Personalized Gene Therapy for Fanconi Anemia: Precision Prime Editing to Target Hematopoietic Stem Cells

Abstract

Fanconi Anemia (FA) is a severe genetic disease that often results in progressive loss of bone marrow function. The FANCA gene is one of the 23 genes that can be mutated in FA patients and is mutated in more than 60% of FA patients. Restoration of this gene has been shown to restore the function of the DNA repair mechanism that is impaired in FA patients. Right now the most used treatment for progressive bone marrow loss in FA patients is blood transplant, which is often accompanied by immunosuppressives and chemotoxic conditioning. In vivo targeting of hematopoietic stem cells with prime editors is an interesting treatment option with minimal off-target effects in the genome. This strategy would prevent adverse effects that are observed after blood transfusions. In this proposal, I describe how I will design a prime editing pegRNA library that can target the most important FANCA mutations. Furthermore, I propose a prime editing screen to find the most efficient prime editing strategy for each patient using patient-derived cells. I keep the cost of this screen as low as possible using a high-throughput model with a sensor-based editing efficiency measurement. A novel virus-like particle will be made to enable HSPC targeting with prime editing machinery. Finally, the most efficient pegRNAs that are found during the personalized screens will be delivered to patient-derived HSPCs to restore the FANCA mutation. Successful and personalized gene therapy for FA patients has the potential to change the lives of a lot of patients who have a very poor prognosis. This proposal aims to prevent progressive bone marrow failure and provide better alternatives for stem cell transplantations. However, in the future personalized prime editing strategies can easily be extended to other cell types using alternative delivery vehicles. Moreover, other FA genes can easily be included in the prime editing library to expand the patient population. In summary, this project provides a solid foundation to treat FA patients in a revolutionary way.