Efficient transduction and lateral spread of a novel AAV virus enhanced by rational design in the retina

Abstract

The present disclosure provides rAAV particles comprising a new capsid variant AAV44.9 (E531D). The present disclosure also provides rAAV particles comprising AAV44.9 (E531D) for treating eyes, including treating retinal diseases. In some specific embodiments, the present disclosure provides rAAV particles comprising AAV44.9 (E531D) capsids, which exhibit enhanced lateral diffusion after subretinal injection into the fovea of ​​a subject, wherein detachment of the fovea is minimized. The present disclosure also provides rAAV particles comprising AAV44.9 (E531D) capsids and polynucleotides encoding heterologous nucleic acid sequences. Also provided are methods of treating comprising administering rAAV particles to a mammal in need thereof, and methods of transducing photoreceptors and RPE cells with rAAV particles.

Description

[0001] Related Applications

[0002] This application claims the benefit of the filing date of U.S. Provisional Application No. 62 / 795,695, filed on January 23, 2019, the entire contents of which are incorporated herein by reference.

[0003] Federally Funded Research

[0004] This invention was made with government support under Grant No. R01 EY024280 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0005] The present invention relates generally to the fields of molecular biology and virology, and in particular to the development of gene therapy vectors and methods for treating retinal diseases. Background Art

[0006] The field of gene therapy has made significant progress through the use of viruses to deliver therapeutic genetic material. Adeno-associated viruses have attracted considerable attention as efficient viral vectors for gene therapy due to their low immunogenicity and ability to efficiently transduce non-dividing cells. AAV has been shown to infect a variety of cell and tissue types, and significant advances have been made over the past decade to adapt this viral system for human gene therapy.

[0007] In the normal "wild-type" form of AAV, AAV DNA is packaged into the viral capsid as a single-stranded molecule of approximately 4600 nucleotides (nt) in length. After the virus infects a cell, the cell's molecular machinery converts the single-stranded DNA into a double-stranded form. Only this double-stranded DNA form can be transcribed into RNA by cellular enzymes, which can then be translated into polypeptides by other cellular pathways.

[0008] Recombinant adeno-associated virus (AAV) vectors have been successfully used for in vivo gene transfer in a variety of preclinical animal models of human disease and have been successfully used for long-term expression of a wide variety of therapeutic genes (Daya and Berns, 2008; Niemeyer et al., 2009; Owen et al., 2002; Keen-Rhinehart et al., 2005; Scallan et al., 2003; Song et al., 2004). AAV vectors have also produced long-term clinical benefits in humans when targeted to immune privileged sites (e.g., for ocular delivery of Leber's congenial amaurosis) (Bainbridge et al., 2008; Maguire et al., 2008; Cideciyan et al., 2008). A major advantage of this vector is its relatively low immune profile, causing only limited inflammatory responses and, in some cases, even inducing immune tolerance to the transgenic product (LoDuca et al., 2009). Nevertheless, when targeting non-immune privileged organs, therapeutic efficiency is limited in humans due to antibodies and CD8+ T cell responses against the viral capsid, while in animal models, adaptive responses to transgenic products have also been reported (Manno et al., 2006; Mingozzi et al., 2007; Muruve et al., 2008; Vandenberghe and Wilson, 2007; Mingozzi and High, 2007).

[0009] When transgenic expression is required in the retinal pigment epithelium (RPE) or photoreceptors (PR), subretinal injection of AAV is usually used. Subretinal injection produces temporary bullous detachment, separating the outer segment of the photoreceptor from the RPE layer. Generally speaking, the subretinal injection bleb in the subject will disappear within the next few days. Subretinal injection may have some harmful effects on photoreceptors, and it is conceivable that such effects are more serious in the retina that has been damaged by the disease. In particular, it has been suggested that foveal detachment in RPE65-LCA patients receiving retinal gene therapy may be harmful (see Jacobson et al., Gene therapy for leber congenital amaurosis caused by RPE65 mutations: safety and efficacy in 15 children and adults followed up to 3 years, Arch Ophthalmol. 2012; 130 (1): 9-24).

[0010] AAV capsids are still needed to improve retinal transduction efficiency and minimize detachment of the fovea during subretinal injections. Summary of the invention

[0011] AAV has become the preferred vector for targeting therapeutic genes to the retina. Both naturally occurring and synthetic AAVs have been identified as showing retinal tropism. Recently, a new AAV capsid serotype 44.9 was isolated from a laboratory stock of simian adenovirus SV15 taken from normal rhesus monkey kidney cell cultures. Reference is made to WO 2016 / 183297, published on November 17, 2016; U.S. Patent Publication No. 2018 / 0355376, published on December 13, 2018; and Novel Adeno-Associated Virus for Gene Therapy, Fed. Reg. 80,149 (August 4, 2015), the entire contents of each of which are incorporated herein in their entirety. AAV44.9 effectively transduces a variety of cell types, including salivary gland cells, hepatocytes, and different types of neurons (e.g., cortical cells, olfactory bulb cells, brain stem cells, and cerebellar Purkinje cells).

[0012] AAV44.9 exhibits in vivo biodistribution comparable to that of AAV9. Intracerebroventricular injections of this capsid have shown transduction levels in the cortex, olfactory bulb, cerebellum, choroid plexus, and brainstem similar to those observed with AAV9. Furthermore, antibody neutralization studies have shown a lower frequency of neutralizing antibodies for AAV44.9 compared to AAV2. And glycan array studies of AAV44.9 have shown binding of the capsid to terminal glucose-containing molecules.

[0013] The amino acid sequence of the capsid protein VP1 of AAV44.9 differs from the amino acid sequence of the capsid protein VP1 of the most closely reported isolate AAVrh.8R at several positions (see Vandenberghe LH et al., Naturally occurring singleton residues in AAV capsid impact vector performance and illustrate structural constraints, Gene Ther. 16: 1416-1418 (2009); Vandenberghe LH, et al., AAV9 targets cone photoreceptors in the nonhuman primate retina, PLoS One 8 (1): e53463 (2013)), two of which are serine residues in variable domain 3. In particular, the amino acid sequence of the capsid protein VP1 of AAV44.9 differs from the amino acid sequence of the capsid protein VP1 of AAVrh.8R at positions 179, 473, and 483.

[0014] The amino acid sequence of the capsid protein VP1 of AAV44.9 differs from the amino acid sequence of the capsid protein VP1 of the closely reported isolate AAVrh.8 (see Gao et al., J. Virol. 78(12):6381-6388 (2004)) at several positions, two of which are serine residues in variable domain 3. In particular, the amino acid sequence of the capsid protein VP1 of AAV44.9 differs from the amino acid sequence of the capsid protein VP1 of AAVrh.8 at positions 179, 473, 483, and 531.

[0015] Rational mutagenesis studies of amino acids in the capsid protein have shown that certain mutations have an inhibitory effect on the gene transfer activity of the vector, particularly the presence of serine and threonine residues in the variable region. Reports suggest that these amino acids increase the surface charge of the particles and target them for degradation in lysosomes, and that substitution with other uncharged amino acids can improve transduction activity. In addition, mutations in residues involved in receptor interaction also have a strong effect on retinal transduction and tropism. In the context of the retina, AAV2 and AAV8 capsids containing surface-exposed tyrosine to phenylalanine (YF) mutations have been shown to display enhanced retinal transduction relative to unmodified capsids.

[0016] In view of the inclusion of additional serine residues in the variable domain 3 of the capsid protein VP1 of AAV44.9 relative to the capsid protein VP1 of AAVrh.8, AAV44.9 has high gene transfer activity in many cell types. WO2016 / 183297 discloses the capsid protein VP1 of AAV44.9, in which the serine at position 470 is replaced by asparagine. When a similar serine residue in the AAV2 capsid is changed, it leads to a substantial increase in the titer of the prepared vector, although it does not change the transduction efficiency (see Aslanidi et al., High-efficiency Transduction of Human Monocyte-derived Dendritic Cells by Capsid-modified Recombinant AAV2 Vectors, Vaccine, 30(26): 3908-3917(2012)). Nevertheless, it is believed that the S470R substitution in AAV44.9 changes the transduction and binding affinity of the AAV44.9 capsid.

[0017] The inventors of the present disclosure used a rational design approach to design a new variant by mutagenizing glutamic acid at residue 531 to aspartic acid. This new serotype variant AAV44.9 (E531D) was evaluated in mice and macaques injected subretinally. It has been hypothesized that amino acid substitutions at positions corresponding to position E530 in the AAV2 capsid (e.g., position 531 in AAVrh.8 and AAV44.9) would alter transduction efficiency. See International Patent Publication No. WO2018 / 156654, published on August 30, 2018, the contents of which are incorporated herein by reference.

[0018] As described herein, it was unexpectedly found that rAAV particles incorporating the AAV44.9 (E531D) capsid variant were able to efficiently transduce rods, cones, and retinal pigment epithelium ("RPE") after subretinal injection. In addition, AAV44.9 (E531D) exhibited enhanced lateral diffusion, transducing photoreceptors and retinal pigment epithelium outside the subretinal injection bleb. The enhanced efficacy and lateral diffusion of AAV44.9 (E531D) make this variant a promising vector for gene therapy targeting the retina.

[0019] Subretinal injections below the cone-rich fovea have been shown to promote loss of central retinal thickness and loss of visual acuity in some treated patients (Phase I / II trial of RPE65-LCA2). See Jacobson et al., Arch Ophthalmol. 2012; 130(1): 9-24. However, subretinal injections of the extrafoveal retina are well tolerated. Therefore, it may be advantageous to use vectors that effectively target foveal cones after extrafoveal subretinal injections, i.e., the fovea does not detach during surgery. Vectors currently used in clinical trials for color blindness and other inherited retinal diseases do not meet this criterion. Transduction of the retina to the injection site is a desirable feature of the new generation of AAV vectors, because transgene expression beyond the initial boundaries of the subretinal bleb may maximize the beneficial gene therapy effects while avoiding some of the deleterious effects of retinal detachment. For example, in human subjects, lateral spread of transduction may allow subretinal injections in the parafoveal region to produce transduction of foveal cells while avoiding the deleterious effects of inducing foveal detachment. Recently, after subretinal delivery of AAV vectors in normal dogs, AAV vectors expressing EGFP showed lateral spread of transgene expression beyond the subretinal injection site. See Breuwer et al., Evaluation of Lateral Spread of Transgene Expression following Subretinal AAV-Mediated Gene Delivery in Dogs, PLoS One, 2013; 8(4): e60218.

[0020] The parafoveal region is the area of ​​the eye surrounding the fovea, approximately 4 degrees eccentricity from the central fixation point. The parafoveal region has the highest density of rods, while also containing a large number of cones. It is a transition zone between the cone- and rod-dominated retina, and is important in the case of diseases where degeneration proceeds from the outer layers to the inner layers of the retina, such as retinitis pigmentosa (RP). The perifoval region is the area surrounding the parafovea, representing the outermost zone of the macula. Like the parafoveal region, the perifoval region plays an important role in the progression of diseases like RP, where retinal degeneration begins in the periphery and progresses to the central retina. The perifoval region is the first area of ​​the macula to undergo degeneration in RP.

[0021] Some aspects of the present disclosure relate to rAAV particles and vectors comprising modified AAV44.9 capsids for treating the eye. In particular, in some embodiments, the present disclosure provides particles comprising AAV44.9 capsids with E531D mutations for treating retinal disorders. In some embodiments, the present disclosure provides particles comprising AAV44.9 (E531D) capsids that exhibit enhanced lateral diffusion after subretinal injection into the fovea, wherein detachment of the fovea is minimized. In some embodiments, the present disclosure provides capsid proteins, e.g., VP1, VP2, or VP3 capsid proteins, comprising the amino acid sequence of any one of SEQ ID NO: 1, 2, or 3, respectively.

[0022] In some embodiments, the present disclosure provides rAAV particles comprising a capsid comprising VP1, VP2, and / or VP3 proteins, wherein the rAAV particle further comprises a polynucleotide comprising a heterologous nucleic acid sequence.

[0023] In some embodiments, the heterologous nucleic acid sequence in the rAAV particle encodes a diagnostic or therapeutic agent, such as a polypeptide, a peptide, a ribozyme, a peptide nucleic acid, a siRNA, RNAi, a guide RNA, an antisense oligonucleotide, an antisense polynucleotide, an antibody, an antigen-binding fragment, or any combination thereof.

[0024] In some specific embodiments, the therapeutic agent a) protects one or more photoreceptor (PR) cells or one or more retinal pigment epithelial ("RPE") cells, b) restores one or more rods and / or cone-mediated functions, c) restores all or part of the visual behavior of one or both eyes, or d) any combination thereof. In some embodiments, after the rAAV particles are initially administered to one or both eyes of a mammal, the generation of the therapeutic agent is substantially continued in one or more photoreceptor cells or one or more RPE cells for a period of at least three months.

[0025] In some embodiments, the heterologous nucleic acid sequence comprises a sequence (e.g., a sequence having at least 80% identity to the target coding sequence) associated with a disease, disorder, or condition such as dominant cone dystrophy; dominant cone-rod dystrophy; Lebel congenital amaurosis; recessive cone dystrophy; recessive cone-rod dystrophy; macular dystrophy; pattern dystrophy; vitelliform dystrophy; central choroidal dystrophy; Stargardt disease; autosomal dominant, autosomal recessive, and X-linked retinitis pigmentosa; retinitis pigmentosa associated with Bardet-Biedl syndrome; X-linked juvenile retinoschisis; color blindness; blue cone monochromacy; and Usher syndrome types I, II, and III. In some embodiments, the heterologous nucleic acid sequence (e.g., a sequence having at least 80% identity to a target coding sequence) comprises a sequence associated with a disease, disorder, or condition such as Duchenne muscular dystrophy, limb-girdle muscular dystrophy, spinal muscular atrophy, glycogen storage disease type II (Pompe Disease), Friedreich's ataxia, mucopolysaccharidosis (MPS) (all forms), lysosomal storage disease (LSD) (all forms), amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Alzheimer's disease.

[0026] In some embodiments, the heterologous nucleic acid sequence has at least 80% identity with the target coding sequence. In some embodiments, the heterologous nucleic acid sequence has at least 95% identity with the target coding sequence. In some embodiments, the heterologous nucleic acid sequence has at least 98% or at least 99% identity with the target coding sequence. In some embodiments, the heterologous nucleic acid sequence has 100% identity with the target coding sequence. In some embodiments, the heterologous nucleic acid sequence comprises a GUCY2D sequence.

[0027] In some embodiments, the heterologous nucleic acid sequence is a replacement coding sequence. In some specific embodiments, the replacement coding sequence is administered to a subject to provide a functional protein, such as GUCY2D, to restore, for example, full or partial photoreceptor function in a subject (e.g., a human). In some embodiments, one or both alleles of a target coding sequence of a subject are silenced by administering rAAV particles comprising a heterologous nucleic acid sequence disclosed herein to a subject (e.g., to a human with dominant cone-rod dystrophy).

