotides (e.g., 80, 81, 82, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides). 82, 83, 84, 85, 86, 87, 88, 89, or 90 nucleotides), 90-100 nucleotides in length (e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length), 100-110 nucleotides in length (e.g., 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 nucleotides in length), and 110-120 nucleotides in length (e.g., 110, 111, 112, 113, 114, 115, 116, 117, 118, or 11...). 9 or 120 nucleotides), 120-130 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),Lengths of 150-160 nucleotides (e.g., 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 160 nucleotides), and lengths of 160-170 nucleotides (e.g., 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, or 170 nucleotides), and lengths of... The length is 170-180 nucleotides (e.g., 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, or 180 nucleotides), and the length is 180-190 nucleotides (e.g., 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, or 190 nucleotides). It has a length of 190-200 nucleotides (e.g., 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 or 200 nucleotides), a length of 200-210 nucleotides, a length of 210-220 nucleotides, a length of 220-230 nucleotides, a length of 230-240 nucleotides, a length of 240-250 nucleotides, a length of 250-260 nucleotides, a length of 260-270 nucleotides, a length of 270-280 nucleotides, a length of 280-290 nucleotides, a length of 290-300 nucleotides, a length of 300-350 nucleotides, a length of 350-400 nucleotides, a length of 400-450 nucleotides, or a length of 450-500 nucleotides. In some embodiments, the target-binding domain is about 150 nucleotides in length. In another embodiment, the target-binding domain may comprise a nucleic acid sequence up to 750 nucleotides in length. In yet another embodiment, the target-binding domain may comprise a nucleic acid sequence up to 1000 nucleotides in length. In yet another embodiment, the target-binding domain may comprise a nucleic acid sequence up to 2000 nucleotides or more in length.

[0168] In some implementations, 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.

[0169] The binding domain can be 80% to 100% complementary to the target intron to enable stable hybridization 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 is selected by those skilled in the art as needed to maintain the trans-splicing molecule and the nucleic acid construct containing the necessary sequence for expression, and within a limit of 3,000 or at most 4,000 nucleotide bases contained in rAAV. The choice of this sequence and the hybridization strength depend on the complementarity and length of the nucleic acid.

[0170] Any of the above-mentioned binding domains may bind to the 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).

[0171] In some embodiments of the invention, the trans-splicing molecule is a 5' trans-splicing molecule characterized by binding a binding domain to intron 19 of ABCA4 (SEQ ID NO: 25) and including a coding domain having functional ABCA4 exons 1-19. In some embodiments, the binding site comprises any one or more of nucleotides 990 to 2,174 of SEQ ID NO: 25 (e.g., any one or more of nucleotides 1,670 to 2,174 of SEQ ID NO: 25, any one or more of nucleotides 1,810 to 2,000 of SEQ ID NO: 25, any one or more of nucleotides 1,870 to 2,000 of SEQ ID NO: 25, or any one or more of nucleotides 1,920 to 2,000 of SEQ ID NO: 25).

[0172] In some embodiments, the trans-splicing molecule is a 5' trans-splicing molecule characterized by a binding domain that binds intron 22 of ABCA4 (SEQ ID NO: 28) and includes a coding domain having functional ABCA4 exons 1-22. In some embodiments, the binding site comprises any one or more of nucleotides 60 to 570, 600 to 800, or 900 to 1350 of SEQ ID NO: 28 (e.g., any one or more of nucleotides 70 to 250 of SEQ ID NO: 28).

[0173] Alternatively, the trans-splicing molecule may be a 3' trans-splicing molecule, characterized by a binding domain that binds to intron 22 (SEQ ID NO: 28) of ABCA4. The trans-splicing molecule may include a coding domain having functional ABCA4 exons 23-50. In some embodiments, the binding site comprises any one or more of nucleotides 1 to 510 or 880 to 1,350 of SEQ ID NO: 28.

[0174] In other embodiments, the trans-splicing molecule is a 5' trans-splicing molecule characterized by a binding domain that binds intron 23 of ABCA4 (SEQ ID NO: 29) and includes a coding domain having functional ABCA4 exons 1-23. In some embodiments, the binding site comprises any one or more of nucleotides 80 to 570 or 720 to 1,081 of SEQ ID NO: 29.

[0175] Alternatively, the trans-splicing molecule may be a 3' trans-splicing molecule, characterized by a binding domain that binds to intron 23 of ABCA4 (SEQ ID NO: 29) and a coding domain having functional ABCA4 exons 24-50. In some embodiments, the binding site comprises any one or more of nucleotides 80 to 1081 of SEQ ID NO: 29 (e.g., any one or more of nucleotides 230 to 1081 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).

[0176] In some embodiments, the trans-splicing molecule is a 5' trans-splicing molecule and is characterized by a binding domain that binds 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 comprises any one or more of nucleotides 600 to 1,250 or 1,490 to 2,660 of SEQ ID NO: 30 (e.g., any one or more of nucleotides 1,000 to 1,200 of SEQ ID NO: 30).

[0177] In other embodiments, the trans-splicing molecule is a 3' trans-splicing molecule characterized by a binding domain that binds intron 24 of ABCA4 (SEQ ID NO: 30) and includes a coding domain having functional ABCA4 exons 25-50. In some embodiments, the binding site comprises any one or more of nucleotides 1 to 250, 300 to 2,100, or 2,200 to 2,692 of SEQ ID NO: 30 (e.g., any one or more of nucleotides 360 to 610 of SEQ ID NO: 30, or any one or more of nucleotides 750 to 1110 of SEQ ID NO: 30).

[0178] CEP290 trans-splicing molecule

[0179] The present invention is characterized by nucleic acid trans-splicing molecules that can be used to treat diseases and conditions associated with CEP290 gene mutations by replacing one or more exons in the CEP290 gene (e.g., the CEP290 gene with a mutation in intron 26). In some embodiments, the nucleic acid trans-splicing molecule is a preRNA trans-splicing molecule (RTM). The design of the trans-splicing molecule allows for the replacement of defective or mutated portions of premRNA with one or more exons of a nucleic acid sequence, such as a functional (e.g., normal) sequence, without mutation. The functional sequence can be wild-type, naturally occurring, or a corrected sequence with some other modifications, such as codon optimization.

[0180] In one implementation, the trans-splicing molecule is configured to correct one or more mutations located on the 5' portion of the CEP290 gene. The trans-splicing molecule provided herein repairs defective genes in subject target cells by replacing defective pre-mRNA gene sequences, thereby generating a functional CEP290 gene capable of transcribing a functional gene product in the cell.

[0181] This invention provides a trans-splicing molecule having a binding domain configured to bind a target CEP290 intron, a splicing domain configured to mediate trans-splicing, and a coding domain having one or more functional CEP290 exons. In the 5' trans-splicing molecule, the coding domain, splicing site, and binding domain are operatively connected in a 5' to 3' direction, such that the trans-splicing molecule is configured to replace the 5' end of an endogenous gene with a coding domain comprising a functional CEP290 exon to correct mutated CEP290 pre-mRNA. In some embodiments, the splicing domain is located within an artificial intron that links the binding domain to the coding domain. The artificial intron may include other elements, such as spacers.

[0182] In some embodiments, the trans-splicing molecule is up to 4,700 nucleotide bases in length (e.g., 3,000 to 4,000 nucleotide bases, 3,100 to 3,800 nucleotide bases, 3,200 to 3,700 nucleotide bases, or 3,300 to 3,500 nucleotide bases, e.g., 3,000 to 3,100 nucleotide bases, 3,100 to 3,200 nucleotide bases, 3,200 to 3,300 nucleotide bases, 3,300 to 3,400 nucleotide bases, etc.). It is 3,400 to 3,500 nucleotide bases long, 3,500 to 3,600 nucleotide bases long, 3,600 to 3,700 nucleotide bases long, 3,700 to 3,800 nucleotide bases long, 3,800 to 3,900 nucleotide bases long, or 3,900 to 4,000 nucleotide bases long, for example, about 2,991 nucleotide bases long, about 3,103 nucleotide bases long, about 3,309 nucleotide bases long, about 3,461 nucleotide bases long, or about 3,573 nucleotide bases long).

[0183] The CEP290 gene targeted by the trans-splicing molecules described herein contains one or more mutations associated with (e.g., causing or related to) diseases such as Leber's congenital amaurosis (LCA 10). An exemplary DNA sequence of the functional (wild-type) human CEP290 gene is given by NCBI reference sequence: NG_008417. The amino acid sequence of an exemplary centrosome protein 290 is given by protein accession number O15078.

[0184] In addition to these publicly available sequences, this includes all subsequently obtained corrected or naturally occurring conserved and nonpathogenic sequences in humans or other mammalian populations. It also includes other conserved nucleotide substitutions or substitutions that cause codon optimization. Sequences provided by the database accession number can also be used to search for homologous sequences in the same or another mammalian organism.

[0185] It is anticipated that the CEP290 nucleic acid sequence and the resulting protein truncated or amino acid fragments can tolerate certain minor modifications at the nucleic acid level, including, for example, modifications to silent nucleotide bases, such as codon preference. In other embodiments, nucleic acid base modifications that alter amino acids are anticipated, for example, to improve the expression of the resulting peptide / protein (e.g., codon optimization). Allelic variations due to the natural degeneracy of the genetic code are also included as possible fragment modifications.

[0186] Modifications to the CEP290 gene also include analogs or modified forms of the protein fragments encoded as described herein. Typically, these analogs differ from the specifically labeled protein by only one to four codon changes. Conserved substitutions are those that occur within amino acid families, relating to the side chains and chemical properties of those amino acids.

[0187] The nucleic acid sequence of the functional CEP290 gene can be derived from any mammal that naturally expresses functional centrosomal protein 290 or its homologs. In other embodiments, the CEP290 gene sequence is modified to enhance expression in target cells. Such modifications include codon optimization.

[0188] CEP290 mutations can be found in the CCHMC Molecular Genetics Laboratory Mutation Database LOVD v.2.0. In particular, mutations in the CEP290 exon are also listed in International Patent Publication No. WO 2017 / 087900, which is incorporated herein by reference. Table 3 above provides information on the size and location of each exon and intron of CEP290.

[0189] In some implementations, the condition associated with the mutation in CEP290 is an autosomal recessive genetic disorder, such as LCA 10.

[0190] Encoding structure field

[0191] In some embodiments, the coding domain of the 5' trans-splicing molecule includes all CEP290 exons (e.g., functional CEP290 exons) at the 5' end of the target CEP290 intron. For example, in an embodiment where the 5' trans-splicing molecule targets CEP290 intron 26, the coding domain includes functional CEP290 exons 2-26. In such embodiments of a 5' trans-splicing molecule having a coding domain including functional CEP290 exons 2-26, the coding domain is approximately 2,991 bp in length. In an embodiment where the 5' trans-splicing molecule targets CEP290 intron 27, the coding domain includes functional CEP290 exons 2-27. In such embodiments of a 5' trans-splicing molecule having a coding domain including 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 includes functional CEP290 exons 2-28. In such embodiments of a 5' trans-splicing molecule having a coding domain including 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 includes functional CEP290 exons 2-29. In such embodiments of a 5' trans-splicing molecule having a coding domain including 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 includes functional CEP290 exons 2-30. In such embodiments of 5' trans-splicing molecules having a coding domain including functional CEP290 exons 2-30, the coding domain is approximately 3,573 bp in length. The aforementioned embodiments of trans-splicing molecules targeting 5'CEP290... Figure 21 As shown in the image.

[0192] In some implementations, the coding structure domain includes 25, 26, 27, 28, or 29 functional CEP290 exons.

[0193] In some embodiments, the coding domain comprises a complementary DNA (cDNA) sequence. For example, one or more functional CEP290 exons within the coding domain may be a cDNA sequence. In some embodiments, the entire coding domain is a cDNA sequence. Alternatively or additionally, all or part of the coding domain or one or more functional CEP290 exons thereof may be naturally occurring sequences (e.g., sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with endogenous CEP290 exons).

[0194] In some embodiments, all or part of the coding domain or one or more functional CEP290 exons thereof are codon-optimized sequences, wherein the nucleic acid sequence has been modified, for example, to enhance expression or stability, without resulting in changes to the encoded amino acids. Codon optimization can be performed, for example, in the manner 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 via recombinant AAV, as described herein, in one embodiment, the coding domain can be a nucleic acid sequence up to 4,000 nucleotides in length (e.g., 3,000 to 4,000 nucleotides, 3,100 to 3,800 nucleotides, 3,200 to 3,700 nucleotides, or 3,300 to 3,500 nucleotides, e.g., 3,000 to 3,100 nucleotides, 3,100 to 3,200 nucleotides, 3,200 to 3,300 nucleotides, 3,300 to 3,400 nucleotides, 3,400 to 3,500 nucleotides, 3,500 to 4,000 nucleotides, 3,100 to 3,800 nucleotides, 3,200 to 3,700 nucleotides, or 3,300 to 3,500 nucleotides, e.g., 3,000 to 4,000 nucleotides, 3,100 to 3,800 nucleotides, 3,200 to 3,700 nucleotides, 3,300 to 3,400 nucleotides, 3,400 to 3,500 nucleotides, 3,500 to 4,000 nucleotides, 3,100 to 3,800 nucleotides, 3,200 to 3,7 ...300 to 3,400 nucleotides, 3,400 to 3,5 3,600 nucleotide bases, with a length of 3,600 to 3,700 nucleotide bases, with a length of 3,700 to 3,800 nucleotide bases, with a length of 3,800 to 3,900 nucleotide bases, or with a length of 3,900 to 4,000 nucleotide bases, for example, with a length of about 3,108 nucleotide bases, with a length of about 3,285 nucleotide bases, with a length of about 3,375 nucleotide bases, with a length of about 3,503 nucleotide bases, with a length of about 3,630 nucleotide bases, with a length of about 3,540 nucleotide bases, with a length of about 3,363 nucleotide bases, with a length of about 3,273 nucleotide bases, with a length of about 3,145 nucleotide bases, or with a length of about 3,018 nucleotide bases).

[0195] Combined structural domain

[0196] The trans-splicing molecule of the present invention is characterized by a binding domain configured to bind to a target CEP290 intron. In one embodiment, the binding domain is a nucleic acid sequence complementary to the sequence of the target CEP290 pre-mRNA (e.g., the target CEP290 intron) to inhibit endogenous target cis-splicing while enhancing trans-splicing between the trans-splicing molecule and the target CEP290 pre-mRNA, for example, to produce a chimeric molecule having a portion of the endogenous CEP290 mRNA and a coding domain having one or more functional CEP290 exons. In some embodiments, the binding domain is in the antisense direction of the target CEP290 intron sequence.

