Molecular Vision 2025; 31:127-141 <http://www.molvis.org/molvis/v31/127>
Received 15 February 2024 | Accepted 27 March 2025 | Published 29 March 2025

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Subretinal delivery of AAV5-mediated human Pde6b gene ameliorates the disease phenotype in a rat model of retinitis pigmentosa

Hee Jong Kim,1,2 Ji Hoon Kwak,3,4 Jun Sub Choi,1,2 Jin Kim,1,2 Seo Yun Moon,1,2 Steven Hyun Seung Lee,1,2 Heuiran Lee,5,6 Keerang Park,1,2 Joo Yong Lee,3,4 So-Yoon Won1,2

The first two authors contributed equally to this work.

1Institute of New Drug Development Research, CdmoGen Co., Ltd., Seoul, Republic of Korea; 2CdmoGen Co., Ltd., Cheongju, Republic of Korea; 3Department of Ophthalmology, Asan Medical Center, University of Ulsan, College of Medicine, Republic of Korea; 4Bio-Medical Institute of Technology, University of Ulsan, College of Medicine, Republic of Korea; 5Department of Microbiology, Asan Medical Center, University of Ulsan College of Medicine, Seoul; 6Bio-Medical Institute of Technology, College of Medicine, University of Ulsan, Seoul, Republic of Korea

Correspondence to: So-Yoon Won, CdmoGen, Institute of New Drug Development Research #1023, A Dong,201, Songpa-daero, Seoul, Seoul 05855, Republic of South Korea; Phone: 82-2-881-5419; FAX: 82432362277; email: sywon@cdmogen.com

Abstract

Purpose: A genetic disorder that affects the beta subunit of cyclic guanosine monophosphate-phosphodiesterase type 6 (PDE6B) in humans leads to autosomal recessive retinitis pigmentosa (RP). This condition causes severe vision loss in early life due to fast deterioration of photoreceptors. This study evaluated the therapeutic potential of subretinal delivery of the adeno-associated virus (AAV)5-mediated human Pde6b gene in an RP rat model caused by Pde6b gene knockout (KO).

Methods: We compared the transduction efficiency and tropism of different AAV serotypes (2, 5 and 8) in Pde6b KO rats and found that AAV5 had the highest and most specific expression in photoreceptors. We injected AAV5-Pde6b into the subretinal space of Pde6b KO rats on postnatal day 21. We assessed the protective effects six weeks postinjection by measuring PDE6B protein expression, photoreceptor structure, retinal morphology and thickness, retinal pigment epithelium integrity and visual function.

Results: AAV5-Pde6b treatment ameliorated the disease phenotype in Pde6b KO rats by restoring PDE6B protein expression, preserving photoreceptor structure, improving retinal morphology and thickness, and maintaining retinal pigment epithelium integrity. Functional analysis of vision by scotopic electroretinogram (ERG) and optokinetic nystagmus revealed that AAV5-Pde6b treatment significantly improved the visual function of Pde6b gene KO rats compared with AAV5-GFP-injected Pde6b KO rats.

Conclusions: Our results demonstrate that AAV5-Pde6b may be a potential therapeutic gene candidate for RP caused by Pde6b mutations.

Introduction

Retinitis pigmentosa (RP) is a hereditary disease that affects the retina, which is the light-sensitive tissue at the back of the eye [1,2]. RP causes the gradual loss of photoreceptor cells responsible for converting light into electrical signals [1,3]. As a result, people with RP experience progressive vision impairment, often leading to legal blindness [1,3]. RP affects about one in every 4,000 people worldwide and is one of the most common causes of inherited blindness [4]. Many genetic factors can cause RP [5], and more than 80 genes linked to nonsyndromic RP, meaning RP that occurs without any other symptoms or conditions, have been identified so far [6].

One of these genes is Pde6b, which encodes the phosphodiesterase 6β (PDE6B) subunit of the PDE6 protein [7]. The PDE6 protein of phosphodiesterase plays a crucial role in the phototransduction process, which is the mechanism by which light is converted into electrical signals in the retina [8,9]. This process begins when photons of light activate Rhodopsin, a photoreceptor protein. Activated Rhodopsin then stimulates the G-protein transducin, activating the enzyme PDE6. PDE6B is a subunit of this enzyme and is essential for its function. Once activated, PDE6 lowers the concentration of cyclic guanosine monophosphate (cGMP) within the cell, leading to closure of cGMP-gated ion channels, resulting in hyperpolarization of the photoreceptor cell and the initiation of a signal to the brain that light has been detected [9,10].