[0028] Exemplary coding sequences of interest include: GUCY2D and Gucy2e, which are associated with dominant cone dystrophy, dominant cone-rod dystrophy, and Leber congenital amaurosis; SPATA7, which is associated with Leber congenital amaurosis; PRPH2, which is associated with Leber congenital amaurosis and autosomal dominant retinal diseases (e.g., retinitis pigmentosa, pattern dystrophy, vitelliform dystrophy, central choroidal dystrophy, and macular dystrophy). GUCY2D encodes the retinal guanylate cyclase 1 (retGC1) enzyme, also known as guanylate cyclase 2D. Mutations in this gene cause Leber congenital amaurosis and cone-rod dystrophy-6 disorder. Gucy2e encodes guanylate cyclase 2E (the mouse homolog of GUCY2D).

[0029] Other target coding sequences may include AIPL1, LCA5, RPGRIP1, CRX, CRB1, NMNAT1, CEP290, IMPDH1, RD3, RDH12, TULP1, KCNJ13, GDF6, and IQCB1 (all associated with Leber congenital amaurosis); BBS1, BBS2, ARL6 / BBS3, BBS4, BBS5, BBS7, TTC8 / BBS8, BBS10, TRIM32 / BBS11, BBS12, CCDCC28B, CEP290, TMEM67, MKS1, and MKKS (all associated with Bardet-Bi edl syndrome (BBS)); RHO, PRPF31, RP1, NRL, and NR2E3 (all associated with autosomal dominant retinitis pigmentosa); RPGR and RP2 (both associated with X-linked retinitis pigmentosa); PDE6A, PDE6B, PDE6G, RP25, CNGA1, CNGB1, and MAK (all associated with autosomal recessive retinitis pigmentosa); RS1 (associated with X-linked juvenile retinoschisis (XLRS)); CNGB3, CNGA3, and GNAT2 (all associated with color blindness); OPN1LW and OPN1MW (both associated with blue cone achromatopsia (blue cone achromatopsia)). cone monochromacy (BCM); CRX, GUCA1A (GCAP1), and GUCA1B (GCAP2) (all associated with dominant cone dystrophy and dominant cone-rod dystrophy); ABCA4 (associated with recessive cone dystrophy, recessive cone-rod dystrophy, macular dystrophy, and fundus xanthophy); PROM1 and ELOVL4 (both associated with fundus xanthophy); MYO7A, USH1C, CDH23, PCDH15, and USH1G (all associated with Usher syndrome type I); USH2A and DFNB31 (both associated with Usher syndrome type II); and CLRN1 (associated with Usher syndrome type III).

[0030] In some embodiments, the heterologous nucleic acid sequence comprises a genomic regulatory sequence of interest (e.g., a locus control region) associated with a disease, disorder, or condition, such as blue cone achromatosis. An exemplary regulatory sequence of interest is the locus control region of the L / M opsin protein, which is associated with blue cone achromatosis.

[0031] In some aspects, the present disclosure provides compositions comprising rAAV particles and a pharmaceutically acceptable carrier, excipient, diluent, and / or buffer.

[0032] In some aspects, the disclosure provides a method for transducing RPE and photoreceptor cells to regulate the expression of heterologous nucleic acid sequences (or transgenes) in a subject, the method comprising administering to a subject (e.g., a human subject) a composition comprising rAAV particles as described herein and a pharmaceutically acceptable carrier, excipient, diluent, and / or buffer. In some aspects, the disclosure provides a method for treating a retinal disease of a subject, the method comprising administering a composition to the eye of the subject.

[0033] In some aspects, the disclosure provides compositions for treating retinal diseases and compositions for preparing medicaments for treating retinal diseases. In some aspects, the disclosure provides compositions comprising rAAV particles as described herein for treating by subretinal or intravitreal administration to one or both eyes of a mammal.

[0034] In some aspects, the present disclosure provides a method for expressing a nucleic acid segment in one or more photoreceptor cells or RPE cells of a mammal, the method comprising: administering rAAV particles as described herein subretinal or intravitreally to one or both eyes of the mammal for a period of time effective to produce the therapeutic agent in one or more PR cells or RPE cells of the mammal, wherein the rAAV particles comprise a polynucleotide comprising at least a first polynucleotide comprising a PR- or RPE-cell-specific promoter operably linked to at least a first heterologous nucleic acid sequence encoding the therapeutic agent.

[0035] The rAAV particles may contain a variety of (two, three, four, five, six, seven, eight, nine or ten) heterologous nucleic acid sequences. In certain embodiments, a variety of heterologous nucleic acid sequences are contained on a single polynucleotide molecule. For example, a variety of heterologous nucleic acid sequences can be used to correct or improve the genetic defects caused by multi-subunit proteins. In a plurality of embodiments, different heterologous nucleic acid sequences can be used to encode each subunit of a protein, or to encode different peptides or proteins. This is ideal for, for example, immunoglobulins, platelet-derived growth factors or dystrophin when the size of the nucleic acid encoding the protein subunit is large. In order for cells to produce multi-subunit proteins, cells are infected with rAAV particles containing each of the different subunits. Alternatively, the different subunits of a protein may be encoded by the same nucleic acid sequence. In a plurality of embodiments, a single heterologous nucleic acid sequence includes nucleic acids encoding each subunit, wherein the nucleic acids for each subunit are separated by an internal ribozyme entry site (internal ribozyme entry site, IRES). When the size of the nucleic acid encoding each subunit is small, for example, when the total size of the nucleic acid encoding the subunit and IRES is less than 5 kilobases, this is ideal.

[0036] As an alternative to IRES, the nucleic acid can be separated by a sequence encoding a 2A peptide that self-cleaves in a post-translational event. This 2A peptide is significantly smaller than an IRES, making it very suitable for use in situations where space is a limiting factor. More commonly, when the heterologous nucleic acid sequence is large and consists of multiple subunits, or two heterologous nucleic acid sequences are delivered together, or rAAV particles carrying one or more desired heterologous nucleic acid sequences or subunits are co-administered to allow them to be concatamerized in vivo to form a single vector genome. In such an embodiment, the first rAAV particle can carry an expression cassette expressing a single heterologous nucleic acid sequence, and the second rAAV particle can carry an expression cassette expressing a different heterologous nucleic acid sequence for co-expression in a host cell. However, the selected heterologous nucleic acid sequence can encode any biologically active product or other product, such as a product ideal for research.

[0037] In some embodiments, the polynucleotide within the rAAV particle comprises regulatory sequences, such as transcription and translation initiation and termination codons, that are specific to the type of host (e.g., bacteria, fungi, plants, or animals) into which the rAAV particle is to be introduced. Preferably, the nucleic acid molecule within the rAAV particle comprises regulatory sequences that are specific to the genus of the host. Most preferably, the molecule comprises regulatory sequences that are specific to the species of the host.

[0038] The polynucleotide within the rAAV particle preferably contains an expression control sequence that provides for expression of one or more heterologous nucleic acid sequences in the host cell, such as a promoter, an enhancer, a polyadenylation signal, a transcription terminator, an internal ribosome entry site (IRES), etc. Exemplary expression control sequences are known in the art and are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, CA. (1990). BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The following drawings constitute part of this specification and are included to illustrate certain aspects of the present invention. The present invention may be better understood by reference to the following description in conjunction with the accompanying drawings, in which like reference numerals represent like elements, and in which:

[0040] Figure 1 Shown are the VP1 -based AAV phylogeny (AAV44.9 in bold) and a table with detailed information on the AAV constructs used for vector preparation.

[0041] Figure 2A , 2B , 2C, 2D, and 2E show qualitative and quantitative analysis of AAV44.9, AAV5, and AAV8(Y731F) at 4 weeks after subretinal injection. Figure 2A A fundus image with an exposure length of 25 is shown. Figures 2B to 2D Flow cytometry dot plots are shown, and Figure 2E Shown is a subretinal injection of 2 × 10 9 Quantitative analysis of the ability of AAV44.9 to transduce rods more efficiently than AAV5 and AAV8(Y731F) after vector genome (vg).

[0042] Figure 3A , 3B and 3C show representative retinal cross-sectional images showing injection of AAV5 (top, Figure 3A )、AAV8(Y731F)(above, Figure 3B )AAV44.9 (above, Figure 3C mCherry expression in photoreceptors and retinal pigment epithelium ("RPE") in the retina of Nrl-GFP transgenic mice 4 weeks after subretinal injection. Figure 3A , 3B The lower panels of 3C and 3C show merged images of nuclear DAPI staining, endogenous GFP expression in rod cells, and mCherry expression in photoreceptor cells as indicated by arrows.

[0043] Figure 4A , 4B and 4C show qualitative and quantitative analysis of AAV44.9(Y731F) and AAV44.9(E531D). Figure 4A The fundus image is shown when the exposure length is 25. Figure 4B Flow cytometry dot plots are shown, and Figure 4C The results show that 2 × 10 9 Quantitative analysis of the finding that AAV44.9(E531D) transduces rod photoreceptors more efficiently than AAV44.9 and AAV44.9(Y731F) after vg.

[0044] Figure 5A and 5B Representative retinal cross-sectional images are shown, showing injection of AAV44.9(Y731F) (top, Figure 5A ) and AAV44.9(E531D) (above, Figure 5B ) in the retinas of Nrl-GFP mice, mCherry expression was mainly in photoreceptors and RPE at 4 weeks after subretinal injection. Figure 5A and 5B The lower panel shows a merged image of nuclear DAPI staining (blue), endogenous GFP expression in rods (green), and mCherry expression in photoreceptor cells (red).

[0045] Fig. 6A and 6B The transduction efficiency of AAV44.9 and AAV44.9(Y731F) in ocular cell lines is shown. AAV44.9(Y731F) showed improved transduction relative to AAV44.9 in the mouse cone photoreceptor cell line ( Fig. 6A However, AAV44.9 is more effective than AAV9(Y731F) in human RPE cell lines ( Figure 6B ).

[0046] Fig. 7A and 7B Qualitative analysis of AAV after intravitreal injection is shown. Fig. 7A Fundus images of AAV2, AAV5, AAV8(Y731F), AAV44.9, AAV44.9(Y731F), and AAV44.9(E531D) are shown (in integration of length 25). Figure 7B Intravitreal injection of 2 × 10 9 vg Variant of fundus image at 4 weeks (integral length 100).

[0047] Fig. 8A , 8B8C and 8D show AAV44.9 containing a cone-preferential chimeric IRBP enhancer-cone transduction protein promoter (IRBP / GNAT2) and a GFP reporter, showing Fig. 8A Shown is GFP expression in cones 4 weeks after SR injection. Figure 8B and 8C Co-staining with cone-arrestin antibody colocalizing with GFP fluorescence is shown.

[0048] Fig. 9A Representative fundus images (red fluorescence filter) of Nrl-GFP mice taken 4 weeks after subretinal injection of AAV44.9 or AAV44.9(E531D) are shown. Vectors were delivered at 2 v×109 vg in 1 uL. Exposure and gain settings were consistent throughout the experiment.

[0049] Fig. 9B The percentage of transduction and the corresponding values ​​of each cell population collected 4 weeks after injection are shown. Fig. 9A ) were dissociated with papain and flow cytometry was performed to quantify the percentage of transduced rods (GFP+mCherry positive) or non-rod cells (mCherry positive), as previously described in Boye et al., Impact of Heparan Sulfate Binding on Transduction of Retina by Recombinant Adeno-Associated Virus Vectors, J. Virol. 2016, 90(8): 4215-4231, the entire contents of which are incorporated herein by reference.

[0050] Fig. 10A Representative fundus images of Nrl-GFP mice taken 4 weeks after subretinal injection of AAVrh.8-mCherry are shown. The vector was injected at 2×10 9 VG delivery. Exposure and gain settings were consistent throughout the experiment.

[0051] Fig. 10B The percentage of transduction and the corresponding values ​​of each cell population taken 4 weeks after injection are shown. Fig. 9A and 10A ) were dissociated with papain and subjected to flow cytometry to quantify the percentage of transduced rods (GFP+mCherry positive) or non-rod cells (mCherry positive).

[0052] Fig.11ARepresentative fundus images of Nrl-GFP mice taken 4 weeks after subretinal injection with lower titers of self-complementary AAV44.9 ("scAAV44.9"), scAAV44.9(E531D), or scAAVrh.8 are shown. The vector was injected at 2×10 8 VG delivery. Exposure and gain settings were consistent throughout the experiment.

[0053] Fig. 11B The percentage of transduction and the corresponding values ​​of each cell population collected 4 weeks after injection are shown. Fig.11A ) were dissociated with papain and subjected to flow cytometry to quantify the percentage of transduced rods (GFP+mCherry positive) or non-rod cells (mCherry positive).

[0054] Fig.12 Qualitative analysis of AAV44.9(E531D) and unmodified AAV44.9 in the context of the cone-specific IRBPe-GNAT2 chimeric promoter 6 weeks after subretinal injection is shown. 12 Fundus images of AAV44.9(E531D)-IRBP / GNAT2-hGFP and AAV44.9-IRBP / GNAT2-hGFP after vg.

[0055] Fig.13 Representative retinal cross-sections of WT mice taken 6 weeks after subretinal injection of AAV44.9(E531D)-IRBP / GNAT2-hGFP are shown. The vector was injected at 2×10 12 vg delivery. Sections were immunostained with antibodies against GFP (green) and cone arrestin (red).

[0056] Fig.14 Representative retinal cross-sections of WT mice taken 6 weeks after subretinal injection of AAV44.9-IRBP / GNAT2-hGFP are shown. The vector was injected at 2×10 12 Vg delivery. Sections were immunostained with antibodies against GFP and cone arrestin as indicated by arrows.

[0057] Fig.15 AAV44.9-hGRK1-GFP and AAV44.9(E531D)-hGRK1-GFP showed enhanced lateral spread and efficacy in macaques injected subretinaly. 12vg vg / mL delivery. The initial boundaries of the bleb and the boundaries of the resulting GFP expression on the day of dosing are marked with white dashed lines. The same vasculature is highlighted with bold dark lines for reference.

[0058] Fig.16 Shown is the extrafoveal subretinal injection of AAV44.9-hGRK1-GFP (1×10 12 Optical coherence tomography (OCT) scans of three subretinal injection blebs produced after 4% paraformaldehyde injection (vg / mL) (see negative contrast fundus image on the left).

[0059] Fig.17 OCT images of macaque retina after subretinal injection of AAV44.9-hGRK1-GFP outside the fovea are shown. Arrows in the SLO image (upper left) indicate the location of the retinal sections shown in the scans in the lower part of the figure. Sections were stained for cone arrestin and DAPI. The percentage of rods / cones expressing GFP is plotted in each area. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

[0060] Fig.18A and 18B Shown are ERG recordings from retinal guanylate cyclase 1 / 2 double knockout (GCdko) mice after subretinal injection of AAV44.9(E531D)-hGRK1-GUCY2e or AAV8(Y731F)-hGRK1-GUCY2e. Fig.18A Shown are the average maximum a-wave and b-wave amplitudes for both scotopic (left) and photopic (right) settings. Fig.18B Representative ERG traces of cones from vehicle-treated eyes or contralateral untreated ("No Tx") eyes are shown.

[0061] Fig.19 OCT images of macaque retina following extrafoveal subretinal injection of AAV44.9(E531D)-hGRK1-GFP are shown. Arrows in the SLO image (upper left) indicate the location of the retinal slice shown in the scan in the lower portion of the figure. The slices were stained for cone arrestin and DAPI. The percentage of rods / cones expressing GFP is plotted in each region.

