Gene therapy compositions and methods for treating retinal diseases

Abstract

The present invention provides recombinant AAV particles containing opsin transgenes for therapy of retinal diseases, as well as related compositions and methods.

Description

[Technical Field]

[0001] The present invention relates to therapeutic compositions and methods for treating retinal diseases through the expression of heterologous genes using an AAV delivery system.

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 485,596, filed February 17, 2023, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing This application contains a Sequence Listing entitled 60650501WO Sequence Listing.xml, created on February 16, 2024, and is 123 kilobytes in size. The Sequence Listing was submitted electronically with the filing of this application and is hereby incorporated by reference in its entirety. [Background technology]

[0004] Gene therapy represents a promising approach to improving and restoring human vision, and there are numerous clinical trials of gene therapy for the treatment of retinal diseases. Adeno-associated virus (AAV)-based systems for the delivery of therapeutic transgenes are utilized by some clinical candidates, in part because the safety profile of AAV has been well characterized. For example, Luxturna™ (BlueTherapeutics) is an AAV2 vector encoding the retinal pigment epithelium-specific protein 65-kD (RPE65), which was approved in 2017 for patients with biallelic RPE65 mutation-associated retinal dystrophy. RPE65 is present in the retinal pigment epithelium (RPE), which is involved in the regeneration of 11-cis retinol in the visual cycle.

[0005] However, there are still significant challenges to the successful translation of any specific therapy into the clinic, including the efficient targeting and expression of therapeutic transgenes in retinal cells.Although much is known about the components required for AAV vector design, it is not possible to know how a specific vector design will function based on previous results using individual vector elements.Instead, it remains unpredictable how a specific combination of elements will function to deliver and express a specific transgene, especially in humans.There is a need for additional AAV vector transgene delivery systems for use in humans in the treatment of retinal diseases.The present invention addresses this need. Summary of the Invention

[0006] The present invention provides AAV vectors and related compositions and methods for the expression of therapeutic transgenes in mammalian and primate eyes, particularly modified channelrhodopsins for improving vision and / or restoring vision in subjects in need thereof. Accordingly, the present invention provides infectious recombinant adeno-associated virus (rAAV) particles, related vector constructs, related compositions, including pharmaceutical compositions, and related methods, including methods for gene therapy of retinal diseases and disorders.

[0007] In one embodiment, the infectious recombinant adeno-associated virus (rAAV) particle comprises (i) a capsid protein having an altered amino acid sequence relative to a native AAV capsid of serotype 2, and (ii) a vector genome consisting of a heterologous polynucleotide comprising, in the 5' to 3' direction, (a) an AAV2 inverted terminal repeat (ITR1), (b) a promoter sequence, (c) a polynucleotide sequence encoding a channelrhodopsin, (d) a polyadenylation sequence, and (e) an AAV2 inverted terminal repeat (ITR2), wherein the heterologous polynucleotide does not encode a fluorescent protein or other reporter protein.

[0008] The rAAV particles may also include those in which the heterologous polynucleotide further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) between the polyadenylation sequence and the ITR2 sequence.

[0009] The rAAV particles may also include where the promoter is selected from a cytomegalovirus (CMV) promoter, an elongation factor 1a (EF1a) promoter, a simian virus 40 (SV40) promoter, a chicken beta-actin promoter, an mGluR6 promoter, and a CAG promoter.

[0010] The rAAV particles may also include cases where the heterologous polynucleotide further comprises an enhancer sequence.

[0011] The rAAV particles may also include those in which the channelrhodopsin comprises the amino acid sequence of ChRown (SEQ ID NO: 5).

[0012] The rAAV particles may also include those in which the channelrhodopsin comprises the amino acid sequence of ChRown (SEQ ID NO: 5), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical thereto.

[0013] The rAAV particles may also include where the polyadenylation signal is the human growth hormone polyadenylation sequence (hGHpA) or the simian virus 40 polyadenylation sequence.

[0014] The rAAV particles may also include where the heterologous polynucleotide comprises pCAG-Chrown-mWPRE-hGHpA (SEQ ID NO: 21) or pCAG-Chrown-hGHpA (SEQ ID NO: 23).

[0015] The rAAV particles may also include those where the promoter is a CAG promoter.

[0016] The rAAV particle may also include a case where the CAG promoter comprises SEQ ID NO:10 and the WPRE element comprises SEQ ID NO:14.

[0017] The rAAV particles may also include where the enhancer is a CMV enhancer or an mGluR6 enhancer.

[0018] The rAAV particles may also comprise where the polyadenylation signal is the human growth hormone polyadenylation sequence (hGHpA) comprising SEQ ID NO:18.

[0019] Other technical features may be readily apparent to those skilled in the art from the following figures, descriptions, and claims.

[0020] In another aspect, provided herein are infectious recombinant adeno-associated virus (rAAV) particles comprising a vector genome comprising a polynucleotide sequence encoding (i) a capsid protein, and (ii) a channelrhodopsin, wherein a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) is positioned downstream of the polynucleotide sequence encoding the channelrhodopsin, and the vector genome does not encode a fluorescent protein, or the capsid protein is of the AAV2 7m8 serotype.

[0021] In some embodiments, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) is located downstream of the polynucleotide sequence encoding the channelrhodopsin. In some embodiments, the WPRE element comprises SEQ ID NO: 14, or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0022] In some embodiments, the vector genome does not encode a fluorescent protein.

[0023] In some embodiments, the capsid protein is AAV2 7m8 serotype. In some embodiments, the capsid protein comprises the amino acid sequence of SEQ ID NO:7.

[0024] In some embodiments, the polynucleotide encoding the channelrhodopsin comprises the channelrhodopsin of SEQ ID NO: 5. In some embodiments, the polynucleotide encoding the channelrhodopsin encodes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 5. In some embodiments, the polynucleotide encoding the channelrhodopsin comprises SEQ ID NO: 17.

[0025] In some embodiments, the vector further comprises an upstream inverted terminal repeat (ITR), wherein the upstream ITR comprises SEQ ID NO: 15. In some embodiments, the vector further comprises a downstream ITR, wherein the downstream ITR comprises SEQ ID NO: 16.

[0026] In some embodiments, the vector further comprises a polyadenylation sequence. In some embodiments, the polyadenylation sequence is the human growth hormone polyadenylation sequence (hGHpA) or the simian virus 40 polyadenylation sequence. In some embodiments, the polyadenylation sequence comprises SEQ ID NO: 18.

[0027] In some embodiments, the vector further comprises a promoter. In some embodiments, the promoter comprises a cytomegalovirus (CMV) promoter, an elongation factor 1a (EF1a) promoter, a simian virus 40 (SV40) promoter, a chicken beta-actin promoter, an mGluR6 promoter, or a CAG promoter. In some embodiments, the promoter comprises SEQ ID NO: 10. In some embodiments, the vector comprises SEQ ID NO: 34. In some embodiments, the vector comprises SEQ ID NO: 29. In some embodiments, the vector comprises an mGluR6 regulatory element region, wherein the mGluR6 regulatory element region comprises a promoter. In some embodiments, the mGluR6 regulatory element comprises at least one, at least two, at least three, or all four of SEQ ID NOs: 24-27. In some embodiments, the mGluR6 regulatory element comprises, from upstream to downstream, intron 4 of the mGluR6 gene, intron 3 of the mGluR6 gene, an mGluR6 enhancer, and a fragment of the mGluR6 promoter. In some embodiments, the mGluR6 regulatory element comprises SEQ ID NO: 28.

[0028] In some embodiments, the vector comprises SEQ ID NO: 30. In some embodiments, the vector comprises SEQ ID NO: 31.

[0029] In some embodiments, the vector comprises, from upstream to downstream, (a) an AAV2 inverted terminal repeat (ITR1), (b) a promoter sequence, (c) a polynucleotide sequence encoding a channelrhodopsin, (d) a polyadenylation sequence, and (e) an AAV2 inverted terminal repeat (ITR2). In some embodiments, the vector comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) located between the polyadenylation sequence and the ITR2 sequence.

[0030] The pharmaceutical composition comprises a plurality of rAAV particles and may also include a pharmaceutically acceptable carrier or excipient.

[0031] The pharmaceutical composition may also include where the composition is formulated for intravitreal injection.

[0032] The pharmaceutical composition may also include when the composition is formulated as an emulsion or suspension. Other technical features may be readily apparent to those skilled in the art from the following figures, descriptions, and claims.

[0033] Also provided is a method for delivering heterologous nucleic acid to a retinal cell, the method comprising contacting the retinal cell with a plurality of rAAV particles as described herein.

[0034] Also provided is a method for treating a retinal disease in a human subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a plurality of rAAV particles described herein. The pharmaceutical composition may also comprise a pharmaceutically acceptable carrier or excipient.

[0035] The method may also include where the rAAV particles are administered by intravitreal injection.

[0036] The method may also include administering the rAAV particles in one or more doses. In embodiments, the one or more doses comprise at least about 1.5 x 10 8 In embodiments, one or more doses contain about 1 x 10 viral genomes (vg). 8 ~1×10 14 Contains the viral genome (vg).

[0037] The method may also include where the rAAV particles are administered in one or more doses per eye.

[0038] The method may also include where the retinal disease is selected from Bardet-Biddell syndrome, choroidal retinal atrophy or degeneration, cone or cone-rod dystrophy, congenital stationary night blindness, Leber's congenital amaurosis (LCA), macular degeneration (MD) including age-related MD (AMD), ocular retinal developmental disorders, ocular atrophy, retinitis pigmentosa, syndromes / systemic diseases associated with retinopathy, Usher syndrome, or other retinopathies including diabetic retinopathy.

[0039] The method may also include a case where the retinal disease is age-related macular degeneration (AMD) or retinitis pigmentosa (RP). Other technical features may be readily apparent to those skilled in the art from the following figures, descriptions, and claims. [Brief explanation of the drawings]

[0040] [Figure 1A] FIG. 1A shows ChRown-mediated spiking activity in response to increasing light intensity; light intensity, measured as photons / cm 2 s, is shown at the top right of each trace. [Figure 1B] FIG. 1B shows ChRown-GFP-mediated spiking activity in response to increasing light intensity; light intensity, measured as photons / cm 2 s, is shown at the top right of each trace. [Figure 1C] Figure 1C shows light-evoked spikes recorded from a single electrode in a ChRown-treated retina. [Figure 1D] FIG. 1D shows light-evoked spikes recorded from a single electrode in a Chrown-GFP-treated retina. [Figure 1E] Figure 1E shows a raster plot of 30 consecutive light-evoked spikes from a single neuron (30 trials and 10 min of recording) in a ChRown-treated retina. [Figure 1F] Figure 1F shows a raster plot of 30 consecutive light-evoked spikes from a single neuron (30 trials and 10 min of recording) in a ChRown-GFP-treated retina. [Figure 1G] Figure 1G shows the mean spike rate histogram of the light-evoked spikes in Figure 1E. [Figure 1H]Figure 1H shows the mean spike rate histogram of the light-evoked spikes in Figure 1F. [Figure 2] Figure 2 shows the results of visual acuity testing using two doses (low, high) of either pCAG-ChRown-GFP-mWPRE-hGHpA (R01) or pCAG-ChRown-mWPRE-hGHpA (R03). Mean ± SD for n = 6 animals / group. [Figure 3A] Figure 3A is a line graph showing the total clinical score from the slit lamp examination. Data are presented as the mean ± SEM. Test articles were vehicle (large circle), AAV2(wt) (square), AAV2(7m8) (triangle), AAV2(7m8) high dose (diamond), AAV8 (small circle), and AAV8 high dose (star). For doses, see Table 3. [Figure 3B] Figure 3B is a line graph showing keratinocyte deposits based on slit lamp examination. Data are presented as mean ± SEM. Test articles were vehicle (small circles), AAV2(wt) (squares), AAV2(7m8) (triangles), AAV2(7m8) high dose (inverted triangles), AAV8 (large circles), and AAV8 high dose (diamonds). See Table 3 for doses. [Figure 3C] Figure 3C is a line graph showing iris injection based on slit lamp examination. Data are presented as mean ± SEM. Test articles were vehicle (small circles), AAV2(wt) (squares), AAV2(7m8) (triangles), AAV2(7m8) high dose (inverted triangles), AAV8 (large circles), and AAV8 high dose (diamonds). See Table 3 for doses. [Figure 3D] Figure 3D is a line graph showing anterior lens capsule deposition based on slit lamp examination. Posterior lens capsule deposition was similar (data not shown). Data are presented as mean ± SEM. Test articles were vehicle (small circles), AAV2(wt) (squares), AAV2(7m8) (triangles), AAV2(7m8) high dose (inverted triangles), AAV8 (large circles), and AAV8 high dose (diamonds). See Table 3 for doses. [Figure 4A]Figure 4A is a line graph showing GFP expression at the foveal location of the eyeball, graded from fluorescent fundus images. Data are shown as mean ± SEM. Test articles were AAV2 (squares), AAV2 7m8 low dose (triangles), AAV2 7m8 high dose (inverted triangles), and AAV8 high dose (circles) at 1 x 1011. [Figure 4B] Figure 4B is a line graph showing GFP expression in peripheral locations of the eye globe, graded from fluorescent fundus images. Data are shown as mean ± SEM. Test articles were AAV2 (squares), AAV2 7m8 low dose (triangles), AAV2 7m8 high dose (inverted triangles), and AAV8 high dose (circles) at 1 x 1011. [Figure 4C] Figure 4C is a line graph showing GFP expression in perivascular locations in the eyeball, graded from fluorescent fundus images. Data are presented as mean ± SEM, and test articles are as shown in Figure 4A. [Figure 5] Figure 5 shows representative infrared and autofluorescence cSLO images of eyes treated with vehicle, AAV2, AAV 7m8 low dose, AAV2 7m8 high dose, AAV8 high dose, and AAV8 low dose, acquired on day 42. GFP expression was restricted across most treatment groups, with the exception of AAV2 7m8 low dose and AAV2 7m8 high dose, where expression ranged from moderate to bright. [Figure 6] Figure 6 is a line graph showing transgene expression (target copies per microgram of RNA) in selected optical tissues of non-human primates at three doses. For details, see Example 6. [Figure 7]Figure 7 shows the relationship between light sensitivity and viral vector dose. Light sensitivity was defined as the lowest light intensity that induced OMR at a grating frequency of 0.042 cycles / degree. Data are presented as mean ± SD (n = 3-4 mice in each group). Light intensity values ​​between groups with a viral dose ≥ 7.5 x 108 vg / eye were not significantly different (p > 0.05, one-way ANOVA). The dashed line indicates the estimated maximum light intensity of the OptoDrum, which was used to measure visual acuity. [Figure 8] Figure 8 shows the relationship between visual acuity and viral vector dose. Visual acuity was determined as the highest grating frequency at 100% contrast that induced OMR. Data are presented as mean ± SD (n = 3-4 mice in each group). Visual acuity values ​​between groups with viral doses ≥ 1.5 x 10 vg / eye were not significantly different (p > 0.05, one-way ANOVA). [Figure 9] Figure 9 shows representative immunofluorescence images of ChRown (fused to GFP) in the whole retina from treated TKO mice. Viral-transduced RGCs were determined by GFP labeling. RGCs were labeled with an antibody against RBPMS, an RGC-specific marker. [Figure 10A] Figure 10A shows the relationship between total RGCs and viral vector dose. RGC density among all treatment groups was not statistically different (p>0.05, one-way ANOVA). [Figure 10B] Figure 10B shows the relationship between viral vector dose and normalized density of transduced RGCs. Data are presented as mean ± SD (n = 4 retinas in each dose group). [Figure 11A] Figure 11A shows a representative Western blot image of ChRown-GFP protein bands from whole retinas labeled using an antibody against GFP. β-actin served as a control. [Figure 11B] Figure 11B shows the relative ECL fluorescence intensity of ChRown-GFP after normalization with the ECL fluorescence intensity of β-actin. Four retinas were used for each virus dose group. Data are presented as mean ± SD (n = 3 experiments). [Figure 12A] Figure 12A shows the relationship between photosensitivity and viral vector dose 2 months after injection. Photosensitivity was defined as the lowest light intensity that induced OMR at a grating frequency of 0.042 cycles / degree. Data are presented as mean ± SD (n = 6 mice in each group). [Figure 12B] Figure 12B shows the relationship between photosensitivity and viral vector dose 6 months after injection. Photosensitivity was defined as the lowest light intensity that induced OMR at a grating frequency of 0.042 cycles / degree. Data are presented as mean ± SD (n = 6 mice in each group). [Figure 13A] Figure 13A shows the relationship between visual acuity and viral vector dose 2 months after injection. Visual acuity was determined as the highest grating frequency at 100% contrast that induced OMR. Data are presented as mean ± SD (n = 6 mice in each group). [Figure 13B] Figure 13B shows the relationship between visual acuity and viral vector dose 6 months after injection. Visual acuity was determined as the highest grating frequency at 100% contrast that induced OMR. Data are presented as mean ± SD (n = 6 mice in each group). [Figure 14A] Figure 14A shows the relationship between light sensitivity and spatial frequency in ChRown-treated TKO mice (n=6). Light sensitivity is defined as the threshold light intensity required to elicit a visual-motor response. [Figure 14B] FIG. 14B shows that light sensitivity assessed at a spatial frequency of 0.042 cycles / degree (approximately the peak sensitivity frequency) was stable up to 10 months after viral vector injection (n=5). [Figure 14C] FIG. 14C shows that vision remained stable for up to 10 months after viral vector injection (n=5). [Figure 14D] FIG. 14D shows the relationship between contrast sensitivity and spatial frequency (n=5). DETAILED DESCRIPTION OF THE INVENTION

[0041] Photoreceptor loss or degeneration is the cause of many human diseases characterized by vision loss, including age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Loss of photoreceptor cells and / or loss of photoreceptor cell function is a major cause of decreased vision, reduced light sensitivity, and blindness in humans. The present invention addresses the need for additional therapeutic agents for the treatment of retinal diseases in which photoreceptor loss or degeneration is a factor by providing therapeutic transgenes encoding photosensitive proteins delivered to retinal cells using the recombinant adeno-associated virus (rAAV) vehicles described herein, as well as related compositions and methods for the treatment of retinal diseases and disorders.

