Dual AAV vectors for treatment of fundus yellow spot disease

Abstract

Disclosed herein are compositions and methods for treating ocular diseases in mammals, and particularly complications associated with fundus yellow spot disease. The present disclosure provides an AAV-based dual vector system that promotes expression of a full-length protein having a coding sequence that exceeds the polynucleotide packaging capacity of a single AAV vector. In particular, described herein are methods and compositions relating to the expression of full length ABCA4 using an AAV-based dual vector system.

Description

[0001] Cross-referencing

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 499,482, filed May 1, 2023, which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] This application is submitted together with an electronic sequence list. The sequence list is provided as a file named 58774-726.601.xml, created on April 25, 2024, with a size of 93,061 bytes. Information in the electronic format of the sequence list is incorporated in its entirety by reference. Background Technology

[0005] Recombinant AAV has emerged as a useful gene delivery vector for treating retinal diseases. However, a limitation of AAV is its relatively small DNA packaging capacity—approximately 4.7 kilobases (KB). Therefore, standard AAV vector systems are unsuitable for treating diseases with large gene mutations or other dysfunctions, such as Stargardt disease. Solutions are needed to package large genes into AAV vector systems and safely deliver gene therapy to patients. Summary of the Invention

[0006] Disclosed are rAAV dual-vector and polynucleotide vector systems and compositions for delivering multiple nucleic acid segments for various gene therapy regimens. Also disclosed are recombinant viral particles comprising any of these rAAV dual-vector and polynucleotide vector systems, isolated host cells, and pharmaceutical compositions. Methods for preparing and using the improved rAAV dual-vector systems disclosed herein in virus-based gene therapy are also provided, particularly for treating and / or improving symptoms of defects in the ATP-binding cassette transporter (ABCA4), including but not limited to methods for treating human macular degeneration. In several aspects, the therapeutic methods and pharmaceutical compositions provided herein are intended for administration to one or both eyes of a subject (e.g., a human or animal subject).

[0007] In some aspects, this document discloses a polynucleotide vector system for providing the ABCA4 gene, comprising a first AAV vector polynucleotide containing a first ABCA4 sequence of the ABCA4 gene and a second AAV vector polynucleotide containing a second ABCA4 sequence of the ABCA4 gene, wherein the ABCA4 gene encodes an ABCA4 protein having at least 95% identity with SEQ ID NO: 2; the first ABCA4 sequence contains exons 1 to 20 of the ABCA4 gene, and the second ABCA4 sequence contains exons 21 to 50 of the ABCA4 gene. In some embodiments, the last nucleotide of the first ABCA4 sequence does not overlap with the first nucleotide of the second ABCA4 sequence. In some embodiments, the last approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides of the first ABCA4 sequence do not overlap with the first approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides of the second ABCA4 sequence. In some embodiments, the first ABCA4 sequence does not contain any of exons 21 to 50 of the ABCA4 gene, and the second ABCA4 sequence does not contain any of exons 1 to 20 of the ABCA4 gene. In some embodiments, the ABAC4 gene has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1. In some embodiments, the first ABCA4 sequence contains a sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 13. In some embodiments, the second ABCA4 sequence comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 15. In some embodiments, the first AAV vector polynucleotide comprises a promoter upstream of the first ABCA4 sequence. In some embodiments, the promoter is the smCBA promoter, CMV promoter, EF-1α promoter, cone repressor protein promoter, human ABCA4 promoter, TαC gene promoter, rhodopsin promoter, cGMP-phosphodiesterase β subunit promoter, human rhodopsin promoter, mouse rhodopsin promoter, hGRK1 promoter, rod-specific IRBP promoter, or VMD2 promoter.In some embodiments, the promoter is an smCBA promoter. In some embodiments, the promoter comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 23. In some embodiments, the first AAV vector polynucleotide comprises a splice donor site. In some embodiments, the splice donor site has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 24. In some embodiments, the second AAV vector polynucleotide comprises a splice acceptor site. In some embodiments, the splice acceptor site has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 25. In some embodiments, the first AAV vector polynucleotide and / or the second AAV vector polynucleotide comprises an alkaline phosphatase (AP) head sequence. In some embodiments, the first AAV vector polynucleotide comprises an AP head sequence, and the AP head sequence comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 22. In some embodiments, the second AAV vector polynucleotide comprises an AP head sequence, and the AP head sequence comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 22. In some embodiments, the second AAV vector polynucleotide comprises a polyadenylation (pA) signal sequence. In some embodiments, the pA signal sequence comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 21. In some embodiments, the first AAV vector polynucleotide and the second AAV vector polynucleotide each comprise a 5' AAV ITR and a 3' AAV ITR.In some embodiments, the 5' AAV ITR comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 26. In some embodiments, the 3' AAV ITR comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 27. In some embodiments, both the 5' AAV ITR and 3' AAV ITR belong to the AAV2 serotype.

[0008] In some respects, recombinant viral particles comprising the first or second AAV vector polynucleotide described herein are described herein. In some embodiments, the recombinant viral particles comprise In some implementations, the recombinant viral particles contain an AAV44.9 (E531D) capsid.

[0009] In some aspects, this document describes isolated host cells comprising the polynucleotide vector system or recombinant viral particles described herein. In some embodiments, the cells are photoreceptor cells, cone cells, rod cells, retinal cells, ganglion cells, retinal pigment epithelial cells, vestibular hair cells, inner ear hair cells, or outer ear hair cells.

[0010] In some aspects, methods for treating or improving a disease or condition in humans or animals are described herein, comprising administering to one or more cells of a human or animal the polynucleotide vector system described herein or the recombinant viral particles described herein, wherein expression of the ABCA4 gene treats or improves the disease or condition and is expressed in one or more cells. In some embodiments, the disease or condition is xanthoma vulgaris. In some embodiments, the treatment provides partial or complete recovery of vision loss. In some embodiments, the polynucleotide vector system is administered via parenteral administration, intravenous administration, intramuscular administration, intraocular administration, intranasal administration, subretinal administration, round window injection, or during cochlear implantation surgery. Attached Figure Description

[0011] To facilitate an understanding of the principles of this disclosure, reference will now be made to the illustrative embodiments or examples illustrated in the accompanying drawings, and they will be described using specific language. However, it should be understood that this is not intended to limit the scope of this disclosure. Any changes and further modifications to the described embodiments, and any further application of the principles of this disclosure as set forth herein, are contemplated as would normally occur to those skilled in the art to which this disclosure pertains.

[0012] Figure 1 A schematic diagram of a non-limiting example of a dual-carrier construct is shown. Dashed boxes indicate constructs that exhibit efficacy both in vitro and in vivo.

[0013] Figure 2 Western blot results from in vitro dual-vector transduction assessment are shown. The first column (from left to right) is the ladder. Columns 2 and 3 are biological replicates of cultured cells transduced with S007 and S008 (transduction alone). Columns 4 and 5 are biological replicates of cultured cells transduced with S009 and S010 (transduction alone). Columns 6 and 7 are biological replicates of cultured cells transduced with S011 and S012 (transduction alone).

[0014] Figures 3A to 3B This demonstrates confirmation that the dual AAV vector produces robust levels of full-length ABCA4 in the retina of Abca4- / - mice. Figure 3A This is an image showing the Western blot results of ABCA4 expression. From left to right, column 1 is the ladder band. Columns 2-5 are biological replicates (single mice) transduced with S009 and S0010. Columns 6-9 are biological replicates (single mice) transduced with S011 and S012. Columns 10-16 are from mice that did not receive the vector. Columns 10-11 are from Abca4+ / - mice. Column 13 is from Abca4- / - mice. Columns 14-16 are from Abca4+ / + mice. Figure 3B This is a graph showing the quantification of ABCA4 expression relative to wild-type (relative to normalized focal adhesion proteins).

[0015] Figure 4 A graph showing image pixel intensity measurements from scanning laser ophthalmoscopy (SLO) images demonstrates that subretinal injection of the S009-S010ABCA4 dual AAV vectors (SEQ ID NO: 5 and 6, respectively) was sufficient to reduce the retinal autofluorescence phenotype in Abca4 - / - knockout mice compared to the contralateral eye injected with the carrier.

[0016] Figures 5A to 5CElectroretinogram (ERG) data are shown, indicating that subretinal injection of the S009-S010 ABCA4 dual AAV carrier was well tolerated in Abca4 - / - mice. Two months post-injection, the ERG response in mice treated with the dual carrier was indistinguishable from that in the contralateral eye where the carrier was injected. Figure 5A The amplitude of the dark-sighted α wave was described. Figure 5B The amplitude of the scotopic b-wave was described. Figure 5C The amplitude of the photopic b wave was described. Invention Details

[0018] The following describes illustrative embodiments of this disclosure. This disclosure provides substances and methods for gene therapy of diseases and conditions, such as macular degeneration. Macular degeneration is a form of age-related macular degeneration. This disease is an autosomal recessive disorder that can cause blindness and affects approximately 1 in 8,000 people. Macular degeneration is associated with biallelic mutations in the gene encoding the ATP-binding cassette transporter ABCA4. ABCA4 is primarily expressed in photoreceptor cells in the retina. In some embodiments, a deficiency in ABCA4 leads to inappropriate transport of vitamin A. In some embodiments, a deficiency in ABCA4 leads to the accumulation of toxic byproducts, such as bisretinoids, in the retina.

[0019] Some aspects of this disclosure relate to AAV-based dual-vector systems that allow the expression of full-length proteins whose coding sequences exceed the multinucleotide packaging capacity of a single AAV vector. This disclosure provides nucleic acid vectors for dual-vector systems (e.g., overlapping vector systems or hybridization vector systems).

[0020] In some embodiments, the vector system of this disclosure employs two discrete AAV vectors, each packaging a relatively large DNA molecule (e.g., about 4.5 to 4.8 Kb) containing a portion of the ABCA4 gene. The two vectors are co-administered to selected recipient cells to reconstruct the full-length ABCA4 gene encoding the biologically active ABCA4 polypeptide. In some embodiments, a portion of the nucleic acid sequence is common to each vector genome (e.g., the common portion contains a non-coding sequence). When co-delivered to suitable cells, the common sequence region facilitates the appropriate concatamerization of the two partial gene cassettes. These gene cassettes then undergo homologous recombination to produce a full-length gene cassette within the cell. The common nucleic acid sequence is then spliced ​​from the resulting RNA. Some non-limiting components of some non-limiting embodiments of the dual-vector system include the use of an AAV inverted terminal repeat (ITR), a small (truncated) form of the chimeric CMV / chicken β-actin promoter (smCBA), a human ABCA4 cDNA sequence, and a mini-bovine growth hormone polyadenylation (pA) signal (mini2pA).

[0021] Dual-carrier system

[0022] In some aspects of this disclosure, the dual-carrier system of this disclosure includes:

[0023] (i) A first AAV vector polynucleotide comprising inverted terminal repeats at each end of the polynucleotide (e.g., the 5' and 3' ends), and a suitable promoter between the inverted terminal repeats, followed by (e.g., the 3' end of the promoter) a partial coding sequence encoding the N-terminal portion of the selected full-length polypeptide, followed by a splicing donor site and introns, and

[0024] (ii) A second AAV vector polynucleotide comprising inverted terminal repeats at each end (5' and 3') of the polynucleotide, and introns between the inverted terminal repeats and splice acceptor sites of the introns, optionally followed by a partial coding sequence encoding the C-terminal portion of the selected full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. In some embodiments, the intron sequences in the first and second AAV vectors comprise alkaline phosphatase homologous recombination sequences (APheads), for example, sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 22.

