Highly efficient transduction and lateral spread in the retina by novel AAV viruses enhanced by rational design

Abstract

The present disclosure provides rAAV particles comprising the new capsid variant AAV44.9(E531D). The present disclosure also provides rAAV particles comprising AAV44.9(E531D) for ocular treatment, including treatment of retinal disorders. In a specific embodiment, the present disclosure provides rAAV particles comprising AAV44.9(E531D) capsids that exhibit enhanced lateral spread after subretinal injection into the fovea of ​​a subject, whereby foveal detachment is minimized. The present disclosure further provides rAAV particles comprising an AAV44.9(E531D) capsid and a polynucleotide encoding a heterologous nucleic acid sequence. Methods of treatment comprising administering the rAAV particles to a mammal in need thereof, and methods of transducing photoreceptor cells and RPE cells with the rAAV particles are also provided.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of the filing date of U.S. Provisional Application No. 62 / 795,695, filed January 23, 2019, the entire contents of which are incorporated by reference.

[0002] Federally funded research 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 the invention.

[0003] FIELD OF THEINVENTION The present invention relates generally to the fields of molecular biology and virology, and specifically to the development of gene therapy vectors and methods for the treatment of retinal diseases. [Background technology]

[0004] 2. Background of the Invention By using viruses to deliver therapeutic genetic material, great progress has been achieved in the field of gene therapy. Due to its low immunogenicity and ability to effectively transduce non-dividing cells, adeno-associated virus has attracted much attention as a highly effective viral vector for gene therapy. AAV has been shown to infect various cell and tissue types, and significant progress has been made over the last decade in adapting this viral system for use in human gene therapy.

[0005] In its normal "wild-type" form, AAV DNA is packaged into the viral capsid as a single-stranded molecule of about 4600 nucleotides (nt) in length. Following infection of a cell by the virus, the cell's molecular machinery converts the single-stranded DNA into a double-stranded form. Only this double-stranded DNA form can be transcribed by cellular enzymes into RNA, which is then translated into polypeptides by further cellular pathways.

[0006] Recombinant adeno-associated virus (AAV) vectors have been successfully used for in vivo gene transfer in numerous preclinical animal models of human disease and 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, for example, in ocular delivery for Leber's congenital amaurosis (Bainbridge et al., 2008; Maguire et al., 2008; Cideciyan et al., 2008). The great advantage of this vector is its relatively low immune profile, which elicits only a limited inflammatory response and, in some cases, even directs immune tolerance to the transgene product (LoDuca et al., 2009). However, therapeutic efficiency is limited in humans due to antibody and CD8+ T cell responses against the viral capsid when targeting immune-privileged organs, while adaptive responses against the transgene product have also been reported in animal models (Manno et al., 2006;Mingozzi et al., 2007;Muruve et al., 2008;Vandenberghe and Wilson, 2007;Mingozzi and High, 2007).

[0007] Subretinal injection of AAV is commonly used when transgene expression in the retinal pigment epithelium (RPE) or photoreceptors (PR) is required. Subretinal injection produces a temporary bullous detachment that separates the photoreceptor outer segments from the RPE layer. Typically, the subretinal injection bleb resolves over the next few days in the subject. Subretinal injection appears to have some deleterious effects on photoreceptors, and such effects are thought to be more severe in retinas already compromised by disease. Specifically, it has been suggested that detaching the fovea in RPE65-LCA patients undergoing 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).

[0008] There remains a need for AAV capsids to increase retinal transduction efficiency and minimize foveal detachment during subretinal injection. Summary of the Invention

[0009] Summary of the Invention AAV has become the vector of choice for targeting therapeutic genes to the retina. Both natural and synthetic AAVs that exhibit retinal tropism have been identified. Recently, a novel AAV capsid serotype 44.9 was isolated from a laboratory stock of simian adenovirus SV15 from normal rhesus monkey kidney cell culture. See WO 2016 / 183297, published November 17, 2016; U.S. Patent Application Publication No. 2018 / 0355376, published December 13, 2018; and Novel Adeno-Associated Virus for Gene Therapy, Fed. Reg. 80, 149 (Aug. 4, 2015), each of which is incorporated herein in its entirety. AAV44.9 efficiently transduces multiple cell types, including salivary gland cells, liver cells, and many types of neurons (eg, cells of the cerebral cortex, olfactory bulb, brain stem, and Purkinje cells of the cerebellum).

[0010] AAV44.9 exhibits in vivo biodistribution comparable to AAV9. Intracerebroventricular injection of this capsid shows transduction levels in the cerebral cortex, olfactory bulb, cerebellum, choroid plexus, and brainstem similar to those observed with AAV9. In addition, antibody neutralization studies suggest a lower frequency of neutralizing antibodies against AAV44.9 compared to AAV2. And glycan array studies of AAV44.9 suggest binding of the capsid to terminal glucose-containing molecules.

[0011] The amino acid sequence of the capsid protein VP1 of AAV44.9 differs from that of the closest reported isolate AAVrh.8R capsid protein VP1 (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)) at several positions, two of which are serine residues in variable domain 3. Specifically, the amino acid sequence of the capsid protein VP1 of AAV44.9 differs at positions 179, 473, and 483 relative to the amino acid sequence of the capsid protein VP1 of AAVrh.8R.

[0012] The amino acid sequence of capsid protein VP1 of AAV44.9 differs from the amino acid sequence of 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. Specifically, the amino acid sequence of capsid protein VP1 of AAV44.9 differs at positions 179, 473, 483, and 531 relative to the amino acid sequence of capsid protein VP1 of AAVrh.8.

[0013] Rational mutagenesis studies of amino acids in the capsid protein suggest that some mutations have an inhibitory effect on the gene transfer activity of the vector, specifically the presence of serine and threonine residues in the variable region. Reports indicate that these amino acids increase the surface charge of the particles, targeting them for degradation in lysosomes, and that substitution with other uncharged amino acids can improve transduction activity. In addition, mutations of residues involved in receptor interactions also have a profound 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 increased retinal transduction relative to unmodified capsids.

[0014] AAV44.9 has high gene transfer activity in several cell types given the inclusion of an additional serine residue in variable domain 3 of the capsid protein VP1 of AAV44.9 relative to the capsid protein VP1 of AAVrh.8. The capsid protein VP1 of AAV44.9 with a substitution of serine at position 470 with asparagine is disclosed in WO 2016 / 183297. When a similar serine residue was modified in the AAV2 capsid, it led to a substantial increase in the titer of the produced vector, but it did not modify 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)). Nonetheless, the S470R substitution in AAV44.9 appears to alter the transduction and binding affinity of the AAV44.9 capsid.

[0015] The inventors of the present disclosure used a rational design approach to create a novel variant by mutagenesing the glutamic acid at residue 531 to aspartic acid. This new serotype variant AAV44.9(E531D) was evaluated in subretinal injected mice and macaques. Amino acid substitutions at positions corresponding to the E530 position in the AAV2 capsid, such as position 531 in AAVrh.8 and AAV44.9, are hypothesized to modify transduction efficiency. See International Patent Application Publication No. WO 2018 / 156654, published on August 30, 2018, the contents of which are incorporated herein by reference.

[0016] As described herein, it has been surprisingly found that rAAV particles incorporating the AAV44.9(E531D) capsid variant can transduce rods, cones, and retinal pigment epithelium ("RPE") efficiently following subretinal injection.In addition, AAV44.9(E531D) exhibits increased lateral spread, allowing transduction of photoreceptors and retinal pigment epithelium outside the subretinal injection bleb.The increased potency and lateral spread of AAV44.9(E531D) make this variant a promising vector for gene therapy targeting the retina.

[0017] Cone-rich subfoveal subretinal injections have been shown to accelerate loss of central retinal thickness as well as loss of vision in some treated patients (Phase I / II study of RPE65-LCA2). See Jacobson et al., Arch Ophthalmol. 2012; 130(1):9-24. Subretinal injections in the extrafoveal retina, however, are well tolerated. It would therefore be advantageous to use a vector that effectively targets the foveal cones following an extrafoveal subretinal injection, i.e., the fovea is not detached during surgery. Vectors currently used in clinical trials for color blindness and other congenital retinal diseases do not meet this criterion. Transduction of the retina at the site of injection is a desirable feature of newer generation AAV vectors, as transgene expression beyond the initial border of the subretinal bleb may avoid some of the deleterious effects of retinal detachment while maximizing beneficial gene therapy effects. For example, in human subjects, lateral spread of transduction could allow subretinal injection in the parafoveal region to result in transduction of foveal cells while avoiding the deleterious effects of inducing foveal detachment. Recently, an EGFP-expressing AAV vector demonstrated lateral spread of transgene expression beyond the subretinal injection site following subretinal delivery of the AAV vector in normal dogs. 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.

[0018] The parafoveal region is a zone of the eye that borders the fovea at approximately 4 degrees eccentricity from the central fixation point. The parafovea has the highest density of rods, yet still contains a large number of cones. It is a transition zone between the cone-dominant and rod-dominant retina, and is important in the context of diseases in which degeneration progresses from the outer retina to the inner retina, such as retinitis pigmentosa (RP). The perifoveal region is a zone that borders the parafovea and corresponds to the outermost band of the macula. Like the parafovea, the perifovea plays an important role in the progression of diseases such as RP, where retinal degeneration begins in the periphery and progresses to the central retina. The perifovea is the first zone of the macula to undergo degeneration in RP.

