AAV-based PDE6b viral vector for treating retinitis pigmentosa containing tissue-specifically expressed PDE6a promoter, and use thereof

Abstract

The present invention relates to: an AAV-based PDE6B viral vector for treating retinitis pigmentosa, the AAV-based PDE6B viral vector containing a tissue-specifically expressed PDE6A promoter; and a use of thereof, and provides a gene therapy for treating retinitis pigmentosa caused by PDE6B gene deficiency. The in vivo therapeutic efficacy of seven types of AAV5-PDE6B vectors was confirmed using an AAV by using a PDE6A promoter that is tissue-specifically expressed in photoreceptor rod cells that develop retinitis pigmentosa. An AAV5-PDE6A-450-PDE6B vector was selected as a candidate due to exhibiting strong tissue-specific expression in photoreceptor rod cells under even off-target conditions, unlike the gene expression characteristics of an AAV5-CMV-PDE6B vector, and was tested so as to be usable in the development of a gene therapeutic agent for treating PDE6B-deficient retinitis pigmentosa patients. Therefore, the present invention, related to AAV5-PDE6B for retinitis pigmentosa treatment and containing a tissue-specifically expressed PDE6A promoter, provides retinitis pigmentosa patients with an important treatment option having improved safety, and can be expected to have fundamental therapeutic effects compared to conventional treatments.

Description

AAV-based PDE6B viral vector for the treatment of retinitis pigmentosa containing a tissue-specifically expressed PDE6A promoter and its use

[0001] The present invention relates to an adeno-associated virus (AAV)-based PDE6B viral vector for the treatment of retinitis pigmentosa comprising a tissue-specifically expressed PDE6A promoter and the use thereof.

[0002] Retinitis Pigmentosa (RP) is a hereditary retinal degenerative disease clinically characterized by night blindness and peripheral vision loss, which progressively impairs visual function. Over time, visual impairment worsens and can eventually lead to blindness; it is the most common hereditary retinal disease worldwide. Mutations in the PDE6B gene are one of the major causes of RP, and a deficiency of the PDE6B protein, which is essential for the function of photoreceptor cells, triggers the disease. PDE6B plays a crucial role in converting light signals into electrical signals in the rod photoreceptor cells of the retina, and defects in this gene cause visual loss. To date, there is no fundamental cure for retinitis pigmentosa, and treatment focuses primarily on symptom relief. Therefore, there is an urgent need for a method to fundamentally treat the disease by correcting the PDE6B gene defect.

[0003] Accordingly, there is a growing need for AAV-based PDE6B gene therapy as an innovative treatment that corrects PDE6B deficiency, expresses normal PDE6B protein with tissue-specific gene expression in retinal photoreceptor cells to prevent disease progression, and remains safe even in off-target situations.

[0004] The present invention aims to provide a pharmaceutical composition for the prevention or treatment of retinitis pigmentosa comprising, as an active ingredient, an adeno-associated virus (AAV) recombinant vector containing a PDE6A promoter gene and a PDE6B gene.

[0005] To solve the above problem, the present invention provides a pharmaceutical composition for the prevention or treatment of retinitis pigmentosa comprising an AAV recombinant vector containing a PDE6A promoter gene and a PDE6B gene as an active ingredient.

[0006] The present invention relates to an AAV-based PDE6B viral vector for the treatment of retinitis pigmentosa containing a tissue-specifically expressed PDE6A promoter and its use, and provides a gene therapy method for treating retinitis pigmentosa caused by a deficiency of the PDE6B gene. Using AAV, the in vivo therapeutic efficacy of seven types of AAV5-PDE6B vectors was confirmed by utilizing a tissue-specifically expressed PDE6A promoter in photoreceptor rod cells undergoing retinitis pigmentosa, along with various other promoters. Specifically, as a result of comparing the degree of inhibition of photoreceptor cell degeneration and restoration of visual function, the two most superior vectors were AAV5-PDE6A-450-PDE6B and AAV5-CMV-PDE6B. When the in vivo therapeutic efficacy of these two vectors was analyzed after subretinal injection at two different dosages, the result was 2.5 x 10⁶ 8 At vg / eye dosage, the therapeutic efficacy of the two vectors is similar, whereas 1x10 9 Analysis showed that AAV5-PDE6A-450-PDE6B had slightly higher therapeutic efficacy at vg / eye dosage. In addition, using AAV5-CMV-PDE6B, 1x10 9As a result of analyzing PDE6B protein expression and the inhibitory effect on retinal degeneration after administering the vg / eye dose at the same timing as previous studies—21-day-old—and at an earlier time—16-day-old, it was confirmed that the test group administered at 16-day-old showed slightly higher protein expression and inhibitory effects on retinal degeneration. Unlike the gene expression characteristics of the AAV5-CMV-PDE6B vector, the AAV5-PDE6A-450-PDE6B vector was selected as a candidate substance because it exhibits strong tissue-specific expression in photoreceptor rod cells even under off-target conditions. As a follow-up study to prepare for clinical trials for its potential use in developing a gene therapy for patients with PDE6B-deficient retinitis pigmentosa, the administration time was set to 16 days, and 1x10 9 After administration at a vg / eye dose, the inhibitory effect on retinal degeneration, changes in the expression of related proteins such as Rhodopsin, Opsin, CNGA1, and CNG3 were confirmed, the recovery of visual function was evaluated (ERG and OKN measurements), and in vitro toxicity was evaluated with increasing dosage. Therefore, the present invention, which relates to AAV5-PDE6B for the treatment of retinitis pigmentosa containing a tissue-specific PDE6A promoter, provides an important treatment option with improved safety for patients with retinitis pigmentosa and allows for the expectation of fundamental therapeutic effects compared to existing treatments.

[0007] Figure 1 shows a schematic diagram of an AAV5 vector with various promoter configurations for PDE6B gene expression.

[0008] Figure 2 shows the results of the analysis of PDE6B protein expression following AAV5-PDE6B gene therapy.

[0009] Figure 3 shows the results confirming the inhibitory effect of AAV5-PDE6B gene therapy on retinal degeneration using various promoters.

[0010] Figure 4 shows the results of retinal function recovery evaluation (ERG measurement) by various AAV5-PDE6B vectors.

[0011] Figure 5 shows the results of the evaluation of visual function recovery (Optokinetic Nystagmus measurement) according to various AAV5-PDE6B vectors.

[0012] Figures 6 and 7 show the results of the evaluation of visual function recovery (ERG and OKN measurements) according to the dosage of the AAV5-PDE6B vector selected for photoreceptor-specific expression.

[0013] Figure 8 shows the results of PDE6B protein expression following AAV5-PDE6B gene therapy containing a PDE6A promoter of photoreceptor tissue-specific expression, the inhibitory effect on retinal degeneration and the evaluation of visual function recovery (ERG and OKN measurements).

[0014] Figure 9 shows the PDE6B and Rhodopsin protein expression according to dosage of the AAV5-PDE6A-450-PDE6B vector for photoreceptor tissue-specific expression, the resulting inhibitory effect on retinal degeneration, and the results of the evaluation of retinal function recovery (ERG measurement).

[0015] Figure 10 shows the results of confirming the expression of PDE6B and Rhodopsin proteins and the resulting inhibitory effect on retinal degeneration according to the administration time of the AAV5-PDE6B vector (16-day-old and 3-week-old).

[0016] Figure 11 shows the results of analyzing protein expression, such as PDE6B, Rhodopsin, and Opsin, following the administration of the AAV5-PDE6A-450-PDE6B vector at 16-day-old.

[0017] Figure 12 shows the results of the analysis of PDE6B function-related protein expression and evaluation of retinal function recovery (ERG and OKN measurements) following the administration of the AAV5-PDE6A-450-PDE6B vector at 16 days of age.

[0018] Figure 13 shows the results of RPE cell area and structural stability recovery following administration of the AAV5-PDE6A-450-PDE6B vector at 16 days of age.

[0019] Figure 14 shows the results of the in vitro toxicity evaluation analysis (Live / Dead Cell Viability / Cytotoxicity Assay, LDH Cytotoxicity Assay) of the AAV5-PDE6A-450-PDE6B vector in ARPE-19 cells.

[0020] The present invention provides a pharmaceutical composition for the prevention or treatment of retinitis pigmentosa comprising, as an active ingredient, an adeno-associated virus (AAV) recombinant vector comprising a PDE6A promoter gene and a PDE6B gene.

