Edward Averbukh

Gene Augmentation Therapy Restores Retinal Function and Visual Behavior in a Sheep Model of CNGA3 Achromatopsia

Achromatopsia is a hereditary form of day blindness caused by cone photoreceptor dysfunction. Affected patients suffer from congenital color blindness, photosensitivity, and low visual acuity. Mutations in the CNGA3 gene are a major cause of achromatopsia, and a sheep model of this disease was recently characterized by our group. Here, we report that unilateral subretinal delivery of an adeno-associated virus serotype 5 (AAV5) vector carrying either the mouse or the human intact CNGA3 gene under the control of the red/green opsin promoter results in long-term recovery of visual function in CNGA3-mutant sheep. Treated animals demonstrated shorter maze passage times and a reduced number of collisions with obstacles compared with their pretreatment status, with values close to those of unaffected sheep. This effect was abolished when the treated eye was patched. Electroretinography (ERG) showed marked improvement in cone function. Retinal expression of the transfected human and mouse CNGA3 genes at the mRNA level was shown by polymerase chain reaction (PCR), and cone-specific expression of CNGA3 protein was demonstrated by immunohistochemisrty. The rescue effect has so far been maintained for over 3 years in the first-treated animals, with no obvious ocular or systemic side effects. The results support future application of subretinal AAV5-mediated gene-augmentation therapy in CNGA3 achromatopsia patients.

Application of the Standard Photodynamic Treatment Protocol for Symptomatic Circumscribed Choroidal Hemangioma

Purpose: To describe the results of photodynamic therapy (PDT) using a standard protocol, developed for treating choroidal neovascularization (CNV), for the treatment of circumscribed choroidal hemangioma (CCH). Methods: A prospective, uncontrolled, consecutive case series of patients with symptomatic CCH that were treated using the standard PDT protocol was evaluated periodically with ophthalmic exams and echography. Results: Nine CCH patients were included in the study. Mean tumor height decreased from 2.7 mm before treatment to 0.8 mm at the end of the follow-up period. Mean visual acuity improved from 6/15 to 6/12. Six patients required one PDT session, two patients two sessions, and one patient three sessions. Side effects included transient visual disturbances in two patients. One patient, who concomitantly presented with age-related macular degeneration, developed CNV. Conclusions: The standard PDT protocol is effective for the treatment of CCH. Complications are uncommon. Further studies should assess the optimal PDT protocol for the treatment of CCH.

A novel day blindness in sheep: epidemiological, behavioural, electrophysiological and histopathological studies.

Four genetically related Improved Awassi sheep flocks had sporadic births of lambs with congenital visual impairments that differed from other known forms of sheep blindness. Pedigree analysis suggested an autosomal recessive mode of inheritance. Behavioural studies of 4-month old affected lambs showed that their day vision (but not night vision) was impaired. Electrophysiological results at this age demonstrated diminished function of cones but not rods. Histopathological and immunohistochemical evaluation of affected retinas from 5-month old lambs revealed both red-green and blue cones, suggesting that the behavioural day blindness and reduced cone electroretinograms reflect cone dysfunction rather than severe cone photoreceptor loss. Awassi day blindness may be a form of achromatopsia.

Bevacizumab for choroidal neovascularization related to inflammatory diseases.

Purpose:The purpose of this study was to report our experience with intravitreal bevacizumab for inflammation-related choroidal neovascularization in two tertiary centers. Methods:This study was a retrospective analysis of patients with choroidal neovascularization related to inflammatory diseases, treated with intravitreal bevacizumab injections (1.25 mg/0.05 mL). Results:Ten eyes of 10 patients (range, 14-78 years; mean age, 44 years) with underlying uveitis were treated with intravitreal bevacizumab for inflammation-related choroidal neovascularization from 2006 to 2008. Mean follow-up time was 13 ± 8 months, and the mean number of injections was 2.7 ± 2. Resolved leakage on fluorescein angiography and resolution of subretinal fluid on optical coherence tomography occurred in all patients, with improvement in visual acuity in 9 of 10 eyes and no change in visual acuity in 1 of 10 eyes. Seven patients received additional treatment based on the underlying condition. Mean macular thickness on optical coherence tomography decreased from 394 ± 116 μm to 254 ± 52 μm (P < 0.01). Mean visual acuity improved from 0.87 ± 0.74 logarithm of the minimum angle of resolution to 0.38 ± 0.63 (P = 0.005). Seven patients reached a visual acuity of 0.2 logarithm of the minimum angle of resolution (Snellen 6/9) or better. Conclusion:Intravitreal bevacizumab is an effective treatment for choroidal neovascularization related to inflammatory diseases when inflammation is controlled.

