Dual AAV vectors for treating Stargardt disease

Abstract

Disclosed herein are compositions and methods for treating mammalian eye diseases, particularly complications associated with Stargardt disease. This disclosure provides an AAV-based dual-vector system that facilitates the expression of full-length proteins whose coding sequences exceed the polynucleotide packaging capacity of individual AAV vectors. In particular, methods and compositions relating to the expression of full-length ABCA4 using an AAV-based dual-vector system are described herein.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Application No. 63 / 499,482, filed on May 1, 2023, which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application is filed with a sequence listing in electronic form. The sequence listing is provided as a file titled 58774 - 726.601.xml, created on April 25, 2024, with a size of 93,061 bytes. The information in the electronic form of the sequence listing is hereby incorporated by reference in its entirety.

Background Art

[0003] Background Recombinant AAV has emerged as a useful gene delivery vehicle for treating retinal diseases. However, one limitation of AAV is its relatively small DNA packaging capacity of approximately 4.7 kilobases (kb). Thus, standard AAV vector systems are not suitable for addressing diseases in which large genes, such as those causing Stargardt disease, are mutated or otherwise dysfunctional. A solution is needed to package large genes into AAV vector systems and safely deliver gene therapy treatments to patients.

Summary of the Invention

[0004] Summary Disclosed are rAAV dual vectors and polynucleotide vector systems and compositions useful for delivering various nucleic acid segments for use in various gene therapy regimens. Further disclosed are recombinant viral particles, isolated host cells, and pharmaceutical compositions comprising any of these rAAV dual vectors and polynucleotide vector systems. Also provided are methods for preparing the improved rAAV dual vector systems disclosed herein, and for using them in virus-based gene therapy, methods for treating and / or achieving remission of symptoms of ATP-binding cassette transporter (ABCA4) defects, including, but not limited to, the treatment of human Stargardt disease. In various aspects, the treatment 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.

[0005] In some embodiments, disclosed herein is a polynucleotide vector system for providing an 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 that is at least 95% identical to 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 and the first nucleotide of the second ABCA4 sequence do not overlap. 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 21-50 of the ABCA4 gene, and the second ABCA4 sequence does not contain any of exons 1-20 of the ABCA4 gene. In some embodiments, the ABAC4 gene is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to Sequence ID No. 1. In some embodiments, the first ABCA4 sequence contains a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 13. In some embodiments, the second ABCA4 sequence contains a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 15. In some embodiments, the first AAV vector polynucleotide contains a promoter upstream of the first ABCA4 sequence.In some embodiments, the promoter is the smCBA promoter, CMV promoter, EF-1 alpha promoter, pyramidal arrestin 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 the smCBA promoter. In some embodiments, the promoter contains a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23. In some embodiments, the first AAV vector polynucleotide contains a splice donor site. In some embodiments, the splice donor site is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24. In some embodiments, the second AAV vector polynucleotide contains a splice acceptor site. In some embodiments, the splice acceptor site is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 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, the AP head sequence comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical sequence to Sequence ID No. 22.In some embodiments, the second AAV vector polynucleotide includes an AP head sequence, the AP head sequence containing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical sequences to SEQ ID NO: 22. In some embodiments, the second AAV vector polynucleotide includes a polyadenylation (pA) signal sequence. In some embodiments, the pA signal sequence contains at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical sequences to SEQ ID NO: 21. In some embodiments, the first AAV vector polynucleotide and the second AAV vector polynucleotide each include a 5'AAV ITR and a 3'AAV ITR. In some embodiments, the 5'AAV ITR contains a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 26. In some embodiments, the 3'AAV ITR contains a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 27. In some embodiments, the 5'AAV ITR and 3'AAV ITR belong to the AAV2 serotype.

[0006] In some aspects, what is described herein is a recombinant viral particle comprising a first AAV vector polynucleotide or a second AAV vector polynucleotide as described herein. In some embodiments, the recombinant viral particle comprises AAV44.9(E531D), AAV7m8, AAV-DJ, AAV2 / 2-MAX, AAVSHh10, AAVSHh10Y, AAV3b, AAVLK03, AAV8BP2, AAV1(E531K), AAV6(D532N), AAV6-3pmut, AAV2G9, AAV44.9, AAVrh.8, AAVrh.8R, or AAVAnc80 capsid. In some embodiments, the recombinant viral particle comprises AAV44.9(E531D) capsid.

[0007] In some aspects, what is described herein is an isolated host cell containing a polynucleotide vector system or recombinant viral particle as described herein. In some embodiments, 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.

[0008] In some aspects, what is described herein is a method for treating or improving a disease or condition in a human or animal, the method comprising administering to one or more cells of a human or animal a polynucleotide vector system or recombinant viral particles described herein, wherein the expression of the ABCA4 gene treats or alleviates the disease or condition and is expressed in one or more cells. In some embodiments, the disease or condition is Stargardt disease. In some embodiments, treatment provides partial or complete recovery of vision loss. In some embodiments, the polynucleotide vector system is administered by parenteral administration, intravenous administration, intramuscular administration, intraocular administration, intranasal administration, subretinal administration, round window injection, or during cochlear implant surgery.

[0009] To facilitate understanding of the principles of this disclosure, references to embodiments or examples described in the drawings will be made, and specific language will be used to describe them. However, it will be understood that this is not intended to limit the scope of this disclosure. Any changes and further modifications to the embodiments described, as well as any further applications of the principles of the disclosure described herein, are intended to be those that a person skilled in the art in which this disclosure relates would ordinarily conceive. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 shows a schematic diagram of a non-limiting example of a dual-vector construct. Dashed boxes indicate constructs that demonstrated efficacy both in vitro and in vivo.

[0011] [Figure 2] Figure 2 shows the results of Western blots from the evaluation of dual-vector transduction in vitro. The first column (from left) is the ladder. Columns 2 and 3 are biological replications (individual transductions) of cultured cells transduced with S007 and S008. Columns 4 and 5 are biological replications (individual transductions) of cultured cells transduced with S009 and S010. Columns 6 and 7 are biological replications (individual transductions) of cultured cells transduced with S011 and S012.

[0012] [Figure 3]Figures 3A and 3B demonstrate that the dual AAV vector produces robust levels of full-length ABCA4 in the retinas of Abca4 - / - mice. Figure 3A is an image of Western blot results showing ABCA4 expression. Starting from the left, column 1 is the ladder. Columns 2-5 are biological replicas (mouse individuals) transduced with S009 and S0010. Columns 6-9 are biological replicas (mouse individuals) transduced with S011 and S012. Columns 10-16 are from mice that were not administered the vector. Columns 10-11 are Abca4 + / - mice. Column 13 is from Abca4 - / - mice. Columns 14-16 are from Abca4 + / + mice. Figure 3B is a graph showing the quantification of ABCA4 expression relative to wild-type (normalized to vinculin).

[0013] [Figure 4] Figure 4 shows a graph of image pixel intensity measurements from scanning laser ophthalmoscopic (cSLO) images, which demonstrates that subretinal injection of the S009-S010 ABCA4 dual AAV vectors (SEQ ID NOs. 5 and 6, respectively) is sufficient to reduce the retinal autofluorescence phenotype features of Abca4 - / - knockout mice compared to the contralateral eye to which the vehicle was injected.

[0014] [Figure 5A] Figures 5A–5C show graphs of electroretinogram (ERG) data demonstrating that subretinal injection of the S009–S010 ABCA4 dual AAV vector is well tolerated in Abca4 - / - mice. The ERG response in dual-vector treated mice is indistinguishable from that of the contralateral eye injected with the vehicle at 2 months post-injection. Figure 5A shows the amplitude of the dark adaptation a wave. [Figure 5B-C]Figures 5A–5C show graphs of electroretinogram (ERG) data demonstrating that subretinal injection of the S009-S010 ABCA4 dual AAV vector is well tolerated in Abca4 - / - mice. The ERG response in dual-vector treated mice is indistinguishable from the contralateral eye that received the vehicle injection two months after injection. Figure 5B shows the amplitude of the dark-adapted b-wave. Figure 5C shows the amplitude of the light-adapted b-wave. [Modes for carrying out the invention]

[0015] Detailed explanation Illustrative aspects of this disclosure are described below. This disclosure provides materials and methods for gene therapy for diseases and conditions such as Stargardt disease. Stargardt disease is a form of macular degeneration. The disease is an autosomal recessive disorder that can lead to blindness and affects 1 in 8,000 people. Stargardt disease is associated with bi-allele 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 defect in ABCA4 results in improper transport of vitamin A. In some embodiments, a defect in ABCA4 results in the accumulation of toxic byproducts such as bisretinoids in the retina.

[0016] Aspects of this disclosure relate to AAV-based dual-vector systems that enable the expression of full-length proteins in which the coding sequence exceeds the polynucleotide packaging capacity of individual AAV vectors. The present invention provides nucleic acid vectors for dual-vector systems (e.g., overlapping vector systems or hybrid vector systems).

[0017] In some embodiments, the vector system of this disclosure employs two separate AAV vectors, each packaging a relatively large DNA molecule (e.g., about 4.5–4.8 Kb) containing a portion of the ABCA4 gene. The two vectors are co-administered to selected recipient cells to reconstruct a full-length ABCA4 gene encoding a biologically active ABCA4 polypeptide. In some embodiments, a portion of the nucleic acid sequence is common to each of the vector genomes (e.g., the common portion contains a non-coding sequence). Upon co-delivery to suitable cells, the common sequence region facilitates the correct concatemerization of the two partial gene cassettes. These gene cassettes then undergo homologous recombination to generate a full-length gene cassette within the cell. From the resulting RNA, the common nucleic acid sequence is then spliced ​​out. Non-limiting components of non-limiting aspects of the dual-vector system include the use of AAV inverted terminal repeat sequences (ITRs), a small (truncated) version of the chimeric CMV / chicken β-actin promoter (smCBA), human ABCA4 cDNA sequences, and miniature bovine growth hormone polyadenylation (pA) signals (mini2pA).

[0018] Dual Vector System In some embodiments of this disclosure, the dual-vector system of this disclosure includes: (i) A first AAV vector polynucleotide comprising an inverted end repeat sequence at each end of the polynucleotide (e.g., the 5' end and the 3' end), a suitable promoter between the inverted end repeat sequences, followed by a partial coding sequence (e.g., at the 3' end of the promoter) encoding the N-terminal portion of a selected full-length polypeptide, followed by a splice donor site and an intron, and (ii) A second AAV vector polynucleotide comprising an inverted end repeat sequence at each end (5' and 3' end) of the polynucleotide, an intron and a splice acceptor site for the intron between the inverted end repeat sequences, optionally followed by a partial coding sequence encoding the C-terminal portion of a selected full-length polypeptide, and optionally followed by a polyadenylation (pA) signal sequence. In some embodiments, the intron sequences in the first and second AAV vectors include sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an alkaline phosphatase homologous recombination sequence (APhead), e.g., sequence number 22.

[0019] In some embodiments, the partition point between the first and second AAV polynucleotide sequences is between exon 19 and exon 20 of the hABCA4 gene. In some embodiments, the partition point between the first and second AAV polynucleotide sequences is between exon 20 and exon 21 of the hABCA4 gene. In some embodiments, the partition point between the first and second AAV vector polynucleotide sequences is between exon 21 and exon 22 of the hABCA4 gene.

