RETGC Gene Therapy

Abstract

Provided herein are expression constructs, viral genomes, and vectors for the expression of retinal membrane-bound guanylyl cyclase 1 (RetGC1), as well as pharmaceutical compositions comprising the vectors disclosed herein.Also provided are methods of using the expression constructs and vectors disclosed herein, including methods of treating a retinal disease in a subject in need thereof, the retinal disease being associated with one or more mutations in the GUCY2D gene, comprising administering to the subject the vectors disclosed herein.

Description

[Technical field]

[0001] The present disclosure relates generally to the fields of molecular biology and medicine. More specifically, the present disclosure provides compositions and methods for gene therapy for the treatment of retinal diseases. [Background technology]

[0002] Retinal membrane-bound guanylyl cyclase (RetGC), located in the disc membrane of photoreceptor outer segments, is one of the key enzymes in photoreceptor physiology, producing cyclic guanosine monophosphate (cGMP), a second messenger of phototransduction in mammalian rods and cones. During photoreceptor excitation and recovery, two RetGC isozymes, RetGC1 and RetGC2 (also known as GC-E and GC-F, or ROSGC1 and ROSGC2, respectively), are tightly regulated by calcium feedback mediated by guanylyl cyclase-activating protein (GCAP).

[0003] Over 100 mutations in GUCY2D, the gene that codes for RetGC, are known to cause two major disorders: autosomal recessive Leber congenital amaurosis type 1 (arLCA or LCA1) or autosomal dominant cone-rod dystrophy (adCRD). In CRD, degeneration begins in the cones, leading to loss of central vision due to the abundance of cones in the macula of the unaffected retina. If degeneration of the cones is followed by degeneration of the rods, CRD can lead to complete blindness. The LCA1 phenotype appears to be even more severe, with loss of photoreceptor function and blindness appearing in early childhood.

[0004] Therefore, there is an urgent need for novel therapies for the treatment of retinal diseases associated with GUCY2D mutations (including but not limited to LCA1 and CRD). Summary of the Invention

[0005] In one aspect, the disclosure provides an expression construct comprising: (a) a promoter sequence that confers expression in photoreceptor cells, and (b) a nucleic acid sequence encoding retinal membrane-bound guanylyl cyclase 1 (RetGC1); wherein the nucleic acid sequence is operably linked to the promoter.

[0006] In one embodiment, the promoter sequence is a rhodopsin kinase (RK) or cytomegalovirus (CMV) promoter sequence.

[0007] In one embodiment, the promoter sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 7. In one embodiment, the promoter sequence comprises SEQ ID NO:7.

[0008] In one embodiment, the promoter sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 8. In one embodiment, the promoter sequence comprises SEQ ID NO:8.

[0009] In one embodiment, the expression construct further comprises a post-transcriptional regulatory element. In one embodiment, the post-transcriptional regulator comprises a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE). In one embodiment, the post-transcriptional regulatory element comprises a sequence that is at least 90% identical to SEQ ID NO: 10. In one embodiment, the post-transcriptional regulatory element comprises SEQ ID NO: 10.

[0010] In one embodiment, the nucleic acid sequence encoding RetGC1 is a coding sequence (cds) from the wild-type RetGC1 (GUCY2D) gene. In one embodiment, the nucleic acid sequence encoding RetGC1 is a codon-optimized sequence. In some embodiments, the nucleic acid sequence encoding RetGC1 comprises a sequence that is at least 90% identical to SEQ ID NO:9. In some embodiments, the nucleic acid sequence encoding RetGC1 comprises SEQ ID NO:9. In some embodiments, the nucleic acid sequence encoding RetGC1 comprises a sequence that is at least 90% identical to SEQ ID NO:13. In some embodiments, the nucleic acid sequence encoding RetGC1 comprises SEQ ID NO:13. In some embodiments, the nucleic acid sequence encoding RetGC1 comprises a sequence that is at least 90% identical to SEQ ID NO:14. In some embodiments, the nucleic acid sequence encoding RetGC1 comprises SEQ ID NO:14. In some embodiments, the nucleic acid sequence encoding RetGC1 encodes a protein that comprises a sequence that is at least 90% identical to SEQ ID NO:12. In some embodiments, the nucleic acid sequence encoding RetGC1 encodes a protein that comprises SEQ ID NO:12.

[0011] In one embodiment, the expression construct further comprises a polyadenylation signal. In an embodiment, the polyadenylation signal comprises a bovine growth hormone polyadenylation (BGH-polyA) signal. In one embodiment, the polyadenylation signal comprises a sequence that is at least 90% identical to SEQ ID NO:11. In one embodiment, the polyadenylation signal comprises SEQ ID NO:11.

[0012] In some embodiments, the expression construct comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-4. In some embodiments, the expression construct comprises a sequence selected from the group consisting of SEQ ID NOs: 1-4.

[0013] In one aspect, a vector is provided that comprises the expression construct disclosed herein. In an embodiment, the vector is a viral vector. In an embodiment, the vector is an adeno-associated virus (AAV) vector. In an embodiment, the vector comprises a genome derived from AAV serotype AAV2. In an embodiment, the vector comprises a capsid derived from AAV 7m8.

[0014] In one aspect, there is provided a pharmaceutical composition comprising a vector disclosed herein and a pharma- ceutically acceptable carrier.

[0015] In one aspect, a method for treating a retinal disease in a subject in need of treatment is provided, wherein the retinal disease is associated with one or more mutations in GUCY2D gene, comprising administering to the subject a vector or pharmaceutical composition as disclosed herein.In some embodiments, the retinal disease is cone-rod dystrophy (CRD) or Leber congenital amaurosis type 1 (LCA1).In one embodiment, the retinal disease is LCA1.

[0016] In one aspect, a method is provided for increasing expression of rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit β (PDE6β) in a subject in need of increased expression of PDE6β, comprising administering to the subject a vector or pharmaceutical composition disclosed herein.

[0017] In one aspect, a method is provided for increasing cyclic guanosine monophosphate (cGMP) levels in photoreceptors in a subject in need thereof, comprising administering to the subject a vector or pharmaceutical composition disclosed herein.

[0018] In embodiments, the vector or pharmaceutical composition is administered by intraocular injection, hi embodiments, the vector or pharmaceutical composition is injected into the central retina of the subject. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows a schematic diagram of the human retina showing the cell layers. [Diagram 2] Wild-type (WT) and RetGC KO iPSC retinal organoids at 20 weeks are shown. Top row: Brightfield images showing whole organoids with outer segment "brush border" at the peripheral edge in both WT and RetGC KO. Middle row. Cone and rod outer and inner segments are stained for cone opsin and rhodopsin. Synapses in the outer plexiform layer (OPL) and inner plexiform layer (IPL) are stained for Ribeye and VGlut. Bipolar cells and amacrine / ganglion cells are stained for PKCa and calretinin. RetGC is localized to photoreceptor outer segments in WT organoids and absent in RetGC KO organoids. [Diagram 3] Total protein expression (Western blot) in whole WT and RetGC KO organoids from day 40 to day 220 in control and RetGC KO organoids (normalized to β-tubulin). [Figure 4] The design of four transgene cassettes packaged into the AAV 7m8 capsid is shown, incorporating the RK and CMV promoters along with the WT GUCY2D gene, with or without the WPRE element and the bovine growth hormone polyadenylation (BGH-polyA) signal. [Diagram 5] Shown are PDE6 staining intensity in WT and transduced RetGC KO organoids. Representative images of retinal organoid outer segments stained for rhodopsin and PDE6β. [Figure 6] Quantitative immunofluorescence for PDE6β staining intensity within rhodopsin-positive outer segments. Each point represents a tile scan of an individual organoid. Staining intensity is expressed as a percentage of WT organoids treated, stained, and imaged on the same block. [Figure 7]Western blot results to determine protein expression of RetGC and β-tubulin (housekeeping) in retinal organoids after transduction with 7m8 vector. Ratiometric, densitometric quantification of the western blot signal of RetGC relative to β-tubulin is shown. [Figure 8] Quantification of cGMP concentration [nM] by FRET assay is shown. Absorbance readings were normalized to total protein [ug]. WT organoids vs. RetGC knockout (non-transduced NT) organoids were compared with organoids transduced with the four vectors (n=7 embryoid bodies (EBs) per experimental group). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Provided herein are expression constructs, viral genomes, and vectors for expression of retinal membrane-bound guanylyl cyclase 1 (RetGC1), as well as methods of using the expression constructs, viral genomes, and vectors to treat retinal diseases associated with one or more mutations in the GUCY2D gene.

