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Red blood cell (RBC) transfusion is one of the most common medical treatments, with more than 10 million units transfused per year in the United States alone. Alloimmunization to foreign Rh proteins (RhD and RhCE) on donor RBCs remains a challenge for transfusion effectiveness and safety. Alloantibody production disproportionately affects patients with sickle cell disease who frequently receive blood transfusions and exhibit high genetic diversity in the Rh blood group system. With hundreds of RH variants now known, precise identification of Rh antibody targets is hampered by the lack of appropriate reagent RBCs with uncommon Rh antigen phenotypes. Using a combination of human-induced pluripotent stem cell (iPSC) reprogramming and gene editing, we designed a renewable source of cells with unique Rh profiles to facilitate the identification of complex Rh antibodies. We engineered a very rare Rh null iPSC line lacking both RHD and RHCE. By targeting the AAVS1 safe harbor locus in this Rh null background, any combination of RHD or RHCE complementary DNAs could be reintroduced to generate RBCs that express specific Rh antigens such as RhD alone (designated D--), Goa+, or DAK+. The RBCs derived from these iPSCs (iRBCs) are compatible with standard laboratory assays used worldwide and can determine the precise specificity of Rh antibodies in patient plasma. Rh-engineered iRBCs can provide a readily accessible diagnostic tool and guide future efforts to produce an alternative source of rare RBCs for alloimmunized patients.

•Human iPSC reprogramming and gene editing can be combined to generate an alternative source of rare RBCs.•Rh-engineered iPSC-derived RBCs provide a novel tool to aid in complex Rh antibody identification.

Blinatumomab is currently approved for use as a single agent in relapsed and refractory acute lymphoblastic leukemia (ALL). Cytotoxicity is mediated via signaling through the T-cell receptor (TCR). There is now much interest in combining blinatumomab with targeted therapies, particularly in Philadelphia chromosome–positive ALL (Ph+ ALL). However, some second- and third-generation ABL inhibitors also potently inhibit Src family kinases that are important in TCR signaling. We combined ABL inhibitors and dual Src/ABL inhibitors with blinatumomab in vitro from both healthy donor samples and primary samples from patients with Ph+ ALL. Blinatumomab alone led to both T-cell proliferation and elimination of target CD19+ cells and enhanced production of interferon-γ (IFN-γ). The addition of the ABL inhibitors imatinib or nilotinib to blinatumomab did not inhibit T-cell proliferation or IFN-γ production. However, the addition of dasatinib or ponatinib inhibited T-cell proliferation and IFN-γ production. Importantly, there was no loss of CD19+ cells treated with blinatumomab plus dasatinib or ponatinib in healthy samples or samples with a resistant ABL T315I mutation by dasatinib in combination with blinatumomab. These in vitro findings bring pause to the excitement of combination therapies, highlighting the importance of maintaining T-cell function with targeted therapies.

Blinatumomab is currently approved for use as a single agent in relapsed and refractory acute lymphoblastic leukemia (ALL). Cytotoxicity is mediated via signaling through the T-cell receptor (TCR). There is now much interest in combining blinatumomab with targeted therapies, particularly in Philadelphia chromosome–positive ALL (Ph+ALL). However, some second- and third-generation ABL inhibitors also potently inhibit Src family kinases that are important in TCR signaling. We combined ABL inhibitors and dual Src/ABL inhibitors with blinatumomab in vitro from both healthy donor samples and primary samples from patients with Ph+ALL. Blinatumomab alone led to both T-cell proliferation and elimination of target CD19+cells and enhanced production of interferon-γ (IFN-γ). The addition of the ABL inhibitors imatinib or nilotinib to blinatumomab did not inhibit T-cell proliferation or IFN-γ production. However, the addition of dasatinib or ponatinib inhibited T-cell proliferation and IFN-γ production. Importantly, there was no loss of CD19+cells treated with blinatumomab plus dasatinib or ponatinib in healthy samples or samples with a resistant ABL T315Imutation by dasatinib in combination with blinatumomab. These in vitro findings bring pause to the excitement of combination therapies, highlighting the importance of maintaining T-cell function with targeted therapies.

