Characterization of anti-AAV2 neutralizing antibody levels in sheep prior to and following intravitreal AAV2.7m8 injection.

Gene augmentation therapy is a promising treatment for incurable, blinding inherited retinal diseases, and intravitreal delivery is being studied as a safe alternative to subretinal injections. Adeno-Associated Viruses (AAV) are commonly-used vectors for ocular gene augmentation therapy. Naturally occurring pre-operative exposure and infection with AAV could result in presence of neutralizing antibodies (NAB's) in patients' serum, and may affect the safety and efficacy of treatment. Our aim was to characterize the humoral response against AAV pre- and post-intravitreal delivery of AAV2.7m8 vectors in a naturally-occurring sheep model of CNGA3 achromatopsia. Serial serum neutralization assays were performed to screen sheep for pre-exiting anti-AAV2 NAB's, and to assess the effect of intravitreal AAV2.7m8 injection on post-operative NAB titers and intraocular inflammation in sheep. The effect of viral dose and transgene type were also assessed. Serological screening revealed pre-operative seropositivity in 21.4% of animals, with age being a risk factor for the presence of anti-AAV2 NAB's. NAB titers increased following intravitreal AAV administration in the majority of sheep. There was no significant difference in the degree of post-operative serum neutralization between pre-operatively seronegative sheep and those with pre-existing antibodies. However, only sheep with pre-existing antibodies presented with signs of post-operative inflammation. We conclude that pre-existing anti-AAV2 NAB's do not affect the level of post-operative NAB titers; however, they increase the risk of post-operative ocular inflammation. Our results could have implications for the management of AAV-mediated ocular gene therapies, a technology being increasingly studied and used in patients.

Advanced Optical Analysis of Divergence Between the Foci of the Neodymium-Doped Yttrium Aluminum Garnet (Nd:YAG) and Aiming Beam Lasers.

Purpose: To evaluate the divergence between the neodymium-doped yttrium aluminum garnet (Nd:YAG) surgical laser and the aiming diode laser beams foci. Design: Optical analysis and measurements were performed using a Volk Goldmann 3-mirror lens with a Nidek YC-1800 Nd:YAG laser apparatus. Subjects: None. Methods: We used the Zemax OpticStudio program for the model of Nd:YAG treatment in a human eye. Additionally, theoretical calculations were performed. Main Outcome Measures: The divergence between the Nd:YAG laser focus and the intersection of the 2 aiming beams inside the eye. Results: Focal points of the 2 laser beams converge 8 mm behind the cornea. Posterior to this point, the intersection of the diode laser aiming beams lies in front of the focal point of the Nd:YAG treatment laser, with distance between the 2 foci progressively increasing up to 305 microns at 24 mm behind the cornea. Conclusions: We report the degree of divergence between the 2 lasers' focal points due to the difference in refraction between the corresponding wavelengths. These results have high practical relevance, as they provide a starting point for increasing the accuracy of Nd:YAG laser treatment, particularly when applied to the posterior segment, thereby minimizing the risk of complications. Current Nd:YAG laser devices have the built-in ability to modify the focal point of the aiming beam along the z-axis, thus providing possibility for an immediate application of our findings in clinical practice. Financial Disclosures: The authors have no proprietary or commercial interest in any materials discussed in this article.

Central Serous Chorioretinopathy Development following Cessation of Terbinafine Treatment.

This report describes a case of central serous chorioretinopathy (CSCR) occurring following cessation of terbinafine treatment. A 51-year-old man presented for a routine ophthalmic examination. He was treated with oral terbinafine for onychomycosis up to 3 months before the presentation. Spectral-domain optical coherence tomography (OCT) showed extrafoveal subretinal fluid in both eyes with small underlying pigment epithelial detachments. There were no additional relevant findings in the patient history or ocular examination. A diagnosis of CSCR was made. After 10 weeks without treatment, OCT demonstrated almost complete resolution of subretinal fluid in both eyes. The exact key ingredients of the perfect storm leading to CSCR in young, healthy individuals are still unknown. Here, we describe, to our knowledge, the first documented case, where the appearance of CSCR was apparently triggered by cessation of antifungal treatment. This unusual case may provoke further research that will bring us closer to understanding the mechanism behind the appearance of CSCR. It may also widen the scope of the routine anamnesis when dealing with patients newly diagnosed with this enigmatic condition.