[0062] Fig. 20A , 20B , 20C and 20D show representative OCT images of the parafoveal region of the macaque retina after extrafoveal subretinal injection of AAV44.9(E531D)-hGRK1-GFP and AAV44.9-hGRK1-GFP. The scale bar is in A = 40 μm, and in B = 20 μm.

[0063] Fig.21A and 21B Shown are representative OCT images of the perifoval region of the macaque retina following these injections. DETAILED DESCRIPTION

[0064] The present disclosure provides an evaluation of the performance of AAV44.9(E531D), a new variant of the capsid serotype AAV44.9, and vectors and particles incorporating this plasmid in subretinal injections in mice and macaques relative to the closely related AAVrh.8 and unmodified AAV44.9, a benchmark vector. As described herein, AAV44.9(E531D) was found to mediate higher retinal transduction relative to unmodified AAV44.9 and AAVrh.8, and both species had significantly higher transduction than benchmark capsids (e.g., AAV5- and AAV8-based vectors).

[0065] Therefore, the present disclosure provides rAAV particles and related compositions and methods comprising the capsid protein of AAV44.9 (E531D). In some embodiments, the rAAV particles contain heterologous nucleic acid sequences, such as nucleic acid sequences encoding therapeutic agents or diagnostic agents. The heterologous nucleic acid sequence can be in the form of a single-stranded (ss) or self-complementary (sc) AAV nucleic acid vector, such as a single-stranded or self-complementary recombinant viral genome.

[0066] The present disclosure also provides rAAV particles and related compositions and methods comprising the capsid protein of AAV44.9 (Y731F). This AAV44.9 capsid variant has a YF mutation at residue 731. In some embodiments, the rAAV particles contain a heterologous nucleic acid sequence, such as a nucleic acid sequence encoding a therapeutic agent or a diagnostic agent. The heterologous nucleic acid sequence can be in the form of a single-stranded (ss) or self-complementary (sc) AAV nucleic acid vector, such as a single-stranded or self-complementary recombinant viral genome.

[0067] Some aspects of the present disclosure relate to vectors comprising an AAV44.9 (E531D) capsid that exhibits enhanced lateral spread following subretinal injection into the fovea, wherein detachment of the fovea (e.g., transient bullous detachment) is minimized. In some embodiments, the present disclosure provides a capsid protein, such as a VP1, VP2, or VP3 capsid protein, comprising an amino acid sequence of SEQ ID NO: 1, 2, or 3.

[0068] In some embodiments, the disclosure provides rAAV particles comprising a capsid comprising VP1, VP2 and / or VP3 proteins, wherein the rAAV particles further comprise a polynucleotide comprising a heterologous nucleic acid sequence. In some embodiments, the rAAV particles comprise a capsid comprising VP1, VP2 and / or VP3 proteins, wherein the VP1 protein comprises the amino acid sequence of SEQ ID NO: 1, the VP2 protein comprises the amino acid sequence of SEQ ID NO: 2, and / or the VP3 protein comprises the amino acid sequence of SEQ ID NO: 3, and wherein the AAV further comprises a polynucleotide comprising a heterologous nucleic acid sequence. The heterologous nucleic acid sequence may be flanked by one or more inverted terminal repeat (ITR) sequences.

[0069] In some embodiments, the present disclosure provides a capsid protein comprising the amino acid sequence of SEQ ID NO: 1, 2 and / or 3.

[0070] In some embodiments, the disclosure provides a nucleic acid, such as a plasmid or a viral vector, comprising a nucleic acid sequence of SEQ ID NO: 4 (which encodes AAV44.9 (E531D) VP1). In some embodiments, the disclosure provides a nucleic acid, such as a plasmid or a viral vector, comprising a nucleic acid sequence of SEQ ID NO: 5 (which encodes AAV44.9 (E531D) VP2). In some embodiments, the disclosure provides a nucleic acid, such as a plasmid or a viral vector, comprising a nucleic acid sequence of SEQ ID NO: 6 (which encodes AAV44.9 (E531D) VP3). In some embodiments, the viral vector is a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the rAAV vector is self-complementary. In some embodiments, the nucleic acid is contained within a cell, such as a mammalian or insect cell.

[0071] The sequences of SEQ ID NOs: 1 to 8 are provided below.

[0072] SEQ ID NO: 1—AAV44.9 (E531D) VP1 amino acid sequence

[0073]

[0074]

[0075] SEQ ID NO: 2—AAV44.9 (E531D) VP2 amino acid sequence

[0076]

[0077] SEQ ID NO: 3—AAV44.9 (E531D) VP3 amino acid sequence

[0078]

[0079] SEQ ID NO: 4—AAV44.9 (E531D) VP1 amino acid sequence

[0080]

[0081] SEQ ID NO: 5—AAV44.9 (E531D) VP2 amino acid sequence

[0082]

[0083] SEQ ID NO:6—AAV44.9(E531D) VP3 amino acid sequence

[0084]

[0085]

[0086] SEQ ID NO: 7—AAV44.9 wild-type VP1 amino acid sequence

[0087]

[0088] SEQ ID NO: 8—AAV44.9 wild-type VP1 nucleotide sequence

[0089]

[0090]

[0091] Methods for producing and using pseudotyped rAAV vectors are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671, 2001; Halbert et al., J. Virol., 74:1524-1532, 2000; Zolotukhin et al., Methods, 28:158-167, 2002; and Auricchio et al., Hum. Molec. Genet., 10:3075-3081, 2001). Methods for producing rAAV particles and heterologous nucleic acids are also known in the art and are commercially available (see, e.g., Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167; and U.S. Patent Publication Nos. US 2007 / 0015238 and US 2012 / 0322861, which are incorporated herein by reference in their entirety; and plasmids and kits available from ATCC and Cell Biolabs, Inc.). For example, a plasmid comprising a heterologous nucleic acid sequence can be combined with one or more helper plasmids (e.g., comprising a rep gene (e.g., encoding Rep78, Rep68, Rep52, and Rep40) and a cap gene (encoding VP1, VP2, and VP3, including a modified VP3 region as described herein) and transfected or permanently integrated into a production cell line so that rAAV particles can be packaged and subsequently purified.

[0092] In some embodiments, one or more helper plasmids include a first helper plasmid comprising a rep gene and a cap gene (e.g., encoding a rAAV capsid protein as described herein) and a second helper plasmid comprising an E1a gene, an E1b gene, an E4 gene, an E2a gene, and a VA gene. In some embodiments, the rep gene is a rep gene derived from AAV2, and the cap gene is derived from AAV44.9 and may include modifications to the gene to produce a modified capsid protein as described herein. Helper plasmids and methods for making such plasmids are known in the art and are commercially available (see, e.g., pDM, pDG, pDP1rs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E / R585E), and pDP8.ape plasmids from PlasmidFactory, Bielefeld, Germany; other products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, CA; and Addgene, Cambridge, MA; pxx6; Grimm et al. (1998), Novel Tools for Production and Purification of Recombinant Adenoassociated Virus Vectors, Human Gene Therapy, Vol. 9, 2745-2760; Kern, A. et al. (2003), Identification of a Heparin-Binding Motifon Adeno-Associated Virus Type 2 Capsids,Journal of Virology,Vol.77,11072-11081.;Grimm et al.(2003),Helper Virus-Free,Optically Controllable,and Two-Plasmid-Based Production of Adeno-associated Virus Vectors of Serotypes 1to6,Molecular Therapy, Vol.7, 839-850; Kronenberg et al.(2005), A ConformationalChange in the Adeno-Associated Virus Type 2Capsid Leads to the Exposure ofHidden VP1N Termini, Journal of Virology, Vol.79, 5296-5303; and Moullier, P. and Snyder, RO (2008), International efforts for recombinant adeno-associatedviral vector reference standards, Molecular Therapy, Vol. 16, 1185-1188). .

[0093] Next, an exemplary, non-limiting method for producing rAAV particles is described. One or more helper plasmids are generated or obtained, which contain the rep and cap ORFs of the desired AAV serotype and the adenovirus VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. The cap ORF may also contain one or more modifications to produce a modified capsid protein as described herein. HEK293 cells (available from ) are transfected with the helper plasmids and a plasmid containing a nucleic acid vector described herein by CaPO4-mediated transfection, lipids, or polymer molecules such as polyethyleneimine (PEI). ). The HEK293 cells are then incubated for at least 60 hours to allow rAAV particles to be produced. Alternatively, in another example, a Sf9-based stable production cell line is infected with a single recombinant baculovirus comprising a heterologous nucleic acid sequence. As another alternative, in another example, a HEK293 or BHK cell line is infected with an HSV comprising a heterologous nucleic acid sequence and optionally one or more helper HSVs comprising adenovirus VA, E2A (DBP) and E4 genes under the transcriptional control of their native promoters as described herein. The HEK293, BHK or Sf9 cells are then incubated for at least 60 hours to allow rAAV particles to be produced. The rAAV particles can then be purified using any method known in the art or described herein, for example, by iodixanol step gradients, CsCl gradients, chromatography or polyethylene glycol (PEG) precipitation.

[0094] The present disclosure also contemplates host cells comprising particles incorporating AAV44.9(E531D) capsids, nucleic acids encoding AAV44.9(E531D) capsids, or rAAV particles as described herein. Such host cells include mammalian host cells, of which human host cells are preferred, and may be isolated, for example in cell or tissue culture. In some embodiments, the host cell is an eye cell.

[0095] In some embodiments, a composition is provided comprising rAAV particles as described herein (e.g., comprising an AAV44.9 (E531D) capsid), and optionally a pharmaceutically acceptable carrier, excipient, diluent, and / or buffer. In some embodiments, the compositions described herein may be administered to a mammal (or subject) in need of treatment. In some embodiments, the subject suffers from or is suspected of suffering from one or more retinal disorders, retinal diseases, or retinal dystrophy. In some embodiments, the subject suffers from or is suspected of suffering from one or more retinal conditions, diseases, and disorders disclosed herein (e.g., cone-rod dystrophy). In some embodiments, the subject has one or more endogenous mutant alleles (e.g., associated with or causing a disease, disorder, or condition of the eye or retina (e.g., cone-rod dystrophy)).

[0096] In some embodiments, a method for transducing mammalian photoreceptor cells or retinal pigment epithelial cells is provided, the method comprising applying rAAV particles as described herein to one or both eyes of a mammal. In some specific embodiments, a method for expressing polynucleotides in one or more photoreceptor cells or RPE cells of a mammal is provided, the method comprising applying rAAV particles as described herein or its composition to one or both eyes of the mammal under the retina or in the vitreous for a period of time that is effective in producing the therapeutic agent in one or more PR cells or RPE cells of the mammal, wherein the rAAV particles include polynucleotides, the polynucleotides include at least the first polynucleotides, and the first polynucleotides include PR or RPE cell-specific promoters that are effectively connected to at least the first heterologous nucleic acid sequence encoding a therapeutic agent.

[0097] In some specific embodiments, the invention provides PR or RPE cell-specific promoters that are effectively connected to at least the first heterologous nucleic acid sequence encoding a therapeutic agent. Exemplary PR or RPE cell-specific promoters may include a) photoreceptor-specific promoters (activated in rods and cones), such as IRBP promoters (hIRPB, IRBP, IRBP241), rhodopsin kinase promoters (hGRK1, GRK1, GRK, RK) and / or chimeric human Retinoschisin-IRBP enhancers (RS / IRPB); cone-specific promoters, such as red / green cone opsin promoters (which may include 2.1 kb (PR2.1) forms or 1.7 kb (PR1.7) forms, see U.S. Patent Publication No. 2018 / 0112231, incorporated herein by reference), cone arrestin promoter (hCAR, CAR), chimeric IRBP enhancer-cone transduction protein promoter (IRBP / GNAT2, IRBPe-GNAT2); rod-specific promoters, such as human rhodopsin promoter (RHO, RHOP, etc.), human NRL promoter (NRL); or RPE-specific promoters, such as RPE65 or Bestrophin / VMD2 (BEST1, BEST, VMD2).

[0098] In some embodiments, the promoter of any of the disclosed rAAV vectors comprises a nucleotide sequence that is at least 95%, at least 98%, at least 99%, or 100% identical to the hGRK1 promoter sequence shown in SEQ ID NO: 11:

[0099]

[0100] Thus, exemplary rAAV vectors described in the present disclosure include AAV44.9(E531D)-hGRK1-GFP, AAV44.9(Y731F)-hGRK1-GFP, AAV44.9(E531D)-IRBP / GNAT2-hGFP, AAV44.9(Y731F)-IRBP / GNAT2-hGFP, AAV44.9(E531D)-hGRK1-GUCY2E, and AAV44.9(Y731F)-hGRK1-GUCY2E.

[0101] In some specific embodiments, the present disclosure provides a constitutive promoter operably linked to at least a first polynucleotide, which may comprise CMV, CBA, CB, smCBA, CBh, or EF1-α.

[0102] In some embodiments, a method is provided involving providing a therapeutically effective amount of a selected therapeutic agent to a mammal in need thereof, the method comprising administering an amount of rAAV particles described herein to one or both eyes of the mammal; and continuing for a period of time effective to provide the therapeutically effective amount of the selected therapeutic agent to the mammal.

[0103] In certain embodiments, the mammal is suspected of having, is at risk of developing, or has been diagnosed with a disease, disorder, or condition such as, but not limited to, dominant cone dystrophy; dominant cone-rod dystrophy; Lebel congenital amaurosis; recessive cone dystrophy; recessive cone-rod dystrophy; macular dystrophy; pattern dystrophy; vitelliform dystrophy; central choroidal dystrophy; fundus xanthophyta, autosomal dominant, autosomal recessive, and X-linked retinal pigment epithelial cell disease. Degeneration; retinitis pigmentosa associated with Bardet-Biedl syndrome; X-linked juvenile retinoschisis; color blindness; blue cone achromatopsia; Usher syndrome types I, II, and III; Duchenne muscular dystrophy; limb-girdle muscular dystrophy; spinal muscular atrophy; glycogen storage disease type II; Friedrich's ataxia; mucopolysaccharidosis (MPS) (all forms); lysosomal storage diseases (LSD) (all forms); amyotrophic lateral sclerosis (ALS); Parkinson's disease; and Alzheimer's disease. In some embodiments, the subject has one or more endogenous mutant alleles associated with or causing a disease, disorder, or condition of the eye or retina, such as genes GUCY2D, GUCY2E, SPATA7, PRPH2, ABCA4, AIPL1, LCA5, RPGRIP1, CRX, CRB1, NMNAT1, CEP290, IMPDH1, RD3, RDH12, TULP1, KCNJ13, GDF6, IQCB1, BBS1, BBS2, ARL6 / BBS3, BBS4, BBS5, BBS7, TTC8 / BBS8, BBS10, TRIM32 / BBS11, BBS12, CCDCC28B, CEP290, TMEM67, MKS1 alleles in MKKS, RHO, PRPF31, RP1, NRL, NR2E3, RPGR, RP2, PDE6A, PDE6B, PDE6G, RP25, CNGA1, CNGB1, MAK, RS1, CNGB3, CNGA3, GNAT2, OPN1LW, OPN1MW, CRX, GUCA1A (GCAP1), GUCA1B (GCAP2), ABCA4, PROM1, and ELOVL4, MYO7A, USH1C, CDH23, PCDH15, USH1G, USH2A, DFNB31, or CLRN1.