[0020] 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 that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 4, 6, or 8, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first AAV vector polynucleotide contains the nucleotide sequence of SEQ ID NO: 3, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second AAV vector polynucleotide contains the nucleotide sequence of SEQ ID NO: 4, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first AAV vector polynucleotide contains the nucleotide sequence of SEQ ID NO: 5, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second AAV vector polynucleotide contains the nucleotide sequence of SEQ ID NO: 6, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.In some embodiments, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 7, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0021] The coding sequences in the first vector and the second vector, when combined, 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.

[0022] In some embodiments, all or part of the intron sequence present at the 3' end of the coding sequence of the first vector is identical or substantially identical to all or part of the intron sequence present at the 5' end of the coding sequence of the second vector.

[0023] In certain embodiments, the intron sequence utilized in any vector system of the present disclosure is the sequence of an intron that naturally exists in the genomic sequence of the gene encoding the selected polypeptide. In some embodiments, the intron comprises an alkaline phosphatase (AP) sequence. In some embodiments, the intron comprises an alkaline phosphatase homologous recombination sequence (APhead). In some embodiments, the alkaline phosphatase homologous recombination sequence comprises the nucleotide sequence of SEQ ID NO: 22, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0024] This specification provides examples of strategies that can overcome the problem of random concatemerization and thereby increase not only the specificity but also the efficiency of these dual-vector platforms. Firstly, the addition of highly recombinogenic sequences, such as those used in the example dual vectors, results in a significant increase in protein expression compared to trans-splicing systems. The finding that AP dual vectors are more efficient than trans-splicing vectors supports the idea that AP sequences direct at least a portion of the concatemerization event in the right direction, and then recombination occurs via this sequence or via the ITR. In particular, the APhead domain can mediate proper head-to-tail concatemerization after recombination of dual vectors in cells. In some embodiments, the example dual-vector system with AP sequences promotes the correct alignment of more concatemers. In some embodiments, the example dual-vector system with AP sequences mediates more efficient expression of ABCA4. In some embodiments, a dual-vector system without AP sequences is used.

[0025] In some embodiments, the intron sequence utilized in the dual-vector system of this disclosure is an intron sequence that is not naturally present in the genomic sequence of a gene encoding a selected polypeptide. In some embodiments, the intron sequence is derived from the MYO7A gene. In certain embodiments, the intron is a synthetic alkaline phosphatase (AP) intron. The intron sequence utilized in the dual-vector system of this disclosure may include splice donor sequences and splice acceptor sequences. In some embodiments, the intron sequence is a recombination-inducible intron sequence (e.g., the AK sequence of an F1 phage). In these embodiments, the dual vector relies on both ITR-mediated concatemerization and AK sequence-mediated homologous recombination for the reconstruction of the full-length expression cassette. Thus, in some embodiments, the intron sequence is the AK sequence of an F1 phage. Accordingly, in some embodiments of the disclosed dual vector, the vector includes one or more AP intron spliceosome recognition sites, e.g., one or more AP splice acceptor (APSA) domains or AP splice donor (APSD) domains. In some embodiments, these vectors include APSA and APSD. In some embodiments, the first vector contains APSA and the second vector contains APSD. In some embodiments, the first vector contains APSD and the second vector contains APSA. See Figure 1. In some embodiments, the vector includes one or more non-AP intron spliceosome recognition sites. In some embodiments, the splice donor contains the nucleotide sequence of SEQ ID NO: 24, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the splice acceptor contains a nucleotide sequence of SEQ ID NO: 25, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0026] Another approach to direct concatemerization is the use of a single-stranded oligonucleotide that can tether the trailing end of the 5' vector to the leading end of the 3' vector. However, this strategy requires the oligonucleotide to be efficiently delivered to the nucleus of the target cell in a timed manner with the dual vector. Finally, head-to-tail concatemerization can be directed using dual vectors that utilize mismatched ITRs, although this process may require further optimization of the AAV packaging mechanism. Thus, in some embodiments, the dual vector pair contains a sequence encoding APhead as part of the AP intron.

[0027] 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 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 intron sequence is the AK sequence of the F1 phage.

[0028] In some embodiments, the partition point between the first and second AAV vector polynucleotide sequences is between exon 21 and exon 22 of the hABCA4 gene. In some embodiments, the partition point between the first and second AAV vector polynucleotide sequences is between exon 20 and exon 21 of the hABCA4 gene. In some embodiments, the partition point between the first and second AAV vector polynucleotide sequences is between nucleic acids 3050 and 3051 of the hABCA4 gene, as numbered in Sequence ID No. 1. In some embodiments, the partition point between the first and second AAV vector polynucleotide sequences is between exon 19 and exon 20 of the hABCA4 gene.

[0029] In some embodiments, the first AAV vector polynucleotide contains a nucleotide sequence encoding the N-terminal portion of ABCA4. In some embodiments, the first AAV vector polynucleotide contains the sequence of SEQ ID NO: 9, SEQ ID NO: 13, or SEQ ID NO: 17, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first AAV vector encodes the N-terminal portion of ABCA4, containing the sequence of SEQ ID NO: 10, SEQ ID NO: 14, or SEQ ID NO: 18, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first AAV vector polynucleotide contains the sequence of SEQ ID NO: 9, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first AAV vector encodes the N-terminal portion of ABCA4, containing the sequence of SEQ ID NO: 10, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first AAV vector polynucleotide contains the sequence of SEQ ID NO: 13, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first AAV vector encodes the N-terminal portion of ABCA4, containing the sequence of SEQ ID NO: 14, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first AAV vector polynucleotide contains the sequence of SEQ ID NO: 17, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.In some embodiments, the first AAV vector encodes the N-terminal portion of ABCA4, which contains the sequence of sequence number 18, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0030] In some embodiments, the second AAV vector polynucleotide contains a nucleotide sequence encoding the C-terminal portion of ABCA4. In some embodiments, the second AAV vector polynucleotide contains the sequence of SEQ ID NO: 11, SEQ ID NO: 15, or SEQ ID NO: 19, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the second AAV vector encodes the C-terminal portion of ABCA4, containing the sequence of SEQ ID NO: 12, SEQ ID NO: 16, or SEQ ID NO: 20, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the second AAV vector polynucleotide contains the sequence of SEQ ID NO: 11, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the second AAV vector encodes the C-terminal portion of ABCA4, containing the sequence of SEQ ID NO: 12, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the second AAV vector polynucleotide contains the sequence of SEQ ID NO: 15, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the second AAV vector encodes the C-terminal portion of ABCA4, containing the sequence of SEQ ID NO: 16, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the second AAV vector polynucleotide contains the sequence of SEQ ID NO: 19, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.In some embodiments, the second AAV vector encodes the C-terminal portion of ABCA4, which contains the sequence of sequence number 20, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0031] In some embodiments, a polynucleotide vector system is provided that includes the following: i) A first AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, a promoter between the inverted terminal repeat sequences, followed by a partial coding sequence encoding the N-terminal portion of the ABCA4 polypeptide, followed by a splice donor site and an intron, and ii) A second AAV vector polynucleotide comprising inverted terminal repeat sequences at each end of the polynucleotide, introns and splice acceptor sites for the introns between the inverted terminal repeat sequences. In some embodiments, between the inverted terminal repeat sequences are introns and splice acceptor sites for the introns, followed by a subcoding sequence encoding the C-terminal portion of the ABCA4 polypeptide, and optionally followed by a polyadenylation (pA) signal sequence.

[0032] In some embodiments, the intron sequences in the first and second AAV vectors include a common polynucleotide sequence, where the common polynucleotide sequence is not part of the coding sequence encoding the ABCA4 polypeptide. In some embodiments, the intron sequences in the first AAV vector and the intron sequences in the second AAV vector include sequences that are at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22.

[0033] In some embodiments, the inverted terminal repeat sequence contains the sequence of SEQ ID NO: 26 or 27, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it. In some embodiments, the promoter contains the sequence of SEQ ID NO: 23, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it. In some embodiments, the N-terminal portion of the ABCA4 polypeptide contains the sequence of SEQ ID NO: 14, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the N-terminal portion of the ABCA4 polypeptide is encoded by a sequence containing the sequence of SEQ ID NO: 13, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the C-terminal portion of the ABCA4 polypeptide contains the sequence of SEQ ID NO: 16, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the C-terminal portion of the ABCA4 polypeptide is encoded by a sequence containing the sequence of SEQ ID NO: 15, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the splice donor site contains the sequence of SEQ ID NO: 24, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the splice acceptor site contains the sequence of SEQ ID NO: 25, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.In some embodiments, the intron contains the sequence of sequence number 22, or a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0034] In some embodiments of the dual vector systems 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.

[0035] The coding sequences in the first and second vectors, when combined, encode a selected full-length polypeptide, or a functional fragment or variant thereof. Thus, in some embodiments, all or part of the intron sequence located at the 3' end of the coding sequence of the first vector is identical or substantially identical to all or part of the intron sequence located at the 5' end of the coding sequence of the second vector, where the intron sequence is not part of the coding sequence encoding the ABCA4 polypeptide.

[0036] Some embodiments of the dual vectors described herein intend a virus or recombinant viral particle comprising a first AAV vector polynucleotide or a second AAV vector polynucleotide as described herein. In certain embodiments, the first AAV vector polynucleotide contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical sequences to SEQ ID NO: 5, and the second AAV vector polynucleotide contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical sequences to SEQ ID NO: 6. In some embodiments, the virus or recombinant viral particle is characterized as an adeno-associated virus (AAV) or an infectious AAV virus particle. In some embodiments, recombinant AAV virus particles contain one or more tyrosine-to-phenylalanine (YF) mutations in the viral or virion capsid protein. A tyrosine-to-phenylalanine (YF) mutation at amino acid position 733 of the viral or virion capsid protein is particularly intended herein (e.g., AAV8 Y733F). Similarly, a tyrosine-to-phenylalanine (YF) mutation at amino acid position 731 of the viral or virion capsid protein is also particularly intended herein (e.g., AAV44.9 (Y731F)).

[0037] In some embodiments, the virus or virion is packaged in AAV5, AAV7, AAV8, AAV9, AAV44.9, AAV44.9(E531D), AAV2(4pMut)ΔHS, AAV2, AAVAnc80, AAVrh.8, AAVrh.8R, AAVrh.10, or AAVrh.74 capsids. In some embodiments, the viral particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV7m8, AAV-DJ, AAV2 / 2-MAX, AAVSHh10, AAVSHh10Y, AAV3b, AAVLK03, AAV8PB2, AAV1(E531K), AAV6(D532N), AAV6-3pmut, AAV2G9, AAV44.9, AAV44.9(E531D), AAVrh.8, AAVrh.8R, and / or AAVAnc80 capsids. In some embodiments, the virions are packaged within the AAV44.9(E531D) capsid variant.

[0038] In some embodiments, the dual polynucleotide vector systems described herein utilize tissue-specific promoters. In some embodiments, the systems utilize promoters that mediate expression in the eye.

[0039] In some embodiments, the dual polynucleotide vector systems described herein use one of the following promoters: cytomegalovirus (CMV) promoter, elongation factor 1-alpha (EF-1-alpha) promoter, pyramidal arrestin promoter, chimeric CMVβ-actin (smCBA) promoter, ABCA4 gene-derived promoter, pyramidal transducin α (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 vitiligo macular degeneration-2 (VMD2) promoter, and combinations thereof. In some embodiments, the polynucleotide vector systems described herein use the human rhodopsin kinase (hGRK1) promoter. In some embodiments, the polynucleotide vector systems use the pyramidal arrestin promoter. In some embodiments, the polynucleotide vector systems use the cytomegalovirus (CMV) promoter. In some embodiments, the polynucleotide vector systems use the elongation factor 1-alpha (EF-1-alpha) promoter.

