Dual AAV-MYO7A vectors with improved safety for the treatment of USH1B

Abstract

Compositions and methods are disclosed for treating mammalian eye diseases, particularly retinal complications associated with Usher Syndrome 1B (USH1B). Further disclosed are compositions and methods for treating mammalian inner ear diseases, particularly inner ear cellular complications associated with Usher Syndrome 1B (USH1B). The present disclosure provides an improved AAV-based dual vector system that facilitates expression of full-length proteins whose coding sequences exceed the polynucleotide packaging capacity of individual AAV vectors. Described herein is a modified hybrid dual vector system that shifts the MYO7A tail domain coding sequence from the front-half vector to the back-half vector by altering the split point (e.g., from between exons 23 and 24 to between exons 21 and 22) to eliminate the generation of truncated MYO7A proteins. Further described herein are improved codon-modified hybrid and overlap vector systems in which putative stop codons and remaining sequences in non-coding sequences are removed.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 63 / 003,774, filed April 1, 2020, the entire disclosure of which is incorporated by reference.

[0002] This invention was made in whole or in part with funds received from the Foundation for Fighting Blindness under Grant No. TA-GT-0419-0774-UFL-GH and from Atsena Therapeutics, Inc. under Contract No. AGR00018211. [Background technology]

[0003] 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—4.7 kilobases (KB). Therefore, standard AAV vector systems are unsuitable for addressing diseases in which large genes are mutated or otherwise dysfunctional, such as Usher syndrome. Solutions are needed to package large genes into AAV vector systems and safely deliver gene therapy treatments to patients. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure relates generally to the fields of molecular biology and virology, and particularly to the development of gene delivery vehicles. Improved rAAV dual vector and polynucleotide vector systems for use in various gene therapy regimens are disclosed, as well as compositions useful for delivering various nucleic acid segments, including those encoding therapeutic proteins, polypeptides, peptides, antisense oligonucleotides, or ribozyme constructs, to selected host cells. Recombinant viral particles, isolated host cells, and pharmaceutical compositions comprising any of these rAAV dual vector and polynucleotide vector systems are also disclosed. Also provided are methods for preparing the improved rAAV dual vector system and using it in various virus-based gene therapies, particularly for the treatment and / or amelioration of symptoms of myosin VII deficiency, including, but not limited to, the treatment of human Usher syndrome type IB. Further provided herein are methods for treating or ameliorating diseases or conditions, including the administration of a rAAV dual vector system encoding the MYO7A protein and resulting in reduced cytotoxicity compared to previously available vector systems. In some aspects, methods are provided in which a vector system is administered, thereby minimizing the amount of truncated MYO7A protein and / or associated cytotoxicity. In some embodiments, the therapeutic polypeptide is not a myosin polypeptide.

[0005] In various aspects, the methods of treatment and pharmaceutical compositions provided herein are intended for administration to one or both eyes of a subject, e.g., a human or animal subject. In further various aspects, the methods of treatment and pharmaceutical compositions provided herein are intended for administration to one or both eyes of a subject, e.g., a human or animal subject.

[0006] The present disclosure provides materials and methods for gene therapy for diseases such as Usher syndrome.Usher syndrome, including type I (e.g., USH1B), type II, and type III, is a condition that causes sensory dysfunction, particularly in the visual, auditory, and vestibular systems.The sensory loss associated with Usher syndrome may be present even at birth and gradually worsens with age.

[0007] USH1B, the most common form of Usher syndrome, is a severe autosomal recessive deafness-blindness disorder caused by mutations in the Myosin VIIa gene. Patients are born deaf due to genetic mutations that cause insufficient expression and / or protein dysfunction of the human myosin VII protein (MYO7A). Blindness results from progressive retinal degeneration that begins within the first decade of life. MYO7A protein is expressed in photoreceptors and the retinal pigment epithelium (RPE) and is involved in opsin transport through photoreceptor cilia and RPE melanosome movement. Studies have shown that defects in the zonula adherens structure surrounding photoreceptor outer segments may be the initial site of disease and may cause the retinal degeneration seen in USH1B patients (Sahly, et al., 2012). However, the coding region of the MYO7A protein is 6534 or 6648 nucleotides long (depending on the isoform), making traditional AAV vector systems unsuitable for gene therapy of USH1B.

[0008] Currently, there are no treatments available for this condition, but gene therapy holds promise for restoring / maintaining function within the visual, auditory, and vestibular systems. Previously, Allocca et al. (2008) published results suggesting that AAV5 serotype vectors were capable of packaging genomes up to 8.9 KB in size and that these vectors expressed full-length proteins when delivered in vivo. In Allocca et al. (2008), the authors expressed full-length MYO7A protein from an AAV5 vector containing hMYO7A driven by a CMV promoter. Subsequent studies confirmed that these "oversized" AAV5 vectors did indeed drive full-length protein expression, but the gene content of each vector capsid was found to be limited to only approximately 5 KB of DNA, rather than the 8.7 KB originally reported by Allocca et al. (2008) (Lai et al., 2010; Dong et al., 2010; Wu et al., 2010). These vector capsids were shown to contain a "heterogeneous mixture" of truncated vector genomes (e.g., mixtures of the two with deletions of the 5' end of the gene, the 3' end of the gene, or internal sequences). Furthermore, these oversized / heterogeneous vectors exhibited poor packaging efficiency (e.g., resulting in low vector titers) and low transduction efficiency (<5 KB) when compared to standard-sized matched reporter vectors (Wu et al., 2010).

[0009] Using a "heterogeneous" system such as those described by Lai et al. (2010), Dong et al. (2010), and Wu et al. (2010), vectors containing portions of the MYO7A transgene were packaged despite the observed poor packaging efficiency, demonstrating proof-of-concept results in the Shaker-1 mouse model of USH1B. The therapeutic results achieved with the heterogeneous AAV-hMYO7A vector were comparable to previous gene replacement results using a lentiviral-based hMYO7A vector (Hashimoto et al., 2007). This lentiviral-MYO7A vector is being developed by Oxford BioMedica in collaboration with Sanofi-Aventis for Phase I / II clinical trials of USH1B and is marketed under the name UshStat® LentiVector®. Lentiviruses are considered a vector platform that is not well suited to infecting postmitotic (e.g., non-dividing) cells. Furthermore, although the vector is suitable for transducing the RPE, numerous studies have shown that it is not effective in transducing adult photoreceptors. Because photoreceptors (PRs) may be the initial site of disease (Sahly, et al., 2012), exclusive targeting of RPE cells with UshStat® may not result in complete or effective therapy, which remains to be seen in human clinical trials.

[0010] Due to the promising reports of excellent safety profiles and efficacy in AAV gene therapy trials for LCA2 / RPE65, there is ongoing interest in creating an AAV-based system for treating USH1B patients. The present inventors have previously characterized an AAV dual vector platform for use in the treatment of USH1B patients, which is also described herein. The original dual vector system (e.g., the "first generation" dual vector system) designed by the present inventors successfully demonstrated that the mRNA generated from this system was 100% accurate to that predicted by correct homologous recombination of the front and back vector pairs, making them useful as gene therapy delivery vector systems. These vectors are described in U.S. Patent Publication Nos. 2019 / 0153050 and 2014 / 0256802, each of which is incorporated herein by reference in its entirety.

[0011] This disclosure is based, at least in part, on the observation that some previous dual vector platforms resulted in the production of truncated MYO7A proteins in cells, which correlated with the production of truncated fragments of the MYO7A protein. Specifically, after injection of a previous first-generation dual vector hybrid system into mouse retina, loss of retinal structure / function was observed, which may have been due to the gain of function exerted by the truncated MYO7A protein containing a portion of the tail domain. The hybrid vector system contains a recombination product sequence and a spliceosome recognition sequence, enabling two pathways by which the two halves of the polynucleotide vector system can combine in cells to generate full-length polynucleotides. Thus, the hybrid vector system is a modular and versatile alternative to simple overlapping and simple trans-splicing dual vector systems. Described herein is a modified dual hybrid vector system that shifts the coding sequence for the MYO7A tail domain (all or part thereof) from the front half vector to the back half vector by altering the split point (e.g., from between exons 23 and 24 to between exons 21 and 22) to eliminate the generation of a truncated MYO7A protein and any associated cytotoxicity (e.g., gain of function toxicity observed in the retina). Additionally, described herein is a modified dual overlap vector system that shifts the coding sequence for the MYO7A tail domain from the front half vector to the back half vector by altering the overlapping coding sequence between the two vector halves.

[0012] Further described herein are codon-modified hybrid and overlap vector systems in which putative stop codons in non-coding sequences are removed. Further described herein are modified overlap vector systems containing altered and / or reduced length overlapping coding sequences between two vectors. Further described herein are modified hybrid vector systems containing reduced length back-half vectors.

[0013] The present disclosure is also based at least in part on improvements to previous first-generation dual-vector overlap systems to increase transduction efficiency in the retina. In some embodiments, the disclosed improvements involve shortening the 5' (front) and / or 3' (back) AAV vectors in the system to increase rAAV particle packaging efficiency.

[0014] In some embodiments, the disclosed rAAV vectors comprise a transgene encoding a MYO7A protein, for example, a human MYO7A protein. In some embodiments, the disclosed rAAV vectors comprise a transgene encoding another protein associated with Usher syndrome. In some embodiments, the disclosed rAAV vectors comprise a transgene encoding another protein associated with another eye or ear disease, disorder, or condition.

[0015] Thus, aspects of the present disclosure provide a modified dual AAV vector system that allows for expression of full-length proteins whose coding sequences exceed the polynucleotide packaging capacity of individual AAV vectors.

[0016] Thus, in some embodiments, a hybrid dual vector system is provided herein. The polynucleotide vector system includes: i) a first AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, and, between the inverted terminal repeat sequences, a promoter followed by a partial coding sequence encoding an N-terminal portion of a myosin polypeptide, followed by a splice donor (SD) site and an intron; and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, and, between the inverted terminal repeat sequences, an intron and a splice acceptor (SA) site for the intron, wherein the intron sequences in the first and second AAV vectors comprise overlapping polynucleotide sequences, and the division point between the first and second AAV vector polynucleotide sequences is between exon 21 and exon 22 of the hMYO7A gene (see Figures 22D and 22E). Provided herein are hybrid polynucleotide systems in which the N-terminal portion of the myosin polypeptide does not include the single-alpha helix (SAH) domain of the myosin polypeptide (e.g., the first AAV vector polynucleotide includes a partial coding sequence that does not encode the SAH domain of the myosin polypeptide). In some embodiments, the overlapping intron sequence includes an alkaline phosphatase intron. Further provided herein are polynucleotide vector systems in which the first AAV vector polynucleotide includes the nucleotide sequence of SEQ ID NO: 33 or 34, and the second AAV vector polynucleotide includes the nucleotide sequence of SEQ ID NO: 32, 35, or 44.

[0017] In another aspect, provided herein is an overlapping dual vector system comprising: 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 an N-terminal portion of a myosin polypeptide, and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, a partial coding sequence encoding a C-terminal portion of a myosin polypeptide between the inverted terminal repeat sequences, wherein the polynucleotide sequences encoding the polypeptide sequences in the first and second AAV vectors comprise overlapping polynucleotide sequences, and the C-terminal portion of the myosin polypeptide comprises a single-alpha helix (SAH) domain of the myosin polypeptide. Further provided herein is a polynucleotide vector system, wherein a first AAV vector polynucleotide comprises a nucleic acid sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:36, and a second AAV vector polynucleotide comprises a nucleic acid sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:38.

[0018] In some embodiments, a polynucleotide vector system comprises: 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 an N-terminal portion of a myosin polypeptide; and ii) a second 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 a C-terminal portion of a myosin polypeptide; wherein the polynucleotide sequences encoding the polypeptide sequences in the first and second AAV vectors are Further provided herein is a polynucleotide vector system comprising overlapping polynucleotide sequences, wherein (i) a first AAV vector polynucleotide comprises a nucleic acid sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 63, 90, or 66, and (ii) a second AAV vector polynucleotide comprises a nucleic acid sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 77 or 80.

[0019] Further provided herein is a polynucleotide vector system comprising: 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 an N-terminal portion of a myosin polypeptide; and ii) a second 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 a C-terminal portion of a myosin polypeptide, wherein the polynucleotide sequences encoding the polypeptide sequences in the first and second AAV vectors comprise overlapping polynucleotide sequences, and wherein (i) the first AAV vector polynucleotide comprises a nucleotide sequence selected from SEQ ID NOs: 63, 90, and 66, and (ii) the second AAV vector polynucleotide comprises a nucleotide sequence selected from SEQ ID NOs: 77 and 80.

[0020] A polynucleotide vector system comprising: 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 an N-terminal portion of a myosin polypeptide; and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, a partial coding sequence encoding a C-terminal portion of a myosin polypeptide between the inverted terminal repeat sequences, wherein the polynucleotide sequences encoding the polypeptide sequences in the first and second AAV vectors are overlapping polynucleotide sequences. Further provided herein is a polynucleotide vector system comprising an amino acid sequence, wherein (i) the first AAV vector polynucleotide encodes an amino acid sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 62, 91, or 65; and (ii) the second AAV vector polynucleotide encodes an amino acid sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 78 or 81.

[0021] Further provided herein is a polynucleotide vector system comprising: i) a first AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, and, between the inverted terminal repeat sequences, a promoter followed by a partial coding sequence encoding an N-terminal portion of a myosin polypeptide; and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, and, between the inverted terminal repeat sequences, a partial coding sequence encoding a C-terminal portion of a myosin polypeptide, wherein the polynucleotide sequences encoding the polypeptide sequences in the first and second AAV vectors comprise overlapping polynucleotide sequences, and the overlapping polynucleotide sequences comprise nucleotide sequences that are at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a sequence selected from any one of SEQ ID NOs: 39 and 52-59.

[0022] Further provided herein is a polynucleotide vector system comprising: 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 an N-terminal portion of a myosin polypeptide; and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, and a partial coding sequence encoding a C-terminal portion of a myosin polypeptide between the inverted terminal repeat sequences, wherein the polynucleotide sequences encoding the polypeptide sequences in the first and second AAV vectors comprise overlapping polynucleotide sequences, and the overlapping polynucleotide sequences comprise sequences encoding any one of SEQ ID NOs: 79 and 82-89.

[0023] Further provided herein is a polynucleotide vector system comprising: i) a first AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, and between the inverted terminal repeat sequences, a promoter followed by a partial coding sequence encoding an N-terminal portion of a myosin polypeptide; and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, and between the inverted terminal repeat sequences, a second intron and a splice acceptor site for the first intron, wherein the nucleotide sequences of the first and second introns (collectively referred to herein as "intron sequences") comprise overlapping polynucleotide sequences, and the first and / or second intron sequences comprise a nucleic acid sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:69 or SEQ ID NO:70.

[0024] Further provided herein is a polynucleotide vector system comprising: i) a first AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, and, between the inverted terminal repeat sequences, a promoter followed by a partial coding sequence encoding an N-terminal portion of a myosin polypeptide, followed by a splice donor site and a first intron; and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, and, between the inverted terminal repeat sequences, a second intron and a splice acceptor site for the first intron, wherein the nucleotide sequences of the first and second introns comprise overlapping polynucleotide sequences, and the division point between the first and second AAV vector polynucleotide sequences is between two exons of a gene encoding a therapeutic protein.

[0025] A polynucleotide vector system comprising: i) a first AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, and, between the inverted terminal repeat sequences, a promoter followed by a partial coding sequence encoding an N-terminal portion of a myosin polypeptide, followed by a splice donor site and a first intron; and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, and, between the inverted terminal repeat sequences, a second intron and a splice acceptor site for the first intron, wherein the nucleotide sequences of the first and second introns overlap. Further provided herein are polynucleotide vector systems comprising polynucleotide sequences selected from the group consisting of: (i) a first AAV vector polynucleotide comprising a nucleic acid sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NOs: 31, 33, 34, and 46; and (ii) a second AAV vector polynucleotide comprising a nucleic acid sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NOs: 32, 35, 44, and 47-49.

[0026] 1. A polynucleotide vector system comprising: i) a first AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, and, between the inverted terminal repeat sequences, a promoter followed by a partial coding sequence encoding an N-terminal portion of a myosin polypeptide, followed by a splice donor site and a first intron; and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, and, between the inverted terminal repeat sequences, a second intron and a splice acceptor site for the first intron, wherein the nucleotide sequences of the first and second introns are over-expressed. Further provided herein is a polynucleotide vector system comprising overlapping polynucleotide sequences, wherein (i) a first AAV vector polynucleotide comprises a nucleic acid sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 33, and (ii) a second AAV vector polynucleotide comprises a nucleic acid sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 32.

[0027] 1. A polynucleotide vector system comprising: i) a first AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, and, between the inverted terminal repeat sequences, a promoter followed by a partial coding sequence encoding an N-terminal portion of a myosin polypeptide, followed by a splice donor site and a first intron; and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, and, between the inverted terminal repeat sequences, a second intron and a splice acceptor site for the first intron, wherein the nucleotide sequences of the first and second introns are over-expressed. Further provided herein is a polynucleotide vector system comprising overlapping polynucleotide sequences, wherein (i) a first AAV vector polynucleotide comprises a nucleic acid sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:34, and (ii) a second AAV vector polynucleotide comprises a nucleic acid sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:35.

[0028] 1. A polynucleotide vector system comprising: i) a first AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, and, between the inverted terminal repeat sequences, a promoter followed by a partial coding sequence encoding an N-terminal portion of a myosin polypeptide, followed by a splice donor site and a first intron; and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, and, between the inverted terminal repeat sequences, a second intron and a splice acceptor site for the first intron, wherein the nucleotide sequences of the first and second introns are over-expressed. Further provided herein is a polynucleotide vector system comprising overlapping polynucleotide sequences, wherein (i) a first AAV vector polynucleotide comprises a nucleic acid sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:34, and (ii) a second AAV vector polynucleotide comprises a nucleic acid sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:44. [Brief explanation of the drawings]

[0029] To promote an understanding of the principles of the present disclosure, reference will now be made to embodiments or examples illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is intended thereby. Any alterations and further modifications of the described embodiments, and any further applications of the principles of the present disclosure as described herein, are contemplated as would normally occur to one skilled in the art to which the disclosure pertains. [Figure 1] FIG. 1 shows the formation of a complete gene cassette from a dual AAV vector by homologous recombination. [Figure 2] FIG. 2 shows a schematic diagram of the two vector components that make up an overlapping dual vector system according to one embodiment of the present disclosure. [Figure 3] Figure 3 shows a schematic diagram of the two vector components that make up an exemplary hybrid dual vector system containing a native intron: the native hMYO7A intron 23 shown in light shading; the splice donor and splice acceptor sequences are shown in darker shading and indicated with (·). [Figure 4] Figure 4 shows a schematic diagram of the two vector components that make up an exemplary hybrid dual vector system containing a synthetic intron. This is an exemplary standard trans-splicing dual vector system, where "intron" refers to the synthetic alkaline phosphatase splice donor and acceptor sites. The synthetic alkaline phosphatase (AP) intron is shown in light shading, while the AP splice donor and splice acceptor sequences are shown in darker shading and with a (·). [Figure 5] Figure 5 shows immunoblots to detect the presence of MYO7A in infected or transfected HEK293 cells. Heterogeneous vectors are compared for all three dual-vector systems. Dual vectors were packaged in either AAV2 or AAV2 (triple mutant) capsids. The triple mutant contains three tyrosine-to-phenylalanine mutations on the capsid surface. For all three dual-vector systems, infections were performed with either a) the front-half (N-terminal) and back-half (C-terminal) vectors or b) the front-half vector alone (to confirm the presence or absence of a truncated protein product expressed from the promoter-containing N-terminal vector). [Figure 6]Figures 6A and 6B show immunoblots to detect the presence of MYO7A in HEK293 cells infected with an exemplary overlapping dual vector system. Results are shown as a time course (lanes 3-7) from 3 to 7 days post-infection and compared to cells transfected with the MYO7A plasmid (lane 1) and an uninfected control (lane 2). The region of interest in Figure 6A is enlarged and shown with higher contrast in Figure 6B. Starting at 3 days post-infection, full-length human MYO7A protein was visible, with peak expression occurring around day 5. [Figure 7] Figures 7A and 7B show retinas from untreated mice and mice subretinally treated with an exemplary overlapping dual vector system. Immunohistochemistry (IHC) was performed using an antibody directed against MYO7A. MYO7A staining and nuclear (DAPI) staining are shown. [Figure 8] Figures 8A-8D show differences in RPE melanosome localization in wild-type versus shaker-1 mice. In wild-type mice, RPE melanosomes migrate apically toward photoreceptor outer segments (Figure 8A), whereas this phenomenon does not occur in mice lacking MYO7A (shaker-1), as seen in (Figure 8B). On the right, high-magnification images of single RPE cells from either wild-type (Figure 8C) or shaker-1 (Figure 8D) mice demonstrate this phenomenon up close. [Figure 9] Figures 9A-9C show that apical migration of RPE melanosomes is restored in shaker-1 mice injected with an exemplary overlapping dual vector system. Electron microscopy shows that melanosomes in untreated shaker-1 mice do not migrate apically (Figure 9A). In shaker-1 mice injected with an exemplary overlapping vector (packaged in AAV2), RPE melanosomes migrate apically toward photoreceptors, which can be seen here in both low- and high-magnification images (Figures 9B and 9C). [Figure 10-1]Figures 10A-10F illustrate the expression of MYO7A from single AAV2 and AAV5 vectors in cultured cells. Figure 10A is a diagram of a viral vector encoding human MYO7A cDNA. Figure 10B is a Western blot of primary RPE cultures from WT eyecups (lane 1), MYO7A-null mice and those infected or uninfected (lane 4) with AAV2-MYO7A (lane 2) or AAV5-MYO7A (lane 3), and MYO7A+ / - mice (lane 5). All lanes were immunolabeled with antibodies against actin (as a loading indicator of relative protein loading) and MYO7A. Figures 10C-10F are immunofluorescence images of primary RPE cell cultures. Cells derived from uninfected MYO7A-null mice (Fig. 10C), MYO7A+ / - mice (Fig. 10D), or MYO7A-null mice infected with either 1x AAV2-MYO7A (Fig. 10E) or 1x AAV5-MYO7A (Fig. 10F). Scale = 10 μm. [Figure 10-2] Same as above. [Figure 11-1]Figures 11A-11M show MYO7A expression from AAV2 and AAV5 dual vectors in vivo. Figures 11A-11E show EM images of MYO7A immunogold labeling of connecting cilia and periciliary MYO7A from rod photoreceptors in MYO7A-null retinas. Figure 11A is a longitudinal and transverse section from an untreated MYO7A-null retina (background labeling only). Figures 11B and 11C are longitudinal and transverse sections from MYO7A-null retinas treated with AAV2-MYO7A (Figure 11B) or AAV5-MYO7A (Figure 11C). Scale = 50 nm. Figures 11D and 11E are transverse sections of connecting cilia from rod photoreceptors in MYO7A-null retinas treated with AAV2-MYO7A (Figure 11D) or AAV5-MYO7A (Figure 11E). Scale = 50 nm. Figures 11F and 11G show EM images of RPE cells from MYO7A-null retinas treated with AAV2-MYO7A (Figure 11F) or AAV5-MYO7A (Figure 11G). Scale = 500 nm. The area indicated by the rectangle is enlarged to show MYO7A immunogold labeling (indicated by the circle) in Figures 11F-1 and 11G-1. Scale = 50 nm. Figures 11H and 11I show EM images of longitudinal and transverse sections of connecting cilia and periciliary tissue from rod (Figure 11H) and cone (Figure 11I) photoreceptors in MYO7A-null retinas treated with AAV2-MYO7A. Sections were double-labeled with MYO7A (12-nm gold) and rod opsin (15-nm gold) antibodies. Rod outer segments were labeled with opsin antibody, and cones were identified by the lack of rod opsin labeling in their outer segments. The section shows just the base of the outer segment. Nearly all of the labeling is MYO7A in the connecting cilia and also in the rods. Scale = 50 nm. Figures 11J-11M are bar graphs showing MYO7A immunogold particle density in rod photoreceptor cilia and pericillia (Figures 11J and 11L) and in the RPE (Figures 11K and 11M) after treatment with various concentrations of AAV2-MYO7A (Figures 11J and 11K) or AAV5-MYO7A (Figures 11L and 11M). n = 3 animals per condition. Bars indicate SEM. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above. [Figure 11-5] Same as above. [Figure 11-6] Same as above. [Figure 11-7] Same as above. [Figure 12-1] Figures 12A-12F show the correction of melanosome localization after subretinal injection with AAV2-MYO7A or AAV5-MYO7A. Optical micrographs showing the presence of melanosomes in the apical processes of the RPE in WT retinas (Figure 12A) and retinas injected with AAV2-MYO7A (Figure 12B) or AAV5-MYO7A (Figure 12C). Further away from the injection site (Figure 12D), melanosomes are present in the apical processes of some RPE cells but not others (arrows indicate apical melanosomes; white lines indicate areas of the apical processes where melanosomes are absent). Figure 12E illustrates a region distal to the injection site, where all RPE cells lacked melanosomes in their apical processes. The brackets on the left indicate RPE apical processes. Scale = 25 μm. Figure 12F is a diagram of the eyecup showing the relative positions of the images shown in Figures 12A-12E. ONH indicates the optic nerve head. [Figure 12-2] Same as above. [Figure 13] Figure 13 shows correction of abnormal opsin levels in the connecting cilia and periciliary regions of rod photoreceptors after subretinal injection with AAV2-MYO7A or AAV5-MYO7A. Bar graphs show opsin immunogold particle density along the length of the connecting cilia. Ultrathin sections of retinas from MYO7A-null and WT mice were stained with rod opsin antibody. MYO7A-null retinas were untreated or treated with either 1x or 1:100 AAV2-MYO7A or AAV5-MYO7A. n = 3 animals per condition. Bars indicate SEM. [Figure 14-1]Figures 14A-14G show the expression of MYO7A from overlapping AAV2-MYO7A dual vectors. Figures 14A-1 and 14A-2 illustrate diagrams of overlapping AAV2-MYO7A dual vectors. The overlapping region contains 1,365 bases. Figure 14B shows Western blots of proteins from primary RPE cultures derived from MYO7A-null mice that were either uninfected (lane 1) or infected with AAV2-MYO7A (overlap dual) (lane 2); primary RPE cultures derived from MYO7A+ / - mice (lane 3); WT eyecups (lane 4); and HEK293 cells transfected with pTR-smCBA-MYO7A (lane 5). All lanes were immunolabeled with anti-MYO7A and anti-actin. Figures 14C-14F show immunofluorescence of cultured RPE cells transduced with AAV2-MYO7A (overlap dual). Figures 14C-14E ​​show primary RPE cultures and ARPE19 (Figure 14F) cells derived from MYO7A-null mice. Scale = 10 μm. Figure 14G is a bar graph showing the distribution of MYO7A immunogold particle density among RPE cells obtained from the retina of MYO7A-null mice injected with AAV2-MYO7A (dual). n = 3 animals. [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 15-1]Figures 15A-15G illustrate the correction of mutant phenotype after subretinal injection with AAV2-MYO7A (overlap dual). Figure 15A shows light microscopy of semithin sections from treated MYO7A-null mouse retinas. The indicated area is near the injection site. The arrows indicate melanosomes in the apical processes. The white lines indicate cells that still exhibit the MYO7A-null phenotype but lack melanosomes in the apical processes. Scale = 50 μm. Figure 15B is a low-magnification immuno-EM image of RPE from a retina treated with AAV2-MYO7A (overlap dual). As in Figure 15A, the white lines indicate areas that still exhibited the MYO7A-null phenotype. The rectangle "c" contains melanosomes in the apical region, indicating corrected RPE cells. Scale = 500 nm. Figures 15C-15E show higher magnification images of the area outlined by the rectangle shown in Figure 15B. MYO7A immunogold particles are indicated by circles. Scale = 50 nm. Figure 15F is a bar graph illustrating MYO7A immunogold particle density measured in RPE cells obtained from MYO7A-null retinas, WT retinas, or MYO7A-null retinas treated with AAV2-MYO7A (overlap dual) and determined as corrected or uncorrected by the location of apical melanosomes. n = 3 animals per condition. Bars indicate SEM. Figure 15G is an immuno-EM image of rod photoreceptor cilia double-labeled with antibodies against MYO7A (small gold particles) and rod opsin (large gold particles). MYO7A labeling is associated with the connecting cilium and periciliary membrane, indicating the expression and correct localization of MYO7A. This region lacks opsin labeling that is restricted to the disc membrane, which is consistent with the wild-type (WT) phenotype and therefore indicates a correction of the mutant phenotype. Scale = 300 nm. [Figure 15-2] Same as above. [Figure 15-3] Same as above. [Figure 16]Figure 16 shows the validation of dual AAV vectors for in vivo delivery of full-length MYO7A. Immunoblots showing MYO7A expression in the retinas of wild-type (C57BL / 6) mice (lane 1), heterozygous shaker-1+ / − mice (lane 2), and shaker-1− / − mice injected with a "simple overlap" MYO7A vector packaged in an AAV8(733) vector. Both the N- and C-terminal vectors of the "simple overlap" system were injected at a concentration of 3 × 1010 vector genomes / μL. The dual AAV vectors mediated expression of MYO7A that was identical in size to that found in WT and shaker-1+ / − mice. β-Actin (visualized in red herein) was used as a loading control to verify that equal amounts of protein were loaded into each well. [Figure 17-1] Figures 17A-17F show AAV-mediated MYO7A expression in ARPE-19 cells using fragmented vectors and a simple overlapping dual-vector system. Cells were transduced with fragmented vectors: 1x AAV2-MYO7A (Figure 17A), AAV5-MYO7A (Figure 17B), and their 1 / 100 dilutions (Figures 17C and 17D), and a simple overlapping dual-vector system: AAV2-MYO7A (dual) (Figure 17F). Non-transduced cells were used as a control (Figure 17E); light, MYO7A; dark, DAPI. Scale = 10 μm. [Figure 17-2] Same as above. [Figure 18-1] Figures 18A-18D show MYO7A expression in the connecting cilia and periciliary areas of rod photoreceptors from MYO7A-null retinas injected with diluted AAV2-MYO7A (Figures 18A and 18B) or AAV5-MYO7A (Figures 18C and 18D); (Figures 18A and 18C) 1:10, (Figures 18B and 18D) 1:100. Scale = 200 nm. [Figure 18-2] Same as above. [Figure 19]Figure 19 shows structural preservation in injected MYO7A-null retinas. Light microscopy of the photoreceptor layer 3 weeks after injection with 10x AAV5-MYO7A. Scale = 15 μm. [Figure 20] Figure 20 shows structural preservation in injected MYO7A-null retinas. Light microscopy of the photoreceptor layer 3 weeks after injection with 1x AAV2-MYO7A. Scale = 10 μm. [Figure 21-1] Figures 21A-21D show correction of abnormal levels of opsin in the connecting cilia and periciliary regions of rod photoreceptors after subretinal injection with AAV2-MYO7A or AAV5-MYO7A. Immuno-EM from WT retina (Figure 21A), MYO7A-null retina treated with 1x AAV2-MYO7A (Figure 21B) or 1x AAV5-MYO7A (Figure 21C), and untreated MYO7A-null retina (Figure 21D) labeled with anti-rod opsin and 12-nm gold-conjugated secondary antibody. Scale = 200 nm. [Figure 21-2] Same as above. [Figure 22]Figures 22A-22E show schematic diagrams of the dual-AAV-vector pairs generated for this study. Figure 22A shows a fragmented AAV (fAAV) vector. Figure 22B shows a simple overlap: the 1365-bp shared between the two vectors is shaded gray. Figure 22C shows a trans-splicing vector. Figure 22D shows an AP hybrid vector: the 270-bp element shared between the two vectors is marked with diagonal shading (1 / 3 AP head as described by Ghosh et al., 2011). Figure 22E shows a natural intron hybrid vector utilizing a 250-bp sequence from MYO7A intron 23. 3'MYO7A is the 3' portion of MYO7A; 5'MYO7A is the 5' portion of MYO7A; AAV is adeno-associated virus; AP is alkaline phosphatase; intron = intron 23 of MYO7A; pA = polyadenylation signal; SA = splice-acceptor site; SD = splice-donor site; and smCBA refers to the (truncated) chimeric cytomegalovirus immediate early / chicken β-actin chimeric promoter. [Figure 23] Figures 23A-23C show cells expressing human MYO7A after infection with the disclosed simple overlap vectors. Figure 23A shows human embryonic kidney (HEK293) cells expressing human MYO7A after infection with the simple overlap vector packaged in AAV2 (MOI of 10,000 for both vectors) (triple YF). Equal amounts of protein were separated by 7.5% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and stained for MYO7A. Figure 23B shows HEK293 cells infected with AAV2 (triple YF) at MOIs of 10,000, 2,000, and 400. Figure 23C is a time course assay of MYO7A expression in HEK293 cells. Cells were harvested 3 to 7 days post-infection. MOI = multiplicity of infection; T = HEK293 cells transfected with full-length MYO7A plasmid; U = untreated HEK293 cells. [Figure 24]Figure 24 shows a comparison of AAV2 and AAV2 (triple YF mutant capsid) based vectors in HEK293 cells. Cells were infected with the AP hybrid and simple overlap MYO7A dual vector platform packaged in AAV2 or AAV2 (triple YF) at an MOI of 10,000 for each vector. [Figure 25-1] Figures 25A-25C show human MYO7A expressed in HEK293 cells. Cells were infected using an AAV2-based vector platform. For each dual-vector system, the corresponding 5' and 3' vectors (or the 5' vector alone) were used for infection. HEK293 cells transfected with the MYO7A plasmid were used as a positive control. Cells were infected with the MYO7A dual-vector pair at an MOI of 10,000 for each vector. Protein samples were analyzed by Western blot using an antibody against MYO7A (Figure 25A). The relative ability of each dual-vector platform to promote reconstitution was compared by quantifying the amount of 5' vector-mediated truncated protein product in the presence or absence of the respective 3' vector (Figure 25B). Full-length MYO7A expression mediated by the dual vectors was quantified relative to the transfection control (Figure 25C). [Figure 25-2] Same as above. [Figure 26-1]Figures 26A-26C demonstrate the ability of the MYO7A dual vector to recombine and properly restore coding sequences. The experimental design is shown in Figure 26A. HEK293 cells were infected with the AAV2-based dual vector platform, RNA was extracted, and gene-specific primers were used to amplify sequences using PCR. Control digests using BglII (B) and PpuMI (P) showed the expected banding pattern shown in Figures 26B-26C. Undigested (U) PCR product served as a control, and a DNA size marker (M) was included for reference. Separately, the products were digested with KpnI and AgeI and then cloned into pUC57 for sequencing of the entire overlapping region. Ten clones per vector platform were analyzed. Sense and antisense reads (1,000 bp each) were obtained using M13 forward and reverse primers specific to the subcloned vector, resulting in overlapping sense and antisense reads of 140 bp (Figure 26C; Table). PCR = polymerase chain reaction. [Figure 26-2] Same as above. [Figure 26-3] Same as above. [Figure 27-1]Figures 27A-27H show dual vector-mediated MYO7A expression in vivo. C57BL / 6J mice were subretinally injected with an AAV2-based dual vector containing a C-terminal hemagglutinin (HA) tag. Retinal protein expression was analyzed by immunohistochemistry and Western blot 4 weeks after injection. Ten-micron frozen retinal cross sections were stained with an antibody against HA and imaged at 10x magnification (Figures 27A, 27C, and 27E) and 60x magnification (Figures 27B, 27D, and 27F). Untreated C57BL / 6J retinas were also stained with an antibody against HA (Figure 27G). Equal amounts of protein were separated on a 4-15% polyacrylamide gel and stained with an HA antibody (Figure 27H). For comparison, endogenous MYO7A from C57BL / 6J retinas was probed with an antibody against MYO7A to confirm that the HA-tagged MYO7A migrated at the appropriate size. RPE - retinal pigment epithelium, IS - inner segment, OS - outer segment, ONL - outer nuclear layer, INL - inner nuclear layer, GCL - ganglion cell layer, PR - photoreceptor. Nuclear (DAPI) staining is indicated using brackets (}). [Figure 27-2] Same as above. [Figure 27-3] Same as above. [Figure 28] Figure 28 shows a representative schematic of how the dual vector system delivers, recombines, and generates a full-length transgene. The MYO7A cDNA is split into two parts, or "halves," and each half is delivered via a separate AAV vector. After coinfection, the gene halves recombine via their shared / overlapping sequences to form the full-length MYO7A. The recombined transgene can then be transcribed and translated into the desired protein product. [Figure 29]Figures 29A-29B show dual vector-mediated MYO7A expression in Myo7a- / - mice. Figure 29A shows the resulting protein expression after simple overlap or AP hybrid dual vector injection. Myo7a- / - mice were injected with a total of 5.0 x 108 vector genomes (vg) (2.5 x 108 vg each) of either simple overlap or AP hybrid dual vectors. All vector expression was driven by the smCBA promoter. Retinas were collected and analyzed 6 weeks after injection. Figure 29B shows quantification of full-length MYO7A expression normalized to vinculin (VCL) for multiple treatment groups. [Figure 30] Figure 30 shows two bar graphs illustrating the dark-adapted b-wave response and mean ONL thickness by treatment at 6 weeks post-injection to evaluate the safety of dual AAV-MYO7A vectors. All treatments were delivered at a total of 5.0 x 10 vg (2.5 x 10 vg each). [Figure 31-1] Figures 31A-31B show that only the front-half hybrid vector produced truncated proteins. Figure 31A is a bar graph showing average MYO7A front-half transcript expression 6 weeks after injection. Figure 31B shows Western blot results 6 weeks after injection using VCL as a loading control. All vectors were delivered at 5.0 x 10 vg. [Figure 31-2] Same as above. [Figure 32] Figure 32 shows two bar graphs illustrating the dark-adapted b-wave response in C57BL / 6J and Myo7a mice 6 weeks after injection. These results show that only the front-half hybrid vector led to loss of retinal function. All vectors were delivered at 5.0 x 10 vg. [Figure 33] Figure 33 shows two bar graphs illustrating the average MYO7A back-half transcript expression and vector genome 6 weeks after injection. These results indicate that the back-half vector does not produce transcripts or truncated proteins. All vectors were delivered at 8.0 x 10 vg. [Figure 34] Figure 34 shows the relocation of the split site from the original site between exons 23 and 24 to a new split site between exons 21 and 22 to prevent the loss of function observed after injection with the front-half hybrid vector. Hybrid vector systems with a new split site between exons 21 and 22 are hereafter referred to as "second generation" hybrids. [Figure 35] Figures 35A-35B show in vitro results after administration of first- and second-generation hybrid vectors in HEK293 cells. Figure 35A shows Western blot results of the original hybrid with a split site between exons 23 and 24 ("Ex23 / 24") and the second-generation hybrid with a split site between exons 21 and 22 ("Ex21 / 22"), using VCL as a loading control. Figure 35B illustrates normalized full-length MYO7A expression. NI indicates "not injected." [Figure 36-1] Figures 36A-36C show in vivo results from subretinal injection of second-generation dual hybrid vectors in Myo7a- / - mice. The total injected dose was 5 x 108 vg. Figure 36A shows Western blot results using VCL as a loading control. Figure 36B shows the p-value for the comparison of full-length protein expression between exons 21 / 22 and 23 / 24 in the hybrid vector system. Figure 36C shows MYO7A expression normalized to WT. Hybrid vectors were encapsidated into AAV5 and AAV8(Y733F) virions and administered, and MYO7A expression associated with each serotype was measured and normalized to WT. [Figure 36-2] Same as above. [Figure 37]Figures 37A-37B show schematic diagrams of exemplary second-generation MYO7A polynucleotide vector systems of the present disclosure. Figure 37A shows a second-generation hybrid vector system with a split site between exons 21 and 22. The AP sequence acts as a polyA and / or splicing signal. Figure 37B shows an overlap vector in which a potential in-frame stop codon has been removed from the 3' end (downstream of the MYO7A N terminal fragment) in the front hybrid vector plasmid. [Figure 38] Figure 38 shows a bar graph depicting results from hybrid AAV-MYO7A injection in vivo. The second-generation Ex21 / 22 hybrid vector expresses comparable amounts of full-length MYO7A compared to the first-generation Ex23 / 24 hybrid vector, but does not result in the production of truncated protein fragments observed in the first-generation Ex23 / 24 hybrid vector. [Figure 39] Figures 39A-39B show in vitro results of expression of truncated MYO7A proteins in HEK293 cells. Figure 39A shows Western blot results of the original hybrid (exon 23 / 24) front, second-generation hybrid (exon 21 / 22) front, and hybrid CMv1 (exon 21 / 22) front. Herein, "CMv1" (or "COv2") refers to the human codon-modified version 1 hybrid vector. Figure 39B is a bar graph showing truncated MYO7A expression normalized to vinculin. [Figure 40] Figure 40 shows Western blot results for the original hybrid front vector, the second generation ex21 / 22 hybrid front vector, the CMv1 ex21 / 22 hybrid front vector, and the CMv2 ex21 / 22 hybrid front vector. As used herein, "CMv2" (sometimes referred to herein as "COv2") refers to the human codon modified version 2 hybrid vector. [Figure 41]Figure 41 shows non-human primate data on the expression of AAV-mediated MYO7A transcripts in the macaque retina and the tolerability of dual AAV5-MYO7A vectors in the subretinally injected macaque retina. Vectors were delivered at a titer of 4 x 10 vg each (8 x 10 vg total). [Figure 42] Figure 42 shows the procedure for removing stop codons from a second-generation hybrid front-half vector ("Myo7a NT-Ex21") to create the CMv1 hybrid front-half vector. The second-generation hybrid front-half vector (relevant fragment of SEQ ID NO: 31 shown as SEQ ID NO: 40) contained three stop codons within the AP intron (potential stop codons shown in red). The restriction enzymes Bsu36I and NheI were used to excise the region containing the stop codons. The excised sequence was then replaced using a Gibson primer set (SEQ ID NO: 42, SEQ ID NO: 43). The new sequence (relevant fragment of SEQ ID NO: 33 shown as SEQ ID NO: 41) had all three potential in-frame stop codons (indicated with an asterisk (*)) removed. In addition to modifying the potential stop codons, two restriction sites, Hpal and Mfel, were added to the new sequence to simplify screening of the resulting clones. [Figure 43] Figure 43 shows the changes made in the second generation overlapping front half vector to create the CMv1 overlapping front half vector. [Figure 44] Figure 44 is a schematic diagram showing a dual AAV vector approach for delivery of MYO7A gene therapy. The MYO7A cDNA is split into two halves, and each half is delivered via a separate AAV vector and delivered in separate AAV particles that are injected simultaneously. The gene halves recombine in each cell to form the full-length MYO7A gene. [Figure 45-1]Figures 45A-45C show two different dual AAV vector platforms driving full-length MYO7A expression. Figure 45A contains a schematic diagram depicting the overlap and hybrid dual AAV vector platforms. Figures 45B and 45C show the amount of MYO7A produced from the original hybrid and overlap vectors after encapsidation in AAV5 or AAV8(Y733F) virions. The VCL marker was used as a control. [Figure 45-2] Same as above. [Figure 46] Figure 46 shows an improved (third generation) overlapping dual vector that exhibits increased packaging efficiency. [Figure 47] Figure 47 shows the overlap length and position. A shorter overlap length results in less shared sequence between the front and back half vectors. [Figure 48] Figures 48A and 48B show overlap vectors compared using the Protein Simple Jess quantification system with a capillary-based Western blot tool. Figure 48A shows the expression of MYO7A and truncated MYO7A from various overlap vectors. Figure 48B shows a table listing the overlap length and the respective ITR-ITR length (bp). [Figure 49] Figures 49A-49B show overlap panel quantification. Overlap vectors containing 687 or 945 bp of overlapping MYO7A sequence produce the same or more full-length MYO7A than the original hybrid vector. Figure 49A shows MYO7A expression normalized to VCL for the original hybrid vector. Figure 49B shows the degree of expression of truncated MYO7A proteins normalized to VCL for the original hybrid vector. [Figure 50-1]Figures 50A-50D show improved hybrid dual vectors that offer reduced cytotoxicity and greater safety. Figure 50A shows Hybrid-V2, in which the "split point" has been altered, resulting in the neck / tail domain not being encoded by the front-half vector. Figure 50B shows a schematic diagram of the hybrid vector. Four potential in-frame stop codons were modified in the Hybrid-V2 front-half vector sequence. All codon modifications were made in the Hybrid-V2 MIN background. Figure 50C shows a table listing the modifications made to each vector and their respective ITR-to-ITR (ITR-ITR) lengths (in bp). Figure 50D shows a comparison of the production of full-length MYO7A and truncated MYO7A fragments relative to the original hybrid vector. [Figure 50-2] Same as above. [Figure 51-1] Figures 51A-51E show hybrid dual vectors with improved safety. Figure 51A shows exemplary vectors representing Hybrid-V2BacMIN, Hybrid-CMv2BacMIN, and Hybrid-V2BacMIN HA and Hybrid-CMv2BacMIN HA. In the hybrid buckMIN vectors, "unnecessary legacy" sequences were removed from the buck-half vectors to ensure that vector size did not exceed packaging capacity. Figure 51B shows a table listing the codon modifications made to each vector and their respective ITR-ITR lengths (bp). Figures 51C-E show that reducing the size of the buck-half vectors leads to increased expression of full-length MYO7A from the hybrid vectors. [Figure 51-2] Same as above. [Figure 51-3] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0030] Exemplary embodiments of the present disclosure are described below. The present disclosure provides materials and methods for gene therapy of diseases and conditions, such as Usher syndrome 1B (USH1B). Aspects of the present disclosure relate to AAV-based dual vector systems that enable expression of full-length proteins whose coding sequences exceed the polynucleotide packaging capacity of individual AAV vectors. Aspects of the present disclosure provide AAV-based dual vector systems for expression in the retina of a subject's eye or hair cells of a subject's inner ear. Accordingly, methods are provided herein for treating ocular and otic conditions and other diseases and disorders associated with USH1B. The present disclosure provides nucleic acid vectors of overlapping vector systems and nucleic acid vectors of hybrid vector systems.

