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Nab Escaping AAV Mutants Isolated from Mouse Muscles
从小鼠肌肉中分离Nab逃脱AAV突变体   

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参见作者原研究论文

本实验方案简略版
Molecular Therapy
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

  1. GeneMate individual 0.2 ml PCR tubes (BioExpress, catalog number: T-3035-1 )
  2. BD Veo insulin syringes with BD Ultra-Fine 6 mm x 31G needle (BD, catalog number: 324910 )
  3. CorningTM 96-Well Clear Bottom Black Polystyrene Microplates (Corning, catalog number: 3904 )
  4. VWR® Tissue Culture 48-well Plate (VWR, catalog number: 10861-560 )
  5. BALB/c mice, typically of 6 weeks old female mice
  6. ElectroMAXTM DH10BTM Cells (Thermo Fisher Scientific, catalog number: 18290015 )
  7. Adherent HEK 293 cells and Huh7 cells
  8. MAX EfficiencyTM DH10BTM Cells (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18297010 )
  9. JBS DNA-Shuffling Kit (Jena Bioscience, catalog number: PP-103 )
  10. DNase I, RNase-free (1 U/µl) (Thermo Fisher Scientific, catalog number: EN0521 )
  11. EDTA
  12. QIAquick PCR Purification Kit (250) (QIAGEN, catalog number: 28106 )
  13. Purified single-stranded AAV (any serotype) (Xiao et al., 1998)
  14. PfuUltra High-Fidelity DNA polymerase (Agilent Technologies, Santa Clara, CA)
  15. Wild type AAV2 plasmid psub201, plasmid pXR2 (RepCap plasmid) and pXX6-80 (a helper plasmid contains the genes E4, E2a and VA from adenovirus)
  16. 1x PBS (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 )
  17. Patient serum from clinical study for Duchenne Muscular Dystrophy (Bowles et al., 2012)
  18. Adenovirus dl309
  19. DNeasy Blood and Tissue Kit (QIAGEN, catalog number: 69504 )
  20. SwaI (New England Biolabs, catalog number: R0604S )
  21. XbaI (New England Biolabs, catalog number: R0145S )
  22. Passive lysis buffer (Promega, catalog number: E1941 )
  23. Luciferase Assay Substrate (Promega, catalog number: E151A )
  24. XenoLight D-Luciferin - Bioluminescent Substrate (PerkinElmer, catalog number: 122799 )
  25. 25 mg/ml D-luciferin substrate (see Recipes)

Equipment

  1. Pipettes
  2. Bio-Rad Thermal Cyclers (Bio-Rad Laboratories, catalog number: 1709703 )
  3. Tabletop centrifuge (Eppendorf, model: 5424 , catalog number: 022620401)
  4. VICTOR Multilabel Plate Reader (PerkinElmer)
  5. Small animal anesthesia system (Xenogen, XGI-8 Gas Anesthesia System)
  6. IVIS Lumina In Vivo imaging system (PerkinElmer)

Software

  1. Living Image software (PerkinElmer)

Procedure

  1. The generation of a new AAV shuffling library
    Note: There are multiple methods that can be used to generate DNA shuffling library, and a handful of kits available from manufacturers. DNA shuffling library can be generated using JBS DNA shuffling kit. The process of generation of AAV shuffling library is shown in Figure 1.


    Figure 1. Flow chart for generation of AAV shuffling library

    1. Amplify the capsid genes by PCR assay using AAV serotypes 1, 2, 3, 6, 8 and 9 mixed in equal ratios as templates. The primers sequences were shown in Table 2 of Li et al. (2008).
    2. Treat a total of 4 μg of the DNA templates by 0.4 U of DNase I at 37 °C for 5 min. Stop DNase activation by adding 1 μl 25 mM EDTA, and then heat inactivation at 75 °C for 10 min.
    3. Purify DNA fragments in size of 100-300 bp using DNA purification Kit following standard procedures.
    4. Denature, re-anneal and repair the purified DNA fragments by Pfu Ultra High-Fidelity DNA polymerase to reassemble random capsid genes in 0.2 ml PCR tube. Run the PCR assay using the program in Table 1 (Li et al., 2008; Yang et al., 2009).

      Table 1. PCR cycling parameter


    5. Clone the capsids from DNA shuffling library into wild-type AAV2 plasmid psub201 to generate AAV capsid DNA library (Li et al., 2008; Li et al., 2016).
    6. Produce AAV shuffling library in HEK293 cells by co-transfection of two plasmids: pXX6-80 and AAV capsid DNA library.
    7. Purify single-stranded AAV with CsCl gradient ultracentrifugation and measure virus titer of shuffled AAV by quantitative PCR (Xiao et al., 1998).