[0197] The 5' trans-splicing molecule typically binds to the target CEP290 intron at the 3' end of the mutation. In one embodiment, the binding domain contains a portion of a sequence complementary to the target CEP290 intron.

[0198] In another implementation, the binding domain targets an intron sequence that is very close to the 3' or 5' splicing signal of the target intron. In yet another implementation, the binding domain sequence can also bind the target intron, in addition to partially adjacent exons.

[0199] Therefore, in certain cases, binding domains specifically bind to the mutated endogenous target pre-mRNA to anchor the coding domain of the trans-splicing molecule to the pre-mRNA, thereby allowing trans-splicing to occur at the correct location in the target CEP290 gene. The nuclear spliceosome processing machinery can then mediate successful trans-splicing of the corrective exon targeting the mutated exon causing the disease.

[0200] In some embodiments, the trans-splicing molecule is characterized by a binding domain comprising a sequence that binds to more than one site on the pre-target mRNA. The binding domain may contain any number of nucleotides necessary to stably bind to the pre-target mRNA to allow trans-splicing with the coding domain. In one embodiment, the binding domain is selected using mFOLD structure analysis for accessible loops (Zuker, Nucleic Acids Res. 2003, 31(13):3406-3415).

[0201] The appropriate length of the target-binding domain can be from 10 to 500 nucleotides. In some embodiments, the length of the binding domain is from 20 to 400 nucleotides. In some embodiments, the length of the binding domain is from 50 to 300 nucleotides. In some embodiments, the length of the binding domain is from 100 to 200 nucleotides. In some embodiments, the binding domain is 10-20 nucleotides long (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides), 20-30 nucleotides long (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides), 30-40 nucleotides long (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides), or 40-50 nucleotides long (e.g., 40, 41, 42, 43, 44). 45, 46, 47, 48, 49, 50 nucleotides), 50-60 nucleotides (e.g., 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides), 60-70 nucleotides (e.g., 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 nucleotides), 70-80 nucleotides (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides), 80-90 nucleotides (e.g., 80, 81, 82, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides), and 80-90 nucleotides (e.g., 80, 81, 82, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides). 82, 83, 84, 85, 86, 87, 88, 89, or 90 nucleotides), 90-100 nucleotides in length (e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length), 100-110 nucleotides in length (e.g., 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 nucleotides in length), and 110-120 nucleotides in length (e.g., 110, 111, 112, 113, 114, 115, 116, 117, 118, or 11...). 9 or 120 nucleotides), 120-130 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),Lengths of 150-160 nucleotides (e.g., 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 160 nucleotides), and lengths of 160-170 nucleotides (e.g., 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, or 170 nucleotides), and lengths of... The length is 170-180 nucleotides (e.g., 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, or 180 nucleotides), and the length is 180-190 nucleotides (e.g., 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, or 190 nucleotides). It has a length of 190-200 nucleotides (e.g., 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 or 200 nucleotides), a length of 200-210 nucleotides, a length of 210-220 nucleotides, a length of 220-230 nucleotides, a length of 230-240 nucleotides, a length of 240-250 nucleotides, a length of 250-260 nucleotides, a length of 260-270 nucleotides, a length of 270-280 nucleotides, a length of 280-290 nucleotides, a length of 290-300 nucleotides, a length of 300-350 nucleotides, a length of 350-400 nucleotides, a length of 400-450 nucleotides, or a length of 450-500 nucleotides. In some embodiments, the target-binding domain is about 150 nucleotides in length. In another embodiment, the target-binding domain may comprise a nucleic acid sequence up to 750 nucleotides in length. In yet another embodiment, the target-binding domain may comprise a nucleic acid sequence up to 1000 nucleotides in length. In yet another embodiment, the target-binding domain may comprise a nucleic acid sequence up to 2000 nucleotides or more in length.

[0202] In some implementations, 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.

[0203] The binding domain can be 80% to 100% complementary to the target intron to enable stable hybridization 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 is selected by those skilled in the art as needed to maintain the trans-splicing molecule and the nucleic acid construct containing the necessary sequence for expression, and within a limit of 3,000 or at most 4,000 nucleotide bases contained in rAAV. The choice of this sequence and the hybridization strength depend on the complementarity and length of the nucleic acid.

[0204] Any of the above-described binding domains may bind to the binding sites of intron 26 (SEQ ID NO: 85; for example, at the mutation site or at the 3' end of the mutation, for example, a substitution mutation 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).

[0205] In certain embodiments of the invention, the trans-splicing molecule is characterized by a binding domain that binds to intron 26 (SEQ ID NO: 85) of CEP290 and includes a coding domain having functional CEP290 exons 2-26. In some embodiments, the binding site comprises any one or more of nucleotides 4,980 to 5,383 of SEQ ID NO: 85. In one embodiment, the binding site comprises any one or more of nucleotides 5,348 to 5,838 of SEQ ID NO: 85 (e.g., any one or more of nucleotides 5,348 to 5,700 of SEQ ID NO: 85, such as any one or more of nucleotides 5,400 to 5,600 of SEQ ID NO: 85, such as any one or more of nucleotides 5,460 to 5,560 of SEQ ID NO: 85, such as at least nucleotide 5,500 of SEQ ID NO: 85).

[0206] In other embodiments, the trans-splicing molecule is characterized by a binding domain that binds to intron 27 (SEQ ID NO: 86) of CEP290 and includes a coding domain having functional CEP290 exons 2-27. In some embodiments, the binding site comprises any one or more of nucleotides 120 to 680, 710 to 2,200, or 2,670 to 2,910 of SEQ ID NO: 86. In some embodiments, the binding site comprises any one or more of nucleotides 790 to 2,100 of SEQ ID NO: 86, such as any one or more of nucleotides 1,020 to 1,630 of SEQ ID NO: 86. In other embodiments, the binding site comprises any one or more of nucleotides 1,670 to 2,000 of SEQ ID NO: 86.

[0207] In some embodiments, the trans-splicing molecule is characterized by a binding domain that binds to intron 28 of CEP290 (SEQ ID NO: 87) and includes a coding domain having functional CEP290 exons 2-28. In some embodiments, the binding site comprises any one or more of nucleotides 1 to 390, 410 to 560, or 730 to 937 of SEQ ID NO: 87. In some embodiments, the binding site comprises any one or more of nucleotides 1 to 200 of SEQ ID NO: 87. In other embodiments, the binding site comprises any one or more of nucleotides 720 to 900 of SEQ ID NO: 87.

[0208] In some embodiments, the trans-splicing molecule is characterized by a binding domain that binds intron 29 of CEP290 (SEQ ID NO: 88) and includes a coding domain having functional CEP290 exons 2-29. In some embodiments, the binding site comprises any one or more of nucleotides 1 to 600, nucleotides 720 to 940, or nucleotides 1370 to 1790 of SEQ ID NO: 88.

[0209] In other embodiments, the trans-splicing molecule is characterized by a binding domain that binds to intron 30 of CEP290 (SEQ ID NO: 89) and includes a coding domain having functional CEP290 exons 2-30. In some embodiments, the binding site comprises any one or more of nucleotides 880 to 1,240 of SEQ ID NO: 89, such as any one or more of nucleotides 950 to 1,240 of SEQ ID NO: 89, such as any one or more of nucleotides 1,060 to 1,240 of SEQ ID NO: 89.

[0210] splice domain

[0211] The following splice domains can be used in any trans-splicing molecule of the present invention (e.g., any ABCA4 trans-splicing molecule or CEP290 trans-splicing molecule described herein).

[0212] The splice domain may include a splice site, a branch point, and / or a polypyrimidine bundle (PPT) to mediate trans-splicing. In some embodiments, the splice domain has a single splice site, meaning that due to the lack of a corresponding splice site, the splice site is capable of trans-splicing but not cis-splicing. In some embodiments, the splice domain of a 3' trans-splicing molecule includes a strongly conserved branch point or branch point sequence, a polypyrimidine bundle (PPT), a 3' splice acceptor (AG or YAG) site, and / or a 5' splice donor site. The splice domain of a 5' trans-splicing molecule does not contain a branch point or PPT but includes a 5' splice acceptor and / or a 3' splice donor splice site.

[0213] The splice domain can be selected by those skilled in the art based on known methods and principles. The splice domain provides the fundamental shared motif for spliceosome recognition. The use of branch points and PPTs follows the shared sequence required for the two phosphate transfer reactions involved in trans-splicing. In one embodiment, the shared sequence for branch points in mammals is YNYURAC (Y = pyrimidine; N = any nucleotide). The polypyrimidine bundle located between the branch point and the splice site receptor is crucial for different branch point utilization and 3' splice site recognition. Shared sequences for the 5' splice donor site and 3' splice region used in RNA splicing are well known in the art. Alternatively, modified shared sequences can be used that retain the ability to function as both the 5' donor splice site and the 3' splice region. Briefly, in one embodiment, the 5' splice site shared sequence is the nucleic acid sequence AG / GURAGU (where / denotes the splice site). In another embodiment, an endogenous splice site corresponding to an exon adjacent to the splice site can be used to maintain any splice regulatory signals.

[0214] In one embodiment, a suitable 5' splice site with a spacer is: 5'-GTA AGA GAG CTCGTT GCG ATA TTA T-3' (SEQ ID NO: 1). In another embodiment, a suitable 5' splice site is AGGT.

[0215] In one embodiment, a suitable 3' trans-splicing molecule branching site is 5'-TACTAAC-3'. In one embodiment, a suitable 3' splicing site is: 5'-TAC TAA CTG GTA CCT CTT CTT TTT TTT CTG CAG-3' (SEQ ID NO: 2) or 5'-CAGGT-3'. In one embodiment, a suitable 3' trans-splicing molecule PPT is: 5'-TGGTAC CTC TTC TTT TTT TTC TG-3' (SEQ ID NO: 3).

[0216] Additional elements or embellishments

[0217] In some embodiments of any trans-splicing molecule of the present invention (e.g., any ABCA4 trans-splicing molecule or CEP290 trans-splicing molecule described herein), the splicing domain is included as part of an artificial intron, which may include one or more additional elements. For example, a spacer region may be included within the artificial intron to separate the splicing domain in the trans-splicing molecule from the target-binding domain. The spacer region may be designed to include features such as (i) a stop codon that acts to block the translation of any unspliced ​​trans-splicing molecule and / or (ii) a sequence that enhances trans-splicing with the target pre-mRNA. The spacer may be between 3 and 25 nucleotides or more, depending on the length of the other components of the trans-splicing molecule and the limitations of rAAV. In one embodiment, a suitable 5' trans-splicing molecule spacer is AGA TCT CGT TGC GAT ATT AT (SEQ ID NO: 4). In one implementation, a suitable 3' spacer is: 5'-GAG AAC ATT ATT ATA GCG TTG CTC GAG-3' (SEQ ID NO: 5).

[0218] Other optional elements of the trans-splicing molecule (e.g., as part of an artificial intron) include small introns that regulate trans-splicing, as well as enhancers of introns or exons (e.g., intron splicing enhancers, such as downstream intron splicing enhancers) or silencers.

[0219] In another embodiment, the trans-splicing molecule further includes (e.g., as part of an artificial intron) at least one safety sequence incorporated into a spacer, binding domain, or other location within the trans-splicing molecule to prevent nonspecific trans-splicing. This is a region of the trans-splicing molecule that covers elements of the 3' and / or 5' splice sites of the trans-splicing molecule through relatively weak complementarity, thereby preventing nonspecific trans-splicing. The trans-splicing molecule is designed such that, upon hybridization to one or more binding / targeting portions of the trans-splicing molecule, the 3' or 5' splice site will be exposed and fully active. Such safety sequences comprise complementary extensions of the cis sequence (or may be a second, separate nucleic acid strand) that bind to one or both sides of the trans-splicing molecule branch point, pyrimidine bundles, the 3' splice site, and / or the 5' splice site (splicing element), or may bind to portions of the splicing element itself. The binding of the secure sequence can be disrupted by the binding of the target-binding region of the trans-splicing molecule to the pre-target mRNA, thereby exposing and activating the splicing element (making it available for trans-splicing into the pre-target mRNA). In another embodiment, the trans-splicing molecule has a 3' UTR sequence or ribozyme sequence added to the 3' or 5' end.

[0220] In one implementation, splicing enhancers, such as sequences referred to as exon splicing enhancers, may also be included in the structure of an artificial intron. Other features may be added to the artificial intron, such as polyadenylation signals that modify RNA expression / stability, or 5' splicing sequences that enhance splicing, additional binding regions, safety self-complementary regions, additional splicing sites, or protecting groups, to modulate molecular stability and prevent degradation. Additionally, a stop codon may be included in the trans-splicing molecule (e.g., as part of an artificial intron) to prevent the translation of the unspliced ​​trans-splicing molecule. Other elements (e.g., 3' hairpin structures, circularized RNA, nucleotide base modifications, or synthetic analogs) may be incorporated into the trans-splicing molecule to promote or facilitate nuclear localization and spliceosome fusion, as well as intracellular stability.

[0221] In some embodiments, the binding of trans-splicing molecules to the target pre-mRNA is mediated by complementarity (i.e., based on the base-pairing characteristics 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 molecules include DNA, RNA, or DNA / RNA hybrid molecules, wherein the DNA or RNA is single-stranded or double-stranded. This document also includes RNA or DNA that can hybridize with one of the aforementioned RNA or DNA, preferably under stringent conditions, such as in 2.5x SSC buffer at 60°C, followed by several washes at a lower buffer concentration, such as 0.5x SSC buffer, at 37°C. These nucleic acids may encode proteins exhibiting lipid phosphatase activity and / or binding to the plasma membrane. When trans-splicing molecules are synthesized in vitro, such trans-splicing molecules can be modified on the base moiety, sugar moiety, or phosphate backbone, for example, to improve the stability of the molecule, hybridization with the target mRNA, transport into cells, cellular stability against enzymatic cleavage, etc. For example, modifying trans-splicing molecules to reduce total charge can enhance cellular uptake of the molecules. Additionally, modifications can be made to reduce sensitivity to nucleases or chemical degradation. Nucleic acid molecules can be synthesized by conjugation with other molecules such as peptides, hybridization-triggered cross-linking agents, transporters, hybridization-triggered cleavage agents, etc.

[0222] Various other well-known modifications to nucleic acid molecules can be introduced as a means of increasing intracellular stability and half-life (see also oligonucleotides above). Possible modifications are known in the art. Modifications that can be made to the structure of synthetic trans-splicing molecules include backbone modifications.

[0223] III. Recombinant AAV molecules

[0224] Any suitable nucleic acid vector can be used in conjunction with this composition and method to design and assemble components of trans-splicing molecules and recombinant adeno-associated virus (AAV). 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. Medic, 2001, 7(l):33-40; and Walther W. and Stein U., Drugs 2000, 60(2):249-71.

