Purpose
To report on the safety of the first 5 cohorts of a gene therapy trial using recombinant equine infectious anemia virus expressing ABCA4 (EIAV-ABCA4) in adults with Stargardt dystrophy due to mutations in ABCA4.
Design
Nonrandomized multicenter phase I/IIa clinical trial.
Methods
Patients received a subretinal injection of EIAVABCA4 in the worse-seeing eye at 3 dose levels and were followed for 3 years after treatment.
Main outcome measures
The primary end point was ocular and systemic adverse events. The secondary end points were best-corrected visual acuity, static perimetry, kinetic perimetry, total field hill of vision, full field electroretinogram, multifocal ERG, color fundus photography, short-wavelength fundus autofluorescence, and spectral domain optical coherence tomography.
Results
The subretinal injections were well tolerated by all 22 patients across 3 dose levels. There was 1 case of a treatment-related ophthalmic serious adverse event in the form of chronic ocular hypertension. The most common adverse events were associated with the surgical procedure. In 1 patient treated with the highest dose, there was a significant decline in the number of macular flecks as compared with the untreated eye. However, in 6 patients, hypoautofluorescent changes were worse in the treated eye than in the untreated eye. Of these, 1 patient had retinal pigment epithelium atrophy that was characteristic of tissue damage likely associated with bleb induction. No patients had any clinically significant changes in best-corrected visual acuity, static perimetry, kinetic perimetry, total field hill of vision, full field electroretinogram, or multifocal ERG attributable to the treatment.
Conclusions
Subretinal treatment with EIAV-ABCA4 was well tolerated with only 1 case of ocular hypertension. No clinically significant changes in visual function tests were found to be attributable to the treatment. However, 27% of treated eyes showed exacerbation of retinal pigment epithelium atrophy on fundus autofluorescence. There was a significant reduction in macular flecks in 1 treated eye from the highest dose cohort. Additional follow-up and continued investigation in more patients will be required to fully characterize the safety and efficacy of EIAV-ABCA4.
ABCA4-related retinopathy (OMIM # 248200 ) results from biallelic mutations in ABCA4 and causes a spectrum of retinal phenotypes including fundus flavimaculatus, juvenile onset macular dystrophy, cone-rod dystrophy with or without foveal sparing, and, in the most severe cases, generalized rod-cone dystrophy. , The juvenile form of the disease and its variants are often referred to collectively as Stargardt disease (SD). ABCA4 -related retinopathy is an autosomal recessive disease, although cases of pseudodominance have been reported, and in addition, several genes can cause a dominant Stargardt-like phenotype. , Although a rare disease, SD is one of the most common inherited retinal dystrophies, affecting approximately 1 in 8000 to 10,000 persons worldwide.
The disease affects both sexes and presents with a trimodal distribution of onset peaking at 7, 23, and 55 years of age. , Juvenile cases present with rapid, progressive loss of central visual acuity, but the rate of disease progression and clinical severity are related to the age of disease onset and specific mutations. Consequent to the central vision loss, most participants also have impaired color vision and may experience photophobia. , In some patients with SD, a delay in dark adaptation may also be detected. , Ophthalmic examination in patients with SD typically reveals atrophic macular lesions with or without the presence of pisciform yellow flecks at the level of the retinal pigment epithelium (RPE). In histopathologic examinations of the eyes of patients with SD, the fleck lesions have been correlated with RPE cells densely packed with lipofuscin. Classically, fluorescein angiography, if performed, demonstrates a “dark choroid,” as where choroidal fluorescence is blocked by the accumulation of lipofuscin. In most cases, peripheral vision is unaffected but can be impaired in the later stages of the disease.
The ABCA4 gene contains 50 exons and encodes ATP-binding cassette, subfamily A (ABC1), member 4, an approximately 250 kDa single-chain glycoprotein, which is localized on the disk margins of the vertebrate photoreceptor (PR) outer segments, and belongs to the ABC transporter superfamily. It has been proposed that ABCA4 participates in the clearance of all- trans -retinal and its Schiff-base conjugate, N-retinylidene phosphatidylethanolamine, intermediates in the formation of A2E from the PRs. Although many published biochemical and animal model studies suggest that the natural substrate of ABCA4 protein is N-retinylidene phosphatidylethanolamine, which spontaneously forms as an all- trans -retinol adduct after the process of light absorption by rhodopsin, the exact mechanism of ABCA4 function is not fully understood. Reported mutations range from single missense changes, which are by far the most common, to more complex gene rearrangements. In terms of ABCA4 protein function, the effects of these mutations range from a total loss of function due to the absence of the protein to production of protein with functionally impaired transporter activity.