[0021] RetGC RetGC catalyzes the synthesis of cGMP in photoreceptor rods and cones, and thus plays an essential role in phototransduction by mediating cGMP replenishment during the visual cycle.

[0022] During photoreceptor excitation and recovery, two RetGC isozymes, RetGC1 and RetGC2 (also known as GC-E and GC-F, or ROSGC1 and ROSGC2, respectively), are tightly regulated by calcium feedback mediated by guanylyl cyclase-activating protein (GCAP).

[0023] The role of RetGC1 is to replenish cGMP levels after light exposure. In the dark, cGMP levels are maintained at a constant rate, keeping cGMP-gated channels open and allowing inward current influx, thereby maintaining partial depolarization of the cell. Light exposure leads to hydrolysis of cGMP and channel closure, resulting in an increase in intracellular Ca. 2+ This promotes a rapid decrease in Ca and hyperpolarization of the cell. 2+ Under these conditions, guanylate cyclase-activating protein (GCAP) stimulates GC1 activity, leading to cGMP synthesis, channel reopening, and restoration of the dark state.

[0024] As the photon passes through the outer segment, it is captured by opsins embedded in the outer segment membrane. The second messenger cGMP is a key component in the signaling step of the visual cycle. The balance of synthesis and degradation in the cytoplasm of the outer segment controls the signaling step of the visual cycle. cGMP is produced from GTP in a reaction catalyzed by RetGC. cGMP is produced by the reaction of Ca 2+ It binds to a channel that allows the influx of ions. Upon phototransduction, cGMP is hydrolyzed to GMP by PDE6, which closes the cGMP channel. This allows Ca 2+ The influx of Ca is inhibited, and Ca is released from the disc membrane. 2+ flows out and its concentration decreases.

[0025] In the phototransduction cycle, photons are absorbed by rhodopsin in rods and cone opsin in cones, where 11-cis retinal is converted to all-trans retinal. All-trans retinal activates the alpha subunit of the G protein transducin, converting GDP to GTP in the process. The generated GTP can then activate the gamma subunit of phosphodiesterase 6 (PDE6), inhibiting cGMP production. This closes the cGMP-gated channel, thus stopping the influx of calcium ions. GCAP in the dark state is bound to calcium ions and therefore cannot associate with RetGC. Ca from GCAP in the light state is converted to GTP by acetylcholine. 2+ Release of GCAP allows GCAP to bind to RetGC and produce cGMP. In parallel, all-trans is inactivated by phosphorylation by rhodopsin kinase and binding to arrestin. GTP bound to the G protein transducin is converted back to GDP. The whole cycle then repeats.

[0026] RetGC1 is encoded by the gene GUCY2D in humans and by the gene Gucy2e in mice, whereas RetGC2 is encoded by the gene GUCY2F in humans.

[0027] Mutations in the GUCY2D gene, which encodes RetGC1, result in severe retinal diseases in humans, primarily autosomal dominant cone-rod dystrophy (adCRD) or autosomal recessive Leber congenital amaurosis type 1 (arLCA). In CRD, degeneration begins in the cones, leading to loss of central vision due to the abundance of cones in the macula of the unaffected retina. If cone degeneration is followed by rod degeneration, CRD can lead to complete blindness. The LCA1 phenotype appears to be even more severe, with loss of photoreceptor function and blindness manifesting in early childhood. Another gene involved in the development of LCA (type 12) is rd3, which encodes the retinal degeneration 3 (RD3) protein, which is an effective inhibitor of GCAP-mediated activation of RetGC1 and is involved in the transport of RetGC1 from the inner to the outer segments of photoreceptors.

[0028] A total of 144 different GUCY2D mutations have been reported. The majority (127 mutations) result in an LCA phenotype in affected patients. LCA-associated mutations are usually recessive and null (mainly frameshift, nonsense, and splicing mutations) and can affect all domains of the RetGC enzyme, whereas CRD mutations are mainly dominant missense and are clustered between E837 and T849 in the "hotspot region" that corresponds to the dimerization domain.

[0029] LCA1 patients present within the first year of life and are routinely reported to have reduced visual acuity, reduced or incapable of recording electroretinogram (ERG) responses, nystagmus, dactyly, and an apparently normal fundus. Reports regarding the extent of photoreceptor degeneration associated with the disease are conflicting. Histopathological analysis of two postmortem retinas (a 26-week-old premature aborted fetus and a 12-year-old donor) revealed signs of photoreceptor degeneration in both rods and cones. Subsequent studies using modern antemortem imaging (i.e., optical coherence tomography) revealed no overt degeneration in patients as old as 53 years of age. More recent studies have shown that, despite the severe visual impairment, LCA1 patients retain normal photoreceptor layer structure, with the exception of abnormalities of the foveal cone outer segments and, in some patients, foveal cone loss.

[0030] In CRD, abnormalities in rod function are less severe than those in cone function and may be detected later in the course of the disease than cone dysfunction. Diagnosis is established by electrophysiological evaluation; functional outcomes vary with the stage of the disease and the age of the individual. A diagnosis of cone-rod dystrophy can be supported by demonstration of peripheral and central visual field loss.

[0031] Expression constructs In one aspect, an expression construct is provided that includes: (a) a promoter sequence that confers expression in photoreceptor cells; and (b) a nucleic acid sequence that encodes retinal membrane-bound guanylyl cyclase (RetGC1), the nucleic acid sequence being operably linked to the promoter. As used herein, "operably linked" refers to both expression control sequences (e.g., promoters) that are contiguous with the coding sequence (cds) of RetGC1, and expression control sequences that act in trans or at a distance to control the expression of RetGC1. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance protein processing and / or secretion.

[0032] Numerous expression control sequences, such as natural, constitutive, inducible and / or tissue-specific sequences, are known in the art and can be utilized to drive the expression of the RetGC1 (GUCY2D) transgene, depending on the type of expression desired. For eukaryotic cells, expression control sequences typically include promoter sequences, enhancer sequences, and polyadenylation sequences that may include splice donor and splice acceptor sites. The polyadenylation sequence is generally inserted after the sequence encoding RetGC1 and before the 3'ITR sequence. Another regulatory component of rAAV useful in the methods disclosed herein is an internal ribosome entry site (IRES). IRES sequences can be used to produce multiple polypeptides from a single gene transcript. IRES (or other suitable sequences) can be used to produce proteins containing multiple polypeptide chains or to express two different proteins from or within the same cell. An exemplary IRES is the poliovirus internal ribosome entry sequence, which supports expression of transgenes in photoreceptors, RPE and ganglion cells. Preferably, the IRES is located 3' to the sequence encoding RetGC1 in the rAAV vector.

[0033] In one embodiment, the promoter sequence comprises a rhodopsin kinase (RK) promoter sequence. In an embodiment, the promoter sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7. In one embodiment, the promoter sequence comprises SEQ ID NO:7.

[0034] In one embodiment, the promoter sequence comprises a cytomegalovirus (CMV) promoter sequence. In an embodiment, the promoter sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8. In one embodiment, the promoter sequence comprises SEQ ID NO:8.

[0035] In some embodiments, the promoter is specific to photoreceptor cells, i.e., the promoter is active in photoreceptor cells but has reduced or no activity in other cell types.

[0036] In one embodiment, the nucleic acid sequence encoding RetGC1 is a coding sequence derived from the wild-type RetGC1 (GUCY2D) gene. In one embodiment, the nucleic acid sequence encoding RetGC1 is a codon-optimized sequence. In some embodiments, the nucleic acid sequence encoding RetGC1 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9. In one embodiment, the nucleic acid sequence encoding RetGC1 comprises SEQ ID NO:9. In some embodiments, the nucleic acid sequence encoding RetGC1 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:13. In one embodiment, the nucleic acid sequence encoding RetGC1 comprises SEQ ID NO:13. In some embodiments, the nucleic acid sequence encoding RetGC1 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14. In one embodiment, the nucleic acid sequence encoding RetGC1 comprises SEQ ID NO: 14. In some embodiments, the nucleic acid sequence encoding RetGC1 encodes a protein that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12. In some embodiments, the nucleic acid sequence encoding RetGC1 encodes a protein that comprises SEQ ID NO: 12.

