Generation of an optimized lentiviral vector encoding a high-expression factor VIII transgene for gene therapy of hemophilia A.
Generation of an optimized lentiviral vector encoding a high-expression factor VIII transgene for gene therapy of hemophilia A.
复制标题
DOI：
10.1038/gt.2012.76
复制
发表时间：
2013-06
期刊：
影响因子：
5.1
通讯作者：
Spencer, H. T.
中科院分区：
文献类型：
--
作者：
Johnston, J. M.;Denning, G.;Doering, C. B.;Spencer, H. T.
We previously compared the expression of several human factor VIII (fVIII) transgene variants and demonstrated the superior expression properties of B domain deleted porcine fVIII. Subsequently, a hybrid human/porcine fVIII molecule (HP-fVIII) comprising 91% human amino acid sequence was engineered to maintain the high-expression characteristics of porcine fVIII. The bioengineered construct then was used effectively to treat knockout mice with hemophilia A. In the current study, we focused on optimizing self-inactivating (SIN) lentiviral vector systems by analyzing the efficacy of various lentiviral components in terms of virus production, transduction efficiency and transgene expression. Specifically, three parameters were evaluated: 1) the woodchuck hepatitis post-transcriptional regulatory element (WPRE), 2) HIV versus SIV viral vector systems, and 3) various internal promoters. The inclusion of a WPRE sequence had negligible effects on viral production and HP-fVIII expression. HIV and SIV vectors were compared and found to be similar with respect to transduction efficiency in both K562s and HEK-293T cells. However, there was an enhanced expression of HP-fVIII by the SIV system, which was evident in both K562 and BHK-M cell lines. To further compare expression of HP-fVIII from an SIV-based lentiviral system, we constructed expression vectors containing the high expression transgene and a human elongation factor-1 alpha (EF1α), cytomegalovirus (CMV) or phosphoglycerate kinase (PGK) promoter. Expression was significantly greater from the CMV promoter, which also yielded therapeutic levels of HP-fVIII in hemophilia A mice. Based on these studies, an optimized vector contains the HP-fVIII transgene driven by a CMV internal promoter within a SIV-based lentiviral backbone lacking a WPRE.
登录
查看更多内容
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
20.3
作者：
Biffi, Alessandra;Bartholomae, Cynthia C.;Montini, Eugenio
通讯作者：
Montini, Eugenio
影响因子：
12.4
作者：
Doering, Christopher B.;Denning, Gabriela;Spencer, H. Trent
通讯作者：
Spencer, H. Trent
影响因子：
56.9
作者：
Cartier, Nathalie;Hacein-Bey-Abina, Salima;Aubourg, Patrick
通讯作者：
Aubourg, Patrick
影响因子：
4.8
作者：
Doering, CB;Healey, JF;Lollar, P
通讯作者：
Lollar, P