Viral vectors comprising the coding region of rdh12 and methods of treating retinal dystrophies

Abstract

The present invention relates to viral vectors comprising the RDH12 coding region and methods of treating retinal dystrophies. Provided are materials, methods and uses for treating ophthalmic conditions such as Leber's congenital amaurosis by administering an effective amount of an adeno-associated virus AAV2, serotype 5 (AAV 2 / 5) or AAV-5 comprising an expressible coding region of human RDH12.

Description

[0001] This application is a divisional application. The original application was filed on November 15, 2018, with application number 201880086649.6 (PCT / US2018 / 061319) and the invention title "Viral vector including RDH12 coding region and method for treating retinal dystrophy".

[0002] Cross-reference to related applications This application claims priority to Provisional U.S. Patent Application No. 62 / 586,624, filed November 15, 2017, which is incorporated herein by reference.

[0003] By incorporating materials submitted electronically. As a separate part of this disclosure, this application contains a sequence list in computer-readable form (file name: 51914A_Seqlisting.txt, created on November 9, 2018 (5,230 bytes, ASCII text file)), the entire contents of which are incorporated herein by reference. Technical Field

[0004] This disclosure relates to a medical treatment method, such as a method for treating a human subject suffering from an ophthalmic condition (e.g., Leber congenital amaurosis) caused by a loss-of-function mutation in at least one gene encoding retinol dehydrogenase 12 protein (RDH12), the method comprising administering to the subject an effective amount of nucleic acid comprising an adeno-associated virus vector comprising human RDH12 complementary DNA (cDNA). Background Technology

[0005] Hereditary retinal diseases (IRDs) are a leading cause of legal blindness in children. Leber congenital amaurosis (LCA) and early-onset severe retinal dystrophy (EOSRD) cause severe visual impairment from birth to a few years of age, accounting for 5% or more of all IRDs combined (Koenekoop et al., 2004). LCA / EOSRD is associated with autosomal dominant and autosomal recessive inheritance patterns, involving the retinal pigment epithelium and rod and cone photoreceptors as primary targets (Weleber et al., 2013). Approximately 10% of LCA / EOSRD is caused by mutations in the gene encoding RDH12 (Kumaran et al., 2017). Given the role of RDH12 in the visual cycle that provides chromophores to photoreceptor cells (Haeseleer et al., 2002; Chen et al., 2012), and RDH12 This constitutes an important therapeutic target. RDH12 It is one of the most important LCA genes.

[0006] Although ophthalmic symptoms such as hereditary retinal diseases are prevalent in both humans and non-human mammals, and the genes encoding retinol dehydrogenases and the effects of mutations in these genes are understood, the role of retinopathy in the development of retinopathy remains unclear. RDH12 There is no known treatment for retinal dystrophy caused by one or more mutations in the retina. Therefore, there remains a need in the art for materials and methods for treating retinal dystrophy such as LCA, as well as for correcting genetic abnormalities that may lead to such dystrophy. Summary of the Invention

[0007] This invention provides an adeno-associated virus (AAV) vector comprising a gene encoding retinol dehydrogenase. RDH12 The coding region of the gene product. Contains RDH12 The AAV carrier can be used to treat retinal dystrophy conditions (such as Leber congenital amaurosis (LCA)), which provides... RDH12 Recombinant constructs with coding regions placed under the control of adjustable or controllable promoters (such as heterologous promoters) are designed to target the lack of wild-type levels (such as those derived from...). RDH12 Subjects with RDH12 activity (caused by mutations in the AAV genome) are provided with complementary retinol dehydrogenase. Despite the advantages of a relatively small genome size, a relatively low tendency to integrate into host DNA, and a relatively low immunogenicity profile, it was a surprising finding that some AAV serotype combinations or pseudotypes produced expression levels of the encoded RDH12 product that were not therapeutically effective and / or exhibited undesirable toxicity when administered to subjects treating retinal dystrophy. Consistent with this finding, certain pseudotypes (such as the AAV2 / 5 pseudotype) were found to exhibit unexpected and surprising levels of effective expression and toxicity profiles consistent with their therapeutic use in treating retinal dystrophy. Subjects treatable using the method of the present invention may include subjects with visual function loss determined by optical coherence tomography (OCT) (e.g., impaired response to electroretinography (ERG) tests) but who retain some photoreceptor cells. Therefore, in one aspect, the present invention provides a method for treating a human subject suffering from an ophthalmic condition (such as Leber congenital amaurosis or LCA, or another clinically defined ophthalmic condition) caused by one or more loss-of-function mutations in a gene encoding the retinol dehydrogenase 12 (RDH12) protein. Specifically, one aspect of this disclosure provides a method for treating a human subject suffering from an ophthalmic condition caused by one or more loss-of-function mutations in a gene encoding the retinol dehydrogenase 12 (RDH12) protein, the method comprising administering an adeno-associated virus vector comprising nucleic acids to at least one eye of the subject, wherein the nucleic acids comprise human... RDH12 DNA (e.g., human DNA) RDH12 cDNA), and wherein RDH12 DNA (e.g., human DNA) RDH12 DNA) encodes a protein that is at least 70%, 80%, 90%, 95%, or 99% identical in length to the protein in SEQ ID NO: 2. In some of the above embodiments, the ophthalmic condition is Leber congenital amaurosis (LCA). In some of the embodiments mentioned above, the ophthalmic condition is Leber congenital amaurosis (LCA). In some embodiments, RDH12 DNA (e.g.) RDH12 cDNA) is expressed under the control of the human rhodopsin kinase 1 (hGRK1) promoter, wherein the hGRK1 promoter comprises or is substantially composed of SEQ ID NO: 3. In some embodiments, the adeno-associated virus vector is AAV-2, serotype-5 (AAV2 / 5), or AAV-5. In some embodiments, RDH12 DNA (e.g.) RDH12 The cDNA comprises at least 60% or 70% of the sequence identical to SEQ ID NO: 1. In some embodiments, the nucleic acid is expressed in approximately 2 × 10⁻⁶ units. 10 Viral genome density per milliliter (vg / mL) up to approximately 2 × 10⁻⁶ 12 Administered at a titer of vg / mL, for example, approximately 2 × 10⁻⁶. 10 Viral genome density per milliliter (vg / mL) up to approximately 2 × 10⁻⁶ 12 A titer of vg / mL, such as approximately 2 × 10⁻⁶. 11 vg / mL or approximately 2 × 10 12 vg / mL. In some embodiments, the nucleic acid is administered into the subretinal space, for example, wherein a microinjection cannula is inserted into the subretinal space.

