Unilateral subretinal administration of HORA-PDE6β in patients with retinitis pigmentosa harbouring mutations in the PDE6β gene: study protocol of HORA-PDE6β-001 phase I/II clinical trial

Abstract

Introduction

This phase I/II clinical trial, titled HORA-PDE6β-001, investigates the safety, tolerability and preliminary efficacy of HORA-PDE6β, a gene therapy for patients with retinitis pigmentosa (RP) caused by mutations in the PDE6β gene. The primary objective is to assess the safety profile of a unilateral subretinal injection of HORA-PDE6β, while secondary objectives include evaluating immune responses, vector shedding and visual function improvements.

Methods and analysis

This trial is conducted at a single centre, the CHU de Nantes, and involves five cohorts, including patients aged 13 years and older. This first-in-human study was approved by the French regulatory authorities on 28 July 2017.

With a total of 19–21 patients enrolled across the cohorts, the study will monitor patients over 5 years, conducting assessments at regular intervals. The investigational product is an adeno-associated virus vector containing the human PDE6β gene, delivered to the retina to address the gene defect that causes RP. This trial aims to identify the optimal dosage for future clinical applications and to explore the therapeutic potential of gene therapy in halting or reversing retinal degeneration.

Ethics and dissemination

The ethics committee gave its approval on 20 July 2017. The results of this study could significantly impact the development of gene therapies for inherited retinal diseases and will be disseminated in scientific conferences and peer-reviewed indexed journals.

Trial registration number

NCT03328130.

WHAT IS ALREADY KNOWN ON THIS TOPIC

• Retinitis pigmentosa affects approximately 1 in 4000 individuals, with about 2% carrying biallelic pathogenic variants in the PDE6β gene—corresponding to a prevalence of roughly 1 in 200 000. No approved treatment is currently available.

WHAT THIS STUDY ADDS

• This first-in-human trial evaluates a gene-specific therapy for PDE6β-related retinitis pigmentosa, providing key insights into its feasibility, safety and preliminary efficacy.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

• The HORA-PDE6β study represents an essential step towards gene-based treatments for inherited retinal diseases. Its findings may guide future trial designs, inform regulatory pathways and accelerate the translation of gene therapy into clinical practice.

Introduction

Retinitis pigmentosa (RP) is a genetically heterogeneous group of inherited retinal diseases, characterised by the progressive degeneration of photoreceptors, leading to vision loss and blindness. RP affects approximately 1 in 3000–4000 individuals worldwide.¹ The disease typically begins with night blindness, followed by narrowing of the peripheral visual field and eventual central vision loss.² Most patients with RP experience a progressive loss of rod photoreceptors, with cone cells affected later, resulting in severe visual impairment.²

Gene therapy has emerged as one of the most promising approaches for the treatment of RP. Adeno-associated virus (AAV) vectors have proven to be highly effective in delivering therapeutic genes to retinal cells with a favourable safety profile.³ Given the numerous challenges posed by the development and approval of these innovative therapies, the only available gene therapy is voretigene neparvovec (Luxturna), which has shown success in improving vision of patients with Leber’s congenital amaurosis (LCA) and RP associated with pathogenic biallelic mutations in RPE65 from all over the world.^4–6 Numerous other gene therapies have been delivered safely in human clinical trials for a wide range of inherited retinal diseases: gene therapy holds the potential to slow or halt the progression of visual impairment in patients, offering hope for a cure in the future.⁷

Mutations in the PDE6β gene, which encodes the β-subunit of rod-specific phosphodiesterase, contribute significantly to autosomal recessive RP. Pathogenic bi-allelic mutations in PDE6β impair phototransduction, leading to the dysfunction and degeneration of rod cells, which are essential for vision in low-light conditions.⁸ Approximately 2% of RP cases are caused by PDE6β mutations.⁹ Petit et al have shown that AAV-based gene therapy can be effective in large animal models, confirming the potential of this therapeutic approach in human clinical trials.¹⁰ Building on the preclinical results from studies in animals^10–15 and on our previous gene therapy clinical trial in patients with RPE65 LCA,¹⁶ the HORA-PDE6β-001 clinical trial was designed to evaluate the safety, immune response and preliminary efficacy of HORA-PDE6β, an AAV-based gene therapy expressing human PDE6β under a photoreceptor-specific promoter (online supplemental figure 1). The study hypothesises that restoring PDE6β function in the retina will slow or halt the progression of visual impairment in patients with PDE6β-associated RP, providing a potential therapeutic solution for this devastating condition.

