干细胞基工程免疫抗HIV感染的人性化小鼠模型
Summary
This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.
Abstract
与基于细胞干基因疗法的快速发展对抗HIV，有迫切要求的用于动物模型来研究所述遗传修饰的细胞的造血细胞分化和免疫功能。人源化的骨髓/肝脏/胸腺（BLT）小鼠模型允许在周围的人的免疫系统，其包括T细胞，B细胞，NK细胞和单核细胞的完整的重建。人胸腺植入物还允许在自体胸腺组织的T细胞的胸腺选择。除了感染艾滋病毒的研究中，模型矗立作为一个强大的工具来研究造血干细胞（HSCs）的细胞的分化，发育和功能。在这里，我们勾勒出人性化的非肥胖型糖尿病（NOD）的建设-严重联合免疫缺陷病（SCID）中的常见γ链基因敲除（Cγ – / – ）-Bone骨髓/肝脏/胸腺（NSG-BLT）小鼠造血干细胞转导与CD4嵌合抗原受体（CD4CAR）慢病毒载体。我们表明，CD4CAR造血干细胞可以成功地分化成多种谱系，并有抗HIV活性。该研究的目的是说明使用NSG-BLT小鼠模型的作为抗HIV工程化免疫的体内模型。值得注意的是，因为慢病毒和人体组织时，实验和手术应该在II级生物安全柜与特殊的预防措施（BSL2 +）设施执行在生物安全等级2（BSL2）。
Introduction
尽管联合抗逆转录病毒治疗的成功，HIV感染仍然是一个终身疾病。抗HIV细胞免疫反应在控制HIV复制非常重要的作用。在干细胞处理的最新进展已经允许基因治疗的飞速发展途径HIV治疗1-3。其结果是，它有一个适当的动物模型，它允许在抗HIV基于细胞的疗法的效力的体内研究是非常重要的。
在动物模型中与HIV的工作是由一个事实，即病毒只感染人类细胞复杂。为了规避这一限制，科学家们开始借助疾病模型像猕猴4,5猴免疫缺陷病毒（SIV）。不幸的是，在此模型中，由于跨物种的固有差异和SIV和HIV之间的差异主要限制。此外，只有高度专业化的设施是C支持工作，非人类灵长类动物，每个猕猴apable需要大量的投资。因此，为模型中的迫切需要，利用人的免疫系统，这是易受HIV感染/发病机制，并且是较少财政望而却步。
非肥胖糖尿病（NOD） -严重联合免疫缺陷（SCID）-commonγ链基因敲除（三γ – / – ）（或NSG）血液/肝脏/胸腺（BLT）的人源化小鼠模型日益被证明是一个重要的工具以研究艾滋病病毒感染。通过注入的造血干细胞（HSC）以及胎胸腺，小鼠能够开发和概括一个人免疫系统1-3。基于干细胞的基因治疗的一种类型涉及'重定向'的外周T细胞重新编程造血干细胞（HSC）能够分化为抗原特异性T细胞为目标的HIV。我们以前曾表明，工程造血干细胞与分子克隆抗HIV特异性T细胞再受体（TCR）对抗原SL9（氨基酸77-85; SLYNTVATL）HIV-1的Gag可以干细胞重定向到形成的抑制人性化的NSG-BLT小鼠模型6 HIV复制成熟T细胞。使用分子克隆的TCR的需要注意的是，它被限制为特定的人白细胞抗原（HLA）亚型，这将限制这种疗法的应用。嵌合抗原受体（CAR），另一方面，可普遍适用于所有的HLA亚型。进行最初的研究利用具有融合于细胞内ζ信令的CD3域人CD4的细胞外和跨膜结构域构成的汽车（称为CD4ζCAR）。 CD4ζCAR上的CD8 T细胞上表达能够识别HIV包膜并引发细胞毒性T细胞应答是类似于由T细胞受体7介导的。我们最近证明人肝星状细胞可以与CD4ζCAR，然后可以分化成多种造血里进行修改neages，包括能够抑制在人源化小鼠模型8 HIV复制的功能的T细胞。在嵌合抗原受体疗法的快速进步癌症9，和有效的宽的中和抗体10-12抗HIV，允许单链抗体车的结构的持续表征，它是可感知的，许多新的候选构建体，除了CD4ζCAR ，将产生与艾滋病毒和其他疾病的干细胞基于基因疗法试验。另外，含有这些抗原特异性的CARs人源化NSG-BLT小鼠模型也可提供一个有用的工具仔细检查体内人T细胞应答。重要的是，我们的协议不同于先前描述的结构的人源化的BLT小鼠13-15在于，造血干细胞在胶状蛋白质混合物代替胎肝中继线16的使用方法。本协议描述：1）humani建设与CD4ζCAR工程捷思BLT小鼠;和2）的遗传修饰的细胞的分化的表征;和3）的经遗传修饰的细胞的功能性表征。
Protocol
Representative Results
Discussion
与汽车和走向临床研究基于HSC工程免疫力蓄势待发，它有一个合适的动物模型来仔细研究这些工程细胞的分化和功能是非常重要的。在这个协议中，我们描述的构建和测试与设计的抗HIV基因修饰的干细胞小鼠人性化的方法。有前移植干细胞的有效转导是重要的。然而，由于T细胞在识别靶细胞增殖的能力，干细胞修饰的低含量足以产生针对HIV复制8健壮响应。