[0008] Another aspect of this disclosure relates to human beings. RDH12 Nucleic acid encoded by DNA (e.g., human DNA) RDH12 cDNA), among which RDH12 DNA (e.g., human DNA) RDH12 DNA (such as cDNA) encodes a protein that is at least 70%, 80%, 90%, 95%, or 99% identical in length to the protein in SEQ ID NO: 2. RDH12 DNA is controlled by the human rhodopsin kinase 1 (hGRK1) promoter. In some embodiments, the hGRK1 promoter comprises or is substantially composed of SEQ ID NO: 3. In some embodiments, the human... RDH12 DNA (e.g., human DNA) RDH12(cDNA) encodes a protein including SEQ ID NO: 2. In some embodiments, the human RDH12 DNA (e.g., human DNA) RDH12 The cDNA is at least 60% or 70% identical to the full length of SEQ ID NO: 1.

[0009] Another aspect of this disclosure is the nucleic acid disclosed herein for treating human subjects suffering from ophthalmic conditions due to one or more loss-of-function mutations in the gene encoding the retinol dehydrogenase 12 (RDH12) protein. In some of the above embodiments, the ophthalmic condition is Leber congenital amaurosis (LCA).

[0010] Another aspect of this disclosure is a viral vector comprising a nucleic acid encoding the RDH12 disclosed herein. In some embodiments, the viral vector is an adeno-associated virus vector. In some embodiments, the adeno-associated virus vector is AAV-2, serotype-5 (AAV2 / 5), or AAV-5.

[0011] Another aspect of this disclosure is the viral vector disclosed herein for treating human subjects suffering from ophthalmic conditions due to one or more loss-of-function mutations in the gene encoding the retinol dehydrogenase 12 (RDH12) protein. In some of the above embodiments, the ophthalmic condition is Leber congenital amaurosis (LCA).

[0012] Another aspect of this disclosure relates to isolated host cells comprising the viral vectors or nucleic acids disclosed herein. In some embodiments, the isolated host cells express the human RDH12 protein.

[0013] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. This document describes the methods and materials used in this invention; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and not limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail.

[0014] Other features and advantages of the invention will become clear from the following detailed description, accompanying drawings, and claims. Attached Figure Description

[0015] Figure 1: Visual circulation and RDH activity in photoreceptor cells. (A) The visual circulation converts vitamin A to 11-cis-retinal (i.e., the chromophore of visual pigment) and recycles all-trans-retinal released after bleaching. (B) Shown is the retinoid flow of the RPE-photoreceptor pair. RDH8 in the outer segment reduces all-trans-retinal. RDH12 in the inner segment reduces all-trans-retinal, 11-cis-retinal, and other toxic short-chain aldehydes. Abbreviations: 11cRAL—11-cis-retinal; 11cROL—11-cis-retinal; AtRAL—all-trans-retinal; AtROL—all-trans-retinal; RCHO—short-chain aldehyde; RCHOH—short-chain alcohol; Rh—rhodopsin; MRh—morphorhodopsin.

[0016] Figure 2 Recombinant RDH12 in injection AAV2 / 5-hGRK1p.hRDH12 Expression and localization in mice. (A) AAV2 / 5- hGRK1p.hRDH12 A schematic diagram of a gene therapy construct, in which a human clone is cloned downstream of the human rhodopsin kinase promoter, between inverted terminal repeat sequences derived from the AAV2 genome. RDH12 cDNA. (B, C) Subretinal injection AAV2 / 5- hGRK1p.hRDH12 (1.3 × 10) 9 Six weeks after administration of PBS (vg) or PBS, the expression of human RDH12 protein in the mouse retina was evaluated using specific antibodies against mouse RDH12 or human RDH12. (B) C57BL / 6J mice injected with PBS Rdh12 - / - Mice and injected AAV2 / 5-hGRK1p.hRDH12 of Rdh12 - / - Protein analysis (Western analysis) was performed on retinal lesions from mice. (C) Immunohistochemical analysis showed that in C57BL / 6J mice, the native mouse Rdh12 (dark gray) was localized to the IS, ONL, and OPL retina, but... Rdh12 - / - There was no localization in mice, but the injection... AAV2 / 5- hGRK1p.hRDH12 The resulting recombinant human RDH12 (light gray) shows C57BL / 6J and Rdh12 - / -Similar localization in mice. Phase-contrast image (left). Abbreviations: ITR—Terminal inverted repeat sequence; hGRK1—Human rhodopsin kinase promoter; SD / SA—Monkey virus 40 splice donor / acceptor site; hRDH12—Human RDH12 cDNA; polyA—Monkey virus 40 polyadenylation signal; RPE—Retinal pigment epithelium; OS—Outer segment; IS—Inner segment; ONL—Outer nuclear layer; OPL—Outer plexiform layer; INL—Inner nuclear layer; IPL—Inner plexiform layer; GCL—Gangella cell layer.

[0017] Figure 3 : AAV2 / 5-hGRK1p. hRDH12 gene replacement therapy recovery RDH12 RDH12 function in defective mice. (A) Analysis by HPLC of mice injected with or without RDH12. AAV2 / 5-hGRK1p.hRDH12 (1.3×10) 9 VG) or PBS C57BL / 6J and Rdh12 - / - Retinal reductase activity in mouse retina. Six weeks post-injection, the formation of all-trans retinol was quantified in a assay using all-trans retinaldehyde as a substrate. Each data point represents the mean ± standard error of at least five independent experiments, pooling data from the retina of three to five mice and performing the assay in triplicate. C57BL / 6J; Rdh12 - / - ; Injected with PBS Rdh12 - / - ; AAV injection Rdh12 - / - (B) Recombinant RDH12 in injection AAV2 / 5-hGRK1p.hRDH12 Assessment in the last 16 weeks Rdh12 - / - Immunohistochemistry of expression in whole mouse retinal sections. Human RDH12 (light gray) is located at IS, ONL, and OPL in the retinal injection region (right side of the image).

[0018] Figure 4 : AAV2 / 5-hGRK1p. hRDH12 gene replacement therapy reduces albinism Rdh12 Photodamage in defective mice. One eye was injected with 5,000 lux two weeks prior to and one week after exposure to 5,000 lux. AAV2 / 5-hGRK1p.hRDH12Furthermore, ERG analysis was performed on mice whose contralateral eyes were not injected. Dark vision (rod separation and combined rod-cone) responses were quantified in groups of 10–13 mice, and the percentage of the initial ERG response remaining after light damage was calculated. Mean results with standard error are shown, along with the significance of differences between injected and uninjected eyes calculated using a two-tailed paired t-test.

