Vascular networks genetically engineered for protein drug delivery

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PROJECT SUMMARY/ABSTRACT Hemophilia A is an inherited bleeding disorder caused by mutations in the F8 gene encoding coagulation factor VIII (FVIII). Current treatment involves repeated i.v. infusions of FVIII concentrates throughout the life of the patient, which creates tremendous discomfort and morbidity. Alternatively, we seek to develop a novel technology for sustained FVIII delivery. Recently, we developed a non-viral ex vivo gene therapy approach for hemophilia A. We used a piggyBac DNA transposon system to insert 70 copies of the F8 gene into human pluripotent stem cells (PSCs). We differentiated these modified F8-PSCs into endothelial cells (iECs; natural producers of FVIII) and demonstrated the production of exceedingly high levels of FVIII. After subcutaneous engraftment of our human F8-iECs into immunodeficient hemophilic (SCID-f8ko) mice, we achieved up to 600% circulating levels of FVIII, effectively correcting the clotting deficiency. Notwithstanding this progress, our open- graft approach has some inherent limitations for translation: 1) immune rejection of non-autologous cells, and 2) concerns over cell dissemination and safety. To address these limitations, we have teamed up with Dr. Minglin Ma (Cornell), who has extensive experience with devices for encapsulation and transplantation of cells in mice and dogs. We propose a technology entailing a novel retrievable encapsulation device. We will assemble our F8-iECs into stable 3D vascular organoids and will then embed multiple organoids into an alginate hydrogel inside a tubular encapsulation device (1-mm diameter; variable length). Based on our preliminary data, we hypothesize that our device will protect the cells from immune rejection and produce FVIII that will reach the bloodstream at therapeutic levels upon implantation into the peritoneal cavity. To test these hypotheses, we propose three Specific Aims. In Aim-1, we will genetically engineer vascular organoids for the production of clinically relevant levels of FVIII. We will develop a new promoterless exon-trap sensor cassette to avoid intra- exon integration of our piggyBac transposon. We will then insert multiple F8 copies into NIH-eligible PSC lines to generate universal clones for high FVIII production. In Aim-2, we will establish an encapsulation device configuration for optimal FVIII production and determine the safety and long-term efficacy in immunocompetent hemophilic mice. We will evaluate cell survival, BDD-FVIII activity in plasma, correction of coagulation deficiency, risk of teratoma formation, and reversibility of the treatment. In Aim-3, we will evaluate the safety and long-term efficacy of our devices in dogs. We will first generate canine-specific FVIII-secreting vascular organoids. We will then transplant our devices (I.P.) in healthy dogs for up to 6 months and evaluate scalability, safety, retrievability, and FVIII production. Lastly, we will test our allogeneic devices in hemophilia A dogs and establish safety and efficacy for up to 1 year. In summary, we propose studies to develop a novel technology to deliver FVIII in hemophilia A. We envision this research could pave the way for future studies in humans.