[0031] For example, several separate AAV-based dual vector systems have been created and are disclosed herein for use in gene replacement therapy, including in the treatment of USH1B in human patients. In certain embodiments, the disclosed vector system uses two separate AAV vectors, each packaging a maximum-sized DNA molecule (e.g., approximately 4.5-4.8 kb). The two vectors are co-administered into selected recipient cells to reconstitute a full-length, biologically active MYO7A polypeptide. In these constructs, a portion of the overlapping nucleic acid sequence is common to each of the vector genomes (see Figure 1). When co-delivered into appropriate cells (Figure 44), the overlapping sequence region facilitates proper concatenation of the two partial gene cassettes. These gene cassettes then undergo homologous recombination to generate a full-length gene cassette within the cell (see Figure 1). Exemplary shared components of exemplary embodiments of the dual vector system include the use of AAV inverted terminal repeats (ITRs), a small (truncated) version of the chimeric CMV / chicken β-actin promoter (smCBA), human MYO7A (hMYO7A) cDNA sequence, and the SV40 polyadenylation (pA) signal.

[0032] In some embodiments, the polynucleotide vectors and vector systems provided herein do not comprise the nucleotide sequences of SEQ ID NOs: 1-4. In exemplary embodiments, the overlap vectors of the present disclosure do not comprise any of SEQ ID NOs: 1 and 2. In exemplary embodiments, the hybrid vectors of the present disclosure do not comprise any of the nucleotide sequences of SEQ ID NOs: 3 and 4. In some embodiments, the vectors of the present disclosure do not comprise the nucleotide sequence of SEQ ID NO: 67 or 71.

[0033] Overlap Vector System In some aspects, overlapping dual AAV vector systems are provided. In some embodiments, the overlapping vector systems of the present disclosure do not produce truncated MYO7A protein fragments after administration to a mouse or subject.

[0034] In one aspect of the present disclosure, the overlap vector system of the present disclosure comprises: i) a first AAV vector polynucleotide comprising inverted terminal repeat sequences at each end (5' and 3' ends) of the polynucleotide, a suitable promoter between the inverted terminal repeat sequences, followed by a partial coding sequence (e.g., 3' of the promoter) encoding the N-terminal portion of a selected full-length polypeptide; and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end (e.g., the 5' and 3' ends) of the polynucleotide, a partial coding sequence encoding a C-terminal portion of a selected full-length polypeptide between the inverted terminal repeat sequences, optionally followed by a polyadenylation (pA) sequence. When combined, the coding sequences in the first and second vectors encode the selected full-length polypeptide or a functional fragment or variant thereof. The polypeptide-encoding sequences in the first and second AAV vectors comprise overlapping sequences.

[0035] In some embodiments of the provided overlap vector systems, the selected full-length polypeptide is a myosin polypeptide. In some embodiments, the myosin polypeptide is human myosin VIIA (hMYO7A). In some embodiments, the myosin polypeptide is human myosin VII B (hMYO7B). In some embodiments, the myosin polypeptide is myosin 7(VII) isoform II. Isoform II(2) of hMYO7A (NM_001127180) encodes a 2175 amino acid protein (250.2 kDa) and lacks an in-frame segment (part of exon 35) in the coding region compared to isoform I (see Chen et al., 1996; Weil et al., 1996). In some embodiments, the myosin polypeptide is another myosin isoform or a functional fragment thereof. In certain embodiments, full-length myosin 7A or isoform II is encoded in the provided vector systems. The peptide sequence of isoform II is shown as SEQ ID NO:8.

[0036] In some embodiments, the selected full-length polypeptide is selected from ABCA4 (Stargardt disease), CEP290 (LCA10), EYS (retinitis pigmentosa), RP1 (retinitis pigmentosa), ALMS1 (Alström syndrome), CDH23 (Usher syndrome 1D), PCDH15 (Usher syndrome 1F), and USHERIN (Usher syndrome 2A). In some embodiments, the selected full-length polypeptide is selected from DMD (Duchenne muscular dystrophy), CFTR (cystic fibrosis), GDE (glycogen storage disease III), DYSF (dysferlinopathy), OTOF (neurosensory nonsyndromic recessive deafness), and F8 (hemophilia A). Diseases and disorders associated with each of these genes are provided in parentheses. In some embodiments, the selected full-length polypeptide is encoded by a gene about 6 kb to about 9 kb in length. In some embodiments, the selected full-length polypeptide is encoded by a gene about 7 kb to about 8 kb in length.

[0037] The present inventors have discovered that the hMYO7A overlap region, e.g., SEQ ID NOS: 39 and 53-59, can be used as overlapping polynucleotide sequences in additional overlapping dual vectors expressing large genes (other than MYO7A). Thus, in some embodiments, overlapping dual vectors expressing portions (or halves) of large genes selected from ABCA4, CEP290, EYS, RPI, ALMS1, CDH23, PCDH15, USH1C, USH1G, USH2A, DNFB31, DMD, CFTR, GDE, DYSF, F8, and DFNB2 contain overlapping regions comprising portions of the hMYO7A gene in their overlapping polynucleotide sequences. These overlapping vectors express large genes other than MYO7A that contain nucleotide sequences that have at least 80%, 85%, 90%, 95%, 98%, or 99% identity to any one of SEQ ID NOS: 39 and 52-59. Such an overlap vector may comprise an overlap region containing the nucleotide sequence of any one of SEQ ID NOs: 39 and 53 to 59, for example, SEQ ID NO: 56 or 57.

[0038] In some embodiments, the selected full-length polypeptide is expressed in one or more photoreceptor cells. In some embodiments, the selected full-length polypeptide is expressed in one or more cells that do not include photoreceptor cells. In some embodiments, the selected full-length polypeptide is expressed in one or more hair cells, for example, hair cells of the auditory system or vestibular system.

[0039] In some embodiments, the C-terminal portion of the selected full-length polypeptide (eg, a myosin polypeptide) comprises the single-alpha helix (SAH) domain of the selected full-length polypeptide.

[0040] In some embodiments, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1 or a functional fragment and / or variant thereof, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2 or a functional fragment and / or variant thereof.

[0041] In some embodiments, first-generation overlap vectors (e.g., an AAV vector polynucleotide comprising the nucleotide sequence of SEQ ID NO:1 or a functional fragment and / or variant thereof and / or a second AAV vector polynucleotide comprising the nucleotide sequence of SEQ ID NO:2 or a functional fragment and / or variant thereof) contain nucleotides 1 to 3644 of the MYO7A cDNA from the ATG in the 5' vector and / or nucleotides 2279 to 6534 in the 3' vector. In some embodiments, fragments are amplified by polymerase chain reaction (PCR) using P1 and P3 and cloned into the 5' vector via NotI and NheI, and into the 3' vector using P3 (AflII) and P4 (KpnI), respectively. The resulting two vector plasmids share 1365 bp of overlapping MYO7A sequence (Figure 2), with the overlap between the sequences ending at the split point between exons 23 and 24.

[0042] In some embodiments, the portion of the coding sequence present at the 3' end of the coding sequence of the first generation overlap vector is identical or substantially identical to the portion of the coding sequence present at the 5' end of the coding sequence of the first generation overlap vector. In specific embodiments, the sequence overlap between the first and second AAV first generation overlap vectors is between about 500 and about 3,000 nucleotides, between about 1,000 and about 2,000 nucleotides, between about 1,200 and about 1,800 nucleotides, or between about 1,300 and about 1,400 nucleotides.

[0043] In certain embodiments, the sequence overlap between the first and second AAV overlap vectors of the present disclosure is 1284 bp, 1027 bp, 1026 bp, 945 bp, 687 bp, 361 bp, 279 bp, or 20 bp in length. In certain embodiments, the sequence overlap between the first and second AAV overlap vectors of the present disclosure is within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of 1284 bp, 1027 bp, 1026 bp, 945 bp, 687 bp, 361 bp, 279 bp, or 20 bp in length. In some embodiments, the sequence overlap is 945 bp, 687 bp, or 361 bp.

[0044] In certain embodiments, the sequence overlap of the first-generation overlap vector system is approximately 1,350 nucleotides. In exemplary embodiments, the sequence overlap of the first-generation overlap vector system is 1,365 nucleotides. In certain embodiments, the overlapping polynucleotide sequence comprises SEQ ID NO:45. In certain embodiments, the encoded polypeptide is wild-type or functional human myosin VIIa (hMYO7A). The amino acid sequences of wild-type and functional hMYO7A polypeptides and the polynucleotides encoding them are known in the art (see, e.g., GenBank Accession Nos. NP_000251 and U39226.1). In certain embodiments, the hMYO7A polypeptide comprises the amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:8, or a functional fragment or variant thereof. In certain embodiments, the hMYO7A polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:5 or SEQ ID NO:7.

[0045] Codon-modified overlap vector In some embodiments of the disclosed overlap vector system, a codon-modified overlap vector is provided. In some embodiments, the first-generation overlap front half vector ("AAV-smCBA-hMYO7A-NT") is shortened. All coding sequences corresponding to the tail domain of MYO7A were removed from the front half vector, thus reducing the size of the overlap region to 361 bp (SEQ ID NO: 39). (This vector does not generate truncated MYO7A fragments containing the tail or SAH domains.) The vector was also modified to remove all potential (or putative) stop codons (see Figure 37B). The resulting vector is the CMv1 overlap front half vector ("AAV-smCBA-hMYO7A-noDimNT-CMv1"). Thus, the overlap vector system of the present disclosure may comprise a CMv1 overlap vector system.

[0046] In some embodiments, overlap vectors with altered (e.g., shortened) overlapping coding sequences are provided. In these embodiments, overlap vectors containing overlapping sequences in the MYO7A gene or another gene that is less than 1365 bp in length are provided. In these systems, the length of the overlapping sequences is reduced to a certain point, thus ensuring that none of the vector genomes overwhelms the packaging capacity of the AAV capsid (4.7-4.9 kb), leading to increased expression of full-length MYO7A. If the overlap length is too small (≦361 bp), full-length MYO7A expression is reduced and a truncated protein appears. Overlap vectors containing 687 or 945 bp of overlapping MYO7A sequence produce full-length MYO7A that is equal to or greater than the original hybrid vector (see Figures 48A, 48B, 49A, and 49B). Such vectors are sometimes referred to herein as "V3" or third-generation overlap vectors. In exemplary embodiments, overlap vectors containing 687 or 945 bp of overlapping MYO7A sequences are exemplified.

[0047] Thus, provided herein are polynucleotide vector systems in which overlapping polynucleotide sequences comprise a nucleotide sequence selected from any one of SEQ ID NOs: 39 and 52-59. In some embodiments, the overlapping polynucleotide sequences comprise a nucleotide sequence selected from any one of SEQ ID NOs: 39, 56, and 57. In exemplary embodiments, the overlapping polynucleotide sequences comprise the sequence of SEQ ID NO: 56 or 57. In some embodiments, the length between the inverted terminal repeat sequences at each end of the first AAV vector polynucleotide is about 4615 nucleotides (nt) or less. In some embodiments, the length between the inverted terminal repeat sequences at each end of the second AAV vector polynucleotide is about 4800 nt or less. In some embodiments, the length between the inverted terminal repeat sequences at each end of the second AAV vector polynucleotide is about 4560 nt.

[0048] Thus, in some embodiments, the polynucleotide vector system of the present disclosure is a CMv1 overlap system. In some embodiments, the vector system is an overlap V2 (second generation) system. In some embodiments, the vector system is a V3 overlap (third generation) system. Any of the disclosed front half overlap vectors can be combined with any of the disclosed back half overlap vectors in the compositions of the present disclosure. The resulting third generation overlap front half vector ("AAV-smCBA-hMYO7A-NTlong-v3") is set forth as SEQ ID NO: 50. The resulting third generation overlap back half vector, including the HA tag, is set forth as SEQ ID NO: 51.

[0049] Thus, in some aspects, 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 an N-terminal portion of a myosin polypeptide; and ii) a polynucleotide vector system comprising a second AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, and a partial coding sequence between the inverted terminal repeat sequences encoding a C-terminal portion of a myosin polypeptide, the polynucleotide sequences encoding the polypeptide sequences in the first and second AAV vectors comprise overlapping polynucleotide sequences; the first AAV vector polynucleotide comprises a nucleotide sequence selected from SEQ ID NOs: 36, 37 and 50; Provided herein is a polynucleotide vector system, wherein the second AAV vector polynucleotide comprises a nucleotide sequence selected from SEQ ID NOs: 38 and 51. In an exemplary embodiment, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 50, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 51. In some embodiments, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 50, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 38. In some embodiments, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 36, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 38.

[0050] In some embodiments, the first AAV vector polynucleotide comprises a partial coding sequence that does not encode the single-alpha helix (SAH) domain of the selected full-length polypeptide. In some embodiments, the first AAV vector polynucleotide of the second-generation overlap vector comprises the nucleotide sequence of SEQ ID NO: 37, or a functional fragment and / or variant thereof, and the second AAV vector polynucleotide of the second-generation overlap vector comprises the nucleotide sequence of SEQ ID NO: 38, or a functional fragment and / or variant thereof.

[0051] In some embodiments, second-generation overlap vectors (e.g., an AAV vector polynucleotide comprising the nucleotide sequence of SEQ ID NO: 37 or a functional fragment and / or variant thereof and / or a second AAV vector polynucleotide comprising the nucleotide sequence of SEQ ID NO: 38 or a functional fragment and / or variant thereof) contain nucleotides from the ATG of the MYO7A cDNA in the 5' vector to nucleotides 1 to 2640 and / or nucleotides 2279 to 6534 in the 3' vector. In some embodiments, fragments are amplified by polymerase chain reaction (PCR) using P1 and P3 and cloned into the 5' vector via NotI and NheI and into the 3' vector using P3 (AflII) and P4 (KpnI), respectively. The resulting two vector plasmids share 361 bp of overlapping MYO7A sequence (Figure 37), with the overlap between the sequences ending at the split point between exons 21 and 22.

[0052] In some embodiments, the overlapping polynucleotide sequence does not include any portion of exon 23 of the hMYO7A gene. In some embodiments, the overlapping polynucleotide sequence does not include the entirety of exon 23 (e.g., 100% of exon 23). In some embodiments, the overlapping polynucleotide sequence includes a portion of exon 17, the entirety of exon 18, the entirety of exon 19, the entirety of exon 20, and a portion of exon 21 of the hMYO7A gene. In some embodiments, the overlapping polynucleotide sequence includes a portion of exon 17, a portion of exon 18, a portion of exon 19, a portion of exon 20, and / or a portion of exon 21 of the hMYO7A gene. As used herein, a "portion" refers to, for example, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47% of the exon and / or intron sequence. , 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% etc.

[0053] In some embodiments, the portion of the coding sequence present at the 3' end of the coding sequence of a first vector of the second-generation overlap vector is identical or substantially identical to the portion of the coding sequence present at the 5' end of the coding sequence of a second vector of the second-generation overlap vector. In certain embodiments, the sequence overlap between the first and second AAV vectors is between about 1 and about 500 nucleotides, between about 100 and about 200 nucleotides, between about 200 and about 300 nucleotides, or between about 300 and about 400 nucleotides.

[0054] In certain embodiments, the sequence overlap of the second-generation overlap vector system is approximately 350 nucleotides. In an exemplary embodiment, the sequence overlap of the second-generation overlap vector system is 361 nucleotides. In certain embodiments, the overlapping polynucleotide sequence comprises SEQ ID NO: 39. In certain embodiments, the encoded polypeptide is wild-type or functional human myosin VIIa (hMYO7A). The amino acid sequences of wild-type and functional hMYO7A polypeptides and the polynucleotides encoding them are known in the art (see, e.g., GenBank Accession Nos. NP_000251 and U39226.1). In certain embodiments, the hMYO7A polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 8, or a functional fragment or variant thereof. In certain embodiments, the hMYO7A polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 7.

[0055] SEQ ID NO: 5 is the nucleotide sequence encoding the human myosin VIIa polypeptide (protein coding sequence is from nucleotides 273 to 6920);

[0056] SEQ ID NO:6 is the amino acid sequence of the human myosin VIIa polypeptide encoded by nucleotides 273 to 6920 of SEQ ID NO:5;

[0057] SEQ ID NO:7 is the nucleotide sequence encoding the human myosin VIIa polypeptide;

[0058] SEQ ID NO: 8 is the amino acid sequence of human myosin VIIa polypeptide (isoform 2);

[0059] CMvl overlapping vector system Some embodiments contemplate overlapping vector systems as described herein in which one or more substitutions have been made in the 3' untranslated region downstream of the MYO7A partial coding sequence and preceding the 3' AAV inverted terminal repeat. In some embodiments, these substitutions are intended to remove potential (or putative) in-frame stop codons. In some embodiments, these substitutions remove one or more putative stop codons in the non-coding sequence. In certain embodiments, the substitutions remove one or more putative stop codons in the 3' untranslated region (e.g., downstream of the MYO7A N-terminal fragment) between the partial coding sequence encoding the C-terminal portion of the polypeptide and the 3' AAV inverted terminal repeat of the second AAV vector polynucleotide. In some embodiments, one or more putative stop codons have been removed and replaced with a "stuffer" sequence (see Figure 43). In some embodiments, the first AAV vector polynucleotide thus comprises a partial coding sequence that does not encode the single-alpha helix (SAH) domain of the selected full-length polypeptide.

[0060] These substitutions result in the creation of a front half vector having a nucleotide sequence comprising SEQ ID NO: 36. In such an embodiment, the overlap vector system of the present disclosure comprises: (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 an N-terminal portion of a selected full-length polypeptide; and (ii) a second AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of the polynucleotide, and a partial coding sequence between the inverted terminal repeat sequences encoding a C-terminal portion of a selected full-length polypeptide; In some embodiments, the second AAV vector polynucleotide is followed by a polyadenylation (pA) signal sequence. When combined, the coding sequences in the first and second vectors encode a selected full-length polypeptide or a functional fragment or variant thereof. The polypeptide-encoding sequences in the first and second AAV vectors comprise overlapping sequences.

[0061] In some embodiments, the C-terminal portion of the selected full-length polypeptide (eg, a myosin polypeptide) comprises the single-alpha helix (SAH) domain of the selected full-length polypeptide.

[0062] In some embodiments, the overlapping polynucleotide sequence comprises SEQ ID NO: 39. In certain embodiments, the sequence overlap between the first and second AAV vectors is between about 1 and about 500 nucleotides, between about 100 and about 200 nucleotides, between about 200 and about 300 nucleotides, or between about 300 and about 400 nucleotides. In certain embodiments, the sequence overlap of the second-generation overlap vector system is about 350 nucleotides. In an exemplary embodiment, the sequence overlap of the second-generation overlap vector system is 361 nucleotides.

[0063] In some embodiments, the overlapping polynucleotide sequence does not include any portion of exon 23 of the hMYO7A gene. In some embodiments, the overlapping polynucleotide sequence does not include the entirety of exon 23 (e.g., 100% of exon 23). In some embodiments, the overlapping polynucleotide sequence includes a portion of exon 17, the entirety of exon 18, the entirety of exon 19, the entirety of exon 20, and a portion of exon 21 of the hMYO7A gene. In some embodiments, the overlapping polynucleotide sequence includes a portion of exon 17, a portion of exon 18, a portion of exon 19, a portion of exon 20, and / or a portion of exon 21 of the hMYO7A gene. As used herein, a "portion" refers to, for example, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, These may include 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0064] In an exemplary embodiment, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 36 or a functional fragment and / or variant thereof, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 38 or a functional fragment and / or variant thereof.