There are currently no approved therapies for SD. The characterization of the molecular basis of SD has opened the door to a therapeutic approach based on gene augmentation. The eye is ideally suited as a target organ for this type of therapy, as it is anatomically separated from the rest of the body, is easy to access, and allows noninvasive diagnostic follow-up of the therapeutic effects in vivo. Moreover, the eye is immune privileged, which is an active process of immune deviation in specific anatomical compartments such as the anterior chamber and subretinal space. These properties along with the relatively low vector dosage requirement within the small compartmentalized space of the subretinal bleb are advantages for limiting immune reactions to gene therapy.
The viral vector used in this investigational product equine infectious anemia virus expressing ABCA4 (EIAV-ABCA4) (SAR422459) is a nonreplicating, integrating, recombinant lentiviral vector based on the EIAV, which is nonpathogenic to humans and has been developed as a vector for therapeutic gene delivery. Subretinal delivery of the investigational product aims to introduce a copy of the normal coding sequence of the human ABCA4 cDNA into the host genome using the human cytomegalovirus promoter to drive expression of normal ABCA4 protein. The EIAV lentiviral vector was pseudotyped with VSV-G envelope protein to allow efficient transduction of the PRs. Reporter gene studies in mice using EIAV-based vectors have demonstrated that expression of the gene product persists for at least 16 months in the mouse eye and greater than 4 years in humans after a single administration. , After subretinal injection of EIAV-ABCA4, cellular transduction is limited to the RPE cells and PR and to a lesser extent other cells of the inner neural retina.
The preclinical safety evaluation program of the EIAV vector assessed the pharmacologic, pharmacokinetic, and toxicologic profiles in mice, rabbits, and nonhuman primates (NHPs). , , In these studies, EIAV-ABCA4 or homologous lentiviral vectors expressing reporter proteins were given by subretinal administration. Despite prior evidence of low lentiviral transduction efficiency of PRs, both of the preclinical studies for this clinical trial showed that EIAV-ABCA4 had good potential. , Subretinal delivery of EIAV-ABCA4 in neonatal (P4-5) Abca4 −/− mice resulted in expression of the ABCA4 protein from 2 weeks after treatment and maintained until the end of the study at 2 months, which correlated with a reduction in A2E accumulation (the levels at 12 months were similar to wild-type). Thus, this study demonstrated that EIAV-ABCA4 was capable of transducing PR neonatal progenitor cells in a meaningful and sustainable manner. Furthermore, reporter gene expression using EIAV-GFP was detected in NHP PRs and RPE; however, this was performed at 1-2 log units higher concentration and absolute dose than the EIAV-ABCA4 dose used in the preclinical studies. The maximum concentration and dose levels for EIAV-ABCA4 in this clinical trial were comparable and established empirically from EIAV-ABCA4 used in NHPs on the basis of the scale of the eye and the historical experience of introducing suspensions into the eye. No agent-related toxicity was noted in any of these preclinical studies.
The current study reports available 3-year results of cohorts 1-5 of an open label phase I/IIa dose escalation safety study of subretinally injected EIAV-ABCA in participants with SD ( www.clinicaltrials.gov , NCT01367444). Data from additional confirmatory cohorts 6-7 are not included in this paper due to the incomplete follow-up period and will be the subject of a subsequent publication.
Materials and methods
Twenty-two participants were recruited from 2 sites (Oregon Health & Science University, Casey Eye Institute, Portland, Oregon, USA, and Centre Hospitalier National d’Ophtalmologie des Quinze-Vingts, Paris, France).The trial conformed to the Declaration of Helsinki for research involving human participants and was approved by the Oregon Health & Science University (OHSU) institutional review board, the Comité de Protection des Personnes Paris Ile de France V, and Regulatory Agencies (FDA and ANSM/HCB). Informed written consent was obtained from all of the participants in the study before the conduct of any study procedures. The summary of the enrolled participants, genetic mutation, and pretreatment mean best-corrected visual acuity (BCVA) is shown in Supplemental Table 1.