Cortical Visual Mapping following Ocular Gene Augmentation Therapy for Achromatopsia.

The ability of the adult human brain to develop function following correction of congenital deafferentation is controversial. Specifically, cases of recovery from congenital visual deficits are rare. CNGA3-achromatopsia is a congenital hereditary disease caused by cone-photoreceptor dysfunction, leading to impaired acuity, photoaversion, and complete color blindness. Essentially, these patients have rod-driven vision only, seeing the world in blurry shades of gray. We use the uniqueness of this rare disease, in which the cone-photoreceptors and afferent fibers are preserved but do not function, as a model to study cortical visual plasticity. We had the opportunity to study two CNGA3-achromatopsia adults (one female) before and after ocular gene augmentation therapy. Alongside behavioral visual tests, we used novel fMRI-based measurements to assess participants' early visual population receptive-field sizes and color regions. Behaviorally, minor improvements were observed, including reduction in photoaversion, marginal improvement in acuity, and a new ability to detect red color. No improvement was observed in color arrangement tests. Cortically, pretreatment, patients' population-receptive field sizes of early visual areas were untypically large, but were decreased following treatment specifically in the treated eye. We suggest that this demonstrates cortical ability to encode new input, even at adulthood. On the other hand, no activation of color-specific cortical regions was demonstrated in these patients either before or up to 1 year post-treatment. The source of this deficiency might be attributed either to insufficient recovery of cone function at the retinal level or to challenges that the adult cortex faces when computing new cone-derived input to achieve color perception.SIGNIFICANCE STATEMENT The possibility that the adult human brain may regain or develop function following correction of congenital deafferentation has fired the imagination of scientists over the years. In the visual domain, cases of recovery from congenital deficits are rare. Gene therapy visual restoration for congenital CNGA3-achromatopsia, a disease caused by cone photoreceptor dysfunction, gave us the opportunity to examine cortical function, to the best of our knowledge for the first time, both before and after restorative treatment. While behaviorally only minor improvements were observed post-treatment, fMRI analysis, including size algorithms of population-receptive fields, revealed cortical changes, specifically receptive field size decrease in the treated eyes. This suggests that, at least to some degree, the adult cortex is able to encode new input.

Endogenous fungal endophthalmitis: risk factors, clinical course, and visual outcome in 13 patients.

AIM: To analyze the risk factors, ophthalmological features, treatment modalities and their effect on the visual outcome in patients with endogenous fungal endophthalmitis (EFE). METHODS: Data retrieved from the medical files included age at presentation to the uveitis clinic, gender, ocular symptoms and their duration before presentation, history of fever, eye affected, anatomical diagnosis and laboratory evidence of fungal infection. Medical therapy recorded included systemic antifungal therapy and its duration, use of intravitreal antifungal agents and use of oral/intravitreal steroids. Surgical procedures and the data of ophthalmologic examination at presentation and at last follow-up were also collected. RESULTS: Included were 13 patients (20 eyes, mean age 58y). Ten patients presented after gastrointestinal or urological interventions and two presented after organ transplantation. In one patient, there was no history of previous intervention. Diagnostic vitrectomy was performed in 16 eyes (80%) and vitreous cultures were positive in 10 of the vitrectomized eyes (62.5%). In only 4 patients (31%), blood cultures were positive. All patients received systemic antifungal therapy. Sixteen eyes (80%) received intravitreal antifungal agent with voriconazole being the most commonly used. Visual acuity (VA) improved from 0.9±0.9 at initial exam to 0.5±0.8 logMAR at last follow-up (P=0.03). A trend of greater visual improvement was noted in favor of eyes treated with oral steroids (±intravitreal dexamethasone) than eyes that were not treated with steroids. The most common complication was maculopathy. Twelve eyes (60%) showed no ocular complications. CONCLUSION: High index of suspicion in patients with inciting risk factors is essential because of the low yield of blood cultures and the good general condition of patients at presentation. Visual prognosis is improved with the prompt institution of systemic and intravitreal pharmacotherapy and the immediate surgical intervention. Oral±local steroids could be considered in cases of prolonged or marked inflammatory responses in order to hasten control of inflammation and limit ocular complications.