[0040] In some embodiments, any vector of the dual polynucleotide vector system described herein may be administered by parenteral administration, such as intravenous, intramuscular, intraocular, or intranasal administration. The vector may be administered in vivo, in vitro, or ex vivo. In certain embodiments, the vectors provided herein may be administered by subretinal injection. The vector may be administered in vivo or ex vivo.

[0041] In some embodiments, any vector from the dual polynucleotide vector systems described herein may be administered to the eye. In certain embodiments, the vector is administered to the target eye by subretinal injection.

[0042] The methods of this disclosure may be used on humans and other animals. Animals intended 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. Where used herein, the terms “patient” and “subject” are used interchangeably and are intended to encompass human and non-human species, including human and non-human cells. Similarly, the in vitro methods of this disclosure may also be performed on cells of one or more human or non-human mammalian species, including human and non-human cells.

[0043] Components of a non-restrictive example of a dual AAV vector Any of the dual polynucleotide vector systems of this disclosure may be used in combination with AAV vector systems known in the art. In treating some diseases, a single administration of the rAAV vector construct may be preferred, while in managing or treating other diseases or conditions, it may be desirable to provide two or more administrations of the vector construct to the patient over a period of administration. In such situations, the AAV vector-based treatment may be delivered one or more times consecutively, daily, weekly, monthly, or at less frequent intervals, as may be required to achieve treatment or remission of one or more symptoms of the disease or disorder being treated. In some embodiments, the vector may be delivered to one or both eyes by administration of infectious adeno-associated virus particles, rAAV virions, or multiple infectious rAAV particles in an amount and duration sufficient to treat or achieve remission of one or more symptoms of the disease or disorder being treated.

[0044] Some aspects of the disclosed vectors intend to be viruses or recombinant viral particles comprising a first AAV vector polynucleotide or a second AAV vector polynucleotide as described herein. In some embodiments, the viruses or recombinant viral particles are characterized as adeno-associated viruses (AAV) or infectious AAV virus particles. In some embodiments, the recombinant AAV virus particles encapsulate one or more tyrosine-to-phenylalanine (YF) mutations in the capsid protein of the virus or virion. The tyrosine-to-phenylalanine (YF) mutation at amino acid position 733 of the capsid protein of the virus or virion is particularly intended herein (e.g., AAV8 Y733F).

[0045] In some embodiments, the virus or virion is packaged in AAV5, AAV7, AAV8, AAV9, AAV44.9, AAV44.9(E531D), AAV2(4pMut)ΔHS, AAV2, AAVAnc80, AAVrh.8, AAVrh.8R, AAVrh.10, or AAVrh.74 capsids. In some embodiments, the viral particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV7m8, AAV-DJ, AAV2 / 2-MAX, AAVSHh10, AAVSHh10Y, AAV3b, AAVLK03, AAV8PB2, AAV1(E531K), AAV6(D532N), AAV6-3pmut, AAV2G9, AAV44.9, AAV44.9(E531D), AAVrh.8, AAVrh.8R, and / or AAVAnc80 capsids. In some embodiments, the virions are packaged within the AAV44.9(E531D) capsid variant.

[0046] In some embodiments, the polynucleotide vector systems described herein utilize tissue-specific promoters. In some embodiments, the systems utilize promoters that mediate expression in the eye.

[0047] In some embodiments, the polynucleotide vector systems described herein use one of the following promoters: cytomegalovirus (CMV) promoter, elongation factor 1-alpha (EF-1-alpha) promoter, pyramidal arrestin promoter, chimeric CMVβ-actin promoter (CBA), truncated CMVβ-actin (smCBA) promoter, human ABCA4 gene-derived promoter, pyramidal transducin 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 vitiligo macular degeneration-2 (VMD2) promoter, and combinations thereof. In some embodiments, the polynucleotide vector systems described herein use the human rhodopsin kinase (hGRK1) promoter. In some embodiments, the polynucleotide vector systems use the pyramidal arrestin promoter. In some embodiments, the polynucleotide vector systems use the cytomegalovirus (CMV) promoter. In some embodiments, polynucleotide vector systems utilize the elongation factor 1-alpha (EF-1-alpha) promoter.

[0048] In certain embodiments, the Disclosure provides rAAV particles derived from a number of different serotypes, including, for example, serotypes selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10. In some embodiments, particles derived from vectors of AAV2, AAV5, and AAV8 serotypes are utilized. In certain embodiments, particles having an AAV8(Y733F) or AAV2(tripleY-F) capsid are used. Accordingly, the Disclosure provides, for example, recombinant AAV particles derived from AAV8(Y733F) or AAV2(tripleY-F) comprising a dual polynucleotide vector system. In some embodiments, the serotype of the AAV vector is not AAV6 or AAV2.

[0049] Additional non-limiting capsids include AAV2, AAV6, and capsids derived from AAV2 and AAV6. Such capsids include AAV7m8, AAV-DJ, AAV2 / 2-MAX, AAVSHh10, AAVSHh10Y, AAV3b, AAVLK03, AAV8PB2, AAV1(E531K), AAV6(D532N), AAV6-3pmut, AAV2G9, AAV2G9, AAV44.9, AAV44.9(E531D), AAVrh.8, AAVrh.8R, and / or AAVAnc80. In some embodiments, the virus or virion is packaged in AAV5, AAV7, AAV8, AAV9, AAV44.9, AAV44.9(E531D), AAV2(4pMut)ΔHS, AAV2, AAVAnc80, AAVrh.8, AAVrh.8R, AAVrh.10, or AAVrh.74 capsids.

[0050] The AAV2 / 2-MAX capsid contains five point mutations: Y272F, Y444F, Y500F, Y730F, and T491V. Both AAVSHh10 and AAV6(D532N) capsids are derivatives of AAV6. Additional capsids suitable for use by the disclosed method include: a capsid containing a non-native amino acid substitution in the amino acid residues of the wild-type AAV2 capsid, wherein the non-native amino acid substitution comprises one or more of Y272F, Y444F, T491V, Y500F, Y700F, Y704F, and Y730F; a capsid containing a non-native amino acid substitution in the amino acid residues of the wild-type AAV6 capsid, wherein the non-native amino acid substitution comprises one or more of Y445F, Y705F, Y731F, T492V, and S663V. In one embodiment, the capsid comprises AAV2G9, which is a variant of AAV2.

[0051] In other embodiments, the capsid comprises a non-native amino acid substitution at amino acid residue 533 or 733 of the wild-type AAV8 capsid, where the non-native amino acid substitution is E533K, Y733F, or a combination thereof. In one embodiment of the disclosed method, the capsid comprises AAV8PB2, which is a variant of AAV8.

[0052] In another embodiment, the capsid comprises non-native amino acid substitutions of the wild-type AAV2 capsid, including one or more of the following mutations: (a)Y444F; (b) Y444F + Y500F + Y730F; (c)Y272F+Y444F+Y500F+Y730F; (d) Y444F + Y500F + Y730F + T491V; or (e)Y272F+Y444F+Y500F+Y730F+T491V.

[0053] In another embodiment, the capsid comprises non-native amino acid substitutions of the wild-type AAV6 capsid, including one or more of the following mutations: (a) Y445F; (b) Y705F + Y731F; (c)T492V; (d) Y705F + Y731F + T492V; (e)S663V; or (f)S663V+T492V.

[0054] Additional capsids suitable for use by the disclosed method are described in International Patent Publication No. WO 2018 / 156654, published on 30 August 2018, which is incorporated herein by reference in its entirety. In certain embodiments, the rAAV particles disclosed herein include one of the following capsids: 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 include a capsid selected from AAV6(3pMut) or AAV2(quadYF+TV). In other embodiments, the rAAV particles of the disclosed method may include any of the capsid variants described in International Patent Publication No. WO 2018 / 156654.

[0055] In certain embodiments, disclosed herein are rAAV particles that may contain a DGE-DF capsid, a P2-V2 capsid, a P2-V3 capsid, a P2-V1 capsid (also known as ME-B), or a P2-V1(Y-F+TV) or ME-B(Y-F+TV) capsid for the enhancement of transduction of the rAAV particles in retinal cells. In other embodiments, the disclosed rAAV particles may include a capsid selected from AAV2(Y444F), AAV2(Y444F+Y500F+Y730F), AAV2(Y272F+Y444F+Y500F+Y730F), AAV2(Y444F+Y500F+Y730F+T491V), and AAV2(Y272F+Y444F+Y500F+Y730F+T491V), AAV6(Y445F), AAV6(Y705F+Y731F), AAV6(Y705F+Y731F+T492V), AAV6(S663V), AAV6(T492V), or AAV6(S663V+T492V).

[0056] The inverted terminal repeat (ITR) sequences used in any AAV vector system of this disclosure may include 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 ITRs 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 number PCT / US2020 / 14838, filed on January 23, 2020, incorporated herein by reference). In some embodiments, the AAV vectors of this disclosure include different AAV ITRs. In non-limiting examples, the vector may include the ITR of AAV2 and the ITR of AAV5. The AAV ITR sequences are well known in the art (see, for example, GenBank acceptance numbers AF043303.1;NC_001401.2;J01901.1;JN898962.1;K01624.1; and K01625.1). In some embodiments, the AAV dual-vector systems disclosed herein can efficiently express therapeutic genes that are larger than those that can normally be packaged in a single AAV vector.

[0057] Accordingly, in some respects, the present disclosure provides viruses or virions comprising either polynucleotides or vectors of the present disclosure. In some embodiments, the virus or virion is an AAV virus. Methods for preparing viruses and virions comprising heterologous polynucleotides or vectors are known in the art. In the case of AAV, cells can be co-infected or co-transfected with an adenovirus or polynucleotide vector comprising an adenovirus gene suitable for AAV helper function.

[0058] In some embodiments, the AAV serotype provides one or more tyrosine-to-phenylalanine (YF) mutations on the capsid surface. In some embodiments, the AAV is the AAV8 serotype (Y733F) having a tyrosine-to-phenylalanine (YF) mutation at position 733.

[0059] In some embodiments, a triple mutant AAV8 vector is used that contains a Tyr-Phe mutation from tyrosine to phenylalanine at positions Y733F, Y500F, and Y730F, respectively. In some embodiments, a triple mutant AAV8 vector is used that contains a Tyr-Phe mutation from tyrosine to phenylalanine at positions Y447F, Y733F, and T494V (e.g., AAV8(Y447F+Y733F+T494F)).

[0060] In some embodiments, the rAAV particles of this disclosure contain a transgene or heterologous nucleic acid that is too large for delivery in a standard AAV system. In some embodiments, the transgene is hABCA4, which encodes 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 described in SEQ ID NO: 1.

[0061] In some embodiments, administration of any of the disclosed polynucleotide vectors to the eye of a subject in need may partially or completely restore vision loss. The transgene may include human ABCA4. In some embodiments, these administrations may reduce lipofuscin accumulation in the macula of the subject.

[0062] In some embodiments, the production of a therapeutic agent encoded by a transgene in any of the disclosed polynucleotide vector systems in ocular cells (such as retinal cells or RPE cells) provides one or more of the following therapeutic endpoints: a) preservation of one or more photoreceptor cells or one or more RPE cells, b) restoration of one or more rod-cell and / or cone-cell-mediated function, c) restoration of visual behavior in one or both eyes, or d) any combination thereof. In certain embodiments, the production of the therapeutic agent in the disclosed method preserves one or more PR cells, such as retinal ganglion cells, bipolar cells, Müller glial cells, astrocyte cells, or RPE cells.