  2. Isolation of Nab escaping AAV mutants from mouse muscles
    1. Mix 1 x 1010 particles of the AAV shuffling library virus in 50 μl PBS with 50 μl undiluted patient serum at 4 °C for 2 h.
    2. Inject the AAV/serum mixture using insulin syringes via intramuscular (i.m.) route into the hind leg muscle of 6-week-old female BALB/c mice (injection volume 100 μl).
    3. Three days post-treatment, inject 107 virus particles (vg) of adenovirus (Adv) dl309 via i.m. into the same muscle to amplify AAV genomes in vivo.
      Note: Any replicable Adv can be used in this step.
    4. Collect muscles from injected mice at 2 days post Adv administration, and extract total DNA using DNeasy Blood and Tissue Kit.
      Note: There are several methods that can be used to isolate DNA from tissues, and a handful of kits available from many manufacturers to do it. Here, we performed a genomic DNA isolation from mouse muscle using DNeasy Blood and Tissue Kit. In this protocol, the isolation of genomic DNA from muscle relies upon the columns and spin steps. Please follow the protocol of the manufacturer.
    5. Amplify AAV mutant capsids by PCR assay using the genomic DNA from the muscle as templates. The sequences of the primers are F1 5’-CAACTCCATCACTAGGGGTTC and R1 5’-CATGGGAAAGGTGCCAGA, which are localized at the AAV2 rep and AAV2 ITR, respectively. Run PCR assay using PfuUltra High-Fidelity DNA polymerase, following the program in Table 2.

      Table 2. PCR cycling parameters


    6. Purify PCR products by the QIAquick PCR Purification Kit.
    7. Digest with SwaI and XbaI and ligate into pXR2 digested with the same endonucleases. Transform the constructed plasmids into a culture of DH10b competent cells.
      Note: The plasmid pXR2 can be any expression plasmid with Rep gene of AAV for further AAV package.
    8. Sequence clones and generate AAV/luc mutant vectors (Xiao et al., 1998).

  3. Nab escaping ability of AAV mutants in vitro
    Note: Cell lines that can be transduced by AAV can be used for neutralizing assay. Add the serum/AAV mixture when the cells are suspended.
    1. Seed Huh7 cells into a 48-well plate at a density of 105 cells for each well.
    2. Incubate two-fold dilutions of the serum (from 1:4 to 1:1,024) with AAV-Luc (1 x 108 vg) for 1 h at 37 °C.
    3. Add the mixture (diluted serum and AAV) into Huh7 cells and incubate for 48 h at 37 °C.
    4. Discard the culture media.
    5. Lyse cells with 100 μl 1x of passive lysis buffer for 30 min.
    6. Transfer the 20 μl lysed medium into 96-well black plate.
    7. Add luciferase substrate (100 μl) into 96-well black plate and briefly mix with lysed medium.
    8. Measure luciferase activity using luminometer reader (VICTOR Multilabel Plate Reader).
    9. Define Nab titers as the highest dilution for which luciferase activity is 50% lower than serum-free controls.

  4. Nab escaping ability of AAV mutants in mice
    1. Incubate 1 x 1010 vg of AAV/luc mutants isolated from muscles with the 5-fold diluted patient serum or PBS for 2 h at 4 °C.
    2. Inject the AAV/serum mixture (total volume 100 μl) in the legs of six-week-old female BALB/c mice via i.m.
    3. Image luciferase expression in mice at 3 weeks post-injection. Anesthetize mice using an isoflurane vaporizer and inject with 120 mg/kg of D-Luciferin substrate intraperitoneally 5 min before imaging. Place the mice inside the camera box of the IVIS system. Start imaging with an exposure capturing in mice.

Data analysis

  1. After the mixture of AAV shuffling library and patient serum was injected into the muscles of the mice, imaging was carried out by the IVIS Lumina In Vivo imaging system, and the photon signal was measured by Living Image software. The results were shown in Figure 1b of the original paper (Li et al., 2016).
  2. The AAV mutant capsids were recovered and sequenced. The results were shown in Figure 1a of the original paper (Li et al., 2016).
  3. The recovered AAV mutant capsids were used to package the AAV mutants. The mixtures of the AAV mutants and patient serum were administrated into muscles of mice. The imaging was carried out by the IVIS Lumina In Vivo imaging system and the photon signal was measured by Living Image software. The results were shown in Figure 2 of the original paper (Li et al., 2016).

