Markus Groppe

Retinal gene therapy in patients with choroideremia: initial findings from a phase 1/2 clinical trial

Summary Background Choroideremia is an X-linked recessive disease that leads to blindness due to mutations in the CHM gene, which encodes the Rab escort protein 1 (REP1). We assessed the effects of retinal gene therapy with an adeno-associated viral (AAV) vector encoding REP1 (AAV.REP1) in patients with this disease. Methods In a multicentre clinical trial, six male patients (aged 35–63 years) with choroideremia were administered AAV.REP1 (0·6–1·0×1010 genome particles, subfoveal injection). Visual function tests included best corrected visual acuity, microperimetry, and retinal sensitivity tests for comparison of baseline values with 6 months after surgery. This study is registered with ClinicalTrials.gov, number NCT01461213. Findings Despite undergoing retinal detachment, which normally reduces vision, two patients with advanced choroideremia who had low baseline best corrected visual acuity gained 21 letters and 11 letters (more than two and four lines of vision). Four other patients with near normal best corrected visual acuity at baseline recovered to within one to three letters. Mean gain in visual acuity overall was 3·8 letters (SE 4·1). Maximal sensitivity measured with dark-adapted microperimetry increased in the treated eyes from 23·0 dB (SE 1·1) at baseline to 25·3 dB (1·3) after treatment (increase 2·3 dB [95% CI 0·8–3·8]). In all patients, over the 6 months, the increase in retinal sensitivity in the treated eyes (mean 1·7 [SE 1·0]) was correlated with the vector dose administered per mm2 of surviving retina (r=0·82, p=0·04). By contrast, small non-significant reductions (p>0·05) were noted in the control eyes in both maximal sensitivity (–0·8 dB [1·5]) and mean sensitivity (–1·6 dB [0·9]). One patient in whom the vector was not administered to the fovea re-established variable eccentric fixation that included the ectopic island of surviving retinal pigment epithelium that had been exposed to vector. Interpretation The initial results of this retinal gene therapy trial are consistent with improved rod and cone function that overcome any negative effects of retinal detachment. These findings lend support to further assessment of gene therapy in the treatment of choroideremia and other diseases, such as age-related macular degeneration, for which intervention should ideally be applied before the onset of retinal thinning. Funding UK Department of Health and Wellcome Trust.

Subretinal Visual Implant Alpha IMS – Clinical trial interim report

A subretinal visual implant (Alpha IMS, Retina Implant AG, Reutlingen, Germany) was implanted in 29 blind participants with outer retinal degeneration in an international multicenter clinical trial. Primary efficacy endpoints of the study protocol were a significant improvement of activities of daily living and mobility to be assessed by activities of daily living tasks, recognition tasks, mobility, or a combination thereof. Secondary efficacy endpoints were a significant improvement of visual acuity/light perception and/or object recognition (clinicaltrials.gov, NCT01024803). During up to 12 months observation time twenty-one participants (72%) reached the primary endpoints, of which thirteen participants (45%) reported restoration of visual function which they use in daily life. Additionally, detection, localization, and identification of objects were significantly better with the implant power switched on in the first 3 months. Twenty-five participants (86%) reached the secondary endpoints. Measurable grating acuity was up to 3.3 cycles per degree, visual acuities using standardized Landolt C-rings were 20/2000, 20/2000, 20/606 and 20/546. Maximal correct motion perception ranged from 3 to 35 degrees per second. These results show that subretinal implants can restore very-low-vision or low vision in blind (light perception or less) patients with end-stage hereditary retinal degenerations.

Visual Acuity after Retinal Gene Therapy for Choroideremia

In this study, the subfoveal injection of a gene-therapy vector carrying nonmutated CHM, the gene that, when mutated, causes a form of blindness called choroideremia, was followed by an improvement in visual acuity in two of six patients at 3.5 years after injection.