The aim of the present paper is to present the study protocol and its amendments made since the trial began, in order to optimise the study design, as requested by the sponsor, regulatory authorities or investigators—an approach, that is, characteristic of first-in-human trials.

Methods and analysis

Trial design

This single centre, first-in-human, open-label, dose-ranging study comprises five consecutive cohorts, including adult patients aged ≥18 years in cohorts #1, #2 (divided into two subcohorts), and #3, and adolescents and young adults aged 13–25 years in cohort #4.

The first patient was enrolled on 6 November 2017. The study is ongoing and recruitment is still open, with completion expected by the end of 2029. The current protocol corresponds to version 17, incorporating amendments implemented by the sponsor, EyeDNA Therapeutics, a wholly owned subsidiary of Coave Therapeutics.

Study objectives

The primary objective of this study is to evaluate the safety of a unilateral subretinal administration of HORA-PDE6β, a novel gene therapy targeting PDE6β-RP. Secondary objectives include assessing the immune response elicited by the recombinant AAV vector, monitoring the potential shedding of the viral vector into bodily fluids, and identifying clinical outcome measures sensitive to change over a 5-year period. The overarching goal is to identify the optimal dose for future clinical investigations and clinical efficacy endpoints that should be used in a future phase III clinical trial.

Planned outcomes

Primary endpoints (safety)

• Comprehensive ophthalmic examination, including assessments of eyelids, muscle movement, cornea, pupils, lens, intraocular pressure and biomicroscopy of the anterior chamber and fundus.

• Assessment of intraocular inflammation using biomicroscopy, laser flare metre and fundus biomicroscopy.

• Evaluation of chorioretinal tolerance using fundus photography, fluorescein and indocyanine green angiographies, and Optical Coherence Tomography (OCT).

• Patient tolerance assessed by a dedicated questionnaire.

• Recording of vital signs.

• Laboratory assessments, including haematology and chemistry.

• Physical examination.

Secondary endpoints (functional tests)

• Quality of life self-evaluation in adult participants.

• Distance and near visual acuity (near visual acuity not assessed for cohort #4).

• Colour vision, visual field testing and full-field ERG (bicolour visual field and multifocal ERG not applicable for cohort #4).

• Pupillometry (not performed in cohort #3), microperimetry, full-field sensitivity threshold (FST) testing (cohort #3 only), mobility testing, dark adaptation testing performed with a vision monitor (Monpack3, Metrovision, Perenchies, France). This examination started with a light adaptation phase lasting approximately 5 min, conducted under Ganzfeld conditions with bright white light illumination (630 cd/m²). This was followed by a period of complete darkness.

During the dark adaptation phase, a light stimulus was presented within a dome at a frequency of one stimulus every 3 s, at four predefined retinal locations corresponding to 10° superior, inferior, temporal and nasal. Stimulus luminance ranged from 318 cd/m² (0 dB) to 0.000003 cd/m² (80 dB). Patients were instructed to press a response button whenever the stimulus was perceived. The dark adaptation phase lasted 40 min, and testing was performed monocularly. Functional MRI is not performed in cohort #4.

Secondary endpoints (morphological and immunological assessments)

• Fundus autofluorescence imaging and OCT.

• Immunological evaluations, including evaluation of humoral and cellular responses to rAAV2/5 and PDE6β, and characterisation of anti-AAV2/5 and anti-PDE6β responses.