[0019] Figure 5 : AAV2 / 5-hGRK1p.hRDH12 It did not significantly affect the homeostatic levels of 11-cis-retinal in the retina. Mice received subretinal injection. AAV2 / 5-hGRK1p.hRDH12 (1.3×10) 9 VG) or PBS, or no injection. After dark adaptation overnight, retinoids were extracted under dark red light and quantified by HPLC analysis. (A) Representative chromatograms for each treatment condition; peaks of cis-11-cis-retinaldehyde oxime, trans-11-cis-retinaldehyde oxime, and cis-all-trans-retinaldehyde oxime are shown. Total retinaldehyde levels of (B) 11-cis-retinaldehyde and (C) all-trans-retinaldehyde at ± standard error for each treatment condition. C57BL / 6J not injected; No injection Rdh12 - / - ; AAV injection Rdh12 - / - ; Injected with PBS Rdh12 - / - ).

[0020] Figure 6 Retinal function is not affected AAV2 / 5-hGRK1p.hRDH12 Adverse effects. C57BL / 6J not injected; not injected Rdh12 - / - Injected AAV2 / 5-hGRK1p.hRDH12 of Rdh12 - / - Mice (up to 2×10) 9 Electroretinogram (ERG) responses in scotopic (rod separation and combination of rod-cone) and photopic (cone-mediated) modes were recorded 6 weeks after treatment (vg). ERG of representative animals in each treatment group is shown, measured 6 weeks after injection.

[0021] Figure 7 Visual pigment localization is not affected AAV2 / 5-hGRK1p.hRDH12 Interference. Evaluation was conducted 16 weeks post-treatment on untreated and injected (1.3 × 10⁻⁶) individuals. 9 vg) Rdh12 - / -Immunohistochemical localization of rhodopsin and cone opsin in mice. Expression of human RDH12 protein in IS and ONL (light gray). Rhodopsin and red / green opsin (dark gray) in eyes injected with AAV2 / 5-hGRK1p.hRDH12. Abbreviations and targets Figure 2 The abbreviations used in the descriptions are the same.

[0022] Figure 8 : The retinal structure will not change due to long-term expression AAV2 / 5-hGRK1p.hRDH12 And damaged. During injection. AAV2 / 5- hGRK1p.hRDH12 (Up to 2×10⁹ VG) 54 weeks later, for C57BL / 6J and Rdh12 - / - Optical coherence tomography (OCT) analysis was performed on the eyes of mice.

[0023] Figure 9 Infiltrating CD68+ macrophages and RDH12 after injection AAV2 / 8-hGRK1p.hRDH12 Expression in the retina. Subretinal injection. AAV2 / 8-hGRK1p.hRDH12 (2 × 10) 9 Immunohistochemical analysis was performed 8 weeks after vg) Rdh12 - / - Evaluation was performed in C57BL / 6J mice. Human RDH12 expression in IS, ONL, and OPL (light gray) and macrophage CD68 labeling (white). Abbreviations and targets Figure 2 The abbreviations used in the descriptions are the same. Detailed Implementation

[0024] Hereditary retinal degeneration is a rare cause of severe vision loss and is a current focus of research in developing targeted gene therapies. Viral vector-mediated somatic cell gene therapy has shown great promise in treating animal models of human retinal degenerative diseases. To date, numerous successful studies have used adeno-associated virus (AAV)-mediated gene delivery to rescue photoreceptor degeneration in small animal models (Ali et al., 2000; Pang et al., 2012; Pawlyk et al., 2010; Pawlyk et al., 2005; Tan et al., 2009) and large animal models (Acland et al., 2001; Alexander et al., 2007; Beltran et al., 2012; Komaromy et al., 2010; Lheriteau et al., 2009). In these cases, the retinal pigment epithelium (RPE), or photoreceptors, is the primary target of transgene expression. Furthermore, some successes have been achieved in phase I clinical trials involving gene therapy for RPE and, more recently, choroiditis (Maclaren et al., 2014) in patients with Leber's congenital amaurosis (LCA) (Bainbridge et al., 2008; Cideciyan et al., 2008; Maguire et al., 2008). Currently, no clinical trials have used AAV-mediated gene replacement therapy to treat patients with RPE. RDH12 Patients with hereditary retinal degeneration caused by mutations.

[0025] In order to develop AAV-mediated gene therapy to treat RDH12 From the mutated individuals, we prepared carriers. RDH12 adeno-associated virus vectors containing cDNA, expressed under the control of the rhodopsin kinase promoter. In published studies of vectors with capsids derived from AAV serotype 5 or AAV serotype 8, we have shown… AAV2 / 5- hGRK1p.RDH12 Subretinal delivery in Rdh12 defects ( Rdh12 - / - Rdh12 in mice leads to the expression of recombinant human RDH12, which is stable, correctly localized, reconstitutes retinaldehyde reductase activity, reduces susceptibility to photodamage, and does not cause adverse reactions. AAV2 / 8-hGRK1p.hRDH12 The observed retinal toxicity. AAV2 / 5-hGRK1p.RDH12 The construct provides a product of RDH12 gene replacement therapy.

[0026] RDH12 Mutations in the gene encoding retinol dehydrogenase 12 (RDH12) lead to severe early-onset retinal degeneration, typically diagnosed as Leber's congenital amaurosis (LCA) or early-onset severe retinal dystrophy (EOSRD). As a member of the short-chain dehydrogenase / reductase family, RDH12 is crucial for reducing retinal produced by the vitamin A visual cycle activity, essential for the photoreceptor cell's light response. When the visual pigment containing the 11-cis retinal chromophore absorbs a photon, 11-cis retinal is isomerized to all-trans retinal, initiating a signal transduction cascade that regulates synaptic signaling. Visual pigment inactivation involves the release of all-trans retinal and its regeneration targeting the 11-cis retinal chromophore. Figure 1 A) It is reduced to all-trans retinol and returned to the retinal pigment epithelium (RPE). When these recycling reactions become ineffective or are disrupted, for example due to aging or genetic diseases, retinaldehyde and its condensation products accumulate in the photoreceptors and retinal pigment epithelium, leading to severe damage to the outer retina (Ben-Shabat et al., 2001; Thompson et al., 2003; Sparrow, 2010; Chen et al., 2012).