[0042] rAAV vehicles advantageously deliver transgenes to retinal cells, which may include retinal photoreceptor cells, retinal ganglion cells, and bipolar cells, as well as retinal amacrine cells, horizontal cells, Muller cells, and retinal pigment epithelial cells.

[0043] In some embodiments, the rAAV vector provides targeted delivery to photoreceptor cells, or a combination of photoreceptor cells, retinal ganglion cells, and bipolar cells. Photoreceptor cells are highly specialized neurons involved in converting light into electrical and chemical signals, and collectively with the activity of ganglion and bipolar cells, transmit these signals to the brain, which generates visual representations. Bipolar cells receive input from photoreceptor cells and pass electrical signals to ganglion cells, whose axons collectively form the optic nerve.

[0044] Photoreceptor cells include rod and cone cells, which contain the light-sensitive proteins rhodopsin and cone opsin. Like other opsins, rhodopsin is a G protein-coupled receptor (GPCR) with seven transmembrane domains embedded in the lipid bilayer of the cell membrane and forming a binding pocket for its ligand, 11-cis-retinal. Signal transduction is initiated when the retina absorbs a photon of light, resulting in its isomerization to all-trans-retinal and activating a series of reactions called the phototransduction cascade. This signal transduction results in the movement of ions across the cell membrane, which then become electrically polarized and generate a series of electrical and chemical signals that are ultimately transmitted to the brain.

[0045] Visual information is processed through the retina via two pathways: the ON pathway, which turns light on, and the OFF pathway, which turns light off. The existence of the ON / OFF pathway is important for enhanced contrast sensitivity. Visual signals within the ON pathway are relayed from ON-type cone bipolar cells to ON-type ganglion cells. Both ON-type cone bipolar cells and ON-type ganglion cells depolarize in response to light. Meanwhile, visual signals within the OFF pathway are conveyed from OFF-type cone bipolar cells to OFF-type ganglion cells. Both OFF-type cone bipolar cells and OFF-type ganglion cells hyperpolarize in response to light. Rod bipolar cells, responsible for the ability to see in dim light (scotopic vision), are ON-type bipolar cells (depolarize in response to light). Rod bipolar cells relay visual signals to ON-type and OFF-type cone bipolar cells through AII amacrine cells (an ON type of retinal cell).

[0046] In some embodiments, the present invention provides a method for treating retinal diseases utilizing the compositions and methods described herein, comprising expressing a channelrhodopsin transgene in retinal cells of a subject with photoreceptor loss or degeneration. Channelrhodopsins are a subfamily of retinylidene rhodopsins originally identified in algae that function as sensory photoreceptors. Heterologous expression in mammalian cells is associated with light-sensitive electrical signaling, calcium influx, and the like.

[0047] Transgene According to various embodiments of the compositions and methods described herein, the transgene is a channelrhodopsin. In some embodiments, the channelrhodopsin is a variant of wild-type Chloromonas osmoticum channelrhodopsin, wherein the variant has improved light sensitivity compared to a reference protein, which may be a native or wild-type protein. The terms "native" and "wild-type" with respect to proteins are used interchangeably and refer to naturally occurring proteins.

[0048] Wild-type Chloromonas ogamas channelrhodopsin is described in Klapoetke et al., 2014 Nat. Methods 11(3):338-46. Chloromonas ogamas channelrhodopsin variants are described in U.S. Patent Nos. 10,392,426 (Klapoetke et al.) and 11,041,004 (Pan et al.).

[0049] The amino acid and nucleotide sequences of wild-type Chloromonas ogamar channelrhodopsin are represented by SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0050] Improved photosensitivity can be demonstrated, for example, by comparing the ion and / or proton flux across a membrane produced by light-activated recombinantly expressed proteins. In this context, "light-activating" refers to light energy that exceeds the threshold for protein activation. In the context of the variant channelrhodopsins described herein, the activation light is about 470 nm. In some embodiments, the activation light has a wavelength of about 450 nm to about 495 nm, which may also be referred to as blue light.

[0051] The variant channelrhodopsin utilized as the transgene was identified by screening a library of rationally designed site-directed mutants in Chloromonas osmosis. The variants described herein were optimized for visual function, including enhanced light sensitivity and favorable channel kinetics, as described in U.S. Patent No. 11,041,004 (Pan et al.) and Ganjawala, T.H., et al., "Improved CoChR Variants Restore Visual Acuity and Contrast Sensitivity in a Mouse Model of Blindness under Ambient Light Conditions," Mol Ther, 2019. 27(6): pp. 1195-1205.

[0052] Unlike previously described channelrhodopsin transgenes, the transgenes utilized in the compositions and methods described herein are advantageously expressed as channelrhodopsin proteins alone, rather than as fusion proteins of channelrhodopsin with fluorescent reporter proteins such as green fluorescent protein (GFP) or similar. All previous preclinical and clinical studies on optogenetic restoration of sensory function have been performed using channelrhodopsins fused to fluorescent proteins at the C-terminus. Furthermore, removal of the fluorescent protein tag has been reported to result in a "significant" reduction in photocurrents in transfected cells (see, e.g., Zerche et al., Mol Ther Methods Clin Dev 2023 Mar 21:29:202-212 and Gauvain et al., Commun Biol. 2021;4:125). Furthermore, although some reports have indicated that fusion proteins resulted in higher transgene expression in certain constructs, presumably by stabilizing the expressed protein against degradation, the inventors unexpectedly discovered no differences in expression, localization, or function between constructs containing a therapeutic transgene alone and constructs containing a fusion protein of a therapeutic transgene and GFP, as discussed in the Examples.

[0053] In embodiments, the transgene comprises a variant channelrhodopsin transgene of SEQ ID NO:3.

[0054] In embodiments, the transgene comprises a variant channelrhodopsin transgene of SEQ ID NO:4.

[0055] In several embodiments, the transgene is a variant of SEQ ID NO:3 or SEQ ID NO:4 having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3 or SEQ ID NO:4.

[0056] In embodiments, the amino acid sequence of the variant channelrhodopsin is represented by SEQ ID NO: 5, referred to herein as ChRown. Unlike other channelrhodopsin-based gene therapies being evaluated for clinical use, ChRown is not a fusion protein comprising a therapeutic transgene fused to a fluorescent reporter protein (FP), such as green fluorescent protein (GFP) or similar.

[0057] In several embodiments, the transgene is a variant of ChRown that has an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:5.

[0058] Generally, transgene sequences can be about 2-5 kb in length, but this size can consist of the transgene as well as additional non-coding sequences or additional copies of the transgene separated by, for example, a ribosomal read-through or internal ribosomal entry site, or "IRES."

[0059] Gene Delivery Vehicles Adeno-associated viruses (AAVs) are non-enveloped viruses of the Parvoviridae family that require a helper virus for propagation and are therefore considered non-pathogenic. Currently, 12 AAV serotypes have been identified, each defined by a unique capsid protein. Different serotypes also exhibit differences in cell tropism, transduction efficiency, and immunogenicity. Serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9 have been reported to exhibit tropism for retinal cells.

[0060] The present invention provides a recombinant adeno-associated virus (rAAV) vehicle for the delivery and expression of therapeutic transgenes targeted to retinal cells. The term "AAV vehicle" refers to an infectious recombinant viral particle comprising (i) a capsid protein encapsulating a vector genome consisting of a heterologous polynucleotide encoding a transgene. The heterologous polynucleotide encoding a transgene may also be referred to as an rAAV vector. The rAAV vector may also contain additional heterologous polynucleotide sequences upstream and downstream of the transgene. For example, as described in more detail below, the rAAV vector may contain non-AAV promoter, enhancer, and termination / polyadenylation sequences. The vector is constructed so that these heterologous sequences are flanked by two AAV inverted terminal repeats (ITRs). The ITRs generally consist of nucleotides 1-145 at the 5' end of the AAV DNA genome and nucleotides 4681-4536 at the 3' end of the AAV DNA genome. The rAAV vector may also include at least 10 nucleotides after the end of the ITR (e.g., part of the "D region").

[0061] In embodiments, the rAAV vector comprises a 5' ITR having the nucleotide sequence of SEQ ID NO:15 and a 3' ITR having the nucleotide sequence of SEQ ID NO:16.

[0062] The capsid is formed from structural proteins, including one to three structural proteins encoded by the AAV Cap open reading frame, designated VP1, VP2, and VP3, respectively. Viral particles are formed during AAV production using a helper plasmid containing the AAV Rep and Cap open reading frames. The Rep reading frame encodes proteins that regulate replication. In several embodiments, the serotype of the rAAV vehicle is AAV2, and the recombinant viral particles contain a modified VP1 capsid protein.

[0063] The rAAV vehicle described herein advantageously infects primate retinal cells and transfers a vector genome containing a therapeutic channelrhodopsin transgene into the retinal cells, where the transgene is expressed at high levels and in the appropriate intracellular structure, i.e., the plasma membrane of the cell, such that the expressed channelrhodopsin protein functions to generate ion flux through the channel in response to activating light. The resulting ion flux leads to depolarization of the neuronal cell, which in turn transmits an electrical signal, thereby generating an electrical signal in response to activating light.

[0064] The term "retinal cells" may include any of the cell types that comprise the retina, including retinal photoreceptor cells, retinal ganglion cells, and bipolar cells, as well as retinal amacrine cells, horizontal cells, Muller cells, and retinal pigment epithelial cells. In some embodiments, the rAAV vector provides targeted delivery to photoreceptor cells or a combination of photoreceptor cells, retinal ganglion cells, and bipolar cells.

[0065] In some embodiments, self-complementary AAV vectors may be used, which feature an inverted repeat genome that can fold into double-stranded DNA (dsDNA) without the need for DNA synthesis or base pairing between multiple vector genomes.

[0066] In some embodiments, the rAAV vehicle comprises a capsid protein having an amino acid sequence that is modified compared to a native AAV capsid sequence (e.g., SEQ ID NO: 37). In this context, the term "modification" refers to the insertion, substitution, or deletion of one or more amino acids relative to the sequence of a serotype 2 AAV capsid.

[0067] In several embodiments, the rAAV vehicle comprises a modified AAV2 VP1 capsid protein having the amino acid sequence of SEQ ID NO:6, wherein the modified AAV2 VP1 comprises a peptide insertion at amino acid position 588 of the wild-type AAV2 capsid sequence, and wherein, excluding the peptide insertion, the amino acid sequence is at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:6.

[0068] In several embodiments, the rAAV vehicle comprises a modified AAV2 VP1 capsid protein having an amino acid sequence comprising an insertion of the peptide represented by SEQ ID NO: 8 (LGETTRP) at an amino acid position corresponding to amino acid position 588 of the wild-type AAV2 VP1 capsid sequence.

[0069] In several embodiments, the rAAV vector comprises a modified AAV2 VP1 capsid protein having the amino acid sequence of SEQ ID NO:38, wherein the modified AAV2 VP1 comprises a peptide insertion at amino acid position 588 of the wild-type AAV2 capsid sequence, and wherein, excluding the peptide insertion, the amino acid sequence is at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:38.

[0070] In several embodiments, the rAAV vehicle comprises a modified AAV2 VP1 capsid protein having an amino acid sequence comprising an insertion of the peptide represented by SEQ ID NO: 9 (LAISDQTKHA) at an amino acid position corresponding to amino acid position 588 of the wild-type AAV2 VP1 capsid sequence.

[0071] In some embodiments, the rAAV vehicle comprises a modified AAV2 VP1 capsid protein having an amino acid sequence containing an amino acid substitution at position Y444, which corresponds to the position in the wild-type AAV2 VP1 capsid sequence. In some embodiments, the amino acid substitution is Y444F.

[0072] In some embodiments, the rAAV vehicle comprises a modified AAV2 VP1 capsid protein having an amino acid sequence comprising an insertion of the peptide represented by SEQ ID NO:8 (LGETTRP) at an amino acid position corresponding to amino acid position 588 of the wild-type AAV2 VP1 capsid sequence, and further comprising a substitution at the amino acid corresponding to amino acid position 588 of the wild-type AAV2 VP1 capsid sequence, and further comprising an amino acid substitution at a position corresponding to position Y444 of the wild-type AAV2 VP1 capsid sequence. In some embodiments, the amino acid substitution is Y444F.

[0073] In some embodiments, the rAAV vehicle comprises a modified AAV2 VP1 capsid protein having an amino acid sequence comprising an insertion of the peptide set forth in SEQ ID NO:9 (LAISDQTKHA) at an amino acid position corresponding to amino acid position 588 of the wild-type AAV2 VP1 capsid sequence, and further comprising an amino acid substitution at a position corresponding to position Y444 of the wild-type AAV2 VP1 capsid sequence. In some embodiments, the amino acid substitution is Y444F.

[0074] In several embodiments, the rAAV vehicle comprises a capsid protein having an altered amino acid sequence compared to a native AAV capsid protein of serotype 2, a polynucleotide comprising an AAV2 terminal repeat sequence in the 5' to 3' direction, a promoter sequence, a polynucleotide sequence encoding an opsin, a polyadenylation sequence, and the AAV2 terminal repeat sequence.

[0075] In some embodiments, the promoter is selected from a cytomegalovirus (CMV) promoter, an elongation factor 1a (EF1a) promoter, a simian virus 40 (SV40) promoter, a chicken beta-actin promoter, an mGluR6 promoter, and a CAG promoter.

[0076] The CAG promoter comprises three elements: (C) a cytomegalovirus (CMV) early enhancer element, (A) the promoter, the first exon and first intron of the chicken beta-actin gene, and (G) the splice acceptor of the rabbit beta-globin gene. In embodiments, the CAG promoter is a modified CAG promoter of SEQ ID NO: 10, or a sequence having at least 95%, at least 98%, or at least 99% identity thereto.

[0077] In some embodiments, the promoter comprises a modified metabotropic glutamate receptor (mGluR6) promoter. In embodiments, the promoter comprises a fragment of the human mGluR6 promoter region identified by SEQ ID NO: 11 or SEQ ID NO: 12, or a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto. In some embodiments, the promoter comprises a fragment of the human mGluR6 promoter region identified by SEQ ID NO: 24, or a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

[0078] In some embodiments, the vector comprises an mGluR6 regulatory element region, wherein the mGluR6 regulatory element region comprises an mGluR6 promoter or a fragment thereof. In some embodiments, the mGluR6 regulatory element region further comprises an enhancer sequence identified by SEQ ID NO: 13, or a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 13. In some embodiments, the mGluR6 regulatory element region further comprises an enhancer sequence identified by SEQ ID NO: 25, or a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 25. In some embodiments, the mGluR6 regulatory element region may further comprise one or more mGluR6 intron sequences identified by one or both of SEQ ID NO: 26 or SEQ ID NO: 27, or a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity thereto.

[0079] In some embodiments, the mGluR6 regulatory element region comprises a fragment of the human mGluR6 promoter, an mGluR6 enhancer, intron 4 of the mGluR6 gene, and / or intron 3 of the mGluR6 gene. In some embodiments, the promoter comprises, from upstream to downstream, intron 4 of the mGluR6 gene, intron 3 of the mGluR6 gene, an mGluR6 enhancer, and a fragment of the mGluR6 promoter. In some embodiments, the promoter comprises, from 5' to 3', SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:25, and SEQ ID NO:24. In some embodiments, the promoter comprises SEQ ID NO:28, or a sequence having at least 95%, at least 98%, or at least 99% identity thereto.

[0080] In some embodiments, the rAAV vehicle comprises a WPRE enhancer element in combination with a CAG promoter.

[0081] In some embodiments, the rAAV vehicle comprises (i) a modified AAV2 VP1 capsid protein having the amino acid sequence of SEQ ID NO:6, wherein the modified AAV2 VP1 comprises a peptide insertion at an amino acid position corresponding to position 588 of the wild-type AAV2 capsid sequence, and has an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:6, excluding the peptide insertion, wherein the peptide insertion comprises SEQ ID NO:8 or SEQ ID NO:9; and (ii) an rAAV vector encoding ChRown of SEQ ID NO:5 operably linked to a modified CAG promoter set forth in SEQ ID NO:10 and a WPRE enhancer set forth in SEQ ID NO:14. In some embodiments, ChRown set forth in SEQ ID NO:5 can be further operably linked to a polyadenylation sequence set forth in SEQ ID NO:18. The polyadenylation sequence set forth in SEQ ID NO:18 can be positioned downstream of the WPRE enhancer according to SEQ ID NO:14.