[0065] In some embodiments, the first AAV vector polynucleotide comprises a partial coding sequence that does not encode the single-alpha helix (SAH) domain of a selected full-length (e.g., myosin) polypeptide, hi some embodiments, the second AAV vector polynucleotide is followed by a polyadenylation (pA) signal sequence.

[0066] In some embodiments, any vector of the overlapping polynucleotide vector system described in this disclosure can be administered parenterally, e.g., intravenously, intramuscularly, intraocularly, intranasally, etc. The vector can be administered in vivo, in vitro, or ex vivo.

[0067] In some embodiments, any vector of the overlapping polynucleotide vector system described herein can be administered to the eye. In certain embodiments, the vector is administered to the eye of a subject by subretinal injection. In some embodiments, any vector of the hybrid polynucleotide vector system described herein can be administered to the ear. In some embodiments, any vector of the hybrid polynucleotide vector system described herein, the polynucleotide vector system, can be administered to the ear of a subject by, for example, round window injection or during cochlear implant surgery.

[0068] SEQ ID NO: 5 is a nucleotide sequence encoding a human myosin VIIa polypeptide (the protein coding sequence is from nucleotides 273 to 6920), the sequence of which is disclosed herein;

[0069] SEQ ID NO:6 is the amino acid sequence of a human myosin VIIa polypeptide encoded by nucleotides 273 to 6920 of SEQ ID NO:5, the sequence of which is disclosed herein;

[0070] SEQ ID NO:7 is a nucleotide sequence encoding a human myosin VIIa polypeptide, the sequence of which is disclosed herein;

[0071] SEQ ID NO: 8 is the amino acid sequence of a human myosin VIIa polypeptide (isoform 2), the sequence of which is disclosed herein.

[0072] Some aspects of the disclosed overlap vectors contemplate 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 comprises SEQ ID NO: 36, and the second AAV vector polynucleotide comprises SEQ ID NO: 38. In some embodiments, the virus or recombinant viral particle is characterized as an adeno-associated virus (AAV) or an infectious AAV viral particle. In some embodiments, the recombinant AAV viral particle comprises one or more tyrosine to phenylalanine (YF) mutations in the viral or virion capsid protein. A tyrosine to phenylalanine (YF) mutation in the viral or virion capsid protein at amino acid position 733 is specifically contemplated herein (e.g., AAV8 Y733F).

[0073] In some embodiments, the virus or virion is packaged in an AAV5, AAV7, AAV8, AAV9, AAV44.9, AAV44.9(E531D), AAV2(4pMut), AAVAnc80, AAVrh.8, AAVrh.8R, AAV9-PHP.B, AAV9-PHP.eB, AAVrh.10, or AAVrh.74 capsid. In some embodiments, the viral particles comprise an 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, AAV9-PHP.B, and / or AAVAnc80 capsid. In exemplary embodiments, the virions are packaged in the AAV44.9(E531D) capsid mutant.

[0074] In some embodiments, the overlapping polynucleotide vector system described herein uses a tissue-specific promoter. In some embodiments, the system uses a promoter that mediates expression in the eye. In some embodiments, the system uses a promoter that mediates expression in the ear.

[0075] In some embodiments, the overlapping polynucleotide vector system described herein uses any one of the following promoters: a cytomegalovirus (CMV) promoter, an elongation factor-1 alpha (EF-1 alpha) promoter, a cone arrestin promoter, a chimeric CMV β-actin promoter (CBA), a truncated chimeric CMV β-actin (smCBA), a promoter derived from the human myosin 7a gene, a promoter derived from the cone transducin (TαC) gene, a rhodopsin promoter, a cGMP-phosphodiesterase β-subunit promoter, a human or mouse rhodopsin promoter, a human rhodopsin kinase (hGRK1) promoter, a synapsin promoter, a glial fibrillary acidic protein (GFAP) promoter, a rod-specific IRBP promoter, an RPE-specific vitelliform macular degeneration-2 (VMD2) promoter, and combinations thereof. In some embodiments, the polynucleotide vector system described herein uses a human rhodopsin kinase (hGRK1) promoter. In some embodiments, the polynucleotide vector system uses a cone arrestin promoter.

[0076] In some embodiments for delivery to the subject's eye (retina), the disclosed overlapping polynucleotide vector system uses a cytomegalovirus (CMV) promoter. In some embodiments, the polynucleotide vector system uses an EF-1 alpha promoter. In some embodiments for delivery to the subject's ear (hair cells), the polynucleotide vector system uses a synapsin or GFAP promoter (see Lee et al., Hearing Research).

[0077] Hybrid Vector System In some aspects, hybrid dual AAV vector systems are provided. These hybrid vector systems deliver higher levels of full-length MYO7A than overlap vectors and produce truncated proteins from front-half vectors. Hybrid front-half vectors lead to reduced retinal function in subretinal-injected mice (see Figures 15B-E). In various embodiments, the hybrid vector systems of the present disclosure do not produce truncated MYO7A protein fragments after administration to mice or subjects.

[0078] Altering the split point, codon-modifying the front-half vector, and / or minimizing the length of the back-half vector leads to production of full-length MYO7A at levels comparable to or greater than those seen with first-generation hybrid vectors. The improved vectors provided herein produce significantly fewer undesired truncated protein products from the front-half vector. In some embodiments, production of truncated proteins is partially or completely eliminated.

[0079] In some aspects of the present disclosure, the hybrid vector system of the present disclosure comprises: (i) a first AAV vector polynucleotide comprising inverted terminal repeats at each end (e.g., the 5' and 3' ends) of the polynucleotide, a suitable promoter between the inverted terminal repeats, followed by a partial coding sequence encoding the N-terminal portion of a selected full-length polypeptide (e.g., 3' of the promoter), followed by a splice donor site and an intron; and (ii) a second AAV vector polynucleotide comprising inverted terminal repeats at each end (e.g., the 5' and 3' ends) of the polynucleotide, an intron and an intron splice acceptor site between the inverted terminal repeats, optionally followed by a partial coding sequence encoding a C-terminal portion of a selected full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence; The intron sequences in the first and second AAV vectors comprise overlapping sequences.

[0080] In some embodiments, the division point between the first and second AAV vector polynucleotide sequences is between exon 21 and exon 22 of the hMYO7A gene.

[0081] In an exemplary embodiment, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 31 or a functional fragment and / or variant thereof, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 32 or a functional fragment and / or variant thereof.

[0082] The coding sequences in the first and second vectors, when combined, encode a selected full-length polypeptide or a functional fragment or variant thereof. In some embodiments, the selected full-length polypeptide is hMYO7A. In some embodiments, the selected full-length polypeptide is hMYO7B. In some embodiments, the selected full-length polypeptide is hMYO7A isoform II. In some embodiments, the polynucleotide sequence corresponding to the tail domain of the MYO7A protein is removed from the first AAV vector polynucleotide.

[0083] It will be appreciated that the present disclosure is not limited to delivery of full-length myosin 7A polypeptides or nucleotides encoding myosin 7A. In some embodiments, the selected full-length polypeptide is selected from ABCA4 (Stargardt disease), CEP290 (LCA10), EYS (retinitis pigmentosa), RP1 (retinitis pigmentosa), ALMS1 (Alström syndrome), CDH23 (Usher syndrome 1D), PCDH15 (Usher syndrome 1F), and USH2A (Usher syndrome 2A). In some embodiments, the selected full-length polypeptide is selected from DMD (Duchenne muscular dystrophy), CFTR (cystic fibrosis), GDE (glycogenosis III), DYSF (dysferlinopathy), OTOF (neurosensory nonsyndromic recessive deafness), and F8 (hemophilia A). In some embodiments, the selected full-length polypeptide is not OTOF.

[0084] In some embodiments, all or a portion 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 a portion of the intron sequence present at the 5' end of the coding sequence of the second vector. In some embodiments, the intron sequence overlap between the first and second AAV vectors is several hundred nucleotides in length. In certain embodiments, the intron sequence overlap is about 50 to about 500 nucleotides in length; or about 200 to about 300 nucleotides in length.

[0085] In certain embodiments, the intron sequence utilized in any vector system of the present disclosure is the sequence of an intron naturally occurring in the genomic sequence of the gene encoding the selected polypeptide. In some embodiments characterized as natural intron hybrid vectors, the intron is intron 23 of the hMYO7A gene. In certain embodiments, the encoded polypeptide is hMYO7A or a functional fragment thereof, and the intron is a partial sequence of the entire intron 23 of the hMYO7A gene. In certain embodiments, the encoded polypeptide is hMYO7A or a functional fragment thereof, and the intron is the entire intron 23 of the hMYO7A gene.

[0086] In some embodiments of the natural intron hybrid vectors described herein, the recombination product sequence of the dual vector system comprises a partial sequence of exon 21, exon 22, and / or exon 23 of the hMYO7A gene. In some embodiments of the natural intron hybrid vectors described herein, the dual vector system comprising a partial sequence of exon 21, exon 22, and / or exon 23 of the hMYO7A gene utilizes a partial sequence of an entire natural intron. In some embodiments, the natural intron is intron 23 of the hMYO7A gene. Thus, in some embodiments, the intron sequence is a partial sequence of the entire intron 23 of the hMYO7A gene. As used herein, a "subsequence" refers to, for example, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120 %, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% etc. In some embodiments, the first and / or second intron sequence comprises a sequence of an intron naturally occurring in the genomic sequence of a gene encoding a myosin polypeptide, hi some embodiments, the intron sequence is a partial sequence of the entire intron 23 of the hMYO7A gene.

[0087] Several strategies have been devised to overcome the problem of random concatemerization and thereby increase the specificity and efficiency of dual vector platforms. First, the addition of a highly recombination product sequence, such as that used in the AP hybrid vector herein, resulted in significantly increased protein expression compared to trans-splicing systems. Ghosh et al. (2011) provide a detailed analysis of the 270-bp AP sequence used in this study, as well as other sequences derived from AP that direct recombination and lead to significant improvements over trans-splicing vectors. The finding that AP hybrid vectors are more efficient than trans-splicing vectors supports the idea that the AP sequence directs at least some of the concatemerization events in the proper orientation, and that recombination then occurs via this sequence or via the ITRs. The AP head domain can mediate proper head-to-tail concatemerization, particularly after dual vector recombination in cells. Nevertheless, when more concatemers are properly aligned, the AP hybrid system mediates more efficient expression of MYO7A. (Another approach to directing concatemerization is the use of single-stranded oligonucleotides that can tether the back end of the 5' vector to the front end of the 3' vector (Hirsch et al., 2009); however, this strategy requires efficient delivery of the oligonucleotide to the nucleus of target cells in coordination with the dual vector.) Finally, dual vectors utilizing mismatched ITRs can be used to direct concatemerization in a head-to-tail orientation (Yan et al., 2005); however, this process may require further optimization of the AAV packaging machinery.

[0088] Thus, in some embodiments, the intron sequence utilized in the vector system of the present disclosure is the sequence of an intron that does not naturally occur in the genomic sequence of the gene encoding the selected polypeptide. In certain embodiments, the intron is a synthetic alkaline phosphatase (AP) intron. The intron sequence utilized in the vector system of the present disclosure can include a splice donor and a splice acceptor sequence. In some embodiments, the intron sequence is the intron sequence of a recombination product (e.g., the AK sequence of F1 phage as shown in Trapani et al. 2014). In these embodiments, the hybrid vectors characterized as second-generation hybrid vectors described herein rely on both ITR-mediated concatemerization and AK sequence-mediated homologous recombination for reconstitution of the full-length expression cassette. Thus, in some embodiments, the intron sequence is the AK sequence of F1 phage. Thus, in some embodiments of the disclosed hybrid vectors, the vectors comprise 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 exemplary embodiments, these vectors comprise an APSA and an APSD. In some embodiments, the front half vector contains an APSA and the back half vector contains an APSD. In some embodiments, the front half vector contains an APSD and the back half vector contains an APSA. See Figures 37A and 45A.

[0089] Thus, in exemplary embodiments, the hybrid vector pair contains a sequence encoding an AP head as part of an AP intron. In some embodiments of the disclosed hybrid vectors, the vector comprises an intron sequence comprising a nucleotide sequence having at least 85%, 90%, 92.5%, 95%, 98%, or 99% identity to either SEQ ID NO: 69 or 70. In some embodiments of the disclosed hybrid vectors, the vector comprises the nucleotide sequence of SEQ ID NO: 69 or 70 (the AP head sequence).

[0090] Polypeptides other than hMYO7A contemplated for delivery using any of the disclosed hybrid vectors include, but are not limited to, harmonin (Uniprot Q9Y6N9), cadherin 23 (Uniprot Q9H251), protocadherin 15 (Uniprot Q96QU1), and usherin (USH2A) (Uniprot 075445). In some embodiments, the selected full-length polypeptide is encoded by a gene about 5 Kb to about 10 Kb in length. In some embodiments, the selected full-length polypeptide is encoded by a gene about 6 Kb to about 9 Kb in length. In some embodiments, the selected full-length polypeptide is encoded by a gene about 7 Kb to about 8 Kb in length. In some embodiments, hybrid dual vectors expressing a portion (or half) of a large gene contain a large gene comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity to any one of SEQ ID NOs: 39 and 52-59, other than MYO7A, and a sequence between the first and second introns of the first and second AAV vector polynucleotides, respectively. In some embodiments, these hybrid vectors contain an intron sequence containing the nucleotide sequence of any one of SEQ ID NOs: 39 and 53-59, e.g., SEQ ID NO: 56 or 57. The large gene can be selected from ABCA4, CEP290, EYS, RP1, ALMS1, CDH23, PCDH15, USH1C, USH1G, USH2A, DNFB31, DMD, CFTR, GDE, DYSF, F8, and DFNB2. These hybrid vectors encoding non-MYO7A (e.g., ABCA4) genes may contain overlapping regions identified as recombination product sequences by the improved overlap vectors provided herein in place of the recombination product AP head sequence / domain. The overlapping regions of these hybrid vectors are flanked by splice acceptor and / or splice donor sequences, such that the overlapping regions are spliced ​​out and do not encode any MYO7A protein.

[0091] In some embodiments, a first generation hybrid vector system contains a split point between exons 23 and 24, and the sequence corresponding to the tail domain of the MYO7A protein is contained within the front half vector represented by SEQ ID NO: 3. The back half vector of this exon 23 / 24 hybrid vector system is set forth in SEQ ID NO: 4. In some embodiments, a second generation hybrid vector system contains a split point located between exons 21 and 22, and the sequence corresponding to the tail domain of the MYO7A protein is removed from the front half vector of the exon 23 / 24 hybrid vector system, thereby resulting in a second generation hybrid front half vector (SEQ ID NO: 31).

[0092] Thus, in the illustrated embodiment, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 31 or a functional fragment and / or variant thereof, the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 32 or a functional fragment and / or variant thereof, and the intron sequence is the AK sequence of F1 phage.

[0093] In some embodiments, the division point between the first and second AAV vector polynucleotide sequences is between exon 21 and exon 22 of the hMYO7A gene. In some embodiments, the division point between the first and second AAV vector polynucleotide sequences is between exon 22 and exon 23 of the hMYO7A gene.

[0094] In some embodiments, the division point between the first and second AAV vector polynucleotide sequences is not between exon 23 and exon 24 of the hMYO7A gene. In exemplary embodiments, the hybrid vector of the present disclosure does not comprise either of the nucleotide sequences of SEQ ID NOs: 3 and 4.

[0095] CMv1 Hybrid Vector System Some embodiments contemplate a hybrid vector system as described herein in which substitutions are made in a non-coding sequence of the vector (e.g., the 3' untranslated region (3'UTR) downstream of the MYO7A partial coding sequence and preceding the 3' AAV inverted terminal repeat of the inverted terminal repeat pair of the first and / or second vector polynucleotide). In some embodiments, the substitutions are located in putative stop codons, thereby eliminating these potential stop codons. In some embodiments, one or more potential stop codons are eliminated by introducing one or more nucleotide substitutions into the alkaline phosphatase (AP) intron splice donor sequence ("AP intron") of the front half vector (e.g., a front half vector comprising SEQ ID NO:31). In some embodiments, three potential stop codons are modified within the alkaline phosphatase intron splice donor sequence of the front half vector. As a result of the modification of these putative stop codons in the AP head sequence, a modified front half vector comprising SEQ ID NO:33 is created. See Figure 42.

[0096] Thus, in some embodiments, the hybrid vector system of the present disclosure comprises: i) a first AAV vector polynucleotide comprising inverted terminal repeats at each end of the polynucleotide, a promoter between the inverted terminal repeats, followed by a partial coding sequence encoding an 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 terminal repeat at each end of the polynucleotide, an intron and an intron splice acceptor site between the inverted terminal repeats, optionally followed by a partial coding sequence encoding the C-terminal portion of a selected full-length polypeptide, followed by a polyadenylation (pA) signal sequence. The intron sequences in the first and second AAV vectors comprise overlapping sequences. In an exemplary embodiment, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 33, or a functional fragment and / or variant thereof, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 32, or a functional fragment and / or variant thereof.

[0097] CMv2 Hybrid Vector System Still other embodiments contemplate a hybrid vector system as described herein in which one additional putative in-frame stop codon has been modified in the front-half vector (e.g., a front-half vector comprising SEQ ID NO: 33). In some embodiments, the modification comprises introducing a substitution into the AP head sequence of the front-half vector that removes the putative stop codon from this sequence. As a result of this modification and removal of this putative stop codon, a further modified front-half vector comprising SEQ ID NO: 34 is created.

[0098] Upon further modification of the putative in-frame stop codon as described herein, some embodiments consider making a complementary change in the back-half vector (e.g., a back-half vector comprising SEQ ID NO: 32), in which the same codon is also modified. Thus, in some embodiments, the modification comprises the introduction of a substitution into the AP head sequence of the back-half vector that removes the putative stop codon from this sequence. As a result of this further stop codon modification, a modified back-half vector comprising SEQ ID NO: 35 is created. See Figure 42.

[0099] As such, the CMv1 vector removes three potential in-frame stop codons in the AP intron, and the CMv2 vector removes these same three potential stop codons in the AP intron and one potential stop codon from the AP head coding sequence.

[0100] Thus, in certain embodiments, the hybrid vector system of the present disclosure comprises: i) a first AAV vector polynucleotide comprising inverted terminal repeats at each end of the polynucleotide, a promoter between the inverted terminal repeats, followed by a partial coding sequence encoding an 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 terminal repeat at each end of the polynucleotide, an intron and an intron splice acceptor site between the inverted terminal repeats, optionally followed by a partial coding sequence encoding the C-terminal portion of a selected full-length polypeptide, followed by a polyadenylation (pA) signal sequence. The intron sequences in the first and second AAV vectors comprise overlapping sequences. In an exemplary embodiment, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 34, or a functional fragment and / or variant thereof, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 35, or a functional fragment and / or variant thereof.

[0101] V2 MIN and CMv2 MIN Hybrid Vector Systems (also known as the CMv2.1 Hybrid Vector System) If the split point is changed from the junction between exons 23 and 24, as in the first-generation hybrid vector system, to the junction between exons 21 and 22, as in the second-generation hybrid vector system, an additional portion of the MYO7A sequence is moved into the back-half second-generation hybrid vector. To this end, in some embodiments, the second-generation back-half hybrid vector approaches, but does not exceed, the AAV packaging limit. Therefore, in some embodiments, a modified back-half vector containing SEQ ID NO: 35 can be further modified to remove any residual (extraneous), non-essential sequences, such as restriction enzyme sites and tag sequences, from the 3' end of the construct. These residual sequences are sometimes referred to as "unseeded legacy" sequences. The resulting modified back-half vector contains SEQ ID NO: 49. This vector system is known as the "V2 MIN" or "V2-back-MIN" hybrid system. In some embodiments, this system contains an HA tag ("V2 MIN HA"). The V2 MIN HA vector is shown as SEQ ID NO: 48. The V2 MIN back-half vector is 122 bp shorter than the first-generation hybrid back-half vector (4981 bp vs. 4861 bp).

[0102] In some embodiments, a vector is provided in which unseeded legacy sequences are removed and one or more substitutions are introduced in non-coding sequences. In some embodiments, these one or more substitutions are placed in putative stop codons. As a result, these potential stop codons are removed. In some embodiments, one or more potential stop codons are removed by introducing one or more nucleotide substitutions into the AP head sequence of the front half vector. In some embodiments, one or more putative stop codons are removed and replaced with a "stuffer" sequence (see Figure 43).

[0103] In some embodiments, three potential stop codons are modified within the alkaline phosphatase intron sequence of the front half vector. In some embodiments, one putative stop codon is modified (removed) by introducing one or more substitutions into the AP head sequence of the front half vector. As a result of modifying these putative stop codons in the AP head sequence, a modified front half vector of SEQ ID NO: 34 is generated.

[0104] In some embodiments, one putative stop codon is similarly modified in the AP head sequence of the back-half vector. As a result of modifying this putative stop codon in the AP head sequence, a modified back-half vector of SEQ ID NO: 44 was generated.

[0105] This vector system is known as the CMv2 MIN system. In some embodiments, this system contains an HA tag ("CMv2 MIN HA"). The CMv2 MIN HA vector is set forth as SEQ ID NO: 47. The CMv2 MIN back-half vector is 121 bp shorter than the first generation hybrid back-half vector (4982 bp vs. 4861 bp).

[0106] Thus, in some embodiments, the hybrid vector system of the present disclosure comprises: i) a first AAV vector polynucleotide comprising inverted terminal repeats at each end of the polynucleotide, a promoter between the inverted terminal repeats, followed by a partial coding sequence encoding an 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 terminal repeat at each end of the polynucleotide, an intron and an intron splice acceptor site between the inverted terminal repeats, optionally followed by a partial coding sequence encoding the C-terminal portion of a selected full-length polypeptide, followed by a polyadenylation (pA) signal sequence. The intron sequences in the first and second AAV vectors comprise overlapping sequences. In exemplary embodiments, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 34, or a functional fragment and / or variant thereof, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 44, or a functional fragment and / or variant thereof. In some embodiments, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 34, or a functional fragment and / or variant thereof, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 47, or a functional fragment and / or variant thereof. In some embodiments, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 34, or a functional fragment and / or variant thereof, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 48, or a functional fragment and / or variant thereof.

[0107] CMv3 MIN Hybrid Vector System Still other embodiments contemplate a hybrid vector system as described herein in which three additional in-frame stop codons have been modified in the front-half vector. In some embodiments, one or more nucleotide substitutions (e.g., three substitutions) are made in the 3'UTR or ITR downstream of the MYO7A coding sequence. Thus, in some embodiments, a front-half vector is provided in which the substitutions remove one in-frame stop codon in the AP head sequence, three in-frame stop codons in the AP intron sequence, and three in-frame stop codons in the 3'UTR sequence. As a result of the modification of these putative stop codons, a further modified front-half vector containing SEQ ID NO: 46 is generated ("AAV-smCBA-hMYO7A-NT-Ex21-APSD-APhead-CMv3" or simply "CMv3 hybrid system"). In an exemplary embodiment, the CMv3 hybrid system is a CMv3 MIN system in which residual unseeded legacy sequences (e.g., restriction enzyme sites) have been removed. In some embodiments, the CMv3 system has an HA tag.

[0108] Thus, in an exemplary embodiment, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 46 or a functional fragment and / or variant thereof, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 35 or a functional fragment and / or variant thereof.

[0109] Thus, in some embodiments, the polynucleotide vector system of the present disclosure is a CMv3 hybrid system. In some embodiments, the vector system is a CMv3 MIN system. In some embodiments, the vector system is a CMv2 system. In some embodiments, the vector system is a CMv2 MIN system. In some embodiments, the vector system is a CMv1 or CMv1 MIN system. In the compositions of the present disclosure, any of the disclosed front half hybrid vectors can be combined with any of the disclosed back half hybrid vectors.

[0110] In an exemplary embodiment, i) a first AAV vector polynucleotide comprising inverted terminal repeats at each end of the polynucleotide, a promoter between the inverted terminal repeats, followed by a partial coding sequence encoding an N-terminal portion of a myosin polypeptide, followed by a splice donor site and an intron; and ii) a second AAV vector polynucleotide comprising inverted terminal repeats at each end of the polynucleotide, an intron and a splice acceptor site for the intron between the inverted terminal repeats; 1. A polynucleotide vector system comprising: the intron sequences in the first and second AAV vectors comprise overlapping polynucleotide sequences; the first AAV vector polynucleotide comprises a nucleotide sequence selected from SEQ ID NOs: 31, 33, 34 and 46; A polynucleotide vector system is provided, wherein the second AAV vector polynucleotide comprises a nucleotide sequence selected from SEQ ID NOs: 32, 35, 44 and 47-49.

[0111] In some embodiments of the hybrid vector systems described herein, the selected full-length polypeptide is a myosin polypeptide. In some embodiments, the myosin polypeptide is human myosin VIIA (hMYO7A). In some embodiments, the myosin polypeptide is human myosin VIIB (hMYO7B). In some embodiments, the myosin polypeptide is myosin 7 (VII) isoform II. In some embodiments, the myosin polypeptide is another myosin isoform or a functional fragment thereof. In certain embodiments, full-length myosin 7A or isoform II is encoded in the provided vector systems.

[0112] In some embodiments, the C-terminal portion of the selected full-length polypeptide (eg, a myosin polypeptide) comprises the single-alpha helix (SAH) domain of the selected full-length polypeptide.

[0113] 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 a portion 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 a portion of the intron sequence present at the 5' end of the coding sequence of the second vector.

[0114] Some embodiments of the hybrid vectors described herein contemplate viruses or recombinant viral particles comprising the first AAV vector polynucleotide or the second AAV vector polynucleotide described herein. In certain embodiments, the first AAV vector polynucleotide comprises SEQ ID NO: 33, and the second AAV vector polynucleotide comprises SEQ ID NO: 32. In some embodiments, the viruses or recombinant viral particles are characterized as adeno-associated viruses (AAV) or infectious AAV viral particles. In some embodiments, the recombinant AAV viral particles comprise one or more tyrosine to phenylalanine (YF) mutations in the viral or virion capsid protein. A tyrosine to phenylalanine (YF) mutation in the viral or virion capsid protein at amino acid position 733 is specifically contemplated herein (e.g., AAV8 Y733F). Similarly, a tyrosine to phenylalanine (YF) mutation in the viral or virion capsid protein at amino acid position 731 is specifically contemplated herein (eg, AAV44.9(Y731F)).

[0115] In some embodiments, the virus or virion is packaged in an AAV5, AAV7, AAV8, AAV9, AAV44.9, AAV44.9(E531D), AAV2(4pMut), AAVAnc80, AAVrh.8, AAVrh.8R, AAV9-PHP.B, AAV9-PHP.eB, AAVrh.10, or AAVrh.74 capsid. In some embodiments, the viral particles comprise an 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 capsid. In an exemplary embodiment for delivery to the retina, the virions are packaged in the AAV44.9(E531D) capsid mutant. In an exemplary embodiment for delivery to hair cells of the ear, virions are packaged in the AAV9-PHP.B capsid mutant.

[0116] In some embodiments, the hybrid polynucleotide vector systems described herein use tissue-specific promoters. In some embodiments, the systems use promoters that mediate expression in the eye. In some embodiments, the systems use promoters that mediate expression in the ear.

[0117] In some embodiments, the hybrid polynucleotide vector system described herein uses any one of the following promoters: a cytomegalovirus (CMV) promoter, an elongation factor-1 alpha (EF-1 alpha) promoter, a cone arrestin promoter, a chimeric CMV β-actin (smCBA) promoter, a human myosin 7a gene-derived promoter, a cone transducin (TαC) gene-derived promoter, a rhodopsin promoter, a cGMP-phosphodiesterase β-subunit promoter, a human or mouse rhodopsin promoter, a human rhodopsin kinase (hGRK1) promoter, a rod-specific IRBP promoter, an RPE-specific vitelloid macular degeneration-2 [VMD2] promoter, and combinations thereof. In some embodiments, the polynucleotide vector system described herein uses a human rhodopsin kinase (hGRK1) promoter. In some embodiments, the polynucleotide vector system uses a cone arrestin promoter. In some embodiments, the polynucleotide vector system uses a cytomegalovirus (CMV) promoter. In some embodiments for delivery to the eye (retina) of a subject, the disclosed overlapping polynucleotide vector system uses a cytomegalovirus (CMV) promoter.

[0118] In some embodiments for delivery to the retina, the hybrid polynucleotide vector system uses the EF-1 alpha promoter. In some embodiments for delivery to the ear (hair cells) of a subject, the polynucleotide vector system uses the synapsin or GFAP promoter (see Lee et al., Hearing Research).

[0119] Each embodiment as contained in the "Hybrid Vectors" section and described herein is specifically contemplated for each hybrid vector system described, e.g., a vector system in which the division point between the first and second AAV vector polynucleotide sequences is between exon 21 and exon 22 of the hMYO7A gene; a vector system in which the division point between the first and second AAV vector polynucleotide sequences is between exon 22 and exon 23 of the hMYO7A gene; a vector system having a front half vector comprising the nucleotide sequence of SEQ ID NO:31 and a back half vector comprising the nucleotide sequence of SEQ ID NO:32; a vector system having a front half vector comprising the nucleotide sequence of SEQ ID NO:33 and a back half vector comprising the nucleotide sequence of SEQ ID NO:32; a vector system having a front half vector comprising the nucleotide sequence of SEQ ID NO:34 and a back half vector comprising the nucleotide sequence of SEQ ID NO:35; and / or a vector system having a front half vector comprising the nucleotide sequence of SEQ ID NO:34 and a back half vector comprising the nucleotide sequence of SEQ ID NO:44.

[0120] In some embodiments, any vector of the hybrid polynucleotide vector system described in this disclosure can be administered parenterally, such as by intravenous, intramuscular, intraocular, intranasal, or intrautricle injection. The vector can be administered in vivo, in vitro, or ex vivo.

[0121] In some embodiments, any vector of the hybrid polynucleotide vector system described herein can be administered to the eye. In certain embodiments, the vector is administered to the eye of a subject by subretinal injection. In some embodiments, any vector of the hybrid polynucleotide vector system described herein can be administered to the ear. In some embodiments, any vector of the hybrid polynucleotide vector system described herein, the polynucleotide vector system, can be administered to the ear of a subject by, for example, round window injection or during cochlear implant surgery.

[0122] The methods of the present disclosure can be used with humans and other animals. Animals contemplated within the scope of the present disclosure include, for example, dogs, cats, rabbits, ferrets, guinea pigs, hamsters, pigs, monkeys or other primates, mice, gerbils, horses, mules, donkeys, bovines, cows, pigs, sheep, and crocodiles. As used herein, the terms "patient" and "subject" are used interchangeably and include such human and non-human species, including human and non-human cells. Similarly, the in vitro methods of the present disclosure can also be performed on cells of one or more human or non-human mammalian species, including human and non-human cells.

[0123] Components of Exemplary Dual AAV Vectors Any of the dual polynucleotide vector systems disclosed herein can be used with AAV vector systems known in the art. While a single administration of an rAAV vector construct may be preferred in the treatment of some diseases, it may be desirable to administer two or more doses of the vector construct to a patient over one or more administration periods in the management or treatment of other diseases or conditions. In such situations, the AAV vector-based therapeutic agent may be provided for one or more consecutive daily, weekly, monthly, or less frequent periods, as may be necessary to achieve treatment or amelioration of one or more symptoms of the disease or disorder being treated. In some embodiments, the vector may be provided to one or both eyes by one or more administrations of infectious adeno-associated virus particles, rAAV virions, or multiple infectious rAAV particles in an amount and for a time sufficient to treat or ameliorate one or more symptoms of the disease or condition being treated.

[0124] In certain embodiments, the present disclosure provides rAAV particles derived from several different serotypes, including, for example, those selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10. In exemplary embodiments, particles derived from AAV2, AAV5, and AAV8 serotype vectors are utilized. In certain embodiments, particles with AAV8(Y733F) or AAV2(triple YF) capsids are used. Thus, the present disclosure provides recombinant AAV particles comprising overlapping and hybrid polynucleotide vector systems, for example, derived from AAV8(Y733F) or AAV2(triple YF). In some embodiments, the serotype of the AAV vector is not AAV6 or AAV2.

[0125] Further exemplary 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, AAV44.9, AAV44.9(E531D), AAVrh.8, AAVrh.8R, AAV9-PHP.B, and / or AAVAnc80. In some embodiments, the virus or virion is packaged in an AAV5, AAV7, AAV8, AAV9, AAV44.9, AAV44.9(E531D), AAV2(4pMut), AAVAnc80, AAVrh.8, AAVrh.8R, AAV9-PHP.B, AAVrh.10, or AAVrh.74 capsid.