[0063] In some embodiments, after initial administration of one or both of the disclosed rAAV polynucleotide vector systems to one or both eyes of a mammal, therapeutic agent production persists substantially for at least 3 months, at least 6 months, at least 9 months, or at least 1 year in one or more photoreceptor cells or one or more RPE cells.

[0064] Thus, the polynucleotide vector systems and compositions of the present disclosure may be used to treat or alleviate symptoms of Stargardt disease in the eye of a subject. In some embodiments, symptoms of Stargardt disease include problems with night vision, problems with color vision, or abnormal accumulation of lipofuscin in the macula of the subject.

[0065] In some embodiments, the Disclosure provides rAAV nucleic acid vectors comprising at least a first nucleic acid segment encoding one or more diagnostic or therapeutic agents that alter, inhibit, reduce, prevent, eliminate, or impair the activity of one or more endogenous biological processes in mammalian cells suitably transformed with the vector of interest. In some embodiments, such diagnostic or therapeutic agents may comprise molecules that selectively inhibit or mitigate the effects of one or more metabolic processes, dysfunctions, impairments, or diseases. In some embodiments, the defect may be caused by injury or trauma to a mammal to which treatment is desired. In other embodiments, the defect may be caused by overexpression of an endogenous biological compound, while in other embodiments, the defect may also be caused by underexpression or even absence of one or more endogenous biological compounds.

[0066] rAAV vector modulo elements Any of the vector systems of this disclosure may include regulatory elements that are functional in the host cell in which the vector is intended to be expressed. These regulatory elements include, for example, promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements.

[0067] Any of the vector systems of this disclosure may include a promoter sequence operably ligated 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, human ABCA4 gene-derived promoters, Roussarcoma virus (RSV) promoters, chimeric CMV / chicken β-actin promoters (CBAs), and truncated forms of CBA (smCBAs). In some embodiments, the promoter includes the smCBA promoter (SEQ ID NO: 23), or a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23. Additional photoreceptor-specific human rhodopsin kinase (hGRK1) promoters, rod-specific IRBP promoters, VMD2 (vitiligo macular degeneration / Best's disease) promoters, RPE-specific vitiligo macular degeneration-2 [VMD2] promoters, and EF1-alpha promoter sequences are also intended to be useful in carrying out various aspects of this disclosure. Non-limiting examples of photoreceptor cell-specific promoters include, but are not limited to, hGRK1, IRBP, rod opsins, NRL, GNAT2e-IRBP, L / M opsins, and pyramidal arrestin promoters.

[0068] In certain embodiments, the promoter is a chimeric CMV-β-actin promoter. In certain 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 certain embodiments, the promoter is a photoreceptor-specific promoter. In further embodiments, the promoter is preferably a cone cell-specific promoter or a rod cell-specific promoter, or any combination thereof. In certain embodiments, the promoter is a promoter of the human ABCA4 gene. In further embodiments, the promoter includes a promoter derived from the cone transducin α (TαC) gene. In certain embodiments, the promoter is a promoter derived from the human GNAT2 gene. 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 [such as the vitiligo macular degeneration-2 (VMD2) promoter (Best1)], or any combination thereof.

[0069] The promoter can be incorporated into the vector using standard techniques known to those skilled in the field of molecular biology and / or virology. Multiple copies of the promoter, and / or multiple different promoters, can be used in the vector of the present disclosure. In one such embodiment, the promoter may be positioned at a distance from the transcription start site that is approximately the same distance from the transcription start site as it is in its native genetic environment, although some variation in this distance is, of course, permissible without substantial reduction in promoter activity. In the implementation of the present disclosure, typically one or more transcription start sites are incorporated into the vector disclosed.

[0070] The vectors of this disclosure may further comprise 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 (IRESs), and / or one or more enhancer elements, or any combination thereof. Transcription termination regions can typically be obtained from the 3' untranslated region of a eukaryotic or viral gene sequence. Transcription termination sequences may be positioned downstream of a coding sequence to provide efficient termination.

[0071] Any of the disclosed polynucleotide vectors may also further include one or more posttranscriptional regulatory sequences or one or more polyadenylation signals, which may include, for example, woodchuck hepatitis virus posttranscriptional regulatory elements (WRPEs), polyadenylation signal sequences, or intron / exon junction / splicing signals, or any combination thereof.

[0072] A signal peptide sequence is an amino-terminal peptide sequence that encodes information that causes a operably linked polypeptide to be positioned at one or more post-translational cellular destinations (e.g., specific organelle compartments, or sites of protein synthesis and / or activity, and even the extracellular environment).

[0073] Enhancers (cis-acting regulatory elements that increase 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 these include, but are not limited to, the CaMV 35S enhancer element, the cytomegalovirus (CMV) initial promoter enhancer element, the SV40 enhancer element, and combinations and / or derivatives thereof. One or more nucleic acid sequences that direct or control the polyadenylation of mRNA encoded by the structural gene of interest may also be optionally included in one or more of the vectors of this disclosure.

[0074] Methods for transduction into host cells and other cells. The present invention provides host cells comprising a vector of a disclosed polynucleotide vector system. In some embodiments, isolated host cells comprising a dual polynucleotide vector system are provided.

[0075] Examples of suitable host cells containing any of the disclosed dual-vector 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), Müller cells, astrocytes, and bipolar cells.

[0076] This disclosure also provides methods for expressing or transducing a selected polypeptide in cells. In certain embodiments, the method comprises incorporating an AAV-based dual vector system as disclosed herein into cells (where the vector system comprises a polynucleotide sequence encoding a selected polypeptide of interest) and expressing the polynucleotide sequence in the cells.

[0077] In some embodiments, the selected polypeptide may be heterologous polypeptide to the cell. In some embodiments, the cell is a mammalian cell, preferably a human cell. In some embodiments, the cell is a human photoreceptor cell, preferably a human photoreceptor cone cell or photoreceptor rod cell. In certain 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 as disclosed herein. In some embodiments, the ABCA4 polypeptide is encoded by the nucleotide sequence shown in SEQ ID NO: 1.

[0078] Accordingly, in some embodiments, the present disclosure provides a method 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., human). In an overall and general sense, such a method comprises administering one or more rAAV particles disclosed herein into one or both eyes of a mammal (e.g., directly subretinal) for a time sufficient to produce a therapeutic agent in one or more PR cells or RPE cells of the mammal, wherein the polynucleotide further comprises at least one first polynucleotide comprising a PR cell or RPE cell-specific promoter operably linked to at least one first heterologous nucleic acid segment encoding the therapeutic agent. 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 astrocyte cells, or a combination thereof.

[0079] Treatment and Transduction Methods In some embodiments, the Disclosure provides methods for treating or relieving diseases or conditions, such as eye diseases, in humans or animals using the gene therapy and AAV-based dual vector systems of the Disclosure. In certain embodiments, the methods of the Disclosure involve administering the vector system of the Disclosure which encodes a polypeptide that provides treatment or remission of a disease or condition. In certain embodiments, the vectors of the Disclosure are provided as AAV viruses or virions. The vector systems can be administered in vivo or ex vivo.

[0080] In some embodiments, the vector systems of the Disclosure are administered in recombinant AAV particles by parenteral administration, such as intravitreous, subretinal, intravenous, intramuscular, intraocular, or intranasal injection. In certain embodiments, the vector systems of the Disclosure are administered to humans or animals by intraocular, intravitreous, or subretinal injection.

[0081] In some aspects, the disease, disorder, or condition to be treated is Stargardt disease. The disclosed dual-vector systems may be introduced into one or more selected mammalian cells using one or more of the methods, which include, but are not limited to, transfection, microinjection, electroporation, lipofection, cell fusion, and calcium phosphate precipitation, as well as bioristic methods. In certain embodiments, the vectors of the Disclosure may be introduced in vivo, for example, by lipofection (e.g., DNA transfection via liposomes prepared from one or more cationic lipids). Synthetic cationic lipids (LIPOFECTIN®, Invitrogen Corp., La Jolla, CA, USA) may be used to prepare liposomes that encapsulate the vectors and facilitate their introduction into one or more selected cells. The vector systems of the Disclosure may also be introduced in vivo as “naked” DNA.

[0082] In a general sense, the methods disclosed include at least the step of administering 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 alleviate one or more symptoms of a mammalian disease, disorder, dysfunction, injury, abnormal condition, or trauma. In some embodiments, the mammal is a human. In some embodiments, the human is a neonat, newborn, infant, or juvenile. In the implementation of the Disclosure, preferred patients are intended to include, for example, humans who have, are suspected of having, are at risk of developing, or have been diagnosed with, one or more retinal disorders, diseases, or dystrophies, including, for example, gene-linked or hereditary retinal disorders, diseases, and dystrophies.

[0083] In some embodiments, the Disclosure provides methods for using the particles, vectors, virions, expression systems, compositions, and host cells described herein in methods for treating or relieving symptoms of various defects in the eye of mammals, and in particular one or more defects in human photoreceptor or RPE cells, or in the preparation of agents for treating or relieving such symptoms. In some embodiments, the subject requiring it is suffering from Stargardt disease. In some embodiments, the subject is suffering from an eye disease or condition that could benefit from treatment with gene therapy comprising the ABCA4 polynucleotide.

[0084] In some embodiments, administration of any of the disclosed vectors, virions, or compositions to a subject requiring them provides partial or complete restoration of melanosome migration in retinal pigment epithelial (RPE) cells. In some embodiments, administration of any of the polynucleotide vector systems, virions, or compositions provides partial or complete restoration of vision loss.

[0085] Such methods may generally involve intravitreal or subretinal administration of one or more of the disclosed particle vectors, virions, host cells, or compositions into one or both eyes of a subject in need, in an amount and for a duration sufficient to treat or alleviate the symptoms of such deficiency in an affected mammal. The methods may also encompass prophylactic treatment of animals suspected of having such a condition, or administration of such compositions to animals at risk of developing such a condition, after diagnosis or prior to the onset of symptoms.

[0086] Pharmaceutical compositions and kits The present invention also provides pharmaceutical compositions comprising the vector system of the present invention in combination with a pharmaceutically acceptable carrier. In the context of the present invention, the dose administered to a patient, particularly a human, should be sufficient to achieve a therapeutic response in the patient over a reasonable time frame without causing lethal toxicity and preferably without causing adverse effects or pathological conditions exceeding 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 concurrent treatment (if any), the frequency of treatment, the success rate, and the severity and stage of the pathological condition.

[0087] The present invention also provides a kit comprising the vector system of the present invention in one or more containers. The kit of the present disclosure may optionally include a pharmaceutically acceptable carrier and / or diluent. In certain embodiments, the kit of the present disclosure may include one or more other components, auxiliaries, or adjuvants described herein. In certain embodiments, the kit of the present disclosure may include instructions or packaging materials describing a method for administering the vector system contained in the kit to a selected mammalian receptor.

[0088] The containers of the disclosed kits may be of any suitable material (e.g., glass, plastic, metal, etc.) and may be of any suitable size, shape, or configuration. In certain embodiments, the vector system of the disclosed herein is provided in the kit as a solid. In other embodiments, the vector system of the disclosed herein is provided in the kit as a liquid or solution. In some embodiments, the kit may comprise one or more ampoules or syringes containing the vector system of the disclosed herein in a suitable liquid or solution form.

