Novel therapeutic approaches to rescue retinal dysfunction in patient-derived photoreceptors

Lebers congenital amaurosis (LCA) is a devastating disease leading to the failure of vision because the light-sensing photoreceptor cells in the eye do not work properly even within the first few years of life. There is currently no cure or treatment for LCA. Inherited genetic mistakes in the AIPL1 gene are one of the causes of LCA. In this study, we want to create a new laboratory system that accurately models the human disease, so that we can test novel therapies to treat LCA caused by faults in the AIPL1 gene. Using state-of-the-art technologies optimised in our group, we will take cells isolated from the urine of LCA patients and reprogramme them to so called induced pluripotent stem cells (iPSC) from which we will grow three dimensional 'optic cups' in a dish in the laboratory, the 'retina in a dish'. This is important, as it is not possible to isolate and culture the light sensitive photoreceptor cells of the retina that are faulty in LCA directly from patients. Therefore, the derivation of these photoreceptor cells from LCA patients via formation of optic cups is critical to model the disease in humans and will be invaluable to validate new AIPL1-targeted therapies that may treat or cure this devastating disease. We will first test a pharmacological approach that targets a class of mutations encountered in up to 60% of all LCA patients with AIPL1 faults. These drugs, which can be safely applied as an eye drop, or as a tablet, target the cell machinery that converts a gene to a protein. We will test whether these drugs can rescue the normal AIPL1 protein from the faulty AIPL1 gene. The rescued AIPL1 protein will be able to perform its normal function in the cell. We will also develop treatments for a different kind of mutation in the AIPL1 gene that interferes with the way different parts of the AIPL1 gene are stitched or 'spliced' together to form the full-length message for the AIPL1 protein. We will test whether our custom-design therapeutic agents can 'override' these splice mutations to form a normal full-length AIPL1 message coding for a normal full-length and functional AIPL1 protein. These novel therapies will be tested on optic cups made from patients harbouring these classes of different AIPL1 mutations. The findings of this research are a critical step towards the application of these novel therapies to treat AIPL1 LCA patients in the clinical setting. The drug that will be used in this study has already been approved by NICE for the treatment of another disease caused by stop mutations, and a similar therapeutic agent targeting splice mutations in another gene causing LCA will shortly enter phase I clinical trials. Hence, the potential for our findings to rapidly enter the clinical phase and make a difference to patients' lives is very high.

AIPL1 mutations cause LCA, the most severe inherited retinal dystrophy, for which there is no cure or treatment. We will combine our expertise in iPSC technology and optic cup differentiation with our understanding of AIPL1 function to model human AIPL1 LCA, to develop and test novel pharmacological and RNA-directed therapeutic strategies targeting the most common classes of AIPL1 mutations. iPSC will be derived from LCA patients homozygous for the c.834G>A(p.W278X) mutation by episomal reprogramming of renal epithelial cells. The c.834G>A mutation will be corrected by genome editing in the patient-derived iPSC using the CRISPR/Cas9 system followed by targeted homologous repair (HDR) with an AIPL1-specific piggyBac transposon. Patient-derived iPSC and control isogenic iPSC will be differentiated to retinal photoreceptor cells via the formation of 3D optic cups. Translational read-through inducing drugs (TRIDs) will be tested for their efficacy for recoding premature termination codons and rescuing the expression and function of full-length AIPL1 in vitro (p.R32X, p.W72X, p.W88X, p.Q163X and p.W278X) and in c.834G>A(p.W278X) patient-derived ocular cups. AIPL1 mutations disrupting a canonical splice donor dinucleotide (c.276+1G>A, c.276+2T>C) or consensus splice donor sequence (c.465G>T, c.642G>C, c.784G>A) will be targeted with modified complementary and exon-specific U1 snRNA. The rescue of transcription of the full-length gene, and expression and function of the AIPL1 protein, will be assessed in vitro and in optic cups derived from an LCA patient compound heterozygous for c.276+1G>A and a patient homozygous for c.624G>C(p.K214N). In situ splice site editing in optic cups will be achieved via direct delivery or viral transduction. Validation of these therapies in vitro and in patient-derived ocular cups will be critical for translation into the clinic, with broader application of our proof-of-concept findings to other inherited retinal dystrophies.

The main beneficiary of the proposed study will be future and current patients with LCA, for which there is currently no treatment or cure. The current proposal will increase understanding of the underlying pathophysiological mechanisms through disease modelling and test novel pharmacological and RNA-directed therapeutic approaches to rescue the pathology. If validated, the results from this study will have a major impact on directing future research towards these therapeutic interventions and drug-development efforts in a clinical setting. We also envision that the data generated will be of direct relevance to academic researchers, clinicians and commercial organizations with an interest in developing therapies for the treatment of other inherited retinal degenerative diseases. The UK has a very strong scientific position in leading the translation of fundamental scientific findings to patient benefit, and the proposed study has the potential to further strengthen that by validating the efficacy and delivery of novel therapeutic tools to pre-clinical disease models through harnessing advances in RNA and genome editing therapies and in induced pluripotent stem cell technology. In particular, the UCL Institute of Ophthalmology has led the way in gene and cell directed therapies for the treatment of retinal disease, including the first-in-human gene therapy trial to treat Leber Congenital Amaurosis caused by deficiency in the RPE65 gene and the first international transplantation strategy for inherited macular dystrophy. Targeted patient groups will benefit from the proposed research through our public engagement programme facilitated by the UCL Public Engagement Unit, which has extensive experience of public and community engagement to increase impact derived from UCL research. This impact will be realized through engagement events, such as the annual Patient Days held at Moorfields Eye Hospital, where patient input and feedback will be used to focus future research priorities.