Risitano: Amyndas: Consultancy; Samsung: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; RA pharma: Research Funding; Pfizer: Speakers Bureau; Apellis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Biocryst: Membership on an entity's Board of Directors or advisory committees; Alexion: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Alnylam: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Achillion: Membership on an entity's Board of Directors or advisory committees. Schrezenmeier:Alexion Pharmaceuticals Inc.: Honoraria, Research Funding. Yonemura:Alexion Pharmaceuticals: Honoraria, Research Funding. Munir:Alexion: Honoraria; F. Hoffmann-La Roche: Consultancy, Other: Medical writing support, furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland. Pavani:Alexion Pharmaceuticals: Current Employment. Wang:Alexion Pharmaceuticals Inc.: Current Employment. Kulagin:Alexion Pharmaceuticals Inc.: Consultancy, Research Funding. Kulasekararaj:Alexion Pharmaceuticals Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sicre de Fontbrune:Alexion Pharmaceuticals Inc.: Honoraria, Research Funding. Röth:Novartis: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria; Biocryst: Consultancy, Honoraria; Apellis: Consultancy, Honoraria; Alexion Pharmaceuticals Inc.: Consultancy, Honoraria, Research Funding.

Naturally occurring, large deletions in the β-globin locus result in hereditary persistence of fetal hemoglobin, a condition that mitigates the clinical severity of sickle cell disease (SCD) and β-thalassemia. We designed a clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) strategy to disrupt a 13.6-kb genomic region encompassing the δ- and β-globin genes and a putative γ-δ intergenic fetal hemoglobin (HbF) silencer. Disruption of just the putative HbF silencer results in a mild increase in γ-globin expression, whereas deletion or inversion of a 13.6-kb region causes a robust reactivation of HbF synthesis in adult erythroblasts that is associated with epigenetic modifications and changes in chromatin contacts within the β-globin locus. In primary SCD patient-derived hematopoietic stem/progenitor cells, targeting the 13.6-kb region results in a high proportion of γ-globin expression in erythroblasts, increased HbF synthesis, and amelioration of the sickling cell phenotype. Overall, this study provides clues for a potential CRISPR/Cas9 genome editing approach to the therapy of β-hemoglobinopathies.

Naturally occurring, large deletions in the β-globin locus result in hereditary persistence of fetal hemoglobin, a condition that mitigates the clinical severity of sickle cell disease (SCD) and β-thalassemia. We designed a clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) strategy to disrupt a 13.6-kb genomic region encompassing the δ- and β-globin genes and a putative γ-δ intergenic fetal hemoglobin (HbF) silencer. Disruption of just the putative HbF silencer results in a mild increase in γ-globin expression, whereas deletion or inversion of a 13.6-kb region causes a robust reactivation of HbF synthesis in adult erythroblasts that is associated with epigenetic modifications and changes in chromatin contacts within the β-globin locus. In primary SCD patient-derived hematopoietic stem/progenitor cells, targeting the 13.6-kb region results in a high proportion of γ-globin expression in erythroblasts, increased HbF synthesis, and amelioration of the sickling cell phenotype. Overall, this study provides clues for a potential CRISPR/Cas9 genome editing approach to the therapy of β-hemoglobinopathies.

Background:Paroxysmal nocturnal hemoglobinuria (PNH) is an ultra-rare, acquired disorder, leading to impaired expression of complement-regulating proteins on the surface of hematopoietic cells. Complement component C5 inhibitors are part of the standard of care for patients with PNH; however, these are generally intravenous treatments. Pozelimab and cemdisiran are investigational agents with a small volume subcutaneous (SC) once every 4 weeks maintenance regimen that may be self-administered. Both inhibit terminal complement through complementary mechanisms of action. Cemdisiran is an N-acetylgalactosamine-conjugated small interfering RNA that suppresses liver production of C5, while pozelimab is a fully human monoclonal antibody inhibitor of C5. The efficacy and safety of the combination of pozelimab and cemdisiran was evaluated in an open-label, single-arm study in patients with PNH who switched from eculizumab therapy (NCT04888507). The completed safety and efficacy data of the optional 52-week open-label extension period (OLEP) are presented.