[0082] In some embodiments, the rAAV vehicle comprises a VP1 capsid protein comprising SEQ ID NO:7 and an rAAV vector encoding ChRown (SEQ ID NO:5) operably linked to a modified CAG promoter (SEQ ID NO:10) and a modified WPRE enhancer (SEQ ID NO:14). In some embodiments, the rAAV vector encoding ChRown operably linked to a modified CAG promoter, a modified WPRE enhancer, and a human growth hormone polyadenylation sequence (hGHpA) is represented by SEQ ID NO:34 (pCAG-Chrown-mWPRE-hGHpA). In some embodiments, the rAAV vector further comprises a 5' ITR. In some embodiments, the 5' ITR is represented by SEQ ID NO:15. In some embodiments, the rAAV vector further comprises a 3' ITR. In some embodiments, the 3' ITR is represented by SEQ ID NO:16. In some embodiments, the rAAV vector comprises or consists of SEQ ID NO:29.

[0083] In some embodiments, the rAAV vehicle comprises a VP1 capsid protein having the amino acid sequence of SEQ ID NO: 38 and an rAAV vector encoding ChRown (SEQ ID NO: 5) operably linked to a modified CAG promoter (SEQ ID NO: 10) and a modified WPRE enhancer (SEQ ID NO: 14). In some embodiments, the rAAV vector encoding ChRown operably linked to a modified CAG promoter, a modified WPRE enhancer, and a human growth hormone polyadenylation sequence (hGHpA) is represented by SEQ ID NO: 34 (pCAG-Chrown-mWPRE-hGHpA). In some embodiments, the rAAV vector further comprises a 5' ITR. In some embodiments, the 5' ITR is represented by SEQ ID NO: 15. In some embodiments, the rAAV vector further comprises a 3' ITR. In some embodiments, the 3' ITR is represented by SEQ ID NO: 16. In some embodiments, the rAAV vector comprises or consists of SEQ ID NO: 29.

[0084] In some embodiments, the rAAV vehicle comprises: (i) a modified AAV2 VP1 capsid protein having the amino acid sequence of SEQ ID NO:6, wherein the modified AAV2 VP1 comprises a peptide insertion at an amino acid position corresponding to position 588 of the wild-type AAV2 capsid sequence, and wherein the modified AAV2 VP1 has an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:6, excluding the peptide insertion, and wherein the peptide insertion comprises SEQ ID NO:8 or SEQ ID NO:9; and (ii) an rAAV vector encoding ChRown of SEQ ID NO:5 operably linked to the mGluR6 promoter set forth in SEQ ID NO:28, the WPRE enhancer set forth in SEQ ID NO:14, and the polyadenylation sequence set forth in SEQ ID NO:18.

[0085] In some embodiments, the rAAV vehicle comprises a VP1 capsid protein comprising SEQ ID NO:7 and an rAAV vector encoding ChRown of SEQ ID NO:5 operably linked to the mGluR6 promoter SEQ ID NO:28 and a modified WPRE enhancer SEQ ID NO:14. In multiple embodiments, the rAAV vector encoding ChRown operably linked to the mGluR6 promoter, a modified WPRE enhancer, and a human growth hormone polyadenylation sequence (hGHpA) is represented by SEQ ID NO:30 (mGluR6-Chrown-mWPRE-hGHpA). In some embodiments, the rAAV vector further comprises a 5' ITR. In some embodiments, the 5' ITR is represented by SEQ ID NO:15. In some embodiments, the rAAV vector further comprises a 3' ITR. In some embodiments, the 3' ITR is represented by SEQ ID NO:16. In some embodiments, the rAAV vector comprises or consists of SEQ ID NO:31.

[0086] In some embodiments, the rAAV vehicle comprises a VP1 capsid protein having the amino acid sequence of SEQ ID NO:38 and an rAAV vector encoding ChRown of SEQ ID NO:5 operably linked to the mGluR6 promoter SEQ ID NO:28 and a modified WPRE enhancer SEQ ID NO:14. In embodiments, the rAAV vector encoding ChRown operably linked to the mGluR6 promoter, a modified WPRE enhancer, and a human growth hormone polyadenylation sequence (hGHpA) is represented by SEQ ID NO:30 (mGluR6-Chrown-mWPRE-hGHpA). In some embodiments, the rAAV vector further comprises a 5' ITR. In some embodiments, the 5' ITR is represented by SEQ ID NO:15. In some embodiments, the rAAV vector further comprises a 3' ITR. In some embodiments, the 3' ITR is represented by SEQ ID NO:16. In some embodiments, the rAAV vector comprises or consists of SEQ ID NO:31.

[0087] In some embodiments, the rAAV vector encoding ChRown comprises any one of CAG-ChRown-GFP-mWPRE-hGHpA (SEQ ID NO: 32), CAG-ChRown-GFP-hGHpA (SEQ ID NO: 33), CAG-ChRown-Mwpre-hGHpA (SEQ ID NO: 34), ChRown-tdTomato-hGHpA (SEQ ID NO: 35), and CAG-ChRown-hGHpA (SEQ ID NO: 36). In some embodiments, the rAAV vector encoding ChRown further comprises a 5' ITR and a 3' ITR. In some embodiments, the 5' ITR is represented by SEQ ID NO: 15, and the 3' ITR is represented by SEQ ID NO: 16.

[0088] Other vectors encoding ChRown include pCAG-Chrown-GFP-mWPRE-hGHpA (SEQ ID NO: 19), pCAG-Chrown-GFP-hGHpA (SEQ ID NO: 20), pChrown-tdTomato-hGHpA (SEQ ID NO: 22), and pCAG-Chrown-hGHpA (SEQ ID NO: 23).

[0089] In some embodiments, the present invention provides gene delivery vehicles in the form of infectious recombinant adeno-associated virus (rAAV) particles comprising: (i) a capsid protein having an altered amino acid sequence relative to a native AAV capsid of serotype 2; and (ii) a vector genome comprising, or consisting of, a heterologous polynucleotide comprising, in the 5' to 3' direction: (a) an AAV2 inverted terminal repeat (ITR1); (b) a promoter sequence; (c) a polynucleotide sequence encoding a channelrhodopsin; (d) a polyadenylation sequence; and (e) an AAV2 inverted terminal repeat (ITR2), wherein the heterologous polynucleotide does not encode a fluorescent protein.

[0090] In some embodiments, the invention provides gene delivery vehicles in the form of infectious recombinant adeno-associated virus (rAAV) particles comprising: (i) a capsid protein having an altered amino acid sequence relative to a native AAV capsid of serotype 2; and (ii) a vector genome comprising, or consisting of, a heterologous polynucleotide comprising the nucleotide sequence of SEQ ID NO: 29, or a sequence having at least 90%, at least 95%, or at least 99% sequence identity thereto. In some embodiments, the capsid protein comprises, or consists of, SEQ ID NO: 38.

[0091] In some embodiments, the invention provides gene delivery vehicles in the form of infectious recombinant adeno-associated virus (rAAV) particles comprising: (i) a capsid protein having an altered amino acid sequence relative to a native AAV capsid of serotype 2; and (ii) a vector genome comprising, or consisting of, a heterologous polynucleotide comprising the nucleotide sequence of SEQ ID NO: 31, or a sequence having at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the capsid protein comprises, or consists of, SEQ ID NO: 38. Manufacturing method

[0092] The rAAV vehicles described herein can be produced by transfecting a suitable cell line, such as HEK293 cells, with two or three plasmids containing (1) the rAAV vector, (2) the AAV Rep and Cap genes, and, optionally, (3) helper genes. Other platforms include the use of mammalian or insect cell lines that stably express one or more viral helper genes, such as E1a and E1b, or the use of baculovirus / insect cell systems, in which one to three baculovirus particles are used to infect insect cells with the rAAV vector containing, for example, the Rep and Cap genes and a transgene. Insect cells can also be engineered to contain the viral Rep and Cap genes or the rAAV vector stably integrated into their genome.

[0093] Cells may be grown as adherent cultures or in suspension. For larger culture volumes, virus can be produced in roller bottles, multilayer adherent culture flasks, continuous perfusion, or WAVE bioreactor systems.

[0094] The method may further include one or more purification and polishing steps downstream of cell culture and virus production. For example, affinity or heparin chromatography may be used to isolate virus from the culture supernatant. Affinity resins may be based on AAV-specific binding proteins, such as scFvs, and single-domain antibodies derived from llamas (camelids). Additionally, benzonase / DNAse treatment of eluted virus may be used to remove extraviral DNA contamination. The method may further include an anion exchange chromatography step or density gradient centrifugation to separate empty AAV particles from particles containing rAAV vectors.

[0095] In some embodiments, the method may include density gradient centrifugation through a density gradient selected from a continuous cesium chloride (CsCl) density gradient and an iodixanol step density gradient. The method may also include one or more of cell lysis and precipitation of cellular DNA and proteins prior to ultracentrifugation through the density gradient.

[0096] Pharmaceutical Composition The present disclosure provides pharmaceutical compositions comprising the rAAV vehicle described herein.In several embodiments, the composition comprises a pharmaceutically acceptable vehicle, diluent, carrier, and / or excipient.Suitable vehicles include aqueous vehicles such as water or buffered saline, such as phosphate buffered saline ("PBS"), or other suitable buffers for maintaining physiological pH, such as acetate buffer, citrate buffer, phosphate buffer, and borate buffer.

[0097] In embodiments, the pharmaceutical compositions may include one or more excipients suitable for ophthalmic use, including tonicity modifiers, viscosity modifiers, cosolvents, and stabilizers.

[0098] Tonicity adjusters include sodium chloride, potassium chloride, dextran, cyclodextrin, mannitol, dextrose, glycerol, sorbitol, boric acid, borax and propylene glycol, and combinations thereof.

[0099] Viscosity modifiers include hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), carboxymethyl cellulose (CMC), methyl cellulose (MC), hydroxyethyl cellulose (HEC), cellulose and its derivatives, polycarbophil, polyoxyethylene glycol (PEG), hyaluronic acid (HA), amylase and its derivatives, amylopectin and its derivatives, dextran and its derivatives, acrylic polymers such as polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and derivatives of polyacrylic acid or polymethacrylic acid, including hydroxymethyl methacrylate (HEMA), carbomers, and combinations thereof.

[0100] Suitable co-solvents include polysorbates, polyoxyethylene glycol (PEG), or propylene glycol, and combinations thereof.

[0101] Suitable stabilizers include sucrose, sorbitol, glycerol, trehalose, or mannitol.

[0102] In some embodiments, the pharmaceutical composition is formulated for administration by a suitable route for transducing retinal cells, including, for example, intravitreal, intraocular, or subretinal injection. The preferred route of administration is intravitreal injection. As disclosed herein, all retinal neurons, including retinal ganglion cells, bipolar cells, horizontal cells, amacrine cells, and photoreceptor cells, are known to be reasonably accessible for intravitreal injection. Intravitreal and / or subretinal injections can provide the necessary access to bipolar cells, particularly in situations where the photoreceptor cell layer is absent due to degeneration. Other routes that may be suitable include periocular routes, such as retrobulbar, subtendinous, or subconjunctival injections, intracameral injections, or suprachoroidal injections.

[0103] In some embodiments, the pharmaceutical composition formulated for intravitreal injection may include one or more of a cosolvent, an isotonicity agent, a buffer, and a stabilizer. The cosolvent may be, for example, polysorbate, polyoxyethylene glycol (PEG), or propylene glycol, and combinations thereof. The isotonicity agent may be, for example, sodium chloride or potassium chloride. The buffer may be a phosphate buffer, such as phosphate-buffered saline or PBS. The stabilizer may be sucrose, sorbitol, glycerol, trehalose, or mannitol.

[0104] In some embodiments, the pharmaceutical composition comprises 1 x 10 per milliliter. 11 ~1×10 15 particles, or 1 x 10 per injection 8 ~1×10 14 The viral genome is formulated to contain a volume of, for example, 50 to 100 microliters.

[0105] Treatment method The present invention provides methods of treating retinal diseases by administering to a human subject in need of therapy for the retinal disease a pharmaceutical composition comprising an rAAV vehicle described herein.

[0106] In embodiments, the retinal disease may be Bardet-Biddell syndrome, chorioretinal atrophy or degeneration, cone or cone-rod dystrophy, macular degeneration (MD) including hereditary MD such as congenital stationary night blindness, Leber's congenital amaurosis (LCA), age-related MD (AMD) and Stargardt disease, ocular-retinal developmental diseases, optic atrophy, retinitis pigmentosa, syndromes / systemic diseases associated with retinopathy, Usher syndrome, or other retinopathies including diabetic retinopathy.

[0107] In embodiments, the retinal disease is age-related macular degeneration (AMD), a common cause of vision loss resulting from the degeneration of photoreceptor cells in the central portion of the retina.

[0108] In embodiments, the retinal disease is Stargardt's disease.

[0109] In some embodiments, the retinal disease is retinitis pigmentosa (RP). RP is a genetic disorder that results in the degeneration of photoreceptor cells in the retina. Vision loss typically begins with damage to rod cells, which are responsible for detecting light and dark, and progresses to cone cells, which are responsible for color vision.

[0110] In some embodiments, the retinal disease is Leber's congenital amaurosis (LCA), which may also be called Leber's hereditary optic neuropathy. LCA is a rare genetic disorder that affects males and results in rapid vision loss due to degeneration of the photoreceptor cells in the retina.

[0111] According to the methods described herein, a therapeutically effective amount of rAAV vehicle is administered to a subject in need of treatment. A therapeutically effective amount is an amount or dose sufficient to treat a disease or disorder, or to achieve a desired therapeutic result, such as improvement or stabilization of a disease or disorder, or one or more clinical symptoms. In some embodiments, a therapeutically effective amount is an amount or dose sufficient to improve one or more of visual acuity, visual field, navigation, motion detection, contrast sensitivity, light sensitivity, object detection, and light / dark discrimination. In some embodiments, a therapeutically effective amount is an amount or dose sufficient to improve activities of daily living and / or quality of life, as determined by standardized questionnaires, patient-reported outcomes (PROs).

[0112] The term "vision" includes the eye's ability to detect light from the external environment and transmit signals to the brain for perception. The compositions and methods described herein can also improve or restore vision, including, for example, improved light detection or perception, improved light sensitivity, improved ability to discern the direction from which light stimuli are coming, improved ability to detect different luminance and contrast levels, improved ability to recognize the shape of visual targets, and improved visual evoked potentials or transmission from the retina to the cortex. Visual restoration may involve placing affected individuals at the lower end of visual function by increasing aspects of vision, such as light sensitivity or visual evoked potentials, without restoring complete vision. Vision improvement may include improvements in visual acuity, contrast sensitivity, color, depth perception, visual field, and the ability to perceive navigation, as well as correlated electrophysiological responses, as described by electroretinograms and visual evoked potentials.

[0113] The degree of visual recovery can be determined through measuring visual acuity before and after administration of a composition containing a therapeutic transgene, including, but not limited to, the methods described herein. Visual acuity can be measured, for example, by (1) a light detection response by a subject after exposure to a light stimulus, (2) a light projection response by a subject after exposure to a light stimulus, (3) a subject's ability to resolve light versus dark patterned visual stimuli, as evidenced by, for example, the presence of demonstrable, reliable eye movements and / or associated head or body movements indicative of target tracking, and / or the presence of a reliable ability to distinguish between patterned visual stimuli and demonstrate such distinctions by verbal or non-verbal means. These may include, for example, by pressing a bar or button and (4) visual acuity, (5) visual field testing, e.g., the Humphrey Visual Field Test and the Full-Field Sensitivity Test (FST), (6) navigation and light intensity measurements, e.g., the Multi-Luminance Mobility Test (MLMT), (7) contrast sensitivity, e.g., qCSF, (8) color vision testing or electrical recordings from the retina and visual cortex, e.g., electroretinogram and visual evoked potentials, and / or dynamic anatomical imaging of the brain (functional magnetic resonance imaging (fMRI)).

[0114] Thus, improvement or restoration of vision may include an increase in the amplitude or dynamic characteristics of the photocurrent or electrical response to light stimulation in retinal cells, an increase in light sensitivity due to a decrease in the threshold light intensity required to initiate a photocurrent or electrical response to light stimulation, an increase in the number or amplitude of light-evoked spikes or spike firing upon light stimulation, or an increase in light responses in the visual cortex (including visual evoked potentials).

[0115] In some embodiments, a therapeutically effective amount is about 100 μl in a volume of about 25 to about 200 μl, or about 50 to 100 microliters per injection. 10 ~about 10 15 The dose contains rAAV infectious units or vector genomes (vg). rAAV infectious units can be measured, for example, according to Del la Camara et al. Accurate Quantification of AAV Vector Genomes by Quantitative PCR, Genes (Basel). 2021 Apr;12(4):601. Vector genome copies can be determined, for example, using a quantitative polymerase chain reaction (qPCR)-based method. AAV serotype 2 reference material (RSM) is available from the American Type Culture Collection. Vector genomes are preferably determined by droplet digital PCR. Other dosages and amounts, preferably within these ranges but possibly outside them, can be selected by a treating professional taking into account the physical condition of the subject (preferably human) being treated, as well as the age, weight, general health, and nature and severity of the particular eye disorder.