[0027] To prevent toxicity, numerous retinol-binding proteins and enzymes are expressed in the retina. RDH12 is a member of a family of short-chain dehydrogenases / reductases that use NADPH to reduce a variety of substrates, including cis and trans-retinal (Haeseleer et al., 2002), C9 aldehydes produced by lipid photooxidation (Belyaeva et al., 2005; Lee et al., 2008; Marchette et al., 2010), and steroid substrates (Keller et al., 2007). Individuals with loss-of-function mutations in the RDH12 gene exhibit a severe retinal degenerative phenotype, typically diagnosed as Leber congenital amaurosis (LCA) (Janecke et al., 2004; Thompson et al., 2005; Perrault et al., 2004; den Hollander et al., 2008; Mackay et al., 2011), for which there is currently no treatment. RDH12 is located in the inner segment of rod and cone photoreceptor cells (Haeseleer et al., 2002; Maeda et al., 2006), where it protects against at least partial photoinduced damage caused by active retinaldehyde (Maeda et al., 2006). Post-exposure all-trans retinaldehyde has been shown to leak from the outer segment of the photoreceptor to the inner segment, and its efficient clearance from the photoreceptor cells requires the activity of RDH8 present in the outer segment and RDH12 present in the inner segment (Chen et al., 2012). Figure 1B). Therefore, RDH12 also has the potential to play an important role in reducing 11-cis-retinal, which is present in amounts exceeding those required for opsin biosynthesis, which can be achieved from the subretinal space into the inner segment (Chen et al., 2012).

[0028] To investigate the potential of gene replacement therapy for LCA / EOSRD caused by RDH12 mutations, we prepared adeno-associated virus vectors carrying human RDH12 cDNA, expressed under the control of a rhodopsin kinase promoter that directs photoreceptor cell-specific expression (Khani et al., 2007; Sun et al., 2010; Young et al., 2003). The DNA constructs were packaged with a capsid derived from AAV serotype 8 that mediates photoreceptor cell transduction with efficient and robust transduction (Allocca et al., 2007; Natkunarajah et al., 2008; Vandenberghe et al., 2011; Vandenberghe et al., 2013), or with a serotype 5 capsid that mediates photoreceptor transduction, but which exhibits slower kinetics and less stable expression compared to the serotype 8 capsid (Yang et al., 2002; Lotery et al., 2003; Allocca et al., 2007; Lebherz et al., 2008).

[0029] In a mouse model lacking Rh12 ( Rdh12 - / - In a comparative study conducted by Kurth et al. (2007), our work showed that the two vectors have significantly different safety characteristics, among which... AAV2 / 5-hGRK1p.hRDH12 It exhibits unexpectedly high levels of effective expression and toxicity characteristics, consistent with its therapeutic use in treating retinal dystrophy.

[0030] Human RDH12 sequence The sequence of an exemplary human RDH12 cDNA encoding all seven translated RDH12 exons (GenBank # NM_152443), consisting of -10 to +980 nucleotides relative to the translation start site, is provided in SEQ ID NO: 1.

[0031] The full-length human RDH12 protein sequence is provided in SEQ ID NO: 2.

[0032] Rhodopsin kinase promoter (hGRK1p) In some embodiments of the methods described herein, a replacement gene construct is used, wherein the human RDH12 cDNA described herein is placed under the control of a human rhodopsin kinase (hGRK1) promoter. In some embodiments, the hGRK1 promoter is approximately 200 base pairs (bp) in length and comprises a short promoter derived from the rhodopsin kinase (RK) hGRK1 gene, which has been shown to drive cell-specific expression in rod and cone cells (Khani et al., 2007; Sun et al., 2010; Young et al., 2003). An exemplary hGRK1 promoter sequence comprises nucleotides -112 to +87 of SEQ ID NO: 3 (Khani et al., 2007).

[0033] Viral delivery vector As mentioned above, the person who will abbreviate RDH12 cDNA is packaged into a delivery vector, such as AAV5 or AAV2 / 5 vector.

[0034] The replacement gene (cDNA) can be administered in any effective vector, such as any formulation or composition capable of efficiently delivering the component gene into cells in vivo. Methods involve inserting the gene into a non-pathogenic, non-replicating viral vector, including recombinant retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids. The viral vector is directly transfected into cells; the plasmid DNA can be delivered naked or by means of, for example, cationic liposomes (liposomes) or derivatized (e.g., antibody conjugates), poly-L-lysine conjugates, grammiomycin S, artificial viral envelopes, or other such intracellular vectors, as well as direct injection of the gene construct or in vivo Ca3(PO4)2 precipitation.

[0035] A preferred method for introducing nucleic acids into cells in vivo is to use a viral vector containing nucleic acids (e.g., cDNA). The advantage of infecting cells with a viral vector is that most target cells can receive the nucleic acids. Furthermore, molecules encoded in the viral vector (e.g., molecules encoded by cDNA contained in the viral vector) are efficiently expressed in cells that have absorbed the viral vector's nucleic acids. Retroviral vectors and adenovirus-derived vectors can be used as recombinant gene delivery systems for transferring exogenous genes in vivo, particularly in humans, across many cell types. However, these vectors cannot convert photoreceptor cells with an efficiency sufficient to make them useful for this application.

[0036] Another viral vector system used for delivering nucleic acids is adeno-associated virus (AAV). AAV is a naturally occurring defective virus that requires another virus (such as adenovirus or herpesvirus) as a helper virus for efficient replication and production of its life cycle (Muzyczka et al., 1992). AAV is also one of the few viruses that can integrate its DNA into non-dividing cells and exhibits a high frequency of stable integration (see, for example, Flotte et al., 1992; Samulski et al., 1989; and McLaughlin et al., 1988). Vectors containing as few as 300 AAV base pairs can be packaged and integrated. The space for exogenous DNA is limited to approximately 4.5 kb. DNA can be introduced into cells using AAV vectors (as described in Tratschin et al. (1985)). AAV vectors have been used to introduce various nucleic acids into different cell types (see, for example, Hermonat et al., 1984; Tratschin et al., 1984a; Tratschin et al., 1984b; Wondisford et al., 1988; and Flotte et al., 1993).

[0037] In a preferred embodiment, the viral delivery vector is a recombinant AAV2 / 5 virus. Prior to administration, the final product can undergo a series of ultrapurification steps to meet clinical grade standards.

[0038] Subject selection Subjects who are candidates for this treatment method include those diagnosed with LCA caused by a mutation in the gene encoding RDH12. Subjects with other clinically defined ophthalmic conditions caused by mutations in the gene encoding RDH12 (e.g., early-onset retinitis pigmentosa) may also be treated using the methods described herein. Methods known in the art can be used to diagnose LCA or other ophthalmic conditions caused by mutations in the gene encoding RDH12.