[0104] In some specific embodiments, a replacement coding sequence is administered to a subject to provide a functional protein, such as GUCY2D or Gucy2e, to, for example, fully or partially restore photoreceptor function in a subject (e.g., a human). In some embodiments, one or two alleles of a target coding sequence of a subject are silenced by administering rAAV particles comprising a heterologous nucleic acid sequence disclosed herein to a subject (e.g., administered to a person with dominant cone-rod dystrophy). In some specific embodiments, endogenous mutant alleles of one or more target coding sequences are silenced or inhibited by administering rAAV particles disclosed herein.

[0105] In some embodiments, the heterologous nucleic acid sequence of any rAAV nucleic acid vector of the present disclosure has a sequence that is at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to a nucleotide sequence selected from SEQ ID NO: 9 or 10. The nucleotide sequences encoding the human GUCY2D gene (SEQ ID NO: 9) and the mouse Gucy2e gene (SEQ ID NO: 10) are shown below.

[0106] GUCY2D:

[0107]

[0108]

[0109]

[0110] Gucy2e:

[0111]

[0112]

[0113]

[0114] In some embodiments, the mammal is a human subject. In some embodiments, the mammal is a non-human primate subject. Non-limiting examples of non-human primate subjects include macaques (e.g., cynomolgus monkeys or rhesus monkeys), marmosets, tamarins, spider monkeys, owl monkeys, vervet monkeys, squirrel monkeys, baboons, gorillas, chimpanzees, and orangutans. Other exemplary subjects include domestic animals, such as dogs and cats; livestock, such as horses, cattle, pigs, sheep, goats, and chickens; and other animals, such as mice, rats, guinea pigs, and hamsters.

[0115] In certain embodiments, methods are provided for subretinal administration of rAAV particles described herein or compositions thereof to the fovea (e.g., foveal cones) of a mammal. In some specific embodiments, detachment of the fovea is minimized during and / or after subretinal administration. In some specific embodiments, subretinal administration of rAAV particles is performed without any detachment of the fovea.

[0116] In some embodiments, the dose of rAAV particles administered to a cell or subject may be about 10 6 Up to 10 14 particles / mL or 10 3 Up to 10 15 particles / mL, or any value in between any range thereof, for example, about 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or 10 14 In one embodiment, administration of more than 10 13 In some embodiments, the dose of rAAV particles administered to a subject may be about 10 6 Up to 10 14 Vector genome (vg) / mL, or 10 3 Up to 10 15 vg / mL, or any value between any ranges thereof, such as about 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or 10 14 In one embodiment, administration of more than 10 13 vg / mL of rAAV particles. Depending on the treatment of the specific disease, disorder or condition to be treated, the rAAV particles can be administered in a single dose or divided into two or more administrations. In some embodiments, 0.0001 mL to 10 mL (e.g., 0.0001 mL, 0.001 mL, 0.01 mL, 0.1 mL, 1 mL, 10 mL) is delivered to the subject in one dose.

[0117] In some embodiments, the rAAV particle titer is 1×10 10 Up to 5×1013 In some embodiments, the rAAV particle titer may be 1×10 10 , 2.5×10 10 , 5×10 10 , 1×10 11 , 2.5×10 11 , 5×10 11 , 1×10 12 , 2.5×10 12 , 5×10 12 , 1×10 13 , 2.5×10 13 , or 5×10 13 vg / mL. In some embodiments, the particle titer is less than 1×10 10 vg / mL. In some embodiments, the rAAV particle titer is greater than 1×10 15 In one embodiment, the rAAV particle titer is greater than 5×10 13 vg / mL. In some embodiments, the rAAV particles are administered by the methods described herein (eg, subretinal or intravitreal).

[0118] Depending on the treatment of the specific disease, disorder or condition to be treated, rAAV particles can be administered in a single dose or divided into two or more administrations. In some embodiments, 1 to 500 microliters of the composition described in this application (e.g., it includes rAAV particles) are administered to one or both eyes of the object. For example, in some embodiments, about 1, about 10, about 50, about 100, about 200, about 300, about 400 or about 500 microliters can be applied to each eye. However, it should be understood that smaller or larger volumes can be applied in some embodiments.

[0119] In some embodiments, the present disclosure provides formulations of one or more rAAV-based compositions disclosed herein in a pharmaceutically acceptable solution for administration to cells or animals, alone or in combination with one or more other therapeutic modalities, and in particular for the treatment of human cells, tissues, and diseases affecting humans.

[0120] If desired, the rAAV particles described herein may also be administered in combination with other agents, such as proteins or polypeptides, or a variety of pharmaceutically active agents, including one or more systemic or local administration of a therapeutic polypeptide, biologically active fragment, or variant thereof. In fact, there is almost no restriction on other components that may also be included, as long as the additional agent does not cause significant adverse effects when in contact with target cells or host tissues. Therefore, in certain cases, rAAV particles may be delivered with a variety of other agents as desired. Such compositions may be purified from host cells or other biological sources, or alternatively, may be chemically synthesized as described herein.

[0121] The formulation of pharmaceutically acceptable excipients and carrier solutions is well within the skill of the art, as is the development of appropriate dosing and treatment regimens for use of the specific compositions described herein in a variety of treatment regimens, including, for example, subretinal, intravitreal, parenteral, intravenous, intranasal, intraarticular, and intramuscular administration and formulations.

[0122] Generally speaking, these formulations may contain at least about 0.1% therapeutic agent (e.g., rAAV particles) or more, but the percentage of one or more active ingredients may of course vary and may conveniently be from about 1% or 2% to about 70% or 80% or more, by weight or volume of the total formulation. Naturally, the amount of therapeutic agent (e.g., rAAV particles) in each therapeutically useful composition may be prepared in such a way that a suitable dose will be obtained in any given unit dose of the compound. Those skilled in the art of preparing such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and therefore a variety of dosages and treatment regimens may be desirable.

[0123] In certain instances, it would be desirable to deliver the rAAV particles as described herein (e.g., comprising AAV44.9(E531D) capsids) in an appropriately formulated pharmaceutical composition disclosed herein subretinal, intraocular, intravitreal, parenterally, subcutaneously, intravenously, intracerebroventricularly, intramuscularly, intrathecally, orally, intraperitoneally, by oral or nasal inhalation, or by direct injection into one or more cells, tissues or organs.

[0124] The pharmaceutical form of the composition suitable for injection (e.g., comprising rAAV particles as described herein) includes a sterile aqueous solution or dispersion. In some embodiments, the form is sterile and is fluid to the extent that there is easy injectability. In some embodiments, the form is stable under manufacturing and storage conditions, and is preserved to resist the contamination of microorganisms (e.g., bacteria and fungi). The carrier can be a solvent or a dispersion medium, which includes, for example, water, saline, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), a suitable mixture thereof, and / or a vegetable oil. Appropriate fluidity can be maintained, for example, by using a coating (e.g., lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant.

[0125] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the rAAV particles described herein are administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, such as mineral oil; vegetable oils, such as peanut oil, soybean oil, and sesame oil; animal oils; or oils of synthetic origin. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers.

[0126] The compositions of the present disclosure can be delivered to the eye by a variety of routes. It can be delivered intraocularly, by topical application to the eye, or by intraocular injection into, for example, the vitreous body (intravitreal injection) or the subretinal (subretinal injection) interphotoreceptor space. In some embodiments, it is delivered to the rod photoreceptor cells. Alternatively, it can be delivered locally by insertion or injection into the tissue surrounding the eye. It can be delivered systemically by oral routes or by subcutaneous, intravenous or intramuscular injection. Alternatively, it can be delivered by catheter or by implant, wherein such implant is made of porous, non-porous or gel material (including membrane, such as silicone rubber membrane or fiber, biodegradable polymer or proteinaceous material). It can be applied before the onset of the disease to prevent it from occurring, such as during surgery on the eye, or immediately after the onset of a pathological disease or during the occurrence of acute or long-term diseases.

[0127] For the use of injectable aqueous solution, for example, if necessary, the solution can be appropriately buffered, and first the liquid diluent can be made isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, intravitreal, subcutaneous and intraperitoneal administration. In this regard, according to the present disclosure, those skilled in the art will know the usable sterile aqueous medium. For example, a dose can be dissolved in 1ml isotonic NaCl solution, then added to 1000ml subcutaneous perfusion fluid or injected in the infusion site of suggestion (see, for example, " Remington's Pharmaceutical Sciences " the 15th edition, pp. 1035-1038 and 1570-1580). According to the condition of the treated object, some changes in dosage will inevitably occur. In any case, the personnel responsible for administration determine the appropriate dose for individual objects. In addition, for people to use, the preparation should meet the sterility, pyrogenicity and general safety and purity standards required by, for example, FDA biological standards.

[0128] Sterile injectable solutions can be prepared by introducing the desired amount of rAAV particles as described herein into a suitable solvent (which has several other ingredients listed above as needed) and then filtering and sterilizing. Generally speaking, dispersions are prepared by introducing a variety of sterilized active ingredients into a sterile carrier, which contains a basic dispersion medium and other ingredients from those listed above as needed. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation method is vacuum drying and freeze drying techniques, which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile filtered solution thereof.

[0129] The amount of a composition (e.g., comprising rAAV particles as described herein) and the time of administration of such a composition will be within the scope of a skilled artisan having the benefit of the present teachings. However, it is likely that administration of a therapeutically effective amount of a disclosed composition can be achieved by a single administration, such as a single injection of a sufficient number of rAAV particles to provide a therapeutic benefit to a patient receiving such treatment. Alternatively, in some cases, it may be desirable to provide multiple or continuous administrations of a composition over a relatively short or relatively extended period of time, which may be determined by the medical personnel supervising the administration of such a composition.

[0130] In some embodiments, after using one or more of compositions described in the application, can keep or recover (for example, partially or entirely) visual acuity.In some embodiments, after using one or more of compositions described in the application, one or more photoreceptor cells or one or more RPE cells can be partially or entirely preserved, and / or the function of one or more of rods and / or cone mediation can be partially or entirely recovered.

[0131] "Treating" a disease as the term is used herein refers to reducing the frequency or severity of at least one sign or symptom of a disease, disorder, or condition (e.g., rod-cone dystrophy) experienced by a subject. The above-described compositions are generally administered to a subject in an effective amount (i.e., an amount capable of producing a desired result). The desired result will depend on the active agent administered. For example, an effective amount of rAAV particles can be an amount of particles capable of transferring a heterologous nucleic acid to a host organ, tissue, or cell.

[0132] The toxicity and efficacy of the compositions used in the methods of the present disclosure can be determined by standard pharmaceutical methods using cells in culture or experimental animals to determine the LD50 (the dose that is lethal to 50% of the population). The dose ratio between toxicity and efficacy is the therapeutic index, and it can be expressed as the ratio LD50 / ED50. Compositions that exhibit large therapeutic indices are preferred. Although those that exhibit toxic side effects can be used, care should be taken to design a delivery system that minimizes the potential damage of such side effects. The dosage of the compositions described herein is generally within the range that includes the ED50 and has little or no toxicity. Depending on the dosage form employed and the route of administration used, the dosage may vary within this range.

[0133] Example

[0134] The following examples are included to demonstrate some preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the examples hereinafter represent techniques that the inventors have found to work well in the practice of the present invention, and therefore can be considered to constitute preferred modes for its practice. However, based on this disclosure, it will be appreciated by those skilled in the art that many changes may be made in the disclosed specific embodiments without departing from the spirit and scope of the present invention, and still similar or similar results may be obtained.

[0135] Example 1 - Therapeutic Molecules for AAV-Based Retinal Gene Therapy

[0136] Retinal transduction and tropism of AAV44.9 were assessed after intravitreal (Ivt) or subretinal (SR) injection. The photoreceptor transduction efficiency of AAV44.9 and benchmark capsids AAV5 and AAV8 (Y731F) was quantified by utilizing a mouse model in which GFP is constitutively expressed in all rod photoreceptors (Nrl-GFP-Smouse).

[0137] We sought to determine whether transduction by AAV44.9 could be improved by adding a YF mutation at residue 731 (Y731F) and by replacing glutamic acid with aspartic acid at position 531 (E531D) alone. As cone photoreceptors are the target of many retinal gene therapies, such as color blindness and cone-rod dystrophy, the ability of AAV44.9 to express transgenes in cone photoreceptors was evaluated.

[0138] Experimental methods

[0139] AAV capsid phylogenetic tree Figure 1 As shown. The VP1 amino acid sequences of AAV were aligned using ClustalW (AlignX-Vector NTI). This alignment was then used to generate a phylogenetic tree using the neighbor-joining method using the EMBL-EBI Simple Phylogeny program (ebi.ac.uk / Tools / phylogeny / simple_phylogeny / ). The resulting tree was visualized using the TreeView program, with AAV5 designated as an outgroup.

[0140] Vector preparation

[0141] Using a triple transfection plasmid-based system, a double-layer cell factory (1,272 cm 2 Self-complementary AAV constructs containing a truncated chimeric CMV-chicken β-actin (smCBA) promoter (sc-smCBA-mCherry) driving mCherry were packaged into AAV44.9, AAV5, and AAV8 (Y731F) in adherent HEK293T cells seeded in a 4% (60% by mass) cell growth area. Cells were harvested and lysed by continuous freeze-thaw cycles. Viruses in the lysate were purified by iodixanol density gradient and buffer exchanged into Alcon BSS supplemented with Tween 20 (0.014%). Viruses were titrated by qPCR against standards and stored at -80°C. The addition of Y731F and E531D substitutions was accomplished by site-directed mutagenesis of the AAV2rep-44.9cap plasmid and confirmed by Sanger sequencing. Additional constructs containing a cone-specific IRBPe-GNAT2 chimeric promoter driving green fluorescent protein (GFP) were packaged in AAV44.9.

[0142] In vitro transduction assay

[0143] ARPE-19 (human retinal pigment epithelial cell line) and 661W (mouse cone cell line) cells were cultured at 1.0×10 4The concentration of individual cells / well was inoculated in 96-well plates. The next day, cells were infected with 10,000p / cells. Three days after infection, fluorescence microscopy was performed under fixed exposure, and cells were separated and quantitatively reported protein expression (mCherry) by fluorescence using flow cytometry. mCherry expression is calculated by multiplying the average mCherry fluorescence by the number of positive cells. The chart only represents the expression level minus the cell level.

[0144] injection

[0145] 2 × 10 cells / ml were delivered intravitreally or subretinally to 4- to 5-week-old Nrl-GFP and C57BL / 6J mice. 9 At least six eyes that received successful injections were analyzed in each experiment.

[0146] Fundus examination

[0147] Fundus examinations were performed 4 weeks after injection using a Micron III camera (Phoenix Research Laboratories, Pleasanton, CA). Bright field and red fluorescence images were collected to visualize retinal health and mCherry expression, respectively. The exposure settings were constant between experiments and are indicated in the figure legends.