Factor VII (FVII) deficiency is a rare autosomal recessive bleeding disorder treated by infusion of fresh-frozen plasma, plasma-derived FVII concentrates and low-dose recombinant activated FVII. Clinical data suggest that a mild elevation of plasma FVII levels (>10% normal) results in improved hemostasis. Research dogs with a G96E missense FVII mutation (FVII-G96E) have <1% FVII activity. By western blot, we show that they have undetectable plasmatic antigen, thus representing the most prevalent type of human FVII deficiency (low antigen/activity). In these dogs, we determine the feasibility of a gene therapy approach using liver-directed, adeno-associated viral (AAV) serotype 8 vector delivery of a canine FVII (cFVII) zymogen transgene. FVII-G96E dogs received escalating AAV doses (2E11 to 4.95E13 vector genomes [vg] per kg). Clinically therapeutic expression (15% normal) was attained with as low as 6E11 vg/kg of AAV and has been stable for >1 year (ongoing) without antibody formation to the cFVII transgene. Sustained and supraphysiological expression of 770% normal was observed using 4.95E13 vg/kg of AAV (2.6 years, ongoing). No evidence of pathological activation of coagulation or detrimental animal physiology was observed as platelet counts, d-dimer, fibrinogen levels, and serum chemistries remained normal in all dogs (cumulative 6.4 years). We observed a transient and noninhibitory immunoglobulin G class 2 response against cFVII only in the dog receiving the highest AAV dose. In conclusion, in the only large-animal model representing the majority of FVII mutation types, our data are first to demonstrate the feasibility, safety, and long-term duration of AAV-mediated correction of FVII deficiency.

Factor VII (FVII) deficiency is a rare autosomal recessive bleeding disorder treated by infusion of fresh-frozen plasma, plasma-derived FVII concentrates and low-dose recombinant activated FVII. Clinical data suggest that a mild elevation of plasma FVII levels (>10% normal) results in improved hemostasis. Research dogs with a G96E missense FVII mutation (FVII-G96E) have <1% FVII activity. By western blot, we show that they have undetectable plasmatic antigen, thus representing the most prevalent type of human FVII deficiency (low antigen/activity). In these dogs, we determine the feasibility of a gene therapy approach using liver-directed, adeno-associated viral (AAV) serotype 8 vector delivery of a canine FVII (cFVII) zymogen transgene. FVII-G96E dogs received escalating AAV doses (2E11 to 4.95E13 vector genomes [vg] per kg). Clinically therapeutic expression (15% normal) was attained with as low as 6E11 vg/kg of AAV and has been stable for >1 year (ongoing) without antibody formation to the cFVII transgene. Sustained and supraphysiological expression of 770% normal was observed using 4.95E13 vg/kg of AAV (2.6 years, ongoing). No evidence of pathological activation of coagulation or detrimental animal physiology was observed as platelet counts, d-dimer, fibrinogen levels, and serum chemistries remained normal in all dogs (cumulative 6.4 years). We observed a transient and noninhibitory immunoglobulin G class 2 response against cFVII only in the dog receiving the highest AAV dose. In conclusion, in the only large-animal model representing the majority of FVII mutation types, our data are first to demonstrate the feasibility, safety, and long-term duration of AAV-mediated correction of FVII deficiency.

Recombinant activated human factor VII (rhFVIIa) is an established hemostatic agent in hemophilia, but its mechanism of action remains unclear. Although tissue factor (TF) is its natural receptor, rhFVIIa also interacts with the endothelial protein C receptor (EPCR) through its γ-carboxyglutamic acid (Gla) domain, with unknown hemostatic consequences in vivo. Here, we study whether EPCR facilitates rhFVIIa hemostasis in hemophilia using a mouse model system. Mouse activated FVII (mFVIIa) is functionally homologous to rhFVIIa, but binds poorly to mouse EPCR (mEPCR). We modified mFVIIa to gain mEPCR binding using 3 amino acid changes in its Gla domain (L4F/L8M/W9R). The resulting molecule mFVIIa-FMR specifically bound mEPCR in vitro and in vivo and was identical to mFVIIa with respect to TF affinity and procoagulant functions. In macrovascular injury models, hemophilic mice administered mFVIIa-FMR exhibited superior hemostatic activity compared with mFVIIa. This was abolished by blocking mEPCR and was absent in ex vivo whole blood coagulation assays, implicating a specific mFVIIa-FMR and endothelial mEPCR interaction. Because mFVIIa-FMR models the TF-dependent and EPCR binding properties of rhFVIIa, our data unmask a novel contribution of EPCR on the action of rhFVIIa administration in hemophilia, prompting the rational design of improved and safer rhFVIIa therapeutics.