[0039] The method described herein may include: identifying subjects (e.g., children, adolescents, or young adults) suffering from LCA or another ophthalmic condition caused by one or more mutations in a gene encoding RDH12, or subjects suspected of having LCA or another ophthalmic condition caused by one or more mutations in a gene encoding RDH12 (e.g., based on the presence of disease symptoms without other obvious cause); obtaining a sample comprising genomic DNA from said subject; and detecting the LCA or another ophthalmic condition using known molecular biology methods. RDH12 The presence of mutations; and selection in leading to LCA or another symptom. RDH12Patients with mutations in all alleles. Symptoms of this condition include macular atrophy, thinning of the fovea, and destruction of the lamellar structure of the retina, leading to early central vision loss and progression to LP (low visual field) vision. Visual field is limited at the earliest age of measurement, and the ERG response becomes irreversible in early adulthood. Testing RDH12 The mutations can include all or part of the mutations in the subjects. RDH12 Genes are sequenced, and the sequences are compared with reference sequences (e.g., GenBank accession number NG_008321.1) to detect mutations. Frameshift mutations, truncation mutations, mutations altering conserved amino acids, mutations affecting transcriptional splicing, or mutations affecting regulatory regions (e.g., promoters) are considered mutations that can lead to the LCA or another ophthalmic condition described herein; functional alterations can be confirmed by expressing the mutant in vitro (e.g., in cultured cells) and measuring, for example, enzyme function. Exemplary homozygous mutations include: Glu127X, Gln189X, Tyr226Cys, Ala269GlyfsX1, and Leu274Pro (all positional references refer to the RDH12 protein sequence of SEQ ID NO: 2). Typical mutations in the heterozygous state of the compound include: Thr49Met / Arg62X; Arg65X / Ala269GlyfsX1; His151D / Thr155Ile; His151D / Arg269GlyfsX1 (Janecke et al., 2004; Schuster et al., 2007) (position refers to the protein sequence of SEQ ID NO: 2).

[0040] Patients with LCA or other ophthalmic conditions caused by at least one RDH12 mutation may be treated with the methods described herein, preferably preserving some photoreceptors and visual function, such as as measured by standard visual function tests or field tests and / or optical coherence tomography (OCT, e.g., spectral domain-OCT (SD-OCT)). The methods described herein may include identifying patients diagnosed with LCA or other ophthalmic conditions caused by at least one RDH12 mutation. RDH12 Subjects with LCA or another ophthalmic symptom caused by mutation and in RDH12 Subjects with at least one confirmed mutation causing their symptoms were tested for visual ability and the presence of remaining central photoreceptors were detected.

[0041] Example The disclosed and claimed subject matter is further described in the following examples, which do not limit the scope of the invention as described in the claims.

[0042] Materials and methods The following materials and methods were used in the experiments disclosed in the examples listed below.

[0043] animal Previously Rdh12 - / - The generation and analysis of mice were described (Kurth et al., 2007). The mice used in this study... Rdh12 - / - Mice were bred through sibling mating between unfertilized males and females housed in our institutional animal facility. The WT mice used in the study were C57BL / 6 mice from The Jackson Laboratory (Wilmington, MA).

[0044] The following transgenic mouse genotypes were used in the study published in this paper: homozygous Rpe65-Met450 (M / M) variants in the C57BL / 6J background. Rdh12 − / − Mice (Kurth, 2007), and homozygous albino mice with the Rpe65-Leu450 (L / L) variant against BALB / c background. Rdh12 − / − Mice (Chrispell, 2009), obtained through breeding. Mice were fed a 12-hour (light) / 12-hour (dark) cycle and euthanized by inhalation of CO2 and subsequent bilateral pneumothorax.

[0045] Construction and generation of plasmids for recombinant AAV2 / 5 and AAV2 / 8 As mentioned earlier, human cDNA was amplified from the human retina via PCR using primers designed to contain the entire RDH12 coding region. RDH12 cDNA was obtained, cloned, and sequenced to verify fidelity (Janecke et al., 2004). To construct the AAV vector, cDNA was prepared from the parent lines. pAAV-hGRK1-hrGFP Inserted into the multiple cloning site of the vector RDH12 cDNA. The obtained pAAV- hGRK1-Rdh12The vectors were packaged into AAV. AAV2 / 5 and AAV2 / 8 pseudovectors were generated by bivariate transfection: (1) an AAV vector plasmid encoding the gene of interest, and (2) an AAV helper plasmid encoding the AAV Rep protein from serotype 2 and the Cap protein from serotype 5 or serotype 8, as well as the adenovirus helper function entering 293T cells. Transfection and purification were performed using published protocols (Nishiguchi et al., 2015). Two days after transfection, cells were lysed by repeated freeze-thaw cycles. After initial removal of cell debris, the nucleic acid components of the virus-producing cells were removed by benzo[a]ase treatment. The recombinant AAV vector particles were purified by affinity chromatography using an AVB matrix, washed in 1x PBS, and concentrated to a volume of 100–150 ml using a Vivaspin 4 (10 kDa) concentrator. The vectors were titrated by qPCR amplification.

[0046] Subretinal injection A group of mice approximately 4 weeks old were placed under general anesthesia via intraperitoneal injection of ketamine (90 mg / kg) / xylanazine (9 mg / kg). A 0.5% prilocaine solution was applied to the cornea as a local anesthetic. The pupil was dilated by topical application of tropicamide (0.5%). Under an ophthalmic surgical microscope, a small incision was made near the limbus with a 30-gauge needle. A 34-gauge blunt needle mounted on a Hamilton syringe was inserted through the incision behind the lens and through the retina. All injections were performed at subretinal locations within the nasal quadrant of the retina. Each eye received up to 2 × 10⁻⁶ injections. 9 vg AAV2 / 5-(hGRK1)-hRDH12 The volume was as high as 2 μL. The RDH12-encoded vector was administered to one eye of each treated mouse, with no injection into the contralateral eye. Fundus examination after injection revealed retinal detachment in most cases exceeding 30%, confirming successful subretinal delivery.

[0047] Antibody The primary antibodies used were: a rabbit anti-Rdh12 polyclonal antibody (CSP) specific to mouse proteins (for 252SPFFKSTSQGAQ263, SEQ ID NO: 4), and a mouse anti-RDH12 monoclonal antibody (2C9) specific to human proteins (for C-284DCKRTWVSPRARNNKT299; SEQ ID NO: 5) (Kurth et al., 2007); a mouse anti-RHO monoclonal antibody (1D4) (MacKenzie et al., 1984); a rabbit anti-RHO polyclonal antibody against denatured proteins; and a rabbit anti-red / green cone opsin polyclonal antibody (Millipore catalog number AB5405).