[0019] Aspects of this disclosure relate to rAAV particles and vectors that contain improved AAV44.9 capsids for ocular treatment. Specifically, this disclosure provides in some embodiments particles that contain AAV44.9 capsids with E531D mutations for the treatment of retinal disorders. In some embodiments, this disclosure provides particles that contain AAV44.9(E531D) capsids that exhibit enhanced lateral spread after subretinal injection into the fovea, where foveal detachment is minimized. In some embodiments, this disclosure provides capsid proteins, for example VP1, VP2 or VP3 capsid proteins that contain any one of the amino acid sequences of SEQ ID NO:1, 2 or 3, respectively.

[0020] In some embodiments, the disclosure provides an rAAV particle comprising a capsid comprising VP1, VP2, and / or VP3 proteins, the rAAV particle further comprising a polynucleotide comprising a heterologous nucleic acid sequence. In some embodiments, the heterologous nucleic acid sequence in the rAAV particle encodes a diagnostic or therapeutic agent, such as a polypeptide, peptide, ribozyme, peptide nucleic acid, siRNA, RNAi, antisense oligonucleotide, antisense polynucleotide, antibody, antigen-binding fragment, or any combination thereof.

[0021] In specific embodiments, the therapeutic agent a) preserves one or more photoreceptor (PR) cells or one or more retinal pigment epithelial ("RPE") cells, b) restores one or more rod- and / or cone-mediated functions, c) fully or partially restores visual behavior in one or both eyes, or d) any combination thereof. In some embodiments, production of the therapeutic agent is sustained in the one or more photoreceptor cells or one or more RPE cells for a period of substantially at least three months following an initial administration of the rAAV particles to one or both eyes of the mammal.

[0022] In some embodiments, the heterologous nucleic acid sequence comprises a sequence (e.g., a sequence having at least 80% identity to a target coding sequence) associated with a disease, disorder, or condition, such as dominant cone dystrophy, dominant cone-rod dystrophy, Leber's congenital amaurosis, recessive cone dystrophy, recessive cone-rod dystrophy, macular dystrophy, pattern dystrophy, vitelliform macular 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 the 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, Pompe disease, Friedreich's ataxia, mucopolysaccharidoses (MPS) (all types), lysosomal storage diseases (LSD) (all types), amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Alzheimer's disease.

[0023] In some embodiments, the heterologous nucleic acid sequence has at least 80% identity to the target coding sequence. In some embodiments, the heterologous nucleic acid sequence has at least 95% identity to the target coding sequence. In some embodiments, the heterologous nucleic acid sequence has at least 98% or at least 99% identity to the target coding sequence. In some embodiments, the heterologous nucleic acid sequence has 100% identity to the target coding sequence. In some embodiments, the heterologous nucleic acid sequence comprises a GUCY2D sequence.

[0024] In some embodiments, the heterologous nucleic acid sequence is a replacement coding sequence. In a specific embodiment, the replacement coding sequence is administered to a subject (e.g., a human) to provide a functional protein, e.g., GUCY2D, to restore photoreceptor function, e.g., fully or partially, to the subject. In some embodiments, administering a rAAV particle comprising a heterologous nucleic acid sequence disclosed herein to a subject (e.g., a human with dominant cone-rod dystrophy) silences one or both alleles of a target coding sequence in the subject.

[0025] Exemplary target coding sequences 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 macular dystrophy, central choroidal dystrophy, and macular dystrophy). GUCY2D encodes the retinal guanylyl cyclase 1 (retGC1) enzyme, also known as guanylate cyclase 2D. Mutations in this gene result in Leber congenital amaurosis and cone-rod dystrophy-6 disorders. Gucy2e encodes guanylate cyclase 2E, the mouse homolog of GUCY2D.

[0026] Additional target coding sequences 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-Biedl 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 vision deficiency); OPN1LW and OPN1MW (both associated with blue-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 Stargardt disease); PROM1 and ELOVL4 (both associated with Stargardt disease); 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).

[0027] In some embodiments, the heterologous nucleic acid sequence comprises a target genomic regulatory sequence (e.g., a locus control region) associated with a disease, disorder, or condition, such as blue-cone monochromacy. An exemplary target regulatory sequence is the locus control region of L / M opsin, which is associated with blue-cone monochromacy.

[0028] In some aspects, the disclosure provides compositions comprising rAAV particles and a pharma- ceutically acceptable carrier, excipient, diluent and / or buffer.

[0029] In some aspects, the disclosure provides a method of transducing RPE and photoreceptor cells to modulate expression of a heterologous nucleic acid sequence (or transgene) in a subject, the method comprising administering to a subject, such as a human subject, an rAAV particle as described herein and a pharma- ceutically acceptable carrier, excipient, diluent, and / or buffer. In some aspects, the disclosure provides a method of treating a retinal disease in a subject, the method comprising administering a composition to an eye of the subject.

[0030] In some aspects, the disclosure provides compositions for use in treating retinal diseases, and for use in the manufacture of medicaments for treating retinal diseases. In some aspects, the disclosure provides compositions comprising rAAV particles as described herein for use in treatment by subretinal or intravitreal administration to one or both eyes of a mammal.

[0031] In some aspects, the disclosure provides a method for expressing a nucleic acid segment in one or more photoreceptor or RPE cells of a mammal, the method comprising: subretinal or intravitreal administration of an rAAV particle as described herein to one or both eyes of the mammal for a time effective to produce a therapeutic agent in one or more PR or RPE cells of the mammal, wherein the rAAV particle comprises 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 a therapeutic agent.

[0032] rAAV particles may contain multiple (2, 3, 4, 5, 6, 7, 8, 9, or 10) heterologous nucleic acid sequences. In some embodiments, multiple heterologous nucleic acid sequences are included on a single polynucleotide molecule. Multiple heterologous nucleic acid sequences can be used, for example, to correct or alleviate genetic defects caused by multi-subunit proteins. In various embodiments, different heterologous nucleic acid sequences can be used to code each subunit of a protein or to code different peptides or proteins. This is desirable when the size of the nucleic acid encoding the protein subunit is large, for example, for immunoglobulins, platelet-derived growth factor, or dystrophin proteins. For cells to produce multi-subunit proteins, the 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 various embodiments, a single heterologous nucleic acid sequence encompasses the nucleic acid encoding each of the subunits, and the nucleic acid for each subunit is separated by an internal ribosome entry site (IRES). This may be desirable when the size of the nucleic acid encoding each of the subunits is small, for example when the combined size of the nucleic acid encoding the subunits and the IRES is less than 5 kilobases.

[0033] As an alternative to IRES, the nucleic acid may be separated by a sequence encoding a 2A peptide, which is self-cleaved in a post-translational event. This 2A peptide is significantly smaller than an IRES and is well suited for use when space is a limiting factor. More frequently, this is the case when the heterologous nucleic acid sequence is large, consists of multiple subunits, or when two heterologous nucleic acid sequences are co-delivered, or when rAAV particles carrying the desired heterologous nucleic acid sequence(s) or subunits are co-administered to form a single vector genome by concatamerizing them in vivo. In such an embodiment, the first rAAV particle may carry an expression cassette expressing a single heterologous nucleic acid sequence, and the second rAAV particle may 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 may code for any biologically active product or other product, for example, a product desired for research.

[0034] In some embodiments, the polynucleotide in the rAAV particle contains regulatory sequences, such as transcriptional and translational initiation and termination codons, that are specific to the type of host into which the rAAV particle is introduced (e.g., bacterial, fungal, plant, or animal). Preferably, the nucleic acid in the rAAV particle contains regulatory sequences that are specific to the genus of the host. Most preferably, the molecule contains regulatory sequences that are specific to the species of the host.

[0035] The polynucleotide present in the rAAV particle preferably includes expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), etc., that provide for expression of the heterologous nucleic acid sequence(s) in a host cell. Exemplary expression control sequences are known in the art and described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

[0036] BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate 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 identify like elements and in which: [Brief description of the drawings]

[0037] [Figure 1] FIG. 1 shows the VP1-based AAV phylogeny (AAV44.9 is shown in bold) and a table with details of AAV constructs for vector production.

[0038] [Figure 2A] Figures 2A, 2B, 2C, 2D, and 2E show qualitative and quantitative analysis of AAV44.9, AAV5, and AAV8(Y733F) at 4 weeks after subretinal injection. Figure 2A shows a fundus image at exposure length 25. [Figure 2B-2C] Figures 2B-2D show flow cytometry scatter plots, and Figure 2E shows quantitative analysis demonstrating that AAV44.9 transduced rods more efficiently than AAV5 and AAV8(Y733F) following subretinal injection with 2 x 10 vector genomes (vg). [Figure 2D] Figures 2B-2D show flow cytometry scatter plots, and Figure 2E shows quantitative analysis demonstrating that AAV44.9 transduced rods more efficiently than AAV5 and AAV8(Y733F) following subretinal injection with 2 x 10 vector genomes (vg). [Figure 2E] Figures 2B-2D show flow cytometry scatter plots, and Figure 2E shows quantitative analysis demonstrating that AAV44.9 transduced rods more efficiently than AAV5 and AAV8(Y733F) following subretinal injection with 2 x 10 vector genomes (vg).

[0039] [Diagram 3] Figures 3A, 3B, and 3C show representative retinal cross-section images showing mCherry expression in photoreceptors and retinal pigment epithelium ("RPE") in Nrl-GFP transgenic mouse retinas injected with AAV5 (upper panel, Figure 3A), AAV8(Y733F) (upper panel, Figure 3B), and AAV44.9 (upper panel, Figure 3C) 4 weeks after subretinal injection. The lower panels of Figures 3A, 3B, and 3C show overlapping images of nuclear DAPI staining, endogenous GFP expression in rod cells, and mCherry expression in photoreceptor cells, as indicated by arrows.