Mutations in Pde6b can impair the function of PDE6B, leading to abnormal levels of cGMP and defective phototransduction. This can cause the rod cells to degenerate and die, reducing night vision and peripheral vision. Over time, this can also affect the cone cells, the photoreceptors that enable color vision and fine details. Mutations in Pde6b account for 5%–8% of cases of autosomal recessive RP, meaning that both copies of the gene must be mutated for the disease to occur [11,12].

Gene therapy delivers healthy copies of defective genes to the retina or modifies existing gene expression, and is a promising approach for RP treatment [1]. Several gene therapy trials are underway for different forms of RP targeting specific mutations or pathways involved in the disease. For example, Luxturna is an approved gene therapy product that corrects mutations in the RPE65 gene, which is responsible for a rare form of RP that affects night vision [13]. A recent study also showed that gene therapy with Nrf2, a transcription factor that regulates antioxidant response, was able to reverse retinal pigment epithelium (RPE) degeneration and protect photoreceptors in a mouse model of RP [14,15]. Gene therapy for RP faces several challenges, such as the diversity of genetic causes, delivery methods, safety and efficacy and ethical issues [16,17].

Adeno-associated viruses (AAVs) are widely used as vectors for retinal gene therapy as they can transduce various cell types and have low immunogenicity [5]. However, various AAV serotypes show differences in expression efficiency depending on the route of administration, and the tropism of each serotype is known to vary depending on the target tissue [18,19]. Therefore, choosing the optimal AAV serotype and delivery method is crucial for therapeutic efficacy. Intravitreal and subretinal injections are two standard gene therapy methods for the retina. Intravitreal injections are performed by inserting a needle through the sclera and into the vitreous humor, the gel-like substance that fills the eye [20]. Subretinal injections create a small retinal detachment and inject the gene therapy under the retina [21]. Both methods have advantages and disadvantages, depending on the type and location of retinal disease, the size and volume of the gene therapy vector, and the potential side effects and complications [22].

Although mouse strains harboring Pde6b mutant alleles (rd1 [Pde6brd1] and rd10 [Pde6brd10]) are considered animal models of RP and are the most widely used models [23,24], rats are considered more suitable models for RP treatment due to their anatomic and physiologic similarities to humans, which may improve the applicability of research findings to human therapies [25,26]. Additionally, their larger size compared with mice minimizes the risk of complications during intricate procedures such as subretinal injections, which are often problematic in animals with smaller eyes [27-31]. We previously developed a stable Pde6b knockout (KO) rat model using clustered regulated interspaced short palindromic repeats (CRISPR)-Cpf1 technology, which causes retinal degeneration [32]. In the present study, we evaluated the efficacy of subretinal delivery of the AAV5-mediated human Pde6b gene (AAV5-Pde6b) in an RP rat model caused by Pde6b gene KO. We compared the transduction efficiency and tropism of AAV serotypes 2, 5 and 8 in Pde6b KO rats, depending on the route of administration. We also assessed the therapeutic effects of clinical phenotype, PDE6B protein expression, retinal morphology and visual function in AAV5-Pde6b treated Pde6b KO rats. Here we aimed to show that subretinal injection of AAV5-Pde6b improves the disease phenotype in Pde6b KO mice and provides proof-of-concept for gene therapy of RP caused by Pde6b mutations.

Methods

Experimental animals

The experiments used 12 six-week-old Sprague-Dawley (SD) rats and 16 three-week-old Pde6b-KO rats, which CRISPR-Cpf1 produced in our previous publishments [32]. SD rats were housed two to three per cage with ad libitum access to water and food during a 12-h light/dark cycle. All reasonable efforts were made to minimize animal suffering and to use the minimum number of animals necessary to perform statistically valid analyses. The study was conducted by the Declaration of Helsinki and approved by the Institutional Review Board of Animal Experiments at the Asan Institute for Life Science (University of Ulsan, College of Medicine; protocol code 2022–12–210, 11 August 2022).

AAV vector production

The human phosphodiesterase 6B gene (PDE6B, NM_000283.4) was used to construct a photoreceptor Pde6b gene insert. The insert was verified by sequencing and then packaged into AAV5 vectors by co-transfecting 293T cells with three plasmids comprising pAAV-CMV-hPde6b (pAAV-Pde6b), pR2C5 and pHelper. The resulting virus, AAV5-Pde6b, was purified and frozen (−80 °C) in sterile tubes for later use. All virus vectors used in this study were obtained from CdmoGen Co., Ltd. (Cheongju, Korea).