[0126] The AAV-DJ capsid is described in Grimm et al., J. Virol., 2008, 5887-5911 and Katada et al., (2019), Evaluation of AAV-DJ vector for retinal gene therapy, PeerJ 7:e6317, each of which is incorporated herein by reference. The AAV7m8 capsid, which is closely related to AAV-DJ, is described in Dalkara, et al. Sci Transl Med. 2013; 5(189):189ra76, which is incorporated herein by reference. The AAV2 / 2-MAX capsid is described in Reid, Ertel & Lipinski, "Improvement of Photoreceptor Targeting via Intravitreal Delivery in Mouse and Human Retina Using Combinatory rAAV2 Capsid Mutant Vectors," Invest. Ophthalmol Vis Sci. 2017; 58:6429-6439, which is incorporated herein by reference. The AAV2 / 2-MAX capsid contains five point mutations: Y272F, Y444F, Y500F, Y730F, and T491V. The AAV1(E531K) capsid is described in Boye et al., "Impact of Heparin Sulfate Binding on Transduction of Retina by Recombinant Adeno-Associated Virus Vectors," J. Virol. 90:4215-4231 (2016), which is incorporated herein by reference.The AAVSHh10 and AAV6(D532N) capsids are both derivatives of AAV6 and are described in Klimczak et al., (2009) A Novel Adeno-Associated Viral Variant for Efficient and Selective Intravitreal Transduction of Rat Muller Cells, PLoS ONE 4(10): e746, which are incorporated herein by reference. The AAV6-3pmut (also known as AAV6(TM6) and AAV6(Y705+Y731F+T492V)) capsid is described in Rosario et al., Microglia-specific targeting by novel capsid-modified AAV6 vectors, Mol Ther Methods Clin Dev. (2016); 13(3):16026 and International Patent Publication No. 2016 / 126857, each of which is incorporated herein by reference.

[0127]

[0013] Additional capsids suitable for use with the disclosed methods include capsids comprising unnatural amino acid substitutions at amino acid residues in a wild-type AAV2 capsid, wherein the unnatural amino acid substitutions include one or more of Y272F, Y444F, T491V, Y500F, Y700F, Y704F, and Y730F; capsids comprising unnatural amino acid substitutions at amino acid residues in a wild-type AAV6 capsid, wherein the unnatural amino acid substitutions include one or more of Y445F, Y705F, Y731F, T492V, and S663V. In certain embodiments, the capsid comprises the AAV2 mutant AAV2G9.

[0128] In other embodiments, the capsid comprises an unnatural amino acid substitution at amino acid residue 533 or 733 of the wild-type AAV8 capsid, wherein the unnatural amino acid substitution is E533K, Y733F, or a combination thereof. The AAV8(Y733F) capsid is described in Doroudchi et al., Amer. Soc. of Gene & Cell Ther. 19(7): 1220-29 (2011). In certain embodiments of the disclosed methods, the capsid comprises AAV8PB2, a mutant of AAV8.

[0129] In other embodiments, the capsid comprises a non-natural amino acid substitution of a 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.

[0130] In other embodiments, the capsid comprises a non-natural amino acid substitution of a 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.

[0131] Additional capsids suitable for use with the disclosed methods are described in International Patent Publication No. WO2018 / 156654, published August 30, 2018, which is incorporated herein by reference in its entirety. In certain embodiments, the disclosed rAAV particles of the invention comprise 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 still other embodiments, the rAAV particles may comprise a capsid selected from AAV6(3pMut) or AAV2(quadYF+TV). In still other embodiments, the rAAV particles of the disclosed methods may comprise any of the capsid variants described in International Patent Publication No. WO2018 / 156654.

[0132] In certain embodiments, disclosed herein are rAAV particles that can include 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) capsid for enhanced transduction of said rAAV particles in cells of the retina. In other embodiments, the disclosed rAAV particles may comprise 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).

[0133] Exemplary inverted terminal repeat (ITR) sequences used in any AAV vector system of the present disclosure can include any AAV ITR. The ITRs used in AAV vectors can be identical or different. In certain embodiments, the ITRs can 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 serotypes 44.9(E531D) and 44.9(Y731F) (see PCT Application No. PCT / US2020 / 14838, filed January 23, 2020, which is incorporated herein by reference). The AAV vectors of the present disclosure can include different AAV ITRs. In a non-limiting example, the vector can include an AAV2 ITR and an AAV5 ITR. AAV ITR sequences are well known in the art (see, e.g., GenBank Accession Nos. AF043303.1, NC_001401.2, J01901.1, JN898962.1, K01624.1, and K01625.1). The AAV dual vector system disclosed herein can efficiently express larger therapeutic genes than can normally be packaged in a single AAV vector.

[0134] Therefore, in some aspects, the present disclosure provides a virus or virion that comprises any of the polynucleotides or vectors of the present disclosure.In certain embodiments, the virus or virion is an AAV virus.The method for preparing the virus and virion that comprises heterologous polynucleotides or vectors is known in the art.In the case of AAV, cells can be co-infected or transfected with adenovirus or polynucleotide vectors that comprise suitable adenovirus genes for AAV helper function.Examples of materials and methods are described in, for example, U.S. Patent No. 8,137,962 and U.S. Patent No. 6,967,018 (each of which is incorporated herein by reference).

[0135] In certain embodiments, the AAV serotype provides one or more tyrosine-to-phenylalanine (YF) mutations on the capsid surface. In certain embodiments, the AAV is an AAV8 serotype with a tyrosine-to-phenylalanine (YF) mutation at position 733 (Y733F). The ability of second-generation hybrid and overlap vectors encapsidated in AAV5 and AAV8(Y733F) virions to produce full-length MYO7A protein is shown in Figures 36A-36C and 45B. AAV8(Y733F) virions outperformed AAV5 virions as measured by Western blot (see Figure 45B).

[0136] In some embodiments, a triple-mutant AAV8 vector is used that contains tyrosine to phenylalanine Tyr-Phe mutations at positions Y733F, Y500F, and Y730F, respectively (see Figure 5). In other embodiments, a triple-mutant AAV8 vector is used that contains tyrosine to phenylalanine Tyr-Phe mutations at positions Y447F, Y733F, and T494V (e.g., AAV8(Y447F+Y733F+T494F)).

[0137] In exemplary embodiments, the rAAV particles of the present disclosure may contain transgenes or heterologous nucleic acids that are too large for delivery in standard AAV systems. Exemplary transgenes include, but are not limited to, rhodopsin, melanopsin, cone opsin, channelrhodopsin, bacterial or archaeal-associated opsins, adrenergic agonists, anti-apoptotic factors, apoptosis inhibitors, cytokine receptors, cytokines, cytotoxins, erythropoietic agents, glutamic acid decarboxylase, glycoproteins, growth factors, growth factor receptors, hormones, hormone receptors, interferons, interleukins, interleukin receptors, kinases, kinase inhibitors, nerve growth factors, netrins, neuroactive peptides, neuroactive peptide receptors, neurogenic factors, neurogenic factor receptors, neuropilins, The gene encoding at least one diagnostic or therapeutic protein or polypeptide is selected from the group consisting of a molecular marker, including a neurotrophic factor, a neurotrophin, a neurotrophin receptor, an N-methyl-D-aspartate antagonist, a plexin, a protease, a protease inhibitor, a protein decarboxylase, a protein kinase, a protein kinase inhibitor, a proteolytic protein, a proteolytic protein inhibitor, a semaphorin, a semaphorin receptor, a serotonin transporter protein, a serotonin uptake inhibitor, a serotonin receptor, a serpin, a serpin receptor, a tumor suppressor, and any combination thereof; and a photosensitive opsin.

[0138] In some embodiments, the transgene is hMYO7A, which encodes a human myosin VIIa polypeptide. In certain embodiments, the hMYO7A polypeptide comprises the amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:8, or a functional fragment or variant thereof. In certain embodiments, the hMYO7A polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:5 or SEQ ID NO:7.

[0139] In some embodiments, the transgene is USH1C, CDH23, PCDH15, and USH1G, all of which are associated with Usher Syndrome Type I. In some embodiments, the transgene is USH2A and DFNB31, both of which are associated with Usher Syndrome II. In some embodiments, the transgene is ABCA4, CEP290, EYS, RP1, ALMS1, CDH23, PCDH15, DFNB2, or USHERIN.

[0140] In some embodiments, administration of any of the disclosed polynucleotide vectors to the eye of a subject in need thereof partially or completely restores vision loss. The transgene may include human MYO7A. These administrations may provide partial or complete restoration of melanosome apical migration in retinal pigment epithelial (RPE) cells.

[0141] In some embodiments, production of a therapeutic agent encoded by a transgene of any of the disclosed polynucleotide vector systems in ocular cells (e.g., retinal cells or RPE cells) provides one or more of the following therapeutic endpoints: a) preserving one or more photoreceptor cells or one or more RPE cells, b) restoring one or more rod- and / or cone-mediated functions, c) restoring visual behavior in one or both eyes, or d) a combination thereof. In certain embodiments, production of a therapeutic agent in the disclosed methods preserves one or more retinal ganglion cells, bipolar cells, Müller glial cells, or astrocyte cells or RPE cells.

[0142] In some embodiments, production of the therapeutic agent persists in one or more photoreceptor cells or one or more RPE cells for substantially at least 3 months, at least 6 months, at least 9 months, or at least 1 year or longer following initial administration of any of the disclosed rAAV polynucleotide vector systems to one or both eyes of a mammal.

[0143] In some embodiments, administration of any of the disclosed polynucleotide vectors to the inner ear of a subject in need thereof partially or completely restores age-related hearing loss. In some embodiments, administration to the inner ear restores age-related hearing loss. The transgene may include human MYO7A. Such administration may provide partial or complete restoration of vestibular function in the inner ear. In some embodiments, any of the disclosed hybrid or overlap vectors may be administered to vestibular hair cells, inner ear hair cells, outer ear hair cells, or a combination thereof.

[0144] In this manner, the disclosed polynucleotide vector systems and compositions can be used to treat or ameliorate symptoms of USH1B (Usher syndrome type 1B) in the eye and / or inner ear. Similarly, administration of the disclosed vector systems and compositions can be used to treat or ameliorate symptoms of autosomal recessive isolated deafness (DFNB2), hearing loss, and / or vision loss. By way of example, administration of the disclosed vector systems and compositions can be used to treat or ameliorate hearing loss associated with insufficient MY07A protein expression (which may be present in USH1B patients). In some embodiments, administration of the disclosed vector systems and compositions can be used to treat or ameliorate age-related hearing loss that occurs in carriers of recessive defective MY07A alleles (i.e., USH1B carriers) or age-related hearing loss as a result of a non-genetic deficiency or lack of MY07A expression.

[0145] As another example, administration of the vector systems and compositions of the present disclosure can provide restoration of melanosome transfer in retinal pigment epithelial (RPE) cells.

[0146] In some embodiments, the present 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 appropriately transformed with the vector of interest. In certain embodiments, such diagnostic or therapeutic agents may comprise molecules that selectively inhibit or reduce the effects of one or more metabolic processes, dysfunctions, disorders, or diseases. In certain embodiments, the defect may be caused by injury or trauma to the mammal for which treatment is desired. In other embodiments, the defect may be caused by overexpression of an endogenous biological compound, while in still other embodiments, the defect may be caused by underexpression or even absence of one or more endogenous biological compounds.

[0147] Regulatory elements of rAAV vectors Any of the vector systems of the present disclosure may contain regulatory elements that are functional in the intended host cells in which the vector is to be expressed. Those skilled in the art can select regulatory elements for use in appropriate host cells, such as mammalian or human host cells. Regulatory elements include, for example, promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements.

[0148] Any of the vector systems of the present disclosure may include a promoter sequence operably linked to a nucleotide sequence encoding a desired polypeptide.Promoters contemplated for use in the present disclosure include, but are not limited to, cytomegalovirus (CMV) promoter, SV40 promoter, human myosin 7a gene-derived promoter, Rous sarcoma virus (RSV) promoter, chimeric CMV / chicken β-actin promoter (CBA) and truncated form of CBA (smCBA) (see, for example, Haire et al. 2006 and U.S. Patent No. 8,298,818, each of which is incorporated herein by reference).Additional photoreceptor-specific, human rhodopsin kinase (hGRK1) promoter, rod-specific IRBP promoter, VMD2 (vitelliform macular degeneration / Best's disease) promoter, RPE-specific vitelliform macular degeneration-2 [VMD2] promoter and EF1-alpha promoter sequences are also contemplated as useful in implementing various embodiments of the present disclosure. Exemplary photoreceptor cell-specific promoters include, but are not limited to, hGRK1, IRBP, rod opsin, NRL, GNAT2e-IRBP, L / M opsin, and cone arrestin promoters.

[0149] 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 is less active or inactive 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 MYO7A gene. In further embodiments, the promoter comprises a promoter derived from the cone transducin alpha (TαC) gene. In certain embodiments, the promoter is a promoter derived from human GNAT2. Other promoters contemplated within the scope of the present disclosure include, but are not limited to, a rhodopsin promoter (human or mouse), a cGMP-phosphodiesterase β-subunit promoter, a retinitis pigmentosa-specific promoter, an RPE cell-specific promoter [e.g., vitelliform macular degeneration-2 (VMD2) promoter (Best1) (Esumi et al., 2004)], or any combination thereof.

[0150] Promoter can be incorporated into vector using standard techniques known to those skilled in the art of molecular biology and / or virology.Multiple copies of promoter and / or multiple separate promoters can be used in the vector of the present disclosure.In one such embodiment, promoter can be positioned at approximately the same distance from the transcription start site as from the transcription start site in its natural gene environment, although some variation in this distance is permitted, without substantially reducing promoter activity.In carrying out the present disclosure, one or more transcription start sites are usually included in the disclosed vector.

[0151] The vector of the present 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 (IRES) and / or one or more enhancer elements, or any combination thereof.Transcription termination regions can usually be obtained from the 3' untranslated region of eukaryotic or viral gene sequences.Transcription termination sequences can be located downstream of the coding sequence to provide efficient termination.

[0152] Any of the disclosed polynucleotide vectors can further comprise one or more post-transcriptional regulatory sequences or one or more polyadenylation signals, including, for example, but not limited to, a woodchuck hepatitis virus post-transcriptional regulatory element (WRPE), a polyadenylation signal sequence, or an intron / exon junction / splicing signal, or any combination thereof.

[0153] A signal peptide sequence is an amino-terminal peptidic sequence that encodes information involved in localization of an operably linked polypeptide to one or more post-translational cellular destinations, including, for example, specific organelle compartments, or to sites of protein synthesis and / or activity, and even to the extracellular environment.

[0154] Enhancers—cis-acting regulatory elements that increase gene transcription—can be included in one of the disclosed AAV-based vector systems. Various enhancer elements are known to those skilled in the relevant art, including, but not limited to, the CaMV 35S enhancer element, the cytomegalovirus (CMV) early promoter enhancer element, the SV40 enhancer element, and combinations and / or derivatives thereof. One or more nucleic acid sequences that direct or regulate the polyadenylation of mRNA encoded by the structural gene of interest can also be included in one or more of the vectors of the present disclosure, as appropriate.

[0155] Host cells and methods for transducing cells The present disclosure provides a host cell comprising a vector of the disclosed polynucleotide vector system. In some embodiments, an isolated host cell comprising an overlapping polynucleotide vector system is provided. In some embodiments, an isolated host cell comprising a hybrid polynucleotide vector system is provided. In certain embodiments, an isolated host cell comprising a second-generation hybrid and an isolated host cell comprising a second-generation overlapping vector are provided.

[0156] Examples of suitable host cells that can contain 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 combinations thereof. Examples of retinal cells include retinal ganglion cells (RGCs), Müller cells, astrocytes, and bipolar cells.

[0157] Further examples of suitable host cells include vestibular hair cells, inner ear hair cells, outer ear hair cells, or any combination thereof.

[0158] The present disclosure also provides a method for expressing or transducing a selected polypeptide in a cell. In certain embodiments, the method includes incorporating into a cell an AAV-based dual vector system as disclosed herein, wherein the vector system encodes the selected polypeptide and includes a polynucleotide sequence of interest, and expressing the polynucleotide sequence in the cell.

[0159] In certain embodiments, the selected polypeptide may be a polypeptide heterologous to the cell. In certain embodiments, the cell is a mammalian cell, preferably a human cell. In certain embodiments, the cell is a human photoreceptor cell, preferably a human photoreceptor cone cell or a photoreceptor rod cell. In certain embodiments, the cell expresses a wild-type, functional, and / or biologically active hMYO7A polypeptide encoded by a nucleic acid segment present in a vector system as disclosed herein. In certain embodiments, the hMYO7A polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:5 or SEQ ID NO:7.

[0160] In certain embodiments, the cell is a photoreceptor cell. In certain embodiments, the cell is a cone cell, preferably a human cone cell or a human rod cell. Such cells can express at least one or more nucleotide sequences provided in the first AAV-based dual vector system of the present disclosure. In certain embodiments, the cell expresses a wild-type, functional, and / or biologically active hMYO7A polypeptide encoded by a nucleic acid segment contained in one or more of the AAV-based vector systems disclosed herein. In certain embodiments, the hMYO7A polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 7.

[0161] Thus, in certain 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., a human). In a general sense, such a method comprises administering (e.g., directly subretinal) to one or both eyes of a mammal one or more of the rAAV particles disclosed herein, wherein the polynucleotide further comprises at least a first polynucleotide comprising a PR- or RPE-cell-specific promoter operably linked to a first heterologous nucleic acid segment encoding a therapeutic agent, for a time effective to produce the therapeutic agent in one or more PR cells or RPE cells of the mammal. In certain embodiments, the therapeutic polynucleotide is stably expressed in photoreceptor cells, retinal pigment epithelial cells, retinal ganglion cells, bipolar cells, Müller glia cells, or astrocyte cells, or a combination thereof. In certain embodiments, the therapeutic polypeptide is stably expressed in vestibular hair cells, inner ear hair cells, or outer ear hair cells.

[0162] Treatment and transduction methods In some aspects, the present disclosure provides a method for treating or ameliorating a disease or condition, such as an eye disease, in a human or animal using a gene therapy and AAV-based dual vector system of the present disclosure. In certain embodiments, the method of the present disclosure comprises administering a vector system of the present disclosure encoding a polypeptide that provides treatment or amelioration of the disease or condition. In certain embodiments, the vector of the present disclosure is provided in an AAV virus or virion. The vector system can be administered in vivo or ex vivo.

[0163] In certain embodiments, the vector system of the present disclosure is administered in recombinant AAV particles by parenteral administration, for example, intravitreal, subretinal, intravenous, intramuscular, intraocular, utricular, or intranasal injection. In some embodiments, the vector system is administered to otic hair cells, for example, by injection into the utricle, one of two gravity-sensitive saccular otolith organs, as described in Lee et al., Hearing Research Vol. 394 (2020) 107882, which is incorporated herein by reference. For example, administration to otic hair cells can be by round window injection or during cochlear implant surgery. In certain embodiments, the vector system of the present disclosure is administered to a human or animal by intraocular, intravitreal, or subretinal injection.

[0164] In some embodiments, the recombinant AAV particles of the present disclosure comprise about 1 x 10 8 vg / ml, 5 × 10 8 vg / ml, 8 × 10 8 vg / ml, 1 × 10 9 vg / ml, 5 × 10 9 vg / ml, 1 × 10 10 vg / ml, 5 × 10 10 vg / ml, 1 × 10 11 vg / ml, 5 × 10 11 vg / ml, 1 × 10 12 vg / ml, 2 × 10 12 vg / ml, 3 × 10 12 vg / ml, 4 × 10 12 vg / ml, approx. 5×10 12 vg / ml, approx. 1×10 13 vg / ml, or approximately 5 × 10 13 In certain embodiments, the rAAV particles are administered by subretinal injection at a titer of 5.0 x 10 vg / ml. 8 vg or 8.0 × 10 8 It is administered in a titer of vg.

[0165] In some embodiments, the subretinal injection is provided in a volume of about 200 μL, about 175 μL, about 160 μL, about 145 μL, about 130 μL, about 115 μL, about 100 μL, about 90 μL, about 80 μL, about 70 μL, about 60 μL, about 55 μL, about 50 μL, about 45 μL, about 35 μL, about 20 μL, about 10 μL, or about 5 μL. In certain embodiments, the injection is provided in a volume of about 50 μL. The dosing regimen and effective amount to be administered can be determined by an ordinary skilled clinician. Administration can be in the form of a single dose or multiple doses. General methods for carrying out gene therapy using polynucleotides, expression constructs and vectors are known in the art (see, e.g., Gene Therapy: Principles and Applications (1999), and U.S. Pat. Nos. 6,461,606, 6,204,251, and 6,106,826, each of which is specifically incorporated herein by reference in its entirety).

[0166] In certain embodiments, the disease, disorder or condition to be treated is Usher syndrome.In some embodiments, the disease or disorder to be treated is autosomal recessive isolated deafness (DFNB2).In other embodiments, the disease, disorder or condition is, for example, age-related macular degeneration (AMD), wet AMD, dry AMD or geographic atrophy.In certain embodiments, the disease or disorder is retinitis pigmentosa or glaucoma.

[0167] The disclosed dual vector system can be introduced into one or more selected mammalian cells using any one or more methods known to those skilled in the art of gene therapy and / or viruses. Such methods include, but are not limited to, transfection, microinjection, electroporation, lipofection, cell fusion, calcium phosphate precipitation, and biolistic methods. In certain embodiments, the vectors of the present disclosure can be introduced in vivo, including, for example, by lipofection (e.g., DNA transfection via liposomes prepared from one or more cationic lipids) (see, e.g., Felgner et al., 1987). Synthetic cationic lipids (Lipofectin®, Invitrogen Corp., La Jolla, CA, USA) can be used to prepare liposomes that encapsulate the vectors, facilitating their introduction into one or more selected cells. The vector system of the present disclosure can be introduced in vivo as "naked" DNA using methods known to those skilled in the art.

[0168] In an overall, general sense, the disclosed methods include administering at least one or more of the rAAV particles disclosed herein to one or both eyes of a mammal in need thereof in an amount and for a time sufficient to treat or ameliorate one or more symptoms of a disease, disorder, dysfunction, injury, abnormal condition, or trauma in the mammal. In some embodiments, the mammal is a human. In some embodiments, the human is a neonate, newborn, infant, or juvenile. In the practice of this disclosure, it is contemplated that suitable patients would 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, but not limited to, genetically related or inherited retinal disorders, diseases, and dystrophies.

[0169] In some aspects, the present disclosure provides methods for using the particles, vectors, viral particles, expression systems, compositions, and host cells described herein in methods for treating or ameliorating symptoms of various defects in the mammalian eye, particularly one or more defects in human photoreceptor or RPE cells, or in the preparation of medicaments for treating or ameliorating symptoms. Exemplary ocular diseases and disorders for treating or ameliorating symptoms (e.g., caused by one or more genetic defects in PR or RPE cells) include retinitis pigmentosa, Leber congenital amaurosis (e.g., LCA10), age-related macular degeneration (AMD), wet AMD, dry AMD, uveitis, Best disease, Stargardt disease, Usher syndrome, geographic atrophy, diabetic retinopathy, retinoschisis, color vision disorders, choroideremia, Bardet-Biedl syndrome, and glycogen storage diseases (ocular manifestations).

[0170] In some embodiments, administration of any of the disclosed vectors, viral particles, or compositions to a subject in need thereof provides partial or complete restoration of melanosome transfer in retinal pigment epithelial (RPE) cells. In exemplary embodiments, administration of any of the polynucleotide vector systems, viral particles, or compositions provides partial or complete restoration of vision loss.

[0171] In some aspects, the present disclosure provides methods for using the particles, vectors, viral particles, expression systems, compositions and host cells described herein in methods for treating or ameliorating symptoms of various defects in mammalian ears, particularly one or more defects in auditory hair cells and vestibular hair cells, or in the preparation of medicaments for treating or ameliorating symptoms.In exemplary embodiments, the subject in need thereof suffers from a disease or disorder selected from Usher syndrome or autosomal recessive isolated hearing loss (DFNB2).In some embodiments, the subject suffers from Usher syndrome type 1B, 1D, 1F or 2A.

[0172] In some embodiments, the subject has an eye disease or condition and / or an ear disease or disorder selected from Stargardt disease, LCA10, retinitis pigmentosa, Alström syndrome, Usher syndrome types 1B, 1D, 1F, or 2A, Duchenne muscular dystrophy, cystic fibrosis, glycogen storage disease III, non-syndromic hearing loss, hemophilia A, or dysferlinopathy.

[0173] Such methods can include intravitreal or subretinal administration of one or more of the disclosed particle vectors, viral particles, host cells, or compositions to one or both eyes of a subject in need thereof in an amount and for a time sufficient to treat or ameliorate the symptoms of such a deficiency in the affected mammal. Methods can also include prophylactic treatment of animals suspected of having such a condition or administration of such compositions to animals at risk of developing such a condition, either after diagnosis or before the onset of symptoms.

[0174] Pharmaceutical Compositions and Kits Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, which are suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, and may, if appropriate, be encapsulated in liposomes. The final dosage form must be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. Prevention of the action of microorganisms can be achieved, if appropriate, by various other antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0175] The present disclosure also provides a pharmaceutical composition comprising the vector system of the present disclosure in combination with a pharmaceutically acceptable carrier. Pharmaceutical compositions adapted for topical or parenteral administration containing a certain amount of compound constitute a preferred embodiment of the present disclosure. The dose administered to a patient, particularly a human, in the context of the present disclosure should be sufficient to achieve a therapeutic response in the patient over a reasonable time frame without fatal toxicity, and preferably with no more than an acceptable level of side effects or morbidity. Those skilled in the art will recognize that the dosage will vary depending on various factors, including the subject's condition (health), the subject's weight, the type of current treatment, if any, the frequency of treatment, the therapeutic ratio, and the severity and stage of the pathological condition.

[0176] The present disclosure also provides a kit comprising the vector system of the present disclosure 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 includes one or more other components, auxiliary agents, or adjuvants as described herein. In certain embodiments, the kit of the present disclosure includes instructions or packaging materials that explain how to administer the vector system contained in the kit to a selected mammalian recipient.

[0177] The containers of the disclosed kits can be made of any suitable material, such as glass, plastic, metal, etc., and can be of any suitable size, shape, or configuration. In certain embodiments, the vector system of the present disclosure is provided in the kit as a solid. In other embodiments, the vector system of the present disclosure is provided in the kit as a liquid or solution. In certain embodiments, the kit can include one or more ampoules or syringes containing the vector system of the present disclosure in a suitable liquid or solution form.

[0178] Further contemplated herein are kits containing premixes of any of the disclosed dual vectors (front-half and back-half vectors). These premixes may be in a single container and / or a single pharmaceutical product in a suitable liquid or solution form.

[0179] The present disclosure also provides for the use of the buffers and compositions disclosed herein in the manufacture of a medicament for the treatment, prevention and / or prophylaxis of a disease, disorder or dysfunction and / or the treatment, prevention or amelioration of symptoms of a disease, disorder, dysfunction, injury or trauma, including but not limited to the treatment, prevention and / or prophylaxis of a disease, disorder or dysfunction and / or the amelioration of one or more symptoms of such disease, disorder or dysfunction.

[0180] The amount of AAV composition and the time of administration of such composition are within the scope of a person skilled in the art who has the benefit of the present teachings.The administration of a therapeutically effective amount of the disclosed composition can be achieved by a single administration, such as a single injection of a sufficient number of infectious particles to provide a therapeutic benefit to a patient undergoing such treatment.Alternatively, in some situations, it may be desirable to provide multiple or sequential administrations of AAV vector composition over a relatively short period of time or over a relatively long period of time, as can be determined by the physician supervising the administration of such composition.

[0181] For example, the number of infectious particles administered to a mammal may be approximately 10, given either as a single dose (or divided into two or more doses, etc.) as may be necessary to achieve therapy for the particular disease or disorder being treated. 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or even higher infectious particles / mL. Indeed, in certain embodiments, it may be desirable to administer two or more different rAAV particle- or vector-based compositions, alone or in combination with one or more other diagnostic agents, drugs, bioactive agents, or the like, to achieve the desired effect of a particular regimen or therapy. For most rAAV vector-based gene therapy regimens, the inventors contemplate that low titers of infectious particles will be required when practicing the disclosed methods of pretreating and co-administering AAV capsids with HA.

[0182] To express a therapeutic agent according to the present disclosure, rAAV particles can be prepared that contain a nucleic acid segment encoding the therapeutic agent under the control of one or more promoters. To place a sequence "under the control of" a promoter, the 5' end of the transcription initiation site of the transcriptional reading frame is generally positioned "downstream" (e.g., 3') of the selected promoter between about 1 and about 50 nucleotides. The "upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded polypeptide. This is what is meant by "recombinant expression" in this context. In some embodiments, the recombinant vector construct comprises a capsid-protein-modified rAAV vector containing an RPE cell-specific or photoreceptor cell-specific promoter operably linked to at least one nucleic acid segment encoding one or more diagnostic and / or therapeutic agents.

[0183] Where the use of such vectors is contemplated for the introduction of one or more exogenous proteins, polypeptides, peptides, ribozymes and / or antisense oligonucleotides into specific cells transfected with the vector, the rAAV particles disclosed herein can be used to deliver one or more exogenous polynucleotides to selected host cells, for example, to one or more selected cells within the mammalian eye.

[0184] In some embodiments, the number of viral particles administered to a subject is approximately 10 6 ~10 14 particles / ml, or 10 3 ~10 15 particles / ml, or any value between any of the ranges, e.g., about 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or 10 14 In one embodiment, the concentration may be 10 particles / ml, etc. 13In some embodiments, the number of viral particles administered to a subject is approximately 10 particles / ml or higher. 6 ~10 14 vector genomes (vgs) / ml, or 10 3 ~10 15 vgs / ml, or any value between any ranges, e.g., about 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or 10 14 vgs / ml, etc. In one embodiment, 10 13 Viral particles may be administered in volumes of 0.0001 ml to 10 ml, e.g., 0.001 ml, 0.01 ml, 0.1 ml, 1 ml, 2 ml, 5 ml, or 10 ml, as needed to achieve therapy for the particular disease or disorder being treated.

[0185] In some embodiments, the present disclosure provides formulations of one or more virus-based compositions disclosed herein in a pharmaceutically acceptable solution for administration to a cell or an animal, alone or in combination with one or more other therapeutic modalities, particularly for therapy of diseases affecting human cells, tissues, and humans.

[0186] If desired, the rAAV particles described herein can also be administered in combination with other drugs, such as proteins or polypeptides or various pharmaceutically acceptable drugs, including one or more systemic or local administrations of therapeutic polypeptides, biologically active fragments or variants thereof.In fact, there is virtually no limit to the other components that can be included, provided that the additional drug does not cause significant adverse effects upon contact with target cells or host tissues.Therefore, rAAV particles can be delivered together with various other drugs as needed in specific cases.Such compositions can be purified from host cells or other biological sources, or can be chemically synthesized as described herein.

[0187] For example, the formulation of pharmaceutically acceptable buffers, excipients and carrier solutions, as well as the development of suitable dosing and treatment regimens for use with the particular compositions described herein in a variety of treatment regimens and formulations, including oral, parenteral, intraocular (e.g., subretinal or intravitreal), intravenous, intranasal, intraarticular, intrautricular, intracochlear and intramuscular administration, are well known to those of skill in the art.

[0188] Typically, these formulations can contain at least about 0.1% therapeutic agent (e.g., rAAV particles) or more, although the percentage of active ingredient(s) can, of course, vary and can conveniently be between about 1 or 2% and about 70% or 80% or more of the total formulation mass or volume. Naturally, the amount of therapeutic agent(s) in each therapeutically useful composition can be prepared in such a way that a suitable dosage is obtained in any given unit dose of compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be taken into account by those skilled in the art of preparing such pharmaceutical formulations, and as such, various dosages and treatment regimens may be desirable.

[0189] The term "excipient" refers to a diluent, adjuvant, carrier, or vehicle into which rAAV particles are administered. Such pharmaceutical excipients can be sterile liquids, such as water and oils, 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. Physiological saline solution and aqueous dextrose and glycerol solutions can also be used as liquid carriers. Exemplary excipients and vehicles include, but are not limited to, HA, BSS, artificial CSF, PBS, lactated Ringer's solution, TMN200 solution, polysorbate 20, and poloxamer 100.

[0190] The amount of rAAV particle composition and the time of administration of such composition are within the scope of those skilled in the art who have the benefit of the present teachings.However, it is likely that the administration of a therapeutically effective amount of the disclosed composition can be achieved by a single administration, such as a single injection of a sufficient number of viral particles to provide a therapeutic benefit to a patient undergoing such treatment.Alternatively, in some situations, it may be desirable to provide multiple or sequential administrations of the composition over a relatively short period of time or over a relatively long period of time, as can be determined by the physician supervising the administration of such compositions.

[0191] Exemplary compositions can include rAAV particles or nucleic acid vectors alone or in combination with one or more additional active ingredients, which can be obtained from natural or recombinant sources or can be chemically synthesized.