Description of the target population and sample selection

Given the stringency of selection criteria and the rarity of PDE6β-associated RP, patients are recruited from across Europe. To facilitate participation, travel expenses, including hotel accommodation, are covered by the study sponsor. In addition, all study documents, including the patient information sheet and informed consent forms, are translated into the patient’s native language to ensure full comprehension and compliance with ethical standards. Eligible patients had genetically confirmed PDE6β-associated RP and met cohort-specific criteria for visual acuity and visual field preservation, together with predefined ophthalmological, systemic and safety-related non-inclusion criteria, as detailed in online supplemental table 1.

Study setting and cohort design

The cohort design is illustrated in figures 1 and 2 and the scaling doses of the treatment across the cohorts are presented in table 1.

Figure 1. Study design for cohort #1 to #3. This figure shows the sequential patient enrolment and dose-escalation strategy for cohorts #1, #2a, #2b and #3. Patients were treated one at a time within each cohort, with transition to the next patient after validation by the DSMC, which analysed the safety data. Arrows indicate DSMC decision points and red markers highlight transitions requiring formal DSMC approval. DSMC, Data and Safety Monitoring Committee.

Figure 2. Study design for cohort #4. This figure summarises the overall progression of the study from adult dose-escalation cohorts (cohorts #1–#3; 15 patients enrolled) to the high-dose paediatric cohort (cohort #4; 4–6 patients). For cohort #4, enrolment followed a stepwise, age-adapted safety strategy: the first two patients were adults (≥18 years), and further enrolment required DSMC review of safety data after completion of post-treatment visits. DSMC recommendations were submitted to the French competent authority (ANSM) to authorise enrolment of paediatric patients (13–17 years). DSMC review timing was adapted according to age (day 7 for adults; day 30 for paediatric patients). Across all cohorts, DSMC safety reviews were conducted after interim analyses, at study completion and ad hoc in case of SAE or SUSAR. ANSM, French competent authority; DSMC, Data and Safety Monitoring Committee; SAE, serious adverse event; SUSAR, suspected unexpected serious adverse reaction.

Table 1. Information related to the proposed concentrations of IMP in each cohort.

Cohort	IMP concentration (vg/mL)	IMP concentration ratio vs the lower IMP concentration	Ratio between IMP concentration vs effective concentration in dogs	Safety margin folds vs NOAEL	Intended volume (µL)	Total dose (vg/eye)
1	4.2×1011	1	0.5	16	800	3.4×1011
2a	8.5×1011	2	1	8	400	3.4×1011
2b	1.6×1012	4	2	4	400	6.4×1011
3	1.6×1012	4	2	4	400	6.4×1011
4	1.6×1012	4	2	4	400	6.4×1011

NOAEL: 6.7×1012 vg/mL (corresponding to a dose of 1×1012 vg).

IMP, investigational medicinal product; NOAEL, No-Observed Adverse Effect Level.

The cohorts were designed to evaluate two escalating dose levels, initially in adult patients with more severe disease, followed by those with less severe disease and finally in paediatric patients, in whom the well-tolerated higher dose could be administered. Interim tolerance analyses allow refinement of the protocol if necessary.

The number of patients was limited to three in the initial cohorts, which primarily assessed tolerance, in line with similar phase I/II gene therapy clinical trials.^{17 18} The number of patients was increased to six in cohorts #3 and #4 to enhance the likelihood of identifying clinical outcome measures sensitive to change and detecting preliminary signs of efficacy in patients with less severe disease.

In practice, two protocol deviations occurred: in cohort #1, one patient did not receive the intended injection volume and in cohort #2b, one patient received an incorrect dose. In both cases, an additional patient was enrolled to preserve cohort evaluability. Consequently, figures 1 and 2 should be interpreted as reflecting the planned allocation scheme rather than the exact number of patients treated.

