Biology subjects

Soupene, E.

Publications and source records attributed to Soupene, E..

3 recordsLinked to original sources

bioRxiv2024

Engineering inducible signaling receptors to enable erythropoietin-free erythropoiesis

Blood transfusion plays a vital role in modern medicine. However, availability is contingent on donated blood, and frequent shortages pose a significant healthcare challenge. Ex vivo manufacturing of red blood cells (RBCs) derived from universal donor O-negative pluripotent stem cells emerges as a solution, yet the high cost of recombinant cytokines required for ex vivo erythroid differentiation remains a major barrier. Erythropoietin (EPO) signaling through the EPO receptor is indispensable to RBC development, and EPO is one of the most expensive components in erythroid-promoting media. Here, we used design-build-test cycles to develop highly optimized small molecule-inducible EPO receptors (iEPORs) which were integrated at a variety of genomic loci using homology-directed repair genome editing. We found that integration of iEPOR at the endogenous EPOR locus in an induced pluripotent stem cell producer line enabled culture with small molecule to yield equivalent erythroid differentiation, transcriptomic changes, and hemoglobin production compared to cells cultured with EPO. Due to the dramatically lower cost of small molecules vs. recombinant cytokines, these efforts eliminate one of the most expensive elements of ex vivo culture media--EPO cytokine. Because dependence on cytokines is a common barrier to ex vivo cell production, these strategies could improve scalable manufacturing of a wide variety of clinically relevant cell types. More broadly, this work showcases how synthetic biology and genome editing may be combined to introduce precisely regulated and tunable behavior into cells, an advancement which will pave the way for increasingly sophisticated cell engineering strategies.

synthetic biology↗

bioRxiv2023

Dual α-globin and truncated erythropoietin receptor knock-in restores hemoglobin production in α-thalassemia major-derived hematopoietic stem and progenitor cells

Alpha-thalassemia is an autosomal recessive disease with increasing worldwide prevalence. The molecular basis is due to mutation or deletion of one or more duplicated -globin genes, and disease severity is directly related to the number of allelic copies compromised. The most severe form, -thalassemia major (TM), results from loss of all four copies of -globin and has historically resulted in fatality in utero. However, in utero transfusions now enable survival to birth. Postnatally, patients face challenges similar to {beta}-thalassemia, including severe anemia and erythrotoxicity due to imbalance of {beta}-globin and -globin chains. While curative, hematopoietic stem cell transplantation (HSCT) is limited by donor availability and potential transplant-related complications. Despite progress in genome editing treatments for {beta}-thalassemia, there is no analogous curative option for patients suffering from -thalassemia. To address this, we designed a novel Cas9/AAV6-mediated genome editing strategy that integrates a functional -globin gene into the {beta}-globin locus in TM patient-derived hematopoietic stem and progenitor cells (HSPCs). Incorporation of a truncated erythropoietin receptor transgene into the -globin integration cassette dramatically increased erythropoietic output from edited HSPCs and led to the most robust production of -globin, and consequently normal hemoglobin. By directing edited HSPCs toward increased production of clinically relevant RBCs instead of other divergent cell types, this approach has the potential to mitigate the limitations of traditional HSCT for the hemoglobinopathies, including low genome editing and low engraftment rates. These findings support development of a definitive ex vivo autologous genome editing strategy that may be curative for -thalassemia. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/555926v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1b63dacorg.highwire.dtl.DTLVardef@18b25a1org.highwire.dtl.DTLVardef@53835corg.highwire.dtl.DTLVardef@d52bf5_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Using human genetics to develop strategies to increase erythropoietic output from genome-edited hematopoietic stem and progenitor cells

Human genetic polymorphisms result in a diversity of phenotypes. Some sequences are pathologic and lead to monogenic diseases, while others may confer beneficial traits. Genome editing is a powerful tool to recreate genotypes found in the population, including the ability to correct pathologic mutations. One of the best characterized naturally occurring mutations causing congenital erythrocytosis arises from a truncation in the erythropoietin receptor (tEPOR) which can result in non-pathogenic hyper-production of red blood cells (RBCs). Using the precision of CRISPR/Cas9 genome editing, we have recreated tEPOR and studied the effect of variations of the genotype on RBC development. We then combined tEPOR with a correction strategy developed for {beta}-thalassemia and demonstrated that coupling the two genome editing events gave RBCs a significant selective advantage. This demonstrates the potential of combining human genetics with the precision of genome editing to enable safer and more effective genome editing therapies for patients with serious genetic diseases.

genetics↗