[0192] Methods 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 disease for the long-term expression of a wide variety of therapeutic genes (Daya and Berns, 2008; Niemeyer et al., 2009; Owen et al., 2002; Keen-Rhinehart et al., 2005; Scallan et al., 2003; Song et al., 2004). AAV vectors have also provided long-term clinical benefit in humans when targeted to immune-privileged sites, such as ocular delivery for Leber congenital amaurosis (Bainbridge et al., 2008; Maguire et al., 2008; Cideciyan et al., 2008). The main advantage of this vector is its relatively low immune profile, inducing only a limited inflammatory response and, in some cases, even directing immune tolerance to the transgene product (LoDuca et al., 2009). Nevertheless, therapeutic efficacy is still maintained in humans when targeted to immune-privileged organs, due to the increased production of antibodies against the viral capsid and CD8 + Although limited due to T cell responses, adaptive responses to transgene products have also been reported in animal models (Manno et al., 2006; Mingozzi et al., 2007; Muruve et al., 2008; Vandenberghe and Wilson, 2007; Mingozzi and High, 2007). These results suggested that immune responses remain a concern for AAV vector-mediated gene transfer.

[0193] Adeno-associated virus (AAV) is considered the vector of choice for ocular gene therapy due to its efficiency, persistence, and low immunogenicity (Daya and Berns, 2008). Identifying vectors capable of transducing PRs via the vitreous has historically relied on identifying which serotypes have natural tropism for this cell type after local delivery. Several serotypes have been used to successfully target transgenes to PRs after subretinal injection (e.g., including AAV2, AAV5, and AAV8). All three have demonstrated efficacy in experiments conducted across multiple mammalian species (e.g., mice, rats, dogs, pigs, and non-human primates) (Ali et al., 1996; Auricchio et al., 2001; Weber et al., 2003; Yang et al., 2002; Acland et al., 2001; Vandenberghe et al., 2011; Bennett et al., 1999; Allocca et al., 2007; Petersen-Jones et al., 2009; Lotery et al., 2003; Boye et al., 2012; Stieger et al., 2008; Mussolino et al., 2011; Vandenberghe et al., 2011).

[0194] Studies comparing their relative efficiencies after subretinal delivery in rodents have shown that both AAV5 and AAV8 transduce PR more efficiently than AAV2, with AAV8 being the most efficient (Yang et al., 2002; Allocca et al., 2007; Rabinowitz et al., 2002; Boye et al., 2011; Pang et al., 2011). AAV2 and AAV8 vectors containing point mutations of surface-exposed tyrosine residues (tyrosine to phenylalanine, YF) have previously been shown to exhibit increased transgene expression in various retinal cell types compared with unmodified vectors after both subretinal and intravitreal injection (Petrs-Silva et al., 2009; Petrs-Silva et al., 2011). Of the vectors initially tested by those authors, the AAV2 triple mutant (named “triple YF”) showed the highest transduction efficiency after intravitreal injection, whereas the AAV2 quadruple mutant (“quad YF”) displayed the novel property of enhanced transduction of the outer retina ( Petrs-Silva et al., 2011 ).

[0195] Further improvements in transduction efficiency have been achieved through directed mutagenesis of surface-exposed threonine (T) or serine (S) residues to unnatural amino acids at one or more of these amino acids. Both YF and TV / TA mutations have been shown to increase efficiency by reducing capsid phosphorylation and subsequent ubiquitination as part of the proteosomal degradation pathway (Zhong et al., 2008; Aslanidi et al., In Press; Gabriel et al., 2013). The transduction profile of intravitreally delivered AAV has been found to be highly dependent on the injection procedure itself. Due to the small size of the mouse eye, transscleral intravitreal injections often result in retinal damage, which may allow for direct delivery of some vectors to the subretinal space.

[0196] Exemplary rAAV nucleic acid vectors useful in accordance with this disclosure include single-stranded (ss) or self-complementary (sc) AAV nucleic acid vectors, such as single-stranded or self-complementary recombinant viral genomes.

[0197] Methods for producing rAAV particles and nucleic acid vectors are also known in the art and commercially available (see, e.g., Zolotukhin et al., Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors, Methods 28 (2002) 158-167; and U.S. Patent Publication Nos. US2007 / 0015238 and US2012 / 0322861, which are incorporated herein by reference; also, plasmids and kits available from the ATCC and Cell Biolabs, Inc.). For example, a plasmid containing the nucleic acid vector sequence can be combined with one or more helper plasmids containing, for example, a rep gene (encoding, e.g., Rep78, Rep68, Rep52, and Rep40) and a cap gene (encoding VP1, VP2, and VP3, including a modified VP3 region as described herein) and transfected into a producer cell line, resulting in packaged rAAV particles that can then be purified.

[0198] In some embodiments, the one or more helper plasmids comprise a first helper plasmid comprising the rep gene and the cap gene and a second helper plasmid comprising the E1a gene, the E1b gene, the E4 gene, the E2a gene, and the VA gene. In some embodiments, the rep gene is derived from AAV2, and the cap gene is derived from AAV2 and comprises modifications to the gene to produce a modified capsid protein as described herein. Helper plasmids and methods for producing such plasmids are known in the art and are commercially available (e.g., pDM, pDG, pDP1rs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E / R585E), pDP8.ape plasmids from PlasmidFactory, Bielefeld, Germany; Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, CA; and other products and services available from Addgene, Cambridge, MA; pxx6; Grimm et al. (1998), Novel Tools for Production and Purification of Recombinant Adenoassociated Virus Vectors, Human Gene Therapy, Vol. 9, 2745-2760; Kern, A. et al. (2003), Identification of a Heparin-Binding Motif on Adeno-Associated Virus Type 2 Capsids, Journal of Virology, Vol. 77, 11072-11081; Grimm et al. (2003), Helper Virus-Free, Optically Controllable, and Two-Plasmid-Based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy, Vol.7, 839-850; Kronenberg et al. (2005), A Conformational Change in the Adeno-Associated Virus Type 2 Capsid Leads to the Exposure of Hidden VP1 N Termini, Journal of Virology, Vol. 79, 5296-5303; and Moullier, P. and Snyder, RO (2008), International efforts for recombinant adeno-associated viral vector reference standards, Molecular Therapy, Vol. 16, 1185-1188).

[0199] An exemplary, non-limiting method for rAAV particle production is described below. One or more helper plasmids containing the rep and cap ORFs of the desired AAV serotype and the adenovirus VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters are generated and obtained. The cap ORF may also contain one or more modifications to generate modified capsid proteins as described herein. HEK293 cells (available from ATCC®) are transfected with the helper plasmid(s) and a plasmid containing the nucleic acid vector 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 allow rAAV particle production. Alternatively, in another example, an Sf9-based producer stable cell line is infected with a single recombinant baculovirus containing the nucleic acid vector. As a further alternative, in another example, HEK293 or BHK cell line is infected with HSV containing nucleic acid vector and optionally one or more helper HSV containing rep and cap ORFs as described herein and adenovirus VA, E2A (DBP) and E4 genes under the transcriptional control of their native promoters.HEK293, BHK or Sf9 cells are then incubated for at least 60 hours to allow rAAV particle production.The rAAV particles can then be purified using any method known in the art or described herein, for example, by iodixanol step gradient, CsCl gradient, chromatography or polyethylene glycol (PEG) precipitation.

[0200] Illustrative Definitions In accordance with this disclosure, polynucleotides, nucleic acid segments, nucleic acid sequences, etc. include, but are not limited to, DNA (including, but not limited to, genomic and / or extragenomic DNA), genes, peptide nucleic acids (PNAs), 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 in whole or in part by the hand of man.

[0201] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although any method and composition similar or equivalent to those described herein can be used in the practice or testing of this disclosure, preferred methods and compositions are described herein.For the purposes of this disclosure, the following terms are defined below:

[0202] As used herein, the terms "nucleic acid" and "polynucleotide sequence" refer to deoxyribonucleotide or ribonucleotide polymers in either single- or double-stranded form and, unless otherwise limited, include known analogs of natural nucleotides that can function in a manner similar to naturally occurring nucleotides. Polynucleotide sequences include both full-length sequences and shorter sequences derived from full-length sequences. It is understood that a particular polynucleotide sequence includes degenerate codons of the native sequence or sequences that may be introduced to provide codon preference in a specified host cell. Polynucleotide sequences within the scope of the present disclosure further include sequences that specifically hybridize to sequences encoding the peptides of the present disclosure. Polynucleotides include both sense and antisense strands as individual strands or in a duplex.

[0203] Fragments and variants of the disclosed polynucleotides can be generated as described herein and tested for function using standard techniques known in the art. Thus, one of ordinary skill in the art can readily prepare and test fragments and variants of the disclosed polynucleotides or polypeptides to determine whether the fragment or variant retains the same or similar functional activity as a full-length or non-mutated polynucleotide or polypeptide, e.g., a myosin VIIa polynucleotide or polypeptide.

[0204] Also within the scope of this disclosure are polynucleotides that have the same or substantially identical nucleotide sequences of the polynucleotides exemplified herein, except for the presence of one or more nucleotide substitutions, additions, or deletions within the sequence of the polynucleotide, so long as these variant polynucleotides retain substantially the same relevant functional activity as the polynucleotides exemplified herein (e.g., they encode a protein having the same amino acid sequence or the same functional activity as one of the polynucleotides specifically exemplified herein).Thus, the polynucleotides disclosed herein should also be understood to include variants and fragments thereof.

[0205] As can be readily understood by those skilled in the art of molecular biology, there may be some variant sequences of genes or polynucleotides found in nature, in addition to variants that can be artificially prepared or synthesized by those skilled in the art in a laboratory environment. The polynucleotides of the present disclosure encompass those specifically exemplified herein as well as any naturally occurring variants thereof, as well as any variants that can be artificially created, so long as the variant retains the desired biological activity.

[0206] Also within the scope of this disclosure are polynucleotides that have the same nucleotide sequences of the polynucleotides exemplified herein, except for nucleotide substitutions, additions, or deletions within the sequence of the polynucleotide, so long as these variant polynucleotides retain substantially the same relevant biological activity as the polynucleotides specifically exemplified herein. Thus, the polynucleotides disclosed herein should be understood to include variants and fragments, as discussed above, of the specifically exemplified sequences.

[0207] The polynucleotides described herein can also be defined in terms of more specific ranges of identity and / or similarity than those exemplified herein. Sequence identity is usually greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and may be greater than 95%. Sequence identity and / or similarity may be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% or greater when compared to the exemplary sequences herein.

[0208] Unless otherwise specified, as used herein, the percent sequence identity and / or similarity of two sequences can be determined using the algorithm of Karlin and Altschul (1990), modified as in Karlin and Altschul (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990). BLAST searches can be performed using the NBLAST program, score=100, word length=12, to obtain sequences with a desired percent sequence identity. To obtain gapped alignments for comparison purposes, gapped BLAST can be used as described (Altschul et al., 1997). When using BLAST and gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) can be used according to published methods.

[0209] The present disclosure also contemplates polynucleotide molecules having sequences sufficiently homologous to the polynucleotide sequences of the present disclosure to permit hybridization with the sequences under standard stringent conditions and methods (Maniatis et al., 1982). As used herein, "stringent" conditions for hybridization are those in which hybridization typically occurs at temperatures above the melting temperature (T) of the DNA hybrid in 6xSSPE, 5xDenhardt's solution, and 0.1% SDS containing 0.1 mg / ml of suitable nonspecifically denatured DNA. m ) and overnight at 20-25 degrees Celsius or less.

[0210] The term "effective amount," as used herein, refers to an amount that is capable of treating or ameliorating a disease or condition, or otherwise producing the intended therapeutic effect.

[0211] The term "operably linked," as used herein, refers to linked nucleic acid sequences that are usually contiguous or substantially contiguous, and, where necessary, contiguous and in reading frame to connect two protein-coding regions. However, because enhancers generally function when separated by several kilobases from the promoter and intron sequences can be of variable length, some polynucleotide elements may be operably linked but not contiguous.

[0212] The term "promoter," as used herein, refers to a region or regions of a nucleic acid sequence that regulates transcription. Exemplary promoters provided herein include, but are not limited to, a CMV promoter, an EF-1 alpha promoter, a cone arrestin promoter, a chimeric CMV β-actin promoter (CBA), a truncated chimeric CMV β-actin (smCBA) promoter, a promoter derived from the human myosin 7a gene, a promoter derived from the TαC gene, a rhodopsin promoter, a cGMP-phosphodiesterase β-subunit promoter, a human or mouse rhodopsin promoter, an hGRK1 promoter, a synapsin promoter, a glial fibrillary acidic protein (GFAP) promoter, a rod-specific IRBP promoter, and a VMD2 promoter.

[0213] The term "regulatory element," as used herein, refers to a region or regions of a nucleic acid sequence that regulate transcription. Exemplary regulatory elements include, but are not limited to, enhancers, post-transcriptional elements, transcriptional control sequences, and the like.

[0214] The terms "substantially corresponding," "substantially homologous," or "substantial identity," as used herein, refer to a nucleic acid or amino acid sequence characteristic of a selected nucleic acid or amino acid sequence that, when compared to a selected reference nucleic acid or amino acid sequence, shares at least about 70 or 75 percent sequence identity. More usually, the selected sequence and the reference sequence share 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. More preferably, more highly homologous sequences often share at least about 96, 97, 98, or greater than 99 percent sequence identity between the selected sequence and the compared reference sequence.

[0215] The percentage of sequence identity may be calculated over the entire length of the sequences to be compared, or may be calculated by excluding small deletions or additions totaling less than about 25 percent of the selected reference sequence. The reference sequence may be a subset of a longer sequence, such as a portion of a gene or adjacent sequence, or a repeated portion of a chromosome. However, in the case of sequence homology between two or more polynucleotide sequences, the reference sequence usually contains at least about 18-25 nucleotides, more usually at least about 26-35 nucleotides, and even more usually at least about 40, 50, 60, 70, 80, 90, or even 100 nucleotides.

[0216] When highly homologous fragments are desired, the degree of percent identity between the two sequences will be at least about 80%, preferably at least about 85%, and more preferably about 90% or 95% or higher, as readily determined by one or more sequence comparison algorithms well known to those of skill in the art, for example, the FASTA program analysis described by Pearson and Lipman (1988).

[0217] The term "subject," as used herein, describes organisms, including mammals, e.g., primates, to which treatment with a composition according to the present disclosure can be provided. Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, humans, non-human primates, such as apes, chimpanzees, monkeys, and orangutans, domestic animals including dogs and cats, and livestock such as horses, cows, pigs, sheep, and goats, or other mammalian species, including, but not limited to, mice, rats, guinea pigs, rabbits, hamsters, etc.

[0218] The term "treatment" and any grammatical variations (e.g., treat, treating and treatment, etc.), as used herein, includes, but is not limited to, alleviating the symptoms of a disease or condition and / or reducing, suppressing, inhibiting, palliating, ameliorating or affecting the progression, severity and / or extent of a disease or condition.

[0219] The term "vector," as used herein, refers to a nucleic acid molecule (usually containing DNA) that is replicable in a suitable host cell or to which another nucleic acid segment can be operably linked to facilitate the replication of the operably linked nucleic acid segment. Exemplary vectors include, but are not limited to, plasmids, cosmids, viruses, etc.

[0220] As used herein, the term "variant" refers to a molecule (e.g., a polynucleotide) having characteristics that deviate from those occurring in nature. For example, a "variant" is 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 polynucleotide. A mutant protein molecule, e.g., a capsid, may contain amino acid sequence modifications relative to the wild-type protein sequence (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, or 15-20 amino acid substitutions) resulting from point mutations introduced into the nucleic acid sequence encoding the capsid protein. These modifications include chemical modifications as well as truncations.

[0221] Mutants of nucleic acid molecules, e.g., polynucleotide vector systems, can contain sequence modifications relative 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 can include truncations at the 5' or 3' end. [Example]

[0222] The following examples are included to demonstrate preferred embodiments of the present disclosure. It should be understood by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the present disclosure, and therefore can be considered to constitute preferred modes for its practice. However, those of skill in the art, in light of the present disclosure, should understand that many changes can be made to the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure. [Example 1]

[0223] Example 1 illustrates an exemplary overlapping dual vector system with an hMYO7A coding overlap. In the exemplary overlapping system (Figure 2), the overlapping DNA sequence shared by both Vector A and Vector B consists of 1350 bp of the coding region of the human MYO7A gene. This is the simplest system of the present disclosure and appears to be highly efficient in terms of full-length gene rearrangement and MYO7A expression. Advantageously, each vector is of standard AAV packaging size, and as such, each packages DNA with high efficiency and is easily adaptable to conventional GMP standards. Such vectors are also easily characterized to allow for the necessary regulatory approval prior to use in humans. [Example 2]

[0224] Example 2 illustrates an exemplary hybrid dual-AAV vector system utilizing hMYO7A intron 23 splicing. In the exemplary system (FIG. 3), the overlapping DNA sequence is composed of the native intron 23 of human MYO7A. Vector A contains the coding sequence (hMYO7ANT) corresponding to the amino-terminal portion of the hMYO7A cDNA relative to intron 23 and the native splice donor site, followed by a 250-bp fragment of intron 23 of hMYO7A (minus the native acceptor site). Vector B contains the carboxyl-terminal portion of the hMYO7A cDNA relative to intron 23 (hMYO7ACT) and a 250-bp fragment of intron 23 of MYO7A (minus the native splice donor site), followed by the native splice acceptor site. Upon co-delivery into a suitable mammalian host cell, the DNA of vectors A and B recombine to form a reconstituted full-length gene cassette. The resulting RNA transcript then "splices out" the native intron. Alternatively, recombination and formation of the gene cassette may occur via the AAV TR. In this case, the RNA transcript "splices out" the native intron 23-TR-intron 23 motif. However, in both cases, the resulting mRNA is that of the reconstituted full-length hMYO7A gene sequence. [Example 3]

[0225] Example 3 demonstrates the in vitro performance of an exemplary overlap vector system containing an hMYO7A-encoding overlap. HEK293 cells were simultaneously infected with vector A and vector B of the overlap dual vector system at a ratio of 10,000:1 vg / cell for each vector (Figure 2). The AAV vector was packaged into AAV2 viral particles containing three YF mutations in the capsid protein (see, e.g., Zhong et al., 2008). As a positive control, cells were transfected with a plasmid containing full-length hMYO7A under the control of smCBA. Protein was collected from cells 3, 4, 5, 6, and 7 days after infection and assayed for its presence in infected cells by immunoblotting using an antibody against MYO7A. The results are shown in Figures 6A and 6B. The area of ​​interest in Figure 6A is enlarged and shown with higher contrast in Figure 6B. Starting at 3 days post-infection, full-length human MYO7A protein was visible, with peak expression of this protein occurring around day 5. [Example 4]

[0226] Example 4 demonstrates the in vivo performance of an exemplary overlap vector system containing the hMYO7A coding overlap. Six-week-old shaker-1 (MYO7A null) mice were subretinally co-injected with 1 μL of the same preparations of Vector A and Vector B used in the in vitro studies above. Both vectors were administered in approximately 1×10 12 MYO7A was delivered at 1000µg / ml. Four weeks after injection, retinas were harvested from treated and untreated eyes, and immunohistochemistry (IHC) was performed using an antibody against MYO7A (see Figures 7A and 7B). Lighter areas indicated MYO7A-specific staining, while darker areas corresponded to nuclear-specific DAPI staining. In treated eyes, MYO7A expression was clearly visible and appeared restricted to photoreceptors, more precisely, to the junction of the photoreceptor inner and outer segments. [Example 5]

[0227] Example 5 shows that AAV dual vectors efficiently deliver oversized genes. material and method Animals. Shaker-1 mice carrying the 4626SB allele, an effective null mutation (Liu et al., 1999; Hasson et al., 1997), were used and maintained on a C57BL6 genetic background and genotyped as described (Liu et al., 1999; Gibbs et al., 2003a). They were maintained on a 12-hour light / 12-hour dark cycle, exposed to 10–50 lux of fluorescent light during the light phase, and treated in accordance with federal and institutional animal care guidelines. Homozygous mutants were distinguished from heterozygous controls by their hyperactivity, head-tossing, and rotational behavior (Gibson et al., 1995), and / or by PCR / restriction digestion assays.

[0228] AAV vector construction. Single-vector platform: An AAV vector plasmid containing a truncated chimeric CMV / chicken β-actin promoter (smCBA) (Haire et al., 2006) and MYO7A cDNA was constructed by removing the entire MYO7A cDNA from pEGFP-C2 by EagI and SalI digestion and then ligating it into pTR-smCBA-GFP, which had been digested with NotI and SalI to remove GFP. The MYO7A cDNA (approximately 6.7 kb) corresponds to isoform 2 of human MYO7A and is identical to that previously described by Hashimoto et al. (2007) and based on the sequence published by Chen et al. (1996) (see SEQ ID NO: 8). MYO7A isoform 2 is 114 kb shorter than isoform 1 (Chen et al., 1996; Weil et al., 1996). Both the MYO7A cDNA and the resulting junction were fully sequenced prior to packaging. All vectors intended for in vitro analysis were packaged separately in wild-type AAV2 or alternatively in the AAV2(triple YF) capsid mutant vector (Petrs-Silva et al., 2011).

[0229] As described above, AAV2-based vectors were selected for in vitro experiments due to their increased transduction efficiency compared to other serotypes (Ryals et al., 2011). All vectors were packaged, purified, and titered using standard methods as previously described (Zolotukhin et al., 2002; Jacobson et al., 2006). Human embryonic kidney (HEK293) cells were transfected by the calcium phosphate method with a vector plasmid carrying the full-length MYO7A coding sequence of variant 2 (the same plasmid used to package the fragmented AAV). These transfected cells were then used as a positive control, demonstrating the appropriate size of full-length MYO7A protein throughout immunoblot analysis. Vector infection was performed in HEK293 cells using the corresponding titer of AAV vector. Briefly, cells were grown to 60-70% confluency. All vectors were diluted in balanced salt solution to achieve the desired multiplicity of infection (MOI). Unless specifically stated, cells were infected with 10,000 genome-containing particles / cell of each vector, resulting in a total MOI of 20,000 for each vector pair. Cells were incubated in medium containing 10% serum at 37°C under 7% CO2 for 3 days post-infection and then analyzed by immunoblotting. Various lots of AAV2-MYO7A and AAV5-MYO7A were infected with 10,000 genome-containing particles / cell. 12 ~10 13 Titers of individual particles / mL were obtained.

[0230] Oligonucleotide sequence. For in vivo studies, a human influenza hemagglutinin (HA) tag was added to the 3' end of the full-length, simple overlap, trans-splicing, hybrid 3' vector by utilizing the unique BamHI site (P19) and replacing the untagged 3' end with an HA-tagged version (P20). All constructs were sequence-confirmed by Sanger sequencing.

[0231] AAV Vector Plasmid Design and Cloning. The full-length coding sequence of MYO7A (human isoform 2; GenBank accession number NM_001127180) was cloned into a vector plasmid containing the strong, ubiquitous CMV / chicken β-actin (smCBA) promoter (Haire et al., 2006), a polyadenylation signal, and the AAV2 ITRs.

[0232] Packaging of this plasmid generated the fAAV vector (Figure 22A). In all systems, the 5' vector shared the smCBA promoter and the 5' portion of MYO7A, while the 3' vector contained the 3' portion of MYO7A and the bovine growth hormone (bGH) polyadenylation signal. The oligonucleotides used for vector construction are listed in Table 1. The simple overlap contained nucleotides 1 to 3644 of the MYO7A cDNA from the ATG in the 5' vector and nucleotides 2279 to 6534 in the 3' vector. The fragments were amplified by polymerase chain reaction (PCR) using oligonucleotides P1 and P3 and cloned into the 5' vector via NotI and NheI, and into the 3' vector using P3 (AflII) and P4 (KpnI), respectively.

[0233] The resulting two vector plasmids share 1365 bp of overlapping MYO7A sequence (Figure 22B). The trans-splicing and hybrid vectors utilize splice junctions composed of either ideal splice donor and acceptor sites derived from the AP coding sequence or the native MYO7A splice junction derived from exons 23 and 24 (Yan et al., 2002). To generate the 5' trans-splicing vector, the splice acceptor site was amplified using oligonucleotides P5 and P6 (NheI), and the amplicon was then used in a second reaction with oligonucleotide P7 (NsiI) to add a portion of the MYO7A coding sequence for cloning.

[0234] The corresponding 3' vectors were similarly generated by amplifying the splice acceptor site using oligonucleotides P8 (AflII) and P9 in the first PCR and adding a portion of the 3' MYO7A coding sequence using oligonucleotide P10 (AgeI) in the second PCR (see Figure 22C). AP hybrid vectors were generated by adding a 270-bp AP overlap sequence to each trans-splicing vector (Ghosh et al., 2011). The sequences were amplified by PCR, with appropriate restriction endonuclease sites added. For the 5' vector oligonucleotides, P11 (NheI) and P12 (SalI) were used, and for the 3' vector, oligonucleotides P13 (NotI) and P14 (AflII) were used (Figure 22D).

[0235] A fourth pair of vectors, the "natural intron hybrid" vectors, were also created to utilize the natural sequence in and surrounding intron 23 of MYO7A as a recombination locus, as well as the subsequent splicing signal. The 5' portion was created by first amplifying intron 23 with oligonucleotides P15 and P16 (NheI), then using the resulting amplicon in a second reaction with oligonucleotide P7 (NsiI) to facilitate cloning. The corresponding 3' vector was constructed by amplifying intron 23 with oligonucleotides P17 and P18 (AflII) and amplifying the resulting amplicon in a second reaction with oligonucleotide P10 (AgeI) (see Figure 22E).

[0236] [Table 1-1] [Table 1-2]

[0237] Oligonucleotides were used to generate the 5' and 3' vectors of the dual vector platform (P1-P20). Oligonucleotides were used to characterize the fidelity of the overlap in the simple overlap, trans-splicing, and AP hybrid vector platforms (P21-P22). Restriction sites used for cloning are underlined, and the introduced hemagglutinin (HA) tag is italicized (P19).

[0238] Dual Vector Platform. Two separate vector plasmids were constructed: Vector A contained the strong, ubiquitous "smCBA" promoter and the MYO7A cDNA encoding the N-terminal portion. Vector B contained the MYO7A cDNA encoding the C-terminal portion and a poly(A) signal sequence. Both vector plasmids contained inverted terminal repeats (ITRs). Using PCR with the full-length MYO7A cDNA as a template, the MYO7A cDNA was roughly split in half, with amplicons encompassing nucleotide positions 1 to 3644 (Vector A) and 2279 to 6647 (Vector B), relative to the ATG start position 1. The resulting two vector plasmids shared 1365 bp of overlapping MYO7A sequence and were 5.0 kb and 4.9 kb in length, respectively. This was well within the size limit of standard AAV vectors. Both vector plasmids were sequence verified and packaged separately by standard AAV production methods (Zolotukhin et al., 2002; Jacobson et al., 2006). The titer of the first lot was 2.5 x 10 of each vector. 12 The first lot contained 4 x 10 particles / mL of each vector. 12 It contained 10 particles / mL.

[0239] Reverse transcription and characterization of overlapping regions. HEK293 cells were infected with the dual vectors, and total RNA was extracted using the RNeasy® kit (Qiagen, Hilden, Germany) according to the manufacturer's recommended protocol. Two micrograms of RNA were then subjected to DNase I (NEB) digestion at 37°C for 30 minutes, followed by heat inactivation at 37°C for 30 minutes and then heat inactivation at 75°C for 10 minutes. Reverse transcription of cDNA was achieved using the SuperscriptIII® kit (Life Technologies, Grand Iceland, NY, USA) according to standard protocols utilizing oligo-dT primers. Two microliters of cDNA was used as a template in PCR using oligonucleotide primers P21 and P22 (see Table 1) (initial denaturation at 95°C for 3 minutes, 35 cycles of 95°C for 45 seconds, 55°C for 45 seconds, 72°C for 12 minutes, and a final 72°C for 15 minutes). The annealing sites of these primers were located 5' and 3' of the cDNA overlap in the simple overlap and hybrid vector pairs (i.e., outside the overlap region), respectively. The 3' primer annealed to a sequence complementary to the bGH polyA. The resulting products were digested with either PpuMI or BglII, separated on a 1.5% agarose gel, and then analyzed by UV screening. Separately, the products were digested with KpnI and AgeI and then cloned into a pUC vector for sequencing of the entire overlap region. Sense and antisense reads were obtained using vector-specific M13 forward and reverse primers, resulting in a 140-bp overlap between the sense and antisense reads. To demonstrate that these methods can detect aberrant sequences (e.g., for quality control), the analysis was repeated using MYO7A sequences with either an artificial insertion (HindIII fill-in at position 2635) or a point mutation (T → C) at position 2381.

[0240] In vitro virus delivery. HEK293A cells (Invitrogen) grown in DMEM with 10% FBS and 1x NEAA and Pen / Strep (Invitrogen) were plated in 6-well plates. The next day, cells were incubated with AAV2- and AAV5-MYO7A at an MOI of 10,000 virus particles / cell in 500 μL of complete medium also containing 40 μM calpain inhibitor (Roche, Pleasanton, CA, USA) at 37°C and 5% CO2. After 2 hours, complete medium was added. The next day, the medium was changed, and the cells were incubated for an additional 48 hours. Alternatively, some cells were transfected with 1 μg of the vector pTR-smCBA-MYO7A and complexed using Lipofectamine 2000 (1:3 ratio) according to the manufacturer's instructions (Invitrogen).

[0241] Primary mouse RPE cells were derived from P14-P16 MYO7A-null animals and cultured in 24-well dishes as described (Gibbs et al., 2003a; Gibbs and Williams, 2003b). After 48 hours in culture, cells were transduced with virus. Cells were incubated in 100 μL of complete medium containing 40 μM calpain inhibitor and 10,000 viral particles / cell from a full-strength AAV stock. After 2 hours, 400 μL of complete medium was added to each well and incubated overnight. The next day, the medium was replaced, and the cells were incubated for an additional 48 hours.

[0242] ARPE19 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM / F-12 with 10% FBS and split into 24-well plates with glass coverslips. Cells were grown to confluency and then transduced in the same manner as primary RPE cells.

[0243] MYO7A expression analysis by Western blot and immunofluorescence. HEK293A and primary mouse RPE cells transduced with AAV-MYO7A were harvested 3 days after transduction. For Western blot analysis, cells were harvested and lysed in 20 mM TRIS, pH 7.4, 5 mM MgCl2, 10 mM NaCl, 1 mM DTT, and 1x protease inhibitor cocktail (Sigma-Aldrich Chemical Co., St. Louis, MO, USA). Equal amounts of total protein were separated on a 7.5% SDS-PAGE gel. After transfer, blots were blocked with 5% nonfat milk and probed with a mouse anti-MYO7A antibody (Developmental Studies Hybridoma Bank, Iowa City, IA, USA) raised against residues 927–1203 of human MYO7A (Soni et al., 2005 ) and a mouse anti-actin antibody (Sigma-Aldrich) as a loading control.

[0244] Three days after infection, immunofluorescence was performed using ARPE19 and primary mouse RPE cells. Cells were fixed with 4% formaldehyde, blocked with blocking solution (0.5% BSA / 0.05% saponin in PBS), and incubated with mouse anti-MYO7A followed by goat anti-mouse Alexa-568 (Molecular Probes, Carlsbad, CA, USA). Coverslips were mounted with mounting medium containing DAPI (Fluorogel II, Electron Microscopy Sciences, Hatfield, PA, USA) and visualized using a confocal system.

[0245] Protein extraction and immunoblotting. Transfected and infected HEK293 cells were harvested, washed twice with PBS, and treated as previously described (Boye et al., 2012) with minor modifications. Cells were lysed by three 30-second pulses of sonication in 200 μL of sucrose buffer (0.23 M sucrose, 2 mM EDTA, 5 mM Tris-HCl, pH 7.5) containing protease inhibitors (Roche, Mannheim, Germany). Unlysed cells and cell debris were removed by centrifugation at 14,000 rpm for 10 minutes. Protein concentrations of the supernatants were measured using BCA (Thermo Fisher Scientific, Rockland, IL, USA). Equal amounts of protein were then loaded onto a 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel (BioRad, Hercules, CA, USA) and transferred onto a PVDF membrane (Millipore, Billerica, MA, USA) in CAPS buffer (pH 11). The blots were then labeled with antibodies against MYO7A (monoclonal antibody against amino acids 11–70 of human MYO7A; Santa Cruz, Dallas, TX, USA; 1:1000) or HA (MMS-101P; Covance, Gaithersburg, MD, USA; 1:500) and β-actin (ab 34731; Abcam, Cambridge, MA, USA; 1:5000). For visualization using the Odyssey system (Li-Cor, Lincoln, NE, USA), anti-mouse and anti-rabbit secondary antibodies conjugated with CW800 and IR680 dyes (Li-Cor), respectively, were used. Semiquantitative densitometric measurements were performed using Odyssey acquisition and analysis software (Li-Cor). Dual-color images were separated in their respective channels and converted to grayscale for presentation purposes. For visualization of protein size, size markers present in one channel of each blot were added to both channels.