[0021] Preferably, the PDE6A promoter gene may consist of a nucleotide sequence represented by SEQ ID NO. 1 or SEQ ID NO. 4, but is not limited thereto.

[0022] Preferably, the adeno-associated virus may be adeno-associated virus 5 (AAV5), but is not limited thereto.

[0023] Preferably, the pharmaceutical composition is 2.5 × 10 8 vg / eye up to 1.0×10 9 It may be administered at a dosage of vg / eye, but is not limited thereto.

[0024] Preferably, the above pharmaceutical composition can restore the expression of PDE6B, Rhodopsin, and Opsin proteins.

[0025] Preferably, the above pharmaceutical composition can restore CNGA1 and CNG3 protein expression.

[0026] Preferably, the pharmaceutical composition is specifically expressed in photoreceptor cells, which can restore retinal potential and improve visual response.

[0027]

[0028] In the present invention, the term "recombinant vector" refers to a gene construct comprising an essential regulatory element operably linked to express a gene insert, which is a vector capable of expressing a target protein or target RNA in a suitable host cell.

[0029] The vector used in the present invention may be a vector comprising linear DNA expressed in human or animal cells, a plasmid vector, or a viral expression vector, or a recombinant viral expression vector comprising a recombinant retrovirus vector, a recombinant adenovirus vector, a recombinant adeno-associated virus (rAAV) vector, a recombinant herpes simplex virus vector, or a recombinant lentivirus vector, and more preferably, the adeno-associated virus may be adeno-associated virus 5 (AAV5), but is not limited thereto.

[0030] The pharmaceutical composition of the present invention may be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredient, and the adjuvants may include solubilizing agents such as excipients, disintegrants, sweeteners, binders, coating agents, leavening agents, lubricants, lubricants, or flavoring agents. The pharmaceutical composition of the present invention may preferably be formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. As acceptable pharmaceutical carriers for compositions formulated as liquid solutions, which are sterile and biocompatible, saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components may be used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. In addition, by additionally adding diluents, dispersants, surfactants, binders, and lubricants, it can be formulated into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets.

[0031] The pharmaceutical formulation forms of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops, or injectable liquids, as well as sustained-release formulations of the active compound. The pharmaceutical composition of the present invention may be administered in a conventional manner via intravenous, intra-arterial, intraperitoneal, intramuscular, intra-arterial, intraperitoneal, intrasternal, transdermal, nasal, inhalation, topical, rectal, oral, ocular, or intradermal routes. The effective amount of the active ingredient in the pharmaceutical composition of the present invention refers to the amount required for the prevention or treatment of a disease. Accordingly, it may be adjusted according to various factors including the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of formulation, the patient's age, weight, general health status, gender and diet, the time of administration, the route of administration and the secretion rate of the composition, the duration of treatment, and concurrently used drugs.

[0032] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0033]

[0034] The operating principle of the present invention is as follows.

[0035] RP caused by a mutation in the PDE6B gene is a disease in which problems occur in the visual signal transmission system because the PDE6B protein does not function properly in photoreceptor rod cells. The AAV5 vector has the advantage of being able to deliver genes to photoreceptor cells in the retina with higher efficiency than other AAV serotypes such as AAV2 or AAV8. In the present invention, the human PDE6B gene was loaded onto an AAV5 vector, and gene expression was induced by the following method.

[0036]

[0037] 1. PDE6A Promoter

[0038] The PDE6A promoter is selectively expressed in rod photoreceptor cells. When the AAV5-PDE6A-PDE6B vector is injected into the retina, the PDE6A promoter is activated, leading to the selective expression of the PDE6B gene in rod photoreceptor cells. This corrects abnormal PDE6B proteins, restoring cell function and preventing the degeneration of retinal cells. In this process, the PDE6A promoter acts primarily on rod photoreceptors to ensure that the PDE6B protein, which plays a crucial role in visual signaling, is expressed in the correct cells. This enhances the efficiency and safety of the treatment and maintains visual function over the long term.

[0039]

[0040] 2. Various gene promoters (RHO, GRK, CMV)

[0041] Since human rhodopsin (RHO) gene promoter (sequence ID: U16824.1), rhodopsin kinase (GRK) gene promoter (sequence ID: AY327580.1), and CMV (Cytomegalovirus) promoter, which is recognized as a universal promoter capable of inducing high levels of gene expression in all cells, have been used as tissue-specific expression promoters in the development of gene therapies to treat PDE6B deletion, AAV5-RHO-PDE6B, AAV5-GRK-PDE6B, and AAV5-CMV-PDE6B vectors were constructed to compare the differences in therapeutic efficacy according to gene expression with the test group vectors.

[0042]

[0043] <Experimental Example>

[0044] The following experimental examples are intended to provide experimental examples that are commonly applied to each embodiment according to the present invention.

[0045]

[0046] 1. Creating AAV Vectors

[0047] The PDE6B gene (NM_000283.4) is an important gene responsible for normal visual function in humans. To produce recombinant AAV5 serotype vectors for gene therapy containing seven promoters inserted with this gene, the pAAV-CMV-PDE6B, pAAV-RHO-PDE6B, pAAV-GRK-PDE6B, pAAV-PDE6A-1000-PDE6B, pAAV-PDE6A-750-PDE6B, pAAV-PDE6A-593-PDE6B, and pAAV-PDE6A-450-PDE6B plasmids were first constructed as shown in Fig. 1. To produce the seven types of AAV5-PDE6B vectors for gene therapy, AAV5 viruses were produced by co-transfecting 293T cells with the pAAV-PDE6B, pR2C5, and pHelper plasmids. The produced viruses were purified in large quantities at CdmoGen Co., Ltd. and then frozen and stored at -80℃.

[0048]

[0049] 2. Virus transmission (subretinal and vitreous injection)

[0050] Rats were anesthetized by intraperitoneal injection of a mixture of alphacalone (20 mg / kg, Alfaxan, Jurox Pty Ltd., NSW, Australia) and xylazine (10 mg / kg, Rompun, Elanco Inc., Greenfield, IN, USA). Treatment was performed on the anesthetized rats under a surgical microscope (Zeiss, Oberkochen, Germany). First, the pupils were dilated using Mydrin-P, and the injection site was thoroughly disinfected. Using a 33G Hamilton syringe, 5 μL of AAV5-PDE6B (2.5 × 10⁶) was injected into the subretinal space and the vitreous cavity, respectively. 8 vg / eye, 1.0 x 10 9vg / eye) was injected. The injection site was located approximately 1 mm posterior to the corneal margin, and care was taken to avoid damaging the lens during the injection. After the injection, the accuracy of the injection was verified using a fundus camera and fluorescence microscope. The concentrated distribution of fluorescence signals on the retina confirmed that vector delivery had been successful. To prevent infection after the injection, Tarivid ointment (Ofloxacin 3 mg / g, Santen Pharmaceuticals, Osaka, Japan) was applied, and appropriate eye care was provided to prevent excessive dehydration during the recovery period.

[0051]

[0052] 3. Histological Analysis and H&E Staining

[0053] After a certain period (6 weeks), the rats were euthanized and their eyes were enucleated. The enucleated eyes were fixed in a 4% PFA (paraformaldehyde) solution at 4°C overnight, and the fixed eye tissue was dehydrated stepwise with a sugar solution and then sectioned to a thickness of 5-10 μm using a cryosectioning machine. The sectioned retinas were treated with PBST (0.5% Triton X-100 in PBS) and then blocked with 5% normal goat serum. A primary antibody reaction was carried out overnight at 4°C using anti-PDE6B antibody (SC-377486, Santa Cruz USA; IF 1:200), anti-GFP antibody (SC-8334, Santa Cruz, USA; IF 1:100), anti-Rhodopsin antibody (ab221664, Abcam, USA; IF 1:100), anti-Phalloidin antibody (P1951, Sigma, USA; IF 1:1000), anti-CNGA1 antibody (ab253296, Abcam, USA; IF 1:100), anti-CNG3 antibody (MBS9233884, MyBioSource, USA; IF 1:100), and anti-Opsin antibody (AB5405, Sigma-Aldrich, USA; IF 1:250), and a secondary antibody reaction was carried out for 4 hours at room temperature using Alexa Fluor auxiliary antibody. The nuclei were visualized using DAPI staining, and slides were mounted using Vectashield. Retinal tissue structures were observed using H&E staining, and photographs were taken at magnifications of 320x and 340x using an Olympus CX41 microscope.

