The impact of minimally oversized adeno-associated viral vectors encoding human factor VIII on vector potency in vivo
The impact of minimally oversized adeno-associated viral vectors encoding human factor VIII on vector potency in vivo
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DOI：
10.1038/mtm.2016.6
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发表时间：
2016-01-01
影响因子：
4.7
通讯作者：
Armentano, Donna
中科院分区：
文献类型：
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作者：
Kyostio-Moore, Sirkka;Berthelette, Patricia;Armentano, Donna
含有超大基因组的重组腺相关病毒（rAAV）载体提供转基因表达，尽管完整基因组的低效率包装。在这里，我们的特点是超大的rAAV 2/8载体（高达5.4 kb）编码人类因子VIII（FVIII）的转录控制下的三个肝脏启动子的属性。所有载体均可在小鼠体内持续产生活性凝血因子VIII 7个月，并且在肝脏中含有相当水平的载体基因组和完整的表达盒。因此，对于5.4kb基因组大小范围，强表达盒对于FVIII生产比载体基因组大小更重要。为了评估稍微过大的载体的效力，将5.1kb AAVrh 8 R/FVIII载体与具有相同表达盒（但含有较小的C1结构域缺失的FVIII）的4.6kb（野生型大小）载体在小鼠中比较3个月。5.1 kb载体的血浆FVIII蛋白和肝脏载体基因组水平比4.6 kb载体低2 - 3倍。两种载体的载体基因组在肝脏中保持相同，主要以高分子量串联环状形式存在。总之，这些结果表明，含有异质包装的载体基因组的稍微过大的载体产生功能性转基因产物，但表现出低两倍至三倍的体内效力。
Recombinant adeno-associated viral (rAAV) vectors containing oversized genomes provide transgene expression despite low efficiency packaging of complete genomes. Here, we characterized the properties of oversized rAAV2/8 vectors (up to 5.4 kb) encoding human factor VIII (FVIII) under the transcriptional control of three liver promoters. All vectors provided sustained production of active FVIII in mice for 7 months and contained comparable levels of vector genomes and complete expression cassettes in liver. Therefore, for the 5.4 kb genome size range, a strong expression cassette was more important for FVIII production than the vector genome size. To evaluate the potency of slightly oversized vectors, a 5.1 kb AAVrh8R/FVIII vector was compared to a 4.6 kb (wild-type size) vector with an identical expression cassette (but containing a smaller C1-domain deleted FVIII) for 3 months in mice. The 5.1 kb vector had twofold to threefold lower levels of plasma FVIII protein and liver vector genomes than that obtained with the 4.6 kb vector. Vector genomes for both vectors persisted equally and existed primarily as high molecular weight concatemeric circular forms in liver. Taken together, these results indicate that the slightly oversized vectors containing heterogeneously packaged vector genomes generated a functional transgene product but exhibited a twofold to threefold lower in vivo potency.