[0148] Measurement of retinal transduction by flow cytometry

[0149] The neural retinas of 4 to 6 Nrl-GFP eyes per group (with RPE manually peeled from the retina) were harvested and dissociated with papain. Flow cytometry was performed on the treated dissociated retinas and untreated controls to quantify the percentage of cells positive for GFP (rod photoreceptors), mCherry (non-rod retinal neurons transduced by rAAV), or both (rod photoreceptors transduced by rAAV). The percentages of rod and non-rod neural retinal cells transduced by each vector were averaged separately.

[0150] Tissue preparation and immunostaining

[0151] Four weeks after injection, eyes were enucleated and fixed overnight in freshly prepared 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) at 4°C. The cornea and lens were removed and the eye cups were incubated in 30% sucrose solution at 4°C overnight. Eyes were embedded in cryostat compound and frozen at -80°C. Sections (12 μm thick) were cut using a cryostat (Leica Microsystem, Buffalo Grove, IL) and transferred to slides. Retinal cryosections were rinsed with 1× phosphate-buffered saline (PBS), blocked with 0.5% Triton-X100 and 1% bovine serum albumin (BSA) for 1 hour each, and then incubated overnight at 4°C with mouse monoclonal anti-cone arrestin antibody (1:1000, kindly provided by Dr. Clay Smith). The next day, the slides were rinsed with 1× PBS and then incubated with Alexa Fluor donkey anti-mouse secondary antibody (1:500) in 1× PBS for 1 h at room temperature and counterstained with DAPI. Images were acquired using a confocal laser scanning microscope (Leica TCS SP8) and a fluorescence microscope (EVOS).

[0152] result

[0153] Rod transduction by AAV44.9 is higher than rod transduction by the benchmark vectors AAV5 and AAV8(Y731F)

[0154] Figures 2A to 2E Qualitative and quantitative analysis of AAV44.9, AAV5, and AAV8(Y731F) at 4 weeks after subretinal injection are shown. Figure 2E The FACS data depicted in Figure 2 show that subretinal injection of 2 × 10 9 After vg, AAV44.9 transduced rods more efficiently than AAV5 and AAV8(Y731F). Figures 3A to 3C Shown are representative retinal cross-sectional images showing mCherry expression in photoreceptors and RPE in the retinas of Nrl-GFP mice injected with AAV5, AAV8(Y731F), and AAV44.9 at 4 weeks after subretinal injection.

[0155] Qualitative and quantitative analysis of AAV44.9(Y731F) and AAV44.9(E531D) is shown in Figures 4A to 4C middle. Figure 4C The FACS data depicted in Figure 2 show that subretinal injection of 2 × 10 9 After vg, AAV44.9(E531D) transduced rod photoreceptors more efficiently than AAV44.9 and AAV44.9(Y731F). Figure 5A and Figure 5BRepresentative retinal cross-sectional images showing mCherry expression primarily in photoreceptors and RPE in the retinas of Nrl-GFP mice injected with AAV44.9(Y731F) and AAV44.9(E531D) 4 weeks after subretinal injection.

[0156] The transduction efficiency of unmodified AAV44.9 and AAV44.9(Y731F) in ocular cell lines is shown in Fig. 6A and Figure 6B AAV44.9(Y731F) showed enhanced transduction relative to AAV44.9 in the mouse cone photoreceptor cell line ( Fig. 6A ), while AAV44.9 was more effective than AAV9(Y731F) in human RPE cell lines ( Figure 6B ).

[0157] Qualitative analysis of AAV capsids after intravitreal injection showed Fig. 7A and Figure 7B Similar to AAV5 and AAV8(Y731F), AAV44.9 and its derivatives did not result in efficient retinal transduction after Ivt injection.

[0158] Fig. 8A AAV44.9 containing the cone-preferential IRBP / GNAT2 promoter and GFP reporter showed GFP expression in cones 4 weeks after SR injection. Figure 8B and Figure 8C Co-staining with cone arrestin antibody and colocalization with GFP fluorescence are also shown.

[0159] The results discussed above indicate that AAV44.9 transduces rods higher than that of unmodified AAV5 and AAV8(Y731F) via the benchmark vectors. Adding the Y731F mutation to AAV44.9 did not improve transduction of photoreceptors in vivo. The results also showed that the E531D mutation greatly enhanced photoreceptor transduction, with AAV44.9(E531D) showing 82% rod transduction compared to 61% rod transduction by unmodified AAV44.9. AAV44.9 efficiently transduces cones, as shown by subretinal delivery of AAV44.9-IRBPe / GNAT2-GFP. Similar to AAV5 and AAV8(Y731F), AAV44.9 and its derivatives do not efficiently transduce the retina following Ivt delivery.

[0160] After subretinal injection, AAV44.9(E531D) outperforms unmodified AAV44.9 and AAVrh.8 in the mouse retina

[0161] Representative fundus images (red fluorescence filter) of Nrl-GFP mice collected 4 weeks after subretinal injection of AAV44.9 or AAV44.9(E531D) (see Fig. 9A ). The vector was 2×10 9 vg delivery. Exposure and gain settings were consistent throughout the experiment. Experiments were repeated twice to confirm the results. Note that the "replicate" experiments were performed with different batches of virus.

[0162] Four weeks after injection, Nrl-GFP mice (from Fig. 9A The retinas of the same mice used in the experiments in , and flow cytometry was performed to quantify the percentage of transduced rods (GFP+mCherry positive) or non-rods (mCherry positive), as described in Boye et al., Impact of Heparan Sulfate Binding on Transduction of Retina by Recombinant Adeno-Associated Virus Vectors, J. Virol. 2016, 90(8): 4215-4231. The percentage of transduction and the corresponding values ​​for each cell population are shown in Fig. 9B Middle. AAV44.9(E531D) transduced a higher percentage of rod photoreceptors relative to unmodified AAV44.9. Note that "replicate" experiments were performed with different batches of virus.

[0163] Representative fundus images of Nrl-GFP mice collected 4 weeks after subretinal injection of AAVrh.8-mCherry (see Fig. 10A ). The vector was 2×10 9 vg delivery. Both GFP and mCherry filter images are included. AAVrh.8 was evaluated due to its structural similarity to both AAV44.9 and AAV44.9(E531D).

[0164] Four weeks after injection, Nrl-GFP mice (from Fig. 9A and Fig. 10A The retinas of the same mice used in the experiments in , and flow cytometry was performed to quantify the percentage of transduced rods (GFP+mCherry positive) or non-rods (mCherry positive). The percentage of transduction and the corresponding values ​​for each cell population are shown in Fig. 10B AAV44.9(E531D) transduced a higher percentage of rod photoreceptors relative to unmodified AAV44.9 or AAVrh.8.

[0165] Representative fundus images of Nrl-GFP mice collected 4 weeks after subretinal injection of low titers of scAAV44.9, scAAV44.9(E531D), or scAAVrh.8 (see Fig.11A ). The vector was 2×10 8 VG delivery. Exposure and gain settings were consistent throughout the experiment.

[0166] Four weeks after injection, Nrl-GFP mice (from Fig.11A The retinas of the same mice used in the experiments in , and flow cytometry was performed to quantify transduced rods (GFP+mCherry positive) or non-rods (mCherry positive). The percentage of transduction and the corresponding values ​​for each cell population are shown in Fig. 11B AAV44.9(E531D) transduced a higher percentage of rod photoreceptors relative to unmodified AAV44.9 or AAVrh.8.

[0167] Qualitative and quantitative analysis of AAVrh.8, AAV44.9, and AAV44.9(E531D) at 4 weeks after subretinal injection showed Fig. 9A and 9B middle. Fig. 9B Depicted is a subretinal injection of 2 × 10 12 FACS plots showing that AAV44.9(E531D) transduces rod photoreceptors more efficiently than AAV44.9 after vg.

[0168] Fig.11A and 11B Qualitative and quantitative analysis of lower titers of AAV44.9, AAV44.9(E531D), and AAVrh.8 at 4 weeks after subretinal injection is shown in . Fig. 11B Depicted is a subretinal injection of 2 × 10 11 FACS plots showing that AAV44.9(E531D) transduced rod photoreceptors more efficiently than AAV44.9(E531D) and AAVrh.8 after vg. Fig.12 Shown are qualitative analyses of AAV44.9(E531D) and AAV44.9 6 weeks after subretinal injection in the presence of the cone-specific IRBPe-GNAT2 chimeric promoter.

[0169] Fig.13 Representative retinal cross-sections of WT mice taken 6 weeks after subretinal injection of AAV44.9(E531D)-IRBP / GNAT2-hGFP are shown. The IRBP / GNAT2 promoter is a cone-specific promoter. The vector was expressed as 2×10 12vg delivery. Sections were immunostained with antibodies against GFP (green) and cone arrestin (red). AAV-mediated GFP expression colocalized with cone arrestin, a cone-specific marker, confirming that the vector efficiently transduced cones. Fig.13 Representative retinal cross-sectional images in show cone arrestin expression in photoreceptors and RPE in the retina of Nrl-GFP mice injected with AAV44.9(E531D) and indicate that AAV44.9(E531D) transduces cones very efficiently.

[0170] Representative retinal cross-sections of WT mice were collected 6 weeks after subretinal injection of AAV44.9-IRBP / GNAT2-hGFP (see Fig.14 ). The IRBP / GNAT2 promoter is a cone-specific promoter. The vector was added at 2×10 12 vg delivery. Sections were immunostained with antibodies against GFP (green) and cone arrestin (red). AAV-mediated GFP expression colocalized with cone arrestin, a cone-specific marker, confirming that the vector efficiently transduced cones.

[0171] Example 2 - Enhanced lateral spread and foveal transduction following subretinal administration of AAV44.9(E531D)-HGRK1-GFP in macaques

[0172] The human rhodopsin kinase (hGRK1) promoter has previously been determined to have exclusive activity in non-human primate rods and cones. Therefore, the ability of the hGRK1 promoter to drive expression of a GFP reporter in macaque eyes in a modified AAV44.9 (E531D) vector was evaluated. The extent of lateral spread from the initial bleb boundary was also evaluated.

[0173] Two rAAV vectors, AAV44.9-hGRK1-GFP and AAV44.9(E531D)-hGRK1-GFP, were administered subretinal to macaques. The vectors were expressed at 1×10 12 A control vector, AAV5-hGRK1-GFP, was also administered to the eye.

[0174] Particles incorporating both modified and unmodified AAV44.9 vectors exhibited enhanced lateral spread and efficacy in subretinal injected macaque subjects (see Fig.15 The initial boundary of the bubble and the boundary of the resulting GFP expression are Fig.15 The same vasculature is highlighted with a bold dark line for reference. AAV44.9(E531D)-mediated GFP expression was visible 1 week after injection. Both AAV44.9 and AAV44.9(E531D) were injected at 1×1012 vg / mL doses were well tolerated in the primate retina. The control vector AAV5 mediated GFP confinement that remained sequestered within the initial injection bleb.

[0175] AAV44.9-hGRK1-GFP (concentration 1×10 12 vg / mL) was injected subretinaly outside the fovea. OCT scans revealed no foveal detachment during the injection ( Fig.16 ). Even without foveal detachment, this extrafoveal subretinal injection transduced even 98% of foveal cones and 100% of central rods (see Fig.17 (upper right of ). The capsid exhibits enhanced lateral diffusion because the boundary of the bubble expands relative to the initial boundary.

[0176] Images were also captured from macaque eyes injected with AAV44.9(E531D)-hGRK1-GFP. Qualitative analysis of monocular images revealed that approximately 50% of foveal cone transduction was mediated by AAV44.9(E531D).

[0177] Three subretinal injections (30 μL each) of AAV44.9(E531D)-hGRK1-GFP were performed in the superior, temporal, and inferior retina outside the fovea of ​​macaque eyes. Retinal sections were stained with antibodies against cone arrestin, and three blinded observers counted GFP-positive cones and rods in five retinal fields in a single plane across the fovea. The results of this administration are shown in Fig.19 OCT scans revealed no detachment of the fovea during injection (see Fig.19 (right image).

[0178] These results show that extrafoveal subretinal injection of AAV44.9(E531D)-hGRK1-GFP in macaques exhibits significant transduction of central cones and rods in the absence of foveal detachment. Peripheral rods and cones were also very efficiently transduced. Thus, extrafoveal subretinal injection results in highly efficient transduction over the entire foveal region.

[0179] like Figures 20A to 20DAs shown, examination of the parafovea after this injection revealed that AAV44.9 (E531D) particles transduced parafoveal cones located both nasal and temporal to the fovea. However, it is noteworthy that parafoveal cone transduction was not achieved with unmodified AAV44.9. This finding is interesting at least because i) parafoveal cones are resistant to transduction by a variety of AAV capsid variants, and ii) the earliest structural and functional losses due to aging and inherited retinal diseases often occur in the parafoveal region. Despite their different abilities to transduce cones in this region, modified and unmodified AAV44.9 vectors effectively transduce parafoveal rods to essentially the same extent.

[0180] like Figures 21A to 21B As shown, examination of the perifoveal region revealed a similar pattern: particles incorporating AAV44.9(E531D) capsids transduced perifoveal cones, but unmodified AAV44.9 did not. Perifoveal rods were efficiently transduced by both capsids in this region. The perifoveal region surrounds the parafoveal region.

[0181] These results further demonstrate that the enhanced lateral spread of transduction provided by the improved AAV44.9(E531D) capsid variant vectors may allow subretinal injections in the parafoveal region to produce transduction of foveal cells while avoiding the deleterious effects of inducing foveal detachment in human subjects.

[0182] Example 3 - Enhanced lateral diffusion following subretinal administration of AAV44.9-GUCY2E-GFP in mice

[0183] The previous example demonstrated foveal transduction of a reporter gene delivered by an improved AAV44.9(E531D) vector. Next, the extent of lateral diffusion following administration of an improved AAV44.9(E531D) vector encoding a therapeutic peptide of interest was determined. The selected therapeutic peptide of interest was Gucy2e, a murine homolog of human guanylate cyclase 2D GUCY2D.

[0184] Two vectors, AAV44.9(E531D)-hGRK1-Gucy2e and AAV8(Y731F)-hGRK1-Gucy2e, were administered subretinally into the eyes of retinal guanylate cyclase 1 / 2 double knockout (GCdko) mice. GCdko mice completely lack retinal function. Since there is no functional retinal guanylate cyclase in these photoreceptors, neither rods nor cones can respond to light. Therefore, gene replacement therapy in GCdko mice can evaluate whether gene replacement / supplementation is successful in both rods and cones. The vectors were administered at 1×10 13vg / mL concentration delivery. The response function of treated and untreated eyes was evaluated by electroretinogram (ERG) measurement. The ERG response from retinal photoreceptor cells is called "a wave", and the electrical response from retinal bipolar cells is called "b wave".

[0185] like Fig.18B As shown, the light response function of cones from eyes treated with AAV vectors was improved relative to that of untreated eyes. These results indicate that both types of photoreceptors are effectively targeted by rAAV particles incorporated into AAV44.9(E531D) capsids and AAV44.9(Y731F) capsids. Fig.18A As shown, after administration of AAV(Y731F) vector, the maximum a and b wave amplitudes were greater than those of AAV44.9(E531D) vector.