[0037] In one embodiment, the expression construct comprises a post-transcriptional regulatory element. In one embodiment, the expression construct comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, the post-transcriptional regulatory element comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:10. In one embodiment, the post-transcriptional regulatory element comprises SEQ ID NO:10.

[0038] In one embodiment, the expression construct comprises a polyadenylation signal. In one embodiment, the expression construct comprises a bovine growth hormone polyadenylation (BGH-polyA) signal. In some embodiments, the polyadenylation signal comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11. In one embodiment, the polyadenylation signal comprises SEQ ID NO:11.

[0039] In one embodiment, the expression construct comprises a nucleic acid comprising one or more inverted terminal repeats (ITRs). In one embodiment, the ITR sequences are derived from AAV serotype 2. In one embodiment, the 5'ITR sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:5. In one embodiment, the 5'ITR sequence comprises SEQ ID NO:5. In one embodiment, the 3'ITR sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:6. In one embodiment, the 3'ITR sequence comprises SEQ ID NO:6.

[0040] vector In one aspect, a recombinant vector and its use for introducing a transgene or expression construct into a cell are provided. In some embodiments, the recombinant vector comprises a recombinant DNA construct that includes additional DNA elements, including a DNA segment that results in replication of the DNA in the host cell and expression of the target gene in the target cell at an appropriate level. Those skilled in the art will understand that the expression control sequences (promoter, enhancer, etc.) are selected based on their ability to promote expression of the target gene in the target cell. As used herein, "vector" refers to a vehicle that includes a polynucleotide to be delivered to a host cell in vitro or in vivo. Non-limiting examples of vectors include recombinant plasmids, yeast artificial chromosomes (YACs), minichromosomes, DNA minicircles, or viruses (including sequences derived from viruses). A vector may also refer to a virion that includes a nucleic acid that is delivered to a host cell either in vitro or in vivo. In some embodiments, a vector refers to a virion that includes a recombinant viral genome, where the viral genome includes one or more ITRs and a transgene.

[0041] In one embodiment, the recombinant vector is a viral vector or a combination of viral vectors.

[0042] In one aspect, a vector is provided that includes any of the expression constructs disclosed herein.

[0043] In one aspect, a vector is provided comprising a nucleic acid comprising: (a) a promoter sequence that confers expression in a photoreceptor cell; and (b) a nucleic acid sequence encoding RetGC1, wherein the nucleic acid sequence encoding RetGC1 is operably linked to the promoter.

[0044] In one embodiment, the promoter sequence comprises an RK promoter sequence. In some embodiments, the promoter sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7. In one embodiment, the promoter sequence comprises SEQ ID NO:7.

[0045] In one embodiment, the promoter sequence comprises a CMV promoter sequence. In some embodiments, the promoter sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8. In one embodiment, the promoter sequence comprises SEQ ID NO:8.

[0046] In some embodiments, the promoter is specific to photoreceptor cells.

[0047] In one embodiment, the nucleic acid sequence encoding RetGC1 is a coding sequence derived from the wild-type RetGC1 (GUCY2D) gene. In one embodiment, the nucleic acid sequence encoding RetGC1 is a codon-optimized sequence. In some embodiments, the nucleic acid sequence encoding RetGC1 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9. In one embodiment, the nucleic acid sequence encoding RetGC1 comprises SEQ ID NO:9. In some embodiments, the nucleic acid sequence encoding RetGC1 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:13. In one embodiment, the nucleic acid sequence encoding RetGC1 comprises SEQ ID NO:13. In some embodiments, the nucleic acid sequence encoding RetGC1 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14. In one embodiment, the nucleic acid sequence encoding RetGC1 comprises SEQ ID NO: 14. In some embodiments, the nucleic acid sequence encoding RetGC1 encodes a protein that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12. In some embodiments, the nucleic acid sequence encoding RetGC1 encodes a protein that comprises SEQ ID NO: 12.

[0048] In one embodiment, the vector comprises a nucleic acid comprising a post-transcriptional regulatory element. In one embodiment, the vector comprises a nucleic acid comprising a WPRE. In some embodiments, the post-transcriptional regulatory element comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10. In one embodiment, the post-transcriptional regulatory element comprises SEQ ID NO: 10.

[0049] In one embodiment, the vector comprises a nucleic acid comprising a polyadenylation signal. In one embodiment, the vector comprises a nucleic acid comprising a BGH-polyA signal. In some embodiments, the polyadenylation signal comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11. In one embodiment, the polyadenylation signal comprises SEQ ID NO:11.

[0050] In one embodiment, the vector comprises a nucleic acid comprising one or more inverted terminal repeats (ITRs). In one embodiment, the ITR sequences are derived from AAV serotype 2. In one embodiment, the 5'ITR sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:5. In one embodiment, the 5'ITR sequence comprises SEQ ID NO:5. In one embodiment, the 3'ITR sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:6. In one embodiment, the 3'ITR sequence comprises SEQ ID NO:6.

[0051] In some embodiments, the vector comprises a nucleic acid comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the sequences of SEQ ID NOs: 1-4. In some embodiments, the vector comprises a nucleic acid comprising a sequence that comprises a sequence selected from the group consisting of SEQ ID NOs: 1-4.

[0052] In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence containing the RK promoter sequence; (b) a nucleic acid sequence encoding RetGC1, wherein the nucleic acid sequence encoding RetGC1 is operably linked to a promoter; (c)WPRE; (d) BGH-polyA signal; and (e) One or more ITRs. In some embodiments, the vector comprises two ITR sequences.

[0053] In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence including a CMV promoter sequence; (b) a nucleic acid sequence encoding RetGC1, wherein the nucleic acid sequence encoding RetGC1 is operably linked to a promoter; (c)WPRE; (d) BGH-polyA signal; and (e) One or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

[0054] In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7; (b) a nucleic acid sequence encoding RetGC1, wherein the nucleic acid sequence encoding RetGC1 is operably linked to a promoter, and the nucleic acid sequence encoding RetGC1 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9, SEQ ID NO:13, or SEQ ID NO:14; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:10; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11; and (e) One or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

[0055] In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8; (b) a nucleic acid sequence encoding RetGC1, wherein the nucleic acid sequence encoding RetGC1 is operably linked to a promoter, and the nucleic acid sequence encoding RetGC1 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9, SEQ ID NO:13, or SEQ ID NO:14; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:10; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11; and (e) One or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

[0056] In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7; (b) a nucleic acid sequence encoding a RetGC1 protein, wherein the RetGC1 protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12, and wherein the nucleic acid sequence encoding the RetGC1 protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:10; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11; and (e) One or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

[0057] In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8; (b) a nucleic acid sequence encoding a RetGC1 protein, wherein the RetGC1 protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12, and wherein the nucleic acid sequence encoding the RetGC1 protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:10; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11; and (e) One or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

[0058] In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising SEQ ID NO:7; (b) a nucleic acid sequence encoding RetGC1, wherein the nucleic acid sequence encoding RetGC1 is operably linked to a promoter, and the nucleic acid sequence encoding RetGC1 comprises SEQ ID NO:9, SEQ ID NO:13, or SEQ ID NO:14; (c) a post-transcriptional regulatory element comprising SEQ ID NO:10; (d) a polyadenylation signal comprising the sequence of SEQ ID NO: 11; and (e) One or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

[0059] In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising SEQ ID NO:8; (b) a nucleic acid sequence encoding RetGC1, wherein the nucleic acid sequence encoding RetGC1 is operably linked to a promoter, and the nucleic acid sequence encoding RetGC1 comprises SEQ ID NO:9, SEQ ID NO:13, or SEQ ID NO:14; (c) a post-transcriptional regulatory element comprising SEQ ID NO:10; (d) a polyadenylation signal comprising the sequence of SEQ ID NO: 11; and (e) One or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

[0060] In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising SEQ ID NO:7; (b) a nucleic acid sequence encoding a RetGC1 protein, the RetGC1 protein comprising SEQ ID NO: 12, the nucleic acid sequence encoding the RetGC1 protein being operably linked to a promoter; (c) a post-transcriptional regulatory element comprising SEQ ID NO:10; (d) a polyadenylation signal comprising the sequence of SEQ ID NO: 11; and (e) One or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

[0061] In one embodiment, a vector is provided that comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising SEQ ID NO:8; (b) a nucleic acid sequence encoding a RetGC1 protein, the RetGC1 protein comprising SEQ ID NO: 12, the nucleic acid sequence encoding the RetGC1 protein being operably linked to a promoter; (c) a post-transcriptional regulatory element comprising SEQ ID NO:10; (d) a polyadenylation signal comprising the sequence of SEQ ID NO: 11; and (e) One or more ITRs. In some embodiments, the nucleic acid comprises two ITR sequences.