[0054]

[0055] 4. Fluorescence Microscopy Analysis

[0056] Immunofluorescence staining data were collected using a Zeiss LSM 780 fluorescence microscope. Protein expression in retinal tissue was analyzed using primary antibodies such as PDE6B, Rhodopsin, Phalloidin, and GFP, as well as auxiliary antibodies Alexa 488 and Alexa 594. Nuclei were visualized using DAPI, and the obtained images were analyzed to confirm the expression levels of PDE6B in the retina and changes in cell structure.

[0057]

[0058] 5. Visual Function Assessment

[0059] The functional recovery of photoreceptors after gene therapy was evaluated using electroretinography (ERG). Mice were acclimatized to a dark environment overnight, after which their response to light stimulation was measured. A-wave and B-wave responses in each eye were recorded using the Ganzfeld ERG system (Phoenix Research Laboratories, OR, USA).

[0060] For the Optokinetic Nystagmus (OKN) test, mice six weeks post-gene therapy were acclimatized to a dark environment for 12 hours before undergoing visual function testing. Visual stimuli were provided using a digital monitor displaying black-and-white striped patterns, positioned 20 cm perpendicular to the mice's visual axis. Visual responses were induced through continuous optodrum imaging. The mice's eye movements were recorded using a digital camera, and the frequency of eye movement in one direction within a dark room was measured at one-minute intervals. The measured frequency of eye movements was used as an indicator to compare visual function before and after treatment, and the difference in visual function between the AAV5-PDE6B injection group and the control group was quantitatively analyzed.

[0061]

[0062] <Example 1> Design of AAV5 vectors with various promoter configurations for PDE6B gene expression

[0063] In this embodiment, the development of a gene therapy was carried out using various promoters, including PDE6A which exhibits tissue-specific expression in photoreceptor cells, including a broadly expressed CMV promoter. A promoter for PDE6B expression was designed from the upstream region (NG_009102.1, NCBI) of the transcription start site (TSS) of PDE6A, and AAV5-PDE6A-PDE6B was produced by loading a normal human PDE6B gene (Human, NM_000283.4) into an AAV viral vector.

[0064] The promoters used in this invention were PDE6A upstream regions of 1000 bp, 750 bp, 593 bp, and 450 bp, and Rhodopsin kinase (GRK1, AY327580), Rhodopsin (Rho, U16824), and CMV promoters were used. The promoter size of all vectors was designed to be 1004 bp, and in the case of the PDE6A promoter, stuffer DNA (SD) was added to the required length to adjust it to the optimal size.

[0065] The promoter sequence of the schematic diagram shown in Figure 1 was prepared as a sequence list.

[0066] In the PDE6A promoter sequence extracted from NG_009102.1, the PDE6A - 1000 bp promoter is described as SEQ No. 1, the PDE6A - 750 bp promoter as SEQ No. 2, the PDE6A - 593 bp promoter as SEQ No. 3, and the PDE6A - 450 bp promoter as SEQ No. 4; in the Rhodopsin promoter sequence extracted from U16824, the RHO promoter is described as SEQ No. 5; in the GRK promoter sequence extracted from AY327580, the GRK promoter is described as SEQ No. 6 and the CMV promoter as SEQ No. 7. The above sequences may have PstI and NcoI at the beginning and end, and some sequences may include Stuffer DNA sequences.

[0067]

[0068] <Example 2> Analysis of PDE6B Protein Expression Following AAV5-PDE6B Gene Therapy

[0069] In this example, the PDE6B gene was expressed in Pde6b-deficient (KO) mice using the AAV5 vector prepared in Example 1, and the expression of the PDE6B protein was analyzed through immunofluorescence staining.

[0070]

[0071] 1. Materials and Methods

[0072] Animal model: 3-week-old (21-day-old) Pde6bKO rats and normal WT rats were used.

[0073] Gene delivery vehicle: Each AAV5 vector consisted of an AAV5 viral vector containing the PDE6B gene, constructed using the following promoter.

[0074] AAV5-CMV-PDE6B

[0075] AAV5-PDE6A-450-PDE6B

[0076] AAV5-PDE6A-593-PDE6B

[0077] AAV5-PDE6A-750-PDE6B

[0078] AAV5-PDE6A-1000-PDE6B

[0079] AAV5-GRK-PDE6B

[0080] AAV5-RHO-PDE6B

[0081]

[0082] Administration method: Each vector was administered into the eyes of Pde6bKO rats via subretinal injection.

[0083] Analysis Method: PDE6B protein expression was confirmed by immunofluorescence staining after administration, and cell nuclei were visualized using DAPI staining.

[0084]

[0085] 2. Results

[0086] (1) Normal rat (WT)

[0087] In the retina of normal WT rats, the PDE6B protein was strongly expressed in the outer segment (OS) of photoreceptor cells, and the layers of the retina were well distinguished by DAPI staining (Fig. 6, WT rat, red: PDE6B, blue: DAPI).

[0088]

[0089] (2) AAV5-CMV-GFP administration group (control group)

[0090] In Pde6bKO mice injected with AAV5-CMV-GFP, PDE6B protein expression was not observed, and the outer layer (OS) of the photoreceptor cells was observed to be degenerated.

[0091]

[0092] (3) AAV5-CMV-PDE6B administration group

[0093] PDE6B protein expression was confirmed in Pde6bKO mice injected with AAV5-CMV-PDE6B.

[0094]

[0095] (4) PDE6A promoter-based AAV5-PDE6B administration group

[0096] The PDE6A-450-PDE6B injection group, utilizing the PDE6A promoter, showed the highest PDE6B protein expression. PDE6B expression was observed in the AAV5-PDE6A-1000-PDE6B administration group. PDE6B expression was not observed in the AAV5-PDE6A-593-PDE6B and AAV5-PDE6A-750-PDE6B administration groups.

[0097]

[0098] (5) GRK and RHO promoter-based AAV5-PDE6B administration group

[0099] In the AAV5-GRK-PDE6B and AAV5-RHO-PDE6B administration groups, PDE6B expression was lower than in the AAV-PDE6B administration group with other promoters.

[0100]

[0101] 3. Conclusion

[0102] In this example, the expression of the PDE6B protein was analyzed after administering AAV5 vectors expressing the PDE6B gene using various PDE6A promoters developed specifically for photoreceptor cells. AAV5-PDE6A-450-PDE6B showed the highest PDE6B expression, while AAV5-CMV-PDE6B showed similar, albeit slightly lower, expression. Although selective expression of the GRK and RHO promoters was confirmed in the photoreceptor cell layer of the retina, they were found to exhibit significantly lower PDE6B expression compared to AAV5-PDE6A-450-PDE6B or AAV5-CMV-PDE6B. Through this, it was confirmed that the degree of PDE6B expression varies depending on the type of promoter in AAV5-PDE6B gene therapy.

[0103]

[0104] <Example 3> Inhibitory effect of AAV5-PDE6B gene therapy using various promoters on retinal degeneration

[0105] In this example, the effect of gene therapy on inhibiting retinal degeneration in a Pde6b-deficient (KO) mouse model was evaluated using an AAV5-PDE6B vector with a photoreceptor-specific promoter developed in association with the expression of the PDE6B protein identified in Example 2. Retinal tissues were analyzed by H&E staining 6 weeks after subretinal injection of each AAV5-PDE6B vector.

[0106]

[0107] 1. Materials and Methods

[0108] Animal model: 3-week-old (21-day-old) Pde6bKO rats and normal WT rats were used.

[0109] Gene delivery method: Each AAV5 vector consists of an AAV5 viral vector containing the PDE6B gene, constructed using the following promoter.

[0110] AAV5-CMV-PDE6B

[0111] AAV5-PDE6A-450-PDE6B

[0112] AAV5-PDE6A-593-PDE6B

[0113] AAV5-PDE6A-750-PDE6B

[0114] AAV5-PDE6A-1000-PDE6B

[0115] AAV5-GRK-PDE6B

[0116] AAV5-RHO-PDE6B

[0117]

[0118] Administration method: Each vector was injected into the eyes of Pde6bKO rats via subretinal injection.

[0119] Analysis method: Six weeks after administration, retinal tissue was observed by H&E staining to analyze and evaluate the structure of the retinal ganglion layer (GCL), inner nucleus layer (INL), outer nucleus layer (ONL), and photoreceptor outer nucleus layer (OS).