Octreotide, a somatostatin analogue, fails to inhibit hypoxia-induced retinal neovascularization in the neonatal rat.

Objective: Octreotide, a somatostatin analogue, has been shown to prevent angiogenesis in diverse in vitro models. We evaluated its effect on retinal neovascularization in vivo, using a neonatal rat retinopathy model. Methods: We used, on alternating days, hypoxia (10% O2) and hyperoxia (50% O2) during the first 14 days of neonatal rats, to induce retinal neovascularization. Half of the rats were injected subcutaneously with octreotide 0.7 μg/g BW twice daily. At day 18 the eyes were evaluated for the presence of epiretinal and vitreal hemorrhage, neovascularization and epiretinal proliferation. Octreotide pharmacokinetics and its effect on serum growth hormone (GH) and insulin-like growth factor I (IGF-I) were examined in 28 rats. Results: Serum octreotide levels were 667 μg/1 two hours after injection, 26.4 μg/1 after nine hours and 3.2 μg/1 after 14 hours. GH levels were decreased by 40% (p = 0.002) two hours after injection but thereafter returned to baseline. IGF-I levels were unchanged two hours after injection and were elevated by 26% 14 hours after injection (p = 0.02). Epiretinal membranes were highly associated with epiretinal hemorrhages (p < 0.001), while retinal neovascularization was notably associated with vitreal hemorrhages (p < 0.001). Conclusions: Twice-daily injections of octreotide failed to produce sustained decrease in serum GH, but produced rebound elevation of serum IGF-I. Accordingly, no statistically significant effect of injections on retinal pathology was noted. This finding, however, does not contradict our assumption that GH suppression may decrease the severity of retinopathy.

Association of Pattern Dystrophy With an HTRA1 Single-Nucleotide Polymorphism

OBJECTIVE To evaluate if adult-onset foveomacular vitelliform dystrophy (AOFVD) and butterfly-shaped pigment dystrophy (BSPD) are associated with risk single-nucleotide polymorphisms (SNPs) for age-related macular degeneration (AMD). METHODS This was a tertiary referral center-based cross-sectional study including 35 consecutive patients with BSPD and AOFVD, 317 patients with AMD, and 159 unaffected individuals. Demographics, clinical information, and ophthalmic imaging studies were collected. Sequencing was performed for the peripherin/RDS and BEST1 genes, and genotyping was performed for SNPs in the genes for complement factor H (CFH) (rs1061170), HTRA1 (rs11200638), and complement component 3 (C3) (rs2231099). RESULTS Adult-onset foveomacular vitelliform dystrophy and BSPD were diagnosed in 24 (68.6%) and 11 (31.4%) of the 35 patients, respectively. The mean (SD) age of patients with pattern dystrophy (PD) was 75.3 (10) years and median visual acuity was 0.7. Pattern dystrophy was associated with the HTRA1 risk allele compared with unaffected individuals (odds ratio, 1.72; 95% CI, 1.11-2.66; P = .03). The HTRA1 SNP showed similar prevalence in patients with AMD and PD. The CFH risk allele was significantly less common in patients with PD compared with patients with AMD (odds ratio, 0.47; 95% CI, 0.28-0.76; P = .002). No mutations in peripherin/RDS or BEST1 were detected. CONCLUSIONS The AOFVD and BSPD phenotypes are associated with an HTRA1 risk SNP. These phenotypes often present in elderly individuals who do not carry peripherin/RDS gene mutations and are associated with retinal pigment epithelium alterations and increased risk for choroidal neovascularization. Further research is required to evaluate if AOFVD and BSPD phenotypes in aged individuals are associated with AMD.

