title: Use of blood outgrowth endothelial cells for gene therapy for hemophilia A.
abstract: A culture of human blood outgrowth endothelial cells (BOECs) was established from a sample of peripheral blood and was transfected using a nonviral plasmid carrying complementary DNA for modified human coagulation factor VIII (B domain deleted and replaced with green fluorescence protein). BOECs were then chemically selected, expanded, cryopreserved, and re-expanded in culture. Stably transfected BOECs were administered intravenously daily for 3 days to NOD/SCID mice at 4 cell dose levels (from 5 x 10(4) to 40 x 10(4) cells per injection). In 156 days of observation, mice showed levels of human FVIII that increased with cell dose and time. Mice in all cell dose groups achieved therapeutic levels (more than 10 ng/mL) of human FVIII, and mice in the 3 highest dose groups acquired levels that were normal (100-200 ng/mL) or even above the normal range (highest observed value, 1174 ng/mL). These levels indicate that the BOECs expanded in vivo after administration. When the mice were killed, it was found that BOEC accumulated only in bone marrow and spleen and that these cells retained endothelial phenotype and transgene expression. Cell doses used here would make scale-up to humans feasible. Thus, the use of engineered autologous BOECs, which here resulted in sustained and therapeutic levels of FVIII, may comprise an effective therapeutic strategy for use in gene therapy for hemophilia A.
authors.affiliation: Department of Medicine, University of Minnesota Medical School, Minneapolis 55455, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Lin
authors.forename: Liming
authors.initials: L
authors.lastname: Chang
authors.forename: Anna
authors.initials: A
authors.lastname: Solovey
authors.forename: John F
authors.initials: JF
authors.lastname: Healey
authors.forename: Pete
authors.initials: P
authors.lastname: Lollar
authors.forename: Robert P
authors.initials: RP
authors.lastname: Hebbel
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism; D017690:Cell Transplantation; D002478:Cells, Cultured / Q000637:transplantation; D018076:DNA, Complementary / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology*; D005169:Factor VIII / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D005240:Feasibility Studies; D015316:Genetic Therapy; D006085:Graft Survival; D049452:Green Fluorescent Proteins; D006467:Hemophilia A / Q000628:therapy*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013045:Species Specificity; D013154:Spleen / Q000166:cytology / Q000378:metabolism; D014162:Transfection; D014183:Transplantation, Heterologous
chemical_list: D018076:DNA, Complementary; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005169:Factor VIII