Recipes

  1. 25 mg/ml D-luciferin substrate
    Prepare 25 mg/ml D-luciferin solution in DPBS

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

  1. Bowles, D. E., McPhee, S. W., Li, C., Gray, S. J., Samulski, J. J., Camp, A. S., Li, J., Wang, B., Monahan, P. E., Rabinowitz, J. E., Grieger. J. C., Govindasamy, L., Agbandje-Mckenna, M., Xiao, X., and Samulski, R. J. (2012) Phase 1 gene therapy for Duchenne muscular dystrophy using a translational optimized AAV vector. Mol Ther 20(2), 443-455.
  2. Li, W., Asokan, A., Wu, Z., Van Dyke, T., DiPrimio, N., Johnson, J. S., Govindaswamy, L., Agbandje-McKenna, M., Leichtle, S., Eugene Redmond, D., Jr., McCown, T. J., Petermann, K. B., Sharpless, N. E. and Samulski, R. J. (2008). Engineering and selection of shuffled AAV genomes: A new strategy for producing targeted biological nanoparticles. Mol Ther 16(7): 1252-1260.
  3. Li, C, Wu S, Albright B, Hirsch M, Li W, Tseng YS, Agbandje-Mackenna M, McPhee S, Asokan A, Samulski, R.J., (2016). Development of patient-specific AAV vectors after neutralizing antibody selection for enhanced muscle gene transfer. Mol Ther 24(1): 53-65.
  4. Xiao, X., Li, J. and Samulski, R. J. (1998). Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol 72(3): 2224-2232.
  5. Yang, L., Jiang, J., Drouin, L. M., Agbandje-McKenna, M., Chen, C., Qiao, C., Pu, D., Hu, X., Wang, D. Z., Li, J. and Xiao, X. (2009). A myocardium tropic adeno-associated virus (AAV) evolved by DNA shuffling and in vivo selection. Proc Natl Acad Sci U S A 106(10): 3946-3951.

简介

中和抗体(Nabs)是腺相关病毒(AAV)载体基因治疗的临床试验中的主要挑战,因为Nab能够抑制患者中的AAV转导。 我们已经成功地分离了几种新型Nab逃逸的AAV嵌合衣壳,通过给予AAV混洗库和患者血清的混合物。 这些AAV嵌合衣壳突变体增强了来自患有高肌肉转导功效的患者血清的Nab逃避。 在该协议中,我们描述了选择Nab逃逸的AAV嵌合衣壳的程序,包括在小鼠肌肉中分离和鉴定Nab逃逸的AAV突变体。

【背景】腺相关病毒(AAV)载体已被用于许多临床前研究和临床试验。使用AAV基因疗法已经接受了许多疾病的最终治疗。然而,在未来的临床试验中,循环中的中和抗体的存在对AAV载体的应用提出了主要挑战。已经探索过许多方法来逃避Nab的活动。在此,我们描述了从AAV改组库中选择Nab逃逸突变体的定向进化方法。

DNA改组是产生不同突变体的强大策略。通过连续几轮的表型选择,DNA改组文库的特点是质量更高,目标多样化。来自AAV改组文库的衣壳突变体的高通量选择已被用作探索具有靶向特定组织和逃避Nabs的能力的AAV突变体的有前途的策略。然而,大多数这些选择方法只是在体外测试;一些研究甚至使用了兔抗AAV2血清或人静脉注射免疫球蛋白。体内选择衣壳突变体的方法可以提供产生更有效的AAV突变体的平台,所述AAV突变体不仅从患者血清中逃脱Nab,而且增强特定组织中的转导。

