Directed Engineering of a High-expression Chimeric Transgene as a Strategy for Gene Therapy of Hemophilia A
Directed Engineering of a High-expression Chimeric Transgene as a Strategy for Gene Therapy of Hemophilia A
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DOI：
10.1038/mt.2009.35
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发表时间：
2009-07-01
影响因子：
12.4
通讯作者：
Spencer, H. Trent
中科院分区：
文献类型：
--
作者：
Doering, Christopher B.;Denning, Gabriela;Spencer, H. Trent
人凝血因子VIII （fVIII）在体外生物合成效率低下，并且已被证明难以使用现有的临床基因转移技术在治疗水平上表达。最近，我们发现猪和某些人/猪杂交fVIII基因的表达比人fVIII高100倍。在这项研究中，我们扩展了这些结果，描述了一种人源化、高表达的人/猪fVIII杂交基因的使用，该基因与人fVIII基因相同89%，并通过慢病毒载体（lv）传递到造血干细胞中，用于血友病a的基因治疗。重组人免疫缺陷病毒载体编码的fVIII嵌合体有效地转导了人胚胎肾(HEK)-293T细胞。用人/猪混合fVIII编码载体转导的细胞比用人fVIII编码载体转导的细胞表达的fVIII水平高6- 100倍。将转导的造血干细胞和祖细胞移植到血友病A小鼠体内，尽管基因修饰的血液单核细胞< 5%，但fVIII的长期表达仍达到治疗水平。此外，猴免疫缺陷病毒（SIV）衍生的载体有效地转导了人造血细胞系K562、EU1、U. 937和Jurkat以及非造血细胞系HEK-293T和HeLa。所有细胞系都表达人/猪杂交fVIII，尽管表达水平不同，K562细胞在造血细胞系中表达水平最高。从这些研究中，我们得出结论，人源化高表达杂交fVIII转基因可用于血友病A的基因治疗应用，与之前取得的成果相比，可显著提高fVIII的表达水平。
Human coagulation factor VIII (fVIII) is inefficiently biosynthesized in vitro and has proven difficult to express at therapeutic levels using available clinical gene-transfer technologies. Recently, we showed that a porcine and certain hybrid human/porcine fVIII transgenes demonstrate up to 100-fold greater expression than human fVIII. In this study, we extend these results to describe the use of a humanized, high-expression, hybrid human/porcine fVIII transgene that is 89% identical to human fVIII and was delivered by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Recombinant human immunodeficiency virus-based vectors encoding the fVIII-chimera efficiently transduced human embryonic kidney (HEK)-293T cells. Cells transduced with hybrid human/porcine fVIII encoding vectors expressed fVIII at levels 6- to 100-fold greater than cells transduced with vectors encoding human fVIII. Transplantation of transduced hematopoietic stem and progenitor cells into hemophilia A mice resulted in long-term fVIII expression at therapeutic levels despite < 5% genetically modified blood mononuclear cells. Furthermore, the simian immunodeficiency virus (SIV)-derived vector effectively transduced the human hematopoietic cell lines K562, EU1, U. 937, and Jurkat as well as the nonhematopoietic cell lines, HEK-293T and HeLa. All cell lines expressed hybrid human/porcine fVIII, albeit at varying levels with the K562 cells expressing the highest level of the hematopoietic cell lines. From these studies, we conclude that humanized-high-expression hybrid fVIII transgenes can be utilized in gene therapy applications for hemophilia A to significantly increase fVIII expression levels compared to what has been-previously achieved.