Delivery of viral vector (intravitreal and subretinal injection)

The rats were anesthetized via intraperitoneal injection of a combination of alfaxalone (20 mg/kg, Alfaxan, Jurox Pty Ltd., NSW, Australia) and xylazine (10 mg/kg, Rompun, Elanco Inc., Greenfield, IN). The intravitreal injections were administered using a 33-G Hamilton syringe (Hamilton, Bonaduz, Switzerland) and the procedure was guided by an operating microscope (Zeiss, Oberkochen, Germany). Pupil dilation was achieved using Mydrin-P, consisting of tropicamide 5 mg/ml and phenylephrine 5 mg/ml (Santen Pharmaceuticals, Osaka, Japan). A sclerotomy was carefully performed approximately 1 mm posterior to the limbus for intravitreal injections using a 33-G Hamilton syringe. This was carried out with due care to avoid causing any damage to the lens. An amount of 5 μl AAV2, -5 or -8-GFP (5.02 × 1010 vg/ml) was administered intravitreally. Subretinal injection required a temporal conjunctival peristome, after which subretinal injection was performed using the same needle and the AAV2, -5 or -8-GFP dose. The accuracy of each injection was assessed via a fundus camera and in vivo fluorescence imaging, as previously described. To prevent intraocular infection and excessive dehydration during recovery after imaging, the eyes were treated with Tarivid ointment (Ofloxacin 3 mg/g, Santen Pharmaceuticals).

Histologic analysis

For those cases with cryosectioned retinas, the eyes were enucleated and fixed overnight with 4% PFA and underwent gradient dehydration with sucrose. Subsequently, the processed eyes were snap-frozen in optimal cutting temperature compound. The retinas were blocked in phosphate-buffered saline with Tween (PBST; 0.5% Triton X-100 in PBS) and treated overnight with 5% normal goat serum with an anti-PDE6B antibody at 4 °C. Following a wash in 0.5% PBST, the samples were incubated for 4 h at room temperature with species-specific Alexa Fluor-coupled secondary antibodies. 4’,6-diamidino-2-phenylindole (DAPI)/Hoechst dyes were used to identify the nucleus. The samples were washed in 0.5% PBST at least five times and mounted with a mounting medium (Vectashield, Vector Laboratories, Burlingame, CA). A Zeiss LSM 780 confocal microscope (Carl Zeiss, Berlin, Germany) was used to obtain immunofluorescence data. Primary antibodies were diluted in an antibody Diluent (Dako, S3022, Agilent-Dako, Canada). We used the following primary antibodies in this study: anti-GFP (sc-8334, 200 mg/ml, Santa Cruz Biotechnology, Dallas, TX; IF: 1:100), anti-PDE6B (GTX33395, Gene Tex, Irvine, CA; IF: 1:100), anti-Rhodopsin (ab221664, Abcam, Cambridge, UK, IF: 1:100) and anti-phalloidin (P1951, 300 units, MilliporeSigma, Burlington, MA; IF: 1:1000). Retinal tissue sections were incubated with Alexa 488- or Alexa 594-conjugated secondary antibodies for fluorescence microscopy and the nuclei were counterstained using DAPI (Thermo Fisher Scientific, Waltham, MA). The retinal tissue sections were stained with hematoxylin and eosin (H&E) and photographed under a microscope (Olympus CX41; Olympus America, Center Valley, PA) at 320× and 340× magnification.

Fundus examination and imaging

Topical anesthesia was achieved using proparacaine hydroxychloride (Alcaine; Alcon, Fort Worth, TX). The eyes were dilated with 1% tropicamide and 2.5% phenylephrine drops (Mydrin-P; Santen Pharmaceuticals) and lubricated with methylcellulose. Fundus photographs were taken using the Micron IV (Phoenix Research Laboratories, Pleasanton, CA), with a wavelength range between 450 and 650 nm; acquired images were stored in Micron IV software (StreamPix; NorPix, Inc., Montreal, QC, Canada).

Optical coherence tomography

Sectional retina images were acquired using image-guided optical coherence tomography (OCT; OCT2; Phoenix Research Laboratories, OR). Six scans centered on the optic nerve were obtained. Retinal thickness and morphology were compared between wild-type (WT) rats and Pde6b KO rats injected with AAV5-GFP or AAV5-Pde6b.