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Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to thank Ms. Jessica Selander in providing artistic assistant in making our figures. This work was funded by grants from the NIAID/NIH, grant no. RO1AI078806, the UCLA Center for AIDS Research (CFAR), grant no. P30AI28697, the California Institute for Regenerative Medicine, grant no. TR4-06845, the American Federation for AIDS Research (amfAR), grant no. #108929-54-RGRL, and the UC Multi-campus Research Program and Initiatives, California Center for Antiviral Drug discovery (CCADD)
Materials
| CD34 microbead kit | miltenyi | 130-046-702 | For sorting human CD34+ progenitor cells |
| Bambanker | Wako | 302-14681 | for freezing cells |
| QIAamp Viral RNA kit | Qiagen | 52904 | For measuring viral load in the serum |
| MACSQuant Flow Cytometer | Miltenyi | For flow analysis | |
| BD LSRFortessa™ | BD biosciences | For flow analysis | |
| Hyaluronidase | Sigma | H6254-500MG | For tissue digestion |
| Deoxyribonuclease I | Worthington | LS002006 | for tissue digestion |
| Collagenase | Life technology | 17104-019 | for tissue digestion |
| CFX Real time PCR detection system | Biorad | For measuring viral load and gene expression | |
| Mice, strain NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ | The Jackson Laboratory | 5557 | For constructing the humanized mice |
| Penicillin Streptomycin (Pen Strep) | Thermo Fisher Scientific | 10378016 | For culturing cells |
| piperacillin/tazobactam | Pfizer | Zosyn | Anti-fungal |
| Amphotericin B (Fungizone antimycotic) | Thermo Fisher Scientific | 15290-018 | Anti-fungal |
| AUTOCLIP Wound Clips, 9 mm – 1000 units | Becton Dickinson | 427631 | For surgery |
| Sterile Poly-Reinforced Aurora Surgical Gowns, 30 per case | Medline | DYNJP2707 | For surgery |
| sutures, 4-0, vicryl | Owens and Minor | 23000J304H | For surgery |
| Alcohol prep pads | Owens and Minor | 3583006818 | For surgery |
| Gloves, surgical, 6 1/2 | Owens and Minor | 4075711102 | For surgery |
| Yssel’s Serum-Free T-Cell Medium | Gemini Bio-products | 400-102 | For CD34+ cell transduction |
| Human Serum Albumin | Sigma-Aldrich | A9511 | For CD34+ cell transduction |
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