Enhanced factor VIII heavy chain for gene therapy of hemophilia A.
Enhanced factor VIII heavy chain for gene therapy of hemophilia A.
复制标题
用于血友病 A 基因治疗的增强因子 VIII 重链。
DOI：
10.1038/mt.2008.292
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发表时间：
2009-03
影响因子：
12.4
通讯作者：
Xiao, Weidong
中科院分区：
文献类型：
--
作者：
Chen, Lingxia;Lu, Hui;Wang, Jinhui;Sarkar, Rita;Yang, Xiao;Wang, Hongli;High, Katherine A.;Xiao, Weidong
使用重组腺病毒相关病毒（AAV）载体的血友病A基因治疗受到因子VIII（FVIII）cDNA的大小的阻碍。先前，已经成功地探索了将FVIII编码序列分成重链（HC）片段和轻链（LC）片段以用于双重重组AAV载体递送。然而，这种方法的主要缺点是“链不平衡”问题，其中LC分泌比HC高约1-2个对数，因此，合成的大多数蛋白质是无功能的。为了改善HC分泌，我们基于LC促进HC分泌的观察构建了替代的FVIII HC。令我们惊讶的是，大多数新的HC分子表现出比传统HC分子（HC 745）增强的表达。优化的HC突变蛋白，HCHL，包括额外的酸性区域3（ar 3）序列，在酶联免疫吸附试验（ELISA）和活化部分凝血活酶时间（aPTT）测定在体外测试中表现出三至五倍的活性。进一步表征表明ar 3序列增加HC分泌，而不是促进HC合成。AAV 8-HCHL+ AAV 8-LC或AAV 8-HC 745 + AAV 8-LC的静脉内递送在血友病A小鼠中实现了表型校正。接受AAV 8-HCHL+ AAV 8-LC的小鼠实现了比AAV 8-HC 745 + AAV 8-LC高三至四倍的HC表达，与FVIII功能测定一致。HCHL由于其增强的表达而应在双AAV载体策略中取代HC 745。
Hemophilia A gene therapy using recombinant adenovirus-associated virus (AAV) vectors has been hampered by the size of the factor VIII (FVIII) cDNA. Previously, splitting the FVIII coding sequence into a heavy-chain (HC) fragment and a light-chain (LC) fragment for dual recombinant AAV vector delivery has been successfully explored. However, the main disadvantage of this approach is a “chain imbalance” problem in which LC secretion is ~1–2 logs higher than that of HC, and therefore, the majority of protein synthesized is nonfunctional. To improve HC secretion, we constructed alternate FVIII HCs based on our observation that LC facilitates HC secretion. To our surprise, most of the new HC molecules exhibited enhanced expression over the traditional HC molecule (HC745). The optimized HC mutein, HCHL, including additional acidic-region-3 (ar3) sequences, exhibited three- to fivefold higher activity in both enzyme-linked immunosorbent assay (ELISA) and activated partial thromboplastin time (aPTT) assay in in vitro testing. Further characterization suggested ar3 sequences increased HC secretion, rather than promoting HC synthesis. Intravenous delivery of AAV8-HCHL+AAV8-LC or AAV8-HC745+AAV8-LC achieved phenotypic correction in hemophilia A mice. Mice receiving AAV8-HCHL+AAV8-LC achieved three- to four-fold higher HC expression than AAV8-HC745+AAV8-LC, consistent with the FVIII functional assays. HCHL should be substituted for HC745 in a dual AAV vector strategy due to its enhanced expression.
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影响因子：
12.4
作者：
Chen, Lingxia;Zhu, Fuxiang;Xiao, Weidong
通讯作者：
Xiao, Weidong
影响因子：
11.1
作者：
Lofqvist, T;Nilsson, IM;Pettersson, H
通讯作者：
Pettersson, H
DOI：
10.1073/pnas.182412299
发表时间：
2002-09-03
影响因子：
11.1
作者：
Gao, GP;Alvira, MR;Wilson, JM
通讯作者：
Wilson, JM
影响因子：
10.4
作者：
Cunningham, MA;Pipe, SW;Kaufman, RJ
通讯作者：
Kaufman, RJ
影响因子：
4.2
作者：
Hauck, B;Xu, RR;Xiao, WD
通讯作者：
Xiao, WD