Gene Therapy for Choroideremia Using an Adeno-Associated Viral (AAV) Vector

Choroideremia is an outer retinal degeneration with a characteristic clinical appearance that was first described in the nineteenth century. The disorder begins with reduction of night vision and gradually progresses to blindness by middle age. The appearance of the fundus in sufferers is recognizable by the characteristic pale color caused by the loss of the outer retina, retinal-pigmented epithelium, and choroidal vessels, leading to exposure of the underlying sclera. Choroideremia shows X-linked recessive inheritance and the choroideremia gene (CHM) was one of the first to be identified by positional cloning in 1990. Subsequent identification and characterization of the CHM gene, which encodes Rab escort protein 1 (REP1), has led to better comprehension of the disease and enabled advances in genetic diagnosis. Despite several decades of work to understand the exact pathogenesis, no established treatments currently exist to stop or even slow the progression of retinal degeneration in choroideremia. Encouragingly, several specific molecular and clinical features make choroideremia an ideal candidate for treatment with gene therapy. This work describes the considerations and challenges in the development of a new clinical trial using adeno-associated virus (AAV) encoding the CHM gene.

Correlation of Optical Coherence Tomography and Autofluorescence in the Outer Retina and Choroid of Patients With Choroideremia.

Purpose To evaluate the relationships between RPE, photoreceptor, and choroidal degeneration in choroideremia. Methods Enhanced-depth imaging optical coherence tomography (EDI-OCT), scanning laser ophthalmoscopy (SLO), and autofluorescence (AF) were performed on 39 patients (78 eyes) with choroideremia. The edges of surviving outer retina on OCT and residual AF were aligned. The distribution of outer retinal tubulations was mapped over a range of ages (16–71 years), and comparison made between pre- and postsubretinal gene therapy. Subfoveal choroidal thickness (SFCT) was compared between 23 choroideremia patients (42 eyes) and 20 age- and refraction-matched male controls (40 eyes). Results The edges of RPE AF aligned with a reduction in outer nuclear layer thickness (Spearmans rho = 0.9992). Correlation was also found between the quality of AF and integrity of ellipsoid zone within islands of surviving retina. Tubulations existed in 71 of 78 (91%) eyes with choroideremia and remained stable following gene therapy. Subfoveal choroidal thickness was reduced at baseline in choroideremia (179.7 ± 17.2 μm) compared with controls (302.0 ± 4.8 μm; P < 0.0001), but did not undergo significant thinning until end-stage retinal degeneration (43.1 ± 6.5 μm). Conclusions The data suggest that RPE loss is the primary cause of photoreceptor degeneration in choroideremia. The choroid is thinner than controls from early stages, in keeping with a mild developmental defect. Photoreceptors appear to lose outer segments following loss of underlying RPE and form tubulations at the edges of degeneration. The preservation of tubulations over time and after subretinal injection would be consistent with these structures maintaining attachment to the inner retina and hence being potentially light responsive (ClinicalTrials.gov, NCT01461213).

The Spectrum of CHM Gene Mutations in Choroideremia and Their Relationship to Clinical Phenotype.

Purpose We report the underlying genotype and explore possible genotypic-phenotypic correlations in a large cohort of choroideremia patients. Methods We studied prospectively a cohort of 79 patients diagnosed within a tertiary referral service for patients with retinal dystrophies. Phenotypic evaluation consisted of clinical examination, including visual acuity and residual retinal area by fundus autofluorescence (FAF). Genotype was established by sequencing. We also investigated whether particular genotypes were associated with more severe phenotypes by performing analysis of covariance (ANCOVA), with visual acuity and FAF as the dependent variables and age as the covariant. Results A total of 74 (94%) of patients in our cohort had causative mutations by sequencing, the majority of which were anticipated to be null. Of these, 35 (47%) had insertions and deletions, 13 (18%) had mutations predicted to affect splicing, and 26 (35%) had single point mutations. In the latter case, 13 of 21 (62%) pedigrees with single point mutations were C to T transitions at C-phosphate-G (CpG) dinucleotides. These mutations were spread across 5 of only 24 CpG dinucleotides in the entire CHM cDNA. Furthermore, these 5 locations are the only sites at which C to T transitions result in a stop codon. No clear evidence was found for genotype–phenotype correlation except in the instance of a patient with a large deletion involving neighbouring sequences. Conclusions In patients with a diagnosis of choroideremia made by a specialty service, there is a high likelihood of establishing a genetic diagnosis. The majority of causative mutations appear to be null and, therefore, may benefit from gene replacement therapy. A disproportionate number of single point mutations observed were C to T transitions, consistent with the evolutionary decay of CpG dinucleotides through methylation and subsequent deamination. Hence, the development of choroideremia in such patients may represent the unwanted consequence of human evolution; de novo mutations are predicted to arise at these sites in future generations. (ClinicalTrials.gov number, NCT01461213.)