Measurement of retinal vessel diameter

We used the Vessel Analysis plugin, which can be added to Fiji to analyze vasculature and allows for the examination and quantitative description of vascular features. The diameters of the retinal vessels were measured at a distance of 1 mm from the optic nerve head in the retina of WT rats and Pde6b KO rats injected with AAV5-GFP or AAV5-Pde6b. The diameters of veins and arteries in one retina were combined to obtain an average value, and the resulting data were converted to micrometers (μm) and displayed on a graph [33].

RNA isolation

RNA was isolated from the retina using the phenol-chloroform extraction method with TRIzol (15,596,026, Thermo Fisher Scientific). Initially, samples were homogenized using a Cordless PELLET PESTLE Motor (749,540-0000, Kimble, Vineland, NJ) with TRIzol. Chloroform was then added to each sample at one-fifth the volume of TRIzol, followed by vortexing for 10 s and centrifugation at 12,000 ×g at 4 °C for 15 min. The aqueous phase was then transferred to a new tube, and isopropanol (109634, Merck, Darmstadt, Germany) was added at half the volume of TRIzol. Samples were vortexed briefly for 10 s, followed by centrifugation at 12,000 ×g at 4 °C for 15 min. Supernatants were removed and the pellets were washed twice with ice-cold 75% ethanol (E7023, Sigma-Aldrich, St. Louis, MI) prepared in DEPC-treated, nuclease-free water (AM9906, Thermo Fisher Scientific). The pellets were then resuspended in nuclease-free water (AM9937, Thermo Fisher Scientific) and the RNA concentration was determined using a spectrophotometer (Epoch Microplate Spectrophotometer, BioTek, Winooski, VT).

cDNA synthesis

Total RNA (1 μg) was incubated with DNase I (18,068,015, Thermo Fisher Scientific) for 10 min at room temperature, followed by incubation with 25 mM EDTA at 65 °C for 10 min. Subsequently, half of the DNase I-treated RNA was incubated at 70 °C for 5 min to eliminate the secondary structure within the RNA template. A reverse transcription master mix (EBT-1542, ELPIS-BIOTECH, Dae-jeon, Korea) was then added to the reaction mixture. The mixture was incubated at 37 °C for 30 min, followed by heating at 94 °C for 5 min to synthesize cDNA.

Quantitative reverse transcription PCR

All quantitative PCR (qPCR) primer/probe sets were obtained from IDT (Coralville, IA). cDNA was subjected to real-time qPCR for human PDE6B and rat β-actin, as described in Table 1. PCR was performed using TaqMan Universal Master mix (4324018, Thermo Fisher Scientific) on a real-time PCR system (StepOnePlus, Thermo Fisher Scientific). The PCR reaction began with a 10-min activation at 95 °C, followed by 40 cycles of 95 °C for 15 s (denaturation) and 60 °C for 1 min (annealing and extension). The assays were duplicated for each sample (n=3/group). Normalization was achieved by subtracting the CT values of β-actin from the CT values of human PDE6B using the formula ΔCT = CT value of human PDE6B − CT value of β-actin. mRNA levels of human PDE6B were calculated relative to β-actin using the ΔCT value. For samples where the human PDE6B value was reported as undetermined in the PCR results, the ΔCT value was recorded as 'undetected'.

Retinal pigment epithelial imaging and morphology analysis

The RPE was stained by phalloidin and flat-mounted for retinal pigment cell morphology analysis. The flat-mounted RPE sheet was examined with an LSM 710 fluorescence confocal microscope (Carl Zeiss Microscopy, Jena, Germany). Images of retinal pigment cells were then taken using Zeiss Zen software, black edition (Carl Zeiss Microscopy). RPE images were analyzed by Fiji (Image J; National Institutes of Health, Bethesda, MD) and CellProfiler (Version 4.2.6, Broad Institute, Inc., Cambridge, MA). CellProfiler automatically analyzed retinal pigment epithelial cell size, shape and solidity. Analysis data for the RPE flat mount of each eye was presented as an average value.