[0225] In some embodiments described herein, a trans-splicing molecule carrying the ABCA4 gene-binding and coding domain is delivered to selected cells requiring treatment with an AAV vector, such as photoreceptor cells. More than 30 naturally occurring AAV serotypes are available. Numerous natural variants exist within the AAV capsid, allowing for the identification and use of AAVs with properties particularly suited to ocular cells. AAV viruses can be engineered using conventional molecular biology techniques to optimize these particles for cell-specific delivery of trans-splicing molecule nucleic acid sequences, to minimize immunogenicity, to modulate stability and particle lifespan, to facilitate efficient degradation, and to ensure accurate delivery to the cell nucleus, among other things.

[0226] The expression of the trans-splicing molecule described herein can be achieved in selected cells via delivery of recombinant engineered AAVs or artificial AAVs containing sequences encoding the desired trans-splicing molecule. The use of AAVs is a common method of exogenous DNA delivery because it is relatively non-toxic, provides 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 stated, the AAV ITR 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 ITR is derived from AAV2. These ITRs or other AAV components can be readily isolated from AAV serotypes using techniques available to those skilled in the art. Such AAVs can be isolated or obtained from academic, commercial, or public sources (e.g., the U.S. Type Culture Collection, Manassas, VA). Alternatively, AAV sequences can be obtained by synthetic or other suitable methods, from references, or from publicly available sequences in databases such as GenBank, PubMed, etc.

[0227] Ideal AAV fragments for assembly into vectors include cap proteins, including vp1, vp2, vp3, and hypervariable regions; rep proteins, including rep78, rep68, rep52, and rep40; and sequences encoding these proteins. These fragments can be readily used in a variety of vector systems and host cells. Such fragments can be used alone, in combination with other AAV serotype sequences or fragments, or in combination with elements of other AAV or non-AAV viral sequences. As used herein, artificial AAV serotypes include, but are not limited to, AAVs having capsid proteins that are not naturally present. Such artificial capsids can be generated by any suitable technique using selected AAV sequences (e.g., fragments of the vp1 capsid protein) combined with heterologous sequences, which may be obtained from different selected AAV serotypes, discontinuous portions of the same AAV serotype, from non-AAV viral sources, or 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 in conjunction with an ITR from an AAV with a different capsid protein, can be used in this invention. In one embodiment, the AAV is AAV2 / 5 (i.e., an AAV having an AAV2 ITR and an AAV5 capsid). In another embodiment, the AAV is AAV2 / 8 (i.e., an AAV having an AAV2 ITR and an AAV8 capsid). In one embodiment, the AAV comprises an AAV8 capsid. Such an AAV8 capsid comprises the amino acid sequence found under NCBI reference sequence: YP_077180.1 (SEQ ID NO: 56). In another embodiment, the AAV8 capsid comprises a capsid encoded by nucleotides 2121 to 4337 of GenBank accession number: AF513852.1 (SEQ ID NO: 57).

[0228] In one embodiment, the AAV includes 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, which is incorporated herein by reference in its entirety. Compared to wild-type AAV8, AAV(b) (SEQ ID NO: 58) contains the amino acid sequence Pro-Glu-Arg-Thr-Ala-Met-Ser-Leu-Pro at amino acid positions 587-595. In another embodiment, the AAV8(b) capsid is encoded by SEQ ID NO: 59.

[0229] In one embodiment, the vector used in the compositions and methods described herein contains at least a sequence encoding a selected AAV serotype capsid, such as the AAV2 capsid or a fragment thereof. In another embodiment, the useful vector contains at least a sequence encoding a selected AAV serotype rep protein, such as the AAV2 rep protein or a fragment thereof. Optionally, such vectors may contain both AAV cap and rep protein. In vectors that provide both AAV rep and cap, both the AAV rep and AAV cap sequences can be of serotype origin, for example, AAV2 origin.

[0230] Alternatively, a vector in which the rep sequence is derived from an AAV serotype different from the serotype providing the cap sequence can be used. In one embodiment, the rep and cap sequences are expressed from separate sources (e.g., separate vectors, or host cells and vectors). In another embodiment, these rep sequences are fused in-frame with cap sequences of different AAV serotypes to form a chimeric AAV vector as described in U.S. Patent No. 7,282,199, which is incorporated herein by reference.

[0231] Suitable recombinant AAV (rAAV) is generated by culturing host cells containing a nucleic acid sequence encoding an AAV serotype capsid protein or a fragment thereof as defined herein; a functional rep gene; a small gene consisting of, for example, an AAV ITR and a trans-splicing molecule nucleic acid sequence; and sufficient helper functions to allow the small gene to be packaged into the AAV capsid protein. Components required for culturing in host cells to package the AAV small gene into the AAV capsid can be trans-provided to the host cells. Alternatively, any one or more desired components (e.g., small gene, rep sequence, cap sequence, and / or helper functions) can be provided by stable host cells that have been engineered to contain one or more desired components using methods known to those skilled in the art.

[0232] In one embodiment, the AAV includes a promoter (or a functional fragment of a promoter). A promoter for use in the rAAV can be selected from a variety of constitutive or inducible promoters, which can express the selected transgene in desired target cells. See, for example, the list of promoters identified in International Patent Publication No. WO 2014 / 012482, which is incorporated herein by reference. In one embodiment, the promoter is cell-specific. The term “cell-specific” refers to a specific promoter selected for the recombinant vector that can direct the expression of the selected transgene in a specific cell type. In one embodiment, the promoter is specific for the expression of the 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 the transgene in retinal pigment epithelium (RPE) cells. In another embodiment, the promoter is specific for the expression of the transgene in ganglion cells. In another embodiment, the promoter is specific for the expression of the transgene in Mueller cells. In another embodiment, the promoter is specific for the expression of the transgene in bipolar cells. In another embodiment, the promoter is specific for transgene expression in horizontal cells. In another embodiment, the promoter is specific for transgene expression in cells without long facets. In yet another embodiment, the transgene is expressed in any of the above-mentioned cells.

[0233] In another implementation, the promoter is a natural promoter for expressing the target gene. 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, cone cell transduced α-subunit, β-phosphodiesterase (PDE) promoter, retinitis pigmentosa promoter, NXNL2 / NXNL1 promoter, RPE65 promoter, chronic retinal degeneration / peripheral protein 2 (Rds / perph2) promoter, and VMD2 promoter.

[0234] Other common regulatory sequences contained in small genes or rAAVs are also disclosed in documents such as WO2014 / 124282, and other documents are incorporated herein by reference. Those skilled in the art can choose from these and other expression control sequences without departing from the scope described herein.

[0235] The AAV small gene may include the trans-splicing molecule and its regulatory sequence described herein, as well as the 5' and 3' AAVITRs. In one embodiment, an ITR of AAV serotype 2 is used. In another embodiment, an ITR of AAV serotype 5 or 8 is used. However, other suitable serotypes of ITRs may be selected. In some embodiments, the small gene is packaged into a capsid protein and delivered to a selected host cell.

[0236] The small gene, rep sequence, cap sequence, and accessory function required to generate rAAV can be delivered to the packaging host cell in the form of any genetic element that can transfer the sequence carried thereon. Selected genetic elements can be delivered by any suitable method, including those described herein. 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, recombination 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 generating rAAV viral particles are well known, and the selection of a suitable method is not a limitation of the invention. See, for example, K. Fisher et al., J. Virol., 1993 70:520-532, and U.S. Patent 5,478,745, each of which is incorporated herein by reference.

[0237] In another embodiment, the trans-splicing molecule small gene is prepared in a proviral plasmid, such as those disclosed in International Patent Publication No. WO 2012 / 158757, which are incorporated herein by reference. Such a proviral plasmid comprises a modular recombinant AAV genome containing operative links, the genome comprising: a wild-type 5'AAV2 ITR sequence flanked by unique restriction sites that allow immediate removal or substitution of said ITR; and a promoter, or photoreceptor-specific promoter / enhancer, of a 49-nucleotide sequence of cytomegalovirus (CMV) sequence upstream of the chicken β-actin sequence, flanked by unique restriction sites that allow convenient removal or substitution of the entire promoter sequence, with unique restriction sites flanking the upstream sequence that allow removal or substitution of only the upstream CMV or enhancer sequence from the promoter sequence. The trans-splicing molecule described herein can be inserted into a polyclonal multi-connector site, wherein the trans-splicing molecule is operatively linked to and regulated by the promoter. The flanking regions of the bovine growth hormone polyadenylated sequence are unique restriction sites that allow for easy removal or substitution of the polyA sequence; the wild-type 3'AAV2 ITR sequence, flanked by unique restriction sites that allow for easy removal or substitution of the 3'ITR, is also part of the plasmid. The plasmid backbone contains elements essential for replication in bacterial cells, such as the kanamycin resistance gene, and is itself flanked by transcription terminator / insulator sequences.

[0238] In one embodiment, the proviral plasmid comprises: (a) a modular recombinant AAV genome containing operational links, the genome comprising: (i) a wild-type 5'AAV2 ITR sequence flanked by unique restriction sites that allow immediate removal or substitution of the ITR; (ii) a promoter comprising (A) a 49-nucleotide CMV sequence upstream of the CMV-chicken β-actin sequence; (b) a photoreceptor-specific promoter / enhancer; or (c) a neuron-specific promoter / enhancer. The promoter is flanked by unique restriction sites that allow immediate removal or substitution of the entire promoter sequence, while the upstream sequence is flanked by unique restriction sites that allow immediate removal or substitution of only the upstream CMV or enhancer sequence from the promoter sequence. Part of the proviral plasmid is also a polyclonal multi-adaptor sequence that allows the insertion of trans-splicing molecules, including any sequence described herein, wherein the trans-splicing molecules are operatively linked to and regulated by a promoter; a bovine growth hormone polyadenylated sequence flanked by unique restriction sites that can be readily removed or substituted for the polyA sequence; and a wild-type 3'AAV2 ITR sequence flanked by unique restriction sites that can be readily removed or substituted for the 3'ITR. The proviral plasmid also contains a plasmid backbone containing elements essential for replication in bacterial cells and a kanamycin resistance gene, flanked by transcription terminator / insulator sequences. The proviral plasmid described herein may also contain a 5.1 kb non-coding λ phage padding sequence in the plasmid backbone to increase backbone length and prevent reverse packaging of the non-functional AAV genome.

[0239] In some embodiments, the proviral plasmid contains multiple copies of the trans-splicing molecule. For example, the present invention is characterized in that the trans-splicing molecule is less than half the packaging limit of AAV, so that it can be repeated once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times, fifteen times, sixteen times, seventeen times, eighteen times, nineteen times, twenty times or more on a single proviral plasmid.

[0240] On the other hand, the promoter of the proviral plasmid is modified to reduce its size, allowing for the insertion of larger trans-splicing molecular sequences into rAAV. In one embodiment, as described in International Patent Publication No. WO 2017 / 087900, the CMV / CBA hybrid promoter, which typically includes non-coding exons and introns totaling approximately 1,000 base pairs, is replaced with a chimeric intron of 130 base pairs, the entire contents of which are incorporated herein by reference.

[0241] These proviral plasmids are then used in conventional packaging methods to produce recombinant viruses expressing the trans-splicing molecule transgene carried by the proviral plasmid. Suitable production cell lines are readily chosen by those skilled in the art. For example, suitable host cells can be selected from any organism, including prokaryotic (e.g., bacterial) cells, and eukaryotic cells, including insect cells, yeast cells, and mammalian cells. In short, the proviral plasmid is transfected into selected packaging cells, where it may be transiently present. Alternatively, a small gene or gene expression cassette with a flanking ITR can be stably integrated into the host cell's genome via chromosome or as an appendage. Suitable transfection techniques are known and readily available for delivering the recombinant AAV genome to host cells. Typically, the proviral plasmid is cultured in host cells expressing cap and / or rep proteins. In the host cells, the small gene consisting of the trans-splicing molecule and the flanking AAV ITR is rescued and packaged into capsid or envelope proteins to form infectious viral particles. Therefore, recombinant AAV infectious particles are generated by culturing packaging cells carrying proviral plasmids in the presence of sufficient viral sequences to allow the viral genome to be packaged into the infectious AAV envelope or capsid.

[0242] IV. Pharmaceutical Compositions and Kits

[0243] This document provides pharmaceutical compositions comprising nucleic acid trans-splicing molecules, proviral plasmids, or rAAVs containing nucleic acid trans-splicing molecules described herein. In some embodiments, the pharmaceutical composition comprises any 5' trans-splicing molecule described herein. In other embodiments, the pharmaceutical composition comprises any 3' trans-splicing molecule described herein. In some embodiments, the pharmaceutical composition comprises a 5' trans-splicing molecule and a 3' trans-splicing molecule, for example, wherein the 5' trans-splicing molecule and the 3' trans-splicing molecule together contain functional ABCA4 exons 1-50 and bind the same target ABCA4 introns.

[0244] Contamination of the pharmaceutical compositions described herein can be assessed using conventional methods, and then formulated into pharmaceutical compositions intended for use in appropriate routes of administration. Other compositions containing trans-splicing molecules, such as naked DNA or as proteins, can be formulated similarly with suitable carriers. Such formulations involve the use of the drug and / or physiologically acceptable mediators or carriers, particularly 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, such as HEPES, to maintain pH at appropriate physiological levels, and optionally, other medicinal agents, pharmaceutical agents, stabilizers, buffers, carriers, adjuvants, diluents, etc.

[0245] In some embodiments, the carrier is a liquid for injection. Exemplary physiologically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free phosphate-buffered saline. Various such known carriers are provided 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 balanced salt solution. In one embodiment, the carrier includes Tween. If the virus is to be stored long-term, it can be frozen in the presence of glycerol or Tween 20.

[0246] In other embodiments, the composition containing the trans-splicing molecule described herein includes a surfactant. A useful surfactant may be included, such as Pluronic F68 (Poloxamer 188, also known as...) These prevent AAV from adhering to inert surfaces, thus ensuring the delivery of the desired dose. As an example, an exemplary composition designed for treating the eye diseases described herein comprises a recombinant gland-associated vector and a pharmaceutically acceptable carrier, the recombinant vector carrying a nucleic acid sequence encoding the 3' trans-splicing molecule described herein under the control of a regulatory sequence expressing the trans-splicing molecule in the ocular cells of a mammalian subject. 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.

