Development of gene therapy for a severe form of inherited retinal degeneration due to defects in AIPL1

Leber‘s Congenital Amaurosis (LCA) is a term used to decribe a heterogeneous group of inherited retinal degenerations that affect children. One form is cause by mutations in a gene called AIPL1 that is expressed in photoreceptors. The retinal disease associated with AIPL1 mutations is progressive and affected children have severe visual impairment when they reach school age. There is currently no effective treatment. The aim of this research project is to use a mouse model of this condition to develop an effective gene therapy protocol that would be applicable to man. By introducing a normal copy of the defective AIPL1 gene we hope to delay or halt the death of photoreceptors and preserve vision. In order to do this we will engineer viral vectors to carry normal copies of the gene to the photoreceptor cells and injecting these into the mouse eye. We shall investigate the factors influencing the efficiency of the gene delivery and assess the outcome by examining the structure of the retina and performing electrical tests of visual function. This project will help us to optimise ocular gene therapy protocols and should facilitate the development of clinical trials. We shall also identify and characterise patients that might benefit from treatment.

Clinical trials of gene therapy in the eye are more readily justifiable for the most severe degenerations that include conditions such as Leber‘s Congenital Amaurosis (LCA). The group in which I work is funded by the Department of Health to conduct a clinical trial of gene therapy for one form of LCA that is caused by a defect in RPE65, a retinal pigment epithelium-specific gene. Defects in at least 11 other genes can give rise to LCA. Among these is the gene that encodes aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1). The gene, which is expressed in both rods and cones, is a specialised chaperone required for the biosynthesis of cGMP phosphodiesterase (PDE), an enzyme involved in phototransduction. The aim of this research project is to investigate the efficacy of gene replacement therapy for the treatment of Type 4 LCA using a transgenic mouse model of the disease and to identify and characterise patients that might benefit from treatment. Since AIPL1 is expressed in photoreceptors, and AAV-2/5 vectors are the most efficient at transducing this cell type, we shall use AAV-2/5 vectors containing mouse and human cDNAs driven by either CMV or photoreceptor cell specific promoters. I have already generated some of these constructs. Following subretinal injection of the vectors into mutant animals, I shall examine the effect on photoreceptor degeneration. Confocal microscopy of sections immunohistologically stained for AIPL1 protein will be used to determine the levels and location of product in the transduced cells. Analysis of retinal structure will be performed using light microscopy determine photoreceptor layer thickness and retinal architecture. The morphology of photoreceptor outer segments will analysed using electron microscopy. We will also the effect of treatment on photoreceptor degeneration and apoptosis by using GFAP and TUNEL staining. Since photoreceptor PDE levels are affected due to AIPL1 role in PDE synthesis, additional immunostaining for PDE will be performed. The ultimate goal of these studies will be to demonstrate the preservation of photoreceptor cell function after gene therapy. ERG will be carried out in treated and untreated animals for up to 1 year after treatment to determine whether there is functional improvement and evidence of sustained protection of photoreceptors. Children with LCA are seen on a regular basis in Professor Moore‘s clinics at Moorfields Eye Hospital and we have already collected DNA from over 150 affected children. The whole of the AIPL1 gene will be sequenced in these subjects and we are confident of identifying at least 10 patients with an AIPL1 mutation. Patients with AIPL1 mutations will undergo detailed phenotyping.