Correlation of Response between Both Eyes to First- and Second-Line Anti-VEGF Therapy in Diabetic Macular Edema.

PURPOSE: To evaluate the anatomical correlation between fellow eyes for bilateral second-line anti-VEGF treatment in eyes with bilateral diabetic macular edema (DME) with incomplete response to first-line bevacizumab therapy. METHODS: Seventy-four eyes (n = 37 patients) with bilateral-DME having incomplete response to first-line bevacizumab therapy that were switched for bilateral treatment with ranibizumab were retrospectively evaluated. Data collected included demographics, visual acuity and macular thickness. We evaluate the correlation for the response of both eyes in terms of macular thickness and visual acuity. RESULTS: The mean±SD age was 76 ± 8 years. The mean±SD number of bevacizumab injections prior the switch was 11.03 ± 5.1 in the first eye (FE) and 10.9 ± 5.2 in the second eye (SE). The central subfield thickness (CST) reduced from 472 ± 171 microns at baseline to 418 ± 161 after the last bevacizumab injection and 365 ± 74 after 3 ranibizumab injections in the FE (p = .016, p = .004, respectively), and from 463 ± 145 microns to 446 ± 123, and 421 ± 103 in the SE (p = .112, p = .001, respectively). There was strong positive correlation between the eyes for the CST reduction under bevacizumab and ranibizumab treatments in each visit. BCVA± SD at baseline was 0.41 ± 0.30 LogMAR in the FE, and 0.42 ± 0.29 in the SE (p = .44). After 3 injections of bevacizumab, the BCVA was 0.37 ± 0.26 and 0.42 ± 0.23 in FE and SE respectively (p = .013, p = .132, respectively). CONCLUSIONS: This study demonstrated a strong anatomical correlation responses between the eyes in patients with bilateral DME for both first-line bevacizumab therapy and second-line ranibizumab therapy. Response to second-line therapy was favorable and correlated among eyes regardless they were from the same or different individuals.

Socioeconomic status and visual outcome in patients with neovascular age-related macular degeneration.

PURPOSE: Visual outcome in patients with neovascular age-related macular degeneration is variable. We aimed to evaluate for association between socioeconomic status visual acuity in neovascular age-related macular degeneration. METHODS: A retrospective single-center study of a consecutive group of neovascular age-related macular degeneration patients was performed. Socioeconomic status was determined for each patient based on the 2008 Israeli census. Medical information was extracted from medical records and included visual acuity and optical coherence tomography parameters. Associations between socioeconomic status and clinical outcomes were analyzed. RESULTS: A total of 233 patients were included in the analysis. A correlation was found between low baseline visual acuity of the first eye diagnosed with neovascular age-related macular degeneration and low socioeconomic status (r = -0.13, p = 0.049; n = 233). The difference between the visual acuity of the lowest and the highest socioeconomic status categories at baseline was approximately 3 ETDRS lines (p = 0.048). Socioeconomic status and baseline visual acuity of the second eye of the same individual with neovascular age-related macular degeneration were not correlated (r = -0.05, p = 0.95). Socioeconomic status was not associated with the number of anti-vascular endothelial growth factor injections of the first or second eye, or the visual acuity outcome of the first or second eye after 1 year of therapy (p = 0.421, p = 0.9, respectively). Central subfield thickness of the first eye at presentation as measured by spectral-domain optical coherence tomography was associated with socioeconomic status (r = -0.31 p = 0.001). CONCLUSION: Individuals of lower socioeconomic status presented at more advanced stage of the disease when developing neovascular age-related macular degeneration in the first eye but not in the second eye. The research underscores the importance of improving referral patterns and awareness for the lowest socioeconomic status classes.

Naturally-occurring myopia and loss of cone function in a sheep model of achromatopsia.

