Haploinsufficiency of Dll4, a vascular-specific Notch ligand, has shown that it is essential for embryonic vascular development and arteriogenesis. Mechanistically, it is unclear how the Dll4-mediated Notch pathway contributes to complex vascular processes that demand meticulous coordination of multiple signalling pathways. Here we show that Dll4-mediated Notch signalling has a unique role in regulating endothelial cell proliferation and differentiation. Neutralizing Dll4 with a Dll4-selective antibody rendered endothelial cells hyperproliferative, and caused defective cell fate specification or differentiation both in vitro and in vivo. In addition, blocking Dll4 inhibited tumour growth in several tumour models. Remarkably, antibodies against Dll4 and antibodies against vascular endothelial growth factor (VEGF) had paradoxically distinct effects on tumour vasculature. Our data also indicate that Dll4-mediated Notch signalling is crucial during active vascularization, but less important for normal vessel maintenance. Furthermore, unlike blocking Notch signalling globally, neutralizing Dll4 had no discernable impact on intestinal goblet cell differentiation, supporting the idea that Dll4-mediated Notch signalling is largely restricted to the vascular compartment. Therefore, targeting Dll4 might represent a broadly efficacious and well-tolerated approach for the treatment of solid tumours.

Cyclic thrombocytopenia (CTP) is a rare disease of periodic platelet count oscillations. The pathogenesis of CTP remains elusive. To study the underlying pathophysiology and genetic and cellular associations with CTP, we applied systems biology approaches to two patients with stable platelet cycling and reciprocal thrombopoietin (TPO) cycling at multiple time points through 2 cycles. Blood transcriptome analysis revealed cycling of platelet-specific genes, which are in parallel with and precede platelet count oscillation, indicating that cyclical platelet production leads platelet count cycling in both patients. Additionally, neutrophil and erythrocyte-specific genes also showed fluctuations correlating with platelet count changes, consistent with TPO effects on hematopoietic progenitors. Moreover, we found novel genetic associations with CTP. One patient had a novel germline heterozygous loss-of-function (LOF) thrombopoietin receptor (MPL) c.1210G>A mutation, and both had pathogenic somatic gain-of-function (GOF) variants in signal transducer and activator of transcription 3 (STAT3). In addition, both patients had clonal T-cell populations that remained stable throughout platelet count cycles. These mutations and clonal T cells may potentially involve in the pathogenic baseline in these patients rendering exaggerated persistent thrombopoiesis oscillations of their intrinsic rhythm upon homeostatic perturbations. This work provides new insights into the pathophysiology of CTP and possible therapies.

Introduction: Pyruvate Kinase Deficiency (PKD) is a rare inherited hemolytic anemia that is caused by mutations in the PKLR gene leading to decreased red cell pyruvate kinase (RPK) activity and impaired erythrocyte metabolism. The disorder is characterized by anemia, reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected individuals. PKD represents a significant unmet medical need as current therapies are palliative and limited to chronic blood transfusions, iron chelation therapy, and splenectomy. The side effects of these supportive treatments include iron overload, end-organ damage and increased infection risks. AG-348, an allosteric activator of RPK, is under evaluation in clinical trials, predominantly in less severely-afflicted transfusion-independent patients. Allogeneic hematopoietic stem cell transplantation (HSCT) has been performed in selected cases and resulted in transfusion independence, suggesting that the disorder may be reversed when an adequate level of hematopoietic stem and progenitor cells (HSPCs) harboring a corrected PKLR gene engraft in the bone marrow (BM). The therapeutic efficacy of allogeneic transplant is limited by the availability of a suitable donor and transplant-associated toxicities. Preclinical studies conducted in a clinically relevant PKD murine model have demonstrated the safety and efficacy of Lin- BM cells transduced with the therapeutic lentiviral vector, PGK-coRPK-WPRE, in ameliorating the PKD phenotype. More specifically, transplantation of transduced cells resulted in increased erythrocyte survival, decreased reticulocytosis, and improvement in the secondary manifestations of hemolytic anemia, including splenomegaly and hepatic iron overload. Based on compelling preclinical data, a global Phase 1 clinical trial RP-L301-0119 (clinicaltrials.gov#NCT04105166) is underway to evaluate the feasibility and safety of lentiviral mediated gene therapy in adults and pediatric subjects with severe PKD. Methods: 6 subjects with severe PKD (defined as having a history of severe and/or transfusion-dependent anemia despite prior splenectomy) will be enrolled in the Phase 1 study; the first 2 subjects will be adults (age ≥18-<50 years), followed by 2 older pediatric subjects (age ≥12-17 years) once initial safety has been established. Subsequently, 2 younger pediatric subjects (age ≥8-11 years) will be enrolled. Peripheral blood (PB) hematopoietic stem cells are collected on two consecutive days via apheresis after mobilization with granulocyte-colony stimulating factor (G-CSF) and plerixafor. CD34+ HSPCs are enriched, transduced with PGK-coRPK-WPRE lentiviral vector (LV), and cryopreserved. Following final release testing of the investigational product (IP), RP-L301, myeloablative conditioning with therapeutic drug monitoring (TDM)-guided busulfan is administered over 4 days. RP-L301 is then thawed and infused. Patients are followed for safety assessments, including replication competent lentivirus (RCL) and insertion site analysis (ISA), and for efficacy parameters including PB and BM genetic correction, decrease in transfusion requirements, clinically significant improvement in anemia, and reduction of hemolysis. Results: An adult female PKD subject (age 31 years) with significant anemia and transfusion requirement has received treatment as of July 2020. Mobilization and apheresis procedures were performed successfully and busulfan conditioning was administered at the target area under the curve (AUC). IP consisted of 3.9×106 CD34+ cells/kg body weight, with a mean vector copy number (VCN) of 2.73. Safety and preliminary efficacy results will be available at the time of presentation. Conclusions: Efficacy in pre-clinical models indicates promising potential for clinical gene therapy in severe PKDHematopoietic stem cell mobilization using G-CSF and plerixafor appears feasible and effective in adult PKD patientsIP was successfully manufactured to meet the required specifications for the Phase 1 clinical study and administered without short-term infusion related complications Disclosures Navarro: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company, Other: SN has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Patents & Royalties, Research Funding. Sevilla:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company. Glader:Agios Pharmaceuticals, Inc.: Consultancy. Beard:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Law:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Zeini:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Choi:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from this company., Patents & Royalties, Research Funding. Rao:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Schwartz:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Segovia:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from the Company., Patents & Royalties, Research Funding.

There are no approved medical therapies for the treatment of pediatric central giant cell granuloma (CGCG), a benign but potentially aggressive tumor of the jaw. Zoledronic acid (ZA), a third-generation bisphosphonate, has been used in CGCG occurring in adults. We describe 4 patients with CGCG treated with ZA, 3 of whom achieved resolution of disease up to 4 years of follow-up. Our experience suggests that ZA may be considered as treatment for pediatric CGCG.