Abstract for laypersons

Retinitis Pigmentosa and Leber’s congenital Amaurosis are two hereditary diseases that can be caused by mutations in a gene called CRB1. Both diseases lead to a graduate vision loss and even blindness starting from early in life to old age. Today, there is no cure or treatment which could stop or prevent the disease. RNA base editing represents a possible new treatment option. The method uses small RNA molecules that correct the disease-causing mutation in the messenger RNA and therefore leaves the genomic DNA of the patient untouched. In this study, we aim to test this new therapeutic approach on a cell culture model which can recreate the organization and makeup of the human retina, so called retinal organoids, in a dish. These retinal organoids are generated from cells which were derived from patients suffering from Retinitis pigmentosa. During the project, we will test RNA base editing for its efficiency to correct the mutation and for potential adverse effects on the retina. Furthermore, we aim to optimize the RNA molecules for an efficient delivery to the retina. If successful, this project could pave the way to a novel treatment option for patients suffering from vision loss and blindness.

Scientific abstract

Retinitis Pigmentosa and Leber’s congenital Amaurosis are two forms of retinal degeneration, which can be caused by mutations in the CRB1 gene. A particularly common mutation is a missense mutation caused by a single nucleotide exchange of guanosine with adenosine. Until today, there is no curative or even significantly stalling treatment. A possible gene therapy using adeno-associated virus (AAV) vectors is hampered by the size of the CRB1 gene, which is exceeding the AAV package capacity, and by the presence of several CRB1 splice variants. An alternative to classic gene therapy approaches is offered by a novel RNA base editing technology. Using tailor-made antisense oligonucleotides (ASOs), it is possible to recruit the endogenous RNA editing protein ADAR (Adenosine Deaminase acting on RNA), to rewrite single adenosine nucleotides into inosine in a site-specific and programmable fashion. As inosine is biochemically interpreted as guanosine this leads to correction of the common disease-causing CRB1 mutation selected. The aim of this study is to develop chemically and pharmacologically optimized ASOs, which could potentially reverse the selected mutation in patients. To show the efficiency of the ASO therapy, we make use of induced pluripotent stem cell lines (iPSC) of two patients who suffer from a severe form of retinitis pigmentosa and carry a homozygous point mutation. These iPSC lines have been created previously and can be differentiated to retinal organoids, which contain all major retinal cell types in a physiological layering and show light sensitivity. In an iterative process, ASOs will be designed and tested on retinal organoids as well as organoid- derived 2D and 3D Müller glia cells enabling a rapid development of effective ASOs. The efficacy of the therapy will be assessed by characterizing the expression level and localization of the CRB1 protein, which are both aberrant due to the mutation. The goal is to identify a lead ASO which shows the desired biological outcome with high potency and specificity. In a next step, this lead ASO will be further pharmacologically optimized and profiled. Here, we will make use of our recently developed retina-on-chip system, which allows physiologically accurate modelling of drug delivery. In a final effort, we will create initial toxicological and immunological data, serving as a first preclinical data set to prepare for a subsequent clinical development. Overall, this project will establish a yet unprecedented ASO therapy which could cure patients affected by a common CRB1 mutation. Furthermore, we aim to showcase an animal testing-free pre-clinical development of this new class of drug compounds.