The consequences of sickle cell disease (SCD) include ongoing hematopoietic stress, hemolysis, vascular damage, and effect of chronic therapies, such as blood transfusions and hydroxyurea, on hematopoietic stem and progenitor cell (HSPC) have been poorly characterized. We have quantified the frequencies of nine HSPC populations by flow cytometry in the peripheral blood of pediatric and adult patients, stratified by treatment and control cohorts. We observed broad differences between SCD patients and healthy controls. SCD is associated with 10 to 20-fold increase in CD34^dim cells, a two to five-fold increase in CD34^bright cells, a depletion in Megakaryocyte-Erythroid Progenitors, and an increase in hematopoietic stem cells, when compared to controls. SCD is also associated with abnormal expression of CD235a as well as high levels CD49f antigen expression. These findings were present to varying degrees in all patients with SCD, including those on chronic therapy and those who were therapy naive. HU treatment appeared to normalize many of these parameters. Chronic stress erythropoiesis and inflammation incited by SCD and HU therapy have long been suspected of causing premature aging of the hematopoietic system, and potentially increasing the risk of hematological malignancies. An important finding of this study was that the observed concentration of CD34^bright cells and of all the HSPCs decreased logarithmically with time of treatment with HU. This correlation was independent of age and specific to HU treatment. Although the number of circulating HSPCs is influenced by many parameters, our findings suggest that HU treatment may decrease premature aging and hematologic malignancy risk compared to the other therapeutic modalities in SCD.

The concentration of circulating hematopoietic stem and progenitor cells has not been studied longitudinally. Here, we report that the proportions of Lin-CD34+38- hematopoietic multipotent cells (HMCs) and of Lin-CD34+CD38+ hematopoietic progenitors cells (HPCs) are highly variable between individuals but stable over long periods of time, in both healthy individuals and sickle cell disease (SCD) patients. This suggests that these proportions are regulated by genetic polymorphisms or by epigenetic mechanisms. We also report that in SCD patients treated with hydroxyurea, the proportions of circulating HMCs and HPCs show a strong positive and negative correlation with fetal hemoglobin (HbF) levels, respectively. Titration of 65 cytokines revealed that the plasma concentration of chemokines CCL2, CCL11, CCL17, CCL24, CCL27, and PDGF-BB were highly correlated with the proportion of HMCs and HPCs and that a subset of these cytokines were also correlated with HbF levels. A linear model based on four of these chemokines could explain 80% of the variability in the proportion of circulating HMCs between individuals. The proportion of circulating HMCs and HPCs and the concentration of these chemokines might therefore become useful biomarkers for HbF response to HU in SCD patients. Such markers might become increasingly clinically relevant, as alternative treatment modalities for SCD are becoming available.

The consequences of Sickle Cell Disease (SCD), including ongoing hematopoietic stress and hemolysis, vascular damage and chronic therapies , such as blood transfusions and Hydroxyurea on hematopoietic stem and progenitor cell (HSPC) have not been characterized. We have quantified the frequencies of nine HSPC populations by flow cytometry in the peripheral blood of pediatric and adult patients stratified by treatment and controls. We observed broad differences between SCD patients and healthy controls. SCD is associated with 10 to 20-fold increase in CD34dim cells, and two to five-fold more CD34bright cells, a depletion in Megakaryocyte-Erythroid Progenitors and an increase in hematopoietic stem cells, when compared to controls. SCD is also associated with abnormal expression of CD235a and by very high levels of expression of the CD49f antigen. These findings were present to varying degrees in all patients, whether or not they were naïve or on chronic therapy. HU treatment tended to normalizes many of these parameters. Chronic stress erythropoiesis, inflammation caused by SCD and hydroxyurea therapy have long been suspected of causing premature aging of the hematopoietic system, and potentially increasing the risk of hematological malignancies. An important finding of this study was that the observed concentration of CD34bright cells and of all the HSPCs decreased logarithmically with time of treatment with HU. This correlation was independent of age and specific to HU treatment. Although the number of circulating HSPCs is influenced by many parameters, our findings suggest that HU treatment may decrease premature aging and hematologic malignancy risk compared to the other therapeutic modalities in SCD.

