Immobilization of FLAG-Tagged Recombinant Adeno-Associated Virus 2 onto Tissue Engineering Scaffolds for the Improvement of Transgene Delivery in Cell Transplants.
Immobilization of FLAG-Tagged Recombinant Adeno-Associated Virus 2 onto Tissue Engineering Scaffolds for the Improvement of Transgene Delivery in Cell Transplants.
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将 FLAG 标记的重组腺相关病毒 2 固定到组织工程支架上，以改善细胞移植中的转基因递送
DOI：
10.1371/journal.pone.0129013
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Ding W
中科院分区：
文献类型：
--
作者：
Li H;Zhang FL;Shi WJ;Bai XJ;Jia SQ;Zhang CG;Ding W
组织工程中基于病毒的基因修饰技术为生产更灵活、更通用的移植生物材料提供了机会。提高效率定位转基因表达是组织工程的关键，也是基于病毒的基因传递的挑战。在本研究中，我们在2型腺相关病毒(AAV)的VP2蛋白的N端加上3×FLAG载体，包装成FLAG标记的重组AAV2嵌合突变体。用N-琥珀酰亚胺基-3-(2-吡啶二硫)丙酸酯(SPDP)将突变的AAVs固定在含有抗FLAG抗体的组织工程支架上。培养的细胞被种植到支架上形成3D移植，然后在体外和体内测试病毒转导。结果表明，与野生型AAV2相比，FLAG标记的AAV2在感染培养细胞时具有相似的转导效率。用抗FLAG抗体固定在PLGA或明胶海绵支架上后，病毒介导的转基因表达显著提高，并且更加定位。我们的数据表明，以AAV衣壳突变为靶点的抗体固定化可能是一种更有效和更精确的转基因传递的实用方法。AAV的固定化在三维组织培养中靶向基因操作的组织工程应用中可能具有诱人的潜力。
The technology of virus-based genetic modification in tissue engineering has provided the opportunity to produce more flexible and versatile biomaterials for transplantation. Localizing the transgene expression with increased efficiency is critical for tissue engineering as well as a challenge for virus-based gene delivery. In this study, we tagged the VP2 protein of type 2 adeno-associated virus (AAV) with a 3×FLAG plasmid at the N-terminus and packaged a FLAG-tagged recombinant AAV2 chimeric mutant. The mutant AAVs were immobilized onto the tissue engineering scaffolds with crosslinked anti-FLAG antibodies by N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP). Cultured cells were seeded to scaffolds to form 3D transplants, and then tested for viral transduction both in vitro and in vivo. The results showed that our FLAG-tagged AAV2 exerted similar transduction efficiency compared with the wild type AAV2 when infected cultured cells. Following immobilization onto the scaffolds of PLGA or gelatin sponge with anti-FLAG antibodies, the viral mediated transgene expression was significantly improved and more localized. Our data demonstrated that the mutation of AAV capsid targeted for antibody-based immobilization could be a practical approach for more efficient and precise transgene delivery. It was also suggested that the immobilization of AAV might have attractive potentials in applications of tissue engineering involving the targeted gene manipulation in 3D tissue cultures.
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