Achromatopsia is an inherited retinal disease characterized by loss of cone photoreceptor function. Day blind CNGA3 mutant Improved Awassi sheep provide a large animal model for achromatopsia. This study measured refractive error and axial length parameters of the eye in this model and evaluated chromatic pupillary light reflex (cPLR) testing as a potential screening test for loss of cone function. Twenty-one CNGA3 mutant, Improved Awassi, 12 control Afec-Assaf and 12 control breed-matched wild-type (WT) Awassi sheep were examined using streak retinoscopy and B-mode ocular ultrasonography. Four CNGA3 mutant and four Afec-Assaf control sheep underwent cPLR testing. Statistical tests showed that day-blind sheep are significantly more myopic than both Afec-Assaf and WT Awassi controls. Day-blind sheep had significantly longer vitreous axial length compared to WT Awassi (1.43 ± 0.13 and 1.23 ± 0.06 cm, respectively, p < 0.0002) and no response to bright red light compared to both controls. Lack of response to bright red light is consistent with cone dysfunction, demonstrating that cPLR can be used to diagnose day blindness in sheep. Day-blind sheep were found to exhibit myopia and increased vitreous chamber depth, providing a naturally occurring large animal model of myopia.

Evaluation of Photoreceptor Transduction Efficacy of Capsid-Modified Adeno-Associated Viral Vectors Following Intravitreal and Subretinal Delivery in Sheep.

Gene augmentation therapy based on subretinal delivery of adeno-associated viral (AAV) vectors is proving to be highly efficient in treating several inherited retinal degenerations. However, due to potential complications and drawbacks posed by subretinal injections, there is a great impetus to find alternative methods of delivering the desired genetic inserts to the retina. One such method is an intravitreal delivery of the vector. Our aim was to evaluate the efficacy of two capsid-modified vectors that are less susceptible to cellular degradation, AAV8 (doubleY-F) and AAV2 (quadY-F+T-V), as well as a third, chimeric vector AAV[max], to transduce photoreceptor cells following intravitreal injection in sheep. We further tested whether saturation of inner limiting membrane (ILM) viral binding sites using a nonmodified vector, before the intravitreal injection, would enhance the efficacy of photoreceptor transduction. Only AAV[max] resulted in moderate photoreceptor transduction following intravitreal injection. Intravitreal injection of the two other vectors did not result in photoreceptor transduction nor did the saturation of the ILM before the intravitreal injection. However, two of the vectors efficiently transduced photoreceptor cells following subretinal injection in positive control eyes. Previous trials with the same vectors in both murine and canine models resulted in robust and moderate transduction efficacy, respectively, of photoreceptors following intravitreal delivery, demonstrating the importance of utilizing as many animal models as possible when evaluating new strategies for retinal gene therapy. The successful photoreceptor transduction of AAV[max] injected intravitreally makes it a potential candidate for intravitreal delivery, but further trials are warranted to determine whether the transduction efficacy is sufficient for a clinical outcome.

PERSISTENT LOCULATED SUBRETINAL FLUID AFTER RHEGMATOGENOUS RETINAL DETACHMENT SURGERY.

PURPOSE: To identify factors associated with persistent subretinal fluid (SRF) after small-gauge pars plana vitrectomy for primary rhegmatogenous retinal detachment. METHODS: This retrospective study included patients from 2 tertiary centers who underwent pars plana vitrectomy for repair of rhegmatogenous retinal detachment between 2013 and 2016. Preoperative and intraoperative parameters were examined for association with development of SRF. RESULTS: Overall, 153 eyes of 153 patients, mean age of 55.2 ± 17.9 years were included. Persistent SRF occurred in 15.0% (n = 23) and was associated with high myopia (65.22 vs. 26.15%, P < 0.001), macula-involving retinal detachment (91.30 vs. 66.15%, P = 0.02), phakic lens status (86.96 vs. 66.15%, P = 0.04), and younger age (47.8 ± 18.7 vs. 56.5 ± 17.5, P = 0.04) while drainage retinotomy was protective (13.04 vs. 34.11%, P = 0.04). In multivariate analysis, high myopia (P = 0.009) and macula-involving retinal detachment (P = 0.004) were associated with SRF, while drainage retinotomy was protective (P = 0.03). Persistent SRF was associated with outer retinal band irregularity (30.4 vs. 9.3%, P = 0.005). There were no significant differences in terms of change in best-corrected visual acuity from presentation (P = 0.70), or final best-corrected visual acuity (P = 0.54). CONCLUSION: Eyes with preoperative high myopia and macular involvement, and those in which a drainage retinotomy was not performed, were more likely to develop persistent SRF.

Consecutive unilateral recording of the two eyes affects dark-adapted ERG responses, when compared to simultaneous bilateral recording.