关键字:AAV, Nab逃脱AAV, 嵌合衣壳, 小鼠肌肉

材料和试剂

  1. GeneMate单独0.2ml PCR管(BioExpress,目录号:T-3035-1)
  2. 带BD Ultra-Fine 6 mm x 31G针头的BD Veo胰岛素注射器(BD,目录号:324910)
  3. Corning TM 96孔透明底部黑色聚苯乙烯微孔板(Corning,目录号:3904)
  4. VWR 组织培养48孔板(VWR,目录号:10861-560)
  5. BALB / c小鼠,通常是6周龄雌性小鼠
  6. ElectroMAX TM DH10B TM细胞(Thermo Fisher Scientific,目录号:18290015)
  7. 贴壁HEK 293细胞和Huh7细胞
  8. MAX效率TM DH10B TM细胞(Thermo Fisher Scientific,Invitrogen TM,产品目录号:18297010)
  9. JBS DNA-Shuffling Kit(Jena Bioscience,目录号:PP-103)
  10. DNase I,不含RNA酶(1U /μl)(Thermo Fisher Scientific,目录号:EN0521)
  11. EDTA
  12. QIAquick PCR纯化试剂盒(250)(QIAGEN,目录号:28106)
  13. 纯化的单链AAV(任何血清型)(Xiao等人,1998)
  14. PfuUltra高保真DNA聚合酶(Agilent Technologies,Santa Clara,CA)
  15. 野生型AAV2质粒psub201,质粒pXR2(RepCap质粒)和pXX6-80(辅助质粒含有来自腺病毒的基因E4,E2a和VA)
  16. 1x PBS(Thermo Fisher Scientific,Gibco TM,目录号:14190144)
  17. 来自Duchenne肌营养不良症临床研究的患者血清(Bowles et ,2012)
  18. 腺病毒dl309
  19. DNeasy血液和组织试剂盒(QIAGEN,目录号:69504)
  20. SwaI(新英格兰生物实验室,目录号:R0604S)
  21. XbaI(新英格兰生物实验室,目录号:R0145S)
  22. 被动裂解缓冲液(Promega,目录号:E1941)
  23. 萤光素酶测定底物(Promega,目录号:E151A)
  24. XenoLight D-荧光素 - 生物发光底物(PerkinElmer,目录号:122799)
  25. 25毫克/毫升的D-荧光素底物(见食谱)

设备

  1. 移液器
  2. Bio-Rad热循环仪(Bio-Rad Laboratories,目录号:1709703)
  3. 台式离心机(Eppendorf,型号:5424,目录号:022620401)
  4. VICTOR Multilabel Plate阅读器(PerkinElmer)
  5. 小动物麻醉系统(Xenogen,XGI-8气体麻醉系统)
  6. IVIS Lumina体内成像系统(PerkinElmer)

软件

  1. 生活图像软件(PerkinElmer)

程序

  1. 一个新的AAV混洗库的生成
    注:有多种方法可用于生成DNA改组文库,以及制造商提供的一些试剂盒。使用JBS DNA改组试剂盒可以产生DNA改组文库。图1显示了AAV改组库的生成过程。


    图1.生成AAV混洗库的流程图

    1. 通过PCR分析扩增衣壳基因,使用以相同比例混合的AAV血清型1,2,3,6,8和9作为模板。引物序列显示在Li等人(2008)的表2中。
    2. 在37℃下用0.4U DNase I处理总共4μgDNA模板5分钟。加入1μl25mM EDTA终止DNase活化,然后在75℃下加热灭活10分钟。
    3. 按照标准程序使用DNA纯化试剂盒纯化大小为100-300bp的DNA片段。
    4. 通过Pfu Ultra High-Fidelity DNA聚合酶变性,重新退火并修复纯化的DNA片段,以在0.2ml PCR管中重新组装随机衣壳基因。使用表1中的程序运行PCR分析(Li等人,2008; Yang等人,2009)。

      表1. PCR循环参数


    5. 将来自DNA改组文库的衣壳克隆到野生型AAV2质粒psub201中以产生AAV衣壳DNA文库(Li等人,2008; Li等人,2016)。
    6. 通过共转染两种质粒pXX6-80和AAV衣壳DNA文库,在HEK293细胞中产生AAV改组文库。
    7. 用CsCl梯度超速离心纯化单链AAV,并通过定量PCR测定改组AAV的病毒滴度(Xiao等人,1998)。

  2. 从小鼠肌肉中分离Nab逃逸的AAV突变体
    1. 在50μlPBS和50μl未稀释的患者血清中,在4℃将1×10 10个AAV改组文库病毒颗粒混合2小时。
    2. 使用胰岛素注射器通过肌内(i.m.)途径注射AAV /血清混合物到6周龄雌性BALB / c小鼠(注射体积100μl)的后腿肌肉中。
    3. 治疗后三天,通过腹膜内注射腺病毒(Adv)dl309的10 7病毒颗粒(vg)。进入相同的肌肉来扩增体内AAV基因组。
      注意:在这一步中可以使用任何可复制的Adv。
    4. 在Adv给药后2天从注射的小鼠收集肌肉,并使用DNeasy血液和组织试剂盒提取总DNA。
      注意:有几种方法可用于从组织中分离DNA,还有许多制造商提供的少数几种方法可用于此方法。在这里,我们使用DNeasy血液和组织试剂盒从小鼠肌肉中分离出基因组DNA。在该协议中,从肌肉中分离基因组DNA依赖于柱和旋转步骤。请遵循制造商的协议。
    5. 通过使用来自肌肉的基因组DNA作为模板的PCR分析来扩增AAV突变体衣壳。引物序列为F1 5'-CAACTCCATCACTAGGGGTTC和R1 5'-CATGGGAAAGGTGCCAGA,分别定位于AAV2 rep和AAV2 ITR。按照表2中的程序使用PfuUltra高保真DNA聚合酶进行PCR测定。