[0089] The present invention also provides the use of the buffers and compositions disclosed herein in the manufacture of a medicament for treating, preventing or relieving symptoms of a disease, disorder, dysfunction, injury or trauma, which includes, but is not limited to, the treatment, prevention and / or prophylaxis of a disease, disorder or dysfunction, and / or remission of one or more symptoms of such disease, disorder or dysfunction.

[0090] To express a therapeutic agent in accordance with this disclosure, rAAV particles may be prepared containing a nucleic acid segment encoding the therapeutic agent under the control of one or more promoters. To place the sequence under the “control” of a promoter, the 5' end of the transcription start site of the transcription reading frame is generally positioned about 1 to about 50 nucleotides “downstream” (e.g., 3' side) of the selected promoter. The “upstream” promoter stimulates the transcription of the DNA and promotes the expression of the encoded polypeptide. This is the meaning of “recombinant expression” in this context. In some embodiments, the recombinant vector construct comprises an rAAV vector in which the capsid protein has been modified, containing a promoter specific to RPE cells or photoreceptor cells, which is operably ligated to at least one nucleic acid segment encoding one or more diagnostic and / or therapeutic agents.

[0091] If such vectors are intended to be used for the introduction of one or more exogenous proteins, polypeptides, peptides, ribozymes, and / or antisense oligonucleotides into specific cells transfected with the vectors, the rAAV particles disclosed herein may be used to deliver one or more exogenous polynucleotides to selected host cells, for example, one or more selected cells in the eye of a mammal.

[0092] In some embodiments, the Disclosure provides formulations of one or more virus-based compositions disclosed herein, either alone or in combination with one or more other therapeutic modalities, in pharmaceutically acceptable solutions for administration to cells or animals, particularly for the treatment of human cells, tissues, and diseases affecting humans.

[0093] In some embodiments, the rAAV particles described herein may be administered in combination with other agents, such as proteins or polypeptides, or various pharmaceutically active agents (including one or more systemic or topical administrations of therapeutic polypeptides, biologically active fragments, or variants thereof).

[0094] Formulations of pharmaceutically acceptable buffers, excipients, and carrier solutions are well known to those skilled in the art, as is the development of suitable administration and treatment regimens for use in various treatment regimens, including, for example, oral, parenteral, intraocular (e.g., subretinal or intravitreous), intravenous, intranasal, intra-articular, intracochlear, and intramuscular administration and formulations.

[0095] The term “excipient” refers to a diluent, adjuvant, carrier, or vehicle administered with rAAV particles. Such medicinal excipients may be sterile liquids such as water and oil, including petroleum oils such as mineral oil, vegetable oils such as peanut oil, soybean oil, and sesame oil, animal oils, or oils of synthetic origin. Saline solution, as well as aqueous solutions of dextrose and glycerol, can also be used as liquid carriers. Non-limiting examples of excipients and vehicles include, but are not limited to, HA, BSS, artificial CSF, PBS, Ringer's lactic acid solution, TMN200 solution, polysorbate 20, and poloxamer 100.

[0096] Non-limiting examples of compositions may include rAAV particles or nucleic acid vectors, either alone or in combination with one or more additional active ingredients obtained from natural or recombinant sources or chemically synthesized.

[0097] Method for producing rAAV particles Recombinant adeno-associated virus (rAAV) vectors have been successfully used for in vivo gene transfer in numerous preclinical animal models of human diseases and for the long-term expression of a wide range of therapeutic genes. AAV vectors have also yielded long-term clinical benefits in humans, such as when targeting immune-privileged sites, for example, delivery to the eye for Leber congenital amaurosis. The main advantages of this vector are its relatively low immune profile, the limited inflammatory response it elicits, and, in some cases, even the induction of immune tolerance to the transgenic product. Nevertheless, therapeutic efficacy when targeting non-immune-privileged organs remains limited in humans due to antibody and CD8+ T cell responses against the viral capsid, while adaptive responses to transgene products have also been reported in animal models. These results suggest that immune responses remain a concern with regard to AAV vector-mediated gene transfer.

[0098] Adeno-associated viruses (AAVs) are considered optimal vectors for ocular gene therapy due to their efficiency, persistence, and low immunogenicity. Historically, identifying vectors capable of introducing PRs via the vitreous humor has relied on identifying which serotypes exhibit intrinsic tropism to this cell type after local delivery. Several serotypes have been used to successfully target transgenes to PRs after subretinal injection (including, e.g., AAV2, AAV5, and AAV8), and all three have demonstrated efficacy in experiments conducted across multiple mammalian species (e.g., mice, rats, dogs, pigs, and non-human primates).

[0099] Studies comparing the relative efficiency after subretinal delivery in rodents show that both AAV5 and AAV8 transduce the retinal retinopathy more efficiently than AAV2, with AAV8 being the most efficient. AAV2 and AAV8 vectors containing a point mutation in a surface-exposed tyrosine residue (tyrosine to phenylalanine, YF) show increased transgene expression in various retinal cell types compared to the unmodified vector after subretinal and intravitreous injection. Of the vectors initially tested by these authors, the triple mutant of AAV2 (named "triple YF") showed the highest transduction efficiency after intravitreous injection, while the quadruple mutant of AAV2 ("quad YF") exhibited enhanced transduction of the outer retina.

[0100] Further improvements in transduction efficiency were achieved by inducing mutagenesis of surface-exposed threonine (T) or serine (S) residues to non-native amino acids in one or more of those amino acids. Both YF and TV / TA mutations have been shown to enhance efficiency by reducing capsid phosphorylation and subsequent ubiquitination as part of the proteasome degradation pathway. The transduction profile of AAV delivered into the vitreous humor has been found to be highly dependent on the injection procedure itself. Due to the small size of mouse eyes, transscleral intravitreous injection is not uncommon to cause retinal damage, which could lead to some vectors being delivered directly into the subretinal space.

[0101] In some embodiments, rAAV nucleic acid vectors useful 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.

[0102] Methods for generating rAAV particles and nucleic acid vectors are commercially available (see plasmids and kits available, e.g., from ATCC and Cell Biolabs, Inc.). For example, a plasmid containing a nucleic acid vector sequence may be combined with one or more helper plasmids, e.g., containing rep genes (e.g., encoding Rep78, Rep68, Rep52, and Rep40) and cap genes (encompassing the modified VP3 region described herein, encoding VP1, VP2, and VP3) to transfect a producer cell line so that rAAV particles can be packaged and subsequently purified.

[0103] In some embodiments, one or more helper plasmids include a first helper plasmid containing a rep gene and a cap gene, and a second helper plasmid containing E1a, E1b, E4, E2a, and VA genes. In some embodiments, the rep gene is a rep gene derived from AAV2, and the cap gene is derived from AAV2 and involves genetic modification to produce the modified capsid protein described herein. Helper plasmids and methods for constructing such plasmids are known in the art and are commercially available (see, for example, the pDM, pDG, pDP1rs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E / R585E), and pDP8.ape plasmids from PlasmidFactory (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).

[0104] Next, a non-limiting method for producing rAAV particles is described. One or more helper plasmids are generated or obtained, each containing rep and cap ORFs for a desired AAV serotype, as well as the adenovirus VA, E2A (DBP), and E4 genes under transcriptional control of their native promoters. The cap ORF may also contain one or more modifications for producing modified capsid proteins as described herein. HEK293 cells (available from ATCC®) are transfected with the helper plasmids and plasmids containing the nucleic acid vectors described herein, via CaPO4-mediated transfection, lipids, or polymer molecules such as polyethyleneimine (PEI). The HEK293 cells are then incubated for at least 60 hours to enable the production of rAAV particles. Alternatively, in another example, an Sf9-based producer stable cell line is infected with a single recombinant baculovirus containing a nucleic acid vector. As a further option, in another example, HEK293 or BHK cell lines are infected with HSV containing a nucleic acid vector, optionally rep and cap ORFs as described herein, and one or more helper HSVs containing the adenovirus VA, E2A(DBP) and E4 genes under transcriptional control of their native promoters. HEK293, BHK, or Sf9 cells are then incubated for at least 60 hours to enable the production of rAAV particles. The rAAV particles can then be purified using any method known in the art or described herein, for example, by iodixanol step gradient, CsCl gradient, chromatography, or polyethylene glycol (PEG) precipitation.

[0105] definition According to this disclosure, polynucleotides, nucleic acid segments, nucleic acid sequences, etc., include, but are not limited to, DNA (including, but not limited to, genomic DNA 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 obtained from natural sources, chemically synthesized, genetically modified, or otherwise prepared or synthesized whole or partially by human hands.

[0106] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Any methods and compositions similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, and preferred methods and compositions are described herein.

[0107] In some embodiments, the terms “nucleic acid” and “polynucleotide sequence” refer to deoxyribonucleotides or ribonucleotide polymers in either single-stranded or double-stranded form, and, unless otherwise limited, include known analogues of native nucleotides that can function in a manner similar to naturally occurring nucleotides. A polynucleotide sequence may include both full-length sequences and shorter sequences derived from full-length sequences. A particular polynucleotide sequence may include degenerate codons of a native sequence(s) or sequence(s), which may be introduced to provide codon preference in a particular host cell. A polynucleotide sequence may include sequences that specifically hybridize with sequences encoding the peptides of this disclosure. A polynucleotide may include both sense and antisense strands, as separate strands or in a double helix.

[0108] The polynucleotide fragments and variants of the present disclosure can be generated as described herein and tested for the presence of function. The polynucleotide or polypeptide fragments and variants of the present disclosure can be tested to determine whether such fragment or variant retains the same or similar functional activity as a full-length or non-variant polynucleotide or polypeptide, such as the ABCA4 polynucleotide or polypeptide.

[0109] Furthermore, polynucleotides having the same or substantially the same nucleotide sequence as the polynucleotides described herein, except for the presence of one or more nucleotide substitutions, additions, or deletions within the polynucleotide sequence, are also within the scope of this disclosure, insofar as these variant polynucleotides retain substantially the same relevant functional activity as the polynucleotides described herein (for example, they encode proteins with the same amino acid sequence or the same functional activity as one of the polynucleotides specifically described herein). Therefore, the polynucleotides disclosed herein should be understood to also include their variants and fragments.

[0110] In addition to variants that can be artificially prepared or synthesized by those skilled in the art in a laboratory setting, a large number of variant sequences of genes or polynucleotides may be found in nature. The polynucleotides of this disclosure include those specifically described herein, as well as any natural variants thereof, and any variants that can be artificially produced (insofar as those variants retain the desired biological activity).

[0111] Furthermore, polynucleotides having the same nucleotide sequence as the polynucleotides described herein, except for substitutions, additions, or deletions of nucleotides within the polynucleotide sequence, are also within the scope of the present invention, insofar as 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 encompass variants and fragments of the sequences specifically described, as discussed above.

[0112] The polynucleotides described herein may also be defined in terms of a more specific range of identity and / or similarity with respect to those described herein. Sequence identity may be greater than 60%, greater than 75%, greater than 80%, greater than 90%, or potentially 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 more with respect to sequences described herein.

[0113] Unless otherwise specified herein, the percent sequence identity and / or similarity of two sequences can be determined using the Karlin and Altschul (1990) algorithm as modified as described by Karlin and Altschul (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs by Altschul et al. (1990). A BLAST search is performed using the NBLAST program (score = 100, word length = 12) to obtain sequences with the desired percent sequence identity. Gapped BLAST can be used to obtain gapped alignments for comparison purposes. When using the BLAST and Gapped BLAST programs, the default parameters of each program (NBLAST and XBLAST) can be used according to the published methods.

