Dual-aav Vector-based Systems And Methods For Delivering Oversized Genes To Mammalian Cells

Abstract

Disclosed are materials and methods for treating diseases of the mammalian eye, and in particular, Usher syndrome 1B (USH1B). The invention provides AAV-based, dual-vector systems that facilitate the expression of full-length proteins whose coding sequences exceed that of the polynucleotide packaging capacity of an individual AAV vector. In one embodiment, vector systems are provided that include i) a first AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide: and ii) a second AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. In another embodiment, the vector system includes i) a first AAV vector polynucleotide comprising an inverted terminal repeat at each end, a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide followed by a splice donor site and intron and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat at each end, followed by an intron and a splice-acceptor site for the intron, followed by a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. The coding sequence or the intron sequence in the first and second AAV vectors preferably includes a sequence region that overlaps.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation of U.S. application Ser. No. 16/265,864, filed Feb. 1, 2019, which is divisional of U.S. application Ser. No. 14/279,142, filed May 15, 2014, which is a continuation of PCT Intl. Patent Appl. No. PCT/US2012/065645 filed Nov. 16, 2012, which claimed priority to U.S. Provisional Patent Appl. No. 61/560,437, filed Nov. 16, 2011 (expired); the contents of each of which is hereby incorporated in its entirety by express reference thereto.

NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

[0003] Not Applicable.

BACKGROUND OF THE INVENTION

Field of the Invention

[0004] The present invention relates generally to the fields of molecular biology and virology, and in particular, to the development of gene delivery vehicles. Disclosed are improved rAAV dual-vector systems, and compositions useful in delivering a variety of nucleic acid segments, including those encoding therapeutic proteins polypeptides, peptides, antisense oligonucleotides, or ribozyme constructs to selected host cells for use in various gene-therapy regimens. Methods are also provided for preparing and using these modified rAAV-dual-vector based systems in a variety of viral-based gene therapies, and in particular, for the treatment and/or amelioration of symptoms of Myosin VII-deficiency, including, without limitation, the treatment of human Usher syndrome.

Description of Related Art

[0005] As has been established by a multitude of successful proof-of-concept studies, and various clinical trials, recombinant AAV has emerged as the most optimal gene delivery vehicle to treat retinal disease. However, one limitation of AAV is its relatively small DNA packaging capacity--approximately 4.7 kilobases (KB). Thus, standard AAV vector systems are unsuitable for addressing diseases in which large genes are mutated or otherwise dysfunctional. An example of such a disease is Usher syndrome.

[0006] The most common form of Usher syndrome, USH1B, is a severe autosomal-recessive, deaf-blindness disorder caused by mutations in the myosinVIIa gene. Blindness occurs from a progressive retinal degeneration that begins after deafness, and after development of the retina. MYO7a protein is expressed in photoreceptors and retinal pigment epithelium (RPE), and is involved in opsin transport through photoreceptor cilia and the movement of RPE melanosomes.

[0007] The coding region for the human Myosin VII protein (MYO7a), however, is 6534 or 6648 nucleotides in length (depending on the allelic variant), making traditional AAV vector systems unsuitable for gene therapy of USH1B.

[0008] Previously, Allocca et al. (2008) published intriguing results suggesting that AAV5 serotype vectors were capable of packaging genomes of up to 8.9 KB in size, and that these vectors expressed full-length proteins when delivered in vivo. In this study, the authors expressed full-length MYO7A protein from an AAV5 vector containing the CMV promoter driving hMyo7a. Subsequent studies, carried out to directly validate the Allocca et al. findings were simultaneously published by three independent groups (Lai et al., 2010; Dong et al., 2010; Wu et al., 2010), and their publication was accompanied by an expert commentary (See, Hirsch et al., 2010). While all three studies confirmed that these `oversized` AAV5 vectors did indeed drive full-length protein expression, the genetic content of each vector capsid was found to be limited only to .about.5 KB of DNA, and not the 8.7 KB originally reported by Allocca et al. (2008). These vector capsids were shown to contain a "heterogeneous mixture" of truncated vector genomes (e.g., the 5'-end of the gene, the 3'-end of the gene, or a mixture of the two, with an internal sequence deletion) (Lai et al., 2010; Dong et al., 2010; Wu et al., 2010). Additionally, these oversized/heterogeneous vectors exhibited poor packaging efficiency (i.e., low-vector titers) and low transduction efficiency when compared to matched reporter vectors of standard size (<5 KB) (Wu et al., 2010).

[0009] Using this `heterogeneous` system, vectors containing portions of the MYO7A transgene were packaged, however, and proof-of-concept results were demonstrated in the shaker-1 mouse model of USH1B. The therapeutic results achieved with the heterogeneous AAV-hMyo7a vectors were comparable to previous gene replacement results using a Lentivirus-based hMyo7a vector (Hashimoto et al., 2007).

[0010] This Lentivirus-Myo7a vector is under development by Oxford BioMedica in collaboration with Sanofi-Aventis for a phase I/II clinical trial of USH1B, marketed under the name UshStat.RTM. LentiVector.RTM.. Lentivirus is regarded as a vector platform that is not well-suited for infecting post-mitotic (i.e., non-dividing) cells. Furthermore, although the vector is suitable for transducing RPE, many studies have shown it to be ineffective at transducing adult photoreceptors. Even though MYO7A is expressed in both cell types, UshStat.RTM. may only be effective at rescuing the RPE phenotype. A study showed that PRs are actually the initial site of disease, so not targeting this cell type effectively may result in zero therapy, although it remains to be seen in the human clinical trial.

[0011] Because of the excellent safety profile and encouraging reports of efficacy in the AAV gene therapy trials for LCA2/RPE65, there has been continuing interest in creating an AAV-based system for treating USH1B patients. However, the current AAV vector for MYO7A, as previously mentioned, is heterogeneous; it is manufactured and purified as a single-virus preparation containing a mixture of viral payloads. This fact, unfortunately, makes it virtually impossible to characterize the vector fully--a requirement for government regulatory review and approval. In order to address this concern directly (i.e., that the vector genome of any AAV-based vectors for USH1B must be fully characterized before gaining Food and Drug Administration (FDA) approval, the inventors have developed an AAV dual-vector-based system to facilitate gene therapy approaches for treating USH1B.

BRIEF SUMMARY OF THE INVENTION

[0012] The subject invention concerns materials and methods for gene therapy of diseases, such as Usher syndrome 1B (USH1B). The inventors have characterized dual vector platforms described herein as to transcript fidelity, and have shown and that mRNA arising from the system is 100% accurate relative to what would be predicted by correct homologous recombination on the front and back vector pairs, making them useful as gene therapy delivery vector systems.

[0013] One aspect of the invention concerns a dual AAV vector system that permits expression of full-length proteins, whose coding sequence exceeds the polynucleotide packaging capacity of an individual AAV vector.

[0014] In one embodiment, a vector system of the invention comprises:

[0015] (i) a first AAV vector polynucleotide comprising an inverted terminal repeat at each end of the polynucleotide, and a suitable promoter followed by a partial coding sequence that encodes an N-terminal part of a selected full-length polypeptide; and

[0016] (ii) a second AAV vector polynucleotide comprising an inverted terminal repeat at each end of the polynucleotide, and a partial coding sequence that encodes a C-terminal part of the selected full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence.

[0017] The coding sequence in the first and second AAV vectors comprises sequence that overlaps, such that part of the coding sequence present at the 3'-end of the coding sequence of the first vector is identical or substantially identical with part of the coding sequence present at the 5'-end of the coding sequence of the second vector. In an illustrative embodiment, the polypeptide encoded is a wild type or biologically-functional human myosin VIIa polypeptide (hmyo7a).

[0018] In another embodiment, a vector system of the invention includes: [0019] (i) a first AAV vector polynucleotide comprising an inverted terminal repeat at each end, a suitable promoter followed by a partial coding sequence that encodes an N-terminal part of a selected full-length polypeptide followed by a splice donor site for an intron and an intron; and [0020] (ii) a second AAV vector polynucleotide comprising an inverted terminal repeat at each end, followed by an intron and a splice acceptor site for the intron, followed by a partial coding sequence that encodes a C-terminal part of the selected full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence.

[0021] The intron sequence in the first and second AAV vectors includes a sequence that overlaps, such that all or part of the intron sequence present at the 3'-end of the coding sequence of the first vector is identical, or substantially identical, with all or part of the intron sequence present at the 5'-end of the coding sequence of the second vector. In one embodiment, the intron is intron 23 of the hmyo7a gene. In a specific embodiment, the polypeptide encoded is a wild type (i.e., functional) human myosin VIIa polypeptide, and the intron is the full intron 23 of the hmyo7a gene.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] For promoting an understanding of the principles of the invention, reference will now be made to the 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 invention is thereby intended. Any alterations and further modifications in the described embodiments, and any further applications of the principles of the invention as described herein are contemplated as would normally occur to one of ordinary skill in the art to which the invention relates. The following drawings form part of the present specification and are included to demonstrate certain aspects of the present invention. The patent or application file contains at least one drawing that is executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

[0023] The invention may be better understood by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numerals identify like elements, and in which:

[0024] FIG. 1 shows the formation of complete gene cassette from dual AAV vectors via homologous recombination;

[0025] FIG. 2 shows a schematic of the two vector components that make up Dual-Vector System 1 in accordance with one aspect of the present invention;

[0026] FIG. 3 shows a schematic of the two vector components that make up Dual-Vector System 2 in accordance with one aspect of the present invention. Native hMyo7a intron 23 in shown in light green; splice donor and splice acceptor sequence are shown in dark green;

[0027] FIG. 4 shows the schematic of the two vector components that make up Dual-Vector System 3 in accordance with one aspect of the present invention. This is an exemplary standard transplicing dual vector pairs with the "intron" in this case referring to the alkaline phosphatase splice donor and acceptor sites. Synthetic alkaline phosphatase (AP) intron is shown in light blue; AP splice donor and splice acceptor sequences are shown in dark blue;

[0028] FIG. 5 shows an immunoblot to detect the presence of MYO7A in infected or transfected HEK293 cells. Heterogeneous vectors (described in introduction) are compared to all three dual-vector systems. Dual vectors were packaged either in AAV2 or in 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 vectors alone (to confirm the presence or absence of a truncated protein product expressed from the promoter-containing N-terminal vectors);

[0029] FIG. 6A and FIG. 6B show immunoblot to detect the presence of myo7A in HEK293 cells infected with Dual-Vector System 1. Results are presented as a time course from 3-7 days post infection (lanes 3-7) and are compared to cells transfected with myo7a plasmid (lane 1) and uninfected control (lane 2). The area inside the white box shown in FIG. 6A is magnified and presented at higher contrast in FIG. 6B. Starting at 3-days' post-infection, full-length human MYO7a protein was visible, with peak expression occurring around day 5;

[0030] FIG. 7A and FIG. 7B show retinas from untreated mice and mice treated subretinally with Dual-Vector System 1. Immunohistochemistry (IHC) was performed using an antibody directed against MYO7A. The `green` is the stain of MYO7A and `blue` corresponds to nuclear, DAPI stain;

[0031] FIG. 8A, FIG. 8B, FIG. 8C, and FIG. 8D show differences in RPE melanosome localization in wild type vs. shaker-1 mice. In wild type mice, RPE melanosome apically migrate towards photoreceptor outer segments (FIG. 8A) whereas this phenomenon fails to occur in mice lacking Myo7a (shaker-1), as seen in (FIG. 8B). To the right is a high magnification image of single RPE cells from either a wild type (FIG. 8C) or shaker-1 (FIG. 8D) mouse showing this phenomenon up close;

[0032] FIG. 9A, FIG. 9B, and FIG. 9C show apical migration of RPE melanosomes is restored in shaker-1 mice injected with Dual-Vector System 1. Electron microscopy reveals that melanosomes of untreated shaker-1 mice do not apically migrate (FIG. 9A). In shaker-1 mice injected with Dual-Vector System 1 (packaged in AAV2), RPE melanosomes migrate apically towards photoreceptors, which can be seen here in both low- and high-magnification images (FIG. 9B and FIG. 9C);

[0033] FIG. 10A, FIG. 10B, FIG. 10C, FIG. 10D, FIG. 10E, and FIG. 10F illustrate the expression of MYO7A from single AAV2 and AAV5 vectors in cultured cells. FIG. 10A is a diagram of the viral vector encoding human MYO7A cDNA. FIG. 10B is a western blot of WT eyecup (lane 1), primary RPE cultures derived from Myo7a-null mice and infected with AAV2-MYO7A (lane 2) or AAV5-MYO7A (lane 3), or not infected (lane 4), and primary RPE cultures derived from Myo7a.sup.+/- mice (lane 5). All lanes were immunolabeled with antibodies against actin (as a loading indicator of relative protein loading) and MYO7A. FIG. 10C, FIG. 10D, FIG. 10E, and FIG. 10F are immunofluorescence images of primary RPE cell cultures. Cells derived from Myo7a-null mice that were not infected (FIG. 10C), from Myo7a.sup.+/- mice (FIG. 10D), or from Myo7a-null mice infected with either 1.times. AAV2-MYO7A (FIG. 10E) or 1.times. AAV5-MYO7A (FIG. 10F). Scale=10 .mu.m;

[0034] FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, FIG. 11E, FIG. 11F, FIG. 11F-1, FIG. 11G, FIG. 11G-1, FIG. 11H, FIG. 11I, FIG. 11J, FIG. 11K, FIG. 11L, and FIG. 11M show the expression of MYO7A from single AAV2 and AAV5 vectors in vivo. FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, and FIG. 11E show EM images of MYO7A immunogold labelling of the connecting cilium and pericilium from rod photoreceptors in a Myo7a-null retina. FIG. 11A is a longitudinal section from an untreated Myo7a-null retina (background label only). FIG. 11B and FIG. 11C are longitudinal sections from Myo7a-null retinas treated with AAV2-MYO7A (FIG. 11B) or AAV5-MYO7A (FIG. 11C). Scale=50 nm. FIG. 11D and FIG. 11E are transverse sections of connecting cilia from rod photoreceptors in Myo7a-null retinas treated with AAV2-MYO7A (FIG. 11D) or AAV5-MYO7A (FIG. 11E). Scale=50 nm. FIG. 11F and FIG. 11G show EM images of RPE cells from Myo7a-null retinas treated with AAV2-MYO7A (FIG. 11F) or AAV5-MYO7A (FIG. 11G). Scale=500 nm. BM=Bruch's membrane, AP=apical processes. Areas indicated by rectangles are enlarged in FIG. 11F-1 and FIG. 11G-1 to show MYO7A immunogold labeling (indicated by circles). Scale=50 nm. FIG. 11H and FIG. 11I show EM image of a longitudinal section of the connecting cilium and pericilium from a rod (FIG. 11H) and a cone (FIG. 11I) photoreceptor in a Myo7a-null retina, treated with AAV2-MYO7A. The section was double-labeled with MYO7A (12-nm gold) and rod opsin (15-nm gold) antibodies. Rod outer segments were labeled with the opsin antibody, while cones were identified by lack of rod opsin labeling in their outer segments. The sections show just the base of the outer segments. Nearly all the label in the connecting cilium is MYO7A, even in the rod. Scale=50 nm. FIG. 11J, FIG. 11K, FIG. 11L, and FIG. 11M are bar graphs indicating MYO7A immunogold particle density in the rod photoreceptor cilium and pericilium (FIG. 11J and FIG. 11K) and in the RPE (FIG. 11L and FIG. 11M), following treatment with AAV2-MYO7A (FIG. 11J and FIG. 11L) or AAV5-MYO7A (FIG. 11K and FIG. 11M) of different concentrations. n=3 animals per condition. Bars indicate SEM;

[0035] FIG. 12A, FIG. 12B, FIG. 12C, FIG. 12D, FIG. 12E, and FIG. 12F show correction of melanosome localization, following subretinal injections with AAV2-MYO7A or AAV5-MYO7A. Light micrographs showing the presence of melanosomes in the apical processes of the RPE in a WT retina (FIG. 12A) and retinas injected with AAV2-MYO7A (FIG. 12B) or AAV5-MYO7A (FIG. 12C). Further away from the injection site (FIG. 12D), melanosomes are present in the apical processes of some RPE cells, but not in others (arrows indicate apical melanosomes; white lines indicate regions where melanosomes are absent from the apical processes). FIG. 12E illustrates a region distant from injection site, where all RPE cells lacked melanosomes in their apical processes. Brackets on left side indicate RPE apical processes. Scale=25 .mu.m. FIG. 12F is a diagram of an eyecup, indicating the relative locations of the images shown in FIG. 12A, FIG. 12B, FIG. 12C, FIG. 12D, and FIG. 12E. Arrow indicates the site of injection; ONH indicates the optic nerve head;

[0036] FIG. 13 shows the correction of abnormal levels of opsin in the connecting cilium and pericilium of rod photoreceptors, following subretinal injections with AAV2-MYO7A or AAV5-MYO7A. The bar graph shows opsin immunogold gold particle density along the length of the connecting cilium. Ultrathin sections of retinas from Myo7a-null and WT mice were stained with rod opsin antibody. The Myo7a-null retinas had been untreated, or treated with either 1.times. or 1:100 AAV2-MYO7A or AAV5-MYO7A. n=3 animals per condition. Bars indicate SEM;

[0037] FIG. 14A-1, FIG. 14A-2, FIG. 14B, FIG. 14C, FIG. 14D, FIG. 14E, FIG. 14F, and FIG. 14G show the expression of MYO7A from the overlapping AAV2-MYO7A dual vectors. FIG. 14A-1 and FIG. 14A-2 illustrate a diagram of the overlapping AAV2-MYO7A dual vectors. The overlapping region contains 1365 bases. FIG. 14B is a Western blot of proteins from primary RPE cultures derived from Myo7a-null mice and not infected (lane 1), or infected with AAV2-MYO7A(dual) (lane 2); primary RPE cultures derived from Myo7a.sup.+/- mice (lane 3); WT eyecup (lane 4); HEK293A cells transfected with pTR-smCBA-MYO7A (lane 5). All lanes were immunolabeled with anti-MYO7A and anti-actin. FIG. 14C, FIG. 14D, FIG. 14E, and FIG. 14F show immunofluorescence of cultured RPE cells transduced with AAV2-MYO7A(dual). FIG. 14C, FIG. 14D, and FIG. 14E show primary RPE cultures derived from Myo7a-null mice and ARPE19 (FIG. 14F) cells. Scale=10 .mu.m. FIG. 14G is a bar graph indicating the distribution of MYO7A immunogold particle density among RPE cells from retinas of Myo7a-null mice, injected with AAV2-MYO7A(dual). n=3 animals;

[0038] FIG. 15A, FIG. 15B, FIG. 15C, FIG. 15D, FIG. 15E, FIG. 15F, and FIG. 15G illustrate correction of mutant phenotypes, following subretinal injections with AAV2-MYO7A(dual). FIG. 15A is the results of light microscopy of a semi-thin section from a treated Myo7a-null mouse retina. The region shown is near the injection site. Arrows indicate melanosomes in the apical processes. White lines indicate cells that still show the Myo7a-null phenotype, with an absence of melanosomes in the apical processes. Scale=50 .mu.m. FIG. 15B is a low-magnification immunoEM image of RPE from a retina treated with AAV2-MYO7A(dual). As in FIG. 15A, the white line indicated a region that still showed the Myo7a-null phenotype. Rectangle `c`, includes melanosomes in the apical region, indicating a corrected RPE cell. Scale=500 nm. FIG. 15C, FIG. 15D, and FIG. 15E show higher-magnification images of regions outlined by the rectangles shown in FIG. 15B. MYO7A immunogold particles were indicated by circles. Scale=50 nm. FIG. 15F is a bar graph illustrating MYO7A immunogold particle density measured in RPE cells from Myo7a-null retinas, WT retinas, or from Myo7a-null retinas treated with AAV2-MYO7A(dual) and determined to be corrected or not corrected by the location of their apical melanosomes. n=3 animals per condition. Bars indicate SEM. FIG. 15G is an immunoEM image of a rod photoreceptor cilium double-labeled with antibodies against MYO7A (small gold particles) and against rod opsin (large gold particles). MYO7A labeling is associated with the connecting cilium and periciliary membrane, indicating expression and correct localization of MYO7A. While this region is devoid of opsin labeling, which is restricted to the disk membranes, it is consistent with the wild type (WT) phenotype, thus indicating correction of the mutant phenotype. Scale=300 nm;

[0039] FIG. 16 shows the validation of dual AAV vectors for delivery of full-length MYO7A in vivo. Immunoblot showing expression of MYO7A (green) in retinas of wild type (C57BL/6) mice (lane 1), heterozygous shaker-1.sup.+/mice (lane 2) and shaker-1.sup.-/- mice injected with `simple overlap` Myo7a vectors packaged in AAV8(733) vectors. Both N-terminal and C-terminal vectors of the `simple overlap` system were injected at a concentration of 3.times.10.sup.10 vector genomes/.mu.L. Dual AAV vectors mediated expression of a MYO7A that was identical in size to that found in WT and shaker-1.sup.+/- mice. .beta.-actin (visualized here in red) was used as a loading control to validate that equal amounts of protein were loaded in each well;

[0040] FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, FIG. 17E, and FIG. 17F show AAV-mediated MYO7A expression in ARPE-19. Cells were transduced with 1.times. AAV2-MYO7A (FIG. 17A), AAV5-MYO7A (FIG. 17B), 1/100 dilutions thereof (FIG. 17C and FIG. 17D) and AAV2-MYO7A (dual) (FIG. 17F). Non-transduced cells were used as a control (FIG. 17E); Red, MYO7A; Blue, DAPI. Scale=10 .mu.m;

[0041] FIG. 18A, FIG. 18B, FIG. 18C, and FIG. 18D show MYO7A expression in the connecting cilium and pericilium of rod photoreceptors from Myo7a-null retinas injected with diluted AAV2-MYO7A (FIG. 18a and FIG. 18B) or AAV5-MYO7A (FIG. 18C and FIG. 18D); (FIG. 18A and FIG. 18C) 1:10, (FIG. 18B and FIG. 18D) 1:100. Scale=200 nm;

[0042] FIG. 19 shows the structural preservation of injected Myo7a-null retinas. Light microscopy of the photoreceptor layer 3 weeks after injection with 10.times. AAV5-MYO7A. Scale=15 .mu.m;

[0043] FIG. 20 shows structural preservation of injected Myo7a-null retinas. Light microscopy of photoreceptor layer 3 months after injection with 1.times. AAV2-MYO7A. Scale=10 .mu.m;

[0044] FIG. 21A, FIG. 21B, FIG. 21C, and FIG. 21D show correction of abnormal levels of opsin in the connecting cilium and pericilium of rod photoreceptors following subretinal injections with AAV2-MYO7A or AAV5-MYO7A. ImmunoEMs from WT retina (FIG. 21A), Myo7a-null retinas treated with 1.times. AAV2-MYO7A (FIG. 21B) or 1.times. AAV5-MYO7A (FIG. 21C), and from an untreated Myo7a-null retina (FIG. 21D) labeled with anti-rod opsin and 12-nm gold-conjugated secondary antibody. Scale=200 nm;

[0045] FIG. 22A, FIG. 22B, FIG. 22C, FIG. 22D, and FIG. 22E show a schematic representation of the dual-AAV-vector pairs created for this study. FIG. 22A is a fragmented vector (fAAV). FIG. 22B shows simple overlap: the 1365-bp shared between the two vectors is shaded gray. FIG. 22C is a trans-splicing vector. FIG. 22D shows an AP hybrid vector: the 270-bp element shared between the two vectors is marked with diagonal gradient shading (1/3 head as described by Ghosh et al., 2011). FIG. 22 shows the native intron hybrid vectors utilizing the natural intron 23 of MYO7A sharing a 250-bp overlapping sequence. 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; smCBA=cytomegalovirus immediate early/chicken .beta.-actin chimeric promoter;

[0046] FIG. 23A, FIG. 23B, and FIG. 23C show human embryonic kidney (HEK293) cells express human MYO7A after infection with simple overlap vectors (MOI of 10,000 for both vectors) packaged in AAV2(tripleY-F). Equal amounts of protein were separated on 7.5% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and stained for MYO7A. FIG. 23A shows HEK293 cells infected with AAV2(tripleY-F) at MOIs of 10,000, 2000, and 400 FIG. 23B is a time-course assay of MYO7A expressed in HEK293 cells. Cells were harvested 3-7 days after infection. In FIG. 23C, HEK293 cells were infected with AAV2 dual vectors; MOI=multiplicity of infection; T=HEK293 cells transfected with full-length MYO7A plasmid; U=untreated HEK293 cells;

[0047] FIG. 24 shows the comparison of AAV2 and AAV2(tripleY.fwdarw.F mutant capsid)-based vectors in HEK293 cells. Cells were infected with single fAAV, AP hybrid, and simple overlap MYO7A dual-vector platforms packaged in AAV2 or AAV2(tripleY.fwdarw.F mutant capsid) at an MOI of 10,000. HEK293 cells transfected with MYO7A plasmid were used as a positive control;

[0048] FIG. 25A, FIG. 25B, and FIG. 25C show human MYO7A expressed in HEK293 cells. Cells were infected with AAV2-based vector platforms. For each of the dual-vector systems, the corresponding 5' and 3' vectors (or the 5' vector alone) were used for infection. HEK293 cells transfected with MYO7A plasmid were used as a positive control. Cells were infected with the MYO7A dual-vector pairs at an MOI of 10,000 for each vector. Protein samples were analyzed on Western blot with an antibody against MYO7A (FIG. 25A). Each platform's relative ability 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 (FIG. 25B). Full-length MYO7A expression mediated by dual vectors was quantified relative to transfection control (FIG. 25C);

[0049] FIG. 26A, FIG. 26B, and FIG. 26C show the characterization of MYO7A dual-vectors' restoration of coding sequence. The experimental plan is shown in FIG. 26A. HEK293 cells were infected with AAV2-based dual-vector platforms, RNA was extracted, and gene-specific primers amplified the sequences using PCT. Control digests with BglII (B) and PpuMI (P) revealed the predicted banding pattern shown in FIG. 26B. Undigested (U) PCR product is shown as control and a DNA size marker for reference (M). Separately, products were digested with KpnI and AgeI, and then cloned into pUC57 for sequencing of the entire overlap region FIG. 26C. Ten clones per vector platform were analyzed. M13 forward- and reverse-primers specific for the subclone vector were used to obtain sense and antisense reads (each .about.1000 bp) resulting in .about.140 bp for which the sense and antisense reads overlapped. PCR=polymerase chain reaction and

[0050] FIG. 27A, FIG. 27B, FIG. 27C, FIG. 27D, FIG. 27E, FIG. 27F, FIG. 27G, and FIG. 27H show the dual vector-mediated MYO7A(HA) expression in vivo. C57BL/6J mice were injected subretinally with AAV2-based dual vectors containing a C' terminal HA tag. Retinal protein expression was analyzed four weeks later by immunohistochemistry and western blot. Ten-micron frozen retinal cross sections were imaged at 10.times. (FIG. 27A, FIG. 27C, and FIG. 27E) and 60.times. (FIG. 27B, FIG. 27D, and FIG. 27F). Equal amounts of protein were separated on a 4-15% polyacrylamide gel and stained with an HA antibody (FIG. 27H). For comparison, endogenous MYO7A from C57BL/6J retina (FIG. 27G) was probed with an antibody against MYO7A to confirm that HA-tagged MYO7A migrated at the appropriate size. RPE-retinal pigment epithelium, IS--inner segments, OS--outer segments, ONL--outer nuclear layer, INL--inner nuclear layer, GCL--ganglion cell layer, PR--photoreceptors.