[0040] [Figure 4A] Figures 4A, 4B, and 4C show qualitative and quantitative analysis of AAV44.9(Y733F) and AAV44.9(E531D). Figure 4A shows fundus images at exposure length 25, Figure 4B shows flow cytometry scatter plots, and Figure 4C shows quantitative analysis demonstrating that AAV44.9(E531D) transduced rod cells more efficiently than AAV44.9 and AAV44.9(Y733F) following subretinal injection at 2x109 vg. [Figure 4B] Figures 4A, 4B, and 4C show qualitative and quantitative analysis of AAV44.9(Y733F) and AAV44.9(E531D). Figure 4A shows fundus images at exposure length 25, Figure 4B shows flow cytometry scatter plots, and Figure 4C shows quantitative analysis demonstrating that AAV44.9(E531D) transduced rod cells more efficiently than AAV44.9 and AAV44.9(Y733F) following subretinal injection at 2x109 vg. [Figure 4C]Figures 4A, 4B, and 4C show qualitative and quantitative analysis of AAV44.9(Y733F) and AAV44.9(E531D). Figure 4A shows fundus images at exposure length 25, Figure 4B shows flow cytometry scatter plots, and Figure 4C shows quantitative analysis demonstrating that AAV44.9(E531D) transduced rod cells more efficiently than AAV44.9 and AAV44.9(Y733F) following subretinal injection at 2x109 vg.

[0041] [Diagram 5] Figures 5A and 5B show representative retinal cross-section images showing mCherry expression primarily in photoreceptors and RPE in AAV44.9(Y733F) (upper panel, Figure 5A) and AAV44.9(E531D) (upper panel, Figure 5B) injected Nrl-GFP mouse retinas 4 weeks after subretinal injection. The lower panels of Figures 5A and 5B show overlaid images of nuclear DAPI staining (blue), endogenous GFP (green) expression in rod cells, and mCherry expression (red) in photoreceptor cells.

[0042] [Figure 6] Figures 6A and 6B show the transduction efficiency of AAV44.9 and AAV44.9(Y733F) in ocular cell lines. AAV44.9(Y733F) showed increased transduction relative to AAV44.9 in mouse cone photoreceptor cell lines (Figure 6A). However, AAV44.9 was more efficient than AAV9(Y733F) in human RPE cell lines (Figure 6B).

[0043] [Figure 7]Figures 7A and 7B show the qualitative analysis of AAV following intravitreal injection. Four weeks after intravitreal injection at 2x109vg, Figure 7A shows fundus images of AAV2, AAV5, AAV8(Y733F), AAV44.9, AAV44.9(Y733F) and AAV44.9(E531D) (at long 25 integration) and Figure 7B shows fundus images of variants (at long 100 integration).

[0044] [Figure 8] Figures 8A, 8B, and 8C show AAV44.9 containing a cone preferential chimeric IRBP enhancer-cone transducin promoter (IRBP / GNAT2) promoter and a GFP reporter, with Figure 8A showing GFP expression in cone cells 4 weeks after SR injection, and Figures 8B and 8C showing co-staining with cone arrestin antibody colocalizing with GFP fluorescence.

[0045] [Figure 9A-1] Figure 9A shows representative fundus images (red fluorescent filter) of Nrl-GFP mice taken 4 weeks after subretinal injection with AAV44.9 or AAV44.9(E531D). Vectors were delivered at 2 x 109 vg in 1 μL. Exposure and gain settings were consistent throughout the experiment. [Figure 9A-2] Figure 9A shows representative fundus images (red fluorescent filter) of Nrl-GFP mice taken 4 weeks after subretinal injection with AAV44.9 or AAV44.9(E531D). Vectors were delivered at 2 x 109 vg in 1 μL. Exposure and gain settings were consistent throughout the experiment.

[0046] [Figure 9B]Figure 9B shows the transduction percentage and corresponding values ​​for each cell population taken 4 weeks after injection. Retinas of Nrl-GFP mice (from Figure 9A) were dissociated with papain and flow cytometry was performed to quantify the proportion of transduced rod (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.

[0047] [Figure 10A] Figure 10A shows a representative fundus image of an Nrl-GFP mouse taken 4 weeks after subretinal injection with AAVrh.8-mCherry. The vector was delivered at 2 x 109 vg in 1 μL. Exposure and gain settings were consistent throughout the experiment.

[0048] [Figure 10B] Figure 10B shows the transduction percentage and corresponding values ​​for each cell population taken 4 weeks after injection. Retinas of Nrl-GFP mice (from Figures 9A and 10A) were dissociated with papain and flow cytometry was performed to quantify the proportion of transduced rod (GFP+mCherry positive) or non-rod cells (mCherry positive).

[0049] [Figure 11A] FIG. 11A shows representative fundus images of Nrl-GFP mice taken 4 weeks after subretinal injection with lower titers of self-complementary AAV44.9 ("scAAV44.9"), AAV44.9(E531D), or scAAVrh.8. Vectors were delivered at 2×108 vg in 1 μL. Exposure and gain settings were consistent throughout the experiment.

[0050] [Figure 11B] Figure 11B shows the transduction percentage and corresponding values ​​for each cell population taken 4 weeks after injection. Retinas of Nrl-GFP mice (from Figure 11A) were dissociated with papain and flow cytometry was performed to quantify the proportion of transduced rod (GFP+mCherry positive) or non-rod cells (mCherry positive).

[0051] [Figure 12] Figure 12 shows a 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. Fundus images for AAV44.9(E531D)-IRBP / GNAT2-hGFP and AAV44.9-IRBP / GNAT2-hGFP following subretinal injection at 2x1012 vg are shown.

[0052] [Figure 13] Figure 13 shows a representative retinal cross section of a WT mouse taken 6 weeks after subretinal injection with AAV44.9(E531D)-IRBP / GNAT2-hGFP. The vector was delivered at 2x1012 vg in 1 μL. The section was immunostained with antibodies raised against GFP (green) and cone arrestin (red).

[0053] [Figure 14] Figure 14 shows a representative retinal cross section of a WT mouse taken 6 weeks after subretinal injection with AAV44.9-IRBP / GNAT2-hGFP. The vector was delivered at 2x1012 vg in 1 μL. Sections were immunostained with antibodies raised against GFP and cone arrestin, as indicated by arrows.

[0054] [Figure 15]Figure 15 shows that AAV44.9-hGRK1-GFP and AAV44.9(E531D)-hGRK1-GFP exhibited enhanced lateral spread and efficacy in subretinal injected macaques. Vectors were delivered at 1x1012 vg vg / mL. The initial border of the bleb on the day of dosing and the border of the resulting GFP expression are outlined with white dotted lines. The identical vasculature is highlighted with a thick dark line for reference.

[0055] [Figure 16] FIG. 16 shows optical coherence tomography (OCT) scans of three subretinal injection blebs created following extrafoveal subretinal injection of AAV44.9-hGRK1-GFP (1×10 vg / mL) in a macaque (see negative contrast fundus image on the left).

[0056] [Figure 17] Figure 17 shows OCT images of macaque retina following extrafoveal subretinal injection of AAV44.9-hGRK1-GFP. The arrow in the SLO image (top left) indicates the location of the retinal cross section shown in the scan in the lower part of the figure. The cross section was stained for cone arrestin and DAPI. The percentage of rods / cones expressing GFP is plotted in each zone. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

[0057] [Figure 18] Figures 18A and 18B show ERG recordings in retinal guanylate cyclase1 / 2 double knockout (GCdko) mice following subretinal injection with AAV44.9(E531D)-hGRK1-GUCY2e or AAV8(Y733F)-hGRK1-GUCY2e. Figure 18A shows the mean maximum a- and b-wave amplitudes under both scotopic (left) and photopic (right) settings. Figure 18B shows representative ERG traces of cones from vector-treated or contralateral untreated ("No Tx") eyes.

[0058] [Figure 19]Figure 19 shows OCT images of macaque retina following extrafoveal subretinal injection of AAV44.9(E531D)-hGRK1-GFP. The arrow in the SLO image (top left) indicates the location of the retinal cross section shown in the scan in the lower part of the figure. The cross section was stained for cone arrestin and DAPI. The percentage of rods / cones expressing GFP is plotted in each zone.

[0059] [Figures 20A-20C] Figures 20A, 20B, 20C, and 20D show representative OCT images of the parafoveal region of the macaque retina following extrafoveal subretinal injection of AAV44.9(E531D)-hGRK1-GFP and AAV44.9-hGRK1-GFP. Scale bars are at A=40 microns, B=20 microns. [Figure 20D] Figures 20A, 20B, 20C, and 20D show representative OCT images of the parafoveal region of the macaque retina following extrafoveal subretinal injection of AAV44.9(E531D)-hGRK1-GFP and AAV44.9-hGRK1-GFP. Scale bars are at A=40 microns, B=20 microns.

[0060] [Figure 21A] 21A and 21B show representative OCT images of the perifoveal region of the macaque retina following these injections. [Figure 21B] 21A and 21B show representative OCT images of the perifoveal region of the macaque retina following these injections. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] Detailed Description The present disclosure provides an evaluation of vector performance in subretinal injected mice and macaques of a novel variant of the capsid serotype AAV44.9, AAV44.9(E531D), and vectors and particles incorporating this plasmid, relative to benchmark vectors (the closely related AAVrh.8 and unimproved AAV44.9). As described herein, in both species, AAV44.9(E531D) was found to mediate higher retinal transduction relative to unimproved AAV44.9 and AAVrh.8, and significantly higher transduction than benchmark capsids (e.g., vectors based on AAV5 and AAV8).

