Wang Hongjie, Georgakopoulou Aphrodite, Nizamis Evangelos, Mok Ka Wai, Eluère Raïssa, Policastro Robert A, Valdmanis Paul N, Lieber André
University of Washington, Department of Medicine, Division of Medical Genetics, Seattle, WA 98195, USA.
Ensoma, Inc., Boston, MA, USA.
Mol Ther Methods Clin Dev. 2024 Aug 13;32(3):101319. doi: 10.1016/j.omtm.2024.101319. eCollection 2024 Sep 12.
We developed an hematopoietic stem cell (HSC) gene therapy approach that does not require cell transplantation. To achieve therapeutically relevant numbers of corrected cells, we constructed HSC-tropic HDAd5/35++ vectors expressing a 3' UTR truncated HMGA2 gene and a GFP reporter gene. A SB100x transposase vector mediated random integration of the tHMGA2/GFP transgene cassette. HSCs in mice were mobilized by subcutaneous injections of G-CSF and AMD3100/Plerixafor and intravenously injected with the integrating tHMGA2/GFP vector. This resulted in a slow but progressive, competitive expansion of GFP PBMCs, reaching about 50% by week 44 with further expansion in secondary recipients. Expansion occurred at the level of HSCs as well as at the levels of myeloid, lymphoid, and erythroid progenitors within the bone marrow and spleen. Importantly, based on genome-wide integration site analyses, expansion was polyclonal, without any signs of clonal dominance. Whole-exome sequencing did not show significant differences in the genomic instability indices between tHGMGA2/GFP mice and untreated control mice. Auto-expansion by tHMGA2 was validated in humanized mice. This is the first demonstration that simple injections of mobilization drugs and HDAd vectors can trigger auto-expansion of transduced HSCs resulting in transgene-marking rates that, theoretically, are curative for hemoglobinopathies.
我们开发了一种无需细胞移植的造血干细胞（HSC）基因治疗方法。为了获得具有治疗意义数量的校正细胞，我们构建了表达3'UTR截短的HMGA2基因和绿色荧光蛋白（GFP）报告基因的嗜HSC的HDAd5/35++载体。SB100x转座酶载体介导tHMGA2/GFP转基因盒的随机整合。通过皮下注射粒细胞集落刺激因子（G-CSF）和AMD3100/普乐沙福动员小鼠体内的造血干细胞，并静脉注射整合型tHMGA2/GFP载体。这导致绿色荧光蛋白阳性外周血单核细胞（GFP PBMCs）缓慢但持续的竞争性扩增，在第44周时达到约50%，并在二次受体中进一步扩增。扩增发生在造血干细胞水平以及骨髓和脾脏内的髓系、淋巴系和红系祖细胞水平。重要的是，基于全基因组整合位点分析，扩增是多克隆的，没有任何克隆优势的迹象。全外显子测序显示tHGMGA2/GFP小鼠与未处理的对照小鼠在基因组不稳定指数上没有显著差异。tHMGA2的自动扩增在人源化小鼠中得到验证。这首次证明，简单注射动员药物和HDAd载体可以触发转导的造血干细胞自动扩增，从而产生理论上对血红蛋白病具有治愈性的转基因标记率。