Study design
In the multicenter study TDU13583 (ClinicalTrial.gov identifier: NCT01367444), 3 doses of EIAV-ABCA4 were evaluated over 7 cohorts, followed for 48 weeks, and then enrolled into the long-term study, LTS13588 (NCT01736592), which follows participants for 15 years. A minimum follow-up period of 3 years was required for inclusion in this interim report, which comprises patients from the first 5 cohorts. Cohorts 1-4 represent the dose escalation phase of the study and consisted of 4 participants each. Cohort 5 consisted of 6 participants, who were treated with the maximum tolerated dose. The main inclusion criteria for all participants enrolled in this study were age equal to or greater than 18 and moderate-to-severe SD with biallelic pathogenic mutations in ABCA4 confirmed by direct sequencing and cosegregation analysis. Additional inclusion criteria for cohorts 1-5 were classified according to Lois and associates ( Table 1 ):
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Group A: BCVA ≤ 20/200 in the worst eye and severe cone-rod dysfunction with no detectable or severely abnormal full field electroretinography (ERG) responses.
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Group B: BCVA ≤ 20/200 in the worst eye with abnormal full field ERG responses.
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Group C: BCVA ≤ 20/100 in the worst eye with abnormal full field ERG responses.
| Cohort | Group | Number of Patients | Dose Level | Dose by Target Strength (TU/eye) | Volume (µL) |
|---|---|---|---|---|---|
| 1 | A | 4 | 1:10 | 1.8 × 10 5 | 300 |
| 2 | B | 4 | 1:10 | 1.8 × 10 5 | 300 |
| 3 | B | 4 | 1:3 | 6 × 10 5 | 300 |
| 4 | B | 4 | Undiluted | 1.8 × 10 6 | 300 |
| 5 | C | 6 | Undiluted | 1.8 × 10 6 | 300 |
Key exclusion criteria were media haze, aphakia or prior vitrectomy, other diseases affecting visual function (eg, glaucoma, optic neuropathy, active uveitis, retinopathy, and maculopathy other than ABCA4 -related retinopathy), myopia greater than 8 diopters spherical equivalent, history of ocular surgery within 6 months, and concomitant systemic disease in which the disease itself, or the treatment for the disease, could alter ocular function. Data Safety Monitoring Board evaluation occurred between cohorts.
Vector and investigational medicinal product
Drug SAR422459 (EIAV-ABCA) is a lentiviral vector system based on the EIAV that was developed and produced for this study by Oxford Biomedica. SAR422459 is a novel, self-inactivating EIAV vector (EIAV-ABCA4) that encodes the ABCA4 gene, driven by the cytomegalovirus constitutive promoter. It is pseudotyped with the vesicular stomatitis virus G protein.
Surgical procedure and postoperative treatment
One study eye was selected. If both eyes were eligible for the study, the worse-seeing eye, as per investigator’s judgment, was selected to be the study eye. Surgery was performed under general anesthesia or local anesthesia with intravenous sedation according to participant, surgeon, and anesthesiologist preferences. A standard 3-port vitrectomy was first performed, and EIAV-ABCA was administered as a subretinal injection of approximately 300 µL through a hydraulically created retinotomy. EIAV-ABCA was prepared in sterile conditions with or without dilution as needed for target dose. A retinotomy was recommended to be performed temporal to the optic nerve and anterior to the major superior vascular arcade. Retinal function and structure based on BCVA, perimetry, and imaging were used in the planning of the desired bleb location. Final retinotomy and bleb locations were determined by the surgeon based on operative conditions, and, for some patients, after discussion with the principal investigator at the other site and the reading center. The surgeon was allowed to repeat the retinotomy if subretinal injection did not succeed at the initial site or if the bleb was spreading in an undesired direction. Tamponade was also allowed at the discretion of the surgeon, and air-fluid exchange was performed in 5 cases.
Perioperative regimen
Investigators were permitted to make treatment decisions guided by postoperative findings. A combination of topical, peribulbar, or systemic administration of corticosteroids was suggested but left at the discretion of surgeons and investigators. As such, all patients received topical corticosteroids for 2 to 6 weeks. In addition, on the day of surgery, 4 patients received 10 mg of intravenous dexamethasone, 4 patients were given a 10 mg injection of periocular dexamethasone, and 1 patient received both intravenous and periocular corticosteroids (Supplemental Table 2).
Surgery video‐recording and intraoperative optical coherence tomography
Bleb position was recorded by video or intraoperative optical coherence tomography (OCT) of the surgical procedure for all participants when possible. The approximate location of the bleb was drawn for all the participants using the surgical video or Retcam images. If the surgical videos or Retcam images were not available, approximate bleb location was identified based on surgical notes.
Laboratory parameters and immunological studies
Laboratory testing to assess safety, vector biodistribution, and any immune responses was performed by collecting blood and urine samples before and after treatment. Standard safety hematology and biochemistry tests as well as urinalysis were performed throughout the study and the follow-up period. EIAV-ABCA distribution in the blood (plasma and buffy coat) and urine was assessed by polymerase chain reaction (PCR). Blood was collected and sera were analyzed for EIAV-ABCA-associated antibodies. The presence of antibodies against any putative antigenic component of the EIAV-based vector was tested, including VSV-G2 envelope protein, neomycin phosphotransferase, ABCA4 protein, and p26 protein (EIAV native capsid protein).