[0062] Thus, the present disclosure provides rAAV particles that comprise the capsid protein of AAV44.9 (E531D) and related compositions and methods.In some embodiments, the rAAV particles comprise heterologous nucleic acid sequences, for example those that code for therapeutic or diagnostic agents.The heterologous nucleic acid sequences can be in the form of single-stranded (ss) or self-complementary (sc) AAV nucleic acid vectors, such as single-stranded or self-complementary recombinant viral genomes.

[0063] The present disclosure further provides rAAV particles comprising the capsid protein of AAV44.9 (Y733F) and related compositions and methods. This AAV44.9 capsid variant has a YF mutation at residue 773. In some embodiments, the rAAV particle comprises a heterologous nucleic acid sequence, for example, one that encodes a therapeutic or diagnostic agent. The heterologous nucleic acid sequence may 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.

[0064] Aspects of the disclosure relate to vectors comprising the AAV44.9(E531D) capsid that exhibit enhanced lateral spread following subretinal injection into the fovea, where foveal detachment (e.g., temporary bullous detachment) is minimized. In some embodiments, the disclosure provides capsid proteins, e.g., VP1, VP2 or VP3 capsid proteins comprising the amino acid sequence of SEQ ID NO:1, 2 or 3.

[0065] In some embodiments, the disclosure provides an rAAV particle comprising a capsid comprising VP1, VP2, and / or VP3 proteins, wherein the rAAV particle further comprises a polynucleotide comprising a heterologous nucleic acid sequence. In some embodiments, the rAAV particle comprises 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.

[0066] In some embodiments, the disclosure provides a capsid protein comprising the amino acid sequence of SEQ ID NO:1, 2, and / or 3.

[0067] In some embodiments, the disclosure provides a nucleic acid, e.g., a plasmid or viral vector, comprising the nucleic acid sequence of SEQ ID NO:4 (encoding AAV44.9(E531D) VP1). In some embodiments, the disclosure provides a nucleic acid, e.g., a plasmid or viral vector, comprising the nucleic acid sequence of SEQ ID NO:5 (encoding AAV44.9(E531D) VP2). In some embodiments, the disclosure provides a nucleic acid, e.g., a plasmid or viral vector, comprising the nucleic acid sequence of SEQ ID NO:6 (encoding AAV44.9(E531D) VP3). In some embodiments, the viral vector is a recombinant adeno-associated virus (AAV) vector. In some embodiments, the rAAV vector is self-complementary. In some embodiments, the nucleic acid is contained within a cell, e.g., a mammalian or insect cell.

[0068] The sequences of SEQ ID NOs: 1-8 are provided below.

[0069] SEQ ID NO:1 - AAV44.9(E531D) VP1 amino acid sequence MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGNGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDTESVPD PQPLGEPPAAPSGLGPNTMASGGGAPMADNNEGADGVGNSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNEGTKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFP ADVFMVPQYGYLTLNNGSQALGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLVRTQTTGTGGTQTLAFSQAGPSSMASQARNWVPGPSYRQQRVSTTTNQNNNSNFAWTGAAKFKLNGRDSLMNPGVAMASHKDDDDRFFPSSGVLIFGKQGAGNDG VDYSQVLITDEEEIKATNPVATEEYGAVAINNQAANTQAQTGLVHNQGVIPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPLTFNQAKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGVYSEPRPIGTRYLTRNL

[0070] SEQ ID NO:2 - AAV44.9(E531D) VP2 amino acid sequence MAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDTESVPDPQPLGEPPAAPSGLGPNTMASGGGAPMADNNEGADGVGNSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDF NRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNEGTKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQALGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLM NPLIDQYLYYLVRTQTTGTGGTQTLAFSQAGPSSMASQARNWVPGPSYRQQRVSTTTNQNNNSNFAWTGAAKFKLNGRDSLMNPGVAMASHKDDDDRFFPSSGVLIFGKQGAGNDGVDYSQVLITDEEEIKATNPVATEEYGAVAINNQA ANTQAQTGLVHNQGVIPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPLTFNQAKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGVYSEPRPIGTRYLTRNL

[0071] SEQ ID NO:3 - AAV44.9(E531D) VP3 amino acid sequence MASGGGAPMADNNEGADGVGNSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNEGTKTIA NNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQALGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLVRTQTTGTGGTQTLAFSQAGPS SMASQARNWVPGPSYRQQRVSTTTNQNNNSNFAWTGAAKFKLNGRDSLMNPGVAMASHKDDDDRFFPSSGVLIFGKQGAGNDGVDYSQVLITDEEEIKATNPVATEEYGAVAINNQAANTQAQTGLVHNQGVI PGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPLTFNQAKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGVYSEPRPIGTRYLTRNL

[0072] SEQ ID NO:4 - AAV44.9(E531D) VP1 nucleic acid sequence

[0073] SEQ ID NO:5 - AAV44.9(E531D) VP2 nucleic acid sequence

[0074] SEQ ID NO:6 - AAV44.9(E531D) VP3 nucleic acid sequence

[0075]

[0076] SEQ ID NO:7 - AAV44.9 wild type VP1 amino acid sequence

[0077] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGNGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDTESVPD PQPLGEPPAAPSGLGPNTMASGGGAPMADNNEGADGVGNSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNEGTKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFP ADVFMVPN VDYSQVLITDEEEIKATNPVATEEYGAVAINNQAANTQAQTGLVHNQGVIPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPLTFNQAKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGVYSEPRPIGTRYLTRNL

[0078] SEQ ID NO:8 - AAV44.9 wild type VP1 nucleic acid sequence

[0079]

[0080] Methods for producing and using pseudotyped rAAV particles 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 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 Application Publication Nos. US 2007 / 0015238 and US 2012 / 0322861, which are incorporated by reference in their entireties; and plasmids and kits are available from ATCC and Cell Biolabs, Inc.). For example, a plasmid containing a heterologous nucleic acid sequence may be combined with one or more helper plasmids, such as those containing the rep gene (encoding, for example, Rep78, Rep68, Rep52 and Rep40) and the cap gene (encoding VP1, VP2, and VP3 (including an improved VP3 region as described herein)), and transfected or permanently integrated into a production cell line such that the rAAV particles are packaged and then purified.

[0081] In some embodiments, the one or more helper plasmids include a first helper plasmid that includes the rep and cap genes (e.g., encoding the rAAV capsid proteins as described herein), and a second helper plasmid that includes the E1a, E1b, E4, E2a, and VA genes. In some embodiments, the rep gene is from AAV2, and the cap gene is from AAV44.9, and may include improvements to the genes to produce improved capsid proteins as described herein. Helper plasmids and methods for making such plasmids are known in the art and commercially available (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 Motif on Adeno-Associated Virus Type IV, Human Gene Therapy, Vol. 9, 2745-2760). 2 Capsids, Journal of Virology, Vol. 77, 11072-11081.;Grimm et al.(See Kronenberg et al. (2003), Helper Virus-Free, Optically Controllable, and Two-Plasmid-Based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy, Vol. 7, 839-850; Kronenberg et al. (2005), A Conformational Change in the Adeno-Associated Virus Type 2 Capsid Leads to the Exposure of Hidden VP1 N Termini, Journal of Virology, Vol. 79, 5296-5303; and Moullier, P. and Snyder, R.O. (2008), International efforts for recombinant adeno-associated viral vector reference standards, Molecular Therapy, Vol. 16, 1185-1188).

[0082] An exemplary, non-limiting method for rAAV particle production is described next. One or more helper plasmids are produced or obtained, containing the rep and cap ORFs for 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 improvements to produce improved capsid proteins as described herein. HEK293 cells (available from ATCC®) are transfected with the helper plasmid(s) and a plasmid containing the nucleic acid vector described herein via CaPO4-mediated transfection, lipids, or polymer molecules such as polyethyleneimine (PEI). The HEK293 cells are then incubated for at least 60 hours to allow rAAV particle production. Alternatively, in another example, a producer stable cell line based on Sf9 is infected with a single recombinant baculovirus containing a heterologous nucleic acid sequence. Alternatively, in another example, HEK293 or BHK cell lines are infected with HSV containing heterologous nucleic acid sequences, and optionally with one or more helper HSV containing the rep and cap ORFs as described herein and the adenovirus VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters.HEK293, BHK, or Sf9 cells are then incubated for at least 60 hours to allow rAAV particle production.rAAV particles can then be purified using any method known in the art or described herein, for example, by iodixanol step gradient, CsCl gradient, chromatography, or polyethylene glycol (PEG) precipitation.

[0083] The present disclosure also contemplates a host cell that comprises a particle incorporating an AAV44.9(E531D) capsid, a nucleic acid encoding an AAV44.9(E531D) capsid, or a rAAV particle as described herein. Such host cells include mammalian host cells, preferably human host cells, and can be isolated, for example, in cell or tissue culture. In some embodiments, the host cell is an ocular cell.

[0084] In some embodiments, compositions are provided that include rAAV particles as described herein (including, for example, AAV44.9(E531D) capsids), and optionally, pharma- ceutically acceptable carriers, excipients, diluents, and / or buffers. In some embodiments, the compositions described herein can be administered to a mammal (or subject) in need of treatment. In some embodiments, the subject has or is suspected of having one or more of a retinal disorder, retinal disease, or retinal dystrophy. In some embodiments, the subject has or is suspected of having one or more of the retinal conditions, diseases, or disorders disclosed herein (for example, cone-rod dystrophy). In some embodiments, the subject has one or more endogenous mutant alleles (for example, those associated with or causing ocular or retinal diseases, disorders, or conditions, such as cone-rod dystrophy).