[0116] In embodiments, the therapeutically effective amount is 1×10 per milliliter 11 ~1×10 15 of viral particles, or 1 x 10 per injection 8 ~1×10 14In some embodiments, the pharmaceutical composition contains 1 x 10 viral genomes per milliliter, for example, in an amount of 50 to 100 microliters. 11 ~1×10 15 of viral particles, or 1 x 10 per injection 8 ~1×10 14 The virus genome is formulated to contain, for example, 50 to 100 microliters.

[0117] In the context of the methods described herein, the subject to be treated is a subject in need of treatment for a retinal disease. The subject is preferably a human, although other mammalian subjects, including non-human primates, are contemplated. The term "patient" refers to a human subject exhibiting one or more clinical symptoms of a retinal disease. In some embodiments, the patient is a human subject diagnosed with a retinal disease.

[0118] "Treatment," "treating," and "treat" describe the management and care of a patient for the purpose of combating retinal disease and include the administration of a composition described herein to alleviate the symptoms or complications of retinal disease, which may include visual restoration, which may be partial visual restoration or improved vision relative to the vision before treatment began. "Treatment" refers to (1) taking steps to obtain a beneficial or desired result, including a clinical result such as improvement or reduction of one or more symptoms of retinal disease; (2) inhibiting retinal disease, e.g., halting or reducing the occurrence or clinical progression of the disease or any one or more of its clinical symptoms; (3) palliating the disease, e.g., causing regression of the disease or its clinical symptoms; or (4) delaying or slowing disease progression.

[0119] In the context of the present methods, the term "administration," when used in reference to the compositions described herein, can refer to direct administration or indirect administration. Indirect administration includes the act of formulating a composition containing the rAAV vehicle described herein. Direct administration includes administration to cells in vitro, administration to cells in vivo, administration to a patient by a medical professional, or self-administration by a patient.

[0120] In several embodiments, including both monotherapy with the compositions described herein and combination therapy with one or more additional therapies or therapeutic agents, administration of the compositions of the invention leads to elimination of symptoms or complications of the disease being treated, but does not require elimination of the disease. In one embodiment, the severity of the symptoms is reduced.

[0121] Example 1: In vitro functional evaluation Tests were conducted to evaluate the in vitro functional efficacy of different rAAV expression plasmids. Transmembrane transport of the expressed transgene was assessed by immunocytochemistry. Electrophysiological recordings were used to assess transgene function. The following five constructs were tested:

[0122] Three constructs produced fusion proteins of the transgene with either the GFP or tdTomato fluorescent reporter protein. pCAG-ChRown-GFP-mWPRE-hGHpA pCAG-ChRown-GFP-hGHpA pChRown-tdTomato-hGHpA

[0123] Two constructs produced the transgene without fusion to a fluorescent protein. pCAG-ChRown-mWPRE-hGHpA pCAG-ChRown-hGHpA

[0124] HEK cell culture, DNA transfection, and patch-clamp recording: HEK293f cells were maintained in Advance Dulbecco's minimum essential medium (Life Technologies, Grand Island, NY) supplemented with 5% fetal bovine serum, 1x minimum essential medium (MEM) non-essential amino acid solution, 100 U / mL penicillin G, and 100 mg / mL streptomycin at 37°C in a humidified atmosphere of 95% air and 5% CO2. HEK293f cells were passaged weekly.

[0125] For patch clamp recordings, HEK 293f cells were seeded on 35 mm dishes. Whole-trans retina (1 μM) was added to the culture medium at the time of cell seeding. Cells were transfected with 1 μg of plasmid using Lipofectamine 2000 (Life Technologies), and patch clamp recordings were performed 2 days after transfection. Recordings in the whole-cell configuration were performed using standard procedures at room temperature (22 °C). The extracellular recording solution contains the following components (in mM): 138 mM NaCl, 1 mM NaHCO3, 0.3 mM Na2HPO4, 5 mM KCl, 0.3 mM KH2PO4, 1.25 mM CaCl2, 0.5 mM MgSO4, 0.5 mM MgCl2, 5 mM HEPES, 22.2 mM glucose, and 0.001% (v / v) phenol red, with the pH adjusted to 7.2 using 0.3 N NaOH. The extracellular solution is based on normal Hank's solution with supplemental 1 mM all-trans retinal buffer. The electrode solution contained the following components (in mM): 110 mM Cs-Cl, 30 mM TEA-Cl, 2 mM MgCl, 0.1 mM CaCl, 10 mM EGTA, and 10 mM HEPES, with the pH adjusted to 7.25 using CsOH. Electrodes were pulled with a vertical pipette puller (Narishige, Japan), coated with silicone, and then polished with a bulb filament (Narishige, Japan). Optical stimuli were generated by a 150-W xenon lamp-based scanning monochromator (TILL Photonics, Germany) with a 10 nm bandwidth. Optical stimuli were coupled to the microscope via an optical fiber. Pulsed optical stimuli were either 10 ms or 1 s. The light intensity was 1.3 × 10 s without a neutral density filter. 17 photon / cm 2 The light intensity was adjusted using neutral density filters (ASI, Applied Scientific Instrumentation, Oregon, USA).

[0126] All data are expressed as mean ± SD, and "n" indicates the number of animals. One-way analysis of variance (ANOVA) was used to determine whether there were statistically significant differences between groups.

[0127] Results: Fluorescence and DIC images of HEK cells after transfection with the transgene-fluorescent protein fusions demonstrated that transmembrane expression was achieved. All five constructs produced light-evoked currents in patch-clamp assays. The off-rates of currents evoked by 10-ms light pulses from each of the five constructs were similar, ranging from 600 to 700 ms. Light-evoked currents in response to 1-s pulses of progressively increasing light intensity yielded current amplitudes and current inactivation characteristics. No apparent differences in expression were observed between transgene-GFP fusion constructs containing the mWPRE and those lacking the mWPRE element.

[0128] Example 2: In vivo functional evaluation in mice In vivo functional evaluation of the test constructs was performed using blinded Opn4 - / - Gnat1 - / - Cnga3 - / - This study was performed in a triple knockout (TKO) transgenic mouse strain. TKO mice lack the visuomotor response (OMR), making them an ideal model system for assessing visual function recovery using visuomotor behavioral assays. The vector construct was injected intravitreally into eyes at a dose of 7.5E+9vg per eye (n=6 per group). Group designations and constructs: R01:pCAG-ChRown-GFP-mWPRE-hGHpA R02:pCAG-ChRown-GFP-hGHpA R03:pCAG-ChRown-mWPRE-hGHpA R04:pChRown-tdTomato-mWPRE-hGHpA R05:pCAG-ChRown-hGHpA

[0129] Viral vector injection: Intravitreal injection of viral vectors was administered to 1-3 month old TKO mice. One hour before the injection experiment, EthiqaXR (buprenorphine, 3.25 mg / kg) was injected intraperitoneally. The animals were then anesthetized with an intraperitoneal injection of a mixture of 100 mg / kg ketamine and 12 mg / kg xylazine. 5 × 10 l in PBS was used. 2 Viral vectors (1.5 μL) diluted to a titer of 1000 mg / mL were injected intravitreally into both eyes of each animal. Viral vectors were injected using a glass micropipette with a programmable Nanoliter Injector III (Drummond Scientific, Broomall, PA, USA). The entry site was approximately 0.5 mm posterior to the sclera.

[0130] Visuomotor response: Visuomotor response (OMR) testing was performed >1 month after viral injection. Two optical motor systems were used: a custom optical motor system (cOMS) (Pan lab, Detroit, USA) and a commercially available OptoDrum (StriaTech, Germany). cOMS was used to measure the threshold light sensitivity required to elicit an OMR, defined as light sensitivity.

[0131] For the cOMS, the light stimulus was generated by a blue LED with a peak wavelength of 470 nm, attached to the inner surface of a wooden cylinder (40 cm diameter x 51 cm height). The light intensity of the LED was controlled by a digital power supply with a voltage output. At each voltage value, the luminous intensity was expressed as μW / cm. 2 The light intensity, in units of 1 W / cm, was measured by a light meter at the center of the platform. 2 =[wavelength / 500]×2.5×10 18 photon / cm 2 Based on the formula for s, photons / cm 2 s. The highest light intensity achievable with a cOMS is 1×10 16 photon / cm 2 It was s.

[0132] A series of interchangeable drums (30 cm diameter x 30 cm height) were created by marking strips on the walls of an acrylic cylinder at spatial frequencies of 0.031, 0.042, 0.064, 0.092, 0.13, and 0.19 cycles per degree. The drums were covered with a light-diffusing film. Drum rotation was controlled by a digital motor and set at 4 rpm or 24 degrees per second. During testing, unrestrained animals were placed on a central platform (6.5 cm diameter) positioned 11.5 cm above the floor. A video camera was mounted above the apparatus for monitoring and capturing animal behavior.

[0133] To determine the threshold light intensity at each grating frequency, each animal's ability to elicit head tracking in response to drum rotation was assessed, starting with a relatively high light intensity. Head tracking was tested in both clockwise and counterclockwise directions. Once tracking was confirmed, the light intensity was systematically reduced to determine the lowest light intensity that still elicited head tracking.

[0134] Immunohistochemistry: For immunohistochemical and Western blot analyses, animals were euthanized by CO2 asphyxiation followed by decapitation. Mouse eyes were enucleated and then fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 20 minutes at room temperature. Immunofluorescence staining was performed on retinal whole mounts. The following primary antibodies were used in this study: rabbit anti-ChRown (1:50, produced by Innovagen AB, Sweden), mouse anti-GFP (1:1000, Neuromab, UC Davis, Davis, CA, USA), and rabbit anti-RBPMS (1:1000, ABN1362, MilliporeSigma, Temecula, CA, USA). Secondary antibodies were conjugated to Alexa 488 (1:600), Alexa 555 (1:1,000), or Alexa 594 (1:500, Thermo Fisher Scientific, Waltham, MA, USA). Fluorescent images were obtained using a ZEISS APOTOME2 light microscope (Apotome; Carl Zeiss Microscopy GmbH, Jena, Germany). Brightness and contrast were adjusted using ZEN3.5 software. RGC counting was performed using stacked images generated in ZEN3.5-3D mode.

[0135] All data are expressed as mean ± SD, and "n" indicates the number of animals. One-way analysis of variance (ANOVA) was used to determine whether there were statistically significant differences between groups.

[0136] Safety / Toxicity: There were no survival safety observations or animal deaths. Histological analysis showed no signs of retinal toxicity.

[0137] Light Sensitivity: The threshold light intensity required to induce OMR at an optimal frequency of 0.042 cycles / degree, defined as light sensitivity, is shown in Table 1. The highest light sensitivity was observed in treatment groups R01 and R03.

[0138] Unexpectedly, treatment groups R01 and R03 showed greater light sensitivity, particularly compared to constructs lacking the mWPRE element, R02 and R05. This was unexpected, at least based on the relative expression of constructs with and without the mWPRE element, which, as noted above, did not differ appreciably. This indicates that expression, while related to function, is not the only factor affecting transgene function. Other factors include proper expression within the cell—in this case, at the cell membrane, as opposed to intracellular expression—and correct protein folding and orientation at the membrane.

[0139] Group R03, treated with a construct containing ChRown but not GFP, showed similar light sensitivity compared to group R01, whose transgene was a ChRown-GFP fusion protein. The similar function observed in treatment groups R01 and R03 was unexpected based on previous literature, e.g., Gauvain, G. et al. Optogenetic therapy: high spatiotemporal resolution and pattern discrimination compatible with vision restoration in non-human primates. Commun. Biol. 4:125 (2021) and Sahel et al. Partial recovery of visual function in a blind patient after optogenetic therapy. Nature Medicine 27:1223-1229 (2021). This indicated that the transgene should be stabilized by fusion with a fluorescent reporter. Such improved stabilization would be expected to result in improved function, which in this case would have resulted in higher light sensitivity for treatment group R01. Even more unexpectedly, treatment group R04, in which the transgene was a fusion protein of ChRown with another fluorescent reporter, tdTomato, exhibited poorer light sensitivity compared to ChRown alone. Fusion proteins, particularly those with tdTomato, have been reported by Guabain and Sahel to stabilize therapeutic transgenes. These results highlight the difficulty of predicting the function of a particular transgene construct based on previous studies using identical or similar genetic elements. [Table 1]

[0140] Immunohistochemistry: TKO mice were euthanized and retinas were isolated for immunostaining. Retinas treated with R01–R04 were labeled with anti-GFP, anti-mCherry, or anti-ChRown (275–288) antibodies. Retinas treated with R01–R05 were also labeled with anti-RBPMS antibodies, a ganglion cell-specific marker. Note that the use of RBPMS antibodies is preferred over DAPI for labeling RGC nuclei because DAPI can also label microglial nuclei and migrated amacrine cells. Robust expression of both ChRown and fusion proteins, ChRown-GFP and ChRown-tdTomato, was observed in inner retinal neurons, primarily RGCs. In this study, GFP was observed on the cell membrane without evidence of intracellular aggregation; however, protein misfolding and aggregation are concerns regarding transgene-FP fusion proteins. Importantly, the ChRown protein itself, but not the fusion protein form, was properly expressed on the cell membrane and dendrites of ganglioneuroma cells, as well as on some horizontal and amacrine cells.

[0141] Example 3: Ex vivo multielectrode array analysis Transplants from TKO mice treated with pCAG-ChRown-GFP-mWPRE-hGHpA (R01) and pCAG-ChRown-mWPRE-hGHpA (R03) were evaluated for retinal electrophysiological responses using multielectrode array analysis.

[0142] Multielectrode array (MEA) recording: MEA recordings were performed according to the method previously described in Bi, A., et al., Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration. Neuron, 2006. 50(1):pp. 23-33. Animals were euthanized by decapitation. Retinas were dissected and placed photoreceptor-side down on a piece of nitrocellulose filter paper (Millipore, Bedford, MA, USA). The mounted retina was placed in an MEA-64 MEA recording chamber with 30 mm diameter electrodes (Multichannel Systems MCS, Reutlingen, Germany) spaced 200 mm apart, with the ganglion cell layer facing the recording electrode. Retinas were continuously perfused with oxygenated extracellular solution at 34°C throughout all experiments. The extracellular solution contained the following components (in mM): 124 NaCl, 2.5 KCl, 2 CaCl, 2 MgCl, 1.25 NaHPO, 26 NaHCO, and 22 glucose with 95% O and 5% CO at pH 7.35. The extracellular solution was made fresh before each recording and bubbled with CO 30 min before tissue dissection. The interval between the onset of each photostimulation was 20 s. Signals were filtered at 200 Hz (low cutoff) and 20 kHz (high cutoff). A threshold of 0.4% was used to detect action potentials, and action potentials from individual neurons were determined using a standard expectation-maximization algorithm to perform T-Dist EM sorting using offline sorter software (Plexon, Dallas, TX, USA). Results were plotted using NeuroExplorer software (Nex Technologies, Madison, AL, USA). The relationship between response amplitude decay and frequency was measured from mean spike rate histograms.

[0143] Light stimuli were generated using a 150 W xenon lamp-based scanning monochromator (TILL Photonics, Germany) with a 10 nm bandwidth. Light intensity was adjusted using a neutral density filter device (ASI, Applied Scientific Instrumentation, Oregon, USA) with a software program control. Light stimuli were projected directly onto the bottom of the recording chamber through an optical fiber.

[0144] Results: Light-dependent transgene-mediated spiking activity of RGCs was observed for both constructs in MEA recordings from retinal whole mounts. Figure 1A shows ChRown-mediated spiking activity. Figure 1B shows the activity of the fusion protein, ChRown-GFP. Photons / cm 2 Light intensity, measured as s, is shown at the top right of each trace. The threshold light sensitivity required to elicit spike activity is 10 13 photon / cm 2 Figures 1C and 1D show light-evoked spikes recorded from a single electrode for ChRown and ChRown-GFP, respectively. Figures 1E and 1F show raster plots of 30 consecutive light-evoked spikes from a single neuron (30 trials and 10 min of recording) for ChRown and ChRown-GFP, respectively. Figures 1G and 1H show mean spike rate histograms for ChRown and ChRown-GFP, respectively. Transgene-mediated spike activity of RGCs was stable during repeated light stimulation.

