PDF(Pubmed)
Abstract：
Hematopoietic stem/progenitor cell (HSPC)-based anti-HIV-1 gene therapy holds great promise to eradicate HIV-1 or to provide long-term remission through a continuous supply of anti-HIV-1 gene-modified cells without ongoing antiretroviral therapy. However, achieving sufficient engraftment levels of anti-HIV gene-modified HSPC to provide therapeutic efficacy has been a major limitation. Here, we report an in vivo selection strategy for anti-HIV-1 gene-modified HSPC by introducing 6-thioguanine (6TG) chemoresistance through knocking down hypoxanthine-guanine phosphoribosyl transferase (HPRT) expression using RNA interference (RNAi). We developed a lentiviral vector capable of co-expressing short hairpin RNA (shRNA) against HPRT alongside two anti-HIV-1 genes: shRNA targeting HIV-1 co-receptor CCR5 and a membrane-anchored HIV-1 fusion inhibitor, C46, for efficient in vivo selection of anti-HIV-1 gene-modified human HSPC. 6TG-mediated preconditioning and in vivo selection significantly enhanced engraftment of HPRT-knockdown anti-HIV-1 gene-modified cells (>2-fold, p < 0.0001) in humanized bone marrow/liver/thymus (huBLT) mice. Viral load was significantly reduced (>1 log fold, p < 0.001) in 6TG-treated HIV-1-infected huBLT mice compared to 6TG-untreated mice. We demonstrated that 6TG-mediated preconditioning and in vivo selection considerably improved engraftment of HPRT-knockdown anti-HIV-1 gene-modified HSPC and repopulation of anti-HIV-1 gene-modified hematopoietic cells in huBLT mice, allowing for efficient HIV-1 inhibition.
摘要：
基于造血干/祖细胞（HSPC）的抗HIV-1基因治疗有望根除HIV-1或通过持续供应抗HIV-1基因修饰细胞而无需持续的抗逆转录病毒治疗来提供长期缓解。然而,实现抗HIV基因修饰的HSPC的足够植入水平以提供治疗功效一直是主要的限制。这里,我们报道了抗HIV-1基因修饰的HSPC的体内选择策略,该策略通过使用RNA干扰(RNAi)敲除次黄嘌呤-鸟嘌呤磷酸核糖转移酶(HPRT)表达,引入6-硫代鸟嘌呤(6TG)化学抗性.我们开发了一种慢病毒载体，能够与两种抗HIV-1基因共表达针对HPRT的短发夹RNA（shRNA）：靶向HIV-1共受体CCR5的shRNA和膜锚定的HIV-1融合抑制剂，C46，用于体内有效选择抗HIV-1基因修饰的人HSPC。6TG介导的预处理和体内选择显着增强了HPRT敲低抗HIV-1基因修饰细胞的植入（>2倍，p<0.0001)在人源化骨髓/肝/胸腺(huBLT)小鼠中。病毒载量显著降低(>1对数倍，与6TG未处理的小鼠相比，在6TG处理的HIV-1感染的huBLT小鼠中p<0.001）。我们证明了6TG介导的预处理和体内选择显着改善了huBLT小鼠中HPRT敲低抗HIV-1基因修饰的HSPC的植入和抗HIV-1基因修饰的造血细胞的再增殖，允许有效的HIV-1抑制。