Electroretinogram record

Animals were dark-adapted overnight and prepared for recording under dim red illumination. General anesthesia was induced by intraperitoneal administration of a 0.6 mL/kg mixture of tiletamine hypochloride and zolazepam hypochloride (Zoletil; Virbac, Carros, France) and 0.4 ml/kg xylazine hydrochloride (Rompun; Bayer Korea, Seoul, Korea). Topical anesthesia was achieved using proparacaine hydroxychloride (Alcaine; Alcon). After anesthesia was complete, the animal was placed in an earthed aluminum recording chamber and the pupils were fully dilated with eye drops containing 1% tropicamide and 2.5% phenylephrine hydrochloride (Mydrin-P; Santen Pharmaceuticals). A drop of methylcellulose was placed on the corneal surface to ensure electrical contact and to maintain corneal integrity. During ERG recording, rats were placed onto a heating pad to maintain appropriate body temperature. ERGs were recorded from the right eye using the Ganzfeld ERG system (Phoenix Research Laboratories, Bend, OR) with a gold wire loop placed on the right cornea. Reference electrodes were placed in the center of the scalp and ground leads were placed in the skin at the base of the tail. Rod-dominated responses to white light flashes over a 4.0–5.0 log-unit range of intensities were recorded. Signals were sampled every 0.5 ms over a response window of 240 ms. For each stimulus condition, responses were computer-averaged; up to 10 records were averaged for the weakest signals.

Optokinetic nystagmus

To examine visual function for six weeks post-treatment, rats underwent 12 h of dark adaptation, followed by visual stimulation using a digital monitor displaying black-and-white bar patterns 20 cm from the rat's eyes. The monitor was positioned perpendicular to the visual axis. Eye movements were recorded using a digital camera after optodrum video stimulation. Eye movement was quantified by counting the number of times the eyes moved in one direction per minute in the dark room.

Statistics and image analysis

Statistics and ImageJ image analysis software were used to quantify the intensity of the immunofluorescence signals and band densities from immunoblots. Data were analyzed using Statistical Package 429 for the Social Sciences (SPSS) software (version 11.0, IBM, Armonk, NY). Statistical significance was assessed using an ANOVA with Student–Newman–Keuls post hoc analysis. Quantitative data were presented as means ± standard error of the mean (SEM) and differences were considered significant at p<0.05.

Results

Noninvasive in vivo green fluorescence protein fluorescence imaging in the retina of Pde6b knockout rats after intravitreal and subretinal injection of AAV2, AAV5 and AAV8 vectors

We compared the efficacy and specificity of three AAV vectors of different serotypes (2, 5 and 8) for delivering genes to the retina of Pde6b KO rats in an RP model. We used AAV vectors carrying the green fluorescence protein (GFP) gene under the control of ubiquitous and cytomegalovirus (CMV) promoters and injected them intravitreally or subretinally (Figure 1A,B). The time course of GFP expression was assessed at two, four, and six weeks postinjection and showed the highest level at six weeks (Figure 1E,H,K,N,Q,T). We used in vivo GFP retinal imaging to monitor the transduction and expression of AAV-mediated GFP in the retina over time. Within the intravitreal injection group, AAV2-GFP-injected Pde6b KO rats showed minimal transduction of the inner nuclear layer (Figure 1C,D,E). In contrast, AAV5-GFP-injected Pde6b KO rats (Figure 1F,G,H) and AAV8-GFP-injected Pde6b KO rats (Figure 1I,J,K) showed a low number of retinal cells expressing fluorescence. However, within the subretinal injection group, all three serotypes successfully transfected the photoreceptor and RPE, surpassing the effect of the intravitreal AAV2-GFP injection, as evidenced by the comparison of fluorescence intensities (Figure 1L-T). Fluorescence increased over time in the subretinal injection group (Figure 1L-T). Interestingly, AAV5-GFP had the highest transduction efficiency and the most significant expression area among the serotypes (Figure 1O,P,Q). These results showed that AAV5-GFP is an appropriate vector for retinal degeneration gene therapy in Pde6b KO rats and that subretinal injection is a better delivery method for achieving the target.

A comparative study of AAV2, AAV5 and AAV8 vectors for retinal gene delivery: evaluating the impact of subretinal and intravitreal injections on efficiency and specificity by immunohistochemistry

To assess the impact of different AAV serotypes (2, 5 and 8) and delivery routes (subretinal and intravitreal injections) on the expression of PDE6B protein in the retina, we performed immunohistochemistry on WT rats (Figure 2A) and Pde6b KO rat retinas (Figure 2B). We quantified AAV2-GFP, AAV5-GFP and AAV8-GFP fluorescence intensity in the outer nuclear layer (ONL), inner and outer segments and RPE in the eyes of WT and Pde6B KO rats (Figure 2D). Subretinal injection had higher expression than intravitreal injection in WT rats (Figure 2C) and Pde6b KO rats (Figure 2D) in the ONL and RPE. These findings demonstrated that AAV5 subretinal injection effectively delivers the Pde6b gene to the retina.