[0246] In vivo virus delivery. Mice were anesthetized using 2.0–3.0% isoflurane inhalation. The animal's pupils were dilated using 1% (wt / vol) atropine sulfate and 2.5% phenylephrine. Local anesthetic (0.5% proparacaine hydrochloride) was also administered. A sclerotomy was performed at the temporal limbus using a 27-Ga needle. A 32-Ga blunt needle (WPI, Sarasota, FL, USA) attached to a microsyringe pump was inserted, and 1 μL of virus solution was injected into the ventral subretinal space of P14–P16 animals. Retinal detachment was visualized under a dissecting microscope and registered as an indication of positive subretinal injection. One microliter of the following AAV8(Y733F)-based vectors was injected subretinally into one eye of C57BL / 6 mice: equally divided (each vector = 1 × 10 13 vg / ml) combined and single fAAV (1 × 10 13 vg / ml), front and back half "hybrid" vectors or equally (each vector = 1 x 10 13 vg / ml) combined front and back half "simple overlap" vector. Subretinal injections were performed as previously described (Timmers et al., 2001). Further analysis was performed only in animals that received comparable successful injections (>60% retinal detachment with minimal surgical complications).

[0247] Light and immunoelectron microscopy of the retina. Eyecups were processed for embedding in either LR White or Epon, and semithin and ultrathin sections were prepared. Semithin sections were stained with toluidine blue and visualized using a Leica confocal system. Ultrathin sections were labeled with purified MYO7A pAb 2.2 (Liu et al., 1997) and monoclonal anti-opsin (1D4, R. Molday), followed by a gold-conjugated secondary antibody (Electron Microscopy Sciences), as previously described (Lopes et al., 2011). Negative control sections, processed simultaneously, included those from MYO7A-null retinas, and WT animals served as positive controls.

[0248] MYO7A immunogold density was determined in sections from age-matched wild-type, MYO7A-null retinas, and from MYO7A-null animals injected with AAV-MYO7A at P14–16 and dissected 3 weeks later. To quantify immunolabeling, all gold particles in full sections of each RPE cell were counted. The area of ​​each cell profile was determined using Image J software. For background labeling, the density of labeling in sections from untreated MYO7A-null animals was measured. Data were presented after subtracting this background labeling.

[0249] The concentrations of MYO7A and opsin immunogold labeling in the connecting cilia of photoreceptor cells were determined by counting gold particles along the longitudinal profile of the connecting cilia and measuring the length of each profile. Analysis and quantification were performed on a minimum of three different retinas from three different animals. Statistical analysis was performed using a one-tailed Student's t-test.

[0250] Six weeks after injection, C57BL / 6 mice were enucleated, and their eyes were processed and immunostained as previously described (Boye et al., 2011) with minor modifications. Retinas were immunostained with a hemagglutinin (HA)-specific antibody (monoclonal Ab clone 12CA5; Roche), counterstained with DAPI, and imaged using a spinning-disk confocal microscope (Nikon Eclipse TE2000 microscope equipped with a Perkin-Elmer Ultraview modular laser system and a Hamamatsu O-RCA-R2 camera). Images were acquired sequentially using a 20x (air) objective. All settings (exposure, gain, laser power) were identical across images. All image analysis was performed using Volocity 5.5 software (Perkin-Elmer, Waltham, MA, USA).

[0251] result AAV-MYO7A single vector preparation. An AAV vector plasmid was engineered to contain a truncated chimeric CMV / chicken β-actin promoter, smCBA (Haire et al., 2006), and a 6.7 kb cDNA encoding the full-length isoform 2 of human MYO7A (NCBI number NM_001127180) (Figure 10A). The smCBA promoter exhibits the same directionality and activity in the mouse retina as that of the full-length CBA promoter (Haire et al., 2006; Pang et al., 2008). Various lots of AAV2-MYO7A and AAV5-MYO7A were used for 10 min. 12 ~10 13 The titer was 10 particles / ml. 12 The concentration of 100 particles / ml was considered the standard concentration (1x) from which dilutions were made. Experiments were performed using virus obtained from three separate preparations. No differences in expression or phenotypic correction were observed between different lots of AAV2-MYO7A or AAV5-MYO7A at a given concentration, as described below.

[0252] MYO7A expression in cell culture. Transduction of primary cultures of MYO7A-null RPE cells with 1× single AAV2-MYO7A or AAV5-MYO7A was comparable to that of WT MYO7A protein by Western blot analysis, demonstrating MYO7A + / -This resulted in the expression of a polypeptide with an apparent mass that was present at levels similar to those found in primary cultures of RPE cells (Figure 10B). Similarly, a single band of the appropriate size was detected in Western blots of HEK293A cells. Immunofluorescence of primary RPE cells showed that the MYO7A protein obtained from 1× single AAV-MYO7A treatment of MYO7A-null cells had a subcellular localization pattern comparable to that of endogenous MYO7A in control cells, indicating the production of a properly targeted protein (Figures 10C-10F). ARPE19 cells were also infected with 1× or diluted (1:100) AAV2-MYO7A or AAV5-MYO7A and compared with untreated cells. An increase in MYO7A immunofluorescence was detected in treated cells, and the subcellular localization of the label was comparable to that in untreated cells (Figures 17A-17F).

[0253] Localization of MYO7A in vivo. Most retinal MYO7A is found in the RPE (Hasson et al., 1995), but the protein is also present in the connecting cilia and periciliary regions of photoreceptor cells (Liu et al., 1997; Williams, 2008). A diagram illustrating this distribution and retinal function of MYO7A has been published in a recent review (Williams and Lopes, 2011).

[0254] Three weeks after injection of 1xAAV2-MYO7A or AAV5-MYO7A into the subretinal space of MYO7A-null mice, retinal tissue was examined by immunoelectron microscopy to test for MYO7A expression. Immunogold labeling was evident in photoreceptor cells, but was localized to connecting cilia and periciliary areas, comparable to that in WT retinas (Figures 11A-E). Labeling was also present throughout RPE cells, particularly in the apical cell body region, as found in WT retinas (Figures 11F, 11F-1, 11G, and 11G-1; see Figures 18A-18D for comparison) (Gibbs et al., 2004; Liu et al., 1997).

[0255] MYO7A has a similar distribution in both rod and cone photoreceptor cells (Liu et al., 1999). To test whether treatment with AAV-MYO7A also affected cone photoreceptor cells, we examined whether MYO7A was present in the ciliary region of cone photoreceptors. Double immunoelectron microscopy (EM) was performed using MYO7A antibodies together with antibodies specific for rod opsin in the treated retinas. Although only a small number of cones were found with aligned connecting cilia in each ultrathin section, MYO7A immunogold labeling was evident in the connecting cilia and periciliary regions of these cones, identified by the lack of rod opsin labeling in their outer segments (in contrast to the surrounding rod outer segments) (Figures 11H and 11I). Thus, AAV2-MYO7A and AAV5-MYO7A can transduce both cone and rod photoreceptor cells.

[0256] Dose-dependent MYO7A expression in photoreceptor and RPE cells. To determine the level of MYO7A expression after treatment with various concentrations of AAV2-MYO7A and AAV5-MYO7A (1x, 1:10, or 1:100 dilutions), MYO7A immunogold labeling was quantified in EM images taken within 1.4 mm of the injection site. Reliable detection of MYO7A in photoreceptor cells, whose distribution is restricted to connecting cilia and periciliary areas, requires higher resolution than that provided by electron microscopy (Liu et al., 1997). Immunogold particle density was measured in images of photoreceptor connecting cilia and periciliary areas shown in full longitudinal sections (from the basal body to the base of the outer segment) or in images showing RPE cells in apical-to-basal sections. Particle density was expressed as particles per cilium length of photoreceptor cells (each connecting cilium is approximately 1.2 µm long) and particles per area of ​​RPE cells (the entire area between the apical and basal surfaces was included). Particle density varies depending on the exposure of epitopes on the surface of the section and, as such, provides a relative linear measure of antigen density under the conditions used here (e.g., grids are etched and labeled in an identical manner, and labeling is not so dense as to be affected by steric hindrance).

[0257] Treatment with 1x AAV2-MYO7A or AAV5-MYO7A resulted in 2.5- to 2.7-fold higher density immunolabeling in photoreceptor cilia compared to that found in WT retina, whereas the 1:10 and 1:100 dilutions resulted in densities of immunolabeling more comparable to WT levels (Figures S11J, S11L, and S19). Quantification of immunogold labeling in the RPE showed that injection of AAV2-MYO7A resulted in 2.7-fold more labeling than WT, with no significant difference observed when using the 1:10 and 1:100 dilutions (Figure S11K). In contrast, the level of MYO7A immunolabeling in the RPE of AAV5-MYO7A-injected retinas varied in relation to viral titer, with all doses of virus achieving 2.2-fold more MYO7A than that found in WT RPE, a 1:10 dilution achieving WT levels, and a 1:100 dilution resulting in, on average, approximately 60% of WT levels (Figure 5M).

[0258] These labeling density counts indicate that 1x AAV-MYO7A resulted in more than double the normal level of MYO7A expression in both photoreceptor and RPE cells. The distribution of MYO7A was unaffected by this overexpression in photoreceptor cells. In RPE cells, the overall distribution of MYO7A was comparable to that in WT, with higher concentrations in the apical cell body region. However, with 1x AAV2-MYO7A or 1x AAV5-MYO7A, the proportion of MYO7A associated with melanosomes was only 55% of that in WT RPE. This difference is likely due to the fact that proteins linking MYO7A to melanosomes, MYRIP and RAB27A (Klomp et al., 2007; Lopes et al., 2007), may have remained at near-WT levels, thus limiting the absolute amount of MYO7A that would associate with melanosomes.

[0259] Despite MYO7A overexpression, no pathology was evident in the retina up to 3 months after injection of 1× (or 1:10) AAV2-MYO7A. However, 10% of AAV5-MYO7A (e.g., 10×) 13 Two of six retinas injected with particles / ml showed evidence of photoreceptor cell loss throughout the retina after 3 weeks (AAV2-MYO7A was not tested at this titer) (Figure 19).

[0260] Correction of melanosome localization in the RPE. In MYO7A-mutant mice, melanosomes are absent from the apical processes of RPE cells (Liu et al., 1998). This mutant phenotype is evident at all neonatal ages and is due to the loss of actin-based transport of melanosomes by myosin 7a motors (Gibbs et al., 2004). Three weeks after injection of 1x AAV2-MYO7A or AAV5-MYO7A into the subretinal space of MYO7A-null mice, melanosomes were observed to be normally distributed in all RPE cells near the injection site (within 1.4 mm) (n = 10 each for AAV2-MYO7A and AAV5-MYO7A) (Figures 12A-12C). Far enough from the injection site, a mixture of corrected and uncorrected RPE cells was evident; in the periphery of the retina, all cells displayed the MYO7A-mutant phenotype, indicating a lack of correction in this region (Figures 12D-12F). Melanosome correction was still evident in retinas fixed 3 months after injection (Figure 20). Correction was also observed in all eyes injected with 1:10 dilution AAV2-MYO7A (n=6) or AAV5-MYO7A (n=6), as well as in all eyes injected with 1:100 dilution AAV2-MYO7A (n=6) or AAV5-MYO7A (n=6); however, when the 1:100 dilution was used, some RPE cells near the injection site were not corrected.

[0261] Correction of opsin distribution. MYO7A-mutant mice have abnormal accumulation of opsin in the connecting cilia of photoreceptor cells, a phenotype evident by immuno-EM using an opsin antibody (Liu et al., 1999). This mutant phenotype suggested that myosin 7a functions in vector-mediated delivery of opsin to outer segments (Liu et al., 1999). Quantification of immunogold opsin labeling in the connecting cilia demonstrated that this phenotype was corrected using 1x AAV2-MYO7A or AAV5-MYO7A (Figure 13 and Figures 21A-21D). This analysis also demonstrated phenotypic correction using a 1:100 dilution, but the data indicated that, regardless of the WT level of MYO7A (Figures 11J and 11L), a full WT phenotype was not achieved (Figure 13), suggesting that a portion of MYO7A may not be fully functional.

[0262] AAV2-MYO7A Dual Vector Preparation. The results described above demonstrate that a single AAV vector can deliver functional MYO7A to RPE and photoreceptor cells in vivo. Because the size of smCBA-MYO7A is approximately 2 kb larger than the nominal carrying capacity of AAV (Grieger and Samulski, 2005), this transduction may involve undefined fragmentation of the smCBA-MYO7A cDNA followed by reassembly of the plus and minus cDNA strands after delivery to cells, as has been shown for other large genes (Dong et al., 2010; Lai et al., 2010; Wu et al., 2010). To assess whether two AAV vectors containing defined overlapping fragments of the MYO7A cDNA (1365 bases) could also mediate full-length MYO7A expression, we developed an AAV2-based dual vector system (Figures 14A-1 and 14A-2). Two separate lots of AAV2-MYO7A (dual vector) were prepared, each containing equal concentrations of AAV2-smCBA-MYO7A (5'-half) and AAV2-MYO7A (3'-half). The titer of the first lot was 2.5 x 10 of each vector. 12The first lot contained 4 x 10 particles / mL. 12 It contained 10 particles / mL.

[0263] MYO7A expression using AAV2 dual vectors. Western blot analysis of primary cultures of MYO7A-null RPE cells infected with either lot of AAV2-MYO7A (dual vector) showed that the cells expressed MYO7A-immunolabeled polypeptides at masses equivalent to those of WT MYO7A (Fig. 14B). However, the expression level of MYO7A in MYO7A-null RPE cells differed from that found with single AAV2 or AAV5 vectors (Fig. 10B), indicating that MYO7A + / - This was significantly less than that found in primary cultures of RPE cells (see lanes 2 and 3 in Figure 14B). Quantitative analysis of Western blots demonstrated that MYO7A-null RPE cells transduced with single vector (1x), AAV2-MYO7A, or AAV5-MYO7A, or with AAV2-MYO7A (dual vector), showed a significant increase in MYO7A + / - They were shown to express MYO7A at levels that were 82%, 111%, and 10%, respectively, of the levels of MYO7A in RPE cells.

[0264] Figure 16 is a Western blot using the same dual vector system as above, but with the AAV8 serotype. Figure 16 shows that the expression levels of MYO7A using the dual vector system were nearly equal to wild-type MYO7A expression levels. Although the reason for the discrepancy is unclear, significantly better results were obtained by us using the dual vector system than those obtained by several external collaborators. Wild-type-like levels of MYO7A expression were observed in Shaker-1 retinas after injection with the dual-AAV8(Y733F) vector. Thus, extremely good expression of MYO7A was achieved using the dual AAV platform.

[0265] Immunofluorescence of primary MYO7A-null RPE cells infected with AAV2-MYO7A (dual vector) showed that a few cells scattered throughout the culture displayed extremely high levels of MYO7A, while all other cells contained only negligible levels (Figures 14C-14E). Cells overexpressing MYO7A typically had altered morphology, suggesting that high levels of MYO7A may be toxic. Similarly, immunofluorescence of ARPE19 cells infected with AAV2-MYO7A (dual vector) resulted in a few cells strongly labeled with MYO7A antibodies, and most of the cells appear to express only endogenous levels of MYO7A (Figures 14F and 17F).

[0266] Immunolabeling of retinas prepared 3 weeks after subretinal injection with either lot of AAV2-MYO7A (dual vector) also revealed only a small number of RPE and photoreceptor cells with clear MYO7A expression, although significant overexpression was not evident in this in vivo experiment. Immunogold particle counting from images of ultrathin sections was used to quantify the level of MYO7A expression in MYO7A-null retinas treated with the second lot of AAV2-MYO7A (dual vector). Within 1.4 mm of the injection site, MYO7A immunolabeling of the connecting cilia and periciliary regions of photoreceptor cells averaged 48% of that in WT retinas: 2.8 particles / μm (n = 3 retinas) compared with 6.5 particles / μm (n = 3 retinas). The average labeling density in apico-basal sections of the RPE was 35% of that in WT retinas: 31 particles / 100 μm (n = 3 retinas). 2 Compared to 11 particles / 100μm 2 However, it is clear that these lower averages were achieved by some cells expressing near-normal amounts of MYO7A and the majority expressing very little; over half of the cells had fewer than 10 particles / 100 μm 2 (Figure 14G).

[0267] Correction of the MYO7A-mutant phenotype using AAV2 dual vectors. Eyes were analyzed for correction of melanosome localization and ciliary opsin distribution within 1.4 mm of the injection site. Using either lot of AAV2-MYO7A (dual vector), some RPE cells (29% [n = 6 retinas] for lot 1 treatment and 35% [n = 9 retinas] for lot 2 treatment) were observed to have normal apical melanosome distribution; however, most cells in this region retained the MYO7A-mutant phenotype, resulting in a mosaic effect that contained a significantly smaller percentage of corrected cells than observed using a 1:100 dilution of either single vector (Figure 15A). The only correction observed in three eyes injected with a 1:10 dilution of AAV2-MYO7A (dual vector) (first lot) was in 18% of RPE cells in one of the retinas. With full-strength AAV2-MYO7A (dual vector) (second lot), opsin immunogold density averaged 3.2 ± 0.4 particles / μm cilium length, reduced from untreated retinas (4.2 ± 0.8 particles / μm; p = 0.003), but still higher than WT levels (1.1 ± 0.2 particles / μm), suggesting that most cells were not corrected.

[0268] Using immunoelectron microscopy, we identified a correlation between phenotypic correction and MYO7A expression levels (determined by the average concentration of immunogold particles in each RPE cell apical-basal section) (Figures 15B-15E). From eyes injected with AAV2-MYO7A (dual vector) (second lot), we found that corrected RPE cells contained an average of 108% of the WT level of MYO7A (the minimum level was 82%). Uncorrected RPE cells contained an average of 26% of the WT level of MYO7A (the maximum level was 92%). These data indicated that higher expression of MYO7A correlated with phenotypic correction (Figure 15F), but also indicated that a portion of the labeled MYO7A protein was not functional, given that melanosomes were normally localized in mice heterozygous for the MYO7A-null allele and had only approximately 50% of the WT level of MYO7A.

[0269] Expression of MYO7A using simple overlap vectors. AAV2-based simple overlap vectors were evaluated in vitro at various MOIs to assess how the concentration of the vector pair correlates with MYO7A expression. Changes in MYO7A levels over time were also assessed in infected cells. HEK293 cells were infected with simple overlap vector pairs packaged in AAV2(triple YF) vectors (Figure 23A). Preliminary coinfection with AAV2(triple YF) simple overlap vectors (MOI of 10,000 for each vector) demonstrated that MYO7A was expressed and that protein migration on gels was identical to that of full-length transfection controls (Figure 23A). Coinfection with each vector at MOIs of 400, 2000, and 10,000 demonstrates that the efficiency of the simple overlap system is proportional to the amount of 5' and 3' vectors used (Figure 23B). MYO7A expression increased as a function of incubation time in HEK293 cells up to 5 days post-injection (FIG. 23C). The visible decrease in expression was due to a reduction in visible cells in the culture vessel at later times.

[0270] Comparison of fAAV-MYO7A to dual-AAV-MYO7A expression and evaluation of AAV serotype efficiency. Previously, fragmented AAV encoding MYO7A was shown to be able to ameliorate the retinal phenotype in shaker1 mice (Colella et al., 2013; Lopes et al., 2013; Trapani et al., 2013). To provide a basis for comparison, dual-AAV vector expression was evaluated in vitro against fAAV. After infection in HEK293 cells, all dual-vector systems expressed MYO7A more efficiently than fAAV (Figure 24). The AP hybrid platform showed the strongest expression, followed by the simple overlap system.

[0271] Other studies have shown that the transduction efficiency and kinetics of AAV2(triple YF) vectors, relative to conventionally sized DNA payloads, are increased both in vitro and in vivo relative to standard AAV2 (Li et al., 2010; Markusic et al., 2010; Ryals et al., 2011). The efficiency of AAV2 versus AAV2(triple YF) dual vectors was directly compared in HEK293 cells. Surprisingly, standard AAV2-mediated MYO7A expression was higher than that observed with titer-matched AAV2(triple YF) (Figure 24). Identical results were obtained when comparing different AAV2 and AAV2(triple YF) dual vector preparations packaged using the same vector plasmid.

[0272] Comparison of the relative efficiency and specificity of full-length MYO7A expression. To quantitatively assess the relative expression efficiency of the dual-vector platforms and to evaluate the specificity of full-length protein, HEK293 cells were infected with either the combined 5' and 3' AAV2-based vector pair or the corresponding 5' vector alone. An additional hybrid vector pair was included, incorporating a native MYO7A intron sequence (intron 23), which served as an overlapping sequence and provided the appropriate splicing signal. All 5' vectors, except for the simple overlap vector, generated small amounts of a defined, sub-full-length peptide detectable by Western blot (Figure 25A). However, the trans-splicing and AP hybrid platforms showed a clear reduction of this unwanted product when the 3' vector was added to the samples (Figure 25A). The native intron hybrid platform also showed this secondary band on Western blot, again suggesting a truncated protein resulting from the 5' vector alone. In contrast to all other platforms tested, the intensity of this band increased with the addition of the 3' vector. The relative ability of each platform to promote reconstitution was compared by quantifying the amount of 5' vector-mediated truncated protein product in the presence or absence of the respective 3' vector (Figure 25B). Full-length MYO7A expression was then quantified relative to the transfection control by Western blot (Figure 25C). AP hybrid-mediated MYO7A was strongest, followed by simple overlap, trans-splicing, and native intron hybrids (Figure 25C).

[0273] Characterization of the overlap / splice region of expressed MYO7A. To characterize the fidelity of mRNA generated from the dual vector, HEK293 cells were infected with the dual vector, RNA was extracted, reverse transcribed, and subjected to PCR using primers binding upstream of the overlap region and in the bGH polyA signal region to generate a 4.5 kb PCR fragment (Figure 26A). An identically treated sample without reverse transcriptase was used as a control for chromosomal DNA contamination. A plasmid containing the full-length MYO7A coding sequence was used as a positive PCR control. Preliminary screening of AAV-mediated MYO7A mRNA was performed by analyzing the pattern of fragment migration on an agarose gel after restriction endonuclease digestion with PpuMI and BglII (Figure 26A). After digestion of the amplicons from each dual-vector platform tested, identical band patterns consistent with the expected pattern (PpuMI: 1591, 876, 556, 548, 541, 238, 168, 42, and 36 bp; BglII: 1335, 1074, 827, 583, 360, 272, and 146 bp) were observed, indicating that no gross changes (deletions / insertions) occurred as a result of either homologous recombination and / or RNA splicing between the vector pairs (Figure 26B). To further characterize the fidelity of the overlap region, a fragment containing the complete overlap region (1829 bp) was restricted and cloned into pUC57 (Figure 26A). Sequencing results of 10 randomly selected clones per vector platform showed that the overlap region was 100% identical to the consensus / predicted MYO7A sequence (Figure 26C). This demonstrated that homologous recombination was accurate in the context of the simple overlap platform. Furthermore, accurate splicing occurred in the context of the trans-splicing vector. Finally, accurate homologous recombination and / or splicing combinations were achieved for the AP hybrid vector.To determine whether this protocol was capable of detecting aberrant sequences in reconstituted MYO7A, sequences containing either a HindIII recognition site at position 2635 (TAGC) or a point mutation at position 2381 (TC) were also generated.

[0274] Dual-vector-mediated MYO7A expression in the mouse retina. To examine MYO7A expression from the two best-performing in vivo dual-vector platforms, C57BL / 6J mice were inoculated with 1 × 10 per eye of the simple overlap and AP hybrid systems packaged in AAV8(733). 10The vector genome was injected subretinal with the vector and analyzed by Western blot and immunohistochemistry 4 weeks later. The AAV8(733)-fAAV-MYO7A vector was also injected to provide a baseline for comparison. To distinguish between endogenous MYO7A and vector-mediated exogenous expression, a sequence coding for an HA tag was added to the C-prime end of the MYO7A cDNA in all constructs. The resulting retinas were immunostained for HA, revealing that the fAAV vector, along with both dual-vector platforms, mediated MYO7A expression in photoreceptors and RPE. A recent report concluded that simple overlap vectors are more efficient for gene transfer to RPE than photoreceptors (Trapani et al., 2013). Simple overlap-mediated MYO7A expression was observed in both RPE and photoreceptors. In contrast to previous results showing "patchy" MYO7A expression mediated by AAV2-based simple overlap vectors (Lopes et al., 2013), we found that the simple overlap vector, when packaged in AAV8(733), mediated MYO7A expression in the majority of RPE and photoreceptor cells. Photoreceptor degeneration / thinning of the outer nuclear layer was evident in eyes injected with the AP hybrid vector system. Despite the observed degeneration, AP hybrid-mediated MYO7A was clearly detected in the remaining PR cell bodies and RPE, sufficient for detection by immunoblotting. Western blot analysis using an HA antibody revealed that simple overlap-mediated MYO7A was present in just detectable amounts. In contrast, fAAV-mediated protein levels were insufficient to be detected in this assay. Immunoblots of WT mouse retinas using an antibody against MYO7A showed that both endogenous MYO7A and dual vector-mediated, HA-tagged MYO7A migrated similarly.

[0275] Consideration This example demonstrates that dual AAV vectors with defined gene payloads can be used to deliver large transgenes in vitro and in vivo. Initial experiments using the simplest of all dual vector platforms demonstrated that the efficiency of AAV2-based simple overlap vectors was proportional to the amount of 5' and 3' vectors used, and that MYO7A expression mediated by this system increased as a function of incubation time in HEK293 cells. Three separate dual vector platforms were then evaluated in vitro and compared to single, fragmented fAAV vectors. All analyzed dual vectors drove higher levels of MYO7A expression than fAAV. Of all platforms tested, a hybrid vector system containing overlapping recombination product sequences and splice donor / acceptor sites derived from the AP gene (AP hybrid) was the most efficient.

[0276] Regarding the specificity of the dual vector platform in expressing the correct sized gene product, it was noted in vitro that an additional band of lower molecular weight was produced by the trans-splicing and hybrid dual vector platforms as detected by immunoblot (the monoclonal antibody used was raised against the amino-terminal MYO7A). Expression of this truncated protein product was significantly more pronounced for infection with the 5' vector alone.

[0277] After entry into the host cell, the viral capsid is removed, releasing the single-stranded DNA payload. The ITRs held by the single strand act as primers for DNA polymerase, generating a double strand. The resulting circular intermediate consists primarily of monomers, which over time convert to multimeric concatemers through intermolecular recombination (Duan et al., 1998; Yang et al., 1999). The dual vector system disclosed herein utilizes this strategy to achieve full-length protein expression. A limiting factor is the fact that the higher recombination product ITRs flanking the expression cassette are naturally identical, leading to random recombination and, consequently, random orientation of the vector parts relative to each other. This random recombination inevitably results in reduced efficiency, as only concatemers with two vector parts in a 5' to 3' orientation can express full-length proteins. This concatemerization over time is consistent with the observation that combining both the 5' and 3' vectors reduces the amount of single-vector product in favor of full-length proteins. Interestingly, the simple overlap system does not generate truncated products even when only the 5' vector is used for infection. In contrast to trans-splicing and hybrid vectors, there is virtually no intervening sequence between the end of the MYO7A coding sequence and the right ITR.

[0278] Notably, in this disclosure, the sequences in the overlap region of all dual vectors tested in vitro were found to be 100% identical to the consensus / predicted MYO7A sequence, indicating accurate homologous recombination and / or splicing in each dual vector platform.

[0279] Consistent with the in vitro results, the highest levels of MYO7A expression were found in the retinas of mice subretinally injected with AAV8-based AP-hybrid vectors (as assessed by probing for HA on Western blots). Notably, truncated proteins were not evident in retinas expressing either simple overlap or AP-hybrid-mediated MYO7A. The reasons for this observed difference remain to be elucidated but may involve differences in DNA repair mechanisms mediating recombination in actively dividing cells versus postmitotic photoreceptors / RPE (Hirsch et al., 2013). Dual vector-mediated MYO7A-HA expression was observed in WT mice in photoreceptors and RPE, locations where MYO7A is thought to have a functional role (Williams and Lopes, 2011). Significant thinning of the outer nuclear layer was observed in eyes injected with the AP-hybrid vector. It has previously been shown that vector-mediated overexpression of MYO7A leads to retinal toxicity (Hashimoto et al. 2007). Given the high transduction efficiency observed in vitro with the AP hybrid platform, the most likely explanation for the observed pathology is excessive production of MYO7A. Despite significant denaturation, significant amounts of AP hybrid-mediated full-length MYO7A-HA were detected by Western blot. Because high vector concentrations were used in these experiments, a simple solution to avoid cytotoxicity might be to reduce the injected vector genome, replace the strong, ubiquitous smCBA promoter with an endogenous or homologous promoter and / or a promoter with reduced strength, or reduce the expression of undesired products, such as the observed protein expressed from the 5' vector alone in vitro. However, it was noted that only full-size MYO7A-HA was evident in Western blots of AP hybrid-treated retinas.

[0280] Aiming to develop AAV-based treatments for USH1B, animal models of this disease have provided a wealth of useful information. Similar to previous observations that fAAV-MYO7A and simple overlap dual vectors were able to restore melanosome migration and opsin localization in shaker1 mice (Lopes et al., 2013), a recent study from an independent laboratory confirmed the utility of the vectors disclosed herein when it reported that they were able to restore an ultrastructural retinal phenotype in an animal model. Notably, shaker1 mice lack retinal degeneration, a severe functional abnormality seen in USH1B patients (Liu et al., 1997). This fact makes in vivo analysis of therapeutic outcomes in shaker1 retinas problematic. Alternative animal models for evaluating treatments for this devastating disease may be useful in adapting this method for human clinical use.

[0281] The results presented here also demonstrated that MYO7A can be efficiently expressed using a dual-AAV vector system. Both platforms containing overlapping elements, i.e., the simple overlap system and the AP hybrid system, were highly efficient. The AP hybrid vector showed the strongest expression of all systems tested, with little truncated protein observable in vitro and none observed in vivo. The simple overlap vector showed good expression and was the most specific (no truncated protein products were observed), even when a 5'-only vector was used to infect cells. AAV has emerged as a preferred clinical vector, efficiently transducing both photoreceptors and RPE. Because we demonstrated that MYO7A sequence fidelity is maintained after recombination and / or splicing in the dual-AAV vector platform, and because only full-length MYO7A was detectable in mouse retinas injected with the dual vector, the dual-AAV vector strategy presented here represents a reasonable option for the treatment of retinal disorders associated with mutations in large genes, such as USH1B.