[0120]

[0121] 2. Results

[0122] (1) Normal rat (WT)

[0123] The retinas of normal WT mice had thick GCL, INL, ONL, and OS layers, and the layer structure was clearly maintained.

[0124]

[0125] (2) AAV5-CMV-GFP administration group (control group)

[0126] In Pde6bKO mice administered with AAV5-CMV-GFP, the ONL was almost completely lost, and severe degeneration was observed in the GCL and INL, which were significantly thinned.

[0127]

[0128] (3) AAV5-CMV-PDE6B administration group

[0129] It was confirmed that ONL was partially preserved in Pde6bKO rats after administration of AAV5-CMV-PDE6B.

[0130]

[0131] (4) PDE6A promoter-based AAV5-PDE6B administration group

[0132] It was confirmed that the thickness of ONL is preserved for AAV5-PDE6A-450-PDE6B and AAV5-PDE6A-1000-PDE6B.

[0133] It was confirmed that AAV5-PDE6A-593-PDE6B and AAV5-PDE6A-750-PDE6B did not affect the thickness preservation of ONL.

[0134]

[0135] 3. Results

[0136] In this example, we analyzed the effect of administering AAV5 vectors, which induced PDE6B gene expression using various PDE6A promoters specific to photoreceptor cells, to Pde6bKO mice on inhibiting retinal degeneration. It was confirmed that the retinal protective effect varied depending on the type of vector. AAV5-CMV-PDE6B, AAV5-PDE6A-450-PDE6B, and AAV5-PDE6A-1000-PDE6B demonstrated inhibition of retinal degeneration, and distinct protective effects were observed in the ONL. In particular, the PDE6A-450 promoter showed superior or equivalent efficacy compared to the CMV promoter. It was confirmed that AAV5-PDE6A-593-PDE6B, AAV5-PDE6A-750-PDE6B, AAV5-RHO-PDE6B, and AAV5-GRK-PDE6B were not effective in inducing selective protective effects in the photoreceptor cell layer.

[0137]

[0138] <Example 4> Evaluation of Retinal Function Recovery by Various AAV5-PDE6B Vectors (ERG Measurement)

[0139] This example was conducted to confirm the recovery of retinal function using an AAV5-PDE6B vector with a photoreceptor-specific promoter identified in Example 3. Various AAV5-PDE6B vectors were administered to Pde6b-deficient (KO) mice, and the recovery of retinal electrophysiological function was evaluated through ERG analysis. Through this, the recovery of retinal function related to the expression of the PDE6B gene was measured.

[0140]

[0141] 1. Materials and Methods

[0142] Animal model: 3-week-old (21-day-old) Pde6bKO rats and normal WT rats were used.

[0143] Administration Method and Gene Delivery Vehicle: An AAV5 viral vector expressing the PDE6B gene was administered via subretinal injection. The vectors used to confirm functional recovery are as follows.

[0144] AAV5-CMV-PDE6B

[0145] AAV5-PDE6A-450-PDE6B

[0146] AAV5-PDE6A-593-PDE6B

[0147] AAV5-PDE6A-750-PDE6B

[0148] AAV5-PDE6A-1000-PDE6B

[0149] AAV5-RHO-PDE6B

[0150] AAV5-GRK-PDE6B

[0151] AAV5-CMV-GFP (control group)

[0152]

[0153] ERG Analysis: Functional recovery of the retina was measured through A-wave and B-wave amplitudes. ERG is a method that evaluates the function of photoreceptors and retinal neural circuits by measuring electrical signals generated in the retina after light stimulation.

[0154]

[0155] 2. Results

[0156] (1) Normal rat (WT)

[0157] The ERG waveform in WT rats had high amplitude, and normal A-wave and B-wave were confirmed.

[0158]

[0159] (2) AAV5-CMV-GFP administration group (control group)

[0160] In Pde6bKO mice administered AAV5-CMV-GFP, ERG signals were almost non-existent and very low in amplitude, indicating a loss of retinal function.

[0161]

[0162] (3) AAV5-CMV-PDE6B administration group

[0163] It was confirmed that A-wave and B-wave amplitudes were partially recovered in Pde6bKO mice inoculated with AAV5-CMV-PDE6B.

[0164]

[0165] (4) PDE6A promoter-based AAV5-PDE6B administration group

[0166] In the AAV5-PDE6A-450-PDE6B and AAV5-PDE6A-1000-PDE6B administration groups, ERG signals were restored, respectively; the AAV5-PDE6A-1000-PDE6B injection group showed no A-wave wavelength but partial recovery of the B-wave signal. No wavelengths were observed in AAV5-PDE6A-593-PDE6B and AAV5-PDE6A-750-PDE6B.

[0167]

[0168] (5) GRK, RHO promoter-based AAV-PDE6B administration group

[0169] In the AAV5-GRK-PDE6B injection group, no A-wave was observed, but the B-wave signal was partially recovered. No wavelength was observed in AAV5-RHO-PDE6B.

[0170]

[0171] 3. Conclusion

[0172] In this example, retinal function in Pde6bKO mice injected with various AAV-PDE6B vectors was evaluated via ERG analysis. As a result, the AAV5-PDE6A-450-PDE6B and AAV5-CMV-PDE6B vectors demonstrated the highest level of retinal function recovery at a similar level, and it was confirmed that A-wave and B-wave amplitudes were restored in particular. AAV5-PDE6A-1000-PDE6B showed partial functional recovery, while the AAV5-GRK-PDE6B promoter-based vector exhibited limited recovery. Based on these results, it was confirmed that the AAV5-PDE6A-450-PDE6B vector is optimal for retinal function recovery through photoreceptor cell tissue-specific expression, thereby enabling photoreceptor cell-specific PDE6B expression.

[0173]

[0174] <Example 5> Evaluation of Visual Function Recovery According to Various AAV5-PDE6B Vectors (Optokinetic Nystagmus Measurement)

[0175] In this example, in addition to the analysis evaluating retinal function recovery of the AAV5-PDE6B vector with a photoreceptor-specific promoter identified in Example 4, the study was conducted to confirm visual function. Various AAV5-PDE6B vectors were administered to Pde6b-deficient (KO) mice, and the recovery of visual function was evaluated by measuring the Optokinetic Nystagmus test (OKN). The OKN indicates the degree of visual function recovery by measuring eye movements that occur in response to visual stimuli.

[0176]

[0177] 1. Materials and Methods

[0178] Animal model: 3-week-old (21-day-old) Pde6bKO rats and normal WT rats were used.

[0179] Administration Method and Gene Delivery Vehicle: An AAV5 viral vector expressing the PDE6B gene was administered via subretinal injection. The vectors used to confirm functional recovery are as follows.

[0180] AAV5-CMV-PDE6B

[0181] AAV5-PDE6A-450-PDE6B

[0182] AAV5-PDE6A-593-PDE6B

[0183] AAV5-PDE6A-750-PDE6B

[0184] AAV5-PDE6A-1000-PDE6B

[0185] AAV5-RHO-PDE6B

[0186] AAV5-GRK-PDE6B

[0187] AAV5-GFP (control group)

[0188]

[0189] OKN Analysis: OKN evaluated the recovery of visual function following PDE6B gene expression through eye movements induced by visual stimuli. Eye movements were expressed as frequency per minute (Frequency / min).

[0190]

[0191] 2. Results

[0192] (1) Control group (AAV5-CMV-GFP administration group)

[0193] In Pde6bKO mice injected with AAV5-CMV-GFP, the OKN response was almost non-existent, which means that visual function was not restored.

[0194]

[0195] (2) AAV5-CMV-PDE6B administration group

[0196] After administration of AAV5-CMV-PDE6B, visual function was partially restored, and the OKN response increased to 8 beats per minute. This indicates that visual function was restored through PDE6B expression.

[0197]

[0198] (3) PDE6A promoter-based AAV5-PDE6B administration group

[0199] The OKN responses differed in the AAV5-PDE6A-450-PDE6B, AAV5-PDE6A-593-PDE6B, AAV5-PDE6A-750-PDE6B, and AAV5-PDE6A-1000-PDE6B administration groups, with AAV5-PDE6A-450-PDE6B showing a maximum recovery of more than 6 times per minute, and AAV5-PDE6A-1000-PDE6B showing 4 times per minute.