These five sequential cohorts, which included patients with genetically confirmed PDE6β-associated retinal degeneration, are as follows:

• Cohort #1: Three adult patients (≥18 years) with a best-corrected visual acuity (BCVA) score of ≤60 Early Treatment Diabetic Retinopathy Study (ETDRS) letters (equivalent to a decimal visual acuity ≤0.32 or 20/63 Snellen) received a subretinal injection of 800 µL containing 4.2×10¹¹ vector genomes (vg)/mL (total dose 3.4×10¹¹ vg) in the worse affected eye.

• Cohort #2 (split into two subcohorts to allow a concentration and dose escalation, added in Amendment 4—Protocol version 8 effective on 30 November 2018):

  • Cohort #2a: Three adult patients (≥18 years) with a BCVA score of ≤75 ETDRS letters (equivalent to a decimal visual acuity ≤0.63 or 20/32 Snellen) received 400 µL containing 8.5×10¹² vg/mL (3.4×10¹¹ vg) in the worse affected eye.

  • Cohort #2b: Three adult patients (≥18 years) with a BCVA score of ≤75 ETDRS letters (equivalent to a decimal visual acuity ≤0.63 or 20/32 Snellen) received 400 µL containing 1.6×10¹² vg/mL (6.4×10¹¹ vg) in the worse affected eye.

• Cohort #3: Six adult patients (≥18 years) with a BCVA score of ≤75 ETDRS letters (equivalent to a decimal visual acuity ≤0.63 or 20/32 Snellen) and preserved central visual field (VF) of at least 10° in all meridians (V4 stimulus) received 400 µL containing 1.6×10¹² vg/mL (6.4×10¹¹ vg) in the worse affected eye.

• Cohort #4: Four to six patients aged 13–25 years with a preserved central visual field of at least 20° in all meridians (V4 stimulus) will receive 400 µL containing 1.6×10¹² vg/mL (6.4×10¹¹ vg). This cohort will be conducted under the regulatory paediatric development framework because it will include adolescent patients aged 13–17 years, although young adult patients aged 18–25 years will also be eligible. This cohort was introduced in Amendment 9 (Protocol Version 12, effective 18 January 2023). According to Amendment 10 (Protocol Version 13, effective 15 March 2023), treatment initiation within cohort #4 was required to start with young adult patients prior to inclusion of adolescent patients.

Study schedule

During the screening phase (120 and 7 days before the surgery), patients undergo a comprehensive baseline assessment. On the day of surgery (D0), the intervention will be performed under general anaesthesia by experienced senior surgeons as follows: standard three-port 25 Gauge vitrectomy, posterior hyaloid staining with triamcinolone, induction of posterior vitreous detachment, peripheral vitrectomy, subretinal injection of the investigational medicinal product, HORA-PDE6β with a 41 Gauge needle, subconjunctival steroidal anti-inflammatory treatment and closure of the sclerotomies.

Following surgery, patients undergo close monitoring during early postoperative visits (days 1, 3 and 7) to assess safety and ocular tolerability. Long-term follow-up continues through day 180 and then annually for 5 years, focusing on safety, efficacy (visual function assessments), immune responses to the AAV vector and vector shedding.

All medications and treatments are permitted, except those specified in the online supplemental table 2. In cases of intraocular inflammation, treatment is initiated promptly and tailored to the type and severity of the inflammation. Ideally, patients receive a topical mydriatic 1 hour before surgery; alternatively, they are premedicated with Mydriaticum 2 mg eye drops administered at 70, 60 and 50 min before surgery, together with Neosynephrine 10% administered at 70 and 50 min before surgery.

The study flow chart is presented in the online supplemental table 3. The study design detailing enrolment and intervention is shown in figure 3, in accordance with Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) recommendations. The SPIRIT checklist, which provides guidance on the minimum content of a clinical trial protocol,¹⁹ has also been uploaded as online supplemental file 1.

Figure 3. Study design. This figure illustrates the study visit schedule from screening and hospitalisation to long-term follow-up. Visits include screening and inclusion (V1), presurgical check-up (V2), surgery and HORA-PDE6β administration (V3), early postoperative follow-up (V4), and subsequent follow-up visits from day 3 to year 4 (V5–V13), with a final visit at year 5 (V14). Study objectives are assessed at 1 year and throughout the 4-year long-term follow-up period. The period of exposure to the investigational medicinal product is indicated. IMP, investigational medicinal product.