BRIEF DESCRIPTION OF THE SEQUENCES

[0051] SEQ ID NO:1 is the nucleotide sequence of an "hMyo7a coding overlap vector A" of the subject invention;

[0052] SEQ ID NO:2 is the nucleotide sequence of an "hMyo7a coding overlap vector B" of the subject invention;

[0053] SEQ ID NO:3 is the nucleotide sequence of an "hMyo7a intron 23 splicing vector A" of the subject invention;

[0054] SEQ ID NO:4 is the nucleotide sequence of an "hMyo7a intron 23 splicing vector B" of the subject invention;

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

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

[0057] SEQ ID NO:7 is a nucleotide sequence that encodes a human myosin VIIa polypeptide;

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

[0059] SEQ ID NO:9 is a synthetic oligonucleotide sequence, designated herein as P1, useful in accordance with one aspect of the present invention;

[0060] SEQ ID NO:10 is a synthetic oligonucleotide sequence, designated herein as P2, useful in accordance with one aspect of the present invention;

[0061] SEQ ID NO:1 is a synthetic oligonucleotide sequence, designated herein as P3, useful in accordance with one aspect of the present invention;

[0062] SEQ ID NO:12 is a synthetic oligonucleotide sequence, designated herein as P4, useful in accordance with one aspect of the present invention;

[0063] SEQ ID NO:13 is a synthetic oligonucleotide sequence, designated herein as P5, useful in accordance with one aspect of the present invention;

[0064] SEQ ID NO:14 is a synthetic oligonucleotide sequence, designated herein as P6, useful in accordance with one aspect of the present invention;

[0065] SEQ ID NO:15 is a synthetic oligonucleotide sequence, designated herein as P7, useful in accordance with one aspect of the present invention;

[0066] SEQ ID NO:16 is a synthetic oligonucleotide sequence, designated herein as P8, useful in accordance with one aspect of the present invention;

[0067] SEQ ID NO:17 is a synthetic oligonucleotide sequence, designated herein as P9, useful in accordance with one aspect of the present invention;

[0068] SEQ ID NO:18 is a synthetic oligonucleotide sequence, designated herein as P10, useful in accordance with one aspect of the present invention;

[0069] SEQ ID NO:19 is a synthetic oligonucleotide sequence, designated herein as P11, useful in accordance with one aspect of the present invention;

[0070] SEQ ID NO:20 is a synthetic oligonucleotide sequence, designated herein as P12, useful in accordance with one aspect of the present invention;

[0071] SEQ ID NO:21 is a synthetic oligonucleotide sequence, designated herein as P13, useful in accordance with one aspect of the present invention;

[0072] SEQ ID NO:22 is a synthetic oligonucleotide sequence, designated herein as P14, useful in accordance with one aspect of the present invention;

[0073] SEQ ID NO:23 is a synthetic oligonucleotide sequence, designated herein as P15, useful in accordance with one aspect of the present invention;

[0074] SEQ ID NO:24 is a synthetic oligonucleotide sequence, designated herein as P16, useful in accordance with one aspect of the present invention;

[0075] SEQ ID NO:25 is a synthetic oligonucleotide sequence, designated herein as P17, useful in accordance with one aspect of the present invention;

[0076] SEQ ID NO:26 is a synthetic oligonucleotide sequence, designated herein as P18, useful in accordance with one aspect of the present invention;

[0077] SEQ ID NO:27 is a synthetic oligonucleotide sequence, designated herein as P19, useful in accordance with one aspect of the present invention;

[0078] SEQ ID NO:28 is a synthetic oligonucleotide sequence, designated herein as P20, useful in accordance with one aspect of the present invention;

[0079] SEQ ID NO:29 is a synthetic oligonucleotide sequence, designated herein as P21, useful in accordance with one aspect of the present invention; and

[0080] SEQ ID NO:30 is a synthetic oligonucleotide sequence, designated herein as P22, useful in accordance with one aspect of the present invention.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0081] Illustrative embodiments of the invention are described below. In the interest of clarity, not all features of an actual implementation are described in this specification. It will of course be appreciated that in the development of any such actual embodiment, numerous implementation-specific decisions must be made to achieve the developers' specific goals, such as compliance with system-related and business-related constraints, which will vary from one implementation to another. Moreover, it will be appreciated that such a development effort might be complex and time-consuming, but would be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure.

[0082] The subject invention concerns materials and methods for genetic therapy of diseases and conditions, such as Usher syndrome 1B (USH1B). One aspect of the invention concerns AAV-based dual-vector systems that provide for expression of full-length proteins whose coding sequence exceeds the polynucleotide packaging capacity of individual AAV vector. In one embodiment, a vector system of the invention includes:

[0083] i) a first AAV vector polynucleotide comprising an inverted terminal repeat at each end (5' and 3' end) of the polynucleotide, and between the inverted terminal repeats a suitable promoter followed by (i.e., 3' to the promoter) a partial coding sequence that encodes an N-terminal part of a selected full-length polypeptide, and

[0084] ii) a second AAV vector polynucleotide comprising an inverted terminal repeat at each end (i.e., the 5'- and 3'-ends) of the polynucleotide, and between the inverted terminal repeats a partial coding sequence that encodes a C-terminal portion of the selected full-length polypeptide, and optionally followed by a polyadenylation (pA) sequence. The coding sequences in the first and second vectors when combined encode the selected full-length polypeptide, or a functional fragment or variant thereof. The polypeptide encoding sequence in the first and second AAV vectors comprises sequence that overlaps.

[0085] In other words, a portion of the coding sequence present at the 3'-end of the coding sequence of the first vector is identical or substantially identical with a portion of the coding sequence present at the 5'-end of the coding sequence of the second vector. In one embodiment, the sequence overlap between the first and second AAV vectors is between about 500 and about 3000 nucleotides; between about 1000 and about 2000 nucleotides; between about 1200 and about 1800 nucleotides; or between about 1300 and about 1400 nucleotides.

[0086] In a specific embodiment, the sequence overlap is about 1350 nucleotides. In an exemplified embodiment, the sequence overlap is approximately 1365 nucleotides. In one embodiment, the polypeptide encoded is wild type or functional human myosin VIIa (hmyo7a). Amino acid sequences of wild type and functional hmyo7a polypeptides, and polynucleotides encoding them, are known in the art (see, for example, GenBank accession numbers NP_000251 and U39226.1). In one embodiment, an hmyo7a polypeptide comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:8, or a functional fragment or a variant thereof. In one embodiment, the hmyo7a polypeptide is encoded by the nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:7. Other polypeptides contemplated include, but are not limited to, harmonin (Uniprot Q9Y6N9), cadherin 23 (Uniprot Q9H251), protocadherin 15 (Uniprot Q96QU1), and usherin (USH2A) (Uniprot O75445). In an exemplified embodiment, 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. In one embodiment, a construct or vector of the invention is administered by parenteral administration, such as intravenous, intramuscular, intraocular, intranasal, etc. In a specific embodiment, a construct or vector is administered by subretinal injection. The construct or vector can be administered in vivo or ex vivo.

[0087] In another embodiment, a vector system of the invention comprises i) a first AAV vector polynucleotide comprising an inverted terminal repeat at each end (i.e., the 5'-end and the 3'-end) of the polynucleotide, and between the inverted terminal repeats a suitable promoter followed by (i.e., 3' to the promoter) a partial coding sequence that encodes an N-terminal part 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 (5'-end and 3'-end) of the polynucleotide, and between the inverted terminal repeats an intron and a splice acceptor site for the intron, followed by a partial coding sequence that encodes a C-terminal part of the selected full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence.

[0088] The coding sequences in the first and second vectors when combined encode the selected full-length polypeptide, or a functional fragment or variant thereof. The intron sequence in the first and second AAV vectors comprises sequence that overlaps. In other words, all or part of the intron sequence present at the 3'-end of the coding sequence of the first vector is identical or substantially identical with all or part of the intron sequence present at the 5'-end of the coding sequence of the second vector. In one embodiment, intron sequence overlap between the first and second AAV vectors is several hundred nucleotides in length. In a specific embodiment, the intron sequence overlap is about 50 to about 500 nucleotides or so in length; alternatively between about 200 and about 300 nucleotides or so in length. In one embodiment, the intron sequence utilized in the vector system of the invention is a sequence of an intron naturally present in the genomic sequence of a gene encoding the selected polypeptide. In one embodiment, the intron is intron 23 of the hmyo7a gene. In a specific embodiment, the polypeptide encoded is hmyo7a and the intron is the full intron 23 of the hmyo7a gene. In an exemplified embodiment, the first AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO:3, or a functional fragment and/or variant thereof, and the second AAV vector polynucleotide comprises the nucleotide sequence of SEQ ID NO:4, or a functional fragment and/or variant thereof. In another embodiment, the intron sequence utilized in the vector system of the invention is a sequence of an intron that is not naturally present in the genomic sequence of a gene encoding the selected polypeptide. In a specific embodiment, the intron is a synthetic alkaline phosphatase (AP) intron. The intron sequences utilized in the vector system of the present invention can comprise splice donor and splice acceptor sequences. In one embodiment, a construct or vector of the invention is administered by parenteral administration, such as intravenous, intramuscular, intraocular, intranasal, etc. In a specific embodiment, a construct or vector is administered by subretinal injection. The construct or vector can be administered in vivo or ex vivo.

[0089] The inverted terminal repeat (ITR) sequences used in an AAV vector system of the present invention can be any AAV ITR. The ITRs used in an AAV vector can be the same or different. In particular embodiments, the ITR may be obtained from an AAV serotype 2 (AAV2) or an AAV serotype 5 (AAV5). An AAV vector of the invention can comprise different AAV ITRs. For example, a vector may comprise an ITR of AAV2 and an ITR of AAV5. AAV ITR sequences are well known in the art (see, for example, GenBank Accession Nos. AF043303.1; NC_001401.2; J01901.1; JN898962.1; K01624.1; and K01625.1).

[0090] The AAV dual-vector systems disclosed herein are able to efficiently express a therapeutic gene that is larger than what may ordinarily be packaged within a single AAV vector.

[0091] The subject invention also concerns a virus or virion comprising a polynucleotide, expression construct, or vector construct of the invention. In one embodiment, the virus or virion is an AAV virus. Methods for preparing viruses and virions comprising a heterologous polynucleotide or construct are known in the art. In the case of AAV, cells can be co-infected or transfected with adenovirus or polynucleotide constructs comprising adenovirus genes suitable for AAV helper function. Examples of materials and methods are described, for example, in U.S. Pat. Nos. 8,137,962 and 6,967,018 (each of which is specifically incorporated herein, by express reference thereto).

[0092] An AAV virus or AAV vector of the invention can be of any AAV serotype, including, but not limited to, serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In exemplary embodiments, AAV2 and AAV5 serotype vectors have been utilized.

[0093] In one embodiment, the AAV serotype provides for one or more tyrosine to phenylalanine (Y.fwdarw.F) mutations on the capsid surface. In a specific embodiment, the AAV is an AAV8 serotype having a tyrosine-to-phenylalanine (Tyr.fwdarw.Phe) mutation at position 733 (Y733F). In FIG. 5, a triple-mutant vector is also contemplated. It contains tyrosine-to-phenylalanine (Tyr.fwdarw.Phe) mutations at positions Y733F, Y500F, and Y730F, respectively, corresponding to the amino acid sequence of the wild-type AAV8-capsid protein, or in one or more of the amino acids corresponding to those sequences in one or more related AAV capids, including, for example, AAV2, AAV5, and the like.

[0094] The subject invention also concerns methods for treating or ameliorating a disease or condition, such as an eye disease, in a human or animal using gene therapy and an AAV-based dual-vector system of the present invention. In one embodiment, a method of the invention comprises administering a vector system of the invention that encodes a polypeptide that provides for treatment or amelioration of the disease or condition. In one embodiment, the vectors of the invention are provided in an AAV virus or virion. The vector system can be administered in vivo or ex vivo. In one embodiment, a vector system of the invention is administered by parenteral administration, such as intravenous, intramuscular, intraocular, intranasal, etc. Administration can be by injection. In a specific embodiment, a vector system of the invention is administered to the human or animal by intraocular subretinal injection. In one embodiment, the disease or condition to be treated is Usher syndrome and the polypeptide provided is a mammalian myosin VIIa protein. In a specific embodiment, the myosin VIIa is a human myosin VIIa polypeptide. In one embodiment, an hmyo7a polypeptide comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:8, or a functional fragment or a variant thereof. In one embodiment, the hmyo7a polypeptide is encoded by the nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:7.

[0095] Other polypeptides contemplated include, but are not limited to, harmonin (Uniprot Q9Y6N9), cadherin 23 (Uniprot Q9H251), protocadherin 15 (Uniprot Q96QU1), and usherin (USH2A) (Uniprot O75445). Dosage regimes and effective amounts to be administered can be determined by ordinarily skilled clinicians. Administration may be in the form of a single dose or multiple doses. General methods for performing gene therapy using polynucleotides, expression constructs, and vectors are known in the art (see, for example, 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 in its entirety by express reference thereto).

[0096] The subject invention also concerns methods for expressing a selected polypeptide in a cell. In one embodiment, the method comprises incorporating in the cell an AAV-based, dual-vector system as disclosed herein, wherein the vector system includes a polynucleotide sequence that encodes a selected polypeptide and of interest, and expressing the polynucleotide sequences in the cell. In certain embodiments, the selected polypeptide may be a polypeptide that is heterologous to the cell. In one embodiment, the cell is a mammalian cell, and preferably, a human cell. In one embodiment, the cell is human a photoreceptor cell, and preferably a human photoreceptor cone cell or a photoreceptor rod cell. In a specific embodiment, the cell expresses a wild type, functional, and/or biologically-active hmyo7a polypeptide that is encoded by a nucleic acid segment present in a vector system as disclosed herein. In one embodiment, the hmyo7a polypeptide is encoded by the nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:7. Other polypeptides contemplated include, but are not limited to, harmonin (Uniprot Q9Y6N9), cadherin 23 (Uniprot Q9H251), protocadherin 15 (Uniprot Q96QU1), and usherin (USH2A) (Uniprot O75445). The cell can be one that is provided in vivo or in vitro.

[0097] The subject invention also concerns one or more mammalian cells that contain one of the AAV-based, dual-vector systems disclosed herein.

[0098] In one embodiment, the cell is a photoreceptor cell. In a specific embodiment, the cell is a cone cell; preferably, it is a human cone cell or a human rod cell. Such cells may express one or more nucleotide sequences provided in at least a first AAV-based, dual-vector system of the invention. In a specific embodiment, the cell expresses a wild type, functional, and/or biologically active hmyo7a polypeptide that is encoded by a nucleic acid segment comprised within one or more of the AAV-based vector systems as disclosed herein. In one embodiment, the hmyo7a polypeptide is encoded by the nucleotide sequence of SEQ ID NO:5 or SEQ ID NO:7. Other polypeptides contemplated include, but are not limited to, harmonin (Uniprot Q9Y6N9), cadherin 23 (Uniprot Q9H251), protocadherin 15 (Uniprot Q96QU1), and usherin (USH2A) (Uniprot O75445).

[0099] Vector systems of the invention can include regulatory elements that are functional in the intended host cell in which the vector is to be expressed. A person of ordinary skill in the art can select regulatory elements for use in appropriate host cells, for example, mammalian or human host cells. Regulatory elements include, for example, promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements.

[0100] A vector of the invention can comprise a promoter sequence operably linked to a nucleotide sequence encoding a desired polypeptide. Promoters contemplated for use in the subject invention include, but are not limited to, cytomegalovirus (CMV) promoter, SV40 promoter, Rous sarcoma virus (RSV) promoter, chimeric CMV/chicken .beta.-actin promoter (CBA) and the truncated form of CBA (smCBA) (see, e.g., Haire et al. 2006 and U.S. Pat. No. 8,298,818, which is specifically incorporated herein in its entirety by express reference thereto). Additional photoreceptor-specific, human rhodopsin kinase (hGRK1) promoter, rod specific IRBP promoter, VMD2 (vitelliform macular dystrophy/Best disease) promoter, and EF1 alpha promoter sequences are also contemplated to be useful in the practice of various aspects of the present invention.

[0101] In a specific embodiment, the promoter is a chimeric CMV-.beta.-actin promoter. In one embodiment, the promoter is a tissue-specific promoter that shows selective activity in one or a group of tissues but is less active or not active in other tissue. In one embodiment, the promoter is a photoreceptor-specific promoter. In a further embodiment, the promoter is preferably a cone cell-specific promoter or a rod cell-specific promoter, or any combination thereof. In one embodiment, the promoter is the promoter for human myosin 7a gene. In a further embodiment, the promoter comprises a cone transducin .alpha. (T.alpha.C) gene-derived promoter. In a specific embodiment, the promoter is a human GNAT2-derived promoter. Other promoters contemplated within the scope of the invention include, without limitation, a rhodopsin promoter, a cGMP-phosphodiesterase .beta.-subunit promoter, a retinitis pigmentosa-specific promoter, an RPE cell-specific promoter [such as a vitelliform macular dystrophy-2 (VMD2) promoter (Best1) (Esumi et al., 2004)], or any combination thereof.

[0102] Promoters can be incorporated into a vector using standard techniques known to those of ordinary skill in the molecular biology and/or virology arts. Multiple copies of promoters, and/or multiple distinct promoters can be used in the vectors of the present invention. In one such embodiment, a promoter may be positioned about the same distance from the transcription start site as it is from the transcription start site in its natural genetic environment, although some variation in this distance is permitted, of course, without a substantial decrease in promoter activity. In the practice of the invention, one or more transcription start site(s) are typically included within the disclosed vectors.

[0103] The vectors of the present invention may further optionally include 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 typically be obtained from the 3' untranslated region of a eukaryotic or viral gene sequence. Transcription termination sequences can be positioned downstream of a coding sequence to provide for efficient termination. Signal peptide sequences are amino-terminal peptidic sequences that encode information responsible for the location of an operably-linked polypeptide to one or more post-translational cellular destinations, including, for example, specific organelle compartments, or to the sites of protein synthesis and/or activity, and even to the extracellular environment.

[0104] Enhancers--cis-acting regulatory elements that increase gene transcription--may also be included in one of the disclosed AAV-based vector systems. A variety of enhancer elements are known to those of ordinary skill in the relevant arts, and include, without limitation, a CaMV 35S enhancer element, a cytomegalovirus (CMV) early promoter enhancer element, an SV40 enhancer element, as well as combinations and/or derivatives thereof. One or more nucleic acid sequences that direct or regulate polyadenylation of the mRNA encoded by a structural gene of interest, may also be optionally included in one or more of the vectors of the present invention.

[0105] The disclosed dual-vector systems may be introduced into one or more selected mammalian cells using any one or more of the methods that are known to those of ordinary skill in the gene therapy and/or viral arts. Such methods include, without limitation, transfection, microinjection, electroporation, lipofection, cell fusion, and calcium phosphate precipitation, as well as biolistic methods. In one embodiment, the vectors of the invention may be introduced in vivo, including, for example, by lipofection (i.e., DNA transfection via liposomes prepared from one or more cationic lipids) (see, for example, Felgner et al., 1987). Synthetic cationic lipids (LIPOFECTIN, Invitrogen Corp., La Jolla, Calif., USA) may be used to prepare liposomes that will encapsulate the vectors to facilitate their introduction into one or more selected cells. A vector system of the invention can also be introduced in vivo as "naked" DNA using methods known to those of ordinary skill in the art.

[0106] Polynucleotides described herein can also be defined in terms of more particular identity and/or similarity ranges with those exemplified herein. The sequence identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and can be greater than 95%. The identity and/or similarity of a sequence can 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 as compared to a sequence exemplified herein.

[0107] Unless otherwise specified, as used herein 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 with the NBLAST program, score=100, word-length=12, to obtain sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described (Altschul et al., 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) can be used in accordance with published methods.

[0108] The subject invention also contemplates those polynucleotide molecules having sequences that are sufficiently homologous with the polynucleotide sequences of the invention to permit hybridization with that sequence under standard stringent conditions and standard methods (Maniatis et al., 1982). As used herein, "stringent" conditions for hybridization refers to conditions wherein hybridization is typically carried out overnight at 20-25.degree. C. below the melting temperature (T.sub.m) of the DNA hybrid in 6.times.SSPE, 5.times. Denhardt's solution, and 0.1% SDS, containing 0.1 mg/mL of a suitable non-specific denatured DNA. Calculation of the melting temperature may be obtained using the standard formula of Beltz et al., (1983):

T.sub.m=81.5.degree. C.+16.6 Log[Na.sup.+]+0.41(% G+C)-0.61(% formamide)-600/length of duplex in base pairs.

[0109] Washes are typically carried out as follows:

[0110] (1) Twice at room temperature for 15 min in 1.times.SSPE, 0.1% SDS (i.e., a low-stringency wash); and

[0111] (2) Once at T.sub.m-20.degree. C. for 15 min in 0.2.times.SSPE, 0.1% SDS (i.e., a moderate-stringency wash).

[0112] As used herein, the terms "nucleic acid" and "polynucleotide sequence" refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides. The polynucleotide sequences include both full-length sequences, as well as shorter sequences derived from the full-length sequences. It is understood that a particular polynucleotide sequence includes the degenerate codons of the native sequence or sequences that may be introduced to provide codon preference in a specific host cell. The polynucleotide sequences falling within the scope of the subject invention further include sequences that specifically hybridize with the sequences coding for a peptide of the invention. The polynucleotide includes both the sense and antisense strands, either as individual strands or in the duplex.

[0113] Fragments and variants of a polynucleotide or polypeptide of the present invention can be generated as described herein and tested for the presence of function using standard techniques known in the art. Thus, an ordinarily skilled artisan can readily prepare and test fragments and variants of a polynucleotide or polypeptide of the invention and determine whether the fragment or variant retains functional activity that is the same or similar to a full-length or a non-variant polynucleotide or polypeptide, such as a myosin VIIa polynucleotide or polypeptide.

[0114] As those skilled in the art can readily appreciate, there can be a number of variant sequences of a protein found in nature, in addition to those variants that can be artificially created by the skilled artisan in the lab. The polynucleotides and polypeptides of the subject invention encompasses those specifically exemplified herein, as well as any natural variants thereof, as well as any variants which can be created artificially, so long as those variants retain the desired functional activity.

[0115] Also within the scope of the subject invention are polypeptides which have the same amino acid sequences of a polypeptide exemplified herein except for amino acid substitutions, additions, or deletions within the sequence of the polypeptide, as long as these variant polypeptides retain substantially the same relevant functional activity as the polypeptides specifically exemplified herein. For example, conservative amino acid substitutions within a polypeptide that do not affect the function of the polypeptide would be within the scope of the subject invention. Thus, the polypeptides disclosed herein should be understood to include variants and fragments, as discussed above, of the specifically exemplified sequences.

[0116] The subject invention further includes nucleotide sequences that encode the polypeptides disclosed herein. These nucleotide sequences can be readily constructed by those skilled in the art having the knowledge of the protein and amino acid sequences that are presented herein. As would be appreciated by one skilled in the art, the degeneracy of the genetic code enables the artisan to construct a variety of nucleotide sequences that encode a particular polypeptide or protein. The choice of a particular nucleotide sequence could depend, for example, upon the codon usage of a particular expression system or host cell.

[0117] Polypeptides having substitution of amino acids other than those specifically exemplified in the subject polypeptides are also contemplated within the scope of the present invention. For example, non-natural amino acids can be substituted for the amino acids of a polypeptide of the invention, so long as the polypeptide having substituted amino acids retains substantially the same activity as the polypeptide in which amino acids have not been substituted. Examples of non-natural amino acids include, but are not limited to, ornithine, citrulline, hydroxyproline, homoserine, phenylglycine, taurine, iodotyrosine, 2,4-diaminobutyric acid, .alpha.-amino isobutyric acid, 4-aminobutyric acid, 2-amino butyric acid, .gamma.-amino butyric acid, .epsilon.-amino hexanoic acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid, .tau.-butylglycine, .tau.-butylalanine, phenylglycine, cyclohexylalanine, .beta.-alanine, fluoro-amino acids, designer amino acids such as .beta.-methyl amino acids, C-methyl amino acids, N-methyl amino acids, and amino acid analogs in general. Non-natural amino acids also include amino acids having derivatized side groups. Furthermore, any of the amino acids in the protein can be of the D--(dextrorotary) or the L--(levorotary) form.

[0118] Amino acids can be generally categorized in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby a polypeptide having an amino acid of one class is replaced with another amino acid of the same class fall within the scope of the subject invention so long as the polypeptide having the substitution retains substantially the same biological activity as a polypeptide that does not have the substitution. Table 1 provides a listing of examples of amino acids belonging to each class.

TABLE-US-00001 TABLE 1 Class of Amino Add Examples of Amino Acids Nonpolar Ala, Val, Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr, Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

[0119] Also within the scope of the subject invention are polynucleotides that have the same, or substantially the same, nucleotide sequence of a polynucleotide 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 (i.e., 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.

[0120] The methods of the present invention can be used with humans and other animals. The other animals contemplated within the scope of the invention include domesticated, agricultural, or zoo- or circus-maintained animals. Domesticated animals include, for example, dogs, cats, rabbits, ferrets, guinea pigs, hamsters, pigs, monkeys or other primates, and gerbils. Agricultural animals include, for example, horses, mules, donkeys, burros, cattle, cows, pigs, sheep, and alligators. Zoo- or circus-maintained animals include, for example, lions, tigers, bears, camels, giraffes, hippopotamuses, and rhinoceroses. As used herein, the terms "patient" and "subject" are used interchangeably and are intended to include such human and non-human species Likewise, in vitro methods of the present invention may also be performed on cells of one or more human or non-human, mammalian species.