[0025] In some embodiments, the splitting point between the first AAV polynucleotide sequence and the second AAV polynucleotide sequence is between exon 19 and exon 20 of the hABCA4 gene. In some embodiments, the splitting point between the first AAV polynucleotide sequence and the second AAV polynucleotide sequence is between exon 20 and exon 21 of the hABCA4 gene. In some embodiments, the splitting point between the first AAV vector polynucleotide sequence and the second AAV vector polynucleotide sequence is between exon 21 and exon 22 of the hABCA4 gene.

[0026] In some embodiments, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 3, 5, or 7, or a functional fragment and / or variant thereof, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 4, 6, or 8, or a functional fragment and / or variant thereof. In some embodiments, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 3, 5, or 7, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 4, 6, or 8, a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 3, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 4, having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 5, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 6, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 7, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0027] When combined, the coding sequences in the first and second vectors encode a selected full-length polypeptide or a functional fragment or variant thereof. In some embodiments, the selected full-length polypeptide is human ABCA4 or hABCA4.

[0028] In some implementations, all or part of the intron sequence at the 3' end of the coding sequence of the first vector is the same as or substantially the same as the intron sequence at the 5' end of the coding sequence of the second vector.

[0029] In some specific embodiments, the intron sequences used in any vector system of this disclosure are intron sequences naturally present in the genomic sequence of the gene encoding the selected polypeptide. In some embodiments, the intron contains an alkaline phosphatase (AP) sequence. In some embodiments, the intron contains an alkaline phosphatase homologous recombination sequence (APhead). In some embodiments, the alkaline phosphatase homologous recombination sequence contains the nucleotide sequence of SEQ ID NO: 22, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0030] This article provides example strategies for overcoming random tandemization problems and thereby improving the specificity and efficiency of these dual-vector platforms. First, the addition of highly recombination-inducing sequences (such as those used herein in example dual-vectors) leads to significantly enhanced protein expression compared to trans-splicing systems. This finding that AP dual-vectors are more effective than trans-splicing vectors supports the idea that the AP sequence directs at least some tandemization events in the correct direction, and recombination subsequently occurs via that sequence or via the ITR. The APhead domain, in particular, mediates appropriate head-to-tail tandemization after dual-vector recombination in the cell. In some embodiments, example dual-vector systems with AP sequences facilitate proper alignment of more tandem bodies. In some embodiments, example dual-vector systems with AP sequences mediate more efficient expression of ABCA4. In some embodiments, dual-vector systems without AP sequences are used.

[0031] In some embodiments, the intron sequences used in the dual-vector system of this disclosure are intron sequences that are not naturally present in the genomic sequence of the gene encoding the selected polypeptide. In some embodiments, the intron sequences are derived from the MYO7A gene. In some specific embodiments, the introns are synthetic alkaline phosphatase (AP) introns. The intron sequences used in the dual-vector system of this disclosure may comprise splice donor and splice acceptor sequences. In some embodiments, the intron sequences are recombinant intron sequences (e.g., the AK sequence of F1 phage). In these embodiments, the dual vectors rely on both ITR-mediated tandemization and AK sequence-mediated homologous recombination to reconstruct the full-length expression cassette. Therefore, in some embodiments, the intron sequences are the AK sequences of F1 phage. Therefore, in some embodiments of the disclosed dual vectors, the vectors comprise one or more AP intron splice recognition sites, such as one or more AP splice acceptor (APSA) domains or AP splice donor (APSD) domains. In some embodiments, these vectors comprise APSA and APSD. In some embodiments, the first half of the vector comprises APSA, and the second half comprises APSD. In some embodiments, the first half of the vector comprises APSD, and the second half comprises APSA. See also Figure 1 In some embodiments, the vector comprises one or more non-AP intron spliceosome recognition sites. In some embodiments, the splice donor comprises the nucleotide sequence of SEQ ID NO: 24, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the splice acceptor comprises the nucleotide sequence of SEQ ID NO: 25, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0032] Another approach to guided tandemization is to use single-stranded oligonucleotides capable of tethering the back end of the 5' vector to the front end of the 3' vector. However, this strategy requires efficient and simultaneous delivery of the oligonucleotides and dual vectors to the nucleus of the target cell. Finally, dual vectors using mismatched ITRs can be used to guide tandemization head-to-tail, although this process may require further optimization of the AAV packaging mechanism. Therefore, in some embodiments, the dual vector pair contains the AP head coding sequence as part of the AP intron.

[0033] Therefore, in some embodiments, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 3, 5, or 7, or a functional fragment thereof and / or a variant thereof, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 4, 6, or 8, or a functional fragment thereof and / or a variant thereof. In some embodiments, the intron sequence is the AK sequence of F1 phage.

[0034] In some embodiments, the splitting point between the first AAV vector polynucleotide sequence and the second AAV vector polynucleotide sequence is between exon 21 and exon 22 of the hABCA4 gene. In some embodiments, the splitting point between the first AAV vector polynucleotide sequence and the second AAV vector polynucleotide sequence is between exon 20 and exon 21 of the hABCA4 gene. In some embodiments, the splitting point between the first AAV vector polynucleotide sequence and the second AAV vector polynucleotide sequence is between nucleic acids 3050 and 3051 of the hABCA4 gene, as indicated in SEQ ID NO: 1. In some embodiments, the splitting point between the first AAV vector polynucleotide sequence and the second AAV vector polynucleotide sequence is between exon 19 and exon 20 of the hABCA4 gene.

[0035] In some embodiments, the first AAV vector polynucleotide comprises a nucleotide sequence encoding the N-terminal portion of ABCA4. In some embodiments, the first AAV vector polynucleotide comprises the sequence of SEQ ID NO: 9, SEQ ID NO: 13, or SEQ ID NO: 17, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the first AAV vector encodes the N-terminal portion of ABCA4, said N-terminal portion comprising the sequence of SEQ ID NO: 10, SEQ ID NO: 14, or SEQ ID NO: 18, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the first AAV vector polynucleotide comprises the sequence of SEQ ID NO: 9 or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the first AAV vector encodes the N-terminal portion of ABCA4, said N-terminal portion comprising the sequence of SEQ ID NO: 10 or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the first AAV vector polynucleotide comprises the sequence of SEQ ID NO: 13 or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the first AAV vector encodes the N-terminal portion of ABCA4, said N-terminal portion comprising the sequence of SEQ ID NO: 14, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the first AAV vector polynucleotide comprises the sequence of SEQ ID NO: 17, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.In some embodiments, the first AAV carrier encodes the N-terminal portion of ABCA4, the N-terminal portion comprising the sequence of SEQ ID NO: 18, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0036] In some embodiments, the second AAV vector polynucleotide comprises a nucleotide sequence encoding the C-terminal portion of ABCA4. In some embodiments, the second AAV vector polynucleotide comprises a sequence of SEQ ID NO: 11, SEQ ID NO: 15, or SEQ ID NO: 19, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the second AAV vector encodes the C-terminal portion of ABCA4, said C-terminal portion comprising a sequence of SEQ ID NO: 12, SEQ ID NO: 16, or SEQ ID NO: 20, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the second AAV vector polynucleotide comprises the sequence of SEQ ID NO: 11, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the second AAV vector encodes the C-terminal portion of ABCA4, said C-terminal portion comprising the sequence of SEQ ID NO: 12, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the second AAV vector polynucleotide comprises the sequence of SEQ ID NO: 15, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the second AAV vector encodes the C-terminal portion of ABCA4, said C-terminal portion comprising the sequence of SEQ ID NO: 16, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the second AAV vector polynucleotide comprises the sequence of SEQ ID NO: 19, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.In some embodiments, the second AAV carrier encodes the C-terminal portion of ABCA4, the C-terminal portion comprising the sequence of SEQ ID NO: 20, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0037] In some implementations, a polynucleotide vector system is provided, comprising:

[0038] i) A first AAV vector polynucleotide, comprising inverted terminal repeats at each end of the polynucleotide, and a promoter between the inverted terminal repeats, followed by a partial coding sequence encoding the N-terminal portion of the ABCA4 polypeptide, then a splicing donor site and introns, and

[0039] ii) A second AAV carrier polynucleotide comprising inverted terminal repeats at each end of the polynucleotide, and introns and intron splice acceptor sites between the inverted terminal repeats. In some embodiments, between the inverted terminal repeats are introns and intron splice acceptor sites, followed by a partial coding sequence encoding the C-terminal portion of the ABCA4 polypeptide, optionally followed by a polyadenylation (pA) signal sequence.

[0040] In some embodiments, the intron sequences in the first and second AAV vectors comprise a common polynucleotide sequence, wherein the common polynucleotide sequence is not part of the coding sequence encoding the ABCA4 polypeptide. In some embodiments, the intron sequences in the first and second AAV vectors comprise a sequence having at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 22.

[0041] In some embodiments, the reverse terminal repeat comprises the sequence of SEQ ID NO: 26 or 27, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the promoter comprises the sequence of SEQ ID NO: 23, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the N-terminal portion of the ABCA4 polypeptide comprises the sequence of SEQ ID NO: 14, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the N-terminal portion of the ABCA4 polypeptide is encoded by a sequence comprising SEQ ID NO: 13 or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 13. In some embodiments, the C-terminal portion of the ABCA4 polypeptide is encoded by a sequence comprising SEQ ID NO: 16 or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 15. In some embodiments, the C-terminal portion of the ABCA4 polypeptide is encoded by a sequence comprising SEQ ID NO: 15 or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 15. In some embodiments, the splice donor site comprises the sequence of SEQ ID NO: 24, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the splice acceptor site comprises the sequence of SEQ ID NO: 25, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some embodiments, the intron comprises the sequence of SEQ ID NO: 22, or a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0042] In some embodiments of the dual-vector system described herein, the selected full-length polypeptide is an ABC transporter polypeptide. In some embodiments, the ABC transporter is a human ABCA polypeptide. In some embodiments, the ABCA polypeptide is ABCA4. In some embodiments, the full-length ABCA4 is encoded in the provided vector system.

[0043] When combined, the coding sequences in the first and second vectors encode a selected full-length polypeptide or a functional fragment or variant thereof. Therefore, in some embodiments, all or part of the intron sequence at the 3' end of the coding sequence in the first vector is identical or substantially identical to all or part of the intron sequence at the 5' end of the coding sequence in the second vector, wherein the intron sequence is not part of the coding sequence encoding the ABCA4 polypeptide.

[0044] Some embodiments of the dual-vector described herein anticipate viral or recombinant viral particles comprising the first or second AAV vector polynucleotide described herein. In some specific embodiments, the first AAV vector polynucleotide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5, and the second AAV vector polynucleotide comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6. In some embodiments, the virus or recombinant viral particle is characterized as adeno-associated virus (AAV) or infectious AAV viral particle. In some embodiments, the recombinant AAV viral particle comprises one or more tyrosine-to-phenylalanine (YF) mutations in the capsid protein of the virus or viral particle. This paper specifically anticipates a tyrosine-to-phenylalanine (YF) mutation at amino acid position 733 in the capsid protein of a virus or viral particle (e.g., AAV8Y733F). Similarly, this paper specifically anticipates a tyrosine-to-phenylalanine (YF) mutation at amino acid position 731 in the capsid protein of a virus or viral particle (e.g., AAV44.9 (Y731F)).

[0045] In some implementations, the virus or virus particles are packaged in the following: In some implementations, the viral particles contain... In some implementations, the viral particles are packaged in an AAV44.9 (E531D) capsid variant.

[0046] In some embodiments, the dual polynucleotide vector system described herein uses a tissue-specific promoter. In some embodiments, the system uses a promoter mediated by the eye.