Measurement: description of the score used

Aqueous flare is quantified by laser flare photometry (Kowa FM-700, Tokyo, Japan), providing objective photon/ms measurements of blood–aqueous barrier disruption. Measurements are performed in a dark room on non-dilated eyes. Anterior chamber inflammation is evaluated using slit-lamp biomicroscopy (Haag-Streit, Switzerland) in accordance with the Standardisation of Uveitis Nomenclature (SUN) Working Group criteria—grading cells in a 1×1 mm beam from grade 0 (no cells) to grade 4+ (>50 cells).²⁰ Vitreous inflammation is assessed by slit-lamp biomicroscopy (Haag-Streit) using the Nussenblatt classification,²¹

Chorioretinal tolerance is evaluated using multimodal imaging. Colour fundus photography is performed using a non-mydriatic TOPCON TRC-NW6S (7-field) and a widefield OPTOS camera following pupil dilation with tropicamide (CibaVision, Novartis, France). For cohorts #3 and #4, four-field ultra-widefield (200°) imaging is acquired using the Clarus 500 (ZEISS) without dilation. Fluorescein and indocyanine green angiography are performed using an HRA system (Heidelberg Engineering, Germany) (7 field images with a 55° lens).Retinal thickness, subretinal or intraretinal fluid, and structural disorganisation are assessed using spectral-domain OCT (SD-OCT; Heidelberg Engineering, Germany) in high-resolution 40 kHz mode, including macular, subretinal bleb, and retinal nerve fibre layer scans. OCT images are analysed locally by trained ophthalmologists and, when required, centrally for standardised, anonymised grading.

Visual function assessment includes distance BCVA measured using ETDRS charts at 4 m under photopic lighting (Precision Vision, USA). When participants are unable to read letters, low-vision categories are recorded as Counting Fingers (CF), Hand Motion (HM), Light Perception (LP) or No Light Perception (NLP). Near vision and reading speed are assessed using MNREAD charts (Minnesota Laboratory).²² Colour vision is evaluated using Ishihara pseudoisochromatic plates under daylight-equivalent illumination²³ and the Farnsworth 15-Hue test²⁴). Visual field testing includes static perimetry using the Humphrey Field Analyzer (Zeiss), manual kinetic perimetry using the Goldmann perimeter (Haag-Streit) with automatic quantitative analysis of V4 isopters and, when feasible, bicolour perimeter performed using the MonCVONE system (Metrovision, Perenchies, France). Bicolour visual field testing was performed in cohorts #1, #2a, #2b and #3, but not in cohort #4. After a 45-minute dark adaptation period under scotopic conditions, retinal sensitivity was assessed during two successive test sessions. The first session explored the extended central retina using 200 ms size V stimuli at a 647 nm (red) wavelength across 65 test points. The second session assessed central and peripheral retinal sensitivity using 200 ms size V stimuli at a 500 nm (blue) wavelength across 121 test points. Tests were conducted monocularly and patients were instructed to press a response button whenever the stimulus was perceived. In addition to documenting constrictions to 20° and 10°, peripheral islands are systematically recorded when present, as they significantly contribute to residual functional vision and mobility performance.²⁵

Electrophysiological function is assessed in accordance with International Society for Clinical Electrophysiology of Vision (ISCEV) standards.²⁶ Full-field and multifocal electroretinography (ERG) are recorded using the Diagnosys Espion system. FST testing is performed in patients with severe visual impairment. Microperimetry (OPKO Health, Miami, USA) is used to assess fixation stability and retinal sensitivity mapping.²⁷ Pupillometry is performed using Vision Monitor MonCyOne (Metrovision, Perenchies, France). A standardised indoor mobility test under controlled luminance conditions captures time to completion and navigation errors as real-world functional vision outcomes.²⁸