[0085] In some embodiments, a method is provided for transducing a mammalian photoreceptor cell or retinal pigment epithelial cell, comprising administering an rAAV particle as described herein to one or both eyes of the mammal. In a specific embodiment, a method is provided for expressing a polynucleotide in one or more photoreceptor cells or RPE cells of a mammal, comprising subretinal or intravitreal administration of an rAAV particle or composition thereof as described herein to one or both eyes of the mammal for a time effective to produce a therapeutic agent in one or more PR or RPE cells of the mammal, wherein the rAAV particle comprises 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 a therapeutic agent.

[0086] In a specific embodiment, the present disclosure provides a PR or RPE cell-specific promoter operably linked to at least a first heterologous nucleic acid sequence encoding a therapeutic agent. Exemplary PR or RPE cell-specific promoters include: a) photoreceptor-specific promoters (active in rod and cone cells), such as IRBP promoter (hIRPB, IRBP, IRBP241), rhodopsin kinase promoter (hGRK1, GRK1, GRK, RK), and / or chimeric human retinoschisin-IRBP enhancer (RS / IRPB); cone-specific promoters, such as red / green cone opsin promoter (which may include a 2.1 kb (PR2.1) version or a 1.7 kb (PR1.7) version); (See U.S. Patent Application Publication No. 2018 / 0112231, incorporated herein by reference), cone arrestin promoter (hCAR, CAR), chimeric IRBP enhancer-cone transducin promoter (IRBP / GNAT2, IRBBe-GNAT2); rod-specific promoters, e.g., human rhodopsin promoter (RHO, RHOP, etc.), human NRL promoter (NRL); or RPE-specific promoters, such as RPE65 or Bestrophin / VMD2 (BEST1, BEST, VMD2).

[0087] 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 sequence of the hGRK1 promoter as set forth in SEQ ID NO:11: GGGCCCCAGAAGCCTGGTGGTTGTTTGTCCTTCTCAGGGGAAAAGTGAGGCGGCCCCTTGGAGGAAGGGGCCGGGCAGAATGATCTAATCGGATTCCAAGCAGCTCAGGGGATTGTCTTTTTCTAGCACCTTCTTGCCACTCCTAA GCGTCCTCCGTGACCCCGGCTGGGATTTAGCCTGGTGCTGTGTCAGCCCCGGTCTCCCAGGGGCTTCCCAGTGGTCCCCAGGAACCCTCGACAGGGCCCGGTCTCTCGTCCAGCAAGGGCAGGGACGGGCCACAGGCCAAGGGC (Sequence number 11)

[0088] Accordingly, exemplary rAAV vectors described in this disclosure include AAV44.9(E531D)-hGRK1-GFP, AAV44.9(Y733F)-hGRK1-GFP, AAV44.9(E531D)-IRBP / GNAT2-hGFP, AAV44.9(Y733F)-IRBP / GNAT2-hGFP, AAV44.9(E531D)-hGRK1-GUCY2E, and AAV44.9(Y733F)-hGRK1-GUCY2E.

[0089] In specific embodiments, the disclosure provides a constitutive promoter operably linked to at least a first nucleic acid sequence, which may include CMV, CBA, CB, smCBA, CBh, or EF1-alpha.

[0090] In some embodiments, methods are provided that involve providing a therapeutically effective amount of a selected therapeutic agent to a mammal in need thereof, the methods comprising administering an rAAV particle as described herein to one or both eyes of the mammal in an amount and for a time effective to provide a therapeutically effective amount of the selected therapeutic agent to the mammal.

[0091] In some embodiments, the mammal is afflicted with a condition that is a deficiency or deficiency in the retina, such as dominant cone dystrophy, dominant cone-rod dystrophy, Leber's congenital amaurosis, recessive cone dystrophy, recessive cone-rod dystrophy, macular dystrophy, pattern dystrophy, vitelliform macular dystrophy, central choroidal dystrophy, Stargardt's disease, autosomal dominant, autosomal recessive and X-linked retinitis pigmentosa, retinitis pigmentosa associated with Bardet-Biedl syndrome, X-linked juvenile retinoschisis, color vision deficiency, blue-cone monochromacy, Usher syndrome Suspected of having, at risk of developing, or diagnosed with a disease, disorder, or condition such as, but not limited to, types I, II, and III, Duchenne muscular dystrophy, limb-girdle muscular dystrophy, spinal muscular atrophy, Pompe disease, Friedreich's ataxia, Mucopolysaccharidoses (MPS) (all types), Lysosomal storage diseases (LSD) (all types), Amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Alzheimer's disease. In some embodiments, the subject is diagnosed with a gene mutation in the 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 or having one or more endogenous mutant alleles associated with or causing an ocular or retinal disease, disorder or condition, such as mutant 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.

[0092] In a specific embodiment, the replacement coding sequence is administered to a subject (e.g., a human) to provide a functional protein, e.g., GUCY2D or Gucy2e, to restore photoreceptor function, for example, completely or partially, to the subject. In some embodiments, administering a rAAV particle comprising a heterologous nucleic acid sequence disclosed herein to a subject (e.g., a human with dominant cone-rod dystrophy) silences one or both alleles of the target coding sequence of the subject. In a specific embodiment, administering a rAAV particle disclosed herein silences or suppresses endogenous mutant alleles of one or more target coding sequences.

[0093] In some embodiments, the heterologous nucleic acid sequence of any of the rAAV nucleic acid vectors of the disclosure has a sequence having at least 95% identity, at least 98%, at least 99% identity, or 100% identity 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.

[0094] GUCY2D:

[0095] Gucy2e:

[0096] 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 or rhesus monkeys), marmosets, tamarins, spider monkeys, night monkeys, vervet monkeys, squirrel monkeys, baboons, gorillas, chimpanzees, and orangutans. Other exemplary subjects include domestic animals such as dogs and cats; livestock animals such as horses, cows, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters.

[0097] In some embodiments, a method is provided for subretinal administration of a rAAV particle or composition thereof described herein to the fovea (e.g., foveal cone cells) of a mammal. In a specific embodiment, foveal detachment is minimized during and / or after subretinal administration. In a specific embodiment, subretinal administration of the rAAV particle is performed in the absence of any foveal detachment.

[0098] In some embodiments, the dose of rAAV particles administered to a subject is 10 6 ~10 14 particles / mL or 10 3 ~10 13 on the order of particles / mL range, or any value therebetween for either range, e.g., about 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 In one embodiment, the concentration may be 10 13 In some embodiments, the number of rAAV particles administered to a subject is greater than 10 particles / mL. 6 ~10 14 Vector genomes (vgs) / mL or 10 3 ~10 15vgs / mL range, or any value therebetween for either range, e.g., about 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 In one embodiment, the concentration may be 10 13 More than vgs / mL of rAAV particles are administered. The rAAV particles can be administered as a single dose, or can be divided into two or more doses as may be required to achieve treatment of the specific disease or disorder being treated. In some embodiments, 0.0001mL to 10mL (e.g., 0.0001mL, 0.001mL, 0.01mL, 0.1mL, 1mL, 10mL) is delivered to the subject in a single dose.

[0099] In some embodiments, the rAAV particle titer is 1×10 10 ~5×10 13 In some embodiments, the rAAV particle titer ranges from 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 In some embodiments, the particle titer can be 1×10 10 In some embodiments, the rAAV particle titer is less than 1×10 15 In one embodiment, the rAAV particle titer is greater than 5×10 13 In some embodiments, the rAAV particles are administered via the methods described herein (e.g., subretinally or intravitreally).

[0100] The rAAV particles can be administered as a single dose or can be divided into two or more administrations as may be required to achieve treatment of a particular disease, disorder or condition. In some embodiments, 1 to 500 microliters of the composition described in this application (e.g., including rAAV particles) is administered to one or both eyes of a subject. 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 administered to each eye. However, it should be understood that in some embodiments, smaller or larger volumes may be administered.

[0101] In some embodiments, the disclosure provides formulations of one or more rAAV-based compositions disclosed herein in a pharma- ceutically acceptable solution for administration to cells or to animals, alone or in combination with one or more other therapeutic modalities, and specifically for the treatment of human cells, tissues, and diseases that affect humans.

[0102] If desired, the rAAV particles described herein can be administered in combination with other agents, such as, for example, proteins or polypeptides or various pharma- ceutical active agents, including systemic or local administration of one or more of a therapeutic polypeptide, biologically active fragment, or variant thereof. There is virtually no limit to the other components that can be included, provided that the additional agents do not cause significant adverse effects when in contact with target cells or host tissues. The rAAV particles can thus be delivered together with various other agents as needed in a particular case. Such compositions can be purified from host cells or other biological sources, or alternatively, can be chemically synthesized as described herein.

[0103] The formulation of pharma- ceutically acceptable excipients and carrier solutions will be well known to those of skill in the art, as will the development of suitable dosing and treatment regimens for use with the particular compositions described herein in a variety of treatment regimens, including, by way of example, subretinal, intravitreal, parenteral, intravenous, intranasal, intraarticular, and intramuscular administration and formulations.

[0104] Typically, these formulations may contain at least about 0.1% or more of a therapeutic agent (e.g., rAAV particles), although the percentage of active ingredient(s) may, of course, conveniently vary between about 1 or 2% and about 70% or 80% or more by weight or volume of the total formulation. Of course, the amount of therapeutic agent(s) (e.g., rAAV particles) in each therapeutically useful composition may be prepared in such a way that a suitable dosage will be obtained for any given unit dose of compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by those skilled in the art of preparing such pharmaceutical formulations, and thus various dosages and treatment regimens may be desirable.