[0121] As one of ordinary skill in the molecular biological arts can readily appreciate, there can be a number of variant sequences of a gene or polynucleotide found in nature, in addition to those variants that may be artificially prepared or synthesized by an ordinary-skilled artisan in a laboratory environment. The polynucleotides of the subject invention encompasses those specifically exemplified herein, as well as any natural variants thereof, as well as any variants which can be created artificially, so long as those variants retain the desired biological activity.

[0122] Also within the scope of the subject invention are polynucleotides which have the same nucleotide sequences of a polynucleotide exemplified herein except for nucleotide substitutions, additions, or deletions within the sequence of the polynucleotide, as 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.

[0123] Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should 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 comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic 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. Optionally, the prevention of the action of microorganisms can be brought about by various other antibacterial and antifungal agents, for example, 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 that delay absorption, for example, aluminum monostearate and gelatin.

[0124] The present invention also concerns pharmaceutical compositions comprising a vector system of the invention in combination with a pharmaceutically acceptable carrier. Pharmaceutical compositions adapted for topical or parenteral administration, comprising an amount of a compound constitute a preferred embodiment of the invention. The dose administered to a patient, particularly a human, in the context of the present invention should be sufficient to achieve a therapeutic response in the patient over a reasonable timeframe, without lethal toxicity, and preferably causing no more than an acceptable level of side effects or morbidity. One skilled in the art will recognize that dosage will depend upon a variety of factors including the condition (health) of the subject, the body weight of the subject, kind of concurrent treatment, if any, frequency of treatment, therapeutic ratio, as well as the severity and stage of the pathological condition.

[0125] The subject invention also concerns kits comprising a vector system of the invention in one or more containers. Kits of the invention can optionally include pharmaceutically acceptable carriers and/or diluents. In one embodiment, a kit of the invention includes one or more other components, adjuncts, or adjuvants as described herein. In one embodiment, a kit of the invention includes instructions or packaging materials that describe how to administer a vector system contained within the kit to a selected mammalian recipient. Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In one embodiment, a vector system of the invention is provided in the kit as a solid. In another embodiment, a vector system of the invention is provided in the kit as a liquid or solution. In certain embodiments, the kits may include one or more ampoules or syringes that contain a vector system of the invention in a suitable liquid or solution form.

[0126] Multiple distinct AAV-based, dual-vector systems have been created and disclosed herein for use in gene-replacement therapies, including, for example, in the treatment of USH1B in human patients. In a specific embodiment, a vector system of the present invention employs two discrete AAV vectors that each packages a maximal-size DNA molecule (i.e., .about.4.5 to 4.8 Kb). The two vectors are co-administered to selected recipient cells to reconstitute the full-length, biologically-active, Myo7a polypeptide. In these constructs, a portion of overlapping nucleic acid sequence is common to each of the vector genomes (see FIG. 1). When co-delivered to suitable cells, the overlapping sequence region facilitates the proper concatamerization of the two partial gene cassettes. These gene cassettes then undergo homologous recombination to produce a full-length gene cassette within the cells (see FIG. 1). Shared components of exemplified embodiments of the dual-vector systems include the use of AAV inverted terminal repeats (TR), the small version of the chimeric CMV/chicken .beta.-actin promoter (smCBA), human Myo7a (hMyo7a) cDNA sequence and the SV40 polyadenylation (pA) signal.

Exemplary Definitions

[0127] In accordance with the present invention, polynucleotides, nucleic acid segments, nucleic acid sequences, and the like, include, but are not limited to, DNAs (including, but not limited to, genomic and/or extragenomic DNAs), genes, peptide nucleic acids (PNAs) RNAs (including, but not limited to, rRNAs, mRNAs, and/or tRNAs), nucleosides, as well as 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.

[0128] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and compositions similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and compositions are described herein. For purposes of the present invention, the following terms are defined below:

[0129] The term "effective amount," as used herein, refers to an amount that is capable of treating or ameliorating a disease or condition or otherwise capable of producing an intended therapeutic effect.

[0130] The term "operably linked," as used herein, refers to that the nucleic acid sequences being linked are typically contiguous, or substantially contiguous, and, where necessary to join two protein coding regions, contiguous and in reading frame. However, since enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous.

[0131] The term "promoter," as used herein, refers to a region or regions of a nucleic acid sequence that regulates transcription.

[0132] The term "regulatory element," as used herein, refers to a region or regions of a nucleic acid sequence that regulates transcription. Exemplary regulatory elements include, but are not limited to, enhancers, post-transcriptional elements, transcriptional control sequences, and such like.

[0133] The term "substantially corresponds to," "substantially homologous," or "substantial identity," as used herein, denote a characteristic of a nucleic acid or an amino acid sequence, wherein a selected nucleic acid or amino acid sequence has at least about 70 or about 75 percent sequence identity as compared to a selected reference nucleic acid or amino acid sequence. More typically, the selected sequence and the reference sequence will have at least about 76, 77, 78, 79, 80, 81, 82, 83, 84 or even 85 percent sequence identity, and more preferably, at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequence identity. More preferably still, highly homologous sequences often share greater than at least about 96, 97, 98, or 99 percent sequence identity between the selected sequence and the reference sequence to which it was compared.

[0134] 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 which total less than about 25 percent or so of the chosen reference sequence. The reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome. However, in the case of sequence homology of two or more polynucleotide sequences, the reference sequence will typically comprise at least about 18-25 nucleotides, more typically at least about 26 to 35 nucleotides, and even more typically at least about 40, 50, 60, 70, 80, 90, or even 100 or so nucleotides.

[0135] When highly-homologous fragments are desired, the extent 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 of the sequence comparison algorithms well-known to those of ordinary skill in the art, such as e.g., the FASTA program analysis described by Pearson and Lipman (1988).

[0136] The term "subject," as used herein, describes an organism, including mammals such as primates, to which treatment with the compositions according to the present invention 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, domesticated animals, including dogs and cats, as well as livestock such as horses, cattle, pigs, sheep, and goats, or other mammalian species including, without limitation, mice, rats, guinea pigs, rabbits, hamsters, and the like.

[0137] The term "treatment" or any grammatical variation thereof (e.g., treat, treating, and treatment etc.), as used herein, includes but is not limited to, alleviating a symptom of a disease or condition; and/or reducing, suppressing, inhibiting, lessening, ameliorating or affecting the progression, severity, and/or scope of a disease or condition.

[0138] The term "vector," as used herein, refers to a nucleic acid molecule (typically one containing DNA) that is capable of replication in a suitable host cell, or one to which another nucleic acid segment can be operatively linked so as to facilitate replication of the operably-nucleic acid segment. Exemplary vectors include, without limitation, plasmids, cosmids, viruses and the like.

[0139] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

EXAMPLES

[0140] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

Dual-AAV Vector System 1: hMyo7A Coding Overlap

[0141] In the case of Dual-Vector System 1 (FIG. 2) the overlapping DNA sequence shared by both vector A and vector B consists of a 1350-bp coding region for the human Myo7a gene. This is the simplest system of the present invention, and appears to be highly efficient in terms of full-length gene reconstitution, and MYO7A expression. Advantageously, each vector is of standard AAV packaging size, and as such, each packages DNA with a high degree of efficiency, and is readily adaptable to conventional GMP standards. Such vectors are also readily characterized to permit requisite regulatory approval prior to use in humans.

Example 2

Dual-AAV Vector System 2: hMyo7A Intron 23 Splicing

[0142] In the case of Dual-Vector System 2 (FIG. 3) the overlapping DNA sequence is composed of the native intron 23 of human Myo7a. Vector A contains the coding sequence corresponding to the amino-terminal portion of the hMyo7a cDNA relative to intron 23 (hMyo7aNT) and the native splice-donor site, followed by the entire intron 23 of hMyo7a (minus the native acceptor site). Vector B contains the carboxy-terminal portion of the hMyo7a cDNA relative to intron 23 (hMyo7aCT), and the full intron 23 of Myo7a (minus the native splice-donor site), followed by the native splice-acceptor site. Upon co-delivery to suitable mammalian host cells, the DNA of vectors A and B recombine to form a reconstituted full-length gene cassette. The resulting RNA transcript will then `splice out` the native intron. Alternatively, recombination and formation of the gene cassette can occur via the AAV TRs. In this case, the RNA transcript will `splice out` the native intron23--TR-intron23 motif. In both cases, however, the resulting mRNA is that of the reconstituted full-length hMyo7a gene sequence.

Example 3

In Vitro Performance of System 1: hMyo7A Coding Overlap

[0143] HEK293 cells were infected simultaneously with vector A and vector B of Dual-Vector System 1 (FIG. 2) at a ratio of 10000:1 vg/cell for each vector. The AAV vectors were then packaged in AAV2 virions that contain three Y.fwdarw.F mutations in the capsid protein (see, for example, Zhong et al., 2008). As a positive control, cells were transfected with plasmid containing full-length hMyo7a under the control of smCBA. Protein was recovered from cells at 3-, 4-, 5-, 6- and 7-days' post-infection, and an antibody directed against MYO7A was used to assay for its presence in the infected cells via immunoblotting. The results are shown in FIG. 6A and FIG. 6B. The area inside the white box is magnified, and presented at higher contrast on the right. Starting at 3-days' post-infection, the full-length human MYO7a protein was visible; peak expression of the protein occurred around Day 5.

Example 4

In Vivo Performance of System 1: hMyo7A Coding Overlap

[0144] Six week old shaker-1 (Myo7a null) mice were sub-retinally co-injected with 1 .mu.L of the same preparations of vector A and vector B of Dual-Vector System 1 used in the above in vitro study. Both vectors contained .about.1.times.10.sup.12 vg/mL. Four weeks' post-injection, retinas from treated and untreated eyes were collected and immunohistochemistry (IHC) was performed using an antibody directed against MYO7A (see FIG. 7A and FIG. 7B). The area in green showed MYO7A-specific staining, while the areas in blue corresponded to the nuclear-specific, DAPI stain. In the treated eye, MYO7A expression was clearly visible, and it appeared to be restricted to photoreceptors--more precisely to the juncture of the photoreceptor inner and outer segments.

Example 5

AAV Dual Vectors Efficiently Deliver Oversized Genes

[0145] Animals. Shaker-1 mice carrying the 4626SB allele, an effective null mutation (Liu et al., 1999; Hasson et al., 1997), were used on the C57BL6 genetic background, and maintained and genotyped as described (Liu et al., 1999; Gibbs et al., 2003a). They were maintained on a 12-hr light/12-hr dark cycle, with exposure to 10-50 lux of fluorescent lighting during the light phase, and were treated according to federal and institutional animal care guidelines. Homozygous mutants were distinguished from the heterozygous controls by their hyperactivity, head-tossing and circling behavior (Gibson et al., 1995), and/or by a PCR/restriction digest assay.

[0146] Construction of AAV Vectors. Single-vector platform: AAV vector plasmid, containing the truncated chimeric CMV/chicken .beta.-actin promoter (smCBA) (Haire et al., 2006) and MYO7A cDNA was constructed by removing the full MYO7A cDNA from pEGFP-C2 by EagI and SalI digest, and then ligating into pTR-smCBA-GFP that had been digested with NotI and SalI to remove GFP. The MYO7A cDNA (.about.6.7 kb) corresponded to isoform 2 of human MYO7A, and was the same as that described previously by Hashimoto et al. (2007), which was 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 junctions were fully sequenced prior to packaging. All vectors intended for in vitro analyses were separately packaged in wild type AAV2, or alternatively in the AAV2(tripleY.fwdarw.F) capsid mutant vector (Petrs-Silva et al., 2011). As noted above, AAV2-based vectors were chosen for the in vitro experiments due to their increased transduction efficiency relative 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 vector plasmid carrying the full-length MYO7A coding sequence of variant 2 (the plasmid used to package fAAV). These transfected cells were then used as a positive control throughout immunoblot analyses to indicate the appropriate size of full-length MYO7A protein. Vector infections were carried out in HEK293 cells with titer-matched AAV vectors. In brief, cells were grown to 60-70% confluency. All vectors were diluted in a balanced salt solution to achieve the desired multiplicity of infection (MOI). If not specifically mentioned, cells were infected at 10,000 genome-containing particles/cell of each vector, resulting in an MOI of 20,000 total for each vector pair. Cells were incubated in medium containing 10% serum for 3 days post-infection at 37.degree. C. under 7% CO.sub.2, and then analyzed via immunoblot. Titers of 10.sup.12 to 10.sup.13 particles/mL were obtained for different lots of AAV2-MYO7A and AAV5-MYO7A.

[0147] Oligonucleotide Sequences. For in vivo studies, a human influenza hemagglutinin (HA) tag was added to the 3' termini of the full-length, simple overlap, trans-splicing, and hybrid 3' vectors by utilizing a unique BamHI site (P19), and replacing the non-tagged 3'-end with an HA-tagged (P20) version. All constructs were sequence verified by Sanger sequencing.

[0148] AAV Vector Plasmid Design and Cloning. The full-length coding sequence of MYO7A (human isoform 2; GenBank Accession No. NM_001127180) was cloned into a vector plasmid containing the strong, ubiquitous CMV/chicken .beta.-actin (smCBA) promoter (Haire et al., 2006), a polyadenylation signal, and the AAV2 ITRs. Packaging of this plasmid generated the fAAV vector (FIG. 22A). In all systems, the 5' vectors shared the smCBA promoter and a 5' portion of MYO7A, whereas the 3' vectors contained a 3'-portion of MYO7A, and a bovine growth hormone (bGH) polyadenylation signal. Oligonucleotides used for vector construction are listed in Table 2. The simple overlap contained nucleotides 1 through 3644 of MYO7A cDNA from the ATG in the 5' vector, and nucleotides 2279 through 6534 in the 3' vector. The fragments were amplified with oligonucleotides P1 and P3 by polymerase chain reaction (PCR) and cloned into the 5' vector via NotI and NheI, and the 3' vector with P3 (AflII) and P4 (KpnI), respectively. The resulting two vector plasmids share 1365 bp of overlapping MYO7A sequence (FIG. 22B). The trans-splicing and hybrid vectors utilize splice junctions composed of either ideal splice donor and acceptor sites derived from AP coding sequence or native MYO7A splice junctions from exons 23 and 24 (Yan et al., 2002). To create 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 part of the MYO7A coding sequence for cloning. The corresponding 3' vector was similarly created by amplifying the splice-acceptor site with oligonucleotides P8 (AflII) and P9 in a first PCR, and adding part of the 3' MYO7A coding sequence with oligonucleotide P10 (AgeI) in a second PCR (see FIG. 22C). The AP hybrid vectors were created by adding 270 bp of AP overlap sequence to the respective trans-splicing vectors (Ghosh et al., 2011). The sequence was amplified by PCR and, in so doing, appropriate restriction endonuclease sites were added. For the 5' vector oligonucleotides P11 (NheI) and P12 (SalI) were used, while oligonucleotides P13 (NotI) and P14 (AflII) were used for the 3' vector (FIG. 22D). A fourth vector pair, "native intron hybrid" vector, was also created to exploit the natural sequence in and around intron 23 of MYO7A as a recombination locus, and subsequent splicing signal. The 5'-portion was created by amplifying intron 23 with oligonucleotides P15 and P16 (NheI) first, and then using the resulting amplicon in a second reaction with oligonucleotide P7 (NsiI) to facilitate cloning. The corresponding 3'-vector was constructed by amplifying the intron 23 with oligonucleotides P17 and P18 (AflII), and the resulting amplicon, with oligonucleotide P10 (AgeI) in a second reaction (see FIG. 22E).

TABLE-US-00002 TABLE 2 OLIGONUCLEOTIDES USED IN THIS STUDY 5'-3' sequence (restriction sites Restriction Oligo underlined) site (SEQ ID NO:) P1 GCGGCGGCCGCCA NotI (SEQ ID NO: 9) CCATGGTGATTCT TCAGCAGGGGGAC P2 GCGGCTAGCGAAG NheI (SEQ ID NO: 10) TTCCGCAGGTACT TGAC P3 GCGCTTAAGCAGG AflII (SEQ ID NO: 11) TCTAACTTTCTGA AGCTG P4 GCGGGTACCTCAC KpnI (SEQ ID NO: 12) TTGCCGCTCCTGG AGCC P5 GGCACCTAGTGGC (SEQ ID NO: 13) TTTGAGGTAAGTA TCAAGGTTACAAG AC P6 GCGGCTAGCTCAG NheI (SEQ ID NO: 14) AAACGCAAGAGTC TTC P7 CTTCTTTGTGCGA NsiI (SEQ ID NO: 15) TGCATCAAG P8 GCGCTTAAGCGAC AflII (SEQ ID NO: 16) GCATGCTCGCGAT AG P9 CGCCCTCGCTCCA (SEQ ID NO: 17) GGTCCTGTGGAGA GAAAGGCAAAG P10 GAACCCGAACCGG AgeI (SEQ ID NO: 18) TCCTTG P11 GCGGCTAGCCCCC NheI (SEQ ID NO: 19) GGGTGCGCGGCG P12 GCGGTCGACGAAA SalI (SEQ ID NO: 20) CGGTCCAGGCTAT GTG P13 GCGGCGGCCGCCC NotI (SEQ ID NO: 21) CCGGGTGCGCGGC G P14 GCGCTTAAGGAAA AflII (SEQ ID NO: 22) CGGTCCAGGCTAT GTG P15 CAGGCACCTAGTG (SEQ ID NO: 23) GCTTTGAGGTACC AGGCTAGGGACAG G P16 GCGGCTAGCCGCC NheI (SEQ ID NO: 24) TGAGCCCAGAAGT TC P17 CGCCCTCGCTCCA (SEQ ID NO: 25) GGTCCTGAAGGAG ACAAGAGGTATG P18 GCGCTTAAGCACC AflII (SEQ ID NO: 26) GCTTGTGTTGATC CTC P19 GCCAGGGAAGGAT BamHI (SEQ ID NO: 27) CCCATG P20 GCGGGTACCTCAT KpnI (SEQ ID NO: 28) GCGTAATCCGGTA CATCGTAAGGGTA CTTGCCGCTCCTG GAGCC P21 AGCTTCGTAGAGT (SEQ ID NO: 29) TTGTGGAGCGG P22 GAGGGGCAAACAA (SEQ ID NO: 30) CAGATG Oligonucleotides were used to make 5' and 3' vectors of the dual-vector platforms (P1-P20). Oligonucleotides were used to characterize the fidelity of the overlap in simple overlap, trans-splicing and AP hybrid vector platforms (P21-P22). Restriction sites used for cloning are underlined and the introduced HA tag is noted in italics (P19).

[0149] Dual-Vector Platform. Two separate vector plasmids were constructed: Vector A contains the strong, ubiquitous "smCBA" promoter and MYO7A cDNA encoding the N-terminal portion. Vector B contains MYO7A cDNA encoding the C-terminal portion and a poly-A signal sequence. Each vector plasmid contained both inverted terminal repeats (ITRs). Using PCR with full-length MYO7A cDNA as a template, the MYO7A cDNA was divided roughly in half with amplicons encompassing nucleotide positions 1 through 3644 (Vector A) and 2279 through 6647 (Vector B) relative to ATG start position 1. The resulting two-vector plasmids shared 1365 bp of overlapping MYO7A sequence, and were 5.0- and 4.9-Kb in length, respectively. This was well within the size limitation of standard AAV vectors. Both vector plasmids were sequence verified and separately packaged by standard AAV production methods (Zolotukhin et al., 2002; Jacobson et al., 2006). The titer of the first lot contained 2.5.times.10.sup.12 particles/mL of each vector, and the second lot contained 4.times.10.sup.12 particles/mL of each vector.

[0150] Reverse Transcription and Characterization of Overlap Region. HEK293 cells were infected with dual vectors, and total RNA was extracted with the RNeasy.RTM. kit (Qiagen, Hilden, Germany) according to the manufacturer's recommended protocol. Two micrograms of RNA was then subjected to DNaseI (NEB) digestion for 30 min at 37.degree. C., followed by heat inactivation at 75.degree. C. for 10 min. Reverse transcription to cDNA was achieved with the SuperscriptIII.RTM. kit (Life Technologies, Grand Island, N.Y., USA) according to the standard protocol utilizing the oligo dT primer. Two microliters of cDNA was used as template in a PCR (95.degree. C. for 3 min initial denature, 35 cycles of 95.degree. C. for 45 sec, 55.degree. C. for 45 sec, 72.degree. C. for 12 min, and a final 72.degree. C. for 15 min) using oligonucleotide primers P21 and P22 (see Table 2). Annealing sites for these primers are located 5' and 3', respectively, of the area of cDNA overlap (in other words, outside the region of overlap) in the simple overlap and hybrid vector pairs. The 3'-primer annealed to sequence that was complimentary to the bGH polyA. Resulting products were digested with either PpuMI or BglII, separated on a 1.5% agarose gel, and subsequently analyzed on a UV screen. Separately, products were digested with KpnI and AgeI, and subsequently cloned into a pUC vector for sequencing of the entire overlap region. M13 forward and reverse primers that were specific for the vector were used to obtain sense and antisense reads resulting in an .about.140 bp overlap of the sense and antisense reads. To demonstrate that these methods were capable of detecting aberrant sequence (i.e., quality control), a MYO7A sequence was generated using either an artificial insertion (HindIII fill-in at position 2635) or a point mutation (T.fwdarw.C) at position 2381, and the analyses were repeated.

[0151] Viral Delivery in Vitro. HEK293A cells (Invitrogen), grown in DMEM with 10% FBS and 1.times. NEAA and Pen/Strep (Invitrogen) were plated in 6 well-plates. The next day cells were incubated, at 37.degree. C. and 5% CO2, with AAV2- and AAV5-MYO7A at an MOI of 10,000 viral particles/cell in 500 .mu.L of complete medium, containing also 40 .mu.M of calpain inhibitor (Roche, Pleasanton, Calif., USA). Two hours later complete medium was added. The next day, the medium was changed and cells were incubated for an additional 48 hrs. Alternatively, some cells were transfected with 1 .mu.g of vector pTR-smCBA-MYO7A, complexed with Lipofectamine 2000 (ratio 1:3), according to the manufacturer's instructions (Invitrogen).

[0152] 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 hrs in culture, cells were transduced with viruses. Cells were incubated in 100 .mu.L of complete medium containing 40 .mu.M of calpain inhibitor, and 10,000 viral particles/cell from full-strength AAV stocks. After 2 hrs, 400 .mu.L of complete medium was added to each well, and incubated overnight. The medium was changed the following day, and cells were incubated for an additional 48 hrs.

[0153] ARPE19 cells (American Type Culture Collection, Manassas, Va., USA) were cultivated 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 were the primary RPE cells.

[0154] MYO7A expression analysis by Western blot and Immunofluorescence. HEK293A and primary mouse RPE cells that were transduced with AAV-MYO7A were collected 3 days post-transduction. For western blot analyses, cells were collected and lysed in 20 mM TRIS, pH 7.4, 5 mM MgCl.sub.2, 10 mM NaCl, 1 mM DTT and 1.times. protease inhibitor cocktail (Sigma-Aldrich Chemical Co., St. Louis, Mo., USA). Equivalent amounts of total protein were separated on a 7.5% SDS-PAGE gel. After transfer, blots were blocked with 5% non-fat milk, and probed with mouse anti-MYO7A antibody, generated against residues 927-1203 of human MYO7A (Developmental Studies Hybridoma Bank, Iowa City, Iowa USA) (Soni et al., 2005), and mouse anti-actin antibody (Sigma-Aldrich) as a loading control.

[0155] Immunofluorescence was performed with ARPE19 and mouse RPE primary cells, 3 days after infection. Cells were fixed in 4% formaldehyde, blocked with blocking solution (0.5% BSA/0.05% saponin in PBS), incubated with the mouse anti-MYO7A followed by goat anti-mouse Alexa-568 (Molecular Probes, Carlsbad, Calif., USA). Coverslips were mounted with mounting medium containing DAPI (Fluorogel II, Electron Microscopy Sciences, Hatfield, Pa., USA) and visualized on a Leica confocal system.

[0156] Protein extraction and immunoblotting. Transfected and infected HEK293 cells were harvested and washed twice in PBS and processed as previously reported with minor modifications (Boye et al., 2012). The cells were lysed by 3.times.30 sec pulses of sonication in 200 .mu.L 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 min. The protein concentration of the supernatant was measured with BCA (Thermo Fisher Scientific, Rockland, Ill., USA). Equal amounts of protein were then loaded on 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels (BioRad, Hercules, Calif, USA) and transferred in CAPS buffer (pH 11) onto PVDF membranes (Millipore, Billerica, Mass.). Blots were then labeled with antibodies against MYO7A (monoclonal antibody raised against amino acids 11-70 of human MYO7A; Santa Cruz, Dallas, Tex., USA; 1:1000) or HA (MMS-101P; Covance, Gaithersburg, Md., USA; 1:500) and .beta.-actin (ab 34731; Abcam, Cambridge, Mass., USA; 1:5000). For visualization with the Odyssey system (Li-Cor, Lincoln, Nebr., USA), an antimouse and an anti-rabbit secondary antibody conjugated with CW800 and IR680 dyes (Li-Cor), respectively, were used. Semiquatitative densitometric measurements were performed with Odyssey acquisition and analysis software (Li-Cor). The dual-color images were separated in their respective channels and converted to gray scale for presentation purposes. Size markers present in one channel of each blot were added to both channels for visualization of protein sizes.

[0157] Viral Delivery in vivo. Mice were anesthetized with 2.0-3.0% isoflurane inhalation. The pupils of the animals were dilated with 1% (wt./vol.) atropine sulfate and 2.5% phenylephrine. A local anesthetic (0.5% proparacaine hydrochloride) was also administered. A sclerotomy in the temporal limbus was performed with a 27-Ga needle. A 32-Ga blunt needle, attached to a microsyringe pump (WPI, Sarasota Fla., USA) was inserted and 1 .mu.L of viral solution was injected into the ventral subretinal space of P14-P16 animals. Retinal detachment was visualized under a dissecting microscope, and registered as indication of a positive subretinal injection. One microliter of the following AAV8(Y733F)-based vectors was injected subretinally in one eye of C57BL/6 mice: single fAAV (1.times.10.sup.13 vg/mL), front and back half "hybrid" vectors combined equally (each vector=1.times.10.sup.13 vg/mL), or front and back half "simple overlap" vectors combined equally (each vector=1.times.10.sup.13 vg/mL). Subretinal injections were performed as previously described (Timmers et al., 2001). Further analysis was carried out only on animals that received comparable, successful injections (>60% retinal detachment with minimal surgical complications).