Recombinant activated human factor VII (rhFVIIa) is an established hemostatic agent in hemophilia, but its mechanism of action remains unclear. Although tissue factor (TF) is its natural receptor, rhFVIIa also interacts with the endothelial protein C receptor (EPCR) through its γ-carboxyglutamic acid (Gla) domain, with unknown hemostatic consequences in vivo. Here, we study whether EPCR facilitates rhFVIIa hemostasis in hemophilia using a mouse model system. Mouse activated FVII (mFVIIa) is functionally homologous to rhFVIIa, but binds poorly to mouse EPCR (mEPCR). We modified mFVIIa to gain mEPCR binding using 3 amino acid changes in its Gla domain (L4F/L8M/W9R). The resulting molecule mFVIIa-FMR specifically bound mEPCR in vitro and in vivo and was identical to mFVIIa with respect to TF affinity and procoagulant functions. In macrovascular injury models, hemophilic mice administered mFVIIa-FMR exhibited superior hemostatic activity compared with mFVIIa. This was abolished by blocking mEPCR and was absent in ex vivo whole blood coagulation assays, implicating a specific mFVIIa-FMR and endothelial mEPCR interaction. Because mFVIIa-FMR models the TF-dependent and EPCR binding properties of rhFVIIa, our data unmask a novel contribution of EPCR on the action of rhFVIIa administration in hemophilia, prompting the rational design of improved and safer rhFVIIa therapeutics.

Several peripheral blood mononuclear cell (PBMC)–derived cell populations can promote angiogenesis, and differences in CD34+ or CD14+ surface expression have been used to separate PBMC subpopulations in this respect. AngiomiRs, microRNAs regulating angiogenesis, are key regulators of angiogenic processes. The present study examines differential angiomiR expression/secretion from CD34+/CD14+, CD34+/CD14−, CD34−/CD14+, and CD34−/CD14− PBMC subsets and their relevance for different proangiogenic properties. Notably, both circulating human CD34+/14+ and CD34+/14− PBMC subsets and their supernatants exerted more potent proangiogenic effects compared with CD34− PBMC subsets. MiR-126 was identified as most differentially expressed angiomiR in CD34+ compared with CD34− PBMC subsets, determined by miR-array and RT-PCR validation. Modulation of miR-126 by anti–miR-126 or miR-mimic-126 treatment resulted in significant loss or increase of proangiogenic effects of CD34+ PBMCs. MiR-126 levels in supernatants of CD34+ PBMC subsets were substantially higher compared with CD34− PBMC subsets. MiR-126 was secreted in microvesicles/exosomes, and inhibition of their release impaired CD34+ PBMCs proangiogenic effects. Notably, high-glucose treatment or diabetes reduced miR-126 levels of CD34+ PBMCs, associated with impaired proangiogenic properties that could be rescued by miR-mimic-126 treatment. The present findings provide a novel molecular mechanism underlying increased proangiogenic effects of CD34+ PBMCs, that is, angiomiR-126 expression/secretion. Moreover, an alteration of angiomiR-126 expression in CD34+ PBMCs in diabetes provides a novel pathway causing impaired proangiogenic effects.

Several peripheral blood mononuclear cell (PBMC)–derived cell populations can promote angiogenesis, and differences in CD34+or CD14+surface expression have been used to separate PBMC subpopulations in this respect. AngiomiRs, microRNAs regulating angiogenesis, are key regulators of angiogenic processes. The present study examines differential angiomiR expression/secretion from CD34+/CD14+, CD34+/CD14−, CD34−/CD14+, and CD34−/CD14−PBMC subsets and their relevance for different proangiogenic properties. Notably, both circulating human CD34+/14+and CD34+/14−PBMC subsets and their supernatants exerted more potent proangiogenic effects compared with CD34−PBMC subsets. MiR-126 was identified as most differentially expressed angiomiR in CD34+compared with CD34−PBMC subsets, determined by miR-array and RT-PCR validation. Modulation of miR-126 by anti–miR-126 or miR-mimic-126 treatment resulted in significant loss or increase of proangiogenic effects of CD34+PBMCs. MiR-126 levels in supernatants of CD34+PBMC subsets were substantially higher compared with CD34−PBMC subsets. MiR-126 was secreted in microvesicles/exosomes, and inhibition of their release impaired CD34+PBMCs proangiogenic effects. Notably, high-glucose treatment or diabetes reduced miR-126 levels of CD34+PBMCs, associated with impaired proangiogenic properties that could be rescued by miR-mimic-126 treatment. The present findings provide a novel molecular mechanism underlying increased proangiogenic effects of CD34+PBMCs, that is, angiomiR-126 expression/secretion. Moreover, an alteration of angiomiR-126 expression in CD34+PBMCs in diabetes provides a novel pathway causing impaired proangiogenic effects.