[0048] Immunoblotting analysis Proteins in retinal homogenates were separated by SDS-PAGE, transferred to a nitrocellulose membrane, blocked, and incubated overnight with a primary antibody. The proteins were washed, incubated with an alkaline phosphatase-conjugated secondary antibody, and developed with 5-bromo-4-chloro-3′-indolylphosphate p-toluidine and nitrotetrazoleonium chloride.

[0049] Histology and immunofluorescence Mice were euthanized, and their eyes were oriented and scored before being enucleated. For cryosections, the lens and anterior segment were removed, the eyes were briefly fixed with 4% paraformaldehyde, washed with PBS, transferred to sucrose / OCT, rapidly frozen, and sectioned at a thickness of 10 μm. For cryoalternative preparation, the whole eye was rapidly frozen in dry ice-cooled isopentane for 30 seconds, then transferred to dry ice-cooled methanol containing 3% glacial acetic acid. The eyes were incubated at 80°C for 48 hours, then overnight at -20°C, embedded in paraffin, and sectioned at a thickness of 6 µm. The paraffin sections were deparaffinized, and the antigen was recovered before immunolabeling by incubation at 90°C in 1 mM EDTA (0.05% Tween 20, pH 8.0) for 30 minutes, as described below. In summary, retinal sections were rinsed with PBS and infiltrated with PBS-T (0.3% Triton X-100); blocked with 1% bovine serum albumin, 10% normal goat serum, and 0.3% Triton X-100; and incubated overnight at 4°C with primary antibodies. After washing, sections were incubated with fluorophore-conjugated secondary antibodies at room temperature for 1 hour. Sections were covered with a ProLongGold gel scaffold containing 4',6-diamino-2-phenylindole (DAPI; Invitrogen) and imaged using a Leica DM6000 fluorescence microscope.

[0050] ERG Record ERG was performed using the Espion e2 recording system (Diagnosys, Lowell, MA) as previously described (Thompson, 2012). Briefly, mice underwent overnight dark adaptation and were anesthetized by intraperitoneal injection of ketamine (93 mg / kg) and ziracetam (8 mg / kg). Pupils were dilated with topical tropicamide (0.5%). Body temperature was maintained at 37°C using a heating pad. Corneal ERG was recorded from both eyes using a gold wire loop with local anesthesia of 0.5% tetracaine and corneal hydration with 2% methylcellulose eye drops. The gold wire loop placed in the mouth served as a reference, and the grounded electrode was located at the tail. The ERG protocol included recording the dark adaptation (scotopic) response to brief white light (for rod-isolated B waves, -2.31 log cd.sm). –2 For the rod-cone combination, the A and B waves are 1.09 log cd.sm –2 ). Response 1.09 log cd.sm –2 Intensity flash (for B-waves with cone dissociation), in white 32 cd.m. –2 Ten minutes after the visual rod suppressed the background, the light-adapted (photopic) ERG was recorded. The response was amplified by a gain of 1,000 from 1.25 to 1000 Hz and digitized at a rate of 2000 Hz. A notch filter was used to eliminate 60 Hz line noise. Responses were computer-averaged and recorded at intervals of 3 to 60 seconds, depending on the stimulus intensity. For statistical analysis, paired t-tests were used to determine if there were significant differences in ERG amplitude between the treated and untreated eyes.

[0051] Photoinduced damage analysis In whitening Rdh12 - / - Injected into one eye of a mouse AAV2 / 5-hGRK1p.hRDH12 (1.3 × 10) 9 Mice were injected with either vg) or an equal volume of PBS, but not into the contralateral eye. Six weeks post-injection, ERG analysis and quantification of dark-vision responses were performed as described above. One week later, mice underwent overnight dark adaptation, their pupils were dilated with tropicamide (0.5%), and they were then placed in a lightbox in a separate transparent tray. Mice were exposed to 5,000 lux for 2 hours, then returned to the feeding room (12 hours dark / 12 hours light (<20 lux)) for 7 days, after which ERG analysis was repeated. The percentage of the original ERG response remaining in each eye after light exposure was calculated, the mean was plotted, and the standard error is shown. A two-tailed paired t-test was used to determine whether the ERG amplitude of the treated eye differed significantly from that of the untreated eye.

[0052] Optical coherence tomography Mice were anesthetized, and pupils were dilated with 0.5% tropicamide. A spectral-domain optical coherence tomography (OCT) system (Bioptigen Envisu R2200 SD-OCT system (Durham, North Carolina, USA)) (volume analysis size 1.4 × 1.4 mm) was placed at the center of the optic nerve head. Systane (Alcon) lubricating droplets were used throughout the imaging process.

[0053] Analysis of retinol content All-trans and 11-cis retinaldehyde were extracted from mouse eyes using a modified method described previously (Bligh and Dyer, 1959). After six weeks, mice were subjected to overnight dark adaptation following injection, then euthanized under dark red light with an excess of CO2, and their eyes were removed and frozen in liquid N2. Each eye was homogenized in 1 mL of chloroform:methanol:hydroxylamine (2 M) (3:6:1) on dark red light and ice and incubated at room temperature for 2 minutes. Next, 200 μL of chloroform and 240 μL of water were added, each sample was vortexed, and centrifuged at 14,000 rpm for 5 minutes. The lower phase was collected, the solvent was evaporated under nitrogen, and the sample was dissolved in hexane. Vitamin A-like compounds in extracts were identified and quantified by high-performance liquid chromatography (HPLC) using a Waters Alliance separation module and a photodiode array detector, with a Supelcosil LC-31 column (25 cm × 4.6 mm × 3 μm) developed with 5% 1,4-dioxane / hexane. Peak identification was performed by comparing the retention times of standard compounds and evaluating the maximum wavelength. Quantification was performed by comparing the peak areas of cis- and trans-11-cis-retinaldehyde oxime at 347 and 351 nm, and the peak areas of cis- and trans-all-trans-retinaldehyde oxime at 357 and 361 nm (Kurth et al., 2007).