[0158] Light Microscopy and Immunoelectron Microscopy of Retinas. Eyecups were processed for embedment in either LR White or Epon, and semithin and ultrathin sections were prepared. Semithin sections were stained with toluidine blue and visualized on 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 gold-conjugated secondary antibodies (Electron Microscopy Sciences), as described previously (Lopes et al., 2011). Negative control sections processed at the same time included those from Myo7a-null retinas, and, as positive control, WT animals were used.

[0159] MYO7A immunogold density was determined on sections of age-matched WT, Myo7a-null retinas and retinas of Myo7a-null animals that had been injected with AAV-MYO7A at P14-16 and dissected three weeks later. For quantification of the immunolabel, all of the gold particles in a complete section of each RPE cell were counted. The area of each cell's profile was determined using ImageJ software. For background labeling, the concentration of label in sections of untreated Myo7a-null animals was measured. Data were expressed with this background labeling subtracted.

[0160] The concentration of MYO7A and opsin immunogold labeling in the connecting cilia of photoreceptor cells was determined by counting gold particles along longitudinal profiles of connecting cilia and measuring the length of each profile.

[0161] Analysis and quantifications were performed in a minimum of three different retinas, from three different animals. Statistical analysis was performed using one-tail Student's t-test.

[0162] Six weeks postinjection, C57BL/6 mice were enucleated and their eyes processed and immunostained as previously described (Boye et al., 2011) with minor modifications. Retinas were immunostained with an antibody specific for HA (monoclonal Ab clone 12CA5; Roche), counterstained with DAPI, and imaged with a spinning disk confocal microscope (Nikon Eclipse TE2000 microscope equipped with Perkin Elmer Ultraview Modular Laser System and Hamamatsu O-RCA-R2 camera). Images were obtained sequentially using a 20.times.(air) objective lens. All settings (exposure, gain, laser power) were identical across images. All image analysis was performed using Volocity 5.5 software (Perkin Elmer, Waltham, Mass., USA).

Results

[0163] AAV-MYO7A single vector preparations. AAV vector plasmid was engineered to contain a truncated chimeric CMV/chicken .beta.-actin promoter, smCBA (Haire et al., 2006) and the 6.7-kb cDNA encoding the full-length isoform 2 of human MYO7A (NCBI #NM_001127180) (FIG. 10A). The smCBA promoter exhibits the same tropism and activity in mouse retinas as that of the full-length CBA promoter (Haire et al., 2006; Pang et al., 2008). Titers of 10.sup.12 to 10.sup.13 particles/mL were obtained for different lots of AAV2-MYO7A and AAV5-MYO7A. A concentration of 10.sup.12 particles/mL was regarded as the standard concentration (1.times.), from which dilutions were made. The experiments were performed with virus obtained from three separate preparations. No differences in expression or phenotype correction, as described below, were observed among the different lots for AAV2-MYO7A or AAV5-MYO7A at a given concentration.

[0164] MYO7A Expression in Cell Culture. Transduction of primary cultures of Myo7a-null RPE cells with 1.times. single AAV2-MYO7A or AAV5-MYO7A resulted in the expression of a polypeptide that, by western blot analysis, had an apparent mass that was comparable to that of WT MYO7A protein, and was present at similar levels to that found in primary cultures of Myo7a.sup.+/- RPE cells (FIG. 10B). Likewise, a single band of appropriate size was detected on western blots of HEK293A cells. Immunofluorescence of the primary RPE cells showed that the MYO7A protein, resulting from 1.times. single AAV-MYO7A treatment of MYO7A-null cells, had a subcellular localization pattern that was comparable to that of endogenous MYO7A in control cells, indicating the generation of appropriately targeted protein (FIG. 10C, FIG. 10D, FIG. 10E, and FIG. 10F). ARPE19 cells were also infected with 1.times. or diluted (1:100) AAV2-MYO7A or AAV5-MYO7A, and compared with non-treated cells. An increase in MYO7A immunofluorescence was detected in the treated cells, and the intracellular localization of the label was comparable to that in untreated cells (FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, FIG. 17E, and FIG. 17F).

[0165] Localization of MYO7A in Vivo. Most retinal MYO7A is found in the RPE (Hasson et al., 1995), however, the protein is also present in the connecting cilium and pericilium of the photoreceptor cells (Liu et al., 1997; Williams, 2008). A diagram illustrating this distribution and the retinal functions of MYO7A has been published in a recent review (Williams and Lopes, 2011).

[0166] Three weeks following injection of 1.times. AAV2-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 label was evident in the photoreceptor cells, where it was localized in the connecting cilium and pericilium, comparable to that in WT retinas (FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, and FIG. 11E). Label was also present throughout the RPE cells, particularly in the apical cell body region (FIG. 11F, FIG. 11F-1, FIG. 11G, and FIG. 11G-1; see FIG. 18A, FIG. 18B, FIG. 18C, and FIG. 18D for controls), as found in WT retinas (Gibbs et al., 2004; Liu et al., 1997).

[0167] 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, it was determined whether MYO7A was also present in the ciliary region of cone photoreceptors. Double immunoEM of treated retinas was performed, using a MYO7A antibody together with an antibody specific for rod opsin. Although there are only a small number of cones with aligned connecting cilia found in each ultrathin section, MYO7A immunogold label was evident in the connecting cilium and periciliary region of these cones, which were identified by lack of rod opsin labeling in their outer segments (in contrast to the surrounding rod outer segments) (FIG. 11H and FIG. 11I). Hence, AAV2-MYO7A and AAV5-MYO7A can transduce cone as well as rod photoreceptor cells.

[0168] Dose-dependent MYO7A expression in photoreceptor and RPE cells. To determine the levels of MYO7A expression following treatment with different concentrations of AAV2-MYO7A and AAV5-MYO7A (1.times., 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 the photoreceptor cells, where its distribution is limited to the connecting cilium and pericilium, requires the higher resolution provided by electron microscopy (Liu et al., 1997). Immunogold particle density was measured in images of the photoreceptor connecting cilium and pericilium, shown in complete longitudinal section (from the basal bodies to the base of the outer segment), and in images showing the RPE cells in apical to basal section. Particle density was expressed as particles per length of cilium for the photoreceptor cells (each connecting cilium is .about.1.2 .mu.m long), and as particles per area for the RPE cells (the entire area between the apical and basal surfaces was included). Particle density is dependent on 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 (i.e., grids were etched and labeled in an identical manner, and the labeling was not so dense as to be affected by steric hindrance).

[0169] Treatment with 1.times. AAV2-MYO7A or AAV5-MYO7A resulted in 2.5-2.7 times the density of immunolabel in the photoreceptor cilium, compared with that found in WT retinas, while the 1:10 and 1:100 dilutions resulted in a density of immunolabel that was more comparable to WT levels (FIG. 11J, FIG. 11L, and FIG. 19). Quantification of immunogold label in the RPE showed that injection of AAV2-MYO7A resulted in 2.7 times more label than in WT, with the 1:10 and 1:100 dilutions showing no significant difference (FIG. 11K). In contrast, the level of MYO7A immunolabel in the RPE of retinas injected with AAV5-MYO7A varied in relation to virus titer, with the full dose virus effecting 2.2-fold more MYO7A than that found in WT RPE, the 1:10 dilution effecting WT levels, and the 1:100 dilution resulting, on average, .about.60% of WT levels (FIG. 11M).

[0170] These counts of labeling density indicate that 1.times. AAV-MYO7A resulted in more than double the normal level of MYO7A expression in both the photoreceptor and RPE cells. The distribution of MYO7A was not affected by this overexpression in the photoreceptor cells. In the RPE cells, the overall distribution of MYO7A was comparable to WT, with a higher concentration in the apical cell body region. However, with 1.times. AAV2-MYO7A or 1.times. AAV5-MYO7A, the proportion of MYO7A that was associated with melanosomes was only 55% of that in WT RPE: This difference is possibly because the proteins that link MYO7A to the melanosomes, MYRIP and RAB27A (Klomp et al., 2007; Lopes et al., 2007), may have remained near WT levels, and thus limited the absolute amount of MYO7A that could associate with the melanosomes.

[0171] Despite the overexpression of MYO7A, no pathology was evident in retinas, up to 3 months after injection of 1.times. (or 1:10) AAV2-MYO7A. However, two out of six retinas injected with 10.sup.13 particles/mL of AAV5-MYO7A (i.e., 10.times.) showed evidence of photoreceptor cell loss across the retina after 3 weeks (AAV2-MYO7A was not tested at this titer) (FIG. 19).

[0172] Correction of melanosome localization in the RPE. In Myo7a-mutant mice, melanosomes are absent from the apical processes of the RPE cells (Liu et al., 1998). This mutant phenotype is evident at all neonatal ages, and is due to loss of actin-based transport of the melanosomes by the myosin 7a motor (Gibbs et al., 2004). Three weeks following injection of 1.times. AAV2-MYO7A or AAV5-MYO7A into the subretinal space of Myo7a-null mice, melanosomes were observed to have a normal distribution in all RPE cells near the site of injection (within 1.4 mm) (n=10 each for AAV2-MYO7A and AAV5-MYO7A) (FIG. 12A, FIG. 12B, and FIG. 12C). Well away from the injection site, a mixture of corrected and uncorrected RPE cells was evident, while, at the periphery of the retina, the cells all exhibited the Myo7a-mutant phenotype, indicating lack of correction in this region (FIG. 12D, FIG. 12E, and FIG. 12F). The correction of melanosomes was still evident in retinas that were fixed 3 months after injection (FIG. 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), although with the 1:100 dilution some of the RPE cells near the site of injection were not corrected.

[0173] Correction of opsin distribution. Myo7a-mutant mice have an abnormal accumulation of opsin in the connecting cilia of the photoreceptor cells, a phenotype that is evident by immunoEM with opsin antibodies (Liu et al., 1999). This mutant phenotype suggested that myosin 7a functions in the vectorial delivery of opsin to the outer segment (Liu et al., 1999). Quantification of immunogold opsin labeling in the connecting cilia, demonstrated that this phenotype was corrected with 1.times. AAV2-MYO7A or AAV5-MYO7A (FIG. 13; FIG. 21A, FIG. 21B, FIG. 21C, and FIG. 21D). This analysis also showed phenotype correction with 1:100 dilutions, although the data indicated that a full WT phenotype was not achieved (FIG. 13), despite WT levels of MYO7A (FIG. 11J and FIG. 11L), suggesting that some of the MYO7A may not be fully functional.

[0174] AAV2-MYO7A dual-vector preparations. The preceding results demonstrate that a single AAV vector is capable of delivering functional MYO7A to the RPE and photoreceptor cells in vivo. Because the size of smCBA-MYO7A is .about.2 kb larger than the nominal carrying capacity of an AAV (Grieger and Samulski, 2005), this transduction may involve undefined fragmentation of the smCBA-MYO7A cDNA followed by reassembly of plus and minus cDNA strands after delivery to the cell as shown for other large genes (Dong et al., 2010; Lai et al., 2010; Wu et al., 2010). To evaluate whether two AAV vectors containing defined, overlapping fragments of MYO7A cDNA (1365 bases) were also capable of mediating full-length MYO7A expression, an AAV2-based dual-vector system (FIG. 14A-1 and FIG. 14A-2) was developed. Two separate lots of the 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 contained 2.5.times.10.sup.12 particles/mL of each vector, and the second lot contained 4.times.10.sup.12 particles/mL.

[0175] MYO7A expression with AAV2 dual vectors. Western blot analysis of primary cultures of Myo7a-null RPE cells, infected with AAV2-MYO7A(dual vector) of either lot, showed that the cells expressed a MYO7A-immunolabeled polypeptide of comparable mass to that of WT MYO7A (FIG. 14B). However, the expression level of MYO7A in the Myo7a-null RPE cells was significantly less than that found in primary cultures of Myo7a.sup.+/- RPE cells (cf. lanes 2 and 3 in FIG. 14B), unlike that found for the single AAV2 or AAV5 vectors (FIG. 10B). Quantitative analysis of western blots showed that Myo7a-null RPE cells, transduced with the single vectors (1.times.), AAV2-MYO7A or AAV5-MYO7A, or with AAV2-MYO7A(dual vector), expressed MYO7A at levels that were 82%, 111%, and 10%, respectively, of the level of MYO7A in Myo7a.sup.+/- RPE cells.

[0176] FIG. 16 is a Western blot using the same dual-vector system as above except in an AAV8 serotype. FIG. 16 shows expression level of myo7A using the dual-vector system that was nearly equivalent to the wild type myo7A expression level. While the reasons for the discrepancy are unclear, much better results were obtained by the inventors using the dual-vector systems than were obtained by several outside collaborators. Wild type like levels of MYO7A expression was observed in shaker-1 retinas following injection with dual-AAV8(Y733F) vectors. Thus, very good expression of MYO7A with the dual AAV platform has been achieved.

[0177] Immunofluorescence of primary Myo7a-null RPE cells, infected with AAV2-MYO7A(dual vector), showed that a few cells scattered throughout the culture exhibited very high levels of MYO7A, but all other cells contained insignificant levels (FIG. 14C, FIG. 14D, and FIG. 14E). The cells overexpressing MYO7A typically had altered morphology, suggesting that the high levels of MYO7A may be toxic. Similarly, immunofluorescence of ARPE19 cells, infected with AAV2-MYO7A(dual vector), resulted in a minority of cells that were labeled intensely with MYO7A antibody, with most of the cells appearing to express only endogenous levels of MYO7A (FIG. 14F and FIG. 17F).

[0178] Immunolabeling of retinas, prepared 3 weeks after subretinal injection with AAV2-MYO7A(dual vector) of either lot, also showed only a few RPE cells and photoreceptor cells with clear MYO7A expression, although significant overexpression was not evident in this in vivo experiment. Immunogold particle counts from images of ultrathin sections were used to quantify the level of MYO7A expression in Myo7a-null retinas that were treated with the second lot of AAV2-MYO7A(dual vector). Within 1.4 mm of the injection site, MYO7A immunolabeling of the connecting cilium and pericilium of the photoreceptor cells was a mean of 48% of that in WT retinas: 2.8 particles/.mu.m (n=3 retinas) compared with 6.5 particles/.mu.m for WT (n=3 retinas). The mean label density in apical-basal sections of the RPE was 35% of that in WT retinas: 11 particles/100 .mu.m.sup.2 compared with 31 particles/100 .mu.m.sup.2 for WT. However, it was clear that these lower means were achieved by some cells expressing near normal amounts of MYO7A and the majority expressing very little; over half the cells had fewer than 10 particles/100 .mu.m.sup.2 (FIG. 14G).

[0179] Correction of Myo7a-mutant phenotypes with AAV2 dual vectors. Eyes were analyzed for correction of melanosome localization and ciliary opsin distribution within 1.4 mm of the injection site. With either lot of AAV2-MYO7A(dual vector), some RPE cells (29% for lot 1 treatment [n=6 retinas], 35% for lot 2 treatment [n=9 retinas]) were observed to have a normal apical melanosome distribution, but most of the cells in this region retained the Myo7a-mutant phenotype, resulting in a mosaic effect (FIG. 15A) that contained a much lower proportion of corrected cells than that observed with a 1:100 dilution of either of the single vectors. The only correction observed in 3 eyes injected with a 1:10 dilution of AAV2-MYO7A(dual vector) (first lot), was in 18% of the RPE cells in one of the retinas. With full-strength of AAV2-MYO7A(dual vector) (second lot), opsin immunogold density averaged 3.2.+-.0.4 particles/.mu.m of cilium length, which was reduced from untreated retinas (4.2.+-.0.8 particles/m; p=0.003), but still greater than WT levels (1.1.+-.0.2 particles/m), suggesting that most cells were not corrected.

[0180] Using immunoelectron microscopy, a correlation between phenotype correction and the expression level of MYO7A was identified (determined by the mean concentration of immunogold particles in an apical-basal section of each RPE cell) (FIG. 15B, FIG. 15C, FIG. 15D, and FIG. 15E). From the eyes injected with AAV2-MYO7A(dual vector) (second lot), it was shown that the corrected RPE cells contained a mean of 108% of the WT level of MYO7A (the minimum level was 82%). RPE cells that were not corrected contained a mean of 26% of the WT level of MYO7A (the maximum level was 92%). While these data showed that higher expression of MYO7A is correlated with phenotype correction (FIG. 15F), it also indicated that some of the labeled MYO7A protein was not functional, given that melanosomes are localized normally in mice that are heterozygous for the Myo7a-null allele and have only .about.50% of the WT level of MYO7A.

[0181] Expression of MYO7A with simple overlap vectors. AAV2-based simple overlap vectors were evaluated in vitro at a variety of MOIs to evaluate how the concentration of vector pairs related to MYO7A expression. How levels of MYO7A changed over time was also evaluated in infected cells. HEK293 cells were infected with simple overlap vector pairs packaged in AAV2(tripleY-F) vector (FIG. 23A). A preliminary co-infection with AAV2(tripleY-F) simple overlap vectors (MOI of 10,000 for each vector) indicated that MYO7A is expressed, and that migration of the protein on gel is identical to a full-length transfection control (FIG. 23A). Coinfection at MOIs of 400, 2000, and 10,000 of each vector shows that the efficiency of the simple overlap system is proportional to the amount of 5' and 3' vectors used (FIG. 23B). MYO7A expression increased as a function of incubation time up to 5 days postinjection in HEK293 cells (FIG. 23C). The visible expression decline was because of a reduction of viable cells in the culture vessel at the later times.

[0182] Comparison of fAAV-MYO7A to dual-AAV-MYO7A expression and evaluation of AAV serotype efficiency. Previously, it was shown that fAAV-MYO7A was able to ameliorate the retinal phenotype of the shaker1 mouse (Colella et al., 2013; Lopes et al., 2013; Trapani et al., 2013). To provide a basis for comparison dual-AAV-vector expression was evaluated relative to fAAV in vitro. After infection in HEK293 cells, all dual-vector systems expressed MYO7A more efficiently than fAAV (FIG. 24). The AP hybrid platform showed the strongest expression, followed by the simple overlap system.

[0183] Other studies have shown, in the context of a conventionally sized DNA payload, that the transduction efficiency and kinetics of AAV2(tripleY-F) vectors are increased relative to standard AAV2 both in vitro and in vivo (Li et al., 2010; Markusic et al., 2010; Ryals et al., 2011). The efficiency of AAV2 versus AAV2(tripleY-F) dual vectors was directly compared in HEK293 cells. Surprisingly, standard AAV2-mediated MYO7A expression was higher than that seen with titer-matched AAV2(tripleY-F) (FIG. 24). Identical results were obtained when comparing different AAV2 and AAV2(tripleY-F) dual-vector preparation packaged with identical vector plasmid.

[0184] Comparison of relative efficiencies and specificity of full-length MYO7A expression. To quantitatively evaluate the relative expression efficiencies of the dual-vector platforms and to assess specificity of full-length protein, HEK293 cells were infected with either the 5' and 3' AAV2-based vector pairs combined or the corresponding 5' vector alone. An additional hybrid vector pair was included that incorporated native MYO7A intronic sequence (intron 23) that served as overlapping sequence and provided appropriate splicing signals. All 5' vectors produced low amounts of a defined, less than full-length peptide detectable on Western blot with the exception of the simple overlap vector (FIG. 25A). However, the trans-splicing and the AP hybrid platforms revealed a distinct decrease of this undesired product when the 3' vector was added to the sample (FIG. 25A). The native intron hybrid platform also showed this secondary band on Western blots, again suggestive of a truncated protein originating from the 5' vector alone. In contrast to all other platforms tested, this band intensity increased with the addition of the 3' vector. Each platform's relative ability 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 (FIG. 25B). Full-length MYO7A expression on Western blot was then quantified relative to transfection control (FIG. 25C). AP hybrid-mediated MYO7A was the strongest followed by simple overlap, trans-splicing, and native intron hybrid (FIG. 25C).

[0185] Characterization of the overlap/splice region of the expressed MYO7A. To characterize the fidelity of the mRNA arising from dual vectors, HEK293 cells were infected with dual vectors and RNA extracted, reverse transcribed, and subjected to PCR utilizing primers binding upstream of the overlap region and in the bGH polyA signal region producing a 4.5 kb PCR fragment (FIG. 26A). An identically treated sample not containing reverse transcriptase was used as control for chromosomal DNA contamination. Plasmid containing the full-length MYO7A coding sequence was used as positive control for PCR. A preliminary screen of AAV-mediated MYO7A mRNA was performed by analyzing the pattern of fragment migration on agarose gel following restriction endonuclease digests with PpuMI and BglII (FIG. 26A). Identical banding patterns, consistent with the predicted 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 following digests of amplicons from each dual-vector platform tested, indicating that no gross alterations (deletions/insertions) occurred as a consequence of either homologous recombination of vector pairs and/or RNA splicing (FIG. 26B). To further characterize the fidelity of the overlap region, a fragment containing the complete overlap area (1829 bp) was restricted and cloned into pUC57 (FIG. 26A). Sequencing results of 10 clones picked at random per vector platform revealed that the overlap region was 100% identical to the consensus/predicted MYO7A sequence (FIG. 26C). This indicated that, in the context of the simple overlap platform, homologous recombination was accurate. Additionally, in the context of trans-splicing vectors, accurate splicing occurred. Finally, for the AP hybrid vectors, a combination of accurate homologous recombination and/or splicing took place. To determine whether this protocol was capable of detecting aberrant sequence in reconstituted MYO7A, a sequence that contained either an insertion of a HindIII recognition site (TAGC) at position 2635 or a point mutation (T-C) at position 2381 was also generated.

[0186] MYO7A expression mediated by dual vectors in mouse retina. To investigate the expression of MYO7A from the two best performing dual-vector platforms in vivo, C57BL/6J mice were subretinally injected with 1.times.10.sup.10 vector genomes per eye of simple overlap and AP hybrid systems packaged in AAV8(733) and analyzed 4 weeks later by Western blot and immunohistochemistry. AAV8(733)-fAAV-MYO7A vector was also injected to provide a basis for comparison. To distinguish between endogenous MYO7A and exogenous expression mediated by vectors, sequence coding for an HA tag was added to the C' terminus of the MYO7A cDNA in all constructs. Resulting retinas were immunostained for HA to reveal that fAAV vector along with both dual-vector platforms mediated expression of MYO7A in photoreceptors and RPE. A recent report concluded that simple overlap vectors were more efficient for gene transfer to the 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 "spotty" MYO7A expression mediated by AAV2-based simple overlap vectors (Lopes et al., 2013), it was found, when packaged in AAV8(733), that simple overlap vectors mediated MYO7A expression in the majority of RPE and photoreceptor cells. Photoreceptor degeneration/outer nuclear layer thinning was apparent in eyes injected with the AP hybrid vector system. Despite the observed degeneration, AP hybrid-mediated MYO7A was clearly detected in residual PR cell bodies and RPE and was sufficient to be detected by immunoblot. By Western blot analysis using HA antibody, simple overlap-mediated MYO7A was present in just detectable amounts. In contrast, fAAV-mediated protein levels were insufficient to be detected in this assay. Using an antibody against MYO7A, immunoblot of WT mouse retina revealed that both endogenous MYO7A and dual-vector-mediated, HA-tagged MYO7A migrated similarly.

Discussion

[0187] In this example, it was shown that dual AAV vectors with defined genetic payloads can be used to deliver a large transgene in vitro and in vivo. The initial experiments using the simplest of all dual-vector platforms revealed that efficiency of AAV2-based simple overlap vectors is 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. Next, three distinct dual-vector platforms were evaluated and compared to single, fragmented fAAV vector in vitro. All dual vectors analyzed drove higher levels of MYO7A expression than fAAV. Of all platforms tested, a hybrid vector system containing overlapping, recombinogenic sequence and splice donor/acceptor sites from the AP gene (AP hybrid) was the most efficient.

[0188] Regarding the specificity with which the dual-vector platforms express the correct-sized gene product, it was noted in vitro that trans-splicing and hybrid dual-vector platforms generated an additional band of lower molecular weight as detected by immunoblot (monoclonal antibody used was raised against the amino terminus MYO7A). The expression of this truncated product was much more pronounced for infections with 5' vectors alone. What might account for this additional band? After entry into the host cell, the virus capsid is removed and the single-stranded DNA payload is released. The ITRs carried by the single strand serve as primer for DNA polymerases to produce a double strand. The resulting circular intermediates consist mainly of monomers that, over time, convert into multimeric concatemers through intermolecular recombination (Duan et al., 1998; Yang et al., 1999). The dual-vector systems created in this study utilize this strategy to achieve full-length protein expression. A limiting factor lies in the fact that the highly recombinogenic ITRs flanking the expression cassettes are identical in nature leading to a random recombination and consequently a random orientation of the vector parts relative to each other. This random recombination inevitably results in reduced efficiency because only concatemers that have the two vector parts in 5'-3' orientation are able to express the full-length protein. This concatemerization over time is consistent with the observation that the amount of single-vector product is reduced in favor of the full-length protein when both 5' and 3' vectors are combined. Interestingly, the simple overlap system does not generate truncated product, even when only the 5' vector is used for infections. In contrast to the trans-splicing and hybrid vectors, there is virtually no intervening sequence between the end of the MYO7A coding sequence and the right-hand ITR. It may be that splice donor sequences enhance the likelihood of truncated product through some as-yet-to-be-determined mechanism.

[0189] A number of strategies have been devised to overcome the issue of random concatemerization and thereby increase specificity as well as efficiency of these dual-vector platforms. First, the addition of a highly-recombinogenic sequence such as that used in the AP hybrid vector here has resulted in significantly increased protein expression compared with the trans-splicing system. 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 improvement over trans-splicing vectors. The finding that AP hybrid vectors are more efficient than trans-splicing vectors supports that the AP sequence directs at least some of the concatemerization events toward the proper orientation with recombination then occurring via this sequence or via the ITRs. Regardless, with more concatemers properly aligned, the AP hybrid system mediates a more-efficient expression of MYO7A. Another approach for directing concatemerization is the use of single-strand oligonucleotides that are capable of tethering the back end of the 5' vector and the front end of the 3' vector together (Hirsch et al., 2009). However, this strategy requires efficient delivery of the oligonucleotide to the nucleus of the target cells timed with the dual vectors. Finally, dual vectors utilizing mismatched ITRs can be used to direct concatemerization in a head-to-tail orientation (Yan et al., 2005), although the process may require further optimization of the AAV packaging machinery.