[0145] Example 4: Mouse pharmacology The OptoDrum system was used to measure visual acuity in animals treated with low or high doses of the following test articles (treatment groups in parentheses): pCAG-ChRown-GFP-mWPRE-hGHpA(R01) pCAG-ChRown-mWPRE-hGHpA(R03) pCAG-GFP (R00, negative control)

[0146] The OptoDrum generates virtual drums with different spatial frequencies on four 24-inch LCD monitors. Animal head tracking detection was based on an automated program. The spatial frequency was systematically increased during the test. Visual acuity was defined as the highest grating frequency that could elicit head tracking. [Table 2]

[0147] Viral vector injection: Intravitreal injection of viral vectors was administered as follows: One hour before the injection experiment, EthiqaXR (buprenorphine, 3.25 mg / kg) was injected intraperitoneally. The animals were then anesthetized with an intraperitoneal injection of a mixture of 100 mg / kg ketamine and 12 mg / kg xylazine. 5 × 10 l in PBS was administered. 2 Viral vectors (1.5 μL) diluted to a titer of 1000 mg / mL were injected intravitreally into both eyes of each animal. Viral vectors were injected using a glass micropipette with a programmable Nanoliter Injector III (Drummond Scientific, Broomall, PA, USA). The entry site was approximately 0.5 mm posterior to the sclera.

[0148] All data are expressed as mean ± SD, and "n" indicates the number of animals. One-way analysis of variance (ANOVA) was used to determine whether there were statistically significant differences between groups.

[0149] There were no survival safety observations or animal deaths.

[0150] Light sensitivity: Thresholds were measured at a grating frequency of 0.042 cycles / degree. Results for the two doses in treatment groups 1 and 3 are shown in Figure 2. No response was elicited in animals administered R00 (negative control). There was no statistically significant difference between the two tested doses.

[0151] Further testing was performed in the TKO mouse model (group size 3–4 animals) to determine approximately 1 × 107 vg / eye~1×10 11 The light sensitivity behavioral response was assessed across a dose range of 1E8 vg / eye. Unexpectedly, given the complex biology of optogenetically restored vision, the dose-response curve followed a smooth exponential function. Furthermore, the data indicate that the minimum dose at which maximal light sensitivity is observed is approximately 1E8 vg, which is the dose at which >50% of retinal ganglion cells can be histologically demonstrated to express ChRown protein. The human equivalent dose (HED) of 1E8 vg / eye (mouse) is 2-6E9 vg / eye (human).

[0152] Example 5: NHP Study A study was conducted in non-human primates (NHPs) to compare the ocular biodistribution and GFP expression of three different AAV capsids (AAV2, AAV8, and AAV2-7m8) after intravitreal (IVT) administration to African green monkeys. In this study, a GFP transgene was fused to ChRown.

[0153] method Each of the five test articles contained the same transgene expression construct and was packaged within either AAV2(wt), AAV8(wt), or AAV2(7M8) viral capsids. As shown in Table 3, 2 x 10 12 vg / mL and 2 × 10 13 Two viral titers of vg / mL were tested. The transgene expression construct was pCAG-ChRown-GFP-mWPRE-hGHpA.

[0154] The control substance was vehicle (1x PBS + 0.001% Pluronic F-68).

[0155] Monkeys underwent baseline screening and were assessed for AAV neutralizing antibody (Nab) seronegativity, complete blood count (CBC), overall well-being, and ocular health by slit-lamp biomicroscopy, ophthalmoscopy, color fundus imaging, confocal scanning laser ophthalmoscopy (cSLO), and optical coherence tomography (OCT). Nab-negative monkeys with >50% transduction at a <1:10 dilution and normal findings were enrolled in the study and assigned to treatment groups (Table 3). For baseline screening and all subsequent procedures (Table 4), anesthesia was achieved with intramuscular ketamine (8 mg / kg) and xylazine (1.6 mg / kg), with pupil dilation using topical 10% phenylephrine, 1% tropicamide, and / or 1% cyclopentolate. [Table 3] [Table 4]

[0156] In Table 4, the number in the first row indicates time in weeks, with the corresponding days being 0, 7, 14, 28, 35, 42, and 43. Administration was by intravitreal injection (OU) into both eyes; X indicates an event occurring in all groups. * 32 animals were screened for Nab and CBC at week 3. B = baseline, week 0, day 0; ** MP (methylprednisolone 8 mg / kg IM) weekly administration; wt = body weight; Ex = physical examination; T = tonometry; SL = slit lamp examination; + Slit lamp examination was performed immediately after administration. C = color fundus imaging; F = fluorescence imaging; OCT = optical coherence tomography; cSLO = confocal scanning laser ophthalmoscopy; Nab = neutralizing antibody; CBC = complete blood count; Necr = necropsy.

[0157] Animals were anesthetized with ketamine / xylazine (8.0 mg / kg ketamine [Covetrus] / 1.6 mg / kg xylazine [Covetrus]) in a sterile mixed cocktail administered for baseline screening and all nonsurgical procedures. Overall well-being was assessed before, during, and after sedation. Temperature within the non-air-conditioned ventilated test enclosure ranged from 22.3 to 33.0 °C throughout the study period. Humidity ranged from 64.0 to 100.0%.

[0158] Topical proparacaine 0.5% was administered, an eye speculum was placed, and the ocular surface was then rinsed with 5% Betadine solution followed by sterile 0.9% saline. In both eyes (OU), according to treatment assignment, intravitreal (IVT) injections were administered using a 31-gauge 0.375-inch needle inserted inferiorly at the level of the ora serrata, approximately 2 mm posterior to the limbus. After injection, topical neomycin, polymyxin, and bacitracin antibiotic ophthalmic ointment were administered.

[0159] Animals received prophylactic corticosteroid treatment with tapering. Topical proparacaine 0.5% was administered, an eye speculum was placed, and the ocular surface was then rinsed with 5% Betadine solution, followed by sterile 0.9% saline. In both eyes (OU), intravitreal (IVT) injections were administered according to treatment assignment using a 31-gauge 0.375-inch needle inserted inferiorly at the level of the ora serrata, approximately 2 mm posterior to the limbus. Topical neomycin, polymyxin, and bacitracin antibiotic ophthalmic ointment were administered after injection.

[0160] Animals received intramuscular (IM) delivery of methylprednisolone (8 mg / kg) on ​​day 0 and then weekly during the survival portion of the study (6 weeks). Monkeys received steroid prorenalin (PRN) according to the following criteria, dictated by scheduled slit lamp examination based on the clinical findings of each individual animal: 1. If water cells (AC) +1 or less were observed, no treatment was administered. 2. If the AC was 2+ or higher, subconjunctival dexamethasone (100 μL of 10 mg / mL) was administered, and the eye was evaluated 48–72 hours later. If the AC decreased (to 2 or lower) and the eye was observed 7 days later, subconjunctival dexamethasone was repeated. 3. Treated as above, if AC of 3 or greater persisted 1 week after subconjunctival dexamethasone, IVT triamcinolone (TMC) (100 μL of 40 mg / mL) was administered and the eye was evaluated within 48–72 hours.

[0161] If at any time point AC or VC (vitreous cells) was 4+, the eye was treated with IVT TMC (100 μL of 40 mg / mL) and evaluated at 48–72 hours.

[0162] At designated time points throughout the study, various clinical observations were performed and scored using a summary score derived from the Nonhuman Primate Ophthalmology Scoring System and study components. Clinical findings included intraocular pressure (IOP) measurements, ocular examination with slit-lamp biomicroscopy, optical coherence tomography (OCT), and confocal scanning laser ophthalmoscopy (cSLO). Additionally, bilateral color anterior and posterior fundus images were captured to detect GFP expression, as well as fluorescent fundus images. Fluorescent photographs were evaluated using a scoring system to define the degree of GFP expression, with quantitative analysis applied as needed, in the foveal, peripheral, and perivascular regions of the eye, with scores of 0 = absent, 1 = trace, 2 = mild, 3 = moderate, 4 = bright, and 5 = strong. Other routine clinical observations included respiratory rate, heart rate, body temperature, chest auscultation, skin integrity, and body weight. Overall well-being was monitored twice daily. Animals were evaluated for signs of ocular inflammation, including swelling, discoloration, wrinkling, and eye rubbing.

[0163] At the designated time points, intraocular pressure (IOP) measurements were collected using a TonoVet (Icare®, Gonk) tonometer set to the canine (d) calibration setting. Three measurements were taken from each eye at each time point, and the mean IOP was defined.

[0164] At the designated time points, both eyes (OU) were examined by slit-lamp biomicroscopy. Qualitative clinical ophthalmologic findings were scored using a summary score derived from the Nonhuman Primate Ophthalmology Scoring System and study components.

[0165] Slit lamp examination of animals in cohort 2 was performed on day 8 instead of day 7, and slit lamp examination of animals in cohort 1 was performed on day 15 instead of day 14. This did not affect the study.

[0166] At the indicated time points, bilateral color anterior and fundus images for detecting GFP expression and fluorescent fundus images were captured using a Topcon TRC-50EX fundus camera equipped with Canon 6D digital imaging hardware and New Vision Fundus Image Analysis System software, with a 50° field of view centered on the fovea. Images were acquired centered on the fovea, with four additional image series acquired from the superior, inferior, nasal, and temporal quadrants. Color fundus photographs were captured at a shutter speed (Tv) of 1 / 25 s, ISO 400, and flash 18. Monochromatic and color fluorescent images were captured using an exciter and barrier filter engagement (480 nm exciter / 525 nm barrier filter), Tv of 1 / 5 s, ISO 3200, and flash 300.

[0167] At designated time points, OCT and cSLO were performed in the OU using a Heidelberg Spectralis OCT HRA (or OCT Plus) with the Hyex TruTrack and AutoRescan follow-up imaging functions, referencing the baseline image. cSLO infrared (IR) and autofluorescence (AF) retinal images were acquired using a 50° lens, centered on the fovea, with additional images acquired in the superior, inferior, nasal, and temporal quadrants. Following the IR and AF images, a global OCT volume scan of the entire macula was performed at high-density scan intervals. Images were qualitatively evaluated using OCT retinal thickness data and quantitative analysis of GFP expression, as needed.

[0168] General well-being was assessed twice daily by cageside observation beginning 1 week prior to dosing. Food consumption and general appetite were assessed by visual inspection of the feed pan and cage floor before each feeding and cage cleaning. Animals were evaluated for signs of ocular inflammation, including swelling, discoloration, wrinkling, and eye rubbing.

[0169] Body weights were obtained at the indicated time points.

[0170] Whole blood (3 mL) was collected via the femoral or saphenous vein at the designated time points. The blood was transferred to vacuum tubes (without anticoagulant) and incubated at room temperature for approximately 1 hour. After centrifugation at 4000 rpm for 10 minutes at 4°C, serum aliquots (approximately 2 aliquots of 0.5 mL per time point) were transferred to Sarstedt cryotubes. The aliquots were stored and shipped at temperatures below -70°C to the sponsor's designated laboratory for Nab analysis.

[0171] At the designated time points, 0.5 mL of blood was transferred directly into K3EDTA lavender top vacuum tubes (Greiner MiniCollect EDTA tubes REF # 450475) and kept on ice until CBC with differential analysis on a Hemavet analyzer.

[0172] After confirming the final image quality before the defined end point, the monkeys were sedated with intramuscular ketamine (8 mg / kg) and xylazine (1.6 mg / kg) and euthanized with sodium pentobarbital (100 mg / kg IV).

[0173] Both eyeballs (OU) were enucleated, excess orbital tissue was trimmed with the optic nerve attached, and then placed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) for 24 hours at room temperature. The anterior segments were then removed, the eyecups flat-mounted, and the retinas collected and suspended in PBS containing 0.05% azide in fully filled 60 mL vials and shipped to the sponsor-designated laboratory in a Credo temperature-controlled container designed to maintain the temperature at 4°C.

[0174] Necropsies were performed and additional tissues were collected: optic nerve OU, optic chiasm, lateral geniculate body (right and left), and visual cortex (right and left) for possible histological pathology and GFP biodistribution analysis, pending findings in living and ocular tissues. Samples were transferred to 10% NBF in labeled vials and maintained at the study facility as per the sponsor's requirements.

[0175] Data generated from protocol-defined endpoints were collated, summarized, and analyzed as descriptive statistics.

[0176] Retinal biodistribution and transgene expression were assessed by repeated in vivo qualitative retinal fluorescence imaging. After confirming final image quality before a defined endpoint, monkeys were sedated and euthanized.

[0177] Both eyeballs (OU) were enucleated, excess orbital tissue was trimmed with the optic nerve attached, and the eyeballs were fixed in paraformaldehyde before the anterior segment of the eyecup was removed. Retinas were collected for immunohistochemistry. Additional tissues were collected, including the optic nerve OU, optic chiasm, lateral geniculate bodies (right and left), and visual cortex (right and left).

[0178] result The test substance was administered according to treatment assignment without any complications. In all cases, administration was by intravitreal injection. There was one animal per group, and for each animal, 1 x 10 11 vg / eye dose administered to the right eye (OD), 1 × 10 12 A dose of 1 × 10 vg / eye was administered to the left eye (OS), but a dose of 1 × 10 vg / eye was administered to both eyes. 1 Excluding groups 14 to 16, which were vg / eye. [Table 5]

[0179] Mean intraocular pressure (IOP) was measured at designated time points from baseline through Day 42. IOP remained within the normal range for eyes within all treatment groups throughout the study. There were no dose-related findings.

[0180] No slit lamp abnormalities were detected, except for intraocular inflammation beginning around day 14. The presence or absence of intraocular inflammation was assessed by slit lamp biomicroscopy in all eyes at the indicated time points and ranged from absent to mild (Figures 3A-3D). There was no evidence of retinal or other ocular pathology. Inflammation was seen in the AAV2-treated group, the AAV2 7m8 low-dose treated group, and the AAV2 7m8 high-dose treated group. Inflammation was absent in the vehicle-treated group and was minimal in the AAV8 low-dose and AAV8 high-dose treated groups. Although there was variability between animals, the level of inflammation appeared to be associated with the degree of retinal GFP fluorescence.

[0181] According to the protocol, on day 21, animals D469, D655, D656, D467, and O9922 received IVT triamcinolone (100 µL of 40 mg / mL) and the eyes were evaluated within 48–72 hours. On day 28, O9988, S1071, O9922, D015, and D655 received IVT triamcinolone (100 µL of 40 mg / mL) and the eyes were evaluated within 48–72 hours. Animal D655 showed the most severe inflammation on day 21 and received additional meloxicam subcutaneously (0.2 mg / kg) for 5 days for analgesia.

[0182] After treatment with triamcinolone, the eyes showed complete resolution of inflammation by 35 and 42 days.

[0183] At designated time points during the study, bilateral color anterior and posterior fundus images and GFP fluorescent fundus images were obtained to assess ocular health and GFP expression. Retinal morphology remained within normal limits. There were no signs of retinal detachment, edema, vasculitis, or other major problems observed in the fundus images.

[0184] GFP fluorescence signal scoring on a scale of 0 to 5 is shown in Figures 4A-4C. No GFP was observed in vehicle-treated eyes or eyes treated with the low dose of AAV8. GFP expression in eyes treated with the high dose of AAV2 7m8 ranged from moderate to bright. Moderate GFP expression was observed in eyes treated with the low dose of AAV2 7m8, while GFP expression was mostly faint in eyes treated with the high dose of AAV8 and AAV2.

[0185] SLO infrared (IR) and autofluorescence (AF) retinal images at 50° were generated throughout the study at the indicated time points. Representative images from each treatment group at day 42 are shown in Figure 5.

[0186] Optical coherence tomography (OCT) was performed to assess retinal structure, thickness, and volume. Retinal thickness and volume remained stable across all treatment groups throughout the study. Overall, mean retinal thickness and volume were lowest in the AAV8 low-dose and AAV2-treated groups compared with the other treatment groups. None of the treatment groups experienced fluctuations in retinal thickness and volume levels from baseline to Day 42.

[0187] Baseline respiratory rates were assessed for heart rate, body temperature, chest acuity, and integumentary integrity. Most animals in this study had respiratory rates higher than the normal range at baseline (16–28 bpm for males and females). All animals with higher respiratory rates were successfully auscultated. These elevations were determined by the veterinarian to be most likely due to elevated respiratory rates prior to sedation and not of clinical concern. Heart rates were within the normal range at baseline for all animals (76–120 bpm for males and 80–140 bpm for females) except for D699, who had a heart rate of 64 bpm. The veterinarian determined that this animal's lower heart rate was likely due to deeper sedation and not of clinical concern. Body temperatures were within the normal range for most animals at baseline (98–101.5°F for males and females) except for D699 (95.4°F) and S1071 (101.8°F).

[0188] General observations twice daily revealed no clinical signs indicative of adverse systemic effects caused by the test substance. All animals were found to be in good general health throughout the study.

[0189] Body weights were obtained at the indicated time points. No significant changes in individual animal body weights were observed over the course of the study.

[0190] Blood was collected, processed, and shipped to a sponsor-designated laboratory for Nab analysis.

[0191] Hematological evaluations were performed at designated time points to assess general animal health. All animals recruited into the study were determined to be healthy by CBC parameters. At baseline, animal D699 had a low hematocrit count and a high mean corpuscular hemoglobin count, which returned to normal by day 42. At day 42, animal D015 had a low platelet count and low procalcitonin level.

[0192] Animals were euthanized at the end of the study and designated ocular and non-ocular tissue samples were collected and processed as needed for biodistribution and transgene expression analysis. conclusion

[0193] This study compared the retinal biodistribution and GFP expression levels of different AAV capsids after intravitreal (IVT) administration in African green monkeys. Achieved transgene expression was significantly higher after a low dose (1 × 10) of AAV2 7m8, as assessed by qualitative observation and semiquantitative scoring of GFP expression. 11 vg / eye) and AAV2 7m8 high dose (1 × 10 12 vg / eye).

