Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy
Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy
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DOI：
10.1073/pnas.182412299
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发表时间：
2002-09-03
影响因子：
11.1
通讯作者：
Wilson, JM
中科院分区：
文献类型：
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作者：
Gao, GP;Alvira, MR;Wilson, JM
通过PCR筛选来自恒河猴的组织中与已知腺相关病毒（AAV）血清型1-6同源的序列的存在。从两种新的AAV（称为AAV 7和AAV 8）分离跨越整个rep-cap ORF的DNA。这些和先前描述的AAV之间的序列比较揭示了衣壳蛋白的最大差异。AAV 7和AAV 8不被针对其它血清型产生的异源抗血清中和。针对AAV 7和AAV 8的中和抗体在人血清中是罕见的，并且当存在时，活性低。通过使用来自AAV 2的rep和反向末端重复序列（ITR）产生由来自AAV 7和AAV 8的衣壳形成的载体，并与由AAV 1、AAV 2和AAV 5的衣壳制成的类似构建的载体进行比较。使用骨骼肌和肝脏定向基因转移的小鼠模型来评估相对载体性能。AAV 7载体在骨骼肌中表现出转基因表达的效率，与用AAV 1观察到的效率相当，AAV 1是用于该应用的最有效的已知血清型。在肝脏中，使用AAV 8的转基因表达比使用其他血清型观察到的高10至100倍。这种提高的效率与载体DNA的持久性增加和转导的肝细胞数量增加相关。用于因子IX的肝定向基因转移的AAV 8载体的效率不受用其它AAV血清型预免疫的影响。基于这些新型非人灵长类动物腺相关病毒的载体应考虑用于人类基因治疗，因为其对针对人类腺相关病毒的抗体的反应性较低，并且因为肌肉中的基因转移效率与使用最已知血清型获得的效率相似，而在肝脏中，基因转移远高于之前描述的。
Tissues from rhesus monkeys were screened by PCR for the presence of sequences homologous to known adeno-associated virus (AAV) serotypes 1-6. DNA spanning entire rep-cap ORFs from two novel AAVs, called AAV7 and AAV8, were isolated. Sequence comparisons among these and previously described AAVs revealed the greatest divergence in capsid proteins. AAV7 and AAV8 were not neutralized by heterologous antisera raised to the other serotypes. Neutralizing antibodies to AAV7 and AAV8 were rare in human serum and, when present, were low in activity. Vectors formed with capsids from AAV7 and AAV8 were generated by using rep and inverted terminal repeats (ITRs) from AAV2 and were compared with similarly constructed vectors made from capsids of AAV1, AAV2, and AAV5. Murine models of skeletal muscle and liver-directed gene transfer were used to evaluate relative vector performance. AAV7 vectors demonstrated efficiencies of transgene expression in skeletal muscle equivalent to that observed with AAV1, the most efficient known serotype for this application. In liver, transgene expression was 10- to 100-fold higher with AAV8 than observed with other serotypes. This improved efficiency correlated with increased persistence of vector DNA and higher number of transduced hepatocytes. The efficiency of AAV8 vector for liver-directed gene transfer of factor IX was not impacted by preimmunization with the other AAV serotypes. Vectors based on these novel, nonhuman primate AAVs should be considered for human gene therapy because of low reactivity to antibodies directed to human AAVs and because gene transfer efficiency in muscle was similar to that obtained with the best known serotype, whereas, in liver, gene transfer was substantially higher than previously described.