Age-dependent effects of RPE65 gene therapy for Leber's congenital amaurosis: a phase 1 dose-escalation trial.

BACKGROUND: Gene therapy has the potential to reverse disease or prevent further deterioration of vision in patients with incurable inherited retinal degeneration. We therefore did a phase 1 trial to assess the effect of gene therapy on retinal and visual function in children and adults with Leber's congenital amaurosis. METHODS: We assessed the retinal and visual function in 12 patients (aged 8-44 years) with RPE65-associated Leber's congenital amaurosis given one subretinal injection of adeno-associated virus (AAV) containing a gene encoding a protein needed for the isomerohydrolase activity of the retinal pigment epithelium (AAV2-hRPE65v2) in the worst eye at low (1.5 x 10(10) vector genomes), medium (4.8 x 10(10) vector genomes), or high dose (1.5 x 10(11) vector genomes) for up to 2 years. FINDINGS: AAV2-hRPE65v2 was well tolerated and all patients showed sustained improvement in subjective and objective measurements of vision (ie, dark adaptometry, pupillometry, electroretinography, nystagmus, and ambulatory behaviour). Patients had at least a 2 log unit increase in pupillary light responses, and an 8-year-old child had nearly the same level of light sensitivity as that in age-matched normal-sighted individuals. The greatest improvement was noted in children, all of whom gained ambulatory vision. The study is registered with ClinicalTrials.gov, number NCT00516477. INTERPRETATION: The safety, extent, and stability of improvement in vision in all patients support the use of AAV-mediated gene therapy for treatment of inherited retinal diseases, with early intervention resulting in the best potential gain. FUNDING: Center for Cellular and Molecular Therapeutics at the Children's Hospital of Philadelphia, Foundation Fighting Blindness, Telethon, Research to Prevent Blindness, F M Kirby Foundation, Mackall Foundation Trust, Regione Campania Convenzione, European Union, Associazione Italiana Amaurosi Congenita di Leber, Fund for Scientific Research, Fund for Research in Ophthalmology, and National Center for Research Resources.

Predicting the pathogenicity of RPE65 mutations.

To assist in distinguishing disease-causing mutations from nonpathogenic polymorphisms, we developed an objective algorithm to calculate an "estimate of pathogenic probability" (EPP) based on the prevalence of a specific variation, its segregation within families, and its predicted effects on protein structure. Eleven missense variations in the RPE65 gene were evaluated in patients with Leber congenital amaurosis (LCA) using the EPP algorithm. The accuracy of the EPP algorithm was evaluated using a cell-culture assay of RPE65-isomerase activity The variations were engineered into plasmids containing a human RPE65 cDNA and the retinoid isomerase activity of each variant was determined in cultured cells. The EPP algorithm predicted eight substitution mutations to be disease-causing variants. The isomerase catalytic activities of these RPE65 variants were all less than 6% of wild-type. In contrast, the EPP algorithm predicted the other three substitutions to be non-disease-causing, with isomerase activities of 68%, 127%, and 110% of wild-type, respectively. We observed complete concordance between the predicted pathogenicities of missense variations in the RPE65 gene and retinoid isomerase activities measured in a functional assay. These results suggest that the EPP algorithm may be useful to evaluate the pathogenicity of missense variations in other disease genes where functional assays are not available.

Safety and efficacy of gene transfer for Leber's congenital amaurosis.

Leber's congenital amaurosis (LCA) is a group of inherited blinding diseases with onset during childhood. One form of the disease, LCA2, is caused by mutations in the retinal pigment epithelium-specific 65-kDa protein gene (RPE65). We investigated the safety of subretinal delivery of a recombinant adeno-associated virus (AAV) carrying RPE65 complementary DNA (cDNA) (ClinicalTrials.gov number, NCT00516477 [ClinicalTrials.gov]). Three patients with LCA2 had an acceptable local and systemic adverse-event profile after delivery of AAV2.hRPE65v2. Each patient had a modest improvement in measures of retinal function on subjective tests of visual acuity. In one patient, an asymptomatic macular hole developed, and although the occurrence was considered to be an adverse event, the patient had some return of retinal function. Although the follow-up was very short and normal vision was not achieved, this study provides the basis for further gene therapy studies in patients with LCA.

Familial cavitary optic disk anomalies: clinical features of a large family with examples of progressive optic nerve head cupping.

PURPOSE: To describe a multigenerational family with autosomal dominant inheritance of cavitary optic nerve head (ONH) anomalies and abnormal ONH vasculature. DESIGN: Description of a single family with inherited eye disease. METHODS: A four-generation pedigree was investigated. Examination included visual acuity, slit-lamp biomicroscopy, intraocular pressure (IOP) measurement, and ophthalmoscopy. Visual fields and fundus photography were obtained when possible. RESULTS: Seventeen clinically affected individuals and two obligate carriers were identified. Most (64.7%) affected persons had bilateral involvement. Visual acuity in affected eyes ranged from 20/20 to no light perception. Although the appearance of affected nerves varied greatly, most lacked a well-formed central retinal artery and instead had multiple radial cilioretinal arteries. Prominent cupping was seen in most affected nerves. Four individuals for whom information was available were treated for glaucoma, but none had documented elevated IOP. Four eyes of two patients demonstrated progressive ONH cupping at normal IOPs. Nine (56.3%) of the 16 individuals for whom we had data had evidence of serous macular detachments; five of these had bilateral macular disease. CONCLUSIONS: A large family with autosomal dominant inheritance of cavitary ONH anomalies and abnormal vasculature is presented. Clinical phenotypes varied markedly. Progressive ONH cupping was documented in four eyes of two patients. Genetic linkage analysis of this family has identified the chromosomal location of a gene responsible for ONH development. This may provide insight into the pathogenesis of glaucomatous ONH damage.