[0194] Intraocular inflammation was observed in most animals without evidence of other ocular pathology and responded to additional topical steroids, showing resolution by the end of the study. Intraocular inflammatory responses are frequently seen in primates after intravitreal AAV. The etiology is unknown, but there is substantial variability between animals, dose-dependent, and in this study, appeared to be related to the amount of retinal GFP expression.

[0195] Low dose 1×10 11 vg / eye, vectors containing the variant VP1 capsid protein 7M8 showed unexpectedly high levels of transduction and expression in NHP retinal ganglion cells. Indeed, this was greater than 1 × 10 11 There appeared to be a ceiling effect as with vg / eye, transduction and expression were not significantly higher.

[0196] This was unexpected for at least two reasons. First, a variant VP1 capsid protein, 7m8, was discovered using directed evolution in mice. It was unexpected that the capsid protein released for mouse retinal transduction would be able to efficiently transduce retinal cells in the primate eye due to structural differences between the mouse and primate eyes. Primate eyes have significant structural differences compared to mouse eyes, including a significant inner limiting membrane, which is known to bind AAV serotypes, including AAV2, and prevent transduction of retinal cells. Therefore, it was surprising that the 7m8 capsid, as shown here, was able to transduce retinal cells in the primate eye with high efficiency.

[0197] Second, the transgene here was a transmembrane protein fused to a fluorescent reporter, GFP. We expected that reduced protein folding, protein trafficking, and transmembrane localization would limit the fluorescent signal. Conversely, abundant fluorescent reporter signal was found, suggesting high levels of transgene expression in primate retinal cells. Considering the possibility that the fluorescent reporter might interfere with transgene expression, we hypothesized that even higher transgene expression could be achieved without the fusion to a fluorescent reporter. This was indeed the case.

[0198] Example 6: NHP Study 2 Quantification of ChRown mRNA in non-human primate (African green monkey) tissues The following describes the quantification of transgene expression in tissue samples taken from rhesus macaques administered low, medium, or high doses of rAAV particles. The viral titers tested were 3 x 10 10 vg / eye, 1×10 11 vg / eye, and 2.35 × 10 11 The transgene expression construct was pCAG-ChRown-mWPRE-hGHpA. These assays were performed in support of a clinical trial. ChRown RNA measurements were determined using an NHP tissue RNA extraction procedure and a RT-quantitative polymerase chain reaction (RT-qPCR) assay.

[0199] For the study, 34 NHPs (20 males and 14 females) were divided into five groups. These five groups were divided based on the dose of drug and tissues collected for biodistribution. NHPs were injected with low, medium, and high doses of drug only into the right eye (OD). For biodistribution analysis, only the right eye (OD) of group 5 was collected. RT-qPCR assays were performed on samples from groups 2 through 4 (six male and three female AGMs per time point), from which 28 and 30 different systemic tissues, respectively, were collected to test the toxicity and biodistribution of ChRown. Group 5 included three males and three female AGMs (per time point), from which 45 tissues (32 ocular and 16 and 13 systemic tissues from each eye) were collected to test the toxicity and biodistribution of ChRown. For each group, samples were collected at two time points (weeks 13 and 25).

[0200] Tissues assayed included the following: eye (aqueous humor, vitreous humor, optic nerve, iris / ciliary body, retina-macula, retina-superior, retina-nasal, retina-inferior, retina-temporal, RPE / choroid-macula, RPE / choroid-superior, RPE / choroid-nasal, RPE / choroid-inferior, RPE / choroid-temporal, sclera), brain (optic chiasm, optic tract-left, lateral geniculate body-left, visual cortex-left, frontal cortex, temporal cortex, hindbrain), dorsal root ganglia (cervical-C3, thoracic-T3, lumbar-L3), gross lesions, heart, kidney (left), liver, lung, lymph nodes (mandibular and mesenteric), ovaries (left and right), pancreas, sciatic nerve, skeletal muscle, small intestine, spinal cord (cervical, thoracic, and lumbar), spleen, stomach, and testis (left). [Table 6]

[0201] RNA was isolated from NHP tissues using the Direct-zol-96 RNA Kit (Zymo Research). Isolated RNA was then quantified via UV / VIS spectroscopy using a Qiagen QIAxpert system. When possible, samples were normalized to 50 ng / μL with nuclease-free water, and 4 μL containing 200 ng of RNA was analyzed via RT-qPCR. If the RNA isolated from a sample could not be normalized to 50 ng / μL, 4 μL of neat RNA was analyzed via RT-qPCR. An RT-qPCR master mix was prepared containing a specially formulated TaqMan Fast Virus 1-Step Master Mix, ChRown gene-specific primers, and a quenched FAM-labeled probe. The RT-qPCR master mix was plated into wells of a 96- or 384-well plate. Linearized plasmid DNA dilutions were added to the wells and used as standards for RT-qPCR analysis. When performing qPCR assays in 384-well plates, an MLP Hamilton was used to dispense the master mix, standards, QCs, NFW, and samples, while manual dispensing of each reagent was used for assays performed in 96-well plates. The plates were then sealed and loaded onto a QuantStudio 7 Flex Real-Time PCR System, where thermal cycling and simultaneous fluorescence measurements were performed. The fluorescence emitted during the RT-qPCR procedure was proportional to the amount of ChRown gene present. Upon completion of the run, the fluorescence data from each sample-containing well was analyzed using a standard curve to determine the amount of ChRown gene present in the sample.

[0202] In addition to assessing transgene expression, NHP tissue samples were also examined for the presence of ChRown DNA. DNA was determined using a validated NHP tissue DNA extraction procedure and quantitative polymerase chain reaction (qPCR) assay. Genomic DNA (gDNA) was isolated from NHP tissues using the KingFisher MagMAX DNA Multi-Sample Ultra 2.0 Kit with extraction buffer. Isolated gDNA was quantified via UV / VIS absorption using a Qiagen QIAxpert system. When possible, samples were normalized to 50 ng / µL with nuclease-free water, and 4 µL containing 200 ng of gDNA was analyzed via qPCR assay. If the gDNA isolated from a sample could not be normalized to 50 ng / µL, 4 µL of undiluted gDNA was analyzed via qPCR. A qPCR master mix containing a specially formulated Taqman Fast Advanced Master Mix, COCHR-3M gene-specific primers, and a quenched FAM-labeled probe was prepared. The qPCR master mix was plated into wells of a 96- or 384-well plate. Linearized plasmid DNA dilutions were added to the wells and used as standards analyzed by the qPCR assay. When performing qPCR assays in 384-well plates, an MLP Hamilton was used to dispense the master mix, standards, QCs, NFW, and samples, while manual dispensing of each reagent was used for assays performed in 96-well plates. The plate was then sealed and loaded into a QuantStudio 7 Flex Real-Time PCR System, where thermal cycling and simultaneous fluorescence measurements were performed. Fluorescence emitted during the qPCR procedure was proportional to the amount of ChRown gene present in the tissue sample. Upon completion of the run, fluorescence data from each sample-containing well was analyzed using a standard curve to determine the amount of ChRown gene present in the sample.

[0203] result Administration by single IVT injection was generally well tolerated: no deaths occurred and no systemic abnormalities were detected up to 3 months.

[0204] Humoral immunity, as measured by serum antidrug antibody (ADA) positive responses, was limited to a total of 6 of 34 animals (including one control animal) and was not dose-related. Similarly, cellular immune responses, as measured by IFN-γ ELISpot analysis of peripheral blood mononuclear cells (PBMCs), showed moderately positive responses in 8 of 34 animals at 3 months. One animal at the medium dose was positive at baseline.

[0205] In untreated eyes, all samples were negative for genomic DNA biodistribution. Non-ocular biodistribution was sporadic and very limited, suggesting that very little vector escaped the eye or CNS.

[0206] In contrast, vector DNA biodistribution was detected in all ocular tissues of vector-treated eyes, including the retina, at all dose levels. The highest levels were observed in the iris / ciliary body, retina, RPE / choroid, and sclera of the treated eye (right). The same tissues from the untreated left eye generally contained below the detection limit for vector DNA, suggesting that the vector did not migrate to the left eye after administration in the right eye. Only a single sample of left RPE / choroid showed approximately 4000 copies per μg of DNA, but this was an isolated finding, as nearly all other tissues from the left eye were below the detection limit.

[0207] Correlating with biodistribution, as shown in Figure 6, high levels of gene expression were seen in all ocular tissues in treated (right) eyes at all dose levels, with the highest expression seen in retinal samples. Several CNS tissues, including the optic chiasm and left optic tract, showed some transgene expression, primarily in the high-dose group. No other significant gene expression was observed in other systemic tissues. No systemic transgene expression was observed. Non-ocular transgene expression was restricted to the contiguous visual pathway of treated eyes (optic chiasm, optic tract, and lateral geniculate body). A single sample of RPE / choroid (1.86 x 10) in high-dose females at this 13-week time point was found to contain 1.86 x 10 4 With the exception of 100 copies / μg RNA, little or no expression was observed in left eye samples.

[0208] conclusion Overall, these results indicate that vector DNA is largely confined to the eye, with no transfer to the untreated (left) eye occurring over a 13-week time frame. Importantly, vector distribution is associated with high levels of transgene expression in the retina and some intraocular tissues of treated eyes up to 13 weeks post-administration.

[0209] Example 7: Relationship of Dosage to Functional Efficacy Studies were performed in a TKO mouse model to assess the relationship between viral dose and functional efficacy of ChRown.

[0210] An AAV2 vector carrying the 7m8(Y444F) variant capsid driven by the CAG promoter was used to express the ChRown-GFP transgene in retinal ganglion cells. The viral vector was delivered to 1.5 × 10 7 vg / eye~1.5×10 10 The doses ranged from 0.01 to 0.01 vg / eye and were injected intravitreally.

[0211] Visual function was assessed by a visuomotor behavioral assay. Light sensitivity was determined as described in Example 2. Visual acuity was measured using an automated virtual system, OptoDrum (StriaTech GmbH, Tübingen, Germany) (Benkner et al., 2013). The system consists of four enclosed LCD monitors with a central animal elevated platform. The monitors display grating stimuli that form a virtual grating cylinder centered on the animal's head. The light intensity of the OptoDrum was fixed and was approximately 30 μW / cm at the center of the platform. 2 The grating stimuli were presented at 100% contrast with a rotation speed of 12 degrees per second. The presentation of the grating stimuli, the change in spatial frequency, and the animal's head tracking were all performed algorithmically by the system. Visual acuity was defined as the highest grating frequency that elicited OMR.

[0212] Figure 7 shows the relationship between viral dose (vg / eye) and light sensitivity. The threshold light intensity was 1.5 x 10 7 to 1.5 x 10 8 vg / eye, 1.5 × 10 15 (±3.2×10 14 , mean ± SD, n = 4) to 3.2 × 10 13 (±6.8×10 12 , n=4) photons / cm 2 The virus dose then rapidly decreased by approximately 50-fold in the 1.5 × 10 8 From 7.5 x 10 8 vg / eye, the threshold light intensity approximately doubles to 1.7 × 10 13 (±2.0×10 12 ;n=4)photons / cm 2 With further dose increases, light sensitivity plateaued, reaching 1.5 × 10 s at the highest dose tested. 10 vg / eye, the threshold light intensity was slightly lower (1.4 × 10 13 ±5.0×10 12 , n=4). Dose ≧7.5×10 8 The threshold light intensities of vg / eye were not statistically different (p>0.05, one-way ANOVA).

[0213] Figure 8 shows the relationship between viral dose and visual acuity. OMR is 1.5 x 10 8 In contrast, OMR was not observed in mice injected with virus doses less than 1.5 × 10 8 Visual acuity was observed in all mice injected with a viral dose of 1.5 × 10 vg / eye or greater. 8 At a dose of 100 mg / eye, the visual acuity was 0.097 ± 0.038 cycles / degree (mean ± SD, n = 4), reaching 0.12–0.13 cycles / degree at higher doses. However, the visual acuity of mice receiving these higher doses was not statistically different (p > 0.05; one-way ANOVA). In contrast, the visual acuity of mice with normal vision (C57BL / 6J) was 0.39 ± 0.018 cycles / degree (mean ± SD, n = 5).

[0214] The data reveal an intriguing pattern between dose and functional efficacy. Light sensitivity increases rapidly, reaching 1.5 × 10 7 ~1.5×10 8 vg / eye dose achieved approximately a 50-fold difference, followed by 7.5 × 10 8 A plateau was approached with dose of <1.5 × 10 vg / eye, and values ​​were not statistically different at higher doses. This pattern is largely consistent with visual acuity assessment. 8 At viral doses of 1.5 × 10 , no OMR was observed, and visual acuity could not be measured. Note that the failure to induce OMR in TKO mice treated with these low doses was simply due to the light intensity required to induce OMR (approximately 30 μW / cm , as shown by the dashed line in FIG. 8 ) exceeding the intensity of the OptoDrum system. On the other hand, viral doses ≥ 1.5 × 10 . 8 In TKO mice treated with , visual acuity was measurable, but the values ​​were not statistically different. Collectively, these results suggest that visual acuity was improved by 1.5 × 10 8 ~7.5×10 8 vg / eye indicates that a saturating viral dose occurred.

[0215] ChRown-GFP expression was observed primarily in RGCs. Viral transduction efficiency in RGCs was examined by immunostaining of retinal whole mounts. Figure 9 shows the results of transfection of 1.5 × 10 7 ~1.5×10 10 Representative images of retinas injected with four different viral doses (vg / eye) are shown. Viral-transduced cells were labeled with an antibody against GFP (green), while RGCs were labeled with an antibody against RBPMS (red), a specific marker for RGCs. Transduced RGCs were identified by co-labeling of GFP and RBPMS. Regarding the results, first, RGC density based on RBPMS labeling did not significantly differ between mice treated with different viral doses (Figure 10A). Meanwhile, the viral transduction efficiency, as the ratio of transduced RGCs to total RGCs, shown in Figure 10B, increased with increasing viral dose. Specifically, approximately 12%, 56%, 69%, and 87% of RGCs were transduced at 1.5 × 10 7, 1.5×10 8 , 7.5×10 8 , and 1.5 × 10 10 Transduction was carried out with a viral dose of vg / eye.

[0216] Viral dose-dependent protein expression of ChRown-GFP in whole retinas by Western blot assay using an antibody against GFP. For this assay, four retinas in each viral dose group were homogenized. Representative Western blot images are shown in Figure 11A. Meanwhile, Figure 11B shows the relationship between viral dose and the relative ECL fluorescence intensity of ChRown-GFP after normalization to the ECL fluorescence intensity of β-actin (mean ± SD, from three experiments). Briefly, protein expression of ChRown-GFP increased with relatively low viral doses, reaching approximately 1.5 × 10 9 vg / eye dose and then decreased with further increases in virus dose.

[0217] These data suggest that there may be a capped or optimal viral dose based on RGC expression to achieve maximum functional efficacy in TKO mouse models. This information would be beneficial for dose design in clinical trials. However, translating this to human studies requires consideration of the differences between mice and humans. To estimate the upper limit of the dose scale, there is a ~1000-fold difference in vitreous volume between mice and humans (5 µL vs. 4.5-5.0 mL). Therefore, to achieve the same viral titer in the human vitreous, the corresponding optimal viral dose would be at least 10 µL. 11 vg / eye. Note that this value is based on the high dose (5 × 10 11vg / eye). However, literature on mouse-to-human dose scaling suggests a lower value, closer to the 38-fold required for mouse-to-human scaling (Schmitt et al., Mol. Pharm., 2019 Oct 7;16(10):4399-4404). Furthermore, AAV transduction efficiency in mice is nearly uniform throughout the retina, whereas transduction efficiency in humans is heterogeneous, as reported in nonhuman primates (NHPs). Specifically, in NHPs, high transduction occurs in the parafoveal and far-peripheral retinal regions. Furthermore, unlike humans, the mouse retina lacks a fovea. How these differences affect the dose-efficacy relationship remains to be determined.

[0218] Example 8 The OptoDrum system was used to measure visual acuity in TKO mice treated with the following test articles (treatment groups in parentheses): AAV2.7m8-mGluR6-GFP-mWPRE-hGHpA (R20, negative control) AAV2.7m8-mGluR6-ChRown-mWPRE-hGHpA(R23) AAV2.7m8-CAG-ChRown-mWPRE-hGHpA(R03)

[0219] Mice were 2-5 months old at the time of injection. Mice receiving the R23 test article were given 1.5 x 10 8 vg, 1.5 × 10 9 vg, or 1.5 × 10 10 vg into both eyes. Mice receiving R20 or R03 were injected with 1.5 × 10 10 vg was injected into both eyes. Injections were performed as described in Example 4.

[0220] Mice were evaluated using the OptoDrum system described in Example 4 at 2 and 6 months post-injection.