[0190] Notably, in this study, it was found that the sequence in the overlap region of all dual vectors tested in vitro was 100% identical to the consensus/predicted MYO7A sequence. This indicates that homologous recombination and/or splicing was accurate in each dual-vector platform.

[0191] Similar to the in vitro results, the highest levels of MYO7A expression was found in retinas of mice subretinally injected with AAV8-based AP hybrid vectors (as assessed by probing for HA on Western blot). Notably, no truncated proteins were evident in retinas expressing either simple overlap or AP-hybrid mediated MYO7A. The reason for this observed difference remains to be elucidated but may involve differences in the DNA repair machinery that mediate recombination in actively dividing cells versus post-mitotic photoreceptors/RPE (Hirsch et al., 2013). Dual-vector-mediated MYO7A-HA expression was observed in the photoreceptors and RPE of WT mice, locations where MYO7A is thought to have a functional role (Williams and Lopes, 2011). In eyes injected with AP hybrid vectors, marked thinning of the outer nuclear layer was observed. It has previously been shown that vector-mediated overexpression of MYO7A leads to retinal toxicity (Hashimoto et al. 2007). Taken together with the high efficiency of transduction observed in vitro for the AP hybrid platform, the most likely explanation for the observed pathology is excessive production of MYO7A. Despite the marked degeneration, significant amounts of AP hybrid-mediated, full-length MYO7A-HA were detected on Western blot. As high concentrations of vectors were used in these experiments, a simple solution to circumvent toxicity could be to reduce vector genomes injected or replace the strong, ubiquitous smCBA promoter with an endogenous or homologous promoter, and/or a promoter with attenuated strength. An alternative explanation for toxicity of the strong AP hybrid platform is expression of undesired products, like the observed protein expressed from the 5' vectors alone in vitro. However, it was noted that only full-size MYO7A-HA was apparent on Western blot of the AP hybrid-treated retina.

[0192] With the goal of developing an AAV-based treatment for USH1B, animal models of this disease have provided an abundance of useful information. Similar to previous observations that fAAV-MYO7A and simple-overlap, dual-vectors were capable of restoring melanosome migration and opsin localization in the shaker1 mouse (Lopes et al., 2013), a recent study by an independent lab confirmed the usefulness of the vectors disclosed herein, when it was reported that they were capable of restoring the ultrastructural retinal phenotypes in the animal model. Notably, shaker1 mice lack retinal degeneration, and the severe functional abnormalities seen in USH1B patients (Liu et al., 1997). This fact renders in vivo analysis of therapeutic outcomes in the shaker1 retina problematic. Alternative animal models for evaluating a treatment for this devastating disease may be useful in adaptation of the present methods to human clinical use.

[0193] These results presented here also demonstrated that MYO7A can be efficiently expressed using dual-AAV-vector systems. The platforms containing overlapping elements, namely, the simple overlap system, and the AP hybrid system were both highly efficient. AP hybrid vectors showed the strongest expression of all systems tested, with little observable truncated protein in vitro and none observed in vivo. Simple overlap vectors showed good expression and were the most specific (no truncated protein products were observed) even when the 5'-only vector was used to infect cells. AAV has emerged as the preferred clinical vector and it efficiently transduces both photoreceptors and RPE. Because it has now been demonstrated that MYO7A sequence fidelity is preserved following recombination and/or splicing of dual-AAV-vector platforms and because only full-length MYO7A was detectable in mouse retinas injected with dual vectors, the dual-AAV-vector strategy presented herein represents a valid option for the treatment of retinal disorders associated with mutations in large genes such as USH1B.

REFERENCES

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[0289] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

[0290] 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.

[0291] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0292] All methods described herein can be performed in any suitable order, unless otherwise indicated herein, or unless otherwise clearly contradicted by context.

[0293] The use of any examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated. No language in the specification should be construed as indicating any element is essential to the practice of the invention unless as much is explicitly stated.

[0294] The description herein of any aspect or embodiment of the invention using terms such as "comprising", "having", "including" or "containing" with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of", "consists essentially of", or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

[0295] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations may be applied to the compositions and/or methods disclosed herein, and/or to the steps or the sequence of steps of the methods described herein without departing from the concept, spirit and/or scope of the invention. More specifically, it will be apparent that certain agents that are chemically- and/or physiologically-related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Sequence CWU 1

1

3017847DNAArtificial sequencethe nucleotide of an "hMyo7a coding overlap vector A" of the subject inventionexon(1164)..(1178) 1cgccaggctg cagggggggg gggggggggg ttggccactc cctctctgcg cgctcgctcg 60ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 120gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttcctcagat 180ctggcgcgcc caattcggta ccctagttat taatagtaat caattacggg gtcattagtt 240catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga 300ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca 360atagggactt tccattgacg tcaatgggtg gactatttac ggtaaactgc ccacttggca 420gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg 480cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc 540tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc 600cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt 660gcagcgatgg gggcgggggg gggggggggg cgcgcgccag gcggggcggg gcggggcgag 720gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga 780aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg 840cgggcgggag tcgctgcgac gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg 900ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc 960ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct 1020gcgtgaaagc cttgaggggc tccgggagct agagcctctg ctaaccatgt tcatgccttc 1080ttctttttcc tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc 1140aaagaattct agcggccgcc acc atg gtg att ctt cag cagggggacc 1188 Met Val Ile Leu Gln 1 5atgtgtggat ggacctgaga ttggggcagg agttcgacgt gcccatcggg gcggtggtga 1248agctctgcga ctctgggcag gtccaggtgg tggatgatga agacaatgaa cactggatct 1308ctccgcagaa cgcaacgcac atcaagccta tgcaccccac gtcggtccac ggcgtggagg 1368acatgatccg cctgggggac ctcaacgagg cgggcatctt gcgcaacctg cttatccgct 1428accgggacca cctcatctac acgtatacgg gctccatcct ggtggctgtg aacccctacc 1488agctgctctc catctactcg ccagagcaca tccgccagta taccaacaag aagattgggg 1548agatgccccc ccacatcttt gccattgctg acaactgcta cttcaacatg aaacgcaaca 1608gccgagacca gtgctgcatc atcagtgggg aatctggggc cgggaagacg gagagcacaa 1668agctgatcct gcagttcctg gcagccatca gtgggcagca ctcgtggatt gagcagcagg 1728tcttggaggc cacccccatt ctggaagcat ttgggaatgc caagaccatc cgcaatgaca 1788actcaagccg tttcggaaag tacatcgaca tccacttcaa caagcggggc gccatcgagg 1848gcgcgaagat tgagcagtac ctgctggaaa agtcacgtgt ctgtcgccag gccctggatg 1908aaaggaacta ccacgtgttc tactgcatgc tggagggtat gagtgaggat cagaagaaga 1968agctgggctt gggccaggcc tctgactaca actacttggc catgggtaac tgcataacct 2028gtgagggccg ggtggacagc caggagtacg ccaacatccg ctccgccatg aaggtgctca 2088tgttcactga caccgagaac tgggagatct cgaagctcct ggctgccatc ctgcacctgg 2148gcaacctgca gtatgaggca cgcacatttg aaaacctgga tgcctgtgag gttctcttct 2208ccccatcgct ggccacagct gcatccctgc ttgaggtgaa ccccccagac ctgatgagct 2268gcctgactag ccgcaccctc atcacccgcg gggagacggt gtccacccca ctgagcaggg 2328aacaggcact ggacgtgcgc gacgccttcg taaaggggat ctacgggcgg ctgttcgtgt 2388ggattgtgga caagatcaac gcagcaattt acaagcctcc ctcccaggat gtgaagaact 2448ctcgcaggtc catcggcctc ctggacatct ttgggtttga gaactttgct gtgaacagct 2508ttgagcagct ctgcatcaac ttcgccaatg agcacctgca gcagttcttt gtgcggcacg 2568tgttcaagct ggagcaggag gaatatgacc tggagagcat tgactggctg cacatcgagt 2628tcactgacaa ccaggatgcc ctggacatga ttgccaacaa gcccatgaac atcatctccc 2688tcatcgatga ggagagcaag ttccccaagg gcacagacac caccatgtta cacaagctga 2748actcccagca caagctcaac gccaactaca tcccccccaa gaacaaccat gagacccagt 2808ttggcatcaa ccattttgca ggcatcgtct actatgagac ccaaggcttc ctggagaaga 2868accgagacac cctgcatggg gacattatcc agctggtcca ctcctccagg aacaagttca 2928tcaagcagat cttccaggcc gatgtcgcca tgggcgccga gaccaggaag cgctcgccca 2988cacttagcag ccagttcaag cggtcactgg agctgctgat gcgcacgctg ggtgcctgcc 3048agcccttctt tgtgcgatgc atcaagccca atgagttcaa gaagcccatg ctgttcgacc 3108ggcacctgtg cgtgcgccag ctgcggtact caggaatgat ggagaccatc cgaatccgcc 3168gagctggcta ccccatccgc tacagcttcg tagagtttgt ggagcggtac cgtgtgctgc 3228tgccaggtgt gaagccggcc tacaagcagg gcgacctccg cgggacttgc cagcgcatgg 3288ctgaggctgt gctgggcacc cacgatgact ggcagatagg caaaaccaag atctttctga 3348aggaccacca tgacatgctg ctggaagtgg agcgggacaa agccatcacc gacagagtca 3408tcctccttca gaaagtcatc cggggattca aagacaggtc taactttctg aagctgaaga 3468acgctgccac actgatccag aggcactggc ggggtcacaa ctgtaggaag aactacgggc 3528tgatgcgtct gggcttcctg cggctgcagg ccctgcaccg ctcccggaag ctgcaccagc 3588agtaccgcct ggcccgccag cgcatcatcc agttccaggc ccgctgccgc gcctatctgg 3648tgcgcaaggc cttccgccac cgcctctggg ctgtgctcac cgtgcaggcc tatgcccggg 3708gcatgatcgc ccgcaggctg caccaacgcc tcagggctga gtatctgtgg cgcctcgagg 3768ctgagaaaat gcggctggcg gaggaagaga agcttcggaa ggagatgagc gccaagaagg 3828ccaaggagga ggccgagcgc aagcatcagg agcgcctggc ccagctggct cgtgaggacg 3888ctgagcggga gctgaaggag aaggaggccg ctcggcggaa gaaggagctc ctggagcaga 3948tggaaagggc ccgccatgag cctgtcaatc actcagacat ggtggacaag atgtttggct 4008tcctggggac ttcaggtggc ctgccaggcc aggagggcca ggcacctagt ggctttgagg 4068acctggagcg agggcggagg gagatggtgg aggaggacct ggatgcagcc ctgcccctgc 4128ctgacgagga tgaggaggac ctctctgagt ataaatttgc caagttcgcg gccacctact 4188tccaggggac aaccacgcac tcctacaccc ggcggccact caaacagcca ctgctctacc 4248atgacgacga gggtgaccag ctggcagccc tggcggtctg gatcaccatc ctccgcttca 4308tgggggacct ccctgagccc aagtaccaca cagccatgag tgatggcagt gagaagatcc 4368ctgtgatgac caagatttat gagaccctgg gcaagaagac gtacaagagg gagctgcagg 4428ccctgcaggg cgagggcgag gcccagctcc ccgagggcca gaagaagagc agtgtgaggc 4488acaagctggt gcatttgact ctgaaaaaga agtccaagct cacagaggag gtgaccaaga 4548ggctgcatga cggggagtcc acagtgcagg gcaacagcat gctggaggac cggcccacct 4608ccaacctgga gaagctgcac ttcatcatcg gcaatggcat cctgcggcca gcactccggg 4668acgagatcta ctgccagatc agcaagcagc tgacccacaa cccctccaag agcagctatg 4728cccggggctg gattctcgtg tctctctgcg tgggctgttt cgccccctcc gagaagtttg 4788tcaagtacct gcggaacttc gctagcgggc actagtccgt cgactgttaa ttaagcatgc 4848tggggagaga tctaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc 4908tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc 4968tcagtgagcg agcgagcgcg cagagaggga gtggccaacc cccccccccc cccccctgca 5028gccctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 5088tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 5148gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 5208atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 5268ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 5328cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 5388tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 5448gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 5508aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 5568tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 5628aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 5688aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc 5748ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 5808ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 5868atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 5928atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 5988tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 6048gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 6108tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 6168gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 6228cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 6288gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 6348atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 6408aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 6468atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 6528aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 6588aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 6648gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 6708gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 6768gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 6828ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 6888ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 6948atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 7008gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt 7068atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg 7128cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt 7188cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag 7248cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga 7308aaataccgca tcaggaaatt gtaaacgtta atattttgtt aaaattcgcg ttaaattttt 7368gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa 7428aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa 7488agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac 7548gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga 7608accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa 7668aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc 7728tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcgcgccatt 7788cgccattcag gctacgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctatta 784727779DNAArtificial sequencethe nucleotide of an "hMyo7a coding overlap vector B" of the subject invention 2aggggggggg gggggggggt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc 60cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg 120agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctcagatc tggcgcgccc 180aattggcttc gaattctagc ggccgctgct taagcaggtc taactttctg aagctgaaga 240acgctgccac actgatccag aggcactggc ggggtcacaa ctgtaggaag aactacgggc 300tgatgcgtct gggcttcctg cggctgcagg ccctgcaccg ctcccggaag ctgcaccagc 360agtaccgcct ggcccgccag cgcatcatcc agttccaggc ccgctgccgc gcctatctgg 420tgcgcaaggc cttccgccac cgcctctggg ctgtgctcac cgtgcaggcc tatgcccggg 480gcatgatcgc ccgcaggctg caccaacgcc tcagggctga gtatctgtgg cgcctcgagg 540ctgagaaaat gcggctggcg gaggaagaga agcttcggaa ggagatgagc gccaagaagg 600ccaaggagga ggccgagcgc aagcatcagg agcgcctggc ccagctggct cgtgaggacg 660ctgagcggga gctgaaggag aaggaggccg ctcggcggaa gaaggagctc ctggagcaga 720tggaaagggc ccgccatgag cctgtcaatc actcagacat ggtggacaag atgtttggct 780tcctggggac ttcaggtggc ctgccaggcc aggagggcca ggcacctagt ggctttgagg 840acctggagcg agggcggagg gagatggtgg aggaggacct ggatgcagcc ctgcccctgc 900ctgacgagga tgaggaggac ctctctgagt ataaatttgc caagttcgcg gccacctact 960tccaggggac aaccacgcac tcctacaccc ggcggccact caaacagcca ctgctctacc 1020atgacgacga gggtgaccag ctggcagccc tggcggtctg gatcaccatc ctccgcttca 1080tgggggacct ccctgagccc aagtaccaca cagccatgag tgatggcagt gagaagatcc 1140ctgtgatgac caagatttat gagaccctgg gcaagaagac gtacaagagg gagctgcagg 1200ccctgcaggg cgagggcgag gcccagctcc ccgagggcca gaagaagagc agtgtgaggc 1260acaagctggt gcatttgact ctgaaaaaga agtccaagct cacagaggag gtgaccaaga 1320ggctgcatga cggggagtcc acagtgcagg gcaacagcat gctggaggac cggcccacct 1380ccaacctgga gaagctgcac ttcatcatcg gcaatggcat cctgcggcca gcactccggg 1440acgagatcta ctgccagatc agcaagcagc tgacccacaa cccctccaag agcagctatg 1500cccggggctg gattctcgtg tctctctgcg tgggctgttt cgccccctcc gagaagtttg 1560tcaagtacct gcggaacttc atccacgggg gcccgcccgg ctacgccccg tactgtgagg 1620agcgcctgag aaggaccttt gtcaatggga cacggacaca gccgcccagc tggctggagc 1680tgcaggccac caagtccaag aagccaatca tgttgcccgt gacattcatg gatgggacca 1740ccaagaccct gctgacggac tcggcaacca cggccaagga gctctgcaac gcgctggccg 1800acaagatctc tctcaaggac cggttcgggt tctccctcta cattgccctg tttgacaagg 1860tgtcctccct gggcagcggc agtgaccacg tcatggacgc catctcccag tgcgagcagt 1920acgccaagga gcagggcgcc caggagcgca acgccccctg gaggctcttc ttccgcaaag 1980aggtcttcac gccctggcac agcccctccg aggacaacgt ggccaccaac ctcatctacc 2040agcaggtggt gcgaggagtc aagtttgggg agtacaggtg tgagaaggag gacgacctgg 2100ctgagctggc ctcccagcag tactttgtag actatggctc tgagatgatc ctggagcgcc 2160tcctgaacct cgtgcccacc tacatccccg accgcgagat cacgcccctg aagacgctgg 2220agaagtgggc ccagctggcc atcgccgccc acaagaaggg gatttatgcc cagaggagaa 2280ctgatgccca gaaggtcaaa gaggatgtgg tcagttatgc ccgcttcaag tggcccttgc 2340tcttctccag gttttatgaa gcctacaaat tctcaggccc cagtctcccc aagaacgacg 2400tcatcgtggc cgtcaactgg acgggtgtgt actttgtgga tgagcaggag caggtacttc 2460tggagctgtc cttcccagag atcatggccg tgtccagcag caggggagcg aaaacgacgg 2520cccccagctt cacgctggcc accatcaagg gggacgaata caccttcacc tccagcaatg 2580ctgaggacat tcgtgacctg gtggtcacct tcctagaggg gctccggaag agatctaagt 2640atgttgtggc cctgcaggat aaccccaacc ccgcaggcga ggagtcaggc ttcctcagct 2700ttgccaaggg agacctcatc atcctggacc atgacacggg cgagcaggtc atgaactcgg 2760gctgggccaa cggcatcaat gagaggacca agcagcgtgg ggacttcccc accgacagtg 2820tgtacgtcat gcccactgtc accatgccac cgcgggagat tgtggccctg gtcaccatga 2880ctcccgatca gaggcaggac gttgtccggc tcttgcagct gcgaacggcg gagcccgagg 2940tgcgtgccaa gccctacacg ctggaggagt tttcctatga ctacttcagg cccccaccca 3000agcacacgct gagccgtgtc atggtgtcca aggcccgagg caaggaccgg ctgtggagcc 3060acacgcggga accgctcaag caggcgctgc tcaagaagct cctgggcagt gaggagctct 3120cgcaggaggc ctgcctggcc ttcattgctg tgctcaagta catgggcgac tacccgtcca 3180agaggacacg ctccgtcaac gagctcaccg accagatctt tgagggtccc ctgaaagccg 3240agcccctgaa ggacgaggca tatgtgcaga tcctgaagca gctgaccgac aaccacatca 3300ggtacagcga ggagcggggt tgggagctgc tctggctgtg cacgggcctt ttcccaccca 3360gcaacatcct cctgccccac gtgcagcgct tcctgcagtc ccgaaagcac tgcccactcg 3420ccatcgactg cctgcaacgg ctccagaaag ccctgagaaa cgggtcccgg aagtaccctc 3480cgcacctggt ggaggtggag gccatccagc acaagaccac ccagattttc cacaaagtct 3540acttccctga tgacactgac gaggccttcg aagtggagtc cagcaccaag gccaaggact 3600tctgccagaa catcgccacc aggctgctcc tcaagtcctc agagggattc agcctctttg 3660tcaaaattgc agacaaggtc atcagcgttc ctgagaatga cttcttcttt gactttgttc 3720gacacttgac agactggata aagaaagctc ggcccatcaa ggacggaatt gtgccctcac 3780tcacctacca ggtgttcttc atgaagaagc tgtggaccac cacggtgcca gggaaggatc 3840ccatggccga ttccatcttc cactattacc aggagttgcc caagtatctc cgaggctacc 3900acaagtgcac gcgggaggag gtgctgcagc tgggggcgct gatctacagg gtcaagttcg 3960aggaggacaa gtcctacttc cccagcatcc ccaagctgct gcgggagctg gtgccccagg 4020accttatccg gcaggtctca cctgatgact ggaagcggtc catcgtcgcc tacttcaaca 4080agcacgcagg gaagtccaag gaggaggcca agctggcctt cctgaagctc atcttcaagt 4140ggcccacctt tggctcagcc ttcttcgagg tgaagcaaac tacggagcca aacttccctg 4200agatcctcct aattgccatc aacaagtatg gggtcagcct catcgatccc aaaacgaagg 4260atatcctcac cactcatccc ttcaccaaga tctccaactg gagcagcggc aacacctact 4320tccacatcac cattgggaac ttggtgcgcg ggagcaaact gctctgcgag acgtcactgg 4380gctacaagat ggatgacctc ctgacttcct acattagcca gatgctcaca gccatgagca 4440aacagcgggg ctccaggagc ggcaagtgag gtaccaaggg cgaattctgc agtcgactag 4500agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc 4560ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga 4620ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca 4680ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggaga gatctgagga 4740ctagtccgtc gactgttaat taagcatgct ggggagagat ctaggaaccc ctagtgatgg 4800agttggccac tccctctctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg 4860ggcgtcgggc gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag 4920tggccaaccc cccccccccc ccccctgcag ccctgcatta atgaatcggc caacgcgcgg 4980ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 5040cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 5100cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 5160accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 5220acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 5280cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 5340acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt 5400atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 5460agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 5520acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 5580gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg 5640gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 5700gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 5760gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 5820acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 5880tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 5940ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 6000catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 6060ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 6120caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 6180ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 6240tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 6300cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 6360aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 6420tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 6480gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 6540cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 6600aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 6660tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 6720tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 6780gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 6840atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 6900taggggttcc gcgcacattt