[0047] In some embodiments, the dual polynucleotide vector system described herein uses any of the following promoters: cytomegalovirus (CMV) promoter, elongation factor-1α (EF-1α) promoter, cone repressor protein promoter, chimeric CMV β-actin (smCBA) promoter, ABCA4 gene-derived promoter, cone transduction protein a (TαC) gene-derived promoter, rhodopsin promoter, cGMP-phosphodiesterase β-subunit promoter, human or mouse rhodopsin promoter, human rhodopsin kinase (hGRK1) promoter, rod-specific IRBP promoter, RPE-specific vitelliform macular dystrophy-2 (VMD2) promoter, and combinations thereof. In some embodiments, the polynucleotide vector system described herein uses the human rhodopsin kinase (hGRK1) promoter. In some embodiments, the polynucleotide vector system uses the cone repressor protein promoter. In some implementations, the polynucleotide vector system uses a cytomegalovirus (CMV) promoter. In some implementations, the polynucleotide vector system uses an elongation factor-1α (EF-1α) promoter.

[0048] In some embodiments, any of the vectors in the dual polynucleotide vector systems described in this disclosure can be administered parenterally, such as intravenously, intramuscularly, intraocularly, or intranasally. The vectors can be administered in vivo, in vitro, or ex vivo. In some specific embodiments, the vectors provided herein can be administered via subretinal injection. The vectors can be administered in vivo or ex vivo.

[0049] In some embodiments, any of the vectors in the dual polynucleotide vector systems described herein can be applied to the eye. In some specific embodiments, the vector is applied to the subject's eye via subretinal injection.

[0050] The methods of this disclosure can be used in humans and other animals. Animals contemplated within the scope of this disclosure include, for example, dogs, cats, rabbits, ferrets, guinea pigs, hamsters, pigs, monkeys or other primates, mice, gerbils, horses, mules, donkeys, burros, cattle, cows, pigs, sheep, and alligators. The terms “patient” and “object” as used herein are used interchangeably and are intended to include such human and non-human species, including human and non-human cells. Similarly, the in vitro methods of this disclosure can also be performed on cells of one or more human or non-human mammalian species, including human and non-human cells.

[0051] Components of non-limiting examples of dual AAV vectors

[0052] Any of the dipolynucleotide vector systems disclosed herein can be used in conjunction with AAV vector systems known in the art. In treating some diseases, a single administration of the rAAV vector construct is preferred, while in managing or treating other diseases or conditions, it may be desirable to provide the patient with two or more administrations of the vector construct over one or more administration periods. In such cases, AAV-based treatment can be continuously provided over one or more periods of daily, weekly, monthly, or lower frequencies, which is necessary to achieve treatment of the treated disease or condition or to improve one or more symptoms of the treated disease or condition. In some embodiments, the vector can be provided to one or both eyes in an amount and duration sufficient to treat the treated disease or condition or to improve one or more symptoms of the treated disease or condition by one or more administrations of infectious adeno-associated virus particles, rAAV viral particles, or multiple infectious rAAV particles.

[0053] Some aspects of the disclosed vectors are contemplated to comprise viral or recombinant viral particles containing the first or second AAV vector polynucleotide described herein. In some embodiments, the virus or recombinant viral particle is characterized as adeno-associated virus (AAV) or infectious AAV viral particle. In some embodiments, the recombinant AAV viral particle contains one or more tyrosine-to-phenylalanine (YF) mutations in the capsid protein of the virus or viral particle. Tyrosine-to-phenylalanine (YF) mutations at amino acid position 733 of the capsid protein of the virus or viral particle are particularly contemplated herein (e.g., AAV8 Y733F).

[0054] In some implementations, the virus or virus particles are packaged in the following: In some implementations, the viral particles contain... In some implementations, the viral particles are packaged in an AAV44.9 (E531D) capsid variant.

[0055] In some embodiments, the polynucleotide vector system described herein uses a tissue-specific promoter. In some embodiments, the system uses a promoter mediated by the eye.

[0056] In some embodiments, the polynucleotide vector system described herein uses any of the following promoters: cytomegalovirus (CMV) promoter, elongation factor-1α (EF-1α) promoter, cone repressor protein promoter, chimeric CMVβ actin promoter (CBA), truncated chimeric CMVβ actin (smCBA) promoter, promoter derived from the human ABCA4 gene, promoter derived from the cone transduction protein a (TαC) gene, rhodopsin promoter, cGMP-phosphodiesterase β subunit promoter, human or mouse rhodopsin promoter, human rhodopsin kinase (hGRK1) promoter, rod-specific IRBP promoter, RPE-specific vitrectomyeloid macular dystrophy-2 [VMD2] promoter, and combinations thereof. In some embodiments, the polynucleotide vector system described herein uses the human rhodopsin kinase (hGRK1) promoter. In some embodiments, the polynucleotide vector system uses the cone repressor protein promoter. In some implementations, the polynucleotide vector system uses a cytomegalovirus (CMV) promoter. In some implementations, the polynucleotide vector system uses an elongation factor-1α (EF-1α) promoter.

[0057] In some specific embodiments, this disclosure provides rAAV particles derived from a variety of different serotypes, including, for example, those selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10. In some embodiments, particles derived from AAV2, AAV5, and AAV8 serotype vectors are used. In some specific embodiments, particles having an AAV8 (Y733F) or AAV2 (triple YF) capsid are used. Therefore, this disclosure provides recombinant AAV particles derived from, for example, AAV8 (Y733F) or AAV2 (triple YF), comprising a dual polynucleotide vector system. In some embodiments, the serotype of the AAV vector is not AAV6 or AAV2.

[0058] Other non-limiting capsids include AAV2, AAV6, and capsids derived from AAV2 and AAV6. Such capsids include... In some implementations, the virus or virus particles are packaged in the following: .

[0059] The AAV2 / 2-MAX capsid contains five point mutations: Y272F, Y444F, Y500F, Y730F, and T491V. Both the AAVSHh10 and AAV6(D532N) capsids are derivatives of AAV6.

[0060] Additional capsids suitable for use with the disclosed methods include: capsids containing non-natural amino acid substitutions at amino acid residues in the wild-type AAV2 capsid, wherein the non-natural amino acid substitutions comprise one or more of Y272F, Y444F, T491V, Y500F, Y700F, Y704F, and Y730F; and capsids containing non-natural amino acid substitutions at amino acid residues in the wild-type AAV6 capsid, wherein the non-natural amino acid substitutions comprise one or more of Y445F, Y705F, Y731F, T492V, and S663V. In some embodiments, the capsid comprises AAV2G9 (a variant of AAV2).

[0061] In other embodiments, the capsid comprises a non-natural amino acid substitution at amino acid residue 533 or 733 of the wild-type AAV8 capsid, wherein said non-natural amino acid substitution is E533K, Y733F, or a combination thereof. In some embodiments of the disclosed method, the capsid comprises AAV8PB2 (a variant of AAV8).

[0062] In other embodiments, the capsid comprises non-natural amino acid substitutions of the wild-type AAV2 capsid, which include one or more of the following mutations:

[0063] .

[0064] In other embodiments, the capsid comprises non-natural amino acid substitutions of the wild-type AAV6 capsid, which include one or more of the following mutations:

[0065] .

[0066] Additional cappings suitable for use with the disclosed methods are described in International Patent Publication No. 2018 / 156654, published August 30, 2018, which is incorporated herein by reference in its entirety. In some specific embodiments, the rAAV particles disclosed herein comprise one of the following cappings: DGE-DF (also known as “V1V4 VR-V”), P2-V2, P2-V3, P2-V1 (also known as ME-B), and P2-V1(Y-F+TV) (also known as ME-B(Y-F+TV)). In other embodiments, the rAAV particles may comprise a capping selected from AAV6 (3pMut) or AAV2 (quadYF+TV). In still other embodiments, the rAAV particles of the disclosed methods may comprise any capping variant described in International Patent Publication No. 2018 / 156654.

[0067] In some specific embodiments, rAAV particles are disclosed herein that may comprise a DGE-DF capsid, a P2-V2 capsid, a P2-V3 capsid, a P2-V1 capsid (also referred to as ME-B), or a P2-V1 (Y-F+TV) or ME-B (Y-F+TV) capsid for enhancing transduction of the rAAV particles in retinal cells. In other embodiments, the disclosed rAAV particles may comprise a capsid selected from:

[0068] .

[0069] The inverted terminal repeat (ITR) sequence used in any AAV vector system of this disclosure may contain any AAV ITR. In some embodiments, the ITRs used in the AAV vectors are the same. In some embodiments, the ITRs used in the AAV vectors are different. In some embodiments, the ITR may be obtained from AAV serotype 2 (AAV2), AAV serotype 5 (AAV5), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 44.9 (AAV44.9), or variants thereof, such as AAV serotype 44.9 (E531D) and 44.9 (Y7331F) (see PCT application No. PCT / US2020 / 14838, filed January 23, 2020, which is incorporated herein by reference). In some embodiments, the AAV vectors of this disclosure contain different AAV ITRs. In one non-limiting example, the vector may contain both the ITR of AAV2 and the ITR of AAV5. AAV ITR sequences are well known in the art (see, for example, GenBank login). In some implementations, the AAV dual-vector system disclosed herein is capable of efficiently expressing therapeutic genes that are larger than those typically packaged in a single AAV vector.

[0070] Therefore, in some aspects, this disclosure provides viruses or viral particles comprising any polynucleotide or vector of this disclosure. In some embodiments, the virus or viral particle is an AAV virus. Methods for preparing viruses and viral particles comprising heterologous polynucleotides or vectors are known in the art. In the case of AAV, cells can be co-infected or transfected with adenovirus or a polynucleotide vector containing adenovirus genes suitable for AAV helper functions.

[0071] In some embodiments, the AAV serotype has one or more tyrosine-phenylalanine (YF) mutations on the capsid surface. In some embodiments, AAV is the AAV8 serotype (Y733F) with a tyrosine-phenylalanine (YF) mutation at position 733.

[0072] In some embodiments, a triple mutant AAV8 vector is used, which contains a tyrosine-to-phenylalanine Tyr-Phe mutation at positions Y733F, Y500F, and Y730F, respectively. In some embodiments, a triple mutant AAV8 vector is used, which contains a tyrosine-to-phenylalanine Tyr-Phe mutation at positions Y447F, Y733F, and T494V (e.g., AAV8(Y447F+Y733F+T494F)).

[0073] In some embodiments, the rAAV particles of this disclosure contain transgenic or heterologous nucleic acids that are too large for delivery in a standard AAV system. In some embodiments, the transgenic gene is hABCA4 encoding a human ABCA4 polypeptide. In some embodiments, the hABCA4 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2 or a functional fragment or variant thereof. In some embodiments, the hABCA4 polypeptide is encoded by the nucleotide sequence listed in SEQ ID NO: 1.

[0074] In some embodiments, application of any of the disclosed polynucleotide vectors to the eye of the subject in need can partially or completely restore vision loss. The transgene may contain human ABCA4. In some embodiments, such application can reduce the accumulation of lipofuscin in the macula of the subject.

[0075] In some embodiments, the generation of a therapeutic agent encoded by a transgene of any of the disclosed polynucleotide vector systems in the cells of the eye (e.g., retinal cells or RPE cells) provides one or more of the following therapeutic endpoints: a) protection of one or more photoreceptor cells or one or more RPE cells; b) restoration of rod and / or cone-mediated function of one or more cells; c) restoration of visual behavior in one or both eyes; or d) any combination thereof. In some specific embodiments, the generation of the therapeutic agent in the disclosed method protects one or more PR cells, such as retinal ganglion cells, bipolar cells, Müller glial cells, or astrocytes or RPE cells.

[0076] In some implementations, after initial administration of any disclosed rAAV polynucleotide vector system to one or both eyes of a mammal, the production of the therapeutic agent persists substantially for a period of at least three months, at least six months, at least nine months, or at least one year or more in one or more photoreceptor cells or one or more RPE cells.

[0077] In this way, the polynucleotide carrier system and compositions thereof of this disclosure can be used to treat or improve symptoms of macular degeneration in the eyes of a subject. In some embodiments, symptoms of macular degeneration include night vision problems, color vision problems, or abnormal accumulation of lipofuscin in the macula of the subject.