[0062] Viral Vectors Viral vectors for expressing a target gene in a target cell, tissue, or organism are known in the art and include, for example, AAV vectors, adenoviral vectors, lentiviral vectors, retroviral vectors, poxvirus vectors, baculovirus vectors, herpes simplex virus vectors, vaccinia virus vectors, or synthetic viral vectors (e.g., chimeric, mosaic, or pseudotyped viruses, and / or viruses containing foreign proteins, synthetic polymers, nanoparticles, or small molecules).

[0063] AAV vectors Adeno-associated viruses (AAV) are small single-stranded DNA viruses that require a helper virus to facilitate efficient replication. The 4.7 kb genome of AAV is characterized by two inverted terminal repeats (ITRs) and two open reading frames that code for the Rep and Cap proteins, respectively. The Rep reading frame codes for four proteins with molecular weights of 78 kD, 68 kD, 52 kD, and 40 kD. These proteins function primarily in AAV replication and rescue and in controlling AAV integration into the host cell chromosome. The Cap reading frame codes for three structural proteins with molecular weights of 85 kD (VP1), 72 kD (VP2), and 61 kD (VP3), which form the virion capsid. More than 80% of the total protein in the AAV virion consists of VP3. Adjacent to the 5' and 3' ends of the rep and cap open reading frames are inverted terminal repeats (ITRs) approximately 145 bp long. The two ITRs are the only cis elements essential for AAV replication, rescue, packaging, and integration of the AAV genome. The entire rep and cap domains can be excised and replaced with therapeutic or reporter transgenes.

[0064] Recombinant adeno-associated virus "rAAV" vectors include any vector derived from any adeno-associated virus serotype. rAAV vectors can have one or more of the AAV wild-type genes deleted in whole or in part, preferably the Rep and / or Cap genes, but retain functional flanking ITR sequences.

[0065] In some embodiments, the viral vector is a rAAV virion that comprises a rAAV genome and one or more capsid proteins, hi some embodiments, the rAAV genome comprises an expression cassette disclosed herein.

[0066] In some embodiments, the viral vectors disclosed herein comprise a nucleic acid comprising an AAV 5'ITR and 3'ITR located 5' and 3', respectively, to the sequence encoding RetGC1. However, in certain embodiments, it may be desirable for the nucleic acid to contain 5'ITR and 3'ITR sequences arranged in tandem, e.g., 5'-3', or head-to-tail, or in another alternative arrangement. In still other embodiments, it may be desirable for the nucleic acid to contain multiple copies of the ITRs, or to have the 5'ITR (or conversely, the 3'ITR) located both 5' and 3' of the sequence encoding RetGC1. The ITR sequences may be located immediately upstream and / or downstream of the heterologous molecule, or intervening sequences may be present. The ITRs need not be wild-type nucleotide sequences, and may be modified (e.g., by insertion, deletion, or substitution of nucleotides), so long as the sequences provide functional rescue, replication, and packaging. The ITRs may be selected from AAV2, or among other AAV serotypes, as described herein.

[0067] In some embodiments, the viral vector is an AAV vector, such as, for example, AAV1 (i.e., an AAV containing AAV1 ITRs and AAV1 capsid protein), AAV2 (i.e., an AAV containing AAV2 ITRs and AAV2 capsid protein), AAV3 (i.e., an AAV containing AAV3 ITRs and AAV3 capsid protein), AAV4 (i.e., an AAV containing AAV4 ITRs and AAV4 capsid protein), AAV5 (i.e., an AAV containing AAV5 ITRs and AAV5 capsid protein), AAV6 (i.e., an AAV containing AAV6 ITRs and AAV6 capsid protein), AAV7 (i.e., an AAV containing AAV7 ITRs and AAV7 capsid protein), AAV8 (i.e., an AAV containing AAV8 ITRs and AAV8 capsid protein), AAV9 (i.e., an AAV9 ITRs and AAV9 capsid protein), AAVrh74 (i.e., an AAV containing AAVrh74 ITRs and AAVrh74 capsid protein), AAVrh.8 (i.e., an AAV containing AAVrh.8 ITRs and AAVrh.8 capsid protein), or AAVrh.10 (i.e., an AAV containing AAVrh.10 ITRs and AAVrh.10 capsid protein).

[0068] In some embodiments, the viral vector is a pseudotyped AAV vector that contains ITRs from one AAV serotype and capsid proteins from a different AAV serotype. In some embodiments, the pseudotyped AAV is AAV2 / 9 (i.e., AAV containing AAV2 ITRs and AAV9 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 10 (i.e., AAV containing AAV2 ITRs and AAV10 capsid proteins).

[0069] In some embodiments, the pseudotyped AAV is AAV2 / 7m8 (i.e., an AAV containing the AAV2 ITRs and the AAV 7m8 capsid protein).

[0070] In some embodiments, the AAV vector contains a recombinant capsid protein, such as a capsid protein that contains a chimera of one or more capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAVrh.8, or AAVrh.10. In embodiments, the capsid is a variant AAV capsid, such as the AAV2 variant rAAV2-retro (SEQ ID NO: 44 from WO2017 / 218842, incorporated herein by reference).

[0071] In one aspect, a viral genome is provided comprising (a) a promoter sequence that confers expression in photoreceptor cells, and (b) a nucleic acid comprising a nucleic acid sequence encoding RetGC1, wherein the nucleic acid sequence encoding RetGC1 is operably linked to the promoter.

[0072] In one embodiment, the promoter sequence comprises an RK promoter sequence. In some embodiments, the promoter sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7. In one embodiment, the promoter sequence comprises SEQ ID NO:7.

[0073] In one embodiment, the promoter sequence comprises a CMV promoter sequence. In some embodiments, the promoter sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8. In one embodiment, the promoter sequence comprises SEQ ID NO:8.

[0074] In some embodiments, the promoter is specific to photoreceptor cells.

[0075] In one embodiment, the nucleic acid sequence encoding RetGC1 is a coding sequence derived from the wild-type RetGC1 (GUCY2D) gene. In one embodiment, the nucleic acid sequence encoding RetGC1 is a codon-optimized sequence. In some embodiments, the nucleic acid sequence encoding RetGC1 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9. In one embodiment, the nucleic acid sequence encoding RetGC1 comprises SEQ ID NO:9. In some embodiments, the nucleic acid sequence encoding RetGC1 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:13. In one embodiment, the nucleic acid sequence encoding RetGC1 comprises SEQ ID NO:13. In some embodiments, the nucleic acid sequence encoding RetGC1 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14. In one embodiment, the nucleic acid sequence encoding RetGC1 comprises SEQ ID NO: 14. In some embodiments, the nucleic acid sequence encoding RetGC1 encodes a protein that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12. In some embodiments, the nucleic acid sequence encoding RetGC1 encodes a protein that comprises SEQ ID NO: 12.

[0076] In one embodiment, the viral genome comprises a nucleic acid comprising a post-transcriptional regulatory element. In one embodiment, the viral genome comprises a nucleic acid comprising a WPRE. In some embodiments, the post-transcriptional regulatory element comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10. In one embodiment, the post-transcriptional regulatory element comprises SEQ ID NO: 10.