[0200]

[0201] (4) GRK and RHO promoter-based AAV5-PDE6B administration group

[0202] AAV5-GRK-PDE6B was observed at 5 times per minute. Although limited recovery was also observed in the AAV5-RHO-PDE6B administration group, the OKN response was relatively lower than that of other promoter-based infusion groups.

[0203]

[0204] 3. Conclusion

[0205] In this embodiment, the effect of AAV5 vectors inducing PDE6B gene expression using various promoters on the recovery of visual function in Pde6bKO mice was evaluated. The results showed that AAV5-CMV-PDE6B exhibited the highest OKN response, indicating that visual function was partially restored.

[0206] However, compared to the CMV promoter expressed in off-target tissues, the fact that the AAV5-PDE6A-450-PDE6B vector, which exhibits tissue-specific expression in photoreceptor rods requiring therapeutic efficacy, demonstrates visual function recovery at the level of the CMV promoter is of great significance in the development of gene therapy. Other vectors showed relatively low or almost no therapeutic efficacy, and the plan was to select the optimal gene therapy vector for treating patients with PDE6B deletion by further varying the administration dosage conditions.

[0207]

[0208] <Example 6> Evaluation of Visual Function Recovery by Dosage of AAV5-PDE6B Vector Selected for Photoreceptor-Specific Expression (ERG and OKN Measurements)

[0209] This example was conducted to confirm the recovery of visual function according to the administration dose of a selected vector among the AAV5-PDE6B vectors having a photoreceptor cell-specific PDE6A promoter identified in Example 5. Various AAV5-PDE6B vectors were administered to Pde6b-deficient (KO) mice at two doses, and evaluated using Electroretinography (ERG) and Optokinetic Nystagmus test (OKN) measurements.

[0210]

[0211] 1. Materials and Methods

[0212] Animal model: 3-week-old (21-day-old) Pde6bKO rats and normal WT rats were used.

[0213] Method of administration and gene delivery vehicle: An AAV5 viral vector expressing the PDE6B gene at two doses, namely 2.5 x 10⁶ 8 vg / eye and 1.0X10 9The procedure was performed via subretinal injection at a vg / eye dosage. The vectors used to confirm functional recovery are as follows.

[0214] AAV5-CMV-PDE6B

[0215] AAV5-PDE6A-450-PDE6B

[0216] AAV5-PDE6A-1000-PDE6B

[0217] AAV5-GRK-PDE6B

[0218] AAV5-CMV-GFP (control group)

[0219]

[0220] ERG Analysis: After dark adaptation in a dark room, the response to light of a specific intensity was measured, and the functional recovery of the retina was measured through A-wave and B-wave amplitudes. For the ERG analysis, the amplitudes of the A-wave and B-wave were measured and each vector was compared and analyzed (Figs. 6A and 6B).

[0221] OKN analysis: After dark adaptation in a dark room, the OKN response to visual stimuli was evaluated by expressing eye movements as the frequency of eye movements per minute (Frequency / min) (Fig. 7).

[0222]

[0223] 2. Results

[0224] (1) Control group (AAV5-CMV-GFP administration group)

[0225] AAV5-CMV-GFP 1x10 9 In Pde6bKO rats administered vg / eye, no ERG or OKN responses were observed. This indicates a loss of visual function.

[0226]

[0227] (2) AAV5-CMV-PDE6B administration group

[0228] AAV5-CMV-PDE6B 2.5X10 8 vg / eye and 1.0X10 9 ERG responses were confirmed at both doses after vg / eye administration, and there was no difference in response between the doses. OKN responses were confirmed at 2-3 times per minute, and 1.0 x 10 9 A slightly higher response was observed in vg / eye, but there was no statistically significant difference.

[0229]

[0230] (3) Selected PDE6A promoter-based AAV5-PDE6B administration group

[0231] AAV5-PDE6A-450-PDE6B, 2.5X10 8 and 1.0X10 9 After vg / eye administration, distinct A-wave and B-wave waveforms were observed in the ERG response at each dose, and 1.0X10 9 Higher ERG responses were observed in eyes administered vg / eye. In the OKN response analysis as well, 2–3 responses per minute were observed, higher than the control group, and 1.0 x 10⁻¹⁰ 9 vg / eye administration is 2.5 x 10 8 Higher responses were observed than with vg / eye, but there was no statistical difference. In the AAV5-PDE6A-1000-PDE6B and AAV5-GRK-PDE6B administration groups, ERG and OKN responses were not different from the control group at all dosages.

[0232]

[0233] 3. Conclusion

[0234] The AAV5-PDE6A-450-PDE6B, based on a selected PDE6A promoter, is 2.5X10 8 vg / eye and 1.0X10 9 Visual function recovery effects were observed at both vg / eye dosages, and there was no difference in the efficacy of visual function recovery between the PDE6A promoter, PDE6A-450, and the CMV promoter. In particular, the PDE6A-450 promoter was 1.0 x 10 9In vg / eye, a larger ERG response was observed compared to the CMV promoter. These results demonstrate that the photoreceptor-specific PDE6A-450 promoter induced PDE6B expression in photoreceptor cells more effectively than the broad-spectrum CMV promoter, implying that the PDE6A promoter-based AAV5-PDE6A-450-PDE6B is effective in treating retinitis pigmentosa caused by PDE6B gene deficiency.

[0235]

[0236] <Example 7> Evaluation of PDE6B protein expression following AAV5-PDE6B gene therapy containing a PDE6A promoter with photoreceptor tissue-specific expression, inhibitory effect on retinal degeneration and recovery of visual ability (ERG and OKN measurements)

[0237] In this example, the experiment was conducted to analyze PDE6B protein expression and evaluate the inhibitory effect on retinal degeneration and the recovery of visual function following administration of the AAV5-PDE6A-450-PDE6B vector, which showed the best efficacy among the AAV5-PDE6A vectors having a photoreceptor cell-specific PDE6A promoter identified in Example 6, along with the AAV5-PDE6A-593-PDE6B and AAV5-PDE6A-750-PDE6B vectors, which required further efficacy evaluation. After administering various AAV5-PDE6B vectors to PDE6B-deficient (KO) mice, PDE6B protein expression, retinal structure analysis, and the recovery of visual function (ERG and OKN measurements) were evaluated.

[0238]

[0239] 1. Materials and Methods

[0240] Animal model: 3-week-old (21-day-old) Pde6bKO rats and normal WT rats were used.

[0241] Administration Method and Gene Delivery Vehicle: An AAV5 virus vector expressing the PDE6B gene was administered via subretinal injection. The vector used for this example is as follows.

[0242] AAV5-CMV-PDE6B

[0243] AAV5-PDE6A-450-PDE6B

[0244] AAV5-PDE6A-593-PDE6B

[0245] AAV5-PDE6A-750-PDE6B

[0246] AAV5-CMV-GFP (control group)

[0247]

[0248] Evaluation time: 6 weeks after administration of the AAV5 virus vector, retinal structure and function were analyzed through H&E staining, immunofluorescence staining for PDE6B protein expression analysis, ERG and OKN measurements.

[0249] Sample collection and analysis: After 6 weeks, H&E staining and fluorescent immunostaining using anti-PDE6B antibody were performed for tissue analysis.

[0250] ERG Analysis: After dark adaptation in a dark room, the response to light of a specific intensity was measured, and the functional recovery of the retina was assessed through A-wave and B-wave amplitudes. The ERG analysis involved measuring the amplitudes of A-waves and B-waves and comparing and analyzing each vector.

[0251] OKN Analysis: After dark adaptation in a dark room, the OKN response to visual stimuli was evaluated by expressing eye movements as the frequency of eye movements per minute (Frequency / min).

[0252]

[0253] 2. Results

[0254] (1) H&E dyeing

[0255] AAV5-CMV-GFP 1x10 9 In Pde6bKO mice administered vg / eye, the thickness of the outer nuclear side (ONL) of the retina was significantly reduced, and the degree of retinal degeneration was severe, resulting in a level of retinal structure collapse (Fig. 8C). In contrast, in Pde6bKO mice administered AAV5-PDE6A-450-PDE6B or AAV5-CMV-PDE6B, the retinal thickness recovered significantly to a normal level, and a retinal structure similar to that of normal mice was maintained (Figs. 8D and 8E). Meanwhile, in Pde6bKO mice administered the other two types of AAV5-PDE6A-PDE6B, it was observed that the maintenance of the retinal structure had deteriorated to a significant degree (Figs. 8F and 8G).