[0078] In some embodiments, this disclosure provides an rAAV nucleic acid vector comprising at least a first nucleic acid segment encoding one or more diagnostic or therapeutic agents that alter, inhibit, reduce, block, eliminate, or attenuate the activity of one or more endogenous biological processes in mammalian cells appropriately transformed with the target vector. In some embodiments, such diagnostic or therapeutic agents may comprise molecules that selectively inhibit or reduce the effects of one or more metabolic processes, dysfunctions, symptoms, or diseases. In some embodiments, the defect may be caused by injury or trauma to the mammal to be treated. In other embodiments, the defect may be caused by overexpression of endogenous biological compounds, and in still other embodiments, the defect may be caused by underexpression or even absence of one or more endogenous biological compounds.

[0079] adjustment elements of rAAV carrier

[0080] Any vector system disclosed herein may contain regulatory elements that function in the intended host cell to express the vector. Regulatory elements include, for example, promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements.

[0081] Any vector system of this disclosure may include a promoter sequence operatively linked to a nucleotide sequence encoding a desired polypeptide. Promoters intended for use in this disclosure include, but are not limited to, cytomegalovirus (CMV) promoters, SV40 promoters, promoters derived from the human ABCA4 gene, Rous sarcoma virus (RSV) promoters, chimeric CMV / chicken β-actin promoters (CBA), and truncated forms of CBA (smCBA). In some embodiments, the promoter comprises the smCBA promoter (SEQ ID NO: 23), or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 23. Other photoreceptor-specific human rhodopsin kinase (hGRK1) promoters, rod-specific IRBP promoters, VMD2 (vitiligoma dystrophy / Best disease) promoters, RPE-specific vitiligoma dystrophy-2 [VMD2] promoters, and EF1-α promoter sequences are also contemplated for use in various aspects of the practice of this disclosure. Some non-limiting examples of photoreceptor-specific promoters include, but are not limited to, hGRK1, IRBP, rod opsin, NRL, GNAT2e-IRBP, L / M opsin, and cone repressor promoters.

[0082] In some specific embodiments, the promoter is a chimeric CMV-β-actin promoter. In some specific embodiments, the promoter is a tissue-specific promoter that exhibits selective activity in one or a group of tissues but low or no activity in other tissues. In some specific embodiments, the promoter is a photoreceptor-specific promoter. In another embodiment, the promoter is preferably a cone cell-specific promoter or a rod cell-specific promoter, or any combination thereof. In some specific embodiments, the promoter is a promoter of the human ABCA4 gene. In another embodiment, the promoter comprises a promoter derived from the cone transduction protein α (TαC) gene. In some specific embodiments, the promoter is a promoter derived from human GNAT2. Other promoters contemplated within the scope of this disclosure include, but are not limited to, rhodopsin promoters (human or mouse), cGMP-phosphodiesterase β-subunit promoters, retinitis pigmentosa-specific promoters, RPE cell-specific promoters [e.g., the vitrectomyces macular dystrophy-2 (VMD2) promoter (Best1)], or any combination thereof.

[0083] Promoters can be incorporated into vectors using standard techniques known to those skilled in the art of molecular biology and / or virology. Multiple copies of the promoter and / or multiple different promoters can be used in the vectors of this disclosure. In one such embodiment, the distance between the promoter and the transcription start site can be approximately the same as its distance from the transcription start site in its natural genetic environment, although some variation in this distance is permitted, without a significant reduction in promoter activity. In practice with this disclosure, one or more transcription start sites are typically included in the disclosed vectors.

[0084] The vectors disclosed herein may also contain one or more transcription termination sequences, one or more translation termination sequences, one or more signal peptide sequences, one or more internal ribosome entry sites (IRES), and / or one or more enhancer elements, or any combination thereof. The transcription termination region is typically derived from the 3' untranslated region of a eukaryotic or viral gene sequence. The transcription termination sequence may be located downstream of the coding sequence to provide effective termination.

[0085] Any disclosed polynucleotide vector may also additionally contain one or more post-transcriptional regulatory sequences or one or more polyadenylation signals, including, for example, but not limited to, woodchuck hepatitis virus post-transcription regulatory elements (WRPE), polyadenylation signal sequences, or intron / exon linkage / splicing signals, or any combination thereof.

[0086] Signal peptide sequences are amino-terminal peptide sequences that encode information responsible for: locating operablely linked polypeptides to one or more translated cellular destinations (including, for example, specific organelle compartments), or to sites of protein synthesis and / or activity, and even to the extracellular environment.

[0087] Enhancers, i.e., cis-regulatory elements that improve gene transcription, may also be included in one of the disclosed AAV-based vector systems. Various enhancer elements are known to those skilled in the art, and include, but are not limited to, the CaMV35S enhancer element, the cytomegalovirus (CMV) early promoter enhancer element, the SV40 enhancer element, and combinations and / or derivatives thereof. One or more nucleic acid sequences that guide or regulate polyadenylation of mRNA encoded by the target structural gene may also optionally be included in one or more vectors of this disclosure.

[0088] Host cells and methods for transducing cells

[0089] This disclosure provides host cells comprising vectors containing the disclosed polynucleotide vector systems. In some embodiments, isolated host cells comprising dual polynucleotide vector systems are provided.

[0090] Examples of suitable host cells comprising any of the disclosed dual-carrier systems include, but are not limited to, photoreceptor cells, cone cells, rod cells, retinal cells (e.g., ganglion cells, retinal pigment epithelial cells), or any combination thereof. Examples of retinal cells include retinal ganglion cells (RGCs), Muller cells, astrocytes, and bipolar cells.

[0091] This disclosure also provides methods for expressing or transducing selected peptides in cells. In some specific embodiments, the methods include incorporating an AAV-based dual-vector system disclosed herein into cells, wherein the vector system comprises a polynucleotide sequence encoding a selected peptide and a target peptide, and expressing the polynucleotide sequence in cells.

[0092] In some embodiments, the selected polypeptide may be a heterologous polypeptide to the cell. In some embodiments, the cell is a mammalian cell, and preferably a human cell. In some embodiments, the cell is a human photoreceptor cell, and preferably a human photoreceptor cone cell or photoreceptor rod cell. In some specific embodiments, the cell expresses a wild-type, functional, and / or biologically active ABCA4 polypeptide encoded by a nucleic acid segment present in the vector system disclosed herein. In some embodiments, the ABCA4 polypeptide is encoded by the nucleotide sequence shown in SEQ ID NO: 1.

[0093] Therefore, in some embodiments, this disclosure provides methods for transducing or expressing a polynucleotide vector system in one or more photoreceptor cells or one or more RPE cells of a mammal (e.g., a human). In a general and general sense, such methods include administering (e.g., direct subretinal administration) one or more rAAV particles disclosed herein to one or both eyes of a mammal, wherein the polynucleotide further comprises at least a first polynucleotide containing a PR- or RPE-cell-specific promoter operatively linked to at least a first heterologous nucleic acid segment encoding a therapeutic agent, the administration lasting for a duration capable of effectively producing the therapeutic agent in one or more PR cells or RPE cells of the mammal. In some embodiments, the therapeutic agent is stably expressed in photoreceptor cells, retinal pigment epithelial cells, retinal ganglion cells, bipolar cells, Müller glial cells, or astrocytes, or combinations thereof.

[0094] Treatment and transduction methods

[0095] In some aspects, this disclosure provides methods for treating or improving diseases or conditions (e.g., eye diseases) in humans or animals using gene therapy and the AAV-based dual-vector system of this disclosure. In some specific embodiments, the methods of this disclosure include administering a vector system of this disclosure encoding a polypeptide that provides treatment or improvement for the disease or condition. In some specific embodiments, the vector of this disclosure is provided in an AAV virus or viral particle. The vector system can be administered in vivo or ex vivo.

[0096] In some embodiments, the vector system of this disclosure is administered in recombinant AAV particles via parenteral administration, such as intravitreal, subretinal, intravenous, intramuscular, intraocular, or intranasal injection. In some specific embodiments, the vector system of this disclosure is administered to humans or animals via intraocular, intravitreal, or subretinal injection.

[0097] In some implementation schemes, the disease, disorder, or condition to be treated is xanthomastia.

[0098] The disclosed dual-vector system can be introduced into one or more selected mammalian cell types using any one or more methods. Such methods include, but are not limited to, transfection, microinjection, electroporation, lipofection, cell fusion, and calcium phosphate precipitation, as well as biolistic methods. In some specific embodiments, the vectors of this disclosure can be introduced in vivo, including, for example, via liposome transfection (e.g., DNA transfection via liposomes prepared from one or more cationic lipids). Synthetic cationic lipids (LIPOFECTIN®, Invitrogen Corp., La Jolla, CA, USA) can be used to prepare liposomes that encapsulate the vector to facilitate its introduction into one or more selected cells. The vector system of this disclosure can also be introduced in vivo as “naked” DNA.

[0099] In a general and general sense, the disclosed method includes at least the following steps: applying one or more of the rAAV particles disclosed herein to one or both eyes of a mammal in need, in an amount and for a duration sufficient to treat or improve one or more symptoms of a disease, condition, dysfunction, injury, abnormality, or trauma in the mammal. In some embodiments, the mammal is a human. In some embodiments, the human is a newborn, infant, toddler, or adolescent. In practice with this disclosure, suitable patients are expected to include, for example, individuals who have, are suspected of having, are at risk of having, or have been diagnosed with one or more retinal diseases, conditions, or malnutrition, including but not limited to genetically related or heritable retinal diseases, conditions, and malnutrition.

[0100] In some aspects, this disclosure provides methods of using the particles, vectors, viral particles, expression systems, compositions, and host cells described herein for treating or improving symptoms of multiple defects in mammalian eyes, particularly symptoms of one or more defects in human photoreceptor cells or RPE cells, or for preparing medicaments for treating or improving symptoms of multiple defects in mammalian eyes, particularly symptoms of one or more defects in human photoreceptor cells or RPE cells. In some embodiments, the subject with this need suffers from macular degeneration. In some embodiments, the subject suffers from an eye disease or condition that may benefit from treatment with gene therapy containing ABCA4 polynucleotides.

[0101] In some embodiments, administration of any disclosed vector, viral particle, or composition to a subject in need provides partial or complete restoration of melanosome migration in retinal pigment epithelium (RPE) cells. In some embodiments, administration of any polynucleotide vector system, viral particle, or composition provides partial or complete restoration of vision loss.

[0102] Such methods typically involve administering one or more of the disclosed particles, vectors, viral particles, host cells, or compositions in an amount and duration sufficient to treat such a defect in diseased mammals or to improve symptoms of such a defect in diseased mammals, either intravitreally or subretinal, to one or both eyes of the subject in need. The methods may also cover prophylactic treatment of animals suspected of having such a condition, or administering such compositions to animals at risk of developing such a condition after diagnosis or before the onset of symptoms.

[0103] Pharmaceutical Compositions and Kits

[0104] This disclosure also provides pharmaceutical compositions comprising a carrier system of this disclosure in combination with a pharmaceutically acceptable carrier. In the context of this disclosure, the dose administered to a patient (particularly a human) should be sufficient to achieve a therapeutic response in the patient within a reasonable timeframe without fatal toxicity, and preferably to cause side effects or morbidity no higher than acceptable levels. In some embodiments, the dose will depend on a variety of factors, including the subject's condition (health), the subject's weight, the type of current treatment (if any), the frequency of treatment, the treatment ratio, and the severity and stage of the pathological condition.

[0105] This disclosure also provides a cartridge containing the carrier system of this disclosure in one or more containers. The cartridge of this disclosure may optionally contain a pharmaceutically acceptable carrier and / or a diluent. In some specific embodiments, the cartridge of this disclosure contains one or more other components, additives, or excipients described herein. In some specific embodiments, the cartridge of this disclosure includes instructions or packaging material describing how to administer the carrier system contained within the cartridge to a selected mammalian recipient.