[0077] In one embodiment, the viral genome comprises a nucleic acid comprising a polyadenylation signal. In one embodiment, the viral genome comprises a nucleic acid comprising a BGH-polyA signal. In some embodiments, the polyadenylation signal comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11. In one embodiment, the polyadenylation signal comprises SEQ ID NO:11.

[0078] In one aspect, the viral genome comprises a nucleic acid comprising one or more inverted terminal repeats (ITRs). In one embodiment, the ITR sequence is derived from AAV serotype 2. In one embodiment, the 5'ITR sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:5. In one embodiment, the 5'ITR sequence comprises SEQ ID NO:5. In one embodiment, the 3'ITR sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:6. In one embodiment, the 3'ITR sequence comprises SEQ ID NO:6.

[0079] In some embodiments, the viral genome comprises a nucleic acid comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the sequences of SEQ ID NOs: 1-4. In some embodiments, the viral genome comprises a nucleic acid comprising a sequence that comprises a sequence selected from the group consisting of SEQ ID NOs: 1-4.

[0080] In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence including the RK promoter sequence; (b) a nucleic acid sequence encoding RetGC1, wherein the nucleic acid sequence encoding RetGC1 is operably linked to a promoter; (c)WPRE; (d) BGH-polyA signal; and (e) One or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

[0081] In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence including a CMV promoter sequence; (b) a nucleic acid sequence encoding RetGC1, wherein the nucleic acid sequence encoding RetGC1 is operably linked to a promoter; (c)WPRE; (d) BGH-polyA signal; and (e) One or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

[0082] In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7; (b) a nucleic acid sequence encoding RetGC1, wherein the nucleic acid sequence encoding RetGC1 is operably linked to a promoter, and the nucleic acid sequence encoding RetGC1 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9, SEQ ID NO:13, or SEQ ID NO:14; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:10; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11; and (e) One or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

[0083] In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8; (b) a nucleic acid sequence encoding RetGC1, wherein the nucleic acid sequence encoding RetGC1 is operably linked to a promoter, and the nucleic acid sequence encoding RetGC1 comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9, SEQ ID NO:13, or SEQ ID NO:14; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:10; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11; and (e) One or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

[0084] In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7; (b) a nucleic acid sequence encoding a RetGC1 protein, wherein the RetGC1 protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12, and wherein the nucleic acid sequence encoding the RetGC1 protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:10; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11; and (e) One or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

[0085] In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8; (b) a nucleic acid sequence encoding a RetGC1 protein, wherein the RetGC1 protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12, and wherein the nucleic acid sequence encoding the RetGC1 protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:10; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11; and (e) One or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

[0086] In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising SEQ ID NO:7; (b) a nucleic acid sequence encoding RetGC1, wherein the nucleic acid sequence encoding RetGC1 is operably linked to a promoter, and the nucleic acid sequence encoding RetGC1 comprises SEQ ID NO:9, SEQ ID NO:13, or SEQ ID NO:14; (c) a post-transcriptional regulatory element comprising SEQ ID NO:10; (d) a polyadenylation signal comprising the sequence of SEQ ID NO: 11; and (e) One or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

[0087] In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising SEQ ID NO:8; (b) a nucleic acid sequence encoding RetGC1, wherein the nucleic acid sequence encoding RetGC1 is operably linked to a promoter, and the nucleic acid sequence encoding RetGC1 comprises SEQ ID NO:9, SEQ ID NO:13, or SEQ ID NO:14; (c) a post-transcriptional regulatory element comprising SEQ ID NO:10; (d) a polyadenylation signal comprising the sequence of SEQ ID NO: 11; and (e) One or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

[0088] In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising SEQ ID NO:7; (b) a nucleic acid sequence encoding a RetGC1 protein, the RetGC1 protein comprising SEQ ID NO: 12, the nucleic acid sequence encoding the RetGC1 protein being operably linked to a promoter; (c) a post-transcriptional regulatory element comprising SEQ ID NO:10; (d) a polyadenylation signal comprising the sequence of SEQ ID NO: 11; and (e) One or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

[0089] In one embodiment, a viral genome is provided that comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising SEQ ID NO:8; (b) a nucleic acid sequence encoding a RetGC1 protein, the RetGC1 protein comprising SEQ ID NO: 12, the nucleic acid sequence encoding the RetGC1 protein being operably linked to a promoter; (c) a post-transcriptional regulatory element comprising SEQ ID NO:10; (d) a polyadenylation signal comprising the sequence of SEQ ID NO: 11; and (e) One or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

[0090] Other viral vectors include adenovirus (AV) vectors, such as those based on human adenovirus type 2 and human adenovirus type 5, which have been rendered replication-deficient by deletion of the E1 and E3 regions. A transcription cassette can be inserted into the E1 region to obtain an E1 / E3 deleted recombinant AV vector. Adenovirus vectors include helper-dependent, large-capacity adenovirus vectors (also known as large-capacity, "gutless" or "gutted" vectors) that do not contain viral coding sequences. These vectors contain cis-acting elements required for viral DNA replication and packaging, mainly inverted terminal repeats (ITRs) and packaging signals (CYs). These helper-dependent AV vector genomes can carry a few hundred base pairs up to approximately 36 kb of foreign DNA.

[0091] Alternatively, other systems such as lentiviral vectors can be used.Lentiviral-based systems can transduce non-dividing cells as well as dividing cells, making them useful for targeting non-dividing cells of the CNS, for example.Lentiviral vectors are derived from human immunodeficiency virus, and like that virus, they can be integrated into host genome, providing the possibility of very long-term gene expression.

[0092] Polynucleotides, including plasmids, YACs, minichromosomes and minicircles, carrying target genes containing expression cassettes can also be introduced into cells or organisms by non-viral vector systems, for example using cationic lipids, polymers, or both as carriers. Conjugated poly-L-lysine (PLL) polymer and polyethyleneimine (PEI) polymer systems can also be used to deliver vectors into cells. Other methods of delivering vectors into cells include the use of hydrodynamic injection, electroporation, and ultrasound, both for cell cultures and organisms. For a review of viral and non-viral delivery systems for gene delivery, see Nayerossadat, N. et al. (Adv Biomed Res. 2012;1:27), which is incorporated herein by reference.

[0093] rAAV virion production The rAAV virions disclosed herein can be constructed and produced using materials and methods described herein, as well as materials and methods known to those of skill in the art. Such engineering methods used to construct any embodiment of the present disclosure are known to those of skill in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989); and International Patent Publication No. WO 95 / 13598. Additionally, methods suitable for producing rAAV cassettes within adenovirus capsids are described in U.S. Patent Nos. 5,856,152 and 5,871,982.

[0094] Briefly, to package rAAV genome into rAAV virions, host cells are used that contain sequences required to express AAV rep and AAV cap or functional fragments thereof, as well as helper genes essential for AAV production. AAV rep and cap sequences are obtained from AAV sources found herein. AAV rep and cap sequences can be introduced into host cells in any manner known to those skilled in the art, including but not limited to transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. In one embodiment, the rep and cap sequences can be transfected into host cells by one or more nucleic acid molecules to stably exist in the cell as episomes. In another embodiment, the rep and cap sequences are stably integrated into the genome of the cell. In another embodiment, the rep and cap sequences are transiently expressed in the host cell. For example, a nucleic acid molecule useful for such transfection comprises, from 5' to 3', a promoter, an optional spacer inserted between the promoter and the start of the rep gene sequence, the AAV rep gene sequence, and the AAV cap gene sequence.

[0095] The rep and cap sequences, together with their expression control sequences, may be provided on a single vector, or each sequence may be provided on its own vector. Preferably, the rep and cap sequences are provided on the same vector. Alternatively, the rep and cap sequences may be provided on a vector containing other DNA sequences that may be introduced into the host cell. Preferably, the promoter used in this construct may be any suitable constitutive, inducible, or native promoter known to those skilled in the art. The molecule providing the rep and cap proteins may be in any form that transports these components into the host cell. Desirably, this molecule is in the form of a plasmid and may contain other non-viral sequences, such as sequences of marker genes. This molecule does not contain AAV ITRs, and generally does not contain AAV packaging sequences. Other viral sequences, particularly adenoviral sequences, are avoided in this plasmid to avoid the occurrence of homologous recombination. This plasmid is desirably constructed so that it can be stably transfected into cells.

