Infusing Factor Viii-Expressing Platelets Or Megakaryocytes As A Novel Therapeutic Strategy For Hemophilia A

Approximately 1:5000 males have the most common inherited form of severe bleeding, hemophilia A, a deficiency of functional coagulation factor VIII. Patients with severe hemophilia A suffer from recurrent bleeding with significant morbidity and mortality with 20-30% of these patients developing antibodies to infused Factor (F) VIII therapy. One area of on-going research for treatments for these patients is ectopically expressing FVIII in megakaryocytes and platelets. This FVIII, termed pFVIII, is stored in alpha granules of platelets and is capable of restoring hemostasis in FVIIInull mice, even in the presence of circulating inhibitors. pFVIII has been proposed to be used for gene therapy for patients with hemophilia A, intractable inhibitors, and life-threatening bleeds. However, prior studies by us have shown that high levels of pFVIII can injure developing megakaryocytes. Combined with the known risk of prolonged thrombocytopenia following bone marrow transplantation, this may limit its utility of this strategy. Because of these limitations, we now propose an alternative therapeutic pFVIII strategy of infusing pFVIII-expressing megakaryocytes or platelets. We envision that such a product would be generated beginning with induced-pluripotent stem cells (iPSCs). iPSC-derived megakaryocytes, termed iMks, that are modified to express pFVIII may then be used to improved hemostasis in problematic inhibitor patients with hemophilia A. As proof-of-principle, we demonstrate that improved hemostasis can be achieved in vitro and in vivo with human pFVIII-expressing murine platelet. Infusion of such platelets can provide several days of improved hemostasis in FVIIInull mice. They were effective in the presence of inhibitors, and the efficacy of pFVIII was enhanced by recombinant factor VIIa. Human pFVIII-expressing iMks also improved hemostasis in vitro and derived platelets from infused human pFVIII-iMks improved hemostasis in FVIIInull mice. These studies indicate the potential therapeutic use of recurrent pFVIII-expressing megakaryocyte or platelet infusions with prolonged hemostatic coverage that may be additive with present-day bypassing agents in hemophilia A patients with clinically relevant neutralizing inhibitors

Genome-wide RNAi Screen Identifies SEC61A and VCP as Conserved Regulators of Sindbis Virus Entry

Alphaviruses are a large class of insect-borne human pathogens and little is known about the host-factor requirements for infection. To identify such factors, we performed a genome-wide RNAi screen using model Drosophila cells and validated 94 genes that impacted infection of Sindbis virus (SINV), the prototypical alphavirus. We identified a conserved role for SEC61A and valosin-containing protein (VCP) in facilitating SINV entry in insects and mammals. SEC61A and VCP selectively regulate trafficking of the entry receptor NRAMP2, and loss or pharmacological inhibition of these proteins leads to altered NRAMP2 trafficking to lysosomal compartments and proteolytic digestion within lysosomes. NRAMP2 is the major iron transporter in cells, and loss of NRAMP2 attenuates intracellular iron transport. Thus, this study reveals genes and pathways involved in both infection and iron homeostasis that may serve as targets for antiviral therapeutics or for iron-imbalance disorders