ccccgaaaag tgccacctga cgtctaagaa accattatta 6960tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg 7020gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt 7080aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc 7140ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt 7200gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggaaattg taaacgttaa 7260tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc 7320cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt 7380tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa 7440aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg 7500gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg 7560acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc 7620tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa 7680tgcgccgcta cagggcgcgt cgcgccattc gccattcagg ctacgcaact gttgggaagg 7740gcgatcggtg cgggcctctt cgctattacg ccaggctgc 777937405DNAArtificial sequencethe nucleotide of an "hMyo7a intron 23 splicing vector A" of the subject inventionexon(1153)..(1167) 3aggggggggg gggggggggt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc 60cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg 120agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctcagatc tggcgcgccc 180aattcggtac cctagttatt aatagtaatc aattacgggg tcattagttc atagcccata 240tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 300cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 360ccattgacgt caatgggtgg actatttacg gtaaactgcc cacttggcag tacatcaagt 420gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 480ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 540catcgctatt accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc 600cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg 660ggcggggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg 720ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt 780tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt 840cgctgcgacg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc 900ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg 960gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc 1020ttgaggggct ccgggagcta gagcctctgc taaccatgtt catgccttct tctttttcct 1080acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattcta 1140gcggccgcca cc atg gtg att ctt cag cagggggacc atgtgtggat 1187 Met Val Ile Leu Gln 1 5ggacctgaga ttggggcagg agttcgacgt gcccatcggg gcggtggtga agctctgcga 1247ctctgggcag gtccaggtgg tggatgatga agacaatgaa cactggatct ctccgcagaa 1307cgcaacgcac atcaagccta tgcaccccac gtcggtccac ggcgtggagg acatgatccg 1367cctgggggac ctcaacgagg cgggcatctt gcgcaacctg cttatccgct accgggacca 1427cctcatctac acgtatacgg gctccatcct ggtggctgtg aacccctacc agctgctctc 1487catctactcg ccagagcaca tccgccagta taccaacaag aagattgggg agatgccccc 1547ccacatcttt gccattgctg acaactgcta cttcaacatg aaacgcaaca gccgagacca 1607gtgctgcatc atcagtgggg aatctggggc cgggaagacg gagagcacaa agctgatcct 1667gcagttcctg gcagccatca gtgggcagca ctcgtggatt gagcagcagg tcttggaggc 1727cacccccatt ctggaagcat ttgggaatgc caagaccatc cgcaatgaca actcaagccg 1787tttcggaaag tacatcgaca tccacttcaa caagcggggc gccatcgagg gcgcgaagat 1847tgagcagtac ctgctggaaa agtcacgtgt ctgtcgccag gccctggatg aaaggaacta 1907ccacgtgttc tactgcatgc tggagggtat gagtgaggat cagaagaaga agctgggctt 1967gggccaggcc tctgactaca actacttggc catgggtaac tgcataacct gtgagggccg 2027ggtggacagc caggagtacg ccaacatccg ctccgccatg aaggtgctca tgttcactga 2087caccgagaac tgggagatct cgaagctcct ggctgccatc ctgcacctgg gcaacctgca 2147gtatgaggca cgcacatttg aaaacctgga tgcctgtgag gttctcttct ccccatcgct 2207ggccacagct gcatccctgc ttgaggtgaa ccccccagac ctgatgagct gcctgactag 2267ccgcaccctc atcacccgcg gggagacggt gtccacccca ctgagcaggg aacaggcact 2327ggacgtgcgc gacgccttcg taaaggggat ctacgggcgg ctgttcgtgt ggattgtgga 2387caagatcaac gcagcaattt acaagcctcc ctcccaggat gtgaagaact ctcgcaggtc 2447catcggcctc ctggacatct ttgggtttga gaactttgct gtgaacagct ttgagcagct 2507ctgcatcaac ttcgccaatg agcacctgca gcagttcttt gtgcggcacg tgttcaagct 2567ggagcaggag gaatatgacc tggagagcat tgactggctg cacatcgagt tcactgacaa 2627ccaggatgcc ctggacatga ttgccaacaa gcccatgaac atcatctccc tcatcgatga 2687ggagagcaag ttccccaagg gcacagacac caccatgtta cacaagctga actcccagca 2747caagctcaac gccaactaca tcccccccaa gaacaaccat gagacccagt ttggcatcaa 2807ccattttgca ggcatcgtct actatgagac ccaaggcttc ctggagaaga accgagacac 2867cctgcatggg gacattatcc agctggtcca ctcctccagg aacaagttca tcaagcagat 2927cttccaggcc gatgtcgcca tgggcgccga gaccaggaag cgctcgccca cacttagcag 2987ccagttcaag cggtcactgg agctgctgat gcgcacgctg ggtgcctgcc agcccttctt 3047tgtgcgatgc atcaagccca atgagttcaa gaagcccatg ctgttcgacc ggcacctgtg 3107cgtgcgccag ctgcggtact caggaatgat ggagaccatc cgaatccgcc gagctggcta 3167ccccatccgc tacagcttcg tagagtttgt ggagcggtac cgtgtgctgc tgccaggtgt 3227gaagccggcc tacaagcagg gcgacctccg cgggacttgc cagcgcatgg ctgaggctgt 3287gctgggcacc cacgatgact ggcagatagg caaaaccaag atctttctga aggaccacca 3347tgacatgctg ctggaagtgg agcgggacaa agccatcacc gacagagtca tcctccttca 3407gaaagtcatc cggggattca aagacaggtc taactttctg aagctgaaga acgctgccac 3467actgatccag aggcactggc ggggtcacaa ctgtaggaag aactacgggc tgatgcgtct 3527gggcttcctg cggctgcagg ccctgcaccg ctcccggaag ctgcaccagc agtaccgcct 3587ggcccgccag cgcatcatcc agttccaggc ccgctgccgc gcctatctgg tgcgcaaggc 3647cttccgccac cgcctctggg ctgtgctcac cgtgcaggcc tatgcccggg gcatgatcgc 3707ccgcaggctg caccaacgcc tcagggctga gtatctgtgg cgcctcgagg ctgagaaaat 3767gcggctggcg gaggaagaga agcttcggaa ggagatgagc gccaagaagg ccaaggagga 3827ggccgagcgc aagcatcagg agcgcctggc ccagctggct cgtgaggacg ctgagcggga 3887gctgaaggag aaggaggccg ctcggcggaa gaaggagctc ctggagcaga tggaaagggc 3947ccgccatgag cctgtcaatc actcagacat ggtggacaag atgtttggct tcctggggac 4007ttcaggtggc ctgccaggcc aggagggcca ggcacctagt ggctttgagg taccaggcta 4067gggacagggg ctccagaggc ccacacacac cgcttgtgtt gatcctccct ccttctgtgc 4127ccttggcctt aaagcccacc cagtccctct gaacagtggg gagcagagat gaactgggtc 4187cgggctgcag gtcccaggtc ctgtccctct ccaacgccct tctcaagttt ttttttttgt 4247tttttttttt tttttttttt ttgagatggg gtcgtaccct gttgctcagg ctggagtgca 4307gtagcgtgat cacagctcac tgcagccttg aacttctggg ctcaggcggc tagcgggcac 4367tagtccgtcg actgttaatt aagcatgctg gggagagatc taggaacccc tagtgatgga 4427gttggccact ccctctctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg 4487gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt 4547ggccaacccc cccccccccc cccctgcagc cctgcattaa tgaatcggcc aacgcgcggg 4607gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 4667ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 4727agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 4787ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 4847caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 4907gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 4967cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta 5027tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 5087gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 5147cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 5207tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 5267tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 5327caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 5387aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 5447cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 5507ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 5567tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 5627atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 5687tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 5747aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 5807catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 5867gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 5927ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 5987aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 6047atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 6107cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 6167gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 6227agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 6287gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 6347caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 6407ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 6467tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 6527aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat 6587catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg 6647tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta 6707agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg 6767gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg 6827tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggaaattgt aaacgttaat 6887attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc 6947gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt 7007ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa 7067accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg 7127tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga 7187cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct 7247agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat 7307gcgccgctac agggcgcgtc gcgccattcg ccattcaggc tacgcaactg ttgggaaggg 7367cgatcggtgc gggcctcttc gctattacgc caggctgc 740547477DNAArtificial sequencethe nucleotide of an "hMyo7a intron 23 splicing vector B" of the subject invention 4cgccaggctg cagggggggg gggggggggg ttggccactc cctctctgcg cgctcgctcg 60ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 120gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttcctcagat 180ctggcgcgcc caattggctt cgaattctag cggccgctgc ttaagcaccg cttgtgttga 240tcctccctcc ttctgtgccc ttggccttaa agcccaccca gtccctctga acagtgggga 300gcagagatga actgggtccg ggctgcaggt cccaggtcct gtccctctcc aacgcccttc 360tcaagttttt ttttttgttt tttttttttt tttttttttt gagatggggt cgtaccctgt 420tgctcaggct ggagtgcagt agcgtgatca cagctcactg cagccttgaa cttctgggct 480caggcgatcc tcccacctga gcttcctgag tagctgggac tccagggcat acctcttgtc 540tccttcagga cctggagcga gggcggaggg agatggtgga ggaggacctg gatgcagccc 600tgcccctgcc tgacgaggat gaggaggacc tctctgagta taaatttgcc aagttcgcgg 660ccacctactt ccaggggaca accacgcact cctacacccg gcggccactc aaacagccac 720tgctctacca tgacgacgag ggtgaccagc tggcagccct ggcggtctgg atcaccatcc 780tccgcttcat gggggacctc cctgagccca agtaccacac agccatgagt gatggcagtg 840agaagatccc tgtgatgacc aagatttatg agaccctggg caagaagacg tacaagaggg 900agctgcaggc cctgcagggc gagggcgagg cccagctccc cgagggccag aagaagagca 960gtgtgaggca caagctggtg catttgactc tgaaaaagaa gtccaagctc acagaggagg 1020tgaccaagag gctgcatgac ggggagtcca cagtgcaggg caacagcatg ctggaggacc 1080ggcccacctc caacctggag aagctgcact tcatcatcgg caatggcatc ctgcggccag 1140cactccggga cgagatctac tgccagatca gcaagcagct gacccacaac ccctccaaga 1200gcagctatgc ccggggctgg attctcgtgt ctctctgcgt gggctgtttc gccccctccg 1260agaagtttgt caagtacctg cggaacttca tccacggggg cccgcccggc tacgccccgt 1320actgtgagga gcgcctgaga aggacctttg tcaatgggac acggacacag ccgcccagct 1380ggctggagct gcaggccacc aagtccaaga agccaatcat gttgcccgtg acattcatgg 1440atgggaccac caagaccctg ctgacggact cggcaaccac ggccaaggag ctctgcaacg 1500cgctggccga caagatctct ctcaaggacc ggttcgggtt ctccctctac attgccctgt 1560ttgacaaggt gtcctccctg ggcagcggca gtgaccacgt catggacgcc atctcccagt 1620gcgagcagta cgccaaggag cagggcgccc aggagcgcaa cgccccctgg aggctcttct 1680tccgcaaaga ggtcttcacg ccctggcaca gcccctccga ggacaacgtg gccaccaacc 1740tcatctacca gcaggtggtg cgaggagtca agtttgggga gtacaggtgt gagaaggagg 1800acgacctggc tgagctggcc tcccagcagt actttgtaga ctatggctct gagatgatcc 1860tggagcgcct cctgaacctc gtgcccacct acatccccga ccgcgagatc acgcccctga 1920agacgctgga gaagtgggcc cagctggcca tcgccgccca caagaagggg atttatgccc 1980agaggagaac tgatgcccag aaggtcaaag aggatgtggt cagttatgcc cgcttcaagt 2040ggcccttgct cttctccagg ttttatgaag cctacaaatt ctcaggcccc agtctcccca 2100agaacgacgt catcgtggcc gtcaactgga cgggtgtgta ctttgtggat gagcaggagc 2160aggtacttct ggagctgtcc ttcccagaga tcatggccgt gtccagcagc aggggagcga 2220aaacgacggc ccccagcttc acgctggcca ccatcaaggg ggacgaatac accttcacct 2280ccagcaatgc tgaggacatt cgtgacctgg tggtcacctt cctagagggg ctccggaaga 2340gatctaagta tgttgtggcc ctgcaggata accccaaccc cgcaggcgag gagtcaggct 2400tcctcagctt tgccaaggga gacctcatca tcctggacca tgacacgggc gagcaggtca 2460tgaactcggg ctgggccaac ggcatcaatg agaggaccaa gcagcgtggg gacttcccca 2520ccgacagtgt gtacgtcatg cccactgtca ccatgccacc gcgggagatt gtggccctgg 2580tcaccatgac tcccgatcag aggcaggacg ttgtccggct cttgcagctg cgaacggcgg 2640agcccgaggt gcgtgccaag ccctacacgc tggaggagtt ttcctatgac tacttcaggc 2700ccccacccaa gcacacgctg agccgtgtca tggtgtccaa ggcccgaggc aaggaccggc 2760tgtggagcca cacgcgggaa ccgctcaagc aggcgctgct caagaagctc ctgggcagtg 2820aggagctctc gcaggaggcc tgcctggcct tcattgctgt gctcaagtac atgggcgact 2880acccgtccaa gaggacacgc tccgtcaacg agctcaccga ccagatcttt gagggtcccc 2940tgaaagccga gcccctgaag gacgaggcat atgtgcagat cctgaagcag ctgaccgaca 3000accacatcag gtacagcgag gagcggggtt gggagctgct ctggctgtgc acgggccttt 3060tcccacccag caacatcctc ctgccccacg tgcagcgctt cctgcagtcc cgaaagcact 3120gcccactcgc catcgactgc ctgcaacggc tccagaaagc cctgagaaac gggtcccgga 3180agtaccctcc gcacctggtg gaggtggagg ccatccagca caagaccacc cagattttcc 3240acaaagtcta cttccctgat gacactgacg aggccttcga agtggagtcc agcaccaagg 3300ccaaggactt ctgccagaac atcgccacca ggctgctcct caagtcctca gagggattca 3360gcctctttgt caaaattgca gacaaggtca tcagcgttcc tgagaatgac ttcttctttg 3420actttgttcg acacttgaca gactggataa agaaagctcg gcccatcaag gacggaattg 3480tgccctcact cacctaccag gtgttcttca tgaagaagct gtggaccacc acggtgccag 3540ggaaggatcc catggccgat tccatcttcc actattacca ggagttgccc aagtatctcc 3600gaggctacca caagtgcacg cgggaggagg tgctgcagct gggggcgctg atctacaggg 3660tcaagttcga ggaggacaag tcctacttcc ccagcatccc caagctgctg cgggagctgg 3720tgccccagga ccttatccgg caggtctcac ctgatgactg gaagcggtcc atcgtcgcct 3780acttcaacaa gcacgcaggg aagtccaagg aggaggccaa gctggccttc ctgaagctca 3840tcttcaagtg gcccaccttt ggctcagcct tcttcgaggt gaagcaaact acggagccaa 3900acttccctga gatcctccta attgccatca acaagtatgg ggtcagcctc atcgatccca 3960aaacgaagga tatcctcacc actcatccct tcaccaagat ctccaactgg agcagcggca 4020acacctactt ccacatcacc attgggaact tggtgcgcgg gagcaaactg ctctgcgaga 4080cgtcactggg ctacaagatg gatgacctcc tgacttccta cattagccag atgctcacag 4140ccatgagcaa acagcggggc tccaggagcg gcaagtgagg taccaagggc gaattctgca 4200gtcgactaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt 4260ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta 4320ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg 4380ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggagag 4440atctgaggac tagtccgtcg actgttaatt aagcatgctg gggagagatc taggaacccc 4500tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgcccggg 4560caaagcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca 4620gagagggagt ggccaacccc cccccccccc cccctgcagc cctgcattaa tgaatcggcc 4680aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 4740cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 4800ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 4860aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 4920acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 4980gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 5040ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac 5100gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 5160cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 5220taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 5280atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga 5340cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 5400cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 5460ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 5520ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 5580tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 5640aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 5700tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 5760gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 5820atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 5880tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 5940ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 6000ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 6060tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 6120ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 6180ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 6240tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 6300gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 6360taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 6420cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa

6480agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 6540gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 6600ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa 6660ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg 6720cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag 6780cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg 6840gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc 6900atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggaaattgt 6960aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa 7020ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt 7080gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa 7140agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag 7200ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt 7260tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg 7320agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc 7380cgcgcttaat gcgccgctac agggcgcgtc gcgccattcg ccattcaggc tacgcaactg 7440ttgggaaggg cgatcggtgc gggcctcttc gctatta 747757465DNAHomo sapiens 5gctctgggca ggagagagag tgagagacaa gagacacaca cagagagacg gcgaggaagg 60gaaagaccca gagggacgcc tagaacgaga cttggagcca gacagaggaa gaggggacgt 120gtgtttgcag actggctggg cccgtgaccc agcttcctga gtcctccgtg caggtggcag 180ctgtaccagg ctggcaggtc actgagagtg ggcagctggg ccccagaact gtgcctggcc 240cagtgggcag caggagctcc tgacttggga ccatggtgat tcttcagcag ggggaccatg 300tgtggatgga cctgagattg gggcaggagt tcgacgtgcc catcggggcg gtggtgaagc 360tctgcgactc tgggcaggtc caggtggtgg atgatgaaga caatgaacac tggatctctc 420cgcagaacgc aacgcacatc aagcctatgc accccacgtc ggtccacggc gtggaggaca 480tgatccgcct gggggacctc aacgaggcgg gcatcttgcg caacctgctt atccgctacc 540gggaccacct catctacacg tatacgggct ccatcctggt ggctgtgaac ccctaccagc 600tgctctccat ctactcgcca gagcacatcc gccagtatac caacaagaag attggggaga 660tgccccccca catctttgcc attgctgaca actgctactt caacatgaaa cgcaacagcc 720gagaccagtg ctgcatcatc agtggggaat ctggggccgg gaagacggag agcacaaagc 780tgatcctgca gttcctggca gccatcagtg ggcagcactc gtggattgag cagcaggtct 840tggaggccac ccccattctg gaagcatttg ggaatgccaa gaccatccgc aatgacaact 900caagccgttt cggaaagtac atcgacatcc acttcaacaa gcggggcgcc atcgagggcg 960cgaagattga gcagtacctg ctggaaaagt cacgtgtctg tcgccaggcc ctggatgaaa 1020ggaactacca cgtgttctac tgcatgctgg agggtatgag tgaggatcag aagaagaagc 1080tgggcttggg ccaggcctct gactacaact acttggccat gggtaactgc ataacctgtg 1140agggccgggt ggacagccag gagtacgcca acatccgctc cgccatgaag gtgctcatgt 1200tcactgacac cgagaactgg gagatctcga agctcctggc tgccatcctg cacctgggca 1260acctgcagta tgaggcacgc acatttgaaa acctggatgc ctgtgaggtt ctcttctccc 1320catcgctggc cacagctgca tccctgcttg aggtgaaccc cccagacctg atgagctgcc 1380tgactagccg caccctcatc acccgcgggg agacggtgtc caccccactg agcagggaac 1440aggcactgga cgtgcgcgac gccttcgtaa aggggatcta cgggcggctg ttcgtgtgga 1500ttgtggacaa gatcaacgca gcaatttaca agcctccctc ccaggatgtg aagaactctc 1560gcaggtccat cggcctcctg gacatctttg ggtttgagaa ctttgctgtg aacagctttg 1620agcagctctg catcaacttc gccaatgagc acctgcagca gttctttgtg cggcacgtgt 1680tcaagctgga gcaggaggaa tatgacctgg agagcattga ctggctgcac atcgagttca 1740ctgacaacca ggatgccctg gacatgattg ccaacaagcc catgaacatc atctccctca 1800tcgatgagga gagcaagttc cccaagggca cagacaccac catgttacac aagctgaact 1860cccagcacaa gctcaacgcc aactacatcc cccccaagaa caaccatgag acccagtttg 1920gcatcaacca ttttgcaggc atcgtctact atgagaccca aggcttcctg gagaagaacc 1980gagacaccct gcatggggac attatccagc tggtccactc ctccaggaac aagttcatca 2040agcagatctt ccaggccgat gtcgccatgg gcgccgagac caggaagcgc tcgcccacac 2100ttagcagcca gttcaagcgg tcactggagc tgctgatgcg cacgctgggt gcctgccagc 2160ccttctttgt gcgatgcatc aagcccaatg agttcaagaa gcccatgctg ttcgaccggc 2220acctgtgcgt gcgccagctg cggtactcag gaatgatgga gaccatccga atccgccgag 2280ctggctaccc catccgctac agcttcgtag agtttgtgga gcggtaccgt gtgctgctgc 2340caggtgtgaa gccggcctac aagcagggcg acctccgcgg gacttgccag cgcatggctg 2400aggctgtgct gggcacccac gatgactggc agataggcaa aaccaagatc tttctgaagg 2460accaccatga catgctgctg gaagtggagc gggacaaagc catcaccgac agagtcatcc 2520tccttcagaa agtcatccgg ggattcaaag acaggtctaa ctttctgaag ctgaagaacg 2580ctgccacact gatccagagg cactggcggg gtcacaactg taggaagaac tacgggctga 2640tgcgtctggg cttcctgcgg ctgcaggccc tgcaccgctc ccggaagctg caccagcagt 2700accgcctggc ccgccagcgc atcatccagt tccaggcccg ctgccgcgcc tatctggtgc 2760gcaaggcctt ccgccaccgc ctctgggctg tgctcaccgt gcaggcctat gcccggggca 2820tgatcgcccg caggctgcac caacgcctca gggctgagta tctgtggcgc ctcgaggctg 2880agaaaatgcg gctggcggag gaagagaagc ttcggaagga gatgagcgcc aagaaggcca 2940aggaggaggc cgagcgcaag catcaggagc gcctggccca gctggctcgt gaggacgctg 3000agcgggagct gaaggagaag gaggccgctc ggcggaagaa ggagctcctg gagcagatgg 3060aaagggcccg ccatgagcct gtcaatcact cagacatggt ggacaagatg tttggcttcc 3120tggggacttc aggtggcctg ccaggccagg agggccaggc acctagtggc tttgaggacc 3180tggagcgagg gcggagggag atggtggagg aggacctgga tgcagccctg cccctgcctg 3240acgaggatga ggaggacctc tctgagtata aatttgccaa gttcgcggcc acctacttcc 3300aggggacaac cacgcactcc tacacccggc ggccactcaa acagccactg ctctaccatg 3360acgacgaggg tgaccagctg gcagccctgg cggtctggat caccatcctc cgcttcatgg 3420gggacctccc tgagcccaag taccacacag ccatgagtga tggcagtgag aagatccctg 3480tgatgaccaa gatttatgag accctgggca agaagacgta caagagggag ctgcaggccc 3540tgcagggcga gggcgaggcc cagctccccg agggccagaa gaagagcagt gtgaggcaca 3600agctggtgca tttgactctg aaaaagaagt ccaagctcac agaggaggtg accaagaggc 3660tgcatgacgg ggagtccaca gtgcagggca acagcatgct ggaggaccgg cccacctcca 3720acctggagaa gctgcacttc atcatcggca atggcatcct gcggccagca ctccgggacg 3780agatctactg ccagatcagc aagcagctga cccacaaccc ctccaagagc agctatgccc 3840ggggctggat tctcgtgtct ctctgcgtgg gctgtttcgc cccctccgag aagtttgtca 3900agtacctgcg gaacttcatc cacgggggcc cgcccggcta cgccccgtac tgtgaggagc 3960gcctgagaag gacctttgtc aatgggacac ggacacagcc gcccagctgg ctggagctgc 4020aggccaccaa gtccaagaag ccaatcatgt tgcccgtgac attcatggat gggaccacca 4080agaccctgct gacggactcg gcaaccacgg ccaaggagct ctgcaacgcg ctggccgaca 4140agatctctct caaggaccgg ttcgggttct ccctctacat tgccctgttt gacaaggtgt 4200cctccctggg cagcggcagt gaccacgtca tggacgccat ctcccagtgc gagcagtacg 4260ccaaggagca gggcgcccag gagcgcaacg ccccctggag gctcttcttc cgcaaagagg 4320tcttcacgcc ctggcacagc ccctccgagg acaacgtggc caccaacctc atctaccagc 4380aggtggtgcg aggagtcaag tttggggagt acaggtgtga gaaggaggac gacctggctg 4440agctggcctc ccagcagtac tttgtagact atggctctga gatgatcctg gagcgcctcc 4500tgaacctcgt gcccacctac atccccgacc gcgagatcac gcccctgaag acgctggaga 4560agtgggccca gctggccatc gccgcccaca agaaggggat ttatgcccag aggagaactg 4620atgcccagaa ggtcaaagag gatgtggtca gttatgcccg cttcaagtgg cccttgctct 4680tctccaggtt ttatgaagcc tacaaattct caggccccag tctccccaag aacgacgtca 4740tcgtggccgt caactggacg ggtgtgtact ttgtggatga gcaggagcag gtacttctgg 4800agctgtcctt cccagagatc atggccgtgt ccagcagcag ggagtgccgt gtctggctct 4860cactgggctg ctctgatctt ggctgtgctg cgcctcactc aggctgggca ggactgaccc 4920cggcggggcc ctgttctccg tgttggtcct gcaggggagc gaaaacgacg gcccccagct 4980tcacgctggc caccatcaag ggggacgaat acaccttcac ctccagtaat gctgaggaca 5040ttcgtgacct ggtggtcacc ttcctagagg ggctccggaa gagatctaag tatgttgtgg 5100ccctgcagga taaccccaac cccgcaggcg aggagtcagg cttcctcagc tttgccaagg 5160gagacctcat catcctggac catgacacgg gcgagcaggt catgaactcg ggctgggcca 5220acggcatcaa tgagaggacc aagcagcgtg gggacttccc caccgactgt gtgtacgtca 5280tgcccactgt caccatgcca ccgcgggaga ttgtggccct ggtcaccatg actcccgatc 5340agaggcagga cgttgtccgg ctcttgcagc tgcgaacggc ggagcccgag gtgcgtgcca 5400agccctacac gctggaggag ttttcctatg actacttcag gcccccaccc aagcacacgc 5460tgagccgtgt catggtgtcc aaggcccgag gcaaggaccg gctgtggagc cacacgcggg 5520aaccgctcaa gcaggcgctg ctcaagaagc tcctgggcag tgaggagctc tcgcaggagg 5580cctgcctggc cttcattgct gtgctcaagt acatgggcga ctacccgtcc aagaggacac 5640gctccgtcaa cgagctcacc gaccagatct ttgagggtcc cctgaaagcc gagcccctga 5700aggacgaggc atatgtgcag atcctgaagc agctgaccga caaccacatc aggtacagcg 5760aggagcgggg ttgggagctg ctctggctgt gcacgggcct tttcccaccc agcaacatcc 5820tcctgcccca cgtgcagcgc ttcctgcagt cccgaaagca ctgcccactc gccatcgact 5880gcctgcaacg gctccagaaa gccctgagaa acgggtcccg gaagtaccct ccgcacctgg 5940tggaggtgga ggccatccag cacaagacca cccagatttt ccacaaggtc tacttccctg 6000atgacactga cgaggccttc gaagtggagt ccagcaccaa ggccaaggac ttctgccaga 6060acatcgccac caggctgctc ctcaagtcct cagagggatt cagcctcttt gtcaaaattg 6120cagacaaggt catcagcgtt cctgagaatg acttcttctt tgactttgtt cgacacttga 6180cagactggat aaagaaagct cggcccatca aggacggaat tgtgccctca ctcacctacc 6240aggtgttctt catgaagaag ctgtggacca ccacggtgcc agggaaggat cccatggccg 6300attccatctt ccactattac caggagttgc ccaagtatct ccgaggctac cacaagtgca 6360cgcgggagga ggtgctgcag ctgggggcgc tgatctacag ggtcaagttc gaggaggaca 6420agtcctactt ccccagcatc cccaagctgc tgcgggagct ggtgccccag gaccttatcc 6480ggcaggtctc acctgatgac tggaagcggt ccatcgtcgc ctacttcaac aagcacgcag 6540ggaagtccaa ggaggaggcc aagctggcct tcctgaagct catcttcaag tggcccacct 6600ttggctcagc cttcttcgag gtgaagcaaa ctacggagcc aaacttccct gagatcctcc 6660taattgccat caacaagtat ggggtcagcc tcatcgatcc caaaacgaag gatatcctca 6720ccactcatcc cttcaccaag atctccaact ggagcagcgg caacacctac ttccacatca 6780ccattgggaa cttggtgcgc gggagcaaac tgctctgcga gacgtcactg ggctacaaga 6840tggatgacct cctgacttcc tacattagcc agatgctcac agccatgagc aaacagcggg 6900gctccaggag cggcaagtga acagtcacgg ggaggtgctg gttccatgcc tgctctcgag 6960gcagcagtgg gttcaggccc atcagctacc cctgcagctg gggaagactt atgccatccc 7020ggcagcgagg ctgggctggc cagccaccac tgactatacc aactgggcct ctgatgttct 7080tccagtgagg catctctctg ggatgcagaa cttccctcca tccacccctc tggcacctgg 7140gttggtctaa tcctagtttg ctgtggcctt cccggttgtg agagcctgtg atccttagat 7200gtgtctcctg tttcagacca gccccaccat gcaacttcct ttgactttct gtgtaccact 7260gggatagagg aatcaagagg acaatctagc tctccatact ttgaacaacc aaatgtgcat 7320tgaatactct gaaaccgaag ggactggatc tgcaggtggg atgagggaga cagaccactt 7380ttctatattg cagtgtgaat gctgggcccc tgctcaagtc taccctgatc acctcagggc 7440ataaagcatg tttcattctc tgaaa 746562215PRTHomo sapiens 6Met Val Ile Leu Gln Gln Gly Asp His Val Trp Met Asp Leu Arg Leu1 5 10 15Gly Gln Glu Phe Asp Val Pro Ile Gly Ala Val Val Lys Leu Cys Asp 20 25 30Ser Gly Gln Val Gln Val Val Asp Asp Glu Asp Asn Glu His Trp Ile 35 40 45Ser Pro Gln Asn Ala Thr His Ile Lys Pro Met His Pro Thr Ser Val 50 55 60His Gly Val Glu Asp Met Ile Arg Leu Gly Asp Leu Asn Glu Ala Gly65 70 75 80Ile Leu Arg Asn Leu Leu Ile Arg Tyr Arg Asp His Leu Ile Tyr Thr 85 90 95Tyr Thr Gly Ser Ile Leu Val Ala Val Asn Pro Tyr Gln Leu Leu Ser 100 105 110Ile Tyr Ser Pro Glu His Ile Arg Gln Tyr Thr Asn Lys Lys Ile Gly 115 120 125Glu Met Pro Pro His Ile Phe Ala Ile Ala Asp Asn Cys Tyr Phe Asn 130 135 140Met Lys Arg Asn Ser Arg Asp Gln Cys Cys Ile Ile Ser Gly Glu Ser145 150 155 160Gly Ala Gly Lys Thr Glu Ser Thr Lys Leu Ile Leu Gln Phe Leu Ala 165 170 175Ala Ile Ser Gly Gln His Ser Trp Ile Glu Gln Gln Val Leu Glu Ala 180 185 190Thr Pro Ile Leu Glu Ala Phe Gly Asn Ala Lys Thr Ile Arg Asn Asp 195 200 205Asn Ser Ser Arg Phe Gly Lys Tyr Ile Asp Ile His Phe Asn Lys Arg 210 215 220Gly Ala Ile Glu Gly Ala Lys Ile Glu Gln Tyr Leu Leu Glu Lys Ser225 230 235 240Arg Val Cys Arg Gln Ala Leu Asp Glu Arg Asn Tyr His Val Phe Tyr 245 250 255Cys Met Leu Glu Gly Met Ser Glu Asp Gln Lys Lys Lys Leu Gly Leu 260 265 270Gly Gln Ala Ser Asp Tyr Asn Tyr Leu Ala Met Gly Asn Cys Ile Thr 275 280 285Cys Glu Gly Arg Val Asp Ser Gln Glu Tyr Ala Asn Ile Arg Ser Ala 290 295 300Met Lys Val Leu Met Phe Thr Asp Thr Glu Asn Trp Glu Ile Ser Lys305 310 315 320Leu Leu Ala Ala Ile Leu His Leu Gly Asn Leu Gln Tyr Glu Ala Arg 325 330 335Thr Phe Glu Asn Leu Asp Ala Cys Glu Val Leu Phe Ser Pro Ser Leu 340 345 350Ala Thr Ala Ala Ser Leu Leu Glu Val Asn Pro Pro Asp Leu Met Ser 355 360 365Cys Leu Thr Ser Arg Thr Leu Ile Thr Arg Gly Glu Thr Val Ser Thr 370 375 380Pro Leu Ser Arg Glu Gln Ala Leu Asp Val Arg Asp Ala Phe Val Lys385 390 395 400Gly Ile Tyr Gly Arg Leu Phe Val Trp Ile Val Asp Lys Ile Asn Ala 405 410 415Ala Ile Tyr Lys Pro Pro Ser Gln Asp Val Lys Asn Ser Arg Arg Ser 420 425 430Ile Gly Leu Leu Asp Ile Phe Gly Phe Glu Asn Phe Ala Val Asn Ser 435 440 445Phe Glu Gln Leu Cys Ile Asn Phe Ala Asn Glu His Leu Gln Gln Phe 450 455 460Phe Val Arg His Val Phe Lys Leu Glu Gln Glu Glu Tyr Asp Leu Glu465 470 475 480Ser Ile Asp Trp Leu His Ile Glu Phe Thr Asp Asn Gln Asp Ala Leu 485 490 495Asp Met Ile Ala Asn Lys Pro Met Asn Ile Ile Ser Leu Ile Asp Glu 500 505 510Glu Ser Lys Phe Pro Lys Gly Thr Asp Thr Thr Met Leu His Lys Leu 515 520 525Asn Ser Gln His Lys Leu Asn Ala Asn Tyr Ile Pro Pro Lys Asn Asn 530 535 540His Glu Thr Gln Phe Gly Ile Asn His Phe Ala Gly Ile Val Tyr Tyr545 550 555 560Glu Thr Gln Gly Phe Leu Glu Lys Asn Arg Asp Thr Leu His Gly Asp 565 570 575Ile Ile Gln Leu Val His Ser Ser Arg Asn Lys Phe Ile Lys Gln Ile 580 585 590Phe Gln Ala Asp Val Ala Met Gly Ala Glu Thr Arg Lys Arg Ser Pro 595 600 605Thr Leu Ser Ser Gln Phe Lys Arg Ser Leu Glu Leu Leu Met Arg Thr 610 615 620Leu Gly Ala Cys Gln Pro Phe Phe Val Arg Cys Ile Lys Pro Asn Glu625 630 635 640Phe Lys Lys Pro Met Leu Phe Asp Arg His Leu Cys Val Arg Gln Leu 645 650 655Arg Tyr Ser Gly Met Met Glu Thr Ile Arg Ile Arg Arg Ala Gly Tyr 660 665 670Pro Ile Arg Tyr Ser Phe Val Glu Phe Val Glu Arg Tyr Arg Val Leu 675 680 685Leu Pro Gly Val Lys Pro Ala Tyr Lys Gln Gly Asp Leu Arg Gly Thr 690 695 700Cys Gln Arg Met Ala Glu Ala Val Leu Gly Thr His Asp Asp Trp Gln705 710 715 720Ile Gly Lys Thr Lys Ile Phe Leu Lys Asp His His Asp Met Leu Leu 725 730 735Glu Val Glu Arg Asp Lys Ala Ile Thr Asp Arg Val Ile Leu Leu Gln 740 745 750Lys Val Ile Arg Gly Phe Lys Asp Arg Ser Asn Phe Leu Lys Leu Lys 755 760 765Asn Ala Ala Thr Leu Ile Gln Arg His Trp Arg Gly His Asn Cys Arg 770 775 780Lys Asn Tyr Gly Leu Met Arg Leu Gly Phe Leu Arg Leu Gln Ala Leu785 790 795 800His Arg Ser Arg Lys Leu His Gln Gln Tyr Arg Leu Ala Arg Gln Arg 805 810 815Ile Ile Gln Phe Gln Ala Arg Cys Arg Ala Tyr Leu Val Arg Lys Ala 820 825 830Phe Arg His Arg Leu Trp Ala Val Leu Thr Val Gln Ala Tyr Ala Arg 835 840 845Gly Met Ile Ala Arg Arg Leu His Gln Arg Leu Arg Ala Glu Tyr Leu 850 855 860Trp Arg Leu Glu Ala Glu Lys Met Arg Leu Ala Glu Glu Glu Lys Leu865 870 875 880Arg Lys Glu Met Ser Ala Lys Lys Ala Lys Glu Glu Ala Glu Arg Lys 885 890 895His Gln Glu Arg Leu Ala Gln Leu Ala Arg Glu Asp Ala Glu Arg Glu 900 905 910Leu Lys Glu Lys Glu Ala Ala Arg Arg Lys Lys Glu Leu Leu Glu Gln 915 920 925Met Glu Arg Ala Arg His Glu Pro Val Asn His Ser Asp Met Val Asp 930 935 940Lys Met Phe Gly Phe Leu Gly Thr Ser Gly Gly Leu Pro Gly Gln Glu945 950 955 960Gly Gln Ala Pro Ser Gly Phe Glu Asp Leu Glu Arg Gly Arg Arg Glu 965 970 975Met Val Glu Glu Asp Leu Asp Ala Ala Leu Pro Leu Pro Asp Glu Asp 980 985 990Glu Glu Asp Leu Ser Glu Tyr Lys Phe Ala Lys Phe Ala Ala Thr Tyr 995 1000 1005Phe Gln Gly Thr Thr Thr His Ser Tyr Thr Arg Arg Pro Leu Lys 1010 1015 1020Gln Pro Leu Leu Tyr His Asp Asp Glu Gly Asp Gln Leu Ala Ala 1025 1030 1035Leu Ala Val Trp Ile Thr Ile Leu Arg Phe Met Gly Asp Leu Pro 1040 1045 1050Glu Pro Lys Tyr His Thr Ala Met Ser Asp Gly Ser Glu Lys Ile 1055 1060