[0106] The container of the disclosed pillbox can be made of any suitable material, such as glass, plastic, metal, etc., and can be of any suitable size, shape, or construction. In some specific embodiments, the carrier system of this disclosure is provided as a solid in the pillbox. In another embodiment, the carrier system of this disclosure is provided as a liquid or solution in the pillbox. In some embodiments, the pillbox may include one or more ampoules or syringes containing the carrier system of this disclosure in a suitable liquid or solution form.

[0107] This disclosure also provides for the use of buffers and the compositions disclosed herein in the preparation of medicaments for the treatment, prevention, or improvement of symptoms of diseases, conditions, dysfunctions, injuries or traumas, including but not limited to the treatment, prevention, and / or prophylaxis of diseases, conditions, or dysfunctions, and / or the improvement of one or more symptoms of such diseases, conditions, or dysfunctions.

[0108] To express therapeutic agents according to this disclosure, rAAV particles containing nucleic acid segments encoding therapeutic agents under the control of one or more promoters can be prepared. To bring the sequence "under the control of a promoter," the 5' end of the transcription start site of the transcription reading frame is typically positioned "downstream" (e.g., at the 3' end) of the selected promoter for about 1 to about 50 nucleotides. The "upstream" promoter stimulates DNA transcription and promotes the expression of the encoded polypeptide. This is the meaning of "recombinant expression" herein. In some embodiments, the recombinant vector construct is one comprising a capsid protein-modified rAAV vector containing an RPE cell-specific promoter or a photoreceptor cell-specific promoter operatively linked to at least one nucleic acid segment encoding one or more diagnostic and / or therapeutic agents.

[0109] When considering the use of such a vector to introduce one or more exogenous proteins, polypeptides, peptides, ribozymes, and / or antisense oligonucleotides into specific cells transfected with the vector, one or more exogenous polynucleotides can be delivered to selected host cells, such as to one or more selected cells in a mammalian eye, using the rAAV particles disclosed herein.

[0110] In some embodiments, this disclosure provides a formulation of one or more of the virus-based compositions disclosed herein, which can be administered, in the form of a pharmaceutically acceptable solution, alone or in combination with one or more other therapeutic modalities, to cells or animals, and particularly for the treatment of diseases, tissues, and human cells affecting humans.

[0111] In some embodiments, the rAAV particles described herein may also be administered in combination with other agents (such as proteins or peptides or multiple pharmaceutically active agents), including one or more systemic or topical applications of therapeutic peptides, bioactive fragments or variants thereof.

[0112] The formulation of pharmaceutically acceptable buffers, excipients, and carrier solutions is well known to those skilled in the art, as is the development of suitable administration and treatment regimens for the use of the particular compositions described herein in a variety of treatment regimens, including, for example, oral, parenteral, intraocular (e.g., subretinal or vitreous), intravenous, intranasal, intra-articular, intracochlear, and intramuscular administration and formulation.

[0113] The term "excipient" refers to a diluent, excipient, carrier, or loading agent applied together with rAAV particles. Such pharmaceutical excipients can be sterile liquids, such as water and oils, including petroleum-based oils such as mineral oils, vegetable oils such as peanut oil, soybean oil, and sesame oil, animal oils, or oils of synthetic origin. Aqueous saline, glucose, and glycerol solutions can also be used as liquid carriers. Some non-limiting examples of excipients and loading agents include, but are not limited to, HA, BSS, artificial CSF, PBS, lactated Ringer's solution, TMN200 solution, polysorbate 20, and poloxamer 100.

[0114] Some non-limiting examples of compositions may include rAAV particles or nucleic acid carriers, alone or in combination with one or more other active ingredients, which may be obtained from natural or recombinant sources or chemically synthesized.

[0115] Preparation method of rAAV particles

[0116] Recombinant adeno-associated virus (rAAV) vectors have been successfully used for in vivo gene transfer in preclinical animal models of various human diseases and for the long-term expression of a wide range of therapeutic genes. AAV vectors have also demonstrated long-term clinical benefits in humans when targeting immune-privileged sites (e.g., for ocular delivery in Leber congenital amaurosis). A key advantage of this vector is its relatively low immune profile, evoking only limited inflammatory responses and, in some cases, even inducing immune tolerance to the transgene. Nevertheless, therapeutic efficacy in humans is limited when targeting non-immune-privileged organs due to antibody and CD8+ T cell responses against the viral capsid, while adaptive responses to the transgene have been reported in animal models. These results indicate that immune response remains a challenge for AAV vector-mediated gene transfer.

[0117] Adeno-associated virus (AAV) is considered the optimal vector for ocular gene therapy due to its efficiency, durability, and low immunogenicity. Identifying vectors capable of transducing PRs via the vitreous has historically relied on identifying which serotypes exhibit a natural tropism for that cell type after local delivery. Several serotypes have been used to successfully target PRs with transgenes after subretinal injection (including, for example, AAV2, AAV5, and AAV8), all three of which have demonstrated potency in experiments conducted across multiple mammalian species, such as mice, rats, dogs, pigs, and non-human primates.

[0118] Studies comparing the relative efficiency of subretinal delivery in rodents showed that both AAV5 and AAV8 transduced PR more efficiently than AAV2, with AAV8 being the most efficient. Previous studies have shown that, following subretinal and intravitreal injection of both, AAV2 and AAV8 vectors containing point mutations (tyrosine to phenylalanine, YF) with exposed tyrosine residues exhibited enhanced transgene expression in various retinal cell types compared to unmodified vectors. Among the vectors initially tested by these authors, the AAV2 triple mutant (named "triple YF") showed the highest transduction efficiency after intravitreal injection, while the AAV2 tetrap mutant ("tetrap YF") exhibited enhanced transduction properties in the outer retina.

[0119] Enhanced transduction efficiency has also been achieved by directionally mutagenizing one or more surface-exposed threonine (T) or serine (S) residues in these amino acids to non-natural amino acids. Both YF and TV / TA mutations have been shown to improve efficiency by reducing capsid phosphorylation and subsequent ubiquitination as part of the proteasome degradation pathway. The transduction profile of intravitreal AAVs has been found to be highly dependent on the injection procedure itself. Due to the small size of mouse eyes, retinal damage from transscleral or intravitreal injection is not uncommon, which can lead to some carriers entering the subretinal space directly.

[0120] In some implementations, the rAAV nucleic acid vectors available according to this disclosure include single-stranded (ss) or self-complementary (sc) AAV nucleic acid vectors, such as single-stranded or self-complementary recombinant viral genomes.

[0121] Methods for producing rAAV particles and nucleic acid vectors are commercially available (see, for example, plasmids and kits available from ATCC and CellBiolabs, Inc.). For example, a plasmid containing a nucleic acid vector sequence can be combined with one or more helper plasmids, such as those containing rep genes (e.g., encoding Rep78, Rep68, Rep52, and Rep40) and cap genes (encoding VP1, VP2, and VP3, including the modified VP3 region described herein), and transfected into a producer cell line for packaging and subsequent purification of rAAV particles.

[0122] In some embodiments, one or more helper plasmids comprise a first helper plasmid containing the rep gene and the cap gene, and a second helper plasmid containing the E1a gene, E1b gene, E4 gene, E2a gene, and VA gene. In some embodiments, the rep gene is a rep gene derived from AAV2, and the cap gene is derived from AAV2 and includes modifications to the genes to produce the modified capsid protein described herein. Helper plasmids and methods for preparing such plasmids are known in the art and are commercially available (see, for example, The pDP8.ape plasmid was obtained from Plasmid Factory, Bielefeld, Germany; other products and services are available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, CA; and Addgene, Cambridge, MA; pxx6.

[0123] A non-restrictive method for rAAV particle generation is described below. One or more helper plasmids are generated or obtained, containing the rep and cap ORF of the desired AAV serotype and the adenoviral VA, E2A (DBP), and E4 genes under transcriptional control of their natural promoters. The cap ORF may also contain one or more modifications to produce the modified capsid protein described herein. HEK293 cells (available from ATCC®) are transfected using the helper plasmids and plasmids containing the nucleic acid vector described herein via CaPO4-mediated transfection, lipid or polymeric molecules such as polyethyleneimine (PEI). The HEK293 cells are then incubated for at least 60 hours to allow rAAV particle generation. Alternatively, in another example, Sf9-based producer stable cell lines are infected with a single recombinant baculovirus containing the nucleic acid vector. As an alternative, in another instance, HEK293 or BHK cell lines are infected with: an HSV containing a nucleic acid vector and optionally one or more helper HSVs containing the rep and cap ORF described herein, as well as adenovirus VA, E2A (DBP), and E4 genes under transcriptional control of their natural promoters. HEK293, BHK, or Sf9 cells are then incubated for at least 60 hours to allow rAAV particle production. The rAAV particles can then be purified using any method known in the art or described herein, such as by stepwise gradient iodixanol, CsCl gradient, chromatography, or polyethylene glycol (PEG) precipitation.

[0124] definition

[0125] According to this disclosure, polynucleotides, nucleic acid fragments, nucleic acid sequences, etc., include but are not limited to DNA (including but not limited to genomic and / or extragenomic DNA), genes, peptide nucleic acids (PNA), RNA (including but not limited to rRNA, mRNA and / or tRNA), nucleosides, and one or more nucleic acid segments that are obtained from natural sources, chemically synthesized, genetically modified, or otherwise wholly or partially synthesized or prepared artificially.

[0126] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While any methods and compositions similar to or equivalent to those described and composed herein may be used in the practice or testing of this disclosure, preferred methods and compositions are described herein.

[0127] In some embodiments, the terms "nucleic acid" and "polynucleotide sequence" refer to deoxyribonucleotides or ribonucleotide polymers in single-stranded or double-stranded form, and unless otherwise limited, encompass known analogs of natural nucleotides that can function in a manner similar to naturally occurring nucleotides. Polynucleotide sequences can include full-length sequences as well as shorter sequences derived from full-length sequences. Specific polynucleotide sequences can include natural sequences or degenerate codons of sequences that can be introduced to provide codon preference in a particular host cell. Polynucleotide sequences can include sequences that specifically hybridize with sequences encoding peptides of this disclosure. Polynucleotides can include both sense and antisense strands, either as a single strand or in a duplex.

[0128] Fragments and variants of the polynucleotides disclosed herein can be generated and the presence of function can be tested as described herein. Fragments and variants of the polynucleotides or peptides disclosed herein can be tested to determine whether the fragment or variant retains the same or similar functional activity as the full-length or non-variant polynucleotide or peptide (e.g., ABCA4 polynucleotide or peptide).

[0129] In addition to one or more nucleotide substitutions, additions, or deletions within the sequence of the polynucleotide, the scope of this disclosure also includes polynucleotides having the same or substantially the same nucleotide sequence as those described herein, provided that these variant polynucleotides retain substantially the same relevant functional activity as those described herein (e.g., encoding a protein having the same amino acid sequence or the same functional activity as one of the polynucleotides specifically described herein). Therefore, the polynucleotides disclosed herein should also be understood to include their variants and fragments.

[0130] In addition to those variants that can be artificially prepared or synthesized by ordinary technicians in a laboratory setting, there are many variant sequences of genes or polynucleotides that exist in nature. The polynucleotides disclosed herein encompass those specifically described herein, as well as any naturally occurring variants and any artificially produced variants, provided that those variants retain the desired biological activity.

[0131] The scope of this disclosure also includes polynucleotides having the same nucleotide sequence as the polynucleotides described herein, except for nucleotide substitutions, additions, or deletions within the polynucleotide sequence, provided that these variant polynucleotides retain substantially the same relevant biological activity as the polynucleotides specifically described herein. Therefore, the polynucleotides disclosed herein should be understood to include variants and fragments of the sequences specifically described above.

