CRISPR-Cas -mediated gene therapy for monogenic blood disorders

Approximately 50 children are born with a hereditary blood disease in Norway every year. Most of these conditions can be cured with bone marrow transplantation, where the diseased bone marrow is replaced with a healthy one from a parent, sibling or unrelated donor. Bone marrow transplant, however, is a difficult and risky procedure, and can cause challenging side effects and even death. Due to the risks, most patients are managed symptomatically. This is problematic, because the disease progresses with time and causes complications such as autoimmune diseases and cancer when the child is older. In this study we developed gene therapy using CRISPR-Cas9 technology to correct genetic mutations in patients' own cells. Our main aim was, and still is, to create a platform for treating various blood disorders in a personalized manner. Throughout the project, we've optimized our gene editing techniques for T cells and stem cells, while also developing protocols to ensure the effectiveness and safety of our approach. Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein - Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the particular cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.

Long term impacts remain unchanged. We have significantly improved CRSIPR-Cas9 gene editing protocols for immune and stem cells, bringing us one step closer to achieving our long-term goals.

AIMS: We aim to develop a CRISPR-Cas-based gene therapy platform for monogenic blood disorders. BACKGROUND: Monogenic blood diseases include primary immunodeficiencies, bone marrow failure syndromes, and disorders of platelet and red blood cell function. The conditions are curative with hematopoietic stem cell transplant, but the procedure carries significant treatment-related complications. The novel CRISPR-Cas system can be used to cure the disease by correcting the causative mutation in harvested patient stem cells, after which the cells can be infused back to the patient. The technique, however, needs revision before clinical use as its efficiency is relatively low and side effects (cancer, immunological phenomena) unknown. WORKPLAN AND PRELIMINARY RESULTS: To increase efficiency for mutation correction, we have optimized the editing properties of Cas9 in various ways. We have attached different editing-enhancing proteins to Cas9, timed editing with cell cycle, and attenuate cell danger signaling during editing. In this study, we will bring all these modifications together into a unified editing platform. We will extensively test the safety of the modified system for off-target cutting and efficiency in hematopoietic cells and mouse models, with the aim of translating the technique to clinical trials within the next 5 years. SIGNIFICANCE: If successful, the method can treat a wide variety of genetic blood diseases and mutations. It is also useful in other therapies that require genetic manipulation of immune cells, such as in cancer immunotherapy.