[0256]

[0257] (2) PDE6B protein expression and histological analysis

[0258] PDE6B protein expression is 1.0 x 10⁻¹⁰ AAV5-PDE6A-450-PDE6B and AAV5-CMV-PDE6B 9 Expression was observed in the photoreceptor tissue of the retina in Pde6bKO mice administered vg / eye (Figs. 8J and 8K). While expression was observed in extensive retinal tissues upon administration of AAV5-CMV-PDE6B, tissue-specific expression was confirmed as strong PDE6B protein expression was observed only in photoreceptor tissues similar to those in normal mice upon administration of AAV5-PDE6A-450-PDE6B. Meanwhile, no expression was detected at all in the AAV5-CMV-GFP administration group (Fig. 8I), and expression was so weak that it could hardly be observed in Pde6bKO mice administered AAV5-PDE6A-593-PDE6B and AAV5-PDE6A-750-PDE6B (Figs. 8L and 8M).

[0259]

[0260] (3) Retinal function analysis (Electroretinography, ERG measurement)

[0261] AAV5-PDE6A-450-PDE6B and AAV5-CMV-PDE6B were 1.0X10 through ERG measurement 9 In Pde6bKO mice administered vg / eye, A-wave and B-wave waveforms similar to those of normal (WT) mice were observed, confirming that function had been restored (Figs. 8R and 8S). Meanwhile, no response was observed in the AAV5-CMV-GFP administration group (Fig. 8Q), and while a slight response was observed in Pde6bKO mice administered AAV5-PDE6A-593-PDE6B and AAV5-PDE6A-750-PDE6B, it was at a very weak level (Figs. 8T and 8U).

[0262]

[0263] (4) Visual function evaluation (Optokinetic Nystagmus Test, OKN measurement)

[0264] AAV5-PDE6A-450-PDE6B and AAV5-CMV-PDE6B 1.0X10 through eye movement measurements 9 It was confirmed that visual function was restored in Pde6bKO mice administered vg / eye (Fig. 8W). Meanwhile, no response was observed in the AAV5-CMV-GFP administration group through OKN measurement, and it was confirmed that the response was very weak in Pde6bKO mice administered AV5-PDE6A-593-PDE6B and AAV5-PDE6A-750-PDE6B.

[0265]

[0266] 3. Conclusion

[0267] AAV5-PDE6A-450-PDE6B, a candidate vector showing potential for gene therapy in retinitis pigmentosa (RP), was found to inhibit retinal degeneration and restore retinal function along with photoreceptor tissue-specific expression similar to that observed in normal mice. However, while AAV5-CMV-PDE6B showed similar therapeutic efficacy when administered, the PDE6B protein expression was observed in somewhat broader retinal tissues, raising the possibility of safety issues due to off-target expression. Accordingly, the conclusion from Example 6 was confirmed once again. In other words, since the PDE6A-450 promoter induced PDE6B expression in photoreceptor tissues more effectively than the broad CMV promoter, it implies that the PDE6A promoter-based AAV5-PDE6A-450-PDE6B is an effective therapeutic vector with improved safety for the treatment of retinitis pigmentosa caused by PDE6B gene deficiency.

[0268]

[0269] <Example 8> Dosage-dependent expression of PDE6B and Rhodopsin proteins of AAV5-PDE6A-450-PDE6B vector with photoreceptor tissue-specific expression, resulting inhibitory effect on retinal degeneration, and evaluation of retinal function recovery (ERG measurement)

[0270] In this example, the expression of PDE6B and Rhodopsin proteins was analyzed according to the dose of the AAV5-PDE6A-450-PDE6B vector having a PDE6A promoter with photoreceptor tissue-specific expression identified in Example 7, and the inhibitory effect on retinal degeneration and recovery of visual function were confirmed. After administering the AAV5-PDE6B vector at two doses to PDE6B-deficient (KO) mice, the expression of PDE6B and Rhodopsin proteins, the structural analysis of the retina, and H&E analysis were performed, along with the recovery of visual function (ERG measurement).

[0271]

[0272] 1. Materials and Methods

[0273] Animal model: 3-week-old (21-day-old) Pde6bKO rats and normal WT rats were used.

[0274] Method of administration and gene delivery vehicle: An AAV5 viral vector expressing the PDE6B gene at two doses, namely 2.5 x 10 8 vg / eye and 1x10 9 It was administered via subretinal injection at a dose of vg / eye. The vector used for this example is as follows.

[0275] AAV5-PDE6A-450-PDE6B

[0276] AAV5-CMV-GFP (control group)

[0277]

[0278] Evaluation time: 6 weeks after administration of the AAV5 virus vector, the recovery of visual function was evaluated by H&E staining, immunofluorescence staining for analysis of PDE6B and Rhodopsin protein expression, and ERG measurement.

[0279] Sample collection and analysis: After 6 weeks, H&E staining and fluorescent immunostaining using anti-PDE6B antibody and anti-Rhodopsin antibody were performed for tissue analysis.

[0280] ERG Analysis: After dark adaptation in a dark room, the response to light of a specific intensity was measured, and the functional recovery of the retina was assessed through A-wave and B-wave amplitudes. The ERG analysis involved measuring the amplitudes of A-waves and B-waves and comparing and analyzing each vector.

[0281]

[0282] 2. Results

[0283] (1) H&E dyeing

[0284] AAV5-CMV-GFP 1x10 9In Pde6bKO mice administered vg / eye, the outer nuclear (ONL) thickness of the retina was significantly reduced, and compared to the severe degree of retinal degeneration where the retinal structure was at a level of collapse (Fig. 9C), 2.5 x 10 8 vg / eye and 1x10 9 In Pde6bKO mice administered AAV5-PDE6A-450-PDE6B at two doses of vg / eye, retinal thickness recovered significantly to a level similar to normal mice, and when a higher dose was administered, the maintenance of retinal structure was superior, maintaining a retinal structure similar to that of normal mice (Figs. 9D and 9E).

[0285]

[0286] (2) PDE6B and Rhodopsin protein expression and histological analysis

[0287] It was confirmed that the expression of PDE6B and Rhodopsin proteins was also expressed specifically in photoreceptor tissues in proportion to the dose of AAV5-PDE6A-450-PDE6B administered (Figs. 9I, 9J, 9M, and 9N). Meanwhile, no expression was observed in the AAV5-CMV-GFP administration group (Figs. 9H and 9L).

[0288]

[0289] (3) Retinal function analysis (ERG measurement)

[0290] Through ERG measurements, A-wave and B-wave waveforms similar to those of normal (WT) mice were observed in Pde6bKO mice administered AAV5-PDE6A-450-PDE6B at two doses, confirming that function had recovered, and it was observed that the A-wave and B-wave waveforms were larger when the high dose was administered (Figs. 9U and 9V).

[0291]

[0292] 3. Conclusion

[0293] AAV5-PDE6A-450-PDE6B exhibited photoreceptor tissue-specific PDE6B and Rhodopsin protein expression similar to that in normal mice, and it was confirmed that expression was proportional to the administered dose, along with inhibitory effects on retinal degeneration and proportional efficacy in the degree of retinal function recovery.

[0294]

[0295] <Example 9> Expression of PDE6B and Rhodopsin proteins according to the timing of administration of AAV5-PDE6B vector (16-day-old or 3-week-old), and the resulting inhibitory effect on retinal degeneration

[0296] In this embodiment, retinal degeneration and retinal structural collapse progress from birth in Pde6b-deficient (KO) mice, and the degree of degeneration progresses very rapidly, especially from 2 weeks of age. Therefore, to analyze whether protein expression and gene therapy efficacy are improved depending on the timing of administration, AAV5-CMV-PDE6B was administered at 16 days of age (16-day-old) or 3 weeks of age (21-day-old), and the expression of PDE6B and Rhodopsin proteins according to the timing of administration was analyzed, and the inhibitory effect on retinal degeneration was evaluated.

[0297]

[0298] 1. Materials and Methods

[0299] Animal models: 16-day-old and 3-week-old Pde6bKO rats and normal WT rats were used.

[0300] Administration Method and Gene Delivery Vehicle: An AAV5 virus vector expressing the PDE6B gene was administered via subretinal injection. The vector used for this example is as follows.

[0301] AAV5-CMV-PDE6B

[0302] AAV5-CMV-GFP (control group)

[0303]

[0304] Evaluation time: 6 weeks after administration of the AAV5 virus vector, H&E staining and immunofluorescence staining were performed and analyzed to analyze PDE6B and Rhodopsin protein expression.