[0132] The polynucleotides described herein may also be defined according to more specific ranges of identity and / or similarity compared to those described herein. Sequence identity may be greater than 60%, greater than 75%, greater than 80%, greater than 90%, or greater than 95%. Sequence identity and / or similarity may be 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater compared to the sequences described herein.

[0133] Unless otherwise stated, the algorithm of Karlin and Altschul (1990), a modification of that in Karlin and Altschul (1993), can be used to determine the percentage sequence identity and / or similarity between the two sequences used in this paper. Such an algorithm is incorporated into the NBLAST and XBLAST procedures of Altschul et al. (1990). A BLAST search can be performed using the NBLAST procedure: score = 100, word length = 12, to obtain sequences with the desired percentage sequence identity. Gapped BLAST can be used to obtain gap alignments for comparative purposes. When using the BLAST and Gapped BLAST procedures, the default parameters of the respective procedures (NBLAST and XBLAST) can be used according to the published methods.

[0134] This disclosure also considers those polynucleotide molecules having sequences sufficiently homologous to the polynucleotide sequence of this disclosure to allow hybridization with that sequence under standard stringent conditions and standard methods. “Stringent” hybridization conditions as used herein refer to conditions in which hybridization is typically performed below the denaturation temperature (T0) of DNA hybridization. m The reaction was carried out overnight at 20 to 25 degrees Celsius in 6×SSPE, 5×Denhardt solution and 0.1% SDS (containing 0.1 mg / mL of suitable nonspecific denatured DNA).

[0135] In some implementations, an effective amount refers to an amount that can treat or improve a disease or condition or otherwise produce the desired therapeutic effect.

[0136] In some implementations, operatively ligated means that the ligated nucleic acid sequences are typically continuous, or substantially continuous, and, when it is necessary to ligate two protein-coding regions, they are continuous and within the reading frame. However, since enhancers typically function thousands of bases apart from promoters, and intron sequences can be of variable length, some polynucleotide elements can be operatively ligated, but not continuous.

[0137] In some implementations, a promoter is one or more regions of a nucleic acid sequence that regulates transcription. Some non-limiting examples of promoters provided herein include, but are not limited to, the CMV promoter, the EF-1α promoter, the cone repressor promoter, the chimeric CMV β-actin promoter (CBA), the truncated chimeric CMV β-actin (smCBA) promoter, the smCBA promoter, promoters derived from the human ABCA4 gene, promoters derived from the TαC gene, rhodopsin promoters, cGMP-phosphodiesterase β-subunit promoters, human or mouse rhodopsin promoters, the hGRK1 promoter, the rod-specific IRBP promoter, and the VMD2 promoter.

[0138] In some implementations, a regulatory element refers to one or more regions of a nucleic acid sequence that regulate transcription. Some non-limiting examples of regulatory elements include, but are not limited to, enhancers, post-transcriptional elements, and transcriptional control sequences.

[0139] In some embodiments, the characteristics are essentially represented by a nucleic acid or amino acid sequence, wherein the selected nucleic acid or amino acid sequence has at least about 70% or about 75% sequence identity compared to a selected reference nucleic acid or amino acid sequence. In some cases, the selected sequence and the reference sequence will have at least about 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or even 85% sequence identity, and more preferably at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% sequence identity. Highly homologous sequences typically share more than at least about 96%, 97%, 98%, or 99% sequence identity between the selected sequence and the reference sequence to which it is compared.

[0140] The percentage of sequence identity can be calculated over the entire length of the sequences to be compared, or by excluding small deletions or additions that total less than approximately 25% of the selected reference sequence. The reference sequence can be a subset of a larger sequence, such as a part of a gene or flanking sequence, or a repetitive portion of a chromosome. In cases of sequence homology between two or more polynucleotide sequences, the reference sequence may contain at least approximately 18 to 25 nucleotides, at least approximately 26 to 35 nucleotides, or at least approximately 40, 50, 60, 70, 80, 90, or even around 100 nucleotides.

[0141] When highly homologous fragments are desired, the degree of percentage identity between two sequences can be at least about 80%, at least about 85%, or about 90% or 95% or higher, as can be easily determined by sequence comparison algorithms (such as FASTA program analysis).

[0142] In some embodiments, the object describes an organism to which it may be provided with treatments according to the compositions disclosed herein, including mammals such as primates. Mammal species that may benefit from the disclosed treatments include, but are not limited to, humans, non-human primates such as apes, chimpanzees, monkeys and orangutans, domestic animals (including dogs and cats) and livestock (e.g., horses, cattle, pigs, sheep and goats) or other mammal species (including, but not limited to, mice, rats, guinea pigs, rabbits, hamsters, etc.).

[0143] In some implementations, treatment or any of its grammatical variations includes, but is not limited to, alleviating the symptoms of a disease or condition; and / or reducing, suppressing, inhibiting, mitigating, improving, or influencing the progression, severity, and / or extent of a disease or condition.

[0144] In some implementations, a vector refers to a nucleic acid molecule (typically a DNA-containing nucleic acid molecule) capable of replicating in a suitable host cell, or a nucleic acid molecule to which another nucleic acid segment can be operatively linked to facilitate replication of the operatively linked nucleic acid segment. Exemplary vectors include, but are not limited to, plasmids, granules, viruses, etc.

[0145] In some embodiments, a variant refers to a molecule (e.g., a capsid polynucleotide) that deviates from characteristics found in nature, for example, the "variant" having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% identity with the wild-type capsid polynucleotide. Variants of protein molecules (e.g., capsids) may contain modifications to the amino acid sequence relative to the wild-type protein sequence (e.g., substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 to 15, or 15 to 20 amino acid substitutions), which are produced by point mutations set in the nucleic acid sequence encoding the capsid protein. These modifications include chemical modifications and truncation.

[0146] Variants of nucleic acid molecules (such as polynucleotide vector systems) may contain sequence modifications relative to the wild-type nucleic acid sequence (e.g., substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 to 15, or 15 to 20 nucleotides). These modifications may include truncation of the 5' end at the 3' end. Example

[0147] The following embodiments are included to illustrate some example implementations of the present disclosure.

[0148] Example 1 describes a non-restrictive dual-AAV vector system spliced ​​using alkaline phosphatase (AP). In Figure 1In each system shown, two vectors are present, each containing an AP homologous domain (APHead). In one of the systems labeled “Dual AAV-ABCA4 SP2 (ex20 / 21)”, vector A contains the coding sequence corresponding to the amino-terminal portion of hABCA4 cDNA up to exon 20 (SEQ ID NO: 13) and a splice donor site (SEQ ID NO: 24), followed by an APHead intron (SEQ ID NO: 22). Vector B contains an APHead intron (SEQ ID NO: 22), followed by a splice acceptor site (SEQ ID NO: 25), and then the C-terminal portion of hABCA4 cDNA starting from exon 21 (SEQ ID NO: 15). After co-delivery to a suitable mammalian host cell, the DNA of vectors A and B recombine to form a reconstructed full-length gene cassette. The resulting RNA transcript then “splices out” the intron (SEQ ID NO: 22). Alternatively, recombination and formation of the gene cassette can occur via AAV ITR. In this case, the RNA transcript will be "spliced" to produce the intron-ITR-intron motif (SEQ ID NO: 29). However, in both cases, the resulting mRNA (SEQ ID NO: 1) is the full-length hABCA4 mRNA, which is then translated into the hABCA4 protein (SEQ ID NO: 2). Similarly, the system labeled "double AAV-ABCA4 SP1 (ex19 / 20)" comprises two vectors—a first vector containing the coding sequence corresponding to the N-terminal portion of the hABCA4 cDNA up to exon 19 (SEQ ID NO: 9), and a second vector containing the coding sequence corresponding to the C-terminal portion of the hABCA4 cDNA starting from exon 20 (SEQ ID NO: 11). Figure 1 The system labeled “Double AAV-ABCA4 SP3 (ex21 / 22)” is also shown, which comprises two vectors: a first vector containing a coding sequence corresponding to the amino-terminal portion of hABCA4 cDNA up to exon 21 (SEQ ID NO: 17), and a second vector containing a coding sequence corresponding to the carboxyl-terminal portion of hABCA4 cDNA starting from exon 22 (SEQ ID NO: 19).

[0149] Example 2 describes the in vitro performance of three non-restrictive dual-vector systems. Each of the three dual AAV-ABCA4 vectors was packaged into AAV44.9 (E531D) (SEQ ID NO: 28) via triple transfection of HEK293 cells: S007 + S008 (SP1, SEQ ID NO: 3 and 4, respectively), S009 + S010 (SP2, SEQ ID NO: 5 and 6, respectively); and S011 + S012 (SP3, SEQ ID NO: 7 and 8, respectively). The resulting vector pairs were used to infect AAVR cells at an MOI of 100,000. Three days post-infection, cells were harvested and total protein was extracted. Protein samples were run on Western blotting and detected with an antibody against ABCA4 (…). Figure 2 The SP2 (S009-S010) and SP3 (S011-S012) dual AAV-ABCA4 vectors can induce high levels of full-length ABCA4 expression in vitro for both vectors.

[0150] Example 3 compared the results of bilateral subretinal injections as described in Examples 1 and 2 with... Figure 1 The three dual-AAV-ABCA4 vector expression cassettes shown demonstrated ABCA4 expression in Abca4- / - knockout mice. Dual-AAV vector-mediated ABCA4 levels were compared with endogenous levels in heterozygous and wild-type mice. Results were... Figure 3A and 3B As shown in the figure, no ABCA4 expression was observed in the retinas of mice injected with S007+S010 samples (SEQ ID NO: 3 and 4; data not shown). S009 / S010 (SEQ ID NO: 5 and 6) produced the highest levels of ABCA4 expression. The ABCA4 levels produced by S009 / S010 ranged from 13% to 69% of the wild-type, with a mean ABCA4 level of 37% of the wild-type. The S011 / S012 vector pair (SEQ ID NO: 7 and 8) produced ABCA4 levels ranging from 15% to 27% of the wild-type, with a mean ABCA4 level of 22% of the wild-type. These results indicate that dual AAV vectors with a split point between exons 20 and 21 produce robust levels of full-length ABCA4 in the retinas of Abca4- / - mice.

[0151] Example 4 illustrates in vivo data from Abca4- / - mice subretinally injected with AAV44.9 (E531D) particles (capsid encoded by SEQ ID NO: 28) containing the S009-S010 carrier pair (SEQ ID NO: 5 and 6). Treatment resulted in reduced autofluorescence in the retinas of Abca4- / - knockout mice. Toxic autofluorescence-bearing retinaldehyde accumulation was observed in the retinas of both patients with xanthomas and Abca4- / - knockout mice, measurable by scanning laser ophthalmoscopy (cSLO). Subretinal injection was performed in one eye of an Abca4- / - knockout mouse. The contralateral eye was injected separately with the carrier. Uninjected wild-type mice were also included as a control. Figure 4 In this study, mean pixel intensity measurements of cSLO images from Abca4- / - mice injected with both vectors and the vector, or uninjected WT controls, were plotted at multiple time points. At all time points following injection, reduced autofluorescence was observed in the vector-injected Abca4- / - eyes compared to the vector-injected eyes. These results suggest that ABCA4 generated from the S009-S010 vector pair can be used to reduce the retinal autofluorescence phenotype in Abca4- / - mice.

