Sustained human factor VIII expression in hemophilia a mice following systemic delivery of a gutless adenoviral vector
Sustained human factor VIII expression in hemophilia a mice following systemic delivery of a gutless adenoviral vector
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DOI：
10.1006/mthe.2001.0510
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发表时间：
2002-01-01
影响因子：
12.4
通讯作者：
Connelly, S
中科院分区：
文献类型：
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作者：
Reddy, PS;Sakhuja, K;Connelly, S
无肠腺病毒载体缺乏所有病毒编码区，与早期代载体相比，显示出降低的细胞毒性、降低的免疫原性和增加的编码能力。使用血友病A，凝血因子VIII（FVIII）的缺陷，作为模型疾病，我们产生并评估了编码人FVIII的无肠载体。FVIII无肠载体生长至高滴度，并从载体种子批次可重复地扩大规模。广泛的病毒DNA分析显示载体基因组没有重排。定量PCR分析表明辅助病毒污染水平<2%，最佳制剂含有0.3%的辅助病毒。我们比较了无肠载体与E1/E2 a/E3缺陷（Av 3）的早期代载体编码相同的FVIII表达盒静脉给药后，血友病A小鼠。无肠载体处理的小鼠显示出10倍更高的FVIII表达水平，并持续至少9个月。相比之下，用Av 3载体处理的小鼠在3个月时显示低于测定灵敏度极限的FVIII水平。通过测量肝酶的血清水平来评估肝毒性，表明在晚于7天的时间点，无肠载体的毒性显著低于Av 3载体。在使用的最高剂量下，两种载体在载体施用后第1天引起肝酶的瞬时10倍增加，表明这种增加是由输入衣壳蛋白的直接毒性引起的。这些数据表明，无肠载体显示增加的FVIII表达的持续时间和水平，并且比类似的早期世代载体的毒性显著更小。
Gutless adenoviral vectors are devoid of all viral coding regions and display reduced cytotoxicity, diminished immunogenicity, and an increased coding capacity compared with early generation vectors. Using hemophilia A, a deficiency in clotting factor VIII (FVIII), as a model disease, we generated and evaluated a gutless vector encoding human FVIII. The FVIII gutless vector grew to high titer and was reproducibly scaled-up from vector seed lots. Extensive viral DNA analyses revealed no rearrangements of the vector genome. A quantitative PCR assay demonstrated helper virus contamination levels of < 2%, with the best preparation containing 0.3% helper virus. We compared the gutless vector with an E1/E2a/E3-deficient (Av3) early generation vector encoding an identical FVIII expression cassette following intravenous administration to hemophilia A mice. Gutless vector-treated mice displayed 10-fold higher FVIII expression levels that were sustained for at least 9 months. In contrast, mice treated with the Av3 vector displayed FVIII levels below the limit of sensitivity of the assay at 3 months. Assessment of hepatotoxicity by measuring the serum levels of liver enzymes demonstrated that the gutless vector was significantly less toxic than the Av3 vector at time points later than 7 days. At the highest dose used, both vectors caused a transient 10-fold increase in liver enzymes I day after vector administration, suggesting that this increase was caused by direct toxicity of the input capsid proteins. These data demonstrate that the gutless vector displayed increased duration and levels of FVIII expression, and was significantly less toxic than an analogous early generation vector.