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

描述(申请人提供)：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，以保护携带HIV-1的细胞免受HIV-1感染或复制。我们现在知道，稳定的长期再繁殖是一个高度复杂的过程，成百上千个具有不同寿命和谱系输出潜力的HSPC在移植后做出贡献。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转基因进行基因修饰作为我们的模型。我们将测试这一假设，即在体内，由艾滋病毒-1或化学手段驱动的选择压力可以正常扩大基因标记细胞的克隆群体，以扭转艾滋病毒-1造成的缺陷。最早的造血干细胞移植研究表明，造血干细胞(HSC)是一个相对单一的群体，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。干细胞克隆可以被分成不同的群体，在利用和谱系承诺方面有着很大的不同。人们可以认为干细胞的再繁殖是成百上千个具有不同特性的移植细胞以高度受控的方式竞争以维持体内平衡的过程。通常认为，为了提供足够的保护细胞来抵御HIV-1的破坏，在体内对基因工程的HSPC或成熟的T细胞进行浓缩是至关重要的。研究表明，当未受保护的细胞被HIV-1杀死时，HIV-1感染会对受保护细胞施加选择压力，使其在人源化小鼠中存活。使用化学选择剂进一步丰富基因修饰的细胞已经在动物中进行了研究。然而，由于HSPC代表着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。除了柏林的一名患者，100%的受者细胞被供体细胞取代，之前的临床研究没有确定足够的“受保护”T细胞水平来控制HIV-1。因此，如果我们要成功地开发一种针对HIV-1的干细胞疗法，我们必须充分研究HIV-1和/或化学制剂在HSPC移植后对基因修饰细胞的富集度和机制。

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.