1065Pro Val Met Thr Lys Ile Tyr Glu Thr Leu Gly Lys Lys Thr Tyr 1070 1075 1080Lys Arg Glu Leu Gln Ala Leu Gln Gly Glu Gly Glu Ala Gln Leu 1085 1090 1095Pro Glu Gly Gln Lys Lys Ser Ser Val Arg His Lys Leu Val His 1100 1105 1110Leu Thr Leu Lys Lys Lys Ser Lys Leu Thr Glu Glu Val Thr Lys 1115 1120 1125Arg Leu His Asp Gly Glu Ser Thr Val Gln Gly Asn Ser Met Leu 1130 1135 1140Glu Asp Arg Pro Thr Ser Asn Leu Glu Lys Leu His Phe Ile Ile 1145 1150 1155Gly Asn Gly Ile Leu Arg Pro Ala Leu Arg Asp Glu Ile Tyr Cys 1160 1165 1170Gln Ile Ser Lys Gln Leu Thr His Asn Pro Ser Lys Ser Ser Tyr 1175 1180 1185Ala Arg Gly Trp Ile Leu Val Ser Leu Cys Val Gly Cys Phe Ala 1190 1195 1200Pro Ser Glu Lys Phe Val Lys Tyr Leu Arg Asn Phe Ile His Gly 1205 1210 1215Gly Pro Pro Gly Tyr Ala Pro Tyr Cys Glu Glu Arg Leu Arg Arg 1220 1225 1230Thr Phe Val Asn Gly Thr Arg Thr Gln Pro Pro Ser Trp Leu Glu 1235 1240 1245Leu Gln Ala Thr Lys Ser Lys Lys Pro Ile Met Leu Pro Val Thr 1250 1255 1260Phe Met Asp Gly Thr Thr Lys Thr Leu Leu Thr Asp Ser Ala Thr 1265 1270 1275Thr Ala Lys Glu Leu Cys Asn Ala Leu Ala Asp Lys Ile Ser Leu 1280 1285 1290Lys Asp Arg Phe Gly Phe Ser Leu Tyr Ile Ala Leu Phe Asp Lys 1295 1300 1305Val Ser Ser Leu Gly Ser Gly Ser Asp His Val Met Asp Ala Ile 1310 1315 1320Ser Gln Cys Glu Gln Tyr Ala Lys Glu Gln Gly Ala Gln Glu Arg 1325 1330 1335Asn Ala Pro Trp Arg Leu Phe Phe Arg Lys Glu Val Phe Thr Pro 1340 1345 1350Trp His Ser Pro Ser Glu Asp Asn Val Ala Thr Asn Leu Ile Tyr 1355 1360 1365Gln Gln Val Val Arg Gly Val Lys Phe Gly Glu Tyr Arg Cys Glu 1370 1375 1380Lys Glu Asp Asp Leu Ala Glu Leu Ala Ser Gln Gln Tyr Phe Val 1385 1390 1395Asp Tyr Gly Ser Glu Met Ile Leu Glu Arg Leu Leu Asn Leu Val 1400 1405 1410Pro Thr Tyr Ile Pro Asp Arg Glu Ile Thr Pro Leu Lys Thr Leu 1415 1420 1425Glu Lys Trp Ala Gln Leu Ala Ile Ala Ala His Lys Lys Gly Ile 1430 1435 1440Tyr Ala Gln Arg Arg Thr Asp Ala Gln Lys Val Lys Glu Asp Val 1445 1450 1455Val Ser Tyr Ala Arg Phe Lys Trp Pro Leu Leu Phe Ser Arg Phe 1460 1465 1470Tyr Glu Ala Tyr Lys Phe Ser Gly Pro Ser Leu Pro Lys Asn Asp 1475 1480 1485Val Ile Val Ala Val Asn Trp Thr Gly Val Tyr Phe Val Asp Glu 1490 1495 1500Gln Glu Gln Val Leu Leu Glu Leu Ser Phe Pro Glu Ile Met Ala 1505 1510 1515Val Ser Ser Ser Arg Glu Cys Arg Val Trp Leu Ser Leu Gly Cys 1520 1525 1530Ser Asp Leu Gly Cys Ala Ala Pro His Ser Gly Trp Ala Gly Leu 1535 1540 1545Thr Pro Ala Gly Pro Cys Ser Pro Cys Trp Ser Cys Arg Gly Ala 1550 1555 1560Lys Thr Thr Ala Pro Ser Phe Thr Leu Ala Thr Ile Lys Gly Asp 1565 1570 1575Glu Tyr Thr Phe Thr Ser Ser Asn Ala Glu Asp Ile Arg Asp Leu 1580 1585 1590Val Val Thr Phe Leu Glu Gly Leu Arg Lys Arg Ser Lys Tyr Val 1595 1600 1605Val Ala Leu Gln Asp Asn Pro Asn Pro Ala Gly Glu Glu Ser Gly 1610 1615 1620Phe Leu Ser Phe Ala Lys Gly Asp Leu Ile Ile Leu Asp His Asp 1625 1630 1635Thr Gly Glu Gln Val Met Asn Ser Gly Trp Ala Asn Gly Ile Asn 1640 1645 1650Glu Arg Thr Lys Gln Arg Gly Asp Phe Pro Thr Asp Cys Val Tyr 1655 1660 1665Val Met Pro Thr Val Thr Met Pro Pro Arg Glu Ile Val Ala Leu 1670 1675 1680Val Thr Met Thr Pro Asp Gln Arg Gln Asp Val Val Arg Leu Leu 1685 1690 1695Gln Leu Arg Thr Ala Glu Pro Glu Val Arg Ala Lys Pro Tyr Thr 1700 1705 1710Leu Glu Glu Phe Ser Tyr Asp Tyr Phe Arg Pro Pro Pro Lys His 1715 1720 1725Thr Leu Ser Arg Val Met Val Ser Lys Ala Arg Gly Lys Asp Arg 1730 1735 1740Leu Trp Ser His Thr Arg Glu Pro Leu Lys Gln Ala Leu Leu Lys 1745 1750 1755Lys Leu Leu Gly Ser Glu Glu Leu Ser Gln Glu Ala Cys Leu Ala 1760 1765 1770Phe Ile Ala Val Leu Lys Tyr Met Gly Asp Tyr Pro Ser Lys Arg 1775 1780 1785Thr Arg Ser Val Asn Glu Leu Thr Asp Gln Ile Phe Glu Gly Pro 1790 1795 1800Leu Lys Ala Glu Pro Leu Lys Asp Glu Ala Tyr Val Gln Ile Leu 1805 1810 1815Lys Gln Leu Thr Asp Asn His Ile Arg Tyr Ser Glu Glu Arg Gly 1820 1825 1830Trp Glu Leu Leu Trp Leu Cys Thr Gly Leu Phe Pro Pro Ser Asn 1835 1840 1845Ile Leu Leu Pro His Val Gln Arg Phe Leu Gln Ser Arg Lys His 1850 1855 1860Cys Pro Leu Ala Ile Asp Cys Leu Gln Arg Leu Gln Lys Ala Leu 1865 1870 1875Arg Asn Gly Ser Arg Lys Tyr Pro Pro His Leu Val Glu Val Glu 1880 1885 1890Ala Ile Gln His Lys Thr Thr Gln Ile Phe His Lys Val Tyr Phe 1895 1900 1905Pro Asp Asp Thr Asp Glu Ala Phe Glu Val Glu Ser Ser Thr Lys 1910 1915 1920Ala Lys Asp Phe Cys Gln Asn Ile Ala Thr Arg Leu Leu Leu Lys 1925 1930 1935Ser Ser Glu Gly Phe Ser Leu Phe Val Lys Ile Ala Asp Lys Val 1940 1945 1950Ile Ser Val Pro Glu Asn Asp Phe Phe Phe Asp Phe Val Arg His 1955 1960 1965Leu Thr Asp Trp Ile Lys Lys Ala Arg Pro Ile Lys Asp Gly Ile 1970 1975 1980Val Pro Ser Leu Thr Tyr Gln Val Phe Phe Met Lys Lys Leu Trp 1985 1990 1995Thr Thr Thr Val Pro Gly Lys Asp Pro Met Ala Asp Ser Ile Phe 2000 2005 2010His Tyr Tyr Gln Glu Leu Pro Lys Tyr Leu Arg Gly Tyr His Lys 2015 2020 2025Cys Thr Arg Glu Glu Val Leu Gln Leu Gly Ala Leu Ile Tyr Arg 2030 2035 2040Val Lys Phe Glu Glu Asp Lys Ser Tyr Phe Pro Ser Ile Pro Lys 2045 2050 2055Leu Leu Arg Glu Leu Val Pro Gln Asp Leu Ile Arg Gln Val Ser 2060 2065 2070Pro Asp Asp Trp Lys Arg Ser Ile Val Ala Tyr Phe Asn Lys His 2075 2080 2085Ala Gly Lys Ser Lys Glu Glu Ala Lys Leu Ala Phe Leu Lys Leu 2090 2095 2100Ile Phe Lys Trp Pro Thr Phe Gly Ser Ala Phe Phe Glu Val Lys 2105 2110 2115Gln Thr Thr Glu Pro Asn Phe Pro Glu Ile Leu Leu Ile Ala Ile 2120 2125 2130Asn Lys Tyr Gly Val Ser Leu Ile Asp Pro Lys Thr Lys Asp Ile 2135 2140 2145Leu Thr Thr His Pro Phe Thr Lys Ile Ser Asn Trp Ser Ser Gly 2150 2155 2160Asn Thr Tyr Phe His Ile Thr Ile Gly Asn Leu Val Arg Gly Ser 2165 2170 2175Lys Leu Leu Cys Glu Thr Ser Leu Gly Tyr Lys Met Asp Asp Leu 2180 2185 2190Leu Thr Ser Tyr Ile Ser Gln Met Leu Thr Ala Met Ser Lys Gln 2195 2200 2205Arg Gly Ser Arg Ser Gly Lys 2210 221576648DNAHomo sapiens 7atggtgattc ttcagcaggg ggaccatgtg tggatggacc tgagattggg gcaggagttc 60gacgtgccca tcggggcggt ggtgaagctc tgcgactctg ggcaggtcca ggtggtggat 120gatgaagaca atgaacactg gatctctccg cagaacgcaa cgcacatcaa gcctatgcac 180cccacgtcgg tccacggcgt ggaggacatg atccgcctgg gggacctcaa cgaggcgggc 240atcttgcgca acctgcttat ccgctaccgg gaccacctca tctacacgta tacgggctcc 300atcctggtgg ctgtgaaccc ctaccagctg ctctccatct actcgccaga gcacatccgc 360cagtatacca acaagaagat tggggagatg cccccccaca tctttgccat tgctgacaac 420tgctacttca acatgaaacg caacagccga gaccagtgct gcatcatcag tggggaatct 480ggggccggga agacggagag cacaaagctg atcctgcagt tcctggcagc catcagtggg 540cagcactcgt ggattgagca gcaggtcttg gaggccaccc ccattctgga agcatttggg 600aatgccaaga ccatccgcaa tgacaactca agccgtttcg gaaagtacat cgacatccac 660ttcaacaagc ggggcgccat cgagggcgcg aagattgagc agtacctgct ggaaaagtca 720cgtgtctgtc gccaggccct ggatgaaagg aactaccacg tgttctactg catgctggag 780ggtatgagtg aggatcagaa gaagaagctg ggcttgggcc aggcctctga ctacaactac 840ttggccatgg gtaactgcat aacctgtgag ggccgggtgg acagccagga gtacgccaac 900atccgctccg ccatgaaggt gctcatgttc actgacaccg agaactggga gatctcgaag 960ctcctggctg ccatcctgca cctgggcaac ctgcagtatg aggcacgcac atttgaaaac 1020ctggatgcct gtgaggttct cttctcccca tcgctggcca cagctgcatc cctgcttgag 1080gtgaaccccc cagacctgat gagctgcctg actagccgca ccctcatcac ccgcggggag 1140acggtgtcca ccccactgag cagggaacag gcactggacg tgcgcgacgc cttcgtaaag 1200gggatctacg ggcggctgtt cgtgtggatt gtggacaaga tcaacgcagc aatttacaag 1260cctccctccc aggatgtgaa gaactctcgc aggtccatcg gcctcctgga catctttggg 1320tttgagaact ttgctgtgaa cagctttgag cagctctgca tcaacttcgc caatgagcac 1380ctgcagcagt tctttgtgcg gcacgtgttc aagctggagc aggaggaata tgacctggag 1440agcattgact ggctgcacat cgagttcact gacaaccagg atgccctgga catgattgcc 1500aacaagccca tgaacatcat ctccctcatc gatgaggaga gcaagttccc caagggcaca 1560gacaccacca tgttacacaa gctgaactcc cagcacaagc tcaacgccaa ctacatcccc 1620cccaagaaca accatgagac ccagtttggc atcaaccatt ttgcaggcat cgtctactat 1680gagacccaag gcttcctgga gaagaaccga gacaccctgc atggggacat tatccagctg 1740gtccactcct ccaggaacaa gttcatcaag cagatcttcc aggccgatgt cgccatgggc 1800gccgagacca ggaagcgctc gcccacactt agcagccagt tcaagcggtc actggagctg 1860ctgatgcgca cgctgggtgc ctgccagccc ttctttgtgc gatgcatcaa gcccaatgag 1920ttcaagaagc ccatgctgtt cgaccggcac ctgtgcgtgc gccagctgcg gtactcagga 1980atgatggaga ccatccgaat ccgccgagct ggctacccca tccgctacag cttcgtagag 2040tttgtggagc ggtaccgtgt gctgctgcca ggtgtgaagc cggcctacaa gcagggcgac 2100ctccgcggga cttgccagcg catggctgag gctgtgctgg gcacccacga tgactggcag 2160ataggcaaaa ccaagatctt tctgaaggac caccatgaca tgctgctgga agtggagcgg 2220gacaaagcca tcaccgacag agtcatcctc cttcagaaag tcatccgggg attcaaagac 2280aggtctaact ttctgaagct gaagaacgct gccacactga tccagaggca ctggcggggt 2340cacaactgta ggaagaacta cgggctgatg cgtctgggct tcctgcggct gcaggccctg 2400caccgctccc ggaagctgca ccagcagtac cgcctggccc gccagcgcat catccagttc 2460caggcccgct gccgcgccta tctggtgcgc aaggccttcc gccaccgcct ctgggctgtg 2520ctcaccgtgc aggcctatgc ccggggcatg atcgcccgca ggctgcacca acgcctcagg 2580gctgagtatc tgtggcgcct cgaggctgag aaaatgcggc tggcggagga agagaagctt 2640cggaaggaga tgagcgccaa gaaggccaag gaggaggccg agcgcaagca tcaggagcgc 2700ctggcccagc tggctcgtga ggacgctgag cgggagctga aggagaagga ggccgctcgg 2760cggaagaagg agctcctgga gcagatggaa agggcccgcc atgagcctgt caatcactca 2820gacatggtgg acaagatgtt tggcttcctg gggacttcag gtggcctgcc aggccaggag 2880ggccaggcac ctagtggctt tgaggacctg gagcgagggc ggagggagat ggtggaggag 2940gacctggatg cagccctgcc cctgcctgac gaggatgagg aggacctctc tgagtataaa 3000tttgccaagt tcgcggccac ctacttccag gggacaacca cgcactccta cacccggcgg 3060ccactcaaac agccactgct ctaccatgac gacgagggtg accagctggc agccctggcg 3120gtctggatca ccatcctccg cttcatgggg gacctccctg agcccaagta ccacacagcc 3180atgagtgatg gcagtgagaa gatccctgtg atgaccaaga tttatgagac cctgggcaag 3240aagacgtaca agagggagct gcaggccctg cagggcgagg gcgaggccca gctccccgag 3300ggccagaaga agagcagtgt gaggcacaag ctggtgcatt tgactctgaa aaagaagtcc 3360aagctcacag aggaggtgac caagaggctg catgacgggg agtccacagt gcagggcaac 3420agcatgctgg aggaccggcc cacctccaac ctggagaagc tgcacttcat catcggcaat 3480ggcatcctgc ggccagcact ccgggacgag atctactgcc agatcagcaa gcagctgacc 3540cacaacccct ccaagagcag ctatgcccgg ggctggattc tcgtgtctct ctgcgtgggc 3600tgtttcgccc cctccgagaa gtttgtcaag tacctgcgga acttcatcca cgggggcccg 3660cccggctacg ccccgtactg tgaggagcgc ctgagaagga cctttgtcaa tgggacacgg 3720acacagccgc ccagctggct ggagctgcag gccaccaagt ccaagaagcc aatcatgttg 3780cccgtgacat tcatggatgg gaccaccaag accctgctga cggactcggc aaccacggcc 3840aaggagctct gcaacgcgct ggccgacaag atctctctca aggaccggtt cgggttctcc 3900ctctacattg ccctgtttga caaggtgtcc tccctgggca gcggcagtga ccacgtcatg 3960gacgccatct cccagtgcga gcagtacgcc aaggagcagg gcgcccagga gcgcaacgcc 4020ccctggaggc tcttcttccg caaagaggtc ttcacgccct ggcacagccc ctccgaggac 4080aacgtggcca ccaacctcat ctaccagcag gtggtgcgag gagtcaagtt tggggagtac 4140aggtgtgaga aggaggacga cctggctgag ctggcctccc agcagtactt tgtagactat 4200ggctctgaga tgatcctgga gcgcctcctg aacctcgtgc ccacctacat ccccgaccgc 4260gagatcacgc ccctgaagac gctggagaag tgggcccagc tggccatcgc cgcccacaag 4320aaggggattt atgcccagag gagaactgat gcccagaagg tcaaagagga tgtggtcagt 4380tatgcccgct tcaagtggcc cttgctcttc tccaggtttt atgaagccta caaattctca 4440ggccccagtc tccccaagaa cgacgtcatc gtggccgtca actggacggg tgtgtacttt 4500gtggatgagc aggagcaggt acttctggag ctgtccttcc cagagatcat ggccgtgtcc 4560agcagcaggg agtgccgtgt ctggctctca ctgggctgct ctgatcttgg ctgtgctgcg 4620cctcactcag gctgggcagg actgaccccg gcggggccct gttctccgtg ttggtcctgc 4680aggggagcga aaacgacggc ccccagcttc acgctggcca ccatcaaggg ggacgaatac 4740accttcacct ccagtaatgc tgaggacatt cgtgacctgg tggtcacctt cctagagggg 4800ctccggaaga gatctaagta tgttgtggcc ctgcaggata accccaaccc cgcaggcgag 4860gagtcaggct tcctcagctt tgccaaggga gacctcatca tcctggacca tgacacgggc 4920gagcaggtca tgaactcggg ctgggccaac ggcatcaatg agaggaccaa gcagcgtggg 4980gacttcccca ccgactgtgt gtacgtcatg cccactgtca ccatgccacc gcgggagatt 5040gtggccctgg tcaccatgac tcccgatcag aggcaggacg ttgtccggct cttgcagctg 5100cgaacggcgg agcccgaggt gcgtgccaag ccctacacgc tggaggagtt ttcctatgac 5160tacttcaggc ccccacccaa gcacacgctg agccgtgtca tggtgtccaa ggcccgaggc 5220aaggaccggc tgtggagcca cacgcgggaa ccgctcaagc aggcgctgct caagaagctc 5280ctgggcagtg aggagctctc gcaggaggcc tgcctggcct tcattgctgt gctcaagtac 5340atgggcgact acccgtccaa gaggacacgc tccgtcaacg agctcaccga ccagatcttt 5400gagggtcccc tgaaagccga gcccctgaag gacgaggcat atgtgcagat cctgaagcag 5460ctgaccgaca accacatcag gtacagcgag gagcggggtt gggagctgct ctggctgtgc 5520acgggccttt tcccacccag caacatcctc ctgccccacg tgcagcgctt cctgcagtcc 5580cgaaagcact gcccactcgc catcgactgc ctgcaacggc tccagaaagc cctgagaaac 5640gggtcccgga agtaccctcc gcacctggtg gaggtggagg ccatccagca caagaccacc 5700cagattttcc acaaggtcta cttccctgat gacactgacg aggccttcga agtggagtcc 5760agcaccaagg ccaaggactt ctgccagaac atcgccacca ggctgctcct caagtcctca 5820gagggattca gcctctttgt caaaattgca gacaaggtca tcagcgttcc tgagaatgac 5880ttcttctttg actttgttcg acacttgaca gactggataa agaaagctcg gcccatcaag 5940gacggaattg tgccctcact cacctaccag gtgttcttca tgaagaagct gtggaccacc 6000acggtgccag ggaaggatcc catggccgat tccatcttcc actattacca ggagttgccc 6060aagtatctcc gaggctacca caagtgcacg cgggaggagg tgctgcagct gggggcgctg 6120atctacaggg tcaagttcga ggaggacaag tcctacttcc ccagcatccc caagctgctg 6180cgggagctgg tgccccagga ccttatccgg caggtctcac ctgatgactg gaagcggtcc 6240atcgtcgcct acttcaacaa gcacgcaggg aagtccaagg aggaggccaa gctggccttc 6300ctgaagctca tcttcaagtg gcccaccttt ggctcagcct tcttcgaggt gaagcaaact 6360acggagccaa acttccctga gatcctccta attgccatca acaagtatgg ggtcagcctc 6420atcgatccca aaacgaagga tatcctcacc actcatccct tcaccaagat ctccaactgg 6480agcagcggca acacctactt ccacatcacc attgggaact tggtgcgcgg gagcaaactg 6540ctctgcgaga cgtcactggg ctacaagatg gatgacctcc tgacttccta cattagccag 6600atgctcacag ccatgagcaa acagcggggc tccaggagcg gcaagtga 664882175PRTHomo sapiens 8Met Val Ile Leu Gln Gln Gly Asp His Val Trp Met Asp Leu Arg Leu1 5 10 15Gly Gln Glu Phe Asp Val Pro Ile Gly Ala Val Val Lys Leu Cys Asp 20 25 30Ser Gly Gln Val Gln Val Val Asp Asp Glu Asp Asn Glu His Trp Ile 35 40 45Ser Pro Gln Asn Ala Thr His Ile Lys Pro Met His Pro Thr Ser Val 50 55 60His Gly Val Glu Asp Met Ile Arg Leu Gly Asp Leu Asn Glu Ala Gly65 70 75 80Ile Leu Arg Asn Leu Leu Ile Arg Tyr Arg Asp His Leu Ile Tyr Thr 85 90 95Tyr Thr Gly Ser Ile Leu Val Ala Val Asn Pro Tyr Gln Leu Leu Ser 100 105 110Ile Tyr Ser Pro Glu His Ile Arg Gln Tyr Thr Asn Lys Lys Ile Gly 115 120 125Glu Met Pro Pro His Ile Phe Ala Ile Ala Asp Asn Cys Tyr Phe Asn 130 135 140Met Lys Arg Asn Ser Arg Asp Gln Cys Cys Ile Ile Ser Gly Glu Ser145 150 155 160Gly Ala Gly Lys Thr Glu Ser Thr Lys Leu Ile Leu Gln Phe Leu Ala 165 170 175Ala Ile Ser Gly Gln His Ser Trp Ile Glu Gln Gln Val Leu Glu Ala 180 185 190Thr Pro Ile Leu Glu Ala Phe Gly Asn Ala Lys Thr Ile Arg Asn Asp 195 200