Systemic safety monitoring includes vital signs, physical examination, 12-lead ECG and comprehensive laboratory testing (haematology, biochemistry and serology), as well as pregnancy testing when applicable. Immunomonitoring encompasses both humoral and cellular immune responses to PDE6β and AAV2/5. Anti-PDE6β and anti-AAV2/5 antibody titres are quantified using ELISA, neutralising antibody activity is assessed by in vitro neutralisation assays and T-cell reactivity is evaluated by IFN-γ ELISpot. HLA typing is performed to explore potential genetic predisposition to immune responses. Samples are collected at baseline (pretreatment), on days 1, 3 and 7, at weeks 2 and 4 and subsequently at months 2, 3, 6, 12 and annually thereafter, in order to capture both acute and persistent responses.

Vector safety is further addressed through biodistribution and shedding analyses, with qPCR-based detection of vector genomes in blood, urine and nasal secretions at the same predefined post-administration time points. Patient-reported outcomes are collected using the National Eye Institute Visual Function Questionnaire NEI VFQ-25 questionnaire for adults²⁹ and a dedicated ocular tolerance questionnaire for all participants.

All domains, methods and grading schemes are summarised in online supplemental table 3.

Following analyses conducted at the end of cohorts #2 and #3, assessments that were excessively time-consuming or of limited added value were removed (see online supplemental table 5). In addition, FST testing was introduced during the course of the protocol as it was not available at study initiation and demonstrated statistically significant outcomes in the Voretigene Neparvovec (Luxturna) clinical study.³⁰

Sample size

The trial aims to recruit between 19 and 21 patients across all cohorts, based on data from similar completed or ongoing gene therapy studies in RP. Patients who receive less than 80% of the intended dose will be replaced to ensure data integrity. Although the sample size is limited, it is appropriate for an early-phase trial focused on assessing safety and exploring potential signals of efficacy. For cohort #4, efforts will be made to include at least three patients aged 13–17 years among the four to six patients planned for this cohort.

Data analysis

The full analysis set (FAS) includes all patients who received treatment. The per-protocol set comprises patients without major protocol deviations that could bias the estimation of treatment effects; all deviations and reasons for exclusion from this set will be documented. The Safety Set includes all patients from the FAS who received treatment, regardless of protocol deviations, and safety analyses will focus on this group.

Efficacy analyses will be performed in evaluable patients, as defined in the statistical analysis plan, and in the per-protocol set. For each functional outcome, changes over time will be assessed to identify clinical outcome measures sensitive to change, and comparisons will then be made between injected and non-injected eyes to detect any potential treatment effect.

The study includes a series of planned interim and final analyses to monitor safety and efficacy outcomes over time. The first interim analysis will be conducted after all patients in cohorts #1 to #3 have completed 1 year of follow-up, followed by a second interim analysis after 2 years. A third and fourth interim analysis will be performed after 1 and 2 years of follow-up, respectively, for all patients in cohort #4. The final analysis is scheduled once all patients in cohort #4 have completed 5 years of follow-up.

Data management

A case report form (CRF) is created for each patient to ensure protocol compliance, support statistical analysis and identify major deviations. Data are collected by the investigator or a clinical research associate and recorded in coded form to maintain patient confidentiality.

Adverse event management

As this study represents a first-in-human administration, adverse events (AEs) and serious AEs (SAEs) will be closely monitored, under the oversight of an independent Data Safety Monitoring Committee (DSMC). The DSMC is composed of five members to avoid voting deadlocks, including two ophthalmologists, one paediatric neurologist, one pharmacologist and one biostatistician.

The DSMC will review protocols, clinical materials and safety data on HORA-PDE6β, providing recommendations on whether to continue, stop or amend the trial (see figures 2 and 3). For cohort #4, the first paediatric patient will be screened following authorisation from the French Competent Authorities (ANSM) and completion of a 7-day safety review of the first two adult patients. Adapted rules for this paediatric population were provided to the DSMC (Amendment 9 Protocol Version 12, effective 18 January 2023).