PURPOSE: Our aim was to compare the electroretinographic (ERG) responses of two eyes obtained by consecutive unilateral recordings to those obtained by a simultaneous bilateral recording in sheep. METHODS: Eight sheep underwent two full-field ERG recordings, using two recording strategies of the standard ISCEV protocol: consecutive unilateral recordings of one eye after the other, and simultaneous bilateral recording of both eyes. The order of recording strategy within an animal (unilateral/bilateral), eye recording sequence in the unilateral session (OD/OS), and amplifier channel assignment for each eye were all randomized. To test whether duration of dark adaptation and/or anesthesia affect the results, the ISCEV protocol was recorded bilaterally in six additional eyes following 38 min of patched dark adaptation, as was done for the second eye recorded in the consecutive unilateral recordings. RESULTS: The second recorded eye in the unilateral session had significantly higher scotopic b-wave amplitudes compared to the first recorded eye and to the bilaterally recorded eyes. A-wave amplitudes of the dark-adapted mixed rod-cone responses to a high-intensity flash were also significantly higher in the second eye compared to the first eye recorded unilaterally and to the bilaterally recorded eyes. Light-adapted responses were unaffected by the recording strategy. When the ISCEV protocol was recorded after 38 min of dark adaptation, the scotopic responses were higher than in the first eyes, and similar to those of the second eyes recorded unilaterally, suggesting that indeed the longer duration of anesthesia and dark adaptation are responsible for the increased scotopic responses of the second eye. CONCLUSIONS: Consecutive unilateral ERG recordings of two eyes result in higher amplitudes of the dark-adapted responses of the eye recorded second, compared to the eye recorded first and to bilaterally recorded eyes. The differences in scotopic responses can be attributed to different duration of dark adaptation and/or anesthesia of the two consecutively recorded eyes. Photopic responses are not affected. Therefore, simultaneous bilateral ERG responses should be recorded when possible, especially for evaluation of scotopic responses.

Six Years and Counting: Restoration of Photopic Retinal Function and Visual Behavior Following Gene Augmentation Therapy in a Sheep Model of CNGA3 Achromatopsia.

Achromatopsia causes severely reduced visual acuity, photoaversion, and inability to discern colors due to cone photoreceptor dysfunction. In 2010, we reported on day-blindness in sheep caused by a stop-codon mutation of the ovine CNGA3 gene and began gene augmentation therapy trials in this naturally occurring large animal model of CNGA3 achromatopsia. The purpose of this study was to evaluate long-term efficacy and safety results of treatment, findings that hold great relevance for clinical trials that started recently in CNGA3 achromatopsia patients. Nine day-blind sheep were available for long-term follow up. The right eye of each sheep was treated with a single subretinal injection of an Adeno-Associated Virus Type 5 (AAV5) vector carrying either a mouse (n = 4) or a human (n = 5) CNGA3 transgene under control of the 2.1-Kb red/green opsin promoter. The efficacy of treatment was assessed periodically with photopic maze tests and electroretinographic (ERG) recordings for as long as 74 months postoperatively. Safety was assessed by repeated ophthalmic examinations and scotopic ERG recordings. The retinas of three animals that died of unrelated causes >5 years post-treatment were studied histologically and immunohistochemically using anti-hCNGA3 and anti-red/green cone opsin antibodies. Passage time and number of collisions of treated sheep in the photopic maze test were significantly lower at all follow-up examinations as compared with pretreatment values (p = 0.0025 and p < 0.001, respectively). ERG Critical Flicker Fusion Frequency and flicker amplitudes at 30 and 40 Hz showed significant improvement following treatment (p < 0.0001) throughout the study. Ophthalmic examinations and rod ERG recordings showed no abnormalities in the treated eyes. Immunohistochemistry revealed the presence of CNGA3 protein in red/green opsin-positive cells (cones) of the treated eyes. Our results show significant, long-term improvement in cone function, demonstrating a robust rescue effect up to six years following a single treatment with a viral vector that provides episomal delivery of the transgene. This unique follow-up duration confirms the safe and stable nature of AAV5 gene therapy in the ovine achromatopsia model.

Safety and Efficacy Evaluation of rAAV2tYF-PR1.7-hCNGA3 Vector Delivered by Subretinal Injection in CNGA3 Mutant Achromatopsia Sheep.

