Erythropoietin (EPO) stimulates erythroid differentiation and maturation. Though the transcriptional regulation of EPO has been well studied, the molecular determinants of EPO secretion remain unknown. Here, we generated a HEK293T reporter cell line that provides a quantifiable and selectable readout of intracellular EPO levels and performed a genome-scale CRISPR screen that identified SURF4 as an important mediator of EPO secretion. Targeting SURF4 with multiple independent sgRNAs resulted in intracellular accumulation and extracellular depletion of EPO. Both of these phenotypes were rescued by expression of SURF4 cDNA. Additionally, we found that disruption of SURF4 resulted in accumulation of EPO in the ER compartment, and that SURF4 and EPO physically interact. Furthermore, SURF4 disruption in Hep3B cells also caused a defect in the secretion of endogenous EPO under conditions mimicking hypoxia, ruling out an artifact of heterologous overexpression. This work demonstrates that SURF4 functions as an ER cargo receptor that mediates the efficient secretion of EPO. Our findings also suggest that modulating SURF4 may be an effective treatment for disorders of erythropoiesis that are driven by aberrant EPO levels. Finally, we show that SURF4 overexpression results in increased secretion of EPO, suggesting a new strategy for more efficient production of recombinant EPO.

The COPII component SEC23A overlaps functionally with its paralogous protein and rescues the SEC23B-deficient CDAII defect.

Human γ-globin is predominantly expressed in fetal liver erythroid cells during gestation from two nearly identical genes, HBG1 and HBG2, that are both perinatally silenced. Reactivation of these fetal genes in adult red blood cells can ameliorate many symptoms associated with the inherited β-globinopathies, sickle cell disease (SCD) and Cooley's anemia. While promising genetic strategies to reactivate the γ-globin genes to treat these diseases have been explored, there are significant barriers to their effective implementation worldwide; alternatively, pharmacological induction of γ-globin synthesis could readily reach the majority of affected individuals. In this study, we generated a CRISPR knockout library that targeted all erythroid genes for which prospective or actual therapeutic compounds already exist. By probing this library for genes that repress fetal hemoglobin (HbF), we identified several novel, potentially druggable, γ-globin repressors, including VHL and PTEN. We demonstrate that deletion of VHL induces HbF through activation of the HIF1α pathway and that deletion of PTEN induces HbF through AKT pathway stimulation. Finally, we show that small molecule inhibitors of PTEN and EZH induce HbF in both healthy and β-thalassemic human primary erythroid cells.

Immune thrombocytopenia (ITP) is an acquired bleeding disorder characterized by IgG-mediated platelet destruction. Current therapies primarily focus on reducing antiplatelet antibodies using immunosuppression or increasing platelet production with TPO mimetics. However, there are no universally safe and effective treatments for patients presenting with severe, life threatening bleeding. IgG-degrading enzyme of S. pyogenes (IdeS), a protease with strict specificity for IgG, prevents IgG-driven immune disorders in murine models, including ITP. In clinical trials, IdeS prevented IgG-mediated kidney transplant rejection; however, the concentration of IdeS used to remove pathogenic antibodies causes profound hypogammaglobulinemia and is immunogenic, which limits its use. Therefore, this study sought to determine whether targeting IdeS to FcγRIIA, a low-affinity IgG receptor on the surface of platelets, neutrophils, and monocytes would be a viable strategy to decrease the pathogenesis of antiplatelet IgG and reduce treatment-related complications of non-targeted IdeS. We generated a recombinant protein conjugate by site-specifically linking the C-terminus of a single-chain variable fragment from a FcγRIIA antibody, clone IV.3, to the N-terminus of IdeS (scIV.3-IdeS). Platelets treated with scIV.3-IdeS had reduced binding of antiplatelet IgG from ITP patients and decreased platelet phagocytosis in vitro, with no decrease in normal IgG. Treatment of mice expressing human FcγRIIA with scIV.3-IdeS reduced thrombocytopenia in a model of ITP and significantly improved the half-life of transfused platelets expressing human FcγRIIA. Together, these data suggest that scIV.3-IdeS can selectively remove pathogenic antiplatelet IgG and may be a potential treatment for patients with ITP and severe bleeding.

1Erythropoietin (EPO), a glycoprotein produced by specialized peritubular fibroblasts in 2 the kidney, is the master regulator of erythropoiesis. EPO is secreted into the plasma in 3 response to tissue hypoxia and stimulates erythroid differentiation and maturation. 4Though the transcriptional regulation of EPO has been well studied, the molecular 5 determinants of EPO secretion remain unknown. Here, we generated a HEK293T 6 reporter cell line that provides a quantifiable and selectable readout of intracellular EPO 7 levels. Using this cell line, we performed a genome-scale CRISPR screen that identified 8 SURF4 as an important mediator of EPO secretion. Targeting SURF4 with multiple 9 independent sgRNAs resulted in intracellular accumulation and extracellular depletion 10 of EPO. Both of these phenotypes were rescued by expression of SURF4 cDNA. 11Additionally, consistent with a role for SURF4 as an ER cargo receptor of EPO, we found 12 that disruption of SURF4 resulted in accumulation of EPO in the ER compartment, and 13 that SURF4 and EPO physically interact. Furthermore, SURF4 disruption in Hep3B 14 cells also caused a defect in the secretion of endogenous EPO, ruling out an artifact of 15 heterologous overexpression. This work suggests that SURF4 functions as an ER cargo 16 receptor that mediates the efficient secretion of EPO. Our findings also suggest that 17 modulating SURF4 may be an effective treatment for disorders of erythropoeisis that 18 are driven by aberrant EPO levels. Finally, we show that SURF4 overexpression results 19 in increased secretion of EPO, suggesting a new strategy for more efficient production of 20 recombinant EPO. 21