Lineage marking in humanized mice to reveal HIV-1 reservoirs

人源化小鼠的谱系标记揭示了 HIV-1 病毒库

• 批准号: 9321958
• 负责人: BENJAMIN K CHEN
• 金额: $ 49.02万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9321958
• 关键词: Acute; Biological Models; CD4 Positive T Lymphocytes; Cell Line; Cell surface; Cells; DNA; Data; Development; Engineering; Experimental Models; Fibroblasts; Gene Expression; Genes; Genetic; Genetic Engineering; Genetic Transcription; Goals; HIV; HIV Infections; HIV therapy; HIV-1; Hematopoietic stem cells; Highly Active Antiretroviral Therapy; Human; Immune; Immune system; Immunodeficient Mouse; Infection; Knowledge; Lentivirus Vector; Measures; Mediating; Methods; Modification; Molecular; Molecular Profiling; Mus; Phenotype; Plasma; Proviruses; Recombinants; Recording of previous events; Reporter; Research; Residual state; Stem cells; Surface; Switch Genes; System; Time; Transplantation; Umbilical Cord Blood; Viral; Viral reservoir; Viremia; Virus; Virus Latency; humanized mouse; in vivo; mouse model; novel; novel strategies; public health relevance; reconstitution; sensor; tool

DESCRIPTION (provided by applicant): When human immunodeficiency virus (HIV) infection is controlled by highly active antiretroviral therapy (HAART), HIV persists in a reservoir of immune cells that are difficult to distinguish from comparable uninfected cells. A key obstacle to eliminating this reservoir is identifying what makes these cells unique. We hypothesize that the developmental history of infected CD4+ T cells is perturbed in ways that can be measured even while HIV is latent. However, we are currently unable to specifically study the reservoir cell states because to determine if virus is present one must activate and alter the cell. To identify these cells an ideal experimental model system would activate an irreversible genetic switch in all HIV infected cells to mark all cells that have survived infection. The use of humanized mice in research provides an opportunity to genetically manipulate human cells that develop and become infected with HIV in an in vivo context. Immunodeficient mice that are xenografted human immune systems can be infected with HIV and display a progressive decline in CD4 T cells that can be treated with highly active antiretroviral therapy. When treatment is withdrawn plasma viremia rebounds. Here we propose to develop a method to engineer humanized mice engrafted with a human immune system that encodes an irreversible genetic switch to mark latent viral reservoirs. Using state of the art lentiviral transduction we will directly transduce hematopoietic stem cells with the HIV sensor construct. A potential shortcoming of this approach is that this method does not result in the uniform modification of all human cells. In preliminary studies, we describe a major breakthrough in stem cell reprogramming whereby human fibroblasts can directly induced to become human hematopoietic stem cells to generate a multilineage reconstitution of immune cells in when transplanted into NOD/SCID/IL2Rgc-/-(NSG) mice. We will combine this novel approach with recombinant tools that encode sensitive and specific HIV activated switches that are developed in clonal fibroblast lines, and these will be reprogrammed into induced hematopoietic stem cells (iHSC) to create novel humanized mice. The goal of this technological proposal is to develop a genetic reporter mouse model where all infected cells express a GFP lineage marker to physically isolate the reservoir for molecular characterization. We will examine the inducibility of the provirus, the surface phenotype genetically marked cells that have survived HIV infection. With a fluorescent tag on the infected cells, it then becomes possible to obtain the transcriptional profile of these cells t better understand the cellular state that maintains viral persistence. Using this unbiased approach, we hope to discover specific genes and/or cell states that would allow us to target or activate this latent reservoir.

描述(申请人提供)：当人类免疫缺陷病毒(HIV)感染通过高效抗逆转录病毒疗法(HAART)控制时，HIV持续存在于免疫细胞的储存库中，这些细胞很难与可比的未感染细胞区分开来。消灭这种细胞的一个关键障碍是确定是什么让这些细胞变得独特。我们假设，被感染的CD4+T细胞的发育历史受到干扰，即使在HIV潜伏的情况下也可以测量到这种干扰。然而，我们目前还不能专门研究病毒储存的细胞状态，因为要确定病毒是否存在，必须激活和改变细胞。为了识别这些细胞，一个理想的实验模型系统将在所有感染艾滋病毒的细胞中激活一个不可逆的遗传开关，以标记所有感染后存活的细胞。人源化小鼠在中国的应用 这项研究提供了一种机会，可以在体内对发育并感染艾滋病毒的人类细胞进行基因操作。移植了人类免疫系统的免疫缺陷小鼠可能会感染艾滋病毒，并表现出CD4T细胞的进行性下降，可以用高效的抗逆转录病毒治疗来治疗。当停止治疗时，血浆病毒血症会反弹。在这里，我们建议开发一种方法来设计植入人类免疫系统的人源化小鼠，这种人免疫系统编码一个不可逆转的遗传开关来标记潜在的病毒库。利用最先进的慢病毒转导技术，我们将利用HIV传感器构建直接转导造血干细胞。这种方法的一个潜在缺点是，这种方法不会导致对所有人类细胞的统一修改。在初步研究中，我们描述了干细胞重新编程方面的一项重大突破，即当移植到NOD/SCID/IL2Rgc-/-(NSG)小鼠体内时，人成纤维细胞可以直接诱导成为人类造血干细胞，从而产生多系重建的免疫细胞。我们将把这一新方法与重组工具结合起来，重组工具编码克隆成纤维细胞系中开发的敏感和特定的HIV激活开关，这些开关将被重新编程为诱导的造血干细胞(IHSC)，以创造新的人源化小鼠。这项技术方案的目标是开发一种遗传报告鼠模型，在该模型中，所有感染细胞都表达GFP谱系标记，以物理隔离该储存库，以便进行分子鉴定。我们将检查前病毒的诱导性，即存活下来的HIV感染的表面表型遗传标记细胞。有了感染细胞上的荧光标记，就有可能获得这些细胞的转录图谱，从而更好地了解维持病毒持久性的细胞状态。使用这种无偏见的方法，我们希望发现特定的基因和/或细胞状态，使我们能够瞄准或激活这个潜在的储存库。