HIV Cure with CCr5 (-) Human IPS Hematopoietic Stem Cells

描述：这项提议的目标是开发一种有效的方法，为艾滋病毒感染者提供“功能治疗”。这种方法是基于这样的观察，即缺乏CCR5表达的受试者对艾滋病毒感染具有高度的抵抗力。我们的假设是，造血CD34干细胞和祖细胞(HSPC)可以通过CCR5基因突变来抵抗HIV感染。这些细胞被移植回自体捐献者，将防止艾滋病毒复制，并起到“治愈”的作用。首先，通过强调非整合载体的策略，纯化的人CD34细胞或来自未感染个体的外周血单核细胞将被转化为诱导多能干细胞(IPS)。然后，这些iPS细胞将被基因改造，使其具有CCR5的Δ32bp自然突变，这与细胞表面缺乏这种受体表达有关。值得注意的是，自然缺乏CCR5表达的细胞被给予了几年后没有艾滋病毒感染证据的“柏林患者”。IPS来源的CCR5突变细胞将转化为CD34细胞(即iPS来源的CD34 HSPC)，然后分化为造血祖细胞。这些细胞将在细胞培养中和移植到人源化小鼠体内后，评估其对艾滋病毒感染的抵抗力和正常细胞功能。同样的程序将对来自艾滋病毒感染者的转基因CD34细胞进行。这些研究旨在优化我们的方法，为HIV感染者提供iPS来源的CD34 HSPC，以防止HIV疾病的进展，并有可能建立一种“功能治疗”。

DESCRIPTION: The objective of this proposal is to develop an effective method for providing a "functional cure" for HIV- infected individuals. The approach is based on the observation that subjects lacking CCR5 expression can be highly resistant to HIV infection. Our hypothesis is that hematopoietic CD34+ stem and progenitor cells (HSPC) can be made resistant to HIV infection via mutation in the CCR5 gene. These cells transplanted back to autologous donors will prevent HIV replication and effect a "cure." First, purified human CD34+ cells or peripheral blood mononuclear cells from uninfected individuals will be converted into induced pluripotent stem (iPS) cells by strategies that emphasize non-integrating vectors. These iPS cells will then be genetically modified to have the Δ32bp natural mutation of CCR5 associated with the absence of this receptor expression on the cell surface. Notably, cells naturally lacking CCR5 expression were given to the "Berlin patient" who has no evidence of HIV infection after several years. The iPS-derived CCR5-mutated cells will be converted into CD34+ cells (i.e. iPS-derived CD34+ HSPC) and then differentiated into hematopoietic progeny cells. These cells will be evaluated for resistance to HIV infection and normal cell function in cell culture and after transplantation into humanized mice. The same procedures will be undertaken with genetically modified CD34+ cells from HIV-infected individuals. These studies are directed at optimizing our approaches for providing iPS- derived CD34+ HSPC to HIV- infected individuals to prevent HIV disease progression and potentially establish a "functional cure."