Use of blood outgrowth endothelial cells for gene therapy for hemophilia A
Use of blood outgrowth endothelial cells for gene therapy for hemophilia A
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DOI：
10.1182/blood.v99.2.457
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发表时间：
2002-01-15
期刊：
影响因子：
20.3
通讯作者：
Hebbel, RP
中科院分区：
文献类型：
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作者：
Lin, Y;Chang, LM;Hebbel, RP
从外周血中分离培养人血生长内皮细胞(BOECs)，用携带修饰的人凝血因子(13区缺失，代之以绿色荧光蛋白)互补DNA的非病毒载体转染BOECs。然后对BOEC进行化学筛选、扩增、冷冻保存，然后在培养中重新扩增。将稳定表达的BOECs按4个细胞剂量水平(每次注射5×10(4)~40×10(4)细胞)静脉注射给NOD/SCID小鼠，连续3d。在156天的观察中，小鼠的人类FVIII水平随着细胞剂量和时间的增加而增加。各细胞剂量组小鼠达到治疗水平(大于10 ng/m L)，3个最高剂量组小鼠达到正常水平(100~200 ng/m L)，甚至高于正常范围(最高观察值1174 ng/m L)。这些水平表明BOECs在给药后在体内扩张。当处死小鼠时，发现BOEC仅在骨髓和脾中蓄积，这些细胞保留了内皮表型和转基因表达。这里使用的细胞剂量将使扩大到人类的规模成为可能。因此，使用工程化的自体BOECs，在这里导致持续和治疗水平的FVIII，可能包括一种有效的治疗策略，用于血友病A的基因治疗。2002年；99：457-462)(C)2002，由美国血液病学会主办。
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 (13 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. (Blood. 2002; 99:457-462) (C) 2002 by The American Society of Hematology.