[0152] Example 5 generated electroretinography (ERG) data from Abca4- / - mice subretinally injected with AAV44.9 (E531D) particles (encoded by SEQ ID NO: 28) containing the S009-S010 dual-carrier pair (SEQ ID NO: 5 and 6). ERG recordings were performed 2 months post-injection under both scotopic (rod-mediated) and photopic (cone-mediated) conditions, and the maximal a-wave (photoreceptor) was recorded. Figure 5A ) and b wave (bipolar cell) Figure 5B (C) Amplitudes were calculated / averaged. ERG recordings were also performed on uninjected wild-type mouse eyes. No significant differences in ERG amplitude were observed between eyes injected with the S009-S010 carrier and those injected with the carrier from Abca4- / - mice. These data suggest that the S009-S010 dual AAV carrier is well tolerated 2 months post-injection and does not cause any reduction in retinal function.

[0153] Equivalent scheme

[0154] Therefore, it should be understood that the foregoing embodiments are merely illustrative examples, and that embodiments of the invention may be practiced in ways different from the specific descriptions and claims within the scope of the appended claims and their equivalents. The inventive embodiments of this disclosure relate to each individual feature, system, article, material, cassette, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, cassettes, and / or methods is included within the scope of this disclosure, provided that such features, systems, articles, materials, cassettes, and / or methods are not inconsistent with each other.

[0155] Unless otherwise expressly stated, the indefinite articles “a” and “an” as used herein in the specification and claims shall be understood to mean “at least one / a type”.

[0156] The phrase “and / or” as used herein in the specification and claims should be understood to mean “any one or both” of the elements so connected, i.e., elements that coexist in some cases and exist separately in others. Multiple elements listed with “and / or” should be understood in the same way, i.e., “one or more” of the connected elements. Other elements may optionally be present in addition to those specifically indicated by the “and / or” clause, whether related to or unrelated to those specifically indicated. Thus, as a non-limiting example, when used in conjunction with open-ended language such as “comprising,” a reference to “A and / or B” may refer only to A in one embodiment (optionally including elements other than B); only to B in another embodiment (optionally including elements other than A); both A and B in yet another embodiment (optionally including other elements); and so on.

[0157] As used herein in the specification and claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when items in a list are separated, “or” or “and / or” should be interpreted as inclusive, i.e., including multiple elements or at least one of the elements in the list, but also including more than one, as well as optional additional items not listed. Only explicitly indicating the opposite terms, such as “only one” or “exactly one”, or when used in a claim, “consisting of”, will refer to including multiple elements or exactly one of the elements in the list. Generally, when preceded by exclusive terms such as “one of,” “one of,” “only one of,” or “exact one of,” the term “or” as used herein should only be interpreted as indicating an exclusive choice (i.e., one or the other, but not both). When used in a claim, “consisting substantially of” should have the ordinary meaning as it is used in the field of patent law.

[0158] As used herein in the specification and in the claims, the phrase "at least one" when referring to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list, but does not necessarily include all elements specifically listed in the list and at least one of each element, and does not exclude any combination of elements in the list. This definition also allows for the optional presence of elements other than those expressly indicated in the list of elements referred to by the phrase "at least one," whether related to or unrelated to those expressly indicated elements. Therefore, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently, "at least one of A and / or B") in one embodiment may refer to at least one A, optionally including more than one A, while B is absent (and optionally including elements other than B); in another embodiment, it may refer to at least one B, optionally including more than one B, while A is absent (and optionally including elements other than A); in yet another embodiment, it may refer to at least one A, optionally including more than one A, and at least one B, optionally including more than one B (and optionally including other elements); and so on.

[0159] It should also be understood that, unless expressly indicated otherwise, in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order of the steps or actions of the method described herein.

[0160] In the claims and the foregoing description, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “constituting,” etc., are to be understood as open-ended, meaning including but not limited to. As described in Section 2111.03 of the U.S. Patent Examination Procedure Manual, only the transitional phrases “constituting of” and “constituting substantially of” should be closed or semi-closed transitional phrases, respectively. It should be understood that embodiments described in this document using open-ended transitional phrases (e.g., “comprising / including”) are also considered in alternative embodiments as “constituting the features described by the open-ended transitional phrase” and “constituting substantially the features described by the open-ended transitional phrase.” For example, if this disclosure describes “a composition comprising A and B,” this disclosure also considers alternative embodiments: “a composition comprising A and B” and “a composition substantially comprising A and B.”

[0161] Implementation Plan

[0162] The following implementation plan is also disclosed in this article:

[0163] 1. A polynucleotide vector system for providing ABCA4 or a functional portion thereof, said polynucleotide vector system comprising...

[0164] i) A first AAV vector polynucleotide comprising inverted terminal repeats at each end of the polynucleotide, and a promoter between the inverted terminal repeats, followed by a partial ABCA4 coding sequence encoding the N-terminal portion of ABCA4, then a splice donor site and a homologous region, and

[0165] ii) A second AAV vector polynucleotide comprising inverted terminal repeats at each end of the polynucleotide, and homologous regions and splice acceptor sites between the inverted terminal repeats, and a portion of the ABCA4 coding sequence encoding the C-terminal portion of ABCA4.

[0166] The homologous regions in the first AAV vector and the homologous regions in the second AAV vector contain overlapping polynucleotide sequences, and

[0167] The split point between the first AAV vector polynucleotide sequence and the second AAV vector polynucleotide sequence is located between exon 20 and exon 21 of the ABCA4 gene.

[0168] 2. The polynucleotide vector system described in Implementation Scheme 1, wherein the homologous regions in the first AAV vector and the homologous regions in the second AAV vector are alkaline phosphatase homologous regions.

[0169] 3. The polynucleotide vector system according to embodiment 1 or 2, wherein the N-terminal portion of ABCA4 has at least 85% sequence identity with SEQ ID NO:14.

[0170] 4. The polynucleotide vector system according to embodiment 1 or 2, wherein the C-terminal portion of ABCA4 has at least 85% sequence identity with SEQ ID NO:16.

[0171] 5. The polynucleotide vector system according to any one of embodiments 1 to 4, wherein the first AAV vector polynucleotide sequence comprises a nucleotide sequence having at least 85% sequence identity with SEQ ID NO: 13.

[0172] 6. The polynucleotide vector system according to any one of embodiments 1 to 5, wherein the first AAV vector polynucleotide sequence comprises a nucleotide sequence having at least 85% sequence identity with SEQ ID NO: 15.

[0173] 7. The polynucleotide vector system according to any one of embodiments 1 to 6, wherein the first AAV vector polynucleotide has at least 85% sequence identity with the nucleotide sequence of SEQ ID NO: 5.

[0174] 8. The polynucleotide vector system according to any one of embodiments 1 to 7, wherein the second AAV vector polynucleotide has at least 85% sequence identity with the nucleotide sequence of SEQ ID NO: 6.

[0175] 9. The polynucleotide vector system according to any one of embodiments 1 to 8, wherein the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 5.

[0176] 10. The polynucleotide vector system according to any one of embodiments 1 to 9, wherein the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 6.

[0177] 11. The polynucleotide vector system according to any one of embodiments 1 to 10, wherein the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 5, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 6.

[0178] 12. The polynucleotide vector system according to any one of embodiments 1 to 11, wherein the length of the polynucleotide sequence shared between the vectors is about 50 to about 500 nucleotides, or about 200 to 300 nucleotides.

[0179] 13. The polynucleotide vector system according to any one of embodiments 1, 3 to 6 or 12, wherein the homologous region comprises an intron sequence.

[0180] 14. The polynucleotide vector system of embodiment 13, wherein the intron sequence comprises an intron sequence naturally present in the genomic sequence of the gene encoding the ABCA4 polypeptide.

[0181] 15. The polynucleotide vector system of embodiment 13 or 14, wherein the intron sequence is the AK sequence of F1 phage.

[0182] 16. The polynucleotide vector system according to any one of embodiments 13 to 16, wherein the intron sequence comprises a synthetic alkaline phosphatase (AP) intron or an intron derived from MYO7A.

[0183] 17. The polynucleotide vector system according to any one of embodiments 1 to 16, further comprising one or more nucleotide substitutions in one or more non-coding regions of the first AAV vector polynucleotide and / or the second AAV vector polynucleotide.

[0184] 18. Any polynucleotide vector system according to embodiment 17, further comprising one or more nucleotide substitutions in one or more non-coding regions of the first AAV vector polynucleotide.

[0185] 19. The polynucleotide vector system of embodiment 18, wherein one or more non-coding sequences comprise an alkaline phosphatase (AP) head sequence.

[0186] 20. The polynucleotide vector system of embodiment 17 or embodiment 18, wherein one or more non-coding sequences contain AP introns.

[0187] 21. The polynucleotide vector system of embodiment 18, wherein one or more non-coding sequences are contained in a 3' untranslated region (UTR) between the ABCA4 partial coding sequence and the 3'AAV inverted terminal repeat.

[0188] 22. The polynucleotide vector system according to any one of embodiments 17 to 21, wherein one or more substitutions are located in one or more presumed in-frame stop codons.

[0189] 23. The polynucleotide vector system of embodiment 22, wherein the substitution is located in one or more presumed in-frame stop codons in the AP head sequence.

[0190] 24. The polynucleotide vector system of embodiment 23, wherein the substitution is located in three putative in-frame stop codons in the AP intron sequence.

[0191] 25. The polynucleotide vector system according to any one of embodiments 1 to 24, wherein the promoter is selected from: CMV promoter, EF-1α promoter, cone repressor protein promoter, smCBA promoter, promoter derived from human ABCA4 gene, promoter derived from TαC gene, rhodopsin promoter, cGMP-phosphodiesterase β subunit promoter, human or mouse rhodopsin promoter, hGRK1 promoter, rod-specific IRBP promoter, VMD2 promoter, and combinations thereof.

[0192] 26. The polynucleotide vector system of embodiment 25, wherein the promoter is a CMV promoter.

[0193] 27. The polynucleotide vector system of embodiment 25, wherein the promoter is the smCBA promoter.

[0194] 28. The polynucleotide vector system of embodiment 25, wherein the promoter is a rhodopsin promoter.

[0195] 29. The polynucleotide system according to any one of embodiments 1 to 29, further comprising one or more nucleotide substitutions to remove one or more putative stop codons from the 3' untranslated region of the second AAV vector polynucleotide, said 3' untranslated region being between a portion of the coding sequence encoding the C-terminal portion of the polypeptide and a 3'AAV inverted terminal repeat sequence.

[0196] 30. The polynucleotide system of embodiment 29, wherein one or more substitutions are located in one or more presumed stop codons.

[0197] 31. The polynucleotide vector system according to any one of embodiments 1 to 30, wherein the second AAV vector polynucleotide is followed by a polyadenylation (pA) signal sequence.

[0198] 32. The polynucleotide vector system according to any one of embodiments 1 to 31, wherein the reverse terminal repeat at each end of the first AAV vector polynucleotide or the second AAV vector polynucleotide comprises 5' AAV ITR and 3' AAV ITR, and wherein the 5' AAV ITR and the 3' AAV ITR are ITRs derived from a single AAV serotype.

[0199] 33. The polynucleotide vector system according to any one of embodiments 1 to 32, wherein each end of the first AAV vector polynucleotide or the second AAV vector polynucleotide contains a reverse terminal repeat comprising 5' AAV ITR and 3' AAV ITR, and wherein the 5' AAV ITR and the 3' AAV ITR are ITRs derived from multiple AAV serotypes.

[0200] 34. The polynucleotide vector system according to any one of embodiments 1 to 28, wherein the AAV inverted terminal repeat (ITR) comprises an ITR from one or more AAV serotypes selected from: AAV serotype 2 (AAV2), AAV serotype 5 (AAV5), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 44.9 (AAV44.9), AAV serotype 44.9 (E531D), and AAV serotype 44.9 (Y733F).

[0201] 35. The polynucleotide vector system of embodiment 34, wherein the AAV serotype is AAV serotype 44.9 (E531D).

[0202] 36. The polynucleotide vector system of embodiment 34, wherein the AAV serotype is AAV2.