[0186] Another major implication of this experiment is that clinical candidate rAAV vectors expressing therapeutic peptides designed to treat inherited retinal diseases (e.g., vectors for delivering the human GUCY2D transgene) incorporated into AAV44.9(E531D) are likely to function in corresponding mouse disease models (e.g., vectors for delivering murine Gucy2e). This is advantageous in situations where multiple batches of a candidate are being developed and evaluated preclinically during multiple stages or phases of manufacturing.

[0187] Other Implementations

[0188] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that those skilled in the art may propose various modifications or variations based thereon, and that these modifications or variations are included in the spirit and scope of the present application and within the scope of the appended claims. All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and set forth herein in its entirety. Unless otherwise indicated herein, references to numerical ranges herein are intended only to be used as a shorthand method of individually referring to each individual value falling within the range, and each individual value is incorporated into the specification as if it were individually cited herein.

[0189] Unless otherwise stated or clearly contradicted by context, descriptions of any aspect or embodiment of the invention herein using terms such as “comprising,” “having,” “including,” or “containing” in reference to one or more elements are intended to provide support for similar aspects or embodiments of the invention “consisting of,” “consisting essentially of,” or “essentially consisting of” the one or more specified elements (e.g., a composition described herein as comprising a specified element is to be understood to also describe a composition consisting of that element, unless stated otherwise or clearly contradicted by context).

