Engineering enhanced erythropoiesis for red blood cell disorders - ABSTRACT In December 2023, the FDA approved the first genome editing therapy, which involves isolation of hematopoietic stem and progenitor cells (HSPCs) from a patient with sickle cell disease, ex vivo CRISPR editing, and re- transplantation into the bone marrow. This represented a landmark achievement for the fields of stem cell transplantation, genome editing, and precision medicine. However, the advent of CRISPR in the clinic has revealed new bottlenecks that limit translation of these potentially curative therapies. Current ex vivo HSC editing relies on chemotherapy- or radiation-based myeloablation to make space for genome-modified HSPCs in the bone marrow, which can lead to prolonged immunosuppression, negative impacts on fertility, and possible malignancy caused by genotoxicity. This represents a major safety concern for patients, however without myeloablation it would not be possible to achieve sufficient engraftment of genetically engineered cells to have a beneficial clinical effect. In addition, cost alone ($2.2M for the FDA-approved therapy) may prevent access for most patients, the vast majority of whom reside in developing nations. Direct in vivo editing of HSPCs emerges as an alternative to time- and cost-intensive ex vivo editing and would avoid myeloablation altogether. However, therapeutic in vivo editing is most effective in the liver, and achieving sufficient in vivo editing in HSPCs remains elusive. To address these challenges, we propose a novel approach: use genome engineering to enhance production of the desired cell type and effectively lower the therapeutic editing threshold. This solution is inspired by a rare condition called benign erythrocytosis, where otherwise healthy individuals have elevated levels of red blood cells and hemoglobin without increased cancer risk or reduced longevity. The proposed strategy aims to recapitulate the effect of naturally occurring mutations in the erythropoietin receptor (EPOR) using genome editing. Here, we will develop multiple strategies to pair the expression of a hypermorphic EPOR with sickle cell disease correction strategies in patient-derived HSPCs. These strategies include base editor-mediated multiplexing editing as well as Cas9/AAV-mediated gene insertion, which will be benchmarked against the FDA- approved therapy. We then will investigate the potential for this technology to mediate reduced or eliminated myeloablation in mouse models. Finally, we will adapt our most effective strategy for direct in vivo editing in HSPCs using a novel reporter mouse model. This approach could allow fewer edited HSPCs in the bone marrow to achieve a therapeutic effect, which could enable ex vivo editing to be curative using reduced-intensity myeloablation or enable direct in vivo editing of HSPCs, bypassing the need for cell isolation and re-transplantation. By drawing inspiration from nature and leveraging cutting-edge genome editing technology, this approach may improve the efficacy and safety of potentially curative treatments while paving the way for more accessible therapeutic options in the future.