The DSMC will review the safety data of each patient aged ≤17 years before authorising subsequent treatments and will recommend suspension of recruitment in the event of newly occurring suspected unexpected serious adverse reactions (SUSARs). The safety observation period is defined as 7 days for patients in cohorts 3 and 4 and 30 days for patients aged under 18 years.

Of note, a predefined list of Adverse Events of Special Interest (AESIs), encompassing medical or scientific significant events requiring close monitoring and rapid reporting to the Sponsor, was established. Ocular AESIs include persistent inflammation, major retinal changes, significant retinal, choroidal or optic nerve damage, loss of visual acuity and endophthalmitis. Other AESIs include hypersensitivity to the investigational medicinal product (IMP) occurring within 24 hours, opportunistic infections within 30 days, new-onset or worsening immunological or haematological disorders, hepatic events (eg, elevations in liver enzymes suggestive of potential drug-induced liver injury), neutropenia, thrombocytopenia and elevated creatinine levels.

Protocol amendments

During the course of this first-in-human trial, protocol amendments were implemented to address safety-related events and to refine study procedures as clinical experience accumulated. These amendments are presented below according to their primary rationale, from the most critical safety-driven changes to subsequent methodological adaptations. Detailed amendment history is provided in online supplemental table 5. Certain protocol adaptations affecting cohort structure are described in detail in the cohort description section and are therefore not repeated here (Amendment 4, Protocol Version 8, effective 30 November 2018). Amendments affecting cohort structure and sequencing, including the introduction of the paediatric cohort and the adult-to-paediatric treatment sequence, are described in the cohort design section and are therefore not repeated here (Amendments 9 and 10, Protocol Versions 12 and 13).

The earliest safety-driven amendment followed the occurrence of a SUSAR, in which a patient developed chorioretinal inflammation 2 months after receiving HORA-PDE6β. In response, and in agreement with the French competent authority (ANSM), the Sponsor required an extension of the interval between the second and third patients within cohorts in order to reinforce safety monitoring (Revision 3, Protocol Version 6, effective 12 February 2018). This amendment affected patient sequencing but did not modify the surgical procedure or the study flowchart.

Subsequent safety-driven amendments directly impacted the surgical procedure and study flowchart. In cohort #1, a subretinal injection volume of 800 µL was initially administered through multiple retinotomies to achieve broad retinal coverage. Following the occurrence of two cases of retinal detachment, the surgical procedure was modified for subsequent cohorts (Amendment 5, Protocol Version 8, effective 30 November 2018). These modifications included a reduction of the injected volume to 400 µL with a corresponding increase in vector concentration to maintain the planned total dose, minimisation of the number of retinotomies, performance of a fluid–air exchange with air tamponade and maintenance of patients in a supine position for 24 hours postoperatively. These changes resulted in an updated surgical workflow and corresponding modifications to the study flowchart.

Following the observation of a chorioretinal inflammatory deposit in a patient from cohort #3, which fully resolved after corticosteroid treatment, a safety-related protocol amendment was implemented.

For cohorts #1, #2a, #2b and #3, postsurgical anti-inflammatory treatment consisted of a subconjunctival corticosteroid injection (dexamethasone 4 mg/mL) administered at the end of surgery, combined with topical steroidal anti-inflammatory eye drops in the study eye for a duration of 5 months.

For patients enrolled in cohort #4, the same local postsurgical regimen was applied (subconjunctival dexamethasone 4 mg/mL and topical steroidal eye drops for 5 months), with the addition of a systematic perioperative oral corticosteroid therapy. Oral prednisone was administered at a dose of 1 mg/kg/day for 3 days prior to surgery and for 7 days postoperatively (maximum daily dose of 40 mg), followed by a tapering regimen over 5 weeks: 0.5 mg/kg/day for 7 days (maximum 20 mg/day), then 0.25 mg/kg/day for 14 days, and finally 0.125 mg/kg/day for 14 days. This modification was incorporated into the study flowchart and formally approved as Amendment 9 (Protocol Version 12, effective 18 January 2023).

