PDF(Pubmed)
Abstract：
Quiescent human hematopoietic stem cells (HSC) are ideal targets for gene therapy applications due to their preserved stemness and repopulation capacities; however, they have not been exploited extensively because of their resistance to genetic manipulation. We report here the development of a lentiviral transduction protocol that overcomes this resistance in long-term repopulating quiescent HSC, allowing their efficient genetic manipulation. Mechanistically, lentiviral vector transduction of quiescent HSC was found to be restricted at the level of vector entry and by limited pyrimidine pools. These restrictions were overcome by the combined addition of cyclosporin H (CsH) and deoxynucleosides (dNs) during lentiviral vector transduction. Clinically relevant transduction levels were paired with higher polyclonal engraftment of long-term repopulating HSC as compared with standard ex vivo cultured controls. These findings identify the cell-intrinsic barriers that restrict the transduction of quiescent HSC and provide a means to overcome them, paving the way for the genetic engineering of unstimulated HSC.
摘要：
静止的人类造血干细胞（HSC）由于其保留的干性和再增殖能力，是基因治疗应用的理想靶标。但是由于它们对基因操纵的抵抗力，它们没有被广泛利用。我们在这里报告了一种慢病毒转导方案的发展，该方案克服了长期重新繁殖的静止HSC中的这种抗性，允许他们有效的遗传操作。机械上,发现静态HSC的慢病毒载体转导被限制在载体进入水平和有限的嘧啶库。通过在慢病毒载体转导期间组合添加环孢菌素H(CsH)和脱氧核苷(dN)克服了这些限制。与标准离体培养的对照相比,临床相关的转导水平与更高的长期再增殖HSC的多克隆移植配对。这些发现确定了限制静止HSC转导的细胞内在屏障，并提供了克服它们的方法。为未受刺激的HSC基因工程铺平了道路。