[0096] Although the molecules providing rep and cap can be transiently transfected into a host cell, it is preferred that the host cell be stably transformed with the sequences necessary to express functional rep / cap proteins in the host cell, e.g., as an episome or by integration into a host cell chromosome. Depending on the promoter controlling expression in such stably transfected host cells, the rep / cap proteins can be expressed transiently (e.g., through the use of an inducible promoter).

[0097] The methods used to construct the embodiments of the present disclosure are conventional genetic or recombinant engineering techniques, as described in the references above. For example, rAAV can be produced using a triple transfection method using either the calcium phosphate method (Clontech) or Effectene reagent (Qiagen, Valencia, Calif.) according to the manufacturer's instructions. See Herzog et al, 1999, Nature Medic., 5(1):56-63 for the method used in the following examples, which uses a plasmid carrying the transgene, a helper plasmid containing AAV rep and cap, and a plasmid providing the adenoviral helper functions of E2A, E4Orf6 and VA. Although the present specification provides examples of specific constructs using the information provided herein, the skilled artisan can select and design other suitable constructs using the selection of spacers, promoters, and other elements including at least one translation start and translation stop signal, and the optional addition of a polyadenylation site.

[0098] rAAV virions are then produced by culturing host cells containing the rAAV viruses described herein, which contain the rAAV genome, AAV rep sequence and AAV cap sequence to be packaged into rAAV virions, under the control of regulatory sequences that induce their expression.Suitable viral helper genes, such as adenovirus E2A, E4Orf6 and VA, among other possible helper genes, can be provided to the culture by various methods known in the art, preferably on separate plasmids.Then, recombinant AAV virions that induce the expression of RetGC1 transgene are isolated from cells or cell cultures in the absence of contaminating helper virus or wild-type AAV.

[0099] The expression of RetGC1 transgene can be measured by methods known in the art. For example, target cells can be infected in vitro and the copy number of transgene in cells can be monitored by Southern blotting or quantitative polymerase chain reaction (PCR). RNA expression level can be monitored by Northern blotting or quantitative reverse transcriptase (RT)-PCR; and protein expression level can be monitored by Western blotting, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or by specific methods detailed in the following examples.

[0100] Pharmaceutical Compositions Provided herein are pharmaceutical compositions comprising any of the vectors disclosed herein and a pharma- ceutically acceptable excipient.

[0101] The rAAV containing the gene encoding RetGC1 is preferably assessed for contamination by conventional methods and then formulated into a pharmaceutical composition suitable for storage and / or administration to a patient.

[0102] Formulation of the vectors disclosed herein involves the use of a pharma- ceutically and / or physiologically acceptable vehicle or carrier, particularly a vehicle or carrier suitable for subretinal injection, such as buffered saline or other buffers, e.g., HEPES, to maintain the pH at an appropriate physiological level.

[0103] The vectors of the present disclosure can be formulated into pharmaceutical compositions. These compositions may contain, in addition to the vector, pharma- ceutically and / or physiologically acceptable excipients, carriers, buffers, stabilizers, antioxidants, preservatives, or other additives known to those skilled in the art. Such materials should be non-toxic and should not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other materials can be determined by one skilled in the art according to the route of administration. Pharmaceutical compositions are typically in liquid form. Liquid pharmaceutical compositions generally include a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Additional carriers are provided in International Patent Publication No. WO00 / 15822, which is incorporated herein by reference. Physiological saline, magnesium chloride, dextrose, or other sugar solutions, or glycols, such as ethylene glycol, propylene glycol, or polyethylene glycol, may be included. In some cases, surfactants may be used, for example pluronic acid (PF68) 0.001%. In some cases, Ringer's solution, lactated Ringer's solution, or Hartmann's solution is used. Preservatives, stabilisers, buffers, antioxidants and / or other additives may be included, as required.

[0104] For delayed release, the vectors may be included in pharmaceutical compositions formulated for sustained release, such as in microcapsules formed from biocompatible polymers or liposome carrier systems by methods known in the art.

[0105] For long term storage of vectors, they may be frozen in the presence of glycerol.

[0106] Treatment method Provided herein is a method for treating a retinal disease in a subject in need of treatment, the retinal disease being associated with one or more mutations in the GUCY2D gene, comprising administering to the subject a vector as disclosed herein.Also provided herein is a method for treating a retinal disease in a subject in need of treatment, the retinal disease being associated with one or more mutations in the GUCY2D gene, comprising administering to the subject a pharmaceutical composition comprising a vector as disclosed herein.Provided herein is a vector for use in a method for treating a retinal disease in a subject in need of treatment, the retinal disease being associated with one or more mutations in the GUCY2D gene.In some embodiments, the subject has a mutation in the GUCY2D gene.

[0107] In some embodiments, the subject is a mammal. As used herein, the term "mammal" is intended to include, but is not limited to, humans, laboratory animals, domestic pets, and livestock. Mammals include, but are not limited to, humans or non-human mammals, such as, for example, cows, horses, dogs, sheep, or cats. Individuals and patients are also of interest herein.

[0108] The terms "treat," "treated," "treating," or "treatment" as used herein refer to therapeutic treatment, the purpose of which is to slow (alleviate) an undesirable physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical outcome. For purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms; reduction in the extent of a condition, disorder, or disease; stabilization (i.e., not worsening) of the pathological condition, disorder, or disease state; delay in onset of a condition, disorder, or disease, or delay in progression of a condition, disorder, or disease; improvement of one or more symptoms of a condition, disorder, or disease state; and remission (partial or complete), or improvement or amelioration of a condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival compared to the expected survival if not receiving treatment. The terms "prevent", "prevention" and the like refer to acting before the onset of an obvious disease or disorder, preventing a disease or disorder from developing, or minimizing the extent of or slowing the progression of a disease or disorder.

[0109] In some embodiments, success of treatment is measured by one or more of the following: visual acuity, electroretinogram (ERG) response, reduction in nystagmus, change in digital eye phenomenon, and histopathological analysis or optical coherence tomography.

[0110] In some embodiments, the retinal disease is cone-rod dystrophy (CRD) or Leber congenital amaurosis type 1 (LCA1). In one embodiment, the retinal disease is LCA1. In one embodiment, the retinal disease is CRD.

[0111] In one aspect, a method is provided that includes: (a) determining whether the subject harbors a mutation in the GUCY2D gene; and (b) if the subject carries a mutation in the GUCY2D gene, administering to the subject a pharmaceutical composition comprising a vector disclosed herein.

[0112] Route and method of administration In some embodiments, the vector or pharmaceutical composition disclosed herein is administered by intraocular injection. In some embodiments, the vector or pharmaceutical composition disclosed herein is administered by direct retinal injection, subretinal injection, or intravitreal injection. In some embodiments, the vector or pharmaceutical composition disclosed herein is administered to the central retina of the subject.

[0113] The dosage of the vector of the present disclosure can be determined according to various parameters, in particular the age, weight and condition of the patient to be treated, the particular eye disorder and the extent to which the disorder has progressed if progressive, the route of administration, and the required regimen. A physician can also determine the route of administration and the dosage required for a particular patient. An effective amount of rAAV carrying a nucleic acid sequence encoding RetGC1 under the control of a promoter sequence is desirably about 1×10 9 ~2×10 12 Between 1 x 10 rAAV genome particles, or 1 x 10 10 ~2×10 11 A genome particle is defined herein as an AAV capsid that contains a single-stranded DNA molecule that can be quantified using sequence-specific methods (such as real-time PCR). In some embodiments, the genome particle ranges from about 1×10 9 ~2×10 12 The rAAV genome particles are provided in a volume of between about 150 and about 800 μl. In some embodiments, the rAAV genome particles are provided in a volume of between about 1 × 10 10 ~2×10 11 The rAAV genome particles are provided in a volume of between about 250 and about 500 μl. Additional doses within these ranges can be selected by the attending physician.