Applied Genetic Technologies Corporation (AGTC) is developing a recombinant adeno-associated virus (rAAV) vector expressing the human CNGA3 gene designated AGTC-402 (rAAV2tYF-PR1.7-hCNGA3) for the treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. The results are herein reported of a study evaluating safety and efficacy of AGTC-402 in CNGA3-deficient sheep. Thirteen day-blind sheep divided into three groups of four or five animals each received a subretinal injection of an AAV vector expressing a CNGA3 gene in a volume of 500 µL in the right eye. Two groups (n = 9) received either a lower or higher dose of the AGTC-402 vector, and one efficacy control group (n = 4) received a vector similar in design to one previously shown to rescue cone photoreceptor responses in the day-blind sheep model (rAAV5-PR2.1-hCNGA3). The left eye of each animal received a subretinal injection of 500 µL of vehicle (n = 4) or was untreated (n = 9). Subretinal injections were generally well tolerated and not associated with systemic toxicity. Most animals had mild to moderate conjunctival hyperemia, chemosis, and subconjunctival hemorrhage immediately after surgery that generally resolved by postoperative day 7. Two animals treated with the higher dose of AGTC-402 and three of the efficacy control group animals had microscopic findings of outer retinal atrophy with or without inflammatory cells in the retina and choroid that were procedural and/or test-article related. All vector-treated eyes showed improved cone-mediated electroretinography responses with no change in rod-mediated electroretinography responses. Behavioral maze testing under photopic conditions showed significantly improved navigation times and reduced numbers of obstacle collisions in all vector-treated eyes compared to their contralateral control eyes or pre-dose results in the treated eyes. These results support the use of AGTC-402 in clinical studies in patients with achromatopsia caused by CNGA3 mutations, with careful evaluation for possible inflammatory and/or toxic effects.

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.

Evaluation of the association of single nucleotide polymorphisms in the PRPH2 gene with adult-onset foveomacular vitelliform dystrophy.

OBJECTIVE: A minority of patients with adult-onset foveomacular vitelliform dystrophy (AFVD) carry mutations in the PRPH2 gene. This gene is highly polymorphic and it was suggested that single-nucleotide polymorphisms (SNPs) in PRPH2 may also be associated with AFVD. We aimed to evaluate for such an association. METHODS: A single center cohort from a tertiary referral center including 52 consecutive patients with a clinical diagnosis of AFVD and 91 unaffected individuals was assessed. Sanger sequencing was performed for the PRPH2, BEST1, and IMPG1/2 genes. Investigation as to the frequency of minor alleles for SNPs in PRPH2 was performed and compared to HapMap and Exome Variant Server (EVS) data. RESULTS: None of the patients carry a mutation in PRPH2, BEST1, or IMPG1/2. Five of 14 known SNPs (rs835, rs361524, rs434102, rs425876, rs390659) in exon 3 of PRPH2 were identified in AFVD patients. A high frequency and percentage of minor alleles of these five SNPs was found in the Israeli AFVD patients and controls compared with European, Chinese, Japanese and African populations identified via HapMap and EVS (p < 0.05). Power calculation suggested that the sample size was sufficient (80%) to rule out an association with an odds ratio above 2.5. CONCLUSIONS: These results suggest that genetic variants in PRPH2 do not compose a major genetic risk factor for AFVD. The Israeli population shows a higher percentage of minor allele frequencies in SNPs in the PRPH2 gene, as compared with other populations. This emphasizes the need for appropriate genetic background when performing SNP association testing.

Characterising the phenotype and progression of sporadic adult-onset foveomacular vitelliform dystrophy.