Macular pigment and lutein supplementation in ABCA4-associated retinal degenerations.

PURPOSE: To determine macular pigment (MP) optical density (OD) in patients with ABCA4-associated retinal degenerations (ABCA4-RD) and the response of MP and vision to supplementation with lutein. METHODS: Patients with Stargardt disease or cone-rod dystrophy and known or suspected disease-causing mutations in the ABCA4 gene were included. All patients had foveal fixation. MPOD profiles were measured with heterochromatic flicker photometry. Serum carotenoids, visual acuity, foveal sensitivity, and retinal thickness were quantified. Changes in MPOD and central vision were determined in a subset of patients receiving oral supplementation with lutein for 6 months. RESULTS: MPOD in patients ranged from normal to markedly abnormal. As a group, patients with ABCA4-RD had reduced foveal MPOD, and there was a strong correlation with retinal thickness. Average foveal tissue concentration of MP, estimated by dividing MPOD by retinal thickness, was normal in patients, whereas serum concentration of lutein and zeaxanthin was significantly lower than normal. After oral lutein supplementation for 6 months, 91% of the patients showed significant increases in serum lutein, and 63% of the patients' eyes showed a significant augmentation in MPOD. The retinal responders tended to be female and to have lower serum lutein and zeaxanthin, lower MPOD, and greater retinal thickness at baseline. Responding eyes had significantly lower baseline MP concentration than did nonresponding eyes. Central vision was unchanged after the period of supplementation. CONCLUSIONS: MP is strongly affected by the stage of ABCA4 disease leading to abnormal foveal architecture. MP could be augmented by supplemental lutein in some patients. There was no change in central vision after 6 months of lutein supplementation. Long-term influences of this supplement on the natural history of these macular degenerations require further study.

Glaucoma phenotype in pedigrees with the myocilin Thr377Met mutation.

OBJECTIVE: To investigate the phenotype and age-related penetrance of primary open-angle glaucoma (POAG) in Australian families with the myocilin mutation Thr377Met. METHOD AND DESIGN: Cross-sectional genetic study. Four unrelated pedigrees carrying the Thr377Met mutation were ascertained from more than 2000 consecutive cases of POAG in the Glaucoma Inheritance Study in Tasmania and from families with glaucoma referred to the study from throughout Australia. Index cases and available family members were examined for signs of glaucoma, and the presence of the GLC1A Thr377Met mutation was ascertained by single-strand conformation polymorphism analysis and subsequent direct sequencing. RESULTS: From the 4 pedigrees carrying the Thr377Met mutation, 23 individuals with either ocular hypertension (OHT) or POAG were found, with a mean +/- SD age at diagnosis of 41.2 +/- 11.5 years, and a mean peak intraocular pressure of 31.7 +/- 9.9 mm Hg. A further 9 mutation carriers older than 18 years were studied who as yet showed no signs of OHT or POAG (6 of these 9 were younger than 30 years). A single individual with POAG was identified who did not carry the Thr377Met mutation. For Thr377Met carriers, age-related penetrance for OHT or POAG was 88% at age 30 years. A positive family history of POAG was present for 3 of the 4 index cases. Thirteen (57%) of the 23 Thr377Met carriers with OHT or POAG had undergone glaucoma drainage surgery. Although the glaucoma in these families appears to be pressure dependent, 2 individuals showed optic disc cupping before detected elevation in intraocular pressure. One family was of British origin, with a different background haplotype from the other 3 families from Greece or Macedonia, who shared a common haplotype. CONCLUSIONS: The GLC1A Thr377Met mutation is associated with POAG that, in the pedigrees studied, had a younger age at onset and higher peak intraocular pressure than in pedigrees with the more common Gln368STOP mutation. In addition, patients with glaucoma with the Thr377Met mutation were more likely to have undergone glaucoma drainage surgery.

Macular pigment and lutein supplementation in choroideremia.

Choroideremia is an incurable X-linked retinal degeneration caused by mutations in the gene encoding Rab escort protein-1. A group of clinically defined and genotyped patients were studied to determine: (1) the degree of rod and cone dysfunction and structural abnormality in the central retina and the level of macular pigment; and (2) the response of macular pigment and foveal vision to a 6 month trial of supplementation with oral lutein (at 20 mg per day). Rod and cone-mediated function was measured with dark-adapted static perimetry; in vivo retinal structure was determined with optical coherence tomography; and macular pigment optical density was measured with heterochromatic flicker photometry. In this cohort of patients (ages 15-65 years), both rod- and cone-mediated central function declined with age as did central retinal thickness. Macular pigment levels did not differ between patients and male control subjects. Supplementation of oral lutein in a subset of patients led to an increase in serum lutein and macular pigment levels; absolute foveal sensitivity did not change. It is concluded that macular pigment density can be augmented by oral intake of lutein in patients with choroideremia. There was no short-term change in the central vision of the patients on the supplement, but long-term influences of lutein supplementation on disease natural history warrant further study.