Georgakopoulou Aphrodite, Li Chang, Kiem Hans-Peter, Lieber André
University of Washington, Department of Medicine, Division of Medical Genetics, Seattle, WA 98195, USA.
Fred Hutchinson Cancer Center, Seattle, WA, USA.
Mol Ther Methods Clin Dev. 2024 Sep 21;32(4):101343. doi: 10.1016/j.omtm.2024.101343. eCollection 2024 Dec 12.
We developed an HSC gene therapy approach that consists of HSC mobilization and intravenous injection of HSC-tropic HDAd vectors. To achieve therapeutically relevant numbers of corrected cells, we incorporated expansion of transduced cells. We used an HDAd vector for a multiplex adenine base editing approach to (1) remove the region within CD33 that is recognized by gemtuzumab ozogamicin (GO) (Mylotarg), and (2) create therapeutic edits within the HBG1/2 promoters to reactivate γ-globin/HbF. studies with HDAd-transduced human CD34 cells showed editing of both targeted sites and a 2- to 3-fold GO-mediated expansion of edited erythroid/myeloid progenitors. After erythroid differentiation, up to 40% of erythrocytes were HbF positive. For studies, mice were transplanted with human CD34 cells. After engraftment, HSCs were mobilized with G-CSF/AMD3100 followed by an intravenous HDAd injection and GO-mediated selection. Two months later, editing in human cells within the bone marrow was significantly higher in GO-treated mice. The percentage of HbF human erythroid cells was 2.5-fold greater compared with untreated mice. These data indicate that GO selection can increase edited erythroid cells.
我们开发了一种造血干细胞（HSC）基因治疗方法，该方法包括HSC动员和静脉注射靶向HSC的重组腺相关病毒（HDAd）载体。为了获得治疗相关数量的校正细胞，我们加入了转导细胞的扩增步骤。我们使用HDAd载体进行多重腺嘌呤碱基编辑，以（1）去除CD33中被吉妥珠单抗奥唑米星（GO，商品名Mylotarg）识别的区域，以及（2）在HBG1/2启动子内进行治疗性编辑以重新激活γ-珠蛋白/HbF。对HDAd转导的人CD34细胞的研究显示，两个靶向位点均发生了编辑，并且编辑后的红系/髓系祖细胞在GO介导下扩增了2至3倍。红系分化后，高达40%的红细胞HbF呈阳性。在研究中，将人CD34细胞移植到小鼠体内。植入后，用粒细胞集落刺激因子（G-CSF）/AMD3100动员HSC，随后静脉注射HDAd并进行GO介导的筛选。两个月后，GO处理的小鼠骨髓中人类细胞的编辑水平显著更高。与未处理的小鼠相比，HbF人类红系细胞的百分比高出2.5倍。这些数据表明，GO筛选可以增加编辑后的红系细胞。