Intravitreal bevacizumab therapy for neovascular age-related macular degeneration associated with poor initial visual acuity

Aim: The aim of the study was to assess the efficacy of intravitreal bevacizumab injections for eyes with neovascular age-related macular degeneration (NVAMD) and poor initial visual acuity (VA). Methods: A retrospective study of 44 consecutive treatment-naïve eyes with NVAMD who had initial VA of 0.1 decimal or worse, and that were treated with intravitreal bevacizumab injections, was undertaken. Charts, optical coherence tomography (OCT) and fluorescein angiograms (FAs) were reviewed for the purpose of the study. Results: Mean lesion size was 3375 (SD 2116) μm, all lesions showed sub- or intra-retinal fluid in OCT, and active neovascularisation comprised 41.6 (SD 17.7)% (range 10–90%) of the lesion area according to FA. The mean follow-up time was 3.9 (SD 5.8) (range 1–21) months. Patients received a mean of 2.6 (SD 2.4) bevacizumab injections (range 1–14), and mean VA improved from 1.85 (SD 0.64) to 1.52 (SD 0.77) LogMAR (p = 0.002). At final examination, nine eyes (20%) had reduced VA, ten eyes (23%) had stable VA and 25 eyes (57%) had improved VA compared with baseline. Following treatment, mean macular thickness was reduced from 332 (SD 98) to 248 (SD 79) μm (p<0.0001). Conclusions: Poor initial VA should not prevent use of bevacizumab in eyes with NVAMD. Selection of patients with signs of active neovascularisation based on ophthalmoscopy, OCT and FA may increase the likelihood of a favourable response to treatment.

Bevacizumab treatment for choroidal neovascularization associated with adult-onset foveomacular vitelliform dystrophy

Purpose To evaluate choroidal neovascularization (CNV) associated with adult-onset foveomacular vitelliform dystrophy (AOFVD) and its response to bevacizumab therapy. Methods Demographics, clinical characteristics, response to bevacizumab therapy, and central foveal thickness (CFT) were retrospectively assessed in 11 eyes with CNV associated with AOFVD. Sixty consecutive patients with neovascular age-related macular degeneration (AMD) were compared to the patients with AOFVD for all clinical characteristics and responses evaluated. Results The mean (±SD) initial logMAR visual acuity (0.7 ± 0.8 vs. 1 ± 0.75), age at onset, number of bevacizumab injections (12.4 ± 10.4 vs 9 ± 6.7), and final logMAR visual acuity (0.87 ± 0.7 vs 1 ± 0.85) were similar between AOFVD and AMD. The mean CFT in AOFVD was reduced from 418 ± 144 μm to 330 ± 64 μm following treatment (p = 0.03). At the final examination, visual acuity had improved in 3 eyes, stabilized in 1 eye, and was reduced in 7 of the AOFVD eyes examined. Conclusions Bevacizumab therapy for AOFVD-associated CNV resulted in reduced foveal thickness, but a guarded visual outcome was still found, due to progression of the vitelliform lesions.

Gene Augmentation Therapy for a Missense Substitution in the cGMP-Binding Domain of Ovine CNGA3 Gene Restores Vision in Day-Blind Sheep

Purpose Applying CNGA3 gene augmentation therapy to cure a novel causative mutation underlying achromatopsia (ACHM) in sheep. Methods Impaired vision that spontaneously appeared in newborn lambs was characterized by behavioral, electroretinographic (ERG), and histologic techniques. Deep-sequencing reads of an affected lamb and an unaffected lamb were compared within conserved genomic regions orthologous to human genes involved in similar visual impairment. Observed nonsynonymous amino acid substitutions were classified by their deleteriousness score. The putative causative mutation was assessed by producing compound CNGA3 heterozygotes and applying gene augmentation therapy using the orthologous human cDNA. Results Behavioral assessment revealed day blindness, and subsequent ERG examination showed attenuated photopic responses. Histologic and immunohistochemical examination of affected sheep eyes did not reveal degeneration, and cone photoreceptors expressing CNGA3 were present. Bioinformatics and sequencing analyses suggested a c.1618G>A, p.Gly540Ser substitution in the GMP-binding domain of CNGA3 as the causative mutation. This was confirmed by genetic concordance test and by genetic complementation experiment: All five compound CNGA3 heterozygotes, carrying both p.Arg236* and p.Gly540Ser mutations in CNGA3, were day-blind. Furthermore, subretinal delivery of the intact human CNGA3 gene using an adeno-associated viral vector (AAV) restored photopic vision in two affected p.Gly540Ser homozygous rams. Conclusions The c.1618G>A, p.Gly540Ser substitution in CNGA3 was identified as the causative mutation for a novel form of ACHM in Awassi sheep. Gene augmentation therapy restored vision in the affected sheep. This novel mutation provides a large-animal model that is valid for most human CNGA3 ACHM patients; the majority of them carry missense rather than premature-termination mutations.

