Purpose

To investigate fundus autofluorescence (AF) characteristics in the Abca4(-/-) mouse, an animal model for AMD and Stargardt disease, and to correlate findings with functional, structural, and biochemical assessments.

Methods

Blue (488 nm) and near-infrared (790 nm) fundus AF images were quantitatively and qualitatively analyzed in pigmented Abca4(-/-) mice and wild type (WT) controls in vivo. Functional, structural, and biochemical assessments included electroretinography (ERG), light and electron microscopic analysis, and A2E quantification. All assessments were performed across age groups.

Results

In Abca4(-/-) mice, lipofuscin-related 488 nm AF increased early in life with a ceiling effect after 6 months. This increase was first paralleled by an accumulation of typical lipofuscin granules in the retinal pigment epithelium (RPE). Later, lipofuscin and melanin granules decreased in number, whereas melanolipofuscin granules increased. This increase in melanolipofuscin granules paralleled an increase in melanin-related 790 nm AF. Old Abca4(-/-) mice revealed a flecked fundus AF pattern at both excitation wavelengths. The amount of A2E, a major lipofuscin component, increased 10- to 12-fold in 6- to 9-month-old Abca4(-/-) mice compared with controls, while 488 nm AF intensity only increased 2-fold. Despite pronounced lipofuscin accumulation in the RPE of Abca4(-/-) mice, ERG and histology showed a slow age-related thinning of the photoreceptor layer similar to WT controls up to 12 months.

Conclusions

Fundus AF can be used to monitor lipofuscin accumulation and melanin-related changes in vivo in mouse models of retinal disease. High RPE lipofuscin may not adversely affect retinal structure or function over prolonged time intervals, and melanin-related changes (melanolipofuscin formation) may occur before the decline in retinal function.

The recent approval in the United States of the first adeno-associated viral (AAV) vector for the treatment of an inherited retinal degeneration validates this approach for the treatment of many other diseases. A major limiting factor continues to be the size restriction of the AAV transgene at under 5 kb. Stargardt disease is the most prevalent form of recessively inherited blindness and is caused by mutations in ABCA4, the gene that codes for ATP-binding cassette transporter protein family member 4, which has a coding sequence length of 6.8 kb. Dual vector approaches increase the capacity of AAV gene therapy, but at the cost of substantially reduced levels of target protein, which may be insufficient to achieve a therapeutic effect. Here we show that the efficacy of recombination of dual vectors is dependent on the length of DNA overlap between two transgenes. With optimized recombination, full-length ABCA4 protein is expressed in the photoreceptor outer segments of Abca4^-/- mice at levels sufficient to reduce bisretinoid formation and correct the autofluorescent phenotype. These observations support a dual vector approach in future clinical trials using AAV gene therapy to treat Stargardt disease.

Stem cell transplantation holds great promise as a potential treatment for currently incurable retinal degenerative diseases that cause poor vision and blindness. Recently, safety data have emerged from several Phase I/II clinical trials of retinal stem cell transplantation. These clinical trials, usually run in partnership with academic institutions, are based on sound preclinical studies and are focused on patient safety. However, reports of serious adverse events arising from cell therapy in other poorly regulated centers have now emerged in the lay and scientific press. While progress in stem cell research for blindness has been greeted with great enthusiasm by patients, scientists, doctors and industry alike, these adverse events have raised concerns about the safety of retinal stem cell transplantation and whether patients are truly protected from undue harm. The aim of this review is to summarize and appraise the safety of human retinal stem cell transplantation in the context of its potential to be developed into an effective treatment for retinal degenerative diseases.

We assessed genotype-phenotype correlations among the visual, auditory, and olfactory phenotypes of 127 participants with Usher syndrome (USH2) (n =80) or nonsyndromic autosomal recessive retinitis pigmentosa (ARRP) (n = 47) due to USH2A variants, using clinical data and molecular diagnostics from the Rate of Progression in USH2A Related Retinal Degeneration (RUSH2A) study. USH2A truncating alleles were associated with USH2 and had a dose-dependent effect on hearing loss severity with no effect on visual loss severity within the USH2 subgroup. A group of missense alleles in an interfibronectin domain appeared to be hypomorphic in ARRP. These alleles were associated with later age of onset, larger visual field area, better sensitivity thresholds, and better electroretinographic responses. No effect of genotype on the severity of olfactory deficits was observed. This study unveils a unique, tissue-specific USH2A allelic hierarchy with important prognostic implications for patient counseling and treatment trial endpoints. These findings may inform clinical care or research approaches in others with allelic disorders or pleiotropic phenotypes.