[0105] In certain situations, it may be desirable to deliver the rAAV particles described herein (e.g., those comprising the AAV44_9(E531D) capsid) in a suitably formulated pharmaceutical composition as disclosed herein, either subretinally, intraocularly, intravitreally, 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.

[0106] Pharmaceutical forms of compositions suitable for injectable use (e.g., those containing rAAV as described herein) include sterile aqueous solutions or dispersions. In some embodiments, the forms are sterile and fluid to the extent that easy syringability exists. In some embodiments, the forms are stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, saline, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.

[0107] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle that is administered with the rAAV particles as described herein. 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 synthetic origin. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers.

[0108] The compositions of the present disclosure can be delivered to the eye through various routes. They can be delivered intraocularly by topical administration to the eye or by intraocular injection, for example, into the vitreous (intravitreal injection) or subretinal (subretinal injection) interphotoreceptor space. In some embodiments, they are delivered to rod photoreceptor cells. Alternatively, they can be delivered locally by insertion or injection into the tissue surrounding the eye. They can be delivered systemically through oral route or by subcutaneous, intravenous or intramuscular injection. Alternatively, they can be delivered by means of a catheter or by means of an implant, where such implants are made of porous, non-porous or gelatinous materials, including membranes such as silastic membranes or fibers, biodegradable polymers, or proteinaceous materials. They can be administered prior to the onset of a condition, for example during eye surgery, to prevent its occurrence, or can be administered immediately after the onset of a pathological condition or during the onset of an acute or prolonged condition.

[0109] For administration of injectable aqueous solutions, for example, the solution may be suitably buffered, and if necessary, the liquid diluent is first made isotonic with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, intravitreal, subcutaneous and intraperitoneal administration. In this context, the sterile aqueous medium that may be used will be known to those skilled in the art in view of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of subcutaneous infusion fluid or injected at the proposed site of injection (see, for example, "Remington's Pharmaceutical Sciences", 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will in any event determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required, for example, by FDA's Office of Biologics standards.

[0110] Sterile injectable solutions can be prepared by incorporating the rAAV particles as described herein in the required amount in a suitable solvent with some of the other ingredients listed above, followed by filter sterilization. In general, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the other ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze-drying techniques, which produce powders of the active ingredients and any additional desired ingredients from their previously sterile-filtered solutions.

[0111] The amount of composition (including, for example, rAAV as described herein), as well as the time of administration of such compositions, will be within the scope of the artisan's ability to predict with the benefit of the teachings of the present invention. However, administration of a therapeutically effective amount of the disclosed compositions may be achieved by a single administration, e.g., a single injection of a sufficient number of rAAV particles to provide a therapeutic benefit to a patient undergoing such treatment. Alternatively, in some circumstances, it may be desirable to provide multiple or successive administrations of the composition, either over a relatively short period of time, or over a relatively long period of time, as may be determined by a physician supervising the administration of such compositions.

[0112] In some embodiments, vision can be maintained or restored (e.g., partially or completely) after administration of one or more compositions described in this application. In some embodiments, vision can be maintained or restored (e.g., partially or completely) after administration of one or more compositions described in this application. One or more photoreceptor cells or one or more RPE cells can be partially or completely preserved, and / or one or more rod- and / or cone-mediated functions can be partially or completely restored.

[0113] As a term, "treating" a disease means reducing the frequency or severity of at least one of the signs or symptoms of a disease, disorder or condition (e.g., cone-rod dystrophy) experienced by a subject. The compositions described above are typically administered to a subject in an effective amount, that is, an amount that can produce a desired result. The desired result will depend on the active agent administered. For example, an effective amount of rAAV particles can be the amount of particles that can transfer heterologous nucleic acid to a host organ, tissue or cell.

[0114] The toxicity and efficacy of the compositions utilized in the methods of the present disclosure were evaluated using LD 50 To determine the dose that is lethal to 50% of the population, the LD2 can be determined by standard pharmaceutical procedures using either cultured cells or experimental animals. The dose ratio between toxicity and efficacy is the therapeutic index, and it is the ratio LD 50 / ED50 The therapeutic index can be expressed by the following formula: ED50=E(x,y)=0. Compositions that exhibit large therapeutic indices are preferred. Those that exhibit toxic side effects may be used, but 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 a range that includes the ED50 with little or no toxicity. Dosage can vary within this range depending on the dosage form used and the route of administration utilized. EXAMPLES

[0115] example The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors as working well in the practice of the invention, and thus may be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, understand that many changes can be made in the specific embodiments disclosed and still obtain like or similar results without departing from the spirit and scope of the invention.

[0116] Example 1 – Therapeutic molecules for AAV-based retinal gene therapy Following intravitreal (Ivt) or subretinal (SR) injection, AAV44.9 retinal transduction and tropism were assessed. By utilizing a mouse model with constitutive expression of GFP in all rod photoreceptors (Nrl-GFP S mice), photoreceptor transduction efficiency was quantified for AAV44.9 as well as the benchmark capsids AAV5 and AAV8(Y733F).

[0117] We sought to determine whether AAV44.9 transduction could be improved by adding a YF mutation at residue 733 (Y733F), and an additional glutamic acid to aspartic acid substitution at position 531 (E531D). Because cone photoreceptors are the target of many retinal gene therapies (e.g., color blindness and cone-rod dystrophies), the ability of AAV44.9 to express transgenes in cone photoreceptors was evaluated.

[0118] Experimental Method The AAV capsid phylogenetic tree is shown in Figure 1. VP1 amino acid sequences for AAV were aligned using ClustalW (AlignX-Vector NTI). The alignment was then used to generate a phylogenetic tree via 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 and AAV5 was designated as the outgroup.

[0119] Vector Production A self-complementary AAV construct (sc-smCBA-mCherry) containing a truncated chimeric CMV-chicken beta actin (smCBA) promoter-driven mCherry was packaged into AAV44.9, AAV5 and AAV8(Y733F) using a triple transfection plasmid-based system in adherent HEK293T (1,272 cm2 cell growth area) seeded in a stacked cell factory. Cells were harvested and lysed by successive freeze-thaw cycles. Virus in the lysate was purified by iodixanol density gradient and buffer exchanged into Alcon BSS supplemented with Tween 20 (0.014%). Virus was titered by qPCR relative to standards and stored at -80°C. Addition of the Y733F and E531D substitutions was accomplished by site-directed mutagenesis of the AAV2rep-44.9cap plasmid and confirmed by Sanger sequencing. An additional construct containing the cone-specific RBPe-GNAT2 chimeric promoter driving green fluorescent protein (GFP) was packaged into AAV44.9.

[0120] In-vitro transduction assay ARPE-19 (human retinal pigment epithelial cell line) and 661W (mouse cone cell line) cells were cultured at 1.0 × 10 4 Cells were seeded at a concentration of 10,000 cells / well. The next day, cells were infected at 10,000p / cell. Three days after infection, fixed exposure fluorescence microscopy was performed, cells were detached, and flow cytometry was used to quantify reporter protein expression (mCherry) via fluorescence. mCherry expression was calculated by multiplying the average mCherry fluorescence by the number of positive cells. Graphs represent expression levels minus cell only levels.

[0121] injection 2 x 10 in 1 μl 9The vector-containing solution of vg was delivered either intravitreally or subretinally into 4-5 week-old Nrl-GFP and C57BL / 6J mice. In each experiment, a minimum of six eyes that received successful injections were analyzed.

[0122] Fundus examination Four weeks after injection, fundus examination was performed using a Micron III camera (Phoenix Research Laboratories, Pleasanton, CA). Brightfield and red fluorescent images were taken to visualize retinal health and mCherry expression, respectively. Exposure settings were constant between experiments and are indicated in the figure legends.

[0123] Measurement of retinal transduction via flow cytometry Neural retinas (retinas were manually stripped of RPE) were harvested from 4–6 Nrl-GFP eyes per cohort and dissociated with papain. Flow cytometry was performed to quantify the percentage of cells that were positive for GFP (rod photoreceptors), mCherry (non-rod retinal neurons transduced by rAAV), or both (rod photoreceptors transduced by rAAV) for treated dissociated retinas and untreated controls. The percentages of rod and non-rod neural retinal cells transduced by each vector were averaged separately.

[0124] Tissue preparation and immunostaining Four weeks after injection, eyes were enucleated and fixed overnight at 4°C in freshly prepared 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). Corneas and lenses were removed, and eye cups were incubated overnight at 4°C in 30% sucrose solution. 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 glass slides. Retinal cryosections were rinsed in 1× phosphate buffered saline (PBS), blocked with 0.5% Triton-X100 and 1% bovine serum albumin (BSA) for 1 h 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, slides were rinsed with 1x PBS and then incubated with Alexa Fluor donkey anti-mouse secondary antibody (1:500) in 1x PBS for 1 hour at room temperature and counterstained with DAPI. Images were acquired using a confocal laser microscope (Leica TCS SP8) and a fluorescence microscope (EVOS).

[0125] result Rod transduction by AAV44.9 is greater than that of the benchmark vectors AAV5 and AAV8(Y733F) Qualitative and quantitative analysis of AAV44.9, AAV5 and AAV8(Y733F) at 4 weeks of subretinal injection is shown in Figures 2A-2E. FACS data depicted in Figure 2E shows that 2 × 10 9 This indicates that AAV44.9 transduced rods more efficiently than AAV5 and AAV8(Y733F) following subretinal injection in vg. Representative retinal cross-section images showing mCherry expression in photoreceptors and RPE in Nrl-GFP mouse retinas injected with AAV5, AAV8(Y733F), and AAV44.9 at 4 weeks after subretinal injection are shown in Figures 3A-3C.