205Asn Ser Ser Arg Phe Gly Lys Tyr Ile Asp Ile His Phe Asn Lys Arg 210 215 220Gly Ala Ile Glu Gly Ala Lys Ile Glu Gln Tyr Leu Leu Glu Lys Ser225 230 235 240Arg Val Cys Arg Gln Ala Leu Asp Glu Arg Asn Tyr His Val Phe Tyr 245 250 255Cys Met Leu Glu Gly Met Ser Glu Asp Gln Lys Lys Lys Leu Gly Leu 260 265 270Gly Gln Ala Ser Asp Tyr Asn Tyr Leu Ala Met Gly Asn Cys Ile Thr 275 280 285Cys Glu Gly Arg Val Asp Ser Gln Glu Tyr Ala Asn Ile Arg Ser Ala 290 295 300Met Lys Val Leu Met Phe Thr Asp Thr Glu Asn Trp Glu Ile Ser Lys305 310 315 320Leu Leu Ala Ala Ile Leu His Leu Gly Asn Leu Gln Tyr Glu Ala Arg 325 330 335Thr Phe Glu Asn Leu Asp Ala Cys Glu Val Leu Phe Ser Pro Ser Leu 340 345 350Ala Thr Ala Ala Ser Leu Leu Glu Val Asn Pro Pro Asp Leu Met Ser 355 360 365Cys Leu Thr Ser Arg Thr Leu Ile Thr Arg Gly Glu Thr Val Ser Thr 370 375 380Pro Leu Ser Arg Glu Gln Ala Leu Asp Val Arg Asp Ala Phe Val Lys385 390 395 400Gly Ile Tyr Gly Arg Leu Phe Val Trp Ile Val Asp Lys Ile Asn Ala 405 410 415Ala Ile Tyr Lys Pro Pro Ser Gln Asp Val Lys Asn Ser Arg Arg Ser 420 425 430Ile Gly Leu Leu Asp Ile Phe Gly Phe Glu Asn Phe Ala Val Asn Ser 435 440 445Phe Glu Gln Leu Cys Ile Asn Phe Ala Asn Glu His Leu Gln Gln Phe 450 455 460Phe Val Arg His Val Phe Lys Leu Glu Gln Glu Glu Tyr Asp Leu Glu465 470 475 480Ser Ile Asp Trp Leu His Ile Glu Phe Thr Asp Asn Gln Asp Ala Leu 485 490 495Asp Met Ile Ala Asn Lys Pro Met Asn Ile Ile Ser Leu Ile Asp Glu 500 505 510Glu Ser Lys Phe Pro Lys Gly Thr Asp Thr Thr Met Leu His Lys Leu 515 520 525Asn Ser Gln His Lys Leu Asn Ala Asn Tyr Ile Pro Pro Lys Asn Asn 530 535 540His Glu Thr Gln Phe Gly Ile Asn His Phe Ala Gly Ile Val Tyr Tyr545 550 555 560Glu Thr Gln Gly Phe Leu Glu Lys Asn Arg Asp Thr Leu His Gly Asp 565 570 575Ile Ile Gln Leu Val His Ser Ser Arg Asn Lys Phe Ile Lys Gln Ile 580 585 590Phe Gln Ala Asp Val Ala Met Gly Ala Glu Thr Arg Lys Arg Ser Pro 595 600 605Thr Leu Ser Ser Gln Phe Lys Arg Ser Leu Glu Leu Leu Met Arg Thr 610 615 620Leu Gly Ala Cys Gln Pro Phe Phe Val Arg Cys Ile Lys Pro Asn Glu625 630 635 640Phe Lys Lys Pro Met Leu Phe Asp Arg His Leu Cys Val Arg Gln Leu 645 650 655Arg Tyr Ser Gly Met Met Glu Thr Ile Arg Ile Arg Arg Ala Gly Tyr 660 665 670Pro Ile Arg Tyr Ser Phe Val Glu Phe Val Glu Arg Tyr Arg Val Leu 675 680 685Leu Pro Gly Val Lys Pro Ala Tyr Lys Gln Gly Asp Leu Arg Gly Thr 690 695 700Cys Gln Arg Met Ala Glu Ala Val Leu Gly Thr His Asp Asp Trp Gln705 710 715 720Ile Gly Lys Thr Lys Ile Phe Leu Lys Asp His His Asp Met Leu Leu 725 730 735Glu Val Glu Arg Asp Lys Ala Ile Thr Asp Arg Val Ile Leu Leu Gln 740 745 750Lys Val Ile Arg Gly Phe Lys Asp Arg Ser Asn Phe Leu Lys Leu Lys 755 760 765Asn Ala Ala Thr Leu Ile Gln Arg His Trp Arg Gly His Asn Cys Arg 770 775 780Lys Asn Tyr Gly Leu Met Arg Leu Gly Phe Leu Arg Leu Gln Ala Leu785 790 795 800His Arg Ser Arg Lys Leu His Gln Gln Tyr Arg Leu Ala Arg Gln Arg 805 810 815Ile Ile Gln Phe Gln Ala Arg Cys Arg Ala Tyr Leu Val Arg Lys Ala 820 825 830Phe Arg His Arg Leu Trp Ala Val Leu Thr Val Gln Ala Tyr Ala Arg 835 840 845Gly Met Ile Ala Arg Arg Leu His Gln Arg Leu Arg Ala Glu Tyr Leu 850 855 860Trp Arg Leu Glu Ala Glu Lys Met Arg Leu Ala Glu Glu Glu Lys Leu865 870 875 880Arg Lys Glu Met Ser Ala Lys Lys Ala Lys Glu Glu Ala Glu Arg Lys 885 890 895His Gln Glu Arg Leu Ala Gln Leu Ala Arg Glu Asp Ala Glu Arg Glu 900 905 910Leu Lys Glu Lys Glu Ala Ala Arg Arg Lys Lys Glu Leu Leu Glu Gln 915 920 925Met Glu Arg Ala Arg His Glu Pro Val Asn His Ser Asp Met Val Asp 930 935 940Lys Met Phe Gly Phe Leu Gly Thr Ser Gly Gly Leu Pro Gly Gln Glu945 950 955 960Gly Gln Ala Pro Ser Gly Phe Glu Asp Leu Glu Arg Gly Arg Arg Glu 965 970 975Met Val Glu Glu Asp Leu Asp Ala Ala Leu Pro Leu Pro Asp Glu Asp 980 985 990Glu Glu Asp Leu Ser Glu Tyr Lys Phe Ala Lys Phe Ala Ala Thr Tyr 995 1000 1005Phe Gln Gly Thr Thr Thr His Ser Tyr Thr Arg Arg Pro Leu Lys 1010 1015 1020Gln Pro Leu Leu Tyr His Asp Asp Glu Gly Asp Gln Leu Ala Ala 1025 1030 1035Leu Ala Val Trp Ile Thr Ile Leu Arg Phe Met Gly Asp Leu Pro 1040 1045 1050Glu Pro Lys Tyr His Thr Ala Met Ser Asp Gly Ser Glu Lys Ile 1055 1060 1065Pro Val Met Thr Lys Ile Tyr Glu Thr Leu Gly Lys Lys Thr Tyr 1070 1075 1080Lys Arg Glu Leu Gln Ala Leu Gln Gly Glu Gly Glu Ala Gln Leu 1085 1090 1095Pro Glu Gly Gln Lys Lys Ser Ser Val Arg His Lys Leu Val His 1100 1105 1110Leu Thr Leu Lys Lys Lys Ser Lys Leu Thr Glu Glu Val Thr Lys 1115 1120 1125Arg Leu His Asp Gly Glu Ser Thr Val Gln Gly Asn Ser Met Leu 1130 1135 1140Glu Asp Arg Pro Thr Ser Asn Leu Glu Lys Leu His Phe Ile Ile 1145 1150 1155Gly Asn Gly Ile Leu Arg Pro Ala Leu Arg Asp Glu Ile Tyr Cys 1160 1165 1170Gln Ile Ser Lys Gln Leu Thr His Asn Pro Ser Lys Ser Ser Tyr 1175 1180 1185Ala Arg Gly Trp Ile Leu Val Ser Leu Cys Val Gly Cys Phe Ala 1190 1195 1200Pro Ser Glu Lys Phe Val Lys Tyr Leu Arg Asn Phe Ile His Gly 1205 1210 1215Gly Pro Pro Gly Tyr Ala Pro Tyr Cys Glu Glu Arg Leu Arg Arg 1220 1225 1230Thr Phe Val Asn Gly Thr Arg Thr Gln Pro Pro Ser Trp Leu Glu 1235 1240 1245Leu Gln Ala Thr Lys Ser Lys Lys Pro Ile Met Leu Pro Val Thr 1250 1255 1260Phe Met Asp Gly Thr Thr Lys Thr Leu Leu Thr Asp Ser Ala Thr 1265 1270 1275Thr Ala Lys Glu Leu Cys Asn Ala Leu Ala Asp Lys Ile Ser Leu 1280 1285 1290Lys Asp Arg Phe Gly Phe Ser Leu Tyr Ile Ala Leu Phe Asp Lys 1295 1300 1305Val Ser Ser Leu Gly Ser Gly Ser Asp His Val Met Asp Ala Ile 1310 1315 1320Ser Gln Cys Glu Gln Tyr Ala Lys Glu Gln Gly Ala Gln Glu Arg 1325 1330 1335Asn Ala Pro Trp Arg Leu Phe Phe Arg Lys Glu Val Phe Thr Pro 1340 1345 1350Trp His Ser Pro Ser Glu Asp Asn Val Ala Thr Asn Leu Ile Tyr 1355 1360 1365Gln Gln Val Val Arg Gly Val Lys Phe Gly Glu Tyr Arg Cys Glu 1370 1375 1380Lys Glu Asp Asp Leu Ala Glu Leu Ala Ser Gln Gln Tyr Phe Val 1385 1390 1395Asp Tyr Gly Ser Glu Met Ile Leu Glu Arg Leu Leu Asn Leu Val 1400 1405 1410Pro Thr Tyr Ile Pro Asp Arg Glu Ile Thr Pro Leu Lys Thr Leu 1415 1420 1425Glu Lys Trp Ala Gln Leu Ala Ile Ala Ala His Lys Lys Gly Ile 1430 1435 1440Tyr Ala Gln Arg Arg Thr Asp Ala Gln Lys Val Lys Glu Asp Val 1445 1450 1455Val Ser Tyr Ala Arg Phe Lys Trp Pro Leu Leu Phe Ser Arg Phe 1460 1465 1470Tyr Glu Ala Tyr Lys Phe Ser Gly Pro Ser Leu Pro Lys Asn Asp 1475 1480 1485Val Ile Val Ala Val Asn Trp Thr Gly Val Tyr Phe Val Asp Glu 1490 1495 1500Gln Glu Gln Val Leu Leu Glu Leu Ser Phe Pro Glu Ile Met Ala 1505 1510 1515Val Ser Ser Ser Arg Gly Ala Lys Thr Thr Ala Pro Ser Phe Thr 1520 1525 1530Leu Ala Thr Ile Lys Gly Asp Glu Tyr Thr Phe Thr Ser Ser Asn 1535 1540 1545Ala Glu Asp Ile Arg Asp Leu Val Val Thr Phe Leu Glu Gly Leu 1550 1555 1560Arg Lys Arg Ser Lys Tyr Val Val Ala Leu Gln Asp Asn Pro Asn 1565 1570 1575Pro Ala Gly Glu Glu Ser Gly Phe Leu Ser Phe Ala Lys Gly Asp 1580 1585 1590Leu Ile Ile Leu Asp His Asp Thr Gly Glu Gln Val Met Asn Ser 1595 1600 1605Gly Trp Ala Asn Gly Ile Asn Glu Arg Thr Lys Gln Arg Gly Asp 1610 1615 1620Phe Pro Thr Asp Ser Val Tyr Val Met Pro Thr Val Thr Met Pro 1625 1630 1635Pro Arg Glu Ile Val Ala Leu Val Thr Met Thr Pro Asp Gln Arg 1640 1645 1650Gln Asp Val Val Arg Leu Leu Gln Leu Arg Thr Ala Glu Pro Glu 1655 1660 1665Val Arg Ala Lys Pro Tyr Thr Leu Glu Glu Phe Ser Tyr Asp Tyr 1670 1675 1680Phe Arg Pro Pro Pro Lys His Thr Leu Ser Arg Val Met Val Ser 1685 1690 1695Lys Ala Arg Gly Lys Asp Arg Leu Trp Ser His Thr Arg Glu Pro 1700 1705 1710Leu Lys Gln Ala Leu Leu Lys Lys Leu Leu Gly Ser Glu Glu Leu 1715 1720 1725Ser Gln Glu Ala Cys Leu Ala Phe Ile Ala Val Leu Lys Tyr Met 1730 1735 1740Gly Asp Tyr Pro Ser Lys Arg Thr Arg Ser Val Asn Glu Leu Thr 1745 1750 1755Asp Gln Ile Phe Glu Gly Pro Leu Lys Ala Glu Pro Leu Lys Asp 1760 1765 1770Glu Ala Tyr Val Gln Ile Leu Lys Gln Leu Thr Asp Asn His Ile 1775 1780 1785Arg Tyr Ser Glu Glu Arg Gly Trp Glu Leu Leu Trp Leu Cys Thr 1790 1795 1800Gly Leu Phe Pro Pro Ser Asn Ile Leu Leu Pro His Val Gln Arg 1805 1810 1815Phe Leu Gln Ser Arg Lys His Cys Pro Leu Ala Ile Asp Cys Leu 1820 1825 1830Gln Arg Leu Gln Lys Ala Leu Arg Asn Gly Ser Arg Lys Tyr Pro 1835 1840 1845Pro His Leu Val Glu Val Glu Ala Ile Gln His Lys Thr Thr Gln 1850 1855 1860Ile Phe His Lys Val Tyr Phe Pro Asp Asp Thr Asp Glu Ala Phe 1865 1870 1875Glu Val Glu Ser Ser Thr Lys Ala Lys Asp Phe Cys Gln Asn Ile 1880 1885 1890Ala Thr Arg Leu Leu Leu Lys Ser Ser Glu Gly Phe Ser Leu Phe 1895 1900 1905Val Lys Ile Ala Asp Lys Val Leu Ser Val Pro Glu Asn Asp Phe 1910 1915 1920Phe Phe Asp Phe Val Arg His Leu Thr Asp Trp Ile Lys Lys Ala 1925 1930 1935Arg Pro Ile Lys Asp Gly Ile Val Pro Ser Leu Thr Tyr Gln Val 1940 1945 1950Phe Phe Met Lys Lys Leu Trp Thr Thr Thr Val Pro Gly Lys Asp 1955 1960 1965Pro Met Ala Asp Ser Ile Phe His Tyr Tyr Gln Glu Leu Pro Lys 1970 1975 1980Tyr Leu Arg Gly Tyr His Lys Cys Thr Arg Glu Glu Val Leu Gln 1985 1990 1995Leu Gly Ala Leu Ile Tyr Arg Val Lys Phe Glu Glu Asp Lys Ser 2000 2005 2010Tyr Phe Pro Ser Ile Pro Lys Leu Leu Arg Glu Leu Val Pro Gln 2015 2020 2025Asp Leu Ile Arg Gln Val Ser Pro Asp Asp Trp Lys Arg Ser Ile 2030 2035 2040Val Ala Tyr Phe Asn Lys His Ala Gly Lys Ser Lys Glu Glu Ala 2045 2050 2055Lys Leu Ala Phe Leu Lys Leu Ile Phe Lys Trp Pro Thr Phe Gly 2060 2065 2070Ser Ala Phe Phe Glu Gln Thr Thr Glu Pro Asn Phe Pro Glu Ile 2075 2080 2085Leu Leu Ile Ala Ile Asn Lys Tyr Gly Val Ser Leu Ile Asp Pro 2090 2095 2100Lys Thr Lys Asp Ile Leu Thr Thr His Pro Phe Thr Lys Ile Ser 2105 2110 2115Asn Trp Ser Ser Gly Asn Thr Tyr Phe His Ile Thr Ile Gly Asn 2120 2125 2130Leu Val Arg Gly Ser Lys Leu Leu Cys Glu Thr Ser Leu Gly Tyr 2135 2140 2145Lys Met Asp Asp Leu Leu Thr Ser Tyr Ile Ser Gln Met Leu Thr 2150 2155 2160Ala Met Ser Lys Gln Arg Gly Ser Arg Ser Gly Lys 2165 2170 2175939DNAArtificial SequenceSynthetic Oligonucleotide 9gcggcggccg ccaccatggt gattcttcag cagggggac 391030DNAArtificial SequenceSynthetic Oligonucleotide 10gcggctagcg aagttccgca ggtacttgac 301131DNAArtificial SequenceSynthetic Oligonucleotide 11gcgcttaagc aggtctaact ttctgaagct g 311230DNAArtificial SequenceSynthetic Oligonucleotide 12gcgggtacct cacttgccgc tcctggagcc 301341DNAArtificial SequenceSynthetic Oligonucleotide 13ggcacctagt ggctttgagg taagtatcaa ggttacaaga c 411429DNAArtificial SequenceSynthetic Oligonucleotide 14gcggctagct cagaaacgca agagtcttc 291522DNAArtificial SequenceSynthetic Oligonucleotide 15cttctttgtg cgatgcatca ag 221628DNAArtificial SequenceSynthetic Oligonucleotide 16gcgcttaagc gacgcatgct cgcgatag 281737DNAArtificial SequenceSynthetic Oligonucleotide 17cgccctcgct ccaggtcctg tggagagaaa ggcaaag 371819DNAArtificial SequenceSynthetic Oligonucleotide 18gaacccgaac cggtccttg 191925DNAArtificial SequenceSynthetic Oligonucleotide 19gcggctagcc cccgggtgcg cggcg 252029DNAArtificial SequenceSynthetic Oligonucleotide 20gcggtcgacg aaacggtcca ggctatgtg 292127DNAArtificial SequenceSynthetic Oligonucleotide 21gcggcggccg cccccgggtg cgcggcg 272229DNAArtificial SequenceSynthetic Oligonucleotide 22gcgcttaagg aaacggtcca ggctatgtg 292340DNAArtificial SequenceSynthetic Oligonucleotide 23caggcaccta gtggctttga ggtaccaggc tagggacagg 402428DNAArtificial SequenceSynthetic Oligonucleotide 24gcggctagcc gcctgagccc agaagttc 282538DNAArtificial SequenceSynthetic Oligonucleotide 25cgccctcgct ccaggtcctg aaggagacaa gaggtatg 382629DNAArtificial SequenceSynthetic Oligonucleotide 26gcgcttaagc accgcttgtg ttgatcctc 292719DNAArtificial SequenceSynthetic Oligonucleotide 27gccagggaag gatcccatg 192857DNAArtificial SequenceSynthetic Oligonucleotide 28gcgggtacct catgcgtaat ccggtacatc gtaagggtac ttgccgctcc tggagcc 572924DNAArtificial SequenceSynthetic Oligonucleotide 29agcttcgtag agtttgtgga gcgg 243019DNAArtificial SequenceSynthetic Oligonucleotide 30gaggggcaaa caacagatg 19

Claims

1.-25. (canceled)

26. A hybrid polynucleotide vector system comprising: i) a first AAV vector polynucleotide comprising a first sequence, a splice donor site, and a first synthetic intron sequence; and ii) a second AAV vector polynucleotide comprising a second sequence, a splice acceptor site, and a second synthetic intron sequence; wherein the first synthetic intron sequence and the second synthetic intron sequence comprise an overlapping polynucleotide sequence.

27. The hybrid polynucleotide vector system of claim 26, wherein the first synthetic intron sequence and the second synthetic intron sequence comprise a synthetic alkaline phosphatase intron.

28. The hybrid polynucleotide vector system of claim 26, wherein the overlapping sequence is about 50 to about 500 nucleotides in length.

29. The hybrid polynucleotide vector system of claim 26, wherein the first synthetic intron sequence and the second synthetic intron sequence comprise part of the human MYO7A gene.

30. The hybrid polynucleotide vector system of claim 26, wherein the first synthetic intron sequence and the second synthetic intron sequence comprise intron 23 of the human MYO7A gene.

31. The hybrid polynucleotide vector system of claim 26, wherein the first synthetic intron sequence comprises the nucleotide sequence of SEQ ID NO: 3.

32. The hybrid polynucleotide vector system of claim 26, wherein the second synthetic intron sequence comprises the nucleotide sequence of SEQ ID NO: 4.

33. The hybrid polynucleotide vector system of claim 26, wherein the first AAV vector polynucleotide and the second AAV vector polynucleotide further comprise an inverted terminal repeat sequence.

34. The hybrid polynucleotide vector system of claim 26 further comprising an AAV capsid.

35. The hybrid polynucleotide vector system of claim 34, wherein the AAV capsid is selected from a group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11.

36. A method of treating a subject in need thereof, the method comprising administering a hybrid polynucleotide vector system comprising: i) a first AAV vector polynucleotide comprising a first sequence, a splice donor site, and a first synthetic intron sequence; and ii) a second AAV vector polynucleotide comprising a second sequence, a splice acceptor site, and a second synthetic intron sequence; wherein the first synthetic intron sequence and the second synthetic intron sequence comprise an overlapping polynucleotide sequence.

37. The method of claim 36, wherein the hybrid polynucleotide vector system is administered by a method selected from intravenous administration, intramuscular administration, intraocular administration, and intranasal administration.

38. The method of claim 36, wherein the overlapping sequence in the first and second AAV vector polynucleotides is about 50 to about 500 nucleotides in length.

39. The method of claim 36, wherein the first synthetic intron sequence and the second synthetic intron sequence comprise part of the human MYO7A gene.

40. The method of claim 36, wherein the first synthetic intron sequence and the second synthetic intron sequence comprises intron 23 of the human MYO7A gene.

41. The method of claim 36, wherein the first synthetic intron sequence comprises the nucleotide sequence of SEQ ID NO: 3.

42. The method of claim 36, wherein the second synthetic intron sequence comprises the nucleotide sequence of SEQ ID NO: 4.

43. The method of claim 36, wherein the hybrid polynucleotide vector system further comprises an AAV capsid.

44. The method of claim 43, wherein the AAV capsid is selected from a group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11.

45. A pharmaceutical composition, comprising a) a hybrid polynucleotide vector system comprising: i) a first AAV vector polynucleotide comprising a first sequence, a splice donor site, and a first synthetic intron sequence; and ii) a second AAV vector polynucleotide comprising a second sequence, a splice acceptor site, and a second synthetic intron sequence; wherein the first synthetic intron sequence and the second synthetic intron sequence comprise an overlapping polynucleotide sequence, and b) a pharmaceutical excipient.

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