[0203] 37. A recombinant viral particle comprising a first AAV vector polynucleotide or a second AAV vector polynucleotide as described in any one of embodiments 1 to 36.

[0204] 38. The recombinant viral particle of embodiment 37, comprising one or more tyrosine-phenylalanine (YF) mutations in the capsid protein of the virus or viral particle.

[0205] 39. The recombinant viral particle of embodiment 37 or embodiment 38, wherein the recombinant viral particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and / or AAV10 capsid.

[0206] 40. The recombinant viral particle according to any one of embodiments 37 to 39, wherein the recombinant viral particle comprises .

[0207] 41. The recombinant viral particle of embodiment 40, wherein the recombinant viral particle comprises an AAV44.9 (E531D) capsid.

[0208] 42. An isolated host cell comprising a polynucleotide vector system according to any one of embodiments 1 to 36 or recombinant viral particles according to any one of embodiments 37 to 41.

[0209] 43. The isolated host cell of embodiment 42, wherein the cell is a photoreceptor cell, cone cell, rod cell, retinal cell, ganglion cell, retinal pigment epithelial cell, vestibular hair cell, inner ear hair cell, outer ear hair cell, or any combination thereof.

[0210] 44. A method for treating or improving a disease or condition in a human or animal, comprising administering to one or more cells of a human or animal a polynucleotide vector system of any one of embodiments 1 to 36 or a recombinant viral particle of any one of embodiments 37 to 41, wherein the ABCA4 or a functional variant thereof provides treatment or improvement for the disease or condition and is expressed in the one or more cells.

[0211] 45. The method of implementation scheme 44, wherein the disease or condition is xanthophyllosis.

[0212] 46. ​​The method of embodiment 44 or 45, wherein the ABCA4 polypeptide comprises the amino acid sequence of SEQ ID NO: 2 or a functional fragment thereof.

[0213] 47. A method of administering the polynucleotide vector system of any one of embodiments 1 to 36 or the recombinant viral particles of any one of embodiments 37 to 41, thereby minimizing the amount of truncated ABCA4 protein produced by administering the polynucleotide vector system.

[0214] 48. A method of administering the polynucleotide vector system of any one of embodiments 1 to 36 or the recombinant viral particles of any one of embodiments 37 to 41, thereby minimizing cytotoxicity resulting from the administration of the polynucleotide vector system.

[0215] 49. The method of any one of embodiments 44 to 48, wherein the administration of the polynucleotide carrier system provides partial or complete restoration of vision loss.

[0216] 50. The method of any one of embodiments 44 to 49, wherein the polynucleotide carrier system is administered via parenteral administration, intravenous administration, intramuscular administration, intraocular administration, intranasal administration, subretinal administration, round window injection, or during cochlear implantation surgery.

[0217] sequence list

[0218] The following sequences are included for clarity and should not be considered as limitations on the disclosed methods and compositions. The AAV vector sequences disclosed herein are non-limiting examples of the disclosed AAV vectors. These sequences include non-limiting examples of components of the disclosed AAV vectors, including, but not limited to, non-limiting examples of coding sequences for the ABCA4 portion, such as SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19; non-limiting examples of ITR sequences, such as the AAV2 ITR sequence; non-limiting examples of promoters, such as the chicken β-actin promoter; non-limiting examples of enhancers, such as cytomegalovirus (CMV) enhancers; non-limiting examples of splicing donor or acceptor sites; non-limiting examples of homologous regions, such as the AP head sequence; non-limiting examples of intron sequences, such as the alkaline phosphatase (AP) intron sequence; and non-limiting examples of polyadenylated sequences, such as the Min2 bGH polyA sequence (SEQ ID NO: 21).

[0219] The nucleotide sequence of human ABCA4 (SEQ ID NO: 1)

[0220]

[0221] The amino acid sequence of human ABCA4 (SEQ ID NO: 2)

[0222]

[0223] First AAV vector, splitting point after exon 19; S007-HF (SEQ ID NO: 3)

[0224]

[0225] Second AAV vector, the splitting point before exon 20; S008-HB (SEQ ID NO: 4)

[0226]

[0227] First AAV vector, splitting point after exon 20; S009-HF (SEQ ID NO: 5)

[0228]

[0229] Second AAV vector, the splitting point before exon 21; S010-HB (SEQ ID NO 6)

[0230]

[0231] First AAV vector, splitting point after exon 21; S011-HF (SEQ ID NO: 7)

[0232]

[0233] Second AAV vector, the splitting point before exon 22; S012-HB (SEQ ID NO: 8)

[0234]

[0235] S007-HF ABCA4 nucleotide sequence only (SEQ ID NO: 9)

[0236]

[0237] S007-HF ABCA4 amino acid sequence only (SEQ ID NO: 10)

[0238]

[0239] S008-HB ABCA4 nucleotide sequence only (SEQ ID NO: 11)

[0240]

[0241] S008-HB ABCA4 amino acid sequence only (SEQ ID NO: 12)

[0242]

[0243] S009-HF ABCA4 nucleotide sequence only (SEQ ID NO: 13)

[0244]

[0245] S009-HF ABCA4 amino acid sequence only (SEQ ID NO: 14)

[0246]

[0247] S010-HB ABCA4 nucleotide sequence only (SEQ ID NO: 15)

[0248]

[0249] S010-HB ABCA4 amino acid sequence only (SEQ ID NO: 16)

[0250]

[0251] S011-HF ABCA4 nucleotide sequence only (SEQ ID NO: 17)

[0252]

[0253] S011-HF ABCA4 amino acid sequence only (SEQ ID NO: 18)

[0254]

[0255] S012-HB ABCA4 nucleotide sequence only (SEQ ID NO: 19)

[0256]

[0257] S012-HB ABCA4 amino acid sequence only (SEQ ID NO: 20)

[0258]

[0259] Min2pA sequence (SEQ ID NO: 21)

[0260]

[0261] AP sequence (SEQ ID NO: 22)

[0262]

[0263] smCBA promoter (SEQ ID NO: 23)

[0264]

[0265] Splice donor (SEQ ID NO: 24)

[0266]

[0267] Scissor receptor (SEQ ID NO: 25)

[0268]

[0269] 5' ITR (SEQ ID NO: 26)

[0270]

[0271] 3' ITR (SEQ ID NO: 27)

[0272]

[0273] AAV44.9 (E531D) (SEQ ID NO: 28)

[0274]

[0275] Spliced ​​sequence (SEQ ID NO: 29)

[0276]

Claims

1. A polynucleotide vector system for providing the ABCA4 gene, said polynucleotide vector system comprising... a. A first AAV vector polynucleotide, comprising the first ABCA4 sequence of the ABCA4 gene, and b. A second AAV vector polynucleotide comprising the second ABCA4 sequence of the ABCA4 gene. The ABCA4 gene encodes an ABCA4 protein that has at least 95% identity with SEQ ID NO: 2; the first ABCA4 sequence contains exons 1 to 20 of the ABCA4 gene, and the second ABCA4 sequence contains exons 21 to 50 of the ABCA4 gene.

2. The polynucleotide vector system of claim 1, wherein the first ABCA4 sequence does not overlap with the second ABCA4 sequence.

3. The polynucleotide vector system of claim 1, wherein the last approximately 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides of the first ABCA4 sequence do not overlap with the first approximately 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides of the second ABCA4 sequence.

4. The polynucleotide vector system of any one of claims 1 to 3, wherein the first ABCA4 sequence does not contain any one of exons 21 to 50 of the ABCA4 gene, and the second ABCA4 sequence does not contain any one of exons 1 to 20 of the ABCA4 gene.

5. The polynucleotide vector system according to any one of claims 1 to 4, wherein the ABAC4 gene has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

1.

6. The polynucleotide vector system of any one of claims 1 to 5, wherein the first ABCA4 sequence comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

13.

7. The polynucleotide vector system of any one of claims 1 to 6, wherein the second ABCA4 sequence comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

15.

8. The polynucleotide vector system of any one of claims 1 to 7, wherein the first AAV vector polynucleotide comprises a promoter upstream of the first ABCA4 sequence.

9. The polynucleotide vector system of claim 8, wherein the promoter is smCBA promoter, CMV promoter, EF-1α promoter, cone repressor protein promoter, human ABCA4 promoter, TαC gene promoter, rhodopsin promoter, cGMP-phosphodiesterase β subunit promoter, human rhodopsin promoter, mouse rhodopsin promoter, hGRK1 promoter, rod-specific IRBP promoter, or VMD2 promoter.

10. The polynucleotide vector system of claim 8, wherein the promoter is the smCBA promoter.

11. The polynucleotide vector system of claim 8, wherein the promoter comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

23.

12. The polynucleotide vector system of any one of claims 1 to 11, wherein the first AAV vector polynucleotide comprises a splicing donor site.

13. The polynucleotide vector system of claim 12, wherein the splicing donor site has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

24.

14. The polynucleotide vector system of any one of claims 1 to 13, wherein the second AAV vector polynucleotide comprises a splice acceptor site.

15. The polynucleotide vector system of claim 14, wherein the splice acceptor site has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

25.

16. The polynucleotide vector system of any one of claims 1 to 15, wherein the first AAV vector polynucleotide and / or the second AAV vector polynucleotide comprises an alkaline phosphatase (AP) head sequence.

17. The polynucleotide vector system of claim 16, wherein the first AAV vector polynucleotide comprises the AP head sequence, and the AP head sequence comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

22.

18. The polynucleotide vector system of claim 16, wherein the second AAV vector polynucleotide comprises the AP head sequence, and the AP head sequence comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

22.

19. The polynucleotide vector system of any one of claims 1 to 18, wherein the second AAV vector polynucleotide comprises a polyadenylation (pA) signal sequence.

20. The polynucleotide vector system of claim 19, wherein the pA signal sequence comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

21.

21. The polynucleotide vector system of any one of claims 1 to 20, wherein the first AAV vector polynucleotide and the second AAV vector polynucleotide each comprise 5' AAV ITR and 3' AAV ITR.

22. The polynucleotide vector system of claim 21, wherein the 5' AAV ITR comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

26.

23. The polynucleotide vector system of claim 21 or claim 22, wherein the 3' AAV ITR comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

27.

24. The polynucleotide vector system of any one of claims 21 to 23, wherein the 5' AAV ITR and the 3' AAV ITR belong to the AAV2 serotype.

25. A recombinant viral particle comprising the first AAV vector polynucleotide or the second AAV vector polynucleotide as described in any one of claims 1 to 24.

26. The recombinant viral particle of claim 25, wherein the recombinant viral particle comprises 。 27. The recombinant viral particle of claim 25, wherein the recombinant viral particle comprises an AAV44.9 (E531D) capsid.

28. An isolated host cell comprising the polynucleotide vector system of any one of claims 1 to 24 or the recombinant viral particles of any one of claims 25 to 27.

29. The isolated host cell of claim 28, wherein the cell is a photoreceptor cell, cone cell, rod cell, retinal cell, ganglion cell, retinal pigment epithelial cell, vestibular hair cell, inner ear hair cell, or outer ear hair cell.

30. A method for treating or improving a disease or condition in a human or animal, comprising administering to one or more cells of the human or animal a polynucleotide vector system of any one of claims 1 to 24 or a recombinant viral particle of any one of claims 25 to 27, wherein expression of the ABCA4 gene treats or improves the disease or condition and is expressed in the one or more cells.

31. The method of claim 30, wherein the disease or condition is xanthophyllosis.

32. The method of claim 30 or claim 31, wherein the treatment provides partial or complete recovery of vision loss.

33. The method of any one of claims 30 to 32, wherein the polynucleotide carrier system is administered via parenteral administration, intravenous administration, intramuscular administration, intraocular administration, intranasal administration, subretinal administration, round window injection, or during cochlear implantation surgery.

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