[0126] Qualitative and quantitative analyses of AAV44.9(Y733F) and AAV44.9(E531D) are shown in Figures 4A-4C. The FACS data depicted in Figure 4C shows that 2 × 10 9 This indicates that AAV44.9(E531D) transduced rods more efficiently than AAV44.9 and AAV44.9(Y733F) following subretinal injection with vg. Representative retinal cross-section images showing mCherry expression primarily in photoreceptors and RPE in Nrl-GFP mouse retinas injected with AAV44.9(Y733F) and AAV44.9(E531D) 4 weeks after subretinal injection are shown in Figure 5A and Figure 5B.

[0127] The transduction efficiencies of unimproved AAV44.9 and AAV44.9(Y733F) in ocular cell lines are shown in Figures 6A and 6B. AAV44.9(Y733F) showed increased transduction relative to AAV44.9 in mouse cone photoreceptor cell lines (Figure 6A), while AAV44.9 was more efficient than AAV9(Y733F) in human RPE cell lines (Figure 6B).

[0128] Qualitative analysis of AAV capsids following intravitreal injection is shown in Figures 7A and 7B. Similar to AAV5 and AAV8(Y733F), AAV44.9 and its derivatives do not lead to efficient retinal transduction following Ivt injection.

[0129] AAV44.9 containing the cone-preferred IRBP / GNAT2 promoter and a GFP reporter shows GFP expression in cone cells in Figure 8A 4 weeks after SR injection. Figures 8B and 8C also show that co-staining with cone arrestin antibody colocalizes with GFP fluorescence.

[0130] The results discussed above show that rod transduction by AAV44.9 was greater than that of the benchmark vectors unimproved AAV5 and AAV8(Y733F). Adding Y733F to AAV44.9 did not improve photoreceptor transduction in vivo. The results also show that the E531D mutation greatly increased photoreceptor transduction, with AAV44.9(E531D) showing 82% rod transduction compared to 61% transduction for unimproved AAV44.9. AAV44.9 efficiently transduces cone cells, as shown by subretinal delivery of AAV44.9-IRBPe / GNAT2-GFP. Similar to AAV5 and AAV8(Y733F), AAV44.9 and its derivatives do not efficiently transduce the retina following Ivt delivery.

[0131] AAV44.9(E531D) outperforms unmodified AAV44.9 and AAVrh.8 in the mouse retina following subretinal injection Representative fundus images (red fluorescent filter) of Nrl-GFP mice were taken 4 weeks after subretinal injection with AAV44.9 or AAV44.9(E531D) (see Figure 9A). Vectors were delivered at 2 x 10 in 1 µL. 9 vg. Exposure and gain settings were consistent throughout the experiment. Experiments were repeated twice to confirm results. Note that the "replicate" experiments were performed with different lots of virus.

[0132] Four weeks after injection, retinas of Nrl-GFP mice (the same mice from the experiment in Figure 9A) were dissociated with papain, and flow cytometry was performed to quantify the proportion of transduced rod (GFP+mCherry positive) or non-rod cells (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 transduction percentage and corresponding values ​​of each cell population are shown in Figure 9B. AAV44.9(E531D) transduced a relatively higher proportion of rod photoreceptors relative to unimproved AAV44.9. Note that the "repeat" experiments were performed with different lots of virus.

[0133] Representative fundus images of Nrl-GFP mice were taken 4 weeks after subretinal injection with AAVrh.8-mCherry (see Figure 10A). The vector was 2 x 10 9 vg. Both GFP and mCherry filtered images are included. AAVrh.8 was evaluated due to its structural similarity to both AAV44.9 and AAV44.9(E531D).

[0134] Four weeks after injection, retinas of Nrl-GFP mice (the same mice from the experiments in Figures 9A and 10A) were dissociated with papain and flow cytometry was performed to quantify the proportion of transduced rod (GFP+mCherry positive) or non-rod cells (mCherry positive). The transduction percentage and corresponding values ​​of each cell population are shown in Figure 10B. AAV44.9(E531D) transduced a relatively higher proportion of cone photoreceptors relative to unmodified AAV44.9 or AAVrh.8.

[0135] Representative fundus images of Nrl-GFP mice were taken 4 weeks after subretinal injection with lower titers of scAAV44.9, scAAV44.9(E531D), or scAAVrh.8 (see Figure 11A). 8 vg. Exposure and gain settings were consistent throughout the experiment.

[0136] Four weeks after injection, retinas of Nrl-GFP mice (the same mice from the experiment in FIG. 11A) were dissociated with papain and flow cytometry was performed to quantify the proportion of transduced rod (GFP+mCherry positive) or non-rod cells (mCherry positive). The transduction percentage and corresponding values ​​of each cell population are shown in FIG. 11B. AAV44.9(E531D) transduced a relatively higher proportion of cone photoreceptors relative to unmodified AAV44.9 or AAVrh.8.

[0137] Qualitative and quantitative analysis of AAVrh.8, AAV44.9 and AAV44.9(E531D) 4 weeks after subretinal injection is shown in Figures 9A and 9B. Figure 9B shows that 2×10 12 1 depicts a FACS plot showing that AAV44.9(E531D) transduced rod cells more efficiently than AAV44.9 following subretinal injection with vg.

[0138] Qualitative and quantitative analysis of AAV44.9, AAV44.9(E531D), and AAVrh.8 at lower titers 4 weeks after subretinal injection are shown in Figures 11A and 11B. Figure 11B shows 2×10 11 Figure 12 depicts FACS plots showing that AAV44.9(E531D) transduced rod cells more efficiently than AAV44.9(E531D) and AAVrh.8 following subretinal injection with vg. Qualitative analysis of AAV44.9(E531D) and AAV44.9 in the context of the cone-specific IRBPe-GNAT2 chimeric promoter 6 weeks after subretinal injection is shown in Figure 12.

[0139] A representative retinal cross section of a WT mouse taken 6 weeks after subretinal injection with AAV44.9(E531D)-IRBP / GNAT2-hGFP is shown in FIG. 13. The IRBP / GNAT2 promoter is a cone-specific promoter. The vector was delivered at 2×10 12 vg. Sections were immunostained with antibodies raised against GFP (green) and cone arrestin (red). AAV-mediated GFP expression colocalized with cone arrestin (a cone-specific marker), confirming that this vector efficiently transduced cones. Representative retinal cross-section images in FIG. 13 show cone arrestin expression in photoreceptors and RPE in Nrl-GFP mouse retinas injected with AAV44.9(E531D) and demonstrate that AAV44.9(E531D) transduces cones very effectively.

[0140] Representative retinal cross sections of WT mice were taken 6 weeks after subretinal injection with AAV44.9-IRBP / GNAT2-hGFP (see FIG. 14). The IRBP / GNAT2 promoter is a cone-specific promoter. The vector was delivered at 2×10 12 vg. Sections were immunostained with antibodies raised against GFP (green) and cone arrestin (red). AAV-mediated GFP expression colocalized with cone arrestin (a cone-specific marker), confirming that this vector efficiently transduces cones.

[0141] Example 2 – Enhanced lateral spread and foveal transduction following subretinal administration of AAV44.9(E531D)-hGRK1-GFP in macaques It was previously determined that the human rhodopsin kinase (hGRK1) promoter has dedicated activity in non-human primate cones and rods. Therefore, the hGRK1 promoter was evaluated for its ability to drive GFP reporter expression in macaque eyes in an improved AAV44.9(E531D) vector. The degree of lateral spread from the initial bleb boundary was also evaluated.

[0142] Two rAAV vectors, AAV44.9-hGRK1-GFP and AAV44.9(E531D)-hGRK1-GFP, were administered subretinal to macaque eyes. The vectors were administered at 1 × 10 12 A control vector, AAV5-hGRK1-GFP, was also administered to the eye.

[0143] Both the improved and unimproved AAV44.9 vectors incorporated particles exhibited enhanced lateral spread and efficacy in subretinal injected macaque subjects (see FIG. 15). The initial border of the bleb and the border of the resulting GFP expression are outlined by white dotted lines in FIG. 15. The identical vasculature is highlighted by a thick dark line for reference. One week after injection, GFP expression mediated by AAV44.9(E531D) was visible. Both AAV44.9 and AAV44.9(E531D) were able to infect 1×10 12 AAV5 was well tolerated in the primate retina at doses of 1000 vg / mL. The control vector, AAV5, mediated restriction of GFP, which remained confined within the original injected bleb.

[0144] AAV44.9-hGRK1-GFP (1 × 10 12 Extrafoveal subretinal injections of 1000 mg ...

[0145] Images were also taken from eyes of macaques injected with AAV44.9(E531D)-hGRK1-GFP, and qualitative analysis in single eyes revealed approximately 50% foveal cone transduction mediated by AAV44.9(E531D).

[0146] 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 ​​the macaque eye. Retinal cross sections were stained with an antibody directed against cone arrestin, and three blinded observers counted the number of GFP-positive cones and rods in five retinal regions across a single plane across the foveal depression. The results of this administration are shown in FIG. 19. OCT scans revealed that the fovea was not detached during the injection (see the right panel of FIG. 19).

[0147] These results show that extrafoveal subretinal injection of AAV44.9(E531D)-hGRK1-GFP in macaques exhibited significant transduction of central cone and rod cells in the absence of foveal detachment. Perifoveal rods and cones were also highly efficiently transduced. Thus, extrafoveal subretinal injection resulted in highly efficient transduction throughout the foveal region.