[0114] The dose may be provided as a single dose, but may be repeated for the fellow eye, or if for any reason (such as surgical complications) the vector did not target the correct area of ​​the retina. The treatment is preferably a single permanent treatment for each eye, but repeated injections, for example with a different AAV serotype, may be considered in the future. Thus, it may be desirable to administer multiple "booster" doses of the pharmaceutical compositions disclosed herein. For example, depending on the duration of the transgene in the target cells of the eye, booster doses may be delivered at six-month intervals, or annually after the initial administration. Such booster doses and the need for them may be monitored by the attending physician, for example, using retinal and visual function tests and visual behavior tests known in the art. Other similar tests may be used to determine the condition of the treated subject over time. Selection of the appropriate test may be performed by the attending physician. Additionally alternatively, the methods disclosed herein may also involve injection of larger volumes of vector-containing solution in single or multiple infections to allow for levels of visual function approaching those seen in wild-type retina.

[0115] Additional methods In one aspect, a method is provided for increasing expression of rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit β (PDE6β) in a subject in need of increased expression of rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit β (PDE6β), comprising administering to the subject a vector disclosed herein. In one aspect, a method is provided for increasing expression of rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit β (PDE6β) in a cell, comprising contacting the cell with a vector disclosed herein.

[0116] In one aspect, a method is provided for increasing cGMP levels in photoreceptors in a subject in need of increasing cGMP levels in photoreceptors, comprising administering to the subject a vector disclosed herein.In one aspect, a method is provided for increasing cGMP levels in photoreceptors in a cell, comprising contacting the cell with a vector disclosed herein.

[0117] Manufactured Products and Kits Kits or articles of manufacture for use in the methods described herein are also provided. In an embodiment, the kit comprises a composition described herein (e.g., a composition for delivery of a RetGC1-encoding transgene) in suitable packaging. Suitable packaging for the compositions described herein (such as injectable ophthalmic compositions) is known in the art and includes, for example, vials (such as sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may further be sterilized and / or sealed.

[0118] Kits are also provided that include the compositions described herein. These kits may further include instructions (or instructions) on how to use the compositions, such as the uses described herein. The kits described herein may further include other materials that are desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts that include instructions for performing administration of the compositions or for performing any of the methods described herein. For example, in some embodiments, the kit includes one or more of a rAAV for expression of a RetGC1-encoding transgene in target cells, a pharma- ceutically acceptable carrier suitable for injection, and a buffer, diluent, filter, needle, syringe, and package insert that includes instructions for performing an injection.

[0119] It should be understood that the present invention is not limited to the specific molecules, compositions, methodologies, or protocols described, which may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention. Furthermore, it should be understood that the disclosure of the present invention herein includes all possible combinations of such specific features. For example, if a specific feature is disclosed in connection with a particular aspect or embodiment of the present invention, or in a particular claim, that feature can also be used in combination with and / or in connection with other specific aspects and embodiments of the present invention, and in the present invention generally, to the extent possible.

[0120] When reference is made herein to a method that includes two or more defined steps, the defined steps may be performed in any order or simultaneously (unless the context excludes this possibility), and the method may include one or more other steps that occur before any of the defined steps, between two of the defined steps, or after all of the defined steps (unless the context excludes these possibilities).

[0121] All other referenced patents and applications are incorporated herein by reference in their entirety. Furthermore, if a definition or use of a term in a reference incorporated herein by reference is inconsistent with or contradicts the definition of that term provided herein, the definition of that term provided herein shall apply and the definition of that term in the reference shall not apply.

[0122] To facilitate a better understanding of the present invention, the following examples of specific embodiments are given, which should not be construed as limiting or defining the entire scope of the invention. EXAMPLES

[0123] Example 1: Generation of RetGC knockout (KO) organoids as an in vitro disease model for retinal diseases associated with mutations in GUCY2D To generate RetGC KO organoids, wild-type (WT) retinal organoids were harvested at several time points during development. GUCY2D mRNA and RetGC protein levels were measured by qPCR and Western blot / immunofluorescence, respectively, with retina-specific markers at various time points during retinal organoid development. WT human fibroblasts were reprogrammed and gene-edited to delete GUCY2D-RetGC using episomal reprogramming factors and CRISPR / CAS9. KO induced pluripotent stem cell (iPSC) clones, along with their unedited (WT) isogenic control lines, were differentiated into retinal organoids. The presence of photoreceptor markers and the absence of RetGC protein were confirmed at the expected time points of development.

[0124] RetGC protein was translocated to photoreceptor outer segments of mammalian retinas. By immunofluorescence, RetGC protein could be detected in the outer segment structures of WT organoids, where it colocalized with rhodopsin. Loss of RetGC protein in mature RetGC KO organoids was confirmed by immunofluorescence and western blot. There was also a significant reduction in GUCY2D (RetGC) mRNA.

[0125] In addition to the loss of RetGC in the outer segments, the phototransduction protein phosphodiesterase-6-beta (PDE6β) was found to be decreased in the outer segments of RetGC KO organoids. PDE6β plays a central role in the phototransduction cycle. Upon light stimulation, cGMP is hydrolyzed to GMP by PDE6β, which closes the cGMP channels in the outer segment discs, resulting in hyperpolarization of photoreceptor cells.

[0126] The above characteristics of RetGC KO organoids indicated that these organoids could be utilized as an in vitro disease model to test the efficacy of RetGC viral vectors in restoring protein levels.

[0127] Example 2: Characterization of RetGC KO organoids We generated RetGC KO and WT retinal organoids from human induced pluripotent cells (hiPSCs) using an established differentiation protocol. The differentiation protocol produced "mature" retinal organoids at day 140 (20 weeks), which could be used for AAV transduction experiments. Mature retinal organoids could be maintained in culture for up to 300 days (43 weeks) without morphologically discernible signs of degeneration.

[0128] The human neural retina is structured with several layers of neural cells, including horizontal cells, bipolar cells, amacrine cells, Müller glia and ganglion cells, photoreceptors, and retinal pigment epithelial cells (Figure 1). The in vitro generated organoids reflect the laminated morphology of the neural retina, with the above retinal cell types arranged in appropriate layers and connected to two synaptic layers.

[0129] WT and RetGC KO organoids were characterized using immunofluorescence, western blot and qPCR techniques. Markers associated with various cell types in the retina were used to identify and describe the similarities in retinal morphology between in vivo human retina and retinal organoids in both WT and RetGC KO cell lines. Figure 2 shows cryosection and immunostaining images of LM opsin and rhodopsin in cone and rod photoreceptors, Ribeye and V Glut in synapses of the outer plexiform layer, PKCα and calretinin in bipolar, horizontal and amacrine cells. Brightfield images show mature organoids with visible "brush borders", which are the outer segments of photoreceptors. The graph in Figure 3 shows an analysis of RetGC protein expression over the time course of retinal organoid development (day 40-day 220). RetGC protein levels are significantly reduced in RetGC KO organoids compared to WT.

[0130] Example 3: Design of vectors to restore RetGC expression in KO organoids Viral vectors containing one of four different expression constructs were designed, as shown in Figure 4. The expression constructs had two different promoters: RK (derived from the photoreceptor-specific rhodopsin kinase promoter) and CMV (derived from the cytomegalovirus). Some of the expression constructs also contained the Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE). All viral genomes were packaged into 7m8 capsids.

[0131] WT and RetGC KO retinal organoids were transduced with four different viral vectors at ages ranging from 140 to 204 days and incubated for 21 days before harvesting and analysis. Transduced organoids were assessed using immunofluorescence, Western blotting, qPCR, and cGMP FRET assays.

[0132] All four AAV 7m8 vectors successfully transduced human photoreceptors in RetGC KO retinal organoids and drove RetGC protein expression as determined by total RetGC protein quantification (Western blot) and mRNA (qPCR). Transgenic RetGC delivered by 7m8 CMV-RetGC and 7m8 RK-RetGC was detectable by immunofluorescence in the correct subcellular compartment of photoreceptor outer segments.

[0133] Example 4: AAV vector-driven RetGC expression restores PDE6β expression in photoreceptor outer segments Figure 5 shows immunostaining of PDE6β in WT, non-transduced and viral vector-transduced retinal organoids. PDE6β was co-stained with rhodopsin protein to establish the presence of outer segments in all organoids and to show how PDE6β protein was reduced in non-transduced controls compared to WT controls. After transduction with viral vector, restoration of PDE6β protein was confirmed.