[0282] Nucleotide sequences of vectors used in Examples 1 to 5 SEQ ID NO: 1 is the nucleotide sequence of the first generation front half vector of the overlap system (i.e., AAV-smCBA-hMYO7A-NTlong; "hMyo7a-coding overlap vector A");

[0283] SEQ ID NO:2 is the nucleotide sequence of the first generation back-half vector of the overlap system (i.e., AAV-hMYO7A-CTlong.HA; "hMyo7a-coding overlap vector B");

[0284] SEQ ID NO: 3 is the nucleotide sequence of the front half vector of the exon 23 / 24 hybrid vector system (i.e., AAV-smCBA-hMYO7ANT-APSD-Ap head);

[0285] SEQ ID NO: 67 (only the N-myosin 7A portion of SEQ ID NO: 3 (AAV-smCBA-hMYO7ANT-APSD-Ap head))

[0286] SEQ ID NO: 68 (only the N-myosin 7A portion encoded by SEQ ID NO: 3 (AAV-smCBA-hMYO7ANT-APSD-Aphead)) MVILQQGDHVWMDLRLGQEFDVPIGAVVKLCDSGQVQVVDDEDNEHWISPQNATHIKPMHPTSVHGVEDMIRLGDLNEAGILRNLLIRYRDHLIYTYTGSILVAVNPYQLLSIYSPEHIRQYTNKKIGEMPPHIFAIADNCYFNMKRNSRDQCCIISGESGAGKTESTKLILQFLAAISGQHSWIEQQVLEATPILEAFGNAKTIRNDNSSRFGKYIDIHFNKRGAIEGAKIEQYLLEKSRVCRQALDERNYHVFYCMLEGMSEDQKKKLGLGQASDYNYLAMGNCITCEGRVDSQEYANIRSAMKVLMFTDTENWEISKLLAAILHLGNLQYEARTFENLDACEVLFSPSLATAASLLEVNPPDLMSCLTSRTLITRGETVSTPLSREQALDVRDAFVKGIYGRLFVWIVDKINAAIYKPPSQDVKNSRRSIGLLDIFGFENFAVNSFEQLCINFANEHLQQFFVRHVFKLEQEEYDLESIDWLHIEFTDNQDALDMIANKPMNIISLIDEESKFPKGTDTTMLHKLNSQHKLNANYIPPKNNHETQFGINHFAGIVYYETQGFLEKNRDTLHGDIIQLVHSSRNKFIKQIFQADVAMGAETRKRSPTLSSQFKRSLELLMRTLGACQPFFVRCIKPNEFKKPMLFDRHLCVRQLRYSGMMETIRIRRAGYPIRYSFVEFVERYRVLLPGVKPAYKQGDLRGTCQRMAEAVLGTHDDWQIGKTKIFLKDHHDMLLEVERDKAITDRVILLQKVIRGFKDRSNFLKLKNAATLIQRHWRGHNCRKNYGLMRLGFLRLQALHRSRKLHQQYRLARQRIIQFQARCRAYLVRKAFRHRLWAVLTVQAYARGMIARRLHQRLRAEYLWRLEAEKMRLAEEEKLRKEMSAKKAKEEAERKHQERLAQLAREDAERELKEKEAARRKKELLEQMERARHEPVNHSDMVDKMFGFLGTSGGLPGQEGQAPSGFE

[0287] SEQ ID NO: 69 (alkaline phosphatase head sequence (AP head) (e.g., AAV-smCBA-hMYO7A-NT-APSD-AP head, AAV-smCBA-hMYO7A-NT-Ex21-APSD-AP head, AAV-smCBA-hMYO7A-NT-Ex21-APSD-AP headCMv1, AAV-AP head-APSA-hMYO7ACTex22.HA-MIN, AAV-AP head-APSA-hMYO7ACTex22-MIN) CCCCGGGTGCGCGGCGTCGGTGGTGCCGGCGGGGCGCCAGGTCGCAGGCGGTGTAGGGCTCCAGGCAGGCGGCGAAGGCCATGACGTGCGCTATGAAGGTCTGCTCCTGCACGCCGTGAACCAGGTGCGCCTGCGGGCCGC GCGCGAACACCGCCACGTCCTCGCCTGCGTGGGTCTCTTCGTCCAGGGGCACTGCTGACTGCTGCCGATACTCGGGGCTCCCGCTCTCGCTCTCGGTAACATCCGGCCGGGCGCCGTCCTTGAGCACATAGCCTGGACCGTTTC

[0288] SEQ ID NO: 70 alkaline phosphatase head sequence (AP head) (e.g., AAV-smCBA-hMYO7A-NT-ex21-APSD-AP head-CMv2, AAV-smCBA-hMYO7A-NT-ex21-APSD-AP head-CMv3, AAV-APhead-APSA-hMYO7ACT-ex22-APSD-CMv2.HA, AAV-APhead-APSA-hMYO7ACT-ex22-APSD-CMv2.HA-MIN, AAV-APhead-APSA-hMYO7ACT-ex22-APSD-CMv2-MIN) CCCCGGGTGCGCGGCGTCGGTGGTGCCGGCGGGGCGCCAGGTCGCAGGCGGTGTAGGGCTCCAGGCAGGCGGCGAAGGCCATGACGTGCGCTATGAAGGTCTGCTCCTGCACGCCGTGAACCAGGTGCGCCTGCGGGCCGC GCGCGAACACCGCCACGTCCTCGCCTGCGTGGGTCTCTTCGTCCAGGGGCACTGCGCACTGCTGCCGATACTCGGGGCTCCCGCTCTCGCTCTCGGTAACATCCGGCCGGGCGCCGTCCTTGAGCACATAGCCTGGACCGTTTC

[0289] SEQ ID NO: 4 is the nucleotide sequence of the back-half vector of the exon 23 / 24 hybrid vector system (i.e., AAV-APhead-APSA-hMYO7ACT.HA);

[0290] SEQ ID NO: 71 Myosin 7A (e.g., AAV-APhead-APSA-hMYO7ACT.HA)

[0291] SEQ ID NO: 72 (Hemagglutinin (HA) tag (AAV-AP head-APSA-hMYO7ACT.HA, AAV-hMYO7A-CTlong.HA) TACCCTTACGATGTACCGGATTACGCATGA [Example 6]

[0292] Example 6 presents a second generation hybrid vector that minimizes expression of a truncated MYO7A protein.

[0293] The hybrid and simple overlapping front-half vectors described in Examples 1-5 contained MYO7A cDNA sequences encoding a portion of the MYO7A protein tail domain. The tail domain of MYO7A is known for its ability to bind other cellular proteins. Examples 1-5 of the present application demonstrate that the original hybrid front-half vector was capable of encoding MYO7A protein (Figures 28-30) (Dyka, et al. 2014). However, after injection of the original hybrid front-half vector into mouse retinas, a partial loss of retinal structure / function was observed. This loss of retinal structure / function was hypothesized to be the result of a gain of function exerted by a truncated MYO7A protein containing the partial tail domain. The truncated MYO7A protein was shown to be produced solely from the front-half vector (Figures 31-32). As a control, the expression levels of any truncated proteins encoded by the back-half vectors were also measured. These results demonstrate that the backhalf vector does not generate truncated products and does not lead to loss of retinal structure / function (Figure 33).

[0294] To eliminate the functional toxicity associated with truncated MYO7A protein fragments, we developed improved second-generation hybrid and simple overlap vectors aimed at eliminating the formation of truncated proteins from the front-half vector. Although the previously developed simple overlap vector did not produce observable amounts of truncated MYO7A protein or show observable loss of structure / function after its injection in mice, we nevertheless developed the second-generation simple overlap vector along with the second-generation hybrid vector. This was done as a precaution to avoid the possibility that the original simple overlap front-half vector might express truncated MYO7A at levels undetectable by immunoblotting and tolerability studies in mice.

[0295] In both the hybrid and simple overlap platforms, the sequence corresponding to the tail domain from the front-half vector was moved to the back-half vector. The MYO7A protein is an actin-based molecular motor, and its N-terminus (head) contains the actin-binding and ATP-binding sites. The 5IQ (neck) contains a single alpha-helix (SAH) stabilized by calmodulin and acting as a lever. The C-terminal (tail) domain of the MYO7A protein determines its functional specificity. Notably, in the hybrid vector, the "split point" was moved from exons 23 / 24 in the original vector to exons 21 / 22 in the second generation (Figures 34 and 50A). As a result, the second-generation vector system has a split point moved from one side of the single alpha-helix (SAH) to the other. This new split between exons 21 and 22 is located between the 5IQ (neck) and the SAH.

[0296] In the simple overlap vector, the amount of overlapping sequence was reduced such that no sequence corresponding to the tail domain remained in the front vector (this new vector construct is hereafter referred to as the "second generation overlap"). The second generation overlap vector therefore contained a shorter segment of overlapping sequence such that the overlap ended at the split point between exons 21 and 22. The second generation hybrid and second generation overlap vectors described herein ensure that no part of the tail domain is encoded by either the hybrid front- or simple overlap front-half vectors. The vector system was modified in this way to reduce the production of truncated MYO7A proteins.

[0297] When second-generation hybrid vectors containing the exon 21 / 22 splitpoint were administered to HEK293 cells in vitro, truncated MYO7A proteins of smaller size corresponding to the alterations in the vector sequence were observed (Figures 35 and 39). - / - When administered to mice by subretinal injection, a smaller size of MYO7A protein was also observed (Figures 36 and 38). The second-generation hybrid vector containing the exon 21 / 22 split point expresses equivalent amounts of full-length MYO7A compared to the original hybrid vector containing the exon 23 / 24 split point (see, e.g., Figures 35 and 36), but does not produce the MYO7A tail protein fragment observed in the original hybrid vector (Figure 38). Overall, it is demonstrated herein that the second-generation hybrid and simple overlap vectors, which do not express the tail domain sequence in the front-half vector, result in a MYO7A protein product that is better tolerated in the retina.

[0298] Front-half AAV-MYO7A vectors (both hybrid and simple overlap) contain a promoter and inverted terminal repeats (ITRs). It is possible that the ITRs, together with the promoter, provide a polyadenylation signal that leads to the generation and maturation of messenger RNA for translation. In the context of hybrid vectors, it is also possible that alkaline phosphatase (AP) splice donor and AP head "intron" sequences may also facilitate mRNA maturation by providing splicing signals. While the exact mechanism for promoting mRNA maturation is unclear, it is observable that hybrid front-half vectors produce truncated proteins (Figures 31-33).

[0299] Although the second-generation hybrid vector is demonstrated herein to result in reduced production of unwanted products, further improvements were made to the vector to reduce the production of truncated MYO7A, thereby further increasing safety and efficiency. In the hybrid vector containing the second-generation exon 21 / 22 split point described above, potential in-frame stop codons located downstream of the MYO7A sequence were removed from the front hybrid vector plasmid (Figure 42). The single-nucleotide substitutions that remove these potential stop codons are designed to utilize the cell's "non-stop" decay mechanism, thereby eliminating spurious RNA before it can be translated. The in-frame stop codons were removed in two steps. First, three potential stop codons within the AP splice donor sequence were removed from the second-generation hybrid vector to create a further improved vector that still contained the exon 21 / 22 split point ("CMv1 hybrid"; SEQ ID NOs: 33 and 32; Figure 42).

[0300] Additional potential in-frame stop codons were located within the AP region of the shared recombination product. Therefore, separate, further improved vectors were generated in which functional improvements were made to both the front-half and back-half second-generation hybrid vectors ("CMv2 hybrid"; SEQ ID NOs: 34 and 35) containing the exon 21 / 22 split point. The CMv2 hybrid front-half vector has three potential stop codons located in the AP splice donor sequence and one potential stop codon located in the AP head recombination product sequence. The CMv2 hybrid back-half vector has the same changes made in the AP head recombination product sequence to correspond to the front-half vector.

[0301] When modified to produce a CMv2 hybrid back-half vector as described herein, the CMv2 hybrid back-half vector approaches, but does not exceed, the packaging limit of the AAV vector construct. To mitigate the possibility that the back-half vector is too large, a construct was designed in which the CMv2 hybrid back-half vector was modified to remove any extraneous sequences present between elements in the construct, as well as the HA sequence. Once modified, the resulting back-half vector is designated the CMv2.1 hybrid back-half vector (SEQ ID NO: 44).

[0302] The CMv1 and CMv2 hybrid vectors were tested in vitro by transfecting these plasmids into HEK293 cells. For comparison, a first-generation hybrid front-half vector ("original") and a second-generation hybrid front-half vector ("ex21 / 22") were also used (Figure 40). It was found that the CMv1 hybrid front-half vector still produced truncated MYO7A. However, the CMv2 hybrid front-half vector did not produce any truncated protein. Vinculin (VCL) was used as a loading control in all samples.

[0303] Corresponding changes were made in the simple overlap vector, albeit without the presence of a truncated protein encoded by this vector (Figure 43). Because the putative stop codon removed in the CMv1 and CMv2 hybrid vectors is not present in the front half of the simple overlap vector, a slightly different modification was made to the simple overlap vector. In the CMv1 overlap vector, the simple overlap vector was modified in the 3' untranslated region between the MYO7A partial coding sequence and the 3' AAV ITR to remove a potential in-frame stop codon ("CMv1 overlap"; SEQ ID NOs: 36 and 38; Figure 43).

[0304] As shown in Figure 41, AAV-mediated MYO7A transcripts were expressed in the macaque retina, demonstrating the tolerability of dual AAV5-MYO7A vectors injected subretinal into the macaque retina, which was evaluated 2 months after injection.

[0305] These data show considerable promise for application of the technology to human patients. [Example 7]

[0306] Example 7 provides a third generation hybrid vector. Third generation overlap vector

[0307] An improved third-generation (V3) overlap vector pair was created by modifying the overlapping region of the MYO7A coding sequence. This V3 overlap pair consists of a front half vector ("AAV-smCBA-hMYO7A-NTlong-v3") containing the nucleotide sequence of SEQ ID NO:50 and a back half vector ("AAV-smCBA-hMYO7A-CTlong-v3.HA") containing the nucleotide sequence of SEQ ID NO:51. This V3 overlap pair contains an N-terminal myosin 7A coding sequence containing SEQ ID NO:66 and a C-terminal myosin 7A coding sequence containing SEQ ID NO:80. These vectors contain shortened overlapping region lengths (see Figures 46 and 47), e.g., overlapping region lengths of 945 bp and 687 bp. Reducing the length of the overlapping region has the effect of reducing the overall size of the vector genome.

[0308] The V3 overlapping dual vector system was shown to result in increased levels of full-length MYO7A protein compared to the original vector, as quantified by the Protein Simple Jess system, as shown in Figures 48A and 49A. The overlapping vector containing 687 bp and 945 bp of overlapping MYO7A sequence provided optimal expression levels.

[0309] As shown in Figure 49B, the V3 overlap vector does not generate appreciable amounts of undesired truncated MYO7A fragments, even when the overlapping region length remains greater than 361 bp. Therefore, reducing the overlap length to a certain point ensures that none of the vector genomes overwhelms the packaging capacity of the AAV capsid (4.7-4.9 kb), leading to increased expression of full-length MYO7A. If the overlap length is too small (≦361 bp), full-length MYO7A expression may be reduced and truncated proteins may appear.

[0310] Due to the shorter length of their overlapping regions, these improved third-generation overlap vectors contain shorter ITR-to-ITR (ITR-ITR) lengths (see Figure 48B). In certain embodiments, the length between the inverted terminal repeats at each end of the first AAV vector polynucleotide is about 4615 nucleotides (nt) or less. The ITR-ITR length in the improved first vector polynucleotide is 4615 nt. In certain embodiments, the length between the inverted terminal repeats at each end of the second AAV vector polynucleotide is about 4800 nt or less. The ITR-ITR length in the improved second vector polynucleotide is about 4560 nt.

[0311] The hMYO7A overlap region, e.g., SEQ ID NOS: 39 and 53-59, can be used as overlapping polynucleotide sequences in additional overlapping dual vectors expressing large genes (other than MYO7A). In particular, overlapping dual vectors expressing large genes other than MYO7A and comprising nucleotide sequences having at least 80%, 85%, 90%, 95%, 98%, or 99% identity to any one of SEQ ID NOS: 39 and 53-59, e.g., SEQ ID NOS: 56 or 57, are disclosed herein. In certain embodiments, these overlapping dual vectors comprise the nucleotide sequence of any one of SEQ ID NOS: 39 and 53-59, e.g., SEQ ID NOS: 56 or 57, and express a large gene selected from ABCA4, CEP290, EYS, RP1, ALMS1, CDH23, PCDH15, USH1C, USH1G, USH2A, DNFB31, DMD, CFTR, GDE, DYSF, F8, and DFNB2. In some embodiments, these overlapping vectors contain two overlapping sequences disclosed herein, for example, mutually exclusive sequences SEQ ID NOs: 39 and 56 or mutually exclusive sequences SEQ ID NOs: 39 and 57.

[0312] rAAV viral particles containing a pair of V3 overlap vectors with overlapping region lengths of 687 bp and 945 bp are packaged and administered to retinal cells. rAAV viral particles containing a pair of V3 overlap vectors with overlapping region lengths of 687 bp and 945 bp are packaged and administered to auditory hair cells.

[0313] Overlap vectors containing the following pairs of nucleotide sequences encoding myosin 7A are evaluated for their ability to produce full-length MYO7A polypeptide in vitro or in vivo: In vitro evaluation can be performed using Protein Simple Jess Western blotting. the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 63 and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 83; the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 63 and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 90; the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 101 and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 83; the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 101 and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 90; the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 66 and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 83; and The first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 66, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO: 90.

[0314] Thus, in some embodiments, provided herein is a polynucleotide vector system wherein a first AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:63, and a second AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:83. In some embodiments, the second AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:90.

[0315] In some embodiments, the first AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 101. In some embodiments, the first AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 66. In some embodiments, provided herein are polynucleotide vector systems, wherein the first AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 66, and the second AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 90.

[0316] The following overlap vectors are evaluated for their ability to produce full-length MYO7A polypeptide in vitro or in vivo. In vitro evaluation can be performed using Protein Simple Jess Western blotting. the first AAV vector comprises the nucleotide sequence of SEQ ID NO: 50 and the second AAV vector comprises the nucleotide sequence of SEQ ID NO: 51; the first AAV vector comprises the nucleotide sequence of SEQ ID NO: 50 and the second AAV vector comprises the nucleotide sequence of SEQ ID NO: 38; the first AAV vector comprises the nucleotide sequence of SEQ ID NO: 1 and the second AAV vector comprises the nucleotide sequence of SEQ ID NO: 38; the first AAV vector comprises the nucleotide sequence of SEQ ID NO: 50 and the second AAV vector comprises the nucleotide sequence of SEQ ID NO: 2; the first AAV vector comprises the nucleotide sequence of SEQ ID NO: 1 and the second AAV vector comprises the nucleotide sequence of SEQ ID NO: 51; the first AAV vector comprises the nucleotide sequence of SEQ ID NO: 36 and the second AAV vector comprises the nucleotide sequence of SEQ ID NO: 2; the first AAV vector comprises the nucleotide sequence of SEQ ID NO: 36 and the second AAV vector comprises the nucleotide sequence of SEQ ID NO: 38; the first AAV vector comprises the nucleotide sequence of SEQ ID NO: 36 and the second AAV vector comprises the nucleotide sequence of SEQ ID NO: 51; the first AAV vector comprises the nucleotide sequence of SEQ ID NO: 37 and the second AAV vector comprises the nucleotide sequence of SEQ ID NO: 38; The first AAV vector comprises the nucleotide sequence of SEQ ID NO: 37 and the second AAV vector comprises the nucleotide sequence of SEQ ID NO: 51.

[0317] Thus, in some embodiments, provided herein is a polynucleotide vector system in which a first AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:50, and a second AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:51. In some embodiments, the second AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:38. In some embodiments, the second AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:2.

[0318] In some embodiments, the first AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In some embodiments, the first AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 36. In some embodiments, the first AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 37. In some embodiments, provided herein is a polynucleotide vector system wherein a first AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:37, and a second AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:38 or SEQ ID NO:51.

[0319] Third-generation hybrid vectors An improved third-generation (CMv3) hybrid system or vector pair was generated by making substitutions in three putative stop codons in the 3' untranslated region (UTR) of the front-half vector of the CMv2 hybrid system. As such, the CMv3 hybrid front-half vector contains substitutions (i.e., removal) of one in-frame stop codon in the AP head sequence, three in-frame stop codons in the AP intron sequence, and three in-frame stop codons in the 3' UTR sequence. In an exemplary embodiment, the CMv3 hybrid system is a CMv3 MIN system in which any remaining unseeded legacy sequences (e.g., restriction enzyme sites) have been removed.

[0320] An exemplary CMv3 hybrid system consists of i) a front half vector comprising the nucleotide sequence of SEQ ID NO:46 ("AAV-smCBA-hMYO7A-NTex21-APSD-APhead-CMv3") and ii) a CMv2 back half vector comprising the nucleotide sequence of SEQ ID NO:35 ("AAV-AP-head-APSA-ex22hMYO7A-CT.HA-CMv2"), a CMv2.1 back half vector comprising the nucleotide sequence of SEQ ID NO:44 ("AAV-APhead-APSA-hMYO7ACTex22-CMv2.1"), or a minimized version of either vector, e.g., CMv2 (or V2-)backMIN comprising the nucleotide sequence of SEQ ID NO:49.

[0321] To generate the hybrid-CMv3 backMIN and hybrid-CMv2 backMIN vectors, "unnecessary legacy" sequences were removed from the back half vectors to ensure that the vector size did not exceed the packaging capacity of the AAV capsid. Due to the removal of unseeded legacy sequences from the back, these improved third-generation hybrid vectors contain shorter ITR-ITR (ITR-ITR) lengths (see Figures 50C and 51B). In certain embodiments, the length between the inverted terminal repeat sequences at each end of the first AAV vector polynucleotide in the hybrid vector is approximately 4279 nucleotides (nt) or less. This has the effect of minimizing the length of the back half vector, thereby improving packaging efficiency. The hybrid-CMv3 backMIN is 122 bp smaller than the related original vector (4981 vs. 4859 bp), and the hybrid-CMv2 backMIN is 121 bp smaller than the related original vector (4982 vs. 4861 bp). For the hybrid-V2BacMIN HA and hybrid-CMV2BacMIN HA vectors, a hemagglutinin (HA) tag was added to the minimized buck half vectors (hybrid-V2BacMIN and hybrid-CMV2BacMIN) to enable detection in normal monkeys.

[0322] The CMv3 hybrid vector system was shown to produce relatively low levels of truncated MYO7A fragments compared to the hybrid CMv1 and hybrid CMv2 vectors, as shown in Figure 50D. Furthermore, pairing the hybrid CMv3 front half vector with the CMv3 MIN back half vector produced levels of full-length MYO7A equal to or greater than those seen with the original (first generation) hybrid vector, as shown in Figure 51C and 51D. This vector pair produced relatively low levels of truncated fragments, as shown in Figure 51E.

[0323] The inventors have also discovered that the hMY07A sequence can be used as an intron sequence to mediate recombination in cells after administration in hybrid dual vectors expressing large genes (other than MYO7A). Such hybrid vectors are generated to contain one or more overlapping regions identified by the improvements to the MYO7A overlap vectors provided herein (such vectors may or may not contain AP head and / or AP intron sequences that would allow insertion of one of these overlapping regions). In particular, disclosed herein are hybrid dual vectors containing sequences between the first and second introns of the first and second AAV vector polynucleotides, respectively, that comprise a nucleotide sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity to any one of SEQ ID NOS: 39 and 52-59. In certain embodiments, hybrid dual vectors contain sequences between the first and second introns of the first and second AAV vector polynucleotides, respectively, comprising the nucleotide sequences of any one of SEQ ID NOS: 39 and 53-59, e.g., SEQ ID NOS: 56 or 57. Additional large genes can be delivered using these hybrid vectors, including ABCA4, CEP290, EYS, RPI, ALMS1, CDH23, PCDH15, USH1C, USH1G, USH2A, DNFB31, DMD, CFTR, GDE, DYSF, F8, and DFNB2. In some embodiments, these hybrid dual vectors contain two overlapping sequences disclosed herein, e.g., the mutually exclusive sequences SEQ ID NOS: 39 and 56, or the mutually exclusive sequences SEQ ID NOS: 39 and 57.

[0324] rAAV viral particles containing a CMv3 hybrid vector pair comprising a SEQ ID NO:46 front half vector and a SEQ ID NO:35 back half vector are packaged and administered to retinal cells. rAAV viral particles containing a CMv3 hybrid vector pair comprising a SEQ ID NO:46 front half vector and a SEQ ID NO:35 back half vector are packaged and administered to auditory hair cells.

[0325] Hybrid vectors containing the following pairs of nucleotide sequences encoding myosin 7A are evaluated for their ability to produce full-length MYO7A polypeptide in vitro or in vivo: the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO:73 and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO:75.

[0326] Thus, in some embodiments, provided herein is a polynucleotide vector system wherein a first AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 73, and a second AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 75.

[0327] Hybrid vector systems containing the following pairs of vector sequences are evaluated for their ability to produce full-length MYO7A polypeptide in vitro or in vivo. the first AAV vector comprises the nucleotide sequence of SEQ ID NO: 31 and the second AAV vector comprises the nucleotide sequence of SEQ ID NO: 32; the first AAV vector comprises the nucleotide sequence of SEQ ID NO: 46 and the second AAV vector comprises the nucleotide sequence of SEQ ID NO: 35; the first AAV vector comprises the nucleotide sequence of SEQ ID NO: 46 and the second AAV vector comprises the nucleotide sequence of SEQ ID NO: 49; the first AAV vector comprises the nucleotide sequence of SEQ ID NO: 34 and the second AAV vector comprises the nucleotide sequence of SEQ ID NO: 47; the first AAV vector comprises the nucleotide sequence of SEQ ID NO: 31 and the second AAV vector comprises the nucleotide sequence of SEQ ID NO: 48; the first AAV vector comprises the nucleotide sequence of SEQ ID NO: 31 and the second AAV vector comprises the nucleotide sequence of SEQ ID NO: 49; the first AAV vector comprises the nucleotide sequence of SEQ ID NO: 33 and the second AAV vector comprises the nucleotide sequence of SEQ ID NO: 32; the first AAV vector comprises the nucleotide sequence of SEQ ID NO: 34 and the second AAV vector comprises the nucleotide sequence of SEQ ID NO: 35; The first AAV vector comprises the nucleotide sequence of SEQ ID NO: 34 and the second AAV vector comprises the nucleotide sequence of SEQ ID NO: 44.

[0328] Thus, in some embodiments, provided herein are polynucleotide vector systems in which a first AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:31, and a second AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:32. In some embodiments, the second AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:48 or SEQ ID NO:49. In some embodiments, the first AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:33.

[0329] In some embodiments, provided herein are polynucleotide vector systems in which a first AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:46, and a second AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:35. In some embodiments, the second AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:49. In some embodiments, the first AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:34.

[0330] In some embodiments, provided herein is a polynucleotide vector system wherein a first AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 34, and a second AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 47. In some embodiments, the second AAV vector polynucleotide comprises a nucleotide sequence that is at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 35 or 44.

[0331] The polynucleotide vector systems of the present disclosure may comprise a nucleotide sequence that is at least 80%, 85%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to any of the following sequences (e.g., any of SEQ ID NOs: 31-38, 44, and 46-51). In some embodiments, the vector comprises a sequence comprising any one of SEQ ID NOs: 31-38, 44, and 46-51. In some embodiments, the vector systems of the present disclosure comprise a nucleotide sequence that contains 1, 2, 3, 4, 5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, or more than 50 nucleotides different from any of the sequences of SEQ ID NOs: 31-38, 44, and 46-51. The disclosed vectors may differ in any of the following vector sequences by the presence or absence of a tag, e.g., an HA tag. The disclosed vectors contain a stretch of 5-10, 10-15, 15-20, 20-25, 25-35, 35-45, 45-60, 60-75, or more than 75 consecutive nucleotides in common with any of SEQ ID NOs: 31-38, 44, and 46-51.

[0332] The myosin 7a-encoding sequence of any of the polynucleotide vectors provided herein can comprise a nucleotide sequence that is at least 80%, 85%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to any of the following hMyosin 7a-encoding sequences in the N-terminal (front half, "-NT") or C-terminal (back half, "-CT") vectors, e.g., SEQ ID NOs: 63, 66, 73, 75, 77, 80, and 90. The disclosed N-terminal and C-terminal myosin 7a-encoding sequences can contain a stretch of 0.5-10, 10-15, 15-20, 20-25, 25-35, 35-45, 45-60, 60-75, or more than 75 contiguous nucleotides in common with any of SEQ ID NOs: 63, 66, 73, 75, 77, 80, and 90.

[0333] The polynucleotide vectors and myosin 7a-encoding sequences provided herein include a nucleotide sequence that differs by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 20-25, or more than 25 nucleotides from any of the following sequences (e.g., any of the vectors of SEQ ID NOs: 31-38, 44, and 46-51; or any of the hMyosin 7a-encoding sequences in the N-terminal (front half, "-NT") or C-terminal (back half, "-CT") vectors set forth as SEQ ID NOs: 63, 66, 73, 75, 77, 80, and 90). The polynucleotide vectors provided herein can comprise a nucleotide sequence that differs from any of the following sequences by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 nucleotides at or near the 5' end of the vector, and can differ by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 nucleotides at or near the 3' end of the vector. The vectors provided herein can comprise truncations of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides at the 5' or 3' end.

[0334] Nucleotide sequences of the vectors provided in Examples 6 and 7 SEQ ID NO:31 is the nucleotide sequence of the second generation hybrid front half vector (i.e., AAV-smCBA-hMYO7A-NT-Ex21-APSD-APhead):

[0335] Array number 73 (hMYO7A-NT (AAV-smCBA-hMYO7A-NT-Ex21-APSD-AP head), (AAV-smCBA-hMYO7A-NT-Ex21-APSD-AP head CMv1), (AAV-smCBA-hMYO7A-NT-Ex21-APSD-AP head CMv2), (AAV-smCBA-hMYO7A-NTex21-APSD-AP head-CMv3))

[0336] Array number 74 (hMYO7A-NT (AAV-smCBA-hMYO7A-NT-Ex21-APSD-AP head), (AAV-smCBA-hMYO7A-NT-Ex21-APSD-AP head CMv1), (AAV-smCBA-hMYO7A-NT-Ex21-APSD-AP head-CMv3)) MVILQQGDHVWMDLRLGQEFDVPIGAVVKLCDSGQVQVVDDEDNEHWISPQNATHIKPMHPTSVHGVEDMIRLGDLNEAGILRNLLIRYRDHLIYTYTGSILVAVNP YQLLSIYSPEHIRQYTNKKIGEMPPHIFAIADNCYFNMKRNSRDQCCIISGESGAGKTESTKLILQFLAAISGQHSWIEQQVLEATPILEAFGNAKTIRNDNSSRFGK YIDIHFNKRGAIEGAKIEQYLLEKSRVCRQALDERNYHVFYCMLEGMSEDQKKKLGLGQASDYNYLAMGNCITCEGRVDSQEYANIRSAMKVLMFTDTENWEISKLLA AILHLGNLQYEARTFENLDACEVLFSPSLATAASLLEVNPPDLMSCLTSRTLITRGETVSTPLSREQALDVRDAFVKGIYGRLFVWIVDKINAAIYKPPSQDVKNSRR SIGLLDIFGFENFAVNSFEQLCINFANEHLQQFFVRHVFKLEQEEYDLESIDWLHIEFTDNQDALDMIANKPMNIISLIDEESKFPKGTDTTMLHKLNSQHKLNANY IPPKNNHETQFGINHFAGIVYYETQGFLEKNRDTLHGDIIQLVHSSRNKFIKQIFQADVAMGAETRKRSPTLSSQFKRSLELLMRTLGACQPFFVRCIKPNEFKKPML FDRHLCVRQLRYSGMMETIRIRRAGYPIRYSFVEFVERYRVLLPGVKPAYKQGDLRGTCQRMEAVLGTHDDWQIGKTKIFLKDHHDMLLEVERDKAITDRVILLQKV IRGFKDRSNFLKLKNAATLIQRHWRGHNCRKNYGLMRLGFLRLQALHRSRKLHQQYRLARQRIIQFQARCRAYLVRKAFRHRLWAVLTVQAYARGMIARRLHQRLRAE

[0337] SEQ ID NO: 32 is the nucleotide sequence of the second generation hybrid backhalf vector (i.e., AAV-APhead-APSA-ex22hMYO7A-CT.HA (with hemagglutinin (HA) tag)):

[0338] 75x22hMYO7A(AAV-APヘッド-APSA-ex22hMYO7A-CT.HA、AAV-APヘッド-APSA-ex22hMYO7A-HA-CT.AACM-2 V-APヘッド-APSA-hMYO7ACTex22-CMv2.1.HA、AAV-APヘッド-APSA-hMYO7ACTex22-CMv2、AAV-APヘッド-APSA-hMYO7ACTex 22.HA-MIN、AAV-APヘッド-APSA-hMYO7ACTex 22-MIN))

[0339] SEQ ID NO: 76, ex22hMYO7A (e.g., AAV-APhead-APSA-ex22hMYO7A-CT.HA, AAV-APhead-APSA-ex22hMYO7A-CT.HA-CMv2, AAV-APhead-APSA-hMYO7ACTex22-CMv2.1.HA)

[0340] SEQ ID NO: 33 is the nucleotide sequence of the CMv1 hybrid front half vector (i.e., AAV-smCBA-hMYO7A-NT-Ex21-APSD-APheadCMv1):

[0341] SEQ ID NO: 34 is the nucleotide sequence of the CMv2 hybrid front half vector (i.e., AAV-smCBA-hMYO7A-NT-Ex21-APSD-APheadCMv2):

[0342] SEQ ID NO: 35 is the nucleotide sequence of the CMv2 hybrid backhalf vector (i.e., AAV-AP-head-APSA-ex22hMYO7A-CT.HA-CMv2):

[0343] SEQ ID NO: 36 is the nucleotide sequence of the CMv1 overlap front half vector (i.e., AAV-smCBA-hMYO7A-noDimNT-CMv1):

[0344] SEQ ID NO:62 (peptide encoded by the N-myosin 7A portion of SEQ ID NO:36 (AAV-smCBA-hMYO7A-noDimNT-CMv1)) MVILQQGDHVWMDLRLGQEFDVPIGAVVKLCDSGQVQVVDDEDNEHWISPQNATHIKPMHPTSVHGVEDMIRLGDLNEAGILRNLLIRYRDHLIYTYTGSILVAVNPYQL LSIYSPEHIRQYTNKKIGEMPPHIFAIADNCYFNMKRNSRDQCCIIISGESGAGKTESTKLILQFLAAISGQHSWIEQQVLEATPILEAFGNAKTIRNDNSSRFGKYIDIH FNKRGAIEGAKIEQYLLEKSRVCRQALDERNYHVFYCMLEGMSEDQKKKLGLGQASDYNYLAMGNCITCEGRVDSQEYANIRSAMKVLMFTDTENWEISKLLAAILHLGN LQYEARTFENLDACEVLFSPSLATAASLLEVNPPDLMSCLTSRTLITRGETVSTPLSREQALDVRDAFVKGIYGRLFVWIVDKINAAIYKPPSQDVKNSRRSIGLLDIFG FENFAVNSFEQLCINFANEHLQQFFVRHVFKLEQEEYDLESIDWLHIEFTDNQDALDMIANKPMNIISLIDEESKFPKGTDTTMLHKLNSQHKLNANYIPPKNNHETQFG INHFAGIVYYETQGFLEKNRDTLHGDIIQLVHSSRNKFIKQIFQADVAMGAETRKRSPTLSSQFKRSLELLMRTLGACQPFFVRCIKPNEFKKPMLFDRHLCVRQLRYSG MMETIRIRRAGYPIRYSFVEFVERYRVLLPGVKPAYKQGDLRGTCQRMEAVLGTHDDWQIGKTKIFLKDHHDMLLEVERDKAITDRVILLQKVIRGFKDRSNFLKLKNA ATLIQRHWRGHNCRKNYGLMRLGFLRLQALHRSRKLHQQYRLARQRIIQFQARCRAYLVRKAFRHRLWAVLTVQAYARGMIARRLHQRLRAEYLWRLEAEKMRLAEEEKL

[0345] SEQ ID NO: 63 (only the N-myosin 7A portion of SEQ ID NO: 36 (AAV-smCBA-hMYO7A-noDimNT-CMv1))

[0346] SEQ ID NO: 37 is the nucleotide sequence of the second generation overlapping front half vector (i.e., AAV-smCBA-hMYO7A-noDIM-NTlong):

[0347] SEQ ID NO: 90 (only the N-myosin 7A portion of SEQ ID NO: 37 (AAV-smCBA-hMYO7A-noDIM-NTlong))

[0348] SEQ ID NO: 91 (only the N-myosin 7A portion of SEQ ID NO: 37 (AAV-smCBA-hMYO7A-noDIM-NTlong)) MVILQQGDHVWMDLRLGQEFDVPIGAVVKLCDSGQVQVVDDEDNEHWISPQNATHIKPMHPTSVHGVEDMIRLGDLNEAGILRNLLIRYRDHLIYTYTGSILVAVNPYQL LSIYSPEHIRQYTNKKIGEMPPHIFAIADNCYFNMKRNSRDQCCIIISGESGAGKTESTKLILQFLAAISGQHSWIEQQVLEATPILEAFGNAKTIRNDNSSRFGKYIDIH FNKRGAIEGAKIEQYLLEKSRVCRQALDERNYHVFYCMLEGMSEDQKKKLGLGQASDYNYLAMGNCITCEGRVDSQEYANIRSAMKVLMFTDTENWEISKLLAAILHLGN LQYEARTFENLDACEVLFSPSLATAASLLEVNPPDLMSCLTSRTLITRGETVSTPLSREQALDVRDAFVKGIYGRLFVWIVDKINAAIYKPPSQDVKNSRRSIGLLDIFG FENFAVNSFEQLCINFANEHLQQFFVRHVFKLEQEEYDLESIDWLHIEFTDNQDALDMIANKPMNIISLIDEESKFPKGTDTTMLHKLNSQHKLNANYIPPKNNHETQFG INHFAGIVYYETQGFLEKNRDTLHGDIIQLVHSSRNKFIKQIFQADVAMGAETRKRSPTLSSQFKRSLELLMRTLGACQPFFVRCIKPNEFKKPMLFDRHLCVRQLRYSG MMETIRIRRAGYPIRYSFVEFVERYRVLLPGVKPAYKQGDLRGTCQRMEAVLGTHDDWQIGKTKIFLKDHHDMLLEVERDKAITDRVILLQKVIRGFKDRSNFLKLKNA ATLIQRHWRGHNCRKNYGLMRLGFLRLQALHRSRKLHQQYRLARQRIIQFQARCRAYLVRKAFRHRLWAVLTVQAYARGMIARRLHQRLRAEYLWRLEAEKMRLAEEEKL

[0349] SEQ ID NO: 38 is the nucleotide sequence of the second generation overlap-back half vector (i.e., AAV-hMYO7A-CTlong-v2.HA).