[0305] Sample collection and analysis: After 6 weeks, H&E staining and fluorescent immunostaining using anti-PDE6B antibody and anti-Rhodopsin antibody were performed for tissue analysis.

[0306]

[0307] 2. Results

[0308] (1) H&E dyeing

[0309] AAV5-CMV-GFP 1x10 9 In Pde6bKO mice administered vg / eye, the outer nuclear (ONL) thickness of the retina was significantly reduced in all cases of administration at 16 days and 3 weeks of age, and the degree of retinal degeneration was severe, with the retinal structure at a level of collapse; in contrast, in Pde6bKO mice administered AAV5-CMV-PDE6B, the retinal thickness recovered to a significant level similar to that of normal mice, and compared to the case administered at 3 weeks of age, the maintenance of retinal thickness and structure was superior in the case administered at 16 days of age, maintaining a retinal structure similar to that of normal mice (Figs. 10A, 10B, and 10C).

[0310]

[0311] (2) PDE6B and Rhodopsin protein expression and histological analysis

[0312] Similar to the H&E staining results, it was confirmed that the expression of PDE6B and Rhodopsin proteins was stronger and superior when administered at 16 days of age compared to when administered at 3 weeks of age (Figs. 10D, 10E, and 10F).

[0313]

[0314] 3. Conclusion

[0315] When the AAV5-CMV-PDE6B vector was administered at 16 days of age, an early stage before severe retinal degeneration progressed, the expression of PDE6B and Rhodopsin proteins was strong, similar to that in normal mice, and consequently, the inhibitory effect on retinal degeneration was found to be superior. These results confirm that this provides important information demonstrating that the timing of treatment for patients with retinitis pigmentosa (RP) can vary significantly depending on when treatment is administered.

[0316]

[0317] <Example 10> Protein expression of PDE6B, Rhodopsin, Opsin, etc. following administration of AAV5-PDE6A-450-PDE6B vector at 16-day-old

[0318] In this example, since protein expression and therapeutic efficacy were confirmed to be superior at 16 days of age compared to 3 weeks of age in Example 9, the expression of PDE6B, Rhodopsin, and Opsin proteins was analyzed after administering AAV5-PDE6A-450-PDE6B, a candidate vector that exhibits photoreceptor tissue-specific expression, to 16-day-old Pde6b-deficient (KO) mice.

[0319]

[0320] 1. Materials and Methods

[0321] Animal model: 16-day-old Pde6bKO rats and normal WT rats were used.

[0322] Method of administration and gene delivery vehicle: 1 x 10⁻¹⁰ AAV5 virus vector expressing the PDE6B gene 9 It was administered via subretinal injection at a vg / eye dosage. The vector used for this example is as follows.

[0323] AAV5-PDE6A-450-PDE6B

[0324] AAV5-CMV-GFP (control group)

[0325]

[0326] Evaluation time: 6 weeks after administration of the AAV5 virus vector, H&E staining and immunofluorescence staining were performed and analyzed to analyze the expression of PDE6B, Rhodopsin, and Opsin proteins.

[0327] Sample collection and analysis: After 6 weeks, H&E staining and fluorescent immunostaining using anti-PDE6B antibody, anti-Rhodopsin antibody, and anti-Opsin antibody were performed for tissue analysis.

[0328]

[0329] 2. Results

[0330] (1) H&E dyeing

[0331] When AAV5-CMV-GFP was administered to 16-day-old Pde6bKO rats, the thickness of the outer nuclear side (ONL) of the retina was significantly reduced, and the degree of retinal degeneration was severe, with the retinal structure at a level of collapse (Fig. 11C). In contrast, in Pde6bKO rats administered AAV5-PDE6A-450-PDE6B at 16-day-old, the retinal thickness recovered to an excellent level similar to that of normal rats (Fig. 11D).

[0332]

[0333] (2) Protein expression and histological analysis of PDE6B, Rhodopsin, and Opsin

[0334] While PDE6B and Rhodopsin protein expression was strongly confirmed (Figs. 11H and 11K), Opsin protein expression was relatively weak but could be confirmed (Fig. 11N).

[0335]

[0336] 3. Conclusion

[0337] When the candidate vector AAV5-PDE6A-450-PDE6B, which exhibits photoreceptor tissue-specific expression, was administered to 16-day-old Pde6b-deficient (KO) mice at an early stage, H&E staining results confirmed that it inhibited retinal degeneration to a level similar to that observed in normal mice. Strong expression of PDE6B and Rhodopsin proteins was observed, and Opsin protein was confirmed to be expressed in photoreceptor tissues, albeit somewhat weakly. These results, along with the results of Example 9, confirmed that the timing of treatment is very important for patients with retinitis pigmentosa (RP).

[0338]

[0339] <Example 11> Analysis of PDE6B function-related protein expression and evaluation of retinal function recovery following administration of AAV5-PDE6A-450-PDE6B vector at 16 days of age (ERG and OKN measurements)

[0340] In this example, the photoreceptor cell tissue-specific expression candidate AAV5-PDE6A-450-PDE6B vector identified in Example 10 was administered to 16-day-old Pde6b-deficient (KO) mice, and the expression of CNGA1 and CNG3 proteins, which are Cyclic nucleotide-gated (CNG) channel subunits that play an important role in PDE6B function and photoreceptor function, was analyzed, and the recovery of visual function (ERG and OKN measurements) was evaluated accordingly.

[0341]

[0342] 1. Materials and Methods

[0343] Animal model: 16-day-old Pde6bKO rats and normal WT rats were used.

[0344] Administration Method and Gene Delivery Vehicle: An AAV5 virus vector expressing the PDE6B gene was administered via subretinal injection. The vector used for this example is as follows.

[0345] AAV5-PDE6A-450-PDE6B

[0346] AAV5-CMV-GFP (control group)

[0347]

[0348] Evaluation time: 6 weeks after administration of the AAV5 viral vector, the degree of recovery of visual function was evaluated through immunofluorescence staining for the analysis of CNGA1 and CNG3 protein expression, ERG, and OKN measurements.

[0349] Sample collection and analysis: After 6 weeks, fluorescent immunostaining using anti-CNGA1 antibody and anti-CNG3 antibody was performed for tissue analysis and protein expression analysis.

[0350] ERG Analysis: After dark adaptation in a dark room, the response to light of a specific intensity was measured, and the functional recovery of the retina was assessed through A-wave and B-wave amplitudes. The ERG analysis involved measuring the amplitudes of A-waves and B-waves and comparing and analyzing each vector.

[0351] OKN Analysis: After dark adaptation in a dark room, the OKN response to visual stimuli was evaluated by expressing eye movements as the frequency of eye movements per minute (Frequency / min).

[0352]

[0353] 2. Results

[0354] (1) CNGA1 and CNG3 protein expression and histological analysis

[0355] It was confirmed that CNGA1 and CNG3 protein expression was specific to photoreceptor tissues when AAV5-PDE6A-450-PDE6B was administered, and CNG3 protein expression was analyzed to be somewhat weak (Figs. 12J and 12M).

[0356]

[0357] (2) Retinal function analysis (ERG measurement)

[0358] In ERG measurements, A-wave and B-wave waveforms similar to those of normal (WT) mice were observed in Pde6bKO mice administered AAV5-PDE6A-450-PDE6B, confirming that visual function had been restored (Figs. 12D, 12E, and 12F). In contrast, when the control group AAV5-CMV-GFP was administered, A-wave and B-wave waveforms were barely detected (Fig. 12C).

[0359]

[0360] (3) Visual function evaluation (OKN measurement)

[0361] AAV5-PDE6A-450-PDE6B 1.0X10 through eye movement measurements 9 It was confirmed that visual function was restored to a degree similar to that of normal mice in Pde6bKO mice administered vg / eye (Fig. 12G). Meanwhile, no response was observed in the AAV5-CMV-GFP administered group through OKN measurement.

[0362]

[0363] 3. Conclusion

[0364] When AAV5-PDE6A-450-PDE6B was administered, CNGA1 and CNG3 proteins, which are important proteins for photoreceptor function, also showed photoreceptor-specific protein expression similar to normal mice, and the recovery of visual function following the recovery of retinal function could be confirmed through ERG and OKN measurements.