[0114] The present invention also envisions a polynucleotide molecule having a sequence whose homology to the polynucleotide sequence of the present disclosure is sufficiently high to allow hybridization with that sequence under standard stringent conditions and standard methods. As used herein, “stringent conditions” for hybridization means that the hybridization is carried out overnight in 6×SSPE, 5×Denhardt solution, and 0.1% SDS containing 0.1 mg / mL of suitable nonspecifically denatured DNA, typically at a temperature 20–25°C lower than the melting temperature (Tm) of the DNA hybrid.

[0115] In some embodiments, an effective dose refers to an amount that can treat or alleviate a disease or condition, or an amount that can produce a differently intended therapeutic effect. In some embodiments, operably ligated means that the ligated nucleic acid sequences are typically contiguous or substantially contiguous, and, if two protein-coding regions need to be joined, contiguous and within the read frame. However, because enhancers generally function at several kilobases away from promoters, and intron sequences can be of varying lengths, some polynucleotide elements may be operably ligated but not contiguous.

[0116] In some embodiments, a promoter is one or more regions of a nucleic acid sequence that control transcription. Non-exclusive examples of promoters provided herein include, but are not limited to, the CMV promoter, the EF-1 alpha promoter, the pyramidal arrestin promoter, the chimeric CMVβ-actin promoter (CBA), the truncated chimeric CMVβ-actin (smCBA) promoter, the smCBA promoter, the human ABCA4 gene-derived promoter, the TαC gene-derived promoter, the rhodopsin promoter, the cGMP-phosphodiesterase β-subunit promoter, the human or mouse rhodopsin promoter, the hGRK1 promoter, the rod-specific IRBP promoter, and the VMD2 promoter.

[0117] In some embodiments, a regulatory element refers to one or more regions of a nucleic acid sequence that regulate transcription. Non-exclusive examples of regulatory elements include, but are not limited to, enhancers, post-transcriptional elements, and transcriptional regulatory sequences.

[0118] In some embodiments, the selected nucleic acid or amino acid sequence substantially represents a feature of a nucleic acid or amino acid sequence, where the selected nucleic acid or amino acid sequence has at least about 70 or about 75 percent sequence identity with respect 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 percent sequence identity, and more preferably at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequence identity. Highly homologous sequences often share at least about 96, 97, 98, or 99 percent or more sequence identity between the selected sequence and the reference sequence with which it is compared.

[0119] The percentage of sequence identity may 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 percent of the selected reference sequences. The reference sequences may be a subset of a larger sequence, such as a gene, a portion of an adjacent sequence, or a repeating portion of a chromosome. In the case of sequence homology of two or more polynucleotide sequences, the reference sequences may contain at least approximately 18–25 nucleotides, at least approximately 26–35 nucleotides, or at least approximately 40, 50, 60, 70, 80, 90, or even 100 nucleotides.

[0120] When highly homologous fragments are desired, the degree of percentage identity between the two sequences may be at least about 80%, at least about 85%, or at least about 90% or 95%, as can be easily determined by sequence comparison algorithms such as FASTA program analysis.

[0121] In some embodiments, "subject" describes organisms, including mammals such as primates, that can be treated with the compositions of the present disclosure. Mammalian species that can benefit from the methods of treatment disclosed include, but are not limited to, humans, apes; chimpanzees; monkeys; and orangutans, domestic animals including dogs and cats, and livestock such as horses, cattle, pigs, sheep and goats, or other mammalian species including, but not limited to, mice, rats, guinea pigs, rabbits, and hamsters.

[0122] In some embodiments, treatment or any grammatical variation thereof (e.g., to treat, to treat, and treatment) includes, but is not limited to, alleviating the symptoms of a disease or condition; and / or reducing, inhibiting, inhibiting, mitigating, easing, or influencing the progression, severity, and / or extent of a disease or condition.

[0123] In some embodiments, a vector refers to a nucleic acid molecule (typically containing DNA) that can replicate in a suitable host cell or to which another nucleic acid segment can be operably ligated to facilitate the replication of an operably ligated nucleic acid segment. Exemplary vectors include, but are not limited to, plasmids, cosmids, viruses, and the like.

[0124] In some embodiments, a variant refers to a molecule (e.g., a capsid polynucleotide) that has characteristics that deviate from those occurring naturally, for example, a “variant” being at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to a wild-type capsid polynucleotide. A variant of a protein molecule (e.g., a capsid) may contain changes in the amino acid sequence (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, or 15-20 amino acid substitutions) compared to the wild-type protein sequence, resulting from point mutations installed in the nucleic acid sequence encoding the capsid protein. These modifications include chemical modifications and truncations.

[0125] Variants of nucleic acid molecules (e.g., polynucleotide vector systems) may contain modifications to the sequence compared to the wild-type nucleic acid sequence (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, or 15-20 nucleotide substitutions). These modifications may include truncations at the 3' or 5' end.

[0126] example The following examples are included to demonstrate the exemplary aspects of this disclosure. Example 1 describes a non-restrictive dual AAV vector system utilizing alkaline phosphatase (AP) splicing. In each system shown in Figure 1, there are two vectors, each containing an AP homologous domain (APHead). In one system labeled "Dual AAV-ABCA4 SP2(ex20 / 21)", vector A contains the coding sequence corresponding to the amino-terminal portion of the hABCA4 cDNA through exon 20 (SEQ ID NO: 13) and the splice donor site (SEQ ID NO: 24), followed by the APHead intron (SEQ ID NO: 22). Vector B contains the APHead intron (SEQ ID NO: 22), followed by the splice acceptor site (SEQ ID NO: 25), and then the carboxyl-terminal portion of the hABCA4 cDNA from the beginning of exon 21 (SEQ ID NO: 15). When co-delivered to a suitable mammalian host cell, the DNA of vectors A and B are recombined to form a reconstructed full-length gene cassette. The resulting RNA transcript will then "splice out" an intron (SEQ ID NO: 22). Alternatively, recombination and formation of the gene cassette can occur via the AAV ITR. In this case, the RNA transcript will "splice out" an intron-ITR-intron motif (SEQ ID NO: 29). However, in either case, the resulting mRNA (SEQ ID NO: 1) is full-length hABCA4, which is then translated into the hABCA4 protein (SEQ ID NO: 2). Similarly, a system labeled "Dual AAV-ABCA4 SP1 (ex19 / 20)" contains two vectors; the first vector contains the coding sequence (SEQ ID NO: 9) corresponding to the amino-terminal portion of the hABCA4 cDNA through exon 19, and the second vector contains the coding sequence (SEQ ID NO: 11) corresponding to the carboxyl-terminal portion of the hABCA4 cDNA from the start of exon 20.Also shown in Figure 1 is a system named "Dual AAV-ABCA4 SP3(ex21 / 22)", which includes two vectors; the first vector contains a coding sequence (SEQ ID NO: 17) corresponding to the amino-terminal portion of the hABCA4 cDNA through exon 21, and the second vector contains a coding sequence (SEQ ID NO: 19) corresponding to the carboxyl-terminal portion of the hABCA4 cDNA from the start of exon 22.

[0127] Example 2 describes the in vitro performance of three non-restrictive dual-vector systems. Each of the three dual AAV-ABCA4 vectors was packaged in AAV44.9(E531D)(SEQ ID NO: 28);S007+S008(SP1, SEQ ID NOs: 3 and 4 respectively);S009+S010(SP2, SEQ ID NOs: 5 and 6 respectively); andS011+S012(SP3, SEQ ID NOs: 7 and 8 respectively) by triple transfection of HEK293 cells. The resulting vector pairs were used at a MOI of 100,000 to infect AAVR cells. Three days post-infection, cells were harvested and total protein was extracted. Protein samples were subjected to immunoblotting and probe detection with antibodies against ABCA4 (Figure 2). Both dual AAV-ABCA4 vector pairs, SP2 (S009-S010) and SP3 (S011-S012), result in high levels of full-length ABCA4 expression in vitro.

[0128] Example 3 compares ABCA4 expression in ABCA4- / - knockout mice obtained from bilateral subretinal injection of three dual AAV-ABCA4 vector expression cassettes, as described in Examples 1 and 2 and shown in Figure 1. The levels of ABCA4 mediated by the dual AAV vectors were compared to endogenous levels in heterozygous and wild-type mice. The results are shown in Figures 3A and 3B. No ABCA4 expression was observed in the retinas of mice injected with the S007+S010 sample (SEQ ID NOs. 3 and 4, data not shown). S009 / S010 (SEQ ID NOs. 5 and 6) produced the highest levels of ABCA4 expression. S009 / S010 produced levels ranging from 13 to 69% of wild-type ABCA4 levels, with an average of 37% of wild-type ABCA4 levels. The S011 / S012 vector pair (SEQ ID NOs: 7 and 8) produced ABCA4 at levels ranging from 15 to 27% of wild-type levels, with an average of 22% of wild-type levels. These results confirm 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.

[0129] Example 4 shows in vivo data from Abca4 - / - mice subretinally injected with AAV44.9 (E531D) (capsid encoded by SEQ ID NO: 28) particles containing the S009-S010 vector pair (SEQ ID NOs: 5 & 6). The treatment reduced autofluorescence in the retina of Abca4 - / - knockout mice. Accumulation of toxic autofluorescent bisretinoids occurred in the retinas of both Stargardt disease patients and Abca4 - / - knockout mice, and these could be measured by scanning laser ophthalmoscope (cSLO). Subretinal injection was performed in one eye of Abca4 - / - knockout mice. The other eye was injected with the solvent only. Wild-type mice that were not injected were also included for comparison. In Figure 4, mean pixel intensity measurements from cSLO images of Abca4 - / - mice injected with the vehicle or uninjected WT controls are graphed at multiple time points compared to Abca4 - / - mice injected with the dual vector. At all post-injection time points, a reduction in autofluorescence was observed in the eyes of Abca4 - / - mice injected with the vector compared to eyes injected with the solvent. These results demonstrate that ABCA4 produced from the S009-S010 vector pair functions to reduce the retinal autofluorescence phenotype in Abca4 - / - mice.

[0130] Example 5 shows electroretinogram (ERG) data from Abca4- / - mice subretinally injected with AAV44.9 (E531D) (capsid encoded by SEQ ID NO: 28) particles containing the S009-S010 dual vector pair (SEQ ID NOs: 5 & 6). ERG recordings were performed 2 months post-injection under dark-adapted (rod-mediated) and light-adapted (cone-mediated) conditions, and the amplitudes of the maximal a-wave (photoreceptor) (Figure 5A) and b-wave (bipolar cell) (Figures 5B-C) were calculated / averaged. ERG recordings were also performed on the eyes of uninjected wild-type mice. No significant difference in ERG amplitude was observed between the eyes of Abca4- / - mice injected with the S009-S010 vector and those injected with the solvent. These data demonstrate that, two months after injection, the S009-S010 dual AAV vector is well tolerated and does not cause any decline in retinal function.

[0131] Equal parts Accordingly, the embodiments described above are presented merely as examples, and it should be understood that embodiments of the invention may be carried out in ways different from those specifically described and claimed, within the scope of the appended claims and their equivalents. The embodiments of the invention of this disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the invention of this disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not contradictory to each other.