[0247] In yet another exemplary embodiment, the composition comprises recombinant AAV2 / 5 pseudoadeno-associated virus carrying a 3' or 5' trans-splicing molecule for ABCA4 gene substitution, the nucleic acid sequence being directed by a promoter to express the trans-splicing molecule in the photoreceptor cells, wherein the composition is formulated together with a vector and other components suitable for subretinal injection. In yet another embodiment, the composition or components used to produce or assemble the composition, including a vector, rAAV particles, a surfactant and / or components for generating rAAV, and suitable laboratory hardware for preparing the composition, can be incorporated into a kit.

[0248] In some cases, the composition comprises a recombinant AAV2 / 5 pseudoadeno-associated virus carrying a 5' trans-splicing molecule for CEP290 gene substitution, the nucleic acid sequence of which directs the expression of the trans-splicing molecule in the photoreceptor cells under the control of a promoter, wherein the composition is formulated together with a vector and other components suitable for subretinal injection. In another embodiment, the composition or components for producing or assembling the composition, including a vector, rAAV particles, a surfactant and / or components for generating rAAV, and suitable laboratory hardware for preparing the composition, may be incorporated into a kit.

[0249] This document also provides a kit containing a first pharmaceutical composition comprising a 5' trans-splicing molecule and a second pharmaceutical composition comprising a 3' trans-splicing molecule, for example, wherein the 5' trans-splicing molecule and the 3' trans-splicing molecule together contain functional ABCA4 exons 1-50 and bind to the same target ABCA4 introns (e.g., wherein the trans-splicing molecule is packaged in any AAV carrier described herein). In some embodiments, the kit includes instructions for mixing the two pharmaceutical compositions prior to administration.

[0250] This article also provides a kit containing a first pharmaceutical composition comprising a 5' trans-splicing molecule that binds to the target CEP290 intron.

[0251] V. Method

[0252] The compositions involving ABCA4 trans-splicing described above can be used as a method for treating diseases or conditions caused by mutations in the ABCA4 gene, such as Stargardt disease (e.g., Stargardt disease 1), including delaying or alleviating symptoms associated with the diseases described herein. Such methods involve contacting a target ABCA4 gene (e.g., ABCA4 pre-mRNA) with a trans-splicing molecule described herein (e.g., one or more of the 3' trans-splicing molecule, 5' trans-splicing molecule, or 3' and 5' trans-splicing molecules) under specific conditions, wherein the coding domain of the trans-splicing molecule can be spliced ​​to the target ABCA4 gene to replace a portion of the target gene with one or more defective or mutated mRNAs of the target gene, thereby correcting ABCA4 expression in target cells. Therefore, the methods and compositions are used to treat ocular diseases / pathologies associated with specific mutations and / or gene expression.

[0253] In one embodiment, the contact involves direct administration to an affected subject. In another embodiment, the contact may occur ex vivo in cultured cells, and the treated ocular cells may be re-implanted into the subject. In one embodiment, the method involves administering rAAV carrying a 3' trans-splicing molecule. In another embodiment, the method involves administering rAAV carrying a 5' trans-splicing molecule. In yet another embodiment, the method involves administering a mixture of rAAV carrying a 3' trans-splicing molecule and rAAV carrying a 5' trans-splicing molecule. These methods comprise administering an effective concentration of any of the compositions described herein to a subject in need.

[0254] In some embodiments, the method includes selecting one or more trans-splicing molecules to treat a subject suffering from a condition associated with a mutation in ABCA4, such as Stargardt disease (e.g., Stargardt disease 1). Such selection may be based on the subject's genotype. In some embodiments, the condition associated with ABCA4 may be an autosomal recessive genetic disorder. In some cases, the subject may be homozygous for or heterozygous for a mutation in ABCA4. Methods for screening and identifying specific mutations in ABCA4 are known in the art.

[0255] In other cases, the compositions involving CEP290 trans-splicing described above can be used as a method for treating diseases or conditions caused by mutations in the CEP290 gene, such as Leber's congenital amaurosis (e.g., LCA 10), including delaying or alleviating symptoms associated with the diseases described herein. Such methods involve contacting a target CEP290 gene (e.g., CEP290 pre-mRNA) with a trans-splicing molecule (e.g., a 5' trans-splicing molecule) under specific conditions, wherein the coding domain of the trans-splicing molecule can be spliced ​​to the target CEP290 gene to replace a portion of the target gene with one or more defective or mutated mRNAs of the target gene, thereby correcting CEP290 expression in target cells. Therefore, the methods and compositions are used to treat ocular diseases / pathologies associated with specific mutations and / or gene expression. The method of the present invention includes correcting a pathogenic point mutation in intron 26 of CEP290 (e.g., nucleotide 1,655 of intron 26) by administering a 5' trans-splicing molecule (e.g., any 5' trans-splicing molecule described herein) or a pharmaceutical composition thereof. Therefore, the present invention provides a method for treating a subject suffering from a disease or condition associated with a mutation in CEP290 (e.g., a disease or condition associated with a mutation in intron 26 of CEP290, e.g., at nucleotide 1,655 of intron 26) by administering a trans-splicing molecule described herein. Any of the aforementioned trans-splicing molecules may be included in a pharmaceutical composition (e.g., a single pharmaceutical composition comprising two molecules, which may be pre-prepared or mixed prior to administration, e.g., as part of a kit).

[0256] In one embodiment, the contact involves direct administration to the affected subject. In another embodiment, the contact may occur ex vivo in cultured cells, and the treated ocular cells may be re-implanted into the subject. In one embodiment, the method involves administering rAAV containing a 5' trans-splicing molecule. These methods comprise administering an effective concentration of any of the compositions described herein to the subject in need.

[0257] In some embodiments, the method includes selecting one or more trans-splicing molecules to treat a subject suffering from a condition associated with a mutation in CEP290, such as LCA10. This selection may be based on the subject's genotype. In some embodiments, the condition associated with CEP290 may be an autosomal recessive genetic disorder. In some cases, the subject may be homozygous for or heterozygous for the mutation in CEP290. Methods for screening and identifying specific mutations in CEP290 are known in the art.

[0258] A single trans-splicing molecule used to correct a single mutation.

[0259] The method of the present invention includes selecting a single trans-splicing molecule based on the location of a single mutation in ABCA4 (e.g., a mutation in one allele of a subject). In the case of autosomal recessive mutations, the activity of a functional protein can be restored by correcting only one of the two mutations, for example, where the second allele has a mutation in another part of the ABCA4 gene that is beyond the reach of a single AAV trans-splicing molecule configured to correct the first mutation.

[0260] Therefore, in some embodiments, the method of the present invention includes selecting a single trans-splicing molecule to correct a single mutation in the 5' portion of a target gene, for example, regardless of the location of the mutation in other alleles. In one embodiment, the mutated exon is exon 1, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exon 1. In one embodiment, exon 1 or exon 2 is mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1 and 2. In one embodiment, one of exons 1, 2, and 3 is mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-3. In one embodiment, one of exons 1, 2, 3, and 4 is mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-4. In one embodiment, one of exons 1, 2, 3, 4, and 5 is mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-5. In one embodiment, one of exons 1, 2, 3, 4, 5, and 6 is mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-6. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, or 7 is mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-7. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, or 8 is mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-8. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, or 9 is mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-9. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 is mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-10. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 is mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-11. In one implementation, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 is mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-12.In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 is mutated, and the target intron is intron 19, 22, 23, or 24, with the coding domain including functional ABCA4 exons 1-13. In another embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 is mutated, and the target intron is intron 19, 22, 23, or 24, with the coding domain including functional ABCA4 exons 1-13. In yet another embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 is mutated, and the target intron is intron 19, 22, 23, or 24, with the coding domain including functional ABCA4 exons 1-14. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 is mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-15. In another embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 is mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-16. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 is mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-17. In another embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 is mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-18. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 is mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-19. In another embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-20.In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-21. In another embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-22. In one embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 is mutated, the target intron is intron 23 or 24, and the coding domain includes functional ABCA4 exons 1-23. In another embodiment, one of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 is mutated, the target intron is intron 24, and the coding domain includes functional ABCA4 exons 1-24.

[0261] Alternatively, in cases where the mutation is located on the 3' portion of the target gene, for example, regardless of the location of the mutation on another allele, a 3' trans-splicing molecule is selected to correct the mutation. 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 includes 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 mutated, the target intron is intron 22 or 23, and the coding domain includes functional ABCA4 exons 24-50. In another embodiment, one of exons 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 25-50. In one embodiment, one of exons 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 26-50. In another embodiment, one of exons 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 27-50. In one embodiment, one of exons 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 28-50. In another embodiment, one of exons 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 29-50.In one embodiment, one of exons 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 30-50. In another embodiment, one of exons 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 31-50. In one embodiment, one of exons 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 32-50. In another embodiment, one of exons 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 33-50. In one embodiment, one of exons 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 34-50. In another embodiment, one of exons 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, 23, or 24, and the coding domain includes functional ABCA4 exons 35-50. In one embodiment, one of exons 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 36-50. In another embodiment, one of exons 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 37-50. In one implementation, one of exons 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 38-50.In one embodiment, one of exons 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 39-50. In another embodiment, one of exons 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 40-50. In yet another embodiment, one of exons 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 41-50. In one embodiment, one of exons 42, 43, 44, 45, 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 42-50. In another embodiment, one of exons 43, 44, 45, 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 43-50. In yet another embodiment, one of exons 44, 45, 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 44-50. In one embodiment, one of exons 45, 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 45-50. In another embodiment, one of exons 46, 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 46-50. In another embodiment, one of exons 47, 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 47-50. In yet another embodiment, one of exons 48, 49, or 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 48-50. In one embodiment, one of exon 49 or 50 is mutated, the target intron is intron 22, 23 or 24, and the coding domain includes functional ABCA4 exon 49 or 50. In one embodiment, exon 50 is mutated, the target intron is intron 22, 23 or 24, and the coding domain includes functional ABCA4 exon 50.

[0262] A single trans-splicing molecule used to correct multiple mutations

[0263] The method of the present invention includes selecting a single trans-splicing molecule based on the location of the mutation in ABCA4 in each allele of the subject when two mutations are located in the 5' or 3' part of a gene, thereby enabling the single trans-splicing molecule that can be packaged in an AAV vector to cross the two mutations and thus correct the two mutations.

[0264] For example, in cases where both mutations occur in the 5' portion of the target gene, a 5' trans-splicing molecule is selected to correct both mutations. In one embodiment, the mutated exon is exon 1 (i.e., both mutations are in exon 1), the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exon 1. In one embodiment, exon 1 and / or exon 2 are mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1 and 2. In one embodiment, one or both of exons 1, 2, and 3 are mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-3. In one embodiment, one or two of exons 1, 2, 3, and 4 are mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-4. In one embodiment, one or two of exons 1, 2, 3, 4, and 5 are mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-5. In one embodiment, one or two of exons 1, 2, 3, 4, 5, and 6 are mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-6. In one embodiment, one or two of exons 1, 2, 3, 4, 5, 6, or 7 are mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-7. In one embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, or 8 are mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-8. In another embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, or 9 are mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-9. In yet another embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 are mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-10. In one implementation, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 are mutated, the target intron is intron 19, 22, 23 or 24, and the coding domain includes functional ABCA4 exons 1-11.In one embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 are mutated, and the target intron is intron 19, 22, 23, or 24, with the coding domain including functional ABCA4 exons 1-12. In another embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 are mutated, and the target intron is intron 19, 22, 23, or 24, with the coding domain including functional ABCA4 exons 1-13. In yet another embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 are mutated, and the target intron is intron 19, 22, 23, or 24, with the coding domain including functional ABCA4 exons 1-13. In one embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 are mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-14. In another embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 are mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-15. 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 mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-16. In another 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 mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-17. 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 mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-18. In another embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 are mutated, the target intron is intron 19, 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-19.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 mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-20. In another embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 are mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-21. 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 mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 1-22. In another embodiment, one or two of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 are mutated, the target intron is intron 23 or 24, and the coding domain includes functional ABCA4 exons 1-23. In one implementation, 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 mutated, the target intron is intron 24, and the coding domain includes functional ABCA4 exons 1-24.

[0265] Additionally, if both mutations occur in the 3' portion of the target gene, a 3' trans-splicing molecule is selected to correct both mutations. In one embodiment, one or both 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 includes functional ABCA4 exons 23-50. In one implementation, 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 mutated, the target intron is intron 22 or 23, and the coding domain includes functional ABCA4 exons 24-50. In one implementation, 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 mutated, the target intron is intron 22, 23, or 24, and the coding domain includes 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 mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 26-50. In another embodiment, one or two of exons 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 27-50. In one implementation, 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 mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 28-50.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 mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 29-50. In another 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 mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 30-50. 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 mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 31-50. In another 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 mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 32-50. 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 mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 33-50. In another 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 mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 34-50. 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 mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 35-50. In another embodiment, one or two of exons 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 36-50.In one embodiment, one or two of exons 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 37-50. In another embodiment, one or two of exons 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 38-50. In one embodiment, one or two of exons 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 39-50. In another embodiment, one or two of exons 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 40-50. In yet another embodiment, one or two of exons 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 41-50. In one embodiment, one or two of exons 42, 43, 44, 45, 46, 47, 48, 49, or 50 are mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 42-50. In another embodiment, one or two of exons 43, 44, 45, 46, 47, 48, 49, or 50 are mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 43-50. In yet another embodiment, one or two of exons 44, 45, 46, 47, 48, 49, or 50 are mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 44-50. In one embodiment, one or two of exons 45, 46, 47, 48, 49, or 50 are mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 45-50. In another embodiment, one or two of exons 46, 47, 48, 49, or 50 are mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 46-50.In one embodiment, one or both of exons 47, 48, 49, or 50 are mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 47-50. In another embodiment, one or both of exons 48, 49, or 50 are mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 48-50. In another embodiment, one or both of exons 49 or 50 are mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exons 49 or 50. In yet another embodiment, exon 50 is mutated, the target intron is intron 22, 23, or 24, and the coding domain includes functional ABCA4 exon 50.

[0266] Two trans-splicing molecules used to correct multiple mutations

[0267] Furthermore, this paper provides a method for correcting multiple mutations within the ABCA4 gene using two trans-splicing molecules (a 5' trans-splicing molecule and a 3' trans-splicing molecule). In some embodiments, the entire ABCA4 gene is substituted upon binding of the two trans-splicing molecules; for example, the 5' trans-splicing molecule and the 3' trans-splicing molecule bind to the same target ABCA4 intron and substitute for exons located upstream and downstream of the target intron, respectively.

[0268] For example, in some embodiments of the invention, the 5' trans-splicing molecule and the 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, the 5' trans-splicing molecule and the 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 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 above combinations of the 5' and 3' trans-splicing molecules can be included in a pharmaceutical composition (e.g., a single pharmaceutical composition comprising the two molecules, which can be pre-prepared or mixed prior to administration, e.g., as part of a kit).

