PDF(Pubmed)
Abstract：
This study lays the groundwork for future lentivirus-mediated gene therapy in patients with Diamond Blackfan anemia (DBA) caused by mutations in ribosomal protein S19 (RPS19), showing evidence of a new safe and effective therapy. The data show that, unlike patients with Fanconi anemia (FA), the hematopoietic stem cell (HSC) reservoir of patients with DBA was not significantly reduced, suggesting that collection of these cells should not constitute a remarkable restriction for DBA gene therapy. Subsequently, 2 clinically applicable lentiviral vectors were developed. In the former lentiviral vector, PGK.CoRPS19 LV, a codon-optimized version of RPS19 was driven by the phosphoglycerate kinase promoter (PGK) already used in different gene therapy trials, including FA gene therapy. In the latter one, EF1α.CoRPS19 LV, RPS19 expression was driven by the elongation factor alpha short promoter, EF1α(s). Preclinical experiments showed that transduction of DBA patient CD34+ cells with the PGK.CoRPS19 LV restored erythroid differentiation, and demonstrated the long-term repopulating properties of corrected DBA CD34+ cells, providing evidence of improved erythroid maturation. Concomitantly, long-term restoration of ribosomal biogenesis was verified using a potentially novel method applicable to patients\' blood cells, based on ribosomal RNA methylation analyses. Finally, in vivo safety studies and proviral insertion site analyses showed that lentivirus-mediated gene therapy was nontoxic.
摘要：
这项研究为未来慢病毒介导的基因疗法在核糖体蛋白S19（RPS19）突变引起的DiamondBlackfan贫血（DBA）患者中奠定了基础。显示新的安全有效疗法的证据。数据显示,与范可尼贫血（FA）患者不同，DBA患者的造血干细胞(HSC)储库没有明显减少,提示这些细胞的收集不应构成对DBA基因治疗的显著限制。随后，开发了2种临床适用的慢病毒载体。在以前的慢病毒载体中，PGK.CoRPS19LV,密码子优化版本的RPS19由磷酸甘油酸激酶启动子（PGK）驱动，已经用于不同的基因治疗试验，包括FA基因治疗。在后一种情况下,EF1α。CoRPS19LV,RPS19表达由延伸因子α短启动子驱动，EF1α(s)。临床前实验显示,用PGK转导DBA患者CD34+细胞。CoRPS19LV恢复红系分化，并证明了校正后的DBACD34+细胞的长期繁殖特性，提供改善红细胞成熟的证据。同时，使用一种适用于患者血细胞的潜在新方法验证了核糖体生物发生的长期恢复，基于核糖体RNA甲基化分析。最后,体内安全性研究和前病毒插入位点分析显示,慢病毒介导的基因治疗是无毒的.