[0350] SEQ ID NO: 77 (C-terminal myosin 7A (e.g., AAV-hMYO7A-CTlong-v2.HA))

[0351] SEQ ID NO: 78 (N-terminal myosin 7A (e.g., AAV-hMYO7A-CTlong.HA)

[0352] In some embodiments, the vectors provided herein comprise a truncated chimeric CBA promoter. The vectors provided herein may comprise a promoter having a nucleotide sequence that differs by 1, 2, or 3 nucleotides from the smCBA promoter set forth as SEQ ID NO: 64. In some embodiments, the vectors provided herein comprise a promoter that is not a smCBA promoter. In some embodiments, the vectors provided herein comprise a promoter selected from the group consisting of a CMV promoter, an EF-1 alpha promoter, a cone arrestin promoter, a human myosin 7a gene-derived promoter, a TαC gene-derived promoter, a rhodopsin promoter, a cGMP-phosphodiesterase β-subunit promoter, a human or mouse rhodopsin promoter, an hGRK1 promoter, a synapsin promoter, a glial fibrillary acidic protein (GFAP) promoter, a rod-specific IRBP promoter, a VMD2 promoter, and combinations thereof. In some embodiments, the promoter is a rhodopsin promoter. In some embodiments, the promoter is a CMV promoter. In some embodiments, the promoter is not a CMV promoter.

[0353] In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the promoter mediates expression in ocular tissue. In some embodiments, the promoter does not mediate expression in ocular tissue. In some embodiments, the promoter mediates expression in hair cells of the auditory system and / or vestibular system.

[0354] SEQ ID NO: 64 smCBA promoter AATTCGGTACCCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAG

[0355] SEQ ID NO: 39 is the nucleotide sequence of the overlapping portion of the second generation of the overlap vector system: CAGGTCTAACTTTCTGAAGCTGAAGAACGCTGCCACACTGATCCAGAGGCACTGGCGGGGTCACAACTGTAGGAAGAACTACGGGCTGATGCGTCTGGGCTTCCTGCGGCTGCAGGCCCTGCACCGCTCCCGGAAGCTGCACCAGCAGTACCGCCTGGCCCGCCAGCGCATCATCCAGTT CCAGGCCCGCTGCCGCGCCTATCTGGTGCGCAAGGCCTTCCGCCACCGCCTCTGGGCTGTGCTCACCGTGCAGGCCTATGCCCGGGGCATGATCGCCCGCAGGCTGCACCAACGCCTCAGGGCTGAGTATCTGTGGCGCCTCGAGGCTGAGAAAATGCGGCTGGCGGAGGAAGAGAAGCTT

[0356] SEQ ID NO: 79 (related protein sequence derived from SEQ ID NO: 39) RSNFLKLKNAATLIQRHWRGHNCRKNYGLMRLGFLRLQALHRSRKLHQQYRLARQRIIQFQARCRAYLVRKAFRHRLWAVLTVQAYARGMIARRLHQRLRAEYLWRLEAEKMRLAEEEKL

[0357] SEQ ID NO:40 is a related fragment of SEQ ID NO:31 in which a potential in-frame stop codon is located in the second generation hybrid front half AP splice donor region (see, eg, Figure 42). CAACGCCTCAGGGCTGAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGAGCTAGCCCC

[0358] SEQ ID NO:41 is a related fragment of SEQ ID NO:33 in which a potential in-frame stop codon in the CMv1 hybrid front half AP splice donor region has been removed (see, e.g., Figure 42). CAACGCCTCAGGGCTGAGGTAAGTATCAAGGTTACAAGACAGGTTAACGGAGACCAATTGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCAGCGCTAGCCCC

[0359] SEQ ID NO: 42 is the forward primer sequence for Gibson primer set 1 (the reverse primer sequence is the reverse complement of the forward primer (not shown)) (see, e.g., Figure 42). CCGCAGGCTGCACCAACGCCTCAGGGCTGAGGTAAGTATCAAGGTTACAAGACAGGTTAACGGAGACCAATTGAAACT

[0360] SEQ ID NO: 43 is the forward primer sequence for Gibson primer set 2 (the reverse primer sequence is the reverse complement of the forward primer (not shown)) (see, e.g., Figure 42). AACGGAGACCAATTGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCAGCGCTAGCCCCCGGGTGCGCGGCG

[0361] SEQ ID NO:44 is the polynucleotide sequence of the CMv2.1 hybrid back-half vector (i.e., AAV-APhead-APSA-hMYO7ACTex22-CMv2.1).

[0362] SEQ ID NO: 45 is the portion of the overlapping sequence derived from the original overlap vector.

[0363] SEQ ID NO: 46 is the polynucleotide sequence of the CMv3 hybrid front half vector (i.e., AAV-smCBA-hMYO7A-NTex21-APSD-APhead-CMv3) (paired with the CMv2 back half vector).

[0364] SEQ ID NO:47 is the polynucleotide sequence of the CMv2.1 hybrid back half vector (i.e., AAV-AP head-APSA-hMYO7ACTex22-CMv2.1.HA) containing an HA tag (to be paired with the CMv2 front half vector).

[0365] SEQ ID NO: 48 is the polynucleotide sequence of the minimized second-generation hybrid back-half vector (i.e., AAV-APhead-APSA-hMYO7ACTex22.HA-MIN) containing an HA tag (paired with the second-generation front-half hybrid vector).

[0366] SEQ ID NO: 49 is the polynucleotide sequence of the minimized second-generation hybrid back-half vector (i.e., AAV-APhead-APSA-hMYO7ACTex22-MIN) that does not contain an HA tag (paired with the second-generation front-half hybrid vector).

[0367] SEQ ID NO: 50 is the polynucleotide sequence of the third generation overlapping front half vector (i.e., AAV-smCBA-hMYO7A-NTlong-v3).

[0368] SEQ ID NO: 66 (only the N-myosin 7A portion of SEQ ID NO: 50)

[0369] SEQ ID NO: 65 (peptide encoded by the N-myosin 7A portion of SEQ ID NO: 50)

[0370] SEQ ID NO: 51 is the polynucleotide sequence of the third generation overlap-back half vector (i.e., AAV-hMYO7A-CTlong-v3.HA).

[0371] SEQ ID NO: 80 (hMYO7A c-terminus (e.g., AAV-hMYO7A-CTlong-v3.HA)

[0372] SEQ ID NO: 81 (hMYO7A c-terminus (e.g., AAV-hMYO7A-CTlong-v3.HA)

[0373] In some embodiments, the overlapping polynucleotide vectors provided herein comprise a region of overlap comprising a nucleotide sequence having 80%, 85%, 90%, 92.5%, 95%, 98%, or 99% sequence identity to any of the overlapping sequences provided herein. In some embodiments, the overlapping region comprises a nucleotide sequence having 80%, 85%, 90%, 92.5%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 39 and 52-59. In some embodiments, the overlapping region comprises a nucleotide sequence having 80%, 85%, 90%, 92.5%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 39 and 53-59. In some embodiments, the overlapping region (or overlapping polynucleotide sequence) comprises a nucleotide sequence having 80%, 85%, 90%, 92.5%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 39, 56, or 57. In some embodiments, the overlapping polynucleotide sequence comprises a nucleotide sequence selected from any one of SEQ ID NOs: 39 and 52-59. The overlap vectors provided herein may comprise overlapping polynucleotide sequences that differ from any one of SEQ ID NOs: 39 and 52-59 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 20-25, or more than 25 nucleotides. In some embodiments, these overlap vectors contain two overlapping sequences disclosed herein, e.g., the mutually exclusive sequences SEQ ID NOs: 39 and 56, or the mutually exclusive sequences SEQ ID NOs: 39 and 57.

[0374] In exemplary embodiments, the overlapping polynucleotide sequence comprises SEQ ID NO: 56. In some embodiments, the overlapping polynucleotide sequence comprises SEQ ID NO: 57. In some embodiments, the overlapping polynucleotide sequence comprises SEQ ID NO: 39. In some embodiments, the overlapping polynucleotide sequence does not comprise SEQ ID NO: 52.

[0375] In some embodiments, the overlapping polynucleotide vectors provided herein comprise regions of overlap (overlapping polynucleotide sequences) that encode proteins having amino acid sequences that comprise 95%, 98%, or 99% or greater sequence identity to any of SEQ ID NOs: 79 and 82-89. In exemplary embodiments, these vectors comprise regions of overlap (overlapping polynucleotide sequences) that encode proteins having the amino acid sequence of any one of SEQ ID NOs: 79 and 82-89. In exemplary embodiments, these vectors comprise regions of overlap (overlapping polynucleotide sequences) that encode proteins having the amino acid sequence of any one of SEQ ID NOs: 79 and 83-89. In some embodiments, the overlapping polynucleotide sequences do not encode the amino acid sequence of SEQ ID NO: 82.

[0376] In various embodiments, the overlapping polynucleotide vectors provided herein contain regions of overlap in polypeptide-coding sequences having lengths of about 1365 bp, 1284 bp, 1027 bp, 1026 bp, 945 bp, 687 bp, 361 bp, 279 bp, or 20 bp. In some embodiments, the overlapping vectors contain regions of overlap less than 1000 bp in length. In some embodiments, the overlapping vectors contain regions of overlap less than 1365 bp. In some embodiments, the overlapping vectors contain regions of overlap less than 700 bp. In some embodiments, the regions of overlap have lengths of between about 20-100 nucleotides, about 100-500 nucleotides, about 100-200 nucleotides, about 200-300 nucleotides, or about 300-400 nucleotides.

[0377] In various embodiments, the overlapping polynucleotide vectors provided herein contain overlapping regions having lengths of exactly 1365 bp, 1284 bp, 1027 bp, 1026 bp, 945 bp, 687 bp, 361 bp, 279 bp, or 20 bp. It is understood that overlapping regions may be mutually exclusive in coding sequences. In some embodiments, the overlapping polynucleotide vector contains one or more overlapping regions, for example, two overlapping regions. In some embodiments, the overlapping vector contains two overlapping regions having lengths of 361 bp and 687 bp.

[0378] In exemplary embodiments, the overlapping polynucleotide vectors provided herein contain overlapping regions having a length of 687 or 945 bp. In some embodiments, these vectors contain overlapping regions of MYO7A sequences having a length of 687 or 945 bp. In some embodiments, the overlapping vectors contain overlapping regions having a length of 361 bp.

[0379] 1365 bp overlap (SEQ ID NO: 52).

[0380] SEQ ID NO: 82 (protein sequence corresponding to SEQ ID NO: 52) RSNFLKLKNAATLIQRHWRGHNCRKNYGLMRLGFLRLQALHRSRKLHQQYRLARQRIIQFQARCRAYLVRKAFRHRLWAVLTVQAYARGMIARRLHQRLRAEYLWRLEAEKMR LAEEEKLRKEMSAKKAKEEAERKHQERLAQLAREDAERELKEKEAARRKKELLEQMERARHEPVNHSDMVDKMFGFLGTSGGLPGQEGQAPSGFEDLERGRREMVEEDLDAALP LPDEDEEDLSEYKFAKFAATYFQGTTTHSYTRRPLKQPLLYHDDEGDQLAALAVWITILRFMGDLPEPKYHTAMSDGSEKIPVMTKIYETLGKKTYKRELQALQGEGEAQLPEG QKKSSVRHKLVHLTLKKKKSKLTEEVTKRLHDGESTVQGNSMLEDRPTSNLEKLHFIIGNGILRPALRDEIYCQISKQLTHNPSKSSYARGWILVSLCVGCFAPSEKFVKYLRNF

[0381] 1284 bp overlap (SEQ ID NO: 53).

[0382] SEQ ID NO: 83 (protein sequence corresponding to SEQ ID NO: 53) GLMRLGFLRLQALHRSRKLHQQYRLARQRIIQFQARCRAYLVRKAFRHRLWAVLTVQAYARGMIARRLHQRLRAEYLWRLEAEKMRLAEEEKLRKEMSAKKAKEEAE RKHQERLAQLAREDAERELKEKEAARRKKELLEQMERARHEPVNHSDMVDKMFGFLGTSGGLPGQEGQAPSGFEDLERGRREMVEEDLDAALPLPDEDEEDLSEYKF AKFAATYFQGTTTHSYTRRPLKQPLLYHDDEGDQLAALAVWITILRFMGDLPEPKYHTAMSDGSEKIPVMTKIYETLGKKTYKRELQALQGEGEAQLPEGQKKSSVR HKLVHLTLKKKKSKLTEEVTKRLHDGESTVQGNSMLEDRPTSNLEKLHFIIGNGILRPALRDEIYCQISKQLTHNPSKSSYARGWILVSLCVGCFAPSEKFVKYLRNF

[0383] 1027 bp overlap (SEQ ID NO: 54).

[0384] SEQ ID NO: 84 (protein sequence corresponding to SEQ ID NO: 54) RSNFLKLKNAATLIQRHWRGHNCRKNYGLMRLGFLRLQALHRSRKLHQQYRLARQRIIQFQARCRAYLVRKAFRHRLWAVLTVQAYARGMIARRLHQRLRAEYLWRLEAEKMRLAEEEKLRKEMSAKKAKEEAERKHQERLAQLAREDAERELKEKEAARRKKELLEQMER ARHEPVNHSDMVDKMFGFLGTSGGLPGQEGQAPSGFEDLERGRREMVEEDLDAALPDEDEEDLSEYKFAKFAATYFQGTTTHSYTRRPLKQPLLYHDDEGDQLAALAVWITILRFMGDLPEPKYHTAMSDGSEKIPVMTKIYETLGKKTYKRELQALQGEGEAQLPEGQ

[0385] 1026 bp overlap (SEQ ID NO: 55).

[0386] SEQ ID NO: 85 (protein sequence corresponding to SEQ ID NO: 55) LAEEEKLRKEMSAKKAKEEAERKHQERLAQLAREDAERELKEKEAARRKKELLEQMERARHEPVNHSDMVDKMFGFLGTSGGLPGQEGQAPSGFEDLERGRREMVEEDLDAALPLPDEDEEDLSEYKFAKFAATYFQGTTTHSYTRRPLKQPLLYHDDEGDQLAALAVWIT ILRFMGDLPEPKYHTAMSDGSEKIPVMTKIYETLGKKTYKRELQALQGEGEAQLPEGQKKSSVRHKLVHLTLKKKSKLTEEVTKRLHDGESTVQGNSMLEDRPTSNLEKLHFIIGNGILRPALRDEIYCQISKQLTHNPSKSSYARGWILVSLCVGCFAPSEKFVKYLRNF

[0387] 945 bp overlap (SEQ ID NO: 56). GGGCTGATGCGTCTGGGCTTCCTGCGGCTGCAGGCCCTGCACCGCTCCCGGAAGCTGCACCAGCAGTACCGCCTGGCCCGCCAGCGCATCATCCAGTTCCAGGCCCGCTGCCGCGCCTATCTGGTGCGCAAGGCCTTCCGCCACCGCCTCTGGGCTGTGCTCACCGTGCAGGCCTATGCCCGGGGCATGATCGCCCGCAGGCTGCACCAACGCCTCAGGGCTGAGTATCTGTGGCGCCTCGAGGCTGAGAAAATGCGGCTGGCGGAGGAAGAGAAGCTTCGGAAGGAGATGAGCGCCAAGAAGGCCAAGGAGGAGGCCGAGCGCAAGCATCAGGAGCGCCTGGCCCAGCTGGCTCGTGAGGACGCTGAGCGGGAGCTGAAGGAGAAGGAGGCCGCTCGGCGGAAGAAGGAGCTCCTGGAGCAGATGGAAAGGGCCCGCCATGAGCCTGTCAATCACTCAGACATGGTGGACAAGATGTTTGGCTTCCTGGGGACTTCAGGTGGCCTGCCAGGCCAGGAGGGCCAGGCACCTAGTGGCTTTGAGGACCTGGAGCGAGGGCGGAGGGAGATGGTGGAGGAGGACCTGGATGCAGCCCTGCCCCTGCCTGACGAGGATGAGGAGGACCTCTCTGAGTATAAATTTGCCAAGTTCGCGGCCACCTACTTCCAGGGGACAACCACGCACTCCTACACCCGGCGGCCACTCAAACAGCCACTGCTCTACCATGACGACGAGGGTGACCAGCTGGCAGCCCTGGCGGTCTGGATCACCATCCTCCGCTTCATGGGGGACCTCCCTGAGCCCAAGTACCACACAGCCATGAGTGATGGCAGTGAGAAGATCCCTGTGATGACCAAGATTTATGAGACCCTGGGCAAGAAGACGTACAAGAGGGAGCTGCAGGCCCTGCAGGGCGAGGGCGAGGCCCAGCTCCCCGAGGGCCAG

[0388] SEQ ID NO: 86 (protein sequence corresponding to SEQ ID NO: 56) GLMRLGFLRLQALHRSRKLHQQYRLARQRIIQFQARCRAYLVRKAFRHRLWAVLTVQAYARGMIARRLHQRLRAEYLWRLEAEKMRLAEEEKLRKEMSAKKAKEEAERKHQERLAQLAREDAERELKEKEAARRKKELLEQMERARHEPVNHSDMVD KMFGFLGTSGGLPGQEGQAPSGFEDLERGRREMVEEDLDAALPDEDEEDLSEYKFAKFAATYFQGTTTHSYTRRPLKQPLLYHDDEGDQLAALAVWITILRFMGDLPEPKYHTAMSDGSEKIPVMTKIYETLGKKTYKRELQALQGEGEAQLPEGQ

[0389] 687 bp overlap (SEQ ID NO: 57). CTGGCGGAGGAAGAGAAGCTTCGGAAGGAGATGAGCGCCAAGAAGGCCAAGGAGGAGGCCGAGCGCAAGCATCAGGAGCGCCTGGCCCAGCTGGCTCGTGAGGACGCTGAGCGGGAGCTGAAGGAGAAGGAGGCCGCTCGGCGGAAGAAGGAGCTCCTGGAGCAGATGGAAAGGGCCCGCCATGAGCCTGTCAATCACTCAGACATGGTGGACAAGATGTTTGGCTTCCTGGGGACTTCAGGTGGCCTGCCAGGCCAGGAGGGCCAGGCACCTAGTGGCTTTGAGGACCTGGAGCGAGGGCGGAGGGAGATGGTGGAGGAGGACCTGGATGCAGCCCTGCCCCTGCCTGACGAGGATGAGGAGGACCTCTCTGAGTATAAATTTGCCAAGTTCGCGGCCACCTACTTCCAGGGGACAACCACGCACTCCTACACCCGGCGGCCACTCAAACAGCCACTGCTCTACCATGACGACGAGGGTGACCAGCTGGCAGCCCTGGCGGTCTGGATCACCATCCTCCGCTTCATGGGGGACCTCCCTGAGCCCAAGTACCACACAGCCATGAGTGATGGCAGTGAGAAGATCCCTGTGATGACCAAGATTTATGAGACCCTGGGCAAGAAGACGTACAAGAGGGAGCTGCAGGCCCTGCAGGGCGAGGGCGAGGCCCAGCTCCCCGAGGGCCAG

[0390] SEQ ID NO: 87 (Protein sequence corresponding to SEQ ID NO: 57) LAEEEKLRKEMSAKKAKEEAERKHQERLAQLAREDAERELKEKEAARRKKELLEQMERARHEPVNHSDMVDKMFGFLGTSGGLPGQEGQAPSGFEDLERGRREMVEEDLDAALPLPDEDEEDLSEYKFAKFAATYFQGTTTHSYTRRPLKQPLLYHDDEGDQLAALAVWITILRFMGDLPEPKYHTAMSDGSEKIPVMTKIYETLGKKTYKRELQALQGEGEAQLPEGQ

[0391] 361 bp overlap (identical to SEQ ID NO: 39) CAGGTCTAACTTTCTGAAGCTGAAGAACGCTGCCACACTGATCCAGAGGCACTGGCGGGGTCACAACTGTAGGAAGAACTACGGGCTGATGCGTCTGGGCTTCCTGCGGCTGCAGGCCCTGCACCGCTCCCGGAAGCTGCACCAGCAGTACCGCCTGGCCCGCCAGCGCATCATCCAGTT CCAGGCCCGCTGCCGCGCCTATCTGGTGCGCAAGGCCTTCCGCCACCGCCTCTGGGCTGTGCTCACCGTGCAGGCCTATGCCCGGGGCATGATCGCCCGCAGGCTGCACCAACGCCTCAGGGCTGAGTATCTGTGGCGCCTCGAGGCTGAGAAAATGCGGCTGGCGGAGGAAGAGAAGCTT

[0392] 279 bp overlap (SEQ ID NO: 58) GGGCTGATGCGTCTGGGCTTCCTGCGGCTGCAGGCCCTGCACCGCTCCCGGAAGCTGCACCAGCAGTACCGCCTGGCCCGCCAGCGCATCATCCAGTTCCAGGCCCGCTGCCGCGCCTATCTGGTGCGCAAGGCCTTCC GCCACCGCCTCTGGGCTGTGCTCACCGTGCAGGCCTATGCCCGGGGCATGATCGCCCGCAGGCTGCACCAACGCCTCAGGGCTGAGTATCTGTGGCGCCTCGAGGCTGAGAAAATGCGGCTGGCGGAGGAAGAGAAGCTT

[0393] SEQ ID NO: 88 (protein sequence corresponding to SEQ ID NO: 58) GLMRLGFLRLQALHRSRKLHQQYRLARQRIIQFQARCRAYLVRKAFRHRLWAVLTVQAYARGMIARRLHQRLRAEYLWRLEAEKMRLAEEEKL

[0394] 20 bp overlap (SEQ ID NO: 59): TGGCGGAGGAAGAGAAGCTT

[0395] SEQ ID NO: 89 (protein sequence corresponding to SEQ ID NO: 59): AEEEKL

[0396] In some embodiments of the disclosed hybrid and overlap vectors, both of the disclosed front half vectors (5' AAV) comprise a left inverted terminal repeat sequence comprising a nucleotide sequence having at least 95% or 98% identity to SEQ ID NO: 60. In some embodiments of the disclosed hybrid and overlap vectors, both of the disclosed front half vectors comprise a left inverted terminal repeat sequence comprising SEQ ID NO: 60. In some embodiments of the disclosed hybrid and overlap vectors, both of the disclosed back half vectors (3' AAV) comprise a right inverted terminal repeat sequence comprising SEQ ID NO: 61. In some embodiments of the disclosed hybrid and overlap vectors, both of the disclosed back half vectors comprise a right inverted terminal repeat sequence comprising a nucleotide sequence having at least 95% or 98% identity to SEQ ID NO: 61. In various embodiments, both of the disclosed dual hybrid and overlap vector pairs comprise a left ITR sequence comprising SEQ ID NO: 60 and a right ITR sequence comprising SEQ ID NO: 61.

[0397] Left ITR sequence (SEQ ID NO: 60). CTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGAGAGGGAGTGGCCAACTCCATCACTAGGGGTT

[0398] Right ITR sequence (SEQ ID NO: 61). AACCCCTAGTGATGGAGTTGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAG

[0399] [Table 2]

[0400] [Table 3-1] [Table 3-2] [Table 3-3]

[0401] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference: [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6]

[0402] equivalent It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or variations will be suggested to those skilled in the art in light thereof, and are to be included within the spirit and scope of the present disclosure and the scope of the appended claims.

[0403] All references cited herein (including publications, patent applications, and patents) are incorporated by reference to the same extent as if each reference was individually and specifically incorporated by reference and was set forth in its entirety herein.

[0404] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise stated herein, and each separate value is incorp...

Claims

1. i) a first AAV vector polynucleotide comprising a promoter followed by inverted terminal repeats at each end of a polypeptide comprising a first partial coding sequence encoding an N-terminal portion of a full-length myosin polypeptide; and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat sequence at each end of a polynucleotide comprising a second partial coding sequence encoding a C-terminal portion of a full-length myosin polypeptide.

1. A polynucleotide vector system comprising: the N-terminal portion of the full-length myosin polypeptide is at least 90% identical to the amino acid sequence of SEQ ID NO:74, and the C-terminal portion of the full-length myosin polypeptide is at least 90% identical to the amino acid sequence of SEQ ID NO:76; the first partial coding sequence does not encode a single-alpha helix (SAH) domain of a full-length myosin polypeptide, and the first partial coding sequence and the second partial coding sequence encode a full-length myosin polypeptide; Polynucleotide vector system.

2. A polynucleotide vector system as described in claim 1, wherein the full-length myosin polypeptide is at least 90% identical to sequence number 8.

3. 3. The polynucleotide vector system of claim 1 or 2, wherein the first partial coding sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO: 73, and the second partial coding sequence comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO:

75.

4. A polynucleotide vector system described in any one of claims 1 to 3, wherein the first AAV vector polynucleotide and the second AAV vector polynucleotide comprise an intron of a full-length myosin polypeptide, an AK sequence of an F1 phage, or an intron of a synthetic alkaline phosphatase (AP).

5. The polynucleotide vector system described in claim 4, wherein the first AAV vector polynucleotide and the second AAV vector polynucleotide comprise an intron of a synthetic AP.

6. A polynucleotide vector system as described in claim 5, wherein the intron of the synthetic AP is at least 90% identical to sequence number 70.

7. A polynucleotide vector system described in any one of claims 1 to 6, wherein a polynucleotide sequence encoding the tail domain of a full-length myosin polypeptide is not present in the first partial coding sequence.

8. 8. The polynucleotide vector system of any one of claims 1 to 7, further comprising one or more nucleotide substitutions in one or more non-coding regions of the first AAV vector polynucleotide, the second AAV vector polynucleotide, or a combination thereof.

9. 9. The polynucleotide vector system according to any one of claims 1 to 8, wherein the promoter is selected from the group consisting of a CMV promoter, an EF-1 alpha promoter, a cone arrestin promoter, an smCBA promoter, a human myosin 7a gene-derived promoter, a TαC gene-derived promoter, a rhodopsin promoter, a cGMP-phosphodiesterase β-subunit promoter, a human or mouse rhodopsin promoter, an hGRK1 promoter, a rod-specific IRBP promoter, a VMD2 promoter, a synapsin promoter, a glial fibrillary acidic protein (GFAP) promoter, and combinations thereof.

10. A polynucleotide vector system described in any one of claims 1 to 9, further comprising one or more nucleotide substitutions to remove one or more putative stop codons in the 3' untranslated region between a second partial coding sequence encoding the C-terminal portion of a full-length myosin polypeptide and an inverted terminal repeat sequence at the 3' end of the polynucleotide comprising the second partial coding sequence.

11. 11. The polynucleotide vector system of claim 10, wherein the one or more nucleotide substitutions are located in one or more putative stop codons.

12. A recombinant viral particle comprising the polynucleotide vector system of any one of claims 1 to 11.

13. The viral particles include AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAV9 capsid, AAV10 capsid, AAV7m8 capsid, AAV-DJ capsid, AAV2 / 2-MAX capsid, AAVSHh10 ...

13. The recombinant viral particle of claim 12, comprising an AAVSHh10Y capsid, an AAV3b capsid, an AAVLK03 capsid, an AAV8BP2 capsid, an AAV1(E531K) capsid, an AAV6(D532N) capsid, an AAV6-3pmut, an AAV2G9 capsid, an AAV44.9 capsid, an AAV44.9(E531D) capsid, an AAVrh.8 capsid, an AAVrh.8R capsid, an AAV9-PHP.B capsid, an AAVAnc80 capsid, or a combination thereof.

14. 14. A recombinant viral particle as described in claim 12 or 13 for use in a method for treating or ameliorating a disease or condition in a human or animal, the method comprising administering the recombinant viral particle to one or more cells of a human or animal, wherein a full-length myosin polypeptide provides treatment or amelioration of the disease or condition and is expressed in the one or more cells.

15. The recombinant viral particle of claim 14, wherein the disease or condition is Usher syndrome.

16. The recombinant viral particle described in claim 15, wherein administering the recombinant viral particle provides partial or complete restoration of melanosome migration in retinal pigment epithelial (RPE) cells.

17. A recombinant viral particle described in any one of claims 14 to 16, wherein administration of the recombinant viral particle results in partial or complete recovery of vision loss in a human or animal.

18. The recombinant viral particle described in claim 14, wherein the disease or condition is autosomal recessive isolated deafness (DFNB2).

19. A recombinant viral particle as described in claim 14, wherein the disease or condition is age-related hearing loss as a result of a non-genetic deficiency or insufficiency of MY07A expression.

20. A recombinant viral particle described in any one of claims 14, 15, 18 or 19, wherein administering the recombinant viral particle provides partial or complete restoration of hearing loss or vestibular function in a human or animal.

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