Ex Vivo Gene Trasfer for Hemophilia A That Enhances Safety and Results in Sustained In Vivo Factor VIII Expression from Lentivirally-Engineered Endothelial Progenitors.

Abstract Hemophilia is an excellent candidate disorder for the use of gene therapy as a treatment modality. However, significant obstacles have been encountered with systemic delivery of viral vectors that have prevented sustained expression of the therapeutic protein. Investigation of alternative gene therapy strategies for hemophilia that enhance safety and facilitate long-term, therapeutic levels of the transgene product is imperative. In this study, we evaluated an ex vivo gene therapy strategy for hemophilia A. Circulating endothelial cell progenitors (blood outgrowth endothelial cells - BOECs) were isolated from canine and mouse blood and transduced with a third generation self-inactivating lentiviral vector encoding the canine FVIII transgene under the transcriptional control of either the CMV promoter or an endothelial cell-specific regulatory element. Transduced BOECs were injected either intravenously (IV) or subcutaneously mixed with Matrigel (SC+Matrigel) into NOD/SCID mice. Canine FVIII antigen levels were assayed at weekly intervals using an Asserachrom VIII:Ag ELISA that detects canine FVIII against a background of normal murine FVIII levels in the NOD/SCID mice. The mean FVIII antigen levels in mice injected with BOECs at 3 weeks following treatment were 37.5 mU/mL and 105.8mU/mL, for IV and SC+Matrigel administration, respectively. These FVIII antigen levels were sustained up to 12 weeks at therapeutic levels (21.3mU/mL and 21.7mU/mL, for IV and SC+Matrigel administration respectively). To evaluate if the observed loss of FVIII expression by 12 weeks post-treatment resulted from transcriptional silencing of the viral promoter, the CMV promoter was replaced with the endothelial cell-specific thrombomodulin (TM) promoter and transduced BOECs were implanted SC with Matrigel. In contrast to results from the CMV-regulated transgene, sustained therapeutic levels of FVIII have been documented for the duration of the study with the TM-regulated construct (34.3 mU/mL at 3 weeks and 22.5 mU/mL at 20 weeks) Immunostaining at 18 weeks after SC implantation of the transduced BOECs, shows that these cells still express FVIII and von Willebrand Factor. Biodistribution analysis by flow cytometry and quantitative PCR demonstrated that SC-implanted BOECs were retained inside the scaffold and were not detected at any other anatomic site. These results indicate that genetically-modified endothelial progenitors implanted in a SC scaffold can provide sustained therapeutic levels of FVIII and are a promising safe delivery vehicle for gene therapy of hemophilia. Currently, these engineered cells have been implanted into immunocompetant mice and FVIII levels are being assessed.

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