Abstract
Neutralizing antibodies (Nabs) are a major challenge in clinical trials of adeno-associated virus (AAV) vector gene therapy, because Nabs are able to inhibit AAV transduction in patients. We have successfully isolated several novel Nab-escaped AAV chimeric capsids in mice by administrating a mixture of AAV shuffled library and patient serum. These AAV chimeric capsid mutants enhanced Nab evasion from patient serum with a high muscle transduction efficacy. In this protocol, we describe the procedures for selection of the Nab-escaped AAV chimeric capsid, including isolation and characterization of Nab-escaping AAV mutants in mice muscle.
Keywords: AAV, Nab-escaping AAV, Chimeric capsid, Mouse muscle
Background
Adeno-associated virus (AAV) vectors have been used in many preclinical studies and clinical trials. Many diseases have received eventual treatment using AAV gene therapy. However, the presence of neutralizing antibodies in circulation poses a major challenge for AAV vector application in future clinical trials. Many approaches have been explored to evade activities of Nab. Herein, we described the approach with directed evolution for selection of Nab-escaping mutants from an AAV shuffling library. DNA shuffling is a powerful strategy for generating diverse mutants. Through successive rounds of phenotypic selection, DNA shuffling libraries were characterized by higher quality and more targeted diversification. High-throughput selection of capsid mutants from AAV shuffling libraries has been used as a promising strategy to explore AAV mutants with the abilities to target specific tissues and evade Nabs. However, most of these selecting methods were only tested in vitro; some studies even used rabbit anti-AAV2 sera or human intravenous immunoglobulin. The approach of in vivo selection of capsid mutants could provide a platform to generate more effective AAV mutants that not only escape Nab from patient serum but also enhance transduction in specific tissues.
Materials and Reagents
Equipment
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Data analysis
Recipes
Acknowledgments
This work was supported by NIH grants R01DK084033 (to C.L. and R.J.S.), P01HL112761, R01AI072176, R01AR064369 and U54AR056953 (to R.J.S). R.J.S. is the founder and a shareholder at Asklepios Biopharmaceutical. He holds patents that have been licensed by UNC to Asklepios Biopharmaceutical, for which he receives royalties. This protocol is adapted from Li et al. (2016).
References
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