Red Blood Cells (RBCs) have long been considered a potentially useful means of delivering drugs to the circulation because delivery through therapeutic RBCs as compared to direct injection in the plasma can lengthen the half-life of the therapeutic agent in the circulation, spatially restrict the drugs to the lumen of the cardio-vascular system, and shield the drug from the immune system. Despite some progress, loading the cells with therapeutically useful cargo remains technically challenging. We have recently developed PSC-RED, a chemically-defined scalable method to differentiate induced pluripotent stem cells (iPSCs) into unlimited numbers of enucleated cultured RBCs. This provides an ideal method to produce therapeutic RBCs since iPSCs can be genetically manipulated with powerful CRISPR-based technologies. ADAMTS13, whose deficiency is responsible for congenital and acquire Thrombotic Thrombocytopenic Purpura (TTP) is a good target as a therapeutic that could be delivered through drug-carrying RBCs because large amounts of plasma concentrate, or more recently recombinant proteins, are necessary to treat TTP. We report here we have produced engineered erythroid cells that contains globin-LCR driven ADAMTS13 fusion transgenes inserted at safe harbor AAVS1, and that these cells express a membrane bound version of an inhibitor-resistant version of ADAMTS13. We show using flow cytometry that the fusion protein is express at high levels, and using a FRET assay that detect cleavage of the von Willebrand cognate site, that the membrane bound ADAMTS13 is enzymatically active. Comparison of enzymatic activity with plasma concentrate suggests that about 50 billion engineered ADAMTS13-cRBCs would be sufficient to deliver an amount of ADAMTS13 equivalent to 2 liters of plasma concentrate. This suggests that a transfusion of about 10 mL of ADAMTS13-RBCs could be therapeutic for congenital and acquired TTP. The number of cRBCs necessary to treat even a few patients is very large. This has been considered a major obstacle to the development of treatment based on in vitro produced RBCs because of the volume of culture that is necessary to produce the cells. We also report that we have developed a culture method based on holo-fiber bioreactors that allows the production of cRBCs at a density of 5.10⁸ cell/mL which is sufficient to produce enough cells to performed small clinical trials.

Thrombotic Thrombocytopenic Purpura (TTP) is caused by congenital or acquired deficiency of ADAMTS13, a metalloproteinase that cleaves von Willebrand Factor (vWF) multimers. Current treatments—plasma exchange and immunosuppression—are costly and associated with significant morbidity therefore, alternative strategies are needed. We developed the kitJak2 platform for producing genetically engineered lab-grown red blood cells (lgRBCs) as drug delivery vectors. We hypothesized that membrane-bound ADAMTS13 displayed on lgRBCs could provide a durable treatment for TTP. To test this, we engineered erythroid cells expressing both wild-type and mutant variants MDTCS fragments of ADAMTS13, conferring resistance to autoantibodies. Flow cytometry and FRET-based assays confirmed robust membrane expression and enzymatic activity. Importantly, mutant MDTCS variants retained catalytic activity in the presence of plasma from TTP patients, whereas wild-type variants were inhibited. For in vivo evaluation, we generated transgenic mice expressing MDTCS ADAMTS13 on their RBC membranes. These mice exhibited normal RBC half-lives and stable, catalytically active ADAMTS13 expression. Using a murine model of TTP—where ADAMTS13 knockout mice challenged with recombinant human vWF (rhvWF) develop thrombocytopenia and schistocytes—we demonstrated that transfusion of ADAMTS13-expressing RBCs significantly mitigated disease, preventing platelet loss and schistocyte formation. This confirms that membrane-bound MDTCS ADAMTS13 cleaves circulating rhvWF under physiological flow conditions in vivo. Finally, employing our KitJak2 platform, we generated human enucleated lgRBCs expressing high levels of catalytically active ADAMTS13. This novel work establishes proof-of-concept that membrane-anchored ADAMTS13-expressing lab- grown RBCs may offer a feasible and effective therapeutic approach for both congenital and acquired TTP.