BACKGROUND/AIMS: Adult-onset foveomacular vitelliform dystrophy (AFVD) is a relatively common macular degeneration which might lead to substantial visual loss. Our purpose was to describe the natural course of genetically evaluated patients with sporadic AFVD. METHODS: A retrospective, consecutive, cohort study included 95 eyes of 51 patients. Mutations in genes previously associated with AFVD (PRPH2, BEST1, IMPG-1 and IMPG-2) were evaluated. Demographics, clinical characteristics, and spectral domain optical coherence tomography features were analysed. Main outcome measures were changes in the best corrected visual acuity (BCVA) and lesion morphology during the follow-up. RESULTS: The mean age (±SD) at diagnosis was 73.8±10.7 years. Mean (±SD) follow-up period was 30.4±16.3 months (range 0-44 months; median 25 months). All patients were genotyped negative for the evaluated mutations. Fifty-three of the eyes were followed for at least 36 months. At baseline these eyes had a mean BCVA (±SD) of 0.27±0.35 LogMAR, and at 36-months BCVA decreased to 0.38±0.35 (p=0.02). At baseline, 23 of these 53 eyes (43.4%) had the vitelliform stage, while only 10 eyes (18.9%) remained at this stage at 36 months (p=0.01). Ellipsoid zone alterations progressed during the follow-up (n=53 eyes) and showed correlation with BCVA reduction (Pearson's correlation coefficient=0.7, p=0.03). CONCLUSIONS: Sporadic AFVD is a slowly progressing macular degeneration of older people. It is associated with visual decline at the rate of approximately one ETDRS line during 3 years. Patients with sporadic AFVD are usually negative for the known mutations previously associated with this phenotype, and present at an age that is higher than described for monogenic AFVD.

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.

Bevacizumab treatment for neovascular age-related macular degeneration in the setting of a clinic: "real life" long-term outcome.

BACKGROUND: To evaluate the long-term outcome of bevacizumab therapy for neovascular age related macular degeneration (NVAMD) in the setting of a clinic. METHODS: Consecutive group of NVAMD patients who were treated in a single 3(rd) referral center with bevacizumab using a loading dosage of 3 monthly injections followed by variable dosing for at least 48 months were retrospectively evaluated. Genotyping was performed for CFH (rs1061170), HTRA1 (rs1200638), and C3 (rs2230199). Main outcome measures included functional and morphological treatment outcomes as well as their risk allele associations. RESULTS: Out of 128 patients who started bevacizumab treatment over 4 years before the study endpoint [mean (± SD): 60 ± 10.9 months], 75 eyes of 67 (52.3%) patients, were still followed. Mean best corrected visual acuity (BCVA) (LogMAR ± SEM) improved from 0.66 ± 0.07 at baseline to 0.48 ± 0.05 (p = 0.012) at 1 year, but deteriorated from the 3(rd) year on and at the final exam reduced to 0.69 ± 0.07 (p = 0.6, compared with initial BCVA). Macular thickness mirrored visual acuity (VA) changes showing initial thinning followed by thickening from the 3(rd) year on. Individuals carrying the CFH risk -allele had a mean thickening (microns ± SEM) of 66.9 ± 70.4 versus a mean thinning of 76.8 ± 22 in non-carriers (p = 0.015). CONCLUSIONS: Bevacizumab therapy for NVAMD using a flexible treatment algorithm in a "real life" clinical setting initially obtained VA gain and thinning of the macula that were maintained for two years, but were lost later on.

Flicker cone function in normal and day blind sheep: a large animal model for human achromatopsia caused by CNGA3 mutation.

PURPOSE: Recently we reported on day blindness in sheep caused by a mutation in the CNGA3 gene, thus making affected sheep a naturally occurring large animal model for therapeutic intervention in CNGA3 achromatopsia patients. The purpose of this study was to characterize flicker cone function in normal and day blind sheep, with the aim of generating a normative data base for ongoing gene therapy studies. METHODS: Electoretinographic (ERG) cone responses were evoked with full-field conditions in 10 normal, 6 heterozygous carriers and 36 day blind sheep. Following light adaptation (10 min, 30 cd/m(2)), responses were recorded at four increasing light intensities (1, 2.5, 5 and 10 cd s/m(2)). At each of these intensities, a single photopic flash response followed by 8 cone flicker responses (10-80 Hz) was recorded. Results were used to generate a normative data base for the three groups. Differences between day blind and normal control animals were tested in two age-matched groups (n = 10 per group). RESULTS: The normal sheep cone ERG wave is bipartite in nature, with critical flicker fusion frequency (CFF) >80 Hz. In all four flash intensities, the single photopic flash a-wave and b-wave amplitudes were significantly lower (p < 0.005), and implicit times significantly delayed (p < 0.0001), in day blind animals. In all four flash intensities, CFF values were significantly lower (p < 0.0001) in day blind sheep. CONCLUSIONS: Cone function is severely depressed in day blind sheep. Our results will provide a normative data base for ongoing gene therapy studies.

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.