[0134] PDE6β staining intensity was significantly reduced in non-transduced RetGC KO compared to WT control retinal organoids; p<0.005 (one-way ANOVA with Kruskal-Wallis test for multiple comparisons). Staining intensity of rhodopsin-positive outer segments was quantified in multiple WT, RetGC KO, and transduced organoids. PDE6β expression was restored to near WT levels in organoids treated with 7m8-CMV-RetGC and 7m8-RK-RetGC. 7m8-CMV-RetGC-WPRE and 7m8-RK-RetGC-WPRE showed improvement compared to KO but did not reach the same levels as the other two vectors (Figure 6 and Table 1).

[0135] [Table 1]

[0136] Example 5: AAV vector-driven RetGC expression restores RetGC protein levels RetGC protein levels were assayed by Western blot. As shown in Figure 7, RetGC expression was higher in EBs transduced with vectors 7m8-CMV-RetGC (30% of WT), 7m8-CMV-WPRE-RetGC (47% of WT), and 7m8-RK-RetGC (27% of WT) compared to non-transduced EBs. For each experimental group, two samples were taken and processed for protein expression analysis.

[0137] Example 6: AAV vector-driven RetGC expression restores total cGMP levels in organoids after light stimulation To measure RetGC activity, a quantitative measurement of cGMP was performed in a competitive assay format using a specific antibody labeled with europium cryptate (donor) and cGMP labeled with d2 reagent (acceptor). The detection principle is based on HTRF® technology. When the dyes are in close proximity, excitation of the donor by a light source (laser or flash lamp) causes a fluorescence resonance energy transfer (FRET) towards the acceptor, which then fluoresces at a specific wavelength (665 nm). Any cGMP present in the sample competes with the binding between the two conjugates, thereby preventing FRET from occurring. The specific signal is inversely proportional to the cGMP concentration.

[0138] WT organoids and KO organoids transduced with the 7m8 vector and non-transduced KO organoids were exposed to light / dark cycles to induce the production of cGMP. The light stimulation protocol used consisted of 5 minutes of white light stimulation and 5 minutes of darkness before dissecting the organoids to isolate the photoreceptors. Samples were dissected and lysed under red light in the presence of IBMX (a PDE inhibitor) as described in the study protocol. The assay determined the cGMP [nM] concentration against a standard curve and the obtained values ​​were normalized to the total protein amount [ug] per sample. Statistical analysis was performed to evaluate the statistical differences between samples compared to the non-transduced KO control (NT).

[0139] As shown in the graph in Figure 8, RetGC KO organoids (NT) had significantly reduced cGMP levels after light stimulation (20% of WT). After transduction, a statistically significant increase in cGMP was found in KO RetGC-GUCY2D organoids transduced with vectors 7m8-CMV-GUCY2D (+76% of WT, p=0.0043) and 7m8-RK-GUCY2D (+37% of WT, p=0.0494). Transduction with both CMV and RK vectors carrying the WPRE element led to an increase in cGMP that was not statistically significant but was on average comparable to that seen in WT samples. The graph shows results from two separate experiments with three or four transduced organoids per group (Figure 8). The observation that total cGMP levels achieved or exceeded WT levels demonstrates the functional efficacy of these above vectors in the context of light-sensitive human photoreceptor phenotypes.

[0140] Overview of Arrays

[0141] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9]

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

Table 2-24

Table 2-25

Table 2-26

[0142] While the foregoing written description of the invention enables one of ordinary skill in the art to make and use what is presently contemplated to be the best mode thereof, those of ordinary skill in the art will understand and recognize the existence of variations, combinations, and equivalents of the specific embodiments, methods, and examples herein.

Claims

1. (a) Rhodopsin kinase (RK) or cytomegalovirus (CMV) promoter (b) A nucleic acid sequence encoding retinal membrane-bound guanylyl cyclase 1 (RetGC1), comprising a sequence that is at least 90% identical to SEQ ID NO: 9, SEQ ID NO: 13, or SEQ ID NO: 14; and (c) A polyadenylation signal, An expression construct comprising the above, wherein the nucleic acid sequence is operably linked to the promoter.

2. The expression construct according to Claim 1, wherein the promoter sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 7 or SEQ ID NO:

8.

3. The expression construct according to Claim 2, wherein the promoter sequence comprises SEQ ID NO:

7.

4. The expression construct according to Claim 2, wherein the promoter sequence comprises SEQ ID NO:

8.

5. The expression construct according to Claim 1, wherein the nucleic acid sequence encoding RetGC1 comprises a sequence that is at least 90% identical to SEQ ID NO: 13 or SEQ ID NO:

14.

6. The expression construct according to Claim 5, wherein the nucleic acid sequence encoding RetGC1 comprises SEQ ID NO:

13.

7. The expression construct according to Claim 5, wherein the nucleic acid sequence encoding RetGC1 comprises SEQ ID NO:

14.

8. The expression construct according to Claim 1, wherein the nucleic acid sequence encoding RetGC1 encodes a protein comprising a sequence that is at least 90% identical to SEQ ID NO:

12.

9. The expression construct according to Claim 8, wherein the nucleic acid sequence encoding RetGC1 encodes a protein comprising SEQ ID NO:

12.

10. The expression construct according to Claim 1, wherein the polyadenylation signal comprises the bovine growth hormone polyadenylation (BGH-polyA) signal.

11. The expression construct according to Claim 1, wherein the polyadenylation signal comprises a sequence that is at least 90% identical to SEQ ID NO:

11.

12. The expression construct according to Claim 11, wherein the polyadenylation signal comprises SEQ ID NO:

11.

13. The expression construct according to Claim 1, further comprising a post-transcriptional regulatory element.

14. The expression construct according to claim 13, wherein the post-transcriptional regulatory element comprises the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

15. The expression construct according to claim 13, wherein the post-transcriptional regulatory element comprises a sequence that is at least 90% identical to SEQ ID NO:

10.

16. The expression construct according to claim 15, wherein the post-transcriptional regulatory element comprises SEQ ID NO:

10.

17. The expression construct according to claim 1, comprising a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 4.

18. The expression construct according to claim 17, comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 4.

19. A vector comprising the expression construct according to any one of claims 1 to 18.

20. The vector according to claim 19, which is a viral vector.

21. The vector according to claim 20, which is an adeno-associated virus (AAV) vector.

22. The vector according to claim 21, comprising a genome derived from AAV serotype AAV2.

23. The vector according to claim 22, comprising a capsid derived from AAV7m8.

24. A pharmaceutical composition comprising the vector according to claim 19 and a pharmaceutically acceptable carrier.

25. A pharmaceutical composition for treating a retinal disease in a subject in need thereof, the pharmaceutical composition comprising the vector according to claim 19, wherein the retinal disease is associated with one or more mutations in the GUCY2D gene.

26. The pharmaceutical composition according to claim 25, wherein the retinal disease is cone-rod dystrophy (CRD) or Leber congenital amaurosis type 1 (LCA1).

27. The pharmaceutical composition according to claim 26, wherein the retinal disease is LCA1.

28. A pharmaceutical composition for increasing the expression of rod cyclic guanosine monophosphate (cGMP)-specific 3',5'-cyclic phosphodiesterase subunit β (PDE6β) in a subject in need of an increase in the expression of rod cyclic guanosine monophosphate (cGMP)-specific 3',5'-cyclic phosphodiesterase subunit β (PDE6β), the pharmaceutical composition comprising the vector according to claim 19.

29. A pharmaceutical composition for increasing the level of cyclic guanosine monophosphate (cGMP) in photoreceptors in a subject in need of an increase in the level of cyclic guanosine monophosphate (cGMP) in photoreceptors, the pharmaceutical composition comprising the vector according to claim 19.

30. The pharmaceutical composition according to claim 25, wherein the vector or the pharmaceutical composition is administered by intraocular injection.

31. The pharmaceutical composition according to claim 30, wherein the vector or the pharmaceutical composition is injected into the center of the retina of the subject.

Images