      表2. PCR循环参数


    6. 通过QIAquick PCR纯化试剂盒纯化PCR产物。
    7. 用SwaI和XbaI消化并连接到用相同核酸内切酶消化的pXR2中。将构建的质粒转化为DH10b感受态细胞的培养物。
      注:质粒pXR2可以是任何表达AAV包装的AAV Rep基因的表达质粒。
    8. 序列克隆并产生AAV / luc突变体载体(Xiao等人,1998)。

  3. AAV突变体在体外的Nab逃避能力
    注意:可以通过AAV转导的细胞系可以用于中和测定。当细胞悬浮时加入血清/ AAV混合物。
    1. 将种子Huh7细胞以10 5个细胞的密度注入每个孔的48孔板中。
    2. 用AAV-Luc(1×10 8 vg)将血清的两倍稀释液(1:4至1:1,024)在37℃孵育1小时。
    3. 将混合物(稀释的血清和AAV)加入Huh7细胞并在37℃孵育48小时。
    4. 丢弃文化媒体。
    5. 用100μl1x的被动裂解缓冲液溶解细胞30分钟。
    6. 将20μl裂解的培养基转移到96孔黑板中。
    7. 将荧光素酶底物(100μl)加入96孔黑色板中,并短暂地与溶解的培养基混合。
    8. 使用光度计读数器(VICTOR Multilabel Plate Reader)测量萤光素酶活性。
    9. 将Nab效价定义为萤光素酶活性比无血清对照低50%的最高稀释度。

  4. NAV突变体在小鼠中的逃避能力
    1. 用4倍的5倍稀释的患者血清或PBS分离肌肉中分离出的1×10 10 vV的AAV / luc突变体2小时。

    2. 注射AAV /血清混合物(总体积100μl)于六周龄雌性BALB / c小鼠的腿内 i.m.
    3. 注射后3周时小鼠中的图像荧光素酶表达。使用异氟醚蒸发器麻醉小鼠,并在成像前5分钟腹膜内注射120mg / kg的D-荧光素底物。把鼠标放在IVIS系统的摄像机箱内。
      使用老鼠曝光捕捉开始成像。

数据分析

  1. 将AAV改组文库和患者血清的混合物注射到小鼠的肌肉中后,通过IVIS Lumina In Vivo成像系统进行成像,并通过Living Image软件测量光子信号。结果显示在原始论文的图1b中(Li等人,2016年)。
  2. AAV突变体衣壳被回收并测序。结果显示在原始论文的图1a中(Li等人,2016年)。
  3. 回收的AAV突变体衣壳用于包装AAV突变体。将AAV突变体和患者血清的混合物施用到小鼠的肌肉中。成像通过IVIS Lumina In Vivo成像系统进行,光子信号由Living Image软件测量。结果显示在原始论文的图2中(Li等人,2016年)。

食谱

  1. 25毫克/毫升D-荧光素底物

    在DPBS中制备25 mg / ml D-荧光素溶液

致谢

这项工作得到了NIH拨款R01DK084033(对C.L.和R.J.S。),P01HL112761,R01AI072176,R01AR064369和U54AR056953(对R.J.S)的支持。 R.J.S.是Asklepios Biopharmaceutical的创始人和股东。他拥有已获得UNC授权给Asklepios生物制药公司的专利,为此他收到版税。该协议改编自Li等人(2016年)。

参考

  1. Bowles,D.E.,McPhee,S.W.,Li,C.,Gray,S.J.,Samulski,J.J.,Camp,A.S.,Li,J.,Wang,B.,Monahan,P.E。,Rabinowitz,J.E.,Grieger。 JC,Govindasamy,L.,Agbandje-Mckenna,M.,Xiao,X。和Samulski,RJ(2012)
  2. Li,W.,Asokan,A.,Wu,Z.,Van Dyke,T.,DiPrimio,N.,Johnson,JS,Govindaswamy,L.,Agbandje-McKenna,M.,Leichtle,S.,Eugene Redmond, D.,Jr.,McCown,TJ,Petermann,KB,Sharpless,NE和Samulski,RJ(2008)。 改组后的AAV基因组的工程和选择:生产靶向生物纳米粒子的新策略。 Mol Ther 16(7):1252-1260。
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  4. Xiao,X.,Li,J。和Samulski,R.J。(1998)。 在缺乏辅助性腺病毒的情况下生产高滴度的重组腺相关病毒载体。 J Virol 72(3):2224-2232。
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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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