[0269] Dosage, monitoring and combination therapy

[0270] The effective concentration of recombinant adeno-associated virus carrying the trans-splicing molecule described herein is approximately 10-1 ppm. 8 Up to 10 13 Between vector genomes (vg / mL). The rAAV infection units were measured according to the description in McLaughlin et al. J. Virol. 1988, 62:1963. In one embodiment, the concentration range is 10. 9 -10 13 vg / mL. In another embodiment, the effective concentration is approximately 1.5 × 10⁻⁶ vg / mL. 11 vg / mL. In one embodiment, the effective concentration is approximately 1.5 × 10⁻⁶ vg / mL. 10 vg / mL. In another embodiment, the effective concentration is approximately 2.8 × 10⁻⁶ vg / mL. 11 vg / mL. In another embodiment, the effective concentration is approximately 5 × 10⁻⁶ vg / mL. 11 vg / mL. In another embodiment, the effective concentration is approximately 1.5 × 10⁻⁶ vg / mL. 12 vg / mL. In another embodiment, the effective concentration is approximately 1.5 × 10⁻⁶ vg / mL. 13 vg / mL.

[0271] The aim is to use the lowest effective dose of the virus (total number of genome copies delivered) to reduce the risk of adverse effects, such as toxicity and other problems related to ocular administration, such as retinal dysplasia and detachment. An effective dose of recombinant adeno-associated virus carrying the trans-splicing molecule described herein is approximately 10 [units of measurement missing] per dose (i.e., per injection). 8 Up to 10 13 One vector genome (vg). In one implementation, the dose is 10 9 and 10 13 Between vg. In another embodiment, the effective dose is approximately 1.5 × 10⁻⁶. 11 vg. In another embodiment, the effective dose is approximately 5 × 10⁻⁶. 11 vg. In one embodiment, the effective dose is approximately 1.5 × 10⁻⁶. 10 vg. In another embodiment, the effective dose is approximately 2.8 × 10⁻⁶. 11 vg. In another embodiment, the effective dose is approximately 1.5 × 10⁻⁶. 12 vg. In another embodiment, the effective concentration is approximately 1.5 × 10⁻⁶. 13 vg. Other doses within these ranges or in other units may be selected by the attending physician taking into account the physical condition of the subject being treated, including the subject's age; the composition being administered and the specific condition; the target cells and the degree of disease development (if progressive).

[0272] The composition can be delivered in volumes of about 50 μL to about 1 mL, including all quantities 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 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 approximately 850 μL. In yet another embodiment, the volume is approximately 1,000 μL.

[0273] In one embodiment, the volume and concentration of the rAAV composition are selected such that it affects only certain anatomical regions containing target cells. In another embodiment, a larger volume and / or concentration of the rAAV composition is used to reach a larger portion of the eye. Similarly, the dosage is adjusted for application to other organs.

[0274] In another embodiment, the present invention provides a method for preventing or inhibiting loss of photoreceptor function or increasing photoreceptor function in a subject. The composition may be administered before or after the onset of disease. For example, photoreceptor function may be assessed using functional studies conventional in the art, such as ERG or visual field measurements. As used herein, “loss of photoreceptor function” refers to a reduction in photoreceptor function compared to a normal, disease-free eye or the same eye at an earlier time point. As used herein, “increased photoreceptor function” refers to improved photoreceptor function or an increase in the number or percentage of functional photoreceptors compared to a diseased eye (with the same eye disease), the same eye at an earlier time point, the untreated portion of the same eye, or the contralateral eye of the same subject.

[0275] For each method described, the treatment can be used to prevent further damage or salvage tissue with mild or advanced disease. As used herein, the term "salvage" refers to preventing the progression of disease, preventing the spread of damage to undamaged cells, or improving damage to damaged cells.

[0276] Therefore, in one embodiment, the composition is applied before the onset of disease. In another embodiment, the composition is applied before the development of symptoms. In yet another embodiment, the composition is applied after the development of symptoms. In still another embodiment, for example, the composition is applied when less than 90% of the target cells are active or remain compared to a reference tissue. In yet another embodiment, for example, the composition is applied when more than 10% of the target cells are active or remain compared to a reference tissue. In another embodiment, the composition is applied when more than 20% of the target cells are active or remain. In yet another embodiment, the composition is applied when more than 30% of the target cells are active or remain.

[0277] In yet another embodiment, any of the above methods are performed in combination with another or a second therapy. This therapy can be any therapy now known or not yet known that helps prevent, halt, or improve these mutations or defects or any effects associated therewith. The second therapy can be administered before, simultaneously with, or after the administration of the aforementioned trans-splicing molecules. In one embodiment, the second therapy relates to a non-specific method for maintaining retinal cell health, such as the administration of neurotrophic factors, antioxidants, or anti-apoptotic agents. Non-specific methods can be achieved by injecting proteins, recombinant DNA, recombinant viral vectors, stem cells, fetal tissue, or genetically modified cells. The latter may include encapsulated transgenic cells.

[0278] In another embodiment, the method includes performing functional and imaging studies to determine the efficacy of the treatment. These studies include electroretinography (ERG) and in vivo retinal imaging, as described in U.S. Patent No. 8,147,823, International Patent Publication Nos. WO 2014 / 011210 or WO2014 / 124282, which are incorporated herein by reference. Additionally, visual field studies, visual field measurements and micro-visual field measurements, motion tests, visual acuity and / or color vision tests may be performed.

[0279] In some implementations, non-invasive retinal imaging and functional studies are desired to identify preserved photoreceptor regions as therapeutic targets. In these implementations, clinical diagnostic tests are employed to determine one or more precise locations for subretinal injections. These tests may include ERG, visual field measurements, topographic mapping of retinal layers and measurement of the thickness of each layer via confocal scanning laser ophthalmoscopy (cSLO) and optical coherence tomography (OCT), topographic mapping of cone density via adaptive optics (AO), functional eye examinations, etc. Given the imaging and functional studies, in some embodiments, one or more injections are performed in the same eye to target different regions of the preserved photoreceptors.

[0280] For use in these methods, the volume and viral titer of each injection are determined separately and may be the same as or different from other injections performed in the same or contralateral eye. In another embodiment, a single, larger volume injection is performed to treat the entire eye. The dosage, administration, and protocol can be determined by the attending physician in accordance with the teachings of this disclosure.

[0281] Example

[0282] This invention is at least in part based on the applicant's discovery that specific introns of ABCA4 and specific regions within these introns provide efficient binding sites for the binding domains of trans-splicing molecules and effectively mediate trans-splicing. The applicant has generated a series of simulated trans-splicing molecules with 150-nucleotide binding domains designed to hybridize to corresponding 150-base-pair binding site sequences of the following introns: (i) target ABCA4 introns (introns 19 and 22-24) and (ii) target CEP290 introns (introns 26-30). Each binding domain in the ABCA4 and CEP290 series is designed to overlap by 140 nucleotides, allowing each intron to be scanned in 10-nucleotide increments between each sequence of tested binding domains. The trans-splicing efficiency across each binding domain of each ABCA4 intron 19 and 22-24 and CEP290 intron 26-30 was quantified. Example 1 describes the ABCA4 screening, and the results are shown in... Figure 1-8 Example 2 describes the screening of CEP290, and the results are shown in... Figure 21-26 .

[0283] Example 1. ABCA4

[0284] This embodiment describes the development of ABCA4 trans-splicing molecules, such as developing ABCA4 cell lines for testing trans-splicing molecules by screening for effective binding sites within specific ABCA4 introns, and testing various ABCA4 trans-splicing molecules to restore ABCA4 protein expression.

[0285] Binding site screening

[0286] Screening of a series of binding domains configured to bind ABCA4 intron 19 (SEQ ID NO: 25) at sequential binding sites revealed a region of the 3' portion of ABCA4 intron 19 that is preferentially active in the trans-splicing of 5' trans-splicing molecules—the region of nucleotides 990 to 2,174 of intron 19. Figure 2Binding sites in the range of nucleotides from 1,670 to 2,174, from 1,810 to 2,000, from 1,870 to 2,000, or from 1,920 to 2,000 have been shown to be particularly efficient in mediating the 5' trans-splicing of intron 19.

[0287] Similarly, suitable binding sites for the 5' trans-splicing molecule within intron 22 were identified. Figure 3 The binding sites in intron 22, ranging from nucleotides 1 to 150 or nucleotides 880 to 1,350, are particularly effective compared to the rest of the intron.

[0288] Figure 4 The results of a similar screening for ABCA4 intron 22 (SEQ ID NO: 28) of 3' trans-splicing molecules are shown. Binding sites with nucleotides 60 to 570, 600 to 800, or 900 to 1350 were identified as preferentially suited for trans-splicing of 3' trans-splicing molecules. In particular, binding domains targeting binding sites in the nucleotide range of 70 to 250 were highly effective in 3' trans-splicing.

[0289] Within intron 23, the relatively effective binding sites for the 5' trans-splicing molecule are those binding sites in the range of nucleotides 80 to 570 or 720 to 1,081 of SEQ ID NO: 29. Figure 5 For 3' trans-splicing molecules, the binding sites are particularly efficient, including those within nucleotides 80 to 1,081 of SEQ ID NO: 29 (e.g., nucleotides 230 to 1,081 of SEQ ID NO: 29, nucleotides 250 to 400 of SEQ ID NO: 29, or nucleotides 690 to 850 of SEQ ID NO: 29), such as... Figure 6 As shown.

[0290] Similar screening at intron 24 (SEQ ID NO: 30) of ABCA4 showed that binding sites in the range of nucleotides 600 to 1,250 or 1,490 to 2,660 are effective in 5' trans-splicing. Figure 7 In particular, the binding sites in the range of 1,000 to 1,200 nucleotides exhibited the highest 5' trans-splicing efficiency. Figure 8 Results of screening for 3' trans-splicing efficiency were shown, revealing that binding sites in the range of nucleotides 1 to 250, 300 to 2,000, or 2,200 to 2,692 (particularly binding sites in the range of nucleotides 750 to 1,110) were the most efficient.

[0291] ABCA4 cell line

[0292] First, cell lines expressing ABCA4 were generated. The ABCA4 gene is known to be expressed only in the active photoreceptors of the retina, and the full-length ABCA4 pre-mRNA and protein are typically undetectable in cultured cells. Therefore, to test the trans-splicing strategy of ABCA4, cells were engineered to express ABCA4 from its native genomic locus on chromosome 1 (1p22.1). Two strategies were employed. In the first case, stable cell lines expressing site-specific (upstream of the ABCA4 transcription start site) DNA-binding TALEN fused with the VP64 viral transactivator were derived. In the second case, a constitutive eukaryotic promoter was directly inserted (using CRISPR / Cas9) into the genomic locus immediately upstream of the ABCA4 transcription start site. The result in both cases was stable cell lines strongly expressing ABCA4 pre-mRNA and protein.

[0293] TALEN cell line

[0294] Design a TALEN targeting a specific domain upstream of the ABCA4 transcription start site and fuse it with the VP-64 transactivator sequence. Figure 9 This combination of three TALENs was transfected into 293 cells, and stable single-cell clones were obtained. Two clones were shown to direct the expression of the ABCA4 protein. Figure 10 ).

[0295] CAG promoter cell lines

[0296] exist Figure 11-13 This paper outlines general strategies for obtaining CAG promoter cell lines. It also includes a site-specific wizard for designing such a wizard. Figure 12A To use homologous arms to insert the CAG promoter and puromycin selection marker ( Figure 12B Purine-resistant cells were cloned, and the required insertions were analyzed by PCR. Several clone lines were selected for further analysis. Figure 14A and 14B The image shows RNA and protein expression in two cell lines (B6 and C3). As confirmed by RNA and protein analysis, both cell lines clearly contain promoter insertions.

[0297] ABCA4 knockout cell line

[0298] Once stable ABCA4 expression is established in cultured cells, knockout ABCA4 expression is generated to test ABCA4 trans-splicing molecules designed to restore ABCA4 protein expression. Typically, guide RNA and Cas9 protein are co-transfected into B6 cells (knocking the CAG promoter into the ABCA4 locus and mediating ABCA4 expression). A second transfection with guide RNA and Cas9 protein is performed after 9 days. The basic design targeting exons 3 and 4 is shown... Figure 15 In this study, single cells were plated using limiting dilutions, and once grown, ABCA4 protein expression was assessed by Western blotting.

[0299] Figure 16 The RNA and protein profiles of single-cell clones obtained after CRISPR / Cas9 treatment are shown, such as... Figure 15 As shown. The degree of RNA and protein excision differs. Clones 17+06 and 17+21 were selected because they exhibited complete ABCA4 protein knockout. Mutation analysis ( Figures 17A-17B (18) confirmed that exons 3 and 4 were targeted and interrupted.

[0300] ABCA4 trans-splicing-mediated protein repair

[0301] Based on the high-throughput binding site screening described above, eight trans-splicing molecules were selected. The methods and results of these studies are described below.

[0302] method

[0303] For Western blot assays, 17+06 or 17+21 cells were seeded in each well of a 12-well plate at a density of 10⁶ cells per well. Each well was transfected with 1 μg of plasmid (RTMx). After 48 hours, cells were collected and membrane preparations were processed using the Mem-PER Plus Membrane Protein Extraction Kit (Thermo Fisher 89842) with 1x HALT™ protease and phosphatase inhibitor mixture (Thermo Fisher 78440) added to all buffers according to the manufacturer's instructions, for analysis via standard Western blot assays. RNA was also processed for analysis, as described below. Membrane lysis buffer was denatured at room temperature with 4x Laemmli sample buffer (Biorad 161-0737) containing 10% reducing agent TCEP 0.5M (Sigma 646547) for 30 minutes. Samples were run on NuPage Precast 3-8% Tris-Acetate gels (ThermoFisher) and proteins were transferred using iBlot 2 Mini PVDF Transfer Packs – run at 25V for 10 minutes. The primary antibody for ABCA4 was Abcam ab72955, a rabbit polyclonal antibody (dilution 1:2500). The secondary antibody was an anti-rabbit antibody (dilution 1:5000). The blot was exposed for different times depending on the signal intensity.