[0190] In light of the present disclosure, all compositions and methods disclosed and claimed herein can be prepared and implemented without undue experimentation. Although the compositions and methods of the present invention have been described according to some preferred embodiments, it is obvious to those skilled in the art that the compositions and methods described herein and the steps or step sequences of the methods can be changed without departing from the concept, spirit and scope of the present invention. More specifically, it is obvious that certain chemical and / or physiologically related agents can replace the agents described herein while obtaining the same or similar results. All of these similar substitutions and modifications that are obvious to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined by the appended claims. Sequence Listing <110> UNIVERSITY OF FLORIDA RESEARCH FOUNDATION, INCORPORATED <120> Efficient transduction and lateral spread of a novel AAV virus enhanced by rational design in the retina <130> U1202.70037WO00 <140> Not Yet Assigned <141> Concurrently Herewith <150> US 62 / 795,695 <151> 2019-01-23 <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 736 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 1 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly 145 150 155 160 Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190 Ala Ala Pro Ser Gly Leu Gly Pro Asn Thr Met Ala Ser Gly Gly Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ser Thr Asn Asp Asn 260 265 270 Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285 Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300 Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile 305 310 315 320 Gln Val Lys Glu Val Thr Thr Asn Glu Gly Thr Lys Thr Ile Ala Asn 325 330 335 Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu 340 345 350 Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro 355 360 365 Ala Asp Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn 370 375 380 Gly Ser Gln Ala Leu Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe 385 390 395 400 Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Thr 405 410 415 Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430 Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Val 435 440 445 Arg Thr Gln Thr Thr Gly Thr Gly Gly Thr Gln Thr Leu Ala Phe Ser 450 455 460 Gln Ala Gly Pro Ser Ser Met Ala Ser Gln Ala Arg Asn Trp Val Pro 465 470 475 480 Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Thr Asn Gln Asn 485 490 495 Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala Ala Lys Phe Lys Leu Asn 500 505 510 Gly Arg Asp Ser Leu Met Asn Pro Gly Val Ala Met Ala Ser His Lys 515 520 525 Asp Asp Asp Asp Arg Phe Phe Pro Ser Ser Gly Val Leu Ile Phe Gly 530 535 540 Lys Gln Gly Ala Gly Asn Asp Gly Val Asp Tyr Ser Gln Val Leu Ile 545 550 555 560 Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Glu 565 570 575 Tyr Gly Ala Val Ala Ile Asn Asn Gln Ala Ala Asn Thr Gln Ala Gln 580 585 590 Thr Gly Leu Val His Asn Gln Gly Val Ile Pro Gly Met Val Trp Gln 595 600 605 Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620 Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu 625 630 635 640 Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650 655 Asp Pro Pro Leu Thr Phe Asn Gln Ala Lys Leu Asn Ser Phe Ile Thr 660 665 670 Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685 Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn 690 695 700 Tyr Tyr Lys Ser Thr Asn Val Asp Phe Ala Val Asn Thr Glu Gly Val 705 710 715 720 Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 735 <210> 2 <211> 599 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 2 Met Ala Pro Gly Lys Lys Arg Pro Val Glu Gln Ser Pro Gln Glu Pro 1 5 10 15 Asp Ser Ser Ser Gly Ile Gly Lys Thr Gly Gln Gln Pro Ala Lys Lys 20 25 30 Arg Leu Asn Phe Gly Gln Thr Gly Asp Thr Glu Ser Val Pro Asp Pro 35 40 45 Gln Pro Leu Gly Glu Pro Pro Ala Ala Pro Ser Gly Leu Gly Pro Asn 50 55 60 Thr Met Ala Ser Gly Gly Gly Ala Pro Met Ala Asp Asn Asn Glu Gly 65 70 75 80 Ala Asp Gly Val Gly Asn Ser Ser Gly Asn Trp His Cys Asp Ser Thr 85 90 95 Trp Leu Gly Asp Arg Val Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu 100 105 110 Pro Thr Tyr Asn Asn His Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser 115 120 125 Gly Gly Ser Thr Asn Asp Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp 130 135 140 Gly Tyr Phe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp 145 150 155 160 Trp Gln Arg Leu Ile Asn Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu 165 170 175 Asn Phe Lys Leu Phe Asn Ile Gln Val Lys Glu Val Thr Thr Asn Glu 180 185 190 Gly Thr Lys Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe 195 200 205 Thr Asp Ser Glu Tyr Gln Leu Pro Tyr Val Leu Gly Ser Ala His Gln 210 215 220 Gly Cys Leu Pro Pro Phe Pro Ala Asp Val Phe Met Val Pro Gln Tyr 225 230 235 240 Gly Tyr Leu Thr Leu Asn Asn Gly Ser Gln Ala Leu Gly Arg Ser Ser 245 250 255 Phe Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr Gly Asn 260 265 270 Asn Phe Gln Phe Ser Tyr Thr Phe Glu Asp Val Pro Phe His Ser Ser 275 280 285 Tyr Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp 290 295 300 Gln Tyr Leu Tyr Tyr Leu Val Arg Thr Gln Thr Thr Gly Thr Gly Gly 305 310 315 320 Thr Gln Thr Leu Ala Phe Ser Gln Ala Gly Pro Ser Ser Met Ala Ser 325 330 335 Gln Ala Arg Asn Trp Val Pro Gly Pro Ser Tyr Arg Gln Gln Arg Val 340 345 350 Ser Thr Thr Thr Asn Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Gly 355 360 365 Ala Ala Lys Phe Lys Leu Asn Gly Arg Asp Ser Leu Met Asn Pro Gly 370 375 380 Val Ala Met Ala Ser His Lys Asp Asp Asp Asp Arg Phe Phe Pro Ser 385 390 395 400 Ser Gly Val Leu Ile Phe Gly Lys Gln Gly Ala Gly Asn Asp Gly Val 405 410 415 Asp Tyr Ser Gln Val Leu Ile Thr Asp Glu Glu Glu Ile Lys Ala Thr 420 425 430 Asn Pro Val Ala Thr Glu Glu Tyr Gly Ala Val Ala Ile Asn Asn Gln 435 440 445 Ala Ala Asn Thr Gln Ala Gln Thr Gly Leu Val His Asn Gln Gly Val 450 455 460 Ile Pro Gly Met Val Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro 465 470 475 480 Ile Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His Pro Ser Pro 485 490 495 Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile 500 505 510 Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Leu Thr Phe Asn Gln Ala 515 520 525 Lys Leu Asn Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val 530 535 540 Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro 545 550 555 560 Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser Thr Asn Val Asp Phe 565 570 575 Ala Val Asn Thr Glu Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr 580 585 590 Arg Tyr Leu Thr Arg Asn Leu 595 <210> 3 <211> 534 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 3 Met Ala Ser Gly Gly Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala 1 5 10 15 Asp Gly Val Gly Asn Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp 20 25 30 Leu Gly Asp Arg Val Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro 35 40 45 Thr Tyr Asn Asn His Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly 50 55 60 Gly Ser Thr Asn Asp Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly 65 70 75 80 Tyr Phe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp Trp 85 90 95 Gln Arg Leu Ile Asn Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn 100 105 110 Phe Lys Leu Phe Asn Ile Gln Val Lys Glu Val Thr Thr Asn Glu Gly 115 120 125 Thr Lys Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr 130 135 140 Asp Ser Glu Tyr Gln Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly 145 150 155 160 Cys Leu Pro Pro Phe Pro Ala Asp Val Phe Met Val Pro Gln Tyr Gly 165 170 175 Tyr Leu Thr Leu Asn Asn Gly Ser Gln Ala Leu Gly Arg Ser Ser Phe 180 185 190 Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn 195 200 205 Phe Gln Phe Ser Tyr Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr 210 215 220 Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln 225 230 235 240 Tyr Leu Tyr Tyr Leu Val Arg Thr Gln Thr Thr Gly Thr Gly Gly Thr 245 250 255 Gln Thr Leu Ala Phe Ser Gln Ala Gly Pro Ser Ser Met Ala Ser Gln 260 265 270 Ala Arg Asn Trp Val Pro Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser 275 280 285 Thr Thr Thr Asn Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala 290 295 300 Ala Lys Phe Lys Leu Asn Gly Arg Asp Ser Leu Met Asn Pro Gly Val 305 310 315 320 Ala Met Ala Ser His Lys Asp Asp Asp Asp Arg Phe Phe Pro Ser Ser 325 330 335 Gly Val Leu Ile Phe Gly Lys Gln Gly Ala Gly Asn Asp Gly Val Asp 340 345 350 Tyr Ser Gln Val Leu Ile Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn 355 360 365 Pro Val Ala Thr Glu Glu Tyr Gly Ala Val Ala Ile Asn Asn Gln Ala 370 375 380 Ala Asn Thr Gln Ala Gln Thr Gly Leu Val His Asn Gln Gly Val Ile 385 390 395 400 Pro Gly Met Val Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile 405 410 415 Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu 420 425 430 Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys 435 440 445 Asn Thr Pro Val Pro Ala Asp Pro Pro Leu Thr Phe Asn Gln Ala Lys 450 455 460 Leu Asn Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu 465 470 475 480 Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu 485 490 495 Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser Thr Asn Val Asp Phe Ala 500 505 510 Val Asn Thr Glu Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg 515 520 525 Tyr Leu Thr Arg Asn Leu 530 <210> 4 <211> 2211 <212> DNA <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 4 atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60 gagtggtggg acttgaaacc tggagccccg aaacccaaag ccaaccagca aaagcaggac 120 gacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180 aagggggagc ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 240 cagcagctca aagcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300 caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360 gccaagaagc gggttctcga acctctcggt ctggttgagg aaggcgctaa gacggctcct 420 ggaaagaaga gaccggtaga gcagtcaccc caagaaccag actcctcatc gggcatcggc 480 aagacaggcc agcagcccgc taaaaagaga ctcaattttg gtcagactgg cgacacagag 540 tcagtccccg acccacaacc tctcggagaa cctccagcag ccccctcagg tctgggacct 600 aatacaatgg cttcaggcgg tggcgctcca atggcagaca ataacgaagg cgccgacgga 660 gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggctggggga cagagtcatc 720 accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caagcaaatc 780 tccaacggca cctcgggagg aagcaccaac gacaacacct actttggcta cagcacccccc 840 tggggtatt ttgacttcaa cagattccac tgccactttt caccacgtga ctggcagcga 900 ctcatcaaca acaattgggg attccggccc aagagactca acttcaagct cttcaacatc 960 caggtcaagg aagtcacgac gaacgaaggc accaagacca tcgccaataa tctcaccagc 1020 accgtgcagg tctttacgga ctcggagtac cagctaccgt acgtgctagg atcagctcac 1080 cagggatgtc tgcctccgtt cccggcggac gtcttcatgg ttcctcagta cggttatcta 1140 actctgaaca atggcagcca ggccctggga cgttcctcct tctactgcct ggagtatttc 1200 ccatcgcaga tgctgagaac cggcaacaac tttcagttca gctacacctt cgaggacgtg 1260 cctttccaca gcagctacgc gcacagccaa agcctggaca ggctgatgaa tcccctcatc 1320 gaccagtacc tgtattacct ggtcagaacg cagaaacg ggactggagg gacgcagact 1380 ctggcattca gccaagcagg ccctagctca atggccagcc aggctagaaa ctgggtgccc 1440 ggaccgagct accggcagcagca gcgcgtctcc acgacaacca accagaacaa caacagcaac 1500 tttgcctgga cgggagctggc caaatttaaa ctgaacggcc gagactctct aatgaacccc 1560 ggcgtggcca tggcttcaca caaggatgac gatgaccggt tcttcccttc tagcggggtc 1620 ctgattttcg gcaagcaagg agccgggaat gatggagtgg attacagcca agtgctgatt 1680 acagatgagg aagaaatcaa ggctaccaac cccgtggcaa cagaggaata tggagcagtg 1740 gccatcaaca accaggccgc taatacgcag gcgcagaccg gactcgtgca caccagggg 1800 gtgattcccg gcatggtgtg gcaagaaga gacgtgtacc tgcagggtcc catctgggcc 1860 aaaattcctc acacggacgg caactttcac ccgtctcccc tgatgggcgg ctttggactg 1920 aagcacccgc ctcctcaaat tctcatcaag aacacaccgg ttccagcgga cccgccgctt 1980 accttcaacc aggccaagct gaactctttc atcacgcagt acagcaccgg acaggtcagc 2040 gtggaaatcg agtgggagct gcagaaagaa aacagcaaac gctggaatcc agagattcag 2100 tacacttcca actactacaa atctacaaat gtggactttg ctgtcaacac ggaaggagtg 2160 tatagcgagc ctcgccccat tggcacgcgc tacctcaccc gtaatctgta a 2211 <210> 5 <211> 1800 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 5 acggctcctg gaaagaagag accggtagag cagtcacccc aagaaccaga ctcctcatcg 60 ggcatcggca agacaggcca gcagcccgct aaaaagagac tcaattttgg tcagactggc 120 gacacagagt cagtccccga cccacaacct ctcggagaac ctccagcagc cccctcaggt 180 ctgggaccta atacaatggc ttcaggcggt ggcgctccaa tggcagacaa taacgaaggc 240 gccgacggag tgggtaattc ctcgggaaat tggcattgcg attccacatg gctgggggac 300 agagtcatca ccaccagcac ccgaacctgg gccctgccca cctacaacaa ccacctctac 360 aagcaaatct ccaacggcac ctcgggagga agcaccaacg acaacaccta ctttggctac 420 agcaccccct gggggtattt tgacttcaac agattccact gccacttttc accacgtgac 480 tggcagcgac tcatcaacaa caattgggga ttccggccca agagactcaa cttcaagctc 540 ttcaacatcc aggtcaagga agtcacgacg aacgaaggca ccaagaccat cgccaataat 600 ctcaccagca ccgtgcaggt ctttacggac tcggagtacc agctaccgta cgtgctagga 660 tcagctcacc agggatgtct gcctccgttc ccggcggacg tcttcatggt tcctcagtac 720 ggttatctaa ctctgaacaa tggcagccag gccctgggac gttcctcctt ctactgcctg 780 gagtatttcc catcgcagat gctgagaacc ggcaacaact ttcagttcag ctacaccttc 840 gaggacgtgc ctttccacag cagctacgcg cacagccaaa gcctggacag gctgatgaat 900 cccctcatcg accagtacct gtattacctg gtcagaacgc agacaaccgg gactggaggg 960 acgcagactc tggcattcag ccaagcaggc cctagctcaa tggccagcca ggctagaaac 1020 tgggtgcccg gaccgagcta ccggcagcag cgcgtctcca cgacaaccaa ccagaacaac 1080 aacagcaact ttgcctggac gggagctgcc aaatttaaac tgaacggccg agactctcta 1140 atgaaccccg gcgtggccat ggcttcacac aaggatgacg atgaccggtt cttcccttct 1200 agcggggtcc tgattttcgg caagcaagga gccgggaatg atggagtgga ttacagccaa 1260 gtgctgatta cagatgagga agaaatcaag gctaccaacc ccgtggcaac agaggaatat 1320 ggagcagtgg ccatcaacaa ccaggccgct aatacgcagg cgcagaccgg actcgtgcac 1380 aaccaggggg tgattcccgg catggtgtgg cagaacagag acgtgtacct gcagggtccc 1440 atctgggcca aaattcctca cacggacggc aactttcacc cgtctcccct gatgggcggc 1500 tttggactga agcacccgcc tcctcaaatt ctcatcaaga acacaccggt tccagcggac 1560 ccgccgctta ccttcaacca ggccaagctg aactctttca tcacgcagta cagcaccgga 1620 caggtcagcg tggaaatcga gtgggagctg cagaaagaaa acagcaaacg ctggaatcca 1680 gagattcagt acacttccaa ctactacaaa tctacaaatg tggactttgc tgtcaacacg 1740 gaaggagtgt atagcgagcc tcgccccatt ggcacgcgct acctcacccg taatctgtaa 1800 gaaggagtgt atagcgagcc tcgccccatt ggcacgcgct acctcacccg taatctgtaa 1800 <210> 6<210> 6 <211> 1605<211> 1605 <212> DNA<212> DNA <213> Artificial sequence<213> Artificial sequence <220><220> <223> Synthetic polypeptide<223> Synthetic polypeptide <400> 6<400> 6 atggcttcag gcggtggcgc tccaatggca gacaataacg aaggcgccga cggagtgggt 60 atggcttcag gcggtggcgc tccaatggca gacaataacg aaggcgccga cggagtgggt 60 aattcctcgg gaaattggca ttgcgattcc acatggctgg gggacagagt catcaccacc 120 aattcctcgg gaaattggca ttgcgattcc acatggctgg gggacagagt catcaccacc 120 agcacccgaa cctgggccct gcccacctac aacaaccacc tctacaagca aatctccaac 180 agcacccgaa cctgggccct gcccacctac aacaaccacc tctacaagca aatctccaac 180 ggcacctcgg gaggaagcac caacgacaac acctactttg gctacagcac cccctggggg 240 ggcacctcgg gaggaagcac caacgacaac acctactttg gctacagcac cccctggggg 240 tattttgact tcaacagatt ccactgccac ttttcaccac gtgactggca gcgactcatc 300 tattttgact tcaacagatt ccactgccac ttttcaccac gtgactggca gcgactcatc 300 aacaacaatt ggggattccg gcccaagaga ctcaacttca agctcttcaa catccaggtc 360 aacaacaatt ggggattccg gcccaagaga ctcaacttca agctcttcaa catccaggtc 360 aaggaagtca cgacgaacga aggcaccaag accatcgcca ataatctcac cagcaccgtg 420 aaggaagtca cgacgaacga aggcaccaag accatcgcca ataatctcac cagcaccgtg 420 caggtcttta cggactcgga gtaccagcta ccgtacgtgc taggatcagc tcaccaggga 480 caggtcttta cggactcgga gtaccagcta ccgtacgtgc taggatcagc tcaccaggga 480 tgtctgcctc cgttcccggc ggacgtcttc atggttcctc agtacggtta tctaactctg 540 tgtctgcctc cgttcccggc ggacgtcttc atggttcctc agtacggtta tctaactctg 540 aacaatggca gccaggccct gggacgttcc tccttctact gcctggagta tttcccatcg 600 cagatgctga gaaccggcaa caactttcag ttcagctaca ccttcgagga cgtgcctttc 660 cacagcagct acgcgcacag ccaaagcctg gacaggctga tgaatcccct catcgaccag 720 tacctgtatt acctggtcag aacgcagaca accgggactg gagggacgca gactctggca 780 ttcagccaag caggccctag ctcaatggcc agccaggcta gaaactgggt gcccggaccg 840 agctaccggc agcagcgcgt ctccaccgaca accaaccaga acaacacag caactttgcc 900 tggacgggag ctgccaaatt taaactgaac ggccgagact ctcttaatgaa ccccggcgtg 960 gccatggctt cacaagga tgacgatgac cggttcttcc cttctagcgg ggtcctgatt 1020 ttcggcaagc aaggagccgg gaatgatgga gtggattaca gccaagtgct gattacagat 1080 ggagaaa tcaaggctac caaccccgtg gcaacagagg aatatggagc agtggccatc 1140 aacaaccagg ccgcttaatac gcaggcgcag accggactcg tgcacaacca gggggtgatt 1200 cccggcatgg tgtggcagaa cagagacgtg tacctgcagg gtcccatctg ggccaaaatt 1260 cctcacacgg acggcaactt tcacccgtct cccctgatgg gcggctttgg actgaagcac 1320 ccgcctcctc aaattctcat caagaacaca ccggttccag cggacccgcc gcttaccttc 1380 aaccaggcca agctgaactc tttcatcacg cagtacagca ccggacaggt cagcgtggaa 1440 atcgagtggg agctgcagaa agaaaacagc aaacgctgga atccagagat tcagtacact 1500 tccaactact acaaatctac aaatgtggac tttgctgtca acacggaagg agtgtatagc 1560 gagcctcgcc ccattggcac gcgctacctc acccgtaatc tgtaa 1605 <210> 7 <211> 736 <212> PRT <213> Unknown <220> <223> AAV44.9 VP1 <400> 7 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly 145 150 155 160 Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190 Ala Ala Pro Ser Gly Leu Gly Pro Asn Thr Met Ala Ser Gly Gly Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ser Thr Asn Asp Asn 260 265 270 Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285 Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300 Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile 305 310 315 320 Gln Val Lys Glu Val Thr Thr Asn Glu Gly Thr Lys Thr Ile Ala Asn 325 330 335 Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu 340 345 350 Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro 355 360 365 Ala Asp Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn 370 375 380 Gly Ser Gln Ala Leu Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe 385 390 395 400 Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Thr 405 410 415 Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430 Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Val 435 440 445 Arg Thr Gln Thr Thr Gly Thr Gly Gly Thr Gln Thr Leu Ala Phe Ser 450 455 460 Gln Ala Gly Pro Ser Ser Met Ala Ser Gln Ala Arg Asn Trp Val Pro 465 470 475 480 Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Thr Asn Gln Asn 485 490 495 Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala Ala Lys Phe Lys Leu Asn 500 505 510 Gly Arg Asp Ser Leu Met Asn Pro Gly Val Ala Met Ala Ser His Lys 515 520 525 Asp Asp Glu Asp Arg Phe Phe Pro Ser Ser Gly Val Leu Ile Phe Gly 530 535 540 Lys Gln Gly Ala Gly Asn Asp Gly Val Asp Tyr Ser Gln Val Leu Ile 545 550 555 560 Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Glu 565 570 575 Tyr Gly Ala Val Ala Ile Asn Asn Gln Ala Ala Asn Thr Gln Ala Gln 580 585 590 Thr Gly Leu Val His Asn Gln Gly Val Ile Pro Gly Met Val Trp Gln 595 600 605 Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620 Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu 625 630 635 640 Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650 655 Asp Pro Pro Leu Thr Phe Asn Gln Ala Lys Leu Asn Ser Phe Ile Thr 660 665 670 Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685 Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn 690 695 700 Tyr Tyr Lys Ser Thr Asn Val Asp Phe Ala Val Asn Thr Glu Gly Val 705 710 715 720 Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 735 <210> 8 <211> 2211 <212> DNA <213> Unknown <220> <223> AAV44.9 VP1 <400> 8 atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60 gagtggtggg acttgaaacc tggagccccg aaacccaaag ccaaccagca aaagcaggac 120 gacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180 aagggggagc ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 240 cagcagctca aagcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300 caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360 gccaagaagc gggttctcga acctctcggt ctggttgagg aaggcgctaa gacggctcct 420 ggaaaagaa gaccggtaga gcagtcaccc caagaaccag actcctcatc gggcatcggc 480 aagacaggcc agcagcccgc taaaaagaga ctcaattttg gtcagactgg cgacacagag 540 tcagtccccg acccacaacc tctcggagaa cctccagcag ccccctcagg tctgggacct 600 aatacaatgg cttcaggcgg tggcgctcca atggcagaca ataacgaagg cgccgacgga 660 gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggctggggga cagagtcatc 720 accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caagcaaatc 780 tccaacggca cctcgggagg aagcaccaac gacaacacct actttggcta cagcacccccc 840 tggggtatt ttgacttcaa cagattccac tgccactttt caccacgtga ctggcagcga 900 ctcatcaaca acaattgggg attccggccc aagagactca acttcaagct cttcaacatc 960 caggtcaagg aagtcacgac gaacgaaggc accaagacca tcgccaataa tctcaccagc 1020 accgtgcagg tctttacgga ctcggagtac cagctaccgt acgtgctagg atcagctcac 1080 cagggatgtc tgcctccgtt cccggcggac gtcttcatgg ttcctcagta cggttatcta 1140 actctgaaca atggcagcca ggccctggga cgttcctcct tctactgcct ggagtatttc 1200 ccatcgcaga tgctgagaac cggcaacaac tttcagttca gctacacctt cgaggacgtg 1260 cctttccaca gcagctacgc gcacagccaa agcctggaca ggctgatgaa tcccctcatc 1320 gaccagtacc tgtattacct ggtcagaacg cagaaacg ggactggagg gacgcagact 1380 ctggcattca gccaagcagg ccctagctca atggccagcc aggctagaaa ctgggtgccc 1440 ggaccgagct accggcagcagca gcgcgtctcc acgacaacca accagaacaa caacagcaac 1500 tttgcctgga cgggagctggc caaatttaaa ctgaacggcc gagactctct aatgaacccc 1560 ggcgtggcca tggcttcaca caaggatgac gaggaccgct tcttcccttc tagcggggtc 1620 ctgattttcg gcaagcaagg agccgggaat gatggagtgg attacagcca agtgctgatt 1680 acagatgagg aagaaatcaa ggctaccaac cccgtggcaa cagaggaata tggagcagtg 1740 gccatcaaca accaggccgc taatacgcag gcgcagaccg gactcgtgca caccagggg 1800 gtgattcccg gcatggtgtg gcagaacaga gacgtgtacc tgcagggtcc catctgggcc 1860 aaaattcctc acacggacgg caactttcac ccgtctcccc tgatgggcgg ctttggactg 1920 aagcacccgc ctcctcaaat tctcatcaag aacacaccgg ttccagcgga cccgccgctt 1980 accttcaacc aggccaagct gaactctttc atcacgcagt acagcaccgg acaggtcagc 2040 gtggaaatcg agtgggagct gcagaaagaa aacagcaaac gctggaatcc agagattcag 2100 tacacttcca actactacaa atctacaaat gtggactttg ctgtcaacac ggaaggagtg 2160 tatagcgagc ctcgccccat tggcacgcgc tacctcaccc gtaatctgta a 2211 <210> 9 <211> 3312 <212> DNA <213> Homo sapiens <400> 9 atgaccgcct gcgcccgccg agcgggtggg cttccggacc ccgggctctg cggtcccgcg 60 tggtgggctc cgtccctgcc ccgcctcccc cgggccctgc cccggctccc gctcctgctg 120 ctcctgcttc tgctgcagcc ccccgccctc tccgccgtgt tcacggtggg ggtcctgggc 180 ccctgggctt gcgaccccat cttctctcgg gctcgccccgg acctggccgc ccgcctggcc 240 gccgccccgcc tgaaccgcga ccccggcctg gcaggcggtc cccgcttcga ggtagcgctg 300 ctgcccgagc cttgccggac gccgggctcg ctggggccg tgtcctccgc gctggcccgc 360 gtgtcgggcc tcgtgggtcc ggtgaaccct gcggcctgcc ggccagccga gctgctcgcc 420 gaaagccg ggatcgcgct ggtgccctgg ggctgcccct ggacgcaggc ggagggcacc 480 acggcccctg ccgtgacccc cgccgcggat gccctctacg ccctgcttcg cgcattcggc 540 tgggcgcgcg tggccctggt caccgccccc caggacctgt gggtggaggc gggacgctca 600 ctgtccacgg cactcagggc ccggggcctg cctgtcgcct ccgtgacttc catggagccc 660 ttggacctgt ctggagcccg ggaggccctg aggaaggttc gggacgggcc cagggtcaca 720 gcagtgatca tggtgatgca ctcggtgctg ctgggtggcg aggagcagcg ctacctcctg 780 gaggccgcag aggagctgggg cctgaccgat ggctccctgg tcttcctgcc cttcgacacg 840 atccactacg ccttgtcccc aggcccggag gccttggccg cactcgccaa cagctcccag 900 cttcgcaggg cccacgatgc cgtgctcacc ctcacgcgcc actgtccctc tgaaggcagc 960 gtgctggaca gcctgcgcag ggctcaagag cgccgcgagc tgccctctga cctcaatctg 1020 cagcaggtct ccccactctt tggcaccatc tatgacgcgg tcttcttgct ggcaaggggc 1080 gtggcagaag cgcgggctgc cgcaggtggc agatgggtgt ccggagcagc tgtggcccgc 1140 cacatccggg atgcgcaggt ccctggcttc tgcggggacc taggaggaga cgaggagccc 1200 ccattcgtgc tgctagacac ggacgcggcg ggagaccggc tttttgccac atacatgctg 1260 gatcctgccc ggggctcctt cctctccgcc ggtacccgga tgcacttccc gcgtggggga 1320 tcagcacccg gacctgaccc ctcgtgctgg ttcgatccaa acaacatctg cggtggagga 1380 ctggagccgg gcctcgtctt tcttggcttc ctcctggtgg ttgggatggg gctggctggg 1440 gccttcctgg cccattatgt gaggcaccgg ctacttcaca tgcaaatggt ctccggcccc 1500 aacaagatca tcctgaccgt ggacgacatc acctttctcc acccacatgg gggcacctct 1560 cgaaaggtgg cccaggggag tcgatcaagt ctgggtgccc gcagcatgtc agacattcgc 1620 agcggcccca gccaacactt ggacagcccc aacattggtg tctatgaggg agacagggtt 1680 tggctgaaga aattcccagg ggatcagcac atagctatcc gcccagcaac caagacggcc 1740 ttctccaagc tccaggagct ccggcatgag aacgtggccc tctacctggg gcttttcctg 1800 gctcggggag cagaaggccc tgcggccctc tgggagggca acctggctgt ggtctcagag 1860 cactgcacgc ggggctctct tcaggacctc ctcgctcaga gagaaataaa gctggactgg 1920 atgttcaagt cctccctcct gctggacctt atcaagggaa taaggtatct gcaccatcga 1980 ggcgtggctc atgggcggct gaagtcacgg aactgcatag tggatggcag attcgtactc 2040 aagatcactg accacggcca cgggagactg ctggaagcac agaaggtgct accggagcct 2100 cccagagcgg aggaccagct gtggacagcc ccggagctgc ttagggaccc agccctggag 2160 cgccggggaa cgctggccgg cgacgtcttt agcttggcca tcatcatgca agaagtagtg 2220 tgccgcagtg ccccttatgc catgctggag ctcactcccg aggaagtggt gcagagggtg 2280 cggagccccc ctccactgtg tcggcccttg gtgtccatgg accaggcacc tgtcgagtgt 2340 atcctcctga tgaagcagtg ctgggcagag cagccggaac ttcggccctc catggaccac 2400 accttcgacc tgttcaagaa catcaacaag ggccggaaga cgaacatcat tgactcgatg 2460 cttcggatgc tggagcagta ctctagtaac ctggaggatc tgatccggga gcgcacggag 2520 gagctggagc tggaaaagca gaagacagac cggctgctta cacagatgct gcctccgtct 2580 gtggctgagg ccttgaagac ggggacacca gtggagcccg agtactttga gcaagtgaca 2640 ctgtacttta gtgacattgt gggcttcacc accatctctg ccatgagtga gcccattgag 2700 gttgtggacc tgctcaacga tctctacaca ctctttgatg ccatcattgg ttcccacgat 2760 gtctacaagg tggagacaat aggggacgcc tatatggtgg cctcggggct gccccagcgg 2820 aatgggcagc gacacgcggc agagatcgcc aacatgtcac tggacatcct cagtgccgtg 2880 ggcactttcc gcatgcgcca tatgcctgag gttcccgtgc gcatccgcat aggcctgcac 2940 tcgggtccat gcgtggcagg cgtggtgggc ctcaccatgc cgcggtactg cctgtttggg 3000 gacacggtca acaccgcctc gcgcatggag tccaccgggc tgccttaccg catccacgtg 3060 aacttgagca ctgtggggat tctccgtgct ctggactcgg gctaccaggt ggagctgcga 3120 ggccgcacgg agctgaaggg caagggcgcc gaggacactt tctggctagt gggcagacgc 3180 ggcttcaaca agcccatccc caaaccgcct gacctgcaac cggggtccag caaccacggc 3240 atcagcctgc aggagatccc acccgagcgg cgacggaagc tggagaaggc gcggccgggc 3300 cagttctctt ga 3312 <210> 10 <211> 3327 <212> DNA <213> Homo sapiens <400> 10 atgagcgctt ggctcctgcc agccggaggg cttcccggcg ccgggttctg tgtccctgcg 60 cggcagtctc cgtccagttt ctcgcgggtc ctgcgctggc caaggcctgg gctaccggga 120 ctcctgctac tgctactgct cccatctcct tctgccctct ctgctgtgtt caaagtgggg 180 gtgctgggcc cctgggcttg cgaccccatc tttgcacggg cccgaccaga cctggctgcg 240 cgtctggccg ccaaccgcct gaatcgtgac tttgctttag acggcggccc ccggttcgag 300 gttgcgctgc tcccagagcc ctgcctgact ccgggctcac taggggctgt gtcctctgcg 360 ctgtctcgag tctctggcct ggtgggtccg gtgaaccccg cagcctgtcg gccagccgaa 420 ctgttggctc aagaagctgg agtagcgctg gtgccctggg gctgccctgg cacgcgggcg 480 gcgggtacta cagccccggc ggtgaccccc gctgcagatg ctctctacgt cctccttaga 540 gcattccgct gggcgcgcgt ggccctgatc actgcacccc aagacctgtg ggtggaggcg 600 ggacgcgctc tgtccacagc actcagggcc cggggtttgc cagttgccct agtgacttcc 660 atggagactt cagaccggtc tggagcccgg gaggccctcg ggaggatccg agatgggcct 720 agagttagag tagtgatcat ggtgatgcac tcggtgctgc tgggcggcga ggagcagcgc 780 tacctactgg aagctgcaga agaactggct ctgactgatg gctccctggt tttcctgccc 840 ttcgacacgc ttcactacgc tttgtctcca ggcccggagg ctctggctgc atttgtcaac 900 agctcccagc tccgcagggc tcacgatgcg gtgctcacac tcacgcgccg ctgtcctcct 960 ggaggcagcg tgcaagacag cctgcgcagg gctcaagaac accaggaact gccccttgac 1020 ctcaacctga agcaggtctc tccgctgttt ggcaccatct atgatgctgt cttcctgttg 1080 gctggggggcg tgaagagagc aagaacagcg gtggggtggtg gctgggtgtc aggtgcatct gtagcccgcc aagtacgggga agcacaagtc tctggctttt gtggggtcct gggaagaacc gaggagccct cctttgtgct gctggacaca gatgcatccg gagaacagtt gttcgcaaca cacctgctag atcctgtctt aggctccctg cgttctgcag ggacccccat gcacttccct 1320 agaggtggac ctgccccggg accagaccct tcctgctggt tcgatccaga tgtgatctgc 1380 aacggagggg tggagccagg cctggtcttt gttggcttcc tcctggtgat agggatgggga 1440 ctgactggag ccttcttggc tcattacttg aggcacaggc tgctacacat gcagatggct tccggcccca acaagatcat cttgacgttg gaagatgtta ctttcctcca cccaccggga ggcagctctc gaaaggtggt ccagggagt agatccagtc tggctacccg gagcgcatca gacattcgca gtgtccccag ccagcccca gagagcacca acgttggcct ctatgagggg 1740. 1740. 1740. 1740. 1740. 1740. 1740. 1740. 1740. 1740. 1740. 1740 aagacagcct tctccaagct tcgagagctc cggcatgaga atgtggctct ctacttggga ctctctcctgg cgggtacagc agacagccct gccaccctg gggaggcat cttggctgtg 1860 gtctcagagc actgtgctcg gggttccctc catgacctcc tggcccag agaataaag 1920 ctggactgga tgttcagtc tccctcctg ctggactca tcaagggaat gagatatctg 1980s caccatcgcg gtgtgccca cgggaggctc aagtcacgga attgcgtggt ggacgggagg 2040 ttcgtgctca aggtgacaga tcatggccat gggcgactgc tggaagcgca aagggtgtta 2100 ccggaacctc ccagtgcaga ggatcagcta tggacagccc cagagcttct tcgggacccc 2160 tccctggagc gccggggaac tctagctggt gatgtcttta gtctggccat catcatgcag 2220 gaggtcgtgt gccgcagcac cccttatgcc atgctggac taacgcccga ggaagtaata 2280 cagagggtgc ggagccctcc tccactgtgt cggcccttgg tgtccatgga ccaggcacccc 2340 atggagtgca tccagctgat gandacaatgc tgggcagc atccagact tcggccttcc 2400 atggacctca cctttgacct gttcagagc atcaacagg gccggacc cacatcatc 2460 gactccatgc ttcggatgct ggagcagtac tctagtacc tggactct gatccgagaa 2520 cgcacagagg agttagagca ggagaagcag aagacagaca ggctgctcac acagatgctg 2580 cctccatctg tggctgaggc cctgaagatg gggacatctg tggagcctga gtactttgaa 2640 gaggtgacac tctacttcag tgacatcgtg ggctttacca ccatttcagc catgagcgag 2700 cctattgagg tggtagacct gcttaatgac ctctatactc tcttcgatgc catcatcggt 2760 gcccatgatg tctataaggt ggaaacaatt ggagatgcat atatggtggc ctccgggctg 2820 ccgcagagga acgggcagcg gcacgctgca gagattgcca acatgtcact ggacatcctc 2880 agtgcagtcg gctccttccg catgcgccat atgcccgagg taccggtgcg catccgcatt 2940 ggtttgcact caggcccgtg cgtggcgggt gtggtgggcc tcaccatgcc tcggtactgc 3000 ctgttcgggg acacggtcaa cactgcctcg agaatggagt ccactggact gccttaccgc 3060 atccacgtta acatgagcac tgttcggatt cttcgcgctc tggaccaagg cttccagatg 3120 gagtgtcgag gccgcacgga gctgaagggc aagggtattg aggacacgta ctggcttgtg 3180 ggcagacttg gcttcaacaa gcccattccc aaaccacctg atctgcagcc aggggccagc 3240 aaccatggta tcagcctgca ggagattccc ccagagagac gcaagaagct ggagaaagcc 3300 aggccaggcc agtttactgg gaagtga 3327 <210> 11 <211> 292 <212> DNA <213> Homo sapiens <400> 11 gggccccaga agcctggtgg ttgtttgtcc ttctcagggg aaaagtgagg cggccccttg 60 gaggaagggg ccgggcagaa tgatctaatc ggattccaag cagctcaggg gattgtcttt 120 ttctagcacc ttcttgccac tcctaagcgt cctccgtgac cccggctggg atttagcctg 180 gtgctgtgtc agccccggtc tcccaggggc ttcccagtgg tccccaggaa ccctcgacag 240 ggcccggtct ctctcgtcca gcaagggcag ggacgggcca caggccaagg gc 292