[0132] When used herein and in the claims, the indefinite articles "a" and "an" should be understood to mean "at least one" unless explicitly stated otherwise. When used herein and in the claims, the phrase "and / or" should be understood to mean "either or both" of the elements thus combined, i.e., elements that exist in some cases as a combination and in others as separate. Any elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements thus combined. In addition to the elements specifically identified by the phrase "and / or," there may be other elements, whether or not they are related to those specifically identified elements. Thus, as a non-restrictive example, when used in combination with open-ended language such as "comprising," in one embodiment it may refer to A only (optionally including elements other than B); in another embodiment it may refer to B only (optionally including elements other than A); and in yet another embodiment it may refer to both A and B (optionally including other elements), and so on.

[0133] Where used herein and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when dividing items in a list, “or” or “and / or” should be interpreted as inclusive, that is, encompassing at least one of a number of elements or a list of elements, but also more than one, and optionally, additional items not listed. Terms that are explicitly indicated as opposite, such as “only one of” or “exactly one of,” or, where used in the claims, “consisting of,” would refer to the inclusion of exactly one element of a number of elements or a list of elements. In general, where used herein, the term “or” should be interpreted as indicating an exclusive choice (i.e., “one or the other, but not both”) only when preceded by terms of exclusivity such as “either,” “one of,” “only one of,” or “exactly one of.” Where used in the claims, “consisting of” should have its usual meaning as used in the field of patent law.

[0134] As used herein and in the claims, the phrase “at least one” in reference 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 of elements, but not necessarily including at least one of each and all of the elements specifically enumerated in the list of elements, nor excluding any combination of elements in the list of elements. This definition also allows for the presence of elements other than those specifically identified in the list of elements to which the phrase “at least one” refers, whether or not they are related to the specifically identified elements. Therefore, as a non-restrictive 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”) could mean: in one embodiment, at least one (or more than one) A such that no B exists (and optionally includes elements other than B); in another embodiment, at least one (or more than one) B such that no A exists (and optionally includes elements other than A); in yet another embodiment, at least one (or more than one) A and at least one (or more than one) B (and optionally including other elements); and so on.

[0135] Furthermore, unless explicitly stated otherwise, it should be understood that in any method claimed herein that includes one or more steps or actions, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are described.

[0136] In the claims and in the above specification, all transitional phrases such as “include,” “encompass,” “carry,” “have,” “contain,” “involve,” “hold,” and “consist of” should be understood to be open-ended, meaning they include but are not limited to them. Only the transitional phrases “consist of” and “essentially consist of” are closed or semi-closed transitional phrases, as described in Section 2111.03 of the U.S. Patent and Trademark Office Manual of Patent Examination Procedure. It should be understood that any use of an open-ended transitional phrase (e.g., “include”) described herein is also intended to be, in alternative embodiments, the features “consist of” and “essentially consist of” described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B,” the disclosure also intends to include the alternative embodiments “a composition comprising A and B” and “a composition essentially consisting of A and B.”

[0137] manner Furthermore, the following aspects are disclosed herein: 1. A polynucleotide vector system for providing ABCA4 or a functional portion thereof, the following: A first AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, a promoter between the inverted terminal repeat sequences, followed by a partial ABCA4 coding sequence encoding the N-terminal portion of the ABCA4 polypeptide, followed by a splice donor site and homologous region, and A second AAV vector polynucleotide containing an inverted terminal repeat sequence at each end of the polynucleotide, homologous regions and splice acceptor sites and a partial ABCA4 coding sequence encoding the C-terminal portion of ABCA4 between the inverted terminal repeat sequences, Includes, Here, homologous regions in the first AAV vector and homologous regions in the second AAV vector contain overlapping polynucleotide sequences, and Here, the partition point between the first AAV vector polynucleotide and the second AAV vector polynucleotide sequence is located between exon 20 and exon 21 of the ABCA4 gene. The aforementioned polynucleotide vector system.

[0138] 2. The polynucleotide vector system according to embodiment 1, wherein the homologous region in the first AAV vector and the homologous region in the second AAV vector are alkaline phosphatase homologous regions.

[0139] 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.

[0140] 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.

[0141] 5. A polynucleotide vector system according to any one of embodiments 1 to 4, wherein the first AAV vector polynucleotide sequence includes a nucleotide sequence having at least 85% sequence identity with SEQ ID NO: 13.

[0142] 6. A polynucleotide vector system according to any one of embodiments 1 to 5, wherein the first AAV vector polynucleotide sequence includes a nucleotide sequence having at least 85% sequence identity with SEQ ID NO: 15.

[0143] 7. A 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.

[0144] 8. A 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.

[0145] 9. A 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.

[0146] 10. A 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.

[0147] 11. A 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.

[0148] 12. A polynucleotide vector system according to any one of embodiments 1 to 11, wherein the length of the polynucleotide sequence shared between the vectors is approximately 50 to approximately 500 nucleotides, or approximately 200 to 300 nucleotides.

[0149] 13. A polynucleotide vector system according to any one of embodiments 1, 3-6, or 12, wherein the homologous region includes an intron sequence.

[0150] 14. A polynucleotide vector system according to embodiment 13, wherein the intron sequence includes a sequence of introns naturally occurring in the genomic sequence of the gene encoding the ABCA4 polypeptide.

[0151] 15. A polynucleotide vector system according to embodiment 13 or 14, wherein the intron sequence is the AK sequence of the F1 phage.

[0152] 16. A 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.

[0153] 17. A 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 a first AAV vector polynucleotide and / or a second AAV vector polynucleotide.

[0154] 18. The 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.

[0155] 19. A polynucleotide vector system according to embodiment 18, wherein one or more non-coding sequences include alkaline phosphatase (AP) head sequences.

[0156] 20. A polynucleotide vector system according to embodiment 17 or embodiment 18, wherein one or more non-coding sequences include an AP intron.

[0157] 21. A polynucleotide vector system according to embodiment 18, wherein one or more non-coding sequences include a 3' untranslated region (UTR) between an ABCA4 subcoding sequence and a 3'AAV reverse terminal repeat sequence.

[0158] 22. A polynucleotide vector system according to any one of embodiments 17 to 21, wherein one or more substitutions are located within one or more presumptive in-frame stop codons.

[0159] 23. The polynucleotide vector system according to embodiment 22, wherein the substitution is located in one or more putative in-frame stop codons in the AP head sequence.

[0160] 24. The polynucleotide vector system according to embodiment 23, wherein the substitution is located in three putative in-frame stop codons in the AP intron sequence.

[0161] 25. A polynucleotide vector system according to any one of embodiments 1 to 24, wherein the promoter is selected from the group consisting of the CMV promoter, EF-1 alpha promoter, pyramidal arrestin promoter, smCBA promoter, human ABCA4 gene-derived promoter, TαC gene-derived promoter, rhodopsin promoter, cGMP-phosphodiesterase β subunit promoter, human or mouse rhodopsin promoter, hGRK1 promoter, rod-specific IRBP promoter, VMD2 promoter, and combinations thereof.

[0162] 26. The polynucleotide vector system according to embodiment 25, wherein the promoter is a CMV promoter.

[0163] 27. The polynucleotide vector system according to embodiment 25, wherein the promoter is an smCBA promoter.

[0164] 28. The polynucleotide vector system according to embodiment 25, wherein the promoter is a rhodopsin promoter.

[0165] 29. A polynucleotide system according to any one of embodiments 1 to 29, further comprising one or more nucleotide substitutions for removing one or more putative stop codons in the 3' untranslated region between a partial coding sequence encoding the C-terminal portion of a polypeptide and the 3' AAV reverse terminal repeat of a second AAV vector polynucleotide.

[0166] 30. A polynucleotide system according to embodiment 29, wherein one or more substitutions are located in one or more putative stop codons.

[0167] 31. A polynucleotide vector system according to any one of embodiments 1 to 30, wherein a polyadenylation (pA) signal sequence follows a second AAV vector polynucleotide.

[0168] 32. A polynucleotide vector system according to any one of embodiments 1 to 31, wherein the inverted terminal repeat sequences at each end of the first AAV vector polynucleotide or the second AAV vector polynucleotide comprise a 5'AAV ITR and a 3'AAV ITR, and the 5'AAV ITR and the 3'AAV ITR are ITRs from a single AAV serotype.

[0169] 33. A polynucleotide vector system according to any one of embodiments 1 to 32, wherein the inverted terminal repeat sequences at each end of the first or second AAV vector polynucleotide include a 5'AAV ITR and a 3'AAV ITR, and the 5'AAV ITR and 3'AAV ITR are ITRs from multiple AAV serotypes.

[0170] 34. A polynucleotide vector system according to any one of embodiments 1 to 28, wherein the AAV inverted terminal repeat sequence (ITR) comprises an ITRS from one or more AAV serotypes selected from the group consisting of 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).

[0171] 35. The polynucleotide vector system according to embodiment 34, wherein the AAV serotype is AAV serotype 44.9 (E531D).

[0172] 36. The polynucleotide vector system according to embodiment 34, wherein the AAV serotype is AAV2.

[0173] 37. Recombinant virus particles comprising a first AAV vector polynucleotide or a second AAV vector polynucleotide as described in any one of embodiments 1 to 36.

[0174] 38. Recombinant viral particles according to embodiment 37, comprising one or more tyrosine-to-phenylalanine (YF) mutations in the capsid protein of a virus or virion.

[0175] 39. Recombinant virus particles according to embodiment 37 or embodiment 38, wherein the recombinant virus particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and / or AAV10 capsids.

[0176] 40. Recombinant virus particles according to any one of embodiments 37 to 39, wherein the recombinant virus particles include AAV7m8, AAV-DJ, AAV2 / 2-MAX, AAVSHh10, AAVSHh10Y, AAV3b, AAVLK03, AAV8BP2, AAV1(E531K), AAV6(D532N), AAV6-3pmut, AAV2G9, AAV44.9, AAV44.9(E531D), AAVrh.8, AAVrh.8R, and / or AAVAnc80 capsid.

[0177] 41. Recombinant virus particles according to embodiment 40, wherein the recombinant virus particles contain the AAV44.9(E531D) capsid.

[0178] 42. Isolated host cells containing a polynucleotide vector system according to any one of embodiments 1 to 36 or a recombinant viral particle according to any one of embodiments 37 to 41.

[0179] 43. Isolated host cells according to embodiment 42, wherein the cells are photoreceptor cells, cone cells, rod cells, retinal cells, ganglion cells, retinal pigment epithelial cells, vestibular hair cells, inner ear hair cells, outer ear hair cells, or any combination thereof.

[0180] 44. A method for treating or improving a disease or symptom in a human or animal, comprising administering to one or more cells of a human or animal a polynucleotide vector system of any of embodiments 1 to 36 or a recombinant viral particle of any of embodiments 37 to 41, wherein ABCA4 or a functional variant thereof provides treatment or remission of a disease or symptom and is expressed in one or more cells.

[0181] 45. The method according to aspect 44, wherein the disease or condition is Stargardt disease.

[0182] 46. ​​The method according to embodiment 44 or 45, wherein the ABCA4 polypeptide comprises the amino acid sequence of SEQ ID NO: 2 or a functional fragment thereof.

[0183] 47. A method for administering any one polynucleotide vector system described in embodiments 1 to 36 or any one recombinant viral particle described in embodiments 37 to 41, wherein the amount of truncated ABCA4 protein produced by the administration of the polynucleotide vector system is minimized.

