Lineage marking in humanized mice to reveal HIV-1 reservoirs

描述（由申请人提供）：当人类免疫缺陷病毒（HIV）感染通过高效抗逆转录病毒治疗（HAART）得到控制时，HIV持续存在于免疫细胞库中，难以与可比的未感染细胞区分开来。消除这种储存库的一个关键障碍是确定是什么使这些细胞独特。我们假设，即使HIV是潜伏的，受感染的CD4 + T细胞的发育史也会受到干扰，可以测量。然而，我们目前无法具体研究储存细胞状态，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，理想的实验模型系统将激活所有HIV感染细胞中的不可逆遗传开关，以标记所有感染后存活的细胞。人源化小鼠的用途 研究提供了在体内环境中遗传操纵发展并感染HIV的人类细胞的机会。作为异种移植的人类免疫系统的免疫缺陷小鼠可以感染HIV，并且显示出可以用高效抗逆转录病毒疗法治疗的CD4 T细胞的进行性下降。当停止治疗时，血浆病毒血症反弹。在这里，我们建议开发一种方法来工程化人源化小鼠移植人类免疫系统，编码一个不可逆的遗传开关，以标记潜伏的病毒水库。使用最先进的慢病毒转导，我们将用HIV传感器构建体直接转染造血干细胞。这种方法的一个潜在缺点是，这种方法不会导致所有人类细胞的均匀修饰。在初步研究中，我们描述了干细胞重编程的一个重大突破，即人成纤维细胞可以直接诱导成为人造血干细胞，以产生免疫细胞的多谱系重建时，移植到NOD/SCID/IL2Rgc-/-（NSG）小鼠。我们将联合收割机这种新方法与重组工具相结合，这些重组工具编码在克隆成纤维细胞系中开发的敏感和特异性HIV激活开关，这些开关将被重编程为诱导造血干细胞（iHSC），以创建新型人源化小鼠。该技术方案的目标是开发一种基因报告小鼠模型，其中所有受感染的细胞表达GFP谱系标记物，以物理分离储库用于分子表征。我们将研究前病毒的诱导作用，前病毒是HIV感染后存活的表面表型遗传标记细胞。在感染的细胞上加上荧光标记，就有可能获得这些细胞的转录谱，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，使我们能够靶向或激活这个潜在的水库。

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 When treatment is withdrawn plasma viremia rebounds.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.