Therapeutic genome editing to treat Stargardt disease – Generation and phenotyping of a porcine model and development of a treatment approach

• Applicant: Professor Dr. Knut Stieger
• Subject Area: Ophthalmology
• Term: from 2019 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 412915769

Stargardt disease, caused by mutations in the ABCA4 gene, is the most common juvenile maculopathy. The disease manifests most often within the macular area with RPE and cone photoreceptor degeneration, succeeded by generalized cone and rod degeneration at later stages. The ABCA4 protein transports retinoids through the photoreceptor disc membrane, thus making them available for entering the visual cycle. Absence of the protein or loss of function mutations cause accumulation of toxic retinoids within the RPE, leading to cell death. Absence of appropriate animal model systems with cone enriched retinae has prevented scientists from gathering more details about the pathogenicity for early cone photoreceptor disease and to develop effective treatment approaches.Therapeutic genome editing employing highly specific endonucleases, such as the CRISPR-Cas9 system, has recently gathered much attention in the gene therapy field. They induce a double strand break at the target site, which is subsequently repaired by the cells own repair mechanisms. Ideally, through the use of a template DNA comprising the wild type sequence, a disease-causing mutation can be corrected during this repair process. Unfortunately, little is known about DNA repair activity in post-mitotic and highly specialized photoreceptors.The minipig represents a unique, biomedical model thanks to its size and comparable physiological parameters with men, as well as its retinal morphology with a cone enriched region. To study ABCA4 associated RPE and cone photoreceptor disease and to develop therapeutic genome editing as treatment approach, we aim at generating a porcine model of Stargardt disease containing the human pathologic V1973X null mutation. To achieve this goal, we will join forces in an interdisciplinary team. We will first generate piglets by CRISPR-Cas9 mediated genome editing in one cell embryos. Upon successful production of founder animals, we will phenotype them by in vivo (electroretinography, optical coherence tomography, behavioral studies) as well as post mortem (retinal morphology, ultra-morphology, immunohistochemistry, proteomics, RNA seq, retinoid end product quantification) analysis methods, and will start generating a colony. We will in parallel study DNA repair activity in porcine photoreceptors and optimize genome editing activity by appropriate interference with DNA repair proteins. We will study gene transfer of all components necessary for genome editing by AAV in wild type pigs prior to employing the optimal composition in the disease model.Data from this project will enable us to continue optimizing the treatment approach in subsequent studies, as well as studying other treatment approaches such as cell transplantation. Our joint interdisciplinary project will develop an efficient and safe therapeutic application in a relevant large animal model that will pave the way towards clinical application in the future.

• DFG Programme: Research Grants
• International Connection: Czech Republic
• Partner Organisation: Czech Science Foundation
• Cooperation Partner: Professor Jan Motlik, Ph.D.