Methodological amendments were subsequently implemented to improve feasibility and clinical relevance of study assessments. Following analyses conducted at the end of cohorts #2 and #3, multifocal ERG was discontinued due to its limited clinical precision and the technical challenges associated with its use in paediatric subjects. MRI was also excluded because of its limited relevance and procedural burden in children.

Additional refinements to eligibility criteria were introduced to better reflect disease severity and progression in advanced cases. In particular, replacement of the visual acuity-based criterion with a visual field-based criterion (≥10 or ≥20 central degrees, depending on the patient) was implemented to improve patient characterisation (Amendment 11, Protocol Version 15, effective 19 July 2023).

Collectively, these amendments illustrate the adaptive nature of the protocol, with safety considerations prioritised throughout and methodological refinements introduced to optimise feasibility and data quality, while preserving the primary objective of safety evaluation in this early-phase gene therapy trial.

Dissemination

Confidentiality is strictly maintained, with patient data anonymised and stored securely in compliance with the General Data Protection Regulation. The study findings will be disseminated through peer-reviewed publications and presentations at scientific conferences, ensuring access for the broader scientific and medical communities while fully protecting participant confidentiality.

Discussion

This study has several notable strengths, including its pioneering approach to gene therapy for PDE6β-associated RP, a condition for which no approved treatment currently exists. The trial integrates long-term assessments of safety, immune response and visual function, together with a DSMC-approved dose-escalation strategy aimed at identifying an optimal dose and therapeutic window.

The adaptive nature of this study protocol can be considered as both a strength and a limitation. On one hand, successive adjustments in response to regulatory and ethical authorities as well as to the practical realities of the trial and patient needs have strengthened scientific rigour, feasibility and alignment with the primary objective of safety. On the other hand, this same flexibility represents a limitation for the analysis of secondary efficacy endpoints, as protocol modifications may introduce heterogeneity across cohorts and over time. The protocol is currently at version 17, and all successive modifications are detailed in online supplemental table 5. Versions 1–4 corresponded to preparatory exchanges with regulatory authorities, with Version 4 being the first effective protocol version (see online supplemental file–protocol version submitted to regulatory authorities). Since then, most amendments have been minor (eg, revised inclusion criteria, changes in sponsor information, changes in co-investigators, interim analyses and adjustments to study duration). However, several major modifications were implemented, including changes to the surgical procedure following cases of retinal detachment (as described above), the addition of cohort #4 involving paediatric patients and the introduction of perioperative corticosteroid therapy for patients in cohort #4.

The study also has limitations, notably its conduct at a single centre in France, which may limit generalisability, and its small sample size, which restricts statistical power. Although the 5-year follow-up period is essential for evaluating long-term safety and durability of effect, it may pose challenges with respect to participant retention and adherence.

Conclusions

The HORA-PDE6β study represents an important step in the development of gene therapy for inherited retinal dystrophies. By focusing on safety, immune response, and functional outcomes, this trial provides a structured framework to inform future therapeutic approaches targeting PDE6β-associated RP. In particular, it aims to identify clinically relevant endpoints that may be considered in subsequent phase III randomised clinical trials designed to evaluate treatment efficacy.

As a first-in-human trial, HORA-PDE6β-001 was designed with particular emphasis on patient safety and scientific rigour from the outset. Nevertheless, as the trial progressed, emerging clinical experience and patient responses necessitated iterative refinements to the protocol. Since its initial submission to the French regulatory authority (ANSM) and the Ethics Committee (CPP), the protocol has undergone several amendments and formal revisions, resulting in the current version 17. These adaptations reflect the dynamic and collaborative nature of early-phase innovative clinical research, incorporating continuous input from the sponsor, investigators and regulatory authorities, and highlight the importance of an adaptive framework in the conduct of first-in-human gene therapy trials.

Data availability statement

Data are available on reasonable request.