[0054] Assay of retinaldehyde reductase activity Six weeks post-injection, the retinaldehyde reductase activity in mouse retinal homogenates was measured. Light-adapted mice were euthanized, and each retina was homogenized in 125 μL of 0.25 M sucrose, 25 mM trihydroxyaminomethane acetate (pH 7), and 1 mM dithiothreitol. The homogenate was centrifuged at 1000 × g for 5 min to remove unbroken cells, and the supernatant was then sonicated on ice with a microtip probe (30 times, 1 second each time). Protein concentrations were determined using a modified Lowry program (Peterson et al., 1977), and RDH12 levels were assessed by Western blotting. Similar samples were pooled, and 20 µg (triples) of each pooled lysate was determined in 200 μM all-trans retinaldehyde and 200 μM NADPH / HEPES buffer (pH 8); the reaction was incubated in a 37°C water bath for 0–45 min (this temperature minimizes thermal isomerization of the retinoid substrate and enzyme inactivation). The formation of all-trans retinol was quantified using normal-phase HPLC and compared with known standards (Chrispell et al., 2009).

[0055] Example 1 AAV-mediated expression of human RDH12 Multiple vectors for RDH12 replacement therapy were developed and tested. Figure 2 Figure A shows the optimal RDH12 vector construct. This construct contains human rhodopsin kinase (GRK1) promoter fragments controlled by the human rhodopsin kinase (GRK1) promoter. RDH12 cDNA. The construct is packaged in AAV2 / 5 serotypes. AAV serotype 5 capsids have been shown to mediate photoreceptor transduction, but with slower kinetics and less stable expression compared to AAV8 capsids (Yang et al., 2002; Lotery et al., 2003; Allocca et al., 2007; Lebherz et al., 2008). Subretinal injection... AAV2 / 5-hGRK1p.hRDH12 (1.3 × 10) 9 Six weeks after vg, the expression of human RDH12 protein in the mouse retina was assessed using antibodies specific to mouse RDH12 or human Rdh12 protein. The levels of RDH12-like vector delivery appeared to be roughly equivalent to the amount of mouse Rdh12. Figure 2 B). Native mouse RDH12 and recombinant human RDH12 were evaluated using species-specific antibodies via indirect immunofluorescence imaging of retinal sections. The localization of endogenous and recombinant RDH12 appeared to be identical, indicating that the protein was being processed normally. Figure 2 C).

[0056] Compared to wild-type mice, Rdh12 - / -The ability of the mouse retina to reduce exogenous retinaldehyde was significantly reduced (Chrispell et al., 2009). Subretinal... Rdh12 - / - Mice were injected with 1.3 × 10 9 vg AAV2 / 5- hGRK1p.hRDH12 Six weeks later, the in vitro retinaldehyde reductase activity in the retinal homogenate was measured. The formation of all-trans retinol was quantified by normal-phase HPLC analysis. Each data point represents the mean ± standard error of at least five independent experiments, with retinas from 3 to 5 mice collected and analyzed in triplicate. (Uninjected) Rdh12 - / - The initial rates of all-trans retinol formation in the retinas of C57BL / 6J mice were 0.013 pmol / min and 0.013 pmol / min, respectively. -1 µg protein -1 and 0.071 pmol minutes -1 µg protein -1 ( Figure 3 A). Rdh12 - / - The residual activity observed in mice reflects a reduction in the presence of other RDH isoforms of all-trans retinaldehyde (Rattner et al., 2000; Haeseleer et al., 2002). In mice injected with AAV2 / 5-hGRK1p.hRDH12... Rdh12 - / - In mice, the all-trans retinol formation rate (0.046 pmol / min) -1 µg protein -1 Significantly higher than those injected with PBS Rdh12 - / - All-trans retinol formation rate in mice (0.016 pmol / min) -1 µg protein - 1 The recovery of retinaldehyde reductase activity (approximately 50%) was consistent with partial retinal transduction (approximately 30%), demonstrating that the vector restored normal activity levels in the transduced photoreceptors. Transgenic expression persisted for over 12 months (the longest time point detected). Immunofluorescence staining showed the effect assessed at 16 weeks post-injection. Rdh12 - / - The extent of mouse retinal transduction and recombinant RDH12 expression—covering approximately one-third of the retinal surface ( Figure 3 B).

[0057] 1.3 × 10⁻⁶ was injected into one eye. 9 Vg or an equal volume of PBS and in the untreated whitening eye of the contralateral eye Rdh12 - / - Evaluation in mice AAV2 / 5-hGRK1p.hRDH12Effects on susceptibility to light-induced damage. ERG analysis of scotopic retinal activity (rod separation and combined rod-cone) was performed 1 week before and 1 week after applying light levels (5,000 lux for 2 hours) that induced significant retinal damage in albino animals to mice. The percentage of residual retinal activity in the carrier-treated eyes was significantly greater than that in the untreated eyes (p ≤ 0.0168). Figure 4 Conversely, in control animals injected with PBS, the percentage of residual scotopic retinal activity in the uninjected eyes was not significantly greater than that in the untreated eyes (p ≥ 0.2255). These findings are related to increased susceptibility to light-induced damage associated with RDH12 deficiency. AAV2 / 5-hGRK1p.hRDH12 The mediated protective effect is consistent.

[0058] The toxicity of the carrier was assessed by the direct effect of human RDH12 activity on retinoid metabolism, and the indirect effects on retinal structure and function are described below.

[0059] RDH12's broad substrate specificity reduces both 11-cis-retinal and all-trans-retinal retinopathy, creating the potential for overexpression or mislocalization of the recombinant protein, thus negatively impacting visual circulation function. The effects of RDH12 injection on retinol content were evaluated using HPLC analysis. AAV2 / 5-hGRK1p.hRDH12 of Rdh12 - / - Visual circulation activity in the eyes of mice, mice injected with PBS, or uninjected C57BL / 6J controls. In the eyes of mice... Rdh12 - / - Subretinal injection in mice AAV2 / 5-hGRK1p.hRDH12 (1.3 × 10) 9 Analysis of retinoids 6 weeks after vg showed that hRDH12 expression did not have a significant effect on the steady-state 11-cis-retinaldehyde level in the retina. Figure 5 A). Representative chromatograms show various controls and indicate the elution times for each retinol class. Total retinaldehyde levels of 11-cis-retinaldehyde and all-trans-retinaldehyde represent the mean ± standard error of at least five independent experiments. Figure 5 B, 5C). After receiving 1.3 × 10 9 vg AAV2 / 5-hGRK1p.hRDH12 And kept in the enclosure (12 hours darkness / 12 hours light (<20 lux)) the C57BL / 6J and Rdh12 - / - ERG response of retinal activity was measured in mice. Scotopic (rod dissociation -2.3 log cd.sm) was measured in representative animals from each treatment group 6 weeks post-injection.–2 Stimulus), combined rod-cone (1.09 log cd.sm) –2 Stimulus) and photopic vision (cone-mediated response, 1.09 log cd.sm –2 ERG (e.g., stimulation) showed that human RDH12 expression had no significant effect on retinal function. Figure 6 ).