A Qualitative and Quantitative Assessment of Fundus Autofluorescence Patterns in Patients With Choroideremia

Purpose We set out to characterize the pattern of fundus autofluorescence (AF) loss in choroideremia (CHM) patients of varying ages and disease severity in order to determine the average rate of progression of this potential disease biomarker. Methods Fifty consecutive CHM patients (100 eyes) attending outpatient clinics at Oxford Eye Hospital underwent analysis with the Heidelberg OCT Spectralis with autofluorescence capabilities. The area of residual AF was traced using Heidelberg Eye Explorer. Bland-Altman analysis was used to calculate the coefficient of repeatability (CR). The degree of AF loss was correlated to different ages and the pattern of residual AF constructed into color-coded maps in order to gain insight into the mechanism of disease progression. Results The CR for measurement of AF area is <1%, indicating that a small change is likely to be significant. Correlation of patient age and area of residual AF produced a clinically relevant index of expected anatomic disease. Progression is 7.7% of the residual area each year (95% confidence intervals 7.0%–8.2%) and follows a logarithmic pattern with age (r = 0.95, P < 0.001). From this we derived the mean half-life of AF as 9 years. Qualitatively, the pattern of remaining AF centered on a point temporal to the fovea. Conclusions The area of residual AF in CHM can be measured reproducibly and shows a distinct pattern of loss. The measured residual area is inversely correlated to age. The ratio of the two variables may provide useful information regarding the rate of progression for any one individual at a given point in time.

Technique of retinal gene therapy: delivery of viral vector into the subretinal space.

PurposeSafe and reproducible delivery of gene therapy vector into the subretinal space is essential for successful targeting of the retinal pigment epithelium (RPE) and photoreceptors. The success of surgery is critical for the clinical efficacy of retinal gene therapy. Iatrogenic detachment of the degenerate (often adherent) retina in patients with hereditary retinal degenerations and small volume (eg, 0.1 ml) subretinal injections pose new surgical challenges.MethodsOur subretinal gene therapy technique involved pre-operative planning with optical coherence tomography (OCT) and autofluorescence (AF) imaging, 23 G pars plana vitrectomy, internal limiting membrane staining with Membrane Blue Dual (DORC BV, Zuidland, Netherlands), a two-step subretinal injection using a 41 G Teflon tipped cannula (DORC) first with normal saline to create a parafoveal bleb followed by slow infusion of viral vector via the same self-sealing retinotomy. Surgical precision was further enhanced by intraoperative OCT (Zeiss Rescan 7000, Carl Zeiss Meditec AG, Jena, Germany). Foveal functional and structural recovery was evaluated using best-corrected Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity, microperimetry and OCT.ResultsTwo patients with choroideremia aged 29 (P1) and 27 (P2) years, who had normal and symmetrical levels of best-corrected visual acuity (BCVA) in both eyes, underwent unilateral gene therapy with the fellow eye acting as internal control. The surgeries were uncomplicated in both cases with successful detachment of the macula by subretinal vector injection. Both treated eyes showed recovery of BCVA (P1: 76–77 letters; P2: 84–88 letters) and mean threshold sensitivity of the central macula (P1: 10.7–10.7 dB; P2: 14.2–14.1 dB) to baseline within a month. This was accompanied by normalisation of central retinal thickness on OCT.ConclusionsHerein we describe a reliable technique for subretinal gene therapy, which is currently used in clinical trials to treat choroideremia using an adeno-associated viral (AAV) vector encoding the CHM gene. Strategies to minimise potential complications, such as avoidance of excessive retinal stretch, air bubbles within the injection system, reflux of viral vector and post-operative vitritis are discussed.