Catalytic domain variants of activated factor VII (FVIIa) with enhanced hemostatic properties are highly attractive for the treatment of bleeding disorders via gene-based therapy. To explore this in a hemophilic mouse model, we characterized 2 variants of murine activated FVII (mFVIIa-VEAY and mFVIIa-DVQ) with modified catalytic domains, based on recombinant human FVIIa (rhFVIIa) variants. Using purified recombinant proteins, we showed that murine FVIIa (mFVIIa) and variants had comparable binding to human and murine tissue factor (TF) and exhibited similar extrinsic coagulant activity. In vitro in the absence of TF, the variants showed a 6- to 17-fold enhanced proteolytic and coagulant activity relative to mFVIIa, but increased inactivation by antithrombin. Gene delivery of mFVIIa-VEAY resulted in long-term, effective hemostasis at 5-fold lower expression levels relative to mFVIIa in hemophilia A mice or in hemophilia B mice with inhibitors to factor IX. However, expression of mFVIIa-VEAY at 14-fold higher than therapeutic levels resulted in a progressive mortality to 70% within 6 weeks after gene delivery. These results are the first demonstration of the hemostatic efficacy of continuous expression, in the presence or absence of inhibitors, of a high-activity gene-based FVIIa variant in an animal model of hemophilia.

Catalytic domain variants of activated factor VII (FVIIa) with enhanced hemostatic properties are highly attractive for the treatment of bleeding disorders via gene-based therapy. To explore this in a hemophilic mouse model, we characterized 2 variants of murine activated FVII (mFVIIa-VEAY and mFVIIa-DVQ) with modified catalytic domains, based on recombinant human FVIIa (rhFVIIa) variants. Using purified recombinant proteins, we showed that murine FVIIa (mFVIIa) and variants had comparable binding to human and murine tissue factor (TF) and exhibited similar extrinsic coagulant activity. In vitro in the absence of TF, the variants showed a 6- to 17-fold enhanced proteolytic and coagulant activity relative to mFVIIa, but increased inactivation by antithrombin. Gene delivery of mFVIIa-VEAY resulted in long-term, effective hemostasis at 5-fold lower expression levels relative to mFVIIa in hemophilia A mice or in hemophilia B mice with inhibitors to factor IX. However, expression of mFVIIa-VEAY at 14-fold higher than therapeutic levels resulted in a progressive mortality to 70% within 6 weeks after gene delivery. These results are the first demonstration of the hemostatic efficacy of continuous expression, in the presence or absence of inhibitors, of a high-activity gene-based FVIIa variant in an animal model of hemophilia.

Hill: Bioverativ: Honoraria; Alexion: Honoraria; Regeneron: Honoraria; Novartis: Honoraria; Roche: Honoraria; Apellis: Honoraria; Akari: Honoraria; Ra Pharma: Honoraria. Piatek:Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dova: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Rigel: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding. Peffault de Latour:Novartis: Consultancy, Honoraria, Research Funding; Amgen: Research Funding; Alexion: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding. Wells:Alexion: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Brodsky:Achillion: Research Funding; Alexion: Membership on an entity's Board of Directors or advisory committees, Other: Grant funding. Nishimura:Alexion: Honoraria, Research Funding; Chugai: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kuriakose:Alexion: Consultancy, Honoraria, Speakers Bureau; Bayer: Consultancy. Pavani:Alexion: Employment. Liu:Alexion: Employment. Ortiz:Alexion: Employment. Schrezenmeier:Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Lee:Achillion: Research Funding; Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Kulasekararaj:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Akari Therapeutics: Consultancy; Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Achilleon: Consultancy; Ra Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.

