Bone marrow stromal cell-mediated gene therapy for hemophilia A: in vitro expression of human factor VIII with high biological activity requires the inclusion of the proteolytic site at amino acid 1648.
Bone marrow stromal cell-mediated gene therapy for hemophilia A: in vitro expression of human factor VIII with high biological activity requires the inclusion of the proteolytic site at amino acid 1648.
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骨髓基质细胞介导的血友病A基因治疗：体外表达具有高生物活性的人因子VIII需要在氨基酸1648处包含蛋白水解位点。
作者：
G. Chiang;H. L. Rubin;V. Cherington;T. Wang;J. Sobolewski;C. A. McGrath;A. Gaffney;S. Emami;N. Sarver;P. Levine;J. Greenberger;D. Hurwitz
为了评估体外骨髓基质细胞（BMSC）系统作为血友病A基因治疗的潜力，我们研究了在体外表达的人因子VIII（hFVIII）在犬BMSC转染质粒载体和转导逆转录病毒载体。载体由hFVIII的B结构域缺失形式组成，其保留或缺失氨基酸1648处的蛋白水解位点。在转染BMSC时，载体支持高达386 mU/10（6）细胞/24小时的类似水平的表达和分泌，即使只有3-9%的细胞表达hFVIII，而42-48%的转染细胞携带质粒载体。当用逆转录病毒载体转导BMSC时，表达hFVIII的细胞百分比高得多（约70%），导致表达和分泌高达1000-4000 mU/10（6）个细胞/24 hr。Western分析表明，具有蛋白水解位点的B结构域缺失形式主要以重链和轻链异源二聚体形式分泌，其类似于血浆中发现的天然形式。相比之下，缺乏蛋白水解位点的hFVIII主要表达为未加工的单个重链-轻链。两种hFVIII形式都被凝血酶正确切割和活化。蛋白水解的hFVIII形式具有>或= 93%的正常生物活性，而未蛋白水解的形式具有始终低于55%的正常生物活性，因此被认为不太适合于治疗应用。这些结果表明，BMSC系统在血友病A的基因治疗中具有潜在的实用性，并强调了选择合适的hFVIII结构用于前瞻性临床应用的重要性。
To evaluate the potential of the ex vivo bone marrow stromal cell (BMSC) system as a gene therapy for hemophilia A, we studied the in vitro expression of human factor VIII (hFVIII) in canine BMSCs following transfection with plasmid vectors and transduction with retroviral vectors. Vectors were composed of B domain-deleted forms of hFVIII that either retain or delete the proteolytic site at amino acid 1648. On transfection of BMSCs, vectors supported expression and secretion of similar levels of up to 386 mU/10(6) cells/24 hr, even though only 3-9% of the cells expressed hFVIII while 42-48% of transfected cells harbored plasmid vector. Much higher percentages (approximately 70%) of cells expressing hFVIII were achieved when BMSCs were transduced by retroviral vectors, resulting in expression and secretion as high as 1000-4000 mU/10(6) cells/24 hr. Western analysis demonstrated that the B domain-deleted forms possessing the proteolytic site were secreted predominantly as heavy and light chain heterodimers that resemble native forms found in plasma. In contrast, the hFVIII lacking the proteolytic site was expressed mostly as unprocessed, single heavy-light chains. Both hFVIII forms were correctly cleaved and activated by thrombin. The proteolyzed hFVIII form possessed > or = 93% normal biological activity while the unproteolyzed form possessed consistently less than 55% normal biological activity and was therefore considered less suitable for therapeutic application. These results demonstrate that the BMSC system has potential utility in gene therapy for hemophilia A and stress the importance of selecting the appropriate hFVIII structure for prospective clinical use.
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DOI：
10.1073/pnas.92.11.4857
发表时间：
1995-05-23
影响因子：
11.1
作者：
PEREIRA, RF;HALFORD, KW;PROCKOP, DJ
通讯作者：
PROCKOP, DJ
影响因子：
3.7
作者：
MARKOWITZ, D;GOFF, S;BANK, A
通讯作者：
BANK, A
影响因子：
20.3
作者：
Debra D. Pittman;E. Alderman;Kathleen N. Tomkinson;Jack H. Wang;Alan R. Giles;Randal J. Kaufman
通讯作者：
Debra D. Pittman;E. Alderman;Kathleen N. Tomkinson;Jack H. Wang;Alan R. Giles;Randal J. Kaufman
抗血友病因子（因子 VIII）的凝血酶激活的蛋白水解要求。
DOI：
10.1073/pnas.85.8.2429
发表时间：
1988
影响因子：
11.1
作者：
Pittman,DD;Kaufman,RJ
通讯作者：
Kaufman,RJ
异种 MHC II 类基因转染的小鼠基质细胞的归巢和免疫原性。
影响因子：
3.3
作者：
Huss,R;Smith,FO;Myerson,DH;Deeg,HJ
通讯作者：
Deeg,HJ