Post ERC: CRISPR-Cas9 for correcting primary immunodeficiencies

Prosjektet vil gjøre meg i stand til å søke ERC Starting Grant igjen i 2021.

Summary of the previous ERC-STG 2020 proposal: Monogenic blood diseases (MBD) are a group of hereditary disorders that cause malfunction of various types of blood cells. Some MBDs can be treated by bone marrow transplant, and some are managed using targeted therapies. However, there are no satisfactory therapies for the majority of MBDs, and patients die in early adulthood. The proposed study aimed to construct a CRISPR-Cas based gene therapy platform for monogenic blood diseases. The goal is a disease-independent system that corrects patient-specific mutations in different blood cell types (stem cells, T cells, NK cells). The corrected autologous T cells or NK cells can ameliorate uncontrolled infections and immune dysregulation and work as a bridge to allogeneic bone marrow transplant in terminally ill patients. By modifying stem cells, the platform can work as a stand-alone curative therapy. The specific aims of the project were the following: 1. Develop safe and efficient CRISPR gene editing platform for primary T cells, NK cells and blood stem cells by a) fusing editing-enhancing mammalian proteins to Cas9; b) binding the repair DNA template to Cas9; c) timing genome editing with cell cycle and d) transiently inhibiting the cellular danger signaling. 2. Evaluate platform safety in vitro. The edited cells are grown in culture and assessed for tumorigenic potential. Furthermore, the cell functionality is assessed by colony-forming assays (stem cells) and cytotoxic lymphocyte function tests (T cells and NK cells). 3. Test the platform in patient cells and animal models by transplanting the edited cells to SCID mice, and determining their capacity to form a functional bone marrow (stem cells) and persist in circulation (T, NK cells). We will also edit a set of patient cells.