INTRODUCTION

Leonard: Amgen: Research Funding. Druker:McGraw Hill: Patents & Royalties; Fred Hutchinson Cancer Research Center: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Monojul: Consultancy; Millipore: Patents & Royalties; Novartis Pharmaceuticals: Research Funding; Oregon Health & Science University: Patents & Royalties; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Meyers Squibb: Research Funding; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Henry Stewart Talks: Patents & Royalties; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Tyner:Constellation: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Gilead: Research Funding; Aptose: Research Funding; Incyte: Research Funding; Genentech: Research Funding; Array: Research Funding; Takeda: Research Funding; AstraZeneca: Research Funding. Lind:Celgene: Research Funding; Janssen Pharmaceutical R&D: Research Funding; Amgen: Research Funding; Fluidigm: Honoraria; Monojul: Research Funding.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Leonard: Amgen: Research Funding. Druker:Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; Millipore: Patents & Royalties; ARIAD: Research Funding; Henry Stewart Talks: Patents & Royalties; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Monojul: Consultancy; Novartis Pharmaceuticals: Research Funding; Bristol-Meyers Squibb: Research Funding; Fred Hutchinson Cancer Research Center: Research Funding; Beta Cat: Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties. Tyner:Gilead: Research Funding; Incyte: Research Funding; Aptose: Research Funding; Janssen: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Array: Research Funding; Takeda: Research Funding; Constellation: Research Funding; Genentech: Research Funding; AstraZeneca: Research Funding. Lind:Fluidigm: Honoraria; Amgen: Research Funding; Celgene: Research Funding; Janssen Pharmaceutical R&D: Research Funding; Monojul: Research Funding.

Introduction:

Introduction:

Introduction:

Introduction:

Introduction: Blinatumomab is a bispecific antibody approved for the use in adults with relapsed acute lymphoblastic leukemia (ALL) but only about 50% of patients respond. A few brief reports have suggested that differences in T cell subsets between responders and non-responders may contribute to the failure of blinatumomab, however our understanding of the immune microenvironment in ALL is still in its infancy. We hypothesize response may be related to an immunosuppressive T cell microenvironment and in this project sought to define this microenvironment using both phenotypic and functional assays.

Introduction:

McBride: Sanofi: Research Funding.

Cradick: CRISPR Therapeutics: Employment. Lundberg:CRISPR Therapeutics: Employment, Equity Ownership. Porteus:CRISPR Therapeutics: Consultancy, Equity Ownership. Mavilio:Adverum Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Consultancy, Research Funding.

Naturally occurring, large deletions in the β-globin locus result in increased fetal hemoglobin (HbF) expression (HPFH, Hereditary Persistence of Fetal Hemoglobin), a condition that mitigates the clinical severity of Sickle Cell Disease (SCD) and β-thalassemia. Here, we integrated BCL11A and GATA1 transcription factor binding site analysis and HPFH mutational data to identify potential HbF silencers in the β-globin locus. Based on this analysis, we designed a CRISPR/Cas9 strategy to disrupt: (i) a 3.5-kb δγ-intergenic region that is specifically deleted in individuals with HPFH. This region contains BCL11A and GATA1 binding sites in adult, HbF-negative primary erythroblasts, thus representing a potential HbF silencer; (ii) a 7.2-kb region, encompassing the 3.5-kb δγ-intergenic element, which is the minimal naturally occurring deletion associated with HPFH ("Corfù" deletion; Chakalova et al., Blood, 2005); (iii) a 13.6-kb genomic region commonly deleted in HPFH, which includes the δ- and β-globin genes and putative intergenic HbF silencers. Targeted deletion or inversion of the 13.6-kb genomic region caused a substantial increase in γ-globin mRNA levels, re-activation of HbF synthesis and a concomitant decrease in HbA expression in adult human erythroid cells (HUDEP-2). Interestingly, deletion of the Corfù region or of the 3.5-kb putative HbF silencer, increased γ-globin gene transcription but failed to produce accumulation of γ-globin mRNA, suggesting a post-transcriptional regulation of γ-globin synthesis in the presence of an intact and active β-globin gene. We then tested re-activation of HbF synthesis in primary adult hematopoietic stem/progenitor cells differentiated towards the erythroid lineage in liquid and clonogenic cultures. Targeting the 13.6-kb genomic region resulted in a high proportion of γ-globin expressing primary erythroblasts, with HbF representing up to 50% of total hemoglobin. Cell morphology, erythroid marker profile, total hemoglobin levels and erythroid maturation were unaffected, consistent with the asymptomatic phenotype of adult HPFH carriers. These data suggest that this region could serve as target for therapeutic genome editing for HbF induction in β-hemoglobinopathies. Overall, this study contributes to the knowledge of the mechanisms underlying fetal to adult Hb switching, and provides clues for a genome editing approach to the treatment of SCD and β-thalassemia.

