Bone marrow illustration
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A novel technique could potentially eradicate the toxic conditioning process required to effectively transplant hematopoietic stem and progenitor cells (HSPCs), which play a crucial role in treating blood disorders like sickle cell disease and β-thalassemia.

Researchers from Stanford University and the University of California, San Francisco (UCSF) utilized CRISPR-Cas9 to genetically modify HSPCs, enabling them to possess a natural human variant that enhances red blood cell production without disrupting HSPC functionality. This study, published in Nature Biomedical Engineering, shows how combining human genetics with precision genome editing can potentially lead to safer and more effective genome-editing therapies for patients with severe genetic diseases, particularly those affecting red blood cells. This study was performed in the labs of M. Kyle Cromer, PhD, and genetic engineering pioneer Matthew Porteus, MD, PhD, scientific founder of CRISPR Therapeutics, academic founder of Graphite Bio (which was acquired by LENZ Therapeutics), and co-founder of Kamau Therapeutics.

Olympic gold medalist’s mutation ups hemoglobin levels

One of the difficulties in HSPC gene therapy is ensuring that enough of the modified cells are successfully implanted to produce a favorable clinical outcome while also minimizing the associated risks. Although achieving clinically significant editing frequencies in HSPCs is possible, the current method requires chemotherapeutic treatments that significantly negatively affect the patient’s health to make room in the bone marrow for the edited HSPCs. However, this method can result in toxicities, such as an elevated susceptibility to cancer.

The study’s lead authors, Sofia E. Luna and Joab Camarena, hypothesized that introducing variants that cause congenital erythrocytosis, a rare noncancerous condition characterized by abnormally high levels of red blood cells and increased hemoglobin, could potentially stimulate the production of red blood cells and decrease toxic conditioning. To achieve this, they utilized the extensively researched genotype for congenital erythrocytosis, first identified in the family of a Finnish Olympic gold medalist in cross-country skiing. In this genotype, the individual’s hemoglobin levels were discovered to exceed the average by more than 50%. The increased hemoglobin level was caused by truncations in the erythropoietin receptor (tEPOR), removing the intracellular inhibitory domain that prevents erythropoietin (EPO) signaling.

Augmenting erythropoiesis using CRISPR technology 

There are big safety concerns about using viruses to move and express tEPOR by randomly inserting themselves into the genomes of billions of HSPCs during bone marrow transplantation, as shown by earlier research. This issue has led to a “black box” warning in the United States for Bluebird Bio’s recently approved LYFGENIA (lovotibeglogene autotemcel), a lentiviral gene therapy drug for sickle cell disease. Moreover, in these cases, viral-mediated delivery requires the expression of tEPOR using a non-native exogenous promoter. This deviates from the inherent regulation of EPOR and poses the risk of unintended consequences, such as the emergence of pathogenic polycythemia. 

So, Luna and Camarena explored multiple CRISPR-based genome-editing strategies to create the tEPOR variant, either by truncating the endogenous EPOR gene or integrating a tEPOR cDNA at safe-harbor loci. To improve the production of red blood cells in genome-edited HSPCs for disease correction, the researchers combined the tEPOR cassette with a previously established strategy for treating β-thalassemia. This approach entails the substitution of the HBA1 gene with a fully operational HBB gene through homology-directed repair (HDR)-based genome editing. They introduced an HBB transgene to restore normal hemoglobin production and increase erythropoietic output from edited HSPCs simultaneously.

To demonstrate the versatility of different tEPOR-introduction methods, the researchers also devised an alternative approach for genome editing. This approach involves simultaneously implementing the original strategy for correcting β-thalassaemia and introducing the EPOR truncation at the natural location in the genome. Both approaches resulted in an increase in the number of genetically modified red blood cells during the process of erythroid differentiation, in contrast to the conventional strategy for correcting β-thalassemia.

The assumed safety of introducing human variants

From a safety standpoint, utilizing CRISPR to integrate natural variations provides the benefit of already undergoing in vivo experimentation in humans. However, it is crucial to recognize that specific genome-editing methods can introduce tEPOR with regulatory mechanisms that are not naturally present, potentially altering the normal functioning of tEPOR. When examining this potential, the authors note that although all cells in patients with congenital erythrocytosis have a truncated EPOR gene, genome-editing therapy will only introduce tEPOR into a specific subset of HSPCs found in the bone marrow. So, it is unlikely that any abnormal outcomes arising from the manifestation of non-native tEPOR, such as a departure from lymphoid or other cellular categories, would lead to cytopenia. This is because many unedited HSPCs remain in the bone marrow after transplantation.

Also, the researchers suggest that by enhancing the generation of erythropoietic output from genetically altered HSPCs, this approach can reduce or eliminate the requirement for risky myeloablation treatments that are presently essential to attaining therapeutic levels of modified HSPCs. Thus, tEPOR expression could be incorporated into any treatment for hematological disorders that necessitate the transplantation of HSPCs. For example, in allogeneic hematopoietic stem cell transplants for red blood cell disorders, it is feasible to employ indel-based genome editing to generate a truncation in the natural EPOR. This alteration can confer a specific benefit to the transplanted erythroid progenitors. So, the approach presented here has the potential to simplify the use of less harmful myeloablative conditioning in situations where mixed chimerism is likely to happen.

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