Survival of the fittest HSPC repopulating clones by anti-HIV-1 gene-modification

项目概述本项目的总体目标是了解通过抗HIV-1转基因对造血干细胞（HSPC）进行基因修饰的后果，从而保护宿主细胞免受HIV-1感染或复制。我们现在知道稳定的长期再生是一个高度复杂的过程，移植后成百上千的具有不同寿命和血统输出潜力的HSPCs做出了贡献。我们将在克隆水平上研究人类再生细胞的行为，并使用抗hiv -1 shRNA转基因基因修饰作为我们的模型。我们将验证由HIV-1或化学手段驱动的体内选择压力可以扩大基因标记细胞的克隆群体，从而逆转HIV-1引起的缺陷。最早的造血干细胞移植研究表明，造血干细胞（HSC）是一个相对均匀的群体，其中任何一个都有可能完全重新填充造血系统。然而，最近，这种观点被证明过于简单化了。干细胞克隆可以聚集成具有广泛不同的利用动力学和谱系承诺的群体。我们可以认为干细胞的再生是一个过程，在这个过程中，成百上千具有不同特性的移植细胞在高度控制的方式下相互竞争，以维持体内平衡。人们普遍认为，为了提供足够的保护细胞来抵御HIV-1的破坏，基因工程HSPC或成熟t细胞的体内富集是至关重要的。研究表明，在人源化小鼠中，当未受保护的细胞被HIV-1杀死时，HIV-1感染会对受保护的细胞施加选择压力，使其存活。利用化学选择剂进一步富集基因修饰细胞已在动物实验中进行了研究。然而，鉴于HSPC代表了如此多样化的克隆群体，人们对HIV-1和化学制剂对不同HSPC克隆施加选择压力的机制和程度知之甚少。除了柏林病人100%的受体细胞被供体细胞取代之外，之前没有临床研究建立足够水平的“受保护”t细胞来控制HIV-1。因此，如果我们要成功地开发一种基于干细胞的HIV-1治疗方法，我们必须充分研究HIV-1和/或化学试剂在移植HSPC后富集基因修饰细胞的程度和机制。

DESCRIPTION (provided by applicant): PROJECT SUMMARY The overall goal of this project is to understand the consequences of gene modification of hematopoietic stem progenitor cell (HSPC) by anti-HIV-1 transgenes which protect the harboring cells from HIV-1 infection or replication. We now know that stable long-term repopulation is a highly complex process whereby hundreds to thousands of HSPCs with differing life-spans and lineage output potentials contribute after transplant. We will investigate the behavior of human repopulating cells at the clonal level and use as our model, gene modification with anti-HIV-1 shRNA transgenes. We will test the hypothesis that in vivo selective pressures driven by HIV-1 or chemical means can expand clonal populations of gene-marked cells normally to reverse deficits caused by HIV-1. The earliest studies of hematopoietic stem cell transplant suggested that hematopoietic stem cells (HSC) were a relatively homogeneous population, any one of which has the potential to fully repopulate the hematopoietic system. However, more recently, this view has proven to be simplistic. Stem cell clones can be clustered into groups with widely differing kinetics of utilization and lineage commitment. One can consider repopulation by the stem cells as a process whereby hundreds or thousands of engrafted cells with differing properties compete in a highly controlled fashion to maintain homeostasis. It is generally thought that in vivo enrichment for the gene engineered HSPC or mature T-cells is critical in order to provide sufficient protected cells to withstand HIV-1 destruction. Studies have shown that HIV-1 infection imposes selection pressure for protected cells to survive in humanized mice when unprotected cells are killed by HIV-1. Further enrichment of gene-modified cells using chemoselection agents have been studied in animals. However, given that HSPC represent such diverse clonal populations, little is understood regarding the mechanism and degree to which HIV-1 and chemical agents exert selective pressure upon different HSPC clones. With the exception of the Berlin patient, where 100% of recipient cells were replaced by donor cells, no previous clinical studies established sufficient levels of "protected" T-cells to control HIV-1. Therefore, if we are to be successful in developing a stem cell-based therapy for HIV-1, we must fully investigate the extent and the mechanism by which HIV-1 and/or chemical agents can enrich for gene-modified cells after transplant of HSPC.