Background

Ivanciu: Bayer Hemophilia Awards Program: Research Funding. Margaritis:Novo Nordisk A/S: Research Funding; Bayer Hemophilia Awards Program: Research Funding.

The human protein C (PC) interaction with the endothelial PC receptor (EPCR) is mediated through the PC Gla domain, via key amino acids Phe4 and Leu8. Specifically, substitution of Leu8 with Val from human prothrombin abolishes the PC-EPCR interaction. The Gla domain of human Factor VII (FVII) shares these positions with PC and, consequently, the EPCR binding capacity. In the mouse, a commonly used in vivo model, the sequence determinants of the Gla domain of mouse PC (mPC) interaction with mouse EPCR (mEPCR) are not known. Remarkably, mouse FVII (mFVII) and its activated form (mFVIIa) have poor affinity for mouse EPCR. We previously described a variant of mFVIIa (mFVIIa-FMR) that contained the Leu4->Phe, Leu8->Met and Trp9->Arg from the mPC Gla domain. We found that this molecule was functionally similar to mFVIIa and could bind mEPCR. Using mFVIIa-FMR as surrogate to study the mPC-mEPCR interaction, we highlighted the importance of the Phe4/Met8/Arg9 in the mPC-mEPCR interaction. We also found that mFVIIa-FMR had enhanced hemostatic properties when infused at 3 mg/kg after FeCl3carotid artery injury in hemophilic mice (vessel occlusion was 2.5 times faster than mFVIIa). In order to further refine whether the mEPCR binding capacity of mPC is coordinated by any/all of Phe4/Met8/Arg9 positions, we previously generated single variants of mPC at these positions using the corresponding amino acids of mFVIIa (that has poor interaction with mEPCR). We found that Phe4 is the sole determinant of specificity of the mPC-mEPCR interaction. Moreover, when Phe4 was placed in mFVIIa, we found that mFVIIa-Phe4 had activity similar to mFVIIa and bound mEPCR on cells (or in solution to soluble mEPCR) with a Kd of ~350nM. This was of similar magnitude to the mEPCR affinity of a mFVIIa variant with the entire mPC Gla domain (~200 nM), indirectly suggesting that Phe4 determines both the specificity and affinity of mPC to mEPCR. Since mFVIIa-FMR showed improved hemostatic properties in vivo as a result of mEPCR binding, enhancing the EPCR-FVIIa binding may generate improved human FVIIa molecules for the treatment of bleeding. Here we wanted to provide proof-of-concept using limited Gla domain modifications. For this, we utilized mFVIIa-Phe4, a minimally modified mFVIIa molecule, described above. Specifically, hemophilia B animals were subjected to a 7.5% FeCl3injury of their carotid artery for 2 minutes; after 10 minutes mice were infused with 3 mg/kg of mFVIIa or mFVIIa-Phe4. Time to vessel occlusion was determined by monitoring blood flow. Hemostatically normal mice occluded in 13.3 ± 3.0 min. We found that infusion of mFVIIa resulted in vessel occlusion at 8.9 ± 1.7 min. However, mice that received mFVIIa-Phe4 reached vessel occlusion within 4.5 ± 2.4 min, ~2.5 times faster than mFVIIa-infused mice (P<0.01). This was similar to that we previously observed with mFVIIa-FMR infusion after injury in hemophilia B mice. Our results suggest the following: (1) Phe4 in the mPC Gla domain confers the specificity and affinity to mEPCR; (2) a single Phe4 substitution in mFVIIa is the only requirement for enhancing its clotting function in vivo. These data reveal another difference between human and mouse systems that may affect EPCR-dependent functions of other vitamin K-dependent proteins. Moreover, our results suggest the possibility that minimally modified variants of FVIIa with respect to EPCR binding may have more favorable hemostatic properties for clinical use.