[0060] Mislocalization of rhodopsin and cone opsin is a good indicator of decreased photoreceptor cell viability (Adamian et al., 2006; Turney et al., 2007; Brunner et al., 2010; Lopes et al., 2010). In the absence of injection... Rdh12 - / - or injection AAV2 / 5-hGRK1p.hRDH12 (1.3 × 10) 9 Immunohistochemical localization of rhodopsin and red / green opsins was evaluated in mice with vg. In mice exhibiting strong transgene expression, no decrease in opsin expression levels or evidence of mislocalization of cone and rod opsins was observed 16 weeks post-treatment; retinal structures remained intact 54 weeks post-injection. Figure 7 ).

[0061] During injection AAV2 / 5-hGRK1p.hRDH12 (2 × 10) 9 (vg) 54 weeks later, for C57BL / 6J and Rdh12 - / - Optical coherence tomography (OCT) analysis was performed on mice, and the results showed no significant changes in retinal layering over a large area, including both sides of the optic nerve. Compared to the uninjected contralateral eye, the retinal structure remained stable for at least one year after injection. Figure 8 ).

[0062] Example 2: Retinal damage caused by AAV2 / 8 Retinal damage in mice injected with AAV2 / 8-hGRK1p.hRDH12.

[0063] A capsid derived from AAV serotype 8 has been shown to mediate efficient and potent transduction in photoreceptor cells (Allocca et al., 2007; Natkunarajah et al., 2008; Vandenberghe et al., 2011; Vandenberghe et al., 2013). Preliminary studies were conducted using AAV2 / 8 serotypes carrying the aforementioned vector construct. At 10 8 -10 9 A dose of one viral genome (vg) was administered via subretinal injection of AAV2 / 8-hGRK1p.hRDH12. Rdh12 - / -In mice, robust expression of recombinant human RDH12 protein was achieved. However, significant retinal damage began to appear in the injected eyes 3 weeks after injection, and in almost all cases, it occurred at 6 weeks post-injection. (via CD68) + The presence of cells was observed in most cases with macrophage infiltration. Figure 9 From weaning to death, towards Rdh12 - / - Continuous systemic administration of cyclosporine (Borel et al., 1976) failed to alleviate retinal damage in mice, suggesting that the immune response to the vector or transgene is not the cause of the damage. This view is consistent with the effect of injection on human RDH12 ( AAV2 / 8-hGRK1p.hRDH12 ) or mouse Rdh12 ( AAV2 / 8-hGRK1p.mRdh12 The results obtained from the wild-type C57BL / 6J mice that were encoded with the construct were consistent, with significant retinal thinning and macrophage infiltration also occurring 6 weeks after injection.

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[0133] Each reference cited in this paper is incorporated herein by reference in its entirety or in its relevant section, as is evident from the context of the citation.

[0134] It should be understood that although the claimed subject matter has been described in conjunction with its detailed description, the foregoing description is intended to illustrate, not limit, the scope of the claimed subject matter, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are all within the scope of the following claims.

Claims

1. Use of an adeno-associated virus vector comprising nucleic acids in the preparation of a medicament for treating a human subject suffering from ophthalmic symptoms caused by one or more loss-of-function mutations in a gene encoding the retinol dehydrogenase 12 (RDH12) protein, said treatment comprising administering the adeno-associated virus vector to at least one eye of said subject, wherein said nucleic acid comprises human... RDH12 DNA, and the person described therein RDH12 The DNA encodes a protein that is at least 70%, 80%, 90%, 95%, or 99% identical in length to the protein in SEQ ID NO:

2.

2. The use according to claim 1, wherein the ophthalmic condition is Leber congenital amaurosis (LCA).

3. The use according to claim 1, wherein... RDH12 DNA expression is controlled by the human rhodopsin kinase 1 (hGRK1) promoter.

4. The use according to claim 3, wherein the hGRK1 promoter comprises SEQ ID NO:

3.

5. The use according to claim 1, wherein the adeno-associated virus vector is AAV-2, serotype-5 (AAV2 / 5), or AAV-5.

6. The use according to claim 1, wherein the RDH12 DNA comprises at least 60% or 70% of the same sequence as SEQ ID NO:

1.

7. The use according to claim 1, further comprising using about 2 × 10 10 The viral genome titer is up to approximately 2 × 10⁻⁶ per milliliter (vg / mL). 12 The nucleic acid was administered at a titer of vg / mL.

8. The use according to claim 1, wherein the nucleic acid is applied to the subretinal space.

9. The use according to claim 8, wherein the microinjection cannula is inserted into the subretinal space.

10. A kind of approach to people RDH12 Nucleic acid encoded by DNA, wherein the human RDH12 The DNA pairs encode proteins that are at least 70%, 80%, 90%, 95%, or 99% identical in length to the full-length of SEQ ID NO: 2, wherein the RDH12 DNA is under the control of the human rhodopsin kinase 1 (hGRK1) promoter.

11. The nucleic acid according to claim 10, wherein the hGRK1 promoter comprises SEQ ID NO:

3.

12. The nucleic acid according to claim 10, wherein the human... RDH12 The DNA encodes a protein including SEQ ID NO:

2.

13. The nucleic acid according to claim 10, wherein the human... RDH12 The DNA is at least 60% or 70% identical to the full length of SEQ ID NO:

1.

14. The nucleic acid according to claims 10 to 13, for treating human subjects suffering from ophthalmic symptoms caused by one or more loss-of-function mutations in the gene encoding the retinol dehydrogenase 12 (RDH12) protein.

15. The nucleic acid according to claim 14, wherein the ophthalmic condition is Leber congenital amaurosis (LCA).

16. A viral vector comprising the nucleic acid according to any one of claims 10 to 15.

17. The viral vector according to claim 16, wherein it is an adeno-associated virus vector.

18. The viral vector of claim 17, wherein the adeno-associated virus vector is AAV-2, serotype-5 (AAV2 / 5), or AAV-5.

19. The viral vector according to any one of claims 16 to 18, for treating human subjects suffering from ophthalmic symptoms caused by one or more loss-of-function mutations in the gene encoding the retinol dehydrogenase 12 (RDH12) protein.

20. The viral vector of claim 19, wherein the ophthalmic condition is Leber congenital amaurosis (LCA).

21. An isolated host cell comprising a viral vector according to any one of claims 16 to 20 or a nucleic acid according to any one of claims 10 to 15.

22. The isolated host cell according to claim 21, wherein the cell expresses human RDH12 protein.

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