Ex vivo gene therapy for hemophilia a that enhances safe delivery and sustained in vivo factor VIII expression from lentivirally engineered endothelial progenitors
Ex vivo gene therapy for hemophilia a that enhances safe delivery and sustained in vivo factor VIII expression from lentivirally engineered endothelial progenitors
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DOI：
10.1634/stemcells.2006-0699
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发表时间：
2007-01-01
期刊：
影响因子：
5.2
通讯作者：
Lillicrap, David
中科院分区：
文献类型：
--
作者：
Matsui, Hideto;Shibata, Masaru;Lillicrap, David
血友病的新治疗策略必须至少与当前治疗一样有效，并证明长期安全性。迄今为止，几个血友病基因转移的小型临床试验未能显示临床前评价的前景。因此，我们希望开发和评估一种新的离体基因转移策略的可行性，其中将来源于祖细胞的细胞工程化以表达因子VIII（FVIII），然后皮下植入以充当FVIII表达的储库。从犬和鼠血液中分离循环血液生长内皮细胞（BOEC），并用编码犬FVIII转基因的慢病毒载体转导。为了提高安全性，将这些细胞皮下植入基质胶支架中，并将该策略的功效与非血友病非肥胖糖尿病/严重联合免疫缺陷小鼠中的工程化BOEC的静脉内递送进行比较。FVIII的治疗水平持续了15周，当巨细胞病毒启动子被血栓调节蛋白调控元件取代时，这些稳定表达的水平延长至20周。在免疫活性血友病小鼠中进行的后续研究（用耐受剂量的FVIII或短暂免疫抑制进行预处理）显示，治疗性FVIII表达持续27周，最终恢复至基线水平。这种转基因表达的丧失似乎是由于植入细胞的消失。用两种耐受化方案中的任一种处理的动物均未产生抗FVIII抗体。生物分布分析表明，BOEC保留在皮下植入物内。这些结果首次表明，植入皮下支架中的遗传修饰的内皮祖细胞可以提供持续的治疗水平的FVIII，并且是血友病A的有希望和安全的治疗方式。
Novel therapeutic strategies for hemophilia must be at least as effective as current treatments and demonstrate longterm safety. To date, several small clinical trials of hemophilia gene transfer have failed to show the promise of preclinical evaluations. Therefore, we wanted to develop and evaluate the feasibility of a novel ex vivo gene transfer strategy whereby cells derived from progenitor cells are engineered to express factor VIII (FVIII) and then implanted subcutaneously to act as a depot for FVIII expression. Circulating blood outgrowth endothelial cells (BOECs) were isolated from canine and murine blood and transduced with a lentiviral vector encoding the canine FVIII transgene. To enhance safety, these cells were implanted subcutaneously in a Matrigel scaffold, and the efficacy of this strategy was compared with i.v. delivery of engineered BOECs in nonhemophilic nonobese diabetic/severe combined immunodeficiency mice. Therapeutic levels of FVIII persisted for 15 weeks, and these levels of stable expression were extended to 20 weeks when the cytomegalovirus promoter was replaced with the thrombomodulin regulatory element. Subsequent studies in immunocompetent hemophilic mice, pretreated with tolerizing doses of FVIII or with transient immunosuppression, showed therapeutic FVIII expression for 27 weeks before the eventual return to baseline levels. This loss of transgene expression appears to be due to the disappearance of the implanted cells. The animals treated with either of the two tolerizing regimens did not develop anti-FVIII antibodies. Biodistribution analysis demonstrated that BOECs were retained inside the subcutaneous implants. These results indicate, for the first time, that genetically modified endothelial progenitor cells implanted in a subcutaneous scaffold can provide sustained therapeutic levels of FVIII and are a promising and safe treatment modality for hemophilia A.