Clinical assessments and study end points
Participants were evaluated at screening, baseline, postoperative day 1, and postoperative weeks 1, 2, 4, 12, 24, 36, 48, year 2, and year 3. Safety and efficacy were assessed for BCVA, ophthalmic examination, multimodal imaging, full field perimetry, and ERG.
Visual acuity
BCVA was measured using the Early Treatment of Diabetic Retinopathy (ETDRS) chart on the electronic visual acuity tester at a distance of 3 m and recorded as the number of letters read. The right eye was tested before the left eye. Participants with poor central vision were instructed to fixate eccentrically or turn their head in any manner that improved BCVA. If the participant employed these maneuvers, the technician ensured that the fellow eye remained covered. Participants were also instructed not to lean forward.
Kinetic and static perimetry
Static (SP) and kinetic perimetry (KP) were performed using the Octopus 900 Pro (Haag-Streit International) with EyeSuite software V.2.2.0 and V 2.3.0. SP was performed using the German Adaptive Thresholding Estimation (GATE) algorithm applied to a custom Octopus 900 grid, the “STGD 185pt GATE V,” extending from 56° nasally to 80° temporally, with the stimulus size V. The background illumination was 10 cd/m 2 (31.4 apostilbs). Volumetric estimate of retinal sensitivity using hill of vision (HOV) was calculated using Visual Field Modeling and Analysis software previously reported by Weleber and associates. KP was collected using the V4e, III4e, and I4e test targets. Test vectors were presented to the participants approximately every 15°, at an angular velocity of 4°/s, and originating approximately 10° outside the age-correlated normal isopter. Any scotomas (nonseeing areas within seeing areas) were mapped using an angular velocity of 2°/s, using all 3 targets where possible. There were cases where the blind spot was not possible to map due to large central scotoma/s. The total seeing area is reported in our results and was calculated for each isopter (seeing area minus any defined scotoma/s if any).
Spectral domain optical coherence tomography
Images were obtained of both eyes with the Heidelberg Spectralis Spectral Domain (SD)-OCT system (Heidelberg Engineering). High-speed 97 horizontal line volume scans centered on the fovea were performed at screening and baseline visits within a 2- to 3-week interval. High-resolution horizontal and vertical line scans were acquired at 30° magnification and centered on the fovea. Manual segmentation, based on previously published algorithms, was performed to correct automated segmentation errors for boundaries of the internal limiting membrane and Bruch’s membrane. , For this analysis, the mean central thickness measurements and macular volume were calculated in the macula by using the central 1 mm circular ETDRS grid.
Fundus photography (FP) and autofluorescence (FAF)
In addition to color FP, FAF images were obtained with the Heidelberg Spectralis HRA (Heidelberg Engineering) using the widefield lens to image the retina at 55°, centered and focused on the macular region at a 488 nm excitation wavelength. Some participants had images taken at 30°. FAF images were taken at different settings, which may have resulted in variable image illumination.
Electroretinography
Retinal function was assessed with full field ERG (ffERG) and multifocal ERG. Electroretinographic responses were recorded according to the International Society for Clinical Electrophysiology of Vision standards. ERG was performed at both sites; one used Burian-Allen electrodes (11 participants) and the other used Dawson, Trick, and Litzkow (DTL) electrodes (11 participants). Only data acquired using the Burian-Allen electrodes were used for analysis.
Certification, training, quality control, and centralized data analysis
For a subset of functional and structural assessments, the Casey Reading Center performed certification of both site staff and equipment. Quality control and analysis was performed by the Casey Reading Center to ensure consistency in testing and data extraction. The tests that received this workflow are as follows: SP and KP, ffERG, SD-OCT, FP, and FAF.