[0148] As shown in Figures 20A-20D, examination of the parafovea following this injection revealed that AAV44.9(E531D) particles transduced parafoveal cones at both nasal and temporal locations relative to the foveal recess. Of note, however, parafoveal cone transduction was not achieved with unmodified AAV44.9. This finding is intriguing at least because i) parafoveal cones are unresponsive to transduction by various AAV capsid variants, and ii) the earliest structural and functional losses due to aging and congenital retinal disease often occur in the parafoveal region. Despite the dissimilarity in their ability to transduce cones in this region, modified and unmodified AAV44.9 vectors efficiently transduced parafoveal rods to a substantially similar extent.

[0149] As shown in Figures 21A-21B, 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 borders the parafoveal periphery.

[0150] These results further demonstrate that the enhanced lateral spread of transduction provided by the improved AAV44.9(E531D) capsid variant vector may allow subretinal injections in the parafoveal region to result in transduction of foveal cells while avoiding the deleterious effects of inducing foveal detachment in human subjects.

[0151] Example 3 – Enhanced lateral spread following subretinal administration of AAV44.9-Gucy2e-GFP in mice Previous examples have demonstrated foveal transduction of a reporter gene delivered by an improved AAV44.9(E531D) vector. The extent of lateral spread following administration of an improved AAV44.9(E531D) vector encoding a therapeutic peptide of interest was next determined. The therapeutic peptide of interest selected was Gucy2e, the murine homolog of human guanylate cyclase 2D, GUCY2D.

[0152] Two vectors, AAV44.9(E531D)-hGRK1-GUCY2e and AAV8(Y733F)-hGRK1-GUCY2e, were administered subretinally into the eyes of retinal guanylate cyclase1 / 2 double knockout (GCdko) mice. GCdko mice have a complete lack of retinal function. Neither rods nor cones are able to respond to light due to the complete absence of functional retinal guanylate cyclase in these photoreceptors. Gene replacement therapy performed in GCdko mice can therefore be evaluated whether gene replacement / replenishment is successful in rod and cone cells simultaneously. The vectors were administered at a concentration of 1 × 1013 The treated and untreated eyes were delivered at a concentration of 0.01 vg / mL. Response function of treated and untreated eyes was assessed by electroretinogram (ERG) measurements. The ERG response from the photoreceptor cells of the retina is referred to as the "a-wave" and the electrical response from the bipolar cells of the retina is referred to as the "b-wave."

[0153] As shown in Figure 18B, the light response function of cone cells from the eyes treated with AAV vectors was improved relative to those of untreated eyes. These results indicate that both classes of photoreceptors were efficiently targeted by rAAV particles incorporating AAV44.9(E531D) capsid and AAV44.9(Y733F) capsid. As shown in Figure 18A, the maximum a- and b-wave amplitudes were greater after administration of AAV(Y733F) vector than that of AAV44.9(E531D) vector.

[0154] Another major significance of this experiment is that the clinical candidate rAAV vectors expressing therapeutic peptides designed to treat congenital retinal diseases incorporating AAV44.9(E531D) (e.g., vectors for delivery of human GUCY2D transgene) are likely to work in the respective mouse models of the disease (e.g., vectors for delivery of mouse Gucy2e). This is advantageous in the context of preclinical development and evaluation of many different candidate drugs during various stages or phases of production.

[0155] Other Aspects It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that in light thereof, various improvements or modifications may be suggested to those skilled in the art, and are to be encompassed within the spirit and scope of the present application and the appended claims.All references cited herein, including publications, patent applications, and applications, 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 in its entirety herein.The recitation of ranges of values ​​herein is intended only to serve as a shorthand method of individually referring to each separate value within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually described herein.

[0156] Descriptions herein of any aspect or embodiment of the invention using terms such as "comprising," "having," "including," or "containing" with reference to an element or elements are intended to provide support for similar aspects or embodiments of the invention that "consist," "consist essentially of," or "substantially comprise" the specified element or elements, unless stated otherwise or clearly contradicted by context (by way of example, a composition described herein as comprising a specific element should be understood to also describe a composition consisting of that element, unless stated otherwise or clearly contradicted by context).

[0157] All compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of this invention have been described in terms of preferred embodiments, it is apparent to those skilled in the art that modifications may be applied to the compositions and methods described herein and in the sequence of steps of the method without departing from the concept, spirit and scope of the invention. More specifically, it is apparent that certain agents that are chemically and / or physiologically related may be substituted for the agents described herein while achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

1. A recombinant adeno-associated virus (rAAV) particle comprising a capsid protein comprising the amino acid sequence of SEQ ID NO:3, the rAAV particle further comprising a polynucleotide comprising a heterologous nucleic acid sequence.

2. The rAAV particle of claim 1, comprising VP1, VP2, and VP3 capsid 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 the VP3 protein comprises the amino acid sequence of SEQ ID NO:

3.

3. The rAAV particle of claim 1 or 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 (PR) cells or retinal pigment epithelial (RPE) cells.

4. The rAAV particle of any one of claims 1 to 3, wherein the heterologous nucleic acid sequence is flanked by one or more inverted terminal repeat (ITR) sequences.

5. An rAAV particle described in any one of claims 1, 2 and 4, wherein the heterologous nucleic acid sequence encodes a diagnostic or therapeutic agent operably linked to a promoter enabling expression of the diagnostic or therapeutic agent in photoreceptor cells or retinal pigment epithelial (RPE) cells.

6. The rAAV particle described in claim 5, wherein the diagnostic or therapeutic agent encodes a polypeptide, peptide, ribozyme, siRNA, antisense oligonucleotide, antisense polynucleotide, antibody, antigen-binding fragment, or any combination thereof.

7. The rAAV particle of any one of claims 1 to 6, wherein the heterologous nucleic acid sequence is operably linked to a human rhodopsin kinase (hGRK1) promoter.

8. A composition comprising an rAAV particle described in any one of claims 1 to 7, and further comprising one or more pharma- ceutically acceptable carriers, buffers, diluents or excipients.

9. A capsid protein comprising the amino acid sequence of SEQ ID NO:1, 2, and / or 3.

10. A nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 4, 5, and / or 6.

11. A plasmid or viral vector comprising the nucleic acid of claim 10.

12. A cell comprising the capsid protein of claim 9 or the nucleic acid of claim 10.

13. The cell of claim 12 which is a mammalian or insect cell.

14. Use of an rAAV particle described in claim 3 or 5 in the manufacture of a medicament for transducing photoreceptor cells or retinal pigment epithelial cells, wherein transduction comprises administering an rAAV particle described in claim 3 or 5 to the eye of a mammal.

15. Use of an rAAV particle described in claim 5 in the manufacture of a medicament for providing a therapeutically effective amount of a therapeutic agent to a mammal, wherein providing comprises administering an rAAV particle described in claim 5 in an amount effective to provide a therapeutically effective amount of the therapeutic agent to the mammal, to one or both eyes of the mammal, for a time effective to provide a therapeutically effective amount of the therapeutic agent to the mammal.

16. 10. Use of an rAAV particle described in any one of claims 1 to 7 or a composition described in claim 8 in the manufacture of a medicament for treating or alleviating one or more symptoms of a disease, disorder, dysfunction, injury, condition, or trauma in a mammal, wherein treating or alleviating comprises subretinal or intravitreal administration of an rAAV particle described in any one of claims 1 to 7 or a composition described in claim 8 in an amount sufficient to treat or alleviate one or more symptoms of the disease, disorder, dysfunction, injury, condition, or trauma in the mammal, to one or both eyes of the mammal for a time sufficient to treat or alleviate one or more symptoms of the disease, disorder, dysfunction, injury, condition, or trauma in the mammal.

17. Use of the rAAV particle of claim 5 in the manufacture of a medicament for expressing a therapeutic agent in a photoreceptor cell or an RPE cell, comprising: The use, wherein expression comprises subretinal or intravitreal administration of the rAAV particle described in claim 5 to one or both eyes of a mammal for a time effective to produce a therapeutic agent in photoreceptor cells or RPE cells of the mammal, wherein the promoter is a photoreceptor cell-specific promoter or an RPE cell-specific promoter.

18. The use according to any one of claims 14 to 17, wherein the mammal is a human.

19. 20. The use of claim 18, wherein the human has, is suspected of having, is at risk of developing, or has been diagnosed with a retinal disorder, retinal disease, retinal dystrophy, or any combination thereof.

20. 18. The use of claim 15 or 17, wherein the therapeutic agent a) preserves photoreceptor cells or RPE cells, b) restores one or more rod and / or cone mediated functions, c) restores visual behavior in one or both eyes, or d) any combination thereof.

21. The use of claim 20, wherein the therapeutic agent persists in photoreceptor cells or RPE cells substantially for a period of at least three months following an initial administration of the rAAV particles to one or both eyes of a mammal.

22. The use of claim 16, wherein the medicament a) preserves photoreceptor cells or RPE cells, b) restores one or more rod and / or cone mediated functions, c) restores visual behavior in one or both eyes, or d) any combination thereof.

23. The use described in claim 22, wherein the therapeutic agent persists in photoreceptor cells or RPE cells substantially for a period of at least three months following an initial administration of the rAAV particles to one or both eyes of a mammal.

24. The rAAV particle of any one of claims 1 to 7, wherein the heterologous nucleic acid sequence comprises GUCY2D.

25. The rAAV particle of claim 1 or 2, 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 set forth in SEQ ID NO:9.

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