[0184] 48. A method for administering a polynucleotide vector system according to any one of embodiments 1 to 36 or a recombinant viral particle according to any one of embodiments 37 to 41, wherein the cytotoxicity resulting from the administration of the polynucleotide vector system is minimized.

[0185] 49. Any one of the methods according to embodiments 44 to 48, wherein administration of a polynucleotide vector system provides recovery of partial or complete vision loss.

[0186] 50. Any one of the methods described in aspects 44 to 49, wherein the polynucleotide vector system is administered by parenteral administration, intravenous administration, intramuscular administration, intraocular administration, intranasal administration, subretinal administration, round window injection, or during cochlear implant surgery.

[0187] array The following sequences are included for clarity and should not be considered to limit the methods and compositions disclosed. The AAV vector sequences disclosed herein are non-limiting examples of the AAV vectors disclosed. These sequences include, but are not limited to, a non-limiting example of components of the disclosed AAV vector, including: non-limiting examples of coding sequences for the ABCA4 portion, e.g., 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, e.g., the AAV2 ITR sequence; non-limiting examples of promoters, e.g., the chicken β-actin promoter; non-limiting examples of enhancers, e.g., the cytomegalovirus (CMV) enhancer; non-limiting examples of splice donor or acceptor sites; non-limiting examples of homologous regions, e.g., the AP head sequence; non-limiting examples of intron sequences, e.g., the alkaline phosphatase (AP) intron sequence; and non-limiting examples of polyadenylated sequences, e.g., the Min2 bGH polyA sequence (SEQ ID NO: 21).

[0188] Human ABCA4 nucleotide sequence (SEQ ID NO: 1)

[0189] Amino acid sequence of human ABCA4 (SEQ ID NO: 2)

[0190] First AAV vector, splitting point after exon-19; S007-Hf (SEQ ID NO: 3)

[0191] Second AAV vector, prior to the split point of exon 20; S008-HB (SEQ ID NO: 4)

[0192] First AAV vector, splitting point after exon 20; S009-HF (SEQ ID NO: 5)

[0193] Second AAV vector, prior to the split point of exon 21; S010-HB (SEQ ID NO: 6)

[0194] First AAV vector, splitting point after exon 21; S011-HF (SEQ ID NO: 7)

[0195] Second AAV vector, prior to the split point of exon 22; S012-HB (SEQ ID NO: 8)

[0196] S007-HF Nucleotide sequence containing only ABCA4 (SEQ ID NO: 9)

[0197] S007-HF amino acid sequence containing only ABCA4 (SEQ ID NO: 10) MGFVRQIQLLLWKNWTLRKRQKIRFVVELVWPLSLFLVLIWLRNANPLYSHHECHFPNKAMPSAGMLPWLQGIFCNVNNPCFQSPTPGESPGIVSNYNNSILARVYRDFQELLMNAPESQHLGRIWTELHILSQFMDTLRTHPERIAGRGIRIRDILKDEETLTLFLIKNIGLSDSVVYLLINSQVRPEQFAHGVPDLALKDIACSEALLERFIIFSQRRGAKTVRYALCSLSQGTLQWIEDTLYANVDFFKLFRVLPTLLDSRSQGINLRSWGGILSDMSPRIQEFIHRPSMQDLLWVTRPLMQNGGPETFTKLMGILSDLLCGYPEGGGSRVLSFNWYEDNNYKAFLGIDSTRKDPIYSYDRRTTSFCNALIQSLESNPLTKIAWRAAKPLLMGKILYTPDSPAARRILKNANSTFEELEHVRKLVKAWEEVGPQIWYFFDNSTQMNMIRDTLGNPTVKDFLNRQLGEEGITAEAILNFLYKGPRESQADDMANFDWRDIFNITDRTLRLVNQYLECLVLDKFESYNDETQLTQRALSLLEENMFWAGVVFPDMYPWTSSLPPHVKYKIRMDIDVVEKTNKIKDRYWDSGPRADPVEDFRYIWGGFAYLQDMVEQGITRSQVQAEAPVGIYLQQMPYPCFVDDSFMIILNRCFPIFMVLAWIYSVSMTVKSIVLEKELRLKETLKNQGVSNAVIWCTWFLDSFSIMSMSIFLLTIFIMHGRILHYSDPFILFLFLLAFSTATIMLCFLLSTFFSKASLAAACSGVIYFTLYLPHILCFAWQDRMTAELKKAVSLLSPVAFGFGTEYLVRFEEQGLGLQWSNIGNSPTEGDEFSFLLSMQMMLLDAAVYGLLAWYLDQVFPGDYGTPLPWYFLLQESYWLGGEGCSTREERALEKTEPLTEETEDPEHPEGIHDSFFEREHPGWVPGVCVKNLVKIFEPCGRPAVDRLNITFYENQITAFLGHNGAGKTTTL

[0198] S008-HB Nucleotide sequence containing only ABCA4 (SEQ ID NO: 11)

[0199] S008-HB Amino acid sequence containing only ABCA4 (SEQ ID NO: 12)

[0200] S009-HF Nucleotide sequence containing only ABCA4 (SEQ ID NO: 13)

[0201] S009-HF amino acid sequence containing only ABCA4 (SEQ ID NO: 14)

[0202] S010-HB Nucleotide sequence containing only ABCA4 (SEQ ID NO: 15)

[0203] S010-HB Amino acid sequence containing only ABCA4 (SEQ ID NO: 16)

[0204] S011-HF nucleotide sequence containing only ABCA4 (SEQ ID NO: 17)

[0205] S011-HF amino acid sequence containing only ABCA4 (SEQ ID NO: 18)

[0206] S012-HB Nucleotide sequence containing only ABCA4 (SEQ ID NO: 19)

[0207] S012-HB amino acid sequence containing only ABCA4 (SEQ ID NO: 20)

[0208] Min2pA sequence (SEQ ID NO: 21) TTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGG

[0209] AP sequence (SEQ ID NO: 22) ccccgggtgcgcggcgtcggtggtgccggcggggggcgccaggtcgcaggcggtgtagggctccaggcaggcggcgaaggccatgacgtgcgctatgaaggtctgctcctgcacgccgtgaaccaggtgcgcctgcgggccgcgcgcgaacaccgccacgtcctcgcctgcgtgggtctcttcgtccaggggcactgcgcactgctgccgatactcggggctcccgctctcgctctcggtaacatccggccgggcgccgtccttgagcacatagcctggaccgtttc

[0210] smCBA promoter (SEQ ID NO: 23) ggtacctagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgcgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggtttaatgacggcttgtttcttttctgtggctgcgtgaaagccttgaggggctccgggagctagagcctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaatt

[0211] Splice donor (SEQ ID NO: 24) gtaagtatcaaggttacaagacaggttaacggagaccaattgaaactgggcttgtcgagacagagaagactcttgcgtttcagcgctagc

[0212] Splice acceptor (SEQ ID NO: 25) taggcacctattggtcttactgacatccactttgcctttctctccacag

[0213] 5'ITR (Sequence ID 26) ttggccactccctctctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct

[0214] 3'ITR (Sequence ID 27) aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaa

[0215] AAV44.9(E531D)(Sequence ID 28) MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGNGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDTESVPD PQPLGEPPAAPSGLGPNTMASGGGAPMADNNEGADGVGNSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNEGTKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFP ADVFMVPN VDYSQVLITDEEEIKATNPVATEEYGAVAINNQAANTQAQTGLVHNQGVIPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPLTFNQAKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGVYSEPRPIGTRYLTRNL

[0216] Spliced ​​sequence (sequence number 29) ccccgggtgcgcggcgtcggtggtgccggcggggggcgccaggtcgcaggcggtgtagggctccaggcaggcggcgaaggccatgacgtgcgctatgaaggtctgctcctgcacgccgtgaaccaggtgcgcctgcgggccgcgcgcgaacaccgccacgtcctcgcctgcgtgggtctcttcgtccaggggcactgcgcactgctgccgatactcggggctcccgctctcgctctcggtaacatccggccgggcgccgtccttgagcacatagcctggaccgtttcgtcgacggatccgcatgctggggagagatctgaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaacagatctgaattcGGcgcgccccccgggtgcgcggcgtcggtggtgccggcggggggcgccaggtcgcaggcggtgtagggctccaggcaggcggcgaaggccatgacgtgcgctatgaaggtctgctcctgcacgccgtgaaccaggtgcgcctgcgggccgcgcgcgaacaccgccacgtcctcgcctgcgtgggtctcttcgtccaggggcactgcgcactgctgccgatactcggggctcccgctctcgctctcggtaacatccggccgggcgccgtccttgagcacatagcctggaccgtttc

Claims

1. A polynucleotide vector system for providing the ABCA4 gene, the following: A first AAV vector polynucleotide containing the first ABCA4 sequence of the ABCA4 gene, and A second AAV vector polynucleotide containing the second ABCA4 sequence of the ABCA4 gene Includes, Here, the ABCA4 gene encodes the ABCA4 protein that is at least 95% identical to SEQ ID NO: 2; the first ABCA4 sequence includes exons 1 to 20 of the ABCA4 gene, and the second ABCA4 sequence includes exons 21 to 50 of the ABCA4 gene. The aforementioned polynucleotide vector system.

2. The polynucleotide vector system according to claim 1, wherein the first ABCA4 sequence and the second ABCA4 sequence do not overlap.

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

4. A polynucleotide vector system according to 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 is identical to SEQ ID NO: 1 by at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

6. The polynucleotide vector system according to any one of claims 1 to 5, wherein the first ABCA4 sequence contains at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the sequence with respect to sequence number 13.

7. The polynucleotide vector system according to any one of claims 1 to 6, wherein the second ABCA4 sequence contains at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the sequence with respect to sequence number 15.

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

9. The polynucleotide vector system according to claim 8, wherein the promoter is the smCBA promoter, CMV promoter, EF-1 alpha promoter, pyramidal arrestin 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 according to claim 8, wherein the promoter is an smCBA promoter.

11. The polynucleotide vector system according to claim 8, wherein the promoter contains a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 23.

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

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

24.

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

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

25.

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

22.

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

22.

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

20. The polynucleotide vector system according to claim 19, wherein the pA signal sequence comprises a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 21.

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

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

26.

23. The polynucleotide vector system according to claim 21 or 22, wherein the 3'AAV ITR contains at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical sequence to sequence number 27.

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

25. Recombinant viral particles comprising a first AAV vector polynucleotide or a second AAV vector polynucleotide as described in any one of claims 1 to 24.

26. The recombinant virus particle according to claim 25, wherein the recombinant virus particle comprises AAV44.9 (E531D), AAV7m8, AAV-DJ, AAV2 / 2-MAX, AAVSHh10, AAVSHh10Y, AAV3b, AAVLK03, AAV8BP2, AAV1 (E531K), AAV6 (D532N), AAV6-3pmut, AAV2G9, AAV44.9, AAVrh.8, AAVrh.8R, or AAVAnc80 capsid.

27. The recombinant virus particle according to claim 25, wherein the recombinant virus particle contains an AAV44.9 (E531D) capsid.

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

29. The isolated host cell according to 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 symptom in a human or animal, comprising administering to one or more cells of a human or animal a polynucleotide vector system according to any one of claims 1 to 24 or a recombinant viral particle according to any one of claims 25 to 27, wherein the expression of the ABCA4 gene is expressed in one or more cells to treat or alleviate the disease or symptom.

31. The method according to claim 30, wherein the disease or condition is Stargardt disease.

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

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

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