Data processing and statistical analysis
The statistical significance of change from the corresponding baseline average was assessed based on previously published test-retest analysis of the entire EIAV-ABCA participants in cohorts 1 through 5. That study established the threshold values for statistically significant changes, referred to as reliability coefficient (RC), for various parameters to be: BCVA letter score (8 letters), KVF isopters I4e, III4e, and V4e (3479, 2488, and 2622 deg , respectively), SVF full volume HOV (V TOT , 14.6 dB-sr), full field ERG 30 Hz flicker amplitude (ffERG, 28.53 mV), and central macular thickness and macular volume (4.43 µm and 0.12 mm 3 , respectively). For central macular thickness and macular volume, RC measurements were taken from 7 participants who used follow-up mode, specifically, 4-1115, 4-0513, 4-0912, 5-1211, 5-1310, 5-1409, and 5-2107 OU. 5-0106 had scan errors in screening OD (treated eye) so that 1 data point was omitted, but the participant was used in the analysis. For 30 Hz flicker amplitude, only participants tested with Burian-Allen electrodes were used for the RC calculation. The RCs are summarized in Supplemental Table 3. Longitudinal plots of changes with statistically significant threshold values were used for presentation. The R statistical language was used to perform all statistical analyses ( http://www.r-project.org ). All zero values were regarded as no data. As such, when pretreatment values were zero or unmeasured, change from baseline was not calculated. Statistical significance was defined as a change that exceeded the RC values.
Clinical significance was determined on a case-by-case basis as a durable change exceeding the RC values, considering the clinical context such as correlation among different modalities and in comparison to the contralateral eye. For 5-0106, 2 experienced masked graders used ImageJ (National Institute of Health) to count the number of flecks for each OCT b-scan from the macular 97-line volume scans at baseline, year 1, and year 3 for both eyes. Flecks were defined as a subretinal hyper-reflective deposit with associated ellipsoid zone (EZ) disruption. Graders were also masked to each other’s results. To determine intergrader agreement, intraclass correlation coefficients with 95% confidence intervals were calculated. In the same subject, area of RPE and EZ loss was quantified using the en face OCT slab projection and manual segmentation methods as described in prior publications. ,
Results
Safety assessments included ophthalmic examination, laboratory assessments, immunology testing, and reporting of ocular and nonocular adverse events (AEs). A screening visit preceded the baseline visit by a maximum of 28 days. These results have been combined as pretreatment for the analysis.
Adverse events
There were 183 AEs reported in the TDU13583 and LTS13588 studies in the period of 3 years after EIAV-ABCA treatment. All 22 patients experienced at least 1 AE. Most of the AEs (163 events or 89%) were mild in intensity, 16 events (9%) were moderate, and 4 were reported as severe (2%). Eighteen of the events were reported as related to the investigational product, 12 of which were considered also related to the surgical procedure. Additional 74 AEs were reported as related to the surgical procedure only and not related to the product. Ophthalmic AEs related to the Investigational Medicinal Product (IMP) or to surgery are summarized in Table 3 . The 3 most common related AEs were conjunctival hemorrhage in 11 participants, intraocular pressure (IOP) increase in 5 participants, and ocular pain in 6 participants. Other AEs reported related to IMP or surgery are presented in Supplemental Table 4.
A total of 5 serious adverse events (SAEs) were reported ( Table 2 ). Of these, 1 ocular SAE was reported as surgery-related in cohort 1. Patient 1-0425 had increased IOP (increased to 35 from 16 mm Hg at baseline). This was thought to be related to the surgical procedure, as it was first reported 1 day after subretinal injection in the study eye (OD). Although IOP elevation initially resolved by week 36 with pressure-lowering eye drops, this was categorized as an SAE, as increased IOP recurred at week 43 (31 mm Hg) and became chronic, requiring long-term control with topical treatment. One nonocular SAE was reported to be IMP-related as a miscarriage in a participant in cohort 5 (5-2107), which occurred 2 years after the study treatment at an estimated 2 weeks of gestation. However, a second pregnancy resulted in a full-term delivery with no reported abnormalities. It is possible that consanquinity may have contributed to the risk of miscarriage. It is unknown if the incidence of miscarriage in our cohort was due to sampling artifact (small in size) or related to the investigational treatment and hence could not be completely ruled out as IMP-related.
| Cohort: Patient ID | AE Description | SAE Onset From Surgery | Outcome | Duration (d) | Severity | Related to IMP | Related to Surgery |
|---|---|---|---|---|---|---|---|
| 1-0425 | Elevated IOP, OD | 8 mo | Resolved | 1479 | Mild | Unrelated | Related |
| 2-0623 | Uterine polyps | 2 y and 10 mo | Resolved | 4 | Severe | Unrelated | Unrelated |
| 2-0623 | Aortic aneurysm | 2 y and 10 mo | Resolved | 102 | Severe | Unrelated | Unrelated |
| 2-0722 | Dyspnea | 2 y and 1 mo | Resolved | 35 | Mild | Unrelated | Unrelated |
| 5-2107 | miscarriage | 2 y | Resolved | 1 | Mild | Related | Unrelated |
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