[0365]

[0366] <Example 12> Evaluation of RPE cell area and recovery of structural stability following administration of AAV5-PDE6A-450-PDE6B vector at 16 days of age

[0367]

[0368] In this example, AAV5-PDE6A-450-PDE6B, a candidate vector for photoreceptor tissue-specific expression, was administered to 16-day-old Pde6b-deficient (KO) mice, and the effect on the size and structural stability (RPE Cell Solidity) of RPE (Retinal Pigment Epithelium) cells was evaluated to assess the efficacy of gene therapy.

[0369]

[0370] 1. Materials and Methods

[0371] Animal model: 16-day-old Pde6bKO rats and normal WT rats were used.

[0372] Administration Method and Gene Delivery Vehicle: An AAV5 virus vector expressing the PDE6B gene was administered via subretinal injection. The vector used for this example is as follows.

[0373] AAV5-PDE6A-450-PDE6B

[0374] AAV5-CMV-GFP (control group)

[0375]

[0376] Evaluation time point and tissue analysis: Six weeks after administration of the AAV5 virus vector, RPE cell structure was visualized via Phalloidin staining, and cell size and structural stability (RPE Cell Solidity) were evaluated.

[0377]

[0378] 2. Results

[0379] (1) Phalloidin staining

[0380] Through Phalloidin staining, it was confirmed that RPE cells were evenly arranged in normal mice (WT) (Figs. 13B and 13E), while in Pde6bKO mice administered AAV5-CMV-GFP, the RPE cell structure was distorted and irregularly distributed, and the cell size was abnormally large (Figs. 13C and 13F).

[0381] Meanwhile, in Pde6bKO mice administered AAV5-PDE6A-450-PDE6B, it was confirmed that the size and arrangement of RPE cells were restored to a level similar to that of normal mice (Figs. 13D and 13G).

[0382]

[0383] (2) Analysis of RPE cell size and structural stability (solidity)

[0384] Graphs 13H, 13I, and 13J show the results of evaluating the size and structural stability (solidity) of RPE cells. In Pde6bKO mice injected with AAV5-CMV-GFP, the size of RPE cells increased significantly, but when AAV5-PDE6A-450-PDE6B was administered, the cell size recovered to a level similar to that of normal mice, and the results of the solidity stability evaluation also showed a recovery to a level similar to that of normal mice.

[0385]

[0386] 3. Conclusion

[0387] In this example, gene therapy with AAV5-PDE6A-450-PDE6B was confirmed to contribute to the restoration of RPE cell size and structural stability in Pde6bKO mice, whereas such restoration was not observed when the control AAV5-CMV-GFP was administered. This example once again demonstrates that structural changes in RPE cells caused by Pde6b deletion can be successfully restored through gene therapy.

[0388]

[0389] <Example 13> In vitro toxicity evaluation of AAV5-PDE6A-450-PDE6B vector in ARPA-19 (Live / Dead Viability / CytotoxicityAssay and LDH-CytotoxicityAssay analysis)

[0390] In this embodiment, to evaluate the cytotoxicity of the AAV5-PDE6A-450-PDE6B vector, which has been confirmed to have therapeutic efficacy in various ways by exhibiting photoreceptor tissue-specific expression showing potential for gene therapy in patients with retinitis pigmentosa, the study was conducted to evaluate the degree of toxicity affecting cells for 7 days after treatment with various concentrations of the AAV5 vector in ARPE-19 cells.

[0391]

[0392] 1. Materials and Methods

[0393] In vitro cells: ARPE-19 (CRL-2302, ATCC, USA) cells were used.

[0394] Vector processing method and gene delivery vehicle: 1 x 10⁻¹⁰ AAV5-PDE6A-450-PDE6B vector in ARPE-19 cells 4 vg / cell ~ 5x10 5 Cytotoxicity was evaluated on day 1 and day 7 after treatment under vg / cell MOI conditions.

[0395] Live / Dead Viability / Cytotoxicity Assay Analysis: Live / Dead Viability / Cytotoxicity Assay Kit (L32250, ThermoFisher Scientific, USA) was used to analyze live and dead cells after fluorescent staining on day 1 and day 7 of treatment with ARPE-19 cells treated with AAV5 vector under various MOI conditions.

[0396] LDH Cytotoxicity Assay Analysis: Using the CyQUANT LDH Cytotoxicity Assay Kit (C20300, ThermoFisher Scientific, USA), ARPE-19 cells treated with AAV5 vector under various MOI conditions were analyzed on day 1 and day 7 to determine if LDH enzyme activity increased due to cytotoxicity using a supernatant.

[0397]

[0398] 2. Results

[0399] (1) Live / Dead Viability / CytotoxicityAssay Analysis Results

[0400] AAV5-PDE6A-450-PDE6B vector 1x10 4 vg / cell ~ 5x10 5 Cytotoxicity was evaluated on live and dead cells on days 1 and 7 after treatment under vg / cell MOI conditions using a fluorescence staining method with a kit, and no cytotoxicity was detected even at the vector treatment concentration with the highest cytotoxicity (Figs. 14C and 14D).

[0401]

[0402] (2) LDH cytotoxicity Assay analysis results

[0403] AAV5-PDE6A-450-PDE6B vector 1x10 4 vg / cell ~ 5x10 5 After treatment under vg / cell MOI conditions, the secreted LDF enzyme activity was analyzed using a kit after collecting the sample on the cell culture supernatant on days 1 and 7, and since there was no increase in LDH enzyme activity, it was evaluated as safe even at the highest vector treatment concentration (Figs. 14A and 14B).

[0404]

[0405] 3. Conclusion

[0406] AAV5-PDE6A-450-PDE6B, a candidate vector with tissue-specific photoreceptor expression and confirmed excellent therapeutic efficacy, was confirmed to have excellent safety through cytotoxicity studies using ARPE-19 cells.

[0407]

[0408] In summary, the therapeutic effects of AAV5-PDE6A-450-PDE6B, which possesses a photoreceptor-specific PDE6A promoter, in PDE6B-deficient retinitis pigmentosa are as follows.

[0409] 1. Restoration of photoreceptor cell function: Normal function of the retina can be restored through the expression of normal PDE6B protein via gene therapy, which leads to the restoration of Rhodopsin protein expression and the subsequent inhibition of retinal degeneration, including photoreceptor cells.

[0410] 2. Protection of retinal structure: The present invention inhibits the degeneration of the retina to maintain the retinal structure and provides the effect of protecting visual function in the long term.

[0411] 3. Improvement of visual function: Normal expression of the PDE6B protein through this gene therapy can prevent visual loss and further contribute to restoring visual function.

[0412] 4. Safety and Efficiency: The gene delivery method using AAV vectors can increase clinical applicability by providing high safety and gene expression efficiency, and is a gene therapy vector that can solve safety issues due to off-targeting through the expression of therapeutic proteins by the PDE6A promoter for photoreceptor tissue-specific gene expression.

[0413]

[0414] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for the prevention or treatment of retinitis pigmentosa comprising, as an active ingredient, an adeno-associated virus (AAV) recombinant vector containing a PDE6A promoter gene; and a PDE6B gene.

2. A pharmaceutical composition for the prevention or treatment of retinitis pigmentosa according to claim 1, characterized in that the PDE6A promoter gene is composed of a nucleotide sequence represented by SEQ ID NO. 1 or SEQ ID NO.

4.

3. A pharmaceutical composition for the prevention or treatment of retinitis pigmentosa according to claim 1, characterized in that the adeno-associated virus is adeno-associated virus 5 (AAV5).

4. In claim 1, the pharmaceutical composition is 2.5 × 10 8 vg / eye up to 1.0×10 9 A pharmaceutical composition for the prevention or treatment of retinitis pigmentosa characterized by being administered at a dosage of vg / eye.

5. A pharmaceutical composition for the prevention or treatment of retinitis pigmentosa, characterized in that, in any one of claims 1 to 4, the pharmaceutical composition restores the expression of PDE6B, Rhodopsin, and Opsin proteins.

6. A pharmaceutical composition for the prevention or treatment of retinitis pigmentosa, wherein, in any one of claims 1 to 4, the pharmaceutical composition is characterized by restoring the expression of CNGA1 and CNG3 proteins.

7. A pharmaceutical composition for the prevention or treatment of retinitis pigmentosa according to any one of claims 1 to 4, wherein the pharmaceutical composition is specifically expressed in photoreceptor cells to restore retinal potential and improve visual response.

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