[0304] For qPCR of RNA samples, RNA was harvested using the RNeasy Plus Mini kit (Qiagen) as described above for qPCR analysis. cDNA was synthesized from 400 ng of RNA in 20 μl of reaction using SuperScript IV VILO Master Mix (Thermo 11756500; diluted 1:4 in water). Native ABCA4 (Thermo commercial assay Hs00979594_m1) spans exons 49-50. Housekeeping genes were used as controls, and RNF20 assay (Thermo commercial assay Hs00219623_m1) was performed. The qPCR primers and probes for the chimeric ABCA4 codon-optimized exon 22-native exon 23 are as follows:

[0305] Probe (FAM)063_ABCA4 co22n23_P1:

[0306] CGTGGACCCTTACAGCAGAAG

[0307] Forward primer 064_ABCA4 co22n23_F1:

[0308] GATCCTGGATGAGCCTAC

[0309] Reverse primer 065_ABCA4 co22n23_R1:

[0310] GGACATGATGATGGTTCTG

[0311] The following are double-stranded qPCR primers and probes with RNF20 assay:

[0312] Probe (VIC)088_RNF20_P2:

[0313] CAGCGACTCAACCGACACTT

[0314] Forward primer 091_RNF20_F2:

[0315] GCAGTGGGATATTGACAA

[0316] Reverse primer 099_RNF20_R5:

[0317] CGAGCATTGATAGTGATTG

[0318] PCT reactions were performed using QuantiFast 2x qPCR Mastermix.

[0319] result

[0320] Trans-splicing molecules binding to introns 19, 22, 23, and 24 were tested. No protein restoration was observed in trans-splicing molecules binding to introns 19 and 24 (data not shown), but trans-splicing molecules binding to introns 22 and 23 resulted in restoration of ABCA4 protein and RNA expression. Figure 19A and 19B (This will be discussed below.)

[0321] Figure 20A This is a Western blot, which shows that the expression of ABCA4 protein is attributed to trans-splicing from two different cell lines (17+06 and 17+21), where a simulated GFP control or 5'A4In22 is tethered to five different binding domains (non-binding domain (NBD) control, 92, 99, 105, 118, and 121, numbered corresponding to...). Figure 3RTM#, in which binding domain 92 binds nucleotides 911-1060 to intron 22, binding domain 99 binds nucleotides 981-1130 to intron 22, binding domain 105 binds nucleotides 1041-1190 to intron 22, binding domain 118 binds nucleotides 1171-1320 to intron 22, and binding domain 121 binds nucleotides 1201-1350 to intron 22 (following a 10-base shift interval across the 150-unit polymer on intron 22, as described above)). Four of these intron 22-binding constructs, 99, 105, 118, and 121, yielded protein recovery, with 105, 118, and 121 showing particularly enhanced recovery, while 118 showed the highest amount of protein expressed in both cell lines. mRNA expression profiles showed a similar pattern, with the 118 construct producing the highest levels of ABCA4 mRNA in both cell lines. Figure 20B The unit is relative to the RNF20 housekeeping gene.

[0322] Figure 20C This is a Western blot showing ABCA4 protein expression, attributed to trans-splicing from two different cell lines (17+06 and 17+21), where the simulated GFP control or 5'A4In23 was tethered to three different binding domains (NBD control, 27, 81, and 85, numbered accordingly). Figure 5 RTM#, in which binding domain 27 binds nucleotides 261-410 to intron 23, binding domain 81 binds nucleotides 801-950 to intron 23, and binding domain 85 binds nucleotides 841-990 to intron 23 (following a 10-base shift interval across the 150-unit polymer on intron 23, as described above). As indicated by the amount of protein expressed in both cell lines, all three intron 23-binding constructs produced trans-splicing. mRNA expression profiles yielded similar results, with all three constructs producing robust ABCA4 mRNA expression in both cell lines. Figure 20D The unit is relative to the RNF20 housekeeping gene.

[0323] In summary, the ABCA4 protein and RNA expression data obtained from trans-splicing molecules binding introns 22 and 23 are correlated with the aforementioned binding domain screening. Specifically, intron 22 binding constructs 105, 118, and 121 and intron 23 binding constructs 27, 81, and 85 are predicted to bind efficiently. Figure 3 and 5Furthermore, current ABCA4 protein recovery data suggest that ABCA4 intron regions containing these construct binding sites are suitable for binding to ABCA4 trans-splicing molecules, thereby conferring protein and RNA reduction capabilities. In this embodiment, 10-20% of protein expression was restored, which is comparable between trans-splicing molecules binding introns 22 and 23. Importantly, since ABCA4-related diseases (e.g., Stargardt's disease) are latent, asymptomatic carriers of these diseases may express lower levels of ABCA4 than normal, and it is undesirable to be bound by theory, as shown here, that partial protein repair may confer meaningful clinical benefits.

[0324] Example 2. CEP290

[0325] A series of binding domains configured to bind CEP290 intron 26 (SEQ ID NO: 85) at sequential binding sites were screened, showing that the 3' region of CEP290 intron 26 was preferentially suited to the region of nucleotides 4,980 to 5,838 of the 5' trans-splicing molecule (trans-splicing of intron 26). Figure 22 The study revealed that binding sites in the range of nucleotides 5,348–5,838, 5,348–5,700, 5,400–5,600, 5,460–5,560, or 5,500 are particularly effective in mediating trans-splicing.

[0326] Figure 23 The results of similar screening for CEP290 intron 27 (SEQ ID NO: 86) are shown. Binding sites identified with nucleotides 120 to 680, 710 to 2,200, and 2,670 to 2,910 are preferentially suited for trans-splicing of 5' trans-splicing molecules. In particular, binding domains targeting binding sites in the range of nucleotides 790 to 2,100, 1,020 to 1,630, or 1,670 to 2,000 are highly efficient in trans-splicing.

[0327] In intron 27 (SEQ ID NO: 87), binding sites in the range of nucleotides 1 to 390 (e.g., nucleotides 1 to 200), nucleotides 410 to 560, or nucleotides 720 to 937 are identified as having relatively high trans-splicing efficiency. Figure 24 ).

[0328] Intron 28 (SEQ ID NO: 88) was similarly characterized and showed relatively effective binding sites in nucleotides 1 to 600, nucleotides 720 to 940, or nucleotides 1370 to 1790. Figure 25 ).