No relevant conflicts of interest to declare.

The Endothelial Protein C Receptor (EPCR) is an important component of the Protein C anticoagulant pathway. The interaction of Protein C (PC) through its Gla domain with EPCR enhances PC activation, thus down-regulating thrombin production. EPCR can also bind human activated Factor VII (FVIIa) and modulate its activity and localization. The residues involved in receptor recognition in both PC and FVIIa are Phe4 and Leu8, located in the first portion of the Gla domain. The importance of Phe8 is indicated by the lack of EPCR binding of a PC variant that contains a Val8 from human prothrombin. Remarkably, the similarity between PC and FVIIa for EPCR binding is lacking in the mouse. Others and we have shown that mouse FVIIa (mFVIIa, which contains a Leu4 and a Leu8) interacts with mouse EPCR (mEPCR) poorly, thus failing to model the spectrum of known human FVIIa properties. In previous work, we generated mFVIIa chimeras that contain parts of the mouse PC (mPC) Gla domain and determined that three residues in the mPC Gla domain can confer mEPCR binding to mFVIIa. Specifically, molecule mFVIIa-FMR that contained the Leu4->Phe, Leu8->Met and Trp9->Arg from mPC was functionally similar to mFVIIa and could bind mEPCR as a true gain-of-function. However, little is known on the contribution of any/all of these positions in mPC binding to mEPCR. Here, we wanted to understand the sequence determinants that dictate this interaction. For this, we generated single amino acid mutants of mPC at position 4, 8 or 9 from the corresponding residues of mFVIIa. Using conditioned medium from transiently transfected cells, we tested the ability of each mPC mutant to bind to mEPCR expressed on the surface of CHO-K1 cells. A single substitution of Phe4 with Leu abolished mEPCR binding of mPC, in contrast to modifications at position 8 (Met to Leu) or 9 (Arg to Trp). The importance of Phe4 for the mPC-mEPCR interaction was confirmed in a reverse experiment modifying mFVIIa (that has poor mEPCR affinity) individually at position 4 (Leu to Phe), 8 (Leu to Met) or 9 (Trp to Arg) according to the mPC sequence. We found that Leu4->Phe was the sole modification that could confer mEPCR binding to mFVIIa. We have previously shown that the interaction of mFVIIa-FMR with mEPCR enhances its hemostatic function (vs. mFVIIa) after administration in hemophilic mice that have undergone injury (Pavani G et al, Blood 2014). To further explore the contribution of position 4 (Leu->Phe [L4F]) in these effects, recombinant mFVIIa-L4F was purified. Titration of mFVIIa-L4F on CHO-K1 cells expressing mEPCR showed a specific and dose-dependent receptor binding, in contrast to mFVIIa, confirming our previous data (see above). Moreover, mFVIIa-L4F showed no difference in clotting activity compared to mFVIIa in a prothrombin time-based assay. In order to compare mFVIIa-L4F to mFVIIa in its ability to generate mouse thrombin, we used a thrombin generation assay using hemophilia B plasma spiked with either procoagulant. We found that addition of either mFVIIa or mFVIIa-L4F generated similar amounts of thrombin at all concentrations tested (3.1 - 25 nM). Therefore, mFVIIa-L4F exhibited similar coagulant activity to mFVIIa but gained mEPCR binding capacity, a feature shared with mFVIIa-FMR. Further experiments are underway to determine whether the single substitution in mFVIIa-L4F is sufficient to recapitulate the enhanced hemostatic properties observed in vivo with mFVIIa-FMR. In conclusion, our findings identify a single amino acid residue (Phe4) in the Gla domain of mouse PC that plays a critical role in the binding to its natural receptor. This property can also be transplanted into mFVIIa, without affecting its coagulant activity. These observations reveal another difference between human and mouse systems and may have implications for EPCR-dependent functions or properties of other vitamin K-dependent proteins.

Abstract 18

Pavani: Bayer: Research Funding. Margaritis:Novo Nordisk A/S: Research Funding; Bayer: Research Funding.

Abstract 2050

Mingozzi: Children's Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties. Anguela:Children's Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties. Wright:Children's Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties. High:Children's Hospital of Philadelphia: Pending patent on technology described, Pending patent on technology described Patents & Royalties.