In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses
In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses
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DOI：
10.1128/jvi.00254-08
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发表时间：
2008-06-01
影响因子：
5.4
通讯作者：
Kay, Mark A.
中科院分区：
文献类型：
--
作者：
Grimm, Dirk;Lee, Joyce S.;Kay, Mark A.
腺相关病毒（AAV）血清型在转导功效和组织向性方面存在很大差异，因此作为人类基因治疗载体具有巨大潜力。然而，实际上，它们在患者中的使用受到普遍的抗 AAV 免疫力或它们在特定目标（例如肝脏中的 2 型 AAV（AAV-2）原型）中表现不足的限制。在这里，我们尝试通过适应性 DNA 家族改组技术融合多种天然 AAV 分离株的理想品质，以创建来自八种不同野生型病毒的混合衣壳的复杂文库。对原代或转化的人肝细胞的选择产生了来自五种起始血清型的杂合体库：2、4、5、8和9。然后使用混合的人抗血清（静脉注射免疫球蛋白[IVIG]）进行更严格的选择，从而选择了单一2型/8型/9型嵌合体AAV-DJ，其与它最接近的天然亲属（AAV-2）相差60个衣壳氨基酸 酸。重组 AAV-DJ 载体在培养物中的表现优于八种标准 AAV 血清型，并且在初始小鼠和 IVIG 免疫小鼠的肝脏中大大超过了 AAV-2。人们发现 AAV-DJ 中的肝素结合域限制了肝脏（和一些其他组织）的生物分布，并影响载体剂量反应和抗体中和。此外，我们报告了通过使用新的 AAV-DJ 衍生的病毒肽展示库，首次成功对 AAV 衣壳进行体内生物淘选。在小鼠肺部连续传代后富集的两种肽介导 AAV-DJ 载体重新靶向不同的肺泡细胞。我们的研究验证了 DNA 家族改组和病毒肽展示是两种强大且兼容的方法，用于人类基因治疗应用的新型 AAV 载体的分子进化。
Adeno-associated virus (AAV) serotypes differ broadly in transduction efficacies and tissue tropisms and thus hold enormous potential as vectors for human gene therapy. In reality, however, their use in patients is restricted by prevalent anti-AAV immunity or by their inadequate performance in specific targets, exemplified by the AAV type 2 (AAV-2) prototype in the liver. Here, we attempted to merge desirable qualities of multiple natural AAV isolates by an adapted DNA family shuffling technology to create a complex library of hybrid capsids from eight different wild-type viruses. Selection on primary or transformed human hepatocytes yielded pools of hybrids from five of the starting serotypes: 2, 4, 5, 8, and 9. More stringent selection with pooled human antisera (intravenous immunoglobulin [IVIG]) then led to the selection of a single type 2/type 8/type 9 chimera, AAV-DJ, distinguished from its closest natural relative (AAV-2) by 60 capsid amino acids. Recombinant AAV-DJ vectors outperformed eight standard AAV serotypes in culture and greatly surpassed AAV-2 in livers of naive and IVIG-immunized mice. A heparin binding domain in AAV-DJ was found to limit biodistribution to the liver (and a few other tissues) and to affect vector dose response and antibody neutralization. Moreover, we report the first successful in vivo biopanning of AAV capsids by using a new AAV-DJ-derived viral peptide display library. Two peptides enriched after serial passaging in mouse lungs mediated the retargeting of AAV-DJ vectors to distinct alveolar cells. Our study validates DNA family shuffling and viral peptide display as two powerful and compatible approaches to the molecular evolution of novel AAV vectors for human gene therapy applications.