[0329] In intron 29 (SEQ ID NO: 89), the 3' portion of the intron is more efficient at mediating 5' trans-splicing compared to the rest of the intron. Figure 26 In particular, binding domains targeting binding sites in the range of nucleotides 95 to 1,240, such as nucleotides 1,060 to 1,240, exhibit the greatest trans-splicing efficiency. sequence list <110> Spotlight Biotechnology Co., Ltd. University of Pennsylvania Board of Trustees <120> trans-splicing molecules <130> 51219-016WO2 <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 constructs <400> 1 gtaagagagc tcgttgcgat attat 25 <210> 2 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 2 tactaactgg tacctcttct tttttttctg cag 33 <210> 3 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 3 tggtacctct tcttttttttt ctg 23 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 4 agatctcgtt gcgatattat 20 <210> 5 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 5 gagaacatta ttatagcgtt gctcgag 27 <210> 6 <211> 135313 <212> RNA <213> Homo sapiens <400> 6 aguccccagu cuuugcuuag gccccuacgu acacaaacug aaccuaguga cccagcaugg 60 ccucuaauuu cucaacacuu cuguacuucu guaaugaua acccaugcuu cucacagauc 120 caugccccaa auuucuguga auaggcccug acuggcccag cuaagaucau gugacugcac 180 augaccaguc cacuuuggca uuaacaagcc uacugcagac ucuucccuug guguuggagu 240 cacuccuaga aaagagcaaa ucuuugagag ccaggcaguc aaccugcugg cagcuuccac 300 ucagccuugg aguuuuucu auguguaacu uucauaaacu gagccuuuuau uauuuauuuu 360 420 480 caaauuuaaa aaucagauau uuuucaucuu acauuaugau gucccaaaac ugccuuuaug 540 cuugugacau agauucauaa ugucuucuca uuccaccugu aaucacuuguu ugaaauaaac 600 auugucuaa ugauaauuuuu ggggacauuc uauuuucuuc agcuuguugc aagugaauug 660 auggugaucu uuugguauug guuucauuau caaauuuauc uccacuccaa aauuacagua 720 auuucaaagu aauuuagucu aauauuuuu ccauagcuuu ucuuccaau agaaacugua 780 aaaaguuaua aauuacuucu cuccacuacu gaauuuugu uugcagaaua acugauguaa 840 guagcagaau gccucuccu aguucaaccc ucaggaauag aagugagaag aucuuaaaa 900 cuucaccauu uuccuugacu uguuuuaau ucugaaugua aaugugaauu gauauggucu 960 1020 gaccacauca auuucauauu cacccugauc auuguauauu auggugaua acuauugaa 1080 ugaauguac aguaucagu aucauuuuug acucacuagg uauauccuca gaauauauug 1140 aaaaaaacuaa acacagcuuu uaaccuuugaca uaauuuuuaa acaacuggag uaaccuuggg 1200 agaaaaaucc uaccaaauau cuaaaaauu gaagaguaa aaagagua auuguccuua 1260 acaucauuaa ucauuagga caugcaauc aaaaccacag ugaaauacca ucucacaccc 1320 uuuaggaugg uggugauaag aaaaaaaaaaaaaaaaaaaggcc aggauggc 1380 aaagcuggaa ccauaggaa ccauaggaaaaagcuagga 1440 auaguauggu aguucuuca aaaaaaaaaaaaaccau uugauucagc aguuucacuc 1500 cuaguauau acccaaaga auugaaagca gaucucaa uauuuguaca cuauguaca 1560 uagcagcauu acucacaaua gccaaaggu ggaaaaacc cgaaugaccc uggauggacg 1620 Aooooaaaaaaaugagg ucuaaacuga caaaaaaaaaaaaaaaaagg uaaaaaagg 1680 aagaaauucu ggccgggcac gguggcucac accuguaaua ccagcacuuu gggaggccaa 1740 ggcaggcaga ucaccugagg uugggaguuu gagaccagcc ugaccacau ggagaaaccu 1800 cauaucuacu uaaaauaaaaaaaaaaaau uagccaagca ugguggcgcc ugccuguauau 1860 Cccagguacu Caguaggcug Aggcagga Aucgcuugaa Caggaagcag Aggugcau 1920 gagcugagau ugcacauca cacuccagcc ugggacaaa gagugaacu gcaucauca 1980 aaaaaaaaaaaaaaaaaaaaaaaaaocugaca caucugcuau ggucuaaau 2040 auguguccu quaaaaaaaaaaaaaaaaaaacccc caaggugaug guauaggag 2100 gugaggcuuu guggagguga ouaggucaug aggguacaac ccucgugaau gggacuagug 2160 cccucauaaa aagaagccca agagagaccc cuuucccuu ccacuggaug aggucacacc 2220 aagaaguuac caucuacaug ucaggaaaua ggcccucacc agacaccaaa ucuauuggca 2280 ccuugaucuu ggacuuccca gccugcaag cugugagaaa uaaguuccug uuguuuauaa 2340 accaccagu uuauggau uuuuuuuagc accucaaca gauuaagaug gcuugcuaca 2400 acauagauga acuuaaaga uuaugcuuuu acouagauucc acuagaugg gguaccaaga 2460 2520 cuggggaguu aguguuuaau ggauacagag uuucaguuuu gcaaaaugaa aaaguucuga 2580 2640 uaaaauaagg uucaaaugau acauuuuauu ucaugugugu gucaaucuca acaaacagau 2700 uuguucaggc aaggaaacug guuagaugcg aauaauacua uuagagcauc aucaauugaa 2760 uauuaacaaa gugcucauag uuuaacuuuc uagcucaagg aagaauggac cauuuugaaa 2820 cuaugacaga acauuacuua uaagcugau gucuuuggga auugggaaagga ggcauauucc 2880 uucacagcu guggcucccc uucagcaacc ucauauacuc uccaagcuuc uuuuccugg 2940 gucaccuguu uaaucacucc cgggacuuaa ucuuccaccu auauguugac cacucacaaa 3000 3060 ugaauacgug uggucagaug ucauagaacu ucagcuucag uaauacaaau gcaaacccu 3120 guuccccca acugccuccu acuccccacu ggccuuccuc uggcauuccc uccucaguua 3180 ugagcaccac cgucucacua gccagccagu caagccccaa acuccaucua gcugacuucu 3240 gccucuuccu caccacccuc uuccaguaac ucaucaggca cugcuguguc ucauuccuuc 3300 cuaucccucc agucccuccc cuucucucca ucauggcugu cacugcaugg uucaggcucu 3360 cuggcucccc ccaaaccacc cccacauugc ugccgaggug aacugacuac ucuuggcagc 3420 cacuggauua aaaucuuuca ucaucuucag caugauaaaa cccauauccu uuagcaugua 3480 acaaggucuu aaugauucug ccagagcuug cuugggggua gccugcacuu gugggccacu 3540 ccagucacuu cacaggugcu caguaaaucu caguugaauc agucaucauc aucaucauca 3600 ucaucaucau caucaucauc aucaauuuuu cagucugguu ccugucuccu uuuccagcau 3660 ccuccauuca uagccucaua gccuucacuc cagccauguu ucacuugugg uuuuccuggg 3720 caagauaagc uauuccuccc ugucuuugca gaguuuaaau gacucacuug uucaaguacc 3780 caccguugcc augugggacc gugagcaaag uacuuaaucu cacuaagcuu cacguuccuc 3840 aucuguaaaa cagcaaauau ggaccucaca aaauuguagu gaggcuaaaa ugaaauaaca 3900 uaugcaaag caguuuaaaaaaaaaacu uauuaaaaa uauuuuuug uaauuucgca 3960 agcuugucuu aaugccauc accuccaagg agccuuuuug cacauaag cagaacuau 4020 cucucuuc uuggaagcuc cucucuc cuccuugcac cuccuuuggg cucucuc cuccuugag 4080 uuauucgagu ucaagucccg uguuuacaac cagaccgcaa acucuauugaa icagcaucc 4140 auuccucucu gugguucucc cuccgcccca uccaggucuc aagggucuag agucuuuca 4200 agagacaca uucugagauu ugaggaggca gagahaaaa guuccacugc gagugccag 4260 ggaggcuucu guuuggggug ucccuugga ucacagaucc cccaccuggu gaugagucaa 4320 cccagcacca ccccauugca gggcuggaau gabaguaaug ggcccaccug cugccucucc 4380 ucauacccgc accccaguca cavauugcaa gugucacg gcucuguccu gcugggccug 4440 gaguguucca gugccuuuuc caucacagca ccaagcagcc acuacuaguc gaucauuuc 4500 agcacaagag auaaacauca uuacccug cuaagcucag agauaaccca acuagcugac 4560 cauaugacu ucagucaua cggagcaua uaaaagacua aaagagggag ggaucacuuc 4620 agaucugccg agugagucga uuggacuuaa agggccaguc aaacccugac ugccggcuca uggcaggcuc uugccgagga caaaugccca gccuauauuu augcaaagag auuuuguucc aaacuuaagg ucaaagauac cuaaagacau cccccucagg aaccccucuc auggaggaga 4860. 4860. 4860. 4860. 4860. 4860. 4860. 4860. 4860. 4860. 4860 acuugugucu uaaggguucu cuuucucucc auaaaaggga gccaacacag ugucggccuc cucucccca cuaagggcu auguguaauu aaaagggauu augcuuugaa ggggaaagu agccuuuaau caccaggaga aggacacagc guccggagcc agaggcgcuc uuaacggcgu uuauguccuu ugcugucuga ggggccucag cucugaccaa ucuggucuuc guguggucau uagcaugggc uucgugagac agauacagcu uuugcucugg aagaacugga cccugcggaa 5220. aaggcaaaag guaacaguua cugucugugg uuuaaaaaug agguguggag caaauaaaca gggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggg 5340. gcagucagug ggcuugucgc cgauuagcac ugaagcagug uuuagcugga cggccuuucu gugggccccu cugacagugc ccuucccagg aagauguguu ucucuguccu cagccacaug 5400 aaaaaucuuuu gccuaccgug ccugucaauc cauugccugc ccgccccucc cccacccccc 5460 guuuuacacc ugccugucca gucuaccgcu cucuagggca uccacgcuga gcagugggaa 5520 gaacuuuaag cccugaagag caggccaaag cgaagcaaga acccccucga acagcuuccc 5580 agcuuaguga ggccuuauuu cauugauucu cugaggcaca 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aucauggaug aagcccuggg uccuguacac 74760 cuuguccagu agacuaauu gcccuauuua aaaaaggcca agccacuuca ggguucaaag 74820 aacuuuugca gcuuuucagu auaaagcaga aauccaggga aucaugaagg aaccuuugca 74880 uucaucuccc auugccuucc uugugccuuuu uuauucucuu cugccuuuc aaauauaaa 74940 uuaguuuuuu cucccaagau gaagcuccu ccuggggcug aggcagagcu guuaucuuca 75000 gggcaauacc ucagauucuc cugguguuga ucuuucuuag ggguggggaa aaaggcugaa 75060 agggcauuug cccacaacac aucuuaggua aaaggcaaccu uauacucaugga accaaacagg 75120 aggccuagcu agaaaguu cuagaagcag ggaaaagcac agacucuuu gugaggucug 75180 agaaagcaaa gaauuccag ggugaaagcg ggggacuccc cuagacuga aguacucucc 75240 caucuguuug uugcucaccu accuauucuu uacuuuguau uauugggccu gggccaggac 75300 uuauccugca agcacugaga uggauguuug uuuucucugg gggauuaguc uuuuuuuuuc 75360 uuuuuuucuu uuguuuug cuuuuguuuu cacuggguca aacaaacaac acuuuaacag 75420 cucagguauu uuucauugua uugacuuguc uaccuguaaa cuuguuaauu uuuacuauaa 75480 auaaaauau cauauauauau augaaaaau ucaacagg gcuugugggc auuuuauuuuu 75540 ucucuacaau cccaacagau acucugccuc uuaagaaaaa aagaaaucau aagaaaaua 75600 ugcuccuuca aaagugaauc acaauaugu uugccaacgg aaggcaaaua uuuuucaccu 75660 gucucauagg cuggacugaa auggauuucu aaaacucucu aaaaccagaa aagagcugag 75720 ugucuccacc caaccucccu ccuuucacag auuaaaaau aaaaaaugga gcccaggaga 75780 cauccaguau cuuccccuau uggucaccug ggacaaaauc uggaacaugc acaugcauug 75840 ccuggcagga acucauucca gugauuaaac ucuucaggag gauguuuccu cuugcuauuu 75900 cauaccuau uugugcaguu ugauagcuag uaaagugauc aaaggaacug uggggcauag 75960 auucaaagu cucaggaa gcagaaauag aagaacagua cuagaggcag caggucccug 76020 accagcaggc cacauccug cugcuccagc acacauccug cacauuuuca gagggggggg 76080 gagagagggg cccugggggg cuguugcauu gagaaucuc gcccugcucc uguaugugca 76140 cugaggccg agagcccuug gaugcugg cuckcuck cuckoo cuckoo cuckoo 76200 cucuggcag acugacuggc cucuggcuc cucuuccccu uccaggaug ccugauaucu 76260 uuuaaaacca augccaagu ogccaaaaaaaaaaaaaaaaackaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaruaaaaaaaaaaaackaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa.uaaaaaaaaaaaaaacke gouuuuuuuuuuuuuuuuuuuuuuuuuugeoooooooooooooooooooooooooooooogugugugu 76320 guucaugcg uguguuuaa ccacacuuca caauuugucc aggcuuguau uaauaccauc 76380 accaggcuca accuggugu uaauuccaag auacuuaaau gcccaucuag gugaauuucu 76440 cagguaaacc auauauuca gcuguaguuu aagcuggcug cccgucauag cacuuugaau 76500 agacuuuuu uuuuuuuuuuuuuuuuuuuuuuuuuga cagagucuca cucugucggc caggcuggag 76560 ugcaguggca cuaucucggc ucacugcaac cuccgccucc cggguucaag cgauucuccu 76620 gccucagccu ccuaguagc ugggauuaca ggugagcgcc accacccg gcuaauuuuu 76680 guauuuuuag uagauacgggg guuucaccau guuggucaga cuggucucga acuccugacc 76740 ucaugauacg ccuacauugg ccuccauag ugcugggauu acggcguga gccaccau 76800 ccggccccug auagacuuu uacucaaggu uaucacauug uuuguauugg 76860 aguaaaougu gccaguggg ggcuaaaga aauaaacuca oouchaauuc aaaccuggoo 76920 uuuuuaaauu uuuuaaaauc acaguuucg aacogugggg cuccucaugg cacauugaga 76980 ggaggagggg aaccucucca agucugaagc uccuguuaua aaucuuccuc uggcaaagou 77040 ugugugauca ggcuugagua ccucacaguc cuagaggcagg ucaaggcug gcuoggaac 77100 ucauuugcuc squirrel squirrel squirrel squirrel squirrel squirrel squirrel squirrel 77160 gguagaguaa cacuggcuuc ugauuggugc aggguguca accagagaag aagagcccu 77220 ggaaaagacc gagccccuaa cagaggaac ggaggaucca gagcacccag aaggaauaca 77280 cgguaaaacc ccgauaaaga auacacagca gaggcgagga aaggcucua agcacugcag 77340 agggchagag cuckoocu cuggshaagg guggaagaa cugacuacucucucucucu 77400 cuguggaaaacc agaucccuuc ucagaggucc aucugcaug guguggaaug 77460 aauggoucag cccagacauu agcgcauauu uccuggagaa agcaauacc aacuaguag 77520 ugugccugug cccuuguuag gcaauuccca agagaguugc acaauugugc ugacuuccga 77580 ggauuuagca agaacauaa cuuggucac ugggacuuaa agcggauaug agcuauaagg 77640 aaagacaaaa auaaaugcuu cuguguccag gggaaag acuccagggg agcugacuac 77700 acucacuua cggcuuacaa aucuagaagg acoucauug aaaccaucag aagccuuucc 77760 ugacagugga aguaccuaa uaauucccuaa acugacgacc cagauuuaca aguuuuguuu 77820 uccuggcuu ugcugcccuc aucuucucuc uuaaacuagu ucuguauuuc ucccaggcu 77880 uuucauuccc uaagcauacg cauuucucug uggccaaau gcucuggguu uagacaggca 77940 gcacagcccc uggcucugc cugacaggggc aggagggu cuggccuuua ucccuccagc 78000 ccacccagg ggcauuuca uaaaaacuaaa gccagagacc ggcagccccu ggcagaguua 78060 gacugcagua caccaugccu neighborhood cuccucca caguggaag ucuaagccaa 78120 aucaggaggc uggggacugg uuccaccuca guugxaggca aggccaggag gcacggauag 78180 aagaaacagu ggacuuuuuc ccchuaggga aagaaugcu uagagcuaca guauaagau 78240 gawaauuaa gcugugccau auagggugaa augaagcagg gauagauggg aggucaggga 78300 gaagugagag cacucgguga gggucugcac uggaggggc augggaa gaaggagggg 78360 agugggguuu gagggauggu gaugaggaag cguggacugc ccuacccacc uauuggaaaa 78420 cugggaguu cugaggagga agagccuua cugaaguca cugaggau cugaggcaag 78480 gugacuaaga gaaoggcggu ccagaaagg ucaugggaga aucugaaggc agaogoogou 78540 uagggaagau gagaaccua agccgcuucc aggaauucau gaggaaugc cccguggacu 78600 guuggcaug agggccuagg accaagguug agcuuggggc macacuccc uauagacagu 78660 gagugcauuc ugacaagcau gggcucuggg uucaauuccc aacucugcca cucaugccua 78720 78780. uguguccuua auaggacgcu ugaugucucu gugucuaagg uuuccuggac uauggaaaug agccuaauaa augucuaccc 78840. 78840. 78840. 78840. 78840. 78840. 78840; 78900. ggcauauagg uaaacguuug augcuaguau uacuauuauu auucuggagu cucumber acggugauag ccgaagccac aggggcaggu gacguuauag gcagaauaca agggccugga gacagagccc uggggccaug uaauuaggca uuauguuuac aucauguuca uuuuuuuucc uccaagacuc cucuuuga cgugagcauc cagggggg uccuggggua ugcgugaaga aucugguaaa gauuuuugag cccuguggcc ggccagcugu ggaccgucug aacaucaccu ucuacgagaa ccagaucacc gcauuccugg gccacaaugg agcuggaa accaccaccu ugugagucuu ccagcagaga agcuggcugc caugcuagcc ugucauuucc uggcuuaguc uuucccuauc agcggcuguc uacucuuucc 79380. 79380. 79380. 79380. 79380. 79380. 79380. 79380. 79380 79440. snow snow snow snow snow snow snow snow snow snow ugugugccgg ggaaguggac auucauucag agaguugag ugacuuuccu gaagccacca covered gcucagcggg ggcaaaagcc aggcaccaca covered covered 79560. covered gcucagcggg ggcaaaagcc snowflake uuccccccau snowflake snowflake aucaagucau ggccacuguc aucaugugca uggagcuau agaguccucc uauuuccuu cucuuucuu ucuuuuuuu uuuuuuuuuu uuuugagaua guaaccauua cccaugcugg agggcagugg ugcgaucuug gcucacugca accuccgccu cccaggauca agcgauucuc ccaccucagc cuccagua ggugggacua caggugcaua ccaccaugcc cagcuaauuuu uuguauuuuuu uuuuuuuuuu uuuuuuuuua guacagacag gguuucacca uguuggccag gcuggucucg aacuccugac 79980. cucaggugau cugcccgccu cagcuuccca aagugcuggg auuacaggcg ugagcgaccg caccaggccg cover uuuucaagga acauuccuuu clothes cauuaggcag gcuucaacau cagcugauga ggguuagugg ucguucugga gaaagugaa aaagaaucag ucucuagagg ggcuugugga guaaccgccu gguaacagaa ggucagggca gggaaggcaa 80160 aggggcucug cgcggaucuc ucagcuccgc aggcgcccca cucuccucca agggacccga 80220 gcgccaucug cugagaggag aacacggccc gccaugguuu cccaaggagc agcagacacg 80280 gaccucgcag ggggcagcga acccacguga cacagucuuc aaguccuuug gagagcccca 80340 ggaagaaca acagcgugua cacccuguga uggaauguuc ucuaggcgg uucaguguga 80400 auggaugug gggccggugc cauucuaauu gguucuguuu cccucuagug guugaucgcg 80460 gagauucgg cuucucauc agcaaguu cagauagccu gagaugguau cagaacucag 80520 gcagagcu ggggugggcg gcccugcauc caucugcuuu cucuccaugc uaacugaauau 80580 ggucagagag cuggaagcaa auuccaggac cccaggcuc cgcaaaggca aacacauuac 80640 uucaucggcu gcugacaugc aacuuccccc agggguuaa acaauguuua auacuaacag 80700 uaauauau uuugaguuuu acuuuuaugcu ggcgcuguuc uaauguugua aguguauuaa 80760 cucauuuaag ccuuacaaca accuaaggac augggaguca uaguucccau uuaaaaaaaa 80820 aaaaaaaaaa agcccaccau ugcucugagg cuuuuuaugu uuuggaucca aagcuaauau 80880 uggugguggu aauucccaug ccuggcuucg aucaauuaau cagcaaaugc cuaggacugc 80940 uuaggguucu ggccuucauc aagaccuuac ccgggcuuua ugaugaugac accuggcuuu 81000 ucaauagcca ugacugcuca cccaggaggc aacgccucga gucaugcacc gaacaccuuu 81060 uauugauccu cuccaacacc aggcuccgug auggcugagc uggggacacc ugugacugca 81120 cgugaacauu uugaggcugg gaaucccaaa ggcccucggc guuggccugg gagcaccaug 81180 aaacaaguag aagcagagaa ggauggcaga gguggcccuc ugcauuaggg ccuggaugua 81240 uacacuggug cuaagggggc cccacagcua auagggguuu gaguuugacu gacagcccca 81300 ggcaggaauc ugugagaguu cucacugaac cugguguggg gguggcccuc cuaaggcaug 81360 uugcuaaagg ccaucucuuc ugccacugac gccuguguuc ugcaggucca uccugacggg 81420 ucuguugcca ccaaccucug ggacugugcu cguuggggga agggacauug aaaccagccu 81480 ggaugcaguc cggcagagcc uuggcaugug uccacaguc 81540 agcgacacag gaacugagac cgcccacucc ccucuccuca ccucugccc cgcccacuu 81600 cucuagagcc cagcucaggg gugccaggcc uggcacagg cagagauaca gacuuauu 81660 ugguucccc uuuguuuaaa guccuuuguc cucuugcag ogagauugu cccugagaau 81720 augggacucu gccucugcug cucagagcug agggcuccuc ccucagaagg gugaggcugc 81780 cucgcucug acagagcagc ugaucgaucc gggagccccu ugugcagccc ugaaguacuu 81840 cucucugggg accaagaca ggagaaccau uguuccuuuu uccuguugaa gccacggccu 81900 gaaggcaaa cuuucagggg ggcuuuucag uacuuuuuu ucccauaa gauaucuuuu 81960 auuucuuauc uagaagcua cgcauaguca uugugaaga aaaaaaaagg agggagg 82...

