Infusion of Coagulation Factor VIII-Containing Induced Pluripotent Stem Cell (iPSC)-Derived Megakaryocytes (iMks) Shows Potential As Hemophilia a Treatment
Authors: Lyde Randolph B , Ahn Hyunsook , Vo Karen K , Jarocha Danuta Jadwiga , Zhai Li , Sullivan Spencer K. , Arruda Valder R , Sabatino Denise E. , Camire Rodney M. , French Deborah , Poncz Mortimer 
Published: October 2019

Abstract
Ectopically expressed factor VIII (FVIII) in megakaryocytes (MKs) and platelets (pFVIII) is stored in a-granules and released at sites of vascular injury by activated platelets (Plts), restoring hemostasis in FVIIInull mice, even in the presence of neutralizing inhibitors. These studies support the idea that unlike therapies that correct plasma levels of FVIII, pFVIII may be a useful therapy in patients with hemophilia A who have intractable inhibitors and significant bleeds. Expressing FVIII in Plts, however, has limitations that make pFVIII gene therapy through bone marrow transplantation (BMT) problematic: 1) pFVIII expressed during megakaryopoiesis can injure the Mks, potentially exacerbating post-BMT thrombocytopenia, and 2) pFVIII's efficacy in joint and intracranial bleeds has yet to be shown, especially in the presence of inhibitors. Due to these limitations we propose an alternative strategy: infusing patient-specific iMks derived from personalized iPSCs and expressing either human B-domain-deleted (BDD) FVIII or variants of FVIII that have greater stability and longer half-lives. Our group has shown that infusing in vitro-grown Mks into mice releases functional Plts in the recipient animals. iPSCs are a renewable source of stem cells that can be pre-screened to select clones that both express high levels of pFVIII and also release high numbers of Plts after differentiation into iMks. As proof-of-principle, iMks were transfected with a self-inactivating lentivirus containing cDNA for 1 of 3 FVIII variants: wildtype BDD FVIII (WT FVIII), a PACE/furin cleavage site FVIII (FVIIIR1645H) variant, and an amino acid 1645 to 1648 deletion FVIII (FVIIIΔ) variant that removes the entire PACE/furin cleavage site. FVIIIR1645H and FVIIIΔ showed greater stability and consequently greater specific activity with no increase in injuring Mks. We previously published that hemophilia A mice expressing pFVIIIR1645H were more hemostatically corrected than comparable mice expressing WT pFVIII. All of the FVIII variant iMks expressed at least a 40-fold higher level of mRNA compared to the non-transduced control (N=6) and integration levels show the same number of viral copies between the groups (N=6). All variants expressed >550 pg FVIII/106 CD42b+ iMKs (N=6). Upon activation with thrombin, transduced Mks released the FVIII into the supernatant. To examine whether this pFVIII injured the developing Mks, baseline PAC-1 binding for Mk activation in culture (N=3), TUNEL staining and Annexin-5 binding for apoptosis (N=4) were analyzed with no differences observed with WT Mks not expressing pFVIII. To test the ability of FVIII-expressing iMks to correct the coagulopathy in hemophilia A, 5x105 iMks were added to FVIIInull murine whole blood (0.11 ml) and evaluated for clot formation using rotational thromboelastometry (ROTEM). Each pFVIII iMk variant showed a decrease in clotting time, clot formation time, and an increase in maximum clot firmness when compared to the non-transduced control (p<0.007 for each, N=4). These FVIII expressing iMks were also tested in vivo in a FeCl3 carotid artery injury murine model. 24 hours prior to infusion, recipient hemophilia A mice were treated with clodronate liposomes to eliminate circulating monocytes and to improve the survival of infused human iMks and their released Plts. Immediately post iMks (5x106)infusion, a 20% FeCl3 solution was applied to the carotid artery for 3 mins and flow rate through the injured vessel was measured for 30 mins. Both WT FVIII and FVIIIR1645H showed a significant decrease in blood flow through the injured vessel from 1.2 ml/min seen in FVIIInull mice receiving control iMks to 0.4 ml/min (p<0.05, N=10). Wild-type mice had a flow rate of 0.13 ml/min. These data indicate that pFVIII within iMKs or their derived Plts expressing FVIII can improve hemostasis in vitro and in vivo. These studies provide the groundwork to examine whether infused iMks pFVIII can improve hemostasis in the setting of inhibitors.

Disclosures
Arruda: Pfizer: Patents & Royalties, Research Funding. Sabatino:Spark Therapeutics: Research Funding. Camire:Bayer: Consultancy; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Pfizer: Consultancy, Patents & Royalties, Research Funding; Novo Nordisk: Research Funding.