[0221] Light sensitivity: Threshold was measured at a grating frequency of 0.042 cycles / degree. Figure 12A shows the results of assessing threshold light intensity two months after injection. Threshold light intensity decreased with increasing doses of R23, reaching a sensitivity equivalent to that of R03 when administered at equivalent doses. No response was elicited in animals administered R20 (negative control). Similar results were observed six months after injection (Figure 12B).

[0222] Visual acuity: Visual acuity was examined using OptoMotry (Cerebral Mechanics Inc.), a computer-based virtual optical motor system. Figure 13A shows a dose-dependent increase in visual acuity for R23 2 months after injection, which persists 6 months after injection. After both 2 and 6 months, 1.5 x 10 10 Mice receiving the vg dose exhibited visual acuity comparable to mice receiving the same dose of R03 (FIGS. 13A and 13B).

[0223] Example 9 Light sensitivity and visual acuity were assessed in TKO mice treated with mGluR6-ChRown-mWPRE-hGHpA (SEQ ID NO: 31) to target ON-type bipolar cells. The vector was an AAV2.7m8 vector containing an additional Y444F substitution. In treated TKO mice, the light intensity required to induce OMR near the optimal spatial frequency (0.042-0.064 cycles / degree in mice) was approximately 5 x 10 12 photon / cm 2 As shown in Figure 14A, the mean visual acuity of the treated TKO mice was 0.19 c / day, compared with 0.40 c / day in mice with normal vision (Figure 14C). Both light sensitivity and visual acuity remained stable for up to 10 months after viral injection (Figures 14B, 14C). Contrast sensitivity measured using the optical motor system described in Example 2 was significantly better at the peak sensitivity spatial frequency, approximately 15% (Figure 14D). The results indicate that the combination of the mGluR6 promoter (SEQ ID NO: 28) and ChRown is an excellent viral vector for optogenetic visual restoration using ON-type bipolar cell targeting.

[0224] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and treatises, are expressly incorporated herein by reference in their entirety for all purposes.

[0225] While the invention disclosed herein has been described in terms of particular embodiments and applications thereof, numerous modifications and variations can be made by those skilled in the art without departing from the scope of the invention as set forth in the claims.

[0226] It will be understood that the present invention is described in various levels of detail in this application. In certain instances, details that are not necessary for those skilled in the art to understand the present invention or that make it difficult for those skilled in the art to appreciate other details may be omitted. The terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting beyond the scope of the appended claims. Unless otherwise defined, technical terms used herein should be understood as commonly understood by those skilled in the art to which this disclosure belongs.

[0227] In the foregoing description and in the claims that follow, it will be understood that: As used herein, the phrases "at least one," "one or more," and "and / or" are open-ended expressions that are both conjunctive and disjunctive in operation. Terms such as "a," "an," "the," "first," and "second" do not exclude a plurality. For example, as used herein, the term "one" ("a" or "an") entity refers to one or more of that entity. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0228] When used before other numerical designations, such as temperature, time, amount, concentration, etc., including ranges, the term "about" indicates approximations that may vary by (+) or (-) 10%, 5%, 1%, or any subrange or subvalue therebetween. Preferably, the term "about" means that the value may vary by ±10%.

[0229] The term "comprises / comprising" does not exclude the presence of other elements, components, features, regions, integers, steps, operations, etc. Moreover, although individual features may be included in several claims, they may be advantageously combined, and inclusion in different claims does not imply that a combination of features is not feasible and / or advantageous. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the claim to the specific materials or steps in the claim "and materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention."

[0230] The term "nucleic acid" refers to a polymer of nucleotides, eg, deoxyribonucleotides or ribonucleotides, and may be used herein as an abbreviation for deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0231] The terms "oligonucleotide," "nucleic acid sequence," and "polynucleotide" are used interchangeably and are intended to include polymeric forms of nucleotides covalently linked together, which may be of various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives, or modifications thereof. Oligonucleotides are typically composed of a sequence of nucleotides containing nucleobases selected from adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U). Thus, the term "polynucleotide sequence" may refer to the textual representation of a polynucleotide molecule, or the term may apply to the polynucleotide molecule itself.

[0232] The term "percent identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence using one of the following sequence comparison algorithms or by visual inspection, are the same or have a specified percentage of amino acid residues or nucleotides. Percent identity can be determined using computer algorithms such as the Basic Local Alignment Search Tool (BLAST), or related tools, including other BLAST-based tools available on the website of the U.S. National Library of Medicine, National Center for Biotechnology Information. BLAST and related algorithms are described, for example, in Altschul et al., 1990, J. Mol. Biol. 215:3,403-410, and Altschul et al., 1997, Nucleic Acids Res. 25:17,3389-402. Unofficial sequence listing

[0233] SEQ ID NO:1 Chloromonas spp. channelrhodopsin protein M L G N G S A I V P I D Q C F C L A W T D S L G S D T E Q L V A N I L Q W F A F G F S I L I L M F Y A Y Q T W R A T C G W E E V Y V C C V E L T K V I I E F F H E F D D P S M L Y L A N G H R V Q W L R Y A E W L L T C P V I L I H L S N L T G L K D D Y S K R T M R L L V S D V G T I V W G A T S A M S T G Y V K V I F F V L G C I Y G A N T F F H A A K V Y I E S Y H V V P K G R P R T V V R I M A W L F F L S W G M F P V L F V V G P E G F D A I S V Y G S T I G H T I I D L M S K N C W G L L G H Y L R V L I H Q H I I I Y G D I R K K T K I N V A G E E M E V E T M V D Q E D E E T V

[0234] Accession No. 2 Chloromonas ogama channelrhodopsin cDNA ATGCTGGGAAACGGCAGCGCCATGTGCCTATCGACCAGTGCTTTTGCCTGGCTTGGACCGACAGCCTGGGAAGCGATACAGAGCAGCTGGTGGCCAACATCCTCCAGTGGTTCGCCTTCGGCTTCAGCATCCTGATCCTGATGTTCTACGCCTACCAGACTTGGAGAGCCACTTGCGGTTGGGAGGAGGTCTACGTCTGTTGCGTCGAGCTGACC AAGGTCATCATCGAGTTCTTCCACGAGTTCGACGACCCCAGCATGCTGTACCTGGCTAACGGACCCGAGTCCAGTGGCTGAGATACGCAGAGTGGCTGCTGACTTGTCCCGTCATCCTGATCCACCTGAGCAACCTGACAGGCCTGAAGGACGACTACAGCAAGCGGACCATGAGGCTGCTGGTGTCAGACGGGGAACCATCGTGTGGGGAGCT ACAAGCGCCATGAGCACAGGCTACGTCAAGGTCATCTTCTTCGTGCTGGGTTGCATCTACGGCGCCAACACCTTCTTCCACGCCGCCAAGGTGTATATCGAGAGCTACCACGTGGTGCCAAAGGGCAGACCTAGAACCGTCGTGCGGATCATGGCTTGGCTGTCTTCCTGTCTTGGGGCATGTTCCCCGTGCTGTTCGTCGGGACCAGAAGGA TTCGACGCCATCAGCGTGTACGGCTCTACCATTGGCCACACCATCATCGACCTCATGAGCAAGAATTGTTGGGGCCTGCTGGACACTATCTGAGAGTGCTGATCCACCAGCACATCATCATCTACGGCGACATCCCGCAAGAAGACCAAGATCAACGTGGCGGGCGAGGAGATGGAAGTGGAGACCATGGTGGACCAGGAGGACGAGACAGTG

[0235] sequence number 3 CoChR L112C M L G N G S A I V P I D Q C F C L A W T D S L G S D T E Q L V A N I L Q W F A F G F S I L I L M F Y A Y Q T W R A T C G W E E V Y V C C V E L T K V I I E F F H E F D D P S M L Y L A N G H R V Q W L R Y A E W L L T C P V I C I H L S N L T G L K D D Y S K R T M R L L V S D V G T I V W G A T S A M S T G Y V K V I F F V L G C I Y G A N T F F H A A K V Y I E S Y H V V P K G R P R T V V R I M A W L F F L S W G M F P V L F V V G P E G F D A I S V Y G S T I G H T I I D L M S K N C W G L L G H Y L R V L I H Q H I I I Y G D I R K K T K I N V A G E E M E V E T M V D Q E D E E T V

[0236] Sequence number 4 CoChR L112C / T139C M L G N G S A I V P I D Q C F C L A W T D S L G S D T E Q L V A N I L Q W F A F G F S I L I L M F Y A Y Q T W R A T C G W E E V Y V C C V E L T K V I I E F F H E F D D P S M L Y L A N G H R V Q W L R Y A E W L L T C P V I C I H L S N L T G L K D D Y S K R T M R L L V S D V G C I V W G A T S A M S T G Y V K V I F F V L G C I Y G A N T F F H A A K V Y I E S Y H V V P K G R P R T V V R I M A W L F F L S W G M F P V L F V V G P E G F D A I S V Y G S T I G H T I I D L M S K N C W G L L G H Y L R V L I H Q H I I I Y G D I R K K T K I N V A G E E M E V E T M V D Q E D E E T V

[0237] Sequence number 5 ChRown M L G N G S A I V P I D Q C F C L A W T D S L G S D T E Q L V A N I L Q W F A F G F S I L I L M F Y A Y Q T W R A T C G W E E V Y V C C V E L T K V I I E F F H E F D D P S M L Y L A N G E R V Q W L R Y A E W L L T C P V I C I H L S N L T G L K D D Y S K R T M R L L V S D V G T I V W G A T S A M S T G Y V K V I F F V L G C I Y G A N T F F H A A K V Y I E S Y H V V P K G R P R T V V R I M A W L F F L S W G M F P V L F V V G P E G F D A I S V Y G S T I G H T I I D L M S K N C W G L L G H Y L R V L I H Q H I I I Y G D I R K T T K I N V A G E E M E V E T M V D Q E D E E T V

[0238] SEQ ID NO: 6 AAV2 VP1 Capsid Protein M A A D G Y L P D W L E D T L S E G I R Q W W K L K P G P P P P K P A E R H K D D S R G L V L P G Y K Y L G P F N G L D K G E P V N E A D A A A L E H D K A Y D R Q L D S G D N P Y L K Y N H A D A E F E R L K E D T S F G G N L G R A V F Q A K K R V L E P L G L V E E P V K T A P G K K R P V E H S P V E P D S S S G T G K A G Q Q P A R K R L N F G Q T G D A D S V P D P Q P L G Q P P A A P S G L G N T M A T G S G A P M A D N N E G A D G V G N S S G N W H C D S T W M G D R V I T T S T R T W A L P T Y N N H L Y K Q I S S Q S G A S N D N H Y F G Y S T P W G Y F D F N R F H C H F S P R D W Q L I N N N W G F R P K R L N F K L F N I Q V K E V T Q N D G T T T I A N N L T S T V Q V F T D S E Y Q L P Y V L G S A H Q G C L P P F P A D V F M V P Q Y G Y L T L N N G S Q A V G R S S F Y C E Y F P S Q M L R T G N N F T F S Y T F E D V P F H S S Y A H S Q S L D R L M N P L I D Q Y L Y Y L S R T N T P S G T T T Q S R L Q F S Q A G A S D I R D Q S R N W L P G P C Y R Q Q R V S K T S A N N N S E Y S W T G AT K Y H L N G R D S L V N P G P A M A S H K D D E E K F F P Q S G V L I F G K Q G S E K T N V D I E K V M I T D E E E I R T T N P V A T E Q Y G S V S T N L Q R N R Q A A T A D V N T Q G V L P G M V W Q D R D V Y L Q G P I W A K I P H T D G H F H P S P L M G G F G L K H P P P Q I L I K N T P V P A N P S T T F S A A K F A S F I T Q Y S T G Q V S V E I E W E Q K E N S K R W N P E I Q Y T S N Y N K S V N V D F T V D T N G V Y S E P R P I G T R Y L T R N L

[0239] SEQ ID NO:7 7M8 (fragment of AAV2 VP1) M A T G S G A P M A D N N E G A D G V G N S S G N W H C D S T W M G D R V I T T S T R T W A L P T Y N N H L Y K Q I S S Q S G A S N D N H Y F G Y S T P W G Y F D F N R F H C H F S P R D W Q R L I N N N W G F R P K R L N F K L F N I Q V K E V T Q N D G T T T I A N N L T S T V Q V F T D S E Y Q L P Y V L G S A H Q G C L P P F P A D V F M V P Q Y G Y L T L N N G S Q A V G R S S F Y C L E Y F P S Q M L R T G N N F T F S Y T F E D V P F H S S Y A H S Q S L D R L M N P L I D Q Y L Y Y L S R T N T P S G T T T Q S R L Q F S Q A G A S D I R D Q S R N W L P G P C Y R Q Q R V S K T S A D N N N S E Y S W T G A T K Y H L N G R D S L V N P G P A M A S H K D D E E K F F P Q S G V L I F G K Q G S E K T N V D I E K V M I T D E E E I R T T N P V A T E Q Y G S V S T N L Q R G N L A L G E T T R P A R Q A A T A D V N T Q G V L P G M V W Q D R D V Y L Q G P I W A K I P H T D G H F H P S P L M G G F G L K H P P P Q I L I K N T P V P A N P S T T F S A A K F A S F I T Q Y S T G Q V S V E I E W E L Q K E N S KRWNPEIQYTSNYNKSVNVDFT VDTNGVYSEPRPIGTRYLTRN L

[0240] SEQ ID NO: 8 Darcala 7m8 peptide LGETTRP

[0241] SEQ ID NO: 9 4DMT peptide id49 from WO 2021 / 22148 LAISDQTKHA

[0242] SEQ ID NO: 10 CAG promoter

[0243] SEQ ID NO: 11 Human mGluR6 1784 bp promoter fragment

[0244] SEQ ID NO:12 Human mGluR6 547bp promoter fragment ccaaagacaagaaaacaggaaaacagacccagagattgggagagggaggggaaggagatgcggggagagccagcaccgccaccccccacactcaggaggggtctccaccctcggagcggtctctcatccctccctagaatccttaaatcctctctcgctcagggcctcggccgcatctgtcacagacttgtcctgaaccgacagcggctggcgcaggtgactggcttggggcgggagcctgggtgtgcgctggggatggaccccgaggaagaggggccaagctgtcgggaagcggcagggctggaggggtggaggcagtggtcgggcgggaccccgggcgacagggttcggcgcttgtaagagcgagacggaggcccgggcaggccggctgagctaactccccagagccgaagtggaaggcgcgccccgagcgccttctccccaggaccccggtgtccctccccgcgccccgagcccgcgctctccttcccccgccctcagagcgctccccgcccctctgtctccccgcagcccgctagacgagccg

[0245] SEQ ID NO:13 Human mGluR6 198bp enhancer gatccttagattatgaaacatttacaattatgaatgaatattagatgttatcaaatgctttttctgcatccatttagataatcatgtttttcctttaatctgttaatgcggtgaattacattaatagatttcctaagtcattaatctgctaaagtgcatttctgggacaaaccagacttggttatgacattgtatgta

[0246] SEQ ID NO:14 WPRE gataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctgatgcgg

[0247] SEQ ID NO: 15 5' ITR ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct

[0248] SEQ ID NO: 16 3' ITR aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcag

[0249] Array No. 17 ChRown DNA atgctgggcaacggatctgctatcgttcctatcgaccagtgcttctgcctggcttggaccgactctctgggctctgataccgaacagctggtggccaatatcctgcagtggttcgccttcggcttcagcattctgatcctgatgttctacgcctaccagacctggcgggccacctgtggctgggaggaagtgtatgtgtgctgcgtggaactgacaaaggtgatcatcgagttcttccacgagttcgacgaccccagcatgctgtacctggccaatggcgagagagtgcagtggctgcggtacgccgagtggctgctgacctgtcctgtcatctgcatccacctgtccaacctgaccggcctgaaggacgattacagcaaaagaaccatgagactgctcgtgtccgatgtgggcaccatcgtgtggggcgctaccagcgctatgagcaccggctacgtgaaggttatcttctttgtgctgggttgcatctatggcgccaacactttcttccacgccgccaaggtgtacatcgagagctaccacgtggtccccaagggcagacctagaacagtggtgcggatcatggcctggctgtttttcctgagctggggaatgtttccagtgctgttcgtggtgggccctgagggatttgatgccatcagcgtgtacggcagcacaatcggccatacaatcattgacctgatgagcaagaactgttggggcctgctgggacactacttgagggtgctgatccaccagcacatcatcatctacggcgacatcagaaagaccaccaaaatcaacgtggccggagaagaaatggaagtcgagacaatggtggaccaagaggacgaggaaacagtg

[0250] Array No. 18 hGHpA gggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaagttgcatcattttgtctgactaggtgtccttctataatattatggggtggaggggggtggtatggagcaaggggcaagttgggaagacaacctgtagggcctgcggggtctattgggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccgcctcctgggttcaagcgattctcctgcctcagcctcccgagttgttgggattccaggcatgcatgaccaggctcagctaatttttgtttttttggtagagacggggtttcaccatattggccaggctggtctccaactcctaatctcaggtgatctacccaccttggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccctgtcctt

[0251] SEQ ID NO: 19 pCAG-ChRown-GFP-mWPRE-hGHpA

[0252] SEQ ID NO: 20 pCAG-ChRown-GFP-hGHpA

[0253] SEQ ID NO: 21 pCAG-ChRown-mWPRE-hGHpA

[0254] SEQ ID NO: 22 pChrown-tdTomato-hGHpA

[0255] SEQ ID NO: 23 pCAG-Chrown-hGHpA

[0256] Array number 24, 500 base pair mGluR6 promoter ttaaaggcagtctaggggagaagcagacccagggagtcagagaggcagagagagaagagagcccttcctccactctcaagctctggagggggtctctgccctcaccctcatccctccccagaatccttaaatcctctagactgtagctctgattttacagctgtcacagactcgtcctactagccagaggttggctcaggtaagcaccactggggaggtagcctagggtgcgctggggtgggtccagaggaagagctgcccagaactgtgggggaaggagcgggaccgaccatcaacagggggacttttcagggagaatgagagcaatcctctggaggcctgggagaggctgctgagttgctggtgcgcgagtcaccaacttttcctgcgctctcggtgtccggccagaatcccgaagtggcagctgagcacggggtggcagcttcgtccgccggctctcaaggcgtcccggtaacttcctttcccgcagtccaggagca

[0257] Array number 25, mGluR6 enhancer gatctccagatggctaaacttttaaatcatgaatgaagtagatattaccaaattgctttttcagcatccatttagataatcatgttttttgcctttaatctgttaatgtagtgaattacagaaatacatttcctaaatcattacatcccccaaatcgttaatctgctaaagtacatctctggctcaaacaagactggttgt

[0258] Array number 26, mGluR6 intron 4 ggtgagtcccccaccccactcatcctccctgatgcttcctgtgtgggatgctcatttccacatttgtctcggagtcccacatgctgagtaactctgagatttgctttaaaatgccatgcaggtaatttaaatgggaaggtctgatccaagtgatgaagtgcagccttgatagcatgcttcctccgccctcccacaggcttccatcttttgtggggtgcccacctccacaccttttcttttagctagagtggtcaagtggacaagctggtcattagcaatcaaggcgtttcagatctggaagtgggtggtgccattatggatcagtgagccctgtattttttgtgcctctgcacaaggtgggtagtgaagccctgtccattacataaccatggcatcccctagccatgacataaagggcagtgaaaaattctttaaggatgccagagctgctttttccatttgtgtgtatgcgtgcaggtgtgtgttgtacatgacacaagtgtatgtgtgtgcatgtggaggcctgaggttgatttcaggaatcatcctcaattctttttctaccttattcactgaggcagggtctgtggagagatcaccgatatggctactgtgggattcccctgtctctgccttcagagccactcctggatacacagtacacctggctcagatggtcaccaccctcct

[0259] SEQ ID NO: 27, mGluR6 intron 3 actccaggccatgagcaactcctcacatctccctaagcccttcctgtcgccctctggagtcttttgttctgggaatgagacaggcttgactggctgaaggttctccgggcctggcctgggaaacacaggaaaacacgactatttttattgttcattgtgggagagagaactggtaggcaaacccaagagcagaaaatgtaccgtgagggacactgccccagtaaaccctgaaacctacattatcctaagccagccaaggttcttttccagcctgggaagttgagcgtgacattggtggctgaatttgtagacagaatggcttctgagtgcccctgacattccccaaaaggaggctctctgcattaatccatttgtctttattataataaaatatccaagtcagggcgttttttaaggaaaagacttatttttaacatcaactcttggaggtgaaagttcaggcagcgtgacaccagctctgctgaggacctagcttgcatcacattttgacaaatgttatggaaagagggagtagagaaggaaagagtgcatggagagaaggaacatcagaaagaagagggacagggttcactctttgatagctattcaccttcacagaattacctcacccttccagaggtcaagagcaacatccccagtgacccaataaccttgcactaagccacacctcttttttattttttatgagacagggcctcactctgtatccctgaacttgctatgtggaccaagctgtcctcttgagtgctgggattaaaggcattcagtatcagggctgg

[0260] SEQ ID NO: 28 mGluR6 promoter

[0261] SEQ ID NO: 29 ITR-CAG-ChRown-mWPRE-hGHpA-ITR

[0262] SEQ ID NO: 30 mGluR6-Chrown-mWPRE-hGHpA

[0263] SEQ ID NO: 31 ITR1-mGluR6-Chrown-mWPRE-hGHpA-ITR2

[0264] SEQ ID NO: 32 CAG-ChRown-GFP-mWPRE-hGHpA

[0265] SEQ ID NO: 33 CAG-ChRown-GFP-hGHpA

[0266] SEQ ID NO: 34 CAG-ChRown-mWPRE-hGHpA

[0267] SEQ ID NO: 35 ChRown-tdTomato-hGHpA

[0268] SEQ ID NO: 36 CAG-ChRown-hGHpA

[0269] SEQ ID NO: 37 Wild-type AAV2 VP1 capsid protein MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGNGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVP DPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPA DVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTN VDIEKVMITDEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0270] SEQ ID NO: 38 AAV2 7m8 VP1 capsid protein MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLALGETTRPARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

Claims

1. 1. An infectious recombinant adeno-associated virus (rAAV) particle comprising: (i) a capsid protein having a modified amino acid sequence relative to a native AAV capsid of serotype 2; and (ii) a vector genome consisting of a heterologous polynucleotide comprising, in the 5' to 3' direction, (a) an AAV2 inverted terminal repeat (ITR1), (b) a promoter sequence, (c) a polynucleotide sequence encoding a channelrhodopsin, (d) a polyadenylation sequence, and (e) an AAV2 inverted terminal repeat (ITR2), wherein the heterologous polynucleotide does not encode a fluorescent protein.

2. 2. The rAAV particle of claim 1, wherein the heterologous polynucleotide further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) between the polyadenylation sequence and the ITR2 sequence.

3. The rAAV particle of claim 2, wherein the WPRE element comprises SEQ ID NO:

14.

4. The rAAV particle of any one of claims 1 to 3, wherein the promoter is selected from a cytomegalovirus (CMV) promoter, an elongation factor 1a (EF1a) promoter, a simian virus 40 (SV40) promoter, a chicken beta-actin promoter, an mGluR6 promoter, and a CAG promoter.

5. The rAAV particle of claim 4, wherein the promoter is a CAG promoter.

6. The rAAV particle of claim 5, wherein the CAG promoter comprises SEQ ID NO:

10.

7. The rAAV particle of claim 4, wherein the promoter is an mGluR6 promoter.

8. The rAAV particle of claim 7, wherein the mGluR6 promoter comprises SEQ ID NO:

24.

9. The rAAV particle of any one of claims 1 to 8, wherein the heterologous polynucleotide further comprises an enhancer sequence.

10. The rAAV particle of claim 9, wherein the enhancer is a CMV enhancer or an mGluR6 enhancer.

11. The rAAV particle of claim 10, wherein the enhancer is an mGluR6 enhancer comprising SEQ ID NO:

25.

12. The rAAV particle according to any one of claims 1 to 11, wherein the promoter comprises, from upstream to downstream, intron 4 of the mGluR6 gene, intron 3 of the mGluR6 gene, an mGluR6 enhancer, and an mGluR6 promoter fragment.

13. The rAAV particle of claim 12, wherein the mGluR6 promoter comprises the nucleotide sequence of SEQ ID NO:

28.

14. The rAAV particle of any one of claims 1 to 13, wherein the channelrhodopsin comprises the amino acid sequence of ChRown (SEQ ID NO: 5).

15. 15. The rAAV particle of any one of claims 1 to 14, wherein the channelrhodopsin comprises the amino acid sequence of ChRown (SEQ ID NO: 5), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical thereto.

16. The rAAV particle of any one of claims 1 to 15, wherein the polyadenylation signal is a human growth hormone polyadenylation sequence (hGHpA) or a simian virus 40 polyadenylation sequence.

17. 17. The rAAV particle of claim 16, wherein the polyadenylation signal is a human growth hormone polyadenylation sequence (hGHpA) comprising SEQ ID NO:

18.

18. 2. The rAAV particle of claim 1, wherein the heterologous polynucleotide comprises pCAG-Chrown-mWPRE-hGHpA (SEQ ID NO: 21) or pCAG-Chrown-hGHpA (SEQ ID NO: 23).

19. 19. The rAAV particle of any one of claims 1 to 18, wherein the capsid protein comprises the amino acid sequence of SEQ ID NO:

7.

20. 20. The rAAV particle of claim 19, wherein the capsid protein further comprises a tyrosine to phenylalanine substitution at a position corresponding to position 444 of the wild-type AAV2 VP1 capsid sequence.

21. A pharmaceutical composition comprising a plurality of rAAV particles according to any one of claims 1 to 20 and a pharmaceutically acceptable carrier or excipient.

22. 22. The pharmaceutical composition of claim 21, wherein the composition is formulated for intravitreal injection.

23. 23. The pharmaceutical composition of claim 21 or 22, wherein the composition is formulated as an emulsion or suspension.

24. 24. A method for treating a retinal disease in a human subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a plurality of rAAV particles of any one of claims 1-20 or the pharmaceutical composition of any one of claims 21-23, and optionally a pharmaceutically acceptable carrier or excipient.

25. 25. The method of claim 24, wherein the rAAV particles are administered by intravitreal injection.

26. 26. The method of claim 24 or 25, wherein the rAAV particles are administered in one or more doses.

27. The one or more doses are at least about 1.5 x 10 ８ 27. The method of claim 26, comprising one viral genome (vg).

28. The one or more doses are about 1 x 10 ８ ~1 x 10 １４ 27. The method of claim 26, comprising a viral genome (vg).

29. 29. The method of any one of claims 24 to 28, wherein the rAAV particles are administered in one or more doses per eye.

30. 30. The method of any one of claims 24 to 29, wherein the retinal disease is selected from Bardet-Biddell syndrome, choroidal retinal atrophy or degeneration, cone or cone-rod dystrophy, congenital stationary night blindness, Leber's congenital amaurosis (LCA), macular degeneration (MD) including age-related MD (AMD), ocular retinal developmental disorders, ocular atrophy, retinitis pigmentosa, syndromes / systemic diseases associated with retinopathy, Usher syndrome, or other retinopathies including diabetic retinopathy.

31. 31. The method of claim 30, wherein the retinal disease is age-related macular degeneration (AMD) or retinitis pigmentosa (RP).

32. 21. A method for delivering heterologous nucleic acid to a retinal cell, comprising contacting the retinal cell with a plurality of rAAV particles of any one of claims 1 to 20.

33. 1. An infectious recombinant adeno-associated virus (rAAV) particle comprising a vector genome comprising a polynucleotide sequence encoding (i) a capsid protein, and (ii) a channelrhodopsin, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) is located downstream of the polynucleotide sequence encoding the channelrhodopsin; the vector genome does not encode a fluorescent protein, or An infectious recombinant adeno-associated virus (rAAV) particle, wherein the capsid protein is AAV2 7m8 serotype.

34. 34. The rAAV particle of claim 33, wherein the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) is located downstream of the polynucleotide sequence encoding the channelrhodopsin.

35. 35. The rAAV particle of claim 33 or 34, wherein the WPRE element comprises SEQ ID NO: 14, or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

36. The rAAV particle of any one of claims 33 to 35, wherein the vector genome does not encode a fluorescent protein.

37. The rAAV particle of any one of claims 33 to 36, wherein the capsid protein is AAV2 7m8 serotype.

38. 38. The rAAV particle of any one of claims 33 to 37, wherein the capsid protein comprises the amino acid sequence of SEQ ID NO:

7.

39. 38. The rAAV particle of any one of claims 33 to 37, wherein the capsid protein comprises or consists of the amino acid sequence of SEQ ID NO:

38.

40. 38. The rAAV particle of any one of claims 33 to 37, wherein the capsid protein further comprises a phenylalanine at a position corresponding to position 444 of the wild-type AAV2 VP1 capsid sequence.

41. The rAAV particle of any one of claims 33 to 40, wherein the polynucleotide encoding the channelrhodopsin encodes the channelrhodopsin of SEQ ID NO:

5.

42. 42. The rAAV particle of any one of claims 33 to 41, wherein the polynucleotide encoding the channelrhodopsin encodes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:

5.

43. 43. The rAAV particle of claim 41 or 42, wherein the polynucleotide encoding the channelrhodopsin comprises SEQ ID NO:

17.

44. The rAAV particle of any one of claims 33 to 43, wherein the vector further comprises an upstream inverted terminal repeat (ITR), and the upstream ITR comprises SEQ ID NO:

15.

45. The rAAV particle of any one of claims 33 to 44, wherein the vector further comprises a downstream ITR, and the downstream ITR comprises SEQ ID NO:

16.

46. The rAAV particle of any one of claims 33 to 45, wherein the vector further comprises a polyadenylation sequence.

47. The rAAV particle of any one of claims 33 to 46, wherein the polyadenylation sequence is a human growth hormone polyadenylation sequence (hGHpA) or a simian virus 40 polyadenylation sequence.

48. 48. The rAAV particle of claim 46 or 47, wherein the polyadenylation sequence comprises SEQ ID NO:

18.

49. The rAAV particle of any one of claims 33 to 48, wherein the vector further comprises a promoter.

50. 50. The rAAV particle of claim 49, wherein the promoter comprises a cytomegalovirus (CMV) promoter, an elongation factor 1a (EF1a) promoter, a simian virus 40 (SV40) promoter, a chicken beta-actin promoter, an mGluR6 promoter, or a CAG promoter.

51. 51. The rAAV particle of claim 49 or 50, wherein the promoter comprises SEQ ID NO:

10.

52. The rAAV particle of any one of claims 33 to 51, wherein the vector comprises SEQ ID NO:

34.

53. The rAAV particle of any one of claims 33 to 52, wherein the vector comprises SEQ ID NO:

29.

54. 51. The rAAV particle of claim 49 or 50, wherein the vector comprises an mGluR6 regulatory element region, and the mGluR6 regulatory element region comprises the promoter.

55. The rAAV particle of claim 54, wherein the mGluR6 regulatory element comprises at least one, at least two, at least three, or all four of SEQ ID NOs: 24-27.

56. 56. The rAAV particle of claim 54 or 55, wherein the mGluR6 regulatory element comprises, from upstream to downstream, intron 4 of the mGluR6 gene, intron 3 of the mGluR6 gene, an mGluR6 enhancer, and a fragment of the mGluR6 promoter.

57. The rAAV particle of claim 55, wherein the mGluR6 regulatory element comprises SEQ ID NO:

28.

58. 58. The rAAV particle of any one of claims 33-49 or 54-57, wherein the vector comprises SEQ ID NO:

30.

59. 59. The rAAV particle of any one of claims 33-49 or 54-58, wherein the vector comprises SEQ ID NO:

31.

60. The rAAV particle of any one of claims 33 to 59, wherein the vector comprises, from upstream to downstream, (a) an AAV2 inverted terminal repeat sequence (ITR1), (b) a promoter sequence, (c) a polynucleotide sequence encoding the channelrhodopsin, (d) a polyadenylation sequence, and (e) an AAV2 inverted terminal repeat sequence (ITR2).

61. 61. The rAAV particle of claim 60, wherein the vector comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) positioned between the polyadenylation sequence and the ITR2 sequence.

62. 62. A pharmaceutical composition comprising a plurality of rAAV particles according to any one of claims 33 to 61 and a pharmaceutically acceptable carrier or excipient.

63. 63. The pharmaceutical composition of claim 62, wherein the composition is formulated for intravitreal injection.

64. 64. The pharmaceutical composition of claim 62 or 63, wherein the composition is formulated as an emulsion or suspension.

65. 65. A method for treating a retinal disease in a human subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a plurality of rAAV particles described in any one of claims 33-61 or the pharmaceutical composition of any one of claims 62-64, and optionally a pharmaceutically acceptable carrier or excipient.

66. 66. The method of claim 65, wherein the rAAV particles are administered by intravitreal injection.

67. 67. The method of claim 65 or 66, wherein the rAAV particles are administered in one or more doses.

68. The one or more doses are at least about 1.5 x 10 ８ 68. The method of claim 67, comprising one viral genome (vg).

69. The one or more doses are about 1 x 10 ８ ~1 x 10 １４ 68. The method of claim 67, comprising a viral genome (vg).

70. 70. The method of any one of claims 65-69, wherein the rAAV particles are administered in one or more doses per eye.

71. 71. The method of any one of claims 65 to 70, wherein the retinal disease is selected from Bardet-Biddell syndrome, choroidal retinal atrophy or degeneration, cone or cone-rod dystrophy, congenital stationary night blindness, Leber's congenital amaurosis (LCA), macular degeneration (MD) including age-related MD (AMD), ocular retinal developmental disorders, ocular atrophy, retinitis pigmentosa, syndromes / systemic diseases associated with retinopathy, Usher syndrome, or other retinopathies including diabetic retinopathy.

72. 72. The method of claim 71, wherein the retinal disease is age-related macular degeneration (AMD) or retinitis pigmentosa (RP).

73. 65. A method of delivering heterologous nucleic acid to a retinal cell, comprising contacting the retinal cell with a plurality of rAAV particles of any one of claims 33-61 or the pharmaceutical composition of any one of claims 62-64.

Images