AAV5-Pde6b treatment inhibits retinal vessel narrowing in Pde6b knockout rats

Analysis of fundus images reveals significant insights into ocular health [34]. We compared the fundus appearance of WT rats and Pde6b KO rats injected with AAV5-GFP or AAV5-Pde6b (Figure 3C–E). WT rats exhibited standard caliber surface blood vessels indicative of normal vascular structures (Figure 3C). However, Pde6b KO rats treated with AAV5-GFP exhibited significant irregularities in vascular size (Figure 3D), suggesting underlying pathological alterations [35]. Interestingly, the Pde6b KO rats that received AAV5-Pde6b showed a mitigated effect on these vascular changes (Figure 3E). Using the Vessel Analysis plugin in Fiji for vasculature analysis has provided significant insights into vascular changes [36]. We found that the increase in complexity of branching patterns in choroidal vessels following AAV5-GFP-injected Pde6b KO rats compared with WT rats highlighted distinct pathological changes (Figure 3F,G). Conversely, AAV5-Pde6b treatment reduced vessel density, suggesting its potential to prevent pathological degeneration (Figure 3H). Furthermore, treatment with AAV5-Pde6b resulted in a noticeable increase in the diameter of retinal vessels in Pde6b KO rats compared with those treated with AAV5-GFP (Figure 3I). These results imply that AAV5-Pde6b treatment may prevent the narrowing of vessels in Pde6b KO rats, suggesting a potential therapeutic effect of AAV5-Pde6b treatment on modulating retinal blood flow.

Restoration of PDE6B protein in Pde6b knockout rats following AAV5-Pde6b treatment

We investigated whether the AAV5-Pde6b gene could restore PDE6B expression in a rat model of Pde6b deficiency. As shown in Figure 4A, the experimental scheme consisted of subretinal injection of AAV5-GFP or AAV5-Pde6b in the eyes of Pde6b KO rats at postnatal day 21 and analysis of retinal function and morphology at six weeks after injection. RT-PCR analysis showed that increased PDE6B expression in the retinas of AAV5-Pde6b-treated KO rats was measured. In contrast, there was no detectable level of PDE6B expression in the retinas of AAV5-GFP-administrated KO rats (Figure 4B). To ascertain the expression of PDE6B protein within the retina's photoreceptor cells, we executed a double immunofluorescence assay, co-staining for PDE6B and Rhodopsin (Figure 4C–H). In WT rats, PDE6B was localized in the photoreceptor outer segment (Figure 4C,F). In Pde6b KO rats injected with AAV5-GFP, no PDE6B expression was detected (Figure 4D,G). In contrast, in Pde6b KO rats injected with AAV5-Pde6b, PDE6B expression was detected in the photoreceptor outer segment (Figure 4E,H). The quantification of PDE6B staining intensity confirmed that AAV5-Pde6b treatment significantly increased the expression of PDE6B in the photoreceptor outer segment compared with AAV5-GFP administration (Figure 4I). We also found that AAV5-Pde6b treatment was able to protect photoreceptors from pathological degeneration in a rat model of Pde6b deficiency. The WT rats had regular Rhodopsin expression in the photoreceptor outer segment (Figure 4C,F,J); however, the Pde6b KO rats that received AAV5-GFP injection had reduced and abnormal Rhodopsin expression (Figure 4D,G,J), whereas the Pde6b KO rats that received AAV5-Pde6b injections had a certain level of Rhodopsin expression in the photoreceptor outer segment (Figure 4E,H,J). The quantification of Rhodopsin immunostaining results showed that AAV5-Pde6b treatment maintained Rhodopsin expression in the photoreceptor structure compared with AAV5-GFP administration (Figure 4I,J). These results proved that AAV5-Pde6b treatment effectively restored PDE6B protein expression and protected photoreceptors from pathological progression in the Pde6b KO rats.

AAV5-Pde6b treatment preserves retinal morphology and photoreceptor function in Pde6b knockout rats

