Nab Escaping AAV Mutants Isolated from Mouse Muscles
从小鼠肌肉中分离Nab逃脱AAV突变体
发布: 2018年05月05日第8卷第9期 DOI: 10.21769/BioProtoc.2841 浏览次数: 6457
评审: Longping Victor TseGeorge William CarnellVikas Duhan
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参见作者原研究论文
The authors used this protocol in:
Feb 2016
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 (AAV)
Nab-escaping AAV (Nab逃脱AAV)
Chimeric capsid (嵌合衣壳)
Mouse muscle (小鼠肌肉)
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
- GeneMate individual 0.2 ml PCR tubes (BioExpress, catalog number: T-3035-1 )
- BD Veo insulin syringes with BD Ultra-Fine 6 mm x 31G needle (BD, catalog number: 324910 )
- CorningTM 96-Well Clear Bottom Black Polystyrene Microplates (Corning, catalog number: 3904 )
- VWR® Tissue Culture 48-well Plate (VWR, catalog number: 10861-560 )
- BALB/c mice, typically of 6 weeks old female mice
- ElectroMAXTM DH10BTM Cells (Thermo Fisher Scientific, catalog number: 18290015 )
- Adherent HEK 293 cells and Huh7 cells
- MAX EfficiencyTM DH10BTM Cells (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18297010 )
- JBS DNA-Shuffling Kit (Jena Bioscience, catalog number: PP-103 )
- DNase I, RNase-free (1 U/µl) (Thermo Fisher Scientific, catalog number: EN0521 )
- EDTA
- QIAquick PCR Purification Kit (250) (QIAGEN, catalog number: 28106 )
- Purified single-stranded AAV (any serotype) (Xiao et al., 1998)
- PfuUltra High-Fidelity DNA polymerase (Agilent Technologies, Santa Clara, CA)
- Wild type AAV2 plasmid psub201, plasmid pXR2 (RepCap plasmid) and pXX6-80 (a helper plasmid contains the genes E4, E2a and VA from adenovirus)
- 1x PBS (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 )
- Patient serum from clinical study for Duchenne Muscular Dystrophy (Bowles et al., 2012)
- Adenovirus dl309
- DNeasy Blood and Tissue Kit (QIAGEN, catalog number: 69504 )
- SwaI (New England Biolabs, catalog number: R0604S )
- XbaI (New England Biolabs, catalog number: R0145S )
- Passive lysis buffer (Promega, catalog number: E1941 )
- Luciferase Assay Substrate (Promega, catalog number: E151A )
- XenoLight D-Luciferin - Bioluminescent Substrate (PerkinElmer, catalog number: 122799 )
- 25 mg/ml D-luciferin substrate (see Recipes)
Equipment
- Pipettes
- Bio-Rad Thermal Cyclers (Bio-Rad Laboratories, catalog number: 1709703 )
- Tabletop centrifuge (Eppendorf, model: 5424 , catalog number: 022620401)
- VICTOR Multilabel Plate Reader (PerkinElmer)
- Small animal anesthesia system (Xenogen, XGI-8 Gas Anesthesia System)
- IVIS Lumina In Vivo imaging system (PerkinElmer)
Software
- Living Image software (PerkinElmer)
Procedure
文章信息
版权信息
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Chai, Z., Samulski, R. J. and Li, C. (2018). Nab Escaping AAV Mutants Isolated from Mouse Muscles. Bio-protocol 8(9): e2841. DOI: 10.21769/BioProtoc.2841.
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分类
免疫学 > 抗体分析 > 抗体功能
微生物学 > 抗微生物试验 > 抗病毒试验
分子生物学 > 蛋白质 > 抗微生物分析
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