Macular hole surgery in patients with end-stage choroideremia.

OBJECTIVE To assess macular hole surgery in patients with end-stage choroideremia with regard to anatomic closure and visual outcome. DESIGN Retrospective, interventional case series. PARTICIPANTS Thirty adult male patients with a diagnosis of advanced choroideremia were reviewed and underwent spectral domain optical coherence tomography (OCT) as part of the screening process for a gene therapy clinical trial. From within that cohort, 3 were identified as having a full-thickness macular hole (FTMH). METHODS A 23-gauge pars plana vitrectomy was performed with peeling of the inner limiting membrane and gas tamponade. Preoperative best-corrected visual acuity ranged from perception of light to 6/24. MAIN OUTCOME MEASURES Prevalence of FTMH in advanced choroideremia, morphologic phenotype characteristics of FTMH in OCT, pre- and postoperative best-corrected visual acuity, and closure rate after surgery. RESULTS The prevalence of FTMH in advanced choroideremia in our cohort was 10%. One hole was associated with significant macular schisis, presumed to be attributable to degeneration of outer retinal layers. Anatomic closure was achieved in all 3 patients and confirmed with spectral domain OCT. Gas tamponade lasted approximately twice as long as might be expected compared with standard FTMH surgery. Objective visual acuity did not improve; however, perceived vision improved in all patients. CONCLUSIONS Although FTMH in choroideremia is a rare finding, it could potentially mask central progression of the disease. Regular screening may help to diagnose holes at an earlier stage when the visual prognosis after surgery may be better. Standard macular hole surgery seems to be effective in gaining anatomic closure, which would be significant for patients who subsequently require macula detachment for subretinal gene therapy. FINANCIAL DISCLOSURE(S) The authors have no proprietary or commercial interest in any of the materials discussed in this article.

Characterizing the Natural History of Visual Function in Choroideremia Using Microperimetry and Multimodal Retinal Imaging.

Purpose Centripetal retinal degeneration in choroideremia (CHM) leads to early visual field restriction and late central vision loss. The latter marks an acute decline in quality of life but visual prognostication remains challenging. We investigated visual function in CHM by correlating best-corrected visual acuity (BCVA), microperimetry and multimodal imaging. Methods Fifty-six consecutive CHM patients attending Oxford Eye Hospital were examined with BCVA, 10–2 microperimetry, optical coherence tomography, and fundus autofluorescence (AF). Microperimetry was repeated in 21 eyes and analyzed with Bland-Altman. Kaplan-Meier survival plots of eyes retaining 20/20 BCVA were created. Intereye symmetry was assessed. Results Microperimetry coefficient of repeatability was 1.45 dB. Survival analysis showed an indistinguishable pattern between eyes (median survival 39 years). Macular sensitivity showed a similar decline in right and left eyes, with half-lives of 13.6 years. Zonal analysis showed faster decline nasal to the fovea. Intereye symmetry was more consistent for microperimetry sensitivity (r = 0.95, P < 0.001) than BCVA (r = 0.42, P = 0.0006). Near normal foveal sensitivity was maintained when the fovea was at least 2500 μm from the advancing edge of AF. Conclusions BCVA is a marker of central degeneration and can provide valuable information about the position of the remaining retina as well as a measure of the impact on daily living. Microperimetry represents the global macular region. Both visual functions showed a high degree of intereye symmetry, particularly in early stages, indicating the fellow eye can provide a suitable control for assessing interventions to one eye. The findings may help to tailor visual prognosis and interpret outcomes of trials.