We investigated the retinal morphology and photoreceptor function of Pde6b KO rats six weeks after receiving AAV5-Pde6b injections, as outlined in Figure 5A. Retinal morphology was observed by H&E staining. As indicated in the representative images, there were apparent differences in the thickness of the ONL layer in the AAV5-GFP-injected Pde6b KO rats compared with that in the WT rats (Figure 5B–D). AAV5-Pde6b-injected Pde6b KO rats partially preserved the structure of the ONL compared with AAV5-GFP-injected Pde6b KO rats (Figure 5C,D). Consistent with these findings, the total retinal thickness of the AAV5-Pde6b-injected Pde6b KO rats was significantly maintained compared with AAV5-GFP-injected Pde6b KO rats (Figure 5E). Representative OCT images of WT rat retinas and Pde6b KO rats injected with AAV5-GFP or AAV5-Pde6b are shown in Figure 5F–H. OCT images reveal that the cumulative thickness of the ONL, IS layer and OS layer did not appear in the Pde6b KO rat compared with WT retinas (Figure 5F,G,I), and this reduction was markedly prevented by AAV5-Pde6b injection (Figure 5H,I). Scotopic ERGs were measured to confirm the protective effect of AAV5-Pde6b treatment on the degenerative retina of Pde6b KO rats. As rod degeneration progressed, A-wave and B-wave amplitudes in AAV5-GFP-injected Pde6b KO rats dramatically decreased compared with WT rats. However, we found that AAV-Pde6b treatment partially restored the A-wave and B-wave amplitudes in Pde6b KO rats compared with AAV5-GFP-injected Pde6b KO rats (Figure 5J,K). We performed optokinetic testing using optokinetic nystagmus as a visual stimulus to assess the visual function of Pde6b KO rats administered with AAV5-Pde6b. We found that AAV5-Pde6b-injected Pde6b KO rats showed significant improvement in optokinetic responses at all spatial frequencies tested compared with those of AAV5-GFP-injected Pde6b KO rats (Figure 5L). These results demonstrate that AAV5-Pde6b treatment improved the visual function of Pde6b KO rats by protecting the photoreceptor function.

AAV5-Pde6b treatment preserved retinal pigment epithelium barrier function in Pde6b knockout rats

The RPE and photoreceptor cells are closely connected in structure and function [37], and evaluating morphological changes in RPE cells in Pde6b KO rats is a critical step in understanding the progression of photoreceptor degeneration. To assess morphological alterations in the RPE cells of Pde6b KO rats, we conducted phalloidin immunostaining on RPE whole mounts, investigating the potential therapeutic effect of AAV5-GFP and AAV5-Pde6b treatment (Figure 6B). In WT rats, RPE cells typically exhibit a consistent size (Figure 6C). In contrast, Pde6b KO rats that received AAV5-GFP treatment demonstrated RPE cells of various sizes, as depicted in Figure 6C. The administration of AAV5-Pde6b in Pde6b KO rats led to a normalization of RPE cell size (Figure 6C). We also analyzed cell solidity (the proportion of the RPE cell area filling a best-fit convex envelope). Our findings revealed that the RPE cells of Pde6b KO rats injected with AAV5-GFP exhibited a marked decrease in solidity compared with WT rats. Conversely, Pde6b KO rats treated with AAV5-Pde6b showed increased cell solidity compared with AAV5-GFP-injected rats (Figure 6D).

Discussion

In this study, we present the promising potential of AAV5-Pde6b under the CMV promoter as a therapeutic gene candidate for treating RP caused by Pde6b deficiency. Our findings demonstrate that AAV5-Pde6b vectors effectively improve photoreceptor response to light as well as the visual function of RP rats. The preservation of both A-wave and B-wave in the scotopic ERG responses, which mirrors the rescue effect observed in H&E staining and fundus imaging, is a significant step forward in the development of gene therapy for RP.

Gene therapy is a promising approach for treating various ocular diseases, such as inherited retinal dystrophies, age-related macular degeneration, glaucoma and corneal disorders [38,39]. Among various gene delivery vectors used for gene therapy, AAVs are the most widely adopted due to their beneficial features of safety, stability and efficiency [5]. Different AAV serotypes have shown different transduction profiles of their specific cell and tissue targets [18,40]. Thus, the optimal AAV serotype and injection route for treating patients with different retinal diseases may maximize the therapeutic effect.

Coave Therapeutics, the French clinical stage biotechnology company, has been developing several gene therapeutic candidates for rare ocular and central nervous system diseases (COAVETX). Although Coave Therapeutics announced positive data from their ongoing Phase I/II clinical trial on CTx-PDE6b (AAV5-Pde6b vectors under the Rhodopsin kinase promoter) in patients with RP on May 31, 2023 (COAVETX), we designed the several novel AAV5-Pde6b vectors under the CMV promoter as well as other ocular tissue-specific promoters to develop more efficient therapeutic gene vectors for treating RP patients with Pde6b mutations. Here, we summarized the potential therapeutic effects of AAV5-Pde6b under the CMV promoter when AAV5-Pde6b was injected in Pde6b KO rats. We demonstrated the therapeutic efficacy of protecting retinal cell layers, including photoreceptors and the RPE monolayer and improving visual function, as confirmed by scotopic ERG responses and optokinetic eye movements in Pde6b KO rats administered with AAV5-Pde6b.