Claims

1. rAAV particles, wherein the rAAV particles comprise the VP1 protein of SEQ ID NO: 1, the VP2 protein of SEQ ID NO: 2, and the VP3 protein of SEQ ID NO:

3.

2. The rAAV particle of claim 1, further comprising a heterologous nucleic acid sequence encoding a therapeutic agent.

3. The rAAV particle of claim 2, wherein the heterologous nucleic acid sequence is operably linked to one or more regulatory sequences that direct expression of the heterologous nucleic acid sequence in photoreceptor cells or retinal pigment epithelial cells.

4. The rAAV particle of claim 2, wherein the heterologous nucleic acid sequence is flanked by one or more inverted terminal repeat (ITR) sequences.

5. The rAAV particle of claim 2, wherein the heterologous nucleic acid sequence is operably linked to a promoter capable of expressing the heterologous nucleic acid sequence in photoreceptors or retinal pigment epithelial cells of a mammalian eye.

6. The rAAV particle of claim 5, wherein the promoter is the human rhodopsin kinase (hGRK1) promoter.

7. The rAAV particle of claim 5, wherein the heterologous nucleic acid sequence encodes a ribozyme, a peptide nucleic acid, a siRNA, an antisense oligonucleotide, an antibody, an antigen-binding fragment, or any combination thereof.

8. The rAAV particle of claim 5, wherein the heterologous nucleic acid sequence encodes a protein.

9. The rAAV particle of claim 5, wherein the heterologous nucleic acid sequence encodes a peptide.

10. A composition comprising the rAAV particles of any one of claims 1 to 9, further comprising one or more pharmaceutically acceptable carriers, buffers, diluents or excipients.

11. A capsid protein consisting of the amino acid sequences of SEQ ID NOs: 1, 2 and 3.

12. A nucleic acid consisting of the nucleic acid sequences of SEQ ID NOs: 4, 5 and 6.

13. The nucleic acid of claim 12, wherein the nucleic acid is contained within a plasmid or a viral vector.

14. A cell comprising the capsid protein of claim 11 or the nucleic acid of claim 12.

15. The cell of claim 14, wherein the cell is a mammalian cell.

16. Use of the rAAV particles of any one of claims 2 to 9 or the composition of claim 10 in the preparation of a medicament for subretinal transduction of mammalian photoreceptor cells or retinal pigment epithelial cells, wherein the subretinal transduction comprises administering the rAAV particles of any one of claims 2 to 9 or the composition of claim 10 subretina to one or both eyes of the mammal.

17. Use of the rAAV particles of any one of claims 2 to 9 or the composition of claim 10 in the preparation of a medicament for providing a therapeutically effective amount of a therapeutic agent to a mammal in need thereof, wherein the providing comprises administering the rAAV particles of any one of claims 2 to 9 or the composition of claim 10 subretinaly to one or both eyes of the mammal; and continuously and effectively providing the therapeutically effective amount of the therapeutic agent to the mammal for a period of time.

18. Use of the rAAV particles of any one of claims 2 to 9 or the composition of claim 10 in the preparation of a medicament for treating or ameliorating a retinal disease in a mammal, wherein the treatment or improvement comprises administering the rAAV particles of any one of claims 2 to 9 or the composition of claim 10 subretinal to one or both eyes of the mammal in an amount and for a period of time, the amount and period of time being sufficient to treat or improve the retinal disease.

19. The use of any one of claims 16 to 18, wherein the mammal is a human.

20. The use of claim 19, wherein the human suffers from or is suspected of suffering from a retinal disease.

21. The use of claim 17, wherein the therapeutic agent produces a) protection of one or more photoreceptor cells or one or more retinal pigment epithelial cells, b) restoration of one or more rod and / or cone mediated functions, c) restoration of visual behavior of one or both eyes, or d) any combination thereof.

22. The use of claim 21, wherein production of the therapeutic agent persists in one or more photoreceptor cells or one or more retinal pigment epithelial cells for a period of at least three months following administration of the rAAV particles into one or both eyes of the mammal.

23. The use of any one of claims 16 to 18, wherein subretinal administration comprises subretinal administration to the fovea of ​​the mammal.

24. The use of claim 23, wherein detachment of the fovea is minimized.

25. The rAAV particle of any one of claims 2 to 9, the composition of claim 10, or the use of any one of claims 16 to 18, wherein the heterologous nucleic acid sequence is selected from GUCY2D, AIPL1, LCA5, RPGRIP1, CRX, CRB1, NMNAT1, CEP290, IMPDH1, RD3, RDH12, TULP1, KCNJ13, GDF6, IQCB1, BBS1, BBS2, ARL6 / BBS3, BBS4, BBS5, BBS7, TTC8 / BBS8, BBS10, TRIM32 / BBS11, BBS12, CCDCC28B, TMEM67, MKS1, MKKS, RHO, PRPF31, RP1, NRL, NR2E3, RPGR and RP2, PDE6B, PDE6G, RP25, CNGA1, CNGB1, MAK, RS1, CNGB3, CNGA3, GNAT2, OPN1LW, OPN1MW, GUCA1A (GCAP1), GUCA1B (GCAP2), ABCA4, PROM1, ELOVL4, MY07A, USH1C, CDF123, PCD5, USFIIG, USH2A, DFNB31, or CLRN1.

26. The rAAV particle of any one of claims 2 to 9, the composition of claim 10, or the use of any one of claims 16 to 18, wherein the heterologous nucleic acid sequence comprises a nucleotide sequence that is at least 95%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO:

9.

27. The rAAV particle of any one of claims 2 to 9, the composition of claim 10, or the use of any one of claims 16 to 18, wherein the heterologous nucleic acid sequence is selected from GUCY2D.

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