Claims

1. A nucleic acid trans-splicing molecule comprising: operably linked in a 3' to 5' orientation: (a) A binding domain configured to bind target ABCA4 intron 23, wherein the binding domain comprises a sequence of 50-300 nucleotides in length and is complementary to a binding site of at least 50 consecutive nucleotides within nucleotides 261 to 410 or nucleotides 801 to 990 of SEQ ID NO:

29. (b) A splice structure domain configured to mediate trans-splicing, wherein the splice structure domain includes a 5' splice site; and (c) Contains the coding structure domain of functional ABCA4 exons 1-23; The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain to endogenous ABCA4 exon 24, thereby replacing endogenous ABCA4 exons 1-23 with functional ABCA4 exons 1-23 and correcting mutations in ABCA4.

2. The nucleic acid trans-splicing molecule according to claim 1, wherein the binding domain binds to the target ABCA4 intron at the 3' end of the mutation, and wherein the mutation is in any one of ABCA4 exons 1-23 or introns 1-23.

3. The nucleic acid trans-splicing molecule according to claim 1, wherein the binding domain is associated with a nucleic acid trans-splicing molecule containing... (a) Nucleotides 261 to 410 of SEQ ID NO: 29; (b) Nucleotides 801 to 950 of SEQ ID NO: 29; or (c) Nucleotides 841 to 990 of SEQ ID NO: 29 The binding sites of at least 50 consecutive nucleotides are complementary.

4. The nucleic acid trans-splicing molecule according to claim 1, wherein the binding domain is complementary to the binding sites of nucleotides 801 to 990 of SEQ ID NO:

28.

5. A nucleic acid trans-splicing molecule comprising: operably linked in a 3' to 5' orientation: (a) A binding domain configured to bind ABCA4 intron 22, wherein the binding domain comprises a sequence of 50-300 nucleotides in length and is complementary to a binding site of at least 50 consecutive nucleotides within nucleotides 880 to 1350 of SEQ ID NO:

28. (b) A splice structure domain configured to mediate trans-splicing, wherein the splice structure domain includes a 5' splice site; and (c) The coding structure domain containing functional ABCA4 exons 1-22; 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 functional ABCA4 exons 1-22.

6. The nucleic acid trans-splicing molecule according to claim 5, wherein the binding domain is associated with a nucleic acid trans-splicing molecule containing... (a) Nucleotides 1041 to 1190 of SEQ ID NO: 28; (b) Nucleotides 1171 to 1320 of SEQ ID NO: 28; or (c) Nucleotides 1201 to 1350 of SEQ ID NO: 28 The binding sites of at least 50 consecutive nucleotides are complementary.

7. The nucleic acid trans-splicing molecule according to claim 1 or 5, wherein the length of the binding domain is 125-150 nucleotides.

8. The nucleic acid trans-splicing molecule according to claim 1 or 5, wherein the length of the binding domain is 150-175 nucleotides.

9. The nucleic acid trans-splicing molecule according to claim 1 or 5, wherein the length of the binding domain is 175-200 nucleotides.

10. The nucleic acid trans-splicing molecule according to claim 1 or 5, wherein the binding domain is 200-250 nucleotides in length.

11. The nucleic acid trans-splicing molecule according to claim 1 or 5, wherein the length of the binding domain is 100-200 nucleotides.

12. The nucleic acid trans-splicing molecule according to claim 1 or 5, wherein the binding domain is 150 nucleotides in length.

13. The nucleic acid trans-splicing molecule according to claim 1 or 5, wherein the binding domain comprises at least 50 consecutive nucleotides complementary to the binding site.

14. The nucleic acid trans-splicing molecule according to claim 1 or 5, wherein the binding domain comprises at least 60 consecutive nucleotides complementary to the binding site.

15. The nucleic acid trans-splicing molecule according to claim 1 or 5, wherein the binding domain comprises at least 70 consecutive nucleotides complementary to the binding site.

16. The nucleic acid trans-splicing molecule according to claim 1 or 5, wherein the binding domain comprises at least 80 consecutive nucleotides complementary to the binding site.

17. The nucleic acid trans-splicing molecule according to claim 1 or 5, wherein the binding domain comprises at least 90 consecutive nucleotides complementary to the binding site.

18. The nucleic acid trans-splicing molecule according to claim 1 or 5, wherein the binding domain comprises at least 100 consecutive nucleotides complementary to the binding site.

19. The nucleic acid trans-splicing molecule according to claim 1 or 5, wherein the binding domain comprises at least 120 consecutive nucleotides complementary to the binding site.

20. The nucleic acid trans-splicing molecule according to claim 1 or 5, wherein the binding domain comprises at least 150 consecutive nucleotides complementary to the binding site.

21. The nucleic acid trans-splicing molecule according to claim 1 or 5, wherein the binding domain comprises at least 200 consecutive nucleotides complementary to the binding site.

22. The nucleic acid trans-splicing molecule according to any one of claims 1, 5 or 6, wherein the binding domain comprises 100 or more consecutive nucleotides complementary to 100 or more nucleotides of the binding site.

23. The nucleic acid trans-splicing molecule of claim 6, wherein the binding domain comprises at least 60 consecutive nucleotides complementary to the binding site.

24. The nucleic acid trans-splicing molecule of claim 6, wherein the binding domain comprises at least 70 consecutive nucleotides complementary to the binding site.

25. The nucleic acid trans-splicing molecule of claim 6, wherein the binding domain comprises at least 80 consecutive nucleotides complementary to the binding site.

26. The nucleic acid trans-splicing molecule of claim 6, wherein the binding domain comprises at least 90 consecutive nucleotides complementary to the binding site.

27. The nucleic acid trans-splicing molecule of claim 6, wherein the binding domain comprises at least 100 consecutive nucleotides complementary to the binding site.

28. The nucleic acid trans-splicing molecule of claim 6, wherein the binding domain comprises at least 120 consecutive nucleotides complementary to the binding site.

29. The nucleic acid trans-splicing molecule of claim 6, wherein the binding domain comprises at least 150 consecutive nucleotides complementary to the binding site.

30. The nucleic acid trans-splicing molecule according to any one of claims 1, 5 or 6, wherein the coding domain is a cDNA sequence.

31. The nucleic acid trans-splicing molecule according to any one of claims 1, 5 or 6, wherein the coding domain comprises a naturally occurring sequence.

32. The nucleic acid trans-splicing molecule according to any one of claims 1, 5 or 6, wherein the coding domain comprises a codon-optimized sequence.

33. The nucleic acid trans-splicing molecule according to any one of claims 1, 5 or 6, wherein the splicing domain is located within an artificial intron, and the artificial intron comprises a spacer sequence.

34. The nucleic acid trans-splicing molecule according to any one of claims 1, 5 or 6, wherein the length is 3,000 to 4,000 nucleotides.

35. The nucleic acid trans-splicing molecule according to any one of claims 1, 5 or 6, wherein the mutation in the ABCA4 gene is associated with Stargardt disease.

36. The nucleic acid trans-splicing molecule of claim 35, wherein the mutation in the ABCA4 gene associated with Stargardt disease is expressed in photoreceptor cells.

37. The nucleic acid trans-splicing molecule according to claim 5, wherein the binding domain is complementary to the binding sites of nucleotides 981 to 1350 of SEQ ID NO:

28.

38. The nucleic acid trans-splicing molecule of claim 37, wherein the binding domain is complementary to the binding sites of nucleotides 1041 to 1350 of SEQ ID NO:

28.

39. A nucleic acid trans-splicing molecule comprising: operably linked in a 3' to 5' orientation: (a) A binding domain configured to bind ABCA4 intron 23, wherein the binding domain comprises a sequence of 50-300 nucleotides in length and is complementary to a binding site of at least 50 consecutive nucleotides within nucleotides 261 to 410 of SEQ ID NO:

29. (b) A splice structure domain configured to mediate trans-splicing, wherein the splice structure domain includes a 5' splice site; and (c) Contains the coding structure domain of functional ABCA4 exons 1-23; The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain to endogenous ABCA4 exon 24, thereby replacing endogenous ABCA4 exons 1-23 with functional ABCA4 exons 1-23.

40. A nucleic acid trans-splicing molecule comprising: operably linked in a 3' to 5' orientation: (a) A binding domain configured to bind ABCA4 intron 23, wherein the binding domain comprises a sequence of 50-300 nucleotides in length and is complementary to a binding site of at least 50 consecutive nucleotides within nucleotides 801 to 950 of SEQ ID NO:

29. (b) A splice structure domain configured to mediate trans-splicing, wherein the splice structure domain includes a 5' splice site; and (c) Contains the coding structure domain of functional ABCA4 exons 1-23; The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain to endogenous ABCA4 exon 24, thereby replacing endogenous ABCA4 exons 1-23 with functional ABCA4 exons 1-23.

41. A nucleic acid trans-splicing molecule comprising: operably linked in a 3' to 5' orientation: (a) A binding domain configured to bind ABCA4 intron 23, wherein the binding domain comprises a sequence of 50-300 nucleotides in length and is complementary to a binding site of at least 50 consecutive nucleotides within nucleotides 841 to 990 of SEQ ID NO:

29. (b) A splice structure domain configured to mediate trans-splicing, wherein the splice structure domain includes a 5' splice site; and (c) Contains the coding structure domain of functional ABCA4 exons 1-23; The nucleic acid trans-splicing molecule is configured to trans-splice the coding domain to endogenous ABCA4 exon 24, thereby replacing endogenous ABCA4 exons 1-23 with functional ABCA4 exons 1-23.

42. A nucleic acid trans-splicing molecule comprising: operably linked in a 3' to 5' orientation: (a) A binding domain configured to bind ABCA4 intron 22, wherein the binding domain comprises a sequence of 50-300 nucleotides in length and is complementary to a binding site of at least 50 consecutive nucleotides within nucleotides 1041 to 1190 of SEQ ID NO:

28. (b) A splice structure domain configured to mediate trans-splicing, wherein the splice structure domain includes a 5' splice site; and (c) The coding structure domain containing functional ABCA4 exons 1-22; 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 functional ABCA4 exons 1-22.

43. A nucleic acid trans-splicing molecule comprising: operably linked in a 3' to 5' orientation: (a) A binding domain configured to bind ABCA4 intron 22, wherein the binding domain comprises a sequence of 50-300 nucleotides in length and is complementary to a binding site of at least 50 consecutive nucleotides within nucleotides 1171 to 1320 of SEQ ID NO:

28. (b) A splice structure domain configured to mediate trans-splicing, wherein the splice structure domain includes a 5' splice site; and (c) The coding structure domain containing functional ABCA4 exons 1-22; 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 functional ABCA4 exons 1-22.

44. A nucleic acid trans-splicing molecule comprising: operably linked in a 3' to 5' orientation: (a) A binding domain configured to bind ABCA4 intron 22, wherein the binding domain comprises a sequence of 50-300 nucleotides in length and is complementary to a binding site of at least 50 consecutive nucleotides within nucleotides 1201 to 1350 of SEQ ID NO:

28. (b) A splice structure domain configured to mediate trans-splicing, wherein the splice structure domain includes a 5' splice site; and (c) The coding structure domain containing functional ABCA4 exons 1-22; 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 functional ABCA4 exons 1-22.

45. The nucleic acid trans-splicing molecule according to any one of claims 39-44, wherein the binding domain comprises a sequence of 50-150 nucleotides in length.

46. ​​The nucleic acid trans-splicing molecule according to any one of claims 39-44, wherein the binding domain comprises a sequence of 50 nucleotides in length.

47. The nucleic acid trans-splicing molecule according to any one of claims 39-44, wherein the binding domain comprises a sequence of 75 nucleotides in length.

48. The nucleic acid trans-splicing molecule according to any one of claims 39-44, wherein the binding domain comprises a sequence of 100 nucleotides in length.

49. The nucleic acid trans-splicing molecule according to any one of claims 39-44, wherein the binding domain comprises a sequence of 125 nucleotides in length.

50. The nucleic acid trans-splicing molecule according to any one of claims 39-44, wherein the binding domain comprises a sequence of 150 nucleotides in length.

51. The nucleic acid trans-splicing molecule according to any one of claims 39-44, wherein the binding domain comprises at least 50 consecutive nucleotides complementary to the binding site.

52. The nucleic acid trans-splicing molecule according to any one of claims 39-44, wherein the binding domain comprises at least 60 consecutive nucleotides complementary to the binding site.

53. The nucleic acid trans-splicing molecule according to any one of claims 39-44, wherein the binding domain comprises at least 70 consecutive nucleotides complementary to the binding site.

54. The nucleic acid trans-splicing molecule according to any one of claims 39-44, wherein the binding domain comprises at least 80 consecutive nucleotides complementary to the binding site.

55. The nucleic acid trans-splicing molecule according to any one of claims 39-44, wherein the binding domain comprises at least 90 consecutive nucleotides complementary to the binding site.

56. The nucleic acid trans-splicing molecule according to any one of claims 39-44, wherein the binding domain comprises at least 100 consecutive nucleotides complementary to the binding site.

57. The nucleic acid trans-splicing molecule according to any one of claims 39-44, wherein the binding domain comprises at least 120 consecutive nucleotides complementary to the binding site.

58. The nucleic acid trans-splicing molecule according to any one of claims 39-44, wherein the binding domain comprises at least 150 consecutive nucleotides complementary to the binding site.

59. The nucleic acid trans-splicing molecule according to any one of claims 39-44, wherein the binding domain comprises 100 or more consecutive nucleotides complementary to 100 or more nucleotides of the binding site.

60. A proviral plasmid comprising a nucleic acid trans-splicing molecule as described in any one of claims 1, 5, or 39-44.

61. An adeno-associated virus (AAV) comprising the nucleic acid trans-splicing molecule as described in any one of claims 1, 5, or 39-44.

62. The AAV of claim 61, wherein the AAV preferentially targets photoreceptor cells.

63. The AAV of claim 61, wherein the AAV comprises AAV5 capsid protein, AAV8 capsid protein, AAV8(b) capsid protein or AAV9 capsid protein.

64. A pharmaceutical composition comprising a nucleic acid trans-splicing molecule as described in any one of claims 1, 5, or 39-44, a proviral plasmid as described in claim 60, or an AAV as described in claim 61.

65. Use of the pharmaceutical composition of claim 64 for the preparation of a medicament for treating a disease associated with a mutation in the ABCA4 gene, wherein the disease is Stargardt disease.

66. The use according to claim 65, wherein the drug is configured for administration via subretinal injection, intravitreal injection or intravenous injection.

67. The use according to claim 66, wherein the drug is configured for administration via subretinal injection or intravitreal injection.

68. The use according to claim 65, wherein the drug exhibits an increase of at least 10% in ABCA4 protein expression after administration.

69. An adeno-associated virus (AAV) comprising an assembled capsid containing a vector genome comprising an AAV 5' ITR, a nucleic acid trans-splicing molecule as described in any one of claims 1, 5, or 39-44, and an AAV 3' ITR, under the operational control of a regulatory sequence.

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