Several log increase in therapeutic transgene delivery by distinct adeno-associated viral serotype vectors
Several log increase in therapeutic transgene delivery by distinct adeno-associated viral serotype vectors
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DOI：
10.1006/mthe.2000.0219
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发表时间：
2000-12-01
影响因子：
12.4
通讯作者：
Walsh, CE
中科院分区：
文献类型：
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作者：
Chao, HJ;Liu, YB;Walsh, CE
我们以前证明，携带人和犬因子IX（FIX）cDNA的rAAV载体可以感染，稳定持久，并分泌功能性人和犬FIX后直接肌肉注射。为了改善FIX蛋白分泌以用于最终的治疗用途，我们着手确定AAV衣壳的改变是否会影响骨骼肌转导和因子IX分泌。研究这个问题的两个原因是（I）针对AAV 2的高滴度中和抗体（NAB）的持续存在和（2）我们先前的研究支持肌纤维类型对AAV 2转导的有限向性。使用相同的CMV/犬因子IX（cFIX）表达盒，我们将该基因组交叉包装到由五种AAV血清型中的每一种产生的病毒体中。在剂量反应试验中，在体外和体内测试了等量的rAAV/cFIX血清型。在组织培养细胞中，分泌到上清液中的FIX抗原水平根据所用的AAV血清型而变化; 2型转导最多，血清型3、1、5和4分别表达较低水平。然而，当使用免疫缺陷的NOD/SCID动物在体内测试相同的病毒时，我们获得了令人惊讶的不同结果。虽然所有血清型至可检测血清水平的发生时间似乎相同，但1型、3型和5型产生的cFIX是2型的100- 1000倍。事实上，转导后12周，1型继续表达平均80 μ g/ml的cFIX水平，随后是5型（6.52 μ g/ml）、3型（3.27 μ g/ml）、4型（258 ng/ml），最后是2型（90 ng/ml）。通过aPTT测量的cFIX的凝血活性支持通过ELISA测量的循环水平，证明分泌的蛋白是功能性的，并且注射组织的RT-PCR与表型特异性转导数据相关。总之，我们发现在将各种rAAV血清型引入小鼠肌肉后，cFIX表达存在显著差异。这些数据对血友病的AAV基因治疗临床试验的设计有直接的影响，也应该扩展到大多数治疗性转基因。
We previously demonstrated that rAAV vectors carrying human and canine factor IX (FIX) cDNA can infect, stably persist, and secrete functional human and canine FIX following direct intramuscular injection. In an attempt to improve FIX protein secretion for eventual therapeutic use, we set out to determine if alteration of the AAV capsid would affect skeletal muscle transduction and factor IX secretion. Two reasons to pursue this question were (I)the persistence of high-titer neutralizing antibody (NAB) to AAV2 and (2) our previous study that supported a restricted tropism of muscle fiber types to AAV2 transduction. Using an identical CMV/canine factor IX (cFIX) expression cassette, we cross-packaged this genome into virions generated from each of the five AAV serotypes. In a dose-response assay, equivalent amounts of rAAV/cFIX serotypes were tested in vitro and in vivo, In tissue culture cells, FIX antigen levels secreted into the supernatant varied depending on the AAV serotype used; type 2 transduced maximally, with serotypes 3, 1, 5, and 4, respectively, expressing lower levels. However, when the same viruses were tested in vivo using immunodeficient NOD/SCID animals, we obtained surprisingly different results. While the time to onset of detectable serum levels appeared the same for all serotypes, types 1, 3, and 5 produced 100- to 1000-fold more cFIX than type 2. In fact, 12 weeks after transduction, type 1 continued to express levels of cFIX on average at 80 mug/ml followed by type 5 (6.52 mug/ml), type 3 (3.27 mug/ml), type 4 (258 ng/ml), and finally type 2 (90 ng/ml). Coagulant activity of cFIX as measured by aPTT supported the circulating levels measured by ELISA demonstrating the secreted protein was functional, and RT-PCR of injected tissue correlated with the serotype-specific transduction data. In summary, we found significant differences in cFIX expression upon introducing various rAAV serotypes into mouse muscle. These data have direct bearing on the design of AAV gene therapy clinical trials for hemophilia and should also extend to most therapeutic transgenes.