The RPE is a single-cell layer between the retina's photoreceptor outer segments (POS) and Bruch’s membrane, and RPE cells maintain retinal health and function [37]. Some of their essential functions include forming the outer blood-retinal barrier (BRB), delivering oxygen and nutrients from the choroid to the outer retina, clearing metabolic waste from the outer retina to the choroid, and regulating the immune response in the subretinal space [41-43]. In RP, the RPE morphology is altered by photoreceptor degeneration and lipofuscin, a waste product of phototransduction, buildup [44,45]. The RPE cells become uneven in shape and size, and show changes in pigmentation [46,47]. These changes can be detected by fundus examination or histological staining of RPE markers such as phalloidin. Our investigation demonstrated that AAV5-Pde6b treatment of Pde6b KO rats suppressed degenerative progression in photoreceptors. In addition, it was confirmed that by preserving the photoreceptor, progressive degeneration of RPE cells adjacent to the photoreceptor, which may lead to secondary impairment, was partially prevented.

In this study, we conducted a short-term efficacy trial (six weeks) to investigate the expression and efficacy of AAV5-CMV-Pde6b in Pde6b KO mice. We are currently working to optimize the AAV5-Pde6b vector with cell-specific promoters and vary dosages to improve precision of the treatment. If a lead compound is identified, we will conduct long-term efficacy trials at six and 12 months. This will provide valuable insight into the durability of therapeutic benefits and potential delayed side effects. This rigorous approach will confirm our initial findings and enrich the scientific community’s understanding of the long-term impact of gene therapy in retinal diseases, ultimately paving the way for clinical applications. The variability in gene therapy’s long-term effects and side effects, contingent upon the method of administration, necessitates vigilant monitoring and assessment during clinical trials. An initial gene therapy injection may show promising results, but its efficacy could wane. Our use of a singular dose of the AAV5-Pde6b vector in treating Pde6b-deficient rats may not represent the optimal approach for the most favorable therapeutic outcome. The literature indicates that varying AAV vector dosages can significantly influence transgene expression and immune reactions, as well as the safety profile [48,49]. Thus, the application and dosage of gene therapy will likely vary based on the type of disease, the extent and progression of retinal degeneration and patient-specific factors such as age, weight and overall health [50,51].

In light of these considerations, future research should delve into the dose–response relationship of our gene therapy to pinpoint the optimal dosage that maximizes efficacy while minimizing toxicity. Such investigations will be pivotal in fine-tuning gene therapy protocols for retinal diseases, ensuring safety and effectiveness in long-term therapeutic applications. In conclusion, our findings suggest that AAV5-mediated subretinal Pde6b gene delivery strongly supports the therapeutic potential of AAV5-Pde6b gene therapy as a promising gene therapy approach for RP caused by Pde6b mutations. The observed results highlight the ability to address the underlying genetic causes and prevent the degenerative progression associated with RP symptoms. Following this study and ongoing investigation, nonclinical studies and early phases of clinical studies may verify its therapeutic efficacy, long-term safety, potential application and clinical applicability as a valuable therapeutic intervention for treating RP patients.

Acknowledgments

Author Contributions: H.J.K., J.H.K., J.K., S.Y.M., and. J.S.C. performed the in vivo experiments and analyzed the data; J.S.C. and J.Y.L. planned and analyzed the mouse models. S.H.S.L., K.P., H.L., and J.Y.L. reviewed and edited the manuscript. J.S.C., K.P., and J.Y.L. provided technical advice about the experimental design; S.Y.W. designed and analyzed the data and wrote the paper. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the Korea Drug Development Fund, funded by the Ministry of Science and ICT, the Ministry of Trade, Industry, and Energy, and the Ministry of Health and Welfare (HN22C0245, Republic of Korea). Declarations: Ethics approval and consent to participate No consent to participate was required for this study. Consent for publication: All authors consented to publication. Conflicts of Interest: H.J.K., J.S.C., J.K., S.Y.M., S.H.S.L., K.P., and S.Y.W. are employees of CdmoGen Co., Ltd., in which S.H.S.L. and K.P. have personal financial interests. No other potential conflicts of interest relevant to this article were reported. Dr. Joo Yong Lee (jylee.retina@gmail.com) and Dr. So-Yoon Won (sywon@cdmogen.com) are co-corresponding authors for this paper.

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