Irido-cilio-choroidal melanoma in a 5-year-old boy treated by Ru-106 brachytherapy.

Uveal melanoma in patients aged 20 years or younger is rare, and comprises approximately 1% (0.6–1.6%) of all uveal melanoma patients. Less than a quarter of these patients are under the age of 10 years. Uveal melanoma in patients 5 years or younger are very rare; most of them have been reported as case reports. Four cases of congenital uveal melanoma have been reported, all of them aggressive tumours and three of them showed extraocular extension. We present a 5-year-old boy with an irido-ciliochoroidal melanoma treated by Ru-106 brachytherapy. A 5-year-old boy was examined in his home country, Hungary, by a general practitioner because of vomiting. In his examination, the physician noticed pigmentation in the iris periphery of the left eye and referred him to an ophthalmologist. On examination a pigmented mass was identified in the nasal side of the anterior uvea and the diagnosis of uveal melanoma was made. Enucleation of the eye was recommended. The child was referred to our ocular oncology clinic for a second opinion and possible treatment. On examination in our clinic, the visual acuity in each eye was 6/6. The child showed no signs of ocular melanosis or cutaneous melanocytic lesions. The right eye was found to be normal. In the left eye, a pigmented area was seen in the nasal periphery of the iris. After mydriasis, a bilobular pigmented mass was seen in the nasal side of the ciliary body, touching the lens which was clear (Fig. 1). High-frequency ultrasound examination showed a solid mass with a small area of cavitation in the ciliary body, involving the iris periphery, anterior chamber angle and anterior choroid. B-scan ultrasonography showed a solid round tumour (Fig. 2) and A-scan showed low internal reflectivity. In B-scan and A-scan ultrasonography the basal diameter of the mass was 12.4 ¥ 8.8 mm and its maximal height 7.4 mm. Although we considered in the differential diagnosis tumours, such as pigmented medulloepithelioma and melanocytoma, the extent of the lesion, involving the iris and choroid and its ultrasound features, led us to the diagnosis of irido-cilio-choroidal melanoma. We decided to treat the boy with Ru-106 brachytherapy. In February 2005, under general anaesthesia, surgery for insertion of a Ru-106 (CCA) (IBt-BEBIG, Berlin, Germany) applicator was carried out, suturing the applicator over the sclera and the corneal periphery to cover the tumour. The medial rectus muscle was disinserted, as its insertion point was over the tumour base. The applicator was left in place for 175 h, irradiating 98 000 cGy (560 cGy per hour) to the tumour base. The apex of the tumour received 4500 cGy (25.7 cGy per hour) (The company’s dose calculations were based on the National Institute for Standards and Technology calibration measurements.). On follow-up examination 4 months after the treatment, a significant decrease in the tumour size was noticed (Fig. 3), a trend that continued for the following year and a half, until the tumour remnants were barely detected, clinically and in ultrasound examination. With time, the sclera over the ciliary body in the treated area showed some thinning, and some fibrovascular tissue growth was noticed in the nasal corneal periphery. The pupil was mildly dragged nasally. The lens showed early posterior subcapsular cataract almost 10 months after the treatment. The lenticular opacity progressed gradually and after 2.5 years the visual acuity deteriorated to finger counting at 1.5 m. The child underwent cataract extraction, performing lens aspiration, posterior capsulorhexis and anterior vitrectomy, and an intraocular lens (IOL) was inserted into the bag. On his latest examination, 4 years after the diagnosis, the visual acuity of the right eye was 6/6 and in the left eye 6/15, corrected to 6/6. Some opacity was seen in the corneal periphery, the pupil was mildly dragged nasally,